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UREA RETENTION MECHANISMS IN THE BRANCHIAL EPITHELniTM OF A
MARINE ELASMOBRANCH, THE S P D N DOGFISH (SQUAXUS ACANTHL4S)
A Thesis
Presented to
The Facdty of Graduate Studies
of
The University of Guelph
by
GLENN ALEXANDER FINES
In partial fiilfillment of requirernents
for the degree of
Master of Science
July, 2000
O Glenn A. Fines, 2000
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ABSTRACT
T m MECECANISMS OF UREA RETENTION IN BRANCHIAL EPITHELIUM OF ELASMOBRANCHS
Glenn Alexander Fines University of Guelph, 2000
Advisors: Dr. J.S. Baliantyne and Dr. PA. Wright
The retention of hi& concentrations of urea in the tissues of marine
elasmobranchs is the key to their osmoregulatory strategy. The rnechanisms responsible
for the low urea permeability of the gill epithelium nom the sphy dogfïsh (Squalus
acanthias) were investigated using enriched basolateral membrane vesicles (BLMV).
Urea uptake &=IO IIM) was sodium dependent and inhibited by phloretin (Iso=0.08
mM) and urea analogs (e.g. thiourea and methylurea). Much of the impermeability of the
impermeability of the BLMV may be due to the very high cholesterol content apparent
fiom the high cholesterol to phospholipid ratio (3.68). The high phosphatidylcholine and
low polyunsaturated fatty acid Levels are also likely to confer increased order to the
bilayer membrane, making it less fluid and less permeable.
Taken together, these findings indicate that low urea permeability in the dogfish
gill is primarily due to an active urea transporter that returns urea to the blood against the
concentration gradient and a unique lipid composition that minimizes diffusion urea
across the basolateral membrane.
ACKNOWLEDGEMENTS
1 gratefidly achowledge the f3nancial support of the Natural Sciences and
Engineering Research Council and the Huntsman Marine Science Centre. I would like to
thank my advisors, Dr. Jim BalIantyne and Dr. Pat Wright for their support and gïving me
the chance to work on this great project I a h want to thank Dr. Glen Van der Kraak for
serving on my advisory cornmittee and all of his helpful comments on my thesis. 1 thank
dl of my labmates (Jason, Natasha, Andy, Marc, J o ~ , Andrea, and Dave) for listening to
my incessant chattering about urea transporters and throwing great parties. 1 wouid like
to thank my family for their support. Finally, 1 wodd Like to thank Jennifer for her
support and encouragement.
TABLE OF CONTENTS
........................................................................................................ Açknowledgements i
.. Table of Contents .......................................................................................................... 11 ... ............................................................................................................... List of Tables iir
.............................................................................................................. List of Figures iv
.............................................. General Introduction .. .................................................. 1
Chapter 1 Active urea transport and an unusual basolateral membrane composition in the gills of a marine elasmobranch .......................................................... 11
........................................................................................................ Introduction -12
........................................................................................ Materials and Methods 15
................................................................................................................ Results -23
........................................................................................................... Discussion 45
Chapter 2 Lipid Composition of the Basolateral Membrane in Gill Epithelium of the ..................................................................... Spiny Dogfish (Squalus acanthias) 56
........................................................................................................ Introduction S7
........................................................................................ Materials and Methods 59
................................................................................................................. Results 61
........................................................................................................... Discussion 65
...................................................................................................... General Discussion 71
.................................................................................................................... References 79
................................................................................................................... Appendix I 95
.................................................................................................................. Appendiv II 96
LIST OF TABLES
Table 1.1. Marker enzyme specific activities and magnitude of purification of basolateral membrane ............................. ,.. ................................................. -24
Table 1.2. Total activity of marker enzymes, percent recovery, and percent contamination in the final basolateral membrane vesicle preparation ............. 25
Table 1.3. Percentage of the basolateral membrane fraction as resealed and the orientation of the vesicles ............................................................................... -26
Table 1.4. Percentage of phospholipid types and total phospholipid and cholesteroi in the basolateral membrane of gill epithelium fiom the spiny dogfish,
........................................ SquaItcs acanthias
Table 1 S. Cho1esterol:phospholipid ratios of representative species fkom difTerent .............................................................................................................. phyla.. .43
Table 1.6 Cornparison of cholesterol to protein ratios in the basolateral membrane of the gill epithelium fiom the spiny dogfïsh and a marine and fieshwater
............................................................................................................. teleost. -44
Table 2.1. Cumulative percentages of individuai fatty acids in gill basolaterd plasma membrane fiom Squalur acanthias ..................................................... 63
Table 2.2. Percentages of individual fatty acids in the major phospholipids fiom the ................................. gill basolateral plasma membrane of Squalus acanthias 64
iii
LIST OF FFGURES
Figure 1.1 Filter optirniration for autofluorescence and non-specinc binding of 14c-
urea for use in urea transport rapid filtration experiments. Blanks involve filtering the radioactive mixture only through the filter. Controls involve adding radioactive mixture to diluted vesicles and immediately filtering through the filter. (Mean t SE, n=2) ............................................................... 28
Figure 1.2 a. Rates of urea uptake at variable urea concentrations, by BLMV fiom the gill of the dogfkh, SquaZus acanthias. (Mean t SE, n=8). b. Expansion of the low end of the [urea] range fiom a. The regression is y = 0a07741n(x) + 0.01 l6 ,Z = 0.9064 (Mean & SE, n=7) c. Lineweaver-Burke transformation of the effect of variable [urea] on uptake rate by BLMV. The regression is y=2.96 12t29.8 lx, 2=0.9778 (Mean f SE, n=7) ................. 30
Figure 1.3 Dose dependent phloretin inhibition of urea uptake in BLMV fiom the gill of the dogfish, Squalus acanîhias (Ise = 0.08 mM) (Mean t SE, n=5) ...... 33
Figure 1.4 Inhibition of urea uptake in BLMV by the urea analogues, acetamide, thiourea, N-methylurea, and NPTU (3 70 mM). (Mean & S .E., n=3). ............ ..3 5
Figure 1.5 ATP (10 mM) stimulation of urea uptake and effects of ouabain (1 mM) and NEM (1 mM) in BLMV fiom the gill of the dogfkh, Squalur acanthias (Mean + SE, n=8). * significant merence fiom control (paired t-test,
............................................................................................................. pcO.05) 37
Figure 1.6 Rate of urea uptake in BLMV from the gill of the dogfish, Squalur acanthias, in the presence of physiologically oriented sodium (225 mM outwardly directed) or potassium gradients (225 mM inwardly directed) (Mean f SE, n=6). * sipifkant dserence fiom control (paired t-test, pcO.005) ** significant difference between sodium and potassium (paired t-
................................................................................................... test, pa).OO5) 39
Figure 1.7 Schematic representation of the proposed ~a+-cou~led, active urea transporter present in the basolateral membrane of the dogfkh gill
...................................................................................................... epithelium. -48
Fig. 1.8 A mode1 for the mechanism of reduced urea permeability of the dogfish gill basolateral membrane due to cholesterol. The cholesterol (red) contributes to a more tightly packed phospholipid bilayer membrane (yellow) thereby
... physically reducing the permeability of the basolateral membrane to urea.. 54
GENERAL INTRODUCTION
The vertebrate class Chondrichthyes (the cartilaginous fishes) is an ancient
lineage that contains two extant subclasses, the Elasmobranchii (sharks, skates, and rays)
and the Holocephali (ratfïsh or chimaeras) (Pough et al. 1996). The Elasmobranchii
evolved fiom the early chondrichthyans, which first appeared during the early Devonian,
approximately 400 million years ago (mya). The most recent radiation of elasmobranchs
appeared by the early Triassic (245 mya), with living families having evolved by the
Jurassic (208 mya) and extant genera appearing in the Cretaceous (144 mya) (Pough et al.
1996). Modem elasmobranchs typically occur in marine environments, dthough some
ascend rivers beyond tidal influence and some are permanent inhabitants of fieshwater
(i.e. Potomoîrygon spp.). The most fascinating characteristic of the elasmobranchs,
however, is the presence of elevated bIood levels of urea (350 to 600mM) (Robertson
1989). In most vertebrates urea is a waste product of amino acid catabolism and is
excreted fiom the body as rapidly as it is produced. Retention of such levels of urea in
elasmobranchs is therefore of considerable interest and has been under investigation for
over a century.
Discoverv of urea in elasmobranchs
Stadeler and Frérichs (1858) were the fist to discover the presence of "colossal
quantities" of urea in extracts fiom the muscle of Raja batis, R. clmata, and the dogfïsh
Scyllurn canicula (=Scylorhinus canicula). Their examination of other animals,
including teleosts and lamprey (Peh.omyzon sp.) revealed only traces of urea. Stadeler
(1 859) extended this observation to the spiny dogfish Spinm acanthias (=Squalus
acanthias) and the torpedos Torpedo marmorata and T. ocellata, and this was later
codbmed by Schultze (1 861) and Rabuteau and Papillon (1873). Knikenberg (1 88 1,
1 886, 1887, 1888) was the fïrst to undertake a systematic examination of the distribution
of urea in vertebrates and demonstrated that large amounts of urea were present not only
in the Selachii and Batoidei (=ElasmobranchÜ), but in the Chimaeroidei (=Holocephali)
as weU, although not in the lungfish (Neoceradohcs). This was an important observation
because of the close relation between the Elasmobranchii and Holocephali. Although the
lungfkh was thought to be extremely primitive, it was more closely affiliated with the
higher bony fishes. This suggested that the presence of elevated blood urea levels was an
evolutionary adaptation shared by ail Chondrichthyans. However, the function of urea in
these organisms and the physiological mechanisms by which the observed uraemic state
(pathological elevation of blood urea in mammals) is brought about, remained to be
described.
Earlv investigations of the function of urea in elasrnobranchs
The involvement of urea in the production of the electric discharge of the electric
organ of Torpedo ocellata was the first proposed function for urea in an elasrnobranch
(Gréhant and Jolyet 1891). They had observed that an increase in the urea content of the
electric organ tissue accompanied the electric discharge. Two years later Rohmann
(1893) reexamined the Gndings of Gréhant and Jolyet (189 1) and concluded that neither
urea nor any other nitrogenous compounds were involved in the production of electricity.
Baglioni (1906% 19 l7b) later confïrmed this by analysing various compounds in the
muscle, electric organ and senun of torpedo. His analyses revealed great difiibility of
urea and its nearly uniform distribution throughout the body of T. ocellata, including the
electric organ.
Another theory for the role of urea in elasmobranchs that was popdar for over 20
years at the tum of the century was that urea was essential for cardiac hct ion. Straub
(1901) found that 3.4% NaCl (the same osmotic pressure as elasmobranch blood) could
not keep the heart beating in good condition. Baglioni (1905) confinned Straub's
observation, having examined the effects of various NaCl solutions upon the heart and
concluded that urea was absolutely essential for cardiac activity. Baglioni (1905)
inferred that all elasmobranch tissues required urea for proper h c t i o n with the ideal
mixture being 2% NaCl and 2.2% urea. Further observations by Baglioni (1 9O6b)
demonstrated that the urine of elasmobranchs contained only low levels of urea, which he
believed to confirm his fïnding that urea was essential to life, by neutralizing the harmfùi
effects of high NaCl levels in the blood. Botazzi (1906), however, cnticized Baglioni's
experiments based on the fact that the red blood cells of elasmobranchs haemolysed in a
urea solution isotonic with 1.3-1.6% NaCl. He argued that a 3.4% NaCl solution was
hypertonie for the elasmobranch heart if the heart was equally permeable to urea because
the total osmolarity of the heart would be made up of both ionic and urea components,
not just ionic components. The key to a urea fiee perfusion medium eluded discovery
(Fühner 1908, De Meyer 19 10, Bornpiani 19 l3), and Baglioni (1 9 Ua) reasserted his
original view that urea was necessary for the maintenance of physiological activity in the
heart and other tissues. He even went so far as to classi@ urea as a hormone, according
to the definition of Bayliss and Starling (1902).
Frédéricq (1922) was the fist to attempt to disprove Baglioni's theory. He began
by confïrmïng that 3.5% NaCl was incapable of maintaining contractility in the ScyZZium
heart, whereas 2% NaCl and 2% urea was. However, he ccntinued by experimenting
with weaker salt solutions and found a urea-fkee solution would maintain a heart beat for
a long t h e when potassium and calcium was present in definite proportions. Frédéricq
concluded that urea plays an important role with regard to the osmotic pressure of the
blood, but is not important osmotically or chernically with regard to the tissues, which are
fkeely permeated by i t Simply stated the current thinking of the time held that urea
fünctioned in an osmotic role across the branchial lamellae, which are Unpermeable to it,
while within the organism it is essentially inert (Smith 1936).
Osmotic role of urea in elasmobranchs
The theory that urea was used for an osmotic hc t ion was hrst alluded to during
the early studies of elasmobranch physiology (Rodier 1 899, 1900, Frédéricq 19O4), but
was not M y embraced until much later (Frédéricq 1922, Duvall and Portier 1923, and
Smith 193 1). It was revealed that elasmobranch blood is normally hypertonie to seawater
@uvall and Portier 1923) and Duvall(1925) estimated that on average, urea is
responsible for 44% of the total osmotic pressure of the blood, and therefore contributes
significantly to the maintenance of the hypertonicity of the blood. This lead to the
conclusion that urea is osmotically important with regard to the extemal environment
(Duvall 1925). The analysis of the blood of fieshwater elasmobranchs revealed that the
chIoride content was 25% lower than in marine elasmobranchs (Thorson et al. 1 967).
Urea content is also rnuch lower, accounting for half of the 50% reduction in total
osmotic pressure of the blood. This discovery led to the conclusion that urea is
physiologically involved in osmotic adaptation, particularly to a marine habitat (Smith
193 1). This has been the accepted function for urea ever since and research has focused
on the mechanisms responsible for enabling the use of urea in this way.
Phvsiolo@cal mechanisms of urea retention
Znitially it was believed that the "rnechanism" responsible for the elevated blood
urea Zevels found in elasmobranchs was rend insufïiciency, or the inability of the kidney
to remove urea fiom the blood (von Schroeder 1890). This theory was short-lived,
however, and was discarded once it was realized that the elevated blood urea levels
played a physiological role (the exact role had yet to be elucidated). Thus began the
search for the mechanisms responsible for urea retention in the elasmobranchs.
It was quickiy determined that the elasmobranch kidney played an active role in
urea retention. Baglioni (1906~) reported that the urea content of elasmobranch urine
rarely rose above one third of that of blood. It was later calculated that approximately
90% of the urea filtered by the glomedus is reabsorbed by the kidney tubule (Clarke and
Smith 1932). The exact site within the kidney and the mechanism responsible for this
reabsorption are uncertain due to conflicting and incomplete evidence for active andor
passive urea reabsorption. Evidence for an active mode of urea reabsorption in the
elasmobranch kidney indudes: 1) the fractional excretion of urea is 0.5% under normal
conditions (similar to active glucose reabsorption) (Kempton 1953), 2) urea reabsorption
is iso-osmotic (Kempton 1953), 3) 95% of filtered urea is reabsorbed, whereas only 35%
of the urea analog thiourea is reabsorbed (Boylan 1967), 4) ~ a + and urea are reabsorbed
at a fked ratio of 1.6 moles of urea per mole of ~ a ' (Schmidt-Nielsen et al. 1972), and 5)
both phloretin and chromate (inhibitors of urea transport in the toad bladder) inhibit urea
reabsorption in the dogfish kidney (Hays et al. 1977). These finding have led to the
proposai of an active urea reabsorption mechanism (Smith 193 1, Kempton 1953,
Schmidt-Nielsen et al. 1972) with loop II of the elasmobranch nephron being implicated
as a possible site for active sodium-linked urea reabsorption (Stolte et al. 1977).
However, there is conflicting evidence which suggests the possibility for the
involvement of a passive urea transport mechanism in the reabsorption of urea in the
elasmobranch kidney, including: 1) failure to detect a transport maximum for urea despite
markedly raising plasma urea concentrations (Kempton 1953), 2) the urea andogs methyl
urea and acetamide do not saturate the urea carrier mechanism (Schmidt-NieIsen and
Rabinowitz 1964), and 3) probenicid, a substance which blocks active urea secretion in
the fiog, does not affect urea reabsorption in the dogfish (Forster and Berglund 1957).
Based on these results, an alternative passive model for urea reabsorption has been
proposed that involves iso-osmotic reabsorption of sodium and water, together with
tubular impermeability to urea in either or both loop II and III of the nephron (E3oyla.n
1972). This model is supported by cytological and histological evidence (Lacey et al.
1975, Endo 1984).
Detailed study of the elasmobranch nephron ultrastructure has revealed a complex
rend counter-current multiplier system that may be involved in fluid regdation and the
passive reabsorption of urea (Lacey et al. 1985). Further evidence supporting the passive
model is the molecular characterization of a facilitated urea transporter fiom the dogfish
kidney, which has a 66% identity to the- rat facilitated urea transporter protein UT-A2
(Smith and Wright 1999). Despite this new molecular data, definitive evidence for the
involvement of either active or passive urea reabsorption in the elasmobranch kidney is
stiil lacking. However, it appears most likely that a combination of counter-current
exchange and facilitated transport is responsible for the highiy efficient reabsorption of
urea by the elasmobranch kidney.
Urea retention bv the elasmobranch d l
Although urea reabsorption by the kidney has been the primary focus of most
researchers, the early studies of the kidney provided the diagnostic tools necessary to
facilitate the examination of the mechanisms involved in the retention of urea by the giU,
an important environmental interface. It was acknowledged early on that the gill was
involved in the retention of urea and that it must have special properties in order to
prevent excess loss of urea across its large s d a c e area. Homer W. Smith (1936) fïrst
proposed that the physicai properties of the gill were such that the gill was rendered
"highly serni-penneable" to urea. Boylan and his colleagues (Boylan and Antkowiak
1962, Boylan et al. 1963) performed the fist detailed examination of the permeability of
the elasmobranch gill. Using their gin-perfüsion technique, Johnson et al. (1964)
measured the rate of loss of urea and water and Farber et al. (1965) measured the rate of
loss of sodium across the dogfish gill and calculated their permeability coefficients
(urea=7.5x 1 ~ - ~ c m / s , &0=7.6~ 1 o 6 c d s , ~ a + = l . 9x 1 O-' cmk). They concluded that the
membranes of the dogfish gill epithelium are generally tighter to each species of solute
than the toad bladder and frog skin. The difference in urea permeability of the dogfish
gdl and the amphibian tissues was much greater than for either H20 or ~ a + . They
concluded that this large difference in urea permeability was due an active transport
process specific for urea. Boylan and Lockwood (1962) tested this hypothesis by
infusing thiourea into dogfish and measuring urea and thiourea loss across the gill.
Previously it had been demonstrated that the kidney rejects thiourea (37% reabsorption
vs. 95% urea reabsorption) (Boylan and Lockwood 1 962). If an active process specific
for urea was present in the gill, they proposed that thiourea should be lost at a greater
rate. However, their measured loss of urea vs. thiourea after thiourea infusion was not
signi£icautly Werent, leading to the conclusion that another process must be at work in
the gill. The effects of urea loading was determined (Boylan and Antkowiak 1962,
Boylan et al. 1963) and it was demonstrated that urea loss increased out of proportion to
the increased gradient. This evidence led these researchers to conclude that a physical
property of the structure of the gill membrane, as opposed to a transport system, was
directly affecthg its permeability. They reasoned that if a transport system was involved,
the loss of urea across the gill should have been proportional to the gradient once the
experimentally increased urea Ievels saturated the transport system. Farber et al. (1 965)
M e r ruled out the likeiihood of an active transport system when they tested the effects
of ouabain and chloromorodrh on the rate of urea loss at the giU and found no
diBeremes fkom the controls. The final conclusion of Boylan (1967) was that the relative
impermeability of the dogfïsh gil1 was due to special characteristics of the membrane
structure and probably not an active transport system.
Focus once again retumed to the elasmobranch kidney and for the next 25 y e m
essentially no M e r research was done to characterize the urea retention mechanisms in
the gill. Wood et al. (1 995) retumed to the gill and demonstrated that contrary to the
earlier studies (Boylan and Antkowiak 1962, Boylan et al. 1963) the infusion of
acetamide and thiourea ~ i g n ~ c a n t l y increased urea efflwc across the gill while the
infusion of urea did not. These results led to the conclusion that urea retention by the gill
was more complex than once thought, and probably involved a transport system (Wood et
al. 1995) in addition to the structural properties of the membrane as proposed by Boylan
(1 967)- This hypothesis was examhed M e r by Part et al. (1 998) with measurements of
the permeability coefficients for water and urea. These values were comparable to those
of Boylan (1 967) and they also demonstrated that phloretin, a non-competitive inhibitor
of urea transport, signifïcantly increased urea efflux at the gills without affecthg water
flux. Part et al. (1998) interpreted these results as providing evidence for a "back
transport" system that is present in the basolateral membrane of the gill epithelial cells
and that the permeability of the apical membrane to urea is very low. The properties of
this transport system and the membrane structure were not examined. Additional
circumstantial evidence for the presence of a urea transport system in the dogfish gill was
provided by Smith and Wright (1 999), when they performed low stringency Northem
analysis of gill tissue showing some cross-reactivity with the dog£ïsh kidney urea
transporter (S hUT).
These studies have provided evidence for the presence of a urea transporter in the
dogfish gill warranting M e r direct detection and characterization of such a system
using vesicles of isolated basolateral membrane. in chapter one 1 describe the methods
used for the isolation of the basolateral membrane fkom the gill epitheliurn of a
representative elasmobranch, the spiny dogfish shark (SquaZus acanthias), the kinetic
characterization of an active urea transporter in the basolateral membrane, and analysis of
the cholesterol content. In chapter two the basolateral membrane preparation was
analyzed for the phospholipid and fatty acid composition using chromatographie rnethods
(thin layer chromatography and gas chromatography). Taken together these studies
contribute to a more complete picture of the mechanisms responsible for the retention of
urea by the elasmobranch giIL
CHAPTER 1: Active urea transport and elevated cholesterol Ievels: Mechanisms
responsible for conferring Iow urea permeability to the elasmobranch gill.
INTRODUCTION
Urea is a molecule found in many organisms with a range of hctions, fiom
acting as a nitrogen source in prokaryotes to a prime waste product in many vertebrates.
In 1858, Stadeler and Frérichs first discovered the presence of "colossal quantities" of
urea in muscle tissue of marine elasmobranch fish (sharks, skates, and rays), sparking
research that has continued for over 140 years. At levels fiom 350 - 600 mM (Robertson
1989), urea functions as an osmolyte in the elasmobranchs, balancing the osmotic
pressure of seawater (Smith 1936). An important unsolved question concems how
elasmobranchs maintain these elevated levels of urea in theu body fluids and tissues,
against an essentially W t e gradient with seawater. Homer W. Smith k t provided
indirect evidence that the elasrnobranch kidney efficiently reabsorbs urea, preventing loss
via the urine (Smith 1936). The giU, with its huge surface area, is consequently the most
important site of urea loss to the environment (Wood et al. 1995). The elasmobranch gill
is relatively impermeable to urea compared to the gills of most teleost fishes (Smith
1936, Boylan 1967), but the mechanism(s) responsible for this impermeability is stiil
unknown.
One possible mechanism that may confer low urea permeabiiity to the
elasmobranch gill is the incorporation of a carrier-mediated urea transport system into the
basolateral membrane. In this case, a urea transport protein would have to be oriented to
return urea to the blood fiom the gill epithelium against the gradient. A variety of urea
transporters have been described fkom tissues of different species including mamrnalian
kidney (Sands et al. 1997), amphibian skin (Ehrenfeld 1998), toadnsh gill (Walsh et al. in
press) and dogfish kidney (Smith and Wright 1999), where thcy perfom various
functions. An experiment using the isolated pemised dogfish head preparation revealed
that phloretin increases urea efflux across the gill (Part et al. 1998), providing
physiological evidence for the presence of a gill urea transporter. In addition, low
stringency Northem analysis of dogfish gili tissue revealed a possible homolog to the
dogfish kidney phloretin-sensitive urea transporter (ShUT) (Smith and Wright 1999).
Taken together, these previous studies provided circumstantial evidence for a specialized
giU urea transport protein.
A second possible mechanism that may confer low urea permeability to the
elasmobranch gill is the incorporation of cholesterol into the phospholipid bilayer
membranes of the gill epithelial celis. Cholesterol is directly correlated with the
permeability of biological membranes to solutes such as urea (Pugh et al. 1989). The low
sodium and water permeability of elasmobranch gills, relative to teleost gills (Boylan
1967), supports the involvement of a general mechanism in conferring an overall
impermeability to the gill, such as the incorporation of cholesterol into the membranes.
The purpose of this study was two-fold: 1) examine the dogfish gill in order to
determine whether an active urea transport system is present in the basolateral membrane
of the gill epithelial ceUs and then characterize its properties if present, 2) examine the
Lipid composition of the dogfish gill basolateral membrane to determine if modifications
to the composition are consistent with the observed extremely low urea permeability.
Rates of urea uptake were measured using a rapid filtration method and resealed vesides
prepared fkom purifïed basolateral membrane. Validation of the purification procedure of
Perry and Flik (1988) for use with elasmobranch gill epithelial tissue was accomplished
by measuring the activities of the enzyme markers for various cellular membranes in the
initiai homogenate and the final basolateral membrane preparation. These measurements
dowed the determination ofthe purification, contamination, resealing, and orientation of
the BLMV. Vesicular volume was rneasured using a radioisotopic method (Brand 1995),
allowing additional c o b a t i o n of vesicle resealing. Urea transport in the BLMV was
estabfished and characterized wing I4c-urea and a rapid filtration technique (Perry and
Flik 1988). Cornpetitive and non-cornpetitive inhibitors of urea transport were tested to
characterize the mode of transport. The energy dependence of urea transport in BLMV
was determined by measuring urea uptake in the presence of ATP, with and without
inhibitors (ouabain and NEM) present. Finally, the ion specincity of urea transport in the
BLMV was determined by incubating vesicles with modified compositions of the intemal
and extemal mediums. The lipid composition and cholesterol content of the BLMV were
detennined using chromatographic and spectrophotometric methods, respectively.
MATERIALS AND METHODS
Experimental Animals
Dogfkh (Squalus acanthias) were obtained by otter trawl in Passamaquoddy Bay,
New Brunswick between mid-July and the end of August 1999 and maintained at the
Huntsrnan Marine Science Centre in 1000 L outdoor tanks under natural photopenod and
supplied with filtered seawater. Dogfish do not feed in captivity and were thus held for
no more than 10 days prior to use. Arctic char (Salvelinus a(pinus) were obtained fiom
stock maintained in the Hagen Aqualab at the University of Guelph. Winter flounder
(Pleuronectes americanus) were O btained b y otter trawl in Passamaquoddy Bay, New
.Brunswick at the end of August 1999, transported to the University of Guelph and
maintained in the Hagen Aqualab.
Gill basolateral plasma membrane vesicles
BLMV were prepared using the method of Perry and Flik (1988), with some
modifications. All steps were performed at W°C. Aduit dogfish were killed by a blow
to the head and the gill arches were removed without being perfûsed. The soft tissue of
the gill arches was scraped nom the cartilaginous tissue aod homogenized with a Dounce
homogenizer first with a loosely and then with a tightly fitting pestle (30 strokes each) in
15 ml of hypotonie homogenization b&er containing (in mM) 25 NaCl, 1 dithiothreitol,
0.5 disodium ethylenedinitrilo tetra-acetic acid (EDTA), 1 N-2-hydroxyethylpiperazine-
N'-2-ethanesulfonic acid (HEPES), 1 Tris[hydroxymethyl]-aminomethane hydrochloride
(Tris-HCl) (pH 8.0), plus 100U/ml Aprotinin. Following homogenization, the volume
was adjusted to a final volume of 50 ml with the same buffer. A sarnple (0.5 ml) was
saved for later enzyme analysis. This homogenate was divided into two centrifuge tubes
and centrifuged at 550 g for 15 min to remove nuclei and cellular debris (Pd. The
supernatant fkom the &st spin CS,) was decanted into a clean centrifuge tube and then
centrifuged at 50000 g for 1 h producing a pellet with a light portion (plasma membranes)
and a dark portion (mitochondria), instead of at 100000 g for 45 min. The light portion
of the pellet was shaken loose with 15 ml (instead of 60 ml) of sucrose b a e r containing
(in mMJ 250 sucrose, 5 MgC12, 5 HEPES, 5 Tris (pH 7.4) and homogenized (Dounce
homogenizer, tight pestle, 100 strokes). This second homogenate was centrifuged at
1000 g for 10 min and then 10000 g for 10 min producing a pellet containing the
remaining contamînating membranes. The supernatant was decanted into a clean
centrifuge tube and centnfuged at 30000 g for 45 min (instead of 30 min) to produce a
linal pellet of enriched basolateral membranes. This final pellet was re-suspended in 0.5
ml of suspension medium containing (in mM) 5 MgC12, 150 NaCl, 20 HEPES, 20 Tris-
HCl (pH 7.4) and was used immediately for enzyme analysis, vesicle volume
deterrninations, urea transport assays, protein concentration determinations, cholesterol
assays, and phospholipid analysis.
Marker e n m e s
Enzyme assays (n=6 individual dogfish) were performed on the initial
homogenate and the final pellet of enriched BLMV to determine the relative purity of the
final preparation and the relative contamination of the &al preparation by other cellular
membranes. Using published methods, N~+,K+-ATP~s~ (McCormick 1993), glucose-6-
phosphatase (Stio et al. 1988). cytochrome C oxidase (Blier and Guderley 1988), and
NMN-adenylyltransferase (Ruggieri et al. 1990) were used as marker enzymes for the
basolateral membrane, endoplasmic reticulurn, iiiner mitochondrial membrane, and
nuclear membrane, respectively. AU measurements were made in duplicate at 25OC in a
temperature controil ed Perkin Elmer Lambda 2 spectrophotometer (Perkin-Elmer Corp.,
Norwallc, CT).
Vesicle resealing and orientation
The extent of resealing and the orientation of the basolateral membranes were
determined using a previously descnbed method (van Heeswijk et al. 1984). The vesicles
were assayed for N~',K+-ATP~s~ activity in the presence and absence of the detergent
digitonin (0.04%) to determine the percentage of reseaied vesicle. To unmask inside-out
(IO) oriented reseaied vesicles, N~+,K+-ATP~s~ activity was determined in the presence
of 0.5 mM ouabain and the difference in K+ stimulated (10 mM KCl) ATP hydrolysis in
untreated and digitonin treated vesicles was determined. Vesicles assayed in EX1
containing medium were pre-incubated with 10 rnM KCI for 20 min on ice. The
percentage of right side-out ( ' O ) vesicles was then calculated as the difference between
resealed and IO vesicles. (See Appendix 1 for more details.) Vesicle resealing was also
determined in BLMV preparations that were fiozen at -80°C, thawed and passed through
a 23-gauge needle and 1 mi syringe ten times to determine the viability of fiozen BLMV
preparations.
BLMV volume measurement
BLMV v o l ~ e s were determined using a previously described method @rand
1995). In brief, 40 pl of BLMV (-0.1 mg of protein) were incubated in 1 ml of medium
containing 10 pI(1 pCi) of 3 ~ z ~ and 10 pl (0.1 pCi) of 1 4 ~ - ~ ~ ~ - 4 0 0 0 for 2 min at 37°C.
The BLMV were sedimented by centrifugation at 12000g for 4 minutes. The supernatant
(500 pi) was then transferred to a scintiilation vial and 15 ml of Scintisafe Econo F
scintillation cocktail (Fisher Scientific, Fair Lawn, NJ) was added. The remainder of the
supernatant was discarded, the pellet was re-suspended in 40p1 of 20% Triton X-100
(v/v) and mixed by vigorous vortex mixing. The base of the centrifuge tube was then cut
off, placed in a scintillation vial with 15 ml of scintillation cocktail and the radioactivity
counted with a liquid scintillation spectrometer (Mode1 121 1 Rack Beta, LKB-Wallac).
Vesicle volume was calculated in pVmg protein as ( 3 ~ z 0 space - 1 4 ~ - ~ ~ ~ - 4 0 0 0
space)/mg protein added (See Appendix II for more details.)
Urea trans~ort assaw
Measurement of the transport of 14c-urea into the BLMV was performed in
duplicate at 10°C by a rapid fi1tration technique as previously described (Perry and Flik
1988). Freshiy prepared BLMV pellets were resuspended at a protein concentration of
0.5 m g h l in resuspension buffer containing (in ) 300 NaCl, 5.2 KC1, 2.7 MgSQ, 5
CaC12, 370 D-mannitol, and 15 Tris-HC1 (pH 7.4) and allowed to equilibrate for one hou
on ice. The BLMV were then collected by centrifugation at 30 000 g for 45 min and
again resuspended at a protein concentration of approximately 6 mg/ml. Thorough
mixing was achieved by passage through a 23-gauge needle (10 times). Transport
experiments were initiated by mixing 10-pl aliquots of BLMV with 40-ul aliquots of the
radioactive elasmobranch incubation medium ( E i î ) (containing 50 pCi/ml 14c-urea),
vortexed and incubated for 15 sec at 10°C. EIM contained (in mM) 300 NaCl, 5.2 KCl,
2.7 MgS04, 5 CaCl*, 370 urea, and 15 Tris-HC1 @H 7.4). Using D-mannitol as an
osmotic substitute, EIM solutions containing urea concentrations of 1 mM to 370 mM
were prepared. The detailed composition of the EIM used for each incubation is
described in the corresponding figure for each experirnent. Incubations were terminated
by the addition of 1 ml of ice-cold stop solution (EIM containing 370 m . urea). The
diluted mixtures were immediately filtered through pre-wetted Millipore Isopore filters
(Millipore, 0.4 p m HTTP type). Filters were washed with two 3 ml aliquots of ice-cold
stop solution and placed in a via1 with 15 ml of Scintisafe Econo F scintillation fluid. The
radioactivity al l each vial was then counted in a liquid scintillation spectrometer (Mode1
12 1 1 Rack Beta, LKB- Wallac or Beckman 6400). Controls for each experiment involved
diiuting 10 pl of BLMV with 1 ml of ice-cold stop solution first, then adding 40 pl of
radioactive mixture and immediately filtering this through pre-wetted Isopore filters.
These filters were then treated as above.
Experiments
Filter control test. Six types of filters were tested for autofluorescence and non-
specific binding of 14c-urea. These were 0.8 p glass fibre (Whatman), 0.45 p PVDF
Durapore (Millipore), 0.6 p. mixed cellulose (Millipore), 0.45 p nylon (Magna), and 0.4 p
polycarbonate Isopore (Mïllipore) filters. Filters were tested for autofluorescence by pre-
wetting them with non-radioactive EIM and then counting as described above. Non-
specific binding of 14c-urea was determined by adding an aliquot of radioactive mixture
to a tube, diluting with 1 ml of ice cold stop solution and then filtering this through each
filter type followed by two 3 ml rinses of ice cold stop solution. The flters were then
counted as described above.
Concentration dependence. Urea uptake was measured over a range of urea
concentrations (1 -3 70 mM). The EIM solutions containhg concentrations of urea less
than 370 mM contained appropriate concentrations of D-mannitol, which fünctions as an
osmotic replacement, thereby elirninating the eEects of osmotic differences. Urea uptake
was measured as described above using each individual EIM solution.
Inhibition assays. Cornpetitive and non-cornpetitive inhibition of urea transport was
examined to M e r define the properties of the transporter. The urea analogues,
acetamide, N-methylurea, thiourea and nitrophenylthiourea were tested. BLMV
were prepared in the same manner as above but individual EIM solutions were made up
by substituting each of these compounds at a concentration of 370 mM in place of urea
and used in place of the mannitol containing re-suspension buffer. The vesicles
containing the respective analogue were then incubated with a radioactive mixture
containing 2.5 mM urea and 3 pCi of 14c-urea and treated as descnbed above. Stock
solutions of the non-competitive inhibitors phloretin (0.03-0.16 mM in ethanol),
amiloride (0.1 mM), bumetanide (0.1 mM in ethanol), N-ethylmaleirnide (NEM) (0.1
mM in ethanol), p-chloromercuriphenylsulfonic acid (pCMPS) (0.1 mM), and
phenylmethylsulfonyl fluoride (PMSF) (0.1 mM in ethanol) were prepared in ethanol or
water at a concentration of 250 mM. These stock solutions were diluted to give the final
concentrations in parentheses above ( h a l ethanol concentrations were less than 0.1%).
Prior to the addition of the radioactive mixture, 10 ul of each inhibitor was added to the
BLMV and vortexed. Then urea uptake was measured as described above.
ATP dependence. ATP dependence of urea uptake was determined by measuring urea
uptake in 15 mM urea EIM containing ATP (10 mM), ATP (10 mM) and ouabain (1
mM), ATP (10 mM) and NEM (1 m m , plus a control. Urea uptake was measured as
described above.
Cation speczficily. Cationic specincity of urea transport was also examhed in the
BLMV, using momed re-suspension buffer and radioactive mixture containing only one
of the following salts NaCl or KCl. BLMV were prepared as descnbed above. The h a l
pellet was resuspended in medium containing (in mM): 15 D-mannitol, 2.7 MgSQ, 5.0
CaC12, 15 Tris-HCl, and 250 NaCl or 25 KCl and 225 N-methyl-D-glucosamine
(NMDG). The incubation medium contained 1 5 mM urea in place of mannitol and 50
pCi/ml 14c-urea. Control incubations used the same medium in which the final pellet
was resuspended. Gradient incubations used (in mM) 25 NaCl and 225 NMDG or 250
KCl for sodium and potassium gradients, respectively.
Protein, phospholipid, and cholesterol rneasurements
The protein concentrations of the BLMV preparations fiom dog£ish, arctic char and
winter flounder were determined by the method of Bradford (1976) using a Bio-Rad kit
(Richmond, CA) with bovine semm albumin as the standard. The basolateral plasma
membrane phospholipids were extracted, separated, and analyzed as previously described
(Bligh and Dyer 1959, Ballantyne et al. 1993). Cholesterol levels of dogfish, arctic char,
and winter flounder BLMV were measured using a commercially available enzymatic
end-point assay kit (Sigma, St. Louis, MO).
Chemicals
Digitonin was obtained fiom The British Drug Houses (Canada) Ltd. (Toronto,
Canada). N-methylurea was obtained fiom Fluka through Sigma-Aldrich Chemicals.
Semicarbazide hydrochioride was obtained fkom Aldrich (Milwaukee, WI). 14c-
polyethylene glycol-4000 was obtained fiom Amersham Life Science (Baie d'Urfé,
Quebec). 3 ~ 2 ~ was obtained fiom Dupont-New England Nuclear (WiImington, DE). 14c-
urea was obtained fiom ICN (Montreal, Quebec). Al1 other chemicals were obtained
fiom either Fisher Scientific (Whitby, ON) or Sigma Chernical (St. Louis, MO) and were
of reagent grade.
Statistical methods
Values are expressed as mean + SE. Statistical cornparisons were made either by
Student's t-test or ANOVA with secondary Student's t-test and considered statistically
significant if pCO.05.
RESULTS
Marker Enzymes
The measurement of the four marker enzymes demonstrated that the basolateral
membrane preparation was highly purifïed and only slightly contaminated by other
membranes (Table 1.1 and Table 1.2). The marker enzyme for basolateral membrane
was enriched 4.12-fold. The specific activities of glucose-6-phosphatase (endoplasmic
reticulum) and cytochrome C oxidase (mitochondria) in the initial homogenate were 0.67
+ 0.25 and 5.72 + 1.98, and 0.07 + 0.07 and 0.17 f 0.06 in the f i a l BLMV preparation,
respectively (Table 1.1). This corresponds to a reduction in endoplasmic reticulum and
inner mitochondrial membrane of 9.49-fold and 34.25-fold, respectively. The nuclear
membrane was siightly enriched in the BLMV preparation with an increase in specific
activity of 1.1 9-fold (Table 1.1). However, the total ac tivity of NMN-adenyl yltransferase
(nuclear membrane) was only 0.55 umol substrate/ h which corresponds to 1.15%
recovery of the initial amount of enzyme and 2.96% contamination (percentage of the
total N~+,K+-ATP~s~ activity (Table 1.2). Endoplasmic reticulum and inner
mitochondrial membrane had recoveries of 0.15% and 0.05% of the initial amount of
enzyme and contributed 1.55% and 4.25% contamination (Table 1.2). Final recovery of
basolateral membrane was 6.15% (Table 1.2), which is similar to other studies (Perry and
F E 1988).
Vesicle Resealing, Orientation. and Volume
The passage of the basolateral membrane preparation through a 23-gauge needle
resulted in the formation of resealed vesicles (Table 1.3), approximately 35% of the
Table 1.1. Marker enzyme specific activities and magnitude of purification of each
membrane
N~+,K+- Glucose-6- Cytochrome C NMN-
ATPase phosphatase Oxidase Adenylyltransferase
umol substrate/h/mgprotein
Homogenate 1.02 k 0.25 0.674 f 0.25 5.72 t 1.98 0.11 kO.10
BLMV 4.20 f 1.71 0.071 + 0.07 0.17 t 0.06 0.13 + 0.10 Magnitude of Purz3cation
+4.12-fold -9.49-fold -33 -65-f01d +1. 194-fold
Values presented as mean t SE (~6).
Table 1.2. Total activity of marker enzymes, percent recovery, and percent contamination
in the final basolateral membrane vesicle preparation
Na-K-ATPase Glucose-6- Cytochrome C NMN-
phosphatase Oxidase Adenyl yltransferase
umol substruteh
Homogenate 302.67 k 35.87 197.39 k 5 1.52 1700.59 -t 481 -92 47.58 + 43.57
BLMV 18.61 t 8.16 0.29 I0 .23 0.79 4 0.47 0.55 + 0.46 Percent Recovery
6.1 5% 0.15% 0.05% 1.16%
Percent Contamination
- 1.56% 4.25% 2.96%
Values presented as mean + SE ( ~ 6 ) .
Table 1.3. Percentage of the basolateral membrane fiaction as resealed vesicles and
vesicle orientation (see text for details).
Percentage of basolateral membrane fiaction
Fresh 65.00 _+ 7.49% 9.41 i 232% 25.59 k 9.83%
Freezefïhaw 87.92 + 3 -92% 6.97 k 3-03% 5-11 -ir 0.89%
Values presented as mean k SE ( ~ 6 ) .
membrane population. This group of resealed vesicles is composed of 9.41% inside-out
and 25.59% right side-out orientation (Table 1.3). Freezing and thawing of the final
BLMV preparation resulted in a decrease in the percentage of resealed vesicles of both
orientations (Table 1.3), despite the passage of the membranes through a 23-gauge needle
&er thawing. The volume of the BLMV was relatively large, 8.60 f 1.78 p l h g protein,
providing additional confirmation of vesicle resealing.
Filter Control Test
None of the nIters tested exhibited any appreciable autofluorescence in the
scintillation cocktail used (Fig. 1.1 ; Blanks). The optimal filter type for use with I4c-urea
was found to be the polycarbonate Isopore filter from Mïllipore (Fig. 1.1). This filter
produced consistently low background counts due to non-specific binding of 14c-urea to
the filter. This accounted for approximately 10% of the total counts in the urea uptake
assays, similar to previous studies (Hopfer et al. 1973).
Concentration Dependence of Urea Uvtake bv BLMV
Urea uptake by BLMV was measured over a range of urea concentrations in the
incubation medium revealing two components of uptake (Fig. 1.2a). At hi&
concentrations (15 - 370 mM) urea uptake is linearily dependent upon the urea
concentration (Fig. 1.2a). However, at low urea concentrations (1 - 15 mM) urea uptake
exhibits saturation-like kinetics (Fig. 1.2b). When these data are transformed using a
Lineweaver-Burke plot, it is revealed that urea uptake at urea concentrations of 1 - 15
mM has a Km of 10.07 mM and V,, of 0.338 poVhr/mg protein (Fig. 1.2~).
Figure 1.1 Filter optimization for autofluorescence (blanks) and non-specific binding of
14c-urea (controls) for use in urea transport rapid filtration experiments. The blanks
represent fïiters only in scintillation cocktail. The controls involve adding radioactive
mixture to pre-diluted vesicles followed by immediate filtration through the filter. (Mean
k SE, n=2).
Figure 1.2 a. Rates of urea uptake at variable urea concentrations, by BLMV fiom the
giii o f the dogfïsh, Squaluî acanthias. (Mean k SE, ~ 8 ) . b. Expansion of the low end of
the [urea] range fiom A. The regression is y = 0.077Ln(x) + 0.0 12,8 = 0 .go6 (Mean + SE, n=7) c. Lineweaver-Burke transformation of the effect of variable [urea] on uptake
rate by B L W . The regression is y=2.96 + 29.8 lx, 1?=0.978 (Mean f SE, ~ 7 ) .
O 50 100 150 200 250 3 0 0 3 5 0 4 0 0
Urea concentration (rriM)
Inhibition of Urea Uptake by BLMV
Urea uptake by BLMV demonstrated sensitivity to the non-competitive inhibitor
phloretin (Fig. 1.3). Phloretin produced a dose-dependent inhibition of urea uptake by
BLMV, with 50 % inhibition occurring at a concentration of 0.08 mM. The use of urea
analogs demonstrated competitive inhibition of urea uptake by BLMV. N-methylurea
and NPTU significantly reduced the rate of urea uptake (pc0.05; Fig. 1.4). Thiourea and
acetamide also tended to reduce the rate of urea uptake although this decrease was not
significantly different fiom the control. The sulfhydryl reagents pCMBS, PMSF, and
NEM did not show any inhibition of urea uptake by BLMV (data not shown).
ATP Deuendence and Inhibition of Urea Uptake by BLMV
Urea uptake was signincantly stimulated by the addition of ATP to the incubation
medium @<0.05; Fig. 1 S). However, upon the addition of ouabain the rate of urea
uptake by BLMV decreased to control levels (Fig. 1.5). The addition of NEM had no
effect on the ATP stimulated urea uptake (Fig. 1.5).
Cation De~endence of Urea Uptake by BLMV
There was no significant difference between urea uptake in medium containing
ody sodium or only potassium ions with no concentration gradient present (Fig. 1.6).
When urea uptake was measured in the presence of a potassium concentration gradient
there was also no significant change in the rate of urea uptake by BLMV. However,
when a sodium concentration gradient was present during urea uptake measurernents, the
rate of urea uptake significantly increased (pC0.0 1 ; Fig. 1.6).
Figure 1.3 Dose dependent phioretin inhibition of urea uptake in BLMV fiom the gill of
the dogtïsh, SquuZus acanthias = 0.08 mM) @dean f SE, n=5).
Phloretin concentration (mM)
Figure 1.4 Inhibition of urea uptake in BLMV by the urea analogues, acetamide, thiourea,
N-methylurea, and NPTU (3 70 mM). (Mean t S E ., n=3)
Figure 1.5 ATP (1 0 mM) stimulation of urea uptake and effects of ouabain (1 mM) and
NEM (1 mM) in BLMV fkom the giII of the dogfish, Squalus ucunthias (Mean * SE, n=8). * significant dserence £iom contrd (paireci t-test, pc0.05)
Figure 1.6 Rate of urea uptake in BLMV fTom the giII of the dogfish, SquuZus acanthim,
in the presence of physiologicdy oriented sodium (225 mM outwardly directed) or
potassium gradients (225 mM inwardly directed) (Mean f SE, n=6). * significant
difference fiom control (paired t-test, p<0.005) ** significant clifference between sodium
and potassium @aired t-test, p<0.005)
j Control i / I Gradient I
Phospholipid composition and choIesterol content
In the BLMV of SquaIus acanthias the dominant phospholipid was PC,
representing 45.6% of the total membrane phospholipids, while PE and PS made up 26.5
and lî.3%, respectively, of the membrane composition (Table 1 -4). Sphingomyelin and
phosphatidylinositol were minor components of the membrane phospholipids. The
mitochondrial phospholipid cardiolipin was present in a smaii amouut, 2.6% of total
membrane phospholipid due to the minor mitochondrial contamination indicated above.
The cholesterol content of BLMV was reiatively high (Table 1.4) resdting in a C: P ratio
of 3.68 & 0.26 (n=8; Table 1.5). The dogfish also had the highest cholesterol to protein
ratio when compared to seawater and fieshwater teleosts (Table 1 A).
Table 1.4. Percentage of phospholipid types and total phospholipid and cholesterol
in the basolateral membrane of gill epithelium fiom the spiny dogfish, SquaZus
Cardiolipin
Phosphatidylcholine
Phosphatidylethanolarnine
Phosphatidylinositol
Phosphatidylserine
Sphingomyelin
PCIPE
TotaI Phospholipid
(nmoVmg protein)
TotaI Cholesterol
( n m o h g protein)
Cholesterol: phospholipid
(moVm01)
Values presented as means & S.E. (n=8). PC, phosphatidylchohe; PE, phosphatidylethanolarnine
Table 1.5 Cholesterol:phospholipid ratios of representative species fkom difEerent
C:P Species Tissue SubceiIular Fraction (mol mol-') Reference
Spiny Dogfish Gill Basolateral Membrane 3.68 i 0.26 Present
Rat (Ratttcs nomegrgreus)
Little Skate (Raja erinacea) IRaUibow Trout (Oncorhynch mykiss)
Squid (Loligo pealei)
Crab (Cancer pagurrrs)
Crab (Carcinus maenus)
Cucumber (Cucu~bitafic@iolia) Fungus (Bohyfis cinerea)
Bacteria (Methylococcus
Epithelium Lens
Intestine
Liver
Intestine
Cerebral and Optic Lobes Steilate Ganglia Leg muscle
Leg muscle
Root
Hypha
Plasma Membrane
Bnish Border Membrane Basolateral Membrane
Basolateral Membrane
Brush Border Membrane
Basolaterai Membrane
Plasma Membrane
Plasma Membrane
Plasma Membrane
Plasma Membrane
Plasma Membrane
Plasma Membrane
Plasma Membrane
mdy B orchman et al. 1989 Molitoris et
al. 1985 Molitoris et al, 1985
Smith and Ploch 199 1
Crockett and Hazel
1995 Crockett
and Hazel 1995
Yamaguchi et al. 1987 Yamaguchi et al, 1987 Cuculescu et al. 1995 Cuculescu et ai. 1995 Bulder et ai. 1991
Kodali et al. 1998 Jahnke
1992 caps ulatus)
9otal stero1:phospholipid ratio
Table 1.6. Cornparison of cholesterol to protein ratios in the basolateral membrane of the
giii epithelium fiom the spiny dogfish and a marine and fieshater teleost.
Fresh Water Arctic Salt Water Arctic Winter Spiny Dogfish
Char ( ~ 8 ) Char (n=8) Flounder (n=6) (n=8)
Mean 0.070 0.050a 0.064~ 0.1 leacd S.E. 0.004 0.004 0.005 0.008
'Signincantly difEerent fkom Fresh Water Arctic Char @<0.005) b~ignificantly different fiom Salt Water Arctic Char @<O.OS) CSignincantly dBerent fiom Salt Water Arctic Char @<0.000005) * ~ i ~ n i n c a n t l ~ different nom Winter Flounder (pc0.0005)
DISCUSSION
Methodolow
The method (Perry and Flik 1988) used to prepare basolateral plasma membranes
Çom the gill epithelium of the spiny dogfish (Squulus acanthias) yielded a specific
enrichment of ~ a f , K'-ATP~s~ indicating selective isolation of basolateral plasma
membranes. Although there was only minor contamination (6%) of membranes fiom
the endoplasrnic reticulum, mitochondria and nucleus, this may have led to a slight
underestimation of urea uptake by the gill BLMV. The final recovery of ~ a * , lC'-~TPase
activity (6.2%) is consistent with previous studies (Perry and Flik 1988), while the
vesicle orientation (IO=9.4%; RO=25.6%) and resealing efficiency (35%) were
somewhat lower than reported values for eel (IO=33%; RO=23%; resealing efficiency
56%) (Flik et al. 1985). The volume measurements c o b e d that the vesicles
successfblly resealed. The decrease in resealing efficiency afier fieezing and thawing of
the final BLMV preparation suggested decreased integrity of the membranes and
therefore onl y fieshly prepared BLMV were used for transport experirnents. Significant
t h e was saved by omitting the g d perfüsion step, used to clear the gills of red blood
cells (Perry and Flik 1988), as there are no urea transporters present in elasmobranch
erythrocyte plasma membranes (Carlson and Goldstein 1997).
Urea Transport
The measurement of urea uptake by enriched BLMV revealed saturation kinetics at
low urea concentrations (KmlO.1 mM, Vm,=0.34 pmoVh/mg protein), suggesting the
presence of carrier-mediated urea transport. The low Km, relative to the urea
concentration in the blood, indicates that the transporter has a relatively high affinity for
urea. This implies that the putative urea transporter acts to "scavenge" intracelldar urea,
actively returning it to the blood and thereby maintaining a low urea concentration within
the gill epithelial cells. The effects of several known inhibitors of urea transport were
examined to M e r characterize the saturable component of urea uptake by the BLMV.
Inhibition by the non-competitive inhibitor phloretin is diagnostic of both facilitated and
secondary active, urea transport systems Wato and Sands 1998, Levine et al. 1973, Smith
and Wright 1999, Walsh et al. 1994). The dose-dependent inhibition of urea uptake in
shark gill BLMV by phloretin in this study is consistent with a previous study on the
isolated perfused dogfish head preparation (Part et al. 1998), which demonstrated that
phloretin &ion signifïcantly increased urea efflux across the gill. The urea analogues
N-methylurea and NPTU also signincantly inhibited urea uptake in shark gill BLMV.
Acetamide and thiourea had a slight, but non-signincant effect on urea uptake rates.
However, these results are inconclusive u t i l the analogue concentrations are optimized,
since studies have shown that under dif5erent conditions urea analogues may or may not
have statistically signincant effects on the branchial urea efflwc in the spiny dogfkh (I?W
et al. 1998, Wood et al. 1995). In mammalian inner medullary collecting duct,
methylurea and thiourea signi6cantly reduced urea permeability, while acetamide did not
(Chou and Knepper 1989). These results demonstrate that urea transporters in difZerent
tissues and animals exhibit dif3erent sensitivities to urea analogues. The inhibition of
urea uptake in shark gill BLMV by phloretin and urea analogues supports previous
hypotheses (Smith and Wright 1999, Wood et al. 1995) for a carrier-mediated urea
transport system in the dogfish shark gill.
Urea uptake is energy dependent in shark gill BLMV. The addition of adenosine
triphosphate (ATP) to the incubation medium sienif?cantly increased the rate of urea
uptake, whereas ouabain, a specific inhibitor of the enzyme N~~,K+-ATP~s~, returned the
rate of ATP-dependent urea uptake to control levels. In the presence of NEM, an
alkylating agent that binds selectively to sulfhydryl groups blocking V-type and P-type
ATPases (e-g. proton pumps) (Ehrenfeld 1998), ATP-dependent urea uptake was
unchanged. This ouabain sensitivity, but NEM insensitivity, of ATP-dependent urea
uptake in shark gill BLMV signifies that urea uptake was indirectly ATP dependent,
coupled to either the sodium or potassium gradients created by N~+,K+-ATP~s~. In the
presence of an outwardly directed sodium gradient, urea uptake was signincantly higher
relative to both the control and potassium gradient experiment. Taken together, these
data confirm that the saturable component of urea uptake in the shark gdi BLMV is due
to a ~ a + - c o u ~ l e d secondary active urea transporter, similar to the type found in the
mammalian kidney (Kato and Sands 1 998).
The dog£ïsh gill urea transporter functions in an antiport fashion (Fig. 1.7), similar
to the transporter described in the rat inner medullary collecting duct, but differs from the
putative sodium-linked urea transporter described fiom the dogfkh kidney (Schmidt-
Nielsen et al. 1972), which is thought to k c t i o n in a symport fashion. By using the
inwardly directed concentration gradient of ~ a + , the dogfkh gill urea transporter actively
pumps urea back into the blood fiom within the cell. This would decrease the
intracellula. urea concentration of the gill epithelial cells, reducing the concentration
gradient for urea across the apical surface of the cells and thus the rate of urea diffusion
or loss across the gill. Depending on the stoichiometry of the exchange, the shark can
Fig. 1.7 Schematic representation of the proposed Na+-coupled, active urea transporter
present in the basolateral membrane of the dogEish gill epithelium. 1 propose that the
active retum of urea to the blood reduces the intracellular urea concentration thereby
reducing the rate of urea diffusion across the apical membrane to the seawater.
(Thickness of the dashed arrows indicate the relative rate of urea diffusion.)
Seawater O mM Urea
Blood 370 mM Urea
Urea
1 I I I
Urea
7
Apical
Basolateral
Urea ~ a + K'
Save up to 5 ATP equivdents (the metabolic cost of synthesizing one urea molecule via
the ornithine-urea cycle including glutamine synthetase) for each irrea molecule returned
to the blood. In the dogfrsh kidney, sodium and urea are reabsorbed at a Itixed ratio of 1.6
moles of urea per mole of ~ a + (Schmidt-Nielsen et ai. 1972). The energetic cost of
transporthg ~ a + via ~a+,lK+-~TPase is 1 ATP for every 3 moles- If a similar ratio of
exchange for sodium and urea were involved in the gill as in the kidney, savings of 4.8
ATP equivdents per molecule of urea retumed to the blood would be achieved. This
means that the metabolic cost associated with urea transport in h e gill is low, but the
metabolic savings are significant.
Gill basolateral membrane composition
In the basolateral membrane of the gill f?om the spiny dogfîsh, SquaZus acanthias,
PC and PE were the main phospholipids, typical of most eukaryotic membranes. The
resulting PC/PE ratio was 1.72. PC stabilizes the membrane as it favors the formation of
a laminar bilayer, while PE destabilizes the membrane by keeping it close to the phase
transition between laminar and hexagonal (HII) phase conformaQions (Thurmond and
Luidblom 1997). In most temperate species the ratio of PC to PE is approxirnately 1.0.
This ratio can be altered according to the enivronmental and physiologica1 conditions
encountered by the organism. For example, cold-acclimated organisms (Arctic char,
molluscs) increase the proportion of PE in their membranes iin order to maintain
membrane fluidity at low temperature (Hazel 1995). This results im a PCPE ratio of less
than one (0.3-0.5). However, in elasmobranchs the cellular membranes face the opposite
problern, increased fluidity due to urea (Barton et al. 1999). Orne adaptation that has
evolved in elasmobranchs to deal with this effect of urea is the presence of
trimethylamine oxide (TMAO). TMAO counteracts the negative effects of urea on both
proteins (Yancey and Somero 1980) and phospholipid membranes (Barton et al. 1999).
Another strategy, which may work in conjunction with TMAO, is increasing the ratio of
PC to PE, which would provide additional stability to the membrane.
The very high cholesterol to phospholipid molar ratio (3.68) reported here is the
highest for native membranes (Table 1.5). Cholesterol decreases the permeability of
biological membranes to urea (Pu& et al. 1989) by inducing an increased order of the
phospholipid molecules that compose the bilayer membrane, allowing them to pack
closer together forming a tighter barrier (Mouritsen and Jmgensen 1994). Cholesterol,
when inserted in the appropriate place in the membrane, also increases membrane
permeability to oxygen (Dumas et al. 1997), therefore the elevated cholesterol levels in
shark gill basolateral membranes would not impair gas exchange. We propose that the
high cholesterol content of shark gill basolateral membrane provide a physical barrier that
retards passive loss of urea at the gill without affecting oxygen permeability (Fig. 1.8).
This hi& C:P molar ratio may also explain the low permeability of the shark gill to water
(Boylan 1967, Part et al. 1998) and sodium (Boylan 1967) relative to teleost fishes. Our
hdings also point to the possibility of similar structural modifications occurring in the
mammalian kidney tubule where urea permeability varies dong the nephron (Sands et al.
1997).
In Perspective
Based on the results of this study we propose that a unique combination of
physiological and structural mechanisms is at least in part responsible for the low urea
permeabiLity of the dofish shark gill. The marine elasmobranch gill is approximately 80
h e s less permeable to urea than the teleost gill resulting in a urea efflux of 270
poVkg/hr (Part et al. 1998). If the elasmobranch gill were as permeable to urea as the
rainbow trout gill, the resulting urea efflux would be immense (10,000 pol/kg/hr) (Part
et al. 1998), because of the enormous blood-to-water gradient. Ushg data from the
present study, it is possible to calculate the relative contribution of active urea transport
to the ciifference between observed and predicted (based on rainbow trout gill urea
permeability) urea efflux rates. The maximal velocity of urea uptake (Vrn~0.34
jmol/h/mg protein) was corrected for vesicle resealing (35%), vesicle orientation (27%
of resealed vesicles), membrane recovery (6.15%), protein level (9 mg/animal), and
animal mass (1.3kg). This results in a total rate of active transport of urea, back into the
blood fiom within the gill epithelium, of 535 ~ o V k g / h . This value is approximately 6%
of the difference in urea permeability between the rainbow bout and elasmobranch gill
(i.e. 10 000 - 270 p o v k g h ) . The remaining 94% may be in part, or in whole, due to
the elevated C:P molar ratio in the basolateral membrane. Thus we envision that the
primary role of the basolateral membrane is to substantially reduce the influx of urea into
the gill epithelial cells, thereby maintaining low intracellular urea concentrations at which
the urea transport system hct ions . The urea transport system actively transports urea
out of the epitheiial cells back into the blood maintaining low intracellular urea
concentrations. The low intracellular urea concentrations achieved by these
conplimentary mechanisms, leads to a reduced diffhion gradient for urea across the
apical membrane and thus a lower effective permeability of the gill to urea. One could
argue, therefore, that high cholesterol levels and active urea transport in the gill
basolateral membranes probably CO-evolved in elasmobranchs enabling the retention of
m a and its use as a key component of their osmoregulatory strategy, while minimising
the energetic cost. However, the hi& non-specific urea uptake by the BLMV suggests
that there are other mechanisms andor structures, particularly the composition of the
apical membrane, which may contribute signifïcantly to the overall low urea permeability
of the elasmobranch gill. Further studies of the elasmobranch gill are thus required to
completely resolve this issue.
Fig. 1.8 A mode1 for the mechanism of reduced urea permeabiiity of the dogfish gill
basolateral membrane due to cholesterol. The cholesterol (dark) contributes to a more
tightly packed phospholipid bilayer membrane (light) thereby physically reducing the
pemeability of the basolateral membrane to urea.
UREA
UREA
CHAPTERZ: A comparison of phospholipid and cholesterol composition of the
basolateral membranes from the gill epithelium of an elasmobranch, the dogfïsh
(SquaZus acan fhias)
INTRODUCTION
Elasmobranchs are unique among marine fish (except for the coelacanth) in that
they retain high leveb of urea (400-600 mM) io their tissues and body fluids for the
purpose of osmoregulation (Smith 193 6). Although urea retention provides an efficient
mechanism for osmutic balance, the chaotropic properties of urea pose another problem,
protein and phospholipid bilayer destabilization. The presence of trirnethylamine oxide
(TMAO) in the blood and tissues counteracts the destabilizing properties of urea on
protein (Yancey and Somero 1980) and membrane integrity (Barton et ai. 1999).
Modification of the phospholipid and fatty acid composition of cellular membranes is
also invoived in maintainhg membrane integrity of fishes under a variety of
environmental conditions (i.e. temperature and salinity) (Hazel and Williams 1990).
Phosphatidylcholine (PC) with a high content of saturated fatty acids (SFA) stabilizes
phospholipid bilayers, while phosphatidylethanolamine (PE) containing more unsaturated
fatty acids, destabilizes phospholipid bilayers in fish (Hazel and Landrey 1988a,b).
Elasmobranch mitochondrial membranes have signincantly higher levels of saturated
fatty acids and signincantly lower levels of polyunsahirated fatty acids (PUFA) relative
to other marine fishes such as the hagfïsh and winter flounder (Glemet and Ballantyne
1996). It has been suggested that these characteristics are adaptations to maintain
mitochondrial membrane i n t e e in the presence of hi& urea concentrations in the
tissues (Glemet and Ballantyne 1996), but other elasmobranch membranes have not been
examined.
The elasmobranch gill, like the gill of al1 fish, is involved in gas exchange,
ionoregulation, and osmoregulation. In addition to these vital fùnctions the elasmobranch
gill must also act as a barrier to urea loss. Compared to the teleost gill, the elasmobranch
gill is approximately 80 times less permeable to urea than the teleost gül (Part et al.
1998). The basolateral plasma membrane of the elasmobranch gli epithelium is in
contact with high levels of urea in blood on the extraceliular side and purportedly low
levels of urea on the intracellular side and may be the main permeabiiity barrier (Wood et
ai. 1995). In chapter one, we identified a ~a+-coupled active urea transport mechaaism
(K,=10 mM) in the basolateral membrane (BLM) of S. acanthiar gills that would act to
scavenge intracehlar urea and retum it to the blood, against the concentration gradient.
In addition, we reported that these same membranes had a cholesterol (C) to phospholipid
(P) molar ratio of 3.68, an extremely hi& value compared to other tissues and organisms
(Table 1.5). A high C:P molar ratio is characteristic of membranes with reduced
permeability to low molecular weight solutes, such as urea (Lande et al. 1995). Taken
together, the urea transport system and hi& C:P molar ratio would facilitate urea
retention in dogfïsh gills. It is also possible that the phospholipid and fatty acid
composition may contribute to the low permeability of the dogfkh gill to urea. The lipid
composition of various epitheiial tissues (Le. mammalian epidermis and trout intestine,
skin, and opercular membrane) has been demonstrated to innuence ~ a + and water
permeability @i Costanzo et al. 1983, Ziboh and Miller 1990, and Ghioni et al. 1997).
The purpose of this study was to characterize the phospholipid and fatty acid
composition of the basolateral plasma membrane fiom the dogfïsh gill epithelium. We
hypothesize that the phospholipid and fatty acid composition of this membrane, coupled
with the high C:P molar ratio, is in part responsible for maintaining membrane integrity
and contributhg to the extremely low permeability of the elasmobranch gill to urea.
METHODS AND MATERIALS
Ex~erimental animals
Spiny dogfish (Squalur acanthiar) were coliected by Otter Trawl in
Passamaquody Bay, New Brunswick fiom mid-July to the end of August 1999 and
maintained at the Huntsman Marine Science Centre in outdoor tanks under natural
photoperiod, and supplied with filtered seawater (lO°C). The dogfish did not feed in
captivity and were heId for a maximum of 10 days prior to experimentation.
Basolateral membrane @LM) preparation
BLM was prepared £kom epithelial tissue scraped fiom the gill arches. Tissue was
homogenized using a Dounce homogenizer in a hypotonie homogenization b a e r
containing (in mM): 25 NaCl, 1 dithiothreitol, 0.5 disodium ethylenedinitrilotetra-acetic
acid (EDTA), 1 N-2-hydroxyethylpiperazine-N'-2-ethanesulc acid (HEPES), 1
Tris [hydroxymethyl] aminomethane hydrochloride (Tris-HCl), pH 8 .O, plus 1 00U/ml
aprotinin. This homogenate was centrifuged at 550 g for 15 min and the supematant
decanted and centnfuged at 50000 g for 1 h. The light portion of the resultuig pellet
(plasma membranes) was homogenized again and centrifûged at 1000 g for 10 min,
10000 g for 10 min and the rernainîng supematant was then centrifuged at 30000 g for 45
min producing a h a 1 pellet of e ~ c h e d basolateral membranes. The BLM were then
fiozen and stored at -80°C for further analysis.
Characterization of basolateral membrane purity
Marker enzymes were measured in both the initial homogenate and in the BLM
preparation. The following were used as markers for cellular membranes: basolateral
membrane - N~+,K+-ATP~s~ (McCormick 1990), endoplasmic reticulum - glucose-6-
phosphatase (Stio et al. 1988), mitochondnal membrane - cytochrome C oxidase @lier
and Guderley 1988), and nuclear membrane - NMN-adenylyltrmsferase (Ruggieri et al.
1990). Al1 measurements were made in duplicate at 25OC.
Analvsis of basolateral membrane phospholipid composition
The BLM phospholipids were extracted, and separated as described by Bligh and
Dyer (1959). Phospholipid separation using thin layer chromatography and fatty acid
analysis using gas chromatography were as described previously (Baliantyne et al. 1993).
Protein measurements
The protein concentrations of the BLM preparations were determined by the
method of Bradford (1976) using a Bio-Rad kit (Richmond, CA) with bovine serum
albumin as the standard.
Chemicals
The lipid standard used (Nu-Chek-Prep., Inc. Elysian, MN) was augmented by the
addition of 15:0, 15:1, and 17:l methylated fatty acids. Solvents were obtained fiom
Fisher Scientific Ltd. (Whitby, Ontario) and were of American Chemical Society-
certified grade. Ail other chemicals were obtained fiom Sigma Chemical Co. (St. Louis,
MO) and were of the highest purity available.
Characterization of basolateral membrane puritv
The BLM was substantially enriched in the basolateral membrane marker N~+,K+-
ATPase, in relation to the marker enzymes for endoplasmic reticulum (glucose-6-
phosphatase), mitochondrial membrane (cytochrome c oxidase), and nuclear membrane
(NMN-adenylyltransferase) (see Table 1.1). This indicated little contamination of the
basolateral membrane with these cellular membranes.
Phos~holipid composition
In the BLM, the dominant phospholipid was PC, representing 46% of the total
membrane phospholipids, while PE and PS made up 27% and 12%, respectively, of the
membrane composition (see Table 1.4). Sphingomyelui and phosphatidylinositol were
minor components of the membrane phospholipids. The phospholipid cardiolipin was
present in a small amount, 2.6% of total membrane phospholipid (see Table 1.4), similar
to the level of mitochondrial contamination indicated by marker enzyme measurements
(see Table 1.2).
Gill basolateral membrane fattv acid composition
The predorninant fatty acids in the cumulative fatty acid composition of
membrane phospholipids were l6:O, 18:0, 18: 1, 20:4n-6, 20:5n-3, 22:6n-3 (Table 2. l),
which accounts for 82% of the cumulative fatty acid composition. Saturated fatty acids
were the prevalent class of fatty acids (38 %) (Table 2.1).
In the BLM, PUFA was the largest class of fatty acids in the phospholipid PE
(Table 2.2). The main individual fatty acids were 20:4n-6, 20511-3, and 22:6n-3 totaling
39% (Table 2.2). PE had the highest levels of an unknown fatty acid (Table 2.2). This
unknown fatty acid was tentatively identïfïed as 16:3 or 16:4 based upon the retention
time relative to stearic acid (Ackman 1962). In PI, SFA and PUFA were the prevalent
classes of fatty acids (Table 2.2). This was due to the large proportions of 18:O and
20:4n-6 (Table 2.2). In PS, SFA and PUFA were the most abundant classes of fatty acids
(Table 2.2) due primarily to 18:0, 20:4n-6 and 22:6n-3 although the monoene 18: 1 was
also prevalent (Table 2.2). In PC, SFA was the dominant fatty acid class (Table 2.2) as a
result of the levels of 16:O (Table 2.2). The monoene 18: 1 was also prevalent in PC
equalling (Table 2.2). Monoenes comprïsed the largest class of fatty acids in SM (Table
2.2). This was due to the levels of 14:l and 24:l (Table 2.2). The saturated fatty acids
14:O and 16:O were also prominent in SM (Table 2.2).
Table 2.1 Cumulative percentages of individual fatty acids in gin basolateral plasma
membrane fiom Squalus acanthiasa
Fatty acid (mol %)
24: 1 1.59 10.19 Total SFA 38.11 I 1.70 Total Monoene 28.02 10.72 Total PUFA 34.88 I 1.61 Total n-3 PUFA 17.86 10.97 Total n-6 PUFA 17.02 k0.80 n-3/n-6 PUFA 1 .O5 I 0.05 Unsaturation indexC 186.48 I 56.58 Mean Chain lengt.hd 18.66 & 0.40
Values presented as mean f S.E.M. (n=8). bvnknown A (9.08 min) eluted between 17: 1 (8.76 min) and l8:O (9.32 min). 'Unsaturation index = Çrnim; where mi is the mole percentage and ni is the number of C-C double bonds in fatty acidi. %lean chain length = Xfci; where 6 is the mole hction and Ci is the number of carbon atoms in fatty acidi.
Table 2.2 Percentages of individual fatty acids in the major phospholipids fiom the gill
basolateral plasma membrane of Squalus ucanthiasa
Fatty Acid Phosphatidyl- Phosphatidyl- Phophatidyl- Phosphatidyl- Sphingomyelin choline ethanolamine inositol serine
1.81 f 0.13 0.40 f 0.11 0.08 I 0.01 0.05 I 0.01 10.68 & 1.63
0.45 I 0-06 0.05 i 0.04 ND 0.50 I 0.25 28.99 rt 2.13 Total SFA 45.91 f 1.20 20.54 I 2.02 45.42 i: 4.87 45.06 I 6.62 38.39 2.34 Total monoenes 29.50 f 0.53 33.28 + 1.41 4.79 I 0.61 18.72 f 2.97 53.78 i: 1.91 Total PUFA 25.16 i: 1-57 47.90 I 2.70 51.15 I 4.75 37.64 k 4.27 8.78 I 1.81 Totaln-3 PUFA 14.48 I 0.87 23.78 I 1.55 14.37 i 1.55 21.27 f 2.65 4.97 i 0.92 Total n-6 PUFA 10.68 f 0.81 24.11 f 1.41 36.78 f 3.98 16.37 k 1.79 3.81 I 1.05 n-3/nd PUFA 1.38 & 0.07 0.99 i- 0.05 0.42 i: 0.05 1.30 1: 0.09 1.56 * 0.27 Unsaturation 144.18*6.38 253.67k11.84 219.31 118.23 193.43f23.09 91.29i8.01 index Mean Chain 17.91 i 0.05 19.33 i 0.11 19.34 I 0.14 19.24 I 0.16 19.27 1: 0.34
values are presented as means f S.E.M. (n=8). ND not detectable. bvnknown A (9.08 min) eluted between 17: 1 (8.76 min) and l8:O (9.32 min).
DISCUSSION
Phospholipid Composition
Zn the basolateral membrane of the dogfkh gili epithelium, PC and PE were the
m a i . phospholipids, typical of vertebrate membranes (Hazel and Williams 1990). These
phospholipids play an important role in regulating the fluidity and permeability of
biological membranes (Hazel and Landrey 1988a). PC stabilizes the membrane as it
favors the formation of a laminar bilayer, while PE destabilizes the membrane by keeping
it close to the phase transition between larninar and hexagonal (HE) phase conformations
(Thurmond and Lindblom 1997). The ratio of PC to PE can be used as an indication of
membrane fluidity and rnay be altered according to the environmental and physiological
conditions encountered by an animd (Hazel and Williams 1990). In rainbow trout this is
clearly demonstrated when the PC/PE ratio dropped fiom 1.71 to 0.78 within 8 hours of
cold acclimation (5OC) and rose fkom 1.3 1 to 2.0 by the second day of warm acclimation
(20°C) (Hazel and Landrey 1988a). Despite inhabiting cold waters (5-10°C), the PC/PE
ratio in dogfish gill basolateral membrane (1.72) is quite high and similar to values seen
in warm acciimated rainbow trout (1.71 @ 20°C) (Hazel and Landrey 1988a) and
European yellow eels (Anguilla anguillu, 1.72 @ 15'C) (Aciemo et al. 1996). The high
PC/PE ratio in the dogfish gill BLM may be a physiological response to the increased
membrane fluidity caused by the hi& levels of urea found within the tissues and body
fluids.
It is also possible that the relatively high PCPE ratio in the BLM contributes to
the low urea pemeability observed in the dogfish gill (Part et al. 1998, Wood et al.
1995). Diet-induced increases of the PC/PE ratio in the bmsh border membranes fkom
rainbow trout can be correlated with decreased sodium permeability, demonstrating that
the capacity for PC molecules to pack closer together than PE molecules results in the
reduction of membrane permeabiiity (Di Costanzo et al. 1983). Active urea transport and
high cholesîerol levels have also been implicated as possible mechanisms involved in
reducing the permeability of the giu to urea (Fines et al. in press). The hi& cholesterol
levels in the BLM of the elasmobranch gill epithelium contributes to the highest reported
cholesterol:phospholipid molar ratio (Table 1.4). Cholesterol decreases the permeability
of biological membranes to urea by induchg increased order of the phospholipid
molecules that comprise the bilayer membrane (Mouritsen and Jmgensen 2994). We
proposed that the high cholesterol levels allow this membrane to firnction as a physical
barrier to the passive loss of urea, appearing as reduced urea permeability. Thus, a high
PC/PE ratio may be complimentary to the high cholesterol levels and also contributes to
the low urea permeability of the dogfish gill.
Fattv Acid Composition
Cumulatively, the most prominent fatty acid class Çom the phospholipids of the
dogfish gill BLM was the saturated fatty acids (38.11%, Table 2.1), similar to the
intestinal mucosa of the marine teleost Anguilla anguilla (39- 17%, Acierno et al. 1996).
Phospholipids containhg saturated fatty acids form tightly packed bilayers with relatively
low membrane fluidity and permeability, while phospholipids containing unsaturated
fatty acids pack less tightly resulting in more permeable membranes (Chen et al. 1971).
The chain length of fatty acids is also important in determining the fluidity and
permeability of a membrane. Longer chain fatty acids (18:O) increase the order of
phospholipid bilayers more than shorter chah fatty acids (1 6:O) thus contributhg more to
overall membrane stability (Chen et ai. 197 1, b e l and Landrey 1988b). Several studies
have demonstrated that fatty acid composition affects membrane permeability including
studies on ~ a + and glucose permeability in the b w h border membrane of trout @i
Costanzo et al. 1983) and channel catfïsh (Houpe et al. 1997), respectively. These studies
clearly demonstrated that modification of the levels of SFA in the phospholipids fiom
these membranes alters the permeability to these two solutes. The high levels of SFA,
both l6:O and 1 8 :O, in the dogfish gill BLM are likely involved in maintaining membrane
stability and the reduction of overall membrane permeability. This is supported by the
fact that phospholipids of mitochondrial membranes fiom fish that do not retain elevated
urea levels (hagfish and flounder) contain significantly higher levels of PUFA when
compared to those of the Little skate, an elasmobranch (Glemet and Ballantyne 1996).
This reflects the adaptation of these membranes to the different intemal solute
environments that occur in each of these fish. This difference between elasmobranchs
and other marine fishes is even more evident when one considers that hagfkh and
flounder occur at the same temperature and salinity as the little skate.
The monoene 18: 1 was cumulatively, the second most prominent fatty acid in the
phospholipids fiom the BLM of the dogfish gill epithelium. This also contributed to the
hi& level of monoenes in the dogfish basolateral membrane, relative to other fishes
(hagfïsh and flounder mitochondria, Glemet and Ballantyne 1996; European eel and sea
bass intestinal mucosa, Aciemo et al. 1996; channel catfish intestinal brush border,
Houpe et al. 1997). The increase in the proportion of monoenes in dogfkh gill(28.02%,
Table 2.1), compared to the eel intestinal mucosa (14.92%, Aciemo et al. 1996),
corresponds to a decrease in PUFA levels (dogfish, 34.88%, Table 2.1; eel, 45.91%,
Acierno et al. 1996). This shift in fatty acid class proportions is primarily due to
decreased 22:6n3 levels (dogfish, 8.27%, Table 2.1; eel, 24.90%, Acierno et al. 1996) and
increased 18:l levels (doash, 18.86%, Table 2.1; eel, 13.00%, Acierno et al. 1996) in
the elasmobranch. The effect of the increased monoene and decreased PUFA ievels is
unclear at this time, but they may contribute to increased membrane stabiiity and reduced
membrane permeability. However, the type of unsaturated fatty acids present in
membranes is of secondary importance to the proportion of saturated fatty acids when
considering the physical properties of a membrane (Hazel and Williams 1990).
The primary PUFA in dogfish gül BLM were 20:4n-6, 20511-3, and 22:6n-3.
Overail, PUFA levels were lower than in the erythrocyte membrane of the little skate
(Barton et al. 1999) and in the liver mitochondrial membranes of the haash , flounder,
and linle skate (Glemet and Ballantyne 1996). PUFA increase the fluidity of biological
membranes due to their "bullcy shape", indicating that the levels are kept low in the
dogfish gill BLM to help maintain membrane stability in the presence of elevated urea
levels. It is important to note that PUFA levels in the mitochondrial membrane of the
little skate liver are higher than those of the dogfkh gill BLM, but are stiil much lower
than in hagfkh or flounder liver mitochondrial membranes (Glemet and Ballantyne
1996). This suggests that in addition to maintaining membrane stability, the very low
PUFA levels of the dogfïsh gili BLM may also be involved in the retention of urea by the
a- Certain PUFA have other roles in membranes. Arachidonic acid (20:4n-6) (AA)
is the principle precursor for the production of eicosanoids (prostaglandins (PG),
leukotrienes, thromboxanes, and hydroxyeicosatetraenoic acids) in fish as it is in
terrestrial mammals (Beil et al. 1986). In teleost fish, the gill is one of the most active
tissues in vitro for PG synthesis (Christ and Van Dorp 1972, Ogata et al. 1978,
Henderson et al. 1985) and PG have been identifïed in gill of carp, trout, plaice, eel,
killifish, Atlantic salmon, and turbot (Ogata et al. 1978, Henderson et al. 1985, Srivastava
and M u d a 1984, Van Praag et al. 1987, Brown et al. 1991, Knight et al. 1995, Bell et
al. 1996). PI is the primary source of AA (Marshall et al. 1982). The dogfish gill is rich
in PI when compared to the mammalian erythrocyte, which is considered to be a rich
source of PI (Simpson and Sargent 1985). Furthermore the PI of the dogfkh has hi&
levels of AA (30.70%, Table 2.2) compared to the gills of rnost teleosts and the
mammalian lung (22.3% and 23.996, respectively; Zabelinskii et al. 1995). Tnus the high
level of PI and AA in the dogfïsh gill suggests that this tissue may be important in PG
biosynthesis as it is in teleosts.
PG play an important role in regulating ion transport in fish. PGE2 decreases ion
transport in opercular skin of the killifish (Van Praag et al. 1987) and PGF2, and PGEi
decrease branchial efflwc of ~ a + and Cl- in seawater adapted mullet without affecting
water efflwc (Pic 1975). The high content of PI rich in AA may indicate that PG are
involved in the regulation of ion and urea in the dogfish gill.
Conclusions
The phospholipid and fatty acid composition of the BLM fiom the dogfish gill
epithelium is characterised by a high PCPE ratio, elevated SFA levels, and reduced
PUFA levels. Ali of these modifications to the BLM lipid composition coupled with the
high cholesterol levels reported eariier (see Table 1.4) would contribute to the
maintenance of membrane stability in the presence of the extremely high urea
concentrations present in elasmobranch tissues. These characteristics also likely result in
decreased permeability to urea in the giil BLM, which is important in minimishg the
rnetabolic cost of replacing urea lost to the extemal environment by diffusion dong the
huge concentration gradient.
GENERAL DISCUSSION
The results reported in the previous chapters indicate that two cornplimentary
mechanisms exist in the elasmobranch gill, active urea transport and a unique lipid
composition, that may contribute to the low urea pemeability. 1 propose that the
modified Lipid composition of the basolateral membrane of the giil epithelium,
particdarly the elevated cholesterol level, increases the stability of the membrane
allowing it to fiinction as the primary barrier to urea diffusion fiom the blood into the
epithelial cells. This property of the basolateral membrane would reduce the rate of urea
diffusion into the epithelial ceiis. Secondarily, the urea transport system wouid function
to scavenge the urea that diffuses into the epithelial cells across the cholesterol-rich
basolateral membrane, transporting it back hto the blood against the concentration
gradient. This system of complimentary mechanisms would result in very low
intracellular urea concentrations in the gill epithelium, which would sigdïcantly reduce
the concentration gradient for urea across the apical membrane of these cells. This would
M e r have the affect of decreasing the rate of urea diffusion across the apical membrane
to the extemal medium, which is ultimately responsible for the low rate of urea loss
observed in the elasmobranch giil. The relatively low Km value (-10 mM) reported for
the urea transporter characterized in this study supports the proposal that the intracellular
urea concentrations of the gill epithelial cells are very low compared to that of the blood.
In this situation, where substrate (urea) levels are proposed to be low, the transporter
would be required to have a high affhity (low Km) for the substrate in order h c t i o n
efficiently. In order to maintain the low intracellular urea concentration, the velocity of
the "back transport" process would be similar to, but ultimately less than the rate of urea
diffusion fiom the blood into the epithelial celIs because the Ioss of urea across the gill is
constant.
The presence of high cholesterol levels in the basolateral membrane of the
elasmobranch gill epithelium is one of the most signincant fïndings of this study. The
interactions between cholesterol and phospholipids are s t i l l not well understood and this
naturally occurring cholesterol-nch membrane provides an ideal mode1 to study the
ability of cholesterol to regulate the fluidity and permeability of membranes. Further
investigations of the organization of cholesterol in these membranes using fluorescent
probes that specifically insert in membrane domains nch in cholesterol, such as lawdan
and prodan, could increase our understanding of how cholesterol exerts different effects
on membrane properties depending on the physiological conditions present.
Previous attempts to identfi the mechanisms responsible for the low urea
permeability of the elasmobranch giil focused ody on the involvement of a urea
transporter (Boylan and Antkowiak 1962, Boylan and Lockwood 1962, Boylan et al.
1963, Payan et al. 1974, Part et al. 1998). The results presented here demonstrate the
importance of utilizing different techniques and approaches to investigate physiological
phenornena. Furthermore, they show that more than one mechanism may be involved in
the design of some physiological processes.
The examination of both membrane proteins and lipids may be usefiil in
investigating other epithelial tissues that exhibit selective permeability to various
electrolytes and solut es (e. g. kidney, bladder, intestine, and gall bladder). Although a
wide range of studies have focused on the protein transporters present in these tissues,
analysis of the phospholipid and fatty acid composition of the membranes may provide
crucial information to the understanding of their selective permeability. For example, the
mammalian kidney tubule, or nephron, is a complex structure composed of many regions
that Vary in their permeability to water, urea, ions, and other solutes. The thin ascending
limb of Henle's loop has relatively high urea permeabiIity mai 1977, Imai and Kokko
1974, Imai et al. 1984) and despite the proposed presence of a urea transporter, none has
been detected. The high urea permeability of this segment is thought to be higher than
cm be explained by simple lipid-phase diffusion, but this is based on calculations using
artificial phospholipid bilayen (Sands et al. 1987). It is possible that in this region of the
nephron there is no transporter present and modifications to the lipid composition of the
cell membranes are solely responsible for the observed urea permeability. Modincations
that would confer high urea permeability include low cholesterol levels, high
phosphatidylethanolamine (PE) levels, low phosphatidylcholine (PC) levels, high
unsaturated fatty acids V A ) levels, low saturated fatty acids (SFA) levels and a shorter
mean fatty acid chah length (Pugh et al. 1989, Hazel and Landrey l988a, 1988b, Hazel
and Williams 1990). In contrast, regions of the nephron that exhibit low urea
pemeability, such as the outer medullary collecting duct (OMCD), the cortical coIlecting
duct, the distal convoluted tubule, the thick ascending limb of Henle's loop, and the thin
descending limb of Henle's loop (reviewed by Gillin and Sands 1993), would likely
exhibit higher cholesterol, PC, and SFA levels and lower PE and UFA levels and a longer
mean fatty acid chah length. These speculations on membrane lipid composition can
only be confirmed by f is t isolating individual nephron segments and directly measuring
the specifïc lipid components. Further investigation of the segments of the nephron could
reveal that a system of structural and transport function, similar to the one 1 have
described for the dogfish gill, operates within the mammalian kidney to control urea
permeability.
Urea transporters are present in many organisms, fiom bacteria to mammals, and
are primarily involved in osmoreguIation and nitrogenou waste management. The
presence of urea transporters in bacteria suggests that these proteins have a long
evolutionary history and have been relatively unrnodi£ïed for hundreds of millions of
years. In some organisms such as bactena, the transporter fimctions to acquire urea fiom
the enviromnent for utilization as a nitrogen source (Jahns 1992, Siewe et al. 1998). In
other organisms the transporters act to recover urea that couid be lost to the environment
by various excretory mechanisms. The facilitated urea transporter present in the
elasmobranch kidney (Smith and Wright 1999) is thought to fiinction in the reabsorption
of urea fkom the urine (Schmidt-Nielsen and Rabinowitz 1964, Schmidt-Nielsen et ai.
1972). In toads and fiogs, active &ansporters are involved in the absorption of urea
through the skin (Garcia-Romeu et al. 1981, Rapoport et al. 1989, Lacoste et al. 1991)
and the reabsorption of urea fkom urine in the bladder to facilitate water conservation
(Levine et al. 1973, Couriaud et al. 1999). On the other hand, the unusual teleost fish,
Opsanu beta (the gulf toadfish) utilize a facilitated transporter to excrete urea in pulses
when stressed (Part et al. 1999). In mammals, a faciiitated transporter in the IMCD
segment of the nephron is believed to contribute to the concentration and excretion of
urea by the kidney (Sands et al. 1987).
Despite the dserent functions and roles of urea transporters in different
organisms, they exhibit remarkable physiological and molecular similarity. Facilitated
and active urea transporters both exhibit dose-dependent inhibition by phloretin and
competitive inhibition by urea analogs such as thiourea, acetamide, and methylurea. The
majority of studies of the molecular characterization of urea transporters have focused on
the facilitated transporters of the mammalian kidney. There are two groups of urea
transporters that have been isolated, types A and B, which are products of two genes
(Sands et al. 1997). The homology of the elasmobranch kidney urea transporter (ShUT)
to both the UTA-2 and UT-B2 urea transporter proteins is approximately 60% (Smith and
Wright 1999). The similarity between the two families (UT-A2 and UT-£32) suggests
that they share a common ancestor. ShUT is not this ancestor because it has a higher
identity with UT-A2 than with UT-B2 due to the lack of residues found only in UT-B2
family members. This led to the suggestion that ShUT is an ancestral form of UT-A2 and
that an ancestral form of UT-B2 may also be present in elasmobranchs but has yet to be
discovered. Regardless, the preliminary molecular evidence for a urea transporter in the
dogfish gill provided by Smith and Wright (1999) has been supported by the
characterization of the urea transporter in this study. These Çidings j u s t e M e r study
of the genes encoding for these proteins in elasmobranch gill tissue in hope of providing
an improved understanding of the evolutionary lineage and expression of urea
transporters in vertebrates.
The results fiom the lipid analysis of the basolateral membrane fiom the dogfkh
gill epithelium has revealed that structural modifications in the membrane, especially the
incorporation of high levels of cholesterol, may be primarily responsible for making the
dogfkh gill relatively impermeable to urea. One may then ask, what came first, the high
levels of urea or the impermeable membrane? Did early elasmobranchs have modified
gill epithelial membranes that facilitated the accumulation of urea within the tissues,
allowing these fish to adapt to the marine environment? Did urea levels increase in
response to saha ter exposure and the necessary changes in the lipid composition of the
membranes evolved in order to adapt to the high levels of urea? I suspect that the fïrst
hypothesis is more likely because the urea permeabiiity of the gills fkom euryhaline
skates (Raja erinacea and Raja radiata) remains constant when the animals are exposed
to dilute (50%) seawater (Payan et al. 1973). Irregardless of which came first, the
beneficial result of this adaptation was that the modifïed membranes assisted in retaining
urea and reduced the energetic costs of this osmoregulatory strategy, making it an
efficient and effective strategy for over 350 million years.
Other aspects of the phospholipid composition of the basolateral membrane fiom
the elasmobranch giil epithelium hint at other functions for this membrane. The
abundance of arachidonic acid (20:4n6) containing phosphatidylinositol (PI) suggests that
the elasmobranch gill plays an important role in the biosynthesis of eicosanoids (e.g.
prostaglandins). In teleostç, prostaglandins are involved in reproduction, particularly
ovulation and spawning behaviou. (Stacey and Goetz 1982), and there is evidence for the
involvement of prostaglandins in regulating osmoregulation (Pic 1 975, Van Praag et al.
1987). It is possible that prostaglandins are involved in regulating osmoregulation and
reproduction in elasmobranchs, which is of great interest due to their unique
osmoregulatory strategy and the range of reproductive strategies in this group. Further
examination of the involvement of the elasmobranch gill in the biosynthesis of
eicosanoids is therefore necessary.
Overall, these investigations have contributed signifïcantly to our understanding
of possible mechanisms responsible for the low urea permeability of the elasmobranch
giu. This is particularly relevant due to the importance of urea in the osmoregdatory
strategy of elasmobranchs and the hi& metabolic cost associated with producing elevated
levels of urea. The metabolic cost of replacing urea that is lost to the environment by
diffusion across the gill is approximately 97.3% less than would be predicted based on
the urea permeability of teleost gills. This translates into savings of 48.65 mm01
ATP/kg/h. Without the retention of urea by the gill due to its low urea permeability, the
metabolic cost of living for elasmobranchs would be almost double (106.35 mm01
ATPlkgh) the estimated total energy requirements of the dogfish (57.70 mm01
ATPlkgh) (Bushnell et al. 1989). However, it should be noted that al l of these values are
calculated and require validation.
The use of purified basolateral membrane vesicles has provided important
information regarding the mechanisms involved in making the elasmobranch gill
relatively impermeable to urea but further characterization of this membrane needs to be
undertaken. Firstly, measwing the urea permeability of protein-fiee vesicles to urea
would provide additional information to support our proposed barrier function of
basolateral membrane phospholipid bilayer. Also effects of temperature, urea
concentration, trimethylamine oxide and other physiological factors on the
fluidity/permeability of this membrane can be determined using flourescent probes.
Probes specifk for cholesterol (laurdan and prodan) could be used to examine the
organization of cholesterol in these membranes in an effort to understand how cholesterol
provides a low urea permeability to the membrane. Secondly, studies should be
conducted to m e r characterize the urea transporter described in chapter one using
vesicles of a single orientation. Lectin affhîty chromatography would pennit the
separation of inside out and right side out vesicles and provide a more precise
detemination of the physiological properties of this transporter (e.g. kinetic parameters,
optimal analogue and inhibitor concentrations, stoichiometry of ~ a + and urea exchange).
Further studies should be undertaken to investigate the intracellular urea
concentration of the gill epithelial ceils. This would help to clar* the mode1 1 have
proposed for the involvement of the basolateral membrane in the retention of urea by the
gill. Measuring the total urea concentration of excised gill filaments and then correcting
for the blood space by measuring hemoglobin content could provide an indirect
determination of intracehlar urea concentration.
Studies on the effect of changes in salinity upon the lipid composition and urea
transport activity in the gill should aiso be conducted as blood urea levels decrease in
dilute seawater and modifications consistent with a homeoviscous response would be
expected. The use of euryhaline elasmobranchs, such as Raja erinacea, and fieshwater
elasmobranchs (Potamot?ygon spp.) may assist in the elucidation of the evolution of
these mechanisms in the elasmobranchs.
Finally, analysis of the lipid composition of the apical membrane would provide
information vital to the complete understanding of the mechanisms involved in
conferring low urea permeability to the elasmobranch gill. This membrane is the last
possible barrier to urea loss across the gill and is very likely to have a modified lipid
composition, not unlike that described for the basolateral membrane in chapter two.
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APPENDIX 1
Schematic representation of the assay of the percentage of inside-out-oriented vesicles. Assuming that resealed vesicles are impermeable to either ATP or ouabain, then N~+,K+- ATPase activity of untreated vesicles reflects the leaky vesicle fiaction. The detergent digitonin (0.04% w/v) stimulates N~+,K+-ATP~s~ activity by permeabilizing the membrane, providing ATP and ouabain access to the interior of the resealed vesicles. The percent ciifference in activity between untreated and digitonin trerited vesicles represents the percentage of resealed vesicles. The percentage of resealed vesicles with an inside-out orientation is obtained by measuring the stimulation of ATP hydrolysis induced by K+ ions of untreated and digitonin-treated vesicles in medium containhg ~ a + and ouabain. During short incubations, K' ions will equilibrate across the membrane quickly while ouabain does not. The N~+,K+-ATP~s~ protein of inside-out vesicles is inaccessible to ouabain and when digitonin is added the membrane barrier for ouabain is removed. This results in a decrease in N~+,K+-ATP~s~ activity which represents the percentage of inside-out vesicles. The difference between total resealed vesicles and inside-out vesicles equals the percentage of right-side out vesicles.
Right-side Out Ouabain
ATP
APPENDIX II
Schematic representation of the assay for measuring vesicle volume. Vesicles are incubated with a radiolabeled probe, such as 3 ~ f l , and then sedimented. The accessible volume of the pellet is calculated nom the specific activity of the probe in the supematant and the total radioactivity in the ellet. The difference in accessible volume (pellet P space) for penneant probes like H20 and probes like 1 4 ~ - ~ ~ ~ - 4 0 0 0 that do not cross the huer membrane equals the volume of the vesicle.
d.p.m. x supematant volume)/supernatant d.p.m. Vesicle Volume = space - "c-PEG-4000 space)/mgprotein added
Incubation Precipitation