characterisation of the 1 adrenergic response ... - thorax · thorax 1994;49:664-669...

Thorax 1994;49:664-669

Characterisation of the 1 adrenergic responsecascade in fetal guinea pig lung

Martha E Lyon, Charles A Lefebvre, Deborah J Davis

AbstractBackground - Suitable models for thestudy of lung development are needed.The suitability of the guinea pig forstudying the role of the P adrenergic re-sponse cascade in fetal lung developmenthas been evaluated.Methods - Radioligand binding assayswith iodine-125 labelled iodopindololwere performed to identify and charac-terise the adrenergic receptors. Todemonstrate that these receptors werefunctional, isoprenaline and forskolinstimulated generation of cyclic AMP(cAMP) in the lung tissue was quantit-ated by radioimmunoassay.Results - The concentration of P recep-tors increased with gestational age from23 fmol/mg at 35 days to 140 fmol/mg at64 days. Competition binding studieswere consistent with a predominance ofP2 receptors. The ability ofisoprenaline tostimulate cAMP generation was greaterduring the saccular phase than duringthe canalicular phase of lung develop-ment. Incorporation of tritium labelledcholine into phosphatidylcholineincreased significantly between thecanalicular and saccular phases.Conclusions - The P adrenergic responsecascade in fetal guinea pig lung exhibitssimilar characteristics to those pre-viously described in fetal human lungand is therefore a good model in which tostudy the effects of P agonists on fetal lungdevelopment.

(Thorax 1994;49:664-669)

Department ofPediatrics andObstetrics/Gynecology,University of Ottawa,Ottawa, OntarioKlH 4M4, CanadaM E LyonC A LefebvreD J Davis

Reprint requests to:Dr D J Davis, Room 8308,Ottawa General Hospital,501 Smyth Road, Ottawa,Ontario KlH 8L6, Canada.

Received 5 July 1993Returned to authors22 October 1993Revised version received8 February 1994Accepted for publication2 March 1994

Inadequate lung development in many prema-ture babies leads to respiratory distress syn-drome, a disorder characterised by surfactantdeficiency, excess alveolar and interstitial lungwater, and morphological immaturity of thelung.' There is clinical and experimental evid-ence which indicates that surfactant synthesisand secretion are mediated, in part, by ad-renergic responses.2' Further studies mayidentify the mechanisms by which the 1B ad-renergic response cascade mediates surfactantsynthesis and secretion and, perhaps, uptakeand utilisation of exogenous surfactant, butappropriate experimental models are needed.Owing to ethical considerations fetal humanlung of appropriate gestational ages is notalways readily available, necessitating the use

of another animal model. Although rabbits andrats are commonly used in studies of lungdevelopment, we felt that fetal guinea pigs

would be a more suitable animal model forseveral reasons. Firstly, the guinea pig has alonger gestation (67-69 days) than rabbits andrats and is also relatively mature at the time ofdelivery.4 Secondly, the proportion of timespent in the different stages of morphologicalmaturation of the lung and the timing of thesestages in relation to delivery in the guinea pig5are more similar to those in the human thanthose in the other small mammals. For ex-ample, the saccular stage of lung developmentbegins by 50 days (75% of gestation) in theguinea pig5 and by 28 weeks (70% of gestation)in the human fetus,6 whereas the rabbit and ratonly reach the saccular stage by 90-95% ofgestation.7 In addition, in both the guinea pig8and the human6 alveolarisation of the lung iswell established before term, and surfactantsynthesis9 and induction of antioxidantenzymes'0 occur relatively late in gestation inboth. The purpose of this study was to charac-terise 1 adrenergic responses in fetal guinea piglung in order to assess the appropriateness ofthis model to study the involvement of the ,Badrenergic response cascade in lung develop-ment.

MethodsANIMALSAll animal experiments were approved by theAnimal Care Committee of the University ofOttawa and conformed with the guidelinesoutlined by the Canadian Council for AnimalCare."

Following a one week acclimatisation timedpregnant 3-4 month old Hartley guinea pigs(Charles River Breeders, Quebec, Canada)were sacrificed by intracardiac injection ofsodium pentobarbitone. The fetuses were deli-vered by hysterotomy and were immediatelyeuthanised with intraperitoneal injection ofsodium pentobarbitone if they showed anysigns of life. The fetuses were weighed andtheir gestational age was assigned using thetable of Draper'2 which reports average fetalweight as a function of gestational age. Thefetal lungs were excised under sterile con-ditions and placed in 50 mmol/l Tris/1 mmol/lEDTA.

MEMBRANE PREPARATIONBecause 13 adrenergic receptors are found incell membranes, membrane particulates wereprepared from the excised lungs; the lungswere minced in 50 mmol/l Tris/1 mmol/lEDTA and filtered through cheesecloth beforecentrifugation at 1200g for 15 minutes. Thesupernatant was then centrifuged at 29 OOOg

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f0 Adrenergic responses in fetal guinea pig lung

for 15 minutes and the resulting pellet waswashed three times in 50 mmol/l Tris/4 mmol/lMgCl2 as previously described.'3 The proteinconcentrations of the particulates were deter-mined according to the method of Bradford'4with bovine serum albumin as the standard.The membrane particulates were frozen in adry ice/ethanol bath and stored at - 70'C untilassayed.

RADIOLIGAND BINDING ASSAYSThere were no previous data on 3 adrenergicreceptors in fetal guinea pig lung so the experi-mental conditions were first validated by aseries of studies designed to assess the optimalincubation time, protein concentration, con-ditions of the incubation and wash buffer, andsubcellular fraction to be assayed. For satura-tion analysis of radioligand binding experi-ments the incubation time must be longenough to ensure that a steady state is reached,but not so long that any decrease in bindingoccurs. This time required to reach steadystate between the radioligand and the ,B recep-tors was determined by incubating 200 pmol/liodine-125 labelled iodopindolol with mem-brane particulates (20 pg protein) in a buffer of50 mmol/l Tris/4 mmol/l MgCl2 (pH 7-4),1 mmol/l ascorbic acid, and either 0 1 mmol/lHCI for total binding or 0 1 mmol/l isoprena-line in 0-1 mmol/l HC1 for non-specific bind-ing, at 30°C for 1-90 minutes. At the pre-scribed times the incubation was stopped bythe addition of 5 ml ice cold 50 mmol/l Tris/4 mmol/l MgCl2; bound and free ligand wereseparated by filtration over Whatman GF/Cfilters. The filters were washed three times andthe radioactivity on the filters was counted todetermine the specific radioligand bindingover time. Apparent steady state between the Preceptors and '25I-iodopindolol was reached by30 minutes as shown by constant specific bind-ing beyond this time. All subsequent experi-ments used an incubation time of 45 minutesto ensure that steady state conditions existed.The optimal protein concentration to per-

form saturation analysis studies was deter-mined by incubating 251I-iodopindolol(200 mol/l) with membrane particulates (0-60 pg protein) as described above. From theseexperiments it was determined that to ensureoptimal detection of the 13 receptors and lessthan 20% non-specific binding a minimum of20 pg membrane protein was required.Using similar methods specific radioligand

binding was determined at incubation temper-atures of 4°C, 22°C, 30°C, and 37°C; maximalspecific radioligand binding was determined tobe at 30°C so this temperature was used insubsequent experiments. Similarly, a total ofthree washes with ice cold buffer, rather thanroom temperature or 37°C buffer, was found tobe optimal and was used in subsequent experi-ments.To determine which subcellular fraction of

fetal guinea pig lung contained the largestconcentration of f receptors, the amount oftotal, non-specific and specific binding of 1251Iiodopindolol to the J3 receptors was monitored

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as described above in the following fractionsobtained from differential centrifugation: (1)1200g pellet (15 minutes at 4°C); (2) 29 OOOgpellet (15 minutes at 4°C); (3) 100 OOOg super-natant (60 minutes at 4°C). The largest 13receptor concentration was located in the29 OOOg pellet so this protein was used for theradioligand binding analyses.Having thus optimised the assay conditions,

saturation analysis experiments were per-formed to determine the concentration of f3adrenergic receptors. In these assays duplicatesamples of each membrane particulate (20 jgprotein) were incubated at 30°C for 45 minutesin 0 125 ml buffer containing 1251I-iodopindolol(0-500 pmol/l) as described above.The dissociation constant (Kd), which de-

scribes the affinity of a ligand for its receptor,was determined from the initial saturation ana-lysis experiments and validated kinetically.'5For these kinetic experiments, koff was deter-mined by incubating '25I-iodopindolol(500 pmol/l) with membrane particulate (20 jigprotein) in 50 mmol/l Tris/4 mmol/l MgCl2 atpH 7A4 for 30 minutes at 30°C. At 30 minutesthe assay volume was increased 100 fold with50 mmol/l Tris/4 mmol/l MgCl2 at pH 7 4 andthe incubation continued for an additional 60minutes. Specific radioligand binding wasmonitored as a function of time before andafter the dilution of the reaction mixture usingthe radioligand binding assay already de-scribed. Kon was calculated as outlined byRoberts. 15

Since 1B receptors exhibit a higher affinity forthe S isomer of a ligand than the R isomer,fetal guinea pig lung preparations were ex-amined for stereoselectivity. Membraneparticulates (20 pg protein) were incubated asdescribed above with '25I-iodopindolol(200 pmol/l) and either S or R propranolol indecreasing concentrations (1 nmol/l to10 pmol/l). The specific binding of '25I-iodo-pindolol to the 13 receptors was monitored byradioligand binding assay described above.

Beta receptor subtype can be determined byagonist competition assays or by competitionassays with specific f31 or 12 antagonists. Forthese assays membrane particulates (20 pg pro-tein) were incubated with '25I-iodopindolol(200 pmol/1) in the presence or absence of 1agonists (isoprenaline, adrenaline, ornoradrenaline), or a selective 01 antagonist(atenolol, 1 pmol/l-10Ipmol/l), or a specific 02antagonist (ICI 118 551, 1 pmol/l-10 pmol/l, agift from Imperial Chemical Industries) for 45minutes at 30°C. The radioligand binding wasmonitored with the assay described above.

cAMP GENERATIONTo be important in fetal lung development,any f3 receptors that are identified by radio-ligand binding assays must be functional. Toassess this we studied the ability of isoprena-line (0-50 pmol/l) and forskolin (0-500 jmol/l)to stimulate cAMP generation in fetal guineapig lung of various gestational ages. Approx-imately 25 mg of lung tissue were incubated ina 0 25 ml volume of 5 mmol/l Tris and 0-9%



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sodium chloride (pH 7 4), 1 mmol/l 3-isobutyl-1-methylxanthine (a non-specific phospho-diesterase inhibitor), and 0-1 mmol/l ascorbicacid, with or without isoprenaline or forskolin,at 37°C for 10 minutes. The reaction wasstopped by adding 0-25 ml of 10% trichloro-acetic acid. The sample was then homogenisedand centrifuged. An ether extraction (5volumes ether: 1 volume sample) was per-formed three times on the supernatant of eachsample. The sample was evaporated to drynessunder a stream of air in a 70°C dry bath. ThiscAMP extract was reconstituted in 0 5 ml of50 mmol/l sodium acetate (pH 4). Cyclic AMPwas then determined by a commercially avail-able radioimmunoassay kit (NEN-Dupont).Under our assay conditions the recovery ofcAMP (50, 25, 10, 5, 1 pmol) was found to beconsistently greater than 80%. The variabilityin recovering 10 pmol cAMP/mg wet weight oflung tissue was 10%. The detection limit ofthis assay was 1 pmol cAMP/mg wet weightand there was a 9% variability in recoveringthis amount of cAMP.

CHOLINE INCORPORATIONFinally we wanted to begin to correlatechanges in the P receptors with evidence ofother events in the lung that are felt to bemediated, at least in part, through the Padrenergic response cascade. As a potentialmeasure of surfactant synthesis, therefore, in-corporation of tritium-labelled choline intophosphatidylcholine was measured. 16 Freshtissue was chopped into 1 mm cubes and in-cubated on a rocking platform at 37°C inWaymouth MB752/1 culture medium (Sigma)with 5 pCi/ml of 3H-choline chloride in anatmosphere of 5% C02/95% air. After fourhours the tissue was washed in 0 9% NaCl andhomogenised using a Teflon homogeniser.The phospholipids were extracted from thehomogenate in a 1:1 chloroform:methanolsolution as described by Folch et al'7 andseparated by thin layer chromatography(TLC) on silica coated TLC plates.'8 TheTLC plates were stained in a solution of cupricacetate in sulphuric acid/ethanol and dried for30 minutes at 1250C.'8 The plates were scrapedand the relative amount of radioactivity in thephosphatidylcholine band was determined byliquid scintillation counting.

DATA ANALYSISThe saturation analysis data were analysed bythe equilibrium binding data analysis (EBDA)program (Elsevier Biosoft) on an IBM com-patible desk top computer. Competition datawere analysed by the GraphPad InPlot pro-gram which provides the best fit final estimatesof fi adrenergic receptor concentration (P..,)and dissociation constant (Kd) from a leastsquares analysis of different site models. Re-sults for receptor concentrations in the dif-ferent subcellular fractions, the cAMP stimu-lation, and choline incorporation data wereanalysed by an analysis of variance followed byScheffe's post hoc test.'9 The data for total 3

receptor concentrations and Kd at differentgestational ages were analysed using a simplelinear regression. Statistical significance wasdefined as p < 0 05. Data throughout are pres-ented as mean (SE).

ResultsSaturation analyses at all gestational agestested (40-64 days) were linear, indicating asingle class of saturable, high affinity bindingsites (fig 1); unfortunately there was insuf-ficient tissue from lungs of fetuses < 40 days ofgestation to do full saturation analyses evenwhen whole litters were pooled. In two fetuses(59 days and 60 days) the dissociationconstants (Kd) determined from saturationanalysis were confirmed kinetically and werevery similar - for example, at 60 daysKd = 37-9 pmol/l from saturation analysisand kinetically kon= 1 33 x 10-9/mol/l/min,koff- 0 042/min, therefore koff/kon = 31 6 pmol/l= Kd; younger fetuses never had sufficienttissue to perform both sets of experimentssimultaneously so we could not validate othersaturation analyses kinetically.

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Figure 1 A typical Scatchard plot of '251I-iodopindololspecific binding to the ,B adrenergic receptors in a 60 daygestation fetal guinea pig lung from saturation analysisexperiments.

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Figure 2 Binding curves for '25I-iodopindolol(200 pmol/l) competing with various adrenergic receptorligands in the presence of different concentrations ofisoprenaline (EZ), adrenaline (*), noradrenaline (A),atenolol (A ), and phentolamine (0 ).

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f Adrenergic responses in fetal guinea pig lung

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Figure 3 Representative binding curvesfwith ICI 118551 in membranes from guinAnalysis of these competition curves andsipercentages of,B, and/I2 receptors and the iages tested were not different.

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120 The 1 receptors we identified demonstrated110 51 Day stereoselectivity for the R and S isomers of80 propranolol; the pharmacologically active S

isomer exhibited a higher affinity for the 160 receptors (EC50= 25 x10 mol/l) than the R

40 * isomer (EC50=2x10 6 mol/l).20 * As an index of 13 receptor subtypes, competi-0 tion assays were performed to monitor the-°,?~oU?ooooa% ability of various adrenergic agents to compete

with '251-iodopindolol. The order of potency of120 the agonist affinities (isoprenaline> adren-110 67 Day aline> noradrenaline) was indicative of a pre-110 dominance of 02 receptors (fig 2). Confirma-80 tion of this observation by competition assays60 with ICI 118 551, a specific 12 antagonist,40 showed that 12 receptors predominated20 \ throughout the time points of gestation20 xstudied (41-67 days). Approximately 65%0 _O , oz , .X ,,, -ts, , (95% CI 57% to 74%) ofthe receptors were02o '00O° ° ° ° ° ° ° ° and 35% (95% CI 26% to 43%) were 1,

185511 (mol/1) (n =11); this did not change with gestation (fig3). The inhibition constant (K) for the high'or'251-iodopindolol (200pmol/l)competing and low affinity sites determined from dis-

tea pig lung of different gestational ages.

even others not shown showed that the placement of'251-iodopindolol binding byinhibition constants (K,) at the gestational ICI 118 551 were 7nmol/l (95% CI 1-08 to

2356 nmol/l) and 1-04pmol/l (95% CI 033 to3-25 gmol/l). The Ki for displacement of iodo-pindolol binding by atenolol was 17pmol/l(95% CI 69 to 42-2pmol/l). These values areconsistent with thosepreviouslydescribedforhuman fetal lung.'3The concentration of total ,Breceptors

progressively increased from 23 fmol/mg at 35days of gestation to 140 fmol/mg at 64 days

...g.+ gestation (fig 4). The mean (SE) Kd did notchange with gestational age (110 (9 7) pmol/l,range 37-178pmol/l, n= 13) (r=0-004,

.. p >0 0 5 ).35 40 45 50 55 60 65 70 To determine whether these receptors were

Gestational age (days) functional, the ability ofP agonists to stimulatecAMP was examined. In the saccular phase of

,hanges in the concentration of/I adrenergic lung development (>50 days) isoprenalineth gestational age in fetal guinea pig lung stimulated 50% more cAMP than was present<00001). The concentration of/i

eceptors was determined by full saturation in the basal state (fig 5). There was no signific-3 specimens and by one point analysis using ant stimulation of cAMP by isoprenaline in theZ5I-iodopindolol as previously described32 in canalicular phase of lung development (40-49sbecause of insufficient tissue to perform ful days) (fig 5). No gestational differences were

observed with forskolin stimulated cAMPgeneration (fig 5). Mean (SE) basal levels ofcAMP were8- 1 (0 86) pmol/mg wet weight oftissue and did not change with gestation.

Finally, incorporation of 3H-choline intophosphatidylcholine, a potential measure ofsurfactant synthesis, was greater in the saccu-lar (5865 (6-3) cpm/mg tissue) than in thecanalicular (34-6 (2) cpm/mg tissue) phase oflung development (p <0-005).

15 F

lo F

5

n -Canalicular Saccular

IsoproterenolCanalicular Saccular

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Figure 5 Mean (SE) cAMP generation in fetal guinea pig lung during thecanalicular and saccular phases of lung development (n = 4). The amount of cAMPstimulated above basal levels is indicated on the vertical axis. *p < 005 between cA2generated during the canalicular and saccular phases.

DiscussionNeonatal respiratory distress syndrome is adisease that is characterised by anatomical im-maturity of the lung, excess lung water, andsurfactant deficiency. The resorption of lungwater and synthesis and secretion of surfactantphospholipids and surfactant associated pro-teins are mediated, in part, by ( adrenergicresponses.2 32024 Beta adrenergic receptors

MP have been identified in fetal rabbit and ratlung,23 as well as in human fetal lung from

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early in the second trimester of gestation.'325Human fetal lung is the ideal model for study-ing the role of the P adrenergic response cas-cade in lung development; however, ethicalconsiderations limit the availability of gesta-tion-appropriate samples. Fetal rabbit lung isone of the most commonly used models inwhich to study P adrenergic responses in fetallung development, despite the predominanceof 13 receptors26 in contrast to the predomin-ance of 12 receptors in human fetal lung.'3Fetal rat lung has also been used. It has similarproportions of 1 receptor subtypes27 to thosefound in humans, but the proportion of timespent in each of the various stages of lungdevelopment in the fetal rat is quite differentfrom that in human fetal lung.7 The 13 adrener-gic response cascade in fetal guinea pig lunghas not been previously reported. However,the relative proportion of 1, to 12 receptors inthe adult guinea pig is similar to that in thehuman lung2829 and the concentration of Preceptors, especially in the alveolar and bron-chial wall epithelium, increases from the new-born to the adult.'0Our studies show that characteristic 13 ad-

renergic receptors are present in fetal guineapig lung. Binding of '251-iodopindolol to Preceptors in fetal guinea pig lung is saturableand of high affinity; these findings are similarto those in fetal human lung.'3 The concentra-tion of P adrenergic receptors increases withgestational age in fetal guinea pig lung in asimilar way to fetal rabbit,'26 fetal rat,327 andfetal human lung." The order of potency ofagonist affinities and competition studies witha specific P2 antagonist indicates a predomin-ance of P2 receptors similar to that in fetalhuman lung" and fetal rat lung,27 but unlikefetal rabbit lung.26To determine whether the P adrenergic

receptors were functional we examined theability of 13 agonists to stimulate cAMP andfound that there was significant isoprenalinestimulated cAMP generation in the saccularphase of lung development. In contrast, therewas no difference detected in the ability offorskolin - a direct activator of adenylate cyc-lase - to stimulate cAMP generation in thesaccular phase compared with the canalicularphase of lung development. Since 13 receptorsare linked to the catalytic moiety of adenylatecyclase via guanine nucleotide regulatory pro-teins (G proteins), it is possible that there is agestational change in the amount or function ofthese G proteins.The synthesis and secretion of both surfact-

ant phospholipids2022 and surfactant associ-ated proteins2324 are controlled in part by Padrenergic mechanisms. In our studies wefound gestational changes in the ability ofisoprenaline to stimulate cAMP in fetal guineapig lung. Interestingly, the gestational agepattern for isoprenaline stimulation of cAMPwas associated with a similar pattern for cho-line incorporation into phosphatidylcholine,which could indicate increased synthesis ofeither surfactant phospholipids or membranephospholipids. It is not known whether similarassociations exist in fetal human lung between

the canalicular and saccular phases of lungdevelopment, nor whether this is important inthe preparation for extrauterine life. In fetalrabbits administration of an irreversible P ad-renergic antagonist (bromacetylalprenolol-methane) during the saccular phase of lungdevelopment decreases both the number ofreceptor binding sites and the phospholipidcontent of lung lavage fluid." We have not yetperformed experiments to determine if block-ing the 13 receptors in fetal guinea pig lung hasany effects on surfactant production.

In conclusion, the P adrenergic responsecascade in fetal guinea pig lung exhibits similarcharacteristics to those previously described infetal human lung. This, combined with severalother aspects of fetal guinea pig development,indicate that fetal guinea pig lung is a bettersmall animal model to study the role of the Padrenergic response cascade in lung develop-ment and adaptation to extrauterine life. Evenin this time of successful surfactant therapy forrespiratory distress syndrome there is stillmuch to learn about the mechanisms ofsurfactant synthesis, release, and recycling inthe newborn. Current evidence points to a rolefor 13 adrenergic responses in these functions;an appropriate animal model will help us tounderstand how better to use these responsesin the prevention and treatment of neonatalrespiratory distress syndrome.

This work was supported by research grants from Physicians'Services Incorporated, the Hospital for Sick Children Founda-tion, and the Children's Hospital of Eastern Ontario ResearchInstitute.

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