J. Anat. (1969), 104, 2, pp. 233-239 233With 7 figuresPrinted in Great Britain

Cholinesterase activity in the supra-orbital saltsecreting gland of the duck

JULIA FOURMAN

Department of Anatomy, University of Leeds

(Received 18 March 1968)

INTRODUCTION

Cholinesterase activity has been found at a number of sites at which sodium trans-port occurs. In the skin of the frog cholinesterase activity increases when sodiumtransport increases and cholinesterase inhibits sodium transport (Kirschner, 1953;Koblick, Goldman & Pace, 1963).The supra-orbital gland of marine birds secretes sodium chloride (Schmidt-

Nielsen, Jorgensen & Osaki, 1958). In the domestic duck the gland is normallyquiescent but secretes if the sodium concentration in the blood is increased (Sco-thorne, 1959). The concentration of sodium in the secretion, which is about threetimes that in the plasma, varies little, hence the amount of sodium secreted is propor-tional to the volume of fluid secreted. The salt gland of the duck is thus a usefulsystem in which to study the role of an enzyme in sodium transport. It was thereforeof interest to use histochemical techniques to compare the distribution and activityof cholinesterase in the secreting and non-secreting supra-orbital glands of the duck.

MATERIALS AND METHODS

Twelve pairs of Aylesbury ducks aged 6-20 weeks were given a normal diet withfree access to water. One of each pair received 1 g NaCl/kg body weight given bystomach tube as a 10% (w/v) solution. Six ducks (Group I) were given three doses ofNaCl at 2 h intervals, and six (Group II) three doses of NaCl/day for 4 d. The birdswere killed 30-60 min after the last dose. The glands of the control ducks producedno visible secretion; those of the ducks given salt, however, produced a copioussecretion (Fig. 1); samples were collected for the estimation of sodium content, butthe volume was not measured.The ducks were killed either by decapitation or by injection of sodium pheno-

barbitone into a wing vein. Blood for serum sodium estimations was collected fromthe severed neck vessels or from a wing vein. The supra-orbital glands were removedand weighed. From the ducks in Group I, weighed portions of the glands and ofneck muscle were taken for the estimation of sodium content (Flear & Florence,1961). In another six ducks, three of which were given three doses of salt, the glandswere perfused with a 2 % solution of Berlin blue to outline the blood vessels. From allducks two portions of gland were taken for histology. One was fixed in 4 % formalinor Zenker-formol solution for paraffin embedding and H & E staining; the other wasfixed overnight at 4 °C in 40 formol saline for histochemistry. Frozen 20 ,am sectionsof gland from each pair of ducks were mounted together on slides and incubated for

234 JULIA FOURMAN

cholinesterase activity by Coupland & Holmes' (1957) modification of the Koellemethod. The substrates were acetylthiocholine iodide or butyrylthiocholine iodide.Some sections were incubated with differential inhibitors; physostigmine 1 x 10-5 Mto inhibit all cholinesterase activity (Barka & Anderson, 1963), 1,5-bis(4:trimethyl-ammonium-phenyl)-pentan-3-one di-iodide (Wellcome Laboratories 62C47)1 x 10-5M to inhibit AChE (Diegenbach, 1965) and N,N-di-isopropylphosphorodianidicfluoride (Mipafox) 2 x 10-5 to inhibit BuChE (Holmstedt, 1957).

Table 1. Group 1. Experimental animals given three doses of 1 g/NaCl/kg body weight

Weight of Na m-equiv./I Na (,u-equiv.100 mg) Na (y-equiv.Body weight paired glands, K 5 A 100 mg)

(g) (mg) Serum Secretion Wet gland Dry gland Wet muscle

Control (6) 1690+129 273 20-9 145 + 2-2 None 5 2 + 0 53 19-3 +254 3-1+02Experimental(6) 1690± 98 336±35 165+6-7** 519+28-8 5-1±0-26 6-5+2-8* 3.80+ 25**

Values are means + standard errors of the means.P for difference between experimental and control means.

*P = 0 1, **P = < 0-05.

Table 2. Group IL Experimental animals given I g/NaCl/kg bodyweight three times a day for 4 days

Weight of paired Na m-equiv./lBody weight glands , A

(g) (mg) Serum Secretion

Controls (6) 1530+70 229+13-3 142+1 NoneExperimental (6) 1520+ 126 451+27* 159+3-5** 533+27-6

Values are means ± standard errors of the means.P for difference between experimental and control means.

*P< 0-01, * *P < 0-001 .

RESULTS

Secreting glands of ducks given salt were engorged with blood. After three doses ofsalt there was no significant increase in weight of the glands; after 4 d of salt admini-stration there was nearly a two-fold increase in the weight (Tables 1 and 2). The serumsodium was significantly increased in both groups of ducks given salt. The supra-orbital gland achieved more than a three-fold concentration of the serum sodium sothat the secretion contained between 408 and 592 m-equiv/l. ofsodium. The concentra-tion of sodium in the secretion was significantly higher (P < 0-05) in the birds givensalt for 4 d (Group II) than in those given only three doses (Group I).

Table 1 shows that there was a significant increase of muscle sodium in the experi-mental group. Secreting glands contain more water than quiescent glands but theincrease of gland sodium in experimental animals was barely significant even whenexpressed as dry weight (Table 1).The histochemical localization of cholinesterase varied with the substrate. With

acetylthiocholine the deposit of sulphide was fine and sharply localized; with butyryl-

Cholinesterases in the salt gland 235

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Fig. 1. Duck given sodium chloride. The secretion of the salt gland is welling from nares.

Fig. 2. Quiescent supra-orbital gland, control duck. Acetylthiocholine iodide.

Fig. 3. Nerves in the intralobular connective tissue. Acetylthiocholine iodide.

JULIA FOURMAN

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236

Cholinsterases in the salt glandthiocholine the deposit was coarser and more diffuse. A positive reaction usingacetylthiocholine substrate occurred in large nerves lying with the main ducts andvessels in the interlobular connective tissue (Fig. 3) and in what appeared to be inter-lacing 'fibres' passing out from the connective tissue around the intralobular ducts torun between the secretory tubules (Figs. 2 and 6). In glands perfused with Berlin bluesome of the filled capillaries were outlined by the deposit, but many were not. Thelocalization was not discrete enough to demonstrate whether the AChE was inendothelial cells or in surrounding structures. The distribution of AChE was thesame in the active as in the quiescent gland and no variation in the density of thedeposit was noted.The positive reaction using butyrylcholinesterase substrate was more variable. In

all glands it was localized around the ducts and tubules in the centre of the lobulesand in the large nerves (Fig. 4). In sections from secreting glands it also appearedwithin the secretory cells; most markedly in sections from birds in Group I (Figs.5 and 7).

Eserine added to the incubating medium completely inhibited the reaction witheither medium. Mipafox reduced the reaction when added to the acetylthiocholinesubstrate. 62C reduced the reaction when added to the butyrylthiocholine substrateand completely inhibited cellular reaction in the secreting glands.

DISCUSSION

The secretory activity, enlargement and vascular engorgement of the supra-orbitalgland of ducks given salt have been described (Scothorne, 1959). In the present study,vascular engorgement was as marked in the active glands that did not increase inweight (Group I) as in those that did (Group II). Mitosis of the secretory cells wasnot seen in paraffin sections from any of the glands. It does not appear that the markedincrease in weight after 4 d salt administration is solely the result either of an increasein blood content or in the number of secretory cells. The present data give no in-formation as to whether it is the result of cellular hypertrophy or an increase ofextracellular water. It seems that the efficiency of the gland is improved by the in-crease in weight that follows activity, for the concentration of sodium in the secretionwas significantly higher in the ducks given salt for the longer period (Group II),although there was no difference in the serum sodium of the two groups.The increase in serum sodium was reflected in an increase in the sodium content of

muscle, yet the increase of sodium in the active glands was barely significant, althoughblood flow is presumably increased (McFarland & Warner, 1966). These glands wereengorged with blood at the time of sampling; but as they had not increased in weightthere was presumably no significant increase of intracellular water. These resultspoint to a remarkable capacity of the gland to removed sodium from the blood.

Fig. 4. Quiescent supra-orbital gland, control duck. Butyrylthiocholine iodide.Fig. 5. Active supra-orbital gland, duck given salt; Group 1. Butyrylthiocholine iodide.Fig. 6. Active supra-orbital gland, duck given salt; Group I. Acetylthiocholine iodide d = duct,r = blood cell in capillary.Fig. 7. Active supra-orbital gland, duck given salt; Group II. Butyrylthiocholine iodide.

237

The localization of the AChE is not as clear-cut as would perhaps appear at firstsight for, contrary to the suggestion of Ash, Pearce & Silver (1966), it is unlikely thatall the AChE-positive 'fibres' which form a network in the centre of the lobule are infact nerve fibres. Studies with fluorescent and silver techniques (unpublished) suggestthat although the gland has a profuse innervation, this is not so extensive as theAChE-positive network might suggest. The form of the network is reminiscent of acapillary plexus, but in the perfused glands the enzyme was associated with only someof the filled vessels. The histochemical picture could result from non-specific stainingof connective tissue surrounding the tubules, but if this was the case the depositswould not be so clearly localized to the more central parts of the lobules and absentfrom the intralobular connective tissue. The histochemical results suggest that in thesalt gland, as in the vasa recta bundles of the mammalian renal medulla, cholineste-rases may be present both in nerves and in some endothelial cells. However, this canbe confirmed only by demonstration of the enzyme at the ultrastructural level.The positive reaction for BuChE which appears within the secreting cells may be

localized to the plasma membrane, which shows many deep infoldings in these cells.(Doyle, 1960). The greater BuChE activity demonstrated in sections from glands after4-d salt loading must be marked, for the glands are enlarged and the absolute contentof the enzyme could be increased without being reflected in the histochemicalappearance. A threefold increase of BuChE in the glands of ducks given salt for 4 dhas been measured by potentiometric titration (Ballantyne & Fourman, 1966).An increase of BuChE activity has been demonstrated to be associated with an

increase in sodium transport in the duck supra-orbital gland and probably also in themammalian kidney (Fourman, 1966). In frog skin and crayfish gills it is the AChEactivity that increases with sodium conductance (Koch, 1954; Kamento, 1961). Thereappears to be no clear-cut functional difference between AChE (true cholinesterase)and BuChE (psudo-cholinesterase); both are found in neurones of the central nervoussystem. In peripheral nerves BuChE is more commonly demonstrated than AChE andboth are found in non-neuronal tissue. The cholinesterases in the membrane of nerveaxons may play some role in the changes in membrane permeability which take placeduring the passage of an impulse (Lewis & Shute, 1965). Cholinesterases could playa similar role in the regulation of membrane permeability in the duck salt gland andin the other salt transporting tissues in which they have been found.

SUMMARY

1. Twelve ducks were given sodium chloride solution which stimulated their supra-orbital glands to secrete a fluid containing approximately a molar salt solution.

2. The distribution of cholinesterases was studied histochemically in these ducksand in twelve controls.

3. Acetylcholinesterase was found in both groups, in a rich nerve plexus and thewalls of a few small intralobular blood vessels.

4. Butyrylcholinesterase was found in both groups in nerves and in ducts, but insecreting glands it was present also within the epithelial cells.

238 JULIA FOURMAN

Cholinesterases in the salt gland 239I am grateful to Professor R. L. Holmes for his interest and encouragement and to

the late Professor P. Fourman for his guidance with the statistics and sodium estima-tions. I would like to express my sincere thanks to Miss S. Woodhead for technicalassistance.

This work was supported by a grant from the Medical Research Council.

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