Schmidt-Nielsen, Jorgensen and Osaki [1958] reported that hyperosmotic

Transcription

1 Q. Jl exp. Physiol. (1969) 54, PLASMA OSMOLALITY AND SALT GLAND SCRTION IN TH DUCK. By R. W. ASH. From the Institute of Animal Physiology, Babraham, Cambridge. (Received for publication 6th July 1968) Secretion by the supra-orbital glands of conscious Aylesbury ducks began when plasma osmolality and [Na+] were increased by 2-8 per cent after oral administration of hyperosmotic NaCl solution. Peak rates of secretion occurred when these parameters were elevated by 8-19 per cent. Oral administration of water in volumes sufficient to decrease plasma osmolality and [Na+] below these values abolished or reduced the flow of secretion. Comparable or larger increases in plasma osmolality produced by hyperosmotic KCI solution administered orally or by intravenous injection of urea or dextrose failed to evoke secretion. Intravenous injection of hyperosmotic sucrose or mannitol increased plasma osmolality and decreased plasma [Na+] but evoked secretory responses which declined although plasma osmolality remained increased. An osmotic component appears to be involved in the secretory mechanism of the salt glands but the relation between secretion and plasma osmolality is not simple and probably depends on the permeability of certain cells to different solutes. TH supra-orbital or nasal glands of marine birds appear to function as an extra-renal mechanism for the preferential excretion of sodium chloride [see Schmidt-Nielsen, 196]. A secretion is evoked from the glands when the birds ingest hyperosmotic NaCl solutions or foodstuffs which contain high concentrations of NaCl. Schmidt-Nielsen, Jorgensen and Osaki [1958] reported that hyperosmotic solutions of NaCl administered either orally or intravenously caused a flow of secretion from the supra-orbital glands of cormorants. An intravenous infusion of hyperosmotic sucrose also induced a secretion which contained a high concentration of NaCl. Schmidt-Nielsen et al. suggested that the secretory mechanism responds to an osmotic load rather than to specific changes in plasma [Na+] or [CI-]. Fange, Schmidt-Nielsen and Robinson [1958] postulated that secretory activity is probably evoked by a reflex mechanism supposedly involving hypothetical osmoreceptors in the brain or in the walls of certain blood vessels. The present experiments are concerned with the relation between plasma osmolality and secretory activity of the supra-orbital glands of the domestic duck. Scothorne [1958] first reported that the duck possesses functional supra-orbital glands which produce a copious secretion in response to administration of hyperosmotic NaCl solution. MATRIALS AND MTHODS Ducks. - Aylesbury drakes weighing kg. and not less than twelve weeks old were fed on concentrate pellets which contained approximately 7 mg. Na+/1 g. 68

2 Salt Gland of Duck 69 dry matter. Fresh tap water was allowed ad libitum but food was withheld for 18 hr. before an experiment. About 1 hr. before an experiment the skin and subcutaneous structures surrounding a superficial vein on the undersurface of a wing were infiltrated with 2 per cent lignocaine hydrochloride and the vein was cannulated. In some experiments both a vein and an adjacent artery were cannulated and the two joined with polythene tubing to form an extra-corporeal arterio-venous loop. Clotting was prevented by an injection of 5, units of heparin IV. The ducks were supported in a sitting position in a wire cage; their wings and feet were secured with adhesive tape. Blood samples. - Approximately 3-5 ml. of venous blood was withdrawn over 1-2 min. into a syringe, transferred to a centrifuge tube and stored at +1 C. until centrifuged. Samples of arterial blood could be quickly obtained by disconnecting the a-v loop. Usually, 2 ml. of -15 M NaCl was infused slowly into the vein after each sample was taken. The blood was centrifuged within 3-4 hr. of being collected and the volume of plasma obtained was about 2 ml. The hematocrit was not measured but it was apparent that the packed cell volume decreased after administration of hyperosmotic solutions and, as may be expected, from repeated withdrawal of the whole blood. Towards the end of an experiment the bills of some ducks lost their pink colour and became cold. Secretion samples. - In the domestic duck the ducts of the salt glands open into the nasal cavity. Secretion accumulates at the external nares, runs to the tip of the bill, and is removed when the duck shakes its head. In most experiments secretion was collected by gently holding the duck's bill over a funnel which drained into a graduated tube. A disadvantage of this method was that occasionally salivary secretions dripped from the mouth and contaminated the salt gland secretion. An alternative method which prevented this was to remove with a Pasteur pipette the salt gland secretion as it accumulated at the nostrils. Administration of fluids. - Hyperosmotic solutions of NaCl, KCI or distilled water were introduced into the alimentary tract through a soft rubber tube passed through the mouth and down the cesophagus. This method will be referred to as oral administration. Hyperosmotic solutions of NaCl, urea, dextrose, sucrose or mannitol were injected intravenously. The term hyperosmotic is used to describe the osmolarity of the solutions with respect to that of plasma. Analysis. - Plasma osmolality was determined cryoscopically using an Advanced Instruments Osmometer calibrated with standard NaCl solutions. Duplicate measurements were made on the same samples of plasma and usually they agreed to within 2 m-osmoles/kg. Appropriate dilutions of plasma and salt gland secretion were made with de-ionized water and the concentrations of Na+ and K+ estimated by flame photometry (vans lectroselenium Ltd.). The potentiometric method of Sanderson [1952] was used to estimate (Cl-] in some samples. RSULTS Secretory activity in the absence of an osmotic stimulus. - As judged by the dry appearance of the external nares, the salt glands of the domestic duck are usually inactive in the absence of an effective osmotic stimulus. In many of the present experiments, however, ml. of clear fluid accumulated at the nostrils of the birds while they were restrained during the insertion of catheters into blood vessels. This apparent secretory activity lasted about 2-5 min. and when the fluid was removed the nostrils often remained dry. Sometimes there was more than one period of secretory activity or the moist appearance of the nostrils persisted. Similar flows of

3 7 Ash secretion were observed on occasion when the ducks became restless or excited. Less frequently small flows occurred during or after withdrawal of a sample of blood from a catheter, but in these instances the birds were not obviously disturbed (see figs. 5 and 6). The [Na+] of the fluid collected from the nostrils was m.equiv./l. Secretory activity in response to hyperosmotic N,aCi. - In seven experiments 2-4 g. of NaCl was administered as a M solution; in six ducks + az c V. cr (D!O 9 5 -Q (D +3 a) C C f 7 a b c c c FIG. 1. Stimulation of secretory activity by hyperosmotic NaCl and abolition of secretion by water. The arrows a and b indicate oral administration of 2 ml. anid 15 ml. respectively of 1 7 M NaCl; c, oral administration of 5 inl. water. secretion was first observed at the nostrils after 3<5-25 min. and thereafter' the flow was virtually continuous. The volumes recorded at consecutive 15 or 3 min. intervals tended to increase progressively and peak rates of flow equivalent to 1-26 ml./hr. were reached within 3-9 min. These high rates of secretion were either maintained for at least 2-5 hr. or declined slowly. Oral administration of distilled water reduced or terminated the response mnore abruptly (fig. 1). The response of one duck to 4 g. of NaCl was comparatively poor; the volume of secretion collected in 9 min. was 9 ml. with a [Cl-] of 263 m.equiv./l. Secretory activity and its relation to plasma osmnolality and Nat. -- In five

4 Salt Gland of Duck 71 experiments samples of venous blood were obtained (i) before oral administration of hyperosmotic NaCl solution, (ii) as soon as secretion appeared at the nostrils after dosing and (iii) at fairly regular intervals thereafter. Secretory activity began 7-25 min. after administration of 2-4 g. of NaCl and coincided with increases in plasma osmolality and [Na+] of 1*4-8*7 per cent and per cent respectively. The flow of secretion, plasma osmolality and [Na+] increased concurrently and this was seen most clearly Na4 * 19 - ~~~~~~~18 ~ ~ ~ ~ ~ su 34Q, ~ ~~~~~~~~~~ ' *- ~~~~~~~~~~~~~~~~~~~~~ IL3441 _# _ * 6 N46 5 > 4 4 X C o.'~~~~~~~~~~~~~~~~ ~~~3 s. t 2 or hr cs ~~~~~~~~~~~~b FIG. 2. Secretory activity in relation to plasma osmolality and [Na+]. Arrow a indicates the oral administration of 4 ml. 1 7 M NaCl. Oral administration of 5 ml. water, arrow b, reduced the flow of secretion and this coincided with a maintained decrease in plasma osmolality and [Na+]. Venous plasma samples. in the experiments in which the plasma parameters and volume of secretion were measured at 15 min. intervals (figs. 2 and 3). Subsequently, the flow of secretion reached a plateau while plasma osmolality and [Na+] continued to increase. Further, in one of the experiments additional increases in plasma osmolality and [Na+] induced by a second osmotic load failed to influence the stable rate of flow of secretion (fig. 3). The plateau and maximal rates of flow corresponded to increases in plasma osmolality and [Na+] of per cent and 8-19 per cent respectively. In one experiment plasma osmolality was increased by 9 per cent and [Na+] by 14 per cent but secretion was not evoked. A maintained reduction in the flow of secretion could be obtained by oral administration of distilled water in volumes sufficient to decrease plasma osmolality and [Na+] to values below those at which secretory activity was apparently maximal (compare fig. 2 and 3). xperiments to

5 72 Ash be described in a later section show that secretion evoked by hyperosmotic NaCl could be abolished by distilled water, provided plasma osmolality and [Na+] were decreased to values below the 'threshold' (see figs 4, 5 and 6). These experiments indicate that the secretory activity of the salt glands is closely associated with plasma osmolality and [Na+] and within limits the magnitude of the response bears some relation to changes in either or both of the plasma parameters. The plateaux may correspond either to 38 * or 2 19 _ c 3 14 >~ 3 I a b c FIG. 3. Secretory activity in relation to plasma osmolality and [Na+]. a, 12 ml. -5 M NaCi administered orally. b, 6 ml. -4 M KCI administered orally. c, 1 ml. water orally. Note that in this experiment oral administration of hyperosmotic KC1 produced a persistent increase in plasma [Na+]. Water failed to influence the flow of secretion or reduce plasma osmolality and [Na+] below values at which secretory activity was apparently at its maximum. Venous plasma samples. the maximum amount of work that the salt glands can perform or they are proportional to the strengths of the stimuli. It is clear that the mechanism excited does not adapt rapidly to the stimulus. A comparison of the effects of NaCi, urea and dextrose - To obtain more information on the relation between plasma osmolality, [Na+] and secretory activity the previous experiments were repeated using smaller quantities of NaCl and the results were compared with those when increases in plasma osmolality were produced by intravenous injections of hyperosmotic urea and dextrose. In six trials on three ducks, secretion flowed from the external nares 8-25 min. after oral administration of 2-5 ml. of 512 M NaCl ( g. NaCl). The onset of secretion coincided with increases in plasma osmolality and [Na+] of per cent. Peak rates of flow were observed within

6 Salt Gland of Duck min. and then flow declined in association with contemporaneous decreases in plasma osmolality and [Na+]. Residual secretory activity was 41~~~~~~~~~~~~~~~~~~~~~~~9 C> 39- ~~~~~~~~~~~~~~~~~~~~~~~~~~7 37 *O h 6 I 3 -O o hr t It lit b c d. f Fia. 4. A comparison of the effects of NaCl and urea on secretory activity. a, 25 ml. 5 M NaCl orally; b, 15 ml. water orally; c, 2 ml. 5 M NaCl orally; d, 4 ml. water orally. e, 2 ml. 3 M urea injected IV; f, 25 ml. 5 M NaCl orally. Urea increased plasma osmolality by 15 per cent but failed to evoke secretion. Venous plasma samples. a. > o 3: s 34 1 Not S z o C to > c e.ā * n*..ie S CA z I Z 3 4! t b c S 6 7 hr f FIG. 5. A comparison of the effects of NaCl and dextrose on secretory activity. a, and c, 2 ml. 5 M NaCl orally; b and d, 3 ml. water orally. At e, 2 ml. 2 M dextrose injected IV. f, 3 ml. 5 M NaCl orally. In the bottom panel, S indicates a 'nonspecifically' induced secretion. abolished by oral administration of distilled water and the return of plasma osmolality and [Na+] to values below threshold (figs. 4 and 5). In two experiments urea (15 ml. of 3 M and 1 ml. of 4 M in one, 2 ml. of 3 M in the other) was injected intravenously over a period of 2-9 min.

7 74 Ash Plasma osmolality was increased by 15 and 25 per cent but there was no flow of nasal secretion; the injections depressed plasma [Na+] temporarily. Oral administration of 25 ml. of -512 M NaCl, during the time when plasma osmolality (and presumably plasma urea concentration) was still elevated by the urea injections, produced a typical response. The flow of secretion began when plasma [Na+] increased to values not markedly different to those at which it occurred with NaCl alone (fig. 4) No- 16 O- 32 _ \ 32 2 'A O O.P.~~~~~~~~~~~~~~~~~~ ) 31 *-:/ 3 - Z15 ~~~~~~~~~~~~~~~~~14 ~~~~~~~~~~~~~~~~~13 ~~~~~~~~~~~-5 3 ~~~~~~~~~~~~~~~~ 4 o- >2.S ~~~~~~~~~~~~~ c~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~ 1~~~ t I I1 v b bc S hr FIG. 6. A comparison of the effects of NaCl and sucrose on secretory activity. a, 25 ml 5 M NaCl orally; b, 5 ml. water orally; c, 1 ml. 1 M sucrose injected IV. S indicates flows of secretion which coincided with blood sampling. Venous plasma samples. Similar experiments were performed with dextrose. Intravenous injections of 2 ml. of 2 M dextrose in two ducks increased plasma osmolality by 11 and 17 per cent and temporarily decreased plasma [Na+]. One duck failed to secrete and the other produced -4 ml. in the 4 min. immediately following the injection. As in the urea experiments, oral administration of hyperosmotic NaCl while plasma osmolality was increased as the result of the dextrose injections produced a typical flow of secretion. Again secretory activity appeared to be independent of changes in total osmolality of the plasma but coincided with increases in plasma [Na+] (fig. 5). The effect of sucrose and mannitol. - In four experiments on three ducks intravenous injections of 1-2 ml. of 1 M sucrose evoked a flow of secretion which commenced within 2 min. of beginning the injection. However, the response in two experiments was small and transient; -6 and 5 ml. of fluid were collected over 1-15 min and then flow ceased. The peak volumes in two ducks were 1 8 and 2-8 ml./15 min. but neither of the responses was

8 Salt Gland of Duck 75 well maintained; in one, secretory activity stopped after 3 min. and in the other it slowed appreciably after 4 min. (fig. 6). Intravenous injections of 15-2 ml. of M. mannitol consistently stimulated a flow of secretion. As with sucrose the response began during the injection and the initial rate of flow was 1 l3-3-6 ml./15 min.; thereafter flow decreased rapidly. Further injections of mannitol again evoked rapid zi o Duck 2 Duck ' 32 -* c A..v Z 4. 4Vc In4 I -L I IA I I I I I t hr a b c FIa. 7. ffect of mannitol on secretory activity. Duck 2: a and b, 2 ml. and 15 ml. respectively of 1-5 M mannitol injected IV; c, 1 ml. water orally. d, 15 ml. 1-7 M NaCl IV. Duck 27: a, b, and c, 2 ml. 1 M mannitol injected IV; d, 1 ml. 1-7 M NaCl injected IV. Venous plasma samples. flows but these responses also diminished in the following 45-6 min. although plasma osmolality remained elevated by 7-11 per cent (fig. 7). Although repeated injections of mannitol decreased plasma [Na+] by as much as 12 per cent, the [Na+] of the secretion was within the range of concentrations observed after the administration of hyperosmotic NaCl. Clearly, increases in plasma [Na+] are not essential for stimulating nasal secretion. A comparison of the effects of hyperosmotic KCl and NaCI. - In the duck, Na+ is the dominant cation in the nasal secretion induced by hyperosmotic NaCl or other effective osmotic stimuli. The nasal glands of the ostrich and certain species of lizards are capable of secreting a fluid which contains c d

9 76 Ash high [K+] and in some circumstances this may exceed [Na+] [Schmidt- Nielsen, Borut, Lee and Crawford, 1963]. Further, the secretion [K+] and the output of Na+ and K+ by the nasal glands of some lizards can be increased by intraperitoneal injections of hyperosmotic NaCl or KCI [Templeton, 1964, 1966]. The aim of the next group of experiments was to see whether increases in plasma osmolality and [K+], brought about by oral 54 i2 c _. O o. -x -. O 3 T.t> C.a. c u hr o ~~b c de FIG. 8. A comparison of the effects of hyperosmotic KCl and NaCl on secretory activity. a, 5 ml. 5 M KCI orally; b, 1 ml. water orally; c, 5 ml. 5 M NaCl orally; d, 15 ml. 5 M NaCl injected IV; e, 12 ml. water orally. Arterial plasma samples. administration of hyperosmotic KCI solution, stimulate activity or influence the composition of nasal secretion in the duck. Two ducks were provided with drinking water containing 27 mm KCI for a week before the experiments and on the day of the experiment 25-3 ml. of 1-3 or 1-6 M KCl was given orally. Both ducks failed to secrete fluid from the external nares in the following 3-4 hr. periods of observation. In contrast to NaCl, the administration of KCI in similar amounts and concentrations was not tolerated. One duck regurgitated fluid 17, 5 and 9 min. after it was dosed with 3 ml. of 1-3 M KCI; the other regurgitated

10 Salt Gland of Duck 77 twice when a second dose of KCI was given 2 hr. after the first. Irreversible cardiac arrest occurred in both ducks after 3-4 hr. Six experiments were performed in which 5-75 ml. of -512 M KCI was given orally and only one of the ducks regurgitated 19 min. after receiving 75 ml. of the solution. Plasma [K+] increased rapidly from L3-4 Y 5 L o L. C o -~ o ~. CN O le 9 o _: I > 17 z U) S 3 + I + cn _. 2 d FIa. 9. A comparison of the effects of hyperosmotic NaCl and KCl on secretory activity. a, 5 ml. 5 M NaCl orally. At b, 7 ml. water was given orally and this markedly reduced but did not completely abolish secretion. c, 5 ml. 5 M KCI orally; d, 8 ml. water orally. Arterial plasma samples. m.equiv./l. to 7-9 m.equiv./l. and this was accompanied by increases in plasma osmolality of per cent. Plasma [Na+] tended to increase by 17-4 per cent and on occasion increases of 6-2 per cent were observed but these changes did not persist. Substantial and continuous flows of nasal secretion were never evoked in any of the experiments, but in three, sporadic flows of ml. occurred and in one other the nostrils were moist (fig. 8). These small samples of fluid contained m.equiv. Na+/l. and m.equiv. K+/L. However, only two samples contained more than 35 m.equiv. K+/L. and these were obtained from the duck which had regurgitated; the possibility cannot be excluded that these samples were contaminated with residual KCI solution in the nostrils. An intravenous injection of 2 ml. of

11 78 Ash x512 M NaCl into one duck 75 min. after oral administration of KCO and when plasma [K+] was 7-5 m.equiv./l. produced an immediate and rapid flow of secretion. This indicated that high plasma [K+] was not inhibiting secretory activity. Oral administration of distilled water 1-2 hr. after the ducks received hyperosmotic KCI decreased plasma osmolality, [K+] and [Na+]. In two experiments plasma osmolality and [Na+] decreased to well below their initial values. At this stage oral administration of 5 ml. of 512 M NaCl subsequently evoked a continuous flow of secretion which began when plasma osmolality and [Na+] were either less than or equal to the values observed before or after K+ administration (fig. 8). In the experiments described above, the effects of hyperosmotic KCI were tested before NaCl. In one further experiment the order of administering the solutions was reversed and under these conditions hyperosmotic KCI produced a substantial response, but it is important to note that the flow of secretion coincided with maintained increases in plasma [Na+] comparable to the values observed with hyperosmotic NaCl (fig. 9). The secretion [K+] after K+ administration was m.equiv./l. compared with m.equiv./l. after Na+ loading. DISCUSSION Clearly, the relation between plasma osmolality and secretory activity of the salt glands is not simple. Comparable increases in total osmolality of the plasma were produced by oral or intravenous administration of NaCl, sucrose, mannitol, KCI, urea and dextrose; but the first three only of these compounds evoked secretion. There is a similarity between these findings and those of Verney [1947] on the osmotic factors which caused a release of antidiuretic hormone in the dog. Verney found that increases in plasma osmotic pressure produced by intracarotid injections of hyperosmotic NaCl, Na2SO4 and sucrose induced an antidiuresis, but dextrose was less effective and urea ineffective. If the specific function of a cell is to detect changes in osmolality of the plasma or extracellular fluid, the cell membrane must be either impermeable to solutes or mechanisms must exist to prevent a net flux of solutes into the cell. Verney postulated that some cells in the supraoptic and paraventricular nuclei of the hypothalamus are completely permeable to urea, less so to dextrose and relatively impermeable to NaCl, Na2SO4 and sucrose. The term 'osmoreceptors' was introduced to describe the properties and function of the cells. In the present work the osmotic stimuli were not confined to a particular region of the body, but the experimental evidence suggests that, irrespective of the site of the cells which respond to osmotic changes, they are permeable to urea, dextrose and KCI. The secretory responses evoked by sucrose and mannitol were transitory compared with those produced by NaCl. For example, repeated IV injections of mannitol caused an initial rapid flow of secretion but this subsided after each injection although plasma osmolality

12 Salt Gland of Duck 79 increased progressively. A possible explanation is that the cells responding to the change in plasma osmolality are relatively permeable to sucrose and mannitol. Consequently the concentrations of these compounds equilibrate across the cell membrane and abolish the osmotic gradient between the cytoplasm and extracellular fluid. After administration of NaCl there were some experiments in which the flow of secretion tended to be related more closely to changes in plasma [Na+] than to the total osmolality of the plasma. Furthermore, the prolonged responses which could be produced by NaCl suggest that the hypothetical receptor cells are either impermeable to Na+ or they are capable of mainltaining a critical difference in concentration of Na+ across the cell membrane. As judged by the results of the present experiments the magnitude of the difference required to evoke secretion appears to be in the range 2-8 per cent. xcitement and experimental procedures which did not involve the application of an osmotic stimulus evoked a secretion in some ducks. A similar phenomenon was reported by Frings and Frings [1959]; they found that the salt glands of albatrosses secreted when the birds were subjected to 'stressful' situations such as forced feeding and when handled for experimental purposes. How these conditions induce secretion is a subject for further investigation but in some of the present experiments it is possible that a secretion evoked 'non-specifically' merged with the response that could be related to changes in plasma osmolality and [Na+]. Nevertheless, it is the copious and continuous flow of secretion in the duck that distinguishes an osmotically induced response from the sporadic secretion caused by 'non-specific' stimuli. RFRNCS FANG, R., SCHMIDT-NILSN, K. and ROBINSON, M. (1958). 'Control of secretion from the avian salt gland'. Amer. J. Physiol. 195, FRINGS, H. and FRINGS, M. (1959). 'Observations on salt balance and behaviour of Laysan and Black-footed albatrosses in captivity.'. The Condor, 61, SANDRSON, R. H. (1952). 'Potentiometric determination of chloride in biological fluids'. Biochem. J. 52, SCHMIDT-NILSN, K., JORGNSN, B. and OsAKI, H. (1958). 'xtra-renal salt excretion in birds'. Amer. J. Physiol. 193, SCHMIDT-NILSN, K. (196). 'The salt secreting gland of marine birds'. Circulation, 21, SCHMIDT-NILSN, K., BORUT, A., L, P., and CRAWFORD,. (1963). 'Nasal salt excretion and the possible function of the cloaca in water conservation'. Science, 142, SCOTHORN, R. J. (1958). 'A histochemical study of the nasal (supra-orbital) gland of the duck'. Nature, Lond. 182, 732. TMPLTON, J. R. (1964). 'Nasal salt excretion in terrestrial lizards'. Comp. Biochem. Physiol. 11, TMPLTON, J. R. (1966). 'Responses of the lizard nasal salt gland to chronic hypersalemia'. Comp. Biochem. Physiol. 18, VRNY,. B. (1947). 'The antidiuretic hormone and the factors which determine its release'. Proc. Roy. Soc. B, 135,