Effects of short-term changes in electrolyte intake on the adrenal steroidogenic responses of juvenile mallard ducks (Anas platyrhynchos)

Camp. Biochem.

Physiol. Vol. 9lA, No. 3. pp. 513-518,1988

0300-9629/88 $3.00 + 0.00 0 1988 Pergamon Press plc

Printedin Great Britain

EFFECTS OF SHORT-TERM CHANGES IN ELECTROLYTE INTAKE ON THE ADRENAL STEROIDOGENIC RESPONSES OF JUVENILE MALLARD DUCKS (ANAS

PLATYRHYNCHOS)

J. L. REDONDO, W. N. HOLMES and JAMEYSCRONSHAW Department

of Biological

Sciences,

University

of California,

Santa

Barbara,

CA 93106, USA

(Received 15 March 1988) Abstract-l. Adjusting the Na+ and K+ intake of juvenile mallard ducks caused the plasma concentrations of corticosterone (B) and aldosterone (Aldo) to increase independently of one another, but none of these changes in electrolyte intake had a significant effect on the deoxycorticosterone (DOC) concentration. 2. With the exception of DOC in birds consuming the control diet, the plasma concentration of each

hormone, regardless of diet, increased significantly following exposure to stress. 3. Stress-induced increases in Aldo concentration were greatest in birds given diets containing concentrations of Na+. 4. Unlike the mammal and some other species responsible for the regulation of both corticosterone

of birds, Na+ may be the primary secretogogue and aldosterone synthesis in the mallard duck.

commercial breeder (Whistling Wings, Hanover, IL) and incubated at 37.4”C and 85% relative humidity. The newly hatched ducklings were maintained for two days in a brooder at 37.4”C before being transferred to -a room at 27°C with a dailv ohotooeriod of 12 hr light (08 : O&20 : 00 hr) and 12 hi iark. *The ducklings were allowed to eat and drink ad libitum, and each day at 08:OO hr the ducklings were given fresh supplies of food (Gamebird Developer, Ralston-Purina) and drinking water (tap water). This regime continued until the birds were four weeks old.

INTRODUCTION

Three adrenal steroid hormones, namely corticosterone, aldosterone and 1 I-deoxycorticosterone, have been unequivocally identified in plasma from the duck (Sandor, 1972). This is a pattern of adrenal steroidogenesis that is common to many mammalian and non-mammalian vertebrates. Unlike the mammalian adrenal cortex, however, the adrenal steroidogenie cells that produce these hormones in the duck are not arranged in histologically distinct layers. Detailed morphometric analysis has revealed the presence of at least two structurally distinct steroidogenie cells in the duck adrenal gland; one of these is located in a narrow zone immediately inside the connective tissue capsule while the other forms a more abundant inner region (Pearce et al., 1978). Although each type of cell is able to synthesize in vitro all of the three corticosteroids found in the plasma of this species, the cells in the inner region of the gland probably constitute the major site of corticosterone synthesis while those in the subcapsular region may be relatively more important as a site of aldosterone synthesis (Holmes and Cronshaw, 1985; Cronshaw et al., 1985). The purpose of these experiments was to define the circumstances under which different patterns of adrenal steroid hormone secretion can be evoked in vivo in juvenile mallard ducks. To this end we have attempted to stimulate the preferential release of each of the corticosteroids synthesized by the adrenal gland by feeding unstressed and stressed birds a series of diets containing different concentrations and proportions of Na+ and K+.

Protocol At 08:OO hr the experimental birds were given an ad libitum supply of one of four different diets containing Na+ and K+ in different concentrations and proportions, while the diet of the control birds remained unchanged. After 48 hr, a 5 ml blood sample was taken from each bird via a leg vein. The bird was then gently restrained, by wrapping it in muslin gauze, and 30 min later a second 5 ml blood sample was taken in the same manner. In view of the sensitivity of these birds to handling (Harvey et al., 1980). the first blood sample was taken within 1.5 min of capture, and experiments were conducted on not more than two birds in a single day. Each blood sample was collected in a heparinized syringe, immediately centrifuged for 5 min at 12,300g and 5°C and stored at -20°C. Since the fertilized eggs were purchased at intervals over a 6-month period, the responses of control birds derived from each shipment of eggs were tested. Also, to ensure that uniformly undisturbed birds were used throughout the study, the ducklings were exposed to, and maintained by, only one person (J.L.R.) from the day of hatching until the day on which they were used experimentally. The composition of the diets fed to the various groups of birds was as follows: (1) Control: Domestic drinking water (2.4mM Na+ and 0.08 mM K + ) and Gamebird Developer food (13.4mM Na+ and 31.6mM K+ per g dry weight). (2) High Na+: Hypertonic saline drinking water equivalent to 60% standard seawater (284mM NaCl in distilled water) and Gamebird Developer food.

MATERIALS AND METHODS

Animals Fertilized

mallard

duck

eggs

were

purchased

from

low

a 513

J. L.

514

REDONDO

et

al.

Table I. Plasma electrolyteconcentrations (mean f SEM) in juvenile mallard ducklings fed diets containing various concentrations and proportions of Na+ and K+ during the preceding 48 hr Diet

Body wt

(n)

(g)

Control (24) High Na+ (15) Low Na+ (8) High K+ (11) High K+ and Low Nat (11)

Osmolality

INa+ (nW

(mOsm)

250.2 * 9.3 222.1”’ + 14.8 214.4’ + 13.2 281.1”’ k 12.6 291.4” * 9.3

308.2 + 2.1 390.2” + 8.9 296.3”’ i 6.7 316.0” k 8.4 288.2** + I.1

[K’l (mM)

142 * 1.7 172” F 3.6 132” f I.0 144”s * 3.3 130.8 + 0.6

4.78 f0.13 4.20” kO.15 4.16** f 0.17 5.36”’ f 0.29 4.94”’ f0.17

The numbers in parentheses represent the number of birds in each group. *P < 0.05, **F’ < 0.01 and ns = not significantly different with respect to the corresponding control value.

(3)

(4) (5)

Low Na+: Glass distilled drinking water and boiled rice (0.9 mM Na+ and 1.4 mM K+ per g dry weight). The rice was rinsed (four times) and boiled in glass distilled water. High K+: 50mM KC1 solution in glass distilled water as drinking water and Gamebird Developer food. High K+/Low Na+: 50mM KC1 solution in glass distilled water as drinking water and boiled rice prepared as in (3).

Sample pur$cation. Plasma samples were thawed at room temperature and a 2.0ml aliquot of each sample was transferred to a new scintillation vial containing 8000 DPM of chromatographically pure [1,2-‘Hl-corticosterone, [1,2-‘HI-aldosterone and [1,2-‘Hl-deoxycorticosterone (Amersham Corporation, Arlington Heights, IL). The samples were allowed to equilibrate for 1 hr at room temperature before extraction with 10 volumes cold anhydrous ethyl ether. The ether phase was separated, dried under nitrogen, redissolved in 0.5 ml benzene :cyclohexane : methanol:water (25: I2.5:4: 1) and purified by gel filtration chromatography using Sephadex LH-20 and the same solvent system. The fractions containing labelled corticosterone, aldosterone and deoxycorticosterone were collected and pooled separately, evaporated to dryness under nitrogen and redissolved in 1.Oml of buffer containing 0.1 M tricine, 0.18 M NaCl and 0.015 M NaN, at pH 8. The radioactivity contained in 0.2 ml of this extract was used to determine the recovery of each steroid and the remainder of sample was stored at -20°C. Hormone analysis. The concentrations of corticosterone, aldosterone and deoxycorticosterone were measured by radioimmunoassay (Nishida et al., 1976; Mayes ef al., 1970; Arnold and James, 1971). Corticosterone antiserum was produced in rabbits immunized with corticosterone-21hemisuccinyl thyroglobulin (ICN Biomedicals Inc., Costa Mesa, CA). Sensitivity for this assay was 15 pg corticosterone per assay tube, intra-assay variation was 5.0% and

inter-assay variation was 12.8%. Aldosterone antiserum was raised in sheep with an aldosterone-3-oxine-bovine serum albumin conjugate (Amel Products Co., New York, NY); sensitivity was 10 pg of aldosterone per assay tube and intraand inter-assay variation was 3.0% and 10.3% respectively. Deoxycorticosterone antiserum was produced in rabbits using a deoxycorticosterone-3-oxine-bovine serum albumin conjugate (Endocrine Sciences, Tarzana, CA); sensitivity was 4 pg deoxycorticosterone per assay tube and intra- and inter-assay variation was 8.3% and 6.8% respectively. Electrolyle analysis. The concentrations of Na+ and K+ in samples of plasma, food and drinking water were determined by flame photometry (Eppendorf) and osmolalities were measured by vapor pressure osmometry (Wesco model 5lOOC). Statistics. Standard curves were fitted by the method of least squares. Mean values were expressed f standard error of the mean (SEM) and were compared either by the Student r-test or by the analysis of variance (ANOVA) followed by Duncan’s multiple range test to identify significant changes (Sokal and Rohlf, 1969). Significant stress-induced changes in plasma hormone concentration were identified using the matched pairs r-test. RESULTS 1 and 2)

Body weights (Tables

body weight of the birds consuming the low Na+ diet was significantly lower than that of the control birds (P c 0.05) whereas no significant change occurred in the group consuming the high Na+ diet. Although the mean terminal body weight of the group given the high K+ diet that contained a “normal” (control) concentration of Na+ did not differ significantly from the controls, both the ANOVA and the Student r-test indicated The

mean

terminal

Table 2. A statistical comparison (ANOVA) of the body weights and the plasma electrolyte concentrations recorded in juvenile mallard ducks maintained on diets containing different concentrations and proportions of Na+ and KC P value Comparison Control vs High Na+ Control vs Low Na+ Control vs High K+ Control vs High K+ and Low Na+ High Na+ vs Low Na+ High Na+ vs High K+ High Na+ vs High K+ and Low Na+ Low Na+ vs High KC Low Na+ vs High K+ and Low Na+ High K+ vs High K+ and Low Na+

Body wt ns <0.05 0s <0.05 ns
Osmolality to.01 ns ns < 0.05
INa+l

[K+l

eo.01 co.01 ns
<0.05 <0.05 <0.05 ns ns
Adrenal steroidogenic responses in duck

515

Table 3. Stressor-induced changes in the plasma corticosteroid concentrations (mean f SEM) of mallard maintained on diets containina different amounts and orooortions of Na+ and K+ Plasma corticosteroid Corticosterone Diet Control (24) High Nat (15) Low Nat

(8) High K+

(11) High K+ and Low Na+ (II)

concentration

ducklings

(ng per ml)

Aldosterone

Deoxycorticosterone

Prestress

Stress

Prestress

Stress

IS.9 f 2.2 27.6” + 2.6 12.9”’ i 3.0 12.1”s f 2.2 I I .O”‘ + 2.2

31.3tt f 2.9 43.7tt f 3.9 41.9tt + 5.3 34.6tt k 5.6 36.8tt f 2.3

0.289 + 0.051 0.251”’ f 0.073 1.5148’ _+0.289 0.273”’ f 0.067 0.577”’ + 0.147

0.914tt ? 0.203 0.823tt f 0.196 2.695t & 0.409 0.704t + 0.145 I .397tt f 0.259

Prestress f * f f k

Stress 0.582”* f 0.068 1.172tt kO.154 1.066tt kO.172 0.630tt k 0.125 1.217tt f 0.227

0.508 0.054 0.594”’ 0.077 0.454”’ 0.079 0.368”’ 0.071 0.375”’ 0.062

*P i 0.05, **I’ < 0.01 and ns = not significantly different with respect to the corresponding prestress value in the control birds. tP < 0.05, ttf < 0.01 and nd = not significantly different with respect to the corresponding value recorded before exposure to stress.

that the group consuming the high K+ diet that contained a relatively low concentration of Na+ had a significantly higher mean body weight than the controls (P < 0.01). When compared using the ANOVA, the mean terminal body weights of the two groups given food containing a high concentration of K+ did not differ significantly from one another, but each was significantly higher than the corresponding value for than those consuming either the high or the low Nat diets (Table 2). Plasma electrolyte concentrations (Tables 1 and 2) No significant variations in the concentrations of Na+, K+ and total osmotically active material were detected among unstressed control birds derived from successive shipments of fertilized eggs. Also, regardless of their diet, no significant changes occurred in plasma osmolality and ion concentrations following exposure of the birds to stress. For the purposes of comparison, therefore, all of the control birds have been considered collectively as a single group, and the electrolyte concentrations measured before and after exposure to stress were averaged for each bird in the control and experimental groups. When compared with the control diet, the high Na+ diet caused significant increases in plasma osmolality (P < 0.01) and Na+ concentration (P < 0.01) and a significant decrease in the plasma K+ concentration (P < 0.01); these changes stimulated extrarenal electrolyte excretion via the nasal salt glands.

In contrast, the low Na+ diet caused the plasma Na+ and K+ concentrations to decrease significantly (P < 0.01) compared with the controls, but these changes were not sufficient to cause a significant change in plasma osmolality. The Student t-test indicated that the total osmotic activity and the Na+ and K+ concentrations in the plasma of birds consuming the high K+ diet did not change with respect to the corresponding values in the control birds; the ANOVA, however, indicated that the plasma K+ concentrations increased significantly in response to consuming the high K+ diet (P < 0.05). In contrast, both the osmolality and the Na+ concentration of the plasma declined significantly with respect to the corresponding control values (P -C0.01) when the birds were given a high K+ diet that contained a relatively low concentration of Na+, but neither the Student t-test nor the ANOVA indicated any change in the plasma K+ concentration. The changes in plasma electrolyte composition that occurred in the groups given each of the experimental diets were also compared statistically with respect to one another; these comparisons are summarized in Table 2. Plasma corticosteroid concentrations (Tables 3 and 4) Before exposure to stress. By changing the Na+ and K+ intake, the plasma concentrations of corticosterone and aldosterone in undisturbed birds were

Table 4. A statistical comparison (ANOVA) of the plasma corticosteroid concentrations recorded in unstressed and stressed juvenile mallard ducks maintained on diets containing different concentrations and proportions of Na+ and K+. Similar comparisons are also made with respect to the hormone concentrations recorded after the birds had been exposed to stress P

Corticosterone Comvarison Control vs High Nat Control vs Low Na+ Control vs High K+ Control vs High K+ and Low Nat High Na+ vs Low Na+ High Na’ vs High K’ High Nat vs High K+ and Low Na’ Low Nat vs High K’ Low Nat vs High K+ and Low Nat High K’ vs High K+ and Low Na+ ’ ns = not significant

value

Aldosterone

Deoxycorticosterone

Prestress

Stress

Prestress

Stress

Prestress

Stress


< 0.05

IIS’

“S

“S

IIS

“S

ns

ns

IIS

OS

“S

IlS

llS

“S

<0.05 <0.05 <0.05

“s “S ns



“S

“S

“S

“S

“S

0.05
“S

“S

“S

to.01 <0.05 “S
nS <



“S

<0.05 <0.05 “S IIS nS

J. L. REWNDO

516

The stress-induced corticosteroid concentrations that occurred in each of the experimental groups were also compared statistically with respect to one another; these comparisons are summarized in Table 4.

-_

I

s

0

2 d 4

q

Unstressed Stressed

et al.

2

DISCUSSION

0

CQntr0l High Na

Low Na

High K

High K LowNa

Fig. 1. The relative abundance of aldosterone expressed as a fraction of the total measured corticosteroid (corticosterone + aldosterone + deoxycorticosterone) in plasma of unstressed and stressed mallard ducklings given diets con-

taining different amounts and proportions of Na+ and K+. *P i 0.05 and **p < 0.01 with respect to the corresponding value in the control birds. + P < 0.05 represents the significance of the stress-induced change. caused to increase independently of one another. Thus, compared with the control birds, those consuming the high Na+ diet had significantly higher plasma corticosterone concentrations (P < 0.01) but unaltered aldosterone concentrations, while those consuming the low Na+ diet had significantly elevated plasma aldosterone concentrations (P < 0.01) and unaltered corticosterone concentrations. Indeed, the increase in plasma aldosterone concentration that occurred in the birds consuming the low Na+ diet was such that this hormone accounted for almost 15% of the total corticosteroid measured in the plasma of the undisturbed birds (Fig. 1). Neither the high nor the low Na+ diets caused significant change in the plasma deoxycorticosterone concentrations. A high K+ diet, regardless of whether it contained either a normal (control) or a low concentration of Na+, caused no significant changes in plasma corticosterone, aldosterone, and deoxycorticosterone concentrations. In birds given the high K+ diet that contained a low concentration of Na+, however, the relative concentrations of these hormones were such that the amount of aldosterone constituted a significantly greater fraction of the total measured corticosteroid (P < 0.05) than it did in the control birds (Fig. 1). The corticosteroid hormone concentrations recorded in each of the experimental groups prior to their exposure to stress were also compared statistically with respect to one another; these comparisons are summarized in Table 4. After exposure to stress. With the exception of deoxycorticosterone concentrations in the control birds, the concentration of each hormone increased significantly in all groups following exposure of the birds to stress. In the two experimental groups given diets containing low concentrations of Na+, however, the stress-induced increases in plasma corticosterone concentration were proportionately greater than the increases in aldosterone (Table 3). Thus, in these instances aldosterone accounted for a significantly smaller fraction of the total measured corticosteroid (Fig. 1).

The mallard duckling clearly possesses a comprehensive system to control the synthesis and release of at least two of the corticosteroids secreted by the adrenal gland. Indeed, these data suggest that eight possible combinations of relative change in plasma corticosterone and aldosterone concentration might be expected to occur if the electrolyte intake of unstressed and stressed mallard ducklings is appropriately changed (Table 5). The circumstances that may require an organism to preferentially change the concentration of one hormone relative to another, however, may not be the same in all species and in this regard the mallard duck is particularly interesting. For example, these birds are able to live either in an environment where there is an abundance of fresh drinking water or in coastal, estuarine and alkali lake environments where they must consume drinking water that is always hyperosmotic relative to their body fluids. When they are living in a freshwater environment aldosterone, through its actions on the renal tubule, the cloaca, and the large intestine, is probably the principal corticosteroid concerned with the regulation of electrolyte balance (Skadhauge, 1978; Holmes et al., 1983). On the other hand, when the mallard duck consumes hyperosmotic drinking water corticosterone becomes the principal mineralregulating corticosteroid by acting simultaneously on at least three target tissues, namely the small intestine, the nasal salt glands, and the kidney (Holmes and Phillips, 1985). In view of these differences it is perhaps not surprising that the circumstances leading to the preferential synthesis and release of each hormone may differ from those seen in mammals. In birds, as in mammals, the ultimate principal regulator of glucocorticoid synthesis is corticotropin (ACTH). The results of the present experiments show that when the unstressed mallard duck consumes a high Na+ diet, the attendant changes in extracellular electrolyte composition cause a specific increase in the plasma corticosterone concentration. This suggests that in the mallard duck either a general increase in extracellular osmolality or specific increases in extracellular concentrations of Na+ and its associated anion, activates the hypophysiotropic reflex. Similar changes in extracellular electrolyte composition do not evoke this response in either mammals or in birds that do not possess functional nasal salt glands (Skadhauge et al., 1983; Inglis et al., 1987; Rosenberg and Hurwitz, 1987). In this regard, it is interesting to note that increases in plasma cortisol and corticosterone concentrations also accompany increases in extracellular osmolality and Na+ concentrations in several species of teleost fishes and amphibians (Hanke, 1985). This type of regulation, therefore may be characteristic of those organisms in which hormones such as cortisol and corticosterone have

Adrenal

steroidogenic

responses in duck

517

Table 5. The possible changes in plasma corticosterone and aldosterone concentration that may occur in juvenile mallard ducklings as a consequence of changing their dietary intake of Na+ and K+ and exposing them to acute stress. These implied changes are based on the Student tests (Table 3) and the ANOVA (Table 4) of the data. Unless otherwise indicated all of the effects relate to achannes in unstressed birds Change in hormone concentration Corticosterone

Aldosterone

UP

UP

UP

Down

UP

Unchanged

Change in diet From

To

Not evoked by specific changes in diet in unstressed birds, but always occurs on exposure to stress regardless of diet. Low Na+

High Na+ High Na+

Control or

High Na+

High K* Down Down

UP Down

High Na+

Low Na+

Low Na+ (Stressed)

High Na+ (Unstressed) OL-

Control (Unstressed)

Low Na+ (Stressed) Down

Unchanged

Control

High Na+ Or

Unchanged Unchanged

UP Down

High Na+

High K+

High K+

Low Na+

Low Na+

Control or

Unchanged

Unchanged

assumed important mineral regulating functions (Hanke et al., 1987). At least two factors are known to act directly on the mammalian adrenal cortex to cause specific increases in aldosterone synthesis, namely K+ and angiotensin II (Bing and Schulster, 1977; Boyd and Mulrow, 1972; Kaplan, 1965; Tait et al., 1972). In addition, elevated extracellular K+ concentrations and low extracellular Na+ concentrations may serve as secretogogues to activate the renin-angiotensin II systems in the kidney and the adrenal cortex (Davis and Freeman, 1976; Reid et al., 1978; Nakamura et al., 1985). However, in view of the extreme hyponatremia needed to stimulate a release of renin, K+ and not Na+ may serve as the primary secretogogue responsible for activating the renin-angiotensin system in mammals (Davis, 1975; Himathongham et al., 1975; Fraser et al., 1979). The situation in the mallard duck is obviously quite different. Only a relatively small decrease in plasma Na+ concentration was necessary to cause a five-fold increase in aldosterone concentration (Tables 1 and 3), whereas increases in plasma K+ concentration were consistently without effect in this respect. There are obviously serious discrepancies between the manner in which the synthesis and secretion of aldosterone is perceived to be controlled in birds and mammals. Furthermore, although a renin-angiotensin system exists in birds (Nishimura, 1980) it has yet to be identified specifically with the control of aldosterone synthesis in the avian adrenal gland in vivo (Gray et al., 1986; Rosenberg and Hurwitz, 1987). Since none of the diets induced a specific increase in plasma deoxycorticosterone concentration, we can say little regarding the circumstances that may lead to preferential synthesis of this hormone in the

Low Na+

High K+

High K+

High K+ plus Low Na+

mallard duckling. A comparison of the plasma deoxycorticosterone concentrations in the two groups of birds consuming the high K+ diets with those consuming the high Na+ diet, however, suggests that high levels of K+ intake may be associated with a suppression of plasma concentrations of this hormone (Table 4). The responses of the mallard ducklings to stress were in general similar to those seen in mammals and other species of birds (Holmes and Phillips, 1976; Harvey et al., 1984; Rosenberg and Hurwitz, 1987). It is interesting to note, however, that the extent of the response was dependent upon electrolyte intake. Thus, the stress-induced concentration of corticosterone was greatest when the Na+ intake was high, while the corresponding value for aldosterone was greatest when the Na+ intake was low; increasing the K+ concentration in the low Nat diet, however, caused the stress-induced concentration of aldosterone to become significantly lower (Tables 3 and 4). Also, although exposure to stress caused no increase in the plasma deoxycorticosterone concentration of the control birds, stress-induced increases occurred in birds consuming all of the experimental diets. Studies in both mammals and birds suggest that stress-induced increases in corticosteroid synthesis reflect the simultaneous actions of ACTH and catecholamines on the adrenal steroidogenic cells, the renin-angiotensin system, and perhaps the hypothalamo-hypophyseal axis (Tilders et al., 1980; Young and Landsberg, 1983; Axelrod and Reisine, 1984; Rees et al., 1985). The stress-induced changes that we have observed in mallard ducklings, however, probably reflect a modified interplay of these factors as they respond appropriately to a set of secretogogues, that seems to differ from those described for the mammalian model.

J. L.

518

bDOND0

Acknowledgements-This

study was supported by grants from the National Science Foundation (PCM-79-15777) and Committee on Research, University of California to James Cronshaw and W. N. Holmes, and by a fellowship to Jose L. Redondo from the Consejo National de Ciencia y Tecnologia, Mexico.

et al.

Holmes W. N. and Phillips J. G. (1985) The avian salt gland. Biol. Rev. 60, 213-256.

Holmes W. N., Wright A. and Gorsline J. (1983) Effects of aldosterone and corticosterone on cloaca1 water and electrolyte excretion of constantly-loaded intact and colostomized ducks (Anas platyrhynchos). Comp. Biochem. Physiol. 74A, 795-805.

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