Breeding condition, temperature, and the regulation of salt and water by pituitary hormones in the red-spotted newt, Notophthalmus viridescens

GENERAL.

AND

COMPARATIVE

ENDOCRINOLOGY

51,

292-302 (1983)

Breeding Condition, Temperatut-6, Water by Pituitary Hormones Notophthalmus

and the Regulation i:n the Red-Spotted

of Salt and Newt,

viridescens

PATRICIA STOCKING BROWN,~TEPHEN C.BROWN,ISABELLE SHEILA M. LEMKE

T. BISCEGLIO,

AND

Department of Biology, Siena College, Loudonville, New York 12211; Department of Biological Sciences, State University of New York, Albany, New York 12222; and Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720

Accepted September 30, 1982 integumental transepithelial potential (TEP) in the Eastern red-spotted newt (Noviridescens) increases linearly with external [Na+] from 0.1 to 10 m&I and is anion independent. Both integumental TEP and osmotic permeability are higher in laboratory-conditioned (LC, terrestrial) than in breeding-condition (BC, aquatic) newts at temperatures of 5-25”. Prolactin (PRL) treatment of LC newts decreased both TEP and rate of water uptake. Arginine vasotocin (AVT) treatment resulted in a substantial increase in water uptake in LC newts, while little or no AVT response was seen in PRL-treated or BC newts. Hypophysectomy (HX) or ergocryptine treatment increased TEP in BC newts, whereas HX + PRL maintained TEP at control levels. Although ergocryptine and HX were without effect on water uptake in BC newts kept at 5” for 9 days, HX + ACTH increased water uptake. HX produced a substantial fall in serum [Na+] in BC newts, while either PRL or ACTH replacement elevated serum [Na+]. Combined ACTH and PRL treatment returned serum [Na+] to control levels. These data suggest that high endogenous prolactin plays a significant role in maintaining serum [Na*] and integumental permeability and transport characteristics in breeding-condition N. viridescens. Although PRL and ACTH are both sodium retaining in the aquatic breeding stage, these two hormones promote opposite effects on the integument; PRL decreases both water uptake and integumental TEP, whereas ACTH (presumably acting through the adrenals) increases water uptake and possibly TEP In vivo tophthalmus

In most amphibians, the environmental transition from water to land which occurs at the end of larval development is both reversed and repeated during the annual breeding migrations of the sexually mature adults. In salamandrid urodeles in particular, such breeding migrations are accompanied by extensive remodeling of the adult integument, during which the rough dry skin of the terrestrial-phase adult is transformed into the smooth slimy skin of the aquatic, breeding-condition animal. For the Eastern red-spotted newt (Notophthalmus viridescelzs), it has been shown that both the migratory activity and the assumption of integumental breeding characteristics are under the control of the adenohypophysial hormone prolactin (Chadwick, 1941). The 292 0016-6480/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

reverse integumental transformation (i.e., from breeding phase to terrestrial phase) can be experimentally elicited by thyroxine administration (Grant and Cooper, 1965; Dent et al., 1973), but will also occur spontaneously when breeding-condition newts are kept in the laboratory at room temperature (Dent et al., 1973; Walters and Greenwald, 1977; Dent, 1982). Although the water/land transitions which occur during breeding in newts are undoubtedly accompanied by physiological adjustments in the control of hydromineral balance, little work has been done directly on this problem. [For example, most previous studies on salamandrids, including our own on N. viridescens (Brown and Brown, 1971, 1973, 1977) and on Taricha torosa (Brown and Brown, 1980;

NEWT HYDROMINERAL

Brown et al., 1981), have been done on terrestrial-phase newts or the equivalent laboratory-conditioned animals.] In addition to the annual water/land transitions accompanying breeding, adult newts in the temperate zone are also exposed to a yearly temperature cycle. The osmoregulatory consequences of this temperature variation are not well known for salamanders, although a number of investigators have reported that frogs and toads are noticeably edematous during the cold weather associated with winter hibernation and spring breeding (see, for example, Jargensen et al., 1978). Correlated with this, observations of seasonal (temperature) variation in skin osmotic permeability (Hevesy et al., 1935; Share and Ussing, 1965; Coiner, 1977; Parsons et al., 1978), kidney function (Jorgensen, 1950; Schmidt-Nielsen and Forster, 1954; Miller et al., 1968), and water turnover (DeHaan and Bakker, 1924) have consistently shown these parameters to be lower in winter than in summer. Furthermore, such changes can be experimentally induced by acute temperature changes in whole-animal or in vitro preparations (Jorgensen, 1950; Jorgensen et al., 1978; Parsons and Lau, 1976). Consideration of potential hormonal involvement in these anuran temperature responses has been limited to possible antidiuretic hormone action on skin permeability. The data (from both in vivo and in vitro experiments) consistently show lower ADH responses in winter/cold-temperature preparations than in summer/warm-temperature ones (Share and Ussing, 1965; Jorgensen, 1950; Parsons et al., 1978). Whether or not ADH could mediate the observed long-term seasonal changes in skin permeability is under current debate (Parsons et al., 1978, Jorgensen et aE., 1978). A hormone more likely to be important in controlhng long-term seasonal and/or temperature-dependent changes in amphibian osmoregulatory performance, in our view, is prolactin. A number of experi-

293

REGULATION

mental studies have shown that prolactin exerts profound effects on the water content of amphibian tissues (Eddy, 1979; Platt and Christopher, 1977) and on epitheliai permeability in a wide variety of animals, including amphibians (see Clarke and Bern, 1980, for recent review). Moreover, a close correlation between ambient temperature and prolactin secretion (high in winter/cold; low in summer/warm) has previously been suggested (Mazzi, 1970). In the absence of a readily available radioimmunoassay (RJA) for amphibian prolactin, salamandrid urodeles offer especially favorable material to examine the possible interaction between water/land transitions, environmental temperature, prolactin secretion, integumental permeability, and hydromineral balance, since newt skin structure gives a reliableif rough-estimate of the presence of endogenous prolactin. We began our investigation of this interaction utilizing breedingcondition N. viridescens and employing laboratory conditioning as a method to induce these animals to transform into the terrestrial phase. The two main questions we address in the present paper are: (1) How does temperature affect the integument& water and ion permeabilities in breeding and terrestrial-phase newts? (2) Are prolactinand possibly ACTH-seasonally important in regulating salamandrid hydromineral balance? MATERIALS

AND METHODS

Animals. N. viridescens (wt range, 2.8-3.9 g) were collected from ponds and lakes in upstate New York and Vermont in May, June, and early July 1980. At the time of capture, all animals were in breeding condition (i.e., possessing the characteristic smooth, mucus-covered skin and tall tail fin). In the laboratory all animals were maintained in aquaria containing artificial pond water (APW; 1.3 m&f NaCl, 0.8 m&l CaQ, 0.1 mM KCl, and 0.2 mM NaHCO,) with half the animals kept at 18-20” and the remainder at 5”. Salamanders were fed liver and mealworms and kept on a 12L:12D photocycle. After 3-4 weeks of such maintenance, animals kept at B-20” had lost weight (mean wt = 1.9 +- 0.1 g), their tail fins had regressed in height, and their skin had become rough. These ani-

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BROWN ET AL.

mals will be referred to as laboratory-conditioned (LC) newts. By contrast, animals kept at 5” maintained their body weight (mean wt = 3.3 f 0.1 g) and retained the tall tail fin and smooth, mucus-covered skin. These animals will be referred to as breeding-condition (BC) newts. Transepithelial potential. In vivo integumental potential difference was measured according to the method of Kirschner (1970). Animals were anesthetized with MS-222, and a Ringer-agar bridge made of PE-50 tubing was inserted through a small hole into the body cavity. Another Ringer-agar bridge was placed in contact with the external bathing solution. The two bridges were then connected to calomel cells, and the potential across the skin was measured with a digital voltmeter. Junctional potentials arising from electrolyte assymetry were corrected according to Bentley (1975). Integumental permeability. Integumental permeability was estimated by measuring water uptake as described previously (Brown and Brown, 1977). Briefly, at the start of each experiment the animals had their urinary bladders emptied and were then blotted and weighed. Next, a latex sleeve was placed over the cloacal opening and the animal immersed in APW. After 1 hr the animals were reweighed and integumental permeability was calculated from the total weight gain (= water uptake). Weight gain before and after arginine vasotocin (AVT) treatment was compared in LC and BC newts at three temperatures. Each animal was injected with 20 ~1 of 0.7% NaCl and the rate of water uptake determined for 1 hr (control period). Animals were then injected with 20 pl AVT (12 mu/g) and the rate of water uptake was measured during a second Ihr period. Serum electrolytes. Blood was collected from the caudal artery into heparinized microcapillary tubes. All tubes were flame-sealed and centrifuged; plasma sodium was determined by flame photometry. Hormone treatment of laboratory-conditioned animals. Intact LC newts were injected daily with 0.7% NaCl (controls), 10 pg bovine prolactin (NIH-P-B4, 18.5 IU/mg), or 1.28 pg porcine ACTH (Sigma, 69 IU/ mg). All injections were in a volume of 10 p,l; prolactin and ACTH were dissolved in 0.7% NaCl. Measurements of TEP were made after 3 weeks of hormone treatment. Hypophysectomy and hormone treatment of breeding-condition animals. Water uptake and TEP measurements were made on all BC newts within 2 days of capture. They were then divided into seven groups. Three were kept intact and given daily 20 pl injections of 10% EtOH (control, CON), 1.0 or 10.0 pg/g a-ergocryptine (ERGO) (Sigma). The ergocryptine was mixed with an equal weight of tartaric acid and dissolved in 10% EtOH. Injections were started 3 days after field collection. Hypophysectomy (HX) of

the remaining four groups was performed on the third or fourth day after collection. Animals in these groups were given daily 20 pl injections of 0.7% NaCl (control), 3 pgig prolactin, 0.4 pg/g ACTH, or 3 &g prolactin f 0.4 pg/g ACTH. All BC animals were kept at 5” during the experiment. Measurements of water uptake, TEP, and serum Na were made 9, 10, and 11 days after HX, ergocryptine, or hormone treatment, respectively. Statistics. One-way analysis of variance (ANOVA) and the Student-Newman-Keuls test (Sokal and Rohlf, 1969) were used to determine treatment effects when multiple treatments were compared. Student’s t test was used when only two experimental groups were compared. Regression analysis was performed according to Sokal and Rohlf (1981).

RESULTS

Transepithelial potential. Initial measurements were made on laboratory-conditioned newts to examine the effects of transport inhibitors and external ions on in vivo TEP. The TEP in N. viridescens appears to be caused predominantly, if not exclusively, by sodium transport since no significant TEP could be measured in animals immersed in sodium-free (choline chloride) solutions. In addition, the in vivo TEP found in newts immersed in sodium-containing solutions could be completely abolished by addition of amiloride (1O-5 M&an effect which could be reversed by replacing the bathing medium with amiloride-free saline. Figure 1 shows that in vivo TEP increased linearly with increasing [Na+], from 0.1 to 10.0 mA4. Over this range, the TEP appears anion independent since there was no significant difference between NaCl and Na,SO, (b = 39 mV for NaCI; 40.5 mV for Na,SO,). Since TEP remained constant (Na,SO,) or actually decreased (NaCl) at external sodium concentrations greater than 10 m&f, all subsequent measurements were made at [Na+lext from 1 to 10 rnM. Preliminary comparisons of the TEP in LC newts (maintained and measured at 20”) with the TEP in BC newts (maintained and measured at 5’) suggested that both laboratory conditioning and higher measurement temperatures might have an effect on

NEWT HYDROMINERAL

295

REGULATION

TABLE 1 (IX) AND In Viva TEP n\i LABORATORY-CONDITIONED BREEDING-CONDITION (BC) NEWTS TREATED FOR 3 WEEKS WITH SALINE (CONTROL), 10 ~&DAY BOVINE PROLACTIN, OR 1.28 I)-&JDAY ACTH, MEASURED IN HI mh4 Na,SO,

80

Treatment

[Na] mM/L

1. In vivo TEP (inside positive) as a function of external sodium concentration in laboratory-conditioned (LC) newts. FIG.

in vivo TEP. We therefore examined the ef-

fects of measurement temperature on TEP in both LC and BC ‘newts. Figure 2 shows that the TEP of LC newts was significantly higher than that of BC newts;at all temperatures tested (Y, P < 0.02; 15”, P < 0.001; 25”, P < 0.01). Although TEP was linearly related to tem+rature in BC newts, TEP tended to level off above 15” in LC animals. The effects of long-term (3-week) hormone treatment of intact LC and BC newts are shown in Table 1. Prolactin treatment reduced in vivo TEP by ca. 25% in both groups. ACTH treatment, by contrast, consistently increased in vivo TEP (ca. lo20%)) although such increases were not statistically significant in the BC newts. Surgical removal of the pituitary from BC newts

LC newts Control Prolactin ACTH BC newts Control Prolactin ACTH Note. 0P < *P < cP <

N

Temp

TEP (mV)

11 12 13

2.5 25 25

82.2 -1- 2.3 63.3 t 4.7” 89.5 f 1.7”

10 10 10

20 20 20

45.8 -c 3.6 33.9 ” 2.3b 56.2 2 5.7

Mean + SE. Differs from control. 0.025. 0.01. 0.005.

(Fig. 3) led to a significant increase vivo TEP (P < 0.01 between control and HX). However, when hypophysectomized BC newts were treated with prolactin, in vivo TEP was indistinguishible from the intact BC controls. Injection of the prolactinrelease inhibitor, ergocryptine, into intact BC newts (Fig. 4) also resulted iti increased in vivo TEP (P < 0.001 between CON and high ERGO). As can be seen from the data, the TEP in newts treated with the higher

100 1 w

60-

CON

TEMPERATURE

VJ

FIG. 2. In vivo TEP as a function of temperature in laboratory-conditioned (LC) and breeding-condition (BC) newts bathed in 10 mM Na$O,.

HX

HX + FRL

FIG. 3. In vivo TEP in intact (CON), ‘hypophysectomized (HX), and prolactin-treated hypaphysectomized (HX + PRL) breeding-condition newts, measured at 15” in 2.5 m&Z NaCl. HX differs from control, P < 0.05.

296

BROWN ET AL.

CON

HX

LOW ERGO

HIGH ERGO

FIG. 4. In vivo TEP in control (CON), hypophysectomized (HX), and ergocryptine (ERGO)-treated intact breeding-condition newts, measured at 5” in 1.6 m&Z NaCl. Difference from control: HX, P < 0.05; high ERGO, P < 0.001.

dose of ergocryptine was not different from that of hypophysectomized animals. Integumental osmotic permeability. In untreated intact BC and LC newts, the rate of water uptake (Fig. 5) increased linearly with increasing temperature between 7 and 26”. However, the rates of increase (slopes)

0'

, 5

I 25

IO TEMPEEATURE

&‘6;

FIG. 5. Water uptake as a function of temperature in laboratory-conditioned (open circles) and breedingcondition (closed circles) newts.

were significantly different (P < 0.001) for the two groups of animals. At the highest temperature examined, water uptake in BC animals was only 43% of that of LC animals . In an attempt to assess further integumental permeability, a standard challenge dose of AVT was administered to BC and LC newts, as well as to LC newts which had been pretreated with prolactin. Figure 6 shows that water uptake increased in response to AVT injection in all untreated LC animals and BC animals at 17 and 25”. However, AVT was ineffective in altering water uptake (osmotic permeability) in BC newts at 8” or in prolactin-treated LC newts at 25”. Rates of water uptake were measured in BC animals 9 days after hypophysectomy or the onset of hormone treatment. Table 2 shows that most of the animal groups maintained the low water permeability characteristic of the BC controls. The group receiving ACTH, however, showed a significant increase (26% above controls; 33% above HX; P < 0.01) in the rate of water uptake. Serum sodium. Figure 7 shows the serum sodium concentrations in BC newts 11 days after hypophysectomy and/or the onset of

TEMPERATURE

(“C)

FIG. 6. Water uptake before and after AVT injection in breeding-condition (BC), laboratory-conditioned (LC), and prolactin-pretreated laboratory-conditioned (LC + PRL) newts as a function of temperature.

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HYDROMINERAL

TABLE 2 WATER UPTAKE (AT 17”) IN BREEDING-CONDITION NEWTS, MEASURED 9 DAYS AFTER HX AND/OR ONSET OF HORMONE TREATMENT

Treatment Control Low ergocryptine High ergocryptine HX HX HX HX

Water uptake Wdhr)

N 10 11 10 11 12

+ PRL + ACTH + PRL + ACTH

27.9 31.5 26.0 26.3 26.9 35.1

i -rk i i

1.3 1.9 2.2 2.3 1.9

t l.f+ 31.6 Z!I 1.9

12 8

Note. Mean _t SE. a Differs from HX, P < 0.01.

hormone treatment. Serum sodium appeared unaffected by ergocryptine treatment, but was decreased to 72% of control values (P < 0.01) by hypophysectomy. Prolactin replacement in hypophysectomized BC newts raised serum sodium to 88% of control values (P < 0.01 between HX and RX + PRL). ACTH raised serum sodium to 93% of controls (P < 0.01 between HX and HX + ACTH), whereas hypophysectomized BC newts treated with both ACTH and prolactin had serum sodium levels not statistically different from intact controls.

CON

LOW ERGO

HIGH ERGO

HX

HX+ PRL

HX+ ACTH

HX+ zt

FIG. 7. Serum sodium concentration in breedingcondition newts after ergocryptine, hypophysectomy, or hormone treatment.

297

REGULATION

DISCUSStON In vivo transepithelial potential. The ini-

tial linear increase of in vivo TEP with increasing external sodium concentrations, observed in laboratory-conditioned N. viridescens, is similar to that reported for other amphibians including Rana pipiens, Ambystoma tigrinum, Amphiuma means, and T. torosa (Dietz et al., 1967; Alvarado and Stiffler, 1970; Kirschner, 1970; Bentley, 1975; Brown et al., 1981). In all of these species, however, TEP declines at external sodium concentrations exceeding IO-20 mM when Cl- is the anion. By contrast, the in vivo TEP of LC newts reached an apparently stable plateau at [Na+lext 2 10 mA4 when sulfate was the anionic form. Similar findings have also been reported for R. pi1970) and T. torosa piens (Kirschner, (Brown, 1977; Brown et al., 1981). These data suggest that saturation of the sodium transport mechanism occurs at [Na+lexr of ca. 10 n-M, which correlates well with radioactive sodium influx measurements on laboratory-conditioned N. viridescens (Wittig and Brown, 1977). The differential response to Cl- and S042- has been interpreted as reflecting greater integument-al permeability (leak conductance} to chloride at high ambient concentrations (Kirschner, 1970). Comparison of LC and BC newts reveals consistently higher in vivo TEP in laboratory-conditioned animals. This is consistent with the findings of Walters and Creenwald (1977), who report higher rates of radioactive sodium influx in laboratoryconditioned N. viridescens than in breedingcondition animals. In addition, Lodi et al. (1978, 1982) report that in another salamandrid, Triturus cristatus, integumental shortcircuit current (in vitro) was higher in summer (terrestrial) newts than in winter (aquatic) /animals. Although both LC, and BC newts showed increasing TEPs with increasing temperatures in acute experiments, the elevations of the temperature curves differed significantly. To our knowl-

298

BROWN

edge, no other studies have examined the effect of temperature on in vivo TEP in amphibians . Long-term prolactin treatment was effective in reducing the high in vivo TEP characteristic of laboratory-conditioned N. viridescens to levels comparable with those found in breeding-condition animals. Data from experiments on other salamandrid urodeles are largely consistent with this. For example, Lodi et al. (1978, 1982) found prolactin to be effective in reducing active sodium transport (XC) across the skin of T. cristatus in the summer, and we earlier found prolactin to be effective in countering the thyroxine-induced increase in SCC in laboratory-conditioned N. viridescens (Brown and Brown, 1973). Moreover, we consistently find prolactin treatment to depress TEP in T. torosa (Brown et al., 1981), although Harlow (1977) could show no effect on TEP or SCC in his experiments. Long-term ACTH treatment of both LC and BC newts elicited a consistent elevation of in vivo TEP. Although few studies of the ACTH-adrenal axis have been done on urodeles, ACTH is presumed to cause adrenal corticoid release in these amphibians. In spite of the numerous studies demonstrating that aldosterone increases XC across anuran membranes, no similar studies have been done on urodeles. In breeding-condition newts maintained at low temperatures, prolactin removal (either by administration of ergocryptine or by hypophysectomy) led to an elevation of in vivo TEP to levels comparable to those found in laboratory-conditioned animals. Prolactin replacement in hypophysectomized BC newts was completely effective in returning TEP to the lower control values, In winter T. cristatus, Lodi et al. (1978, 1982) found both hypophysectomy and bromocryptine injections to be effective in elevating SCC. In these experiments also, prolactin treatment of hypophysectomized newts returned SCC to control levels. Although in earlier experiments with T. to-

ET AL.

rosa (Brown et al., 1981) we found hypophysectomy to be without effect on in vivo TEP, the experimental animals were laboratory conditioned and presumably had low levels of endogenous prolactin. Consistent with this interpretation is the fact that prolactin treatment of these animals significantly lowered TEP Although the effects of long-term prolactin treatment or hypophysectomy on integumental transport appear consistent in salamandrid urodeles, this is not true for other amphibians. For example, in Desmognathus fuscus (Brown et al., 1979), hypophysectomy lowered TEP and SCC and neither prolactin nor corticosterone acted as an effective replacement. Moreover, Myers et al. (1961) report that in R. pipiens hypophysectomy also led to a decrease of integumental TEP and SCC-an effect which persisted for 3 weeks. Integumental osmotic permeability. The differences in rates of water uptake by LC and BC newts were modest at low temperatures (5-W), but substantial at higher ones. At 16”, LC newts showed an osmotic influx equal to 153% of their body wtiday vs 56% body wtfday in BC newts; at 25”, the respective values were 197 and 83%. Prolactin treatment of LC newts reduced water uptake to rates comparable to those of BC animals. These differences in osmotic permeability were paralleled by the magnitudes of response to AVT administration. Given the same challenge dose, LC newts showed a greater increase in water uptake at all temperatures. BC newts at 8” and LC newts pretreated with prolactin at 25” showed no response. As in frogs and toads, therefore, Notophthalmus shows reduced integumental osmotic permeability, rate of water turnover, and AVT response when kept at low environmental temperatures. In addition, the data indicate that prolactin treatment alone will produce the same effects, even when animals are maintained at warm temperatures. Other work on salamandrid urodeles is consistent with this. For example, we found that prolactin treatment

NEWT

HYDROMINERAL

also reduced AVT responsiveness in laboratory-conditioned T. torosa (Brown and Brown, 1982) and Lodi et al. (1982) report that prolactin treatment reduced the high osmotic permeability of summer T. cristatus. The mechanism by which prolactin reduces the AVT response in newts remains unexplained at present. It is possible, for example, that prolactin may cause a decrease in target tissue AVT sensitivity by a specific mechanism such as down-regulation of AVT receptors. Alternatively, a more general mechanism of decreasing osmotic permeability may be involved, since we have also found prolactin to counteract the thyroxine-induced increase in integumental permeability in N. viridescens (Brown and Brown, 1973). In breeding-condition newts, rates of water uptake remained at low control levels after long-term (IO-day) ergocryptine treatment, hypophysectomy, or hypophysectomy + prolactin replacement. Only those hypophysectomized newts receiving ACTH showed significantly altered (increased) rates of water uptake. These data suggest the possibility that ACTH (presumably acting through adrenal steroids) may be involved in maintaining high skin permeability in laboratory-conditioned or terrestrial-phase animals. In salamandrid urodeles such as N. viridescens, therefore, prolactin appears to act to maintain low integumental osmotic permeability in the aquatic breeding phase, while adrenal steroids may act to maintain high integumental permeability in the terrestrial (or equivalent laboratory-conditioned) phase. An analogous system of hormonal control over integumental permeability has been shown to exist in several euryhaline and anadromous teleosts (Bern, 1975 ; Clarke and Bern, 1980). Serum sodium concentration. Hypophysectomy of breeding-condition newts led to a 30% drop in serum sodium levels. This decline is nearly twice that found in hypophysectomized laboratory-conditioned newts reported earlier (Brown and Brown,

REGULATION

299

1973). The significant increases in serum sodium caused by prolactin and ACTH, alone and in combination, argue for important roles for these hormones in regulating salt balance in aquatic breeding-phase newts. In light of these findings, the failure of our ergocryptine treatment to alter serum sodium levels was somewhat surprising. Possibly once-a-day injection of this inhibitor is simply insufficient to depress prolactin release for an entire 24-hr period. C ‘her evidence suggesting that prolactin has a sodium-retaining effect in urodeles has been found in T. cristatus (Sampietro and Vercelli, 1968)) larval A. tigrinum (Wittouck, 1975), and neotenic A. gracile (Brewer et al., 1980) and Necturus macuEosus (Pang and Sawyer, 1974; Gallagher, 1974). By contrast, prolactin replacement failed to maintain serum sodium after hypophysectomy in laboratory-conditioned N. viridescens (Brown and Brown, 1973), D. fuscus (Brown et al., 1979), R. pipierts (Crim, 1972), and T. torosa (Grim, 1972; Brown et al., 1981). It should be noted that in the latter two species, prolactin treatment actually caused further reduction in serum sodium levels. The earlier dataprincipally on larval or neotenic hh9stoma or Necturus-have led a number of investigators to conclude that prolactin may only have a sodium-retaining action in larval or neotenic stages of urodeles (Nicoll, 1974; Bern, 1975; Platt and Christopher, 1977; Brown et al., 1979; Brewer et al., 1980). However, the data presented’here suggest that prolactin may also have a sodiumretaining effect in aquatic breeding-phase salamandrids. Whether or not prolactin has a comparable effect in other adult amphibians that return to water during the breeding season has not been investigated. Salamandrids may, in fact, be unique, since apparently no other adult amphibians are known to undergo the major structural changes in the integument during their seasonal reproductive activity. Such mo~rphological changes in breeding salamandrids may be

300

BROWN ET AL.

accompanied by physiological “reversion’” to a larval condition where prolactin has some sodium-retaining effect. In addition, the data clearly show that ACTH (presumably acting through the adrenals) is also an effective sodium-retaining agent in breeding-condition newts. ACTH has not previously been reported to maintain serum sodium in urodeles, although Middler et al. (1969) found that cortisol (but not ACTH) could maintain serum sodium in B. marinus. The fact that serum sodium was completely restored to control levels by a combination of prolactin + ACTH replacement suggests the possibility of interaction between these hormones. Recent demonstrations that prolactin can stimulate adrenal steroid secretion in mammals (Ogle and Kitay, 1979), birds (Carsia et al., 1981), and fish (Fleming and Ball, 1972; Hanson and Fleming, 1979) suggest that such interaction may be a common feature of vertebrates and, hence, deserving of further investigation in amphibians. In summary, it appears that adult N. viridescens regularly alternate between two distinct seasonal states of osmoregulatory control. In terrestrial-phase (or equivalent laboratory-conditioned) newts, integumental TEP and osmotic permeability are high at a time when endogenous prolactin levels are presumably low. Treatment with prolactin at this stage causes significant reduction in both TEP and osmotic permeability. When newts are in the aquatic phase ( = breeding condition), integumental ion transport and osmotic permeability are low at a time when endogenous prolactin levels are presumably high. At this stage, experimental treatments designed to lower prolactin levels (i.e., HX and/or ergocryptine injection) markedly increase TEP but do not alter water uptake; prolactin replacement completely reverses the effect of HX on TEP These data indicate that TEP and osmotic permeability are independently controlled, with osmotic permeability being less dependent on the continual presence of pi-

tuitary hormones. The effects of ACTH on the integument were always opposite to those elicited by prolactin. Nevertheless, both hormones were effective in elevating serum sodium levels following HX. This suggests that prolactin helps to maintain body fluid salt concentration by reducing osmotic water influx, whereas ACTH may act by increasing the rate of salt retention. ACKNOWLEDGMENTS We wish to thank Professor M. Manning of the Medical College of Ohio for supplying AVT and Dr. H. A. Bern for his kind interest and support during the later stages of this work at Berkeley. This work was supported in part by NSF Grant PCM-79-22793, NSF Fellowship SPl-8013095 (P.S.B.), NIH Fellowship L-F32AM-06371-01 (S.C.B.), and the William and Flora Hewlett Foundation of Research Corporation.

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Bern, H. A. (1975). Prolactin and osmoregulation. Amer. Zool. 15, 937-948. Brewer, K. J., Hoyt, B. J., and McKeown, B. A. (1980). The effects of prolactin, corticosterone and ergocryptine on sodium balance in the urodele, Ambystoma 203-208.

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Brown, P. S. (1977). Comparison of transepithelial potential differences in plethodontid and salamandrid urodeles. Amer. Zool. 17, 878. Brown, P S., and Brown, S. C. (1971). Growth and metabolic effects of prolactin and growth hormone in the red-spotted newt, Notophthalmus viridestens. J. Exp. Zool. 178, 29-34. Brown, P. S., and Brown, S. C. (1973). Prolactin and thyroid hormone interactions in salt and water balaxe in the newt, Notophthalmus viridescens. Gen. Comp.

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20, 456-466.

Brown, P S., and Brown, S. C. (1977). Water balance responses to dehydration and neurohypophysial peptides in the salamander, Notophthalmus viridescens.

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