Sodium appetite elicited by furosemide: effects of differential dietary maintenance

BEHAVIORAL BIOLOGY, 10,313-327 (1974), Abstract No. 3243R

Sodium Appetite Elicited by Furosemide: Effects of Differential Dietary Maintenance

J O H N E. J A L O W I E C *

Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545

Rats were maintained on two diets differing in sodium content and were injected later with the diuretic, furosemide, to induce acute sodium loss. Rats maintained on a sodium deficient diet ingested more sodium following diuretic injection than animals maintained on a sodium replete diet and showed behavioral overcompensation for the deficit established by natriuresis. In contrast, the salt intake of diuretic-injected rats maintained on the sodium replete diet was precisely sufficient to restore sodium balance to control levels. Maintenance on a sodium deficient diet did not reduce sodium balance substantially and furosemide-induced sodium loss produced similar degrees of hypovolemia and hyponatremia when water was available regardless of dietary maintenance. However, marked urine sodium retention accompanied by enhanced excretion of potassium during maintenance on the sodium deficient diet suggested that mineralocorticoid activity was enhanced prior to diuretic treatment. The greater intakes of hypertonic (0.51 M) saline in the diuretic-injected rats maintained on the sodium deficient diet were attributed to potentiation of the sodium appetite elicited by sodium loss by high endogenous mineralocorticoid levels, since maintenance on the sodium deficient diet did not elicit sodium appetite in the absence of sodium depletion.

INTRODUCTION T h e s o d i u m a p p e t i t e elicited b y s o d i u m d e f i c i e n c y in t h e rat can be m e d i a t e d b y decreases in p l a s m a v o l u m e ( h y p o v o l e m i a ) a n d s o d i u m c o n c e n t r a t i o n ( h y p o n a t r e m i a ) as well as increases in a l d o s t e r o n e ( S t r i c k e r a n d Wolf, 1966; W o l f a n d H a n d a l , 1966). A l t h o u g h e a c h o f these c o n c o m i t a n t s o f s o d i u m d e f i c i e n c y c o u l d b e n a t r o r e x i g e n i c ( s o d i u m a p p e t i t e s t i m u l a t i n g ) by t h e m s e l v e s ( S t r i c k e r a n d Wolf, 1969; T o s t e s o n , D e F r i e z , A b r a m s , G o t t s c h a l k *This study was supported, in part, by Grant APA-248 from the National Research Council of Canada awarded to E. M. Stricker and NIMH training Grant 5 TOI MH 10625. I would like to thank Dr. E. M. Stricker for helpful criticism and support and Dr. R. A. Malo of Hoechst Pharmaceuticals of Montreal for generously supplying the furosemide (Lasix) powder. 313 Copyright © 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

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and Landis, 1951; Wolf and Handal, 1966), no single stimulus appears to be necessary. For example, an iso-osmotic reduction in plasma volume can elicit a salt appetite in both intact and adrenalectomized rats (Stricker and Wolf, 1966; Wolf and Stricker, 1967). In addition, although aldosterone treatment enhances salt intake in sodium replete rats (Wolf and Handal, 1966), adrenalectomized rats show clear evidence of salt appetite (Jalowiec and Stricker, 1973; Richter, 1936). Recently, Stricker and Wolf (1969) have linked the three sufficient stimuli above to a hypothetical reservoir receptor which would in turn mediate salt appetite when the reservoir was depleted. However, few attempts have been made to examine the possible interactions between hypovolemia, hyponatremia and increased mineralocorticoid levels which might be important in the elicitation of salt appetite. For example, sodium appetite began slowly after uncomplicated hypovolemia (8-12 hr) following considerable water intake (Stricker and Jalowiec, 1970),whereas the appetite was evident 3-5 hr after formalin-induced hypovolemia with concurrent hyponatremia (Jalowiec and Stricker, 1970a). Similarly, sodium appetite of rats recovered from most of the vascular effects of formalin injection was greatest when hyponatremia was accompanied by indications of high endogenous mineralocorticoid levels (Jalowiec and Stricker, 1970b) suggesting that hyponatremia might potentiate the natrorexigenic effect of high mineralocorticoid levels. The present study describes the salt appetite induced by acute loss of sodium in urine and examines the effect of differential dietary maintenance on the salt appetite elicited by diuresis and natriuresis. Briefly, rats were maintained on diets either sodium replete or sodium deficient, sodium loss in urine was induced by injection of furosemide, and subsequent salt intake and sodium balance was measured.

METHODS Sub/ects and Pre-treatment Maintenance

Male albino rats (Sprague-Dawley), weighing 300-350 g at the start of the experiment, were housed in individual metabolism cages. The laboratory was continuously illuminated and temperature controlled (24-26°C). Initially, all animals were permitted free access to demineralized water and 0.51 M NaC1 solution, available from graduated tubes (-+ 1.0 ml) with stainless steel drinking nozzles, and pelleted Purina Laboratory Chow (Na+ replete; 150.0 mEq/kg). Procedure

Pure furosemide powder (4-chloro-N-2-furylmethyl-5-sulfamoylanthranillic acid) was mixed with a slightly alkaline (pH = 7.4) 0.15 M sodium

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bicarbonate solution since it precipitates as a carboxylic acid at a pH below 7.0. Furosemide appears to inhibit renal sodium reabsorption by interfering with active sodium transport in the distal tubule (Dirks and Seely, 1969), the ascending loop of Henle (Morgan, Tadokoro, Martin and Berliner, 1970) and the proximal tubule (Brenner, Keimowitz, Wright and Berliner, 1969). The site of action in the loop of Henle is unique among diuretics and accounts for the superior natriuretic and diuretic properties of furosemide. Preliminary studies were conducted to determine the optimal dose required to produce maximal sodium loss. Forty-six rats were given single intraperitoneal injections of 1.0 ml of either O, 1.0, 2.5, 5.0, 10.0 or 20.0 mg of furosemide. Following injections, the rats were deprived of food and fluid and urine excretions were collected every half-hour for 2 hr as described below. This 2-hr collection period was sufficient since the onset of the diuresis was very fast (3-5 min) and approximately 75% of the total urine volume and sodium loss occurred within the first hour. The rats excreted little urine during the second hour and generally were anuric for the next several hours. In addition, urine sodium concentrations began to decrease while potassium concentrations increased suggesting the onset of sodium retention mediated by increased mineralocorticoid activity (Johnson, 1954; Simpson and Tait, 1952). Generally, maximal sodium loss was produced by 10.0 mg injections (Fig. 1). Smaller doses clearly elicited a diuresis and natriuresis but the losses were less 21-

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reliable. Doubling the optimal dose did not increase the urine volume or sodium content appreciably indicating that a response "ceiling" had been reached. Adverse behavioral reactions were never seen following any of the administered doses. Six days prior to furosemide injection, an additional 40 rats, previously maintained on the sodium replete diet (NaR) with water and 0.51 M NaC1 solution, were divided randomly into two equal groups. One group remained on the NaR regimen while the other was given access to a sodium deficient diet (NaD). This pelleted diet was especially prepared (General Biochemical Co.) to contain less than 0.00009 mEq of Na+/kg and normal potassium levels (114.0 mEq/kg) as assayed in Purina Laboratory Chow but was otherwise comparable to the supplier's sodium deficient test diet for rats derived from Hartroft and Eisenstein (1957). Body weights were recorded to insure that the new diet had not led to anorexia. In addition, continuous sodium balance was calculated for the NaD rats by analysing daily urine samples for sodium content and subtracting total urine sodium loss from sodium intake (Jalowiec and Stricker, 1970a). Fecal sodium was found to be negligible. Cage interiors were sponged daily to avoid possible accumulation of salts even though cage rinses yielded no measurable amounts of sodium. On the morning of the seventh day, all animals were weighed and then deprived of food and 0.51 M NaC1 solution but not water. Ten rats from each dietary program were injected with 10.0 mg of furosemide. The remaining animals received 1.0 ml injections of the vehicle. Urine was collected via funnels with feces screens into graduated tubes (-+ 0.1 ml) and water intakes were monitored hourly for 12hr and then at 24hr. Urine sodium and potassium concentrations were determined by flame photometry (Instrumentation Laboratories). The sodium replacement test began at the end of the 24-hr period when the drinking tube containing 0.51 M NaC1 was returned to the cages. Intakes and excretions were recorded subsequently at 5, 10, 15, 20, 30, 60, 120 and 180 rain, whereupon the salt drinking test and urine collections were terminated and food returned to the animals' cages.

Blood Analyses In order to determine the intravascular effects of furosemide injection, 75 rats were used for blood sampling. Uninjected control rats were bled after 6 days of NaR-maintenance (n = 5) or NaD-maintenance (n = 8). Additional NaR and NaD-maintained rats ( n = 15, 15) were injected with furosemide and allowed access to water for 2, 6 or 24 hr before being bled. Other NaRmaintained rats (n = 2 2 ) w e r e injected with furosemide and water deprived before bleeding. Finally, NaD rats were bled 24 hr after furosemide following 15 min or 2 hr of access to 0.51 M NaC1 (n = 5, 5). All rats were anesthetized

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POTENTIATION OF SODIUM APPETITE

with ether and blood samples were withdrawn from the abdominal aortas and immediately analysed for hematocrit (microcapillary tubes), plasma sodium and potassium concentrations (flame photometer), plasma protein content (refractometer) and plasma osmolality (Advanced Instruments Osmometer).

RESULTS Maintenance on a sodium deficient diet clearly resulted in marked sodium retention and high urinary potassium/sodium ratios (see vehicleinjected, NaD-maintained group; Fig. 2). These indirect indicators of high endogenous mineralocorticoid activity are supported by direct measurements of secretory rates from other laboratories (Marieb and Mulrow, 1965). F e w animals ingested enough 0.51 M NaCI solution during the maintenance period to prevent a slight negative sodium balance (mean sodium balance of 20 rats = -0.45 -+ 0.1 mEq). Generally, all of this loss occurred within the first two days of NaD-maintenance. The following four days were characterized by almost total sodium retention with extremely low urine sodium concentrations (less than 2.0 mEq/1). These results suggested that NaD-maintained rats began 160--

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the test on the seventh day with high endogenous mineralocorticoid levels but only minor negative sodium balance. In contrast, NaR-maintained (vehicleinjected) rats showed normally variable urine sodium concentrations and low urine potassium/sodium ratios (Fig. 2) suggesting no sodium retention and low levels of mineralocorticoid activity (n.b. urine was not collected during NaR-maintenance because food particles contaminated samples). Injection of furosemide resulted in significant increases in water intake and urine excretion in both NaR and NaD-maintained rats when compared to their respective controls (all P ' s < . 0 0 1 ) ; two-tailed t test; Fig. 3). The enhanced water intake was apparent within two hours after injection and appeared during the period of maximum diuresis. Water intakes were comparable in both furosemide-injected groups during the initial 12 hr, but after 60--

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24 hr, NaD-maintained rats had ingested significantly more water than NaRmaintained rats (P < .01). A similar difference in 24 hr intake was seen in the vehicle-injected groups ( P < . 0 5 ) and thus, can probably be ascribed to an effect of NaD-maintenance rather than some aspect of diuretic treatment. Urine volume losses were similar for both furosemide-treated groups. The diuresis was complete within 2 hr and was followed by an extended period of approximately normal urine flow. Corresponding to the large increases in urine volume after furosemide injection, urine sodium loss was significantly increased during the initial 12 hr (both P ' s < .001; as indicated by negative sodium balance in Fig. 4). Interestingly, sodium loss was much greater for the NaR-maintained group ( P < .001) and this difference was apparent 3 hr after furosemide injection. A similar difference was seen between the two vehicle-injected groups but was not evident until after 24-hr food deprivation (Fig. 4). The vehicle-injected rats maintained on the NaD-regimen lost virtually no sodium during the 24-hr test as might be expected, since they began the test with evidence of sodium retention. In contrast, their NaR-maintained counterparts slowly excreted approximately 1.0 mEq Na + (Fig. 4). These differences in sodium loss were the result of the greater urine sodium concentrations of NaR-maintained rats (Fig. 2) since their urine volumes were similar to those of NaD-maintained rats in both furosemide and control conditions (Fig. 3). Furosemide-injected animals showed a brief initial period of slightly elevated urine sodium levels which soon decreased to the low levels seen in animals that retain sodium actively (Jalowiec and Stricker, 1970a). Note that for the NaD-maintained rats 6,0-~thicJe Furosemide

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this natriuresis represents an increase from absolutely minimal concentrations followed by a rapid return to those low levels. Calculations of urinary potassium/sodium ratios following injections indicated that mineralocorticoid activity (Johnson, 1954; Simpson and Tait, 1952) was evident in all groups except the NaR-maintained rats injected with the vehicle (Fig. 2), i.e., urinary sodium levels decreased as urinary potassium levels increased resulting in enhanced ratios. NaD-maintained rats injected with the vehicle excreted urine with very low sodium concentrations and high potassium levels and the ratios depicted in Fig. 2 for this group reflect the concentrations of potassium in their urine. In contrast, NaR-maintained rats injected with the vehicle excreted urine with similar concentrations of potassium and sodium so that their ratios were approximately 1.0 indicating negligible sodium retention or mineralocorticoid activity. Both furosemideinjected groups initially excreted urines with comparable concentrations of potassium and sodium but as sodium concentrations decreased, potassium concentrations increased resulting in gradually increasing potassium/sodium ratios. Rate and degree of increase were greater in the furosemide-injected, NaD-maintained group because urine sodium concentrations decreased to lower levels, not because potassium levels were higher then those seen in the furosemide-injected, NaR-maintained rats. Note that comparisons between vehicle and furosemide-treated rats (but not between both furosemide-injected groups or both vehicle,injected groups) would be tenuous since urine volumes were markedly different (Fig. 3). The furosemide-treated groups began to drink immediately when the 0.51M NaC1 solution was returned 24 hr after injection (Fig. 4). More rapid salt drinking was seen in the rats previously maintained on the sodium deficient diet. At the end of 3 hr, both furosemide-injected groups had significantly increased their salt intakes in comparison to their respective controls (both P's < .001), but the NaD-maintained rats ingested substantially more salt than the NaR-maintained group ( P < .05). These 3-hr intakes represent essentially all of the sodium appetite elicited by furosemide since intakes over the following 2 l hr when food was available were small (mean intake approx. 1.5 and 0.7 mEq Na + for the NaD and NaR-maintained groups). There was no evidence of a sodium appetite in either of the control groups despite the previously noted indications of enhanced mineralocorticoid activity in rats maintained on the sodium deficient diet. The rapid salt intakes of the furosemide-injected rats clearly restored sodium balance during the replacement tests (Fig. 4). However, for the rats previously maintained on the NaD-regime, sodium intake was grossly in excess (200%) of the sodium deficit established by natriuresis. This behavioral over-compensation was evident within the initial 30 min of salt access and was not appreciably reduced during the next 2.5 hr by enhanced sodium excretion. Nevertheless, gradual increases in urine sodium concentrations suggested that

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sodium retention was waning (Fig. 2). The sodium intake of the NaR-maintained rats was more appropriate to their sodium deficit since it restored sodium balance to the level tolerated by their vehicle-injected controls (the significance of the negative level is considered in the following discussion). With regard to the two control groups, sodium balance increased only slightly in the NaD-maintained rats and remained essentially unchanged in the NaR-maintained group. Water intakes did not increase substantially in either of the vehicle-injected groups during the replacement test but furosemideinjected rats showed some increased water intake following 0.51M NaC1 drinking (Fig. 3).

Blood Analyses The animals used to examine the intravascular effects of furosemide injection showed sodium deficits and fluid intakes similar to those just described. Analyses of aortic blood samples from non-injected rats demonstrated that differential dietary maintenance had no marked effect on the blood parameters measured (Figs. 5 and 6). Furosemide injection evoked little change in plasma sodium concentration unless the animals were water deprived (Fig. 5). The significant increases in plasma sodium (P < .05) in water-deprived 152 --

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Fig. 5. Mean plasma sodium and potassium concentrations after furosemide injection in rats maintained on either the sodium replete or sodium deficient diet. Open, inverted triangles represent values from water deprived rats. Arrow denotes salt access. Vertical lines = _+ SEM.

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Fig. 6. Mean hematocrit, plasma protein and osmolality values after furosemide injection. Symbols as in Fig. 5. rats were probably due to the dehydrating effects of the extensive loss of hypotonic urine induced by furosemide. Nevertheless, these animals experienced sodium deficits comparable to those previously described. Plasma potassium concentrations were dep~ressed, but not significantly, following furosemide perhaps as a result of potassium loss in urine. However, the diuresis and natriuresis rapidly produced hypovolemia. Both hematocrit and plasma protein concentrations were increased within 2 h r of treatment ( P < .02; P < .05 respectively) and remained elevated for 24 hr (Fig. 6). The ingestion of water had little effect on hypovolemia since water alone cannot restore plasma volume (Stricker and Wolf, 1967). In accordance with the absence of substantial alterations in plasma electrolytes, no reliable changes were evident in plasma osmolality (Fig. 6). Blood samples from furosemide-injected NaD rats permitted access to 0.51 M NaC1 (24 hr post-injection) provided information on the intravascular effects of sodium replacement. Plasma sodium levels were reduced somewhat after 24 hr water drinking, but returned to no-injection control levels within 15min of 0.51 M NaC1 drinking and were significantly elevated after 2 h r (/9< .05; Fig. 5). Plasma osmolalities were increased also after 2 hr salt access (Fig. 6). These changes may not be the result of absorption of sodium from the gastrointestinal tract, but instead may reflect movement of extracellular water into the gut in response to the hypertonicity of ingested fluid (O'Kelly,

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Falk and Flint, 1958). Plasma volume was clearly restored after 2 hr access to 0.51M NaC1 since plasma protein and hematocrit had returned to normal (Fig. 6).

DISCUSSION In many respects, the vascular effects of furosemide injection were reminiscent of changes observed following polyethylene glycol (PG) treatment. Subcutaneous PG injection results in uncomplicated hypovolemia by drawing isotonic vascular fluid into an interstitial edema (Stricker, 1966). Furosemide injection produced hypovolemia also but the fluid loss was external and plasma sodium concentration was increased due to the hypotonic diuresis. Both of these treatments can be contrasted to formalin injection which results in hyponatremia as well as hypovolemia by producing an interstitial edema (Jalowiec and Stricker, 1970a). All three of these treatments enhance water drinking but, whereas the thirst following PG or formalin injection has been attributed to hypovolemic stimuli (Jalowiec and Stricker, 1970a; Stricker, 1966), the thirst observed after furosemide injection may have been elicited by a complex interaction of hypovolemic and osmotic (cellular dehydration due to hypernatremia) stimuli (Corbit, 1968). The balance studies demonstrated that furosemide-evoked sodium loss was a powerful stimulus for sodium appetite and thirst as well as sodium retention once the initial natriuresis was complete. In view of previous suggestions that sodium appetite in rats is not closely related to sodium deficiency (Falk, 1965; Falk and Lipton, 1967; Jalowiec and Stricker, 1970a; Novakova and Cort, 1966; Wolf, 1964), furosemide-injected rats were expected to over-compensate for the deficits established by the natriuresis. Surprisingly, behavioral over-compensation was evident only in the group maintained on the sodium deficient diet prior to injection (Fig. 4). The degree of over-drinking in these animals provides a striking contrast to the precise sodium replacement behavior of sheep (Denton, Orchard and Weller, 1969). In addition, note that the over-compensation was evident within the first 30 min of salt access. Plasma sodium values from animals bled after 15 rain of 0.51 M NaC1 access indicated that some restoration of extracellular sodium levels might have begun shortly after salt ingestion. However, the rapidity with which over-drinking of salt occurred suggests that previously postulated cues for satiation of sodium appetite such as oropharyngeal metering (Denton and Sabine, 1961), taste (Nachman and Valentino, 1966), or gut distention and absorption of ingested sodium (Mook, 1963; 1969) are not effective enough to prevent behavioral over-compensation. In this regard, the present study increases the plausibility of a reservoir receptor for sodium need whose activity would rapidly signal sodium appetite but would diminish only slowly

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consequent to restoration of reservoir sodium (Stricker and Wolf, 1969). A rapid and precise satiety mechanism may not be a vital requirement since excessive ingestion of sodium would not be harmful unless complicated by renal failure and the absence of water. Furosemide injection also enhanced sodium intake in rats maintained on the sodium replete diet, but the ingestion was more gradual and significantly less than that of NaD-maintained animals despite a significantly greater sodium deficit during the preceding 24 hr (Fig. 4). If net sodium loss were the only determinant of consequent sodium intake, then NaR-maintained rats should have ingested more sodium, not less, than the NaD-maintained group. The explanation of this paradox is uncertain, but could involve differences in sodium balance between the NaD and NaR-maintained rats at the start of sodium deprivation. NaR-maintained rats may have had an excess of body sodium in comparison to NaD-maintained animals which were in zero or slightly negative sodium balance. Assuming a daily food intake of 20-25 g, the rats maintained on the sodium replete diet (150mEq Na+/kg) had been ingesting 3.0-3.8 mEq Na+/day necessitating a substantial daily excretion. This obligatory loss, represented in part by the gradual excretion of approximately 1.0 mEq Na + by the vehicle-injected, NaR-maintained group during sodium deprivation, could have been accelerated by furosemide injection. Thus, differences in sodium loss between the NaR and NaD-maintained groups can perhaps be accounted for by an obligatory sodium loss required of the NaR animals. Further, the negative sodium balance of the vehicle-injected NaR group that developed during sodium deprivation would represent a level comparable to the sodium balance of the NaD-maintained rats at the start of sodium depletion. Yet in contrast to the over-compensation noted in the sodium replacement behavior of the NaD-maintained rats, the salt intake of NaR-maintained rats injected with furosemide was precisely sufficient to re-establish sodium balance at the level tolerated by control rats (Fig. 4). If this level can be considered the balance point, then it would appear that under certain circumstances, sodium deficient rats behave like sodium deficient sheep and precisely replace their sodium deficits. Acute sodium loss elicited gross behavioral over-compensation for the established deficit in rats maintained on a sodium deficient diet but resulted in apparently precise replacement behavior in rats maintained on a sodium replete diet. What is the explanation for this difference? Previous reports demonstrated that maintenance on a sodium deficient diet increased mineralocorticoid secretory rates (Marieb and Mulrow, 1965). In the present study, low urine sodium concentrations and enhanced urinary K+/Na+ ratios suggested that NaD-maintained rats had high mi~aeralocorticoid activity even before furosemide injection while the NaR-maintained group showed no such evidence until well after injection (5 hr). Since furosemide treatment had similar effects on blood volume and plasma sodium whether rats received NaR

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or NaD-maintenance, high endogenous mineralocorticoid levels may have been responsible for the potentiated sodium appetite and consequent over-compensation seen in rats maintained on the sodium deficient diet. Briefly, acute sodium deficiency accompanied by high levels of mineralocorticoid elicited a greater sodium appetite than either high mineralocorticoid levels alone (NaDmaintained, vehicle-injected rats) or sodium deficiency with delayed and perhaps smaller increases in mineralocorticoids (NaR-maintained, furosemideinjected). The role of mineralocorticoids as primary stimuli for sodium appetite is controversial in view of the marked salt appetite of adrenalectomized rats (Jalowiec and Stricker, 1973; Richter, 1936) and the natrorexigenic effects of hypovolemia and hyponatremia in adrenalectomized rats (Wolf and Steinbaum, 1965; Wolf and Stricker, 1967). Although dose-response studies have shown that physiological injections of aldosterone (the most potent mineralocorticoid in the rat) can augment salt intake (Wolf and Handal, 1966), some authors consider any exogenous dose to be unphysiological (Denton, Nelson, Orchard and Weller, 1969). In addition, rats in the present study with evidence of high mineralocorticoid levels but no excessive sodium deficiency (vehicle-injected, NaD-maintained) did not show a sodium appetite. Nevertheless, it remains possible to assume a primary role for mineralocorticoids in the elicitation of salt appetite. Since endogenous secretory rates could not be measured directly it is conceivable that mineralocorticoid levels were not maximized by six days of maintenance on a sodium deficient diet and thus were not sufficient to elicit sodium appetite until additional stimulation by acute sodium loss. Accordingly, NaD-maintenance may have evoked a high but not natrorexigenic level of mineralocorticoids, acute sodium loss in NaR-maintained rats may have elicited a higher level capable of enhancing salt intake, and finally, acute sodium loss in NaD-maintained rats could have elicited the highest level thereby evoking the most pronounced sodium appetite. However, calculated urinary K+/Na + ratios in the present study do not necessarily support such an explanation since these indirect measures of mineralocorticoid activity should not be interpreted in a quantitative manner. Instead, these results suggest that the role of mineralocorticoids in sodium appetite is as a potentiator of the natrorexigenic effects of sodium deficiency (i.e., hypovolemia, hyponatremia, reservoir sodium deficits) and emphasize the importance of the interaction between the effects of sodium deficiency in eliciting sodium appetite. Similar kinds of interaction have been reported recently (Jalowiec and Stricker, 1970b; Stricker and Jalowiec, 1970) and together with the present work suggest that salt ingestion would be faster and more extensive if two or more natrorexigenic stimuli were operating concurrently or in sequence. Behavioral over-compensation could be the result of potentiation of the natrorexigenic effect of one stimulus by another and, as recent studies have demonstrated (Jalowiec and Stricker, 1973), can occur even in the absence of increased mineralocorticoid activity.

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