Ethanol-Induced Changes in Plasma Proteins ...

Copyright !993 by the American Psychological Association, Inc. 0735-7044/93/S3.00

Behavioral Neuroscience 1993, Vol. 107. No. 2. 339-345

Ethanol-Induced Changes in Plasma Proteins, Angiotensin II, and Salt Appetite in Rats Douglas A. Fitts and Roxane G. Hoon Sodium depletion in rats elicits a sodium appetite that results from a cerebral action of angiotensin II (ANG II) and aldosterone. Alcohol also activates the renin-angiotensin system, but the mechanism is poorly understood and not related to sodium excretion. In this study, 2.5 g/kg ip ethanol produced a 20% decline in plasma volume and plasma protein concentration in 1-2 hr and elicited salt appetite beginning in 3-4 hr. Blockade of ANG II synthesis in the brain and periphery with the angiotensin-converting enzyme inhibitor captopril eliminated the thirst and salt appetite. Peripheral captopril alone enhanced fluid intake, which indicated that alcohol elevated renin secretion. Ethanol-induced suppression of hepatic plasma protein secretion and the consequent fall in plasma colloid osmotic pressure apparently resulted in hypovolemia and renin secretion, which then produced thirst and salt appetite through an action of ANG II on the brain.

hypovolemia, or a drop in blood pressure, but direct effects of alcohol have not been ruled out (Puddey et al., 1985). In any case, the hypovolemia and renin secretion associated with acute ethanol administration parallel the conditions that elicit salt appetite. We examined the possibility that these consequences of alcohol administration can elicit salt appetite in rats. In an initial experiment we examined the effects of different doses of alcohol on salt appetite, and in a second experiment we examined the effects of the angiotensin-converting enzyme (ACE) inhibitor captopril on the alcohol-induced salt appetite. Direct and indirect determinations of plasma volume and body water distribution were observed in order to verify the changes in hydration after the ethanol injections.

After a depletion of sodium, the peptide hormone angiotensin II (ANG II) acts peripherally to constrict arterioles, to enhance sodium retention at the kidney, and to induce aldosterone secretion. ANG II also acts by way of circumventricular organs in the brain to provoke vasopressin secretion and to activate the sympathetic nervous system. These hormonal actions result in a retention of the remaining water and sodium in the body and a maintenance of blood pressure. ANG II also induces thirst and, along with aldosterone, salt appetite, although it is not clear in every case whether the ANG II that supports these responses is of circulatory or neural origin (Dalhouse, Langford, Walsh, & Barnes, 1986; Fitts, Tjepkes, & Bright, 1990; Sakai, Chow, & Epstein, 1990). Acute alcohol administration activates the renin-angiotensin system, although the effect is more robust after chronic alcohol intake (Collins, Brosnihan, Zuti, Messina, & Gupta, 1992; Linkola, Tikkanen, Fyhrquist, & Rusi, 1980; Nieminen, Linkola, Fyhrquist, Tikkanen, & Forslund, 1985; Puddey, Vandongen, Beilin, & Rouse, 1985; Stott et al., 1987; Wright, Morseth, Abhold, & Harding, 1986). There is also evidence that some of the normal functions of ANG II are blunted in the presence of ethanol. For example, at moderate blood ethanol concentrations, the usual relation between plasma ANG II and aldosterone secretion appears to be broken (Collins et al., 1992; Nieminen et al., 1985). The stimulus for renin secretion after acute ethanol administration is probably a change in fluid-electrolyte balance,

General Method Animals Male rats of the Long-Evans strain, which weighed 300-500 g, were used. They were maintained in hanging wire mesh cages with continuous access to food and tap water except as specified in the experiments. The temperature was maintained at 23-25 °C, and the lights in the room were on 12 hr each day. The rats had continuous access to 0.3-M NaCI solution in addition to water for 1 week before the experiments. During this access period the positions of the tubes were alternated daily to minimize the development of position expectancies. The numbers of rats used are given in the Method of each experiment.

Douglas A. Fitts and Roxane G. Hoon, Department of Psychology, University of Washington. This research was supported by National Institutes of Health Research Grant NS22274 and by a small grant from the Alcoholism and Drug Abuse Institute of the University of Washington to Douglas A. Fitts. We thank J. B. Simpson and S. C. Woods for their comments on an earlier version of the article and S. J. Lucania of the Bristol-Myers Squibb Research Institute for the donation of captopril. We acknowledge the technical help of Tom Moore and Julie Reiman. Correspondence concerning this article should be addressed to Douglas A. Fitts, Department of Psychology, NI-25, University of Washington, Seattle, Washington 98195.

Fluid Intakes Water and 0.3-M NaCI solution were measured with inverted 100-ml graduated cylinders fitted with rubber stoppers and drinking spouts. Intakes were recorded to the nearest milliliter.

Drugs Captopril, a specific inhibitor of ACE, was mixed fresh immediately before each experiment with sterile isotonic saline as the vehicle. 339

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DOUGLAS A. FITTS AND ROXANE G. HOON

Assays 8

Hematocrit was determined by the microhematocrit method, plasma protein concentrations were determined by refractometry unless it is otherwise stated that the concentrations were determined by the Lowry micromethod (Sigma Chemical [St. Louis] kit), blood ethanol was determined by the enzymatic method (Sigma Chemical kit), and plasma sodium concentration and osmolality were measured by flame photometry and freezing point depression, respectively. Plasma osmolality was corrected for ethanol in the blood by the preparation of a standard curve on which blood ethanol was regressed against the change in osmolality in 4 rats of the same strain, gender, and weight as the experimental animals. Known amounts of ethanol were added to blood samples and left undisturbed 5 min before analysis of blood ethanol, plasma osmolality, plasma protein, hematocrit, and plasma sodium. Regression of the means of the measured ethanol concentrations on the actual ethanol concentrations yielded a regression equation with a slope and intercept not significantly different from 1.0 and 0.0: Measured Alcohol = 0.939 (actual alcohol) + 1.047; r(3) = .9994. Regression of the changes in sample osmolality, that is, from no ethanol to a given amount of added ethanol, on the measured blood ethanol concentration yielded a linear equation: Change in Osmolality = 0.2639 (blood ethanol concentration) + 1.197; r(3) = .9980. All sample data fell within the limits of linearity, 0 and 234 mg/dl, and the equation was used to correct all sample plasma osmolality values. The hematocrits, plasma proteins, and plasma sodium concentrations were highly stable across all blood ethanol concentrations, which confirmed that the mere addition of ethanol to blood does not affect these determinations. Means and standard errors of the variables in the unadulterated blood were: plasma osmolality, 294 ± 1.9 mOsm/kg; plasma protein, 5.8 ± 0.1 g/dl; hematocrit, 42.8% ± 0.5%; and plasma sodium, 132 ± 1 mmol/L.

7

D Water •

D 0.3 M NaCI

6

Fluid

5

Intake

.

ml/7 hr

3

I

'#

f1 1 1 PI

2 1

| 1

0

M%

Vehicle

m

%%?

vm, 1.0

1

^

2.5

Dose of Ethanol (g/kg, IP) Figure 1. Water and 0.3-M NaCl intakes 7 hr after an ip injection of 1.0 or 2.5 g/kg ethanol in 10 ml/kg volume or of an equal volume of the isotonic saline vehicle. (Saline intake was significantly increased at the 2.5 g/kg dose relative both of the other groups.)

cantly elevated at the 2.5 g/kg dose compared either with the vehicle group or with the 1.0 mg/kg group (p < .01). The main effect for total fluid intake (water and saline), F(2, 24) = 5.89, p < .01, reflected an elevated intake of both fluids by the 2.5 g/kg group.

Statistics Data were analyzed with an analysis of variance appropriate to the individual designs. Planned comparisons were tested with Fisher's least significant difference test if an F ratio was significant and with the Bonferroni correction if a ratio was not. Unplanned comparisons were made with the Newman-Keuls test. A probability of less than .05 was required for significance.

Experiment 1: Dose Response Test of Ethanol-Induced Salt Appetite The purpose of this experiment was to determine whether the hypovolemia or renin secretion associated with acute ethanol administration could elicit salt appetite. Method In this initial test, 27 rats were given injections of 0, 1.0, or 2.5 g/kg of ethanol in a 10 ml/kg volume with sterile isotonic saline as the vehicle. Treatments were randomly assigned with 9 rats per group. Water and 0.3-M saline solutions were available immediately, and intakes were recorded at 7 hr after the injections. The end of the drinking period was 1 hr after the colony lights were turned off.

Results The results of the dose-response test are given in Figure 1. Planned comparisons revealed that saline intake was signifi-

Experiment 2: Captopril Blockade Test The purpose of this experiment was to determine whether alcohol-induced salt appetite depends on ANG II synthesis. Method Surgery and injections. Some rats were fitted with chronic indwelling cerebral guide cannulas targeted for the left lateral ventricle under stereotaxic procedures. Each cannula was constructed from 23-gauge stainless steel tubing obturated with stainless steel wire. The cannula was affixed to the skull with stainless steel screws and methyl methacrylate cement. Equi-Thesin anesthetic (Drug Services of the University of Washington Hospital, Seattle) was used for anesthesia (0.35 ml/100 g ip), Betadine solution (Purdue Frederick, Norwalk, CT) was applied topically both before and after implantation, and Gentamicin sulfate injection (Elkins-Sinn, Cherry Hill, NJ; 0.2 ml im) was given to control postsurgical infection. The rats were placed in a warm environment until they were awake enough to return to their cages. The rats were allowed to recover for 2 weeks before they were used in experiments. On the days of experiments that called for intracranial injections, the obturator was removed from the guide cannula, and a 31-gauge injector was inserted to a point 0.5 mm beyond the tip of the guide. This injector was connected to a Hamilton 25-ji.l syringe with a meter of PE-10 polyethylene tubing. The entire system was filled with isotonic saline except for the immediate injector end that was filled with a captopril solution or the sterile isotonic saline vehicle separated from the rest of the fluid by a 0.5-u.l bubble of air. As soon as the

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ALCOHOL AND SALT APPETITE injector was inserted, the rat was placed into a holding cage while a 2-ia.l injection was made over 10 s. Procedure. In this test, 53 rats with lateral ventricular cannulas were randomly assigned to four treatment groups that included a vehicle group, an alcohol only group, a universal captopril group, and a peripheral captopril group. All rats received three injections in the following order: a 2 ml/kg ip injection, a 2-(J intracranial injection, and a 10 ml/kg ip injection. The first ip injection and the intracranial injection were given consecutively, and the second ip injection was given 10 min later. The vehicle group received only control saline treatments in all three injections. The alcohol only group received control saline injections in the first ip and in the intracranial injections but then had a 2.5 g/kg injection of alcohol in the second ip injection. Thus, the vehicle and alcohol groups should replicate the basic finding of salt appetite as demonstrated by the 2.5 g/kg group in Experiment 1. The peripheral captopril group received an ip injection of 5 mg/kg captopril followed by a vehicle injection in the ventricles and an alcohol injection 10 min later; this dose of captopril is sufficient to completely block peripheral ACE without blocking all of the enzyme in the circumventricular organs (Fitts & Masson, 1990; Thunhorst, Fitts, & Simpson, 1989). However, central ACE within the blood-brain barrier is unaffected because of poor permeability of captopril through the barrier (Barry, Jarden, Paulson, Graham, & Strandgaard, 1984). The universal captopril group received an ip injection of 100 mg/kg captopril followed by 25 jig of captopril in the ventricles and an alcohol injection 10 min later; these doses block all of the ACE in the periphery, in the circumventricular organs, and in the rest of the brain within the blood-brain barrier. Thus, all groups except the vehicle group received alcohol, and the two captopril groups differed from the alcohol only group by having a pretreatment of captopril designed to block ACE either in the periphery alone (peripheral captopril group) or both in the periphery and in the brain (universal captopril group). Water and saline were withheld for the first 3 hr of the experiment. Between 1 and 3 hr after the injection (average 2 hr), a random sample of 4 rats from each group was taken from their cages and sampled for 5.0 ml of blood by cardiac puncture under rapid halothane anesthesia. A 100-|j,l sample of blood was pipetted into 0.9 ml of dilute trichloroacetic acid to stop enzyme activity and was frozen for later analysis of blood ethanol concentration. Two capillary tubes were filled with blood for immediate hematocrit determination, and the remaining blood was centrifuged at 3,000 rpm for 5 min to extract plasma. Plasma osmolality and protein were measured immediately. The residual plasma was then frozen for later determination of sodium concentration. Three hours after the initial treatments, the remaining 37 rats received a second booster round of blocking or control treatments exactly as previously described except that no additional alcohol or vehicle injection was given. Water and saline solutions were then supplied on the cages, the initial readings were taken, and the rats were left alone to drink for 4-6 hr, including 2-3 hr of the night cycle. The sample sizes for these groups were: vehicle control, n = 8; alcohol only, n — 12; universal captopril, n = 10; and peripheral captopril, n = l. At the end of the drinking period, 7 rats per group were exsanguinated and assayed as described for the 2-hr group. This included either the entire group or a random sample if the group was larger than 7. The water and salt intakes of all rats were measured in the random order that they appeared on the rack of cages, so the contribution of the differing time intervals on the drinking and blood values was randomly distributed across the different groups.

Results Drinking data. The behavioral data from Experiment 2 are shown in Figure 2. The basic finding of elevated water and salt

Fluid Intake ml/7-9 hr

3

Vehicle

Alcohol Only

Univer. CAP

Periph. CAP

Figure 2. Water and saline intakes after vehicle or 2.5 g/kg ip ethanol injections combined with two injections 3 hr apart of ip and lateral ventricular treatments with captopril (CAP). (Vehicle = no alcohol or CAP; alcohol only = alcohol but no CAP; univer. CAP = alcohol plus 100 mg/kg CAP ip and 25 ng CAP LV; periph. CAP = alcohol and 5 mg/kg CAP ip. Alcohol alone enhanced saline intake, but universal blockade abolished the excess drinking resulting from alcohol injection. Peripheral blockade alone enhanced total fluid intake.)

intake after 2.5 g/kg alcohol was replicated in the contrast between the vehicle and alcohol only groups, Bonferroni f(33) = 3.62,p < .05. Water intake was considerably smaller in this experiment compared with Experiment 1, a probable reflection of the hydrating effects of the injected isotonic saline, but the hypertonic saline intakes were virtually identical between experiments. The universal captopril group that had captopril both peripherally and centrally showed no thirst or salt appetite responses to alcohol treatment, and the data from this group resembled those of the vehicle control group, Bonferroni t(33) = 3.40, p < .05, versus the elevated alcohol intake of the alcohol only group. The peripheral captopril group that had a lower dose of captopril ip and no central captopril showed a greatly elevated total fluid intake compared with vehicle controls or with the universal captopril group (main effect of total fluid), F(3, 33) = 4.12, p < .05 (NewmanKeuls test,p < .05). Most of the saline intake in this group was accounted for by a single rat's intake of 16 ml, which was the largest intake by any rat. Blood and plasma data. The results of the blood and plasma analyses are given in Table 1. The hematocrits of all three groups of alcohol-injected rats were greatly elevated compared with the vehicle control at both time points (main effect), F(3, 36) = 7.34, p < .01. Plasma protein values were reduced in the three alcohol groups at both times compared with control, but the effect with time was complex (interaction), F(3, 36) = 9.02, p < .01. At the 7- to 9-hr sampling, plasma protein increased significantly in the vehicle group from the earlier time point, which suggests ongoing recovery from plasma dilution after the saline injection, but the alcohol

342


only group and the peripheral captopril groups both further reduced protein concentration. These were the two groups that drank increased amounts of water and saline prior to blood sampling. Plasma osmolality corrected for blood ethanol content was elevated at both time periods compared with controls in the alcohol only and universal captopril groups but not in the peripheral captopril group (main effect), F(3, 36) = 16.40,/> < .01. Furthermore, at the 7- to 9-hr time period, the alcohol only group had a significantly decreased osmolality from the earlier time point, possibly a result of drinking. However, the universal captopril group did not change significantly with time. Plasma sodium concentrations were reduced in the universal captopril group at both time periods, although the concentration increased significantly from the earlier to the later time points (interaction), F(3, 36) = 3.50, p < .05. By contrast, the sodium concentration of the peripheral captopril group declined between the 1- to 3-hr and 7- to 9-hr time periods, probably as a function of the high water intake by the group. Blood ethanol concentrations were significantly elevated in the universal captopril group at both time periods compared with the vehicle control group according to the main effect, F(2, 27) = 5.07, p < .05. The interaction was not significant, and a Bonferroni contrast with only 4 subjects per group was not powerful enough to detect a difference in the peripheral captopril group at 1-3 hr despite the fact that this group had the highest overall blood ethanol concentration.

Experiment 3: Plasma and Blood Volume Determinations The hematocrits and plasma protein concentrations in Table 1 changed in opposite directions after the ethanol injections despite the fact that these two measures typically vary directly with each other and inversely with the plasma volume. However, acute ethanol administration causes a 40%-50% decline in hepatic plasma protein secretion that begins at 1 hr and increases through at least 3 hr of intoxication (Donohue, Chaisson, & Zetterman, 1991; Tiernan & Ward, 1986; reviewed in Poso, 1987). Therefore, the hematocrit but not the plasma protein readings should reflect a decline in plasma volume after ethanol. In this experiment we measured plasma volumes and plasma protein concentrations directly in order to validate the indirect measures with the microhematocrit and refractometry methods. Method Thirty-seven rats were used in each part of this experiment. Rats were randomly assigned to groups for direct determination of plasma volume by a modification of the Evans-Blue dye dilution technique developed by Wang (1959) or for direct determination of plasma proteins with the modified Lowry micromethod (Sigma Chemical, Procedure No. 690). Groups of rats received either isotonic saline ip or 2.5 g/kg ethanol in isotonic saline vehicle in an equal 10 ml/kg volume. Plasma volumes were measured 1 hr later in 5 saline- and 6 ethanol-

Table 1 Blood and Plasma Data for Experiment 2 Treatment Variable

Vehicle (sober)

Alcohol alone

Universal CAP

Peripheral CAP

1-3 hr n

% hematocrit PP(g/dl) POsm (mOsm/kg) PNa(mmol/L) EEC (mg/dl)

4 40.9 ± 1.6 5.3 ± 0.1 307 ± 1 132 ± 1 —

4 52.1 ± 2.2t 4.9 ± 0.2* 321 ± 3t 128 ± 2 180 ± 14

4 53.6 ± 5.6^: 4.6 ±0.1* 330 ± 4t 122 ± 2* 213 ± 8$

51.6 4.8 310 132 218

4 ± 4.2$ ± 0.1* ±4 ± 1 ±5

7 ± 1.9$ ± 0.1* ± 4$ ± l*t ± 21$t

49.6 4.4 300 128 12

7 ± 0.9$ ± 0.1 *t ±3 ± 1* ± 5t

7-9 hr n

% hematocrit PP(g/dl) POsm (mOsm/kg) PNa(mmol/L) BEC (mg/dl)

7 44.1 ± 0.4 5.6 ± O.lt 300 ± 2 132 ± 1 —

7 49.5 ± 2.5+ 4.1 ±0.1*t 305 ± 2$t 130 ± 2 2 ± It

50.2 4.6 318 127 44

Note. PP = plasma protein concentration; POsm = plasma osmolality; PNa = plasma sodium concentration; BEC = blood ethanol concentration. Plasma osmolalities were corrected for ethanol. Blood and plasma values 1-3 hr and 7-9 hr after ip injection of 2.5 g/kg ethanol in 10 ml/kg volume or isotonic saline vehicle followed by two blocking injections with captopril (CAP) 3 hr apart in the following groups: Vehicle = no alcohol or CAP; alcohol only = alcohol but no CAP; universal CAP = alcohol and CAP both ip (100 mg/kg each injection) and LV (25 u,geach injection); and peripheral CAP = alcohol and ip CAP (5 mg/kg CAP each injection). The rats in the 7-9 hr groups were allowed to drink water and 0.3-M NaCI solution 3 hr after injection of alcohol. "p < .05 (vs. Vehicle for same time period), tp < .05 (vs. previous time period). $/? < .05 (vs. vehicle in main effect of groups or vs. alcohol group in BEC).

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ALCOHOL AND SALT APPETITE injected rats. Protein was assayed from blood collected by cardiac puncture under halothane anesthesia either 1 or 2 hr later (sample sizes for saline and ethanol groups: at 1 hr, «s = 6 and 7; at 2 hr, ns = 5 and 8).

For plasma volume determinations, rats were anesthetized with Equi-Thesin, 0.35 ml/100 g in control rats and 0.15 ml/100 g in ethanol-treated rats. Both external jugular veins were exposed, and an initial blood sample of 1.1 ml was drawn from the left vein with a 26-gauge heparinized needle and a glass syringe. This sample was used to determine hematocrit, and the plasma was used as a blankJfor the spectrophotometer (Coleman Junior, Maywood, IL), as a plasma dye standard, and to determine plasma protein in the refractometer. After the initial blood sample was taken, a weighed volume of approximately 0.25 ml of Evans Blue dye (T-1824, 0.5%) was injected into the same vein with a 0.25-ml glass syringe and a 26-gauge needle. Blood samples of 1.0 ml were drawn from the contralateral vein at 10, 30, and 60 min to determine plasma dye concentration with a spectrophotometer at 605 nm. The concentration at Time 0 was divided into the amount of dye injected to calculate absolute plasma volume. This volume was then expressed as ml/100 g of body weight. Blood volume was estimated with Wang's (1959) formula: Blood Volume = [PlasmaVolume x 100] H- [100 - Hematocrit x 0.95 x 0.74].

counterparts from arterial blood in cannulated rats anesthetized with halothane (Table 1). Plasma protein. Plasma protein concentrations were measured directly with a modification of the Lowry micromethod and compared with the refractometer readings and with hematocrits. The data for 1 and 2 hr are shown in the upper part of Table 2. The hematocrit was significantly elevated at both times. The correlation between the refractometer readings and_the Lowry assay was .89 (df = 24), and the regression line (see note in Table 2) showed that the actual protein concentrations were about 24% higher than the refractometer readings across the linear range. Both determinations revealed a significant drop in protein concentration by the ethanol group at 2 hr, but the Lowry method was more sensitive in this experiment for detecting the change at 1 hr. These data confirm that plasma volume declines within the first 1-2 hr after an ethanol injection, as previously suggested in the hematocrit measurements. Plasma protein concentrations fall as rapidly as the hematocrit values rise, a dissociation that results from a drastic reduction in hepatic secretion of plasma proteins after the ethanol injection (Donohue et al., 1991; Poso, 1987; Tiernan & Ward, 1986).

Discussion

Results Plasma volume. The results of Experiment 3 are shown at the bottom of Table 2. Of the 6 alcohol-injected rats, 2 died because of an interaction between the alcohol and the anesthetic before the preparation was complete. Thus, the sample sizes were 5 for the saline-injected group and 4 for the alcohol-injected group. The decrease in plasma volume with ethanol was significant, t(l) = 4.44, p < .01. Compared with the controls, the alcohol group lost 23% of plasma volume. Blood volumes were 5.49 ± 0.10 and 4.77 ± 0.32 ml/100 g in the saline and alcohol groups, respectively, which was a 13% decline in the alcohol group, t(4) = 2.38, p < .05. As in Experiment 2, the hematocrits, t(l) = 8.51, p < .01, and plasma protein concentrations, t(l) = 2.62, p < .05, were significantly different in opposite directions. Hematocrits from venous blood in previously unoperated rats anesthetized with Equi-Thesin were about 9 percentage points higher than their

Two experiments of this study have shown that an ip load of 2.5 g/kg of ethanol will produce salt appetite in rats. This dose of ethanol produced hind-limb ataxia and loss of righting response in all of the animals. Sleeping time was not evaluated, but the alcohol-injected animals in the second experiment were ambulatory by the second blocking or control injections at 3 hr when the fluids were returned to the cages. Informal observations in Experiment 1 showed that virtually no drinking took place before this 3-hr mark, and most took place after 4 or 5 hr. All drinking experiments included at least 1 hr of darkness at the end of the drinking phase, but the importance of this variable was not evaluated. A number of alcoholinjected rats clearly began drinking before the lights went out. Captopril treatments had different effects on alcoholinduced drinking by these rats depending on the location and dose of the captopril injections. Universally blocked rats

Table 2 Plasma Proteins by Refractometry or Lowry Method, Hematocrit, and Plasma Volume in Vehicle or Ethanol-Injected Rats of Experiment 3

Treatment Saline Ethanol Saline Ethanol Saline Ethanol

n 6 7 5 8 5 4

Time after ethanol (in hr) 1 1 2

2 1 1

% hematocrit 45.3 54.9 46.8 56.3 50.0 61.0

± 0.3 ± 0.7* ± 0.5 ± 0.8* ± 1.1 ± 0.5*

Plasma protein (refraction; g/dl) 5.7 ±0.1 5.6 ± 0.2 5.7 ± 0.1 4.7 ± 0.1*t 5.1 ± 0.8 4.5 ± 0.2t

Plasma protein (Lowry;

Plasma volume

g/dl)

(ml/ 100 g)

7.3 ± 0.3 6.7 ± 0.2* 7.3 ± 0.1

5.9 ± 0.2** — —

— — — 3.54 ± 0.06

2.73 ± 0.20*

Note. The correlation between protein methods for 26 rats was .89 (p < .001) Lowry = 1.239 (refraction) + 0.048. Lowry method samples used heart blood, and plasma volume samples used jugular vein blood. *p < .01 (vs. above saline control), tp < .05 (vs. above saline control). $p < .01 (vs. ethanol at 1 hr).

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presumably had no ANG II formation cither centrally or peripherally (Evered, Robinson, & Richardson, 1980), and this treatment completely blocked the alcohol-induced drinking of water and saline. This observation suggests that the reninangiotensin system is responsible for alcohol-induced salt appetite. Water drinking could also result from ANG II. although the elevated consumption of 0.3-M NaCl solution itself could account for some of this water drinking. An alternative interpretation may be that the captopril somehow made these rats sick or otherwise nonspecifically disinclined to drink and that the reduced drinking is a trivial function of this debilitation. Arguments can be drawn from several sources to support the conclusion that the universal captopril treatments given here were not debilitating. First, the intakes of these rats dropped only to the vehicle control level, not to zero. This suggests at least a nominal inclination to drink. The rats did not appear obviously different from other alcohol-injected rats when handled for blood sampling or booster injections. Previous experiments have shown that peripheral or central captopril injections in the doses used here did not reduce thirst or body weight when the mechanism of the thirst treatments did not involve the renin-angiotensin system, such as hypertonic NaCl or deoxycorticosterone acetate injections (Elfont & Fitzsimons, 1985; Thunhorst et al., 1989). The captopril-treated rats drank as much as control animals after these treatments and so were not debilitated. In a close parallel with this study, rats in this laboratory received infusions of captopril at 0.25 mg/hr peripherally and 25 u,g/hr in the lateral ventricles for 12 days without a reduction in body weight or water intake, and their blood chemistries were similar to control animals (Fitts, in press). Thus, captopril alone in the absence of high blood ethanol levels does not have effects of its own opposite to ethanol's that could account for these effects (see also Elfont & Fitzsimons, 1985; Evered & Robinson, 1984; Evered ct al., 1980; Thunhorst, Fitts. & Simpson, 1987, 1989). Some changes in blood parameters in response to alcohol were observed in the universally blocked group of this study. In relation to the alcohol only group, the universally blocked group had lower plasma sodium at both times, higher blood alcohol at both times, and higher plasma protein concentrations at 7-9 hr despite the very low values in all alcohol-treated groups. The latter phenomenon almost certainly resulted from the fact that the alcohol only and peripheral captoprii groups drank a large amount of fluid that caused further dilution of plasma protein. In fact, this measure is a functional verification that the saline was actually ingested by the alcohol only rats. The peripherally blocked captopril rats did not suppress fluid intake; instead, they increased it compared even with the alcohol only group. This paradoxical effect of peripheral ACE inhibition results from an increased conversion of ANG I to ANG II in the circumventricular organs of the brain (Fitts & Masson, 1990; Fitts et al., 1990; Thunhorst et al., 1987, 1989). This synthesis is possible because of the much higher concentrations of ACE in these organs versus the concentrations in the lung and elsewhere (Saavedra, Fernandez-Pardal, & Chevillard, 1982) where most peripheral ANG II is generated. Thus, it is possible to block all of the enzyme in the lung with a relatively low dose of captopril without blocking all of the enzyme in the circumventricular organs. Unblocked enzyme

remains in the circumventricular organs to enhance local ANG II synthesis (Fitts & Masson, 1990; Thunhorst et al., 1989) and to activate dipsogenic receptors (Fitts & Masson, 1989). The mechanism operates only when a dehydration or experimental thirst or salt appetite manipulation involves renin secretion, or at least when it does not involve an active suppression of renin secretion as during hypertonic saline or mineralocorticoid injections (Elfont & Fitzsimons. 1985; Evered & Robinson, 1984). Thus, the enhanced fluid intake by the peripherally blocked group in this study strongly suggests the presence of renin secretion as hypothesized in the introduction. The exaggerated fluid intake by the peripherally blocked group provides evidence that alcohol-treated rats can respond without an intact peripheral renin-angiotensin system. It may otherwise be argued that the universally blocked group failed to respond because of a severe hypotension caused by the alcohol in the absence of a peripheral rcnin-angiotensin system to support blood pressure. Rats without this peripheral system do not lack the ability to drink. The blood ethanol levels of the universal captopril group suggest a reduced elimination or delayed absorption of ethanol. A reduction in the rate of ethanol absorption has previously been noted in rats treated with captopril (Spinosa, Perlanski, Lccnen, Stewart, & Grupp, 1988). Although the effect was not significant, the peripheral captopril group had an even higher mean blood ethanol level at 1-3 hr than the universal captopril group, which suggests that delayed absorption may result from peripheral rather than central captopril treatment in the short term. The fact that this peripheral captopril group drank even more total fluid than the alcohol only group suggests that the difference in absorption of ethanol is not an important determinant in the universal captopril rats" disinclination to drink. The stimulus for renin secretion during acute ethanol intoxication may be diuresis-related dehydration, hypotension, or even a direct effect of ethanol (Puddey et al., 1985), such as on the juxtaglomerular cells. However, some evidence implicates hypovolemia as a stimulus for renin secretion. Elevated hematocrits and reduced plasma volumes were observed in rats forced to drink a 10% alcohol solution for 6 days (Wright & Donlan, 1979). The carcass water of these rats was also reduced, which suggests that the hypovolemia reflected a chronic total body water loss that resulted from ethanolinduced diuresis and reduced absolute water intake. In our study reduced plasma volume occurred 1 hr after an ip ethanol injection, at which time hematocrits were increased and plasma protein concentrations were decreased. This rapidly developing hypovolemia occurred despite a general decrease in sodium excretion after acute ethanol administration (Fitts, 1986; Sargent, Simpson & Beard, 1980), so the loss of plasma must occur because of an extravasation of fluid rather than a natriuresis. The most logical explanation for the loss of plasma into the extravascular fluid involves the large loss of plasma protein mass, as demonstrated here by reduced plasma protein concentrations even within a contracted plasma space. This reduction of plasma protein mass after ethanoi may result from a combination of a 40%-50% inhibition of hepatic plasma protein secretion, an enhanced catabolism of presecretory proteins in the autophagic vacuoles and lysosomcs of hepato-

ALCOHOL AND SALT APPETITE cytes (Donohue et al., 1991; Poso, 1987; Tiernan & Ward, 1986), and possibly an ethanol-induced extravasation of the plasma protein itself. This drastic loss of plasma proteins leads directly to hypovolemia by reducing the plasma oncotic pressure in the venular capillaries, which, in turn, reduces the influx of interstitial fluids into the vasculature. This mechanism resembles the classical view of hypoproteinemic edema formation of cirrhosis and nephrosis. Hypovolemia then stimulates the renin-angiotensin system, which increases blood pressure and elicits thirst and salt appetite. Hypovolemia and an activation of the renin-angiotensin system are well-described effects of acute alcohol injection. Similarly, the massive reduction of protein synthesis by the liver, including plasma proteins, is well documented. We know of no evidence for an extravasation of plasma proteins during alcohol intoxication, but this seems probable because it is doubtful that a reduction in protein secretion alone could account for this rapid and massive a loss of plasma proteins after an injection of ethanol. To our knowledge, this is the first report to suggest that the reduction in plasma protein mass after alcohol, whatever the origin, may be the cause of the hypovolemia and renin secretion. In conclusion, salt appetite after an ip injection of alcohol results from a synthesis of ANG II somewhere in the rat brain. The peripheral stimulus for the secretion of renin during the intoxication is most probably the rapidly developing hypovolemia that results from a reduction in plasma oncotic pressure secondary to an ethanol-induced reduction in plasma protein concentration. The experiments emphasize the importance of hypovolemia per se rather than an elevation of plasma protein concentrations or sodium depletion in the generation of angiotensin II and salt appetite.

References Barry, D. I., Jarden, J. O., Paulson, O. B., Graham, D. I., & Strandgaard, S. (1984). Cerebrovascular aspects of converting enzyme inhibition: I. Effects of intravenous captopril in spontaneously hypertensive and normotensive rats. Journal of Hypertension, 2, 589-597. Collins, G. B., Brosnihan, K. B., Zuti, R. A., Messina, M, & Gupta, M. K. (1992). Neuroendocrine, fluid balance, and thirst responses to alcohol in alcoholics. Alcoholism: Clinical and Experimental Research, 16, 228-233. Dalhouse, A. D., Langford, H. G., Walsh, D., & Barnes, T. (1986). Angiotensin and salt appetite: Physiological amounts of angiotensin given peripherally increase salt appetite in the rat. Behavioral Neurosdence, 100, 597-602. Donohue, T. M., Jr., Chaisson, M. L., & Zetterman, R. K. (1991). Plasma protein catabolism in ethanol- and colchicine-treated liver slices. Alcohol: Clinical and Experimental Research, 15, 7-12. Elfont, R. M., & Fitzsimons, J. T. (1985). The effect of captopril on sodium appetite in adrenalectomized and deoxycorticosteronetreated rats. Journal of Physiology, 365, 1-12. Evered, M. D., & Robinson, M. M. (1984). Increased or decreased thirst caused by inhibition of angiotensin-converting enzyme in the rat. Journal of Physiology, 348, 573-588. Evered, M. D., Robinson, M. M., & Richardson, M. A. (1980). Captopril given intracerebroventricularly, subcutaneously or by gavage inhibits angiotensin-converting enzyme activity in the rat brain. European Journal of Pharmacology, 68, 443-449. Fitts, D. A. (1986). Ethanol in cardiomyopathic hamsters: Na and

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water excretion and righting response. Pharmacology, Biochemistry and Behavior, 24, 967-973. Fitts, D. A. (in press). Angiotensin and captopril increase alcohol intake. Pharmacology, Biochemistry and Behavior. Fitts, D. A., & Masson, D. B. (1989). Forebrain sites of action for drinking and salt appetite to angiotensin or captopril. Behavioral Neurosdence, 103, 865-872. Fitts, D. A., & Masson, D. B. (1990). Preoptic angiotensin and salt appetite. Behavioral Neurosdence, 104, 643-650. Fitts, D. A., Tjepkes, D. S., & Bright, R. O. (1990). Salt appetite and lesions of the ventral part of the ventral median preoptic nucleus. Behavioral Neurosdence, 104, 818-827. Linkola, J., Tikkanen, I., Fyhrquist, F., & Rusi, M. (1980). Renin, water drinking, salt preference and blood pressure in alcohol preferring and alcohol avoiding rats. Pharmacology, Biochemistry and Behavior, 12, 293-296. Nieminen, M. M., Linkola, J., Fyhrquist, F., Tikkanen, I., & Forslund, T. (1985). Renin-aldosterone axis in ethanol intoxication: Effect of A II infusion. International Journal of Clinical Pharmacology, Therapy and Toxicology, 23, 137-140. Poso, A. R. (1987). Ethanol and hepatic protein turnover. Alcohol & Alcoholism, Suppl. 1, 83-90. Puddey, I. B., Vandongen, R., Beilin, L. J., & Rouse, I. L. (1985). Alcohol stimulation of renin release in man: Its relation to the hemodynamic, electrolyte, and sympatho-adrenal responses to drinking. Journal of Clinical Endocrinology and Metabolism, 61, 37-42. Saavedra, J. M., Fernandez-Pardal, J., & Chevillard, C. (1982). Angiotensin-converting enzyme in discrete areas of the rat forebrain and pituitary gland. Brain Research, 245, 317-325. Sakai, R. R., Chow, S. Y., & Epstein, A. N. (1990). Peripheral angiotensin II is not the cause of sodium appetite in the rat. Appetite, 15, 161-170. Sargent, W. Q., Simpson, J. R., & Beard, J. D. (1980). Twenty-fourhour fluid intake and renal handling of electrolytes after various doses of ethanol. Alcoholism: Clinical and Experimental Research, 4, 74-83. Spinosa, G., Perlanski, E., Leenen, F. H. H., Stewart, R. B., & Grupp, L. A. (1988). Angiotensin converting enzyme inhibitors: Animal experiments suggest a new pharmacological treatment for alcohol abuse in humans. Alcoholism: Clinical and Experimental Research, 12, 65-70. Stott, D. J., Ball, S. G., Inglis, G. C., Davies, D. L., Fraser, R., Murray, G. D., & Mclnnes, G. T. (1987). Effects of a single moderate dose of alcohol on blood pressure, heart rate and associated metabolic and endocrine changes. Clinical Science, 73, 411-416. Thunhorst, R. L., Fitts, D. A., & Simpson, J. B. (1987). Separation of captopril effects on salt and water intake by subfornical organ lesions. American Journal of Physiology, 252, R409-R418. Thunhorst, R. L., Fitts, D. A., & Simpson, J. B. (1989). Angiotensinconverting enzyme in subfornical organ mediates captopril-induced drinking. Behavioral Neurosdence, 103, 1302-1310. Tiernan, J. M., & Ward, L. C. (1986). Acute effects of ethanol on protein synthesis in the rat. Alcohol & Alcoholism, 21, 171-179. Wang, L. (1959). Plasma volume, cell volume, total blood volume and F cells factor in normal and splenectomized Sherman rats. American Journal of Physiology, 196, 188-192. Wright, J. W., & Donlan, K. (1979). Reversal of ethanol induced body dehydration with prolonged consumption in rats. Pharmacology, Biochemistry and Behavior, 10, 31-36. Wright, J. W., Morseth, S. L., Abhold, R. H., & Harding, J. W. (1986). Elevations in plasma angiotensin II with prolonged ethanol treatment in rats. Pharmacology, Biochemistry and Behavior, 24, 813-818. Received September 8, 1992 Revision received October 20, 1992 Accepted October 20, 1992 •