Marked increases in the consumption of concentrated NaCl solution were elicited in rats by daily injection of the synthetic mineralocorticoid, deoxycorticosterone acetate (DOCA). DOCA-treated rats drank different volumes of NaCl solution depending on its concentration (between 0.15 M and 0.50 M), with less consumed (in milliliters) the more concentrated the fluid was. In consequence, total Na+ intake (in milliequivalents) was roughly similar in all groups. Gastric emptying of Na+ also diminished as the concentration of the ingested NaCl solution increased, and the delivery of Na+ to the small intestine was remarkably similar in all groups. Cumulative volume of ingested fluid in the stomach and small intestine was very closely related to intake (in milliliters) of the concentrated NaCl solutions. Systemic plasma Na+ levels did not increase until after rats stopped consuming concentrated NaCl solution, although they were elevated at the onset of water ingestion. The situation appeared to be different when 0.15 M NaCl was consumed. This isotonic solution emptied and was absorbed relatively rapidly, and DOCA-treated rats drank larger amounts of it throughout a 1-h test period than when they drank concentrated NaCl solutions. Collectively, these findings suggest that saline consumption by DOCA-treated rats may be inhibited by two presystemic factors, one related to the volume of ingested fluid (i.e., distension of the stomach and small intestine) and one related to its concentration (i.e., elevated osmolality of fluid in the small intestine and/or in adjacent visceral tissue).
- gastric emptying
in laboratory rats, salt appetite often is elicited in association with a physiological need for Na+, as after bilateral adrenalectomy (20), injection of a diuretic agent (11), or subcutaneous colloid treatment (33). However, maintenance of rats for 1 wk on NaCl-deficient diet stimulates salt appetite even though Na+ deficits are minimal due to marked increases in aldosterone secretion (34). In addition, a robust salt appetite can be obtained without inducing Na+ deficiency (19) by daily injection of the synthetic mineralocorticoid, deoxycorticosterone acetate (DOCA).
When salt appetite is stimulated in rats by injection of a diuretic agent or by subcutaneous colloid treatment and animals are given ad libitum access to concentrated NaCl solution, ingestion of saline repairs the induced Na+ deficits and thereby eliminates the salt appetite. The same effects occur in adrenalectomized rats, although bodily Na+ deficits and salt appetite recur because of the animals' chronic inability to retain Na+ in urine (29). DOCA-treated rats similarly drink large volumes of saline in numerous small bouts throughout the day (29) even though there are no bodily Na+ deficits to repair; instead, the rats appear to have a continuous salt appetite that is temporarily inhibited each time they consume NaCl solution.
DOCA-treated rats also consume large volumes in one lengthy initial bout when given access to NaCl solution for a limited time each day (5, 22). The present experiments sought to identify the physiological factors that terminate NaCl ingestion under such conditions. The general approach taken resembles that used in recent studies of water consumption by rats after overnight water deprivation. Thirst was found to be inhibited by a presystemic signal associated with distension of the stomach and small intestine (10). Thus, by analogy, it seemed reasonable that an early inhibition of salt appetite might be mediated by a similar signal resulting from rapid increases in gastrointestinal distension when DOCA-treated rats drink NaCl solution. The present studies tested this hypothesis in rats given various concentrations of NaCl solution to drink. A second goal of these experiments was to determine whether the concentration of the ingested NaCl solution would affect intake and gastric emptying and thereby allow the delivery of Na+ to the small intestine to be modulated.
Adult male Sprague-Dawley rats (Harlan Laboratories) weighing 300–400 g on the day of terminal experiments were housed singly in cages in the Department of Neuroscience at the University of Pittsburgh (experiments 1A, 2A, and 2B) or in the Biomedical Research Facility at Florida State University (experiment 1B). Funnels were attached beneath the floor of the cages in experiment 1A to permit collection of urine. Both colony rooms were maintained at a constant temperature (22–23°C) and with a fixed light-dark cycle (lights off from 7 PM to 7 AM). All rats had >1 wk of ad libitum access to pelleted laboratory chow (5001; Purina) and tap water at all times except where noted. A single bottle of NaCl solution also was available for a limited time each day (see below), during which time food was not present.
Experimental protocols were reviewed by and received approval from the Institutional Animal Care and Use Committees of the University of Pittsburgh and Florida State University.
Rats were given subcutaneous injections of DOCA (2 mg in 0.4 ml sesame oil) each morning for 7–10 days, by which time a significant salt appetite (in comparison to baseline intakes of NaCl solution) developed and stabilized. Prior to each injection, food was removed from the cages and a NaCl solution was made available for 15–60 min per day, after which food was returned until the next morning. Water was available continuously during this training period.
Experiment 1A determined the effects of DOCA treatment on the intakes of NaCl solution and water. On 35 successive days, 7 rats were injected with DOCA at 9:30 AM, and immediately thereafter they were given access to a NaCl solution and water, but not food, for 1 h. (Food and water, but not saline, were available ad libitum at all other times.) The drinking fluids were presented in graduated burettes (±0.1 ml), and readings were taken at 5, 10, 15, 30, and 60 min of the test period. After the initial training period, during which 0.30 M NaCl was available, the solutions used were 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M, 0.40 M, and 0.50 M NaCl, given in ascending order, with a new solution presented every 6th day. The intakes reported were taken on the 5th day of testing and every 5th day thereafter. On those same days, at 15, 30, and 60 min of the test session, voided urine was collected in calibrated tubes, its volume (±0.1 ml) was recorded, and then the tubes were covered with Parafilm; Na+ concentrations were measured <1 h later (±1 meq/l) using a Na+-sensitive electrode (Beckman Coulter, Synchron EL-ISE model 4410, Brea, CA).
Experiment 1B used a similar protocol to obtain more detailed information about the effects of DOCA treatment on the intakes of four of those NaCl solutions (i.e., 0.15 M, 0.20 M, 0.30 M, and 0.40 M NaCl) and of water. Eight rats were given ad libitum access to Purina chow powder, water, and 0.30 M NaCl solution for 1 wk to accustom them to the special cages in which the behavioral measurements were made (23). On the front of these cages a 4 × 4-cm opening had been made to accommodate a jar containing powdered food. The fluid bottles were supported by a rack on the back of the cage. The drinking spouts were recessed ∼0.5 cm from the cage, and the rat had access to the drinking fluid by licking through a slot in the cage wall. Licks on each drinking spout activated a contact circuit and were transmitted to a microprocessor and recorded in consecutive 6-s time bins throughout the 1-h sessions.
Rats were injected with DOCA each morning at 9 AM for 22 days. During the first 10 days of this period, rats had access to 0.30 M NaCl from 9 AM to 5 PM, to food from 5 PM to 9 AM, and to water continuously. Most of the saline intake occurred during the first hour in which it was available. Detailed experimental observations during those 1-h periods began on day 11. Before each daily session, the food was removed and fresh drinking fluids were provided. On days 11–13, 0.15 M NaCl was given instead of 0.30 M NaCl. Over the next 9 days, a different NaCl solution was available. Specifically, each rat was given 0.20 M NaCl on days 14–16, 0.30 M NaCl on days 17–19, and 0.40 M NaCl on days 20–22. At 5 PM each day, the saline solution was removed, and food was returned to the cages where it remained until 9 AM on the following morning.
An analysis program allowed for the establishment of drinking bouts and ingestive episodes. The criteria for saline or water drinking bouts were operationally defined as requiring a minimum of eight licks, and the bout was terminated when no licking occurred for a period of 5 min. There were no set maximums to these bouts and the volumes consumed varied widely. Often, a rat would initiate a water bout sooner than 5 min after terminating a saline bout (or vice versa). We designated such a pair of bouts (or, in one case, two pairs of bouts) as an ingestive episode. Using these criteria, we recorded the number and size of each drinking bout and ingestive episode during each test session for each rat.
The volumes of fluid consumed during the 1-h test session were obtained by weighing the bottle before and after the session; the difference (in grams) was taken to be the volume ingested (in milliliters). Intakes at intermediate times were computed by multiplying the ingested volume by the number of licks at each minute and then dividing that product by the number of licks in each session (159 licks were required to consume 1 ml regardless of which NaCl solution the rats drank; r = 0.95, P < 0.001, for the pairings of cumulative licks and intakes for all rats and all solutions). These computations assumed that the volume consumed per lick was constant throughout the test session.
Results presented are for intakes by the eight rats on the 3rd day of exposure to each fluid; that is, on days 13, 16, 19, and 22. More specifically, data analysis focused on the collective 74 saline drinking bouts (17, 19, 21, and 17, respectively) and 41 water drinking bouts (0, 5, 17, and 19, respectively) that occurred on those 4 days. Note that the 1-h intakes of each saline solution differed by <10% during the 3 days in which it was available.
Experiment 2A determined the effects of DOCA-induced salt intake on gastric and small intestinal fluid volumes and on systemic plasma Na+ concentration (pNa), in separate groups of rats drinking one of the same seven NaCl solutions that were used in experiment 1A. Specifically, rats drank either 0.10 M (n = 7), 0.15 M (n = 14), 0.20 M (n = 6), 0.25 M (n = 5), 0.30 M (n = 9), 0.40 M (n = 8), or 0.50 M NaCl (n = 5). For each animal, the same concentration of saline was available in burettes from the first day of training through the terminal experiment. Daily access to saline during the training period was gradually reduced from 3 h on the 1st day to 15 min on the last day, by which time the rats drank saline promptly and with few brief pauses until they stopped; immediately thereafter, saline was removed from the cages and food and water were returned. After the 7–10 day training period, a terminal experiment was conducted that was identical to the training tests except in two details. First, the saline solution was colored with a few drops of dark green food dye (McCormick, Hunt Valley, MO), which allowed the ingested fluid to be readily visible in the small intestine (see below). Second, the drinking tests were terminated in one of two ways, in each subgroup: a total of 32 rats were allowed to drink (usually for >6 min) until they paused for a cumulative total of 2 min, whereas the other 22 rats were allowed to drink for variable amounts of time (0.50–7.25 min) before being interrupted by the experimenter. The former animals were used to provide information about the distribution of ingested fluid at the time that drinking ceased; the results of experiment 1B had indicated that little further drinking occurred during the remainder of the test period (none within 10 min) after a cumulative pause of 2 min. In contrast, data from the latter animals were used to determine whether distension of the stomach and small intestine increased in tandem or sequentially while the DOCA-treated rats drank saline and whether the ingested fluid affected pNa before drinking ceased. In either case, rats were killed by decapitation within 10 s after the test ended, and fluid intakes (±0.1 ml) and times spent drinking (±1 s) were recorded. Note that decapitated rats were held horizontally during blood collection to minimize the likelihood that gastric fluid would enter the esophagus and contaminate the blood sample. In fact, in no case did green dye appear in the esophagus of these animals.
Trunk blood was collected in ice-cold heparinized tubes (143 USP sodium heparin; Becton Dickinson, Franklin Lakes, NJ) and kept on ice until stomachs and intestines were obtained. The abdomen was opened and hemostats were placed at the junction of the stomach with the small intestine, at the junction of the stomach with the esophagus, and at the most distal site of visible dye in the small intestine, in that order. This portion of the surgical procedure took less than 2 min. Each stomach was removed from the carcass, the intestinal distance containing the dye was measured (±1 cm), and that segment of the intestine was removed. The excised tissues were stripped of adhering blood vessels and connective tissue, and the stomach contents and small intestines were placed in separate beakers. The beakers were covered with Parafilm until they were placed in an oven and dried to constant weight at 60°C for 1–2 days. Finally, the blood was centrifuged (10,000 g for 5 min at 4°C), plasma was harvested, pNa was measured (±1 meq/l) using the above-mentioned Na+-sensitive electrode, and plasma protein concentration was measured (±0.1 g/dl) using a refractometer.
To summarize, our goal was to analyze in each of 54 rats all blood samples for plasma concentrations of Na+ and protein, gastric chyme, and small intestine (separately) for dry matter and water, and intestinal distance traversed by the ingested fluid. These analyses sum to a total of 378 individual measurements, of which eight were lost due to procedural errors.
In experiment 2B, three additional groups (n = 5 in each) were tested using a two-bottle drinking test in which either 0.20 M, 0.25 M, or 0.30 M NaCl solution was available with water. The general procedures were the same as those described in experiment 2A. On the terminal day all rats consumed saline first, as usual; they were killed by decapitation either when they began to drink water (for 3 s, before they had consumed appreciable amounts) or, if they did not drink water, after 15 min of cumulative pause time while drinking saline. Plasma concentrations of Na+ and protein were measured in these 15 animals, as described above.
The percent of gastric emptying was computed as the difference between the measured fluid intake and the estimated amount of ingested fluid that remained in the stomach, divided by the intake and then multiplied by 100. To estimate the amount of ingested saline that remained in the stomach, it was necessary to distinguish that fluid from water already present in the gastric chyme. Accordingly, nine other DOCA-treated rats were trained as above with 0.30 M NaCl, but on the test day they were denied access to saline and were decapitated soon after the DOCA treatment; their blood (n = 9), stomachs (n = 5), and small intestines (n = 5) were removed and treated as described above. A scatterplot of the stomach liquids of individual animals (in milliliters, on the y-axis) expressed as a function of stomach solids (in grams, on the x-axis) was prepared, and the associated trendline, y = 1.31x + 0.33 (r = 0.95, P < 0.01), was used to correct for the amount of fluid associated with gastric solids in each rat tested. This equation is almost identical to the one obtained previously in this laboratory using a large group of rats feeding ad libitum (31). Note that of 61 DOCA-treated rats in which suitable measurements were made (i.e., 52 in experiment 2A plus the nine just mentioned that were denied access to NaCl solution), only eight had >0.5 g of stomach solids; thus in 53 animals, the associated volumes subtracted from measured gastric water were ≤0.98 ml. These calculations assumed that food residues present in the stomach at the start of the drinking test, as well as the associated water in the gastric chyme, remained in the stomach during the brief period of testing.
Another small correction in gastric emptying was required for the fluid in the stomach that was derived from orogastric secretions instead of ingestion. Thus five additional DOCA-treated rats were fitted with indwelling stainless-steel gastric fistulae (1.3 cm long, with a 1-cm diameter flange at each end), using procedures described previously (16), and subsequently they were housed singly in polypropylene cages (48 × 25 × 20 cm). Each fistula was plugged with a removable stainless steel screw except during experimental sessions. After 4–7 days of recovery from surgery, rats were acclimated to manipulations of the fistula and to drinking in specially constructed test boxes that permitted drainage of the ingested fluid and its collection in a calibrated tube (16). Drinking fluid was available in a calibrated burette attached to one end of the cage, but no food was available during the test period. On the test day, rats were DOCA-treated as above and given either 0.15 M (n = 2), 0.30 M (n = 2), or 0.50 M NaCl (n = 1) to drink, and intakes and drained fluid were measured (±0.1 ml) every 5 min for 20 min. A scatterplot of the data was prepared displaying drained fluid volumes (on the y-axis, in milliliters) as a function of fluid intake (on the x-axis, in milliliters); each of the 20 individual data points (i.e., four points for each of 5 rats) were observed to fall along the trendline, y = 1.126x + 0.084 (r = 0.99, P < 0.001), which had been derived previously using 23 rats drinking water or 0.15 M NaCl after overnight water deprivation (30). In other words, these data collectively indicate that an average of 12.6% more fluid drained from the rats' stomachs than they had consumed, regardless of whether they drank water or saline and were stimulated to drink by water deprivation or DOCA treatment. Thus we estimated that 12.6% of the fluid not associated with food in the stomachs of the DOCA-treated rats was derived from orogastric secretions, and the remaining 87.4% was derived from ingested saline.
The small intestines of 5 DOCA-treated rats denied access to saline, mentioned above, were empty of visible food and fluid. These tissues were removed at arbitrary lengths, ranging from 35 to 90 cm, and dried to constant weight to determine their liquid content per centimeter. This value (0.069 ± 0.006 ml/cm) was multiplied by the length of intestinal tissue that contained dyed liquid when DOCA-treated rats drank saline and that product was subtracted from the total water volume in each intestinal segment to yield the volumes of fluid in the intestinal lumen. Mean distension of the small intestine was computed as the estimated fluid in the lumen (in milliliters) divided by the measured length of intestinal segment that contained the dye (in centimeters). Finally, the amount of fluid that had been absorbed from the small intestines was computed as the fluid intake (including 12.6% orogastric secretions) minus the sum of the gastric water content (corrected for food-associated water) and the volume contained in the intestinal lumen. Assuming that the orogastric secretions were emptied and absorbed in the same way as the ingested fluid, the amount of absorbed fluid derived from ingested fluid therefore equals 100/112.6 (88.8%) times the total absorbed fluid.
All data are presented in scatterplots or as means ± SE. Statistical reliability of observed differences in fluid intake was determined using a two-way ANOVA with repeated-measures analysis. A one-way ANOVA with Tukey's post hoc analysis was used to determine significance at specific time points. Regression equations were calculated by the method of least squares, and significance was determined using Pearson's correlation coefficients or curvilinear methods when the data were better fit to logarithmic or exponential functions. P < 0.05 was considered to be statistically significant.
DOCA-treated rats began to drink as soon as the NaCl solution was made available. As shown in Fig. 1A, the rats drank saline continuously at first and consumed similar volumes after 5 min regardless of fluid concentration (all P values = ns). By 10–15 min, however, intake of all five concentrated solutions had begun to level off (i.e., at least two-thirds of the 60-min volumes were consumed by then), whereas intake of the two most dilute solutions had not. By 30 min, intake of 0.40 M and 0.50 M NaCl had become significantly lower than that of the five other solutions (all P values of <0.05). By the end of the 1-h test session, rats drank the largest volumes of 0.10 M–0.25 M NaCl, less 0.30 M NaCl, and the smallest volumes of 0.40 M and 0.50 M NaCl (Table 1; all P values of <0.05 between these subgroups).
A somewhat different picture was seen when NaCl intakes were expressed in milliequivalents of Na+ rather than milliliters of solution. As shown in Fig. 1B, ingestion of the five concentrated NaCl solutions slowed considerably after 15 min, but the range of intakes was relatively small throughout the test session (most P values = ns). Ingestion of 0.15 M NaCl followed the same general pattern, although it was relatively slow during the first 10 min (all P values of <0.05 compared with the intakes of the concentrated solutions), but Na+ consumption increased to the level of most of the concentrated solutions by the end of the 1-h test (P < 0.001 compared with the intakes of 0.25 M and 0.50 M NaCl). In contrast, ingestion of 0.10 M NaCl increased especially slowly, and by 10 min and for the remainder of the test, Na+ consumption was significantly below that of each concentrated NaCl solution (all P values of <0.001). In other words, DOCA-treated rats tended to drink similar amounts of Na+ (in milliequivalents) throughout the 1-h test regardless of fluid concentration unless the solution was too dilute.
Water intakes were delayed in onset for at least 10–15 min and occurred only when DOCA-treated rats drank the concentrated NaCl solutions. The volumes of water consumed then were directly related to the saline concentration. That is, they ingested the most water when they drank 0.50 M NaCl (all P values of <0.05), the least when they drank 0.20 M NaCl (all P values of <0.05), and intermediate amounts of the other three solutions (Table 1).
DOCA-treated rats excreted 5–7 ml of urine during the 1-h test session, mostly in the second 30 min. The mean urine Na+ concentrations were 150–190 meq/l when the rats drank the concentrated NaCl solutions and were lower when they drank the other two fluids (Table 1; all P values of <0.01). The net amounts of ingested Na+ and water that were retained were computed by subtracting voided urine contents from ingested amounts. The ratio of Na+ balance to water balance increased in proportion to the concentration of NaCl solution that was ingested and was >150 mM except when 0.10 M NaCl was consumed (Table 1).
DOCA-treated rats were given one of four NaCl solutions to drink together with water, and their intakes during the 1-h test sessions were electronically monitored and thus permitted a detailed analysis. The volumes consumed and patterns of intake generally were similar to those observed in experiment 1A. Thus the rats invariably drank saline immediately and consumed the largest volumes of 0.15 M and 0.20 M NaCl, less 0.30 M NaCl, and even less 0.40 M NaCl (all P values of <0.05). Rats drinking one of the three concentrated NaCl solutions began to drink water between 15 and 35 min, on average (P values = ns between groups), and subsequently they consumed both saline and water in additional bouts during the remainder of the session. The concentration of the fluid mixture consumed once water intake began always was dilute: 119, 110, and 79 mM NaCl, after 0.20 M, 0.30 M, and 0.40 M NaCl, respectively (Fig. 2). When 0.30 M or 0.40 M NaCl was available, the rats drank both saline and water in 22% and 31%, respectively, of all drinking episodes, whereas there were no multibout episodes when 0.20 M NaCl was available. Table 2 indicates that the more concentrated the NaCl solution, the smaller the size of the saline bouts (both initial bouts and subsequent bouts) and the more numerous and larger the water bouts.
DOCA-treated rats drank 0.10 M-0.40 M NaCl in an initial drinking bout that lasted 10.3 ± 0.3 min, whereas they drank 0.50 M NaCl for much less time (3.5 ± 0.3 min; P < 0.001). As shown in Fig. 3A, they drank 0.10 M–0.30 M NaCl solution at very similar, rapid rates during this bout (1.8 ± 0.1 ml/min; r = 0.95, P < 0.001) while they drank 0.40 M and 0.50 M NaCl at steady but significantly slower rates (1.1 ± 0.1 ml/min, P < 0.001; r = 0.97, P < 0.001), due to more frequent short pauses within the bout. When saline intakes were plotted in milliequivalents of Na+ instead of ml (Fig. 3B), the rate at which rats drank 0.20–0.50 M NaCl (0.45 ± 0.02 meq/min; r = 0.92, P < 0.001) was almost twice as fast (P < 0.001) as the rate at which they drank 0.10 and 0.15 M NaCl (0.25 ± 0.01 meq/min; r = 0.97, P < 0.001).
As expected, the amount of fluid found in the stomachs of DOCA-treated rats at the end of their initial drinking bout increased in proportion to the volume of fluid that they had ingested. However, this amount varied because the rate of gastric emptying differed considerably depending on the concentration of NaCl solution (Fig. 4, Table 3). Specifically, 0.40 M and 0.50 M NaCl emptied more slowly than 0.10–0.25 M NaCl (all P values < 0.05), each of which emptied at rates that were not significantly different from one another.
Less than 0.5 g of solid matter was found in the gastric chyme of most DOCA-treated rats. Nonetheless, as shown in Fig. 5, gastric emptying clearly slowed in proportion to the amount of gastric solids in most groups. This interaction was much stronger when rats consumed 0.40 M or 0.50 M NaCl solution than when they drank 0.20–0.30 M NaCl; it was milder when they drank 0.15 M NaCl, and it was not apparent when they ingested 0.10 M NaCl.
The difference between the fluid intake and the measured volume of gastric water provided an estimate of the volume of fluid that emptied from the stomach. The concentration of that fluid was estimated by adding dilute orogastric secretions (∼12.6% of fluid intake) to the fluid intake, which thereby diluted the concentration of fluid in the stomach (albeit slightly). Fig. 6 displays this estimate of Na+ (in milliequivalents) emptied from the stomach of DOCA-treated rats plotted as a function of time. The significant correlation between these two variables (r = 0.88, P < 0.001) suggests that Na+ (in milliequivalents) emptied at a stable rate regardless of the concentration of NaCl solution that was ingested.
The volume of fluid in the small intestine increased in proportion to the volume of ingested fluid, regardless of which saline solution was consumed (Fig. 7; r = 0.93, P < 0.001). Because the ingested fluid was dyed green, it was easy to determine how far the emptied fluid had traveled in the small intestine while the rats drank. That distance was logarithmically related to the fluid volume in the intestinal lumen (Fig. 8A; r = 0.96, P < 0.001). Intestinal volume also produced proportionate increases in the mean distension of the small intestine (Fig. 8B; r = 0.98, P < 0.001). As expected from those results, intestinal distance and mean intestinal distension were exponentially related to one another (r = 0.84, P < 0.001). This relation (not shown) indicated that there was little visible distension when the first 60 cm of small intestine proximal to the pylorus were filled, but distension increased markedly at greater distances. Note that the rats whose intestinal distance was above (or below) the trendline in Fig. 8A were the animals whose mean intestinal distension was comparably below (or above) the trendline in Fig. 8B (r = 0.94, P < 0.001).
When DOCA-treated rats consumed 0.20–0.50 M NaCl, the total volume of fluid found in the stomach and small intestine [i.e., gastrointestinal (GI) fill] was highly correlated with and approximately equal to the volume consumed (Fig. 9; r = 0.98, P < 0.001). Similarly, GI fill was highly correlated with intake of 0.10 M and 0.15 M NaCl (Fig. 9; r = 0.98, P < 0.001), although with these solutions, not as much fluid was found in the gastrointestinal tract as had been consumed. The estimated volumes of missing fluid, presumably absorbed (since dyed fluid was not found in the cecum), increased in proportion to fluid intake, ranging from ∼2.1 ml when rats drank 5 ml of dilute fluid to ∼6.5 ml when they drank 20 ml. Note that the GI fill associated with the largest intakes of 0.20–0.40 M NaCl was somewhat smaller than the GI fill associated with the largest intakes of 0.10 or 0.15 M NaCl.
Systemic pNa did not increase significantly while DOCA-treated rats were ingesting concentrated NaCl solutions (Table 4). Similarly, the plasma protein concentrations were comparable in all groups and resembled those of untreated control rats in this laboratory (6.1 g/dl).
When given access to both saline and water, DOCA-treated rats were observed to drink 0.20 M, 0.25 M, or 0.30 M NaCl steadily at the same rate as in experiment 2A (1.7 ± 0.1 ml/min, P = ns). When they stopped, 11 of the 15 rats tested (n = 4, 3, 4 in each group, respectively) began to consume water after a cumulative pause time of 5.9 ± 0.9 min, whereas the test was terminated in the four other rats when the cumulative pause time reached 15 min. In these 15 animals, ingestion of concentrated NaCl solutions significantly increased systemic pNa above control values in DOCA-treated rats not given access to NaCl solution (Table 4).
It has long been known that DOCA treatment produces a marked increase in consumption of NaCl solutions by rats (19). Presumably, the steroid mimics an important feature of Na+ deficiency and thereby induces salt appetite, and the intended goal of the NaCl consumption is to correct a perceived deficit in body fluids. But there are no such deficits, of course, and the induced NaCl intake soon stops. The present study was designed to determine whether the volume and concentration of the NaCl solution ingested by DOCA-treated rats influenced the intake and gastric emptying of Na+ (in milliequivalents). There were four novel findings that deserve emphasis. First, DOCA-treated rats drank 0.15–0.50 M NaCl solution in larger amounts the less concentrated it was, which resulted in a relatively consistent rate at which Na+ was consumed. Second, gastric emptying of ingested NaCl also was inversely related to the concentration of saline solution, which resulted in a consistent rate at which ingested Na+ was delivered to the small intestine. Third, the induced intake of NaCl solution was inhibited when the concentration of the fluid entering the small intestine was hypertonic. And fourth, the volume of fluid consumed was closely related to the cumulative volume of ingested fluid that was found in the stomach and small intestine when the test was terminated, which allows the possibility that gastrointestinal distension provides another early signal that inhibits saline consumption.
DOCA-treated rats drank large volumes of concentrated NaCl solutions rapidly in each experiment. In the initial drinking bout, maximal intakes of 14–19 ml of 0.20 M or 0.25 M NaCl, of 11–13 ml of 0.30 M NaCl, and of 4–10 ml of 0.40 M or 0.50 M NaCl compute to 2–4 meq Na+, which is equivalent to about one-third of the extracellular Na+ in the animals. Observed drinking rates of 1.7–2.1 ml/min when rats consumed 0.10–0.25 M NaCl are comparable to the high rates at which water is ingested by dehydrated rats (10), which suggests that the DOCA-treated rats were similarly motivated to consume saline. Because rats are known to drink at the rate of 6–7 licks/s (24), and in the present experiments they required 159 licks to obtain 1 ml of saline, it can be estimated that the animals drank for 39–56 s of each minute of the test, on average, with the remaining time distributed in brief pauses.
Inspection of Fig. 1A indicates that DOCA-treated rats drank concentrated NaCl solution during an initial bout that lasted up to 10–15 min, but they did not maintain their strong motivation to consume saline subsequently. Furthermore, very different volumes of NaCl solution were ingested by the end of the 1-h test session; the most concentrated solutions were consumed in the smallest volumes. Consequently, the NaCl intake in 60 min remained within a narrow range (4.5–6.5 meq Na+) despite the broad range of concentrations (Figs. 1B). The intake of 0.10 M NaCl was an exception to this finding, perhaps because the solution is so dilute that very large intakes (i.e., 45–65 ml) would have been required for the same amount of Na+ (in milliequivalents) to be consumed.
Gastric emptying of NaCl generally paralleled saline intake and also was inversely related to the concentration of the ingested NaCl solution within the range of 0.15–0.50 M. Rats emptied Na+ more slowly when the concentration was relatively high, and consequently, the delivery of Na+ to the small intestine was remarkably stable regardless of which NaCl solution was ingested (Fig. 6). These results suggest that visceral osmoreceptors (or Na+-receptors) detected the concentration of NaCl solution passing through the intestinal lumen and/or adjacent tissue (3, 17) and regulated gastric emptying accordingly. As such, the findings resemble the differential gastric emptying of glucose solutions in rats after a glucose load, which is known to allow a steady rate of glucose delivery to the small intestine (13, 16), although it contrasts with the nonsteady rate of gastric emptying while hungry rats feed (13).
Gastric emptying of ingested NaCl solution was additionally decreased in proportion to the amount of solid matter (presumably, residual food) in the stomach. This effect was greatest when rats drank 0.50 M NaCl, smaller when they drank 0.20–0.25 M NaCl, and still smaller when they drank 0.15 M NaCl (Fig. 5). Note that in an earlier study in which intakes were monitored as in experiment 1B (29), DOCA-treated rats with ad libitum access to food and fluids were observed to consume 0.50 M NaCl predominantly when they ate. The present findings suggest the sense of this strategy: the accumulation of food in the stomach is likely to have considerably reduced gastric emptying of the strongly concentrated saline solution, thereby allowing more saline and less water to be consumed. Presumably, the putative visceral osmoreceptors detect the combined influence of osmolytes from the food and the ingested NaCl solution and increase inhibition of gastric emptying.
Once ingested fluid enters the small intestine, it may influence saline consumption in several ways. First, it can raise systemic plasma osmolality (pOsm) and thereby activate cerebral osmoreceptors, which can inhibit salt appetite directly (25, 36), as well as by stimulating thirst as a competing drive. In this regard, ingested saline was observed to travel deep into the small intestine before it began to stretch the intestinal wall, as also has been found when water and dilute NaCl solutions are ingested by dehydrated rats (10). As a consequence of these effects, the small intestine accommodated an increasing volume of the ingested fluid while providing an expanded surface area from which that fluid might affect systemic pOsm. However, no statistically significant changes in systemic pNa were observed while DOCA-treated rats consumed concentrated solutions or immediately after they stopped drinking (Table 4). A 2–3% increase in systemic pNa (Table 4), which is just above the threshold for the stimulation of thirst in rats (7), was observed later when water intake began.
Although there was no evidence of increases in systemic pOsm in association with an early inhibition of salt appetite, the presence of concentrated NaCl solution in the small intestine might activate visceral osmoreceptors (or Na+ receptors) that inhibit further consumption of saline. Such receptors have been proposed to stimulate thirst and neurohypophyseal secretion of vasopressin and oxytocin via vagal signals to the caudal brain stem (3, 14, 15, 27). In this regard, previous studies found that substantial increases in water intake occurred in thirsty rats either after unmyelinated visceral afferents were destroyed by systemic administration of the neurotoxin capsaicin (5) or after area postrema lesions (6), as if an early postingestive inhibitory signal had been disrupted. The contribution of these sensory neurons to the control of salt appetite was suggested by the parallel findings that capsaicin pretreatment increased the amount of 0.40 M NaCl consumed by DOCA-treated rats in an initial drinking bout (5) and that focal lesions of the area postrema markedly increased spontaneous ingestion of 0.50 M NaCl (4, 28). In addition, saline infusion into the hepatic portal vein decreased intake of 0.50 M NaCl solution by rats made Na+-deficient with a diuretic (40, 41), and this effect was eliminated by hepatic vagotomy (40).
On the other hand, the consumption of saline by DOCA-treated rats in the present experiments was inhibited even when the animals ingested 0.10 M or 0.15 M NaCl solution, which cannot elevate either systemic or intestinal Na+ concentrations. Thus some other inhibitory signal must be involved, instead of or in addition to one related to the concentration of the ingested fluid. That signal may reflect the volume of ingested fluid. In this regard, the amount of fluid consumed did not generally predict the volume of fluid contained in the stomach because the latter volume was strongly influenced by the concentration of the ingested NaCl solution (Fig. 4). However, the amount of fluid consumed was highly correlated with the volume of fluid contained in the small intestine regardless of which NaCl solution was consumed (Fig. 7). It also was highly correlated with the combined volume of fluid contained in the stomach and small intestine (Fig. 9), although two regression lines were observed. GI fill amounted to ∼100% of the fluid intakes while concentrated NaCl solutions were consumed (presumably, whatever absorption occurred was counterbalanced by an osmotic movement of water into the small intestine), whereas GI fill was ∼80% of the fluid intakes while rats consumed 0.10 M or 0.15 M NaCl. Similar percentages to the latter were reported recently when dehydrated rats ingested water or dilute NaCl solution, and this gastrointestinal distension was proposed to provide an early stimulus that inhibits thirst (10). Perhaps it provides an inhibitory stimulus in the control of salt appetite, as well. In any case, because DOCA-treated rats that drank 0.10 M or 0.15 M NaCl generally consumed more saline (in milliliters) in an uninterrupted initial drinking bout than rats that drank more concentrated solutions (Fig. 9), it seems plausible that both the volume and the concentration of the ingested NaCl solution provided signals inhibiting further consumption of saline.
Fifteen years ago, Tordoff and colleagues (39) reported the results of detailed investigations in which the early consequences of NaCl consumption were studied in rats pretreated with a diuretic drug. Specifically, the Na+-deficient rats were allowed to consume fixed amounts (i.e., 2, 4, or 6 ml) of 0.50 M NaCl solution, and measurements of gastric and small intestinal Na+ contents were made 15–120 min after the onset of saline intake. A main finding was that gastric emptying of Na+ varied in proportion to the volume of saline consumed, ranging from 0.45 to 0.77 meq in 15 min. These amounts were not unlike the values seen in the present study (Fig. 6); the rat drinking the largest volume (4.3 ml) of 0.5 M NaCl emptied 0.66 meq Na+ in 4.3 min, a rate that likely slowed considerably when drinking stopped. However, by examining Na+ intakes over a broader range of volumes and concentrations of NaCl solution than were used in the previous study, and at earlier time points, a consistent pattern of effects on gastrointestinal Na+ content emerged in the present experiments that was related to the volume and concentration of the fluid consumed by DOCA-treated rats. It is not yet known whether this difference in outcome of the two studies reflects a difference in the treatment used to elicit salt appetite or in the experimental design used to study it.
To summarize, DOCA-treated rats appear to stop drinking NaCl solution in response to inhibitory signals arising from the volume and the concentration of the ingested fluid. Specifically, we propose that the rats drink concentrated NaCl solution until they are inhibited from further ingestion by two signals acting in concert, one associated with gastrointestinal distension and the other associated with increases in the osmolality (or Na+ concentration) of intestinal fluid. The concentrated NaCl solutions leave the stomach and small intestine relatively slowly, but ultimately pOsm increases and the rats become thirsty and start drinking water, a behavioral response that complements the excretion of concentrated urine. However, this water intake further increases gastrointestinal distension and inhibits fluid ingestion. Thus these animals drank little saline after an initial bout. The situation appears to be different when 0.10 M or 0.15 M NaCl is consumed, since GI fill may provide the only signal that inhibits further fluid intake. These dilute solutions empty and are absorbed relatively rapidly, which reduces inhibition without increasing pOsm and thereby allows recurrent fluid ingestion. Future experiments are needed to evaluate these hypotheses and to determine whether they extend to other models of NaCl appetite in rats.
The present report does not address the issue of why DOCA-treated rats initiate NaCl ingestion but why they stop. The mechanism by which mineralocorticoids stimulate salt appetite in rats is unsettled (see Refs. 26 and 43, for reviews). Administered DOCA likely does not serve simply as a precursor to aldosterone because it can enhance salt appetite in adrenalectomized rats (44). Instead, one popular idea is that mineralocorticoids provide an excitatory signal by acting directly in the brain, usually in concert with ANG II (8). In this regard, Geerling and colleagues (9) recently discovered a unique group of neurons in the caudal brainstem that express both the mineralocorticoid receptor and the enzyme 11-β-hydroxysteroid dehydrogenase type 2 (which makes the cells selectively responsive to mineralocorticoids rather than to glucocorticoids as well). These neurons were activated by DOCA treatment and by dietary NaCl deprivation. However, they also were activated in NaCl-deprived adrenalectomized rats, which lack aldosterone, thus indicating the presence of additional factors that influence the central control of salt appetite. In this regard, adrenalectomy, dietary NaCl deprivation, and DOCA treatment are each known to lower activity in centrally projecting oxytocinergic neurons (21, 32, 36), which appear to have an important role in inhibiting salt appetite in rats (37, 38). Thus DOCA could provide an excitatory stimulus for salt appetite while also reducing activity in a central inhibitory system.
The present results indicate that inhibition of 0.20–0.50 M NaCl consumption by DOCA-treated rats is associated with several developments, namely, gastrointestinal distension, perfusion of the small intestine by hypertonic saline, and an increase in systemic pNa. It seems relevant that all three of these effects are known to stimulate pituitary oxytocin secretion (18, 35, 36) and concurrent activation of parvocellular oxytocinergic neurons that appear to be involved in the central inhibition of salt appetite in rats (42). Conversely, destruction of central oxytocin receptors eliminates the inhibition of salt appetite that results from systemic plasma hyperosmolality but, curiously, not the inhibition that results from hypernatremia (1, 2). Thus, an additional inhibitory system, perhaps involving atrial natriuretic peptide, should also be considered in the central control of salt appetite (26).
This research was supported in part by a grant from the National Institute of Mental Health (MH-25140).
The authors are grateful for the expert technical assistance of April Protzik, and the helpful comments of Michael Bykowski, Reza Manesh, Carrie Smith, and Jennifer Vaughan. Portions of this work were submitted by M. A. Bushey in partial fulfillment of the requirements for the B.Phil. from the Honors College at the University of Pittsburgh. A preliminary version of this report was presented at the annual meeting of the Society for the Study of Ingestive Behavior in Pittsburgh, PA, in July 2005.
Present address of M. A. Bushey: School of Medicine, The Johns Hopkins University, 733 N. Broadway, Baltimore, MD 21205
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