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Am J Physiol Regul Integr Comp Physiol 273: R1474-R1483, 1997;
0363-6119/97 $5.00
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Vol. 273, Issue 4, R1474-R1483, October 1997

Decreases in arterial pressure activate oxytocin neurons in conscious rats

Jennifer C. Schiltz1, Gloria E. Hoffman2, Edward M. Stricker1, and Alan F. Sved1

Departments of 1 Neuroscience and 2 Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Hemorrhage and nonhypotensive hypovolemia are known to increase plasma levels of oxytocin (OT) and vasopressin (VP) in rats. The present experiments demonstrated that secretion of OT and VP also are stimulated by acute drug-induced hypotension. Injection of hydralazine abruptly decreased arterial blood pressure in conscious rats and induced Fos expression, a marker of neuronal activation, within OT and VP neurons in the hypothalamus. Hydralazine also elicited substantial increases in plasma levels of both OT and VP. Injection of chlorisondamine similarly elicited acute hypotension and increased plasma levels of OT and VP. Furthermore, when the hypotensive effect of chlorisondamine was blunted by coinfusion of phenylephrine, the induced increases in OT and VP were markedly attenuated. Across all treatments, arterial blood pressure was inversely related to plasma levels of OT and VP. Plasma osmolality was not increased by hydralazine, nor was there evidence of gastric malaise, two known stimuli for OT secretion in rats. These results suggest that arterial hypotension increases neurohypophysial release of OT and VP in conscious rats.

vasopressin; chlorisondamine; supraoptic nucleus; Fos

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

HEMORRHAGE IS A KNOWN stimulus for pituitary secretion of oxytocin (OT) in rats (20, 21, 28) and dogs (38). Consistent with these studies, hemorrhage increases the electrophysiological activity of hypothalamic magnocellular OT neurons (24) and induces Fos expression, a marker of increased neuronal activity, in these neurons (8, 28). Injection of a hyperoncotic solution of polyethylene glycol (PEG), which reduces plasma volume without affecting arterial blood pressure (AP), is also a stimulus for secretion of OT in rats (34). In addition, OT secretion induced by injection of PEG is potentiated by concurrent hypotension produced by alpha 2-adrenergic receptor blockade (32), suggesting that OT neurons are responsive to decreases in AP during hypovolemia. Furthermore, it is well known that hemorrhage and nonhypotensive hypovolemia stimulate pituitary vasopressin (VP) secretion (29, 34), suggesting that both OT and VP neurons respond to these stimuli.

The responses of OT and VP neurons to changes in AP, however, are not believed to be similar. Putative OT neurons within the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus do not appear to alter their firing rate in response to brief increases in AP in anesthetized rats (2, 25), in contrast to the well-characterized inhibition of VP neurons under such conditions (15, 25). Indeed, this difference in response to increases in AP is a key criterion used to distinguish between OT and VP neurons during electrophysiological recordings of magnocellular neurons projecting to the posterior pituitary (25) and has led to the notion that OT neurons are insensitive to arterial baroreceptor stimulation. However, a recent study (14) in chloralose-anesthetized rats suggests that OT and VP release evoked by electrical stimulation of the parabrachial nucleus may be blunted by the increase in AP that accompanies the stimulation.

Few studies have addressed the question of whether OT neurons respond to decreased AP under normovolemic conditions, and these have provided conflicting results. Minimal Fos expression in OT magnocellular neurons has been found in response to nitroprusside-induced hypotension in anesthetized rats, in contrast to robust Fos expression in VP neurons (30). On the other hand, removal of arterial baroreceptor inputs to the brain by surgical sinoaortic denervation has been reported to increase plasma levels of OT in conscious rats (20, 23). These latter findings, coupled with our recent observation that hydralazine (HDZ)-induced hypotension in conscious rats is associated with Fos expression in hypothalamic loci containing magnocellular OT neurons (13), have prompted us to evaluate more fully the response of OT neurons to decreases in AP. The present results indicate that OT neurons, like VP neurons, are stimulated by hypotension in conscious rats.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals

Adult male Sprague-Dawley rats (Zivic-Miller Laboratories, Zelienople, PA) weighing 275-400 g were used in these experiments. Before the experiments, they were housed individually in wire-mesh cages in a temperature-controlled room (22-23°C). Lights were on between 8:00 AM and 8:00 PM. Rats were given free access to tap water and Purina rat chow.

Experimental Protocols

Three different protocols were used in these studies. The initial experiment examined HDZ-induced Fos expression within OT and VP neurons in the hypothalamus. The second set of experiments examined the effect of hypotensive drugs on plasma levels of OT and VP. The third experiment determined whether these hypotensive drugs produced gastric malaise, and thereby would produce a conditioned taste aversion when paired with a novel drinking solution.

Fos studies. The brain tissue for the Fos study was obtained from rats that were used in an experiment that has been reported previously (13). Briefly, rats were instrumented with arterial and venous catheters and allowed to recover for 5 days before study. Groups of rats were given HDZ (10 mg/kg iv, n = 4) or isotonic saline (1 ml/kg iv, n = 4). Heart rate (HR) and mean arterial pressure (MAP) were monitored for a 30-min baseline period before the drug treatment and 90 min after the drug infusion. Then, rats were anesthetized with Nembutal (50 mg/kg ip) and perfused via the ascending aorta with sodium nitrate followed by a fixative containing 4% paraformaldehyde and 2.5% acrolein (13, 17). Brains were removed, sectioned at 25 µm on a freezing-stage microtome, and stored in a cryoprotectant solution until they were stained for Fos and either OT or VP.

Immunocytochemical localization of Fos was performed as described previously (13, 17) using a rabbit polyclonal antiserum directed to the NH2-terminal region (dilution 1:45,000; Oncogene Sciences, Manhasset, NY) and an Elite ABC protocol with peroxidase staining (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA). After the tissue was stained for Fos, sections were stained for VP-neurophysin or OT using a dual-peroxidase staining procedure (17). The mouse monoclonal VP-neurophysin antibody was used at a dilution of 1:2,000 and was provided by Dr. A. J. Silverman (New York, NY), and the OT antibody was used at a dilution of 1:200,000 and was provided by Dr. M. Morris (Winston-Salem, NC).

The total number of OT or VP neurons in the PVN, SON, or accessory magnocellular nuclei was counted in each brain using bright-field microscopy. The entire rostral to caudal extent of each nucleus was examined bilaterally from a 1-in-12 series of 25-µm sections. The total number of OT and VP neurons was counted, and it was noted whether or not each one was Fos positive; the total numbers of Fos-positive neurons within the PVN and SON have been reported previously (13).

Hormone release studies. One arterial catheter (PE-50 tubing filled with heparinized saline) and one venous catheter (polyvinyl tubing, 0.58 mm ID, 0.965 mm OD; Bolab, Lake Havasu, AZ) were implanted in the abdominal aorta and vena cava via the right femoral artery and vein, respectively, in rats anesthetized with halothane. The distal ends of the catheters were tunneled subcutaneously along the back and exited at the nape of the neck. A nylon jacket connected to a tether and swivel system protected the catheters and allowed intravenous drug administration, blood sampling, and continuous recording of MAP and HR in conscious, unrestrained rats. Rats were placed individually in round, Plexiglas cages (26 cm ID) and allowed to recover overnight. The next day, the arterial catheter was connected to a pressure transducer (Gould P23), and MAP and HR were continuously monitored (Grass model 7 Physiograph) except for 2-min periods when blood samples were collected through the arterial catheter. Baseline MAP and HR were recorded for 30-60 min before a baseline blood sample was withdrawn from the arterial catheter. This 1.5-ml sample was immediately replaced with an equal volume of saline. Thirty minutes later, rats were injected intravenously with either isotonic saline (1 ml/kg, n = 8) or HDZ (10 mg/kg, n = 8; Sigma Chemical, St. Louis, MO). Additional 1.5-ml blood samples were taken 10, 30, and 90 min after the drugs had been administered. Immediately following the collection of each blood sample, the blood volume was replaced with an equal volume of heparinized saline containing the red blood cells from the previous sample. Blood samples were centrifuged (10,000 g, 1 min) soon after they were collected, and a 0.7-ml aliquot of plasma was removed from each sample and stored at -80°C for subsequent radioimmunoassay of OT and VP.

Another series of experiments used similar procedures except that each rat had an additional catheter (Tygon, 0.01 mm ID, 0.03 mm OD; Fisher Scientific, Pittsburgh, PA) inserted into a femoral vein. Rats were injected with chlorisondamine (2 mg/kg, n = 24; Ciba-Geigy, Summit, NJ). In eleven of these rats, an intravenous infusion of phenylephrine (2-10 µl/min of a 0.5 mg/ml solution) was begun immediately after the injection of chlorisondamine and adjusted throughout the experiment to maintain MAP at approximately the control value of 125 mmHg. Control rats received an intravenous injection of isotonic saline (1 ml/kg, n = 6).

The OT and VP contents of the plasma samples were determined by radioimmunoassay following extraction using C18 Sep-Pak Vac Cartridges (1 ml, 50 mg; Waters, Milford, MA). Plasma samples and solutions were pulled through the columns using a vacuum manifold. First, each C18 cartridge was washed with 3 ml of methanol followed by 3 ml of distilled water. Then, the plasma sample (0.7 ml of plasma diluted in an equal volume of 8% acetic acid) was run through the cartridge. Next, the column was washed with 3 ml of 4% acetic acid, and finally OT and VP were eluted with 2 ml of a 3:1 solution of acetonitrile and 4% acetic acid. The extract was frozen, dried using a Speed Vac (Savant Instruments), and then dissolved in 700 µl of buffer (50 mM NaPO4, 25 mM EDTA, 0.9% NaCl, 0.5% bovine serum albumin, 0.1% sodium azide); 200-µl aliquots were used for the radioimmunoassays. The initial incubation, performed at 4°C for 48 h, contained ~4,500 counts/min of 125I-labeled OT or VP (New England Nuclear-DuPont, Boston, MA) and a rabbit polyclonal antibody to either OT (final dilution 1:300,000) or VP (final dilution 1:260,000) in a volume of 400 µl. The specificities of these antibodies, which were generously donated by Dr. J. Fernstrom (Pittsburgh, PA), have been described previously (10). After this primary incubation, antibodies were precipitated using a second antibody procedure; after overnight incubation with normal rabbit serum (final dilution 1:600; Jackson Immunoresearch, West Grove, PA) and goat anti-rabbit serum (final dilution 1:120; Linco, St. Charles, MO), tubes were centrifuged (3,000 g, 30 min), the supernatant fluid was aspirated, and the remaining pellets were counted in a gamma counter (Packard-Auto Gamma Scintillation Spectrometer). Values of OT and VP were calculated from standard curves generated with known amounts of synthetic OT and VP (Bachem, Torrance, CA) that were run through the extraction procedure. In these assays, 10% displacement of binding of the labeled peptide was ~6.8 pg/ml for OT and 2.5 pg/ml for VP. Recovery of OT and VP in this extraction procedure is ~85% (e.g., in four assays used for the data included in these studies, recovery of OT was 85 ± 5%), and intra- and interassay variations were <10%.

Plasma osmolality was measured from 50-µl plasma samples by freezing-point depression using a micro-Osmette osmometer (Precision Systems, Natick, MA).

Behavioral studies. Because OT secretion in rats has been associated with treatments that cause nausea and gastric malaise in humans and produce conditioned taste aversions in rats (36), we determined whether hypotensive drugs could produce a conditioned taste aversion in a forced choice, repeated trial, conditioned taste aversion protocol (11). Twelve rats weighing 275-300 g at the start of the study were used in this experiment. Access to drinking water was restricted to 30 min each day. After several days on this schedule, when their intakes had reached stable values, rats were given access to a novel 0.1% saccharin solution instead of water. Immediately after this 30-min test, rats were injected intraperitoneally with either isotonic saline (1 ml/kg, n = 4), HDZ (10 mg/kg, n = 4), or chlorisondamine (2 mg/kg, n = 4). On the two subsequent days, rats again had access to water for 30 min/day, but on the third day they were given access to saccharin solution instead of water and, after the test period, they were injected intraperitoneally with the same drugs as before. This protocol continued (i.e., rats were given saccharin solution to drink and received drug treatments every 3 days, with water intake occurring on the intervening 2 days) until intake of the saccharin solution had been paired with the drugs a total of four times.

Five to seven days after the fourth trial, these 12 rats were anesthetized with halothane and instrumented with arterial and venous catheters as described above. The following day, they were injected intraperitoneally for the fifth time with the same drug they had been receiving (i.e., either saline, HDZ, or chlorisondamine), and MAP and HR were monitored. In addition, arterial blood samples were withdrawn 30 min before the drugs were given and 30 min after the injection, for measurement of plasma OT and VP concentrations as described above.

Statistical Analysis

Values are presented as means and SE. Data from the Fos experiment were evaluated by two-way analysis of variance (ANOVA), with treatment and loci as the two main factors. MAP and HR data in all experiments were evaluated with a repeated-measures ANOVA. OT and VP values were analyzed by repeated-measures ANOVA in the HDZ study and in the conditioned taste aversion study. Differences between groups were determined at each time point by using Fisher's least-significant difference tests. To estimate plasma peptide levels integrated over the entire sampling period when rats were given chlorisondamine and phenylephrine, baseline values for each rat were subtracted from the values measured in the 10-, 30-, and 90-min blood samples, and the three remaining values were added together. These cumulative values for OT and VP were then analyzed for each group using one-way ANOVA. OT and VP values in all experiments were log transformed before ANOVA to normalize the variance. All statistical analyses were performed using Systat for Windows (Systat for Windows, version 5; Systat, Evanston, IL).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Fos Expression in Response to HDZ

As reported previously (13), intravenous infusion of isotonic saline did not alter AP and did not elicit Fos expression in neurons of the SON or PVN, whereas HDZ reduced MAP from 119 ± 3 to 72 ± 7 mmHg and caused robust Fos expression in these hypothalamic nuclei. In the present study, both OT and VP magnocellular neurons within the PVN and SON were found to express Fos after HDZ treatment (Fig. 1). Within the PVN, parvocellular OT neurons, in addition to magnocellular neurons, appeared to express Fos in response to HDZ (Fig. 1D). Fos-containing parvocellular OT neurons were most prominent along the dorsal edge of the PVN and within the ventral aspect of the medial parvocellular division (Fig. 1D). The percent of OT and VP neurons that expressed Fos in response to HDZ were counted and compared by a two-way ANOVA (Table 1). Overall, a similar fraction of OT and VP neurons expressed Fos in response to HDZ (P > 0.05 for the main factors in the ANOVA). However, within the SON a significantly greater percentage of VP neurons expressed Fos than did OT neurons (P < 0.05).


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  		Fig. 1.   Staining of Fos and vasopressin (VP) or oxytocin (OT) in hypothalamic neurons. Photomicrographs of representative sections from the supraoptic nucleus (SON) (A, C, and E) and paraventricular nucleus (PVN) at the level of the posterior magnocellular nucleus (B, D, and F) of a rat treated with hydralazine (HDZ). Numerous Fos-positive neurons appear with nuclear dark blue-black staining within SON and PVN neurons. Cells labeled for VP (A and B) or OT (C-F) have a light gray reaction product in the cytoplasm. Note that the double-labeled OT neurons along the dorsal edge and ventromedial edge of the PVN in D are likely to be parvocellular neurons. E and F are magnified views of C and D. The scale bar is 100 µm in panels A-D and 10 µm in E and F. Table 1 shows counts of double-labeled OT and VP neurons.

                              
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  		Table 1.   Percentage of cells double-labeled with VP or OT and Fos in the PVN, SON, or accessory magnocellular neurons after hydralazine

OT and VP Release in Response to Hypotension

To determine whether Fos expression in OT neurons was associated with pituitary secretion of OT, plasma concentrations of OT were measured at several times following HDZ in a separate group of rats. Intravenous injection of saline had little effect on MAP or HR (Fig. 2, A and B), and it did not significantly alter plasma OT or VP levels at 10, 30, or 90 min after the injection (Fig. 3). HDZ elicited a rapid and sustained decrease in MAP (Fig. 2A) and tachycardia (Fig. 2B) and substantial increases in plasma OT and VP levels, compared with either baseline levels of the hormones or with the hormone levels in rats injected with saline (Fig. 3). The time course for the OT response was similar to that for VP; concentrations of OT and VP both were elevated within 10 min of HDZ treatment, they were higher at 30 min, and they remained elevated at 90 min. HDZ did not significantly change plasma osmolality (Table 2).


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  		Fig. 2.   Arterial pressure and heart rate responses to intravenous injection of HDZ. Mean arterial pressure (MAP; A) and heart rate responses (B) were measured in conscious rats after injection of saline (square , n = 8) or HDZ (bullet , n = 8). MAP is significantly lower in rats treated with HDZ compared with saline-treated rats within 5 min of injection. HDZ also significantly elevated heart rate within 5 min of injection. Arrows in A and B indicate time of injection. bpm, Beats/min.


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  		Fig. 3.   OT and VP responses to intravenous injection of HDZ. Plasma levels of OT (A) and VP (B) were measured in conscious rats at different times after injection of saline (open bars, n = 8) or HDZ (filled bars, n = 8). OT and VP were significantly elevated in plasma at 10, 30, and 90 min after HDZ injection. * P < 0.05 compared with saline-injected rats at that time or to baseline values within group.

                              
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  		Table 2.   Plasma osmolality in rats injected intravenously with saline or hydralazine

To determine whether a different hypotensive drug also would elicit an increase in plasma OT levels, the effects of chlorisondamine were examined in a separate group of rats. Whereas intravenous injection of isotonic saline had no significant effect on MAP or HR or on plasma levels of OT or VP, chlorisondamine decreased MAP within 1 min (Fig. 4), and by 10 min significant increases in plasma levels of OT and VP were observed (Fig. 5). A sustained intravenous infusion of phenylephrine prevented the chlorisondamine-induced hypotension, except for a 1- to 2-min period immediately following the injection of chlorisondamine when the infusion of phenylephrine was being established (Fig. 4). In these rats, plasma levels of OT and VP were significantly lower than the levels observed in rats receiving chlorisondamine alone, but they were still significantly elevated compared with the levels in saline-injected rats (Fig. 5).


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  		Fig. 4.   Arterial pressure and heart rate responses to intravenous injection of saline, chlorisondamine, or chlorisondamine and phenylephrine. MAP (A) and heart rate responses (B) were measured in conscious rats after injection with saline (square , n = 6), chlorisondamine (black-triangle, n = 13), or chlorisondamine combined with an infusion of phenylephrine (bullet , n = 11). Arrows indicate time of injection. Chlorisondamine induced a significant decrease in arterial pressure within 1 min of injection. Heart rate in response to chlorisondamine was significantly reduced by 10 min after injection. In rats treated with chlorisondamine and phenylephrine, MAP was significantly lower than in saline-treated rats at 1 min after infusions began, but MAP in these rats was not different from MAP in saline-treated rats by 10 min after infusions.


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  		Fig. 5.   OT and VP responses to intravenous injection with saline, chlorisondamine, or chlorisondamine and phenylephrine. Plasma levels of OT (A) and VP (B) were measured in conscious rats at different times after saline (n = 6), chlorisondamine (Chlor, n = 13), or a combination of chlorisondamine and phenylephrine (Chlor + Phe, n = 11). Plasma levels of OT and VP were significantly elevated in chlorisondamine-treated rats compared with levels in saline-treated rats. Plasma OT and VP levels were significantly less in the group given chlorisondamine and phenylephrine than in the group given chlorisondamine alone; however, they were also different from the group given saline. * P < 0.05 between groups.

To examine the relationship between the magnitude of hypotension and plasma levels of OT and VP, plasma hormone levels were plotted as a function of MAP in response to saline, HDZ, chlorisondamine, or chlorisondamine and phenylephrine in each rat (Fig. 6). The 30-min values were selected for this analysis because they represented the peak increase in plasma OT and VP levels. For both OT and VP, there was a significant inverse relationship between plasma concentration and MAP that could be fitted with an exponential equation (Fig. 6). In addition, there was a highly significant correlation between the plasma levels of the two hormones (Fig. 7; r = 0.75, P < 0.001), with VP levels increasing approximately twice as much as OT levels in response to hypotension.


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  		Fig. 6.   Relationship between MAP and plasma levels of OT or VP. Plasma levels of OT (A) and VP (B) were plotted as a function of MAP 30 min after intravenous injection of isotonic saline (+), chlorisondamine (square ), hydralazine (triangle ), or chlorisondamine and phenylephrine (bullet ). An exponential line fit the relationship for each hormone (r = 0.64 for OT, P < 0.001, and r = 0.75 for VP, P < 0.001).


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  		Fig. 7.   Relationship between OT and VP in response to isotonic saline, HDZ, chlorisondamine, or chlorisondamine and phenylephrine. Plasma levels of OT were plotted as a function of plasma levels of VP in individual rats 30 min after intravenous injection with isotonic saline (+, 1 ml/kg), hydralazine (triangle , 10 mg/kg), chlorisondamine (square , 2 mg/kg), or chlorisondamine and phenylephrine (bullet ). A straight line OT = 19.9 + 0.48(VP) described the relation (r = 0.75, P < 0.001).

Absence of Conditioned Taste Aversion to Saccharin

The ingestion of 0.1% saccharin was not influenced by a single pairing of saccharin solution with either HDZ or chlorisondamine (Fig. 8, trial 1). Furthermore, three additional pairings of 0.1% saccharin with HDZ or chlorisondamine at 3-day intervals did not elicit a significant decrease in saccharin intake (Fig. 8). In contrast to the results observed here with HDZ or chlorisondamine, a single pairing of saccharin solution with lithium chloride, a known nauseogenic agent, in this paradigm completely eliminated saccharin intake (7). Water ingestion during the intervening days was not significantly different among the groups, nor was was it different from the amount of saccharin solution consumed on the test days (Fig. 8).


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  		Fig. 8.   Intakes of a 0.1% saccharin solution by rats on successive trials in a conditioned taste aversion paradigm. Groups of rats were allowed 30-min access to tap water each day. On the day indicated by "novel," rats were given their first access to 0.1% saccharin to drink instead of water. Immediately after the ingestion period, they were injected with saline (bullet , 1 ml/kg ip), HDZ (black-triangle, 10 mg/kg ip), or chlorisondamine (square , 2 mg/kg ip). On the two subsequent days, they were given water during the 30-min test period. On the third day (1st trial), they were given only the saccharin solution to drink and, at the end of the drinking period, they were injected with the drugs as before. This protocol was repeated 4 times over 12 successive days. A repeated-measures analysis of variance found no effect of drug (P > 0.05) but a significant effect of trial (P < 0.05), which likely reflects the tendency for the intakes in all groups to increase over the trials and the greater intake of the saline group on the third trial. On the first trial day, 1 rat in the HDZ group drank much less saccharin solution than the other rats, which led to the large SE values for that group on that day. Average water intakes on the intervening days throughout the study were comparable among groups (saline group = 22.9 ± 0.8 ml, HDZ group = 24.0 ± 0.8 ml, chlorisondamine group = 22.2 ± 0.7 ml) and were not different from the intakes of saccharin solution by each group.

One week after the end of the conditioned taste aversion protocol, the same rats were injected intraperitoneally with either saline, HDZ, or chlorisondamine to assess the effects of these treatments on MAP and plasma hormone levels. Similar to the results with acute intravenous administration of these hypotensive drugs, HDZ and chlorisondamine significantly decreased MAP (Table 3) and significantly elevated plasma levels of OT and VP (Table 4).

                              
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  		Table 3.   MAP and HR in rats injected intraperitoneally with saline, HDZ, or chlorisondamine

                              
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  		Table 4.   Plasma levels of OT and VP in rats injected intraperitoneally with saline, HDZ, or chlorisondamine

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The major finding of these studies is that hypothalamic OT neurons are activated by drugs that decrease arterial pressure in conscious rats. HDZ, which decreases AP via a direct action on vascular smooth muscle cells (12), evoked Fos expression in OT-containing neurons in the SON and PVN, and increased plasma levels of OT. Chlorisondamine, a drug that lowers AP by blocking nicotinic transmission in autonomic ganglia (12), also elicited a significant increase in plasma OT. In addition, the increase in plasma levels of OT elicited by chlorisondamine was attenuated by counteracting the hypotension with infusion of a vasoconstricting agent, phenylephrine, suggesting that the OT release resulted from the hypotension produced by chlorisondamine. The increase in plasma OT concentration induced by these drugs was significantly correlated with the magnitude of the hypotension, providing further support for the hypothesis that the OT response was evoked by the decrease in AP.

This is the first report that a decrease in AP can activate magnocellular OT neurons in the hypothalamus of conscious rats, as measured by either evoked expression of Fos or by increases in plasma OT levels. However, several previous studies are consistent with the notion that pituitary OT is released in response to hypotension. Hypotension potentiates the increase in plasma OT levels elicited by PEG-induced hypovolemia (32). Bisset et al. (3) reported that OT-like immunoreactivity increased in urine in response to nitroprusside-induced hypotension in ethanol-anesthetized, water-loaded rats. In addition, plasma levels of OT increased in response to intravenous administration of corticotropin-releasing hormone, possibly as a result of the hypotensive effect of this treatment (5). Finally, sinoaortic denervation has been reported to increase plasma OT levels (20, 23), suggesting that decreased arterial baroreceptor afferent activity, as normally accompanies hypotension, can stimulate OT release.

On the other hand, some reported results appear to be inconsistent with the hypothesis that OT neurons are stimulated by decreases in AP. For example, Shen et al. (30) reported that hypotension evoked by intravenous infusion of nitroprusside in rats anesthetized with ketamine and xylazine was associated with increased Fos expression in <10% of OT-containing magnocellular neurons. Similarly, the electrophysiological activity of OT neurons was not altered when occlusion of the inferior vena cava lowered AP to 50 mmHg in pentobarbital sodium-anesthetized rats (22). However, it is possible that the anesthetics used in these studies interfered with hypotension-induced activation of OT neurons. In support of this possibility, Xiong and Hatton (39) recently found fewer OT neurons activated in response to hypertonic saline in anesthetized rats compared with those observed in conscious rats.

The mechanism by which HDZ or chlorisondamine stimulates OT neurons in rats may relate to one of several signals generated by the induced hypotension. One such signal is a decrease in afferent nerve activity from arterial baroreceptors. It is presumably this mechanism that mediates hypotension-evoked VP release (29), and two studies have reported that acute removal of arterial baroreceptor input results in an increase in OT release (20, 23). Circulating levels of angiotensin II, which increase in response to hypotension, are also capable of stimulating OT neurons (9) and may therefore help to mediate the hypotension-induced increase in plasma OT levels. Signals arising from renal afferent nerves additionally may play a role in this response (31).

The increase in plasma OT levels observed in response to HDZ or chlorisondamine cannot be explained by other known stimuli for OT release. Substantial OT release in male rats is known to occur in response to hypovolemia (28, 34), gastric distension (26), hypo- and hyperthermia (21), hyperosmolality (33), and nausea (37). The design of the present study precludes hypovolemia and gastric distension as being the primary stimulus for the observed OT release. Also, it is unlikely that HDZ or chlorisondamine induced a change in body temperature that was large enough to account for the increase in plasma OT levels in the present experiments. Increased plasma osmolality cannot be the primary stimulus for OT release in these studies because HDZ treatment did not significantly alter plasma osmolality. Furthermore, it is interesting to note that infusions of hypertonic saline produce plasma elevations of OT and VP in an approximate ratio of 3 to 1 (33), which is consistent with the known effect of OT as a natriuretic hormone (36). In contrast, the ratio of OT to VP released in response to HDZ or chlorisondamine is ~0.5 to 1.

Nausea or gastric malaise also seem unlikely to be the stimulus for OT release in response to HDZ or chlorisondamine. Nauseogenic drugs, such as lithium chloride or apomorphine, cause conditioned taste aversions in rats (11); in a paradigm similar to the one used in the present study, a single pairing of saccharin with lithium chloride completely eliminated intake of the novel test solution (7). However, neither HDZ nor chlorisondamine was observed to elicit a conditioned taste aversion even with repeated pairing of the drugs with the novel test solution, suggesting that these drugs do not produce gastric malaise or nausea.

The physiological consequence of hypotension-induced OT release into the circulation remains uncertain. OT can act as a vasoconstrictor in some vascular beds, but seemingly only at very high concentrations (1). On the other hand, OT increases blood flow in the kidney in conscious rats (1), and thereby may contribute to the maintenance of renal perfusion pressure during hypotension. OT can also dilate blood vessels in the brain (6), which might help to maintain cerebral perfusion during periods of hypotension. It also has been suggested that OT contributes to the maintenance of AP during hemorrhage in dogs, possibly by elevating plasma renin (4), and it is conceivable that it also participates in this way during isovolemic hypotension in rats. Indeed, a recent study suggests that intravenous infusion of OT can elevate plasma renin levels in anesthetized rats (19). Further work is needed to establish the functional consequences of elevated plasma OT levels during conditions of hypotension or hypovolemia in rats.

In addition to activation of magnocellular OT neurons, HDZ-induced hypotension was associated with Fos expression in parvocellular OT neurons in the PVN. Activation of these neurons may contribute to the cardiovascular compensations to hypotension because OT injected into brain or spinal cord CSF has been shown to increase AP, HR, and sympathetic nervous system activity (27). Activation of parvocellular OT neurons also has been associated with inhibition of NaCl intake in rats (35), and hypotension has been shown to inhibit salt appetite (35).

The present studies also demonstrated that hypotension induced by HDZ or chlorisondamine was associated with Fos expression in the majority of hypothalamic magnocellular VP neurons. Furthermore, the elevated plasma levels of VP observed in response to HDZ and chlorisondamine confirm several reports that pharmacologically induced decreases in AP stimulate VP release in rats (16, 18). The present data extend these previous observations to show that there is an inverse exponential relationship between AP and plasma VP in rats as exists between plasma volume and plasma VP levels (34).

In summary, the present results indicate that in conscious rats hypothalamic OT neurons are activated by HDZ and, furthermore, that pituitary OT release is stimulated by hypotension induced either by HDZ or chlorisondamine. It appears that the OT release is specific to the decrease in AP because the magnitude of the increase in plasma OT concentration is highly correlated with MAP. Whether this response is mediated by baroreceptor afferent signals, hormonal signals, or other secondary consequences of reduced arterial pressure remains to be clarified, as does the physiological significance of OT release during hypotension.

    ACKNOWLEDGEMENTS

The expert photographic assistance provided by Tom Waters is greatly appreciated.

    FOOTNOTES

These studies were supported by grants from the National Institutes of Health (HL-55687 to A. F. Sved, NS-28477 to G. E. Hoffman, and MH-25140 to E. M. Stricker). J. C. Schiltz was supported by a fellowship from the American Heart Association (Pennsylvania Affiliate) and the Provost's Development Fund (University of Pittsburgh).

Together with other work, these studies were submitted by J. C. Schiltz to the Department of Neuroscience in partial fulfillment of the requirements for her PhD degree.

Present address of J. C. Schiltz: The Salk Institute, Laboratory of Neuronal Structure and Function, PO Box 85800, San Diego, CA 92037.

Present address of G. E. Hoffman: Dept. of Anatomy and Neurobiology, Univ. of Maryland School of Medicine, Baltimore, MD 21201.

Address for reprint requests: Alan F. Sved, Dept. of Neuroscience, 446 Crawford Hall, Univ. of Pittsburgh, Pittsburgh, PA 15260.

Received 29 April 1997; accepted in final form 10 July 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Regul Integr Compar Physiol 273(4):R1474-R1483
0363-6119/97 $5.00 Copyright © 1997 the American Physiological Society


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