INTRODUCTION

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A NUMBER OF STUDIES have shown that arginine vasopressin (AVP) dose dependently suppresses lumbar (23) and renal (3, 18, 25) sympathetic nerve activity (RSNA) and heart rate (HR) (14, 18). This sympathoinhibitory action is observed not only by exogenously administered AVP but also by endogenously released AVP (2, 10). Three possible mechanisms have been postulated: 1) a baroreceptor mechanism, by which AVP may enhance a sensitivity of the arterial baroreceptors (1) and reflexively suppress the sympathetic efferent nerve activity; 2) a direct action, by which AVP may suppress postganglionic neuronal activity at the site of the sympathetic ganglions (11); and 3) a central mechanism, by which AVP may act directly on the central nervous system to modulate the autonomic nervous system and finally suppress the peripheral sympathetic nerve activity (25, 26). However, it is believed that the central mechanism is the most prominent, because APX completely abolishes the AVP-induced sympathoinhibitory action in conscious animals (26), even though all other receptors and effectors remain intact. Therefore, it is postulated that the circulating hormone AVP acts on the area postrema (AP) to modify autonomic functions, categorizing this response as a neurohumoral interaction.

The suppression of RSNA due to the AVP-induced neurohumoral interaction may result in increasing urinary output, since RSNA strongly modifies the renal reuptake of Na⁺ and H₂O (5). Therefore, it is hypothesized that AVP-induced sympathoinhibition might cause increasing urinary output and produce less body fluid balance. The sympathoinhibition due to the interaction, however, has only been observed in short-term experiments having a duration of a few minutes to a few hours (3, 18, 23, 25, 26). The long-term effects (several days) have not yet been determined in rabbits. The purpose of the present study was to examine the long-term effects of AVP-induced neurohumoral interaction via the area postrema (AP) on body fluid balance, mean arterial blood pressure (MAP), and HR in rabbits.

	METHODS

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Surgery. Fifteen New Zealand White rabbits weighing 2.21-3.07 kg (2.795 ± 0.082 kg) were used in this study. The rabbits were anesthetized with a subcutaneous injection of an anesthetic mixture [(in mg/kg body wt) 43 ketamine, 3.6 chlorpromazine, and 8.6 xylazine], intubated and mechanically ventilated with room air, and placed in a stereotaxic head holder. With the use of sterile surgical procedures, a midoccipital incision was made and the atlantooccipital membrane was cut exposing the fourth ventricle. With the aid of a surgical microscope, the AP was identified and then removed via suction in eight rabbits (APX) and left intact (Int) in seven rabbits (sham lesioned, Int rabbits). After a 4-wk recovery period, the rabbits were reanesthetized, and Silastic catheters were implanted in the abdominal aorta via the right femoral artery for measurement of arterial blood pressure and in the right external jugular vein for infusion of AVP. A 1-wk recovery period was allowed before the experimental protocol was initiated. Catheters were heparinized every 2 days until the experiments were completed. Rabbits were treated postoperatively with ampicillin (6.0 mg/kg) and nalbuphine (2.0 mg/kg) at the day of and after the surgery and with dexamethasone (1.0 mg/kg) at the day of the open head surgery. Before entering the study, the rabbits were housed in a standard holding room and were exposed on a daily basis to the laboratory environment until fully acclimated. All procedures were conducted in accordance with institutional and National Institutes of Health guidelines.

Experimental protocol. One week before the study was initiated, the rabbits were transferred to individual metabolic cages (18 in. wide × 17.25 in. tall × 24 in. deep, Hoeltge, Cincinnati, OH) in an isolated room with a 12:12-h day-night cycle (lights on at 7:00 AM). Standard rabbit chow (Teklad hi-fiber rabbit diet 015), distilled water, and saline (0.9% NaCl solution) were provided ad libitum. A venous catheter was extended and connected to a cannula swivel that was fixed on trailing equipment. The second extension tube from the cannula swivel ran out of the cage and was plugged until the infusion experiment started. The extension tubes were covered by a lightweight coiled spring to prevent dislodging and any destruction. Food, water, and saline intake and urinary volume (UV) and its Na⁺ concentration, as well as MAP and HR, were measured daily. After stable baseline measurements were obtained, the following experimental protocol was initiated: 5 control days, followed by 5 days of AVP infusion (0.1 mU · kg¹ · min¹), and ending with 3 recovery days. AVP was infused using the trailing line with a Razel model A-99 MR syringe pump. A 3-ml blood sample was collected through the arterial line four times from each rabbit: once during the control period, twice (2nd and 4th days) during the AVP infusion period, and 7 days after cessation of AVP infusion. Hematocrit, plasma osmolality, plasma Na⁺ concentration, and plasma AVP concentration were determined from each sample. On completion of the experiment, each rabbit was anesthetized, and the brain was removed for histological evaluation to confirm the AP lesion.

H₂O and Na⁺ balance measurements. Daily H₂O balance was calculated as the difference between 24-h water plus saline intake and UV. Daily Na⁺ balance was calculated as the difference between daily Na⁺ intake and urinary Na⁺ excretion (U_Na). Total Na⁺ intake was calculated as the sum of the Na⁺ content of the food (Na⁺ concentration of the food times amount of food) and the Na⁺ amount of saline ingested (Na⁺ concentration of the saline times the volume of saline consumed). U_Na was determined from the daily UV and urine Na⁺ concentration, plus residual Na⁺ collected by rinsing the funnels. The Na⁺ concentrations of saline, urine, and a homogenate of the food were measured with standard flame photometry (Nova Biomedical, Waltham, MA).

Cardiovascular measurements. Each day an arterial line was extended and connected to a Cobe CDXIII pressure transducer. The extension tube was taped up with trailing equipment of the venous line. The position of the transducer remained fixed at heart level on the sitting rabbit's side. After the rabbits were stabilized, baseline MAP and HR were measured for 60-90 min in conscious and in free-moving situations. MAP was obtained by a filter with a 2-s time constant. HR was determined using a Beckman cardiotachometer coupler triggered by the arterial pressure pulse. The analog signals of MAP and HR were digitized with an analog-to-digital converter (MacLab, World Precision Instruments) and sampled at 10 Hz. These data were displayed using a Macintosh microcomputer and recorded to disk. The averages of MAP and HR were then calculated for each recording period.

AVP measurement. A radioimmunoassay technique was used to determine AVP concentrations (18). This involved extraction of AVP from the plasma through a Sep-Pak C₁₈ cartridge (Waters). The recovery of 1.0 µU AVP from the plasma extraction was 85 ± 1%. The antiserum was a rabbit-raised specific antiserum to AVP and did not cross-react with lysine-vasopressin, oxytocin, arginine-vasotocin, or angiotensin I and II. The lowest level of detection was 0.05 µU/tube.

Other measurements. Plasma Na⁺ concentrations were measured by standard flame photometry (Nova Biomedical). Plasma osmolality was measured by freezing-point depression (Precision Systems, Natick, MA). Hematocrit was measured by the capillary method.

Histological examinations. The size and location of APX was examined histologically by methods described elsewhere (26). Briefly, each rabbit was anesthetized, and the brain was perfused with saline followed by 10% buffered Formalin. The brain stem was removed and stored in buffered Formalin with 10% sucrose. The fixed brain stem was embedded in paraffin, sectioned coronally (10 µm thick), slide mounted, and then stained for Nissl substance (cresyl violet stain). The sections were then carefully examined via light microscopy to determine the completeness and location of the lesion. Only the rabbits whose AP was completely destroyed were assigned to the APX group (n = 8).

Statistical analysis. Data were expressed as means ± SE. All statistical analyses were performed with a commercially available statistical package for Macintosh personal computer (StatView-J, vs. 4.11 and SuperANOVA, vs. 1.11, Abacus Concepts). The significance of the effect of AVP infusion and/or the lesion of the AP on each variable was analyzed using two-way repeated-measures analysis of variance (ANOVA) followed by a contrast (4). P < 0.05 was considered significant.

	RESULTS

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Urinary responses to AVP infusion. AVP infusion suppressed UV in both APX and Int rabbits. However, the suppression of urinary output by AVP was significantly less in the APX rabbits (60.7 ± 5.7% of the control average) than in the Int rabbits (40.5 ± 5.6%; Fig. 1, A and C). AVP infusion suppressed U_Na in Int rabbits but did not affect APX rabbits. U_Na values during AVP infusion in APX rabbits were higher than those in Int rabbits (Fig. 1, B and D).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Daily urinary volume (UV; A and C) and daily urinary Na+ excretion (UNa; B and D). * P < 0.05 compared with control;  P < 0.05 between intact [Int; A and B (n = 7)] and area postrema lesioned [APX; C and D (n = 8)] rabbits.

Food intake. Averaged food intake in Int rabbits was 228 ± 17, 198 ± 25, and 234 ± 24 g/day in the control, AVP-infusion, and recovery period, respectively. There was no significant difference among these three values. Averaged food intake in APX rabbits was 203 ± 9, 209 ± 18, and 203 ± 20 g/day in the control, AVP-infusion, and recovery period, respectively. Also, there was no significant difference among these three values. Two-way ANOVA did not show any significant difference in any of the values of the three periods between Int and APX groups.

Water balance and body weight. Int rabbits drank more distilled water than saline during the control or AVP-infusion period (Fig. 2, A and B), whereas APX rabbits drank more saline than distilled water in each period, respectively (Fig. 2, C and D). Total H₂O intake, calculated from the combined water and saline volume, showed no significant difference during each period between the two groups (Int vs. APX: 212 ± 31 vs. 204 ± 20 in the control, 121 ± 10 vs. 114 ± 24 in the infusion period, and 247 ± 32 vs. 250 ± 56 ml/day in the recovery period). AVP infusion suppressed distilled water, saline, and total H₂O intake in both Int and APX groups.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Daily water intake (A and C) or saline intake (B and D). * P < 0.05 compared with control;  P < 0.05 between Int (A and B; n = 7) and APX (C and D; n = 8) rabbits.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Daily H2O balance (A and C) and daily Na+ balance (B and D). H2O balance was calculated as H2O intake minus UV.  P < 0.05 between Int (A and B; n = 7) and APX (C and D; n = 8) rabbits.

Sodium balance. The AP lesion significantly increased positive Na⁺ balance in the control period (Fig. 3, B and D). Subsequent AVP infusion did not significantly alter the daily Na⁺ balance in either the Int or the APX rabbits. However, daily Na⁺ balance tended to be suppressed by AVP infusion in the APX rabbits. To clarify this finding, we calculated, for each rabbit, a cumulative value of daily Na⁺ balances for 5 days during the control or AVP-infusion period, respectively. The cumulative values showed a significant difference between the control (+19.7 ± 7.0 meq/5 days) and AVP-infusion (+4.6 ± 6.6 meq/5 days) periods in the APX rabbits, but not in the Int rabbits (+1.36 ± 4.5 vs. 1.47 ± 8.2 meq/5 days, respectively). However, no significant difference was found in cumulative Na⁺ balance during the AVP infusion period between the Int and APX rabbits.

Plasma Na⁺ concentration, hematocrit, and plasma osmolality. AVP infusion decreased plasma Na⁺ concentration in both groups. However, the fall in plasma Na⁺ concentration was greater in the Int rabbits than in the APX rabbits (Table 1). This same trend was also seen in hematocrit and plasma osmolality (Table 1).

MAP and HR. The average 5-day control values of daily MAP was 60.0 ± 2.0 mmHg in Int rabbits and 62.2 ± 0.8 mmHg in APX rabbits. There was no significant difference between the two. The change in MAP (daily MAP minus the averaged 5-day control data of MAP) was not altered by the AVP infusion in either Int or APX rabbits (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Changes in body weight (BW; A), mean arterial pressure (MAP; B) and heart rate (HR; C). bpm, Beats/min. * P < 0.05 compared with control;  P < 0.05 between Int and APX rabbits.

Histological examinations. Figure 5 illustrates an intact AP from a sham-lesioned rabbit (Fig. 5A) and a medulla from an APX rabbit (Fig. 5B). In the APX rabbit, the entire AP is ablated with major portions of the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus, and the entire hypoglossal nucleus still intact. Four other rabbits had lesions comparable to the one shown in Fig. 5, whereas the remaining three rabbits had larger lesions showing damage to the medial portions of the NTS. These three rabbits produced the same baroreflex HR responses to phenylephrine infusion as those of Int rabbits. Rabbits with incomplete or abnormal baroreflex responses were not included in our analysis.

View larger version (141K): [in this window] [in a new window]   Fig. 5.   Coronal sections of rabbit medulla at the level of the obex in Int (A) and APX (B) rabbits. AP, area postrema; NTS, nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus; XII, hypoglossal nucleus.

Plasma AVP concentration. Blood samples for AVP determination were obtained from six of seven Int rabbits and six of eight APX rabbits. In both groups, infusion of AVP significantly increased plasma AVP concentration (Table 1). No significant difference was found in plasma AVP concentrations in the control, AVP, or recovery period between the Int and APX rabbits. The AVP dosage increased the plasma AVP concentration from 0.37 to 5.25 µU/ml in these 12 rabbits. These plasma AVP concentrations are similar to those observed in hyperosmolar states induced by Na⁺ loading (16, 17, 19).

	DISCUSSION

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In the present study, we compared the effects of prolonged AVP infusion on urinary output of H₂O and Na⁺, body fluid balance, MAP, and HR between Int and APX rabbits. The direct effects of AVP on the kidney of both groups of rabbits should be identical regardless of whether the AP is intact or absent. We therefore believe that the differences in the effects between the two groups indicate an AP-mediated action of AVP and, thus, a neurohumoral interaction. During AVP infusion, Int rabbits demonstrated a lower urinary output of H₂O and Na⁺ than APX rabbits. AVP also produced an increase in body weight and decreased the plasma Na⁺ concentration, plasma osmolality, and HR in Int rabbits but not in APX rabbits. These results suggest that prolonged AVP acts to enhance water retention via a neurohumoral interaction in the AP.

Previous data from short-term AVP infusions (minutes to hours) showed that AVP interacted with AP neurons to suppress RSNA (26), independently of the pressor effects of AVP (3, 18). RSNA is strongly involved in the renal tubular reuptake of Na⁺ and H₂O (5). Therefore, the AVP-induced suppression of RSNA observed during short-term infusions might possibly suppress the neurogenic reuptake of H₂O and Na⁺, thereby increasing UV and U_Na. Based on these data, it could be hypothesized that UV and U_Na might be higher in Int rabbits than in APX rabbits even when AVP is infused long term.

A long-term infusion of AVP in the Int animals resulted in a lower UV and U_Na than observed in the APX animals (Fig. 1). This leads to the possibility that the long-term interaction of AVP with the presence of the AP may actually result in a suppression of UV and U_Na and therefore enable the body to retain H₂O and Na⁺. This phenomenon seems to contradict our hypothesis, discussed above. However, since the urinary output is strongly influenced by H₂O or Na⁺ intake, it is important to examine daily balances of H₂O and Na ions.

There was no significant difference in total H₂O intake (water + saline intake) between Int and APX animals. During AVP infusion, however, daily H₂O balance was less in the APX animals than in the Int animals (Fig. 3, A and C). This result indicates that Int animals excreted less H₂O by long-term AVP than APX animals did, which means that our original hypothesis for H₂O balance was incorrect.

Sodium metabolism is more complicated than H₂O metabolism. APX rabbits preferred saline to distilled water for drinking in both the control and AVP-infusion periods (Fig. 2), indicating that APX increased sodium appetite, an effect previously noted in the rat (7, 13, 15). This resulted in significantly increased Na⁺ balance in APX rabbits during the control period (Fig. 3D). These conditions may attenuate the AVP-induced hyponatremia in APX rabbits compared with that in Int rabbits. We do not have any data to explain the Na⁺ homeostasis in such an animal with increased Na⁺ appetite, but it can be assumed that the animal might increase Na⁺ excretion not only in urine, but also in intestinal fluid, saliva when grooming, or skin scurf, therefore resulting actually in zero balance if calculated from intake minus total output. The AVP infusion decreased the cumulative Na⁺ balance from a previously increased level to a nearly standard level in the APX rabbits. This phenomenon may partly come from the result that the AVP infusion did not suppress U_Na excretion in the APX rabbits. These results suggest that the AVP infusion suppressed Na⁺ retention from a preinfusion level in the APX rabbits. Therefore, our original hypothesis for Na⁺ balance was also incorrect.

The present study showed that AVP infusion in the Int rabbits suppressed urinary H₂O and Na⁺ excretions and resulted mainly in water retention, compared with the APX rabbits. This result suggests that long-term effects of AVP mediated by the AP suppress urinary H₂O excretion, which allows the body to retain H₂O.

Several researchers have reported characteristics of H₂O and Na⁺ metabolism in APX animals. Edwards and Ritter (6) have reported that ad libitum H₂O intake values of Int and APX animals are not different, but that an exaggerated response to exogenous angiotensin II is observed in APX animals. APX increases Na⁺ appetite (7, 13, 15), and APX animals remain in a positive Na⁺ balance (7, 13). It is thought that increased sodium appetite is not secondary to excessive urinary loss of Na⁺ and H₂O (7). These reports were consistent with the results in the control period of this study. Miselis et al. (15) have reported that APX causes permanent weight loss. In the present study, no significant difference was observed in body weight between Int and APX rabbits. The difference in findings may be due to the minimal damage in the NTS of APX rabbits in this study. Furthermore, our rabbits were allowed to recover for a minimum of 4 wk after surgical ablation. We believe that this recovery time may have allowed these APX rabbits to develop a new homeostatic state. Pawloski et al. (21) have reported that AVP infusion (0.2 and 2.0 ng · kg¹ · min¹) in APX rats did not change the daily H₂O balance. However, their data indicated that daily H₂O balances in APX rats were usually lower than those in control rats. If they had calculated accumulated values of daily H₂O balances for 10 days in each group of rats, they might have obtained a significant difference between control and APX rats.

The mechanisms by which the AP may modulate urinary H₂O and Na⁺ were not examined in this study. Iovino et al. (12) have suggested that the AP plays an important role in AVP secretion from the hypothalamic vasopressinergic neurons of the supraoptic nucleus. They demonstrated that APX animals showed an attenuated increase in plasma AVP concentration in response to a hypertonic NaCl challenge. This evidence leads to the possibility that APX animals might have lower plasma AVP concentration than normal animals when they are taking food or when other external or internal stimuli occur. As shown in the present study, APX rabbits conserved less H₂O inside the body compared with Int rabbits. Thus, Iovino's suggestion could explain our data. In the present study, however, our plasma AVP examination did not show any significant difference between the Int and APX rabbits, although we did not examine plasma AVP responses to daily stimuli.

Although no difference in MAP was observed between the two groups of animals in this study, we cannot rule out the possibility that differences in MAP between the two groups may contribute to the observed alterations in Na⁺ and H₂O excretion, because MAP was measured only 60 to 90 min/day under the resting condition, whereas Na⁺ and H₂O balance reflected a 24-h period. However, if only the difference in MAP causes the alterations in urinary excretion, then both Na⁺ and H₂O should be influenced and result in isotonic changes in body fluid balance, since pressure diuresis has been reported to produce closely isotonic excretion of Na⁺ and water (22). In our case, based on the data of Na⁺ and water balances, body weight, and plasma Na concentration, water retention was observed in Int rabbits, whereas relative euhydration was observed in APX rabbits. No isotonic differences were observed in body fluid balance between the two. Therefore, we believe that a circulatory factor may not be a main cause of the differences in urinary output. The other mechanisms are discussed in the following.

There are no data showing whether the sympathoinhibitory effects of AVP, observed in short-term experiments, are maintained during chronic increases in circulating AVP. AVP acts on the V₁ receptors in the AP (9). Long-term stimulation of V₁ receptors in the AP may result in the involvement of other areas in the central nervous system, such as vagal motor centers, hypothalamus, and/or other autonomic premotor neurons. All of these central sites may modulate adrenocortical functions, adrenomedullary functions, systemic and renal vasomotor nerve activities, and cardiac functions. These new physiological circumstances might induce neurohumoral influences to cause the suppression of urinary H₂O and Na ions by AVP-induced neurohumoral interaction via the AP. Finally, since the AP was surgically removed in our study, we cannot rule out the possibility that the effects with AVP were mediated by neurohumoral interactions with other areas of the brain containing neurons whose axons pass through the AP. However, no differences have been seen in the RSNA responses to AVP administrations between both rabbits with surgical (26) and kainic acid-induced (25) lesions of AP. We believe that the effects of the neurohumoral interaction in other areas of the brain on RSNA are minimal.

The present study showed that chronic APX did not alter the resting levels of MAP and HR in the control period, as seen in other studies using rabbits (26). This suggests that the AP has no direct effect on the controlling functions of resting MAP or HR in rabbits. However, in studies using rats, it has been reported by some (24) that APX results in chronic hypotension and bradycardia, whereas others (21) report no change in MAP or HR by a similar dose of AVP to this study. The reason for the differences is not clear; however, it is likely related to the duration of the studies and the postoperative recovery period following the AP lesion or a species difference.

In this study, we did not systematically evaluate the role of the AP in the regulation of MAP and HR during chronic infusion of AVP. However, chronic infusion of AVP did not produce any increase in the resting MAP in either the Int or the APX rabbits. In the Int rabbits especially, AVP infusion produced body fluid retention. It is assumed that a chronically increased body fluid might be masked by the capacitance vessels and the extracellular and intracellular spaces, resulting in no increase in the resting MAP. Although AVP-induced neurohumoral interaction via the AP might be expected to modulate the resting MAP based on data from the acute AVP infusions (3, 18, 26), our results suggest that this interaction is not sufficient to overcome other compensatory systems regulating MAP. APX with the infusion of AVP abolished the decrease in HR. Several mechanisms may potentially explain this phenomenon. First, acute decreases in HR may involve an action of AVP at the AP, resulting in decreases in sympathetic outflow and increases in vagal outflow. Second, it is possible that decreases in HR during chronic AVP infusion in the Int rabbits are related to an expanded volume. Third, Int rabbits demonstrated a decrease in plasma Na⁺ concentration during AVP infusion. Hyponatremia has previously been shown to produce slowing of the sinoatrial pacemaker rhythm (8, 20).

Perspectives