INTRODUCTION

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EXCITATORY AMINO ACID (EAA) receptors exist on vagal afferent neurons (4, 13, 16), the adrenal medulla (27, 29), and the pituitary gland (28). The application of the EAA analogs, kainic acid (KA) and N-methyl-D-aspartate (NMDA), to nodose ganglia of rats causes the degeneration of vagal baroreceptor and cardiopulmonary afferent cell bodies (15, 24). In addition, glutamate stimulates the release of catecholamines from isolated canine adrenal glands (18). The presence of EAA receptors on these structures may allow circulating EAAs to alter afferent function and the release of adrenal and pituitary hormones. Indeed, the systemic injection of KA and NMDA produces relatively transient increases in mean arterial blood pressure (MAP) and heart rate (HR) followed by a bradycardia in conscious normotensive rats (14, 19, 20). This pressor response is reduced markedly by the ₁-adrenoceptor antagonist prazosin, whereas the residual response is abolished by a V₁ arginine vasopressin (AVP)-receptor antagonist (14, 19, 20). The tachycardia is blocked by -adrenoceptor blockade, whereas the bradycardia is virtually eliminated by blockade of muscarinic receptors (14, 19, 20). After ganglion blockade, KA and NMDA produce pressor responses that are markedly attenuated by blockade of V₁ AVP receptors but produce no tachycardia (14, 19, 20). These results suggest that the increases in MAP and HR produced by KA and NMDA are caused by elevated sympathetic nerve activity rather than the direct release of catecholamines from sympathetic nerve terminals. Moreover, it appears that KA and NMDA cause the release of AVP from the pituitary. The EAA analog-induced increases in sympathetic nerve activity and AVP release may involve inhibition of the baroreceptor reflex (23), inasmuch as KA suppresses carotid sinus nerve activity (7). In addition, the EAA analogs may release AVP by direct actions on glutamate receptors in the pituitary (28). The bradycardia produced by NMDA and KA may involve a centrally mediated increase in vagal efferent nerve activity or a direct release of acetylcholine from vagal nerve terminals (14, 19, 20).

The circumventricular organs of the brain include the subfornical organ (SFO), the organum vasculosum of the lamina terminalis (OVLT), and the area postrema (1, 2, 10). These so-called sensory circumventricular organs are free of a blood-brain barrier, respond to changes in the circulating levels of a variety of factors, and transmit information reflecting levels of such factors to the brain (see Ref. 10 for review and discussion). In particular, the SFO and OVLT monitor blood hormone levels to adjust arterial blood pressure by changes in sympathetic vasomotor drive. Electrolytic lesions of the area around the anteroventral region of the third ventricle (AV3V) destroy the OVLT and fibers of passage from the SFO (10). AV3V lesions attenuate the pressor responses produced by systemic injections of angiotensin II (6), renin (2), ₂-melanocyte-stimulating hormone (3), and serotonin (17) but not those produced by norepinephrine or the ₁-adrenoceptor agonist phenylephrine (2, 3). This suggests that AV3V lesions do not diminish the vasoconstrictor actions of neural- or adrenal-derived catecholamines. Lesions of the AV3V region also attenuate the pressor responses produced by changes in NaCl concentrations in the cerebral ventricles (11). The pressor effects produced by central injections of serotonin also involve the AV3V region (21). Moreover, lesions of the AV3V region prevent the development of several forms of hypertension (1, 2, 8, 22).

The cardiovascular responses produced by systemically injected KA and NMDA may involve the direct stimulation of EAA receptors in the AV3V region (5). Alternatively, the AV3V region may simply play a role in mediating these responses. The aim of this study was to test the hypothesis that the AV3V region is required for the full expression of the cardiovascular responses produced by systemically administered EAA analogs in conscious freely moving rats.

	MATERIALS AND METHODS

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Rats. Male Sprague-Dawley rats (250-300 g; n = 15) were used in these studies. The experimental procedures were approved by the University of Iowa Institutional Animal Care and Use Committee.

Sham and electrolytic lesions of the AV3V region. The rats were anesthetized with an Equithesin-like anesthetic cocktail (0.33 ml/100 g body wt; composed of 0.97 g of pentobarbital sodium and 4.25 g of chloral hydrate/100 ml distilled water) and secured in a Kopf 900 stereotaxic apparatus with the skull leveled between bregma and lambda. An electrode (24-gauge nichrome wire insulated except at the tip) was lowered on the midline 0.3 mm caudal to bregma to a depth of 7.5 mm from the dura. Anodal current (2-3 mA) was passed for 25-30 s (rectal cathode). In sham-lesioned rats, the electrode was lowered to 1.0 mm above the target tissue, and no current was passed. After 4 wk of recovery, the rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and catheters (PE-50) were placed into the left femoral artery to measure pulsatile pressure (PP) and MAP. Catheters were also placed into the left femoral vein to inject drugs. The rats were allowed at least 4 days to recover before being used in experiments.

Experimental protocols. On the day of experimentation, the arterial catheter was connected to a Beckman Dynograph-coupled pressure transducer (Cobe Laboratories) for measurement of PP and MAP. HR was determined from PP by a cardiotachometer. The rats received bolus injections of KA (1 mg/kg iv), NMDA (1 mg/kg iv), and AVP (10 ng/kg iv) before and 15-20 min after administration of the ganglion blocker chlorisondamine (CLX; 5 mg/kg iv) and again after the administration of the V₁ AVP-receptor antagonist [-mercapto-,-cyclopentamethylenepropionyl¹,O-Me-Tyr²,Arg⁸]vasopressin (AVPX, 20 µg/kg iv). Injections of KA, NMDA, or AVP were given 5 min apart. MAP and HR values were allowed to return to preinjection values before the next test agent was injected.

Histology. At the end of the experiments, the rats were deeply anesthetized with pentobarbital sodium (100 mg/kg iv) and perfused transcardially with 0.1% PBS followed by 10% Formalin. The brains were removed and stored in fixative until frozen sections (40 µm) were cut and stained for Nissl substance with cresyl violet.

Drugs. KA and NMDA were obtained from Sigma (St. Louis, MO). AVP and AVPX were obtained from Peninsula Laboratories (Belmont, CA). The Equithesin-like anesthetic was prepared by the University of Iowa Hospitals and Clinics Pharmacy. CLX was obtained from Ciba-Geigy (Summit, NJ).

Statistics. Data are expressed as means ± SE of the actual values and the arithmetic changes in these values. The means ± SE of the area under the curves for the total change in MAP are also presented. The area under the curve values were determined to examine whether the overall responses produced by KA and NMDA were different in AV3V-lesioned compared with sham-lesioned rats. The data were analyzed by repeated-measures ANOVA followed by Student's modified t-test with the Bonferroni correction for multiple comparisons between means with the modified error mean square term from the ANOVA (12, 25, 26). A value of P < 0.05 was taken to denote statistical difference.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Verification of lesions. The accuracy and completeness of the electrolytic lesions were established as described previously (17). The lesions shared a common area of damage to the periventricular tissue surrounding the optic recess. The lesions consistently incorporated the preoptic periventricular nuclei, the ventral portion of the median preoptic nucleus, and the OVLT. Some bilateral damage was observed at the medial edge of the medial preoptic nuclei.

Body weights of the sham- and AV3V-lesioned rats. The mean body weights of the sham-lesioned (n = 7) and AV3V-lesioned (n = 8) rats were 401 ± 8 and 332 ± 10 g, respectively (P < 0.05).

Effects of CLX and AVPX on resting MAP and HR values of sham- and AV3V-lesioned rats. Resting MAP and HR values of sham- and AV3V-lesioned rats before ganglionic blockade with CLX (5 mg/kg iv) and the subsequent administration of AVPX (20 µg/kg iv) are summarized in Table 1. MAP and HR values for the KA, NMDA, and AVP studies are shown. Resting MAP values of the AV3V-lesioned rats were similar to those of the sham-lesioned rats (P > 0.05 for all comparisons). CLX lowered MAP to similar levels in sham- and AV3V-lesioned rats (P > 0.05 for all comparisons). AVPX produced small falls in MAP that were equivalent in sham- and AV3V-lesioned rats (P > 0.05 for all comparisons). Resting HR values of AV3V-lesioned rats were higher than those of sham-lesioned rats (P < 0.05 for all comparisons). CLX produced similar arithmetic falls in HR in sham- and AV3V-lesioned rats (P > 0.05 for all comparisons). As such, the resting HR of the CLX-treated AV3V-lesioned rats was higher than that of the CLX-treated sham-lesioned rats (P < 0.05 for all comparisons). AVPX did not affect HR in sham- or AV3V-lesioned rats (P > 0.05 for all comparisons).

Effects of KA on MAP in sham- and AV3V-lesioned rats. The maximal changes in MAP produced by KA (1 mg/kg iv) in sham- and AV3V-lesioned rats before and after administration of CLX and again after administration of AVPX are summarized in Fig. 1, top. Before ganglion blockade, KA produced pressor responses in sham- and AV3V-lesioned rats (P < 0.05 for both responses). The pressor responses in AV3V-lesioned rats were smaller than in sham-lesioned rats (P < 0.05). After administration of CLX, KA increased MAP in sham-lesioned rats (P < 0.05) but not in AV3V-lesioned rats (P > 0.05). The pressor response produced by KA in CLX-treated sham-lesioned rats was abolished by AVPX. The total pressor responses produced by KA (1 mg/kg iv) in sham- and AV3V-lesioned rats before and after administration of CLX and again after administration of AVPX are summarized in Fig. 1, bottom. Before administration of CLX, the total changes in MAP were smaller in AV3V-lesioned than in sham-lesioned rats (P < 0.05). As mentioned above, KA did not increase MAP in CLX-treated AV3V-lesioned rats, and KA did not affect MAP after administration of AVPX in either group.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Top: summary of maximal changes in mean arterial blood pressure (MAP) produced by kainic acid (KA, 1 mg/kg iv) in sham-lesioned rats (n = 7) and in rats with electrolytic lesion of anteroventral region of 3rd ventricle (AV3V lesion, n = 8) before and after administration of chlorisondamine (CLX, 5 mg/kg iv) and subsequent administration of vasopressin V1-receptor antagonist [-mercapto-,-cyclopentamethylenepropionyl1,O-Me-Tyr2,Arg8]vasopressin (AVPX, 20 mg/kg iv). Bottom: summary of total changes in MAP produced by KA in these rats. Each value represents mean ± SE of arithmetic and total changes in MAP. * P < 0.05 vs. baseline.  P < 0.05, AV3V lesion vs. sham lesion. ns, Nonsignificant response.

Effects of NMDA on MAP in sham- and AV3V-lesioned rats. The maximal changes in MAP produced by NMDA (1 mg/kg iv) in sham- and AV3V-lesioned rats before and after administration of CLX and again after administration of AVPX are summarized in Fig. 2, top. Before ganglion blockade, NMDA increased MAP in sham- and AV3V-lesioned rats (P < 0.05 for both responses). The pressor responses in AV3V-lesioned rats were smaller than in sham-lesioned rats (P < 0.05). After administration of CLX, NMDA increased MAP in sham- and AV3V-lesioned rats (P < 0.05 for both responses). The pressor responses in AV3V-lesioned rats were smaller than in sham-lesioned rats (P < 0.05). The pressor responses produced by NMDA in CLX-treated sham- and AV3V-lesioned rats were abolished by AVPX. The total pressor response produced by NMDA (1 mg/kg iv) in sham- and AV3V-lesioned rats before and after administration of CLX and again after administration of AVPX are summarized in Fig. 2, bottom. Before ganglion blockade, the total changes in MAP were smaller in AV3V-lesioned than in sham-lesioned rats before and after ganglion blockade (P < 0.05 for both comparisons). NMDA did not affect MAP after administration of AVPX in either group (P > 0.05 for both responses). In a separate group of rats (n = 8), the cardiovascular effects of the above doses of KA and NMDA were determined before and after administration of saline and again after another injection of saline. The cardiovascular effects of KA and NMDA were similar before and after the injection of saline (P > 0.05 for all comparisons, data not shown). Moreover, the cardiovascular effects of KA and NMDA in ganglion-blocked rats (n = 6) were similar before and after administration of saline (P > 0.05 for all comparisons, data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Top: summary of changes in MAP produced by N-methyl-D-aspartate (NMDA, 1 mg/kg iv) in sham-lesioned (n = 7) and AV3V-lesioned rats (n = 8) before and after administration of CLX (5 mg/kg iv) and subsequent administration of AVPX (20 mg/kg iv). Bottom: summary of total changes in MAP produced by NMDA in these rats. Each value represents mean ± SE of arithmetic and total changes in MAP. * P < 0.05 vs. baseline.  P < 0.05, AV3V lesion vs. sham lesion. ns, Nonsignificant response.

Effects of AVP on MAP of sham- and AV3V-lesioned rats. The maximal changes in MAP produced by AVP (10 ng/kg iv) in sham- and AV3V-lesioned rats before and after administration of CLX and again after administration of AVPX are summarized in Fig. 3, top. Before ganglion blockade, AVP produced pressor responses in sham- and AV3V-lesioned rats (P < 0.05 for both responses). The pressor responses in AV3V-lesioned rats were similar to those in sham-lesioned rats (P > 0.05). After administration of CLX, AVP increased MAP in sham- and AV3V-lesioned rats (P < 0.05 for both responses). The pressor responses produced by AVP were smaller in AV3V-lesioned than in sham-lesioned rats (P < 0.05). As can be seen, the pressor response produced by AVP was substantially greater after ganglion blockade compared with before treatment in both sham- and AV3V-lesioned rats (P < 0.05 for both responses). The total pressor responses produced by AVP (10 ng/kg iv) in sham- and AV3V-lesioned rats before and after administration of CLX and again after administration of AVPX are summarized in Fig. 3, bottom. The total changes in MAP were smaller in AV3V-lesioned than in sham-lesioned rats before and after ganglion blockade (P < 0.05 for both comparisons). AVP did not affect MAP after administration of AVPX in either group (P > 0.05 for both responses). In separate rats (n = 8), we determined the cardiovascular effects of the above dose of AVP before and after administration of saline and again after another injection of saline. The cardiovascular effects of AVP were similar before and after the injection of saline (P > 0.05 for all comparisons, data not shown). Moreover, the cardiovascular effects of AVP in ganglion-blocked rats (n = 6) were similar before and after administration of saline (P > 0.05 for all comparisons, data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Top: summary of changes in MAP produced by arginine vasopressin (10 ng/kg iv) in sham-lesioned (n = 7) and AV3V-lesioned rats (n = 8) before and after administration of CLX (5 mg/kg iv) and subsequent administration of AVPX (20 mg/kg iv). Bottom: summary of total changes in MAP produced by arginine vasopressin. Each value represents mean ± SE of arithmetic and total changes in MAP. * P < 0.05 vs. baseline.  P < 0.05, AV3V lesion vs. sham lesion. ns, Nonsignificant response.

Effects of KA, NMDA, and AVP on HR. The effects of KA, NMDA, and AVP on the HR of sham- and AV3V-lesioned rats are summarized in Table 2. In sham-lesioned rats, KA and NMDA produced initial increases in HR that were temporally related to the pressor responses produced by the EAA analogs. The increases in HR were followed by falls in HR. Resting MAP values were not different from preinjection values during the bradycardia (P > 0.05 for all comparisons, data not shown). The KA- and NMDA-induced increases and decreases in HR in AV3V-lesioned rats were smaller than in sham-lesioned rats (P < 0.05 for all comparisons). The pressor responses produced by AVP were associated with pronounced falls in HR that were similar in sham- and AV3V-lesioned rats (P > 0.05 for both comparisons). KA, NMDA, and AVP did not affect HR in ganglion-blocked rats before or after administration of AVPX (P > 0.05 for all responses, data not shown).

	DISCUSSION

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The present study confirms that the resting MAP values of AV3V-lesioned rats are not different from those of sham-lesioned rats (17). The principal finding of this study was that the pressor responses produced by KA and NMDA were markedly smaller in AV3V-lesioned rats compared with sham-lesioned rats. In naive rats, the pressor responses produced by the EAA analogs are mainly caused by an increase in sympathetic nerve activity and, to a lesser extent, the release of AVP (14, 19, 20). Previous studies have reported that the resting plasma osmolalities of AV3V-lesioned rats are higher than those of sham-lesioned rats (1, 17). This finding is consistent with the established role of the AV3V region in the regulation of fluid and salt homeostasis (1, 2). As such, it is possible that the hyperosmolality or other nonspecific effects of AV3V lesions may suppress peripheral vascular responsiveness to sympathetic activity or circulating catecholamines. However, the pressor responses produced by systemic injections of norepinephrine and the ₁-adrenoceptor agonist phenylephrine are not affected by lesions of the AV3V region (2, 3). This suggests that the diminished pressor responses produced by the EAA analogs in AV3V-lesioned rats were not caused by a loss of vasoconstrictor potency of neurogenic- or adrenal-derived catecholamines. However, although the maximal pressor responses to AVP were similar in sham- and AV3V-lesioned rats, the total increases in MAP were slightly smaller in AV3V-lesioned rats (see below). As such, the diminished vasoconstrictor effectiveness of AVP may have contributed to the diminished response to KA and NMDA in AV3V-lesioned rats. Taken together, these findings suggest that the AV3V region plays a vital role in the increases in sympathetic nerve activity produced by systemically injected EAA analogs.

The present study did not establish the mechanisms by which AV3V lesions diminish the pressor responses produced by the EAA analogs. Systemically injected EAA analogs may activate sympathetic nerve activity by the inhibition of baroreceptor reflex function (23), inasmuch as the application of KA to the isolated rabbit carotid sinus markedly reduces the responses of carotid baroreceptor afferents to graded increases in carotid sinus pressure (7). Accordingly, AV3V lesions may prevent the centrally mediated activation of preganglionic sympathetic motor nerves in response to the inhibition of the baroreceptor reflex. It is also possible that systemically injected EAA analogs may increase sympathetic nerve activity by direct activation of EAA receptors in the AV3V region (5) and especially the SFO and OVLT, which are free of a blood-brain barrier (10). Whatever the mechanism, the present findings are consistent with the evidence that the AV3V region plays an important role in the expression of the pressor response to a variety of centrally (11, 21) and systemically injected compounds (2, 3, 6, 17) and that AV3V lesions attenuate the development of various forms of experimental hypertension, including those dependent on an increase in sympathetic drive (1, 2, 8, 22).

The present study confirms that the pressor responses produced by the systemic injection of EAA analogs in conscious ganglion-blocked rats are abolished by a V₁ AVP-receptor antagonist (14, 19, 20). The finding that the AVP-induced pressor response was not associated with a fall in HR in CLX-treated rats suggests that the dose of CLX effectively abolished ganglionic transmission. Moreover, the dose of AVPX used in this study completely abolished the substantial increases in MAP produced by AVP. Taken together, these findings suggest that the systemic injection of EAA analogs causes the release of AVP from the pituitary. The pronounced pressor response to KA and NMDA in ganglion-blocked sham- and AV3V-lesioned rats is probably caused by the exaggerated effects of AVP in these rats (see Fig. 3). It is possible that the blood-borne EAAs directly release AVP by the stimulation of glutamate receptors in the pituitary (28). However, the pressor responses produced by the EAA analogs were markedly diminished in ganglion-blocked AV3V-lesioned rats, which suggests that systemically injected EAA analogs do not directly release AVP from the pituitary. Rather, these results suggest that EAA analogs promote the centrally mediated release of AVP and that the AV3V region plays a critical role in this process. The AVP-mediated pressor response produced by KA in CLX-treated rats was completely abolished by the AV3V lesions. In contrast, NMDA produced a small but significant increase in MAP. This suggests that the AV3V region has a more important role in the expression of the cardiovascular effects of KA than NMDA.

An important finding of this study was that pressor activity of AVP was smaller in AV3V-lesioned rats than in sham-lesioned rats. Before ganglion blockade, AVP produced a pressor response in AV3V-lesioned rats that was similar in magnitude to that in sham-lesioned rats. However, the total pressor responses were smaller in AV3V-lesioned rats. After ganglion blockade, the magnitude of the AVP-induced pressor response was less in AV3V-lesioned than in sham-lesioned rats. The total pressor response was also less in AV3V-lesioned than in sham-lesioned rats. These results suggest that the pressor responses to systemically injected AVP are dependent, in part, on the integrity of the AV3V region. We have not determined the mechanisms underlying the loss of response to AVP in AV3V-lesioned rats, although we have found that circulating levels of AVP are similar in sham- and AV3V-lesioned rats (9). It is possible that AVP-mediated changes in autonomic nerve activity are dependent on the AV3V region (1, 2). Moreover, it is possible that the changes in plasma osmolality or other undetermined effects of AV3V lesions may cause the downregulation of AVP receptors in vascular smooth muscle. Because KA did not produce a pressor response in ganglion-blocked AV3V-lesioned rats, it is unlikely that the loss of response to AVP completely accounts for the absence of response to KA. However, NMDA produced a minor increase in MAP in ganglion-blocked AV3V-lesioned rats. The loss of response to AVP in AV3V-lesioned rats may have contributed to the diminished responses to NMDA.

The KA- and NMDA-induced pressor responses were associated with a tachycardia. This tachycardia is mainly a result of an increase in cardiac sympathetic nerve activity (14, 19, 20). The increases in HR produced by KA and NMDA were substantially smaller in the AV3V-lesioned compared with the sham-lesioned rats. However, these results are difficult to interpret because the resting HR values of the AV3V-lesioned rats were higher than those of the sham-lesioned rats. The KA- and NMDA-induced tachycardia was followed by a sustained bradycardia. This bradycardia is mainly a result of an increase in cardiovagal activity (14, 19, 20). In addition, this bradycardia occurs when the KA- and NMDA-induced pressor responses have completely subsided (14, 19, 20), which suggests that the bradycardia is not caused by activation of the baroreceptor reflex. The KA- and NMDA-induced bradycardia was substantially smaller in AV3V-lesioned compared with sham-lesioned rats. The mechanisms by which KA and NMDA produce a vagally mediated bradycardia have not been established. However, the present findings suggest that the falls in HR produced by the EAA analogs are dependent on the integrity of the AV3V region.

In summary, the pressor responses produced by systemic injection of KA and NMDA were diminished in AV3V-lesioned rats. These findings provide further evidence that the AV3V region regulates sympathetic nerve activity and AVP release (1, 2). In sham-lesioned rats, the KA- and NMDA-induced pressor responses are caused by an increase in sympathetic nerve activity and the release of AVP. The smaller pressor responses produced by the EAA analogs in AV3V-lesioned rats may be caused by a diminished increase in sympathetic nerve activity and a minimal release of AVP. Lesions of the AV3V region also attenuated the vagally mediated decreases in HR produced by KA and NMDA. Taken together, it appears that the AV3V region plays a role in the expression of the cardiovascular responses produced by systemic injections of EAA analogs.