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Am J Physiol Regul Integr Comp Physiol 285: R1231-R1239, 2003. First published July 24, 2003; doi:10.1152/ajpregu.00028.2003
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NEUROHUMORAL CONTROL OF CIRCULATION AND HYPERTENSION

Responses to GABA-A receptor blockade in the hypothalamic PVN are attenuated by local AT1 receptor antagonism

Qing Hui Chen and Glenn M. Toney

Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900

Submitted 21 January 2003 ; accepted in final form 17 July 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Blockade of GABA-A receptors in the hypothalamic paraventricular nucleus (PVN) has been repeatedly shown to increase arterial blood pressure (ABP), heart rate (HR), and sympathetic nerve activity (SNA), but the mechanism(s) that underlies this response has not been determined. Here, we tested whether full expression of the response requires activation of local ANG II AT1 receptors. ABP, HR, and renal SNA responses to PVN microinjection of bicuculline methobromide (BIC; 0.1 nmol) were recorded before and after microinjection of vehicle (saline); losartan (or L-158809), to block local AT1 receptors; or PD123319, to block AT2 receptors. After PVN microinjection of vehicle or PD123319 (10 nmol), BIC significantly (P < 0.05) increased mean arterial pressure (MAP), HR, and renal SNA. However, PVN microinjection of 2 and 20 nmol of losartan dose dependently reduced responses to PVN-injected BIC, with the 20-nmol dose nearly abolishing MAP (P < 0.005), HR (P < 0.05), and renal SNA (P < 0.005) responses. Another AT1 receptor antagonist, L-158809 (10 nmol), produced similar effects. Neither losartan nor L-158809 altered baseline parameters. Responses to PVN injection of BIC were unchanged by losartan (20 nmol) given intravenously or into the PVN on the opposite side. MAP, HR, and renal SNA responses to PVN microinjection of L-glutamate (10 nmol) were unaffected by PVN injection of losartan (20 nmol), indicating that effects of losartan were not due to nonspecific depression of neuronal excitability. We conclude that pressor, tachycardic, and renal sympathoexcitatory responses to acute blockade of GABA-A receptors in the PVN depend on activation of local AT1 receptors.

bicuculline methobromide; angiotensin II; sympathetic nerve activity; arterial pressure; paraventricular nucleus

{gamma}-AMINOBUTYRIC ACID (GABA) is the dominant inhibitory neurotransmitter in the mammalian brain. One region where GABA plays a key role in regulating cardiovascular function is the hypothalamic paraventricular nucleus (PVN). Here, GABAergic terminals account for nearly 50% of all synapses (10), and ongoing GABAergic activity results in a high level of functional inhibitory "tone." The latter is evident from numerous studies in which local GABA-A receptor blockade has been shown to produce dramatic increases in arterial blood pressure (ABP), heart rate (HR), and sympathetic nerve activity (SNA) in conscious and anesthetized rats (24, 25, 38, 39).

The fact that acute blockade of GABA-A receptors in the PVN produces prompt cardiovascular and autonomic responses indicates that a source of excitation is also present that is capable of increasing action potential discharge in PVN autonomic neurons. This is the case, because inhibition of neuronal discharge by GABA-A receptor activation results from membrane hyperpolarization caused by an increase in Cl- conductance through the GABA-A receptor channel (12). Consequently, GABA-A receptor blockade by itself only reduces membrane Cl- conductance and thus only removes GABA-mediated membrane hyperpolarization. In the absence of convergent excitation, GABA-A receptor blockade would not be expected to depolarize neuronal membrane potential to the threshold for spike activation. Thus the goal of the present study was to identify a source of excitation that contributes to responses produced by GABA-A receptor blockade in the PVN.

On the basis of neurochemical data, it is clear that a number of excitatory neurotransmitters impinge on the PVN that could serve as a source of local excitation. These include such classical transmitters as glutamate (18), acetylcholine (29), and norepinephrine (16) as well as neuropeptides like corticotrophin-releasing factor, met-enkephalin, and angiotensin II (ANG II) (14, 23, 27). Although many candidate sources of excitation are present in the PVN, this study focused on the role of ANG II in the response to removal of GABAergic inhibition. This was prompted by evidence that ANG II-containing inputs to PVN arise from important cardiovascular and body fluid regulatory regions of the forebrain lamina terminalis (23, 26) and by electrophysiological studies, which indicate that actions of ANG II in the PVN contribute to increased neuronal discharge in response to elevated plasma ANG II (22). What is not yet clear is whether sympathetic-regulatory neurons of the PVN receive tonic ANG II inputs and whether these contribute to ongoing PVN neuronal activity (7).

Nevertheless, functional studies have demonstrated that physiological (11) and pathological (40) conditions that increase circulating ANG II also increase PVN neuronal activity (40) as well as the SNA response to acute PVN GABA-A receptor blockade (11). Collectively, these data raise the possibility that interactions between ANG II and GABA could participate in establishing PVN neuronal excitability. That GABA-A (10, 13, 15, 17) and AT1 receptors (28, 32, 34) are both expressed in autonomic regions of the PVN is consistent with this possibility. The present study investigated local ANG II-GABA interactions by recording pressor, tachycardic, and renal sympathoexcitatory responses to acute GABA-A receptor blockade in the PVN before and after local administration of ANG II AT1 and AT2 receptor antagonists. Some of these results have been presented in abstract form (5).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Experiments were performed in 32 male Sprague-Dawley rats (350-450 g) anesthetized with a mixture of {alpha}-chloralose (80 mg/kg) and urethane (800 mg/kg) given intraperitoneally. An adequate depth of anesthesia was indicated by the absence of pedal and corneal reflexes. An arterial catheter was inserted into the aorta via a femoral artery and was connected to a pressure transducer to measure ABP. HR was obtained from the R-wave of the ECG (lead I). The left femoral vein was also catheterized and used to administer drugs. After tracheal cannulation, rats were paralyzed with gallamine triethiodide (25 mg·kg-1·h-1 iv) and artificially ventilated with oxygen-enriched room air. After paralysis, anesthesia was monitored by the stability of HR and ABP, and supplements equal to 10% of the initial dose were given when needed. End-tidal PCO2 was continuously monitored and maintained within normal limits (35-40 mmHg) by adjusting ventilation rate (80-100 beats/min) and/or tidal volume (2.0-3.0 ml). Body temperature was held at 37°C with a water-circulating pad. All experimental and surgical procedures were approved by the Institutional Animal Care and Use Committee of The University of Texas Health Science Center at San Antonio.

Recording renal SNA. A flank incision was made to expose the left kidney. A renal nerve bundle was isolated from surrounding tissue, mounted on a stainless steel wire electrode (A-M systems, 0.127 mm outer diameter), and covered with a silicon-based impression material (Coltene, light body) to insulate the recording from body fluids. The recorded signal was directed to an alternating current amplifier equipped with half-amplitude filters (band pass: 100-1,000 Hz) and a 60-Hz notch filter. The processed signal was rectified, integrated (30-ms time constant), and digitized at a frequency of 1,000 Hz and stored on computer disk.

Microinjection of drugs. Animals were placed in a stereotaxic head frame, and the skull was leveled between bregma and lambda. A section of skull was removed, and a single-barreled glass micropipette was lowered vertically into the left PVN using a piezoelectric microdrive (Burleigh Instruments). The following stereotaxic coordinates were used (in mm): caudal to bregma, 1.6-2.0; lateral to midline, 0.5-0.7; and ventral to dura, 7.0-7.5. The final position of the microinjector was the site that produced a pressor response to bicuculline methobromide (BIC) of >=15 mmHg and was typically located on the initial placement or with only one adjustment to a deeper (50-100 µm) site (8). Injected compounds included the GABA-A receptor antagonist BIC (0.1 nmol), the AT1 receptor antagonist losartan (2 or 20 nmol) or L-158809 (10 nmol), and the AT2 receptor antagonist PD123319 (10 nmol). Vehicle (pH-buffered saline) and AT receptor antagonist compounds were injected at two sites located ~0.1 mm rostral and caudal to each BIC microinjection site to ensure more complete and uniform coverage. Drugs were injected over a period of ~2 min in a volume of 100 nl per site with a pneumatic pump (WPI).

Experimental protocol. Animals were allowed to stabilize for 1 h after surgery, at which time the response to PVN-microinjected BIC was tested. ABP, HR, and renal SNA were allowed to rise to a maximum (~5-10 min) before removal of the injector pipette. Recorded variables typically returned to baseline within 30 min. Next, a glass pipette containing the AT1 receptor antagonist losartan was lowered into the PVN and, depending on the experiment, either a 2- or 20-nmol dose was injected. The BIC microinjector was then reintroduced into the PVN, and responses were tested again 20 and 120 min later. To ensure that effects of losartan were due to its action at AT1 receptors, separate experiments were performed using a second, chemically distinct AT1 receptor antagonist, L-158809. In a separate group of animals, the role of local AT2 receptors in mediating responses to PVN-microinjected BIC was tested by recording responses before and after PVN injection of the AT2 receptor antagonist PD123319. To control for possible nonspecific effects of the injected volume, vehicle injections of saline were performed, and effects on ABP, HR, and renal SNA responses to BIC injected into the PVN were determined. To determine whether effects of losartan could be attributed to a peripheral site of action, i.e., following leakage into the blood, 20 nmol of losartan was delivered intravenously and effects on the pressor, tachycardic, and renal sympathoexcitatory response to PVN-microinjected BIC were determined. To control for possible effects of losartan caused by its diffusion out of the PVN, effects of losartan injected into the PVN were tested on responses to BIC delivered into the contralateral PVN. Finally, to further exclude the possibility of nonspecific actions of losartan to reduce neuronal excitability, effects of PVN-microinjected losartan were tested on responses evoked by L-glutamate (10 nmol; Sigma) injected into the PVN.

Histology. To mark the microinjection site, the final injection of BIC was dissolved in vehicle containing Chicago Sky Blue dye (2%). Brains were removed and postfixed for 24 h at 4°C in 0.1 M phosphate-buffered saline containing 4% paraformaldehyde. Tissue containing the hypothalamic PVN was cut into 40-µm-thick coronal sections, and microinjection sites were identified under bright-field microscopy.

Data analysis. Mean arterial pressure (MAP) was determined by adding one-third of the pulse pressure to the diastolic pressure. Systolic and diastolic pressures were determined from the raw ABP signal. Renal SNA was determined as an average of the rectified integrated signal. Baseline values were obtained by averaging over a 3-min period immediately before each treatment. MAP, HR, and renal SNA responses to BIC in the PVN were measured by averaging a 60-s period centered on the maximal response. MAP, HR, and renal SNA responses to BIC injected into the PVN were measured before, 20 min after, and 120 min after microinjection of vehicle, losartan, L-158809, or PD123319 into the PVN.

Responses to BIC in the PVN were compared before and after each microinjected compound by use of a repeated measures analysis of variance (ANOVA). For analyses that yielded a significant interaction, pair-wise comparisons were made using the Bonferroni multiple comparison test. To ensure that recorded variables returned to baseline between treatments, baseline values were similarly compared. Summary data in the text and figures are expressed as means ±SE. Differences were considered statistically significant at a critical value of P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Histological examination of brain tissue revealed that microinjector tips were consistently lowered to within 50-100 µm of the dorsal border of the PVN. On the basis of dye diffusion (Fig. 1), microinjected volume uniformly impacted the dorsal cap region of the PVN and extended occasionally into the ventrolateral sub-nucleus without rupturing the wall of the third ventricle. Although the distribution of injected dye indicated that AT1 and AT2 receptor antagonists may have spread to sites located slightly (~50 µm) rostral and/or caudal to the PVN, this did not appear to account for any observed efficacy, since treatment with antagonists was equally effective in instances where the injected volume was confined within the boundaries of the PVN. In addition, AT1 receptor antagonists microinjected deliberately at sites rostral, caudal, and lateral to PVN did not significantly alter pressor and renal SNA responses to BIC injected into the PVN (data not shown).



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  		Fig. 1. Schematic drawings of the rat hypothalamus in coronal section. Shaded areas indicate regions of the hypothalamus exposed to dye coinjected with bicuculline methobromide (BIC). The shape of each area was determined by tracing the outline of the dye observed on each 40-µm-thick section through the paraventricular nucleus (PVN). Then, tracings of the outermost distribution of dye were made by overlying areas from similar rostral-caudal sections taken from different brains. Thus the areas shown are larger than the dye distribution observed in any single brain, but represent the widest possible distributions of injectate for the entire group of BIC-treated animals. Note that injections were largely confined to the PVN without significant invasion of the perifornical, anterior, or dorsomedial hypothalamic areas. AH, anterior hypothalamic area; 3V, third cerebral ventricle; f, fornix; RCh, retrochiasmatic area.

 

Effect of PVN-microinjected compounds on MAP, HR, and renal SNA. Microinjection into the PVN of either vehicle (saline), the AT1 receptor antagonist losartan or L-158809, or the AT2 receptor antagonist PD123319 failed to acutely alter resting values of MAP, HR, and renal SNA (Table 1). Recorded variables were unchanged at 20 min postinjection of vehicle, losartan, and PD123319 and remained stable throughout the 120-min postinjection period. In the case of treatment with L-158809, recorded variables were unchanged 20 min after injection, with HR and renal SNA remaining stable throughout the 120-min experimental period. A small but significant fall in MAP (P < 0.05) was noted in this group at the 120-min time point. Overall, data indicate that the preparation was stable and remained responsive throughout the experimental period.


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  		Table 1. Effect of PVN-microinjected compounds on resting MAP, HR, and renal SNA

 

Effect of AT1 and AT2 receptor blockade in the PVN on responses to PVN-microinjected BIC. Microinjection of BIC (0.1 nmol) into the PVN significantly (P < 0.05) increased MAP, HR, and renal SNA. Vehicle treatment did not alter either the magnitude or the time course of BIC-evoked responses (Fig. 2), which began within ~20 s, reached a peak within 5-10 min, and returned gradually to baseline within 20-30 min.



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  		Fig. 2. Arterial blood pressure (ABP; bottom), renal sympathetic nerve activity (renal SNA; middle), and heart rate (HR; top) responses to microinjection of the GABA-A receptor antagonist BIC into hypothalamic PVN before (control), 20 min (20' post), and 120 min (120' post) after injection of saline vehicle into the ipsilateral PVN. Each 100-nl injection was performed over a period of ~2 min. Note that ABP, renal SNA, and HR were each markedly increased after each administration of BIC into the PVN. Compared with the control response (left), the magnitude of BIC-evoked responses was unaltered by prior vehicle injection (middle) and did not significantly change with time (right). All tracings were recorded in the same animal. bpm, Beats/min.

 

The role of local AT1 receptors in the response to BIC in the PVN was examined by testing responses before and after delivering the AT1 receptor-selective antagonist Losartan into the ipsilateral PVN. Figure 3 shows an example of the response to BIC injected into the PVN after microinjection of two different doses of losartan: 2 and 20 nmol. Note that the 2-nmol dose appears to reduce responses to BIC (Fig. 3A), whereas responses were nearly prevented by the 20-nmol dose (Fig. 3B). Summary data in Fig. 4 indicate that although MAP (34 ± 5 to 30 ± 3 mmHg), HR (32 ± 9 to 23 ± 5 beats/min), and renal SNA (68 ± 14 to 34 ± 7% increase) responses to BIC injected into the PVN (n = 4) tended to be reduced 20 min after microinjecting a 2-nmol dose of losartan, effects did not reach statistical significance. In a separate group of six animals, the 20-nmol dose of losartan significantly attenuated ABP (26 ± 2 to 3 ± 3 mmHg; P < 0.005), HR (21 ± 5 to 1 ± 3 beats/min; P < 0.05), and renal SNA (60 ± 3 to 9 ± 4% increase; P < 0.005) responses to PVN-microinjected BIC. Also shown in Fig. 4 are data from five other animals in which PVN microinjection of L-158809, a chemically distinct AT1 receptor antagonist, similarly reduced BIC-evoked increases in MAP (36 ± 2 to 11 ± 1 mmHg; P < 0.005), HR (35 ± 8 to 17 ± 6 beats/min; P < 0.05), and renal SNA (62 ± 8 to 16 ± 6% increase; P < 0.005). Responses to BIC in the PVN recovered to ~80% of the control value within 120 min. Figure 5, left, shows that the same dose of losartan that effectively eliminated BIC-evoked responses when injected into the PVN was without significant effect when administered intravenously (MAP, 23 ± 5 vs. 32 ± 8 mmHg; HR, 14 ± 0.5 vs. 11 ± 2 beats/min; renal SNA, 63 ± 16 vs. 89 ± 26% increase)(n = 3). Figure 5, middle, also demonstrates that pressor (38 ± 11 to 38 ± 4 mmHg), tachycardic (36 ± 10 to 37 ± 3 beats/min), and renal sympathoexcitatory (72 ± 2 to 65 ± 4% increase) responses to BIC injection in the PVN were unaltered when BIC and losartan were injected on opposite sides (n = 3). Combined, these data indicate that effects of losartan were specific to the PVN and were not likely due to leakage into the peripheral circulation. That PVN injection of losartan (20 nmol) failed to alter the increase in either MAP (9 ± 2 vs.7 ± 1 mmHg), HR (12 ± 2 vs. 10 ± 2 beats/min), or renal SNA (28 ± 3 vs. 24 ± 5% increase) produced by PVN microinjection of L-glutamate (10 nmol, n = 3) indicates that the efficacy of losartan was also not due to a generalized reduction in neuronal excitability (Fig. 5). In parallel experiments (n = 3), effects of PVN microinjection of the AT2 receptor antagonist PD123319 (10 nmol) were tested. This treatment had no effect on either baseline parameters or on BIC-evoked increases in MAP (31 ± 5 to 31 ± 5 mmHg), HR (21 ± 3 to 22 ± 5 beats/min), or renal SNA (63 ± 7 to 58 ± 6% increase).



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  		Fig. 3. Effect of prior microinjection of the AT1 receptor antagonist losartan into the hypothalamic PVN on ABP (bottom), renal SNA (middle), and HR (top) responses to microinjection of the GABA-A receptor antagonist BIC into the ipsilateral PVN. Note that ABP, renal SNA, and HR each increased markedly after control PVN administration of BIC (A and B, left). A: responses to PVN-injected BIC were reduced after delivery of a 2-nmol dose of losartan into the PVN. B: a 20-nmol dose of PVN-injected losartan nearly abolished ABP, renal SNA, and HR responses to subsequent microinjection of BIC. Losartan effects were maximal within ~20 min, and responses to PVN-injected BIC nearly recovered within 120 min of losartan microinjection. Each 100-nl injection was performed over a period of ~2 min. Tracings in A and B were recorded in 2 different animals.

 


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  		Fig. 4. Group data showing the average increase ({triangleup}) in mean arterial pressure (MAP; bottom), renal SNA (middle), and HR (top) in response to microinjection of BIC into the hypothalamic PVN. Responses before (control) and 20 min and 120 min after ipsilateral PVN microinjection of saline vehicle (n = 5), 2 nmol losartan (n = 4), 20 nmol losartan (n = 6), and 10 nmol L-158809 (n = 5) are shown. Values are means ± SE. *P < 0.05 vs. baseline. **P < 0.05 vs. control. {dagger}P < 0.005 vs. control.

 


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  		Fig. 5. ABP (bottom), renal SNA (middle), and HR (top) responses to microinjection of BIC into the hypothalamic PVN before (open bars) and 20 min after (solid bars) losartan (20 nmol), administered either intravenously (left; n = 3) or into the PVN on the side contralateral to the BIC injection site (middle; n = 3). Pressor, renal sympathoexcitatory, and tachycardic responses to PVN microinjection of L-glutamate were unaffected by prior ipsilateral PVN microinjection of losartan (20 nmol)(right; n = 3).

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Acute blockade of GABA-A receptors in the hypothalamic PVN was shown in the present study to produce significant and highly reproducible increases in ABP, HR, and renal SNA (24, 25, 38, 39). The method used to activate PVN neurons was disinhibition by local microinjection of the GABA-A receptor antagonist BIC. Tissue distribution of BIC was estimated by determining the spread of coinjected dye. On this basis, it appears that injection sites were largely confined to the PVN. It should be noted, however, that although the distributions of dye and BIC likely overlap, they might not be identical. Still, the conclusion that drug effects were confined to the PVN is consistent with results showing that BIC injections deliberately placed lateral to the PVN failed to elicit characteristic cardiovascular and sympathetic responses (8, 38). This is important because GABA-A receptor blockade in the adjacent dorsomedial hypothalamus has been reported to increase arterial pressure and sympathetic activity (37). Effects of BIC also were not likely due to leakage into the peripheral circulation, since previous studies reported that direct intravenous injection of a fivefold higher dose of BIC failed to alter ongoing MAP or renal SNA (38).

In the present study, we postulated that ANG II-containing inputs to the PVN might be an important source of excitatory drive that contributes to neuronal activation during GABA-A receptor blockade. Consistent with this hypothesis, responses to BIC injection into PVN were significantly attenuated by prior blockade of local AT1, but not AT2, receptors. It should be emphasized that losartan did not attenuate responses to BIC injected into the PVN by an action outside the nucleus, since the same dose of losartan injected systemically or into the contralateral PVN was without effect. Still, it should be mentioned that effects of AT1 receptor antagonists in the present study were observed at higher doses than used in some other studies (20, 33). For example, Tagawa and Dampney (33) reported that a 1-nmol dose of losartan or L-158809 injected into the rostral ventrolateral medulla (RVLM) attenuated both the renal sympathetic and cardiovascular response to BIC injected into the PVN. It is important to emphasize, however, that the efficacy of losartan in the RVLM and the PVN may be different such that dose effects in their study and ours may not be directly comparable. Moreover, data reported in their study were limited to a 1-nmol dose, and this only partially blocked responses to BIC injected into PVN. Whether a larger dose would have been more effective is not known. In the present study, graded doses of losartan (2 and 20 nmol) were used that produced dose-dependent effects, and the 20-nmol dose nearly abolished both the renal sympathetic and cardiovascular response to BIC injected into the PVN.

Of course, dose-dependent inhibitory effects cannot entirely rule out nonselective actions that lead to neuronal depression. To address this, we used both losartan and L-158809, which are chemically distinct receptor antagonists whose only known common pharmacological property is antagonism of AT1 receptors (4, 30). These two drugs produced similar effects, thereby supporting the conclusion that AT1 receptor activation likely contributes to responses evoked by BIC injected in the PVN. Similarly, experiments showed that PVN injection of losartan failed to alter cardiovascular and sympathetic responses to L-glutamate delivered into the PVN, again indicating that actions were not due to a nonspecific reduction in PVN neuronal excitability. Taken together, data from the present study support the conclusion that blockade of AT1 receptors in the PVN effectively attenuates responses to subsequent blockade of local GABA-A receptors.

It is important to note that effects of GABA-A receptor blockade were diminished in the present study when losartan and BIC were injected into the same PVN site (see Figs. 3 and 4). In contrast, responses to injected BIC were unaltered when losartan was delivered into the PVN on the opposite side (Fig. 5). These findings suggest that responses to acute GABA-A receptor blockade in the PVN involve ANG II activation of local AT1 receptors. It should also be emphasized that the highest dose of losartan used in this study (20 nmol) effectively abolished BIC-evoked responses, thereby suggesting that local ANG II actions may not merely contribute to the response but might be required for suprathreshold depolarization and PVN spike activation during GABA-A receptor blockade. Clearly, intracellular electrophysiological recording studies are needed to fully test this possibility.

Although the present study provides evidence that AT1 receptors play a role in mediating cardiovascular and sympathetic responses to PVN neuronal disinhibition by GABA-A receptor blockade, tonic actions of ANG II in the PVN do not appear to support resting arterial pressure and SNA. This conclusion is supported by data from the present study showing that losartan injected into PVN by itself failed to alter resting ABP, HR, or renal SNA. This is reminiscent of studies showing that blockade of AT1 receptors in the RVLM also has no effect on resting ABP or SNA (9, 20). Whether the lack of a resting effect of losartan injected into the PVN and RVLM reflects a lack of tonic ANG II input to these important sympathetic control regions is not presently known. Still, data indicate that under conditions such as heart failure and hypertension, actions of ANG II in the PVN (40) and RVLM (9, 20) appear to contribute to the tonic neuronal activity. Thus available evidence indicates that ANG II in the PVN and RVLM may subserve similar sympathetic regulatory functions, and future studies will be needed to further test this possibility.

What is clear from the present results is that the lack of a baseline effect of PVN-injected losartan cannot be explained by depolarizing actions of its counter ion potassium (30), since resting parameters were also unaltered by L-158809, which is not a potassium salt (4). It should be emphasized that the lack of effect of acute AT1 receptor blockade in the PVN on baseline parameters does not necessarily mean that ANG II inputs to the PVN are tonically inactive. For example, it is possible that tonic actions of ANG II in the PVN could be masked either by the dominance of local GABAergic inhibition or by downstream synaptic influences. As noted above, the former possibility will require that intracellular electrophysiological studies be performed. The latter possibility is supported indirectly by evidence showing that forebrain-directed intracarotid artery injection of the angiotensin-converting enzyme inhibitor captopril significantly reduces ongoing renal SNA (35). This effect was reported to occur only in sinoaortic baroreceptor-denervated animals, indicating that baroreceptor reflex buffering could mask tonic excitation mediated by the brain renin-angiotensin system (RAS) (6, 35). Whether excitatory effects of brain RAS involve ANG II actions in the PVN remains to be fully established, although available evidence supports this possibility (14, 22, 26).

Another alternative explanation for the lack of effect of losartan injected into PVN on resting parameters is provided by in vitro electrophysiological studies showing that PVN neurons receive tonic inhibitory input from local GABAergic neurons (2, 3, 21). Indeed, evidence indicates that autonomic regions of the PVN are targeted by both ANG II- (14, 23) and GABA-containing fibers (3, 31) and express both AT1 (28, 32, 34) and GABA-A (10, 13, 15, 17) receptors. This raises the possibility that ANG II could have excitatory effects on both output neurons of the PVN and local inhibitory neurons. Under conditions where excitatory and inhibitory effects in the PVN are balanced, AT1 receptor blockade by itself could fail to significantly alter the net activity of PVN output neurons.

One mechanism whereby excitatory and inhibitory influences could be maintained in relative balance has been suggested by recent in vitro electrophysiological studies (2, 19, 21). Indeed, Latchford and Ferguson (21) have shown that ANG II activates a local "inhibitory feedback" circuit in the PVN. This was demonstrated by observing that ANG II not only excites magnocellular neurons but also produces a concurrent increase in the frequency of inhibitory postsynaptic potentials in the same cell. Arginine vasopressin (19) and N-methyl-D-aspartate (NMDA) receptor activation (2) has been reported to produce similar effects. Interestingly, depolarization responses to ANG II were shown to be enhanced during blockade of GABA-A receptors, suggesting that ANG II activates a local system of GABAergic neurons that "feed back" on the ANG II-responsive cell. That local GABAergic interneurons are involved was demonstrated by showing that treatment with tetrodotoxin to prevent action potential formation eliminated the ability of ANG II to increase miniature inhibitory postsynaptic currents (21). What remains to be determined is whether such a local inhibitory system governs the activity of sympathetic regulatory neurons in the PVN (7). Likewise, it is not clear whether such a system dampens neuronal responses to tonic ANG II input, thereby contributing to the failure of local AT1 receptor blockade to alter resting ABP, HR, and renal SNA as observed in the present study. This important issue warrants additional study.

Future studies also should take into account electrophysiological data indicating that, like PVN neuronal responses to ANG II, depolarization by NMDA receptor activation is also augmented during reduced GABA-A receptor tone (2). These data are consistent with recent findings from our laboratory showing that cardiovascular and renal sympathetic responses to PVN-injected BIC are dramatically reduced by prior blockade of local NMDA/nonNMDA receptors (8). Overall, it appears from available evidence that both ANG II and excitatory amino acid inputs to the PVN activate GABAergic interneurons, and thus both inputs appear capable of "autoregulating" PVN neuronal excitability.

In conclusion, the present study demonstrates that blockade of ANG II AT1 receptors in the PVN is without effect on resting MAP, HR, and renal SNA but significantly reduces pressor, tachycardic, and renal sympathoexcitatory responses to PVN GABA-A receptor blockade. To gain a more complete understanding of the regulatory processes that govern PVN autonomic function, studies are needed to determine the source of ANG II input and to clarify the organization of local neuronal networks that control PVN neuronal responses to removal of GABA-A receptor tone.

Perspectives

The hypothalamic PVN is increasingly recognized as an important contributor to cardiovascular function both under normal conditions and in disease. Because GABA is the dominant inhibitory neurotransmitter in the PVN, a popular hypothesis is that reduced GABAergic control of PVN sympathetic regulatory neurons underlies the increase in SNA observed in diseases such as hypertension (1, 25) and heart failure (38). To explore this hypothesis more fully, it is critical to understand the cellular and integrative mechanisms by which such plasticity contributes to PVN neuronal excitability and sympathetic drive. It is necessary, therefore, to establish how transmitter interactions regulate the strength of GABAergic control. This study focused on possible interactions with ANG II in mediating the response to acute GABA-A receptor blockade. Although the present data suggest that ANG II plays a vital role in determining the suprathreshold activity of PVN neurons during GABA-A receptor blockade, it is important to emphasize that local treatment with AT1 receptor antagonists alone did not alter resting parameters of cardiovascular function, including renal SNA. Thus additional studies will be necessary to determine whether ANG II input to the PVN is tonically active (7) and whether this contributes to the level of sympatho-adrenal activity present under physiological conditions (11) or in diseases such as arterial hypertension (1, 17, 25, 36) and congestive heart failure (38, 40).


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This project was supported by National Heart, Lung, and Blood Institute Grant HL-56834 and was conducted during the tenure of an American Heart Association Established Investigator Award (0140161N) granted to G. M. Toney. Q. H. Chen was supported by a postdoctoral fellowship (0225072Y) from the American Heart Association, Texas Affiliate.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge M. Cato and B. Guler for excellent technical assistance. We also thank Drs. S. D. Stocker, L. P. La-Grange, and J. R. Haywood for useful discussions of this work and Dr. L. C. Daws for critical review of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. Toney, Dept. of Physiology-7756, The Univ. of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900 (E-mail: toney{at}uthscsa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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