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Enhanced angiotensin-mediated responses in the nucleus tractus solitarii of spontaneously hypertensive rats
Brain Research Bulletin 60 (2003) 209–214
Enhanced angiotensin-mediated responses in the nucleus tractus solitarii of spontaneously hypertensive rats Nobuhide Katsunuma, Kazuyoshi Tsukamoto∗ , Satoru Ito, Katsuo Kanmatsuse Second Department of Internal Medicine, Nihon University School of Medicine, 30-1, Oyaguchi-Kamichou, Itabashi-ku, Tokyo 173-8610, Japan Received 9 September 2002; received in revised form 16 December 2002; accepted 19 December 2002
Abstract Studies using an AT1 receptor antagonist, losartan, demonstrated that depressor and bradycardic responses to angiotensin II (Ang II) injection into the nucleus tractus solitarii (NTS) are mediated via those receptors. We further characterized Ang II-evoked cardiovascular responses in this nucleus in spontaneously hypertensive rats (SHR) using a new, selective AT1 receptor antagonist, valsartan. In ␣-chloralose-anesthetized Sprague–Dawley (S-D) rats, Wistar–Kyoto (WKY) rats, and SHR, unilateral injection of Ang II into the NTS decreased arterial pressure (AP) and heart rate (HR). This response was eliminated by preinjection of valsartan. Depressor responses were much greater in SHR than in WKY rats. In normotensive rats, bilateral valsartan injection did not alter baseline AP or HR, or baroreceptor reflex index (BRI) calculated as the maximal change in HR (bpm) divided by phenylephrine- or nitroprusside-induced maximal change in mean AP (mmHg). In SHR, this treatment did not alter baseline HR and BRI, but significantly increased AP. Preinjection of valsartan did not alter injected glutamate effects in any strain. Thus, stimulation of AT1 receptors within the NTS contributes to cardiovascular regulation independently of the baroreceptor reflex and the glutamatergic system. This angiotensinergic system in SHR acts tonically to reduce AP. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Angiotensin II; Valsartan; AT1 receptor; Nucleus tractus solitarii; SHR
1. Introduction The nucleus tractus solitarii (NTS), specifically the region in which baroreceptor afferent nerves terminate, plays an important role in cardiovascular control. The NTS contains a large variety of putative neurotransmitters, many of which have been reported to influence cardiovascular regulation. By autoradiographic methods, AT1 receptors have been shown to predominate within the NTS [2,9,11,14,20]. Further, previous studies in anesthetized rats [5,8] have shown that unilateral injection of angiotensin II (Ang II) into the NTS elicits a decrease in arterial pressure (AP) and heart rate (HR). Since these cardiovascular responses were prevented by prior injection of an AT1 receptor antagonist losartan into the NTS, the Ang II-evoked response may be mediated via AT1 receptors [8]. However, losartan also binds to a non-Ang II receptor site in rat liver and in other tissues [4]. When a different AT1 receptor antagonist, candesartan, was injected bilaterally into the NTS, baseline AP
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and HR showed little change, nonetheless, this treatment significantly increased sensitivity for baroreceptor reflex control of renal sympathetic nerve activity and HR [13]. On the other hand, the previous studies have suggested that the Ang II-evoked response is mediated at least in part by substance P using similar pharmacologic methods [5,17]. However, details of the angiotensinergic system within the NTS remain unclear. In the present study, we aimed to further characterize the effect of Ang II injected into the NTS using a new and highly selective AT1 receptor antagonist, valsartan. A previous study using genetically hypertensive rats (SHR) of the Okamoto strain determined that the depressor response to injection of Ang II into the caudal ventrolateral medulla (CVLM) was exaggerated in these animals [15]. Nevertheless, responses evoked by Ang II injection into the rostral ventrolateral medulla (RVLM) were similar in SHR and Wistar–Kyoto (WKY) rats [15]. In contrast, another study found that injection of a nonselective Ang II antagonist, sarthran, into the RVLM or CVLM enhanced responses in SHR [16], suggesting that Ang II-related inhibitory neurotransmission from CVLM to the RVLM may not be
0361-9230/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0361-9230(03)00013-3
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sufficient to offset the greater intrinsic pressor activity of the RVLM in SHR. Another previous study demonstrated an antagonistic Ang II/␣2 receptor interaction within the NTS in WKY rats that is altered in SHR [6]. These findings suggest that the differences in brainstem angiotensinergic systems may be related to hypertension in SHR, again indicating a need to further characterize the Ang II-evoked cardiovascular response in the NTS in SHR.
2. Materials and methods Experiments were conducted on adult male Sprague– Dawley (S-D) rats (300–480 g), WKY rats, and spontaneously hypertensive rats (SHR; 16–20 weeks; Charles River, Japan). Animals were housed in groups of 2–3 in hanging wire mesh cages in temperature-controlled rooms with a fixed 12 h/12 h light/dark cycle for at least 2 weeks prior to experiments. Food (MF; Oriental Yeast Co., Ltd) and tap water were available ad libitum. Rats were anesthetized with halothane (2% in 100% O2 , administered via a nasal cone). A cannula (PE 50 tubing filled with heparinized saline) was inserted into the right femoral artery for recording AP to determine mean arterial pressure (MAP) and HR (Grass Model 7 Physiograph). A second cannula was placed in the right femoral vein for administering drugs. The trachea was cannulated and the rats were artificially ventilated with 2% halothane in 100% O2 (Harvard Small Animal Respirator) following administration of a muscle relaxant (d-tubocurarine, 0.5 mg/kg, i.v., supplemented hourly with 0.2 mg/kg, i.v.). The rats were placed in a stereotaxic instrument (Kopf) with the incisor bar positioned 11 mm below the interaural line. The dorsal surface of the brainstem was exposed by limited craniotomy and, with the aid of a surgical microscope, the area postrema was visualized. Anesthesia with ␣-chloralose was administered (60 mg/kg, i.v., supplemented hourly with 20 mg/kg, i.v.), and halothane was discontinued. Rats were ventilated with 100% O2 throughout the remainder of the experiment. Injections of drug solutions or vehicle were made into the NTS using single-barrel glass micropipettes pulled to an outer diameter of 40–50 m. The tip of the micropipette was positioned 0.5 mm rostral to the calamus scriptorius, 0.5 mm lateral from the midline, and 0.5 mm deep with respect to the surface of the brainstem for injection into the NTS. All drugs were dissolved in artificial cerebrospinal fluid (aCSF) and delivered in a volume of 100 nl. A PicoPump was used for injections (WPI, New Haven, CT). The volume of drug injected into the NTS was carefully monitored by observing the movement of the fluid meniscus in the calibrated glass pipette. Bilateral injections were made one side at a time, with approximately 30 s separating the two injections. In experiments where one agent was injected followed by injection of another agent, the pipette was removed and replaced for the subsequent injection.
At the conclusion of each experiment, the animal was anesthetized with urethane (1.5 g/kg, i.v.) and perfused intracardially with saline followed by 10% buffered formalin. The brainstem was removed, frozen, and sectioned (40 m) using a microtome. Sections were affixed to glass slides and stained with cresyl violet. Only animals whose pipette tracks were centered in the medial subnucleus of the NTS at the level of the area postrema were included in the data analysis. Animals with evidence of bleeding in the NTS were excluded from data analysis. An injection including 100 nl of Fast Green dye was made into the NTS prior to sacrifice. Injection sites were similar to those in previous experiments. Valsartan was obtained from Novartis Pharma AG (Switzerland). All other chemicals used in these experiments were purchased from Sigma Chemical Co. (St. Louis, MO). Data are expressed as the mean ± S.E.M. Data were compared by analysis of variance (ANOVA) followed by Scheffe’s F test.
3. Results 3.1. Studies in S-D rats Unilateral injection of a 0.1- or 1-pmol dose of Ang II into the NTS of S-D rats anesthetized with chloralose elicited similarly significant depressor and bradycardic responses (0.1 pmol versus 1 pmol: −29 ± 2∗ mmHg and −20 ± 6∗ bpm versus −26 ± 4∗ mmHg and −25 ± 5∗ bpm, ∗ P < 0.05 versus baseline MAP and HR, n = 7). These cardiovascular responses were rapid in onset and lasted less than 2 min. Injection of vehicle did not significantly alter baseline MAP or HR (2 ± 1 mmHg and 0 ± 2 bpm, P > 0.1 versus baseline MAP and HR, n = 6). In contrast, a larger dose of Ang II (30 pmol) elicited a significantly smaller depressor and bradycardic response (change from baseline, −13 ± 3∗ mmHg and −12 ± 3∗ bpm, ∗ P < 0.05 versus baseline MAP and HR, n = 6) than the responses elicited by either 0.1- or 1-pmol doses of Ang II. To determine the ability of the AT1 receptor antagonist, valsartan, to effectively and selectively block the above action of Ang II, responses to a 0.1-pmol dose of Ang II or other depressor substances were tested 3–7 min after injection of valsartan or aCSF with no drugs as a control into the same site. Neither unilateral nor bilateral injection of valsartan (10 pmol) altered baseline MAP or HR (unilateral versus bilateral injection, changes of 1 ± 1 mmHg and 0 ± 0 bpm versus 5 ± 3 mmHg and 4 ± 5 bpm, P > 0.1 versus baseline MAP and HR, n = 6–8). A prior unilateral injection of valsartan (10 pmol) completely prevented the depressor and bradycardic response to Ang II (0.1 pmol; with previous aCSF as a control, the change with Ang II was −30 ± 2 mmHg and −20 ± 6 bpm; with prior valsartan, the change with Ang II was −3 ± 4∗ mmHg and −2 ± 2∗ bpm, ∗ P < 0.05 versus control, n = 6; Fig. 1). In contrast to the effects of prior valsartan (10 pmol) injection on responses
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Fig. 1. Effect of valsartan on depressor (A) and bradycardic responses (B) elicited by unilateral injection of glutamate (Glu) or angiotensin II (Ang II) into the NTS of S-D rats. Either Glu (150 pmol) or Ang II (0.1 pmol) significantly decreased the baseline MAP and HR approximately 3–7 min after pretreatment with aCSF (0.1 l) containing no drug. Depressor and bradycardic responses produced by Glu approximately 3–7 min after pretreatment with valsartan (10 pmol) in aCSF were similar to those after pretreatment with aCSF alone (after aCSF, −38 ± 3 mmHg and −29 ± 5 bpm; after valsartan, −32 ± 3 mmHg and −20 ± 2 bpm, n = 8). However, pretreatment with valsartan completely abolished the cardiovascular responses to Ang II seen after pretreatment with aCSF alone (after aCSF, −30 ± 2 mmHg and −20 ± 6 bpm; after valsartan, −3 ± 4∗ mmHg and −2 ± 2∗ bpm, ∗ P < 0.05 versus pretreatment with aCSF, n = 8).
elicited by Ang II, prior unilateral injection of valsartan (10 pmol) into the NTS did not alter depressor or bradycardic responses elicited by injection of glutamate (150 pmol) into the NTS (with previous aCSF as a control, −38 ± 3 mmHg and −29 ± 5 bpm; with prior valsartan, −32 ± 3 mmHg and −20 ± 2 bpm, P > 0.1 versus control, n = 7; Fig. 1). This dose of glutamate elicited similar depressor and bradycardic responses to those elicited by Ang II, representing a maximally effective dose of glutamate [22]. 3.2. Studies in SHR and WKY rats Baseline MAP in chloralose-anesthetized SHR rats was greater than in WKY rats (SHR versus WKY rats: 173 ± 9∗ mmHg versus 108 ± 4 mmHg, ∗ P < 0.05 versus WKY rats, n = 6), but HR was similar in both strains (SHR versus WKY rats: 347 ± 18 bpm versus 332 ± 12 bpm, P > 0.1 versus WKY rats, n = 6). Ipsilateral injection of Ang II (0.1 pmol) 3–7 min after unilateral injection of aCSF into the NTS in both strains elicited decreases in MAP and HR
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Fig. 2. Effect of valsartan on depressor (A) and bradycardic responses (B) elicited by unilateral injection of Ang II into the NTS in SHR and WKY. In both strains, Ang II (0.1 pmol) significantly decreased the baseline MAP and HR approximately 3–7 min with aCSF (0.1 l) containing no drug. Depressor responses were greater in SHR than in WKY (SHR versus WKY, −71 ± 10∗ mmHg versus −36 ± 6 mmHg, ∗ P < 0.05 versus WKY, n = 6–7). Bradycardic responses were not significantly different between two strains (SHR versus WKY, −37 ± 6 bpm versus −64 ± 16 bpm). Depressor and bradycardic responses produced by Ang II were completely abolished approximately 3–7 min after pretreatment with valsartan (10 pmol) in aCSF in both strains (SHR versus WKY: MAP, −4±2∗ mmHg versus −3±2∗ mmHg; HR, −1±2∗ bpm versus −5 ± 2∗ bpm, ∗ P < 0.05 versus pretreatment with aCSF for each strain, n = 6–7). Abbreviations: SHR, spontaneously hypertensive rat; WKY rat, Wistar–Kyoto rat.
(Fig. 2). However, these depressor responses in SHR were greater than those in WKY rats (SHR versus WKY rats: −71 ± 10∗ mmHg versus −36 ± 6 mmHg, ∗ P < 0.05 versus WKY rats, n = 6; Fig. 2); the maximal decrease in MAP elicited by Ang II in SHR was approximately twice that in WKY rats. Expressed as a percent change from baseline MAP, differences in depressor responses were less striking (−37 ± 4% in SHR and −34 ± 6% in WKY rats, P > 0.1). Decreases in HR evoked by ipsilateral injection of Ang II into the NTS after unilateral injection of aCSF did not differ significantly between strains (SHR versus WKY rats: −37 ± 6 bpm versus −64 ± 16 bpm, P > 0.1, n = 6; Fig. 2). Percent change was also similar (SHR versus WKY rats: −11 ± 2% versus −17 ± 4%, n = 6). Unilateral injection of valsartan (10 pmol) into the NTS did not alter baseline MAP or HR in either strain. In both strains, prior injection
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Fig. 4. Effect of bilateral injection of valsartan into the NTS in SHR and WKY. Bilateral injection of valsartan (10 pmol) into the NTS did not alter baseline MAP in WKY. This treatment significantly increased baseline MAP (SHR versus WKY, 21 ± 4∗ mmHg versus 5 ± 3 mmHg, ∗ P < 0.05 versus the SHR baseline MAP and between strains, n = 5). Treatment did not alter baseline HR in either strain.
Fig. 3. Effect of valsartan on depressor (A) and bradycardic (B) responses elicited by unilateral injection of Glu into the NTS of SHR and WKY. In both strains, Glu (150 pmol) significantly decreased the baseline MAP and HR approximately 3–7 min after pretreatment with aCSF (0.1 l) containing no drug or valsartan (10 pmol). Depressor responses produced by Glu were similar in both strains after pretreatment with aCSF (SHR versus WKY, −43 ± 9 mmHg versus −41 ± 5 mmHg, n = 7), but bradycardic responses were smaller in SHR than in WKY (SHR versus WKY, −31±8∗ bpm versus −55±6 bpm, ∗ P < 0.05 from WKY, n = 7). Depressor responses produced by Ang II were also similar in both strains (SHR versus WKY, −39 ± 5 mmHg versus −34 ± 5 mmHg, n = 7), but bradycardic responses were smaller in SHR than in WKY after pretreatment with valsartan (SHR versus WKY, −21 ± 3∗ bpm versus −53 ± 9 bpm, ∗ P < 0.05 between strains, n = 7).
of valsartan (10 pmol) into the NTS prevented depressor and bradycardic responses elicited by subsequent injection of 0.1 pmol of Ang II (MAP in SHR versus WKY rats: −4 ± 2 mmHg versus −3 ± 2 mmHg; HR: −1 ± 2 bpm versus −5 ± 2 bpm, P > 0.1 versus baseline MAP and HR, n = 6; Fig. 2). The same pretreatment did not alter the depressor or bradycardic response elicited by subsequent injection of glutamate (150 pmol) in either strain (SHR versus WKY rats: with aCSF as a control, −43 ± 9 mmHg and −31 ± 8∗ bpm versus −41 ± 5 mmHg and −55 ± 6 bpm; with prior valsartan, −39 ± 5 mmHg and −21 ± 3∗ bpm versus −34 ± 5 mmHg and −53 ± 9 bpm, ∗ P < 0.05 versus WKY rats, n = 6; Fig. 3). In SHR, bilateral injection of valsartan (10 pmol) into the NTS increased MAP, a response that was not observed in WKY rats (SHR versus WKY rats: 21 ± 4∗ mmHg versus 5 ± 3 mmHg, ∗ P < 0.05 versus WKY rats and baseline MAP, n = 4; Fig. 4). Expressed as percent change from baseline, the responses were 14 ± 3∗ % in SHR
and 2 ± 3% in WKY (∗ P < 0.05). The increase in MAP elicited by bilateral injection of valsartan in SHR rats lasted 15–20 min, and MAP returned to baseline values within 30 min in all SHR. Valsartan (10 pmol) did not alter baseline HR in either strain (SHR versus WKY rats: −3 ± 12 bpm versus −14 ± 6 bpm, P > 0.1, n = 4). The baroreceptor reflex index (BRI) was calculated as the maximal change in HR (bpm) divided by the maximal change in MAP (mmHg) elicited by intravenous injection of either phenylephrine (2 g/kg) or nitroprusside (5 g/kg). In both strains, BRI was determined before (control) and 3–7 min after bilateral injection of valsartan (10 pmol) into the NTS. Although control BRI was less in SHR than in WKY rats, bilateral injection of valsartan (10 pmol) did not alter BRI in either strain (P > 0.05). Phenylephrine (2 g/kg) increased MAP to the same extent in both strains (SHR versus WKY rats: 41 ± 4 mmHg versus 34 ± 2 mmHg, P > 0.1, n = 6) while nitroprusside (5 g/kg) elicited a similar decrease in MAP in the two strains (SHR versus WKY rats: −56 ± 8 bpm versus −42 ± 3 bpm, P > 0.1, n = 6; Table 1).
Table 1 Effect of valsartan on the baroreceptor reflex in SHR and WKY rats BRI tested with phenylephrine
BRI tested with nitroprusside
Control
After valsartan
Control
1.0 ± 1.7 ± 0.2
0.8 ± 0.2 0.7 ± 0.1a 1.2 ± 0.2 1.3 ± 0.2
SHR 0.9 ± WKY 1.3 ± 0.1
0.1a
0.2a
After valsartan
The baroreceptor reflex index (BRI) was calculated as the maximal change in HR (bpm) divided by the maximal change in MAP (mmHg) elicited by intravenous injection of either phenylephrine (2 g/kg) or nitroprusside (5 g/kg). BRI was determined in chloralose-anesthetized SHR and WKY rats before (control) and approximately 3–7 min after bilateral injection of valsartan (10 pmol) into NTS. Valsartan did not alter BRI in either strain although BRI was less in SHR than in WKY rats (a P < 0.05, n = 6). In these rats, baseline MAP was 153 ± 3 in SHR and 102 ± 3 in WKY rats. HR was 347 ± 18 in SHR and 332 ± 12 in WKY rats. Phenylephrine increased MAP to the same extent in both rat strains (41 ± 4 in SHR and 34 ± 2 in WKY) while nitroprusside elicited a similar decrease in MAP in the two strains (−56 ± 8 in SHR and −42 ± 3 in WKY).
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4. Discussion The present results included three main findings. First, unilateral injection of Ang II into the NTS in S-D rats, WKY rats, or SHR elicited a depressor and bradycardic response which was eliminated by prior injection of an AT1 receptor selective antagonist, valsartan, at the same site in the NTS. Nevertheless, prior injection of valsartan did not alter cardiac responses elicited by injection of glutamate. Second, the depressor response elicited by injection of Ang II was significantly greater in SHR than in WKY rats. Third, although bilateral injection of valsartan into the NTS of the normotensive rats such as S-D rats and WKY rats did not alter baseline AP, this treatment elicited an increase in AP in SHR. In addition, although the baroreflex was less sensitive in SHR than in WKY rats, bilateral injection of valsartan into the NTS did not alter the baroreflex sensitivity in either strain. Thus, these results suggest that exogenous Ang II injected into the NTS acts on AT1 receptors to reduce AP independently of the glutamatergic system and baroreflexes. Furthermore, this system is altered within the NTS of SHR, apparently acting tonically to reduce AP. Autoradiographic studies have shown that AT1 receptors but not AT2 receptors are present in the NTS of adult rats [2,9,11,14,20]. In agreement, a pharmacologic study demonstrated that injection into the NTS of an AT1 receptor antagonist (losartan, 100 pmol in 30 nl aCSF) but not an AT2 receptor antagonist (CGP-42112A) prevented the response evoked by Ang II (0.2 pmol in 30 nl aCSF) [8]. This suggested that Ang II injected into the NTS elicits hypotension and bradycardia via AT1 receptors [8]. However, losartan additionally binds to a non-Ang II site, as shown in rat liver and in other tissues [4]. Moreover, some previous studies have demonstrated an interaction between Ang II receptors and ␣2 -adrenoceptors in the NTS [6,7], while in others the cardiovascular effects of Ang II in the NTS were mediated partly by substance P, without direct interaction between receptors [5]. Considering the complicating factors above, we selected valsartan to further characterize the cardiovascular role of AT1 receptors in the NTS. Valsartan is a potent, specific, highly selective AT1 receptor antagonist without any agonist activity [3]; it has a greater affinity for the AT1 receptors than losartan (approximately five-fold) [4]. Furthermore, while valsartan can bind to the non-Ang II losartan binding site, it does so with 10,000 times less affinity than for the AT1 receptor [4]. Therefore, this antagonist was felt to be more informative than losartan for NTS studies. We found that prior injection of valsartan in doses that completely block the effect of Ang II did not alter cardiovascular responses elicited by glutamate, in agreement with previous results [8]. The angiotensinergic system in the NTS, then, acts independently of the glutamatergic system. Bilateral injection of a dose of valsartan into the NTS capable of completely abolishing the cardiovascular responses elicited by Ang II did not alter baseline AP and HR in normotensive
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rats such as S-D rats and WKY rats. Thus, stimulation of NTS AT1 receptors in normotensive rats is not involved in tonic regulation of cardiovascular function. Antisense oligonucleotide inhibition of expression of the angiotensinogen and AT1 receptor genes in the brainstem has been found to lower high blood pressure in SHR [10]. Thus, the angiotensinergic system in the NTS also may be related to the pathogenesis of hypertension in SHR. Indeed, in the present study, the depressor effect of Ang II injected into the NTS is considerably greater in SHR than in WKY rats. As in normotensive rats, this effect of Ang II in SHR was abolished by prior injection of valsartan. These results suggest that the enhanced effect of Ang II in SHR is mediated solely by AT1 receptors within the NTS. When expressed as an absolute change in MAP, the depressor responses elicited by glutamate were similar in normotensive rats and hypertensive rats, as shown previously [22]. However, the magnitude of the response elicited by Ang II in SHR was approximately twice that of responses elicited either by Ang II in normotensive rats or by glutamate in all strains including SHR. Furthermore, bilateral injection of valsartan into the NTS of normotensive rats did not alter baseline AP and HR, while the same treatment in SHR elicited a significant increase in AP. Since prior injection of valsartan did not alter the effect of glutamate in any rat strain, the results indicate that the anigiotensinergic system within the NTS not only acts independently of the glutamatergic system but also is altered in SHR. In these hypertensive animals, AT1 receptors within the NTS are called into play in tonic cardiovascular regulation, presumably in response to the hypertensive state. The enhanced depressor response to Ang II demonstrated in SHR differed from the uniform depressor response induced in all strains via NTS neurons stimulated by glutamate. As one possible explanation for the difference, previous studies have shown that the glutamatergic system within the NTS is required for the baroreflex [12], while the cholinergic system is not [21]. Yet, when injected into the NTS in WKY rats and SHR, either glutamate or acetylcholine elicits a similar depressor response [22]. In the present observations, stimulation of AT1 receptors in the NTS phasically regulated AP in normotensive rats, but also tonically regulated AP in SHR. Based on these observations, the response to Ang II in the NTS of SHR may include both baroreflex-independent and -dependent components, while the same treatment in normotensive rats stimulates only the baroreflex-independent pathway, resulting in a smaller overall response to Ang II. However, since our present study did not, unfortunately, indicate that bilateral injection of valsartan into the NTS attenuates baroreflex sensitivity in SHR, we need to confirm the relation between baroreflex function and AT1 receptors within the NTS using other methods. Previous studies comparing WKY rats and SHR [15,16] have shown different responses of angiotensinergic system in some brainstem nuclei. Although Ang II injected into the RVLM elicits a similar depressor response in both strains,
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the same treatment in the CVLM elicits a greater depressor response in SHR than in WKY rats [15]. Injection of a nonselective Ang II antagonist, sarthran, into the RVLM or CVLM elicits greater responses in SHR [16]. In another previous study [1], a tonic inhibitory pathway from the NTS to the RVLM include a relay through the CVLM, probably via a glutamatergic projection from the NTS to the CVLM. On the other hand, whether or not the baroreflex-independent pathway from the NTS to the RVLM is relayed through the CVLM is uncertain. However, the enhanced responses to Ang II in the CVLM in SHR may support the hypothesis that enhanced responses to Ang II in SHR involve inhibition of both baroreflex-independent and -dependent pathways in the NTS. Since a diminished response to glutamate in the CVLM has been shown in SHR [18,19], these contrasting conclusions suggest that, in the NTS or CVLM in SHR, the physiology of the angiotensinergic system differs from that of the glutamatergic system. In summary, our present data indicate that the cardiovascular response elicited by injection of exogenous Ang II into the NTS was abolished by prior injection of a selective AT1 receptor antagonist, valsartan. Thus, Ang II injected into the NTS acts at AT1 receptors to reduce AP and HR. Bilateral injection of valsartan into the NTS did not alter baseline AP and HR in normotensive rats, but this treatment increased AP in SHR. On the other hand, this treatment did not alter baroreceptor reflex sensitivity in any rat strain. These results suggest that stimulation of AT1 receptors in the NTS elicits a decrease in AP and HR independently of the baroreflex and glutamatergic systems. In addition, the angiotensinergic system in SHR is altered to tonically oppose elevated AP. References [1] S.K. Agarwal, A.J. Gelsema, F.R. Calaresu, Inhibition of rostral VLM by baroreceptor activation is relayed through caudal VLM, Am. J. Physiol. 258 (1990) R1271–R1278. [2] G.P. Aldred, S.Y. Chai, K. Song, J. Zhuo, D.P. MacGregor, F.A.O. Mendelsohn, Distribution of angiotensin II receptor subtypes in the rabbit brain, Regul. Pept. 44 (1993) 119–130. [3] L. Criscione, M. de Gasparo, P. Buhlmayer, S. Whitebread, H.R. Ramjoue, J. Wood, Pharmacological profile of valsartan: a potent, orally active, nonpeptide antagonist of the angiotensin II AT1 -receptor subtype, Br. J. Pharmacol. 110 (1993) 761–771. [4] M. de Gasparo, S. Whitebread, Binding of valsartan to mammalian angiotensin AT1 receptors, Regul. Pept. 59 (1995) 303–311. [5] D.I. Diz, D.L. Fantz, I.F. Benter, S.M. Bosch, Acute depressor actions of angiotensin II in the nucleus of the solitarii tract are mediated by substance P, Am. J. Physiol. (Regul. Integr. Comp. Physiol. 42) 273 (1997) R28–R34.
[6] D.R. Fior, S. Yang, U. Ganten, D. Ganten, K. Fuxe, Evidence for a differential modulation of the alpha-2 adrenoceptors by angiotensin II in the nucleus tractus solitarii of the spontaneously hypertensive and the Wistar–Kyoto normotensive rats, Brain Res. 679 (1995) 168–177. [7] D.R. Fior, S. Yang, P. Hedlund, J.A. Narvaez, L.F. Agnati, K. Fuxe, Evidence of an antagonistic angiotensin II/␣2 -adrenoceptor interaction in the nucleus tractus solitarii, Eur. J. Pharmacol. 262 (1994) 271–282. [8] J.E. Fow, D.B. Averill, K.L. Barnes, Mechanisms of angiotensin-induced hypotension and bradycardia in the medial solitary tract nucleus, Am. J. Physiol. (Heart Circ. Physiol. 36) 267 (1994) H259–H266. [9] D.R. Gehlert, S.L. Gackenheimer, D.A. Schober, Autoradiographic localization of subtypes of angiotensin II antagonist binding in the rat brain, Neuroscience 44 (2) (1991) 501–514. [10] R. Gyruko, D. Wielbo, M.I. Phillips, Antisense inhibition of AT1 receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin, Regul. Pept. 49 (1993) 167–174. [11] D.P. Healy, R. Rettig, T. Nguyen, M.P. Printz, Quantitative autoradiography of angiotensin II receptors in the rat solitary-vagal area effects of nodose gangionectomy or sinoaortic denervation, Brain Res. 484 (1989) 1–12. [12] T. Kubo, M. Kihara, Unilateral blockade of excitatory amino acid receptors in the nucleus tractus solitarii produces an inhibition of baroreflexes in rats, Naunyn Schmiedeberg’s Arch. Pharmacol. 343 (1991) 317–322. [13] K. Matsumura, D.B. Averill, C.M. Ferrario, Angiotensin II acts at AT1 receptors in the nucleus of the solitary tract to attenuate the baroreceptor reflex, Am. J. Physiol. (Regul. Integr. Comp. Physiol. 44) 275 (1998) R1611–R1619. [14] F.A.O. Mendelsohn, A.M. Allen, J. Clevers, D.A. Denton, E. Tarjan, M.J. McKinley, Localization of angiotensin II receptor binding in rabbit brain by in vitro autoradiography, J. Comp. Neurol. 270 (1988) 372–384. [15] H. Muratani, D.B. Averill, C.M. Ferrario, Effect of angiotensin II in ventrolateral medulla of spontaneously hypertensive rats, Am. J. Physiol. (Regul. Integr. Comp. Physiol. 29) 260 (1991) R977–R984. [16] H. Muratani, C.M. Ferrario, D.B. Averill, Ventrolateral medulla in spontaneously hypertensive rats: role of angiotensin II, Am. J. Physiol. (Regul. Integr. Comp. Physiol. 33) 264 (1993) R388–R395. [17] L. Qu, A.J. McQueeney, K.L. Barnes, Presynaptic or postsynaptic location of receptors for angiotensin II and substance P in the medial solitary tract nucleus, J. Neurophysiol. 75 (1996) 2220–2228. [18] J.K. Smith, K.W. Barron, Cardiovascular effects of l-glutamate and tetrodotoxin microinjected into the rostral and caudal ventrolateral medulla in normotensive and spontaneously hypertensive rats, Brain Res. 506 (1990) 1–8. [19] J.K. Smith, K.W. Barron, GABAergic responses in ventrolateral in spontaneously hypertensive rats, Am. J. Physiol. 258 (1990) R450– R456. [20] K. Song, A.M. Allen, G. Paxinos, F.A.O. Mendelsohn, Mapping of angiotensin II receptor subtype heterogeneity in rat brain, J. Comp. Neurol. 316 (1992) 467–484. [21] K. Tsukamoto, Y. Ming, A.F. Sved, Effect of atropine injected into the nucleus tractus solitarius on the regulation of blood pressure, Brain Res. 648 (1994) 9–15. [22] K. Tsukamoto, A.F. Sved, Enhanced ␥-aminobutyric acid-mediated responses in nucleus tractus solitarius of hypertensive rats, Hypertension 22 (6) (1993) 819–825.
Enhanced angiotensin-mediated responses in the nucleus tractus solitarii of spontaneously hypertensive rats Nobuhide Katsunuma, Kazuyoshi Tsukamoto∗ , Satoru Ito, Katsuo Kanmatsuse Second Department of Internal Medicine, Nihon University School of Medicine, 30-1, Oyaguchi-Kamichou, Itabashi-ku, Tokyo 173-8610, Japan Received 9 September 2002; received in revised form 16 December 2002; accepted 19 December 2002
Abstract Studies using an AT1 receptor antagonist, losartan, demonstrated that depressor and bradycardic responses to angiotensin II (Ang II) injection into the nucleus tractus solitarii (NTS) are mediated via those receptors. We further characterized Ang II-evoked cardiovascular responses in this nucleus in spontaneously hypertensive rats (SHR) using a new, selective AT1 receptor antagonist, valsartan. In ␣-chloralose-anesthetized Sprague–Dawley (S-D) rats, Wistar–Kyoto (WKY) rats, and SHR, unilateral injection of Ang II into the NTS decreased arterial pressure (AP) and heart rate (HR). This response was eliminated by preinjection of valsartan. Depressor responses were much greater in SHR than in WKY rats. In normotensive rats, bilateral valsartan injection did not alter baseline AP or HR, or baroreceptor reflex index (BRI) calculated as the maximal change in HR (bpm) divided by phenylephrine- or nitroprusside-induced maximal change in mean AP (mmHg). In SHR, this treatment did not alter baseline HR and BRI, but significantly increased AP. Preinjection of valsartan did not alter injected glutamate effects in any strain. Thus, stimulation of AT1 receptors within the NTS contributes to cardiovascular regulation independently of the baroreceptor reflex and the glutamatergic system. This angiotensinergic system in SHR acts tonically to reduce AP. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Angiotensin II; Valsartan; AT1 receptor; Nucleus tractus solitarii; SHR
1. Introduction The nucleus tractus solitarii (NTS), specifically the region in which baroreceptor afferent nerves terminate, plays an important role in cardiovascular control. The NTS contains a large variety of putative neurotransmitters, many of which have been reported to influence cardiovascular regulation. By autoradiographic methods, AT1 receptors have been shown to predominate within the NTS [2,9,11,14,20]. Further, previous studies in anesthetized rats [5,8] have shown that unilateral injection of angiotensin II (Ang II) into the NTS elicits a decrease in arterial pressure (AP) and heart rate (HR). Since these cardiovascular responses were prevented by prior injection of an AT1 receptor antagonist losartan into the NTS, the Ang II-evoked response may be mediated via AT1 receptors [8]. However, losartan also binds to a non-Ang II receptor site in rat liver and in other tissues [4]. When a different AT1 receptor antagonist, candesartan, was injected bilaterally into the NTS, baseline AP
∗
Corresponding author. Tel.: +81-3-3972-8111; fax: +81-3-3972-1098.
and HR showed little change, nonetheless, this treatment significantly increased sensitivity for baroreceptor reflex control of renal sympathetic nerve activity and HR [13]. On the other hand, the previous studies have suggested that the Ang II-evoked response is mediated at least in part by substance P using similar pharmacologic methods [5,17]. However, details of the angiotensinergic system within the NTS remain unclear. In the present study, we aimed to further characterize the effect of Ang II injected into the NTS using a new and highly selective AT1 receptor antagonist, valsartan. A previous study using genetically hypertensive rats (SHR) of the Okamoto strain determined that the depressor response to injection of Ang II into the caudal ventrolateral medulla (CVLM) was exaggerated in these animals [15]. Nevertheless, responses evoked by Ang II injection into the rostral ventrolateral medulla (RVLM) were similar in SHR and Wistar–Kyoto (WKY) rats [15]. In contrast, another study found that injection of a nonselective Ang II antagonist, sarthran, into the RVLM or CVLM enhanced responses in SHR [16], suggesting that Ang II-related inhibitory neurotransmission from CVLM to the RVLM may not be
0361-9230/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0361-9230(03)00013-3
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sufficient to offset the greater intrinsic pressor activity of the RVLM in SHR. Another previous study demonstrated an antagonistic Ang II/␣2 receptor interaction within the NTS in WKY rats that is altered in SHR [6]. These findings suggest that the differences in brainstem angiotensinergic systems may be related to hypertension in SHR, again indicating a need to further characterize the Ang II-evoked cardiovascular response in the NTS in SHR.
2. Materials and methods Experiments were conducted on adult male Sprague– Dawley (S-D) rats (300–480 g), WKY rats, and spontaneously hypertensive rats (SHR; 16–20 weeks; Charles River, Japan). Animals were housed in groups of 2–3 in hanging wire mesh cages in temperature-controlled rooms with a fixed 12 h/12 h light/dark cycle for at least 2 weeks prior to experiments. Food (MF; Oriental Yeast Co., Ltd) and tap water were available ad libitum. Rats were anesthetized with halothane (2% in 100% O2 , administered via a nasal cone). A cannula (PE 50 tubing filled with heparinized saline) was inserted into the right femoral artery for recording AP to determine mean arterial pressure (MAP) and HR (Grass Model 7 Physiograph). A second cannula was placed in the right femoral vein for administering drugs. The trachea was cannulated and the rats were artificially ventilated with 2% halothane in 100% O2 (Harvard Small Animal Respirator) following administration of a muscle relaxant (d-tubocurarine, 0.5 mg/kg, i.v., supplemented hourly with 0.2 mg/kg, i.v.). The rats were placed in a stereotaxic instrument (Kopf) with the incisor bar positioned 11 mm below the interaural line. The dorsal surface of the brainstem was exposed by limited craniotomy and, with the aid of a surgical microscope, the area postrema was visualized. Anesthesia with ␣-chloralose was administered (60 mg/kg, i.v., supplemented hourly with 20 mg/kg, i.v.), and halothane was discontinued. Rats were ventilated with 100% O2 throughout the remainder of the experiment. Injections of drug solutions or vehicle were made into the NTS using single-barrel glass micropipettes pulled to an outer diameter of 40–50 m. The tip of the micropipette was positioned 0.5 mm rostral to the calamus scriptorius, 0.5 mm lateral from the midline, and 0.5 mm deep with respect to the surface of the brainstem for injection into the NTS. All drugs were dissolved in artificial cerebrospinal fluid (aCSF) and delivered in a volume of 100 nl. A PicoPump was used for injections (WPI, New Haven, CT). The volume of drug injected into the NTS was carefully monitored by observing the movement of the fluid meniscus in the calibrated glass pipette. Bilateral injections were made one side at a time, with approximately 30 s separating the two injections. In experiments where one agent was injected followed by injection of another agent, the pipette was removed and replaced for the subsequent injection.
At the conclusion of each experiment, the animal was anesthetized with urethane (1.5 g/kg, i.v.) and perfused intracardially with saline followed by 10% buffered formalin. The brainstem was removed, frozen, and sectioned (40 m) using a microtome. Sections were affixed to glass slides and stained with cresyl violet. Only animals whose pipette tracks were centered in the medial subnucleus of the NTS at the level of the area postrema were included in the data analysis. Animals with evidence of bleeding in the NTS were excluded from data analysis. An injection including 100 nl of Fast Green dye was made into the NTS prior to sacrifice. Injection sites were similar to those in previous experiments. Valsartan was obtained from Novartis Pharma AG (Switzerland). All other chemicals used in these experiments were purchased from Sigma Chemical Co. (St. Louis, MO). Data are expressed as the mean ± S.E.M. Data were compared by analysis of variance (ANOVA) followed by Scheffe’s F test.
3. Results 3.1. Studies in S-D rats Unilateral injection of a 0.1- or 1-pmol dose of Ang II into the NTS of S-D rats anesthetized with chloralose elicited similarly significant depressor and bradycardic responses (0.1 pmol versus 1 pmol: −29 ± 2∗ mmHg and −20 ± 6∗ bpm versus −26 ± 4∗ mmHg and −25 ± 5∗ bpm, ∗ P < 0.05 versus baseline MAP and HR, n = 7). These cardiovascular responses were rapid in onset and lasted less than 2 min. Injection of vehicle did not significantly alter baseline MAP or HR (2 ± 1 mmHg and 0 ± 2 bpm, P > 0.1 versus baseline MAP and HR, n = 6). In contrast, a larger dose of Ang II (30 pmol) elicited a significantly smaller depressor and bradycardic response (change from baseline, −13 ± 3∗ mmHg and −12 ± 3∗ bpm, ∗ P < 0.05 versus baseline MAP and HR, n = 6) than the responses elicited by either 0.1- or 1-pmol doses of Ang II. To determine the ability of the AT1 receptor antagonist, valsartan, to effectively and selectively block the above action of Ang II, responses to a 0.1-pmol dose of Ang II or other depressor substances were tested 3–7 min after injection of valsartan or aCSF with no drugs as a control into the same site. Neither unilateral nor bilateral injection of valsartan (10 pmol) altered baseline MAP or HR (unilateral versus bilateral injection, changes of 1 ± 1 mmHg and 0 ± 0 bpm versus 5 ± 3 mmHg and 4 ± 5 bpm, P > 0.1 versus baseline MAP and HR, n = 6–8). A prior unilateral injection of valsartan (10 pmol) completely prevented the depressor and bradycardic response to Ang II (0.1 pmol; with previous aCSF as a control, the change with Ang II was −30 ± 2 mmHg and −20 ± 6 bpm; with prior valsartan, the change with Ang II was −3 ± 4∗ mmHg and −2 ± 2∗ bpm, ∗ P < 0.05 versus control, n = 6; Fig. 1). In contrast to the effects of prior valsartan (10 pmol) injection on responses
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Fig. 1. Effect of valsartan on depressor (A) and bradycardic responses (B) elicited by unilateral injection of glutamate (Glu) or angiotensin II (Ang II) into the NTS of S-D rats. Either Glu (150 pmol) or Ang II (0.1 pmol) significantly decreased the baseline MAP and HR approximately 3–7 min after pretreatment with aCSF (0.1 l) containing no drug. Depressor and bradycardic responses produced by Glu approximately 3–7 min after pretreatment with valsartan (10 pmol) in aCSF were similar to those after pretreatment with aCSF alone (after aCSF, −38 ± 3 mmHg and −29 ± 5 bpm; after valsartan, −32 ± 3 mmHg and −20 ± 2 bpm, n = 8). However, pretreatment with valsartan completely abolished the cardiovascular responses to Ang II seen after pretreatment with aCSF alone (after aCSF, −30 ± 2 mmHg and −20 ± 6 bpm; after valsartan, −3 ± 4∗ mmHg and −2 ± 2∗ bpm, ∗ P < 0.05 versus pretreatment with aCSF, n = 8).
elicited by Ang II, prior unilateral injection of valsartan (10 pmol) into the NTS did not alter depressor or bradycardic responses elicited by injection of glutamate (150 pmol) into the NTS (with previous aCSF as a control, −38 ± 3 mmHg and −29 ± 5 bpm; with prior valsartan, −32 ± 3 mmHg and −20 ± 2 bpm, P > 0.1 versus control, n = 7; Fig. 1). This dose of glutamate elicited similar depressor and bradycardic responses to those elicited by Ang II, representing a maximally effective dose of glutamate [22]. 3.2. Studies in SHR and WKY rats Baseline MAP in chloralose-anesthetized SHR rats was greater than in WKY rats (SHR versus WKY rats: 173 ± 9∗ mmHg versus 108 ± 4 mmHg, ∗ P < 0.05 versus WKY rats, n = 6), but HR was similar in both strains (SHR versus WKY rats: 347 ± 18 bpm versus 332 ± 12 bpm, P > 0.1 versus WKY rats, n = 6). Ipsilateral injection of Ang II (0.1 pmol) 3–7 min after unilateral injection of aCSF into the NTS in both strains elicited decreases in MAP and HR
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Fig. 2. Effect of valsartan on depressor (A) and bradycardic responses (B) elicited by unilateral injection of Ang II into the NTS in SHR and WKY. In both strains, Ang II (0.1 pmol) significantly decreased the baseline MAP and HR approximately 3–7 min with aCSF (0.1 l) containing no drug. Depressor responses were greater in SHR than in WKY (SHR versus WKY, −71 ± 10∗ mmHg versus −36 ± 6 mmHg, ∗ P < 0.05 versus WKY, n = 6–7). Bradycardic responses were not significantly different between two strains (SHR versus WKY, −37 ± 6 bpm versus −64 ± 16 bpm). Depressor and bradycardic responses produced by Ang II were completely abolished approximately 3–7 min after pretreatment with valsartan (10 pmol) in aCSF in both strains (SHR versus WKY: MAP, −4±2∗ mmHg versus −3±2∗ mmHg; HR, −1±2∗ bpm versus −5 ± 2∗ bpm, ∗ P < 0.05 versus pretreatment with aCSF for each strain, n = 6–7). Abbreviations: SHR, spontaneously hypertensive rat; WKY rat, Wistar–Kyoto rat.
(Fig. 2). However, these depressor responses in SHR were greater than those in WKY rats (SHR versus WKY rats: −71 ± 10∗ mmHg versus −36 ± 6 mmHg, ∗ P < 0.05 versus WKY rats, n = 6; Fig. 2); the maximal decrease in MAP elicited by Ang II in SHR was approximately twice that in WKY rats. Expressed as a percent change from baseline MAP, differences in depressor responses were less striking (−37 ± 4% in SHR and −34 ± 6% in WKY rats, P > 0.1). Decreases in HR evoked by ipsilateral injection of Ang II into the NTS after unilateral injection of aCSF did not differ significantly between strains (SHR versus WKY rats: −37 ± 6 bpm versus −64 ± 16 bpm, P > 0.1, n = 6; Fig. 2). Percent change was also similar (SHR versus WKY rats: −11 ± 2% versus −17 ± 4%, n = 6). Unilateral injection of valsartan (10 pmol) into the NTS did not alter baseline MAP or HR in either strain. In both strains, prior injection
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Fig. 4. Effect of bilateral injection of valsartan into the NTS in SHR and WKY. Bilateral injection of valsartan (10 pmol) into the NTS did not alter baseline MAP in WKY. This treatment significantly increased baseline MAP (SHR versus WKY, 21 ± 4∗ mmHg versus 5 ± 3 mmHg, ∗ P < 0.05 versus the SHR baseline MAP and between strains, n = 5). Treatment did not alter baseline HR in either strain.
Fig. 3. Effect of valsartan on depressor (A) and bradycardic (B) responses elicited by unilateral injection of Glu into the NTS of SHR and WKY. In both strains, Glu (150 pmol) significantly decreased the baseline MAP and HR approximately 3–7 min after pretreatment with aCSF (0.1 l) containing no drug or valsartan (10 pmol). Depressor responses produced by Glu were similar in both strains after pretreatment with aCSF (SHR versus WKY, −43 ± 9 mmHg versus −41 ± 5 mmHg, n = 7), but bradycardic responses were smaller in SHR than in WKY (SHR versus WKY, −31±8∗ bpm versus −55±6 bpm, ∗ P < 0.05 from WKY, n = 7). Depressor responses produced by Ang II were also similar in both strains (SHR versus WKY, −39 ± 5 mmHg versus −34 ± 5 mmHg, n = 7), but bradycardic responses were smaller in SHR than in WKY after pretreatment with valsartan (SHR versus WKY, −21 ± 3∗ bpm versus −53 ± 9 bpm, ∗ P < 0.05 between strains, n = 7).
of valsartan (10 pmol) into the NTS prevented depressor and bradycardic responses elicited by subsequent injection of 0.1 pmol of Ang II (MAP in SHR versus WKY rats: −4 ± 2 mmHg versus −3 ± 2 mmHg; HR: −1 ± 2 bpm versus −5 ± 2 bpm, P > 0.1 versus baseline MAP and HR, n = 6; Fig. 2). The same pretreatment did not alter the depressor or bradycardic response elicited by subsequent injection of glutamate (150 pmol) in either strain (SHR versus WKY rats: with aCSF as a control, −43 ± 9 mmHg and −31 ± 8∗ bpm versus −41 ± 5 mmHg and −55 ± 6 bpm; with prior valsartan, −39 ± 5 mmHg and −21 ± 3∗ bpm versus −34 ± 5 mmHg and −53 ± 9 bpm, ∗ P < 0.05 versus WKY rats, n = 6; Fig. 3). In SHR, bilateral injection of valsartan (10 pmol) into the NTS increased MAP, a response that was not observed in WKY rats (SHR versus WKY rats: 21 ± 4∗ mmHg versus 5 ± 3 mmHg, ∗ P < 0.05 versus WKY rats and baseline MAP, n = 4; Fig. 4). Expressed as percent change from baseline, the responses were 14 ± 3∗ % in SHR
and 2 ± 3% in WKY (∗ P < 0.05). The increase in MAP elicited by bilateral injection of valsartan in SHR rats lasted 15–20 min, and MAP returned to baseline values within 30 min in all SHR. Valsartan (10 pmol) did not alter baseline HR in either strain (SHR versus WKY rats: −3 ± 12 bpm versus −14 ± 6 bpm, P > 0.1, n = 4). The baroreceptor reflex index (BRI) was calculated as the maximal change in HR (bpm) divided by the maximal change in MAP (mmHg) elicited by intravenous injection of either phenylephrine (2 g/kg) or nitroprusside (5 g/kg). In both strains, BRI was determined before (control) and 3–7 min after bilateral injection of valsartan (10 pmol) into the NTS. Although control BRI was less in SHR than in WKY rats, bilateral injection of valsartan (10 pmol) did not alter BRI in either strain (P > 0.05). Phenylephrine (2 g/kg) increased MAP to the same extent in both strains (SHR versus WKY rats: 41 ± 4 mmHg versus 34 ± 2 mmHg, P > 0.1, n = 6) while nitroprusside (5 g/kg) elicited a similar decrease in MAP in the two strains (SHR versus WKY rats: −56 ± 8 bpm versus −42 ± 3 bpm, P > 0.1, n = 6; Table 1).
Table 1 Effect of valsartan on the baroreceptor reflex in SHR and WKY rats BRI tested with phenylephrine
BRI tested with nitroprusside
Control
After valsartan
Control
1.0 ± 1.7 ± 0.2
0.8 ± 0.2 0.7 ± 0.1a 1.2 ± 0.2 1.3 ± 0.2
SHR 0.9 ± WKY 1.3 ± 0.1
0.1a
0.2a
After valsartan
The baroreceptor reflex index (BRI) was calculated as the maximal change in HR (bpm) divided by the maximal change in MAP (mmHg) elicited by intravenous injection of either phenylephrine (2 g/kg) or nitroprusside (5 g/kg). BRI was determined in chloralose-anesthetized SHR and WKY rats before (control) and approximately 3–7 min after bilateral injection of valsartan (10 pmol) into NTS. Valsartan did not alter BRI in either strain although BRI was less in SHR than in WKY rats (a P < 0.05, n = 6). In these rats, baseline MAP was 153 ± 3 in SHR and 102 ± 3 in WKY rats. HR was 347 ± 18 in SHR and 332 ± 12 in WKY rats. Phenylephrine increased MAP to the same extent in both rat strains (41 ± 4 in SHR and 34 ± 2 in WKY) while nitroprusside elicited a similar decrease in MAP in the two strains (−56 ± 8 in SHR and −42 ± 3 in WKY).
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4. Discussion The present results included three main findings. First, unilateral injection of Ang II into the NTS in S-D rats, WKY rats, or SHR elicited a depressor and bradycardic response which was eliminated by prior injection of an AT1 receptor selective antagonist, valsartan, at the same site in the NTS. Nevertheless, prior injection of valsartan did not alter cardiac responses elicited by injection of glutamate. Second, the depressor response elicited by injection of Ang II was significantly greater in SHR than in WKY rats. Third, although bilateral injection of valsartan into the NTS of the normotensive rats such as S-D rats and WKY rats did not alter baseline AP, this treatment elicited an increase in AP in SHR. In addition, although the baroreflex was less sensitive in SHR than in WKY rats, bilateral injection of valsartan into the NTS did not alter the baroreflex sensitivity in either strain. Thus, these results suggest that exogenous Ang II injected into the NTS acts on AT1 receptors to reduce AP independently of the glutamatergic system and baroreflexes. Furthermore, this system is altered within the NTS of SHR, apparently acting tonically to reduce AP. Autoradiographic studies have shown that AT1 receptors but not AT2 receptors are present in the NTS of adult rats [2,9,11,14,20]. In agreement, a pharmacologic study demonstrated that injection into the NTS of an AT1 receptor antagonist (losartan, 100 pmol in 30 nl aCSF) but not an AT2 receptor antagonist (CGP-42112A) prevented the response evoked by Ang II (0.2 pmol in 30 nl aCSF) [8]. This suggested that Ang II injected into the NTS elicits hypotension and bradycardia via AT1 receptors [8]. However, losartan additionally binds to a non-Ang II site, as shown in rat liver and in other tissues [4]. Moreover, some previous studies have demonstrated an interaction between Ang II receptors and ␣2 -adrenoceptors in the NTS [6,7], while in others the cardiovascular effects of Ang II in the NTS were mediated partly by substance P, without direct interaction between receptors [5]. Considering the complicating factors above, we selected valsartan to further characterize the cardiovascular role of AT1 receptors in the NTS. Valsartan is a potent, specific, highly selective AT1 receptor antagonist without any agonist activity [3]; it has a greater affinity for the AT1 receptors than losartan (approximately five-fold) [4]. Furthermore, while valsartan can bind to the non-Ang II losartan binding site, it does so with 10,000 times less affinity than for the AT1 receptor [4]. Therefore, this antagonist was felt to be more informative than losartan for NTS studies. We found that prior injection of valsartan in doses that completely block the effect of Ang II did not alter cardiovascular responses elicited by glutamate, in agreement with previous results [8]. The angiotensinergic system in the NTS, then, acts independently of the glutamatergic system. Bilateral injection of a dose of valsartan into the NTS capable of completely abolishing the cardiovascular responses elicited by Ang II did not alter baseline AP and HR in normotensive
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rats such as S-D rats and WKY rats. Thus, stimulation of NTS AT1 receptors in normotensive rats is not involved in tonic regulation of cardiovascular function. Antisense oligonucleotide inhibition of expression of the angiotensinogen and AT1 receptor genes in the brainstem has been found to lower high blood pressure in SHR [10]. Thus, the angiotensinergic system in the NTS also may be related to the pathogenesis of hypertension in SHR. Indeed, in the present study, the depressor effect of Ang II injected into the NTS is considerably greater in SHR than in WKY rats. As in normotensive rats, this effect of Ang II in SHR was abolished by prior injection of valsartan. These results suggest that the enhanced effect of Ang II in SHR is mediated solely by AT1 receptors within the NTS. When expressed as an absolute change in MAP, the depressor responses elicited by glutamate were similar in normotensive rats and hypertensive rats, as shown previously [22]. However, the magnitude of the response elicited by Ang II in SHR was approximately twice that of responses elicited either by Ang II in normotensive rats or by glutamate in all strains including SHR. Furthermore, bilateral injection of valsartan into the NTS of normotensive rats did not alter baseline AP and HR, while the same treatment in SHR elicited a significant increase in AP. Since prior injection of valsartan did not alter the effect of glutamate in any rat strain, the results indicate that the anigiotensinergic system within the NTS not only acts independently of the glutamatergic system but also is altered in SHR. In these hypertensive animals, AT1 receptors within the NTS are called into play in tonic cardiovascular regulation, presumably in response to the hypertensive state. The enhanced depressor response to Ang II demonstrated in SHR differed from the uniform depressor response induced in all strains via NTS neurons stimulated by glutamate. As one possible explanation for the difference, previous studies have shown that the glutamatergic system within the NTS is required for the baroreflex [12], while the cholinergic system is not [21]. Yet, when injected into the NTS in WKY rats and SHR, either glutamate or acetylcholine elicits a similar depressor response [22]. In the present observations, stimulation of AT1 receptors in the NTS phasically regulated AP in normotensive rats, but also tonically regulated AP in SHR. Based on these observations, the response to Ang II in the NTS of SHR may include both baroreflex-independent and -dependent components, while the same treatment in normotensive rats stimulates only the baroreflex-independent pathway, resulting in a smaller overall response to Ang II. However, since our present study did not, unfortunately, indicate that bilateral injection of valsartan into the NTS attenuates baroreflex sensitivity in SHR, we need to confirm the relation between baroreflex function and AT1 receptors within the NTS using other methods. Previous studies comparing WKY rats and SHR [15,16] have shown different responses of angiotensinergic system in some brainstem nuclei. Although Ang II injected into the RVLM elicits a similar depressor response in both strains,
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the same treatment in the CVLM elicits a greater depressor response in SHR than in WKY rats [15]. Injection of a nonselective Ang II antagonist, sarthran, into the RVLM or CVLM elicits greater responses in SHR [16]. In another previous study [1], a tonic inhibitory pathway from the NTS to the RVLM include a relay through the CVLM, probably via a glutamatergic projection from the NTS to the CVLM. On the other hand, whether or not the baroreflex-independent pathway from the NTS to the RVLM is relayed through the CVLM is uncertain. However, the enhanced responses to Ang II in the CVLM in SHR may support the hypothesis that enhanced responses to Ang II in SHR involve inhibition of both baroreflex-independent and -dependent pathways in the NTS. Since a diminished response to glutamate in the CVLM has been shown in SHR [18,19], these contrasting conclusions suggest that, in the NTS or CVLM in SHR, the physiology of the angiotensinergic system differs from that of the glutamatergic system. In summary, our present data indicate that the cardiovascular response elicited by injection of exogenous Ang II into the NTS was abolished by prior injection of a selective AT1 receptor antagonist, valsartan. Thus, Ang II injected into the NTS acts at AT1 receptors to reduce AP and HR. Bilateral injection of valsartan into the NTS did not alter baseline AP and HR in normotensive rats, but this treatment increased AP in SHR. On the other hand, this treatment did not alter baroreceptor reflex sensitivity in any rat strain. These results suggest that stimulation of AT1 receptors in the NTS elicits a decrease in AP and HR independently of the baroreflex and glutamatergic systems. In addition, the angiotensinergic system in SHR is altered to tonically oppose elevated AP. References [1] S.K. Agarwal, A.J. Gelsema, F.R. Calaresu, Inhibition of rostral VLM by baroreceptor activation is relayed through caudal VLM, Am. J. Physiol. 258 (1990) R1271–R1278. [2] G.P. Aldred, S.Y. Chai, K. Song, J. Zhuo, D.P. MacGregor, F.A.O. Mendelsohn, Distribution of angiotensin II receptor subtypes in the rabbit brain, Regul. Pept. 44 (1993) 119–130. [3] L. Criscione, M. de Gasparo, P. Buhlmayer, S. Whitebread, H.R. Ramjoue, J. Wood, Pharmacological profile of valsartan: a potent, orally active, nonpeptide antagonist of the angiotensin II AT1 -receptor subtype, Br. J. Pharmacol. 110 (1993) 761–771. [4] M. de Gasparo, S. Whitebread, Binding of valsartan to mammalian angiotensin AT1 receptors, Regul. Pept. 59 (1995) 303–311. [5] D.I. Diz, D.L. Fantz, I.F. Benter, S.M. Bosch, Acute depressor actions of angiotensin II in the nucleus of the solitarii tract are mediated by substance P, Am. J. Physiol. (Regul. Integr. Comp. Physiol. 42) 273 (1997) R28–R34.
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