Enhanced serotonin-mediated responses in the nucleus tractus solitarius of spontaneously hypertensive rats

Brain Research 863 (2000) 1–8 www.elsevier.com / locate / bres

Research report

Enhanced serotonin-mediated responses in the nucleus tractus solitarius of spontaneously hypertensive rats a b, a a Kazuyoshi Tsukamoto , Alan F. Sved *, Satoru Ito , Kazutoshi Komatsu , a Katsuo Kanmatsuse a

Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo 173 -8610, Japan b Department of Neuroscience, University of Pittsburgh, Pittsburgh PA 15260, USA Accepted 19 January 2000

Abstract Previous studies have demonstrated that injection of serotonin into the nucleus tractus solitarius (NTS) elicits hypotension and bradycardia in rats. The present study sought to further characterize this response and to examine the role of serotonergic mechanisms in the NTS in cardiovascular regulation in spontaneously hypertensive (SHR) rats. Injections of picomole amounts of serotonin into the NTS of chloralose-anesthetized normotensive Sprague–Dawley (S–D) or Wistar–Kyoto (WKY) rats produced hypotension and bradycardia that were eliminated by prior injection into the NTS of the selective 5HT 2 antagonist sarpogrelate. Bilateral injection of sarpogrelate did not alter blood pressure or reflex changes in heart rate in response to phenylephrine-induced increases in blood pressure or nitroprusside-induced decreases in blood pressure. In SHR rats, the depressor response produced by injection of serotonin into the NTS was markedly larger than in WKY rats, and was larger than depressor responses previously reported for other excitatory substances injected into the NTS. In SHR rats bilateral injection of sarpogrelate produced an increase in blood pressure, although it did not alter baroreceptor-evoked changes in heart rate. These results provide further support for the hypothesis that stimulation of 5HT 2 receptors in the NTS contributes to cardiovascular regulation independent of the baroreceptor reflex. Furthermore, this serotonergic system is altered in SHR rats, apparently acting tonically to reduce blood pressure.  2000 Elsevier Science B.V. All rights reserved. Themes: Endocrine and autonomic regulation Topics: Cardiovascular regulation Keywords: Sarpogrelate; Baroreceptor reflex; Hypertension; Brain stem

1. Introduction The nucleus tractus solitarius (NTS), the projection site in the brain stem of baroreceptor afferent nerves, as well as other visceral afferent nerves, plays an important role in cardiovascular control. The NTS contains a large variety of putative neurotransmitters, many of which have been Abbreviations: AP, arterial pressure; CVLM, caudal ventrolateral medulla; DOB, 2,5-dimethoxy-3-bromoamphetamine; DON, 2,5-dimethoxy-3-nitroamphetamine; 5-HIAA, 5-hydroxyindoleacetic acid; 5HT, 5-hydroxytriptamine; MAP, mean arterial pressure; NTS, nucleus tractus solitarius; Sarpo, sarpogrelate, MCI-9042; SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto rat. *Corresponding author. Tel.: 11-412-624-6996; fax: 11-412-6249198. E-mail address: [email protected] (A.F. Sved)

reported to influence cardiovascular regulation [2,12]. Previous studies in rats have noted that injection of serotonin (5-hydroxytryptamine; 5HT) into the NTS, specifically the region in which baroreceptor afferent nerves terminate, elicits a decrease in arterial pressure (AP) and heart rate (HR) [1,4,9,11,16,22]. Although the response to 5HT appears very similar to that of baroreceptor afferent nerve stimulation, blockade of 5HT receptors does not appear to alter baroreceptor reflex responses. Indeed, despite a number of studies [1,11,22] the physiological relevance of 5HT in the NTS to cardiovascular regulation remains unclear. Pharmacologically, small doses of 5HT injected into the NTS decrease AP and HR in anesthetized rats [4,9,11,16,22], as well as in conscious rats [1]. Although some studies support the hypothesis that the response is

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mediated via 5HT 2 receptors [1,16,22], other studies indicate that the response is mediated by 5HT 1 receptors possibly of the 5HT 1B subtype [4,9]. Thus, some researchers find that ketanserin, a 5HT receptor antagonist with specificity for 5HT 2 receptors but also with some affinity for 5HT 1 receptor types, blocks the response to 5HT injected into the NTS. In contrast, other researchers find that the response to 5HT is not blocked by ketanserin [4,9], possibly reflecting the higher doses of 5HT used in these latter studies. Similarly, some investigators find that 5HT 2 receptor agonists, such as 2,5-dimethoxy-3-bromoamphetamine (DOB) and 2,5-dimethoxy-3-nitroamphetamine (DON), injected into the NTS elicit responses similar to those produced by 5HT [16]; on the other hand, other investigators report that 5HT 1 receptor agonists, but not 5HT 2 receptor agonists, produce hypotension and bradycardia when injected into the NTS [4]. Thus, one goal of the present study was to examine the effect on this response, of a new and highly selective 5HT 2 receptor antagonist, sarpogrelate (Sarpo, MCI-9042) [14,18,20]. Previous studies using genetically hypertensive rats of the Okamoto strain (SHR) have indicated that the depressor response to injection of 5HT into the NTS is exaggerated [19]. A similarly exaggerated depressor response has also been reported following injection into the NTS of a selective 5HT 2 receptor agonist [15]. A second purpose of these experiments was, therefore, to further characterize this 5HT-evoked cardiovascular response in SHR rats.

2. Materials and methods Adult male Sprague–Dawley (S–D), Wistar–Kyoto (WKY), and spontaneously hypertensive (SHR) rats (Charles River, Japan), 16–20 weeks of age and weighing between 300 and 480 g were used in these experiments. Animals were housed in groups of 2–3 in hanging wire mesh cages in temperature-controlled rooms with a fixed 12 h light–dark cycle for at least 2 weeks prior to experiments. Food (MF; Oriental Yeast Co.) and tap water were available ad libitum. For measuring arterial pressure (AP) and heart rate (HR) during injections of substances into the NTS, rats were prepared as previously described in detail [27]. Briefly, rats were initially anesthetized with halothane and cannulas were inserted into a femoral artery and a femoral vein. The trachea was cannulated and the rat was connected to a ventilator. The rat was placed into a stereotaxic instrument and the dorsal surface of the medulla was exposed [27]. After completion of all surgery, the rat was injected with alpha-chloralose (60 mg / kg iv) and the halothane was terminated. Additional chloralose (20 mg / kg iv) was administered hourly. Rats were injected with tubocurarine (0.5 mg / kg, supplemented hourly with 0.2 mg / kg) and ventilated with 100% oxygen for the remainder of the experiment. Injections of solutions into the NTS were

made using single-barrel glass micropipettes; coordinates for injections into the NTS were 0.5 mm rostral to the caudal tip of the area postrema, 0.5 mm lateral to the midline, and 0.5 mm below the dorsal surface of the brain stem. All injections were in a volume of 100 nl artificial CSF vehicle administered during 2–5 s. Bilateral injections were made one side at a time, with approximately 30 s separating the two injections. In experiments in which an antagonist was injected followed by the injection of an agonist, the pipette was removed and replaced for the subsequent injections; typically the agonist was injected 5–10 min after injection of the antagonist, although the time delay was only 1–2 min in some experiments. In experiments in which rats received multiple injections (e.g., multiple doses of 5HT), at least 10 min of stable baseline MAP was recorded between injections. In some experiments, baroreceptor reflex responses were tested before injection of Sarpo and then again approximately 2 min after bilateral injection of Sarpo into the NTS. Baroreceptor reflexes were tested by measuring the maximal change in HR produced in response to increasing AP with a bolus injection of phenylephrine (2 mg / kg iv) or decreasing AP with a bolus injection of sodium nitroprusside (5 mg / kg iv). These doses of phenylephrine and nitroprusside were selected because they alter MAP by approximately 40 mmHg. The maximal change in HR was divided by the maximal change in MAP to arrive at a baroreceptor reflex index (BRI) expressed as bpm / mm Hg. At the conclusion of the experiment, approximately 20 nl of 1% Fast green dye was injected into the NTS for histological verification of the center of the microinjection site. All injection sites were centered in the medial subnucleus of the NTS at the level of the rostral area postrema. Sarpo was obtained from Mitusbishi Chemical Corp. (Japan). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Data are expressed as mean6S.E.M. Data were analyzed by ANOVA, followed by Scheffe’s test. Dose-response data were analyzed by one-way ANOVA in S–D rats and by 2-way ANOVA in SHR and WKY rats. Other responses in SHR and WKY rats were compared by t-test.

3. Results

3.1. Studies in Sprague–Dawley rats Unilateral microinjection of doses of 5HT within the pmol range into the NTS of S–D rats anesthetized with chloralose produced a decrease in AP and HR (Fig. 1A), as previously noted by other investigators [1,16,22]. The response was dose-dependent, rapid in onset, and transient, lasting less than 1 min. Injection of vehicle did not significantly alter MAP (262 mmHg) or HR (2261bpm) (n57). The smallest dose to elicit a statistically significant

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Fig. 1. Typical polygraph records of the effect of pretreatment with Sarpo on the cardiovascular responses to 5HT, glutamate, and acetylcholine injected into the NTS. Panel A shows the response in a rat in which 20 pmol 5HT was injected unilaterally into the NTS before and 2 min after injection of CSF vehile into the NTS. In a group of rats treated this manner 5HT injection decreased MAP 4469 mm Hg before injection of CSF and 3968 mm Hg afterwards (n59). Panel B shows responses to 5HT before and after injection of 200 pmol Sarpo into the NTS. In a group of rats treated in this manner, 5HT decreased MAP 4267 mm Hg before Sarpo and 461 mm Hg afterwards (n55; P,0.05) The effects of Sarpo on the response to glutamate (Panel C) and acetylcholine (Panel D) are also presented. In 5 rats, glutamate decreased MAP 3663 mm Hg and this was not altered by pretreatment with Sarpo. Similarly, in 5 rats ACh injection decreased MAP by 3166 mm Hg and this was not altered by Sarpo pretreatment.

decrease in MAP was 1 pmol (21364 mmHg, n58, P,0.05 compared to either baseline values or vehicle injections), whereas the minimal dose to elicit the maximal response, a decrease in MAP of 4466 mmHg and a bradycardia of 104648 bpm (n56), was 10 pmol. To determine the ability of the 5HT 2 receptor anatagonist Sarpo to effectively and selectively block this action of 5HT, the responses to a supramaximal dose of 5HT or other depressor substances were tested before and 5–10 min after injection of Sarpo into the same site. Sarpo, injected unilaterally at a dose of 200 pmol, did not alter baseline AP or HR but completely prevented the depressor

and bradycardic response to 20 pmol of 5HT (Fig. 1B); 100 pmol Sarpo attenuated the response to 5HT by 70% (5HT decreased MAP by 3969 mmHg before Sarpo and 1164 mmHg afterwards; n55; P,0.05). In other rats (n55), 200 pmol Sarpo completely blocked the response to 5HT when the antagonist was injected 1–2 min prior to the agonist. In contrast to the effects of Sarpo on the responses elicited by 5HT, injection of 200 pmol of Sarpo into the NTS did not alter the depressor and bradycardic responses elicited by injection of 150 pmol of glutamate (Fig. 1C) or 440 pmol of acetylcholine (Fig. 1D) into the NTS. These doses of glutamate and acetylcholine elicit similar AP and

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HR responses to those produced by 5HT, and, like 20 pmol 5HT, represent maximally effective doses of these substances [28]. Bilateral injection of 200 pmol of Sarpo into the NTS did not alter MAP (11667 mmHg before vs. 11465 mmHg after, n56, P.0.1) and HR (400625 bpm before vs. 366617 bpm after, n56, P.0.1). In addition, bilateral injection of Sarpo into the NTS did not significantly alter baroreceptor evoked changes in HR, assessed by reflex changes in HR in response to either increases in AP evoked by phenylephrine or decreases in AP evoked by nitroprusside. Using phenylephrine to elicit reflex responses, the BRI was 2.860.8 bpm / mmHg before injection of Sarpo and 3.360.9 bpm / mmHg after injection of Sarpo (n56, P.0.1); when tested with nitroprusside, the BRI was 1.060.2 and 1.460.5 bpm / mmHg before and after injection of Sarpo (n56, P.0.1), respectively.

3.2. Studies in SHR and WKY rats Baseline MAP in chloralose-anesthetized SHR rats was greater than in WKY rats, whereas HR was similar in the two strains (Table 1). Unilateral injection of 5HT into the NTS of SHR and WKY rats elicited decreases in AP and HR. At all doses tested, 5HT elicited a larger decrease in MAP in SHR than in WKY rats (Table 1, Fig. 2). In WKY and SHR rats, the dose-response relationship for 5HTevoked depressor responses was shifted to the left compared to Sprague–Dawley rats, and in WKY rats the smallest dose tested, 1 pmol, elicted a maximal response (Table 1). The maximal decrease in MAP elicited by 5HT in SHR rats was approximately double that in WKY rats. In contrast, decreases in HR evoked by injection of 5HT into the NTS were similar between SHR and WKY rats (Table 1).

As in Sprague–Dawley rats, unilateral injection of 200 pmol Sarpo into the NTS did not alter baseline MAP or HR in SHR and WKY rats, but did prevent the depressor and bradycardic response elicited by subsequent injection of 20 pmol of 5HT (Fig. 2). Bilateral injection of 200 pmol Sarpo into the NTS of SHR increased MAP by approximately 15 mmHg (Table 2), a response that was not observed in WKY rats. The increase in MAP elicited by bilateral injections of Sarpo in SHR rats lasted 15–20 min, and MAP returned to baseline values within 30 min in all rats. Sarpo did not alter HR in SHR rats, whereas it elicited a small bradycardia in WKY rats. Baroreceptor reflex-evoked changes in HR were also tested before and after bilateral injection of Sarpo into the NTS in WKY and SHR rats. As previously noted by others [6,7,13], the sensitivity of the baroreceptor reflex was attenuated in SHR rats compared to WKY rats. However, Sarpo did not alter baroreceptor reflex changes in HR in either rat strain (Table 3).

4. Discussion Injection of pmole doses of serotonin into the NTS evoked a rapid and transient decrease in AP and HR, in full agreement with previous studies [9,11,16,22]. The present results show that this cardiovascular response elicited by 5HT can be completely prevented by Sarpo, a selective 5HT 2 receptor antagonist. Thus the present studies are consistent with previous work showing that selective 5HT 2 receptor agonists (e.g., 2,5-dimethoxy-3bromo-amphetamine, DOB) injected into the NTS also lower AP and HR [15,16]. Studies using the 5HT receptor antagonists ketanserin, ritanserin, and piremperone have also suggested that the response to 5HT is mediated via

Table 1 Effect of 5HT injected into the NTS on blood pressure and heart rate in SHR and WKY rats a 5HT

Mean arterial pressure (mm Hg) Baseline

Change

0 pmol

SHR (n55) WKY (n55)

15968 b 11369

2161 163

1 pmol

SHR (n57) WKY (n56)

15368 b 11365

5 pmol

SHR (n57) WKY (n56)

20 pmol

SHR (n56) WKY (n56)

a

Heart rate (bpm) % change

Baseline

060 060

360616 368611

Change

24565 b c d e 22866 c e d

23064 e d 22566 e d

362612 366613

253613 c e d 270623 c e d

21564 e d 22067 e d

15067 b 11962

26165 c b e d 23368 c e

24265 b e d 22868 e

357610 356610

290626 c e 270620 c e

22567 e 22066 e

154611 b 11867

26263 c b e 23465 c e

24164 b e 22864 e

368610 364617

2100625 e 296615 c e

22766 e 22764 e

364 2365

% change 060 060

Different doses of 5HT were injected unilaterally into the NTS in chloralose-anesthetized SHR and WKY rats. Data are expressed as the maximal change in MAP and heart rate that occured within 1 min of the injection. Each dose tested elicited a significant change MAP and heart rate in each strain. b Indicates significant differences between SHR and WKY rats, P,0.05. c Indicates significant change from baseline, P,0.05. d Indicates significant difference from the next smaller dose, P,0.05. e Indicates significant change from vehicle (0 pmol), P,0.05.

K. Tsukamoto et al. / Brain Research 863 (2000) 1 – 8

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Fig. 2. Typical polygraph records of the effects of injection of 5HT into the NTS of SHR and WKY rats. 5HT (20 pmol) was injected unilaterally into the NTS before and approximately 2 min after injection of Sarpo (200 pmol) into the same site. In WKY rats (n55) Sarpo reduced the 5HT-evoked depressor response from 3063 mm Hg to 664 mm Hg (P,0.05). In SHR rats (n55) the 5HT evoked depressor response was 5563 mm Hg before injection of Sarpo and 866 mm Hg after Sarpo injection (P,0.05).

5HT 2 receptors [16,22], although this finding has not been observed by all investigators [4,9], possibly due to the higher doses of 5HT used in these latter studies. Sarpo appears to be a highly selective 5HT 2A antagonist [14,18,20], although it is about 10-fold less potent than ketanserin. This difference in potency between Sarpo and ketanserin is consistent with the doses used in this study; previous studies showing that ketanserin blocked the responses to 20 pmol of 5HT injected into the NTS used 10 pmol of ketanersin [16,22] whereas in the present study 200 pmol of Sarpo was needed to fully block the effects of that dose of 5HT. The specificity of Sarpo for 5HT 2A

receptors is based on a variety of receptor binding assays and functional tests. Among 5HT receptor subtypes, Sarpo displaces with high affinity only the binding of drugs that bind to 5HT 2A receptors [18]; Sarpo also displaced the binding of iodocyanopindolol, although with 100-fold lower affinity, suggesting that Sarpo may also bind to 5HT 1B receptors. Binding assays and functional tests also suggest that Sarpo has a weak affinity for alpha-adrenergic receptors, with an affinity at least 100-fold less than that for 5HT 2A receptors [20]. Ketanserin can also bind to alpha-adrenergic receptors, and the selectivity of ketanserin for 5HT 2 receptors compared to alpha-adrenergic

Table 2 Effect on blood pressure and heart rate of bilateral injection of Sarpo into the NTS in SHR and WKY rats a Mean arterial pressure (mm Hg)

Heart rate (bpm)

Baseline

Change with vehicle

Baseline

SHR (n55) WKY (n55)

16267 c 11865

2262 2262

364615 360615

SHR (n59) WKY (n59)

16266 c 11665

Change with Sarpo 1764 b c d 465

358616 353610

a

Change with vehicle 2462 2367 Change with Sarpo 2464 22467 c d

Sarpo (200 pmol) was injected bilaterally into the NTS in chloralose-anesthetized SHR and WKY rats. Data are expressed as the maximal change in MAP or HR occuring within 3 min of the injection on the second side of the NTS. b Indicates significant change from baseline. c Indicates significant difference between SHR and WKY rats, P,0.05. d Indicates significant change from vehicle, P,0.05.

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Table 3 Effect of Sarpo on the baroreceptor reflex in SHR and WKY rats a BRI tested with phenylephrine

SHR (n56) WKY (n55)

BRI tested with nitroprusside

Control

After Sarpo

Control

After Sarpo

1.360.2* 2.360.3

1.060.3* 2.160.2

0.760.1 1.660.3

0.760.1* 1.760.4

a

The baroreceptor reflex index (BRI) was calculated as the maximal change in HR (bpm) divided by the maximal change in MAP (mm Hg) elicited by intravenous injection of either phenylephrine (2 mg / kg) or nitroprusside (5 mg / kg). BRI was determined in chloralose-anesthetized SHR and WKY rats before (control) and approximately 5–10 min after injection of Sarpo (200 pmol) into NTS bilaterally. Sarpo did not alter BRI in either strain although BRI was less in SHR than in WKY rats (* P,0.05). In these rats, baseline MAP was 15066 in SHR and 10864 in WKY rats, HR was 372614 in SHR and 326626 in WKY rats. Phenylephrine increased MAP to the same extent in both rat strains (3765 in SHR and 3062 in WKY) whereas nitroprusside elicited a similar decrease in MAP in the two strains (25364 in SHR and 24365 in WKY).

receptors is not as great as it is with Sarpo. Also, unlike ketanserin, which has a relatively high affinity for histamine H 1 receptors, Sarpo shows very little binding to H 1 receptors [20]. Thus, based on the pharmacological selectivity of Sarpo, these experiments provide additional data in support of the position that the cardiovascular effects of 5HT injected into the NTS are mediated by 5HT 2 receptors, possibly 5HT 2A receptors. Injection of Sarpo into the NTS, in doses that completely block the effect of exogenous 5HT, did not alter cardiovascular responses elicited by either glutamate or acetylcholine, two other agents that elicit a similar depressor and bradycardic response. Similarly, glutamateevoked depressor responses are not blocked by metergoline [26], a less selective 5HT antagonist. Prior studies had noted that the response to 5HT injected into the NTS resembled the response to baroreceptor stimulation, and we have observed that the effects of aortic depressor nerve stimulation and injection of 5HT into the NTS on the pattern of regional blood flow are similar (Ito and Sved, unpublished). Nonetheless, Sarpo injected into the NTS, did not alter baroreceptor evoked changes in HR, in agreement with previous studies using other 5HT antagonists [1,11,22]. Thus, 5HT 2 receptors do not appear to be essential for baroreceptor reflex-mediated changes in HR and do not tonically influence the baroreceptor reflex. However, it remains possible that stimulation of 5HT 2 receptors under conditions of increased 5HT release may facilitate reflex responses and therefore produce responses that resemble those of the reflex. In SHR rats, the depressor effect of 5HT injected into the NTS is considerably larger than that observed in WKY rats. This enhanced depressor response to injection of 5HT [19] or the specific 5HT 2 agonist DOB [15] has been noted previously. Like in Sprague–Dawley rats, the response to 5HT in SHR rats was completely blocked by Sarpo,

indicating that it is mediated solely by 5HT 2 receptors. The larger 5HT-evoked depressor response in SHR rats compared to WKY rats is apparent whether the data are expressed as absolute change in AP, which we have previously argued may be the most appropriate way to analyze this type of data [27], or as percent change in AP. The enhanced depressor response to 5HT injected into the NTS in SHR relative to WKY is particularly interesting in light of the similarity of depressor effects of other agents injected into the NTS. Expressed as an absolute change in MAP, glutamate and acetylcholine evoked depressor responses are similar in the two strains [27]. Furthermore, the maximal depressor response elicited by electrical stimulation of the aortic depressor nerve is not different between strains [5,29]. Thus, the effect of 5HT is somehow different from these other treatments that elicit depressor responses via stimulation of NTS neurons. The maximal depressor response to 5HT in SHR rats is also surprising in terms of its absolute magnitude, a decrease in MAP of approximately 65 mmHg. In contrast, maximal stimulation of the aortic depressor nerve decreases MAP by only 40–50 mmHg in both strains [5,29]; other substances that elicit a decrease in MAP when injected into the NTS of SHR rats also decrease MAP to a lesser extent, typically 40–50 mmHg. Even a maximallyeffective dose of glutamate, which should excite all NTS neurons, elicits a depressor response that, in SHR rats, is smaller than the maximal effect of 5HT (P,0.05, comparing present data to those published previously by Tsukamoto and Sved [27]). In WKY rats, all of these treatments result in a similar maximal decrease in MAP. Thus, the magnitude of the depressor response elicited in SHR rats by injection of 5HT into the NTS suggests that the response contains components different from the baroreceptor reflex. In normotensive rats, the depressor response elicited by 5HT, as well as glutamate and aortic depressor nerve stimulation, involves a neural pathway including the caudal ventrolateral medulla (CVLM) [21,24]. The CVLM has been suggested to mediate baroreceptor-evoked and NTS-evoked depressor responses as well as depressor responses that are independent of the baroreceptor reflex and the NTS [25]. Thus, it is possible that the enhanced depressor response to 5HT in SHR rats is mediated via baroreceptor-independent circuitry involving the NTS and CVLM. Such a mechanism would predict that stimulation of the CVLM should elicit an enhanced depressor response in SHR rats, and such a response has been reported [8,17,23]. If such a mechanism involving baroreceptorindependent circuits in CVLM that can be driven from the NTS is responsible for the enhanced response to 5HT in SHR rats, how can injection of glutamate into the NTS, which should also excite this pathway, not elicit a similar response? One possible explanation would be that glutamate excites inhibitory as well as excitatory projections from the NTS. Indeed, injection of glutamate into the NTS

K. Tsukamoto et al. / Brain Research 863 (2000) 1 – 8

should stimulate the chemoreceptor reflex and therefore act to increase MAP. There is, however, another way to consider these data. Ganglionic blockade decreases MAP to a greater extent in SHR than WKY rats; in rats prepared similarly to those in the present study, MAP decreased by 9369 mm Hg in SHR rats (n57) compared to 4964 mmHg in WKY rats (n55) (P,0.05, Ito and Sved, unpublished observation). Thus, expressed as a percent of maximal decrease in blood pressure produced by inhibition of sympathetic outflow, the depressor response to injection of 5HT into the NTS is approximately 65–70% in both strains. In contrast, the response to stimulation of the aortic depressor nerve or injection of glutamate or acetylcholine into the NTS is comparatively greater in WKY rats than in SHR rats. This way of expressing the results is consistent with the limited data available regarding the effects of these treatments on sympathetic nerve activity. Thus, stimulation of the aortic depressor nerve or injection of glutamate into NTS elicits a smaller percent inhibition of sympathetic nerve activity in SHR rats than in WKY rats [19,29] whereas injection of 5HT into the NTS elicits a similar percent decrease in renal sympathetic nerve activity in both strains [19]. Nonetheless, comparing between SHR and WKY rats the depressor response to 5HT injected into the NTS is quantitatively different from that elicited by either stimulation of the aortic depressor nerve or injection of glutamate or acetylcholine into the NTS. Although the bradycardic response to injection of 5HT into the NTS appears similar between SHR and WKY rats, this response also deserves comment. Stimulation of the aortic depressor nerve or injection of glutamate or acetylcholine into the NTS elicits a decrease in HR in SHR rats that is significantly smaller than the response in WKY rats [27,29]. Thus, as with 5HT induced changes in AP, the HR response in SHR rats relative to that elicited from stimulation of baroreceptor afferents or injection of glutamate or acetylcholine into the NTS is different than in WKY rats. Interpretation of the HR responses might be most consistent with analysis of AP responses based on their magnitude relative to that produced by ganglionic blockade, as described above. Thus, it may be that compared to the control, WKY strain, decreases in AP and HR elicited by baroreceptor reflex circuitry at the level of the NTS might be decreased in SHR whereas the response to serotonin, working on a background of elevated sympathetic outflow and AP, is normal. However, not only is the response to 5HT injected into the NTS larger in SHR rats compared to WKY rats, serotonergic mechanisms in the NTS in SHR rats appear to be tonically active. In contrast to the response observed in normotensive Sprague–Dawley and WKY rats, bilateral injection of Sarpo into the NTS of SHR rats elicited a significant and consistent increase in blood pressure. Nevertheless, Sarpo did not appear to alter baroreceptor reflex-evoked responses. Thus, in SHR rats, under baseline

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conditions serotonin appears to stimulate serotonin receptors in the NTS. Previous biochemical data suggest that serotonergic mechanisms may be altered in the NTS of SHR rats. Kuolu et al. [10] noted that levels of 5HT and its major metabolite (5HIAA), as well as in vivo tryptophan hydroxylation rate, in the NTS were elevated in 4-week old SHR rats compared to WKY rats. However, in 14-week old rats these differences between SHR and WKY rats were not apparent [10]. Extracellular fluid levels of 5HIAA, monitored by microdialysis, did not differ between strains [3], although the increase in response to baroreceptor stimulation may have been greater in SHR rats. In summary, the present data demonstrate that a 5HT 2 receptor mediated depressor mechanism in the NTS is altered in SHR rats. The response, which may relate to baroreceptor-independent mechanisms of blood pressure regulation, is tonically active and more pronounced in SHR rats compared to WKY rats.

Acknowledgements These studies were supported in part by a grant from Nihon University School of Medicine and a grant from the National Institutes of Health of the United States (HL55687 to AFS).

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