A study to investigate capsaicin-induced pressure response in vagotomized rats.

OBJECTIVES: Capsaicin is used to evoke pulmonary C reflexes and produces complex pressure responses along with apnea/tachypnea, and bradycardia. In the present study, the mechanisms involved in capsaicin-induced pressure responses were explored.
MATERIALS AND METHODS: Tracheal, jugular venous, and femoral artery cannulations were performed in anesthetized adult rats. Blood pressure, respiratory excursions, and electrocardiogram were recorded. Cardiorespiratory reflex changes evoked by jugular venous injection of capsaicin (10 μg/kg) were recorded in vagotomized and antagonist pretreated animals.
RESULTS: Capsaicin produced triphasic pressure response exhibiting immediate hypotension, intermediate recovery, and delayed hypotension. Time-matched respiratory changes showed apnea, bradypnea, and tachypnea, respectively. Bradycardia occurred at immediate and intermediate phases. After vagotomy, immediate hypotension was abolished; the intermediate recovery was potentiated as hypertensive response; and the delayed hypotension persisted. In case of respiration, the immediate bradypnea persisted and delayed tachypnea was abolished; while heart rate changes at immediate and intermediate phases were abolished. Antagonists of α1-adrenoceptor (prazosin or terazosin, 0.5 mg/kg), β-adrenoceptor (propranolol, 1 mg/kg), AT1 receptor (losartan, 10 mg/kg) and Ca(2+) channel (diltiazem, 1 mg/kg) failed to block the capsaicin-induced intermediate hypertensive response in vagotomized animals.
CONCLUSIONS: These observations implicate the existence of mechanisms other than adrenergic, angiotensinergic, or Ca(2+) channel-dependent mechanisms for mediating the capsaicin-induced intermediate hypertensive response in vagotomized animals.

Introduction

Capsaicin is a nociceptive substance present in Capsicum annuum and is a potent stimulant of transient receptor potential vanilloid type 1 (TRPV1) receptors present on C fibers.[12] Intravenous injection of capsaicin produces reflex changes in cardiorespiratory parameters manifesting as apnea, bradycardia, and triphasic pressure response in anesthetized animals.[23456] Capsaicin-induced triphasic pressure response exhibits as immediate decrease, intermediate increase, and delayed prolonged decrease in blood pressure (BP).[78910] Vagotomy abolished the immediate fall in pressure (1st phase of response) while potentiating the intermediate increase of pressure.[10] The immediate hypotensive response therefore appears to be vagally mediated; however, the mechanism underlying the capsaicin-induced intermediate increase of pressure is poorly understood.[78910] The increase in pressure can be due to the activation of adrenergic or angiotensinergic mechanisms. The actions of adrenoceptors either at α- or β-receptors are mediated through G-protein-coupled receptors (GPCR) which in turn increases the intracellular calcium level via Ca2+ channels.[11] Angiotensin-II, another potent vasoconstrictor, also activates GPCR stimulating PIP3-mediated pathway for the opening of L-type of Ca2+ channels.[121314] Increased intracellular Ca2+ in turn increases arteriolar muscle tone and also activates TRPV1 receptors.[131516] Further, TRPV1 receptor is shown to regulate intracellular Ca2+ levels independently.[16] Thus, Ca2+ plays a vital role in TRPV1-mediated actions and its role in capsaicin-induced pressure response is not known. Therefore, the present study was undertaken to explore the involvement of Ca2+ channel for the hypertensive response induced by capsaicin. Further, the involvement of adrenoceptors and angiotensinergic receptors for these responses was also delineated.

Materials and Methods

Animals

The study protocol was approved by ethics committee of the Institute (Dean/2007-08/1442) for conducting animal experiments. Adult female rats of Charles Foster strain weighing 176 ± 14 g were used. The animals were housed in a temperature, humidity, and light (12 h:12 h light dark period) controlled room with ad libitum food and water.

Dissection and Recording

The methods for dissection and recording of cardiorespiratory parameters were as described earlier.[1718] Briefly, animals were anesthetized with urethane (1.5 g/kg i.p.). An additional dose of urethane (0.1–0.15 g/kg i.p.) was injected whenever required as assessed by the corneal and withdrawal reflexes. Trachea, jugular vein, and femoral artery were cannulated. Tracheal cannulation was used to keep the respiratory tract patent; jugular venous cannulation for capsaicin/antagonist administration, and femoral artery cannulation for recording BP via Statham transducer. Eletrocardiographic potentials were recorded by connecting the needle electrodes in standard limb lead-II configuration. Respiratory movements were recorded by securing a thread to the skin over the xiphisternum to a force-displacement transducer. All the recordings were taken on a computerized chart recorder.

Drugs and Solutions

Capsaicin, prazosin (α1-adrenoceptor antagonist), propranolol (β-adrenoceptor antagonist), and losartan (AT1 receptor antagonist) were obtained from Sigma Chemical Company St. Louis, MO, USA. Dilztiazem (L-type of Ca2+ channel antagonist) was from WAKO pure Chemical Industries Ltd, Osaka, Japan. Terazosin (α1-adrenoceptor antagonist) was from Abbott India Ltd, Mumbai. Stock solution of capsaicin (1 mg/mL) was prepared in ethanol. Stock solution of other drugs was made in distilled water and diluted with normal saline at the time of administration. The volume of the injections was kept at 0.1 mL.

Experimental Protocols

The animals were divided broadly into two groups: In Group-I (n = 12) after obtaining the initial recordings [respiration, electrocardiogram (ECG), and BP], capsaicin (10 μg/kg) was administered intravenously as bolus injection. The arterial pressure, respiration, and heart rate (HR) were recorded for 1 min. Subsequently, bilateral vagotomy was performed and 10 min later capsaicin (10 μg/kg) response was obtained as before.

In Group-II (n = 17), capsaicin (10 μg/kg)-induced reflex response was recorded initially and after bilateral vagotomy as described in Group-I. Subsequently, prazosin (0.5 mg/kg), terazosin (0.5 mg/kg), propranolol (1 mg/kg), losartan (10 mg/kg), or diltiazem (1 mg/kg) was injected and 15 min later capsaicin (10 μg/kg) reflex response was recorded. The dosage of various antagonists was selected from the earlier reports.[192021]

Statistical Analysis

Peak changes in mean arterial pressure (MAP) (mm Hg) from initial at different phases after capsaicin were computed and expressed as percentage. The time-matched respiratory frequency (RF) and HR at immediate (0–10 s), intermediate (10–20 s), and delayed (45–60 s) phases of BP response were computed after capsaicin administration. The responses were normalized to the respective initial values (before capsaicin administration). The data were pooled to obtain mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to compare the significant difference between values before vagotomy, after vagotomy or after pretreatment with antagonists in vagotomized animals. Student-Newman-Keuls test was applied for multiple comparisons at each phase of response. In addition, Student's t test was used to compare the paired responses before and after vagotomy. P < 0.05 was considered significant.

Results

A typical triphasic response pattern in arterial BP at 10 μg/kg of capsaicin was observed with preliminary experiments. Therefore, this concentration was used for further experiments.

Capsaicin-induced Pressure Response

Injection of capsaicin (10 μg/kg) produced changes in MAP in a time-dependent manner. There was immediate fall (by 50%) after a latency of 1.3 ± 0.1 s and lasted for 5–6 s. Subsequently, the pressure recovered and peaked slightly above the initial level [Figure 1]. The latency and duration of this response was 6.1 ± 0.4 s and 10 s, respectively. Thereafter, there was a delayed fall in pressure (by 30%) with a latency of 17.7 ± 1.4 s and persisted for longer time. Thus, the MAP response can be categorized as immediate hypotensive, intermediate recovery, and a delayed hypotensive response. The time-matched respiratory responses to the pressure responses manifested as bradypnea in immediate and intermediate phases and tachypnea in delayed phase, respectively [Figure 1]. Bradycardia was observed at immediate and intermediate phases [Figure 1].

Figure 1

Capsaicin-induced cardiorespiratory responses before and after vagotomy. The original tracings of an experiment showing the capsaicin (10 μg/kg)-induced changes in blood pressure (BP); respiration (Resp); and heart rate (ECG), before and after vagotomy are presented in the left panel. Vertical dashed line indicates the point of injection of capsaicin (10 μg/kg). Horizontal line (time scale) = 5 s. Recording of BP at 10 times slower speed is shown in inset A and B for before and after vagotomy, respectively. In inset A, the triphasic BP response (immediate hypotension, intermediate recovery, and delayed hypotension) after capsaicin is shown and in inset B, the potentiation of intermediate hypertensive response is indicated by an arrow. The mean ± SEM values (n= 12) of MAP, RF, and HR as % of initial at immediate (Immed), intermediate (Inter), and delayed phases are presented in the bar diagrams. * (P< 0.05, Student's t-test for paired observations)

Capsaicin-induced Pressure Response Following Vagotomy

The initial values of MAP, RF, and HR before vagotomy were 97 mm Hg, 66 breaths/min and 352 beats/min, respectively [Table 1]. After bilateral vagotomy, resting RF was decreased by 50% (P < 0.05), while there was no alteration in MAP or HR. In vagotomized animals, capsaicin-induced intermediate hypertensive response was potentiated (44% increase; P < 0.05) [Figure 1]. However, the bradycardiac response was abolished but bradypneic response persisted at this phase. In these animals, capsaicin-induced immediate hypotensive and bradycardiac responses were not seen while bradypnea persisted. The delayed hypotensive response persisted even after vagotomy, while tachypnea and bradycardia were abolished [Figure 1].

Table 1

Comparison of MAP, RF, and HR in animals before and after vagotomy and after respective antagonist

Since, intermediate hypertensive response was potentiated after vagotomy; further, experiments were performed to understand the mechanisms involved in the potentiation of intermediate hypertensive response.

Effect of α1-Adrenoceptor Antagonists on Capsaicin-induced Hypertensive Response in Vagotomized Animals

In vagotomized animals, prazosin (0.5 mg/kg) per se decreased the resting MAP significantly (P < 0.05) [Table 1], but HR and RF were not altered from the pre-prazosin values. However, prazosin pretreatment did not reverse the intermediate hypertensive response produced by capsaicin [Figure 2]. Similarly, the HR and RF changes at various phases after prazosin treatment remained similar to the responses in vagotomized animals [Figure 2].

Figure 2

The original tracings of blood pressure (BP), respiration (Resp), and electrocardiogram (ECG) before, after vagotomy and after prazosin in vagotomized animals are presented on the left. Vertical dashed line indicates the point of capsaicin administration. Horizontal line (time scale) = 5 s for all. The mean ± SEM values (n = 3) of MAP, RF, and HR at immediate (Immed), intermediate (Inter), and delayed phases are presented in the bar diagrams. Before indicates before vagotomy; + VagX indicates after vagotomy; and + Prazosin indicates prazosin after vagotomy. * (P < 0.05, One-way ANOVA followed by Student-Newman-Keuls test).

In terazosin-treated group, capsaicin-induced triphasic pressure response before and after vagotomy were similar to the earlier group [Figure 1]. In vagotomized animals, terazosin (0.5 mg/kg) per se decreased the resting MAP significantly (P < 0.05) [Table 1], but HR and RF were not different from the pre-terazosin values. After terazosin pretreatment, the intermediate hypertensive response produced by capsaicin was not blocked (P < 0.05) [Table 2].

Table 2

Capsaicin-induced MAP responses in animals before, after vagotomy, and after vagotomy + terazosin

Effect of β-Adrenoceptor Antagonist on Capsaicin-induced Hypertensive Response in Vagotomized Animals

In vagotomized animals, propranolol (1 mg/kg) per se did not produce alterations in the resting MAP, HR, and RF [Table 1]. Propranolol pretreatment did not block the intermediate hypertensive response produced by capsaicin [Figure 3]. The RF and HR changes at various phases after propranolol remained similar in vagotomized animals [Figure 3].

Figure 3

The original tracings of BP, respiration (Resp), and ECG before, after vagotomy and after propranolol in vagotomized animals are presented on the left. Vertical dashed line indicates the point of capsaicin administration. Horizontal line (time scale) = 5 s for all. The mean ± SEM values (n = 3) of MAP, RF, and HR at immediate (Immed), intermediate (Inter), and delayed phases are presented in the bar diagrams. Before indicates before vagotomy; + VagX indicates after vagotomy; and + Propran indicates propranolol after vagotomy. *(P < 0.05, One-way ANOVA followed by Student- Newman-Keuls test).

Effect of AT1 Receptor Antagonist on Capsaicin-induced Hypertensive Response in Vagotomized Animals

In vagotomized animals, losartan (10 mg/kg) per se decreased the resting MAP significantly [Table 1]. Losartan pretreatment did not block the intermediate hypertensive response produced by capsaicin [Figure 4]. The HR and RF changes at various phases after losartan remained similar to the responses in vagotomized animals [Figure 4].

Figure 4

The original tracings of BP, respiration (Resp), and ECG before, after vagotomy and after losartan in vagotomized animals are presented on the left. Vertical dashed line indicates the point of capsaicin administration. Horizontal line (time scale) = 5 s for all. The mean ± SEM values (n = 4) of MAP, RF, and HR at immediate (Immed), intermediate (Inter), and delayed phases are presented in the bar diagrams. Before indicates before vagotomy; + VagX indicates after vagotomy; and + Losart indicates losartan after vagotomy. (*) indicates significant difference from the before values at each phase (P < 0.05, One-way ANOVA followed by Student-Newman-Keuls test)

Effect of Ca2+ Channel Antagonist on Capsaicin-induced Hypertensive Response in Vagotomized Animals

In vagotomized animals, diltiazem (1 mg/kg) did not produce alterations in the resting MAP, HR, and RF [Table 1]. After diltiazem pretreatment, the intermediate hypertensive response produced by capsaicin was not reversed to the initial level [Figure 5]. The HR and RF changes at various phases after diltiazem remained similar to the responses in vagotomized animals [Figure 5].

Figure 5

The original tracings of BP, respiration (Resp), and ECG before, after vagotomy and after diltiazem in vagotomized animals are presented on the left. Vertical dashed line indicates the point of capsaicin administration. Horizontal line (time scale) = 5 s for all. The mean ± SEM values (n = 3) of MAP, RF, and HR at immediate (Immed), intermediate (Inter), and delayed phases are presented in the bar diagrams. Before indicates before vagotomy; + VagX indicates after vagotomy; and + Dilt indicates diltiazem after vagotomy. *(P< 0.05, One-way ANOVA followed by Student-Newman-Keuls test)

Discussion

The present study while confirming the triphasic BP response produced by capsaicin furhter reveals that the immediate hypertensive response is vagally mediated but not the intermediate and delayed responses. In contrast, the intermediate hypertensive response was potentiated and manifested as hypertensive response after vagotomy which was not mediated through the Ca2+ channel-dependent mechanism.

The immediate phase of the triphasic pressure response occurred with a short latency and was abolished after vagotomy. These observations support for the vagally mediated reflex response. The intermediate hypertensive response persisted even after vagotomy rather it got potentiated. This can be due to the increased cardiac activity or due to increased peripheral vascular tone. Intermediate response was associated with bradycardia (HR-response) before vagotomy and after vagotomy the HR response was abolished while the hypertensive response was potentiated. Thus, it indicates the noninvolvement of HR changes for the potentiation of hypertensive response [Figure 1]. In the absence of pacemaker involvement, hypertensive response can be due to the increased peripheral vasoconstriction produced by sympathetic overactivity. In a study elsewhere, the hypertensive response to capsaicin was not attenuated in spinalized rats.[8] Thus, indicating the noninvolvement of medullary sympathetic drive. Further, the noninvolvement of α-adrenoceptors and angiotensinergic mechanisms has been mentioned elsewhere with no supporting data.[10] The present results exclude the involvement of α1-adrenoceptors and β-adrenoceptors. A study elsewhere also demonstrated that phentolamine failed to block the hypertensive response with capsaicin.[8] Thus, the intermediate hypertensive response induced by capsaicin is not due to α1 or β adrenoceptors. In the absence of sympathetic involvement, angiotensin-II, a potent vasoconstrictor, can be expected to produce capsaicin-induced hypertension. However, our results exclude the involvement of AT1-receptor for mediating the response. Similar to angiotensin-II, endothelin-1 is another potent vasoconstrictor. It has been reported that capsaicin induces the release of endothelin from nerve terminals.[22] Therefore, the possibility of endothelin involvement cannot be ruled out for mediating the capsaicin-induced pressure response.

Studies have shown that acute responses to capsaicin involve TRPV1 receptors via calcium-dependent or independent mechanisms.[16] Calcium-calmodulin modulates the TRPV1 activation by capsaicin and is also involved in smooth muscle contractions.[1316] Recently, it has been shown that TRPV1 channels are involved in regulating muscle tone largely through Ca2+ influx.[1516] Therefore, we tested the involvement of calcium by blocking L-type of calcium channel. Our data with diltiazem excludes the involvement of L-type of Ca2+ channels. TRPV1 receptor itself is a nonselective cation channel with high permeability for calcium.[23] Further, the capsaicin-induced activation of TRPV1 receptors is known to increase the influx of Ca2+ into the endothelial cells.[23] Even in the absence of Ca2+ influx, the lyophillic TRPV1 agonists may act directly on the endogenous TRPV1 receptors located on the endoplasmic reticulum to increase the intracellular calcium level which may be responsible for capsaicin-induced contractions (tonic activity).[24]

The hypertensive response can also be due to activation of peripheral chemoreceptors because of hypoxemia resulting from long-lasting apnea. Stimulation of chemoreceptors produces hyperventilation and hypertension. However in the intermediate phase, there was hypoventilation and hypertension, which is contradictory to chemoreceptor stimulation. This is supported by an earlier study where capsaicin-induced hypertension was potentiated after carotid sinus nerve transection.[25] Thus, the hypertensive response may not be mediated by chemoreceptors.

Capsaicin-induced hypertension can also be due to the decreased vasodilatation induced by the depletion of calcitonin gene-related peptide (CGRP) as reported.[2627] They demonstrated hypertension in rats pretreated (4 days before) with very high concentration of capsaicin (50 mg/kg).[24] In contrast to these observations, our data show that the latency for hypertensive response is too short (6 s) to produce the depletion of vasodilator substances. Further, the concentration of capsaicin used in the present study is too small (5000 times). In addition, the hypertensive response was transient and was followed by a delayed hypotensive response. Thus, depletion of CGRP for the capsaicin-induced intermediate hypertensive response is unlikely. However, in acute experiments similar to the present study, CGRP is shown to be released from the capsaicin-sensitive sensory nerves and produces transient hypotension, which may be accounted for delayed hypotensive response.[28]

Taken together, our study demonstrates that capsaicin produces initial hypotensive, intermediate recovery, and delayed hypotensive response as reported earlier.[10] The initial hypotensive response is vagally mediated, while the intermediate and delayed responses are not mediated through vagus. The capsaicin-induced hypertensive response is also not mediated through adrenergic, angiotensinergic, and Ca2+ channel-dependent mechanisms. In the absence of vagal or sympathetic mechanisms, the direct action of capsaicin on smooth muscle may be a possibility. Supporting this speculation, it has been shown that capsaicin produces contraction of vascular smooth muscle in vitro.[2930]