Effect of Substance P on Venous Tone in Conscious Rats

Abstract

Substance P is an undecapeptide that belongs to the mammalian tachykinin family. We investigated the effects of substance P on venous tone in conscious rats through measurement of mean circulatory filling pressure. The effects of substance P (10 and 30 nmol/kg, IV) and the vehicle (0.9% NaCl) on mean arterial pressure, heart rate, and mean circulatory filling pressure were examined in 2 groups each of conscious rats: rats without or with ganglionic blockade through pretreatment with mecamylamine (10 mg/kg, IV) and norepinephrine (4 μg/kg/min, IV). In the unblocked rats, both doses of substance P reduced mean arterial pressure (−19 ± 1 and −35 ± 2 mm Hg) and increased heart rate (+113 ± 14 and +115 ± 23 beats/min). The high dose of substance P also decreased mean circulatory filling pressure (−1.7 ± 0.3 mm Hg). In the ganglionic-blocked rats, both doses of substance P decreased mean arterial pressure (−48 ± 6 and −73 ± 6 mm Hg) and mean circulatory filling pressure (−2.9 ± 0.4 and −4.2 ± 0.8 mm Hg) but did not affect HR. In conclusion, substance P is a vasodilator peptide that has a prominent venodilator action.

Substance P is an undecapeptide that belongs to the mammalian tachykinin family, which also includes neurokinin A and neurokinin B.¹ In addition, the mammalian tachykinin family includes the recently identified hemokinin 1² and endokinins A, B, C, and D.³ The expression of substance P and its mRNA are widely abundant in both the central and peripheral nervous systems.⁴ Substance P-like immunoreactivities have been found in various blood vessels such as the feline cerebral artery,⁵ the rat superior mesenteric artery,⁶ the rat portal vein,⁷ and human ovarian veins.⁸ The actions of substance P are mediated by three distinct G protein-coupled receptors: NK-1, NK-2, and NK-3. Substance P is the preferred ligand for the NK-1 receptor, whereas neurokinin A and neurokinin B are the preferred ligands for NK-2 and NK-3 receptors, respectively.¹ The actions of tachykinins include smooth muscle contraction, vasodilatation, pain transmission, and neurogenic inflammation.⁹

The venous system plays a crucial role in regulating cardiac output. In isolated veins, substance P induced either relaxation^8-10 or contraction.^11-13 Substance P induced in situ dilatation of the human hand vein.¹⁴ The aim of the present study was to determine the effect of substance P on body venous tone through measurement of mean circulatory filling pressure in conscious rats. Mean circulatory filling pressure is the pressure that would occur throughout the circulation if all pressures were brought to an equilibrium.¹⁵ Mean circulatory filling pressure was shown experimentally to be directly proportional to venous return.¹⁵ The effect of substance P on venous tone was also examined in rats devoid of autonomic activity through pretreatment of mecamylamine followed by the restoration of vasomotor tone via the continuous infusion of norepinephrine. Norepinephrine was used because vasodilator drugs have been shown to have greater influence on mean circulatory filling pressure after the elevation of venous tone.^16,17

METHODS

Animals

Male Sprague-Dawley rats (350-450 g) were anesthetized with halothane (4% in air for induction and 1.5% for maintenance). A polyethylene (PE50) catheter was introduced into the left femoral artery to record mean arterial pressure by a pressure transducer (PD23DB, Gould Statham, CA). Heart rate was derived electronically from the upstroke of the arterial pulse pressure (MP150, Biopac Systems, Inc.). The vehicle or drugs were administered through a catheter inserted into the right femoral vein. The left femoral vein was also cannulated to allow the insertion of a catheter into the inferior vena cava for the measurement of central venous pressure by another pressure transducer (PD23DB, Gould Statham). A saline-filled, balloon-tipped catheter was advanced into the right atrium through the right external jugular vein. The proper positioning of the balloon was tested by transiently inflating the balloon, which, when correctly placed, resulted in a simultaneous decrease in mean arterial pressure to 20-25 mm Hg and an increase in central venous pressure within 5 seconds of circulatory arrest. All cannulas were filled with heparinized saline (25 IU/mL) and tunneled to the back of the neck, exteriorized, and secured. The rats were allowed 6 hours to recover from the effects of surgery and anesthesia before further use. All experimental procedures conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were reviewed by the Animal Care Committee of the University of British Columbia.

Measurement of Mean Circulatory Filling Pressure

The method for determining mean circulatory filling pressure has been described in detail elsewhere.¹⁸ Briefly, steady-state readings of mean arterial pressure and central venous pressure were noted at 4-5 seconds after a temporary stopping of the circulation by inflation of the atrial balloon. To correct for the incomplete equilibration of arterial and venous pressures during circulatory arrest, mean circulatory filling pressure was calculated by the following equation: mean circulatory filling pressure = VPP + 1/60(FAP − VPP), where FAP and VPP denote the final arterial pressure and venous plateau pressure, respectively, and 1/60 represents the ratio of arterial to venous compliance.

Experimental Protocol

The rats were divided into four groups (n = 6 each). Mean arterial pressure, heart rate, and central venous pressure were continuously monitored and displayed on a data acquisition system (Acknowledge 3.7, Biopac Systems, Inc). The rats were given 30 minutes to stabilize before baseline values of mean arterial pressure, heart rate, and mean circulatory filling pressure were obtained. Afterward, two groups of rats were given IV injections of substance P (10 and 30 nmol/kg) or an equivalent volume of the vehicle (0.9% NaCl) at dose intervals of 20 minutes. Pressure and heart rate measurements were taken at the plateau phase of the depressor response. Two other groups were pretreated with mecamylamine (10 mg/kg, IV bolus), and measurements were taken 8 minutes later. This dose of mecamylamine was previously shown to completely block the autonomic ganglia for 2 hours.¹⁹ Afterward, norepinephrine was infused continuously (4 μg/kg/min), and measurements were taken at 10 minutes after the initiation of infusion. This was followed by injections of substance P or the vehicle as described for the previous two groups of rats.

Statistical Analysis

All data are presented as mean ± SEM. The data were analyzed by one-way (between groups of rats) or two-way (within the same group) repeated-measures analysis of variance followed by multiple comparisons of group data using the Tukey test (SigmaStat statistical software), with P < 0.05 selected as the criterion for statistical significance.

Drugs

Substance P (Sigma, St Louis, MO) was dissolved in distilled water and kept in aliquots at −20°C until the day of the experiment, when it was diluted in normal saline (0.9% NaCl). Mecamylamine and norepinephrine (Sigma, St Louis, MO) were also dissolved in normal saline.

RESULTS

Effect of Mecamylamine and Norepinephrine on Mean Arterial Pressure, Heart Rate, and Mean Circulatory Filling Pressure

There are no significant differences in baseline readings of mean arterial pressure, heart rate, and mean circulatory filling pressure among the four groups of rats (Table 1). Mecamylamine significantly decreased mean arterial pressure, mean circulatory filling pressure, and heart rate in groups III and IV. The subsequent infusion of norepinephrine increased mean arterial pressure and mean circulatory filling pressure in both groups. Heart rate was significantly increased by norepinephrine in group III but not group IV.

TABLE 1: Baseline Values of Mean Arterial Pressure, Heart Rate, and Mean Circulatory Filling Pressure in Conscious Rats (n = 6 Per Group)

Effect of Substance P on Mean Arterial Pressure, Heart Rate, and Mean Circulatory Filling Pressure

The vehicle did not significantly alter mean arterial pressure, heart rate, or mean circulatory filling pressure in either the intact (unblocked) or ganglionically blocked rats, which served as time controls (Fig. 1).

FIGURE 1: Effects (mean ± SEM, n = 6 per group) of the low (L) and high (H) doses of substance P (10 and 30 nmol/kg, shaded columns) or an equal volume of vehicle (V1 and V2; 0.9% NaCl, white columns) on mean arterial pressure (MAP), heart rate (HR), and mean circulatory filling pressure (MCFP) in conscious, intact rats and rats pretreated with mecamylamine (Mec, 10 mg/kg, IV) and norepinephrine (NA, 4 μg/kg/min, IV). All points are changes from the baseline. ^aSignificantly different (P < 0.05) from the corresponding changes in the vehicle group. ^bSignificantly different (P < 0.05) from the corresponding changes elicited by substance P in the intact rats.

In the intact rats, both doses of substance P significantly reduced mean arterial pressure and increased heart rate; however, only the high dose of substance P significantly decreased mean circulatory filling pressure (Fig. 1).

In the ganglionically blocked rats, both doses of substance P decreased mean arterial pressure and mean circulatory filling pressure but did not affect heart rate (Fig. 1). The decreases in mean arterial pressure and mean circulatory filling pressure by both doses of substance P were significantly greater, and the changes in heart rate were markedly attenuated, in the ganglionically blocked rats relative to the corresponding responses in the intact rats (Fig. 1).

DISCUSSION

In this study, IV injections of substance P decreased mean arterial pressure in conscious rats that were intact or treated with mecamylamine and norepinephrine. The depressor response was greater in the rats pretreated with mecamylamine and norepinephrine, and this was likely related to the higher baseline mean arterial pressure (greater vasoconstrictor tone), secondary to the infusion of norepinephrine, and caused by lack of reflex activation of the sympathetic nervous system as demonstrated by the absence of reflex tachycardia. Substance P has been reported to directly stimulate the sympathetic ganglia.²⁰ Therefore, ganglionic blockade with mecamylamine would be expected to block the direct ganglionic stimulatory action of substance P as well as the reflex sympathetic activation that would follow a depressor response. The depressor action of substance P has been reported.^21-23 In the present study, the hypotensive effect of substance P was accompanied by tachycardia that was abolished after treatment with mecamylamine. These results indicate that substance P has no direct chronotropic action on the heart. Substance P was reported to have no effect on cardiac work of the isolated rat heart preparation.²⁴ Furthermore, substance P caused dose-dependent bradycardia in the isolated perfused guinea pig heart through activation of cholinergic neurons.²⁵

Substance P reduced mean circulatory filling pressure in the intact rat. This shows that it has a prominent venodilator action in vivo. The venodilator action of substance P was augmented in rats pretreated with mecamylamine and norepinephrine. Mecamylamine was administered to block reflex venoconstriction induced by hypotension, and norepinephrine was continuously infused to increase baseline venous tone to facilitate detection of venodilator responses.¹⁸ Previously, it has been shown that the direct venodilator effect of the well-established venodilator nitroglycerin was not apparent except following blockade of reflex venoconstriction with the ganglionic blocker hexamethonium.²⁶

Substance P has been shown to decrease blood pressure and cardiac output in anesthetized rats.²¹ The mechanism responsible for the reduction of cardiac output (or venous return) was unclear because there are no published studies that examined the effect of substance P on the venous system. In vitro studies have shown that substance P relaxed the human saphenous,¹⁰ ovarian,⁸ and omental veins,²⁷ and it contracted the rabbit mesenteric and jugular veins.^11-13 In addition, substance P induced venodilatation of the human dorsal hand veins and has been suggested to be the most potent venodilator known.¹⁴ Removal of the endothelium attenuated relaxation induced by substance P in human ovarian veins⁸ and monkey mesenteric veins,²⁸ indicating that the venodilator response is endothelium dependent. Furthermore, N^G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, inhibited the vasodilator responses evoked by substance P in the human ovarian vein⁸ and the human isolated omental arteries and veins,²⁹ indicating involvement of the NO/L-arginine pathway. It has also been shown that substance P caused an endothelium-dependent increase in the level of cGMP in the human saphenous vein.³⁰ On the contrary, Strobel et al¹⁴ have shown that substance P-induced venodilatation in human hand veins is through mediation of the ATP-dependent potassium channels but not the NO/L-arginine pathway. The opening of tetraethylammonium (TEA)-sensitive K⁺ channels has also been suggested to be the mechanism by which substance P caused relaxation of the human isolated omental arteries and veins.²⁹ There are no reported studies on the effect of endothelial dysfunction on substance P-induced dilatation of veins. It has been shown in human coronary arteries that endothelial dysfunction associated with hypertension, hypercholesterolemia, and atherosclerosis reduces substance P-induced vasodilatation of arteries.^31,32 In conscious rats, substance P appears to have venodilator efficacy similar to those of nociceptin,¹⁷ calcitonin gene-related peptide,¹⁶ adenosine,³³ and hexamethonium,³⁴ which were able to concurrently reduce mean arterial pressure and mean circulatory filling pressures in intact rats.

In conclusion, substance P is an efficacious vasodilator peptide that decreases both mean arterial pressure and mean circulatory filling pressure in conscious, unrestrained rats. The vasodepressor effect of substance P is accompanied by reflex tachycardia that is abolished by ganglion blockade.

ACKNOWLEDGMENT

This work is supported by the Heart & Stroke Foundation of British Columbia & the Yukon.