Evidence for Functional Responses to Sensory Nerve Stimulation of Rat Small Mesenteric Veins AMRITA AHLUWALIA and PATRICK VALLANCE

Sensory C-fibers have been implicated in the control of vascular tone and are believed to be predominantly arteriolar in the microvasculature. There have been no direct investigations into the effects of C-fiber activation in venous microvessels. Therefore, we have investigated the effects of neuropeptides and activation of sensory C-fibers in rat small mesenteric veins. Small secondor third-order veins were dissected from the rat mesentery and mounted in a tension myograph for measurement of reactivity. Neither substance P or calcitonin generelated peptide (CGRP) relaxed precontracted veins. However, substance P caused a concentration-dependent contraction. The curve was shifted to the right in a concentration-dependent manner by the tachykinin neurokinin1 receptor antagonist RP 67,580 (0.1–1 mM). To activate sensory C-fibers, capsaicin was applied. Capsaicin had no contractile activity in these vessels but caused concentration-dependent relaxation. This response was significantly attenuated in veins taken from animals in which C-fibers had been largely destroyed (P , .001, n 5 5) and in vessels that had been pretreated with the vanilloid receptor blocker ruthenium red (P , .01, n 5 5). Endothelial denudation (n 5 6) also abolished the response, but the nitric oxide synthase inhibitor N-monomethyl-L-arginine (100 mM, n 5 5) did not inhibit the response; N-nitro-L-arginine methyl ester (100–300 mM, n 5 4) did inhibit the response. The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one also significantly attenuated the response (n 5 5). The cyclooxygenase inhibitor indomethacin (5 mM, n 5 5) and the CGRP receptor antagonist CGRP8–37 (1 mM) were without effect. These results demonstrate that capsaicin, a selective Cfiber activator, relaxes small veins in an endothelium-dependent but CGRPand substance P-independent manner, and they demonstrate that the venous side of the microcirculation responds directly to sensory stimulation. Sensory nerves are involved in the control of vascular tone and appear to play an important role in certain types of vascular inflammation. The proinflammatory nature of neuropeptides released from sensory C-fibers has been demonstrated in several models of inflammation, and neurogenic components have been identified in inflammatory disease states, including rheumatoid arthritis (Levine et al., 1985) and asthma (Barnes, 1986). Activation of sensory C-fibers may be achieved in several ways, including application of the agent capsaicin, the active ingredient of ‘chili pepper’ (Jancso et al., 1967), which selectively activates sensory C-fibers (for reviews, see Holzer, 1991; Dray, 1992), or direct electrical stimulation of nerve fibers. C-fibers are located within the arteriolar side of the microcirculation (e.g., Fleming et al., 1989; Baluk et al., 1992), and activation of these nerves results in the release of the neuropeptides CGRP and SP (Holzer, 1991). It is generally assumed that C-fibers do not innervate the venous side of the microcirculation (e.g., McDonald, 1988; Baluk et al., 1992). Indeed, although there are studies demonstrating the innervation of larger veins, such as human saphenous vein (Herbst et al., 1992), there is no immunohistochemical evidence demonstrating SP or CGRP immunoreactivity in the walls of small veins. When arteriolar C-fibers are activated, CGRP is thought to act at the arteriolar level to produce vasodilatation, whereas SP travels downstream to increase venular permeability. Together, these effects produce greater net edema than does either alone. This synergism has been attributed to an increase in the hydrostatic pressure (due to selective arteriolar dilatation), coupled with increased movement of fluid out of the permeable venules. Synergism between CGRP and SP has been demonstrated in the skin of rabbits and rats (Brain and Williams, 1985; Gamse and Saria, 1985). However, this has not been a universal finding, and other studies have demonstrated that CGRP has an inhibitory effect (Raud et al., 1991) or no effect (Ekblom et al., 1993) on the response to agents that increase venular permeability. These studies have all relied upon exploring the effects of the neuropeptides in vivo or in intact Received for publication April 18, 1996. 1 This work and A.A. were supported by The Wellcome Trust. ABBREVIATIONS: CGRP, calcitonin gene-related peptide; L-NAME, N-nitro-L-arginine methyl ester; NK, neurokinin; L-NMMA, N-monomethylL-arginine; NO, nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; SP, substance P. 0022-3565/97/2819-0009$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 281, No. 9 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 281:9–14, 1997 9 at A PE T Jornals on July 9, 2017 jpet.asjournals.org D ow nladed from perfused vascular beds in vitro. In these integrated experimental systems, it is difficult to distinguish between direct effects of neurogenic stimulation on the component parts of the vasculature. To overcome this problem, we have undertaken studies of isolated microvessels in vitro. We previously demonstrated that C-fiber activation in small arteries relaxes constricted vessels and inhibits the contractile responses to sympathetic stimulation (Ahluwalia and Vallance, 1996a). We have now investigated the effects of neuropeptides and capsaicin on the reactivity of isolated small veins of the microcirculation of the rat mesentery. These results have been presented in preliminary form to The British Pharmacological Society (Ahluwalia and Vallance, 1996b).