Capsaicin and vanilloid receptors in the perfused rat hindlimb : mechanisms of action

Previous studies with the vanilloid spice principle capsaicin have shown a biphasic V02 response, with increased perfusion pressure (PP or vasoconstriction), in the perfused rat hindlimb that has led to suggestions of vanilloid receptor subtypes (VNI/VN2) in this preparation (Colquhoun et al., 1995). The present study attempted to further define the role of vanilloid receptors by the use of selective competitive (capsazepine or CPZ) and non-competitive (ruthenium red) vanilloid antagonists. CPZ inhibited capsaicin-mediated effects in the perfused hindlimb in a competitive manner with an affinity, estimated by Schild plot analysis, comparable to that seen in other preparations. Submicromolar concentrations of CPZ selectively inhibited the increased VO2 produced by the putative VNI receptor, and inhibited all effects at higher concentrations.. Submicromolar concentrations of ruthenium red, a vanilloid cation channel blocker, selectively inhibited the putative VN2 receptor-mediated effects of capsaicin (strong vasoconstriction and inhibition of V02). These observations, showing different sensitivity to blockade by CPZ and ruthenium red, further support the presence of two vanilloid receptor/ion channel subtypes in the rat hindlimb. Tetrodotoxin (TTX) failed to attenuate any changes produced by capsaicin, suggesting that the mechanism of action of capsaicin in the rat hindlimb may differ from other tissues where TTX-sensitive an insensitive cellular mechanisms operate. Moreover, 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), shown to bind to vanilloid receptors in a non-cooperative manner, induced very similar effects to capsaicin and provided preliminary evidence that the biphasic effects of capsaicin on VO2 are not likely to be due to cooperativity of binding to vanilloid receptors. The role of capsaicin-sensitive neurons and their peptide neurotransmitters in capsaicin-induced responses in the perfused hindlimb were also studied. Non-peptide antagonists for tachykinin NK1 and NK2 receptors (CP-99,994 and SR 48968) selectively blocked the stimulation of VO2 produced by submicromolar concentrations of capsaicin. Furthermore, infused substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) stimulated VO2 and induced mild vasoconstriction with a rank order of potency NKA>NICB>SP. These data support a role for the tachykinins, acting primarily at NK2 receptors, in capsaicin-induced stimulation of V02. Infused calcitonin gene-related peptide (CGRP) did not alter basal VO2 or PP. However, the CGRP receptor antagonist CGRP(8_37) potentiated both V02 and PP responses to capsaicin in the rat hindlimb indicating a vasodilator role for endogenously released CGRP. The stimulation of V02 and PP increase produced by low concentrations of infused capsaicin were absent one day after capsaicin pretreatment supporting a role for capsaicin-sensitive neurons in these responses. Conversely, the inhibition of V02 and increased PP produced by micromolar concentrations of capsaicin were potentiated 1, 7 and 14 days after capsaicin pretreatment and may either support a vasodilator role for endogenously released neuropeptides (e.g. CGRP), which are known to be depleted by capsaicin pretreatment, or upregulation of VN2 receptors. The vascular actions of CGRP, SP and NKA were further examined in the perfused rat hindlimb under noradrenalin (NOR)-induced vasoconstriction. CGRP caused strong, sustained dilatation that was nitric oxide (NO)-independent, while SP and NKA elicited transient dilatation that was partly NO-dependent. Conversely, a low concentration of capsaicin elicited strong vasoconstriction in the NA-stimulated perfused hindlimb. The metabolic effects of these agents was unclear from these studies given the strong V02-stimulating effect of NOR. The studies undertaken support the notion that at least two vanilloid receptor subtypes mediate the vascular and metabolic effects of capsaicin in the perfused rat hindlimb. The effects of capsaicin in this tissue may result from a combination of dilatation and V02 stimulation induced by endogenous sensory neuropeptides, and the predominant vasoconstrictor effect of capsaicin via stimulation of specific vanilloid receptors. These findings underline the complex nature of capsaicin's novel actions in perfused rat skeletal muscle.