The effects of nonselective nitric-oxide synthase (NOS) inhibitors [N--nitro-L-arginine methyl ester (L-NAME) and N--nitro-L-arginine (L-NNA)] and specific neuronal NOS (nNOSon adrenergic nerve-mediated vasoconstriction were studied in rat perfused mesenteric vascular beds without endothelium. Perfusion of L-NAME, L-NNA, or L-VNIO markedly augmented vasoconstrictor responses to periarterial nerve stimulation (PNS; 2-8 Hz) without affecting vasoconstriction induced by exogenously injected norepinephrine (NE). Addition of L-arginine, a precursor for the synthesis of nitric oxide (NO), reversed the augmentation of the PNS response by L-NAME. The PNS (8 Hz)-evoked NE release in the perfusate was increased by L-NAME perfusion. In preparations treated with capsaicin [a depleter of calcitonin gene-related peptide (CGRP)-containing nerves], L-NAME did not augment vasoconstrictor responses to PNS or NE injection. Combined perfusion of CGRP(8-37) (a CGRP receptor antagonist) and L-NAME induced additive augmentation of the vasoconstrictor response to PNS but did not affect the response to NE injection. In preparations with active tone produced by methoxamine and in the presence of guanethidine, L-NAME perfusion did not affect the vasodilator response induced by PNS. Immunostaining of the mesenteric artery showed the presence of nNOS-like immunopositive nerve fibers, which were absent in arteries pretreated with capsaicin. These findings suggest that NO, which is released from perivascular capsaicin-sensitive nerves, presynaptically inhibits neurogenic NE release to modulate adrenergic neurotransmission.

1 Previous studies showed that nicotine induces adrenergic nerve-dependent vasodilation that is mediated by endogenous calcitonin gene-related peptide (CGRP) released from CGRP-containing (CGRPergic) nerves. The mechanisms underlying the nicotine-induced vasodilation were further studied. 2 Rat mesenteric vascular beds without endothelium were contracted by perfusion with Krebs solution containing methoxamine, and the perfusion pressure was measured with a pressure transducer.3 Perfusion of nicotine (1-100 mM) for 1 min caused concentration-dependent vasodilation. Capsazepine (vanilloid receptor-1 antagonist; 1-10 mM) and ruthenium red (inhibitor of vanilloid response; 1-30 mM) concentration-dependently inhibited the nicotine-induced vasodilation without affecting the vasodilator response to exogenous CGRP.4 Nicotine-induced vasodilation was not inhibited by treatment with 3,4-dihydroxyphenylalanine (DOPA) receptor antagonist (L-DOPA cyclohexyl ester; 0.001-10 mM), dopamine D1 receptorselective antagonist (SCH23390; 1-10 mM), dopamine D2 receptor antagonist (haloperidol; 0.1-0.5 mM), ATP P2x receptor-desensitizing agonist (a,b-methylene ATP; 1-10 mM), adenosine A 2 receptor antagonist (8(p-sulfophenyl)theophylline; 10-50 mM) or neuropeptide Y (NPY)-Y1 receptor antagonist (BIBP3226; 0.1-0.5 mM). 5 Immunohistochemical staining of the mesenteric artery showed dense innervation of CGRP-and vanilloid receptor-1-positive nerves, with both immunostainings appearing in the same neuron. The mesenteric artery was also densely innervated by NPY-positive nerves. Double immunostainings showed that both NPY and CGRP immunoreactivities appeared in the same neuron of the artery. 6 These results suggest that nicotine acts on presynaptic nicotinic receptors to release adrenergic neurotransmitter(s) or related substance(s), which then stimulate vanilloid receptor-1 on CGRPergic nerves, resulting in CGRP release and vasodilation.

Nicotine stimulates presynaptic nicotinic acetylcholine receptors in perivascular adrenergic nerves and releases unknown transmitter(s) that activate transient receptor potential vanilloid-1 (TRPV1) located on calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves, resulting in vasodilation. The present study investigated a potential transmitter transmitting between perivascular adrenergic nerves and CGRPergic nerves. Rat mesenteric vascular beds without endothelium were contracted by perfusion with Krebs' solution containing methoxamine, and the perfusion pressure and pH levels of the perfusate were measured. Nicotine perfusion for 1 min induced concentration-dependent vasodilation and lowered pH levels, which were abolished by cold-storage denervation of preparations, guanethidine (adrenergic neuron blocker), and mecamylamine (nicotinic ␣ 3 ␤ 4 -acetylcholine receptor antagonist). Capsazepine (TRPV1 antagonist) blunted nicotine-induced vasodilation, but had no effect on the reduction of pH. Injection of hydrochloric acid (HCl) and perfusion of Krebs' solution at low pH (6.0 -7.2) induced vasodilation. HClinduced vasodilation was inhibited by cold-storage denervation, capsazepine, capsaicin (CGRP depletor), and CGRP(8 -37) (CGRP receptor antagonist). Perfusion of adrenergic transmitter metabolites (normetanephrine and 3-methoxydopamine), but not of other metabolites, induced vasodilation, which was not inhibited by capsaicin treatment. Immunohistochemical staining of mesenteric arteries showed dense innervation of CGRP-and TRPV1-immunopositive nerves, with both immunostainings appearing in the same neuron. Mesenteric arteries were densely innervated by neuropeptide Y-immunopositive nerves, which coalesced with CGRP-immunopositive nerves. Scanning and immunoscanning electron microscopic images showed coalescence sites of different perivascular fibers before they intruded into smooth muscles. These results indicate that nicotine initially stimulates adrenergic nerves via nicotinic ␣ 3 ␤ 4 -receptors to release protons and thereby induces CGRPergic nerve-mediated vasodilation via TRPV1.It is widely accepted that vascular tone is maintained mainly by sympathetic adrenergic nerves via the release of the neurotransmitter norepinephrine. However, accumulating evidence reveals that nonadrenergic, noncholinergic (NANC) vasodilator nerves also play a role in regulation of vascular tone. We have demonstrated that the rat mesenteric artery has dense innervation of calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves that release a transmitter, CGRP, causing vasodilation (Kawasaki et al., 1988). Recently, we reported that nitric oxide (NO)-containing nerves innervating rat mesenteric arteries are involved in modulation of adrenergic neurotransmission (Hatanaka et al., 2006). The rat mesenteric artery is also densely innervated by adrenergic nerves, which contain the main neuro-

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The aim of this study was to investigate age-related changes in the density of calcitonin gene-