Abstract-It has been suggested in isolated porcine cerebral arteries that stimulation by nicotine of ␣ 7 -nicotinic acetylcholine receptors (␣ 7 -nAChRs) on sympathetic nerves, but not direct stimulation of parasympathetic nitrergic nerves, caused nitrergic neurogenic dilation. Direct evidence supporting this hypothesis has not been presented. The present study, which used in vitro tissue bath and confocal microscopy techniques, was designed to determine whether choline, a selective agonist for ␣ 7 -nAChRs, induced sympathetic-dependent nitrergic dilation of porcine basilar arterial rings. Choline and several nAChR agonists induced exclusive relaxation of basilar arterial rings without endothelium. The relaxation was blocked by tetrodotoxin, nitro-L-arginine, guanethidine, and ␤ 2 -adrenoceptor antagonists. Furthermore, the relaxation was blocked by methyllycaconitine and ␣-bungarotoxin (preferential ␣ 7 -nAChR antagonists) and mecamylamine but was not affected by dihydro-␤-erythroidine (a preferential ␣ 4 -nAChR antagonist). Confocal microscopic study demonstrated that choline and nicotine induced significant calcium influx in cultured porcine superior cervical ganglionic cells but failed to affect calcium influx in cultured sphenopalatine ganglionic cells, providing direct evidence that choline and nicotine did not act directly on the parasympathetic nitrergic neurons. The increased calcium influx in superior cervical ganglionic cells was attenuated by ␣-bungarotoxin and methyllycaconitine but not by dihydro-␤-erythroidine. These results support our hypothesis that activation of ␣ 7 -nAChRs on cerebral perivascular sympathetic nerves causes calcium influx and the release of norepinephrine, which then act on presynaptic Key Words: choline Ⅲ ␣ 7 -nicotinic acetylcholine receptors Ⅲ cerebral neurogenic vasodilation Ⅲ nitric oxide Ⅲ sympathetic-parasympathetic interaction T he ␣-bungarotoxin (␣-BGTX)-sensitive nicotinic acetylcholine receptor (nAChR), which is one of the predominant nAChR subtypes in the brain, consists of the ␣ 7 -subunit, possesses calcium permeability, and exhibits rapid desensitization. 1 Choline, which is a precursor of acetylcholine (ACh) synthesis and a product of ACh hydrolysis by acetylcholinesterase, has been shown to act as a relatively selective agonist for ␣ 7 -nAChRs in the central nervous system. 2,3 By activating presynaptic ␣ 7 -nAChRs, choline has been shown to elicit the release of neurotransmitters, including norepinephrine (NE). 2,4 The presence of nAChRs on sympathetic adrenergic nerve terminals is also well established. 5,6 However, the functional significance of choline and ␣ 7 -nAChRs in the perivascular neurons has not been clarified.We reported recently that nicotine-induced NO-mediated neurogenic vasodilation in porcine basilar arteries and feline middle cerebral arteries was dependent on intact perivascular sympathetic adrenergic innervation. 7-9 Results from pharmacological studies using nicotinic receptor antagonists further suggest that nicotine acts on ␣ 7 -nAChRs ...

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Background-Perivascular adipose tissue (PVAT)-derived relaxing factor (PVATRF) significantly regulates vascular tone. Its chemical nature remains unknown. We determined whether palmitic acid methyl ester (PAME) was the PVATRF and whether its release and/or vasorelaxing activity decreased in hypertension. Methods and Results-Using superfusion bioassay cascade technique, tissue bath myography, and gas chromatography/mass spectrometry, we determined PVATRF and PAME release from aortic PVAT preparations of Wistar Kyoto rats and spontaneously hypertensive rats. The PVAT of Wistar Kyoto rats spontaneously and calcium dependently released PVATRF and PAME. Both induced aortic vasorelaxations, which were inhibited by 4-aminopyridine (2 mmol/L) and tetraethylammonium 5 and 10 mmol/L but were not affected by tetraethylammonium 1 or 3 mmol/L, glibenclamide (3 mol/L), or iberiotoxin (100 nmol/L). Aortic vasorelaxations induced by PVATRF-and PAME-containing Krebs solutions were not affected after heating at 70°C but were equally attenuated after hexane extractions. Culture mediums of differentiated adipocytes, but not those of fibroblasts, contained significant PAME and caused aortic vasorelaxation. The PVAT of spontaneously hypertensive rats released significantly less PVATRF and PAME with an increased release of angiotensin II. In addition, PAME-induced relaxation of spontaneously hypertensive rats aortic smooth muscle diminished drastically, which was ameliorated significantly by losartan. Conclusions-We found that PAME is the PVATRF, causing vasorelaxation by opening voltage-dependent K ϩ channels on smooth muscle cells. Diminished PAME release and its vasorelaxing activity and increased release of angiotensin II in the PVAT suggest a noble role of PVAT in pathogenesis of hypertension. The antihypertensive effect of losartan is attributed partly to its reversing diminished PAME-induced vasorelaxation. (Circulation. 2011;124:1160-1171.) Key Words: angiotensin II Ⅲ fatty acids Ⅲ hypertension Ⅲ potassium ion channels Ⅲ vasomotor tone Ⅲ vasorelaxation Ⅲ losartan T he systemic blood vessels are surrounded by various amounts of perivascular adipose tissue (PVAT). Since the first report by Soltis and Cassis in 1991 that PVAT attenuated contraction of aortic rings to norepinephrine, 1 it has been well accepted that the anticontractile effect of PVAT is due to release of relaxing factor(s) from these adipocytes. 2 The vasodilation induced by PVAT-derived relaxing factor (PVATRF) is independent of the endothelium, cyclooxygenase, or cytochrome P450 pathway. 2,3 The general consensus is that PVATRFinduced vasodilation is due to opening of potassium channels on the smooth muscle cells. [2][3][4][5] The chemical identity of the PVATRF, however, remains unknown. Clinical Perspective on p 1171Our recent studies using superfusion bioassay cascade technique have demonstrated release of an endogenous potent vasodilator, palmitic acid methyl ester (PAME), from the superior cervical ganglion and retina of the rat. 6,7 Because PAME is hydro...

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.

We previously reported that nicotine-induced nitric oxide (NO)-mediated cerebral neurogenic vasodilation was dependent on intact sympathetic innervation. We hypothesized that nicotine acted on sympathetic nerve terminals to release norepinephrine (NE), which then acted on adrenoceptors located on the neighboring nitric oxidergic (NOergic) nerve terminals to release NO, resulting in vasodilation. The adrenoceptor subtype in mediating nicotine-induced vasodilation in isolated porcine basilar arterial rings denuded of endothelium was therefore examined pharmacologically and immunohistochemically. Results from using an in vitro tissue bath technique indicated that propranolol and preferential beta(2)-adrenoceptor antagonists (ICI-118,551 and butoxamine), in a concentration-dependent manner, blocked the relaxation induced by nicotine (100 microM) without affecting the relaxation elicited by transmural nerve stimulation (TNS, 8 Hz). In contrast, preferential beta(1)-adrenoceptor antagonists (atenolol and CGP-20712A) did not affect either nicotine- or TNS-induced relaxation. Results of double-labeling studies indicated that beta(2)-adrenoceptor immunoreactivities and NADPH diaphorase reactivities were colocalized in the same nerve fibers in basilar and middle cerebral arteries. These findings suggest that NE, which is released from sympathetic nerves upon application of nicotine, acts on presynaptic beta(2)-adrenoceptors located on the NOergic nerve terminals to release NO, resulting in vasodilation. In addition, nicotine-induced relaxation was enhanced by yohimbine, an alpha(2)-adrenoceptor antagonist, which, however, did not affect the relaxation elicited by TNS. Prazosin, an alpha(1)-adrenoceptor antagonist, on the other hand, did not have any effect on relaxation induced by either nicotine or TNS. The predominant facilitatory effect of beta(2)-adrenoceptors in releasing NO may be compromised by presynaptic alpha(2)-adrenoceptors.