Although dihydropyridines are widely used for the treatment of vasospasm, their effectiveness is questionable, suggesting that other voltage-dependent calcium channels (VDCCs) contribute to control of cerebrovascular tone. This study therefore investigated the role of dihydropyridine-insensitive VDCCs in cerebrovascular function. Using quantitative PCR and immunohistochemistry, we found mRNA and protein for L-type (Ca(V)1.2) and T-type (Ca(V)3.1 and Ca(V)3.2) channels in adult rat basilar and middle cerebral arteries and their branches. Immunoelectron microscopy revealed both L- and T-type channels in smooth muscle cell (SMC) membranes. Using patch clamp electrophysiology, we found that a high-voltage-activated calcium current, showing T-type channel kinetics and insensitivity to nifedipine and nimodipine, comprised approximately 20% of current in SMCs of the main arteries and approximately 45% of current in SMCs from branches. Both components were abolished by the T-type antagonists mibefradil, NNC 55-0396, and efonidipine. Although nifedipine completely blocked vasoconstriction in pressurized basilar arteries, a nifedipine-insensitive constriction was found in branches and this increased in magnitude as vessel size decreased. We conclude that a heterogeneous population of VDCCs contributes to cerebrovascular function, with dihydropyridine-insensitive channels having a larger role in smaller vessels. Sensitivity of these currents to nonselective T-type channel antagonists suggests that these drugs may provide a more effective treatment for therapy-refractory cerebrovascular constriction.

1 The e ects of L-cysteine were tested in rat aortic rings on responses to nitric oxide free radical (NO . ), nitroxyl (NO 7 ) derived from Angeli's salt and endothelium-derived relaxing factor (EDRF) activated by acetylcholine, ATP and the calcium ionophore A23187. Concentrations of 300 mM or less of L-cysteine had no e ect on responses. 2 Relaxations produced by exogenous NO . (0.25 ± 2.5 mM) were markedly prolonged and relaxations produced by sodium nitroprusside (0.001 ± 0.3 mM) were enhanced by 1 and 3 mM Lcysteine. The enhancements by L-cysteine of responses to NO . and sodium nitroprusside may be attributed to the formation of S-nitrosocysteine. 3 Relaxations mediated by the nitroxyl anion (0.3 mM) donated from Angeli's salt were more prolonged than those produced by NO . , and nitroxyl-induced relaxations were reduced by L-cysteine (1 and 3 mM). 4 EDRF-mediated relaxations produced by acetylcholine (0.01 ± 10 mM), ATP (3 ± 100 mM) and the calcium ionophore A23187 (0.1 mM) were signi®cantly reduced by 3 mM L-cysteine. 5 The similarity between the inhibitory e ects of L-cysteine on responses to EDRF and on those to nitroxyl suggests that a component of the response to EDRF may be mediated by nitroxyl anion.

In this review we discuss the contribution of NO, prostacyclin and endothelium-derived relaxing factor--endothelium-derived hyperpolarizing factor, or EDHF, to vascular function. We also explore the hypotheses (1): that tissues can store NO as nitrosothiols (RSNOs) and (2) that such RSNO stores can be modulated by physiological and pathophysiological processes. Notably in the microcirculation, EDHF appears to play an important role in the regulation of vascular tone. Leading candidates for EDHF include extracellular potassium (K+), an epoxygenase product, hydrogen peroxide and/or a contribution from myoendothelial gap junctions. Data from our laboratory indicate that in mouse vessels, different endothelium-dependent vasodilators, such as acetylcholine and protease-activated receptor (PAR) agonists, release different endothelium-derived relaxing factors. The combination of two K-channel toxins, apamin and charybdotoxin, inhibits EDHF activity in most protocols. Endothelial dysfunction is considered as the major risk factor and a very early indicator of cardiovascular disease including the cardiovascular complications of type I & types II diabetes. Impaired endothelium-dependent vasodilatation results primarily from a decreased synthesis of endothelium-derived nitric oxide (NO) and/or an increase in the production of reactive oxygen species such as superoxide. We have shown that the administration of tetrahydrobiopterin, an important co-factor for nitric oxide synthase (NOS) partially restores endothelial function (1) in leptin-deficient mice (db/db) with spontaneous type II diabetes, as well as (2) in human vascular tissue harvested for coronary artery bypass grafting (CABG). These data suggest that a deficiency in the availability of tetrahydrobiopterin plays an important role in vascular dysfunction associated with Type II diabetes. In addition, changes in the contribution of EDHF occur in vascular tissue from the db/db mice suggesting a compensatory increase in EDHF production; whether this alteration in EDHF production is physiological or pathophysiological remains controversial.

Opinions on the role of reactive oxygen species (ROS) in the vasculature have shifted in recent years, such that they are no longer merely regarded as indicators of cellular damage or byproducts of metabolism--they may also be putative mediators of physiological functions. Hydrogen peroxide (H2O2), in particular, can initiate vascular myocyte proliferation (and, incongruously, apoptosis), hyperplasia, cell adhesion, migration, and the regulation of smooth muscle tone. Endothelial cells express enzymes that produce ROS in response to various stimuli, and H2O2 is a potent relaxant of vascular smooth muscle. H2O2 itself can mediate endothelium-dependent relaxations in some vascular beds. Although nitric oxide (NO) is well recognized as an endothelium-derived dilator, it is also well established, particularly in the microvasculature, that another factor, endothelium-derived hyperpolarizing factor (EDHF), is a significant determinant of vasodilatory tone. This review primarily focuses on the hypothesis that H2O2 is an EDHF in resistance arteries. Putative endothelial sources of H2O2 and the effects of H2O2 on potassium channels, calcium homeostasis, and vascular smooth muscle tone are discussed. Furthermore, given the perception that ROS can more likely elicit cytotoxic effects than perform signalling functions, the arguments for and against H2O2 being an endogenous vasodilator are assessed.

1 The current study examined the hypothesis that endothelial production of hydrogen peroxide (H 2 O 2 ) mediates relaxations to acetylcholine (ACh) in aorta and small mesenteric arteries (SMA) from mice. 2 Relaxations to ACh (0.01 -10 mM) and H 2 O 2 (0.1 -1000 mM) were produced in aorta and SMA isolated from wild-type C57BL/6 mice and type II diabetic mice (db/db). In SMA, relaxations to ACh were produced in the presence of N o -nitro-L-arginine methyl ester (100 mM) and indomethacin (Indo, 10 mM). [4,3-]quinoxalin-1-one (10 mM) significantly reduced ACh-induced relaxations in SMA, abolished responses in aorta, but had no effect on relaxations induced by H 2 O 2 . Catalase (2500 U ml À1 ) abolished responses to H 2 O 2 , but did not alter relaxations to ACh in the SMA and only caused a small rightward shift in responses to ACh in the aorta. 4 ACh-, but not H 2 O 2 -, mediated relaxations were significantly reduced by tetraethylammonium (10 mM), the combination of apamin (1 mM) and charybdotoxin (100 nM), and 25 mM potassium chloride (KCl). Higher KCl (60 mM) abolished relaxations to both ACh and H 2 O 2 . Polyethylene glycolated superoxide dismutase (100 U ml À1 ), the catalase inhibitor 3-amino-1,2,4-triazole (3-AT, 50 mM) and treatment with the copper chelator diethyldithiolcarbamate (3 mM) did not affect relaxations to ACh. 5 H 2 O 2 -induced relaxations were endothelium-independent and were not affected by ethylene diamine tetraacetic acid (EDTA 0.067 mM), 4-aminopyridine (1 mM), ouabain (100 mM) and barium (30 mM), 3-AT or Indo. 6 Although the data from this study show that H 2 O 2 dilates vessels, they do not support the notion that H 2 O 2 mediates endothelium-dependent relaxations to ACh in either aorta or SMA from mice.