Background The endogenous nucleoside adenosine plays an important role in the regulation of vascular tone, especially during ischemia. Experimental data derived from animal m od els suggest that nitric oxide (NO) contributes to the vasodilator effect of adenosine. The primary purpose of this investigation was to determine whether the endothelial release of NO contributes to adenosine-induced vasodilation in humans.Methods and Results Venous occlusion plethysmography was used to assess the forearm blood flow (FBF) responses to graded intra-arterial infusions of adenosine (1.5 to 500 ju,g/ min). Dose-response curves were constructed before and dur ing intra-arterial infusion of the N O synthase inhibitor N a-

Abstract-Bradykinin (BK) stimulates endothelial cells to release a number of relaxing factors, such as NO, prostanoids (PGs), and an endothelium-derived hyperpolarizing factor (EDHF). However, the contributions of NO, PG, and EDHF in the vascular relaxation to BK vary with species and anatomic origin of blood vessels used. Therefore, the present study was designed to investigate the contributions of NO, PG, and EDHF in vasodilation caused by BK in human forearm resistance vessels. Forearm blood flow (FBF) was recorded with venous occlusion plethysmography in healthy nonsmoking subjects. At first, studies were performed to validate the NO clamp technique for its ability to inhibit endogenous NO generation. Brachial artery infusion of serotonin (0.6, 1.8, and 6 ng ⅐ 100 mL forearm volume [FAV] Ϫ1 ⅐ min Ϫ1) caused significant forearm vasodilation (2.6 to 4.6 mL ⅐ 100 mL FAV Ϫ1 ⅐ min Ϫ1), which is known to be NO mediated. Indeed, during the NO clamp, cumulative doses of serotonin caused no vasodilation (2.4 to 2.6 mL ⅐ 100 mL FAV Ϫ1 ⅐ min Ϫ1 ), indicating that the generation of endogenous NO was completely blocked. Thereafter, the vasodilative actions of BK were investigated. Brachial artery infusion of BK (50, 100, and 200 ng ⅐ 100 mL FAV Ϫ1 ⅐ min Ϫ1 ) caused significant forearm vasodilation in all studies (from 3.1 to 20.4 mL ⅐ 100 mL FAV Ϫ1 ⅐ min Ϫ1). After the inhibition of cyclooxygenase and NO synthase activity through the use of aspirin and the NO-clamp technique, BK increased FBF in a similar manner (3.9 to 18.9 mL ⅐ 100 mL FAV Ϫ1 ⅐ min Ϫ1 ), indicating that the vasodilative actions of BK are independent of NO and PG generation. However, vasodilation caused by the 2 lower doses of BK were significantly attenuated after K Ca channel activity was blocked with tetraethylammonium chloride (0.1 mg ⅐ 100 mL FAV Ϫ1 ⅐ min Ϫ1), suggesting that in the lower dose range, BK mediates vasodilation through the opening of vascular potassium channels. In conclusion, BK is a potent vasodilator peptide in human forearm resistance vessels, causing vasodilation through hyperpolarization of the vascular wall independent of NO and PG production. In addition, the NO-clamp technique is a valid instrument to investigate the contribution of NO in the vasodilative response to different agents. , and an endothelium-derived hyperpolarizing factor (EDHF). 1-3 However, the contributions of NO, PG, and EDHF to the vascular relaxation to BK vary with species and anatomic origin of the blood vessels used. 4,5 In vitro studies performed on isolated blood vessels demonstrate that the stimulation of NO release by acetylcholine is more prominent in larger arteries. In contrast, the contribution of EDHF to endothelium-dependent relaxation by BK is significant larger in microvessels. 6 These findings become relevant when interpreting the results of in vivo studies that measure hemodynamic parameters such as blood pressure and vascular resistance. For example, Mügge et al 7 investigated in vivo the response to acetylcholine in the perfused rabbit...

Abstract-Hydrochlorothiazide has been shown to exert direct vasodilator effects by activation of calcium-activated potassium (K Ca ) channels in human and guinea pig isolated resistance arteries. Since hydrochlorothiazide binds to and inhibits the enzyme carbonic anhydrase and because K Ca channel activation is pH sensitive, we investigated the role of intracellular and extracellular carbonic anhydrase in the vascular effects of thiazide diuretics. Small arteries were isolated from guinea pig mesentery and studied by use of a microvascular myograph technique. In some experiments, tone and intracellular pH (pH i ) were measured simultaneously with 2Ј,7Ј-bis(2-carboxyethyl)-5(6)Ј-carboxyfluorescein (BCECF-AM). Bendroflumethiazide, a thiazide diuretic with minimal inhibitory effects on carbonic anhydrase, had little effect on noradrenaline-induced tone (16Ϯ8% relaxation) compared with hydrochlorothiazide (74Ϯ12% relaxation). In contrast to hydrochlorothiazide, the action of bendroflumethiazide was unaffected by 100 nmol/L charybdotoxin, a selective blocker of K Ca channels. All inhibitors of carbonic anhydrase relaxed noradrenaline-induced tone in a concentration-dependent manner, and this effect was blocked by charybdotoxin. Key Words: hydrochlorothiazide Ⅲ carbonic anhydrase inhibition Ⅲ muscle, smooth, vascular Ⅲ pH i Ⅲ potassium channels T hiazide diuretics were developed in the 1950s by chemical modification of carbonic anhydrase inhibitors. Although their blood pressure-lowering effects have been well documented, their mechanism of action is still not fully resolved. The principal site of action of thiazides is the early segment of the distal nephron, where they inhibit a luminal transmembrane-coupled NaCl transport system. In the long term, thiazides act by reducing peripheral resistance rather than by their diuretic effects, 1 and therefore a direct vascular effect has been proposed.Previous studies in isolated human and guinea pig resistance arteries have established a direct vasodilator activity of hydrochlorothiazide. This vasorelaxant response to hydrochlorothiazide was abolished by charybdotoxin and iberiotoxin, both selective blockers of large-conductance Ca 2ϩ -activated potassium (K Ca ) channels, but not by inhibitors of other vascular K ϩ channels. 2 On the basis of the fact that thiazide-like drugs such as cicletanine and diazoxide lead to hyperpolarization in vascular smooth muscle cells 3 and the fact that hydrochlorothiazide increases 86 Rb efflux as a marker of K ϩ efflux, 2,4 it was proposed that hydrochlorothiazide opens K Ca channels, thereby leading to K ϩ efflux and membrane hyperpolarization. The resultant closure of voltage-dependent Ca 2ϩ channels leads to a fall in [Ca 2ϩ ] i and vasorelaxation. 5 In addition to [Ca 2ϩ ] i , the open state probability of the K Ca channel is also modulated by intracellular pH (pH i ). Channel opening is inhibited by intracellular acidosis in carotid body cells, 6 while in isolated blood vessels intracellular alkalinization leads to relaxation asso...