Nitric oxide (NO) released from endothelial cells or exogenous nitrates is a potent dilator of arterial smooth muscle; however, the molecular mechanisms mediating relaxation to NO in the microcirculation have not been characterized. The present study investigated the relaxant effect of nitrovasodilators on microvessels obtained from the rat mesentery and also employed whole cell and single-channel patch-clamp techniques to identify the molecular target of NO action in myocytes from these vessels. Both sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) relaxed phenylephrine-induced contractions by approximately 80% but were significantly less effective in relaxing contractions induced by 40 mM KCl. Relaxation to SNP was also inhibited by the K(+)-channel blocker tetraethylammonium or by inhibition of the activity of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG). These results suggest that SNP stimulated K+ efflux by opening K+ channels via PKG-mediated phosphorylation. Perforated-patch experiments revealed that both SNP and SNAP increased outward currents in microvascular myocytes, and single-channel studies identified the high-conductance Ca(2+)- and voltage-activated K+ (BKCa) channel as the target of nitrovasodilator action. The effects of nitrovasodilators on BKCa channels were mimicked by cGMP and inhibited by blocking the activity of PKG. We conclude that stimulation of BKCa-channel activity via cGMP-dependent phosphorylation contributes to the vasodilatory effect of NO on microvessels and that a direct effect of NO on BKCa channels does not play a major role in this process. We propose that this mechanism is important for the therapeutic effect of nitrovasodilators on peripheral resistance and arterial blood pressure.

Insulin resistance (IR) syndrome is associated with impaired vascular relaxation; however, the underlying pathophysiology is unknown. Potassium channel activation causes vascular smooth muscle hyperpolarization and relaxation. The present study determined whether a reduction in large conductance calcium- and voltage-activated potassium (BK(Ca)) channel activity contributes to impaired vascular relaxation in IR rats. BK(Ca) channels were characterized in mesenteric microvessels from IR and control rats. Macroscopic current density was reduced in myocytes from IR animals compared with controls. In addition, inhibition of BK(Ca) channels with tetraethylammonium (1 mM) or iberiotoxin (100 nM) was greater in myocytes from control (70%) compared with IR animals (approximately 20%). Furthermore, activation of BK(Ca) channels with NS-1619 was three times more effective at increasing outward current in cells from control versus IR animals. Single channel and Western blot analysis of BK(Ca) channels revealed similar conductance, amplitude, voltage sensitivity, Ca2+ sensitivity, and expression density between the two groups. These data provide the first direct evidence that microvascular potassium currents are reduced in IR and suggest a molecular mechanism that could account for impaired vascular relaxation in IR.

Abstract-Angiotensin II (Ang II) is one of the most potent vasoconstrictor substances, yet paradoxically, Ang II may dilate certain vascular beds via an undefined mechanism. Ang II-induced vasoconstriction is mediated by the AT 1 receptor, whereas the relative expression and functional importance of the AT 2 receptor in regulating vascular resistance and blood pressure are unknown. We now report that Ang II induces relaxation of mesenteric microvessels and that this vasodilatory response was unaffected by losartan, an AT 1 receptor antagonist, but was inhibited by PD123,319, a selective antagonist of AT 2 receptors. In addition, reverse transcriptase-polymerase chain reaction studies revealed high amounts of AT 2 receptor mRNA in smooth muscle from these same microvessels. Ang II-induced relaxation was inhibited by either tetraethylammonium or iberiotoxin, suggesting involvement of the large-conductance, calcium-and voltage-activated potassium (BK Ca ) channel. Subsequent whole-cell and single-channel patch-clamp studies on single myocytes demonstrated that Ang II increases the activity of BK Ca channels. As in our tissue studies, the effect of Ang II on BK Ca channels was inhibited by PD123,319, but not by losartan. In light of these consistent findings from tissue physiology, molecular studies, and cellular/molecular physiology, we conclude that Ang II relaxes microvessels via stimulation of the AT 2 receptor with subsequent opening of BK Ca channels, leading to membrane repolarization and vasodilation. These findings provide evidence for a novel endothelium-independent vasodilatory effect of Ang II. Key Words: angiotensin II Ⅲ receptors, angiotensin Ⅲ potassium channels Ⅲ patch-clamp techniques M any complications of hypertension and congestive heart failure are ameliorated by interdiction of the renin-angiotensin system, and agents that attenuate the effects of angiotensin II (Ang II) are now a first-line pharmacological antihypertensive intervention. Ang II induces constriction, hypertrophy, and proliferation of vascular smooth muscle via the AT 1 receptor. It is generally assumed that the therapeutic effects of AT 1 antagonists are due to direct receptor blockade; however, a secondary effect of these agents is increased plasma levels of Ang II, 1 which may then activate AT 2 receptors. This receptor bears only 34% sequence homology to the AT 1 receptor 2,3 and mediates growth inhibition 4 and apoptosis 5 of vascular smooth muscle. In contrast, very little is known about the role of AT 2 receptors in regulating blood pressure. Both Ang II receptors are expressed in the vasculature, but AT 2 receptor expression is heterogeneous with respect to vascular bed, species, and developmental stage. 6 Nonetheless, evidence suggests that AT 2 receptors modulate vascular contractility. AT 2 receptors mediate endotheliumdependent vasodilation of renal arterioles. 7 Furthermore, AT 2 -deficient mice exhibit an increased pressor response to Ang II, 8 whereas overexpression of AT 2 receptors enhances Ang II-induced endothe...

Ergul, Adviye, Vera Portik-Dobos, Ararat D. Giulumian, Mariela M. Molero, and Leslie C. Fuchs. Stress upregulates arterial matrix metalloproteinase expression and activity via endothelin A receptor activation. Am J Physiol Heart Circ Physiol 285: H2225-H2232, 2003. First published July 3, 2003 10.1152/ajpheart.00133.2003.-Degradation of the extracellular matrix proteins by matrix metalloproteinases (MMP) is an important regulatory step in the vascular remodeling process. Recent studies demonstrated that ETA receptors regulate cardiac MMP activity and fibrosis in DOCA-salt hypertension. However, little is known about endothelin (ET)-1 regulation of vascular MMP activity in hypertension. Thus early changes in ET-1-mediated regulation of MMP activity were measured in borderline hypertensive rats that develop impaired vasorelaxation and hypertension with chronic exposure to stress. Experiments were performed after 10 days of exposure to the behavioral stressor, air-jet stress, but before the onset of stress-induced hypertension. Study groups were 1) control (n ϭ 8); 2) air-jet stress for 10 days (n ϭ 8); 3) control plus ETA antagonist ABT-627 (n ϭ 4), and 4) air-jet stress plus ET A antagonist (n ϭ 4). MMP activity in the thoracic aorta was assessed by gelatin zymography. MMP protein and tissue ET-1 levels were evaluated by immunohistochemistry, and ET receptor density was determined by immunoblotting. Exposure to stress caused a twofold increase in plasma ET-1 levels (P Ͻ 0.05), and there was increased ET-1 staining at the tissue level. Total MMP activity and expression of MMP-2 and MMP-9 were increased in the stress group. ETA receptor antagonism prevented the increase in MMP expression and activation in the stress group. These results provide evidence that the MMP system is activated before the development of hypertension and ET-1 mediates these early events in vascular remodeling.endothelin-1; ABT-627 ENDOTHELIN-1 (ET-1) contributes to blood pressure elevation as well as cardiac, vascular, and renal complications in several experimental models, including DOCA-salt hypertensive rat, DOCA-salt-treated spontaneously hypertensive rats (SHRs), Dahl salt-sensitive rats, and aldosterone-infused rats (18,21,24,25). In DOCA-salt hypertensive rats, enhanced myocardial remodeling and fibrosis associated with increased fibronectin, matrix metalloproteinase (MMP) activity, and proinflammatory mediators occur in the left ventricle and ET A receptor antagonism inhibits these changes (1, 2). In clinical hypertension, ET-1 is increased in African-American hypertensive patients who present with increased incidence of cardiovascular complications, including left ventricle hypertrophy and stroke (7,8). Although the exact mechanism is not clear, it has been proposed that African-Americans experience chronic sympathetic system activation due to more recurrent exposure to social and environmental stress, which contribute to the increased incidence of hypertension and related complications in this patient population (9). A recent stud...

In borderline hypertensive rats (BHR), behavioral stress produces hypertension, which has been attributed to increases in sympathetic nervous system activity and peripheral changes in vascular structure. However, the mechanisms mediating development of stress-induced hypertension have not been well defined. Experiments were designed to determine hemodynamic effects and changes in small mesenteric artery (≈300 μm) vascular reactivity in response to 10 days of air-jet stress (2 h/day) in BHR and in Wistar-Kyoto (WKY) rats. The acute stress-induced increase in mean arterial pressure (AP) was impaired in WKY rats compared with BHR on day 1, and habituation developed to the increase in AP in BHR, but not WKY rats. Conversely, WKY rats adapted to the stress-induced tachycardia to a larger extent than BHR. The mechanisms mediating endothelium-dependent relaxation to acetylcholine (ACh) were altered in small mesenteric arteries isolated from WKY rats and BHR after 10 days of air-jet stress. Inhibition of nitric oxide synthase activity had a significantly larger inhibitory effect on ACh-induced relaxation in vessels from stressed compared with control BHR. Also, cyclooxygenase products contributed to ACh-induced relaxation of small mesenteric arteries from stressed WKY rats, but not control WKY rats. Endothelium-independent relaxation to nitroprusside was impaired in vessels from stressed WKY rats, but not stressed BHR. Finally, contraction to phenylephrine was impaired in vessels from stressed BHR, but not WKY rats. In conclusion, changes in vascular reactivity induced by air-jet stress appear to correlate with, and may contribute to, the differential hemodynamic adaptations to stress observed in WKY rats and BHR.