Christopher G. Sobey

Potassium Channel Function in Vascular Disease

Potassium ion (K(+)) channel activity is a major regulator of vascular muscle cell membrane potential (E(m)) and is therefore an important determinant of vascular tone. There is growing evidence that the function of several types of vascular K(+) channels is altered during major cardiovascular diseases, such as chronic hypertension, diabetes, and atherosclerosis. Vasoconstriction and the compromised ability of an artery to dilate are likely consequences of defective K(+) channel function in blood vessels during these disease states. In some instances, increased K(+) channel function may help to compensate for increased vascular tone. Endothelial cell dysfunction is commonly associated with cardiovascular disease, and altered activity of nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor could also contribute to changes in resting K(+) channel activity, E(m), and K(+) channel-mediated vasodilatation. Our current knowledge of the effects of disease on vascular K(+) channel function almost exclusively relies on interpretation of data obtained by using pharmacological modulators of K(+) channels. As further progress is made in the development of more selective drugs and through molecular approaches such as gene targeting technology in mice, specific K(+) channel abnormalities and their causes in particular diseases should be more readily identified, providing novel directions for vascular therapy.

Increased NADPH-Oxidase Activity and Nox4 Expression During Chronic Hypertension Is Associated With Enhanced Cerebral Vasodilatation to NADPH In Vivo

Background and Purpose— We examined the importance of NADPH-oxidase in reactive oxygen species production in cerebral arteries and its effect on vascular tone in vivo. Furthermore, we investigated whether chronic hypertension affects function or expression of this enzyme in cerebral vessels. Methods— Superoxide generation was detected in isolated rat basilar arteries with the use of lucigenin-enhanced chemiluminescence. mRNA expression of NADPH-oxidase subunits was assessed by real-time polymerase chain reaction. Basilar artery diameter was measured with the use of a cranial window preparation in anesthetized rats. Results— NADPH-stimulated superoxide production was 2.3-fold higher in arteries from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto rats (WKY) and could be blocked by the NADPH-oxidase inhibitor diphenyleneiodonium. Higher NADPH-oxidase activity was also reflected at the molecular level as mRNA expression of the NADPH-oxidase subunit Nox4 was 4.1-fold higher in basilar arteries from SHR versus WKY. In contrast, expression of Nox1, gp91phox, p22phox, and p47phox did not differ between strains. Application of NADPH to basilar arteries caused larger vasodilatation in SHR than WKY. Vasodilatation to NADPH could be attenuated by diphenyleneiodonium, as well as diethyldithiocarbamate (Cu2+/Zn2+–superoxide dismutase inhibitor), catalase (H2O2 scavenger), or tetraethylammonium (BKCa channel inhibitor). Conclusions— Activation of NADPH-oxidase in cerebral arteries generates superoxide, which is dismutated by Cu2+/Zn2+–superoxide dismutase to H2O2. H2O2 then elicits vasodilatation via activation of BKCa channels. Upregulation of Nox4 during chronic hypertension is associated with elevated cerebral artery NADPH-oxidase activity.

SUBARACHNOID HAEMORRHAGE : WHAT HAPPENS TO THE CEREBRAL ARTERIES?

1. Subarachnoid haemorrhage (SAH) is a unique disorder and a major clinical problem that most commonly occurs when an aneurysm in a cerebral artery ruptures, leading to bleeding and clot formation. Subarachnoid haemorrhage results in death or severe disability of 50‐70% of victims and is the cause of up to 10% of all strokes. Delayed cerebral vasospasm, which is the most critical clinical complication that occurs after SAH, seems to be associated with both impaired dilator and increased constrictor mechanisms in cerebral arteries. Mechanisms contributing to development of vasospasm and abnormal reactivity of cerebral arteries after SAH have been intensively investigated in recent years. In the present review we focus on recent advances in our knowledge of the roles of nitric oxide (NO) and cGMP, endothelin (ET), protein kinase C (PKC) and potassium channels as they relate to SAH.

Role of Potassium Channels in Regulation of Cerebral Vascular Tone

Several types of potassium (K+) channels are present in cerebral blood vessels. Opening or closure of these ion channels may have significant effects on membrane poxad tential, which is a major determinant of entry of extraxad cellular calcium and thus vascular tone, Because of the ionic properties of the cell membrane at rest, the change in activity of only a few K+ channels is sufficient to change membrane potential significantly and alter vasxad cular tone (Nelson and Quayle, 1995; Quayle et aI., 1997). This review will focus on recent findings regardxad ing the characteristics and functional importance of K+ channels in the cerebral circulation.

Evidence for Selective Effects of Chronic Hypertension on Cerebral Artery Vasodilatation to Protease-Activated Receptor-2 Activation

BACKGROUND AND PURPOSEnProtease-activated receptor-2 (PAR-2) can be activated after proteolysis of the amino terminal of the receptor by trypsin or by synthetic peptides with a sequence corresponding to the endogenous tethered ligand exposed by trypsin (eg, SLIGRL-NH(2)). PAR-2 mediates nitric oxide (NO)-dependent dilatation in cerebral arteries, but it is unknown whether PAR-2 function is altered in cardiovascular diseases. Since hypertension selectively impairs NO-mediated cerebral vasodilatation in response to acetylcholine and bradykinin, we sought to determine whether PAR-2-mediated vasodilatation is similarly adversely affected by this disease state.nnnMETHODSnWe studied basilar artery responses in Wistar-Kyoto rats (WKY) (normotensive) and spontaneously hypertensive rats (SHR) in vivo (cranial window preparation) and in vitro (isolated arterial rings). The vasodilator effects of acetylcholine, sodium nitroprusside, and activators of PAR-2 and protease-activated receptor-1 (PAR-1) were compared in WKY versus SHR. Immunohistochemical localization of PAR-2 was also assessed in the basilar artery.nnnRESULTSnIncreases in basilar artery diameter in response to acetylcholine were 65% to 85% smaller in SHR versus WKY, whereas responses to sodium nitroprusside were not different. In contrast to acetylcholine, vasodilatation in vivo to SLIGRL-NH(2) was largely preserved in SHR, and SLIGRL-NH(2) was approximately 3-fold more potent in causing vasorelaxation in SHR versus WKY in vitro. In both strains, responses to SLIGRL-NH(2) were abolished by N(G)-nitro-L-arginine, an inhibitor of NO synthesis. Activators of PAR-1 had little or no effect on the rat basilar artery. PAR-2-like immunoreactivity was observed in both the endothelial and smooth muscle cells of the basilar artery in both strains of rat.nnnCONCLUSIONSnThese data indicate that NO-mediated vasodilatation to PAR-2 activation is selectively preserved or augmented in SHR and may suggest protective roles for PAR-2 in the cerebral circulation during chronic hypertension.

Flow-induced cerebral vasodilatation in vivo involves activation of phosphatidylinositol-3 kinase, NADPH-oxidase, and nitric oxide synthase

Reactive oxygen species (ROS) such as superoxide (O•−2) and hydrogen peroxide (H2O2) are known cerebral vasodilators. A major source of vascular ROS is the flavin-containing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. Activation of NADPH-oxidase leads to dilatation of the basilar artery in vivo via production of H2O2, but the endogenous stimuli for this unique vasodilator mechanism are unknown. Shear stress is known to activate both NADPH-oxidase and phosphatidylinositol-3 kinase (PI3-K) in cultured cells. Hence, this study used a cranial window preparation in anesthetized rats to investigate whether increased intraluminal blood flow could induce cerebral vasodilatation via the activation of NADPH-oxidase and/or PI3-K. Bilateral occlusion of the common carotid arteries to increase basilar artery blood flow caused reproducible, reversible vasodilatation. Topical treatment of the basilar artery with the NADPH-oxidase inhibitor diphenyleneiodonium (DPI) (0.5 and 5 μmol/L) inhibited flow-induced dilatation by up to 50% without affecting dilator responses to acetylcholine. Treatment with the H2O2 scavenger, catalase similarly attenuated flow-induced dilatation, suggesting a role for NADPH-oxidase-derived H2O2 in this response. The nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) partially reduced flow-induced dilatation, and combined treatment with a ROS inhibitor (DPI or catalase) and l-NAME caused a greater reduction in flow-induced dilatation than that seen with any of these inhibitors alone. Flow-induced dilatation was also markedly inhibited by the PI3-K inhibitor, wortmannin. Increased O•−2 production in the endothelium of the basilar artery during acute increases in blood flow was confirmed using dihydroethidium. Thus, flow-induced cerebral vasodilatation in vivo involves production of ROS and nitric oxide, and is dependent on PI3-K activation.

Activation of Protease-Activated Receptor-2 (PAR-2) Elicits Nitric Oxide–Dependent Dilatation of the Basilar Artery In Vivo

BACKGROUND AND PURPOSEnProtease-activated receptors (PARs) are a family of G-protein-coupled receptors activated by a tethered ligand amino acid sequence within the amino terminal that is revealed by site-specific proteolysis. In the vascular endothelium, activation of PAR-2 by treatment with trypsin or by using the amino acid ligand sequence (SLIGRL) produces endothelium-dependent relaxation of isolated noncerebral vascular segments. In this study, we first tested whether PAR-2 activation produces cerebral vasodilatation in vivo and then examined whether PAR-2-mediated vasodilatation is dependent on the production of nitric oxide.nnnMETHODSnConcentration-dependent vasodilator effects of the PAR-2 agonist peptide SLIGRL and trypsin were examined on the basilar artery using a cranial window in anesthetized rats. In addition, the vasodilator effects of SLIGRL, acetylcholine (ACh), and sodium nitroprusside (SNP) were examined in the absence and presence of N(G)-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ), an inhibitor of soluble guanylate cyclase.nnnRESULTSnBaseline diameter of the basilar artery averaged 239+/-4 microm. Under control conditions, SLIGRL (10(-6) to 10(-4) mol/L) and trypsin (0.01 to 10 U/mL) produced concentration-dependent vasodilator responses. In time-control experiments, SLIGRL (3 x 10(-6) and 10(-5) mol/L), ACh (10(-6) and 10(-5) mol/L), and SNP (10(-8) and 10(-7) mol/L) elicited reproducible dilatation of the basilar artery. In another group of rats, L-NNA (10(-4) mol/L) markedly inhibited dilator responses to both SLIGRL (13+/-3% versus 1+/-1% and 39+/-7% versus 11+/-2%; both P<0.05) and ACh (8+/-1% versus 0+/-0% and 13+/-2% versus 3+/-1%; both P<0.05). By contrast, responses to SNP were significantly augmented after treatment with L-NNA (P<0.05 versus control), indicating that inhibitory effects of L-NNA were specific for responses mediated by endogenous nitric oxide. Furthermore, in another group ODQ (10(-5) mol/L) inhibited responses to SLIGRL to a degree similar to that seen with L-NNA, consistent with a mechanism of PAR-2-mediated vasodilatation that involves activation of guanylate cyclase by nitric oxide.nnnCONCLUSIONSnTo the best of our knowledge, this study is the first to examine whether PAR-2-mediated vasodilatation is functional in cerebral arteries and is also the first to directly assess the effects of PAR-2 activation on vascular tone in vivo. The results suggest that activation of PAR-2 is an effective and powerful vasodilator mechanism in cerebral arteries in vivo. Cerebral vasodilator responses to PAR-2 activation are mediated by nitric oxide and are likely to be endothelium dependent.

Vasorelaxant and antioxidant activity of the isoflavone metabolite equol in carotid and cerebral arteries

BACKGROUND AND PURPOSEnEquol is the main active intestinal metabolite of the isoflavone daidzein and is postulated to be responsible for the cardiovascular benefits of soy. Cerebral vascular effects of equol are unknown. We compared the vasorelaxant and antioxidant effects of equol and daidzein in carotid and basilar artery of normal and hypertensive rats.nnnEXPERIMENTAL APPROACHnRelaxant responses to equol and daidzein were measured in the isolated carotid artery and in the basilar artery in vivo. Effects of nitric oxide synthase (NOS) inhibition, high extracellular K(+), endothelial removal and gender on responses to equol were investigated in carotid arteries. Antioxidant activity was assessed as the reduction of NADPH-induced superoxide levels. Hypertension was induced using angiotensin II (0.7 mg/kg per day for 14 days).nnnKEY RESULTSnIn normotensive rats, equol displayed vasorelaxant activity similar to daidzein. The relaxant effect of equol was independent of an intact endothelium, NOS activity, K(+) channels and gender. In the basilar artery, where superoxide levels are higher, equol exerted weak antioxidant effects, whereas effects of daidzein were insignificant. During hypertension, equol-induced vasorelaxation was preserved, whereas relaxant responses to daidzein were impaired.nnnCONCLUSIONS AND IMPLICATIONSnEquol possesses substantial vasodilator and weak antioxidant activity in cerebral arteries, with similar activity to daidzein, whereas in hypertension the vasorelaxant response to equol, but not daidzein, is preserved. However, daidzein possesses comparable direct vascular effects with equol, without the need for intestinal conversion to equol. Nevertheless, equol may represent a more useful therapeutic agent during cerebral vascular disease.

Inhibitory effect of 4‐aminopyridine on responses of the basilar artery to nitric oxide

Voltage‐dependent K+ channels are present in cerebral arteries and may modulate vascular tone. We used 200u2003μM 4‐aminopyridine (4‐AP), thought to be a relatively selective inhibitor of voltage‐dependent K+ channels at this concentration, to test whether activation of these channels may influence baseline diameter of the basilar artery and dilator responses to nitric oxide (NO) and cyclic GMP in vivo. Using a cranial window in anaesthetized rats, topical application of 4‐AP to the basilar artery (baseline diameter=240±5u2003μm, mean±s.e.mean) produced 10±1% constriction. Sodium nitroprusside (a NO donor), acetylcholine (which stimulates endothelial release of NO), 8‐bromo cyclic GMP (a cyclic GMP analogue), cromakalim (an activator of ATP‐sensitive K+ channels) and papaverine (a non‐NO, non‐K+ channel‐related vasodilator) produced concentration‐dependent vasodilator responses that were reproducible. Responses to 10 and 100u2003nM nitroprusside were inhibited by 4‐AP (20±4 vs 8±2% and 51±5 vs 33±5%, respectively, n=10; P<0.05). Responses to acetylcholine and 8‐bromo cyclic GMP were also partially inhibited by 4‐AP. In contrast, 4‐AP had no effect on vasodilator responses to cromakalim or papaverine. These findings suggest that NO/cyclic GMP‐induced dilator responses of the basilar artery are selectively inhibited by 4‐aminopyridine. Responses to nitroprusside were also markedly inhibited by 10u2003μM 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (an inhibitor of soluble guanylate cyclase; 16±4 vs 1±1% and 44±7 vs 7±1%; n=10; P<0.05). Thus, dilator responses of the rat basilar artery to NO appear to be mediated by activation of soluble guanylate cyclase and partially by activation of a 4‐aminopyridine‐sensitive mechanism. The most likely mechanism would appear to be activation of voltage‐dependent K+ channels by NO/cyclic GMP.

Evidence That Estrogen Suppresses Rho-Kinase Function in the Cerebral Circulation In Vivo

Background and Purpose— Premenopausal women are less susceptible to cardiovascular diseases than men or postmenopausal women. Such disease states are often associated with increased vascular RhoA/Rho-kinase activity and decreased activity of nitric oxide (NO). This study tested whether female gender is associated with lower Rho-kinase activity or higher NO activity in cerebral arteries in vivo and whether estrogen contributes to any such gender differences. Methods— Changes in basilar artery diameter were measured with the use of a cranial window preparation in anesthetized Sprague-Dawley rats. Some female rats were ovariectomized (OVX) and treated subcutaneously daily for 14 days with vehicle (dimethyl sulfoxide) or 17β-estradiol. Vascular expression of RhoA or Rho-kinase was assessed by Western blotting. Results— The Rho-kinase inhibitor Y-27632 was selectively ≈3-fold more potent as a cerebral vasodilator in males versus females. Expression of total RhoA or Rho-kinase did not differ between males and females. In OVX rats, vasodilator responses to Y-27632 resembled responses in males. Treatment of OVX rats with 17β-estradiol normalized the vasodilator effects of Y-27632 to be equivalent to responses in intact female controls. The NO synthase inhibitor N-nitro-l-arginine methyl ester caused ≈50% greater constriction of the basilar artery in females versus males, but responses in OVX rats treated with either vehicle or 17β-estradiol did not differ from those recorded in intact females. Conclusions— These data indicate that vascular Rho-kinase function is suppressed in females because of the effects of estrogen, whereas the higher NO activity in females is estrogen independent.