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Background and Purpose: Reactive oxygen species (ROS) may play a critical role in the regulation of vascular tone and development of vascular diseases, such as stroke. NAD(P)H oxidase is a major source of ROS in vascular cells, including endothelial cells. It has been considered that Nox2 and Nox4 are exclusively expressed among Nox homologues in the endothelial cells of noncerebral blood vessels. However, the precise molecular identity of the NAD(P)H oxidase in the endothelial cells of the cerebral arteries is not fully understood. We examined the expression of Nox homologues and their activation mechanism in the endothelial cells of the cerebral arteries., Methods: We isolated and cultured basilar artery endothelial cells (BAECs) of Sprague-Dawley rats. Expression of NAD(P)H oxidase was examined by reverse-transcription-polymerase chain reaction (RT-PCR) and immunohistological staining., Results: RT-PCR disclosed abundant expression of Nox4 with marginal Nox2 in BAEC. In addition, Nox1 was expressed highly both at mRNA and protein levels in BAECs. Immunohistological staining also showed the prominent expression of Nox1 in the endothelial cells of the basilar artery. With respect to the cytosolic components of NAD(P)H oxidases, BAECs expressed p67phox and, to a lesser extent, p47phox, Noxo1, and Noxa1. Both NADH and NADPH induced superoxide production of the BAEC membranes. The phagocyte-type cytosolic components, p47phox and p67phox, significantly enhanced the NADH-induced superoxide production of the BAEC membranes, whereas the components failed to increase the NADPH-induced superoxide production., Conclusions: Nox1 is highly expressed in the endothelial cells of the cerebral arteries along with Nox2 and Nox4, and the endothelial NAD(P)H oxidase of the cerebral arteries may have a unique activation mechanism by the phagocyte-type cytosolic components.

Background and Purpose: During cerebral ischemia, both hypoxia and hypercapnia appear to produce marked dilatation of the cerebral arteries. Hypercapnia and hypoxia may be accompanied by extracellular and intracellular acidosis, which is another potent dilator of cerebral arteries. However, the precise mechanism by which acidosis produces dilatation of the cerebral arteries is not fully understood. The objective of the present study was to examine the mechanisms by which intracellular acidosis produces dilatation of the basilar artery in vivo., Methods: Using a cranial window in anesthetized rats, we examined responses of the basilar artery to sodium propionate, which was used to cause intracellular acidosis specifically. Expression of subunits of potassium channels was determined by reverse transcription and polymerase chain reaction (RT-PCR)., Results: Topical application of propionate increased diameter of the basilar artery in a concentration-related manner. Propionate-induced dilatation of the artery was attenuated by glibenclamide, an inhibitor of ATP-sensitive potassium channels. However, inhibitors of nitric oxide synthase (N(G)-nitro-L-arginine), large-conductance calcium-activated potassium channels (iberiotoxin), and cyclooxygenase (indomethacin) did not affect the vasodilatation. Expression of mRNA for SUR2B and Kir6.1 was detected, with the use of RT-PCR, in the cultured basilar arterial muscle cells., Conclusions: The findings suggest that intracellular acidification may produce dilatation of the basilar artery through activation of ATP-sensitive potassium channels in vivo. Kir6.1/SUR2B may be the major potassium channels that mediate propionate-induced dilatation of the artery.

1. Ionic channels appear to play an important role in contractile responses of the cerebral arteries and, thereby, contribute to the regulation of cerebral circulation. In the present study, we investigated the role of large-conductance Ca(2+)-activated K+ (BK(Ca)) channels in the regulation of cerebral arterial tone during chronic hypertension. 2. Ring segments of the basilar artery from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were placed in bath chambers and the isometric tension of each ring was measured. 3. Application of inhibitors of BK(Ca) channels, namely tetraethylammonium (TEA; > or = 0.1 mmol/L) and charybdotoxin (CTX; > or = 0.1 nmol/L), produced spontaneous contraction with rhythmic oscillation in the basilar artery from SHR. 4. The oscillatory contraction was not induced by 5-hydroxytryptamine (0.01-10 micromol/L) or depolarization by external high K+ (20-60 mmol/L). 5. The rhythmic contraction was completely abolished by either the removal of external Ca(2+) or the application of nicardipine (10 nmol/L). 6. The oscillation was not affected by the substitution of external Cl(-) by various equimolar anions (i.e. acetate, benezenesulphonate, bromide and isethianate). 7. The amplitude of the oscillation was dose-dependently increased by the vasodilators forskolin and sodium nitroprusside, as well as by stimulation of the endothelium with histamine and acetylcholine, whereas the frequency was decreased. 8. In contrast, the oscillation was eliminated by depletion of Ca(2+) stores by caffeine. Neither TEA (10 mmol/L) nor CTX (10 nmol/L) produced any significant contraction of the basilar artery in WKY rats. 9. These results suggest that BK(Ca) channels may play an important role in regulating the resting tone of the cerebral artery in SHR.

Objectives: Several factors mediating vascular responses appear to play an important role in the increased resistance of cerebral blood vessels during hypertension. The objective of this study was to elucidate the mechanisms by which hypertension increases the basal tone of the basilar artery in vivo., Methods: Using a cranial window, we examined effects of inhibitors of L-type voltage-dependent calcium channels (nicardipine) and Rho-associated kinase (Y-27632) on the baseline diameter of the basilar artery in spontaneously hypertensive rats (SHR) and compared to the responses in normotensive Wistar-Kyoto (WKY) rats., Results: Topical application of nicardipine (10(-8), 10(-7) and 10(-6) mol/l) produced dilatation of the basilar artery. Nicardipine-induced vasodilatation was enhanced in SHR compared to WKY rats. Nicardipine (10-6 mol/l) dilated the artery in WKY rats and SHR by 9 +/- 2 and 24 +/- 4%, respectively. Topical application of Y-27632 (10(-7), 10(-6) and 10(-5) mol/l) produced dilatation of the basilar artery in WKY rats in a concentration-related manner. The vasodilatation produced by Y-27632 was markedly enhanced in SHR compared to WKY rats. Y-27632 (10(-5) mol/l) dilated the artery in WKY rats and SHR by 14 +/- 2 and 45 +/- 6%, respectively. We also tested the effects of inhibitors of nitric oxide synthase (NG-nitro-l-arginine methyl ester, l-NAME), ATP-sensitive potassium channels (glibenclamide) and large-conductance calcium-activated potassium channels (tetraetyhlammonium, TEA). l-NAME and, to a lesser extent, glibenclamide produced similar constriction of the basilar artery in both strains. TEA did not affect the baseline diameter of the basilar artery in WKY rats and produced small but significant vasoconstriction in SHR., Conclusions: These results suggest that the activity of L-type calcium channels and Rho-associated kinase is enhanced in the basilar artery during hypertensionin vivo. The enhanced contractility may contribute to the increased resistance of the basilar artery during chronic hypertension in vivo.

We tested the hypothesis that activation of Na(+)/H(+) exchanger is involved in dilator responses of the basilar artery to endothelium-dependent vasodilators in vivo. Using a cranial window in anesthetized rats, we examined responses of the basilar artery to acetylcholine and bradykinin. Topical application of acetylcholine and bradykinin increased diameter of the basilar artery in a concentration-related manner. Because N(G)-nitro-L-arginine, an inhibitor of nitric oxide synthase, almost abolished vasodilator responses to acetylcholine and bradykinin, vasodilatation produced by the agonists appears to be mediated primarily by nitric oxide. 5-N,N-Hexamethyleneamiloride, an inhibitor of Na(+)/H(+) exchanger, did not affect baseline diameter of the basilar artery, but inhibited vasodilatation in response to acetylcholine and bradykinin, without affecting vasodilatation produced by sodium nitroprusside. FR183998, another inhibitor of Na(+)/H(+) exchanger, also attenuated acetylcholine-induced dilatation of the basilar artery without affecting vasodilatation in response to sodium nitroprusside. Monomethylamine hydrochloride, which produces intracellular alkalinization, enhanced acetylcholine-induced dilatation of the basilar artery in the presence of 5-N,N-hexamethyleneamiloride. These results suggest that intracellular alkalinization produced by activation of Na(+)/H(+) exchanger may enhance nitric oxide production in the basilar arterial endothelium and thereby contribute to dilator responses of the artery in vivo.

Background and Purpose: We tested the hypothesis that activation of phosphatidylinositol (PI) 3-kinase is involved in dilator responses of the basilar artery to acetylcholine in vivo., Methods: Responses of the basilar artery were measured by the cranial window technique in anesthetized rats. To examine the role of PI 3-kinase in acetylcholine-induced calcium signaling, we measured intracellular free calcium concentration ([Ca(2+)](i)) of cultured rat basilar arterial endothelial cells using a fluorescent calcium indicator, indo 1., Results: -Topical application of acetylcholine (10(-6), 10(-5.5), and 10(-5) mol/L) increased the diameter of the basilar artery by 8+/-1%, 14+/-2%, and 24+/-3%, respectively. An inhibitor of PI 3-kinase, wortmannin (10(-8) mol/L), did not change the baseline diameter of the artery. In the presence of wortmannin, acetylcholine (10(-6), 10(-5.5), and 10(-5) mol/L) dilated the artery only by 3+/-2%, 6+/-2%, and 12+/-2%, respectively. Thus, wortmannin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05 versus control). Wortmannin had no effect on dilatation of the artery in response to a nitric oxide donor, sodium nitroprusside. LY294002, another inhibitor of PI 3-kinase, also inhibited dilator response of the basilar artery to acetylcholine. Acetylcholine produced an increase in [Ca(2+)](i) of the endothelial cells. Genistein, an inhibitor of tyrosine kinase, markedly attenuated acetylcholine-induced calcium influx to the cells; however, wortmannin had no effect on acetylcholine-induced calcium changes., Conclusions: These results suggest that acetylcholine-induced dilatation of the basilar artery is mediated, at least in part, by activation of PI 3-kinase in vivo. Acetylcholine-induced [Ca(2+)](i) changes of the endothelial cells may not be mediated by activation of the kinase. PI 3-kinase as well as [Ca(2+)](i) may play an important role in the acetylcholine-induced nitric oxide production of the basilar arterial endothelial cells.

In smooth muscle cells isolated from cerebral blood vessels, histamine activates Cl- channels through an elevation of intracellular Ca2+. We investigated whether Cl- currents were also evoked by adenosine triphosphate (ATP) or caffeine in isolated smooth muscle cells from the rabbit basilar artery using the perforated patch-clamp technique. Bath application of 10 microM ATP or 1 mM caffeine (holding potential -60 mV) activated transient inward currents. With prolonged bath application of 10 microM ATP or 1 mM caffeine, oscillatory inward current were sporadically generated. At a holding potential of -40 mV, transient Cl- currents were induced by 10 microM histamine, 10 microM ATP, and 1 mM caffeine, following activation of a K+ current. At -10 or -20 mV, histamine predominantly activated the K+ current. A repetitively activated outward current was induced by membrane depolarization. These results suggest that oscillations in intracellular Ca2+ induced by histamine, ATP, and caffeine caused Cl(-)-current activation at the resting membrane potential. This Cl- current may depolarize the membrane and, thus activate voltage-dependent currents, including a Ca(2+)-dependent K+ current. Both the Ca(2+)-dependent K+ and Cl- currents induced by various stimuli may contribute to the modulation of Ca2+ influx by reinforcing membrane depolarization.

The membrane current evoked by histamine in isolated smooth muscle cells from rabbit basilar artery was investigated using the perforated-patch technique. When 10 microM histamine was applied in the bath at a holding potential of -60 mV, an inward current (79.2 +/- 55.8 pA) was transiently activated. An outward current was additionally evoked by 10 microM histamine when the membrane was held at -40 mV or less negative potentials. The outward but not the inward current was completely blocked by 100 nM charybdotoxin. A higher concentration of histamine (30 microM) failed to produce the inward current (3.4 +/- 4.8 pA) when Cl- concentration in the pipette was reduced. The apparent reversal potential of the inward current induced by histamine in physiological salt solution, in high-tetraethylammonium (TEA+) solution (bath), or in low-Cl- solution (pipette) was -6.3 +/- 4.4, -7.5 +/- 4.9, or -45.8 +/- 8.5 mV, respectively. Niflumic acid (100 microM) reversibly blocked the inward current, which was also blocked by 10 microM pyrilamine but not by 10 microM cimetidine. When histamine was continuously applied in the bath, spontaneous transient inward currents were generated. Removal of external Ca2+ or addition of 1 microM nicardipine or 2 mM caffeine reduced the amplitude of the histamine-induced inward current. These results suggest that histamine induces an inward current via H1 receptors at the resting membrane potential, possibly due to activation of Cl- currents. The Cl- inward current might be generated by elevation of intracellular Ca2+ via histamine receptors. The inward current may also contribute to control of the Ca2+ influx via a change in the membrane potential.