Kinoshita, Hiroyuki

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, NG-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall. (Circ Res. 1997;80:327-335.)

Background and PurposeConcentration of hydrogen ions is an important regulator of cerebral arterial tone under physiological and pathological conditions. Previous studies demonstrated that in cerebral arteries, relaxations to hypercapnia are due to decrease in extracellular pH. The present study was designed to determine the role of potassium channels in mediation of cerebral arterial relaxations induced by extracellular acidosis.MethodsRings of canine basilar arteries without endothelium were suspended for isometric force recording. Acidosis (pH 7.3 to 7.0) was produced by incremental addition of hydrochloric acid (1.0N). The concentration of hydrogen ions was continuously monitored with a pH meter.ResultsDuring contractions to UTP, acidosis (pH 7.3 to 7.0) induced pH-dependent relaxations. These relaxations were abolished in arteries contracted by potassium chloride (20 mmol/L). A nonselective potassium channel inhibitor, BaCl2(10−3and 10−4mol/L), and an ATP-sensitive potassium channel inhibitor, glyburide (5×10−6mol/L), significantly reduced relaxations to acidosis. Furthermore, BaCl2(10−3mol/L) and glyburide (5×10−6mol/L) abolished relaxations to an ATP-sensitive potassium channel opener, cromakalim (10−8to 3×10−5mol/L). However, these potassium channel inhibitors did not affect relaxations to a voltage-dependent calcium channel inhibitor, diltiazem (10−8to 10−4mol/L), and glyburide (5×10−6mol/L) did not alter relaxations to a nitric oxide donor, SIN-1 (10−9to 10−4mol/L). A calcium-activated potassium channel inhibitor, charybdotoxin (10−7mol/L), and a delayed rectifier potassium channel inhibitor, 4-aminopyridine (10−3mol/L), did not affect relaxations to acidosis.ConclusionsThese results suggest that extracellular acidosis causes relaxations of cerebral arteries in part by activation of potassium channels. ATP-sensitive potassium channels appear to contribute to acidosis-induced decrease in cerebral arterial tone.