Nitric oxide synthase and guanylate cyclase levels in canine basilar artery after subarachnoid hemorrhage

Abstract

Endothelium-dependent vasodilation may be impaired during cerebral vasospasm following subarachnoid hemorrhage. Under normal circumstances nitric oxide (NO) released by endothelial cells induces relaxation of smooth muscle by activating the soluble form of guanylate cyclase within muscle cells. In this study the levels of both endothelial NO synthase, the enzyme that produces NO, and soluble guanylate cyclase were determined in canine basilar arteries in a double-hemorrhage model using Western blot immunoassays. Thirty dogs were assigned to three groups: Group D0, control; Group D2, dogs sacrificed 2 days after cisternal injection of blood; and Group D7, dogs given double cisternal injections of blood and sacrificed 7 days after the first injection. Constriction of the basilar artery was confirmed by arterial angiography. Portions of the affected arteries or the corresponding region in control animals were solubilized for sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting. A specific monoclonal antibody against endothelial NO synthase was used. The extract from basilar arteries showed two bands on the blots: 135 kD, characteristic of endothelial NO synthase, and 120 kD, which may be a degradation product of the enzyme. The densitometer values of the bands were presented as percentages of D0 control values. Although the total signal in the D7 group was less than that of the D0 control group (D2, 97% +/- 22%; D7, 78% +/- 40%), it was not statistically significant. The proportion of the 135-kD form decreased between Groups D0 and D7, but the difference was not significant. A single major band corresponding to the alpha-subunit of soluble guanylate cyclase was seen at 70 kD in the basilar artery extracts. The signals of D2 and D7 samples were 69% +/- 40% and 25% +/- 18%, respectively. There was a significant difference between D7 and D0 (p < 0.001). The reduced expression of soluble guanylate cyclase may be related to the impairment of endothelium-dependent vasodilation in vasospasm.