SULFHYDRYL INVOLVEMENT IN NITRIC-OXIDE SEQUESTRATION AND NITRIC-OXIDEINDUCED GUANYLYL CYCLASE ACTIVATION IN VASCULAR SMOOTH-MUSCLE

In the present study, the role of vascular smooth muscle sulhydryl gro ups was investigated with respect to sequestration of nitric oxide (NO ) and activation of soluble guanylyl cyclase by NO. Vascular smooth mu scle 100 000 x g supernatant (soluble) fraction was prepared in phosph ate buffer, using the medial layer of bovine pulmonary artery. The sol uble fraction was incubated with 100 pmol NO for 5 min in a sealed fla sk at 37 degrees C under anerobic conditions in the presence or absenc e of the sulfhydryl reagent, N-ethylmaleimide (NEM, 5 mM). NO sequestr ation by the soluble fraction was measured as an indicator of NO bindi ng. Total thiol content was measured in the soluble fraction with and without exposure to NEM. Guanylyl cyclase activity was measured in the soluble fraction with and without exposure to NO and a combination of NO and NEM. NEM decreased total thiol content in the soluble fraction from 103.59 nmol/mL to undetectable levels, and decreased guanylyl cy clase activity to below basal levels. The percentage of NO sequestered by the soluble fraction was inhibited by NEM by approximately 25% fro m a control value of 26.52 +/- 9.39 to 18.72 +/- 8.52, n = 13, p < 0.0 5. The data indicate that sulfhydryl groups are essential for guanylyl cyclase activation by NO, and are also involved in the sequestration of NO by the vascular smooth muscle soluble fraction.