1 The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca 2+ ] i and divalent cation in¯ux was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10 77 M), NO (10 710 ± 10 76 M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca 2+ ] i and cation in¯ux as indicated by fura-2¯uorimetry. 2 Zaprinast (10 74 M), increased NO-stimulated levels of cyclic GMP by 3 ± 20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca 2+ ] i reached after administration of NO, the initial decreases in [Ca 2+ ] i initiated by NO were not signi®cantly dierent in magnitude or duration from those that occurred in the absence of zaprinast. ] i or cation in¯ux caused by higher concentrations of NO (10 77 ± 10 76 M) were unaected. Relaxation of intact rabbit aorta rings to NO (10 77 ± 10 75 M) also persisted in the presence of ODQ without a signi®cant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation in¯ux caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no signi®cant eect on the decrease caused by NO. 4 Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to aect the decrease in [Ca 2+ ] i and cation in¯ux via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute signi®cantly to the decrease in [Ca 2+ ] i and smooth muscle relaxation to NO.

The response to nitric oxide of intracellular free Ca2+ levels, measured by fura 2 fluorimetry, and cyclic GMP, measured by RIA, was evaluated on smooth muscle cells of the thoracic aorta in primary culture from normal and cholesterol-fed rabbits. Relaxation to acetylcholine and nitric oxide was also determined in isolated rings of aorta. After 10 weeks of high-cholesterol diet, the intact aorta relaxed less to both acetylcholine and nitric oxide. In cultured cells from hypercholesterolemic rabbits, intracellular Ca2+ oscillated, and the mean Ca2+ levels were approximately twofold greater than in normal aortic cells. Nitric oxide failed to affect basal Ca2+ in either cell type. The peak and sustained rise in intracellular Ca2+ induced by angiotensin II (10(-7) mol/L) were similar in the two cell types. However, nitric oxide (10(-10) to 10(-6) mol/L) decreased the sustained Ca2+ levels to a significantly smaller extent in cells from cholesterol-fed rabbits. In addition, in cells from hypercholesterolemic rabbits, nitric oxide added before angiotensin II inhibited to a smaller degree the transient increase in intracellular free Ca2+ caused by angiotensin II in the nominal absence of extracellular Ca2+, as well as the increase in Ca2+ associated with the addition of extracellular Ca2+. Measurements of fura 2 quenching caused by Mn2+ influx confirmed that nitric oxide inhibited the entry of extracellular divalent cations significantly less in cells from hypercholesterolemic rabbits. Basal levels of cyclic GMP were significantly less than normal, and nitric oxide increased levels of cyclic GMP to a significantly smaller degree in cells from cholesterol-fed rabbits. These data indicate a substantial resistance to nitric oxide action in aortic smooth muscle cells of cholesterol-fed rabbits. This observation is consistent with the notion that resistance of smooth muscle cells to nitric oxide contributes to abnormal endothelium-dependent vasodilation during hypercholesterolemia and can play a role in the pathogenesis of atherosclerosis.