Nitric oxide (NO)-induced relaxation is assodated with increased levels ofcGMP in vascular smooth muscle cells. However, the mechanism by which cGMP causes relaxation is unknown. This study tested the hypothesis that activation of Ca-sensitive K (Kc.) chanels, mediated by a cGMPdependent protein kinase, is responsible for the relaxation occurring in response to cGMP. In rat pulmonary artery rings, cGMP-dependent, but not cGMP-independent, relaxation was inhibited by tetraethylammonium, a classical K-channel blocker, and charybdotoxin, an inhibitor of Kca channels. Increasing extrcellular K concentration also inhibited cGMPdependent relaxation, without reducing vascular smooth muscle cGMP levels. In whole-cell patch-clamp experiments, NO and cGMP increased whole-cell K current by activating Kc. channels. This effect was mimicked by intracellular administration of (Sp)-guanosine cyclic 3',5'-phosphorothioate, a preferential cGMP-dependent protein kinase activator. Okadaic acid, a phosphatase inhibitor, enhanced whole-cell K current, consistent with an important role for channel phosphorylation in the activation of NO-responsive Kc channels. Thus NO and cGMP relax vascular smooth muscle by a cGMP-dependent protein kinase-dependent activation of K channels. This suggests that the final common pathway shared by NO and the nitrovasodilators is cGMP-dependent K-channel activation.Nitric oxide (NO) and nitrovasodilators cause vasodilatation by activating guanylate cyclase and increasing cGMP in vascular smooth muscle (VSM) (1). The mechanism by which cGMP reduces vascular tone has been uncertain. Several experiments suggest that cGMP-mediated vasodilation is associated with changes in membrane potential. (i) KCl, which depolarizes VSM cells, inhibits endothelium-dependent vasodilatation (2). (ii) NO itself hyperpolarizes VSM in many (3-5), but not all (6, 7), studies. Finally, agents that increase cGMP can activate K channels (8-10). K-channel activity is the main determinant of membrane potential, and K efflux resulting from K-channel opening causes hyperpolarization, inhibits voltage-gated Ca channels, and promotes relaxation (Fig. 1).The current investigation evaluated two hypotheses: (i) K-channel activation is essential for cGMP-induced VSM relaxation and (ii) increases in cGMP activate K channels by stimulating cGMP-dependent protein kinase (cGK).To precisely characterize the role of NO/cGMP-activated K channels in vascular relaxation, it is necessary to combine studies of vascular tone [isolated pulmonary artery (PA) rings] and electrophysiology (whole-cell patch-clamp studies of PA VSM). These studies prove that NO and agents that increase cGMP cause relaxation in large part by a cGKmediated activation of Ca-sensitive K (Kca) channels. MATERIALS AND METHODSDrugs. Drugs and reagents were from Sigma and were dissolved in normal saline unless otherwise stated. Bath concentrations of solvents were <0.1% and all vehicles were tested to exclude nonspecific effects. Saturated NO solutions (2-3 mM) were prepared...

Previous studies have suggested that nitric oxide (NO) plays a role in regulation of renal vascular tone and sodium handling.We questioned whether the effects of NO synthase inhibition on renal function are direct or due to increased renal perfusion pressure (RPP) and whether stimulation of endogenous NO activity plays a role in adaptation to increased dietary salt intake. Intrarenal arterial infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) in control rats resulted in decreased glomerular filtration rate, renal vasoconstriction, natriuresis, and proteinuria. When RPP was held at basal levels with a suprarenal aortic snare, L-NMMA had similar hemodynamic effects but decreased sodium excretion and did not induce proteinuria. Exposure of rats to high salt intake (1% NaCl drinking water) for 2 wk induced increased serum concentration and urinary excretion of the NO decomposition products, NO2 + NO3. Urinary NO2 + NO3 and sodium excretion were significantly correlated. Compared with controls, chronically salt-loaded rats also demonstrated enhanced renal hemodynamic responses to NO synthase inhibition. We conclude that the endogenous NO system directly modulates renal hemodynamics and sodium handling and participates in the renal adaptation to increased dietary salt intake. Enhanced NO synthesis in response to increased salt intake may facilitate sodium excretion and allow maintenance of normal blood pressure. (J. Clin. Invest. 1993. 91:642-650.)

Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin and is released and/or synthesized by platelets, macrophages, endothelial cells, and rat mesangial cells. In the present investigation, we found that human glomerular mesangial cells in culture release a PDGF-like protein which competes for 125I-PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts. The competing and to a lesser extent the mitogenic activities present in the conditioned medium are partially recognized by an anti-PDGF antibody. Northern blot analysis of poly(A)+ RNA from human mesangial cells demonstrates the expression of both PDGF A- and B-chain mRNAs. PDGF also binds to mesangial cells in a specific manner and stimulates DNA synthesis and cell proliferation. These data suggest that a PDGF-like protein secreted by mesangial cells or released from platelets, monocytes, or endothelial cells during glomerular inflammation may function as an autocrine or a paracrine growth factor for these cells. The biological role of PDGF in mediating proliferative and other inflammatory events in the glomerulus remains to be identified.

Escherichia coli endotoxin (LPS) can induce the clinical syndrome of septic shock and renal cortical necrosis and can stimulate nitric oxide (NO) production from macrophages, vascular smooth muscle, and glomerular mesangial cells in vitro. NO is an endogenous vasodilator, which also inhibits platelet aggregation and adhesion. We therefore sought to determine whether LPS would stimulate NO production in vivo and, if so, whether this NO would modulate endotoxin-induced glomerular thrombosis. The stable NO endproducts, NO2 and NO3, were measured in serum and urine collections from rats during baseline and after injection of LPS, with or without substances that modulate NO synthesis. The urinary excretion of NO2/NO3 was 1,964±311 nm/8 h during the baseline and increased to 6,833±776 nm/8 h after a single intraperitoneal injection of0.1 mg/kg LPS (P < 0.05). The serum concentration of NO2/ NO3 also significantly increased after LPS injection. Both the urine and serum stimulation was significantly prevented by the NO synthesis inhibitor, N,,-nitro-L-arginine methyl ester (L-NAME). L-Arginine, given with LPS + L-NAME significantly restored the NO2/NO3 levels in the urine. Ex vivo incubation oftissues from rats treated with LPS demonstrated NO production by the aorta, whole kidney, and glomeruli, but not cortical tubules. Histological examination of kidneys from rats given either LPS or L-NAME alone revealed that 2 and 4.5% of the glomeruli contained capillary thrombosis, respectively. In contrast, rats given LPS + L-NAME developed thrombosis in 55% of glomeruli (P < 0.001), which was significantly prevented when L-arginine was given concomitantly. We conclude that LPS stimulates endogenous production of NO in vivo and that this NO is critical in preventing LPS-induced renal thrombosis. (J. Clin. Invest. 1992. 90:1718-1725

Endogenous nitric oxide plays an important role in modulation of renal hemodynamics and sodium handling, with increased nitric oxide production inducing renal vasodilation and natriuresis. In the normal rat, nitric oxide activity increases as an adaptive response to increased dietary salt intake, perhaps facilitating natriuresis and thus blood pressure homeostasis. We hypothesized that impaired nitric oxide synthetic ability would result in sensitivity to the pressor effects of high dietary salt intake. Four groups of normal Sprague-Dawley rats were observed for eight weeks: Control, 0.4% NaCl chow and tap water; Salt, 4% NaCl chow and tap water; NAME, 0.4% NaCl chow and water containing the nitric oxide synthase inhibitor, L-nitro-arginine-methylester; Salt+NAME, 4% NaCl chow and water containing L-nitro-arginine-methylester. Compared to Controls, Salt rats demonstrated a significant increase in urinary excretion rate of the stable nitric oxide metabolites, NO2 and NO3, and had no increase in blood pressure. Furthermore, Salt rats had no functional or structural evidence of renal injury. In contrast, Salt+NAME rats demonstrated a significantly higher blood pressure than NAME rats, and urinary NO2 and NO3 excretion rate did not increase despite high salt intake. After eight weeks, Salt+NAME rats had significantly impaired renal function and proteinuria. We conclude that adaptive changes in endogenous NO production play a critical role in sodium and blood pressure homeostasis. Furthermore, impaired nitric oxide synthase activity may be a pathogenetic factor in the development of salt-sensitive hypertension.