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Myosin phosphatase target subunit 1 (MYPT1) is the regulatory subunit of myosin light chain phosphatase (MLCP). It plays a critical role in vasodilatation induced by cGMP-elevating agents such as nitric oxide (NO). The present study was performed to determine the role of MYPT1 in the development of tolerance of the pulmonary artery to NO. Incubation of isolated porcine pulmonary arteries for 24 or 48 h with DETA NONOate (DETA NO) significantly reduced protein levels of MYPT1 and the leucine zipper-positive (LZ+) isoform of MYPTI but not that of PPlcS. The extent of reduction in total MYPT1 protein level was comparable to that of MYPTI (LZ+). The decrease in MYPT1 protein caused by 48-h DETA NO incubation was prevented by ODQ, an inhibitor of guanylyl cyclase, and by inhibitors of proteasomes (MG-132 and lactacystin) but was not affected by the inhibitor of protein synthesis, cycloheximide. A reduction in MYPT1 protein was also obtained with 8-bromo-cGMP, but this was prevented by Rp-8bromo-PET-cGMP [inhibitor of cGMP-dependent protein kinase (PKG)]. Incubation for 48 h with DETA NO also reduced dephosphorylation of myosin light chain and relaxation of the artery in response to DETA NO, which was prevented by MG-132. These results suggest that the reduction in MYPT1 protein contributes to the development of tolerance of pulmonary arteries to NO. This may result from increased degradation of MYPT1 after prolonged PKG activation. myosin light chain phosphatase; guanosine 3',5'-cyclic monophosphate-dependent protein kinase; vasodilatation doi: 10.1152/ajplung.00340.2009.

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response of this vessel to acutely applied NO and to the [alpha]-adrenoreceptor agonist phenylephrine. Two-hour treatment with the NO donor (Z)-l-[N-(2-amino-ethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETANO) decreased both NO and phenylephrine responsiveness. Twenty-four-hour treatment with DETA-NO resulted in a further reduction in NO responsiveness but no further reduction in phenylephrine responsiveness. Acute addition of soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) had no effect on phenylephrine responsiveness in PA not treated with DETA-NO. ODQ treatment fully restored phenylephrine responsiveness in PA treated with DETA-NO. sGC[[beta].sub.1] subunit protein levels in PA tissue homogenate were 48.6 [+ or -] 6.9, 51.6 [+ or -] 3.5, and 41.3 [+ or -] 2.8 ng/mg total protein for freshly prepared and 2-h and 24-h NO-treated PA, respectively. Steady-state tissue cGMP was not significantly different in control versus NO-treated PA. sGC specific activity in the absence of added NO was measured in PA homogenate and was 0.29 [+ or -] 0.02, 1.38 [+ or -] 0.12, and 0.53 [+ or -] 0.08 [micro]mol cGMP x [min.sup.-1] x mg [sGC.sup.-1], in freshly prepared and 2-h and 24-h NO treated PA, respectively. Ten-minute Hb treatment completely normalized sGC basal activity in homogenates prepared from DETA-NO-treated PA, which was 0.23 [+ or -] 0.02, 0.18 [+ or -] 0.03, and 0.25 [+ or -] 0.04 [micro]mol cGMP x [min.sup.-1] x mg [sGC.sup.-1], in freshly prepared and 2-h and 24-h NO-treated PA, respectively. The kinetics of the Hb reversal of NO-mediated sGC persistent activation do not support sGC covalent modification as the activation mechanism. We conclude that prolonged NO exposure results in a persistently increased sGC specific activity, which accounts for the observed [alpha]-adrenoreceptor agonist hyporesponsiveness. nitric oxide; guanosine 3',5'-cyclic monophosphate; [alpha]-adrenoreceptor agonist

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-[alpha] or ER-[beta], and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-[alpha] agonist propylpyrazole triol (PPT) and/or the selective ER-[beta] agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings (n = 3-10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine ([10.sup.-8]M - [10.sup.-5]M) and hypoxia ([Po.sub.2] 35-45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine ([10.sup.-8]M - [10.sup.-4]M) and sodium nitroprusside ([10.sup.-9]M - [10.sup.-5]M). PPT or DPN ([10.sup.-9]M - 5 x [10.sup.-5]M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor [N.sup.[omega]]-nitro-t.-arginine methyl ester (L-NAME) ([10.sup.-4]M). Selective ER-[alpha] activation (PPT, 5 x [10.sup.-5]M) rapidly ( propylpyrazole triol; diarylpropiolnitrile; phenylephrine; hypoxic pulmonary vasoconstriction; nongenomic effects

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or [Ca.sup.2+]-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the L-arginine-endothelial NO synthase (eNOS) and L-argininearginase pathways. Incubation of PAEC with different concentrations of UA (2.5-15 mg/dl) for 24 h did not affect L-[[sup.3]H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for L-arginine. An inhibitor of arginase (S)(2boronoethyl)-L-cysteine prevented UA-induced reduction of A23 187stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC. nitric oxide; endothelial dysfunction; pulmonary hypertension

Caveolin-1, the structural and signaling protein of caveolae, is an important negative regulator of endothelial nitric oxide synthase (eNOS). We observed that mice lacking caveolin-1 ([Cavl.sup.-/-]) had twofold increased plasma NO levels but developed pulmonary hypertension. We measured pulmonary vascular resistance (PVR) and assessed alterations in small pulmonary arteries to determine the basis of the hypertension. PVR was 46% greater in [Cavl.sup.-/-] mice than wild-type (WT), and increased PVR in [Cavl.sup.-/-] mice was attributed to precapillary sites. Treatment with [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) to inhibit NOS activity raised PVR by 42% in WT but 82% in [Cavl.sup.-/-] mice, indicating greater NO-mediated pulmonary vasodilation in [Cavl.sup.-/-] mice compared with WT. Pulmonary vasculature of [Cavl.sup.-/-] mice was also less reactive to the vasoconstrictor thromboxane A2 mimetic (U-46619) compared with WT. We observed redistribution of type I collagen and expression of smooth muscle [alpha]-actin in lung parenchyma of [Cavl.sup.-/-] mice compared with WT suggestive of vascular remodeling. Fluorescent agarose casting also showed markedly decreased density of pulmonary arteries and artery filling defects in [Cavl.sup.-/-] mice. Scanning electron microscopy showed severely distorted and tortuous pulmonary precapillary vessels. Thus caveolin-1 null mice have elevated PVR that is attributed to remodeling of pulmonary precapillary vessels. The elevated basal plasma NO level in [Cavl.sup.-/-] mice compensates partly for the vascular structural abnormalities by promoting pulmonary vasodilation. nitric oxide; pulmonary hypertension; fluorescent angiography; corrosion casting