Long-lasting blood pressure lowering effects of nitrite are NO-independent and mediated by hydrogen peroxide, persulfides, and oxidation of protein kinase G1α redox signalling.

AIMS: Under hypoxic conditions, nitrite (NO2-) can be reduced to nitric oxide (NO) eliciting vasorelaxation. However, nitrite also exerts vasorelaxant effects of potential therapeutic relevance under normal physiological conditions via undetermined mechanisms. We, therefore, sought to investigate the mechanism(s) by which nitrite regulates the vascular system in normoxia and, specifically, whether the biological effects are a result of NO generation (as in hypoxia) or mediated via alternative mechanisms involving classical downstream targets of NO [e.g. effects on protein kinase G1α (PKG1α)]. METHODS AND RESULTS: Ex vivo myography revealed that, unlike in thoracic aorta (conduit vessels), the vasorelaxant effects of nitrite in mesenteric resistance vessels from wild-type (WT) mice were NO-independent. Oxidants such as H2O2 promote disulfide formation of PKG1α, resulting in NO- cyclic guanosine monophosphate (cGMP) independent kinase activation. To explore whether the microvascular effects of nitrite were associated with PKG1α oxidation, we used a Cys42Ser PKG1α knock-in (C42S PKG1α KI; 'redox-dead') mouse that cannot transduce oxidant signals. Resistance vessels from these C42S PKG1α KI mice were markedly less responsive to nitrite-induced vasodilation. Intraperitoneal (i.p.) bolus application of nitrite in conscious WT mice induced a rapid yet transient increase in plasma nitrite and cGMP concentrations followed by prolonged hypotensive effects, as assessed using in vivo telemetry. In the C42S PKG1α KI mice, the blood pressure lowering effects of nitrite were lower compared to WT. Increased H2O2 concentrations were detected in WT resistance vessel tissue challenged with nitrite. Consistent with this, increased cysteine and glutathione persulfide levels were detected in these vessels by mass spectrometry, matching the temporal profile of nitrite's effects on H2O2 and blood pressure. CONCLUSION: Under physiological conditions, nitrite induces a delayed and long-lasting blood pressure lowering effect, which is NO-independent and occurs via a new redox mechanism involving H2O2, persulfides, and PKG1α oxidation/activation. Targeting this novel pathway may provide new prospects for anti-hypertensive therapy.

Redox signaling and splicing dependent change in myosin phosphatase underlie early versus late changes in NO vasodilator reserve in a mouse LPS model of sepsis.

Microcirculatory dysfunction may cause tissue malperfusion and progression to organ failure in the later stages of sepsis, but the role of smooth muscle contractile dysfunction is uncertain. Mice were given intraperitoneal LPS, and mesenteric arteries were harvested at 6-h intervals for analyses of gene expression and contractile function by wire myography. Contractile (myosin and actin) and regulatory [myosin light chain kinase and phosphatase subunits (Mypt1, CPI-17)] mRNAs and proteins were decreased in mesenteric arteries at 24 h concordant with reduced force generation to depolarization, Ca(2+), and phenylephrine. Vasodilator sensitivity to DEA/nitric oxide (NO) and cGMP under Ca(2+) clamp were increased at 24 h after LPS concordant with a switch to Mypt1 exon 24- splice variant coding for a leucine zipper (LZ) motif required for PKG-1α activation of myosin phosphatase. This was reproduced by smooth muscle-specific deletion of Mypt1 exon 24, causing a shift to the Mypt1 LZ+ isoform. These mice had significantly lower resting blood pressure than control mice but similar hypotensive responses to LPS. The vasodilator sensitivity of wild-type mice to DEA/NO, but not cGMP, was increased at 6 h after LPS. This was abrogated in mice with a redox dead version of PKG-1α (Cys42Ser). Enhanced vasorelaxation in early endotoxemia is mediated by redox signaling through PKG-1α but in later endotoxemia by myosin phosphatase isoform shifts enhancing sensitivity to NO/cGMP as well as smooth muscle atrophy. Muscle atrophy and modulation may be a novel target to suppress microcirculatory dysfunction; however, inactivation of inducible NO synthase, treatment with the IL-1 antagonist IL-1ra, or early activation of α-adrenergic signaling did not suppressed this response.

Soluble guanylate cyclase is required for systemic vasodilation but not positive inotropy induced by nitroxyl in the mouse.

Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO·), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-ß subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1α(C42S) mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1α is not a mechanism for HNO-mediated vasodilation.

Atrial natriuretic peptide-mediated inhibition of microcirculatory endothelial Ca2+ and permeability response to histamine involves cGMP-dependent protein kinase I and TRPC6 channels.

OBJECTIVE: Histamine increases microvascular endothelial leakage by activation of complex calcium-dependent and -independent signaling pathways. Atrial natriuretic peptide (ANP) via its cGMP-forming guanylyl cyclase-A (GC-A) receptor counteracts this response. Here, we characterized the molecular mechanisms underlying this interaction, especially the role of cGMP-dependent protein kinase I (cGKI). APPROACH AND RESULTS: We combined intravital microscopy studies of the mouse cremaster microcirculation with experiments in cultured microvascular human dermal endothelial cells. In wild-type mice, ANP had no direct effect on the extravasation of fluorescent dextran from postcapillary venules, but strongly reduced the histamine-provoked vascular leakage. This anti-inflammatory effect of ANP was abolished in mice with endothelial-restricted inactivation of GC-A or cGKI. Histamine-induced increases in endothelial [Ca(2+)]i in vitro and of vascular leakage in vivo were markedly attenuated by the Ca(2+)-entry inhibitor SKF96365 and in mice with ablated transient receptor potential canonical (TRPC) 6 channels. Conversely, direct activation of TRPC6 with hyperforin replicated the hyperpermeability responses to histamine. ANP, via cGKI, stimulated the inhibitory phosphorylation of TRPC6 at position Thr69 and prevented the hyperpermeability responses to hyperforin. Moreover, inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil prevented the edematic actions of histamine in wild types but not in mice with endothelial GC-A or cGKI deletion. CONCLUSIONS: ANP attenuates the inflammatory actions of histamine via endothelial GC-A/cGMP/cGKI signaling and inhibitory phosphorylation of TRPC6 channels. The therapeutic potential of this novel regulatory pathway is indicated by the observation that sildenafil improves systemic endothelial barrier functions by enhancing the endothelial effects of endogenous ANP.

The cyclic GMP-dependent protein kinase Iα suppresses kidney fibrosis.

Cyclic guanosine monophosphate (cGMP) is synthesized by nitric oxide or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions in the kidney. Hence, integration of cGMP signaling by cGMP-dependent protein kinases (cGKs) might play a critical role in renal physiology; however, detailed renal localization of cGKs is still lacking. Here, we performed an immunohistochemical analysis of cGKIα and cGKIß isozymes in the mouse kidney and found both in arterioles, the mesangium, and within the cortical interstitium. In contrast to cGKIα, the ß-isoform was not detected in the juxtaglomerular apparatus or medullary fibroblasts. Since interstitial fibroblasts play a prominent role in interstitial fibrosis, we focused our study on cGKI function in the interstitium, emphasizing a functional differentiation of both isoforms, and determined whether cGKIs influence renal fibrosis induced by unilateral ureter obstruction. Treatment with the guanylyl cyclase activators YC1 or isosorbide dinitrate showed stronger antifibrotic effects in wild-type than in cGKI-knockout or in smooth muscle-cGKIα-rescue mice, which are cGKI deficient in the kidney except in the renal vasculature. Moreover, fibrosis influenced the mRNA and protein expression levels of cGKIα more strongly than cGKIß. Thus, our results indicate that cGMP, acting primarily through cGKIα, is an important suppressor of kidney fibrosis.

Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I.

AIMS: Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. METHODS AND RESULTS: To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to ß-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca(2+)]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca(2+)i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca(2+)i-handling, and contractility via cGKI. CONCLUSION: These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca(2+)i handling and contractility.