Enhancement of cGMP-dependent pathway activity ameliorates hyperglycemia-induced decrease in SIRT1-AMPK activity in podocytes: Impact on glucose uptake and podocyte function.

Hyperglycemia significantly decreases 3',5'-cyclic guanosine monophosphate (cGMP)-dependent pathway activity in the kidney. A well-characterized downstream signaling effector of cGMP is cGMP-dependent protein kinase G (PKG), exerting a wide range of downstream effects, including vasodilation and vascular smooth muscle cells relaxation. In podocytes that are exposed to high glucose concentrations, crosstalk between the protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) decreased, attenuating insulin responsiveness and impairing podocyte function. The present study examined the effect of enhancing cGMP-dependent pathway activity on SIRT1-AMPK crosstalk in podocytes under hyperglycemic conditions. We found that enhancing cGMP-dependent pathway activity using a cGMP analog was associated with increases in SIRT1 protein levels and activity, with a concomitant increase in the degree of AMPK phosphorylation. The beneficial effects of enhancing cGMP-dependent pathway activity on SIRT1-AMPK crosstalk also included improvements in podocyte function. Based on our findings, we postulate an important role for SIRT1-AMPK crosstalk in the regulation of albumin permeability in hyperglycemia that is strongly associated with activity of the cGMP-dependent pathway.

M. tuberculosis PknG manipulates host autophagy flux to promote pathogen intracellular survival.

The eukaryotic-type protein kinase G (PknG), one of the eleven eukaryotic type serine-threonine protein kinase (STPK) in Mycobacterium tuberculosis (Mtb), is involved in mycobacterial survival within macrophages, presumably by suppressing phagosome and autophagosome maturation, which makes PknG an attractive drug target. However, the exact mechanism by which PknG inhibits pathogen clearance during mycobacterial infection remains largely unknown. Here, we show that PknG promotes macroautophagy/autophagy induction but inhibits autophagosome maturation, causing an overall effect of blocked autophagy flux and enhanced pathogen intracellular survival. PknG prevents the activation of AKT (AKT serine/threonine kinase) via competitively binding to its pleckstrin homology (PH) domain, leading to autophagy induction. Remarkably, PknG could also inhibit autophagosome maturation to block autophagy flux via targeting host small GTPase RAB14. Specifically, PknG directly interacts with RAB14 to block RAB14-GTP hydrolysis. Furthermore, PknG phosphorylates TBC1D4/AS160 (TBC1 domain family member 4) to suppress its GTPase-activating protein (GAP) activity toward RAB14. In macrophages and in vivo, PknG promotes Mtb intracellular survival through blocking autophagy flux, which is dependent on RAB14. Taken together, our data unveil a dual-functional bacterial effector that tightly regulates host autophagy flux to benefit pathogen intracellular survival.Abbreviations: AKT: AKT serine/threonine kinase; ATG5: autophagy related 5; BMDMs: bone marrow-derived macrophages; DTT: dithiothreitol; FBS: fetal calf serum; GAP: GTPase-activating protein; MOI: multiplicity of infection; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; OADC: oleic acid-albumin-dextrose-catalase; PC, phosphatidylcholine; PH: pleckstrin homology; PI3K: phosphoinositide 3-kinase; PknG: protein kinase G; PtdIns(3,4,5)P3: phosphatidylinositol(3,4,5)-trisphosphate; SQSTM1: sequestosome 1; STPK: serine-threonine protein kinase; TB: tuberculosis; TBC1D4: TBC1 domain family member 4; TPR: tetratricopeptide repeat; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Deranged myofilament phosphorylation and function in experimental heart failure with preserved ejection fraction.

AIMS: Heart failure (HF) with preserved ejection fraction (HFpEF) is a major cause of morbidity and mortality. Key alterations in HFpEF include increased left ventricular (LV) stiffness and abnormal relaxation. We hypothesized that myofilament protein phosphorylation and function are deranged in experimental HFpEF vs. normal myocardium. Such alterations may involve the giant elastic protein titin, which contributes decisively to LV stiffness. METHODS AND RESULTS: LV tissue samples were procured from normal dogs (CTRL) and old dogs with hypertension-induced LV hypertrophy and diastolic dysfunction (OHT/HFpEF). We quantified the expression and phosphorylation of myofilament proteins, including all-titin and site-specific titin phosphorylation, and assessed the expression/activity of major protein kinases (PKs) and phosphatases (PPs), myofilament calcium sensitivity (pCa(50)), and passive tension (F(passive)) of isolated permeabilized cardiomyocytes. In OHT vs. CTRL hearts, protein kinase-G (PKG) activity was decreased, whereas PKCα activity and PP1/PP2a expression were increased. Cardiac MyBPC, TnT, TnI and MLC2 were less phosphorylated and pCa(50) was increased in OHT vs. CTRL. The titin N2BA (compliant) to N2B (stiff) isoform-expression ratio was lowered in OHT. Hypophosphorylation in OHT was detected for all-titin and at serines S4010/S4099 within titin-N2Bus, whereas S11878 within proline, glutamate, valine, and lysine (PEVK)-titin was hyperphosphorylated. Cardiomyocyte F(passive) was elevated in OHT, but could be normalized by PKG or PKA, but not PKCα, treatment. CONCLUSIONS: This patient-mimicking HFpEF model is characterized by titin stiffening through altered isoform composition and phosphorylation, both contributing to increased LV stiffness. Hypophosphorylation of myofilament proteins and increased calcium sensitivity suggest that functional impairment at the sarcomere level may be an early event in HFpEF.

Modulation of cardiac ERG1 K(+) channels by cGMP signaling.

Different K(+) currents have been implicated in the myocardial action potential repolarization including the I(Kr). ERG1 alpha subunits, identified as the molecular correlate of I(Kr), have been shown to form heteromultimeric channels in the heart and their activity is modulated by a complex interplay of signal transduction events. Using electrophysiological techniques, we examined the effects of the cGMP-analogue 8-Br-cGMP on rat and guinea-pig papillary action potential duration (APD), on the biophysical properties of heterologously expressed homo- and heteromeric ERG1 channels, and on cardiac I(Kr). 8-Br-cGMP prolonged APD by about 25% after pharmacological inhibition of L-type Ca(2+) currents and I(Ks). The prolongation was completely abolished by prior application of the hERG channel blocker E-4031 or the protein kinase G (PKG) inhibitor Rp-8-Br-cGMPS. Expression analysis revealed the presence of both ERG1a and -1b subunits in rat papillary muscle. Both 8-Br-cGMP and ANP inhibited heterologously expressed ERG1b and even stronger ERG1a/1b channels, whereas ERG1a channels remained unaffected. The inhibitory 8-Br-cGMP effects were PKG-dependent and involved a profound ERG current reduction, which was also observed with cardiac AP clamp recordings. Measurements of I(Kr) from isolated mouse cardiomyocytes using Cs(+) as charge carrier exhibited faster deactivation kinetics in atrial than in ventricular myocytes consistent with a higher relative expression of ERG1b transcripts in atria than in ventricles. 8-Br-cGMP significantly reduced I(Kr) in atrial, but not in ventricular myocytes. These findings provide first evidence that through heteromeric assembly ERG1 channels become a critical target of cGMP-PKG signaling linking cGMP accumulation to cardiac I(Kr) modulation.

NMDA receptor activation increases free radical production through nitric oxide and NOX2.

Reactive oxygen species (ROS) and nitric oxide (NO) participate in NMDA receptor signaling. However, the source(s) of the ROS and their role in the increase in cerebral blood flow (CBF) induced by NMDA receptor activation have not been firmly established. NADPH oxidase generates ROS in neurons, but there is no direct evidence that this enzyme is present in neurons containing NMDA receptors, or that is involved in NMDA receptor-dependent ROS production and CBF increase. We addressed these questions using a combination of in vivo and in vitro approaches. We found that the CBF and ROS increases elicited by topical application of NMDA to the mouse neocortex were both dependent on neuronal NO synthase (nNOS), cGMP, and the cGMP effector kinase protein kinase G (PKG). In mice lacking the NADPH oxidase subunit NOX2, the ROS increase was not observed, but the CBF increase was still present. Electron microscopy of the neocortex revealed NOX2 immunolabeling in postsynaptic somata and dendrites that also expressed the NMDA receptor NR1 subunit and nNOS. In neuronal cultures, the NMDA-induced increase in ROS was mediated by NADPH oxidase through NO, cGMP and PKG. We conclude that NADPH oxidase in postsynaptic neurons generates ROS during NMDA receptor activation. However, NMDA receptor-derived ROS do not contribute to the CBF increase. The findings establish a NOX2-containing NADPH oxidase as a major source of ROS produced by NMDA receptor activation, and identify NO as the critical link between NMDA receptor activity and NOX2-dependent ROS production.

cGMP-dependent protein kinase type I inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes.

Cardiac myocyte apoptosis during ischemia and reperfusion (I/R) is tightly controlled by a complex network of stress-responsive signaling pathways. One pro-apoptotic pathway involves the interaction of the scaffold protein TAB1 with p38 mitogen-activated protein kinase (p38 MAPK) leading to the autophosphorylation and activation of p38 MAPK. Conversely, NO and its second messenger cGMP protect cardiac myocytes from apoptosis during I/R. We provide evidence that the cGMP target cGMP-dependent protein kinase type I (PKG I) interferes with TAB1-p38 MAPK signaling to protect cardiac myocytes from I/R injury. In isolated neonatal cardiac myocytes, activation of PKG I inhibited the interaction of TAB1 with p38 MAPK, p38 MAPK phosphorylation, and apoptosis induced by simulated I/R. During I/R in vivo, mice with a cardiac myocyte-restricted deletion of PKG I displayed a more pronounced interaction of TAB1 with p38 MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared with wild-type littermates. Notably, adenoviral expression of a constitutively active PKG I mutant truncated at the N terminus(PKGI-DeltaN1-92) did not inhibit p38 MAPK phosphorylation and apoptosis induced by simulated I/R in vitro, indicating that the N terminus of PKG I is required. As shown by co-immunoprecipitation experiments in HEK293 cells, cGMP-activated PKG I, but not constitutively active PKG I-DeltaN1-92 or PKG I mutants carrying point mutations in the N-terminal leucine-isoleucine zipper, interacted with p38 MAPK, and prevented the binding of TAB1 to p38 MAPK. Together, our data identify a novel interaction between the cGMP target PKG I and the TAB1-p38 MAPK signaling pathway that serves as a defense mechanism against myocardial I/R injury.

Cyclic GMP-dependent protein kinase Ialpha inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells.

Vascular smooth muscle contractile state is regulated by intracellular calcium levels. Nitric oxide causes vascular relaxation by stimulating production of cyclic GMP, which activates type I cGMP-dependent protein kinase (PKGI) in vascular smooth muscle cells (VSMC), inhibiting agonist-induced intracellular Ca2+ mobilization ([Ca2+]i). The relative roles of the two PKGI isozymes, PKGIalpha and PKGIbeta, in cyclic GMP-mediated inhibition of [Ca2+]i in VSMCs are unclear. Here we have investigated the ability of PKGI isoforms to inhibit [Ca2+]i in response to VSMC activation. Stable Chinese hamster ovary cell lines expressing PKGIalpha or PKGIbeta were created, and the ability of PKGI isoforms to inhibit [Ca2+]i in response to thrombin receptor stimulation was examined. In Chinese hamster ovary cells stably expressing PKGIalpha or PKGIbeta, 8-Br-cGMP activation suppressed [Ca2+]i by thrombin receptor activation peptide (TRAP) by 98 +/- 1 versus 42 +/- 5%, respectively (p <0.002). Immunoblotting studies of cultured human VSMC cells from multiple sites using PKGIalpha- and PKGIbeta-specific antibodies showed PKGIalpha is the predominant VSMC PKGI isoform. [Ca2+]i following thrombin receptor stimulation was examined in the absence or presence of cyclic GMP in human coronary VSMC cells (Co403). 8-Br-cGMP significantly inhibited TRAP-induced [Ca2+]i in Co403, causing a 4-fold increase in the EC50 for [Ca2+]i. In the absence of 8-Br-cGMP, suppression of PKGIalpha levels by RNA interference (RNAi) led to a significantly greater TRAP-stimulated rise in [Ca2+]i as compared with control RNAi-treated Co403 cells. In the presence of 8-Br-cGMP, the suppression of PKGIalpha expression by RNAi led to the complete loss of cGMP-mediated inhibition of [Ca2+]i. Adenoviral overexpression of PKGIbeta in Co403 cells was unable to alter TRAP-stimulated Ca2+ mobilization either before or after suppression of PKGIalpha expression by RNAi. These results support that PKGIalpha is the principal cGMP-dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/cyclic GMP pathway.

The sulindac derivatives OSI-461, OSIP486823, and OSIP487703 arrest colon cancer cells in mitosis by causing microtubule depolymerization.

Exisulind (sulindac sulfone) and three highly potent derivatives, OSI-461 (CP461), OSIP486823 (CP248), and OSIP487703, inhibit growth and induce apoptosis in SW480 human colon cancer cells, with IC(50)s of 200, 2, 0.1, and 0.003 micromol/L, respectively. The latter three compounds, but not exisulind, induce marked M-phase cell cycle arrest in these cells. This effect seems to be independent of the known ability of these compounds to cause activation of protein kinase G. When tested at twice their IC(50) concentration for growth inhibition, OSI-461, OSIP486823, and OSIP487703 cause depolymerization of microtubules in interphase cells, inhibit spindle formation in mitotic cells, and induce multinucleated cells. In vitro tubulin polymerization assays indicate that all three compounds interact with tubulin directly to cause microtubule depolymerization and/or inhibit de novo tubulin polymerization. These results suggest that the dual effects of OSI-461, OSIP486823, and OSIP487703 on impairment of microtubule functions and protein kinase G activation may explain the potent antiproliferative and apoptotic effects of these compounds in cancer cells.

Inhibition of cGMP-dependent protein kinase reverses phenotypic modulation of vascular smooth muscle cells.

We have previously shown that type I cGMP-dependent protein kinase (PKG) can alter the phenotype of cultured vascular smooth muscle cells (VSMCs). Although the expression of contractile proteins in VSMCs has been shown to be modulated with the induction of PKG, experiments in which PKG inhibition brings about reduced expression of contractile markers have not been performed. To more thoroughly examine the role of PKG in the expression of contractile proteins, recombinant adenovirus containing the PKG coding sequence (AD-PKG) was used to induce gene expression and morphologic changes in adult rat aortic VSMCs. Cells expressing PKG, but not control adenovirus-infected cells, began to express a specific marker protein for the contractile phenotype, smooth muscle myosin heavy chain (SMMHC), within 48 hours of PKG induction. The morphology of the AD-PKG-infected cells began to change from a fibroblastic phenotype to a spindle-shaped phenotype within 72 hours after PKG induction. The specific cell-permeable PKG inhibitory peptide DT-2, but not control peptides, reversed the biochemical and morphologic changes associated with PKG expression. These results suggest that PKG expression and activity in cultured VSMCs is capable of altering the VSMC phenotype. These data also verify the intracellular action of DT-2 and reveal uptake and dynamic properties of this PKG-inhibiting peptide.

eNOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells.

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.

BK(Ca) channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy.

We investigated the influence of pregnancy on large-conductance calcium-activated potassium channel (BK(Ca)) activity (NP(o)) and on channel expression in membranes of isolated human myometrial smooth muscle cells. NP(o) in inside-out patches was higher in pregnant myometria (PM) compared with nonpregnant myometria (NPM), and the half-maximal activation potential was shifted by 39 mV to more negative potentials. This effect was not due to an enhanced BK(Ca) channel expression. In the presence of cAMP kinase (PKA) or cGMP kinase (PKG), NP(o) increased in patches from PM but decreased in those from NPM. Western blot analysis and use of a specific PKG inhibitor (1 microM KT-5823) verified the existence of a partially active membrane-associated PKG. Inhibition of PKA by 100 nM PKI, the inhibitory peptide of PKA, had no effect on NP(o). 8-p-Chlorophenylthio-cGMP (8-pCPT-cGMP) hyperpolarized cells from PM. This effect was abolished by iberiotoxin, a specific blocker of BK(Ca) channels. It is concluded that an endogenous, membrane-bound PKG in myometrial cells specifically enhances BK(Ca) channel activity during pregnancy and thus may contribute to uterine quiescence during pregnancy.

Phosphorylation of the NC-1.1 receptor and regulation of natural cytotoxicity by protein kinase C and cyclic GMP-dependent protein kinase.

Natural cytotoxicity (NC) against cancer involves receptor-ligand interactions between lymphohemopoietic cells that mediate NC against tumor cells. The only candidate for a receptor on cells mediating NC is NC-1.1, identified using mAb 1C4. In this study we showed that mAb 1C4 blocked NC-1.1+ cell conjugation to WEHI-164 tumor cells, indicating that NC-1.1 is a surface protein required for cell-cell interaction. Affinity-purified NC-1.1 was a 45-kDa monomeric protein. It was a good in vitro substrate for cyclic GMP (cGMP)-dependent protein kinase (PKG) and protein kinase C (PKC) and a relatively poor substrate for cAMP-dependent protein kinase (PKA). Phosphopeptide mapping revealed one phosphopeptide phosphorylated by PKG and PKA, and two additional peptides phosphorylated by PKC. Phosphorylation by PKG or PKA abolished phosphorylation at the PKC sites, while coincubation of NC-1.1 with both PKG and PKC reduced phosphorylation of all sites. NC-1.1 was also a phosphoprotein after immunoprecipitation from intact spleen cells and its phosphorylation was increased after cell stimulation with PKC or PKG activators (phorbol esters or 8-bromo-cGMP). The possible consequences of intracellular signaling were tested in functional assays for NC. Phorbol ester activation of spleen cells increased NC, while 8-bromo-cGMP and 8-bromo-cAMP had little effect. However, coincubation with both phorbol ester and either 8-bromo-cGMP or 8-bromo-cAMP virtually abolished NC without affecting cell conjugation. These results suggest that NC-1.1 is a receptor for a ligand on certain tumor cells and reveal that key intracellular signaling pathways involving PKC, PKG, and PKA interact to effect a coordinated control of NC.

Regulation of L-type calcium channels by cyclic nucleotides and phosphorylation in smooth muscle cells from rabbit portal vein.

In a previous study, we demonstrated that a high concentration (> or = 1 microM) of isoproterenol (ISO) produced a dual effect on L-type Ca2+ current (ICa(L)) in vascular smooth muscle (VSM) cells from the portal vein: an initial stimulatory action followed by a sustained inhibition. The first stimulatory phase was fast (presumably more direct) and may reflect G-protein gating of the Ca2+ channels. The second inhibitory phase was slower (presumably more indirect) and may be mediated by the adenylate cyclase/cAMP pathway. In order to define further the mechanism for the ISO inhibition of ICa(L), the effects of cyclic nucleotides and their related protein kinases were examined in freshly isolated single smooth muscle cells from the rabbit portal vein using the whole-cell voltage clamp technique. To isolate ICa(L), the pipette solution contained high Cs+ (to block K+ outward current), and the bath contained physiological salt solution. Upon extracellular application of membrane-permeable cAMP and cGMP analogs (8-Br-cAMP and 8-Br-cGMP, 3 mM), ICa(L) was significantly inhibited by 27.9 +/- 5.0 and 33.5 +/- 4.8%, respectively. Forskolin (100 microM) also depressed ICa(L). The protein kinase inhibitor, H-7, prevented the inhibitory effects of both cyclic nucleotides and forskolin. In addition, intracellular application (via the patch pipettes) of cAMP-dependent protein kinase (PK-A, catalytic subunit; 1.76 microM) and cGMP-dependent protein kinase (PK-G, 50 nM, pre-activated by 10 microM cGMP) significantly inhibited the peak amplitude of ICa(L) by 45.5 +/- 10 and 43.2 +/- 6.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphoinositide metabolism in airway smooth muscle.

Agonist-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate, which generates inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol, is thought to be one of the major mechanisms underlying pharmacomechanical coupling in airway smooth muscle. This article is a review of the currently available information on phosphoinositide and inositol 1,4,5-trisphosphate metabolism in this tissue and includes data on inositol 1,4,5-trisphosphate-induced Ca2+ release and the receptor mediating this effect. The final section outlines the potential mechanisms underlying physiological regulation of phosphoinositide metabolism by other second-messenger pathways operative in this tissue.