Rho kinase contributes to basal vascular tone in humans: role of endothelium-derived nitric oxide

Our objective was to determine the role of the Rho-associated kinase (ROK) for the regulation of FBF (FBF) and to unmask a potential role of ROK for the regulation of endothelium-derived nitric oxide (NO). Moreover, the effect of fasudil on the constrictor response to endothelin-1 was recorded. Regarding background, phosphorylation of the myosin light chain (MLC) determines the calcium sensitivity of the contractile apparatus. MLC phosphorylation depends on the activity of the MLC kinase and the MLC phosphatase. The latter enzyme is inhibited through phosphorylation by ROK. ROK has been suggested to inhibit NO generation, possibly via the inhibition of the Akt pathway. In this study, the effect of intra-arterial infusion of the ROK inhibitor fasudil on FBF in 12 healthy volunteers was examined by venous occlusion plethysmography. To unmask the role of NO, fasudil was infused during NO clamp. As a result, fasudil markedly increased FBF in a dose-dependent manner from 2.34 +/- 0.21 to 6.96 +/- 0.93 ml/100 ml forearm volume at 80 mug/min (P < 0.001). At 1,600 mug/min, fasudil reduced systolic, diastolic, and mean arterial pressure while increasing heart rate. Fasudil abolished the vasoconstrictor effect of endothelin-1. The vascular response to fasudil (80 mumol/min) was blunted during NO clamp (104 +/- 18% vs. 244 +/- 48% for NO clamp + fasudil vs. fasudil alone; data as ratio between infused and noninfused arm with baseline = 0%, P < 0.05). In conclusion, 1) basal peripheral and systemic vascular tone depends on ROK; 2) a significant portion of fasudil-induced vasodilation is mediated by NO, suggesting that vascular bioavailable NO is negatively regulated by ROK; and 3) the constrictor response to endothelin involves the activation of ROK.     

TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase. An essential role in smooth muscle inhibitory neurotransmission

Potassium channels activated by membrane stretch may contribute to maintenance of relaxation of smooth muscle cells in visceral hollow organs. Previous work has identified K(+) channels in murine colon that are activated by stretch and further regulated by NO-dependent mechanisms. We have screened murine gastrointestinal, vascular, bladder, and uterine smooth muscles for the expression of TREK and TRAAK mRNA. Although TREK-1 was expressed in many of these smooth muscles, TREK-2 was expressed only in murine antrum and pulmonary artery. TRAAK was not expressed in any smooth muscle cells tested. Whole cell currents from TREK-1 expressed in mammalian COS cells were activated by stretch, and single channel recordings showed that the stretch-dependent conductance was due to 90 pS channels. Sodium nitroprusside (10(-6) or 10(-5) m) and 8-Br-cGMP (10(-4) or 10(-3) m) increased TREK-1 currents in perforated whole cell and single channel recordings. Mutation of the PKG consensus sequence at serine 351 blocked the stimulatory effects of sodium nitroprusside and 8-Br-cGMP on open probability without affecting the inhibitory effects of 8-Br-cAMP. TREK-1 encodes a component of the stretch-activated K(+) conductance in smooth muscles and may contribute to nitrergic inhibition of gastrointestinal muscles.     

Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor,improves wound healing through cGMP/cGMP-dependent protein kinase

Nitric oxide (NO) donors have been shown to improve wound healing, but the mechanism is not well defined. Here we show that the novel NO donor nitrosyl-cobinamide (NO-Cbi) improved in vitro wound healing in several cell types, including an established line of lung epithelial cells and primary human lung fibroblasts. On a molar basis, NO-Cbi was more effective than two other NO donors, with the effective NO-Cbi concentration ranging from 3 to 10 μM, depending on the cell type. Improved wound healing was secondary to increased cell migration and not cell proliferation. The wound healing effect of NO-Cbi was mediated by cGMP, mainly through cGMP-dependent protein kinase type I (PKGI), as determined using pharmacological inhibitors and activators, and siRNAs targeting PKG type I and II. Moreover, we found that Src and ERK were two downstream mediators of NO-Cbi's effect. We conclude that NO-Cbi is a potent inducer of cell migration and wound closure, acting via cGMP, PKG, Src, and extracellular signal regulated kinase (ERK).     

A role for coccidian cGMP-dependent protein kinase in motility and invasion

The coccidian parasite cGMP-dependent protein kinase is the primary target of a novel coccidiostat, the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine (compound 1), which effectively controls the proliferation of Eimeria tenella and Toxoplasma gondii parasites in animal models. The efficacy of compound 1 in parasite-specific metabolic assays of infected host cell monolayers is critically dependent on the timing of compound addition. Simultaneous addition of compound with extracellular E. tenella sporozoites or T. gondii tachyzoites inhibited [3H]-uracil uptake in a dose-dependent manner, while minimal efficacy was observed if compound addition was delayed, suggesting a block in host cell invasion. Immunofluorescence assays confirmed that compound 1 blocks the attachment of Eimeria sporozoites or Toxoplasma tachyzoites to host cells and inhibits parasite invasion and gliding motility. Compound 1 also inhibits the secretion of micronemal adhesins (E. tenella MIC1, MIC2 and T. gondii MIC2), an activity closely linked to invasion and motility in apicomplexan parasites. The inhibition of T. gondii MIC2 adhesin secretion by compound 1 was not reversed by treatment with calcium ionophores or by ethanol (a microneme secretagogue), suggesting a block downstream of calcium-dependent events commonly associated with the discharge of the microneme organelle in tachyzoites. Transgenic Toxoplasma strains expressing cGMP-dependent protein kinase mutant alleles that are refractory to compound 1 (including cGMP-dependent protein kinase knock-out lines complemented by such mutants) were used as tools to validate the potential role of cGMP-dependent protein kinase in invasion and motility. In these strains, parasite adhesin secretion, gliding motility, host cell attachment and invasion displayed a reduced sensitivity to compound 1. These data clearly demonstrate that cGMP-dependent protein kinase performs an important role in the host-parasite interaction.     

Prolonged treatment of porcine pulmonary artery with nitric oxide decreases cGMP sensitivity and cGMP-dependent protein kinase specific activity

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response to acutely applied NO, cGMP analog, or atrial natriuretic peptide (ANP). Twenty-four-hour treatment with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) resulted in >10-fold decrease in the response to acutely applied DETA-NO. In parallel with this, the relaxant response to acutely applied cGMP analog, beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-PET-cGMPS), and ANP decreased. The reduction in ANP responsiveness in PA was not associated with a reduction in cGMP levels evoked by 10(-6) M ANP. Twenty-four hours in culture and treatment with DETA-NO decreased total cGMP-dependent protein kinase (cGKI) mRNA level compared with that in freshly prepared PA (1.05 +/- 0.12, 0.42 +/- 0.08, and 0.11 +/- 0.01 amol/mug, respectively). Total cGKI protein levels were decreased to a lesser extent by 24 h in culture and further decreased by 24-h DETA-NO treatment compared with that in freshly prepared PA (361 +/- 33, 272 +/- 20, and 238 +/- 25 ng/mg total protein, respectively). Maximal cGMP-stimulated phosphotransferase activity was reduced in 24-h cultured and DETA-NO-treated PA (986 +/- 84, 815 +/- 81, and 549 +/- 78 pmol P(i).min(-1).mg soluble protein(-1)), but the cGMP concentration resulting in 50% of maximal phosphotransferase activity was not. cGKI specific activity (maximal cGMP-activated phosphotransferase activity/ng cGKI) was significantly reduced in PA treated with DETA-NO for 24 h compared with freshly prepared and 24-h cultured PA (1.95 +/- 0.22, 2.64 +/- 0.25, and 2.85 +/- 0.28 pmol P(i).min(-1).ng cGKI(-1), respectively). We conclude that prolonged NO treatment induces decreased acute NO responsiveness in PA in part by decreasing cGMP sensitivity. It does so by decreasing both cGKI expression and cGKI specific activity.     

Akt pathway mediates a cGMP-dependent survival role of nitric oxide in cerebellar granule neurones

Apoptotic death results from disrupting the balance between anti-apoptotic and pro-apoptotic cellular signals. The inter- and intracellular messenger nitric oxide is known to mediate either death or survival of neurones. In the present work, cerebellar granule cells were used as a model to assess the survival role of nitric oxide and to find novel signal transduction pathways related to this role. It is reported that sustained inhibition of nitric oxide production induces apoptosis in differentiated cerebellar granule neurones and that compounds that slowly release nitric oxide significantly revert this effect. Neuronal death was also reverted by a caspase-3-like inhibitor and by a cyclic GMP analogue, thus suggesting that nitric oxide-induced activation of guanylate cyclase is essential for the survival of these neurones. We also report that the Akt/GSK-3 kinase system is a transduction pathway related to the survival action of nitric oxide, as apoptosis caused by nitric oxide deprivation is accompanied by down-regulation of this, but not of other, kinase systems. Conversely, treatments able to rescue neurones from apoptosis also counteracted this down-regulation. Furthermore, in transfection experiments, overexpression of the Akt gene significantly decreased nitric oxide deprivation-related apoptosis. These results are the first evidence for a mechanism where endogenous nitric oxide promotes neuronal survival via Akt/GSK-3 pathway.     

ATP analog specificity of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phosphorylase kinase

The ATP analog specificities of the homogeneous cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase have been compared by the ability of 27 analogs to compete with ATP in the protein kinase reaction. Although the data suggest general similarities between the ATP sites of the two homologous cyclic-nucleotide-dependent protein kinases, specific differences especially in the adenine binding pocket are indicated. These differences in affinity suggest potentially useful ATP analog inhibitors of each kinase. For example, apparent autophosphorylation of the purified regulatory subunit of the cAMP-dependent protein kinase is blocked by nebularin triphosphate, suggesting that the phosphorylation is catalyzed by trace contamination of cGMP-dependent protein kinase. Some of the ATP analogs have also been tested using phosphorylase b kinase in order to compare this enzyme with the cyclic-nucleotide-dependent enzymes. All three protein kinases have high specificity for the purine moiety of ATP, and lower specificity for the ribose or triphosphate. The similarity between the ATP site of phosphorylase b kinase to that of the cyclic-nucleotide-dependent protein kinases suggests that it is related to them. The ATP analog specificities of enzymes examined in this study are different from those reported for several unrelated ATP-utilizing enzymes.     

Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs

Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to beta-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.     

Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase.

Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation.     

Natriuretic peptide receptor-A negatively regulates mitogen-activated protein kinase and proliferation of mesangial cells: role of cGMP-dependent protein kinase

We have examined the effect of atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (NPRA) on mitogen-activated protein kinase/extracellular signal-regulated kinase 2 (MAPK/ERK2) activity in rat mesangial cells overexpressing NPRA. Agonist hormones such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), angiotensin II (ANG II), and endothelin-1 (ET-1) stimulated 2.5- to 3.5-fold immunoreactive MAPK/ERK2 activity in these cells. ANP inhibited agonist-stimulated activity of MAPK/ERK2 by 65-75% in cells overexpressing NPRA, whereas in vector-transfected cells, its inhibitory effect was only 18-20%. NPRA antagonist A71915 and KT5823, a specific inhibitor of cGMP-dependent protein kinase (PKG) completely reversed the inhibitory effect of ANP on MAPK/ERK2 activity. ANP also inhibited the PDGF-stimulated [(3)H]thymidine uptake by almost 70% in cells overexpressing NPRA, as compared with only 20-25% inhibition in vector-transfected cells. These results demonstrate that ANP/NPRA system negatively regulates MAPK/ERK2 activity and proliferation of mesangial cells in a PKG-dependent manner.     

Physiological role of cGMP and cGMP-dependent protein kinase in the cardiovascular system

Dedicated to Professor Dr. H.-D. Söling, Göttingen (FRG), on the occasion of his sixtieth birthday.     

Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha

cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways. To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-Ialpha as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein). Although the N-terminal region (amino acids 1-66) of cGK-Ialpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Ibeta, cGK-II, or cAMP-dependent protein kinase. GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids. Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42. Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-Ialpha is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-Ialpha and -Ibeta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Ialpha in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo. Finally, we demonstrated that the kinase-deficient mutant of cGK-Ialpha stably associates with GKAP42 and that binding of cGMP to cGK-Ialpha facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-Ialpha and that cGK-Ialpha may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.     

cGMP-dependent protein kinases: properties, regulation and biological role

The data available in literature on properties, methods of regulation and biological role of cGMP-dependent protein kinases are analyzed and generalized. Evidence on distribution of these enzymes in different tissues and intracellular compartments is presented. Physicochemical properties of cGMP-dependent protein kinases from different sources are analyzed. Peculiarities of their primary structure, regulation of their activity at the expense of autophosphorylation as well as in the presence of the modulator protein, metal ions and other factors are considered. These data permit postulating importance of cGMP-dependent phosphorylation in some physiological and biochemical processes.     

A role for cGMP-dependent protein kinase II in AMPA receptor trafficking and synaptic plasticity

Regulated trafficking of AMPA receptors (AMPARs) is an important mechanism that underlies the activity-dependent modification of synaptic strength. Trafficking of AMPARs is regulated by specific interactions of their subunits with other proteins. Recently, we have reported that the AMPAR subunit GluR1 binds the cGMP-dependent kinase type II (cGKII) adjacent to the kinase catalytic site, and that this interaction is increased by cGMP. In this complex, cGKII phosphorylates GluR1 at serine 845 (S845), a site known to be phosphorylated also by PKA. S845 phosphorylation leads to an increase of GluR1 on the plasma membrane. In neurons, cGMP is produced by soluble guanylate cyclase (sGC), which is activated by nitric oxide (NO). Calcium flux through the NMDA receptor (NMDAR) activates neuronal nitric oxide synthase (nNOS), which produces NO. Using a combination of biochemical and electrophysiological experiments, we have shown that trafficking of GluR1 is under the regulation of NO, cGMP and cGKII. Moreover, our study indicates that the interaction of cGKII with GluR1, which is under the regulation of the NMDAR and NO, plays an important role in hippocampal plasticity.     

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.