Activation Of cGMP-dependent Protein Kinase Research Articles

Activity-Dependent Regulation of Surface Glucose Transporter-3

Glucose transporter 3 (GLUT3) is the main facilitative glucose transporter in neurons. Glucose provides neurons with a critical energy source for neuronal activity. However, the mechanism by which neuronal activity controls glucose influx via GLUT3 is unknown. We investigated the influence of synaptic stimulation on GLUT3 surface expression and glucose import in primary cultured cortical and hippocampal neurons. Synaptic activity increased surface expression of GLUT3 leading to an elevation of intracellular glucose. The effect was blocked by NMDA receptor (NMDAR) and neuronal nitric oxide synthase (nNOS) inhibition. The Akt inhibitor I (Akt-I) blocked NMDAR-induced GLUT3 surface expression while a nNOS-phosphomimetic mutant (S1412D) enhanced GLUT3 expression at cell surface. These results suggest that NMDAR/Akt-dependent nNOS phosphorylation is coupled to GLUT3 trafficking. We demonstrated that activation of cGMP-dependent protein kinase (cGK) increased the surface expression of GLUT3, which was repressed by Rp-8-pCPT-cGMPS, a potent cell-permeable inhibitor of cGKs. These studies characterize the molecular basis for activity-dependent increases in surface GLUT3 after stimulation of the NMDARs. NMDAR-induced increase in surface GLUT3 represents a novel pathway for control of energy supply during neuronal activity that is critical for maintaining glucose homeostasis during neuronal transmission.

Ethanol Attenuates Contraction of Primary Cultured Rat Airway Smooth Muscle Cells

Airway smooth muscle cells are the main effector cells involved in airway narrowing and have been used to study the signaling pathways involved in asthma-induced airway constriction. Our previous studies demonstrated that ethanol administration to mice attenuated methacholine-stimulated increases in airway responsiveness. Because ethanol administration attenuates airway responsiveness in mice, we hypothesized that ethanol directly blunts the ability of cultured airway smooth muscle cells to shorten. To test this hypothesis, we measured changes in the size of cultured rat airway smooth muscle (RASM) cells exposed to ethanol (100 mM) after treatment with methacholine. Ethanol markedly attenuated methacholine-stimulated cell shortening (methacholine-stimulated length change = 8.3 ± 1.2% for ethanol versus 43.9 ± 1.5% for control; P < 0.001). Ethanol-induced inhibition of methacholine-stimulated cell shortening was reversible 24 hours after removal of alcohol. To determine if ethanol acts through a cGMP-dependent pathway, incubation with ethanol for as little as 15 minutes produced a doubling of cGMP-dependent protein kinase (PKG) activity. Furthermore, treatment with the PKG antagonist analog Rp-8Br-cGMPS (10 μM) inhibited ethanol-induced kinase activation when compared with control-treated cells. In contrast to the effect of ethanol on PKG, ethanol pretreatment did not activate a cAMP-dependent protein kinase. These data demonstrate that brief ethanol exposure reversibly prevents methacholine-stimulated RASM cell contraction. In addition, it appears that this effect is the result of activation of the cGMP/PKG kinase pathway. These findings implicate a direct effect of ethanol on airway smooth muscle cells as the basis for in vivo ethanol effects.

Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway

Nitric oxide (NO), a modulator of several physiological processes, is involved in different human sperm functions. We have investigated whether NO may stimulate the motility of human spermatozoa via activation of the soluble guanylate cyclase (sGC)/cGMP pathway. Sperm samples obtained by masturbation from 70 normozoospermic patients were processed by the swim-up technique. The kinetic parameters of the motile sperm-rich fractions were assessed by computer-assisted sperm analysis. After a 30-90 min incubation, the NO donor S-nitrosoglutathione (GSNO) exerted a significant enhancing effect on progressive motility (77, 78, and 78% vs 66, 65, and 62% of the control at the corresponding time), straight linear velocity (44, 49, and 48 μm/s vs 34, 35, and 35.5 μm/s), curvilinear velocity (81, 83, and 84 μm/s vs 68 μm/s), and average path velocity (52, 57, and 54 μm/s vs 40, 42, and 42 μm/s) at 5 μM but not at lower concentrations, and in parallel increased the synthesis of cGMP. A similar effect was obtained with the NO donor spermine NONOate after 30 and 60 min. The GSNO-induced effects on sperm motility were abolished by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (a specific sGC inhibitor) and mimicked by 8-bromo-cGMP (8-Br-cGMP; a cell-permeating cGMP analog); the treatment with Rp-8-Br-cGMPS (an inhibitor of cGMP-dependent protein kinases) prevented both the GSNO- and the 8-Br-cGMP-induced responses. On the contrary, we did not observe any effect of the cGMP/PRKG1 (PKG) pathway modulators on the onset of hyperactivated sperm motility. Our results suggest that NO stimulates human sperm motility via the activation of sGC, the subsequent synthesis of cGMP, and the activation of cGMP-dependent protein kinases.

Redox regulation of guanylate cyclase and protein kinase G in vascular responses to hypoxia.

The production of cGMP by the soluble form of guanylate cyclase (sGC) in bovine pulmonary arteries (BPA) is controlled by cytosolic NADPH maintaining reduced thiol and heme sites on sGC needed for activation by NO, and the levels of Nox oxidase-derived superoxide and peroxide that influence pathways regulating sGC activity. Our recent studies in BPA suggest that the activities of peroxide metabolizing pathways in vascular smooth muscle potentially determine the balance between sGC stimulation by peroxide and a cGMP-independent activation of cGMP-dependent protein kinase (PKG) by a disulfide-mediated subunit dimerization. Cytosolic NADPH oxidation also appears to function in BPA through its influence on protein thiol redox control as an additional mechanism promoting vascular relaxation through PKG activation. These processes regulating PKG may participate in decreases in peroxide and increases in NADPH associated with contraction of BPA to hypoxia and in cytosolic NADPH oxidation potentially mediating bovine coronary artery relaxation to hypoxia.

Simply say yes to NO? Nitric oxide (NO) sensor-based assessment of coronary endothelial function

This editorial refers to ‘First evaluation of real-time nitric oxide changes in the coronary circulation in patients with non-ischaemic dilated cardiomyopathy using a catheter-type sensor’, by S. Takarada et al. doi:10.1093/eurheartj/ehq156 In 1980 Furchgott and Zawadzki published their observation on how removal of the endothelium abolishes acetylcholine-induced relaxation of vascular smooth muscle cells, which ignited a passionate search for an endothelium-derived vasorelaxing factor.1 Yet who would have thought in the beginning that the search was for a gas! Seven years later, Ignarro and co-workers identified nitric oxide (NO) as the endothelium-derived vasorelaxing factor.2 In the same decade, Murad's laboratory pointed out that NO activates the cytosolic (soluble) isoenzyme form of guanylate cyclase which increases cGMP synthesis and leads to the activation of cGMP-dependent protein kinase and ultimately reduces intracellular calcium concentration and elicits vascular smooth muscle relaxation.3 Closing the loop on endothelium-dependent vasorelaxation with these findings and discovering ‘nitric oxide as a signaling molecule in the cardiovascular system’, Robert Furchgott, Louis Ignarro, and Ferid Murad were awarded the Nobel Prize in Physiology/Medicine in 1998. NO's role in the physiology and pathology of the cardiovascular system is quite manifold and extends beyond its anatomical boundaries (Figure 1). In synoptic terms, NO is a multifunctional molecule that exerts anti-atherosclerotic properties in the vasculature and contributes to primarily the diastolic function of the myocardium.4,5 Abnormalities in the endogenous NO system have been found in various pathological conditions of the cardiovascular system including hypertension, atherosclerosis, and cardiomyopathies. … *Corresponding author. Tel: +1 507 255 4152, Fax: +1 507 255 2550, Email: lerman.amir{at}mayo.edu

Nitric oxide-sensitive guanylyl cyclase is the only nitric oxide receptor mediating platelet inhibition.

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling cascade is involved in the precise regulation of platelet responses. NO released from the endothelium is known to activate NO-sensitive guanylyl cyclase (NO-GC) in platelets. By the generation of cGMP and subsequent activation of cGMP-dependent protein kinase (PKG), NO-GC mediates the reduction of the intracellular calcium and inhibits platelet adhesion and aggregation. However, NO has been postulated to influence these platelet functions also via cGMP-independent mechanisms. We studied the effect of NO on platelets lacking NO-sensitive guanylyl cyclase with regards to aggregation, adhesion, calcium mobilization and bleeding time. Here, we show that NO signaling leading to inhibition of agonist-induced platelet aggregation is totally abrogated in platelets from mice deficient in NO-GC (GCKO). Even at millimolar concentrations none of the several different NO donors inhibited collagen-induced aggregation of GCKO platelets. In addition, NO neither affected adenosine 5'-diphosphate (ADP)-induced adhesion nor thrombin-induced calcium release in GCKO platelets. Although the NO-induced cGMP signal transduction was totally abrogated cyclic adenosine monophosphate (cAMP) signaling was still functional; however, cGMP/cAMP crosstalk was disturbed on the level of phosphodiesterase type 3 (PDE3). These in vitro data are completed by a reduced bleeding time indicating the lack of NO effect in vivo. We conclude that NO-GC is the only NO receptor in murine platelets mediating the inhibition of calcium release, adhesion and aggregation: lack of the enzyme leads to disturbance of primary hemostasis.

S2057 Diabetes-Induced Changes in the Expression of Proteins Involved in the Regulation of Sustained Mlc 20 Phosphorylation and cGMP Levels in Gastrointestinal Smooth Muscle

The gastrointestinal complications of diabetes mellitus include nausea, vomiting, increase in intragastric pressure and delay in gastric emptying. Several studies showed that the changes in gastric emptying in diabetes are associated with the loss of enteric neurons and interstitial cells of Cajal, and dysfunction of neuronal nitric oxide synthase activity. We have previously shown that in gastrointestinal smooth muscle sustained MLC20 phosphorylation and contraction are dependent on inhibition of MLC phosphatase, which is mediated by RhoA-dependent pathway involving phosphorylation of MYPT1 (at Thr696) by Rho kinase and CPI-17 (at Thr38) by PKC. Smooth muscle relaxation is dependent on stimulation of cAMP and cGMP levels and activation of cAMPand cGMP-dependent protein kinases. The levels of cAMP and cGMP are regulated mainly by phosphodiesterase (PDE) 4D5 and PDE5, respectively. We hypothesized that diabetes induces changes in the expression of proteins involved in the maintenance of smooth muscle tone and regulation of cGMP levels. Aim. To determine the expression of PDE5, CPI-17 and Rho kinase as well as agonist-induced Rho kinase activity in gastric smooth muscle from control and diabetic animal models.Methods. Gastric smooth muscle was isolated from spontaneously type II diabetic ob/ob mice and type I diabetic rats. Type I diabetes was induced in rats by intravenous injection of streptozotocin. The rats were kept for 9 weeks and induction of diabetes was ascertained by the determination of blood glucose concentrations. Expression of CPI-17, Rho kinase and PDE5 was measured by immunoblot analysis and acetylcholine-induced Rho kinase activity was measured by immunokinase assay. Results. Expression of CPI-17, Rho kinase and PDE5 are significantly higher in gastric smooth muscle from both models of diabetic animals than from control animals. Consistent with the increased expression of Rho kinase, acetylcholine-induced Rho kinase activity was also significantly higher in smooth muscle from both models of diabetic animals than from control animals. Conclusion. This study identified mechanisms for increased smooth muscle tone and decreased smooth muscle relaxation in diabetes that are directly attributable to changes in smooth muscle physiology. The mechanisms involved increased expression of proteins involved in the regulation of sustained MLC20 phosphorylation (CPI-17 and Rho kinase) and cGMP levels (PDE5). Upregulation CPI-17 and Rho kinase could contribute to increased intragastric pressure, whereas upregulation of PDE5 could contribute to rapid hydrolysis of cGMP and decreased relaxation leading to delay in gastric emptying.

PKG inhibits TCF signaling in colon cancer cells by blocking β-catenin expression and activating FOXO4

Activation of cGMP-dependent protein kinase (PKG) has anti-tumor effects in colon cancer cells but the mechanisms are not fully understood. This study has examined the regulation of beta-catenin/TCF signaling, as this pathway has been highlighted as central to the anti-tumor effects of PKG. We show that PKG activation in SW620 cells results in reduced beta-catenin expression and a dramatic inhibition of TCF-dependent transcription. PKG did not affect protein stability, nor did it increase phosphorylation of the amino-terminal Ser33/37/Thr41 residues that are known to target beta-catenin for degradation. However, we found that PKG potently inhibited transcription from a luciferase reporter driven by the human CTNNB1 promoter, and this corresponded to reduced beta-catenin mRNA levels. Although PKG was able to inhibit transcription from both the CTNNB1 and TCF reporters, the effect on protein levels was less consistent. Ectopic PKG had a marginal effect on beta-catenin protein levels in SW480 and HCT116 but was able to inhibit TCF-reporter activity by over 80%. Investigation of alternative mechanisms revealed that cJun-N-terminal kinase (JNK) activation was required for the PKG-dependent regulation of TCF activity. PKG activation caused beta-catenin to bind to FOXO4 in colon cancer cells, and this required JNK. Activation of PKG was also found to increase the nuclear content of FOXO4 and increase the expression of the FOXO target genes MnSOD and catalase. FOXO4 activation was required for the inhibition of TCF activity as FOXO4-specific short-interfering RNA completely blocked the inhibitory effect of PKG. These data illustrate a dual-inhibitory effect of PKG on TCF activity in colon cancer cells that involves reduced expression of beta-catenin at the transcriptional level, and also beta-catenin sequestration by FOXO4 activation.

Transcriptional Regulation of cGMP-Dependent Protein Kinase II (cGK-II) in Chondrocytes

The C-type natriuretic peptide/natriuretic peptide receptor-B/cGMP pathway plays an important role in the regulation of endochondral ossification. In chondrocytes, the physiological effect of cGMP is mediated primarily by the activation of cGMP-dependent protein kinase II (cGK-II). In this study, we investigated the transcriptional regulation of cGK-II in chondrocytes. The expression pattern of cGK-II transcripts was examined during chondrogenic differentiation of ATDC5 cells. cGK-II mRNA was not detectable in undifferentiated cells, but increased dramatically prior to differentiation to the hypertrophic stage. To analyze the transcriptional regulation of cGK-II, the 5'-flanking region of the mouse cGK-II gene was isolated and characterized. The promoter activity of the cGK-II gene decreased markedly following deletion and mutagenesis of the putative Nkx-binding site between nucleotide positions -292 and -286. These results suggest that the homeobox gene Nkx family is critical for the transcriptional regulation of cGK-II during chondrogenesis.

Intracellular Mechanisms Responsible for PKG Stimulation of ATP-Sensitive Potassium Channels

The activity of the ATP-sensitive potassium (KATP) channel, a high-fidelity metabolic sensor that couples cellular metabolic status to membrane excitability, is modulated by a variety of post-translational mechanisms. Findings obtained from our recent studies suggest that activation of cGMP-dependent protein kinase (PKG) enhances the function of neuronal KATP channels via intracellular signaling and that generation of reactive oxygen species (ROS) is required for PKG stimulation of the channel. In the present study we further investigated the potential role of intracellular calcium and camodulin in mediating the modulatory effect of PKG and ROS.

Degradation of leucine zipper-positive isoform of MYPT1 may contribute to development of nitrate tolerance

A depressed cGMP-dependent protein kinase (PKG) activity is implicated in nitrate tolerance. The present study determines whether the leucine zipper-positive (LZ+) isoform of myosin phosphatase target subunit 1 (MYPT1), a key target protein for PKG actions, is involved in the development of nitrate tolerance. Nitrate tolerance in in vitro preparations was obtained by a 24 h incubation with nitroglycerin (NTG). Nitrate tolerance in in vivo preparations was obtained by subcutaneous injection of mice with NTG, and the aortas were used. Protein levels of total MYPT1, MYPT1 (LZ+), PP1Cdelta, myosin light chain (MLC), and phosphorylated MLC were determined by Western blot analysis. Isometric vessel tension was determined by an organ chamber technique. Protein levels of MYPT1 (LZ+), but not of PP1Cdelta, were significantly reduced in in vitro and in vivo nitrate-tolerant arteries. The decrease in the MYPT1 (LZ+) protein level of coronary artery was also induced by a nitric oxide donor and a cGMP analogue, which was prevented by the inhibitors of soluble guanylyl cyclase and PKG. The decrease in MYPT1 (LZ+) protein levels was not affected by the inhibitor of protein synthesis, but was prevented by the inhibitors of proteasomes. The diminished inhibition of dephosphorylation of MLC as well as the attenuated relaxation of porcine coronary artery and mouse aorta to NTG was improved by proteasome inhibitors. This study demonstrates that a reduction in the protein level of MYPT1 (LZ+) is involved in nitrate tolerance. This may result in part from a proteasome-dependent degradation of MYPT1 (LZ+).

Abstract 5642 Thrombospondin-1, via CD47, Blocks Endothelial-Dependent Arterial Relaxation by Limiting Endothelial Nitric Oxide Synthase Activation

Introduction: The secreted protein thrombospondin-1 (TSP1) is an important negative regulator of nitric oxide/cGMP signaling in vascular cells. TSP1, signaling through its necessary receptor CD47, limits activation of soluble guanylate cyclase and cGMP-dependent protein kinase. Extending these discoveries we recently defined a role for TSP1 in the acute control of blood pressure. However, it is not clear if the inhibitory role TSP-CD47 signaling has on blood pressure is secondary to a direct effect on arterial relaxation. To investigate this we tested the hypothesis that TSP1 directly limits arterial vasorelaxation by limiting activation of endothelial nitric oxide synthase (eNOS). Methods: TSP1-CD47 regulation of eNOS activation was studied in endothelial cells and arteries. Vasorelaxation was assessed ex vivo in arteries from wild type, TSP1 and CD47 null mice. Endothelial dependent changes in blood pressure were studied in transgenic mice. Results: TSP1 and CD47 null arteries demonstrated significantly greater dilation in response to physiologic activation of eNOS by acetylcholine compared to wild type vessels. Treating the luminal endothelial compartment of both wild type and TSP1 null arteries (but not CD47 null) with TSP1 inhibited vessel relaxation to acetylcholine and potentiated vasoconstriction to phenylephrine. TSP1 and a recombinant fragment of the signature domain that specifically binds CD47 potently blocked both acetylcholine-stimulated eNOS activation and changes in intracellular calcium in cultured endothelial cells. In vivo, acetylcholine-stimulated hypotention in TSP1 null mice was blocked by intravenous replacement of TSP1. Conclusion: Circulating TSP1, through CD47, limits endothelial dependent arterial relaxation and demonstrates a role for circulating TSP1 as a hypertensive agent supporting blood pressure through continuous modulation of both endogenous NO production and signaling.

Role of Ca 2+ /Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase 1 in Mediating Cardiomyocyte Hypertrophy

Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca(2+)/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca(2+)/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Herein, we investigate the role of Ca(2+)/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. These results elucidate a novel role for Ca(2+)/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca(2+) and cGMP signaling cross-talk during cardiac hypertrophy.

Expression of constitutively active cGMP-dependent protein kinase inhibits glucose-induced vascular smooth muscle cell proliferation

Previously, we have demonstrated that cGMP-dependent protein kinase (PKG) activity is downregulated in vessels from diabetic animals or in vascular smooth muscle cells (VSMCs) exposed to high-glucose conditions, contributing to diabetes-associated vessel dysfunction. However, whether decreased PKG activity plays a role in hyperglycemia-induced proliferation of VSMCs is unknown. In this report, high-glucose-mediated decreased PKG activity in VSMCs was restored by transfection of cells with expression vector for the catalytic domain of PKG-I (PKG-CD, constitutive active PKG). The effect of glucose on cell proliferation was determined. Our data demonstrated that high glucose exposure stimulated VSMC proliferation and G1 to S phase progression of the cell cycle, which was inhibited by restoration of PKG activity. Expression of constitutively active PKG inhibited G1 phase exit in VSMCs under high glucose conditions, which was accompanied by an inhibition of retinoblastoma protein (Rb) phosphorylation (a key switch for G1 to S phase cell cycle progression). Glucose-induced cyclin E expression and cyclin E-cyclin-dependent kinase 2 activity was also reduced by expression of PKG-CD in VSMCs. Moreover, expression of PKG-CD suppressed glucose-induced p27 degradation. These data demonstrate that restoring the high-glucose-mediated decrease in PKG activity in VSMCs inhibits glucose-induced abnormal VSMC proliferation occurring upstream of Rb phosphorylation. Our work provides the first direct evidence linking decreased PKG activity to high glucose-induced proliferation and cell cycle progression in VSMCs, suggesting that strategies to increase PKG activity might be useful in preventing abnormal VSMC proliferation in diabetic patients and might provide treatments for diabetes-associated proliferative vascular diseases.

CGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

BackgroundThe Na+/Cl--dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear.ResultsIn this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIα specifically associates with hSERT.ConclusionOur findings provide evidence of a physical and compartmentalized association between SERT and PKGIα that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIα supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.

Regulation of smooth muscle-specific gene expression by cGMP-dependent protein kinase

Background Vascular smooth muscle cells (VSMCs) in normal adult blood vessels exhibit a differentiated, phenotype, but in atherosclerotic disease and after acute vascular injury, they de-differentiate into a proliferative, synthetic phenotype; the latter is characterized by decreased smooth muscle (SM)-specific gene expression and decreased cGMP-dependent protein kinase (PKG) activity [1,2]. Prolonged in vitro culture of primary aortic VSMCs, or exposure of pulmonary VSMCs to hypoxia also leads to de-differentiation associated with loss of PKG, and restoring PKG activity increases SM-specific gene expression and re-differentiates cells into the contractile phenotype, but the mechanism(s) whereby PKG regulates SM genes are poorly understood [2,3].

Soluble Guanylate Cyclase

Arthur Kornberg, who shared with Severo Ochoa the 1959 Nobel Prize for Physiology or Medicine, stated in his autobiography that he had never met a dull enzyme.1 We strongly believe that soluble guanylate cyclase (sGC; E.C.4.6.1.2) is no exception to this. Article see p 2781 Nitric oxide (NO) plays an important physiological role as a signaling molecule as well as a cytotoxic agent. It is produced by 3 distinct NO synthases, specifically endothelial, neuronal, and inducible NO synthase. In the vasculature, endothelial NO synthase plays an important role in vascular homeostasis because NO generated in endothelial cells promotes vascular smooth muscle cell relaxation and inhibits platelet aggregation. Importantly, bioavailability of NO can be reduced in cardiovascular diseases, a condition which has been termed “endothelial dysfunction.” This phenomenon provides a rationale for therapeutic intervention. The primary target of NO is sGC, which is a heterodimeric enzyme consisting of an α- and β-subunit and a prosthetic heme group, which is located in the β-subunit. The heme group contains a ferrous iron atom (Fe2+). Binding of NO to this iron changes the conformation of the enzyme and leads to a >100-fold increase in catalytic rate (ie, the conversion of guanosine triphosphate to the second messenger cyclic guanosine monophosphate [cGMP]). The NO-induced cGMP signal modulates intracellularly the activity of several effector molecules: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cGMP-gated ion channels. Of note, both heme iron oxidation (Fe3+) and heme removal render sGC unresponsive to NO (Figure). These 2 conditions could therefore provide a mechanism for “vascular smooth muscle cell dysfunction” in cardiovascular disease states, which are often characterized by marked vasoconstriction either systemically or in specific organs. Figure. Schematic illustrating 3 different forms of soluble guanylate cyclase and their respective responsiveness to nitrovasodilators, sGC stimulators (eg, BAY 41–2272), and sGC …

Type II cGMP-dependent Protein Kinase Mediates Osteoblast Mechanotransduction

Continuous bone remodeling in response to mechanical loading is critical for skeletal integrity, and interstitial fluid flow is an important stimulus for osteoblast/osteocyte growth and differentiation. However, the biochemical signals mediating osteoblast anabolic responses to mechanical stimulation are incompletely understood. In primary human osteoblasts and murine MC3T3-E1 cells, we found that fluid shear stress induced rapid expression of c-fos, fra-1, fra-2, and fosB/DeltafosB mRNAs; these genes encode transcriptional regulators that maintain skeletal integrity. Fluid shear stress increased osteoblast nitric oxide (NO) synthesis, leading to activation of cGMP-dependent protein kinase (PKG). Pharmacological inhibition of the NO/cGMP/PKG signaling pathway blocked shear-induced expression of all four fos family genes. Induction of these genes required signaling through MEK/Erk, and Erk activation was NO/cGMP/PKG-dependent. Treating cells with a membrane-permeable cGMP analog partly mimicked the effects of fluid shear stress on Erk activity and fos family gene expression. In cells transfected with small interfering RNAs (siRNA) specific for membrane-bound PKG II, shear- and cGMP-induced Erk activation and fos family gene expression was nearly abolished and could be restored by transducing cells with a virus encoding an siRNA-resistant form of PKG II; in contrast, siRNA-mediated repression of the more abundant cytosolic PKG I isoform was without effect. Thus, we report a novel function for PKG II in osteoblast mechanotransduction, and we propose a model whereby NO/cGMP/PKG II-mediated Erk activation and induction of c-fos, fra-1, fra-2, and fosB/DeltafosB play a key role in the osteoblast anabolic response to mechanical stimulation.

Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia

Exposure to prolonged hypoxia can result in pulmonary vascular remodeling and pulmonary hypertension. Hypoxia induces pulmonary vascular smooth muscle cell (PVSMC) proliferation and vascular remodeling by affecting cell adhesion and migration and secretion of extracellular matrix proteins. We previously showed that acute hypoxia decreases cGMP-dependent protein kinase (PKG) activity in PVSMC and that PKG plays a role in maintaining the differentiated contractile phenotype in normoxia. In this study, we investigated the effect of hypoxia on PVSMC adhesion and migration and the role of PKG in these functions. Ovine fetal pulmonary artery SMC were incubated in normoxia (Po(2) approximately 100 Torr) or hypoxia (Po(2) approximately 30-40 Torr) or treated with the PKG inhibitor DT-3 for 24 h in normoxia. To further study the role of PKG in the modulation of adhesion and migration, PVSMC were transiently transfected with a full-length PKG1alpha [PKG-green fluorescent protein (GFP)] or a dominant-negative construct (G1alphaR-GFP). Cell adhesion to extracellular matrix proteins was determined, and integrin-mediated adhesion was assessed by alpha/beta-integrin-mediated cell adhesion array. Exposure to hypoxia (24 h) and pharmacological inhibition of PKG1 by DT-3 significantly promoted adhesion mediated by alpha(4)-, beta(1)-, and alpha(5)beta(1)-integrins to fibronectin, laminin, and tenacin and also resulted in increased cell migration. Likewise, inhibition of PKG by expression of a dominant-negative PKG1alpha construct increased cell adhesion and migration, comparable to that induced by hypoxia. Dynamic actin reorganization associated with integrin-mediated cell adhesion is partly regulated by the actin-binding protein cofilin, the (Ser3) phosphorylation of which inhibits its actin-severing activity. We found that increased PKG expression and activity is associated with decreased cofilin (Ser3) phosphorylation, implying a role for PKG in the modulation of cofilin activity and actin dynamics. Together, these findings identify cGMP/PKG1 signaling as central to the functional differences between PVSMC exposed to normoxia versus hypoxia.

ERK phosphorylation mediates sildenafil-induced myocardial protection against ischemia-reperfusion injury in mice

Sildenafil, a selective inhibitor of phosphodiesterase type 5, induces powerful protection against myocardial ischemia-reperfusion injury through activation of cGMP-dependent protein kinase (PKG). We further hypothesized that PKG-dependent activation of survival kinase ERK may play a causative role in sildenafil-induced cardioprotection via induction of endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and Bcl-2. Our results show that acute intracoronary infusion of sildenafil in Langendorff isolated mouse hearts before global ischemia-reperfusion significantly reduced myocardial infarct size (from 29.4 +/- 2.4% to 15.9 +/- 3.0%; P < 0.05). Cotreatment with ERK inhibitor PD98059 abrogated sildenafil-induced protection (31.8 +/- 4.4%). To further evaluate the role of ERK in delayed cardioprotection, mice were treated with sildenafil (ip) 24 h before global ischemia-reperfusion. PD98059 was administered (ip) 30 min before sildenafil treatment. Infarct size was reduced from 27.6 +/- 3.3% in controls to 7.1 +/- 1.5% in sildenafil-treated mice (P < 0.05). The delayed protective effect of sildenafil was also abolished by PD98059 (22.5 +/- 2.3%). Western blots revealed that sildenafil significantly increased phosphorylation of ERK1/2 and GSK-3beta and induced iNOS, eNOS, Bcl-2, and PKG activity in the heart 24 h after treatment. PD98059 inhibited the enhanced expression of iNOS, eNOS, and Bcl-2 and the phosphorylation of GSK-3beta. PD98059 had no effect on the sildenafil-induced activation of PKG. We conclude that these studies provide first direct evidence that PKG-dependent ERK phosphorylation is indispensable for the induction of eNOS/iNOS and Bcl-2 and the resulting cardioprotection by sildenafil.