Expression Of cGMP-dependent Protein Kinase Research Articles

Addendum to: Expression of cGMP-dependent protein kinase, PKGIα, PKGIβ, and PKGII in malignant and benign breast tumors.

Addendum to: Expression of cGMP-dependent protein kinase, PKGIα, PKGIβ, and PKGII in malignant and benign breast tumors.

Lei-gong-gen formula granule attenuates hyperlipidemia in rats via cGMP-PKG signaling pathway

Ethnopharmacological relevanceLei-gong-gen formula granule (LFG) is a folk prescription derived from Zhuang nationality, the largest ethnic minority among the 56 nationalities in China. It is composed of three herbs, namely Centella asiatica (L.) Urb., Eclipta prostrata (L.) L., Smilax glabra Roxb. It has been widely used as health protection tea for many years to prevent cardiovascular and cerebrovascular diseases such as hyperlipidemia and hypertension. Aim of the studyThis study validated the lipid-lowering effect of LFG in a hyperlipidemia rat model. Then we employed network pharmacology and molecular biological approach to identify the active ingredients of LFG, corresponding targets, and its anti-hyperlipidemia mechanisms. Materials and methodsHyperlipidemia rat model was established by feeding male Sprague-Dawley rats with high-fat diet for two weeks. LFG (two doses of 10 and 20 g/kg) was administered orally to hyperlipidemia rat model for 4 weeks, twice per day. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) were monitored in rats pre and post-treatment. Hematoxylin-eosin staining was applied to observe the pathology and lipid accumulation of liver. We then performed network pharmacology analysis to predict the ingredients, their associated targets, and hyperlipidemia associated targets. Pathway analysis with significant genes was carried out using KEGG pathway. These genes and proteins intersectioned between compound targets and hyperlipidemia targets were further verified with samples from hyperlipidemia rats treated with LFG using Real-time RT-PCR and Western Blot. ResultsLFG attenuated hyperlipidemia in rat model, and this was characterized with decreased serum levels of TC, LDL-C, liver wet weight, and liver index. LFG alleviated the hepatic steatosis in hyperlipidemia rats. Network pharmacology analysis identified 53 bioactive ingredients from LFG formula (three herbs), which link to 765 potential targets. 53 hyperlipidemia associated genes were retrieved from public databases. There were 10 common genes between ingredients-targets and hyperlipidemia associated genes, which linked to 20 bioactive ingredients. Among these 10 genes, 3 of them were validated to be involved in LFG's anti-hyperlipidemia effect using Real-time RT-PCR, namely ADRB2 encoding beta-2 adrenergic receptor, NOS3 encoding nitric oxide synthase 3, LDLR encoding low-density lipoprotein receptor. The cGMP-PKG signaling pathway was enriched for hyperlipidemia after pharmacology network analysis with ADRB2, NOS3, and LDLR. Interestingly, expression of cGMP-dependent protein kinase (PKG) was downregulated in hyperlipidemia rat after LFG treatment. Molecular docking study further supported that ferulic acid, histidine, p-hydroxybenzoic acid, and linalool were potential active ingredients for LFG's anti-hyperlipidemia effect. LC-MS/MS analysis confirmed that ferulic acid and p-hydroxybenzoic acid were active ingredients of LFG. ConclusionLFG exhibited the lipid-lowering effect, which might be attributed to downregulating ADRB2 and NOS3, and upregulating LDLR through the cGMP-PKG signaling pathway in hyperlipidemia rat. Ferulic acid and p-hydroxybenzoic acid might be the underlying active ingredients which affect the potential targets for their anti-hyperlipidemia effect.

Comparative proteomic analysis of olfactory rosettes in anadromous Coilia nasus and resident Coilia nasus

The Japanese grenadier anchovy ( Coilia nasus ) undergoes upstream migration to spawning annually but can also be observed in freshwater resident populations. It has been hypothesized that anadromous adult C. nasus may utilize olfactory cues to locate spawning grounds. We firstly performed a comparative proteome analysis about olfactory rosettes in two populations to hunt for the proteomic changes. Among 5 408 identified proteins, 1 515 proteins (629 up-regulated and 886 down-regulated) were differentially expressed. Especially, several proteins and pathways associated with olfactory signaling were found to be significantly differential. Compared with resident C. nasus, the expressions of Golf protein and the sodium/calcium exchanger were significantly up-regulated in anadromous C. nasus. The expression of adenylate cyclase and regulator of G-protein signaling (RGS) were decreased. Our findings suggest a decrease in the expression of cGMP-dependent protein kinase (PKG) in anadromous C. nasus compared to resident C. nasus. The expression of Calmodulin (CaM) was increased and CaM-dependent protein kinase II (CaMKII) was decreased. In addition, KEGG pathway enrichment analysis of up-regulated proteins indicated statistically significant difference not only in olfactory transduction but also in the cGMP-PKG signal pathway. Furtherly, we sought out some proteins expressed in the same trend occurring in DEGs (differentially expressed genes) and DEPs (differentially expressed proteins) by doing the integrative analysis of proteome and transcriptome in olfactory rosettes of C. nasus.

Notch activation augments nitric oxide/soluble guanylyl cyclase signaling in immortalized ovarian surface epithelial cells and ovarian cancer cells

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.

Expression of cGMP-dependent protein kinase, PKGIα, PKGIβ, and PKGII in malignant and benign breast tumors

Cyclic GMP-dependent protein kinases (PKG) constitute a small family of enzymes that are encoded by two genes. Two major forms of PKG have been identified in mammalian cells, PKG I and PKG II. In addition, there are two splice variants of PKG I, which are designated as Iα and Iβ. There are increasing evidences that PKG can play an important role in the inhibition of cell proliferation and induction of apoptosis. In our previous studies, the inhibitory effects of cGMP/PKG on the cell growth were indicated using breast cancer cell lines. Accordingly, the present study was designed to compare the expression levels of three PKG isoforms in normal, benign, and malignant breast tissues. The expression level of PKG isoforms was assayed using quantitative real-time RT-PCR. The correlation between relative expression of PKG isoforms and clinicopathological characteristics were also analyzed. Downregulation of PKG isoforms was observed in the malignant and benign tumors when compared to those of respective normal tissues. No significant correlation was found between PKGIα, PKGIβ, and PKGII expression and clinicopathological features. The present study is the first to evaluate the expression level of PKG isoforms PKGIα, PKGIβ, and PKGII in the malignant and benign breast tumors. Reduction in the PKG expression is an important evidence to support the antitumor activity of this enzyme in vivo.

Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase and myocardin

We have previously reported that in ovine fetal pulmonary venous smooth muscle cells (FPVSMC), decreased expression of cGMP-dependent protein kinase (PKG) by hypoxia could explain hypoxia-induced SMC phenotype modulation. In this study, we investigated the role of myocardin, a possible downstream effector of PKG, in SMC phenotype modulation induced by 1 and 24 h of hypoxia. Hypoxia for 1 h induced the phosphorylation of E-26-like protein 1 (Elk-1), indicating a quick activation of Elk-1 after hypoxia. Either hypoxia (1 h) or treatment with DT-3, a PKG inhibitor, increased associations of Elk-1 with myosin heavy chain (MHC) gene and serum response factor (SRF), which was paralleled by a decrease in association of myocardin with MHC gene and SRF. Exposure to hypoxia of FPVSMC for 24 h significantly decreased the promoter activity of multiple SMC marker genes, downregulated protein and mRNA expression of myocardin, and upregulated mRNA expression of Elk-1, but had no significant effects on the phosphorylation of Elk-1. Inhibition of myocardin by siRNA transfection downregulated the expression of SMC marker proteins, while overexpression of myocardin prevented the hypoxia-induced decrease in expression of SMC marker proteins. Inhibition of PKG by siRNA transfection downregulated the expression of myocardin, but upregulated that of Elk-1. Overexpression of PKG prevented hypoxia-induced effects on protein expression of myocardin and Elk-1. These data suggest that PKG induces displacement of myocardin from SRF and upregulates myocardin expression, thus activating the SMC genes transcription. The inhibitory effects of hypoxia on PKG may explain hypoxia-induced SMC phenotype modulation by decreasing the effects of PKG on myocardin.

Cyclic GMP specifically suppresses Type-Iα cGMP-dependent protein kinase expression by ubiquitination

Type I cGMP-dependent protein kinase (PKG-I) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell-specific gene expression. Expression of PKG-I in cultured smooth muscle cells depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin-I and tumor necrosis factor-α, which are known to stimulate Type II NO synthase (iNOS) expression. We report here that the suppression of PKG-I protein levels in smooth muscle cells is triggered by the ubiquitin/26S proteasome pathway. Incubation of vascular smooth muscle cells with phosphodiesterase-resistant cyclic GMP analogs (e.g., 8-bromo-cGMP) decreases PKG-I protein level in a time- and concentration-dependent manner. To study this process, we tested the effects of 8-Br-cGMP on PKG-I protein level in Cos7 cells, which do not express endogenous type I PKG mRNA. 8-Br-cGMP induced the ubiquitination and down-regulation of PKG-Iα, but not PKG-Iβ. Treatment of cells with the 26S proteasome inhibitor, MG-132, increased ubiquitination of PKG. Blocking PKG-I catalytic activity using the cell-permeant specific PKG-I inhibitor, DT-2, inhibited cGMP-induced PKG-I ubiquitination and down-regulation, suggesting that PKG catalytic activity and autophosphorylation were required for suppression of PKG-I level. Mutation of the known autophosphorylation sites of PKG-Iα to alanine uncovered a specific role for autophosphorylation of serine-64 in cGMP-dependent ubiquitination and suppression of PKG-I level. The results suggest that chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and degradation of PKG-Iα in smooth muscle.

Novel Options for the Pharmacological Treatment of Chronic Anal Fissure – Role of Botulin Toxin

A chronic anal fissure (CAF) is commonly referred to as an ischemic ulcer. For many years it was thought that sphincteroctomy produces anal sphincter relaxation, enhances microcirculation and promotes CAF healing. The latest studies have shown that fissure healing does not appear to be dependent on reduction in mean resting anal pressure. Our description of the process of CAF healing is based on understanding the balance between nitric oxide (NO) concentration and a level of oxidative and nitroxidative stress in wounds, which is responsible for contraction of smooth muscles (also anal sphincters), endothelial/skeletal muscle cell remodelling and proliferation. Pharmacological sphincterotomy with botulinum toxin (BTX) has an effect on motor endplate but it also has an influence on nitric oxide synthase (NOS) and other agents. Hypoxia in contracted anal sphincters induces vasoconstriction, in part, by decreasing endothelial NOS expression. Clostridium botulinum C3 exoenzyme - Rho-kinase inhibitor reverses this vasoconstriction. CAF is a site where the haemostatic mechanisms are activated. Rho inactivator C3-transferase from Clostridium botulinum abolished thrombin - stimulated endothelial cell contraction. Attenuated biotransformation of Glyceryl trinitrate (GTN) by mitochondrial aldehyde dehydrogenase and suppression of cGMP-dependent protein kinase expression may play a key role in understanding the problem of synergistic action of BTX and GTN. BTX and GTN are different forms of pharmacological sphincterectomies. This mechanism could explain the potentiate effect of BTX action after NO donors application for CAF. The application of BTX releases the blockage in GTN bioactivation in smooth muscle cells and suppresses basal continuous sympathetic activity, causing modulation of anal sphincters. It is responsible for CAF healing.

Modulation of Pulmonary Vascular Smooth Muscle Cell Phenotype in Hypoxia: Role of Myocardin and ELK‐1

Hypoxia triggers pulmonary vascular remodeling, which is associated with vascular smooth muscle cell (SMC) phenotype modulation from a contractile to a synthetic state. Altered expression of cGMP‐dependent protein kinase (PKG) after hypoxia is implicated in SMC phenotype modulation. But the mechanism of SMC phenotype modulation after hypoxia at a gene transcriptional level is not clear. In this study, we found that in pulmonary vascular SMC, hypoxia (24h) significantly down‐regulated protein and mRNA expression of Myocardin, but up‐regulated mRNA expression of E‐26‐like protein 1 (Elk‐1). Acute hypoxia (1h) also induced phosphorylation of ELK‐1. Data from chromatin immunoprecipitation assay showed that under hypoxia conditions, less Myocardin and more ELK‐1 was bound to serum response factor (SRF), the transcription factor that is involved in SMC specific gene transcription. These data indicate that hypoxia activates ELK‐1 and inactivates Myocardin, thus inhibiting SRF function, suppressing SMC specific gene transcription and promoting SMC modulation to a de‐differentiated, synthetic state. Inhibition of PKG by PKG siRNA down‐regulated the expression of Myocardin, but up‐regulated the expression of ELK‐1, indicating that PKG may modulate SMC phenotype via regulation of Myocardin and ELK‐1.

Dysregulation of cGMP‐dependent protein kinase 1 (PKG‐1) impairs erectile function in diabetic rats: influence of in vivo gene therapy of PKG1α

To investigate the expression of cGMP-dependent protein kinase 1 (PKG1)alpha and PKG1beta in the corpus cavernosum, and to evaluate the effect of adenoviral gene transfer of PKG1alpha to the erectile compartment on erectile function in a rat model of diabetes. Diabetic (DM; induced by streptozotocin) male Sprague Dawley rats were transfected with adenoviruses (AdCMVbetagal or AdCMVPKG1alpha, in 10 rats each) 2 months after the induction of DM. Intracavernosal pressure (ICP) during stimulation of the cavernosal nerve (CN) was assessed, and compared with mean arterial pressure (MAP). Erectile tissue was harvested for Western blot analysis, immunohistochemistry and total PKG activity. Ten age-matched rats without DM served as the control. Compared to controls, AdCMVbetagal-transfected DM rats had significantly lower peak ICP responses, ICP/MAP ratios, and filling rates during CN stimulation. In DM rats transfected with AdCMVPKG1alpha, peak ICP, ICP/MAP ratios and filling rates were significantly better than in DM rats transfected with the reporter gene. As assessed by Western blot and immunohistochemistry, expression of PKG1alpha and PKG1beta was lower in corporal tissue from DM AdCMVbetagal-transfected rats than in controls. PKG1alpha expression was improved after AdCMVPKG1alpha gene therapy. Total PKG activity was lower in DM rat corporal tissue than in controls, and PKG1alpha gene transfer significantly improved DM corporal PKG activity to a value greater than in the control. PKG1alpha and PKG1beta activities are reduced in the erectile tissue of the diabetic rat, and gene transfer of PKG1alpha to the penis restored PKG activity and erectile function in vivo in diabetic rats. Gene therapy procedures targeting PKG1alpha might be an interesting future therapeutic approach to overcome diabetic erectile dysfunction resistant to oral pharmacotherapy.

Regulation of cGMP-dependent Protein Kinase Expression by Rho and Krüppel-like Transcription Factor-4

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.

Exisulind and guanylyl cyclase C induce distinct antineoplastic signaling mechanisms in human colon cancer cells

The nonsteroidal anti-inflammatory drug sulindac is metabolized to sulindac sulfone (exisulind), an antineoplastic agent that inhibits growth and induces apoptosis in solid tumors. In colon cancer cells, the antineoplastic effects of exisulind have been attributed, in part, to induction of cyclic guanosine 3',5'-monophosphate (cGMP) signaling through inhibition of cGMP-specific phosphodiesterases, which elevates intracellular cGMP, and novel expression of cGMP-dependent protein kinase (PKG) Ibeta, the presumed downstream effector mediating apoptosis. Here, inhibition of proliferation and induction of cell death by exisulind was dissociated from cGMP signaling in human colon cancer cells. Accumulation of intracellular cGMP produced by an exogenous cell-permeant analogue of cGMP or a potent agonist of guanylyl cyclase C yielded cytostasis without cell death. Surprisingly, the antiproliferative effects of induced cGMP accumulation were paradoxically less than additive, rather than synergistic, when combined with exisulind. Further, although exisulind induced expression of PKG Ibeta, it did not elevate intracellular cGMP and its efficacy was not altered by inhibition or activation of PKG I. Rather, PKG I induced by exisulind may mediate desensitization of cytostasis induced by cGMP. Thus, cytotoxic effects of exisulind are independent of cGMP signaling in human colon cancer cells. Moreover, combination therapies, including exisulind and agents that induce cGMP signaling, may require careful evaluation in patients with colon cancer.

Stage‐ and Cell‐Specific Expression of Soluble Guanylyl Cyclase Alpha and Beta Subunits, cGMP‐Dependent Protein Kinase I Alpha and Beta, and Cyclic Nucleotide‐Gated Channel Subunit 1 in the Rat Testis

Several studies suggest that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) modulate testicular function. In this study, we examined the expression of cGMP-dependent protein kinase G-I (PKG-I), and cyclic nucleotide-gated channel 1 (CNG-1), 2 known mediators of cGMP action, and the expression of soluble guanylyl cyclase (sGC) subunits in the rat testis. Immunohistochemical analysis revealed that the alpha subunit of sGC was expressed in the blood vessels and Leydig cells of adult rat testes. In addition, the sGC alpha subunit was observed in the acrosomal structures of spermatids undergoing the middle and later stages of spermiogenesis, but not in mature spermatozoa. Similar localization and expression patterns were seen for the sGC beta subunit, indicating coexpression of the sGC subunits. PKG-I was expressed in blood vessels and in the acrosomal region of spermatids during the early and middle stages of spermiogenesis but was not observed in Leydig cells or in mature spermatozoa. In contrast to sGC and PKG-I, CNG-1 was expressed only in cytoplasm and the residual bodies of late-stage (17-19) spermatids, with no staining observed in blood vessels and Leydig cells. These results demonstrate that sGC, PKG-I, and CNG-1 are expressed in a stage- and cell-specific manner in the rat testis. The distinct temporal patterns of expression of these components of cGMP signaling pathways suggest different physiological roles for sGC, PKG-I, and CNG-1 in spermiogenesis and steroidogenesis.

Regulation of cGMP-dependent Protein Kinase Expression by Soluble Guanylyl Cyclase in Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the alpha1 and beta1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.

Epithelio-mesenchymal transition in a neoplastic ovarian epithelial hybrid cell line

A hybrid cell line, IOSE-Ov29, was created through fusion of cells from the human ovarian adenocarcinoma line OVCAR3 and the non-tumorigenic SV40 Tag-transfected human ovarian surface epithelial line IOSE-29. OVCAR3 cells exhibit a differentiated epithelial phenotype, whereas line IOSE-29 expresses mesenchymal characteristics that were acquired in culture by epithelio-mesenchymal transition. Microsatellite analysis, comparative genomic hybridization (CGH), and MFISH showed the genotype of the IOSE-Ov29 cells to contain components of both parent cell lines, but to be predominantly OVCAR3 derived. IOSE-Ov29 resembled OVCAR3 and differed from IOSE-29 as shown by its unlimited life span, tumorigenicity, epithelial morphology, keratin, occludin, E-cadherin and CA125 expression, increased expression of kinases of the PI3K pathway, and loss of cGMP-dependent protein kinase expression. IOSE-29-derived properties included SV40 Tag expression, growth inhibition by activin, collagen type III secretion, increased adhesion and spreading on tissue culture plastic, and increased growth rate. Proliferation of all three lines was stimulated by FSH and ATP and inhibited by GnRH I and GnRH II. Interestingly, IOSE-Ov29 was more anchorage independent than either parent line and was the only line that invaded Matrigel in Boyden chambers and formed invasive branches in collagen gels. The results indicate that IOSE-Ov29 is an IOSE-29/OVCAR3 hybrid, which differs from both parent lines genetically and phenotypically. Unexpectedly, fusion with the non-tumorigenic IOSE-29 cells enhanced malignancy-associated characteristics of OVCAR3, presumably as a result of the expression of IOSE-29-derived mesenchymal properties that are usually acquired by carcinoma cells through epithelio-mesenchymal transition during metastatic progression.

Downregulation of cGMP-dependent protein kinase expression by inflammatory cytokines in vascular smooth muscle cells.

NO and cGMP have antigrowth and anti-inflammatory effects on the vessel wall in response to injury. It is well established that after vascular injury proinflammatory cytokines are involved in vascular wall remodeling. The purpose of this study was to ascertain the signaling mechanisms involved in cGMP-dependent protein kinase (PKG) suppression by inflammatory cytokines in primary bovine aortic vascular smooth muscle cells (VSMC). Interleukin (IL)-Ibeta, tumor necrosis factor (TNF)-alpha, and LPS decreased the mRNA and protein levels of PKG in VSMC. IL-Ibeta, TNF-alpha, and LPS increased inducible nitric oxide synthase (iNOS) expression and cGMP production. Treatment of cells with selective inhibitors of iNOS or soluble guanylate cyclase (sGC) reversed the downregulation of PKG expression induced by cytokines and LPS. The NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) and 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), a NO-independent sGC activator, decreased PKG mRNA and protein expression in bovine aortic VSMC. Cyclic nucleotide analogs [8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP) and 8-(4-chlorophenylthio)adenosine 3,5'-cyclic monophosphate (CPT-cAMP)] also suppressed PKG mRNA and protein expression. However, CPT-cAMP was more effective than CPT-cGMP in decreasing PKG mRNA levels. Selective inhibition of PKA with the Rp isomer of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphorothioate (Rp-8p-CPT cAMPS) prevented the downregulation of PKG by LPS. In contrast, the Rp isomer of 8-(4-chlorophenylthio)guanosine 3,5'-cyclic monophosphorothioate (Rp-8p-CPT cGMPS; inhibitor of PKG) had no effect on LPS-induced inhibition of PKG mRNA and protein expression. These studies suggest that cross-activation of PKA in response to iNOS expression by inflammatory mediators downregulates PKG expression in bovine aortic VSMC.

Increased effects of C-type natriuretic peptide on cardiac ventricular contractility and relaxation in guanylyl cyclase A-deficient mice.

The natriuretic peptides (NPs), atrial (ANP), B-type (BNP), and C-type (CNP) natriuretic peptides as well as their respective receptor-guanylyl cyclases (GC-A for ANP and BNP, and GC-B for CNP) are expressed in the heart. However, the local role of NPs in the regulation of cardiac contractility and the mutual interactions of NPs remain controversial. In the present study we evaluated the effects of ANP and CNP on cardiac function of wild-type (GC-A +/+) and GC-A-deficient (GC-A -/-) mice. The effects of NPs and their molecular mechanisms were assessed in the isolated perfused mouse working heart preparation. In GC-A +/+ hearts, CNP exerted a biphasic action: an immediate increase in inotropy and lusitropy, followed by a slowly developing negative inotropic effect. These effects were mimicked by the cGMP-analogue, 8-pCPT-cGMP. In contrast, ANP did not affect cardiac function. In GC-A -/- hearts, the immediate contractile responses to CNP and 8-pCPT-cGMP were significantly enhanced. CNP increased cardiac cGMP levels and stimulated phospholamban (PLB) phosphorylation; the effect on PLB, but not cGMP, was enhanced in GC-A -/- hearts. In addition, cardiac expression of cGMP-dependent protein kinase (cGK I) was significantly increased in GC-A -/- mice. CNP exerts a biphasic, initially positive inotropic and lusitropic, then negative inotropic effect in isolated working mouse hearts. A putative mechanism contributing to the immediate contractile responses is cGMP/cGK I-dependent phosphorylation of PLB and subsequent activation of the sarcoplasmic reticulum Ca(2+)-pump. ANP has no direct effects on cardiac contractility but chronic absence of its receptor, GC-A, results in increased responsiveness to CNP.

Expression of cGMP-dependent Protein Kinase in Human Atrium

We have shown that cGMP-dependent protein kinase (PKG) mediates stimulation of L-type calcium current by cGMP in rabbit atrial myocytes. The human atrium may have similar PKG-dependent regulation of calcium current. To elucidate the significance of PKG in cardiac function, we have isolated human PKG type I α cDNA (+1 to 2016), determined the nucleotide sequence and analyzed specific expression of PKG in human atrium. We obtained full-length cDNA of PKG type Iα from human atrial RNA using reverse transcriptase-polymerase chain reaction (RT-PCR). The coding region of human cardiac PKG I α showed 99.9% homology to previously published human PKG I α except for base No. 1983. At this position G was substituted for T and this resulted in an amino acid substitution from Leu649to Phe649. The cloned PKG I α cDNA was expressed in COS cells and the expressed PKG showed cGMP-stimulated PKG enzyme activity and immunoreactivity. Ribonuclease protection assay, Western blot analysis, and PKG enzyme activity assays in homogenates from human atrial tissue demonstrated the presence of PKG mRNA and protein in human atrial tissue. Immunofluorescence staining confirmed that PKG is highly expressed in human atrial myocytes. These findings suggest that PKG is highly expressed in human atrium and that PKG-dependent phosphorylation may be important in regulation of calcium channel activity in human atrial myocytes.

Expression of cGMP-dependent protein kinase I and its substrate VASP in neointimal cells of the injured rat carotid artery.

Neointimal fibroproliferative lesions after balloon angioplasty remain a major clinical problem, frequently leading to restenosis of initially successfully dilated coronary arteries. The cGMP-dependent protein kinase type I (cGMP-PK I) and its substrate vasodilator-stimulated phosphoprotein (VASP), molecular targets of the atrial natriuretic factor (ANF) and nitric oxide (NO) signalling pathways, are likely to be involved in various aspects of vascular wall regulation and restenosis formation. To investigate the occurrence of cGMP-PK I and VASP in neointimal cells in situ, we performed immunohistochemistry and immunoblotting experiments on denuded rat carotid arteries. Although the soluble cGMP-PK I showed a homogeneous distribution throughout the neointima, VASP apparently was more concentrated in smooth muscle cells (SMCs) lining the artery lumen, possibly reflecting enhanced growth factor stimulation of luminal SMCs. The membrane-associated cGMP-PK type II could not be detected in both the non-injured vessel wall and the restenotic tissue. The presence of both cGMP-PK I and VASP, major regulators of the actin cytoskeleton and cell motility, in neointimal tissue suggest that this emerging signal transduction pathway could be a target for the regulation and control of restenosis.

MRNA expression of CGMP-dependent protein kinase 1 and 2 in the rat brain

mRNA expression of CGMP-dependent protein kinase 1 and 2 in the rat brain