Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The spasmolytic and anti-spasmogenic activity of beta-adrenoceptor agonists on airways smooth muscle is thought to involve activation of the cyclic AMP/cyclic AMP-dependent protein kinase (PKA) cascade. Here we have tested the hypothesis that PKA mediates the anti-spasmogenic activity of isoprenaline and other cyclic AMP-elevating agents in guinea-pig isolated trachea by utilizing a number of cell permeant cyclic AMP analogues that act as competitive 'antagonists' of PKA. 2. Anion-exchange chromatography of guinea-pig tracheae resolved two peaks of PKA activity that corresponded to the type I ( approximately 5%) and type II ( approximately 93%) isoenzymes. 3. Pre-treatment of tracheae with zardaverine (30 microM), vasoactive intestinal peptide (VIP) (1 microM) and the non-selective activator of PKA, Sp-8-CPT-cAMPS (10 microM), produced a non-parallel rightwards shift in the concentration-response curves that described acetylcholine (ACh)-induced tension generation. The type II-selective PKA inhibitor, Rp-8-CPT-cAMPS (300 microM), abolished this effect. 4. Pre-treatment of tracheae with Sp-8-Br-PET-cGMPS (30 microM) produced a non-parallel rightwards shift of the concentration-response curves that described ACh-induced tension generation. The selective cyclic GMP-dependent protein kinase (PKG) inhibitor, Rp-8-pCPT-cGMPS (300 microM), abolished this effect. 5. Pre-treatment of tracheae with isoprenaline (1 microM) produced a 10 fold shift to the right of the ACh concentration-response curve by a mechanism that was unaffected by Rp-8-Br-cAMPS (300 microM, selective inhibitor of type I PKA), Rp-8-CPT-cAMPS (300 microM) and Rp-8-pCPT-cGMPS (300 microM). 6. We conclude that the anti-spasmogenic activity of Sp-8-CPT-cAMPS, zardaverine and VIP in guinea-pig trachea is attributable to activation of the cyclic AMP/PKA cascade whereas isoprenaline suppresses ACh-induced contractions by a mechanism(s) that is independent of PKA and PKG.
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PMID:Evidence that the anti-spasmogenic effect of the beta-adrenoceptor agonist, isoprenaline, on guinea-pig trachealis is not mediated by cyclic AMP-dependent protein kinase. 1149 3

In studies on human platelets, nitroprusside (NP) alone at 1-10 micromol/l increased platelet cyclic AMP (cAMP) by 40-70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo-oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2',5'-dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen-induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP-dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Ibeta isoenzyme. An inhibitor of this enzyme (Rp)-beta-phenyl-1,N2-etheno-8-bromoguanosine 3',5'-cyclic-monophosphorothioate, diminished the inhibition of collagen-induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen-induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP-dependent protein kinases had supra-additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.
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PMID:Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside. 1202 40

P-selectin is rapidly translocated from platelet alpha-granules following activation. Intracellular cyclic AMP (cAMP) is a potent inhibitory pathway that results in global downregulation of platelet activation. While cAMP-dependent protein kinase (PKA) has long been considered as the main mediator of cAMP-dependent effects, no study has yet evaluated its effect on P-selectin expression in human platelets. Pretreatment of thrombin-stimulated platelets with forskolin resulted in a concentration- dependent inhibition of P-selectin expression that correlated with adenylyl cyclase activity. Inhibition of PKA with H-89 reversed cAMP-induced inhibition of P-selectin while cGMP-dependent protein kinase (PKG) inhibition with KT5823 significantly potentiated cAMP-dependent inhibition of P-selectin. Similar results were also observed in a platelet/neutrophil binding assay. In conclusion, cAMP-induced inhibition of P-selectin expression is, in large part, mediated through activation of PKA. PKG appears to be solicited for P-selectin expression when cAMP levels are elevated which suggest a cAMP/PKG-dependent pathway of platelet activation.
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PMID:Differential regulation of P-selectin expression by protein kinase A and protein kinase G in thrombin-stimulated human platelets. 1257 12

Hepatotoxicity of allyl alcohol involves its bioactivation to acrolein and subsequent protein sulfhydryl loss and lipid peroxidation. However, the links between these events and hepatocellular death are not known. The purpose of these studies was to examine whether specific signal transduction pathways are associated with allyl alcohol toxicity in hepatocytes. Inhibition or augmentation of cyclic AMP and/or protein kinase A (PKA) by Rp-Ado-3N,5N-cyclic monophosphorothioate triethylamine salt or 3-isobutyl-1-methylxanthine had no effect on allyl alcohol-induced cell death. H-7, an inhibitor of PKA, PKC, and PKG, partially inhibited cell killing by allyl alcohol, whereas chelerythrine chloride, a nonselective PKC inhibitor, almost completely abolished allyl alcohol cytotoxicity. Neither 2,2N,3,3N,4,4N-hexahydroxy-1,1N,-biphenyl-6,6N-dimethanol-dimethyl ether, a selective PKC alpha and beta inhibitor, nor bisindolylmaleimide I, an inhibitor of PKC alpha, beta, and epsilon, had any effect on allyl alcohol cytotoxicity. In contrast, rottlerin, a selective PKCdelta inhibitor, blocked hepatocellular killing by allyl alcohol. Cytoprotection by chelerythrine chloride and rottlerin was not the result of inhibition of bioactivation of allyl alcohol because each inhibitor also prevented cell death from acrolein. Western blotting and immunohistochemical techniques revealed that allyl alcohol stimulated phosphorylation and translocation of PKCdelta to hepatocyte membranes (i.e., activation), and this activity was inhibited by rottlerin. Cell death appeared to occur via oncotic necrosis rather than apoptosis based on single-stranded DNA ELISA and propidium iodide staining. Together, these results indicate that activation of PKCdelta is a critical, early event in initiating hepatocyte injury and death from allyl alcohol.
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PMID:Allyl alcohol activation of protein kinase C delta leads to cytotoxicity of rat hepatocytes. 1275 90

Calcium induces transcriptional activation of the fos promoter by activation of the cyclic AMP response element (CRE)-binding protein (CREB), and in some cells its effect is enhanced synergistically by cyclic GMP (cGMP) through an unknown mechanism. We observed calcium-cGMP synergism in neuronal and osteogenic cells which express type II cGMP-dependent protein kinase (G-kinase); the effect on the fos promoter was mediated by the CRE and proportional to G-kinase activity. Dominant negative transcription factors showed involvement of CREB- and C/EBP-related proteins but not of AP-1. Expression of C/EBP-beta but not C/EBP-alpha or -delta enhanced the effects of calcium and cGMP on a CRE-dependent reporter gene. The transactivation potential of full-length CREB fused to the DNA-binding domain of Gal4 was increased synergistically by calcium and cGMP, and overexpression of C/EBP-beta enhanced the effect, while a dominant negative C/EBP inhibited it. With a mammalian two-hybrid system, coimmunoprecipitation experiments, and in vitro binding studies, we demonstrated that C/EBP-beta and CREB interacted directly; this interaction involved the C terminus of C/EBP-beta but occurred independently of CREB's leucine zipper domain. CREB Ser(133) phosphorylation was stimulated by calcium but not by cGMP; in cGMP-treated cells, (32)PO(4) incorporation into C/EBP-beta was decreased and C/EBP-beta/CRE complexes were increased, suggesting regulation of C/EBP-beta functions by G-kinase-dependent dephosphorylation. C/EBP-beta and CREB associated with the fos promoter in intact cells, and the amount of promoter-associated C/EBP-beta was increased by calcium and cGMP. We conclude that calcium and cGMP transcriptional synergism requires cooperation of CREB and C/EBP-beta, with calcium and cGMP modulating the phosphorylation states of CREB and C/EBP-beta, respectively.
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PMID:Synergism between calcium and cyclic GMP in cyclic AMP response element-dependent transcriptional regulation requires cooperation between CREB and C/EBP-beta. 1277 52

Ethanol exposure in airway epithelium increases cyclic AMP (cAMP)-dependent protein kinase (PKA) activity. Activation of PKA and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) has been shown to increase ciliary beat frequency (CBF) in bovine bronchial epithelial cells (BBECs). We have shown that biologically relevant concentrations of ethanol stimulate increases in CBF in a nitric oxide-dependent manner, mediated through elevated cAMP levels and subsequent PKA activation. This ethanol-driven rapid and transient increase in CBF occurs 15 to 30 min after exposure to 100 mM ethanol. However, after prolonged exposure to 100 mM ethanol (>/=6 h), CBF and the catalytic activity of PKA return to baseline levels. We hypothesize that cyclic nucleotide-dependent phosphodiesterase (PDE) activity attenuates the duration of ethanol-stimulated ciliary motility. The effect of ethanol on the PDE activity in BBECs was determined through direct assay of catalytic activity. When BBECs were incubated with 100 mM ethanol, significant increases in cAMP levels occurred within 1 h, with corresponding increases in PKA activity. Treatment of BBECs with 100 mM ethanol increased cAMP-PDE activity significantly by 4 h. 3-Isobutyl-1-methylxanthine, Ro 20-1724, and rolipram inhibited ethanol-stimulated cAMP-PDE activity. These agents inhibited ethanol-stimulated cAMP-PDE activity and increased the magnitude of ethanol-stimulated PKA activity observed under the same conditions. These findings support the idea that acute exposure (<6 h) to ethanol increases cAMP levels, and the associated increase in PKA activation is regulated by cAMP-dependent PDE, specifically PDE4. Other compensatory mechanisms however, may be responsible for the down-regulation of PKA, which occurs after chronic epithelial exposure (>/=6 h) to ethanol.
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PMID:Ethanol increases phosphodiesterase 4 activity in bovine bronchial epithelial cells. 1461 9

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using conventional patch-clamp technique, whole-cell currents were evoked in freshly isolated smooth-muscle cells from rat mesenteric resistance arteries by elevation of intracellular calcium with either 10 mM caffeine, 1 microM BAY K8644, 0.4 microM ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 microM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP or with a peptide inhibitor of PKG, or with the nonhydrolysable ATP analogue AMP-PNP. Under biionic conditions, the anion permeability sequence of the channel was SCN- > Br- > I- > Cl- > acetate > F- >> aspartate, but the conductance sequence was I- > Br- > Cl- > acetate > F- > aspartate = SCN-. The current had no voltage or time dependence. It was inhibited by nickel and zinc ions in the micromolar range, but was unaffected by cobalt and had a low sensitivity to inhibition by the chloride channel blockers niflumic acid, DIDS, and IAA-94. The properties of this current in mesenteric artery smooth-muscle cells differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under conditions of high calcium in the patch-pipette solution, a current similar to the latter could be identified also in the mesenteric artery smooth-muscle cells. We conclude that smooth-muscle cells from rat mesenteric resistance arteries have a novel cGMP-dependent calcium-activated chloride current, which is activated by intracellular calcium release and which has characteristics distinct from other calcium-activated chloride currents.
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PMID:A cyclic GMP-dependent calcium-activated chloride current in smooth-muscle cells from rat mesenteric resistance arteries. 1471 79

The present study describes the single channel properties of a novel cGMP-activated Ca(2+)-dependent Cl(-) channel in rat mesenteric artery smooth muscle cells. Single channel currents were recorded in cell-attached patches in the presence of 8 Br cGMP in response to the addition of caffeine or noradrenaline and in both outside-out and inside-out patches when the internal patch surface was bathed in cGMP and Ca(2+). The channels were permeable to Cl(-) ions with an anion permeability sequence of SCN(-) (1.7) > Cl(-) (1.0) > I(-) (0.6). Single channel mean open probability (NP(o)) was independent of voltage and the channels displayed three conductance levels of 15, 35 and 55 pS. cGMP was required for channel activation and the single channel NP(o) increased sharply with raised [Ca(2+)](i), maximal activation occurring at a [Ca(2+)](i) of about 100 nM. The relationship between NP(o) and cGMP concentration was voltage independent and could be fitted by the Hill equation giving a K(d) of about 3 microM and a Hill coefficient (n(H)) of 3. cGMP- and Ca(2+)-dependent channel currents were inhibited by 10 microM ZnCl(2) but niflumic acid, an inhibitor of Ca(2+)-activated Cl(-) channels, had no effect. Inhibition of cGMP-dependent protein kinase activity by the cGMP-dependent protein kinase inhibitor KT5823 or replacement of ATP by AMP-PNP reduced NP(o), while activation of cGMP-dependent protein kinase by guanosine 3', 5'-cyclic monophosphate, beta-phenyl-1, N(2)-etheno-8-bromo-sodium salt (8 Br PET cGMP) produced a significant increase in single channel NP(o). It is likely that these single channel currents underlie the noradrenaline-activated inward current important for vasomotion in these resistance arteries.
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PMID:Single cGMP-activated Ca(+)-dependent Cl(-) channels in rat mesenteric artery smooth muscle cells. 1472 80

Activation of the arterial baroreceptors induces expression of the proto-oncogene c-fos in the nucleus tractus solitarii (NTS), the terminal site of baroreceptor afferents in the medulla oblongata. This induced expression is an intracellular event that is crucial to long-term maintenance of stable blood pressure. Using Sprague-Dawley rats maintained under propofol anesthesia, we evaluated the role and delineated the underlying molecular mechanisms of nitric oxide (NO) in this process. Baroreceptor activation induced by 30 min of sustained hypertension significantly and sequentially increased the level of cyclic GMP-dependent protein kinase I (PKG-I), phosphorylated cyclic AMP response element-binding protein (pCREB), c-fos mRNA, and Fos protein in the NTS. All of these up-regulated expressions were significantly attenuated in animals that were pretreated immediately before baroreceptor activation with bilateral microinjection into the NTS of a selective neuronal nitric-oxide synthase (nNOS) inhibitor, 7-nitroindazole (2.5 pmol), or a soluble guanylyl cyclase (sGC) inhibitor, 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (1 nmol). Bilateral NTS microinjection of a cell-permeable cGMP analog, 8-bromoguanosine-3',5'-cyclic monophosphate (10 nmol) significantly elevated the level of pCREB or c-fos mRNA in the NTS. On the other hand, the up-regulated CREB phosphorylation or c-fos induction evoked in the dorsomedial medulla by baroreceptor activation was significantly antagonized by NTS application of a cell-permeable cGMP antagonist, (R)p-8-bromoguanosine-3',5'-cyclic monophosphorothioate (5 nmol), or a PKG inhibitor, (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H,-2,7b,11a-trizadizo-benzo(a,g)cycloocta(c,d,e)-trinden-1-one (1 nmol). We conclude that NO derived from nNOS in the NTS on baroreceptor activation may participate in c-fos expression via phosphorylation of CREB in a process that engages the sGC/cGMP/PKG-I signaling cascade.
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PMID:Nitric oxide regulates c-fos expression in nucleus tractus solitarii induced by baroreceptor activation via cGMP-dependent protein kinase and cAMP response element-binding protein phosphorylation. 1474 73

Nitric oxide (NO)/cyclic GMP (cGMP)-mediated mechanisms have a pivotal function in reducing the tone of the penile smooth musculature during normal erectile responses. The cyclic AMP (cAMP) signaling pathway is also involved in the adjustment of smooth muscle contractility, and suggestions for interactions between cGMP- and cAMP-mediated mechanisms have been presented. Using activators of the cGMP- or the cAMP-pathway, as well as inhibitors of protein kinase A (PKA; cAMP-dependent kinase) and protein kinase G (PKG; cGMP-dependent kinase), the present study was undertaken to further delineate the functional relation between these pathways in the penis. In addition, the distribution of PKA and some cAMP-binding phosphodiesterases (cAMP-PDEs) were investigated in human erectile tissue. Functional experiments were performed on isolated human corpus cavernosum (HCC). The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10 microM), or the PKG, Rp-8-pCPT-cGMPS (10 microM), on relaxation induced by the cumulative administration of sodium nitroprusside (SNP), forskolin, sildenafil or tadalafil (IC351) were studied in preparations of HCC precontracted with 1 microM norepinephrine (NE). Using immunohistochemical procedures, the presence of immunoreactivity for cAMP-PDEs PDE3, PDE4, and PDE4A, as well as for PKA was investigated in specimens of HCC from which preparations were also used in the functional experiments. Forskolin, SNP, sildenafil, and IC 351 dose-dependently reversed NE-induced tension of isolated HCC preparations. The relaxing effects of SNP were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS. In contrast, relaxation induced by forskolin, sildenafil and tadalafil were significantly reversed by both Rp-8-pCPT-cGMPS and Rp-8CPT-cAMPS. Abundant immunoreactivity for PDE3 and PKA was observed in the corpus cavernosum smooth muscle cells. Immunoreactivity for PDE4 was also detected in the smooth musculature and in the cytoplasm of endothelial cells lining the cavernous sinusoids, as well as in nerve fibres interspersing the trabecular stroma. The present results support the hypothesis of interactions between cGMP- and cAMP-mediated signals in the HCC, and suggest that the effects of inhibitors of PDE5 on isolated erectile tissue may also partly or indirectly include actions of the cAMP second messenger system. The exact mechanism by which such an interaction occurs is not clear, but it may involve altered activity of the cGMP-inhibited PDE3 brought about by a change in the intracellular levels of cGMP by the inhibition of PDE5. This will in turn lead to increasing levels of cAMP, facilitating the interaction of cAMP with the PKA. The immunoreactivity specific for PDE3, PDE4, PDE4A and PKA registered in HCC section is also in support of an important role for the cAMP/PKA-system for penile smooth muscle function.
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PMID:Interactions between cGMP- and cAMP-pathways are involved in the regulation of penile smooth muscle tone. 1504 18


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