Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Conventional inhibitors of cyclic AMP-dependent protein kinase lack membrane-permeability or selectivity, or both. The Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, Rp-cAMPS, is a novel membrane-permeable antagonist of cyclic AMP. We have assessed the ability of this compound to distinguish between cyclic AMP-dependent and cyclic AMP-independent contractile responses elicited in ventricular cardiomyocytes isolated from the hearts of adult rats. Cardiomyocytes were stimulated to contract at 0.5 Hz in the presence of calcium ion (2 mM) and adenosine deaminase (5 units/ml). Contractile shortening was expressed as maximum shortening relative to prestimulus cell length (delta L%). In the presence of a maximally-effective concentration of isoprenaline (100 nM), which acts by a cyclic AMP-dependent mechanism, Rp-cAMPS inhibited the contractile response in a concentration-dependent and time-dependent manner. Following preincubation for 30 min with Rp-cAMPS (100 microM), the contractile response to isoprenaline (100 nM) was 14% of that elicited in the absence of this inhibitor. An incubation time of 30 min was chosen for all subsequent studies. Rp-cAMPS (< or = 200 microM) inhibited the contractile response to isoprenaline (100 nM) significantly and in a concentration-dependent manner, but failed to inhibit the contractile responses elicited by phenylephrine (2 microM) and calcium ion (7 mM) which act by cyclic AMP-independent mechanisms. In the presence of Rp-cAMPs (200 microM), the contractile response to isoprenaline (100 nM) was 24% of that in the absence of inhibitor. Rp-cAMPS was used subsequently to investigate the contractile-coupling mechanisms associated with some novel inotropic agents. Rp-cAMPS (< or = 200 microM) also inhibited the contractile responses to secretin (20 nM) and VIP (20 nM) significantly. In the presence of Rp-cAMPS (200 microM), the contractile response elicited by secretin (20 nM) was 19% of that in the absence of inhibitor, while that elicited by VIP (20 nM) was abolished completely. Rp-cAMPS (< or = 200 microM) failed to inhibit the contractile response elicited by CGRP (1 nM). In summary, Rp-cAMPS is a membrane-permeable, selective antagonist of cyclic AMP in ventricular cardiomyocytes and can be used, in conjunction with the bioassay of the intracellular accumulation of cyclic AMP, to distinguish between cyclic AMP-dependent and cyclic AMP-independent contractile coupling mechanisms in these cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Use of the cyclic AMP antagonist, Rp-cAMPS, to distinguish between cyclic AMP-dependent and cyclic AMP-independent contractile responses in rat ventricular cardiomyocytes. 789 68

The studies discussed in this review demonstrate that phosphorylation is an important mechanism for the regulation of ligand-gated ion channels. Structurally, ligand-gated ion channels are heteromeric proteins comprised of homologous subunits. For both the AChR and the GABA(A) receptor, each subunit has a large extracellular N-terminal domain, four transmembrane domains, a large intracellular loop between transmembrane domains M3 and M4, and an extracellular C-terminal domain (Fig. 1B). All the phosphorylation sites on these receptors have been mapped to the major intracellular loop between M3 and M4 (Table 1). In contrast, glutamate receptors appear to have a very large extracellular N-terminal domain, one membrane hairpin loop, three transmembrane domains, a large extracellular loop between transmembrane domains M3 and M4, and an intracellular C-terminal domain (Fig. 1C). Most phosphorylation sites on glutamate receptors have been shown to be on the intracellular C-terminal domain, although some have been suggested to be on the putative extracellular loop between M3 and M4 (Table 1). A variety of extracellular factors and intracellular signal transduction cascades are involved in regulating phosphorylation of these ligand-gated ion channels (Fig. 2). Once again, the AChR at the neuromuscular junction is the most fully understood system. Phosphorylation of the AChR by PKA is stimulated synaptically by the neuropeptide CGRP and in an autocrine fashion by adenosine released from the muscle in response to acetylcholine. In addition, acetylcholine, via calcium influx through the AChR, appears to activate calcium-dependent kinases including PKC to stimulate serine phosphorylation of the receptor. Presently, agrin is the only extracellular factor known to stimulate phosphorylation of the AChR on tyrosine residues. For glutamate receptors, non-NMDA receptor phosphorylation by PKA is stimulated by dopamine, while NMDA receptor phosphorylation by PKA and PKC can be induced via the activation of beta-adrenergic receptors, and metabotropic glutamate or opioid receptors, respectively. In addition, Ca2+ influx through the NMDA receptor has been shown to activate PKC. CaMKII, and calcineurin, resulting in phosphorylation of AMPA receptors (by CaMKII) and inactivation of NMDA receptors (at least in part through calcineurin). In contrast to the AChR and glutamate receptors, no information is presently available regarding the identities of the extracellular factors and intracellular signal transduction cascades that regulate phosphorylation of the GABA(A) receptor. Surely, future studies will be aimed at further clarifying the molecular mechanisms by which the central receptors are regulated. The presently understood functional effects of ligand-gated ion channel phosphorylation are diverse. At the neuromuscular junction, a regulation of the AChR desensitization rate by both serine and tyrosine phosphorylation has been demonstrated. In addition, tyrosine phosphorylation of the AChR or other synaptic components appears to play a role in AChR clustering during synaptogenesis. For the GABA(A) receptor, the data are complex. Both activation and inhibition of GABA(A) receptor currents as a result of PKA and PKC phosphorylation have been reported, while phosphorylation by PTK enhances function. The predominant effect of glutamate receptor phosphorylation by a variety of kinases is a potentiation of the peak current response. However, PKC also modulates clustering of NMDA receptors. This complexity in the regulation of ligand-gated ion channels by phosphorylation provides diverse mechanisms for mediating synaptic plasticity. In fact, accumulating evidence supports the involvement of protein phosphorylation and dephosphorylation of AMPA receptors in LTP and LTD respectively. There has been a dramatic increase in our understanding of the nature by which phosphorylation regulates ligand-gated ion channels. However, many questions remain unanswered. (AB
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PMID:Regulation of ligand-gated ion channels by protein phosphorylation. 1021 14

Adrenomedullin (AM), a hypotensive peptide isolated from human pheochromocytoma, inhibits the proliferation of mesangial cells (MC) induced by mitogens such as platelet-derived growth factor. Quite recently, we have demonstrated that transmural pressure applied to cultured MC increased DNA synthesis and cell proliferation through protein kinase C and tyrosine kinase pathways. However, the modulatory effect of AM on pressure-induced cell proliferation is as yet unknown. In the present study, we examined the effect of AM on transmural pressure-induced DNA synthesis in cultured rat MC. Pressure was applied to cells placed in a sealed chamber using compressed helium. Application of pressure resulted in an increase in [(3)H]thymidine incorporation (approximately 2.0-fold). AM clearly inhibited pressure-induced DNA synthesis in a concentration-dependent manner. This inhibition was paralleled by an increase in cellular cAMP levels evoked by AM. Forskolin and dibutyryl cAMP mimicked the inhibitory effect of AM. The protein kinase A inhibitor H-89 significantly attenuated the effect of AM. Human AM(22-52)-NH(2), a putative AM receptor antagonist, reversed the inhibitory effects of AM more potently than did human CGRP(8-37), a calcitonin gene related peptide receptor antagonist. Our results suggest that AM, by acting mainly on AM-sensitive receptors, inhibits pressure-induced DNA synthesis in cultured rat MC through activation of protein kinase A. AM may play a protective role against MC proliferation in certain pathological conditions.
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PMID:Adrenomedullin inhibits transmural pressure induced mesangial cell proliferation through activation of protein kinase A. 1057 97

The present study documents that adrenomedullin (AM), a vasoactive peptide originally identified in pheochromocytoma tissue and present in the testis, in vitro affects the function of testicular peritubular myoid cells (TPMC), a contractile cell type located in the seminiferous tubule wall. AM stimulated cAMP production by cultured TPMC taken from 16-day-old rats, and this effect was completely inhibited by the AM antagonist AM-(22-52) and partially by the CGRP (calcitonin gene-related peptide) antagonist CGRP-(8-37). Studies on TPMC contractile activity documented that AM inhibits TPMC contraction induced by endothelin-1 (ET-1) and that its effect is antagonized by AM-(22-52). Neutralizing AM produced by TPMC with the addition of anti-AM antibody induced a significant increase of ET-1-induced contraction. When exposed to the protein kinase A inhibitor H-89, AM inhibitory activity on ET-1-induced TPMC contraction was suppressed, whereas the nitric oxide synthase inhibitor N:(G)-nitro-L-arginine methyl esther did not modify AM activity. In conclusion, our study indicates that AM stimulates cAMP production and inhibits the contraction induced by ET-1 in TPMC in vitro, and that AM produced by TPMC has an autocrine effect. We propose that AM may have a role in the control of seminiferous tubule contraction.
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PMID:Adrenomedullin inhibits the contraction of cultured rat testicular peritubular myoid cells induced by endothelin-1. 1115 65

In this study we investigated the effect of adrenomedullin (AM) on fMLP-mediated activation of human neutrophils. AM partially, but significantly, suppressed fMLP-induced upregulation of CD11b expression. The inhibitory effects of AM upon fMLP-induced upregulation of CD11b expression were completely blocked by CGRP [8-37], a CGRP receptor antagonist. AM significantly increased cAMP content in neutrophils and SQ-22,536, an adenylate cyclase inhibitor, and KT-5720, a PKA inhibitor, significantly blocked the inhibitory effects of AM upon fMLP-induced upregulation of CD11b expression. This study indicates that binding of AM to the CGRP receptor suppresses fMLP-induced upregulation of CD11b expression of human neutrophils by increasing intracellular cAMP levels. AM may play an important role in the regulation of inflammatory processes, especially in the binding of neutrophils to vascular endothelial cells and subsequent neutrophil emigration evident in acute pulmonary inflammation.
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PMID:Adrenomedullin suppresses fMLP-induced upregulation of CD11b of human neutrophils. 1140 11

Responses to human CGRP, adrenomedullin (ADM), and proadrenomedullin NH2-terminal 20 peptide (PAMP) were studied in small human thymic arteries. CGRP, ADM, and PAMP produced concentration-dependent vasodilator responses in arteries preconstricted with the thromboxane mimic U-46619. Responses to ADM and PAMP were attenuated, whereas responses to CGRP were not altered by endothelial denudation. Inhibitors of nitric oxide synthase and guanylyl cyclase attenuated responses to ADM and PAMP but not to CGRP. The CGRP1 receptor antagonist CGRP(8-37) attenuated responses to CGRP and ADM but not to PAMP. Responses to CGRP were reduced by SQ-22536 and Rp-cAMPS, inhibitors of adenylyl cyclase and PKA. These data suggest that responses to CGRP and ADM are mediated by CGRP(8-37)-sensitive receptors and that the endothelial ADM receptor induces vasodilation by a nitric oxide-guanylyl cyclase mechanism, whereas a smooth muscle CGRP receptor signals by a cAMP-dependent mechanism. A different endothelial receptor recognizes PAMP and signals by a nitric oxide-dependent mechanism.
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PMID:Responses to human CGRP, ADM, and PAMP in human thymic arteries. 1252 88

We have reported previously that bradykinin (BK) induces potent and reproducible concentration-dependent contractions of the pig iris sphincter (PIS) muscle in vitro through the activation of BK B(2) receptors. Here we attempted to investigate additional mechanisms by which BK induces contraction of the PIS in vitro. BK-mediated contraction of the PIS relied largely on the external Ca2+ influx by a mechanism sensitive to the L-, N- and P-type of Ca2+ channel selective blockers. Likewise, BK-induced contraction of the PIS was greatly inhibited by the CGRP-(8-37), NK(2) or NK(3) receptor antagonists (SR 48968, SR 142801), and to a lesser extent by the NK(1) antagonist (FK 888). Capsaicin desensitization of PIS or capsazepine pre-incubation also significantly reduced BK-mediated contraction in the PIS. Furthermore, KT 5720 or GF 109203X (the protein kinase A and C inhibitors, respectively) also significantly inhibited BK-mediated contraction. Taken together, these results indicate that BK-mediated contraction of the PIS seems to be mediated primarily by the release of CGRP and tachykinins from sensory nerve fibers, and relies largely on extracellular Ca2+ influx via activation of L-, N- and P-type of Ca2+ channels. Finally, these responses are mediated by activation of both protein kinase A- and C-dependent mechanisms.
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PMID:New evidence on the mechanisms underlying bradykinin-mediated contraction of the pig iris sphincter in vitro. 1449 83

In contrast to vascular muscles, the contribution of a hypotensive peptide adrenomedullin (AM) to the regulation of visceral smooth muscles is obscure. The content, synthesis, and effects of AM on the muscular tone in rat ileum were explored. It was found that there was immunoreactive AM (301 pg/mg of protein) and AM mRNA expression (162 fg/pg actin mRNA) in the ileum and that AM evoked relaxation in ileal strips (Ki = 0.85 nM) precontracted with serotonin. Antagonists of both AM (AM(22-52)) and calcitonin gene-related peptide (CGRP(8-37)) receptors did not affect this AM-induced relaxation, whereas it was suppressed by a selective blocker of beta3-adrenoreceptor (SR 59230A). The AM-induced relaxation was accompanied by a production of cAMP. Antagonists of protein kinases A (KT 5720 and H-7) and an inhibitor of the ATP-dependent K(+)-channels (glibenclamide) attenuated the effect of AM. We suggest that AM is a local regulator of the ileal tone, with an inhibitory action on muscle contraction. AM may activate the beta3-adrenoceptors, resulting in protein kinase A activation, which in turn opens the ATP-dependent K(+)-channels.
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PMID:The inhibitory effect of adrenomedullin in the rat ileum: cross-talk with beta3-adrenoceptor in the serotonin-induced muscle contraction. 1456 72

Blood vessels are surrounded by variable amounts of adipose tissue. We showed earlier that adventitial adipose tissue inhibits rat aortic contraction by release of a transferable factor, adventitium-derived relaxing factor (ADRF), which activates smooth muscle K(+) channels. However, little is known about the mechanisms of ADRF release. Using isolated rat aortic rings and isometric contraction measurements, we show that ADRF release depends on extracellular [Ca(2+)] (EC(50) approximately 4.7 mM). ADRF effects do not involve neuronal presynaptic N-type Ca(2+) and Na(+) channels or vanilloid, cannabinoid, and CGRP receptors. ADRF release is strongly inhibited by the protein tyrosine kinase inhibitors genistein and tyrphostin A25. In contrast, daidzein, an inactive genistein analog, and the protein tyrosine kinase inhibitor ST638 had no effect. Protein kinase A inhibition by H89 also inhibited ADRF release, whereas the protein kinase G inhibitor KT-5823 had no effect. We propose that ADRF release is Ca(2+) dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. Furthermore, ADRF release does not depend on perivascular nerve endings.
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PMID:Mechanisms of ADRF release from rat aortic adventitial adipose tissue. 1464 61

Adrenomedullin (AM) is a potent vasoactive peptide and plays an important role in cardiovascular function. In this study, we delivered the AM gene locally into the heart, using a catheter-based technique to investigate the signaling mechanism mediated by AM in protection against cardiomyocyte apoptosis induced by acute ischemia/reperfusion. After adenovirus-mediated gene delivery, highly efficient and specific expression of luciferase, green fluorescent protein, or recombinant human AM was identified in the left ventricle. Delivery of the AM gene 5 days before ischemia/reperfusion attenuated myocardial apoptosis identified by in situ dUTP nick-end labeling and DNA laddering, and the effect was blocked by the AM antagonist human calcitonin gene-related peptide (CGRP 8 to 37). AM gene transfer increased phosphorylation of Akt and glycogen synthase kinase (GSK-3beta) but reduced GSK-3beta and caspase-3 activities in the heart. The effects of AM on GSK-3beta and caspase-3 activities were blocked by CGRP (8-37) and by adenovirus containing dominant-negative Akt (DN-Akt). Furthermore, in cultured cardiomyocytes, AM also attenuated apoptosis induced by hypoxia/reoxygenation, which was accompanied by increased phospho-GSK-3beta but reduced GSK-3 and caspase-3 activities. GSK-3 and caspase-3 activities were both blocked by Ad.DN-Akt and lithium, whereas only caspase-3 was inhibited by its inhibitor Z-VAD. The effects of AM on anti-apoptosis and promoting cell viability were blocked by DN-Akt but not by constitutively active Akt, lithium, or Z-VAD. These results indicate that AM protects against cardiomyocyte apoptosis induced by ischemia/reperfusion injury through the Akt-GSK-caspase signaling pathway.
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PMID:Adrenomedullin protects against myocardial apoptosis after ischemia/reperfusion through activation of Akt-GSK signaling. 1466 48


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