Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Our results demonstrate that the novel vasoactive regulatory peptide adrenomedullin is a potent mitogen for Swiss 3T3 cells. Acting via a specific adrenomedullin receptor, it stimulates a dose-dependent increase in DNA synthesis in synergy with insulin. Additionally, adrenomedullin stimulates further progression through the cell cycle resulting in cell proliferation, an effect that was further enhanced by the presence of insulin. Adrenomedullin rapidly induces accumulation of intracellular cAMP but does not stimulate an increase in intracellular Ca2+, activation of protein kinase C, or tyrosine phosphorylation of intracellular substrates. Adrenomedullin-stimulated mitogenesis is markedly enhanced in Swiss 3T3 cells stably transfected with a constitutively activated Gs alpha, which are highly sensitive to agents that elevate cAMP, and is inhibited by the PKA inhibitor H-89. Adrenomedullin is, thus, identified as a novel mitogenic regulatory peptide acting via cAMP.
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PMID:Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells. 854 9

We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22 +/- 0.04 nM (mean +/- S.E.M.) and a concentration of binding sites (Bmax) of 0.95 +/- 0.19 pmol/mg of protein (mean +/- S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8-37) competed weakly at this site (IC50 > 10 and 601 +/- 298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13 +/- 0.01 nM; Bmax = 0.83 +/- 0.10 pmol/mg of protein) where both CGRP(8-37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15 +/- 0.12 and 8.68 +/- 0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site-ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8-37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.
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PMID:A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors. 876 78

Smooth muscle cells isolated from the caecal circular smooth muscle layers of the guinea pig were used to determine whether adrenomedullin and guanylin can inhibit the contractile response produced by 10(-9) M cholecystokinin octapeptide (CCK-8). In addition, to elucidate each intracellular mechanisms, we examined the effects of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate guanylate cyclase, and an inhibitor of soluble guanylate cyclase on the adrenomedullin- or guanylin-induced relaxation of the caecal circular smooth muscle cells. Both adrenomedullin and guanylin inhibited the contractile response produced by CCK-8 in a dose-dependent manner, with IC50 values of 0.12 nM and 2.4 pM, respectively. An inhibitor of cAMP-dependent protein kinase significantly inhibited the relaxation produced by adrenomedullin. In contrast, an inhibitor of particulate guanylate cyclase and an inhibitor of soluble guanylate cyclase did not have any significant effect on the relaxation produced by adrenomedullin. On the other hand, an inhibitor of particulate guanylate cyclase significantly inhibited the guanylin-induced relaxation, although an inhibitor of cAMP-dependent protein kinase and an inhibitor of soluble guanylate cyclase did not have any significant effect on the guanylin-induced relaxation. In this study, we first demonstrated the direct inhibitory effects of adrenomedullin via cAMP system and guanylin via particulate guanylate cyclase system on the isolated caecal circular smooth muscle cells.
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PMID:Direct inhibitory effect of adrenomedullin and guanylin on isolated caecal circular smooth muscle cells of guinea pig. 932 27

Activation of cAMP signaling pathway was shown to inhibit some pathobiologic processes in mesangial cells (MC). We investigated whether adrenomedullin (ADM), a potent agonist of adenylate cyclase, is synthesized in MC and whether it can, via cAMP, suppress the generation of reactive oxygen metabolites (ROM) and proliferation of cells in glomeruli. With the use of an immunohistologic technique ADM was detected in mesangial and microvascular areas of rat glomeruli. MC grown in primary culture synthesized ADM, and the synthesis was stimulated by TNF alpha and IL-1 beta but not by PDGF and EGF. ADM inhibited ROM generation in MC dose-dependently and caused in situ activation of protein kinase A (PKA). In macrophages (cell line J774) ROM generation was about four times higher than in MC and was inhibited by ADM in a similar way as in MC. The rate of MC proliferation, measured by [3H]-incorporation, and the activity of mitogen-activated protein kinase (MAPK) stimulated by PDGF and EGF were dose-dependently inhibited by ADM; the maximum inhibition (at 10 nM ADM) was about -80%. Mitogenesis of MC and MAPK activity when stimulated to a similar extent by endothelin (ET-1) was inhibited by ADM to a significantly (P < 0.01) lesser degree (-30%). Further, ADM inhibited PDF-stimulated mitogenesis and activation of MAPK in cultured vascular smooth muscle cells (VSMC). The inhibition of PDGF-activated MAPK by ADM in VSMC was reversed by the protein kinase A (PKA) inhibitor, H89. Taken together, results indicate the adrenomedullin (ADM) generated in mesangial cells (MC) can suppress, via activation of the cAMP-protein kinase A (PKA) signaling pathway, reactive oxygen metabolites (ROM) generation in MC and infiltrating macrophages as well as mitogen-activated protein kinase (MAPK)-mediated mitogenesis in MC and vascular smooth muscle cells (VSMC). We suggest that introglomerular ADM may serve as a cytoprotective autoacoid that suppresses pathobiologic processes evoked by immuno-inflammatory injury of glomeruli.
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PMID:Cytoprotective effects of adrenomedullin in glomerular cell injury: central role of cAMP signaling pathway. 932 30

This study was designed to investigate the synthesis and action of adrenomedullin in the rat adrenal gland. The results obtained from in situ hybridization and immunocytochemical studies suggest that adrenomedullin is synthesized not only in the medulla, but also within the zona glomerulosa of the rat adrenal cortex. Findings from in situ hybridization and binding studies also suggested that specific adrenomedullin receptors are expressed in the zona glomerulosa, and that low levels are present in the inner zones of the cortex. The Kd of the zona glomerulosa adrenomedullin receptor (5.5 nmol/l) suggests that it may respond to locally produced adrenomedullin rather than circulating concentrations of the peptide, which are in a lower range. It was found that adrenomedullin acted on zona glomerulosa cells in vitro to stimulate aldosterone release and cAMP formation, but in this tissue did not stimulate inositol phosphate turnover. The effect of adrenomedullin on aldosterone secretion was significantly attenuated by a protein kinase A inhibitor, suggesting that cAMP mediates the effects of adrenomedullin on aldosterone secretion. Adrenomedullin did not significantly affect the response of zona glomerulosa cells to stimulation by either ACTH or angiotensin II. Adrenomedullin did not affect the release of catecholamines, either adrenaline or noradrenaline, by intact adrenal capsular tissue. These data suggest that both adrenomedullin and its specific receptor are expressed in the rat adrenal zona glomerulosa, leading to the hypothesis that adrenomedullin may have an autocrine/paracrine role in the regulation of the rat adrenal zona glomerulosa.
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PMID:Local production and action of adrenomedullin in the rat adrenal zona glomerulosa. 958 4

To examine whether adrenomedullin (AM), a novel vasodilator peptide, acts as a growth modulator in the vasculature, the effects of AM on protein tyrosine phosphorylation, mitogen-activated protein kinase (MAPK) activation, protooncogene expression, DNA synthesis, and cell proliferation were studied in cultured rat vascular smooth muscle cells (VSMC). AM and calcitonin gene-related peptide (CGRP), although weaker than AM, stimulated DNA synthesis and cell proliferation of quiescent VSMC, whose effects were inhibited by a CGRP receptor antagonist, CGRP-(8-37). AM induced a rapid increase in MAPK activity, followed by the expression of the immediate early protooncogene c-fos. AM-induced MAPK activation and cell proliferation were completely blocked by protein tyrosine kinase inhibitors (genistein and ST638). Moreover, AM rapidly induced tyrosine phosphorylation of several proteins (approximately 120, approximately 90, and approximately 50 kDa) and transiently increased association of a tyrosine-phosphorylated protein (approximately 120 kDa) and Shc with the glutathione-S-transferase-Grb2 fusion protein. A MAPK kinase inhibitor (PD98059) also reduced the AM-induced MAPK activation, c-fos messenger RNA expression, and cell proliferation. Although AM has been shown to induce vasodilation through cAMP production in VSMC, a cAMP antagonist (Rp-cAMP-thionate) and a protein kinase A inhibitor (KT5720) failed to block AM-induced MAPK activation and DNA synthesis. Moreover, 8-bromo-cAMP and forskolin did not affect the MAPK activity. AM had no effect on either the intracellular Ca2+ concentration or inositol 1,4,5-trisphosphate formation. In addition, a protein kinase C inhibitor (GF109203X) did not inhibit the AM-induced MAPK activation. These data suggest that in addition to its vasodilatory effect through the cAMP-dependent pathway, AM exerts its mitogenic activity via protein tyrosine kinase-mediated MAPK activation in quiescent rat VSMC.
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PMID:Adrenomedullin as a novel growth-promoting factor for cultured vascular smooth muscle cells: role of tyrosine kinase-mediated mitogen-activated protein kinase activation. 968 93

Adrenomedullin activates receptor-mediated adenylate cyclase to cause vasorelaxation. To elucidate whether desensitization of adenylate cyclase coupled to vascular adrenomedullin receptors occurs, we studied the adenylate cyclase activity after treatment with rat adrenomedullin in cultured rat aortic vascular smooth muscle cells. Cyclic AMP (cAMP) generation induced by adrenomedullin was markedly decreased by pretreatment with adrenomedullin: a maximal reduction (approximately 80%) was induced after 2 h and persisted during 24 h. Desensitization was independent of protein kinase A, protein kinase C, protein tyrosine kinase or receptor sequestration, because pretreatment with either isoproterenol, forskolin, tetradecanoylphorbol acetate, cytochalasin D, or colchicine did not affect the adrenomedullin-stimulated cAMP response. Furthermore, preincubation with inhibitors for these protein kinases prior to pretreatment with adrenomedullin failed to affect the adrenomedullin-induced decrease in cAMP response following the second stimulation with adrenomedullin. The present results provide the evidence for the existence of desensitization of adenylate cyclase coupled to vascular adrenomedullin receptors.
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PMID:Down-regulation of adenylate cyclase coupled to adrenomedullin receptor in vascular smooth muscle cells. 971 78

Recent evidence (1) suggests that the related peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) bind to the same heptahelical transmembrane receptor, with receptor specificity being determined by a receptor associated modifying protein (RAMP). If correct, this hypothesis would predict that each peptide should desensitize the cellular response to subsequent stimulation by itself or the other peptide. We have therefore studied the patterns of desensitization of these receptors in SK-N-MC cells. SK-N-MC cells were stimulated for 20 minutes in either serum free medium alone (control) or SFM containing AM 10(-8) M or CGRP 10(-7) M. Cells were then incubated for a further 20 minutes in SFM containing a second agonist and 1 mM isobutyryl methylxanthine (IBMX), before harvesting and assay for cAMP. Pre-exposure of cells to CGRP or AM decreased cAMP generation in response to subsequent stimulation with CGRP by 58% (+/-14) and 42% (+/-14) (SD) respectively. Pre-incubation of cells with 100 nM H-89 abolished this effect, indicating that desensitization was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with AM by pre-exposure to AM or CGRP. These results suggest that CGRP and AM receptors exhibit different patterns of desensitization in SK-N-MC cells: a finding with significant implications for the RAMP hypothesis.
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PMID:Desensitization of CGRP and adrenomedullin receptors in SK-N-MC cells: implications for the RAMP hypothesis. 988 66

Adrenomedullin is a potent vasodilatory peptide that increases cAMP in a number of different systems including rat mesangial cells. Since mesangial cells play a significant role in glomerular matrix production, we evaluated the effects and molecular mechanisms of adrenomedullin action on hyaluronic acid release, an important extracellular matrix component. Adrenomedullin increased hyaluronic acid release in mesangial cells in a concentration-dependent manner. Forskolin, an adenylate cyclase activator, and dibutyryl-cAMP, a cell permeable cAMP analog, also increased hyaluronic acid release significantly. Adrenomedullin-stimulated hyaluronic acid release was inhibited by the adrenomedullin receptor antagonist, adrenomedullin-(22-52). Inhibition of protein kinase A with H89 [[N-[2-(( p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride)]], a potent protein kinase A inhibitor did not affect adrenomedullin-stimulated hyaluronic acid release; however, H89 [[N-[2-(( p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride]] inhibited forskolin- and dibutyryl-cAMP-induced hyaluronic acid production. In addition, SB203580 [[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-im idazole), a P38 mitogen-activated protein kinase (P38 MAPK) inhibitor attenuated adrenomedullin-, forskolin-, and dibutyryl-cAMP-stimulated hyaluronic acid release. Hyaluronic acid release induced by adrenomedullin, forskolin and dbcAMP was also inhibited by wortmannin [[1S-(1alpha, 6balpha, 9abeta, 11alpha, 11bbeta)]-11-(Acetyloxy)-1, 6b, 7, 8, 9a, 10, 11, 11b-octahydro-1-(methoxymethyl)-9a, 11b-dimethyl-3H-furo[4, 3, 2-de]indeno[4, 5-h]-2-benzopyran-3, 6, 9-trione]. We conclude that adrenomedullin, forskolin and dbcAMP cause an increase in hyaluronic acid release in rat mesangial cells through a pathway that involves activation of wortmannin-sensitive kinase and P38 MAPK. Although cAMP stimulation and protein kinase A activation can induce hyaluronic acid release. adrenomedullin-stimulated hyaluronic acid release appears to be independent of protein kinase A activation. These data provide the first demonstration of the involvement of P38 MAPK- and wortmannin-sensitive kinase pathways in the stimulation of hyaluronic acid production by rat mesangial cells.
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PMID:Mechanism of adrenomedullin-stimulated hyaluronic acid release in rat mesangial cells. 1033 8

Adrenomedullin is a recently discovered vasodilatory peptide that has been shown to be a potent activator of adenylate cyclase in a variety of cell systems, including rat mesangial cells. The major aim of the present study was to determine the regulation of rat mesangial cell proliferation (using [3H]thymidine incorporation as an index), apoptosis (using nucleosome-associated cytoplasmic DNA fragmentation as an index) and mitogen-activated protein kinase (MAPK) cascade, specifically extracellular signal-regulated kinase (ERK), jun-amino terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, by adrenomedullin-stimulated cyclic AMP-protein kinase-A pathway. Adrenomedullin increased cAMP levels significantly above basal and the response was inhibited by the adrenomedullin receptor antagonist, adrenomedullin-(22-52). Adrenomedullin also decreased [3H]thymidine incorporation and increased nucleosome-associated cytoplasmic DNA fragmentation, in a concentration-dependent fashion. Both these responses were receptor mediated as, adrenomedullin-(22-52) inhibited these effects. The decrease in proliferation and increase in apoptosis were both mimicked by forskolin, a direct adenylate cyclase activator. Adrenomedullin-mediated decrease in proliferation and increase in apoptosis were inhibited by H89 [[N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride]], a potent protein kinase-A inhibitor. Associated with the changes in proliferation and apoptosis, adrenomedullin decreased ERK2 activity, and increased JNK1 and P38 MAPK activities. All these kinase activities, except the increase in JNK1 activity could be simulated using forskolin. In addition, only adrenomedullin-mediated changes in ERK2 and P38 MAPK activities were inhibited by H89 while, adrenomedullin-stimulated JNK1 was not consistently inhibited by the protein kinase-A inhibitor. These results suggest that adrenomedullin might play an important role in mesangial cell turnover and that although adrenomedullin-mediated responses are primarily cAMP-dependent, it does not preclude the involvement of cAMP-independent pathways.
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PMID:Regulation of glomerular mesangial cell proliferation in culture by adrenomedullin. 1037 18


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