Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rap1 antibody. The identity between the cAMP phosphoprotein and the rap1 GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 microM was found to: (1) inhibit to the same degree (40%) both Ca(2+)-ATPase activity and the Ca2+ transport function mediated by the Ca(2+)-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rap1 GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.
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PMID:Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes. 131 May 90

Annexin V is a protein of unknown biological function that undergoes Ca(2+)-dependent binding to phospholipids located on the cytosolic face of the plasma membrane. Preliminary results presented herein suggest that a biological function of annexin V is the inhibition of protein kinase C (PKC). In vitro assays showed that annexin V was a specific high-affinity inhibitor of PKC-mediated phosphorylation of annexin I and myosin light chain kinase substrates, with half-maximal inhibition occurring at approximately 0.4 microM. Annexin V did not inhibit epidermal growth factor receptor/kinase phosphorylation of annexin I or cAMP-dependent protein kinase phosphorylation of the Kemptide peptide substrate. Since annexin V purified from both human placenta and recombinant bacteria inhibited protein kinase C activity, it is not likely that the inhibitor activity was associated with a minor contaminant of the preparations. The following results indicated that the mechanism of inhibition did not involve annexin V sequestration of phospholipid that was required for protein kinase C activation: similar inhibition curves were observed as phospholipid concentration was varied from 0 to 800 micrograms/mL; the extent of inhibition was not significantly affected by the order of addition of phospholipid, substrate, or PKC, and the core domain of annexin I was not a high-affinity inhibitor of PKC even though it had similar Ca2+ and phospholipid binding properties as annexin V. These data indirectly indicate that inhibition occurred by direct interaction between annexin V and PKC. Since the concentration of annexin V in many cell types exceeds the amounts required to achieve PKC inhibition in vitro, it is possible that annexin V inhibits PKC in a biologically significant manner in intact cells.
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PMID:Inhibition of protein kinase C by annexin V. 131 Jun 21

In various species, including humans, 5-hydroxytryptamine (5-HT) has been shown to exert positive chronotropic and inotropic cardiac effects through different types of receptors. The goal of the present study was to investigate the regulation by 5-HT of voltage-gated Ca2+ channels in human atrial myocytes and to characterize the receptor involved. Cardiomyocytes isolated enzymatically and mechanically were voltage-clamped using the whole-cell configuration of the patch-clamp technique. Extracellular perfusion of 5-HT increased Ca2+ current (ICa) amplitude with a EC50 (0.1 microM) similar to that observed with isoprenaline. The effects of 5-HT were blocked by the addition of protein kinase A inhibitor in the pipette. In addition, the effects of 5-HT, isoprenaline, and intracellular cAMP on ICa were not additive. These results support the hypothesis that the inotropic effect of 5-HT in human atrial myocytes is related to an increase of ICa via an elevation of intracellular cAMP levels and stimulation of cAMP-dependent protein kinase. The effects of 5-HT were not blocked by antagonists of 5-HT1 (methiothepin), 5-HT2 (ketanserin), or 5-HT3 (ICS 205-930 at a low concentration) receptors. The benzamide derivatives renzapride and zacopride and the azabicyclobenzimidazolone derivative BIMU 8 increased ICa, but less efficiently than did 5-HT or 5-methoxytryptamine. Moreover, ICS 205-930 at high concentrations (greater than 1 microM) completely antagonized the effects of 5-HT. Thus, the pharmacology of the 5-HT receptor involved in an increase of ICa in human atrial myocytes resembles that recently described for the 5-HT4 receptor. In atrial myocytes dissociated from rat, rabbit, guinea pig, or frog, 5-HT at high concentrations had no effect on Ca2+ currents, suggesting that the distribution of 5-HT4 receptors in cardiac tissues is species dependent.
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PMID:Serotonin increases calcium current in human atrial myocytes via the newly described 5-hydroxytryptamine4 receptors. 131 10

Mefloquine (alpha-(2-piperidyl)-2,8-bis(trifluoromethyl)-4-quinolinemethanol) , an antimalarial drug, has been shown to inhibit human neutrophil functions, particularly oxygen-dependent bactericidal activity. Since calcium- and phospholipid-dependent protein kinase C (PKC) has a central role in the regulation of this function, we hypothesized that its activity might be altered by mefloquine. We found that mefloquine directly inhibited PKC in a dose-dependent manner, with an IC50 of 45 microM. This inhibition appeared to be non-competitive with respect to ATP, histone and phosphatidylserine. In addition, mefloquine inhibited the binding of [3H]phorbol 12,13 dibutyrate to PKC, indicating that it interacts with the regulatory domain of PKC. By contrast, mefloquine had little or no effect on neutrophil cAMP-dependent protein kinase or its catalytic subunit. Phorbol myristate acetate-induced protein phosphorylation in intact neutrophils was also inhibited by preincubation with mefloquine at concentrations similar to those inhibiting superoxide anion production. These data suggest that inhibition of neutrophil functions by mefloquine may be due to the inhibition of cellular PKC and that mefloquine could have further biological effects in situations in which PKC is involved.
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PMID:Inhibition of human neutrophil protein kinase C activity by the antimalarial drug mefloquine. 131 82

Ca2+ pumps are essential for removing cytosolic Ca2+ either across the plasma membrane (PM) or into internal organelles such as the sarcoplasmic reticulum (SR). Four genes (PMCA1, PMCA2, PMCA3 and PMCA4) have been reported to encode the PM Ca2+ pumps and three (SERCA1, SERCA2 and SERCA3) to encode the SR Ca2+ pumps. The PM Ca2+ pumps are stimulated by calmodulin, the SR Ca2+ pumps encoded by SERCA1 and SERCA2 are stimulated by phospholamban while the product of SERCA3 may be regulated directly by cAMP-dependent protein kinase. Alternative splicing of the primary transcripts of several of these genes has been reported to occur in a tissue selective manner and for others to alter during ontogeny. For the PM Ca2+ pump, alternative RNA splicing may result in isoforms with altered cyclic nucleotide dependent protein kinase sensitivity. The diversity in distribution of Ca2+ pump isoforms and their regulatory factors when coupled with different Ca2+ entry mechanisms allows for tissue selectivity and plasticity in stimulus-response coupling. The roles of various Ca2+ pump isoforms, the rationale behind their tissue selective expression and the plasticity in this expression are among the new challenges to researchers in this field.
Cell Calcium 1992 Jan
PMID:Calcium pump isoforms: diversity, selectivity and plasticity. Review article. 131 40

Phosphorylation of the rat brain ryanodine receptor was studied using a monoclonal antibody, Ry-1, against the cardiac ryanodine receptor. A large polypeptide with the same SDS-PAGE mobility as that of the canine cardiac receptor was detected in rat brain membranes by immunoblotting. The brain ryanodine receptor was solubilized from the microsomal membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and more than 85% of the solubilized receptor was immunoprecipitated by Ry-1. Immunoprecipitated receptors were phosphorylated by cAMP-dependent protein kinase. The ryanodine receptor was also expressed in cultured fetal rat brain neurons and was phosphorylated by treating the cells with dibutyryl cAMP. The number of cells showing a caffeine-induced Ca2+ transient was increased significantly in the phosphorylating condition. These results suggest that the Ca channel activity of the brain ryanodine receptor is regulated by cAMP-dependent phosphorylation.
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PMID:Cyclic AMP-dependent phosphorylation of the rat brain ryanodine receptor. 131 34

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca2+ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca2+ current (ICa) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p less than or equal to 0.05). Analysis of voltage-dependent steady-state activation (m infinity) curves revealed a 0.70 +/- 0.27 charge increase in the activation-gate valency (zm) following 8-Br-cAMP perfusion. Similar responses were observed when Ba2+ was the charge carrier, where zm was increased by 1.33 +/- 0.34 charges (n = 8). The membrane potential for half activation (V1/2) was also significantly shifted 6 mV more negative for IBa (mean, n = 8). The time course for IBa (and ICa) activation was well described by second-order m2 kinetics. The derived time constant for activation (tau m) was voltage-dependent, and the tau m/V relation shifted negatively after 8-Br-cAMP treatment. Ca2+ channel gating rates were derived from the tau m and m infinity 2 values according to a Hodgkin-Huxley type m2 activation process. The forward rate (alpha m) for channel activation was increased by 8-Br-cAMP at membrane potentials greater than or equal to 0 mV, and the backward rate (beta m) decreased at potentials less than or equal to + 10 mV. Time-dependent inactivation of ICa consisted of a slowly decaying component (tau h approximately 300 ms) and a "non-inactivating" steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.
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PMID:Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells. 131 68

We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase activity associated with the GH receptor. To gain insight into the role of GH-dependent tyrosine kinase activity in signaling by GH, we investigated the possibility that GH might stimulate MAP kinase, a serine/threonine/tyrosine kinase thought to be a common element in tyrosine kinase-initiated response cascades. Treatment of 3T3-F442A fibroblasts with 100 ng/ml GH results in a 3-6-fold increase in the ability of cell-free extracts to phosphorylate MAP-2 and myelin basic protein. GH-stimulated kinase activity is unaffected by heparin, H7, or cAMP-dependent protein kinase inhibitor peptide, partially reduced by staurosporin and inhibited by fluoride and calcium ions, indicating that the kinase is not protein kinase C or A, casein kinase, or a calcium/calmodulin-dependent protein kinase. Based on gel permeation chromatography, the molecular mass of the GH-stimulated MAP kinase is approximately kDa. Furthermore, anti-phosphotyrosine antibodies revealed the GH-dependent appearance of two phosphotyrosine-containing proteins in cell-free lysates of GH-treated cells that co-migrate with proteins recognized by anti-MAP kinase antibodies. The GH-dependent increase in MAP kinase activity displays a biphasic time course and is dependent on the concentration of GH applied to the cells. GH-dependent MAP kinase activity, partially purified by Mono-Q chromatography, is inactivated by treatment with alkaline phosphatase. Addition of H7 to the cells prior to the addition of GH has no effect, whereas addition of H8 increases MAP kinase activity in control cells with no effect in GH-treated cells, indicating that protein kinase C is unlikely to be an intermediary in the GH-dependent stimulation of MAP kinase activity. These findings indicate that signaling by GH in 3T3-F443A cells may, at least in part, utilize a kinase cascade similar to those that have been proposed for other membrane receptors with associated tyrosine kinase activity.
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PMID:Stimulation by growth hormone of MAP kinase activity in 3T3-F442A fibroblasts. 131 28

The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.
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PMID:Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B. 131 36

We have investigated the effects of isoproterenol (ISO) and forskolin on carbachol(CCh)- and fluoroaluminate (AlF4-)-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, myo-inositol 1,4,5-trisphosphate (IP3) production, 1,2-diacylglycerol, measured as phosphatidic acid (PA) formation, and contraction in the bovine iris sphincter smooth muscle. The data from these studies can be summarized as follows. (1) CCh (20 microM) stimulated significantly PIP2 hydrolysis, IP3 production, PA formation, and contraction. (2) Addition of ISO (0.1-25 microM), which raises the tissue cAMP level, to muscle precontracted with CCh attenuated PIP2 hydrolysis, IP3 production, PA formation and contraction in a time- and dose-dependent manner. (3) AlF4- (10 microM) induced a slow but progressive hydrolysis of PIP2, accompanied by parallel production of IP3, formation of PA, and contraction of the smooth muscle. The effects of AlF4- were dose-dependent and inhibited by deferoxamine, an Al3+ ion chelator. (4) Both forskolin (1-25 microM), which directly stimulates adenylate cyclase, and ISO inhibited the responses induced by AlF4- (10 microM) in a dose-dependent manner. (5) NaF (1-5 mM) had no effect on the activity of phospholipase C (PLC), purified from bovine iris sphincter. Furthermore, phosphorylation of the enzyme by catalytic subunit of protein kinase A had no inhibitory effect on PLC activity against PIP2. In conclusion, neither the muscarinic receptor nor PLC are the target sites for cAMP inhibition; instead the putative G-protein, which couples the activated muscarinic receptor to PLC, may be phosphorylated by cAMP-dependent protein kinase. This could attenuate the stimulation of PLC by the G-protein, thus resulting in inhibition of PIP2 hydrolysis and consequently leading to muscle relaxation. These results demonstrate cross-talk between the cAMP and IP3-Ca2+ second messenger systems and suggest that this could constitute a regulatory mechanism for the process of contraction-relaxation in smooth muscle.
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PMID:Effects of isoproterenol and forskolin on carbachol- and fluoroaluminate-induced polyphosphoinositide hydrolysis, inositol trisphosphate production, and contraction in bovine iris sphincter smooth muscle: interaction between cAMP and IP3 second messenger systems. 131 46


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