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)

1. Putrescine has been implicated in modulating cytoplasmic calcium concentration and is correlated with selective neuronal vulnerability in cerebral ischaemia. In order to determine whether putrescine modulates voltage-activated calcium channels, whole-cell and single channel patch clamp experiments were performed with N1E-115 mouse neuroblastoma cells. 2. L-type calcium channel currents showed a 34 +/- 21% increase (n = 6 cells) during external application of 1 mM putrescine. There was no change in the kinetics of the current and no shift in the current-voltage relationship along the voltage axis. 3. T-type calcium channel currents were not affected by 1 mM putrescine. 4. The effect of putrescine on single L-type calcium channels was studied using the cell-attached configuration of the patch clamp technique. Putrescine (5 mM) applied to the bathing solution, but not present in the pipette, caused an increase in open time of the single channel current without changing the conductance of the channel. In 345 depolarizing steps compiled from three cells, the number of channel openings longer than 3 ms increased from six to seventy-six, and the number of channel openings longer than 9 ms increased from zero to twenty-seven. This single channel study supports the hypothesis that putrescine acts on the L-type channel from the inside of the cell. 5. External application of 1 mM spermine and 1 mM spermidine had no effect on T- and L-type calcium channels. Thus, the effect of putrescine is probably not mediated by the higher polyamines. 6. In order to test whether the effect of putrescine is mediated by a second messenger, specific protein kinase C and cyclic AMP-dependent protein kinase inhibitors, staurosporine and KT5720, respectively, were applied prior to putrescine. When cells were preconditioned with 200 nM staurosporine, the increase of the L-type calcium current by 1 mM putrescine was inhibited. By contrast, 200 nM KT5720 did not inhibit the putrescine effect. Therefore, the increase of L-type channel currents by putrescine may be mediated by protein kinase C but not the cyclic AMP-dependent protein kinase. 7. The putrescine-induced enhancement of the L-type calcium channel activity may play an important role in calcium-induced neurotoxicity.
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PMID:The effect of polyamines on voltage-activated calcium channels in mouse neuroblastoma cells. 839 76

1. The whole-cell voltage clamp technique was used to record calcium currents in the somatic membrane of rat cultured dorsal root ganglion neurones. 2. Neurones were enzymatically isolated from animals of three age groups (neonatal, 2-7 days; adult, 7 months; and old, 30 months) and maintained in primary culture 3-14 days. 3. The neurones isolated from neonatal and old rats showed two distinct types of Ca2+ currents, a low-threshold transient current and a high-threshold sustained current, whereas neurones from old rats showed only a high-threshold calcium current. 4. The density of the high-threshold calcium current was 28.4 +/- 6.3 pA/pF (mean +/- S.E.M., n = 54) in neonatal, 39.1 +/- 7.2 pA/pF (n = 62) in adult and 11.0 +/- 4.6 pA/pF (n = 64) in old dorsal root ganglion neurones. 5. We found no difference in elementary high-threshold Ca2+ current characteristics in neurones from different age groups. The single-channel conductance was (with 60 mM Ca2+ in the recording pipette) 16.0 +/- 2.7 pS (mean +/- S.E.M., n = 9) in neonatal, 16.2 +/- 1.7 pS (n = 11) in adult and 16.4 +/- 1.2 pS (n = 12) in old neurones. 6. Current-voltage relations and kinetics of high-threshold calcium currents showed no detectable age-dependent difference. 7. The run-down of high-threshold calcium currents in dorsal root ganglion neurones from old rats was practically insensitive to intracellular administration of cyclic AMP and ATP. The same intervention caused a significant deceleration of Ca2+ current run-down in the majority of neonatal and in some adult cells. 8. We suggest that the disappearance of the low-threshold calcium current and reduction of high-threshold calcium current with ageing is due to a depression of calcium channel expression during late ontogenesis. The decrease of sensitivity of high-threshold calcium channels to phosphorylation by cyclic AMP-dependent protein kinase in aged neurones could also be a reason for altered turnover between silent and functional pools of calcium channels, which may underlie the age-dependent decline in the density of high-threshold calcium channels.
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PMID:Calcium currents in aged rat dorsal root ganglion neurones. 839 26

Phosphorylation by protein kinase A is thought to be involved in voltage-dependent facilitation of calcium channels. Here we have shown that the subunit complex of a cloned human cardiac calcium channel, expressed in Xenopus oocytes, responds to voltage-dependent facilitation by an approximately 50% increase of the calcium channel peak current. The removal of all protein kinase A consensus sequences by site-directed mutagenesis decreased but did not eliminate the response to prepulse facilitation. Moreover, Rp-cAMP-S, an inhibitor of protein kinase A, could not prevent facilitation of the wild-type calcium channel currents. Similarly, AMP-PNP a nonhydrolyzable analog of ATP, while significantly decreasing the whole-cell current amplitude, failed to reduce the response to double-pulse facilitation. Therefore, we conclude that the voltage-dependent facilitation of cloned calcium channel currents is not due to enhancement of phosphorylation, but probably to some type of voltage-induced conformational change in the channel.
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PMID:Lack of involvement of protein kinase A phosphorylation in voltage-dependent facilitation of the activity of human cardiac L-type calcium channels. 861 75

Parafollicular (PF) cells secrete 5-hydroxytryptamine in response to increased extracellular Ca2+ ([Ca2+]e). This stimulus causes Cl- channels in PF secretory vesicles to open, leading to vesicle acidification. PF cells express a plasmalemmal heptahelical receptor (CaR) that binds Ca2+, Gd3+, and Ba2+. We now report that the CaR mediates vesicle acidification. Ca2+, Gd3+, and Ba2+ induced vesicle acidification, which was independent of channel-mediated Ca2+ entry. Agonist-induced vesicle acidification was blocked by pertussis toxin, inhibitors of phosphatidylinositol-phospholipase C, calmodulin, NO synthase, guanylyl cyclase, or protein kinase G. PF cells contained NO synthase immunoreactivity, and vesicles were acidified by NO donors and dibutyryl cGMP. [Ca2+]e, and Gd3+ mobilized thapsigargin-sensitive internal Ca2+ stores. [35S]G alpha i and [35S]G alpha q were immunoprecipitated from PF membranes incubated with agonists in the presence of [35S]adenosine 5'-O-(thiotriphosphate). Labeling of G alpha i but not G alpha q was antagonized by pertussis toxin. Vesicles acidified in response to activation of protein kinase C; however, protein kinase C inhibition blocked calcium channel- but not CaR-dependent acidification. We propose the following signal transduction pathway: CaR -> Gi -> phosphatidylinositol-phospholipase C -> inositol 1,4,5-trisphosphate -> [Ca2+]i -> Ca2+/calmodulin -> NO synthase -> NO -> guanylyl cyclase -> cGMP -> protein kinase G -> opens vesicular Cl- channel.
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PMID:Acidification of serotonin-containing secretory vesicles induced by a plasma membrane calcium receptor. 862 45

In contrast to excitable tissues where calcium channels are well characterized, the nature of the B lymphocyte calcium channel is unresolved. Here, we demonstrate by single cell analysis of freshly isolated rat B cells that the anti-immunoglobulin (Ig)-induced calcium influx takes place through a channel which shares pharmacologic and serologic properties with the L-type calcium channel found in excitable tissues. It is sensitive to the dihydropyridines nicardipine and Bay K 8644, to calciseptine, and to an anti-peptide antibody raised against the alpha1 subunit of the L-type calcium channel, but is voltage-insensitive. Anti-alpha1 and anti-alpha2 antibodies stain B but not T lymphocytes. Application of a cGMP agonist, measurement of cGMP levels in anti-Ig-stimulated B cells, and examining the effect of a guanylyl cyclase inhibitor on the anti-Ig response show that cGMP mediates the influx. This possibly involves a cGMP-dependent protein kinase. The anti-Ig-induced response is not abolished by prior treatment of B cells with a high dose of thapsigargin. These findings undermine the widely held belief of a categorical divide between excitable and non-excitable tissue calcium channels, demonstrate the limitations of the capacitative calcium influx theory, and point to a distinction between the calcium response mechanisms utilized by B and T lymphocytes.
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PMID:Anti-Ig-induced calcium influx in rat B lymphocytes mediated by cGMP through a dihydropyridine-sensitive channel. 863 46

5-Hydroxytryptamine (5-HT) stimulates corticosteroid secretion from adrenal cells through activation of 5-HT4 receptors positively coupled to adenylyl-cyclase. In the present study, we investigated in frog adrenocortical cells the effect of 5-HT4 receptor agonists on cytosolic calcium concentration ([Ca2+]i) and determined the sequence of events associated with 5-HT4 receptor agonist zacopride (10[-8] to 10[-5]M each in the vicinity of cultured adrenocortical cells caused a dose-dependent increase in [Ca2+]i. Preincubation of the cells with the selective 5-HT4 receptor antagonist [1-[2-(methylsulfonylamino)ethyl]-4- piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate maleate totally blocked the 5-HT-induced stimulation of [Ca2+]i. Chelation of extracellular calcium with ethylene glycol bis (beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (10 MM) suppressed the stimulatory effect of 5-HT on [Ca2+]i. Conversely, thapsigargin, an inhibitor of calcium ATPase activity, had no effect on the [Ca2+]i rise. The calcium influx induced by 5-HT4 receptor agonists was not affected by nifedipine and omega-conotoxin GVIA but was totally blocked by pimozide, a T-type calcium channel antagonist. The [Ca2+]i response to zacopride was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and markedly reduced by the protein kinase A inhibitor adenosine-3',5'-cyclic monophosphorothioate. We studied in perifused frog adrenal slices the involvement of [Ca2+]i rise and cAMP formation in the mechanism of action of 5-HT4 receptor agonists. Zacopride-induced steroidogenesis was significantly reduced in the presence of adenosine-3'5'-cyclic monophosphorothioate or after suppression of calcium in the perifusion medium. The stimulatory effect of zacopride on corticosteroid secretion was not affected by nifedipine and omega-conotoxin GVIA but was significantly inhibited by pimozide. Taken together, these data indicate that activation of 5-HT4 receptors in adrenocortical cells causes stimulation of adenylyl cyclase and subsequently increases calcium influx through a T-type calcium channel. Both the increased in cAMP formation and the calcium rise are involved in the stimulatory effect of 5-HT on corticosteroid secretion.
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PMID:Activation of 5-hydroxytryptamine4 receptors causes calcium influx in adrenocortical cells: involvement of calcium in 5-hydroxytryptamine-induced steroid secretion. 864 88

A phosphodiesterase (PDE) III inhibitor, amrinone, inhibited both the negative inotropic actions of verapamil and nicardipine in guinea pig ventricular papillary muscle; this effect was canceled by the protein kinase A inhibitor H-89. The PDE IV inhibitor 1,3-di-n-butyl-7-(2'-oxopropyl)xanthine (denbufylline), which elicited a negative inotropic action by itself, attenuated the action of verapamil up to 10 microM, without any interaction with nicardipine. The attenuation by denbufylline was not influenced by H-89. This suggests that in the ventricular papillary muscle, denbufylline acts on some verapamil-sensitive site(s) in the membrane and interferes with the calcium channel function without involvement of its PDE inhibitory activity.
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PMID:A xanthine derivative denbufylline inhibits negative inotropic response to verapamil in guinea pig ventricular papillary muscles, independent of its phosphodiesterase inhibitory activity. 869 37

1. Ranolazine has protective effects against ischaemia as exemplified by a reduction of the associated enzyme release and an attenuation of the fall of ATP and other metabolic changes. It has been suggested that ranolazine may affect GTP-binding proteins involved in the beta-adrenergic protein kinase A (PKA) cascade by interacting with Gs. Calcium channel currents are stimulated by this cascade but the effect of ranolazine upon them is not known. The whole cell patch clamp technique was used to examine the action of ranolazine on basal calcium channel currents and those stimulated by activation at various steps in the PKA cascade. 2. Ranolazine had only a small effect on the basal calcium current (100 microM caused 11.3% inhibition), but markedly attenuated the beta-adrenoceptor stimulated current (20 nM isoprenaline increased current by 2.3 fold, 10 microM ranolazine inhibited this increase by 47.6%). When the PKA cascade was activated downstream to the receptor by either G-protein activation with Gpp[NH]p or adenylate cyclase activation with forskolin, the calcium current showed a sensitivity to ranolazine similar to the basal current. Activation of the PKA cascade via H2 receptors gave rise to currents which showed an intermediate sensitivity to ranolazine. Ranolazine inhibition of ICa persisted during muscarinic attenuation of beta-adrenoceptor activation. 3. The results indicate that ranolazine, at concentrations which have significantly beneficial effects during ischaemic episodes, only greatly affects whole cell calcium current when facilitated by beta-adrenoceptor or histamine receptor activation. Ranolazine would appear to act at the receptor level, rather than at the GTP-binding or Gs/adenylate cyclase level. An additional smaller effect is also present, which may be mediated by a direct effect on the channel, or components closely associated with it.
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PMID:Effects of ranolazine on L-type calcium channel currents in guinea-pig single ventricular myocytes. 873 23

1. External application of the unsaturated fatty acid arachidonic acid (AA) to frog ventricular cells caused a large inhibition (approximately 85%) of the L-type calcium current (ICa,L) previously stimulated by the beta-adrenergic agonist isoprenaline (Iso). The concentration producing half-maximal inhibition (K1/2) was 1.52 microM. The inhibitory effect did not affect the peak current-voltage relationship but produced a negative shift in the inactivation curve. 2. The inhibitory effect of AA also occurred in cells internally perfused with cAMP and non-hydrolysable analogues of cAMP. These data suggest that AA is acting by a mechanism located beyond adenylyl cyclase and does not involve changes in intracellular cAMP levels. 3. AA also inhibited the calcium current stimulated by internal perfusion with the catalytic subunit of protein kinase A (PKA), suggesting that AA acts downstream of channel phosphorylation. 4. The inhibitory effect of AA on the isoprenaline- or cAMP-stimulated ICa,L is largely reduced in cells internally perfused with the thiophosphate donor analogue of ATP, ATP gamma S, or protein phosphatase 1 and 2A inhibitors like microcystin (MC) or okadaic acid (OA). External application of the phosphatase inhibitor calyculin (Caly) also reduced the AA effect. These data suggested that the AA effect on ICa,L involves activation of protein phosphatase activity. 5. The effect of AA on ICa,L was not affected by staurosporine, an inhibitor of protein kinases. It was also unaffected in cells internally perfused with GTP gamma S. These results suggest that neither a PKC- nor a G-protein-mediated mechanism are likely to be involved in the effect of AA on ICa,L. 6. A saturated fatty acid, myristic acid (MA), had no inhibitory effect on the isoprenaline-stimulated Ca2+ current, whereas, in the same cells arachidonic acid produced approximately 85% inhibition of ICa,L. 7. The inhibitory effect of AA was not affected by exposing the cells to indomethacin (Indo), an inhibitor of the metabolism of AA by cyclo-oxygenase, nor nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway. However, the non-metabolizable analogue of AA, 5,8,11,14-eicosatetraynoic acid (ETYA), was without effect on the isoprenaline-stimulated ICa,L. 8. These results suggest that AA inhibits ICa,L via a mechanism which involves, in part, stimulation of protein phosphatase activity. This process could provide a new mechanism in the modulation of calcium channel activity.
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PMID:Effect of arachidonic acid on the L-type calcium current in frog cardiac myocytes. 873 95

Full length L-type calcium channel alpha 1 subunits are rapidly phosphorylated by protein kinase A (PK-A) in vitro and in vivo at sites located in their long carboxyl terminal tails. In skeletal muscle, heart, and brain the majority of biochemically isolated alpha 1 subunits lacks these phosphorylation sites due to posttranslational proteolytic processing. Truncation may therefore modify the regulation of channel activity by PK-A. We combined site-directed mutagenesis and heterologous expression to investigate the extent to which putative cAMP-dependent phosphorylation sites in the C-terminus of alpha 1 subunits from skeletal muscle, heart, and brain are phosphorylated in vitro. The full length size form of wild-type and mutant calcium channel alpha 1 subunits was obtained at high yield after heterologous expression in Saccharomyces cerevisiae. Like in fetal rabbit myotubes [Rotman, E.I., et al. (1995) J. Biol. Chem. 270, 16371-16377], the rabbit skeletal muscle alpha 1 C-terminus was phosphorylated at serine residues 1757 and 1854. In the carboxyl terminus of alpha 1S from carp skeletal muscle and alpha 1C from rabbit heart a single serine residue was phosphorylated by PK-A in vitro. The C-terminus of alpha 1D was phosphorylated at more than one site. Employing deletion mutants, most of the phosphorylation ( > 70%) was found to occur between amino acid residues 1805 and 2072. Serine 1743 was identified as additional phosphorylation site in alpha 1D. We conclude that in class S and C calcium channels the most C-terminal phosphorylation sites are substrate for PK-A in vitro, whereas in class D calcium channels phosphorylation also occurs at a site which is likely to be retained even after posttranslational truncation.
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PMID:Identification of PK-A phosphorylation sites in the carboxyl terminus of L-type calcium channel alpha 1 subunits. 875 18


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