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. The intracellular mechanisms of serotonin (5-HT) response were investigated in dissociated rat hippocampal pyramidal neurons using the nystatin-perforated patch technique. 2. Under voltage-clamp conditions, 5-HT evoked outward currents (I5-HT) with an increase in membrane conductance at a holding potential of -40 mV. The outward current reversed at the K+ equilibrium potential, which shifted 59.4 mV with a 10-fold change in extracellular K+ concentration. 3. The first application of 5-HT on neurons perfused with Ca(2+)-free external solution induced outward currents of I5-HT but the amplitude was diminished dramatically with successive applications. Pretreatment with the membrane-permeant Ca2+ chelator 1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) also diminished the I5-HT amplitude. 4. Pretreatment with pertussis toxin (PTX) had no effect on I5-HT. 5. The I5-HT was not cross-desensitized with the caffeine-induced outward current but with outward current mediated by the muscarinic acetylcholine receptor. Pretreatment with Li+ significantly enhanced the I5-HT, indicating that I5-HT is involved in the elevation of intracellular free Ca2+ released from inositol triphosphate (IP3)-sensitive Ca2+ store sites but not from the caffeine-sensitive ones. 6. The calmodulin (CaM) antagonists, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited I5-HT in a concentration-dependent manner. 7. The Ca2+/CaM-dependent protein kinase II inhibitor 1-[N,O-Bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosil]-4-phenylpiperazine depressed the I5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Serotonin-operated potassium current in CA1 neurons dissociated from rat hippocampus. 849 47

The tibialis anterior (TA) muscles of 6-month-old and 24-month-old male Wistar rats, after being characterized, at the fast motor unit level, for twitch properties, were dissected and processed by a procedure [Margreth A., Damiani E., Tobaldin G. Biochem Biophys Res Commun 1993; 197: 1303-1311] aimed at obtaining a representative total membrane fraction comprising 70-80% of the total muscle content of sarcoplasmic reticulum (SR) and transverse tubule (TT) membranes (about 20 mg protein/g). Skeletal muscle membranes were analyzed for protein composition, and the content and functional properties of specific components of the free and junctional subcompartments of the SR and of junctional TT. Our results, while confirming a twitch prolongation in TA of old rats, do not demonstrate any associated age-related change concerning: (a) the overall number and functional properties of Ca2+ pumps, as characterized by kinetic parameters, Ca(2+)-dependency, and the protein isoform specificity of SR Ca(2+)-ATPase; (b) the number of functional junctional SR Ca(2+)-release channels, on the basis of Bmax values for high-affinity binding of [3H]-ryanodine to skeletal muscle membranes at optimal Ca2+; (c) the overall muscle dihydropyridine receptor/ryanodine receptor (RyR) ratio. We conclude from these findings, and the additional negative evidence for changes in membrane density of specific components of junctional SR, including 60 kDa Ca(2+)-calmodulin protein kinase, that this membrane domain, like the Ca(2+)-pump domain of the SR, are in no way basically altered at early stages of the aging process, as investigated here. Because of that, we allege particular significance to the occurrence of age-related, specific abnormalities in regulation of RyR in rat TA. The main supportive evidence is as follows: (a) an increased sensitivity to Ca2+ of the RyR of old muscle, and, more importantly; (b) an increased sensitivity to caffeine of [3H]ryanodine binding to the RyR at optimal Ca2+ and also optimal for the activity of the Ca(2+)-release channel. The results reported here also demonstrate that there are two classes of caffeine sites in rat TA muscle, as defined by differences in EC50 values at resting (pCa 7) and at high Ca2+ (pCa 4-5), that sites involved in stimulation of [3H]-ryanodine binding to the RyR are distinguished by a higher affinity (caffeine below mM), and that only these sites undergo age-related changes. Thus, although the underlying age-related abnormality of the RyR remains to be elucidated, it appears to satisfy the requirement for being regarded as a specific change, which in itself might argue for its being fundamentally related to the twitch prolongation of the muscle.
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PMID:Age-related abnormalities in regulation of the ryanodine receptor in rat fast-twitch muscle. 865 53

Of two neurosecretory PC12 cell clones that respond to NO donors and 8-bromo-cGMP with similar increases in cADP-ribose and that possess molecularly similar Ca2+ stores, only one (clone 16A) expresses the type 2 ryanodine receptor, whereas the other (clone 27) is devoid of ryanodine receptors. In PC12-16A cells, activation of the NO/cGMP pathway induced slow [Ca2+]i responses, sustained by release from Ca2+ stores. In contrast, PC12-27 cells were insensitive to NO donors. Likewise, in PC12-16A cells preincubated with NO donors, Ca2+ stores were partially depleted, as revealed by a test with thapsigargin, whereas those in clone 27 were unchanged. The NO-induced Ca2+ release was increased synergistically by caffeine, and the corresponding store depletion was magnified by ryanodine. The specificity for the NO/cGMP pathway was confirmed by the effects of two blockers of cGMP-dependent protein kinase I, while the role of cADP-ribose was demonstrated by the effects of its antagonist, 8-amino-cADP-ribose, administered to permeabilized cells. These results demonstrate in neurosecretory cells a ryanodine receptor activation pathway similar to that known in sea urchin oocytes. The signaling events described here could be of great physiological importance, especially in the nervous system.
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PMID:The type 2 ryanodine receptor of neurosecretory PC12 cells is activated by cyclic ADP-ribose. Role of the nitric oxide/cGMP pathway. 866 43

Intracellular calcium is considered to play a major role in secretory responses of various exocrine cell types. We examined whether xanthine derivatives can inhibit Ca2+ mobilization and entry in secretory cells in the airways. Therefore, the inhibitory effect of xanthines in the intracellular Ca2+ concentration ([Ca2+]i) in the isolated submucosal acinar cells of the guinea-pig nasal septum was investigated by means of fluorescence ratio microscopy. The inhibitory effects on Ca2+ release from stores was examined in Ca(2+)-free conditions. Effects on Ca2+ entry were estimated by two different protocols; 1) the sustained phase in a long-term application of acetylcholine (ACh) and 2) the [Ca2+]i overshoot following removal of ACh in Ca(2+)-free conditions. Xanthine derivatives, 3-isobutyl-1-methyl-xanthine (IBMX), caffeine, and theophylline, significantly inhibited the increase in [Ca2+]i evoked by ACh; both mobilization from internal Ca2+ stores and Ca2+ entry from the external space. The rank order of potency of these xanthine derivatives was IBMX > theophylline > caffeine. The addition of dibutyryl-cyclic adenosine monophosphate (cAMP) and forskolin to nasal gland acinar cells failed to inhibit the ACh-evoked increase in [Ca2+]i. Furthermore, a protein kinase A inhibitor, H-89, did not affect the inhibitory effect of the xanthine derivatives. The action of xanthines on the present acinar cells did not involve Ca(2+)-induced Ca2+ release (CICR) or an interaction with purinergic receptors. Thus, xanthines have a direct inhibitory effect both on Ca2+ release and entry in nasal gland acinar cells, and might thereby have antisecretory activity within the airways.
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PMID:Xanthine derivatives inhibit the increase in intracellular Ca2+ concentration induced by acetylcholine in nasal gland acinar cells of the guinea-pig. 866 8

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.
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PMID:Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes. 964 49

Stimulation of portal vein myocytes with noradrenaline (NA) in the presence of a voltage-dependent Ca2+ channel blocker, evoked a transient increase in the concentration of free cytosolic Ca2+, due to inositol 1,4,5-trisphosphate mediated Ca2+ release, followed by activation of a Ca2+ entry pathway. Combining patch-clamp and indo-1 measurements we have tested the effects of various pharmacological agents on this Ca2+ entry following NA-induced Ca2+ release in order to determine the mechanism involved. Only the guanylate cyclase inhibitor LY-83583 specifically inhibited the maintained Ca2+ entry during NA stimulation. This inhibition was reversed by dibutyryl cGMP (DB-cGMP) or 8-bromo cGMP. Under control conditions, addition of DB-cGMP to the external solution was without effect. Thapsigargin and caffeine each depleted the intracellular Ca2+ store but did not evoke Ca2+ entry in venous myocytes under control conditions. However, application of DB-cGMP or NA after Ca2+ store depletion induced by caffeine or thapsigargin caused a rise in [Ca2+]i by activation of a Ca2+ entry pathway. The effect of cGMP seems to involve phosphorylation since cGMP-activated protein kinase inhibitors KT-5823 and H-8 blocked the NA-induced Ca2+ entry. Our results thus suggest that the activation of the voltage-independent Ca2+ entry by NA involves an increase in cellular cGMP.
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PMID:Activation of voltage-independent Ca2+ entry by noradrenaline involves cGMP in vascular myocytes. 874 49

Adenosine A1 receptor densities were increased in cultured LLC-PK1 and OK cells by chronic treatment with the adenosine receptor antagonists 1,3,7-trimethylxanthine (caffeine, 1 mM) and 1,3-dimethyl-8-cyclopentylxanthine [cyclopentyltheophylline (CPT), < or = 0.4 mM], respectively. The A1 receptor number per cell was increased twofold by 10-day treatments with 1 mM caffeine or 0.1 mM CPT, and the sodium-coupled glucose uptake was augmented twofold by 1 mM caffeine and sevenfold by 0.1 microM CPT (higher doses of CPT were progressively less stimulatory). Glucose uptake was blocked by acute (2-h) treatment with CPT, adenosine deaminase, or calphostin C. Caffeine (1 mM) or CPT (> or = 0.1 mM) inhibited cell proliferation for the first 10 days, then cell growth assumed a normal proliferative rate despite continued presence of antagonist. Cytosolic protein kinase C (PKC) beta-isoform immunoactivity and PKC-beta II mRNA were elevated at least twofold during 10 days of 0.1 mM CPT or 1 mM caffeine treatment. The sustained elevation in sodium-glucose symport and PKC activity observed with adenosine receptor antagonists was similar to acute (2-h) effects of the adenosine A1 agonist R(-)-N6-phenylisopropyladenosine (R-PIA, 0.1-1 microM). Moreover, cell proliferation was increased by adenosine (0.1 microM R-PIA), whereas Na-K-adenosinetriphosphatase activity was unaltered with chronic antagonist or acute adenosine treatments. Caffeine treatment may have some non-adenosine A1 receptor-mediated actions, because it slightly (30%) augmented protein kinase A activity. It is concluded that chronic exposure of proximal tubule cells to caffeine or CPT augments PKC and sodium-glucose transport but retards cell proliferation mainly via adenosine A1 receptor-mediated mechanisms.
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PMID:Upregulated renal adenosine A1 receptors augment PKC and glucose transport but inhibit proliferation. 877 86

Current responses to caffeine in single hepatocytes dissociated from adult rat liver were investigated with the conventional whole cell patch-recording configuration. Caffeine produced a sustained inward current (Icaf) with increasing conductance at a holding potential of -40 mV. The reversal potential of Icaf was close to the Cl- equilibrium potential. Icaf was not affected by the internal perfusion of 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or Cs+, whereas the Ca(2+)-activated K+ outward current elicited by A-23187 was inhibited by intracellular BAPTA or Cs+. A 1 mM 3-isobutyl-1-methylxanthine (IBMX) was about equipotent to 1 mM caffeine in inducing the current. Icaf was not modulated by the external application of N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), a cyclic nucleotide-dependent protein kinase inhibitor, or intracellular perfusion with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). It was concluded that caffeine induced an increase in membrane Cl- conductance without utilizing the rise of intracellular free Ca2+ or adenosine 3',5'-cyclic monophosphate (cAMP) and without mediating G protein, suggesting the possible existence of caffeine receptor-Cl- channel complexes on liver plasma membrane.
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PMID:Caffeine-induced chloride current in dissociated rat hepatocytes. 877 13

1. Whole-cell patch-clamp recording were employed to study facilitation of Ca2+ currents and excessive Ca2+ tail currents evoked by strong and long-lasting conditioning depolarizations in skeletal myoballs cultured from newborn rats. 2. Paired-pulse facilitation and excessive tail currents showed the same voltage dependence, becoming prominent at conditioning potentials above +30 mV. 3. Recruitment of excessive tail currents and facilitation occurred with the same time dependence (time constant (tau), approximately 200 ms to approximately 1 s), accelerating with the depolarization strength of conditioning pulses. 4. Reversal of Ca2+ current facilitation during the repolarization period between conditioning and test pulses was time- and voltage dependent. The time window of recruitment of facilitated Ca2+ currents narrowed considerably at more negative repolarization potentials (tau: approximately 10 ms at -100 mV, but approximately 1.5 at 0 mV). 5. Neither omission of internal ATP nor perfusion of the cells with the peptide inhibitor of protein kinase A (PKI) had significant effects on Ca2+ current facilitation, although internal perfusion with ATP gamma S slowly suppressed the facilitation currents by about 30%. External application of either ryanodine or caffeine under control conditions selectively and significantly suppressed the facilitated Ca2+ currents by about 30-40%. 6. We propose that facilitation of Ca2+ currents and excessive tail currents are consequences of a common mechanism linked to ryanodine receptors.
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PMID:Silent calcium channels generate excessive tail currents and facilitation of calcium currents in rat skeletal myoballs. 881 12

1. Raising the intracellular [Ca2+] for 10 s at 23 degrees C abolished depolarization-induced force responses in mechanically skinned muscle fibres of toad and rat (half-maximal effect at 10 and 23 microM, respectively), without affecting the ability of caffeine or low [Mg2+] to open the ryanodine receptor (RyR)/Ca2+ release channels. Thus, excitation-contraction coupling was lost, even though the Ca2+ release channels were still functional. Coupling could not be restored in the duration of an experiment (up to 1 h). 2. The Ca(2+)-dependent uncoupling had a Q10 > 3.5, and was three times slower at pH 5.8 than at pH 7.1. Sr2+ caused similar uncoupling at twenty times higher concentration, but Mg2+, even at 10 mM, was ineffective. Uncoupling was not noticeably affected by removal of ATP or application of protein kinase or phosphatase inhibitors. 3. Confocal laser scanning microscopy showed that the transverse tubular system was sealed in its entirety in mechanically skinned fibres and that its integrity was maintained in uncoupled fibres. Electron microscopy revealed distorted or severed triad junctions and Z-line aberrations in uncoupled fibres. 4. Only when uncoupling was induced at a relatively slow rate (e.g. over 60 s with 2.5 microM Ca2+) could it be prevented by the protease inhibitor leupeptin (1 mM). Immunostaining of Western blots showed no evidence of proteolysis of the RyR, the alpha 1-subunit of dihydropyridine receptor (DHPR) or triadin in uncoupled fibres. 5. Fibres which, whilst intact, were stimulated repeatedly by potassium depolarization with simultaneous application of 30 mM caffeine showed reduced responsiveness after skinning to depolarization but not to caffeine. Rapid release of endogenous Ca2+, or raised [Ca2+] under conditions which minimized the loss of endogenous diffusible myoplasmic molecules from the skinned fibre, caused complete uncoupling. Taken together, these results suggest that Ca(2+)-dependent uncoupling can also occur in intact fibres. 6. This Ca(2+)-dependent loss of depolarization-induced Ca2+ release may play an important feedback role in muscle by stopping Ca2+ release in localized areas where it is excessive and may be responsible for long-lasting muscle fatigue after severe exercise, as well as contributing to muscle weakness in various dystrophies.
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PMID:Raised intracellular [Ca2+] abolishes excitation-contraction coupling in skeletal muscle fibres of rat and toad. 884 31


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