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. Whole-cell patch-clamp technique was used to study the beta-adrenergic and cholinergic regulation of the inwardly rectifying K+ conductance (gK1) in isolated guinea-pig ventricular myocytes. 2. In Cl(-)-free solutions or in the presence of 9-anthracenecarboxylic acid or Co2+, bath-applied isoprenaline (Iso) partially inhibited the steady-state whole-cell conductance (gss) calculated from the steady-state current (Iss)-voltage (Iss-V) curve at membrane voltages (Vm) negative to the equilibrium potential for potassium (EK). Iss was also inhibited at Vm positive to EK when the extracellular [K+] was 20 mM. The Iso-sensitive component of gss exhibited the characteristics of the inwardly rectifying K+ conductance (gK1). 3. The Iso-induced inhibition of gK1 was reversible, concentration dependent, blocked by propranolol, mimicked by both forskolin and dibutyryl cAMP, and prevented by including a cAMP-dependent protein kinase (PKA) inhibitor in the pipette solution. These findings suggest that PKA mediates the Iso-induced inhibition of gK1. 4. The apparent dissociation constant (KD) for the concentration dependence of Iso-induced inhibition was 0.035 microM and the Hill coefficient was approximately 1.0. A maximal Iso concentration (1 microM) inhibited gK1 by 40 +/- 4.1% (mean +/- S.E.M.; n = 13). 5. Bath application of acetylcholine (ACh, 0.1 microM or more) antagonized the Iso-induced (1 microM) inhibition of gK1; [ACh] > 1.0 microM antagonized 88 +/- 2.1% (n = 10) of the inhibition. ACh increased the KD for Iso to inhibit Iso-sensitive gK1 and also reduced the maximal Iso-induced inhibition. 6. ACh-induced antagonism could be abolished by pre-incubating myocytes with pertussis toxin (PTX), suggesting that a muscarinic receptor-coupled, PTX-sensitive G protein, Gi, is involved. 7. ACh (10 microM) also antagonized approximately 70% of the dibutyryl cyclic AMP (1 mM)-induced inhibition of gK1 (n = 3), suggesting that the ACh-induced antagonism involves more than simply inhibiting the Iso-mediated activation of adenylyl cyclase via the activated Gi. 8. Intracellularly applied okadaic acid (OkA, 1 microM) did not alter gK1 (control = 134 +/- 5.1 nS vs. OkA = 136 +/- 6.1 nS), but the Iso-induced decrease in gK1 was less (P < 0.001) with OkA present (42.1 +/- 2.4 nS, n = 5) than when absent (54.0 +/- 2.2 nS, n = 10). However, ACh (10 microM) failed to antagonize Iso-induced inhibition with OkA present, suggesting involvement of a protein phosphatase.
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PMID:beta-adrenergic and cholinergic modulation of the inwardly rectifying K+ current in guinea-pig ventricular myocytes. 747 26

The two-microelectrode voltage-clamp technique was used to monitor K+ channel activity in Xenopus oocyte follicular cells, which are electrically coupled to the oocyte itself by gap junctions. Endogenous vasodilators such as calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), prostaglandin E2 (PGE2) and adenosine activate glibenclamide-ATP-sensitive K+ (KATP) channels in Xenopus oocyte follicular cells. The mechanism of action of CGRP was studied in detail. CGRP effects undergo a rapid desensitization. CGRP acts via CGRPI receptors. Its effects are antagonised by the amino-truncated CGRP analog hCGRP(8-37). The second messenger for CGRP activation of KATP channels is cAMP. Phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine enhances the CGRP response while adenyl cyclase inhibition by either 2',5'-dideoxyadenosine or progesterone nearly completely depresses the CGRP response. Vasoconstrictors such as ACh and angiotensin II also have receptors in follicular cells. ACh strongly inhibits the CGRP activation of K+ channels as it inhibits the activation of KATP channels by P1060, but angiotensin II does not. It is concluded that as in vascular smooth muscle cells, CGRP and probably other hyperpolarizing vasodilators open KATP channels in follicular cells by protein kinase A activation.
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PMID:CGRP-induced activation of KATP channels in follicular Xenopus oocytes. 753 Aug 40

In guinea pig ventricle, the protein kinase A-regulated Cl- current (ICl) is conducted by an alternatively spliced isoform of the cystic fibrosis transmembrane conductance regulator. We studied muscarinic regulation of this current using the whole-cell configuration of the patch-clamp technique. Acetylcholine (ACh) antagonized activation of ICl activated by 1 microM isoproterenol (ISO) in a concentration-dependent manner. The concentration of ACh that produced a half-maximal effect (K1/2) was 36 nM, the slope factor was 1.1, and the relative magnitude of the Cl- conductance at maximally effective concentrations of ACh (Gmin) was 21% of that observed in the presence of ISO alone. In the presence of 100 nM atropine, a competitive antagonist at the muscarinic receptor, the K1/2 value for ACh inhibition of ICl was increased to 4.3 microM, but the slope factor and Gmin were not affected, which indicated that the dissociation constant (KB) for atropine was < 1 nM. ACh-induced inhibition of the ISO-activated ICl was also blocked by the quaternary ammonium compound tetraethylammonium (TEA). Like atropine, TEA increased the K1/2 value for ACh inhibition of ICl without affecting the slope factor or Gmin. Schild analysis confirmed that TEA is also a competitive antagonist at the muscarinic receptor, with a KB value of 137 microM. However, tetramethylammonium (TMA), a structurally related compound, acted as an agonist at the muscarinic receptor. TMA inhibited ICl activated by 1 microM ISO with a K1/2 value of 342 microM, a slope factor of 0.87 and a Gmin value of 17%. Increasing the concentration of ISO shifted the K1/2 value for both ACh and TMA inhibition of ICl to higher concentrations and increased Gmin, without significantly affecting the slope factor. These results indicate that muscarinic regulation of ICl depends on the level of beta adrenergic stimulation in a functionally uncompetitive manner. They also suggest that TMA acts like ACh, a full agonist at the muscarinic receptor. Furthermore, we conclude that quaternary ammonium compounds, which are often used as ion substitutes and direct ion channel blockers, should be used with caution because of the significant and diverse effects they exert at muscarinic receptors.
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PMID:Muscarinic regulation of the cardiac CFTR Cl- current by quaternary ammonium compounds. 753 45

Acetylcholine receptors at the neuromuscular junction of innervated vertebrate muscle (called Rs AChRs) have a stable degradation rate (t1/2 approximately 8-12 days) which accelerates after denervation to a half-life of approximately 3 days, but can be restabilized by reinnervation or by cAMP. We examined the mechanism by which cAMP regulates the Rs degradation rate. When dibutyryl cAMP (DB-cAMP) was applied to denervated mouse diaphragms in organ culture, it stabilized the accelerated degradation rate of the Rs. We found that this stabilization is reversible upon removal of the DB-cAMP, is cAMP specific and is mediated by intracellular cAMP. A major observation of this study is that the cAMP-induced stabilization of Rs AChRs is via protein kinase A (PKA), since H89, a PKA inhibitor, blocked the DB-cAMP induced stabilization of Rs, and H85, an analog of H89, which does not inhibit PKA but does inhibit other kinases as efficiently as H89, did not prevent the DB-cAMP-induced stabilization of Rs degradation. These results suggest that the cAMP messenger system via a PKA-dependent pathway could be among the mechanisms whereby the nerve regulates AChR degradation.
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PMID:Protein kinase A regulates the degradation rate of Rs acetylcholine receptors. 755 4

In bovine adrenal zona fasciculata (AZF) cells, angiotensin II (AII) may stimulate depolarization-dependent Ca2+ entry and cortisol secretion through inhibition of a novel potassium channel (IAC), which appears to set the resting potential of these cells. Aspects of the signaling pathway, which couples AII receptors to membrane depolarization and secretion, were characterized in patch clamp and membrane potential recordings and in secretion studies. AII-mediated inhibition of IAC, membrane depolarization, and cortisol secretion were all blocked by the AII type I (AT1) receptor antagonist losartan. These responses were unaffected by the AT2 antagonist PD123319. Inhibition of IAC by AII was prevented by intracellular application of guanosine 5'-O-2-(thio)-diphosphate but was not affected by pre-incubation of cells with pertussis toxin. Although mediated through an AT1 receptor, several lines of evidence indicated that AII inhibition of IAC occurred through an unusual phospholipase C (PLC)-independent pathway. Acetylcholine, which activates PLC in AZF cells, did not inhibit IAC. Neither the PLC antagonist neomycin nor PLC-generated second messengers prevented IAC expression or mimicked the inhibition of this current by AII. IAC expression and inhibition by AII were insensitive to variations in intracellular or extracellular Ca2+ concentration. AII-mediated inhibition of IAC was markedly reduced by the non-hydrolyzable ATP analog adenosine 5'-(beta, gamma-imino)triphosphate and by the non-selective protein kinase inhibitor staurosporine. The protein phosphatase antagonist okadaic acid reversibly inhibited IAC in whole cell recordings. These findings indicate that AII-stimulated effects on IAC current, membrane voltage, and cortisol secretion are linked through a common AT1 receptor. Inhibition of IAC in AZF cells appears to occur through a novel signaling pathway, which may include a losartan-sensitive AT1 receptor coupled through a pertussis-insensitive G protein to a staurosporine-sensitive protein kinase. Apparently, the mechanism linking AT1 receptors to IAC inhibition and Ca2+ influx in adrenocortical cells is separate from that involving inositol trisphosphate-stimulated Ca2+ release from intracellular stores. AII-stimulated cortisol secretion may occur through distinct parallel signaling pathways.
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PMID:Losartan-sensitive AII receptors linked to depolarization-dependent cortisol secretion through a novel signaling pathway. 767 18

Acetylcholine (Ach) activates the muscarinic K+ current in atrial cells via the inhibitory GTP binding protein. After activation, the whole-cell K+ current decreases rapidly (rapid desensitization) to approximately half of the initial current within approximately 20 seconds. The mechanism of this rapid desensitization was investigated in adult rat and guinea pig atrial cells. Whole-cell voltage-clamp and patch-clamp techniques were used to study the K+ current. In voltage-clamped whole cells, ACh activated a K+ current that desensitized rapidly during the initial approximately 20 seconds followed by a slower decrease over several minutes. The rapid K+ current desensitization (a rapid decrease in channel open probability) was also observed at the single-channel level in cell-attached patches and was associated with a progressive shortening of the channel open time and prolongation of the closed time. These changes in channel current and kinetics were abolished by removal of the cytoplasm (by forming inside-out patches) and were partially inhibited by phosphatase inhibitors, suggesting an involvement of cytosolic phosphatase(s) in K+ current desensitization. In inside-out patches with ACh in the pipette and GTP in the bath, the open time of muscarinic K+ channels and channel open probability were increased by 1 mM Mg(2+)-ATP (but not by the nonhydrolyzable analogue, adenylylimidodiphosphate) and decreased by alkaline phosphatase. These results suggest that the rapid K+ current desensitization in adult rat or guinea pig atrial cells is produced by changes in the gating kinetics of the K+ channel, possibly mediated via membrane-associated protein kinase and cytosolic phosphatase(s).
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PMID:Mechanism of rapid desensitization of muscarinic K+ current in adult rat and guinea pig atrial cells. 768 54

The actions of angiotensin II (ANG II) were examined in the spontaneously active cells isolated from the rabbit sinoatrial node, using the nystatin-permeabilized, whole cell, patch-clamp method. At 30 nM, ANG II significantly lowered the spontaneous firing rate of the action potentials from 212 +/- 21 to 172 +/- 32 beats/min, with a concomitant reduction in the action potential amplitude. The voltage-clamp experiments showed that ANG II inhibited the L-type Ca2+ current (ICa) with a dissociation constant (Kd) of approximately 4 nM and a maximal inhibition of 30%. The inhibition was blocked by an AT1-receptor antagonist CV11974. Acetylcholine (ACh) at 10 microM reduced the ICa by 42 +/- 12%, and ANG II did not cause any further inhibition in the presence of ACh. At 100 nM, ANG II reduced the ICa by only 12% in the presence of 2 microM isoproterenol, and a similar inhibition was observed with 0.1 microM ACh. ANG II did not affect the dibutyryl adenosine 3',5'-cyclic monophosphate-stimulated ICa. Protein kinase C activator 12-O-tetra-decanoylphorbol-13-acetate did not mimic ANG II in the effects on ICa, and preincubation of the cells with calphostin C, a protein kinase C inhibitor, did not attenuate the ANG II effect. ANG II exerts a negative chronotropic effect in the pacemaker cells as its direct action through a pathway involving adenosine 3',5'-cyclic monophosphate-dependent protein kinase.
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PMID:Angiotensin II inhibition of L-type Ca2+ current in sinoatrial node cells of rabbits. 790 Aug 59

The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up of four types of subunits in the stoichiometry alpha 2 beta gamma delta. These subunits have been shown to be differentially phosphorylated by cAMP-dependent protein kinase (PKA) protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that phosphorylation of the AChR in vitro and in vivo regulates the rate of desensitization of the receptor. In this study we have used site-specific mutagenesis and patch-clamp techniques to examine the role of phosphorylation in the regulation of desensitization of the AChR expressed in Xenopus oocytes Expression of wild-type AChR in Xenopus oocytes results in the constitutive phosphorylation of the AChR on the gamma and delta subunits. This phosphorylation is apparently due to the high basal level of PKA in oocytes since a specific peptide inhibitor of PKA completely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significantly enhanced by forskolin or cAMP analogs or by coexpression with the catalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichiometry. Using site-specific mutagenesis, the sites of phosphorylation were determined to be serines 353 and 354 on the gamma subunit and serines 361 and 362 on the delta subunit. To examine the functional properties of wild-type and mutant receptors lacking phosphorylation sites, we used patch-clamp techniques to measure the responses of out-side-out patches to repetitive pulses of ACh using a rapid perfusion system. Wild-type and mutant receptors showed rapid concentration-dependent activation and desensitization to applied agonist. The time constant of desensitization of ensemble mean currents ranged from several hundred milliseconds at low ACh concentrations to 100-200 msec at saturating concentrations. The desensitization time constants for mutant receptors lacking all phosphorylation sites were significantly slower than wild-type phosphorylated receptors at all concentrations of ACh tested. In addition, mutant receptors that had the serine residues changed to glutamate residues in order to mimic the negative charge of the phosphorylated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These results provide further support that phosphorylation of the nicotinic ACh receptor regulates rate of desensitization.
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PMID:Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes. 802 70

Although neuronal nicotinic ACh receptors (nAChR) play a key role in synaptic transmission and information transfer in the nervous system, little is known about the molecular mechanisms that govern the expression of the multiple subunits that form the receptors and determine their functional properties. Using electrophysiological and molecular biological approaches, we have investigated the NGF-mediated regulation of nAChR expression in rat pheochromocytoma (PC12) cells and protein kinase A (PKA)-deficient PC12 cells. We report that NGF treatment increases steady state levels of mRNA encoding the alpha 3, alpha 5, alpha 7, beta 2, and beta 4 subunits, increases the occurrence of ACh-induced single-channel activity in excised patches, and increases ACh-induced macroscopic current density, all by mechanisms independent of PKA activity.
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PMID:Nerve growth factor increases nicotinic ACh receptor gene expression and current density in wild-type and protein kinase A-deficient PC12 cells. 812 Jun 17

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.
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PMID:Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity. 813 39


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