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)

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.
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PMID:Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells. 298 84

We have determined whether the process of agonist-mediated phosphorylation of the muscarinic receptor correlates with the process of muscarinic receptor desensitization in chick cardiac tissue. Exposure of ventricular slices to the agonist carbachol under conditions previously shown to lead to large increases in muscarinic receptor phosphorylation (Kwatra, M. M., and Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432) resulted in decreased affinity of the muscarinic receptor for agonists. The agonist oxotremorine mimicked and the antagonist atropine prevented the effects of carbachol on receptor phosphorylation and agonist affinity. The time courses and concentration dependences for agonists to induce phosphorylation of the muscarinic receptor and decreases in agonist affinity were similar. Treatment of chick atria with acetylcholine under conditions which led to receptor phosphorylation resulted in decreased sensitivity of these preparations to the negative inotropic effect of carbachol. Taken together, the results support the concept that phosphorylation of cardiac muscarinic receptors may be related to the process of receptor desensitization. The mechanism by which agonists induce receptor phosphorylation was also investigated. The phosphorylated amino acids formed in response to agonists were serine and threonine. The protein kinase C activator phorbol myristate acetate had no effect on receptor phosphorylation or agonist affinity, nor did it prevent the effects of carbachol on either of these parameters. Receptor phosphorylation also was unaffected by the calmodulin antagonists W-7 and W-13, by elevation of cyclic nucleotides, and by agonists which activate other cardiac receptor systems. The results suggest that the phosphorylation of cardiac muscarinic receptors requires agonist occupancy of the receptor and/or may involve the participation of a selective protein kinase.
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PMID:Correlation of agonist-induced phosphorylation of chick heart muscarinic receptors with receptor desensitization. 368 Feb 52

High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands 3H-N-methylscopolamine (3H-NMS), 3H-quinuclidinyl benzilate (3H-QNB), and 3H-pirenzepine. 3H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, 3H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by 3H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for 3H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure. In conclusion, both M1 and M2 muscarinic receptor subtypes and an adenylate cyclase system linked to at least some of these receptors are present as intrinsic components of myelin. The possibility that some of these muscarinic receptors may be involved in regulation of phosphinositide metabolism and the protein kinase activities of myelin is considered.
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PMID:Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin. 369 57

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

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

Our previous work on atrial myocytes suggested that the effect of acetylcholine (ACh) to increase K+ conductance can be potentiated by prior loading of the sarcoplasmic reticulum (SR) with Ca2+. The present study, therefore, sought to determine whether prior exposure to isoproterenol (ISO) potentiates ACh-induced increases in K+ conductance and the underlying mechanisms. A nystatin-perforated patch whole-cell configuration was used to record from cat atrial myocytes. Voltage-clamp ramps (40 mV/s) were used to assess total membrane conductance. The experimental protocol consisted of two consecutive 30-second ACh exposures (ACh1 and ACh2) separated by a 6-minute recovery period in ACh-free solution. In general, experimental interventions, such as exposure to ISO, were imposed during the period between ACh1 and ACh2 to determine their effects on the response to ACh2 in relation to ACh1. Under control conditions, K+ conductances induced by ACh1 and ACh2 were not different from one another with or without activation of L-type Ca2+ current (ICa,L) during the recovery period. When 1 mumol/L ISO plus ICa,L activation was imposed during the recovery period, ACh2 induced a significantly larger increase in K+ conductance than ACh1. The ACh2-induced K+ current potentiated by ISO was time independent and selectively blocked by 10 mumol/L glibenclamide and therefore identified as ATP-sensitive K+ current (IK,ATP). The effect of ISO to induce ACh2-activated IK,ATP was mimicked by 1 mumol/L forskolin or 200 mumol/L 8-(4-chlorophenylthio)-cAMP, but not by 0.5 mumol/L BAY K 8644, and was selectively abolished by (1) 5 mumol/L thapsigargin or 1 mumol/L ryanodine, agents that prevent accumulation of SR Ca2+, (2) inhibition of L-type Ca2+ current (ICa,L) by 1 mumol/L nisoldipine or zero external Ca2+, (3) 50 mumol/L Rp-cAMPs, an inhibitor of cAMP-dependent protein kinase A, or (4) 2 mumol/L propranolol. Atropine (1 mumol/L) abolished all ACh-induced currents. Moreover, ACh2-activated IK,ATP was selectively blocked by 0.2 mumol/L pirenzepine, an M1 muscarinic receptor antagonist, or 0.1 mumol/L calphostin C, a selective inhibitor of protein kinase C. AFDX116 (100 mumol/L), an M2 muscarinic receptor antagonist, blocked the conventional ACh-activated K+ current and revealed ACh2-activated IK,ATP. These results indicate that prior exposure to ISO potentiates ACh-induced increases in K+ current via ACh-activated IK,ATP.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:beta-Adrenergic stimulation induces acetylcholine to activate ATP-sensitive K+ current in cat atrial myocytes. 764 26

The effect of ethanol on muscarinic receptor-stimulated formation of inositol 1,4,5-trisphosphate was studied in human neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced a biphasic increase of inositol 1,4,5-triphosphate with an initial peak after 10 sec declining to a plateau phase of elevation above basal levels, which was sustained for at least 5 min in the presence of agonist. The peak, but not the plateau phase, was concentration-dependently decreased by exposure to ethanol. Maximal inhibition was obtained within 30 sec of exposure to ethanol. Ethanol caused an increase in the EC50 value of carbachol for the initial rate of inositol 1,4,5-trisphosphate formation, measured after 10 sec of stimulation, from 98 microM in the absence to 196 microM in the presence of 100 mM ethanol. The potencies of pirenzepine and hexahydro-sila-difenidol hydrochloride for inhibiting [3H]quinuclidinyl benzilate binding and inositol 1,4,5-trisphosphate formation suggest that both phases are mediated via the muscarinic M1 receptor. Phorbol 12-myristate 13-acetate inhibited both phases of inositol 1,4,5-trisphosphate formation, whereas okadaic acid and modulators of cAMP-dependent protein kinase were without any effect. There was no inhibitory effect of ethanol when protein kinase C was inhibited by H7 and calphostin C, indicating that the ethanol effect is dependent on protein kinase C activity.
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PMID:Ethanol inhibits the peak of muscarinic receptor-stimulated formation of inositol 1,4,5-trisphosphate in neuroblastoma SH-SY5Y cells. 766 67

The inhibitory pathway of cardiac cAMP-dependent protein kinase regulated Cl- conductance was investigated using the whole-cell configuration of patch-clamp techniques in single guinea pig ventricular myocytes. Pertussis toxin-sensitive G proteins (Gi), mediating the signal transductions between muscarinic receptors and adenylate cyclase, have a substantial tonic activity even in the absence of muscarinic receptor modulators. Muscarinic agonists or antagonists (like atropine) either increase or decrease this basal activity of Gi by altering the proportion of active and inactive forms of the receptors. Similar to L-type Ca-channel currents, the Cl- conductance showed a transient over-recovery upon cessation of brief muscarinic receptor stimulation by carbachol (CCh) (rebound). Atropine alone enhanced the Cl- conductance elicited by low concentrations of Iso (reverse agonist). After washout of atropine, the over-suppression of the conductance was observed as a mirror image of CCh-induced rebound (reverse rebound). Both types of rebound became prominent when cell dialysis with pipette solutions containing 100 microM GTP was minimized with high-resistance pipettes. Endogenous GTP is therefore an intracellular modulator, and not simply a mediator, of Gi-dependent signal transduction.
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PMID:Inhibitory pathway of cardiac PKA-dependent Cl- conductance via pertussis toxin-sensitive G proteins. 775 28

In many tissues, inwardly rectifying K channels are coupled to seven-helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.
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PMID:A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes. 776 82

Activation of muscarinic receptors has been shown to inhibit L-type calcium conductances by mechanisms sensitive to pertussis toxin (PTX). In this study we show that agonist stimulation of the m4 muscarinic receptor leads to an increase in an L-type calcium conductance in the AtT-20 pituitary cell line, by a PTX-sensitive mechanism. The amplitude of the dihydropyridine (DHP)-sensitive or L-type calcium current was increased by acetylcholine (ACh), with no shift in the voltage dependence. This action of ACh was completely inhibited by PTX pre-treatment. Forskolin, cAMP and phorbol 12,13-dibutyrate reduced, while RpcAMPs, an inhibitor of cAMP-dependent protein kinase (PKA), increased the L-type calcium conductance. We propose that the m4 muscarinic receptor activates the L-type calcium channel by inhibition of adenylyl cyclase resulting in reduced cAMP levels and, hence, reduced PKA activity. This novel increase in calcium current via the m4 muscarinic receptor appears to reflect the coupling with an L-type channel of the D class, due to the sensitivity of the L-type calcium conductance to both DHPs and omega-conotoxin, and, thus, is distinct from the skeletal muscle and cardiac L-type channels of the C class previously studied.
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PMID:Enhancement of an L-type calcium current in AtT-20 cells; a novel effect of the m4 muscarinic receptor. 779 45


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