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)

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

1. Wide-tipped, low-resistance (approximately 1 M omega) pipettes were used to record the whole-cell Cl- current activated by cAMP-dependent protein kinase (PKA) in guinea-pig ventricular myocytes internally dialysed with or without GTP. Without GTP in the pipette, the response to 1 microM-isoprenaline declined with time and eventually disappeared, usually within approximately 20 min of rupturing the membrane and beginning cell dialysis. 2. This rundown of the isoprenaline response occurred more quickly with wider, lower-resistance pipette tips. 3. After complete rundown of the isoprenaline response, histamine (10 microM), another agonist known to elicit the Cl- current, also had no effect, but extracellular forskolin (1 microM) or intrapipette cAMP (1 mM) could still readily elicit the Cl- current. 4. In contrast, with 100 microM-GTP in the pipette, the response to 1 microM-isoprenaline was well maintained for periods greater than 20 min. But, if GTP was then withdrawn from the pipette, a rundown of the isoprenaline response was seen comparable to that in the experiments begun with GTP-free pipette solution. Moreover, in experiments begun without pipette GTP, the addition of 100 microM-GTP to the pipette solution, after the response to isoprenaline had disappeared, was able to restore that Cl- current response. 5. With GTP in the pipette, the forskolin-induced Cl- current could be suppressed by concurrent exposure to carbachol (10 microM). That inhibition was not seen in myocytes pretreated with pertussis toxin. In untreated myocytes dialysed with GTP-free pipette solution, after disappearance of the isoprenaline response, the muscarinic receptor-mediated inhibition was itself abolished. 6. We confirm that both beta-adrenoceptor-mediated activation of the Cl- current by isoprenaline, and muscarinic receptor-mediated inhibition of the forskolin-induced Cl- current, are mediated by G proteins, and conclude that the disappearance of both receptor-mediated responses during whole-cell recording with GTP-free pipette solution reflects the fall of cellular [GTP] below the level required to maintain G protein-dependent signal transduction.
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PMID:Pipette GTP is essential for receptor-mediated regulation of Cl- current in dialysed myocytes from guinea-pig ventricle. 133 50

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.
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PMID:Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance. 137 58

1. Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. The effect of internal perfusion with the hydrolysis-resistant GTP analogue, GppNHp (5'guanylylimidodiphosphate), on basal ICa and ICa stimulated with forskolin or isoprenaline was examined to gain insight into the role of G proteins in ICa regulation. 2. Without added guanine nucleotides, isoprenaline stimulated ICa approximately 14-fold with an EC50 of 0.09 microM. Forskolin stimulated ICa approximately 10-fold with an EC50 of 0.30 microM. 3. Internal 30 microM-GppNHp produced an approximately 80% decrease in ICa elevated by 0.3 microM-isoprenaline or 3 microM-forskolin. The inhibition of isoprenaline stimulation was due to a decrease in the maximal stimulation from approximately 14-fold to approximately 14-fold without a significant change in the EC50. In contrast, the reduction in forskolin stimulation was due to a 22-fold increase in the EC50 to 11.4 microM, with little change in maximal stimulation. 4. The inhibition of stimulated ICa by GppNHp is likely to be mediated by a G protein, because the effects of GppNHp are irreversible, and are blocked by excess GTP. ICa is affected similarly by GppNHp and by ACh. This suggests that GppNHp activates the same G protein that is normally activated by ACh, but activation by GppNHp occurs in the absence of agonist occupation of the muscarinic receptor. 5. The increase in the EC50 for forskolin produced by internal GppNHp was reversed by exposure to isoprenaline, which itself did not affect ICa amplitude. On average, exposure to isoprenaline in the presence of GppNHp caused an irreversible 81-fold decrease in the EC50 for forskolin to 0.14 microM. Stimulation of ICa by forskolin after internal GppNHp and exposure to isoprenaline was completely blocked by the protein kinase A inhibitor PKI(5-22). 6. These effects do not involve the phospholipase C system, because they are not mimicked by phorbol esters or internal inositol 1,4,5-trisphosphate (IP3) and are not blocked by bromophenacyl bromide or neomycin. 7. Direct effects of G proteins on ICa were not evident, because internal perfusion with PKI(5-22) completely inhibited isoprenaline- or forskolin-stimulated increases in ICa, and neither ACh nor internal GppNHp (30-500 microM) affected basal ICa or ICa elevated by internally perfused cyclic AMP. 8. These results suggest that the predominant site of action of the inhibitory G protein activated by either GppNHp or ACh is adenylyl cyclase. Furthermore, the internally perfused frog cardiomyocytes may provide a useful approach for probing the detailed interactions of G proteins, forskolin, and adenylyl cyclase in an intact cell.
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PMID:Regulation of Ca2+ current in frog ventricular cardiomyocytes by 5'-guanylylimidodiphosphate and acetylcholine. 165 25

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Muscarinic receptor stimulation increases inositol-phospholipid metabolism and inhibits cyclic AMP accumulation in PC12 cells. 254 58

The preceding discussion documents the diverse ways in which monoclonal antibodies have contributed to neuroscience research. They provide highly specific reagents to membrane-associated proteins, such as pumps, channels, receptors, and cell-adhesion molecules, that are useful for purifying these proteins, studying their structures at high resolution, and mapping their distributions. In many cases, the specific reagents were obtained using only partially purified antigens. Monoclonal antibodies to cytoskeletal proteins, organelles, and protein kinases have revealed that specific molecules are concentrated in anatomically distinct regions of the cell. A protein kinase has been shown to be a major postsynaptic constituent in many synapses. Individual proteins, such as actin, tubulin, and calmodulin appear to have different antigenic epitopes shielded in different parts of the cell. Monoclonal antibodies have provided a diversity of cell-type-specific reagents in both vertebrate and invertebrate nervous systems. They seem likely to be useful in identifying functionally related subpopulations of neurons and describing neural cell lineages. They will also serve to identify molecules that are important in regulating cell migration in the cerebellum, in marking cell position in the retina, and directing axon growth. This review also documents many purposes for which monoclonal antibodies are poorly suited or must be used with caution: A monoclonal antibody to a protein does not always reveal every place where that molecule is located. Pre- or post-translational microheterogeneity can expose different epitopes on the protein, such as may occur on the Na+-channel. Other proteins within the cell may shield antigenic sites on proteins such as calmodulin. Monoclonal antibodies can bind to epitopes on unrelated molecules (Nigg et al 1982, Lane & Koprowski 1982). This is revealed in some cases as multiple bands on immunoblots. Some cross-reactivity, however, may have a functional basis. For example, structural homology is clearly the basis for the antigenic epitopes that are shared among the five classes of intermediate filaments (Pruss et al 1981). The epitope that appears to be shared between the muscarinic and alpha 1-adrenergic receptors may be conserved because the two receptors modulate common effectors. The cross-reactivity between these receptors was only recognized because very specific and sensitive assays exist for each. It is quite possible that these same antibodies also bind sites on many other types of receptors. Mapping the distribution of this epitope may therefore have little relationship to the actual distribution of the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Applications of monoclonal antibodies to neuroscience research. 258 Apr 71

It was found that serotonin relaxed desensitization and decreased acetylcholine-induced potassium current. Effects of serotonin were connected with serotonin-activated adenylate cyclase, adenosine 3':5'-cyclic monophosphate (cAMP) and phosphorylation of muscarinic receptor proteins by cAMP-dependent protein kinase.
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PMID:[Serotonin modulation of the muscarinic cholinoreceptor status of the neurons in the mollusk Planorbarius corneus]. 260 68

Recently we showed that the chick heart muscarinic acetylcholine receptor is a phosphoprotein in intact cells and that treatment with agonists results in a striking increase in receptor phosphorylation [J. Biol. Chem. 261:12429-12432 (1986)]. Furthermore, we showed that the agonist-induced increase in the phosphorylation of chick heart muscarinic receptors correlates with receptor desensitization [J. Biol. Chem. 262:16314-16321 (1987)]. We have now extended studies of receptor phosphorylation to mammalian cardiac muscarinic receptors, in order to test the concept that phosphorylation is of general importance in the regulation of muscarinic receptor function. We have determined that, in intact porcine atria, M2 muscarinic receptors are phosphoproteins and that treatment with the agonist carbachol markedly increases receptor phosphorylation, to 4-6 mol of phosphate/mol of protein. Phosphorylation occurs on serine and threonine residues. Activation of either protein kinase C or cAMP-dependent protein kinase did not mimic the effect of agonists on receptor phosphorylation. These results are very similar to those seen with the chick heart muscarinic receptors. To determine whether the porcine and the chick cardiac muscarinic receptors represent similar or different proteins, we undertook detailed pharmacological studies and, in addition, prepared peptide maps of purified muscarinic receptors from chick heart and porcine atria. Our data show that there are marked differences in the pharmacological properties of the chick and the porcine cardiac muscarinic receptors. The peptide maps of the porcine and chick heart muscarinic receptors are also different, suggesting that muscarinic receptors in chick and porcine cardiac cells differ in their primary structure. Taken together, the data show that porcine and chick cardiac muscarinic receptors possess pharmacological and structural differences, but both receptors undergo agonist-mediated phosphorylation in intact cardiac cells. These data support the possibility that receptor phosphorylation may be of general importance in the regulation of muscarinic receptors.
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PMID:The porcine heart M2 muscarinic receptor: agonist-induced phosphorylation and comparison of properties with the chick heart receptor. 272 67

Previous studies have demonstrated that muscarinic cholinergic receptors (mAChR) become markedly phosphorylated when intact cardiac cells are stimulated with a muscarinic agonist. This process appears to be related to the process of receptor desensitization. However, the mechanism of agonist-induced phosphorylation of mAChR is not known. In situ phosphorylation studies suggested that agonist-induced phosphorylation of mAChR may involve the participation of a receptor-specific kinase and/or require agonist occupancy. These observations regarding phosphorylation and desensitization of mAChR are similar to observations made for beta-adrenergic receptors. Recent studies have indicated that homologous desensitization of beta-adrenergic receptors may be due to the phosphorylation of these receptors by a novel protein kinase that only recognizes the agonist-occupied form of the receptors. As muscarinic receptors are structurally homologous to beta-adrenergic receptors, we have initiated studies to identify the protein kinase responsible for the phosphorylation of muscarinic receptors by determining whether the chick heart muscarinic receptor would serve as a substrate for the beta-adrenergic receptor kinase (beta-AR kinase). We report that the purified and reconstituted chick heart muscarinic receptor serves as an excellent substrate in vitro for the beta-AR kinase. Phosphorylation of mAChR receptors by the beta-AR kinase was only observed in the presence of a muscarinic receptor agonist and was prevented in the presence of antagonist. Both the extent of phosphorylation (3-4 mol of P/mol of receptor) and the phosphoamino acid composition of the mAChR after incubation in vitro with beta-AR kinase were similar to the characteristics of agonist-induced phosphorylation of mAChR in situ.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation of chick heart muscarinic cholinergic receptors by the beta-adrenergic receptor kinase. 276 1

The effect of TSH, carbachol (CC), and ATP on intracellular calcium concentration ([Ca2+]i) in primary cultures of dog thyroid cells was examined using the fluorescent Ca2+ indicator fura-2. TSH caused an increase in [Ca2+]i at 37 C, but not 22 C, while it increased cAMP formation in these cells at both 22 and 37 C. CC and ATP increased [Ca2+]i at both 22 and 37 C. The CC-induced increase in [Ca2+]i was under muscarinic receptor control, and it was biphasic, with an initial spike followed by a sustained increase at a lower level. TSH and ATP were weaker agonists compared to CC, since maximal doses of TSH (100-500 mU/ml) and ATP (100-500 microM) increased [Ca2+]i by 40-70% over basal levels, compared to a 2- to 4-fold increase in [Ca2+] induced by maximal doses of CC (10-50 microM). The TSH-induced increase in [Ca2+]i was transient, returning to basal levels within 1-2 min after application of the agonist. All three agents were able to transiently increase [Ca2+]i to be internal stores. In the presence of the inorganic Ca2+ channel blockers La3+, Ni2+, and Co2+, the peak [Ca2+]i change was little affected, while the persistent response to CC and ATP was blocked, indicating dependence of this phase on influx of Ca2+. Paradoxically, these channel blockers abolished the effect of TSH on [Ca2+]i. TSH stimulation of cAMP formation was also inhibited 80-90% by these blockers, but not in Ca2+-free/EGTA buffer. These results suggest that the Ca2+ channel blockers may have actions in addition to inhibition of Ca2+ entry in these cells. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl] specifically blocked both the initial and sustained increase induced by CC, while having no effect on ATP or TSH-induced [Ca2+]i, suggesting that TMB-8 may not be a general antagonist of Ca2+ mobilization. Activators of protein kinase-C, such as phorbol esters or an analog of diacylglycerol, inhibited the [Ca2+]i rise induced by all the three agonists used, indicating a regulatory role of protein kinase-C activation on [Ca2+]i in these cells. In FRTL-5 cells, [Ca2+]i was also increased by TSH and ATP, but not by CC. ATP, however, was a more effective agonist than in dog thyroid cells, while TSH increased [Ca2+]i by a similar magnitude in both cell types. The results of the present study demonstrate that TSH, albeit of lesser potency than CC, increases [Ca2+]i by causing intracellular Ca2+ mobilization in cultured dog thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Intracellular Ca2+ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells. 279 72


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