Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The signal transduction pathways of the dopamine-D1 receptor were investigated in two cell types stably transfected with the human D1 receptor cDNA, rat pituitary GH4C1 cells (GH4-hD1), and mouse Ltk-fibroblast cells (L-hD1). In both GH4-hD1 and L-hD1 cell lines, stimulation of the dopamine-D1 receptor induced a marked increase in cAMP accumulation. In addition, dopamine potentiated activation of L-type voltage-dependent calcium channels in a cAMP-dependent manner in GH4-hD1 cells. However, in L-hD1 cells, dopamine increased cytosolic free calcium concentrations ([Ca++]i) by mobilization of intracellular calcium rather than by calcium influx. This effect was correlated with a dopamine-induced enhancement of phospholipase C activity in L-hD1 cells. Pretreatment (24 h) with cholera toxin (CTX) was used to maximally activate the GTP-binding protein (G protein) Gs, causing a maximal elevation of cAMP levels and uncoupling the D1 receptor from Gs. The described actions of dopamine in both cell lines were abolished by pretreatment with CTX, indicating that CTX substrates (e.g. Gs) may mediate these actions. The blockade by CTX was not due to CTX-induced elevation of cAMP, since pretreatment with forskolin or 8-bromo-cAMP to activate cAMP-dependent protein kinase did not inhibit dopamine actions nor alter basal [Ca++]i. Pretreatment (1-3 h) of L-hD1 cells with forskolin (10 microM) or 8-bromo-cAMP (5 mM) altered neither the basal activity of phospholipase C nor basal [Ca++]i in L-hD1 cells but greatly enhanced the dopamine-induced increase of phosphatidyl inositol turnover and [Ca++]i. From these results we conclude that: 1) the dopamine-D1 receptor induces multiple and cell-specific signals, including elevation of cAMP levels in both GH and L cells, cAMP-dependent activation and potentiation of opening of L-type voltage-dependent calcium channel in GH cells, and a novel phosphatidyl inositol-linked mobilization of cellular calcium in L cells; 2) coupling of the D1 receptor to these responses involves CTX-sensitive proteins, possibly Gs; and 3) acute preactivation of cAMP-dependent protein kinase can markedly enhance, rather than attenuate, certain pathways of dopamine-D1 transmembrane signaling.
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PMID:Cholera toxin-sensitive 3',5'-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A. 128 71

The complete amino acid sequence of the L-type calcium channel alpha 1 subunit from the carp (Cyprinus carpio) white skeletal muscle was deduced by cDNA cloning and sequence analysis. The open reading frame encodes 1852 amino acids (Mr 210,060). A 155-amino acid COOH-terminal sequence (after the fourth internal repeat) is evolutionarily preserved (90% homology) and may represent an important functional domain of L-type calcium channels. The photolabeled, membrane-bound, and purified carp alpha 1 subunits have masses of 211 and 190 kDa. The purified channel could not be phosphorylated by cAMP-dependent protein kinase. Two glycoproteins (alpha 2 subunits) are associated with the alpha 1 subunit and change their apparent masses from 235 and 220 kDa to 159 kDa upon reduction of disulfide bonds. Nucleic acid hybridization with alpha 2 cDNA revealed an 8.0-kilobase transcript in carp skeletal muscle. Evidence for a copurification of subunits similar in size to mammalian beta or gamma subunits was not obtained.
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PMID:Calcium channels from Cyprinus carpio skeletal muscle. 184 62

Activation of the cardiac beta-adrenergic receptor stimulates cAMP levels and activates cAMP-dependent protein kinase. The kinase phosphorylates the calcium channel and enhances thereby the availability and the number of channels that are opened during depolarization. The increased calcium influx leads then to a positive inotropic response. The calcium channel can be identified in vitro by organic calcium channel blockers, which bind stereoselectively to a high affinity, low capacity site localized in sarcolemma and junctional sarcoplasmic reticulum. This binding site has been purified from skeletal muscle microsomes. The purified receptor contains three peptides of Mr 165, 55, and 32 kDa in stoichiometric amounts. The high affinity binding sites for dihydropyridines and phenylalkylamines are localized on the 165 kDa peptide. This peptide is phosphorylated up to 2 mol/mol by cAMP-dependent protein kinase. Reconstitution of the purified receptor yields a calcium channel that has many properties of the cardiac L-type calcium channel. It is suggested that these properties are confined to a 165 kDa peptide in skeletal muscle and to a 183 kDa peptide in cardiac muscle.
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PMID:The biochemical properties of L-type calcium channels. 246 31

beta-Adrenergic stimulation of ventricular heart cells results in the enhancement of two important ion currents that regulate the plateau phase of the action potential: the delayed rectifier potassium channel current (IK) and L-type calcium channel current (ICa). The temperature dependence of beta-adrenergic modulation of these two currents was examined in patch-clamped guinea pig ventricular myocytes at various steps in the beta-receptor/cyclic AMP-dependent protein kinase pathway. External applications of isoproterenol and forskolin were used to activate the beta-receptor and the enzyme adenylate cyclase, respectively. Internal dialysis of cyclic 3',5'-adenosine monophosphate (cAMP) or the catalytic subunit of cAMP-dependent protein kinase (CS), as well as the external addition of 8-chlorphenylthio cAMP (CPT-cAMP) was applied to increase intracellular levels of cAMP and CS. Isoproterenol-mediated increases in IK, but not ICa, were found to be very temperature dependent over the range of 20-37 degrees C. At room temperature (20-22 degrees C) isoproterenol produced a large (threefold) enhancement of ICa but had no effect on IK. In contrast, at warmer temperatures (30-37 degrees C) both currents increased in the presence of this agonist and the kinetics of IK were slowed at -30 mV. A similar temperature sensitivity also existed after exposure to forskolin, CPT-cAMP, cAMP, and CS, suggesting that this temperature sensitivity of IK may arise at the channel protein level. Modulation of IK during each of these interventions was accompanied by a slowing in IK kinetics. Thus, regulation of cardiac potassium channels but not calcium channels involves a temperature-dependent step that occurs after activation of the catalytic subunit of cAMP-dependent protein kinase.
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PMID:Beta-adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents. 247 62

It is well established that the inotropic effect of beta-adrenergic agonists is mediated by the stimulation of adenylyl cyclase activity and the subsequent phosphorylation of specific proteins by cAMP-dependent protein kinase. The L-type calcium channel is believed to be one of the proteins phosphorylated; the phosphorylation of calcium channels is believed to increase calcium entry into myocytes, which is, at least in part, responsible for the positive inotropic effect. The present studies show that the cAMP-elevating effect of isoproterenol is increased as extracellular calcium is lowered and that calcium channel blockers potentiate the cAMP-elevating effect of isoproterenol in the presence in extracellular calcium. This effect is not dependent on effects on cAMP catabolism and is not specific for beta-adrenergic receptors, because the cAMP-elevating effect of forskolin is similarly affected. Measurements of adenylyl cyclase activity in cardiac membranes show that submicromolar Ca2+ concentrations directly inhibit adenylyl cyclase activity. These results demonstrate that increased entry of Ca2+ via L-type calcium channels in response to beta-adrenergic receptor stimulation acts as a negative regulator of the effect of beta receptor stimulation on adenylyl cyclase activity.
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PMID:Calcium entry via L-type calcium channels acts as a negative regulator of adenylyl cyclase activity and cyclic AMP levels in cardiac myocytes. 769 67

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

Cyclic AMP-mediated phosphorylation of calcium channel subunits was studied in vitro and in vivo in preparations from dog heart. Calcium channels in native cardiac membranes were phosphorylated by cAMP-dependent protein kinase (PKA) solubilized with digitonin and subsequently immunoprecipitated using a polyclonal antibody generated against the deduced carboxy-terminal sequence of the cardiac beta subunit. A 62 kDa protein was identified as the major PKA-substrate in the immunoprecipitates. In the intact myocardium, this putative beta subunit was found to be phosphorylated in response to cAMP elevating agents. In contrast, no phosphorylation of a protein with an electrophoretic mobility similar to the alpha 1 subunit was detected, although 1,4-dihydropyridine receptor sites were recovered in the immunoprecipitates. Thus, we suggest that PKA-mediated phosphorylation of the beta subunit is the major mechanism for beta-adrenergic regulation of cardiac L-type calcium channel activity.
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PMID:Phosphorylation of the L-type calcium channel beta subunit is involved in beta-adrenergic signal transduction in canine myocardium. 825

We investigated age-related changes in the binding sites of muscarinic acetylcholine, forskolin, adenosine 3',5'-cyclic monophosphate (cAMP), and of a voltage-dependent L-type calcium channel blocker in the gerbil brain using receptor autoradiography. [3H]Quinuclidinyl benzilate (QNB), [3H]forskolin, [3H]cAMP, and [3H]PN200-110 were used to label muscarinic receptors, adenylate cyclase, cAMP-dependent protein kinase, and L-type calcium channels, respectively. In middle-aged animals (16-month-old gerbils), [3H]QNB, [3H]PN200-110, [3H]forskolin, and [3H]cAMP binding sites were elevated in the hippocampal region compared with that of young gerbils (4 weeks old). Further, a significant elevation in [3H]forskolin binding was seen in the nucleus accumbens. In contrast, [3H]QNB, [3H]PN200-110, and [3H]forskolin binding sites were reduced in the cerebellum, neocortex and thalamus, and hypothalamus in middle-aged animals, respectively. [3H]cAMP binding was not altered in other regions except for an elevation in the hippocampus. Thus, the age-related alterations in receptor binding may proceed by different mechanisms in various brain regions. Chronic vinconate treatment partly modulated the age-related alterations in [3H]QNB, [3H]forskolin, and [3H]cAMP binding in the hippocampus, but not that of [3H]PN200-110. Vinconate also regulated the age-related changes in [3H]forskolin binding in the nucleus accumbens. These results indicate that the age-related alterations in the binding sites of muscarinic acetylcholine, forskolin, cAMP, and L-type calcium channel blocker occur in particular in the hippocampus. Further, they suggest that a novel vinca alkaloid derivative, vinconate, can partly modulate age-related changes in these binding sites.
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PMID:Effect of vinconate against regional age-related changes in the gerbil brain. 838 45

L-type calcium channels mediate long-lasting calcium currents which are modulated by protein phosphorylation. Using site-directed anti-peptide antibodies, we show that the alpha 1 subunit of the neuronal class C L-type calcium channel from rat brain exists in two size forms. The longer form, LC2, with an apparent molecular mass of 210-235 kDa was phosphorylated in vitro by cAMP-dependent protein kinase (cA-PK), but the shorter form, LC1, with an apparent molecular mass of 190-195 kDa was not a substrate for cA-PK. In contrast, LC1 and LC2 are both substrates for protein kinase C (PKC), calcium- and calmodulin-dependent protein kinase II, and cGMP-dependent protein kinase (cG-PK). The site-directed anti-peptide antibody CNC2 was produced against the COOH-terminal end of the class C L-type alpha 1 subunit as predicted by molecular cloning and sequencing of cDNA. CNC2 recognized LC2 but not LC1 by immunoblotting and immunoprecipitated only LC2 phosphorylated by either cA-PK or PKC. These results indicate that LC1 is truncated at its COOH-terminal end with respect to LC2 and that cA-PK preferentially phosphorylates sites in the COOH-terminal region of the alpha 1 subunit that are present in LC2 but not LC1. The selectivity of cA-PK for phosphorylation of the COOH-terminal region of LC2 suggests that the channel activities of the two alpha 1 subunit size forms may be differentially regulated by neurotransmitters and hormones which act through cAMP-dependent mechanisms, while both alpha 1 subunit isoforms may be modulated by PKC, cG-PK, and calcium- and calmodulin-dependent protein kinase II.
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PMID:Differential phosphorylation of two size forms of the neuronal class C L-type calcium channel alpha 1 subunit. 839 38

The molecular basis of the regulation of cardiac L-type calcium channel activity by cAMP-dependent protein kinase (cA-PK) remains unclear. Direct cA-PK-dependent phosphorylation of the bovine ventricular alpha1 subunit in vitro has been demonstrated in microsomal membranes, detergent extracts and partially purified (+)-[3H]PN 200-110 receptor preparations. Two 32P-labeled phosphopeptides, derived from cyanogen bromide cleavage, of 4.7 and 9.5 kDa were immunoprecipitated specifically by site-directed antibodies against the rabbit cardiac alpha1 subunit amino acid sequences 1602-1616 and 1681-1694, respectively, consistent with phosphorylation at the cA-PK consensus sites at Ser(1627) and Ser(1700). No phosphopeptide products consistent with phosphorylation at three other C-terminal cA-PK consensus phosphorylation sites (Ser(1575), Ser(1848) and Ser(1928)) were identified using similar procedures suggesting that these sites are poor substrates for this kinase. Ser(1627) and Ser(1700) may represent sites of cA-PK phosphorylation involved in the physiological regulation of cardiac L-type calcium channel function.
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PMID:Cyclic AMP-dependent protein kinase phosphorylates residues in the C-terminal domain of the cardiac L-type calcium channel alpha1 subunit. 866 19


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