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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanisms by which guanosine 3',5'-cyclic monophosphate (cGMP) modulates the contraction induced by ATP were investigated in small mesenteric resistance arteries of the rat. The nitric oxide donors 3-morpholinosydnonimine (SIN-1, 10 microM) and sodium nitroprusside (SNP, 10 microM) increased cGMP but not adenosine 3',5'-cyclic monophosphate (cAMP) content of the tissue. SIN-1, SNP, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 100 microM) inhibited the myosin light chain phosphorylation and the contractile response to ATP. Both effects were completely reversed by the selective inhibitor of cGMP protein kinase, Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (30 microM). The sensitivity to Ca2+ of arteries permeabilized with Staphylococcus aureus alpha-toxin (4,000 hemolytic units/ml) was not affected by 8-BrcGMP. The two nitric oxide donors and 8-BrcGMP decreased the rise in intracellular Ca2+ induced by ATP. The vasodilator agents abolished the contractile response to the exogenous calcium in vessels that were exposed to 3 mM ATP after depletion of intracellular Ca2+ stores. Thapsigargin (1 microM), an inhibitor of the sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase, reversed the inhibitory effect of the vasodilator agents when the contraction induced by ATP was elicited in the presence of the Ca2+ entry blocker nitrendipine (1 microM) or in Ca(2+)-free medium. These results show that cGMP inhibits ATP-induced contraction by decreasing intracellular Ca2+ concentration in small resistance arteries. They indicate that this effect results from decreased Ca2+ influx and enhanced Ca2+ sequestration through a thapsigargin-sensitive pump via activation of a cGMP protein kinase.
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PMID:Effects of cGMP on calcium handling in ATP-stimulated rat resistance arteries. 790 Aug 76

Rabbit portal veins were permeabilized using Staphylococcus aureus alpha-toxin, and adenosinetriphosphatase (ATPase) was measured as the formation of [3H]ADP, [3H]AMP, and [3H]adenosine from [3H]ATP in the solution bathing the muscle. The resting ATPase (1.96 +/- 0.15 mM/min, n = 13) is approximately 5-10 times higher than that measured in Triton X-100-permeabilized muscles (0.28 +/- 0.01 mM/min, n = 4), with nucleotide accumulating as ADP, AMP, and adenosine. The ATPase activity is also seen when the intact muscle is incubated in a Krebs solution containing 1 mM MgATP (2.76 +/- 0.10 mM/min, n = 73). This suggests that it is due primarily to an ecto-ATPase. The ectoenzyme is capable of hydrolyzing both ATP and ADP, and in both cases there is a higher rate at 3 than at 1 mM nucleotide. The high resting ATPase compromises the control of nucleotide concentrations within the permeabilized tissue even in the presence of an ATP-regenerating system consisting of phosphocreatine (PCr, 35mM) and creatine kinase (1 mg/ml). Treatment of the intact muscle with the ectonucleotidase inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) followed by alpha-toxin permeabilization and inclusion of sodium azide in subsequent solutions reduces the ecto-ATPase by approximately 70%. Addition of PCr and creatine kinase then results in the maintenance of high [ATP] and low [ADP] in the muscle, and importantly, there are no significant changes in [ATP], [ADP], [adenosine/AMP], or the ADP-to-ATP ratio upon activation of the muscle in pCa 4.5. In general, the force output in high Ca2+ increased as the metabolic profile of the muscle improved. When ATPase was measured as the appearance of [32P]Pi from [32P]PCr and [gamma-32P]ATP, the alpha-toxin-permeabilized muscle subjected to the above treatment showed only approximately 30% higher total ATPase under activated conditions compared with the freeze-glycerinated Triton-treated portal vein. The suprabasal ATPase is similar in both preparations. We conclude that the reduction of the basal ATPase by the DIDS-azide treatment permits both rigorous control of nucleotide contents and accurate measurement of ATPase activity in alpha-toxin-permeabilized smooth muscle.
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PMID:Metabolic characteristics of alpha-toxin-permeabilized smooth muscle. 802 97

To determine cross-bridge properties during agonist-stimulated contractions, steady-state force and relative steady-state stiffness were recorded at rest (pCa 9) and during both full (pCa 4) and partial (pCa 7) Ca2+ activations of isolated single alpha-toxin permeabilized vascular smooth muscle cells. For pCa 4 and pCa 7, agonist (1 microM histamine) activation resulted in significant (P < 0.05) increases in both force and stiffness. The agonist-induced increase of steady-state force was significantly (P < 0.05) greater than that of stiffness; at pCa 4, there was a 48% increase for force vs. 17% for stiffness, and, at pCa 7, there was a 160% increase for force vs. 57% for stiffness. The increase in force and stiffness after agonist prestimulation implies that the number of attached cross bridges has increased. However, after agonist prestimulation, we found that the increase of force was greater (P < 0.05) than that of stiffness, resulting in a greater force at any given level of stiffness. Thus these data indicate that agonist activation, presumably via activation of a G protein, increases the relative force per attached cross bridge, possibly by modulating the kinetics of the actomyosin adenosinetriphosphatase to increase in the relative population of cross bridges in force-producing states [actinomyosin (AM) or AM.ADP].
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PMID:Agonist activation modulates cross-bridge states in single vascular smooth muscle cells. 843 Jul 60

It is generally believed that histamine-stimulated gastric acid secretion involves a transient elevation of intracellular Ca2+ and the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) cascade through phosphorylation, whose actions ultimately effect the fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing vesicles to the apical plasma membrane of parietal cells. To dissect the signaling events underlying gastric acid secretion, we have developed a permeabilized gastric gland model using Staphylococcus alpha-toxin. The advantage of this model is its ability to retain cytosolic components that are required for the secretory machinery. Here we show that acid secretion in alpha-toxin-permeabilized glands is a cAMP-dependent process, reaching a maximal stimulation at 100 microM cAMP. The cAMP-elicited acid secretion, as monitored by the accumulation of the weak base aminopyrine (AP), required functional mitochondria or exogenously supplied ATP. Maximal stimulation elicited by cAMP for AP uptake by permeabilized glands was 51-85% of intact glands. Moreover, secretory activity was potentiated by 0.1 mM ATP. The recruitment of H(+)-K(+)-ATPase-rich tubulovesicles into the apical plasma membrane was measured using biochemical and morphological assays, thus validating the cell activation processes in response to cAMP. From this permeabilized model, [gamma-32P]ATP was used to directly phosphorylate target proteins. A number of proteins whose phosphorylation-dephosphorylation is specifically modulated by cAMP were found. These studies establish the first permeabilized gland model in which the resting-to-secreting transition can be triggered and show that cAMP-mediated phosphorylation is correlated with secretory activity.
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PMID:Stimulation of gastric acid secretion by cAMP in a novel alpha-toxin-permeabilized gland model. 876 31

Gastric glands isolated from rabbit stomach were permeabilized with Staphylococcus aureus alpha-toxin. Acid secretion by parietal cells, as measured by the accumulation of weak base, was inhibited by incubation with alpha-toxin but could be restored by addition of exogenous ATP (1 mM). The permeable glands were found to retain acid secretory responses to receptor-linked secretagogues, histamine and carbachol, as well as to intracellular mediators, forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate, indicating the presence of intact, functional intracellular coupling mechanisms. Both basal and stimulated acid secretion by the permeable glands were blocked by the Mg2+ chelator, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA; 5 mM), whereas CDTA had no effect on nonpermeabilized glands. These results are interpreted to show that alpha-toxin permeabilizes parietal cells to moderate sized molecules without causing a loss of critical intracellular components. The acid secretory responses to histamine and carbachol persisted in media containing low ( < 50 nM) levels of free Ca2+ buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (0.5 mM), indicating that changes in bulk Ca2+ are not required for these responses. Inclusion of the nonhydrolyzable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM), resulted in inhibition of spontaneous acid secretion, blocked responses to all agents tested, and inhibited stimulated acid secretion. GTP gamma S had no effect on nonpermeabilized glands. No effects on acid secretion by either permeable or nonpermeable glands were observed with GTP, guanosine diphosphate, or guanosine 5'-O-(2-thiodiphosphate). GTP gamma S had no effect on H+ gradient formation by gastric membrane vesicles, showing that it does not inhibit the gastric H(+)-K(+)-adenosinetriphosphatase directly. These results are interpreted to show that GTP gamma S interacts at a postreceptor site to inhibit or reverse a critical step in stimulus-secretion coupling in parietal cells. In contrast to the effect on parietal cells, GTP gamma S was found to stimulate pepsinogen secretion by alpha-toxin-permeabilized chief cells. The differential effects of GTP gamma S on acid and pepsinogen secretions suggest unique roles for GTP binding proteins in these two secretory processes. The use of alpha-toxin-permeabilized gastric glands should prove useful in defining the stimulus-secretion coupling mechanisms involved in both acid and pepsinogen secretions.
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PMID:Differential effects of GTP gamma S on acid and pepsinogen secretion by permeable gastric glands. 876 3

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.
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PMID:Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase. 877 38

The dopamine DA1 receptor transduces its signal via adenylyl cyclase and phospholipase C in the renal proximal tubule, which has been suggested to be defective at the level of receptor-G protein coupling in spontaneously hypertensive rats (SHR). We prepared basolateral membranes from Wistar-Kyoto (WKY) rats and SHR to determine the coupling of DA1 receptor with G proteins, especially G(q/11). Fenoldopam, a DA1-receptor agonist, produced a time- and concentration-dependent stimulation in 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding in WKY rats. Fenoldopam-induced (10 microM) stimulation was significantly inhibited by a DA1-receptor antagonist, Sch-23390. Specific antibodies against COOH terminals of G(S)alpha and G(q/11)alpha produced 50-60% and 40-50% inhibition, respectively, in fenoldopam stimulation of [35S]GTPgammaS binding. Western analysis of basolateral membranes with these antibodies revealed the presence of G(S)alpha (45 kDa) and G(q/11)alpha (42 kDa). Fenoldopam stimulation of [35S]GTPgammaS binding was significantly attenuated in SHR compared with WKY rats. Parathyroid hormone stimulation of [35S]GTPgammaS binding was similar in SHR and WKY rats, whereas stimulation by phenylephrine was significantly reduced in SHR. Densitometric quantification of 42-kDa band showed a reduced amount in SHR, whereas the density of 45-kDa band was not significantly different compared with WKY rats. We provide the direct evidence showing the coupling of DA1 receptor with G(q/11)alpha and G(S)alpha and propose that, in addition to a defect in the receptor-G protein coupling, a reduced amount of G(q/11)alpha observed in the hypertensive animals may also contribute to the diminished dopamine-induced inhibition of Na+-K+-adenosinetriphosphatase in SHR.
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PMID:Renal dopamine DA1 receptor coupling with G(S) and G(q/11) proteins in spontaneously hypertensive rats. 908 77

alpha-Toxin-permeabilized gastric glands represent a functional model in which acid secretion can be elicited by either adenosine 3',5'-cyclic monophosphate (cAMP) or ATP, with proven morphological and functional transition between resting and secretory states [X. Yao, S. M. Karam, M. Ramilo, Q. Rong, A. Thibodeau, and J. G. Forte. Am. J. Physiol. 271 (Cell Physiol. 40): C61-C73, 1996.] In this study we use alpha-toxin-permeabilized rabbit gastric glands to study energy metabolism and the interplay between nucleotides to support acid secretion, as indicated by the accumulation of aminopyrine (AP). When permeabilized glands were treated with a phosphodiesterase inhibitor, the secretory response to cAMP was inhibited, whereas the secretory response to ATP was potentiated. This implied that 1) ATP provided support not only as an energy source but also as substrate for adenylate cyclase, 2) activation of acid secretion by cAMP needed ATP, and 3) ATP and cAMP exchanged rapidly inside parietal cells. To address these issues, we tested the action of adenine nucleotides in the presence and absence of oxidizable substrates. All adenine nucleotides, including AMP, ADP, ATP, and cAMP, could individually enhance the glandular AP accumulation in the presence of substrates, whereas only a high concentration of ATP (5 mM) was able to support secretory activity in substrate-free buffer. Moreover, ATP could maintain 75-80% of maximal secretory activity in phosphate-free buffer; cAMP alone could not support secretion in phosphate-free buffer. In glands and in H(+)-K(+)-adenosinetriphosphatase-rich gastric microsomes, we showed the operation of adenylate kinase, creatine kinase, and ATP/ADP exchange activities. These enzymes, together with endogenous adenylate cyclase and phosphodiesterase, provide the recycling of nucleotides essential for the viability of alpha-toxin-permeabilized gastric glands and imply the importance of nucleotide recycling for energy metabolism in intact parietal cells.
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PMID:Nucleotide metabolism by gastric glands and H(+)-K(+)-ATPase-enriched membranes. 945 79

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