Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Defects in platelet cytoplasmic Ca++ mobilization have been postulated but not well demonstrated in patients with inherited platelet secretion defects. We describe studies in a 42-year-old white woman, referred for evaluation of easy bruising, and her 23-year-old son. In both subjects, aggregation and 14C-serotonin secretion responses in platelet-rich plasma (PRP) to adenosine diphosphate (ADP), epinephrine, platelet activating factor (PAF), arachidonic acid (AA), U46619, and ionophore A23187 were markedly impaired. Platelet ADP and adenosine triphosphate (ATP), contents and thromboxane synthesis induced by thrombin and AA were normal. In quin2-loaded platelets, the basal intracellular Ca++ concentration, [Ca++]i, was normal; however, peak [Ca++]i measured in the presence of 1 mmol/L external Ca++ was consistently diminished following activation with ADP (25 mumol/L), PAF (20 mumol/L), collagen (5 micrograms/mL), U46619 (1 mumol/L), and thrombin (0.05 to 0.5 U/mL). In aequorin-loaded platelets, the peak [Ca++]i studied following thrombin (0.05 and 0.5 U/mL) stimulation was diminished. Myosin light chain phosphorylation following thrombin (0.05 to 0.5 U/mL) stimulation was comparable with that in the normal controls, while with ADP (25 mumol/L) it was more strikingly impaired in the propositus. We provide direct evidence that at least in some patients with inherited platelet secretion defects, agonist-induced Ca++ mobilization is impaired. This may be related to defects in phospholipase C activation. These patients provide a unique opportunity to obtain new insights into Ca++ mobilization in platelets.
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PMID:Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects. 275 41

The data presented in this report show that N-ethylmaleimide (NEM) is a powerful inhibitor of thrombin-induced platelet aggregation. NEM increased guanosine 3', 5'-cyclic monophosphate (cGMP) and adenosine 3', 5'-cyclic monophosphate (cAMP) levels in intact cells. The inhibition of cAMP high-affinity phosphodiesterase and cGMP phosphodiesterase was implicated in the elevation of the cyclic nucleotides. NEM dose dependently blocked the thrombin-stimulated, but not the phorbol myristate acetate-dependent phosphorylation of the protein kinase C substrate pleckstrin. Myosin light chain phosphorylation was also inhibited by NEM. In addition, the sulphydryl reagent inhibited Ca2+ mobilisation induced by thrombin. The data indicate that phospholipase C activation by thrombin is interrupted by NEM at the level of receptor-mediated signal transduction.
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PMID:N-ethylmaleimide inhibition of thrombin-induced platelet aggregation. 1048 31

Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma-thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction.
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PMID:Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. 1074 61

Myosin is an ATPase, able to form filaments with actin, thus initiating smooth muscle contraction (conversion of chemical energy into mechanical energy). Myosin activity is regulated by cytosolic calcium, via a calcium-calmodulin-MLCK-dependent phosphorylation. Extrusion of cytosolic calcium via calcium pumps (in the plasma membrane and sarcoplasmic reticulum) and via a sodium-calcium exchange allow smooth muscle cells to maintain their resting state. Constrictor agonists (hormones, neurotransmitters or drugs) act at membrane receptors inducing: (i) a fast and transient calcium mobilization from the sarcoplasmic reticulum, via phospholipase C (PLC) stimulation and inositol triphosphate (IP3) production or via a "calcium-induced calcium release" mechanism and opening of calcium channels in the sarcoplasmic reticulum and (ii) a slow and maintained mobilization of extracellular calcium, via the opening of voltage-dependent calcium channels in plasma membranes. Smooth muscle relaxation is ensured by a phosphatase which hydrolyzes phosphorylated myosin and decreases the calcium sensitivity of the contractile apparatus. Calcium signal is regulated at that level by: (i) protein kinase C, tyrosine kinase and arachidonic acid which inhibit phosphatase activity and (ii) cyclic AMP (cAMP) and cyclic GMP (cGMP) which enhance phosphatase activity. A second regulatory site is situated at the level of the non-contractile calcium compartment, which buffers signal transduction and where cGMP and/or cAMP enhance calcium extrusion mechanisms.
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PMID:[Cellular mechanisms of smooth muscle contraction]. 1093 9

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca(2+) sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr(695) of the MLCP regulatory subunit (MYPT1) and at Thr(38) of the MLCP inhibitor protein CPI-17 results in inhibition of MLCP activity. We have previously demonstrated that CPI-17 Thr(38) phosphorylation plays an important role in G-protein-mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site- and phospho-specific antibodies, phosphorylation of MYPT1 Thr(695) and CPI-17 Thr(38) was examined along with MYPT1 Thr(850), which is a non-inhibitory Rho-kinase site. We found that both CPI-17 Thr(38) and MYPT1 Thr(850) were phosphorylated in response to agonists or GTPgammaS concurrently with contraction and myosin phosphorylation in alpha-toxin-permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr(695) did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho-kinase inhibitor Y-27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr(850) and CPI-17 Thr(38), respectively, in intact VD while MYPT1 Thr(695) phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr(695) is independent of stimulation of G-proteins, Rho-kinase or PKC. In the phasic PV, phosphorylation of CPI-17 Thr(38) may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI-17 concentration, probably utilizes other Ca(2+) sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI-17.
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PMID:Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle. 1256 12

Ca(2+) sensitivity of arterial contractility is governed by regulating myosin phosphatase activity in response to agonist stimuli. CPI-17, a myosin phosphatase inhibitor phosphoprotein, is phosphorylated concomitantly with agonist-induced contractile Ca(2+) sensitization in mammalian artery. CPI-17 has not been detected in chicken artery, but is readily detectable in pigeon artery. To evaluate a role of CPI-17, we compared contractility of the arteries of 'CPI-17-deficient' chicken with those of CPI-17-rich rabbit and pigeon, and studied the effect of CPI-17-reconstitution in chicken artery. Other major regulatory/contractile proteins for Ca(2+) sensitization are expressed in both chicken and rabbit arteries. Agonists, such as an alpha(1)-agonist and endothelin-1, produced significant contraction in arteries of all species under physiological Ca(2+)-containing conditions. Depletion of Ca(2+) abolished these contractions in chicken but partially inhibited them in rabbit and pigeon arteries. Unlike CPI-17-rich tissues, chicken arteries exerted little Ca(2+) sensitization in response to alpha(1)-agonist or endothelin-1. GTPgammaS produced a slight Ca(2+) sensitizing effect in chicken artery, but this was significantly smaller compared with CPI-17-rich tissues. A PKC activator (PDBu) did not generate but rather reduced a contraction in both intact and alpha-toxin-permeabilized chicken artery in contrast to a large contraction in CPI-17-rich arteries. Myosin light chain phosphorylation was reduced by PDBu in chicken but elevated in rabbit artery. Addition of recombinant CPI-17 into beta-escin-permeabilized chicken artery restored PDBu-induced and enhanced GTPgammaS-induced Ca(2+) sensitization. Thus, CPI-17 is essential for G protein/PKC-mediated Ca(2+) sensitization in smooth muscle.
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PMID:CPI-17-deficient smooth muscle of chicken. 1509 Jun 8

Partial bladder outlet obstruction (PBOO) alters the function of the whole bladder and produces specific alterations in the contractility of the bladder smooth muscle cell. The goal of this study was to test the hypothesis that PBOO affects smooth muscle contraction at the level of the receptor- and G protein-dependent increase in myofilament Ca2+ sensitivity. To address this question, we used alpha-toxin-permeabilized strips of bladder smooth muscle from control animals and animals subjected to 2 wk of PBOO. Increasing free [Ca2+] increased force in permeabilized strips from control animals; the addition of 10 microM carbachol and 10 microM GTP increased both the Ca2+ sensitivity of the contractions and the maximal levels of force attained. In contrast, although increases in [Ca2+] increased force in permeabilized strips from PBOO animals, the addition of carbachol and GTP had no additional effects. Myosin light chain phosphorylation levels increased with [Ca2+], and although they tended to be higher in strips from PBOO animals, they did not reach statistical significance. Assessment of G protein activity from both animal models suggests this is not a site responsible for the loss of carbachol and GTP enhancement of myofilament Ca2+ sensitivity. The addition of phorbol dibutyrate increased the Ca2+ sensitivity of force development in strips from both animal models, suggesting that an alteration in PKC signaling is not involved. Our results are consistent with the hypothesis that PBOO decreases receptor-mediated myofilament calcium sensitization and that the site of action is downstream from either the G proteins or PKC.
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PMID:Partial bladder outlet obstruction abolishes the receptor- and G protein-dependent increase in calcium sensitivity in rabbit bladder smooth muscle. 1516 3

The development of luminal organs begins with the formation of spherical cysts composed of a single layer of epithelial cells. Using a model three-dimensional cell culture, this study examines the role of a cytoskeletal motor, myosin II, in cyst formation. Caco-2 and SK-CO15 intestinal epithelial cells were embedded into Matrigel, and myosin II was inhibited by blebbistatin or siRNA-mediated knockdown. Whereas control cells formed spherical cysts with a smooth surface, inhibition of myosin II induced the outgrowth of F-actin-rich surface protrusions. The development of these protrusions was abrogated after inhibition of F-actin polymerization or of phospholipase C (PLC) activity, as well as after overexpression of a dominant-negative ADF/cofilin. Surface protrusions were enriched in microtubules and their formation was prevented by microtubule depolymerization. Myosin II inhibition caused a loss of peripheral F-actin bundles and a submembranous extension of cortical microtubules. Our findings suggest that inhibition of myosin II eliminates the cortical F-actin barrier, allowing microtubules to reach and activate PLC at the plasma membrane. PLC-dependent stimulation of ADF/cofilin creates actin-filament barbed ends and promotes the outgrowth of F-actin-rich protrusions. We conclude that myosin II regulates the spherical shape of epithelial cysts by controlling actin polymerization at the cyst surface.
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PMID:Myosin II regulates the shape of three-dimensional intestinal epithelial cysts. 1846 May 84