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)

We studied the functions of betagamma-subunits of G(i/o) protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl(-) currents in oocytes expressing beta(2)-adrenoceptor and the protein kinase A-dependent Cl(-) channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [d-Ala(2), d-Leu(5)]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing beta(2)-adrenoceptor-CFTR and 5-HT(1A) receptor (5-HT(1A)R), delta-opioid receptor, or GABA(B) receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT(1A)R. The 5-HT-induced enhancement of G(s)-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin alpha (G(t)alpha). The 5-HT-induced enhancement was further augmented by coexpression of the Gbetagamma-activated form of adenylate cyclase (AC) type II but not AC type III. Thus betagamma-subunits of G(i/o) protein contribute to the enhancement of G(s)-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT(1A)R or delta-opioid receptor alone. They elicited Ca(2+)-activated Cl(-) currents in oocytes coexpressing these receptors with the Gbetagamma-activated form of phospholipase C (PLC)-beta2 but not with PLC-beta1. These currents were inhibited by pretreatment with PTX and coexpression of G(t)alpha, suggesting that betagamma-subunits of G(i/o) protein activate PLC-beta2 and then cause intracellular Ca(2+) mobilization. Our results indicate that betagamma-subunits of G(i/o) protein participate in diverse intracellular signals, enhancement of G(s)-coupled receptor-mediated responses, and intracellular Ca(2+) mobilization.
 ...
PMID:Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system. 1515 2

We examined the effects of intracellular Cl- concentration ([Cl-]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+ transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71-85, 2002). Treatment with cAMP (100 microM 8-cpt-cAMP plus 100 microM IBMX) stimulated ENaC-mediated Na+ absorption as well as Cl- secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in alpha-toxin-permeabilized monolayers to have the anion selectivity sequence NO3- > Br- > Cl- > I-. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS-Isc) driven by an apical-to-basolateral Na+ concentration gradient] was progressively inhibited by increasing the [Cl-] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl-] in the apical solution. This inhibitory effect of [Cl-]i occurred regardless of the presence or absence of net Cl- transport. However, from fluorometric measurements using the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na+ absorption and Cl- secretion, produced a decrease of [Cl-]i from 76 +/- 14 to 36 +/- 8 mM (P = 0.03). Thus it might be expected that activation of Cl- secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [Cl-]i from 15 to 145 mM decreased AS-Isc from 24.5 +/- 1.0 to 10.2 +/- 1.6 microA/cm2. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 +/- 0.16 to 1.32 +/- 0.17 fmol/cm2 and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 +/- 1.2 to 8.2 +/- 1.4 microA/fmol.
 ...
PMID:Inhibition of ENaC by intracellular Cl- in an MDCK clone with high ENaC expression. 1516 4

The mechanisms by which uridine triphosphate (UTP) stimulates ATP release from Schwann cells cultured from the sciatic nerve were investigated using online bioluminescence techniques. UTP, a P2Y(2) and P2Y(4) receptor agonist, stimulated ATP release from Schwann cells in a dose-dependent manner with an ED(50) of 0.24 microm. UTP-stimulated ATP release occurs through P2Y(2) receptors as it was blocked by suramin which inhibits P2Y(2) but not P2Y(4) receptors. Furthermore, positive immunostaining of P2Y(2) receptors on Schwann cells was revealed and GTP, an equipotent agonist with UTP at rat P2Y(4) receptors, did not significantly stimulate ATP release. UTP-stimulated ATP release involved second messenger pathways as it was attenuated by the phospholipase C inhibitor U73122, the protein kinase C inhibitor chelerytherine chloride, the IP(3) formation inhibitor lithium chloride, the cell membrane-permeable Ca(2+) chelator BAPTA-AM and the endoplasmic reticulum Ca(2+)-dependent ATPase inhibitor thapsigargin. Evidence that ATP may be stored in vesicles that must be transported to the cell membrane for exocytosis was found as release was significantly reduced by the Golgi-complex inhibitor brefeldin A, microtubule disruption with nocodazole, F-actin disruption with cytochalasin D and the specific exocytosis inhibitor botulinum toxin A. ATP release from Schwann cells also involves anion transport as it was significantly reduced by cystic fibrosis transmembrane conductance regulator inhibitor glibencamide and anion transporter inhibitor furosemide. We suggest that UTP-stimulated ATP release is mediated by activation of P2Y(2) receptors that initiate an IP(3)-Ca(2+) cascade and protein kinase C which promote exocytosis of ATP from vesicles as well as anion transport of ATP across the cell membrane.
 ...
PMID:Secretion of ATP from Schwann cells in response to uridine triphosphate. 1565 52

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl- channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl- ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl- channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl- channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl- concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl- concentration was increased in Xenopus oocytes in the presence of a high bath Cl- concentration, which inhibited ENaC without changing surface expression of alpha beta gammaENaC. In contrast, a 5 mM bath Cl- concentration reduced the cytosolic Cl- concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl- into oocytes and in inside/out macropatches by exposure to high cytosolic Cl- concentrations. The effect of Cl- was mimicked by Br-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl(-)-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.
 ...
PMID:Cl- interference with the epithelial Na+ channel ENaC. 1602 56

The intracellular signaling pathways responsible for extracellualr uridine-5'-triphosphate (UTPo)-induced chloride (Cl-) currents (I(Cl.UTP)) were studied in mouse ventricular myocytes with the whole-cell clamp technique. UTPo (0.1 to 100 microM) activated a whole-cell current that showed a time-independent activation, a linear current-voltage relationship in symmetrical Cl- solutions, an anion selectivity of Cl- > iodide > aspartate, and an inhibition by a thiazolidinone-derived specific inhibitor (CFTR(inh)-172, 10 microM) of cystic fibrosis transmembrane conductance regulator (CFTR), but not by a disulfonic stilbene derivative (DIDS, 100 microM), these properties matching those of CFTR Cl- channels. The potency order of nucleotides for an activation of the Cl- current was UTP = ATP > uridine-5'-diphosphate (UDP) = ADP. Suramin (100 microM), a P2Y receptor antagonist, strongly inhibited the UTPo -activation of the Cl- current, whereas pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 microM), another P2Y receptor antagonist, induced little inhibition of I(Cl.UTP). The activation of I(Cl.UTP) was sensitive to protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, intracellular GDPbetaS (nonhydrolyzable GDP analogue) or anti-Gq/11 antibody. UTPo failed to activate the Cl- current when the cells were dialyzed with nonhydrolyzable ATP analogues (ATPS or AMP-PNP) without ATP, suggesting that ATP hydrolysis is a prerequisite for the current activation. I(Cl.UTP) was persistently activated with a mixture of ATPgammaS + ATP in the pipette, suggesting the involvement of phosphorylation reaction in the current activation process. Our results strongly suggest that I(Cl.UTP) is due to the activation of CFTR Cl- channels through Gq/11-coupled P2Y2 receptor-PLC-PKC signaling and ATP hydrolysis in mouse heart.
 ...
PMID:Regulation of extracellular UTP-activated Cl- current by P2Y-PLC-PKC signaling and ATP hydrolysis in mouse ventricular myocytes. 1729 97

The activities of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel and the amiloride-sensitive epithelial Na(+) channel (ENaC) are acutely coordinated in the sweat duct. However, the mechanisms responsible for cross-talk between these ion channels are unknown. Previous studies indicated that luminal pH of sweat ducts varies over 3 pH units and that the cytoplasmic pH affects both CFTR and ENaC. Therefore, using basolaterally alpha-toxin-permeabilized apical membrane preparations of sweat ducts as an experimental system, we tested the hypothesis that the cytosolic pH may mediate the cross-talk between CFTR and ENaC. We showed that while luminal pH had no effect, cytosolic pH acutely affected ENaC activity. That is, acidic pH inhibited, while basic pH activated, ENaC. pH regulation of ENaC appears to be independent of CFTR or endogenous kinase activities because basic pH independently stimulated ENaC (1) in normal ducts even when CFTR was deactivated, (2) in CF ducts that lack CFTR in the plasma membranes and (3) after blocking endogenous kinase activity with staurosporine. Considering the evidence of Na(+)/H(+) exchange (NHE) activity as shown by the expression of mRNA and function of NHE in the basolateral membrane of the sweat duct, we postulate that changes in cytosolic Na(+) ([Na(+)]( i )) may alter cytosolic pH (pH( i )) as salt loads into the cell during electrolyte absorption. These changes may play a role in coordinating the activities of ENaC and CFTR during transepithelial salt transport.
 ...
PMID:Effect of cytosolic pH on epithelial Na+ channel in normal and cystic fibrosis sweat ducts. 1893 3

Cystic fibrosis (CF) is caused by mutations in the gene producing the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl(-) channel. Its dysfunction limits Cl(-) secretion and enhances Na+ absorption, leading to viscous mucus in the airway. Ca2+-activated Cl(-) channels (CaCCs) are coexpressed with CFTR in the airway surface epithelia. Increases in cytosolic Ca(2+) activate the epithelial CaCCs, which provides an alternative Cl(-) secretory pathway in CF. We developed a screening assay and screened a library for compounds that could enhance cytoplasmic Ca2+, activate the CaCC, and increase Cl(-) secretion. We found that spiperone, a known antipsychotic drug, is a potent intracellular Ca2+ enhancer and demonstrated that it stimulates intracellular Ca2+, not by acting in its well-known role as an antagonist of serotonin 5-HT2 or dopamine D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Spiperone activates CaCCs, which stimulates Cl(-) secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro and in CFTR-knockout mice in vivo. In conclusion, we have identified spiperone as a new therapeutic platform for correction of defective Cl(-) secretion in CF via a pathway independent of CFTR.
 ...
PMID:Spiperone, identified through compound screening, activates calcium-dependent chloride secretion in the airway. 1898 51

It has been reported that the cystic fibrosis transmembrane conductance regulator (CFTR) can be activated through cAMP- and protein kinase A-independent pathways involving GTP-binding proteins and an unknown kinase. In this study, we further examined how G protein-coupled pathways regulate CFTR. We demonstrate that stimulation of purinergic P2Y(2) receptors in CFTR-expressing oocytes and in airway epithelial cells activates CFTR Cl(-) currents. Activation of CFTR Cl(-) currents via P2Y(2) was inhibited by CFTR(inh)-172 and was independent of intracellular Ca(2+), protein kinase C, or calmodulin-dependent kinase (CAMK). However, activation of CFTR was suppressed by inhibition of phospholipase C and by the nonselective protein kinase inhibitor staurosporine. Activation of CFTR through P2Y(2) receptors was enhanced when G(i) proteins were inhibited by pertussis toxin. Inhibition of protein kinase A and of protein kinases downstream of P2Y(2) receptors such as mitogen-activated protein kinases, tyrosine kinase, or c-src kinase did not interfere with activation of CFTR. The present results demonstrate an antagonistic regulation of CFTR by P2Y(2) receptors: CFTR is inhibited by stimulation of G(i) proteins and is activated by stimulation of G(q/11)/PLC and an unknown downstream protein kinase.
 ...
PMID:CFTR is activated through stimulation of purinergic P2Y2 receptors. 1900 11

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl(-) conductance (gCFTR) in the apical membrane is completely deactivated following alpha-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through alpha-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10(-8) M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo.
 ...
PMID:PKA mediates constitutive activation of CFTR in human sweat duct. 1986 88

Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.
...
PMID:CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction. 2582 45


<< Previous 1 2 3 Next >>