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)

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel has been identified in the cardiac muscle of a number of mammalian species, including humans. The goal of this study was to begin quantifying the structural requirements necessary for arylaminobenzoate block of the CFTR channel. The cardiac cAMP-dependent Cl- current (ICl) was measured using the whole-cell arrangement of the patch-clamp technique in guinea pig ventricular myocytes during stimulation of protein kinase A with forskolin. At drug concentrations below the IC50 value for channel block, reduction of ICl by the arylaminobenzoates occurred in a strongly voltage-dependent manner with preferential inhibition of the inward currents. At higher drug concentrations, block of both the inward and outward ICl was observed. Increasing the length of the carbon chain between the benzoate and phenyl rings of the arylaminobenzoates resulted in a marked increase in drug block of the channel, with IC50 values of 47, 17, and 4 microM for 2-benzylamino-5-nitro-benzoic acid, 5-nitro-2-(2-phenylethylamino)-benzoic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), respectively. Increasing the carbon chain length further with the compound 5-nitro-2-(4-phenylbutylamino)-benzoic acid, caused no additional increase in the potency of drug block (IC50 = 4 microM). Inhibition of ICl by the arylaminobenzoates was modulated by the pH of the external solution; increasing the pH from 7.4 to 10.0 greatly weakened NPPB block, whereas decreasing the pH to 6.4 enhanced block. In addition, block of ICl was observed during intracellular dialysis of NPPB, and this action was not affected by raising the external pH.
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PMID:Arylaminobenzoate block of the cardiac cyclic AMP-dependent chloride current. 949 22

P2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca2+]i, activation of phospholipases C (PLC) and A2 (PLA2), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na+,K+,Cl- cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier ( approximately 50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca2+]i that was abolished by a chelator of Ca2+i, BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+, Cl- cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4, 5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na+,K+, Cl- cotransport activity. Inhibitors of PLA2 (AACOCF3), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na+,K+,Cl- cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na+ channels (amiloride), Cl- channels (NPPB) and Na+/H+ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na+,K+,Cl- cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na+,K+,Cl- cotransport and suggest the presence of a novel P2-receptor-coupled signaling mechanism.
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PMID:ATP-induced inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells: lack of involvement of known purinoceptor-coupled signaling pathways. 991 50

1. The contribution of ClC-2 protein to the inwardly rectifying Cl- conductance in cultured porcine choroid plexus epithelial cells was investigated using Western analysis and whole-cell current recordings. 2. Inwardly rectifying currents were elicited by hyperpolarizing voltage at a potential more negative than -50 mV in the presence of intracellular protein kinase A (PKA). The relative halide selectivity estimated from the shift in the reversal potential (Erev) was I- > Br- > Cl- > F-. 3. Extracellular vasoactive intestinal peptide (VIP) activated the same currents in a dose-dependent manner with a half-maximal concentration of 167.3 nM. H-89 (a PKA inhibitor) interfered with the current activation by VIP. 4. The Cl- channel was inhibited by external Cd2+, Ba2+or H+, but only weakly inhibited by known Cl- channel blockers including glibenclamide, NPPB, DIDS and anthracene-9-carboxylic acid (9AC). 5. A specific antibody to ClC-2 detected a 79 kDa protein in porcine choroid plexus cells, which was reduced in cells treated with antisense oligodeoxynucleotide for ClC-2. Both PKA and VIP failed to activate the inwardly rectifying Cl- currents in cells transfected with the antisense oligodeoxynucleotide, while they activated the currents in cells transfected with GFP alone or the control oligodeoxynucleotide randomized from antisense oligonucleotide. 6. It is concluded that ClC-2 protein contributes to the inwardly rectifying Cl- conductance in porcine choroid plexus epithelial cells.
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PMID:The chloride channel ClC-2 contributes to the inwardly rectifying Cl- conductance in cultured porcine choroid plexus epithelial cells. 1069 77

Secretin stimulates bicarbonate secretion from pancreatic duct cells, but what influence secretin exerts on intestinal tissues remains to be clarified. The aim of this study is to examine effects of secretin on ion transport in intestinal epithelial Caco-2 cells. We mounted monolayers of Caco-2 cells grown on permeable supports for 21-28 d in a Ussing chamber and measured short-circuit currents (I(sc)). Addition of secretin (5-100 nM) to the basolateral solution dose-dependently induced biphasic increases of I(sc) (transient and sustained phase). Dibutyryl cyclic AMP (200 microM), forskolin (10 microM), and 3-isobutyl-1-methylxanthine (IBMX, 1 mM) also induced I(sc) responses similar to the administration of secretin. Addition of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 100 microM) or benzamil (100 microM) to the apical solution markedly reduced the secretin-induced I(sc) increase in the transient phase. A selective antagonist of cAMP-dependent protein kinase (PKA), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89, 1 microM), and a membrane permeable Ca(2+) chelator, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM, 10 microM) reduced the secretin-induced I(sc). Basolateral addition of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 1 mM) suppressed the sustained phase I(sc) increase. Secretin also induced alkalinization of the apical solution (DeltapH, 0.053 +/- 0.013). The alkalinization did not occur when DIDS (1 mM) was added to the basolateral solution or Na(+) was removed from the solutions. Taken together, our observations suggest: (1) secretin stimulates a benzamil-sensitive Na(+) influx and an NPPB-sensitive Cl(-) efflux across the apical membrane through PKA-dependent and Ca(2+)-sensitive pathways; and (2) secretin also induces alkalinization of the apical solution through the activation of a DIDS-sensitive Na(+)-HCO(3)(-) cotransport in the basolateral membrane of Caco-2 cells.
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PMID:Activation of transepithelial ion transport by secretin in human intestinal Caco-2 cells. 1088 Aug 78

In guinea pig gallbladder epithelium, a secretion of fluid, secondary to an electrogenic secretion of Cl(-) and HCO(-)(3), is elicited in the presence of a high intracellular concentration of adenosine 3'-5'-cyclic monophosphate (cAMP). The aim of this study was to analyze the effects of secretagogues on the activity of anionic channels in isolated epithelial cells using the patch-clamp technique and measuring the electrical potential difference of the cellular membrane (pd(cm)). In cell-attached configuration, with the microelectrode filled with a solution of N-methylglucamine-Cl, or in inside-out configuration (symmetrical solution), it was possible to demonstrate the presence of an 18-pS Cl(-) channel with linear current/voltage (I/V) relationship and voltage independence; this channel is not activated by cAMP (cell-attached configuration). In inside-out configuration (symmetrical solution), another anionic channel with a conductance of 2.8 pS, voltage independence, and a linear I/V relationship was also identified. This channel was stimulated by cAMP (cell-attached configuration) and by PKA + ATP + cAMP (inside-out configuration). The channel was inhibited by NPPB (10(-5) M), but not by other anionic inhibitors. Measurements of the pd(cm) value suggested that in isolated cells, as in whole tissue, cAMP activates conductance for both Cl(-) and HCO(-)(3). The selectivity of the channel was gluconate < SO(2-)(4) < Cl(-) < Br(-) < I(-) < HCO(-)(3) < SCN(-) and the P(HCO(3))/P(Cl) was 2.6. Some features of the channel resemble those of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and RT-PCR performed on mRNA from isolated epithelial cells detected the presence of a CFTR homologue mRNA. The results obtained indicate that this channel is responsible for the HCO(-)(3) conductance activated by cAMP.
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PMID:An anion channel in guinea pig gallbladder epithelial cells is highly permeable to HCO(-)(3). 1100 23

Effects of HCO(3)(-) on protein kinase C (PKC)- and protein kinase A (PKA)-induced anion conductances were investigated in Necturus gallbladder epithelial cells. In HCO(3)(-)-free media, activation of PKC via 12-O-tetradecanoylphorbol 13-acetate (TPA) depolarized apical membrane potential (V(a)) and decreased fractional apical voltage ratio (F(R)). These effects were blocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a Cl(-) channel blocker. In HCO(3)(-) media, TPA induced significantly greater changes in V(a) and F(R). These effects were blocked only when NPPB was present in both mucosal and basolateral compartments. The data suggest that TPA activates NPPB-sensitive apical Cl(-) conductance (g(Cl)(a)) in the absence of HCO(3)(-); in its presence, TPA stimulated both NPPB-sensitive g(Cl)(a) and basolateral Cl(-) conductance (g(Cl)(b)). Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased V(a) and F(R); however, these changes were not affected by external HCO(3)(-). We conclude that HCO(3)(-) modulates the effects of PKC on g(Cl)(b). In HCO(3)(-) medium, TPA and IBMX also induced an initial transient hyperpolarization and increase in intracellular pH. Because these changes were independent of mucosal Na(+) and Cl(-), it is suggested that TPA and IBMX induce a transient increase in apical HCO(3)(-) conductance.
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PMID:Effect of HCO(3)(-) on TPA- and IBMX-induced anion conductances in Necturus gallbladder epithelial cells. 1102 86

Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A (PKA) and ATP regulated Cl- channel. Studies using mostly ex vivo systems suggested diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and glybenclamide inhibit CFTR Cl- conductance (CFTR GCl). However, the properties of inhibition in a native epithelial membrane have not been well defined. The objective of this study was to determine and compare the inhibitory properties of the aforementioned inhibitors as well as the structurally related anion-exchange blockers (stilbenes) including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) in the microperfused intact and basilaterally permeabilized native sweat duct epithelium. All of these inhibitors blocked CFTR in a dose-dependent manner from the cytoplasmic side of the basilaterally permeabilized ducts, but none of these inhibitors blocked CFTR GCl from the luminal surface. We excluded inhibitor interference with a protein kinase phosphorylation activation process by "irreversibly" thiophosphorylating CFTR prior to inhibitor application. We then activated CFTR GCl by adding 5 mM ATP. At a concentration of 10(-4) M, NPPB, DPC, glybenclamide, and DIDS were equipotent and blocked approximately 50% of irreversibly phosphorylated and ATP-activated CFTR GCl (DIDS = 49 +/- 10% > NPPB = 46 +/- 10% > DPC = 38 +/- 7% > glybenclamide = 34 +/- 5%; values are mean +/- SE expressed as % inhibition from the control). The degree of inhibition may be limited by inhibitor solubility limits, since DIDS, which is soluble to 1 mM concentration, inhibited 85% of CFTR GCl at this concentration. All the inhibitors studied primarily blocked CFTR from the cytoplasmic side and all inhibition appeared to be independent of metabolic and phosphorylation processes.
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PMID:Effect of anion transport blockers on CFTR in the human sweat duct. 1220 48

Changes in the volume of rat alveolar type II cells (AT-II cells) induced by terbutaline, a beta(2)-agonist, were measured using video-enhanced contrast microscopy. The changes consisted of three phases: initial cell shrinkage, cell swelling, and gradual cell shrinkage. The initial cell shrinkage was Ca(2+)-dependent and was inhibited by quinine (a K+ channel blocker). The subsequent cell swelling was cAMP-dependent and was inhibited by amiloride (a Na+ channel blocker). The final cell shrinkage was cAMP-dependent and was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl- channel blocker). Thus, terbutaline-induced cell volume changes were regulated by both Ca2+ and cAMP. Accumulation of cAMP alone, however, induced the Ca2+ -dependent cell shrinkage of AT-II cells and H-89 (a PKA inhibitor) inhibited terbutaline-induced cell volume changes. This suggests that cAMP accumulation stimulates the Ca2+ signal during terbutaline stimulation. In conclusion, terbutaline stimulates not only Na+ influx, but also K+ and Cl- release mediated via cAMP accumulation in rat AT-II cells, which induces the triphasic cell volume changes.
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PMID:Terbutaline-induced triphasic changes in volume of rat alveolar type II cells: the role of cAMP. 1261 62

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl- cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl- transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl- channels on microdissected mouse DCTs using the patch-clamp technique. A channel of approximately 9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NPo in cell-attached patches was not modified when tubules were preincubated in the presence of 10-5 M forskolin, but the channel was inhibited by phorbol ester (10-6 M). In addition, NPo was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NPo increased threefold), or pH increased from 6.4 to 8.0 (NPo increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl- relative permeability was 0.09, and the anion selectivity sequence Cl(-)--I(-) > Br(-)--NO3(-) > F(-). Intracellular NPPB (10-4 M) and DPC (10-3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl- channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.
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PMID:A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel. 1266 33

Single bronchiolar ciliary cells were isolated from rat lungs. The beta(2)-adrenergic regulation of ciliary beat frequency (CBF) was studied using video-optical microscopy. Terbutaline (a beta(2)-adrenergic agonist) increased CBF in a dose-dependent manner, and it also decreased the volume of the ciliary cells. These terbutaline actions were inhibited by a PKA inhibitor (H-89) and mimicked by forskolin, IBMX and DBcAMP. Ion transport inhibitors were used to isosmotically manipulate the volume of the terbutaline-stimulated bronchiolar ciliary cells. Amiloride (1 microM) and bumetanide (20 microM) potentiated cell shrinkage and the CBF increase, and they shifted the terbutaline dose-response curve to the lower-concentration side. Quinidine (500 microM), in contrast, increased cell volume and suppressed the CBF increase. Moreover, a KCl solution containing amiloride (1 microM) and strophanthidin (100 microM) increased cell volume and suppressed the CBF increase, and then the subsequent removal of either amiloride or strophanthidin decreased cell volume and further increased CBF. NPPB (10 microM) or glybenclamide (200 microM) had no effect on the action of terbutaline. Thus, in terbutaline-stimulated ciliary cells, cell shrinkage enhances the CBF increase; in contrast, cell swelling suppresses it. However, the results of direct manupulation of cell volume by applying osmotic stresses (hyperosmotic shrinkage or hyposmotic swelling) were the opposite of the findings of the isosmotic experiments: hyposmotic cell swelling enhanced the CBF increase, while isosmotic swelling suppressed it. These results suggest that isosmotic and non-isosmotic volume changes in terbutaline-stimulated bronchiolar ciliary cells may trigger different signalling pathways. In conclusion, terbutaline increases CBF and decreases the volume of rat bronchiolar ciliary cells via cAMP accumulation under isosmotic conditions, and the isosmotic cell shrinkage enhances the CBF increase by increasing cAMP sensitivity.
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PMID:Beta 2-adrenergic regulation of ciliary beat frequency in rat bronchiolar epithelium: potentiation by isosmotic cell shrinkage. 1459 91


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