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)

PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Nao and 0.7 mM for Ko, is absolutely dependent on Clo and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 approximately 650 nM) or calyculin A (EC50 approximately 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF on cotransport rate is biphasic, with an initial rapid approximately 2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 approximately 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (> 2 days) leads to neurite formation and a maintained approximately 2-fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.
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PMID:Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells. 814 46

Swelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K+ (86Rb+) influx suppressed by 10 microM bumetanide. Bumetanide-sensitive transport of 86Rb+ is dependent on extracellular K+, Na+ and Cl- both in iso-osmotic conditions and under hypo-osmotic shock, supporting the notion that it is mediated by Na+,K+,2Cl- cotransport. Inhibitors of protein kinase C (10 microM polymyxin B and l microM staurosporine) had no significant effect on basal cotransport but reduced its hypotonic stimulation by 70-80%. Similar results were obtained with calmodulin antagonist R24571 (10 microM), indicating Ca2+/calmodulin-dependence of the process. Influence of polymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl- cotransport was also suppressed by protein kinase C activator PMA (l microM). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 microM vanadate, 5 mM fluoride and 0.5 microM okadaic acid) activated greatly the bumetanide-sensitive 86Rb+ uptake in isotonic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmodulin-dependent staurosporine/polymyxin B-sensitive protein kinase other than protein kinase C in swelling-induced activation of Na+,K+,2Cl- cotransport in glial cells.
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PMID:Swelling-induced activation of Na+,K+,2Cl- cotransport in C6 glioma cells: kinetic properties and intracellular signalling mechanisms. 897 7

Bumetanide is well known for its ability to inhibit the nonconductive Na+-K+-2Cl- cotransporter. We were surprised in preliminary studies to find that bumetanide in the contraluminal bath also inhibited NaCl absorption in the human sweat duct, which is apparently poor in cotransporter activity. Inhibition was accompanied by a marked decrease in the transepithelial electrical conductance. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is richly expressed in the sweat duct, we asked whether bumetanide acts by blocking this anion channel. We found that bumetanide 1) significantly increased whole cell input impedance, 2) hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membrane potential, 4) increased the ratio of apical-to-basolateral membrane resistance, and 5) decreased transepithelial Cl- conductance (GCl). These results indicate that bumetanide inhibits CFTR GCl in both cell membranes of this epithelium. We excluded bumetanide interference with the protein kinase A phosphorylation activation process by "irreversibly" phosphorylating CFTR [by using adenosine 5'-O-(3-thiotriphosphate) in the presence of a phosphatase inhibition cocktail] before bumetanide application. We then activated CFTR GCl by adding 5 mM ATP. Bumetanide in the cytoplasmic bath (10(-3) M) inhibited approximately 71% of this ATP-activated CFTR GCl, indicating possible direct inhibition of CFTR GCl. We conclude that bumetanide inhibits CFTR GCl in apical and basolateral membranes independent of phosphorylation. The results also suggest that >10(-5) M bumetanide cannot be used to specifically block the Na+-K+-2Cl- cotransporter.
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PMID:Bumetanide blocks CFTR GCl in the native sweat duct. 988 39

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl- secretory response of the mouse jejunum using the Ussing short-circuit current (Isc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in Isc (EC50 41 +/- 1 microM). Pretreating tissues with 0.25 microM forskolin reduced the concentration-dependent increase in Isc by DCEBIO and increased the EC50 (53 +/- 5 microM). Bumetanide blocked (82 +/- 5%) the DCEBIO-stimulated Isc consistent with Cl- secretion. DCEBIO was a more potent stimulator of Cl- secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated Isc by >80% indicating the participation of CFTR in the DCEBIO-stimulated Isc response. Clotrimazole reduced DCEBIO-stimulated Isc by 67 +/- 15%, suggesting the participation of the intermediate conductance Ca2+-activated K+ channel (IKCa) in the DCEBIO-activated Isc response. In the presence of maximum forskolin (10 microM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in Isc of 43 +/- 5 microA/cm2 and then falling to a steady-state response of 17 +/- 10 microA/cm2 compared with DCEBIO control tissues (61 +/- 6 microA/cm2). The forskolin-stimulated Isc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated Isc, providing evidence that DCEBIO increased Cl- secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl- secretion of the mouse jejunum and that DCEBIO targets components of the Cl- secretory mechanism.
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PMID:DCEBIO stimulates Cl- secretion in the mouse jejunum. 1613 45

Previously, we demonstrated that genistein stimulated Cl(-) secretion in the mouse jejunum (Baker MJ and Hamilton KL, Am J Physiol Cell Physiol 287: C1636-C1645, 2004); however, the mode of action of genistein still remains unclear. Here, we examined the activation of Cl(-) secretion by the modulation of phosphodiesterases (PDEs) by genistein (75 microM) in the mouse jejunum with the Ussing short-circuit current (I(sc)) technique. Drugs tested included theophylline (10 mM), a nonspecific PDE inhibitor; 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MM-IBMX; 100 microM), erythro-9-(2-hydroxyl-3-nonyl)-adenine (EHNA; 40 microM), milrinone (100 microM), and rolipram (40 and 100 microM), which are specific inhibitors of PDE1-PDE4, respectively. Theophylline stimulated a bumetanide-sensitive I(sc), indicative of Cl(-) secretion, and abolished genistein's stimulatory action on I(sc). Neither 8-MM-IBMX nor EHNA altered the basal I(sc) nor did these PDE inhibitors affect the stimulatory action of genistein on the I(sc) of the mouse jejunum. Rolipram had no effect on basal I(sc), but it reduced the genistein-stimulated I(sc) compared with time-matched control tissues. Milrinone stimulated a concentration-dependent increase in I(sc). Bumetanide (10 microM) inhibited 60 +/- 4% of milrinone-induced I(sc). Pretreating tissues with milrinone prevented genistein from stimulating I(sc), and pretreatment with genistein reduced the effect of milrinone on I(sc). H89 (50 microM), a PKA inhibitor, reduced the milrinone-stimulated I(sc). Likewise, H89 reduced the genistein-stimulated I(sc). Here, we demonstrate, for the first time, that genistein activates Cl(-) secretion of the mouse jejunum via inhibition of a PDE3-dependent pathway.
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PMID:Genistein stimulates electrogenic Cl- secretion via phosphodiesterase modulation in the mouse jejunum. 1953 15