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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human respiratory tract is constantly exposed to polycyclic aromatic hydrocarbons (PAHs) through inhalation of atmospheric pollutants. We examined the effects of three PAHs (benzo[a]pyrene, anthracene, and fluoranthene) on the airway ion transport, which is essential for lung defense and normal airway function, using human airway epithelia (Calu-3). These three PAHs had no significant effect on the basal short-circuit current (I(sc)). However, fluoranthene (1-100 microM) applied in the apical compartment potentiated I(sc) in response to cAMP-related agents (isoproterenol, forskolin, and 8-bromo-cAMP). The effects of fluoranthene were unaffected by ellipticine, a PAH receptor antagonist. Estimation of the anionic composition of I(sc) revealed that isoproterenol increased both HCO(3)(-) and Cl(-) transport in the control, whereas it potentiated only Cl(-) transport in the presence of fluoranthene. The fluoranthene-induced modulations of these anion transporters were counteracted by charybdotoxin (ChTx, a hIK-1 channel blocker). Fluoranthene gradually augmented the ChTx-sensitive K(+) current (I(K)) across the basolateral membrane, accompanied by a sustained increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)). In the presence of fluoranthene, however, a much larger hIK-1-dependent I(K) was identified by the application of 8-bromo-cAMP without concomitant elevation of [Ca(2+)](i). These results suggest that fluoranthene switches from cAMP-dependent HCO(3)(-) secretion to Cl(-) secretion through the hIK-1 channel, whose sensitivity to protein kinase A may be up-regulated by the sustained [Ca(2+)](i) elevation produced by this chemical.
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PMID:Effects of fluoranthene, a polycyclic aromatic hydrocarbon, on cAMP-dependent anion secretion in human airway epithelia. 1461 Feb 44

Human NBC3 is an electroneutral Na(+)/HCO(3)(-) cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO(3)(-) metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO(2) + H(2)O left arrow over right arrow HCO(3)(-) + H(+) in many tissues. We investigated whether NBC3, like some Cl(-)/HCO(3)(-) exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K(d) = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 +/- 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 microM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH(i)) recovery rate by 31 +/- 3, or 38 +/- 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH(i) recovery rate by 69 +/- 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [gamma-(32)P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO(3)(-) transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.
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PMID:Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA. 1473 10

The sensitivity of the Cl(-)/HCO(3)(-) exchanger to dopamine D(1)- and D(2)-like receptor stimulation in immortalized renal proximal tubular epithelial cells from the spontaneous hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) was examined. The activity of the Cl(-)/HCO(3)(-) exchanger (in pH U/s) in SHR cells (0.00191) was greater than in WKY cells (0.00126). The activity of Cl(-)/HCO(3)(-) exchanger was exclusively observed at the apical cell side and probably occurs through the SLC26A6 anion transporter that is expressed in both WKY and SHR cells. Stimulation of D(1)-like receptors with SKF-38393 markedly attenuated the HCO(3)(-)-dependent intracellular pH recovery in WKY cells but not in SHR cells. Stimulation of D(2)-like receptors with quinerolane did not alter Cl(-)/HCO(3)(-) exchanger activity in both WKY and SHR cells. The selective D(1)-like receptor antagonist SKF-83566 prevented the effect of SKF-38393. Both WKY and SHR cells responded to dibutyryl-cAMP (DBcAMP) with inhibition of the Cl(-)/HCO(3)(-) exchanger, and downregulation of PKA (overnight exposure to DBcAMP) abolished the inhibitory effect of both DBcAMP and SKF-38393 in WKY cells. Both SHR and WKY cells responded to forskolin with increases in the formation of cAMP. However, only WKY responded to SKF-38393 with increases in the formation of cAMP that was prevented by SKF-83566. It is concluded that WKY cells respond to D(1)-like dopamine receptor stimulation with inhibition of the apical Cl(-)/HCO(3)(-) (SLC26A6) exchanger and SHR cells have a defective D(1)-like dopamine response.
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PMID:Defective D1-like receptor-mediated inhibition of the Cl-/HCO3- exchanger in immortalized SHR proximal tubular epithelial cells. 1497 1

We have previously demonstrated the presence of a Na(+)-K(+)-2Cl cotransporter in cultured bovine corneal endothelial cells (CBCEC) and determined that this cotransporter is located in the basolateral membrane. This transporter may contribute to volume regulation and transendothelial fluid transport. We have now investigated factors regulating the activity of the cotransporter. This activity was assessed by measuring the bumetanide-sensitive (86)Rubidium ((86)Rb) uptake in (86)Rb-containing solutions. Data were normalized to protein content determined with a Lowry protein assay. We investigated the regulation by extracellular and intracellular ion concentrations, by osmotic gradients, and by second messengers. Our results indicate that extracellular Na+ and K+ each are required for activation of the cotransporter and activate with first-order kinetics at half-maximally effective concentrations (k(1/2)) of 21.1 and 1.33 mM, respectively. Extracellular Cl- is also required for cotransport activation, but shows higher order kinetics; the k(1/2) for Cl- is 28.1 mM and the Hill coefficient 2.1. HCO(3)(-) exerts a modulating effect on cotransporter activity; at 0 HCO(3)(-) the bumetanide-sensitive K(+) uptake is reduced by 30% compared to that at 26 mm HCO(3)(-). Manipulations of the intracellular [Cl-] by preincubation in Cl- -free solution or inhibition of Cl- efflux resulted in increased uptake at low [Cl-](i) and decreased uptake at high [Cl-](i). To assess the role of protein kinases in the regulation of cotransport, we have determined the effect of protein kinase inhibitors. H-89 and KT5270, inhibitors of PKA, inhibit cotransport almost completely, while calphostin C, an inhibitor of PKC, produces a small activation of cotransport. The tyrosine kinase inhibitor genistein reduced K+ uptake while its inactive analog daidzein was without effect. The calmodulin kinase inhibitor KN-93 was without effect. We also investigated the effects of phosphatase inhibitors. Calyculin A (k(1/2)=21 nM) and okadaic acid (k(1/2)=915 nM) produced approximate doubling of K+ uptake, suggesting that phosphatase 1 is dominant. We also investigated the role of the cytoskeleton and its activation. Reduction of Ca(i)(2+) by preincubation in Ca2+ -free medium as well as by exposure to W-7, an inhibitor of the binding of Ca(2+) to calmodulin, reduced K+ uptake. Consistent with this, ML-7, a relatively specific inhibitor of the Ca2+ -calmodulin activated myosin light chain kinase, inhibited cotransport by 40%. The Ca2+ -calmodulin activated myosin light chain kinase contributes to the modulation of the cytoskeleton by regulating the actin-myosin interaction. Consistent with the above, disruption of the actin polymerization by cytochalasin D led to a decrease in K+ uptake. We conclude that extracellular Na+, K+ and Cl- are requirements for the function of the CBCEC Na(+)-K(+)-2Cl(-) cotransporter, while intracellular Cl- and extracellular HCO(3)(-) modulate its activity. Several protein kinases, including PKA, PKC, tyrosine kinase, and myosin light chain kinase, modulate the K+ uptake. Another modulating pathway for cotransport involves the state of the cytoskeleton.
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PMID:Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells. 1593 33

CFTR channels conduct HCO(3)(-) in addition to Cl(-) in airway epithelial cells. A defective HCO(3)(-)-transporting function of CFTR may underlie the pathogenesis of cystic fibrosis. In the present study, we have investigated whether a HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) is functionally coupled with CFTR and thus forms an autoregulatory mechanism for HCO(3)(-) transport in human airway epithelial Calu-3 cells. A reverse transcriptase-polymerase chain reaction showed that transcripts of both full-length and truncated sACs are present in Calu-3 cells. Truncated sAC protein is the predominant, if not the only, isoform expressed in Calu-3 cells. HCO(3)(-) stimulated a modest increase in cAMP production, and the increase was sensitive to 2-hydroxyestradiol (2-HE), a sAC inhibitor, but not to SQ22,536, a blocker of conventional transmembrane adenylyl cyclases. These results suggest that sAC is functional in Calu-3 cells. Adding 25 mM HCO(3)(-) to the bath stimulated CFTR-mediated whole cell currents in the absence, but not in the presence, of 2-HE. In cell-attached membrane patches, 25 or 50 mM HCO(3)(-) in the bath markedly increased the product of channel number and open probability of CFTR, and this activation was attenuated by 2-HE. These findings demonstrate that sAC signaling pathway is involved in the regulation of CFTR function in human airway epithelium and thereby provides a link between the level of intracellular HCO(3)(-)/CO(2) and the modulation of HCO(3)(-)-conductive CFTR function by cAMP/PKA.
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PMID:Regulation of CFTR channels by HCO(3)--sensitive soluble adenylyl cyclase in human airway epithelial cells. 1595 23

Endothelin (ET) and nitric oxide (NO) modulate ion transport in the kidney. In this study, we defined the function of ET receptor subtypes and the NO guanylate cyclase signaling pathway in mediating the adaptation of the rabbit cortical collecting duct (CCD) to metabolic acidosis. CCDs were perfused in vitro and incubated for 3 h at pH 6.8, and bicarbonate transport or cell pH was measured before and after acid incubation. Luminal chloride was reversibly removed to isolate H(+) and HCO(3)(-) secretory fluxes and to raise the pH of beta-intercalated cells. Acid incubation caused reversal of polarity of net HCO(3)(-) transport from secretion to absorption, comprised of a 40% increase in H(+) secretion and a 75% decrease in HCO(3)(-) secretion. The ET(B) receptor antagonist BQ-788, as well as the NO synthase inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME), attenuated the adaptive decrease in HCO(3)(-) secretion by 40%, but only BQ-788 inhibited the adaptive increase in H(+) secretion. There was no effect of inactive d-NAME or the ET(A) receptor antagonist BQ-123. Both BQ-788 and l-NAME inhibited the acid-induced inactivation (endocytosis) of the apical Cl(-)/HCO(3)(-) exchanger. The guanylate cyclase inhibitor LY-83583 and cGMP-dependent protein kinase inhibitor KT-5823 affected HCO(3)(-) transport similarly to l-NAME. These data indicate that signaling via the ET(B) receptor regulates the adaptation of the CCD to metabolic acidosis and that the NO guanylate cyclase component of ET(B) receptor signaling mediates downregulation of Cl(-)/HCO(3)(-) exchange and HCO(3)(-) secretion.
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PMID:Endothelin and nitric oxide mediate adaptation of the cortical collecting duct to metabolic acidosis. 1670 53

Although aldosterone influences a variety of cellular processes through nongenomic mechanisms, the significance of nongenomic pathways for aldosterone-induced regulation of epithelial function is not understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO(3)(-) absorption in the medullary thick ascending limb (MTAL) through a nongenomic pathway. This inhibition is mediated through a direct cellular action of aldosterone to inhibit the apical membrane NHE3 Na(+)/H(+) exchanger. The present study was designed to identify the intracellular signaling pathway(s) responsible for this aldosterone-induced transport regulation. In rat MTALs perfused in vitro, addition of 1 nM aldosterone to the bath decreased HCO(3)(-) absorption by 30%. This inhibition was not mediated by cAMP/PKA and was not prevented by inhibitors of PKC or PI3-K, pertussis toxin, or rapamycin. The inhibition of HCO(3)(-) absorption by aldosterone was largely eliminated by the MEK/ERK inhibitors U-0126 and PD-98059. Aldosterone increased ERK activity 1.8-fold in microdissected MTALs. This ERK activation is rapid (</=5 min) and is blocked by U-0126 or PD-98059 but is unaffected by spironolactone or actinomycin D. Pretreatment with U-0126 to block ERK activation prevented the effect of aldosterone to inhibit apical NHE3. These data demonstrate that aldosterone inhibits NHE3 and HCO(3)(-) absorption in the MTAL through rapid activation of the ERK signaling pathway. The results identify NHE3 as a target for nongenomic regulation by aldosterone and establish a role for ERK in the acute regulation of NHE3 and its epithelial absorptive functions.
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PMID:Aldosterone inhibits apical NHE3 and HCO3- absorption via a nongenomic ERK-dependent pathway in medullary thick ascending limb. 1675 29

The Na(+)-driven Cl(-)/HCO(3)(-) exchanger (NCBE) plays an important role in the regulation of intracellular pH (pH(i)). We previously identified two variants of NCBE from rat brain of which the variant with a carboxyterminal PSD-95/Dlg/ZO-1 (PDZ) motif (rb2NCBE) colocalized with the actin cytoskeleton. Increased rb2NCBE activity by PKA inhibition and reduction by forskolin and cAMP agonist suggest PKA regulation of NCBE. Disruption of actin filaments also decreased rb2NCBE activity. EBP50 and FLAG-rb2NCBE were reciprocally co-immunoprecipitated from rb2NCBE transfected cells. It is concluded that NCBE activity is inhibited by PKA and depends on the integrity of the actin cytoskeleton within a multiprotein complex at the plasma membrane.
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PMID:Regulation of the rat brain Na+ -driven Cl-/HCO3 - exchanger involves protein kinase A and a multiprotein signaling complex. 1691 13

Tg737(orpk) mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737(orpk) mutants is associated with defects in ion transport, we compared the steady-state pH(i) and Na(+)-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737(orpk) mutant and wild-type mice. The data indicate that Tg737(orpk) mutant choroid plexus epithelium have lower pH(i) and higher Na(+)-dependent HCO(3)(-) transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na(+)-dependent HCO(3)(-) transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737(orpk) mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH(i) and ion transport activity.
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PMID:Altered pH(i) regulation and Na(+)/HCO3(-) transporter activity in choroid plexus of cilia-defective Tg737(orpk) mutant mouse. 1718 27

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO(2)/HCO(3)(-) during exacerbations of airway diseases, but the role of CO(2)/HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO(2)/HCO(3)(-)-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO(2)/HCO(3)(-)-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO(2)/HCO(3)(-) increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.
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PMID:Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP. 1759 88


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