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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation in human airway epithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short-circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and ATP. CFTR as the current carrier for HCO3- was identified by 1) stimulation by cAMP, 2) ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing mutated delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 monolayers, cytosolic addition of 100 microM cAMP stimulated apical HCO3- currents from -9.4 +/- 1.6 to -31.1 +/- 3.9 microA/cm2 (n = 18), and apical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 microA/cm2 (n = 27). Average relative permselectivity for HCO3- vs. Cl- was approximately 15%. Absence of cytosolic ATP resulted in loss of cAMP stimulation of HCO3- and Cl- currents. Genistein (50 microM), which has been proposed to inhibit phosphatases controlling apical CFTR, as well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) further activated cAMP-stimulated HCO3- and Cl- currents. Activated currents remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channel blockers glibenclamide (300 microM) and N-phenylanthranilic acid (5 mM), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 microM), inhibited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker effects were absent in human CF tracheal cells homozygous for the delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in CFT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maximize CFTR activity in airway epithelial cells.
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PMID:cAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia. 914 51

The role of Na(+)-K(+)-2Cl- cotransport in ion and fluid transport of the corneal endothelium was examined by measuring changes in corneal hydration and uptake of 86Rb by the endothelial cell layer. Isolated, intact rabbit corneas maintain normal hydration when they are superfused at the endothelial surface with bicarbonate (HCO3-)-Ringer solutions as a result of equilibrium between active ion and fluid transport out of the stromal tissue and leak of fluid into stromal tissue from the aqueous humor. Furosemide and bumetanide did not alter this equilibrium when they were added to the superfusion medium. Uptake of 86Rb by the endothelium of the incubated cornea was increased 25% by bumetanide, but uptake in the presence of ouabain (70% less than that of controls) was not changed by bumetanide. In Na(+)-free medium, uptake of 86Rb was reduced by 58%, but it was unchanged in Cl(-)-free medium. Calyculin A, a protein phosphatase inhibitor and activator of Na(+)-K(+)-Cl- cotransport, was without effect on 86Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes was significantly different when bumetanide was present in the media. It is concluded that Na(+)-K(+)-2Cl- cotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport of this cell layer. Its presence in cultured endothelial cells may reflect the reported importance of this protein in growth, proliferation, and differentiation.
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PMID:Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na(+)-K(+)-2Cl- cotransport. 937 32

Bicarbonate excretion in bile is a major function of the biliary epithelium. It is driven by the apically located Cl-/HCO3- exchanger which is functionally coupled with a cAMP-dependent Cl- channel (CFTR). A number of hormones and/or neuropeptides with different mechanisms and at different intracellular levels regulate, in concert, the processes underlying bicarbonate excretion in the biliary epithelium. Secretin induces a bicarbonate rich choleresis by stimulating the activity of the Cl-/HCO3- exchanger by cAMP and protein kinase A mediated phosphorylation of CFTR regulatory domain. Protein phosphatase 1/2A are involved in the run-down of secretory stimulus after secretin removal. Acetylcholine potentiates secretin-choleresis by inducing a Ca(++)-calcineurin mediated "sensitization" of adenyl cyclase to secretin. Bombesin and vasoactive intestinal peptide also enhance the Cl-/HCO3- exchanger activity, but the intracellular signal transduction pathway has not yet been defined. Somatostatin and gastrin inhibit basal and/or secretin-stimulated bicarbonate excretion by down-regulating the secretin receptor and decreasing cAMP intracellular levels induced by secretin.
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PMID:Hormonal regulation of bicarbonate secretion in the biliary epithelium. 962 62

The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pHi). Furthermore, we characterized the effects on pHi regulation of exposure to 1 microM ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2',7' Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pHi was 7.1 in NaCl (nominally HCO3-free) Ringers. Inhibition of NHE with 100 microM dimethylamiloride (DMA) rapidly decreased pHi by 0.37 units. Similarly, selective inhibition of V-type H+-ATPase with 10 microM bafilomycin A1 decreased pHi by 0.22 units. Following acid loading in NaCl Ringers with a 20 mm NH4Cl prepulse, pHi recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 microM DMA or 20 microM bafilomycin A1. Based on decreases in H+ efflux resulting from selective inhibition of NHE and V-type H+ pump activity, NHE activity accounts for 76% of the pHi recovery following acid loading. Under control conditions, ET-1 (1 microM) had no effect on pHi whereas ET-1 completely suppressed pHi recovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ETA because in the presence of BQ-123 (10 microM), a selective ETA receptor antagonist, pHi recovery was completely restored. Suppression of pHi recovery also occurred following stimulation of protein kinase C (PKC) with 10(-7) m phorbol myristate (PMA) whereas 10(-7) m 4 alpha phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pHi recovery after inhibition of PKC with 0.5 microM calphostin C suggesting that the inhibition of pHi recovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca2+-dependent calmodulin stimulated CaM II kinase with KN-62 (10 microM) reversed the suppression of pHi recovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 microM phenylarsine oxide, 10 nm okadaic acid, 10 microM cyclosporin A1 or 20 microM BAPTA, also obviated the suppression of pHi recovery by ET-1. Therefore ETA receptor mediated inhibition of pHi regulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H+ pump. This alteration may prevent them from becoming adequately stimulated to elicit pHi recovery in response to acid loading.
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PMID:ETA receptor mediated inhibition of intracellular pH regulation in cultured bovine corneal epithelial cells. 965 2

1. We examined the effects of noradrenaline on steady-state intracellular pH (pHi) and the recovery of pHi from internal acid loads imposed by the NH4+ prepulse technique in hippocampal CA1 neurones acutely dissociated from adult rats. 2. Under nominally HCO3--free conditions, acid extrusion was accomplished by a Na+-dependent mechanism, probably the amiloride-insensitive variant of the Na+-H+ exchanger previously characterized in both fetal and adult rat hippocampal neurones. In the presence of external HCO3-, acid extrusion appeared to be supplemented by a Na+-dependent HCO3--Cl- exchanger, the activity of which was dependent upon the absolute level of pHi. 3. Noradrenaline evoked a concentration-dependent and sustained rise in steady-state pHi and increased rates of pHi recovery from imposed intracellular acid loads. The effects of noradrenaline were not dependent upon the presence of external HCO3- but were blocked by substituting external Na+ with N-methyl-D-glucamine, suggesting that noradrenaline acts to increase steady-state pHi by increasing the activity of the Na+-H+ exchanger. 4. The effects of noradrenaline on steady-state pHi and on rates of pHi recovery from imposed acid loads were mimicked by beta1- and beta2-, but not alpha-, adrenoceptor agonists. The beta-adrenoceptor antagonist propranolol blocked the ability of noradrenaline to increase both steady-state pHi and rates of pHi recovery from acid loads. 5. The effects of noradrenaline on steady-state pHi and on pHi recovery rates following acid loads were not dependent on changes in [Ca2+]i. However, the effects of noradrenaline were blocked by pre-treatment with the adenylate cyclase inhibitor 2',5'-dideoxyadenosine and the cAMP-dependent protein kinase inhibitors Rp-adenosine-3',5'-cyclic monophosphorothioate (sodium salt; Rp-cAMPS) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H-89). 6. Forskolin, an activator of endogenous adenylate cyclase, and 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, mimicked the ability of noradrenaline to increase both steady-state pHi and rates of pHi recovery from imposed acid loads, as did Sp-cAMPS, a selective activator of cAMP-dependent protein kinase. The effect of forskolin on steady-state pHi was blocked by pre-treatment with Rp-cAMPS whereas the effect of Sp-cAMPS was enhanced by pre-treatment with the protein phosphatase inhibitor, okadaic acid. 7. Noradrenaline also increased steady-state pHi and rates of pHi recovery from imposed acid loads in cultured postnatal rat hippocampal neurones. In this preparation, the effects of noradrenaline were occluded by 18-24 h pre-treatment with cholera toxin. 8. We conclude that noradrenaline increases the activity of the Na+-H+ exchanger in rat hippocampal neurones, probably by inducing an alkaline shift in the pHi dependence of the antiport, thereby raising steady-state pHi. The effects of noradrenaline are mediated by beta-adrenoceptors via a pathway which involves the alpha-subunit of the stimulatory G-protein Gs (Gsalpha), adenylate cyclase, cAMP and the subsequent activation of cAMP-dependent protein kinase which, in turn, may phosphorylate the exchange mechanism.
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PMID:Effects of noradrenaline on intracellular pH in acutely dissociated adult rat hippocampal CA1 neurones. 976 38

Bicarbonate/CO(2), a physiological effector of sperm capacitation, has been shown to induce a rapid and reversible change in the lipid architecture of the plasma membrane of live boar sperm: the change is detectable as an increase in the cells' ability to bind the fluorescent dye merocyanine, a characteristic which implied an increase in lipid packing disorder (Harrison et al. 1996. Mol Reprod Dev 45:378-391). Evidence suggested that cAMP may act as a second messenger in the system, and we have therefore investigated this cAMP-dependency in more detail. Bicarbonate stimulates cAMP levels within 1 min in a dose-dependent fashion, prior to parallel increases in merocyanine binding. Although the potent somatic cell adenylyl cyclase activator forskolin is unable to induce significant increases in cAMP or merocyanine binding, increases in merocyanine binding are inducible in a dose-dependent fashion by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate, a cAMP analogue highly specific in its ability to stimulate protein kinase A; moreover, the bicarbonate-induced membrane change is inhibited by H89, a specific protein kinase A inhibitor. Neither bisindolylmaleimide I (protein kinase C inhibitor) nor lavendustin A (protein tyrosine kinase inhibitor) are inhibitory. In the presence of low levels of the potent phosphodiesterase inhibitor papaverine, increases in merocyanine binding are enhanced by okadaic acid and (more effectively) by calyculin (both protein phosphatase inhibitors). We conclude that boar sperm plasma membrane lipid architecture is controlled via a target protein that is dynamically phosphorylated by cAMP-dependent protein kinase and dephosphorylated by protein phosphatase type 1. Mol. Reprod. Dev. 55:220-228, 2000.
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PMID:cAMP-dependent protein kinase control of plasma membrane lipid architecture in boar sperm. 1061 62

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.
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PMID:Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology. 1088 79

Because poorly motile sperm samples can often be stimulated by treatments that increase intracellular levels of cyclic adenosine monophosphate (cAMP), it has been supposed that such samples are unable to maintain an adequate supply of the cyclic nucleotide with which to activate protein kinase A (PKA). To investigate this hypothesis, we incubated boar sperm samples with bicarbonate (a stimulator of adenylyl cyclase) and compared its effect with that of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (cBIMPS, a highly permeable and stable cAMP analog). Videomicroscopy assessment of motility was followed by computer analysis of the sperm tracks to produce motility descriptor values for many individual cells in each sample, whence "cluster" analysis of these data identified groups of spermatozoa that differed in motility characteristics. Bicarbonate stimulation of motility was characterized by an increase in the linearity (LIN) and progressive velocity of part of the sperm population only. The size of this "fast linear" subpopulation varied considerably between ejaculates. However, treatment with cBIMPS did not induce significantly more "fast linear" sperm than treatment with bicarbonate. In further experiments investigating the role of protein kinases in motility control, bicarbonate stimulation was greatly inhibited by H89 (a specific inhibitor of PKA), whereas GF109203X and lavendustin A (inhibitors of protein kinase C [PKC] and protein tyrosine kinase [PTK], respectively) had essentially no effect. While inclusion of the protein phosphatase inhibitor calyculin stimulated motility, it failed to increase the overall percentage of "fast linear sperm" induced by bicarbonate. We conclude that intersperm and interejaculate differences in boar sperm motility are not due to inadequacy in cAMP supply or to ineffective PKA activity.
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PMID:Bicarbonate stimulation of boar sperm motility via a protein kinase A-dependent pathway: between-cell and between-ejaculate differences are not due to deficiencies in protein kinase A activation. 1206 64

Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO(2)-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing. We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10(7) cells), achieving more than 70% yield of membrane proteins. Phosphopeptide enrichment by titanium dioxide chromatography followed by capillary liquid chromatography-tandem mass spectrometry allowed us to assign 703 unique phosphorylation sites in 376 phosphoproteins. Our experiments revealed that treatment of cell cultures with three different types of protein phosphatase inhibitors produces distinct phosphopeptide populations and an increase of 10-40% of the number of detected and sequenced phosphoserine, phosphothreonine and phosphotyrosine containing peptides. In summary, our analytical strategy enables functional phosphoproteomic analysis of stem cell differentiation and cell surface biomarker discovery using very low amounts of starting material.
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PMID:TiO(2)-based phosphoproteomic analysis of the plasma membrane and the effects of phosphatase inhibitor treatment. 1857 22

To analyze the cardiac functions of AE3, we disrupted its gene (Slc4a3) in mice. Cl(-)/HCO3(-) exchange coupled with Na+-dependent acid extrusion can mediate pH-neutral Na+ uptake, potentially affecting Ca2+ handling via effects on Na+/Ca2+ exchange. AE3 null mice appeared normal, however, and AE3 ablation had no effect on ischemia-reperfusion injury in isolated hearts or cardiac performance in vivo. The NKCC1 Na+-K+-2Cl(-) cotransporter also mediates Na+ uptake, and loss of NKCC1 alone does not impair contractility. To further stress the AE3-deficient myocardium, we combined the AE3 and NKCC1 knock-outs. Double knock-outs had impaired contraction and relaxation both in vivo and in isolated ventricular myocytes. Ca2+ transients revealed an apparent increase in Ca2+ clearance in double null cells. This was unlikely to result from increased Ca2+ sequestration, since the ratio of phosphorylated phospholamban to total phospholamban was sharply reduced in all three mutant hearts. Instead, Na+/Ca2+ exchanger activity was found to be enhanced in double null cells. Systolic Ca2+ was unaltered, however, suggesting more direct effects on the contractile apparatus of double null myocytes. Expression of the catalytic subunit of protein phosphatase 1 was increased in all mutant hearts. There was also a dramatic reversal, between single null and double null hearts, in the carboxymethylation and localization to the myofibrillar fraction, of the catalytic subunit of protein phosphatase 2A, which corresponded to the loss of normal contractility in double null hearts. These data show that AE3 and NKCC1 affect Ca2+ handling, PLN regulation, and expression and localization of major cardiac phosphatases and that their combined loss impairs cardiac function.
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PMID:Impaired cardiac contractility in mice lacking both the AE3 Cl-/HCO3- exchanger and the NKCC1 Na+-K+-2Cl- cotransporter: effects on Ca2+ handling and protein phosphatases. 1877 25


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