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 effects of anoxia on intracellular pH (pH(i)) were examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H(+)-sensitive fluorophore, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. During perfusion with HCO/CO(2)- or HEPES-buffered media (pH 7.35) at 37 degrees C, 5- or 10-min anoxic insults were typified by an intracellular acidification on the induction of anoxia, a subsequent rise in pH(i) in the continued absence of O(2), and a further internal alkalinization on the return to normoxia. The steady-state pH(i) changes were not consequent on changes in [Ca(2+)](i) and, examined in the presence of HCO, were not significantly affected by (DIDS). In the absence of HCO, the magnitude of the postanoxic alkalinization was attenuated when external Na(+) was reduced by substitution with N-methyl-D-glucamine (NMDG(+)), but not Li(+), suggesting that increased Na(+)/H(+) exchange activity contributes to this phase of the pH(i) response. In contrast, 100-500 microM Zn(2+), a known blocker of H(+)-conductive pathways, reduced the magnitudes of the internal alkalinizations that occurred both during and following anoxia. The effects of NMDG(+)-substituted medium and Zn(2+) to reduce the increase in pH(i) that occurred after anoxia were additive. Consistent with the steady-state pH(i) changes, rates of pH(i) recovery from internal acid loads imposed immediately after anoxia were increased, and the application of Zn(2+) and/or perfusion with NMDG(+)-substituted medium slowed pH(i) recovery. Reducing extracellular pH from 7.35 to 6.60, or reducing ambient temperature from 37 degrees C to room temperature, also attenuated the increases in steady-state pH(i) observed during and after anoxia and reduced rates of pH(i) recovery from acid loads imposed in the immediate postanoxic period. Finally, inhibition of the cAMP/protein kinase A second-messenger system reduced the magnitude of the rise in pH(i) after anoxia in a manner that was dependent on external Na(+); conversely, activation of the system with isoproterenol increased the postanoxic alkalinization, an effect that was attenuated by pretreatment with propranolol, Rp-cAMPS, or when NMDG(+) (but not Li(+)) was employed as an external Na(+) substitute. The results suggest that a Zn(2+)-sensitive acid efflux mechanism, possibly a H(+)-conductive pathway activated by membrane depolarization, contributes to the internal alkalinization observed during anoxia in adult rat CA1 neurons. The rise in pH(i) after anoxia reflects acid extrusion via the H(+)-conductive pathway and also Na(+)/H(+) exchange, activation of the latter being mediated, at least in part, through a cAMP-dependent signaling pathway.
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PMID:Intracellular pH response to anoxia in acutely dissociated adult rat hippocampal CA1 neurons. 1197 62

Na(+)-HCO(3)(-) cotransporters play an important role in intracellular pH regulation and transepithelial HCO(3)(-) transport in various tissues. Of the characterized members of the HCO(3)(-) transporter superfamily, NBC1 and NBC4 proteins are known to be electrogenic. An important functional property of electrogenic Na(+)-HCO(3)(-) cotransporters is their HCO(3)(-):Na(+) coupling ratio, which sets the transporter reversal potential and determines the direction of Na(+)-HCO(3)(-) flux. Recent studies have shown that the HCO(3)(-):Na(+) transport stoichiometry of NBC1 proteins is either 2:1 or 3:1 depending on the cell type in which the transporters are expressed, indicating that the HCO(3)(-):Na(+) coupling ratio can be regulated. Mutational analysis has been very helpful in revealing the molecular mechanisms and signaling pathways that modulate the coupling ratio. These studies have demonstrated that PKA-dependent phosphorylation of the COOH terminus of NBC1 proteins alters the transport stoichiometry. This cAMP-dependent signaling pathway provides HCO(3)(-) -transporting epithelia with an efficient mechanism for modulating the direction of Na(+)-HCO(3)(-) flux through the cotransporter.
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PMID:Structural determinants and significance of regulation of electrogenic Na(+)-HCO(3)(-) cotransporter stoichiometry. 1237 62

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCD(i)) and terminal (hIMCD(t)) inner medullary regions of human kidneys. AVP, PGE(2), and forskolin increased cAMP in both hIMCD(i) and hIMCD(t) cells. The effects of AVP and PGE2 were greatest in hIMCD(i); however, forskolin increased cAMP to the same extent in hIMCD(i) and hIMCD(t). Basal short-circuit current (I(SC)) of hIMCD(i) monolayers was 1.4 +/- 0.5 microA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE(2), and forskolin increased I(SC); the current was reduced by blocking PKA, apical Cl- channels, basolateral NKCC1 (a Na+ - K+ - 2Cl- cotransporter), and basolateral Cl-/HCO(3)(-) exchangers. In fluid transport studies, hIMCD(i) monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCD(t) monolayers, basal current was not different from zero and cAMP had no effect on I(SC). We conclude that AVP and PGE2 stimulate cAMP-dependent Cl- secretion by hIMCD(i) cells, but not hIMCD(t) cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.
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PMID:Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells. 1238 81

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na(+)-dependent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO(3)(-) transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO(3)(-)-transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.
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PMID:The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3. 1240 79

The HCO(3)(-) : Na(+) cotransport stoichiometry of the electrogenic sodium bicarbonate cotransporter kNBC1 determines the reversal potential (E(rev)) and thus the net direction of transport of these ions through the cotransporter. Previously, we showed that phosphorylation of kNBC1-Ser(982) in the carboxy-terminus of kNBC1 (kNBC1-Ct), by cAMP-protein kinase A (PKA), shifts the stoichiometry from 3 : 1 to 2 : 1 and that binding of bicarbonate to the cotransporter is electrostaticaly modulated. These results raise the possibility that phosphorylated kNBC1-Ser(982), or other nearby negatively charged residues shift the stoichiometry by blocking a bicarbonate-binding site. In the current study, we examined the role of the negative charge on Ser(982)-phosphate and three aspartate residues in a D986NDD custer in altering the stoichiometry of kNBC1. mPCT cells expressing kNBC1 mutants were grown on filters and mounted in an Ussing chamber for electrophysiological studies. Enhanced green fluorescence protein (EGFP)-tagged mutant constructs expressed in the same cells were used to determine the phosphorylation status of kNBC1-Ser(982). The data indicate that both kNBC1-Asp(986) and kNBC1-Asp(988), but not kNBC1-Asp(989), are required for the phosphorylation-induced shift in stoichiometry. A homologous motif (D887ADD) in the carboxy-terminus of the anion exchanger AE1 binds to carbonic anhydrase II (CAII). In isothermal titration calorimetry experiments, CAII was found to bind to kNBC1-Ct with a K(D) of 160 +/- 10 nM. Acetazolamide inhibited the short-circuit current through the cotransporter by 65 % when the latter operated in the 3 : 1 mode, but had no effect on the current in the 2 : 1 mode. Acetazolamide did not affect the cotransport stoichiometry or the ability of 8-Br-cAMP to shift the stoichiometry. Although CAII does not affect the transport stoichiometry, it may play an important role in enhancing the flux through the transporter when kNBC1-Ser(982) is unphosphorylated.
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PMID:Regulation of the sodium bicarbonate cotransporter kNBC1 function: role of Asp(986), Asp(988) and kNBC1-carbonic anhydrase II binding. 1241 14

The contributions of HCO(3)(-)-dependent, DIDS-sensitive mechanisms to the maintenance of steady-state pH(i), and the regulation of their activities by cAMP-dependent protein kinase (PKA), were investigated in CA1 neurons with the H(+)-sensitive fluorophore, BCECF. The addition of HCO(3)(-)/CO(2) to neurons with "low" (pH(i) < or = 7.20) and "high" (pH(i) > 7.20) initial pH(i) values under Hepes-buffered conditions, increased and decreased steady-state pH(i), respectively. Conversely, under HCO(3)(-)/CO(2)-buffered conditions, DIDS caused pH(i) to decrease and increase in neurons with low and high initial pH(i) values, respectively. In the presence, but not the absence, of HCO(3)(-), the PKA inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS; 50 microM) evoked DIDS-sensitive increases and decreases in pH(i) in neurons with low and high initial pH(i) values, respectively. In contrast, in neurons with low initial pH(i) values, activation of PKA with the Sp isomer of cAMPS (Sp-cAMPS; 25 microM) elicited increases in pH(i) that were smaller in the presence than in the absence of HCO(3)(-), whereas in neurons with high initial pH(i) values, Sp-cAMPS-evoked rises in pH(i) were larger in the presence than in the absence of HCO(3)(-); the differences between the effects of Sp-cAMPS on pH(i) under the different buffering conditions were attenuated by DIDS. Consistent with the possibility that changes in the activities of HCO(3)(-)-dependent, DIDS-sensitive mechanisms contribute to the steady-state pH(i) changes evoked by the PKA modulators, in neurons with initial pH(i) values < or = 7.20, Rp-cAMPS concurrently inhibited Na(+)-independent Cl(-)-HCO(3)(-) exchange and stimulated Na(+)-dependent Cl(-)-HCO(3)(-) exchange; in contrast, Sp-cAMPS concurrently stimulated Na(+)-independent Cl(-)-HCO(3)(-) exchange and inhibited Na(+)-dependent Cl(-)-HCO(3)(-) exchange. Data from a limited number of neurons with initial pH(i) values > 7.20 suggested that the directions of the reciprocal changes in anion exchange activities (inhibition or stimulation) evoked by Rp- and Sp-cAMPS may be opposite in cells with low vs. high resting pH(i) values. Taken together, the results indicate that the effects of modulating PKA activity on steady-state pH(i) in rat CA1 neurons under HCO(3)(-)/CO(2)-buffered conditions reflect not only changes in Na(+)-H(+) exchange activity but also changes in Na(+)-dependent and Na(+)-independent Cl(-)-HCO(3)(-) exchange activity that, in turn, may be dependent upon the initial pH(i).
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PMID:Regulation of Cl--HCO3- exchangers by cAMP-dependent protein kinase in adult rat hippocampal CA1 neurons. 1248 90

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of "sperm capacitation." We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO(3)(-)/cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO(3)(-) has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO(3)(-) but not Cl(-) induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO(3)(-)-dependent hyperpolarization was not observed when Na(+) was replaced by the non-permeant cation choline(+). Replacement of Na(+) by choline(+) also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO(3)(-) current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na(+) suggest that a Na(+)/HCO(3)(-) cotransporter is present in mouse sperm and is coupled to events regulating capacitation.
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PMID:Involvement of a Na+/HCO-3 cotransporter in mouse sperm capacitation. 1249 93

We examined the effect of several protein kinase inhibitors, such as staurosporine for protein kinase C (PKC), H-89 for protein kinase A (PKA) and genistein for tyrosine kinase (TK) on acid-induced duodenal bicarbonate secretion (DBS) in rats. HCO(-)(3) secretion was measured using the pH-stat method. Mucosal acidification was performed by perfusing the duodenal loop for 10 min with pH 2.2 HCl. Indomethacin, staurosporine and genistein were added to acidified saline and then perfused, respectively. In some cases, genistein and phorbol 12-myristate 13-acetate (PMA) were added to the luminal solution to examine the effect on basal duodenal HCO(-)(3) secretion. PGE(2) (PKA pathway) and PMA (PKC pathway) stimulate basal DBS. Indomethacin, H-89, staurosporine and genistein inhibit acid-induced DBS, indicating involvement of the cyclooxygenase, PKA, PKC and TK pathways.
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PMID:Role of protein kinases on acid-induced duodenal bicarbonate secretion in rats. 1256 54

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO(3)(-) and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25 degrees C. Local perfusion with HCO(3)(-) produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO(3)(-) begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8+/-0.2 seconds at 15 mM HCO(3)(-) and 17.5+/-0.4 seconds at 1 mM HCO(3)(-). Removal of external HCO(3)(-) allows a slow return to basal beat frequency over approximately 10 minutes. Increases in beat symmetry accompany the accelerating action of HCO(3)(-). As in our past work, HCO(3)(-) also facilitates opening of voltagegated Ca(2+) channels, increasing the depolarization-evoked rate of rise of intracellular Ca(2+) concentration by more than fivefold. This action also is detectable at 1 mM HCO(3)(-) and occurs with an apparent halftime of approximately 60 seconds at 15 mM HCO(3)(-). The dual actions of HCO(3)(-) respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO(3)(-) on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 micro M cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca(2+) concentration from 17+/-4 to 41+/-6 nM second(-1). However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO(3)(-), perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO(3)(-) produces during sperm activation.
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PMID:Bicarbonate actions on flagellar and Ca2+ -channel responses: initial events in sperm activation. 1258 48

In this report, we describe the cloning, cellular localization, and functional characteristics of Na(+)/H(+) exchanger 1 (NHE1) from red blood cells of the winter flounder Pseudopleuronectes americanus (paNHE1). The paNHE1 protein localizes primarily to the marginal band and exhibits a 74% similarity to the trout beta-NHE, and 65% to the human NHE1 (hNHE1). Functionally, paNHE1 shares characteristics of both beta-NHE and hNHE1 in that it is activated both by manipulations that increase cAMP and by cell shrinkage, respectively. In accordance, the paNHE1 protein exhibits both protein kinase A consensus sites as in beta-NHE and a region of high homology to that required for shrinkage-dependent activation of hNHE1. After shrinkage-dependent activation of paNHE1 and resulting activation of a Cl(-)/HCO(3)(-) exchanger, their parallel operation results in net uptake of NaCl and osmotically obliged water. Activation of paNHE1 by cAMP is at least additive to that elicited by osmotic shrinkage, suggesting that these stimuli regulate paNHE1 by distinct mechanisms. Finally, exposure to the serine/threonine phosphatase inhibitor calyculin A potently activates paNHE1, and this activation is also additive to that induced by shrinkage or cAMP.
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PMID:Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage, cAMP, and calyculin A. 1273 9


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