Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NHE3, a cloned intestinal and renal brush border Na+/H+ exchanger, has previously been shown to be both stimulated and inhibited by different protein kinases/growth factors. For instance, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by protein kinase C. In the present study, we used a series of NHE3 C terminus truncation mutants to identify separate regions of the C-terminal cytoplasmic tail responsible for stimulation and inhibition by protein kinases/growth factors. Five NHE3 C terminus truncation mutant stable cell lines were generated by stably transfecting NHE3 deletion cDNAs into PS120 fibroblasts, which lack any endogenous Na+/H+ exchanger. Using fluorometric techniques, the effects of the calcium/calmodulin (CaM) inhibitor W13, calcium/CaM kinase inhibitor KN-62, phorbol myristate acetate, okadaic acid, FGF, and fetal bovine serum on Na+/H+ exchange were studied in these transfected cells. Inhibition of basal activity of full-length NHE3 is mediated by CaM at a site C-terminal to amino acid 756; this CaM effect occurs through both kinase dependent and independent mechanisms. There is another independent inhibitory domain for protein kinase C between amino acids 585 and 689. In addition, there are at least three stimulatory regions in the C-terminal domain of NHE3, corresponding to amino acids 509-543 for okadaic acid, 475-509 for FGF, and a region N-terminal to amino acid 475 for fetal bovine serum. We conclude that separate regions of the C terminus of NHE3 are involved with stimulation or inhibition of Na+/H+ exchange activity, with both stimulatory and inhibitory domains having several discrete subdomains. A conservative model to explain the way these multiple domains in the C terminus of NHE3 regulate Na+/H+ exchange is via an effect on associated regulatory proteins.
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PMID:Separate C-terminal domains of the epithelial specific brush border Na+/H+ exchanger isoform NHE3 are involved in stimulation and inhibition by protein kinases/growth factors. 777 26

N-terminal peptides of parathyroid hormone (PTH) and PTH-related peptide (PTHRP) elicit a wide variety of biological responses in target cells, including the inhibition of Na+/H+ exchanger NHE3 activity in renal cells. This response is believed to be mediated by ligand binding to a common receptor (i.e. PTH/PTHRP receptor type I) and activation of cAMP-dependent and/or Ca2+/phospholipid-dependent protein kinases (PKA and PKC, respectively). However, the mechanism of action of these N-terminal peptides is now unclear because of recent data reporting the existence of additional receptor isoforms. Therefore, to directly examine the ligand binding and signaling characteristics of the PTH/PTHRP receptor type I and its ability to elicit a biological response, cDNAs encoding the rat type I receptor and the rat NHE3 isoform were transfected into Chinese hamster ovary (AP-1) cells that lack endogenous expression of these proteins. Competition binding assays using [125I-Tyr36]PTHRP-(1-36)-NH2 radioligand indicated that several biologically active human N-terminal PTH and PTHRP fragments (PTH-(1-34), PTH-(3-34), PTH-(28-42), PTH-(28-48), and PTHRP-(1-34)) were capable of binding to the type I receptor. Both PTH-(1-34) and PTHRP-(1-34) stimulated adenylate cyclase and PKC activities in these cells, whereas PTH-(3-34), PTH-(28-42), and PTH-(28-48) selectively enhanced only PKC activity. PTHRP-(1-16), a biologically inert fragment, was incapable of binding to this receptor and influencing either the PKA or PKC pathway. Furthermore, all the analogues with the exception of PTHRP-(1-16) inhibited NHE3 activity. Inhibition of PKC by the potent antagonist chelerythrine chloride abolished the depression of NHE3 activity by PTH-(3-34), PTH-(28-42), and PTH-(28-48) but did not alleviate the effects of PTH-(1-34). Likewise, antagonism of PKA by H-89 was unable to prevent the inhibition caused by PTH-(1-34). However, inhibition of both PKA and PKC by the nonselective protein kinase antagonist H-7 abolished the reduction of NHE3 activity by PTH-(1-34). These data indicate that discrete N-terminal analogues of PTH and PTHRP can interact with the classical PTH/PTHRP receptor type I and activate PKA and/or PKC. Activation of either signaling pathway independently leads to inhibition of NHE3.
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PMID:Structurally diverse N-terminal peptides of parathyroid hormone (PTH) and PTH-related peptide (PTHRP) inhibit the Na+/H+ exchanger NHE3 isoform by binding to the PTH/PTHRP receptor type I and activating distinct signaling pathways. 866 42

Endothelin-1 (ET-1) binding to ETB receptors increases the activity of the apical membrane Na+/H+ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPETB6 cells, a clonal cell line of OKP cells that overexpresses ETB receptors, ET-1-induced increases in Na+/H+ antiporter activity are mediated 50% by Ca2(+)-dependent pathways and 50% by tyrosine kinase pathways. ET-1 induces tyrosine phosphorylation of proteins of 68, 110, 125, 130, and 210 kDa. ET-1-induced tyrosine phosphorylation is mediated by the ETB receptor and is not dependent on increases in cell Ca2+ or protein kinase C. The 68-, 110-, 125-, and 130-kDa phosphoproteins are cytosolic, whereas the 210-kDa phosphoprotein is an integral membrane protein. Immunoprecipitation studies showed that the 68-kDa protein is paxillin and the 125-kDa protein is p125FAK (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125FAK, and p130 but did not prevent tyrosine phosphorylation of p210 and did not prevent ET-1-induced increases in Na+/H+ antiporter activity. Thus 50% of ETB receptor-induced Na+/H+ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ETB receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.
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PMID:Role of tyrosine kinase pathways in ETB receptor activation of NHE3. 884 5

NHE3 is most likely the isoform involved in renal reabsorption of HCO3- and Na+. The functional properties of the "cloned" NHE3 isoform, including its transport regulation by extra- and intracellular stimuli, have so far been studied using non-epithelial expression systems. In the present report we stably transfected NHE3 cDNA (rabbit isoform) into Madin-Darby canine kidney cells (MDCK) cells and compared the sensitivity to inhibitors and the regulation of the Na+/H+-exchanger by vasotocin in NHE3 transfectants to that of the intrinsic basolateral Na+/H+-exchanger in untransfected and control transfected MDCK cells. By Southern blot analysis we documented that the NHE3 transcript is expressed in NHE3 transfectants. Na+/H+-exchange activity, measured as sodium-dependent recovery of intracellular pH from an acid load using 2', 7'-bis(carboxymethyl)-5(6)-carboxy-fluorescein (BCECF), was equally present at the basolateral cell surface of all cell lines; however, NHE3 transfectants demonstrated transport activity in the apical membrane that was significantly higher than that in untransfected or control transfected MDCK cells. Studies with ethylisopropylamiloride (EIPA) have shown that there is a similar sensitivity to inhibitors of the apical and/or basolateral Na+/H+-exchanger in transfected and untransfected MDCK cell lines. In contrast, the apical Na+/H+-exchanger (as compared to the basolateral Na+/H+-exchanger) of NHE3 transfectants was found to be relatively insensitive to the inhibitor HOE 694. Vasotocin decreased the activity of the apical Na+/H+-exchanger in NHE3 transfectants and stimulated the activity of the basolateral Na+/H+-exchanger in transfected (with NHE3 or pMAMneo) and untransfected MDCK cells. Phorbol ester, as expected, increased the activity of the Na+/H+-exchanger in the basolateral membrane of all cell lines; also, it stimulated transport activity at the apical cell surface of NHE3 transfectants. No change of Na+/H+-exchange activities was seen in studies with 8-bromo-cAMP. The PKC inhibitor calphostin C completely suppressed regulation of the apical and/or basolateral Na+/H+-exchanger by vasotocin, it partially blocked activation of the apical Na+/H+-exchanger in NHE3 transfectants by phorbol 12-myristate 13-acetate (PMA), and completely blocked stimulation of basolateral Na+/H+-exchanger by PMA. Consistent with a V1 receptor action, the effects of vasotocin in NHE3 transfectants and in MDCK cells were blocked by the V1 receptor antagonist, d(CH2)5Tyr(Me)-AVP, but were not reproduced by the V2 receptor agonist desmopressin. It is concluded that NHE3 in the apical membrane of NHE3-transfected MDCK cells contributes to the differential regulation of the apical and basolateral Na+/H+-exchanger by vasotocin; NHE3 is inhibited and endogenous Na+/H+-exchange activity is stimulated by vasotocin via V1 receptor activation of the protein kinase C pathway.
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PMID:Regulation of the transfected Na+/H+-exchanger NHE3 in MDCK cells by vasotocin. 909 65

We examined the regulation of the Na+/H+ exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinal C2/bbe cells, which spontaneously differentiate and have little basal apical NHE activity. Unidirectional apical membrane 22Na+ influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of 5-(N,N-dimethyl)amiloride-inhibitable flux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHE activity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulated by serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3 exhibited similar patterns of regulation, suggesting that the second messenger effects observed were not secondary to effects on cell pH. Thus, in contrast to their expression in nonepithelial cells, NHE2 and NHE3 expressed in an epithelial cell line behave similarly to endogenously expressed intestinal apical membrane NHEs. We conclude that physiological regulation and function of epithelium-specific NHEs are dependent on tissue-specific factors and/or conditional requirements.
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PMID:Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe. 973 Sep 53

Angiotensin II is a key element in regulating the volume of extracellular liquid. It acts indirectly through aldosterone secretion by adrenals and directly on the renal tubule too: It regulates luminal Na+/H+ antiporters (NHE3 and possibly NHE2) after binding to membrane AT1 receptors located both on the basolateral and on the apical side of the cells. The main site of Ang II action is proximal tubule, mainly the S1 segment which has a high level of AT1 receptors. Circulating Ang II concentrations (10(-12) to 10(-10) M), increased NaCl, water and NaHCO3 reabsorption via NHE3 in the proximal tubule. There is also a synthesis of Ang II within the cells of proximal tubule, which is secreted within the lumen where the physiological concentration is stable 10(-8) M, i.e. 100 to 1000 times higher than the circulating concentration. Luminal ANG II originating from kidney has a physiological autocrine function on NaCl, water and probably NaHCO3 reabsorption, since inhibiting Ang II synthesis, by conversion enzyme inhibition, or effect, by AT1 receptor antagonists, induces a reduction of proximal tubule reabsorption. The stimulatory effects of circulating and intrarenal Ang II seem to be explained by protein kinase C stimulation and possibly by a reduction of cAMP production or by a stimulation of a non-receptor tyrosine kinase. When pharmacological doses of Ang II (> 10(-8) M) are applied in the peritubular or the luminal medium of isolated microperfused proximal tubule in vitro, a paradoxical inhibition of NHE3 was observed. These effects appear to involve arachidonic acid metabolites through the cytochrome P450 pathway and possibly a rise in cytosolic free Ca++. The physiological significance of these supraphysiological effects are unknown.
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PMID:[Effect of angiotensin ii on Na+/H+ exchangers of the renal tubule]. 985 78

1. Adenosine influences the vectorial transport of Na+ and HCO3- across kidney epithelial cells. However, its action on effector proteins, such as the Na+-H+ exchanger NHE3, an epithelial brush border isoform of the Na+-H+ exchanger (NHE) gene family, is not yet defined. 2. The present study was conducted in Xenopus laevis distal nephron A6 epithelia which express both an apical adenosine receptor of the A1 type (coupled to protein kinase C (PKC)) and a basolateral receptor of the A2 type (coupled to protein kinase A (PKA)). The untransfected A6 cell line expresses a single NHE type (XNHE) which is restricted to the basolateral membrane and which is activated by PKA. 3. A6 cell lines were generated which express exogenous rat NHE3. Measurements of side-specific pHi recovery from acid loads in the presence of HOE694 (an inhibitor with differential potency towards individual NHE isoforms) detected an apical resistant Na+-H+ exchange only in transfected cell lines. The sensitivity of the basolateral NHE to HOE694 was unchanged, suggesting that exogenous NHE3 was restricted to the apical membrane. 4. Stimulation of the apical A1 receptor with N 6-cyclopentyladenosine (CPA) inhibited both apical NHE3 and basolateral XNHE. These effects were mimicked by the addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and partially prevented by the PKC inhibitor calphostin C which also blocked the effect of PMA. 5. Stimulation of the basolateral A2 receptor with CPA inhibited apical NHE3 and stimulated basolateral XNHE. These effects were mimicked by 8-bromo-cAMP and partially prevented by the PKA inhibitor H89 which entirely blocked the effect of 8-bromo-cAMP. 6. In conclusion, CPA inhibits rat NHE3 expressed apically in A6 epithelia via both the apical PKC-coupled A1 and the basolateral PKA-coupled A2 adenosine receptors.
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PMID:Adenosine inhibits the transfected Na+-H+ exchanger NHE3 in Xenopus laevis renal epithelial cells (A6/C1). 1006 8

Acute hormonal modulation of NHE3 activity is partly mediated by kinases, including protein kinase C (PKC). We examined the role of NHE3 phosphorylation in regulating its activity in response to PKC activation by phorbol 12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblasts transfected with NHE3, PMA increased NHE3 activity and phosphorylation. When six potential PKC target serines were mutated, NHE3 phosphorylation was drastically reduced and PMA failed to regulate NHE3 phosphorylation or function. To examine whether NHE3 phosphorylation is sufficient for functional regulation by PKC, we exploited the heterogeneous response of NHE3 activity to PMA in individual clones of transfectants. Clones with stimulatory, inhibitory, or null responses to PMA were observed. Despite the diverse functional response, changes in NHE3 phosphorylation as revealed by tryptic phosphopeptide maps were similar in all clones. We conclude that although phosphorylation appears to be necessary, it is insufficient to mediate PKC regulation of NHE3 function and factors extrinsic to the NHE3 protein must be involved.
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PMID:Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation. 1032 70

Expression of endogenous Na(+)/H(+) exchangers (NHEs) NHE3 and NHE1 at the apical (AP) and basolateral (BL) membrane domains was investigated in three clones (ATCC, PF-11, and TC-7) derived from the human adenocarcinoma cell line Caco-2. In all three clones, NHE1 was the only isoform detected at the BL domain during 3 to 22 postconfluent days (PCD). In clone PF-11, the BL NHE1 activity increased up to 7 PCD and remained stable thereafter. Both NHE1 and NHE3 were found at the AP domain at 3 PCD and contributed 67 and 33% to the total AP Na(+)/H(+) exchange, respectively. The AP NHE3 activity increased significantly from 3 to 22 PCD, from 93 to 450 microM H(+)/s, whereas AP NHE1 activity decreased from 192 to 18 microM H(+)/s during that time. Similar results were obtained with the ATCC clone, whereas very little AP NHE3 activity was observed in clone TC-7. Surface biotinylation and indirect immunofluorescence confirmed these results and also suggested an increase in the number of cells expressing NHE3 being the major mechanism of the observed overall increase in NHE3 activity in PF-11 and ATCC clones. Phorbol 12-myristate 13-acetate (PMA, 1 microM) acutely inhibited NHE3 activity by 28% of control, whereas epidermal growth factor (EGF, 200 ng/ml) stimulated the activity by 18%. The effect of PMA was abolished by the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, suggesting involvement of PKC in the PMA-induced inhibition of NHE3. Similar magnitude of inhibition by PMA and stimulation by EGF was observed at 7 and 17 PCD, suggesting the development of regulatory mechanisms in the early postconfluent period. Taken together, these data suggest a close similarity of membrane targeting and regulation of endogenous NHE3 between Caco-2 cells and native small intestinal epithelial cells and support the usefulness of some Caco-2 cell clones as an in vitro model for studies on physiology of NHE3 in the intestinal epithelium.
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PMID:Development of an endogenous epithelial Na(+)/H(+) exchanger (NHE3) in three clones of caco-2 cells. 1044 43

Until recently, studies to characterize the intestinal epithelial Na(+)/H(+) exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na(+)/H(+) exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH(i) only approximate Na(+)/H(+) exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used (22)[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca(++) involves changes to both K(Na) and V(max). In contrast, the same two second messengers inhibit NHE3 by a decrease in V(max) exclusively. Phorbol ester activation of protein kinase C alters both V(max) and K(Na) of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways.
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PMID:Regulation of intestinal epithelial brush border Na(+)/H(+) exchanger isoforms, NHE2 and NHE3, in C2bbe cells. 1048 97


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