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  • 1
    ISSN: 1432-2013
    Keywords: Adenylate cyclase ; Phosphodiesterase ; cAMP ; Protein kinase A ; Protein kinase C ; Na+/H+ exchange ; Endothelin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Endothelin-1 (ET-1) controls multiple aspects of kidney function. In this study we have analysed the effects of ET-1 on apical Na+/H+ exchange activity in opossum kidney (OK) cells. ET-1 (at 10−10 M and 10−8 M) activated Na+/H+ exchange activity within 5 min of exposure. ET-1 (10−8 M) prevented PTH-induced (parathyroid hormone; 10−8 M) inhibition of Na+/H+ exchange activity; it also abolished transport inhibition in response to 10−3 M IBMX (isobutylmethylxanthine) and 3×10−7 M TPA (phorbol 12-myristate 13-acetate), but had no effect on the 8-bromo-cAMP-induced (10−4 M) decrease of transport rate. Basal cAMP content, IBMX- and PTH-stimulated cAMP production were unaffected by ET-1 (10−8 M). The stimulatory action of ET-1 (10−8 M) on Na+/H+ exchange activity was prevented by calphostin C (10−8 M). These data document that OK cells might serve as a useful in vitro model for analysis of cellular mechanisms involved in endothelin action; proteine kinase C activation seems to participate in the observed endothelin effects.
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  • 2
    ISSN: 1432-2013
    Keywords: Key words NHE isoforms ; Heterologous expression ; Amiloride sensitivity ; Vasopressin receptor signalling ; Renal epithelia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  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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  • 3
    ISSN: 1432-2013
    Keywords: Na/Pi cotransport ; Microtubules ; Nocodozole ; OK cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The role of microtubules and actin microfilaments in adaptive changes of the apical Na-dependent transport of phosphate (Pi) was investigated in opossum kidney (OK) cells. Up-regulation of Na/Pi cotransport was achieved by incubating OK cells in a medium containing 0.1 mM Pi; down-regulation of Na/Pi cotransport was provoked by refeeding adapted cells with 2 mM Pi. Up-regulation of Na/Pi cotransport was found to be inhibited by approximately 50% after a pretreatment of the cells with the microtubule disrupting agents nocodozole and colchicine; indirect immunofluorescence indicated complete depolymerization of the microtubular network. No inhibition of the adaptive response was observed after treatment of the cells with cytochalasin B to depolymerize actin microfilaments. In adapted cells, depolymerization of microtubules by nocodozole led to a reversibility of Na/Pi cotransport similar to that observed after refeeding adapted cells with 2 mM Pi. No effects of the microtubule disrupting drugs were observed on Na/l-glutamic acid transport. Depolymerization of microtubules did not prevent parathyroid-hormone-mediated inhibition of Na/Pi cotransport. It is concluded that microtubules are (at least in part) involved in the correct insertion of newly synthesized apical Na/Pi cotransport systems and that microtubules are not involved in the internalization of Na/Pi cotransport systems.
    Type of Medium: Electronic Resource
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