Regulation of Na(+)-Ca2+ exchange in cultured human mesangial cells.
Na(+)-Ca2+ exchange contributes to regulation of cytosolic free Ca2+ levels ([Ca2+]i) of cultured human mesangial cells following phospholipase C stimulation, as shown by larger responses to vasoconstrictors such as angiotensin II (ANG II) or endothelin 1 in Na(+)-free media. In turn, previous activation of phospholipase C by vasoconstrictors significantly enhances the amplitude of the [Ca2+]i elevation induced by Na+ withdrawal. We studied the mechanisms of upregulation in monolayer cultures loaded with the fluorescent Ca(2+)-sensitive probe fura-2. The exchanger was stimulated by insulin and inhibited by chronic exposure to serum. A rise of [Ca2+]i was not sufficient per se to enhance exchange activity, as prior elevation of [Ca2+]i with the ionophores ionomycin or 8-bromo-A23187 failed to augment the response to Na+ withdrawal. Protein kinase C (PKC) activation by phorbol 12-myristate-13-acetate (PMA), alone or in combination with a rise of [Ca2+]i, potently inhibited basal and vasoconstrictor-enhanced Na(+)-Ca2+ exchange. Suppression of the effects of ANG II was not due to frustrated phospholipase C activation by PMA, because addition of PMA after ANG II also inhibited Na(+)-Ca2+ exchange. PKC downregulation by 24-h pretreatment with PMA or inhibition with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or staurosporine did not prevent activation by ANG II. The exchanger was markedly potentiated by Na+ loading the cells with gramicidin D or reducing extracellular K+. ANG II failed to stimulate Na(+)-Ca2+ exchange when added in the absence of extracellular Na+. Therefore vasoconstrictors promote Na(+)-Ca2+ exchange by a mechanism independent of [Ca2+]i and PKC while presumably linked to Na+ influx.
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Cattedra di Nefrologia, Università Degli Studi di Roma La Sapienza, Italy.
Pugliese
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Cinotti
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D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003470:Culture Media; D016232:Endothelins / Q000494:pharmacology; D016257:Fura-2; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism*; D006096:Gramicidin / Q000494:pharmacology; D006706:Homeostasis; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D000470:Alkaloids; D003470:Culture Media; D016232:Endothelins; D007328:Insulin; D007476:Ionophores; D007546:Isoquinolines; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D006096:Gramicidin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D012964:Sodium; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2