Differential regulation of H⁺-ATPases in MDCK-C11 cells by aldosterone and vasopressin

Abstract

The objective of the present work was to characterize the biochemical activity of the proton pumps present in the C11 clone of Madin-Darby canine kidney (MDCK) cells, akin to intercalated cells of the collecting duct, as well as to study their regulation by hormones like aldosterone and vasopressin. MDCK-C11 cells from passages 78 to 86 were utilized. The reaction to determine H+-ATPase activity was started by addition of cell homogenates to tubes contained the assay medium. The inorganic phosphate (P(i)) released was determined by a colorimetric method modified from that described by Fiske and Subbarow. Changes in intracellular calcium concentration in the cells was determined using the Ca2+-sensing dye fluo-4 AM. Homogenates of MDCK-C11 cells present a bafilomycin-sensitive activity (vacuolar H+-ATPase), and a vanadate-sensitive activity (H+/K+-ATPase). The bafilomycin-sensitive activity showed a pH optimum of 6.12. ATPase activity was also stimulated in a dose-dependent fashion as K+ concentration was increased between 0 and 50 mmol x L(-1), with an apparent K(m) for the release of P(i) of 0.13 mmol x L(-1) and Vmax of 22.01 nmol x mg(-1) x min(-1). Incubation of cell monolayers with 10(-8) mol x L(-1) aldosterone for 24 h significantly increased vacuolar H+-ATPase activity, an effect prevented by 10(-5) mol x L(-1) spironolactone. Vacuolar H+-ATPase activity was also stimulated by 10(-11) mol x L(-1) vasopressin, an effect prevented by a V1 receptor-specific antagonist. This dose of vasopressin determined a sustained rise of cytosolic ionized calcium. We conclude that (i) homogenates of MDCK-C11 cells present a bafilomycin-sensitive (H+-ATPase) activity and a vanadate-sensitive (H+/K+-ATPase) activity, and (ii) vacuolar H+-ATPase activity is activated by aldosterone through a genomic pathway and by vasopressin through V1 receptors.

Genomic and nongenomic stimulatory effect of aldosterone on H+-ATPase in proximal S3 segments

“…This stimulatory effect we observed is only due to aldosterone since in the presence of ethanol alone (1-h preincubation), in a similar concentration we used to prepare the hormonal solution, the basal pH i and the pHirr were not different from the control values. Our present results are in accordance with data indicating that aldosterone (10 Ϫ8 M for 24 h) increases H ϩ -ATPase activity in MDCK-C11 cells (7) and that this hormone (10 nM, 15-min preincubation) causes a rapid stimulatory effect on H ϩ -ATPase activity in isolated outer medullary collecting ducts (38) and increases the activity of vacuolar H ϩ -ATPase in turtle urinary bladder (1). However, the present study is the first demonstration, to our knowledge, that aldo- Values are means Ϯ SE; N/n ϭ the no.…”

“…The present data are compatible with our recent demonstration of an aldosterone nongenomic (15-or 1-min preincubation) and genomic (l-h preincubation) effect on the Na ϩ /H ϩ exchanger in S2 (25) and S3 (19) segments of rat proximal tubule. The genomic aldosterone-induced regulation of the H ϩ -ATPase seems to be a mineralocorticoid specific effect, since our data showed that spironolactone inhibits the aldosterone (1-h preincubation) effect on H ϩ -ATPase, as had been observed in MDCK-C11 cells (7). The present results are in accordance with our finding of MR in the S3 segment by RT-PCR (19).…”

Mechanisms Underlying Rapid Aldosterone Effects in the Kidney

“…These early studies showed that aldosterone stimulated H + secretion independently of Na + transport as a result of trafficking of proton pumps stored in cytosolic vesicles into the apical membrane of intercalated cells via a PKCdependent signaling pathway (94). Morphological changes in intercalated cell number and apical membrane area were also found to be a (96). The sensitivity of the aldosterone effect to spironolactone in the MDCK cell line would indicate a genomic pathway.…”

Mineralocorticoids Stimulate the Activity and Expression of Renal H+,K+-ATPases

“…The observed body weight gain in DOCP-treated mice is consistent with the known effect of DOCP to enhance Na ϩ and fluid volume retention. By the fourth day, DOCP treatment resulted in hypernatremia, an effect that started to wane by 8 …”