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      Aldosterone Rapidly Activates Na +/H + Exchange in M-1 Cortical Collecting Duct Cells via a PKC-MAPK Pathway

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          Background: In this study, the mechanism of the rapid non-genomic effect of aldosterone on Na<sup>+</sup>/H<sup>+</sup> exchanger (NHE)-mediated intracellular pH (pH<sub>i</sub>) recovery from an acid load in murine M-1 cortical collecting duct cells was assessed. Methods: Spectrofluorescence microscopy and Western blot analysis was carried out and NH<sub>4</sub>Cl was used to induce the acid load. Results: Aldosterone (10 n M) induced a rapid (<5 min) concentration-dependent increase in pH<sub>i</sub> recovery in M-1 cells, an effect mimicked by its precursor deoxycorticosterone (1 n M). This response was unaffected by the mineralocorticoid receptor (MR) antagonist spironolactone (10 µ M) but was significantly reduced by the NHE antagonists 5′-(N-ethyl- N-isopropyl)amiloride (EIPA) (20 µ M) and cariporide (1 µ M). The PKC inhibitor chelerythrine chloride (1 µ M) significantly attenuated the aldosterone-induced increase in NHE1 activity. HBDDE (80 µ M), a PKC<sub>α</sub> inhibitor, inhibited the rapid aldosterone effect whereas rottlerin (15 µ M), a PKC<sub>δ</sub> antagonist, did not. The glucocorticoid receptor agonists hydrocortisone (1 µ M) and dexamethasone (100 n M) decreased NHE activity, whereas the synthetic mineralocorticoid fludrocortisone (1 n M) had no significant effect. MAPK inhibition using PD98059 (25 µ M) significantly attenuated the rapid aldosterone effect; Western blot analysis showed that aldosterone activation of ERK 1/2 was unaffected by pretreatment with spironolactone but was inhibited following chelerythrine chloride. Conclusion: Aldosterone causes a rapid non-genomic increase in NHE1 activity in M-1 cells via a PKC<sub>α </sub>/MAPK pathway independent of the classical MR.

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          Most cited references 13

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          Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium.

          Estrogens and growth factors such as epidermal growth factor (EGF) act as mitogens promoting cellular proliferation in the breast and in the reproductive tract. Although it was considered originally that these agents manifested their mitogenic actions through separate pathways, there is a growing body of evidence suggesting that the EGF and estrogen-mediated signaling pathways are intertwined. Indeed, it has been demonstrated recently that 17beta-estradiol (E2) can induce a rapid activation of mitogen-activated protein kinase (MAPK) in mammalian cells, an event that is independent of both transcription and protein synthesis. In this study, we have used a pharmacological approach to dissect this novel pathway in MCF-7 breast cancer cells and have determined that in the presence of endogenous estrogen receptor, activation of MAPK by E2 is preceded by a rapid increase in cytosolic calcium. The involvement of intracellular calcium in this process was supported by the finding that the presence of EGTA and Ca2+-free medium did not affect the activation of MAPK by E2 and, additionally, that this response was blocked by the addition of the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate. Cumulatively, these data indicate that the estrogen receptor, in addition to functioning as a transcription factor, is also involved, through a nongenomic mechanism, in the regulation of both intracellular calcium homeostasis and MAPK-signaling pathways. Although nongenomic actions of estrogens have been suggested by numerous studies in the past, the ability to link estradiol and the estrogen receptor to a well defined signaling pathway strongly supports a physiological role for this activity.
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            Characterization of a mouse cortical collecting duct cell line.

            A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepithelial resistance (885.7 +/- 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 +/- 3.5 mV. The associated short-circuit current (SCC) averaged 71.8 +/- 10.3 microA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 +/- 0.9 and B = 127.4 +/- 0.4; [K+], L = 8.6 +/- 0.6 and B = 2.1 +/- 0.3; [Cl], L = 68.6 +/- 5.8 and B = 101.8 +/- 6.6; [HCO3], L = 15.5 +/- 1.5 and B = 8.6 +/- 1.2; while pH was 7.16 +/- 0.03 at the luminal and 6.94 +/- 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+.(ABSTRACT TRUNCATED AT 250 WORDS)
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              Rapid aldosterone signaling in vascular smooth muscle cells: involvement of phospholipase C, diacylglycerol and protein kinase C alpha.

              Rapid in vitro effects of aldosterone (ALDO) on intracellular sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in human mononuclear leukocytes and rat vascular smooth muscle cells (VSMC). These nongenomic effects are signaled through membrane receptors with a high affinity for aldosterone, but not for hydrocortisone. Effects of ALDO on the production of diacylglycerol (DAG) and protein kinase C alpha (PKC) were measured in VSCM by enzymatic assay and immunoblotting. DAG production was stimulated twofold by ALDO (> or = 1 nM) within 30 sec while hydrocortisone was inactive at concentrations of up to 1 microM. The inhibitors of phospholipase C, neomycin and U-73122 completely blocked this effect. PKC translocation from cytosol to membranes by ALDO occurred within 5 min, the extent of this effect was comparable to that of angiotensin II. These data demonstrate rapid intracellular signaling for ALDO in VSMC through phospholipase C, DAG and PKC in addition to calcium and inositol-1,4,5-trisphosphate as determined earlier.

                Author and article information

                Nephron Physiol
                Nephron Physiology
                S. Karger AG
                January 2005
                27 December 2004
                : 99
                : 1
                : p1-p9
                aDepartment of Physiology, University College Cork, Cork, and bMolecular Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
                81796 Nephron Physiol 2005;99:p1–p9
                © 2005 S. Karger AG, Basel

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                Page count
                Figures: 8, References: 25, Pages: 1
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/81796
                Original Paper


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