The small G protein K-Ras2A is rapidly induced by aldosterone in A6 epithelia. In these Xenopus sodium reabsorbing cells, aldosterone rapidly activates preexisting epithelial Na+channels (XENaC) via a transcriptionally mediated mechanism. In the Xenopus oocytes expression system, we tested whether the K-Ras2A pathway impacts on XENaC activity by expressing XENaC alone or together withXK-Ras2A rendered constitutively active (XK-Ras2AG12V). As a second control,XENaC-expressing oocytes were treated with progesterone, a sex steroid that induces maturation of the oocytes similarly to activated Ras. Progesterone or XK-Ras2AG12Vled to oocyte maturation characterized by a decrease in surface area and endogenous Na+ pump function. In both conditions, the surface expression of exogenous XENaC′s was also decreased; however, in comparison with progesterone-treated oocytes,XK-ras2AG12V-coinjected oocytes expressed a fivefold higher XENaC-mediated macroscopic Na+ current that was as high as that of control oocytes. Thus, the Na+ current per surface-expressedXENaC was increased byXK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting thatXK-Ras2AG12V increased the mean activity ofXENaCs at the oocyte surface. These observations raise the possibility that XK-Ras2A, which is the first regulatory protein known to be transcriptionally induced by aldosterone, could play a role in the control of XENaC function in aldosterone target cells.

Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK‐N‐MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. Expression of the EpoR in SK‐N‐MC cells was detected by reverse transcription‐PCR, Western blot and immunofluorescence analysis. Patch‐clamp studies of SK‐N‐MC cells confirmed the expression of T‐type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. Confocal laser scanning microscopy analysis of SK‐N‐MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T‐type Ca2+ channel blocker flunarizine. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T‐type voltage‐dependent Ca2+ channels.

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