ABSTRACT
We have examined the respective influence of aldosterone, vasopressin and cell sodium delivery on Na+,K+-ATPase expression. The level of expression of the mRNA encoding for the alpha1- and beta1-subunits of Na+,K+-ATPase was evaluated in cortical collecting duct (CCD) cells from rats under different aldosterone status, in cells from the rat CCD cell line RCCD1 treated or not with vasopressin and in CCD cells from mice inactivated or not for the a-subunit of the epithelial sodium channel. The amount of mRNA was determined by in situ hybridization. Both aldosterone and vasopressin up-regulate transcripts encoding for the alpha1-subunit of Na+,K+-ATPase while beta1 is unaltered. Interestingly, when cell sodium entry was largely reduced (alphaENaC knock-out mice), the amount of transcripts encoding for the alpha1-subunit of Na+,K+-ATPase was significantly decreased in spite of high plasma aldosterone concentrations. No effect was observed on beta1-subunit. Altogether, these results suggest a coordinated hormonal and ionic control of Na+,K+-ATPase expression by different transcriptional pathways (steroid-receptor, cAMP-dependent and Na+dependent) in CCD cells. These regulations affect only alpha1-subunit of Na,K+-ATPase but not beta1.
Subject(s)
Aldosterone/pharmacology , Kidney Tubules, Collecting/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Vasopressins/pharmacology , Adrenalectomy , Animals , Cell Line , Epithelial Sodium Channels , In Situ Hybridization , Kidney Cortex/drug effects , Kidney Cortex/enzymology , Kidney Tubules, Collecting/drug effects , Kidney Tubules, Collecting/physiology , Male , Mice , Mice, Knockout , Protein Subunits , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Renal Agents/pharmacology , Sodium Channels/genetics , Sodium Channels/metabolism , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
In the cochlea, endolymph is a K-rich and Na-poor fluid. The purpose of the present study was to check the presence and to assess the role of epithelial Na channel (ENaC) in this organ. alpha-, beta-, and gamma-ENaC subunit mRNA, and proteins were detected in rat cochlea by RT-PCR and Western blot. alpha-ENaC subunit mRNA was localized by in situ hybridization in both epithelial (stria vascularis, spiral prominence, spiral limbus) and nonepithelial structures (spiral ligament, spiral ganglion). The alpha-ENaC-positive tissues were also positive for beta-subunit mRNA (except spiral ganglion) or for gamma-subunit mRNA (spiral limbus, spiral ligament, and spiral ganglion), but the signals of beta- and gamma-subunits were weaker than those observed for alpha-subunit. In vivo, the endocochlear potential was recorded in guinea pigs under normoxic and hypoxic conditions after endolymphatic perfusion of ENaC inhibitors (amiloride, benzamil) dissolved either in K-rich or Na-rich solutions. ENaC inhibitors altered the endocochlear potential when Na-rich but not when K-rich solutions were perfused. In conclusion, ENaC subunits are expressed in epithelial and nonepithelial cochlear structures. One of its functions is probably to maintain the low concentration of Na in endolymph.
Subject(s)
Cochlea/chemistry , Epithelial Cells/chemistry , Protein Subunits , RNA, Messenger/analysis , Sodium Channels/analysis , Action Potentials/drug effects , Action Potentials/physiology , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Cochlea/drug effects , Cochlea/physiology , Diuretics/pharmacology , Endolymph/chemistry , Epithelial Cells/drug effects , Epithelial Cells/physiology , Guinea Pigs , Male , RNA, Messenger/physiology , Rats , Rats, Long-Evans , Sodium Channels/drug effects , Sodium Channels/physiology , Sodium Chloride/pharmacologyABSTRACT
The classical short-term effect (within minutes) of arginine vasopressin (AVP) consists in increasing sodium, chloride and water transport in kidney cells. More recently, long-term actions (several hours) of the hormone have been evidenced on water and sodium fluxes, due to transcriptional enhancement in the expression of their transporters. The present study demonstrates that AVP is also responsible for a long-term increase in net chloride secretion. In the RCCD(1) rat cortical collecting duct cell line, 10(-8) M AVP induced, after several hours, an increase in net (36)Cl(-) secretion. This delayed effect of AVP was inhibited by basal addition of 10(-4) M bumetanide and apical addition of 10(-4) M glibenclamide, suggesting chloride entry at the basal membrane through a Na(+)/K(+)/2Cl(-) and apical secretion through a chloride conductance. An original acute cell permeabilization method was developed to allow for entry of antibodies directed against the regulatory region (R) of the cystic fibrosis transmembrane regulator (CFTR) into the cells. This procedure led to a complete and specific blocking of the long-term net chloride secretion induced by AVP. Finally, it was observed that CFTR transcripts steady-state level was significantly increased by AVP treatment. Besides the well-documented short-term effect of AVP on chloride transport, these results provide evidence that in RCCD(1) cells, AVP induces a delayed increase in transepithelial net chloride secretion that is mediated by a Na(+)/K(+)/2Cl(-) co-transporter and CFTR.
Subject(s)
Arginine Vasopressin/pharmacology , Chlorides/metabolism , Kidney Cortex/metabolism , Kidney Tubules, Collecting/metabolism , Animals , Carrier Proteins/physiology , Cells, Cultured , Chlorides/physiology , Chlorine/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Kidney Cortex/drug effects , Kidney Tubules, Collecting/drug effects , Membrane Proteins/physiology , Potassium/metabolism , RNA, Messenger/metabolism , Radioisotopes , Rats , Sodium/metabolism , Sodium-Potassium-Chloride SymportersABSTRACT
The amiloride-sensitive epithelial sodium channel (ENaC) is a main determinant of sodium absorption in renal and colonic epithelial cells. Surprisingly, it is also expressed in non-transporting epithelia such as the epidermis. To gain insight into the putative role of ENaC in keratinocytes, we have evaluated its expression in human skin and in cultured human keratinocytes. Our results indicate that (1) ENaC is expressed in the epidermis and in cultured keratinocytes, at the mRNA and at the protein levels, (2) the ratio of expression of the different ENaC subunits is drastically modified at the protein level during cell growth and differentiation, with a selective upregulation of the &bgr; subunit, (3) no transepithelial sodium transport function is apparent in cultured keratinocytes, but patch-clamp recordings indicate the existence of functional sodium channels with properties similar to those of the cloned ENaC and (4) ENaC inhibition does not alter keratinocyte proliferation, but it significantly decreases the frequency of dome formation in confluent keratinocyte cultures. These results document for the first time the characteristics of ENaC subunit expression in human keratinocytes, and suggest that ENaC may be important during differentiation.
Subject(s)
Keratinocytes/cytology , Keratinocytes/metabolism , Sodium Channels/genetics , Sodium Channels/metabolism , Sodium/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Biological Transport/drug effects , Biological Transport/physiology , Cell Differentiation/physiology , Cell Size/physiology , Cells, Cultured , Electric Conductivity , Epidermal Cells , Epithelial Sodium Channels , Gene Expression/physiology , Hair Follicle/chemistry , Hair Follicle/metabolism , Humans , Keratinocytes/chemistry , Patch-Clamp Techniques , Sodium Channels/analysisABSTRACT
We have examined whether arginine vasopressin (AVP) can induce a long-term modulation of transepithelial ion transport in addition to its well known short-term effect. In the RCCD1 rat cortical collecting duct cell line, an increase in both short-circuit current and 22Na transport was observed after several hours of 10(-8) M AVP treatment (a concentration above the in vivo physiological range). This delayed effect was partially prevented by apical addition of 10(-5) M amiloride and was blocked by 10(-6) M actinomycin D and 2 x 10(-6) M cycloheximide. The amounts of mRNA encoding the alpha1 (not beta1) subunit of Na+/K+-ATPase and the beta and gamma (not alpha) subunits of the amiloride-sensitive epithelial Na+ channel were significantly increased by AVP treatment. The increase in mRNA was blocked by actinomycin D, not by amiloride, suggesting a Na+-independent increase in the rate of transcription of these subunits. The translation rates of the alpha1 subunit of Na+/K+-ATPase and the beta and gamma subunits of the rat epithelial sodium channel increased significantly, whereas the translation rates of the other subunits remained unchanged. Finally, the number of Na+ channels present in the apical membrane of the cells increased, as demonstrated by enhanced specific [3H]phenamil binding.