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[Na.sup.+], [K.sup.+]-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant a1R-[Na.sup.+], [K.sup.+]-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on [K.sup.+] ([sup.86]Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-[Na.sup.+], [K.sup.+]-ATPase. α2R mRNA in transfected cells was ~8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10 µmol/L ouabain led to complete inhibition of [sup.86]Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of 86Rb influx in α1R-transfectd cells was observed at 1000 µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3 µmol/L ouabain, α1R- cells survived after 24 h incubation with 1000 µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-[Na.sup.+], [K.sup.+]-ATPase subunit mRNA and immunoreactive protein does not contribute to [Na.sup.+]/[K.sup.+] pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2- [Na.sup.+], [K.sup.+]-ATPase on [Na.sup.+]/[K.sup.+] pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells. Key words: [Na.sup.+], [K.sup.+]-ATPase, α1- and α2-subunits, transfection, transcription, activity, renal epithelium, cell death. La [Na.sup.+], [K.sup.+]-ATPase est un heterodimere compose des sous-unites α1-α4 catalytiques et β1- β3 regulatrices. Dans un article recent, nous avons indique qu'une transfection la [Na.sup.+], [K.sup.+]-ATPase α1R resistante a l'ouabaine protege les cellules C7-MDCK epitheliales renales exprimant l'isoforme α1S sensible a l'ouabaine contre l'action cytotoxique de l'ouabaine. Pour examiner le role de la sous-unite α2 dans le transport des ions et l'action cytotoxique de l'ouabaine, nous avons compare l'effet de la ouabaine sur l'influx [K.sup.+] ([sup.86]Rb) et la survie des cellules C7-MDKC soumises a une transfection stable avec la [Na.sup.+], [K.sup.+]-ATPase α1R et α2R. Dans les cellules transfectees, l'ARNm de α2R a ete d'un facteur ~8 plus abondant que l'ARNm de o 1R, alors que la teneur en proteine immunoreactive de α2R a ete d'un facteur 5 plus faible que celle de la proteine endogene de l'isoforme α1S. Une concentration d'ouabaine de 10 µmol/L a mene a une inhibition complete de l'influx de [sup.86]Rb dans les cellules transfectees α2R et temoins, alors que l'inhibition maximale de l'influx de [sup.86]Rb dans les cellules transfectee α1R a ete observee a une concentration d'ouabaine de 1000 µmol/L. L'apoptose massive des cellules transfectees α2R et temoins exposees a 3 µmol/L d'ouabaine n'a pas ete observee dans les cellules α1R, lesquelles ont survecu apres une incubation de 24 h avec 1000 µmol/L d'ouabaine. Ainsi, nos resultats montrent que contrairement ce qu'on observe pour l'α1R, la presence de l'ARNm et de la proteine immunoreactive de la [Na.sup.+], [K.sup.+]-ATPase α2R, ne contribue pas a l'activite de la pompe [Na.sup.+]/[K.sup.+] et ne protege pas les cellules C7-MDCK contre l'action cytotoxique de l'ouabaine. Les resultats donnent aussi a penser que l'absence d'effet de la [Na.sup.+], [K.sup.+]-ATPase α2R sur l'activite de la pompe [Na.sup.+]/[K.sup.+] et la survie des cellules pourrait etre attribuee a une faible efficacite de sa traduction et (ou) de son transport dans la membrane plasmique des cellules epitheliales renales. Mots-cles : [Na.sup.+], [K.sup.+]-ATPase, sous-unites α1R et α2R, transfection, transcription, activite, epithelium renal, mort cellulaire. [Traduit par la Redaction], [Na.sup.+], [K.sup.+]-ATPase, a heterodimer consisting of catalytic α-and regulatory β-subunits, plays a key role in the maintenance of electrochemical monovalent cation gradients across the plasma membrane in all types of [...]

High salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodeling, cardiac hypertrophy, and stroke incidence. In this review, we discuss the molecular origins of primary sensors involved in the phenomenon of salt sensitivity. Based on the analysis of literature data, we conclude that the kidneys and central nervous system (CNS) are two major sites for salt sensing via several distinct mechanisms: 1) [[Cl.sup.-]] sensing in renal tubular fluids, primarily by [Na.sup.+]-[K.sup.+]-[Cl.sup.-] cotransporter (NKCC) isoforms NKCC2B and NKCC2A, whose expression is mainly limited to macula densa cells; 2) [[Na.sup.+]] sensing in cerebrospinal fluid (CSF) by a novel isoform of [Na.sup.+] channels, [Na.sub.x], expressed in subfornical organs; 3) sensing of CSF osmolality by mechanosensitive, nonselective cation channels (transient receptor potential vanilloid type 1 channels), expressed in neuronal cells of supraoptic and paraventricular nuclei; and 4) osmolarity sensing by volume-regulated anion channels in glial cells of supraoptic and paraventricular nuclei. Such multiplicity of salt-sensing mechanisms likely explains the differential effects of [Na.sup.+] and [Cl.sup.-] loading on the long-term maintenance of elevated blood pressure that is documented in experimental models of salt-sensitive hypertension. sodium; chloride; osmolality; plasma; tubular fluid; cerebrospinal fluid; salt intake

We have shown previously that the hypertension-related, calcium-regulated gene (HCaRG) is involved in the control of renal cell proliferation and differentiation (Devlin AM, Solban N, Tremblay S, Gutkowska J, Schurch W, Orlov SN, Lewanczuk R, Hamet P, and Tremblay J. Am J Physiol Renal Physiol 284: F753-F762, 2003). To determine whether HCaRG plays a role in kidney repair after injury, we extended our studies on the cellular function of HCaRG by comparing cell migration of two kidney cell lines [HEK293 and Madin-Darby canine kidney (MDCK)-C7] stably transfected with the plasmid alone or with a plasmid containing HCaRG cDNA. HCaRG-expressing HEK293 cells, which undergo lower proliferation, migrated faster than control cells and presented greater adhesiveness to the extracellular matrix. Faster migration was also observed for the MDCK-C7 cells, after they were stably transfected with HCaRG eDNA. HCaRG overexpression induced major morphological changes in HEK293 cells, including the formation of lamellipodia. Expression microarrays of HCaRG-expressing HEK293 cells revealed the elevated expression of several genes known to he involved in cell migration and lamellipodia formation, including transforming growth factor-[alpha] (TGF-[alpha]), galectins, autotaxins and fibronectin. These cells exhibited augmented synthesis and release of activated TGF-[alpha]. Conditioned medium from HCaRG-expressing cells stimulated the migration and induced significant morphological changes in control cells, in part, through activation of the TFG-[alpha]/EGF receptor. Together, these data support a role for HCaRG in kidney repair after injury through its effect on renal cell migration and TGF-[alpha] secretion. hypertension-related, calcium-regulated gene; cell motility; kidney repair; lamellipodia; microarrays; kidney 293 cells; Madin-Darby canine kidney cells

Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones ([alpha]-intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 [micro]M ATP or 100 [micro]M UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive [P.sub.2Y2-], [P.sub.2Y4-], and [P.sub.2Y6-] purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive [P.sub.2]-purinoceptors or a unique [P.sub.2Y]-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1. [P.sub.2]-purinoceptors; Na-K-Cl cotransporter; C7- and C11-Madin-Darby canine kidney cells

We recently identified a novel calcium-regulated gene, HCaRG, that is highly expressed in the kidney and maps to a chromosomal locus determining kidney weight in rats. The mRNA levels of HCaRG negatively correlate with the proliferative status of the kidney cells. To investigate its role in renal epithelial cellular growth directly, we studied the human embryonic kidney cell line (HEK-293) stably transfected with either plasmid alone or plasmid containing rat HCaRG. [[sup.3]H]thymidine incorporation was significantly lower in HCaRG clones. Although HCaRG clones exhibited some enhanced susceptibility to cell death, this was not the primary mechanism of reduced proliferation. Cell cycle analysis revealed a [G.sub.2]M phase accumulation in HCaRG clones that was associated with upregulation of p[21.sup.Cip1/WAF1] and downregulation of p[27.sup.Kip1]. HCaRG clones had a greater protein content, larger cell size, and re]eased 4.5- to 8-fold more of an atrial natriuretic peptide-like immunoreactivity compared with controls. In addition, HCaRG clones demonstrated the presence of differentiated junctions and a lower incidence of mitotic figures. Genistein treatment of wild-type HEK-293 cells mimicked several phenotypic characteristics associated with HCaRG overexpresssion, including increased cell size and increased release of atrial natriuretic peptide. Taken together, our results suggest that HCaRG is a regulator of renal epithelial cell growth and differentiation causing [G.sub.2]M cell cycle arrest. novel gene; p[21.sup.Cip/WAF1; p[27.sup.Kip1]; atrial natriuretic peptide; human embryonic kidney 293 cells; hypertension-related, calcium-regulated gene

Changes in cell volume in vascular smooth muscle cells (VSMC) have been suggested to be associated with ion transport pathways involved in regulatory volume increase (RVI). Herein, it was shown that bumetanide-inhibited ion transport contributes to the regulation of VSMC cell volume after cell shrinkage. These changes in cell volume were mediated by adenosine 3',5'-cyclic monophosphate-dependent pathways.