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Regulation of Na+,K(+)-ATPase and the Na+/K+/Cl- co-transporter in the renal epithelial cell line NBL-1 under osmotic stress.

ABSTRACT: The long-term adaptation of the Na+,K(+)-ATPase to hypertonicity was studied using the bovine renal epithelial cell line NBL-1. Na+,K(+)-ATPase activity measured in intact cells as the ouabain-sensitive fraction of Rb+ uptake was stimulated (40% above controls) after incubating the cells in hypertonic medium. This stimulation was not correlated with significant changes in the amount of Na+,K(+)-ATPase alpha 1 subunit protein. Nevertheless, the amount of alpha 1 but not beta 1 subunit mRNA progressively increased after hypertonic shock (3-4-fold above basal values). These results suggest that the alpha 1 subunit gene is modulated by medium osmolarity, although this does not necessarily involve enhanced translation of the mRNA into active alpha 1 protein. Indeed, the increase in the biological activity of the Na+,K(+)-ATPase is abolished when the electrochemical Na+ transmembrane gradient is depleted by monensin, which is consistent with a post-translational effect on the activity of the sodium pump. A furosemide-sensitive component of Rb+ uptake, attributable to Na+/K+/Cl- co-transporter activity, was very low when cells were cultured in a regular medium, but was greatly induced after hypertonic shock. This induction could not be blocked by cycloheximide. Colcemide addition slightly reduced the absolute increase in Na+/K+/Cl- co-transporter activity, while cytochalasin B significantly potentiated the effect triggered by hypertonic shock. It is concluded: (i) that in NBL-1 cells the alpha 1 but not the beta 1 subunit of the Na+,K(+)-ATPase is encoded by an osmotically sensitive gene, and (ii) that the Na+/K+/Cl- co-transporter, although an osmotically sensitive carrier, is induced by a mechanism that is independent of protein synthesis but may rely, in an undetermined manner, on the structure of the cytoskeletal network.

SUBMITTER: Ferrer-Martinez A

PROVIDER: S-EPMC1217774 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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Project description:Chronic exposure to nicotine during the first postnatal week in rats, a developmental period that corresponds to the third trimester of human gestation, results in sexually dimorphic long-term functional defects in the adult hippocampus. One potential cause could be the sex-specific differences in the maturation of GABA(A) receptor-mediated responses from excitatory to inhibitory, which depends on the expression of the Na(2+)/K(+)/Cl(-) co-transporter 1 (NKCC1) and the K(+)/Cl(-) co-transporter 2 (KCC2). In the rat hippocampus, this switch occurs during the first and second postnatal week in females and males, respectively, and is regulated by nicotinic receptor activation. Excitatory GABAergic signaling can increase brain-derived neurotrophic factor (BDNF) expression, which might exacerbate sex differences by impacting synaptogenesis. We hypothesized that chronic neonatal nicotine (CNN) exposure differentially regulates the expression of these co-transporters and BDNF in males and females. We use quantitative isotopic in situ hybridization to examine the expression of mRNAs for NKCC1, KCC2, BDNF, and NMDA receptor subunit 2A (NR2A) and NMDA receptor subunit 2B (NR2B) in the postnatal day (P) 5 and 8 rat hippocampi in both sexes that were either control-treated or with 6mg/kg/day nicotine in milk formula (CNN) via gastric intubation starting at P1. In line with prolonged GABAergic excitation, we found that at P5 males had significantly higher mRNA expression of NKCC1 and BDNF than females. CNN treatment resulted in a significant increase in KCC2 and BDNF mRNA expression in male but not female hippocampus (p<0.05). Males also had higher expression of NR2A and lower expression of NR2B at P5 compared to females (p<0.05). At P8, there were neither sex nor treatment effects on mRNA expression, indicating the end of a critical period for sensitivity to nicotine. These results suggest that differential maturation of GABA(A)R-mediated responses result in sex-specific sensitivity to nicotine during early postnatal development, potentially explaining the differential long-term effects of CNN on hippocampal function.

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Regulation of Na+,K(+)-ATPase and the Na+/K+/Cl- co-transporter in the renal epithelial cell line NBL-1 under osmotic stress.

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