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Role of Na+,K+-ATPase in cell adhesion and cell volume regulation : mutagenesis of Na+,K+-ATPase and transfection in embryonic kidney cell line

thesis
posted on 2024-09-03, 05:40 authored by Roger Belusa

The regulation of Na+,K+-ATPase activity by hormones and neurotransmittors via intracellular signaling molecules such as kinases and phosphatases, occurs in several physiological situations in vivo. Alterations of Na+,K+ATPase activity as a result of pathophysiological mechanisms have been shown to be involved in the final outcome of the pathological process.

By affecting Na+,K+-ATPase activity and regulation we have shown that the Na+,K+ATPase is involved in several vital cellular mechanisms. We also have demonstrated that the regulation of Na+,K+-ATPase activity is dependent on the developmental phase of the rat kidney cortex proximal convoluted tubule cells. In both infant and adult rat PCT aracidonic acid metabolites stimulated PKC and inhibited Na+,K+-ATPase activity in a dose dependent manner. Using different inhibitors of arachidonic acid (AA) metabolizing enzymes and semiquantitative RT- PCR we could reveal that rat PCT Na+,K+-ATPase activity in infant and adult animals is inhibited by two different AA signaling pathways. In adult rat PCT the main signaling pathway is thru the cytochrome P450 monooxygenase and in infant rat PCT the 12lipoxygenase is predominant.

Mutating serine 23 into an alanine in the rat Na+,K+-ATPase alpha1 subunit abolished the inhibitory effect of phorbolester PDBu on Na+,K+-ATPase activity. Both at basal conditions and during PKC stimulation we could record significant differences in intracellular Na+ and pH between wild type and S23A alpha1 subunit expressing cells. Cellular morphology was drastically altered in cells expressing S23A alpha1 subunit. They appeared more rounded with fewer cell extensions and less adhesive.

We could also demonstrate a physiological significant correlation between Na+,K+-ATPase activity and cell attachment to fibronectin. Subtotal inhibition of Na+,K+-ATPase activity (approximately 30% inhibition) was sufficient to change cell attachment to fibronectin. A ouabain resistant Na+,K+-ATPase alpha 1 subunit (leucine 799 to cystein substitution) expressing cells was unaffected even at high concentrations of ouabain. To influence Na+,K+- ATPase activity by other means than ouabain we used alterations in extracellular V. Decreasing the extracellular K+ concentration, decreased cell attachment, and in parallel increasing K+ concentration reactivated cell attachment. A transient increase in calcium was seen during the first 15-20 minutes of attachment in untreated cells, but cells treated with ouabain exhibit a sustained increase. This different response in intracellular calcium can be one major explanation for the impaired attachment to fibronectin when cells are exposed to ouabain. We could also detect a significant decrease in auto-phosphorylation in FAK in the early phase of attachment to fibronectin, when cells were treated with ouabain.

Using the ouabain resistant form of Na+,K+-ATPase we have been able to study its involvement in the regulatory volume decrease (RVD) process, and study the relationship between ouabain (cardiac glycoside) sensitivity and K+ affinity. The leucine 799 to cystein mutation produced an almost ouabain resistant enzyme, and a decreased K+ affinity in the mutated enzyme was recorded. A dose-dependent decrease in RVD was seen in cells exposed to ouabain. Even an immediate effect of ouabain on RVD was seen. When ouabain was added at the same time point as the start of the incubation with hypoosmolar solution, RVD was still significantly decreased in cells exposed to ouabain.

My overall hypothesis is that the hormonal regulation of the Na+,K+-ATPase activity has physiological effects on cell phenotype important during e.g. the development of an organ.

List of scientific papers

I. Li D, Belusa R, Nowicki S, Aperia A (2000). "Arachidonic acid metabolic pathways regulating activity of renal Na(+)-K(+)-ATPase are age dependent. " Am J Physiol Renal Physiol 278(5): F823-9
https://pubmed.ncbi.nlm.nih.gov/10807595

II. Belusa R, Wang ZM, Matsubara T, Sahlgren B, Dulubova I, Nairn AC, Ruoslahti E, Greengard P, Aperia A (1997). "Mutation of the protein kinase C phosphorylation site on rat alpha1 Na+,K+-ATPase alters regulation of intracellular Na+ and pH and influences cell shape and adhesiveness. " J Biol Chem 272(32): 20179-84
https://pubmed.ncbi.nlm.nih.gov/9242694

III. Belusa R, Aizman O, Andersson RM, Aperia A (2001). "Changes in Na+,K+-ATPase activity influence cell attachment to fibronectin." Am J Physiol (Submitted)

IV. Anderss RM, Belusa R, Uhlen P, Brismar H, Aperia A (2001). "The function of M5-M6 segment of Na+,K+-ATPase catalytic subunit is of importance for cell volume regulation." Am J Physiol (Submitted)

History

Defence date

2001-06-14

Department

  • Department of Women's and Children's Health

Publication year

2001

Thesis type

  • Doctoral thesis

ISBN-10

91-628-4874-7

Number of supporting papers

4

Language

  • eng

Original publication date

2001-05-24

Author name in thesis

Belusa, Roger

Original department name

Department of Women's and Children's Health

Place of publication

Stockholm

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