Extracellular acidosis minimizes actin cytoskeletal alterations during ATP depletion.
Extracellular acidosis has been shown to be protective during ischemia in renal tubule cells. However, the mechanism of protection remains unknown. Since ischemia leads to disruption and polymerization of the cortical actin cytoskeleton, we hypothesized acidosis may better preserve the actin cytoskeleton during ischemia. Therefore, the purpose of our studies was to examine the effect of pH on the integrity of the actin cytoskeleton during ATP depletion and ATP repletion. To do this, we used an in vitro model of reversible ATP depletion in LLC-PK1 cells at extracellular pH values (pHo) of 6.9, 7.4, and 7.9. Immunofluorescent studies with rhodamine-phalloidin demonstrated more marked redistribution and clumping of cortical actin at pHo 7.9 and 7.4 vs. 6.9 after 90 min of chemical anoxia. After 15 min of ATP depletion, G-actin, quantified by the deoxyribonuclease assay, decreased from 53.7 +/- 0.8 to 43.2 +/- 1.5 microgram/mg protein at pHo 6.9 vs. 37.6 +/- 1.8 microgram/mg protein at pHo 7.4 (P < 0.001). After 60 min, there still was significantly less conversion of G-actin to F-actin at pHo 6.9 vs. 7.4, with a decrease from 55.9 +/- 2.0 to 39.6 +/- 2.0 micrograms/mg protein at 6.9 vs. 35.8 +/- 2.4 at 7.4 micrograms/mg protein (P < 0.05). Furthermore, extracellular acidosis during the phase of ATP repletion resulted in more rapid normalization of cellular G-actin levels (95 +/- 3% of control vs. 82 +/- 2% for pH 6.9 vs. 7.4, respectively, P < 0.01). Together, these findings indicate the actin cytoskeleton is better preserved in an acidic environment during ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
Fish
E M
EM
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Molitoris
B A
BA
D000138:Acidosis; D000199:Actins / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000704:Analysis of Variance; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology*; D002999:Clone Cells; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D007684:Kidney Tubules; D007700:Kinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D000199:Actins; D000968:Antimycin A; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase