Membrane depolarization by isotonic or hypertonic KCl: differential effects on mRNA levels of tyrosine hydroxylase and dopamine beta-hydroxylase mRNA in PC12 cells.
Membrane depolarization is an important and common manipulation used to study the result of enhanced neuronal activity on adaptive changes, including alterations in gene expression. In this study, the effect of elevated KCl, under isotonic and hypertonic conditions, on the changes in mRNA levels of the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) was compared. Treatment of PC12 pheochromocytoma cells for several hours with 50 mM KCl, under conditions where osmolarity was maintained, induced TH mRNA levels several fold, without changing DBH mRNA levels (Kilbourne and Sabban, 1990). In contrast, 50 mM KCl added to culture media without adjusting the osmolarity did not alter TH mRNA levels for up to 24 h. Longer continuous exposure to this hypertonic depolarization condition reduced TH mRNA levels to about 10% of control levels. DBH mRNA levels also declined when PC12 cells were treated from 12 h to 5 days with hypertonic 50 mM KCl. The effect appeared to be specific, since actin mRNA levels were elevated about 2-fold with these same hypertonic treatments. As a control for osmotic changes, 50 mM NaCl was used and did not alter TH or DBH mRNA levels. Viability of the cells was maintained and total protein synthesis was reduced somewhat after 12 h of exposure to hypertonic 50 mM KCl, and remained relatively constant for as long as 4 days. Thus, there appears to be an interaction between osmolarity and elevated KCl since very different results of the effects of membrane depolarization on the mRNA levels for the catecholamine biosynthetic enzymes were obtained depending on the osmolarity of the cultures. The extent of elevation of TH mRNA with isotonic KCl was also dependent on cell density. At high cell densities, membrane depolarization no longer induced TH mRNA levels. The results of this study indicate the experimental parameters which can be crucial in studies of membrane depolarization.
Kilbourne
E J
EJ
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
McMahon
A
A
Sabban
E L
EL
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D004299:Dopamine beta-Hydroxylase / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D006982:Hypertonic Solutions; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D016716:PC12 Cells; D011189:Potassium Chloride / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
D006982:Hypertonic Solutions; D012333:RNA, Messenger; D011189:Potassium Chloride; D014446:Tyrosine 3-Monooxygenase; D004299:Dopamine beta-Hydroxylase