Differential expression of multiple glutaminase mrnas in llc-pk 1
-f+ cells.
Porter, Dave, William R. Hansen, Lynn Farrell, and Norman P. Curthoys.
Department of Biochemistry and Molecular Biology, Colorado State
University, Fort Collins, CO 80523, USA
APStracts 2:0057F, 1995.
LLC-PK1-F+ cells are porcine proximal tubule-like cells which have
been used to model the renal ammoniagenic response to metabolic
acidosis. A 3.2-kb porcine GA cDNA (pGA201) containing 528 bp of
coding sequence and 2.7 kb of 3' untranslated region was cloned and
sequenced. Probes derived from both porcine and rat GA cDNAs were
used to characterize the expression of putative GA mRNAs in LLC-PK1
-F+ cells. Two larger putative GA mRNAs (approximately 5.0 and 4.5 kb
in length) were resolved and a smaller 2.5 kb species was also
observed. The level of the 5.0-kb mRNA is detectable in freshly split
LLC-PK1-F+ cells and increases as the cells reach confluence. In
contrast, the amount of the 4.5-kb GA mRNA is greatest in freshly
split cells and decreases gradually as the cells approach confluence.
The levels of the 5.0- and 2.5-kb mRNAs are also affected by
refeeding the cells and the 2.5-kb mRNA accumulates to high levels if
cells are retained in the same media for 4 days. Exposure to acidic
media had little or no effect on the levels of GA mRNAs expressed in
confluent or post-confluent cells, whereas in growing and
undifferentiated cells, this treatment did affect the level of the
4.5-kb mRNA. Thus, the putative GA mRNA species are differentially
expressed. Given this complexity, a careful assessment of GA mRNA
species, of basal expression and of growth conditions are essential
for a meaningful analysis of GA mRNA levels in cultured cells.
Received 27 December 1994; accepted in final form 6 April 1995.
APS Manuscript Number F456-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 25 April 1995.