Single-cell rt-pcr analysis of clc-2 mrna expression in ureteric
bud tip.
Huber, Stephan, Bernd Schrsppel, Matthias Kretzler, Detlef
Schlsndorff, and Michael Horster.
Physiologisches Institut and Medizinische Poliklinik, Ludwig
-Maximilians-UniversitSt, 80336 M[diaeresis]ynchen, Germany
APStracts 5:0037F, 1998.
Embryonic epithelia at the tip of the ureteric bud (UB) face the
interspace between epithelial and mesenchymal cells and are
fundamentally involved in reciprocal signaling during early
nephrogenesis. To characterize their membrane conductive proteins,
patch clamp and single-cell RT-PCR techniques were applied to
embryonic rat UBs (day E17) microdissected from the outer cortex.
Cells at the UB tip had a high whole cell conductance (14 +/- 2 nS/10
pF; n = 8). The main fractional conductance resembled that of Ca
-activated Cl channels in non-epithelial cells, by its time-dependent
activation at depolarizing and inactivation at hyperpolarizing
voltages. A second Cl-selective current fraction, by contrast,
activated slowly during strong hyperpolarization suggestive of a ClC
-2-mediated conductance. To determine the origin of this current,
cytoplasm was harvested into the patch pipette, RNA was reverse
transcribed, and cDNA encoding the GAPDH housekeeper gene or the ClC
-2 Cl channel was amplified by polymerase chain reaction (PCR). GAPDH
and ClC-2 PCR products were identified in 23 and 8 (out of a total of
57) single-cell cDNA samples, respectively. ClC-2 PCR products with
two different lenghts were obtained which might be due to two
alternatively spliced ClC-2 mRNA isoforms. This first and combined
approach by patch clamp and single-cell RT-PCR techniques to
embryonic epithelia indicates that (i) cells at the ureteric bud tip
express a phenotype remarkably different from that of post-embryonic
collecting duct principal cells. (ii) ClC-2 is likely to have a key
role in early nephrogenesis.
Received 29 April 1997; accepted in final form 5 February 1998.
APS Manuscript Number F145-7.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 19 February 1998