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1 Faculty of Life Sciences, The University of Manchester, Manchester, Greater Manchester, United Kingdom
* To whom correspondence should be addressed. E-mail: Craig.smith{at}manchester.ac.uk.
In this study we engineered an MDCK type I cell line to stably express the mouse urea transporter UT-A2. Monolayers of MDCK-mUT-A2 cells had a basal phloretin-inhibitable urea permeability of 8.4x10-6 ± 0.3 cm/sec. Treatment of MDCK-mUT-A2 monolayers with AVP led to a rapid dose dependent increase in trans-monolayer phloretin-inhibitable urea flux. The temporal pattern of response was markedly different from that observed for MDCK cells expressing rat UT-A1. Exposure of MDCK-mUT-A2 cells to either 10µM forskolin or 250µM 8-bromo cAMP also increased urea flux rate. Inclusion of the PKA inhibitor H89 (10µM), had no effect on the forskolin-stimulated increase in urea flux across MDCK-mUT-A2 monolayers. Treatment with either 10µM CPA or 1mM ATP also caused an increase in UT-A2 mediated urea flux, although these responses where transient compared to those induced by AVP or elevated cAMP. Taken together these results show for the first time that UT-A2 is acutely sensitive to AVP, cAMP and increased intracellular calcium.
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