Expression of TRPC in A7r5 vascular smooth muscle cells with and without vasopressin-regulated non-capacitative CA2+ entry

E. J. A. Taylor, I Pino de la Huerga & C. W. Taylor, Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, U.K.

In A7r5 cells, vasopressin reciprocally regulates capacitative (CCE) and non-capacitative (NCCE) Ca2+ entry pathways (Moneer et al., 2002), but several cell lines have been identified in which vasopressin neither inhibits CCE nor activates NCCE. Members of the transient receptor potential canonical (TRPC) family of cation channels have been suggested to contribute to the channels responsible for both CCE and NCCE (Clapham et al., 2000). Our aim was to establish whether changes in expression of any of the 7 TRPC genes were associated with the loss of reciprocal regulation of CCE and NCCE by vasopressin in A7r5 cells.

cDNA was synthesised from RNA isolated directly from frozen A7r5 cells, in which the behaviour of CCE and NCCE had been characterized immediately before freezing. For quantitative PCR, each reaction included LUX primers for a specific TRPC and primers for a housekeeping gene (ß-actin), to allow calibration. For every PCR reaction, the cycle threshold (CT) for each TRPC product was normalized to CT for the housekeeping (HK) product (∆CT=CTTRPC - CTHK) (Livak et al., 2001). Assuming 100% efficiency for each PCR cycle, ∆CT can be converted to a relative RNA expression level (REL) from: REL=2-∆CT (Livak et al., 2001).

Results from 4 cells lines with normal reciprocal regulation of CCE and NCCE and 2 without, suggest that loss of reciprocal regulation of the two Ca2+ entry pathways is associated with much reduced expression of TRPC 2, 3 and 6, with no change in expression of TRPC1 (Table). We suggest that cells in which vasopressin reciprocally regulates CCE and NCCE appear more likely to express hetero-tetrameric TRPC channels (Clapham et al., 2001) and that these may required for reciprocal regulation of the two Ca2+ entry pathways by vasopressin.

Expression of TRPC RNA in A7r5 cells with and without vasopressin-regulated NCCE. Three independent PCR reactions were performed for each cell line. Results are means ±S.E.means from 4 (with NCCE) or 2 (without NCCE) cell lines.

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Livak, K. J. et al., (2001). Methods 25, 402-408.

Supported by the Wellcome Trust (072084).