Using the video imaging technique this group has previously reported that regulatory volume decrease (RVD) in ZR-75-1 cells is Ca²⁺ dependent. RVD was inhibited in the absence of external Ca²⁺ and in the presence of voltage-gated Ca²⁺ channel inhibitors, nifedipine (L-type) and flunarizine (T-type; Ashes et al. 2002). These results suggested that external Ca²⁺ entered the cell via voltage-gated L- and/or T-type Ca²⁺ channels. In this study, the expression of L- and T-type channels was investigated in ZR-75-1 cells using reverse transcriptase-polymerase chain reaction (RT-PCR). The α1C and α1D are subunits of the L-type Ca²⁺ channel. The α1C subunit is expressed in cardiac muscle and the α1D subunit in the CNS and endocrine cells, where it may have a role in stimulus-secretion coupling. The α1G, α1H and α1I subunits have been cloned and display biophysical and pharmacological properties of native T-type Ca²⁺ channels when overexpressed in Xenopus oocytes or HEK-293 cells (Randall & Benham 1999).

Total RNA was isolated using the Promega SV Total RNA isolation system. The purity and yield of the total RNA was determined spectrophotometrically and sample integrity was confirmed by agarose gel electrophoresis. Primer sequences were designed for L-type subunits, α1C and α1D, and T-type subunits, α1G, α1H and α1I, using the primer design program ‘Primer3’ (www-genome.wi.mit.edu). BLAST (Basic Local Alignment Search Tool; www.ncbi.nlm.nih.gov/BLAST) was used to check for sequence alignment and to ensure minimal homology of the primer sequences with other members of the α1-subunit family. Upstream and downstream primer sequences were designed to anneal in different exons to ensure that if contaminated by DNA, genomic products would be larger than those amplified from RNA. Primers for β-actin (Viana et al. 1995) were used as positive control as β-actin is ubiquitously expressed. RT-PCR was carried out using the Promega Access RT-PCR system.

Agarose gel electrophoresis of the total RNA yielded the 28S and 18S ribosomal RNA subunits indicating that the isolated RNA was intact and had not degraded. Using the respective primers, fragments corresponding to the expected sizes for α1D, α1G and α1H subunits were obtained.

These results indicate that α1D, α1G and α1H subunit mRNA is expressed in detectable amounts in ZR-75-1 cells. These data support previous work by this group indicating that both L- and T-type voltage-gated Ca²⁺ channels may play a role in Ca²⁺ entry in these cells.