All experiments were carried out at 35�C37?��C. The solutions used were of the following composition (mmol?L?1): Hanks��: 130 Na+, 5.8 K+, 135 Cl?, 4.16 HCO3?, 0.34 HPO32?, 0.44 H2PO4?, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO42?, 10 dextrose, 2.9 sucrose, 10 HEPES, pH adjusted to 7.4 with NaOH; Ca2+-free Hanks�� solution (for cell dispersal): NaCl (125), KCl (5.36), glucose,10 sucrose (2.9), NaHCO3 (15.5), KH2PO4 (0.44), Na2HPO4 (0.33), N-[2-Hydroxyethylpiperazine]-N��-[2-ethanesulfonic acid] (HEPES; 10), pH adjusted to 7.4 with NaOH. Krebs�� solution: NaCl (120), KCl (5.9), NaHCO3 (1.2), glucose (5.5) CaCl2 (12.5), MgCl2,6 pH maintained at 7.4 by bubbling with 95% O2�C5% CO2. Perforated patch solution: CsCl (133), MgCl2 (1.0), EGTA (0.5), HEPES, Bay 11-7085
(10) pH adjusted to 7.2 with CsOH. Drugs used were nifedipine (Abcam, Cambridge, UK), FPL 64176 (Tocris Bioscience, Bristol, UK) and Bay K 8644+ (Sigma). Summary data are presented as the mean?��?SEM. Statistical differences in experiments were compared using Students paired t-test, or one-way anova as appropriate, taking the P?<?0.05 level as significant. Throughout, n refers to the number of cells in each experimental series. In each case these were obtained from a minimum of three different colon samples. Tissue samples were stored at ?20?��C in RNAlater <a href="http://www.selleckchem.com/products/Temsirolimus.html
">Akt inhibitor (QIAGEN, Valencia, CA, USA) until use. Immediately prior to isolation of the RNA, tissue samples were transferred to a 1.5?mL tube, snap frozen in liquid nitrogen, and pulverized to yield a dry powder. RNA was isolated from these samples using the RNeasy mini kit (QIAGEN), with DNase treatment included, and eluted with RNase-free water. RNA concentration was determined using a nanodrop Vadimezan datasheet
spectrophotometer (Thermo scientific, Waltham, MA, USA) and the purified RNA was then stored at ?80?��C. Prior to cDNA synthesis, the RNA was denatured for 5?min at 70?��C and then transferred to ice. RNA was reverse transcribed using the Superscript VILO cDNA synthesis kit (Invitrogen, Life Technologies Ltd, Paisley, UK) according to the manufacturers�� instructions. Real-time quantitative PCR (qPCR) was performed on a QUANTICA real-time PCR system (TECHNE, Bibby Scientific Ltd, Staffordshire, UK) using the SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies Ltd, Paisley, UK) with CACNA1C gene-specific primers (forward 5��-CCCCGAAACATGAGCATGCCC-3�� and reverse 5��-CGCCAGTAGCGGCTGAACTTTGAC-3��). Each primer was designed to span a particular exon�Cexon boundary present in all major CACNA1C transcripts. Quantitect human GAPDH primers (QIAGEN) were used for amplification of GAPDH as a reference gene for use in sample normalization. The cycling conditions were as follows: an initial 10-min denaturation at 95?��C was followed by 40 cycles of: denaturation at 95?��C for 15?s, annealing at 55?��C for 15?s, extension at 68?��C for 1?min. Tissue samples were analyzed in triplicate. In addition, no-template controls were included for both primer sets.