All procedures were carried out in accordance with current EU legislation and with the approval of Dundalk Institute of Technology Animal Ethics Committee. Male C57BL/6 wild-type (WT) mice aged 10–16 wks old were humanely killed by intraperitoneal injection of pentobarbitone (100 mg/kg). Vasa deferentia were removed and placed in Krebs’ solution. Each vas deferens was pinned to a Sylgard-coated dissection dish containing Krebs’ solution. Adherent fat, blood vessels and connective tissue were carefully removed by sharp dissection under a dissecting microscope. A small syringe with a small-gauge needle was carefully inserted into the prostatic end of the vas deferens to flush out semen. The vas deferens was cut into epididymal and prostatic segments, approximately 10–15 mm in length for isometric tension recording or transferred to Ca2+-free Hanks’ solution for enzymatic digestion.

Vas deferens segments were cut into 1 mm3 pieces and stored in Ca2+-free Hanks’ solution for 15 min at 4˚C prior to cell dispersal. Tissue pieces were incubated in dispersal medium containing (per 5 ml) of Ca2+-free Hanks’ solution: 10 mg collagenase (Sigma type 1 A), 1 mg proteinase (Sigma type XXIV), 10 mg bovine serum albumin (Sigma) and 10 mg trypsin inhibitor (Sigma) for 7–8 min at 37˚C. Tissue was then transferred to Ca2+-free Hanks’ solution and stirred for a further 7–8 min to release single smooth muscle cells. These were plated in Petri dishes containing 100 µM Ca2+ Hanks’ solution and allowed to settle in glass bottomed Petri dishes until they had stuck down.

Longitudinal segments of vas deferens were mounted in water-jacketed organ baths, perfused with warmed Krebs’ solution, adjusted to 5 mN tension, and equilibrated for 40 min. Isometric contractions were recorded using a Myobath system, and data acquired using DataTrax2 software (WPI). Electrical field stimulation (EFS) was used to excite transmural nerves and was applied via two platinum electrode wires (5 mm length, 2.5 mm apart) by a MultiStim system-D330 stimulator (Digitimer Ltd, England). Two different EFS protocols were used in this study. The first examined the effect of EFS at a single frequency (4 Hz), applied for a duration of 1 s at 100 s intervals and the second used a range of frequencies (1, 2, 4, 8 and 16 Hz) for durations of 30 s at 20 min intervals. Mean contraction amplitude of contractions evoked by the former protocol was obtained by averaging peak contraction amplitude of ten EFS-induced contractions before and during drug-addition (when they had their maximal effect). Drugs were added directly to the organ bath, where they were diluted in Krebs’ solution to their final concentration. GF109203X is regarded as a potent inhibitor of PKC with IC50 values in the nanomolar range [18]. However, at concentrations above 1 µM GF109203X, and other structurally-related PKC inhibitors, have been reported to have non-selective effects including inhibition of nicotinic and muscarinic acetylcholine receptors [19, 20]) and voltage-dependent Na+ channels [21]. We opted to use GF109203X at a concentration of 1 µM to maximise its inhibitory effects on PKC while minimising the risk of non-selective effects associated with higher concentrations.

The perforated patch configuration of the whole cell patch clamp technique was used to record ATP-induced currents from freshly isolated vas deferens smooth muscle cells (VDSMC) or HEK-293 cells transiently transfected with human P2X1 (NM_002558, Origene Technologies) plasmid construct (200 ng ml− 1) using Lipofectamine 2000 (Invitrogen). Electrical access between the pipette and cell interior was achieved by inclusion of the pore forming compound amphotericin B (420 µg/mL) in the pipette solution. Voltage clamp commands were delivered via an Axopatch 1D patch clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) connected to a Digidata 1440 A Digitizer (Axon Instruments) interfaced to a computer running pClamp software (Axon Instruments). During experiments, the dish containing the cells was superfused with Hanks’ solution. In addition, the cell under study was continuously superfused by means of a close delivery system consisting of a pipette (tip diameter 200 μm) placed approximately 200 μm away. This could be switched, with a dead-space time of < 5 s, to a solution containing a drug. Cells were held at -60 mV and ATP (1 µM) was applied for five second durations. P2X1 currents were reproducible at four and seven-minute intervals for VDSMC and HEK-293 cells, respectively, and two reproducible responses to ATP were obtained under control conditions in each experiment prior to addition of drugs.

α,β-meATP (10 µM, Tocris), prazosin hydrochloride (100 nM, Abcam), Phorbol 12,13-dibutyrate (PDBu, 1 µM Sigma-Aldrich), GF109203X (1 µM Merck), ATP (1 µM, Sigma-Aldrich), phenylephrine (3 µM Sigma-Aldrich).

Solutions used were of the following composition (mM): Krebs’ solution: 120 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 NaH2PO4·2H2O, 5.5 glucose, 1.2 MgCl2, and 2.5 CaCl2. pH was adjusted to 7.4 by bubbling the solution with 95% O2–5% CO2. Hanks’ Solution: NaCl (125.0), KCl (5.4), Glucose (10.0), Sucrose (2.9), NaHCO3 (4.2), KH2PO4 (0.4), NaH2PO4 (0.3), MgCl2.6H2O (0.5), CaCl2.2H2O (1.8), MgSO4 (0.4), HEPES (10.0). pH to 7.4 using NaOH. Ca2+-free Hanks’ Solution: NaCl (125), KCl (5.36), Glucose (10), Sucrose (2.9), NaHCO3 (15.5), Na2HPO4 (0.33), KH2PO4 (0.44), HEPES-free acid (10). pH to 7.4 with NaOH. Perforated patch pipette solution: CsCl (133), MgCl2 (1.0), EGTA (0.5), HEPES (10), pH adjusted to 7.2 with CsOH.

Experimental series were obtained from three or more animals; n refers to the number of tissue segments or cells studied and N to the number of animals. Data were analysed using Prism software (GraphPad). Summary data are presented as mean ± SEM. Statistical comparisons were performed on original (non-normalised) data using either Student’s paired t-test or, if three experimental groups were compared, ANOVA followed by Tukeys’ post hoc test, with p < 0.05 considered statistically significant.