Cystometry

Immediately following each metabolic cage experiment, cystometry was performed. Deep anaesthesia was induced with 3% isoflurane and maintained during the entirety of the cystometry investigations. The surgical procedure followed previous similar studies [20]. Briefly, the urinary bladder was exposed by laparotomy and the femoral artery and vein were catheterized to monitor blood pressure and administer drugs (MeCh and ATP), respectively. After the catheterization of the femoral blood vessels, a pressure sensing catheter and a cannula were placed in the urinary bladder via a midline incision in the bladder dome and subsequently fixed with a ligature. Saline was infused via the cannula to induce simulated micturition cycles. The bladder was emptied before each infusion and was filled until the rat voided (approx. 30–40 s). The change in intravesical pressure that occurred during bladder filling (ΔP), volume change in the bladder (ΔV), voiding time and non-voiding contractions (NVCs), defined as increases in intravesical pressure more than 10 mmHg over baseline pressure without any voiding, were noted during the experiments. For standardization purposes, NVCs were counted during a two hour period, from the beginning of the first induced micturition cycle and onwards. Bladder compliance was calculated by dividing ΔV by ΔP. Simulated micturition cycles were performed five times before and after concentration–response series of the cholinergic agonist MeCh (1, 2 and 5 µg * kg⁻¹ i.v.) and the purinergic agonist ATP (5, 10 and 100 µg * kg⁻¹ i.v.). Each concentration–response series was performed two times. Calculations were performed on the average responses during the induced micturition cycles and the concentration–response series.