Initial Electrophysiological Study Of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Cation Channel On ICCs In The Adult Guinea Pig Bladder

As we all know that spontaneous action potential(AP) can be recorded from muscular tissue of bladder, even if its nerval control is removed. But till now its mechanism is unknown to us.It has been confirmed the existence of the ICC in bladder on early study. The Ih current was recorded so simply. All of these results could not confirm which kind of role the ICC may play on functions.The Ih current is regarded as "pacemaker current" on the study of gastrointestinal tract and sinus node. HCN channels are the molecular basis of the Ih current. Our series of preliminary studies have prompted the existence of HCN channels on ICC in bladder. However, its electrophysiological characteristics have not yet been clear. Additionally, the method of using cultured ICCs was unsatisfactory in electrophysiological experiments. For the most main reason, It's very difficult to clamp patch on cultured ICCs. The test parameters could not meet the actual that is under physiological conditions.So the aim of our study: To find the method to get better ICCs by acute separation.To observe the expression of HCN by by c-Kit and HCN antigen using double-labeled immunofluorescence (IF). To get the basic electrophysiologic parameters of HCN. To provide the theoretical basis and experimental basis in further research.Main results and conclusions:1. We have got the stable and reliable method for better ICCs by acute separation. The Program for its enzymatic digestion:â…¡Collagenase 2.0mg/ml; Trypsin inhibitor 2.0mg/ml ;Papain 1.0mg/ml; BSA 1.0mg/ml. The time of digestion is 15 min.2. We have confirmed the existence of HCN by c-Kit and HCN antigen using double-labeled immunofluorescence (IF).3. Whole-cell patch clamp recorded a inward current which began to slowly active between -90mV～-140mV in voltage and time dependent manner without deactivation. The current range is 300-800pA. Ih current were blocked by 20μM ZD7288 and 500uM Cs⁺.4. The reversal potential of Ih was 42±2mV, Na+ versus K+(PNa/PK) was 0.42, and membrane capacitance was 10-40pF. The extracellular ZD7288 blocked Ih current with concentration-dependent, and its IC50 was 24.7±6.5uM. the blocking effect of ZD7288 is not reversible. The extracellular Cs⁺ blocked Ih current with concentration-dependent, and its IC50 was 232.5±24.8μM. the blocking effect of Cs⁺ is reversible.5. Both the extracellular Na⁺ and K⁺ could impact activation and conductance of HCN with concentration-dependent. There were no significant effect on Ih current by adding extracellular drug such as TTX, TEA, and Ba²⁺.6. The extracellular Ca2+ could significantly affect the activation time of HCN with concentration-dependent. The intracellular cAMP could significantly affect the activation time of HCN with concentration-dependent.