Cyclic 3',5'-Adenosine Monophosphate Transiently Stimulates the in vitro Fluid Transport Across the Isolated Porcine Ciliary Body Epithelium via Protein Kinase A

摘要

Purpose: : To study the effects of cyclic 3',5'-adenosine monophosphate (cAMP) signalling pathway on the fluid flow (FF) and potential difference (PD) across the porcine ciliary body epithelium (CBE) measured by the modified Ussing-type fluid chamber. Methods: : A complete annulus of the CBE preparation was excised and mounted in a modified Ussing-type fluid chamber. The two hemi-chambers were connected to different external vessels. On one side, a bubbling reservoir fitted with a pair of Ag/AgCl electrodes containing Ringer solution was connected. On the other side, the hemi-chamber was a close compartment fitted with another pair of Ag/AgCl electrodes and a graduated capillary. The capillary level reflected the change of fluid volume inside the chamber and the rate of FF was calculated accordingly. The PD was monitored via one pair of Ag/AgCl electrodes by a voltage-current clamp unit, and tissue resistance was determined by passing a fixed (10 ?μA) current across the CBE preparation. The effects of several cAMP-regulating agents on FF and PD were examined. Results: : Forskolin (10 ?μM, n = 5), an activator of adenylate cyclase, hyperpolarized the PD by 100% (P 0.005) and increased the FF by 42% (P 0.005). The cAMP analogue 8-Br-cAMP (100 ?μM, n = 5) hyperpolarized the PD by 190% (P 0.005) and stimulated the FF by 56% (P 0.005). The non-specific inhibitor of phosphodiesterases, 3-isobutyl-1-methylxanthine (IBMX, 1 mM, n = 6) only slightly hyperpolarised the PD by 22% (P 0.005) but caused no significant change in the FF (P = 0.74). The pre-treatment of protein kinase A (PKA) inhibitor, H-89 (50 ?μM, n = 5) partially blocked the cAMP-stimulated PD and FF by 65% (P 0.001) and 79% (P 0.01) respectively. Conclusions: : The fluid formation by the CBE can be stimulated by cAMP/PKA signalling pathway. This pathway likely modulates the transport of ion across the CBE.