Unoprostone Isopropyl and Its Primary Metabolite M1 Activate BK and ClC-2 Channels and Prevent ET-1-Induced Smooth Muscle Effects

Abstract

Purpose: : The aim of this study was to determine the molecular and cellular effects of the IOP lowering anti-glaucoma agent unoprostone isopropyl (UI), its primary metabolite that lacks the isopropyl group (metabolite M1) and trans-unoprostone isopropyl (TU), the latter of which does not lower IOP. Effects on large conductance Ca²⁺-activated K⁺ (BK) channels, ClC-2 Cl^- channels, EP and FP receptors, [Ca²⁺]_i, [cAMP]_i and [cGMP]_i were determined as well as effects on plasma membrane potential and protection against ET-1-induced [Ca²⁺]_i increases in vascular smooth muscle. These were compared to effects of latanoprost (LP) and travoprost (TP).

Methods: : Whole cell patch clamp was used to study effects on BK and ClC-2 Cl^- channels. [Ca²⁺]_i was measured with indo-1, [cAMP]_i and [cGMP]_i with ELISAs and plasma membrane potential using diBAC(4)5.

Results: : It was previously reported that UI activated BK channels with EC₅₀=0.6 ± 0.2 nM. In the present study, UI activated ClC-2 Cl^- channels with EC₅₀=0.56 ± 0.07 nM and M1 activated BK and ClC-2 Cl^- channels with EC₅₀s of 0.6 ± 0.06 and 0.48 ± 0.11 nM, respectively, but TU had no effect. UI and M1 had no agonist activity on EP or FP receptors (EC₅₀>1.25 µM), but M1 had no significant pharmacological activity on the FP receptor (EC₅₀=600 nM). LP and TP but not UI, M1 or TU increased [Ca²⁺]_i, [cAMP]_i, and [cGMP]_i. UI and M1 caused IbTX-sensitive, AL8810-insensitive membrane hyperpolarization in smooth muscle cells and prevented ET-1-induced [Ca²⁺]_i increases. TU had no effects. In contrast, LP and TP caused IbTX-insensitive, AL8810-sensitive depolarization, and did not protect against ET-1-induced [Ca²⁺]_i increases.

Conclusions: : UI and M1, but not TU, LP or TP activate BK K⁺ and ClC-2 Cl^- channels without EP or FP receptor, [Ca²⁺]_i, [cAMP]_i, or [cGMP]_i involvement. UI and M1, unlike TU, LP and TP, cause membrane hyperpolarization and protect against ET-1-induced [Ca²⁺]_i increases. The high affinity activation of the ion channels by UI and M1 (which lower IOP) and the lack of effect of TU (which does not lower IOP) suggest that UI and M1 actions are stereo specific and responsible for the pharmacological activity of UI and M1.

Keywords: ion channels • electrophysiology: non-clinical • receptors: pharmacology/physiology