Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576, USA.
Invest Ophthalmol Vis Sci. 2012 Aug 7;53(9):5178-89. doi: 10.1167/iovs.11-9046.
Effects of cis-unoprostone isopropyl, its primary metabolite M1, trans-unoprostone isopropyl, latanoprost free acid, and fluprostenol were studied on Ca(2+)-activated K(+) (BK) channels, plasma membrane potential, cAMP, cGMP, and steady state Ca(2+), and protection against endothelin-1 (ET-1)-induced steady state Ca(2+) increases in human cortical neuronal (HCN-1A), trabecular meshwork (HTMC), and pulmonary artery smooth muscle (PASMC) cells. Effects on recombinant human prostaglandin (PG) receptors were determined.
BK channel currents were measured using whole-cell patch clamp; cAMP, cGMP with ELISAs; Ca(2+) with indo-1; plasma membrane potential using diBAC(4)(3); and PG receptor effects with PG receptor-expressing cells and FLIPR fluo-4 Ca(2+) assays.
Unoprostone isopropyl and M1 activated sustained iberiotoxin (IbTX)-sensitive, AL-8810 (FP receptor antagonist)-insensitive BK channel currents with EC(50)s of 0.51 ± 0.03 nM (n = 5) and 0.52 ± 0.03 nM (n = 6) in HTMCs; 0.61 ± 0.06 nM (n = 8) and 0.46 ± 0.04 nM (n = 5) for M1 in HCN-1A cells and PASMC, respectively. They caused AL-8810-insensitive, IbTX-sensitive membrane hyperpolarization at 10 nM; up to 100 nM had no effect on or decreased cAMP, cGMP, and Ca(2+); and prevented ET-1-induced Ca(2+) increases. In contrast, 10 nM latanoprost free acid and fluprostenol caused membrane depolarization; increased cAMP, cGMP, and Ca(2+); and did not prevent ET-1-induced Ca(2+) increases. Trans-unoprostone isopropyl had no effects. Unoprostone isopropyl (1.25 μM) had no effect on PG receptors, and neither did M1, except for activating the FP receptor with EC(50) = 557.9 ± 55.2 nM (n = 4).
Prostones, unoprostone isopropyl and M1, are potent AL-8810-insensitive, stereospecific BK channel activators, without cAMP, cGMP, or Ca(2+) involvement, and prevent ET-1-induced steady state Ca(2+) increases in HTMCs.
研究顺式乌诺前列酮异丙酯、其主要代谢物 M1、反式乌诺前列酮异丙酯、拉坦前列素游离酸和氟前列醇对 Ca(2+)激活的 K(+)（BK）通道、质膜电位、cAMP、cGMP和稳态 Ca(2+)的影响，以及对内皮素-1（ET-1）诱导的人皮质神经元（HCN-1A）、小梁网（HTMC）和肺动脉平滑肌（PASMC）细胞稳态 Ca(2+)增加的保护作用。测定了对重组人前列腺素（PG）受体的影响。
使用全细胞膜片钳测量 BK 通道电流；使用 ELISA 测量 cAMP、cGMP；使用 indo-1 测量 Ca(2+)；使用 diBAC(4)(3)测量质膜电位；使用表达 PG 受体的细胞和 FLIPR fluo-4 Ca(2+)测定法测定 PG 受体的作用。
乌诺前列酮异丙酯和 M1 激活了持续的 iberiotoxin（IbTX）敏感、AL-8810（FP 受体拮抗剂）不敏感的 BK 通道电流，在 HTMC 中的 EC(50)分别为 0.51 ± 0.03 nM（n = 5）和 0.52 ± 0.03 nM（n = 6）；在 HCN-1A 细胞和 PASMC 中，M1 的 EC(50)分别为 0.61 ± 0.06 nM（n = 8）和 0.46 ± 0.04 nM（n = 5）。在 10 nM 时，它们引起 AL-8810 不敏感、IbTX 敏感的膜超极化；高达 100 nM 对 cAMP、cGMP和 Ca(2+)没有影响或降低；并防止 ET-1 诱导的 Ca(2+)增加。相比之下，10 nM 拉坦前列素游离酸和氟前列醇引起膜去极化；增加 cAMP、cGMP和 Ca(2+)；并且不能防止 ET-1 诱导的 Ca(2+)增加。反式乌诺前列酮异丙酯没有作用。乌诺前列酮异丙酯（1.25 μM）对 PG 受体没有影响，M1 也没有影响，除了以 EC(50) = 557.9 ± 55.2 nM（n = 4）激活 FP 受体。
前列腺素、乌诺前列酮异丙酯和 M1 是强效的 AL-8810 不敏感、立体特异性 BK 通道激活剂，不涉及 cAMP、cGMP或 Ca(2+)，并防止 ET-1 诱导的 HTMC 中稳态 Ca(2+)增加。
