Unoprostone isopropyl and metabolite M1 activate BK channels and prevent ET-1-induced [Ca²⁺]i increases in human trabecular meshwork and smooth muscle.
OBJECTIVE
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](i), [cGMP](i), and steady state [Ca(2+)](i), and protection against endothelin-1 (ET-1)-induced steady state [Ca(2+)](i) 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.


METHODS
BK channel currents were measured using whole-cell patch clamp; [cAMP](i), [cGMP](i) with ELISAs; [Ca(2+)](i) 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.


RESULTS
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](i), [cGMP](i), and [Ca(2+)](i); and prevented ET-1-induced [Ca(2+)](i) increases. In contrast, 10 nM latanoprost free acid and fluprostenol caused membrane depolarization; increased [cAMP](i), [cGMP](i), and [Ca(2+)](i); and did not prevent ET-1-induced [Ca(2+)](i) 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).


CONCLUSIONS
Prostones, unoprostone isopropyl and M1, are potent AL-8810-insensitive, stereospecific BK channel activators, without [cAMP](i), [cGMP](i), or [Ca(2+)](i) involvement, and prevent ET-1-induced steady state Ca(2+) increases in HTMCs.
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576, USA. John.Cuppoletti@uc.edu
John
J
Cuppoletti
Danuta H
DH
Malinowska
Kirti P
KP
Tewari
Jayati
J
Chakrabarti
Ryuji
R
Ueno
D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D000242:Cyclic AMP; D006152:Cyclic GMP / Q000378:metabolism; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000187:drug effects*; D008564:Membrane Potentials / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism
D002120:Calcium Channel Agonists; D019332:Endothelin-1; D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; C121227:AL 8810; C072630:isopropyl unoprostone; C064719:iberiotoxin; D015237:Dinoprost; D000242:Cyclic AMP; D006152:Cyclic GMP; D002118:Calcium