dinoprost and iberiotoxin

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.. 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.. 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).. 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.

Topics: Calcium; Calcium Channel Agonists; Cell Membrane; Cyclic AMP; Cyclic GMP; Dinoprost; Dose-Response Relationship, Drug; Endothelin-1; Humans; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Muscle, Smooth; Patch-Clamp Techniques; Peptides; Trabecular Meshwork

Unoprostone isopropyl (unoprostone) is a docosanoid currently used as an antiglaucoma agent. Unoprostone is known to have neuroprotective effects and to activate large conductance Ca²⁺-activated K⁺ (BK) channels. Recently, unoprostone has been tested in clinical studies as a therapeutic agent for retinitis pigmentosa (RP) and studies have demonstrated an improvement in retinal sensitivity and in the protection of central retinal sensitivity with its use. However, the mechanism of action underlying unoprostone's protective effect in RP is not fully known. It is well known that the pathogenesis of RP can be accelerated by oxidative stress or light irradiation. Therefore, the current study investigated the effects and the underlying mechanism of action of unoprostone on oxidative stress- and light irradiation-induced damage in photoreceptor and retinal pigment epithelial cultures.. The study used the mouse retinal cone-cell line 661W to investigate the effects of unoprostone and its major metabolite, unoprostone-free acid (M1), on oxidative stress- or light irradiation-induced cell death, and a human retinal pigment epithelial cell line (ARPE-19), was used to investigate the effects on light-induced disruption of phagocytotic function in a latex bead assay. Additionally, we examined whether the effects of unoprostone and M1 were mediated by BK channels using iberiotoxin, a selective inhibitor of BK channels.. Unoprostone and M1 protected against light- or H₂O₂-induced cell death in 661W cells, and against light-induced phagocytotic dysfunction in ARPE-19 cells. Additionally, iberiotoxin inhibited the protective effects of unoprostone and M1.. These findings indicate that unoprostone has protective effects on oxidative stress- and light irradiation-induced damage in vitro and that these effects are mediated by activation of BK channels. This confirms that unoprostone represents a promising therapeutic agent for the treatment of RP and other retinal diseases.

Topics: Animals; Antioxidants; Calcium; Cell Death; Cell Line; Dinoprost; Epithelial Cells; Humans; Hydrogen Peroxide; Large-Conductance Calcium-Activated Potassium Channels; Light; Mice; Oxidative Stress; Peptides; Phagocytosis; Potassium Channel Blockers; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium

Effects of unoprostone isopropyl (unoprostone), a prostaglandin metabolite analog; latanoprost, a PGF(2alpha) analog; and PGF(2alpha) were examined in HCN-1A cells, a model system for studies of large conductance Ca(2+) activated K(+)(BK) channel activator-based neuroprotective agents. Unoprostone and latanoprost, both used as anti-glaucoma agents, have been suggested to act through FP receptors and have neuroprotective effects. Ion channel activation, plasma membrane polarization, [Ca(2+)](i) changes and protection against long-term irreversible glutamate-induced [Ca(2+)](i) increases were studied. Unoprostone activated iberiotoxin (IbTX)-sensitive BK channels in HCN-1A cells with an EC(50) of 0.6+/-0.2 nM and had no effect on Cl(-) currents. Unoprostone caused IbTX-sensitive plasma membrane hyperpolarization that was insensitive to AL8810, an FP receptor antagonist. In contrast, latanoprost and PGF(2alpha) activated a Cl(-) current sensitive to [Ca(2+)](i) chelation, tamoxifen and AL8810, and caused IbTX-insensitive, AL8810-sensitive membrane depolarization consistent with FP receptor-mediated Ca(2+) signaling Cl(-) current activation. Latanoprost and PGF(2alpha), but not unoprostone, increased [Ca(2+)](i). Unoprostone, PGF(2alpha) only partially, but not latanoprost protected HCN-1A cells against glutamate-induced Ca(2+) deregulation. These findings show that unoprostone has a distinctly different mechanism of action from latanoprost and PGF(2alpha). Whether unoprostone affects the BK channel directly or an unidentified signaling mechanism has not been determined.

Topics: Antihypertensive Agents; Calcium; Calibration; Cell Line; Chelating Agents; Chloride Channels; Dinoprost; Glutamic Acid; Humans; Ion Channel Gating; Latanoprost; Membrane Potentials; Peptides; Potassium Channels, Calcium-Activated; Prostaglandins F, Synthetic; Receptors, Prostaglandin; Tamoxifen

To clarify the vasodilatory mechanism of unoprostone isopropyl (unoprostone), a PG F2alpha related compound used for treatment of glaucoma, we have investigated the effect of this drug and its metabolites on isolated rabbit ciliary artery in vitro.. Under the dissecting microscope, ciliary arteries were prepared from albino rabbit eyes and mounted in a myograph system. The effects of unoprostone isopropyl and other agents were investigated using isometric tension recording methods.. Unoprostone induced a dose-dependent relaxation in ciliary arteries that were pre-contracted with high-K solution, 10 microM histamine or 10 microM PG F2alpha. Neither unoprostone metabolite M1 or M2 had a relaxant effect on the precontracted vessels. Relaxation was unaffected by inhibition of adenylyl cyclase with SQ 22536, guanylyl cyclase with ODQ, or maxi-K channels with iberiotoxin. Pretreatment with unoprostone did not affect histamine-induced transient contractions in Ca2+ -free solution. However, SKF96365, a general Ca2+ channel blocker, evoked relaxation similar to unoprostone with respect to amplitude and rate of onset.. Unoprostone, but not its metabolites M1 and M2, relaxed pre-contracted rabbit ciliary artery. The mechanism of vascular smooth muscle relaxation by unoprostone differs from that of IOP reduction and does not depend on adenylyl cyclase, guanylyl cyclase, or maxi-K channels. Relaxation may be mediated by inhibition of Ca2+ entry, possibly through capacitative Ca2+ channels.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Antihypertensive Agents; Ciliary Arteries; Dinoprost; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Histamine; Isometric Contraction; Large-Conductance Calcium-Activated Potassium Channels; Male; Muscle, Smooth, Vascular; Oxadiazoles; Peptides; Potassium Channels, Calcium-Activated; Quinoxalines; Rabbits; Vasodilation

Although PGF(2alpha) affects contractility of vascular smooth muscles, no studies to date have addressed the electrophysiological mechanism of this effect. The purpose of our investigation was to examine the direct effects of PGF(2alpha) on membrane potentials, Ca(2+)-activated K(+) (K(Ca)) channels, delayed rectifier K(+) (K(V)) channels, and L-type Ca(2+) channels with the patch-clamp technique in single rabbit middle cerebral arterial smooth muscle cells (SMCs). PGF(2alpha) significantly hyperpolarized membrane potentials and increased the amplitudes of total K(+) currents. PGF(2alpha) increased open-state probability but had little effect on the open and closed kinetics of K(Ca) channels. PGF(2alpha) increased the amplitudes of K(V) currents with a leftward shift of the activation and inactivation curves and a decrease in the activation time constant. PGF(2alpha) decreased the amplitudes of L-type Ca(2+) currents without any significant change in threshold or apparent reversal potentials. This study provides the first finding that the direct effects of PGF(2alpha) on middle cerebral arterial SMCs, at least in part, could attenuate vasoconstriction.

Topics: Animals; Calcium; Calcium Channels, L-Type; Cell Separation; Chelating Agents; Delayed Rectifier Potassium Channels; Dinoprost; Egtazic Acid; Female; In Vitro Techniques; Male; Membrane Potentials; Middle Cerebral Artery; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Peptides; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Potassium Channels, Voltage-Gated; Rabbits

In the present study mechanism of inhibitory effects of capsaicin on the contractility of rabbit coronary artery were studied by measurement of isometric tension and intracellular Ca2+ concentration. Capsaicin (1 microM to 30 microM) relaxed the coronary artery pre-contracted with prostaglandin (PG) F2alpha (1 microM) in a concentration-dependent manner. The PGF2alpha-induced increase in intracellular Ca2+ concentration was also inhibited. The effects of capsaicin were readily reversed by washing capsaicin from the bath. Capsaicin-induced relaxation was not attenuated by pretreatment with capsazepine (1 microM), a blocker of vanilloid receptor or ruthenium red (1 microM), a blocker of non-selective cation channel. Previous exposure to a high concentration of capsaicin (100 microM) or repeated application of capsaicin did not eliminate the relaxation response to subsequent application of capsaicin. Increasing the external K+ concentration to 80 mM significantly attenuated the capsaicin-induced relaxation with simultaneous change in intracellular Ca2+ concentration. Pretreatment with iberiotoxin (100 nM), a blocker of Ca2+-activated K+ channel, only partially inhibited the capsaicin-induced relaxation. However, application of 4-aminopyridine (4-AP, 1 mM), a blocker of delayed rectifier K+ current significantly inhibited the capsaicin-induced relaxation with concomitant attenuation of the effect on intracellular Ca2+ concentration. These results indicate that capsaicin may have a direct relaxing effect on the smooth muscle contractility, and relaxation may be due to activation of the 4-AP-sensitive, delayed rectifier K+ channels in the rabbit coronary artery.

Topics: 4-Aminopyridine; Animals; Calcium; Capsaicin; Coronary Vessels; Dinoprost; Drug Interactions; Fluorescent Dyes; Fura-2; In Vitro Techniques; Isometric Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Peptides; Potassium Channel Blockers; Potassium Channels; Rabbits; Ruthenium Red

The action of endothelin in small intestinal resistance vessels of the guinea pig was studied by examining submucosal arteriole vasoactivity in vitro and electrical properties of mesenteric arteriole smooth muscle cells. Endothelin-1 (ET-1) constricted submucosal arterioles with a half-maximal effective concentration of 170 pM. ET-3 caused detectable constriction with a minimum of 20 nM. The ET-1 response was prolonged, with a time to 90% relaxation of 41 +/- 2.8 min after washout. The ETA antagonist BQ-123 (200 nM) decreased the sensitivity to ET-1 approximately 40-fold. Arterioles preconstricted with prostaglandin F2 alpha did not relax when superfused with ET-1, ET-3, or an ETB agonist, IRL-1620, and pretreatment with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine was ineffective in countering ET-1-induced constriction, indicating the absence of functional ETB receptors. Resting membrane potential in isolated cells was characterized by transient hyperpolarizing spikes (THs). ET-1 (20 nM) increased TH frequency and caused the emergence of a larger amplitude population. Under voltage clamp, spontaneous transient outward currents (STOCs) were seen that reversed at the K+ equilibrium potential. ET-1 increased STOC frequency and amplitude. Iberiotoxin (IBTX; 200 nM), a maxi-K+ channel antagonist, blocked the ET-1-induced THs and reduced STOC activity. IBTX or tetraethylammonium increased the rate and extent of ET-1-induced arteriole constriction. We suggest that ET-1-induced vasoactivity of ileal resistance arterioles involves ETA receptor-mediated early activation of maxi-K+ channels that serves to counter strong constriction.

Topics: Animals; Arginine Vasopressin; Arterioles; Dinoprost; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-3; Endothelins; Guinea Pigs; Ileum; Intestinal Mucosa; Kinetics; Large-Conductance Calcium-Activated Potassium Channels; Male; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth; Muscle, Smooth, Vascular; Norepinephrine; Peptide Fragments; Peptides; Peptides, Cyclic; Potassium Channels; Potassium Channels, Calcium-Activated; Receptor, Endothelin A; Scorpion Venoms; Tetraethylammonium; Time Factors; Vasoconstriction