dinoprost and fura-2-am

This study analyzes additional mechanisms behind the ocular hypotensive effect of prostaglandin F (PGF) receptor (FP receptor) agonists PGF2alpha and fluprostenol (fluprostenol-isopropyl ester [travoprost]), which reduce intraocular pressure (IOP) in patients with glaucoma probably by enhancing uveoscleral flow. The trabecular meshwork (TM) is actively involved in IOP regulation through contractile mechanisms. Contractility of TM is induced by endothelin (ET)-1, a possible pathogenic factor in glaucoma. The involvement of FP receptor agonists in the ET-1 effects on TM function was studied.. The effects of FP receptor agonists on contractility of bovine TM (BTM) were investigated using a force-length transducer. The effects of PGF2alpha on intracellular Ca2+ ([Ca2+]i) mobilization in cultured cells were measured using fura-2AM. The expression of the FP receptor protein was examined using Western blot analysis.. The ET-1-induced (10(-8) M) contraction in isolated BTM was inhibited by PGF2alpha (10(-6) M) and fluprostenol (10(-6) M). This effect was blocked by FP receptor antagonists. Carbachol-induced contraction or baseline tension was not affected by PGF2alpha or fluprostenol. In cultured TM cells, ET-1 caused a transient increase in [Ca2+]i that was reduced by PGF2alpha. No reduction occurred in the presence of the FP receptor antagonist Al-8810. Western blot analysis revealed the expression of the FP receptor in native and cultured TM.. FP receptor agonists operate by direct interaction with ET-1-induced contractility of TM. This effect is mediated by the FP receptor. Thus, FP receptor agonists may decrease IOP by enhancing aqueous humor outflow through the TM by inhibiting ET-1-dependent mechanisms.

Topics: Animals; Blotting, Western; Calcium; Cattle; Cells, Cultured; Cloprostenol; Dinoprost; Endothelin-1; Fura-2; Muscle Contraction; Muscle, Smooth; Receptors, Prostaglandin; Trabecular Meshwork; Travoprost

The main purpose of this study was to check whether phyto- and endogenous estrogens influence calcium ion mobilization [Ca(2+)](i) in bovine endometrial cells and whether this action is connected with biological effects i.e. prostaglandin (PG)F(2alpha) production. In our study we used two calcium measurement methods by comparing the microscopic method with widely used quantitative - spectrofluorometric method of [Ca(2+)](i) measurement. We also wanted to confirm whether visualization of calcium ion [Ca(2+)](i) in cells using microscopic method supported by micro image analysis (Micro Image Olympus system) reflects real, qualitative changes in the ion concentration. In both methods a cell-permeable form of fluorescent [Ca(2+)](i) indicator Fura-2 was used. Cultured bovine endometrial epithelial and stromal cells influenced by phorbol-2-myristate-13-acetate (PMA; positive control), estradiol 17-beta (E(2); endogenous estrogen) and active metabolites of phytoestrogens (environmental estrogens) were used as a model to study PGF(2alpha) secretion and [Ca(2+)](i) mobilization in the cells. Equol and para-ethyl-phenol in doses of 10(-8)-10(-6) M increased PGF(2alpha) concentration both in epithelial and stromal cells (P<0.05). In both methods, equol and para-ethyl-phenol did not cause intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P>0.05). Both methods revealed that only E(2) and PMA induced intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P<0.05). The results of both methods were highly correlated (P<0.001; r=0.82 for epithelial cells and r=0.89 for stromal cells). In conclusion, both methods gave approximately the same results and showed that phytoestrogens, in contrast to PMA and E(2), did not cause intracellular [Ca(2+)](i) mobilization in endometrial cells. The obtained results proved that the [Ca(2+)](i) visualization method supported by micro image analysis can produce similar results to the spectrofluorometric method.

Topics: Animals; Calcium; Cattle; Dinoprost; Endometrium; Epithelial Cells; Equol; Estradiol; Female; Fluorescent Dyes; Fura-2; Isoflavones; Microscopy, Fluorescence; Phenols; Phytoestrogens; Spectrometry, Fluorescence; Stromal Cells; Tetradecanoylphorbol Acetate

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

To investigate the presence of specific prostaglandin receptors in primary cultures of human ciliary muscle cells by measuring agonist-stimulated intracellular calcium mobilization.. The ciliary muscle cells, cultured from postmortem human ciliary muscle explants, were first characterized by anti-desmin and anti-smooth muscle alpha-actin antibodies. Increase in intracellular calcium concentrations, in fura 2-AM loaded human ciliary muscle cells stimulated by prostaglandin receptor agonists, were determined with a digital fluorescence imaging system.. The resting intracellular calcium concentration in the fura 2-AM loaded cells was 60.0 +/- 6.0 nM. The threshold concentration of PG receptor agonists needed to increase the concentration of intracellular calcium was 10(-8) M. The stimulation of these cells by PGF2 alpha, 17-phenyl trinor PGE2, and U46619, the FP, EP1, and TP receptor agonists, resulted in the dose-dependent increase of intracellular calcium.. The results of the present study suggest that EP1, FP, and TP receptors are present in human ciliary muscle cells.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Actins; Adult; Calcium; Ciliary Body; Desmin; Dinoprost; Dinoprostone; Fluorescent Dyes; Fura-2; Humans; Middle Aged; Muscle, Smooth; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Thromboxane A2