iik7 and luzindole

Melatonin (N-acetyl-5-methoxytryptamine) is documented as a hormone involved in the circadian regulation of physiological and neuroendocrine function in mammals. Herein, the effects of melatonin on the functions of porcine granulosa cells in vitro were investigated. Porcine granulosa cells were cultivated with variable concentrations of melatonin (0, 0.001, 0.01, 0.1, 1.0, and 10ng/mL) for 48h. Melatonin receptor agonist (IIK7) and antagonist (Luzindole, 4P-PDOT) were used to further examine the action of melatonin. The results showed optimum cell viability and colony-forming efficiency of porcine granulosa cells at 0.01ng/mL melatonin for 48-h incubation period. The percentage of apoptotic granulosa cells was significantly reduced by 0.01 and 0.1ng/mL melatonin within the 48-h incubation period as compared with the rest of the treatments. Estradiol biosynthesis was significantly stimulated by melatonin supplementation and suppressed for the progesterone secretion; the minimum ratio of progesterone to estradiol was 1.82 in 0.01ng/mL melatonin treatment after 48h of cultivation. Moreover, the expression of BCL-2, CYP17A1, CYP19A1, SOD1, and GPX4 were up-regulated by 0.01ng/mL melatonin or combined with IIK7, but decreased for the mRNA levels of BAX, P53, and CASPASE-3, as compared with control or groups treated with Luzindole or 4P-PDOT in the presence of melatonin. In conclusion, the study demonstrated that melatonin mediated proliferation, apoptosis, and steroidogenesis in porcine granulosa cells predominantly through the activation of melatonin receptor MT2 in vitro, which provided evidence of the beneficial role of melatonin as well as its functional mechanism in porcine granulosa cells in vitro.

Topics: Animals; Apoptosis; Cells, Cultured; Female; Gene Expression Regulation; Granulosa Cells; Isoindoles; Melatonin; Receptor, Melatonin, MT2; Swine; Tryptamines

We have investigated the effect of melatonin and its analogues on rabbit corneal epithelial wound healing.. New Zealand rabbits were anaesthetised and wounds were made by placing Whatman paper discs soaked in n-heptanol on the cornea. Melatonin and analogues (all 10 nmol) were instilled. Wound diameter was measured every 2 hours by means of fluorescein application with a Topcon SL-8Z slit lamp. Melatonin antagonists (all 10 nmol) were applied 2 hours before the application of the n-heptanol-soaked disc and then every 6 hours together with melatonin. To confirm the presence of MT2 receptors in corneal epithelial cells immunohistochemistry, Western blot and RT-PCR assays in native tissue and in rabbit corneal epithelial cells were performed. The tear components were extracted then processed by HPLC to quantify melatonin in tears.. Migration assays revealed that melatonin and particularly the treatment with the MT2 agonist IIK7, accelerated the rate of healing (p < 0.001). The application of the non-selective melatonin receptor antagonist luzindole and the MT2 antagonist DH97 (but not prazosin), prevented the effect of melatonin on wound healing (both p < 0.001). Immunohistochemistry, Western blot and RT-PCR assays showed the presence of MT2 melatonin receptor in corneal epithelial cells. In addition, we have identified melatonin in tears and determined its daily variations.. These data suggest that MT2 receptors are implicated in the effect of melatonin on corneal wound healing regulating migration rate. This suggests the potential use of melatonin and its analogues to enhance epithelial wound healing in ocular surface disease.

Topics: Animals; Antioxidants; Blotting, Western; Burns, Chemical; Cell Line; Epithelium, Corneal; Eye Burns; Eye Proteins; Heptanol; Immunohistochemistry; Isoindoles; Male; Melatonin; Rabbits; Receptor, Melatonin, MT2; Reverse Transcriptase Polymerase Chain Reaction; Tears; Tryptamines; Wound Healing

Melatonin, the MT(2) melatonin receptor agonist IIK7 [N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethanamine], and the putative MT(3) melatonin receptor agonist 5-MCA-NAT [5-methoxycarbonylamino-N-acetyltryptamine] have previously been shown to reduce intraocular pressure (IOP) in ocular normotensive rabbits. To gain a better understanding of the structure-activity relationship of compounds that activate MT(2) and MT(3) receptors mediating reductions in IOP, novel melatonin analogs with rationally varied substitutions were synthesized and tested for their effects on IOP in ocular normotensive rabbits (n = 160). All synthesized melatonin analogs reduced IOP. The best-effect lowering IOP was obtained with the analogs INS48848 [methyl-1-methylene-2,3,4,9-tetrahydro-1H-carbazol-6-ylcarbamate], INS48862 [methyl-2-bromo-3-(2-ethanamidoethyl)-1H-indol-5-ylcarbamate], and INS48852 [(E)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-3-phenylprop-2-enamide]. These compounds produced dose-dependent decreases in IOP that were maximal at 0.1 mM (total dose of 0.259 μg for INS48848, 0.354 μg for INS48862, and 0.320 μg for INS48852) and 1 mM (total dose of 2.59 μg for INS48848, 3.54 μg for INS48862, and 3.20 μg for INS48852), with maximal reductions of 36.0 ± 4.0, 24.0 ± 1.5, and 30.0 ± 1.5% for INS48848, INS48862, and INS48852, respectively. Studies using melatonin receptor antagonists (luzindole, prazosin, and DH97 [N-pentanoyl-2-benzyltryptamine]) indicated that INS48862 and INS48852 activate preferentially a MT(2) melatonin receptor and suggest that INS48848 may act mainly via a MT(3) receptor. The most effective compounds were also well tolerated in a battery of standard ocular surface irritation studies. The implication of these findings to the design of novel drugs to treat ocular hypertension is discussed.

Topics: Animals; Dose-Response Relationship, Drug; Drug Design; Eye; Glaucoma; Intraocular Pressure; Isoindoles; Melatonin; Ocular Hypertension; Rabbits; Receptor, Melatonin, MT2; Receptors, Melatonin; Structure-Activity Relationship; Time Factors; Tryptamines