title: Intracellular signaling in human iridial fibroblasts and iridial melanocytes in response to prostaglandins, endothelin, isoproterenol, and other pharmacological agents.
abstract: OBJECTIVE
  The receptor-coupled signal transduction systems present in isolated human iridial fibroblasts (HIF) and in human iridial melanocytes (HIM) were investigated. Cell responsiveness to numerous prostaglandins (PGs), and other compounds of interest, was profiled in order to better understand their involvement in the iridial hyper-pigmentation process observed during treatment of elevated intraocular pressure with FP-receptor against PG analogs.
  
  
  METHODS
  [(3)H]-inositol phosphates ([(3)H]-IPs) generated in the cells were measured by ion-exchange chromatography followed by liquid scintillation spectroscopy. cAMP generated in the cells was quantified using an enzyme immunoassay.
  
  
  RESULTS
  HIF cells exhibited a robust phosphoinositide (PI) hydrolysis response to FP-class PG analogs, such as cloprostenol (potency, EC(50) = 2.4 ± 0.5 nM, n = 5), fluprostenol (EC(50) = 5.3 ± 0.6 nM, n = 3), PGF(2α) (EC(50) = 54 ± 18 nM, n = 5), and latanoprost acid (EC(50) = 121 ± 17 nM, n = 4). Other PGs exhibited the following potencies (EC(50)) for stimulating [(3)H]-IPs accumulation in HIF cells: PGD(2) EC(50) = 327 ± 195 nM, n =3; PGE(2) EC(50) = 550 ± 50 nM, n = 3; and two TP-receptor agonists (I-BOP, EC(50) = 23 ± 8 nM, n = 3; U-46619 EC(50) = 1.1 ± 0.4 µM, n = 3). Endothelin-1 (ET-1) and histamine increased [(3)H]-IPs production in HIF and HIM cells. HIM cells exhibited minimal PI turnover response to cloprostenol, latanoprost acid, latanoprost, PGF(2α), PGE(2), and histamine, but there were robust responses to ET-1 (EC(50) = 4.6 nM, n = 2) and an ET(B)-receptor agonist (BQ-3020, EC(50) = 5 nM, n = 2) that were blocked by an ET(B)-antagonist (BQ-788, IC(50) = 21 ± 6 nM, n = 3). In the adenylyl cyclase activation assay, numerous PGs (1 and 10 µM) stimulated cAMP production in HIF cells yielding the following rank order of efficacy: PGI(2) > PGE(2) > misoprostil > isoproterenol = BW245C > PGD(2) = PGF(2α) = fluprostenol. In HIM cells, PGE(2) (EC(50) = 1.3 ± 0.3 nM) and isoproterenol (β-agonist; EC(50) = 89 ± 13 nM) potently and efficaciously stimulated cAMP production and ICI-118851 (β(2)-antagonist) attenuated the effects of isoproterenol. However, latanoprost acid, latanoprost, ET-1, and BW245C (DP-receptor agonist) were relatively less efficacious than isoproterenol and PGE(2) in HIM cells at stimulating cAMP production.
  
  
  CONCLUSIONS
  These studies have shown that while HIF cells express FP prostaglandin and histamine receptors coupled to phospholipase C to produce [(3)H]-IPs, the HIM cells lack such functionally active FP-receptors. In contrast, HIF and HIM cells express functional ET-1 receptors coupled to [(3)H]-IPs production and both cell-types respond to PGE(2), BW245C, and isoproterenol by generating cAMP. It is concluded that human iridial fibroblasts and melanocytes respond differently to PGs and histamine, but in the same manner to ET-1, isoproterenol and BW245C. This may have relevance to the intercellular communication within the iris relative to the melanogenic processes.
authors.affiliation: Pharmaceutical Research, Alcon Research, Ltd., Fort Worth, Texas 76134, USA. naj.sharif@alconlabs.com
authors.forename: Najam A
authors.initials: NA
authors.lastname: Sharif
authors.forename: Julie Y
authors.initials: JY
authors.lastname: Crider
mesh_terms: D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D007498:Iris / Q000166:cytology*; D007545:Isoproterenol / Q000494:pharmacology*; D008544:Melanocytes / Q000187:drug effects* / Q000378:metabolism; D011453:Prostaglandins / Q000494:pharmacology*; D044022:Receptor, Endothelin A / Q000378:metabolism; D011968:Receptors, Histamine / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000378:metabolism; D012588:Scintillation Counting; D015398:Signal Transduction
chemical_list: D019332:Endothelin-1; D007295:Inositol Phosphates; D011453:Prostaglandins; D044022:Receptor, Endothelin A; D011968:Receptors, Histamine; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; D000242:Cyclic AMP; D007545:Isoproterenol