The long-term goal of this research is to elucidate the regulatory mechanisms involved in arachidonic acid (AA) liberation and eicosanoid biosynthesis in the iris-ciliary body (ICB) and other ocular tissues. The central hypothesis to be tested is that phospholipase A2 (PLA2) activation is a major source for AA release in the ICB. This represents a logical pursuit of the findings obtained during the current budget period. ICB is the major source of eicosanoid synthesis in the eye. The investigators and others have reported that activation of the ICB with various agonists, including carbachol, PGE2alpha, substance P, endothelin-1, and PAF results in the rapid hydrolysis of polyphosphoinositides into IP3 and DAG, the two second messengers, and the concomitant liberation from membrane phospholipids of AA for eicosanoid biosynthesis. The investigators have found that the generation of these phospholipid-derived second messengers and the formation of cAMP and their physiological consequences to be species dependent. While the involvement of phospholipases (A2,C,D) in AA release has been documented in several tissues, including ICB, the precise mechanism underlying the stimulated release of this polyunsaturated fatty acid remains undefined. This is also true of the phospholipid source of AA. There is strong evidence from the investigators own laboratory on the ICB and from other investigators working with different tissues that PLA2, which acts on phospholipids to liberate AA and lysophospholipid, plays a central role in the control of AA required for eicosanoid biosynthesis. Based on these findings the investigators now propose to address the following specific aims: (1) Investigate the AA content of the major membrane phospholipids, phospholipases (A2,C,D) activities, and the effects of various agonists on AA release and eicosanoid biosynthesis in ICB and trabecular meshwork of different mammalian species. (2) Investigate the mechanisms involved in agonist-induced AA release and phospholipid source of AA in the iris sphincter, and AA release from phospholipids in membranes isolated from iris sphincters. Endogenous release of AA will be monitored with GLC. (3) Investigate the regulatory role of protein kinase C in AA release and PG synthesis in the iris sphincter. Both PKC inhibitors and down-regulation of PKC with PDBu will be employed. (4) Purify and characterize phospholipase(s) A2 from rabbit ICB. Activators and inhibitors of PLA2 will be employed. An attempt will be made to reconstitute the purified PLA2 with G-proteins. (5) Investigate the mechanism of the effects of in vitro and in vivo PG receptor desensitization on basal and on agonist-induced AA release and PG synthesis, IP3 production, cAMP formation, and on contraction in the iris sphincter. The proposed studies will yield important basic information about the physiological regulation of AA release from membrane phospholipids, the link between receptor activation and AA release, the profile of PGs produced in response to each agonist, the mechanism of PG receptor desensitization and its cross desensitization of other receptors, and the role of phospholipases (A2,C,D) in stimulus- dependent release of AA in ocular tissues. Understanding the biochemical basis for the regulation of AA liberation and PG synthesis will make their pharmacological manipulation more plausible, and will lead to the development of more effective anti-glaucoma and anti-inflammatory drugs.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY004387-18
Application #
2888129
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1981-12-01
Project End
2002-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
18
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Medical College of Georgia (MCG)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Augusta
State
GA
Country
United States
Zip Code
30912
Kaddour-Djebbar, I; Ansari, H R; Akhtar, R A et al. (2005) Species differences in the effects of prostanoids on MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in bovine and cat iris sphincter smooth muscle. Prostaglandins Leukot Essent Fatty Acids 72:49-57
Ansari, Habib R; Kaddour-Djebbar, Ismail; Abdel-Latif, Ata A (2004) Involvement of Ca2+ channels in endothelin-1-induced MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in rabbit iris sphincter smooth muscle. Cell Signal 16:609-19
Ansari, Habib R; Kaddour-Djebbar, Ismail; Abdel-Latif, Ata A (2004) Effects of prostaglandin F2alpha, latanoprost and carbachol on phosphoinositide turnover, MAP kinases, myosin light chain phosphorylation and contraction and functional existence and expression of FP receptors in bovine iris sphincter. Exp Eye Res 78:285-96
Sharif, Naj A; Crider, Julie Y; Husain, Shahid et al. (2003) Human ciliary muscle cell responses to FP-class prostaglandin analogs: phosphoinositide hydrolysis, intracellular Ca2+ mobilization and MAP kinase activation. J Ocul Pharmacol Ther 19:437-55
Ansari, Habib R; Davis, Angela M; Kaddour-Djebbar, Ismail et al. (2003) Effects of prostaglandin F2alpha and latanoprost on phosphoinositide turnover, myosin light chain phosphorylation and contraction in cat iris sphincter. J Ocul Pharmacol Ther 19:217-31
Husain, Shahid; Kaddour-Djebbar, Ismail; Abdel-Latif, Ata A (2002) Alterations in arachidonic acid release and phospholipase C-beta(1) expression in glaucomatous human ciliary muscle cells. Invest Ophthalmol Vis Sci 43:1127-34
Ansari, H R; Husain, S; Abdel-Latif, A A (2001) Activation of p42/p44 mitogen-activated protein kinase and contraction by prostaglandin F2alpha, ionomycin, and thapsigargin in cat iris sphincter smooth muscle: inhibition by PD98059, KN-93, and isoproterenol. J Pharmacol Exp Ther 299:178-86
Abdel-Latif, A A (2001) Cross talk between cyclic nucleotides and polyphosphoinositide hydrolysis, protein kinases, and contraction in smooth muscle. Exp Biol Med (Maywood) 226:153-63
Husain, S; Abdel-Latif, A A (2001) Effects of prostaglandin F(2alpha)and carbachol on MAP kinases, cytosolic phospholipase A(2)and arachidonic acid release in cat iris sphincter smooth muscle cells. Exp Eye Res 72:581-90
Ali, N; Yousufzai, S Y; Abdel-Latif, A A (2000) Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. Cell Signal 12:491-8

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