Chung, S. M.

Klintworth, G. K.

Lapetina, E. G.

Proia, A. D.

English

CU FIND (Campbell University, Catherine W. Wood School of Nursing)

Campbell University CU FIND Osteopathic Medicine, Jerry M. Wallace School of Faculty Research and Publications 6-1986 Cholinergic stimulation of phosphatidic acid formation by rat cornea in vitro A. D. Proia S. M. Chung G. K. Klintworth E. G. Lapetina Follow this and additional works at: https://cufind.campbell.edu/medicine_school  Part of the Other Medicine and Health Sciences Commons Recommended Citation Proia, A. D.; Chung, S. M.; Klintworth, G. K.; and Lapetina, E. G., "Cholinergic stimulation of phosphatidic acid formation by rat cornea in vitro" (1986). Osteopathic Medicine, Jerry M. Wallace School of. 517. https://cufind.campbell.edu/medicine_school/517 This Article is brought to you for free and open access by the Faculty Research and Publications at CU FIND. It has been accepted for inclusion in Osteopathic Medicine, Jerry M. Wallace School of by an authorized administrator of CU FIND. For more information, please contact long@campbell.edu. Cholinergic Stimulation of Phosphotidic AcidFormation by Rat Cornea In VitroAlan D. Proia,* Sophio M. Chung,* Gordon K. Klinrworrh,* and Eduardo G. LaperinotThe cornea has one of the highest acetylcholine (ACh) concentrations of any tissue but the function ofthe ACh has remained enigmatic. During studies on corneal arachidonic acid metabolism, we observedthat ACh stimulates formation of labeled phosphatidic acid in rat corneas whose phospholipids wereprelabeled with [14C]arachidonate. ACh did not affect the metabolism of free [l4C]arachidonate.(l4C]Arachidonyl-phosphatidic acid formation was doubled after 10 min of incubation in the presence ofACh concentrations of 10" M or greater. The stimulation by ACh could be completely blocked byatropine and scopolamine and partially blocked by d-tubocurarine. These studies suggest that intact ratcornea has muscarinic cholinergic receptors and that the enzymes of the inositol phospholipids pathwayare present since phosphatidic acid is an obligatory intermediate in that cycle of reactions. InvestOphthalmol Vis Sci 27:905-908, 1986Bovine and rabbit corneas have one of the highestacetylcholine concentrations of any body tissue.1'2Choline acetyltransferase, the enzyme responsible foracetylcholine synthesis, has been demonstrated in cor-neal epithelium of rabbits, cows, and humans, but littleor no enzyme activity could be detected in feline cor-neal epithelium.3 The function of the acetylcholine isuncertain, but proposed roles have included sensorymediation and regulation of epithelial sodium iontransport.1 Recently, Cavanagh, and Colley showedthat cultured rabbit corneal epithelial cells have mus-carinic cholinergic receptors4 and that addition of ace-tylcholine to cultures increases intracellular guanosine-3',5'-cyclic monophosphate concentrations and en-hances [3H]thymidine and [14C]leucine incorporation.5In the pancreas, parotid gland, brain, and other tis-sues, cholinergic stimulation results in inositol phos-pholipid hydrolysis6-7; this hydrolysis has been impli-cated in the generation of calcium signals that triggercellular processes such as secretion, contraction, andmetabolism.6"8 Phosphatidylinositol hydrolysis formsdiacylglycerol which is subsequently phosphorylatedFrom the Departments of Ophthalmology and Pathology,* DukeUniversity Medical Center, Durham, North Carolina and Departmentof Molecular Biology,f The Wellcome Research Laboratories, Bur-roughs Wellcome Co., Research Triangle Park, North Carolina.This work was supported in part by National Eye Institute Grants1 R01 EY00147, 1 R01 EYO5883, and 1 P30 EY05722. A portionof this work was performed while Dr. Proia was a Research to PreventBlindness-Dolly Green Scholar.Submitted for publication: December 20, 1984.Reprint requests: Alan D. Proia, MD, PhD, Duke University EyeCenter, Durham, NC 27710.to produce phosphatidic acid.6 8 Diacylglycerol acti-vates protein kinase C, a potential regulator of cell pro-liferation and differentiation.9 Phosphatidic acid is aputative calcium ionophore.810"16 In the experimentsreported here, we have examined the effect of cholin-ergic stimulation on phosphatidic acid formation byrat cornea in vitro.Materials and MethodsMaterials[-14C]Arachidonic acid (specific activity 60.1 mCi/mmol) was purchased from Amersham; ArlingtonHeight, IL, and used without further purification. Bo-vine albumin (essentially fatty acid free; Cohn fractionV), acetylcholine bromide, eserine (free base), atropine(free base), scopolamine hydrochloride, and d-tubo-curarine chloride were from Sigma Chemical Co.; St.Louis, MO.Preparation of CorneasCorneas were excised from enucleated eyes of 200-250-gm Sprague Dawley male rats (Harlan SpragueDawley Inc.; Indianapolis, IN) and were weighed aftergentle blotting to remove adherent fluid. Rats werecared for and treated in accordance with the ARVOResolution on the Use of Animals in Research.Radiolabeling of Corneal PhospholipidsIsolated corneas were incubated for 3 hr at 37°C in0.5 ml of modified Tyrode's solution,17 pH 7.4, con-taining 1.8 mM CaCl2, 1.0% albumin, and 1.0 /iCi[14C]arachidonic acid. After labeling the phospho-905Downloaded from iovs.arvojournals.org on 08/19/2019906 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1986 Vol. 27S 150enE^ 100-o50-•H1 1-5 10Time (min)15Fig. 1. Effect of incubation time on acetylcholine stimulation ofphosphatidic acid (PA) formation. Rat corneas whose phospholipidswere prelabeled with [I4C]AA were incubated with ( • • ) orwithout (O O) 1.0 mM acetylcholine (ACh) for varying timeperiods. Data are expressed as femtomoles of [I4C]PA formed permilligram of cornea (wet weight). Each point represents the mean(±SEM) of three (1 and 3 min), four (5 and 15 min), or nine (10min) determinations.lipids, the majority of the free arachidonate was re-moved by washing four times with 1.0 ml of ice-coldmodified Tyrode's solution. Using this procedure, thinlayer chromatography showed that 65-80% of the[l4C]arachidonate was usually in the phospholipidfraction, 15-30% in the free arachidonate fraction, andapproximately 5% in triacylglycerols.Phospholipase Activity AssayLabeled corneas (washed) were incubated at 37°Cin 0.5 ml of modified Tyrode's solution, pH 7.4, con-taining 0.1% albumin to "trap" potential arachidonatemetabolites.18 Our routine assay contained 1.0 mMacetylcholine and 50 pM eserine (to inhibit acetylcho-linesterase). In control experiments, there was no dif-ference in [l4C]arachidonyl-phosphatidic acid forma-tion when corneas were incubated with or without 50fiM eserine, in the absence of acetylcholine. In addition,incubation of radiolabeled corneas with 10 ixM atropine(in the absence of acetylcholine) did not alter phos-phatidic acid formation. Incubations were terminatedby adding 1.8 ml of chloroform:methanol (1:2) andfive drops of concentrated formic acid (final pH about3.0). After standing at room temperature for 30 minwith occasional mixing, the lipids were extracted usingan adaptation of the Bligh and Dyer procedure.19Thin Layer Chromatography (TLC)Chloroform extracts were dried under N2 gas andapplied to silica gel G TLC plates (LK5D Linear-Kplates, Whatman Chemical Separation Inc.; Clifton,NJ) using chloroform. TLC plates were used withoutprior heat activation. TLC plates were developed usingthe organic phase of ethyl acetate:isooctane:acetic acid:water (90:50:20:100) acidified by adding 1.0 ml glacialacetic acid to each 100 ml of organic phase.20'21 RFvalues were phospholipids = 0-0.04, phosphatidic acid= 0.09, 12-HETE(12S-hydroxy-5,8,10,14-eicosatetra-enoic acid) = 0.67, arachidonic acid = 0.75, and tria-cylglycerol = 0.86. TLC bands were visualized usingautoradiography with Kodak (Rochester, NY) XAR-5 film, and fractions were scraped and quantitated usinga liquid scintillation counter. For each set of assays,controls consisting of corneas boiled for 5 mins wereassayed; background radioactivity in the control phos-phatidic acid fraction was subtracted from the otherassays.ResultsUsing rat corneas whose phospholipids were prela-beled with [14C]arachidonic acid (AA), [l4C]ara-chidonyl-phosphatidic acid (PA) formation was in-creased 86% after 10 min of incubation in the presenceof 1.0 mM ACh (Fig. 1). There were no significantdifferences in the [14C]AA incorporation into the phos-pholipid, 12-HETE, or triacylglycerol fractions whencontrols were compared with samples incubated in thepresence of ACh.Our routine assays were performed using 1.0 mMACh which was sufficient to give maximal stimulationof PA formation (Fig. 2). The stimulation by ACh couldbe completely blocked by the muscarinic antagonistsatropine and scopolamine and partially blocked by thenicotinic antagonist d-tubocurarine (Fig. 3). The stim-ulation by ACh in the presence of d-tubocurarine wassignificantly less than that observed with ACh alonebut was significantly greater than control assays inwhich ACh was absent.Acetylcholine did not significantly affect the for-mation of PA when corneas were incubated with free[I4C]AA (Fig. 4). Metabolism of [14C]AA into phos-pholipids, 12-HETE, and triacylglycerol was not af-fected by the presence of ACh.DiscussionThese studies demonstrate that ACh stimulates for-mation of labeled phosphatidic acid from phospholip-Downloaded from iovs.arvojournals.org on 08/19/2019No. 6 CHOLINERGIC STIMULATION OF CORNEAL PHOSPHATIDATE SYNTHESIS/Proio er ol. 90710"ACh Concentrat ion ( M )10-2Fig. 2. Concentration curve of ACh stimulation of PA synthesis.Rat corneas whose phospholipids were prelabeled with [14C]AA wereincubated for 10 minutes in the presence of varying concentrationsof ACh. Each point is the mean (±SEM) of four assays.ids prelabeled with [I4C]AA and suggest that this pro-cess is mediated by muscarinic cholinergic receptors.Cholinergic stimulation of PA formation in pancreas,22avian salt gland,23 brain synaptosomes,24 and smoothmuscle2526 is associated with increased phosphatidyl-inositol (PI) turnover. PI hydrolysis yields diacylglyc-erol and some investigators have hypothesized that thisis the primary reaction stimulated by ACh and the rate-limiting step for PA production.27 According to thisview, increased PA formation is a "marker" for theactivity of the inositol phospholipids pathway. How-ever, other authors have proposed that cholinergicstimulation of radioisotopic labeling of PA is due todiacylglycerol kinase activation28 or reflects an in-creased availability of diacylglycerol resulting fromstimulation of phosphatidic acid phosphohydrolaseactivity.29 Our results indicate that ACh was not stim-ulating phosphatidic acid phosphohydrolase activity inrat cornea since that would have decreased [I4C]PAlevels and not increased them as observed. Studies withpurified enzyme preparations from rat cerebral cortexhave indicated that cholinergic activation of diacyl-glycerol kinase is unlikely.30 The presence of a phos-pholipase C activity catalyzing phosphodiesteraticcleavage of phosphatidylinositol in rat cornea31 is inaccord with the cholinergic stimulation of PA forma-tion being mediated via the PI cycle, but our data donot exclude that possibility that PA is being formed byphospholipase C cleavage of other phospholipids.120+10080604020illilliControl ACh ACh+Tubo ACh + Atr ACh + ScopFig. 3. Effect of nicotinic and muscarinic cholinergic antagonistson ACh stimulation of PA formation. Rat corneal phospholipidswere prelabeled with [I4C]AA and the corneas were then incubatedfor 10 min in buffer alone (control) or in the presence of either 1.0mM ACh, 1.0 mM ACh + 10 nM d-tubocurarine (Tubo), 1.0 mMACh + 10 fiM atropine (Atr), or 1.0 mM ACh + 10 nM scopolamine(Scop). Results are the mean (±SEM) of four assays except for AChalone (five assays).Phosphatidic acid has at least two potential roles incellular metabolism: as a calcium ionophore810"16 andas a source of arachidonic acid.32 The importance ofphosphatidic acid for the release of arachidonic acidhas been inferred from studies in platelets.32 In thesecells, production of phosphatidic acid precedes for-Time (min)Fig. 4. Incorporation of free [14C]AA into PA in the presence orabsence of 1.0 mM ACh. Rat corneas were incubated for either 5 or10 min with 1.0 mM ACh and 1.0 /iCi [14C]AA. Data are shown as[I4C]PA formation (mean ± SEM) for triplicate assays.Downloaded from iovs.arvojournals.org on 08/19/2019908 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / June 1986 Vol. 27mation of arachidonate during thrombin activation,21and there is a phospholipase A2 activity specific forphosphatidic acid.32 The possibility that phosphatidicacid is a source of arachidonic acid for metabolism byrat cornea is currently being investigated in our labo-ratory.Attempts to detect corneal cholinergic receptors us-ing radioligand-binding assays have yielded positiveresults with cultured rabbit corneal epithelial cells,4 butnegative results using a membrane fraction preparedfrom rabbit corneas.33 Our data are consistent with thepresence of muscarinic cholinergic receptors on ratcornea and suggest that they are involved in regulationof the phosphatidylinositol cycle. By modulating thiscycle, ACh may regulate production of inflammatorymediators from arachidonic acid and ACh may be im-portant in regulating cell proliferation during reepi-thelialization.Key words: acetylcholine, cornea, phosphatidic acid, cholin-ergic receptors, phosphatidylinositolReferences1. Fogle JA and Neufeld AH: The adrenergic and cholinergic cornealepithelium. Invest Ophthalmol Vis Sci 18:1212, 1979.2. Williams JD and Cooper JR: Acetylcholine in bovine cornealepithelium. Biochem Pharmacol 14:1286, 1965.3. Mindel JS and Mittag TW: Choline acetyltransferase in oculartissues of rabbits, cats, cattle, and man. Invest Ophthalmol 15:808, 1976.4. Colley AM and Cavanagh HD: Binding of [3H]dihydroalprenololand [3H]quinuclidinyl benzilate to intact cells of cultured cornealepithelium. Metab Pediatr Syst Ophthalmol 6:75, 1982.5. Cavanagh HD and Colley AM: Cholinergic, adrenergic, and PGE,effects on cyclic nucleotides and growth in cultured corneal ep-ithelium. Metab Pediatr Syst Ophthalmol 6:63, 1982.6. Michell RH: Inositol phospholipids and cell surface receptorfunction. Biochem Biophys Acta 415:81, 1975.7. Brown JH and Brown SB: Does phosphoinositide hydrolysismediate 'inhibitory' as well as 'excitatory' muscarinic responses?Trends Pharmacol Sci 5:417, 1984.8. Putney JW Jr: Recent hypotheses regarding the phosphatidyl-inositol effect. Life Sci 29:1183, 1981.9. Nishizuka Y: The role of protein kinase C in cell surface signaltransduction and tumour promotion. Nature 308:693, 1984.10. Gerrard JM, Butler AM, Peterson DA, and White JG: Phos-phatidic acid releases calcium from a platelet membrane fractionin vitro. Prostagland Med 1:387, 1978.11. Putney JW Jr, Weiss SJ, Van De Walle CM, and Haddas RA:Is phosphatidic acid a calcium ionophore under neurohumoralcontrol? Nature 284:345, 1980.12. Salmon DM and Honeyman TW: Proposed mechanism of cho-linergic action in smooth muscle. Nature 284:344, 1980.13. Serhan C, Anderson P, Goodman E, Dunham P, and WeissmanG: Phosphatidate and oxidized fatty acids are calcium ionophores.Studies employing arsenazo III in liposomes. J Biol Chem 256:2736, 1981.14. Imai A, Ishizuka Y, Kawai K, and Nozawa Y: Evidence forcoupling of phosphatidic acid formation and calcium influx inthrombin-activated human platelets. Biochem Biophys ResCommun 108:752, 1982.15. Benton AM, Gerrard JM, Michiel T, and Kindom SE: Are ly-sophosphatidic acids or phosphatidic acids involved in stimulusactivation coupling in platelets? Blood 60:642, 1982.16. Lapetina EG and Watson SP: The relative importance of inositol1,4,5-trisphosphate, 1,2-diacylglycerol and phosphatidic acid inmediating human platelet activation by thrombin. Proc IUPHAR9th Internat Cong Pharmacol 1984 London, MacMillan Press,1984, pp 127-131.17. Siess W, Cuatrecasas P, and Lapetina EG: A role for cyclooxy-genase products in the formation of phosphatidic acid in stim-ulated human platelets: differential mechanisms of action ofthrombin and collagen. J Biol Chem 258:4683, 1983.18. Isackson PC, Raz A, Hsueh W, and Needleman P: Lipases andprostaglandin biosynthesis. In Advances in Prostaglandin andThromboxane Research, Vol 3, Galli, C, Galli, G, and Porcellati,G, editors. New York, Raven Press, 1978, pp. 113-120.19. Lapetina EG, Schmitges CJ, Chandrabose K, and CuatrecasasP: Cyclic adenosine 3',5' monophosphate and prostacyclin inhibitmembrane phospholipase activity in platelets. Biochem BiophysRes Commun 76:828, 1977.20. Hong SL and Levine L: Inhibition of arachidonic acid releasefrom cells as the biochemical action of anti-inflammatory cor-ticosteroids. Proc Natl Acad Sci USA 73:1730, 1976.21. Lapetina EG and Cuatrecasas P: Stimulation of phosphatidicacid production in platelets precedes the formation of arachi-donate and parallels the release of serotonin. Biochim BiphysActa 573:394, 1979.22. Hokin-Neaverson M: Acetylcholine causes a net decrease inphosphatidylinositol and a net increase in phosphatidic acid inmouse pancreas. Biochem Biophys Res Commun 58:763, 1974.23. Hokin MR and Hokin LE: Studies on the carrier function ofphosphatidic acid in sodium transport. I. The turnover of phos-phatidic acid and phosphoinositide in the avian salt gland onstimulation of secretion. J Gen Physiol 44:61, 1960.24. Fisher SK and Agranoff BW: Calcium and the muscarinic syn-aptosomal phospholipid labeling effect. J Neurochem 34:1231,1980.25. Abdel-Latif AA, Owen MP, and Matheny JL: Adrenergic andcholinergic stimulation of 32P-labeling of phospholipids in rabbitiris muscle. Biochem Pharmacol 25:461, 1976.26. Jafferji SS and Michell RH: Muscarinic cholinergic stimulationof phosphatidylinositol turnover in the longitudinal smoothmuscle of guinea-pig ileum. Biochem J 154:653, 1976.27. Durell J, Garland JT, and Friedel RO: Acetylcholine action: bio-chemical aspects. Science 165:862, 1969.28. Hokin MR and Brown DF: Inhibition by 7-hexachlorocyclo-hexane of acetylcholine-stimulated phosphatidylinositol synthesisin cerebral cortex slices and of phosphatidic acid-inositol trans-ferase in cerebral cortex particulate fractions. J Neurochem 16:475, 1969.29. Schacht J and Agranoff BW: Stimulation of hydrolysis of phos-phatidic acid by cholinergic agents in guinea pig synaptosomes.J Biol Chem 249:1551, 1974.30. Lapetina EG and Hawthorne JN: The diglyceride kinase of ratcerebral cortex. Biochem J 122:171, 1971.31. Chung SM, Proia AD, KJintworth GK, Watson SP, and LapetinaEG: Deoxycholate induces the preferential hydrolysis of phos-phoinositides by human platelet and rat corneal phospholipaseC. Biochem Biophys Res Commun 129:411, 1985.32. Lapetina EG: Regulation of arachidonic acid production: roleofphospholipasesCand A2. Trends Pharmacol Sci 3:115, 1982.33. Olsen JS and Neufeld AH: The rabbit cornea lacks cholinergicreceptors. Invest Ophthalmol Vis Sci 18:1216, 1979.Downloaded from iovs.arvojournals.org on 08/19/2019

Cholinergic stimulation of phosphatidic acid formation by rat cornea in vitro

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Cholinergic stimulation of phosphatidic acid formation by rat cornea in vitro

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