Serotonin-stimulated cyclic AMP synthesis in the rabbit corneal epithelium Arthur H. Neufeld, Sally. Ledgard, Marcia M. Jumblatt, and Stephen D. Klyce* Serotonin increases the level of cyclic AMP in incubated rabbit corneas; the concentration of agonist producing half-maximal stimulation is approximately 1.5 fxm. Nialamide, an inhibitor of monoamine oxidase, potentiates the response to serotonin but not to epinephrine. Amitriptxjline, an inhibitor of neuronal uptake of serotonin, does not potentiate the stimulation of cyclic AMP synthesis. Lysergic acid diethylaynide, but not timolol, antagonizes the response to serotonin; the half-maximal inhibitory concentration is approximately 6 nm lysergic acid, diethylamide. A comparison of the time course of the increase in cyclic AMP synthesis after addition of serotonin or epinephrine to the incubation media indicates that serotonin, but not epinephrine, must penetrate a barrier to its free diffusion. We conclude that the corneal epithelium contains specific serotonergic receptors that, upon activation, cause the synthesis of cyclic AMP, which mediates the stimulation of chloride transport (c.f companion article, Klyce et al. 7 ). The serotonergic receptors must be at a location posterior to the (3-adrenergic receptors, which are on the anterior surface of the apical cells. (INVST OPHTHALMOL VIS SCI 23:193-198, 1982.) Key words: serotonin, epinephrine, cyclic AMP, cornea, epithelium, chloride transport, lysergic acid diethylamide, timolol, rabbit Serotonin (5-hydroxytryptamine) is released from specific nerve terminals as a neurotransmitter 1 and from mast cells as an inflammatory mediator. 2 Various regions of the central nervous system have serotonin-containing nerves and at least two types of sero- From the ye Research Institute of Retina Foundation and Department of Ophthalmology, Harvard Medical School, Boston, Mass., and *Lions ye Research Laboratories, Louisiana State University ye Center, New Orleans, La. This work was supported in part by U.S.P.H.S. grants Y02360 and Y-03311, and by Biomedical Research Support grant 5S07RR05527. Dr. Neufeld is the recipient of Research Career Development Award Y- 00114. Submitted for publication Aug. 20, 1981. Reprint requests: Arthur H. Neufeld, Ph.D., ye Research Institute, 20 Staniford St., Boston, Mass. 02114. Portions of this work were reported at the Annual Meeting of the Association for Research in Vision and Ophthalmology, May 2, 1981, Sarasota, Fla. tonergic receptors. 3 ' 4 Stimulation by serotonin of the high-affinity receptors causes the activation of adenylate cyclase and the synthesis of cyclic AMP; stimulation of the lowaffinity receptors causes behavioral changes in the animal. 5 Both types of serotonergic receptors are inhibited by D-lysergic acid diethylamide (LSD), a specific serotonergic receptor antagonist. Binding of serotonin to these receptors accomplishes the synaptic function of this neurotransmitter. Peripherally, the release of serotonin from mast cells during degranulation implies a role as an inflammatory mediator. The action of serotonin on peripheral tissues includes vasoconstriction and platelet aggregation. 6 The preceding article 7 demonstrates serotonin-stimulated chloride transport in the rabbit cornea and implicates neural control of this process. In this article we have characterized the serotonergic response pathway in the rabbit cornea pharmacologically and bio- 0146-0404/82/080193+06$00.60/0 1982 Assoc. for Res. in Vis. and Ophthal., Inc. 193
194 Neufeld et al. Invest. Ophthalmol. Vis. Sci. August 1982 160n 140 - PI 50 T c 40-30- o a O) ^. CL < O "o a 120-100- 20;; CONT onial 10~ 7 10~ 6 10~ 5 10-4 M [SROTONIN] Fig. 2. Serotonin-dependent cyclic AMP synthesis in the absence (CONT) and presence of 0.1 mm nialamide (NIAL). All points are the mean ± S..M. of six to 14 determinations. 5 10 15 20 25 30 MIN Fig. 1. Time courses of basal (BAS), epinephrine- (PI), and serotonin- (SR) stimulated cyclic AMP synthesis in the presence of IBMX. Agonists were added at 0 min. All points are the mean ± S..M. of six to eight determinations. chemically by studying the ability of the neurotransmitter to stimulate the synthesis of cyclic AMP. Because of the abundance of /3-adrenergic receptors in the cornea, 8 we compared the serotonergic response with the adrenergic response to epinephrine. Materials and methods New Zealand white male rabbits (1.5 to 2.4 kg) were killed with an intravenous overdose of so- dium pentobarbital. The corneas were excised, quartered, and immersed in buffer (100 mm NaCl, 20 mm NaH 2 PO 4, 6.9 mm dextrose, 4.5 mm KC1, 0.8 mm MgCl 2, and 0.4 mm CaCl 2, ph 7.6) at 37 C for 15 min. Tissues were transferred to buffer containing 0.5 mm 3-isobutyl-l-methylxanthine (IBMX) for 5 min and incubated for 5 to 15 min in the presence or absence of the following agents at the concentrations indicated: nialamide (10" 4 M), amitriptyline (KT 5 M), LSD (10~ 9 M to 10~ 5 M), timolol maleate (10~ 9 M to 10~ 5 M), serotonin (10~ 7 M to 10~ 4 M), and epinephrine (10~ 9 M to 10~ 5 M). Immediately after the incubation period, corneas were extracted and assayed for cyclic AMP by radioimmunoassay as previously described 9 and for protein by the method of Lowry et al. 10 IBMX, epinephrine, nialamide, and serotonin were from Sigma Chemical Co., St. Louis, Mo.; LSD was from Sandoz Pharmaceuticals,. Hanover, N.J.; timolol maleate and amitryptiline were from Merck, Sharp & Dohme, West Point, Pa. The components for the radioimmunoassay of cyclic AMP were from Collaborative Research. Results In vitro incubation of full-thickness corneas in the presence of serotonin or epinephrine increased the level of cyclic AMP in the tis-
Volume 23 Number 2 Serotonin-stimulated cyclic AMP synthesis 195 30-r [SROTONIN ] Fig. 3. Serotonin-dependent cyclic AMP synthesis in the absence (CONT) and presence of 10 /u,m amitriptyline (AMI). All points are the mean ± S..M. of six determinations. sue, but the time course of synthesis and accumulation of cyclic AMP in response to the two agonists was quite different. As demonstrated in Fig. 1, the level of cyclic AMP did not change in the absence of exogenous agonist. Addition of serotonin to the incubation media stimulated the synthesis to a maximum of about 25 pmol cyclic AMP/mg protein/15 min. After reaching this maximum, there was a decline in the amount of cyclic AMP in the tissue over the next 15 min. However, after the addition of epinephrine to the incubation media, the level of cyclic AMP increased rapidly. Within the first 5 min, large amounts of cyclic AMP were synthesized and accumulated to reach a maximum of 125 pmol cyclic AMP/mg protein/5 min. No additional accumulation occurred over the next 25 min, and there may have been some decline in the level of cyclic AMP between 15 and 30 min after addition of epinephrine. Thus, not only the time course of synthesis of cyclic AMP but also the amount of cyclic AMP synthesized over time in response to epinephrine is quite different than the amount synthesized in response to serotonin. The cornea has substantially less ability to respond to serotonin with the synthesis of cyclic AMP than it does to respond to epinephrine. As shown in Fig. 2, in the presence of 0.5 [PINPHRIN] Fig. 4. pinephrine-dependent cyclic AMP synthesis in the absence (CONT) and presence of 0.1 mm nialamide (N1AL). All points are the mean ± S..M. of four to six determinations. mm IBMX, serotonin at concentrations between 1 and 100 /JLM caused a dose-dependent synthesis and accumulation of cyclic AMP. The apparent concentration for half-maximal stimulation was 1.5 JU-M serotonin, with maximal accumulation occurring at about 10 /xm. Nialamide (0.1 mm) potentiated the synthesis of cyclic AMP in response to serotonin. In the absence of serotonin, nialamide did not cause an increase in the level of cyclic AMP. Fig. 3 demonstrates that amitriptyline (10 /xm) did not potentiate the synthesis of cyclic AMP in response to serotonin. Comparing Fig. 2 with Fig. 4, the stimulation of the synthesis of cyclic AMP by epinephrine in this tissue was quite different than that by serotonin. Cyclic AMP synthesis and accumulation occurred over a range from 10 to 100 nm epinephrine; the apparent concentration for half-maximal stimulation was 60 nm epinephrine. Saturating concentrations of epinephrine caused the synthesis of much more cyclic AMP than saturating concentrations of serotonin. Furthermore, epi-
196 Neufeld et al. Invest. Ophthalmol. Vis. Sci. August 1982 50-r 100 T " 40 o Q. O) 30-10- ocont LSD 10 " 7,-6 10" 6 10~ ~ 5 10" 4 [SROTONIN] Fig. 5. Serotonin-dependent cyclic AMP synthesis in the absence (CONT) and presence of 10 /AM LSD. All points are the mean ± S..M. of four to 14 determinations. M 10 10" 10 [LSD] Fig. 6. Inhibition of serotonin-dependent cyclic AMP synthesis by LSD. Percent inhibition was calculated by dividing serotonin-dependent cyclic AMP synthesis in the presence of LSD by that in the absence of LSD. All points are the mean ± S..M. of six determinations. nephrine-stimulated synthesis of cyclic AMP was not sensitive to potentiation by nialamide. Fig. 5 illustrates that the specific serotonergic antagonist, LSD, prevented the synthesis and accumulation of cyclic AMP in the cornea in response to serotonin. In the absence of serotonin, LSD did not cause any change in the level of cyclic AMP. The concentration dependence of inhibition by LSD is demonstrated in Fig. 6. The apparent concentration for half-maximal inhibition of serotonin-stimulated synthesis of cyclic AMP was 6 nm LSD. To determine whether the mechanism by which serotonin increases the level of cyclic AMP is by stimulation of/3-adrenergic receptors, corneas were stimulated with serotonin in the presence of timolol. As indicated in Fig. 7, 10 /xm timolol did not antagonize the synthesis of cyclic AMP in response to serotonin concentrations between 1 and 100 /xm. Discussion The cornea contains a serotonergic response pathway that activates the synthesis of cyclic AMP and stimulates chloride transport. 7 This pathway must be located in the corneal epithelium, so that serotonin stimulates the same physiologic response as catecholamines do via the adrenergic pathway. 11 " 13 The stimulation of the synthesis of cyclic AMP by serotonin is specific. Serotonin acts at serotonergic receptors, as demonstrated by the inhibition of the response by LSD, a specific serotonergic antagonist. LSD has no agonistic activity in the cornea. Serotonin does not act at the /3-adrenergic receptors because timolol does not antagonize the ability of serotonin to stimulate the synthesis of cyclic AMP. However, timolol does block serotonin-stimulated chloride transport. 7 Thus timolol may inhibit biochemical steps that are necessary for the activation of chloride transport but are beyond the synthesis of cyclic AMP. Although the serotonergic receptors have not been characterized for this tissue, the receptors appear similar to the low-affinity serotonergic receptors found in the central nervous system. 3 " 5 The apparent concentration for half-maximal activation of the synthesis of cyclic AMP of 1.5 fxm serotonin is similar to the affinity constant for serotonin at the sero-
Volume 23 Number 2 Serotonin-stimulated cyclic AMP synthesis 197 tonin-2 receptors (2.7 /LtM), as determined by radioligand binding assays of membranes prepared from brain tissues. 3 In the brain, the serotonergic receptors linked to adenylate cyclase are the high-affinity receptors (K D = 2.7 nm serotonin), 5 and whether the low-affinity receptors are linked to adenylate cyclase is unclear. Like the brain, the corneal epithelium may contain more than one type of serotonergic receptor. However, until radioligand binding assays are performed, we conclude that the corneal epithelium contains low-affinity, serotonin-2-like receptors that are linked to adenylate cyclase in this tissue. At saturating concentrations serotonin causes much less synthesis of cyclic AMP and a smaller increase in chloride transport than does epinephrine. Perhaps the density of serotonergic receptors in corneal epithelium is considerably less than that of /3-adrenergic receptors. Nialamide is a potent inhibitor of monoamine oxidase. 14 Although nialamide does not stimulate the synthesis of cyclic AMP, the drug alone does stimulate chloride transport to some degree. At this time we cannot account for this discrepancy. The potentiation of the serotonergic stimulation of the synthesis of cyclic AMP by nialamide indicates that serotonin is susceptible to degradation by monoamine oxidase. The physiologic evidence that serotonin is more effective at stimulating chloride transport when delivered to the endothelial surface suggests that serotonin does not cross the apical tight junctions and must approach the epithelium from the posterior side. 7 Thus, as exogenous serotonin diffuses through the tissue to the serotonergic receptors, monoamine oxidase degrades the transmitter. Monoamine oxidase would be present in mitochondria in the corneal epithelial cells and in the corneal nerve terminals. However, the inhibitor of neuronal uptake of serotonin, amitriptyline, does not potentiate serotonergic stimulation. Therefore the monoamine oxidase that metabolizes the serotonin is not in the nerves and must be in the epithelial cells. pinephrine, which is also a substrate for o a. CD 40 T 30-20 10-6 10-5 10,-4 M [SROTONIN] Fig. 7. Serotonin-dependent cyclic AMP synthesis in the absence (CONT) and presence of 10 /u-m timolol (TIM). All points are the mean ± S..M. of 10 to 13 determinations. monoamine oxidase, is not potentiated by nialamide. Thus, as epinephrine diffuses to the /3-adrenergic receptors, this transmitter does not pass monoamine oxidase-rich areas in the tissue. Furthermore, the time courses for increases in cyclic AMP after serotonin and epinephrine, as well as the time courses for stimulation of chloride transport by these two agents, are quite different. In both cases the response to serotonin reaches a maximum more slowly than the response to epinephrine, indicating a longer diffusion time for serotonin to reach the serotonergic receptors. Because of the different susceptibilities of serotonin and epinephrine to degradation by monoamine oxidase and because of the different time courses of the responses, we conclude that the /3-adrenergic receptors are on the anterior membrane of the apical cells and the serotonergic receptors are on cell membranes deeper within the epithelium. The chloride entry step is probably located on the basolateral membranes of the wing or basal cells, and thus chloride diffuses anteriorly within the epithelial cells via gap junctions. pinephrine stimulates chloride transport, via mediation by cyclic AMP, by causing a conductance change to chloride of
198 Neufeld et al. Invest. Ophthalmol. Vis. Sci. August 1982 the anterior membrane of the apical cells. 15 For serotonin to act deeper within the epithelium than epinephrine but to stimulate chloride transport by causing a conductance change similar to epinephrine in the apical cells, the serotonergic receptors must be located on the posterior membranes of the apical cells or on the basolateral membranes of the wing and/or basal cells. The latter location would require diffusion of cyclic AMP within the cell syncytium to the apical cells. In sum, the corneal epithelium contains at least two receptor pathways that, upon activation, cause the synthesis of cyclic AMP and stimulate chloride transport. The /3-adrenergic pathway is located on the anterior surface of the apical cells at a greater density than the serotonergic pathway, which is located deeper within the epithelium. The physiologic significance of having two activation pathways for stimulating chloride transport is unclear. RFRNCS 1. Fuller RW: Pharmacology of central serotonin neurons. Ann Rev Pharmacol Toxicol 20:111, 1980. 2. yre P: Histamine H2 receptors in the sheep bronchus and cat trachea: the action of burimamide. Br J Pharmacol 48:321, 1973. 3. Peroutka SK and Snyder SH: Multiple serotonin receptors: differential binding of [ 3 H]5-hydroxytryptamine, [ 3 H]lysergic acid diethylamide and [ 3 H]spiroperidol. Mol Pharmacol 16:687, 1979. 4. Pedigo NW, Yamamura HI, and Nelson DL: Discrimination of multiple [ 3 H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in the rat brain. J Neurochem 36:220, 1981. 5. Peroutka SJ, Lebovitz RM, and Snyder SH: Two distinct central serotonin receptors with different physiological functions. Science 212:827, 1981. 6. Douglas WW: Autacoids. In The Pharmacological Basis of Therapeutics. Gilman AG, Goodman LS, and Gilman A, editors, ed. 6. New York, 1980, MacMillan Publishing Co., pp. 590-629. 7. Klyce SD, Palkama KA, Harkonen M, Marshall WS, Huhtaniitty S, Mann KP, and Neufeld AH: Neural serotonin stimulates chloride transport in the rabbit corneal epithelium. INVST OPHTHALMOL VIS SCI 23:181, 1982. 8. Neufeld AH, Zawistowski KA, Page D, and Bromberg BB: Influences on the density of j8-adrenergic receptors in the cornea and iris-ciliary body of the rabbit. INVST OPHTHALMOL Vis Sci 17:1069, 1978. 9. Jumblatt MM, Fogle JA, and Neufeld AH: Cholera toxin stimulates adenosine 3'5'-monophosphate synthesis and epithelial wound closure in the rabbit cornea. INVST OPHTHALMOL Vis Sci 19:1321, 1980. 10. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265, 1951. 11. Klyce SD, Neufeld AH, and Zadunaisky JA: The activation of chloride transport by epinephrine and Db cyclic AMP in the cornea of the rabbit. INVST OPHTHALMOL 12:127, 1973. 12. Chalfie M, Neufeld AH, and Zadunaisky JA: Action of epinephrine and other cyclic AMP-mediated agents on the chloride transport of the frog cornea. INVST OPHTHALMOL 11:644, 1972. 13. Candia OA, Bently PJ, and Cook PI: Stimulation by amphotericin B of active Na transport across amphibian cornea. Am J Physiol 226:1438, 1974. 14. Axelrod J: Noradrenaline rate and control of its biosynthesis. Science 173:598, 1971. 15. Klyce SD and Wong RKS: Site and mode of adrenaline action of chloride transport across the rabbit corneal epithelium. J Physiol 266:777, 1977.