Effects of Calcitonin Gene-Related Peptide in the Eye

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1 Investigative Ophthalmology & Visual Science, Vol. 29, No. 7, July 1988 Copyright Association for Research in Vision and Ophthalmology Effects of Calcitonin Gene-Related Peptide in the Eye A Study in Rabbirs and Cars Olli Oksob*t and Johan Srjernschanrz:}: Calcitonin gene-related peptide (CGRP) has recently been demonstrated in sensory neurons of the eye. The purpose of the present study was to determine the effects of exogenous CGRP in the rabbit and cat eye. CGRP was injected intracamerally and the intraocular pressure was measured in cannulated eyes. The pupil diameter and the aqueous humor protein concentration were also measured. Indomethacin was used to prevent prostaglandin synthesis and tetrodotoxin (TTX) to block nerve conductance. In the rabbit eye, CGRP caused iridial hyperemia, a breakdown of the blood-aqueous barrier and increased intraocular pressure. These responses were dose-related. The increase in IOP as well as the breakdown of the blood-aqueous barrier could not be blocked with TTX or indomethacin. In cats CGRP caused a decrease in IOP and had only slight effect on the aqueous humor protein concentration. Neither in rabbits nor in cats had CGRP any detectable effect on the pupil size. Intracameral injection of 0.1 Mg (7.4 X 10~ n moles) substance P together with 0.1 ng (2.6 X 10"" moles) CGRP in rabbits caused maximal miosis but did not potentiate the intraocular effects of CGRP only. These results indicate that CGRP has marked vascular effects in the rabbit eye, causing a breakdown of the blood-aqueous barrier and increased IOP. The mechanism of this phenomenon does not involve prostaglandins neither nerve conduction, implying most likely a direct effect on the vascular smooth muscle. The mechanism of the decrease of IOP in cats remains unknown. Invest Ophthalmol Vis Sci 29: ,1988 The eye typically reacts to noxious stimuli with miosis, hyperemia, breakdown of the blood-aqueous barrier and increased intraocular pressure (IOP). The mediators behind these responses are not known but generally, prostaglandins and sensory neurons are thought to be the most important factors. 1 " 6 When the iris is traumatized directly, prostaglandins seem to be the key factor 1-3 probably exerting most of their effects through an activation of sensory neurons. 4 " 6 In nonperforated eyes the noxious impulse is thought to be propagated into the eye through an axon reflex mechanism. 7 In these cases prostaglandins seems to be less important. Calcitonin gene-related peptide (CGRP), a novel 37-amino acid polypeptide encoded within the calcitonin gene, 8 has been shown to occur in the nervous system. 9 CGRP has histochemically been localized to sensory neurons in the anterior segment of the eye. 10 " 13 Substance P (SP) or a closely related tachykinin has been shown to be involved in the miotic From The Research Department of Pharmaceutical Company Oy Star Ab, Tampere, Finland, and the fdivision of Pharmacology, Department of Pharmacy, University of Helsinki, Helsinki, Finland. X Present address: Pharmacia Ophthalmics Ab, Uppsala, Sweden. Submitted for publication: July 6, 1987; accepted March 2, Reprint requests: Olli Oksala, Pharmaceutical Company Oy Star Ab, P.O. Box 33, Tampere, Finland. component part of the acute irritative response of the rabbit eye 5614 " 17 but the vascular effects of SP in the anterior uvea of the rabbit have been reported to be much less marked In the rabbit eye SP can be released from sensory nerves by trigeminal nerve stimulation 14 and recently it was shown that CGRP can also be released into the aqueous humor of rabbits in a similar way. 19 Thus, as a strong vasodilatator, CGRP could very well be involved in the acute irritative response in the eye causing hyperemia, a breakdown of the blood-aqueous barrier and ocular hypertension. In previous studies CGRP has been demonstrated immunohistochemically and biochemically in the eye and the effects of exogenous CGRP have been demonstrated in the rabbit eye The purpose of the present study was to analyze further the effects of exogenous CGRP in the rabbit eye and to investigate the effects of CGRP in the cat eye with special reference to the irritative response. Materials and Methods Albino rabbits weighing kg and cats weighing 2-3 kg of either sex were used. The rabbits were anaesthetized with % urethane (2 g/kg b.w.). The anaesthesia of the cats was initiated with intramuscular injection of a combination of ketamine ( mg/kg b.w.) and xylazine (0.5 mg/kg b.w.) and maintained with intravenous infusion of ketamine. In both rab- 1006

No. 7 EFFECTS OF CGRP IN THE EYE / Oksalo and Srjernschonrz 1007 bits and cats the level of the anaesthesia was such that the corneal reflex was only weak or absent.

2 No. 7 EFFECTS OF CGRP IN THE EYE / Oksalo and Srjernschonrz 1007 bits and cats the level of the anaesthesia was such that the corneal reflex was only weak or absent. All the animals received indomethacin 10 mg/kg b.w. intravenously min before the cannulation of the eyes. The animals were kept warm by a thermostated heating pad. All the animals were tracheotomized to assure adequate ventilation and some of them were artificially ventilated. In all experiments the ph, P C o 2 and P O2 of the arterial blood were checked routinely a few times during the course of the experiment (ABL ; Radiometer, Copenhagen, Denmark). Artificial ventilation was adjusted to give normal or close to normal values for Pco 2 > Po 2 an d ph. Both in rabbits and cats a femoral artery was cannulated and connected to a pressure transducer for blood pressure monitoring and in cats a femoral vein was also cannulated for administration of the anaesthetic. The eyes were cannulated with 27 gauge needles connected to polyethylene tubings. Great care was taken not to touch the iris at any time during the experiment. The intraocular pressure and the blood pressure were measured with Statham P-50 pressure transducers (Gould, Inc., Oxnard, CA) connected to a Grass Model 79-D polygraph (Quincy, MA). Intracameral injections were carried out with Hamilton precision syringes. A volume of 5 /ul was injected which caused a transient increase in IOP of 2-6 mm Hg lasting from 2 to 5 min. An intravenous infusion of a 6% Dextran solution (Pharmacia, Uppsala, Sweden) diluted (1:1) with isotonic saline was used whenever needed to sustain the blood pressure. The infusion was started as soon as the mean arterial blood pressure fell below a level of about 70 mm Hg and was continued as long as necessary. The total amount of dextran solution did not exceed 50 ml. The pupil diameter was measured with a transparent millimeter ruler under standard laboratory light. Iridial hyperemia was estimated macroscopically in albino animals. The hyperemia of the experimental eye was compared with that of the contralateral control eye, and was graded slight, medium or strong. At the end of the experiment, paracentesis was performed with a 27 gauge needle and aqueous humor was stored at -50 until analyzed for protein concentration. Calcitonin gene-related peptide (a-cgrp (rat); Peninsula Laboratories, Belmont, CA), substance P (Peninsula), and tetrodotoxin (Sigma Chemical Company, St. Louis, MO) were dissolved in isotonic saline and stored at -50 C. Experiments With Intracameral Injection of CGRP in Normal Eyes In these experiments the eyes were cannulated with two needles, one for intraocular pressure measurement and one for administration of CGRP/vehicle. CGRP at Mg (2.6 X 10~ ll -1.3 X 10" 8 moles) was injected into the experimental eye after a stable baseline IOP was obtained (5- min after the second cannulation), while the other eye served as a control, receiving isotonic saline only. Experiments With Intracameral Injection of CGRP in TTX Pretreated Eyes In these experiments an additional needle was used for TTX administration. One microgram (3.1 X 10~ 9 moles) of TTX was injected intracamerally bilaterally 10 min before the CGRP administration. Only rabbits were used. The effects of TTX denervation were estimated in six eyes by stimulating the preganglionic part of the sympathetic trunk electrically (10 Hz, 2 msec, 1 ma) before and after the TTX injection. Before the injection stimulation caused rapidly mydriasis, the pupil diameter reaching 10.1 ± 0.4 mm. The TTX injection itself caused a slight mydriasis, the pupil diameter being 8.7 ± 0.4 mm after TTX adminitration compared with 6.2 ± 0.1 mm prior to TTX injection. Ten minutes after the injection, sympathetic stimulation had no effect at all on the pupil size. All animals underwent in addition light stimulation in order to verify the blockade of cholinergic miosis. It was assumed that the unmyelinated sensory neurons are blocked in a similar way to the cholinergic and adrenergic neurons. The control eye received the vehicle only. Experiments With Intracameral Injection of CGRP andsp These experiments were performed only in rabbits. A mixture of 0.1 ^g (7.4 X 10"" moles) of SP and 0.1 Hg (2.6 X 10"" moles) of CGRP was injected intracamerally into four eyes. The control eye received the vehicle only. The results are expressed as the arithmetical mean value ± SEM, and were statistically evaluated with the student t-test for paired data by comparing the experimental eye with the control eye. This research was carried out according to the guidelines of the ARVO Resolution on the Use of Animals in Research. Results Intracameral injection of CGRP into the rabbit eye caused hyperemia, a breakdown of the bloodaqueous barrier and increased intraocular pressure. CGRP at 0.1 /ig (2.6 X 10"" moles) caused slight hyperemia in the iris; a dose of 1-5 fig (2.6 X 10" X 10" 9 moles) caused strong hyperemia. Intracameral injection of ^g (2.6 X 10~"-1.3

3 1008 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1988 Vol i B * * TUm(mln) X) mmhfl 35 * *!? TP t Tim* (min) Tlnwdnln) Fig. 1. Effect ofintracameral injection of 0.1 /xg(a, n = 6), 0.5 ^g(b, n = 6), l-2/xg(c, n = 5) and 5 ng(d, n = 5) CGRP on the intraocular pressure in rabbits. Mean arterial blood pressure indicated by BP in the lower part of the respective picture. Experimental eyes indicated by solid line and control eyes by dotted line. Bars indicate SEM. * = P < 0.05, ** = P < 0.01 and *** = P < EXPERIMENTAL EYE I R A B B I T CGRP 0.1 uglcgrp 0.5 ug cgrp 1-2 ug CGRP 5 ug Illllll CONTROL EYE Fig. 2. Effect ofintracameral injection of CGRP on the aqueous humor protein concentration in rabbits and cats. Bars indicate SEM. ** = P< X 10 9 moles) CGRP caused a dose-dependent increase in IOP (Fig. 1) and in the aqueous humor protein concentration (Fig. 2). Intracameral injection of 10 ng (2.6 X 10~ 12 moles) had no effect on the IOP or aqueous humor protein concentration. CGRP had no effect on the pupil size. In cats intracameral injection of 5 tig (1.3 X 10~ 9 moles) CGRP caused a decrease in IOP (Fig. 3). There were no macroscopic signs of ocular irritation in cats, but a tendency towards an increase (statistically insignificant) in the aqueous humor protein concentration was detected (Fig. 2). CGRP injected intracamerally in as high a dose as 50 Mg (1.3 X 10~ 8 moles) in three cats caused only a slight increase in the aqueous humor protein concentration and did not elevate IOP. Pretreatment of rabbit eyes with 1 /*g (3.1 X 10~" 9 moles) intracameral TTX, 10 min before injection of

4 No. 7 EFFECTS OF CGRP IN THE EYE / Oksalo. and Srjernschonrz 1009 mmhg 35 mmhg 1T BP """* T I- I Fig. 3. Effect of intracameral injection of 5 fig CGRP on the intraocular pressure in cats. Experimental eyes indicated by solid line and control eyes by dotted line. Bars indicate SEM. Mean arterial blood pressure indicated as in Figure 1, (n = 6). * = P < Fig. 4. Effect of intracameral injection of 1 ug CGRP on the intraocular pressure in rabbits which received an intracameral injection of TTX bilaterally 10 min prior to CGRP. Experimental eyes indicated by solid line and control eyes by dotted line. Bars indicate SEM. Mean arterial blood pressure indicated as in Figure 1, (n = 6). * = P < 0.05, ** = P< tig (2.6 X lo" 10 moles) CGRP did not block the effects of CGRP on IOP or the aqueous humor protein concentration (Figs. 4, 5). TTX itself induced hyperemia and a slight but insignificant rise in IOP and the protein concentration of the aqueous humor. Intracameral injection of a mixture of 0.1 ug (7.4 X 10-" moles) SP and 0.1 /*g (2.6 X lo" 11 moles) CGRP induced maximal miosis but had no additional effect on IOP (Fig. 6), the hyperemia or the aqueous humor protein concentration (4 ± 58 mg/100 ml in the experimental eyes and 92 ± 6 mg/100 ml in the contralateral control eyes) as compared with the corresponding dose of CGRP only. If anything, the increase in IOP was smaller than with 0.1 ug CGRP only. Discussion Recent studies demonstrate that CGRP in the anterior uvea is confined to sensory neurons, 10 " 13 is released by stimulation of the trigeminal nerve, 19 and is a strong vasodilatator in the eye Thus, this potent polypeptide is a good candidate for one of the mediators of part of the irritative response of the eye, and possibly of antidromic vasodilatation in general. It seems likely that the threshold dose of CGRP to induce a change in the parameters studied is in the range of ng (2.6 X 10~ X 10" 11 moles) when administered intracamerally in the rabbit. With a dose of 5 tig the upper limit was reached with a severe drop in blood pressure and contralateral effects. Less statistical significance was obtained in this group, eg, as compared with the 1 ug group (Fig. 2). Intracameral administration of as much as 100 /xg (7.4 X 10~ 8 moles) of SP has been shown to have only little effect on the vasculature of the anterior uvea and the blood-aqueous barrier in urethane anaesthetized rabbits. 18 Thus, compared with SP, CGRP seems to be of the order of magnitude 1000 times more potent as a vasodilatator in the rabbit eye. This mg/100 ml o ** n=6 III > ip TTX 1 ug CGRP 1 ug m< EXPERIMENTAL EYE IH CONTROL EYE n=3 i JDIIil TTX 1 ug NaCl Fig. 5. Effect of intracameral injection of 1 fig CGRP and isotonic NaCl on the aqueous humor protein concentration in rabbits which received an intracameral injection of TTX bilaterally 10 min prior to CGRP. Bars indicate SEM. ** = P < 0.01.

1010 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1988 Vol. 29 mmhg 35i 15 10- - -10 10 40 50 60 70 Fig. 6. Effect of intracameral injection of a mixture of 0.1 ng SP and 0.

5 1010 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1988 Vol. 29 mmhg 35i Fig. 6. Effect of intracameral injection of a mixture of 0.1 ng SP and 0.1 ng CGRP on the intraocular pressure in rabbits. Experimental eyes indicated by solid line and control eyes by dotted line. Bars indicate SEM. Mean arterial blood pressure indicated as in Figure 1, (n = 4). estimation is in relatively good agreement with estimations of the vasodilatatory effects of SP and CGRP in the feline tooth pulp. 21 The experiments performed in TTX-pretreated rabbits suggest that the effect of exogenous CGRP is not dependent on nerve conduction in the uvea. Of course, it cannot be excluded that CGRP may exert an effect through a prejunctional receptor directly coupled to substances stored in the nerve terminals. Strongly irritating agents, such as capsaicin and compound 48/80 seem to act through such a mechanism, 22 whereas physiologic compounds such as prostaglandin E, histamine and bradykinin seem to require conduction in sensory nerves for function. 617 Since both cholinergic and adrenergic neurons were blocked it is reasonable to assume that the unmyelinated sensory neurons were also blocked. Thus, it is probable that the effect of CGRP is a direct one on the vascular smooth muscle. Although TTX in itself caused vasodilatation which could have rendered the eye more sensitive to edema and barrier breakdown, the fact remains that TTX did not induce any marked barrier breakdown but blocked nerve conduction. Control experiments with intracameral injection of isotonic saline only, in TTX-pretreated eyes, confirmed that the injection per se had no effect on the blood-aqueous barrier (Fig. 5). The effect of intracameral injection of CGRP in cats was surprising, since the effect on the bloodaqueous barrier was very modest, and there was a decrease in IOP. Even when CGRP was injected in a dose as high as 50 ^g (1.3 X 10 8 moles) intracamerally there was no increase in IOP and no significant barrier breakdown. The mechanism behind the decrease in IOP remains unknown, and merits further studies. Since the cat eye is considerably larger than the rabbit eye it can be assumed that intracameral injection of 5 ng CGRP in the cat eye corresponds to about 1 Mg in the rabbit. Thus, it seems that there is a species difference in the response to CGRP between cats and rabbits. Since CGRP and SP coexist in sensory neurons of the anterior uvea, 1113 and SP has very little effect in the cat eye, 23 it may simply be that the same is true for CGRP and that other systems are more important in sensory mechanisms in the cat eye. Simultaneous injection of SP with CGRP did not appreciably increase the response to CGRP in rabbits; in fact, the increase in IOP and aqueous humor protein concentration was less than with CGRP only. This may be due to a decreased access of CGRP to the site of action due to instantaneous miosis. It should also be noted that there was a relatively strong decrease in systemic blood pressure of this group of animals after administration of the SP-CGRP mixture (Fig. 6). Thus, it seems that SP in the dose used does not potentiate the vascular effects of CGRP to any greater extent. Recently it was shown that low doses of CGRP potentiated the miotic effect of a low dose of SP as well as the weak vascular effects of SP. 19 With the doses used in the present study it was not possible to show any potentiating effect of CGRP on the miotic effect of SP since the effect of SP was already maximal. The objective was rather to see if SP potentiates the vascular effects of CGRP. Thus, any substantial interaction between SP and CGRP in the anterior uvea of the rabbit does not seem to exist at the dose levels used. The present study confirms that CGRP is a very potent vasodilatator in the rabbit anterior uvea, causing breakdown of the blood-aqueous barrier. This effect does not seem to be mediated by neural elements or prostaglandins secondarily. In the cat, on the contrary, CGRP seems to have little inflammatory effect, if any, and a decrease was observed in IOP. Key words: calcitonin gene-related peptide, intraocular pressure, aqueous humor protein concentration, bloodaqueous barrier, irritative response, nociception, rabbit, cat Acknowledgment We would like to thank Ms. Tuija Pauhu for technical assistance. References 1. Cole DF and Unger WG: Prostaglandins as mediators for the response of the eye to trauma. Exp Eye Res 17:357, 1973.

No. 7 EFFECTS OF CGRP IN THE EYE / OKsolo and Srjernschanrz 1011 2. Neufeld AH, Jampol LM, and Sears ML: Aspirin prevents the disruption of the blood aqueous barrier in rabbit. Nature 238:158, 1972.

6 No. 7 EFFECTS OF CGRP IN THE EYE / OKsolo and Srjernschanrz Neufeld AH, Jampol LM, and Sears ML: Aspirin prevents the disruption of the blood aqueous barrier in rabbit. Nature 238:158, Jampol LM, Neufeld AH, and Sears ML: Pathways for the response of the eye to injury. Invest Ophthalmol 14:184, Butler JM and Hammond BR: Effect of sensory denervation on the response of the rabbit eye to bradykinin and PGEi. Trans Ophthalmol Soc UK 97:668, Butler JM and Hammond BR: The effects of sensory denervation on the responses of the rabbit eye to prostaglandin Ei, bradykinin and substance P. Br J Pharmacol 69:495, Mandahl A and Bill A: Ocular responses to antidromic trigeminal stimulation, intracameral prostaglandin E, and E 2, capsaicin and substance P. Acta Physiol Scand 112:331, Butler JM, Unger WG, and Cole DF: Axon reflex in ocular injury: Sensory mediation of the response of the rabbit eye to laser irradiation of the iris. Q J Exp Physiol 65:1981, Rosenfeld MG, Mermod J-J, Amara SG, Swanson LW, Sawchenco PE, Rivier J, Vale WW, and Evans RM: Production of a novel neuropeptide by the calcitonin gene via tissue specific RNA processing. Nature 4:129, Gibson SJ, Polak JM, Bloom SR, Sabate IM, Mulderry PM, Ghatei MA, McGregor GP, Morrison JFB, Evans RM, and Rosenfeld MG: Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight species. J Neurosci 4:3101, Unger WG, Terenghi G, Ghatei MA, Ennis KW, Butler JM, Zhang SQ, Too HP, Polak JM, and Bloom SR: Calcitonin gene-related polypeptide as a mediator of the neurogenic ocular injury response. J Ocular Pharmacol 1:189, Wahlestedt C, Hakanson R, Beding B, Brodin E, Ekman R, and Sundler F: Intraocular effects of CGRP, a neuropeptide in sensory nerves. In Tachykinin Antagonists, Hakanson R and Sundler F, editors. Amsterdam, New York, Oxford, Elsevier, 1985, pp Stone RA, Kuwayama Y, Terenghi G, and Polak JM: Calcitonin gene-related peptide: Occurrence in corneal sensory nerves. Exp Eye Res 43:279, Terenghi G, Polak JM, Ghatei MA, Mulderry PK, Butler JM, Unger WG, and Bloom SR: Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the mammalian eye. J Comp Neurol 233:506, Bill A, Stjernschantz J, and Mandahl A: Substance P: Release on trigeminal nerve stimulation, effects in the eye. Acta Physiol Scand 106:371, Stjernschantz J, Sears ML, and Stjernschantz L: Intraocular effects of substance P in the rabbit. Invest Ophthalmol Vis Sci :53, Leander S, Hakanson R, Rosell S, Folkers K, Sundler F, and Tornquist K: A Specific substance P antagonist blocks smooth muscle contractions induced by non-cholinergic, non-adrenergic nerve stimulation. Nature 294:467, Mandahl A: Release of substance P and its role in the acute inflammatory response in the rabbit eye. Acta Universitatis Upsaliensis 470, (Academic dissertation), Stjernschantz J, Sears ML, and Mishima H: Role of substance P in the antidromic vasodilatation, neurogenic plasma extravasation and disruption of the blood-aqueous barrier in the rabbit eye. Naunyn Schmiedebergs Arch Pharmacol 321:329, Wahlestedt C, Beding B, Ekman R, Oksala O, Stjernschantz J, and Hakanson R: Calcitonin gene-related peptide in the eye: Release by sensory nerve stimulation and effects associated with neurogenic inflammation. Regul Pept 16:107, Lowry OM, Rosebrough NJ, Farr AJ, and Randall RS: Protein measurement with folin phenol reagent. J Biol Chem 193:265, Gazelius B, Edwall B, Olgart L, Lundberg JM, Hokfelt T, and Fischer JA: Vasodilatatory effects and coexistence of calcitonin gene-related peptide (CGRP) and substance P in sensory nerves of cat dental pulp. Acta Physiol Scand 1:33, Mandahl A and Bill A: Effects of substance P antagonist (D- Arg 1, D-Pro 2, D-Trp 79, Leu")-SP on the miotic response to substance P, antidromic trigeminal nerve stimulation, capsaicin, prostaglandin E, compound 48/80, and histamine. Acta Physiol Scand 117:139, Unger WG and Tighe J: The response of the isolated iris sphincter muscle of various mammalian species to substance P. Exp Eye Res 39:465, 1984.

Calcitonin Gene-Related Peptide Immunoreactive Nerves in Human and Rhesus Monkey Eyes

Investigative Ophthalmology & Visual Science, Vol. 29, No. 2, February 1988 Copyright Association for Research in Vision and Ophthalmology Calcitonin Gene-Related Peptide Immunoreactive Nerves in Human