Cholecystokinin contracts isolated human and monkey iris sphincters; a study with CCK receptor antagonists

European Journal of Pharmacology, 211 (1992) 183-187

183

© 1992 Elsevier Science Pubhshers B.V All rights reserved 0014-2999/92/$05 00

EJP 52275

Cholecystokinin contracts isolated human and monkey iris sphincters; a study with CCK receptor antagonists B i r g i t t a A l m e g ~ r d , J o h a n S t j e r n s c h a n t z ~ a n d A n d e r s Bill Department of Physiology and Medtcal Btophystcs, Uppsala Untverstty and l Glaucoma Research Laboratortes, Kabt Pharmacta OphthalmtcsAB, Uppsala, Sweden

Recewed 19 August 1991, revised MS received 15 October 1991, accepted 12 November 1991

The contractde effects of cholecystokinin (CCK) on iris sphincter and ciliary muscles from monkey and human eyes were studied using an isolated smooth muscle bath. The ability of the CCK A receptor antagonists, lorglumlde and loxiglumide, to inhibit CCK-8s-lnduced contraction was also examined. Various neuropeptides reported to be present in capsaicin-sensitwe sensory neurons were also screened for contractile effect. CCK contracted isolated human and monkey ~rls sphincters at nM concentrations. Both antagonists caused a rightward shift of the dose-response curve for CCK-8s on the monkey iris sphincter. The ciliary muscle from both species failed to contract in response to CCK-8s. Of the eight other neuropeptldes screened on the monkey iris sphincter, only [ArgS]vasopressin ehcited a weak contraction when used in p~M concentrations. These results indicate that the primate lrldlal sphincter muscle exhibits a high sens~tw~ty to CCK, and that CCK A receptor antagomsts effectwely block the CCK-induced contraction Cholecystokinin (CCK), Iris sphincter muscle; Lorglumide; Loxlglumide; Ciliary muscle

1. Introduction

Non-muscarinic miosis, a p h e n o m e n o n encountered in ocular trauma and surgery, has been most extensively studied in the rabbit eye. Depending on the type of noxious stimuli applied, the miosis has been suggested to be mediated by prostaglandins a n d / o r a neurogenic component. Scratching of the iris causes release of prostaglandins while chemical treatment with formaldehyde does not trigger prostaglandin release (Cole and Unger, 1973) but seems to involve a sensory component in the miotic response (Butler et al., 1979). T r e a t m e n t with local anesthetics has an inhibitory effect on the miosis caused by irritation of the iris in rabbits and on surgically induced miosis in humans (Butler et al., 1979; Van Rij et al., 1984). This effect is considered to be due to the anesthetic action on sensory nerves which may inhibit the release of a miotic substance. In the rabbit eye, substance P is released into the aqueous humor upon trigeminal nerve stimulation and produces miosis when given intracamerally (Bill et al., 1979). However, substance P has no miotic effect in the monkey eye (Mandahl et al., 1980). Iso-

lated human iris sphincters also do not contract in response to the peptide (Unger and Tighe, 1984), Recently it was found that cholecystokinin (CCK) is a potent miotic in the monkey eye but not in rat, cat, rabbit or guinea-pig eye (Bill et al., 1990). The purpose of the present study was to investigate the contractile effect of CCK in the human and monkey iridial sphincter and ciliary muscles with an in vitro method. Since the receptors on monkey iris seem to be of the peripheral (CCK A) receptor type, we have also investigated the blocking effect of two potent and selective C C K A receptor antagonists: lorglumide (Makovec et al., 1987) and loxiglumide (Setnikar et al., 1987). Eight other neuropeptides, described to be present in sensory neurons (see Maggi and Meli, 1988), were screened in addition for contractile effect on the monkey iris sphincter. Preliminary reports of part of this study have been presented (Almeg~rd et al., 1990, 1991).

2. Materials and m e t h o d s 2.1. Source o f eyes

Correspondence to: B. Almeg~rd, Department of Physiology and Medical Biophysics, Box 572, S-751 23 Uppsala, Sweden Tel. 46.18 17 44.70, fax 46.18 17.49.38

The human eyes used in this study were eleven donor eyes obtained from a local hospital in Sweden and from the Eye bank of Arhus, Denmark. The time o

184 elapsed after death was 12-32 h and the age of the donors ranged from 47 to 88 years. One additional eye was obtained from a patient with malignant melanoma in the choroid and was used within 2 h after enucleation. Cynomolgus monkey eyes were obtained from local research establishments and were enucleated immediately after death. The eyes were kept in a phosphate buffer or saline on ice until used, but no longer than for 7 h. The monkeys had previously been used in other experiments. The iris was used only if it was considered normal.

2.2. Preparatton of muscle strips One iris sphincter muscle strip was prepared from each eye by making a radial cut and then cutting completely around the pupil approximately 1-1.5 mm from the pupillary margin. In some experiments a strip of the ciliary muscle, about 15 mm long and 3 mm wide, was dissected in a circular direction. A silk thread was tied to each end and the preparation was mounted in a 10-ml organ bath; one thread was tied to a glass anchoring rod and the other end to a force displacement transducer (Grass FT 03) connected to a polygraph (Grass Model 7D Polygraph). A resting tension of 75 mg (for iris sphincters) or 150 mg (for ciliary muscle strips) was applied and contractions were measured isometrically. The organ bath contained a modified Krebs solution of the following composition (mM): NaCI 132.5, N a H C O 3 16.1, KCI 4.7, MgC1 1.0, N a H 2 P O 4 1.4, CaCI 2 2.4 and glucose 7.8. Indomethacin, 2.8 /zM, was present in all experiments. Atropine, 0.2 #M, and propanolol, 6.8 /zM, were present in all experiments on iris sphincter muscles with sulphated CCK-8 (CCK-8s) and in some of the experiments with CCK-33 and the other neuropeptides tested. The temperature was kept at 3 7 ° C and the buffer solution was bubbled with a O 2 - C O 2 ( 9 3 % - 7 % ) gas mixture.

ments with lorglumide, dose-response curves for two or three concentrations of the antagonist were made using the same monkey iris. In two experiments, the CCK-8s dose-response was performed after CCK-33 dose-response determinations. The contractile effect of CCK-33 was determined on seven monkey iris sphincters, two of which had previously been used for CCK-8s dose-response determinations. The contractile effect of calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), somatostatin, corticotropin-releasing factor (CRH), galanin, [ArgS]vasopressin, bombesin and substance P was examined by adding cumulative doses according to table 1. Strips of ciliary muscle were treated with cumulative doses of CCK-8s, added to the bath at 2-3 min intervals. Carbachol was added thereafter to establish the viability of the muscle.

2.4. Drugs CCK-8s, CCK-33, bombesin, substance P, CGRP, VIP, somatostatin, CRH, galanin, [ArgS]vasopressin and lorglumide (D,L-4-(3,4-dichlorobenzoylamino)-5(dipentylamino)-5-oxo-pentanoic acid; Rotta Research Laboratorium S.p.A.) were purchased from Peninsula Laboratories Inc. (Belmont, CA, USA). Loxiglumide (D,L-4-(3,4-dichlorobenzoylamino)-5-(N-3-methoxypropylpentylamino)-5-oxo-pentanoic acid) was kindly supplied by Rotta Research Laboratorium S.p.A. (Monza, Italy). Indomethacin, atropine, propranolol and carbachol were purchased from Sigma (St. Louis, MO, USA). The peptides, carbachol, atropine and the sodium salt of loxiglumide were dissolved in physiological saline, propranolol was dissolved in distilled water and lorglumide was dissolved in isotonic sodium bicarbonate. Indomethacin was dissolved using 0.13 M Na2CO 3 in water with the pH adjusted to 7.5 by titration with a saturated KH2PO 4 solution.

2.3. Experimental procedure 2.5. Evaluation of data In experiments with CCK-8s, an initial period of stabilization of 40-60 min for human iris sphincters and 20-90 min (mostly 40-60 min) for monkey iris sphincters was allowed before CCK-8s was added. The peptide was added to the bath in cumulative doses at 4-5 min intervals until a maximal contraction was achieved. The strip was then washed several times and allowed to return to baseline. The antagonist was added and 10-15 min elapsed before a new CCK dose-response relationship was determined. In most experiments 28 mM K - R i n g e r solution was added at the end. I-I~gher K concentrations were found to give less response. Four to seven dose-response determinations were made for each dose of antagonist. In some experi-

The ECso values were calculated using a non-linear regression model (PROC NLIN, SAS Institute Inc.). The data were fitted to the equation R = m a x / 1 + eA0og EC50-1ogC) where R is the response expressed as percentage of the maximal response obtained with agonist in the absence of antagonist. C is the agonist concentration. Max, A and ECso are parameters determined by the regression model. The pA 2 values were determined from Schild plots (Arunlakshana and Schild, 1959). The log (dose-ratio ( D R ) - 1 ) of the agonist was plotted against the log concentration of the antagonist. The D R was calculated using the EC50 values in the presence and in the absence of antago-

185 Response (%)

Response (%)

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°°-" 25

75.

10-9 10-s CCK (M)

10 -7

0 10~11 10~10

10-s

Fig. 1. Dose-response curve for CCK-induced contraction of monkey ms sphincters. ( ,x ) CCK-33 (n = 7), (©) CCK-8s (n = 26). The contrachon is expressed as percentage of the maximum CCK-lnduced contrachon of each preparation. The values represent the m e a n s + S.E.M

10.9

10-8 10.7 CCK-8s (M)

10.6

10-5

10.4

Fig. 3 Respons of monkey ins sphincters to CCK-8s in the absence ( o ) and presence of loxiglumlde, (o) 0 1 txM (n = 4), ( * ) 3 1 ~ M (n = 5 ) , ( [ ] ) 9 . 3 ~ M (n = 4) and ( t , ) 31 # M (n = 4 )

Response (%) 100

nist. The line was obtained by linear regression using the least squares method. The results were expressed as means + S.E.M.

~'

75

~"

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•

50"

3. Results

o

Monkey iris sphincters contracted in response to CCK-8s and CCK-33 with an ECs0 of 5.2 nM (n = 26) and 7.1 nM (n = 7), respectively (fig. 1). The maximal response obtained with CCK-8s was 69.4 + 4.0% (n = 25) of the K-evoked response. CCK-8s also induced contractions of human iris sphincter muscles with an ECs0 of 2.0 nM (n = 12) (fig. 2). The maximal response obtained with CCK-8s was 86.6 + 11.4% (n = 10) of the K-evoked response. The responses had the shape of a stable plateau.

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3.2. Effects of CCK antagonists Loxiglumide and lorglumide caused a rightward shift of the d o s e - r e s p o n s e curve for CCK-8s on the monkey iris sphincters (figs. 3 and 4). A Schild plot analysis yielded a straight line with a slope of 0.95 + 0.42 and log (dose-ratio- 1) .3

~,,~,,f .,.

10 5

Fig 4. Response of monkey iris sphincters to CCK-8s m the absence ( 0 ) and presence of Iorglumlde, (13) 0.1 p,M (n =7), ( * ) 1 1 /zM (n = ?) and (zx) 3 3 / z M (n = 4).

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3.1. Effects of CCK-8s and CCK-33

Res

/

25

10-10

10.9

10"9 10"7 CCK-8s (M)

10"6

10.5

Fig. 2. Response of human iris sphincters to CCK-8s in the absence ( © , n = 12) and presence ( • , n = 6) of the CCK antagonist, loxiglumide (3 1 p,M). The contraction is expressed as percentage of the

maximum contraction obtained with CCK-8s alone in each preparation. The values represent the means +_S.E M.

•

i

-7

.

i

•

-6 Antagonist(leg M)

i

-5

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Fig. 5. Schdd plot of the antagonistic action of lorglumide ( [ ] ) and loxiglumlde ( • ) on CCK-mduced miosls on the monkey iris sphincter. Each point represents four to seven dose-response determinations. The dose ratio was calculated using the ECs0 values in the presence and absence of antagonist. The lines were obtained by regresston analysis

186 TABLE 1 T h e c o n t r a c t i l e e f f e c t o f p e p t l d e s , l o c a l i z e d to s e n s o r y n e u r o n s , o n t h e m o n k e y iris s p h i n c t e r . D o s e s w e r e a d d e d c u m u l a t i v e l y + i n d i c a t e s c o n s t r i c t i o n o f t h e ~rls s p h i n c t e r m u s c l e , - i n d i c a t e s lack o f c o n s t r i c t i o n Peptlde

Concentration m bath (M)

Response

n

CCK-8s CCK-33 [ArgS]vasopressln SubstanceP Galanln CGRP Bombesln

9 x 1 0 - 1 1 _ 9 x 10 7 2 x 1 0 - 1 2 - 3 x 10 - 7 4 x 1 0 - 1 ° - 7 x 10 - 6 7x10 -s -7x10 6 1 x 10-111-1 x 10 - 6 7x10 s _4x10-6 3 x 10 1 0 - - 3 X 10 6

-I+ + a _ _

26 7 5 4 1-4 4 2

Corticotropln releasing factor VIP Somatostatln

8x10 ll-8x 10-7 1 x 1 0 - I 0 _ 1 x 10 6 2 x 10-111_2 x 1 0 - 6

_ _

_

2 2 2

d C o n t r a c t i o n in p r e s e n c e o f h i g h e s t c o n c e n t r a t i o n

1.02_+ 0.21 for lorglumide and loxiglumide, respectively. The pA 2 values calculated were 7.63 and 7.31, respectively (fig. 5). Loxiglumide also caused a rightward shift of the dose-response relationship for CCK8s on human iris sphincters (fig. 2).

3.3. Effects on the cthary muscle Ciliary muscles from six monkeys and seven humans did not contract significantly in response to 46-910 nM CCK-8s, but responded to carbachol, 10-100/zM, with a marked contraction.

3.4. Effects of other sensory neuropeptides tested Table 1 shows that of the other eight peptides tested only [ArgS]vasopressin caused a small contraction at /zM concentrations. The response was 27% of a 46 nM CCK-8s-evoked contraction.

4. Discussion

We have recently demonstrated that CCK causes a non-muscarinic miosis in the monkey eye, 1.3 nM CCK-8s producing 50% of the maximal effect (Bill et al., 1990). In order to verify the effect in the human iris sphincter muscle and to be able to examine further the contractile effect on the monkey iris sphincter muscle and ciliary muscles from both species, we now used an isolated smooth muscle bath. Consistent with out findings in vivo, CCK-8s contracted the monkey iris sphincter muscle in vitro at concentrations of the same order of magnitude. CCK-8s also contracted the isolated human iris sphincter muscle. The sensitivity of the human iris sphincter muscle to CCK-8s was approxi-

mately the same as that of the monkey iris, indicating that the monkey eye should be a relevant model for further studies of this phenomenon. Two types of CCK receptors have been described: CCK A and CCK B receptors. The A-type is present in the alimentary system, but also in some isolated areas of the brain. The CCK B receptors are widely distributed in the brain (Moran et al., 1986). The receptors can be distinguished by their affinity towards different fragments of CCK. In vivo it was found that the receptors present on the monkey iris were of the peripheral (A-)type; CCK-8s was 1000 times more potent than the non-sulphated CCK-8 fragment and the response was antagonized by the CCK A receptor antagonist, lorglumide (Bill et al., 1990). Two antagonists were examined in this study: lorglumide and loxiglumide. Lorglumide is the most potent CCK antagonist among the recently discovered class of pentanoic acid derivatives (Makovec et al., 1987), with a high affinity for peripheral CCK receptors. The calculated pA z value in longitudinal muscles of guinea-pig ileum was 7.77 (Barth6 et al., 1987). Loxiglumide is a closely related compound with somewhat lower potency. The pA 2 value calculated for loxiglumide was 6.8 in rat pancreas acini (Otsuki et al., 1989). On the monkey iris sphincter both antagonists caused a rightward shift of the CCK-8s concentrationresponse relationship. A Schild plot yielded a slope that was close to unity for lorglumide and loxiglumide, indicating competitive antagonism. It would seem that the pA 2 values obtained for these antagonists with the iris are in reasonably good agreement with those obtained with other tissues. The CCK-induced contraction of human iris sphincters was also antagonized by loxiglumide. The presence of CCK-like immunoreactivity (CCKLI) in primary sensory neurons has been described by several investigators (Lundberg et al., 1978; Tuchscherer and Seybold, 1985; Gibbins et al., 1987). In the eye, CCK-LI has been demonstrated in the rat iris (Bj6rklund et al., 1985) and in the trigeminal ganglion of guinea-pigs (Kuwayama et al., 1987). One can speculate that if CCK is released from sensory nerves during ocular trauma such as surgery, it may be involved in the non-muscarinic miosis frequently encountered during ocular surgery. CCK-8s failed to cause a significant contraction of ciliary muscle strips, a result which is consistent with previous findings; CCK could not affect the ciliary muscle enough to cause an increase in outflow facility (Almeg~rd and Andersson, 1990). Eight neuropeptides described to be present in capsaicin-sensitive sensory neurons (see Maggi and Meli, 1988) were screened for contractile effects on the monkey iris sphincter. Of these peptides only [Arg s] vasopressin caused a small response. However, it is very

187

doubtful whether this peptide has any physiological significance in non-muscarinic miosis, since a high concentration (/zM) was necessary to elicit a weak response. These results thus indicate that CCK was the only one of the neuropeptides tested toward which the human and monkey iris sphincter muscle exhibited any sensitivity in the physiological range. In conclusion, CCK causes contraction of isolated human and monkey iris sphincters with an ECs0 at nM concentrations. The contraction of the monkey iris sphincter was blocked competitively by lorglumide and loxiglumide.

Acknowledgements Eyes were provided by the courtesy of Professor Nlels Ehlers (,~rbus), Dr. Ulf Stenevi (Lund), Dr. Peter Naeser (Uppsala), Ms Isabelle Rudolfsson and Ms. Blrgltta Norlind (Procordm OraTech AB, Goteborg). We would like to thank Dr Gbran Sel6n and Dr. Goran Sperber for help with the computer analyses and valuable discussions We would also hke to thank Mrs Mantha Karlsson for technical assistance

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