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Effect of somatostatin and galanin on isolated rabbit iris sphincter and dilator muscles
Experimental Eye Research 77 (2003) 609–614 www.elsevier.com/locate/yexer
Effect of somatostatin and galanin on isolated rabbit iris sphincter and dilator muscles Kazutsuna Yamajia,*, Takeshi Yoshitomib, Shiro Usuia a
Laboratory for Neuroinformatics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan b Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan Received 4 February 2003; accepted in revised form 26 June 2003
Abstract The neuropeptides somatostatin and galanin are present in the iris and may modulate pupil diameter. We examined the effects of somatostatin and galanin on isolated rabbit iris dilator and sphincter smooth muscles that were mounted in an organ bath. An isometric transducer recorded changes in tension in response to electric field stimulation (100 Hz, 0.3 m sec in duration, 10 V in strength) delivered by a pair of platinum plate electrodes. The dilator muscle response to field stimulation was not changed by either peptide, even at the highest concentrations examined. The sphincter muscle response consisted of two components: a fast component mediated by acetylcholine and slow component mediated by substance P. Both somatostatin and galanin attenuated the cholinergic component in a dose-dependent manner (from 0.3 nM to 0.1 mM ) but had no effect on responses mediated by substance P. Galanin was more effective (attenuation of 43% at 0.1 mM ) compared with somatostatin (attenuation of 16% at 0.1 mM ) in reducing the cholinergic response. Neither peptide affected the contraction induced by acetylcholine (1 mM ). Therefore both peptides inhibited cholinergic transmission in the sphincter muscle, although the degree of inhibition by each was different. We conclude that somatostatin and/or galanin may induce mydriasis by attenuating cholinergic neurotransmitter release. q 2003 Elsevier Ltd. All rights reserved. Keywords: somatostatin; galanin; rabbit; smooth muscle; iris sphincter; iris dilator; isometric contraction
1. Introduction The complex innervation of the mammalian iris is being gradually clarified. Control of the pupil has long been attributed to classical autonomic neurotransmitters such as noradrenalin and acetylcholine. Recently, a variety of biologically active peptides released from trigeminal (Haruno et al., 1996; Nishiyama et al., 1982; Yoshitomi et al., 2002) and facial nerves (Hayashi and Masuda, 1982) have also been found to modulate pupil diameter. Somatostatin, first detected in ovine hypothalamus (Brazeau et al., 1973), is also present in the iris/ciliary body of rat (Mori et al., 1997), rabbit, sheep and cow (Elbadri et al., 1991), and recently it was found in the iris of monkey (Firth * Corresponding author. Dr K. Yamaji, Laboratory for Neuroinformatics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 3510198, Japan. E-mail address: [email protected] (K. Yamaji). 0014-4835/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. DOI:10.1016/S0014-4835(03)00177-5
et al., 2002). Galanin is also present in the iris of rat (Stromberg et al., 1987), pig (Stone et al., 1988), cat (Grimes et al., 1994) and monkey (Firth et al., 2002). Somatostatin and galanin may be of trigeminal (Firth et al., 2002; Stromberg et al., 1987) or sympathetic origin (Wright et al., 1989; Grimes et al., 1994). Ekblad et al. (1985) reported that administration of galanin to isolated rabbit iris sphincter muscle inhibits the response evoked by electrical stimulation, although it does not inhibit the carbachol-stimulated contraction. Their results imply a mydriatic effect of galanin; however, pupil size was not affected by galanin in vivo (Almegard and Andersson, 1990). Further investigation of the effects of these peptides on the sphincter and dilator muscles is necessary because the pupil is regulated by a push-pull interaction of both. This study clarifies the functional role of somatostatin and galanin on the iris sphincter and dilator muscles in vitro by means of the isometric tension recording method.
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2. Materials and methods 2.1. Isolation and incubation of muscle specimens All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male albino rabbits weighting 2 – 3 kg were euthanized by injecting an overdose of pentobarbital sodium (Abbott Laboratories, North Chicago, IL, USA) into the marginal ear vein. The eyes were immediately enucleated and placed in Krebs solution composed of (mM ): NaCl, 94.8; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 25.0; and glucose, 11.7 and gassed with 95% O2 and 5% CO2. After removal of the cornea, a ring-shaped iris sphincter muscle specimen (1 mm in width) or radialshaped dilator muscle specimen (45 8 in sector) was prepared according to the method previously reported (Yamaji et al., 2003). The ends of each specimen were tied with silk thread for mounting in an organ bath (1.5 ml). One end of the specimen was connected to the isometric tension transducer (Nihon Kohden Co., TB-612T), and the other end was secured to a hook at the bottom of the organ bath. The organ bath was perfused continuously (0.15 ml/s) with oxygenated Krebs solution warmed to 37 8C. The initial lengths of sphincter and dilator muscle specimens were adjusted to 10 mm and 5 mm respectively, allowing
maximum contraction to occur (Yamaji et al., 2003). After at least 1 hr of equilibration, the experiments were performed. 2.2. Electrical field stimulation experiments Electrical field stimulation was applied through a pair of platinum electrodes with 11 mm separation and placed in the organ bath so that the current pulse passed transversely across the tissue. The sphincter muscle response to electric stimulation consists of fast and slow components (Ueda et al., 1982). Stimulation with 100 pulses (10 V at 100 Hz) elicits both fast and slow contractions while stimulation with 10 pulses elicits only the fast contraction (Yoshitomi et al., 2002). For the dilator muscle, only the fast component occurs regardless of the strength of the stimulation (Yoshitomi et al., 2002). We used 100 pulses to produce effective contractions of this muscle. To determine if either somatostatin or galanin selectively affected fast or slow contractions, each peptide was added to the perfusion medium 15 min before the onset of field stimulation with 100 pulses. Control responses were recorded before peptide application and after washing out (1 hr) the peptide. Dose response curves (0.3 nM –0.1 mM ) for somatostatin and galanin were generated for the sphincter muscle. Prior
Fig. 1. Effect of somatostatin and galanin on sphincter muscle response evoked by electric field stimulation. Stimulation with 100 pulses was employed to produce the biphasic response. Each peptide was applied 15 min before stimulation. Light and dark gray in lower figure show the fast and slow components respectively. Mean ^ SD, n ¼ 6;*P , 0:05; * * P , 0:01; and N.S. ¼ P . 0:05:
K. Yamaji et al. / Experimental Eye Research 77 (2003) 609–614
to each dose of peptide, the sphincter was stimulated for 20 min with 10 pulses every 1.5 min to achieve control contractions. The peptide was then added to the perfusion medium, and the contractions were recorded for another 20 min with the same stimulation. A washout period of at least 1 hr followed each peptide, and then the control contractions were repeated followed by the next concentration of peptide. 2.3. Direct contraction experiments These experiments were performed to investigate if somatostatin and galanin affect the postjunctional site. Control contractions were initiated with 1 mM acetylcholine. This concentration was chosen because the median effective concentration (EC50) for contractile response to acetylcholine in this tissue is approximately 0.3 mM (Ueda et al., 1981; Yoshitomi et al., 2002). The 1 mM acetylcholine also enables to produce similar contractile amplitude to that of evoked by electric stimulation which was applied in the previously mentioned experiments. After a washout period of at least 1 hr, 0.1 mM somatostatin or galanin was added to the Krebs solution 15 min before contraction was again stimulated by acetylcholine. After
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reaching the maximum contraction, which took about 2 min, the peptide was washed out for at least 1 hr. Control contractions by the agonist were repeated after the peptideapplied contraction. 2.4. Chemicals The following drugs and chemicals were used in this study: somatostatin and galanin (Peptide Institute, Inc., Osaka, Japan) and acetylcholine (Wako Chemical Inc., Osaka, Japan). Peptides were prepared in aliquots and stored at 2 30 8C.
3. Results 3.1. Electrical field stimulation experiments The sphincter muscle specimens produced a biphasic response to stimulation with 100 pulses. Somatostatin (0.1 mM ) and galanin (0.1 mM ) significantly attenuated the fast component of the response but had no effect on the slow component (Fig. 1). The fast and slow components correspond to the cholinergically and substance P mediated
Fig. 2. Effect of somatostatin and galanin on sphincter (A) and dilator (B) muscles. Stimulation (10 pulse) was applied every 1.5 min. The peptide was present continuously after t ¼ 0 min: Each concentration was applied to the same specimen after at least a 1 hr washout period. Somatostatin, left; galanin, right.
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contractile responses, respectively (Ueda et al., 1981). Thus each peptide inhibited only the cholinergic response. To examine the effect further, 10-pulse stimulation was applied to elicit only the cholinergic response (Fig. 2(A)). For somatostatin, the attenuation reached its maximum at 5 min and then gradually diminished for another 5 min until it became stable. In contrast, attenuation of the contraction by galanin was relatively stable throughout its application. Comparison of the steady state revealed that the attenuation by galanin was greater than that by somatostatin. This difference was clear in the doseresponse curves (Fig. 3). The maximum attenuation and EC50 estimated from the sigmoidal function were 16.11% and 3.38 nM for somatostatin and 42.83% and 14.21 nM for galanin. The effect of cyclosomatostatin, a commercially available somatostatin receptor antagonist, on this tissue was also examined. The cyclosomatostatin was added to the perfusion medium 20 min before the application of somatostatin. The attenuation caused by 0.1 mM somatostatin (14.2 ^ 4.5%, n ¼ 6Þ did not show a significant difference ðP . 0:1Þ after application of 1 mM cyclosomatostatin (13.6 ^ 2.7%, n ¼ 6Þ: The effects of somatostatin and galanin on contraction of the iris dilator muscle by field stimulation were also tested. At concentrations of up to 0.1 mM , neither peptide had an effect on either the basal tone or the contraction amplitude evoked by electric stimulation (Fig. 2(B), Fig. 4). 3.2. Direct contraction experiments In the field stimulation experiments, only the response of the sphincter muscle was affected by the peptides. The attenuation of contraction in those experiments could be due to the activation of peptide receptors at either ganglionic or
Fig. 3. Dose-response relationship of somatostatin and galanin on the fast component of sphincter muscle responses to electric field stimulation. ‘Amplitude (%)’ in each specimen was calculated as the ratio of the response before (100%) and after drug application. For each experiment, 5 responses were measured before and after drug application. Solid curves were determined by fitting the sigmoidal function to the experimental data using a non-linear optimization method. Somatostatin (O), galanin ( ), Mean ^ SD, n ¼ 6:
†
post-ganglionic sites. Therefore, the effect of each peptide on 1 mM acetylcholine induced contractions was examined. Neither peptide resulted in a change in the response amplitude induced by acetylcholine (Fig. 5).
4. Discussion Previous reports showed that isolated iris sphincter muscle in rabbit (Ueda et al., 1982) and monkey (Almegard et al., 1992) were not affected by somatostatin in concentrations ranging from 0.1 nM to 10 mM and from 0.2 nM to 2 mM , respectively. These results are not inconsistent with our findings because they observed the effects on basal tone. We also found that somatostatin did not affect the sphincter basal tone (Fig. 2(A)). Because somatostatin clearly attenuated the contraction of rabbit iris sphincter stimulated by field current, our results also suggest that it may inhibit the monkey iris sphincter muscle. The absence of somatostatin effects on dilator muscle basal tone and contraction evoked by electric stimulation is consistent with the finding that somatostatin is less common in the dilator muscle (Firth et al., 2002). The attenuation of sphincter contraction in response to field stimulation and the absence of attenuation for direct cholinergic stimulation suggests that the effects of somatostatin and galanin are due to inhibition of prejunctional cholinergic transmission, not inhibition of postjunctional acetylcholine sensitivity. However, administration of somatostatin to the anterior chamber reportedly does not affect the pupil size in rabbit (Hernandez et al., 1985) and monkey (Almegard and Bill, 1993). In these experiments, the measurements were taken with a caliper that may lack sufficient sensitivity and accuracy to detect any effect. Precise measurement using a TV pupillometer may be necessary to reveal in situ effects since the in vitro effect is relatively small. Another possibility for the discrepancy between in vivo and in vitro studies may result from the delivery of the somatostatin to the anterior chamber by cannulation (Hernandez et al., 1985; Almegard and Bill, 1993). This can elicit an irritative response, particularly in the rabbit (Bito et al., 1982). Miosis is one of the typical symptoms of ocular inflammation and may override the mydriatic effect by somatostatin in the in vivo studies. The rapid degradation of the peptides in vivo should also be pointed out as one of the reasons. This may cause the lack of effect of somatostatin on pupil size. Cyclosomatostatin, a somatostatin receptor antagonist, had no effect on attenuation induced by somatostatin in the sphincter muscle. Five distinct somatostatin receptor types (SSTR1 – 5) were found to date (Hoyer et al., 1994). Cyclosomatostatin seems especially effective for SSTR2 (Hocart et al., 1999). SSTR4 is the major subtype in rat iris/ ciliary body, however, SSTR4 is expressed in posterior epithelium and dilator muscle of the iris (Mori et al., 1997). Concerning other subtypes, the effect is unclear because
K. Yamaji et al. / Experimental Eye Research 77 (2003) 609–614
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Fig. 4. Effect of somatostatin and galanin on dilator muscle response to electric field stimulation. For each experiment, 5 responses were measured before and after drug application. Light gray, basal tone before contraction; dark gray, response to 100 pulse stimulation. Mean ^ SD, n ¼ 6; N.S. ¼ P . 0:05:
only SSTR4 expression was examined by in situ hybridization. Further investigation is necessary to characterize the subtype in the iris sphincter region. Galanin attenuated the cholinergic neurotransmitter release at a prejunctional site. This is consistent with a previous report (Elbadri et al., 1991). We also showed that galanin had no effect on the dilator muscle where it is present, but in low amounts (Firth et al., 2002), which supports our findings. Thus, the attenuation of sphincter contraction and absence of any effect on the dilator
muscle leads us to conclude that galanin may induce mydriasis. Substance P (Nishiyama et al., 1982), calcitonin generelated peptide (Haruno et al., 1996) and pituitary adenylate cyclase-activating peptide (Yoshitomi et al., 2002) are released from the trigeminal nerve and have the potential to induce miosis. These peptides are considered to be the mediators of the ocular inflammatory response (Yoshitomi et al., 2002). According to the histological studies, the origin of somatostatin and galanin is different among mammalian
Fig. 5. Effect of somatostatin and galanin on sphincter muscle responses to 1 mM acetylcholine. The peptide was applied 15 min before acetylcholine contraction. Neither peptide had a detectable effect. Mean ^ SD, n ¼ 6; N.S. ¼ P . 0:05:
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species. Somatostatin is likely to be of trigeminal origin in monkey (Firth et al., 2002) but of sympathetic origin in rat (Wright et al., 1989). In case of galanin, the trigeminal origin is suggested in rat (Stromberg et al., 1987), pig (Stone et al., 1988) and monkey (Firth et al., 2002) but the sympathetic origin is noted in cat (Grimes et al., 1994). If somatostatin and galanin originate from trigeminal neurons in rabbit, then their effects on pupillary response would be different from other neuropeptides. To investigate the reason for a different role of each peptide for pupillary control mechanism is our future study.
References Almegard, B., Andersson, S.E., 1990. Outflow facility in the monkey eye: effects of calcitonin gene-related peptide, cholecystokinin, galanin, substance P and capsaicin. Exp. Eye Res. 51, 685 –689. Almegard, B., Bill, A., 1993. C-terminal calcitonin gene-related peptide fragments and vasopressin but not somatostatin-28 induce miosis in monkeys. Eur. J. Pharmacol. 250, 31–35. Almegard, B., Stjernschantz, J., Bill, A., 1992. Cholecystokinin contracts isolated human and monkey iris sphincters; a study with CCK receptor antagonists. Eur. J. Pharmacol. 211, 183 –187. Bito, L.Z., Nichols, R.R., Baroody, R.A., 1982. A comparison of the miotic and inflammatory effects of biologically active polypeptides and prostaglandin E2 on the rabbit eye. Exp. Eye Res. 34, 325 –337. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., Guillemin, R., 1973. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179, 77–79. Ekblad, E., Hakanson, R., Sundler, F., Wahlestedt, C., 1985. Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations. Br. J. Pharmacol. 86, 241 –246. Elbadri, A.A., Shaw, C., Johnston, C.F., Archer, D.B., Buchanan, K.D., 1991. The distribution of neuropeptides in the ocular tissues of several mammals: a comparative study. Comp. Biochem. Physiol. C 100, 625– 627. Firth, S.I., Kaufman, P.L., De Jean, B.J., Byers, J., Marshak, D.W., 2002. Innervation of the uvea by galanin and somatostatin immunoreactive axons in macaques and baboons. Exp. Eye Res. 75, 49 –60.
Grimes, P.A., McGlinn, A.M., Koeberlein, B., Stone, R.A., 1994. Galanin immunoreactivity in autonomic innervation of the cat eye. J. Comp. Neurol. 348, 234–243. Haruno, I., Yoshitomi, T., Harada, Y., Katori, M., Ishikawa, S., 1996. Calcitonin gene-related peptide induced relaxation of the rabbit iris dilator muscle. Curr. Eye Res. 15, 105–110. Hayashi, K., Masuda, K., 1982. Effects of vasoactive intestinal polypeptide (VIP) and cyclic-AMP on the isolated sphincter pupillae muscles of the albino rabbit. Jpn. J. Ophthalmol. 26, 437 –442. Hernandez, D.E., Simons, K.B., Spampinato, D., Rioux, F., St-Pierre, S., 1985. Pupillary effects of neurotensin: structure-activity relationships. Neuropeptides 6, 561–568. Hocart, S., Jain, R., Murphy, W., Taylor, J., Coy, D., 1999. Highly potent cyclic disulfide antagonists of somatostatin. J. Med. Chem. 42, 1863–1871. Hoyer, D., Lubbert, H., Bruns, C., 1994. Molecular pharmacology of somatostatin receptors. Naunyn Schmiedebergs Arch. Pharmacol. 350, 441 –453. Mori, M., Aihara, M., Shimizu, T., 1997. Differential expression of somatostatin receptors in the rat eye: SSTR4 is intensely expressed in the iris/ciliary body. Neurosci. Lett. 223, 185–188. Nishiyama, A., Mochizuki, M., Masuda, K., 1982. Effects of substance P on the isolated iris sphincter muscle of the albino rabbit. Jpn. J. Ophthalmol. 26, 29–36. Stone, R.A., McGlinn, A.M., Kuwayama, Y., 1988. Galanin-like immunoreactive nerves in the porcine eye. Exp. Eye Res. 46, 457 –461. Stromberg, I., Bjorklund, H., Melander, T., Rokaeus, A., Hokfelt, T., Olson, L., 1987. Galanin-immunoreactive nerves in the rat iris: alterations induced by denervations. Cell. Tissue Res. 250, 267–275. Ueda, N., Muramatsu, I., Hayashi, H., Fujiwara, M., 1982. Trigeminal nerve: the possible origin of substance P-nergic response in isolated rabbit iris sphincter muscle. Life Sci. 31, 369–375. Ueda, N., Muramatsu, I., Sakakibara, Y., Fujiwara, M., 1981. Noncholinergic, nonadrenergic contraction and substance P in rabbit iris sphincter muscle. Jpn. J. Pharmacol. 31, 1071–1079. Wright, L.L., Luebke, J.I., 1989. Somatostatin-, vasoactive intestinal polypeptide- and neuropeptide Y-like immunoreactivity in eye- and submandibular gland-projecting sympathetic neurons. Brain Res. 494, 267 –275. Yamaji, K., Yoshitomi, T., Usui, S., Ohnishi, Y., 2003. Mechanical properties of the rabbit iris smooth muscles. Vision Res. 43, 479 –487. Yoshitomi, T., Yamaji, K., Ishikawa, H., Ohnishi, Y., 2002. Effect of pituitary adenylate cyclase-activating peptide on isolated rabbit iris sphincter and dilator muscles. Invest. Ophthalmol. Vis. Sci. 43, 780 –783.
Effect of somatostatin and galanin on isolated rabbit iris sphincter and dilator muscles Kazutsuna Yamajia,*, Takeshi Yoshitomib, Shiro Usuia a
Laboratory for Neuroinformatics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan b Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan Received 4 February 2003; accepted in revised form 26 June 2003
Abstract The neuropeptides somatostatin and galanin are present in the iris and may modulate pupil diameter. We examined the effects of somatostatin and galanin on isolated rabbit iris dilator and sphincter smooth muscles that were mounted in an organ bath. An isometric transducer recorded changes in tension in response to electric field stimulation (100 Hz, 0.3 m sec in duration, 10 V in strength) delivered by a pair of platinum plate electrodes. The dilator muscle response to field stimulation was not changed by either peptide, even at the highest concentrations examined. The sphincter muscle response consisted of two components: a fast component mediated by acetylcholine and slow component mediated by substance P. Both somatostatin and galanin attenuated the cholinergic component in a dose-dependent manner (from 0.3 nM to 0.1 mM ) but had no effect on responses mediated by substance P. Galanin was more effective (attenuation of 43% at 0.1 mM ) compared with somatostatin (attenuation of 16% at 0.1 mM ) in reducing the cholinergic response. Neither peptide affected the contraction induced by acetylcholine (1 mM ). Therefore both peptides inhibited cholinergic transmission in the sphincter muscle, although the degree of inhibition by each was different. We conclude that somatostatin and/or galanin may induce mydriasis by attenuating cholinergic neurotransmitter release. q 2003 Elsevier Ltd. All rights reserved. Keywords: somatostatin; galanin; rabbit; smooth muscle; iris sphincter; iris dilator; isometric contraction
1. Introduction The complex innervation of the mammalian iris is being gradually clarified. Control of the pupil has long been attributed to classical autonomic neurotransmitters such as noradrenalin and acetylcholine. Recently, a variety of biologically active peptides released from trigeminal (Haruno et al., 1996; Nishiyama et al., 1982; Yoshitomi et al., 2002) and facial nerves (Hayashi and Masuda, 1982) have also been found to modulate pupil diameter. Somatostatin, first detected in ovine hypothalamus (Brazeau et al., 1973), is also present in the iris/ciliary body of rat (Mori et al., 1997), rabbit, sheep and cow (Elbadri et al., 1991), and recently it was found in the iris of monkey (Firth * Corresponding author. Dr K. Yamaji, Laboratory for Neuroinformatics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 3510198, Japan. E-mail address: [email protected] (K. Yamaji). 0014-4835/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. DOI:10.1016/S0014-4835(03)00177-5
et al., 2002). Galanin is also present in the iris of rat (Stromberg et al., 1987), pig (Stone et al., 1988), cat (Grimes et al., 1994) and monkey (Firth et al., 2002). Somatostatin and galanin may be of trigeminal (Firth et al., 2002; Stromberg et al., 1987) or sympathetic origin (Wright et al., 1989; Grimes et al., 1994). Ekblad et al. (1985) reported that administration of galanin to isolated rabbit iris sphincter muscle inhibits the response evoked by electrical stimulation, although it does not inhibit the carbachol-stimulated contraction. Their results imply a mydriatic effect of galanin; however, pupil size was not affected by galanin in vivo (Almegard and Andersson, 1990). Further investigation of the effects of these peptides on the sphincter and dilator muscles is necessary because the pupil is regulated by a push-pull interaction of both. This study clarifies the functional role of somatostatin and galanin on the iris sphincter and dilator muscles in vitro by means of the isometric tension recording method.
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2. Materials and methods 2.1. Isolation and incubation of muscle specimens All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male albino rabbits weighting 2 – 3 kg were euthanized by injecting an overdose of pentobarbital sodium (Abbott Laboratories, North Chicago, IL, USA) into the marginal ear vein. The eyes were immediately enucleated and placed in Krebs solution composed of (mM ): NaCl, 94.8; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 25.0; and glucose, 11.7 and gassed with 95% O2 and 5% CO2. After removal of the cornea, a ring-shaped iris sphincter muscle specimen (1 mm in width) or radialshaped dilator muscle specimen (45 8 in sector) was prepared according to the method previously reported (Yamaji et al., 2003). The ends of each specimen were tied with silk thread for mounting in an organ bath (1.5 ml). One end of the specimen was connected to the isometric tension transducer (Nihon Kohden Co., TB-612T), and the other end was secured to a hook at the bottom of the organ bath. The organ bath was perfused continuously (0.15 ml/s) with oxygenated Krebs solution warmed to 37 8C. The initial lengths of sphincter and dilator muscle specimens were adjusted to 10 mm and 5 mm respectively, allowing
maximum contraction to occur (Yamaji et al., 2003). After at least 1 hr of equilibration, the experiments were performed. 2.2. Electrical field stimulation experiments Electrical field stimulation was applied through a pair of platinum electrodes with 11 mm separation and placed in the organ bath so that the current pulse passed transversely across the tissue. The sphincter muscle response to electric stimulation consists of fast and slow components (Ueda et al., 1982). Stimulation with 100 pulses (10 V at 100 Hz) elicits both fast and slow contractions while stimulation with 10 pulses elicits only the fast contraction (Yoshitomi et al., 2002). For the dilator muscle, only the fast component occurs regardless of the strength of the stimulation (Yoshitomi et al., 2002). We used 100 pulses to produce effective contractions of this muscle. To determine if either somatostatin or galanin selectively affected fast or slow contractions, each peptide was added to the perfusion medium 15 min before the onset of field stimulation with 100 pulses. Control responses were recorded before peptide application and after washing out (1 hr) the peptide. Dose response curves (0.3 nM –0.1 mM ) for somatostatin and galanin were generated for the sphincter muscle. Prior
Fig. 1. Effect of somatostatin and galanin on sphincter muscle response evoked by electric field stimulation. Stimulation with 100 pulses was employed to produce the biphasic response. Each peptide was applied 15 min before stimulation. Light and dark gray in lower figure show the fast and slow components respectively. Mean ^ SD, n ¼ 6;*P , 0:05; * * P , 0:01; and N.S. ¼ P . 0:05:
K. Yamaji et al. / Experimental Eye Research 77 (2003) 609–614
to each dose of peptide, the sphincter was stimulated for 20 min with 10 pulses every 1.5 min to achieve control contractions. The peptide was then added to the perfusion medium, and the contractions were recorded for another 20 min with the same stimulation. A washout period of at least 1 hr followed each peptide, and then the control contractions were repeated followed by the next concentration of peptide. 2.3. Direct contraction experiments These experiments were performed to investigate if somatostatin and galanin affect the postjunctional site. Control contractions were initiated with 1 mM acetylcholine. This concentration was chosen because the median effective concentration (EC50) for contractile response to acetylcholine in this tissue is approximately 0.3 mM (Ueda et al., 1981; Yoshitomi et al., 2002). The 1 mM acetylcholine also enables to produce similar contractile amplitude to that of evoked by electric stimulation which was applied in the previously mentioned experiments. After a washout period of at least 1 hr, 0.1 mM somatostatin or galanin was added to the Krebs solution 15 min before contraction was again stimulated by acetylcholine. After
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reaching the maximum contraction, which took about 2 min, the peptide was washed out for at least 1 hr. Control contractions by the agonist were repeated after the peptideapplied contraction. 2.4. Chemicals The following drugs and chemicals were used in this study: somatostatin and galanin (Peptide Institute, Inc., Osaka, Japan) and acetylcholine (Wako Chemical Inc., Osaka, Japan). Peptides were prepared in aliquots and stored at 2 30 8C.
3. Results 3.1. Electrical field stimulation experiments The sphincter muscle specimens produced a biphasic response to stimulation with 100 pulses. Somatostatin (0.1 mM ) and galanin (0.1 mM ) significantly attenuated the fast component of the response but had no effect on the slow component (Fig. 1). The fast and slow components correspond to the cholinergically and substance P mediated
Fig. 2. Effect of somatostatin and galanin on sphincter (A) and dilator (B) muscles. Stimulation (10 pulse) was applied every 1.5 min. The peptide was present continuously after t ¼ 0 min: Each concentration was applied to the same specimen after at least a 1 hr washout period. Somatostatin, left; galanin, right.
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contractile responses, respectively (Ueda et al., 1981). Thus each peptide inhibited only the cholinergic response. To examine the effect further, 10-pulse stimulation was applied to elicit only the cholinergic response (Fig. 2(A)). For somatostatin, the attenuation reached its maximum at 5 min and then gradually diminished for another 5 min until it became stable. In contrast, attenuation of the contraction by galanin was relatively stable throughout its application. Comparison of the steady state revealed that the attenuation by galanin was greater than that by somatostatin. This difference was clear in the doseresponse curves (Fig. 3). The maximum attenuation and EC50 estimated from the sigmoidal function were 16.11% and 3.38 nM for somatostatin and 42.83% and 14.21 nM for galanin. The effect of cyclosomatostatin, a commercially available somatostatin receptor antagonist, on this tissue was also examined. The cyclosomatostatin was added to the perfusion medium 20 min before the application of somatostatin. The attenuation caused by 0.1 mM somatostatin (14.2 ^ 4.5%, n ¼ 6Þ did not show a significant difference ðP . 0:1Þ after application of 1 mM cyclosomatostatin (13.6 ^ 2.7%, n ¼ 6Þ: The effects of somatostatin and galanin on contraction of the iris dilator muscle by field stimulation were also tested. At concentrations of up to 0.1 mM , neither peptide had an effect on either the basal tone or the contraction amplitude evoked by electric stimulation (Fig. 2(B), Fig. 4). 3.2. Direct contraction experiments In the field stimulation experiments, only the response of the sphincter muscle was affected by the peptides. The attenuation of contraction in those experiments could be due to the activation of peptide receptors at either ganglionic or
Fig. 3. Dose-response relationship of somatostatin and galanin on the fast component of sphincter muscle responses to electric field stimulation. ‘Amplitude (%)’ in each specimen was calculated as the ratio of the response before (100%) and after drug application. For each experiment, 5 responses were measured before and after drug application. Solid curves were determined by fitting the sigmoidal function to the experimental data using a non-linear optimization method. Somatostatin (O), galanin ( ), Mean ^ SD, n ¼ 6:
†
post-ganglionic sites. Therefore, the effect of each peptide on 1 mM acetylcholine induced contractions was examined. Neither peptide resulted in a change in the response amplitude induced by acetylcholine (Fig. 5).
4. Discussion Previous reports showed that isolated iris sphincter muscle in rabbit (Ueda et al., 1982) and monkey (Almegard et al., 1992) were not affected by somatostatin in concentrations ranging from 0.1 nM to 10 mM and from 0.2 nM to 2 mM , respectively. These results are not inconsistent with our findings because they observed the effects on basal tone. We also found that somatostatin did not affect the sphincter basal tone (Fig. 2(A)). Because somatostatin clearly attenuated the contraction of rabbit iris sphincter stimulated by field current, our results also suggest that it may inhibit the monkey iris sphincter muscle. The absence of somatostatin effects on dilator muscle basal tone and contraction evoked by electric stimulation is consistent with the finding that somatostatin is less common in the dilator muscle (Firth et al., 2002). The attenuation of sphincter contraction in response to field stimulation and the absence of attenuation for direct cholinergic stimulation suggests that the effects of somatostatin and galanin are due to inhibition of prejunctional cholinergic transmission, not inhibition of postjunctional acetylcholine sensitivity. However, administration of somatostatin to the anterior chamber reportedly does not affect the pupil size in rabbit (Hernandez et al., 1985) and monkey (Almegard and Bill, 1993). In these experiments, the measurements were taken with a caliper that may lack sufficient sensitivity and accuracy to detect any effect. Precise measurement using a TV pupillometer may be necessary to reveal in situ effects since the in vitro effect is relatively small. Another possibility for the discrepancy between in vivo and in vitro studies may result from the delivery of the somatostatin to the anterior chamber by cannulation (Hernandez et al., 1985; Almegard and Bill, 1993). This can elicit an irritative response, particularly in the rabbit (Bito et al., 1982). Miosis is one of the typical symptoms of ocular inflammation and may override the mydriatic effect by somatostatin in the in vivo studies. The rapid degradation of the peptides in vivo should also be pointed out as one of the reasons. This may cause the lack of effect of somatostatin on pupil size. Cyclosomatostatin, a somatostatin receptor antagonist, had no effect on attenuation induced by somatostatin in the sphincter muscle. Five distinct somatostatin receptor types (SSTR1 – 5) were found to date (Hoyer et al., 1994). Cyclosomatostatin seems especially effective for SSTR2 (Hocart et al., 1999). SSTR4 is the major subtype in rat iris/ ciliary body, however, SSTR4 is expressed in posterior epithelium and dilator muscle of the iris (Mori et al., 1997). Concerning other subtypes, the effect is unclear because
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Fig. 4. Effect of somatostatin and galanin on dilator muscle response to electric field stimulation. For each experiment, 5 responses were measured before and after drug application. Light gray, basal tone before contraction; dark gray, response to 100 pulse stimulation. Mean ^ SD, n ¼ 6; N.S. ¼ P . 0:05:
only SSTR4 expression was examined by in situ hybridization. Further investigation is necessary to characterize the subtype in the iris sphincter region. Galanin attenuated the cholinergic neurotransmitter release at a prejunctional site. This is consistent with a previous report (Elbadri et al., 1991). We also showed that galanin had no effect on the dilator muscle where it is present, but in low amounts (Firth et al., 2002), which supports our findings. Thus, the attenuation of sphincter contraction and absence of any effect on the dilator
muscle leads us to conclude that galanin may induce mydriasis. Substance P (Nishiyama et al., 1982), calcitonin generelated peptide (Haruno et al., 1996) and pituitary adenylate cyclase-activating peptide (Yoshitomi et al., 2002) are released from the trigeminal nerve and have the potential to induce miosis. These peptides are considered to be the mediators of the ocular inflammatory response (Yoshitomi et al., 2002). According to the histological studies, the origin of somatostatin and galanin is different among mammalian
Fig. 5. Effect of somatostatin and galanin on sphincter muscle responses to 1 mM acetylcholine. The peptide was applied 15 min before acetylcholine contraction. Neither peptide had a detectable effect. Mean ^ SD, n ¼ 6; N.S. ¼ P . 0:05:
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species. Somatostatin is likely to be of trigeminal origin in monkey (Firth et al., 2002) but of sympathetic origin in rat (Wright et al., 1989). In case of galanin, the trigeminal origin is suggested in rat (Stromberg et al., 1987), pig (Stone et al., 1988) and monkey (Firth et al., 2002) but the sympathetic origin is noted in cat (Grimes et al., 1994). If somatostatin and galanin originate from trigeminal neurons in rabbit, then their effects on pupillary response would be different from other neuropeptides. To investigate the reason for a different role of each peptide for pupillary control mechanism is our future study.
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