Double Reciprocal Innervotions in Dog Iris Sphincter and Dilator Muscles

Transcription

1 Double Reciprocal Innervotions in Dog Iris Sphincter and Dilator Muscles Takeshi Yoshitomi and Yushi Iro Neuro-effector transmission and mechanical responses in smooth muscles of the dog iris were studied, using tension recording and microelectrode methods. Electrical stimulations evoked an initial phasic contraction followed by relaxation in both the iris sphincter and dilator muscles. Atropine selectively suppressed phasic contraction of the sphincter and relaxation of the dilator muscle, while guanethidine selectively blocked relaxation of the sphincter and contraction of the dilator muscle. Pharmacological investigations revealed distributions of a r excitatory (mediating contractions) and a 2 -inhibitory (mediating relaxations) adrenoceptors in addition to /^-inhibitory adrenoceptors in the sphincter muscles, and a- excitatory and 0-inhibitory adrenoceptors in the dilator muscle. These results indicate that the iris sphincter and dilator muscles receive double reciprocal innervations by the cholinergic and adrenergic nervous systems. Norepinephrine (NE) or carbachol did not modify membrane potential of the smooth muscle cells in either muscle tissue, yet these agents evoked muscle relaxation or contraction, respectively; in the sphincter muscle. Reversed sequences of mechanical responses were observed in the dilator. Cafree solution reduced the resting tension and blocked the agonist-induced contraction in both muscle tissues. Excess-[K] 0 solution dose-dependently depolarized the muscle membrane, and evoked combined mechanical responses of relaxation and contraction which were blocked by adrenergic and cholinergic blocking agents, mainly due to NE or acetylcholine (ACh) released from the nerve terminals in both muscle tissues. In the sphincter muscle, excess- K] 0 solution evoked a phasic contraction in the presence of these blocking agents. Specific mechanical features of the dog iris in relation to excitation-contraction coupling were given attention. Invest Ophthalmol Vis Sci 27:83-91, 1986 It has been considered that the mammalian iris sphincter or dilator muscles are innervated by cholinergic or adrenergic excitatory (mediating contractions) nerve fibers, respectively, and miosis or mydriasis is the result of contraction of iris sphincter or dilator muscles due to activation of these excitatory nerve fibers. 1 Histological investigations revealed that there are adrenergic or cholinergic innervations in the sphincter and dilator muscle, respectively, in various species including monkey, 2 guinea-pig, 3 cat and rabbit, 4 and human. 5 Physiological investigations also revealed that the sphincter muscle is innervated in part by inhibitory (mediating relaxations) adrenergic nerve fibers in the cat 67 and the rat, 8 and that the dilator muscle is also innervated by inhibitory cholinergic nerve fibers in the feline, 9 bovine, 10 and human." In an attempt to clarify the functional roles of cholinergic or adrenergic innervations in iris dilator or sphincter muscles, as related to miosis or mydriasis, From the Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan. Submitted for publication: February 13, Reprint requests: T. Yoshitomi, MD, Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan. we did comparative studies on the effect of nerve stimulation on the dog iris sphincter and dilator muscles. The effects of exogenously applied cholinergic and adrenergic agents or excess [K] o or Ca-free solution on the electrical and mechanical properties of the smooth muscle cells were also observed with regard to responses to field stimulation. We now report evidence that the dog iris sphincter and dilator muscles are functionally innervated by both cholinergic and adrenergic nerve fibers, and that a r excitatory and a 2 - and ^-inhibitory adrenoceptors are distributed on these muscle cells. In addition, differences in the mechanical properties between these muscles and other visceral smooth muscles were discussed. Materials and Methods Mongrel dogs of either sex (1-2 yr of age, kg) were anaesthetized with an iv administration of pentobarbitone (30 mg/kg), and bled from the femoral artery. The eyes were immediately enucleated and kept in oxygenated Krebs solution. Under microscopic observations, the cornea was removed, the iris carefully dissected out, and specimens ( mm wide and 3-4 mm long) were prepared from the sphincter and dilator muscles. 83

2 84 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1986 Vol. 27 iris sphinctermuscle atroplne(1<r 6 M) guanethidlne(5x10-^m) a2- a3- iris dilator muscle 1 guanethldlne(5x10-6 M) atropine b2< 50mg 30mg Fig. 1. Effects of field stimulation (0.4 sec in duration and 50 V in strength) on mechanical activities of dog iris sphincter (al-a3) and dilator muscle (bl-b3). Dots indicate application of field stimulation, and numbers of field stimulations indicated below the dots are the stimulus number at 20 Hz. The observed in Krebs solution (a 1 and b 1); in the presence of atropine (10~ 6 M) with (a3) or without (a2) guanethidine (5 X 10~ 6 M) (a2 and a3); in the presence of guanethidine (5 X 10" 6 M) with (b3) or without (b2) atropine (10" 6 M) (b2 and b3). To measure the development of isometric tension, a strip of sphincter or dilator muscle was mounted in a 0.9-ml organ bath through which the test solution, at a temperature of 34 C, flowed continuously (0.3 ml/sec). The preparations were placed vertically, the ends tied with silk thread, and one end of the strip was tied to a mechano-transducer (Nihon-Koden Ltd., Tokyo; RCA5734) and the other to a hook at the bottom of the bath. The resting tension was mg. To investigate neural effects on the motility of these muscles, electrical field stimulations were applied through a pair of electrodes consisting of silver plates, separated by 5 mm and placed so that a current pulse would pass transversely across the tissue. 12 Single and repetitive stimulations at 20 Hz were applied, using a current pulse of msec in duration and V in strength. To record the membrane potential, the muscle preparations ( mm wide and 5-6 mm long) were mounted in an organ bath with a volume of 2 ml through which the solution flowed continuously at a rate of 3 ml/min at 34 C. The conventional glass micro-electrode with a resistance of MQ and filled with 3 M KC1 was inserted from the posterior surface. Modified Krebs solution of the following ionic concentration was used (mm): Na , K + 5.9, Mg , Ca , Cr 134.0, HCO 3 " 15.5, H 2 PO 4 " 1.2 and glucose The solution was aerated with 97% O 2 and 3% CO 2, and the ph was adjusted to Excess- [K] o solutions was prepared by replacing equimolar NaCl with KC1, isotonically. The Ca-free 2 mm EGTA-containing solution was prepared by replacing CaCl 2 with equimolar MgCl 2 and adding 2 mm EGTA. The following drugs were used: tetrodotoxin (TTX, Sankyo, Tokyo), atropine sulphate (Daiichi, Tokyo), phentolamine (Ciba-Geigy, Basel, Switzerland), propranolol (Sumitomo, Osaka, Japan), timolol (Timoptol, Merk-Banyu-Japan, Tokyo), guanethidine, sulfate, yohimbine, carbachol (Tokyo Kasei, Tokyo), prazosin (Pfizer, Basel, Switzerland), ethyleneglycolbis (jft-aminoethylether)-n,n'-tetraacetic acid (EGTA, Dozin Kumamoto, Japan), atenolol (ICI, Macclesfield, Cheshire, England), and norepinephrine (Sigma; St. Louis, MO 63178). This research was carried out according to the guidelines of the ARVO Resolution on the Use of Animals in Research. Results Effects of Electrical Field Stimulation on the Mechanical Properties of Dog Iris Sphincter and Dilator Muscles When specimens of both iris sphincter and dilator muscle, (under a resting tension of 10 to 20 mg) were mounted in the organ bath, the tissue gradually relaxed to a steady tone after 0.5 to 1-hr superfusion with Krebs solution. Spontaneous contraction of either muscle did not occur throughout the experiment. Application of a single field stimulation (0.4 msec in duration and 50 V in strength) evoked a biphasic muscle response, ie, initial phasic contraction followed by long lasting relaxation in both muscles (Fig. 1). In the sphincter muscle, the amplitude of phasic contraction was larger than that of relaxation, while the dominant response in the dilator muscle was relaxation. When repetitive stimulations (5 or 10 stimuli at 20 Hz) were applied, amplitudes of phasic contraction and relaxation of the sphincter or the phasic contraction of the dilator muscle were enhanced, in proportion to the number of stimuli. These biphasic responses were completely blocked by application of 10~ 7 M tetrodotoxin (TTX), thereby indicating that the mechanical responses were of neurogenic origin. Figure 1 also shows the effect of atropine or guanethidine on the mechanical responses of sphincter or dilator muscles evoked by electrical field stimulation.

3 No. 1 INNERVATIONS IN DOG IRIS MUSCLES / Yoshiromi ond Iro 85 iris sphincter muscle phentolamlne phentolamine timolol(10~ 5 M) (10-5M) propranolol timolol(10" 5 M) 'Phentolamlne (10" 5 M) timolol(10" 6 M) d2'.y^" prazosin(10" 6 M) d3 "V yohimbine(5x10-6 M) 1min. d'2i d'3 d'4 - f H norepinephrine (10' 5 M) 1min. 10mg 10mg Fig. 2. Effects of various adrenergic blocking agents on the mechanical responses of the dog iris sphincter evoked by electrical field stimulations (0.4 sec in duration, 50 V in strength, 10 stimuli at 20 Hz) (a-d), or exogenously applied norepinephrine (10~ 5 M) (d'), after pretreatment with atropine (10~ 6 M). In the presence of atropine, electrical field stimulations or exogenously applied norepinephrine (10~ 5 M) evoked long lasting relaxation (al, bl, cl, dl, d'l). Effects of 10" 5 M phentolamine (al-a3) in the absence (a2) or presence of 10" 5 M propranolol (a3); bl-b3: effects of phentolamine (10~ 5 M) in the absence (b2) or presence of 10~ 5 M timolol (b3); cl-c3: effects of timolol (10~ 5 M) in the absence (c2) or presence of 10" 5 M phentolamine (c3); dl-d4, dl'-d4': effects of timolol (10~ 6 M), prazosin (1O~ 6 M) and yohimbine (5 X 10~ 6 M) on the muscle relaxation evoked by electrical field stimulations (d 1 -d4) or exogenously applied norepinephrine (dl'-d4'). dl-d4 and dl'-d4' were recorded from the same preparation. These observations indicate that the iris sphincter muscles of the dog receive cholinergic excitatory and adrenergic inhibitory innervations and that the dilator muscles receive cholinergic inhibitory and adrenergic excitatory innervations. Effects of a and /? Adrenergic Blocking Agents on Mechanical Responses of the Iris Muscles To investigate properties of the adrenoceptors distributed on the smooth muscle cells of iris sphincter or dilator muscle cells, the effects of a and 0 adrenergic blocking agents were observed in the presence of atropine (10" 6 M) (Fig. 2, 3). In the sphincter muscle, effects of phentolamine (10~ 5 M) on the amplitude of muscle relaxation evoked by field stimulation varied from one preparation to another; ie, in four out of six preparations, this agent slightly decreased the amplitude of the relaxation (Fig. 2, al vs a2) and in the others (2 out of 6 preparations; Fig. 2, bl vs b2) increased the amplitude. Propranolol (10~ 5 M) had no effect on the amplitude of the relaxation, in the presence or absence of phentolamine (Fig. ris dilator muscle atroplne(10" M) Neither atropine nor guanethidine altered the resting tension of the muscles. When the sphincter muscle was exposed to atropine (10~ 6 M), the generation of phasic contraction was blocked and only the long lasting relaxation (30-45 sec) occurred in response to the field stimulation. This muscle relaxation disappeared after 30-min treatment with guanethidine (5 X 10~ 6 M). In the dilator muscle, treatment with guanethidine (5 X 10~ 6 M) suppressed the generation of phasic contraction but had no effect on the muscle relaxation. The increase in the stimulus number (5 or 10 stimuli at 20 Hz, Fig. 1, b2) did not enhance the amplitude of the muscle relaxation, thereby indicating that the threshold level for the activation of inhibitory nerve fibers was low compared with that of the excitatory ones, and a single stimulus was sufficient to fully activate the inhibitory nerve fibers in the musculature. Additional application of atropine (10~ 6 M) abolished the muscle relaxation induced by field stimulation..phento!amlne(1o" 5 M) phentolamlne(5x10~ 9 M) phentolamlne(10~ 4 M) Fig. 3. Effects of various adrenergic blocking agents on the mechanical response of the dog iris dilator evoked by electrical field stimulations (1-30 stimuli at 20 Hz), in the presence of atropine (10~ 6 M). al-a3: mechanical responses evoked by 1,5, 10 stimuli at 20 Hz in Krebs solution, respectively; bl-b3: effects of atropine (10" 6 M): cl-c4, d3-d4: effects of 10~ 5 or 5 X 10" 5 M phentolamine in the presence of atropine (10" 6 M); c3-c4: effects of 10" 4 M phentolamine; O-f4: effects of 10~ 5 M timolol on muscle relaxation evoked by repetitive field stimulations in the presence of 10" 4 M phentolamine and 10~ 6 M atropine.

4 86 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / January 1986 Vol. 27 1Q' 7 noropincphrinc carbachol relative tension B LOT 0 norepinephrine carbachol II Fig. 4. (A) effects of various concentrations of norepinephrine (O) or carbachol ( ) on the resting membrane potential of the smooth muscle cells of iris sphincter. Each point indicates the mean value and vertical bars indicte 2 X S.D. (n = 13-66). (B) relationships between the concentration of carbachol ( ) or norepinephrine (O) and the relative amplitude of muscle contraction (plus) or relaxation (minus). The amplitude of muscle contraction evoked by 3 X 10~ 5 M carbachol was taken as a relative amplitude of+ 1.0 (n = 6). 2a). Figures 2b, c show the effect of timolol (10~ 5 M), a non-selective jff-adrenergic receptor blocking agent, on the amplitude of relaxation in the presence or absence of phentolamine. Application of timolol (10~ 5 M) reduced the amplitude of muscle relaxation to about 50% of the initial value (Fig. 2, cl vs c2). However, increases in the concentration (5 X 10~ 5 M) did not abolish generation of the muscle relaxation. The combined applications of timolol and phentolamine (10~ 5 M) led to cessation of the generation of muscle relaxation (Fig. 2, c3). Figure 2d shows the effects of various adrenergic blocking agents on the muscle relaxation induced by exogenously applied NE (10~ 5 M) or electrical field stimulation, observed in the same preparation. Timolol (10~ 6 M) reduced the amplitude of muscle relaxation induced by both NE and field stimulation to about 50% ofthe initial value. In the presence of timolol, NE evoked a small but discrete phasic contraction which was superimposed on the long lasting relaxation (Fig. 2, d'2). Application of prazosin (10 6 M) in the presence of timolol abolished the phasic contraction and potentiated the muscle relaxation evoked by NE or electrical field stimulation (Fig. 2, d2 vs d3 or d'2 vs d'3). Additional application of yohimbine (5 X 10" 6 M) (timolol + prazosin + yohimbine) greatly reduced the amplitude of relaxation induced by NE or electrical field stimulation (Fig. 2, d3 vs d4 or d'3 vs d'4), thereby indicating the presence of «i excitatory, a 2 and 0 inhibitory adrenoceptors in the sphincter muscle. In an attempt to classify the subtype of 3-adrenoceptors distributed in the sphincter muscle, we used atenolol (a j8,-selective adrenergic antagonist). In the presence of atropine, atenolol (10~ 5 M) or combined applications of atenolol (10~ 5 M) and phentolamine (10~ 5 M) reduced the amplitude of muscle relaxation to 45.8 ± 8.4 (±S.D. n = 4) or to 17.5 ± 7.8% (±S.D. n = 4) of the value. These observations indicate that the dog iris sphincter muscle contains fr -subtype adrenergic receptors. Figure 3 shows effects of atropine (10~ 6 M) and a and /3 adrenergic blocking agents on the mechanical responses of the dilator muscle evoked by field stimulation. Atropine (10~ 6 M) selectively blocked the muscle relaxation, and in the presence of atropine, field stimulation only evoked the phasic contraction. Amplitudes of the phasic contraction were reduced by phentolamine (10~ 5 M) or prazosin (10~ 6 M) to about 20% of the value. With increased concentrations (10~ 4 M), phentolamine blocked the generation of the phasic contraction. However, when repetitive stimulations (10 or 30 stimuli at 20 Hz) were applied in the presence of atropine (10" 6 M) and phentolamine (10~ 4 M), the muscle relaxation which occurred (Fig. 3, e3 and e4) was suppressed by application of timolol (10~ 5 M) (Fig. 3, O and f4). These results indicate that the dilator muscle possesses a-excitatory and ^-inhibitory adrenoceptors. In the presence of atropine (10~ 6 M) and phentolamine (10~ 5 M), 10~ 5 M atenolol reduced the amplitude of muscle relaxation to 24.8 ± 9.9 (±S.D. n = 4) percent of the value, thereby indicating the distribution of j8i-adrenoceptors on this muscle membrane. However, the presence or absence of j8 2 -adrenoceptors in the sphincter and dilator muscles was not investigated. Effects of Cholinergic and Adrenergic Agents on Electrical and Mechanical Properties of the Dog Iris Muscles To investigate properties of adrenergic or cholinergic receptors in the iris muscles, effects of cholinomimetic or adrenomimetic agents on electrical and mechanical properties of smooth muscle cells of the iris sphincter or dilator were observed. For this we used microelectrode or isometric tension recording methods.

5 No. 1 INNEIWATIONS IN DOG IRIS MUSCLES / Yoshiromi ond Iro 87 The resting membrane potential of iris sphincter muscle ranged between -52 to -62 mv, with a mean value of ± 2.4 mv (Mean ± S.D., n = 123). Figure 4A shows the effect of carbachol or NE on the resting membrane potential of this muscle. Both agents in concentrations up to 3 X 10~ 5 M had no effect on the resting membrane potential, yet carbachol or NE evoked contraction or relaxation of the sphincter muscle, respectively. The minimum concentrations of carbachol and NE required to evoke the contraction or relaxation were in a range between 10" 9-10~ 8 M and 10~ 7-10~ 6 M, and the maximum response was observed at the concentration of 3 X 10~ 5 M, in both cases. Figure 4B shows the dose-response relationships observed by applications of carbachol or NE, where the amplitude of 3 X 10~ 5 M carbachol-induced contraction was normalized as 1.0. Figure 5 A shows the effects of carbachol or NE on SOmg 30mg Fig. 6. Effects of Ca-free solution containing 2 mm EGTA on carbachol or norepinephrine-induced contraction of the dog iris sphincter (a 1, a2) and dilator muscles (b 1, b2). Mechanical responses (al) induced by exogenously applied carbachol (1O~ 5 M) in Krebs solution. Effects (a2) of Ca-free solution on the resting tension or the carbachol-induced contraction of the sphincter. Mechanical responses (bl) evoked by exogenously applied norepinephrine (3 X 10~ 5 M) in Krebs solution. Effects (b2) of Ca-free solutions on the resting tension or the norepinephrine-induced contraction of the dilator. Horizontal baras indicate application of carbachol or norepinephrine. Iris dilator A n.p. mv relative 1O~ 7 tension 0 norepinephrine carbachol O norepinephrine carbachol I I 1O~ S 3x10' S M the resting membrane potential of the dilator muscles. The mean value of the resting membrane potential of the iris dilator muscle was ± 2.1 mv (Mean ± S.D., n = 139, ranging between 46 mv to 54 mv). Carbachol (3 X 10" 5 M) or NE (3 X 10" 5 M) did not modify the membrane potential, although these agents did evoked muscle relaxation or contraction, respectively. Figure 5B shows the dose-response relationship observed by applications of carbachol or NE, where the amplitude of muscle relaxation evoked by 3 X 10~ 5 M carbachol was taken as the relative amplitude of 1.0. The minimum concentrations of carbachol and NE required to evoke muscle relaxation and contraction were in ranges between 10~ 9-10~ 8 M and KT 7-1CT 6 M, respectively. Effects of Ca-free 2 mm EGTA-containing Solution on Agonist-Induced Contraction of Iris Muscles 1 I. Fig. 5. (A) effects of various concentrations of norepinephrine (O) or carbachol ( ) on the resting membrane potential of smooth muscle cells of the iris dilator. Each point indicates the mean value and vertical bars indicate 2 X S.D. (n = 16-46). (B) relationships between the concentration of carbachol ( ) or norepinephrine (O) and the relative amplitude of muscle contraction (plus) or relaxation (minus). The amplitude of muscle relaxation evoked by 3 X 10~ 5 M carbachol was taken as a relative amplitude of -1.0 (n = 6). To investigate the source of Ca ++ contributing to activations of contractile proteins, we examined the effects of Ca-free solution containing 2 mm EGTA on the agonist-induced contraction in sphincter and dilator muscles. As shown in Figure 6, application of carbachol (10~ 5 M) or NE (3 X 10~ 5 M), for a short period, evoked contraction in the sphincter and dilator muscles, respectively, Treatment of the tissue with Ca-free solution containing 2 mm EGTA gradually reduced the resting tension to a certain level, and in the presence of Cafree solution, carbachol or NE did not evoke a mechanical response. The resting tension gradually reverted to the original level after the tissue was rinsed in normal Krebs solution.

6 88 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / January 1986 Vol. 27 Iris sphincter muscle 10.2mM a1 tropln*o0~*m) tropln.<10~*m) ph*ntolamln«(10~ 8 M) tlmolol(io~ s M) Fig. 7. Effects of excess- [K] o solutions on mechanical properties of the dog iris sphincter in the absence or presence of cholinergic and adrenergic blocking agents. Mechanical responses (a, b, c) to field stimulations (10 stimuli at 20 Hz) observed in Krebs solution (a), in the presence of atropine (10~ 6 M) (b), or atropine (10~ 6 M) + phentolamine (10~ 5 M) + timolol (10~ 5 M) (c). Effects of various concentrations of [K] o solutions (ala4), under treatment with atropine (bl-b4) or atropine + phentolamine + timolol (cl-c4), respectively. Effect of Excess [K] o on the Electrical and Mechanical Properties of the Iris Muscles Figure 7 shows an example of the effects of various excess-[k]o solutions on mechanical responses of the iris sphincter muscle. Application of 10.2 or 39.2 mm [K] o solution evoked a tonic contracture (Fig. 7, al and a2) and with increased concentrations (77.1 or 118 mm); the phasic contraction was followed by relaxation (Fig. 7, a3 and a4). The maximum amplitude of the phasic contraction was observed in 77.1 mm [K] o solution. In the presence of atropine (10" 6 M), 10.2 or 39.2 mm [K] o solution also evoked a tonic contracture; however, relaxation with a small initial phasic contraction occurred with 77.1 and 118 mm [K] o (Fig. 7, b3 and b4). The muscle relaxation evoked by excess- [K] o solution containing atropine was suppressed by additional application of phentolamine (10~ 5 M) and timolol (10~ 5 M) (Fig. 7c). Thus, in the presence of atropine, phentolamine and timolol, 77.1 or 118 mm [K] o solutions evoked a phasic contraction. These results indicate that the phasic contraction and relaxation induced by excess-[k] 0 solutions are mainly due to ACh or NE released from cholinergic or adrenergic nerve terminals, although excess-[k] 0 solutions also evoked phasic contraction in the iris sphincter muscle. Contrary to the sphincter muscles, relatively low concentrations of excess-[k] o solution (10.2 or 39.2 mm) evoked muscle relaxation in the iris dilator muscles. With increased [K] o concentrations (77.1 or 118 mm) initial relaxation and following phasic contractions occurred (Fig. 8, a3 and a4). Application of atropine (10~ 6 M) abolished the muscle relaxation evoked by excess-[k] 0 solutions, and enhanced the amplitude of phasic contractions. Additional applications of phentolamine (10~ 5 M) and timolol (10~ 5 M) led to cessation of phasic contractions, and there was no mechanical response in response to excess-[k] 0 solutions (Fig. 8, cl-c4). To investigate the effect of excess-[k] 0 solutions on the membrane potential in the presence or absence of atropine, phentolamine and timolol, the microelectrode method was used. In both muscle tissues, combined applications of atropine (10~ 6 M), phentolamine (10~ 5 M), and timolol (10~ 5 M) did not modify membrane potentials recorded at any given concentration of [K] o. As shown in Figure 9, excess-[k] 0 solutions dose-dependently depolarized the membrane in both muscles. The potential change induced by a tenfold increase in [K] o concentration plotted on a log scale was 48 or 43 mv in the sphincter on dilator muscle, respectively. These results clearly show discrepancies between the electrical membrane phenomena and mechanical responses of the iris sphincter and dilator muscles. Discussion Innervation of Sphincter and Dilator Muscles of the Iris Our results provide the first evidence that iris sphincter and dilator muscles receive functional, double reciprocal innervations by cholinergic and adrenergic nervous systems. Cholinergic activation contracts the sphincter and relaxes the dilator muscle to produce miosis. On the other hand, activation of the adrenergic nervous system contracts the dilator and relaxes the sphincter, and mydriasis occurs. Thus, cholinergic and adrenergic inhibitory innervation of dilator and sphincter muscles may play a physiological role in supporting miosis and mydriasis, respectively. These events are mainly led by cholinergic and adrenergic excitatory innervation in the iris muscles.

7 No. 1 INNERVATIONS IN DOG IRIS MUSCLES / Yoshiromi ond Iro 89 iris dilator muscle atroplnedo" 6^ atroplnedo^m) phentolamlne( 10~ 5 M) llmolol(10" 6 M) 77.1mM a3 Fig. 8. Effects of [K] o solutions on mechanical properties of the dog iris dilator muscle in the absence or presence of cholinergic and adrenergic blocking agents. Mechanical responses (a, b, c) to field stimulations (10 stimuli at 20 Hz) observed in Krebs solution (a), in the presence of atropine (10~ 6 M (b) or atropine (10~ 6 M) + phentolamine (10~ 5 M) + timolol (10~ 5 M) (c), respectively, Effects of various [K] o solutions (ala4), under treatment with atropine (10~ 6 M) (bl-b4) or atropine + phentolamine (10~ 5 M) + timolol (10~ 5 M) (cl-c4), respectively. In isolated sphincter muscles of the rat, it was noted that electrical field stimulation in the presence of atropine or exogenously applied NE or isoproterenol induced a weak contraction or relaxation. 8 The present experiments showed that a x -excitatory and «2 -inhibitory adrenoceptors as well as 0-inhibitory adrenoceptors are present in the dog iris sphincter muscle. These observations would explain why exogeneously applied NE produced different mechanical responses of the iris sphincter muscle, in different preparations, ie contraction, relaxation or slight relaxation followed by contraction. 6 In the bovine iris sphincter, it was reported that sotalol but not propranolol suppressed the muscle relaxation by isoproterenol, 13 but later it was found that propranolol (5 X 10~ 6 M) abolished the muscle relaxation evoked by the nerve stimulation in the presence of atropine. 14 In the present experiments, propranolol had not effect, yet timolol, a non-selective 0-adrenergic antagonist which is five to ten times more potent than propranolol, 15 abolished the relaxation of the muscle. The 01 -antagonist, atenolol, greatly reduced the amplitude of muscle relaxation in the presence of atropine, suggesting the presence of 0,-adrenoceptors in the dog iris sphincter. In the iris dilator muscle, there is considerable evidence for a-adrenergic excitatory innervation. 1 '" 16 ' 17 Cholinergic inhibitory innervations were found in rat, 17 cat, 6 human 1 ' and bovine dilator muscle. 10 Our study on the dog iris dilator muscle revealed a-excitatory and /3-inhibitory adrenoceptors, in addition to cholinergic inhibitory innervation. These results show that the iris sphincter and dilator muscles receive double reciprocal innervations by cholinergic and adrenergic nervous systems and which probably play physiological roles in miosis and mydriasis. However, the physiological significance of a x-excitatory adrenoceptors in the sphincter and /^-inhibitory adrenoceptors in the dilator is not clear. Mechanical Specificities of Iris Muscles The resting membrane potentials were about -57 mv and 50 mv in the sphincter and dilator, respectively, and excess-[k] 0 solutions depolarized the membrane, dose-dependently. However, exogenously applied carbachol or NE, in concentrations below 3 X 10~ 5 M did not modify the membrane potentials in either muscle tissue. Furthermore, we did not observe any change in the membrane potential of the smooth muscle cells of iris sphincter or dilator muscles with electrical field stimulations, using microelectrode or double sucrose-gap methods (unpublished observations by T. Yoshitomi and Y. Ito). Thus, electrical field stimulation may not produce excitatory or inhibitory junction potentials in dog iris muscles. In various smooth muscle cells, however, membrane depolarization or hyperpolarization is not a prerequisite for generation of contraction or relaxation. For example, in pulmonary or mesenteric arteries, low concentrations of NE induce contraction without depolarization of the membrane; this phenomena was termed pharmacomechanical coupling In guinea pig or cat trachea,

8 90 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / January 1986 Vol. 27 R.P. ^ Q iris sphincter -20 mv -20 R.P mv r-w iris dilator O atropine (10" 6 M) phentolamine (10~ 5 M) timolol(10~ 5 M) O atropine (1O" 6 M) phentolaminedo" M) f timolol(10 M) mm(k) Q mm(k) Fig. 9. Effects of excess-[k] 0 solutions on the resting membrane potential of the sphincter (A) and dilator muscles (B) in the absence (O) or presence of atropine (10~ 6 M) + phentolamine (10~ 5 M) + timolol (10~ 5 M) ( ). Each point indicates the mean value and vertical bars indicate 2 X S.D. (n = 9-48). activation of intrinsic non-adrenergic non-cholinergic inhibitory nerve fibers by electrical stimulation produces no change in the electrical membrane property of smooth muscle cells, yet does produce muscle relaxation. 20 In porcine or canine coronary arteries, ACh has no effect on membrane potential and resistance but does evoke contraction 21 or relaxation. 22 In smooth muscle cells, the sources of Ca ++ contributing to activation of contractile proteins are of extra- and intra-cellular origin. 19 In the present experiments, Ca-free (EGTA-containing) solution reduced the resting tension, and agonist-induced contractions were not observed in Ca-free solutions in either muscle tissue. This means that the resting tension of the muscle is directly led by the influx of Ca ++ and that the receptor operated calcium channel utilizes mainly extracellular Ca ++. These observations differ from those obtained with airway smooth muscle cells, where ACh or caffeine-induced contractions were largely unaffected in Ca-free (2 mm EGTA-containing) solution. 23 The [K]o-induced contractions or relaxations of dog dilator muscle are probably mainly due to NE or ACh released from nerve terminals by excess-[k] 0, as has been proposed for human and bovine dilator muscles. 10 " However, in dog sphincter muscles, excess- [K] o (77.1 and 118 mm) solution evoked phasic contraction in the presence of atropine, phentolamine, and timolol indicating that this response is due to membrane depolarization and suggesting the presence of voltage dependent Ca ++ channels in the sphincter muscle. Similarly, in rabbit iris sphincter, excess-[k] 0 solutions produced substance P-operated and voltage dependent muscle contractions. 24 Therefore, in smooth muscle cells of the dilator muscle, excess-[k] 0 might evoke minute and rapid contractions in the presence of atropine, phentolamine and timolol, and these contractions would not be detectable in the present recording system due to accompanying noise. The lack of response to agonists in Ca-free solution and the diminished sensitivity to excess-[k] 0 in the mechanical response of sphincter and dilator muscles of the dog iris indicate poorly developed Ca-store sites (mainly sarcoplasmic reticulum) and sparsely distributed, voltage-dependent Ca channels activated by excess [K] o in these muscle tissues, in comparison to other visceral smooth muscles. 19 Key words: iris sphincter muscle, iris dilator muscle, cholinergic innervation, adrenergic innervation, reciprocal innervations Acknowledgments We are grateful to Prof. H. Kuriyama for continuous encouragement and to M. Ohara for reading the manuscript. References 1. Nolte J: Iris and pupil. In Physiology of the Human Eye and Visual System, Records Raymond E, editor. Hagerstown, Harper &Row, 1979, pp Nomura T and Smelser GK: The identification of adrenergic and cholinergic nerve endings in the trabecular meshwork. Invest Ophthalmol 13:525, Nishida S and Sears M: Fine structural innervation of the dilator muscle of the iris of the albino guinea-pig studied with permanganate fixation. Exp Eye Res 8:292, Laties AM and Jacobowitz D: A histochemical study of the adrenergic and cholinergic innervation of the anterior segment of the rabbit eye. Invest Ophthalmol 3:502, Hutchins JB and Hollyfield JG: Autoradiographic identification of muscarinic receptors in human iris smooth muscle. Exp Eye Res 38:515, Schaeppi U and Koella WP: Adrenergic innervation of cat iris sphincter. Am J Physiol 207:273, van Alphen GWHM, Kern R, and Robinette S: Adrenergic re-

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