Effects of acetylcholine and carbachol on the iris sphincter

We compared the effects of acetylcholine and carbachol on iris sphincters. The muscle strips (mostly bovine tissues) were isolated and incubated in a 0.2ml organ bath. Results revealed an approximately ten-thousandfold difference in potency between carbachol and acetylcholine, in the bovine iris. Acetylcholine and/or endogenous chemical agents were spontaneously released from bovine tissue. Acetylcholinesterase activity in this tissue strongly inhibited the acetylcholine action, in order to protect the nerve terminals from random depolarization due to the released acetylcholine. Among several species (bovine, rabbit, hamster), Ach action was the least in the bovine iris sphincter muscle.     

Desensitization of the isolated bovine iris sphincter muscles by pilocarpine

We investigated the effect of pilocarpine on the bovine iris sphincter. This muscle contracted in a dose-dependent manner from 2 × 10^–6 M, with a maximal response at 3 × 10^–3 M. The ED₅₀ values was (1.6 ± 0.3) × 10^–4M. Pilocarpine generated a desensitization while desensitization was not significant in the case of other cholinergic agents such as acetylcholine (Ach) and carbachol. Desensitization was profoundly increased in the presence of Ach, neostigmine or eserine: the responses to the second and third trials of pilocarpine were reduced to approximately 8–10% or 30% of the corresponding first responses. Pilocarpine reportedly releases transmitters and alters choline-acetyltransferase activity.These results taken together suggest that either variable Ach synthesis, inhibitory transmitter release or possible toxic action in high concentrations may be involved in the pilocarpine-induced responses of the bovine iris sphincter muscle. The desensitization and parital agonist-antagonist action of pilocarpine could not explain the characteristics of the bovine iris sphincter muscle.     

Gene transfer by adenovirus in rabbit iris sphincter muscle

The effect of insertion of an exogenous gene on smooth muscle function in rabbit iris sphincter muscle was investigated. An adenoviral vector encoding the bacterial LacZ gene (AdLacZ, 10(7) pfu) and viscoelastics were injected into the posterior chamber of eyes of albino rabbits. Three days after injection, the effects of acetylcholine (Ach), carbachol (Carb), substance P (SP) and electrical field stimuli on isolated iris sphincter were investigated using isometric tension-recording methods. X-Gal histostaining showed that iris sphincter smooth muscle cells were transfected in 7 of 11 muscle strips. Contraction-response curves for Ach, Carb or SP were not different from control. We conclude that the iris sphincter muscle can be gene-transfected by posterior chamber infusion of an adenoviral vector with viscoelastics. Adenovirus-mediated gene transfer per se had no measurable effect on tension development.     

Direct effects of muscarinic agents on the outflow pathways in human eyes

PURPOSE: Recent studies demonstrating the presence of muscarinic receptors and contractile-like cells in the trabecular meshwork tissue and/or cell cultures from human eyes suggest the possibility that there may be a direct effect of muscarinic agonists on outflow facility. The present studies were conducted to determine whether muscarinic agonists could change outflow facility in perfused human ocular anterior segments, which lack an intact ciliary muscle. METHODS: Human eyes were dissected and perfused according to previously described methods. A steady state baseline facility was established for 90 minutes, after which up to four sequential concentrations ranging from 10(-9) to 10(-3) M of pilocarpine, aceclidine, or carbachol were added to the perfusion medium. In other studies, 10(-6) M atropine was perfused alone followed by 10(-7) M carbachol with 10(-6) M atropine, whereas fellow control eyes received carbachol alone. Outflow facility was measured for 60 minutes after each drug addition. The outflow facility measurement in each eye after drug administration was compared with the baseline measurement. RESULTS: Outflow facility increased from baseline facility in eyes treated with pilocarpine, aceclidine, or carbachol at lower concentrations (10(-9) to 10(-6) M) but remained unchanged at higher concentrations (10(-4) to 10(-2) M). The effects of carbachol at 10(-7) M were completely blocked by atropine. CONCLUSIONS: Muscarinic agonists increase outflow facility in human eyes by a direct stimulation of the outflow tissues in the absence of an intact ciliary muscle. This effect is biphasic, occurring at concentrations of 10(-6) M and lower with no effect at higher concentrations.     

Autoradiographic identification of muscarinic receptors in human iris smooth muscle

We have used the specific, irreversible muscarinic ligand [³H]-propylbenzilylcholine mustard to localize putative muscarinic cholinergic receptors in the smooth muscle tissue of the human iris. Analysis of autoradiograms from labeled irides reveals high grain densities over the iris sphincter muscle, consistent with the well-known pharmacology of this muscle. In addition, a smaller but significant population of muscarinic binding sites was seen in the iris dilator muscle as well. Grain densities in both muscles are substantially reduced in control tissue treated with relatively high concentrations of the muscarinic antagonists quinuclidinyl benzilate (QNB) and atropine. This is, to our knowledge, the first report of autoradiographic localization of putative muscarinic receptors in the human iris.     

Double reciprocal innervations in dog iris sphincter and dilator muscles

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 alpha 1-excitatory (mediating contractions) and alpha 2-inhibitory (mediating relaxations) adrenoceptors in addition to beta-inhibitory adrenoceptors in the sphincter muscles, and alpha-excitatory and beta-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. Ca-free 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.     

Pre-synaptic actions of noradrenaline on the dog ciliary muscle tissue

Effects of noradrenaline (NA) on the electrical and mechanical properties of the dog ciliary muscle were investigated using microelectrode- and isometric tension recording methods. Electrical-field stimulation to the tissue evoked excitatory junctional potentials (e.j.ps) followed by twitch contractions. Both responses were abolished by tetrodotoxin (TTX, 10(-7) M) or atropine (10(-6) M), thereby indicating that these responses are cholinergic-related. Exogenously applied NA (greater than 10(-8) M) suppressed the amplitude of e.j.ps and the following twitch contraction evoked by field stimulations, with no change in the resting tension, membrane potential and input membrane resistance of the smooth muscle cells. Exogenously applied carbachol dose-dependently contracted the ciliary muscle. However, NA (10(-5) M) did not affect the carbachol-induced contraction, thereby indicating that NA acts on the nerve terminals and reduces the amount of transmitter released from the terminals. Inhibitory effects of NA on the twitch contraction were antagonized by yohimbine (10(-6) M), but not by prazosin (10(-6) M) or timolol (10(-6) M). Guanethidine (5 X 10(-6) M) had no effect on the contraction evoked by field stimulation. These results provide evidence that exogenously applied NA activates the alpha 2-adrenoceptor located on cholinergic nerve terminal, hence the excitatory neuro-effector transmission is suppressed. These actions may explain the decrease in near point of accommodation observed with clinical application of adrenergic agents.     

Endothelin-1 stimulates phosphatidylinositol hydrolysis in the iris/ciliary complex and is a potent constrictor of the sphincter muscle.

Endothelin-1 (0.5-10 nM) produced a concentration-related contraction of the isolated iris sphincter muscle. The contraction rate of the muscle was slower for endothelin than carbachol, but endothelin was also more potent than carbachol, although the maximum contraction size was greater for carbachol. The endothelin response was unaffected by atropine whereas the carbachol effect was abolished. Endothelin-1 and carbachol stimulate the accumulation of inositol phosphates in a concentration-related way. The endothelin response was unaffected by atropine, prazosin or ketanserin, but the carbachol effect was specifically antagonized by atropine. It is suggested that activation of endothelin-1 receptors to stimulate phosphatidylinositol hydrolysis may be the initial phase in the contraction response of the iris sphincter muscle.     

Effect of pituitary adenylate cyclase-activating peptide on isolated rabbit iris sphincter and dilator muscles

PURPOSE: Pituitary adenylate cyclase-activating peptide (PACAP) is a sensory neuropeptide in the eye that is released by noxious stimuli and considered to be a mediator of the neurogenic ocular injury response, including miosis. The purpose of this study was to clarify the functional role of PACAP in iris sphincter and dilator muscles. METHODS: Iris sphincter and dilator muscles were isolated from rabbit eyes, and the effect of PACAP on mechanical responses of these muscles using isometric tension-recording methods was investigated. RESULTS: The iris sphincter responded to electric field stimulation with contractions composed of fast twitch and subsequent slow components. Both PACAP 27 and PACAP 38 enhanced the twitch response, but neither had an effect on the slow response. The effect of both PACAPs on the twitch response was dose dependent. Neither PACAP had an effect on the amplitude of contraction evoked by exogenously applied Ach. For the iris dilator muscle, PACAP 27 inhibited the contractions induced by field stimulation or phenylephrine, whereas PACAP 38 had no effect. CONCLUSIONS: Both PACAP 27 and PACAP 38 enhance cholinergic transmission in sphincter muscle. The PACAP 27 induces relaxation of the dilator muscle by a direct effect on the muscle itself. The PACAP released during an ocular inflammatory response may induce miosis by the enhancement of cholinergic stimulation of the iris sphincter and by direct relaxation of the dilator muscles.     

The response of the isolated iris sphincter muscle of various mammalian species to substance P

Contractile responses of the isolated iris sphincter muscle from the rabbit, cow, pig, cat, dog, baboon and man to substance P were compared under isotonic conditions using carbachol as a reference standard. Iris preparations from the rabbit, pig and cow showed strong and consistent responses to substance P whilst those from the cat, dog, baboon and human eyes did not contract.     

Effect of somatostatin and galanin on isolated rabbit iris sphincter and dilator muscles

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 μm) but had no effect on responses mediated by substance P. Galanin was more effective (attenuation of 43% at 0.1 μm) compared with somatostatin (attenuation of 16% at 0.1 μm) 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.     

Action of biologically active peptides on monkey iris sphincter and dilator muscles

Biologically active peptides modulate pupillary responsiveness in many non-primate mammals. We examined the action of seven different peptides on iris sphincter and dilator muscles of rhesus monkey. Iris sphincter and dilator muscle preparations from rhesus monkeys were mounted in an organ bath, and tension changes were recorded by an isometric transducer. Electrical field stimulation (100Hz, 0.3 msec, 10V) was applied through a pair of platinum plate electrodes. Monkey iris sphincter and dilator muscles produced simple cholinergic and adrenergic excitatory responses respectively to electrical field stimulation. Strong field stimulation did not elicit slow Substance P (SP) mediated contractions like those in rabbit iris sphincter. Exogenously applied pituitary adenylate cyclase-activating peptide (PACAP) enhanced in a concentration-dependent manner (0.3 nM-0.1 microm) the sphincter response to field stimulation, while neuropeptide Y (NPY) and somatostatin (SRIF) attenuated it. These three peptides did not affect sphincter contractions induced by acetylcholine, and therefore were acting at presynaptically. SP, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL) had no effect (at 0.1 microm) on iris sphincter. None of seven exogenously applied peptides had an effect on monkey iris dilator muscle. The innervation of primate irises may be relatively simple compared to non-primates because each of the peptides in this study can modulate miosis or mydriasis in non-primate mammals. Future studies will be expected on the functional significance of species differences in iridial innervation.     

Response of bovine intra-ocular muscles to transmural stimulation in the presence of various prostaglandins

Effects of prostaglandins (PGs) E1, E2 and F2 alpha on the electrically stimulated and non-stimulated smooth muscles of bovine intra-ocular tissues were investigated in vitro. PGs E1, E2 and F2 alpha contracted the sphincter and these contractions were not antagonized by either cholinergic or adrenergic blocking agents. PGF2 alpha was much weaker than PGE1 or PGE2. PGs in high concentrations produced an irreversible contracture of the sphincter muscle. In contrast, the dilator muscle contracted with PGF2 alpha, but not PGE1 or PGE2. The ciliary muscle did not respond to these PGs or to indomethacin. Electrical field stimulation with short pulses produced excitation of the intrinsic nerves. The responses produced were not altered in amplitude by the application of any dose of PGs or indomethacin, although indomethacin did lower the tone of the sphincter. PGs did not influence the amplitude of the acetylcholine-induced responses of the intra-ocular muscles. These results indicate that in each of the bovine intra-ocular muscles, PGs play a minor role in neuromuscular transmission both prejunctionally and postjunctionally. Thus, PG-induced contractions of the bovine intra-ocular muscles are considered to occur by a direct action on the muscle rather than on the nerves.     

Effects of acetylcholine and norepinephrine on incorporation of [32P]orthophosphate into phospholipids of rabbit iridial processes and iris smooth muscle

We have investigated: (a) phospholipid composition, inclusive of higher inositides, of rabbit iridial processes and iris smooth muscles; (b) ³²P_i incorporation into their respective phospholipids, and (c) the effects of muscarinic cholinergic and adrenergic agonists and antagonists on ³²P labelling of phospholipids of the iridial processes and iris smooth muscle. (1) Phosphatidylcholine, phosphatidylethanolamine, their respective plasmalogens and sphingomyelin, were found to be the major phospholipids in the iridial processes and iris smooth muscle. They constituted about 85% of the total phospholipids of these ocular tissues. (2) Both iridial processes and iris smooth muscles rapidly incorporated ³²P_i and [1-¹⁴C]-arachidonic acid into their respective phospholipids, however this incorporation amounted to only 20% of that found for the whole iris-ciliary body. This could suggest a metabolic interrelationship between the iridial processes and the smooth muscle of the iris. (3) Addition of acetylcholine and norepinephrine to the iridial processes and iris smooth muscle increased ³²P labelling of phosphatidic acid and phosphatidylinositol of the tissues. The increase in phospholipid labelling was higher in the iridial processes as compared to the iris smooth muscle. The effect of acetylcholine was blocked by atropine and that of norepinephrine was blocked by phentolamine and prazosin but not by yohimbine. This suggests that the observed effects of these neurotransmitters on phospholipid phosphorylation in the iridial processes and iris smooth muscle are mediated through muscarinic cholinergic and α₁-adrenergic receptors, respectively. The data presented provide additional support for the concept that in the iris-ciliary body the neurotransmitter-induced ³²P labelling of phosphoinositides is probably linked to the functional activities of this tissue.     

The effects of physostigmine on the response characteristics of the cat visual evoked potential

Steady—state pattern visual evoked potentials were recorded from the surface of the cat primary visual cortex before and after the intravenous administration of physostigmine, an agent that blocks the enzyme responsible for the breakdown of synaptically released acetylcholine. Under pentobarbital anesthesia, physostigmine increased the amplitude and changed the phase of the second response harmonic of the visual evoked potential, whereas the amplitude and phase of the fourth harmonic were not affected. These effects persisted for 15 to 45 minutes and were blocked by prior treatment with scopolamine or atropine. In addition, scopolamine or atropine administered 5 to 10 minutes after physostigmine returned the visual evoked potential to the baseline state. In comparison, when nitrous oxide was used, physostigmine caused a marked reduction in visual evoked potential amplitude, an effect that was reversed by subsequent atropine. These results indicate that the cholinergic system influences the visual evoked potential via a muscarinic pathway and that this influence is strongly affected by the anesthetic regimen used.Abbreviations Ach acetylcholine - LGN lateral geniculate nucleus     

Effects of β antagonists on mechanical properties in rabbit ciliary artery

· Background: We set out to clarify the mechanisms involved in the effectiveness of β-adrenergic receptor antagonists on the ocular circulation. · Methods: The effects of three β antagonists, timolol, betaxolol and carteolol, on the isolated rabbit ciliary artery were investigated in vitro using isometric tension recording methods. · Results: Phenylephrine dose-dependently contracted ciliary artery smooth muscle, and bunazosin (1 μM) shifted this dose-response curve to the right. Isoproterenol, on the other hand, had no effect up to the concentration of 1 mM. Betaxolol and timolol had no effect on the ciliary artery. However, carteolol dose-dependently contracted this muscle from a concentration of 1 μM. After precontraction by excess-[K]₀ solutions, application of betaxolol or timolol dose-dependently provoked relaxation; the minimum concentration of betaxolol or timolol required to generate the relaxation was 100 μM and 300 μM, respectively. Carteolol did not generate relaxation at concentrations up to 1 mM. After pretreatment with L-NAME (300 μM), the amplitude of relaxation induced by 10 μM carbachol was reduced to 33.0±20.2%, while betaxolol- or timolol-induced relaxation was unchanged. Diltiazem (10 μM) induced relaxation which was not inhibited by pretreatment with L-NAME. · Conclusion: Betaxolol and timolol could directly relax rabbit ciliary artery in vitro at relatively high concentrations, and relaxation was not due to NO released from the preparation. Presumably, this relaxation occurs through action similar to Ca antagonists. However, the clinical importance of this effect is not yet clear. Carteolol had no relaxant effect in vitro. Received: 20 October 1998 Revised version received: 4 January 1999 Accepted: 5 January 1999     

Effects of prostaglandin F2alpha, latanoprost and carbachol on phosphoinositide turnover, MAP kinases, myosin light chain phosphorylation and contraction and functional existence and expression of FP receptors in bovine iris sphincter

A potential role for myosin light chain kinase (MLCK) in regulating intraocular pressure and outflow function has recently been reported in living monkey eye and rabbit eye. There is little information about the effects of the ocular hypotensive agents, prostaglandin F2alpha (PGF2alpha) and latanoprost on this signaling pathway in ocular tissues. The aim of this study was to determine the agonist activity of PGF2alpha, latanoprost and carbachol (CCh) on the MLCK pathway in isolated bovine iris sphincter and furthermore to investigate the existence of the FP receptor in this tissue. In the present studies on the MLCK pathway four signal transduction mechanism assays were employed, phosphoinositide (PI) turnover, p42/p44 MAP kinase phosphorylation and activation, MLC phosphorylation and contraction. In the studies on the existence of the FP receptor in the bovine iris sphincter, the pharmacology and expression of the FP receptor protein, using a polyclonal anti-FP-receptor antibody and Western blot analysis, were determined. The data obtained on the MLCK pathway showed that the three agonists stimulated the biochemical and pharmacological responses in a concentration and time-dependent manner and that the order of potency and efficacy is PGF2alpha>latanoprost>CCh. The EC50 values in the PI turnover, MAP kinase phosphorylation, MLC phosphorylation and contraction assays were for PGF2alpha: 9, 42, 200 and 140 nM, respectively, for latanoprost: 13, 59, 250 and 828 nM, respectively, and for CCh: 22, 200, 630 and 910 nM, respectively. Wortmannin, a selective inhibitor of MLCK, dose-dependently inhibited MLC phosphorylation and contraction induced by PGF2alpha, demonstrating a close relationship between activation of the MLCK pathway and contraction. The pharmacological studies showed that in the concentration range of 1 nM to 10 microM, the FP-receptor agonists caused concentration-response curves with the following order of potencies: 17-phenyl trinor PGF2alpha (bimatoprost acid)>PGF2alpha>cloprostenol>latanoprost>latanoprost acid>bimatoprost amide>fluprostenol. Immunoblot analysis of the FP receptor demonstrated expression of the prostaglandin FP receptor protein in this smooth muscle. These results clearly indicate that the MLCK signaling pathway is involved in the FP-receptor function of the bovine iris sphincter and furthermore demonstrate that functional FP receptors exist and are expressed in this tissue.     

Changes in isolated ciliary muscle caused by repeated instillation of carbachol ointment in rabbits and effect of topically applied amlexanox

PURPOSE: We investigated changes in ciliary muscle caused by continual contraction in rabbits and evaluated the efficacy of topically applied amlexanox, which relaxes the ciliary muscle, on such changes. SUBJECTS AND METHODS: After topical application of carbachol ointment 5 times daily for 2 weeks, the contractile responses of isolated ciliary muscles to carbachol were measured isometrically, and the ciliary smooth muscle fibers were stained with phosphotungstic acid and hematoxylin and observed histologically. 1% amlexanox solution was instilled 5 minutes before every instillation of carbachol ointment. RESULTS: Repeated topical carbachol ointment caused decreases in both contractile responses of isolated ciliary muscles to carbachol and number of ciliary smooth muscle fibers stained by phosphotungstic acid and hematoxylin. Amlexanox inhibited these changes. CONCLUSION: We found that continual contraction of the ciliary muscle caused functional and histological changes in it. These changes are thought to occur in some diseases which cause excessive contraction of the ciliary muscle. Topical amlexanox might be useful for these diseases.     

Role of prostaglandins in the cooling-induced constriction of the iris sphincter

Effects of prostaglandins and/or indomethacin on the cooling induced contraction of the bovine iris sphincter muscle were studied in vitro. Cooling of the bathing solution contracted the bovine iris sphincter and the muscle became sensitive to carbachol. Lowering the temperature elevated the muscle tone, while with pretreatment of indomethacin, the elevation was markedly inhibited. Further application of prostaglandins (PGs) such as 10(-8)-3 X 10(-8) M PGE2 reproduced the response induced by low temperature. Effects of cimetidine, methysergide and naloxone were also studied. Whether PGs are exogenously applied or endogenously produced, PGs are responsible for the induction of such responses.     

电针干预对透镜诱导型近视豚鼠视网膜和视皮层中BDNF、NGF及AchE表达的影响

目的 探讨电针干预对透镜诱导型近视(LIM)豚鼠视网膜和视皮层中脑源性神经营养因子(BDNF)、神经生长因子(NGF)及乙酰胆碱酯酶(AchE)表达变化的影响.方法 将90只2周龄雄性豚鼠随机分为正常对照(NC)组、LIM组和LIM+电针治疗(LIM+EA)组.NC组豚鼠正常饲养,不进行干预;LIM组和LIM+EA组豚...