M3-Type Muscarinic Receptors Predominantly Mediate Neurogenic Quick Contraction of Bovine Ciliary Muscle

• 1.The present experiments were designed to investigate which subtypes of muscarinic receptors are involved in the neurogenic quick contraction of bovine ciliary muscle in connection to quick eye focal accommodation.
• 2.Transmural electrical stimulation (TES) produced a transient contraction, which was abolished in the presence of 3×10⁻⁷ M tetrodotoxin and 10⁻⁶ M atropine, but greatly augmented by 3×10⁻⁷ M physostigmine.
• 3.The exogenously applied acetylcholine (ACh: 10⁻⁹ to 3×10⁻⁶ M) produced a concentration-dependent contraction, which was competitively antagonized by 10⁻⁶ M atropine and augmented by 3×10⁻⁷ M physostigmine, but unaffected by 3×10⁻⁷ M tetrodotoxin.
• 4.The magnitude and time to peak of the maximal contraction produced by TES were significantly greater (1267.5±86.0 mg, P<0.005) and shorter (9.0±0.2 sec, P<0.005) than corresponding values (97.0±9.9 mg and 20.3±2.1 sec, respectively) of the phasic contraction caused by exogenously applied 10⁻⁵ M ACh, at which concentration the agonist caused the maximal contraction. The velocity (140.6±7.8 mg/sec) of the transient contraction caused by TES was approximately 28-fold greater than that of the phasic contraction caused by ACh (5.1±0.9 mg/sec).
• 5.The contractions produced by TES were greatly attenuated by 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) as an M₃ antagonist and slightly by pirenzepine as an M₁ antagonist (20.2±7.9% inhibition at the highest concentration), but not by methoctramine (MET) as an M₂ antagonist. The IC₅₀ value (−log M) for 4-DAMP was determined to be 7.17±0.14.
• 6.Scatchard plot analysis of [³H]-quinuclidinylbenzilate (QNB) binding revealed that the binding sites constituted a single population with a K_d of 31.2±0.8 pM and a B_max of 895.5±93.2 fmol/mg protein. The activity in inhibiting [³H]-QNB binding was most potent with 4-DAMP (-log K_i=7.98±0.02), but less potent with pirenzepine (−log K_i=6.43±0.04) and MET (−log K_i=7.32±0.16). 4-DAMP was approximately 35- and 5-fold more potent than pirenzepine and MET in terms of −log K_i values, respectively, suggesting the predominant localization of M₃ receptor subtypes in the bovine ciliary muscle membrane.
• 7.These results suggest that TES produces a neurogenic quick contraction of the bovine ciliary muscle, which would be mediated mainly by ACh released from the intramural nerve terminals and subsequent excitation of M₃ receptor subtypes localized on the ciliary muscle cells, and that neurogenic quick contraction of the ciliary muscle is possibly involved in part in eye focal accommodation.