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3H-noradrenaline release from mouse iris–ciliary body: role of presynaptic muscarinic heteroreceptors

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Abstract

Sympathetic neurotransmitter release and its modulation by presynaptic muscarinic heteroreceptors were studied in mouse iris–ciliary bodies. Tissue preparations were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. Firstly, experimental conditions were defined, allowing study of presynaptic sympathetic inhibition in mouse iris–ciliary body. If tissue was stimulated four times with 36 pulses/3 Hz, tritium overflow peaks were reliably and reproducibly measured. As expected, these stimulation conditions led to marked α2-autoinhibition as indicated by the release-enhancing effect of the α2-antagonists phentolamine and rauwolscine. To ensure autoinhibition-free 3H-noradrenaline release, which is optimal for studying presynaptic sympathetic inhibition, α2-receptors were blocked in all subsequent experiments. Under these conditions, evoked tritium overflow was almost completely abolished in the presence of the sodium channel blocker tetrodotoxin, indicating a neuronal origin of 3H-noradrenaline release. Secondly, muscarinic inhibition of 3H-noradrenaline release was characterized using the conditions described above (36 pulses/3 Hz; phentolamine 1 μM and rauwolscine 1 μM throughout). The muscarinic receptor agonist oxotremorine M decreased evoked tritium overflow in a concentration-dependent manner with an IC50 of 0.33 μM and maximal inhibition of 51%. The concentration–response curve of oxotremorine M was shifted to the right by the muscarinic antagonists ipratropium and methoctramine, whereas pirenzepine was ineffective. The observed rank order of antagonist potencies, ipratropium > methoctramine > pirenzepine, which is typical for the M2 subtype, indicates that presynaptic muscarinic receptors on sympathetic axons of mouse iris–ciliary bodies are predominantly M2. Finally, inhibition of 3H-noradrenaline release by endogenously secreted acetylcholine was investigated. Longer pulse trains, 120 pulses/3 Hz and 600 pulses/5 Hz, were used and the cholinesterase inhibitor physostigmine was added to the superfusion medium to increase synaptic levels of endogenous acetylcholine. Under these conditions, ipratropium approximately doubled the evoked overflow of tritium, indicating that endogenously released acetylcholine can activate presynaptic muscarinic heteroreceptors. In conclusion, the present experiments establish measurement of the electrically induced release of 3H-noradrenaline from mouse iris–ciliary bodies. As in other species, noradrenaline release in this preparation was subject to presynaptic muscarinic inhibition. Our results also indicate that the presynaptic muscarinic receptors on sympathetic axons in mouse iris–ciliary body are predominantly M2. Moreover, these receptors can be activated by both exogenous agonists and endogenously released acetylcholine and, hence, may operate physiologically in the interplay between the parasympathetic and sympathetic nervous system.

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Authors and Affiliations

  1. DA Ophthalmology, Novartis Institutes for BioMedical Research, PO Box WSJ-386.746, 4002, Basel, Switzerland

    Michel Bernhard, Caroline Keller, Mirko Haslebacher, George N. Lambrou & Anne-Ulrike Trendelenburg

  2. CNRS UMR7034, Université Louis Pasteur, 67404, Illkirch, France

    Michel Bernhard & Kenneth Takeda

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  1. Michel Bernhard
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  2. Kenneth Takeda
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  3. Caroline Keller
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  5. George N. Lambrou
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  6. Anne-Ulrike Trendelenburg
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Correspondence to Anne-Ulrike Trendelenburg.

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Bernhard, M., Takeda, K., Keller, C. et al. 3H-noradrenaline release from mouse iris–ciliary body: role of presynaptic muscarinic heteroreceptors. Naunyn-Schmiedeberg's Arch Pharmacol 370, 305–313 (2004). https://doi.org/10.1007/s00210-004-0972-z

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