Studies on nitric oxide in autonomic neurotransmission

Abstract

The aim of the present thesis was to study nitric oxide (NO) as a mediator in autonomic neurotransmission in the enteric nervous system, including studies on the mechanism for neuronal release of NO, as well as the effects of NO on intestinal smooth muscle and enteric neurones. Furthermore, the signal transduction pathway for the evoked effects of NO and nitrergic neurotransmission were investigated. Nerve-induced NO/NO2- release in isolated guinea-pig colon was quantified by chemiluminescence technique in order to identify mechanisms involved in neuronal NO release. A considerable part of the nerve-mediated NO release likely results from muscarinic M1 receptor activation. Hence, M1 receptor activation was shown to evoke a smooth muscle relaxation in rat small intestine, which was demonstrated to be mediated via a nerve-dependent nitrergic mechanism. The dependence on vesicular exocytosis for nerve-induced NO release was investigated by use of botulinum toxin B (BTX B), which is known to cleave the synaptic vesicle protein synaptobrevin/VAMP and inhibit calcium-mediated exocytotic release of neurotransmitters. Pre-treatment with BTX B blocked the exatatory cholinergic and tachykininergic neurotransmission as well as the acetylcholine release in the guinea-pig colon, whereas the inhibitory nitrergic neurotransmission and nerve-induced NO/NO2 release were unaffected. This indicates that neuronal release of NO is non-vesicular. The nitrergic neurotransmission was shown to constitute a substantial portion of the inhibitory neurotransmission, evoking smooth muscle relaxation in guinea-pig colon. The NO-dependent relaxation was shown to be mediated exclusively through the soluble guanylyl cyclase transduction pathway. NO may also modulate excitatory cholinergic and tachykininergic neuroeffector responses in guinea-pig ileum. The inhibitory modulation by NO was to a large extent a postjunctional effector-modulation on the smooth muscle, but a prejunctional neuromodulation may also exist. In addition to the inhihtory effect of NO in intestine, NO was shown to evoke an excitatory response resulting in smooth muscle contraction in the guinea-pig small intestine. The contractile response to NO was likely mediated via activation of excitatory cholinergic and tachykininergic neurones, via a pathway utilising the soluble guanylyl cyclase. An additional inhibitory transmission, operating in parallel with NO, was also shown to evoke smooth muscle relaxation in guinea-pig colon. This transmission had a more rapid onset in comparison with the nitrergic transmission, but was independent of the soluble guanylyl cyclase transduction pathway. Similar to NO, this principle seemed independent of synaptic vesicular exocytosis. In conclusion, the present study lends support to the hypothesis that NO is released from enteric neurones upon nerve-activation as a consequence of direct nerve depolarisation as well as via M1 muscarinic receptor activation. Nerve-mediated NO release in itself appears to be independent of synaptic vesicular exocytosis, but may be influenced by other vesicularly stored transmitters. Evoked effects by NO in intestine include smooth muscle relaxation, modulation of excitatory neurotransmission as well as excitatory effects on intestinal smooth muscle through neuronal activation. Thus, NO is proposed as a neurotransmitter with possible dual effects on gastrointestinal motility.