ABSTRACT
Cholinergic modulation of nociceptive transmission through both nicotinic and muscarinic receptors in the spinal cord represents an important mechanism in pain signaling. However, what neuronal elements release acetylcholine and how release might change in response to deafferentation are unclear. The present studies demonstrated Ca++- and K+-dependent release of [3H]-acetylcholine from slices of regional areas of rat spinal cord. That [3H]-acetylcholine was synthesized from [3H]-choline was demonstrated by the lack of [3H]-acetylcholine release following incubation with either the choline uptake inhibitor hemicholinium or the choline acetyltransferase inhibitor bromoacetylcholine. Rats treated neonatally with capsaicin or with spinal nerve ligation as adults showed a significantly decreased K+-stimulated release of [3H]-acetylcholine from dorsal horn but not ventral horn lumbar spinal cord slices. In rats subjected to dorsal rhizotomy, while basal release from lumbar dorsal spinal cord slices was reduced, K+-stimulated [3H]-acetylcholine release, while decreased, was not significantly different compared with controls. The data presented here show that there are regional differences in the release of acetylcholine from spinal cord and that this release can be modulated by chemical or surgical deafferentation. These results also indicate that the source of acetylcholine in the dorsal cord originates mainly from resident somata and their collaterals, interneurons and/or descending terminals, with only very minor contributions coming from primary afferents. The present data help to further elucidate the role of acetylcholine in spinal signaling, particularly with respect to the effects of nerve injury and nociceptive neurotransmission.
Subject(s)
Acetylcholine/metabolism , Pain/metabolism , Spinal Cord/metabolism , Acetylcholine/pharmacology , Afferent Pathways/injuries , Afferent Pathways/metabolism , Afferent Pathways/surgery , Animals , Capsaicin/pharmacology , Cholinergic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hemicholinium 3/pharmacology , Ligation , Male , Organ Culture Techniques , Pain/physiopathology , Rats , Rats, Sprague-Dawley , Rhizotomy , Spinal Cord/drug effects , Spinal Nerves/drug effects , Spinal Nerves/injuries , Spinal Nerves/surgeryABSTRACT
Many of the physiological hallmarks associated with neurogenic inflammatory processes in cutaneous tissues are similarly present within orofacial structures. Such attributes include the dependence upon capsaicin-sensitive sensory neurons and the involvement of certain inflammatory mediators derived therein, including calcitonin gene-related peptide (CGRP). However, there are also important differences between the trigeminal and spinal nervous systems, and the potential contributions of neurogenic processes to inflammatory disease within the trigeminal system have yet to be fully elucidated. We present here a model system that affords the ability to study mechanisms regulating the efferent functions of peptidergic terminals that may subserve neurogenic inflammation within the oral cavity. Freshly dissected buccal mucosa tissue from adult, male, Sprague-Dawley rats was placed into chambers and superfused with oxygenated, Krebs buffer. Serial aliquots of the egressing superfusate were acquired and analysed by radioimmunoassay for immunoreactive CGRP (iCGRP). Addition of the selective excitotoxin, capsaicin (10-300 microm), to the superfusion buffer resulted in a significant, concentration-dependent increase in superfusate levels of iCGRP. Similarly, release of iCGRP from the buccal mucosa could also be evoked by a depolarizing concentration of potassium chloride (50 mm) or by the calcium ionophore A23187 (1 microm). The specific, capsaicin receptor antagonist, capsazepine (300 microm), completely abolished the capsaicin-evoked release of iCGRP while having no effect whatsoever on the potassium-evoked release. Moreover, capsaicin-evoked release was dependent upon the presence of extracellular calcium ions and was significantly, though incompletely, attenuated by neonatal capsaicin denervation. Collectively, these data indicate that the evoked neurosecretion of iCGRP in response to capsaicin occurs via a vanilloid receptor-mediated, exocytotic mechanism. The model system described here should greatly facilitate future investigations designed to identify and characterize the stimuli that regulate the release of CGRP or other neurosecretory substances in isolated tissues. This system may also be used to elucidate the role of these mediators in the aetiology of inflammatory processes within the trigeminal field of innervation.
