[3H]saxitoxin as a marker for canine deep muscular plexus neurons

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Abstract
The objectives of this study are 1) to examine the potential of [3H]saxitoxin binding as a marker for the neuronal membranes in canine small intestinal muscle membrane preparations, 2) to develop a synaptosomal preparation from deep muscular plexus, and 3) to partially characterize [3H]saxitoxin binding to this fraction. A purified synaptosomal fraction, relatively low in the smooth muscle plasma membrane marker enzyme 5''-nucleotidase but enriched in [3H]saxitoxin binding (2,592 fmol/mg), was obtained on sucrose density gradient. Vasoactive intestinal peptide immunoreactivity was also highest (51.82 pmol/mg protein) in this fraction. The binding was rapid at 20.degree. C with quick and complete dissociation after the addition of excess unlabeled tetrodotoxin (TTX). Scatchard analysis of the saturation binding data revealed a single population of binding sites (Bmax = 5,705 fmol/mg protein). The affinity constants calculated from the kinetic and saturation data were in close agreement (Kd = 0.26 and 0.69 nM, respectively). TTX completed for the binding (Ki = 2.1 nM), whereas veratridine and guanidinium hydrochloride did not. Monovalent and divalent cations had differential effects on the binding.