Release of bombesin-like immunoreactivity from synaptosomal membranes isolated from the rat ileum.

Abstract

In the enteric nervous system, direct effects on peptidergic neurotransmitter release are difficult to assess since the neuronal network predisposes to numerous interactions between the various transmitter systems. The aim of the present study was to examine the release of bombesin-like immunoreactivity from isolated nerve synapses of the enteric nervous system. Enriched synaptosomal fractions were obtained by using homogenized tissue from rat ileum, which was subjected to various steps of differential and sucrose density centrifugation. Specific binding of [3H]saxitoxin served as a marker for neuronal membranes. For comparison, the content of bombesin-like immunoreactivity was determined. Both the enriched synaptosomal fraction (mitochondrial fraction II or P2) and the purified synaptosomal fraction (F2), obtained after discontinuous sucrose density centrifugation, showed substantial enrichment of the neuronal marker [3H]saxitoxin and bombesin-like immunoreactivity. The basal release of bombesin-like immunoreactivity was 52 +/- 17 pg/mg (100%). KCl-evoked depolarization (65 mM) significantly stimulated the release of bombesin-like immunoreactivity to 142.2% (P < 0.05, n = 17). The release was abolished in Ca(2+)-free medium. Stimulation of the release of bombesin-like immunoreactivity was also observed in the presence of the Ca2+ ionophore A-23187 (10(-6) M: 129%, P < 0.05, n = 17), supporting the role of Ca2+ in the release process. Cholinergic stimulation with carbachol elicited a significant dose-dependent release of bombesin-like immunoreactivity (10(-8) M: 106%, 10(-7) M: 175%, P < 0.05, 10(-6) M: 156%, P < 0.05, 10(-5) M: 115%, n = 14), which was reduced by atropine (10(-6) M: 99%, P < 0.01, n = 14). The basal value was 67 +/- 9 pg/mg (100%). The different effects of the muscarinic M1 receptor antagonist pirenzepine, which stimulated release of bombesin-like immunoreactivity in combination with carbachol 10(-6) M (10(-6) M: 123%, n = 10), and of the muscarinic M2 receptor antagonist AFDX 116, which attenuated release of bombesin-like immunoreactivity evoked by carbachol (10(-5) M: 66%, P < 0.01, 10(-6) M: 88%, n = 10), strongly suggest modulation of the release of bombesin-like immunoreactivity at the presynaptic receptor site through an excitatory muscarinic M2 receptor. The basal value was 46 +/- 9 pg/mg (100%). In summary, bombesin-like immunoreactivity can be released from these synaptosomes by both depolarization with KCl in a Ca(2+)-dependent manner and by cholinergic stimulation. The synaptosomes of intrinsic nerves of the gut offer an approach to study the release of neuropeptides and neurotransmitters at the subcellular level independent of the ganglionic network.