Release of somatostatin-like immunoreactivity from enriched enteric nerve varicosities of rat ileum

Abstract

Synaptosomes were isolated from rat ileum by various steps of differential centrifugation. The peptide content for somatostatin-like immunoreactivity was used as marker for neuronal membranes. The enriched synaptosomal fraction (P2) showed a good enrichment of somatostatin content (4-fold) in comparison to the post-nuclear supernatant. The basal release of somatostatin-like immunoreactivity was 26 ± 3 pg/mg tissue protein. KCl-evoked depolarization (65 mM) caused a significant increase of somatostatin-like immunoreactivity release (72 ± 11 pg/mg, n = 12, P < 0.001) compared to basal release. In Ca 2+-free medium the evoked release of somatostatin-like immunoreactivity was abolished. A substantial increase of somatostatin-like immunoreactivity release (52 ± 7 pg/mg, n = 12, P < 0.05) was also observed in the presence of the Ca 2+ ionophore A-23187. The cholinergic agonist carbachol elicited a dose-dependent release of somatostatin-like immunoreactivity (10 −7 M: 54 ± 8 pg/mg, 10 −6 M: 63 ± 6 pg/mg, 10 −5 M: 53 ± 5 pg/mg, n = 12, P < 0.001), which was blocked by atropine (10 −6 M: 35 ± 6 pg/mg, n = 12, P < 0.001), but not by hexamethonium. Other presynaptic modulating substances such as serotonin, the selective neurokinin-B agonist [βAsp 4,MePhe 7]neurokinin B-(4–10), neurotensin, cholecystokinin-8, caerulein and pentagastrin had no stimulatory effect on release of somatostatin-like immunoreactivity. In summary, somatostatin-like immunoreactivity can be released from enteric synaptosomes by both depolarization with KCl and cholinergic stimulation via a muscarinic mechanism. The synaptosomes of intrinsic nerves offer an approach to study release of neuronal somatostatin on the subcellular level.