RELEASE OF BOMBESIN-LIKE IMMUNOREACTIVITY FROM SYNAPTOSOMAL MEMBRANESISOLATED FROM THE RAT ILEUM

In the enteric nervous system, direct effects on peptidergic neurotran smitter release are difficult to assess since the neuronal network pre disposes to numerous interactions between the various transmitter syst ems. The aim of the present study was to examine the release of bombes in-like immunoreactivity from isolated nerve synapses of the enteric n ervous system. Enriched synaptosomal fractions were obtained by using homogenized tissue from rat ileum, which was subjected to various step s of differential and sucrose density centrifugation. Specific binding of [H-3]saxitoxin served as a marker for neuronal membranes. For comp arison, 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 discon tinuous sucrose density centrifugation, showed substantial enrichment of the neuronal marker [H-3]saxitoxin and bombesin-like immunoreactivi ty. The basal release of bombesin-like immunoreactivity was 52 +/- 17 pg/mg (100%). KCl-evoked depolarization (65 mM) significantly stimulat ed the release of bombesin-like immunoreactivity to 142.2% (P < 0.05, n = 17). The release was abolished in Ca2+-free medium. Stimulation of the release of bombesin-like immunoreactivity was also observed in th e 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 rele ase 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 wa s reduced by atropine (10(-6) M: 99%, P < 0.01, n = 14). The basal val ue was 67 +/- 9 pg/mg (100%). The different effects of the muscarinic M(1) receptor antagonist pirenzepine, which stimulated release of bomb esin-like immunoreactivity in combination with carbachol 10(-6) M (10( -6) M: 123%, n = 10), and of the muscarinic M(2) receptor antagonist A FDX 116, which attenuated release of bombesin-like immunoreactivity ev oked by carbachol(10(-5) M: 66%, P < 0.01, 10(-6) M: 88%, n = 10), str ongly suggest modulation of the release of bombesin-like immunoreactiv ity at the presynaptic receptor site through an excitatory muscarinic M(2) 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 Ca2+-dependent manner and by cho linergic stimulation. The synaptosomes of intrinsic nerves of the gut offer an approach to study the release of neuropeptides and neurotrans mitters at the subcellular level independent of the ganglionic network .