Ion-induced release of LHRH, TRH and α-MSH from hypothalamic synaptosomes and its inhibition by vinblastine

Homogenates of rat hypothalamic tissue were fractionated by means of discontinuous sucrose density gradient centrifugation. Immunoreactive luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and α-melanocyte stimulating hormone (α-MSH) were found to be concentrated in the synaptosome-enriched fraction. This fraction was suspended in 0.32 M sucrose and the release of the three peptides was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration, and the concentration of each peptide in the ultrafiltrate was determined by radioimmunoassay. When the synaptosomal fraction was incubated at 30° C in 0.32 M sucrose containing either 60 mM K⁺-2 mM Ca²⁺ or 140 mM Na⁺ alone a release of LHRH, TRH, and α-MSH occurred. Of the total content 30-50% of LHRH but only about 10% of TRH and α-MSH was releasable. When the synaptosome preparation was preincubated for 30 min at 30°C with 10^-4 M vinblastine. K⁺- as well as Na⁺-induced release of LHRH, THR, and α-MSH was inhibited, and the stimulatory effect of each cation was almost totally blocked by preincubation with 5×10^-4 and 10^-3 M vinblastine. The inhibitory action of vinblastine (5×10^-4 M) did not affect the oxidation of glucose to CO₂ by the synaptosomes. The results of the present investigation demonstrate that synaptosome-enriched fractions of hypothalamic origin are more stable with respect to LHRH, TRH, and α-MSH release during incubation in isotonic sucrose than they are in ionic solutions, and that the peptides are released by a vinblastine-sensitive mechanism.