Release of immunoreactive alpha-MSH by synaptosome-enriched fractions of homogenates of hypothalami

Abstract

Immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be concentrated in a synaptosome-enriched fraction prepared by differential centrifugation of rat hypothalamic homogenates. The release of the hormone from this preparation was investigated. After incubation, the synaptosomes were isolated by ultrafiltration and alpha-MSH in the ultrafiltrate was determined by radioimmunoassay. Particle-bound alpha-MSH, recovered by extraction with acid ethanol, and alpha-MSH released from the synaptosome preparation, were immunologically similar to synthetic alpha-MSH and had an accompanying melanotropic activity. Less than 10% of the particle-bound alpha-MSH was released during incubation in 0.32 M sucrose. However, in the presence of 2 mM Ca2+, alpha-MSH release increased with increasing concentrations (30-150 mM) of K+. The stimulatory effect of 60 mM K+ was complete within 2 min and was potentiated by increasing Ca2+ concentrations over the range of 0 to 2 mM. K+-induced release of alpha-MSH was independent of temperature from 1 to 30 degrees C, and neither glucose (10 mM) nor dopamine (10(-10)-10(-2) M) had any effect on the release of the peptide. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains a releasable pool of immunoreactive alpha-MSH that is mobilized by depolarizing concentrations of K+ in a Ca2+-dependent manner.