A SUBCELLULAR POOL OF HYPO‐OSMOTICALLY RESISTANT PARTICLES CONTAINING THYROTROPIN RELEASING HORMONE, α‐MELANOCYTE STIMULATING HORMONE, AND LUTEINIZING HORMONE RELEASING HORMONE IN THE RAT HYPOTHALAMUS

Abstract— A 10% homogenate of male rat hypothalami, prepared in 0.32M‐sucrose‐10μM‐CaCl₂, was diluted either with one volume of 0.32M‐sucrose‐10μM‐CaCl₂ (iso‐osmotic) or with 10μM‐CaCl₂ (hypo‐osmotic). A 900 g supernatant fluid fraction (0.9 K‐S) was prepared from the diluted homogenates and fractionated on continuous sucrose density gradients under non‐equilibrium and equilibrium conditions. In the iso‐osmotic 0.9 K‐S thyrotropin releasing hormone (TRH), α‐melanocyte stimulating hormone (α‐MSH), and luteinizing hormone releasing hormone (LHRH) were each found to be sequestered in two populations of particles which were different in size but similar in density. In the hypo‐osmotic 0.9 K‐S, TRH, α‐MSH, and LHRH were each found to be sequestered in a single population of particles. In their sedimentation properties (as judged by differential, non‐equilibrium, and equilibrium density centrifugations), the hypo‐osmotically resistant particles and the small particles present in the iso‐osmotic 0.9 K‐S were identical. However, in their peptide content, the two sets of particles differed from each other. If the total quantity of particle‐bound peptides recovered after gradient centrifugation of the iso‐osmotic 0.9 K‐S is taken as 100%, one finds that the amount of TRH, α‐MSH. and LHRH recovered in the small particles is 39%, 50%, and 39%. respectively, whereas the amount of TRH, a‐MSH, and LHRH recovered in the hypo‐osmotically resistant particles is 42%, 68%, and 67%, respectively. This increase in the quantity of peptides sequestered in the small particles occurred concomitantly with the disappearance of peptides from the large synaptosome‐like particles. It is estimated that within the large synaptosome‐like particles 7% of the TRH, 35% of the a‐MSH, and 45% of the LHRH are associated with hypo‐osmotically resistant particles. Ultrastructural analysis of purified hypo‐osmotically resistant particles containing TRH. α‐MSH. or LHRH revealed a predominance of membrane‐bounded packets of electron‐dense material.