Demonstration of a temperature-dependent association of thyrotropin releasing hormone, α-melanocyte stimulating hormone, and luteinizing hormone releasing hormone with subneuronal particles in hypothalamic synaptosomes

A. Barnea, W. B. Neaves, G. Cho, J. C. Porter

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Abstract

Thyrotropin releasing hormone (TRH), α-melanocyte stimulating hormone (α-MSH), and luteinizing hormone releasing hormone (LHRH) exist in two subcellular pools in hypo-osmotically shocked homogenates of rat hypothalami prepared at 4°C: a stable pool of particle-bound peptides and a labile pool of soluble peptides. In the present study, we investigated the possibility that within the labile pool these peptides are associated with subneuronal particles (SNP) in such a manner that dissociation occurs under hypo-osmotic conditions at 4°C, whereas a stable association is maintained at 25°C. Adult male rat hypothalami were homogenized in 10 vol of 0.32 M-sucrose-10 μM-CaCl2. Hypo-osmotic shock of the homogenate was performed by dilution with an equal volume of 10 μM-CaCl2, whereas non-shocked controls were diluted with 0.32 M-sucrose-10 μM-CaCl2. A 900 g supernatant fluid was prepared from the diluted homogenates and fractionated on continuous sucrose density gradients. The amount of immunoreactive TRH, α-MSH, and LHRH sequestered in hypo-osmotically stable SNP (peptide-SNP complexes) were determined and compared to the total particle-bound peptides recovered from the gradients after fractionation of the non-shocked control homogenates. In their sedimentation properties and ultrastructural profiles, the peptide-SNP complexes prepared at 25°C were similar to those prepared at 4°C. However, in peptide content, a marked difference was noted between peptide-SNP complexes prepared at 25°C and at 4°C. The peptide-SNP complexes prepared at 25°C contained 82% of the total particle-bound TRH, 101% of the α-MSH, and 87% of the LHRH. On the other hand, peptide-SNP complexes prepared in an identical manner but at 4°C contained only 42% of the TRH, 68% of the α-MSH, and 67% of the LHRH. The difference between the amounts of peptides associated with SNP at 25°C and at 4°C was taken as an indication of the existence of TRH-, α-MSH-, and LHRH-SNP complexes which are stable under hypo-osmotic conditions at 25°C but which are solubilized at 4°C. It is suggested that these temperature-sensitive peptide-SNP complexes comprise the labile pool of TRH, α-MSH, and LHRH in hypothalamic synaptosomes.

Original languageEnglish (US)
Pages (from-to)1125-1134
Number of pages10
JournalJournal of Neurochemistry
Volume31
Issue number5
StatePublished - Dec 1 1978

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ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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