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Secretion of melanin-concentrating hormone and neuropeptide-EI from cultured rat hypothalamic cells
1Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037.
Abstract:
Rat melanin-concentrating hormone (MCH) is a homolog of the peptide first isolated from salmon pituitary glands which produces melanosome aggregation within melanophores of teleost fish as well as interacting with the hypothalamic-pituitary axis. We have previously characterized the rat and human MCH counterparts as identical 19-amino acid peptides showing approximately 70% peptide sequence identity to salmon MCH. Immunoreactivity for MCH has been found in high concentrations within cell bodies of the dorsolateral hypothalamus, with projections to the hippocampus, brainstem, posterior pituitary, and cerebral cortex. We have adapted a cultured cell model for studying MCH secretory responses of hypothalamic cells obtained from 7-day-old rats. MCH and the MCH precursor-derived peptide neuropeptide glutamic acid isoleucine (NEI) were secreted from these cells after 2 days of culture and for up to 22-24 days of culture. The secreted peptides were confirmed by HPLC analysis and RIA to be identical to MCH isolated from rat hypothalamic tissue and to the sequences of MCH and NEI predicted from the MCH precursor. MCH and NEI secretion was stimulated 3- to 5-fold by 8-bromo-cAMP and 8-bromo-cGMP. Dexamethasone produced a dose-dependent increase in cell content of both MCH and NEI and an increase in MCH secretion. The present study is the first to demonstrate the existence of the predicted peptide NEI in a biological system and indicates that cultured neonatal rat hypothalamic cells are a useful model for the study of MCH/NEI release in vitro.
Insights
Researchers developed a new in vitro model using neonatal rat hypothalamic cells to study melanin-concentrating hormone (MCH) and neuropeptide glutamic acid isoleucine (NEI) secretion. This model confirms NEI
Area of Science:
- Neuroendocrinology
- Peptide Hormone Research
- Cellular Signaling
Background:
- Melanin-concentrating hormone (MCH) is a peptide hormone involved in the hypothalamic-pituitary axis.
- Rat and human MCH are 19-amino acid peptides with sequence similarity to salmon MCH.
- MCH is found in high concentrations in the rat hypothalamus with projections to various brain regions.
Purpose of the Study:
- To establish and validate a cultured cell model for studying MCH and MCH precursor-derived peptide (NEI) secretion from rat hypothalamic cells.
- To investigate the secretion patterns and regulation of MCH and NEI in vitro.
- To confirm the existence of the predicted peptide NEI in a biological system.
Main Methods:
- Primary culture of hypothalamic cells from 7-day-old rats.
- High-Performance Liquid Chromatography (HPLC) and Radioimmunoassay (RIA) for peptide identification and quantification.
- Stimulation of peptide secretion using 8-bromo-cAMP, 8-bromo-cGMP, and dexamethasone.
Main Results:
- Cultured hypothalamic cells secreted MCH and NEI for up to 22-24 days.
- Secreted peptides were confirmed to be identical to rat hypothalamic MCH and NEI.
- MCH and NEI secretion was stimulated by cAMP and cGMP analogs, and dexamethasone increased cell content and MCH secretion.
Conclusions:
- Neonatal rat hypothalamic cells in culture provide a viable model for studying MCH/NEI release in vitro.
- This study provides the first evidence for the existence of the predicted peptide NEI in a biological system.
- The findings support the role of MCH and NEI in hypothalamic function and neuroendocrine regulation.