Characterization of the neurosecretory activity of hypothalamic beta-endorphin-containing neurons in primary culture.
To determine the neurosecretory activity of hypothalamic beta-endorphin (beta EP)-containing neurons, rat fetal hypothalamic cells were mechanically dispersed and maintained in primary cultures for periods up to 24 days; their electrophysiological properties and regulation by depolarization, calcium and sodium channel-active agents were studied. Under culture conditions, the majority of the cells were immunopositive to neurofilament antibody, and a significant number (7-10%) were reactive to beta EP antibody. Cultured cells were often electrically excitable and possessed voltage-activated ionic conductances. In culture, there was a progressive increase in immunoreactive beta EP (IR-beta EP) in both cells and media, reaching maximum values at 12-16 days. The majority of IR-beta EP in both cells and media corresponded to [125I]beta EP on gel chromatography and was similar to the form previously found in the hypothalamus. These findings suggest viability of the beta EP neurons and continuing synthesis of IR-beta EP during the culture period. To evaluate the influence of membrane depolarization on IR-beta EP release, the cells were challenged with 56 mM potassium. This treatment induced a significant increase in medium IR-beta EP. The depolarization-induced IR-beta EP release was dependent upon calcium, since a calcium channel blocker, verapamil (0.1 microM), prevented the release; also a calcium ionophore, A23187 (1 microM), stimulated IR-beta EP release in the cultures. Activation of the sodium channel by veratridine (100 microM) also increased the medium content of IR-beta EP, and this effect was blocked by tetrodotoxin (1 microM). These results suggest that the beta EP neurons in primary culture respond to the well defined physiological challenges and that the culture system can be used in determining the regulation of hypothalamic beta EP activity.
Sarkar
D K
DK
Department of Veterinary, Washington State University, Pullman 99164.
Sakaguchi
D S
DS
D000818:Animals; D002478:Cells, Cultured; D004594:Electrophysiology; D007031:Hypothalamus / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D009490:Neurosecretory Systems / Q000166:cytology / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology; D001615:beta-Endorphin / Q000378:metabolism*
D001615:beta-Endorphin; D014701:Veratridine; D011188:Potassium