We have investigated the nature of the immunoreactive somatostatin (SS) and thyrotropin-releasing hormone (TRH) produced by long-term capillary perfusion cultures of rat cortical and hypothalamic cells. Dispersed cortical and hypothalamic cells from 17-day-old fetal rats were injected on to the outer surfaces of separate capillary membrane perfusion systems. Recirculating nutrient medium (Minimum Essential Medium with added glucose, antibiotics and 10% fetal calf serum) was then perfused via the capillary lumen at a rate of 1.5 ml/min and was changed three times weekly. Medium reservoirs, gaseous exchange coils and capillary columns were maintained in a 95% air/5% CO2 environment with 100% humidity. After 6 and 12 days in continuous perfusion, both cortical and hypothalamic cells demonstrated immunoreactive SS release following 60 mM K+ depolarization (5- to 7-fold increase from basal secretion levels of 15-20 pg/3 x 107 cells/10 min). This response was clearly calcium dependent since it was abolished dring washes with Ca2+-free Krebs-Ringer bicarbonate solution. Affinity purified material from pooled neuronal perfusates showed three distinct peaks of somatotropin release-inhibiting factor (SRIF) immunoreactivity following polyacrylamide gel chromatography on Biogel-P10. The dominant form coeluted with synthetic tetradecapeptide-somatostatin (SS-14) and a smaller amount (c̄ 30%) coeluted with synthetic SS-28. (The SS-14 antibody used showed equimolar cross reactivity with SS-28). A larger form of immunoreactive material was also detected with an apparent molecular weight of about 11,500 daltons. Hypothalamic and cortical perfusates produced similar electrophoretic patterns of immunoreactive SS (ISS). Immunoreactive TRH-like material was also detected in pooled cortical and hypothalamic perfusates giving a calculated average basal secretion rate of 0.4-0.7 pg/107 cells/10 min. HPLC of the affinity purified material showed similar retention times to synthetic TRH. These data demonstrate that fetal hypothalamic and cortical cells can produce both SRIF and TRH in this capillary perfusion system over prolonged periods of time. This permits repeated acute and chronic studies on the same cells. In addition, SS-28 release by both cortical and hypothalamic cells supports the view that this peptide may be a functionally important component of somatostatinergic neuron secretion.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Endocrine and Autonomic Systems
- Cellular and Molecular Neuroscience