Characterisation of Somatostatin and TRH Release by Rat Hypothalamic and Cerebral Cortical Neurons Maintained on a Capillary Membrane Perfusion System

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Abstract
The nature of the immunoreactive somatostatin (SS) and TRH produced by long-term capillary perfusion cultures of rat fetal cortical and hypothalamic cells was investigated. Dispersed cortical and hypothalamic cells from 17-day-old fatal 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 capillay lumen at a rate of 1.5 ml/min and was changed 3 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 .times. 107 cells/10 min). This response was clearly Ca dependent since it was abolished during washes with Ca2+-free Krebs-Ringer bicarbonate solution. Affinity purified material from pooled neuronal perfusates showed 3 distinct peaks of somatotropin release-inhibiting factor (SRIF) immunoreactivity following polyacrylamide gel chromatography on Biogel-P10. The dominant form coeluted with synthetic tetradecapeptide-SS (SS-14) and a smaller amount (.hivin.c 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 MW of .apprx. 11,500 daltons. Hypothalamic and cortical perfusates produced similar electrophoretic patterns of immunoreactive SS. 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 [high performance liquid chromatography] of the affinity purified material showed similar retention times to synthetic TRH. The 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. 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.