Intracellular signal transduction systems involved in the release of luteinizing hormone-releasing hormone from the mediobasal hypothalamus of male rats

Abstract

Two signal transduction systems, adenylate cyclase (AC) and phosphoinositides (PI), have been implicated in the release of luteinizing hormone-releasing hormone (LHRH). A potential linkage between the systems may involve the conversion of diacylglycerol (DAG) to phosphatidic acid (PA) and its subsequent metabolism to arachidonic acid. Consequently, this study was conducted to: 1) validate of the use of rat mediobasal hypothalamic (MBH) slices in a perifusion culture system to study LHRH release and 2) investigate the involvement of and possible linkage between AC and PI in LHRH release. MBH from male rats were placed in 500 p.1 perifusion chambers and perifused at a rate of 25 μl/min with Krebs-Ringer bicarbonate media (5.5 mM glucose) at 37° C under an atmosphere of 95% 02:5% CO2- Effluent fractions were collected into tubes on ice over 30 min intervals. Treatment compounds were introduced between 210 and 330 min of perifusion, while control tissues were left untreated. LHRH content (pg/30 min) of the fractions was determined by validated radioimmunoassay. Area under the LHRH release curve was determined using the trapezoidal rule. Sliced MBH (4 MBH units/chamber) demonstrated greater (P<.05) depolarization-induced LHRH release when compared with intact MBH. All subsequent experiments utilized 3 sliced MBH per chamber. Tissues from adult male rats did not respond (P>.1) to either 60mM K+ or 3 μm PGE2 compared to controls. MBH tissues obtained from immature rates responded (P<.05) to 60 mM K+, but were unresponsive (P>.1) to 3 μM PGE2 treatment. However, a 30 μM dose of PGE2 induced (P<.01) LHRH release from the MBH of immature rates. Tissues sliced in a transverse plane released more (P<.01) LHRH in response to 60 mM K+ than those sliced in a sagittal plane. These data have indicated that the perifusion of sliced (transverse plane) MBH from immature male rats is a valid system to study intracellular transduction systems involved in LHRH release...