Release of radioimmunoassayable LHRH from hypothalamic fragments of adult male rats was measured using an in vitro incubation system. Each flask, containing three tissue fragments in 0.5 ml Krebs-Ringer bicarbonate glucose buffer, was incubated for 15 min, followed by a 30-min incubation during which either basal release or the effect of catecholamines on LHRH output was evaluated. In some cases, tissues employed consisted of a fragment of medial basal hypothalamus which included the median eminence (ME), the arcuate and ventromedial nuclei, and surrounding structures (MBH-ME unit). In other experiments, the ME was separated from the rest of the MBH by a rnicrodissection procedure and both fragments were incubated in separate flasks. Basal LHRH release from the different fragments was readily detectable, during both the preincubation and incubation periods. LHRH release from the ME, was 10 to 15-fold higher than that from the MBH or the MBH-ME unit. This increased release from the ME was not due to a different rate of degradation in the absence of the MBH, since release of LHRH by coincubates of ME and MBH fragments was similar to the combined release of ME and MBH incubated in separate flasks. The amount of LHRH released during a 30-min incubation represented 0.8% and 0.26% of the initial tissue content in ME and MBH fragments, respectively. Catecholamines (CAs), added in vitro at doses ranging from 0.6–600 μM, did not significantly alter LHRH output from the MBH-ME unit. However, when the ME was incubated in the presence of CAs, either norepinephrine (NE) or dopamine (DA) significantly stimulated LHRH release in a dose-related manner. The minimal effective dose for both CAs was μM. Epinephrine was ineffective at any of the doses tested. LHRH release from MBH fragments was stimulated only by the highest dose (600 μM) of either NE or DA. The specificity of the effect of the CAs was tested by the use of receptor blockers. Pimozide added in vitro (10-6 M) completely blocked the effect of DA on LHRH release from the ME, without altering the stimulatory action of NE. Conversely, addition of phentolamine (10-6 M) prevented the effect of NE but not that of DA. To further test the specificity of the DA effect on the ME, animals were pretreated with diethyldithiocarbamate, an inhibitor of dopamine β-hydroxylase, to prevent transformation of DA to NE. Under those circumstances, DA was still able to increase LHRH release. The effect of DA on LHRH release from the ME was even more evident if animals were treated with a-methyl p-tyrosine in addition to diethyldithiocarbamate to deplete endogenous stores of DA. These results indicate that LHRH terminals in the ME are sensitive to the stimulatory action of both NE and DA and suggest that both amines may play a modulatory role in LHRH release from this structure.
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