活性アミンのACTH放出に及ぼす影響:その2, in vitroでのACTH放出活性と下垂体前葉のcyclic 3′, 5′ adenosine monophosphate含量に及ぼす効果との相関について

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In the first report, we demonstrated that the biogenic amines noradrenaline (NA), dopamine (DA) and serotonin (5HT), can act directly on the rat anterior pituitary to increase ACTH release in vitro, using a revised in vitro method for the assay of corticotropin releasing factor (CRF) activity developed in our laboratory. Cyclic 3', 5' adenosine monophosphate (cyclic AMP) has been implicated as a mediator of many hormonal actions, including the effect of hypothalamic releasing hormones on the release of pituitary hormones. Therefore, ACTH release induced by biogenic amines in vitro may be mediated through cyclic AMP. Based on these considerations, we studied the effect of biogenic amines on the cyclic AMP content in the rat anterior pituitary and considered the correlation of ACTH release and cyclic AMP increase stimulated in vitro by biogenic amines. Furthermore, we also considered the correspondence between the changes in the two parameters induced by some amine inhibitors and dexamethasone (DX). After two paired rat hemipituitaries were preincubated in 2 ml of Krebs-Ringer bicarbonate buffer (KRB) containing 0.2% glucose and 10-2M theophylline at 37°C for 30min. under a moist atmosphere of 95% 02 and 5% CO2, the medium was replaced with 2 ml of fresh medium containing the test substances and ascorbic acid (1 mg/ml) to prevent oxidative degradation of amines. Incubation was then carried out for an additional 15 min. Paired controls were employed throughout. The incubation was terminated by freezing the incubated pituitaries on alminium foil immersed in dry ice and acetone. Frozen tissues were homogenized in ice cold 6% trichloroacetic acid and denatured protein was removed by centrifugation. Precipitates were dissolved in 1 N NaOH and assayed for protein by Lowry's method8). Supernatants were washed with cold water-saturated ether and cyclic AMP was determined by the competitive protein binding assay of Gilman6) using the binding protein obtained from rat liver according to the method of Kumon et al.5) or by the radioimmunoassay of Steiner et al.7) Both methods gave comparable values using the same tissue. The results were expressed as nanomoles/gram of tissue protein or percentage of paired control. The system and method for measuring ACTH releasing activity were the same as described previously1). The paired comparison Student's t test was used for statistical analysis. The following results were obtained : 1) NA increased cyclic AMP content in the pituitary at a concentration of 4 × 10-4M which was the same concentration at which it stimulated ACTH release from the pituitary in vitro. However, it was ineffective at 4 × 10-5M, which was also the case in stimulating ACTH release. Furthermore, when reserpine pretreated rat pituitaries were used, (17 hrs before decapitation, 1 mg/rat, i.p.) a concentration of 4 × 10-5M of NA, which was capable of stimulating ACTH release, also increased pituitary cyclic AMP content. These effects of NA on both pituitary ACTH release and cyclic AMP content were inhibited by the β blocker propranolol, but not by the a blocker phentolamine. In spite of addition from the preincubation period, DX had no effect on either increasing ACTH release or cyclic AMP in response to NA. 2) 5HT increased both pituitary ACTH release and cyclic AMP at a concentration of 3.3 × 10-4M. Even at a lower concentration (3.3 × 10-5M), 5HT stimulated the ACTH release significantly and was also found to tend to increase the cyclic AMP content to 145% of that of the control although this was not significant. These effects of 5HT on both pituitary ACTH release and cyclic AMP content were inhibited by the same concentration of the serotonergic blocker methysergide,
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活性アミンのACTH放出に及ぼす影響:その2, in vitroでのACTH放出活性と下垂体前葉のcyclic 3′, 5′ adenosine monophosphate含量に及ぼす効果との相関について

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