抄録
A radioimmunoassay has recently been developed for the quantitation of opioid peptides such as methionine-enkephalin and leucine-enkephalin isolated from the adrenal medulla. However, the current assay system still has some problems including immunological cross-reactivity which is often encountered, as well as proteolytic degradation of the peptides during the extraction procedure from the tissue.We have performed a series of basic experiments to improve the assay system of the opioid peptides present in adrenal medulla by overcoming the problems described above, and applied the improved method to an in vitro analysis of enkephalin secretion from the adrenal medulla.
The present methionine-enkephalin assay system proved to be satisfactory with respect to intra-assay variation and recovery ratio, with minimal cross-reactivity with leucine-enkephalin. However, leucine-enkephalin assay system showed significant degree of cross-reactivity with methionine-enkephalin, which could be reduced from 20% to as low as 1% by oxydizing the samples with hydrogen peroxide before the test.
We have also shown that spontaneous degradation of enkephalins during the extraction from a tissue could be prevented by the inclusion of peptidase inhibitors such as amastatin, puromycin or phosphoramidon in the extraction procedure. Boiling of tissue homogenate in acetic acid also proved useful in preventing an eventual loss of enkephalin immuno-reactivity. On the contrary, extraction with sucrose solution or hydrochloric acid resulted in low immunoreactivity.
Finally, we examined the kinetics of opioid secretion from the adrenal medulla in vitro, using our improved assay method. A continuous flow incubation system was developed for this purpose, in which secretory response of adrenal medulla to nicotine was characterized by serial fluorimetric assay of catecholamines and a radioimmunoassay of enkephalins in the effluent medium. During the course of perifusion, the initial release of a large volume of catecholamines and enkephalins decreased in one hour and a half to the baseline level and remained at the level thereafter. In 2 hours after the start of perifusion, both catecholamines and enkephalins output rose abruptly after the addition of nicotine and returned promptly to the baseline after its withdrawal. Thus, we have shown that enkephalins were co-released from the adrenal medulla with catecholamines in response to nicotine by perifusion experiment.
