In vitro release of [⁵Met] and [⁵Leu]-enkephalins from the rat brain crude synaptosomal (P₂) fraction: Ca²⁺-dependency of K⁺-stimulation and effects of various drugs

Abstract

The rat brain P₂ fraction was suspended in Krebs Ringer bicarbonate buffer containing 20 μM bacitracin and incubated at 37°C for 10 min under an atmosphere of 95% O₂-5% CO₂. Incubation was terminated by centrifugation at 4°C and 10, 000×g for 10 min. The supernatant was designated as the S₁ fraction, and from the pellet, the S₂ to S₄ fractions were collected by repeated suspension, incubation, and centrifugation. The radioimmunoassays of each S fraction revealed the spontaneous release of [⁵Met] and [⁵Leu]-enkephalins at the ratio of 2 to 1. The peptide contents gradually decreased from S₁ to S₄, but the release tended to become constant in S₃ and S₄. Thus, the effects of some ions and drugs on the release were compared at the stage of obtaining the S₃ fraction. The release of both peptides were significantly stimulated in 50 mM KCl buffer, and the stimulatory effect appears to be dependent on Ca²⁺ concentration. Veratrine and A23187 were also effective stimulants of the release. On the other hand, neither spontaneous nor K⁺-stimulated release of enkephalins was affected by morphine (1 μM), naloxone (1 μM), kyotorphin (1 or 10 μM), and Li⁺ (50 mM). Similar results were obtained with the release of ³H-noradrenaline taken up in vitro by the P₂ fraction. The usability of the P₂ fraction as an in vitro model for the study of stimulus-coupled release of enkephalins was discussed with some limitations found herein.