Reserpine-induced alterations in the processing of proenkephalin in cultured chromaffin cells. Increased amidation.

I Lindberg

doi:10.1016/s0021-9258(18)66568-8

Abstract

We have used antisera directed towards eight different portions of the proenkephalin molecule to examine the processing rates and patterns of proenkephalin-derived peptides in chromaffin cell cultures in the presence and absence of reserpine. Reserpine treatment produced profound effects on the molecular weight profile of nearly all enkephalin-containing peptides. Increased production of low molecular weight immunoreactive [Met5]enkephalin, [Leu5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, and [Met5]enkephalin-Arg6-Phe7 was observed in reserpine-treated cultures; immunoreactivity corresponding to several intermediate sized enkephalin-containing peptides such as Peptide B and the high molecular weight [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive peptide was decreased. The production of two amidated opioid peptides, amidorphin and metorphamide, was greatly accelerated in the presence of reserpine. The increased levels of low molecular weight enkephalins could not be accounted for by assuming decreased basal release. These results indicate that reserpine treatment is able to increase the extent of post-translational processing of proenkephalin within chromaffin cells.

Highlights

We have used antisera directed towards eigdhiftfer- immunoreactive [Met5]enkephalin, Eidenet al. [6] have sugent portions of the proenkephalin molectuoleexamine gested that reserpine is able to increase the production of the processing rates and patterns ofproenkephalin- [Met'lenkephalin from proenkephalin
Opioid peptides of the enkephalinfamily are widely distrib- rapidly increases the levels of a variety of smaller proenkeuted within the central and peripheral nervous system and phalin-derived peptides, some of which require further postare thought to participateas neurotransmitters/neuromodu- translational processing such as amidation
Immunoreactivitycorrespondingto [Leu'lenkephalin and [Met'lenkephalin was eluted at the position expected for the medium was examined by removing medium from the cells at varying authentic pentapeptides

Summary

MATERIALS AND METHODS

Preparation of Chromffin Cell Cultures-Chromaffin cell cultures were prepared frombovine adrenalglands(obtained from a local slaughterhouse) following the method of Wilson et 01. [8] with the following modifications. Wilson et al [4,5]first demonstrated that reserpine treat- with warm 0.5% collagenase (Sigma,Type 1A) in place of mechanical ment of cultured adrenal chromaffin cells is able to increase perfusion; fresh collagenase was used each time. The collagenasedigestion was terminated by washing the cells three times with Locke's solution tially attributed to an increased biosyntohfepsrios enkephalin containing 2% bovineserumalbumin Following the Percoll gradient step, the dissociated cells were shown that reserpine treatment of chromaffin cell cultures washed three times withLocke'ssolution and were finallyresuseffectively decreases levels of proenkephalin mRNA. Based pended in a minimal volume of Dulbecco's modified Eagle's medium on their findinogf increased amountsof low molecular weight (Gibco) containing 10% heated fetal calf serum (Gibco), 10 mM HEPES,' 40 mg/liter gentamycin, 10 p~ cytosine arabinoside, 100. In order to compare the profile of immunoreactive enkephalins present

RESULTS

DISCUSSION

A Chronic Reserpine