Processing of chromogranins in chromaffin cell culture: effects of reserpine and alpha-methyl-p-tyrosine.
Bovine chromaffin cell cultures were treated with either reserpine or alpha-methyl-p-tyrosine for up to 10 days. Afterwards the cells were harvested and the degree of proteolytic processing of secretogranin II, chromogranin A and chromogranin B was determined by immunoblotting and HPLC followed by RIA. There was a significant increase in the proteolysis of all three chromogranins after 4-6 days in the presence of reserpine. The small peptides formed in the presence of reserpine in vitro are also produced in vivo. A similar effect was observed with alpha-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, but the response took up to 10 days to develop. Both drugs decreased catecholamine levels but reserpine was more effective, reaching a high degree of depletion after 4 days. In addition, experiments in vitro indicate that low millimolar amounts of either adrenaline (IC50 5.2 mM) or noradrenaline (IC50 2.4 mM) can significantly impair the proteolytic activity of recombinant murine prohormone convertase 1 when assayed with synthetic fluorogenic and/or peptidyl substrates. We conclude that a lowering of catecholamine levels in chromaffin granules leads to a concomitant increase in proteolytic processing of all secretory peptides. Apparently within chromaffin granules the endoproteases are inhibited by catecholamines and thus their removal leads to increased proteolysis.
Wolkersdorfer
M
M
Department of Pharmacology, University of Innsbruck, Austria.
Laslop
A
A
Lazure
C
C
Fischer-Colbrie
R
R
Winkler
H
H
D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D053379:Chromogranin A; D002864:Chromogranins / Q000096:biosynthesis* / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004837:Epinephrine / Q000494:pharmacology; D008781:Methyltyrosines / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000494:pharmacology; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification; D012110:Reserpine / Q000494:pharmacology*; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors; D019805:alpha-Methyltyrosine
D053379:Chromogranin A; D002864:Chromogranins; D004791:Enzyme Inhibitors; D008781:Methyltyrosines; D011506:Proteins; D019805:alpha-Methyltyrosine; D012110:Reserpine; D014446:Tyrosine 3-Monooxygenase; D009638:Norepinephrine; D004837:Epinephrine