ENDOPLASMIC-RETICULUM CA2-TRANSPORT OF PROINSULIN IN THE PANCREATIC BETA-CELL( IS IMPORTANT FOR THE PROTEOLYTIC PROCESSING AND INTRACELLULAR)

The role of intracellular Ca2+ in the proteolytic processing and intra cellular transport of secretory granule proproteins was investigated b y pulse-chase radiolabelling of isolated rat islets of Langerhans. The conversion of proinsulin was inhibited by depletion of medium Ca2+ wi th EGTA and by blocking the transport of Ca2+ into cells with the Ca2-channel antagonists verapamil, nifedipine and NiCl2. Proinsulin conve rsion was also reduced by the endoplasmic reticulum Ca2+-ATPase inhibi tor thapsigargin, indicating that the process requires transport of Ca 2+ into the endoplasmic reticulum. This was supported by the finding t hat proinsulin processing was inhibited when Ca2+ was depleted before or during pulse-labelling, but not after transport of the protein to p ost-endoplasmic-reticulum compartments. Similarly, the inhibition of p roinsulin processing was reversed by re-introduction of medium Ca2+ ar ound the time of radiolabelling, but not after 15 min of chase incubat ion. Ca2+ depletion also decreased proteolytic maturation of the proho rmone convertases PC1, PC2 and carboxypeptidase H. Secretion experimen ts suggested that the rate and extent of proinsulin transport into sec retory granules were inhibited marginally by Ca2+ depletion, whereas t hose of the convertases were markedly impeded. Inhibition of proinsuli n conversion by Ca2+ depletion was thus not simply related to the Ca2-dependencies of mature PC1 and PC2, but also to a requirement for end oplasmic reticulum Ca2+ in proteolytic maturation of the convertases a nd in their transfer to secretory granules. The results also suggest t hat the Ca2+ required for prohormone processing in the granules enters the secretory pathway via the endoplasmic reticulum.