Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

Publisher Website
Google Scholar
Abstract
Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction (SF2) was a highly purified preparation of rough endoplasmic reticulum (RER); the less-dense fraction (SF1) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was .apprx. 6.8 for the RER (SF2) and .apprx. 7.5 for SF1. Initial rate measurements were used to determine the affinity of the RER uptake system for free Ca2+. An apparent Km of 0.16 .+-. 0.06 .mu.M and Vmax of 21.5 .+-. 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 .mu.M-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 .mu.M-carbamylcholine, 45Ca2+ uptake was increased in the RER. The increased uptake was due to a higher Vmax with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1. In conclusion, 2 distinct nonmitochondrial, ATP-dependent Ca-uptake systems were demonstrated in rat pancreatic acini. One of these is located in the RER, but the precise location of the other has not been determined. The Ca2+-transporting activity in the RER may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.