Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland

Abstract

Ca2+ transport was studied by using basolateral plasma membrane vesicles from rat parotid gland prepared by a Percoll gradient centrifugation method. In these membrane vesicles, there were two Ca2+ transport systems; Na+/Ca2+ exchange and ATP-dependent Ca2+ transport. An outwardly directed Na+ gradient increased Ca2+ uptake. Ca2+ efflux from Ca2+-preloaded vesicles was stimulated by an inwardly directed Na+ gradient. However, Na+/Ca2+ exchange did not show any 'uphill' transport of Ca2+ against its own gradient. ATP-dependent Ca2+ transport exhibited 'uphill' transport. An inwardly directed Na+ gradient also decreased Ca2+ accumulation by ATP-dependent Ca2+ uptake. The inhibition of Ca2+ accumulation was proportional to the external Na+ level. Na+/Ca2+ exchange was inhibited by monensin, tetracaine and chlorpromazine, whereas ATP-dependent Ca2+ transport was inhibited by orthovanadate, tetracaine and chlorpromazine. Oligomycin had no effect on either system. These results suggest that in the parotid gland cellular free Ca2+ is extruded mainly by an ATP-dependent Ca2+ transport system, and Na+/Ca2+ exchange may modify the efficacy of that system.

Characterization of Calcium Translocation across the Plasma Membrane of Primary Osteoblasts Using a Lipophilic Calcium-sensitive Fluorescent Dye, Calcium Green C18

“…Sealed inside-out vesicles took up Ca 2ϩ , implying that the direction of pumping in cells was outward, as has been found for numerous other cell types (Carafoli, 1987;Garrahan and Rega, 1990). The uptake rate by osteoblast plasma membrane vesicles was 9.9 Ϯ 2.3 nmol/mg of protein/min, which falls in the range reported for several other vesicle studies, including red blood cells (Caride et al, 1983), parotid gland (Takuma et al, 1985), neutrophils (Ochs and Reed, 1983), and liver (Kraus-Friedmann et al, 1982). Na ϩ / Ca 2ϩ exchange also exists in osteoblasts (Krieger, 1992;Short et al, 1994) and may contribute in a minor way to the Ca 2ϩ efflux observed, as subsequently discussed.…”

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

“…A previous report from our laboratory (Takuma et al, 1985) showed that inverted basolateral plasma membrane vesicles accumulate Ca 2+ in the presence of ATP. The Ca 2+ accumulated in the vesicles could be discharged by imposing an inwardly directed Na § gradient, indicating the presence of a Na § 2+ exchange mechanism in the same membrane vesicles and also confirming the plasma membrane origin of the ATP-dependent Ca 2+ transporter.…”

“…PREPARATION OF BASOLATERAL MEMBRANE VESICLES BLMV were prepared as described previously (Takuma et al, 1985), with some minor modifications. The procedure, in brief, was as follows.…”

“…The pellet obtained was rehomogenized (by hand) in the sucrose medium using a Teflon| homogenizer, mixed with Percoll | (12% vol/vol) and centrifuged at 41,700 x g for 30 min. The basolateral membrane fraction was collected (Takuma et al, 1985) and washed three times with 100 mM mannitol, 0.1 mg PMSF, 1 mM DTT, 10 mM Tris-HEPES, pH 7.5, by centrifuging at 49,000 x g for 15 rain. Membranes were finally washed in a medium containing 300 mM mannitol, 1 mM DTT, 10 mM Tris-HEPES, pH 7.5, and resuspended in a minimum volume of the same medium.…”

“…Moreover, oq-adrenoreceptor and muscarinic receptor activation, which result in considerable efflux of Ca 2+ and fluid secretion, elicit only small levels of protein secretion. It can therefore be suggested that cytosolic Ca 2+ homeostasis in these receptor-stimulated conditions would be established by the concerted activities of ATP-dependent Ca 2+ transport systems located in the endoplasmic reticulum (Kanagasuntheram & Teo, 1982;Immelmann & Soling, 1983) and in the basolateral plasma membrane (Takuma et al, 1985;Helman et al, 1986). The latter system is likely the key Ca2+-transporting mechanism mediating Ca 2+ efflux from this cell (Takuma et al, 1985).…”