Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine.
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; 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 approx. 6.8 for the rough endoplasmic reticulum ( SF2 ) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM 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 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. 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, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum 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.
Project description:Rat parotid gland homogenates were fractionated into mitochondrial, heavy microsomal and light microsomal fractions by differential centrifugation. ATP-dependent 45Ca2+ uptake by the subcellular fractions paralleled the distribution of NADPH-cytochrome c reductase, an enzyme associated with the endoplasmic reticulum. The highest rate of Ca2+ uptake was found in the heavy microsomal fraction. Ca2+ uptake by this fraction was dependent on the presence of ATP and was sustained at a linear rate by 5 mM-oxalate. Inhibitors of mitochondrial Ca2+ transport had no effect on the rate of Ca2+ uptake. Na+ and K+ stimulated Ca2+ uptake. At optimal concentrations. Na+ stimulated Ca2+ uptake by 120% and K+ stimulated Ca2+ uptake by 260%. Decreasing the pH from 7.4 to 6.8 had little effect on Ca2+ uptake. The Km for Ca2+ uptake was 3.7 microM free Ca2+ and 0.19 mM-ATP. Vanadate inhibited Ca2+ uptake; 60 microM-vanadate inhibited the rate of Ca2+ accumulation by 50%. It is concluded that the ATP-dependent Ca2+ transport system is located on the endoplasmic reticulum and may play a role in maintaining intracellular levels of free Ca2+ within a narrow range of concentration.
Project description:The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.
Project description:The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 microM intracellular) of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 microM intracellular) of GTP-S- or guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 microM) prevented the inhibition of Ca2+ inflow by GTP-S- and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4(-) (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5))3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.
Project description:A method is described for preparing isolated rat submandibular acini by collagenase digestion followed by mechanical dispersion. As assessed by Trypan Blue exclusion, phase contrast microscopy, ATP content and release of mucins and lactate dehydrogenase, the acini are morphologically and functionally intact. Secretory function of isolated acini was similar to that of intact tissue in terms of time-course, dose dependence and degree of stimulation of mucin release by adrenergic secretagogues. Mucin release was increased to the same extent (approx. 3-4-fold) by either isoproterenol or noradrenaline at a maximally effective concentration (10 microM). Stimulation of mucin release by isoproterenol (10 microM), noradrenaline (10 microM) or adrenaline (10 microM) was inhibited by propranolol (30 microM) but not by phentolamine (30 microM). Isoproterenol (10 microM) increased both 45Ca2+ uptake and efflux from the acini, which was shown to represent a net release of calcium. However, there was a delay (approx. 10 min) in onset of stimulation of 45Ca2+ mobilization which was not apparent in isoproterenol stimulation of mucin release. Our results indicate that increases in intracellular calcium mobilization in response to a beta-adrenergic secretagogue do not trigger mucin secretion from rat submandibular acini.
Project description:Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], arising from hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], is proposed as the link between membrane-receptor activation and mobilization of Ca2+ from intracellular sites in hormone-secreting cells. The location of Ins(1,4,5)P3-sensitive membranes was investigated in cultured neonatal beta-cells. Membranes were obtained after lysis of cells attached to positively charged Sephadex. After lysis the presence of the enzyme markers 5'-nucleotidase, glucose-6-phosphatase, NADH-cytochrome c reductase, UDP-galactosyltransferase and succinate dehydrogenase indicated the mixed nature of the preparation. After sonication, however, UDP-galactosyltransferase and succinate dehydrogenase activities were undetectable, but 4.8% of total cellular glucose-6-phosphatase and 3.4% of total cellular NADH-cytochrome c reductase remained with 5'-nucleotidase in the preparation, indicating endoplasmic-reticulum association. ATP-dependent 45Ca2+ accumulation was shown in this preparation (410 +/- 24 pmol/mg of protein at 150 nM free Ca2+) and was inhibited by vanadate (100 microM). Ca2+ release was effected by Ins(1,4,5)P3, with half-maximal release at 0.5 +/- 0.14 microM-Ins(1,4,5)P3, t1/2 11.2 +/- 1.1 s. GTP- and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG)-promoted release of 45Ca2+ was demonstrated in this preparation, but the kinetics of release (half-maximal Ca2+ release at 5.4 +/- 0.7 microM, with t1/2 77.3 +/- 6.9 s, and at 51.1 +/- 4.2 microM, with t1/2 19.0 +/- 2.2 s, for GTP and p[NH]ppG respectively), and the ability of neomycin sulphate to block p[NH]ppG-induced release only, are indicative of separate release mechanisms after treatment with these agents. A close association between plasma membrane and elements of the endoplasmic reticulum is indicated in this model, providing a possible mechanism for local alterations in free Ca2+ in the sub-plasma-membrane region.
Project description:In single, Fura 2-loaded RBL-2H3 cells, antigen and thapsigargin depleted the same intracellular pool of Ca2+ in the absence of external Ca2+; provision of external Ca2+ induced immediate increases in levels of free Ca2+ ([Ca2+]i). These increases were dependent on the presence of external Ca2+ and, presumably, on influx of Ca2+ across the cell membrane. Both stimulants enhanced intracellular accumulation of 45Ca2+ through ostensibly similar mechanisms because accumulation was blocked to similar extents by various multivalent cations or by depolarization with K+. Because thapsigargin blocked reuptake of Ca2+ into inositol 1,4,5-trisphosphate sensitive stores, uptake occurred independently of the refilling of these stores. Uptake was dependent instead on sequestration of 45Ca2+ in a pool of high capacity that was insensitive to thapsigargin, caffeine, GTP and inositol 1,4,5-trisphosphate but sensitive to ionomycin and mitochondrial inhibitors. The existence of an inositol 1,4,5-trisphosphate-insensitive pool was also apparent in permeabilized cells; at 0.1 microM [Ca2+]i, uptake of 45Ca2+ was largely confined (> 80%) to the inositol 1,4,5-trisphosphate-sensitive pool, but at 2 microM [Ca2+]i uptake was largely (> 60%) into the inositol 1,4,5-trisphosphate-insensitive pool. Provision of mitochondrial inhibitors along with thapsigargin to block uptake into both pools, did not impair the thapsigargin-induced increase in [Ca2+]i or influx of Ca2+, as indicated by changes in Fura 2 fluorescence, but did block the intracellular accumulation of 45Ca2+. The studies illustrate the utility of simultaneous measurements of [Ca2+]i and 45Ca2+ uptake for a full accounting of Ca2+ homoeostasis as exemplified by the ability to distinguish between influx and mitochondrial uptake of Ca2+.
Project description:Depletion of the Ins(1,4,5)P3-sensitive intracellular Ca2+ store of vascular endothelial cells after selective inhibition of the endoplasmic-reticulum (ER) Ca2+ pump by thapsigargin or 2,5-di-t-butylhydroquinone (BHQ) increases Ca2+ influx from the extracellular space in the absence of phosphoinositide hydrolysis. One model to account for these results suggests a close association between the internal store and the plasmalemma, allowing for the vectorial movement of Ca2+ from the extracellular space to the ER. Furthermore, recent evidence suggests that Ins(1,4,5)P3-induced Ca2+ release from intracellular stores is regulated by the free cytosolic Ca2+ concentration ([Ca2+]i). Thus agonist-induced Ca2+ entry may directly regulate Ca2+ release from internal stores. To test these hypotheses, we examined the effect of 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF 96365), an inhibitor of Ca2+ influx, on unidirectional 45Ca2+ efflux (i.e. retrograde radioisotope flux via the influx pathway) and on [Ca2+]i as measured by fura-2. Bradykinin produced a transient increase in [Ca2+]i, reflecting release of Ca2+ from internal stores, and a sustained increase indicative of Ca2+ influx. In the absence of agonist, 45Ca2+ efflux was slow and monoexponential with time. Addition of BK dramatically increased 45Ca2+ efflux; 50-60% of the 45Ca2+ associated with the cell monolayer was released within 2 min after addition of bradykinin. Both the bradykinin-induced change in [Ca2+]i and the stimulation of 45Ca2+ efflux was completely blocked by loading the cells with the Ca2+ chelator BAPTA. At a supermaximal concentration of bradykinin (50 nM), SKF 96365 (50 microM) inhibited the rise in [Ca2+]i attributed to influx without affecting release from internal stores. At a threshold concentration of bradykinin (2 nM), SKF 96365 blocked influx, but stimulated Ca2+ release from internal stores, as indicated by increases in both the transient component of the fura-2 response and 45Ca2+ efflux. Thapsigargin (200 nM) and BHQ (10 microM) produced an increase in 45Ca2+ efflux that was completely blocked by SKF 96365 or by cytosolic loading with BAPTA. These results suggest the existence of a restricted sub-plasmalemmal space that is defined by an area of surface membrane which contains the Ca(2+)-influx pathway but is devoid of Ca2+ pumps, and by a section of ER that is rich in thapsigargin-sensitive Ca(2+)-pump units.(ABSTRACT TRUNCATED AT 400 WORDS)
Project description:The effects of glucagon and vasopressin, singly or together, on cytosolic free Ca2+ concentration [( Ca2+]i) and on the 45Ca2+ efflux were studied in isolated rat liver cells. In the presence of 1 mM external Ca2+, glucagon and vasopressin added singly induced sustained increases in [Ca2+]i. The rate of the initial fast phase of the [Ca2+]i increase and the magnitude of the final plateau were dependent on the concentrations (50 pm-0.1 microM) of glucagon and vasopressin. Preincubating the cells with a low concentration of glucagon (0.1 nM) for 2 min markedly accelerated the fast phase and elevated the plateau of the [Ca2+]i increase caused by vasopressin. In the absence of external free Ca2+, glucagon and vasopressin transiently increased [Ca2+]i and stimulated the 45Ca2+ efflux from the cells, indicating mobilization of Ca2+ from internal store(s). Preincubating the cells with 0.1 nM-glucagon accelerated the rate of the fast phase of the [Ca2+]i rise caused by the subsequent addition of vasopressin. However, unlike what was observed in the presence of 1 mM-Ca2+, glucagon no longer enhanced the maximal [Ca2+]i response to vasopressin. In the absence of external free Ca2+, higher concentrations (1 nM-0.1 microM) of glucagon, which initiated larger increases in [Ca2+]i, drastically decreased the subsequent Ca2+ response to vasopressin (10 nM). At these concentrations, glucagon also decreased the vasopressin-stimulated 45Ca2+ efflux from the cells. It is suggested that, in the liver, glucagon accelerates the fast phase and elevates the plateau of the vasopressin-mediated [Ca2+]i increase respectively by releasing Ca2+ from the same internal store as that permeabilized by vasopressin, probably the endoplasmic reticulum, and potentiating the influx of extracellular Ca2+ caused by this hormone.
Project description:Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.
Project description:Ca2+ uptake into the intracellular stores of permeabilized hepatocytes was entirely dependent on ATP and substantially inhibited by either ionomycin or thapsigargin, although both were required for complete inhibition. Unidirectional efflux of 45Ca2+ after removal of ATP from cells loaded to steady state (1.60+/-0.12 nmol/10(6) cells) was monoexponential and occurred with a half-time of 103+/-10 s. However, the 45Ca2+ content of the stores did not return to their pre-ATP level, but reached a plateau at 0.12+/-0.04 nmol/10(6) cells. A similar amount of Ca2+ was trapped within the stores when Ca2+ uptake was prevented by thapsigargin and chelation of Ca2+; at all temperatures between 2 degreesC and 37 degreesC; and after stores had first been loaded with unlabelled Ca2+. Simultaneous addition of inositol 1,4,5-trisphosphate (InsP3) and inhibition of Ca2+ uptake reduced the amount of trapped Ca2+ to a level consistent with InsP3 rapidly and more completely emptying a fraction of the stores that could be only partially emptied by the passive leak. After dilution of the specific activity of the 45Ca2+ under conditions that maintained the steady-state activities of the pumps and leaks, the stores rapidly lost their entire 45Ca2+ content. We conclude that the channel responsible for mediating the leak of Ca2+ abruptly closes when the luminal [Ca2+] of the intracellular stores falls below a critical threshold corresponding to about 7% of their steady-state loading. Whereas InsP3 is capable of completely emptying a fraction of the stores, regulation of the passive leak by luminal [Ca2+] is likely to prevent it from completely emptying them; such regulation may ensure that the many other functions of Ca2+ within the endoplasmic reticulum are not compromised.