Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.     

Calcium uptake by subcellular fractions of human umbilical artery.

Two different mechanisms for the active accumulation of Ca2+ by subcellular fractions of human umbilical artery are described. One, located in the mitochondrial fraction, was induced by exogenous ATP or respiratory substrates (ADP and succinate) and was inhibited by azide. The other, located in the microsomal fraction, was induced by ATP and potentiated by oxalate, but not inhibited by azide. Increasing ATP concentrations up to 4-5 mM increased microsomal Ca2+ accumulation, whereas increasing ATP concentration above 2-3 mM caused inhibition of mitochondrial Ca2+ uptake. Although changing pH from 7.4 to 7.2 had no effect on mitochondrial Ca2+ accumulation, it doubled microsomal uptake. Neither adenosine 3',5'-monophosphate nor guanosine 3',5'-monophosphate in the presence or absence of protein kinase and kinase modulator affected Ca2+ uptake by or phosphorylation of the subcellular fractions. Partially purified protein kinases from umbilical and beef skeletal muscle contained a component(s) distinguishable from the kinase on the basis of its heat stability that enhanced ATP-induced Ca2+ uptake by mitochondrial fractions from the umbilical artery. It is suggested that alterations in Ca2+ sequestration induced by changes in ATP concentration and intracellular pH in mitochondrial and microsomal fractions, respectively, could play a role in the control of arterial patency and closure with changes in PO2.     

Calcium fluxes in isolated pancreatic acini: effects of secretagogues

45Ca2+ exchange and total calcium content were measured in isolated mouse pancreatic acini. 45Ca2+ uptake could be described as the sum of a constant and a single exponential kinetic component; about 60% of total acinar calcium was exchangeable. Stimulation by bethanechol increased 45Ca2+ uptake, but the time course of uptake could be fit only by the addition of a more rapid kinetic component without any change in the total exchangeable Ca2+. 45Ca2+ washout after 1-h loading could be fit as the sum of two exponential components. Stimulation increased the rate of 45Ca2+ washout with the appearance of a third and more rapid kinetic component. There was not, however, a good correspondence between the exponential constants measured in uptake and washout protocols in unstimulated acini. Exponential constants were also affected by the concentration of calcium in the medium, further indicating the presence of nonlinearities in 45Ca2+ exchange. The dose-response relationships were similar for bethanechol stimulation of 45Ca2+ uptake and amylase release, whereas stimulation of 45Ca2+ washout reached a maximum at a higher concentration of bethanechol. Both 45Ca2+ uptake and analytical measurement of total Ca2+ showed a rapid drop in acinar Ca2+ content followed by a gradual reuptake on stimulation by bethanechol. It is concluded that the initial primary effect of secretagogues is to increase Ca2+ efflux, which is interpreted to be the result of release of sequestered calcium into the cytosol.     

FK506 induces biphasic Ca2+ release from microsomal vesicles of rat pancreatic acinar cells

The effect of the immunosuppressant drug FK506 on microsomal Ca2+ release was investigated in rat pancreatic acinar cells. When FK506 (0.1-200 microM) was added to the microsomal vesicles at a steady state of ATP-dependent 45Ca2+ uptake, FK506 caused a dose-dependent and a biphasic release of 45Ca2+. Almost 10% of total 45Ca2+ uptake was released at FK506 concentrations up to 10 microM (Km=0.47 microM), and 60% of total 45Ca2+ uptake was released at FK506 concentrations over 10 microM (Km=55 microM). Preincubation of the vesicles with cyclic ADP-ribose (cADPR, 0.5 microM) increased the FK506 (< or =10 microM)-induced 45Ca2+ release (Ozawa T, Biochim Biophys Acta 1693: 159-166, 2004). Preincubation with heparin (200 microg/ml) resulted in significant inhibition of the FK506 (30 microM)-induced 45Ca2+ release. Subsequent addition of inositol 1,4,5-trisphosphate (IP3, 5 microM) after FK506 (100 microM)-induced 45Ca2+ release did not cause any release of 45Ca2+. These results indicate that two types of FK506-induced Ca2+ release mechanism operate in the endoplasmic reticulum of rat pancreatic acinar cells: a high-affinity mechanism of Ca2+ release, which involves activation of the ryanodine receptor, and a low-affinity mechanism of Ca2+ release, which involves activation of the IP3 receptor.     

Adenosine triphosphate dependent calcium uptake by subcellular fractions from bovine neurohypophyses.

Bovine neurohypophyses were fractionated by differential and density gradient ultracentrifugation and the Ca-2+ uptake and ATPase activities in the microsomal, mitochondrial and secretory granule fractions were studied. The microsomal and mitochondrial fractions accumulated Ca-2+ in the presence of ATP. The accumulation by the latter per mg protein was at least twice as large as by the former. This Ca2+ accumulation was accompanied by liberation of inorganic phosphate (Pi). In the presence of sodium azide (2 mM) Ca-2+ uptake and Pi liberation were inhibited in the mitochondrial, but not in the microsomal fraction. Further studies of the microsomal fractions revealed that the ATP-dependent Ca-2+ uptake and Pi liberation activities were temperature and pH-dependent and required Mg-2+. Both activities were stimulated by very low concentrations of Ca-2+ (1-10 muM) and were inhibited by EGTA (2 mM). N-ethylmaleimide (2 mM) inhibited both the Ca-2+ uptake and ATPase activities of the microsomal fraction. These results suggest the presence of a membrane ATPase that is stimulated by both Ca-2+ and Mg-2+. It is suggested that the observed Ca-2+ uptake activities are involved in maintaining a low axoplasmic free Ca-2+ concentration, thus playing an important role in the release mechanism of vasopressin by the neurosecretory terminals.     

A comparative study of the calcium accumulation by mitochondria and microsomes isolated from the smooth muscle of the guinea-pig taenia coli

Summary The calcium uptake by mitochondria and microsomes isolated from the guinea-pig taenia coli was studied at physiological Ca²⁺ concentrations, buffered by Ca-EGTA mixtures. The Ca accumulation by the mitochondria was measured from the difference between the amount of Ca taken up in the presence and in the absence of a specific mitochondrial inhibitor. The Ca uptake by the microsomes was determined in a solution containing oxalate and a mitochondrial inhibitor. It was calculated from the difference in Ca uptake measured with and without ATP. By using this procedure, the necessity of extensive purification of the isolated fractions was avoided.The (Ca²⁺) for half-maximal transport in the mitochondria is 7×10^–6 M. At (Ca²⁺) lower than 2×10^–7 M, Ca is taken up in an energy-dependent way.In the microsomes the apparentK _m for Ca is 7×10^–7 M. Accumulation is still stimulated by ATP at a (Ca²⁺) as low as 4×10^–8 M.The results show that the rate of Ca uptake by the cell organelles corresponding to the microsomal vesicles is sufficiently fast to explain the speed of relaxation of the taenia coli. The results also suggest that these cell organelles are more important than the mitochondria in regulating the cytoplasmic Ca concentration.     

Calcium uptake in preterminal central synapses: Importance of mitochondria

Summary Energy dependent ⁴⁵Ca²⁺ uptake in the synaptosomal preparation from guinea pig cortex has been investigated. ⁴⁵Ca²⁺ uptake was stimulated by ATP, the absolute value of uptake being dependent on the extent of synaptosomal disruption caused by osmotic shock. A quantitative comparison of microsomal and mitochondrial ATP-dependent ⁴⁵Ca²⁺ uptake showed that only mitochondria had a large enough capacity to account for the Ca uptake levels observed in the synaptosomal preparation. ATP-stimulated ⁴⁵Ca²⁺ uptake in mitochondria, intact and shocked synaptosomes was inhibited by atractyloside, DNP, oligomycin and ruthenium red but unaffected by antimycin A and rotenone. This was interpreted as evidence that mitochondria were responsible for ATP-dependent synaptosomal Ca²⁺ uptake, the increase in uptake seen on osmotic lysis being due to the deocclusion of intraterminal mitochondria. Synaptosomal and mitochondrial ⁴⁵Ca²⁺ uptake was also stimulated by the mitochondrial respiratory substrate glutamate; this uptake was sensitive to antimycin A, DNP, rotenone and ruthenium red but insensitive to atractyloside or oligomycin thus indicating it was of mitochondrial origin. No change in glutamate-dependent ⁴⁵Ca²⁺ uptake was seen on osmotic lysis of the synaptosomes as the expected increase due to the release of occluded mitochondria was counterbalanced by the damaging effect of hypo-osmotic shock on the glutamate-stimulated ⁴⁵Ca²⁺ uptake process.     

A (Ca(2+)-Mg2+)ATPase from Schistosoma mansoni is coupled to an active transport of calcium.

The (Ca(2+)-Mg2+)ATPase activity in microsomes of Schistosoma mansoni is fully inhibited by vanadate (I50 = 2.5 microM). 45Ca2+ is accumulated within microsomal vesicles in an ATP-dependent process that is enhanced 5-fold in the presence of 40 mM phosphate. Accumulated 45Ca2+ is rapidly released by 5 microM of the Ca2+ ionophore A23187 (t1/2 less than or equal to 6 s). (Ca(2+)-Mg2+)ATPase activity and Ca2+ uptake share the same subcellular distribution pattern and similar Ca2+ sensitivities (K0.5 = 0.39 microM and 0.15 microM, respectively). The substrate selectivity is high for both ATPase activity and Ca2+ transport. These results indicate the presence of an active transport of Ca2+ coupled to the (Ca(2+)-Mg2+)ATPase activity previously described in this parasite. A plasma membrane localization and physiological role in calcium homeostasis are suggested.     

缺血等因素对大鼠心肌线粒体Ca^2＋转运的影响

本研究检测了体外缺血等因素对心肌线粒体Ca~(2+)转运的影响。结果表明,缺血明显抑制线粒体Ca~(2+)-ATP酶活性及~(45)Ca摄取率。缺血10分钟时,线粒体Ca~(2+)-ATP酶活性开始下降,40分钟时显著降低,~(45)Ca摄取率的变化也呈类似趋势。去甲肾上腺素及cAMP对线粒体Ca~(2+)-ATP酶活性,~(45)Ca摄取率无任何影响。无机磷对线粒体Ca~(2+)-ATP酶有轻微的抑制作用,二磷酸腺苷对谚酶活性有明显抑制作用,其IC_(50)值为2.5mmol/L。     

Adenosine Triphosphate Dependent Calcium Uptake by Subcellular Fractions from Bovine Neurohypophyses

Bovine neurohypophyses were fractionated by differential and density gradient ultracentrifugation and the Ca²⁺ uptake and ATPase activities in the microsomal, mitochondrial and secretory granule fractions were studied. The microsomal and mitochondrial fractions accumulated Ca²⁺ in the presence of ATP. The accumulation by the latter per mg protein was at least twice as large as by the former. This Ca²⁺ accumulation was accompanied by liberation of inorganic phosphate (P_i). In the presence of sodium azide (2 mM) Ca²⁺ uptake and P_i liberation were inhibited in the mitochondrial, but not in the microsomal fraction. Further studies of the microsomal fraction revealed that the ATP-dependent Ca²⁺ uptake and P_i liberation activities were temperature and pH-dependent and required Mg²⁺. Both activities were stimulated by very low concentrations of Ca²⁺ (1–10 μM) and were inhibited by EGTA (2 mM). N-ethyl-maleimide (2 mM) inhibited both the Ca²⁺ uptake and ATPase activities of the microsomal fraction. These results suggest the presence of a membrane ATPase that is stimulated by both Ca²⁺ and Mg²⁺. It is suggested that the observed Ca²⁺ uptake activities are involved in maintaining a low axoplasmic free Ca²⁺ concentration, thus playing an important role in the release mechanism of vasopressin by the neuro-secretory terminals.     

Brain microsomal calcium accumulation in rats with increasing age: involvement of thapsigargin-sensitive Ca2+-ATPase

The alteration in calcium content and Ca2+-ATPase activity in the brain microsomes of rats with increasing age was investigated. Brain microsomal calcium content was significantly increased in aged rats (50 weeks old) as compared with that of young rats (5 weeks old). Such an increase was not seen in the brain mitochondria of aged rats. Increasing age caused a significant elevation of Ca2+-ATPase activity in the brain microsomes. The microsomal Ca2+-ATPase activity of young rats was significantly decreased by the addition of thapsigargin (10-7-10-5 M), a specific inhibitor of microsomal Ca2+ pump enzyme (Ca2+-ATPase), in the enzyme reaction mixture. The inhibitory effect of thapsigargin (10-5 M) was also seen in the brain microsomes of aged rats. Moreover, the effect of calcium (5 and 10 microM) addition in elevating Ca2+-ATPase activity was not revealed in the microsomes of aged rats, whereas the metal could increase the enzyme activity in young rats, suggesting an involvement of activatory factor on the enzyme with increasing age. The present study demonstrates that ageing induces an increase in thapsigargin-sensitive Ca2+-ATPase activity and a corresponding elevation of calcium content in the brain microsomes. This finding suggests a cellular mechanism by which ageing causes calcium accumulation in brain.     

Mechanism of action of magnesium on acetylcholine-evoked secretory responses in isolated rat pancreas.

This study investigates the effects of magnesium (Mg2+) on acetylcholine (ACh)-evoked secretory responses and calcium (Ca2+) mobilization in the isolated rat pancreas. ACh induced marked dose-dependent increases in total protein output and amylase release from superfused pancreatic segments in zero, normal (1 x 1 mM) and elevated (10 mM) extracellular Mg2+. Elevated Mg2+ attenuated the ACh-evoked secretory responses compared to zero and normal Mg2+. In the absence of extracellular Ca2+, but presence of 1 mM-EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid), ACh elicited a small transient release of protein from pancreatic segments compared to a larger and more sustained secretion in the absence of both Ca2+ and Mg2+. Incubation of pancreatic segments with 45Ca2+ resulted in time-dependent uptake with maximum influx of 45Ca2+ occurring after 20 min of incubation period. ACh stimulated markedly the 45Ca2+ uptake compared to control tissues. In elevated extracellular Mg2+ the ACh-induced 45Ca2+ influx was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. ACh also evoked dose-dependent increases in cytosolic free Ca2+ concentrations ([Ca2+]i) in pancreatic acinar cells loaded with the fluorescent dye Fura-2 AM. In elevated Mg2+ the ACh-induced cytosolic [Ca2+]i was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. These results indicate that Mg2+ can influence ACh-evoked secretory responses possibly by controlling both Ca2+ influx and release in pancreatic acinar cells.     

Protein synthesis inhibitors enhance secretagogue sensitivity of in vitro rat pancreatic acini

Isolated rat pancreatic acini were treated with cycloheximide and amylase release was measured. This agent increased the sensitivity to both synthetic octapeptide of cholecystokinin (CCK8) and carbamylcholine, the major secretagogues known to utilize Ca2+ as a second messenger. The mechanism of the cycloheximide effect was via inhibition of protein synthesis, as indicated by the following: 1) the concentration of cycloheximide used inhibited leucine incorporation by greater than 90%; 2) this effect was not instantaneous but increased up to a 2-h pretreatment; and 3) a similar effect was obtained with puromycin, a chemically different inhibitor of protein synthesis. Cycloheximide acted on the steps by which secretagogues mobilize cellular Ca2+ because the dose-response curve for 45Ca2+ efflux was shifted to the same extent as that for amylase release, whereas the dose-response curve for amylase release induced by the Ca2+ ionophore A23187 was not altered. The results suggest, therefore, that a rapidly turning-over protein present in pancreatic acinar cells exerts an inhibitory influence on Ca2+ mobilization by secretagogues.     

Effect of acetylcholine stimulation on calcium and sodium uptake by the isolated rat pancreas.

1. The role of Ca2+ and Na+ ions in 'stimulus-secretion' coupling in the isolated uncinate pancreas of 4-week-old rats has been examined using radioisotope tracer techniques. The amount of 45Ca2+ and 22Na+ (mumole/g wet wt.) taken up by unstimulated glands was compared to that of glands in which amylase release was stimulated by ACh(10(-5) M) at various incubation times. 2. The amount of 45Ca taken up by the glands within 60 min of incubation was not found to be increased by the presence of ACh(10(-5) M). In fact, during short incubation periods the 45Ca uptake was significantly less in the stimulated glands than the unstimulated glands. 3. Presence of ACh(10(-5) M) did cause a significant initial increase in 22Na uptake lasting up to 20 min from onset of stimulation. 4. These results indicate that the rise in intracellular Ca2+ concentration which is suggested by an increase in 45Ca efflux during the action of pancreatic secretagogues, is not a consequence of increased 45Ca uptake by the pancreas; but they indicate that an initial action of ACh could be to elicit an increase in Na+ influx and that Na+ ions are likely to be involved in the action of ACh on pancreatic acinar cells.     

Effect of lanthanum on pancreatic protein synthesis in streptozotocin-diabetic rats

The role of extracellular Ca2+ in mediating the stimulatory effect of cholecystokinin octapeptide (CCK8) on [3H]phenylalanine incorporation into protein was studied in isolated pancreatic acini from streptozotocin-diabetic rats. The stimulatory effect of CCK8 (10(-10) M) on [3H]phenylalanine incorporation was completely abolished by preincubating acini with either 10(-4) M lanthanum or 10(-3) M manganese. At these concentrations neither compound altered the basal rate of amino acid incorporation, and both compounds inhibited CCK-mediated Ca2+ influx without affecting either basal or CCK-mediated 45Ca2+ efflux. Lanthanum (10(-4) M) also blocked the stimulatory effect of the cholinergic analogue carbachol (10(-5) M) on amino acid incorporation but did not alter the stimulatory effect of insulin (1.67 x 10(-8) M). Vasoactive intestinal polypeptide and 12-O-tetradecanoyl-phorbol-13-acetate failed to increase the incorporation of [3H]phenylalanine into acinar protein. These findings suggest that CCK and other pancreatic secretagogues that act via Ca2+ enhance protein synthesis by increasing cell membrane permeability to Ca2+ and provide additional evidence that this may be an important mechanism by which CCK regulates pancreatic exocrine function.     

Effects of glucose on 45Ca2+ uptake by pancreatic islets as studied with the lanthanum method.

1. Fluxes of 45Ca2+ were studied in pancreatic islets from non-inbred ob/ob-mice. Because La3+ blocked the transmembrane fluxes of 45Ca2+ in islet cells, incubations aimed at measuring glucose-induced changes of the intracellular Ca2+ were ended by washing the islets with 2 mM-La3+ for 60 min. 2. Uptake of 45Ca2+ progressed for 2 hr; the intracellular concentration of exchangable Ca2+ was about 7 m-mole/kg dry wt., as estimated from the isotope distribution at apparent equilibrium in islets exposed to 3 mM D-glucose. Raising the D-glucose concentration to 20 mM enhanced the 45 Ca2+ uptake whether or not the islets had first been equilibrated with the isotope. The stimulatory effect of D-glucose was observed in Tris buffer containing no anions but Cl- as well as in polyanionic bicarbonate buffer. The effect could not be reproduced with equimolar L-glucose. 3. The rate of 45Ca2+ release was the same whether the islets had been pre-loaded in the presence of 3 or 20 mM D-glucose. Thus the 45Ca2+ that had been taken up in response to 20 mM D-glucose appeared to be released much more slowly than the bulk of intracellular 45Ca2+. The release of 45Ca2+ was not significantly influenced by D-glucose during the release period. Incubation for 30 min was require for half of the radioactivity to be released. 4. The rates of insulin secretion were about the same in uni-anionic Tris buffer as in polyanionic bicarbonate buffer. A marked insulin secretory response to 20 mM D-glucose was observed in either buffer. 5. It is concluded that 20 mM D-glucose causes a net uptake of Ca2+ from the extracellular fluid into the interior of the beta-cells. This uptake is probably not regulated at the level of the plasma membrane but more likely reflects an increased affinity of some intracellular phase or compartment for the ion. Because the observed uptake and release of intracellular 45Ca2+ are slow processes in comparison with the rapid effects of extracellular Ca2+ on insulin secretion, insulin secretion may also depend on a more superficial and La3+-displacable Ca2+ pool.     

Histamine-evoked amylase secretion is associated with small changes in calcium mobilization in isolated guinea-pig pancreas.

An investigation was made of the effect of histamine on amylase secretion and calcium mobilization in isolated guinea-pig pancreatic segments. The effect of acetylcholine (ACh) was also examined for comparison. Histamine evoked a dose-dependent increase in amylase output from superfused segments. It had small transient effects on both 45Ca influx and efflux, and elevated cytosolic free Ca2+ concentration in acini. The equivalent dose of ACh evoked a greater amylase output and greater changes in Ca2+ mobilization. The results suggest that histamine may have a physiological role in exocrine secretion of the guinea-pig pancreas but is less potent than ACh.     

Effects of Ca2+ channel agonist-antagonist enantiomers of dihydropyridine 202791 on insulin release, 45Ca uptake and electrical activity in isolated pancreatic islets

This is the first study using the selective agonist/antagonist stereoisomers of dihydropyridine 202791 to investigate stimulus-secretion coupling in pancreatic islet cells. We studied effects of the (+)(Ca2+ channel agonist) and (-)(Ca2+ channel antagonist) forms of the dihydropyridine, on 45calcium net uptake, insulin secretion, and membrane potential measured in rodent islets. The antagonist partially inhibited glucose-induced insulin secretion and Ca2+ uptake; however, the potassium-induced Ca2+ uptake was completely inhibited. The antagonist did not completely block glucose-evoked spike activity. Addition of the agonist enhanced insulin release and Ca2+ uptake in the presence of 5.6 mM-glucose, but did not increase insulin release or Ca2+ uptake in 16.7 mM-glucose. In the presence of tetraethylammonium (TEA), (+)202791 increased and (-)202791 decreased the duration of glucose-induced action potentials. The results again confirm the presence of a dihydropyridine-sensitive Ca2+ channel in pancreatic B-cells. In addition these data suggest that in these cells there is activation of a dihydropyridine-insensitive Ca2+ entry in the presence of glucose.     

Enhanced Ca2+ uptake by mouse erythrocytes in malarial (Plasmodium berghei) infection

Erythrocytes from Plasmodium berghei-infected mice on incubation either in plasma or artificial isotonic media showed an increase in uptake of 45Ca2+ compared with erythrocytes from uninfected mice. Infected cells (55% parasitaemia) incubated in plasma from normal or infected mice gave uptake rates of 9.8 and 8.1 nmol h-1 per 10(10) cells, assuming equilibrium between added 45Ca2+ and plasma Ca2+. Uptake rates of erythrocytes from infected mice were increased in the presence of glucose, with a rate of 15.0 nmol h-1 per 10(10) cells (52-58% parasitaemia) at 5 mM glucose, compared with 1.5 nmol h-1 per 10(10) cells in the absence of glucose. The enhancement of 45Ca2+ uptake was more pronounced with increasing parasitaemia, and in the fraction relatively enriched with erythrocytes carrying mature parasites. It is likely, therefore, that the enhancement is due to changes in membrane permeability accompanying parasite development. Enhanced haemolysis accompanied 45Ca2+ uptake of erythrocytes carrying mature parasites, but not of those carrying young parasites or uninfected erythrocytes. The possible role of an altered Ca2+ status in erythrocyte pathophysiology during malarial infection is discussed.     

Effect of atropine, ouabain, antimycin A, and A23187 on "trigger Ca2+ pool" in exocrine pancreas

45Ca2+ fluxes have been analyzed in dispersed acinar cells prepared from rat pancreas. Sudden addition of carbamylcholine (CCh) to 45Ca2+-preloaded acinar cells at quasi-steady state for 45Ca2+ resulted in a quick 45Ca2+ release followed by a slower 45Ca2+ reuptake with net accumulation of 45Ca2+. Subsequent sudden addition of atropine caused a further transient increase in cellular 45Ca2+ followed by a slow decrease to a steady-state value. 45Ca2+ release could not be evoked a second time by pancreozymin when prestimulated with CCh. However, if CCh stimulation was abolished by an interposed step of atropine, restimulation by cholecystokinin-pancreozymin was possible. Addition of A23187 or antimycin A to cells induced a fast decrease in cellular 45Ca2+. This effect was not additive to the CCh effect. In ouabain-pretreated cells, the CCh-induced sudden loss of cellular 45Ca2+ was blocked by 60%. The following slow reuptake of 45Ca2+ was blocked completely. Subsequent addition of atropine caused a fast uptake of cellular 45Ca2+ with no secondary decline. The data are consistent with the following model: acetylcholine releases Ca2+ from a cellular "trigger pool" into the cytosol located in or near the cell membrane. Then Ca2+ is extruded from the cell via Ca2+ pumps partly by a Na+-dependent Ca2+ transport system (quick phase of 45Ca2+ release). Subsequently, due to increased Ca2+ permeability of the plasma membrane as induced by acetylcholine, Ca2+ influx occurs and Ca2+ is taken up from the cytosol into intracellular Ca2+ pools (slow 45Ca2+ reuptake phase). Atropine causes refilling of the trigger Ca2+ pool and return of the increased Ca2+ permeability of the plasma membrane back to the unstimulated state.