Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca2+ and Mg2+ ions bound to membranes. Acini, preloaded by incubation with CTC (100 microM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 min of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secretagogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca(2+)-complexed CTC.     

Redistribution of intracellular Ca2+ in mitogen-stimulated human peripheral blood lymphocytes

The fluorescent probe chlortetracycline (CTC) was used to investigate redistribution of intracellular Ca2+ in concanavalin A (Con A)-stimulated human peripheral blood lymphocytes. The addition of the mitogen to CTC-equilibrated lymphocytes induced (within 10 to 15 minutes) a Con A-concentration dependent decrease in CTC fluorescence indicating the release of membrane-bound Ca2+. The effect was independent of the level of extracellular Ca2+ and could be observed in the presence of EGTA; it was suppressed by the metabolic inhibitors FCCP, antimycin and sodium cyanide. Analysis of the excitation spectra of CTC fluorescence indicated that the observed effect is caused by redistribution of intracellular Ca2+ rather than Mg2+. Thus the lectin interaction with the lymphocyte plasma membrane results in Ca2+ release into the cytosol from the intracellular stores.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

CHLOROTETRACYCLINE-ASSOCIATED FLUORESCENCE CHANGES DURING CALCIUM UPTAKE AND RELEASE BY RAT BRAIN SYNAPTOSOMES1

Abstract– The fluorescent divalent metal chelate-probe, chlorotetracycline (CTC), was used as a dynamic monitor of calcium association with rat brain snynaptosomes. The determined fluorescence excitation and emission maxima, 412 nm and 522 nm respectively, were used to monitor membrane-calcium interactions as a function of various parameters. Positive correlations were observed between increased or decreased fluorescence quantum yield and the uptake of both CTC and ⁴⁵Ca by synpatosomes. The divalent metal ionophore A23187 enhanced fluorescence as well as probe and ⁴⁵Ca uptake. Whereas, the polar chelator, EGTA, markedly reduced fluorescence, and the synaptosomal bound CTC and ⁴⁵Ca. The CTC fluorescence changes also demonstrated the saturable manner in which ⁴⁵Ca bound synaptosomes. At concentrations greater than 100μg/ml, CTC bound to the synaptosomes in a manner which quenched fluorescence at 522 nm. Also, CTC, at concentrations above 15 μg/ml, enhanced the uptake of ⁴⁵Ca. At CTC concentrations between 10 and 15 μg/ml the quenching and iono-phoretic properties of the probe were minimized without affecting the capability of using the probe to visualize calcium interactions with synaptosomal membranes. Also, at a low CTC concentration (12.5 μg/ml) the inhibition of calcium uptake by increasing monovalent ion concentrations was clearly demonstrated.     

Chlorotetracycline fluorescence is a quantitative measure of the free internal Ca2+ concentration achieved by active transport. In situ calibration and application to bovine cardiac sarcolemmal vesicles

Chlorotetracycline (CTC) fluorescence is shown to be a competent and quantitative measure of the free internal calcium concentration, [Ca2+]i, obtained by ATP supported active uptake by bovine cardiac sarcolemmal (SL) vesicles. The fluorescence response of CTC to [Ca2+]i is calibrated by pre-equilibrating the vesicles with known Ca2+ concentrations and then diluting into a Ca2+-free medium containing CTC. The experiments show that CTC comes into equilibrium with the internal Ca2+ more rapidly than the latter can passively leak from the vesicles. The amplitude of the fluorescence increase is proportional to the Ca2+ concentration with which the vesicles are pre-equilibrated. This constitutes a calibration procedure for the use of CTC fluorescence as a quantitative measure of the free internal Ca2+ concentrations achieved in active transport. This method is applied to the determination of the average free Ca2+ concentrations achieved in ATP-energized uptake with sarcolemmal vesicles. Under optimal conditions an initial rate of 13 mM/min (37 nmol/mg/min) is observed. Uptake reaches a maximum corresponding to 70 mM (179 nmol/mg). Half-maximal values are obtained after 5 min of reaction. The mechanism of the CTC response to free internal Ca2+ concentration is discussed and is compared with measurements of vesicle-associated 45Ca2+.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.     

Salinity affects intracellular calcium in corn root protoplasts

Previous work with the fluorescent Ca probe chlorotetracycline (CTC) showed that salinity displaces Ca from membranes of root cells. Using a variety of indirect approaches, we studied whether salinity displaces Ca from the cell surface or from internal membranes of corn (Zea mays L. cv Pioneer 3377) root protoplasts. Preloading the cells with supplemental Ca counteracted subsequent NaCl effects on CTC fluorescence. CTC quenching by exogenous EGTA was not competitive with CTC quenching by NaCl. The Ca channel reagent (+)-202-791 had significant interactions with the effect of NaCl on CTC fluorescence. The effect of NaCl on CTC fluorescence was attenuated by pretreatment with Li, but was restored by inositol. Salinity increased Na influx, decreased Ca influx, and increased Ca efflux from the cells. Fluorescence anisotropy indicated that NaCl decreased the fluidity of the external face of the plasmalemma but increased the fluidity of cell membranes in general. Our results suggest that salinity displaces Ca associated with intracellular membranes through activation of the phosphoinositide system and depletion of intracellular Ca pools.     

Intracellular free calcium content increase in the erythrocytes treated with the cryoprotective medium based on polyethylene glycol 1500 (PEG-1500)

Changes in intracellular free calcium content ([Ca2+]i) in human erythrocytes treated with the cryoprotective medium based on low toxic polymer--polyethylene glycol 1500 (PEG-1500) and then transferred to physiologic salt solution containing 2 mM CaCl2 were studied using fluorescent calcium probe--fura-2. A method of [Ca2+]i calculation with allowance for haemolysis of the cells during the experiment was proposed. It was shown that ignorance of the cell haemolysis resulted in significantly higher [Ca2+]i values obtained. Significant time-dependent increase of [Ca2+]i in the cells treated with PEG-1500 cryoprotective medium at +4 degrees C as well as at +22 degrees C (without freezing) and then transferred in the 2 mM CaCl2 containing physiological salt solution at +37 degrees C was observed. Freezing-thawing of the cells treated with the PEG-1500 cryoprotective medium enhanced haemolysis and further accumulation of calcium in the cells. The results of the study prove that the use of PEG-1500-based cryoprotective medium which does not require washing for human erythrocytes will be accompanied by progressive destruction (haemolysis) of the cells in the blood vessels and may have some negative consequences connected with [Ca2+]i increase in the cryopreserved erythrocytes.     

Maintenance of intracellular calcium in Escherichia coli

Recently a series of fluorescent calcium indicator dyes have been developed for measurement of free intracellular calcium in eukaryotic cells. Here we report the use of one such dye, fura-2, for the study of intracellular calcium levels in the prokaryote Escherichia coli. Cells of E. coli were loaded with the membrane-permeable acetoxymethyl ester of fura-2, which was cleaved intracellularly to give the free pentaacid. The concentration of free [Ca2+]i in unstarved cells was maintained at 90 +/- 10 nM, irrespective of the Ca2+ concentration in the extracellular medium. Cells of a strain lacking the H+-translocating ATPase were depleted of endogenous energy reserves and loaded with calcium. In this strain oxidative phosphorylation is uncoupled, so ATP is not produced by respiration. In starved cells [Ca2+]i varied from 0.2 to 0.7 microM when the loading Ca2+ concentration varied from 10 microM to 10 mM. Addition of glucose lowered the Ca2+ levels to 90 nM. Addition of respiratory substrates as energy donors produced cyanide-sensitive efflux. Total cell Ca2+ increased in parallel to the extracellular calcium, but the pool of free calcium did not equilibrate with the total cellular pool. These results demonstrate that 1) the pool of total Ca2+ in the bacterial cell is large and responds to extracellular calcium, 2) the free [Ca2+]i is independent of extracellular calcium, and 3) energy in the form of a proton motive force is required for maintenance of the free intracellular pool of calcium.     

Effects of Ca2+-ATPase inhibitors, ionomycin, and pharmacological modulators of ryanodine receptor on calcium release from intracellular pools and on oscillatory contractile behavior in Physarum polycephalum

Changes in calcium levels in organelles of the plasmodium of the myxomycete Physarum polycephalum were analyzed using the fluorescent calcium indicator chlortetracycline (CTC). Both the Ca2+-ATPase inhibitor 2,5;-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (100 microM) and the calcium ionophore ionomycin (1 microM) induce a significant decrease in fluorescence level (by 30%) in CTC-stained microplasmodia; this is caused by release of calcium from intracellular storage compartments. An activator of ryanodine receptors, caffeine (10-50 mM), is less effective on Ca2+ release than BHQ or ionomycin, and their inhibitor, ryanodine (100 microM), almost completely blocks the response to caffeine, but only slightly decreases the effects of BHQ or ionomycin. Procaine, another inhibitor of ryanodine receptors, at 10 mM concentration completely abolishes both the BHQ and the ionomycin responses, but 50 mM is necessary to block the effect of 25 mM caffeine. These results suggest that both the BHQ- and the ionomycin-dependent Ca2+ releases occur through the ryanodine receptor and are to be considered as calcium-induced Ca2+ release (CICR). Both the ionomycin and the BHQ responses persist in the presence of Cd2+, which blocks Ca2+ channels of the plasmalemma. In most cases, Cd2+ itself induces release of Ca2+ from the CTC-stained calcium pool; the more effective Cd2+ is, the less the following ionomycin or BHQ responses occur. This indicates that Ca2+ entry through plasmalemma plays no significant role in the ionomycin- or BHQ-evoked initiation of CICR, and that the Cd2+- and BHQ/ionomycin-depleted Ca2+ stores overlap.     

Prolonged exercise induces structural changes in SR Ca(2+)-ATPase of rat muscle.

Sarcoplasmic reticulum (SR) isolated from the deep red portion of the gastrocnemius muscle of Sprague-Dawley rats after a single bout of prolonged exercise was shown to have depressed Ca(2+)-stimulated Mg(2+)-dependent ATPase activity over a temperature range of 15 to 42.5 degrees C when compared to SR obtained from control muscle. Inclusion of the calcium ionophore, A23187, failed to restore the depressed ATPase activity from SR of exercised muscle to control values, but it did normalize the stimulatory effect of temperature on ATPase activity. This depression was also manifested as an increased activation energy when the data were converted to an Arrhenius plot. SR vesicles from both groups showed no differences or discontinuities in plots of steady-state fluorescence anisotropy. When the binding characteristics of the fluorescent probe, fluorescein isothiocyanate (FITC), were analyzed, SR vesicles prepared from exercised muscle displayed a 40% reduction in binding capacity with no apparent change in Kd. These findings support the conclusion that a single bout of exercise induces a structural change in the Ca(2+)-ATPase protein of rat red gastrocnemius muscle that is not a direct result of gross lipid alterations or increased muscle temperature.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Suppression of high-pressure-induced hemolysis of human erythrocytes by preincubation at 49 degrees C.

When human erythrocytes were preincubated at 37-52 degrees C under atmospheric pressure before exposure to a pressure of 200 MPa at 37 degrees C, the value of hemolysis was constant (about 43%) up to 45 degrees C but became minimal at 49 degrees C. The results from anti-spectrin antibody-entrapped red ghosts, spectrin-free vesicles, and N-(1-pyrenyl)iodoacetamide-labeled ghosts suggest that the denaturation of spectrin is associated with such behavior of hemolysis at 49 degrees C. The vesicles released at 200 MPa by 49 degrees C-preincubated erythrocytes were smaller than those released by the treatment at 49 degrees C or 200 MPa alone. The size of vesicles released at 200 MPa was independent of preincubation temperature up to 45 degrees C, and the vesicles released from 49 degrees C-preincubated erythrocytes became smaller with increasing pressure up to 200 MPa. Thus, hemolysis and vesiculation under high pressure are greatly affected by the conformation of spectrin before compression. Since spectrin remains intact up to 45 degrees C, the compression of erythrocytes at 200 MPa induces structural changes of spectrin followed by the release of large vesicles and hemolysis. On the other hand, in erythrocytes that are undergoing vesiculation due to spectrin denaturation at 49 degrees C, compression produces smaller vesicles, so that the hemolysis is suppressed.     

Amine weak bases disrupt vesicular storage and promote exocytosis in chromaffin cells

The vesicular contents in bovine chromaffin cells are maintained at high levels owing to the strong association of its contents, which is promoted by the low vesicular pH. The association is among the catecholamines, Ca2+, ATP, and vesicular proteins. It was found that transient application of a weak base, methylamine (30 mM), amphetamine (10 microM), or tyramine (10 microM), induced exocytotic release. Exposure to these agents was also found to increase both cytosolic catecholamine and intracellular Ca2+ concentration, as measured by amperometry and fura-2 fluorescence. Amphetamine, the most potent amine with respect to evoking exocytosis, was found to be effective even in buffer without external Ca2+; however, the occurrence of spikes was suppressed when BAPTA-acetoxymethyl ester was used to complex intracellular Ca2+. Amphetamine-induced spikes in Ca2+-free medium were not suppressed by thapsigargin or ruthenium red, inhibitors of the sarco(endo)plasmic reticulum Ca2+-ATPase and mitochondrial Ca2+ stores. Atomic absorption measurements of amphetamine- and methylamine-treated vesicles reveal that intravesicular Ca2+ stores are decreased after a 15-min incubation. Taken together, these data indicate that amphetamine and methylamine can disrupt vesicular stores to a sufficient degree that Ca2+ can escape and trigger exocytosis.     

Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

Arachidonic acid increases intracellular calcium in erythrocytes

Recently, we have measured in erythrocytes a voltage-modulated and dihydropyridine-inhibited calcium influx. Since arachidonic acid and other polyunsaturated fatty acids influence the activities of most ion channels, we studied their effects on the erythrocyte Ca(2+) influx. It was measured on fresh erythrocytes, isolated from healthy donors, using the fluorescent dye Fura 2 as indicator of [Ca(2+)](i). AA (5-50 microM) and EPA (20-30 microM) stimulated a concentration-dependent increase in [Ca(2+)](i), deriving from extracellular calcium (1 mM), without affecting the intra- and extracellular pH and membrane voltage. The Ca(2+) influx rate varied from 0.5 to 3 nM Ca(2+)/s in the presence of AA and from 0.9 to 1.7 nM Ca(2+)/s with EPA. The Ca(2+) influx elicited by AA and EPA was not inhibited by dihydropyridines, while cyclooxygenase inhibitors were effective and PGE1 or PGE2 did not produce any effect. We conclude that AA could activate an erythrocyte voltage-independent Ca(2+) transport via an intermediate product of cyclooxygenase pathway; however, a direct interaction with the membrane lipid-protein cannot be excluded.     

Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature

Vesiculation of intact erythrocytes can be induced by decreasing their intracellular pH and then heating the red cell suspension to a critical temperature value. While at intracellular pH 6 vesiculation begins at 45 degrees C, further decrease in the intracellular pH lowers the critical temperature. In addition, the critical temperature value can be modified by varying the length of the interval between titration and heating as well as by changing the temperature during this interval. The vesicles are large (1-3.5 micron in diameter), haemoglobin-containing and completely free of skeletal proteins. Pretreatment of the cells with diamide and 2,4-dinitrophenol had no substantial effect on vesiculation, while N-ethylmaleimide, chlorpromazine and wheat germ agglutinin proved to be inhibitory. Increasing the osmolarity of the incubation medium markedly decreased the critical temperature: red cells suspended in a solution of 600 mosM NaCl vesiculated at 42 degrees C instead of 45 degrees C when the intracellular pH was decreased to 6. We propose that the vesiculation is due to a purely physicochemical molecular mechanism which affects the state and dimension of the membrane skeleton. We also discuss the possible role of an altered haemoglobin-membrane interaction in preventing low pH-induced intramembrane particle aggregation in the membrane skeleton-free vesicles.     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : I. Characterization of a Ca-Pumping ATPase Associated with the Endoplasmic Reticulum

Calcium transport was examined in microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue using chlorotetracycline as a fluorescent probe. This probe demonstrates an increase in fluorescence corresponding to calcium accumulation within the vesicles which can be collapsed by the addition of the calcium ionophore A23187. Calcium uptake in the microsomal vesicles was ATP dependent and completely inhibited by orthovanadate. Centrifugation of the microsomal membrane fraction on a linear 15 to 45% (w/w) sucrose density gradient revealed the presence of a single peak of calcium uptake which comigrated with the marker for endoplasmic reticulum. The calcium transport system associated with endoplasmic reticulum vesicles was then further characterized in fractions produced by centrifugation on discontinous sucrose density gradients. Calcium transport was insensitive to carbonylcyanide m-chlorophenylhydrazone indicating the presence of a primary transport system directly linked to ATP utilization. The endoplasmic reticulum vesicles contained an ATPase activity that was calcium dependent and further stimulated by A23187 (Ca(2+), A23187 stimulated-ATPase). Both calcium uptake and Ca(2+), A23187 stimulated ATPase demonstrated similar properties with respect to pH optimum, inhibitor sensitivity, substrate specificity, and substrate kinetics. Treatment of the red beet endoplasmic reticulum vesicles with [gamma-(32)P]-ATP over short time intervals revealed the presence of a rapidly turning over 96 kilodalton radioactive peptide possibly representing a phosphorylated intermediate of this endoplasmic reticulum associated ATPase. It is proposed that this ATPase activity may represent the enzymic machinery responsible for mediating primary calcium transport in the endoplasmic reticulum linked to ATP utilization.     

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.