Biochimica et Biophysica Acta (BBA) - Biomembranes
Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum
Abstract
Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4–6 μmol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 μmol/mg, but stayed constant at about 0.7 μmol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium.
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Physiological characterization of the Na<sup>+</sup>/Ca<sup>2+</sup> exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii
2002, Comparative Biochemistry and Physiology - A Molecular and Integrative PhysiologyThe purpose of this study was to physiologically characterize the basolateral Na+/Ca2+ exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na+-dependent Ca2+ uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na+-dependent Ca2+ uptake rate into BLMV was temperature insensitive. Na+-dependent Ca2+ uptake rate was dependent upon free Ca2+ with saturable Michaelis–Menten kinetics determined as follows: hepatopancreas, maximal uptake rate (Jmax)=2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate (Km)=0.69 μM Ca2+; antennal gland, Jmax=13.2 nmol/mg per min, Km=0.59 μM Ca2+. The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na+-dependent Ca2+ uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 μM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 μM in hepatopancreas and was stimulatory at 1000 μM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na+-dependent Ca2+ uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca2+ ATPase (PMCA), the NCX has 70-fold greater Jmax (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater Jmax and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca2+ efflux in intermolt.
Regulation of Ca<sup>2+</sup> transport by sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase at limiting [Ca<sup>2+</sup>]
1999, Biochimica et Biophysica Acta - BiomembranesThe factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 μM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47±0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254–263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49±1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible ‘pump/leak’ mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.
The cardiac content of sarcoplasmic reticulum in the rat determined by calcium uptake rate, calcium oxalate capacity, ryanodine binding and thapsigargin titration
1998, Journal of Molecular and Cellular CardiologyThe amount of cardiac sarcoplasmic reticulum in rat hearts was estimated by comparing marker activities in the isolated SR fraction with their activities in the homogenate. Four distinguishable markers were measured: the oxalate-supported rate of calcium uptake, the calcium oxalate capacity,3H-ryanodine binding and the thapsigargin equivalents. The calcium uptake rate and capacity and thapsigargin equivalents were determined in the presence and absence of SR Ca2+channel blockade with high concentrations of ryanodine. All of these activities are believed to be located only in the SR. However, the calculation of the heart content of SR was somewhat different for the four markers. The calcium uptake rate gave 8.4 mg SR protein per g tissue in the absence of ryanodine, and 9.6 mg per g in its presence; calcium oxalate capacity gave similar numbers, 9.9 mg per g in the absence of ryanodine and 8.0 mg per g in its presence. The thapsigargin titration gave similar equivalent with or without ryanodine, indicating that the homogenate contained about 8.0 mg of SR per g tissue. Using3H-ranodine binding as a marker, the cardiac content of SR was calculated to be 16.7 mg per g. These differences are attributed to the non-ideal behavior of these markers. Some of the Ca2+uptake activity is not thapsigargin sensitive, and some of the3H-ryanodine binding does not fractionate with the SR Ca2+uptake activity.
Effect of ischemia on the fraction of ryanodine-sensitive cardiac sarcoplasmic reticulum
1997, Journal of Molecular and Cellular CardiologyThe effect of 15 min of global, normothermic ischemia on cardiac sarcoplasmic reticulum (SR) was investigated using the Ca2+uptake rate and3H-ryanodine binding of ventricular homogenates and isolated SR vesicles. Ischemia did not affect ryanodine binding in the homogenate, while it increased it in the isolated SR vesicles. Although ischemia decreased the homogenate oxalate-supported Ca2+uptake rate, measured in the presence of high ryanodine to close the ryanodine-sensitive efflux pathway (+RY), its decrease of the Ca2+uptake rate, measured in the absence of ryanodine (−RY), was more marked. This finding was also observed in the isolated SR. Although inhibition of the Ca-ATPase and its coupled Ca2+uptake by thapsigargin proportionately decreased SR Ca2+uptake −RY and +RY, ischemia decreased the Ca2+uptake −RY proportionately more. This result suggested that there was a greater fraction of Ca2+uptake activity in ryanodine-sensitive vesicles after ischemia. However, ischemia also reduced the yield of SR activity in the isolated SR fraction and the results could potentially be due to differential selection of ryanodine-sensitive and ryanodine-insensitive SR in the isolation procedure. We directly tested the hypothesis that ischemia changes the fraction of Ca2+uptake activity in the ryanodine-sensitive vesicles by estimating the Ca-oxalate capacity measured +RY and −RY. Ischemia decreased the capacity −RY much more than +RY in the homogenate, indicating that more of the SR volume and Ca2+uptake activity was in the ryanodine-sensitive vesicles after ischemia.
Kinetic characterization of sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase
1996, Biomembranes: A Multi-Volume TreatiseSarcoplasmic reticulum Ca2+-ATPase is a membranous enzyme, which couples the energetically down-hill ATP hydrolysis to the up-hill transport of Ca2+ from the muscle cytoplasm—in which the free Ca2+ concentration is in the submicromolar range—to the luminal compartment of sarcoplasmic reticulum—in which the free Ca2+ concentration is in the submillimolar or millimolar range. This enzyme belongs to the class of P-type ATPases—that is, ATPases whose catalytic cycle involves formation of a covalent phosphoenzyme. An effect on the apparent Ca2+ dependence of the transport velocity is obtained without the modification of the equilibrium properties of the Ca2+-binding sites because of the interplay among the various kinetic constants in the ATPase catalytic cycle. The complete cycle of Ca2+ transport and ATP hydrolysis by sarcoplasmic reticulum ATPase is reversible. The proportion of ATPase with bound substrate controls the apparent rate of phosphorylation.
Pulmonary microsomes contain a Ca<sup>2+</sup>-transport system sensitive to oxidative stress
1995, BBA - BioenergeticsA variety of events, including inhalation of atmospheric chemicals, trauma, and ischemia-reperfusion, may cause generation of reactive oxygen species in the lung and result in airways constriction. The specific metabolic mechanisms that translate oxygen radical production into airways constriction are yet to be identified. In the lung, calcium homeostasis is central to release of bronchoactive and vasoactive chemical mediators and to regulation of smooth muscle cell contractility, i.e. airway constriction. In the present work, we characterized Ca2+-transport in the microsomal fraction of mouse lungs, and determined how reactive oxygen species, generated by Fe2+/ascorbate and H2O2/hemoglobin, affected Ca2+ transport. The microsomal fraction of pulmonary tissue accumulated 90 ± 5 nmol Ca2+/mg protein by an ATP-dependent process in the presence of 15 mM oxalate, and 16 ± 2 nmol Ca2+ in its absence. In the presence of oxalate, the rate of Ca2+ uptake was 50 ± 5 nmol Ca2+/min per mg protein at pCa 5.9(37°C). The Ca2+-ATPase activity was 50–60 nmol Pi/min per mg protein (pCa 5.9, 37°C) in the presence of alamethicin. Inhibitors of mitochondrial H+-ATPase had no effect on the Ca2+ transport. Half-maximal activation of Ca2+ transport was produced by 0.4–0.5 μM Ca2+. Endoplasmic reticulum Ca2+-pump (SERC-ATPase) was found to be predominantly responsible for the Ca2+-accumulating capacity of the pulmonary microsomes. Incubation of the microsomes in the presence of either Fe2+/ascorbate or H2O2/hemoglobin resulted in a time-dependent accumulation of peroxidation products (TBARS) and in inhibition of the Ca2+ transport. The inhibitory effect of Fe2+/ascorbate on Ca2+ transport strictly correlated with the inhibition of the Ca2+-ATPase activity. These results are the first to indicate a highly active microsomal Ca2+ transport system in murine lungs which is sensitive to endogenous oxidation products. The importance of this system to pulmonary disorders exacerbated by oxidative chemicals remains to be studied.