Kinetics Of Calcium Uptake Research Articles

Equilibrium interactions of biomimetic DNA aptamers produce intrafibrillar calcium phosphate mineralization of collagen

Native and biomimetic DNA structures have been demonstrated to impact materials synthesis under a variety of conditions but have only just begun to be explored in this role compared to other biopolymers such as peptides, proteins, polysaccharides, and glycopolymers. One selected DNA aptamer has been explored in calcium phosphate and calcium carbonate mineralization, demonstrating sequence-dependent control of kinetics, morphology, and crystallinity. This aptamer is here applied to a biologically-relevant bone model system that uses collagen hydrogels. In the presence of the aptamer, intrafibrillar collagen mineralization is observed compared to negative controls and a positive control using well-studied poly-aspartic acid. The mechanism of interaction is explored through affinity measurements, kinetics of calcium uptake, and kinetics of aptamer uptake into the forming mineral. There is a marked difference observed between the selected aptamer containing a G-quadruplex secondary structure compared to a control sequence with no G-quadruplex. It is hypothesized that the equilibrium interaction of the aptamer with calcium-phosphate precursors and with the collagen itself leads to slow kinetic mineral formation and a morphology appropriate to bone. This points to new uses for DNA aptamers in biologically-relevant mineralization systems and the possibility of future biomedical applications. Statement of significanceCollagen is the protein structural component that mineralizes with calcium phosphate to form durable bone. Crystalline calcium phosphate must be infused throughout the collagen fiber structure to produce a strong material. This process is assisted by soluble proteins that interact with both calcium phosphate precursors and the collagen protein and has been proposed to follow a polymer-induce liquid precursor (PILP) model. Further understanding of this model and control of the process through synthetic, biomimetic molecules could have significant advantages in biomedical, restorative procedures. For the first time, synthetic DNA aptamers with specific secondary structures are here shown to influence and direct collagen mineralization. The mechanism of this process has been studied to demonstrate an important equilibrium between the DNA aptamer, calcium phosphate precursors, and collagen.

Interactive effects of copper and calcium in Daphnia pulex

<p>Many freshwater habitats around the world suffered dramatic water chemistry changes over the last century mostly due to anthropogenic activities, including an overall reduction in pH due to high sulfur emissions and unsustainable forestry practices. One consequence of this change in water chemistry is a drop in available calcium concentration, which creates problems for aquatic organisms that rely on dissolved calcium to build their exo- or endoskeletons and reinforce their carapace during regular molts. <em>Daphnia</em> populations in shield lakes in northern Ontario are also exposed to other stressors, including copper, which persists at high concentrations in many of these freshwater lakes and ponds due to mining and other human activities. Copper toxicity on animals is influenced by the availability of other competing ions, such as calcium. Using our newly developed high throughput toxicity screening system, we show that mortality of <em>Daphnia pulex</em> increases with exposure to low calcium (0.05 mg L<sup>-1</sup>) and high copper (300 µg L<sup>-1</sup>). When these two stressors were combined, we found that copper was less toxic at high calcium concentrations, indicating a protective effect of calcium against copper toxicity. We then established basic calcium uptake kinetics in <em>D. pulex</em> using radioactive tracer <sub>45</sub>Ca and provide evidence that copper, at environmentally relevant concentrations, competes with calcium uptake based on K<sub>m</sub> and V<sub>max</sub>. Our data show that both calcium decline and copper increase in aquatic ecosystems may negatively impact natural <em>Daphnia</em> populations, and that interactions between these two metals may occur in natural environments that result in fitness consequences for zooplankton. </p>

Inhibition of miR-25 improves cardiac contractility in the failing heart

Heart failure is characterized by a debilitating decline in cardiac function1, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy2,3. microRNAs (miRs) are dysregulated with heart failure4,5 but whether they control contractility or constitute therapeutic targets remain speculative. Using high throughput, functional screening of the human microRNAome, we identified miRs that suppress intracellular calcium handling in heart muscle by interacting with mRNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a. Of 875 miRs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas AAV9-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 dramatically halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggests that it might be targeted therapeutically to restore function.

Exercise Training Decreases Arrhythmias But Is Not Associated With Improved Mitochondrial Calcium Retention Capacity

PURPOSE: To determine if 10 days of exercise training decreases cardiac arrhythmias in the isolated female rat heart, and to determine if mitochondrial adaptations are involved. We hypothesized that exercise would promote an anti-arrhythmic phenotype characterized by improved calcium tolerance, improved ROS scavenging by superoxide dismutase (SOD), and delayed mitochondrial permeability transition pore opening. METHODS: Female Sprague-Dawley rats were randomly assigned to either the sedentary handling control (Sed) or the exercise trained (Ex) group. Animals in the Ex group were placed on a motorized treadmill at speeds of 15/30/15 meters per minute for 15/30/15 minutes respectively. Animals in the Sed group were handled and placed on the treadmill each day. Exercise duration was 10 days. Twenty-four hours after the last session hearts were excised via thoracotomy and either instrumented for isolated heart experiments or used for mitochondrial isolations. Isolated hearts were exposed to global ischemia for 30 minutes, followed by 30 minutes of reperfusion. RESULTS: Ex led to a significant decrease in the severity of arrhythmias by: 1) reducing the arrhythmia score (5.7 ± 0.9 and 3.0 ± 0.6, for Sed and Ex group respectively, P < 0.05) and 2) decreasing the time spent in a ventricular arrhythmia (11 ± 3 and 2 ± 1 minutes, for Sed and Ex group respectively, P 0.05). However, during reperfusion coronary flow was significantly preserved in the Ex group (P < 0.05, RM ANOVA). Western blots for both SOD isoforms showed that neither SOD1 nor SOD2 protein levels were increased following Ex. In isolated mitochondria, the calcium retention capacity in Ex animals was significantly lower than Sed counterparts (433 ± 33 and 343 ± 10 nmoles of calcium, for Sed and Ex respectively, P < 0.05). CONCLUSIONS: Our results suggest that Ex causes intrinsic changes in the female heart that decrease the severity of arrhythmias without increases in protein expression of SOD1 or SOD2. Surprisingly, mitochondria of trained animals were more sensitive to calcium-induced opening of the mitochondrial permeability transition pore. Future experiments investigating the kinetics of calcium uptake and extrusion in isolated mitochondria will improve our understanding of how mitochondrial calcium handling influences the propensity for arrhythmias.

Uptake Kinetics and Absorption of Calcium in Apple Matrices

The purpose of this paper was to study the kinetics of calcium uptake of blanched and not blanched apple tissue during impregnation treatments under vacuum or/and at atmospheric pressure. The relative absorption of calcium in rats, regarding calcium carbonate as reference substance, was also analyzed as a first approach. Apple impregnation is conducted (with and without previous blanching in saturated vapor) in isotonic glucose aqueous solutions containing calcium gluconate and lactate. A 50 mm Hg vacuum pressure is applied for a time t1 (0—180 min) or atmospheric pressure is used for a process time t2 (0—7.5 h). Calcium uptake by apple tissue is influenced by system pressure, blanching, and impregnation time, reaching 500—4600 μg Ca/g according to process variables. Preliminary data of true calcium absorption ranges between 74 and 86% regarding calcium carbonate.

D-galactosamine induced hepatocyte apoptosis is inhibited in vivo and in cell culture by a calcium calmodulin antagonist, chlorpromazine, and a calcium channel blocker, verapamil.

Studies were conducted in C57BL/6N Crj male mice and in cultured hepatocytes to clarify the relationship between galactosamine (GaIN) induced apoptosis and [Ca2+]i kinetics. Chlorpromazine (CPZ), a Ca(2+)-calmodulin antagonist, and verapamil (VR), a Ca(2+)-channel blocker each inhibited GaIN-induced DNA fragmentation and the appearance of apoptotic bodies. The kinetics of calcium uptake were evaluated using a calcium analyzer with the acetoxymethyl ester of fura-PE3 (fura-PE3/AM, 2.5 microM) as the calcium reporter. An increase in [Ca2+]i was detected in the cultured hepatocytes within 3 hours after treatment with 20 mM GaIN; this increase was inhibited by pretreatment with either 20 microM CPZ or 30 microM VR. Ca2+ imaging by confocal laser scanning microscopy showed that increase in [Ca2+]i after treatment with GaIN was initially localized around nuclei, while [Ca2+]i signals were later diffuse and observed throughout the cytoplasm. The activities of lactate dehydrogenase (LDH) and serum glutamate-pyruvate transaminase (sGPT), used as indicators of plasma membrane damage and leakage, however, were not reduced by pretreatment with CPZ or VR. From these findings, we infer that the DNA fragmentation in GaIN-induced hepatocyte apoptosis is associated with an elevation in the perinuclear concentration of Ca2+, but GaIN-induced necrotic cell death is triggered through pathway(s) that are insensitive to blockage of Ca2+ influx and therefore appear to occur independently of elevation in [Ca2+]i. These results help to clarify the role of calcium flux in hepatocyte apoptosis and necrosis induced by exposure to hepatotoxins in vivo and in vitro.

WATER RELATIONS, CALCIUM AND POTASSIUM CONCENTRATION IN FRUITS AND LEAVES DURING ANNUAL GROWTH IN MATURE KIWIFRUIT PLANTS

WATER RELATIONS, CALCIUM AND POTASSIUM CONCENTRATION IN FRUITS AND LEAVES DURING ANNUAL GROWTH IN MATURE KIWIFRUIT PLANTS

Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus

The toxic effects of Cd2+ on Ca2+ influx kinetics in developing tilapia (Oreochromis mossambicus) larvae were evaluated. Addition of 20 µg l-1 of Cd2+ to the environment of 0 and 3 day-old larvae competitively inhibited the Ca2+ uptake within 4h resulting in a great increase in Km values for Ca2+ influx (19.3 and 17.4 fold, respectively) as compared with their respective controls. Consequently, the actual Ca2+ influx of larvae in solutions of 0.2 mM Ca2+ are suppressed by 32–45%. Also, 3 day-old larvae were more sensitive to internally accumulated Cd2+ than 0 day-old larvae. Although the Ca2+ influx in 0 and 3 day-old larvae may be restored to the levels of their respective controls with 24h of being transferred to a 20 µg l-1 Cd2+ solution, total body Ca2+ content was significantly reduced in 3 day-old larvae. Increased Ca2+ uptake efficiency ensures sufficient Ca2+ for normal growth. However, rapid increase in Ca2+ influx after hatching also leads to higher Cd2+ uptake. Exposure to Cd2+ will lead to a drop in body Ca2+ content resulting in retardation of larval growth. Therefore, we conclude that if Ca2+ uptake is interfered with at this critical stage of development, larvae will not be able to maintain normal levels of body Ca2+ and will show signs of Cd2+ poisoning.

Effect of environmental calcium levels on calcium uptake in tilapia larvae Oreochromis mossambicus

Effects of environmental calcium concentrations on the survival, growth, body calcium content and calcium uptake kinetics in developing tilapia (Oreochromis mossambicus) larvae were studied. Fertilized eggs were incubated in high- and low-calcium artificial freshwater (0.88-0.96 mmol l(-1) vs. 0.02-0.03 mmol l(-1) CaCl2 or CaSO4) until 3 days after hatching. Tilapia larvae showed similar hatching rates and wet weights in either high- or low-calcium medium, indicating neither the development nor the growth in tilapia larvae was affected by the environmental calcium levels. The body calcium content in low-calcium groups was about 90-95% that of high-calcium groups, No matter what calcium source was used (CaCl2 or CaSO4), acclimation to low calcium medium caused a stimulation of calcium uptake (measured in 0.2 mmol l(-1) calcium),i.e., 1.2-1.3 fold higher than that of high calcium groups. This enhanced calcium uptake capacity was characterized by a 50% decrease in Km and a 25% increase in Jmax. Effect of different calcium salts on calcium influx was significant only in low calcium level,i.e., calcium influx in low-CaCl2 group higher than that in low-CaSO4 group. These results suggest that tilapia larvae are able to modulate their calcium uptake mechanism to maintain normal body calcium content and growth in environments with different levels of calcium.

Mechanisms for zinc acclimation in freshwater rainbow trout

A sublethal effect of zinc exposure in freshwater fish is the inhibition of calcium uptake. However, calcium also inhibits zinc uptake and this inhibition has been found to be competitive (Spry D.J. & Wood C.M. (1989). J. Exp. Biol., 142, 425–446). The subject of the acclimation of aquatic animals to metals is well researched, but there is, as yet, no simple answer as to how this acclimation is accomplished. The objectives of the present study were to characterize the interactions of zinc on calcium uptake kinetics, and to investigate if a restoration of calcium influx was associated with zinc acclimation. In addition, the possible involvement of metallothionein and a reduced zinc influx in the acclimation process was studied.

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca(2+) uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 μM CaCl2 solution (pH 4.5) for 3 d. Subsequently, net Ca(2+) fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca(2+)-selective microelectrode. The kinetics of Ca(2+) uptake into cells of the root apex, for external Ca(2+) concentrations from 20 to 300 μM, were found to be quite similar for both cultivars in the absence of external Al; Ca(2+) transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 μM total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca(2+) absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca(2+) uptake over the entire Ca(2+) concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca(2+) uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 μM) caused the K m for Ca(2+) uptake to increase with each rise in Al concentration, from approx. 100 μM in the absence of Al to approx. 300 μM in the presence of 10 μM Al, while having no effect on the V max. The same Al exposures had little effect on the kinetics of Ca(2+) uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca(2+) transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca(2+)-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.

Calcium uptake kinetics into brush-border membrane vesicles: Higher Vmax in the spontaneously hypertensive rat than in normotensive control

Arterial hypertension in the spontaneously hypertensive rat (SHR) is associated with an abnormal Ca 2+ homeostasis, compared with its normotensive control, the Wistar Kyoto rat (WKY). In particular, epithelial Ca 2+ transport is perturbed, with intestinal absorption and renal tubular reabsorption being decreased in the adolescent and adult SHR. In the present study we examined Ca 2+ uptake into isolated duodenal brush-border membrane vesicles (BBMV) in 12–14 week-old male rats. This uptake can be separated in a non-saturable and a saturable component. The latter follows Michaelis-Menten kinetics. Vmax of this component was found to be significantly higher (p<0.05) in SHR than in WKY (0.58±0.19 versus 0.35±0.06 nmol/mg protein × 10 sec, mean±SD) whereas Km did not differ. Thus, the defect in the intestinal Ca 2+ absorption previously identified in the SHR of this age is not due to a decrease in Ca uptake at the level of the duodenal brush-border membrane, but is most likely located in the baso-lateral membrane.

Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane

Direct measurements of phosphorylation of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) have shown that the lifetime of the first phosphorylated intermediate in the Ca2+ transport cycle, E1 approximately P, increases with decreasing [Mg2+] (Dupont, Y. 1980. Eur. J. Biochem. 109:231-238). Previous x-ray diffraction work (Pascolini, D., and J.K. Blasie. 1988. Biophys. J. 54:669-678) under high [Mg2+] conditions (25 mM) indicated that changes in the profile structure of the SR membrane could be responsible for the low-temperature transient trapping of E1 approximately P that occurs at temperatures below 2-3 degrees C, the upper characteristic temperature th for lipid lateral phase separation in the membrane. We now present results of our study of the Ca2+ uptake kinetics and of the structure of the SR membrane at low [Mg2+] (less than or equal to 100 microM). Our results show a slowing in the kinetics of both phases of the Ca2+ uptake process and an increase in the duration of the plateau of the fast phase before the onset of the slow phase, indicating an increase in the lifetime (transient trapping) of E1 approximately P. Calcium uptake kinetics at low [Mg2+] and moderately low temperature (approximately 0 degree C) are similar to those observed at much lower temperatures (approximately -10 degrees C) at high [Mg2+]. The temperature-induced structural changes that we observed at low [Mg2+] are much more pronounced than those found to occur at higher [Mg2+]. Also, at the lower [Mg2+] the upper characteristic temperature th for lipid lateral phase separation was found to be higher, at approximately 8-10 degrees C. Our studies indicate that both temperature and [Mg2+] affect the structure and the functionality (as measured by changes in the kinetics of Ca2+ uptake) of the SR membrane. Membrane lipid phase behavior and changes in the Ca2+ ATPase profile structure seem to be related, and we have found that structural changes are responsible for the slowing of the kinetics of the fast phase of Ca2+ uptake, and could also mediate the effect that [Mg2+] has on E1 approximately P lifetime.

Low molecular weight factor in bovine caudal epididymal fluid that stimulates calcium uptake in caput spermatozoa.

Secretions from the mammalian epididymis contain proteins that bind to developing sperm and are presumed to play a role in sperm maturation. The biochemical functions in sperm of most of these proteins are not known. In this report we describe the presence of a low molecular weight compound in bovine caudal epididymal luminal fluid (CF) that has a potent stimulatory effect on calcium (45Ca2+) uptake in immature caput epididymal spermatozoa. The studies were initially undertaken to characterize the effect of the protein caltrin, present in bovine seminal plasma (BSP), on calcium uptake into caput spermatozoa. Caltrin is known to block calcium influx into mature bovine sperm. Unexpectedly, the kinetics of calcium uptake into caput sperm showed a biphasic response when treated with BSP, namely, a stimulation of uptake at 1 to 5 min and inhibition of uptake after this time. Since caudal sperm do not show this biphasic response, we reasoned that BSP contained a factor derived from CF that must interact with developing sperm before the binding of caltrin to sperm can prevent further calcium uptake. We first demonstrated that preincubation of caput sperm with CF eliminated the biphasic calcium uptake effect induced in caput sperm by BSP and that caudal fluid alone had a potent stimulatory effect on calcium uptake in caput sperm. Half-maximal stimulation (fivefold over control) occurred at a caudal fluid protein concentration of 0.27 mg/ml. Partial purification of the factor indicates that it is of low molecular weight (MW approximately 1,000), but further chemical characterization has not been carried out and its epididymal site of origin is not known. The results indicate that the regulation of intracellular calcium levels in sperm differs in immature and mature bovine sperm in that an epididymal factor promotes calcium uptake during epididymal maturation, and the seminal fluid protein caltrin prevents it at ejaculation.

Polyamines Stimulate Mitochondrial Calcium Transport in Rat Brain

The effects of the polyamines spermine and spermidine on rat brain mitochondrial calcium transport were examined using a variety of techniques for measuring the kinetics of calcium uptake and the buffering capabilities of isolated mitochondria. Spermine both increased the rate of calcium accumulation and decreased the set-point to which isolated mitochondria buffer free calcium concentration. In the presence of physiological concentrations of sodium and magnesium, spermine lowered the extramitochondrial calcium level to approximately 0.3 microM, a value close to the resting intracellular calcium concentration. The effect of polyamines was concentration dependent, with a half-maximal effect of spermine observed at approximately 0.1-0.4 mM (respiratory substrate dependent), whereas spermidine was approximately 10 times less potent. Calcium transport by hippocampal mitochondria was stimulated markedly more by spermine than was calcium transport by mitochondria isolated from brainstem. The stimulatory effect of spermine was not due to an increase in the transport of respiratory substrates inside the mitochondria nor to an effect on the enzymes using these respiratory substrates. An examination of the effect of spermine on the kinetics of calcium uptake indicated that spermine increased calcium uptake maximally at low calcium concentrations. Beyond that level, the stimulatory effect of spermine decreases, and spermine can even inhibit calcium uptake. These results are in good agreement with previous reports on the effects of polyamines on calcium transport in mitochondria from peripheral tissue. They support the hypothesis that spermine increases the rate of calcium uptake by mitochondria by increasing the affinity of the uniporter for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal maturation: calcium transport by basolateral membranes.

The overall characteristics of calcium transport across the intestine have been defined using in vivo perfusion techniques and in vitro everted gut sacs. However, calcium transport represents three separate processes: entry at the brush border membranes, movement across the cytoplasm, and exit at the basolateral membranes (BLM). Studies in adult animals indicated that the active step in calcium transport is located at the BLM. The present studies describe for the first time the maturational aspects of calcium transport across the BLM of the enterocyte. We utilized a percoll density gradient to prepare enriched BLMs from suckling and adolescent rats. Calcium uptake into BLMs represented mainly transport into the intravesicular space. Calcium transport in both age groups was driven by ATP; the calcium transport in the presence and absence of ATP was significantly greater in suckling rats compared to adolescent rats. Kinetics of calcium uptake calculated from uptake values in the presence of ATP minus no ATP conditions showed a Km of 0.06 +/- 0.01 and 0.03 +/- 0.01 microM for adolescent and suckling rats respectively (p less than 0.05). Vmax was 1.5 +/- 0.1 and 0.8 +/- 0.08 nmol/mg protein/min for adolescent and suckling rats respectively (p less than 0.01). Calcium/sodium exchange mechanisms was also present in both age groups. However, the magnitude of sodium-dependent calcium exchange was smaller in suckling rats compared to adolescent rats. These data suggest that calcium exit at the BLMs of enterocytes of suckling and adolescent rats occurs by an ATP-dependent and a calcium/sodium exchange mechanism. Both mechanisms exhibit maturational changes.

Calcium uptake kinetics in relation to conidiation in submerged cultures of Penicillium cyclopium

Uptake of calcium ions by Penicillium cyclopium in submerged culture was biphasic. The ion underwent rapid initial binding to the mycelium with a dissociation constant (KD) value of 0.114 mM-Ca2+ and binding capacity (Yt) of 3.5 nmol-Ca2+ (mg dry wt)−1. Metabolismindependent chemical adsorption of calcium was followed by metabolism-dependent active transport with a Km value of 1.88 m m and Vmax of 8.9 nmol-Ca2+ (mg dry wt)−1 min−1. Time-course experiments showed that exposures of mycelium to Ca2+ for periods as brief as 30 s were accompanied by substantial Ca2+ binding, but negligible active transport of the ion, and resulted in conidiation identical to that in cultures continuously exposed to Ca2+. The relationships between calcium binding and the mechanisms of conidiation in P. cyclopium are discussed.

Potentiation by chlordecone of the defect in hepatic microsomal calcium sequestration induced by carbon tetrachloride

The effect of carbon tetrachloride (CCl4) on the capacity of hepatic microsomes to sequester calcium was studied following pretreatment of rats with chlordecone. Chlordecone pretreatment alone had no effect on the kinetics of calcium uptake by hepatic microsomes. It was found, however, that chlordecone pretreatment of rats potentiated by sixfold the potency of CCl4 to suppress microsomal calcium sequestration capacity when measured one hour after CCl4 administration.

Uptake of extracellular calcium by neonatal neutrophils.

Neutrophils from human neonates exhibit abnormalities of plasma membrane and cytoskeletal dependent functions such as chemotaxis, deformability, and lectin capping. The uptake of extracellular calcium by neutrophils is a key early event in neutrophil activation and in the optimal performance of these functions. We found that neonatal and control neutrophils acquire extracellular radioactive calcium comparably under most experimental conditions. No differences (P greater than 0.05) were seen between neonatal and control neutrophils following stimulation with soluble sodium fluoride, N-formyl-methionyl-L-leucyl-L-phenylalanine, dimethylsulfoxide, and opsonized zymosan particles. However, the uptake of calcium by resting (unstimulated) neutrophils was significantly less (P less than 0.0001) by neonatal cells. The kinetics of calcium uptake by neonatal and control neutrophils were similar both at rest and following stimulation for 15 min with sodium fluoride. Although additional studies will be necessary to completely define the ionic events involved in the chemotaxis of neonatal neutrophils, it seems unlikely that the abnormal mobility of these cells is due simply to an inability of neonatal neutrophils to acquire extracellular calcium in response to cellular stimulation.

Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate.

The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.