Enhanced Calcium Uptake Research Articles

Cumulative depletions of extracellular calcium in rabbit ventricular muscle monitored with calcium-selective microelectrodes.

Transient changes of extracellular free calcium in rabbit ventricular muscle under nonsteady state conditions were measured with double-barreled calcium microelectrodes. Resumption of stimulation after a rest interval produces a cumulative decrease of extracellular free calcium often by more than 10% (with bulk extracellular free calcium = 0.2 mM). The extracellular free calcium returns to the bulk value as a new steady state is achieved. The changes of extracellular free calcium recorded presumably represent net calcium uptake and loss by cardiac muscle cells. These cumulative extracellular free calcium depletions are blocked by 0.5 mM cobalt and 1 microM nifedipine and are increased to 167 +/- 11% of control by the calcium agonist Bay k 8644 (1 microM) and to 620 +/- 150% of control by increasing stimulus frequency from 0.2-2 Hz. Caffeine (10 mM) inhibits the cumulative extracellular free calcium depletions, probably by rendering the sarcoplasmic reticulum unable to accumulate calcium. It is proposed that the extracellular free calcium depletions recorded represent, in large part, calcium which has entered the cells and has been taken up by the sarcoplasmic reticulum (which had become depleted of calcium during the rest interval). Nifedipine and cobalt inhibit these cumulative depletions presumably by preventing the calcium entry which could subsequently be accumulated by the sarcoplasmic reticulum. The net cellular calcium uptake produced by such a post-rest stimulation protocol can also be inhibited by 1-3 microM acetylstrophanthidin and reduction of extracellular sodium to 70 mM. Acetylstrophanthidin and low extracellular sodium would be expected to shift the sodium-calcium exchange in favor of increased calcium uptake, which may, in turn, prevent the loss of sarcoplasmic reticulum calcium during the rest interval. This would limit the amount of calcium which the sarcoplasmic reticulum could take up with subsequent activation. In contrast to the results with caffeine, ryanodine (1 microM) increases the magnitude and rate of calcium uptake after a rest interval, indicative of a fundamental difference in the actions of caffeine and ryanodine. When stimulation is stopped in the presence of ryanodine, extracellular free calcium increases much faster than in control. This suggests that ryanodine may enhance calcium uptake by the sarcoplasmic reticulum during repetitive stimulation and may enhance calcium efflux from the sarcoplasmic reticulum during quiescence. These experiments provide insight into transsarcolemmal calcium movements and certain aspects of cellular calcium regulation.

Calcium uptake during insulin-aggravated ischemic myocardial contracture in the rat heart.

Perfusion of isolated, nondiabetic rat hearts with high concentrations of regular insulin (10 mU/ml) produced earlier onset of myocardial contracture during ischemia than control perfusions without insulin. High insulin concentrations also increased total myocardial calcium content (p less than .001) and myocardial Ca-45 uptake postischemia (p less than .05). Insulin perfusion before ischemia did not enhance calcium uptake. These results support the hypothesis that insulin may increase calcium movement across myocardial cell membranes, which may lead to increased ischemic contracture. Insulin-aggravated myocardial ischemia in cardiothoracic surgery and cardiac resuscitation deserves further investigation.

Evidence of regulation of calcium uptake by phytochrome in maize protoplasts

Red light stimulated calcium uptake in maize leaf protoplasts upto 140% over the dark control. The stimulation was maximally noticed after 120 seconds of incubation and was reversed by far red light. 5-Hydroxytryptamine also enhanced calcium uptake in dark, upto the red level. A possible role of phosphoinositide for signal transduction in calcium uptake for phytochrome mediated responses in higher plants is suggested.

Agents that increase cellular cyclic AMP or calcium stimulate prolactin release from the 235-1 pituitary cell line

The 235-1 pituitary tumor clone was utilized to study prolactin secretion after perturbing cyclic AMP and calcium metabolism. Cellular cyclic AMP levels were elevated after treatment with PGE 1, cholera toxin, forskolin, isobutyl-methylxanthine as well as dibutryl cyclic AMP; these cyclic AMP responses were associated with increased prolactinx release. Ionophore A23187 and maitotoxin, which enhance calcium uptake into cells, also amplified prolactin secretion. In contrast, the calmodulin antagonists penfluridol and W7 reduced basal prolactin release. These data support the hypothesis that cyclic AMP, calcium and calmodulin can participate in prolactin release from 235-1 cells.

Stimulation of calcium uptake in rat calvaria by prostacyclin

Treatment of newborn rat calvaria discs with a variety of unsaturated fatty acids led to a 50% enhancement of calcium uptake. Arachidonic acid was effective at lower concentrations than cis-vaccenic or oleic acid, while trans-vaccenic acid and saturated fatty acids did not enhance calcium uptake. Cyclooxygenase inhibitors indomethacin and acetylsalacylic acid abolished the enhancement of calcium uptake seen in response to cis-vaccenic acid and inhibited calcium uptake by otherwise untreated bones. Prostacyclin was found to produce up to 2 fold stimulation of calcium uptake with an EC 50 of approximately 0.1 μM. No statistically significant stimulation of calcium uptake was seen in response to PGE 2 or PGF 1α up to 25 μM, whilw slight stimulation was produced by 6-keto PGF 1α but only at concentrations of 10 μM. Prostacyclin production by calvaria was demonstrated and was stimulated over 50% by cis-vaccenic acid. These results suggest that not only is enhanced prostacyclin production responsible for elevation of calcium uptake in response to unsaturated fatty acids, but also that prostacyclin may be an important regulator of bone calcium homeostasis.

The effect of streptozocin-induced diabetes on the plasma membrane calcium uptake activity of rat liver.

The effect of streptozocin (STZ)-induced diabetes on the plasma membrane calcium uptake of rat liver was investigated. Plasma membrane preparations from diabetic rats showed a 2-3-fold increase in calcium uptake activity over controls 3-4 wk after the initial injections. Such an increase can be either reversed or blocked by treating the diabetic rats with exogenous insulin or administering nicotinamide 15 min before and 3 h after the STZ injection, respectively. The activity of 5'-nucleotidase and the [3H]ouabain binding to the plasma membranes were similar in samples from both the control and diabetic rats. These findings made it unlikely that preferential enrichment of plasma membranes or increased proportion of inside-out vesicles was the cause of the enhanced calcium uptake activity in membranes from diabetic animals. In addition, the effect of diabetes on the calcium uptake activity did not diminish even when the assay was performed in the presence of 2.5 microM ruthenium red, an inhibitor of calcium uptake by mitochondria, or when oxalate was omitted from the assay, suggesting that the effect was specifically on the plasma membrane pump. The enhanced calcium uptake activity was a result of an increase in the Vmax (58.8 versus 113.1 pmol calcium/mg protein/min for control and diabetic rats, respectively). No significant change in Km for calcium was detected.

Humoral immunostimulation: X. Cytochalasin B stimulates complement-dependent calcium uptake in antibody-treated cells

Cytochalasin B exerted a biphasic effect upon the uptake of calcium in L cells treated with anti-L-cell antibody and complement. At a high concentration (1 × 10 −4 M), cytochalasin B inhibited the antibody- and complement-induced stimulation of calcium uptake into L cells; whereas, at a lower concentration (1 × 10 −6 M), the drug enhanced uptake. Cytochalasin B-induced stimulation of calcium uptake was exclusively dependent upon the concentration of the antibody-antigen-activated complement system since the drug did not enhance calcium uptake in cells treated with antibody alone nor in cells treated with antibody and heat-inactivated complement. Moreover, when special serums deficient or depleted of certain complement components were used as sources of complement, there was a loss of the enhancement of calcium uptake. Thus, guinea pig serums deficient in C4 or C3-C9, human serums deficient in Cl r, C2, C2,4 or C4, and rabbit serums deficient in C3-C9 or C6 all failed to permit enhanced calcium uptake in antibody-treated cells in the presence of 1 × 10 −6 M cytochalasin B in contrast to normal guinea pig, human, or rabbit serums. Stimulation of calcium uptake in antibody- and complement-treated cells by cytochalasin B was exquisitely dependent upon cellular energy sources since the stimulation was abrogated when uptake studies were performed in the presence of the metabolic inhibitors, dinitrophenol and iodoacetate. Enhanced calcium uptake by cytochalasin B was not due to simple increased intracellular volume of treated cells nor to increased antibody (IgG) binding. Likewise, antibody and complement did not alter either high- or low-affinity binding of Cytochalasin B to cells. Substrate saturation studies suggested that the enhanced calcium uptake in the presence of cytochalasin B was due to an increase in the V max of transport with utilization of the same K m. CB did not appear to produce this effect upon calcium transport through its well-known effects upon glucose transport since CB augmented calcium uptake in the absence as well as in the presence of glucose. In summary, it appears that cytochalasin B modulates calcium uptake in antibody-treated L cells via interaction with an intact complement pathway and that the effect is due to an energy-requiring activation of the components of a transport system.

A possible mechanism for relaxation of rat uterine smooth muscle by nicardipine hydrochloride (YC-93), a new potent vasodilator.

A new potent vasodilator, nicardipine hydrochloride inhibited oxytocin-induced contraction of rat uterus dose-dependently with an increase in the intracellular cyclic AMP level at the onset of relaxation. Dibutyryl cyclic AMP and papaverine, an inhibitor of cyclic AMP phosphodiesterase (PDEase), also inhibited the contraction. Nicardipine inhibited competitively PDEase in homogenates of rat uterus which exhibited apparently two Km values for cyclic AMP (3.6 micro M and 67.3 micro M) with the Ki of 5.3 micro M and 13.2 micro M, respectively, but had no effect on adenylate cyclase. Nicardipine enhanced calcium uptake by rat uterine microsomes, at concentrations which inhibited oxytocin-induced contraction in the same manner as cyclic AMP. The maximal stimulation by nicardipine of the microsomal calcium uptake was identical substantially to that by cyclic AMP, and both were not additive. Cyclic AMP was also accumulated during the uptake reaction in the presence of nicardipine. On the contrary, neither myosin ATPase nor microsomal Ca2+-dependent ATPase was inhibited directly by nicardipine. These results suggest that the inhibition of oxytocin-induced contraction of rat uterus by nicardipine may be due to an enhancement of microsomal calcium uptake, mediated by cyclic AMP accumulated through the inhibition of PDEase.

A POSSIBLE MECHANISM FOR RELAXATION OF RAT UTERINE SMOOTH MUSCLE BY NICARDIPINE HYDROCHLORIDE (YC-93), A NEW POTENT VASODILATOR

A new potent vasodilator, nicardipine hydrochloride inhibited oxytocin-induced contraction of rat uterus dose-dependently with an increase in the intracellular cyclic AMP level at the onset of relaxation. Dibutyryl cyclic AMP and papaverine, an inhibitor of cyclic AMP phosphodiesterase (PDEase), also inhibited the contraction. Nicardipine inhibited competitively PDEase in homogenates of rat uterus which exhibited apparently two Km values for cyclic AMP (3.6 μM and 67.3 μM) with the Ki of 5.3 μM and 13.2 μM, respectively, but had no effect on adenylate cyclase. Nicardipine enhanced calcium uptake by rat uterine microsomes, at concentrations which inhibited oxytocin-induced contraction in the same manner as cyclic AM P. The maximal stimulation by nicardipine of the microsomal calcium uptake was identical substantially to that by cyclic AMP, and both were not additive. Cyclic AMP was also accumulated during the uptake reaction in the presence of nicardipine. On the contrary, neither myosin ATPase nor microsomal Ca2+-dependent ATPase was inhibited directly by nicardipine. These results suggest that the inhibition of oxytocin-induced contraction of rat uterus by nicardipine may be due to an enhancement of microsomal calcium uptake, mediated by cyclic AMP accumulated through the inhibition of PDEase.

Neutrophil aggregation: evidence for a different mechanism of action by phorbol myristate acetate.

AbstractChemotactic factors, the ionophore A23187, and arachidonic acid aggregate polymorphonuclear neutrophils. They appear to act in part by causing neutrophils to take up extracellular calcium and to metabolize arachidonic acid. Hence, the aggregation response to these stimuli requires extracellular calcium, is enhanced by cytochalasin B (which enhances calcium uptake in stimulated cells), and is inhibited by arachidonate antimetabolites. Here, phorbol myristate acetate was also found to aggregate neutrophils. However, this response was relatively insensitive to extracellular calcium, cytochalasin B, and arachidonate antimetabolites. The mechanism of action of the phorbol ester, therefore, may not require calcium influx nor arachidonate metabolism. This suggests that neutrophil aggregation can occur through at least two different mechanisms. Other neutrophil responses (e.g., release of specific granule enzymes and oxidative metabolism) may also proceed through these same mechanisms. Phorbol myristate a...

Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcoplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg 2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol P i/mg protein were incorporated in 10 min at 30°C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25–100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occured on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca 2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca 2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.

Transient 45Ca uptake and release in isolated rat-liver cells during recovery from deenergized states.

1. Aerobic incubation of isolated rat liver cells--after dilution from the anaerobic stock suspension--transiently brings about a state, during which a reversible calcium uptake can be observed on addition of a respiratory substrate. Uptake varies greatly and can reach more than 50 nmol/mg protein, but declines to zero on prolonged preincubation, especially at higher temperature. Repeated additions of succinate or 3-hydroxybutyrate evoke new calcium transients. If ATP is simultaneously added, if greatly potentiates succinate-initiated reversible uptake. 2. If rotenone is present during the preincubation phase, calcium transients are strongly enhanced. Uptake is blocked by uncouplers and respiratory inhibitors, indicating the involvement of mitochondria. 3. Calcium uptake is not accompanied by increased oxygen consumption. The actual respiration cannot account sufficiently for the energy need of calcium uptake. Participation of cytoplasmic ATP is likely, as inhibitors of adenine nucleotide translocase affect uptake. 4. Lanthanum enhances calcium uptake in contrast to its action on mitochondria. 5. Pulse-labeling experiments indicate that the calcium taken up is removed from a rapidly exchangeable calcium pool by withdrawal into the mitochondria as a deep compartment. 6. Calcium uptake is accelerated either by increasing the phosphate level or by high temperature. It is prolonged by low temperature, high pH or high ATP concentration. Calcium release accelerates with increasing temperature, decreasing pH and a further rise in phosphate concentration. 7. The dependency on phosphate and temperature reveals a delicately poised equilibrium of uptake and release. At ambient temperature, phosphate increases uptake up to a concentration of 0.5 mM. Higher concentrations accelerate both uptake and release. At lower temperature, the accelerating effect on uptake predominates. A temperature shift during incubation results in adaptation of the calcium equilibrium to the new temperature, i.e. release of calcium at high temperature, uptake at low temperature. 8. Oxidizing metabolites inhibit succinate-stimulated calcium uptake and promote release of previously accumulated calcium. An increased sensitivity to phosphate is established. 9. With respect to isolated mitochondria, isolated liver cells appear to be a more realistic model for studying the physiological mechanism of mitochondrial calcium release, since compartmental constraints and regulations are maintained.

Role of cyclic AMP in rat aortic microsomal phosphorylation and calcium uptake

The role of adenosine 3',5'-monophosphate (cyclic AMP)-dependent membrane phosphorylation in the regulation of microsomal calcium transport in rat aortic smooth muscle was studied. Cyclic AMP-dependent protein kinase augmented the phosphorylation of serine residues in a microsomal protein component with a molecular weight of about 44,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the majority of 32P incorporation was in serine residue(s). The phosphorylated protein had stability characteristics of a phosphoester. The phosphorylated substrate was not extracted from the trichloroacetic acid (TCA) precipitate with organic solvents or by suspension in hot TCA; and the demonstrated hydroxylamine insensitivity suggested that the substrate was not lipid or nucleic acid. Intrinsic phosphoprotein phosphatase cleaved the labeled phosphate from the cyclic AMP-stimulated microsomes in the first 5 min of incubation. Microsomes phosphorylated in the presence of 1 micron cyclic AMP or 1 micron cyclic AMP plus 0.1 mg/ml protein kinase exhibited enhanced calcium uptake. We suggest that reversible phosphorylation of microsomal membranes may play an important role in the regulation of aortic microsomal calcium transport by cyclic AMP.

Embryonic chick intestine in organ culture: interaction of adenylate cyclase system and vitamin D3-mediated calcium absorptive mechanism.

Embryonic chick duodenum can be maintained in organ culture in contact with a serum-free medium with excellent preservation of mucosal structure. The presence of vitamin D3 in the culture medium induces de novo synthesis of a calcium-binding protein (CaBP) and stimulates uptake and mucosal to serosal transport of calcium in the cultured intestine. Using this system, it was found that vitamin D3 in the culture medium elevated duodenal cyclic AMP concentration after only 3hr of incubation. This response was prior to induction of CaBP and stimulation of calcium accumulation. Following a decline to control levels at 6 hr, there was a continuous rise in duodenal cyclic AMP, paralleling CaBP synthesis and enhanced calcium uptake, throughout the 48-hr culture period. Addition of either dibutyryl cyclic AMP (dbcAMP) or theophylline to the medium at lmM stimulated 45Ca accumulation by the intestine regardless of the presence of vitamin D3,although stimulation was greatest in the presence of the sterol, and the eff...

Ultrastructural changes in the plasma membrane and sarcoplasmic reticulum of myocardial cells during hibernation

The ultrastructure of myocardial cellular membranes in control ground squirrels was compared with that of hibernating ground squirrels. In the control ground squirrels, the cell coat portion of the plasma membrane averaged 249.9 Å in thickness and was less electronopaque than that of the hibernating ground squirrels, whose coat averaged 352.0 Å. Electron and light microscopic stains indicate that this coat contains a significant proportion of acid mucopolysaccharides. The sarcoplasmic reticulum is more evident, and the lateral sacs of the reticulum are more electron-opaque during hibernation. It is suggested that these changes may enhance calcium uptake and thereby facilitate excitation-contraction coupling during hibernation.