Inhibition of Na+-coupled solute transport by calcium in brush border membrane vesicles

Intracellular Ca⁺⁺ is known to influence Na⁺ flux in luminal membranes. Abnormally elevated Ca⁺⁺ levels in some cells is believed to be the primary pathophysiologic defect in cystic fibrosis (CF). This in turn is thought to alter Na⁺ transport which accounts for certain clinical manifestations of this disease. Two Na⁺-dependent intestinal transport mechanisms have been reported to be suppressed or missing in CF. To examine whether alterations in cell Ca⁺⁺ may account for these findings, studies were performed to examine the influence of Ca⁺⁺ on Na⁺-solute co-transport across intestinal luminal membranes. Purified brush border membrane vesicles prepared from rat small bowel were preincubated in either Ca⁺⁺-free buffer or buffer containing 2.5 mM CaCl₂. Ca⁺⁺ loaded vesicles showed marked inhibition of Na⁺ co-transport of taurocholic acid, taurochenodeoxycholic acid, glucose and valine when compared to controls. The uptake of Na⁺ was also significantly reduced by intravesicular Ca⁺⁺. These data demonstrate that intravesicular Ca⁺⁺ inhibits Na⁺-coupled solute transport as well as Na⁺ influx across intestinal brush border membranes. These data suggest that intracellular Ca⁺⁺ may suppress Na⁺-dependent solute absorption in the intestine. Results presented here further support the theory that elevated intracellular Ca⁺⁺ may account for intestinal malabsorption and other altered transport phenomena reported in CF.     

Ca++ fluxes in isolated cells of rat pancreas. Effect of secretagogues and different Ca++ concentrations

Summary Secretagogues of pancreatic enzyme secretion, the hormones pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, and caerulein as well as the Ca⁺⁺-ionophore A 23187 stimulate⁴⁵Ca efflux from isolated pancreatic cells. The nonsecretagogic hormones adrenaline, isoproterenol, secretin, as well as dibutyryl cyclic adenosine 3,5-monophosphate and dibutyryl cyclic guanosine 3,5-monophosphate have no effect on⁴⁵Ca efflux. Atropine blocks the stimulatory effect of carbamylcholine on⁴⁵Ca efflux completely, but not that of pancreozymin. A graphical analysis of the Ca⁺⁺ efflux curves reveals at least three phases: a first phase, probably derived from Ca⁺⁺ bound to the plasma membrane; a second phase, possibly representing Ca⁺⁺ efflux from cytosol of the cells; and a third phase, probably from mitochondria or other cellular particles. The Ca⁺⁺ efflux of all phases is stimulated by pancreozymin and carbamylcholine. Ca⁺⁺ efflux is not significantly effected by the presence or absence of Ca⁺⁺ in the incubation medium. Metabolic inhibitors of ATP production, Antimycin A and dinitrophenol, which inhibit Ca⁺⁺ uptake into mitochondria, stimulate Ca⁺⁺ efflux from the isolated cells remarkably, but inhibit the slow phase of Ca⁺⁺ influx, indicating the role of mitochondria as an intracellular Ca⁺⁺ compartment. Measurements of the⁴⁵Ca⁺⁺ influx at different Ca⁺⁺ concentrations in the medium reveal saturation type kinetics, which are compatible with a carrier or channel model. The hormones mentioned above stimulate the rate of Ca⁺⁺ translocation.The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca⁺⁺ transport most likely at the level of the cell membrane and that Ca⁺⁺ exchange diffusion does not contribute to the⁴⁵Ca⁺⁺ fluxes.With the technical assistance of C. Hornung.     

Biological activities of cystic fibrosis serum. III. CF serum induced uptake of 45Ca++ by rabbit tracheal explants.

Cystic Fibrosis (CF) serum and its isolated component IgG fraction produce an increased uptake of ⁴⁵Ca⁺⁺ in rabbit tracheal explants when compared to control serum and its isolated IgG fraction. Heterozygote serum also produced an increased uptake of ⁴⁵Ca⁺⁺ but not to the same extent as CF serum. The calcium channel blocker D600 inhibited the CF serum induced uptake of ⁴⁵Ca⁺⁺ indicating that CF serum may be acting on the plasma membrane to produce changes in calcium permeability in rabbit tracheal explants.     

Release of endogenous dopamine from tuberoinfundibular neurons

Release of endogenous dopamine (DA) from arcuate-periventricular nucleus-median eminence fragments has been analyzed in an $\text{in vitro}$ static incubation system.Exposure of these hypothalamic fragments to increasing concentrations of K⁺ ions produced a dose-dependent release of endogenous DA. The highest rate of K⁺-stimulated DA efflux occurred in the first 10 minutes, thereafter it progressively decline reaching prestimulated levels at 30 minutes. If two consecutive depolarizing stimuli of 40 mM KCl were applied to the same hypothalamic fragment, after a 40 minutes rest period, an equivalent release of endogenous DA occurred. Removal of Ca⁺⁺ ions from the incubation medium containing the Ca⁺⁺ chelator EGTA caused a decrease of basal DA efflux and completely prevented the K⁺-induced release of DA.Furthermore when verapamil, a blocker of Ca⁺⁺ entrance, was added to the incubation medium in a concentration of 50 μM, the K⁺-induced DA efflux was completely counteracted, whereas spontaneous release was unmodified.Finally nomifensine, a potent blocker of DA uptake, added $\text{in vitro}$ in a final concentration of 10 μM, significantly reinforced K⁺-induced release of endogenous DA. Since nomifensine did not modify basal DA release, this study confirmed its prevalent uptake blocking property rather than its releasing action on DA.     

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

Active calcium ion uptake by inside-out and right side-out vesicles of red blood cell membranes

The relationship between active extrusion of Ca⁺⁺ from red cell ghosts and active uptake of Ca⁺⁺ by isolated red cell membrane fragments was investigated by studying the Ca⁺⁺ uptake activities of inside-out and right side-out vesicles. Preparations A and B which had mainly inside-out and right side-out vesicles, respectively, were isolated from red cell membranes and were compared with respect to Ca⁺⁺ adenosine triphosphatase (ATPase) and ATP-dependent Ca⁺⁺ uptake activities. Preparation A had nearly eight times more inside-out vesicles and took up eight times more ⁴⁵Ca in the presence of ATP compared to preparation B. Separation of the ⁴⁵Ca-labeled membrane vesicles by density gradient centrifugation showed that the ⁴⁵Ca label was localized to the inside-out vesicle fraction. In addition, the ⁴⁵Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of ⁴⁵Ca loss was not influenced by the presence of EGTA, but was slowed in the presence of La⁺⁸ (0.1 mM) in the efflux medium. The results presented here support the thesis that the active uptake of Ca⁺⁺ by red cell membrane fragments is due to the active transport of Ca⁺⁺ into inside-out vesicles.     

A comparative study of the role of mitochondria and the sarcoplasmic reticulum in the uptake and release of Ca++ by the rat diaphragm

The respective importance of mitochondria and of sarcoplasmic reticulum in the uptake and maintenance of Ca⁺⁺ by the isolated rat diaphragm has been compared. Diaphragms were incubated at 30° in conditions optimal for Ca⁺⁺ uptake either by isolated mitochondria or by sarcoplasmic reticulum: more Ca⁺⁺ was taken up from the “mitochondrial” medium. For maximal uptake, Pi and Mg⁺⁺ were necessary; substitution of NaCl and KC1 with sucrose had no effect on the uptake. The uptake was markedly inhibited by uncouplers of oxidative phosphorylation, by respiratory inhibitors, and by lowering the temperature of the incubation medium to 0°; it was not affected by oligomycin, aurovertin, DCCD, nor by inhibitors of Ca⁺⁺ transport in the isolated sarcoplasmic reticulum (ergotamine, ergobasinine, caffeine). The lack of effect of caffeine was not due to lack of penetration into the muscle. Permeability barriers for ergotamine and ergobasinine could not be excluded. The maintenance of Ca⁺⁺ by the diaphragm was optimal in a medium contaming Pi and Mg⁺⁺. Uncoupling agents and respiratory inhibitors accelerated the rate and extent of release of Ca⁺⁺ by the diaphragm. Lowering the temperature of the incubation medium to 0°, or addition of oligomycin, aurovertin, DCCD, had no effect on the release. The release of Ca⁺⁺ was also unaffected by ergotamine, ergobasinine, caffeine. The results suggest a role for mitochondria in the uptake and maintenance of Ca⁺⁺ by the isolated diaphragm.     

Differences in Ca2+ mobilization induced by alpha-adrenergic agonist and phosphatidic acid in cultured hepatocytes

In an attempt to elucidate the relationship between phosphatidylinositol breakdown and alpha-adrenergic responses, effects of phosphatidic acid and phosphatidylinositol related metabolites on Ca²⁺ mobilization and glucose output in cultured hepatocytes were examined. Norepinephrine induced the net ⁴⁵Ca²⁺ efflux from preloaded cells and stimulated glucose output via alpha-adrenergic receptor stimulation, whereas phosphatidic acid caused ⁴⁵Ca²⁺ uptake to cells and did not stimulate glucose output. Myo-inositol-monophosphate, diglyceride and arachidonic acid, which are released by phosphatidylinositol breakdown, had no effect on ⁴⁵Ca²⁺ efflux and glucose output in cells. These results suggest that phosphatidic acid and phosphatidylinositol related metabolites can not mimic the alpha-adrenergic actions in cultured hepatocytes.     

Action of insulin and cell calcium: Effect of ionophore A23187

Summary We have measured the effects of the carboxylic Ca⁺⁺ ionophore A23187 on muscle tension, resting potential and 3-O-methylglucose efflux. The ionophore produces an increase in tension that is dependent on external Ca⁺⁺ concentration since (a) the contracture was blocked by removing external Ca⁺⁺ and (b) its size was increased by raising outside Ca⁺⁺. Neither resting potential nor resting and insulin-stimulated sugar efflux were modified by the ionophore. These data imply that the action of insulin is not mediated by increasing cytoplasmic [Ca⁺⁺]. Additional support for this conclusion was obtained by testing the effects of caffeine on sugar efflux. This agent, which releases Ca⁺⁺ from the reticulum, did not increase resting sugar efflux and inhibited the insulin-stimulated efflux. Incubation in solutions containing butyrated derivatives of cyclic AMP or cyclic GMP plus theophylline did not modify the effects of insulin on sugar efflux. Evidence suggesting that our experimental conditions increased the cytoplasmic cyclic AMP activity was obtained.     

Rapid alteration in Ca++ content and fluxes in phorbol 12-myristate 13-acetate treated myoblasts

The tumor promoter phorbol 12-myristate 13-acetate rapidly induces alterations in both Ca⁺⁺ content and transport in cultured differentiated chick myoblasts. At 4 ng/ml (6nM), the promoter caused a 25 ± 12% decrease in total intracellular Ca⁺⁺ within 5 h after its addition. Measurement of ⁴⁵Ca⁺⁺ transport at this time revealed a 15 ± 6% decrease in the rate constants for both efflux and influx. Values of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the cytosolic Ca⁺⁺ pool in control and treated cells were 9.1 and 10.7 min, respectively, for efflux and 8.6 and 10.4 min, respectively, for influx. Ca⁺⁺ influx was decreased maximally within 90 sec after promoter addition. No effect was observed on ⁸⁶Rb⁺ uptake or intracellular concentration at equilibrium. The Ca⁺⁺ response is among the most rapid yet reported and may play a primary role in altering cellular metabolism.     

Mediation of beta-adrenergic stimulated amylase release from mouse parotid gland

Amylase released from mouse parotid fragments by the β-adrenergic agonist, isoproterenol, was associated with l) enhanced ⁴⁵Ca⁺⁺ efflux and 2) a dependence on the extracellular Na⁺ concentration. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on ⁴⁵Ca⁺⁺ efflux. In the absence of extracellular sodium isoproterenol and monensin failed to significantly release ⁴⁵Ca⁺⁺. Complete inhibition of isoproterenol stimulated amylase release occurred when 75 per cent or greater of the extracellular Na⁺ was replaced by sucrose; carbachol stimulated amylase release was not affected. Tetracaine (0.2 mM to 1.0 mM) inhibited both isoproterenol and carbachol stimulated amylase release and inhibited the ⁴⁵Ca⁺⁺ uptake induced by carbachol. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on amylase release; this effect was significantly reduced in the absence of extracellular Na⁺. It is proposed that a primary step in the release of amylase form mouse parotid gland in response to β-adrenergic stimulation is an increased influx of Na⁺ followed by release of intracellularly stored calcium.     

Role of external potassium in the calcium-induced potassium efflux from human red blood cell ghosts

Summary The exposure of red cell ghosts to external Ca⁺⁺ and K⁺ leads to a rapid net K⁺ efflux. Preincubation of the ghosts for various lengths of time in the absence of K⁺ in the external medium prior to a challenge with maximally effective concentrations of Ca⁺⁺ and K⁺ renders the ghosts unresponsive to that challenge with a half-time of about 7–10 min. Preincubation at a range of K⁺ concentrations for a fixed length of time (60 min) prior to the challenge revealed that K⁺ concentrations of about 500 m or more suffice to maintain the K⁺ channel in a maximally responsive state for at least 60 min. These K⁺ concentrations are considerably lower than the K⁺ concentrations required to make the responsive channel respond with a maximal rate of K⁺ efflux. Thus, external K⁺ is not only necessary to induce the permeability change but also to maintain the transport system in a functional state.The presence of Mg⁺⁺ or ethylenediamine-tetraacetic acid (EDTA) in the K⁺-free preincubation media preserves the responsiveness to a challenge with Ca⁺⁺ plus K⁺. In contrast to external K⁺, the presence of external Ca⁺⁺ does not reduce but rather enhances the loss of responsiveness. An excess of EDTA prevents the effects of Ca⁺⁺ while washes with EDTA after exposure to Ca⁺⁺ do not reverse them.In red cell ghosts that contain Ca⁺⁺ buffers, the transition from a responsive to a nonresponsive state incubation in the absence of external K⁺ is enhanced. The effects of incubation in the presence of Ca⁺⁺ in K⁺-free media are reversed; external Ca⁺⁺ now reduces the rate at which the responsiveness is lost. The loss of responsiveness after incubation in K⁺-free media prior to a challenge with external K⁺ and internal Ca⁺⁺ does also take place when K⁺-efflux from red cell ghosts is measured by means of⁴²K⁺ into media that have the same K⁺ concentrations as the ghost interior. This confirms that the effects of K⁺-free incubation are due to the modification of the K⁺-selective channel rather than to an inhibition of diffusive Cl^–-efflux.Abbreviation used in text TRIS Tris (hydroxymethyl) aminomethan This paper is dedicated to the memory of Walther Wilbrandt.     

Ca++ fluxes in resealed synaptic plasma membrane vesicles

The effect of the monovalent cations Na⁺, Li⁺, and K⁺ on Ca⁺⁺ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K₂/succinate and ⁴⁵CaCl₂, rapidly released approximately 60% of the intravesicular Ca⁺⁺ when exposed to NaCl or to the Ca⁺⁺ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca⁺⁺. The EC₅₀ for Na⁺ activation of Ca⁺⁺ efflux from the vesicles was approximately 6.6mM. exposure of the Ca⁺⁺-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca⁺⁺ from the vesicles, but the Na⁺-induced efflux could still be detected above this K⁺ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid ⁴⁵Ca uptake with an estimated EC₅₀ for Ca⁺⁺ of 7–10 μM. This Ca⁺⁺ uptake was blocked by dissipation of the Na⁺ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na⁺Ca⁺⁺ exchange process and of a K⁺ - sensitive Ca⁺⁺ flux across the membranes.     

Effect of lipid on the access of ATP and calcium to the delipidated Ca++-ATPase of intestinal brush border membrane.

The delipidated Ca⁺⁺-ATPase prepared from intestinal brush border membranes showed a higher activity of Ca⁺⁺-independent ATPase, a lower Km value for ATP and a higher Km value for Ca⁺⁺ than its original membrane Ca⁺⁺-ATPase. The addition of phosphatidylcholine re-activated the delipidated Ca⁺⁺-ATPase to approximately 89 % of its original membrane Ca⁺⁺-ATPase activity but did not restore the affinity for Ca⁺⁺. This phospholipid raised the Km value for ATP but had little effect on the Km value for Ca⁺⁺. Palmitic acid elevated the Km value for Ca⁺⁺ but did not change the Km value for ATP. Kinetic analyses of these data suggest that the hydrocarbon chain of phosphatidylcholine is an important rate-limiting factor for the access of Ca⁺⁺ to the enzyme and the polar head groups of phosphorylcholine and ester bond may be the factor for the access of ATP.     

Calcium efflux from human erythrocyte ghosts

Summary The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with⁴⁵Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that⁴⁵Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1mm Ca in the external solution ([Ca⁺⁺] _o ), the rate constant of passive Ca efflux (k min^–1, fraction of⁴⁵Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min^–1. There was no further increase at higher [Ca⁺⁺] _o . The relation between the rate constant of Ca efflux and [Ca⁺⁺] _o is thus characterized by saturation kinetics. The passive transfer system for Ca could also be activated by Sr. The alkali metal ions Na, K and Li did not seem to have any significant influence on passive Ca transfer. The passive Ca efflux was slightly inhibited by Mg and strongly inhibited by Pb. Under most experimental conditions, a fraction of 15 to 50% of the intracellular Ca seemed to be inexchangeable. The inexchangeable fraction decreased with increasing [Ca⁺⁺] _o and increased with increasing [Ca⁺⁺] _i . It was not influenced by alkali metal ions, CN or Pb, but it could be completely removed from the cells by the addition of 0.1mm Mersalyl to the incubation medium or by hemolysis with addition of a detergent. The active ATP-dependent Ca transport differed characteristically from passive transfer; the rate constant decreased with increasing [Ca⁺⁺] _o , and the inexchangeable Ca fraction increased with increasing [Ca⁺⁺] _o . The experimental results suggest that there exists a carrier-mediated Ca–Ca exchange diffusion in the erythrocyte membrane and that only a fraction of the ghost cell population participates in the Ca exchange diffusion.     

Calcium ion fluxes across the external surface ofPhysarum polycephalum

Summary There is no significant change in Ca⁺⁺ efflux rate from plasmodia during chemotactic responses to several sugars, whereas substantially increased Ca⁺⁺ efflux caused by EDTA does not significantly affect movement. Evidently the Ca⁺⁺ fluxes controlling movement take place inside the organism, and chemotaxis probably involves a second messenger.     

Effects of adenine nucleotide translocase inhibitors on dinitrophenol-induced Ca2+ efflux from pig heart mitochondria

Bongkrekic acid and atractyloside, inhibitors of adenine nucleotide translocase, do not inhibit Ca²⁺ uptake and H⁺ production by pig heart mitochondria. However, bongkrekic acid, but not atractyloside, inhibits dinitrophenol-induced Ca²⁺ efflux and H⁺ uptake. Conversely, ruthenium red blocks Ca²⁺ uptake and H⁺ production but does not prevent dinitrophenol-induced Ca²⁺ efflux and H⁺ uptake by mitochondria. These results suggest that mitochondrial Ca²⁺ uptake and release exist as two independent pathways. The efflux of Ca²⁺ from mitochondria is mediated by a bongkrekic acid sensitive component which is apparently not identical to the ruthenium red sensitive Ca²⁺ uptake carrier.     

Effects of newly arachidonic acid metabolites on microsomal Ca binding, uptake and release

The 5,6- 8,9-; 11,12- and 14,15-epoxyeicosatrienoic acids and their respective hydration products, the vic-doisl, recently reported as metabolites of arachidonic acid in rat liver microsomes, were examined for effect on release of 45Ca from canine aortic smooth muscle miscrosomes. At 10⁻⁶ M, the diols had no effect, but the 5,6-; 11,12- and 14,15-epoxyacids increased the loss of ⁴⁵Ca. Further studies with the 14,15-epoxyacid demonstrated a dose-dependent decrease of Ca⁺⁺ uptake (ATP present) in canine aortic microsomes in 0.03 mM Ca⁺⁺, whereass Ca⁺⁺ binding (ATP absent) was not affected. Ca⁺⁺ uptake, binding and release in rat liver microsomes was similarly affected by the 14,15-epoxyacid, the major epoxyeicosatrienoic acid derivative produced by rat liver miscrosomal incubations. It is suggested that a alterations in Ca⁺⁺ metabolism might be a possible mechanism of actions for these derivatives of arachidonic acid.     

Calcium transport and cellular distribution in quiescent and serum-stimulated primary cultures of bone cells and skin fibroblasts

Primary cultures of bone cells and skin fibroblasts were examined for their Ca⁺⁺ content, intracellular distribution and Ca⁺⁺ fluxes. Kinetic analysis of ⁴⁵Ca⁺⁺ efflux curves indicated the presence of three exchangeable Ca⁺⁺ compartments which turned over at different rates: a “very fast turnover” (S₁), a “fast turnover” (S₂), and a “slow turnover” Ca⁺⁺ pool (S₃). S₁ was taken to represent extracellular membrane-bound Ca⁺⁺, S₂ represented cytosolic Ca⁺⁺, and S₃ was taken to represent Ca⁺⁺ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca⁺⁺ as compared with cultured fibroblasts. Most of this extra Ca⁺⁺ was localized in the “slow turnover” intracellular Ca⁺⁺ pool (S₃). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca⁺⁺: (1) In both types of cells serum caused an increase in the amount of Ca⁺⁺ in the “very fast turnover” Ca⁺⁺ pool, and an increase in the rate constant of ⁴⁵Ca⁺⁺ efflux from this pool, indicating a decrease in the strength of Ca⁺⁺ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca⁺⁺ in the “slow turnover” Ca⁺⁺ pool (S₃), an increase in the rates of Ca⁺⁺ efflux from the cells to the medium, and from S₃ to S₂, as well as a decrease in the rate of influx into S₃. (3) In bone cells the amount of Ca⁺⁺ in S₃ remained high in “serum activated” cells, the rate of efflux from S₃ to S₂ increased, and the rate of influx into S₃ also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca⁺⁺ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca⁺⁺ in bone cells and fibroblasts, in parallel with measurements of ³H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca⁺⁺ which was followed by a biphasic increase lasting until cell division.     

Mechanisms for Intracellular Calcium Regulation in Heart : I. Stopped-Flow Measurements of Ca++ Uptake by Cardiac Mitochondria

Initial velocities of energy-dependent Ca⁺⁺ uptake were measured by stopped-flow and dual-wavelength techniques in mitochondria isolated from hearts of rats, guinea pigs, squirrels, pigeons, and frogs. The rate of Ca⁺⁺ uptake by rat heart mitochondria was 0.05 nmol/mg/s at 5 µM Ca⁺⁺ and increased sigmoidally to 8 nmol/mg/s at 200 µM Ca⁺⁺. A Hill plot of the data yields a straight line with slope n of 2, indicating a cooperativity for Ca⁺⁺ transport in cardiac mitochondria. Comparable rates of Ca⁺⁺ uptake and sigmoidal plots were obtained with mitochondria from other mammalian hearts. On the other hand, the rates of Ca⁺⁺ uptake by frog heart mitochondria were higher at any Ca⁺⁺ concentrations. The half-maximal rate of Ca⁺⁺ transport was observed at 30, 60, 72, 87, 92 µM Ca⁺⁺ for cardiac mitochondria from frog, squirrel, pigeon, guinea pig, and rat, respectively. The sigmoidicity and the high apparent K_m render mitochondrial Ca⁺⁺ uptake slow below 10 µM. At these concentrations the rate of Ca⁺⁺ uptake by cardiac mitochondria in vitro and the amount of mitochondria present in the heart are not consistent with the amount of Ca⁺⁺ to be sequestered in vivo during heart relaxation. Therefore, it appears that, at least in mammalian hearts, the energy-linked transport of Ca⁺⁺ by mitochondria is inadequate for regulating the beat-to-beat Ca⁺⁺ cycle. The results obtained and the proposed cooperativity for mitochondrial Ca⁺⁺ uptake are discussed in terms of physiological regulation of intracellular Ca⁺⁺ homeostasis in cardiac cells.