Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg²⁺ linear with time and dependent on coupled respiration. No net Ca²⁺ release occurred under these conditions, while a concomitant release of K⁺ was observed. Mg²⁺ efflux mediated either by P_i or low concentrations of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by P_i and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg²⁺ which was associated also with a release of endogenous Ca²⁺. Under these conditions EGTA completely prevented Mg²⁺ and K⁺ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg²⁺ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca²⁺ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg²⁺ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg²⁺ efflux on phosphate transport is explained by the phosphate requirement for Ca²⁺ movement.Abbreviations Diamide diazenedicarboxylic acidbis-dimethylamide - FCCP p-trifluoromethoxyphenylhydrazone - EGTA ethylene glycol-bis-(2-amino ethyl ether)-N,N-tetracetic acid - P_i inorganic phosphate - Ruthenium red Ru₂(OH)₂Cl₄ · 7NH₃ · 3H₂O - state 4 controlled state of respiration in the presence of substrate - RCI respiratory control index - NEM N-ethyl maleimide A partial and preliminary report of these results has been published inBiochem. Biophys. Res. Comm.,78 (1977) 23.     

Abnormal lysosomal hydrolases excreted by cultured fibroblasts in I-cell disease (mucolipidosis II).

The characteristics of rat liver mitochondria swelling induced by diamide, an oxidizing agent for thiol groups, and by Ca ions are very similar. In both cases the swelling, which is initiated by addition of 0.5–1 mM phosphate or acetate, is prevented by FCCP, antimycin A, EGTA, Mg⁺⁺ and ruthenium red. Diamide potentiates the swelling action of Ca⁺⁺, while DTE potentiates that of Mg⁺⁺. The additive effects of calcium and diamide on rat liver mitochondria have been correlated with their synergic action in promoting the release of mitochondrial Mg⁺⁺. The results strongly indicate that some of the effects of diamide are mediated by a mobilization of endogenous divalent ions and that the antagonism between Ca⁺⁺ and Mg⁺⁺ is closely correlated with the redox state of membrane bound thiol groups.     

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.     

Effect of oligomycin on NADH oxidation and its coupled phosphorylation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum

Summary NADH oxidation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum is significantly stimulated by the addition of phosphate (Pi) and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. K _m values for Pi in NADH oxidation and phosphorylation are 10^–3 m and 8×10^–4 m, respectively. These K _m values are almost the same as in corresponding photophosphorylation and oxidative phosphorylation catalyzed with chromatophores. As in the case of NADH oxidation with chromatophores, NADH oxidation with the particulate fraction has an optimal pH at 7.5 without additions, which is shifted to 6.9 by the addition of Pi and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. The optimal pH for coupled phosphorylation is 6.9. 10 g per ml of oligomycin can suppress stimulation of NADH oxidation by Pi, or by the energy trapping system, and prevent the shift of optimal pH. The particulate fraction can catalyze Pi-incorporation into glucose-6-phosphate without externally added ATP, so that Pi-incorporation is inhibited by oligomycin. From these findings, it is concluded that NADH oxidation in the particulate fraction is tightly coupled to phosphorylation.     

Calcium efflux from platelet vesicles of the dense tubular system. Analysis of the possible contribution of the Ca2+ pump

The ATP dependent Ca²⁺ uptake of platelet vesicles was inhibited by the two hydrophobic drugs trifluoperazine (TFP) and propranolol (PROP). Inhibition was significantly lowered when Pi was used instead of oxalate as a precipitant agent. When the ATPase ligands substrate (Mg²⁺ and Pi) were absent of the efflux medium, a slow release of Ca²⁺ which did not couple with ATP synthesis (passive Ca²⁺ efflux) was observed. Both, TFP and PROP enhanced the passive Ca²⁺ efflux. This enhanced efflux was partially inhibited only when Mg²⁺ and Pi were added together to the efflux reaction media, but it was not affected by spermidine, ruthenium red or thapsigargin (TG). The Ca²⁺ ionophores A23187 and ionomycin, also enhanced passive Ca²⁺ efflux. However, in this case, Ca²⁺ efflux was inhibited just by inclusion of Mg²⁺ to the medium. Ca²⁺ efflux promoted by Triton X-100 was not affected by either Mg²⁺ or Pi, included together or separately into the efflux medium. The ATP Pi measured in the presence of Triton X-100 and millimolar Ca²⁺ concentrations was inhibited by both TFP and PROP, but not by Ca²⁺ ionophores up to 4 M. The data suggest that the observed enhancement of passive Ca²⁺ efflux promoted by TFP and PROP could be attributed to a direct effect of these drugs over the platelet Ca²⁺ pump isoforms (Sarco Endoplasmic Reticulum Calcium ATPase, SERCA_2b and SERCA₃) themselves, as it was reported for the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA₁).     

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.     

The effect of Mg ++ on the conformation of the Ca ++ -binding component of troponin

Mg⁺⁺ like Ca⁺⁺ induces a conformational change in the Ca⁺⁺-binding component of troponin. However, this change is only 36 % of the change in fluorescence intensity and 80 % of the change in optical rotation induced by Ca⁺⁺. The apparent binding constant of Mg⁺⁺ to the Ca⁺⁺-binding component is 5 × 10³ M⁻¹, much smaller than that of Ca⁺⁺. Circular dichroism measurements show that these changes are simple helix-coil transitions. Unlike the Ca⁺⁺-induced conformational change, the Mg⁺⁺-induced change cannot be propagated to other muscle proteins, and therefore has no physiological meaning.     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

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.     

Levels of ornithine decarboxylase activation used as a simple marker of metal induced growth arrest in tissue culture.

Exposure of proliferating cells to specific water solube metal compounds at 0.1 or 1.0 mM concentrations inhibited cell growth and also depressed the induction of ornithine decarboxylase, an enzyme which is tightly coupled to the initiation of cell growth. Salts of Co⁺⁺, Ni⁺⁺, Cu⁺⁺, Cr⁺⁶ and Cd⁺⁺ significantly reduced incorporation of radiolabeled leucine, thymidine or uridine into trichloroacetic acid insoluble material, inhibited the doubling of Chinese hamster ovary cells, and blocked the the induction of ornithine decarboxylase. The addition of similar concentrations of other metals such as Fe⁺⁺, K⁺, Mg⁺⁺, Pb⁺⁺, Ca⁺⁺ or Sn⁺⁺ had no effect on ODC induction and also did not inhibit the other parameters associated with cell proliferation which were measured. These results suggest that ornithine decarboxylase induction can be used as a marker of metal induced growth arrest.     

The migration of divalent cations in mitochondria visualized by a fluorescent chelate probe

Summary The use of the fluorescent chelate probe, chlorotetracycline, in mitochondria is described. The probe shows a high fluorescence in the presence of mitochondria which may be ascribed to binding of the probe to membrane-associated Ca⁺⁺ and Mg⁺⁺. The fluorescence excitation and emission spectra are diagnostic of binding of the probe to Ca⁺⁺ in coupled mitochondria and Mg⁺⁺ in uncoupled mitochondria. The fluorescence polarization spectra are diagnostic of the cations having a moderately high mobility in the membrane environment. The effects of exogenous EDTA and of endogenous Mn⁺⁺ indicate that the probe is primarily visualizing actively accumulated Ca⁺⁺ on the inner surface of the inner membrane. By employing the Ca⁺⁺ transport inhibitor, Tb⁺⁺⁺, the fluorescence changes associated with metabolic alterations are shown to arise partly from cation transport and partly through alterations in the binding properties of the inner surface of the membrane. Chlorotetracycline is a probe for divalent cations associated with the membrane and is of general utility in the study of cation migrations in cellular and subcellular systems.     

Glutamate-supported calcium movements in rat liver mitochondria effects of anions and pH

Ca²⁺ efflux from rat liver mitochondria in the presence of glutamate is stimulated by a decrease in pH from 7.3 to 6.8 and the rate is dependent on the phosphate concentration. During Ca²⁺ (13 μm) uptake and release at low pH (+ phosphate), swelling is minimal, but a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. The depolarization (but not Ca²⁺ efflux) is reversed by ruthenium red. An absolute requirement for phosphate to support Ca²⁺ efflux is demonstrated by using acetate or lactate to support Ca²⁺ uptake (efflux is depressed at pH 6.8). Preincubation with mersalyl, to block phosphate movements, with subsequent phosphate addition preceeding Ca²⁺ uptake also inhibits efflux. β-Mercaptoethanol then stimulates efflux concomittent with membrane repolarization. Ca²⁺ efflux is not a simple result of collapse of ΔpH since nigericin inhibits phosphate transport and Ca²⁺ release. Following Ca²⁺ uptake at pH 6.8, respiratory inhibition occurs, but oxygen consumption coupled to ATP synthesis can be stimulated by succinate (+ rotenone). Addition of succinate allows reuptake of Ca²⁺, reduction of pyridine nucleotides, and repolarization of the membrane potential. Respiratory inhibition is also seen with nigericin, but no Ca²⁺ efflux is observed. Coupled respiration with glutamate is seen at pH 6.8 following Ca²⁺ uptake in the presence of lactate with subsequent addition of phosphate to promote Ca²⁺ efflux. We conclude that Ca²⁺ efflux is not a consequence of respiratory inhibition, but is mediated solely by phosphate movements. The inhibitory effect of Mg²⁺ on Ca²⁺ efflux is probably due to Mg²⁺-dependent inhibition of the Ca²⁺ diffusion potential so that the compensatory increase in ΔpH due to membrane depolarization does not occur and phosphate entry is slowed.     

The participation of phosphate in the formation of a carrier for the transport of Mg++ and Mn++ ions into yeast cells

During the absorption of phosphate by yeast, the cells acquire the capacity to absorb Mn⁺⁺ and Mg⁺⁺, a capacity which is retained even after phosphate is no longer present in the medium. Cells pretreated with phosphate and then washed, slowly lose their ability to absorb Mn⁺⁺, the rate of loss depending on the temperature and on the metabolic state. The fermentation of sugars induces a very rapid loss of absorptive capacity, whereas the respiration of ethyl alcohol, lactate, or pyruvate has little effect. Inhibitor studies with sodium acetate, redox dyes, and arsenate, reveal parallel effects on Mn⁺⁺ absorption, and on phosphate absorption. It is concluded that the synthesis of a carrier for the transport of Mg⁺⁺ and Mn⁺⁺ involves a phosphorylation step closely coupled with reactions involved in the absorption of phosphate.     

Retention of Ca2+ by rat liver and rat heart mitochondria: Effect of phosphate,Mg2+, and NAD(P) redox state

Parallel measurements of Ca²⁺ uptake, oxygen consumption, endogenous Mg²⁺ efflux, and swelling in rotenone-poisoned rat liver and rat heart mitochondria showed that heart mitochondria is much more resistant to uncoupling by Ca²⁺ in the presence of phosphate than rat liver mitochondria. The extent of Mg²⁺ efflux and swelling induced by Ca²⁺ accumulation are much less pronounced in heart mitochondria. Uncoupling and swelling in liver mitochondria seem to result from the loss of membrane-bound Mg²⁺ as a consequence of Ca²⁺ recycling across the membrane as induced by phosphate. Exogenous Mg²⁺ protects liver mitochondria against the deleterious effects of Ca²⁺ by inhibiting a ruthenium red-insensitive Ca²⁺ efflux induced by phosphate. Phosphate does not induce recycling of Ca²⁺ in heart mitochondria. On the other hand, heart mitochondria respiring on NAD-linked substrates or with succinate in the absence of rotenone behave like liver mitochondria with respect to the alterations caused by Ca²⁺ recycling. In heart mitochondria the recycling of Ca²⁺ is related to the redox state of pyridine nucleotides, which suggests that the ruthenium red-insensitive efflux of Ca²⁺ is subject to metabolic control. In addition it has been observed that Sr²⁺does not undergo cyclic movements across the membrane. The data indicate that the efflux pathway is more specific for Ca²⁺ than the ruthenium red-sensitive influx transporter.     

Effects of different ions on resting polarization and on the mass receptor potential of carotid body chemosensors

The carotid body and its own nerve were removed from cats anesthetized with sodium pentobarbital and placed in an air gap system; the carotid body was bathed in modified Locke's solution equilibrated with 50% O₂ in N₂, pH 7.43 at 35°C. The sensory discharges, changes in “resting” receptor polarization and the mass receptor potential evoked by ACh or NaCN were recorded with nonpolarizable electrodes placed across the gap. Receptor potentials and sensory discharges evoked by ACh showed an appreciable increase in amplitude and frequency when the preparation was bathed in eserinized Locke. Eserine did not change appreciably the responses evoked by NaCN. Excessive depolarization elicited by either ACh or NaCN was accompanied by sensory discharge block. Removal of K⁺ ions from the bathing solution induced receptor hyperpolarization and an increase in the amplitude of the evoked receptor potentials. An increase of K⁺ concentration had the opposite effect. Reduction of Na⁺ or NaCl to one half, or total removal of this salt, induced an initial reduction and later disappearance of the sensory discharges, some receptor hyperpolarization and a reduction in the amplitude of the evoked receptor potentials. Reduction or removal of Ca⁺⁺ produced receptor depolarization, a marked depression of the evoked receptor potentials, an increase in the frequency of the sensory discharges and a reduction in the amplitude of the nerve action potentials. High Ca⁺⁺ or Mg⁺⁺ had little or no effect on action potential amplitude or resting polarization, but decreased sensory discharge frequency and the evoked receptor potentials. Total or partial replacement of Ca⁺⁺ with Mg⁺⁺ induced complex effects: (1) receptor depolarization which occurred in low Ca⁺⁺, was prevented by addition of Mg⁺⁺ ions; (2) the amplitude of the evoked receptor potentials was depressed; (3) the nerve discharge frequency was reduced as it was in high Mg⁺⁺ solutions; and (4) the amplitude of the nerve action potentials was reduced as it was in low Ca⁺⁺ solutions. Temperature had a marked effect on the chemoreceptors since a t high temperatures the receptors were depolarized and the discharge frequency increased. The baseline discharge and responses evoked by ACh or NaCN were depressed at low temperatures. The results are discussed in terms of possible receptor mechanisms influenced by the different ions.     

Cation dependence of guinea pig epidermal cell spreading

Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg⁺⁺ plus Ca⁺⁺ or Mg⁺⁺ alone. Supplementing with Ca⁺⁺ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Zn⁺⁺, Ni⁺⁺), showed that only Mg⁺⁺ and Mn⁺⁺, and to a lesser extent, Ca⁺⁺, supported cell spreading. In contrast to Mg⁺⁺, however, Mn⁺⁺ could support spreading in the absence of whole serum if serum albumin were present. Although Mn⁺⁺ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg⁺⁺ plus serum, equal concentrations of Ca⁺⁺ completely blocked the Mn⁺⁺ effect. In contrast to the increasing cell spreading, which occurred in Mg⁺⁺-containing medium with time, cell death occurred in Mn⁺⁺-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn⁺⁺-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg⁺⁺ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn⁺⁺ and Mg⁺⁺-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.     

Calcium dependent action potentials in skeletal muscle fibres of a beetle larva, Xylotrupes dichotomus

The ionic requirement for generating action potentials in ventral longitudinal muscle fibers dissected from beetle larvae was examined by conventional electrophysiological techniques. Muscle fibers that generated only graded responses in physiological saline were able to generate an all-or-none action potential when the potassium permeability of the membrane was inhibited by tetraethylammonium⁺ added to the saline. The peak of the action potential thus elicited was intimately related to the external Ca⁺⁺ concentration. The action potential was blocked by Co⁺⁺ which is known as a competitive inhibitor of Ca-spikes. Neither tetrodotoxin (3 μM) nor a Na-free condition effectively blocked the generation of the action potential. Mg⁺⁺ induced a shift in the peak of the action potential; this was, however, due to the stabilizing action of Mg⁺⁺ but not due to the penetration of Mg⁺⁺ through the muscle membrane. No action potential was elicited in the muscle fiber when immersed in a Ca-free, EGTA saline even when a high concentration of either Mg⁺⁺, Na⁺, or tetraethylammonium⁺ was present. The action potential of the larval muscle fiber was thus concluded to be a Ca-spike, through the channel of which Na⁺ or Mg⁺⁺ did not penetrate.     

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.     

Characterization of Mg Inhibition of Mitochondrial Ca Uptake by a Mechanistic Model of Mitochondrial Ca Uniporter

Ca²⁺ is an important regulatory ion and alteration of mitochondrial Ca²⁺ homeostasis can lead to cellular dysfunction and apoptosis. Ca²⁺ is transported into respiring mitochondria via the Ca²⁺ uniporter, which is known to be inhibited by Mg²⁺. This uniporter-mediated mitochondrial Ca²⁺ transport is also shown to be influenced by inorganic phosphate (Pi). Despite a large number of experimental studies, the kinetic mechanisms associated with the Mg²⁺ inhibition and Pi regulation of the uniporter function are not well established. To gain a quantitative understanding of the effects of Mg²⁺ and Pi on the uniporter function, we developed here a mathematical model based on known kinetic properties of the uniporter and presumed Mg²⁺ inhibition and Pi regulation mechanisms. The model is extended from our previous model of the uniporter that is based on a multistate catalytic binding and interconversion mechanism and Eyring's free energy barrier theory for interconversion. The model satisfactorily describes a wide variety of experimental data sets on the kinetics of mitochondrial Ca²⁺ uptake. The model also appropriately depicts the inhibitory effect of Mg²⁺ on the uniporter function, in which Ca²⁺ uptake is hyperbolic in the absence of Mg²⁺ and sigmoid in the presence of Mg²⁺. The model suggests a mixed-type inhibition mechanism for Mg²⁺ inhibition of the uniporter function. This model is critical for building mechanistic models of mitochondrial bioenergetics and Ca²⁺ handling to understand the mechanisms by which Ca²⁺ mediates signaling pathways and modulates energy metabolism.     

Bile acid uptake and calcium flux in brush border membrane vesicles

Our laboratory has recently reported that intestinal bile acid malabsorption in cystic fibrosis (CF) is a primary mucosal cell defect. Others have suggested that elevated intracellular Ca⁺⁺ levels in other cell types in CF may represent a common primary dysfunction in Ca⁺⁺ efflux in these cells. We examined the possibility that intestinal bile acid absorption and Ca⁺⁺ efflux in mucosal cells may be linked physiologically. Brush border membrane vesicles (BBMV) prepared from guinea pig ileum served as the experimental model to test this hypothesis. Ca⁺⁺ (2.5×10^?3M) present in the incubation medium did not alter the uptake of taurocholic acid (TCA) by BBMV. Also, TCA uptake into BBMV preloaded with Ca⁺⁺ was not significantly different from that in BBMV not previously loaded with Ca⁺⁺. Furthermore, with TCA present in the incubation medium, Ca⁺⁺ efflux from preloaded BBMV was not altered. These data suggest that ileal TCA uptake, as measured by BBMV, is not dependent upon either intra- or extravesicular Ca⁺⁺. Also, Ca⁺⁺ efflux from BBMV is unaffected by TCA uptake. Although separate lines of evidence suggest that intestinal bile acid malabsorption and reduced plasma membrane Ca⁺⁺ flux are primary defects in CF, we conclude that in the normal intestine these functions are independent physiological processes.