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.     

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.     

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.     

EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA

Rat liver mitochondria allowed to accumulate maximal amounts of Ca⁺⁺ and HPO₄⁼ ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca⁺⁺ and HPO₄⁼ from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca⁺⁺-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca⁺⁺-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca⁺⁺-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca⁺⁺ and phosphate from the mitochondria into the medium.     

Binding of calcium and zinc to the acetylcholine receptor purified from Torpedo Californica

Binding of [⁶⁵Zn⁺⁺] and [⁴⁵Ca⁺⁺] to the acetylcholine (ACh)-receptor, purified from the $\text{Torpedo}$ electric organ, was studied by equilibrium dialysis. Whereas [⁶⁵Zn⁺⁺] bound to 56 nmoles of sites per mg protein with a dissociation constant of 2.5 × 10⁻⁶M, no binding of [⁴⁵Ca⁺⁺] at concentrations up to 10⁻³M could be detected with this method. However, the binding of [acetyl-³H]choline to the receptor was blocked equally by very high Zn⁺⁺ or Ca⁺⁺ concentrations, and the K_i for this low affinity binding was 7 × 10⁻³M. The high affinity binding of [⁶⁵Zn⁺⁺] to the receptor was blocked best by Cd⁺⁺ then Co⁺⁺ and Mn⁺⁺, but least by Mg⁺⁺ and Ca⁺⁺. When the purified ACh-receptor itself was analyzed for the presence of cations by atomic absorption, it was discovered that 4.7% of its weight was due to bound Ca⁺⁺ that could not be removed even by extensive dialysis. When Ca⁺⁺-free solutions (containing 1 mM EDTA) were used during purification, 0.6% of the molecular weight of the receptor was still due to bound Ca⁺⁺. This was equivalent to 15 moles of Ca⁺⁺ for each mole of ACh bound at saturation. It is suggested that the source of this Ca⁺⁺ is endogenous, and that it is tightly bound to the ACh-receptor molecule.     

Intracellular Calcium Binding and Release in Frog Heart

The capacities and affinities of intracellular calcium-binding sites have been studied in frog ventricles, in which the concentration of Ca⁺⁺ in the sarcoplasm can be controlled as a result of treatment with EDTA. The total calcium content of calcium-depleted and nondepleted muscles at rest and muscles generating considerable tension was 0.8, 1.4, and 5.4 µmol/g of muscle, respectively. Net movement of calcium into or out of the cells occurred without change in tension when the sarcoplasmic concentration of Ca⁺⁺ was either of two values, less than 10^-7 M or approximately 5 x 10^-7 M. These data can be explained by the presence of two groups of intracellular calcium sinks which compete with the contractile proteins, one with a capacity of about 0.6 µmol/g and an affinity constant greater than 10⁷ M^-1 and a second with a capacity of 4.0 µmol/g and an affinity constant of about 2 x 10⁶ M^-1. The higher affinity calcium is released by anoxia, oligomycin, or abrupt changes in sarcoplasmic Ca⁺⁺. Muscles soaked in Sr-Ringer's contain electron densities in the sarcoplasmic reticulum and to a lesser extent in the mitochondria.     

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.     

Proton permeability and the regulation of potassium permeability in mitochondria by uncoupling agents

Summary The addition of agents that uncouple electron transfer from energy conservation (uncouplers) to state 4 mitochondria causes the following ion movements: K⁺ is extruded from the mitochondria in association with phosphate and possibly other anions, but not H⁺. Endogenous Ca⁺⁺ is extruded from the mitochondria, and H⁺ moves in to counter-balance the Ca⁺⁺ movement; some phosphate movement may be associated with Ca⁺⁺ extrusion. The rate and extent of K⁺ extrusion induced by uncoupler is dependent on the concentrations of external phosphate and divalent ions. Phosphate induces K⁺ extrusion, while Mg⁺⁺ and Mn⁺⁺ inhibit it. TheV _max of K⁺ transport is 300 moles K⁺/g protein per min. The K _m for FCCP-induced potassium extrusion is 0.25 M at pH 7.4. The inhibitory effect of Mg⁺⁺ is noncompetitive with respect to uncoupler concentration but competitive with respect to phosphate concentration. The experimental evidence does not support the existence of high H⁺ permeability in the presence of uncoupler. A correlation is observed between the rate of K⁺ extrusion and the energy reserves supplied from the high energy intermediate. The action of uncoupler in inducing K⁺ permeability is considered to arise through its action in depleting the energy reserves of mitochondria rather than through a specific activating effect of permeability by the uncoupler itself. The relationship of membrane potential to regulation of K⁺ permeability is discussed.     

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.     

Divalent cation-activated ATP hydrolysis by mitochondrial ATP'ase — Mechanism for energy depletion in ischemic reperfused myocardium

Recovery of high-energy compounds by ischemic myocardium is believed to be important for its return to normal functioning. While it has been previously shown that oxidative phosphorylation is markedly reduced in mitochondria isolated from ischemic myocardium in the presence of all substrates, alterations in ATPase activity have not been confirmed. This study demonstrates that, although the rate of ATP hydrolysis produced by mitochondria isolated from 2-hr ischemic myocardium does not significantly differ from that produced by non-ischemic mitochondria, the rate produced by 2-hr ischemic, 2 hr reperfused mitochondria is significantly higher. Also, Ca⁺⁺ content was observed to be higher in reperfused than in non-reperfused ischemic mitocheondria. The addition of EDTA, EGTA, or oligomycin to the reperfused ischemic mitochondria resulted in the inhibition of ATPase activity. These results indicate that mitochondrial ATPase in ischemic myocardium is activated by Ca⁺⁺ ions and that ischemic reperfused myocardium may contain mitochondria with uncontrolled ATPase activity such that high energy phosphate supplies are excessively depleted when the cells are reperfused.     

Preparation of metaphase chromatin of Physarumpolycephalum without the loss of repressed RNA synthesis

A novel method for the preparation of intact chromatin from the slime mold $\text{Physarum}\text{polycephalum>}$ which retains the $\text{in}\text{vivo}$ property of RNA synthesis is described. Preparations from G₂-cells were highly active, while those from metaphase-cells were inactive. The plasmodial cells were disrupted by gentle homogenization on a polyethylene sieve in a neutral isotonic sucrose medium containing Mg⁺⁺, deoxycholate and EGTA, a Ca⁺⁺-chelating agent. The nuclei were lysed in a hypotonic buffer without use of EDTA and chromatin was precipitated by centrifugation after addition of Mg⁺⁺.     

Studies on the Active Transport of Calcium in Human Red Cells

The Ca⁺⁺ transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca⁺⁺ from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of ⁴⁵Ca infused inside red cells indicated that over 95% of all Ca⁺⁺ in the cells was transported into media in 20 min incubation under the optimum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP¹, CTP, and UTP were about equally effective, GTP somewhat less effective, and ITP least effective among the nucleotides tested. The Ca⁺⁺ transport does not appear to involve exchange of Ca⁺⁺ with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca⁺⁺ transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca⁺⁺ transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.     

The Initiation of Spike Potential in Barnacle Muscle Fibers under Low Intracellular Ca++

Electrical properties of the muscle fiber membrane were studied in the barnacle, Balanus nubilus Darw. by using intracellular electrode techniques. A depolarization of the membrane does not usually produce an all-or-none spike potential in the normal muscle fiber even though a mechanical response is elicited. The intracellular injection of Ca⁺⁺-binding agents (K₂SO₄ and K salt of EDTA solution, K₃ citrate solution, etc.) renders the fiber capable of initiating all-or-none spikes. The overshoot of such a spike potential increases with increasing external Ca concentration, the increment for a tenfold increase in Ca concentration being about 29 mv. The threshold membrane potential for the spike and also for the K conductance increase shifts to more positive membrane potentials with increasing [Ca⁺⁺]_out. The removal of Na ions from the external medium does not change the configuration of the spike potential. In the absence of Ca⁺⁺ in the external medium, the spike potential is restored by Ba⁺⁺ and Sr⁺⁺ but not by Mg⁺⁺. The overshoot of the spike potential increases with increasing [Ba⁺⁺]_out or [Sr⁺⁺]_out. The Ca influx through the membrane of the fiber treated with K₂SO₄ and EDTA was examined with Ca⁴⁵. The influx was 14 pmol per sec. per cm² for the resting membrane and 35 to 85 pmol per cm² for one spike. From these results it is concluded that the spike potential of the barnacle muscle fiber results from the permeability increase of the membrane to Ca⁺⁺ (Ba⁺⁺ or Sr⁺⁺).     

Lipids and lipolytic enzyme activities of rat heart mitochondria

The lipid composition and lipolytic enzyme activities in rat cardiac mitochondria were examined. Subsarcolemmal mitochondria were prepared by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by exposure of the remaining low-speed pellet to the protease, nagarse. These procedures are known to yield two functionally different populations of mitochondria. However, their phospholipid contents and compositions were identical, as were the positional distributions of the constituent fatty acids. Of the ethanolamine phospholipids, 20% were plasmalogens, and about 2% of the choline phospholipids consisted of this alkenylacyl species. Both subsarcolemmal and interfibrillar mitochondria contained a Ca²⁺-activated phospholipase A₂, as evidenced by the Ca²⁺-dependent release of unsaturated fatty acids and lysophosphatidylethanolamine from endogenous lipids. Ruthenium red prevented the activation of this enzyme by Ca²⁺, indicating that the activity is located in the matrix space or associated with the inner surface of the inner membrane. Both mitochondrial fractions produced free fatty acids and lysophosphatidylethanolamine in the absence of free Ca²⁺ apparently due to an outer membrane phospholipase A₁. The activity of this enzyme decreased with time, particularly in interfibrillar mitochondria, providing that Ca²⁺ was absent. Nagarse treatment of subsarcolemmal mitochondria resulted in a preparation with the same phospholipase A₁ properties as interfibrillar mitochondria. The possibility that differences in phospholipase A₁ properties account for some of the functional variations between the two mitochondrial types is discussed.     

Microvillar Ca++ signaling: A new view of an old problem

Proceeding from the recent finding that the main components of the Ca⁺⁺ signal pathway are located in small membrane protrusions on the surface of differentiated cells, called microvilli, a novel concept of cellular Ca⁺⁺ signaling was developed. The main features of this concept can be summarized as follows: Microvilli are formed on the cell surface of differentiating or resting cells from exocytic membrane domains, growing out from the cell surface by elongation of an internal bundle of actin filaments. The microvillar tip membranes contain all functional important proteins synthesized such as ion channels and transporters for energy-providing substrates and structural components, which are, in rapidly growing undifferentiated cells, distributed over the whole cell surface by lateral diffusion. The microvillar shaft structure, a bundle of actin filaments, forms a dense cytoskeletal matrix tightly covered by the microvillar lipid membrane and represents an effective diffusion barrier separating the microvillar tip compartment (entrance compartment) from the cytoplasm. This diffusion barrier prevents the passage of low molecular components such as Ca⁺⁺ glucose and other relevant substrates from the entrance compartment into the cytoplasm. The effectiveness of the actin-based diffusion barrier is modulated by various signal pathways and effectors, most importantly, by the actin-depolymerizing/reorganizing activity of the phospholipase C (PLC)-coupled Ca⁺⁺ signaling. Moreover, the microvillar bundle of actin filaments plays a dual role in Ca⁺⁺ signaling. It combines the function of a diffusion barrier, preventing Ca⁺⁺ influx into the resting cell, with that of a high-affinity, ATP-dependent, and IP₃-sensitive Ca⁺⁺ store. Activation of Ca⁺⁺ signaling via PLC-coupled receptors simultaneously empties Ca⁺⁺ stores and activates the influx of external Ca⁺⁺. The presented concept of Ca⁺⁺ signaling is compatible with all established data on Ca⁺⁺ signaling. Properties of Ca⁺⁺ signaling, that could not be reconciled with the basic principles of the current hypothesis, are intrinsic properties of the new concept. Quantal Ca⁺⁺ release, Ca⁺⁺-induced Ca⁺⁺ release (CICR), the coupling phenomen between the filling state of the Ca⁺⁺ store and the activity of the Ca⁺⁺ influx pathway, as well as the various yet unexplained complex kinetics of Ca⁺⁺ uptake and release can be explained on a common mechanistic basis. J. Cell. Physiol. 180:19–34, 1999. © 1999 Wiley-Liss, Inc.     

Cytochrome P450 in the liver mitochondrial outer membrane of 20-methyl cholanthrene or Aroclor treated rabbits.

Outer mitochondrial membrane was isolated from swelled rabbit liver mitochondria and precipitated with Ca⁺⁺ at a concentration that does not precipitates microsomes. After induction with 20-methyl cholanthrene or Aroclor the outer membrane shows the presence of cytochrome P₄₅₀ and glucose 6-phosphatase.     

A study of the intracellular transport of calcium in rat heart

The distribution of in vivo injected ⁴⁵Ca⁺⁺ in the subcellular fractions of rat heart has been studied. Most of the radioactivity of the cell was found to be associated with the subcellular organelles; only a small fraction was recovered in the soluble phase. Mitochondria contained the greatest part of the total radioactivity associated with the subcellular organelles. After injection of ⁴⁵Ca⁺⁺ the specific activity of the mitochondrial calcium pool was several times higher than that of the calcium of the sarcoplasmic reticulum. Pentachlorophenol has been administered to rats to uncouple oxidative phosphorylation in heart mitochondria in vivo and its effect on the distribution of ⁴⁵Ca⁺⁺ in the heart studied. Under these conditions, it has been found that mitochondria contained much less ⁴⁵Ca⁺⁺ than the controls; this decrease was paralleled by an increase of the radioactivity associated with the microsomes and with the final supernatant. Experiments in which ⁴⁵Ca⁺⁺ was added to heart homogenates at 0° indicated that ⁴⁵Ca⁺⁺ also became bound to mitochondria and the other subcellular structures at 0°. However, PCP had no effect on the distribution of radioactivity among the subcellular fractions under these conditions. The results suggest that (1) energy-linked movements of Ca⁺⁺ take place in mitochondria of the intact rat heart, (2) a part of the uptake of ⁴⁵Ca⁺⁺ by mitochondria does not depend on metabolism, and, (3) the movements of Ca⁺⁺ in heart mitochondria in vivo are probably more active than those in the sarcoplasmic reticulum.     

Effect of exogenous ATP on the volume of TA3 ascites tumor cells

When exogenous ATP is added to suspensions of TA₃ ascites tumor cells suspended in Ca⁺⁺ and Mg⁺⁺ free media, a significant increase in cell volume can be measured. This increase is reversible upon addition of Ca⁺⁺ and/or Mg⁺⁺ back to the media. The enlargement of these cells is temperature sensitive and specific for ATP; no other nucleotides, EDTA or ouabain were effective. The evidence suggest that this phenomena may be due to an alteration in membrane permeability and that the regulation of membrane permeability is an energy dependent process.     

Induction of permeability of the inner membrane of yeast mitochondria

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a non-specific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochon-dria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca²⁺-dependent pores, neither the classical Ca²⁺/P_i-dependent, cyclosporin A-sensitive pore even under deenergization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca²⁺ concentrations (in the presence of the Ca²⁺ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Ultrastructural distribution of Ca++ within neurons

Summary We used the oxalate-pyroantimonate technique to determine the ultrastructural distribution of Ca⁺⁺ in neurons of the rat sciatic nerve. The content of the precipitate was confirmed by X-ray microanalysis and appropriate controls. In the cell bodies of the dorsal root ganglia, Ca⁺⁺ precipitate was found in the Golgi, mitochondria, multivesicular bodies and large vesicles of the cytoplasm but not in lysosomes, and was prominently absent from regions of rough endoplasmic reticulum and ribosomes. It was seen in the nucleus but not in the nuclear bodies or nucleolus.Within the axon itself, Ca⁺⁺ precipitate was also found sequestered in mitochondria and smooth endoplasmic reticulum. In addition Ca⁺⁺ precipitate found diffusely throughout the axoplasm exhibited a discrete and heterogeneous distribution. In myelinated fibers the amount of precipitate decreased predictably in the axoplasm beneath the Schmidt-Lanterman clefts and in the paranodal regions at the nodes of Ranvier. This correlated with the presence of dense precipitate in the Schmidt-Lanterman clefts them-selves and in the paranodal loops of myelin.Intracytoplasmic ionic Ca⁺⁺ is maintained at 10^–7 M by balanced processes of influx, sequestration and extrusion. The irregular distribution of Ca⁺⁺ precipitate in the axoplasm of myelinated fibers suggests that there may be specific regions of preferential efflux across the axolemma.