The nature of the electron spin resonance signal during aerobic uptake of Mn2+ in mitochondria from rat liver

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high-energy membrane sites or to complexes of Mn2+ with inorganic phosphate. Identification of the spin exchange signal with a Mn-Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N-ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Pi is generated in the matrix, in the presence of N-ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal. The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50-60 nmol X mg protein-1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations. The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93-99] that the spin exchange signal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.     

Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes.

The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.     

Ca2+ channels and intracellular Ca2+ stores in neuronal and neuroendocrine cells

Changes in [Ca2+]i are essential in modulating a variety of cellular functions. In no other cell type does the regulation of [Ca2+]i reach the level of sophistication observed in cells of neuronal origin. Because of its physicochemical characteristics, the fluorescent Ca2+ indicator Fura-2 has become extremely popular among neuroscientists. The use of this probe, however, has generated a number of problems, in particular, extracytosolic trapping and leakage from intact cells. In the first part of this contribution we briefly discuss the practical application of Fura-2 to the study of [Ca2+]i in primary cultures of neurons and astrocytes. In the second part, we review some recent data (mainly from our laboratories) obtained in neurons and neuroendocrine cells, concerning the regulation of different types of Ca2+ channels and the role and mechanism of intracellular Ca2+ mobilization. The experimental evidence supporting the existence of a previously unrecognised organelle, the calciosome, that we hypothesize represents the functional equivalent in non-muscle cells of sarcoplasmic reticulum, will also briefly be discussed.     

An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes

The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.     

Leptinotoxin-h Action in Synaptosomes, Neurosecretory Cells, and Artificial Membranes: Stimulation of Ion Fluxes

Leptinotoxin-h (LPTx), a neurotoxin (otherwise designated beta-leptinotarsin-h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry 23, 734-741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+-containing Ringer medium, at concentrations in the 10(-11) - 10(-10) M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage-dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx; and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+-free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (approximately 10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono- and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of alpha-latrotoxin (alpha-LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit alpha-LTx binding.     

Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping, deplete cellular ATP and inhibit respiration of isolated mitochondria.

3,3'-Dipropylthiodicarbocyanine iodide, a widely used fluorescent probe of membrane potential, was found to inhibit anti-Ig antibody, induced capping of mouse lymphocytes. The dye also lowered the cell ATP content. Experiments with isolated mitochondria revealed that the probe had a potent inhibitory action at site I of the respiratory chain. This mitochondrial blockade helps to explain the ATP depletion and blockade of capping, and gives cause for caution in the use of this dye as a probe of cell membrane potential. Three related dicarbocyanine dyes had similar toxic effects, but two cyanine dyes with much longer alkyl side chains, which have been used as probes of membrane fluidity, did not.     

Dicumarol, an inhibitor of ADP-ribosylation of CtBP3/BARS, fragments golgi non-compact tubular zones and inhibits intra-golgi transport

Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex.     

Beta-adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor

-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by -adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of -adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types. adenovirus; protein kinase A; phosphodiesterase; L-type Ca²⁺ channel     

Dynamic Properties of an Inositol 1,4,5-Trisphosphate– and Thapsigargin-insensitive Calcium Pool in Mammalian Cell Lines

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate– and thapsigargin-insensitive Ca²⁺ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca²⁺ pool is released into the cytoplasm by the Ca²⁺ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate–sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca²⁺ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca²⁺ concentration elicited by activation of Ca²⁺ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca²⁺ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca²⁺ uptake was excluded since ionomycin is inefficient in releasing Ca²⁺ from acidic pools and Ca²⁺ accumulation/release in/from this store was unaffected by monensin or NH₄Cl, drugs known to collapse organelle acidic pH gradients. Ca²⁺ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca²⁺–ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca²⁺–ATPases. The cytological nature and functional role of this Ca²⁺ storage compartment are discussed.The cytosolic free Ca²⁺ concentration ([Ca²⁺]_i)¹ of eukaryotic cells rests in the range of 50–200 nM, i.e., at a very low level, if compared to the Ca²⁺ concentration of physiological media (2 mM). However, the total cellular Ca²⁺ content is closer to this latter value (1–3 mmol/l of cell water). In other words, eukaryotic cells sequester large amounts of Ca²⁺ mainly by uptake inside intracellular Ca²⁺ stores (∼90%) (for reviews see Pozzan et al., 1994; Clapham, 1995).The complexity of intracellular Ca²⁺ stores has been intensively investigated in recent years (for reviews see Meldolesi et al., 1990; Pozzan et al., 1994; Simpson et al., 1995). Attention has been focused mainly on Ca²⁺ stores that are highly dynamic because of their ability to rapidly take up and release Ca²⁺. Ca²⁺ sequestration into these pools depends on Ca²⁺–ATPases, known as sarco/endoplasmic reticulum Ca²⁺–ATPases (SERCAs) (Burk et al., 1989; Bobe et al., 1994; Wuytack et al., 1994). All the SERCA isoforms share the property of being selectively inhibited by thapsigargin (Tg), a tumor-promoting sesquiterpene lactone (Lytton et al., 1991). Tg acts with both high affinity, at nanomolar concentrations, and high specificity, with virtually no effect on the Ca²⁺– or Na⁺/K⁺– ATPase of the plasmalemma.Other drugs, such as 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBHQ) and cyclopiazonic acid (CA), also block SERCAs, albeit with a significantly lower affinity (Mason et al., 1991). Ca²⁺ release, on the other hand, depends mainly on two types of Ca²⁺ release channels named inositol 1,4,5-trisphosphate (InsP₃) and ryanodine receptors (for reviews see Mikoshiba, 1993; Sorrentino and Volpe, 1993; Ehrlich, 1995). These channels are expressed in variable proportions in different cell types and couple extracellular stimuli to the release of Ca²⁺, with possible ensuing generation of Ca²⁺ waves and spikes (for reviews see Amundson and Clapham, 1993; Taylor, 1994; Bootman and Berridge, 1995). The relationship between these types of Ca²⁺-release channels is still largely debated. The ryanodine-sensitive channel is also activated by caffeine, and ryanodine- and caffeine-sensitive stores are generally regarded to comprise the same pool (Zacchetti et al., 1991; Barry and Cheek, 1994; but also see Giannini et al., 1992; McNulty and Taylor, 1993).In the vast majority of cell types so far investigated, the InsP₃- (and/or the ryanodine-) sensitive stores almost completely overlap with those sensitive to Tg (Zacchetti et al., 1991; Gamberucci et al., 1995) and are thus referred to also as Tg-sensitive Ca²⁺ pools. From the cytological point of view, the InsP₃-/Tg-sensitive Ca²⁺ pool is identified with the ER or with a subfraction of it (Hashimoto et al., 1988).The complexity of the relationships between the InsP₃- and ryanodine/caffeine-sensitive stores does not cover the entire issue of intracellular Ca²⁺ pool heterogeneity. Other types of Ca²⁺ pools are known to exist, the size of which varies considerably among different cell types. These latter Ca²⁺ stores account for roughly half of all sequestered Ca²⁺ (Chandra et al., 1991; Fasolato et al., 1991; Shorte et al., 1991; Bastianutto et al., 1995; Mery et al., 1996). They have been identified through the increase in [Ca²⁺]_i upon application of Ca²⁺ ionophores, after depletion of the Tgsensitive pool with a combination, or a sequence, of InsP₃generating agonists, Tg, and caffeine. These residual Tginsensitive pools appear rather heterogeneous in terms of cytological identity and pharmacological sensitivity. Part of these pools shows an acidic lumenal pH and is discharged only by a combination of a Ca²⁺ ionophore and of agents that collapse internal acidic pH gradients (such as monensin and NH₄Cl). ⁴⁵Ca²⁺ labeling of Tg-insensitive pools is slower than that of the Tg-sensitive store, and, for this reason, they have been generally indicated as slowly exchanging Ca²⁺ pools (Fasolato et al., 1991). As far as their identification is concerned, the acidic pool seems largely identifiable with secretory compartments and lysosomes, while very little is known yet about the rest of the Tg-insensitive store.Here we demonstrate that a nonacidic, InsP₃- and Tg- insensitive Ca²⁺ pool rapidly accumulates large amounts of Ca²⁺ when high and sustained increases of [Ca²⁺]_i are induced by opening of voltage- or store-operated Ca²⁺ channels. This Ca²⁺ storage compartment is insensitive to mitochondrial uncouplers and appears diffusely distributed in the cell cytosol. The possibility is discussed that this low-affinity, high-capacity Ca²⁺ pool represents a previously unidentified subcompartment of the ER expressing a Tg-insensitive Ca²⁺–ATPase.     

The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum.

In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.