Intracellular calibration of the fluorescent Mg2+ indicator furaptra in rat ventricular myocytes

Single ventricular myocytes enzymatically isolated from rat hearts were loaded with the Mg2+ indicator furaptra, and the relationship between the fluorescence ratio signal (R) and the intracellular free concentration of Mg2+ ([Mg2+]i) was studied in situ at 25 degrees C. After the application of ionophores (ionomycin, monensin, nigericin and valinomycin), an immediate change in furaptra R was noted, followed by a slow change in R that reached a steady level in 2-4 h. The direction of the early change in R that accompanied rigor contraction was independent of the extracellular Mg2+ concentration ([Mg2+]o), and was consistent with the breakdown of ATP and release of bound Mg2+. The intracellular calibration curve was constructed from the steady levels of R obtained at various [Mg2+]o between 0 and 47 mM. The dissociation constant of intracellular furaptra was estimated to be 5.3 mM, which was 44% higher than that determined in salt solutions (3.7 mM). The basal [Mg2+]i of rat ventricular myocytes calculated with the intracellular curve averaged 0.91 mM.     

Intracellular free magnesium and its regulation, studied in isolated ferret ventricular muscle with ion-selective microelectrodes

Intracellular free magnesium ([Mg2+]i) was measured in isolated ferret papillary muscles using ion-selective microelectrodes filled with the new magnesium sensor ETH 5214. This new sensor, unlike its predecessor ETH 1117, does not react to marked changes in K+, Na+ or pH. Reducing Ca2+ from 20 microM to around 10 nM also did not affect the response so these electrodes are ideally suited to study intracellular Mg2+ and its regulation. The mean value for the [Mg2+]i from thirty-two experiments (forty-two impalements) was 0.85 mM, confirming previous estimates from this laboratory. Intracellular Mg2+ is not passively distributed and the possibility that Mg2+ is transported out of the cell by a Na(+)-Mg2+ exchanger was investigated. An increase in [Mg2+]o caused an increase in [Mg2+]i, as did stepwise reduction in the [Na+]o. However, this increase in [Mg2+]i on Na+ reduction also occurred in Mg2(+)-free solution suggesting that the increase in [Mg2+]i was due to the increase in intracellular Ca2+ on Na+ reduction. Moreover, increasing [Na+]i by strophanthidin did not change the [Mg2+]i and on increasing [Mg2+]o there was no reduction in the [Na+]i. Blocking ATP production lead to small increases in the [Mg2+]i. These results are not consistent with a Na(+)-Mg2+ exchanger as being the main outward transport mechanism for Mg2+ in this tissue.     

Myoplasmic Mg2+ concentration in Xenopus muscle fibres at rest, during fatigue and during metabolic blockade.

Intracellular free Mg2+ concentration ([Mg2+]i) was measured in isolated single fibres of Xenopus muscle using the fluorescent Mg2+ indicator furaptra. In resting muscle the [Mg2+]i was 1.7 mM in a Mg(2+)-free Ringer solution. There was no significant change in [Mg2+]i over 2 h in Mg(2+)-free Ringer solution. Elevating extracellular [Mg2+] to 40 mM for 5 min caused a small rise (0.13 mM) in [Mg2+]i. There was no detectable rise in [Mg2+]i after 5 min in Na(+)-free Ringer solution. These results suggest that the membrane is relatively impermeable to Mg2+ and that there was no detectable Na(+)-Mg2+ exchange over 5 min. When muscle fibres were fatigued by repeated tetani continued until force declined to about 40% of control, [Mg2+]i showed characteristic changes. During the early period of fatigue when force first showed a small decline and then became almost stable, [Mg2+]i was unchanged; during the final period of fatigue when force declined more rapidly, [Mg2+]i increased by 0.8 mM. Recovery of [Mg2+]i took about 30 min. Recovery of force was complex: tetanic force first declined (post-contractile depression) and then slowly recovered to control. Since the minimum force occurred at about the time when [Mg2+]i had recovered, it seems unlikely that post-contractile depression is caused by elevated [Mg2+]i. Rigor, produced by inhibiting oxidative phosphorylation and glycolysis, was associated with a larger increase (1.6 mM) in [Mg2+]i than fatigue. The rise in [Mg2+]i during fatigue and metabolic blockade could be explained as release of Mg2+ normally bound to ATP. A model of the metabolic changes and the resulting increase in [Mg2+]i explains our results reasonably well.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Concentration, compartmentation and metabolic function of intracellular free Mg2+.

Intracellular total Mg2+ and free Mg2+ are compartmentalized between cell organelles and within the cytosol. Different values of [Mg2+]i in the cytosol of the same cell type were measured by various investigators. A main reason for the differences is the uncertainty of the dissociation constants used for the Mg furaptra complex in the fluorescence method and for MgATP when 31P NMR was employed. The more realistic KD values of Mg furaptra and MgATP measured under in situ conditions are higher than the KDs used by most investigators. The [Mg2+]is obtained and the KDs used by various authors were presented. The role of intracellular Mg2, in metabolic functions and the action of various effectors on [Mg2+]i and [Ca2+] was reviewed. Intracellular Mg2+ may have a permissive role supporting the effector-induced mechanisms that are mediated by Ca2+ as a second messenger.     

Cytoplasmic free concentrations of Ca2+ and Mg2+ in skeletal muscle fibers at rest and during contraction

This review summarizes estimates for cytoplasmic-free concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) at rest and during contraction of skeletal muscles, from which substantial quantitative information about them has been accumulated. Although the estimates of resting [Ca2+]i in the literature widely differ, which is because of the variety of difficulties related to different methodologies used, recent studies suggest that estimates of resting [Ca2+]i of approximately 0.05-0.1 microM are likely to be correct. Following action potential propagation, the Ca2+ release from the sarcoplasmic reticulum causes a transient rise of [Ca2+]i (Ca2+ transient). The large peak amplitude and brief time course of the Ca2+ transients have been established only recently by studies with low-affinity Ca2+ indicators developed in the past decade. These technical improvements in [Ca2+]i measurements have made it possible to study relationships between [Ca2+]i and force in intact muscle fibers. In the second part of this review, various estimates of [Mg2+]i in the resting muscle are discussed. Relatively recent estimates of the [Mg2+]i level appear to be about 1.0 mM. Using the current knowledge of concentrations and reaction properties of intracellular Ca2+-Mg2+ binding sites, we constructed a model for dynamic Mg2+ movement following Ca2+ transients. The model predicts that with a train of action potentials, the sustained rise of [Ca2+]i produces an elevation of [Mg2+]i of about 200 microM.     

Mechanisms of Mg2+ influx, efflux and intracellular 'muffling' in leech neurones and glial cells.

Mg2+ is known to influence conductance and gating properties of a multitude of ion channels and is thus able to modulate synaptic transmission. Therefore, a tight regulation of the intracellular free Mg2+ concentration ([Mg2+]i) in neurones and glial cells is crucial for maintaining the functions of central nervous systems. [Mg2+]i is regulated through the balance of Mg2+ influx and Mg2+ efflux, together with heavy damping of [Mg2+]i changes through intracellular buffering and sequestration. To investigate the mechanisms involved in [Mg2+]i regulation, neurones and glial cells from the central nervous system of the leech Hirudo medicinalis proved to be an ideal model system. The present article summarizes the evidence for a Mg2+ influx pathway which is distinct from that for Ca2+, for a dual regulation of Mg2+ efflux (a 1 Na+/1 Mg2+ antiport and a Na(+)-independent Mg2+ efflux mechanism), for pH-dependent Mg2+ buffering through ATP and other intracellular Mg2+ binding components and for the involvement of mitochondria in intracellular Mg2+ sequestration.     

Na+/Mg2+ antiport in non-erythrocyte vertebrate cells.

Experiments, results and conclusions on Na+/Mg2, antiport in lymphocytes, HL60 cells, Ehrlich ascites tumor cells, platelets, pancreatic acinar cells, sublingual acini, hepatocytes, ruminal epithelial cells, kidney cells, smooth muscle cells, heart muscle cells and skeletal muscle cells were reviewed. Only in a few experiments was the Mg2+ efflux via the Na+/Mg2+ antiport studied directly by measuring the alterations in [Mg2+]o and intracellular Na+ content. In most cell types, the Na+/Mg2+ antiport was investigated indirectly by measuring [Mg2+], and [Na+]i after changing the physiological Mg2+ homeostasis by effectors or by loading the cells with Mg2+. The effects of inhibitors and incubation in Na+ -free medium on the alterations in [Mg2+]i and [Na+]i were taken as evidence of the Na+/Mg2+ antiport. By these methods, the Na+/Mg2+ antiport was found in all investigated mammalian cell types. As far as they have been studied, the kinetic properties and regulation of Na+/Mg2+ antiport are reviewed.     

Subcellular localization of glutamate-stimulated intracellular magnesium concentration changes in cultured rat forebrain neurons using confocal microscopy

Glutamate can stimulate increases in intracellular magnesium concentration ([Mg2+]i) and induce neurotoxicity, both independent of Ca2+ changes. Although Mg2+ is essential within the cell, very little is known about how it is regulated, especially in neurons. Therefore we used the fluorescent indicator, magindo-1 and confocal microscopy to examine possible intracellular pools of Mg2+ in cultured neurons that can be dynamically regulated by glutamate. The magindo-1 fluorescence signal was present throughout the cell body and extends into the neuronal processes. The magindo-1 405 nm/490 nm ratio signal was similar in the cytoplasm and nucleus, suggesting that resting [Mg2+]i is uniform across the neuron. The addition of 100 microM glutamate/10 microM glycine in an extracellular Ca2+- and Na+-free buffer stimulated an increase in [Mg2+]i in both the nuclear and cytoplasmic regions of similar magnitude and duration. This glutamate exposure also stimulated a [Mg2+]i increase in neuronal processes which was inhibited by the N-methyl-D-aspartate receptor antagonist, MK-801 (10 microM). The glutamate-stimulated [Mg2+]i increase in both the cell body and neuronal processes was dependent on the extracellular Mg2+ concentration. These findings suggest glutamate-stimulated [Mg2+]i changes may not only impact cytoplasmic processes, but also directly trigger nuclear events involved, for example, in neuronal injury.     

Effects of ATP and its analogues on [Ca2+]i dynamics in the rabbit corneal epithelium.

Adenosine-5'-triphosphate (ATP) plays a pivotal role in various tissues as an extracellular transmitter. ATP released from nerve endings and/or damaged cells may elicit reactions in adjacent cells. To identify such reactions, we investigated the dynamics of the intracellular calcium ion concentrations ([Ca2+]i) in the rabbit corneal epithelium during ATP-stimulation. Intact epithelial sheets isolated from corneal tissue were loaded with Fura-2, and [Ca2+]i dynamics in each cell layer were analyzed using a digital imaging system (Argus 50/CA). Normal architecture was preserved, suggesting that functional integrity remained intact. Perfusion with HEPES-buffered Ringer's solution containing ATP (10 microM) and uridine-5'-triphosphate (UTP; 10 microM) caused a biphasic [Ca2+]i increase in the superficial layer that manifested itself as a rapid initial spike followed by a long-lasting plateau phase. Adenosine-5'-diphate (10 microM) elevated the [Ca2+]i level, but induced only the initial spike, which was smaller than those induced by ATP and UTP. Adenosine (10 microM) did not elicit any [Ca2+]i changes in the epithelial cells. Suramin (10 microM; a P2 receptor antagonist) blocked the ATP-induced [Ca2+]i increase, whereas the P2X receptor agonists, alpha, beta-methylene ATP (10 microM), 2-methyl-thio ATP (10 microM) and Benzoylbenzoyl ATP (10 microM), did not elicit any increases in [Ca2+]i. In the basal cell layer, ATP-induced [Ca2+]i dynamics were biphasic, while oscillatory fluctuations of [Ca2+]i were induced in the wing cells of the mid layer of the corneal epithelium by ATP stimulation. Ca2+ oscillations were sometimes synchronized among adjacent wing cells, but these waves did not propagate to other cell layers. These results suggest that extracellular ATP elicits a [Ca2+]i increase mainly via P2Y receptors. In addition, synchronized Ca2+ oscillation in the wing cell layer indicates that intracellular events may spread to neighboring cells within the layer.     

Na+/Mg2+ antiport in erythrocytes of spontaneously hypertensive rats: role of Mg2+ in the pathogenesis of hypertension.

Total Mg2+ content in plasma and erythrocytes did not significantly differ between WKY and SHR. Mg2+ efflux via Na+/Mg2+ antiport was 10% lower in non Mg(2+)-loaded erythrocytes of SHR than in WKY, and 16% lower in Mg(2+)-loaded erythrocytes of SHR. The activation of Na+/Mg2+ antiport in erythrocytes by Cl-, as tested by substitution of Cl- with SCN-, and the regulation of Na+/Mg2+ antiport by protein kinases, as tested by PMA and staurosporine, showed no differences between WKY and SHR. The reduction of Na+/Mg2+ antiport was explained by a reduction in the number of Na+/Mg2+ antiporter molecules in SHR erythrocytes. Mg2+ efflux in KCl medium by K+/Mg2+ antiport via the unspecific choline exchanger was not significantly reduced in SHR and was equally affected by PMA and staurosporine in WKY and SHR. An explanation for some controversial results, unchanged or reduced concentration of Mg2+ in serum, total Mg2+ and free Mg2+ in erythrocytes of SHR and patients with essential hypertension was proposed. The role of Na+/Mg2+ antiport and [Mg2+]i in the pathogenesis of experimental and clinical hypertension was discussed.     

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes)

 The effect of adenosine triphosphate (ATP) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca²⁺]_i increase, which was dose dependent in the 2.5–50 μM range (EC₅₀=4.3 μM). The ATP-evoked [Ca²⁺]_i rise was not modified during the first minute following the removal of external Ca²⁺. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca²⁺]_i increase. Caffeine (10 mM) and ryanodine (1 μM) did not affect the ATP-induced [Ca²⁺]_i rise. The pituicytes responded to various P₂ purinoceptor agonists with the following order of potency: ATP=ATP[γ-S]=2-MeSATP≥ADP, where ATP[γ-S] is adenosine 5′-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5′-triphosphate. Adenosine, AMP, α,β-methylene adenosine-5′-triphosphate (α,β-MeATP), β,γ methylene adenosine-5′-triphosphate (β,γ-MeATP) and uridine 5′-triphosphate (UTP) were ineffective. The P₂ purinoceptor antagonists blocked the ATP-evoked [Ca²⁺]_i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The ATP-evoked [Ca²⁺]_i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 μM) abolished the ATP-evoked [Ca²⁺]_i rise, whereas its inactive stereoisomer U-73343 (0.5 μM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca²⁺]_i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P_2Y receptor. Received: 24 September 1998 / Received after revision: 10 December 1998 / Accepted: 18 December 1998     

Evidence for P2Y-type ATP receptors on the serosal membrane of frog skin epithelium

The present study presents the first evidence for P2Y-type adenosine 5'-triphosphate (ATP) receptors on the basolateral membranes of frog skin epithelial cells. Cytosolic calcium ([Ca2+]i) was measured with fura-2 and Calcium-Green-1 using epifluorescence microscopy and confocal laser scanning microscopy respectively. In the presence of Ca2+ in the solutions ATP increased [Ca2+]i. The increase in [Ca2+]i was due to the agonist activity of ATP and not to the activity of the potential products of ATP metabolism, i.e. adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) or adenosine, as shown by a comparison of the magnitude of the increases in [Ca2+]i caused by the various compounds. The rise in [Ca2+]i was predominantly monophasic at low ATP concentrations (below 100 microM). At higher concentrations the initial spike was followed by a plateau phase. In the absence of Ca2+ in the extracellular solution ATP caused Ca2+ release from intracellular stores. This could be inhibited by pre-treatment of the tissue with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. The nucleotide uridine 5'-triphosphate (UTP) had similar effects on [Ca2+]i although the plateau level of the [Ca2+]i response was higher with this P2Y agonist. Confocal laser scanning microscopy showed that all cell layers of the epithelium responded to ATP. Our data indicates that serosal ATP acts on serosal P2Y-type receptors in frog skin epithelium. This is the first evidence of a phospholipase C-coupled receptor in this tissue.     

ATP-induced [Ca2+]i changes in the human corneal epithelium

ATP, when leaked from damaged cells, is capable of eliciting responses in neighboring cells. A better understanding of the mechanism of this response is essential for designing therapeutic strategies for disease, there have been only a limited number of studies on the effect of ATP on the human cornea. We examined ATP-induced intracellular Ca2+ ([Ca2+]i) changes in the human corneal epithelia, cultured to near confluence. Cells were loaded with the Ca2+ indicators, Indo-1 or Fluo-4, and [Ca2+]i was monitored. ATP was found to induce an increase in [Ca2+]i, which was initiated from the perinuclear region and the nuclear envelope per se, and then propagated gradually towards the periphery. Intranuclear Ca2+ was momentarily increased. UTP elicited an identical response, but adenosine and alpha, beta-methylene ATP had no effect. Pretreatment with U73122 or thapsigargin inhibited the ATP-induced increase in [Ca2+]i. When a cell was topically stimulated with ATP, the [Ca2+]i increase spread beyond the cell boundary. The intercellular communications that accompanied the [Ca2+]i changes were inhibited by octanol. We conclude that extracellular ATP in the human cornea caused the mobilization of Ca2+ from intracellular Ca2+ stores (e.g. the endoplasmic reticulum and nuclear envelope) via P2Y purinoceptors of the epithelial cell. The response to ATP appears to spread to neighboring regions through gap junctions in the epithelium.     

H2O2 enhances Ca2+ release from osteoblast internal stores

The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.     

The intracellular concentration of free magnesium in extensor digitorum longus muscles of the rat

Magnesium-sensitive microelectrodes were used to measure the intracellular concentration of free Mg2+, [Mg2+]f, in rat extensor digitorum longus muscles in vitro at 30 degrees C. The intracellular activities of Na+ and K+ were also determined so that allowance could be made for the interference from these ions with the Mg2+ electrode response. The mean value for [Mg2+]f based on twenty-six measurements in twelve muscles was 0.47 mM.     

ATP-induced Ca2+ response mediated by P2U and P2Y purinoceptors in human macrophages: signalling from dying cells to macrophages

The activation of macrophages by various stimuli leading to chemotactic migration and phagocytosis is known to be mediated by an increase in intracellular Ca2+ concentration ([Ca2+]i). We measured changes in [Ca2+]i using a Ca2+ imaging method in individual human macrophages differentiated from freshly prepared peripheral blood monocytes during culture of 1-2 days. A transient rise in [Ca2+]i (duration 3-4 min) occurred in 10-15 macrophages in the vicinity of a single tumour cell that was attacked and permeabilized by a natural killer cell in a dish. Similar Ca2+ transients were produced in 90% of macrophages by application of supernatant obtained after inducing the lysis of tumour cells with hypo-osmotic treatment. Ca2+ transients were also evoked by ATP in a dose-dependent manner between 0.1 and 100 microm. The ATP-induced [Ca2+]i rise was reduced to less than one-quarter in Ca2+-free medium, indicating that it is mainly due to Ca2+ entry and partly due to intracellular Ca2+ release. UTP (P2U purinoceptor agonist) was more potent than ATP or 2-chloro-ATP (P2Y agonist). Oxidized ATP (P2Z antagonist) had no inhibitory effect. Both cell lysate- and ATP-induced Ca2+ responses were inhibited by Reactive Blue 2 (P2Y and P2U antagonist) to the same extent, but were not affected by PPADS (P2X antagonist). Sequential stimuli by cell lysate and ATP underwent long-lasting desensitization in the Ca2+ response to the second stimulation. The present study supports the view that macrophages respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [Ca2+]i rise via P2U and P2Y receptors.     

Mitochondrial modulation of Ca2+ -induced Ca2+ -release in rat sensory neurons

Ca2+ -induced Ca2+ -release (CICR) from ryanodine-sensitive Ca2+ stores provides a mechanism to amplify and propagate a transient increase in intracellular calcium concentration ([Ca2+]i). A subset of rat dorsal root ganglion neurons in culture exhibited regenerative CICR when sensitized by caffeine. [Ca2+]i oscillated in the maintained presence of 5 mM caffeine and 25 mM K+. Here, CICR oscillations were used to study the complex interplay between Ca2+ regulatory mechanisms at the cellular level. Oscillations depended on Ca2+ uptake and release from the endoplasmic reticulum (ER) and Ca2+ influx across the plasma membrane because cyclopiazonic acid, ryanodine, and removal of extracellular Ca2+ terminated oscillations. Increasing caffeine concentration decreased the threshold for action potential-evoked CICR and increased oscillation frequency. Mitochondria regulated CICR by providing ATP and buffering [Ca2+]i. Treatment with the ATP synthase inhibitor, oligomycin B, decreased oscillation frequency. When ATP concentration was held constant by recording in the whole cell patch-clamp configuration, oligomycin no longer affected oscillation frequency. Aerobically derived ATP modulated CICR by regulating the rate of Ca2+ sequestration by the ER Ca2+ pump. Neither CICR threshold nor Ca2+ clearance by the plasma membrane Ca2+ pump were affected by inhibition of aerobic metabolism. Uncoupling electron transport with carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone or inhibiting mitochondrial Na+/Ca2+ exchange with CGP37157 revealed that mitochondrial buffering of [Ca2+]i slowed oscillation frequency, decreased spike amplitude, and increased spike width. These findings illustrate the interdependence of energy metabolism and Ca2+ signaling that results from the complex interaction between the mitochondrion and the ER in sensory neurons.     

Extracellular magnesium deficiency induces contraction of arterial muscle: role of PI3-kinases and MAPK signaling pathways

The present study investigated the actions of extracellular Mg2+ ([Mg2+]o) deficiency on isolated rat aortae and rat aortic smooth muscle cells (RASMC). Exposure of isolated, intact rat aortic rings to Mg(2+)-free or Mg(2+)-deficient medium (0.15-0.6 mM) produced endothelium-independent, concentration-dependent contractions: the lower the [Mg2+]o, the stronger the contraction. Pre- or post-incubation of the vessels with low concentrations of U0126, SB-203580, PD-98059, wortmannin, LY-294002, or a SH2 domain inhibitor peptide suppressed [Mg2+]o deficiency-induced contractions significantly. The concentrations of these antagonists required for half-maximal inhibition (IC50) were not very different from the inhibitory constants (Ki) for these drugs. A variety of specific pharmacological antagonists of several known endogenously-formed vasoconstrictors did not inhibit or attenuate the contractions induced by low [Mg2+]o. Mg(2+)-free medium induced a 6- to 7-fold increase in intracellular Ca2+ ([Ca2+]i) in RASMC. Pre- or post-treatment of the cells with U0126, SB-203580, PD-98059, wortmannin, LY-294002, or a SH2 domain inhibitor peptide markedly inhibited the increments in ([Ca2+]i) in RASMC induced by exposure to Mg(2+)-free medium. The present findings suggest that Mg(2+)-deficiency-induced contractions of rat aortae are associated with activation of several cellular signal pathways, such as mitogen-activated protein kinase, phosphatidylinositol-3 (PI3) kinases, and SH2 domain-containing proteins.