Integrating cytosolic calcium signals into mitochondrial metabolic responses

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.     

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.     

Contribution of Na+/Ca2+ exchanger in maintaining [Ca2+]c at a stable state in rat pancreatic islets

The effects of lowering extracellular Na+ concentration [Na+]o, on cytosolic Ca2+ concentration, [Ca2+]c were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. The total replacement of extracellular Na+ (Na+o) by equimolar N-methyl-D-(--)-glucamine caused a rapid rise in [Ca2+]c, and partial replacement of Na+o resulted in correlative rises in [Ca2+]c in accordance with the magnitude of reduced [Na+]o. The rise in [Ca2+]c induced by Na+o removal was strongly inhibited in the Ca2+o-deficient environment or by Ni2+. The [Ca2+]c rise, however, remained almost unchanged in the presence of nifedipine or SK&F 96365, and was enhanced by the addition of ouabain. The electrochemical gradients for Ca2+ (delta mu Ca2+) and Na+ (delta mu Na+) were calculated to be 39.08 and 12.8 kJ/mol, respectively, in this study, indicating a stoichiometry of 3Na+: 1 Ca2+. These results indicate that, in rat pancreatic islets, the rise in [Ca2+]c induced by lowering [Na+]o is mainly due to Ca2+ entry medicated by the Na+/Ca2+ exchanger operating with the stoichiometry of 3Na+:1 Ca2+, and that the Na+/Ca2+ exchanger plays an important role in maintaining stable-state [Ca2+]c.     

Histamine-induced calcium released from cultured human mucosal microvascular endothelial cells from nasal inferior turbinate

Changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human mucosal microvascular endothelial cells (HMMECs) from nasal inferior turbinate were measured using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy. Histamine caused a transient increase in intracellular free Ca2+ in cell populations and in individual cells, followed by a decrease to a sustained elevation. Histamine (100 microM) elevated [Ca2+]i in HMMECs up to 563 +/- 20 nM from a resting level of 60 +/- 45 nM (means +/- SD, n = 31). Promethazine (a histamine H1 receptor antagonist) inhibited [Ca2+]i increase during histamine stimulation, whereas cimetidine (a H2 receptor antagonist) and thioperamide (a H3 receptor antagonist) showed no inhibition. These results suggest that the histamine increase [Ca2+]i in HMMECs induces both a Ca2+ release from stores and a Ca2+ influx through activation of the H1 receptor.     

Mitochondrial deenergization underlies neuronal calcium overload following a prolonged glutamate challenge

The purpose of our work was to study the relationship between glutamate (GLU)-induced mitochondrial depolarization and deterioration of neuronal Ca2+ homeostasis following a prolonged GLU challenge. The experiments were performed on cultured rat cerebellar granule cells using the fluorescent probes, rhodamine 123 and fura-2. All the cells, in which 100 microM GLU (10 microM glycine, 0 Mg2+) induced only relatively slight mitochondrial depolarization (1.1-1.3-fold increase in rhodamine 123 fluorescence), retained their ability to recover [Ca2+]i following a prolonged GLU challenge. In contrast, the cells in which GLU treatment induced pronounced mitochondrial depolarization (2-4-fold increase in rhodamine 123 fluorescence), exhibited a high Ca2+ plateau in the post-glutamate period. Application of 3-5 mM NaCN or 0.25-1 microM FCCP during this Ca2+ plateau phase usually failed to produce a further noticeable increase in [Ca2+]i. Regression analysis revealed a good correlation (r2 = 0.88 +/- 0.03, n = 19) between the increase in the percentage of rhodamine 123 fluorescence and the post-glutamate [Ca2+]i. Collectively, the results obtained led us to conclude that the GLU-induced neuronal Ca2+ overload was due to the collapse of the mitochondrial potential and subsequent ATP depletion.     

The role of the mitochondria in the clearance of cytosolic calcium

The magnitude and space-temporal profile of the intracellular Ca2+ transients are determined both by the mechanism that decrease and increase calcium levels in the cytoplasm. By the use of cocktails with different content of specific inhibitors of the extrusion and sequester mechanisms, the ability of mitochondrial Ca2+ transport to limit the elevation in free cytosolic Ca2+ concentration, following an imposed Ca2+ load was reexamined, indicating variable data with respect to various cells. In chromaffin cells, inhibition of mitochondrial Ca2+ accumulation with protonophore, dramatically modifies the shape of the [Ca2+]c response, indicating that mitochondrial Ca2+ uptake is an important mechanism for clearance of large Ca2+ loads. By contrast, using digital imaging in the presence of specific mitochondria inhibitors to investigate the [Ca2+]c responses of cerebellar granule cells in which ATP generation has been totally separated from mitochondrial Ca2+ transport, indicates surprising results: it was confirmed that mitochondria in these cells accumulate Ca2+ entering the cell in response to plasma membrane depolarization, but specific abolition of mitochondrial Ca2+ accumulation without ATP depletion significantly decreases the bulk cytoplasmic Ca2+ transients generated by elevated KCl levels, whereas the response in greatly increased when protonophore are present and ATP/ADP ratios are allowed to collapse. The results suggest that nonmitochondrial ATP-dependent transport pathways are primarily responsible for removing excess Ca2+ from the cytoplasm. Far from restricting the elevation in [Ca2+]c in response to a Ca2+ load, functional mitochondria may enhance the elevation in the bulk cytoplasm. The existent conflict of data, suggests the need for a new reevaluation of the role of mitochondria in Ca2+ clearance, and the possibility that mitochondria contribute to, rather than protect against, excitoxicity has to be investigated.     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem. 4. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+-dependent elevation of [Ca2+]j, but had no effect on the extracellular Ca2+-independent elevation of [Ca2+], caused by ET-1 (10-7M).5. Bradykinin (10-7 M) or ATP (10- 5M) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+-independent and extracellular Ca2+-dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.6. From these findings we conclude that, in porcine endotheliel cells in situ, ET-1 elevates [Ca2+]i as are result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+ .The Ca2+ influx is regulated by an IAP-sensitive G-protein, while the release of Ca2+ from the intracellular store is not.     

Changes in mitochondrial Ca2+ homeostasis in primary sensory neurons of diabetic mice

Changes in neuronal Ca2+ homeostasis were studied on freshly isolated dorsal root ganglion neurons of adult control mice and mice with streptozotocin (STZ)-induced diabetes. The cytoplasmic free Ca2+ concentration ([Ca2+]in) was measured using indo-1 based microfluorimetry. The participation of mitochondria in [Ca2+]in homeostasis was determined by investigation of changes which occurred after addition of mitochondrial protonophore (CCCP) to the extracellular solution. In control cells 10 microM CCCP applied before membrane depolarization induced an increase of the amplitude of depolarization-induced [Ca2+]in transients and disappearance of their delayed recovery, indicating the participation of mitochondria in fast uptake of Ca2+ ions from the cytosol during the peak of the transient and subsequent slow release them back during its decay. In diabetic animals the increase of the peak transient amplitude under the action of CCCP became diminished in small (nociceptive) neurons and the delayed elevation of [Ca2+]in disappeared in both large and small neurons. It is concluded that in diabetic conditions substantial changes occur in the Ca2+ homeostatic functions of mitochondria, manifested by decreased Ca2+ uptake in small neurons and depressed Ca2+ release into the cytosol in all types of neurons.     

Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+]c, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+]sr) is much higher than in the other compartments (> 100 microM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (approximately 1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2+, the kinetics and amplitudes of the changes in [cation2+]sr evoked by the various stimuli could also be directly analyzed.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neurons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca2+]i) in the neurons was low (50-100 nM). Depolarization of the neurons with 50 mM K+ resulted in rapid elevation of [Ca2+]i to 500-1,000 nM showing recovery to baseline [Ca2+]i over several minutes. The increases in [Ca2+]i caused by K+ depolarization were completely abolished by the removal of extracellular Ca2+, and were reduced by approximately 80% by the 'L-type' Ca2+ channel blocker, nimodipine (1 microM). [Ca2+]i was also increased by the excitatory amino acid L-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNQX and DNQX. In the absence of extracellular Mg2+, large fluctuations in [Ca2+]i were observed that were blocked by removal of extracellular Ca2+, by tetrodotoxin (TTX), or by antagonists of N-methyl D-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg2+ and TTX, NMDA caused dose-dependent increases in [Ca2+]i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca2+]i in the absence of extracellular Ca2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca2+ stores. Thapsigargin (2 microM) had little effect on [Ca2+]i, or on the recovery from K+ depolarization. Removal of extracellular Na+ had little effect on basal [Ca2+]i or on responses to high K+, suggesting that Na+/Ca2+ exchange mechanisms do not play a significant role in the short-term control of [Ca2+]i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca2+]i; however, [Ca2+]i recovered as normal from high K+ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca2+]i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca2+]i associated with action potential activity was measured. The amplitude of the [Ca2+]i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca2+]i from the modest Ca2+ loads imposed on the neuron by action potential trains follows a simple exponential decay (tau = 3-5 s).     

Na+/Ca2+ exchanger modulates kainate-triggered Ca2+ signaling in Bergmann glial cells in situ

The role of sodium-calcium exchanger in calcium homeostasis in Bergmann glial cells in situ was investigated by monitoring cytoplasmic calcium ([Ca2+]i) and sodium ([Na+]i) concentrations. The [Ca2+]i and [Na+]i transients were measured either separately by using fluorescent indicators fura-2 and SBFI, respectively, or simultaneously using the indicators fluo-3 and SBFI. Since the removal of extracellular Na+ induced a relatively small (approximately 50 nM) elevation of [Ca2+]i, the Na+/Ca2+ exchanger seems to play a minor role in regulation of resting [Ca2+]i. In contrast, kainate-triggered [Ca2+]i increase was significantly suppressed by lowering of the extracellular Na+ concentration ([Na+]o). In addition, manipulations with [Na+]o dramatically affected the recovery of the kainate-induced [Ca2+]i transients. Simultaneous recordings of [Ca2+]i and [Na+]i revealed that kainate-evoked [Ca2+]i transients were accompanied with an increase in [Na+]i. Moreover, kainate induced significantly larger [Ca2+]i and smaller [Na+]i transients under current-clamp conditions as compared to those recorded when the membrane voltage was clamped at -70 mV. The above results demonstrate that the Na(+)-Ca2+ exchanger is operative in Bergmann glial cells in situ and is able to modulate dynamically the amplitude and kinetics of [Ca2+]i signals associated with an activation of ionotropic glutamate receptors.     

Long-term activation of capacitative Ca2+ entry in mouse microglial cells

The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.     

Fluorescent monitoring of Jurkatt cell intracellular magnesium during metabolic poisoning

Divalent cation movement characterizes the final common pathway of cellular death from ischemic or metabolic injury. The influx of calcium is an essential step in cellular death. We hypothesized that intracellular magnesium levels may change during the progression to cellular death. Verapamil-sensitive changes in free ionized intracellular Mg2+ ([Mg2+[i) and Ca2+ ([Ca2+]i) levels were estimated in transformed T-lymphocytes exposed to metabolic inhibitors. Separate experiments used a Mg(2+)-sensitive fluoroprobe, fura-2 (Ex 1,344, Ex 2,376, Em 500), and a Ca(2+)-sensitive fluoroprobe, fura-2 (Ex 1,340, Ex 2,380, Em 510). Chemical anoxia (sodium cyanide 1 mM, iodoacetic acid 10 mM) caused a gradual increase in [Ca2+]i (control 126 +/- 13 nM) to > 1 mM by 10 min. This increase in [Ca2+]i was not affected by verapamil treatment. In separate experiments, [Mg2+]i levels were monitored during chemical anoxia. The specificity of mag-fura for Mg2+ over Ca2+ was reflected in the absence of a response to the lymphocyte Ca2+ mobilizer OKT-3. Uncorrected control [Mg2+]i levels (.4 +/- .1 mM) were not affected by the combined cyanide-iodoacetate treatment. A small increase in mag-fura-2 fluorescence was noted, probably due to binding of Ca2+ to the fluoroprobe when [Ca2]i exceeded 1 mM. Elimination of Ca2+ from the extracellular buffer increased the resting estimate of intracellular [Mg2+] to 1.6 + .1 mM. These results indicate that 1) extracellular Ca2+ can interfere with the fluorescent determination of intracellular magnesium concentration, and 2) intracellular free Mg2+ concentrations do not change in this cell line during chemical anoxia.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Insulin exocytosis and glucose-mediated increase in cytoplasmic free Ca2+ concentration in the pancreatic beta-cell are independent of cyclic ADP-ribose

Stimulation of pancreatic beta-cells by glucose gives rise to an increase in the cytoplasmic free calcium concentration ([Ca2+]i) and exocytosis of insulin. Cyclic adenosine 5'-diphosphate ribose (cADPR), a metabolite of beta-NAD+, has been reported to increase [Ca2+]i in pancreatic beta-cells by releasing Ca2+ from inositol 1,4,5-trisphosphate-insensitive intracellular stores. In the present study, we have examined the role of cADPR in glucose-mediated increases in [Ca2+]i and insulin exocytosis. Dispersed ob/ob mouse beta-cell aggregates were either pressure microinjected with fura-2 salt or loaded with fura-2 acetoxymethyl ester, and [Ca2+]i was monitored by microfluorimetry. Microinjection of beta-NAD+ into fura-2-loaded beta-cells did not increase [Ca2+]i nor did it alter the cells' subsequent [Ca2+]i response to glucose. Cells microinjected with the cADPR antagonist 8NH2-cADPR increased [Ca2+]i in response to glucose equally well as those injected with cADPR. Finally, the ability of cADPR to promote exocytosis of insulin in electropermeabilized beta-cells was investigated. cADPR on its own did not increase insulin secretion nor did it potentiate Ca2+-induced insulin secretion. We conclude that cADPR neither plays a significant role in glucose-mediated increases in [Ca2+]i nor interacts directly with the molecular mechanisms regulating exocytosis of insulin in normal pancreatic beta-cells.     

Mechanisms of neuronal calcium homeostasis destabilization caused by hyperstimulation of glutamate receptors

The present paper summarizes the data obtained in studying the mechanisms of glutamate-induced deterioration of neuronal Ca2+ homeostasis. In the cultured mammalian central neurons, a short-term (< 1 min) glutamate (GLU, 100 mu) challenge is known to induce a readily reversible (transient) neuronal [Ca2+]i increase. In contrast, a long-term (15-30 min) GLU exposure leads to the appearance of high [Ca2+]i plateau or to the partial recovery of the increased [Ca2+]i. Experiments show that impaired [Ca2+]i recovery in the postglutamate period cannot be explained by the increased [Ca2+]i permeability of the neuronal membrane, as earlier considered. Moreover, a sustained elevation of [Ca2+]i during and after chronic GLU application is associated with a progressive decrease in Ca2+ permeability. The major cause of GLU-induced Ca2+ overload is the mitochondrial depolarization resulted from excessive Ca2+ influx into the mitochondria, the generation of free radicals and the opening of a "giant pore" in the inner mitochondrial membrane. This in turn suppresses both ATP synthesis and Ca2+ electrophoretic uptake into the mitochondrial matrix. In combination with [Ca2+]i-dependent acidification, this leads to the suppression of Ca2+ release from the cell via Na+/Ca2+ exchanger and Ca2+/H+ pump of the neuronal membrane. Therefore, [Ca2+]i recovery following a long-term GLU treatment becomes strongly or even irreversibly compromised.     

Pindolol does not act only on 5-HT1A receptors in augmenting antidepressant activity in the mouse forced swimming test

Hypocapnia produces cerebral vasoconstriction. The mechanisms involved in hypocapnia-induced elevation of vascular smooth muscle tone remain unclear. We addressed the hypothesis that, in cerebrovascular smooth muscle, increases in extracellular pH (pHo) cause increases in Ins(1,4,5)P3 and cytosolic calcium ([Ca2+]c). Superfused primary cultures of piglet cerebral microvascular smooth muscle cells were exposed to artificial CSF (aCSF) of control (pHo 7. 4, PCO2 36 mm Hg), metabolic alkalosis (pHo 7.7, PCO2 36 mm Hg), or respiratory alkalosis (pHo 7.7, PCO2 19 mm Hg). Intracellular pH (pHi) and [Ca2+]c were measured, using BCECF and fura-2, respectively, with dual wavelength spectroscopy. Ins(1,4,5)P3 was determined by a protein binding assay. Both metabolic and respiratory acidosis treatments increased pHi from the control value of about 7.2 to 7.35. Metabolic and respiratory alkalosis increased Ins(1,4,5)P3, as we showed previously. Metabolic and respiratory alkalosis increased [Ca2+]c about 80% and 110%, respectively. Neither Ins(1,4,5)P3 nor [Ca2+]c increased in cells treated with aCSF that produced control pHo with increased pHi (7.3). In contrast, when pHo increased (7.7), but pHi was maintained at control (7.2), Ins(1,4,5)P3 increased from 123 pmol/well to 307 pmol/well and [Ca2+]c increased 46%. However, the increase of [Ca2+]c was less than with either respiratory or metabolic alkalosis. Thus, hypocapnia-induced cerebral vasoconstriction could involve production of Ins(1,4,5)P3 with resultant elevation in [Ca2+]c. While the Ins(1,4,5)P3 signal appears to be dependent on an increase in extracellular pH, a role for intracellular pH cannot be completely excluded.     

The presence of Human Papillomavirus (HPV) in microinvasive in situ spinocellular carcinoma of the oral cavity. Preliminary report

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.     

New light on mitochondrial calcium

The possibility of specifically addressing recombinant probes to mitochondria is a novel, powerful way to study these organelles within living cells. We first showed that the Ca(2+)-sensitive photoprotein aequorin, modified by the addition of a mitochondrial targeting sequence, allows to monitor specifically the Ca2+ concentration in the mitochondrial matrix ([Ca2+]m) of living cells. With this tool, we could show that, upon physiological stimulation, mitochondria undergo a major rise in [Ca2+]m, well in the range of the Ca2+ sensitivity of the matrix dehydrogenases, in a wide variety of cell types, ranging from non excitable, e.g., HeLa and CHO, and excitable, e.g., cell lines to primary cultures of various embryological origin, such as myocytes and neurons. This phenomenon, while providing an obvious mechanism for tuning mitochondrial activity to cell needs, appeared at first in striking contrast with the low affinity of mitochondrial Ca2+ uptake mechanisms. Based on indirect evidence, we proposed that the mitochondria might be close to the source of the Ca2+ signal and thus exposed to microdomains of high [Ca2+], hence allowing the rapid accumulation of Ca2+ into the organelle. In order to verify this intriguing possibility, we followed two approaches. In the first, we constructed a novel aequorin chimera, targeted to the mitochondrial intermembrane space (MIMS), i.e., the region sensed by the low-affinity Ca2+ uptake systems of the inner mitochondrial membrane. With this probe, we observed that, upon agonist stimulation, a portion of the MIMS is exposed to saturating Ca2+ concentrations, thus confirming the occurrence of microdomains of high [Ca2+] next to mitochondria. In the second approach, we directly investigated the spatial relationship of the mitochondria and the ER, the source of agonist-releasable Ca2+ in non-excitable cells. For this purpose, we constructed GFP-based probes of organelle structure; namely, by targeting to these organelles GFP mutants with different spectral properties, we could label them simultaneously in living cells. By using an imaging system endowed with high speed and sensitivity, which allows to obtain high-resolution 3D images, we could demonstrate that close contacts (< 80 nm) occur in vivo between mitochondria and the ER.