Perinuclear, perigranular and sub-plasmalemmal mitochondria have distinct functions in the regulation of cellular calcium transport

We have identified three distinct groups of mitochondria in normal living pancreatic acinar cells, located (i) in the peripheral basolateral region close to the plasma membrane, (ii) around the nucleus and (iii) in the periphery of the granular region separating the granules from the basolateral area. Three-dimensional reconstruction of confocal slices showed that the perigranular mitochondria form a barrier surrounding the whole of the granular region. Cytosolic Ca(2+) oscillations initiated in the granular area triggered mitochondrial Ca(2+) uptake mainly in the perigranular area. The most intensive uptake occurred in the mitochondria close to the apical plasma membrane. Store-operated Ca(2+) influx through the basolateral membrane caused preferential Ca(2+) uptake into sub-plasmalemmal mitochondria. The perinuclear mitochondria were activated specifically by local uncaging of Ca(2+) in the nucleus. These mitochondria could isolate nuclear and cytosolic Ca(2+) signalling. Photobleaching experiments indicated that different groups of mitochondria were not luminally connected. The three mitochondrial groups are activated independently by specific spatiotemporal patterns of cytosolic Ca(2+) signals and can therefore participate in the local regulation of Ca(2+) homeostasis and energy supply.     

The distribution of the endoplasmic reticulum in living pancreatic acinar cells

Studies on pancreatic acinar cells provided the original evidence for the Ca(2+) releasing action of inositol 1,4,5-trisphosphate (IP(3)). Ironically, this system has presented problems for the general theory that IP(3) acts primarily on the endoplasmic reticulum (ER), because the IP(3)-elicited Ca(2+) release occurs in the apical pole, which is dominated by zymogen granules (ZGs) and apparently contains very little ER. Using confocal and two-photon microscopy and a number of different ER-specific fluorescent probes, we have now investigated in detail the distribution of the ER in living pancreatic acinar cells. It turns out that although the bulk of the ER, as expected, is clearly located in the baso-lateral part of the cell, there is significant invasion of ER into the granular pole and each ZG is in fact surrounded by strands of ER. This structural evidence from living cells, in conjunction with recent functional studies demonstrating the high Ca(2+) mobility in the ER lumen, provides the framework for a coherent and internally consistent theory for cytosolic Ca(2+) signal generation in the apical secretory pole, in which the primary Ca(2+) release occurs from ER extensions in the granular pole supplied with Ca(2+) from the main store at the base of the cell by the tunnel function of the ER.     

Vesicular Ca(2+) -induced secretion promoted by intracellular pH-gradient disruption

The actions of the protonophore CCCP on intracellular Ca2+ regulation and exocytosis in chromaffin cells have been examined. Simultaneous fura-2 imaging and amperometry reveal that exposure to CCCP not only perturbs mitochondrial function but that it also alters vesicular storage of Ca2+ and catecholamines. By disrupting the pH gradient of the secretory vesicle membrane, the protonophore allows both Ca(2+) and catecholamine to leak into the cytosol. Unlike the high cytosolic Ca2+ concentrations resulting from mitochondrial membrane disruption, Ca2+ leakage from secretory vesicles may initiate exocytotic release. In conjunction with previous studies, this work reveals that catalytic and self-sustained vesicular Ca(2+) -induced exocytosis occurs with extended exposure to weak acid or base protonophores.     

Agonist-evoked mitochondrial Ca2+ signals in mouse pancreatic acinar cells

In the present study we have investigated cytosolic and mitochondrial Ca(2+) signals in isolated mouse pancreatic acinar cells double-loaded with the fluorescent probes fluo-3 and rhod-2. Stimulation of pancreatic acinar cells with 500 nm acetylcholine caused release of Ca(2+) from intracellular stores and produced cytosolic Ca(2+) signals in form of Ca(2+) waves propagating from the luminal to the basal cell pole. The increase in the cytosolic Ca(2+) concentration was followed by Ca(2+) uptake into mitochondria. Between onset of cytosolic and mitochondrial Ca(2+) signals there was a delay of 10.7 +/- 0.4 s. Ca(2+) uptake into mitochondria could be inhibited with Ruthenium Red and carbonyl cyanide m-chlorophenylhydrazone, whereas 2,5-di-tert-butylhydroquinone, which inhibits sarco(endo)plasmic reticulum Ca(2+) ATPases, did not prevent Ca(2+) accumulation in mitochondria. Carbonyl cyanide m-chlorophenylhydrazone-induced Ca(2+) release from mitochondria could only be observed after a preceding stimulation of the cell with a physiological agonist or by treatment with 2, 5-di-tert-butylhydroquinone, indicating that under resting conditions mitochondria do not contain releasable Ca(2+) ions. Analysis of the propagation rate of acetylcholine-induced Ca(2+) waves revealed that inhibition of mitochondrial Ca(2+) uptake did not accelerate spreading of cytosolic Ca(2+) signals. Our experiments indicate that in the early phase of secretagogue-induced Ca(2+) signals, mitochondria behave as passive Ca(2+)-buffering elements and do not actively suppress spreading of Ca(2+) signals in pancreatic acinar cells.     

Two different but converging messenger pathways to intracellular Ca(2+) release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate

Hormones and neurotransmitters mobilize Ca(2+) from the endoplasmic reticulum via inositol trisphosphate (IP(3)) receptors, but how a single target cell encodes different extracellular signals to generate specific cytosolic Ca(2+) responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca(2+) spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca(2+) signals. We show that IP(3), cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca(2+) spiking by activating common oscillator units composed of IP(3) and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP(3) receptors, whereas cholecystokinin responses are triggered via a different but converging pathway with NAADP and cyclic ADP-ribose receptors. Cholecystokinin potentiates the response to acetylcholine, making it global rather than local, an effect mediated specifically by cyclic ADP-ribose receptors. In the apical pole there is a common early activation site for Ca(2+) release, indicating that the three types of Ca(2+) release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.     

Mitochondrial Ca2+ uptake is important over low [Ca2+]i range in arterial smooth muscle

Mitochondrial Ca(2+) uptake is usually thought to occur only when intracellular Ca(2+) concentration ([Ca(2+)](i)) is high. We investigated whether mitochondrial Ca(2+) removal participates in shaping [Ca(2+)](i) signals in arterial smooth muscle over a low [Ca(2+)](i) range. [Ca(2+)](i) was measured using fura 2-loaded, voltage-clamped cells from rat femoral arteries. Both diazoxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized the mitochondria. Diazoxide application increased resting [Ca(2+)](i), suggesting that Ca(2+) is sequestered in mitochondria. Over a low [Ca(2+)](i) range, diazoxide and CCCP slowed Ca(2+) removal rate, determined after a brief depolarization. When [Ca(2+)](i) was measured during sustained depolarization to -30 mV, CCCP application increased [Ca(2+)](i). When Ca(2+) transients were repeatedly evoked by caffeine applications, CCCP application elevated resting [Ca(2+)](i). Caffeine-induced Ca(2+) transients were compared before and after CCCP application using the half decay time, or time required to reduce increase in [Ca(2+)](i) by 50% (t((1/2))). CCCP treatment significantly increased t((1/2)). These results suggest that Ca(2+) removal to mitochondria in arterial smooth muscle cells may be important at a low [Ca(2+)](i).     

Relative contribution of the Na(+)/Ca(2+) exchanger, mitochondria and endoplasmic reticulum in the regulation of cytosolic Ca(2+) and catecholamine secretion of bovine adrenal chromaffin cells

The relative importance of mitochondria, the Na(+)/Ca(2+) exchanger (NCX) and the endoplasmic reticulum (ER) in the regulation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were examined in bovine chromaffin cells using fura-2 for average [Ca(2+)](i) and amperometry for secretory activity, which reflects the local Ca(2+) concentration near the exocytotic sites. Chromaffin cells were stimulated by a high concentration of K(+) when the three Ca(2+) removal mechanisms were individually or simultaneously inhibited. When the mitochondrial Ca(2+) uptake was inhibited, the [Ca(2+)](i) decayed at a significantly slower rate and the secretory activity was higher than the control cells. The NCX appears to function only in the initial phase of [Ca(2+)](i) decay and when the ER Ca(2+) pump is blocked. Similarly, the ER had a significant effect on the [Ca(2+)](i) decay and on the secretion only when the NCX was blocked. Inhibition of all three mechanisms leads to a substantial delay in [Ca(2+)](i) recovery and an increase in the secretion. The results suggest that the three mechanisms work together in the regulation of the Ca(2+) near the Ca(2+) channels and exocytotic sites and therefore modulate the secretory activity. When Ca(2+) diffuses away from the exocytotic sites, the mitochondrial Ca(2+) uptake becomes the dominant mechanism.     

Stable Golgi-mitochondria complexes and formation of Golgi Ca(2+) gradients in pancreatic acinar cells

We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca(2+) gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer edge of the secretory granular region of individual acinar cells. We also assessed Golgi positioning in acinar cells located within intact pancreatic tissue using two-photon microscopy and found a similar localization. The mitochondria segregate the Golgi from lateral regions of the plasma membrane, the nucleus, and the basal part of the cytoplasm. The Golgi is therefore placed between the principal Ca(2+) release sites in the apical region of the cell and the important Ca(2+) sink formed by the peri-granular mitochondria. During acetylcholine-induced cytosolic Ca(2+) signals in the apical region, large Ca(2+) gradients form over the Golgi (decreasing from trans- to cis-Golgi). We further describe a novel, close interaction of the peri-granular mitochondria and the Golgi apparatus. The mitochondria and the Golgi structures form very close contacts, and these contacts remain stable over time. When the cell is forced to swell, the Golgi and mitochondria remain juxtaposed up to the point of cell lysis. The strategic position of the Golgi (closer to release sites than the bulk of the mitochondrial belt) makes this organelle receptive to local apical Ca(2+) transients. In addition the Golgi is ideally placed to be preferentially supplied by ATP from adjacent mitochondria.     

Calcium buffering activity of mitochondria controls basal growth hormone secretion and modulates specific neuropeptide signaling

Goldfish somatotropes contain multiple functionally distinct classes of non-mitochondrial intracellular Ca(2+) stores. In this study, we investigated the role of mitochondrial Ca(2+) handling in the control of hormone secretion. Inhibition of mitochondrial Ca(2+) uptake with 10 microM ruthenium red (RR) and 10 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) caused a small and reversible increase in cytosolic [Ca(2+)]. Despite relatively modest global Ca(2+) signals, RR and CCCP stimulated robust GH secretion under basal culture conditions. CCCP-stimulated hormone release was abolished in cells pre-incubated with 50 microM BAPTA-AM, suggesting that elevations in cytosolic [Ca(2+)] mediate this release of GH. Both caffeine-sensitive intracellular Ca(2+) stores and L-type Ca(2+) channels can be the source of the Ca(2+) buffered by mitochondria in somatotropes. The stimulatory effect of RR on caffeine-stimulated GH release was enhanced dramatically in the presence of ryanodine, pointing to a complex interaction between these three Ca(2+) stores. Inhibition of mitochondrial Ca(2+) uptake with RR augmented GH release evoked by only one of the two endogenous gonadotropin-releasing hormones. Thus, we provide the first evidence that mitochondrial Ca(2+) buffering is differentially involved in specific agonist Ca(2+) signaling pathways and plays an important role in the control of basal GH release.     

Ca2+ homeostasis and exocytosis in carotid glomus cells: role of mitochondria

In oxygen sensing carotid glomus (type 1) cells, the hypoxia-triggered depolarization can be mimicked by mitochondrial inhibitors. We examined the possibility that, other than causing glomus cell depolarization, mitochondrial inhibition can regulate transmitter release via changes in Ca(2+) dynamics. Under whole-cell voltage clamp conditions, application of the mitochondrial inhibitors, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or cyanide caused a dramatic slowing in the decay of the depolarization-triggered Ca(2+) signal in glomus cells. In contrast, inhibition of the Na(+)/Ca(2+) exchanger (NCX), plasma membrane Ca(2+)-ATPase (PMCA) pump or sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump had much smaller effects. Consistent with the notion that mitochondrial Ca(2+) uptake is the dominant mechanism in cytosolic Ca(2+) removal, inhibition of the mitochondrial uniporter with ruthenium red slowed the decay of the depolarization-triggered Ca(2+) signal. Hypoxia also slowed cytosolic Ca(2+) removal, suggesting a partial impairment of mitochondrial Ca(2+) uptake. Using membrane capacitance measurement, we found that the increase in the duration of the depolarization-triggered Ca(2+) signal after mitochondrial inhibition was associated with an enhancement of the exocytotic response. The role of mitochondria in the regulation of Ca(2+) signal and transmitter release from glomus cells highlights the importance of mitochondria in hypoxic chemotransduction in the carotid bodies.     

Role of mitochondria in Ca(2+) homeostasis of mouse pancreatic acinar cells

The effects of mitochondrial Ca(2+) uptake on cytosolic Ca(2+) concentration ([Ca(2+)](c)) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca(2+) indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Delta[Ca(2+)](c)=46 +/- 6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca(2+) transient, produced a larger cytosolic calcium elevation (Delta[Ca(2+)](c)=84 +/- 6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca(2+)](m)) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca(2+) uniporter), the decay time-constant of the Tg/ACh-induced Ca(2+) response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca(2+) indicator rhod-2 revealed large increases in [Ca(2+)](m) in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10nM ACh elicited large global increases in [Ca(2+)](c), compared to control cells in which ACh-induced Ca(2+) signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca(2+) homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.     

Contributions of intracellular compartments to calcium dynamics: implicating an acidic store

Many cells show a plateau of elevated cytosolic Ca(2+) after a long depolarization, suggesting delayed Ca(2+) release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic beta-cells show a thapsigargin-sensitive plateau ('hump') of Ca(2+) after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na(+)-Ca(2+) exchange and by only 18% upon blocking ryanodine or IP(3) receptors together. Further, the time course of ER Ca(2+) measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine beta-napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca(2+) into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in beta-cells. We conclude that the dipeptide compromises dense-core secretory granules and that these granules comprise an acidic calcium store in beta-cells whose loading and/or release is sensitive to thapsigargin and which releases Ca(2+) after cytosolic Ca(2+) elevation.     

Role of mitochondria in intracellular calcium signaling in primary and secondary sensory neurones of rats

The participation of different calcium-regulated mechanisms in the generation of cytosolic Ca(2+) transients during neuronal excitation has been compared in isolated large and small primary (dorsal root ganglia (DRG)) and secondary (spinal dorsal horn (DH)) rat sensory neurones. As it was shown before in murine primary sensory neurones the application of mitochondrial protonophore CCCP by itself induced only small elevation of [Ca(2+)](i). However, its preceding application substantially increased the peak amplitude of depolarization-induced transients. Application of CCCP immediately after termination of the depolarizing pulse induced in both types of primary neurones a massive release of Ca(2+) from mitochondria into the cytosol. In secondary neurones application of CCCP by itself induced a substantial release of Ca(2+) from the mitochondria, but its preceding application resulted in only an insignificant increase in the peak amplitude of depolarization-triggered calcium transients. Application of CCCP immediately after termination of depolarization elicited a small release of Ca(2+), which became more pronounced when the application was delayed. Preceding application of CCCP increased the amplitude of the transients induced by caffeine-triggered Ca(2+) release from the endoplasmic reticulum in secondary neurones and did not affect those in large primary neurones. These findings may be explained by substantial differences in the density and distribution of mitochondria in the cytosol of primary and secondary sensory neurones. This suggestion was confirmed electronmicroscopically, showing a much lower density of mitochondria near plasmalemma in secondary sensory neurones and predominant clustered location of mitochondria beneath the plasmalemma in the primary cells. The possible functional importance of these differences is discussed.     

Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria

Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local [Ca(2+)] control that operates between IP(3)receptor Ca(2+)release channels (IP(3)R) and mitochondrial Ca(2+)uptake sites, and displays functional similarities to synaptic transmission. Activation of IP(3)R by IP(3)is known to evoke quantal Ca(2+)mobilization that is associated with incremental elevations of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)). Here we report that activation of IP(3)R by adenophostin-A (AP) yields non-quantal Ca(2+)mobilization in mast cells. We also show that the AP-induced continuous Ca(2+)release causes relatively small [Ca(2+)](m)responses, in particular, the sustained phase of Ca(2+)release is not sensed by the mitochondria. Inhibition of ER Ca(2+)pumps by thapsigargin slightly increases IP(3)-induced [Ca(2+)](m)responses, but augments AP-induced [Ca(2+)](m)responses in a large extent. In adherent permeabilized cells exposed to elevated [Ca(2+)], ER Ca(2+)uptake fails to affect global cytosolic [Ca(2+)], but attenuates [Ca(2+)](m)responses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca(2+)pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca(2+)uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca(2+)release through the IP(3)R or during gradual Ca(2+)influx to the junction between ER and mitochondria.     

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

Polarity in intracellular calcium signaling.

The concentration of free calcium ions (Ca(2+)) in the cytosol is precisely regulated and can be rapidly increased in response to various types of stimuli. Since Ca(2+) can be used to control different processes in the same cell, the spatial organization of cytosolic Ca(2+) signals is of considerable importance. Polarized cells have advantages for Ca(2+) studies since localized signals can be related to particular organelles. The pancreatic acinar cell is well-characterized with a clearly polarized structure and function. Since the discovery of the intracellular Ca(2+)-releasing function of inositol 1,4,5-trisphosphate (IP(3)) in the pancreas in the early 1980s, this cell has become a popular study object and is now one of the best-characterized with regard to Ca(2+) signaling properties. Stimulation of pancreatic acinar cells with the neurotransmitter acetylcholine or the hormone cholecystokinin evokes Ca(2+) signals that are either local or global, depending on the agonist concentration and the length of the stimulation period. The nature of the Ca(2+) transport events across the basal and apical plasma membranes as well as the involvement of the endoplasmic reticulum (ER), the nucleus, the mitochondria, and the secretory granules in Ca(2+) signal generation and termination have become much clearer in recent years.     

Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis

Pituitary gonadotropes transduce hormonal input into cytoplasmic calcium ([Ca(2+)](cyt)) oscillations that drive rhythmic exocytosis of gonadotropins. Using Calcium Green-1 and rhod-2 as optical measures of cytoplasmic and mitochondrial free Ca(2+), we show that mitochondria sequester Ca(2+) and tune the frequency of [Ca(2+)](cyt) oscillations in rat gonadotropes. Mitochondria accumulated Ca(2+) rapidly and in phase with elevations of [Ca(2+)](cyt) after GnRH stimulation or membrane depolarization. Inhibiting mitochondrial Ca(2+) uptake by the protonophore CCCP reduced the frequency of GnRH-induced [Ca(2+)](cyt) oscillations or, occasionally, stopped them. Much of the Ca(2+) that entered mitochondria is bound by intramitochondrial Ca(2+) buffering systems. The mitochondrial Ca(2+) binding ratio may be dynamic because [Ca(2+)](mit) appeared to reach a plateau as mitochondrial Ca(2+) accumulation continued. Entry of Ca(2+) into mitochondria was associated with a small drop in the mitochondrial membrane potential. Ca(2+) was extruded from mitochondria more slowly than it entered, and much of this efflux could be blocked by CGP-37157, a selective inhibitor of mitochondrial Na(+)-Ca(2+) exchange. Plasma membrane capacitance changes in response to depolarizing voltage trains were increased when CCCP was added, showing that mitochondria lower the local [Ca(2+)](cyt) near sites that trigger exocytosis. Thus, we demonstrate a central role for mitochondria in a significant physiological response.     

Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion

Fertilization increases both cytosolic Ca(2+) concentration and oxygen consumption in the egg but the relationship between these two phenomena remains largely obscure. We have measured mitochondrial oxygen consumption and the mitochondrial NADH concentration on single ascidian eggs and found that they increase in phase with each series of meiotic Ca(2+) waves emitted by two pacemakers (PM1 and PM2). Oxygen consumption also increases in response to Ins(1,4,5)P(3)-induced Ca(2+) transients. Using mitochondrial inhibitors we show that active mitochondria sequester cytosolic Ca(2+) during sperm-triggered Ca(2+) waves and that they are strictly necessary for triggering and sustaining the activity of the meiotic Ca(2+) wave pacemaker PM2. Strikingly, the activity of the Ca(2+) wave pacemaker PM2 can be restored or stimulated by flash photolysis of caged ATP. Taken together our observations provide the first evidence that, in addition to buffering cytosolic Ca(2+), the egg's mitochondria are stimulated by Ins(1,4,5)P(3)-mediated Ca(2+) signals. In turn, mitochondrial ATP production is required to sustain the activity of the meiotic Ca(2+) wave pacemaker PM2.     

Localization and regulation of Ca2+ entry and exit pathways in exocrine gland cells

Studies of Ca2+ transport pathways in exocrine gland cells have been useful, chiefly because of the polarized nature of the secretory epithelial cells. In pancreatic acinar cells, for example, Ca2+ reloading of empty intracellular stores can occur solely via Ca2+ entry through the basal part of the plasma membrane. On the other hand, the principal site for intracellular Ca2+ release-with the highest concentration of inositol 1,4,5-trisphosphate (IP(3)) receptors-is in the apical secretory pole close to the apical plasma membrane. This apical part of the plasma membrane contains the highest density of Ca2+ pumps and is therefore the principal site for Ca2+ extrusion. On the basis of the known properties of Ca2+ entry and exit pathways in exocrine gland cells, the mechanisms controlling Ca2+ exit and entry are discussed in relation to recent direct information about Ca2+ transport into and out of the endoplasmic reticulum (ER) and the mitochondria in these cells.     

Intracellular mechanisms of hypoxia-induced calcium increase in rat sensory neurons

Elevation of cytosolic level of Ca(2+) was measured by spatial screening of freshly isolated dorsal root ganglion neurons loaded with Fura-2AM after subjecting them to a moderate hypoxic solution (pO(2)=10-40 mmHg). Short exposure of neurons to hypoxia resulted in a reversible elevation of intracellular Ca(2+) to about 120% in the cell center and to 80% in the cell periphery. Such elevation could be almost completely eliminated by removal of Ca(2+) or Na(+) from external medium or application of nifedipine, an L-type calcium channel blocker. Remarkable antihypoxic efficiency (58%) was achieved by preapplication of mitochondrial protonophore CCCP. A conclusion is made that in sensory neurons the hypoxia-induced elevation of cytosolic Ca(2+) is induced by combined changes of function in three cell substructures: voltage-operated L-type Ca(2+) and Na(+) channels and Ca(2+) accumulation by mitochondria. Mitochondria are important for spatial difference in the hypoxia-induced Ca(2+) elevation due to their specific location in these neurons.