Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells

As amply documented by electrophysiology, depolarisation in Paramecium induces a Ca(2+) influx selectively via ciliary voltage-dependent Ca(2+)-channels, thus inducing ciliary beat reversal. Subsequent downregulation of ciliary Ca(2+) has remained enigmatic. We now analysed this aspect, eventually under overstimulation conditions, by quenched-flow/cryofixation, combined with electron microscope X-ray microanalysis which registers total calcium concentrations, [Ca]. This allows to follow Ca-signals within a time period (> or =30ms) smaller than one ciliary beat ( approximately 50ms) and beyond. Particularly under overstimulation conditions ( approximately 10(-5)M Ca(2+) before, 0.5mM Ca(2+) during stimulation) we find in cilia a [Ca] peak at approximately 80ms and its decay to near-basal levels within 110ms (90%) to 170ms (100% decay). This [Ca] wave is followed, with little delay, by a [Ca] wave into subplasmalemmal Ca-stores (alveolar sacs), culminating at approximately 100ms, with a decay to original levels within 170ms. Also with little delay [Ca] slightly increases in the cytoplasm below. This implies rapid dissipation of Ca(2+) through the ciliary basis, paralleled by a rapid, transient uptake by, and release from cortical stores, suggesting fast exchange mechanisms to be analysed as yet. This novel type of coupling may be relevant for some phenomena described for other cells.     

Refilling of cortical calcium stores in Paramecium cells: in situ analysis in correlation with store-operated calcium influx

This is the first thorough study of refilling of a cortical calcium store in a secretory cell after stimulation in which we combined widely different methodologies. Stimulation of dense-core vesicle ("trichocysts") exocytosis in Paramecium involves a Ca(2+) -influx" superimposed to Ca(2+) -release from cortical stores ("alveolar sacs" (ASs)). In quenched-flow experiments, membrane fusion frequency rose with increasing [Ca(2+)](o) in the medium, from approximately 20-25% at [Ca(2+)](o) < or = 0.25 microM to 100% at [Ca(2+)](o) between 2 and 10 microM, i.e. close to the range of estimated local intracellular [Ca(2+)] during membrane fusion. Next, we analyzed Ca(2+)-specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on Ca(2+) -signaling. In double trigger experiments, with BAPTA in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical Ca(2+) -signal (due solely to Ca(2+) released from cortical stores) recovered with t(1/2) approximately 65 min. When ASs were analyzed in situ by X-ray microanalysis after different trigger times (+Ca(2+)(o)), t(1/2) for store refilling was similar, approximately 60 min. These values are similar to previously measured 45Ca(2+) -uptake by isolated ASs. In sum we find, (i) exogenous Ca(2+) increases exocytosis/membrane fusion performance with EC(50)=0.7 microM, (ii) Ca(2+) -signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical calcium stores goes on over hours and thus is much slower than expected.     

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.     

Calcium regulates independently ciliary beat and cell contraction in Paramecium cells

Intracellular Ca(2+) concentration is a well-known signal regulator for various physiological activities. In many cases, Ca(2+) simultaneously regulates individual functions in single cells. How can Ca(2+) regulate these functions independently? In Paramecium cells, the contractile cytoskeletal network and cilia are located close to each other near the cell surface. Cell body contraction, ciliary reversal, and rises in ciliary beat frequency are regulated by intracellular Ca(2+) concentration. However, they are not always triggered simultaneously. We injected caged calcium into Paramecium caudatum cells and continuously applied weak ultraviolet light to the cells to slowly increase intracellular Ca(2+) concentration. The cell bodies began to contract just after the start of ultraviolet light application, and the degree of contraction increased gradually thereafter. On the other hand, cilia began to reverse 1.4s after the start of ultraviolet application and reversed completely within 100ms. Ciliary beat frequency in the reverse direction was significantly higher than in the normal direction. These results indicate that cell body contraction is regulated by Ca(2+) in a dose-dependent manner in living P. caudatum. On the other hand, ciliary reversal and rise in ciliary beat frequency are triggered by Ca(2+) in an all-or-none manner.     

Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale: preparative and analytical aspects as exemplified with paramecium cells

We analyzed preparative and analytical aspects of the dynamic localization of Ca(2+) during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (approximately 100-nm thickness) or cilia (approximately 250-nm diameter). We used widely different standards to calibrate the ratio of CaK(alpha) net counts in relation to actual ?Ca. Calibration curves showed a linear relationship and a detection limit of ?Ca = 2 mM, while ?Ca in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr(2+) can rapidly be substituted for Ca(2+) in the medium before and during stimulation, thus allowing one to determine Me(2+) fluxes. With our "model" cell, Paramecium, we showed that, upon stimulation (causing rapid Ca(2+) mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.     

My favorite cell--Paramecium

A Paramecium cell has a stereotypically patterned surface, with regularly arranged cilia, dense-core secretory vesicles and subplasmalemmal calcium stores. Less strikingly, there is also a patterning of molecules; for instance, some ion channels are restricted to certain regions of the cell surface. This design may explain very effective and selective responses, such as that to Ca(2+) upon stimulation. It enables the cell to respond to a Ca(2+) signal precisely secretion (exocytosis) or by changing its ciliary activity. These responses depend on the location and/or type of signal, even though these two target structures co-exist side-by-side, and normally only limited overlap occurs between the different functions. Furthermore, the patterning of exocytotic sites and the possibility of synchronous exocytosis induction in the sub-second time range have considerably facilitated analyses, and thus led to new concepts of exocytotic membrane fusion. It has been possible to dissect complicated events like overlapping Ca(2+) fluxes produced from external sources and from internal stores. Since molecular genetic approaches have become available for Paramecium, many different gene products have been identified only some of which are known from "higher" eukaryotes. Although a variety of basic cellular functions are briefly addressed to demonstrate the uniqueness of this unicellular organism, this article focuses on exocytosis regulation.     

Veratridine induces a Ca2+ influx,cyclic GMP formation,and backward swimming inParamecium tetraurelia wildtype cells and Ca2+ current-deficient pawn mutant cells

Summary Veratridine opens voltage-dependent Na⁺ channels in many metazoans. InParamecium, which has voltage-dependent Ca²⁺ channels and a Ca/K action potential, no such Na⁺ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca²⁺. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba²⁺, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba²⁺ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca²⁺ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na⁺ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca²⁺ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca²⁺ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier.     

Ciliary reorientation is evoked by a rise in calcium level over the entire cilium

Internal Ca2+ levels control the pattern of ciliary and flagellar beating in eukaryotes. In ciliates, ciliary reversal is induced by a rise in intra-ciliary Ca2+, but the mechanism by which Ca2+ induces reversal is not known. We injected the fluorescent Ca2+ indicator Calcium Green into a ciliate Didinium nasutum and observed the intra-ciliary Ca2+ level during the initial reversed stroke preceding spontaneous cyclic reversed beating. In D. nasutum, Ca2+ rose throughout the length of the cilia undergoing initial reversed stroke. Electron microscopy with a combined oxalate-pyroantimonate method showed Ca2+ deposits distributed throughout the reversed cilia. We injected caged Ca2+ into D. nasutum and irradiated the base or mid region of the cilia with UV to locally increase Ca2+ concentration. Uncaging Ca2+ in the middle of the cilia produced reversal distally, but not proximally to the site of Ca2+ release. These results strongly suggest that not only Ca2+ influx sites, but also Ca2+ binding sites and vectoral bending machineries for ciliary reversal, are distributed throughout the cilium.     

Light sensitivity of the ciliate Tetrahymena vorax induced by the fluorescent dye acridine orange.

The ciliate Tetrahymena vorax is normally insensitive to light. However, after uptake of acridine orange, blue light evokes instant backward swimming. The dye accumulates mainly in posterior vacuoles, with half-maximal uptake after 1 min. Illumination for 10 s induced a depolarisation of approximately 15 mV lasting less than 2 s, followed by a sustained hyperpolarisation of approximately 20 mV. Deciliated cells displayed a similar response. The hyperpolarisation was linked to reduced membrane resistance, showed a reversal potential of approximately -55 mV and was blocked by 1 mmol l(-1) TEA. The rate of rise of electrically evoked Ca(2+)-spikes was reduced during the hyperpolarisation, which is compatible with elevated cytosolic Ca(2+) concentration. This suggests that the hyperpolarisation may be caused by activation of Ca(2+)-sensitive K(+) channels. The depolarisation was abolished in Ca(2+)-free medium, whereas the hyperpolarisation was unaffected. Illumination for 2 s, or prolonged stimulation restricted to the anterior part of the cell, induced depolarisation only. Illumination of the posterior part caused delayed hyperpolarisation with no preceding depolarisation. We conclude that the induced backward swimming is associated with Ca(2+) influx through anterior channels, while Ca(2+) released from intracellular stores activates K(+) channels responsible for the delayed hyperpolarisation.     

Role of proton gradients and vacuola H(+)-ATPases in the refilling of intracellular calcium stores in exocrine cells

Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca(2+)](i)), a key regulatory factor for many cellular processes. A pivotal feature of these Ca(2+) signals is the release of Ca(2+) from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated with the endoplasmic reticulum. Although the mechanisms of calcium release and calcium influx have been extensively studied, the biology of the Ca(2+) stores is poorly understood. The presence of heterogeneous calcium pools in cells has been previously reported [1] [2] [3]. Although recent technical improvements have confirmed this heterogeneity [4], knowledge about the mechanisms underlying Ca(2+) transport within the stores is very scarce and rather speculative. A recent study in polarized exocrine cells [5] has revealed the existence of Ca(2+) tunneling from basolateral stores to luminal pools, where Ca(2+) is initially released upon cell activation. Here, we present evidence that, during stimulation, Ca(2+) transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools driven by proton gradients generated by vacuolar H(+)-ATPases. This finding unveils a new aspect of the machinery of Ca(2+) stores.     

Glucose induces synchronous mitochondrial calcium oscillations in intact pancreatic islets

Mitochondria shape Ca(2+) signaling and exocytosis by taking up calcium during cell activation. In addition, mitochondrial Ca(2+) ([Ca(2+)](M)) stimulates respiration and ATP synthesis. Insulin secretion by pancreatic beta-cells is coded mainly by oscillations of cytosolic Ca(2+) ([Ca(2+)](C)), but mitochondria are also important in excitation-secretion coupling. Here, we have monitored [Ca(2+)](M) in single beta-cells within intact mouse islets by imaging bioluminescence of targeted aequorins. We find an increase of [Ca(2+)](M) in islet-cells in response to stimuli that induce either Ca(2+) entry, such as extracellular glucose, tolbutamide or high K(+), or Ca(2+) mobilization from the intracellular stores, such as ATP or carbamylcholine. Many cells responded to glucose with synchronous [Ca(2+)](M) oscillations, indicating that mitochondrial function is coordinated at the whole islet level. Mitochondrial Ca(2+) uptake in permeabilized beta-cells increased exponentially with increasing [Ca(2+)], and, particularly, it became much faster at [Ca(2+)](C)>2 microM. Since the bulk [Ca(2+)](C) signals during stimulation with glucose are smaller than 2 microM, mitochondrial Ca(2+) uptake could be not uniform, but to take place preferentially from high [Ca(2+)](C) microdomains formed near the mouth of the plasma membrane Ca(2+) channels. Measurements of mitochondrial NAD(P)H fluorescence in stimulated islets indicated that the [Ca(2+)](M) changes evidenced here activated mitochondrial dehydrogenases and therefore they may modulate the function of beta-cell mitochondria. Diazoxide, an activator of K(ATP), did not modify mitochondrial Ca(2+) uptake.     

HCO(3)(-) ions modify the role of PKC isoforms in the modulation of rat mast cell functions

PKC and the intracellular calcium signal are two well-known intracellular signaling pathways implicated in the induction of mast cell exocytosis. Both signals are modified by the presence or absence of HCO(3)(-) ions in the external medium. In this work, we studied the regulation of the exocytotic process by PKC isozymes and its relationship with HCO(3)(-) ions and PKC modulation of the calcium entry. The calcium entry, induced by thapsigargin and further addition of calcium, was inhibited by PMA, a PKC activator, and enhanced by 500 nM GF109203X, which inhibits Ca(2+)-independent PKC isoforms. PMA inhibition of the Ca(2+) entry was reverted by 500 and 50 nM GF109203X, which inhibit Ca(2+)-independent and Ca(2+)-dependent isoforms, respectively, and G?6976, a specific inhibitor of Ca(2+)-dependent PKCs. Thus, activation of Ca(2+)-dependent and Ca(2+)-independent PKC isoforms inhibit Ca(2+) entry in rat mast cells, either in a HCO(3)(-)-buffered or a HCO(3)(-)-free medium. PMA, GF109203X, G?6976 and rottlerin, a specific inhibitor of PKC delta, were also used to study the role of PKC isoforms in the regulation of exocytosis induced by thapsigargin, ionophore A23187 and PMA. The results demonstrate that Ca(2+)-dependent PKC isoforms inhibit exocytosis in a HCO(3)(-)-dependent way. Moreover, Ca(2+)-independent PKC delta was the main isoform implicated in promotion of Ca(2+)-dependent mast cell exocytosis in the presence or absence of HCO(3)(-). The role of PKC isoforms in the regulation of mast cell exocytosis depends on the stimulus and on the presence or absence of HCO(3)(-) ions in the medium, but it is independent of PKC modulation of the Ca(2+) entry.     

Rundown of secretion after depletion of intracellular calcium stores in bovine adrenal chromaffin cells

In this study, the relationship between intracellular calcium stores and depolarization-evoked stimulation was examined in bovine chromaffin cells, using changes in membrane capacitance to monitor both exocytosis and endocytosis. Cells were voltage-clamped using the perforated whole-cell patch configuration to minimize alterations in intracellular constituents. Control cells exhibited reproducible secretory responses each time the cell was stimulated. However, the same stimulation protocol elicited progressively smaller secretory responses in cells where their intracellular calcium store was emptied by thapsigargin. Transient elevation of the intracellular calcium concentration with a brief histamine treatment enhanced subsequent secretory responses in control but not in thapsigargin-treated cells. A series of depolarizations to -20 mV, which allowed small amounts of Ca(2+) influx but which by itself did not trigger catecholamine secretion, enhanced subsequent exocytosis in both control and thapsigargin-treated cells. Caffeine-pretreated cells exhibited a rundown in the secretory response that was similar to that produced by thapsigargin. These results suggest that brief elevations of [Ca(2+)](i) could enhance subsequent secretory responses. In addition, the data suggest that intracellular calcium stores are vital for the maintenance of exocytosis during repetitive stimulation.     

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca²⁺] _o (extracellular Ca²⁺ concentration) is between about 10^–4 to 10^–3 m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca²⁺ _o or increasing [Ca²⁺] _o to 10^–2 m inhibit exocytotic membrane fusion. Veratridine does not release Ca²⁺ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na⁺ _o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca²⁺] _o ) in the somatic, i.e., nonciliary cell membrane and that a Ca²⁺ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4.     

A novel function of Noc2 in agonist-induced intracellular Ca2+ increase during zymogen-granule exocytosis in pancreatic acinar cells

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca(2+)-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 μM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca(2+)](i)-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca(2+)](i) increases.     

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.     

Centrin is essential for the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels

Voltage-gated Ca(2+) channels play a critical role in controlling Ca(2+) entry in various cells. Ciliary reversal in Paramecium depends on the Ca(2+) influx through voltage-gated Ca(2+) channels on the ciliary membrane. One of the voltage-gated Ca(2+) channel mutants in Paramecium caudatum, cnrC, neither produces Ca(2+) action potentials nor responds to any depolarizing stimuli. Here, we report that the cnrC(+) gene product is P. caudatum centrin (Pccentrin1p), a member of the Ca(2+)-binding EF-hand protein superfamily. The Pccentrin1p gene of cnrC was found to contain a single-base deletion, a mutation that caused the loss of the fourth EF-hand of Pccentrin1p. Moreover, the wild-type Ca(2+) channel function was impaired by Pccentrin1p gene silencing, leading to the loss of current-evoked Ca(2+) action potentials and stimulated ciliary reversal. These results demonstrate that Pccentrin1p is indispensable for the activity of the voltage-gated Ca(2+) channels that control ciliary reversal in Paramecium.     

Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca2+ ionophore on mitochondria in intact cells

We have used fluorescence digital imaging techniques to explore the actions of two groups of Ca(2+) ionophores: (i). ferutinin, an electrogenic naturally occurring ionophore, and (ii). the neutral ionophores 4-BrA23187 and ionomycin, on cytosolic [Ca(2+)] ([Ca(2+)](c)), mitochondrial [Ca(2+)] ([Ca(2+)](m)) and mitochondrial membrane potential (deltapsi(m)) in HepG2 cells and primary hippocampal neurones in culture. 4-BrA23187 and ionomycin promoted the equilibration of [Ca(2+)] gradients between cellular compartments, including ER, mitochondria and cytosol. Thus, [Ca(2+)](c) and [Ca(2+)](m) increased together and then recovered in parallel on removal of the ionophore. In contrast, following a rise in [Ca(2+)](c) in response to ferutinin, [Ca(2+)](m) remained elevated for prolonged periods after the recovery of [Ca(2+)](c) levels despite washout of the compound. Both groups of Ca(2+) ionophores caused some mitochondrial depolarisation, although this was highly variable in degree. Mitochondrial depolarisation induced by ionomycin and 4-BrA23187 was often modest, independent of cyclosporin A (CsA), was suppressed in the absence of extracellular Ca(2+) and was enhanced by pre-incubation of cells with the inhibitor of the mitochondrial Ca(2+)/2Na(+)-exchanger, CGP37157, suggesting that the change in potential reflects the prior state of mitochondrial calcium loading. The mitochondrial depolarisation induced by ferutinin was not influenced by CGP37157 but was completely blocked by CsA, suggesting that it reflects opening of the mitochondrial permeability transition pore (mPTP). We suggest that ferutinin may provide a very valuable tool to promote mitochondrial calcium overload experimentally and to promote calcium-dependent opening of the mPTP.     

Calcium signalling and pancreatic cell death: apoptosis or necrosis?

Secretagogues, such as cholecystokinin and acetylcholine, utilise a variety of second messengers (inositol trisphosphate, cADPR and nicotinic acid adenine dinucleotide phosphate) to induce specific oscillatory patterns of calcium (Ca(2+)) signals in pancreatic acinar cells. These are tightly controlled in a spatiotemporal manner, and are coupled to mitochondrial metabolism necessary to fuel secretion. When Ca(2+) homeostasis is disrupted by known precipitants of acute pancreatitis, for example, hyperstimulation or non-oxidative ethanol metabolites, Ca(2+) stores (endoplasmic reticulum and acidic pool) become depleted and sustained cytosolic [Ca(2+)] elevations replace transient signals, leading to severe consequences. Sustained mitochondrial depolarisation, possibly via opening of the mitochondrial permeability transition pore (MPTP), elicits cellular ATP depletion that paralyses energy-dependent Ca(2+) pumps causing cytosolic Ca(2+) overload, while digestive enzymes are activated prematurely within the cell; Ca(2+)-dependent cellular necrosis ensues. However, when stress to the acinar cell is milder, for example, by application of the oxidant menadione, release of Ca(2+) from stores leads to oscillatory global waves, associated with partial mitochondrial depolarisation and transient MPTP opening; apoptotic cell death is promoted via the intrinsic pathway, when associated with generation of reactive oxygen species. Apoptosis, induced by menadione or bile acids, is potentiated by inhibition of an endogenous detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), suggesting its importance as a defence mechanism that may influence cell fate.     

Involvement of protein kinase A in the regulation of intracellular free calcium and phosphoinositide turnover in rat myometrium

Preincubation of Fura 2-loaded rat myometrial cells with H-8, an inhibitor of protein kinase A, for 1 h reversed the inhibitory effects of 8-(4-chlorophenylthio)-cAMP (CPTcAMP) on the oxytocin-stimulated increase in (Ca2+)i (intracellular free calcium), with an EC50 of 47 microM. H-8 also prevented the inhibition by relaxin and isoproterenol of the oxytocin-induced increase in (Ca2+)i. The EC50 of H-8 in reversing the relaxin effect was 42 microM. H-8 reversal of the effect of relaxin on (Ca2+)i was evident both in the absence of extracellular calcium and in cells pretreated with pertussis toxin. H-8 also reversed the inhibitory effects of relaxin and CPTcAMP on the oxytocin-induced increase in [3H]inositol phosphate formation and [3H]phosphoinositide hydrolysis. Preincubation of myometrial cells for 1 h with H-7, another protein kinase inhibitor, only partially attenuated the inhibition by relaxin and CPTcAMP of the oxytocin-induced increase in (Ca2+)i and [3H]inositol phosphate formation at concentrations 4-5 times greater than those of H-8. Acute (15-min) exposure to phorbol myristate acetate (1.0 microM) did not affect basal (Ca2+)i or the oxytocin-stimulated increases in (Ca2+)i or inositol phosphate formation. These results imply a regulatory role for protein kinase A in the inhibition of the oxytocin-induced increase in (Ca2+)i and inositol phosphate formation by relaxants.