Imaging of intracellular Ca waves induced by muscarinic receptor stimulation in rat parotid acinar cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca²⁺, the increase in [Ca²⁺]_i evoked by a high concentration (10 IM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca²⁺ wave toward the basolateral region. The [Ca²⁺]_i reached the maximum high level throughout the cells 1–2 s after stimulation. As Ca²⁺ was eliminated from the extracellular medium, the Ca²⁺ wave was a result of Ca²⁺ release from intracellular stores. The magnitude and velocity of the Ca²⁺ wave decreased with decreasing concentration of CCh, and the increase in [Ca²⁺]_i induced by low CCh concentrations (≤ 0.5 μM) was always larger in the apical region of acinar cells than in the basal region. The Ca²⁺ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca²⁺ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca²⁺ pump, caused a slow and homogeneous increase in [Ca²⁺]_i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca²⁺]_i suggesting that the inositol-1,4,5-trisphosphate (InsP₃) and ThG-sensitive Ca²⁺ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP₃ is essential to trigger the Ca²⁺ wave and that the development of the Ca²⁺ wave may be attributed to regional differences in InsP₃ sensitivity of Ca²⁺ stores. The agonist-induced Ca²⁺ wave is probably a general phenomenon in exocrine acinar cells.     

Peculiarities of phospholipase C-dependent release of CA2+ from intracellular stores upon activation of choline and purine receptors in myocytes of the guinea-pig small intestine

Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 μM carbachol, CCh, as well as 10.0 and 100.0 μM ATP) induced a rise in the [Ca²⁺]_i. Carbachol evoked an increase in the [Ca²⁺]_i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise in the [Ca²⁺]_i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca²⁺]_i in myocytes. The absence of the slow component in the [Ca²⁺]_i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is not related to activation of the entry of Ca²⁺ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca²⁺]_i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 μM), completely blocked rises in the [Ca²⁺]_i induced by both CCh and ATP; therefore, the release of Ca²⁺ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the difference in the release of Ca²⁺ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional effects of the above-mentioned neurotransmitters on smooth muscles. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 3–10, January–February, 2006.     

Alpha-adrenergic agonist and endothelin-1 induced intracellular Ca response in the presence of a Ca entry blocker in cultured rat ventricular myocytes

Previously we demonstrated that stimulation of cultured neonatal rat ventricular myocytes by either α₁-adrenergic agonist or endothelin-1 resulted in a rapid formation of total inositolphosphates, although the levels of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate did not rise significantly. The aim of this study was to examine whether stimulation by α₁-adrenergic agonist and endothelin-1 could still elicit phosphatidylinositol cycle mediated intracellular Ca²⁺ mobilization in these cells. The intracellular free Ca²⁺ concentration ([Ca ²⁺]i) was measured by single cell imaging dual wavelength fluorescence microscopy in Fura-2-loaded cardiomyocytes. The interference of agonist induced [Ca²⁺]; responses by the beat to beat variation of [Ca²⁺]i was prevented by arresting the cells with the Ca²⁺ entry blocker diltiazem (10 μM). The [Ca²⁺]i response (expressed as % of baseline ratio of fluorescence intensities of Fura-2 at 340 nm and 380 nm excitation wavelength), induced by phenylephrine (10⁻⁴ M) and endothelin-1 (10⁻⁸ M) was small, up to 20% of baseline after 9–20 min. In contrast, Ca²⁺-influx induced by incubation in Na⁺-free buffer caused a steep increase of [Ca²⁺]; up to 150% of baseline after 30 s.Analysis of single cells following stimulation with phenylephrine or endothelin-1 showed heterogeneity with respect to a rise in [Ca²⁺. However, if rapid Ca²⁺-influx was induced by incubation in Na⁺-free buffer, [Ca²⁺]i responses in individual myocytes occurred homogeneously.It is concluded that the α₁-adrenergic agonist and endothelin-1induced [Ca²⁺]i responses are delayed in time, small and quite heterogeneous among cells. The findings are in agreement with earlier observations which revealed no detectable overall increase of the Ca²⁺ releasing inositolphosphates under these conditions and suggest that other second messengers, such as 1,2-diacylglycerol, are involved in the agonist mediated Ca²⁺ signals.     

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca²⁺-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca²⁺ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca²⁺]_i due to intracellular Ca²⁺ release and Ca²⁺ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca²⁺]_i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca²⁺]_i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca²⁺ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca²⁺ store depletion in these cells, it is not clear how the cells decode individual Ca²⁺ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca²⁺]_i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca²⁺ entry generates [Ca²⁺]_i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca²⁺ entry generates sustained [Ca²⁺]_i elevation at high [CCh] and contributes to frequency of [Ca²⁺]_i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca²⁺ dependent gene expression pathways, consistent with the different patterns of [Ca²⁺]_i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca²⁺]_i generated by Orai1, independent of global [Ca²⁺]_i changes or TRPC1-mediated Ca²⁺ entry. In contrast, Ca²⁺ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca²⁺ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca²⁺-dependent gene expression pathways.     

Okadaic acid induces the release of Ca from intracellular stores in ECV304 endothelial cells

The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca²⁺ ([Ca²⁺]_c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca²⁺]_c both in the presence and absence of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, depletion of the intracellular Ca²⁺ stores with either ATP (1 μM) or thapsigargin (100 nM) prevented any further release of Ca²⁺ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca²⁺), a prior release of Ca²⁺ induced by okadaic acid reduced the magnitude of the response to ATP (1 μM). Taken together these observations indicate that okadaic acid induces Ca²⁺ release from the agonist-sensitive pool. The okadaic acid-induced Ca²⁺ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca²⁺]_c oscillations, which at their peak resulted in 40–100% cells, at any one time, having an elevated [Ca²⁺]_c. The response appeared to propagate between adjacent cells and the elevation of [Ca²⁺]_c. appeared initially in the cell periphery. In adherent cells, the release of Ca²⁺ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca²⁺ from the agonist- and thapsigargin-sensitive intracellular stores.     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

Implication of the nucleus in excitation contraction coupling of heart cells

In the present study, Fluo-3 Ca²⁺ measurement and confocal microscopy techniques were used in order to localize cytosolic [ ]_c and nuclear [ ]_n free Ca²⁺ distribution in resting and spontaneously contracting single heart cells from 10-day-old chick embryos. In resting single cells, the concentration of Ca²⁺ in the cytoplasm was lower than that in the nucleus. Increasing cytosolic free Ca²⁺ from 100–1600 nM gradually increased [Ca²⁺]_n with a maximum capacity near 1200 nM. Results from Fura-2 microfluorometry and Fluo-3 confocal microscopy suggest a potential cross talk between the increase of cytosolic free Ca²⁺ and the uptake and release of Ca²⁺ by the nucleus during spontaneous contraction of single myocytes. Calcium waves in spontaneously contracting cells were found to spread from one cell to the next with the nucleus acting as a fluorescent beacon in which Ca²⁺ levels remained elevated for several milliseconds even after cytosolic Ca²⁺ had returned to near basal values. These results strongly suggest that the nucleus plays a negative and positive feedback role in controlling cytosolic free Ca²⁺ concentration during excitation-contraction coupling in heart cells.     

Axial stretch enhances sarcoplasmic reticulum Ca leak and cellular Ca reuptake in guinea pig ventricular myocytes: Experiments and models

Cardiac cellular calcium (Ca²⁺) handling is the well-investigated mediator of excitation–contraction coupling, the process that translates cardiac electrical activation into mechanical events. The reverse—effects of mechanical stimulation on cardiomyocyte Ca²⁺ handling—are much less well understood, in particular during the inter-beat period, called ‘diastole’. We have investigated the effects of diastolic length changes, applied axially using a pair of carbon fibres attached to opposite ends of Guinea pig isolated ventricular myocytes, on the availability of Ca²⁺ in the main cellular stores (the sarcoplasmic reticulum; SR), by studying the rest-decay of SR Ca²⁺ content [Ca²⁺]_SR, and the reloading of the SR after prior depletion of Ca²⁺ from the cell.Cells were loaded with Fura-2 AM (an indicator of the cytosolic ‘free’ Ca²⁺ concentration, [Ca²⁺]_i), and pre-conditioned by field-stimulation (2 Hz) at 37 °C, while [Ca²⁺]_i transients and sarcomere length (SL) were recorded simultaneously. After reaching a steady state in the behaviour of observed parameters, stimulation was interrupted for between 5 and 60 s, while cells were either held at resting length, or stretched (controlled to cause a 10% increase in SL, to aid inter-individual comparison). Thereafter, each cell was returned to its original resting length, followed by swift administration of 10 mM of caffeine (in Na⁺/Ca²⁺-free solution), which causes the release of Ca²⁺ from the SR (caffeine), but largely prevents extrusion of Ca²⁺ from the cytosol to the cell exterior (Na⁺/Ca²⁺-free solution). By comparing the [Ca²⁺]_i in cells exposed/not exposed to diastolic stretch of different duration, we assessed the rest-decay dynamics of [Ca²⁺]_SR. To assess SR reloading after initial Ca²⁺ depletion, the same stretch protocol was implemented after prior emptying of the cell by application of 10 mM of caffeine in normal Tyrode solution (which causes Ca²⁺ to be released from the SR and extruded from the cell via the Na⁺/Ca²⁺ exchanger; NCX).Axial stretch enhanced the rate of both rest-decay and reloading of [Ca²⁺]_SR. Application of 40 μM streptomycin, a blocker of stretch-activated ion channels, did not affect the stretch-induced increase in SR reloading. This behaviour was reproduced in a computer simulation study, using a modified version of the 2006 Iribe–Kohl–Noble model of single cardiac myocyte Ca²⁺ handling, suggesting that stretch increases both Ca²⁺ leak from the SR and Ca²⁺ influx via the sarcolemma. This may have important implications for the mobilisation of Ca²⁺ in stretched cells, and could contribute to the regional ‘matching’ of individual cardiomyocyte contractility to dynamic, and regionally varying, changes in mechanical loads, such as diastolic pre-load, of cardiac tissue.     

Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract: The mechanisms involved in Ca²⁺ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca²⁺]_i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca²⁺]_i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca²⁺]_i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP₃) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca²⁺ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca²⁺]_i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP₃ receptors, appear to be crucial to the mAChR-mediated [Ca²⁺]_i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP₃-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP₃ receptors may not contribute significantly to NMDA-evoked elevation of [Ca²⁺]_i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca²⁺ stores is proposed.     

Distinct Mechanisms of [Ca2+]i Oscillations in HSY and HSG Cells: Role of Ca2+ Influx and Internal Ca2+ Store Recycling

This study examined [Ca²⁺]_i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca²⁺]_i and K _Ca currents in both cell types. Low IP₃, but not thapsigargin (Tg), induced [Ca²⁺]_i oscillations, whereas Tg blocked CCh-stimulated [Ca²⁺]_i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca²⁺ from HSY cells (i) did not affect CCh-stimulated [Ca²⁺]_i oscillations or internal Ca²⁺ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca²⁺]_i into oscillations. CCh- or thapsigargin-induced Ca²⁺ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca²⁺ pump (SERCA) and inositoltrisphosphate receptors (IP₃Rs) than HSG cells. These data demonstrate that [Ca²⁺]_i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca²⁺ from, and release of Ca²⁺ into, the cytosol by the SERCA and IP₃R activities, respectively. In HSY cells, Ca²⁺ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca²⁺]_i. In HSG cells, [Ca²⁺]_i oscillations directly depend on Ca²⁺ influx; Ca²⁺ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca²⁺]_i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca²⁺ stores. Received: 30 October 2000/Revised: 26 February 2001     

Generation of repetitive Ca transients by bombesin requires intracellular release and influx of Ca through voltage-dependent and voltage independent channels in single HIT cells

In the present study, the bombesin-induced changes in cytosolic free Ca²⁺ ([Ca²⁺]_i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca²⁺ transients. The average frequency of the Ca²⁺ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min⁻¹ (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca²⁺ transient followed by a sustained plateau or by a decrease to basal levels. In Ca²⁺- free medium, bombesin caused only one or two Ca²⁺ transients and withdrawal of extracellular Ca²⁺ abolished the Ca²⁺ transients. The voltage-dependent Ca²⁺ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca²⁺ transients and stopped the Ca²⁺ transients in some cells. Thapsigargin caused a sustained rise in [Ca²⁺]_i) in the presence of extracellular Ca²⁺ while in its absence the rise in [Ca²⁺]_i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca²⁺], by about 50%. Depletion of intracellular Ca²⁺ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca²⁺-free medium caused an agonist-independent increase in [Ca²⁺]_i) when extracellular Ca²⁺ was restored, which was larger than in control cells that had been incubated in Ca²⁺-free medium for the same period of time. This rise in [Ca²⁺]_i and the thapsigargin-induced increase in [Ca²⁺]_i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca²⁺ pool enhances Ca²⁺ influx through VDCC and voltage-independent Ca²⁺ channels (VICC). In conclusion, the bombesin-induced Ca²⁺ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca²⁺ transients. Intracellular Ca²⁺ is mobilized during each Ca²⁺ transient, but Ca²⁺ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca²⁺ response. Ca²⁺ influx in whole or part is activated by a capacitative Ca²⁺ entry mechanism.     

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca²⁺]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca²⁺]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca²⁺ probe. Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca²⁺]i in both small and large luteal cells, having an effect at concentrations as low as 5μM; and was maximally effective at 50μM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca²⁺]i, with NDGA exerting a greater stimulation of [Ca²⁺i than indomethacin. Prostaglandin F_2α (PGF_2α) as well as prostaglandin E₂ (PGE₂) increased [Ca²⁺ in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca²⁺]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF_2α and PGE₂) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca²⁺]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

Ca²⁺ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca²⁺-dependent enzymes. However, excessive mitochondrial Ca²⁺ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca²⁺ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca²⁺ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca²⁺ levels ([Ca²⁺]_c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca²⁺ content (Ca^2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca²⁺]_c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca²⁺ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca²⁺m that was not further increased during prolonged incubations, despite a sustained [Ca²⁺]_c elevation. By contrast, thapsigargin induced a net Ca²⁺ accumulation in mitochondria that continued for up to 30 min and reached Ca^2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca²⁺ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca²⁺ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca²⁺ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca²⁺ through artificial means, enabling mitochondria to function as a Ca²⁺ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)     

Signal percolation through plants and the shape of the calcium signature

Plants respond to almost any kind of external stimulus with transients in their cytoplasmic free calcium concentration ([Ca²⁺]_c). A huge variety of kinetics recorded by optical techniques has been reported in the past. This variety has been credited the specificity needed to explain how information about incoming stimuli is evaluated by the organism and turned into the right physiological responses which provide advantages for survival and reproduction. A physiological response often takes place away from the site of stimulation. This requires cell-to-cell communication. Hence, responding cells are not necessarily directly stimulated but rather receive an indirect stimulus via cell-to-cell communication. It appears unlikely that the ‘[Ca²⁺]_c signature’ in the primarily stimulated cell is conveyed over long distances via cell-to-cell communication from the ‘receptor cells’ to the ‘effector cells’. Here, a novel aspect is highlighted to explain the variety of [Ca²⁺] kinetics seen by integrating methods of [Ca²⁺]_c recording. Plants can generally be seen as cellular automata with specific morphology and capable for cell-to-cell communication. Just a few rules are needed to demonstrate how waves of [Ca²⁺]_c-increases percolate through the organism and thereby deliver a broad variety of ‘signatures’. Modelling intercellular signalling may be a possible way to find explanations for different kinds of signal transmission, signal amplification, wave formation, oscillations and stimulus-response coupling. The basic examples presented here show that care has to be taken when interpreting cellular ‘[Ca²⁺]_c signatures’ recorded by optical techniques which integrate over a big number of cells or even whole plants.Key words: cellular automata, cell-to-cell communication, cytoplasmic calcium, modelling, percolation, signature     

Taurine prevents intracellular calcium overload during calcium paradox of cultured cardiomyocytes

Summary The effect of taurine on the cellular distribution of [Ca²⁺]_i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca²⁺ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca²⁺-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca²⁺]; rapidly fell to a level of 3–6 × 10^–8M. Subsequent restoration of medium calcium increased [Ca²⁺]_i to level 4–7 times normal. Large increases in [Ca²⁺]_i were observed in most cells and were associated with the development of contracture and bleb formation.Taurine pretreatment (20mM) caused no significant effect on [Ca²⁺]_i during Ca²⁺ depletion. However, it inhibited excessive accumulation of [Ca²⁺]_i during the Ca²⁺ repletion. Moreover, taurine treated cells recovered their Ca²⁺-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment.     

Imaging cytosolic free-calcium distribution and oscillations in pollen tubes with confocal microscopy: A comparison of different dyes and loading methods

Summary A steep, oscillating tip-focused gradient in cytosolic free calcium ([Ca²⁺]_c) has been implicated in pollen tube growth. Further understanding of the biological causes and consequences of these processes relies on the precise imaging of [Ca²⁺]_c during the different growth phases. In this work, the minimum technical requirements for confocal [Ca²⁺]_c imaging ofAgapanthus umbellatus pollen tubes were examined. A range of dyes, dye forms, and loading methods were compared. Non-ratio and ratio imaging were critically analysed, in terms of the detection of the [Ca²⁺]_c gradient and its fluctuations over time. Both ratiometric and nonratiornetric methods detected relative changes in [Ca²⁺]_c. However, visualisation of the [Ca²⁺]_c gradient, with an accurate spatial definition, was only possible with ratiometric methods. The gradient observed in this study ranged from 1.8 M (tip) to 180–220 nM (basal level), within the first 4–10 m. Apical [Ca²⁺]_c fluctuations with an amplitude between 415 nM and 1.8 M showed a period of 40 to 75 s. All protocols for dye-loading proved to have strengths and weaknesses. Thus, the choice of a dye and its loading procedure should consider the required imaging period, extent of sequestration, effect on cell performance and viability, ease of loading procedure, and aim of the study. The present study constitutes an examination of the [Ca²⁺]_c gradient in pollen tubes by these criteria.Abbreviations CLSM confocal laser scanning microscope - [Ca²⁺]_c cytosolic free calcium - PT pollen tube Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Elevations of intracellular calcium reflect normal voltage-dependent behavior,and not constitutive activity,of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca²⁺ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca²⁺ influx and determining the bulk average cytoplasmic Ca²⁺ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca²⁺ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca²⁺ current (I_Ca) and evoked a rise in [Ca²⁺] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca²⁺] increase ([Ca²⁺]_c) was approximately uniform, whereas that of the subplasma membrane space ([Ca²⁺]_PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca²⁺ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca²⁺ channels were not normally constitutively active. The repetitive localized [Ca²⁺]_PM rises (“persistent Ca²⁺ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca²⁺ channels regulate the bulk average [Ca²⁺]_c. A dihydropyridine blocker of Ca²⁺ channels, nimodipine, which blocked I_Ca and accompanying [Ca²⁺]_c rise, reduced neither the resting bulk average [Ca²⁺]_c (at −70 mV) nor the rise in [Ca²⁺]_c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca²⁺ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca²⁺]_c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca²⁺].     

Changes produced in intracellular calcium concentration and transmembrane currents by iontophoretic injection of cAMP in Helix pomatia neurons

Transmembrane currents and changed [Ca²⁺]_in produced by iontophoretic injection of cAMP were investigated in voltage clampedHelix pomatia neurons. The Fura-2 fluorescence probe technique was used to measure [Ca²⁺]_in. Injection of cAMP was found to produce a protracted rise in the latter at a membrane potential range of –40 to –100 mV in conjunction with transmembrane inward current. Duration of the changes in [Ca²⁺]_in largely dependent on neuronal size and varied between 50 and 500 sec (parameters for neurons with somata of around 100 and 40 µm respectively). In a medium with Ca²⁺ replaced by Mg²⁺ (as well as after addition of EDTA, a calcium chelator) both transmembrane current and the pattern of increase in [Ca²⁺]_in remained unchanged. Inward current usually declined substantially but degree of change in [Ca²⁺]_in remained the same when Na⁺ was eliminated from the solution by replacing its Tris⁺. Addition of 2 mM Cd²⁺ to the external medium hardly affected current level and increase in [Ca²⁺]_in. Neither procaine, a local anesthetic, nor ryanodine (which inhibits release of calcium from the intracellular store) changed the cAMP effects observed. A concentration of 1 mM La³⁺ depressed both inward current and the [Ca²⁺]_in increase. Findings would imply the occurrence of cAMP-dependent release of calcium from the intracellular store in the neurons tested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 396–402, May–June, 1989.     

Short-term regulation of cytosolic Ca2+, cytosolic pH and vacuolar pH under NaCl stress in the charophyte alga Nitellopsis obtusa

Isolated characean internodal cells of Nitellopsis obtusa can be stored in artificial pond water for many days, but they cannot survive in 100mol m^?3 NaCl solution unless more than several mol m^?3 Ca²⁺ is added. Short-term effects of NaCl stress on the cytosolic concentration of Ca²⁺ ([Ca²⁺]_c), cytosolic pH (pH_c) and vacuolar pH (pH_v) were studied in relation to the external concentration of Ca²⁺ ([Ca²⁺]_e). Changes in [Ca²⁺]_c were measured with light emission from a Ca²⁺-sensitive photoprotein, semisynthetic fch-aequorin which had been injected into the cytosol. Both pH_c and pH_v were measured with double-barrelled pH-sensitive microelectrodes. When internodal cells were treated with 100 mol m^?3 NaCl (0–1 mol m^?3 NaCl (0.1 mol m^?3 [Ca²⁺]_e), [Ca²⁺]_c increased and then recovered to the original level within 60 min. The time course of the transient change in [Ca²⁺]_c was not influenced by the level of [Ca²⁺]_c (0.1 and 10 mol m^?3). In some cases, the transient increase in [Ca²⁺]_c was induced only by increasing external osmotic pressure with sorbitol. In response to treatment with 100 mol m^?3 NaCl (0.1 mol m^?3 [Ca²⁺]_c), pH_c decreased by 0.1–0.2 units after 10min but recovered after 30–60 min, while pH_v increased by 0.4–0.5 units after 2–50 min and tended to recover after 60 min. The initial changes in both pH_c and pH_v were suppressed when [Ca²⁺]_e was raised from 0.1 to 10mol m^?3. These results show that the charophyte alga Nitellopsis can regulate [Ca²⁺]_c, pH_c and pH_v under NaCl stress in the short term and that the protective effect of Ca²⁺ on salinity stress is apparently unrelated to perturbation of Ca²⁺ and pH homeostasis.