Cell transformation induces a cytoplasmic Ca2+ oscillator in Madin-Darby canine kidney cells

Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP ₃)-sensitive Ca²⁺ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca²⁺-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca²⁺]_i showed that Ca²⁺ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca²⁺ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca²⁺ release through transformation. Oscillations are primarily due to the activity of an InsP ₃-sensitive cytosolic Ca²⁺ oscillator.     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

Global,synchronous oscillations in cytosolic calcium and adherence in bradykinin‐stimulated Madin‐Darby canine kidney cells

Aims and Methods: Intercellular Ca²⁺ oscillations are a universal mode of signalling in both excitable and non‐excitable cells. Here, we study the relationship between Ca²⁺ signalling and coherent changes in adhesion properties by measuring the transepithelial impedance across bradykinin‐stimulated Madin‐Darby canine kidney (MDCK) cell layers grown on a microelectrode. During hormone stimulation, the impedance is found to oscillate, reflecting that the cells undergo morphological/adhesive alterations with high spatio‐temporal organization. The experiments are supplemented with parallel, digital imaging fluorescence microscopy of bradykinin‐induced single‐cell Ca²⁺ oscillations. Results: In agreement with previous experiments, MDCK cells are found to elicit synchronous, multicellular Ca²⁺ oscillations in response to hormone stimulus. The periods of the Ca²⁺ oscillations and the electrical fluctuations are found to coincide. Further, blocking of gap junctions by 18α‐glycyrrhetinic acid causes a loss of synchrony in Ca²⁺ signals and inhibition of impedance oscillations, emphasizing the importance of gap junctions in the signal transduction process. Conclusion: Based on these observations it is concluded that the co‐ordinated adhesive changes in MDCK cells are a direct consequence of synchronized Ca²⁺ oscillations. Calcium signalling represents an efficient way of organizing physiological responses in a tissue. A possible functional implication of the structural changes might be to modulate transportation of various substances across the cell sheet.     

Nonselective cation channels are essential for maintaining intracellular Ca2+ levels and spontaneous firing activity in the midbrain dopamine neurons

Intracellular Ca²⁺ and Ca²⁺-permeable ion channels are important in regulating the firing activity and pattern of midbrain dopamine neurons, but the role of Ca²⁺-permeable nonselective cation channels (NSCCs) on spontaneous firing activity is unclear. Therefore, we investigated how Ca²⁺-permeable NSCCs modulate spontaneous firing activity and cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in acutely isolated midbrain dopamine neurons of the rat. Applications of voltage-dependent Ca²⁺ channels antagonists failed to abolish spontaneous firing activity completely, but they decreased firing rate and [Ca²⁺]_c. However, a blockade of NSCCs by 2-APB or SKF96365 more potently suppressed spontaneous firings with a depolarization of membrane potential and strong decreases in basal [Ca²⁺]_c levels. The depolarization of membrane potentials was attenuated by intracellular dialysis with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). NSCCs blockers inhibited oscillatory potentials and decreased basal [Ca²⁺]_c in the presence of tetrodotoxin. Apamin, a small-conductance Ca²⁺-activated K⁺ channel inhibitor, depolarized membrane potentials and enhanced firing rates. From these data, we conclude that NSCCs not only make up the tonic Ca²⁺ entry pathways to uphold basal [Ca²⁺]_c levels but also contribute to generation of spontaneous firings, thereby regulating spontaneous firing activities of the midbrain dopamine neurons.     

Confocal imaging of calcium release events in single smooth muscle cells

Localized [Ca²⁺]_i transients (‘sparks’) first directly detected in cardiac myocytes were considered to represent ‘elementary’ Ca²⁺-release events playing a key role during excitation–contraction coupling ( Cheng et al. 1993 ). In this study we employed confocal [Ca²⁺]_i imaging to characterize subcellular calcium signalling in fluo-3 loaded visceral and vascular smooth muscle cells. In some experiments membrane potential of the myocyte was controlled using whole-cell patch clamp technique and changes in membrane current were recorded simultaneously with [Ca²⁺]_i imaging. Some local [Ca²⁺]_i transients were very similar to ‘Ca²⁺ sparks’ observed in heart, i.e. lasting ≈200 ms with a peak fluorescence ratio of 1.75 ± 0.23 (mean ± SD, n = 33). Ca²⁺ sparks were found to occur in certain preferred locations in the cell, termed frequent discharge sites. Other events were faster and smaller, lasting only ≈40 ms with a peak normalized fluorescence of 1.36 ± 0.09 (mean ± SD, n = 28). A high correlation between spontaneous transient outward currents and spark occurrence was observed. Proliferating waves of elevated [Ca²⁺]_i initiated during membrane depolarization seem to arise from spatio-temporal recruitment of local Ca²⁺-release events. The spatial non-uniformity of sarcoplasmic reticulum and ryanodine receptor distribution within the cell may account for the existence of ‘frequent discharge sites’ and the wide variation in the Ca²⁺ wave propagation velocities observed.     

Discrimination of Ca2+-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities

Summary We have developed a procedure to discriminate actomyosin-type ATPase activity from Ca²⁺-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca²⁺-induced tension and accompanying ATPase activity. When they were treated with an alkaline CyDTA-containing solution of low ionic strength which was reported to remove troponin C, the fibres showed a considerable amount of Ca²⁺-dependent ATPase activity, in spite of having little or no Ca²⁺-induced isometric tension. The residual ATPase activity is ascribed to the Ca²⁺-ATPase activity of SR, because it is completely abolished by 1% CHAPS treatment for 10 min. This conclusion is also supported by the finding that the Ca²⁺-dependence of the ATPase activity is very similar to that of Ca²⁺-ATPase of SR isolated from rabbit skeletal muscle, and that the estimated activity is consistent with the reported values of direct determinations. On the other hand, treatment with a detergent such as CHAPS or Triton X-100 removes SR activities (ATPase and Ca-uptake), leaving Ca²⁺-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca²⁺-ATPase activity of SR may be minimal in total steady-state ATPase activity of mechanically skinned mammalian skeletal muscle fibres. Successive CyDTA and CHAPS treatments eliminated both Ca²⁺-induced tension and ATPase activity, which were recovered by the addition of troponin C. Using these procedures, we also examined the effect of cyclopiazonic acid (CPA) which was reported to be a specific inhibitor of Ca²⁺-ATPase of SR. Ca²⁺-ATPase activity of SR in skinned fibres was inhibited completely by 10 m CPA and held to one-half by about 0.2 m. This effect was only partially reversible. CPA at 10 m or higher concentrations showed Ca²⁺-sensitizing action on myofibrils, which was readily reversible. CPA at 3 m inhibited almost completely the Ca²⁺-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.     

The nuclear envelope of resting C6 glioma cells is able to release and uptake Ca2+ in the absence of chemical stimulation

 Many agonists evoke events in the cell nucleus through the control of Ca²⁺ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca²⁺. However, the release of Ca²⁺ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca²⁺ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca²⁺-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca²⁺ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca²⁺ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca²⁺ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 μM), a specific Ca²⁺-ATPase inhibitor, a long-lasting Ca²⁺ release was observed at the nuclear envelope. Moreover, most of the released Ca²⁺ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca²⁺ store that possesses the ability to discharge and sequestrate Ca²⁺; and (2) the Ca²⁺-releasing channels are present in the inner nuclear membrane. Received: 21 July 1997 / Received after revision: 22 September 1997 / Accepted: 23 September 1997     

The effects of interfering with GTP-binding proteins on the activation mechanism of calcium release-activated calcium current

 In electrically non-excitable cells, Ca²⁺ entry is mediated predominantly by the store-operated Ca²⁺ influx pathway, which is activated by emptying the intracellular Ca²⁺ stores. Just how the Ca²⁺ content of the stores is communicated to the activity of store-operated Ca²⁺ channels in the plasma membrane is unclear. It has been suggested that, in some cell types, the link is accomplished by either a small or a heterotrimeric GTP-binding protein, which is inhibited by guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) and, in some cases, pertussis toxin. Using the whole-cell patch-clamp technique to directly measure the store-operated Ca²⁺ current I _CRAC (Ca²⁺-release-activated Ca²⁺ current) in RBL cells, we report that manipulations designed to interfere with GTP-binding protein activity (dialysis with GTP[γ-S], exposure to pertussis toxin) routinely fail to affect the activation of I _CRAC. However, these agents alter the activity of a K⁺ current in the same cells, demonstrating biological activity. Furthermore, activation of I _CRAC does not seem to require the presence of a pre-existing diffusible messenger in the cytoplasm to any appreciable extent because the current reaches the same amplitude irrespective of the whole-cell dialysis time. We conclude that neither a mobile pre-existing molecule nor a GTP-dependent step is necessary for the activation of I _CRAC in RBL-1 cells. Received: 9 September 1998 / Received after revision and accepted: 2 November 1998     

Induction of Ca2+ oscillations by selective,U73122-mediated,depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca²⁺ sequestering and releasing properties of internal Ca²⁺ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca²⁺ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P ₃]-sensitive, but not the Ins(1,4,5)P ₃-insensitive, Ca²⁺ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca²⁺ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av), which was largely independent of external Ca²⁺. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonistevoked [Ca²⁺]_i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca²⁺-ATPase activity resulted in a [Ca²⁺]_i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P ₃- casu quo agonist-sensitive internal Ca²⁺ store, whereas thapsigargin affects both the Ins(1,4,5)P ₃-sensitive and -insensitive Ca²⁺ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca²⁺]_i. The U73122-evoked oscillations were abolished in the absence of external Ca²⁺. The ability of U73122 to generate external Ca²⁺-dependent Ca²⁺ oscillations suggests that depletion of the agonistsensitive store leads to an increase in Ca²⁺ permeability of the plasma membrane and that the Ins(1,4,5)P ₃-insensitive Ca²⁺ pool is necessary for the Ca²⁺ oscillations.     

Alginate‐coated magnetic nanoparticles for noninvasive MRI of extracellular calcium

Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca²⁺), are essential ingredients for life. Despite the tremendous importance of Ca²⁺ for the well‐being of living systems, the noninvasive determination of the changes in Ca²⁺ levels in general, and extracellular Ca²⁺ levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca²⁺ levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca²⁺ concentrations in the range of 250 µm to 2.5 mm , even in the presence of competitive ions. The alginate‐coated magnetic NPs (MNPs) aggregate in the presence of Ca²⁺, which, in turn, affects the T₂ relaxation of the water protons in their vicinity. The new alginate‐coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca²⁺ levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca²⁺ levels in vivo. These results demonstrate that alginate‐coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca²⁺ levels in myriad in vitro and in vivo biomedical applications. Copyright © 2014 John Wiley & Sons, Ltd.     

Acetylcholine induces Ca2+ oscillations via m3/m4 muscarinic receptors in the mouse oocyte

Changes in intracellular Ca²⁺ concentration are required for the activation of mammalian oocytes. They are caused mainly by Ca²⁺ release from the endoplasmic reticulum (ER) via InsP ₃ receptors (InsP ₃R). Several studies have reported that acetylcholine (ACh) is capable of triggering early activation events in mouse oocytes over-expressed with the m1 muscarinic ACh receptor (m1AChR). Here we examined which subtypes of the mAChR (m1 to m4) are involved in the generation of Ca²⁺ oscillations in native mouse oocytes. ACh (10 M) elicited regular Ca²⁺ oscillations similar to those induced by sperm in their temporal characteristics. The Ca²⁺ oscillations were abolished by application with atropine, the mAChR inhibitor. Within 1 min after treatment of ACh, intracellular Fluo-3 fluorescence intensity increased from 794±119 to 2023±755 (increase to 250% of original value), indicating a strong rise of cytosolic Ca²⁺ concentration. 4-DAMP mustard and Tropicamide, specific antagonists of m3AChR and m4AChR, completely abolished ACh-induced Ca²⁺ oscillations. In the ovulated oocytes, the expression of m3/m4 AChR was clearly detected by RT-PCR analysis. Furthermore, ACh-induced Ca²⁺ oscillations were also abolished or decreased by PLC inhibitors (U73122 or D609) and an InsP ₃-receptor antagonist (xestospongin C), confirming that ACh generates Ca²⁺ oscillations via the PLC-InsP ₃ (PI) pathway. These results strongly suggest that m3/m4AChR is coupled to the generation of Ca²⁺ oscillations mainly via the PI pathway in mouse oocytes.     

Effect of 10‐day cast immobilization on sarcoplasmic reticulum calcium regulation in humans

This study investigated the effects of 10‐day lower limb cast immobilization on sarcoplasmic reticulum (SR) Ca²⁺ regulation. Muscle biopsies were analysed in eight healthy females for maximal rates of SR Ca²⁺ release, Ca²⁺ uptake and Ca²⁺ ATPase activity at control, during immobilization at day 3 (IM 3), day 6 (IM 6) and day 10 (IM 10). Quadriceps muscle cross‐sectional area (CSA) and 1‐repetition maximum (1RM) leg extension strength were measured to determine the extent of muscle size and strength adaptations. Muscle CSA and strength decreased following 10 days of immobilization (11.8 and 41.6%, respectively, P < 0.01). A decrease in SR Ca²⁺ uptake rate (analysed per g wet wt) was found at IM 3 (13.2%, P=0.05), with a further decrease at IM 10 (19.8% from control, P < 0.01). At IM 10, a decrease in SR Ca²⁺ uptake rate (per mg protein) also occurred (19.9%, P < 0.01). Sarcoplasmic reticulum Ca²⁺ ATPase activity and rate of Ca²⁺ release were not altered with 10 days of immobilization. This study observed a decrease in SR Ca²⁺ uptake rate, muscular atrophy and strength loss over 10 days of immobilization in humans.     

Genotype–Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel

Type 1 ryanodine receptor (RYR1) is a Ca²⁺ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca²⁺‐induced Ca²⁺ release (CICR), resulting in abnormal Ca²⁺ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live‐cell Ca²⁺ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca²⁺ content, and an increased resting cytoplasmic Ca²⁺ level. The three parameters for CICR (Ca²⁺ sensitivity for activation, Ca²⁺ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [³H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca²⁺ sensitivity for activation in a site‐specific manner. Genotype–phenotype correlations were explained well by the near‐atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.     

Initiation site of Ca(2+) entry evoked by endoplasmic reticulum Ca(2+) depletion in mouse parotid and pancreatic acinar cells

Purpose

In non-excitable cells, which include parotid and pancreatic acinar cells, Ca²⁺ entry is triggered via a mechanism known as capacitative Ca²⁺ entry, or store-operated Ca²⁺ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca²⁺ stores, which acts as an important factor triggering Ca²⁺ entry. However, both the mechanism of store-mediated Ca²⁺ entry and the molecular identity of store-operated Ca²⁺ channel (SOCC) remain uncertain.

Materials and Methods

In the present study we investigated the Ca²⁺ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.

Results

Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca²⁺-ATPase, in an extracellular Ca²⁺ free state, and subsequent exposure to a high external calcium state evoked Ca²⁺ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca²⁺ channel, completely blocked Tg-induced Ca²⁺ entry. Microfluorometric imaging showed that Tg-induced Ca²⁺ entry started at a basal membrane, not a apical membrane.

Conclusion

These results suggest that Ca²⁺ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.     

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

 We have previously shown that a new type of K⁺ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl^- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K⁺ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/l, n = 26). Reduction of extracellular Ca²⁺ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SK_Ca). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K⁺ channel noise caused by the very small K⁺ channel increased. Activity of non-selective cation channels (NS_cat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NS_cat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca²⁺ activated and required >10 μmol/l Ca²⁺ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SK_Ca was also Ca²⁺ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca²⁺ (cs, n = 12). SK_Ca was inhibited by Ba²⁺ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca²⁺ activity ([Ca²⁺]_i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K⁺ concentration to 30 mmol/l, reduced [Ca²⁺]_i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca²⁺-sensitive K⁺ channel. This channel is activated promptly by very small increments in [Ca²⁺]_i and is inactivated by a fall in [Ca²⁺]_i induced by forskolin. Received: 15 April 1996 / Received after revision and accepted: 17 June 1996     

Differential actions of exogenous and intracellular spermine on contractile activity in smooth muscle of rat portal vein

Effects of the naturally occurring polyamine spermine on electrical and contractile properties of the rat portal vein were studied. 1 mM spermine nearly abolished spike activity and spontaneous contractions and decreased the intracellular Ca²⁺ concentration ([Ca²⁺],). The phasic force responses to 0.1 and 1 μM phenylephrine were partially inhibited, but not the sustain plateau contraction caused by 5 /IM phenylephrine. The Ca²⁺-force relation in high-K⁺ (128 mM)-depolarized veins was shifted to the right, EC₅₀ for Ca²⁺ increasing from 0.50 ± 0.03 mM (control, n= 8) to 0.65 ± 0.06 and to 0.94 ± 0.03 at 1 (n – 4) and 10 (n = 3) mM spermine, respectively. However, at a Ca²⁺ concentration of 2.5 mM, giving maximal force, there was no effect of spermine (1 mM) on either force or [Ca²⁺],. Whereas extracellular spermine thus reduced contractile activity at moderate levels of stimulation, increased intracellular concentration of spermine potentiated the force response to Ca²⁺. Intracellular loading of spermine by reversible permeabilization increased its concentration by 2–3 times. The spontaneous activity and response to phenylephrine were unchanged. However, the Ca²⁺-force relation of depolarized veins was shifted to the left, EC₅₀ decreasing from 0.51 ± 0.04 mM in controls (n= 7) to 0.36 ± 0.02 mM in the loaded veins (n= 9). Spermine increased Ca²⁺-activated force in portal veins permeabilized with β-escin. The degree of potentiation was consistent with observed effects in spermine-loaded intact veins. The results suggest that spermine at physiological intracellular concentration may contribute to the determination of Ca²⁺ sensitivity in vascular smooth muscle cells.     

Prolonged exercise potentiates sarcoplasmic reticulum Ca<Superscript>2+</Superscript> uptake in rat diaphragm

The effects of a single bout of prolonged treadmill exercise [mean=81 (13) min] on sarcoplasmic reticulum (SR) Ca²⁺ release, uptake and ATPase activity were determined in the costal region of rat diaphragm (D) and red gastrocnemius (RG). Glycogen depletion measurements made immediately following exercise suggested that treadmill running substantially recruited the fibers throughout both muscles. SR Ca²⁺ ATPase activity, measured in isolated SR vesicles, decreased in the RG by 33% but remained unchanged in D in response to the exercise bout. This effect in RG was matched by a 37% decline in Ca²⁺ uptake and a 28% depression in Ca²⁺ release when measured in muscle homogenates. Conversely, Ca²⁺ uptake increased between 157% and 263% in the D in the absence of any change in Ca²⁺ release. These data show that the attenuation of SR function that has been consistently observed in limb muscle over the last several decades is absent in diaphragm despite the fact that its fibers appear to experience sufficient activity to deplete their glycogen. In fact, the large increase in Ca²⁺ uptake in D shows that prolonged activity actually potentiates the ability of SR vesicles to sequester Ca²⁺ in the absence of any increase in energy cost. Thus, it appears necessary to re-evaluate the role of exercise in regulating Ca²⁺ sequestration by the SR as different muscles may respond in ways that are dictated by their function. Electronic Publication     

ATP-induced Ca2+ signals in bronchial epithelial cells

 Ca²⁺-dependent Cl^– secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca²⁺ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP ₃) receptors and not ryanodine receptors are involved in intracellular Ca²⁺ release. The expression pattern of the three InsP ₃ receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca²⁺ signals in the intact cell, consisting of abortive Ca²⁺ spikes or fully developed [Ca²⁺] rises and intracellular Ca²⁺ waves, were indicative of positive feedback of Ca²⁺ on the InsP ₃ receptors. Low Ca²⁺ concentrations stimulated and high Ca²⁺ concentrations inhibited InsP ₃-induced Ca²⁺ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca²⁺-binding site between amino acid residues 2077 and 2101 in the type-2 InsP ₃ receptor and between amino acids 2030 and 2050 in the type-3 InsP ₃ receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP ₃ receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca²⁺ and that this phenomenon contributes to the ATP-induced Ca²⁺ signals in intact 16HBE14o- cells. Recieved: 11 September 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998     

Permissive role of calcium on regulatory volume decrease in freshly isolated mouse cholangiocytes

Calcium (Ca²⁺) pathways are important in cell volume regulation in many cells, but its role in volume regulatory processes in cholangiocytes is unclear. Thus, we have investigated the role of Ca²⁺ in regulatory volume decrease (RVD) in cholangiocytes using freshly isolated bile duct cell clusters (BDCCs) from normal mouse. No significant increase in [Ca²⁺]_i was observed during RVD, while ionomycin and ATP showed significant increases. Confocal imaging also showed no significant changes in the levels or distributions of intracellular Ca²⁺ during RVD. Cell volume study by quantitative videomicroscopy indicated that removal and chelation of extracellular Ca²⁺ by ethylene glycol-bis (β-aminoethyl ether)-N,N,N-tetraacetic acid (EGTA) or administration of nifedipine did not affect RVD but verapamil significantly inhibited the RVD. Moreover, Ca²⁺ agonists or inhibitors of Ca²⁺ release from intracellular stores had no significant effect on RVD. However, 1,2-bis (2-aminophenoxy) ethane-N,N,N′N′-tetraacetic acid-AM (BAPTA-AM) showed significant decreases in [Ca²⁺]_i and significantly inhibited RVD, which was reversed with coadministration of valinomycin, suggesting that BAPTA-AM-induced inhibition is due to potassium conductance or other cellular processes requiring permissive [Ca²⁺]_i. These findings indicate that an increase in [Ca²⁺]_i or extracellular Ca²⁺ is not required for RVD but Ca²⁺ has a permissive role in RVD of mouse cholangiocytes.     

"In vivo" monitoring of neuronal network activity in zebrafish by two-photon Ca(2+) imaging

The zebrafish larva is a powerful model for the analysis of behaviour and the underlying neuronal network activity during early stages of development. Here we employ a new approach of "in vivo" Ca²⁺ imaging in this preparation. We demonstrate that bolus injection of membrane-permeable Ca²⁺ indicator dyes into the spinal cord of zebrafish larvae results in rapid staining of essentially the entire spinal cord. Using two-photon imaging, we could monitor Ca²⁺ signals simultaneously from a large population of spinal neurons with single-cell resolution. To test the method, Ca²⁺ transients were produced by iontophoretic application of glutamate and, as observed for the first time in a living preparation, of GABA or glycine. Glycine-evoked Ca²⁺ transients were blocked by the application of strychnine. Sensory stimuli that trigger escape reflexes in mobile zebrafish evoked Ca²⁺ transients in distinct neurons of the spinal network. Moreover, long-term recordings revealed spontaneous Ca²⁺ transients in individual spinal neurons. Frequently, this activity occurred synchronously among many neurons in the network. In conclusion, the new approach permits a reliable analysis with single-cell resolution of the functional organisation of developing neuronal networks.