Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca²⁺]_i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca²⁺]_i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca²⁺]_i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca²⁺]_i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca²⁺]_i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca²⁺]_i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca²⁺]_i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca²⁺]_i, creating [Ca²⁺]_i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca²⁺]_i uptake destroyed the polarized response of ENaC to [Ca²⁺]_i. Overall, our data suggest that ENaC is regulated by [Ca²⁺]_i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca²⁺]_i sequestration.     

Shear stress-induced Ca2+ mobilization in MDCK cells is ATP dependent,no matter the primary cilium

Primary cilium has emerged as mechanosensor to subtle flow variations in epithelial cells, but its role in shear stress detection remains controversial. To probe the function of this non-motile organelle in shear stress detection by cells, we compared calcium signalling responses induced by shear stress in ciliated and unciliated MDCK cells. Cytosolic free Ca²⁺ ([Ca²⁺]_i) was measured using Fura-PE3 video imaging fluorescence microscopy in response to shear stress due to laminar flow (385 μl s^?1). Our results show that both unciliated and ciliated MDCK cells are shear stress sensitive via ATP release and autocrine feedback through purinergic receptors. However, purinergic calcium signals differed in response intensity and receptor subtypes. In unciliated cells, shear stress-induced elevation in [Ca²⁺]_i was predominantly mediated through P2X receptors (P2XR). In contrast, calcium mobilization in ciliated MDCK cells resulted from P2YRs and store-operated Ca²⁺-permeable channels besides P2XRs. These findings lend support to the hypothesis that ATP release in response to shear stress is independent of the primary cilium and that transduction of mechanical strain into a specific biochemical responses stems on the mobilization of different sets of purinergic receptors.     

[Ca2+]i Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia

Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca²⁺]_i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca²⁺]_i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca²⁺]_i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y₂ receptor, HlyA readily triggered [Ca²⁺]_i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca²⁺]_i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y₂ receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y₂ receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y₂ receptors is the main mediator of HlyA-induced [Ca²⁺]_i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response.     

The effects of extracellular nucleotides on [Ca2+]i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

HKC‐8 cells are a human‐derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca²⁺]_i signalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca²⁺]_i in HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca²⁺]_i that was dependent on an intracellular source of Ca²⁺, while prolonged activation of P2Y receptors induced a rise in [Ca²⁺]_i that was dependent on intra‐ and extracellular sources of Ca²⁺. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca²⁺ entry in coupling to activation of P2Y in HKC‐8 cells. U73221, an inhibitor of PI‐PLC, did not inhibit the initial ATP‐induced response; whereas D609, an inhibitor of PC‐PLC, caused a significant decrease in the initial ATP‐induced response, suggesting that P2Y receptors are coupled to PC‐PLC. Although P2X were present in HKC‐8, The P2X agonist, α,β me‐ATP, failed to cause a rise in [Ca²⁺]_i. However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca²⁺]_i. Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca²⁺]_i elevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.     

Effect of Celecoxib on Ca2+ Fluxes and Proliferation in MDCK Renal Tubular Cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca^{2 +} concentration ([Ca^{2 +}]_i) and proliferation was examined by using the Ca^{2 +}-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca^{2 +}]_i in a concentration-dependent manner. Celecoxib-induced [Ca^{2 +}]_i increase was partly reduced by removal of extracellular Ca^{2 +}. Celecoxib-induced Ca^{2 +} influx was independently suggested by Mn^{2 +} influx-induced fura-2 fluorescence quench. In Ca^{2 +}-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca^{2 +}-ATPase, caused a monophasic [Ca^{2 +}]_i increase, after which celecoxib only induced a tiny [Ca^{2 +}]_iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca^{2 +}]_i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca^{2 +}]_i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca^{2 +}]_i increase in renal tubular cells by stimulating both extracellular Ca^{2 +} influx and intracellular Ca^{2 +} release and is highly toxic to renal tubular cells in vitro.     

Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells

Abstract— ATP-induced changes in the intracellular Ca²⁺concentration ([Ca²⁺]_i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca²⁺indicator fura-2, we have shown that the [Ca²⁺]_i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Ca^z+]_i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′-thiotriphosphate and 5′-adenylyl-β, γ-imidodiphosphate, could not trigger significant [Ca²⁺]_i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β-methylene-ATP, β,γ-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca²⁺]_i at 3 mM. In the absence of extracellular Ca²⁺, the effect of ATP was inhibited totally, but could be restored by the addition of Ca²⁺ to the cells. Upon removal of Mg²⁺, the maximum increase in [Ca²⁺]_i induced by ATP was enhanced by about 42%. Ca²⁺-channel blockers partially inhibited the ATP-induced [Ca²⁺]_i rise. The ATP-induced [Ca²⁺]_i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca²⁺]_i rise completely. No heterologous desensitization of [Ca²⁺]_i rise was observed between ATP and bradykinin. The magnitude of the [Ca²⁺]_i rise induced by ATP increased between 1.5 and 3.1 times when external Na⁺was replaced with Tris, N-methyl-d -glucamine, choline, or Li⁺. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca²⁺]_i to the basal level in Na⁺-containing or Na⁺-free Tris solution. Our results suggest that ATP stimulates Ca²⁺influx via at least two pathways: ion channels that are permeable to Ca²⁺ and Na⁺, and pores formed by ATP^4-.     

Calcium and the regulation of mammalian ciliary beating

Summary This report summarizes our recent work on the role of intracellular Ca²⁺ ([Ca²⁺]_i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca²⁺]_i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 M acetylcholine (ACh). CBF was recorded during the first and [Ca²⁺]_i during the second stimulation. ACh increased [Ca²⁺]_i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca²⁺]_i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 M thapsigargin, an inhibitor of the endoplasmic-reticulum Ca²⁺-ATPase, showed transient [Ca²⁺]_i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca²⁺]_i and CBF. Finally, changing extracellular Ca²⁺-concentrations induced corresponding changes in [Ca²⁺]_i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca²⁺]_i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca²⁺]_i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca²⁺]_i to ACh as well as they allowed direct assessment of the coupling between [Ca²⁺]_i and CBF. Simultaneous measurements revealed that [Ca²⁺]_i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca²⁺]_i by 1–3 ciliary beat cycles (ca. 150–450 ms).     

ATP-Induced Oscillations of Cytosolic Ca2+ Activity in Cultured Astrocytes from Rat Brain are Modulated by Medium Osmolarity Indicating a Control of [Ca2+]i Oscillations by Cell Volume

Oscillations of cytosolic Ca²⁺ activity ([Ca²⁺]_i) induced by stimulation with ATP in rat astrocytes in primary cultures were analysed. Astrocytes, prepared from the brains of newborn rats, loaded with the fluorescent Ca²⁺ indicator fura-2/AM, were continuously stimulated with ATP (10 M). ATP caused a large initial [Ca²⁺ peak, followed by regular [Ca²⁺]_i oscillations (frequencies 1–5/min). Astrocytes were identified by glial fibrillary acidic protein staining of cells after [Ca²⁺]_i recording. The oscillations were reversibly blocked by the P₂ purinoceptor antagonist suramin (30 M). Influx of extracellular Ca²⁺ and mobilization of Ca²⁺ from intracellular stores both contributed to the oscillations. The effects of hypertonic and hypotonic superfusion medium on ATP-induced [Ca²⁺]_i oscillations were examined. Hypertonic medium (430 mOsm) reversibly suppressed the ATP-induced oscillations. Hypotonic medium (250 mOsm), in spite of having heterogeneous effects, most frequently induced a rise in [Ca²⁺]_i, or reversibly increased the frequency of the oscillations. Thus, a change in cell volume might be closely connected with [Ca²⁺]_i oscillations in astrocytes indicating that [Ca²⁺]_i oscillations in glial cells play an important role in regulatory volume regulation in the brain.     

Changes in Intracellular Ca2+ and Energy Levels During In Vitro Ischemia in the Gerbil Hippocampal Slice

Abstract: The time course of the decline in energy levels during an in vitro ischemia-like condition was compared with changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod-2. During the on-line monitoring of [Ca²⁺]_i, the slices were perfused with an in vitro ischemia-like medium (33°C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region-specific and acute [Ca²⁺]_i elevations were observed in CA1 ～4 min after onset of the in vitro ischemia-like condition and also in the inner portion of the DG with a delay of 10–40 s. The change in ATP levels was related to the increase in [Ca²⁺]_i. ATP levels in all subregions gradually decreased before the acute [Ca²⁺]_i elevation. Concomitant with the acute [Ca²⁺]_i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high-energy-charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca²⁺]_i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region-specific increase in [Ca²⁺]_i, which activates a Ca²⁺-ATP-driven pump and produces a subsequent fall in neuronal ATP content.     

Negative Regulation of CFTR Activity by Extracellular ATP Involves P2Y2 Receptors in CFTR-expressing CHO Cells

Extracellular nucleotides exert autocrine/paracrine effects on ion transport by activating P2 receptors. We studied the effects of extracellular ATP and UTP on the cystic fibrosis transmembrane conductance regulator (CFTR) channel stably expressed in Chinese Hamster Ovary cells (CHO-BQ1 cells). CFTR activity was measured using the (¹²⁵I) iodide efflux technique and whole-cell patch-clamp recording in response to either forskolin or xanthine derivatives. Using RT-PCR and intracellular calcium concentration ([Ca²⁺]_i) measurement, we showed that CHO-BQ1 cells express P2Y2 but not P2Y4 receptors. While ATP and UTP induced similar increases in [Ca²⁺]_i, pre-addition by one of these two agonists desensitized the response for the other, suggesting that ATP- and UTP-induced [Ca²⁺]_i increases were mediated by a common receptor, which was identified as the P2Y2 subtype. CFTR activity was reduced by ATP and UTP but not by ADP or adenosine applications. This inhibitory effect of ATP on CFTR activity was not due to a change in cAMP level. Furthermore, CFTR activation by forskolin or IBMX failed to promote [Ca²⁺]_i increase, suggesting that CFTR activation did not generate an ATP release large enough to stimulate P2Y2 receptors. Taken together, our results show that endogenous P2Y2 receptor activation downregulates CFTR activity in a cAMP-independent manner in CHO cells. B. Marcet and V. Chappe contributed equally to this work.     

Platelet-Activating Factor Evokes Ca2+ Transients After the Blockade of Ryanodine Receptor by Dantrolene in RAW 264.7 Macrophages

In the present study we studied platelet-activating factor (PAF)-, and ATP-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) using RAW 264.7 macrophages filled with fura-2/AM and imaged with fluorescence video microscopy. We found that the prevalence of detectable [Ca²⁺]_i responses to PAF application was significantly higher in the presence of dantrolene. Dantrolene itself significantly decreased basal [Ca²⁺]_i of macrophages compared to control cases after a 20-min incubation period. In the dantrolene-treated cells even the peak [Ca²⁺]_i in response to PAF (as an average of all cells) was below the baseline of control suggesting that decreased [Ca²⁺]_i plays a permissive role in the Ca²⁺ rise induced by PAF in macrophages. In contrast to the effect of PAF, neither the amplitude of response to ATP nor the frequency of responding cells changed significantly during dantrolene treatment in our experiments. These cells were able to respond to a standard immune stimulus as well: lipopolysaccharide (LPS) was able to increase [Ca²⁺]_i. Our data indicate that the effectiveness of PAF to increase [Ca²⁺]_i in RAW 264.7 macrophages depends on the resting [Ca²⁺]_i. It has also been shown in this study that PAF and ATP differently regulate Ca²⁺ homeostasis in macrophages during inflammatory response and therefore they possibly differently modulate cytokine production by macrophages.     

Ca influx and ATP release mediated by mechanical stretch in human lung fibroblasts

One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca²⁺]_i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca²⁺]_i. The stretch-induced [Ca²⁺]_i elevation was attenuated in Ca²⁺-free solution. In contrast, the increase of [Ca²⁺]_i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd³⁺, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca²⁺]_i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca²⁺ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.     

Activation of protein kinase C inhibits ATP-induced [Ca2+]i elevation in rat osteoblastic cells: Selective effects on P2Y and P2U signaling pathways

Extracellular ATP elicits transient elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in osteoblasts through interaction with more than one subtype of cell surface P₂-purinoceptor. Elevation of [Ca²⁺]_i arises, at least in part, by release of Ca²⁺ from intracellular stores. In the present study, we investigated the possible roles of protein kinase C (PKC) in regulating these signaling pathways. [Ca²⁺]_i of indo-1-loaded UMR-106 osteoblastic cells was monitored by spectrofluorimetry. In the absence of extracellular Ca²⁺, ATP (100 μM) induced transient elevation of [Ca²⁺]_i to a peak 57 ± 7 nM above basal levels (31 ± 2 nM, means ± S. E. M., n = 25). Exposure of cells to the PKC activator 12-O-tetradecanoyl-β-phorbol 13-acetate (TPA, 100 nM) for 2 min significantly reduced the amplitude of the ATP response to 13 ± 4 nM (n = 11), without altering basal [Ca²⁺]_i. Inhibition was half-maximal at approximately 1 nM TPA. The Ca²⁺ response to ATP was also inhibited by the PKC activators 1,2-dioctanoyl-sn-glycerol or 4β-phorbol 12, 13-dibutyrate, but not by the control compounds 4α-phorbol or 4α-phorbol 12, 13-didecanoate. Furthermore, exposure of cells to the protein kinase inhibitors H-7 or staurosporine for 10 min significantly attenuated the inhibitory effect of TPA. However, these protein kinase inhibitors did not prolong the [Ca²⁺]_i response to ATP alone, indicating that activation of PKC does not account for the transient nature of this response. When the effects of other nucleotides were examined, TPA was found to cause significantly greater inhibition of the response to the P_2Y-receptor agonists, ADP and 2-methylthioATP, than the response to the P_2U-receptor agonist, UTP. These data indicate that activation of PKC selectively inhibits the P_2Y signaling pathway in osteoblastic cells. In vivo, endocrine or paracrine factors, acting through PKC, may regulate the responsiveness of osteoblasts to extracellular nucleotides. © 1995 Wiley-Liss, Inc.     

An Interaction Between ATP and High K+: Mutual Impairment of ATP- and High K+-Evoked [Ca2+]i Increase in NG 108–15 Cells

The interaction between ATP- and high K⁺-evoked increase in intracellular free calcium concentration ([Ca²⁺]_i) was investigated to gain an insight into the mechanism of interaction of ATP with voltage-sensitive calcium channels. [Ca²⁺]_i was measured in the neuronal model, neuroblastoma × glioma hybrid cells (NG 108–15), using the fluorescence indicator fura-2. In the presence of 1.8 mM extracellular Ca²⁺, ATP induced a rapid, concentration-dependent increase in [Ca²⁺]_i. High K⁺ (50 mM) evoked a [Ca²⁺]_i rise from 109 ± 11 nM to 387 ± 81 nM (n = 16). The application of either of these two [Ca²⁺]_i-increase provoking agents in sequence with the other caused impairment of the latter effect. The mutual desensitization of the responses to ATP and high K⁺ strongly suggests that both agents rely at least in part on the same source of Ca²⁺ for elevation of [Ca²⁺]_i in NG 108–15 cells.     

Mechanisms of calcium transport in the giant axon of the squid and their physiological role

The giant axon of the squid has been extensively used as a model for studying Ca regulation in excitable cells. Different techniques (extrusion, injection and dialysis) have been employed to characterize Ca fluxes across the axon membrane. Since both Ca efflux and influx are markedly dependent on [Ca²⁺]_i, considerable effort has been dedicated to determine the resting value of the [Ca²⁺]_i. Results from different laboratories indicate that the [Ca²⁺]_i, in a normal fibre, range from 20–100 nM. Under dialysis conditions (internal control), with an imposed [Ca²⁺]_i of 80 nM, Ca influx is balanced by an outward Ca movement of about 40 f/CS. Ca extrusion occurs through two parallel transport systems: one having a high affinity for [Ca²⁺]_i, dependent on ATP, not affected by Na_i, Na_o, Ca_o, Mg_o and inhibited by internal vanadate (uncoupled component), the other, more prominent at relatively high [Ca²⁺]_i, does not require ATP, is inhibited by Na_i activated by Na_o and not inhibited by vanadate. (Na_o-dependent component). The existence of these two systems provide the axon with an effective way to maintain in the long term a constant low [Ca²⁺]_i in spite of short term fluctuations due to increased Ca influx during nervous activity.     

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.     

Regulation of Intracellular Ca2+ by CFTR in Chinese Hamster Ovary Cells

In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl⁻ secretion. As recently proposed, beside its role of Cl⁻ channel, CFTR may regulate the activity of other channels such as a Ca²⁺-activated Cl⁻ channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers, that CFTR can act as a regulator of intracellular [Ca²⁺] _i ([Ca²⁺] _i ), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca²⁺] _i in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with an adenovirus-LacZ (CHO-LacZ). The transient [Ca²⁺] _i increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca²⁺] _i in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca²⁺] _i response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation by ATP released from the cell and regulation of [Ca²⁺] _i by CFTR in CHO epithelial cell membranes. Received: 5 April 1999/Revised: 28 June 1999     

Temperature dependency of calcium responses in mammary tumour cells

Changes of intracellular calcium concentration ([Ca²⁺]_i) induced by the extracellular application of ATP and bradykinin in mouse mammary tumour cells (MMT060562) were investigated by image analysis of fluo-3 fluorescence at 24°C and 35°C. ATP (0·1–100 μM ) and bradykinin (0·1 nM –1 μM ) induced the increase of [Ca²⁺]_i at both temperatures and Ca²⁺-depletion did not affect these [Ca²⁺]_i responses. Both [Ca²⁺]_i responses became more sensitive at 35°C than at 24°C. A clear latency of [Ca²⁺]_i increased after the application of the agonists was observed, and it changed with the concentration of the agonist. As concentrations of ATP or bradykinin became lower, the latency and rise time became longer. At higher concentrations, the latency and rise time approached a constant value. The latency shortened remarkably at 35°C. These results suggested the involvement of a regenerative or threshold process in the [Ca²⁺]_i responses in mammary tumour cells. © 1997 John Wiley & Sons, Ltd.     

Lysosomal exocytosis of ATP is coupled to P2Y2 receptor in marginal cells in the stria vascular in neonatal rats

Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague–Dawley rats were established. P2Y₂ receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Ca²⁺, whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y₂R-PLC-IP3 pathway to evoke Ca²⁺ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca²⁺]_I; this effect was partly inhibited by P2Y₂R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca²⁺]_i was significantly lower than that after treatment with P2Y₂R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca²⁺ triggers Ca²⁺ release from ER Ca²⁺ stores. Baseline [Ca²⁺]_i declined after treatment with the Ca²⁺ chelator 50 μM bis-(aminophenolxy) ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca²⁺ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca²⁺ triggers Ca²⁺ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca²⁺ signals indirectly via the P2Y₂R-PLC-IP3 pathway.