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.     

Vibration of osteoblastic cells using a novel motion-control platform does not acutely alter cytosolic calcium,but desensitizes subsequent responses to extracellular ATP

Low-magnitude high-frequency mechanical vibration induces biological responses in many tissues. Like many cell types, osteoblasts respond rapidly to certain forms of mechanostimulation, such as fluid shear, with transient elevation in the concentration of cytosolic free calcium ([Ca²⁺]_i). However, it is not known whether vibration of osteoblastic cells also induces acute elevation in [Ca²⁺]_i. To address this question, we built a platform for vibrating live cells that is compatible with microscopy and microspectrofluorometry, enabling us to observe immediate responses of cells to low-magnitude high-frequency vibrations. The horizontal vibration system was mounted on an inverted microscope, and its mechanical performance was evaluated using optical tracking and accelerometry. The platform was driven by a sinusoidal signal at 20–500 Hz, producing peak accelerations from 0.1 to 1 g. Accelerometer-derived displacements matched those observed optically within 10%. We then used this system to investigate the effect of acceleration on [Ca²⁺]_i in rodent osteoblastic cells. Cells were loaded with fura-2, and [Ca²⁺]_i was monitored using microspectrofluorometry and fluorescence ratio imaging. No acute changes in [Ca²⁺]_i or cell morphology were detected in response to vibration over the range of frequencies and accelerations studied. However, vibration did attenuate Ca²⁺ transients generated subsequently by extracellular ATP, which activates P2 purinoceptors and has been implicated in mechanical signaling in bone. In summary, we developed and validated a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Vibration did not elicit acute elevation of [Ca²⁺]_i, but did desensitize responses to later stimulation with ATP.     

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.     

Histamine Induces ATP Release from Human Subcutaneous Fibroblasts,via Pannexin-1 Hemichannels,Leading to Ca2+ Mobilization and Cell Proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca²⁺ ([Ca²⁺]_i) and ATP release from human subcutaneous fibroblasts via H₁ receptor activation. Histamine-induced [Ca²⁺]_i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca²⁺]_i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with ¹⁰Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca²⁺]_i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H₁ receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y₁ receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y₁ receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca²⁺]_i mobilization and cell growth through the cooperation of H₁ and P2 (probably P2Y₁) receptors.     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

Effect of ATP/ADP/phosphate potential on the maximal steady-state uptake of Ca2+ by skeletal sarcoplasmic reticulum

The ability of the Ca²⁺-Mg²⁺ ATPase pump of skeletal SR to produce and maintain a Ca²⁺ gradient was studied as a function of the ATP/ADP/P_i ratio. The internal free Ca²⁺ concentration [Ca²⁺]_i was monitored by changes in fluorescence of CTC. Increasing ADP concentrations in the medium reduce the maximal [Ca²⁺]_i concentration achieved. The inclusion or the omission of 4×10^–4 M P_i or doubling the absolute ATP and ADP concentrations at a constant ATP/ADP ratio does not affect the level obtained. The level depends primarily on the ATP/ADP ratio. The [Ca²⁺] concentration shows a 1.5 power dependence on the ATP/ADP ratio. Further, [Ca²⁺]_i achieved at steady state does not depend on whether the pump had been working in the forward or the reverse direction prior to testing. Analysis shows that the levels of Ca²⁺ achieved are much lower than the levels predicted thermodynamically under the assumption of ideal coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2:1. Under this condition the osmotic energy of the [Ca²⁺]_i/[Ca²⁺]_o ratio was shown to be 48% as large as the free energy of hydrolysis of ATP, giving an overall thermodynamic efficiency of 48%. Analysis shows that maximal steady-state uptake is determined by the balance between the rates of uptake by the pump and rates of leak processes (intrinsic or extrinsic to the pump). Comparison with other studies shows that the [Ca²⁺]_i achieved results in trans-inhibition of the pump by tying up the Ca²⁺ translocator in the inwardly oriented phosphorylated form. The absence of an effect of P_i can be taken as evidence that the dissociation of Ca²⁺ from the inwardly oriented translocator on the phosphoylated enzyme must precede the dephosphorylation of the enzyme.     

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.     

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.     

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.     

Glucagon receptor mediates calcium signaling by coupling to Gαq/11 and Gαi/o in HEK293 cells

Glucagon induces intracellular Ca²⁺ ([Ca²⁺]_i) elevation by stimulating glucagon receptor (GCGR). Such [Ca²⁺]_i signaling plays important physiological roles, including glycogenolysis and glycolysis in liver cells and the survival of β-cells. Previous studies indicated that phospholipase C (PLC) might be involved in glucagon-mediated [Ca²⁺]_i response. Other studies also debated whether cAMP accumulation mediated by GCGR/Gα_s coupling contributes to [Ca²⁺]_i elevation. But the exact mechanisms remain uncertain. In the present study, we found that glucagon induces [Ca²⁺]_i elevation in HEK293 cells expressing GCGR. Removing extracellular Ca²⁺ did not affect glucagon-stimulated [Ca²⁺]_i response. But depleting the intracellular Ca²⁺ store by thapsigargin completely inhibited glucagon-induced [Ca²⁺]_i response. Experiments with forskolin and adenylyl cyclase inhibtor revealed that cAMP is not the cause of [Ca²⁺]_i response. Further studies with Gα_q/11 RNAi and pertussis toxin (PTX) indicated that both Gα_q/11 and Gα_i/o are involved. Combination of Gα_q/11 RNAi and Gα_i/o inhibition almost completely abolished glucagon-induced [Ca²⁺]_i signaling.     

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.     

Intracellular Free Calcium Dynamics in Stretch-Injured Astrocytes

Abstract: We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca²⁺]_i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura-2 microphotometry. Our results indicate that astrocyte [Ca²⁺]_i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury-induced [Ca²⁺]_i elevation is not sustained and returns to near-basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca²⁺]_i returns to normal after injury, we have identified persistent injury-induced alterations in calcium-mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate-sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor-mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate-linked metabotropic glutamate receptor agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid with an elevation in [Ca²⁺]_i. We hypothesize that changes in calcium-mediated signaling pathways, rather than an absolute elevation in [Ca²⁺]_i, is responsible for some of the pathological consequences of traumatic brain injury.     

Functional Expression of TRPM8 and TRPA1 Channels in Rat Odontoblasts

Odontoblasts produce dentin during development, throughout life, and in response to pathological conditions by sensing stimulation of exposed dentin. The functional properties and localization patterns of transient receptor potential (TRP) melastatin subfamily member 8 (TRPM8) and ankyrin subfamily member 1 (TRPA1) channels in odontoblasts remain to be clarified. We investigated the localization and the pharmacological, biophysical, and mechano-sensitive properties of TRPM8 and TRPA1 channels in rat odontoblasts. Menthol and icilin increased the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Icilin-, WS3-, or WS12-induced [Ca²⁺]_i increases were inhibited by capsazepine or 5-benzyloxytriptamine. The increase in [Ca²⁺]_i elicited by allyl isothiocyanate (AITC) was inhibited by HC030031. WS12 and AITC exerted a desensitizing effect on [Ca²⁺]_i increase. Low-temperature stimuli elicited [Ca²⁺]_i increases that are sensitive to both 5-benzyloxytriptamine and HC030031. Hypotonic stimulation-induced membrane stretch increased [Ca²⁺]_i; HC030031 but not 5-benzyloxytriptamine inhibited the effect. The results suggest that TRPM8 channels in rat odontoblasts play a role in detecting low-temperature stimulation of the dentin surface and that TRPA1 channels are involved in sensing membrane stretching and low-temperature stimulation. The results also indicate that odontoblasts act as mechanical and thermal receptor cells, detecting the stimulation of exposed dentin to drive multiple cellular functions, such as sensory transduction.     

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.     

α-Parvalbumin reduces depolarizationminduced elevations of cytosolic free calcium in human neuroblastoma cells

We investigated whether the expression of human α-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca²⁺]_i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM.Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K⁺. During this period, [Ca²⁺]_i was monitored by video microfluorimetry using the Ca²⁺ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca²⁺]_i The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca²⁺]_i than non-parvalbumin-expressing cells. Resting [Ca 2+], did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca²⁺]_i in neuroblastoma cells can be suppressed by parvalbumin.     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

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-.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.