Effects of bradykinin on Ca2+ mobilization and prostaglandin E2 release in human periodontal ligament cells.

In fura-2-loaded human periodontal ligament (HPDL) cells, bradykinin induced a rapidly transient increase and subsequently sustained increase in cytosolic Ca²⁺ ([Ca²⁺]_i). When external Ca²⁺ was chelated by EGTA, the transient peak of [Ca²⁺]_i was reduced and the sustained level was abolished, implying the Ca²⁺ mobilization consists of intracellular Ca²⁺ release and Ca²⁺ influx. Thapsigargin, a specific Ca²⁺-ATPase inhibitor for inositol 1,4,5-trisphosphate (1,4,5-1P₃)-sensitive Ca²⁺ pool, induced an increase in [Ca²⁺]_i in the absence of external Ca²⁺. After depletion of the intracellular Ca²⁺ pool by thapsigargin, the increase in [Ca²⁺]_i induced by bradykinin was obviously reduced. Bradykinin also stimulated formation of inositol polyphosphates including 1,4,5-IP₃. These results suggest that bradykinin stimulates intracellular Ca²⁺ release from the 1,4,5-1P₃-sensitive Ca²⁺ pool. Bradykinin stimulated prostaglandin E₂ (PGE₂) release in the presence of external Ca²⁺, but not in the absence of external Ca²⁺. Ca²⁺ ionophore A23187 and thapsigargin evoked the release of PGE₂ in the presence of external Ca²⁺ despite no activation of bradykinin receptors. These results indicate that bradykinin induces Ca²⁺ mobilization via activation of phospholipase C and PGE₂ release caused by the Ca²⁺ influx in HPDL cells.     

Pleiotropic Effects of Bitter Taste Receptors on [Ca2+]i Mobilization,Hyperpolarization, and Relaxation of Human Airway Smooth Muscle Cells

Asthma is characterized by airway inflammation and airflow obstruction from human airway smooth muscle (HASM) constriction due to increased local bronchoconstrictive substances. We have recently found bitter taste receptors (TAS2Rs) on HASM, which increase [Ca²⁺]_i and relax the muscle. We report here that some, but not all, TAS2R agonists decrease [Ca²⁺]_i and relax HASM contracted by G-protein coupled receptors (GPCRs) that stimulate [Ca²⁺]_i. This suggests both a second pathway by which TAS2Rs relax, and, a heterogeneity of the response phenotype. We utilized eight TAS2R agonists and five procontractile GPCR agonists in cultured HASM cells. We find that heterogeneity in the inhibitory response hinges on which procontractile GPCR is activated. For example, chloroquine inhibits [Ca²⁺]_i increases from histamine, but failed to inhibit [Ca²⁺]_i increases from endothelin-1. Conversely, aristolochic acid inhibited [Ca²⁺]_i increases from endothelin-1 but not histamine. Other dichotomous responses were found when [Ca²⁺]_i was stimulated by bradykinin, angiotensin, and acetylcholine. There was no association between [Ca²⁺]_i inhibition and TAS2R subtype, nor whether [Ca²⁺]_i was increased by G_q- or G_i-coupled GPCRs. Selected studies revealed a correlation between [Ca²⁺]_i inhibition and HASM cell-membrane hyperpolarization. To demonstrate physiologic correlates, ferromagnetic beads were attached to HASM cells and cell stiffness measured by magnetic twisting cytometry. Consistent with the [Ca²⁺]_i inhibition results, chloroquine abolished the cell stiffening response (contraction) evoked by histamine but not by endothelin-1, while aristolochic acid inhibited cell stiffening from endothelin-1, but not from histamine. In studies using intact human bronchi, these same differential responses were found. Those TAS2R agonists that decreased [Ca²⁺]_i, promoted hyperpolarization, and decreased HASM stiffness, caused relaxation of human airways. Thus TAS2Rs relax HASM in two ways: a low-efficiency de novo [Ca²⁺]_i stimulation, and, a high-efficiency inhibition of GPCR-stimulated [Ca²⁺]_i. Furthermore, there is an interaction between TAS2Rs and some GPCRs that facilitates this [Ca²⁺]_i inhibition limb.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

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

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

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

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

Characterization of voltage operated R-type Ca2+ channels in modulating somatostatin receptor subtype 2- and 3-dependent inhibition of insulin secretion from INS-1 cells

Somatostatin (SST) inhibits Ca²⁺ entry into pancreatic B-cells via voltage-operated Ca²⁺ channels (VOCCs) of L-type, leading to the suppression of insulin secretion. Activation of R-type channels increases insulin secretion. However, the role of R-type Ca²⁺ channels (Ca_V2.3) in mediating the effects of SST on insulin secretion has not been so far investigated. Here, we identify the SST-receptor subtypes (SSTR) expressed on insulin-producing INS-1 cells by RT-PCR and by functional assays. The role of R-type channels in regulating [Ca²⁺]_i in response to SST-treatment was detected by cell fluorescence imaging and patch-clamp technique. INS-1 expressed SSTR2 and SSTR3 and agonists (ag.) selective for these receptors reduced 10 nM exendin-4/20 mM glucose-stimulated insulin secretion. Surprisingly, SST and SST2-ag. transiently increased [Ca²⁺]_i. Subsequently, these agonists led to a decrease in [Ca²⁺]_i below the basal levels. In contrast, SST3-ag. failed to induce a transient peak of [Ca²⁺]_i. Instead, a persistent minor suppression of [Ca²⁺]_i was detected from 25 min. R-type channel blocker SNX-482 altered [Ca²⁺]_i in SST- and SST2-ag.-treated cells. Notably, the inhibition of insulin secretion by SST and SST2-ag., but not SST3-ag. was attenuated by SNX-482. Taken together, SST and SSTR2 regulate [Ca²⁺]_i and insulin secretion in INS-1 cells via R-type channels. In contrast, the R-type calcium channel does not mediate the effects of SST3-ag. on insulin secretion. We conclude that R-type channels play a major role in the inhibition of insulin secretion by somatostatin in INS-1 cells.     

Regulatory Volume Decrease and Intracellular Ca2+ in Murine Neuroblastoma Cells Studied with Fluorescent Probes

The possible role of Ca²⁺ as a second messenger mediating regulatory volume decrease (RVD) in osmotically swollen cells was investigated in murine neural cell lines (N1E-115 and NG108-15) by means of novel microspectrofluorimetric techniques that allow simultaneous measurement of changes in cell water volume and [Ca²⁺]_i in single cells loaded with fura-2. [Ca²⁺]_i was measured ratiometrically, whereas the volume change was determined at the intracellular isosbestic wavelength (358 nm). Independent volume measurements were done using calcein, a fluorescent probe insensitive to intracellular ions. When challenged with ∼40% hyposmotic solutions, the cells expanded osmometrically and then underwent RVD. Concomitant with the volume response, there was a transient increase in [Ca²⁺]_i, whose onset preceded RVD. For hyposmotic solutions (up to ∼−40%), [Ca²⁺]_i increased steeply with the reciprocal of the external osmotic pressure and with the cell volume. Chelation of external and internal Ca²⁺, with EGTA and 1,2-bis-(o -aminophenoxy) ethane-N,N,N ′,N ′-tetraacetic acid (BAPTA), respectively, attenuated but did not prevent RVD. This Ca²⁺-independent RVD proceeded even when there was a concomitant decrease in [Ca²⁺]_i below resting levels. Similar results were obtained in cells loaded with calcein. For cells not treated with BAPTA, restoration of external Ca²⁺ during the relaxation of RVD elicited by Ca²⁺-free hyposmotic solutions produced an increase in [Ca²⁺]_i without affecting the rate or extent of the responses. RVD and the increase in [Ca²⁺]_i were blocked or attenuated upon the second of two ∼40% hyposmotic challenges applied at an interval of 30–60 min. The inactivation persisted in Ca²⁺-free solutions. Hence, our simultaneous measurements of intracellular Ca²⁺ and volume in single neuroblastoma cells directly demonstrate that an increase in intracellular Ca²⁺ is not necessary for triggering RVD or its inactivation. The attenuation of RVD after Ca²⁺ chelation could occur through secondary effects or could indicate that Ca²⁺ is required for optimal RVD responses.     

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

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

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

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

Caffeine-Induced Ca2+ Transients and Exocytosis in Paramecium Cells. A Correlated Ca2+ Imaging and Quenched-Flow/Freeze-Fracture Analysis

Caffeine causes a [Ca²⁺] _i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca²⁺]^rest _i ∼50 to 80 nm, [Ca²⁺]^act _i rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9–28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca²⁺ spread during the subsequent ≥2 sec. Chelation of Ca²⁺ _o considerably attenuated [Ca²⁺] _i increase. Therefore, caffeine may primarily mobilize cortical Ca²⁺ pools, superimposed by Ca²⁺ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca²⁺-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca²⁺] _i signals were reduced by [Ca²⁺] _o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca²⁺] _i signal was generated by caffeine stimulation (with Ca²⁺ _i and Ca²⁺ _o available). We conclude the following. (i) Caffeine can mobilize Ca²⁺ from cortical stores independent of the presence of Ca²⁺ _o . (ii) To yield adequate signals for normal exocytosis, Ca²⁺ release and Ca²⁺ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca²⁺] _i increase entails caffeine-mediated access of Ca²⁺ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca²⁺] _i -values for membrane fusion and contents discharge. Received: 23 May 1997/Revised: 18 August 1997     

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.     

Characteristics of cytosolic Ca2+ elevation induced by muscarinic receptor activation in single adrenal chromaffin cells of the guinea pig

In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca²⁺]_i) followed by a sustained rise. In Ca²⁺-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca²⁺]_i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca²⁺]_i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca²⁺]_i and inhibited the following [Ca²⁺]_i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca²⁺]_i. Muscarine-induced sustained [Ca²⁺]_i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca²⁺]_i rise and Mn²⁺ influx in response to muscarine were suppressed by a voltage-dependent Ca²⁺ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca²⁺ entry, but also Ca²⁺ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca²⁺ channels may function as one of the Ca²⁺ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.     

Effect of caffeine on mucus secretion and agonist-dependent Ca2+ mobilization in human gastric mucus secreting cells

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca²⁺]_i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca²⁺]_i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [³H]glucosamine. Caffeine by itself failed to increase [Ca²⁺]_i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca²+]_i rise, resulting in inhibiting activation of Ca²⁺-dependent K⁺ current (I_K.Ca) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca²⁺]_i rise and activation of I_K.Ca induced by A23187 or inositol trisphosphate (IP₃). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca²⁺]_i rise in human gastric epithelial cells, probably through the blockade of receptor-IP₃ signaling pathway, which may affect the mucin secretion. © 1997 Elsevier Science B.V. All rights reserved.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

The effects of metabolic inhibition on intracellular pH and Ca

We have investigated the effects of inhibiting aerobic and/or anaerobic metabolism on contraction, intracellular calcium and pH in single rat ventricular myocytes. Inhibition of aerobic metabolism alone (with CN) had little effect on these variables. However, if anaerobic glycolysis was also inhibited, then the application of CN decreased systolic [Ca³⁺]_i and increased diastolic [Ca²⁺]_i. There was also a development of a diastolic contracture which lagged behind the increase of diastolic [Ca²⁺]_i. These events were accompanied by an intracellular acidosis. The acidosis was shown to depress contraction and perhaps to account for the fact that diastolic [Ca²⁺]_i increased before the contracture.     

Intracellular Ca2+ Homeostasis in Trypomastigotes of Trypanosoma cruzi

ABSTRACT Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca²⁺ concentration([Ca²⁺]_i) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca²⁺]_o (preloaded cells) increased [Ca²⁺]_i < 20 nM whereas total cell Ca²⁺ increased by 1.5 to 2.0 pmole/cell. This amount of Ca²⁺ would be expected to increase [Ca²⁺]_i to > 10 μM suggesting active sequestration of Ca²⁺. We tested the hypothesis that maintenance of [Ca²⁺]_i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca²⁺]_i through well characterized pathways in higher eukaryotic cells were employed. [Ca²⁺], responses in the presence or absence of [Ca²⁺]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca²⁺]_i homeostasis. In the presence of 0.5 mM [Ca²⁺]_o, the ability of several agents to increase [Ca²⁺]_i was magnified in the order ionomycin ? nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca²⁺], observed only in response to monensin. Manoalide, an inhibitor of phospholipase A₂, enhanced the accumulation of [Ca²⁺]_i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca²⁺]_i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca²⁺ may involve phospholipase A₂ activity. A Na⁺/Ca²⁺ exchange mechanism did not appear to contribute to Ca²⁺ homeostasis.     

Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells

Ca²⁺ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca²⁺ concentration in cytosol ([Ca²⁺]_i) and nuclei ([Ca²⁺]_n). After calibration, [Ca²⁺]_n was constantly higher than [Ca²⁺]_i before and after the chelation of extracellular Ca²⁺ suggesting an active Ca²⁺ accumulation system on nuclear membrane. [Ca²⁺]_n was also constantly higher than [Ca²⁺]_i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1μM). The temporal change of [Ca²⁺]_n and [Ca²⁺]_i was identical, and [Ca²⁺]_i gradient towards the nucleus and peripheral or central [Ca²⁺]_n rise was observed after these stimulations. From these results, [Ca²⁺]_n is not only regulated by the active Ca²⁺ accumulation system on nuclear membrane at rest but also the generation of Inositol-triphosphate. FCS caused heterogeneous [Ca²⁺]_n or [Ca²⁺]_i rise from cell to cell; single spike or oscillatory change of [Ca²⁺]_n and [Ca²⁺]_i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca²⁺, suggesting that FCS stimulated [Ca²⁺]_n and [Ca²⁺]_i rise solely depending on Ca²⁺ influx from extracellular medium. The higher concentration of [Ca²⁺]_n and heterogeneous [Ca²⁺]_n rise may have important roles in nuclear-specific cellular responses. © 1996 Wiley-Liss, Inc.