Sodium calcium exchanger operates in the reverse mode in metastatic human melanoma cells.

Cytosolic Ca2+ ([Ca2+]cyt) is important in the regulation of several cellular functions involved in metastasis. We hypothesize that distinct [Ca2+]cyt regulation explains the acquisition of a more metastatic phenotype. To test this hypothesis, we used highly and lowly metastatic human melanoma cells and [Ca2+]cyt was monitored using Fura—2AM and fluorescence spectroscopy. Stimulation with ATP elicited a sustained increase in [Ca2+]cyt in highly metastatic cells, but a transient increase in lowly metastatic cells. Na+ substitution revealed Na+/Ca2+ exchanger (NCX) activity in reverse mode in highly, but not in lowly metastatic cells. In highly metastatic cells, addition of Na+ in the plateau phase of [Ca2+]cyt increase elicited with ATP, in the absence of Na+, resulted in a rapid return to basal, indicating that NCX can operate in both reverse and forward modes. Inhibition and knockdown of NCX, using KB—R7943 and siRNA NCX—1 respectively, supported the significance of NCX in [Ca2+]cyt regulation in highly metastatic cells. Stimulation with UTP triggered a rapid increase in highly metastatic cells [Ca2+]cyt, but not in lowly metastatic cells suggesting that highly and lowly metastatic cells exhibit distinct purinergic receptors. These data indicate that following agonist—stimulation, NCX operates preferentially in the reverse mode to enable a sustained [Ca2+]cyt increase in highly metastatic cells. The forward mode of NCX operation to extrude Ca2+ is preferred in lowly metastatic cells. The acquisition of a more metastatic phenotype involves a switch in NCX activity from forward to reverse mode that is favorable to maintain elevated [Ca2+]cyt in response to agonist stimulation.

Vacuolar H+-ATPase is down-regulated by the angiogenesis-inhibitory pigment epithelium-derived factor in metastatic prostate cancer cells.

The Vacuolar H+-ATPases (V-ATPases), a multi-subunits nanomotor present in all eukaryotic cells resides in the endomembranes of exocytotic and endocytotic pathways. Plasmalemmal V-ATPases have been shown to be involved in tumor cell metastasis. Pigment epithelium-derived factor (PEDF), a potent endogenous inhibitor of angiogenesis, is down-regulated in prostate cancer cells. We hypothesized that the transduction of PEDF in prostate cancer cells will down-regulate V-ATPase function; that in turn will decrease the expression of the V-ATPase accessory protein ATP6ap2 and a-subunit isoforms that target V-ATPase to the cell surface. To test these hypotheses, we used the human androgen-sensitive prostate cancer cells LNCaP, and its castration-refractory-derivative CL1 that were engineered to stably co-express the DsRed Express Fluorescent Protein with or without PEDF. To determine if PEDF down-regulates the function of V-ATPase, we measured the rate of proton fluxes (JH+) of the cytosolic and endosome/lysosome compartments. The mRNA levels for subunit-a isoforms and the ATP6ap2 were measured using quantitative reverse transcription-PCR. The results showed that PEDF expression decreased the rate of JH+ in metastatic CL1 cells without affecting JH+ in non-metastatic LNCaP cells, when studying pH(cyt). Interestingly, PEDF did not affect JH+ in endosomes/lysosomes either in metastatic cells or in non-metastatic cells. We also showed that PEDF significantly decreases the levels of a4 isoform and ATP6ap2 in metastatic CL1 cells, without affecting the levels of a4 isoform in the non-metastatic LNCaP cells. These data identify PEDF as a novel regulator of V-ATPase suggesting a new way by which PEDF may inhibit prostate tumor growth.

V-ATPase regulates communication between microvascular endothelial cells and metastatic cells.

To metastasize distant organs, tumor cells and endothelial cells lining the blood vessels must crosstalk. The nature of this communication that allows metastatic cells to intravasate and travel through the circulation and to extravasate to colonize different organs is poorly understood. In this study, we evaluated one of the first steps in this process­the proximity and physical interaction of endothelial and metastatic cells. To do this, we developed a cell separator chamber that allows endothelial and metastatic cells to grow side by side. We have shown in our previous studies that V-ATPases at the cell surface (pmV-ATPase) are involved in angiogenesis and metastasis. Therefore, we hypothesized that the physical proximity/interaction between endothelial and metastatic cells expressing pmV-ATPase will increase its activity in both cell types, and such activity in turn will increase pmV-ATPase expression on the membranes of both cell types. To determine pmV-ATPase activity we measured the proton fluxes (JH+) across the cell membrane. Our data indicated that interaction between endothelial and metastatic cells elicited a significant increase of JH+ via pmV-ATPase in both cell types. Bafilomycin, a V-ATPase inhibitor, significantly decrease JH+. In contrast, JH+ of the non-metastatic cells were not affected by the endothelial cells and vice-versa. Altogether, our data reveal that one of the early consequences of endothelial and metastatic cell interaction is an increase in pmV-ATPase that helps to acidify the extracellular medium and favors protease activity. These data emphasize the significance of the acidic tumor microenvironment enhancing a metastatic and invasive phenotype.

Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling.

The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.

Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells.

Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3--based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells.

Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency.

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH(4)), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH(4) levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH(4) levels with sepiapterin increased NO production, suggesting that BH(4) deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH(4). GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.

pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines.

A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.

pH and drug resistance. II. Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs.

Resistance to chemotherapeutic agents is a major cause of treatment failure in patients with cancer. The primary mechanism leading to a multidrug-resistant phenotype is assumed to be plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein (P-gp). However, acidic intracellular organelles can also participate in resistance to chemotherapeutic drugs. In this study, we investigated, both experimentally and theoretically, the effect of acidic vesicle turnover on drug resistance. We have developed a general model to account for multiple mechanisms of resistance to weakly basic organic cations, e.g. anthracyclines and Vinca alkaloids. The model predicts that lower cytosolic concentrations of drugs can be achieved through a combination of high endosomal turnover rates, a low endosomal pH, and an alkaline-inside pH gradient between cytosol and the extracellular fluid. Measured values for these parameters have been inserted into the model. Computations using conservative values of all parameters indicate that turnover of acidic vesicles can be an important contributor to the drug-resistant phenotype, especially if vesicles contain an active uptake system, such as H+/cation exchange. Even conservative estimates of organic cation-proton antiport activity would be sufficient to make endosomal drug extrusion a potent mechanism of resistance to weakly basic drugs. The effectiveness of such a drug export mechanism would be comparable to drug extrusion via drug pumps such as P-gp. Thus, turnover of acidic vesicles can be an important factor in chemoresistance, especially in cells that do not overexpress plasma membrane-bound drug pumps like P-glycoprotein.

Improved blood substitute: evaluation of its effects on human endothelial cells.

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

Interactions between benzylamiloride and fura-2: studies in vitro and in cardiac myocytes.

Amiloride derivatives are commonly used inhibitors of Na+/H+- and Na+/Ca2+-exchange. Because they are fluorescent molecules the use of benzylamiloride (BZA), an inhibitor of Na+/Ca2+ exchange, in conjunction with Fura-2, a commonly used fluorescent Ca2+ indicator, might complicate interpretation of fluorescence data obtained. In vitro data show that BZA decreases the Fura-2 fluorescence at all useful wavelengths in a concentration-dependent manner. The Fura-2 ratio 340/380 (used to estimate intracellular Ca2+ ([Ca2+]in)) also decreased with increasing BZA concentrations. The Stern-Volmer relation suggests that this phenomenon is due to either static or dynamic quenching. Varying temperatures from 4 to 37 degreesC did not alter Stern-Volmer constants, consistent instead with fluorescence resonance energy transfer (FRET). The in situ relevance of these interactions was evaluated in adult rat cardiac myocytes which exhibit Na+/Ca2+ exchange reflected by rapid [Ca2+]in increase following Na+ removal. Pretreatment with BZA >/= 25 microM decreased the magnitude of Fura-2 changes induced by Na+ removal. Analysis of the individual Fura-2 useful wavelengths indicated that >/= 25 microM BZA altered the Fura-2 signal in a manner consistent with the quenching effects noted in vitro. Together, these data show that BZA interacts with Fura-2 in vitro and in situ and suggest caution when interpreting Fura-2 fluorescence data derived in conjunction with BZA.

Distinct regulation of pHin and [Ca2+]in in human melanoma cells with different metastatic potential.

We investigated whether alterations in the mechanisms involved in intracellular pH (pHin) and intracellular calcium ([Ca2+]in) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pHin and [Ca2+]in simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steady-state pHin and [Ca2+]in between these cell types were not significantly different. Treatment of cells with NH4Cl resulted in larger pHin increases in highly than in poorly metastatic cells, suggesting that C8161 cells have a lower H+ buffering capacity than A375P. NH4Cl treatment also increased [Ca2+]in only in C8161 cells. To determine if the changes in [Ca2+]in triggered by NH4Cl treatment were due to alterations in either H+- or Ca2+-buffering capacity, cells were treated with the Ca2+-ionophore 4Br-A23187, to alter [Ca2+]in. The magnitude of the ionophore-induced [Ca2+]in increase was slightly greater in C8161 cells than in A375P. Moreover, A375P cells recover from the ionophore-induced [Ca2+]in load, whereas C8161 cells did not, suggesting that A375P may exhibit distinct [Ca2+]in regulatory mechanisms than C8161 cells, to recover from Ca2+ loads. Removal of extracellular Ca2+ ([Ca2+]ex) decreased [Ca2+]in in both cell types at the same extent. Ionophore treatment in the absence of [Ca2+]ex transiently increased [Ca2+]in in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca2+-ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady-state [Ca2+]in only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca2+ stores for [Ca2+]in homeostasis is greater in highly than in poorly metastatic cells. Bafilomycin treatment, to inhibit V-type H+-ATPases, corroborated our previous results that V-H+-ATPases are functionally expressed at the plasma membranes of highly metastatic, but not in poorly metastatic cells (Martínez-Zaguilán et al., 1993). Collectively, these data suggest that distinct pHin and [Ca2+]in regulatory mechanisms are present in poorly and highly metastatic human melanoma cells.

Mag-Fura-2 (Furaptra) exhibits both low (microM) and high (nM) affinity for Ca2+.

Based on studies using high-affinity Ca2+ probes (dissociation constant (Kd) = 0.15-0.3 microM), steady-state [Ca2+]in is believed to be in the nanomolar range in most cells. However, probes with lower affinity indicate that [Ca2+]in may increase to micromolar levels during activation of specific cell functions, e.g., contraction. These conclusions rely on accurate knowledge of the Kd of the dyes for Ca2+. Mag-Fura-2 (also known as Furaptra) is a low-affinity Ca2+ indicator (Kd ca. 50 microM) which has been used for such studies. In the present work, Mag-Fura-2 is shown to respond to changes in cytosolic Ca2+ in the submicromolar range. In vitro, and in situ titration of Mag-Fura-2 in A7r5 cells, demonstrate that Mag-Fura-2 exhibits both high- and low-affinity for Ca2+. Moreover, pH affects both high and low affinity Ca2+ binding site. Since Mag-Fura-2 has been used to study Ca2+ within specific subcellular compartments, the present observations indicate that knowledge of factors such as ambient pH of these compartments is required to accurately interpret Ca2+ responses. Furthermore, the sensitivity of Mag-Fura-2 at submicromolar levels must be considered for accurate determination of Ca2+ in specific compartments believed to exhibit high micromolar levels of Ca2+.

Effects of oxidants on properties of fluorescent calcium indicators.

An increasing number of studies use calcium-sensitive fluorescent dyes to address the relationship between elevated levels of intracellular calcium and free-radical-mediated damage in a variety of pathophysiological phenomena. The present study evaluates the effects of reactive oxygen species on the spectral properties of widely used calcium probes such as Fura-2 and Fluo-3. We found that both Fura-2 and Fluo-3 are rapidly inactivated by hydroxyl radicals and enzymatically inactivated by peroxidase/H2O2. This results in a decrease in the dynamic range of sensitivity of both dyes to Ca2+, as well as in a decrease in the affinity of Fluo-3 for Ca2+. The data suggest that oxidation of the calcium probes affects the measurement of calcium in vitro and may alter the interpretation of in vivo data since the absence of or small changes in the calcium fluorescence signal can be the result of probe deactivation by free oxygen radicals rather than the lack of actual Ca2+ changes.

Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy.

Described is a microscopic spectral imaging approach to monitor pH and Ca2+ simultaneously from combined spectra of multiple ion indicators. Emitted light from a cell is focused onto a grating spectrograph and spectra are imaged with a cooled charge-coupled device camera. The combined spectral output of fura 2 and SNARF-1 was analyzed to follow changes in intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) simultaneously and to correct the Ca2+ signal for concurrent changes in pHi. Responses of individual hamster insulinoma (HIT-T15) cells to effectors of ion homeostasis were heterogeneous. Treatment with NH4Cl increased pHi and transiently increased [Ca2+]i. Removal of NH4Cl induced cytosolic acidification concomitant with either no change or sustained increases in [Ca2-]i. Glucose treatment generally resulted in rapid and sustained increases in both [Ca2+]i and pHi but also heterogeneous pHi and [Ca2+]i responses. Corrections of the fura 2 signal for pH were important for following Ca2+ transitions elicited by NH4Cl but were less important for glucose-induced responses. The spectral imaging microscope provides a sensitive method for simultaneous measurements of pHi and [Ca2+]i in single cells.

Selection of fluorescent ion indicators for simultaneous measurements of pH and Ca2+.

The advent of fluorescent ion sensitive indicators has improved our understanding of the mechanisms involved in regulating pHi and [Ca2+]i homeostasis in living cells. However, changes in [Ca2+]i can alter pHi regulatory mechanisms and vice versa, making assignment of either ion to a particular physiological response complex. A further complication is that all fluorescent Ca2+ indicators are sensitive to protons. Therefore, techniques to simultaneously measure these two ions have been developed. Although several combinations of pH and Ca2+ probes have been used, few systematic studies have been performed to assess the validity of such measurements. In vitro analysis (i.e. free acid forms of dyes) indicated that significant quenching effects occurred when using specific dye combinations. Fura-2/SNARF-1 and MagFura-2/SNARF-1 probe combinations were found to provide the most accurate pH and [Ca2+] measurements relative to Fluo-3/SNARF-1, Ca2+-Green-1/SNARF-1, or BCECF/SNARF-1. Similar conclusions were reached when probes were calibrated after loading into cells. The magnitude of interactions between pH and Ca2+ probes could be a factor which may limit the use of certain specific combinations. Loading of probes that exhibit interactions into distinct intracellular compartments (i.e. separated by a biological membrane) abolished the quenching effects. These data indicate that interactions between the probes used to simultaneously monitor pH and Ca2+ must be considered whenever probe combinations are used.

Acidic pH enhances the invasive behavior of human melanoma cells.

As a consequence of poor perfusion and elevated acid production, the extracellular pH (pHex) of tumors is generally acidic. Despite this, most in vitro experiments are still performed at the relatively alkaline pHex of 7.4. This is significant, because slight changes in pHex can have profound effects on cell phenotype. In this study we examined the effects of mildly acidic conditions on the in vitro invasive potential of two human melanoma cell lines; the highly invasive C8161, and poorly invasive A375P. We observed that culturing of either cell line at acidic pH (6.8) caused dramatic increases in both migration and invasion, as measured with the Membrane Invasion Culture System (MICS). This was not due to a direct effect of pH on the invasive machinery, since cells cultured at normal pH (7.4) and tested at acidic pH did not exhibit increased invasive potential. Similarly, cells cultured at acidic pH were more aggressive than control cells when tested at the same medium pH. These data indicate that culturing of cells at mildly acidic pH induces them to become more invasive. Since acid pH will affect the intracellular pH (pHin) and intracellular calcium ([Ca2+]in), we examined the effect of these parameters on invasion. While changes in [Ca2+]in were not consistent with invasive potential, the changes in pHin were. While these conditions decrease the overall amount of gelatinases A and B secreted by these cells, there is a consistent and significant increase in the proportion of the activated form of gelatinase B.

Regulation of endoplasmic reticulum-Ca-ATPase by glycolysis in eukaryotic cells.

This paper reviews work by our and other laboratories that explores the coupling between glycolysis and endoplasmic reticulum (ER)-Ca-ATPases in regulating Ca2+ homeostasis in several cell types. Changes in intracellular Ca2+ [(Ca2+]in) link interaction between hormones and cell surface receptors with the initiation of specific cellular functions. Thus, changes in [Ca2+]in mediate signal transduction mechanisms that modulate many physiological functions including cell growth, muscle cell contractility, and exocytosis in secretory cells. In most eukaryotic cells, total cellular Ca2+ is in the millimolar range, yet only a fraction (i.e., nanomolar) is free in the cytosol. Cells use both active and 'passive' mechanisms to maintain [Ca2+]in within a narrow range. Active mechanisms include plasma membrane and endoplasmic/sarcoplasmic reticulum (ER/SR)-Ca-ATPases, Ca2+ channels (inositol trisphosphate- and voltage-sensitive), and Na+/Ca2+ exchangers. 'Passive' mechanisms include Ca(2+)-binding proteins (e.g., calsequestrin, calmodulin, calreticulin). The relative contribution of active and 'passive' mechanisms to [Ca2+]in homeostasis in a given cell is not known. Ca2+ might move among several intracellular compartments, including the ER/SR, mitochondria, nucleus, Golgi apparatus, endosomes and lysosomes. The ubiquitous distribution of ER-Ca-ATPases in these intracellular organelles suggests a major role of this pump in Ca2+ homeostasis, but the importance of intracellular compartments to [Ca2+]in homeostasis is not well understood. Glucose has been suggested to have a role in regulating some of these ion transport processes. Thus, the increased cell metabolism that follows glucose stimulation is associated with altered [Ca2+]in homeostasis. The precise mechanisms by which glucose or its metabolites modulate [Ca2+]in homeostasis are unknown but might involve regulation of ER-Ca-ATPases.

Modulation of intracellular Ca2+ by glucose in MDCK cells: role of endoplasmic reticulum Ca(2+)-ATPase.

Intracellular free calcium ([Ca2+]i) has multiple functional roles in renal epithelia, including mediating ligand- and volume-activated K+ and Cl- channels, modulating the permeability of apical membrane to Na+, and regulating tubuloglomerular feedback. We investigated glucose effects on intracellular pH (pHi) and [Ca2+]i in Madin-Darby canine kidney (MDCK) cells using fluorescent probes, SNARF-1 and fura 2, respectively. The addition of glucose decreased both pHi and [Ca2+]i in a dose-dependent fashion. Thapsigargin (TG) and cyclopiazonic acid (CPA), well-known endoplasmic reticulum (ER) Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) inhibitors, abolished the glucose-induced [Ca2+]i decrease. Without glucose, 1 microM TG induced a sustained elevation in [Ca2+]i, which increased further with glucose addition, whereas 15 microM CPA induced a transient increase in [Ca2+]i that was not affected by further addition of glucose. The sustained elevation in [Ca2+]i induced by TG was dependent on extracellular Ca2+. TG-induced [Ca2+]i increase was modulated by glucose, i.e., at higher glucose concentrations, TG induced a larger and more rapid rise in [Ca2+]i. We conclude that glucose has dual effects on [Ca2+]i regulation. Glucose alone reduces [Ca2+]i by activating ER-type Ca(2+)-ATPase, since this phenomenon is TG and CPA sensitive. In the presence of TG, glucose increases [Ca2+]i probably by increasing Ca2+ entry. Our data suggest a model in which TG activates capacitative Ca2+ entry by depletion of the ER Ca2+ pool. Glucose increases TG-induced [Ca2+]i elevation by further enhancing capacitative Ca2+ entry.