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.

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.

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.

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.

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.

NIH3T3 cells transfected with the yeast H(+)-ATPase have altered rates of protein turnover.

NIH3T3 cells transfected with the yeast plasma membrane H(+)-ATPase (RN1a line) or transfected with a low-activity mutant H(+)-ATPase (N-Mut line) were used to examine the relationship between cytosolic pH (pHcyt) and protein turnover. At an extracellular pH (pHex) of 7.15, NIH3T3 and N-Mut cells have a pHcyt of 7-7.1 and a vacuolar pH (pHvac) of 6.3, whereas in RN1a cells both the pHcyt and the pHvac are 0.3 unit more alkaline. Rates of protein synthesis and degradation are optimum at pHex 7.2 and are much more sensitive to pH changes in RN1a cells than in NIH3T3 cells. However, irrespective of pH, rates of protein degradation in RN1a cells are always less than those measured in NIH3T3 cells. Rates of protein synthesis are the same for sparse cultures of RN1a and NIH3T3 cells and show a density-dependent decline in NIH3T3 cells but remain high in RN1a cells even at high cell densities. These data indicate that the elevation of pHcyt caused by transformation with the H(+)-ATPase has no direct effect on protein synthesis. On the other hand, rates of protein degradation are consistently lower in RN1a cells than in NIH3T3 or N-Mut cells. Basal rates of protein degradation, measured in medium containing 10 mM 3-methyladenine or 10% serum or 1 microM insulin, as well as the autophagic response to serum or insulin withdrawal, are both significantly lower in RN1a cells. These data indicate that transformation with the H(+)-ATPase has a direct effect on rates of protein degradation, possibly through an elevation of pH. The higher pHvac will directly effect lysosomal protein breakdown and the higher pHcyt may be permissive for maintenance of low basal rates of protein breakdown. Overall, we conclude that transformation with the H(+)-ATPase provides a permissive environment for high rates of protein synthesis and low rates of protein degradation that result in high rates of growth and the tumor phenotype.

Phenobarbital induces cytosolic acidification in an established liver epithelial cell line.

Phenobarbital (PB), a long-acting barbiturate, is also a known tumor promoter. The mechanism responsible for the promoting effect of PB has not yet been elucidated. Changes in intracellular pH (pHin) have been associated with both early and late events of multistage carcinogenesis. We conducted this study to evaluate whether PB alters pH homeostasis. Adult rat liver (ARL) epithelial cells were cultured for 48 h on glass coverslips, loaded with the intracellular pH indicator SNARF-1, and perfused with various concentrations of PB to evaluate its effect on pHin. Our results show that PB treatment of cultured cells induces a concentration-dependent cytosolic acidification. These results indicate that its ability to decrease pHin may be involved in the mechanism of tumor promotion by PB.

Differential regulation of Ca2+ homeostasis in ovine large and small luteal cells.

This study was undertaken to characterize differences in Ca2+ homeostasis between small and large ovine luteal cells. Although increasing extracellular pH (pHex) resulted in increases in intracellular calcium ([Ca2+]in) in both cell types, the large cells exhibited a greater sensitivity, suggesting that distinct [Ca2+]in regulatory mechanisms with distinct pH optima are operating in the two cell types. The sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitors thapsigargin (TG) and cyclopiazonic acid (CPA) increased [Ca2+]in in both cell types. Treatment of small cells with CPA resulted in transient increases in [Ca2+]in, whereas CPA produced sustained increases in [Ca2+]in in large cells. In small cells, pretreatment with CPA prevented further increases in [Ca2+]in in response to TG and vice versa. In large cells, TG pretreatment precluded further increases in [Ca2+]in with either prostaglandin F2 alpha (PGF2 alpha) or CPA. In contrast, after CPA pretreatment, PGF2 alpha or TG induced further increases in [Ca2+]in in large cells. This suggests that a TG-sensitive, CPA-insensitive Ca2+ pool is present in large cells but not in small cells. Neither Na+ removal nor KCl addition affected [Ca2+]in in either cell type, indicating that neither the Na+/Ca2+ exchanger nor voltage-dependent Ca2+ channels are involved in Ca2+ homeostasis in these cells. Addition of the calcium antagonist, LaCl3 (La3+), produced a gradual increase in [Ca2+]in in large cells but no changes in [Ca2+]in in small cells. Additionally, treatment with increasing concentrations of 4-bromo-A23187 resulted in titratable increases in [Ca2+]in that are greater in large than small cells, suggesting that small cells possess a higher Ca(2+)-buffering capacity than large cells. Progesterone secretion by large cells was significantly inhibited at alkaline pHex. In the presence of PGF2 alpha, progesterone secretion exhibited a distinct pH optimum of 7.0. In contrast, pHex did not affect secretion of progesterone in small cells under any of the conditions tested. TG, CPA, and La3+ all reduced secretion of progesterone in large, but not small, cells. These results demonstrate that ovine large and small luteal cells differ in regulation of [Ca2+]in homeostasis, and that treatments that increase [Ca2+]in decrease progesterone secretion in large cells but have no effect in small cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of glucose on pHin and [Ca2+]in in NIH-3T3 cells transfected with the yeast P-type H(+)-ATPase.

NIH-3T3 cells transfected with yeast H(+)-ATPases (RN1a cells) are tumorigenic (Perona and Serrano, 1988, Nature, 334:438). We have previously shown that RN1a cells maintain a chronically high intracellular pH (pHin) under physiological conditions. We have also shown that RN1a cells are serum-independent for growth, maintain a higher intracellular Ca2+ ([Ca2+]in), and glycolyze more rapidly than their non-transformed counterparts (Gillies et al., Proc. Natl. Acad. Sci., 1990, 87:7414; Gillies et al., Cell. Physiol. Biochem., 1992, 2:159). The present study was aimed to understand the interrelationships between glycolysis, pHin, and [Ca2+]in in RN1a cells and their non-transformed counterparts, NIH-3T3 cells. Our data show that the higher rate of glycolysis observed in RN1a cells is due to the presence of low affinity glucose transporters. Consequently, the higher rate of glycolysis is exacerbated at high glucose concentration in RN1a cells. Moreover, the maximal velocity (Vmax) for glucose utilization is up to sixfold higher in RN1a cells than in the NIH-3T3 cells, suggesting that the number of glucose transporters is higher in RN1a than NIH-3T3 cells. Glucose addition to NIH-3T3 cells results in modest decreases in both pHin and [Ca2+]in. In contrast, RN1a cells respond to glucose with a large decrease in pHin, followed by a large decrease in [Ca2+]in. The decrease in [Ca2+]in observed upon glucose addition is likely due to activation of Ca(2+)-ATPase by glycolysis, since the Ca2+ decrease is abolished by the Ca2+ ATPase inhibitors thapsigargin and cyclopiazonic acid. Glucose addition to ATP-depleted cells results in a decrease in [Ca2+]in, suggesting that ATP furnished by glycolysis is utilized by this pump.

Furazolidone increases thapsigargin-sensitive Ca(2+)-ATPase in chick cardiac myocytes.

Furazolidone is a nitrofuran antibiotic that causes dilated cardiomyopathy in turkeys and chicks and serves as an important model of human dilated cardiomyopathy. The mechanism by which furazolidone produces cardiac injury remains unknown. We investigated the hypothesis that furazolidone alters Ca2+ homeostasis in cardiac muscle cells. Myocytes harvested from 7-day-old chick embryos were treated with furazolidone (0.02, 0.1, and 1 mM) for 24-52 h and then coloaded with seminaphthorhodafluor-1 (SNARF 1) and fura 2 to measure simultaneously intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i), respectively. Furazolidone did not affect steady-state [Ca2+]i levels in cardiac myocytes. Na+ removal was associated with a rapid increase in [Ca2+]i due to the Na+/Ca2+ exchanger, which was similar in control and furazolidone-treated cells. The rate of [Ca2+]i recovery after Na+ removal was significantly increased in the furazolidone-treated cells compared with controls. In most cells, recovery from Ca2+ load is accomplished by the activity of Ca(2+)-adenosinetriphosphatases (ATPases). Thapsigargin, inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, prevented the furazolidone-induced changes. These results demonstrate that furazolidone increases the activity of thapsigargin-sensitive Ca(2+)-ATPase without affecting Na+/Ca2+ exchange. These data enhance our understanding of the mechanism of furazolidone-induced injury in cardiac myocytes and may be useful in determining mechanisms of injury in dilated cardiomyopathy.

NIH 3T3 cells transfected with a yeast H(+)-ATPase have altered sensitivity to insulin, insulin growth factor-I, and platelet-derived growth factor-AA.

The role of intracellular pH (pHin) in the regulation of cell growth in both normal and transformed cells is a topic of considerable controversy. In an effort to study this relationship NIH 3T3 cells were stably transfected with the gene for the yeast H(+)-ATPase, constitutively elevating their pHin. The resulting cell line, RN1a, has a transformed phenotype: The cells are serum independent for growth, clone in soft agar, and form tumors in nude mice. In the present study, we further characterize this system in order to understand how transfection with this proton pump leads to serum-independent growth, using defined media to investigate the effects of specific growth factors on the transfected and parental NIH 3T3 cells. While both cell lines show similar growth increases in response to platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF), they respond differently to insulin, insulin-like growth factor-I (IGF-I) and PDGF-AA. RN1a cells exhibit increased growth at nanomolar concentrations of insulin but the parental cells had only a relatively minor response to insulin at 10 microM. Both cell lines showed some response to IGF-I in the nanomolar range but the response of RN1a cells was much larger. Differences in insulin and IGF-I receptor number alone could not explain these results. The two cell lines also respond differently to PDGF-AA. RN1a cells are relatively insensitive to stimulation by PDGF-AA and express fewer PDGF alpha receptors as shown by Northern blots and receptor-binding studies. We propose a unifying hypothesis in which the H(+)-ATPase activates a downstream element in the PDGF-AA signal transduction pathway that complements insulin and IGF-I signals, while leading to downregulation of the PDGF alpha receptor.

Prostaglandin F2 alpha releases calcium from a thapsigargin-sensitive pool in ovine large luteal cells.

Thapsigargin (TG) and A23187 were used to examine the regulation of cytosolic free calcium (Cai2+) in ovine large and small luteal cells. Thapsigargin (50 nM) induced a sustained increase of Cai2+ in fura 2-acetoxymethyl ester (AM)-loaded cells (large = 1.32 +/- 0.07-fold, small = 1.45 +/- 0.07-fold, P < 0.05). A23187 (1 microM) induced a rapid transient increase of Cai2+ (large = 1.37 +/- 0.07-fold, small = 1.46 +/- 0.10-fold, P < 0.05). In large cells, 0.5 microM prostaglandin F2 alpha (PGF2 alpha) increased Cai2+ 1.54 +/- 0.11-fold. Pretreatment with 50 nM TG abolished the PGF2 alpha-induced calcium response. Pretreatment with PGF2 alpha attenuated (P < 0.05) the TG-induced Cai2+ increase. Progesterone secretion was significantly (P < 0.05) inhibited by incubation with 50 nM TG, 1 microM A23187, and 0.5 microM PGF2 alpha in large but not small cells. These data suggest that PGF2 alpha releases calcium from a TG-sensitive intracellular calcium pool in ovine large luteal cells.

Vacuolar-type H(+)-ATPases are functionally expressed in plasma membranes of human tumor cells.

Mammalian cells generally regulate their intracellular pH (pHi) via collaboration between Na(+)-H+ exchanger and HCO3- transport. In addition, a number of normal mammalian cells have been identified that express H(+)-adenosinetriphosphatases (ATPases) in their plasma membranes. Because tumor cells often maintain a high pHi, we hypothesized that they might functionally express H(+)-ATPases in their plasma membranes. In the first phase of the present study, we screened 19 normal and tumorigenic human cell lines for the presence of plasmalemmal H(+)-ATPase activity using bafilomycin A1 to inhibit V-type H(+)-ATPase and Sch-28080 to inhibit P-type H(+)-K(+)-ATPase. Bafilomycin A1 decreased pHi in the six tumor cell lines with the highest resting pHi in the absence of HCO3-. Sch-28080 did not affect pHi in any of the human cells. Simultaneous measurement of pH in the cytoplasm and in the endosomes/lysosomes localized the activity of bafilomycin to the plasma membrane in three cell lines. In the second phase of this study, these three cell lines were shown to recover from NH4(+)-induced acid loads in the absence of Na+. This recovery was inhibited by N-ethylmaleimide, bafilomycin A1, and ATP depletion and was not significantly affected by vanadate, Sch-28080, or hexamethyl amiloride. These results indicate that a vacuolar type H(+)-ATPase is expressed in the plasma membrane of some tumor cells.