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J G Fitz

Publications and source records attributed to J G Fitz.

At least 37 records · Page 2Linked to original sources
PubMed1999

Evidence for Gd(3+) inhibition of membrane ATP permeability and purinergic signaling.

Extracellular ATP functions as an important autocrine and paracrine signal that modulates a broad range of cell and organ functions through activation of purinergic receptors in the plasma membrane. Because little is known of the cellular mechanisms involved in ATP release, the purpose of these studies was to evaluate the potential role of the lanthanide Gd(3+) as an inhibitor of ATP permeability and to assess the physiological implications of impaired purinergic signaling in liver cells. In rat hepatocytes and HTC hepatoma cells, increases in cell volume stimulate ATP release, and the localized increase in extracellular ATP increases membrane Cl(-) permeability and stimulates cell volume recovery through activation of P(2) receptors. In cells in culture, spontaneous ATP release, as measured by a luciferin-luciferase-based assay, was always detectable under control conditions, and extracellular ATP concentrations increased 2- to 14-fold after increases in cell volume. Gd(3+) (200 microM) inhibited volume-sensitive ATP release by >90% (P < 0.001), inhibited cell volume recovery from swelling (P < 0.01), and uncoupled cell volume from increases in membrane Cl(-) permeability (P < 0.01). Moreover, Gd(3+) had similar inhibitory effects on ATP release from other liver and epithelial cell models. Together, these findings support an important physiological role for constitutive release of ATP as a signal coordinating cell volume and membrane ion permeability and suggest that Gd(3+) might prove to be an effective inhibitor of ATP-permeable channels once they are identified.

Adenosine Triphosphate↗
PubMed1998

Phosphatidylinositol 3-kinase contributes to cell volume regulation through effects on ATP release.

Regulated changes in cell volume represent a signal that modulates a broad range of cell and organ functions. In HTC hepatoma cells, increases in volume are coupled to membrane ion permeability through a pathway involving (i) ATP efflux, (ii) autocrine stimulation of P2 receptors, and (iii) increases in anion permeability and Cl- efflux, contributing to recovery of volume toward basal values. Based on recent evidence that cell volume increases also stimulate phosphoinositide kinases, the purpose of these studies was to determine if phosphatidylinositol 3-kinase (PI 3-kinase) modulates these pathways. Exposure of cells to hypotonic buffer (20 or 40% less NaCl) caused an initial increase in cell volume and stimulated a rapid increase in ATP release. Subsequent opening of Cl- channels was followed by recovery of cell volume toward basal values, despite the continuous presence of hypotonic buffer. Inhibition of PI 3-kinase with wortmannin (Ki = 3 nM) significantly inhibited both the rate of volume recovery and activation of Cl- currents; similar results were obtained with LY294002 (10 microM). Additionally, current activation was inhibited by intracellular dialysis with antibodies specific for the 110-kDa catalytic subunit of PI 3-kinase. Since release of ATP is a critical element in the volume-regulatory pathway, the role of PI 3-kinase on volume-stimulated ATP release was assessed. Both wortmannin and LY294002 decreased basal and volume-stimulated ATP permeability but had no effect on the current response to exogenous ATP (10 microM). These findings indicate that PI 3-kinase plays a significant role in regulation of cell volume and suggest that the effects are mediated in part through modulation of cellular ATP release.

Adenosine Triphosphate↗
PubMed1998

Activation of protein kinase Calpha couples cell volume to membrane Cl- permeability in HTC hepatoma and Mz-ChA-1 cholangiocarcinoma cells.

Physiological increases in liver cell volume lead to an adaptive response that includes opening of membrane Cl- channels, which is critical for volume recovery. The purpose of these studies was to assess the potential role for protein kinase C (PKC) as a signal involved in cell volume homeostasis. Studies were performed in HTC rat hepatoma and Mz-ChA-1 human cholangiocarcinoma cells, which were used as model hepatocytes and cholangiocytes, respectively. In each cell type, cell volume increases were followed by: 1) translocation of PKC from cytosolic to particulate (membrane) fractions; 2) a 10- to 40-fold increase in whole-cell membrane Cl- current density; and 3) partial recovery of cell volume. In HTC cells, the volume-dependent Cl- current response (-46 +/- 5 pA/pF) was inhibited by down-regulation of PKC (100 nmol/L phorbol 12-myristate 13-acetate for 18 hours [PMA]; -1.97 +/- 1.5 pA/pF), chelation of cytosolic Ca2+ (2 mmol/L EGTA; -5.3 +/- 4.0 pA/pF), depletion of cytosolic adenosine triphosphate (ATP) (3 U/mL apyrase; -12.58 +/- 1. 45 pA/pF), and by the putative PKC inhibitor, chelerythrine (25 micromol/L; -7 +/- 3 pA/pF). In addition, PKC inhibition by chelerythrine and calphostin C (500 nmol/L) prevented cell volume recovery from swelling. Similar results were obtained in Mz-ChA-1 biliary cells. These findings indicate that swelling-induced activation of PKC represents an important signal coupling cell volume to membrane Cl- permeability in both hepatic and biliary cell models.

Alkaloids↗
PubMed1998

Stimulation of cyclic guanosine monophosphate production by natriuretic peptide in human biliary cells.

BACKGROUND & AIMS: Guanosine 3',5'-cyclic monophosphate (cGMP), whose production is stimulated by the interaction of nitric oxide, natriuretic peptides, and guanylin with their respective guanylate cyclases, activates secretion through ion channels in several epithelia. Cl- channels have been identified in the apical membrane of biliary epithelial cells. The aim of this study was to investigate the production of cGMP and its effects on Cl- permeability in biliary epithelial cells. METHODS: Halide efflux measurement, whole-cell patch clamp recording, radioimmunoassay, and reverse-transcription polymerase chain reaction using two human biliary cell lines (H69 and Mz-ChA-1) were performed. RESULTS: In cells equilibrated with 125I, bromo-cGMP stimulated halide efflux by 22%. In whole-cell patch clamp recordings, the addition of cGMP intracellularly, or of atrial natriuretic peptide extracellularly, stimulated inward currents at negative membrane potentials, consistent with Cl- efflux through open channels. In H69 cells, atrial and C-type natriuretic peptides stimulated production of cGMP. Mz-ChA-1 responded only to atrial natriuretic peptide. Both cell lines expressed messenger RNA for the guanylate cyclase type A receptor and the guanylate cyclase free-clearance receptor. CONCLUSIONS: These data suggest that natriuretic peptide stimulates cGMP production in human biliary epithelial cells, which in turn may regulate ductular bile formation through the opening of Cl- channels.

Atrial Natriuretic Factor↗
PubMed1998

Inhibition of Na+/H+ exchange stimulates CCK secretion in STC-1 cells.

It has been demonstrated that K+ channel regulation of membrane potential is critical for control of CCK secretion. Because certain K+ channels are pH sensitive, it was postulated that pH affects K+ channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na+/H+ exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na+/H+ exchange inhibited Na+-dependent H+ efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca2+ concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca2+ channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca2+. To determine whether the effects of EIPA and Na+ depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10-50 mM KCl in the bath. Maneuvers to lower intracellular pH (pHi), including reducing extracellular pH (pHo) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K+ permeability, we measured outward currents carried by K+, using whole cell patch techniques. K+ current was significantly inhibited by lowering pHo to 7.0. These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na+/H+ exchange and membrane potential may be functionally linked, where inhibition of Na+/H+ exchange lowers pHi and depolarizes the membrane, perhaps through inhibition of pH-sensitive K+ channels. In turn, K+ channel closure and membrane depolarization open voltage-dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ and CCK release. The effects of pHi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.

Amiloride↗
PubMed1997

Hepatocellular ATP-binding cassette protein expression enhances ATP release and autocrine regulation of cell volume.

In a model liver cell line, recovery from swelling is mediated by a sensitive autocrine pathway involving conductive release of ATP, P2 receptor stimulation, and opening of membrane Cl- channels (Wang, Y., Roman, R. M., Lidofsky, S. D., and Fitz, J. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12020-12025). However, the mechanisms coupling changes in cell volume to ATP release are not known. Based on evidence that certain ATP-binding cassette (ABC) proteins may function as ATP channels or channel regulators, we evaluated the potential role of ABC proteins by comparing ATP release and volume regulation in rat HTC and HTC-R hepatoma cells, the latter of which overexpress Mdr proteins. In both cell types, Cl- current activation (ICl-swell) and volume recovery following swelling were dependent on conductive ATP efflux. The rate of volume recovery was approximately 6-fold faster in HTC-R cells compared with HTC cells. This effect is likely due to enhanced ABC protein-dependent ATP release since (i) ICl-swell and cell volume recovery were eliminated by inhibition of P-glycoprotein transport (20 microM verapamil and 15 microM cyclosporin A); (ii) swelling-induced Cl- current density was similar in both cell types (approximately -50 pA/pF; not significant); and (iii) ATP conductance measured by whole-cell techniques was increased approximately 3-fold in HTC-R cells compared with HTC cells. Moreover, HTC-R cells exhibited enhanced survival during hypotonic stress. By modulating ATP release, hepatic ABC proteins may play a key role in the cellular pathways coupling changes in cell volume to ion permeability and secretion.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Cytosolic Ca2+ and protein kinase Calpha couple cellular metabolism to membrane K+ permeability in a human biliary cell line.

Cholangiocytes represent an important target of injury during the ischemia and metabolic stress that accompanies liver preservation. Since K+ efflux serves to minimize injury during ATP depletion in certain other cell types, the purpose of these studies was to evaluate the effects of ATP depletion on plasma membrane K+ permeability of Mz-ChA-1 cells, a model human biliary cell line. Cells were exposed to dinitrophenol (50 microM) and 2-deoxyglucose (10 mM) as the standard model of metabolic injury. Whole-cell and single K+ channel currents were measured using patch clamp techniques; and intracellular [Ca2+] ([Ca2+]i) was estimated by calcium green-1 fluorescence. Metabolic stress increased [Ca2+]i, and stimulated translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosolic to particulate cell fractions. The same maneuver increased membrane K+ permeability 40-70-fold as detected by (a) activation of K+selective whole cell currents of 2,176+/-218 pA (n = 34), and (b) opening of apamin-sensitive K+ channels with a unitary conductance of 17.0+/-0.2 pS. PKCalpha translocation and channel opening appear to be related since stress-induced K+ efflux is inhibited by chelation of cytosolic Ca2+, exposure to the PKC inhibitor chelerythrine (25 microM) and downregulation of PKC by phorbol esters. Moreover, K+ currents were activated by intracellular perfusion with recombinant PKCalpha in the absence of metabolic inhibitors. These findings indicate that in biliary cells apamin-sensitive K+ channels are functionally coupled to cell metabolism and suggest that cytosolic Ca2+ and PKCalpha are selectively involved in the response.

2,4-Dinitrophenol↗
PubMed1997

Regulation of cation-selective channels in liver cells.

In liver cells, cation-selective channels are permeable to Ca2+ and have been postulated to represent a pathway for receptor-mediated Ca2+ influx. This study examines the mechanisms involved in the regulation of these channels in a model liver cell line. Using patch-clamp recording techniques, it is shown that channel open probability is a saturable function of cytosolic [Ca2+], with half-maximal opening at 660 nm. By contrast, channel opening is not affected by membrane voltage or cytosolic pH. In intact cells, reduction of cytosolic [Cl-], a physiological response to Ca2+-mobilizing hormones and cell swelling, is also associated with an increase in channel opening. Finally, channel opening is inhibited by intracellular ATP through a mechanism that does not involve ATP hydrolysis. These findings suggest that opening of cation-selective channels is coupled to the metabolic state of the cell and provides a positive feedback mechanism for regulation of receptor-mediated Na+ and Ca2+ influx.

Animals↗
PubMed1997

Characterization of a swelling-activated anion conductance in homozygous typing cell hepatoma cells.

Liver cell volume and intracellular ion concentrations are maintained within a narrow physiologic range by regulated changes in membrane ion permeability. These studies of homozygous HTC hepatoma cells, a model liver cell line, evaluate the relationship between cell volume and membrane ion permeability, and assess the possibility that cell swelling allows the efflux of the intracellular osmolite taurine through the opening of a conductive pathway. Cell swelling induced by exposure to hypotonic solutions (203 mOsm) caused a rapid increase in cell volume, followed by recovery toward basal values. Volume recovery was inhibited by Cl- depletion or by exposure to the putative Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) (25 micromol/L). Swelling increased the efflux rates of 36Cl (181% +/- 15%, P < .01) and 125I (310% +/- 21%, P < .01). In whole cell patch clamp recordings, cell swelling induced by 1) exposure to hypotonic solution or 2) intracellular perfusion with hypertonic sucrose-containing solutions activated an anion-selective current which was outwardly rectified and showed time-dependent inactivation at depolarizing potentials. The current density at -80 mV increased proportionally with increases in the transmembrane osmotic gradient from basal values of -1 pA/pF to maximal values of 70 pA/pF with 100 mmol/L sucrose in the pipette. Basal taurine permeability was low, but cell swelling increased the efflux of [1,2-3H]taurine to 1,587% +/- 172% of basal levels (P < .05). Intracellular perfusion with hypertonic solutions activated currents carried by anionic taurine, with an estimated taurine/Cl- permeability ratio of .88 +/- .17 for whole cell currents. These studies demonstrate that the HTC membrane anion permeability is closely coupled to changes in cell volume, and that the recovery from swelling depends upon activation of anion-selective conductance pathways permeable to both Cl- and taurine.

Animals↗
PubMed1997

Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes.

To evaluate whether ATP in bile serves as a signaling factor regulating ductular secretion, voltage-clamp studies were performed using a novel normal rat cholangiocyte (NRC) model. In the presence of amiloride (100 microM) to block Na+ channels, exposure of the apical membrane to ATP significantly increased the short-circuit current (Isc) from 18.2 +/- 5.9 to 52.8 +/- 12.7 microA (n = 18). The response to ATP is mediated by basolateral-to-apical Cl- transport because it is inhibited by 1) the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (1 mM), diphenylanthranilic acid (1.5 mM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 microM) in the apical chamber, 2) the K+ channel blocker Ba2+ (5 mM), or 3) the Na(+)-K(+)-2Cl- cotransport inhibitor bumetanide (200 microM) in the basolateral chamber. Other nucleotides stimulated an increase in Isc with a rank order potency of UTP = ATP = adenosine 5'-O-(3)-thiotriphosphate, consistent with P2u purinergic receptors. ADP, AMP, 2-methylthioadenosine 5'-triphosphate, and adenosine had no effect. A cDNA encoding a rat P2u receptor (rP2uR) was isolated from a liver cDNA library, and functional expression of the corresponding mRNA in Xenopus laevis oocytes resulted in the appearance of ATP-stimulated currents with a similar pharmacological profile. Northern analysis identified hybridizing mRNA transcripts in NRC as well as other cell types in rat liver. These findings indicate that exposure of polarized cholangiocytes to ATP results in luminal Cl- secretion through activation of P2u receptors in the apical membrane. Release of ATP into bile may serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.

Adenosine Triphosphate↗
PubMed1996

Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation.

Recovery of cell volume in response to osmotic stress is mediated in part by increases in the Cl- permeability of the plasma membrane. These studies evaluate the hypothesis that ATP release and autocrine stimulation of purinergic (P2) receptors couple increases in cell volume to opening of Cl- channels. In HTC rat hepatoma cells, swelling induced by hypotonic exposure increased membrane Cl- current density to 44.8 +/- 7.1 pA/pF at -80 mV. Both the rate of volume recovery and the increase in Cl- permeability were inhibited in the presence of the ATP hydrolase apyrase (3 units/ml) or by exposure to the P2 receptor blockers suramin and Reactive Blue 2 (10-100 microM). Cell swelling also stimulated release of ATP. Hypotonic exposure increased the concentration of ATP in the effluent of perfused cells by 170 +/- 36 nM in the presence of a nucleotidase inhibitor (P < 0.01). In whole-cell recordings with ATP as the charge carrier, cell swelling increased membrane current density approximately 30-fold to 16.5 +/- 10.4 pA/pF. These findings indicate that increases in cell volume lead to efflux of ATP through opening of a conductive pathway consistent with a channel, and that extracellular ATP is required for recovery from swelling. ATP may function as an autocrine factor that couples increases in cell volume to opening of Cl- channels through stimulation of P2 receptors.

Adenosine Triphosphate↗
PubMed1996

Metabolic stress opens K+ channels in hepatoma cells through a Ca2+- and protein kinase calpha-dependent mechanism.

These studies of a model liver cell line evaluate the mechanisms responsible for regulated release of K+ ions during metabolic stress. Metabolic inhibition of HTC hepatoma cells by exposure to 2, 4-dinitrophenol (50 microM) and 2-deoxy-D-glucose (10 mM) stimulated outward currents carried by K+ of 974 +/- 75 pA at 0 mV (n = 20, p < 0.001). Currents were inhibited by chelation of intracellular Ca2+ or exposure to apamin (50 nM), an inhibitor of SKCa channels. In cell-attached recordings from intact cells, removal of metabolic substrates (25/28 cells) or exposure to metabolic inhibitors (32/40 cells) opened K+-selective channels with a conductance of 6.5 +/- 0. 2 pS. Channels had an open probability of 0.31 +/- 0.08 and opened in bursts averaging 3.55 +/- 0.27 ms in duration (n = 6). Metabolic stress was associated with rapid translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosol to membrane; and down-regulation of PKCalpha by phorbol esters or exposure to the PKC inhibitor chelerythrine (10 microM) each inhibited currents. Moreover, intracellular perfusion with purified PKCalpha activated currents in a Ca2+- and concentration-dependent manner. These findings indicate that metabolic stress leads to opening of apamin-sensitive SKCa channels in hepatoma cells through a Ca2+- and PKC-dependent mechanism and suggest that PKCalpha may be selectively involved in the response. This mechanism functionally couples the metabolic state of cells to membrane K+ permeability and represents a potential target for modification of liver injury associated with ischemia and preservation.

2,4-Dinitrophenol↗
PubMed1996

Hepatocellular transport: role of ATP-binding cassette proteins.

The interest of mammalian biologists in ATP-binding cassette (ABC) proteins is relatively recent. However, ABC proteins are widespread in distribution and have long been known to play an important transport role in prokaryotes. The review includes a brief overview of the structure, regulation, and varied functions of ABC proteins in different cell types as well as a synopsis of the emerging role of ABC proteins in human biology and disease. The review then focuses on the established (canalicular secretion of organic cations by the multidrug resistance, or MDR 1, gene product; ductular secretion of fluid and electrolytes mediated by CFTR), probable (biliary phospholipid secretion by the MDR 2 gene product; secretion of non-bile acid organic anions by the multidrug resistance protein, or MRP), and possible (bile acid secretion; biliary secretion of the signaling molecule, ATP; hormone transport) roles of known and novel ABC proteins in hepatobiliary secretion.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1996

Adenosine nucleotides in bile.

Activation of purinergic receptors by ATP stimulates Cl- efflux in biliary epithelial cells. To determine whether purinergic agonists are present under physiological conditions, we have assayed mammalian bile for nucleotides and assessed whether hepatoma and cholangiocarcinoma cell lines are capable of nucleotide release. Bile samples were collected from human, rat, and pig donors and assayed for nucleotide concentrations by luminometry. ATP, ADP, and AMP were present in bile from each species, and the average total nucleotide concentration in human bile was 5.21 +/- 0.91 microM (n = 16). In an in vitro model of HTC rat hepatoma cells or Mz-ChA-1 cholangiocarcinoma cells on a superfused column, nucleotides were present in the effluent from each cell type. Addition of alpha, beta-methyleneadenosine 5'-diphosphate (50 microM) to inhibit 5'-nucleotidase activity increased AMP concentrations two- to threefold. Exposure to forskolin (100 microM) or ionomycin (2 microM) stimulated nucleotide release from cholangiocarcinoma but not hepatoma cells. These studies indicate that adenosine nucleotides are present in bile in concentrations sufficient to activate purinergic receptors. Purinergic receptor activation by local nucleotide release might constitute an autocrine and/or paracrine mechanism for modulation of biliary secretion.

5'-Nucleotidase↗
PubMed1996

Regulation of cell volume in a human biliary cell line: activation of K+ and Cl- currents.

The mechanisms responsible for recovery from cell swelling were evaluated in Mz-ChA-1 cells from human cholangiocarcinoma, a model biliary cell line. Exposure to hypotonic buffer (40% less NaCl) rapidly increased relative cell volume to 1.35 +/- 0.10 as measured by a Coulter Multisizer, followed by regulatory volume decrease to 1.08 +/- 0.03 by 30 min. The same maneuver increased 86Rb (69 +/- 17%) and 125I (422 +/- 58%) efflux in cell monolayers. 86Rb efflux was selectively inhibited by Ba2+ [half-maximal inhibitory concentration (IC50) approximately 1.5 mM], and 125I by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) (IC60 approximately 50 microM). Inhibition of these conductive pathways partially inhibited recovery from swelling. Membrane conductance measured by whole cell patch-clamp analysis increased in 57 of 57 cells during swelling due to activation of both K+ and Cl- conductances in most cells. K+ currents (75% of cells, 881 +/- 150 pA at 0 mV) were nearly linear and Ba2+ sensitive; Cl- currents (70% of cells, 2,696 +/- 244 pA at +60 mV) were outwardly rectified, showed time-dependent inactivation at depolarizing potentials, and were inhibited by NPPB. Chelation of cytosolic Ca2+ decreased swelling-induced isotope efflux, prevented activation of macroscopic K+ and Cl- currents, and blocked volume recovery. These studies indicate that biliary cells are able to regulate cell volume during osmotic stress by activation of separate K+ and Cl- conductances through a mechanism that depends in part on Ca(2+)-sensitive signaling pathways.

Bile Ducts↗