Macroporous thin membranes for cell transplant in regenerative medicine.

The aim of this paper is to present a method to produce macroporous thin membranes made of poly (ethyl acrylate-co-hydroxyethyl acrylate) copolymer network with varying cross-linking density for cell transplantation and prosthesis fabrication. The manufacture process is based on template techniques and anisotropic pore collapse. Pore collapse was produced by swelling the membrane in acetone and subsequently drying and changing the solvent by water to produce 100 microns thick porous membranes. These very thin membranes are porous enough to hold cells to be transplanted to the organism or to be colonized by ingrowth from neighboring tissues in the organism, and they present sufficient tearing stress to be sutured with surgical thread. The obtained pore morphology was observed by Scanning Electron Microscope, and confocal laser microscopy. Mechanical properties were characterized by stress-strain experiments in tension and tearing strength measurements. Morphology and mechanical properties were related to the different initial thickness of the scaffold and the cross-linking density of the polymer network. Seeding efficiency and proliferation of mesenchymal stem cells inside the pore structure were determined at 2 h, 1, 7, 14 and 21 days from seeding.

Evidence for multidrug resistance-1 P-glycoprotein-dependent regulation of cellular ATP permeability.

The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)1 family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release approximately 3-fold. The MDR1 inhibitors cyclosporine A (10 microm) and verapramil (10 microm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl- current density from -33.1 +/- 12.5 pA/pF to -2.0 +/- 0.3 pA/pF (-80 mV, p < or = 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd3+ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways.

ClC-2 chloride channels contribute to HTC cell volume homeostasis.

Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.

Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation.

These studies provide evidence that cystic fibrosis transmembrane conductance regulator (CFTR) potentiates and accelerates regulatory volume decrease (RVD) following hypotonic challenge by an autocrine mechanism involving ATP release and signaling. In wild-type CFTR-expressing cells, CFTR augments constitutive ATP release and enhances ATP release stimulated by hypotonic challenge. CFTR itself does not appear to conduct ATP. Instead, ATP is released by a separate channel, whose activity is potentiated by CFTR. Blockade of ATP release by ion channel blocking drugs, gadolinium chloride (Gd(3+)) and 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), attenuated the effects of CFTR on acceleration and potentiation of RVD. These results support a key role for extracellular ATP and autocrine and paracrine purinergic signaling in the regulation of membrane ion permeability and suggest that CFTR potentiates ATP release by stimulating a separate ATP channel to strengthen autocrine control of cell volume regulation.

Volume-sensitive purinergic signaling in human hepatocytes.

BACKGROUND/AIMS: Purinergic signaling potentially contributes to many liver functions. Therefore, the purpose of these studies was to characterize adenosine 5'-triphosphate (ATP) release from human hepatocytes, and to determine the role of extracellular ATP in the autocrine regulation of Cl- permeability and cell volume homeostasis. METHODS: Release of ATP (luciferase-luciferin assay), Cl- currents (whole-cell patch clamp), and cell volume (Coulter Multisizer) were measured in human hepatocytes within 12 h of isolation. RESULTS: Hepatocyte swelling increased bioluminescence from basal values of 11.21+/-0.45 to 178.29+/-44.49 and 492.15+/-89.41 arbitrary light units following 20 and 40% buffer dilutions, respectively (p<0.001), representing an increase in extracellular ATP from approximately 10 to >300 nM. Whole-cell Cl- currents activated during exposure to hypotonic buffer (15% less mosmol, 126.34+/-36.49 pA/pF) and ATP (10 microM, 71.92+/-15.48 pA/pF) exhibited outward rectification, time-dependent inactivation at depolarizing potentials, and sensitivity to the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Removal of extracellular ATP (apyrase) prevented volume-sensitive current activation. Exposure to hypotonic buffer (30% less mosmol) increased mean relative volume to 1.092+/-0.004 by 2.5 min, and volume recovery (1.019+/-0.002 by 30 min) was abolished by NPPB, apyrase, and the P2 receptor antagonist suramin. CONCLUSIONS: These findings indicate that human hepatocytes exhibit constitutive and volume-dependent ATP release, which is a critical determinant of membrane Cl- permeability and cell volume regulation. ATP release may represent an extracellular signaling pathway that couples the cellular hydration state to important hepatic functions.

Bivariate animal model estimates of genetic, phenotypic, and environmental correlations for production, reproduction, and somatic cells in Jerseys.

Correlations were obtained between 18 response variables of a Jersey herd (Florida Agricultural Experiment Station, Gainesville) for 374 first lactations. Estimates were from the use of multivariate, derivative-free, restricted maximum likelihood procedures with the simplex method of partial maximization. Estimates agreed closely with those obtained previously by other methods in this and other dairy populations. All correlations between yields were high and positive; those between yields and days from parturition to first service were negative and near zero. Correlations between yields and somatic cell scores were moderate and negative; those between yields and constituent percentages in general were negative, except for the yield and percentage of the same constituent. Genetic correlations between chloride content and somatic cells and between measures of somatic cells were 1.0. Results suggest that single-trait selection for milk yield should result in correlated increases in constituent yields with slight decreases in percentage composition of constituents and somatic cell counts.

Domain-specific purinergic signaling in polarized rat cholangiocytes.

In cholangiocytes, adenine nucleotides function as autocrine/paracrine signals that modulate ductular ion transport by activation of purinergic receptors. The purpose of these studies was to identify cellular signals that modulate ATP release and nucleotide processing in polarized normal rat cholangiocytes. In Ussing chamber studies, selective exposure of the apical and basolateral membranes to ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) stimulated increases in short-circuit current. Apical purinergic receptor agonist preference was consistent with the P2Y(2) subtype. In contrast, basolateral ADP was more potent in stimulating transepithelial currents, consistent with the expression of different basolateral P2 receptor(s). Luminometric analysis revealed that both membranes exhibited constitutive ATP efflux. Hypotonic exposure enhanced ATP release in both compartments, whereas decreases in ATP efflux during hypertonicity were more prominent at the apical membrane. Increases in intracellular cAMP, cGMP, and Ca(2+) also increased ATP permeability, but selective effects on apical and basolateral ATP release differed. Finally, the kinetics of ATP degradation in apical and basolateral compartments were distinct. These findings suggest that there are domain-specific signaling pathways that contribute to purinergic responses in polarized cholangiocytes.

Functional interactions between oxidative stress, membrane Na(+) permeability, and cell volume in rat hepatoma cells.

BACKGROUND & AIMS: Oxidative stress leads to a rapid initial loss of liver cell volume, but the adaptive mechanisms that serve to restore volume have not been defined. This study aimed to evaluate the functional interactions between oxidative stress, cell volume recovery, and membrane ion permeability. METHODS: In HTC rat hepatoma cells, oxidative stress was produced by exposure to H(2)O(2) or D-alanine plus D-amino acid oxidase (40 U/mL). RESULTS: Oxidative stress resulted in a rapid decrease in relative cell volume to 0.85 +/- 0.06. This was followed by an approximately 100-fold increase in membrane cation permeability and partial volume recovery to 0.97 +/- 0.05 of original values. The volume-sensitive conductance was permeable to Na(+) approximately K(+) >> Tris(+), and whole-cell current density at -80 mV increased from -1.2 pA/pF at 10(-5) mol/L H(2)O(2) to -95.1 pA/pF at 10(-2) mol/L H(2)O(2). The effects of H(2)O(2) were completely inhibited by dialysis of the cell interior with reduced glutathione, and were markedly enhanced by inhibition of glutathione synthase. CONCLUSIONS: These findings support the presence of dynamic functional interactions between cell volume, oxidative stress, and membrane Na(+) permeability. Stress-induced Na(+) influx may represent a beneficial adaptive response that partially restores cell volume over short periods, but sustained cation influx could contribute to the increase in intracellular [Na(+)] and [Ca(2+)] associated with cell injury and necrosis.

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.

The lipid products of phosphoinositide 3-kinase contribute to regulation of cholangiocyte ATP and chloride transport.

ATP stimulates Cl(-) secretion and bile formation by activation of purinergic receptors in the apical membrane of cholangiocytes. The purpose of these studies was to determine the cellular origin of biliary ATP and to assess the regulatory pathways involved in its release. In Mz-Cha-1 human cholangiocarcinoma cells, increases in cell volume were followed by increases in phophoinositide (PI) 3-kinase activity, ATP release, and membrane Cl(-) permeability. PI 3-kinase signaling appears to play a regulatory role because ATP release was inhibited by wortmannin or LY294002 and because volume-sensitive current activation was inhibited by intracellular dialysis with antibodies to the 110 kDa-subunit of PI 3-kinase. Similarly, in intact normal rat cholangiocyte monolayers, increases in cell volume stimulated luminal Cl(-) secretion through a wortmannin-sensitive pathway. To assess the role of PI 3-kinase more directly, cells were dialyzed with the synthetic lipid products of PI 3-kinase. Intracellular delivery of phosphatidylinositol 3, 4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate activated Cl(-) currents analogous to those observed following cell swelling. Taken together, these findings indicate that volume-sensitive activation of PI 3-kinase and the generation of lipid messengers modulate cholangiocyte ATP release, Cl(-) secretion, and, hence, bile formation.

Endogenous ATP release regulates Cl- secretion in cultured human and rat biliary epithelial cells.

P2Y receptor stimulation increases membrane Cl- permeability in biliary epithelial cells, but the source of extracellular nucleotides and physiological relevance of purinergic signaling to biliary secretion are unknown. Our objectives were to determine whether biliary cells release ATP under physiological conditions and whether extracellular ATP contributes to cell volume regulation and transepithelial secretion. With the use of a sensitive bioluminescence assay, constitutive ATP release was detected from human Mz-ChA-1 cholangiocarcinoma cells and polarized normal rat cholangiocyte monolayers. ATP release increased rapidly during cell swelling induced by hypotonic exposure. In Mz-ChA-1 cells, removal of extracellular ATP (apyrase) and P2 receptor blockade (suramin) reversibly inhibited whole cell Cl- current activation and prevented cell volume recovery during hypotonic stress. Moreover, exposure to apyrase induced cell swelling under isotonic conditions. In intact normal rat cholangiocyte monolayers, hypotonic perfusion activated apical Cl- currents, which were inhibited by addition of apyrase and suramin to bathing media. These findings indicate that modulation of ATP release by the cellular hydration state represents a potential signal coordinating cell volume with membrane Cl- permeability and transepithelial Cl- secretion.

Genetic trends for milk yield of Jerseys and correlated changes in productive and reproductive performance.

Estimates of genetic trends in 24 measures of milk and constituent yields, somatic cell counts, and reproduction were obtained from 935 records of 374 Jerseys in a single herd. Data were obtained from a designed project for single-trait selection from 1969 through 1987. One line was subjected to selection solely for milk yield and included 259 cows; an unselected control line included 115 cows. Estimates of trends were based on differences in linear phenotypic trends between lines for first lactations, all lactations, and for 305-d and total records. The genetic changes in milk yield for these four data sets were 1.22 to 1.48%/yr (36.8 to 41.0 kg per cow yr) and 0.54 to 1.64%/yr for five constituent yields. Except for the percentages of minerals plus lactose, all constituent percentages decreased by 0.05 to 0.60%/yr. The ratios of protein to fat and solids-not-fat to fat increased 0.30 to 0.54%/yr, respectively. The number of services required per conception increased (0.17%) in first parity records and in all data (0.69%). The intervals from parturition to first estrus and from parturition to first service decreased in first lactation (1.19 and 0.82%) annually but increased (1.25 and 0.01%) in all data. Age of heifers at first estrus decreased by 0.44% annually. Most of the five measures of somatic cells decreased in first lactations but increased for all data. Estimates of realized genetic correlations of 14 measures of constituent yield and composition (four correlations each) agreed well with values expected from the literature. The results quantified change in milk yield, constituent yields and percentages, reproductive performance, and somatic cell counts in a single herd and should prove useful in the development of selection programs for dairy cattle.

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.

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.

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.

Alanine uptake activates hepatocellular chloride channels.

Cells involved in the retrieval and metabolic conversion of amino acids undergo significant increases in size in response to amino acid uptake. The resultant adaptive responses to cell swelling are thought to include increases in membrane K+ and Cl- permeability through activation of volume-sensitive ion channels. This viewpoint is largely based on experimental models of hypotonic swelling, but few mammalian cells experience hypotonic challenge in vivo. Here we have examined volume regulatory responses in a physiological model of cell-swelling alanine uptake in immortalized hepatocytes. Alanine-induced cell swelling was followed by a decrease in cell volume that was temporally associated with an increase in membrane Cl- currents. These currents were dependent both on alanine concentration and Na+, suggesting that currents were stimulated by Na+-coupled alanine uptake. Cl- currents were outwardly rectifying, exhibited an anion permeability sequence of I- > Br- > Cl-, and were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid, features similar to those reported for a widely distributed class of volume-sensitive anion channels evoked by experimental hypotonic stress. These findings suggest that volume-sensitive anion channels participate in adaptive responses to amino acid uptake and provide such channels with a new physiological context.

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.