Guinea Pig Hepatocytes Research Articles

Activation of the plasma membrane chloride channel by protein kinase C in isolated guinea‐pig hepatocytes.

1. To assess the nature of the underlying mechanism of noradrenaline-induced increase of Cl- conductances in hepatocytes, macroscopic and unitary currents through noradrenaline-induced Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, cell-attached and excised inside-out configurations of the patch-clamp technique. 2. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 0.1 microM, bath application of noradrenaline activated the time-independent membrane currents under whole-cell voltage-clamp conditions. The current was similarly activated by phorbol ester (PMA), an activator of protein kinase C (PKC), while a specific protein kinase C inhibitor, H-9, reversed PMA activation of the current. The inactive phorbol ester, 4 alpha-phorbol 12-myristate, 13-acetate (alpha PMA), failed to activate the channel. 3. The reversal potential of the PMA-activated current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration, indicating that the current was Cl- selective. Bath application of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) partially inhibited both the noradrenaline- and PMA-induced currents. 4. In single channel recordings from cell-attached patches, bath application of noradrenaline or PMA induced unitary current activity, the averaged slope conductance of which was 10.1 +/- 1.5 pS (mean +/- S.D.; n = 12) in the noradrenaline-induced current and 9.7 +/- 1.3 pS (n = 7) in the PMA-induced current. The open time distribution was moderately well fitted by a single exponential function with mean open lifetime of 88.5 +/- 10.6 ms (n = 10), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 24.4 +/- 5.8 ms (n = 10) and for the slow component of 316.9 +/- 49.2 ms (n = 10). 5. Bath application of purified PKC to excised inside-out patches activated the channel. The PKC selective inhibitor, PKC(19-36), and DIDS inhibited the PKC-activated channel. 6. These results suggest that PKC can phosphorylate the channel protein or a related structure leading to the activation of Cl- channels in guinea-pig hepatocytes.

P-410 Direct activation of the chloride channel in guinea-pig hepatocytes by protein kinase C

P-410 Direct activation of the chloride channel in guinea-pig hepatocytes by protein kinase C

Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes

The influence of 1-D-myo-inositol 1,4,5-trisphosphate (InsP3) breakdown by InsP3 5-phosphatase in determining the time course of Ca2+ release from intracellular stores was investigated with flash photolytic release of a stable InsP3 derivative, 5-thio-InsP3, from a photolabile caged precursor. The potency and Ca(2+)-releasing properties of the biologically active D isomers of 5-thio-InsP3 and InsP3 itself were compared by photolytic release in guinea pig hepatocytes. After a light flash, cytosolic free calcium concentration ([Ca2+]i) showed an initial delay before rising quickly to a peak and declining more slowly to resting levels, with time course and amplitude generally similar to those seen with photolytic release of InsP3. Differences were a three- to eightfold lower potency of 5-thio-InsP3 in producing Ca2+ release, much longer delays between photolytic release and Ca2+ efflux with low concentrations of 5-thio-InsP3 than with InsP3, and persistent reactivation of Ca2+ release, producing periodic fluctuations of cytosolic [Ca2+]i with high concentrations of 5-thio-InsP3 but not InsP3 itself. The lower potency of 5-thio-InsP3 may be a result of a lower affinity for closed receptor/channels or a lower open probability of liganded receptor/channels. The longer delays with 5-thio-InsP3 at low concentration suggest that metabolism of InsP3 by 5-phosphatase may reduce the concentration sufficiently to prevent receptor activation and may have a similar effect on InsP3 concentration during hormonal activation. The maximal rate of rise of [Ca2+]i, the duration of the period of high Ca2+ efflux, and the initial decline of [Ca2+]i are similar with5-thio-lnsP3 and lnsP3, indicating that lnsP3 breakdown is not important in terminating Ca2+ release. The second activation ofInsP3 receptors with 5-thio-lnsP3 and particularly the sustained periodic fluctuations of [Ca2+]i indicate persistence of 5-thio-lnsP3,suggesting that InsP3 breakdown prevents reactivation of InsP3 receptors. The photochemical properties of 1-(2-nitrophenyl)-ethyl caged 5-thio-lnsP3 are photolytic quantum yield = 0.57 (cf. 0.65 for caged InsP3) and rate of photolysis = 87 s-I (half-life approximately 8 ms; cf. 3 ms for caged lnsP3; pH7.1; ionic strength, 0.2 M; 21 OC). Caged 5-thio-lnsP3 at concentrations up to 360 pM did not activate lnsP3 receptors to produce Ca2+ release or block Ca2+ release by free 5-thio-lnsP3.

Species Differences in the Clonal Expansion of Hepatocytes in Response to the Coaction of Epidermal Growth Factor and Nafenopin, a Rodent Hepatocarcinogenic Peroxisome Proliferator

Species Differences in the Clonal Expansion of Hepatocytes in Response to the Coaction of Epidermal Growth Factor and Nafenopin, a Rodent Hepatocarcinogenic Peroxisome Proliferator. James, N.H. and Roberts, R. A. (1995). Fundam. Appl. Toxicol. 26, 143-149.Peroxisome proliferators are members of the nongenotoxic family of rodent hepatocarcinogens. There exist substantial species differences in response to peroxisome proliferators among mammalian species. We have reported previously that peroxisome proliferators can synergize with epidermal growth factor (EGF) to promote the clonal expansion of rat hepatocytes associated with the early stages of hepatocarcinogenesis. The aim of the present study was to determine whether responsiveness in this in vitro assay reflected the known species differences in response to peroxisome proliferators. The process of tumorigenicity was modeled in the soft agar cloning assay since growth in soft agar is thought to reflect the early stages of tumorigenesis. This is because clonal expansion under these conditions requires the cells to survive, to undergo mitosis, and to escape from the contact-dependent growth associated with normal cell behavior. The data presented here show that mouse hepatocytes are able to undergo clonal expansion in soft agar in response to nafenopin and EGF giving a three to fourfold increase in colony numbers over control. This result is comparable to the fivefold increase in rat hepatocyte colony numbers that we have reported previously. In contrast, hamster, guinea pig, and human hepatocytes did not respond to the concerted action of EGF and nafenopin despite their ability to respond to EGF as a mitogen in monolayer culture. These data demonstrate that the clonal expansion of rodent hepatocytes in soft agar in response to peroxisome proliferators and EGF displays the same species differences as other pleiotropic responses to these compounds and is likely therefore to be relevant to the process of hepatocarcinogenesis.

Fenofibrate: Metabolism and Species Differences for Peroxisome Proliferation in Cultured Hepatocytes

Fenofibrate: Metabolism and Species Differences for Peroxisome Proliferation in Cultured Hepatocytes. Cornu-Chagnon, M.-C., Dupont, H., and Edgar, A. (1995). Fundam. Appl. Toxicol. 26, 63-74.The hypolipidemic agent fenofibrate, which is a peroxisome proliferator in some rodents in vivo, was studied in cultured hepatocytes for its metabolism and effects on enzymatic induction related to peroxisome proliferation so as to lead to a better understanding of the mechanisms involved in peroxisome proliferation. [14C]-Fenofibrate was completely metabolized within 24 hr by primary cultures of rat hepatocytes and the metabolic pattern corresponded to that found in vivo. The main products were fenofibric acid and its glucuronidated form. Carbonyl reduction of fenofibric acid also occurred. The metabolic pattern of [14C]fenofibric acid was nearly the same as that of fenofibrate. Fenofibrate, fenofibric acid, and its reduced metabolite all induced peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation activity (PCOA) in rat hepatocytes, whereas derivatives lacking the carboxyl group were nearly inactive. The known species differences with respect to sensitivity to peroxisome proliferators in vivo was mirrored in cultured cells because fenofibric acid did not induce peroxisomal PCOA in primary culture of guinea pig hepatocytes nor in the human hepatoma cell line HepG2. The mechanistic association between the induction of CYP4A1-catalyzed lauric acid ω-hydroxylase (LAH) activity and peroxisomal PCOA induction was investigated. Fenofibric acid concomitantly induced LAH activity and peroxisomal PCOA in rat hepatocytes. Specific inhibition of LAH activity (-52%) by 10-undecynoic acid partially prevented induction of peroxisomal PCOA (-32%). The putative role of dicarboxylic acids, the oxidation product of ω-hydroxymonocarboxylic acids, in PCOA induction was further substantiated by the observed induction of peroxisomal PCOA by 1-12-dodecanedioic acid. We conclude that (1) fenofibric acid is the possible proximate peroxisome proliferator of fenofibrate in rat hepatocytes, (2) cultured hepatocytes reflect in vivo sensitivity to fenofibrate with respect to peroxisome proliferation, and (3) there is some evidence that the catalytic activity of the CYP4A1 enzyme mediates, at least in part, peroxisomal PCOA induction.

Species Differences in the Clonal Expansion of Hepatocytes in Response to the Coaction of Epidermal Growth Factor and Nafenopin, a Rodent Hepatocarcinogenic Peroxisome Proliferator

Peroxisome proliferators are members of the nongenotoxic family of rodent hepatocarcinogens. There exist substantial species differences in response to peroxisome proliferators among mammalian species. We have reported previously that peroxisome proliferators can synergize with epidermal growth factor (EGF) to promote the clonal expansion of rat hepatocytes associated with the early stages of hepatocarcinogenesis. The aim of the present study was to determine whether responsiveness in this in vitro assay reflected the known species differences in response to peroxisome proliferators. The process of tumorigenicity was modeled in the soft agar cloning assay since growth in soft agar is thought to reflect the early stages of tumorigenesis. This is because clonal expansion under these conditions requires the cells to survive, to undergo mitosis, and to escape from the contact-dependent growth associated with normal cell behavior. The data presented here show that mouse hepatocytes are able to undergo clonal expansion in soft agar in response to nafenopin and EGF giving a three- to fourfold increase in colony numbers over control. This result is comparable to the fivefold increase in rat hepatocyte colony numbers that we have reported previously. In contrast, hamster, guinea pig, and human hepatocytes did not respond to the concerted action of EGF and nafenopin despite their ability to respond to EGF as a mitogen in monolayer culture. These data demonstrate that the clonal expansion of rodent hepatocytes in soft agar in response to peroxisome proliferators and EGF displays the same species differences as other pleiotropic responses to these compounds and is likely therefore to be relevant to the process of hepatocarcinogenesis.

Cyclic GMP activates an influx of Ca 2+ across the plasma membrane of guinea pig hepatocytes

Cyclic GMP activates an influx of Ca 2+ across the plasma membrane of guinea pig hepatocytes

Properties of single potassium channels in guinea pig hepatocytes.

The patch-clamp technique of cell-attached and inside-out configurations was used to study the single potassium channels in isolated guinea pig hepatocytes. The single potassium channels in isolated guinea pig hepatocytes were recorded at different K+ concentrations. A linear single-channel current-voltage relationship was obtained at the voltage range of -80 to -20 mV with slope conductance of 70 +/- 6 pS (n = 10). Under symmetrical high K+ concentration of 148 mM in the cell-attached patch membrane, the I-V curve exhibited a mild inward rectification at potentials positive to + 20 mV. The values of reversal potential was +5 +/- 2 mV (n = 10). When the external potassium concentration ([K+]o) was decreased to 74 mM and 20 mM, the slope conductance was decreased to 48 +/- 2 pS (n = 4) and 24 +/- 3 pS (n = 3), respectively. The reversal potential was changed by 58 mV for a tenfold change in [K+]o, indicating that this channel was highly selective for K+. Open probabilities (Po) of the channel were 73-93% without apparent voltage dependence. The distributions of open time of the channels were fitted to two exponentials, while those of closed time were fitted to three exponentials, exhibiting no voltage dependence. The success rate of K+ channel activity to be recorded was 28% at room temperature, and there were no increases in the success rate nor in the channel opening probabilities at a temperature of 34-36 degrees C. Po in inside-out patches was not changed by application of 1 microM Ca2+ nor 1 mM Mg2+ to the internal side of patch membranes. It is concluded that a novel type of the K+ channels in guinea pig hepatocytes had different properties of slope conductance, channel kinetics, and sensitivity to [Ca2+]i, from those in other species.

Hexokinase and glucokinase binding in permeabilized guinea-pig hepatocytes

The release of glucokinase (hexokinase IV) from digitonin-permeabilized hepatocytes from rat, guinea pig or mouse liver is inhibited by physiological concentrations of Mg2+ (> 0.25 mM). Preincubation of hepatocytes with fructose increases glucokinase release during permeabilization in the presence of Mg2+ but decreases glucokinase release in the absence of Mg2+, suggesting that fructose causes translocation of glucokinase from the Mg(2+)-dependent site. Glucose (25 mM) and sorbitol (1 mM) also induce translocation of glucokinase from the Mg(2+)-dependent site in guinea-pig, as in rat hepatocytes, but glucose is less effective than fructose or sorbitol, and the concentrations of fructose and sorbitol that cause half-maximal activation (A50) are 3-fold and 20-fold higher, respectively, in guinea-pig than in rat hepatocytes (170 microM and 257 microM, compared with 61 microM and 13 microM). Dihydroxyacetone and glycerol have no effect on fructose-induced or sorbitol-induced translocation in guinea-pig hepatocytes, in contrast with the potentiation and inhibition, respectively, by these substrates in rat hepatocytes. Some, but not all, of the differences between rat and guinea-pig hepatocytes could be due to the more reduced cytoplasmic NADH/NAD+ redox state in guinea-pig cells. The activity of low-Km hexokinases accounts for 30% of total hexokinase activity (low-Km hexokinases + glucokinase) in guinea-pig hepatocytes. Of the low-Km hexokinase activity, approx. 30% is released in the presence of Mg2+, 9% shows Mg(2+)-dependent binding and 60% shows Mg(2+)-independent binding. There was no substrate-induced translocation of low-Km hexokinase activity, indicating that translocation is specific for hexokinase IV.

Metabolism of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in isolated liver cells from guinea pig, hamster, mouse, and rat.

The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant compound of the aminoimidazoazaarens (AIA) group of mutagens/carcinogens isolated from the crust of fried and broiled meat, was examined in freshly isolated hepatocytes from untreated rat, mouse, hamster, and guinea pig. Activation was evaluated by the total level of covalent binding of PhIP to macromolecules. Rat hepatocytes had the lowest rate of metabolism, both to reactive and detoxified metabolites. The products were identified as 4'-PhIP-sulfate, PhIP-glucuronide, and N(OH)-PhIP-glucuronide. The ring hydroxylation rate was much greater in mouse hepatocytes, the main products being 4'-PhIP-sulfate and 4-hydroxy-PhIP. The level of covalent binding in the mouse hepatocytes exceeded those of the rat and guinea pig at high doses of PhIP. An extensive metabolism was seen in guinea pig hepatocytes, the major products being 4'-PhIP-sulfate, 4'-O-PhIP glucuronide, PhIP-glucuronide, and N(OH)-PhIP-glucuronide. In addition, several other unknown metabolites were formed. However, the amount of covalent binding in guinea pig hepatocytes was similar to that in rat hepatocytes. Covalent binding of PhIP metabolites was highest in hamster hepatocytes. Three of the main metabolites were identified as 4'-PhIP-sulfate, 4'-O-PhIP-glucuronide, and PhIP-glucuronide, but several unknown PhIP metabolites also were formed. Only minor amounts of N(OH)-PhIP-glucuronide were produced in the hamster. The present study shows that both the direct detoxification of PhIP and further conjugation of the 2-hydroxylamino-PhIP to reactive and/or detoxified metabolites are important for the resulting covalent binding.

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

Characterization of the calcium-sensitive voltage-gated delayed rectifier potassium channel in isolated guinea pig hepatocytes.

The voltage-dependent K+ channel was examined in enzymatically isolated guinea pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. The resting membrane potential in isolated hepatocytes was -25.3 +/- 4.9 mV (n = 40). Under the whole-cell voltage-clamp, the time-dependent delayed rectifier outward current was observed at membrane potentials positive to -20 mV at physiological temperature (37 degrees C). The reversal potential of the current, as determined from tail current measurements, shifted by approximately 57 mV per 10-fold change in the external K+ concentration. In addition, the current did not appear when K+ was replaced with Cs+ in the internal and external solutions, indicating that the current was carried by K+ ions. The envelope test of the tails demonstrated that the growth of the tail current followed that of the current activation. The ratio between the activated current and the tail amplitude was constant during the depolarizing step. The time course of growth and deactivation of the tail current were best described by a double exponential function. The current was suppressed in Ca(2+)-free, 5 mM EGTA internal or external solution (pCa > 9). The activation curve (P infinity curve) was not shifted by changing the internal Ca2+ concentration ([Ca2+]i). The current was inhibited by bath application of 4-aminopyridine or apamin. alpha 1-Adrenergic stimulation with noradrenaline enhanced the current but beta-adrenergic stimulation with isoproterenol had no effect on the current. In single-channel recordings from outside-out patches, unitary current activity was observed by depolarizing voltage-clamp steps whose slope conductance was 9.5 +/- 2.2 pS (n = 10). The open time distribution was best described by a single exponential function with the mean open lifetime of 18.5 +/- 2.6 ms (n = 14), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 2.0 +/- 0.3 ms (n = 14) and that for the slow component of 47.7 +/- 5.9 ms (n = 14). Ensemble averaged current exhibited delayed rectifier nature which was consistent with whole-cell measurements. In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The concentration of Ca2+ at the half-maximal activation was 0.031 microM. These results suggest that guinea pig hepatocytes possess voltage-gated delayed rectifier K+ channels which are modified by intracellular Ca2+.

Comparable 30-kDa apamin binding polypeptides may fulfill equivalent roles within putative subtypes of small conductance Ca(2+)-activated K+ channels.

Apamin, a peptide neurotoxin from bee venom, blocks small conductance Ca(2+)-activated K+ channels in central synapses and peripheral tissues. Using 125I-apamin, single classes of high affinity binding sites (Kd 1-3 pM) were identified on plasma membranes from rat, rabbit, guinea pig, and bovine brain and from rabbit, guinea pig, and bovine liver. Binding was sensitive to scyllatoxin, dequalinium, gallamine, and d-tubocurarine but not to charybdotoxin, toxin I, or mast cell degranulating peptide. In contrast, saturable binding of 125I-apamin to rat liver plasma membranes was virtually undetectable, thereby providing a correlation with the ability to measure apamin-sensitive Ca(2+)-activated potassium currents in rabbit and guinea pig hepatocytes but not in rat hepatocytes. In agreement with membrane binding studies, homobifunctional cross-linkers identified apparently identical 33-kDa 125I-apamin binding polypeptides on brain plasma membranes from all species and analogous but distinct polypeptides on plasma membranes from rabbit, guinea pig, and bovine liver. None of these affinity-labeled polypeptides were detectable on plasma membranes from rat liver. Affinity labeling was abolished on both liver and brain membranes by apamin, scyllatoxin, dequalinium, gallamine, and d-tubocurarine. These results indicate that comparable approximately 30-kDa polypeptides may fulfill equivalent functional roles within putative subtypes of apamin-sensitive small conductance Ca(2+)-activated K+ channels.

Ascorbate Indirectly Stimulates Fatty Acid Utilization in Primary Cultured Guinea Pig Hepatocytes by Enhancing Carnitine Synthesis

L-Ascorbic acid is required for the synthesis of L-carnitine, which is essential for the oxidation of long-chain fatty acids. The purpose of this study was to investigate the role of ascorbate on the oxidation of long-chain fatty acids in primary cultured guinea pig hepatocytes. The hepatocytes were incubated in medium containing carnitine in the presence or absence of fatty acids. Exogenous fatty acids had no influence on the uptake of total carnitine into cells, but they lowered the free carnitine and consequently raised the concentration of short-chain acyl carnitine. Furthermore, carnitine supplementation of the medium in the presence of fatty acids led to a decrease of triglycerides in cells and an increase in the secretion of beta-hydroxybutyrate. These changes were also induced by the supplementation of the medium with both ascorbate and the precursor of carnitine (gamma-butyrobetaine) in the presence of fatty acids, although either ascorbate of gamma-butyrobetaine alone had no effect. In addition, increasing the concentration of supplemental ascorbate resulted in an enhancement of ketogenesis and a decrease of triglyceride accumulation. These results suggest that ascorbate enhances carnitine synthesis, which in turn stimulates beta-oxidation of fatty acids.

Different biotransformation of morphine in isolated liver cells from guinea pig and rat

The biotransformation of morphine was characterized in freshly isolated parenchymal and non-parenchymal liver cells from rats and guinea pigs in suspension culture to establish an in vitro model for morphine metabolism. Liver cells were prepared by a collagenase perfusion technique, and separated by differential centrifugation. Morphine metabolism was investigated at different concentrations (1, 5, 100 and 200 μM). Samples were taken repeatedly during 2–4 hr of incubation, and subsequently analysed on a HPLC system employing both UV and electrochemical detection. In suspensions of hepatocytes from both animal species morphine-3-glucuronide (M3G) was the major metabolite of morphine, and was formed at comparable rates at all concentrations examined. Guinea pig hepatocytes in addition produced considerable quantities of morphine-6-glucuronide (M6G), whereas this metabolite was detected only intracellularly in minor quantities in rat hepatocytes. The ratio between the two morphine glucoronides (M3G/M6G) in suspensions of guinea pig hepatocytes was approximately 4:1. N-Demethylation of morphine was more pronounced per mg cell protein in rat hepatocytes compared to guinea pig cells. Metabolic activity towards morphine was not detected in non-parenchymal cells of the two species. The morphine glucuronidation pattern found in guinea pig hepatocytes resembles to a greater extent than that found in rat hepatocytes the pattern found in in vivo studies of humans. It was concluded that isolated guinea pig parenchymal cells appeared to be a promising in vitro system for studies of morphine glucuronidation, and to observe metabolism in general.

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes.

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.

Effect of propaquizafop and its free-acid derivative on lauric acid hydroxylation and peroxisomal β-oxidation in primary cultured rat, mouse, guinea pig and marmoset hepatocytes

The effects of the phenoxyisopropionic acid derivative propaquizafop and its free-acid derivative on microsomal lauric acid hydroxylase and peroxisomal fatty acyl-CoA β-oxidation have been investigated in primary cultured hepatocytes obtained from various species and compared with those induced by bezafibrate. The hepatocyte cultures were incubated with these compounds for 48 hr at concentrations between 0.1–100μ m. No signs of cytotoxicity were observed as shown by the lack of lactate dehydrogenase (LDH) release into the culture medium. Lauric acid 12-hydroxylase was found to be strongly induced after treatment of rat and mouse hepatocytes with all three compounds, but remained largely unaffected in guinea pig and marmoset hepatocytes. Concomitantly, peroxisomal fatty acyl-CoA β-oxidation was found to be induced in rat but not in mouse hepatocytes after treatment for 48 hr. However, clearly increased β-oxidation activities could also be observed in mouse hepatocytes after a 72-hr incubation period. In contrast, only marginally increased β-oxidation activities were recorded, if at all, in guinea pig and marmoset hepatocytes. The results demonstrate that the effects of propaquizafop and its free-acid derivative in hepatocytes from four species are very similar to those produced by the known peroxisome proliferator (PP) bezafibrate. This is in accordance with the known difference in susceptibility of various species to PP, for example, rats and mice being most responsive whereas guinea pigs and primates including humans are far less responsive or even unresponsive.

Effects of the peroxisome proliferator mono(2-ethylhexyl)phthalate in primary hepatocyte cultures derived from rat, guinea pig, rabbit and monkey: Relationship between interspecies differences in biotransformation and peroxisome proliferating potencies

Primary hepatocyte cultures derived from rat, rabbit, guinea pig and monkey have been treated in vitro with metabolites of di(2-ethylhexyl)phthalate, i.e. mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate (metabolite V) and mono(2-ethyl-5-oxohexyl)-phthalate (metabolite VI). In rat hepatocyte cultures MEHP and metabolite VI were equally potent in inducing peroxisome proliferation, while metabolite V was much less potent. In rat hepatocytes a 50% increase in both peroxisomal palmitoyl-CoA oxidase activity and microsomal lauric acid ω-hydroxylation activity was found after treatment with 5–15 μM MEHP. In guinea pig, rabbit and monkey hepatocyte cultures, a 50% increase in peroxisomal palmitoyl-CoA oxidase activity was found after treatment with 408–485 μM MEHP. No induction of lauric acid ω-hydroxylation activity was found. These results indicate that peroxisome proliferation can be induced by MEHP in rabbit, guinea pig and monkey hepatocytes, but that these species are at least 30-fold less sensitive to peroxisome proliferation induction than rats. The proposed mechanistic inter-relationship between induction of lauric acid ω-hydroxylation activity and peroxisome proliferation is found in rat hepatocytes, but not in hepatocytes of the other three species. Treatment of guinea pig hepatocyte cultures with MEHP resulted in an increase in triglyceride concentrations in the hepatocytes. In rat and rabbit hepatocyte cultures, triglyceride concentrations were much less altered by MEHP. In monkey hepatocytes a decrease in hepatic triglyceride concentration was found after treatment with MEHP. These effects are in agreement with in vivo effects observed before. After treatment of primary hepatocyte cultures with MEHP, high concentrations of ω- and (ω-1)-hydroxylated metabolites of MEHP were found in media from rat, rabbit and guinea pig cultures. The formation of these metabolites did not decline in time. During treatment the metabolite profile in media from rat hepatocyte cultures moved towards ω-hydroxy metabolites of MEHP. In media from monkey hepatocyte cultures the lowest concentrations of hydroxylated metabolites were determined. No major species differences were found in the potency to form oxidized MEHP metabolites, and thus no unique metabolite differences were found, which could explain the species differences in sensitivity for peroxisome proliferation.

Dequalinium: a potent inhibitor of apamin-sensitive K + channels in hepatocytes and of nicotinic responses in skeletal muscle

The bisquaternary compound dequalinium has been tested for its ability to inhibit the loss of K + which angiotensin II causes in guinea-pig hepatocytes and which occurs through apamin-sensitive Ca 2+-activated K + (SK Ca) channels. Dequalinium blocked angiotensin II-evoked K + loss with an IC 50 of 1.5 ± 0.1 μM and also inhibited 125I-monoiodoapamin binding with a K I of 1.1 ± 0.1 μM. It is the most active non-peptide SK Ca blocker so far described. The neuromuscular blocking agent vecuronium was also tested, and proved to be considerably less effective (IC 50, 4.5 ± 0.3 μM; K I, 3.6 ± 0.5 μM). Dequalinium was also examined for its actions at nicotinic receptors in skeletal muscle and was found to be a potent, non-competitive antagonist of carbachol contractions of the frog rectus abdominis. In the frog cutaneus pectoris muscle, end-plate depolarizations induced by carbachol became smaller and more transient in the presence of dequalinium at 10 nM. However, contractions of the frog sartorius and rat diaphragm in response to nerve stimulation were inhibited only by concentrations > 1 μM. These apparently discrepant effects of dequalinium on nicotinic responses could be explained either by open channel block of slow onset or by ‘stabilization’ of the desensitized state of the receptor. The potency of dequalinium will make it a useful agent for the study of nicotinic receptors as well as of SK Ca channels.

The contribution of pyruvate cycling to loss of [6-3H]glucose during conversion of glucose to glycogen in hepatocytes: effects of insulin, glucose and acinar origin of hepatocytes

1. During conversion of [6-3H,U-14C]glucose to glycogen in liver, loss of 6-3H can occur either by cycling via pyruvate (between glycolysis and gluconeogenesis) or by other mechanisms. We used mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, to determine the extent to which pyruvate cycling contributes to loss of 6-3H during glucose conversion to glycogen in hepatocytes. 2. Mercaptopicolinate increased the 3H/14C ratio in glycogen during incubation of rat, guinea pig, pig and human hepatocytes with [6-3H,U-14C]glucose. The increase in the 3H/14C ratio in glycogen caused by mercaptopicolinate was greater in periportal than in perivenous rat hepatocytes, indicating that cycling of glucose via pyruvate is more prominent in cells with a higher gluconeogenic relative to glycolytic capacity. 3. The effect of mercaptopicolinate on the 3H/14C ratio in glycogen was observed both in the absence and in the presence of insulin, indicating that stimulation of glycogen synthesis by insulin is not associated with inhibition of pyruvate cycling. In rat and guinea pig but not in pig hepatocytes, the effects of mercaptopicolinate on the 3H/14C ratio in glycogen were greater at 10-15 mM glucose than at 30 mM glucose, suggesting diminished cycling via pyruvate at high glucose concentrations. 4. Insulin increased the loss of 6-3H during stimulation of conversion of glucose to glycogen in hepatocytes from all species. This was due in part to an increase in pyruvate cycling and in part to other mechanisms that are not inhibited by mercaptopicolinate. 5. These results suggest that pyruvate cycling is a significant, but not exclusive, component of the loss of 6-3H in the hepatocyte during glucose conversion to glycogen. The extent of pyruvate cycling is dependent on the acinar origin of the hepatocytes and on the glucose concentration and presence of insulin.