Biomedical subjects

J W DePierre

Publications and source records attributed to J W DePierre.

At least 19 recordsLinked to original sources

PubMed2005

Bacterial lipopolysaccharide both renders resistant mice susceptible to mercury-induced autoimmunity and exacerbates such autoimmunity in susceptible mice.

The initiation and severity of systemic autoimmune diseases are influenced by a variety of genetic and environmental factors, in particular bacterial infections and products. Here, we have employed bacterial lipopolysaccharide (LPS), which non-specifically activates the immune system, to explore the involvement of innate immunity in mercury-induced autoimmunity in mice. Following treatment of mouse strains resistant [DBA/2 (H-2(d))] or susceptible [SJL(H-2(s))] to such autoimmunity with mercuric chloride and/or LPS or with physiological saline alone (control), their immune/autoimmune responses were monitored. Resistant DBA/2 mice were rendered susceptible to mercury-induced autoimmunity by co-administration of LPS, exhibiting pronounced increases in the synthesis of IgG1 and IgE, high titres of IgG1 deposits in the kidneys and elevated circulating levels of IgG1 antibodies of different specificities. Furthermore, the percentages of the T cells isolated from the spleens of DBA/2 mice exposed to both mercury and LPS that produced pro-inflammatory cytokines were markedly increased by in vitro stimulation with phorbol myristate acetate (PMA) and ionomycin, which was not the case for splenic T cells isolated from mice receiving mercuric chloride, LPS or saline alone. In addition, exposure of susceptible SJL mice to mercury in combination with LPS aggravated the characteristic features of mercury-induced autoimmunity, including increased synthesis of IgG1 and IgE, the production of IgG1 anti-nucleolar antibodies (ANolA) and the formation of renal deposits of IgG1. In summary, our findings indicate that activation of the innate immune system plays a key role in both the induction and severity of chemically induced autoimmunity.

Animals↗

PubMed2002

Tricyclic antidepressants prevent the differentiation of monocytes into macrophage-like cells in vitro.

The investigation was designed to determine whether the two tricyclic antidepressant agents (TCAs) clomipramine and imipramine and the selective reuptake inhibitor citalopram affect differentiation of human monocytes to macrophage-like cells (MAC-LCs). We established primary adherent cultures of peripheral blood monocytes and monitored their morphology, capacity for phagocytosis and antigen expression during transformation to MAC-LCs. As expected, maturation of monocytes to MACs is accompanied by changes in morphology, elevated expression of the antigens CD16 and CD51 and an increase in the percentage of phagocytic cells. Treatment of cells with the TCAs clomipramine (40 micromol/L) or imipramine (100 micromol/L) and with citalopram (100 micromol/L), for 11 days resulted in the following observations: (1) monocytes treated with TCAs never developed the morphology characteristic of the MAC-LCs; (2) TCAs reduced the percentage of phagocytic cells; (3) TCAs had little influence on the expression of CD14, CD16, CD51, and HLA-DR. However, when added after monocyte differentiation into MAC-LCs, citalopram and clomipramine no longer reduced the percentage of phagocytic cells and these effects were not simply due to irreversible cytotoxicity. Thus clomipramine, imipramine, and citalopram inhibit differentiation of human monocytes into MAC-LCs in vitro, but in a reversible manner.

Antidepressive Agents, Tricyclic↗

PubMed2001

Expression of rat aldehyde reductase AKR7A1: influence of age and sex and tissue-specific inducibility.

The regulation of the aldo-keto reductase AKR7A1 was examined in the livers of male and female rats during development by using Western blots, and its contribution to carbonyl metabolism was assessed by using enzyme assays. Hepatic levels of AKR7A1 are low in fetal rats and rise to a peak at around 6 weeks of age in animals of both sexes. Higher levels of the enzyme are found in adult male rat liver than in adult female rat liver. The reductase, therefore, appears to be subject to sex-specific regulation. The effect of growth hormone in mediating this difference in expression was examined by using hypophysectomized animals whose serum growth hormone levels had been feminized by continuous administration. Results demonstrate that such treatment leads to a reduction in AKR7A1 expression. AKR7A1 was found to be constitutively expressed in rat tissues such as liver, kidney, small intestine, and testis, but it was not detected in nasal mucosa, skeletal muscle, heart, adrenal gland, brain, or spleen. However, AKR7A1 was inducible by the synthetic antioxidant ethoxyquin in liver, kidney, and small intestine, but not in the other tissues examined. These results show that levels of this important detoxication enzyme vary considerably according to age and sex and that dietary antioxidants can also influence its level in several tissues.

Age Factors↗

PubMed2001

Further evidence for the involvement of inhibition of cell proliferation and development in thymic and splenic atrophy induced by the peroxisome proliferator perfluoroctanoic acid in mice.

We recently demonstrated that severe thymic and splenic atrophy occur upon dietary treatment of mice with potent peroxisome proliferators (PPs), e.g. perfluorooctanoic acid (PFOA), WY-14,643, nafenopin, and di(2-ethylhexyl)phthalate (DEHP). In the present study, we investigated this phenomenon further employing a relative inert PP, PFOA. Comparison of the dose-dependencies and time-courses indicated that the peroxisome proliferative effect occurred prior to atrophy of both the thymus and spleen. However, following withdrawal of PFOA from the diet, the weight of the thymus and spleen rapidly returned to normal within 10 and 5 days, respectively, in contrast to the more persistent peroxisome proliferation. Furthermore, the changes in thymus and spleen weight upon PFOA treatment and the following withdrawal from diet paralleled the changes in total thymocyte and splenocyte counts, respectively. It was found previously that the decreases in the thymocyte populations present in the S and G2/M phases, as well as in the number of CD4+CD8+ cells upon PFOA treatment, were the most dramatic, perhaps reflecting inhibition of thymocyte proliferation in connection with thymocyte development. Here, the recovery of thymocytes began with increases in the populations in these same phases of the cell cycle, with CD4+CD8+ cells recovering most rapidly, lending further support to our previous hypothesis. The possible relationship of these immunotoxic effects of PPs to the changes they cause in fatty acid metabolism is discussed.

Animals↗

PubMed2001

Reactive oxygen species and mitochondria mediate the induction of apoptosis in human hepatoma HepG2 cells by the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid.

We have previously shown that one of the most potent rodent hepatocarcinogens, perfluorooctanoic acid (PFOA), induces apoptosis in human HepG2 cells in a dose- and time-dependent manner. In this study we have investigated the involvement of reactive oxygen species (ROS), mitochondria, and caspase-9 in PFOA-induced apoptosis. Treatment with 200 and 400 microM PFOA was found to cause a dramatic increase in the cellular content of superoxide anions and hydrogen peroxide after 3 h. Measurement of the mitochondrial transmembrane potential (Delta Psi(m)) after PFOA treatment showed a dissipation of Delta Psi(m) at 3 h. Caspase-9 activation was seen at 5 h after treatment with 200 microM PFOA. In order to evaluate the importance of these events in PFOA-induced apoptosis, cells were cotreated with PFOA and N-acetylcysteine (NAC), a precursor of glutathione, or Cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MPT pore). NAC reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced ROS. In addition, CsA also reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced opening of the MPT pore. In summary, we have delineated a ROS and mitochondria-mediated pathway for induction of apoptosis by PFOA.

Acetylcysteine↗

PubMed2001

Regulation of the GST Mu-1 isoenzyme in Y1 cells by adrenocorticotropic hormone is primarily transcriptional.

In earlier experiments, adrenocorticotropic hormone (ACTH) was shown to decrease the level of glutathione transferase M1 (murine glutathione transferase mu-1) (mGSTM1), as well as of the corresponding mRNA, in a murine adrenocortical cell line. In the present study, the effect of ACTH on mGSTM1 gene transcription was examined using two techniques. First, a cDNA that coded for the mGSTM1 subunit but lacked the corresponding promoter sequences was transfected into the adrenocortical cell line, and the effect of ACTH on the level of the corresponding transcript was compared to that of endogenous mGSTM1 mRNA. The other technique used was nuclear run-on transcription, where the rate of transcription of endogenous mGSTM1 mRNA in ACTH-treated cells was compared to that in untreated control cells. These experimental approaches indicated that the rate of transcription of the mGSTM1 gene is regulated by ACTH in adrenocortical cells.

Adrenal Glands↗

PubMed2000

Effects of vitamin A deficiency on selected xenobiotic-metabolizing enzymes and defenses against oxidative stress in mouse liver.

Male and female C57B1/6 mice were rendered vitamin A-deficient, and the effects of this deficiency on certain xenobiotic-metabolizing enzymes and defenses against oxidative stress were examined. Vitamin A deficiency significantly increased the levels of DT-diaphorase, glutathione transferase, and catalase in the hepatic cytosolic fraction from male mice (5.2-, 1.6-, and 3.5-fold, respectively), as well as from female mice (4.8-, 3.3-, and 2.4-fold, respectively). In the hepatic mitochondrial fraction (containing peroxisomes) from male animals, the activities of urate oxidase and catalase were increased 3.4- and 1.7-fold, respectively. The activity of catalase in the mitochondrial fraction from female mice was not affected by vitamin A deficiency, whereas the activity of peroxisomal urate oxidase was increased 2.9-fold. The hepatic level of ubiquinone was increased somewhat. The significance of the increases observed here is presently unclear, but it may be speculated that vitamin A and/or its metabolites are somehow involved in the down-regulation of these proteins. Another possibility is that these enzymes are increased as a result of hepatic oxidative stress caused by vitamin A deficiency. However, vitamin A deficiency had no effect on the activity of superoxide dismutase in this study, whereas the activity of glutathione peroxidase was slightly decreased (27%) in the hepatic cytosolic fraction from male mice. In addition, the hepatic level of alpha-tocopherol was decreased dramatically in the vitamin A-deficient animals.

Animals↗

PubMed2000

Conjugation of 1-naphthol in primary cell cultures of rat ovarian cells.

The present study concerns conjugation of 1-naphthol in primary cultures of rat ovarian cells. Two phase II enzymes catalyzing conjugation, i.e. phenol sulfotransferase (P-SULT) and phenol UDP-glucuronosyltransferase (P-UGT), were measured using 1-naphthol as substrate. The rates of conjugation by the different cell types of the rat ovary were the same at low concentrations and short incubation times. However, after 20 h of incubation the rate of conjugation in cells isolated from ovaries enriched in corpora lutea (CL) exceeded the rate in cells isolated from ovaries enriched in preovulatory follicles. In addition, when the granulosa cells were removed from the preovulatory follicles, the rate of conjugation was 1.7-fold higher, i.e. in the theca/stroma cells. When the cells were incubated with 1-[14C]naphthol and conjugates were subsequently separated by thin-layer chromatography, naphthyl glucuronide was the only conjugate observed. Pentachlorophenol (PCP), a commonly used inhibitor of P-SULT, inhibited 1-naphthol conjugation 50% in cell cultures, as well as in microsomal preparations. alpha-Naphthoflavone (ANF) and ellipticine (ELP), both cytochrome P450 (CYP) inhibitors, affected the conjugation of 1-naphthol in different ways; ANF did not affect P-UGT activity in microsomal preparations, but inhibited 1-naphthol conjugation in cell cultures by as much as 90%. On the other hand, ELP inhibited the conjugation of 1-naphthol up to 99% in the cell cultures, but only 75% in microsomal fractions. Testosterone (TST) and estradiol inhibited this activity approximately equal 50% in both of these experimental systems. Clomiphene citrate (CLF), a drug used to induce ovulation and demonstrating both estrogenic and antiestrogenic effects, did not influence the conjugation of 1-naphthol significantly in the cell cultures. The present findings demonstrate that P-UGT is by far the major enzyme conjugating 1-naphthol in the rat ovary and that commonly used inhibitors of P-SULT and CYPs also inhibit P-UGT activity, either directly or via other mechanisms.

Animals↗

PubMed2000

Antidepressant-induced lipidosis with special reference to tricyclic compounds.

Cationic amphiphilic drugs, in general, induce phospholipid disturbances. Tricyclic, as well as other antidepressants belong to this group. In experimental animals, antidepressants induce lipid storage disorders in cells of most organs, a so-called generalized phospholipidosis. This disorder is conveniently detected by electron microscopic examination revealing myelin figures. Myelin figures or myeloid bodies are subcellular organelles containing unicentric lamellar layers. The lipidotic induction potency during in vivo is related to the apolarity of the compound. Metabolism of phospholipids takes place within the cell continuously. Several underlying mechanisms may be responsible for the induction of the phospholipid disturbance. For instance, it has been suggested that the compounds bind to phospholipids and such binding may alter the phospholipid's suitability as a substrate for phospholipases. Free TCA or metabolites thereof may also inhibit phospholipases directly, as has been demonstrated for sphingomyelinase in glioma and neuroblastoma cells. Both these mechanisms might result in phospholipidosis. Interaction between drug and phospholipid bilayer has been investigated by nuclear magnetic resonance technique. There seems to be large differences in the sensitivities amongst different organs. Steroid-producing cells of the adrenal cortex, testis and ovaries are in particular susceptible to drug-induced lipidosis. The so-called foam cells are lung macrophages located in the interstitium which become densely packed with myelin figures during TCA exposure. It requires about 3-6 weeks of treatment to develop this converted cell. In cell cultures however, phospholipidosis is demonstrated already after 24 h only. It appears that the cells that undergo TCA-induced lipidosis may recover after withdrawal of the drug. The time required to achieve complete recovery ranges from 3-4 weeks to several months, depending on the organ affected. Little is known about the functional significance of lipidosis. Even if TCA and other antidepressants show other effects, it has not been possible to exclusively relate it to phospholipidosis. However, few attempts have been made to correlate the physiological effects of TCAs in experimental animals to the morphological changes associated with phospholipidosis. There is an increasing evidence however, that cationic amphiphilic drugs may have effects on immune function, signal transduction and receptor-mediated events, effects that to some extent might be related to disturbances in phospholipid metabolism.

Animals↗

PubMed2000

Biocompatibility of microplates for culturing epithelial renal cells evaluated by a microcalorimetric technique.

In the present study we have developed a microcalorimetric procedure which allows convenient investigation of biocompatibility in a microsystem. We examined the biocompatibility of a porcine renal epithelial tubule cell line LLC-PK1 and a human primary renal epithelial tubule cell (RPTEC) with microplates composed of three different materials, i.e. Thermanox, transparent film and titanium. All three materials showed equal biocompatibility with LLC-PK1 cells, judging from the attainment of steady-state power curves and the same rate of heat production per cell (2.5 microW / microg DNA). The human renal cells were poorly biocompatible with the Thermanox and transparent film. However, on titanium the RPTEC cell did adhere, as demonstrated by a steady-state power curve. The human cells also showed a higher metabolic activity (3.0 microW / microg DNA), than did LLC-PK1 cells cultured on the same type of microplates. In research on biocompatibility there is a need for alternatives to experimental animal investigations. The present technique allows studies of cellular interactions with different biomaterials in a rapid and standardized manner and may therefore prove to be a useful screening procedure.

Journal Article↗

PubMed1999

Levels and subcellular distributions of detoxifying enzymes in the ovarian corpus luteum of the pregnant and non-pregnant pig.

The levels and subcellular distribution of enzymes involved in defenses against reactive oxygen superoxide dismutase (SOD; E.C.1.15.1.1), glutathione peroxidase (GPX; E.C.1.11.1.9), catalase (CAT; E.C.1.11.1.6), and DT-diaphorase (DT; E.C.1.6.99.2) and of the conjugating enzymes glutathione transferase (GST; E.C.2.5.1.18) and p-sulphotransferase (p-ST; E.C.2.8.2.1) in the corpus luteum of ovaries from pregnant and non-pregnant pigs were investigated. In addition, non-protein thiols and glutathione reductase (GRD; E.C.1.6.4.2) were examined in the same manner. The total cytosolic activities of CAT, DT, GRD, and p-ST were significantly increased, whereas total GST activity was decreased in the pregnant corpus luteum compared to the corresponding activities in non-pregnant corpus luteum. In the case of the mitochondrial fraction from pregnant corpus luteum, GPX and GRD displayed significant increases in specific activity. Upon subfractionation of the mitochondrial fraction (i.e. mitoplast preparation), SOD activity was distributed equally between the mitoplasts and the supernatant. CAT and GPX activities were mainly recovered in the supernatant, while the major GRD activity pelleted with the mitoplasts. Microsomes from pregnant corpus luteum demonstrated increased specific GPX activity and decreased SOD activity compared to the non-pregnant corpus luteum. No differences in the non-protein thiol levels in the cytosolic, mitochondrial, or microsomal fractions from the corpus luteum were observed between non-pregnant and pregnant sows.

Animals↗

PubMed1999

Changes in the generation of reactive oxygen species and in mitochondrial membrane potential during apoptosis induced by the antidepressants imipramine, clomipramine, and citalopram and the effects on these changes by Bcl-2 and Bcl-X(L).

In order to investigate the molecular mechanism of the antineoplastic effects exerted by the antidepressive agents imipramine, clomipramine, and citalopram, we examined the effects of these compounds on cell viability, generation of reactive oxygen species (ROS), and mitochondrial membrane potential (DeltaPsi(m)) in human acute myeloid leukemia HL-60 cells. Our results indicate that exposure to these compounds causes a loss in cell viability by activating the apoptotic process, as identified by electron microscopy, DNA gel electrophoresis, and flow cytometry. The increased generation of ROS induced by these drugs was a relatively early event and preceded the loss of DeltaPsi(m). Overexpression of the antiapoptotic protein Bcl-2 or Bcl-X(L) prevents antidepressant-induced apoptosis, as well as loss of DeltaPsi(m), but does not affect the generation of ROS.

Antidepressive Agents↗

PubMed1999

Expression of glutathione transferase isoenzymes in the porcine ovary in relationship to follicular maturation and luteinization.

The expression of different isoenzymes of glutathione transferase (GST), i.e. the cytosolic subunits GSTA1/A2, A3, A4, A5, M1/2, M2 and P1, T2, and the microsomal GST in follicles of different sizes and in corpora lutea from porcine ovary, was investigated by Western blotting. No immunoreactivity was obtained with anti-rat GSTT2 or anti-rat microsomal GST polyclonal antibodies. In contrast, GSTA1/A2, A3, A4, A5, M1/2, M2 and P1 are all expressed in the cytosol from porcine ovaries. In general, the highest levels of these GST isoenzymes were present in the cytosol from corpora lutea, in agreement with measurements of activity towards 1-chloro-2,4-dinitrobenzene. Immunoreactivity with anti-rat GSTP1 was only obtained with follicles. The cytosolic GSTs from follicles and corpora lutea were affinity purified on glutathione-Sepharose and separated by reversed-phase high-performance liquid chromatography in order to quantitate the different subunits. A peak corresponding to the class pi subunit was present in follicles. This peak was also seen with corpora lutea, although at very low level. There were four peaks containing class mu subunits. The remaining peaks were concluded to contain the class alpha subunits, except for two peaks which are suggested to contain proteins other than GSTs. The levels of the different subunits were quantitated on the basis of the areas under the peaks and the relative amounts in follicles of different sizes and in corpora lutea corresponded well with the Western blot analysis.

Animals↗

PubMed1999

The antidepressants imipramine, clomipramine, and citalopram induce apoptosis in human acute myeloid leukemia HL-60 cells via caspase-3 activation.

Some widely used antidepressants such as imipramine, clomipramine, and citalopram have been found to possess antineoplastic effects. In the present study, these compounds were found to induce apoptotic cell death in human acute myeloid leukemia HL-60 cells. Apoptosis induced by the antidepressants was identified by electron microscopy and conventional agarose gel electrophoresis and was quantitated by propodium iodide staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) via flow cytometry. Treatment with apoptosis-inducing concentrations of the antidepressants (80 microM imipramine, 35 microM clomipramine, or 220 microM citalopram) caused induction of caspase-3/caspase-3-like activity, which was monitored by the cleavage of poly(ADP-ribose) polymerase (PARP), the loss of the 32 kD caspase-3 (CPP32) precursor, and the cleavage of the fluorescent CPP32-like substrate PhiPhiLux. Pretreatment with a potent caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk) inhibited antidepressant-induced CPP32/CPP32-like activity and apoptosis. Furthermore, activation of caspase induced by the antidepressants was preceded by the hypergeneration of intracellular reactive oxygen species (ROS). These results suggested that the antidepressants may induce apoptosis via a caspase-3-dependent pathway, and induction of apoptosis by the antidepressants may provide a clue for the mechanism of their antineoplastic effects.

Antidepressive Agents↗

PubMed1999

Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells.

The effects of perfluorooctanoic acid (PFOA), a potent hepatocarcinogen and peroxisome proliferator in rodents, on human cells have not yet been examined. In the present study we demonstrate that treatment of human hepatoblastoma HepG2 cells with PFOA induces apoptosis, as well as perturbs the cell cycle. This apoptosis was characterized by electron microscopy, which revealed typical nucleosomal fragmentation (also observed as a 'DNA ladder' upon electrophoresis on agarose) and was quantitated using propidium iodide staining of cellular DNA and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. This process was dose- and time-dependent: apoptosis became manifest with 200 microM and maximal (45% of the cells) upon exposure to 450 microM PFOA for 24 h. Electrophoresis of the DNA from HepG2 cells exposed to 500 microM PFOA for 24 h or to 400 microM PFOA for 48 h revealed a smear typical of non-specific degradation. These findings indicate that in the presence of high concentrations of PFOA for long times, HepG2 cells undergo primary and secondary necrosis. Quantitation of trypan blue exclusion supported this conclusion. Flow cytometric analysis revealed that the cell cycle of HepG2 cells was perturbed by exposure to 50-150 microM PFOA. A 50 microM concentration resulted in a significant increase in the proportion of G(2)/M cells and, simultaneously, a decrease in the number of cells in the S phase, whereas treatment with 100 or 150 microM PFOA increased the proportion of cells in the G(0)/G(1) phase and decreased the number of cells in the G(2)/M and S phases. Simultaneous flow cytometric analysis of apoptosis-associated DNA strand breaks using the TUNEL procedure and of propidium iodide staining of cellular DNA revealed DNA breaks in HepG2 cells exposed to 150 microM PFOA, prior to nuclear fragmentation.

Apoptosis↗

PubMed1998

Modulation of apoptosis induced by tricyclic antidepressants in human peripheral lymphocytes.

We have recently reported that the tricyclic antidepressants (TCAs) imipramine, clomipramine, and citalopram induce apoptosis in human peripheral lymphocytes. This system is well suited for studies on the pathophysiology/physiology of apoptosis regulation. Apoptosis was determined using both DNA gel electrophoresis and flow cytometric analysis. TCA-induced apoptosis in lymphocytes was monitored in the presence of the protein synthesis inhibitor cycloheximide (CHX), the RNA synthesis inhibitor actinomycin D (Act D), the antioxidant reduced glutathione (GSH), the nuclease inhibitor aurintricarboxylic acid (ATA), the cytokine interleukin-2 (IL-2), and the immunostimulator linomide. CHX and Act D failed to prevent and actually enhanced TCA-induced apoptosis in lymphocytes, indicating that protein and RNA syntheses are not required for this process. Exogenous IL-2, GSH, and ATA protected the lymphocytes from apoptosis induced by TCAs in a dose-dependent manner, whereas linomide had no effect on TCA-induced apoptosis under our in vitro conditions. Our data demonstrate that TCA-induced apoptosis in human lymphocytes shares many common features with other stimuli-induced apoptotic processes.

Antidepressive Agents, Tricyclic↗

PubMed1998

Rapid one-step isolation of mouse liver catalase by immobilized metal ion affinity chromatography.

A novel and rapid procedure for the isolation of catalase from mouse liver, after prior treatment with the peroxisome proliferator perfluorooctanoic acid was developed using immobilized metal ion affinity chromatography involving chelation with zinc ions. The purification developed is simple, rapid (requiring 3 hours from cytosol or peroxisomal matrix to homogeneous proteins), reproducible, and yields virtually complete overall recovery of catalase activity. This procedure makes catalase from a variety of tissues and physiological and environmental conditions more readily available for study.

Animals↗

PubMed1998

Application of microcalorimetry for recording basal metabolic and Na+, K+-ATPase activity in LLC-PK1 cells, a model for the renal tubular epithelial cell.

In the present study we have employed a microcalorimetric procedure to measure the heat generated by a porcine renal tubule cell line (LLC-PK1) and its Na+, K+-ATPase. Microplates with an area of 2.2 cm2 were found to be optimal in terms of producing sufficient heat and a steady-state power curve. We compared the rate of heat production by cells in suspension and on monolayers and found a much lower value in suspension, that is, 1.42+/-0.2 versus 2.54+/-0.19 microW/microg DNA. Ouabain, the specific Na+, K+-ATPase inhibitor, caused a reduction in this heat output. The maximal inhibition in cell suspensions was 40% and remained unchanged with as much as 100 microM ouabain, the highest concentration tested. With cells cultured on microplates, ouabain in the concentration interval 0.1-3 microM caused a 25% inhibition of heat output. With 25-100 microM ouabain, a 50% inhibition was observed and at higher concentrations, no further inhibition occurred. Furthermore, upon removal of ouabain, full recovery of the Na+, K+-ATPase was observed, a process that could easily be monitored by using cell monolayers cultured on microplates.

Animals↗