Phenotypic control of gap junctional communication by cultured alveolar epithelial cells.

We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed approximately 3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.

Strain differences in adrenal microsomal steroid metabolism in guinea pigs.

We recently reported that CYP2D16, a xenobiotic-metabolizing P450 isozyme, was expressed at higher levels in adrenal microsomes from inbred Strain 13 guinea pigs than in those from outbred English Short Hair (ESH) animals. Studies were done to determine if there also were strain differences in adrenal microsomal steroid metabolism. In both inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zone preparations of the adrenal cortex, 21-hydroxylase activities were greater in microsomes from ESH than from Strain 13 guinea pigs. By contrast, 17alpha-hydroxylase activities were similar in the two strains. In both strains, 21-hydroxylase activities were greater in inner than outer zone microsomes, but the opposite was found for 17alpha-hydroxylase activities (outer>inner). Northern and Western analyses revealed higher levels of CYP21 mRNA and protein in adrenals from ESH than Strain 13 guinea pigs, but there were no strain differences in CYP17 mRNA or protein concentrations. Despite the zonal differences in adrenal 17alpha-hydroxylase and 21-hydroxylase activities, CYP17 and CYP21 mRNA and protein levels were similar in the inner and outer zones within each strain of guinea pig. The results demonstrate strain differences in microsomal steroid metabolism that are explained by differences in CYP21 expression. By contrast, the zonal differences in steroid hydroxylase activities may be attributable to post-translational mechanisms.

Strain differences in adrenal CYP2D16 expression in guinea pigs. Relationship to xenobiotic metabolism.

Experiments were done to determine the mechanisms responsible for differences in adrenal microsomal xenobiotic metabolism between Strain 13 and English Short-Hair (ESH) guinea pigs. The rates of adrenal xenobiotic metabolism (bufuralol 1'-hydroxylase, benzo[a]pyrene hydroxylase, benzphetamine N-demethylase) were 2-3 times greater in microsomes from the Strain 13 animals. In both strains, xenobiotic-metabolizing activities were far greater in the inner zone (zona reticularis) than in the outer zones (zona fasciculata and zona glomerulosa) of the adrenal cortex. Northern blot analyses of total adrenal RNA with a CYP2D16 cDNA as the probe revealed significantly greater amounts of CYP2D16 mRNA in the Strain 13 guinea pigs. In addition, SDS-PAGE and Western blotting of adrenal microsomes demonstrated higher concentrations of CYP2D16 protein in Strain 13 than in ESH animals. Expression of CYP2D16 was predominantly in the inner zone of the adrenal, coinciding with the major site of xenobiotic metabolism. The results demonstrated higher levels of expression of CYP2D16 in adrenal glands from Strain 13 than from ESH guinea pigs, which may account for the strain differences in adrenal xenobiotic metabolism. Strain 13 guinea pigs should serve as a good experimental model for further studies on the regulation of adrenal CYP2D16.

Differential effects of adrenocorticotropin in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex.

Studies were performed to compare the effects of ACTH treatment in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex. In untreated animals, CYP2D16 protein and messenger RNA (mRNA) expression as well as xenobiotic-metabolizing activities (bufuralol 1'-hydroxylase, benzphetamine N-demethylase, and benzo(a)pyrene hydroxylase) were far greater in the inner (zona reticularis) than the outer (zona fasciculata plus zona glomerulosa) zones of the cortex. ACTH treatment for 3 or 7 days significantly decreased the rates of xenobiotic metabolism in both the inner and outer adrenal zones. Western and Northern blot analyses revealed that adrenal CYP2D16 protein and mRNA concentrations were significantly decreased by ACTH. In contrast to its inhibitory effects on CYP2D16, ACTH treatment increased steroid 17 alpha-hydroxylase activity in the adrenal inner zone, but did not affect outer zone activity. Microsomal CYP17 protein concentrations were not affected by ACTH despite increases in CYP17 mRNA levels in both zones. The results indicate that ACTH causes down-regulation of adrenal CYP2D16, probably at the transcriptional level. Thus, modulation of CYP2D16 by ACTH is opposite that for the steroidogenic P450 isozymes, suggesting unique regulatory mechanisms. In addition, the data suggest that posttranscriptional mechanisms contribute to ACTH regulation of 17 alpha-hydroxylase activity in the guinea pig adrenal cortex.

Adenine nucleotide metabolism by chondrocytes in vitro: role of ATP in chondrocyte maturation and matrix mineralization.

The objective of the investigation was to explore the notion that chondrocytes in the growth plate secrete nucleotides and that these compounds are used to regulate cell maturation and matrix mineralization. Chondrocytes were isolated from the cephalic region of chick embryo sterna and maintained in culture until confluent. To promote expression of the mature phenotype, cultures were then treated with retinoic acid. During the culture period, medium was removed and analyzed for nucleotides using a modified reverse-phase high-performance liquid chromatography (HPLC) procedure. We found that culture medium, conditioned by the chondrocytes, contained significant quantities of nucleotides. Moreover, the nucleotide concentrations were similar in magnitude to levels reported for media conditioned by other cell types. In terms of species, adenosine diphosphate (ADP) was the major nucleotide present in the conditioned medium; adenosine monophosphate (AMP) was present, but at a lower concentration than ADP. To examine the possibility that adenosine triphosphate (ATP) was released by the cultured chondrocytes, but was rapidly degraded into ADP and AMP, we examined the kinetics of ATP breakdown by chondrocytes. We found that chondrocytes degraded over 70% of exogenous ATP within 15 minutes. Similar experiments performed with ADP and AMP indicated that these nucleotides were also degraded by the cells, but at a slower rate than ATP. To determine whether the extracellular nucleotides modulate cartilage development, we examined the effect of exogenous ATP on four major determinants of chondrocyte function: alkaline phosphatase activity, cell proliferation rate, anaerobic metabolism, and mineral deposition. We found that ATP caused only minimum alterations in cell number and alkaline phosphatase activity; however, it increased the lactate content of the medium probably by stimulating anaerobic glycolysis. We noted that ATP had a significant effect on the amount and type of mineral deposited into chondrocyte cultures. Compared with untreated controls, ATP stimulated formation of a small amount of poorly crystallized calcium phosphate. The results of the study show for the first time that chondrocytes release nucleotides into the extracellular milieu. Although they are rapidly degraded, they serve to regulate both mineral formation and energy metabolism.

Inactivation of adrenal cytochromes P450 by 1-aminobenzotriazole. Divergence of in vivo and in vitro actions.

Recent investigations demonstrated that administration of 1-aminobenzotriazole (ABT) to rats caused adrenal gland enlargement. Studies were done to pursue the mechanism(s) involved. Preliminary experiments revealed that the adrenal enlargement caused by ABT was associated with a decline in plasma corticosterone concentrations, suggesting inhibition of adrenal steroidogenesis. Indeed, a single injection of ABT (25 or 50 mg/kg body weight) to rats caused concentration-dependent declines (60-80%) in adrenal mitochondrial and microsomal cytochrome P450 (P450) concentrations. The decreases in adrenal P450 levels exceeded those in hepatic microsomes. Accompanying the declines in adrenal P450 concentrations were decreases in steroid hydroxylase activities. Mitochondrial 11 beta-hydroxylase and cholesterol side-chain cleavage activities and microsomal 21-hydroxylase activity were diminished markedly (60-90%) by ABT treatment. In contrast, activity of adrenal 3 beta-hydroxysteroid dehydrogenase-isomerase was not affected by ABT, indicating specificity for P450-dependent reactions. Incubation of adrenal microsomes or mitochondria in vitro with ABT plus an NADPH-generating system had no effect on P450 concentrations or on steroid hydroxylase activities. Similar incubations with hepatic microsomes caused declines in P450 levels and in the rates of P450-mediated xenobiotic metabolism. The results demonstrate that ABT is a potent inhibitor of adrenal steroid hydroxylases in vivo, but the in vitro studies indicate that the mechanism of action differs from that on other P450 isozymes. The absence of inhibitor effects in vitro suggests that an extra-adrenal metabolite of ABT is responsible for the in vivo inactivation of steroidogenic enzymes.

Retinoic acid induces a shift in the energetic state of hypertrophic chondrocytes.

In the epiphyseal growth plate, chondrocyte maturation is accompanied by dramatic alterations in energy metabolism. To explore the relationship between these two events, we used retinoic acid (RA) to promote chondrocyte maturation in culture. The specific question that was addressed was, does RA treatment of cultured chondrocytes in vitro induce a change in energy status similar to that seen in hypertrophic chondrocytes in vivo. Maturing chondrocytes isolated from the cephalic region of day 18 chick embryo sterna were allowed to grow for 7-14 days in monolayer until confluent and then treated with 10-300 nM RA. Immature chondrocytes from the caudal region of sternum were grown in parallel and served as control cells for the study. We found that in maturing cephalic cell cultures, RA had a rapid and profound effect on oxidative metabolism. The retinoid caused a reduction in the energy charge ratio (ECR) and the ATP/ADP ratio and a sharp decrease in cell ATP levels. Maximum inhibition was observed when the RA concentration was 10-35 nM. Compared with the adenine nucleotides, creatine phosphate levels were decreased to a lesser extent by RA, although there was substantial inhibition of creatine kinase activity. We expected to find a compensatory elevation in glycolytic activities; however, the lactate levels in the medium of the treated cells indicated that anaerobic glycolysis was depressed. In contrast to the cephalic chondrocytes, when caudal cell cultures were treated with RA, lactate formation was stimulated and there were minimal effects on oxidative metabolism. To determine the mechanism of inhibition of glycolysis, we measured the activity of pyruvate kinase in RA-treated cephalic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunotoxic effects of mercuric compounds on human lymphocytes and monocytes. III. Alterations in B-cell function and viability.

The major goal of the study was to determine the effects of high and low levels of mercury on human B-cells. Following treatment of B-cells with HgCl2 (0-1000 ng) and MeHgCl2 (0-100 ng), their activation by mitogens was evaluated. Both forms of mercury caused a dose dependent reduction in B-cell proliferation in the presence or absence of monocytes. MeHgCl was approximately 10 times more potent than HgCl2. Mercury also inhibited the ability of these cells to synthesize IgM and IgG. Analysis of the expression of activation markers indicated that CD69, an early marker of cell activation, was not effected by mercury. In comparison, B-cell expression of the low affinity IgE receptor and the transferrin receptor were significantly reduced. Of particular interest, cells activated by mitogen for 48 hr became refractory to the immunotoxic effects of mercury. When exposed to high levels of HgCl2 (0.5-10 micrograms/ml) and MeHgCl (0.05-1 micrograms/ml), there was minimal reduction in B-cell viability at 1-4 hr, however, after exposure to mercury for 24 hr, cell death was apparent. MeHgCl was approximately 5-10 times more potent than HgCl2. Electron microscopic analysis revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation and condensation of nucleoplasm. Both forms of mercury caused a rapid and sustained elevation in the intracellular levels of Ca++. The results of this investigation clearly show that mercury-containing compounds are immunomodulatory; moreover, the decrease in B-cell function indicates that this metal is immunotoxic at very low exposure levels. Furthermore, the cytotoxic events are consistent with the notion that mercury initiates changes associated with programmed cell death.

Developmental regulation of creatine kinase activity in cells of the epiphyseal growth cartilage.

During the process of endochondral bone formation, the maturing chondrocyte exhibits profound changes in energy metabolism. To explore the mechanism of energy conservation in cartilage we examined the expression of creatine kinase, an enzyme that catalyzes the formation of ATP in tissues under oxygen stress. Measurement of creatine kinase activity and cytochemical assessment of enzyme distribution clearly showed that the level of enzyme activity was related to chondrocyte maturation. Thus, as the cells hypertrophied, there was a progressive increase in creatine kinase activity. Similarly, an elevation in creatine kinase activity was noted in chondrocyte cultures as the cells assumed an hypertrophic state. When cartilage calcification was disturbed by rickets, there was a decrease in enzyme activity in the hypertrophic region. Studies were performed to examine the creatine kinase isozyme profile of cells of the epiphysis. In resting and proliferating cartilage, the isoform was MM. In hypertrophic cartilage, the predominant isoforms were MB and BB. In terms of the creatine phosphate content, the highest values were seen in the proliferative region; lower amounts were present in hypertrophic and resting cartilage; and no creatine phosphate was detected in calcified cartilage. These data suggest that turnover of creatine phosphate is greatest in the mineralized region of the epiphysis. The results of these investigations point to creatine kinase as being under developmental control. The activity of the enzyme in cartilage cells should serve as a marker of developmental events associated with chondrocyte proliferation, hypertrophy, and mineralization.

Ascorbic acid regulates multiple metabolic activities of cartilage cells.

Bones grow in length because of the activities of cartilage cells in the epiphyseal growth plate. We have examined selected events that occur in the growth cartilage by the use of cultured epiphyseal cells; we have also evaluated the influence of ascorbate on these activities. Our studies indicate that 1) ascorbate induces the expression of a unique collagen isoform, type X collagen; 2) ascorbate stimulates alkaline phosphatase activity of maturing chondrocytes; and 3) ascorbate regulates the energy status of the maturing chondrocyte. We have found that in the presence of ascorbate there is a change in oxidative activity. Thus, lactate formation is inhibited, there is an increase in the adenylate energy charge ratio, and there is an elevation in the activity of isocitrate dehydrogenase. The results of these studies point to multiple effects of vitamin C on chondrocyte maturation involving changes in protein synthesis and energy metabolism.

Superoxide dismutase and catalase activities in the growth cartilage: relationship between oxidoreductase activity and chondrocyte maturation.

Superoxide dismutase (SOD) and catalase are enzymes that protect cells from radical attack. Catalase disproportionates hydrogen peroxide, and SOD is an oxidoreductase that serves to dismutate the superoxide anion. The objective of this communication was to measure the activity of these disproportionating enzymes in the chick tibial growth cartilage and to relate enzyme activity to chondrocyte maturation and tissue calcification. Analytic techniques were optimized for the measurement of both enzymes; particular care was taken to ensure that the values obtained were due to SOD and catalase, not to the presence of other oxidases or contaminants. Catalase and SOD had similar profiles of activity in cartilage. For both enzymes, the highest levels of activity were observed in premineralized cartilage; as chondrocytes matured there was a progressive decrease in the activity of SOD and catalase. Comparison of chondrocyte SOD activity with nonmineralizing tissues indicated that the activity of cultured cartilage cells was low. We also measured the SOD activity of avascular chondrodystrophic cartilage and found it to be less than that of proliferating cartilage. When cartilage was electrofocused, three SOD isozymes were detected. The pI of the major isozyme corresponded to the copper-zinc isoform. We suggest that the observed changes in enzymatic activity are dependent on a number of cartilage-specific factors that include the vascular supply, the local production of oxygen radicals by chondrocytes, and the oxidative state of the tissue.

Pentose phosphate shunt metabolism by cells of the chick growth cartilage.

We have measured the activity of the pentose shunt pathway in the chick growth cartilage. Measurement of D-[1-14C] glucose and D-[6-14C] glucose metabolism by chondrocytes indicated that pentose phosphate shunt activity was low. However, when the cells were stimulated with phenazine methosulfate (PMS) and t-butyl hydroperoxide, a significant elevation in shunt activity was observed. This activity was further increased by dithiothreitol. Enzymatic and substrate requirements of the shunt pathway were examined and related to morphology of the tissue. It was found that as chondrocytes mature, there is increased glucose-6-phosphate dehydrogenase activity, and decreased quantities of glucose-6-phosphate and NADPH. While these investigations indicated that shunt activity was maximum in hypertrophic cartilage, the results of cytochemical studies suggested that the activity was greatest in those cells that were most removed from the O2 supply. Experiments were performed to examine O2 requirements of chondrocytes in relationship to the pentose phosphate shunt. First, using a phosphorescence quenching technique, total O2 uptake by these cells was found to be constant over a large part of the physiological range of O2 tensions. Over the same range, when stimulated by PMS, O2 uptake by CN- treated cells was increased. In the 1-5 microM O2 range, non-mitochondrial O2 consumption decreased more slowly than total respiration. Finally, the observation that NADPH directly stimulated chondrocyte O2 consumption suggest that cartilage cells may be able to form O2 metabolites.

Adenine, guanine, and inosine nucleotides of chick growth cartilage: relationship between energy status and the mineralization process.

The major aim of this investigation was to measure the nucleotide content of the developing chick epiphysis and to relate changes in nucleotide levels to chondrocyte maturation and the development of mineralization. Using a cryostat, sections of cartilage were isolated from the proximal head of the tibial growth cartilage, care being taken to preserve the metabolic integrity of the tissue. Sections were identified microscopically, pooled, and the nucleotide and nucleoside content of each sample determined by HPLC. Procedures used for the study were shown to minimize degradation of nucleotides. Their effectiveness was assessed through an evaluation of the rapid freezing technique and by examination of the effects of apatite on the recovery of endogenous and added nucleotides. Analysis of nucleotide levels in the growth cartilage indicated that chondrocytes undergo a profound change in energy metabolism during development and maturation. Thus, in the premineralized resting and proliferative zones, ATP and, to a lesser extent, GTP values were high, suggesting that the chondrocytes obtained metabolic energy through both glycolytic and mitochondrial oxidative processes. In the hypertrophic zone and in calcified cartilage, there was a profound decrease in the ATP concentration and a corresponding fall in the energy charge and the ATP/ADP ratios. The nucleotide levels in this zone indicated that there was increased reliance on nonoxidative metabolism. Measurement of nucleoside levels in premineralized cartilage suggested that there was little resynthesis of nucleotides through the salvage pathway. These observed changes in nucleotide values are consistent with earlier observations concerning chondrocyte redox and the low pO2 tension of the hypertrophic zone.2+off