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The expression of the rate-limiting enzyme of haem degradation, haem oxygenase-1 (HO-1), can be induced by various stimuli, including lipopolysaccharide, tumour necrosis factor alpha and NO. The NO signal can be transmitted by cGMP, therefore this study was aimed at testing the activation of the HO-1 gene by cGMP. In primary cultures of rat hepatocytes, both HO-1 mRNA and protein were induced by the NO donor sodium nitroprusside and 8-bromo-cGMP. The HO-1 mRNA induction by cGMP was prevented by the specific protein kinase G inhibitor KT5823. The cGMP-dependent HO-1 mRNA induction was dose-dependent and transcriptionally regulated, as determined by studies with actinomycin D and a nuclear run-on assay. Cycloheximide lowered the cGMP-dependent induction of HO-1 mRNA to about one half. Luciferase reporter constructs driven by about 800 bp of the 5'-flanking region of the rat HO-1 gene were transiently transfected into primary rat hepatocytes; 8-bromo-cGMP caused a 6-fold induction, which was obliterated by deletion and mutation of the cAMP response element/activator protein-1 (CRE/AP-1) (-665/-654) site. Thus HO-1 induction by cGMP appears to be stimulated by the protein kinase G pathway and may be mediated mainly via a CRE/AP-1 element in the rat HO-1 promoter.

Heme oxygenase-1 (HO-1) is the inducible form of the rate-limiting enzyme of heme degradation; it regulates the cellular content of heme. To investigate the role of the cAMP-dependent protein kinase (PKA) signaling pathway on hepatic HO-1 gene expression, primary rat hepatocyte cultures were treated with the PKA-stimulating agents dibutyryl-cAMP (Bt2cAMP), forskolin, and glucagon. HO-1 mRNA levels were induced by these agents in a time-dependent manner with a transient maximum after 6 hr of treatment. The induction of HO-1 was dose dependent, reaching a maximum at concentrations of 250 muM Bt2cAMP and 50 nM glucagon, respectively. The accumulation of HO-1 mRNA correlated with increased levels of HO-1 protein as determined by Western blot analysis. The Bt2cAMP-dependent induction of HO-1 mRNA expression was prevented by pretreatment with the PKA inhibitor KT5720 but not with the protein kinase G inhibitor KT5823. HO-1 mRNA induction by CdCl2 and heme was differentially affected by Bt2cAMP. Up-regulation of the HO-1 gene by Bt2cAMP occurred on the transcriptional level as determined by nuclear run-off assay and blocking of the Bt2cAMP-dependent induction of HO-1 mRNA by actinomycin D. Treatment with Bt2cAMP increased the half-life of HO-1 mRNA from 4.7 to 5.5 hr. Taken together, the results of the current study demonstrate that HO-1 gene expression is induced by activation of the cAMP signal transduction pathway via a transcriptional mechanism in primary rat hepatocyte cultures.

Glucagon is the major hormone activating glycogenolysis and gluconeogenesis both localized in the periportal, more aerobic zone of the liver. Accordingly, the glucagon receptor (GcgR) mRNA was found to be predominantly expressed in this area. In hepatocyte cultures high glucose concentrations as reached after a meal induced GcgR mRNA under arterial but not venous pO2. The induction by glucose was partially antagonized by insulin and unaffected by glucagon. The modulation by 02 of the glucose-dependent induction would contribute to the zonated expression of GcgR mRNA.

Hepatocytes around the afferent (periportal) vessels differ from those around the efferent (perivenous) vessels in their contents of key enzymes, and therefore have different metabolic capacities. Thus, the model of "metabolic zonation" proposes that the periportal cells produce glucose via glycogenolysis and gluconeogenesis and that the perivenous cells utilize glucose via glycogen synthesis and glycolysis. The periportal and perivenous cells receive different signal patterns, because substrates including oxygen and hormones are degraded and products and mediators are formed during passage of blood through the liver. The different signal patterns should be important for both short-term regulation of metabolic rates and for long-term induction and maintenance of the enzyme equipments by control of gene expression. From the periportal to the perivenous zone, the concentration of the signal oxygen falls corresponding to a drop from about 13 (arterial) to 9 (mixed periportal) and then to 4 (hepatovenous) volume% gas atmosphere. For short-term regulation of metabolism, in perivenous-like cells net glucose production measured over a period of two hours was observed below 2%, net glycogen synthesis above 4%, and net lactate utilization above 6% oxygen. In periportal-like cells net glucose formation and net lactate utilization increased sharply from anoxia to 6% oxygen and then only moderately. For long-term regulation of gene expression, the glucagon (cAMP)-dependent activation of the PCK gene was modulated by oxygen. The transcriptional rate, the abundance of mRNA and the enzyme activity were increased to higher levels under arterial rather than under venous oxygen. Conversely, the insulin-dependent activation of the glucokinase gene was negatively modulated by oxygen. A heme protein appeared to be involved in oxygen sensing, since CO mimicked the effects of oxygen on the PCK gene. Hydrogen peroxide was produced by hepatocytes as a function of oxygen tension; exogenously added, it mimicked the effects of oxygen on PCK gene induction. Therefore, the heme protein containing an oxygen sensor could be a peroxide producing oxidase. It is not known at present whether the same oxygen sensor is also involved in the short-term regulation by oxygen of hepatic carbohydrate metabolism. Transfection of PCK promoter-CAT gene constructs into primary hepatocytes showed that oxygen modulated PCK gene activation in the region of -277/+73. This modulation was not mediated by isolated cAMP responsive elements.

The glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene within two hours is modulated by O2 in rat hepatocytes. It was the aim of the present study to test if this short-term modulation by O2 of the glucagon induction might be influenced by long-term culture of hepatocytes for 24 hours under different O2 tensions prior to glucagon induction. Cells were precultured for 24 hours at arterial O2 (16% O2) or venous O2 (8% O2), then induced within two to four hours with 1 nM glucagon each at arterial or venous O2. In arterial O2 precultured cells PCK mRNA and activity were induced to 100% at arterial O2 and to about 60% at venous O2. In venous O2 precultured cells PCK mRNA and activity were induced only to about 70% at arterial O2 and to about 60% at venous O2. Transfected PCK promoter (-2500)-CAT constructs were activated by glucagon with the same long-term modulatory effects of oxygen as the endogenous PCK gene. Gel mobility shift assays with nuclear extracts prepared from hepatocytes and a PCK promoter fragment ranging from -149 to -42 bp revealed one complex with a higher DNA binding activity when extracts of cells precultured for 24 hours under venous O2 as compared to arterial O2 were used. Therefore, the short-term modulation by O2 of PCK gene activation by glucagon was widely lost during preculture at low O2. This diminution of O2 sensitivity of PCK induction may be due to a nuclear protein or proteins which are induced by perivenous O2 tensions and bind to the PCK promoter.

Liver glucokinase (GK) is localized predominantly in the perivenous zone. GK mRNA was induced by insulin maximally under venous O2 partial pressure (pO2) and only half-maximally under arterial pO2. CoCl2 and desferrioxamine mimicked venous pO2 and enhanced the insulin-dependent induction of GK mRNA under arterial pO2. H2O2 mimicked arterial pO2 and reduced insulin-induced GK mRNA under venous pO2 to the lower arterial levels. Thus the zonal O2 gradient in liver seems to have a key role in the heterogenous expression of the GK gene.

Heme proteins acting as oxidases which produce H2O2 have been proposed to function as O2 sensors. In order to find out whether the modulation by O2 of PCK gene activation and the stimulation of the ALD A gene by venous O2 operate via H2O2, the effects of different concentrations of H2O2 and catalase as H2O2 scavenger were studied in rat hepatocyte cultures under different O2 tensions. Primary hepatocytes were treated with 0.1 nM glucagon, 50 microM H2O2 and/or 100 micrograms/ml catalase each at arterial O2 or venous pO2. PCK mRNA was induced by glucagon maximally under arterial O2 and only half maximally under venous O2. ALD A mRNA was induced only by venous O2. H2O2 enhanced the induction of PCK mRNA to similar levels under venous O2 tensions and the induction of ALD A mRNA under both O2 was completely inhibited. Addition of catalase antagonized the actions of H2O2 completely. These findings support the hypothesis that an H2O2-generating heme protein is involved in the O2 sensing system regulating gluconeogenic and glycolytic gene expression in response to O2.

The enormous number of different liver functions are carried out by parenchymal and four main types of nonparenchymal cells, either alone or in cooperation. Although the liver tissue is uniform on the level of histology, it is heterogenous on the level of morphometry and histochemistry. This heterogeneity is related to the blood supply; cells located in the upstream or periportal zone differ from those in the downstream or perivenous zone in their equipment with key enzymes, translocators, receptors, and subcellular structures and therefore have different functional capacities. This is the basis of the model of metabolic zonation, according to which glucose release from glycogen and via gluconeogenesis, amino acid utilization and ammonia detoxification, protective metabolism, bile formation, and the synthesis of certain plasma proteins such as albumin and fibrinogen occur mainly in the periportal area, whereas glucose utilization, xenobiotic metabolism, and the formation of other plasma proteins such as alpha 1-antitrypsin or alpha-fetoprotein occur predominantly in the perivenous zone. The mor- phologic and functional heterogeneity is the result of zonal differences in the activation of the cellular genome caused by gradients in oxygen, substrate, hormone, and mediator levels, in innervation, as well as in cell-to-cell and cell-to-biomatrix interactions.

The glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene is modulated by oxygen. It was proposed that heme proteins might function as O2 sensors; their actions are impaired after replacement of the central Fe2+ ion by Co2+ and inhibition of heme synthesis by succinylacetone (SA). Therefore, the effects of CoCl2 and SA, alone and in combination, on the glucagon-dependent induction of PCK activity and PCK mRNA were investigated at different physiological oxygen tensions in primary rat hepatocyte cultures. The cells were exposed to 50 microM CoCl2 and/or 2 mM SA from 4-24 h. After addition of fresh media without CoCl2 or SA, PCK was induced with 1 nM glucagon. PCK activity and PCK mRNA were elevated to 100% at 16% O2 and to about 65% at 8% O2. CoCl2 reduced these increases to about 45% at 16% O2 and to about 35% at 8% O2. SA lowered the inductions to about 50% and 40% each at 16% and 8% O2. CoCl2 plus SA diminished the elevations to about 5% at both oxygen tensions. In the presence of CoCl2 and/or SA, ornithine decarboxylase induction by insulin was not impaired; lactate dehydrogenase did not leak from the cells, which in electron microscopical inspections had normal cell structures. These findings support the hypothesis that a heme protein is involved in the activation of the PCK gene and that it acts as an O2 sensor.