W. J. C. Geerts

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level.

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.

High protein diet induces pericentral glutamate dehydrogenase and ornithine aminotransferase to provide sufficient glutamate for pericentral detoxification of ammonia in rat liver lobules.

Abstract The liver plays a central role in nitrogen metabolism. Nitrogen enters the liver as free ammonia and as amino acids of which glutamine and alanine are the most important precursors. Detoxification of ammonia to urea involves deamination and transamination. By applying quantitative in situ hybridization, we found that mRNA levels of the enzymes involved are mainly expressed in periportal zones of liver lobules. Free ammonia, that is not converted periportally, is efficiently detoxified in the small rim of hepatocytes around the central veins by glutamine synthetase preventing it from entering the systemic circulation. Detoxification of ammonia by glutamine synthetase may be limited due to a shortage of glutamate when the nitrogen load is high. Adaptations in metabolism that prevent release of toxic ammonia from the liver were studied in rats that were fed diets with different amounts of protein, thereby varying the nitrogen load of the liver. We observed that mRNA levels of periportal deaminating and transaminating enzymes increased with the protein content in the diet. Similarly, mRNA levels of pericentral glutamate dehydrogenase and ornithine aminotransferase, the main producers of glutamate in this zone, and pericentral glutamine synthetase all increased with increasing protein levels in the diet. On the basis of these changes in mRNA levels, we conclude that: (a) glutamate is produced pericentrally in sufficient amounts to allow ammonia detoxification by glutamine synthetase and (b) in addition to the catalytic role of ornithine in the periportally localized ornithine cycle, pericentral ornithine degradation provides glutamate for ammonia detoxification.

Isomyosin expression patterns in tubular stages of chicken heart development: a 3-D immunohistochemical analysis

SummaryThe 3-D distribution of atrial and ventricular isomyosins is analysed in tubular chicken hearts (stage 12+ to 17 (H/H)) using antibodies specific for adult chicken atrial and ventricular myosin heavy chains, respectively.At stage 12+ (H/H) all myocytes express the atrial isomyosin; furthermore, all myocytes except those originally situated in the dorsolateral wall of the sinu-atrium coexpress the ventricular isomyosin as well. Moreover, it appears that recently incorporated myocardial cells at both ends of the heart tube start with a coexpression of both isomyosins. From stage 14 (H/H) onwards a regional loss of expression of one of either isomyosins is observed in the atrial and ventricular compartment. In this way the single isomyosin expression types that are characteristic for the adult working myocardium of the atria and ventricles arise. So, the isomyosin expression patterns are, unexpectedly, hardly useful to discriminate the different heart parts of the tubular heart. The ventricle, defined by its adult type of isomyosin expression, is even not detectable before stage 14 (H/H). Interestingly, interconnected coexpression areas, which may be precursor conductive tissues, are still present at stage 17 (H/H) in the outflow tract, the ventricular trabeculae, the atrio-ventricular transitional zone and in the sinuatrium.The pattern of isomyosin coexpression was found to correlate with a peristaltoid contraction and a slow conduction velocity, whereas single expression areas correlate with a synchronous contraction and a relatively fast conduction velocity.The possible implications of the changing isomyosin pattern for the differentiation of the tubular myocardium, in particular in relation to the development of the conductive tissues, will be discussed.

Image analysis and image processing as tools to measure initial rates of enzyme reactions in sections: distribution patterns of glutamate dehydrogenase activity in rat liver lobules.

To analyze regional differences in the activity of glutamate dehydrogenase in rat liver in situ, we developed an image recording and processing system for monitoring the formation of a colored final reaction product in time. All absorbance measurements of test and control reactions in time in consecutive sections were used to fit the data to a quadratic curve, with the derivative at t = 0 representing the initial velocity of formazan formation. The images of sections incubated for test and control reactions were topographically matched with an affine transformation using the positions of vessels as fiducials. Specific enzyme activity was calculated by subtracting the coefficients representing the initial velocity at corresponding locations in the test and control reactions and appeared to be 8 and 4 mumoles glutamate converted per min per cm3 of tissue at 20 degrees C in pericentral and periportal zones of fasted female rats, respectively. Those values are in agreement with biochemical data. The ability to construct two-dimensional images of cellular distribution patterns of enzyme activity in liver lobules is particularly useful for the study of metabolic zonation in this organ.

The local expression of adult chicken heart myosins during development

SummaryImmunofluorescence studies were performed on serial sections of three days embryonic chicken hearts using antibodies specific for adult atrial and ventricular myosin heavy chains respectively.The anti-ventricular myosin serum reacted with the entire myocardium showing a decreasing intensity going from the truncus arteriosus to the atrial part; however, the antiatrial myosin serum reacted weakly with the myocardium of the atrial part.Two other interesting observations were made, i) the anti-atrial myosin serum reacted with non-myocardial cells the cardiac jelly, ii) both antisera reacted with a thin myocardial layer, extending from the ventral wall of the atrial part via the medio-dorsal wall of the atrio-ventricular canal to the dorsal wall of the ventricular part

The local expression of adult chicken heart myosins during development. II. Ventricular conducting tissue.

SummaryThe development of the ventricular conducting tissue of the embryonic chicken heart has been studied using a previous finding that morphologically recognizable atrial conducting tissue coexpresses the atrial and the ventricular myosin isoforms. It is found that, by these criteria, at 9 days part of the ventricular conduction system consists of a myocardial ring located around the infundibula of the aorta and truncus pulmonalis. Part of this ring is formed by the retro-aortic root branch. The ring continues via the septal branch into the atrioventricular bundle and its branches, that all express both myosin isoforms. The retroaortic root branch could be traced back as a part of the myocardial wall of the truncus arteriosus at the 4 days embryonic stage.At the 16th day of development, the septal branch, atrioventricular bundle and left and right bundle branches no longer express the atrial isomyosin, but two bundles originating from the septal branch still express both isomyosins, one being the retro-aortic root branch, the other being only immunologically recognizable and directed to the ventral side of the truncus pulmonalis; this latter we call the pulmonary root branch. Both bundles are remnants of the myocardial ring.

Gender-dependent regulation of glutamate dehydrogenase expression in periportal and pericentral zones of rat liver lobules.

We studied the level(s) at which glutamate dehydrogenase (GDH; EC 1.4.1.2) expression is regulated in the livers of fed male and female rats. The cellular content of GDH mRNA, protein, and enzyme activity was determined quantitatively using image analysis for measurement of the absorbance in consecutive serial sections that were processed for in situ hybridization, immunohistochemistry, and enzyme histochemistry. In both males and females, GDH protein and activity patterns were similar, with pericentral values being twice as high as periportal values. GDH mRNA distribution patterns in female liver lobules reflected those of GDH protein and activity, but GDH mRNA distribution patterns in male rat livers were found to be homogeneous owing to a more than twofold lower cellular mRNA content in pericentral zones than in female rats. We conclude that gender affects GDH expression selectively in pericentral zones at posttranscriptional and pretranslational levels.

Creatine kinase isozyme expression in embryonic chicken heart

SummaryThe distribution pattern of creatine kinase (EC 2.7.3.2) isozymes in developing chicken heart was studied by immunohistochemistry.Creatine kinase M, which is absent from adult heart, is transiently expressed between 4 and 11 days of incubation. During that period, numerous muscular cells in the roof and septum of the atrium, in the interventricular septum and on top of the trabeculae cordis and at the rim of the outflow tract stain strongly with a polyclonal antibody that is specific for the M subunit. In the ventricle and outflow tract, the M-positive cells are found mainly subendocardially and in the right half, at the transition of conducting and working myocytes.Creatine kinase B, which is the predominant adult isozyme, is initially expressed to a high concentration in a small group of disperse myocardial cells in the upstream part of the inflow tract.When compared to the expression pattern of cardiac myosin heavy chains, the observed creatine kinase expression pattern suggests that M-positive cells are mainly found in areas that participate in the formation of cardiac conductive tissue, whereas B-positive cells are first found in areas that are involved in the generation of cardiac rhythm.

The dynamics of local kinetic parameters of glutamate dehydrogenase in rat liver

Kinetic parameters of glutamate dehydrogenase (GDH, EC 1.4.1.2) for glutamate were determined in periportal and pericentral zones of adult male and female rat liver lobules under normal fed conditions and after starvation for 24 h. GDH activity was measured as formazan production over time against a range of glutamate concentrations in serial cryostat sections using image analysis. Captured gray value images were transformed to absorbance images and local initial velocities (Vini) were calculated. A hyperbolic function was used to describe the relationship between substrate concentration and local Vini. Under fed conditions, Vmax values were similar in male and female rats (8±2 and 16±2 μmol min−1 cm−3 liver tissue in periportal and pericentral zones, respectively). Starvation increased Vmax, especially in pericentral zones of females (to 27±1 μmol min−1 cm−3 liver tissue). Under fed conditions, the affinity of GDH for glutamate was similar in male and female rats (2.5±0.5 mM and 3.5±0.8 mM in periportal and pericentral zones, respectively). Starvation had no effect on Km values in male rats, but in female rats affinity for glutamate decreased significantly in both zones (Km values of 4.0±0.1 mM and 8.6±0.8 mM, respectively). These local changes in the kinetic parameters of GDH indicate that conversion of glutamate to α-oxoglutarate cannot be predicted on the basis of GDH concentrations or zero-order activity in the different zones of liver lobules alone.

Differences in erythropoiesis in normal chicken and quail embryos

SummaryUsing antibodies against the fetal and adult forms of α- and β-globin, it has been shown that erythropoiesis in the para-aortic foci (PAF) constitutes a major species-specific difference between chicken and quail embryos. In quail embryos, para-aortic foci are rare, small and rather heterogeneous with regard to their erythropoietic and haemopoietic cell composition. In contrast, the PAFs in chicken embryos are abundant and consist of large numbers of erythropoietic cells.In both species a time difference (approximately 1 day) is observed between the first expression of the fetal α- and β-globin and the adult α- and β-globin in erythropoietic cells. Adult erythropoiesis in both species can be detected first in the stalk of the yolk sac; this is similar to the situation in mammalian and amphibian species. From this time onward the number of circulating adult erythrocytes increases steadily. Whereas in chicken, large intraembryonic foci that can serve as sources for these adult cells arise concomitantly, no such foci can be detected in quail embryos, suggesting that the quail yolk sac is a major source for these adult red blood cells.