A radioimmunoassay for the N-terminal propeptide of rat procollagen type III. Application to the study of the uptake of the N-terminal propeptide of procollagen type III in isolated perfused rat liver.

Antibodies against a synthetic peptide representing the 14 C-terminal amino acids of the N-terminal propeptide of rat and bovine procollagen type III were raised in rabbits and used to develop a radioimmunoassay. N-Terminal propeptide of procollagen type III, purified from calf skin, served as standard and tracer material. The IC50 of the standard inhibition curve was 2.1 micrograms/l, the lower limit of detection about 0.4 microgram/l, interassay variation was 8.5% and the intraassay variation 6.6% in typical experiments. Three peaks of antigenicity were detected in rat serum after gel chromatography. One peak coeluted with purified N-terminal propeptide of procollagen type III, one peak contained material approximately twice this size, and one peak eluted close to the void volume of the column. The antigen concentration in rat serum decreased in an age dependent manner. Rat, bovine, sheep and minipig serum antigen was sufficiently crossreactive to allow the application of the assay to these species, whereas human, goat and guinea pig samples were not. The degradation product Col 1, causing non-parallel inhibition in commercially available assays for human samples was not recognized since it does not contain the epitope represented by the synthetic peptide. The assay was used to study the half-life of bovine and endogenous N-terminal propeptide of procollagen type III in isolated perfused rat liver. [125I]-labeled antigen was cleared rapidly from the perfusate (t1/2 less than 5 min). The bovine antigen was removed from the perfusate with a half-life of 15 +/- 4 min. Endogenous propeptide was perfused for 120 min with little change in concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of prolyl hydroxylation and procollagen processing in chick-embryo calvaria by a derivative of pyridine-2,4-dicarboxylate. Characterization of the diethyl ester as a proinhibitor.

The biochemical and morphological consequences of procollagen prolyl 4-hydroxylase inhibition by pyridine-2,4-dicarboxylic acid (2,4-PDCA) and its diethyl ester (diethyl-2,4-PDC) were studied in chick-embryo calvaria, which predominantly synthesize type I collagen. Half-maximal inhibition of tissue hydroxyproline formation required 650 microM-2,4-PDCA, whereas the Ki with respect to chicken prolyl 4-hydroxylase in vitro was 2 microM. In contrast, half-maximal inhibition was caused by 10 microM-diethyl-2,4-PDC in the intact calvaria, although chicken prolyl 4-hydroxylase in vitro was not inhibited even at 1 mM. The collagenous material produced in the presence of diethyl-2,4-PDC showed an altered 'melting' profile and a lowering of the transition temperature by 10 degrees C, indicating misalignment and thermal instability of its triple-helical structure. Amount and electrophoretic mobility of procollagen type I chains were increased in a dose-dependent manner. The amounts of partially processed species and alpha-chains were decreased, without change in mobility. This marked effect on procollagen-collagen conversion in the intact calvaria suggests that the underhydroxylated collagenous material generated in the presence of diethyl-2,4-PDC is resistant to or acts as endogenous secondary inhibitor of type I procollagen N-proteinase. Electron microscopy of treated calvaria cells showed dilated rough endoplasmic reticulum and numerous phagolysosomes, indicating intracellular retention and lysosomal degradation of the newly synthesized underhydroxylated collagenous material. In summary, these results identify 2,4-PDCA and diethyl-2,4-PDC as the first prolyl 4-hydroxylase-directed inhibitor/proinhibitor pair that affects intra- and extra-cellular events during collagen formation.

Beneficial effects of inhibitors of prolyl 4-hydroxylase in CCl4-induced fibrosis of the liver in rats.

S 0885 and HOE 077 inhibit CCl4-induced liver fibrosis in rats, as shown by significantly reduced hydroxyproline content of the liver and improved liver histology. Mortality of drug-treated animals is significantly diminished. Serum collagen parameters correlate well with the hydroxyproline content of the liver and can be used as noninvasive markers for the fibrotic process. HOE 077 is a proinhibitor, which by itself does not inhibit prolyl 4-hydroxylase. HOE 077 is well absorbed from the gastrointestinal tract. It is taken up by rat liver and is converted to the active metabolites. At a concentration of 1 mM, HOE 077 does not affect collagen synthesis in human fibroblasts, bovine chondrocytes and chicken calvaria. At therapeutic doses the compound does not reduce collagen content of kidney, lung, aorta, femur epiphysis, skin and tendon of the rat, validating the high specifity of the liver selective prodrug/inhibitor conversion. From animal experiments, a human daily dose of 0.5-1 g can be extrapolated.

Increased deposition of basement membrane macromolecules in specific vessels of the spontaneously hypertensive rat.

The genetically determined chronic hypertension manifested in the Spontaneously Hypertensive Rat (SHR) serves as a useful animal model for the study of human essential hypertension. One of the earliest morphologic changes observed in the vasculature of the SHR is the development of a thickened subendothelial space (SES). Neither the biochemical composition nor the anatomic distribution of this early subendothelial deposit has been definitively determined. By combining morphologic, immunologic, and molecular biological approaches, it was demonstrated that by 15 weeks the acellular subendothelial thickening in the vasculature of the SHR results at least in part from the increased synthesis and deposition of basement membrane macromolecules. Moreover, rather than being manifest systemically, this early connective tissue lesion appears to be localized primarily to the aorta and major branches off the aortic arch.

Assessment of disease activity in essential cryoglobulinemia by serum levels of a basement membrane antigen, laminin.

When compared with 40 normal controls, 46 patients with essential cryoglobulinemia had higher serum concentrations of a basement membrane antigen, laminin fragment P1 (LP1). Serum LP1 values were more pronounced in patients with visceral involvement, compared with those with skin vasculitis only. Asymptomatic patients or those with arthralgia usually had normal values. Increased disease activity was associated with a significant increase in LP1 (P less than 0.01). For evaluation of disease activity, determination of serum LP1 was a more sensitive measure than any complement factor assay.

Serum concentration of laminin, and course of the disease in patients with various malignancies.

Serum concentration of laminin was measured radioimmunologically in 96 patients suffering from various malignancies. Laminin levels were significantly elevated in patients with carcinomas and leukemias, but not in patients with sarcomas or lymphomas when compared with healthy controls. A good correlation could be found between serum laminin concentration and response to therapy in patients with carcinoma and leukemia. Elevated laminin levels were associated with a progressive course of the tumor condition. Furthermore, a close correlation has been detected between serum concentrations of laminin and carcinoembryonic antigen (CEA) in patients with carcinoma of the colon. The serum laminin level seems to be a valuable parameter for observation of the course of certain malignancies.

Type IV collagen antigens in serum of rats with experimental fibrosis of the liver.

Specific and sensitive radioimmunoassays for the N-terminal (7S-collagen) and C-terminal (NC 1) crosslinking domains of type IV collagen were used to study the effects of prolonged treatment of rats with CCl4 on the concentrations of these antigens in serum. After four to six weeks of CCl4 treatment a highly significant increase of type IV collagen antigen levels in serum was to be seen. These elevated levels exhibited a significant correlation to the increased amounts of hydroxyproline in liver samples, when the samples were taken during CCl4 treatment. Analysis of the molecular weight distribution of serum antigens by gelfiltration revealed for 7S-collagen a bimodal distribution, whereas NC 1 eluted mainly as one peak. This peak coeluted with the high molecular weight peak of 7S-collagen, suggesting that this peak might represent intact type IV collagen. The lower molecular weight peak of 7S-collagen antigen eluted at a position which was comparable to standard 7S-collagen. Determination of type IV collagen antigens in serum might be of diagnostic usefulness for the follow up and treatment control of hepatic fibrosis.

Radioimmunoassay of laminin in serum and its application to cancer patients.

In this sensitive radioimmunoassay for laminin fragment P1 (purified from pepsin extracts of human placenta), monovalent antibody fragments obtained from rabbit antisera against the laminin component are used. The inhibition assay showed a low intra- and interassay variability, and the inhibition curves for various serum samples had parallel slopes. Molecular-sieve chromatography demonstrated heterogeneity of the laminin antigen in serum. For quantification we defined arbitrary units based on the amount of antigenic material present in normal human serum. Sera from 361 tumor patients showed increased values in about half of the cases. Correlation with increased concentration of carcinoembryonic antigen was poor (r = 0.259). The assay may be useful for diseases involving basement-membrane metabolism.

Type IV collagen antigens in serum of diabetic rats: a marker for basement membrane collagen biosynthesis.

The nature of type IV collagen antigens in the serum of streptozotocin diabetic rats was studied using radioimmunoassays for the N-terminal (7S-collagen) and C-terminal domain of type IV collagen. Type IV collagen antigen crossreacting with antibodies to the C-terminal domain was elevated from 32.0 +/- 5.36 ng/ml (n = 10) in serum of normal rats to 94.9 +/- 24.5 ng/ml (n = 10, P less than 0.0001) in serum of streptozotocin diabetic rats and could be normalized to 40.1 +/- 8.30 ng/ml (n = 18) by insulin treatment. Molecular sieve chromatography of serum demonstrated a high molecular weight fraction containing the C-terminal and N-terminal domains and smaller material containing only the N-terminal domain. Degradation of the high molecular weight material by collagenase indicates that it consists of intact collagen type IV. Its relative proportion increased from 42% to 54% 4 weeks after diabetes induction. Together with unaltered clearance rates of 7S collagen in normal and diabetic rats, the data suggest that the increase of collagen type IV antigens in diabetic states reflects increased synthesis of collagen type IV.

Distinctive roles of oleate and glucagen in gluconeogenesis.

1. The effect of oleate on the subcellular distribution of ATP, 2-oxoglutarate, glutamate, citrate, malate and phosphoenolpyruvate was studied in hepatocytes from rats starved for 48 h by applying a modified digitonin method. The results markedly differ from those observed after glucagon [Siess, E. A., Brocks, D. G., Lattke, H. K., and Wieland, O. H. (1977) Biochem J. 166, 225-235]. Total cellular amounts and the distribution of ATP and 2-oxoglutarate remained unchanged. In the mitochondrial matrix glutamate was increased, while mitochondrial phospho-enolpyruvate was decreased. Citrate and malate were increased both in the mitochondrial and cytosolic space. 2. In contrast to the effect of glucagon, gluconeogenesis from dihydroxyacetone, fructose or glutamine was not stimulated by oleate. Gluconeogenesis from propionate was even inhibited by the fatty acid. 3. The stimulation by glucagon of glucose production from dihydroxyacetone or fructose was undiminished in biotin-deficient hepatocytes. Glucose formation from lactate, however, was stimulated only in biotin-substituted hepatocytes. 4. The results indicate that oleate stimulates gluconeogenesis by increasing pyruvate carboxylase activity (EC 6.4.1.1), whereas glucagon displays a more complex mode of action.

Regulation of net triacylglycerol synthesis by metabolic substrates in isolated rat liver cells.

Triacylglcerol metabolism was studied in isolated rat hepatocytes using a fully enzymatic method to masure cellular glyceride-glycerol. With this method 40 and 50 MUmol triacylglycerol were found per g cellular protein in liver cells from fed and starved rats, respectively, comparable to values obtained after organic solvent extraction and alkaline hydrolysis of neutral lipids. Carbohydrate refeeding of animals increased triacylglycerol levels in hepatocytes to 80 mumol. Upon incubation without fatty acids a 15% decrease in cellular triacylglycerol was found in 60 min. When 1mM oleate or palmitate were added cellular triacylglycerol increased. The rates of net triacylglycerol increased. The rates of net triacylglycerol synthesis were not significantly different with oleate and palmitate. Starvation reduced the rates from both fatty acids, whereas carbohydrate refeeding led to a marked increase in net triacylglycerol synthesis. Besides 20mM glucose, 5mM L-lactate and 5mM fructose stimulated triacylglycerol synthesis from fatty acids. THe stimulatory effect of lactate was higher in hepatocytes from starved animals, so that the differences in triacylglycerol synthesis between liver cells from fed and starved rats were abolished. Fatty acids taken up and not recovered in newly formed triacylglycerol were released as ketone bodies. When radioactive lectate was offered to cells from starved rats, label incorporated into neutral lipids was exclusively recovered from the glycerol moiety of triacylglycerols. 5mM ethanol which alone increased fatty acid esterification, reduced the stimulatory effect of lactate but increased the effect of fructose on net triacylglycerol formation. These findings indicate that esterification rate in liver cells from starved rats can be limited by availability of alpha-glycerophosphate, which is provided by glyceroneogenesis. The possible physiological significance of these findings is discussed with regard to liver cell heterogeneity and nutritional adaptation of liver triacylglycerol formation.

Validity of the digitonin method for metabolite compartmentation in isolated hepatocytes.

1. A modification of the digitonin method of Zuurendonk & Tager (1974) (Biochim. Biophys. Acta 333, 393-399) (i.e. the 'convaentional' method) was developed that allows the fractionation of isolated hepatocytes at -5 degrees C (i.e. 'low-temperature' method). 2. With respect to compartmentation of adenine nucleotides, glutamate and citrate, the two methods yielded very similar results. 3. In contrast, the mitochondrial amounts of aspartate and malate, as revealed by the low-temperature method, were about twice as high as those found by the conventional procedure. No change in the total cellular content occurred. 4. With n-butylmalonate and glisoxepid present in the conventional digitonin medium, significantly higher amounts of malate and aspartate respectively were found in the mitochondrial pellets. The results obtained by the low-temperature method, however, were not influenced by the these inhibitors. 5. It is concluded that under the conventional conditions of cell fractionation no appreciable redistribution of adenine nucleotides, glutamate and citrate occurs.

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats.

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.

Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats.

In hepatocytes from 48 h-starved rats identical glucagon dose-response curves were obtained for the stimulation of gluconeogenesis from lactate, for ketogenesis and for the decreasing of the C5-dicarboxylate pool. Glucagon (20 nM) caused a 5-fold increase in 3-hydroxybutyrate formation, but decreased acetoacetate production 50% of that of the control. In hepatocytes from biotin-deficient rats glucagon no longer stimulated gluconeogenesis from lactate, but still produced its effects on the mitochondrial redox state and the C5-dicarboxylate pool. The results suggest that the primary site of the hormone action on gluconeogenesis is located within the mitochondria rather than in the cytosol.

Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate.

1. The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation. 2. In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40-50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate. 3. In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25%. Other metabolites remain unchanged. After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70%. The [NAD(+)]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same. Glucagon shifts this ratio and also that of the [NADP(+)]/[NADPH] couple towards a more reduced state. 4. In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50%. Other metabolites are not changed by glucagon. Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values. The [NAD(+)]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former. 5. Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent. Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon. 6. The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis.