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.

Summary. 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< 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.Key words: Basement membrane, collagen type IV, serum analysis, diabetes.Basement membrane thickening during the course of diabetes mellitus is a well established fact [1]. In vivo studies with radioactively labelled precursor amino acids have demonstrated increased glomerular basement membrane synthesis in diabetic rats [2][3][4]. Such experiments are, however, laborious and of limited usefulness for foUowup studies. Recently radioimmunochemical methods have become available to study basement membrane metabolism by measuring serum concentrations of basement membrane proteins like 7S-collagen, the N-terminal cross-linking domain of type IV collagen, and laminin or laminin fragments [5]. Earlier studies have shown increased serum levels of 7S-collagen in streptozotocin diabetic rats which are normalized by insulin treatment [6]. It remains unknown whether the increased serum levels are due to (a) a higher rate of synthesis of basement membrane collagen; (b) different clearance rates of 7S-collagen in diabetic and normal rats; or (c) an increased degradation of basement membrane collagen. In this study we report on experiments which favour the first interpretation. Materials and methods AnimalsNormally-fed male Wistar rats of the strain HOE WISKF (Hoechst AG, Frankfurt/Main, FRG), initially weighing 200-250 g, were used throughout the study. Diabetes was induced by injection of freshly dissolved streptozotocin (70mg/kg, Calbiochem, Frankfurt/Main, FRG) into the tail vein. Rats treated with insulin were subcutaneously injected with 9 IU Insulin (Long Insulin, Hoechst AG. Frankfurt/ Main, FRG) twice daily (1~ of the dose in the morning, % of the dose in the late afternoon). MethodsThe N-terminal and C-terminal domains of type IV collagen, 7S-collagen and non-collagenous domain t (NCt) were isolated from a transplantable murine tumour (EHS), which produced basement membrane material. The material was purified ...

Dimethylnitrosamine (DMN)-induced liver fibrosis was used as an experimental model to study the relationship between serum concentrations of the N-terminal propeptide of type III procollagen [S-Pro(III)-N-P] and the N-terminal (S-7S) and C-terminal (S-NC1) domains of type IV collagen and hepatic concentrations of type III and IV collagen mRNAs. Increases in S-Pro(III)-N-P, and especially in the two type IV collagen-related antigens, were found to be early events in the formation of DMN-induced hepatic fibrosis. The mean concentration of S-Pro(III)-N-P was 120% of the control mean on day 7 of DMN treatment, 230% on day 14 and 250% on day 21. The corresponding values for S-7S were 260, 950 and 1100% and, for S-NC1, 310, 820 and 1000%. All these changes were very similar to those found in the hepatic concentrations of the respective mRNAs. These data support a previous suggestion that an enhanced production of basement-membrane (type IV) collagen is an early event in the development of the DMN-induced hepatic fibrosis. The results also indicate that S-7S and S-NC1 are very sensitive indicators of changes in type IV collagen metabolism. Data obtained in gel-filtration experiments for these three serum antigens were consistent with the suggestion that all three antigens are mainly derived from the synthesis of the respective collagens.

No abstract

No abstract