Early and delayed effects of hydrocortisone and onapristone on intestinal brush-border enzymes and their sialylation and on thymus growth in suckling rats.

The antigestagen-antiglucocorticoid onapristone (ZK 98.299) was tested on three glucocorticoid-sensitive systems after hydrocortisone (HC) administration to suckling male rats, by determining onapristone (ZK)-induced inhibition of HC-provoked (1) increase of activities of intestinal brush-border enzymes, (2) desialylation of brush-border components and (3) thymolysis. HC acetate (75 mg/kg body weight (b.w.)) was injected s.c. on postnatal days 9 and 10, and ZK (150 mg/kg b.w.) on days 9, 10 and 11. The animals were killed on day 12 for assessing the early effect, or on days 15-17 for determining the delayed effect of HC and ZK. In all three systems the glucocorticoid effects were antagonized by ZK. The most sensitive to HC were systems 1 and 3, which exhibited both the early and the delayed effects. The most sensitive to the counteraction of ZK against administered HC was system 1, where HC was antagonized in both its early and delayed effects, whereas only delayed antagonistic action against administered HC was found in system 2. ZK alone had an early inhibitory effect on the activities of several brush-border enzymes and produced an early increase in thymus weight, accompanied by an increased DNA-protein ratio. No delayed effects of ZK alone on the three systems were observed.

The effect of an anti-glucocorticoid (ZK 98299) on thymus evolution and on hydrocortisone-induced thymolysis, intestinal brush-border enzymes and their desialylation in suckling rats.

1. The action in Onapristone infant male rats displays short-term and delayed effects. 2. Suppression of intestinal brush-border enzymes and increase of thymus mass were observed only immediately after 3-day treatment with Onapristone. After an additional 3 days its effect disappeared. There was no immediate or delayed effect of Onapristone on the desialylation of brush-border enzymes. 3. In the short-term and delayed effects, Onapristone suppressed the HC-provoked induction of several intestinal brush-border enzymes, especially alpha-glycosidases. In the delayed effect the drug also suppressed thymolysis induced by the exogeneously given glucocorticoid, and suppressed the HC-induced desialylation of a brush-border enzyme DP IV, which serves as a marker of the desialylation process. 4. These experiments seem to support a conclusion that the postnatal development of intestinal brush-border enzymes and the development of thymus in infant rats are controlled by endogeneously secreted glucocorticoids. 5. The control of sialylation of intestinal brush-border proteins by endogeneously secreted glucocorticoids during the postnatal development of the rat remains debatable.

Development of gastrointestinal functions.

Data are summarized about digestion and absorption of carbohydrates, lipids and proteins during mammalian perinatal development including human fetuses. Corresponding with the high fat intake in suckling rats, absorption of triglycerides was found to be approximately 2-3 times higher in suckling than in adult rats. Carnitine contents of the small intestinal mucosa of rats decrease postnatally, reaching adult levels at the time of weaning. Other studies suggested that gluconeogenesis may occur in the small intestine in the neonatal period. The intestinal mucosa of infant rats produces ketones; it was suggested that ketone production is to a large extent due to a breakdown of long-chain fatty acids. Studies dealing with the development of colonic sodium transport in rats are described. Other studies on the developing colon showed that the proximal colon resembles ileum during the early postnatal period. Developmental changes of the "specialization" of intestinal segments are reviewed. In all studies attention is given to the maturative effects of hormones of the adrenal cortex and thyroid gland (88 references).

Sialic acid--marker of development and differentiation.

The authors discuss the role of structural diversity of various sialic acids in glycoconjugates, i. e. in glycoproteins and glycolipids of cell membranes and of body fluids under various physiologic and pathologic conditions, as summarized in recent reviews. Furthermore they report the conclusions of their own experiments regarding desialylation of intestinal brush-border enzymes during postnatal development in the rat and its induction after hydrocortisone injection. The mechanism of desialylation was investigated in the differentiating cells of Lieberkuhn crypts and at the levels of sialyltransferase transcription and of glucocorticoid receptors. These results are correlated with the findings of other authors and compared with the effect of dexamethasone in organ cultures of the fetal rat intestine.

Dipeptidyl peptidase IV in cell proliferation and differentiation.

In this short review we discuss the present knowledge of a dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) physiological role, paying special attention to its involvement in complex processes of the cell cycle. Our own results and recent experimental project are overviewed in this respect too.

gamma-Glutamyltranspeptidase and dipeptidyl peptidase IV activity in the serum of normal and hepatoma-bearing chickens and in the plasma membranes from liver and hepatoma Mc-29.

Investigations on the activity of gamma-glutamyltranspeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) in the serum of healthy chickens and those bearing hepatoma Mc-29, and in liver and hepatoma plasma membranes were carried out. There was no difference in the serum enzyme activities of control and tumor-bearing chickens but the activity of GGT was twice higher and that of DPP IV 20 times lower in hepatoma plasma membranes than in chicken liver plasma membranes. Using thin-layer analytical isoelectric focusing in agarose gels it was established that the pI range of GGT from host serum and hepatoma plasma membranes was shifted to more acidic values. This could be interpreted as a specific feature for this enzyme considered as a tumor marker.

Dipeptidyl peptidase IV in the human lung and spinocellular lung cancer.

The isoelectric point and proportions of soluble and membrane bound dipeptidyl peptidase IV (DPP-IV) in human lung and spinocellular lung cancer tissue were tested. It was found that soluble DPP-IV is relatively less frequent in the cancer than in normal lung tissue. We demonstrated multiple molecular forms of DPP-IV in normal and cancer lung tissues, differing probably not only in the degree of sialylation. DPP-IV from lung cancer tissue consists of more basic molecular forms than that from normal lung tissue. These results suggest that the molecular properties of DPP-IV in normal and cancerous lung tissues may be different.

Regulation of sialylation of intestinal brush-border enzymes and of sialyltransferase activity in organ cultures by dexamethasone.

To investigate the direct effect of glucocorticoids on sialylation of intestinal brush-border hydrolases, explants of fetal and suckling rat intestine were maintained in serum-free or serum-containing organ culture with or without dexamethasone (Dx). Glucoamylase and dipeptidyl peptidase IV developed in organ culture from 18-day-old fetuses persisted in highly sialylated forms for 8 days irrespective of Dx presence, parallel in vivo development leading to less sialylated forms at the age of 6 days. In postnatal cultures the Dx-stimulated glucoamylase appeared in a new highly sialylated form never seen after the hormone application in vivo. These findings are in agreement with the elevation of bound sialyltransferase (ST) of cultured intestine in protein-free media. In serum-containing medium Dx stimulated the formation and release of soluble ST into the culture medium.

Actinomycin D suppresses hydrocortisone-induced changes in distribution and sialylation of brush-border enzymes in jejunal villi and crypts.

To investigate the regulatory role of hydrocortisone in the postnatal decline of sialylation of intestinal brush-border hydrolases, we applied actinomycin D, the well-known inhibitor of transcription, simultaneously with the hormone to 9-day-old rats. On day 12 actinomycin D inhibited hydrocortisone-induced changes in the profile of enzyme activities along the villus-crypt axis and suppressed the appearance of asialylated forms of enzymes manifested precociously in the differentiating crypt cells under the effect of the hormone. These findings were correlative with sialyltransferase activity along the villus-crypt axis.

Effect of sodium molybdate on the thyroxine-binding affinity of transport cytosol proteins.

The presence of sodium molybdate during tissue homogenization is known to increase the number of cytosol binding sites for glucocorticoids, progesterone, androgens and oestrogens. We wondered whether a phenomenon similar to this stabilization of steroid receptors would also occur in thyroxine-binding cytosol protein. We found that the presence of sodium molybdate (10 mmol/l) in rat adenohypophyseal cytosol increased its thyroxine-binding capacity by up to 96%. In the case of binding protein cytosol minus molybdate, Ka = 5.5 X 10(9) l.mol-1, whereas for cytosol plus molybdate Ka(1) = 6.0 X 10(9) l.mol-1 and Ka(2) = 3.0 X 10(10) l.mol-1. Cytosol prepared without molybdate did not contain a binding protein class with a higher Ka. The effect is stereo-specific and the LT4 bond is not displaced by DT4.

Tumour inhibitory effects of TCGF/IL-2/-containing preparations.

Supernatants from ConA-stimulated rat spleen cell cultures and from cultures of PMA-stimulated murine lymphoma subline EL-4TF were found to contain TCGF and to inhibit growth of a transplantable, MC-induced sarcoma MC11 in syngeneic mice. Tumour-inhibitory effects of the supernatants were dependent on local and repeated administration. Prior to use of the supernatants obtained from PMA-stimulated EL-4TF cell cultures, the dialysable PMA had to be removed; contamination with PMA was found to abolish the tumour-inhibitory effect of the supernatants and to produce enhancement of tumour growth. A significant tumour-inhibitory effect has also been obtained with partially purified TCGF prepared from culture supernatants of cloned EL-4TF cells by ammonium sulphate precipitation, ion-exchange (FPLC) chromatography, and AcA 44 Ultrogel filtration.

Effect of hydrocortisone on the desialylation of intestinal brush-border enzymes of the rat during postnatal development.

Hydrocortisone acetate or hemisuccinate (75 mg/kg body mass) applied to rats i.m. and/or s.c. on the 9th and 10th postnatal days causes a precocious decrease of sialic acid content of the small intestinal brush-border membrane. On the 15th postnatal day the bound sialic acid of the whole membrane fraction drops to almost half of the values of control animals and to one third of the control values for the papain-solubilized membrane proteins. The hydrocortisone effect is manifested on isoelectric focusing zymograms by a faster increase of pI of the solubilized brush-border enzymes on the 12th and 15th postnatal days.

Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum.

The heterogeneity of dipeptidyl peptidase IV (EC 3.4.14.5) was investigated in normal human serum. Thin-layer analytical isoelectric focusing revealed the presence of multiple molecular forms of the enzyme, their isoelectric points being in the pH range of 3.30-4.25. The maximum of enzyme activity appeared around pH 3.50. After treatment with neuraminidase the pI shifted to 4.70-5.40 with two maxima at pH 5.00 and 5.15. The Triton X-100 solubilized as well as the papain-treated-Triton X-100 solubilized enzyme from the whole human adult jejunal biopsy were also found to be heterogeneous. They focused--both before and after neuraminidase treatment--at pH values different from those of the enzyme of normal human serum. There was almost no pI shift after neuraminidase treatment of the intestinal enzyme from adult enterobiopsy. Electrophoresis in continuous polyacrylamide gradient gels as well as gel chromatography on Bio-Gel A-1.5m revealed two molecular forms of dipeptidyl peptidase IV in normal human serum. The estimated relative molecular mass of the major enzyme form was 250 000 in both the separation techniques used. On the other hand, the apparent relative molecular mass of the minor enzyme form was 450 000 as assessed by gradient gel electrophoresis, and 550 000, when estimated by gel chromatography. The Km values for glycyl-L-proline-4-nitroanilide as substrate with the major and minor forms of the serum enzyme were 1.60 +/- 0.39 X 10(-4) mol/l and 1.60 +/- 0.13 X 10(-4) mol/l, respectively. Our results indicate that the dipeptidyl peptidase IV in normal human serum is a heterogeneous enzyme as far as its charge and molecular size are concerned.

An assay of dipeptidyl peptidase IV activity in human serum and serum of pregnant women with glycyl-L-proline-1-naphthylamide and other glycyl-L-proline-arylamides as substrates.

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.