Alterations in hyaluronan synthesis during developing joint cavitation.

The mechanisms essential for generating diarthrodial joint cavities between skeletal elements in developing limbs remain enigmatic. Histochemical localization of hyaluronan (HA) at joint interzones concomitant with cavitation led to the postulation that HA may be pivotal in this process. HA synthesis involves the transfer of UDP-glucuronate and UDP-N-acetyl glucosamine to nascent HA by HA synthase. Uridine diphosphoglucose dehydrogenase (UDPGD) activity is responsible for the prior conversion of UDP-glucose to UDP-glucuronate. We have assessed the relationship between the appearance of HA and enzyme activities (quantitatively where possible) involved in HA synthesis during metatarsophalangeal joint development in embryonic chicks. Microspectrophotometric assessment of UDPGD activity using an in situ biochemical assay indicated that cells immediately adjacent to forming cavities contained increased UDPGD activity, which was subsequently maintained after cavitation. Immunocytochemistry showed that high levels of expression of HA synthase were localized to these same cells. In addition, radiolabeled sulfate autoradiography showed that cells bordering developing cavities incorporated relatively little sulfate, suggesting that UDP-glucuronate is utilized in the synthesis of undersulfated or non-sulfated glycosaminoglycans. These results indicate that the differentiation of cells bordering presumptive spaces may involve alterations associated specifically with differential synthesis of HA, which appears to be a primary event in joint cavity formation.

Impairment of UDP-glucose dehydrogenase and glucuronidation activities in liver and small intestine of rat and guinea pig in vitro by piperine.

The effects of piperine, a major ingredient of black pepper, on UDP-glucose dehydrogenase (UDP-GDH) and glucuronidation potentials of rat and guinea pig liver and intestine were studied. Piperine caused a concentration-related strong inhibition of UDP-GDH (50% at 10 microM) reversibly and equipotently, in both tissues. Partially purified rat liver UDP-GDH was used to obtain the kinetic values at pH optima of 9.4 and 8.6. At pH 9.4: KmUDP-glucose = 15 microM, Vmax = 5.2 nmol NADH/min/mg protein, Ki = 6 microM. With NAD, a Ki of 16 microM was obtained. At pH 8.6: Km = 35 microM, Vmax = 7.5 nmol, Ki = 15 microM. In all of these cases, piperine caused non-competitive inhibition. Data from structure-activity comparisons of piperine analogs indicated that the presence of conjugated double bonds in the side chain of the molecule is a factor in piperine inhibition. However, the UDP-glucuronic acid (UDPGA) contents were decreased less effectively by piperine in isolated rat hepatocytes compared with enterocytes of guinea pig small intestine. Piperine at 50 microM caused a marginal decrease of UDPGA in hepatocytes when the rate of glucuronidation of 3-hydroxybenzo[a]pyrene (3-OH-BP) decreased by about 40%. The decrease obtained at 10 microM piperine in intestinal cells was comparable to that obtained at 50-100 microM in hepatocytes. UDP-glucuronosyltransferase (UGT) activities towards 3-OH-BP (UGT1A1) and 4-OH-biphenyl (UGT2B1) were also determined. Piperine did not affect the rate of glucuronidation of 4-OH-biphenyl in rat liver, whereas that of 3-OH-BP was impaired significantly. In guinea pig small intestine, both these activities were inhibited significantly requiring less than 25 microM piperine to produce a more than 50% inhibition of UGT(s). The results suggested that (i) piperine is a potent inhibitor of UDP-GDH, (ii) inhibition is offered exclusively by the conjugated double bonds of the molecule, and (iii) piperine exerts stronger effects on intestinal glucuronidation than in rat liver.

Giant cells in arthritic synovium.

OBJECTIVES: Giant cells are commonly present in inflamed synovium, often in close association with the intimal layer. The nature of these multinucleate cells has been reassessed using new cytochemical and immunochemical techniques. METHODS: Cryostat sections of non-inflamed, rheumatoid arthritic and osteoarthritic synovia were analysed for the presence of CD68 and non-specific esterase, markers associated with macrophages; activity of uridine diphosphoglucose dehydrogenase, associated with fibroblast-like synoviocytes; and tartrate resistant acid phosphatase and the vitronectin receptor subunit CD51, associated with osteoclasts. RESULTS: Giant cells were not seen in non-inflamed tissue. In diseased tissue giant cells in the intimal layer fell into two major groups: CD68 negative or dull cells with high uridine diphosphoglucose dehydrogenase (UDPGD) activity suggestive of true synoviocyte polykaryons; and CD68 positive cells with low UDPGD activity suggestive of macrophage polykaryons. The two groups were seen in samples from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA), but the former were more prominent in OA and the latter in RA. Most CD68 positive giant cells also showed tartrate resistant acid phosphatase activity and prominent expression of CD51. As such they were histochemically indistinguishable from osteoclasts, but their bone resorbing capacity remains unknown. CONCLUSIONS: Giant cells in arthritic synovium appear to be of two types, one related to true synoviocytes and one to macrophages.

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.

Uridine diphosphoglucose dehydrogenase activity in normal and osteoarthritic human chondrocytes.

The activity of uridine diphosphoglucose dehydrogenase (UDPGD), a key enzyme in the synthesis of proteoglycans was measured by a quantitative cytochemical method in normal and in osteoarthritic (OA) human cartilage. Normal adult chondrocytes showed low UDPGD activity, which was about half the activity of glucose-6-phosphate dehydrogenase of the same specimens. No significant increase in UDPGD activity was noted in OA chondrocytes. The lack of significantly enhanced UDPGD activity in OA indirectly agrees with studies showing normal 35S uptake in this disease and might explain in part the inability of chondrocytes to cope with continuous proteoglycan loss.

Mechanism of decreased acetaminophen glucuronidation in the fasted rat.

The mechanism by which an acute fast decreases the glucuronidation of hepatotoxic doses of acetaminophen in the rat was examined. Fasting did not depress the level of the enzyme, glucuronyl transferase, or the basal level of the co-substrate, UDP-glucuronic acid (UDPGA). Administration of a hepatotoxic dose of acetaminophen rapidly depleted UDPGA levels in both fed and fasted rats to the same nadir. Fed and fasted rats differed in that the rate of repletion of UDPGA levels was markedly slower in fasted rats. The total hepatic levels of UDP-glucose dehydrogenase and its cofactor, NAD+, were not decreased by fasting. In fasted rats, hepatic levels of the UDPGA precursor, UDP-glucose, were approximately 60% those of fed rats both before and after a hepatotoxic dose of acetaminophen. In fed rats, acetaminophen induced a marked depletion of hepatic glycogen levels and a dramatic increase in blood glucose levels. Acetaminophen induced a similar marked increase in blood glucose levels in fasted rats in spite of the fact that they lacked hepatic glycogen. It is concluded that the fasting-induced decrease in the glucuronidation of hepatotoxic doses of acetaminophen results from decreased production of UDPGA. The decreased synthetic capacity for UDPGA does not appear to be due to the inability of the liver to produce glucose units per se, but rather to the fasting-induced altered activities of the enzymes of carbohydrate metabolism which, in turn, alter the fate of glucose-6-phosphate derived from gluconeogenesis.

Hepatic UDP-glucuronic acid regulation during acetaminophen biotransformation in rats.

Acetaminophen (AA) glucuronidation is capacity limited in several species after administration of high doses and previous data indicate that this phenomenon is due probably to a decrease in the concentration of the reaction cosubstrate UDP-glucuronic acid in liver. The rate-limiting determinant in UDP-glucuronic acid synthesis during AA glucuronidation is not known. The objective of the present study was to determine whether UDP-glucuronic acid synthesis during AA biotransformation is restricted by the supply of UDP-glucose or is limited by UDP-glucose dehydrogenase activity. Adult male Sprague-Dawley rats were injected with 600 mg/kg i.p of AA and liver was obtained 30, 60, 120 and 240 min later for quantitation of UDP-glucose, glycogen and UDP-glucuronic acid. AA was found to decrease markedly UDP-glucuronic acid concentration in liver 30, 60 and 120 min after injection (28, 52 and 58% of control values, respectively). In contrast, hepatic UDP-glucose levels were not altered after 30 min, but were decreased to 55 and 68% of control values 60 and 120 min after AA administration. Glycogen concentrations were decreased at the 30-min time interval only (78% of control). Therefore, maximal depletion of UDP-glucuronic acid occurred when UDP-glucose levels were not affected. UDP-glucose dehydrogenase is subject to product inhibition by NADH and UDP-glucuronic acid and it is possible that NADH accumulates during rapid utilization of UDP-glucuronic acid. Consequently, the effects of AA on cytosolic NADH/NAD ratios in liver were examined by determining the lactate/pyruvate ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of dietary protein quantity on the activity of UDP-glucuronyltransferase and its physiological significance in drug metabolism.

Low dietary protein has been shown to induce the activity of rat hepatic UDP-glucuronyltransferase (UDPGTase) as measured in vitro. The assay of UDPGTase in vitro is hampered by the need to solubilize the microsomal membrane, without destroying the physiological significance of the measurements. The present work was to determine the effect of dietary protein on the activity of UDPGTase and on the activity of UDP-glucose dehydrogenase. Chloral hydrate induced sleeping time was used as a bioassay for UDPGTase, confirming the physiological significance of the in vitro analysis. Sixty male rats were maintained on three different protein levels (7.5, 15, and 45%) for 16 days. Fifteen rats from each group were sacrificed and hepatic UDPGTase, cytochrome P-450, UDP-glucose dehydrogenase, and alcohol dehydrogenase were assayed. Five rats from each group were dosed with 7.5% chloral hydrate (4.8 mL/kg body weight) to measure sleeping time. Rats on 7.5% dietary protein had significantly higher UDPGTase activity than rats fed either 15 or 45% protein diets. These differences in enzyme activity in vitro correlated with the differences in chloral hydrate sleeping time. Dietary protein was not found to affect the activity of UDP-glucose dehydrogenase as measured in vitro.

Effects of Fructus gardeniae extract on hepatic function.

This article discusses the effects of Fructus Gardeniae extract on hepatic function. Fructus Gardeniae extract manifested no hepatotoxic effects on rats, as shown by alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase studies. Fructus Gardeniae extract failed to activate the UDP-glucuronyltransferase system; whereas in hyperbilirubinemic state the enzyme was activated, presumably by substrate induction. Fructus Gardeniae extract increased the activity of UDP-glucose dehydrogenase, which would result in an increase in availability of UDP-glucuronic acid intracellularly, BSP clearance study showed an unexpected impairment of hepatic uptake of the dye after extract treatment. The action mechanism involved in lowering of serum bilirubin level by Fructus Gardeniae extract may well be complex; it is probably acting on a locus other than glucuronyl transferase.