David S. Newcombe

Cartilage cyclic nucleotide phosphodiesterase: Inhibition by anti-inflammatory agents

Abstract Soluble 3′,5′-nucleotide phosphodiesterase (PDE) activity is described in chicken epiphyseal and articular cartilage. Kinetic studies of these enzymes demonstrate a high and low K m for the substrates, adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Epiphyseal and articular PDE activities are inhibited by those anti-inflammatory agents which are potent inhibitors of the enzyme, prostaglandin synthetase (PS). Specificity of this inhibition is indicated by the activity of these agents against the low K m enzyme. Other anti-inflammatory agents with significantly less potency as PS inhibitors or with no activity against prostaglandin synthetase are found to be either inactive or relatively less potent as inhibitors of cartilage PDE activity. A variety of other anti-inflammatory or anti-rheumatic agents, which are not known to affect prostaglandin synthetase activity, are poor inhibitors of cartilage PDE activity. These data provide insight into the mechanism of action of certain anti-inflammatory agents and into the relationships between prostaglandins and inflammatory reactions.

Hydrocortisone inhibition of the bradykinin activation of human synovial fibroblasts

Human synovial fibroblasts in culture respond to bradykinin with a 20-fold increment in intracellular cyclic AMP concentrations, however bradykinin does not directly activate adenylate cyclase activity in a particulate fraction derived from these cells. Bradykinin evokes a release of labeled arachidonic acid and prostaglandins E and F from synovial fibroblasts pre-labeled with 3H-arachidonic acid. Hydrocortisone inhibits the bradykinin induced increment in cyclic AMP and the release of arachidonic acid and prostaglandins E and F from synovial fibroblasts. Indomethacin, which also inhibits the cyclic AMP response to bradykinin, has no effect on the release of arachidonic acid from synovial fibroblasts. Indomethacin does, however, inhibit the quantity of prostaglandins released into the medium. These studies support the hypothesis that bradykinin does not activate human synovial fibroblast adenylate cyclase, but presumably activates a phospholipase whose products in turn result in the synthesis of prostaglandins. These and other investigations also suggest that a product(s) of the prostaglandin pathway causes the increment in cyclic AMP.

The effect of anti-inflammatory agents on human synovial fibroblast prostaglandin synthetase

Human synovial fibroblast prostaglandin synthetase activity is inhibited by many different non-steroidal anti-inflammatory agents. Aspirin, indomethacin and phenylbutazone significantly inhibit both PGE1, PGE2 and PGF1alpha and PGF2alpha synthesis; whereas penicillamine and aurothioglucose are more potent inhibitors of the F prostaglandins. Histidine and antimalarials do not inhibit, to a significant degree, human synovial prostaglandin synthetase activity. Hydrocortisone has no direct effect on prostaglandin synthetase activity. No changes in synthetase activity are observed when synovial cells are incubated with hydrocortisone, and the prostaglandin synthetase system subsequently isolated and assayed. The proposed inhibitory effects of hydrocortisone on prostaglandin production by synovium may be the resulf of an alteration of enzyme substrate or cofactor concentration rather than a direct effect on prostaglandin synthetase.

Human synovial fibroblasts: the relationships between cyclic AMP, bradykinin, and prostaglandins

Human synovial fibroblasts in culture respond to bradykinin (8×10−9 M) with an increment in intracellular cyclic AMP concentration. These bradykinin (BK) concentrations are comparable to levels of the nonapeptide found in pathological synovial effusions. The cyclic AMP response to BK is enhanced by a heat stable factor(s) in fetal calf serum (FCS) and by the addition of arachidonic acid (AA) to monolayer cultures incubated in serum-free media.Synovial fibroblasts initially treated with BK are refractory to rechallenge with this agent as measured by the absence of an increment in cyclic AMP. These BK refractory cells do respond with significant increment in cyclic AMP to challenge with prostaglandin E1 (PGE1). Cells that have become refractory to PGE1 stimulation respond to BK. This suggests that a receptor or activator system different from the one for PGE1 and PGE2 exists for BK.When both BK and PGE1 are incubated together with synovial fibroblasts, the cyclic AMP response elicited is more than additive as compared to the response of each hormone separately. Indomethacin (IM) inhibits the BK evoked cyclic AMP response unless cell cultures are pretreated with PGE1. The PGE1 analog, 7-oxa-13-prostynoic acid, is a better inhibitor of the cyclic AMP response induced by BK than by PGE1. BK does not elicit a cyclic AMP response solely by elaborating PGE1, yet the prostaglandin pathway and its products seem to have a role in the degree of the cyclic AMP response to BK challenge.

Ethanol metabolism and uric acid

Abstract Despite the relationship between gout and alcohol consumption, which has been propounded by the laity and physicians for over a century, the biochemical mechanisms for altered uric acid metabolism after ethanol ingestion have only recently been determined. The primary cause for the occurrence of hyperuricemia in this setting is an increased generation of NADH as a result of the oxidation of ethanol to acetaldehyde, which increases the conversion of pyruvate to lactate. The latter metabolite then can alter the transtubular transport of uric acid with the production of hyperuricemia and a concomitant decrease in urinary uric acid excretion. That an alteration in the NADH NAD ratio occurs with ethanol ingestion can be indirectly inferred by the changes in serotonin, galactose, and norepinephrine metabolism. Additional factors which may effect urate metabolism with chronic ethanol ingestion include the syndrome, alcoholic hyperlipemia, the fat-dependent ketogenic effect produced by alcohol, and possibly, a poor nutritional status associated with starvation and ketoacidosis. Ketoacids effect the tubular transport of uric acid in a manner similar to lactate. Both delirium tremens with its increased lactate production secondary to muscular hyperactivity and “withdrawal” seizures may result in hyperuricemia as well. The author would suggest that these alterations in the uric acid pool as a result of either acute or chronic alcohol intake could provide the pathophysiologic conditions to initiate acute gouty arthritis in the genetically prone individual.

Endocrinopathies and uric acid metabolism

Abstract Evidence for the presence of uric acid abnormalities in acromegaly is equivocal, and the hyperuricemia seen post-adrenalectomy is related to the primary hypertensive state rather than to adrenal dysfunction. Hyperparathyroidism, hypoparathyroidism, hypothyroidism, and hyperthyroidism have all been documented to have alterations in uric acid metabolism. In these endocrinopathies, abnormalities of renal function appear to be the primary mechanistic factors in the pathogenesis of these changes in urate metabolism. However, nonrenal factors and parathyroid and thyroid hormones have not been fully evaluated as to their roles in altered purine metabolism. The relationship between carbohydrate intolerance and hyperuricemia or gout represents an extremely complex problem. It is clear that diabetic ketoacidosis is associated with increased serum uric acid concentrations concomitant with a decreased urinary urate excretion, and the mechanism for these derangements is well established. Despite the accumulated evidence favoring an increased incidence of carbohydrate intolerance in gouty patients, the actual incidence and the mechanism which relates carbohydrate and purine abnormalities remains elusive. Even though many factors such as race, obesity, age, and other parameters may effect both carbohydrate and uric acid metabolism, recent data point toward insulin deficiency and not insulin resistance as the cause for carbohydrate intolerance in gout.

A spectrophotometric assay for hypoxanthine-guanine phosphoribosyltransferase.

Abstract The present paper describes a new spectrophotometric assay for HGPRTase activity which is more rapid than and as sensitive as the isotopic assays for this enzyme and which avoids the use of high-voltage electrophoresis and liquid scintillation counting. A simple technique using thin-layer chromatography for the separation of the nucleotide, inosinic acid, from its free base, hypoxanthine, is proposed in preference to the presently employed separation systems of high-voltage electrophoresis or column chromatography.

The effect of 7-oxa-13 prostynoic acid on the mechanism of action of bradykinin in human synovial fibroblasts.

Bradykinin, a potent inflammatory mediator, induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the synthesis and release of 3H-arachidonic acid and 3H-E prostaglandins from these cells pre-labeled in their phospholipids. Fetal calf serum in the media also stimulates the synthesis and release of these labeled lipids from pre-labeled human synovial fibroblasts and potentiates the bradykinin-induced cyclic AMP response. The PGE1 analogue, 7-oxa-13 prostynoic acid, completely abrogates both the bradykinin-induced cyclic AMP response and the bradykinin- and fetal calf serum-evoked release of labeled E-prostaglandins from pre-labeled cells. In serum-free media, the prostaglandin antagonist stimulated the release of 3H-arachidonic acid from pre-labeled human synovial fibroblasts and did not inhibit the bradykinin-induced release of this lipid.

Cyclic AMP and Thyroid Hormone: Inhibition of Epiphyseal Cartilage Cyclic 3′,5′-Nucleotide Phosphodiesterase Activity by L-Triiodothyronine

Summary The mechanism for the alteration in cartilage growth and metabolism present both in childhood thyroid hormone deficiency states and in hyperthyroid children has not been defined. Since thyroid hormone has a direct effect on the adenylate cyclase-cyclic 3′,5′-nucleotide phos-phodiesterase system in certain tissues, the effect of thyroid hormones on soluble cyclic 3′,5′-nucleotide phosphodiesterase (PDE) activity prepared from chicken epiphyseal (growth) cartilage was examined. L-Triiodothyronine at a concentration of 1 × 10−4 M resulted in 69% inhibition of soluble cartilage cyclic 3′,5′-nuc-leotide phosphodiesterase activity. At identical concentrations L-thyroxine, D-thyroxine, D-triiodothyronine, and L-diiodothyronine demonstrated only 42, 41, 38, and 20% inhibition of phosphodiesterase activity, respectively. The concentration of L-triiodothyronine that resulted in 50% inhibition of phosphodiesterase activity was 3.5 × 10−5 M. This investigation demonstrates a significant inhibitory effect of L-triiodothyronine on cartilage 3′,5′-nucleotide phosphodiesterase activity, whereas other thyroid analogs tested were considerably less active as inhibitors of this enzyme. These findings suggest that cyclic nucleotide metabolism may be involved in regulating the effects of the most potent thyroid hormone, L-tri-iodothyronine, on epiphyseal cartilage. This investigation was supported in part by National Institutes of Health Grant No. R01 AM 16151 and an Arthritis Clinical Research Center Grant.

Regulation of bradykinin-induced cyclic AMP response by quinacrine and prostaglandin E2 and F2α in human synovial fibroblasts

Bradykinin induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the release of [3H] arachidonic acid and [3H]-E prostaglandins from human synovial fibroblasts prelabeled in their phospholipids. Both these bradykinin-induced reactions are inhibited by quinacrine, an inhibitor of phospholipase A activity. The cyclic AMP response of human synovial fibroblasts to bradykinin is potentiated by prostaglandin E2 and inhibited by prostaglandin F2α. These data emphasize the critical role of the prostaglandin system in reactions induced by bradykinin and suggest mechanisms by which inflammatory reactions due to bradykinin may be modulated.