Adenylate cyclase inhibition by hormones. The Mg2+ hypothesis

In washed anterior pituitary membranes, there is enough GTP to occupy Ns and therefore to obtain activation of adenylate cyclase by vasointestinal peptide. GTP concentrations needed to obtain adenylate cyclase inhibition by dopamine (above 5 X 10- M) stimulate the adenylate cyclase. The dopamine effect is a blockade of this stimulation. We propose that at least in this system, Ni does not inhibit but stimulates the adenylate cyclase and that inhibitory hormones block this stimulation. We also demonstrate in several adenylate cyclase systems that hormones produced adenylate cyclase inhibition by lowering their Mg affinity A general model for adenylate cyclase activation and inhibition is proposed.     

The effect of prostaglandins on cultured lapine articular chondrocytes

The effect of 8 prostaglandins (PG) on growth and sulfate incorporation by monolayer and spinner-cultured rabbit articular chondrocytes has been measured. PGA₁, PGB₁, PGE₁ and PGE₂ reduced synthesis of sulfated glycosaminoglycans (GAG) but the PGF series did not. PGA₁ was the most potent, being effective at a concentration of 2.5 μg/ml [6.8 μM] while the others required 25 μg/ml. These compounds had no effect on degradation of GAG. All 8 PGs augmented growth slightly but significantly at 2.5 μg/ml. At the higher concentration, PGA₁ was highly cytotoxic, and PGB₁ as well as PGE₂ reduced cell growth. The cytotoxicity of PGA₁ was also observed in two additional types of cultured connective tissue cells, but the inhibition of sulfated-GAG synthesis by PGA₁ and PGB₁ was confined to the chondrocytes. The response of cultured chondrocytes to exogenous PGs, albeit at apparently unphysiologically high concentrations, together with other evidence, suggests that these compounds may conceivably play a direct role in cartilage metabolism in vivo.     

Adenylate cyclase responsiveness of human insulinomas

Adenylate cyclase activity was assayed in a crude particulate fraction of one benign and one malignant human insulinoma. Adenylate cyclase of both tumours responded to 5'-guanylyl-imidodiphosphate, sodium fluoride, glucagon and prostaglandin E2, and in addition the adenylate cyclase of the benign tumour responded to isoprenaline. Glucose and prostaglandin I2 (prostacyclin) did not stimulate the adenylate cyclase in either tumour, although prostaglandin I2 stimulated insulin secretion in cultures of the benign tumour. The in vitro responsiveness of the adenylate cyclase to glucagon did not correlate closely with the effect of glucagon on insulin secretion in vivo.     

Cerebral endothelial cell culture II. Adenylate cyclase response to prostaglandins and their interaction with the adrenergic system

The response of endothelial adenylate cyclase (AC) to prostaglandins (PGE₁, PGE₂, PGF_1α, PGF_2α, PGD₂ and PGI₂) and the relationship of PGE₂ to adrenergic systems were investigated in cerebrovascular endothelial cultures. E-type prostaglandins and PGI₂ were more effective in stimulating endothelial AC (EC₅₀ = 3 × 10^?7M, and 3 × 10^?7M, respectively) than prostaglandins of the F-series and PGD₂ which activated AC at high doses only. A modulation of endothelial AC response to either PGE₂ or norepinephrine (NE) was observed in the presence of both agents in the system. It was manifested by a dose-dependent NE inhibition of the PGE₂-stimulated formation of cAMP, which was partially restored by phentolamine. Alpha and β-adrenergic agonists (α, clonidine and 6-fluoronorepinephrine; β, isoproterenol) also partly blocked while forskolin and PGE₂ synergistically stimulated the production of cAMP in the endothelial cultures. These findings strongly suggest that the interaction of prostaglandins and α- and β-adrenergic agonists with the AC system in cerebrovascular endothelium may play a role in the regulation of the cerebral microcirculation and/or blood pressure.     

Adenylate cyclase stimulation by trypsin.

Adenylate cyclase activity of a rat embryo fibroblast cell line (F111) is markedly increased by brief treatment with 1:300 trypsin. The degree of stimulation depends upon the length of time the cells are treated and the concentration of trypsin. Crystalline trypsin produced a stimulation similar to that obtained with 1:300 trypsin. Further, the addition of soybean trypsin inhibitor blocked the stimulation of adenylate cyclase by 1:300 trypsin. Trypsin-treated adenylate cyclase responds to PGE1, but there is no increase over that of untreated enzyme. This result and the increase in fluoride-stimulated levels of activity suggest that the trypsin is acting upon the catalytic unit of the enzyme.     

Expression of prepro-enkephalin in human articular chondrocytes is linked to cell proliferation.

This study shows that cultured human articular chondrocytes express high levels of 1.4 kb prepro-enkephalin mRNA. Chondrocytes store met-enkephalin intracellularly and secrete this neuropeptide in mature as well as in precursor form. Gene expression is inducible by serum factors. High levels of prepro-enkephalin mRNA are detected in proliferating chondrocytes but not in confluent, contact-inhibited cells. Phorbol myristate acetate and dibutyryl cyclic AMP, but not dexamethasone, increase levels of prepro-enkephalin mRNA. Furthermore, transforming growth factor beta (TGF beta) and platelet derived growth factor (PDGF) upregulate gene expression, whereas retinoic acid, which inhibits chondrocyte proliferation, suppresses both basal and induced gene expression. Using in situ hybridization it is shown that only 1-3% of primary chondrocytes express prepro-enkephalin mRNA, whereas 52 +/- 12% of subcultured cells are strongly positive. Analysis of DNA synthesis, by autoradiography of incorporated [3H]thymidine, shows that these numbers correspond to the percentage of cells in S-phase of the cell cycle. In cultures of primary chondrocytes TGF beta promotes the formation of cartilage nodules and stimulates proliferation of adherent cells. This is associated with high levels of prepro-enkephalin mRNA in proliferating cells but not in contact-inhibited cells in cartilage nodules. In contrast, formation of cartilage nodules, proliferation and the expression of enkephalin are suppressed by interleukin-1 beta. In summary, expression of prepro-enkephalin in human articular chondrocytes is differentially controlled by cartilage regulatory factors and closely associated with cell proliferation.     

Adenylate cyclase from rat-liver plasma membrane: inhibition by mersalyl and other mercurial derivatives.

The adenylate cyclase activity from a rat liver plasma membrane preparation was inhibited by low concentrations (1-10 muM) of the mercurial diuretic mersalyl. Complete inhibition was obtained with 0.1 mM mersalyl. Similar effects were observed whether the adenylate cyclase preparation was assayed in the presence of 10 muM GTP, 0.1 muM glucagon, 10 mM NaF or without any addition. The effect of mersalyl was not due to inhibition of the regenerating system present in the incubation medium, since the effect of mersalyl was preserved and even enhanced in its absence. The inhibition brought about by mersalyl was due to both a decrease of the maximal velocity of the reaction and of the affinity of the enzyme for the substrate. It was immediate, and irreversible spontaneously, but it was reversed by the simultaneous additions of 2-mercaptoethanol, in a dose-dependent fashion. Other -SH reagents were found to have an effect equal to, or lower than, that of mersalyl. Mersalyl had no effect upon Mg2+-ATPase, although it inhibited the (Na+-K+) activated ATPase. Since mersalyl is known to be a 'non-penetrant' reagent, it is postulated that a catalytically important, mercurial-sensitive, part of adenylate cyclase is at the surface of the plasma membrane. This view is supported by the following facts: (a) mersalyl acted with a similar dose-response curve upon an intact as well as a detergent-dispersed cyclase preparation while no effect was observed upon a solubilized Mg2+-ATPase preparation; (b) a covalent p-chloromercuribenzoate-Sephadex preparation (but not its supernatant) inhibited the cyclase from intact membranes. It is proposed that mercurial derivatives, by their relative specificity of action (no effect on Mg2+-ATPase), can serve as useful probes in the elucidation of the multicomponent structure of the cyclase system.     

Adenylate cyclase and membrane fluidity

Summary The relationships between membrane fluidity as induced by drug addition and the stimulation of adenylate cyclase by hormones (mainly catecholamines), GTP, Gpp(NH)p and NaF are reviewed. In particular, the data corresponding to pigeon erythrocyte membranes are reviewed and compared with other data published in the literature. A brief summary of the theories involved in fluidity measurements and their significance at the molecular level is also given for anisotropy of fluorescence and electron spin resonance.One of the conclusions is that the cationic drugs and neutral alcohols by perturbing preferentially the inner half-layer of the bilayer induced in pigeon erythrocyte membrane correlated multiphasic changes on fluidity and adenylate cyclase activity.This and other experimental data concerning the regulation of the adenylate cyclase are discussed in regard to a new interpretation of cyclase stimulation: the repressor hypothesis. In cell membrane the catalytic unit C is repressed by its association with a repressor complex made of the hormone receptor R and the regulatory protein N. The activation of cyclase activity is the dissociation of the catalytic unit C from the repressor complex R.N according to the equilibrium: R.N.C (inactive) R.N + C (active). Hormones, metal ions (magnesium), and nucleotides (GTP) are the allosteric ligands which shift this equilibrium towards the dissociation. state with the liberation of the active form, membrane-bound, C unit. Gpp(NH)p, fluoride and forskolin will also shift the equilibrium toward the right. GDP and free receptors favour the associated repressed state of the system.     

Anabolic and catabolic responses of human articular chondrocytes to varying oxygen percentages

Introduction  

Oxygen is a critical parameter proposed to modulate the functions of chondrocytes ex-vivo as well as in damaged joints. This article investigates the effect of low (more physiological) oxygen percentage on the biosynthetic and catabolic activity of human articular chondrocytes (HAC) at different phases of in vitro culture.     

Cytokine regulation of facilitated glucose transport in human articular chondrocytes.

Glucose serves as the major energy substrate and the main precursor for the synthesis of glycosaminoglycans in chondrocytes. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. This study examines molecular regulation of facilitated glucose transport in normal human articular chondrocytes by proinflammatory cytokines. IL-1beta and TNF-alpha, and to a lesser degree IL-6, accelerate facilitated glucose transport as measured by [(3)H]2-deoxyglucose uptake. IL-1beta induces an increased expression of glucose transporter (GLUT) 1 mRNA and protein, and GLUT9 mRNA. GLUT3 and GLUT8 mRNA are constitutively expressed in chondrocytes and are not regulated by IL-1beta. GLUT2 and GLUT4 mRNA are not detected in chondrocytes. IL-1beta stimulates GLUT1 protein glycosylation and plasma membrane incorporation. IL-1beta regulation of glucose transport in chondrocytes depends on protein kinase C and p38 signal transduction pathways, and does not require phosphoinositide 3-kinase, extracellular signal-related kinase, or c-Jun N-terminal kinase activation. IL-1beta-accelerated glucose transport in chondrocytes is not mediated by endogenous NO or eicosanoids. These results demonstrate that stimulation of glucose transport represents a component of the chondrocyte response to IL-1beta. Two classes of GLUTs are identified in chondrocytes, constitutively expressed GLUT3 and GLUT8, and the inducible GLUT1 and GLUT9.     

Interleukin-1 beta enhances the response of human articular chondrocytes to extracellular ATP.

It has been observed that both interleukin-1 (IL-1) and extracellular ATP stimulate the production of prostaglandin E (PGE) by human articular chondrocytes in monolayer culture. The combined effects of recombinant human IL-1 beta and ATP were therefore studied using these cells. IL-1 beta rapidly enhanced the response to a maximally effective concentration of ATP (100 microM). On continuous exposure of the cells to the cytokine, its effect was greatest after approx. 24 h and tended to decline thereafter. The enhancement of the response to 100 microM ATP by IL-1 beta was dose-dependent. Removal of IL-1 beta prior to treating the cells with 100 microM ATP did not affect the degree of enhancement of the response. The effect of the cytokine on the response to suboptimal concentrations of extracellular ATP was also tested. IL-1 beta lowered the minimum concentration of ATP required to elicit an increase in the production of PGE by human articular chondrocytes. These findings are of interest, since they indicate a synergistic interaction between a cytokine and a purinergic agonist. Moreover, since both the sensitivity of the cells to extracellular ATP and the maximum response to this agent were enhanced, it is possible that IL-1 modulates more than one step in the process of P2-purinoceptor-mediated stimulation of PGE production. These observations may be relevant to the pathogenesis of some forms of arthritis.     

Recombinant human midkine stimulates proliferation of articular chondrocytes

Objectives: Midkine, a heparin‐binding growth factor, promotes population growth, survival and migration of several cell types, but its effect on articular chondrocytes remains unknown. The aim of this study was to investigate its role on proliferation of articular chondrocytes in vitro and in vivo. Materials and methods: Bromodeoxyuridine incorporation and MTT assays were performed to examine the proliferative effect of recombinant human midkine (rhMK) on primary articular chondrocytes. Activation of extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K) was analysed using western blot analysis. Systemic and local delivery of rhMK into mice and rats was preformed to investigate the proliferative effect of rhMK in vivo, respectively. Histological evaluation, including measurement of articular cartilage thickness, cell density, matrix staining and immunostaining of proliferating cell nuclear antigen was carried out. Results: rhMK promoted proliferation of articular chondrocytes cultured in a monolayer, which was mediated by activation of ERK and PI3K. The proliferative role of rhMK was not coupled to dedifferentiation of culture‐expanded cells. Consistent with its action in vitro, rhMK stimulated proliferation of articular chondrocytes in vivo when it was administered subcutaneously and intra‐articularly in mice and rats, respectively. Conclusion: Our results demonstrate that rhMK stimulates proliferation of primary articular chondrocytes in vitro and in vivo. The results of this study warrant further examination of rhMK for treatment of animal models of articular cartilage defects.