Adenosine mediates transforming growth factor-beta 1 release in kidney glomeruli of diabetic rats

Up regulation of the transforming growth factor-beta 1 (TGF-β1) axis has been recognized as a pathogenic event for progression of glomerulosclerosis in diabetic nephropathy. We demonstrate that glomeruli isolated from diabetic rats accumulate up to sixfold more extracellular adenosine than normal rats. Both decreased nucleoside uptake activity by the equilibrative nucleoside transporter 1 and increased AMP hydrolysis contribute to raise extracellular adenosine. Ex vivo assays indicate that activation of the low affinity adenosine A_2B receptor subtype (A_2BAR) mediates TGF-β1 release from glomeruli of diabetic rats, a pathogenic event that could support progression of glomerulopathy when the bioavailability of adenosine is increased.     

Heart design: free ADP scales with absolute mitochondrial and myofibrillar volumes from mouse to human

Our aim was to estimate a number of bioenergetic parameters in the beating mouse, rat and guinea pig heart in situ and compare the values to those in hearts of mammals over a 2000-fold range in body mass. For the mouse, rat and guinea pig heart, we report a phosphorylation ratio of 1005±50 (n=16), 460±32 (n=10) and 330±22 (n=5) mM⁻¹ and a free cytosolic [ADP] concentration of 13, 18 and 22 μM, respectively. When each parameter was plotted against body mass, they scaled closely to the quarter power (−0.28, r=0.99 and −0.23, r=0.97). A similar regression slope was found when the inverse of free [ADP] was plotted against absolute mitochondrial (slope=−0.26, r=0.99) and myofibrillar volumes (slope=−0.24, r=0.99). The similar slopes indicate that the ratio of absolute mitochondria and myofibrillar volumes in the healthy mammalian heart is a constant, and independent of body size. In conclusion, our study supports the hypothesis that the mammalian heart has a number of highly conserved thermodynamic and kinetic parameters that obey quarter-power laws linking the phosphorylation ratio, ATP turnover rates, free [ADP] and absolute mitochondrial volumes to body size. The results are discussed in terms of possible mechanisms and potential deviations from these laws in some disease states.     

Nerve Growth Factor Effect on Adenosine Transport in Cultured Chromaffin Cells

Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.     

Ultrastructural localization of adenosine triphosphatase activity in HeLa cells at various stages of the cell cycle

Summary HeLa cells in a monolayer culture were synchronized to S, G₂ and mitotic phases by use of excess (2.5 mM) deoxythymidine double-block technique. The localizations of Ca⁺⁺-activated adenosine triphosphatase (ATPase) at different phases of the cell cycle were studied using light and electron-microscopic histochemical techniques, and microphotometric comparisons of the densities of reaction products. Enzyme reaction product was always localized in the endoplasmic reticulum, nuclear membrane, mitochondria and Golgi apparatus, but there were qualitative and quantitative differences related to the phases of the cell cycle. In S phase the activity was mainly concentrated in a perinuclear area of the cytoplasm whereas in G₂ and mitosis the activity was scattered throughout the cell. The total activity per cell was maximal in G₂, was less in S phase and least in mitosis. Activity in the mitochondria and endoplasmic reticulum was distinctly less in mitosis than in other phases of the cell cycle. The mitochondrial ATPase differed from the ATPase at other sites in ion dependence and sensitivity to oligomycin. The results suggest that there may be several distinct ATPases in proliferating cells.     

Purinergic modulation of cardiac activity in the flounder during hypoxic stress

Summary The interaction of adrenaline and adenosine was examined in cardiac tissue of the flounderPlatichthys flesus.When applied alone both agents increased contractility in both auricular and ventricular myocardial strips. This positive inotropic effect was associated with a small depolarization in the tissues examined by the sucrose gap technique. Simultaneous application of adrenaline and adenosine gave an inhibition of the control responses seen with either agent alone in both auricle and ventricle.Radiocalcium flux studies on ventricular tissue showed that influx was increased by adrenaline or adenosine alone above control values, but when applied together radiocalcium influx was reduced. Radiocalcium efflux from cardiac microsomes was stimulated by challenge with adrenaline or adenosine alone. This stimulation was not seen following simultaneous challenge by both agents.The effect of adrenaline on responses of hypoxic flounder hearts was less than that seen in normoxic hearts. This situation was reversed by pretreatment with the purine receptor blocker caffeine. Caffeine pretreatment also reduced the positive inotropic effect seen in normoxic hearts challenged with adenosine.TLC studies gave strong evidence that hearts perfused with hypoxic salines produced both adenosine and adrenaline.The results are discussed as evidence for a mechanism of heart regulation which the flounder may use as a defence against severe acute hypoxic stress.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Synchrotron-excited time-resolved fluorescence spectroscopy of adenosine,protonated adenosine and 6N, 6N-dimethyladenosine in aqueous solution at room temperature

The fluorescence behavior of adenosine in neutral solution has been studied by time-resolved spectroscopy using synchrotron excitation and timecorrelated single photon counting, and by decay time measurements. Three emissions have been identified and correlated with three excitation spectra. The assignment of these transitions has been made by comparison with similar measurements on ⁶N, ⁶N-dimethyladenosine (6 DMA), and on adenosine in acid solution (ADO H⁺). It is proposed that two of the transitions of adenosine which correlate with 6DMA originate from coplanar and orthogonal rotational conformers of the amino group. The other transition, correlating with ADO H⁺ may originate either from the ³H-imino tautomer, or from a differently solvated rotational conformer.A partial presentation of this work has been made at the Second Congress of the European Society for Photobiology Padova, Italy, 6–10 September 1987     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate. I. Equilibrium model

The thermodynamic treatment of the disproportionation reaction of adenosine 5′-diphosphate to adenosine 5′-triphosphate and adenosine 5′-monophosphate is discussed in terms of an equilibrium model which includes the effects of the multiplicity of ionic and metal bound species and the presence of long range electrostatic and short range repulsive interactions. Calculated quantities include equilibrium constants, enthalpies, heat capacities, entropies, and the stoichiometry of the overall reaction. The matter of how these calculations can be made self-consistent with respect to both calculated values of the ionic strength and the molality of the free magnesium ion is discussed. The thermodynamic data involving proton and magnesium-ion binding data for the nucleotides involved in this reaction have been evaluated.     

Cortical membrane-trafficking during the meiotic resumption of Xenopus laevis oocytes

Summary The giant mucous cells in the skin of the terrestrial banana slug Ariolimax columbianus secret intact granules containing mucins. Electron microscopy, after ultrarapid freezing and freeze-substitution in osmium, shows that the secreted granules are bounded by two distinct membranes, presumably derived from the Golgi apparatus and the plasmalemma. Relatively stable, intact granules can be obtained in great quantity in our in vitro system. Rapid lysis of the granules was induced by adenosine triphosphate. At much higher concentrations, adenosine diphosphate and 5-adenylimido-diphosphate also caused lysis. Other nucleotides and related compounds, as well as 1,4,5-inositol triphosphate and molluscan neurotransmitters and neuropeptides, had no effect on the granules. The stability of secreted granules varied with the ionic composition of the isosmotic medium in which they were suspended. When the predominant cation in the medium was potassium, and calcium was also present, granules lysed if exposed to shear stress (stirring of the suspension). This did not occur if sodium was the major cation present. None of the other ions in the suspension media had detectable effects on the stability of the granules.     

Release of Purines, Noradrenaline, and GABA from Rat Hippocampal Slices by Field Stimulation

Labelled adenine, noradrenaline (NA), and gamma-aminobutyric acid (GABA) were taken up by the transversely cut hippocampal slice. [3H]NA and [14C]GABA were retained as such, [3H]- (or [14C]-) adenine mainly as adenine nucleotides. There was a spontaneous overflow of all three types of compounds ranging from 0.1 (GABA) to 0.21 (NA) %/min. The rate of [3H]NA overflow increased rapidly during electrical field stimulation. The release rate was well maintained over a 15-min period. The rate of [14C]GABA release also increased rapidly but it was not maintained over a 15-min period even if uptake and/or metabolism was inhibited by nipecotic acid (1 mM) and aminooxyacetic acid (AOAA, 0.1 mM). The bulk of the purines was released after the stimulation period. For all compounds the amounts released were frequency- and calcium-dependent. At a frequency of 3 Hz a 10 V stimulation was sufficient to cause a maximal [3H]NA release and 20 V to cause maximal [14C]GABA release, but 14C-purine release was increased further by increasing the voltage to 40 V. The evoked purine release was inhibited by a nucleoside uptake inhibitor (dipyridamole). On stimulation of [3H]NA-labelled slices the released radioactivity was composed of greater than 95% unchanged NA. The specific activities of NA in the slice and in the superfusate were practically identical. In [3H]adenine-labelled slices the released radioactivity was composed of adenosine, inosine, and hypoxanthine, but the activity in the slice of ATP, ADP, and AMP.(ABSTRACT TRUNCATED AT 250 WORDS)