Upregulation of inducible NO synthase by exogenous adenosine in vascular smooth muscle cells activated by inflammatory stimuli in experimental diabetes

BACKGROUND: Adenosine has been shown to induce nitric oxide (NO) production via inducible NO synthase (iNOS) activation in vascular smooth muscle cells (VSMCs). Although this is interpreted as a beneficial vasodilating pathway in vaso-occlusive disorders, iNOS is also involved in diabetic vascular dysfunction. Because the turnover of and the potential to modulate iNOS by adenosine in experimental diabetes have not been explored, we hypothesized that both the adenosine system and control of iNOS function are impaired in VSMCs from streptozotocin-diabetic rats. METHODS: Male Sprague-Dawley rats were injected with streptozotocin once to induce diabetes. Aortic VSMCs from diabetic and nondiabetic rats were isolated, cultured and exposed to lipopolysaccharide (LPS) plus a cytokine mix for 24 h in the presence or absence of (1) exogenous adenosine and related compounds, and/or (2) pharmacological agents affecting adenosine turnover. iNOS functional expression was determined by immunoblotting and NO metabolite assays. Concentrations of adenosine, related compounds and metabolites thereof were assayed by HPLC. Vasomotor responses to adenosine were determined in endothelium-deprived aortic rings. RESULTS: Treatment with adenosine-degrading enzymes or receptor antagonists increased iNOS formation in activated VSMCs from nondiabetic and diabetic rats. Following treatment with the adenosine transport inhibitor NBTI, iNOS levels increased in nondiabetic but decreased in diabetic VSMCs. The amount of secreted NO metabolites was uncoupled from iNOS levels in diabetic VSMCs. Addition of high concentrations of adenosine and its precursors or analogues enhanced iNOS formation solely in diabetic VSMCs. Exogenous adenosine and AMP were completely removed from the culture medium and converted into metabolites. A tendency towards elevated inosine generation was observed in diabetic VSMCs, which were also less sensitive to CD73 inhibition, but inosine supplementation did not affect iNOS levels. Pharmacological inhibition of NOS abolished adenosine-induced vasorelaxation in aortic tissues from diabetic but not nondiabetic animals. CONCLUSIONS: Endogenous adenosine prevented cytokine- and LPS-induced iNOS activation in VSMCs. By contrast, supplementation with adenosine and its precursors or analogues enhanced iNOS levels in diabetic VSMCs. This effect was associated with alterations in exogenous adenosine turnover. Thus, overactivation of the adenosine system may foster iNOS-mediated diabetic vascular dysfunction

Ability of γδ T cells to modulate the Foxp3 T cell response is dependent on adenosine.

Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases

The role of the neuromodulator adenosine in alcohols actions.

The interaction between the neuromodulator adenosine and adenosine receptors on the surface of neurons modifies the neurons responses to neurotransmitters. The activated adenosine receptors alter the levels of small signaling molecules (i.e., second messengers) in the cells. Depending on the receptors and cells involved, these changes can make it easier or more difficult for neurotransmitters to excite the cell. Adenosines activity is regulated by proteins called nucleoside transporters, which carry adenosine into and out of the cell. Alcohol interferes with the function of the adenosine system. For example, both acute and chronic alcohol exposure affect the function of the adenosine-carrying nucleoside transporters, thereby indirectly altering the second-messenger levels in the cells. Through this mechanism, adenosine may mediate some of alcohols effects, such as intoxication, motor incoordination, and sedation

A depletable pool of adenosine in area CA1 of the rat hippocampus

Adenosine plays a major modulatory and neuroprotective role in the mammalian CNS. During cerebral metabolic stress, such as hypoxia or ischemia, the increase in extracellular adenosine inhibits excitatory synaptic transmission onto vulnerable neurons via presynaptic adenosine A1 receptors, thereby reducing the activation of postsynaptic glutamate receptors. Using a combination of extracellular and whole-cell recordings in the CA1 region of hippocampal slices from 12- to 24-d-old rats, we have found that this protective depression of synaptic transmission weakens with repeated exposure to hypoxia, thereby allowing potentially damaging excitation to both persist for longer during oxygen deprivation and recover more rapidly on reoxygenation. This phenomenon is unlikely to involve A1 receptor desensitization or impaired nucleoside transport. Instead, by using the selective A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine and a novel adenosine sensor, we demonstrate that adenosine production is reduced with repeated episodes of hypoxia. Furthermore, this adenosine depletion can be reversed at least partially either by the application of exogenous adenosine, but not by a stable A1 agonist, N6-cyclopentyladenosine, or by endogenous means by prolonged (2 hr) recovery between hypoxic episodes. Given the vital neuroprotective role of adenosine, these findings suggest that depletion of adenosine may underlie the increased neuronal vulnerability to repetitive or secondary hypoxia/ischemia in cerebrovascular disease and head injury

Expanding a fluorescent RNA alphabet: synthesis, photophysics and utility of isothiazole-derived purine nucleoside surrogates.

A series of emissive ribonucleoside purine mimics, all comprised of an isothiazolo[4,3-d]pyrimidine core, was prepared using a divergent pathway involving a key Thorpe-Ziegler cyclization. In addition to an adenosine and a guanosine mimic, analogues of the noncanonical xanthosine, isoguanosine, and 2-aminoadenosine were also synthesized and found to be emissive. Isothiazolo 2-aminoadenosine, an adenosine surrogate, was found to be particularly emissive and effectively deaminated by adenosine deaminase. Competitive studies with adenosine deaminase with each analogue in combination with native adenosine showed preference for the native substrate while still deaminating the isothiazolo analogues

A population of immature cerebellar parallel fibre synapses are insensitive to adenosine but are inhibited by hypoxia

The purine adenosine plays an important role in a number of physiological and pathological processes and is neuroprotective during hypoxia and ischemia. The major effect of adenosine is to suppress network activity via the activation of A1 receptors. Here we report that in immature cerebellar slices, the activation of A1 receptors has variable effects on parallel fibre synaptic transmission, ranging from zero depression to an almost complete abolition of transmission. Concentration–response curves suggest that the heterogeneity of inhibition stems from differences in A1 receptor properties which could include coupling to downstream effectors. There is less variation in the effects of adenosine at parallel fibre synapses in slices from older rats and thus adenosine signalling appears developmentally regulated. In the cerebellum, hypoxia increases the concentration of extracellular adenosine leading to the activation of A1 receptors (at adenosine-sensitive parallel fibre synapses) and the suppression of glutamate release. It would be predicted that the synapses that were insensitive to adenosine would be less depressed by hypoxia and thus maintain function during metabolic stress. However those synapses which were insensitive to adenosine were rapidly inhibited by hypoxia via a mechanism which was not reversed by blocking A1 receptors. Thus another mechanism must be responsible for the hypoxia-mediated depression at these synapses. These different mechanisms of depression may be important for cell survival and for maintenance of cerebellar function following oxygen starvation

Automatic instrument for chemical processing to detect microorganism in biological samples by measuring light reactions

An automated apparatus is reported for sequentially assaying urine samples for the presence of bacterial adenosine triphosphate (ATP) that comprises a rotary table which carries a plurality of sample containing vials and automatically dispenses fluid reagents into the vials preparatory to injecting a light producing luciferase-luciferin mixture into the samples. The device automatically measures the light produced in each urine sample by a bioluminescence reaction of the free bacterial adenosine triphosphate with the luciferase-luciferin mixture. The light measured is proportional to the concentration of bacterial adenosine triphosphate which, in turn, is proportional to the number of bacteria present in the respective urine sample

Stability of Diluted Adenosine Solutions in Polyolefin Infusion Bags

Background Intravenous or intracoronary adenosine is used in the cardiac catherization lab to achieve maximal coronary blood flow and determine fractional flow reserve. Objective To determine the stability of adenosine 10 and 50 µg/mL in either 0.9% sodium chloride injection or 5% dextrose injection in polyolefin infusion bags stored at 2 temperatures, refrigeration (2°C-8°C) or controlled room temperature (20°C-25°C). Methods Adenosine 10 µg/mL and 50 µg/mL solutions were prepared in 50 mL polyolefin infusion bags containing 0.9% sodium chloride injection or 5% dextrose injection and stored at controlled room temperature or under refrigeration. Each combination of concentration, diluent, and storage was prepared in triplicate. Samples were assayed using stability-indicating, reversed-phase high-performance liquid chromatography immediately at time 0 and at 24 hours, 48 hours, 7 days, and 14 days. Stability was defined as retaining 90% to 110% of the initial adenosine concentration. The samples were also visually inspected against a light background for clarity, color, and the presence of particulate matter. Results After 14 days, all samples retained 99% to 101% of the initial adenosine concentration. No considerable change in pH or visual appearance was noted. The stability data indicated no significant loss of drug due to chemical degradation or physical interactions during storage. Conclusion Adenosine solutions of 10 and 50 µg/mL were stable for at least 14 days in 50 mL polyolefin infusion bags of 0.9% sodium chloride injection or 5% dextrose injection stored at controlled room temperature and refrigerated conditions

Biosensor measurement of purine release from cerebellar cultures and slices

We have previously described an action-potential and Ca2+-dependent form of adenosine release in the molecular layer of cerebellar slices. The most likely source of the adenosine is the parallel fibres, the axons of granule cells. Using microelectrode biosensors, we have therefore investigated whether cultured granule cells (from postnatal day 7–8 rats) can release adenosine. Although no purine release could be detected in response to focal electrical stimulation, purine (adenosine, inosine or hypoxanthine) release occurred in response to an increase in extracellular K+ concentration from 3 to 25 mM coupled with addition of 1 mM glutamate. The mechanism of purine release was transport from the cytoplasm via an ENT transporter. This process did not require action-potential firing but was Ca2+dependent. The major purine released was not adenosine, but was either inosine or hypoxanthine. In order for inosine/hypoxanthine release to occur, cultures had to contain both granule cells and glial cells; neither cellular component was sufficient alone. Using the same stimulus in cerebellar slices (postnatal day 7–25), it was possible to release purines. The release however was not blocked by ENT blockers and there was a shift in the Ca2+ dependence during development. This data from cultures and slices further illustrates the complexities of purine release, which is dependent on cellular composition and developmental stage

Enzymatic regeneration of adenosine triphosphate cofactor

Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes