Expeditious synthesis and biological evaluation of new C-6 1,2,3-triazole adenosine derivatives A1 receptor antagonists or agonists.

The synthesis of new C-6 1,2,3-triazole adenosine derivatives via microwave assisted 1,3-dipolar cycloaddition as key step is described. The binding on membranes of cells that over express A(1) adenosine receptors (A(1)AR) was also evaluated. Among them, four compounds increased cAMP production, in a dose-dependent manner acting as antagonists of the A(1)AR, while two compounds act as agonists.

Design, synthesis and biological evaluation of a bivalent micro opiate and adenosine A1 receptor antagonist.

The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A(1) adenosine and mu opiate receptors with a K(i) of 0.8+/-0.05 and 0.7+/-0.03 microM, respectively. This hybrid molecule increases cAMP production in cells that over express the mu receptor as well as those over expressing the A(1) adenosine receptor and reverses the antalgic effects of mu and A(1) adenosine receptor agonists in animals.

Prognostic value of ischaemia-modified albumin in patients with non-ST-segment elevation acute coronary syndromes.

BACKGROUND: Ischaemia-modified albumin (IMA) is a new sensitive diagnostic biochemical marker of myocardial ischaemia. The purpose of the study was to analyse the prognostic value of IMA in patients admitted for non-ST-segment elevation acute coronary syndromes (NSTE ACS). METHODS: Consecutive patients admitted for NSTE ACS in our institution were prospectively included. IMA, cardiac troponin I (TnI) and C-reactive protein (CRP) were measured in all patients within 3h of last chest pain. The clinical combined endpoint was major adverse cardiac events (MACE) including cardiac death, nonfatal myocardial infarction (MI) and recurrent ischaemia leading to urgent revascularization. The independent prognostic impact of IMA on occurrence of the combined endpoint during hospitalization and at 1 year was tested by a logistic regression model and was systematically adjusted for other known clinical and biological predictors. RESULTS: Seventy-nine patients were enrolled. Nine (11.4%) patients experienced the combined endpoint during hospitalization and 16 (20.2%) during 1-year follow-up. Median IMA level was significantly higher in patients with MACE during hospitalization (115 [93-126]U/mL versus 100 [42-138]U/mL; p=0.007) and at 1 year (114 [93-126]U/mL versus 97 [42-138]U/mL; p<0.001). After adjustment for conventional prognostic risk factors, IMA remained an independent predictor of MACE both during hospitalization (odds ratio [OR]: 1.08; 95% confidence interval [CI]: 1.01-1.16; p=0.03) and at 1 year (hazards ratio [HR]: 1.07; 95% CI: 1.03-1.12; p=0.003). CONCLUSION: Baseline levels of IMA were associated with both short- and long-term cardiovascular (CV) events in patients admitted for NSTE ACS.

Adenosine A2A receptor hyperexpression in patients with severe SIRS after cardiopulmonary bypass.

OBJECTIVE: Adenosine (ADO) is an endogenous nucleoside, which has been involved in blood pressure failure during severe systemic inflammatory response syndrome (severe SIRS) after cardiac surgery with cardiopulmonary bypass (CPB). Adenosine acts via its receptor subtypes, namely A1, A2A, A2B, or A3. Because A2A receptors are implicated in vascular tone, their expression might contribute to severe SIRS. We compared adenosine plasma levels (APLs) and A2A ADO receptor expression (ie, B, K, and mRNA amount) in patients with or without postoperative SIRS. PATIENTS: : This was a prospective comparative observational study. Forty-four patients who underwent cardiac surgery involving CPB. Ten healthy subjects served as controls. MEASUREMENTS AND RESULTS: Among the patients, 11 presented operative vasoplegia and postoperative SIRS (named complicated patients) and 33 were without vasoplegia or SIRS (named uncomplicated patients). Adenosine plasma levels, K, B, and mRNA amount (mean +/- SD) were measured on peripheral blood mononuclear cells. Adenosine plasma levels, B, and K were significantly higher in complicated patients than in uncomplicated patients (APLs: 2.7 +/- 1.0 vs 1.0 +/- 0.5 micromol l, P < 0.05; B: 210 +/- 43 vs 65 +/- 26 fmol/mg, P < 0.05; K: 35 +/- 10 vs 2 +/- 1 nM, P < 0.05). In uncomplicated patients, APLs remain higher than in controls (1 +/- 0.5 vs 0.6 +/- 0.25 micromol/L; P < 0.05). Mean arterial pressure was inversely correlated to APLs (R = -0.58; P < 0.001) and B (R = -0.64; P < 0.001) leading to an increased requirement of vasoactive drugs during the postoperative period in vasoplegic patients. CONCLUSIONS: High expression of A2A ADO receptor and high APLs may be a predictive factor of postoperative severe SIRS after CPB.

Release of markers of myocardial damage evaluated in the coronary sinus during cardiac surgery.

Myocardial damage is a frequent complication of cardiac surgery by direct mechanical trauma during the surgical procedure and by myocardial ischemia, which occurs during the cardiopulmonary bypass (CBP). Because the concentrations of biomarkers in the blood collected from the coronary sinus are the best witness of the myocardial damages, we measured the levels of specific cardiac troponin I (c-TnI) and nonspecific (adenosine, myoglobin) markers of left ventricular damages in the coronary sinus of patients during cardiac surgery. Thirty patients who underwent aortic valve replacement for aortic stenosis were included. Blood samples were collected in the coronary sinus and in the radial artery at the beginning (T0), at the end of the CBP (T1), and then 24 hours later (T2). At T0 and T1, adenosine, c-TnI, and myoglobin levels were significantly higher in the coronary sinus than in the radial artery (T0: adenosine: mean +27%; c-TnI: +41%; myoglobin: +28%; T1: adenosine: mean +58%; c-TnI: +58%; myoglobin: +25%; p < .001). These parameters were significantly higher in the coronary sinus at T1 compared with T0 (adenosine: +50%; c-TnI: +229%; myoglobin: +94%; p < .01) and in the radial artery (adenosine: +21%; c-TnI: +194%; myoglobin: +98%; p < .01). At T2, c-TnI further increased. Damages to the myocardium during cardiac surgery are minimal in our surgical conditions but are probably underestimated when using markers measured at the peripheral level. However, most of the damages appear several hours after the surgery.

Increased expression of adenosine A2A receptors in patients with spontaneous and head-up-tilt-induced syncope.

BACKGROUND: Adenosine may play a role in the triggering of neurocardiogenic syncope, but no information on adenosine receptors is available at the present time. OBJECTIVE: The purpose of this study was to investigate whether adenosine A2A receptors expression is altered in patients with neurocardiogenic syncope. METHODS: Adenosine plasma levels (APLs), the expression of A2A receptors, were measured (mean +/- standard error of the mean) during tilt testing. Expression of receptors was assessed on mononuclear cells using a selective receptor ligand. RESULTS: At baseline, the APLs of 16 patients with a positive test were higher than those of 17 patients with a negative test and of those of a control group (2.10 +/- 0.30 vs. 0.40 +/- 0.05 and 0.41 +/- 0.06 muM, respectively; P <.0001). The number of receptors was higher in patients tested positive than in patients tested negative or in the control group (122 +/- 10 vs. 38 +/- 4 and 44 +/- 4 fmol/g of proteins, respectively; P <.0001). No difference was found in the affinity or synthesis among the three groups. CONCLUSION: This study showed an increased number and an up-regulation of adenosine A2A receptors in patients with spontaneous syncope and a positive head-up tilt, which in the context of high APLs may play a role in the recurrence of syncopal episodes.

Influence of haemodialysis and left ventricular failure on peripheral A(2A) adenosine receptor expression.

BACKGROUND: Haemodialysis (HD) sometimes accelerates left ventricular failure (LVF). As adenosine (ADO) is strongly implicated in cardiovascular functions, particularly via A(2A) receptor activation and as changes of peripheral A(2A) receptors mirror changes occurring in the cardiovascular system, we examined the influence of HD and LVF on both ADO plasma concentration and the expression of A(2A) receptors (i.e. Bmax, K(D) and mRNA amount) of peripheral blood mononuclear cells. METHODS: This cross-sectional study included 61 chronic renal failure (CRF) patients: 41 without LVF (24 haemodialysed and 17 undialysed) and 20 with LVF (9 haemodialysed and 11 undialysed). Ten LVF patients without CRF and 10 healthy subjects were also examined. RESULTS: (i) Bmax values of CRF patients without LVF were significantly decreased in undialysed patients compared with haemodialysed patients, and compared with controls (69 +/- 25 vs 98 +/- 33 vs 180 +/- 60 fmol/mg of protein, P < 0.05). Bmax values of CRF patients with LVF were lower in undialysed patients than in haemodialysed patients (60 +/- 27 vs 101 +/- 27 fmol/mg of protein, P < 0.05). Bmax values of LVF patients without CRF were lower than in controls (51 +/- 19 vs 180 +/- 60 fmol/mg of protein). (ii) A(2A) mRNA expression was increased in haemodialysed patients compared with controls (20.2 +/- 0.75 vs 17.6 +/- 1.3, P < 0.05). (iii) ADO plasma levels were high in haemodialysed patients and further increased during the HD sessions. CONCLUSION: The number of A(2A) receptors was decreased by CRF with or without LVF. However, this decrease was less important in haemodialysed patients. The changes in peripheral A(2A) receptor expression suggest a significant inflammatory response to HD and heart or kidney failure. Whether these changes do reflect alterations in cardiomyocytes needs further investigation.

Relationship between A2A adenosine receptor expression and intradialytic hypotension during hemodialysis.

BACKGROUND: Intradialytic hypotension (IDH) is a common complication of hemodialysis sessions (HDSs). Adenosine may contribute to the drop in blood pressure during IDH events because it has hypotensive effects. As A(2A) adenosine receptor expression is essential for blood pressure control, we compared the expression of A(2A) receptors (Bmax, K(D), and messenger ribonucleic acid [mRNA] levels) in peripheral blood mononuclear cells from IDH and non-IDH patients and from controls. We also evaluated adenosine plasma levels (APLs). METHODS: We included 10 hemodialyzed patients with at least three IDH events per month. We also included 11 hemodialyzed patients with no history of IDH events and 10 healthy subjects as controls. RESULTS: IDH patients had higher Bmax values than non-IDH patients (mean before HDS, +86%; after HDS, +112%), whereas non-IDH patients had lower Bmax values than controls (mean -72%). K(D) values were not significantly different between patients and controls. The levels of mRNA increased significantly during HDS but without an increase in receptor expression on the cell membranes. APLs were higher in hemodialyzed patients than in controls. CONCLUSION: We found that A(2A) receptors are more expressed in IDH patients than in non-IDH patients, whereas APL was high in all patients. Both high APL and a relative increase in A(2A) receptor expression may favor IDH events.

Influence of the dialysis membrane on markers of tissue ischemia.

BACKGROUND: Hemodialysis (HD) is often accompanied by adverse effects, such as tissue ischemia. We have already observed an increase in plasma adenosine (ADO) levels during HD sessions, which may be the result of tissue ischemia. Here we evaluate the influence of the dialysis membrane on two sensitive and early markers of ischemia: ADO and ischemia-modified albumin (IMA). METHODS: We included in the study 50 patients with end-stage renal failure, 39 hemodialyzed (mean age 61+/-24 years; 24 male; membranes: 23 synthetic and 16 cellulose based) and 11 undialyzed (mean age 55+/-12 years; 6 male), and 10 healthy subjects (mean age 47+/-11 years; 4 male). We compensated for hemoconcentration during HD by measuring either the IMA to albumin (Alb) or the ADO to Alb ratio. RESULTS: Under basal conditions, the IMA to Alb ratio was not significantly different in patients and controls and HD did not significantly modify this ratio. Conversely, the ADO to Alb ratio (mean+/-SD in micromol/g) was higher in patients before HD compared with either undialyzed patients or controls (before HD: 0.077+/-0.02; undialyzed patients: 0.026+/-0.11; controls: 0.022+/-0.01). During HD, there was a significant increase in the ADO to Alb ratio (before HD: 0.077+/-0.02; after HD: 0.09+/-0.029; p<.01). We found no significant difference in the IMA to Alb or ADO to Alb ratio using either synthetic or cellulose-based membranes. CONCLUSIONS: We concluded that ADO is a more sensitive marker of ischemia than IMA and that, under our HD conditions, the ischemia caused by HD was very weak, independent of the dialysis membrane.

CD26 modulates nociception in mice via its dipeptidyl-peptidase IV activity.

BACKGROUND: CD26 is a multifunctional cell surface glycoprotein expressed by T and B cells. It exhibits a dipeptidyl-peptidase activity (DPP-IV) that cleaves the penultimate proline from the N-terminus of polypeptides, thereby regulating their activity and concentration. METHODS: Using CD26-/- mice resulting from targeted inactivation of the gene, we examined the consequences of a DPP-IV defect on behavioural response to nociceptive stimuli and concentration of the pain modulator peptides substance P (SP) and endomorphin 2, two DPP-IV substrates. RESULTS: CD26 inactivation induced a three-fold decrease in circulating endopeptidase activity while that found in brain extracts was normal, albeit very weak. CD26-/- mice had high SP concentrations in plasma (3.4+/-1 pg/ml versus 1.5+/-0.3 pg/ml, P<10(-3)) but not in brain extracts (35+/-12 pg/ml versus 32+/-9 pg/ml, P>0.05). Endomorphin-2 levels in the two groups were in the same range for plasma and brain extracts. CD26-/- mice displayed short latencies to nociceptive stimuli (hot plate test: 6.6+/-1.2 s versus 8.6+/-1.5 s, P<10(-4); tail pinch test: 3.1+/-0.6 s versus 4.2+/-0.8 s, P<10(-3)). Administration of an SP (NK1) receptor antagonist or DPP-IV to CD26-/- mice normalised latencies. DPP-IV inhibitors decreased latencies only in CD26+/+ mice. CONCLUSIONS: Our observations represent the first fundamental evidence showing that DPP-IV influences pain perception via modulation of the peripheral SP concentration. Our work also highlights the role of peripheral NK1 receptors in nociception.

The impact of the fourth disulfide bridge in scorpion toxins of the alpha-KTx6 subfamily.

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.

Kinetic study of adenosine concentrations and the expression of adenosine deaminase in mononuclear cells during hemodialysis.

BACKGROUND: We previously showed that high intralymphocytic adenosine (Ado) concentrations are found in hemodialyzed patients due to the reduced activity of mononuclear cell adenosine deaminase (MCADA). These abnormalities contribute to the immune defect observed in HD patients. The kinetics of these abnormalities and the causes of the low MCADA activity, however, have not been investigated. Here, we addressed this question. Since interferon gamma (IFNgamma) partially modulates MCADA, we also evaluated the effect of IFNgamma on MCADA activity in vitro. METHODS AND RESULTS: The study included 12 patients (eight men and four women) who were observed from the first to the 36th hemodialysis (HD) session, and eight healthy subjects (controls). MCADA activity and Ado concentrations were normal before the first HD session. Ado concentrations progressively increased from the first (10.5 +/- 3.1 pmol/10(7) cells) to the fourth session (26.7 +/- 3 pmol/10(7) cells), before stabilizing at a high level. MCADA activity increased transiently until the second session (2.2 +/- 0.5 IU/10(7) cells before HD vs. 2.8 +/- 0.6 IU/10(7)cells), and then decreased and stabilized at a low level (1.0 +/- 0.5 IU/10(7)cells). The amount of MCADA mRNA transiently increased until the third session (mRNA MCADA/mRNA beta-actin: 0.6 +/- 0.2 vs. 0.8 +/- 0.2), and then decreased to 0.3 +/- 0.1 at the 36th session. MCADA activity underwent a dose-dependent increase after IFNgamma stimulation. CONCLUSION: HD affects the transcription of the gene encoding MCADA after just three HD sessions, leading to decreased MCADA activity and increased plasma concentration of Ado.

Supraspinal antinociceptive effects of mu and delta agonists involve modulation of adenosine uptake.

BACKGROUND: The modulation of extracellular adenosine concentration by opioids provides evidence that the antinociceptive effects of these compounds involve endogenous adenosine. The aim of this study was to determine whether there is a relation between the inhibition of brain synaptosome adenosine uptake by opioid agonists and the analgesic effects of these compounds. METHODS: The authors used the hot plate and tail-pinch tests to evaluate in mice (C57BL/6 females; weight, 25-30 g) the effects of caffeine, a nonspecific adenosine receptor antagonist, on the antinociceptive effect induced by the intracerebroventricular administration of oxymorphone as a mu agonist, SNC80 as a delta agonist, or U69593 as a kappa agonist. They also investigated the effect of these opioid receptor agonists on the uptake of adenosine by whole brain synaptosomes. RESULTS: Caffeine decreased the analgesic effects induced by oxymorphone or SNC80 but not those induced by U69593. Oxymorphone and SNC80 inhibited adenosine uptake by brain cells, but U69593 did not. CONCLUSION: The antinociceptive effects obtained with mu or delta (but not kappa) agonists administered supraspinally were indicative of the involvement of modulation of adenosine uptake.

Evolution of maurotoxin conformation and blocking efficacy towards Shaker B channels during the course of folding and oxidation in vitro.

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channels, including the voltage-gated Shaker B subtype. In the present study, we have investigated over 80 h: (1) the time-course of folding of synthetic MTX (sMTX) by CD analysis; (2) the kinetics of disulphide bridge formation by MS; and (3) the potency of MTX in blocking Shaker B currents during the combined process of its in vitro folding and oxidation. From the CD data, we show that stable secondary structures of sMTX evolve sequentially over time, with the appearance of the alpha-helix within 5 h, followed by the formation of the beta-sheet within 22 h. Using MS analysis, the sMTX intermediates were also found to appear sequentially from the least (one-disulphide-bridged sMTX) to the most oxidized species (native-like, four-disulphide-bridged sMTX). The time course of formation of secondary structures coincides mainly with the occurrence of one-disulphide-bridged sMTX for the alpha-helix and two- or three-disulphide-bridged sMTX for the beta-sheet. On-line electrophysiological recordings, which measure sMTX blocking efficacy on K(+) currents during its folding and oxidation, were performed on Shaker B channels expressed in Xenopus oocytes. Unexpectedly, the results demonstrate that sMTX is highly potent at the initial stage of oxidation, whereas its blocking activity can be transiently and dramatically reduced at later stages during the course of folding/oxidation before it reaches full bioactivity. These data suggest that formation of disulphide bridges can both physically stabilize and alter the bioactive three-dimensional structure of sMTX.