L. Ceccatelli

Changes of peripheral A2A adenosine receptors in chronic heart failure and cardiac transplantation

Peripheral blood mononuclear cells of chronic heart failure (CHF) patients produce great amounts of pro‐inflammatory cytokines, indicating that circulating cells are activated and could mirror changes occurring in inflammatory cells infiltrating the failing heart. Adenosine is a regulatory metabolite acting through four membrane receptors that are linked to adenylyl cyclase: activation of the A2A receptor subtype has been reported to inhibit cytokine release. Changes of the adenosinergic system may play a role in CHF development. Here we report an increase of A2A receptor expression, density, and coupling to adenylyl cyclase in blood circulating cells of CHF patients. A2A receptor up‐regulation was also found in the explanted hearts of these patients, suggesting that changes of peripheral adenosine receptors mirror changes occurring in the disease target organ. In a cohort of patients followed longitudinally after heart transplantation, alterations of peripheral A2A adenosine receptor progressively normalized to control values within 6 months, suggesting that improvement of cardiac performance is accompanied by progressive restoration of a normal adenosinergic system. These results validate the importance of the A2A receptor in human diseases characterized by a marked inflammatory/immune component and suggest that the evaluation of this receptor in peripheral blood cells may be useful for monitoring hemodynamic changes and the efficacy of pharmacological and non‐pharmacological treatments in CHF patients.

Nitroprusside in vitro inhibits platelet aggregation and intracellular calcium translocation. Effect of haemoglobin

The biologically final active compound of nitrovasodilators is now supposed to be nitric oxide (NO), a labile substance identical to EDRF. The effects of nitroprusside on platelet functions were studied in vitro. Platelet aggregation induced by several stimuli (ADP, collagen, arachidonic acid and PAF) was inhibited by increasing concentrations of the drug (1-50 uM); interestingly, the potency of nitroprusside is higher when PAF is employed as stimulating agent in comparison with the other agonists (ED50 = 2 uM for ADP, 2.5 uM for A.A., 4.5 uM for collagen and 0.3 uM for PAF-induced aggregations). The concomitant addition of haemoglobin is able to reverse the inhibitory effect of nitroprusside, according to the view that haemoglobin possesses a high affinity for NO, thus antagonizing the effect of this compound. Nitroprusside was also able to inhibit intracellular calcium translocation, as studied with the Quin 2 technique, induced by PAF and arachidonic acid. Fron these observations the hypothesis may be suggested that nitroprusside inhibits platelet functions by mimicking the endogenous NO, and that the intracellular calcium metabolism is involved in the inhibitory activity of the drug.

Experimental Model of Short-Time Exercise-Induced Preconditioning in POAD Patients:

Regular physical exercise improves walking performance in patients affected with periph eral obliterative arterial disease (POAD). The mechanisms underlying the phenomenon are still controversial. In order to verify the hypothesis that physical conditioning of lower limbs on a treadmill and ischemic preconditioning of the heart could share some biolog ical aspects, 14 POAD subjects underwent a training program on the treadmill consisting of five repeated submaximal exercises at five-minute and two-hour intervals preceding the maximal tolerance test. Moreover, a protocol with two daily submaximal walking exercises over one week was also performed. Pain-free and total walking distance were measured before and after they performed the program. Moreover, plasma levels of adenosine and adenosine triphosphate (ATP) were measured and polymorphonuclear (PMN) leukocyte activity was studied together with rheologic parameters. Pain-free distance was prolonged by 15.4% and 14.3%, and total distance was prolonged by 23.1% and 26.9%, in the exercises with five-minute and two-hour intervals, respectively. After one week of daily exercises, the onset of pain and the end of the test were delayed by 24% and 43.7%, respectively. An improvement in blood rheology and a reduced PMN reactivity were also observed with the three protocols, associated with an increase in plasma levels of adenosine and ATP. Similarly to ischemic preconditioning in the heart, the possibility is suggested that an adenosine-mediated mechanism may contribute to the development of physical condi tioning in treadmill-trained POAD patients.

Long-term plasma levels of leptin and adiponectin in Rett syndrome.

Objective  Rett syndrome is a progressive neurological disorder affecting almost exclusively females after age 6 months and characterised by acquired microcephaly, psychomotor retardation, growth failure, purposeless hand movements, autistic‐like behaviour and wide‐based and stiff legged gait. Leptin and adiponectin, peptides secreted by adipose tissue, are involved in the regulation of body weight and energy expenditure.

Variations of plasma leptin and adiponectin levels in autistic patients.

Autism is a neurodevelopmental disorder with pathogenesis not completely understood. Although a genetic origin has been recognized, it has been hypothesized a role for environmental factors, immune dysfunctions, and alterations of neurotransmitter systems. In young autistic patients we investigated plasma leptin and adiponectin levels over a year period. Thirty-five patients, mean age at the basal time 14.1+/-5.4 years, were enrolled. Controls were 35 healthy subjects, sex and age matched. Blood samples were withdrawn in the morning at the baseline and 1 year after. In patients leptin concentrations significantly increased, while adiponectin did not significantly change. Leptin values in patients were significantly higher than those found in controls at each time; adiponectin values did not differ at each time between patients and controls. Since patients were not obese, we could hypothesize that leptin might participate to clinical manifestations other than weight balance. The role of adiponectin in autism is still debatable.

Adenosine inhibits polymorphonuclear leukocyte in vitro activation: a possible role as an endogenous calcium entry blocker.

Adenosine is able to in vitro inhibit FMLP-dependent activation of polymorphonuclear leukocytes as evaluated by enzyme release, superoxide anion generation and chemiluminescence production. The inhibiting effect is more relevant when A23187 is employed as stimulating agent. In this case the effect is significantly reversed by increasing concentrations of extracellular calcium. Since A23187-dependent activation is strictly dependent on Ca++ influx into the cell, the hypothesis is suggested that adenosine could act by regulatory mechanisms involving membrane calcium transport.

Benzodiazepines Inhibit in Vitro Free Radical Formation from Human Neutrophils Induced by Fmlp and A23187.

Diazepam (DZP) inhibited in vitro in a concentration-dependent manner superoxide anion generation and chemiluminescence from human neutrophils stimulated by the formylated oligopeptide FMLP and by the calcium ionophore A23187. The dose-dependent inhibitory effect of DZP on A23187-dependent superoxide generation in the presence of Ca++ 0.6 mM was highly antagonized by increasing extracellular Ca++ concentration to 1.5 mM and to 2.0 mM. Ro 5-4864, a specific ligand for peripheral type benzodiazepine (BZ) binding site, inhibited superoxide generation induced by FMLP, while clonazepam (CNZ), which is selective for brain sites, did not possess any activity. Ro 15-1788, a central type BZ receptor antagonist, did not show any antagonistic activity on DZP-dependent inhibition. A new physiological property for substances presenting an affinity for peripheral type BZ binding sites is supposed. The inhibitory effect of BZ on neutrophil functions seemed to be associated with a Ca++-involving mechanism.

Pharmacokinetics and pharmacodynamics of neutrophil‐associated ciprofloxacin in humans

To study the possibility that the penetration of the antibiotic ciprofloxacin into polymorphonuclear leukocytes (PMN) may be associated with some changes in cell reactivity.

Statins discontinuation in compliant chronic users induces atherothrombotic profile despite baseline clinical setting and treatments.

Statins offer important benefits for the large populations of individuals at high risk for cardiovascular and cerebrovascular events in both primary and secondary preventions [1]. Due to the positive clinical impact, the concern of statins withdrawal on putative rebound vascular events has been suggested and evaluated both in terms of biological mechanisms or clinical settings as extensively reviewed [2– 3]. Furthermore, although from retrospective analysis, several observations have confirmed an increased events rate especially when statins were withdrawn during acute phase of acute vascular stress like coronary syndromes [4–5], major vascular surgery [6–7] and ischemic stroke [8], also in a randomized study [9]. Still unsolved is whether statin withdrawal syndrome does exist directly or could be related mainly to the specific atherothrombotic risk related to the behaviour of baseline disease as suggested by the discordant data from the retrospective analysis of the TNT study in patients with stable coronary disease [10]. The aim of the present study was to evaluate the putative effects of statins withdrawal due to several causes on atherothrombotic and inflammatory activation as well as lipid profile in subjects in primary prevention for cardiovascular risk on established statin therapy for at least one year. The patients population consisted of ninety-six subjects treated for familial dyslipidaemia (group A); one-hundred-forty subjects treated for dyslipidaemia and hypertension (group B) and/or clinical atherosclerosis (including ASA 100 mg/day, group C) [11–12] that were evaluated at seven (T7), fourteen (T14), thirty (T30) and sixty (T60) days after statin withdrawal for lipid profile including oxidized-LDL (ox-LDL); hs-CRP, soluble CD40 ligand (sCD40L), platelet P-selectin (pCD62+) and endogenous thrombin potential (ETP). Patients gave written informed consent to blood collection after ethic committee approval according to the guidelines of ICJME as for a previous protocol [13–14]. Fifty healthy subjects matched for gender and age were evaluated as controls for measurable parameters. Total cholesterol, LDLC, high-density lipoprotein cholesterol (HDL-C), triglycerides, ox-LDL, hsCRP andpCD62+expressionwere evaluated as previously described [14]. Apo-A and Apo-B were determined by immunonephelometry

Adenosine system and cell calcium translocation: interference of calcium channel blockers.

Adenosine is able to inhibit in vitro neutrophil functions induced by formyl-methionyl-leucyl-phenylalanine (FMLP) and A23187, but not phorbol 12-myristate 13-acetate (PMA). The inhibiting activity on A23187 is reversed by increasing extracellular Ca2++ concentration. The calcium entry blocker flunarizine shows an activity very similar to that of adenosine. Both adenosine and flunarizine prevent Ca++ influx into activated neutrophils as detected by the fluorescent Ca++ chelator Quin-2. Finally, flunarizine binds to the neutrophil membrane and adenosine competitively inhibits flunarizine binding as assessed by 1H-Nuclear Magnetic Resonance (1H-NMR) technique, thus indicating that the two agents share a common binding site on the cell membrane.