Differential effect of adenosine on DNA synthesis in lymphoid and myeloid cell lines.

In this report, the effects of adenosine on the promyelocytic cell line HL-60 and on T-lymphocytic clones are compared. According to previous reports, adenosine induces a dose-dependent inhibition of DNA synthesis in T-lymphocytes. Conversely, adenosine dose-dependently enhances DNA synthesis in HL-60 cells, as documented with [3H]thymidine uptake studies and flow cytometric cell-cycle analysis. Unlike its effect on lymphocytes, the adenosine effect on HL-60 cells does not seem to be mediated by receptor binding, but it appears to be correlated with an intracellular mechanism following active uptake. Despite the different effects exerted by adenosine on lymphocytes and myeloid cells, a purinergic pathway appears to be more generally involved in the regulation of some phases of cell growth.

Selective 1H-NMR relaxation investigations of membrane-bound drugs in vitro. 3. Calcium-entry blockers and adenosine.

Selective proton relaxation rates (SPRR) were measured for selected protons of nimodipine or diltiazem in the presence of neutrophils, allowing detection of binding to the cell membrane. Fast exchange exchange of drug molecules between bound and free environments was shown to be the main factor determining the enhancement of SPRR, whereas viscosity effects could be neglected. The SPRR enhancement was almost completely cancelled out by the presence of adenosine as a cosolute in a dose-dependent fashion, leading to the suggestion that the endogenous mediator 'adenosine' affects binding of calcium-entry blockers to the neutrophil surface.

QTc interval prolongation during infusion with dipyridamole or adenosine.

The aim of our study was to discover whether there was a relationship between the QTc interval prolongation on the standard 12-lead electrocardiogram (ECG) and provoked myocardial ischemia. Since the increase of adenosine plasma levels, obtained either with adenosine or dipyridamole (an adenosine reuptake inhibitor) infusion, has been used to test the coronary artery reserve in patients affected by coronary artery disease, the QTc interval modifications during dipyridamole or adenosine echocardiographic stress test were evaluated. Twenty-five patients admitted to our Institute for evaluation of chest pain of suspected myocardial origin underwent an echocardiographic dipyridamole stress test (0.84 mg/kg over 10 min) after discontinuation of antianginal treatment. Of these patients, 10 underwent an echocardiographic adenosine stress test (scalar doses of 50, 75, 100, 140 micrograms/kg/min) after 48-72 h. The Bazett formula was used to evaluate the QTc interval. After dipyridamole and adenosine administration, a significant prolongation of the QTc interval was observed only in those patients who had positive test results. Our data suggested that QTc interval prolongation during pharmacological stress tests might be considered a marker of myocardial ischemia.

Pharmacological preconditioning of ischemic heart disease by low-dose dipyridamole.

Fourteen patients affected with coronary artery disease underwent two consecutive dipyridamole echocardiographic stress tests, in basal conditions and after repeated low doses of intravenous dipyridamole, following the observation that pulse increases in adenosine plasma levels due to repeated intravenous administration of dipyridamole mimic the mechanism of ischemic preconditioning. Echocardiographic, electrocardiographic, haemodynamic parameters, and adenosine plasma levels were measured. After the second test, six patients were completely negative, and in those eight still positive the onset of dyssynergy was delayed.

Angioimmunoblastic lymphadenopathy with dysproteinemia complicated by Kaposi's sarcoma.

A case of clinically and morphologically typical angioimmunoblastic lymphadenopathy (AILD) in a 68-year-old man during a prolonged antibiotic treatment for urinary infection is presented. Lymph node biopsy at first showed findings suggestive of an exhaustion of the germinal center immunological activity (like those characterizing angiofollicular lymph any clear transition into malignant lymphoma. The course of the disease was characterized by the occurrence of opportunistic infections (toxoplasmosis, herpes zoster), and finally by the onset of a cutaneous Kaposi's sarcoma. The possible relation of AILD to Kaposi's sarcoma is discussed, and the main clinical and morphological data of the case of AILD (about 200) reported in the literature are reviewed.

Defibrotide inhibits Ca2+ dependent neutrophil activation: implications for its pharmacological activity in vascular disorders.

Defibrotide (DEF) is a polydeoxyribonucleotide agent provided with profibrinolytic and antithrombotic properties. Moreover, an antiischemic, cardioprotective effect of the drug has recently been demonstrated in experimental animals. Increasing evidence exists of the important role played by neutrophils in the development of tissue damage during chronic and acute ischemia and in the early phases of reperfusion. In order to evaluate whether the overall cytoprotective effect of DEF could be based, at least in part, on a neutrophil-involving mechanism, the authors studied the in vitro effects of the drug on human neutrophil activation triggered by several specific stimuli. The drug dose-dependently (10-100 microM) inhibited enzyme release, superoxide anion generation, and chemiluminescence induced in neutrophils by the chemoattractant oligopeptide fMLP and by the divalent cation ionophores A23187 and ionomycin. The increase of extracellular calcium concentration from 0.5 to 2.0 mM antagonized the inhibitory effect of DEF. The use of the fluorescent probe Fura 2/AM led them to show that DEF is able to reduce the cytosolic free calcium increase following specific stimulation by affecting extracellular calcium entrance. Such a behavior resembles that of calcium-antagonistic drugs, thus suggesting that DEF works, at least in part, similarly to calcium entry blockers. Such an activity on cell calcium translocation could represent the underlying molecular mechanism of cytoprotection. Finally, the inhibitory action on neutrophil functions may play a role in tissue protection during ischemic injury.

In vivo and in vitro evidence of an adenosine-mediated mechanism of calcium entry blocker activities.

Drugs such as dipyridamole (200 micrograms/kg/min), an adenosine uptake inhibitor, and theophylline (300 micrograms/kg/min), an adenosine receptor antagonist, respectively increased and decreased postischemic hyperemia in normal subjects, as well as in POAD patients. Moreover, dipyridamole pretreatment was able to antagonize the reduction of peak flow induced by nifedipine, and the potentiating effect of flunarizine on postischemic hyperemia was affected significantly by theophylline, thus suggesting a possible interference of calcium entry blocker drugs with the endogenous adenosine system. In a cellular model (polymorphonuclear leukocytes--PMN) the inhibitory effect of calcium entry blockers on stimulated functions (degranulation and free radical production) was highly antagonized by theophylline. Finally, a 1H-NMR spectroscopy study showed a binding interaction between adenosine and flunarizine on the cell membrane. An adenosine-receptor coupling to the calcium entry blocker channels is suggested.

Hemodynamic and hemorheologic effects of i.v. Dilevalol in hypertensive patients.

The authors evaluated the effect of Dilevalol infusion on blood pressure, heart rate, central hemodynamics, and rheologic parameters in hospitalized inpatients affected with mild or moderate hypertension. After a dose-finding phase and a washout period of one week, 10 patients aged fifty to seventy-two-years (median 61.5) were given either a single dose of Dilevalol 60 mg or placebo, and seven days later they underwent the other treatment, according to a single-blind, crossover design. Central hemodynamic measurements were performed by means of M-mode echocardiography, and hemorheologic parameters were evaluated by means of strain-gauge plethysmography. The maximal increase in lower extremity flow at rest had been obtained with the infusion of 60 mg Dilevalol during dose-finding, and so this dose was chosen for the second part of the study. The infusion of Dilevalol significantly increased rest flow and decreased blood viscosity, but the changes in central, parameters were not considered clinically relevant, although statistically significant. Blood pressure decreased without significant changes in heart rate. Thus, the acute administration of Dilevalol reduced blood pressure, without affecting heart rate and central hemodynamics, confirming the vasodilating effect of the drug. A significant improvement was also shown on blood viscosity in these hypertensive patients.

Experimental model of short-time exercise-induced preconditioning in POAD patients.

Regular physical exercise improves walking performance in patients affected with peripheral 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 biological 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 conditioning in treadmill-trained POAD patients.

Action of cinnarizine on the hyperviscosity of blood in patients with peripheral obliterative arterial disease.

Cinnarizine, a drug capable of improving blood flow, was studied for its action on blood viscosity and its main components in patients affected by peripheral obliterative arterial diseases (POAD). Both acute and chronic administration of the drug diminished the increased whole-blood viscosity in patients, without affecting plasma and serum viscosity, hematocrit, plasma fibrinogen concentration, and plasma osmolality. Since cinnarizine also led to a significant increase of peripheral muscular blood flow, it was hypothesized that this action may be due to an increased deformability of the red cells, and may play a considerable role in the therapeutic approach to POAD.

Adenosine and chronic ischemia of the lower limbs.

Adenosine is an endogenous nucleoside with multiple biological properties which plays a central role in the pathophysiology of tissue ischemia. Adenosine signals an imbalance between oxygen demand and supply, and it initiates responses to redress such a discrepancy. Besides its vasodilating properties, adenosine possesses anti-platelet and anti-neutrophil activities and provides cytoprotection. Adenosine is presumably the main mediator of the preconditioning phenomenon. During ischemia of the lower limbs, adenosine plays a physiological role by inducing vasodilatation and by preventing microcirculatory failure. Exercise training prolongs claudication distance possibly by inducing pulse increases of adenosine and consequently skeletal muscle preconditioning. Moreover, the adenosine increase which follows the administration of some drugs, such as buflomedil and propionylcarnitine, opens new perspectives in the management of leg ischemia. In fact, the concept arises of an ischemic (exercise-dependent) or pharmacologic preconditioning in the treatment of patients with claudication.

[Effects of diphtheria and tetanus toxins on liver regeneration in the rat]. / Effetti delle tossine difterica e tetanica sulla rigenerazione epatica nel ratto.

The effects of diphtheria toxin and tetanus toxin on the regenerating rat liver were studied. The following parameters were used: liver regeneration percentage, mitotic index, protein content and serum complement C3 fraction level. Diphtheria toxin does not affect the liver regeneration, that what confirms the poor sensitivity of the rat to this toxin. Tetanus toxin inhibits the cellular division, while causes an increase in the cell protein content, resulting in hepatocyte hypertrophy. Both toxins cause a fall of the serum complement C3 fraction.

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.

Effects of dipyridamole and adenosine on vasoactive peptides calcitonin gene-related peptide and atrial natriuretic peptide in humans: role of sympathetic activation.

1. It has been observed that dipyridamole (DIP) administration produces equivalent cardiovascular effects at lower systemic adenosine (ADO) plasma concentrations than those obtained with exogenous ADO infusion. This observation led to the identification of DIP for additional 'ischaemia-inducing' mechanisms, possibly based on sympathetic activation. 2. In turn, exogenous ADO administration has proven to elicit a complex neurohumoral response, including an increase in the plasma concentration of catecholamines, associated with augmented levels of the vasoactive peptides calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP). More particularly, increases in CGRP seem to be dependent on sympathetic activation, while changes in ANP do not. 3. In order to clarify some aspects of the activity of DIP on neurohumoral systems, the effects of administration of DIP and ADO on plasma levels of noradrenaline (NA), CGRP and ANP were studied in healthy volunteers. Haemodynamic parameters were also monitored. 4. Infusion of exogenous ADO produced plasma levels of ADO as high as 1893+/-386 nmol/L, together with a significant increase in plasma levels of CGRP, ANP and NA. Similarly, the infusion of DIP produced augmented plasma concentrations of the examined parameters, with a peak plasma ADO concentration of 470+/-49 nmol/L. 5. At a given ADO plasma concentration of 450+/-10 nmol/L, the increase in CGRP and NA levels with DIP infusion was significantly higher than that observed following the infusion of ADO, whereas the increase in the plasma concentration of ANP following DIP infusion was very similar to that seen following ADO infusion. 6. The physiological background of these findings is based on evidence that DIP displays a greater sympathoexcitatory activity than does exogenous ADO and only the increase in plasma CGRP seems to be mediated, although indirectly, by beta-adrenoceptor stimulation. The exact mechanism of DIP-dependent sympathetic activation remains to be elucidated.

Inhibition by cinnarizine of the properdin-dependent activation of complement.

Cinnarizine (1-benzhydryl-4-cinnamylpiperazine dihydrochloride), a well-known antagonist of vasoactive agents, has previously been shown to inhibit the activation of complement at the level of the fourth component. Cinnarizine was now tested in a system of complement activation by the properdin-dependent alternative pathway. At the concentration of 7.10(-5) M the compound completely inhibited the hemolytic reaction, which is the consequence of human complement activation in the presence of inulin or zymosan. This in vitro effect of cinnarizine was abolished by the addition of 1 mM MgCl2. These findings extend the anticomplementary activity of cinnarizine to the properdin pathway and support its interaction with the magnesium-dependent steps of the activation sequence.

Pharmacodynamics of ticlopidine in man in relation to plasma and blood cell concentration.

In 6 normal volunteers given single oral doses of 250,500 and 1000 mg ticlopidine (T), the peak plasma level of unchanged drug was reached after about 2 h. There was no correlation between the plasma T level and its inhibitory effect on platelet function, expressed as % inhibition of ADP-induced aggregation. By means of HPLC and GC/MS significant concentrations of T were demonstrated in washed red cells, platelets and neutrophils, with a marked difference in the time course of the appearance of cell-associated drug. The time course of platelet-associated T very accurately fitted that of the antiaggregatory activity. After subacute oral administration (250 mg b.d. for 7 days), the maximum effect on platelet function was observed after 3 to 4 days, when a significant concentration of platelet-associated T had been reached. The pharmacological effect persisted as long as drug was detectable in platelet. An in vitro study strongly suggested that the antiaggregating effect was retained by treated washed platelets but not by treated plasma. It is suggested that the platelet compartment represents the pharmacological target of T via a specific uptake system.

Adenosine blocks calcium entry in activated neutrophils and binds to flunarizine-sensitive calcium channels.

Adenosine is able to prevent Ca+(+) influx into activated neutrophils as detected by the specific fluorescent indicator Quin 2. Such an effect is shown in a similar fashion by the calcium entry blocker flunarizine. The binding interaction between flunarizine and the neutrophil membrane as well as the flunarizine-adenosine antagonism are shown by the 1H-NMR technique, thus supporting evidence of a competition between the agents at the same or a nearby site on the cell membrane.

Conformation and dynamics of the chemotactic peptide formyl-L-methionyl-L-leucyl-L-phenylalanine in solution.

Several conformational and dynamic features of the chemotactic peptide formyl-L-methionyl-L-leucyl-L-phenylalanine in solution have been delineated by investigations of NMR and IR spectroscopic parameters. Both 1D and 2D NMR experiments have been performed for detection of scalar and dipolar proton-proton connectivities, whereas 13C and 1H relaxation parameters have been interpreted in terms of molecular dynamics. The main conformation appeared to be unfolded with the three hydrophobic side chains extending in divergent directions with respect to the backbone. The existence of relatively weak intermolecular hydrogen bonds was demonstrated, involving the formamide end group, with increase in the hydrophobicity of the external surface.

Modulation of venous endothelial activity and transcellular calcium transport by defibrotide: the adenosine hypothesis.

Defibrotide is a polydeoxyribonucleotide that possesses profibrinolytic and cytoprotective activities. These properties have been associated with its capacity to induce the release of prostacyclin and tissue plasminogen activator (t-PA) from endothelial cells. In the present study, the bolus administration of defibrotide in humans was able to induce (100-800 mg) a dose-dependent decrease in plasminogen activator inhibitor (PAI) (from 19.4 +/- 7.11 to 7.20 +/- 6.41 AU/mL) and an increase in t-PA (from 3.70 +/- 0.96 to 4.50 +/- 1.20 IU/mL) and in the stable prostacyclin derivative 6-keto-PGF1 alpha (from 18.83 +/- 3.83 to 26.75 +/- 8.48 pg/0.1 mL) in the venous blood. In a second part of the study, defibrotide has been shown to inhibit dose-dependently (10-100 microns) neutrophil activation in vitro: it decreased lysosomal enzyme release and superoxide anion and chemiluminescence production induced by the oligopeptide fMLP and the ionophores A23187 and ionomycin. The increase in extracellular calcium concentration from 0.5 to 2 mm antagonized the inhibitory effect of the drug. Defibrotide was able to reduce the cytosolic free calcium increase induced by specific stimuli by blunting calcium entry. Such an inhibitory activity of defibrotide was antagonized by theophylline, an adenosine receptor antagonist. The study confirms some pharmacological activities of defibrotide (release of t-PA and prostacyclin in vivo), and it also suggests that the compound blocks Ca2+ entry into the cells, possibly by interfering with the adenosine receptors.