Differentiating multi-MeV, multi-ion spectra with CR-39 solid-state nuclear track detectors.

The development of high intensity petawatt lasers has created new possibilities for ion acceleration and nuclear fusion using solid targets. In such laser-matter interaction, multiple ion species are accelerated with broad spectra up to hundreds of MeV. To measure ion yields and for species identification, CR-39 solid-state nuclear track detectors are frequently used. However, these detectors are limited in their applicability for multi-ion spectra differentiation as standard image recognition algorithms can lead to a misinterpretation of data, there is no unique relation between track diameter and particle energy, and there are overlapping pit diameter relationships for multiple particle species. In this report, we address these issues by first developing an algorithm to overcome user bias during image processing. Second, we use calibration of the detector response for protons, carbon and helium ions (alpha particles) from 0.1 to above 10 MeV and measurements of statistical energy loss fluctuations in a forward-fitting procedure utilizing multiple, differently filtered CR-39, altogether enabling high-sensitivity, multi-species particle spectroscopy. To validate this capability, we show that inferred CR-39 spectra match Thomson parabola ion spectrometer data from the same experiment. Filtered CR-39 spectrometers were used to detect, within a background of ~ 2 × 1011 sr-1 J-1 protons and carbons, (1.3 ± 0.7) × 108 sr-1 J-1 alpha particles from laser-driven proton-boron fusion reactions.

Design and commissioning of a neutron counter adapted to high-intensity laser matter interactions.

The advent of multi-PW laser facilities world-wide opens new opportunities for nuclear physics. With this perspective, we developed a neutron counter taking into account the specifics of a high-intensity laser environment. Using GEANT4 simulations and prototype testings, we report on the design of a modular neutron counter based on boron-10 enriched scintillators and a high-density polyethylene moderator. This detector has been calibrated using a plutonium-beryllium neutron source and commissioned during an actual neutron-producing laser experiment at the LULI2000 facility (France). An overall efficiency of 4.37(59)% has been demonstrated during calibration with a recovery time of a few hundred microseconds after laser-plasma interaction.

Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, ß-arrestin translocation and chemotaxis assays.

BACKGROUND AND PURPOSE: Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR1 antagonists in myeloma cells. EXPERIMENTAL APPROACH: We compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [(125)I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through ß-arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, we also evaluated the compounds for their ability to alter ß-arrestin translocation. KEY RESULTS: There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G-protein-dependent and -independent functional responses. CONCLUSIONS AND IMPLICATIONS: Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of ß-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization.

Modulation of PAR(1) signalling by benzimidazole compounds.

BACKGROUND AND PURPOSE: Recently, a small molecule (Q94) was reported to selectively block PAR(1) /Gα(q) interaction and signalling. Here, we describe the pharmacological properties of Q94 and two analogues that share its benzimidazole scaffold (Q109, Q89). Q109 presents a modest variation from Q94 in the substituent group at the 2-position, while Q89 has quite different groups at the 1- and 2-positions. EXPERIMENTAL APPROACH: Using human microvascular endothelial cells, we examined intracellular Ca(2+) mobilization and inositol 1,4,5-trisphosphate accumulation as well as isoprenaline- or forskolin-stimulated cAMP production in response to thrombin. KEY RESULTS: Q89 (10 µM) produced a leftward shift in the thrombin-mediated intracellular Ca(2+) mobilization concentration-response curve while having no effect on the E(max) . Both Q94 (10 µM) and Q109 (10 µM) reduced intracellular Ca(2+) mobilization, leading to a decrease in E(max) and an increase in EC(50) values. Experiments utilizing receptor-specific activating peptides confirmed that Q94 and Q109 were selective for PAR(1) as they did not alter the Ca(2+) response mediated by a PAR(2) activating peptide. Consistent with our Ca(2+) results, micromolar concentrations of either Q94 or Q109 significantly reduced thrombin-induced inositol 1,4,5-trisphosphate production. Neither Q94 nor Q109 diminished the inhibitory effects of thrombin on cAMP production, indicating they inhibit signalling selectively through the G(q) pathway. Our results also suggest the 1,2-disubstituted benzimidazole derivatives act as 'allosteric agonists' of PAR(1) . CONCLUSIONS AND IMPLICATIONS: The Q94 and Q109 benzimidazole derivatives represent a novel scaffold for the development of new PAR(1) inhibitors and provide a starting point to develop dual signalling pathway-selective positive/negative modulators of PAR(1) .

Effects of NCX 4050, a new NO donor, in rabbit and human corpus cavernosum.

The effects of NCX 4050, a drug belonging to a new class of NO donors, was investigated in isolated preparations of human and rabbit corpus cavernosum (CC) and in human foetal corpora cavernosa (hfCC) smooth muscle cells. In strips of rabbit CC, NCX 4050 (0.001-100 microM) induced a concentration-dependent relaxation which was influenced neither by Nw-nitro-l-arginine-methyl-ester (l-NAME; 100 microm) nor by endothelium deprivation. The NCX 4050-induced relaxation was significantly reduced by the guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microm) and enhanced by a specific phosphodiesterase 5 inhibitor, sildenafil (300 nm). Moreover, NCX 4050 (0.01-1 microm), induced a concentration-dependent potentiation of the relaxant response induced by electrical field stimulation (EFS) in rabbit preparations pre-treated with guanethidine and indomethacin. The relaxant effect of NCX 4050 was similar to that obtained by increasing concentrations (0.001-100 microm) of sodium nitroprusside (SNP) in either rabbit or human preparations. To further investigate the activity of NCX 4050 on human corpora cavernosa, we exposed cultured hfCC smooth muscle cells to increasing concentrations of NCX 4050 and SNP. We found that both compounds dose-dependently reduced cell proliferation. The antiproliferative effect of all the concentration tested of NCX 4050 was completely blocked by ODQ (1 microm). These results suggest that in rabbit and human corpora cavernosa NCX 4050 acts by activating guanylate cyclase activity, induces smooth muscle relaxation and quiescence. Our results provide a rationale for a possible future use of NCX 4050 in the pharmacotherapy of erectile dysfunction linked to an impaired release of NO from the endothelium.

alpha(1D)-adrenoceptors cause endothelium-dependent vasodilatation in the rat mesenteric vascular bed.

The vasodilator activity of alpha(1)-adrenoceptor agonists was tested in the rat mesenteric vascular bed (MVB), and the mechanism involved was investigated in cultured endothelial cells isolated from the bovine coronary vascular bed. In preparations preconstricted by U46619, noradrenaline and phenylephrine induced a slight relaxant effect at nanomolar concentrations. This effect was abolished in endothelium-denuded preparations and in preparations pretreated with 100 microM N(omega)-nitro-L-arginine methyl ester plus 3 microM indomethacin. Both the phospholipase C inhibitor U73122 and the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin inhibited the vasorelaxant effect of phenylephrine. The cellular level of inositol monophosphate (IP(1)) in bovine endothelial cells doubled after a 15-min exposure to 0.03 to 0.1 nM phenylephrine. The activity of cNOS was significantly increased following exposure to the same concentrations of phenylephrine. Both chloroethylclonidine and the selective alpha(1D)-adrenoceptor antagonist BMY 7378 reduced, in a concentration-dependent manner, the relaxant effect induced by phenylephrine, whereas the selective alpha(1A)-adrenoceptor antagonist (+)-niguldipine was ineffective. BMY 7378 also blocked the cNOS activation induced by phenylephrine. Conversely, the increase in perfusion pressure induced by micromolar concentrations of phenylephrine was blocked by 1 nM (+)-niguldipine, but was unaffected by BMY 7378. These findings demonstrate that nanomolar concentrations of phenylephrine, which are devoid of any contractile effect, induced a slight endothelium-dependent vasorelaxation in the rat MVB through the stimulation of alpha(1D)-adrenoceptors, located on endothelial cells, which act through phospholipase C stimulation, followed by IP(1) generation, and nitric-oxide synthase activation. Conversely, the increase in perfusion pressure induced by micromolar concentrations of phenylephrine is attributable to the stimulation of alpha(1A)-adrenoceptors.

Lack of nitric oxide- and guanosine 3':5'-cyclic monophosphate-dependent regulation of alpha-thrombin-induced calcium transient in endothelial cells of spontaneously hypertensive rat hearts.

While the expression and/or activity of endothelial nitric oxide synthase (eNOS) has been characterized in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rat (WKY) hearts, in coronary endothelial cells (ECs) from both strains, the effect of NO on intracellular calcium concentration ([Ca(2+)](i)) is still unknown. Coronary microvascular ECs were isolated from SHR and WKY and characterized. Immunocytochemistry and Western blot analysis showed that eNOS was similarly expressed in ECs from both strains. Measuring [Ca(2+)](i) by imaging analysis of fura-2-loaded cells, we demonstrated that alpha-thrombin (3-180 U l(-1)) induced a superimposable dose-dependent calcium transient in ECs from both strains. In WKY ECs, S-nitroso-N-acetyl-DL-penicillamine (SNAP) dose-dependently (10 - 100 microM) and 0.1 microM atrial natriuretic factor (ANF) reduced the maximum and the decay time of alpha-thrombin-induced calcium transient. The inhibitory effects of SNAP and ANF were prevented by blocking cyclic GMP-dependent protein kinase. Non selective eNOS inhibitors prolonged the decay time of alpha-thrombin-induced calcium transient, while the selective inducible NOS inhibitor 1400 W was ineffective. SNAP (100 microM) and 0.1 microM ANF increased cyclic GMP content up to 22.9 and 42.3 fold respectively. In SHR ECs, alpha-thrombin-induced calcium transient was not modified by SNAP, ANF or eNOS inhibition. SNAP (100 microM) and 0.1 microM ANF increased cyclic GMP content up to 9. 3 and 51 fold respectively. In WKY ECs, SNAP dose-dependently (10 - 100 microM) reduced also bradykinin-induced calcium transient, while in SHR ECs was ineffective. We concluded that in SHR ECs, the cyclic GMP-dependent regulation of calcium transient is lost.

Inositol phosphate metabolism and nitric-oxide synthase activity in endothelial cells are involved in the vasorelaxant activity of nebivolol.

Nebivolol is a recently developed beta-blocker provided with vasodilator properties. Because the mechanism of the putative endothelium-dependent effect of this beta-adrenoceptor blocker has not been completely elucidated, the aim of this study was to investigate the effects of nebivolol on an isolated resistance vascular bed and on cell messengers and constitutive nitric-oxide synthase activity (cNOS) in endothelial cells. Experiments were carried out using the rat mesenteric vascular bed and cultured bovine coronary postcapillary venular endothelial cells from bovine heart (CVEC). In mesenteric vascular bed preconstricted by 30 microM noradrenaline and 0.3 microM U46619, dl-nebivolol induced a concentration-dependent relaxing effect at concentrations between 3 and 30 microM; this effect was changed to a concentration-dependent vasoconstrictor response either in endothelium-denuded preparations or in intact preparations pretreated with 100 microM N(omega)-nitro-L-arginine methyl ester plus 3 microM indomethacin. The vasorelaxant effect of dl-nebivolol in preconstricted preparations was completely blocked by pretreatment either with the phospholipase C inhibitor U73122 (1 microM) or with the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (1 microM) for 30 min. The cellular level of the inositol trisphosphate metabolite inositol monophosphate in coronary postcapillary venular endothelial cells was not affected by dl-nebivolol in the concentration range 100 nM to 1 microM, but it was concentration dependently increased after exposure for 15 min to 10 and 30 microM dl-nebivolol. The activity of cNOS was almost doubled after a 5-min exposure to 10 microM dl-nebivolol and was significantly impaired by thapsigargin and N(omega)-nitro-L-arginine methyl ester treatment, although it was unaffected by N(omega)-nitro-D-arginine methyl ester. These findings demonstrate that nebivolol, in micromolar concentrations, induces vasorelaxation through activation of inositol phosphate metabolism and stimulation of cNOS activity in endothelial cells.

Plasma and platelet ascorbate pools and lipid peroxidation in insulin-dependent diabetes mellitus.

BACKGROUND: As diabetes mellitus represents a situation in which production of peroxides is increased, the aim of this study was to investigate the relationship between plasma and platelet levels of ascorbic acid (AA)/dehydroascorbic acid (DHA) and those of malonyldialdehyde (MDA), an indirect marker of lipoperoxides, both assayed using high-performance liquid chromatography (HPLC), in 59 patients with insulin-dependent diabetes mellitus (IDDM) compared with 51 healthy control subjects matched for sex, age, smoking habits, as well as for dietary intake of energy, alcohol and vitamin C. RESULTS: Mean plasma and platelet MDA were significantly higher in the patients affected with IDDM than in control subjects. Moreover, the diabetic group was characterized by a huge decrease in plasma AA [8.45 +/- 5.5 mumol L-1 (SD) vs. 33.4 +/- 7.6 mumol L-1, P = 0.0001], mirrored by a significant increase in plasma DHA (11.9 +/- 3.9 mumol L-1 vs. 3.9 +/- 2.5 mumol L-1, P = 0.0001). No detectable DHA was observed in the platelets from both diabetic and control subjects, whereas AA was significantly increased in platelets from diabetic patients compared with control subjects (42.6 +/- 7.4 vs. 34.8 +/- 5.1 nmol 10(-9) platelets, P = 0.0001). Platelet AA in the diabetic group was significantly inversely correlated with glycated haemoglobin (r = -0.34; P = 0.04) and directly with plasma AA (r = 0.39; P = 0.02), the sum of plasma AA + DHA (r = 0.44; P = 0.009) and with platelet MDA (r = 0.38; P = 0.02). CONCLUSION: (a) The ratio plasma AA/DHA is significantly lowered in IDDM in association with an increase in MDA levels; (b) only AA is detected in platelets, being augmented in the diabetic group; (c) plasma ascorbate depletion does not reflect platelet levels of AA; and, finally, (d) metabolic control, as well as intracellular lipoperoxides, modulates platelet AA in IDDM.

Calcium waves in unstimulated left ventricular cardiomyocytes isolated from aged spontaneously hypertensive and normotensive rats.

In this work, we described the incidence and the characteristics of calcium waves in cardiomyocytes isolated from aged normotensive rats (Wistar Kyoto, WKY) and age-matched spontaneously hypertensive rats (SHR) using imaging analysis of fura-2-loaded left ventricular cardiomyocytes. Left ventricular cardiomyocytes were isolated by enzymatic digestion from hearts of 18-20 month old WKY and aged-matched SHR. Intracellular calcium concentration did not differ in either strain, whereas the incidence of cells presenting calcium waves was greater in cardiomyocytes isolated from SHR. Moreover, cardiomyocytes isolated from SHR were significantly longer than those isolated from WKY. The calcium wave frequency was lower in SHR cardiomyocytes, while the velocity of the calcium waves was similar in both strains. Our results suggest that alterations in the calcium handling of SHR may contribute to the increased incidence of arrhythmias described in SHR hearts.

Iloprost antagonizes the increase in internal calcium concentration induced by alpha-thrombin in human platelets: a study of desensitization.

We studied the interaction between the synthetic prostacyclin analog iloprost and the aggregating agent alpha-thrombin by measuring the internal calcium ion concentration ([Ca(2+)]i) of human fura-2-loaded platelets. Iloprost (0.003-100 micrograms/l) did not modify the resting calcium level; when added 2 minutes before exposure of the platelets to a submaximally active concentration of alpha-thrombin (10 U/l), iloprost dose-dependently antagonized the increase in [Ca(2+)]i. To evaluate if iloprost retained this antagonistic effect even after a prolonged contact, which is well known to cause a "desensitization" phenomenon, platelets were preincubated with iloprost (35 micrograms/l) for 3 hours. After washout, the effect of newly added iloprost (0.01-100 micrograms/l) on the alpha-thrombin-induced increase in [Ca(2+)]i was tested. Iloprost was still able to antagonize the increase in [Ca(2+)]i induced by alpha-thrombin in "desensitized" platelets; however, the dose-inhibitory response curve was significantly shifted to the right when compared with that obtained in control platelets (i.e., platelets preincubated for 3 hours with iloprost's solvent), and the resulting IC50 was significantly higher: 1.78 versus 0.2 micrograms/l (p < 0.001). Since the maximal inhibitory effect of iloprost could also be reached under these experimental conditions, we conclude that iloprost retains its ability to antagonize the increase in [Ca(2+)]i induced by alpha-thrombin in desensitized platelets.

No-mimetic furoxans: arylsulphonylfuroxans and related compounds.

A new class of methylfuroxans and methylfurazans arylthio, arylsulphinyl and arylsulphonyl substituted, characterized by vasodilating and antiaggregatory properties, is described. Vasodilating activity, tested on rabbit aortic rings percontracted with 1 microM noradrenaline, is enhanced by N-oxidation of the furazan ring and is maximized by increase of the sulphur atom oxidation level. Compounds 4-methyl-3-(p-methoxyphenylsulphonyl) furoxan 6c, 3-phenyl-4-phenylsulphonylfuroxan 10, 4-phenyl-3-phenylsulphonylfuroxan 11 and 3,4-bis(phenyl-sulphonyl)furoxan 12 (EC50 values ranging between 0.055-1.07 microM), seem to be promising since they show the highest potency as well as maximal efficacy, causing complete reversal of noradrenaline induced contraction. The structure-activity relationship, observed in the platelet aggregation test, is substantially similar to that reported for the vasodilating activity, in line with the general profile of these drugs as putative NO-mimetic derivatives.

Release of cytochromes from hypoxic and reoxygenated guinea pig heart.

Isolated, perfused hearts from guinea pigs were subjected to hypoxia for 30 minutes followed by reoxygenation for 30 minutes. Cellular damage was assessed by measuring the release of the cytoplasmic enzyme lactate dehydrogenase and the mitochondrial markers cytochrome c and cytochrome oxidase. The release of the enzymes was correlated with electron microscopy. Hypoxia induced an increase in the release of lactate dehydrogenase and cytochrome c. During reoxygenation, the release of lactate dehydrogenase was exacerbated while that of cytochrome c decreased, suggesting a partial recovery of the mitochondria. Cytochrome oxidase was not detectable in the extracellular space during hypoxia or reoxygenation. It is suggested that cytochrome c is a specific marker for damage to mitochondria caused by hypoxia and its loss may affect respiratory chain function.

Effects of L- and D-arginine and some related esters on the cytosolic mechanisms of alpha-thrombin-induced human platelet activation.

1. In Fura-2 preloaded human platelets, the increase in cytosolic calcium induced by alpha-thrombin was reduced by some L- and D-arginine ester compounds the IC50 (microM) values of which were 7.4 for TAEE, 56.9 for BAEE, 77.6 for TAME, 560 for T(d)AME, 656.3 for L-ArgOMe and 2206.7 for D-ArgOMe. alpha-tosyl-L-Arginine, L- and D-arginine were inactive. 2. The inhibitory activity of the L-arginine esters was not modified when platelets were pretreated with 100 microM N omega-monomethyl-L-arginine. 3. The L-arginine esters did not increase cyclic GMP content in platelets either in the presence or absence of indomethacin and apyrase at rest and after alpha-thrombin stimulation. 4. The kinetic parameters of platelet Na+/H+ antiporter (amiloride-inhibitable, evaluated after cytosolic nigericin-induced acidification) were modified by L- and D-arginine esters, while the native amino acids were ineffective. 5. The inhibitory effects of the L- and D-arginine esters on platelet activation appear to be mainly due to their inhibitory effect on Na+/H+ antiporter.

Plasma retinol and alpha-tocopherol concentrations in insulin-dependent diabetes mellitus: their relationship to microvascular complications.

Plasma alpha-tocopherol and retinol, both assayed by an HPLC method, have been evaluated in a group of 60 patients affected by insulin-dependent (type 1) diabetes mellitus, stratified according to the presence of retinopathy and nephropathy diagnosed by an urinary albumin excretion rate ranging between 20 and 200 micrograms/min (microalbuminaria) or > 200 micrograms/min (macroalbuminuria), all of whom were compared with 26 healthy controls strictly matched for age and sex. Plasma lipids and age were positively correlated with plasma retinol and alpha-tocopherol in both diabetic and control subjects. Either plasma retinol or its ratio to cholesterol were significantly and independently reduced in the younger subset of diabetics, as compared to controls, independently from other confounding variables, while plasma alpha-tocopherol was unchanged in diabetic subjects and in healthy controls. Retinopathy was not associated with altered levels of both plasma alpha-tocopherol or retinol. The presence of increased urinary albumin excretion was associated with higher plasma levels of alpha-tocopherol and, only for macroalbuminuria, of retinol. However, after processing the data by a multivariate model, nephropathy was characterized by an increase only in plasma alpha-tocopherol. In conclusion, according to our findings, plasma retinol is significantly decreased in younger insulin-dependent diabetic patients while alpha-tocopherol is significantly altered in diabetic patients with nephropathy.

Taurine antagonizes the increase in intracellular calcium concentration induced by alpha-adrenergic stimulation in freshly isolated guinea-pig cardiomyocytes.

The effect of taurine on phenylephrine alpha-adrenergic action was studied in freshly-isolated guinea-pig ventricular myocytes. Intracellular calcium concentration was measured at nearly physiological extracellular CaCl2 in both cell suspension using quin-2 (mean intracellular calcium concentration 154.0 +/- 8.0 nM, n = 24), and single myocyte with fura-2 (mean intracellular calcium concentration 159.0 +/- 20.0 nM, n = 23). Phenylephrine increased intracellular calcium concentration in both preparations. In cell suspensions in the presence of 10(-6) M propranolol and at 2.2 mM extracellular calcium concentration, phenylephrine dose-dependently (3 x 10(-7)-10(-5) M) increased intracellular calcium concentration, its effect being abolished in the presence of phentolamine. Taurine 20 mM in the incubation fluid increased taurine content in the cells from 110 +/- 7 nmol/mg to 317 +/- 49 nmol/mg of total proteins. In the range 0.5-20 mM, taurine concentration-dependently reduced the phenylephrine-induced intracellular calcium increase in cell suspensions and 20 mM taurine decreased the effect of 10(-5) M phenylephrine (measured in the presence of 10(-6) M propranolol) by about 80%. Beta-Alanine (20 mM) did not modify the phenylephrine effect. Our data show that the "protective" effect of taurine in in vitro cardiac preparations and in cardiomyocyte isolation procedures is due to its effect on cardiomyocyte intracellular calcium ion concentration, and that taurine specifically antagonizes alpha-adrenoceptor activation.

Immunoquantitation of cytochrome C in cardiac perfusate.

An indirect three step ELISA has been assessed in order to detect the possible release of cytochrome c, a mitochondrial protein, from isolated and perfused guinea-pig heart. The ELISA described in this study is sufficiently sensitive and accurate to measure extracellular cytochrome c.