Gian Pietro Sgaragli

Potentiation of mitochondrial Ca2+ sequestration by taurine

The effects of taurine (2-aminoethanesulphonic acid) and its analogues, 2-aminoethylarsonic acid, 2-hydroxyethanesulphonic (isethionic) acid, 3-aminopropanesulphonic acid, 2-aminoethylphosphonic acid, and N,N-dimethyltaurine, were studied on the transport of Ca2+ by mitochondria isolated from rat liver. Taurine enhanced Ca2+ uptake in an apparently saturable process, with a Km value of about 2.63 mM. Taurine behaved as an uncompetitive activator of Ca2+ uptake, increasing both the apparent Km and Vmax values of the process. This effect was not modified in the presence of cyclosporin A (CsA). N,N-Dimethyltaurine also stimulated Ca2+ uptake at higher concentrations, but there was no evidence that the process was saturable over the concentration range used (1-10 mM). Aminoethylarsonate was a weak inhibitor of basal Ca2+ uptake, but inhibited that stimulated by taurine in an apparently competitive fashion (Ki = 0.05 mM). The other analogues had no significant effects on this process. Taurine either in the presence or the absence of CsA had no effect on Ca2+ release induced by 200 nM ruthenium red. Thus, the mechanism of taurine-enhanced Ca2+ accumulation appears to involve stimulation of Ca2+ uptake via the uniport system rather than inhibition of Ca2+ release via the ion (Na+/Ca2+ and/or H+/Ca2+) exchangers or by taurine modulating the permeability transition of the mitochondrial inner membrane. Overall, these findings indicate an interaction of taurine with an as yet unidentified mitochondrial site which might regulate the activity of the uniporter. The unique role of taurine in modulating mitochondrial Ca2+ homeostasis might be of particular importance under pathological conditions that are characterised by cell Ca2+ overload, such as ischaemia and oxidative stress.

Rectal temperature and prostaglandin E2 increase in cerebrospinal fluid of conscious rabbits after intracerebroventricular injection of hemoglobin

Abstract Fever accompanies subarachnoid hemorrhage (SAH) in the majority of patients. In a previous study, hemoglobin (Hb) was shown to catalyze in vitro, under aerobic conditions, the conversion of arachidonic acid to prostaglandin E2 (PGE2) and prostaglandin F2α. The aim of the present work was to assess whether this pathway also operates in vivo and to provide a mechanism to explain post-SAH fever. To this end, PGE2 concentration was determined in cerebrospinal fluid (CSF) of conscious rabbits chronically cannulated in the lateral ventricle and cisterna magna, following intracerebroventricular (i.c.v.) injection of 10 µg or 100 µg of commercial rabbit bicrystallized Hb as a model of SAH. Before i.c.v. injection, Hb solutions were filtered on a polimixin-B column to remove substantially, by over 90%, endotoxin-like substances. Results show that in nine rabbits injection of 10 µg Hb did not significantly modify body temperature or significantly alter CSF PGE2 content. On the contrary, in nine rabbits, injection of 100 µg Hb produced a significant increase in core temperature which was accompanied by a significant increase in CSF PGE2. When data related to these two parameters from the 9 control and 18 Hb-treated rabbits were analyzed as a single group, a linear, positive, and highly significant correlation was found. These findings indicate that, once Hb is released into the subarachnoid space during SAH, it enhances CSF PGE2 content and elicits hyperthermia, thus offering an explanation for the fever that is an aggravating condition in most SAH patients.

Potentiation of intracellular Ca2+ mobilization by hypoxia-induced NO generation in rat brain striatal slices and human astrocytoma U-373 MG cells and its involvement in tissue damage

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic–ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low‐oxygen and Ca2+‐free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia‐induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine–NO and inhibited by N‐ω‐nitro‐L‐arginine methyl ester (L‐NAME) or by the NO scavenger 2‐phenyl‐4,4,5,5 tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO). A membrane‐permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U‐373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 ± 13.1% compared to normoxic controls and this effect was inhibited by L‐NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L‐NAME. Although heparin plus RR or RY antagonized hypoxia‐induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO–Ca2+ signalling might be a potential therapeutic target in hypoxia‐induced neuronal degeneration.

Cyclooxygenase activity of hemoglobin

Publisher Summary When hemoglobin (Hb) diffuses into the extracellular space of living tissues, which may occur during hemolysis or when erythrocytes escape from vessels, its catalytic potential toward several endogenous or exogenous compounds can be activated. There has been much attention focused on the capacity of Hb to metabolize polyunsaturated fatty acids (PUFAs), giving rise to the formation of products that may be important in the pathogenesis of systemic (intravascular hemolysis) or local (micro- or macrohemorrhages, inflammation, hemorrhagic infarcts) conditions in which erythrocyte lysis occurs. At low concentrations, Hb catalyzes a quasi-lipoxygenase reaction on linoleic acid with remarkably high substrate specificity. The fatty acid cyclooxygenases, which may be regarded as a special type of lipoxygenase that initiate the biosynthesis of prostaglandins and thromboxanes, require heme for their activity. Hb is a good candidate for exerting cyclooxygenase activity owing to its peroxidative properties already described in the chapter. There is also some discussion about the cyclooxygenase activity of Hb, which has been made possible by the use of either physicochemical or biological analytical method.

Cytochrome P450-dependent N-dealkylation of L-deprenyl in C57BL mouse liver microsomes : effects of in vivo pretreatment with ethanol, phenobarbital, β-naphthoflavone and L-deprenyl

The monoamine oxidase inhibitor L-deprenyl [(-)-deprenyl, selegiline] is an effective therapeutic agent for improving early symptoms of idiopathic Parkinsons disease. It appears to exert this action independently of its inhibition of monoamine oxidase B (MAO-B) and some of its metabolites are thought to contribute. Cytochrome P450 (CYP) activities are known to give rise to L-deprenyl metabolites that may affect the dopaminergic system. In order to clarify the interactions of L-deprenyl with these enzymes, C57BL mice were treated with L-deprenyl, ethanol, phenobarbital or beta-naphthoflavone to induce different CYP isozymes. After preincubation of L-deprenyl with liver microsomes from control or treated mice, the metabolites were analysed by a GLC method. L-deprenyl (10 mg/kg i.p. for 3 days) caused a significant decrease in total CYP levels (0.315+/-0.019, L-deprenyl; 0.786+/-0.124, control, nmol/mg protein) and CYP2E1-associated p-nitrophenol hydroxylase activity (0.92+/-0.04 vs. 1.17+/-0.06 nmol/min/mg). Both phenobarbital and ethanol increased the N-depropynylation activity towards L-deprenyl that leads to the formation of methamphetamine (4. 11+/-0.64, phenobarbital; 4.77+/-1.15, ethanol; 1.77+/-0.34, control, nmol/min/mg). Ethanol alone increased the N-demethylation rate of L-deprenyl, that results in formation of nordeprenyl (3.99+/-0.68, ethanol; 1.41+/-0.31, control, nmol/min/mg). Moreover, the N-dealkylation pathways of deprenyl are inhibited by 4-methylpyrazole and disulfiram, two CYP2E1 inhibitors. None of the other treatments modified L-deprenyl metabolism. These findings indicate that mainly CYP2E1 and to a lesser extent CYP2B isozymes are involved in L-deprenyl metabolism. They also suggest that, by reducing CYP content, L-deprenyl treatment may impair the metabolic disposition of other drugs given in combination regimens.

Rat tissue concentrations of chlorimipramine, chlorpromazine and their N-demethylated metabolites after a single oral dose of the parent compounds.

A single oral dose of 90 mg kg−1 chlorimipramine or chlorpromazine, corresponding to 54.5 or 55.9 μmol, respectively, was given to male Sprague‐Dawley rats and concentrations of parent drugs and their N‐desmethyl metabolites measured by gas chromatography in plasma and major organs (brain, liver, lung, kidney, heart, spleen and peritoneal fat).

L-type Ca2+ channel current characteristics are preserved in rat tail artery myocytes after one-day storage

To develop a cheap and simple method of storing for 24‐h vascular tissue and single myocytes while preserving therein the biophysical and pharmacological characteristics of L‐type Ca2+ channels and contractile activity.

Effects of 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) on rat aorta smooth muscle

To characterise the pharmacological activity of 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) on vascular smooth muscle, the different effects of BHQ on rat aorta were investigated under several experimental conditions. In aortic rings at rest or depolarised with 80 mM K+ in the presence of 1 microM nifedipine, BHQ evoked a slow tonic contraction which was antagonised by 1 mM Ni2+. Depolarised rings contracted in response to addition of 1 mM Ca2+, with an EC50 value of 32.4+/-1.0 mM for K+. At 20 mM K+, Ca2+-induced contraction was enhanced by BHQ. This effect was antagonised by 1 mM Ni2+, but not by 1 microM nifedipine. By contrast, at 40, 80 and 128 mM K+, BHQ antagonised Ca2+-induced contraction. This effect was partially reversed by 1 microM methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyri dine-5-carboxylate (Bay K 8644) or by increasing extracellular Ca2+ concentration. In the presence of nifedipine and Ni2+, depolarised rings (80 mM K+) contracted in response to addition of 1 microM phenylephrine; this response was fast and then slowly decreased. When the preparations were preincubated with BHQ, the phenylephrine-induced contraction was transient and antagonised in a concentration-dependent manner by BHQ. These results indicate that the myotonic effect of BHQ on rat aortic rings depends on activation of Ca2+ influx via a Ni2+-sensitive pathway, whereas its myolytic activity is due either to antagonism of Ca2+ entry via L-type Ca2+ channels or depletion of intracellular Ca2+ stores.

Myorelaxant activity of 2-t-butyl-4-methoxyphenol (BHA) in guinea pig gastric fundus

This study investigates the mechanism whereby the antioxidant 2-t-butyl-4-methoxyphenol (BHA) relaxes guinea pig gastric fundus smooth muscle. In circular smooth muscle strips, 10 microM cyclopiazonic acid, a specific inhibitor of sarcoplasmic reticulum Ca2+-ATPase, induced a prolonged rise in tension which depended on the presence of extracellular Ca2+. BHA (pIC50 = 5.83), sodium nitroprusside (6.85), isoproterenol (7.69) and nifedipine (8.02), but not 2,6-di-t-butyl-4-methoxyphenol (DTBHA) (up to 30 microM), relaxed muscle strips contracted with cyclopiazonic acid. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyri dine-5-carboxylate (Bay K 8644) (1 microM) antagonised the nifedipine- but not the BHA-induced relaxation. Nifedipine and isoproterenol (10 microM) caused a decrease in spontaneous tone, but did not counteract the subsequent rise in tension elicited by 10 microM cyclopiazonic acid. Conversely, 100 microM BHA and 100 microM sodium nitroprusside not only significantly reduced spontaneous tone but also markedly impaired the response of the muscles to cyclopiazonic acid. DTBHA failed to show either effect. When added to preparations completely relaxed by 100 microM BHA, 10 mM tetraethylammonium still elicited nifedipine-sensitive tonic and phasic contractions in the presence or absence of 10 microM cyclopiazonic acid. BHA and DTBHA inhibited, in a concentration-dependent manner, the Ca2+-promoted contraction of strips depolarised by 10 mM tetraethylammonium. The BHA antagonism showed a non-competitive profile while that of DTBHA was competitive. In muscle strips at rest, 10 microM BHA caused a significant increase in tissue cAMP concentration, leaving cGMP unmodified. To conclude, the myorelaxant action of BHA on gastric fundus smooth muscle appears to be mediated partly by an increase in cAMP levels and partly by inhibition of Ca2+ influx from the extracellular space.

2,5-Di-t-butyl-1,4-benzohydroquinone induces endothelium-dependent relaxation of rat thoracic aorta

The aim of this work was to clarify the mechanism by which 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) induces relaxation of rat thoracic aorta. In particular, the role of endothelium-derived nitric oxide (NO) was investigated. BHQ concentration dependently (0.1-10 microM) relaxed rat aorta rings precontracted with phenylephrine. This effect was dependent on the intactness of the endothelium, suppressed by preincubation with 100 microM N(omega)-nitro-L-arginine methyl ester and antagonised by 3-30 microM methylene blue. The 10 microM BHQ-induced relaxation, however, was followed by the gradual and slow return to phenylephrine-induced tone. Superoxide dismutase (250 U/ml) increased the BHQ-induced relaxation, while preincubation with 3 mM diethyldithiocarbamate inhibited it in a time-dependent fashion. BHQ gave rise to superoxide anion formation which was markedly inhibited by the addition of superoxide dismutase (250 U/ml), either in the presence or in the absence of aorta rings. The non-specific blocker of Ca2+ channels, Ni2+, concentration dependently attenuated the BHQ relaxing effect. BHQ did not modify the relaxation induced by the NO donor 3-morpholino-sydnonimine in endothelium-deprived rings. In conclusion, BHQ induces endothelium-dependent relaxation and gives rise, by auto-oxidation, to the formation of superoxide anion. The former effect results from the enhanced synthesis of NO rather than from its enhanced biological activity; NO synthase is presumed to be stimulated by BHQ-induced activation of Ca2+ influx through Ni2+-sensitive Ca2+ channels.