Xanthine oxidoreductase inhibitor topiroxostat ameliorates podocyte injury by inhibiting the reduction of nephrin and podoplanin / El inhibidor de la xantina oxidorreductasa, topiroxostat, mejora la lesión de los podocitos al inhibir la reducción de nefrina y podoplanina

Background: Topiroxostat, an inhibitor of xanthine oxidoreductase (XOR) was shown to reduce urinary albumin excretion of hyperuricemic patients with chronic kidney disease. However, its pharmacological mechanism is not well understood. In this study, we examined the effects of topiroxostat on glomerular podocytes. Podocyte is characterized by foot process and a unique cell-cell junction slit diaphragm functioning as a final barrier to prevent proteinuria. Methods: The effects of topiroxostat on the expressions of podocyte functional molecules were analysed in db/db mice, a diabetic nephropathy model, anti-nephrin antibody-induced rat podocyte injury model and cultured podocytes treated with adriamycin. Results: Topiroxostat treatment ameliorated albuminuria in db/db mice. The expression of desmin, a podocyte injury marker was increased, and nephrin and podocin, key molecules of slit diaphragm, and podoplanin, an essential molecule in maintaining foot process were downregulated in db/db mice. Topiroxostat treatment prevented the alterations in the expressions of these molecules in db/db mice. XOR activity in kidney was increased in rats with anti-nephrin antibody-induced podocyte injury. Topiroxostat treatment reduced XOR activity and restored the decreased expression of nephrin, podocin and podoplanin in the podocyte injury. Furthermore, topiroxostat enhanced the expression of podoplanin in injured human cultured podocytes. (AU)

Antecedentes: El topiroxostat, un inhibidor de la xantina oxidorreductasa (XOR), mostró reducir la excreción de albúmina en la orina de pacientes hiperuricémicos con enfermedad renal crónica. Sin embargo, su mecanismo farmacológico no se conoce con exactitud. En este estudio examinamos los efectos del topiroxostat en los podocitos glomerulares. El podocito se caracteriza por unas prolongaciones en forma de pie y un diafragma de hendidura de unión célula-célula único que funciona como barrera final en la prevención de la proteinuria. Métodos: Se analizaron los efectos del topiroxostat en las expresiones de las moléculas funcionales de los podocitos en ratones db/db, en un modelo de nefropatía diabética, en un modelo de lesión podocitaria inducida por anticuerpos antinefrina en ratas y en podocitos cultivados tratados con adriamicina. Resultados: El tratamiento con topiroxostat mejoró la albuminuria en ratones db/db. La expresión de la desmina, un marcador de lesión podocitaria, estaba aumentada, y la nefrina y la podocina, moléculas clave del diafragma de hendidura, y la podoplanina, una molécula esencial en el mantenimiento de las prolongaciones en forma de pie, estaban atenuadas en los ratones db/db. El tratamiento con topiroxostat evitó alteraciones en las expresiones de estas moléculas en los ratones db/db. La actividad de la XOR en el riñón se incrementó en ratas con lesión podocitaria inducida por anticuerpos antinefrina. El tratamiento con topiroxostat redujo la actividad de la XOR y restauró la disminución de la expresión de nefrina, podocina y podoplanina en la lesión podocitaria. Además, el topiroxostat aumentó la expresión de podoplanina en podocitos humanos cultivados lesionados. (AU)

Changes in ERK1/2 phosphorylation in the rat striatum and medial prefrontal cortex following administration of the adenosine A1 receptor agonist and antagonist.

The extracellular signal-regulated kinase (ERK) is enriched in the central nervous system, including the dopamine responsive regions such as the striatum and medial prefrontal cortex (mPFC). The kinase is sensitive to changing cellular and synaptic input and is implicated in the regulation of synaptic transmission and plasticity. In this study, the role of a Gαi/o protein-coupled adenosine A1 receptor in the regulation of ERK1/2 was investigated in the rat brain in vivo. We found that an A1 agonist CPA after an intraperitoneal injection reduced ERK1/2 phosphorylation in the nucleus accumbens (NAc) and mPFC. In contrast, a single dose of an A1 antagonist DPCPX induced a rapid and transient increase in ERK1/2 phosphorylation in the caudate putamen (CPu), NAc, and mPFC. Pretreatment with a dopamine D1 receptor antagonist SCH23390 abolished the DPCPX-induced ERK1/2 phosphorylation in the striatum and mPFC. Coadministration of DPCPX and a D1 agonist SKF81297 at a low dose induced a greater elevation of ERK1/2 phosphorylation. Activation or blockade of A1 receptors had no effect on total ERK1/2 expression in the striatum and mPFC. These results reveal an existence of an inhibitory linkage from adenosine A1 receptors to ERK1/2 in striatal and mPFC neurons. This inhibitory linkage seems to form a dynamic balance with positive dopamine D1 receptor signaling to control the ERK1/2 pathway.

The xanthine derivative KMUP-1 inhibits models of pulmonary artery hypertension via increased NO and cGMP-dependent inhibition of RhoA/Rho kinase.

BACKGROUND AND PURPOSE: KMUP-1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP-1 on acute and chronic pulmonary artery hypertension (PAH) in rats. EXPERIMENTAL APPROACH: We measured pulmonary vascular contractility, wall thickening, eNOS immunostaining, expressions of ROCK II, RhoA activation, myosin phosphatase target subunit 1 (MYPT1) phosphorylation, eNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG) and phosphodiesterase 5A (PDE-5A), blood oxygenation and cGMP/cAMP, and right ventricular hypertrophy (RVH) in rats. KEY RESULTS: In rings of intact pulmonary artery (PA), KMUP-1 relaxed the vasoconstriction induced by phenylephrine (10 microM) or the thromboxane A(2)-mimetic U46619 (0.5 microM). In endothelium-denuded PA rings, this relaxation was reduced. In acute PAH induced by U46619 (2.5 microg x kg(-1) x min(-1), 30 min), KMUP-1 relaxed vasoconstriction by enhancing levels of eNOS, sGC and PKG, suppressing those of PDE-5A, RhoA/ROCK II activation and MYPT1 phosphorylation, and restoring oxygenation in blood and cGMP/cAMP in plasma. Incubating smooth muscle cells from PA (PASMCs) with KMUP-1 inhibited thapsigargin-induced Ca(2+) efflux and angiotensin II-induced Ca(2+) influx. In chronic PAH model induced by monocrotaline, KMUP-1 increased eNOS and reduced RhoA/ROCK II activation/expression, PA wall thickening, eNOS immunostaining and RVH. KMUP-1 and sildenafil did not inhibit monocrotaline-induced PDE-5A expression. CONCLUSION AND IMPLICATIONS: KMUP-1 decreased PAH by enhancing NO synthesis by eNOS, with consequent cGMP-dependent inhibition of RhoA/ROCK II and Ca(2+) desensitization in PASMCs. KMUP-1 has the potential to reduce vascular resistance, remodelling and RVH in PAH.

Antagonist selective modulation of adenosine A1 and A3 receptor pharmacology by the food dye Brilliant Black BN: evidence for allosteric interactions.

Allosteric binding sites on the adenosine receptor family represent potential therapeutic targets for a number of conditions involving metabolic stress. This study has identified Brilliant Black BN as a novel allosteric modulator of the adenosine A(1) and A(3) receptors. In addition to being a food dye and pharmaceutical excipient, Brilliant Black BN is commonly used within calcium mobilization assays to quench extracellular fluorescence. Brilliant Black BN (5-500 microM) had no significant effect on the calcium mobilization stimulated by the nonselective adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine in Chinese hamster ovary cells stably transfected with the human adenosine A(1) or A(3) receptor. Likewise, calcium mobilization and radioligand binding assays found that Brilliant Black BN (5-500 microM) did not significantly influence the antagonism mediated by 8-cyclopentyl-1,3-dipropylxanthine (100 nM) at the A(1) receptor. In contrast, the affinity of N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acetamide (MRS1220) at the A(3) receptor and xanthine amine congener (XAC) and XAC-X-BY630 at the A(1) and A(3) receptors was significantly decreased in the presence of 500 muM Brilliant Black BN. A reduction in XAC potency at the A(1) and A(3) receptor was achieved within 1 min of Brilliant Black BN addition, despite receptors having been pre-equilibrated with antagonist. Dissociation kinetics of the fluorescent XAC derivative, XAC-X-BY630, revealed that the decrease in affinity is probably due to a significant increase in dissociation rate of the antagonist in the presence of Brilliant Black BN. Taken together, these results suggest that Brilliant Black BN can act allosterically to modify ligand affinity at A(1) and A(3) receptors.

Inhibition of hepatitis B virus gene expression and replication by helioxanthin and its derivative.

Chronic hepatitis B virus (HBV) infection continues to be an important worldwide cause of morbidity and mortality. All the currently approved therapeutic drugs have their limitations: interferon-alpha (IFN-alpha) has limited efficacy and a high incidence of adverse effects; nucleoside analogues are very efficient HBV DNA inhibitors, but resistance occurs eventually. Therefore, it is important to develop new non-nucleoside/nucleotide agents with different modes of action that can be used for antiviral combination therapy. Here, we report on a novel class of compounds, helioxanthin and its derivative 5-4-2, which had potent anti-HBV activities in HepG2.2.15 cells, with the EC50s of 1 and 0.08 microM, respectively. The lamivudine-resistant HBV, L526M/M550V double mutant strain, was also sensitive to helioxanthin and 5-4-2. This class of compounds not only inhibited HBV DNA, but also decreased HBV mRNA and HBV protein expression. The EC50 of HBV DNA inhibition was consistent with the EC50 of HBV 3.5 Kb transcript inhibition, which was 1 and 0.09 microM for helioxanthin and 5-4-2 respectively. Western blot analysis of cell lysate from HepG2.2.15 cells showed that the core protein expression decreased in a dose-dependent manner after drug treatment. In conclusion, helioxanthin and 5-4-2 are potentially unique new anti-HBV agents, which possess a different mechanism of action from existing therapeutic drugs. Both compounds inhibited HBV RNA and protein expression in addition to inhibiting HBV DNA.

Losartan and atenolol on hypertension induced by adenosine receptor blockade.

1. The prolonged infusion of 1,3-dipropyl-8-sulfophenylxanthine (DPSPX), a non-selective antagonist of adenosine receptors, induces hypertension, an increase in plasma renin activity and morphological cardiovascular changes. 2. The aim of this work was to evaluate the effects of losartan, a selective AT1 receptor antagonist, and atenolol, a beta-adrenoceptor antagonist, on DPSPX-induced hypertension. 3. Male Wistar rats (250-300 g, n = 4-6) were treated for 1 or 4 weeks with: saline i.p.; DPSPX (90 microg kg(-1) h(-1)) i.p.; losartan (15 mg kg(-1) day(-1)) p.o.; atenolol (25 mg kg(-1) day(-1)) p.o.; DPSPX (90 microg kg(-1) h(-1)) i.p. + losartan (15 mg kg(-1) day(-1)) p.o.; DPSPX (90 microg kg(-1) h(-1)) i.p. + atenolol (25 mg kg(-1) day(-1)) p.o. Blood pressure was measured by the 'tail-cuff' method in conscious animals. Fragments of the mesenteric and tail arteries were processed for morphological study and the mean diameter of the vascular smooth muscle cells was determined. 4. DPSPX increased blood pressure. Losartan and atenolol prevented this rise but had no effect on blood pressure of control rats. DPSPX-treated groups showed hypertrophy of the vascular smooth muscle cells and proliferation of subintimal cells. Losartan but not atenolol prevented these changes. Losartan had no effect on the vascular morphology of control rats, while treatment with atenolol for 4 weeks induced hypertrophy of the vascular smooth muscle cells. 5. Both losartan and atenolol counteract the development of DPSPX-induced hypertension but only losartan prevents the alterations in vascular morphology.

Acceleration of hypertensive cerebral injury by the inhibition of xanthine-xanthine oxidase system in stroke-prone spontaneously hypertensive rats.

It is well-known that, in ischemic cerebral injury, a free radical and its byproducts are generated by xanthine-xanthine oxidase system and eliminated by scavengers such as superoxide dismutase (SOD), catalase, uric acid and ascorbic acid. To investigate the possible involvement of the xanthine-xanthine oxidase system in hypertensive cerebral injury, we examined chronological changes in uric acid level in the cerebral cortex and the effects of the inhibition of xanthine oxidase or catalase using stroke-prone spontaneously hypertensive rats (SHRSP). In young SHRSP, uric acid content was lower than age-matched Wistar-Kyoto rats (WKY), but in mature SHRSP strongly exposed to oxidative stress uric acid content had risen dramatically. Administration of allopurinol, an inhibitor of xanthine oxidase, caused a marked decrease in uric acid content. In these SHRSP, cerebral injury was much more intense compared to the control group. On the other hand, administration of aminotriazole, an inhibitor of catalase, did not affect the brain pathology of SHRSP, in spite of a mild reduction in tissue uric acid content. These results suggest that the xanthine-xanthine oxidase system is not the major source of free radical generation in hypertensive cerebral injury. Moreover, the results also suggest that tissue uric acid may have a key role for the incidence of hypertensive cerebral injury in SHRSP.

De novo analysis of receptor binding affinity data of 8-ethenyl-xanthine antagonists to adenosine A1 and A2a receptors.

The receptor binding affinity data to adenosine A1 and A2a receptors of a wide series of 8-ethenyl-xanthine derivatives has been analyzed by means of the Free-Wilson model. The analysis of the individual group contributions (aij) shows the importance of the presence of an ethenyl moiety at position 8 on the xanthine ring for obtaining selective A2a antagonists. The different aij values of the substituents for the adenosine. A1 receptor do not correlate with the corresponding ones for the A2a receptor, indicating the possibility to obtain A1 and A2a selective compounds. The presence of aromatic substituents at the 8-ethenyl group, such as 3,5-(OCH3)2-phenyl, permits to obtain strongly A2a selective compounds (affinity ratio of up to 100); moreover, it appears that 8-ethenyl-xanthinic derivatives cannot have high selectivity for the adenosine A1 receptor (affinity ratio < or = 10).

Adenosine A2A receptors facilitate 45Ca2+ uptake through class A calcium channels in rat hippocampal CA3 but not CA1 synaptosomes.

In the hippocampus, the neuromodulatory role of adenosine depends on a balance between inhibitory A1 responses and facilitatory A2A responses. Since the presynaptic effects of hippocampal inhibitory A1 adenosine receptors are mostly mediated by inhibition of Ca2+ channels, we now investigated whether presynaptic facilitatory A2A adenosine receptors would modulate calcium influx in the hippocampus. The mixed A1/A2 agonist, 2-chloroadenosine (CADO; 1 microM) inhibited veratridine (20 microM)-evoked 45Ca2+ influx into hippocampal synaptosomes of the CA1 or CA3 areas by 24.2 +/- 4.5% and 17.2 +/- 5.8%, respectively. In the presence of the A, antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), the inhibitory effect of CADO (1 microM) on 45Ca2+ influx was prevented in CA1 synaptosomes, but was converted into a facilitatory effect (14.2 +/- 6.7%) in CA3 synaptosomes. The A2A agonist, CGS 21680 (3-30 nM) facilitated 45Ca2+ influx in CA3 synaptosomes, with a maximum increase of 22.9 +/- 3.9% at 10 nM, and was virtually devoid of effect in CA1 synaptosomes. This facilitatory effect of CGS 21680 (10 nM) in CA3 synaptosomes was prevented by the A2A antagonist 8-(3-chlorostyryl)caffeine (CSC; 200 nM), but not by the A1 antagonist, DPCPX (20 or 100 nM). The facilitatory effect of CGS 21680 on 45Ca2+ uptake by CA3 synaptosomes was prevented by the class A calcium channel blocker, omega-agatoxin-IVA (200 nM). These results indicate that presynaptic adenosine A2A receptors facilitate calcium influx in the CA3 but not the CA1 area of the rat hippocampus through activation of class A calcium channels.

Cardiac vagal afferent stimulation by free radicals during ischaemia and reperfusion.

1. Myocardial ischaemia and reperfusion can evoke excitation of cardiac vagal afferent nerve endings and activation of a cardiogenic depressor reflex (Bezold-Jarisch effect). We postulate that oxygen free radicals, which are well known to be produced during ischaemia and reperfusion, contribute to this excitation. 2. Activity from vagal afferent fibres in rats, whose endings were located in the walls of all four chambers of the heart, was recorded in response to topical application of pro-oxidant chemicals to the surface of the heart. Activity was also recorded from vagal afferent fibres, whose endings were located in the left ventricle, in response to occlusion of the left anterior coronary artery (LAC) for 30 min and subsequent reperfusion. A majority of the recorded fibres were classified as chemosensitive C-fibre endings due to their irregular discharge under resting conditions, their activation in response to the topical application of capsaicin (1-10 micrograms) to the surface of the heart encompassing the receptive field and their conduction velocities. 3. Topical application of either H2O2 or xanthine/xanthine oxidase to the heart activated 50% of the chemosensitive endings and did not directly affect cardiac mechanoreceptors. This effect was reproducible, dose-dependent and was not due to [H+]. 4. Administration of the superoxide radical scavenging enzyme, superoxide dismutase (20000 U/kg, i.v.), decreased the response of fibres to xanthine/xanthine oxidase but had no effect on the activation caused by H2O2. The antioxidants deferoxamine (20 mg/kg, i.v.) or dimethylthiourea (10 mg/kg, i.v.), which scavenge the hydroxyl radical, abolished the responses to xanthine/xanthine oxidase and H2O2. Administration of indomethacin (5 mg/kg, i.v.) had no effect on the afferent response to H2O2. 5. In response to ligation of the left anterior coronary (LAC), the activity of chemosensitive endings within the ischaemic zone increased within the first 2 min of occlusion. Endings outside the ischaemic zone were not affected at the beginning of ischaemia. Reperfusion activated only chemosensitive endings responsive to topical H2O2. These reperfusion-sensitive endings were located both within and outside the ischaemic zone of the left ventricle. 6. Indomethacin (5 mg/kg, i.v.) prevented activation of chemosensitive endings at the beginning of LAC occlusion regardless of their sensitivity to H2O2 but had no effect on the response to reperfusion. Conversely, deferoxamine (20 mg/kg, i.v.) had no effect on the activation of chemosensitive fibres at the onset of ischaemia, whereas it completely prevented activation at reperfusion. 7. We propose that there are two different mechanisms that activate chemosensitive afferent vagal fibres in the rat heart during ischaemia and reperfusion. The first causes excitation of these endings at the onset of ischaemia and is mediated by prostaglandin synthesis within the ischaemic zone. The second mechanism leads to a more widespread activation of chemosensitive afferents in the left ventricle during prolonged ischaemia and at the moment of reperfusion and is mediated by oxygen free radical formation.

Vasoactive intestinal polypeptide prevents injury of pulmonary vascular permeability due to xanthine with xanthine oxidase.

Hyperpermeability is a crux of pathogenesis of sudden lung edema in many pulmonary disorders, especially in acute lung injury and adult respiratory distress syndrome (ARDS). Using our modified method for assessment of pulmonary vascular permeability, we observed the effects of xanthine with xanthine oxidase (X-XO) perfused in rat pulmonary artery and the protection of vasoactive intestinal polypeptide (VIP) against the injury of pulmonary vascular permeability. After addition of xanthine oxidase in the perfusate reservoir containing xanthine, 125I-albumin leak index (125IALI) was remarkably increased while peak airway pressure (Paw) was not significantly increased, and perfusion pressure of pulmonary artery (Ppa) and lung wet/dry weight ratio (W/D) were only slightly increased. Xanthine plus xanthine oxidase also increased thromboxane B2 (TX B2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) in the perfusate. Treatment with VIP obviously reduced or totally prevented all signs of injury. Simultaneously, VIP also diminished or abolished the associated generation of arachidonate products. The results indicated that VIP has potent protective activity against injury of pulmonary vascular permeability and may be a physiological modulator of inflammatory damage to vascular endothelium associated with toxic oxygen metabolites.

Oxidative denaturation of lens protein: prevention by pyruvate.

The denaturation of lens proteins as apparent by the generation of protein carbonyl in the presence of active oxygen and the prevention of such denaturation by pyruvate were studied. Active oxygen was generated by the action of xanthine oxidase on xanthine under aerobic conditions. Rat lens protein when incubated with xanthine and xanthine oxidase produced significant amounts of the carbonyl derivative. The formation of such carbonyl was substantially inhibited by pyruvate. In addition, the keto acid also was found to stimulate the utilization of glucose through HMP shunt, a mechanism known to transport reducing equivalents from glucose to peroxide. The results suggest that pyruvate exerts a beneficial effect in attenuating the age-related protein modifications and consequent physiological impairments. These studies are also considered useful from the therapeutic point of view.

Dextran sulphates protect porcine arterial endothelial cells from free radical injury.

1. The ability of dextran sulphate to protect cultured porcine arterial endothelial cells injured by addition of xanthine and xanthine oxidase (X/XO) or hydrogen peroxide to cell medium was examined using a variety of drug preparations. Cell damage was assessed by determining cell viability (by trypan blue exclusion) and release of lactate dehydrogenase into the medium. 2. Dextran sulphates of average molecular weight (M(r)) 5000, 8000 (hydrogenated or unhydrogenated) at 0.05, 0.5, 5 and 50 micrograms ml-1 medium, added 24 h prior to X/XO, protected cells, whereas dextran sulphate M(r) 500,000 was protective only at 0.5 microgram ml-1. 3. None of the dextran sulphates used showed any toxic effect on cells in concentrations up to 500 micrograms ml-1 medium. 4. When the duration of pretreatment with dextran sulphate M(r) 8000 was varied, 6 h was required for a protective effect on cells damaged by X/XO, which was enhanced with durations of 16 and 24 h. 5. Dextran sulphates had a similar protective effect on cells damaged by hydrogen peroxide. 6. This study suggest that dextran sulphates may prevent conditions resulting from free radical injury.

[Antioxidative action of soyasaponin monomers I, A1, A2, and their inhibitory effects on action potential and beating of myocardiocytes in culture].

Ventricular myocardiocytes from newborn Wistar rats were cultured. Soyasaponin monomers I, A1, A2 2 micrograms.ml-1 added to the culture medium, like nimodipine 1.25 microgram.ml-1, decreased the number of spontaneously beating clusters and the action potential parameters of myocardiocytes, which restored after washing out and were reversed by Ca2+ 80 micrograms.ml-1 or epinephrine 10 micrograms.ml-1, indicating the Ca2+ channel blockade action of the saponin monomers. Adding xanthine 0.42 mmol.L-1 and xanthine oxidase 5.4 nmol.L-1 into culture medium led to the increase of free radical content of the myocardiocytes and the decrease of action potential parameters, which were reversed by I 2 micrograms.ml-1, indicating the antioxidative action of saponin monomer I.

Pharmacological modulation of the spasmogenic response to methylxanthines in guinea-pig trachea.

The spasmogenic activity of methylxanthines was evaluated in guinea-pig isolated trachea treated with indomethacin (2.8 microM) and cooled to 20 degrees C. The contraction elicited by caffeine or theophylline (10 mM) was reduced in the presence of ouabain (10 microM), amiloride (100 microM), staurosporine (1 microM), H-7 (50 microM), polymyxin B (500 microM), K(+)-free solution, low Na+ (25 mM) medium or Ca(2+)-free (EGTA 0.1 mM) solution but was unaltered in the presence of verapamil (10 microM) or vanadate (10-100 microM). These results suggest that tracheal spasm to methylxanthines predominantly involves Ca2+ release from intracellular stores with a minor component due to extracellular Ca2+ entry through verapamil-insensitive pathways. A Na+/Ca2+ exchange process and the activation of protein kinase C may be also involved.

Evaluation of the effects of felodipine, verapamil and hydralazine on the survival rate of rats subjected to lethal effects of oxygen free radicals.

Intravenous administration of xanthine (X: 0.225 mg/kg, i.v.) plus xanthine oxidase (XO: 3.0 units/kg, i.v.) to anesthetized rats resulted in a rapid fall in the arterial pressure and a mortality rate of over 80% during 120 min observation period. Pretreatment of the rats with superoxide dismutase (SOD) or SOD plus catalase significantly enhanced survival rate to 60% confirming that the toxicity after [X + XO] administration is due to the generation of oxygen free radicals. Pretreatment of the rats with either felodipine, a dihydropyridine calcium antagonist or verapamil, a structurally different Ca(2+)-channel blocker was most effective in promoting survival rate to 90%; in contrast, hydralazine, an arteriolar dilator but not a calcium antagonist, was ineffective in significantly enhancing survival. In the vehicle treated groups, mortality of the rats after [X + XO] administration was associated with significant increases in serum creatine phosphokinase (CPK) levels; both the calcium antagonists as well as hydralazine prevented any significant changes in CPK levels. Since only the calcium antagonists but not hydralazine were effective in providing significant protection against mortality, the data suggests that CPK may not be a reliable indicator to predict prevention of lethal toxicity induced by free radicals. Hence, the observation that calcium antagonists can promote survival would suggest that calcium overload may be the ultimate mediator of tissue toxicity. These observations can account for the remarkable efficacy of various calcium antagonists in preventing ischemia-reperfusion induced damage to organs, such as heart and kidneys, in which a role for free radicals has been postulated.

Postsynaptic dopamine/adenosine interaction: II. Postsynaptic dopamine agonism and adenosine antagonism of methylxanthines in short-term reserpinized mice.

Caffeine and its first-stage metabolites (paraxanthine, theophylline and theobromine) caused a significant potentiation of the locomotor activity induced by bromocriptine, 5 mg/kg, in mice pretreated with reserpine, 5 mg/kg (4h prior to the start of motor activity recordings). None of these substances significantly enhanced locomotor activity in reserpinized mice when administered alone. The rank order of potency was caffeine greater than paraxanthine greater than theophylline greater than theobromine. A high dose of a D-2 antagonist (sulpiride 100 mg/kg) caused a marked inhibition of the locomotor activity induced by bromocriptine, 5 mg/kg, plus 25 mg/kg of caffeine, paraxanthine or theophylline. However, a high dose of a D-1 antagonist (SCH-23390 1 mg/kg) caused a significant decrease of the locomotor activity induced by bromocriptine 5 mg/kg, plus 25 mg/kg of caffeine or paraxanthine, but did not change the locomotor activity caused by bromocriptine, 5 mg/kg, plus theophylline 25 mg/kg. The inhibitory effect of 5'-(N-ethyl)carboxamido-adenosine (NECA), 0.025 mg/kg, on bromocriptine-induced locomotor activation in reserpinized mice was reversed by the simultaneous administration of 10, 25 and 50 mg/kg of caffeine, paraxanthine or theophylline. The rank order of potency for reversal was theophylline greater than paraxanthine = caffeine. We suggest that methylxanthines act postsynaptically by potentiating the effects of D-2 stimulation and that this potentiation can be produced by D-1 agonism (paraxanthine or caffeine) and by adenosine antagonism (theophylline, paraxanthine or caffeine), most probably involving A-2 receptors.

[A comparison of the efficacy of aminophylline and doxapram in preventing idiopathic apnea in preterm newborn infants]. / Comparazione dell'efficacia dell'Aminofillina e del Doxapram nella prevenzione della apnea idiopatica dei neonati pretermine.

Doxapram is an analeptic of the respiratory system that has been used in the last few years for the treatment of idiopathic apnea spells in infants who show resistance to methylxantine. In this study we have compared the efficacy of aminophylline and doxapram for the prevention of idiopathic apnea spells in two groups of preterm infants comparable for gestational age, birthweight and postnatal age. The two drugs resulted to be effective in preventing the spells of apnea in 66% and 60% of the cases respectively. In the cases in which there was a partial or negative response, the association of the two substances resulted in a noticeable reduction of the apnea spells. The positive effect of the association of aminophylline and doxapram is probably due to the different action mechanism on the stimulation of the respiratory system.

The effect of oxidant stress on diamide-treated human granulocytes.

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xamthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.

[Effects of various drugs on the lipolytic actions caused by catecholamines and methylxanthine derivatives in white adipose tissues. 1. Effects of procaine and xylocaine (author's transl)].

Effects of procaine and xylocaine on the lipolytic actions caused by catecholamines and methylxanthine derivatives in white adipose tissues from rats were investigated. Both procaine and xylocaine remarkably inhibited the lipolyses caused by norepinephrine, epinephrine, caffeine and theophylline. Xylocaine inhibited the lipolysis more strongly than procaine, and also inhibited the basal lipolysis. The inhibition by either procaine or xylocaine appeared 60 minutes after the addition of the norepinephrine-induced lipolytic action. The antilipolytic action of procaine was evident in medium containing 2 mM EDTA instead of Ca2+, and its antilipolytic action was accelerated by increasing Ca2+ concentration in the medium. From these positive results, we suggest that both procaine and xylocaine have an antilipolytic effect, and this effect is closely dependent on the Ca2+ concentration in the medium.