Pauli Vuorinen

Inhibition by nitric oxide-donors of human polymorphonuclear leucocyte functions.

1 The study was designed to test the hypothesis that nitric oxide (NO)‐releasing compounds increase guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) production in human polymorphonuclear leucocytes (PMNs) and concomitantly inhibit PMN functions, i.e. leukotriene B4 (LTB4) synthesis, degranulation, chemotaxis and superoxide anion (O2−) release. The effects of two new NO‐releasing compounds, GEA 3162 and GEA 5024 were compared to 3‐morpholino‐sydnonimine (SIN‐1) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP). 2 GEA 3162 and GEA 5024 (1–100 μm) inhibited Ca ionophore A23187‐induced LTB4 and β‐glucuronidase release, chemotactic peptide FMLP‐induced chemotaxis and opsonized zymosan‐triggered chemiluminescence dose‐dependently in human PMNs. SIN‐1 and SNAP were weaker inhibitors. 3 Cellular cyclic GMP production was increased after exposure to NO‐donors concomitantly with the inhibition of PMN functions. No alterations in the levels of adenosine 3′:5′‐cylic monophosphate (cyclic AMP) were detected. 4 The results suggest that NO, possibly through increased cyclic GMP, inhibits the activation of human PMNs and may thus act as a local modulator in inflammatory processes.

Nitric oxide donor GEA 3162 inhibits endothelial cell-mediated oxidation of low density lipoprotein

The effect of a nitric oxide (NO) donor GEA 3162 on the endothelial cell (EC)‐mediated oxidation of low density lipoprotein (LDL) was studied. In comparison to LDL incubated with EC without GEA 3162, the presence of GEA 3162 inhibited LDL oxidation by EC, as indicated by the following findings. (a) The degradation rate of LDL in macrophages was reduced to control levels. (b) The electrophoretic mobility of LDL decreased in a dose‐dependent manner, (c) The concentrations of thiobarbituric acid‐reactive substances and hydroperoxide‐derived hydroxy fatty acids were lower. (d) The breakdown of apolipoprotein B was reduced. The results indicate that GEA 3162 prevents EC‐mediated oxidation of LDL.

Nitric oxide-donating properties of mesoionic 3-aryl substituted oxatriazole-5-imine derivatives.

1 The nitric oxide (NO)‐releasing properties of two new mesoionic 3‐aryl substituted oxatriazole‐5‐imine derivatives (GEA 3162 and GEA 3175) were characterized and compared with the known NO‐donors 3‐morpholino‐sydnonimine (SIN‐1) and S‐nitroso‐N‐acetylpenicillamine (SNAP). 2 GEA 3162, GEA 3175, SIN‐1 and SNAP inhibited adenosine 5′‐diphosphate‐induced platelet aggregation (IC50 values 0.18, 0.39, 3.73 and 2.12 μm, respectively). All four compounds induced a dose‐dependent and more than 4 fold increase in cyclic GMP in platelets. The increase in cyclic GMP concentration was potentiated more than 1.5 fold by a phosphodiesterase inhibitor, zaprinast (10 μm) and inhibited 38–97% by oxyhaemoglobin (10–45 μm). 3 All of the four compounds studied converted oxyhaemoglobin to methaemoglobin and formed a paramagnetic NO‐haemoglobin complex. All but GEA 3175 formed nitrite and nitrate in phosphate buffer. During a 40 min incubation, GEA 3162, SIN‐1 and SNAP (100 μm) produced 50–70 μm NO2−+NO3− as determined by high performance liquid chromatography. The release of NO and NO2 by GEA 3175 was increased 140 fold in the presence of human plasma (0.14 and 19.7 ppb in the absence and presence of 1% human plasma, respectively) as analyzed by ozone chemiluminescence. 4 The results suggest that the mesoionic 3‐aryl substituted oxatriazole‐5‐imine derivatives GEA 3162 and GEA 3175 as well as SIN‐1 and SNAP release nitric oxide.

Contractions induced by potassium‐free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats

1 Vascular contractions induced by K+‐free solution and relaxation responses following the return of K+ to the organ bath were studied in mesenteric arterial rings from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto rats (WKY) with particular focus on the role of vascular adrenergic nerve‐endings and endothelium. 2 In endothelium‐denuded rings the omission of K+ from the incubation medium resulted in gradual contractions, the rate of which was slower in SHR than WKY. Nifedipine (1 μm) inhibited the contractions more effectively in SHR than WKY. 3 Adrenergic denervation in vitro with 6‐hydroxydopamine reduced the contractions induced by the K+‐free medium in endothelium‐denuded rings. The remaining contractions after denervation were markedly greater in SHR than WKY. 4 The presence of intact vascular endothelium attenuated the K+‐free contractions in both strains, the attenuation being smaller in SHR than WKY. NG‐nitro‐l‐arginine methyl ester (l‐NAME, 0.1 mm) and methylene blue (10 μm), but not indomethacin (10 μm), abolished the attenuating effect of endothelium on the K+‐free contractions. l‐Arginine (1 mm) reversed the effect of l‐NAME in WKY but not in SHR. 5 The re‐addition of K+ after full K+‐free contractions dose‐dependently relaxed the rings. The rate of this K+‐induced relaxation was significantly slower in SHR than WKY at all K+ concentrations (0.1–5.9 mm) studied, whether the endothelium or functioning adrenergic nerve‐endings were present or not. Ouabain (1 mm) totally inhibited the K+ relaxation in SHR but only partially in WKY. 6 Vascular smooth muscle contractions induced by high concentrations of potassium were comparable between the strains. The EC50 for noradrenaline‐induced contractions was lower in SHR than WKY, but the maximal forces did not differ significantly. 7 In conclusion, the contractile response in K+‐free solution more clearly differentiates vascular rings from SHR and WKY than the responses induced by the classical contractile agents noradrenaline and high concentrations of potassium. The depressant effect of the presence of intact endothelium on the K+‐free contractions, which was smaller in SHR than WKY, is mediated via the endothelium‐derived relaxing factor. Neurotransmitter release from vascular adrenergic nerve‐endings participates less in the K+‐free contractile response in SHR than WKY. Moreover, the contractile response is more dependent on calcium entry through nifedipine‐sensitive calcium channels in SHR than WKY. The greater K+‐free contractions of denervated endothelium‐denuded rings and the reduced K+ relaxation rate in SHR when compared to WKY suggest increased cell membrane permeability and decreased activity of vascular Na+, K+‐ATPase, respectively, in this type of genetic hypertension.

Endothelium-dependent and -independent effects of exogenous ATP, adenosine, GTP and guanosine on vascular tone and cyclic nucleotide accumulation of rat mesenteric artery

1 . The effects of exogenous guanosine 5′‐triphosphate (GTP) and guanosine on vascular tone and cyclic nucleotide accumulation of noradrenaline‐precontracted endothelium‐intact and endothelium‐denuded rat mesenteric artery rings were compared with the effects of the known purinoceptor agonists adenosine 5′‐triphosphate (ATP) and adenosine. 2 . GTP (10 μm−1 mm) dose‐dependently relaxed endothelium‐intact mesenteric artery rings by producing a rapid initial response followed by sustained relaxation resembling the relaxant response to acetylcholine. GTP also slightly relaxed endothelium‐denuded artery rings. The acetylcholine‐ and GTP‐induced relaxations of endothelium‐intact rings were attenuated by NG‐nitro l‐arginine methyl ester (l‐NAME, 330 μm) which attenuation was reversed with l‐arginine (1 mm). 3 . Guanosine (10 μm−1 mm) relaxed both endothelium‐intact and ‐denuded artery rings in a dose‐dependent manner. The relaxations were more pronounced in endothelium‐intact preparations and were only slightly attenuated by l‐NAME (330 μm). 4 . ATP (1 μm−1 mm) and adenosine (10 μm−1 mm) dose‐dependently relaxed endothelium‐intact and ‐denuded artery rings. The responses were more pronounced in endothelium‐intact vascular preparations. 5 . GTP (100 μm) and guanosine (100 μm) increased guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) accumulation in both endothelium‐intact and ‐denuded artery rings corresponding to the relaxations observed. The concentrations of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) were not affected. 6 . ATP (100 μm) increased cyclic GMP concentration of endothelium‐intact artery rings. The concentrations of cyclic AMP were not affected by ATP (100 μm) and adenosine (100 μm) in endothelium‐intact and ‐denuded vascular preparations. 7 . These results provide evidence that exogenous GTP and guanosine relax precontracted endothelium‐intact and ‐denuded rat mesenteric artery rings by increasing cyclic GMP accumulation. The response to GTP of endothelium‐intact rings can mainly be explained by the release of endothelium‐derived relaxing factor (EDRF), but that of guanosine is only partly due to EDRF, and is a combination of endothelium‐dependent and ‐independent effects. The endothelium‐independent response of GTP and guanosine is a direct, unknown effect on smooth muscle and guanylate cyclase.

Inhibition of human lymphocyte proliferation by nitric oxide-releasing oxatriazole derivatives.

The effects of novel nitric oxide (NO)-releasing oxatriazole derivatives GEA 3162 and GEA 3175 were studied on cell proliferation and cGMP synthesis in human peripheral blood mononuclear cells stimulated with a lectin mitogen concanavalin A. GEA 3162 (1-30 microM) and GEA 3175 (3-30 microM) inhibited mononuclear cell proliferation in a dose-dependent manner being more potent than the earlier known NO-donor S-nitroso-N-acetylpenicillamine. The inhibitory action was more pronounced when submaximally stimulating concentrations of concanavalin A (0.1 and 1 microg/ml) were used and no inhibition was seen when concanavalin A concentrations were increased up to 10 microg/ml. The antiproliferative concentrations of GEA 3162, GEA 3175 and S-nitroso-N-acetylpenicillamine induced a rapid and transient increase in cGMP production in mononuclear cells cultured in the presence of concanavalin A. Both the antiproliferative action and the increased cGMP production were attenuated when red blood cells were added into the cultures indicating that NO is responsible for both of these actions. An analogue of cGMP, 8-bromo-cGMP (0.1-3 mM) reduced concanavalin A-induced proliferation in a dose-dependent manner suggesting that cGMP may be involved in the antiproliferative action of NO-donors. NO-releasing compounds have immunosuppressive actions which offer therapeutic possibilities and should be kept in mind as potential adverse events when these compounds are used in other indications.

Effects of cysteine and nitroglycerin on bovine heart guanylate cyclase and on tissue cyclic GMP and lactate of rat atria

The effects of thiols on guanylate cyclase activation by nitroglycerin were studied in bovine heart and the effects of cysteinee and nitroglycerin on the tissue levels of cyclic GMP and lactate were studied in beating rat atria. Cysteine (2.5 X 10(-3) M) together with nitroglycerin (1 X 10(-3) M), increased 15-fold the activity of guanylate cyclase. In hypoxia, nitroglycerin (1 X 10(-3) M) together with cystein (5 X 10(-3) M) increased cyclic GMP and decreased the tissue lactate level. It is concluded that cysteine potentiates the effect of nitroglycerin on cyclic GMP formation even in integrated cell systems with an intact physiological function.

The effects of cyclic AMP and cyclic GMP on redox state and energy state in hypoxic rat atria

The effects of 8-bromo-cAMP (10(-4)M) and 8-bromo-cGMP (10(-4)M) on tissue lactate, NADH, creatine phosphate (CP), ATP, ADP and AMP were studied in hypoxic (50% oxygen saturation) spontaneously beating rat atria. CP/ATP ratio and energy charge (EC) were also calculated. In hypoxic rat atria there was a significant increase in tissue lactate, NADH, ADP and AMP and a decrease in CP, CP/ATP ratio and EC. 8-bromo-cGMP abolished these hypoxia-induced changes. The levels of lactate and NADH decreased and those of CP, ATP, ADP and EC increased after administration of 8-bromo-cGMP. 8-bromo-cAMP increased the level of lactate during hypoxia, but did not affect the other hypoxia-induced changes. The present work indicates that cyclic GMP can change the redox state and energy state in a more favourable direction for the hypoxic rat heart.

Effects of P1 and P2Y purinoceptor antagonists on endothelium-dependent and -independent relaxations of rat mesenteric artery to GTP and guanosine

1 Guanosine 5′‐triphosphate (GTP) and guanosine can relax both endothelium‐intact and ‐denuded arterial preparations. In the present work the P1 and P2Y purinoceptor antagonists, 8‐phenyltheophylline and reactive blue 2, respectively, were used to study the mechanisms of relaxation responses induced by GTP, guanosine, adenosine 5′‐triphosphate (ATP) and adenosine in noradrenaline‐precontracted rat mesenteric artery rings. 2 GTP (10 μm – 1 mm) dose‐dependently relaxed endothelium‐intact mesenteric artery rings and also induced moderate relaxation responses in endothelium‐denuded preparations. Pretreatment of the rings with 8‐phenyltheophylline (10 μm) or reactive blue 2 (10 μm) did not attenuate the relaxant effect of GTP. 3 Guanosine (10 μm – 1 mm) relaxed both endothelium‐intact and ‐denuded artery rings in a dose‐dependent manner. The presence of 8‐phenyltheophylline or reactive blue 2 had no effects on guanosine‐induced relaxations. 4 ATP‐induced (0.1 μm – 0.1 mm) relaxation of endothelium‐intact artery rings was attenuated by reactive blue 2 while 8‐phenyltheophylline was ineffective. ATP also relaxed endothelium‐denuded artery rings and this relaxation was inhibited by 8‐phenyltheophylline, but not by reactive blue 2. 5 Adenosine‐induced (10 μm–1 mm) relaxation of endothelium‐intact and ‐denuded artery rings was attenuated by the presence of 8‐phenyltheophylline, but not of reactive blue 2. 6 In conclusion, the endothelium‐dependent and ‐independent relaxations of rat mesenteric arteries to GTP and guanosine are not mediated via P1 and P2Y purinoceptors. Therefore, these results support our previous suggestion on the presence of a novel guanine nucleotide‐specific receptor, a putative PG receptor, on both endothelial and smooth muscle cells, which may participate in the regulation of arterial tone.

Induction of beta-2-microglobulin release in vitro by chronic lymphocytic leukaemia cells: relation to total protein synthesis

The in vitro production of beta2-M by B-CLL cells from 27 patients was investigated. In all cases, low spontaneous beta2-M release was observed. The production of beta2-M was enhanced to various extents when induced with 13 different stimulants and their combinations including IL-2, TNFalpha, SAC and TPA. Beta2-M release was 3.8-fold (range from 1.9 to 9.2-fold) in cultures stimulated with TPA (10 ng/ml), compared with the spontaneous release, and even faster if TNFalpha or IL-2 were added. A strong correlation was revealed between beta2-M production and the total protein synthesis of leukaemic cells when the latter was assessed using 14C-L-leucine incorporation. Hence, both beta2-M release and leucine incorporation are promising activation markers for CLL B-lymphocytes.