Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts

1. The functional role of the nitric oxide (NO)/guanosine 3':5'-cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts. 2. Rat isolated hearts were perfused at constant pressure with Krebs-Henseleit buffer for 25 min (baseline), then made ischaemic by reducing coronary flow to 0.2 ml min(-1) for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored. 3. At baseline, heart rate was 287+/-12 beats min(-1), coronary flow was 12.8+/-0.6 ml min(-1), left ventricular developed pressure (LVDevP) was 105+/-4 mmHg and left ventricular end-diastolic pressure 4.6+/-0.2 mmHg in vehicle-treated hearts (control; n=12). Baseline values were similar in all treatment groups (P>0.05). 4. In normoxic perfused hearts, 1 microM N(G)-nitro-L-arginine (L-NOARG) significantly reduced coronary flow from 13.5+/-0.2 to 12.1+/-0.1 ml min(-1) (10%) and LVDevP from 97+/-1 to 92+/-1 mmHg (5%; P<0.05, n=5). 5. Ischaemic contracture was 46+/-2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), unaffected by low concentrations of nitroprusside (1 and 10 microM) but reduced to approximately 30 mmHg (approximately 25%) at higher concentrations (100 or 1000 microM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L-NOARG reduced contracture at 1 microM to 26+/-3 mmHg (23%), but increased it to 63+/-4 mmHg (59%) at 1000 microM (n=6). Dobutamine (10 microM) exacerbated ischaemic contracture (81+/-3 mmHg; n = 7) and the cyclic GMP analogue Sp-8-(4-p-chlorophenylthio)-3',5'-monophosphorothioate (Sp-8-pCPT-cGMPS; 10 microM) blocked this effect (63+/-11 mmHg; P<0.05 vs dobutamine alone, n=5). 6. At the end of reperfusion, LVDevP was 58+/-5 mmHg, i.e. 55% of pre-ischaemic value in control hearts, significantly increased to approximately 80% by high concentrations of nitroprusside (100 or 1000 microM) or L-NOARG at 1 microM, while a high concentration of L-NOARG (1000 microM) reduced LVDevP to approximately 35% (P<0.05 vs control; n=6). 7. Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P<0.05; n=12); nitroprusside at 1 microM had no sustained effect, but increased cyclic GMP approximately 6 fold at 1000 microM; L-NOARG (1 or 1000 microM) was without effect (n=6). Nitroprusside (1 or 1000 microM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no effect (n=6). 8. In conclusion, the cardioprotective effect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not affected by low concentrations of NO synthase inhibitors, their beneficial effect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.     

Fencamfamine modulates sodium, potassium-ATPase through cyclic AMP and cyclic AMP-dependent protein kinase in rat striatum

Dopamine (DA) and fencamfamine (FCF) modulatory action on Na,K-ATPase and Mg-ATPase activity were evaluated in rat striatum. DA and FCF induced a decrease in Na,K-ATPase, without affecting Mg-ATPase activity. The effect of FCF was dose-dependent from 10 to 100 microM, with an IC50 of 4.7 x 10(-5) M. Furthermore, the effect of FCF (100 microM) increasing AMPc levels, but not GMPc, was nonadditive with that of DA (10 microM), which is consistent to a common site of action. The 8-bromo-cyclic AMP also induced a specific reduction in the Na,K-ATPase activity. The reduction of Na,K-ATPase induced by FCF (100 microM) was blocked by either SCH 23390 or sulpiride, which are D1 and D2 receptor antagonists. The decrease in striatal NA,K-ATPase activity induced by FCF was blocked by KT 5720, a selective inhibitor of cyclic AMP-dependent protein kinase (PKA), but not by KT 5823, a selective inhibitor of cyclic GMP-dependent protein kinase (PKG). Otherwise, KT 5720 or KT 5823 did not produce any change in Na,K-ATPase or Mg-ATPase activity. These data suggest that FCF reduces Na,K-ATPase activity through cyclic AMP-dependent changes in protein phosphorylation via a PKA mechanism.     

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O2.- release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an IC50 value of 60.7+/-8.2 microM. A comparable effect was also demonstrated in the inhibition of O2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O2.- generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca2+]i, but it accelerated the rate of [Ca2+]i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetra hydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinde n-1-one, (KT 5720), a PKA inhibitor, the inhibition of O2.- generation by YT-1, as well as by prostaglandin E1 (PGE1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE1. Moreover, the ability of PGE1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.     

Accuracy of the inverse Womersley method for the calculation of hemodynamic variables

OBJECTIVE: The role of phosphodiesterase III inhibition and calcium sensitization in the cardiac actions of levosimendan, (R)-[[4-(1,4,5, 6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono]propane dinitrile, was studied. METHODS: Various heart preparations were used to investigate positive inotropy, chromotropy, coronary flow and calcium sensitivity of contractile proteins. The cAMP- and cGMP-dependent protein kinases (PKA and PKG) were inhibited by KT5720 and KT5823, respectively. Furthermore, the synthesis of cAMP was stimulated by forskolin and increased phosphorylation of troponin I was induced by isoprenaline. RESULTS: In Langendorff guinea-pig heart, levosimendan (0.01-1 microM) and milrinone (0.1-10 microM) increased the left ventricular systolic peak pressure almost to the same extent. In the presence of KT5720 (1 microM) milrinone was devoid of positive inotropic activity. In contrast, KT5720 did not antagonize the inotropic effect of levosimendan at < or = 0.03 microM (-up to the EC50 of levosimendan). The effects of levosimendan and milrinone on heart rate and coronary flow were not affected by KT5720. The PKG inhibitor, KT5823 (1 microM), on the other hand, potentiated the levosimendan-induced increase in coronary flow while it had no effect on the increase induced by milrinone. The mechanical parameters were not affected by KT5823. In the papillary muscle, the positive inotropic effect of milrinone but not that of levosimendan was potentiated by forskolin (0.1 microM). In contrast to milrinone, the positive inotropy by levosimendan was decreased by isoprenaline pretreatment (0.1 microM; 3 min). In line with this, the calcium-sensitizing effect of levosimendan was decreased in skinned fibers prepared from isoprenaline-treated hearts. CONCLUSIONS: Our results indicate that the cardiac effects of levosimendan at its therapeutically relevant concentrations were not mediated through PKA or PKG and its positive inotropy is therefore most probably due to the previously reported troponin-C-mediated calcium sensitization of contractile proteins.     

Preeclampsia, lipid peroxidation, and calcium adenosine triphosphatase activity of red blood cell ghosts

1. The effects of oxatriazole-type (GEA 3162 and GEA 5624) nitric oxide (NO) donors on mitogenesis and proliferation were studied in vascular smooth muscle cell (VSMC) culture. The effects of the GEA-compounds were compared with well-known NO-donors 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). 2. All NO-donors released NO and increased the production of cyclic GMP concentration-dependently. The production of cyclic GMP was inhibited by the guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). 3. The NO-donors inhibited basal and serum-induced DNA synthesis concentration-dependently. The GEA-compounds were needed in concentrations 10 times lower than SIN-1 and SNAP. GEA 3162, SIN-1 and SNAP were also able to inhibit serum-induced cell proliferation. GEA 5624 was ineffective. The antimitogenic effect of NO-donors was not reduced by inhibiting the guanylate cyclase. 4. These results suggest that NO inhibits serum-induced DNA synthesis and proliferation of VSMC by a cyclic GMP-independent mechanism. The oxatriazole-type NO-donor GEA 3162 was found to be a more potent inhibitor of mitogenesis and cell proliferation than SIN-1 and SNAP.     

A study of effects of mouthwash on the human oral mucosae: with special references to sites, sex differences and smoking

Nitric oxide has been shown to decrease myocardial contractility and O2 consumption. This study was designed to evaluate the hypothesis that nitric oxide-mediated increases in cyclic GMP require elevated cyclic AMP to produce cardiac depression. Using isolated, Langendorff-perfused rat hearts, we determined the effects of intracoronary nitroprusside (NP, 1 and 10 mM) in the absence and presence of isoproterenol (ISO, 10(-8) M) on cardiac function, O2 consumption, cyclic GMP and cyclic AMP. ISO, with and without NP, increased cyclic AMP (from 287 +/- 21 to 477 +/- 33 pmol/g) without altering cyclic GMP. Left-ventricular pressure increased from 97 +/- 12 to 178 +/- 9 mm Hg and dP/dtmax from 1,786 +/- 275 to 4,049 +/- 354 mm Hg/s. NP increased cyclic GMP (from 4 to 30 pmol/g) in both the absence and presence of ISO, but NP did not alter cyclic AMP. Without ISO, NP insignificantly altered left-ventricular pressure; however, in the presence of ISO, NP significantly decreased left-ventricular pressure by -25 +/- 4 mm Hg and decreased dP/dtmax by -619 +/- 142 mm Hg/s. Isoproterenol increased O2 consumption, but the changes with NP were not significant. When this study was repeated in the presence of LY83583, a guanylate cyclase inhibitor, NP still produced cardiac depression in the presence of ISO. Therefore, cardiodepressant effects of NP were only observed against a background of inotropic stimulation with ISO. However, effects of NP on contractility were unrelated to increases in cyclic GMP or cyclic GMP-induced changes in cyclic AMP.     

Organization and transcription of the gene encoding potato UDP-glucose pyrophosphorylase

Fenspiride inhibits the calcium signal evoked by the inflammatory peptide formyl-Met-Leu-Phe (fMLP) in peritoneal macrophages, but at concentrations (approximately 1 mM) far above the therapeutic range (approximately 1 microM). Here, in rat alveolar macrophages, high fenspiride concentrations (1 mM) were required to inhibit the calcium signals evoked by the calcium agonist Bay K8644 or by ionomycin. Moreover, fenspiride (1 mM) was a poor inhibitor of the cell membrane depolarization induced by gramicidine D. By contrast, fenspiride blocked Na+-H+ antiport activation by (i) fMLP with an IC50 = 3.1 +/- 1.9 nM and (ii) PMA (phorbol 12-myristate 13-acetate) with an IC50 = 9.2 +/- 3.1 nM. Finally, protein kinase C (PKC) activity of macrophage homogenate was not significantly modified by 10 or 100 microM fenspiride (at 100 microM: 2.57 +/- 1.60 vs. 2.80 +/- 1.71 pmol/10(6) cells/min). In conclusion, fenspiride inhibits fMLP- and PMA-induced pH signals in rat alveolar macrophages, probably by acting distally on the PKC transduction signal. This pH antagonistic action may be relevant for the antiinflammatory mechanism of fenspiride and requires further investigation.     

Effect of intracerebroventricular and intravenous administration of nitric oxide donors on blood pressure and heart rate in anaesthetized rats

1. The effects of nitric oxide (NO) releasing substances, sodium nitroprusside, 3-morpholino sydnonimine (SIN-1) and a novel oxatriazole derivative, GEA 3162, on blood pressure and heart rate were studied after peripheral or central administration in anaesthetized normotensive Wistar rats. 2. Given as cumulative intravenous injections, both nitroprusside and GEA 3162 (24-188 nmol kg-1) induced short-lasting and dose-dependent decreases in mean arterial pressure, while SIN-1 decreased blood pressure only slightly even after larger doses (94-3000 nmol kg-1). Heart rate increased concomitantly with the hypotensive effect of the NO-releasing substances. 3. Cumulative intracerebroventricular administration of GEA 3162 (24-188 nmol kg-1) induced a dose-dependent hypotension with slight but insignificant increases in heart rate. In contrast, intracerebroventricular nitroprusside induced little change in blood pressure, while a large dose of SIN-1 (3000 nmol kg-1, i.c.v.) slightly increased mean arterial pressure. However, intracerebroventricular nitroprusside and SIN-1 increased heart rate at doses that did not significantly affect blood pressure. 4. To determine whether the cardiovascular effects of GEA 3162 were attributable to an elevation of cyclic GMP levels, pretreatments with methylene blue, a putative guanylate cyclase inhibitor, were performed. This substance failed to attenuate the cardiovascular effects of peripherally or centrally administered GEA 3162, suggesting that the effects were independent of guanylate cyclase. 5. In conclusion, the centrally administered NO-donor, GEA 3162, induced a dose-dependent. hypotensive response without significant changes in heart rate. Furthermore, intracerebroventricular injections of nitroprusside and SIN-1 increased heart rate without affecting blood pressure. These results suggest that NO released by these drugs may affect central mechanisms involved in cardiovascular regulation independently of cyclic GMP.     

In vivo microdialysis study of a specific inhibitor of soluble guanylyl cyclase on the glutamate receptor/nitric oxide/cyclic GMP pathway

1. Nitric oxide (NO) is known to stimulate soluble guanylyl cyclase, thereby eliciting an elevation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in target cells. Recently, a selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), has been identified and characterized in vitro. We have investigated the in vivo effects of ODQ on the glutamate receptor/NO/ cyclic GMP pathway by monitoring extracellular cyclic GMP during microdialysis of the cerebellum or the hippocampus of freely-moving adult rats. 2. Intracerebellar administration of ODQ (1-100 microM) via the microdialysis probe inhibited, in a concentration-dependent manner, the basal extracellular level of cyclic GMP. The maximal inhibition, measured after a 20 min perfusion with 100 microM ODQ, amounted to 80% and persisted unchanged as long as ODQ was perfused. When ODQ was removed from the perfusion stream after 20 min, the levels of cyclic GMP started to recover, suggesting reversibility of guanylyl cyclase inhibition by ODQ. 3. The cyclic GMP response evoked in the cerebellum by NMDA (200 microM) or by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 microM) was largely attenuated by 100 microM ODQ. The pattern of the inhibition curves suggests competition for guanylyl cyclase between ODQ and the NO generated by NMDA or AMPA receptor activation. 4. ODQ (100 microM) prevented the elevation of extracellular cyclic GMP levels provoked by intracerebellar infusion of the NO generator S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). The inhibition of the SNAP effect was rapidly relieved when ODQ was removed from the perfusion fluid. However, ODQ (100 microM) was unable to affect the cyclic GMP response elicited by 5 mM SNAP, in keeping with the proposed idea that ODQ binds to the "NO receptor' in a reversible and competitive manner. 5. Infusion of ODQ (10, 100 or 300 microM) into the hippocampus of freely-moving rats diminished the basal extracellular level of cyclic GMP. The maximal inhibition amounted to 50% and was produced by 100 microM ODQ. 6. The cyclic GMP response observed when 1 mM SNAP was perfused in the hippocampus, similar in percentage terms to that seen in cerebellum, was dramatically reduced during co-infusion of 100 microM ODQ. 7. ODQ appears to act in vivo as a selective, reversible and possibly competitive inhibitor of the soluble guanylyl cyclase targeted by NO. This enzyme may generate most (about 80%) of the cyclic GMP found under basal conditions in the extracellular space of the cerebellum. In the hippocampus, about 50% of the basal cyclic GMP does not seem to originate from the ODQ-sensitive soluble guanylyl cyclase.     

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.     

High selectivity for inhibition of phosphodiesterase III and positive inotropic effects of MCI-154 in guinea pig myocardium

MCI-154 (0.3-100 microM) exerted a concentration-dependent positive inotropic effect in isolated guinea pig papillary muscles (EC50 0.8 microM). The efficacy of MCI-154 (253% of predrug value) was 1.7-fold higher than that of saterinone but comparable to that of milrinone. Carbachol markedly reduced the increase in force of contraction (FOC) of MCI-154. In intact contracting papillary muscles, the positive inotropic effect was accompanied by an increase in cyclic AMP content to 0.78 +/- 0.09 pmol/mg wet weight (n = 10), corresponding to 150% of the basal value (0.51 +/- 0.05 pmol/mg wet weight, n = 21) in the presence of submaximal cyclic AMP phosphodiesterase (PDE) isoenzyme III inhibiting concentrations of MCI-154 (30 microM). MCI-154 (1-1,000 microM) concentration-dependently inhibited the activity of PDE III from homogenates of guinea pig myocardium. The IC50 was 3.8 microM. PDE I, II, and IV were not significantly affected up to 100 microM (PDE I and IV) and up to 1,000 microM (PDE II). In comparison, milrinone and saterinone were PDE III/IV-selective PDE inhibitors. Rolipram inhibited PDE IV only. IBMX and theophylline were nonselective PDE inhibitors. MCI-154 had only a marginal positive chronotropic effect. The frequency of spontaneously beating right auricles from guinea pig heart was increased by 8.7% at most (n = 5). MCI-154 increased Ca2+ sensitivity in chemically skinned porcine ventricular muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two replicons in phage-plasmid P4

1 Characteristics of cyclic GMP- and cyclic AMP-mediated relaxation in aortic segments of rats with chronic heart failure (CHF) and the effects of chronic treatment with an angiotensin I converting enzyme (ACE) inhibitor, trandolapril, were examined 8 weeks after coronary artery ligation. 2 Cardiac output indices of coronary artery-ligated and sham-operated rats were 125+/-8 and 189+/-10 ml min(-1) kg(-1), respectively (P<0.05), indicating the development of CHF at this period. 3 The maximal relaxant response of aortic segments to 10 microM acetylcholine in rats with CHF and sham-operated rats was 64.0+/-5.7 and 86.9+/-1.9%, respectively (P<0.05), whereas the relaxant response to sodium nitroprusside (SNP) remained unchanged. Tissue cyclic GMP content in rats with CHF was lower than that of sham-operated rats. 4 In endothelium-intact segments of rats with CHF, the maximal relaxant response to 10 microM isoprenaline (44.5+/-6.7%) was lower that sham-operated rats (81.3+/-2.5%, P<0.05) and the concentration-response curve for NKH477, a water-soluble forskolin, was shifted to the right without a reduction in the maximal response. Isoprenaline-induced relaxation of aortic segments was attenuated by NG-nitro-L-arginine methyl ester (L-NAME) in sham-operated rats, but not in rats with CHF. Relaxation to 30 microM dibutyryl cyclic AMP in rats with CHF (26.8+/-2.7%) was lower than that in sham-operated rats (63.4+/-11.8%, P<0.05). 5 Trandolapril (3 mg kg(-1) day(-1)) was orally administered from the 2nd to 8th week after the operation. Aortic blood flow of rats with CHF (38.5+/-3.6 ml min(-1)) was lower than that of sham-operated rats (55.0+/-3.0 ml min(-1)), and this reduction was reversed (54.1+/-3.4 ml min(-1)) by treatment with trandolapril. The diminished responsiveness described above was normalized in the trandolapril-treated rat with CHF (i.e., the maximal relaxation to acetylcholine, 94.7+/-1.0%; that to isoprenaline, 80.5+/-2.8%; that to dibutyryl cyclic AMP, 54.7+/-6.2%). However, aortic segments of trandolapril-treated rats with CHF, L-NAME did not attenuate isoprenaline-induced relaxation and the tissue cyclic GMP level was not fully restored, suggesting that the ability of the endothelium to produce NO was still partially damaged. 6 The results suggest that vasorelaxation in CHF, diminished mainly due to dysfunction in endothelial nitric oxide (NO) production and cyclic AMP-mediated signal transduction, was partially restored by long-term treatment with trandolapril. The mechanism underlying the restoration may be attributed in part to prevention of CHF-induced endothelial dysfunction.     

Involvement of endogenous nitric oxide in the mechanism of bradykinin-induced peripheral hyperalgesia

1. When NG-nitro-L-arginine methyl ester (L-NAME, 0.1-10 nmol) or NG-monomethyl-L-arginine (L-NMMA, 10 nmol-1 mumol) was intradermally administered with bradykinin (BK, 3 nmol) into the instep of rat hind-paws, a dose-related suppression of BK-induced hyperalgesia, assessed by the paw-pressure test, was produced. 2. L-Arginine (1 mumol) but not D-arginine (1 mumol) reversed the suppressive effects of L-NAME (10 nmol) and L-NMMA (1 mumol) on BK-induced hyperalgesia. 3. Concomitant intradermal administration of BK (3 nmol) with haemoglobin (1 nmol) significantly suppressed BK-induced hyperalgesia in the paw-pressure test. The BK-induced hyperalgesia was abolished by concomitant intradermal administration of either a guanylate cyclase inhibitor, methylene blue (10 nmol), or LY83583 (1 nmol). In addition, KT5823 (1 nmol) or Rp-8-bromoguanosine-3':5'-cyclic monophosphothioate (Rp-8-Br-cGMPS; 1 nmol), an inhibitor of cyclic GMP-dependent protein kinase, also significantly suppressed BK-induced hyperalgesia. 4. The carrageenin-induced hyperalgesia was significantly attenuated by L-NAME in a dose-dependent manner. 5. L-Arginine (1 mumol), sodium nitroprusside (1 mumol), dibutyryl cyclic GMP (1 mumol) or 8-bromo cyclic GMP (1 mumol) all failed to produce any significant relieving effect on the nociceptive threshold of rodent hind-paws. Concomitant administrations of each agent with a sub-threshold dose (0.1 nmol) of BK induced significant hyperalgesia. 6. Rp-adenosine 3':5'-cyclic monophosphothioate (Rp-cAMPS; 1 nmol), an inhibitor of cyclic AMP-dependent protein kinase, significantly suppressed BK-induced mechanical hyperalgesia. Concomitant administration of forskolin (1 nmol) with 8-bromo cyclic GMP (100 nmol) induced significant hyperalgesia. 7. In the superfusion experiment of a blister base on the instep of rodent hind-paws, intradermally administered BK (3 nmol) significantly increased the outflow of both cyclic GMP and cyclic AMP from the blister base. Concomitant administrations of L-NAME (10 nmol) with BK significantly reduced the BK-induced outflow of cyclic GMP without affecting the cyclic AMP content. 8. These results suggest that the NO-cyclic GMP pathway is involved in the mechanism of BK-induced hyperalgesia, and an activation of both cyclic GMP-and cyclic AMP-second messenger system plays an important role in the production of peripherally induced mechanical hyperalgesia.     

Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide

1. The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10(-1) M), NO (10(-10) - 10(-6) M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura-2 fluorimetry. 2. Zaprinast (10(-4) M), increased NO-stimulated levels of cyclic GMP by 3-20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. 3. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10(-5) M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10(-10) - 10(-8) M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10(-7) - 10(-6) M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10(-7) - 10(-5) M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. 4. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.     

Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells

1. Extracellular ATP (EC50=146+/-57 microM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells. 2. The order of agonist potency was: ATPgammaS (adenosine 5'-O-[3-thiotriphosphate]) > or = BzATP (2'&3'O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate) > or = dATP > ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: beta,gamma methylene ATP > or = 2-methylthioATP > ADP > or = Ap4A (P1, P4-di(adenosine-5') tetraphosphate) > or = Adenosine > UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP-dependent activation of adenylyl cyclase. 3. Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPgammaS (EC50=30.4+/-6.9 microM) was a full agonist. However, adenosine 5'-O-[1-thiotriphosphate] (ATPalphaS; EC50=45+/-15 microM) and adenosine 5'-O-[2-thiodiphosphate] (ADPbetaS; EC50=33.3+/-5.0 microM) were partial agonists. 4. ADPbetaS (IC50=146+/-32 microM) and adenosine 5'-O-thiomonophosphate (AMPS; IC50=343+/-142 microM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 microM). Consistent with its partial agonist activity, ADPbetaS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors. 5. The broad spectrum P2 receptor antagonist, suramin (500 microM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 microM) and 8-sulpho-phenyltheophylline (8-SPT; 100 microM) were without effect. 6. Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997). 7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.     

Effect of cyclic GMP reduction on regional myocardial mechanics and metabolism in experimental left ventricular hypertrophy

We tested the hypotheses that decreased myocardial cyclic GMP levels produced by intracoronary injection of methylene blue would increase local myocardial work and O2 consumption while decreasing intracellular cyclic GMP and that the relation between work, O2 consumption, and cyclic GMP may be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. In 8 control and 8 LVH open-chest anesthetized dogs, 1 mg/kg/min methylene blue was infused into the left anterior descending coronary artery (LAD); the circumflex region (CFX) served as control area. Regional work was calculated as the integrated product of force (miniature transducer) and segment shortening (sonomicrometry). Regional myocardial O2 consumption was calculated from flow measurements (radioactive microspheres), and regional O2 saturations (microspectrophotometry). A radioimmunoassay was used to determine intracellular level of cyclic GMP in the myocardium. Global hemodynamics and blood gases were unchanged by methylene blue in both control and LVH animals. Intracoronary methylene blue increased regional work from 762 +/- 129 to 1,451 +/- 307 g center dot mm/min in controls and from 912 +/- 173 to 1581 +/- 253 g center dot mm/min in the LVH groups. No significant changes in CFX regional work were observed. Regional blood flow, O2 extraction, and O2 consumption remained unchanged after injection of methylene blue in both control and LVH animals. The basal levels of cyclic GMP in the LVH group were fivefold higher than that in controls. In both groups, cyclic GMP levels were significantly decreased by methylene blue and to a greater extent in the LVH animals (from 6.16 +/- 1.2 to 3.34 +/- 0.44 pmol/g) than in the control animals (from 1.32 +/- 0.20 to 1.09 +/- 0.19 pmol/g). Therefore, intracoronary methylene blue increased regional myocardial work equally in control and LVH hearts without affecting regional metabolism (i.e., increased efficiency). For the same increased mechanical function, the hypertrophic myocardium exhibited a greater reduction in cyclic GMP pool size.     

Effects of selective phosphodiesterase 3 inhibition in the perfused liver of the rat after endotoxin treatment

1. This study was designed to investigate the role of rat phosphodiesterase 3 (RPDE3) in regulation of liver metabolism in sepsis. We studied the effects of the phosphodiesterase 3 inhibitor (PDI), enoximone, alone and in combination with regulating factors of hepatic carbohydrate metabolism and bile secretion in the perfused liver of rats treated 4 h earlier with endotoxin. In addition, cyclic AMP and cyclic GMP levels were determined in the effluate and bile by radio immunoassay methods. 2. After endotoxin treatment, infusion of enoximone at three concentrations (1 microM, 10 microM) resulted in an increased glucose output from -1.4 +/- 0.9 to 7.8 +/- 2.5 mumol l-1 20 min-1. Bile acid-independent bile flow increased also, in a dose-dependent manner. 3. In untreated livers, cyclic AMP release increased in the effluate from 1000 +/- 73 fmol g-1 min-1 to 1710 +/- 143 fmol g-1 min-1 when enoximone (10 microM) was administered. In bile from untreated livers, the level of cyclic AMP was also significantly increased by enoximone. After endotoxin treatment, the enoximone (10 microM) effect on cyclic AMP levels in effluate and bile was greatly reduced. Levels of cyclic GMP in the effluate and bile appeared unchanged in the presence of enoximone. 4. During co-infusion of glucagon (1 nM) and enoximone (10 microM), cyclic nucleotide levels in the effluate and bile of livers after endotoxin treatment were determined. In the effluate, cyclic AMP release increased from 827 +/- 144 fmol g-1 min-1 to 17802 +/- 2821 fmol g-1 min-1 when glucagon was administered. The presence of enoximone enhanced cyclic AMP further to 41696 +/- 920 fmol g-1 min-1. The same changes in cyclic AMP release were found in bile. Levels of cyclic GMP in the effluate and bile were not significantly affected by the administration of glucagon and the PDI. 5. Glucose release was determined during glucagon, sympathetic nerves stimulation and phenylephrine administration in the presence and absence of enoximone. The addition of enoximone to glucagon increased glucose release by 8.2 +/- 2.8 mumol g-1 20 min-1, without alteration of lactate balance. The PDI enhanced the glycogenolytic effects of nerve stimulation and of phenylephrine, accompanied by a reduction in lactate production. 6. Enoximone significantly enhanced the bile acid independent bile flow after glucagon, nerves stimulation and after administration of phenylephrine. Bile acid secretion was unaffected by the PDI. The vasoconstrictor effect of nerve stimulation was reduced by the PDI. 7. We conclude that endotoxin treatment reduces the ability of the PDI, enoximone, to increase cyclic AMP release in the perfused liver. The significant increase in cyclic AMP release after stimulation with glucagon and enoximone favours the view that RPDE3 is involved in the degradation of cyclic AMP in the liver after exposure to endotoxin. Additionally, the inhibition of the RPDE3 results in glucose release, vasodilatation and choleresis in endotoxin pretreated livers.     

The nitric oxide donor SIN-1 protects endothelial cells from tumor necrosis factor-alpha-mediated cytotoxicity: possible role for cyclic GMP and heme oxygenase

In cultured endothelial cells, incubation with TNF-alpha (50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10-150 microM) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5, -tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30 microM). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10-100 microM). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1 microM) or 1H--1,2,4-oxadiazole-4, 3-a-quinoxalin-1-one (ODQ, 0.1 microM), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25 microM), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-alpha, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.     

Trazodone is a potent agonist at 5-HT2C receptors mediating inhibition of the N-methyl-D-aspartate/nitric oxide/cyclic GMP pathway in rat cerebellum

The effects of trazodone on the cyclic GMP elevation elicited by N-methyl-D-aspartate in rat cerebellar slices were analyzed. Trazodone inhibited in a concentration-dependent manner (EC50 = 0.82 nM) the cyclic GMP response evoked by 0.1 microM N-methyl-D-aspartate. The inhibition was near complete at 10 nM trazodone. The effect of 10 nM trazodone was unaffected by 0.3 microM spiperone or rauwolscine, antagonists with selectivity for the 5-HT(serotonin)2A or the 5-HT2B subtype, respectively, but it was totally prevented by 0.01 microM mesulergine, a 5-HT2A/5-HT2B/5-HT2C receptor antagonist. Trazodone was potently counteracted (IC50 = 2.7 nM) by the selective 5-HT2B/5-HT2C receptor antagonist N-(1-methyl-5-indolyl)-N-(3-pyridil) urea HCl and, less potently (IC50 = 95 nM), by ketanserin, a 5-HT2A/5-HT2C receptor blocker. It is concluded that trazodone behaves as a potent full agonist at the 5-HT2C receptor mediating inhibition of the cerebellar N-methyl-D-aspartate/nitric oxide/cyclic GMP system.     

Hypothermia during or after severe perinatal asphyxia prevents increase in cyclic GMP-related nitric oxide levels in the newborn rat striatum

The striatum is rich in nitric oxide synthase (NOS). It is present in a dense fiber network and in a few medium-sized non-spiny interneurons. Previous work showed chronic overexpression of NOS in the rat striatum after a severe perinatal asphyctic (SPA) insult. This was prevented by hypothermia. We investigated whether the overexpression of NOS was accompanied by increased NOS activity. As nitric oxide (NO) is a potent activator of the soluble isoform of guanylyl cyclase, we measured striatal 3',5'-cyclic monophosphate (cyclic GMP) synthesis in 10-day-old (P10) rat pups that were subjected to SPA during normothermia or hypothermia during or after the insult. Cyclic GMP levels in striatal tissue from control pups were approximately 25.8 pmol/mg protein and in the SPA group approximately 38.1 pmol/mg protein (p<0.01). Hypothermia, during as well as after insult, prevented this increase of cyclic GMP. Nomega-nitro-L-arginine (L-NAME) (0.1 mM) decreased cyclic GMP levels in control, SPA and hypothermia treated pups to similar low levels (approximately 8% of level without L-NAME). Sodium nitroprusside (SNP) stimulated cyclic GMP showed no differences between the four groups. This indicates that high cyclic GMP levels in the striatum of rats subjected to SPA are caused by increased NOS activity. Hypothermia after an asphyctic insult could be a promising treatment.