Inhibitors of the activity of poly (ADP-ribose) synthetase reduce the cell death caused by hydrogen peroxide in human cardiac myoblasts

1. Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS). Inhibitors of PARS activity reduce the degree of reperfusion injury of the heart in vivo and in vitro. Here we investigate the role of PARS in the cell death of human cardiac myoblasts caused by hydrogen peroxide.
2. Exposure of human cardiac myoblasts to hydrogen peroxide caused a time- and concentration-dependent reduction in mitochondrial respiration (cell injury), an increase in cell death (LDH release), as well as an increase in PARS activity.
3. The PARS inhibitors 3-aminobenzamide (3 mM), 1,5-dehydroxyisoquinoline (300 μM) or nicotinamide (3 mM) attenuated the cell injury and death as well as the increase in PARS activity caused by hydrogen peroxide (3 mM; 4 h for cell injury/death, 60 min for PARS activity) in human cardiac myoblasts. In contrast, the inactive analogues 3-aminobenzoic acid (3 mM) or nicotinic acid (3 mM) were without effect.
4. The iron chelator deferoxamine (1–10 mM) caused a concentration-dependent reduction in the cell injury and death caused by hydrogen peroxide in these human cardiac myoblasts.
5. Thus, the cell injury/death caused by hydrogen peroxide in human cardiac myoblasts is secondary to the formation of hydroxyl radicals and due to an increase in PARS activity. We therefore propose that activation of PARS contributes to the cell injury/cell death associated with oxidant stress in the heart.

     

Comparison of antioxidant activities of aminoguanidine,methylguanidine and guanidine by luminol-enhanced chemiluminescence

1. The objective of this study was to investigate the ability of aminoguanidine, methylguanidine and guanidine to inhibit free radicals or metabolites generated by either stimulated human leucocytes or cell-free systems using luminol-enhanced chemiluminescence (CL).
2. Aminoguanidine (0.1 μM–10 mM), methylguanidine (10 μM–10 mM) and guanidine (10 μM–10 mM) produced concentration-dependent inhibition (96±0.1%, n=7, 59±1.3%, n=6, and 62±3%, n=6, P<0.05 at 10 mM, respectively) in FMLP-stimulated leucocytes CL.
3. In cell-free experiments, hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), hydroxyl radical and peroxynitrite-induced CL responses were initiated by hydrogen peroxide (3.5 mM), NaOCl (50 μM), FeSO₄ (40 nM) and peroxynitrite (20 nM), respectively. Aminoguanidine, methylguanidine and guanidine produced concentration-dependent inhibition in H₂O₂-(69±0.7%, n=7, 26±1%, n=6, and 15±0.5%, n=6, at 1 mM, respectively) and HOCl-(84±0.3%, n=6, 50±1%, n=6, and 29±1%, n=7, at 1 mM, respectively) induced luminol CL. Peroxynitrite-induced CL was markedly attenuated in a concentration-dependent manner by aminoguanidine (99±0.1%, n=6, at 10 mM), methylguanidine (5±0.2%, n=6, at 10 mM) and guanidine (27±0.4%, n=7, at 10 mM). However, inhibition with aminoguanidine was found to be more marked than with methylguanidine and guanidine. Aminoguanidine (95±0.5%, n=6, at 1 mM) and methylguanidine (25±1%, n=6, at 1 mM), but not guanidine (2±1%, n=6, at 1 mM), significantly decreased ferrous iron-induced CL.
4. Collectively, these data suggest that aminoguanidine and a high concentration (⩾0.1 mM) of methylguanidine have direct scavenging activities against H₂O₂, HOCl, hydroxyl radical and peroxynitrite. Guanidine, at a high concentration (⩾0.1 mM), scavenges H₂O₂, HOCl and peroxynitrite, but not the hydroxyl radical. These direct scavenging properties may contribute to inhibitory effects of these compounds on human leucocyte CL.

     

Metabotropic glutamate receptor subtypes mediating slow inward tail current (IADP) induction and inhibition of synaptic transmission in olfactory cortical neurones

1. The pharmacological features of the pre- and postsynaptic metabotropic glutamate receptors (mGluRs) present in the guinea-pig olfactory cortex, were examined in brain slices in vitro by use of a conventional intracellular current clamp/voltage clamp recording technique.
2. Bath-application of trans-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (50 μM) produced a sustained membrane depolarization, increase in cell excitability and induction of a post-stimulus inward (afterdepolarizing) tail current (I_ADP) (measured under ‘hybrid'' voltage clamp) similar to those evoked by the muscarinic receptor agonist oxotremorine-M (OXO-M, 2 μM).
3. L-Glutamate (0.25–1 mM, in the presence of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μM DL-amino-5-phosphono valeric acid (DL-APV)) or the broad spectrum mGluR agonists 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 μM), 1S,3S-ACPD (50 μM), ibotenate (Ibo; 25 μM, in the presence of 100 μM DL-APV), the selective mGluR I agonists (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG, 10 μM), (S)-3-hydroxyphenylglycine ((S)-3HPG, 50 μM), or quisqualate (10 μM, in the presence of 20 μM CNQX), but not the mGluR II agonist 2S,1′S,2′S-2-(2′-carboxycyclopropyl)-glycine (L-CCGI, 1 μM) or mGluR III agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 1 mM), were all effective in producing membrane depolarization and inducing a post-stimulus I_ADP. Unexpectedly, the proposed mGluR II-selective agonist (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)-glycine (DCG-IV, 10 μM, in the presence of 100 μM DL-APV) was also active.
4. The excitatory effects induced by 10 μM 1S,3R-ACPD were reversibly antagonized by the mGluR I/II antagonist (+)-α-methyl-4-carboxyphenylglycine ((+)-MCPG, 0.5–1 mM), as well as the selective mGluR I antagonists (S)-4-carboxyphenylglycine ((S)-4CPG) and (S)-4-carboxy-3-hydroxyphenyl glycine ((S)-4C3HPG) (both at 1 mM), but not the nonselective mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3, 1 mM) or the selective mGluR III antagonist (S)-α-methyl-L-AP4 (MAP4, 1 mM).
5. The excitatory postsynaptic potentials (e.p.s.ps), induced by single focal stimulation of cortical excitatory fibre tracts, were markedly reduced by 1S,3R-ACPD or L-AP4 (both at 10 μM), and by the selective mGluR II agonists (mGluR I antagonists) (S)-4CPG or (S)-4C3HPG (both at 1 mM) but not (S)-3,5-DHPG or (S)-3HPG (both at 100 μM).
6. The inhibitory effects of 1S-3R-ACPD, but not L-AP4, were reversibly blocked by (+)-MCPG (1 mM), whereas those produced by L-AP4, but not 1S,3R-ACPD, were blocked by the selective mGluR III antagonist MAP4 (1 mM).
7. It is concluded that a group I mGluR is most likely involved in mediating excitatory postsynaptic effects, whereas two distinct mGluRs (e.g. group II and III) might serve as presynaptic inhibitory autoreceptors in the guinea-pig olfactory cortex.

     

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery

1. The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Suramin (1 μM–1 mM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi-effective concentrations of α,β-methyleneATP (α,β-meATP) (5 μM), 2-methylthioATP (2-meSATP) (100 μM) and adenosine 5′-triphosphate (ATP) (1 mM) in a concentration-dependent manner. Responses to α,β-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.
3. Contractions evoked by uridine 5′-triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM).
4. Desensitization of the P2X₁-receptor by α,β-meATP abolished contractions evoked by 2-meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control.
5. Responses to α,β-meATP (5 μM) and 2-meSATP (100 μM) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response.
6. ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β-meATP (5 μM), 2-meSATP (100 μM) and ATP (1 mM) in a concentration-dependent manner.
7. These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X₁-receptors and G-protein-coupled P2Y-receptors.

     

Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA).
2. Contraction to noradrenaline (NA, 1 nM–1 μM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N^G-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml⁻¹).
3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquinazoline methanesulphonate) (0.1–10 μM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent.
4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM–3 μM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03–30 μM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue.
5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml⁻¹)-sensitive vasorelaxation to exogenous Cu/Zn SOD (200 u ml⁻¹) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml⁻¹).
6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.

     

Regulation of citrulline recycling in nitric oxide-dependent neurotransmission in the murine proximal colon

1. We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis.
2. Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 μM). Electrical field stimulation (EFS, 0.3–20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component).
3. L-Nitro-arginine-methyl ester (L-NAME, 100 μM) selectively abolished the slow component of i.j.ps. The effects of L-NAME were reversed by L-arginine (0.2–2 mM) but not by D-arginine (2 mM). Sodium nitroprusside (an NO donor, 1 μM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps.
4. L-Citrulline (0.2 mM) also reversed the effects of L-NAME, and this action was maintained during sustained exposures to L-citrulline (0.2 mM). This may reflect intraneuronal recycling of L-citrulline to L-arginine.
5. Higher concentrations of L-citrulline (e.g. 2 mM) had time-dependent effects. Brief exposure (15 min) reversed the effects of L-NAME, but during longer exposures (30 min) the effects of L-NAME gradually returned. In the continued presence of L-citrulline, L-arginine (2 mM) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of L-citrulline, the ability to generate arginine from citrulline was reduced.
6. Aspartate (2 mM) had no effect on i.j.ps, the effects of L-NAME, or the actions of L-citrulline in the presence of L-NAME. L-Citrulline (0.2–2 mM) alone had no effect on i.j.ps under control conditions.
7. S-methyl-L-thiocitrulline (10 μM), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by L-arginine (2 mM), but not by L-citrulline (2 mM).
8. These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of L-citrulline to L-arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of L-citrulline are elevated.

     

Loss of endothelium-derived nitric oxide in rabbit aorta by oxidant stress: restoration by superoxide dismutase mimetics

1. Structurally distinct superoxide dismutase (SOD) mimetics were examined for their ability to protect nitric oxide (NO) from destruction by oxidant stress in rabbit aorta.
2. These were the spin traps, PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide), tempol (4-hydroxy 2,2,6,6,-tetramethylpiperidine-1-oxyl) and tiron (4,5-dihydroxy-1,3-benzene-disulphonic acid), the metal salts, CuSO₄ and MnCl₂, and the metal-based agents CuDIPS (Cu (II)-[diisopropylsalicylate]₂) and MnTMPyP (Mn (III) tetrakis [1-methyl-4-pyridyl]porphyrin).
3. Oxidant stress was generated in isolated aortic rings by inactivating endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA; 60 min) either alone at 3 mM or at 0.3 mM in combination with superoxide generation using xanthine oxidase (XO; 4.8 mu ml⁻¹) and hypoxanthine (HX; 0.1 mM).
4. Acetylcholine (ACh)-induced relaxation was inhibited by DETCA (3 mM, 60 min) and was not restored by exogenous SOD (250 u ml⁻¹), suggesting the oxidant stress was intracellular. MnTMPyP (600 μM and 1 mM) and MnCl₂ (100 μM) were the only agents to reverse the blockade of ACh-induced relaxation.
5. Addition of XO/HX to DETCA (0.3 mM)-treated tissues powerfully impaired ACh-induced relaxation and exogneous SOD (250 u ml⁻¹) fully reversed the blockade, suggesting the oxidant stress was extracellular. CuDIPS (0.1–3 μM), CuSO₄ (0.3–3 μM), MnCl₂ (1–100 μM) and MnTMPyP (100–600 μM) also reversed blockade powerfully, tempol (30 μM–1 mM) and tiron (0.3–10 mM) reversed blockade weakly and PTIYO (10–300 μM) enhanced the blockade.
6. Thus, MnTMPyP was the only SOD mimetic to restore NO-dependent relaxation in conditions of both extracellular and intracellular oxidant stress. This agent may, therefore, provide a lead in the development of SOD mimetics for the treatment of pathologies associated with oxidant stress.

     

Presynaptic and postsynaptic actions of halothane at glutamatergic synapses in the mouse hippocampus

1. Whole-cell patch-clamp recordings in adult mouse hippocampal slices were used to test the mechanism by which the volatile anesthetic halothane inhibits glutamate receptor-mediated synaptic transmission. Non-N-methyl-D-aspartate (nonNMDA) and NMDA receptor-mediated currents in CA1 pyramidal cells were pharmacologically isolated by bath application of D,L-2-amino-5-phosphonovaleric acid (APV; 100 μM) or 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 5 μM), respectively.
2. Halothane blocked both nonNMDA and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) to a similar extent (IC₅₀ values of 0.66 and 0.57 mM, respectively).
3. Partial blockade of the EPSCs by lowering the extracellular concentration of calcium ([Ca²⁺]_o), but not by application of CNQX (1 μM), was accompanied by an increase in paired-pulse facilitation (PPF). Halothane-induced blockade of the EPSCs also was associated with an increase in PPF.
4. The effects of halothane on α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA receptor-mediated currents induced by agonist iontophoresis, were compared. AMPA-induced currents were blocked with an IC₅₀ of 1.7 mM. NMDA-induced currents were significantly less sensitive to halothane (IC₅₀ of 5.9 mM).
5. The effect of halothane on iontophoretic AMPA dose-response curves was tested. Halothane suppressed the maximal response to AMPA without affecting its EC₅₀, suggesting a noncompetitive mechanism of inhibition.
6. All effects of halothane were reversible upon termination of the exposure to the drug.
7. These data suggest that halothane blocks central glutamatergic synaptic transmission by presynaptically inhibiting glutamate release and postsynaptically blocking the AMPA subtype of glutamate receptors.

     

Nitric oxide-related cyclic GMP-independent relaxing effect of N-acetylcysteine in lipopolysaccharide-treated rat aorta

1. We have recently demonstrated the formation of protein-bound dinitrosyl-iron complexes (DNIC) in rat aortic rings exposed to lipopolysaccharide (LPS) and shown that N-acetylcysteine (NAC) can promote vasorelaxation in these arteries, possibly via the release of nitric oxide (NO) as low molecular weight DNIC from these storage sites. The aim of the present study was to investigate further the mechanism of the relaxation induced by NAC in LPS-treated vessels.
2. In rings incubated with LPS (10 μg ml⁻¹ for 18 h) and precontracted with noradrenaline (NA, 3 μM) plus N^ω-nitro-L-arginine methylester (L-NAME, 3 mM), the relaxation evoked by NAC (0.1 to 10 mM) was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 μM, a selective inhibitor of soluble guanylyl cyclase) but not affected by Rp-8-bromoguanosine 3′5′-cyclic monophosphorothioate (Rp-8BrcGMPS, 60 μM a selective inhibitor of cyclic GMP-dependent protein kinase). Tetrabutylammonium (TBA, 3 mM, as a non selective K⁺ channels blocker) or elevated concentration of external KCl (25 or 50 mM) significantly attenuated the NAC-induced relaxation. Selective K⁺ channels blockers (10 μM glibenclamide, 0.1 μM charybdotoxin, 0.5 μM apamin or 3 mM 4-aminopyridine) did not affect the NAC-induced relaxation. The relaxing effect of NAC (10 mM) was not associated with an elevation of guanosine 3′ : 5′ cyclic monophosphate (cyclic GMP) in LPS-treated rings.
3. In aortic rings precontracted with NA (0.1 μM), low molecular weight DNIC (with thiosulphate as ligand, 1 nM to 10 μM) evoked a concentration-dependent relaxation which was antagonized by ODQ (1 μM) and Rp-8BrcGMPS (150 μM) but not significantly affected by TBA (3 mM) or by the use of KCl (50 mM) as preconstricting agent. The relaxation produced by DNIC (0.1 μM) was associated with an 11 fold increase in aortic cyclic GMP content, which was completely abolished by ODQ (1 μM).
4. Taken together with our previous data, the main finding of the present study is that the vascular relaxation induced by NAC in LPS-treated aorta, although probably related to NO through an interaction via preformed NO stores, was not mediated by activation of the cyclic GMP pathway. It may involve the activation of TBA-sensitive K⁺ channels. The differences in the mechanism of relaxation induced by NAC and by exogenous DNIC suggest that the generation of low molecular weight DNIC from protein-bound species does not play a major role in the NAC-induced relaxation observed in LPS-treated rat aorta. In addition, it is suggested that ODQ may display other properties than the inhibition of soluble guanylyl cyclase.

     

Recovery by ascorbate of impaired nitric oxide-dependent relaxation resulting from oxidant stress in rat aorta

1. In this study we investigated the ability of ascorbate to protect nitric oxide from destruction by superoxide anion.
2. Ascorbate produced concentration-dependent relaxation of rings of rat aorta, comprising two components: the first, seen at 1–300 μM, reached a maximum of 45.3±2.8%, and was abolished by endothelial removal or treatment with L-NAME (100 μM), demonstrating involvement of nitric oxide. The second occurred at concentrations of 1 mM and above and was associated with falls in the pH of the bathing fluid.
3. Pretreatment with ascorbate at concentrations up to 3 mM had no effect on the relaxation to acetylcholine (10 nM–10 μM) on endothelium-containing rings or adenosine (0.1 μM–3 mM) on endothelium-denuded rings.
4. An oxidant stress was applied to aortic rings, comprising inhibition of endogenous Cu/Zn superoxide dismutase by diethyldithiocarbamate (0.1 mM) followed by generation of superoxide anion by hypoxanthine (0.1 mM/xanthine oxidase (16 u ml⁻¹). This reduced maximal acetylcholine-induced relaxation from 96.7±1.3% to 42.4±3.5% (P<0.001). Treatment with ascorbate (30 μM–3 mM) reversed this blockade in a concentration-dependent manner.
5. Our findings show that ascorbate has the ability to protect nitric oxide from destruction by superoxide anion. This action is seen with ascorbate at levels normally present in plasma, suggesting that this antioxidant may exert a tonic protective effect on nitric oxide within the vasculature.

     

Role of ATP in fast excitatory synaptic potentials in locus coeruleus neurones of the rat

1. Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). The pressure application of α,β-methylene ATP (α,β-meATP) caused reproducible depolarizations which were depressed by suramin (30 μM) and abolished by suramin (100 μM). Pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10, 30 μM) also concentration-dependently inhibited the α,β-meATP-induced depolarization, although with a much slower time-course than suramin. Almost complete inhibition developed with 30 μM PPADS. Reactive blue 2 (30 μM) did not alter the effect of α,β-meATP, while reactive blue 2 (100 μM) slightly depressed it.
2. Pressure-applied (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also depolarized LC neurones. Kynurenic acid (500 μM) depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM) abolished the response to AMPA. Suramin (100 μM) potentiated the AMPA effect.
3. Pressure-applied noradrenaline hyperpolarized LC neurones. Suramin (100 μM) did not alter the effect of noradrenaline.
4. Focal electrical stimulation evoked biphasic synaptic potentials consisting of a fast depolarization (p.s.p.) followed by a slow hyperpolarization (i.p.s.p.). A mixture of D(−)-2-amino-5-phosphonopentanoic acid (AP-5; 50 μM), CNQX (50 μM) and picrotoxin (100 μM) depressed both the p.s.p. and the i.p.s.p. Under these conditions suramin (100 μM) markedly inhibited the p.s.p., but did not alter the i.p.s.p. In the combined presence of AP-5 (50 μM), CNQX (50 μM), picrotoxin (100 μM), strychnine (0.1 μM), tropisetron (0.5 μM) and hexamethonium (100 μM), a high concentration of suramin (300 μM) almost abolished the p.s.p. without changing the i.p.s.p.
5. In the presence of kynurenic acid (500 μM) and picrotoxin (100 μM), PPADS (30 μM) depressed the p.s.p. Moreover, the application of suramin (100 μM) to the PPADS (30 μM)-containing medium failed to cause any further inhibition. Neither PPADS (30 μM) nor suramin (100 μM) altered the i.p.s.p.
6. It was concluded that the cell somata of LC neurones are endowed with excitatory P2-purinoceptors. ATP may be released either as the sole transmitter from purinergic neurones terminating at the LC or as a co-transmitter of noradrenaline from recurrent axon collaterals or dendrites of the LC neurones themselves.

     

Interaction between Ca2+, K+, carbamazepine and zonisamide on hippocampal extracellular glutamate monitored with a microdialysis electrode

1. Multiple components of hippocampal glutamate release were examined by study of Ca²⁺- and K⁺-evoked hippocampal extracellular glutamate release using an in vivo microdialysis glutamate biosensor in urethane-anaesthetized rats. In addition, the effects of the antiepileptic drugs, carbamazepine (CBZ) and zonisamide (ZNS) perfused through the probe on glutamate release were assessed.
2. Basal glutamate levels were below detection limits (∼0.1 μM). An increase in extracellular KCl (from 2.7 to 50 and 100 mM) increased extracellular hippocampal glutamate levels to 9.2±1.4 and 20.0±2.6 μM, respectively, calculated from the area under curve (AUC) for 60 min.
3. This KCl-evoked glutamate release consisted of three components: an initial transient rise, a late gentle rise, and late multiple phasic transient rises.
4. An increase in or removal of extracellular CaCl₂ levels respectively enhanced and reduced the 50 mM KCl-evoked hippocampal glutamate release (AUC for 60 min) from 9.2±1.4 to 12.4±2.1 and 5.8±0.9 μM.
5. Perfusion with 100 μM CBZ or 1 mM ZNS inhibited both the 50 mM KCl-evoked hippocampal glutamate release (AUC for 60 min) from 9.2±1.4 to 5.5±1.1 and to 5.8±1.3 μM, respectively, as well as the stimulatory effects of Ca²⁺ on KCl-evoked hippocampal glutamate release.
6. These results suggest that both CBZ and ZNS may reduce epileptiform events by inhibiting excitatory glutamatergic transmission.

     

Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells

1. Nucleotide-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml⁻¹) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. Most experiments were carried out on non-proliferating microglial cells. ATP (100 nM–1 mM), ADP (10 nM–10 mM) and UTP (1 μM–100 mM), but not uridine (100 μM–10 mM) produced a slow outward current at a holding potential of 0 mV. The effect of UTP (1 mM) did not depend on the presence of extracellular Mg²⁺ (1 mM). The outward current response to UTP (1 mM) was similar in non-proliferating and proliferating microglia.
2. In non-proliferating microglial cells, the ATP (10 μM)-induced outward current was antagonized by suramin (300 μM) or reactive blue 2 (50 μM), whereas 8-(p-sulphophenyl)-theophylline (8-SPT; 100 μM) was inactive. By contrast, the current induced by UTP (1 mM) was increased by suramin (300 μM) and was not altered by reactive blue 2 (50 μM) or 8-SPT (100 μM).
3. The current response to UTP (1 mM) disappeared when K⁺ was replaced in the pipette solution by an equimolar concentration of Cs⁺ (150 mM). However, the effect of UTP (1 mM) did not change when most Cl⁻ was replaced with an equimolar concentration of gluconate⁻ (145 mM). The application of 4-aminopyridine (1 mM) or Cs⁺ (1 mM) to the bath solution failed to alter the UTP (1 mM)-induced current. UTP (1 mM) had almost no effect in a nominally Ca²⁺-free bath medium, or in the presence of charybdotoxin (0.1 μM); the inclusion of U-73122 (5 μM) or heparin (5 mg ml⁻¹) into the pipette solution also blocked the responses to UTP (1 mM). By contrast, the effect of ATP (10 μM) persisted under these conditions.
4. I-V relations were determined by delivering fast voltage ramps before and during the application of UTP (1 mM). In the presence of extracellular Cs⁺ (1 mM) and 4-aminopyridine (1 mM) the UTP-evoked current crossed the zero current level near−75 mV. Omission of Ca²⁺ from the Cs⁺ (1 mM)- and 4-aminopyridine (1 mM)-containing bath medium or replacement of K⁺ by Cs⁺ (150 mM) in the pipette solution abolished the UTP current.
5. Replacement of GTP (200 μM) by GDP-β-S (200 μM) in the pipette solution abolished the current evoked by UTP (1 mM).
6. When the pipette solution contained Cs⁺ (150 mM) instead of K⁺ and in addition inositol 1,4,5,-trisphosphate (InsP₃; 10 μM), an inward current absolutely dependent on extracellular Ca²⁺ was activated after the establishment of whole-cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100 mV, as measured by fast voltage ramps.
7. A rise of the internal free Ca²⁺ concentration from 0.01 to 0.5 μM on excised inside-out membrane patches produced single channel activity with a reversal potential of 0 mV in a symmetrical K⁺ solution. The reversal potential was shifted to negative values, when the extracellular K⁺ concentration was decreased from 144 to 32 mM. By contrast, a decrease of the extracellular Cl⁻ concentration from 164 to 38 mM did not change the reversal potential.
8. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP₃. The depletion of the intracellular Ca²⁺ pool appears to initiate a capacitative entry of Ca⁺ from the extracellular space. This Ca²⁺ then activates a Ca²⁺-dependent K⁺ current.

     

Corrigendum

1. Epidemiological and experimental data have shown that homocysteine may provoke vascular lesions and that moderate homocysteinaemia may constitute an independent risk factor for vascular disease. It is now documented that homocysteine damages human endothelial cells in culture, possibly by producing hydrogen peroxide in an oxygen-dependent reaction.
2. In this study, we have examined the direct effect of this sulphur amino acid on pancreatic vascular resistance. Experiments were performed on the vascular bed of the rat isolated pancreas perfused at constant pressure; thus, any change in pancreatic vascular resistance resulted in a change in the flow rate. D,L-Homocysteine perfused for one hour at three different concentrations (200 μM, 2 mM, 20 mM) did not induce any significant change in the flow rate per se. Homocysteine infusion for 30 min at a concentration of 200 μM or 2 mM abolished the endothelium-dependent vasodilatation induced by acetylcholine (0.05 μM), but did not modify adenosine (1.5 μM)-induced vasodilatation.
3. The effect of D,L-homocysteine (200 μM or 2 mM) cannot be ascribed to a direct antimuscarinic effect since 30 min pretreatment of rat ileum with these concentrations did not significantly change the contractile effect of increasing concentrations of acetylcholine (0.015–15 μM).
4. Preincubation of human umbilical vein endothelial cells with D,L-homocysteine (0.2–5.0 mM) had no significant effect on overall cell number or viability during 18 h of incubation; the endothelial cells exposed to concentrations up to 5 mM exhibited a spindle-shaped, whirled pattern. This pattern was reversed 48 h after the removal of homocysteine. A cytotoxic effect was seen after 18 h incubation in 10 mM D,L-homocysteine.
5. In conclusion, an acute infusion of homocysteine altered acetylcholine endothelium-induced vasodilatation, whereas the adenosine vasodilatator effect was insensitive to the deleterious action of homocysteine in vitro.

     

Depolarization counteracts glucocorticoid inhibition of adenohypophysical corticotroph cells

1. In AtT20 mouse corticotroph tumour cells large conductance Ca²⁺-activated K⁺-channels (BK-channels) have an essential role in the early glucocorticoid inhibition of adrenocorticotrophin (ACTH) secretion evoked by corticotrophin-releasing factor. The present study examined whether or not BK-channels are also pivotal to glucocorticoid inhibition of normal rat anterior pituitary cells.
2. A membrane-permeant, non-metabolizable cyclic AMP analogue, 8-(4-Chlorophenylthio)adenosine-3′,5′-cyclic-monophosphate (CPT-cAMP) was used as the primary secretagogue stimulus, as this mimics the increase of intracellular cyclic AMP caused by corticotrophin-releasing factor, but is not subject to the complex Ca²⁺-dependent regulation of cyclic AMP metabolism that is evident in corticotroph cells.
3. Experiments in AtT20 cells showed that ACTH secretion stimulated by 1 mM CPT-cAMP was suppressed to 34±1.5% (n=12) of the control stimulus by a maximal dose of 100 nM dexamethasone. The ACTH secretion evoked by the combination of 1 mM CPT-cAMP with either 5 μM (−)BayK8644 (L-type Ca²⁺-channel activator) or 5 mM TEA (K⁺-channel blocker) was respectively 69.1±7.6% and 69.3±11.8% of control after 2 h preincubation with 100 nM dexamethasone (P<0.05 vs CPT-cAMP). The ACTH response elicited by 5 μM (−)BayK8644 and 5 mM TEA given together was completely resistant to inhibition by 100 nM dexamethasone. Furthermore, TEA and (−)BayK8644 given together synergistically stimulated ACTH release in combination with 0.1 mM or 1 mM CPT-cAMP, and these ACTH responses were not inhibited by 100 nM dexamethasone.
4. In primary cultures of rat anterior pituitary cells, TEA (up to 20 mM), charybdotoxin (30 nM) or apamin (100 nM) failed to modify the glucocorticoid inhibition of 0.1 mM CPT-cAMP-induced ACTH release. The combination of 5 mM TEA and 5 μM (−)BayK8644 elicited a small but significant increase in ACTH secretion but did not modify the inhibition of 0.3 mM CPT-cAMP-induced ACTH secretion by 100 nM dexamethasone.
5. In primary cultures of rat anterior pituitary cells, depolarization of the membrane potential with 40 mM KCl enhanced the ACTH response to CPT-cAMP and markedly reduced the maximal inhibitory effect of dexamethasone to 55±1.2% as well as that of corticosterone to 33±2.1% vs 100±2.5% and 100±1.9% inhibition respectively, when 0.1 mM CPT-cAMP was used alone. Introduction of 5 μM (−)BayK8644 with 40 mM KCl in this system had no additional effect on glucocorticoid inhibition.
6. No glucocorticoid inhibition of ACTH release to any of the stimuli applied was observed in cells pretreated with the mRNA synthesis inhibitor, 5,6-dichloro-furanosyl-benzimidazole riboside (DRB) (0.1 mM) or the protein synthesis blocker, puromycin (0.1 mM).
7. In summary, early glucocorticoid inhibition of stimulated ACTH release by cultured rat anterior pituitary cells was dependent on the synthesis of new mRNA and protein. Depolarization of the membrane potential potentiated CPT-cAMP-induced ACTH secretion in AtT20 cells as well as cultured rat corticotrophs and this was associated with a resistance to the early inhibitory effect of glucocorticoids. Glucocorticoid inhibition in rat anterior pituitary corticotrophs was unaltered by TEA, charybdotoxin as well as apamin, and hence it is unlikely to involve predominantly BK-or SK-type Ca²⁺-activated K⁺-channels.
8. These results support the thesis that a prime target of glucocorticoid feedback inhibition in anterior pituitary corticotrophs is the membrane potential and indicate that glucocorticoid-induced proteins regulate the activities of several distinct plasma membrane ion channels.

     

Effects of the Aconitum alkaloid mesaconitine in rat hippocampal slices and the involvement of α- and β-adrenoceptors

1. The effects of mesaconitine, the main alkaloid contained in Aconiti tuber, were investigated by use of extracellular recordings of stimulus-evoked population spikes and field excitatory postsynaptic potentials (e.p.s.ps) in the CA1 region of rat hippocampal slices.
2. At a concentration of 10 nM, mesaconitine evoked excitations, which were manifested as an increase in the amplitude of the orthodromic spike and the appearance of multiple spikes following the first postsynaptic spike, without affecting the magnitude of paired-pulse facilitation. The increase in spike amplitude was persistent and was not reversed by up to 90 min of washout. At concentrations of 30 and 100 nM, the alkaloid produced a biphasic effect, that is an excitation followed by an inhibition without having any effect upon the field e.p.s.p. At concentrations above 100 nM, mesaconitine suppressed the orthodromic population spike and the field e.p.s.p.
3. The excitatory effect was also observed when electrical stimulation was stopped completely during the application of mesaconitine (10 nM) and during the first 15 min of washout.
4. The enhancement of the population spike and the appearance of multiple spikes induced by mesaconitine (10–100 nM) were blocked by pretreatment with the β-adrenoceptor antagonists propranolol (1 μM) and timolol (1 μM), whereas the inhibitory effect was blocked by the α-adrenoceptor antagonists yohimbine (1 μM) and phentolamine (10 μM). However, when the β-adrenoceptor antagonist timolol was added 10 min after the application of mesaconitine, it failed to block the long-lasting enhancement of the spike amplitude and the appearance of multiple population spikes.
5. Application of the selective β-adrenoceptor agonist isoprenaline (500 nM) to the hippocampal slices induced an increase in the amplitude of the orthodromic population spike and elicited 2–3 additional spikes. Mesaconitine (10 nM) did not further potentiate this enhancement of the spike amplitude when added after a 15 min pretreatment with isoprenaline.
6. Perfusion of forskolin, which directly activates adenylate cyclase, enhanced the population spike. Mesaconitine had no additional effect when applied after pretreatment with forskolin.
7. It is concluded that the excitatory effects evoked by lower concentrations of the plant alkaloid mesaconitine are mediated by stimulation of β-adrenoceptors and the consequent activation of intracellular processes which lead to the long-lasting changes in excitability.

     

Potassium channel activation and relaxation by nicorandil in rat small mesenteric arteries

1. We used whole-cell patch clamp to investigate the currents activated by nicorandil in smooth muscle cells isolated from rat small mesenteric arteries, and studied the relaxant effect of nicorandil using myography.
2. Nicorandil (300 μM) activated currents with near-linear current-voltage relationships and reversal potentials near to the equilibrium potential for K⁺.
3. The nicorandil-activated current was blocked by glibenclamide (10 μM), but unaffected by iberiotoxin (100 nM) and the guanylyl cyclase inhibitor LY 83583 (1 μM). During current activation by nicorandil, openings of channels with a unitary conductance of 31 pS were detected.
4. One hundred μM nicorandil had no effect on currents through Ca²⁺ channels recorded in response to depolarizing voltage steps using 10 mM Ba²⁺ as a charge carrier. A small reduction in current amplitude was seen in 300 μM nicorandil, though this was not statistically significant.
5. In arterial rings contracted with 20 mM K⁺ Krebs solution containing 200 nM BAYK 8644, nicorandil produced a concentration-dependent relaxation with mean pD₂=4.77±0.06. Glibenclamide (10 μM) shifted the curve to the right (pD₂=4.32±0.05), as did 60 mM K⁺. LY 83583 caused a dose-dependent inhibition of the relaxant effect of nicorandil, while LY 83583 and glibenclamide together produced greater inhibition than either alone.
6. Metabolic inhibition with carbonyl cyanide m-chlorophenyl hydrazone (30 nM), or by reduction of extracellular glucose to 0.5 mM, increased the potency of nicorandil.
7. We conclude that nicorandil activates K_ATP channels in these vessels and also acts through guanylyl cyclase to cause vasorelaxation, and that the potency of nicorandil is increased during metabolic inhibition.

     

Insulin-releasing action of the novel antidiabetic agent BTS 67 582

1. BTS 67 582 (1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate) is a novel antidiabetic agent with a short-acting insulin-releasing effect. This study examined its mode of action in the clonal B-cell line BRIN-BD11.
2. BTS 67 582 increased insulin release from BRIN-BD11 cells in a concentration-dependent manner (10⁻⁸ to 10⁻⁴ M) at both non-stimulating (1.1 mM) and stimulating (16.7 mM) concentrations of glucose.
3. BTS 67 582 (10⁻⁴ M) potentiated the insulin-releasing effect of a depolarizing concentration of K⁺ (30 mM), whereas the K⁺ channel openers pinacidil (400 μM) and diazoxide (300 μM) inhibited BTS 67 582-induced release.
4. Suppression of Ca⁺ channel activity with verapamil (20 μM) reduced the insulin-releasing effect of BTS 67 582 (10⁻⁴ M).
5. BTS 67 582 (10⁻⁴ M) potentiated insulin release induced by amino acids (10 mM), and enhanced the combined stimulant effects of glucose plus either the fatty acid palmitate (10 mM), or agents which raise intracellular cyclic AMP concentrations (25 μM forskolin and 1 mM isobutylmethylxanthine), or the cholinoceptor agonist carbachol (100 μM).
6. Inhibition of glucose-stimulated insulin release by adrenaline or noradrenaline (10 μM) was partially reversed by BTS 67 582 (10⁻⁴ M).
7. These data suggest that the insulin-releasing effect of BTS 67 582 involves regulation of ATP-sensitive K⁺ channel activity and Ca²⁺ influx, and that the drug augments the stimulant effects of nutrient insulin secretagogues and agents which enhance adenylate cyclase and phospholipase C. BTS 67 582 may also exert insulin-releasing effects independently of ATP-sensitive K⁺ channel activity.

     

Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage-sensitive calcium channels

1. Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[³H]-arginine to L-[³H]-citrulline.
2. Elevation of extracellular K⁺ to 20, 35 or 60 mM concentration-dependently augmented L-[³H]-citrulline production. The response to 35 mM KCl was abolished by N^G-nitro-L-arginine (100 μM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl₂ concentration up to 10 mM also prevented the K⁺ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity.
3. However, the effect of high K⁺ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2-3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine.
4. The 35 mM K⁺ response was insensitive to ω-conotoxin GVIA (1 μM) and nifedipine (100 μM), but could be prevented in part by ω-agatoxin IVA (0.1 and 1 μM). The inhibition caused by 0.1 μM ω-agatoxin IVA (∼30%) was enhanced by adding ω-conotoxin GVIA (1 μM) or nifedipine (100 μM). Further inhibition (up to above 70%) could be observed when the three Ca²⁺ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 μM) the K⁺-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 μM ω-conotoxin GVIA plus 100 μM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX.
5. It was concluded that elevation of extracellular [K⁺] causes NO synthase activation by external Ca⁺ entering cells mainly through channels of the P/Q-type. Other Ca²⁺ channels (L- and N-type) appear to contribute when P/Q-channels are blocked.

     

The spasmogenic effects of vanadate in human isolated bronchus

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 μM–3 mM) produced concentration-dependent, well-sustained contraction. Its −logEC₅₀ was 3.74±0.05 (mean±s.e.mean) and its maximal effect was equivalent to 97.5±4.2% of the response to acetylcholine (ACh, 1 mM).
2. Vanadate (200 μM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 μM), zileuton (10 μM), a mixture of atropine, mepyramine and phentolamine (each at 1 μM), or by mast cell degranulation with compound 48/80.
3. Vanadate (200 μM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 μM) or to a Ca²⁺-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 μM) in Ca²⁺-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca²⁺-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca²⁺ stores. In such tissues cyclopiazonic acid (CPA; 10 μM) prevented Ca²⁺-induced recovery of vanadate-induced contraction.
4. Tissue incubation in K⁺-rich (80 mM) PSS, K⁺-free PSS, or PSS containing ouabain (10 μM) did not alter vanadate (200 μM)-induced contraction. Ouabain (10 μM) abolished the K⁺-induced relaxation of human bronchus bathed in K⁺-free PSS. This action was not shared by vanadate (200 μM). The tissue content of Na⁺ was increased and the tissue content of K⁺ was decreased by ouabain (10 μM). In contrast, vanadate (200 μM) did not alter the tissue content of these ions. Tissue incubation in a Na⁺-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 μM).
5. Vanadate (200 μM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 μM), staurosporine (1 μM) and calphostin C (1 μM). Genistein (100 μM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate.
6. Vanadate (0.1–3 mM) and ACh (1 μM–3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca²⁺-free medium either alone or in combination with ryanodine (10 μM).
7. In human cultured tracheal smooth muscle cells, histamine (100 μM) and vanadate (200 μM) each produced a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i).
8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 μM) in human bronchus was associated with cellular depolarization.
9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca²⁺ from an intracellular store. The Ca²⁺ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca²⁺]_i that are close to basal.