Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A₁ receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12–14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe.p.s.p.) recorded extracellularly from the stratum radiatum in the CA1 area.
2. The concentration-response curve for the inhibitory effects of the selective adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA; 2–50 nM), on the fe.p.s.p. slope (EC₅₀=12.5 (9.2–17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 μM) (EC₅₀=27.2 (21.4–34.5) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 μM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM). DHPG (10 μM) itself had an inhibitory effect of 20.1±1.9% (n=4) on the fe.p.s.p. slope.
3. The concentration-response curves for the inhibitory effects of CPA (2–20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG-I; 1 μM), or in the presence of the non-selective mGluR agonist (which activates both group I and II mGluR), (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate (ACPD; 100 μM). L-CCG-I had no consistent effects and ACPD (100 μM) decreased by 19.4±1.8% (n=4) the fe.p.s.p. slope.
4. The concentration-response curve for the inhibitory effects of CPA (2–100 nM) on the fe.p.s.p. slope (EC₅₀=8.2 (6.9–9.6) nM) was displaced to the right by the group III mGluR selective agonist, L-2-amino-4-phosphonobutyrate (L-AP4; 25 μM) (EC₅₀=17.7 (13.1–21.9) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by L-AP4 (25 μM) was blocked in the presence of the mGluR antagonist (selective for the group III mGluR), (R,S)-α-methyl-4-phosphonophenylglycine (MPPG; 200 μM).
5. Both the direct effect of DHPG on synaptic transmission and the attenuation of the inhibitory effect of CPA (10 nM) were prevented in the presence of the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C.
6. The attenuation by L-AP4 (25 μM) of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope was also prevented by the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C. But this effect seemed to be distinct from the direct effect of L-AP4 (25 μM) on synaptic transmission, which was not modified by the protein kinase C selective inhibitors.
7. We conclude that agonists of metabotropic glutamate receptors (Groups I and III) are able to attenuate the inhibitory effects of adenosine A₁ receptor activation in the hippocampus. This interaction may have pathophysiological relevance in hypoxia, in which there is marked release of both excitatory amino acids and the important endogenous neuroprotective substance, adenosine.

     

Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones

1. The interactions between N-methyl-D-aspartate (NMDA) and metabotropic glutamate receptors (mGluRs) were investigated in striatal slices, by utilizing intracellular recordings, both in current- and voltage-clamp mode.
2. Bath-application (50 μM) or focal application of NMDA induced a transient membrane depolarization, while in the voltage-clamp mode, NMDA (50 μM) caused a transient inward current. Following bath-application of the non-selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 μM), NMDA responses were reversibly potentiated both in current (197±15% of control) and voltage-clamp experiments (200±18% of control).
3. Bath-application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG, 10–300 μM) resulted in a dose-dependent potentiation of NMDA-induced membrane depolarization (up to 400±33% of control). This potentiation was either prevented by preincubation with (RS)-α-methyl-4-carboxyphenylglycine (RS-α-MCPG, 300 μM), or blocked when applied immediately after 3,5-DHPG wash-out.
4. Neither (2S,1′S,2′S)2-(2′-carboxycyclopropyl)glycine (L-CCG I, up to 100 μM) nor (2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV, 1 μM), agonists for group II mGluRs caused any change in NMDA responses. Likewise, L-serine-O-phosphate (L-SOP, 30 μM), agonist for group III mGluRs, did not affect the NMDA-induced depolarization.
5. The enhancement of the NMDA responses was mimicked by phorbol-12,13-diacetate (PDAc, 1 μM) which activates protein kinase C (PKC). The 3,5-DHPG-mediated potentiation of the NMDA-induced depolarization was prevented by preincubation with staurosporine (100 nM) or calphostin C (1 μM), antagonists of PKC.
6. Electrophysiological responses to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor activation were not affected by agonists for the three-classes of mGluRs.
7. The present data suggest that group I mGluRs exert a positive modulatory action on NMDA responses, probably through activation of PKC. This functional interaction in the striatum appears of crucial importance in the understanding of physiological and pathological events, such as synaptic plasticity and neuronal death, respectively.

     

Effects of L-NG-nitro-arginine on noradrenaline induced contraction in the rat anococcygeus muscle

1. The influence of L-N^G-nitro-arginine (L-NOARG, 30 μM) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.
2. Noradrenaline (0.1–100 μM) contracted the muscle in a concentration-dependent manner. L-NOARG (30 μM) had no effect on noradrenaline responses.
3. Phenoxybenzamine (Pbz 0.1 μM) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC₅₀ control: 6.92±0.09; pEC₅₀ Pbz: 5.30±0.10; n=20).
4. The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.
5. Contractile responses to noradrenaline after partial α₁-adrenoceptor alkylation with phenoxybenzamine (0.1 μM) were clearly enhanced by L-NOARG.
6. The potentiating effect of L-NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μM) L-arginine but not by (100 μM) D-arginine.
7. These results indicate that spontaneous release of NO by nitrergic nerves can influence the α₁-adrenoceptor-mediated response to exogenous noradrenaline.

     

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.

     

The interaction of diadenosine polyphosphates with P2X-receptors in the guinea-pig isolated vas deferens

1. The site(s) at which diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP₄A) and diadenosine 5′, 5′′′-P¹,P⁵-pentaphosphate (AP₅A) act to evoke contraction of the guinea-pig isolated vas deferens was studied by use of a series of P₂-receptor antagonists and the ecto-ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (300 nM–30 μM), suramin (3–100 μM) and pyridoxal-5′-phosphate (P-5-P) (3–1000 μM) inhibited contractions evoked by equi-effective concentrations of AP₅A (3 μM), AP₄A (30 μM) and α,β-methyleneATP (α,β-meATP) (1 μM), in a concentration-dependent manner and abolished them at the highest concentrations used.
3. PPADS was more potent than suramin, which in turn was more potent than P-5-P. PPADS inhibited AP₅A, AP₄A and α,β-meATP with similar IC₅₀ values. No significant difference was found between IC₅₀ values for suramin against α,β-meATP and AP₅A or α,β-meATP and AP₄A, but suramin was more than 2.5 times more potent against AP₄A than AP₅A. P-5-P showed the same pattern of antagonism.
4. Desensitization of the P_2X1-receptor by α,β-meATP abolished contractions evoked by AP₅A (3 μM) and AP₄A (30 μM), but had no effect on those elicited by noradrenaline (100 μM).
5. ARL 67156 (100 μM) reversibly potentiated contractions evoked by AP₄A (30 μM) by 61%, but caused a small, significant decrease in the mean response to AP₅A (3 μM).
6. It is concluded that AP₄A and AP₅A act at the P_2X1-receptor, or a site similar to the P_2X1-receptor, to evoke contraction of the guinea-pig isolated vas deferens. Furthermore, the potency of AP₄A, but not AP₅A, appears to be inhibited by an ecto-enzyme which is sensitive to ARL 67156.

     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Involvement of 5-hydroxytryptamine7 receptors in inhibition of porcine myometrial contractility by 5-hydroxytryptamine

1. 5-Hydroxytryptamine (5-HT; 1 nM–100 μM) concentration-dependently inhibited the amplitude and frequency of spontaneous contractions in longitudinal and circular muscles of the porcine myometrium. The circular muscle (EC₅₀; 68–84 nM) was more sensitive than the longitudinal muscle (EC₅₀; 1.3–1.44 μM) to 5-HT. To characterize the 5-HT receptor subtype responsible for inhibition of myometrial contractility, the effects of 5-HT receptor agonists on spontaneous contractions and of 5-HT receptor antagonists on inhibition by 5-HT were examined in circular muscle preparations.
2. Pretreatment with tetrodotoxin (1 μM), propranolol (1 μM), atropine (1 μM), guanethidine (10 μM) or L-NAME (100 μM) failed to change the inhibition by 5-HT, indicating that the inhibition was due to a direct action of 5-HT on the smooth muscle cells.
3. 5-CT, 5-MeOT and 8-OH-DPAT mimicked the inhibitory response of 5-HT, and the rank order of the potency was 5-CT>5-HT>5-MeOT>8-OH-DPAT. On the other hand, oxymethazoline, α-methyl-5-HT, 2-methyl-5-HT, cisapride, BIMU-1, BIMU-8, ergotamine and dihydroergotamine had almost no effect on spontaneous contractions, even at 10–100 μM.
4. Inhibition by 5-HT was not decreased by either pindolol (1 μM), ketanserin (1 μM), tropisetron (10 μM), MDL72222 (1 μM) or {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (10 μM), but was antagonized by the following compounds in a competitive manner (with pA₂ values in parentheses): methiothepin (8.05), methysergide (7.92), metergoline (7.4), mianserin (7.08), clozapine (7.06) and spiperone (6.86).
5. Ro 20-1724 (20 μM) and rolipram (10 μM) significantly enhanced the inhibitory response of 5-HT, but neither zaprinast (10 μM) nor dipyridamole (10 μM) altered the response of 5-HT.
6. 5-HT (1 nM–1 μM) caused a concentration-dependent accumulation of intracellular cyclic AMP in the circular muscle.
7. From the present results, the 5-HT receptor, which is functionally correlated with the 5-HT₇ receptor, mediates the inhibitory effect of 5-HT on porcine myometrial contractility. This inhibitory response is probably due to an increase in intracellular cyclic AMP through the activation of adenylate cyclase that is positively coupled to 5-HT₇ receptors.

     

Pharmacological characterization of type 1α metabotropic glutamate receptor-stimulated [35S]-GTPγS binding

1. The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [³⁵S]-guanosine 5′-[γ-thio]triphosphate ([³⁵S]-GTPγS) binding assay.
2. L-Glutamate increased the rate of [³⁵S]-GTPγS binding in a concentration-dependent manner (−logEC₅₀ (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [³⁵S]-GTPγS, 1 μM GDP, 10 mM MgCl₂, 100 mM NaCl and 100 μg membrane protein ml⁻¹. The L-glutamate (100 μM)-stimulated increase in [³⁵S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM).
3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K_D) for [³⁵S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (B_max) of 361±30 fmol mg⁻¹ protein.
4. Metabotropic glutamate receptor agonists, quisqualate (−logEC₅₀ (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [³⁵S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists.
5. (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [³⁵S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (K_D) of 34.0±7.8 μM to be calculated for this mGluR antagonist.
6. Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml⁻¹, 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [³⁵S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [³⁵S]-GTPγS binding by the residual PTX-insensitive G-protein population.
7. The modulation of [³⁵S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

     

Effect of some cyclooxygenase inhibitors on the increase in guanosine 3′:5′-cyclic monophosphate induced by NO-donors in human whole platelets

1. The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3′:5′-cyclic monophosphate (cyclic GMP) induced by nitric oxide-donor agents was tested in human whole platelets and in platelet crude homogenate.
2. In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide-donors (NO-donors) sodium nitroprusside (NaNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a dose-dependent way, its IC₅₀ being 13.7 μM and 15.8 μM, respectively.
3. Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO-donors in a dose-dependent way (IC₅₀=32.7 μM, NaNP and 25.0 μM, SNAP), while acetylsalicylic acid (up to 1000 μM) and diclofenac (up to 100 μM) were ineffective.
4. However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production.
5. Indomethacin (10 μM), indoprofen (30 μM), diclofenac (100 μM) and acetylsalicylic acid (1000 μM) showed a comparable efficacy in inhibiting platelet thromboxane B₂ (TXB₂) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO-donors was not mediated by inhibition of cyclooxygenase.
6. In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP.
7. The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO-donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen.

     

Pharmacological characterization of endothelin-induced rat pulmonary arterial dilatation

1. The aim of study was to characterize endothelin (ET)-induced vasodilatation in isolated extrapulmonary rat arteries (EPA) and in intrapulmonary arteries (IPA) preconstricted with 1 μM phenylephrine.
2. The ET-3 (1 nM–100 nM)- and ET-1 (10 nM–100 nM)-induced transient vasodilatations in EPA were more potent than those in IPA. The vasodilatation induced by ET-3 (100 nM) was larger than that induced by ET-1 (100 nM).
3. Both the ET_B antagonist, BQ788 (3 μM) and or endothelium denudation, but not the ET_A antagonist, BQ123 (3 μM), abolished the vasodilatation induced by ET-1 or ET-3 (100 nM each) in EPA and in IPA. The ATP-sensitive K+channel blocker, glibenclamide (20 μM) and the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine (L-NMMA, 1 mM) suppressed the ET-induced vasodilatation in EPA and in IPA.
4. We conclude that the vasodilatation induced by endothelins is markedly reduced in rat isolated IPA, and suggest that the endothelial ET_B-mediated vasodilatation varies depending on rat pulmonary arterial regions. Furthermore, ET_B-mediated vasodilatation involves activation of ATP-sensitive K⁺ channels and of nitric oxide synthase in rat isolated EPA and IPA.

     

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.

     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)_A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels.
2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.
3. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC₅₀ values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABA_A, 5-HT₃, nicotinic ACh and P2_X receptors, respectively.
4. Ciprofloxacin (10–3000 μM) inhibited GABA_A-mediated responses in the vagus nerve with an IC₅₀ (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABA_A-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times.
5. Responses mediated by 5-HT₃, nicotinic ACh and P2_X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).
6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC₅₀ of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC₅₀ of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.
7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA_A receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA_A receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA_A receptor.

     

Properties of the pore-forming P2X7 purinoceptor in mouse NTW8 microglial cells

1. We have used whole-cell patch clamping methods to study and characterize the cytolytic P2X₇ (P2_Z) receptor in the NTW8 mouse microglial cell line.
2. At room temperature, in an extracellular solution containing 2 mM Ca²⁺ and 1 mM Mg²⁺, 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate (Bz-ATP; 300 μM), or ATP (3 mM), evoked peak whole cell inward currents, at a holding potential of −90 mV, of 549±191 and 644±198 pA, respectively. Current-voltage relationships generated with 3 mM ATP reversed at 4.6 mV and did not display strong rectification.
3. In an extracellular solution containing zero Mg²⁺ and 500 μM Ca²⁺ (low divalent solution), brief (0.5 s) application of these agonists elicited larger maximal currents (909±138 and 1818±218 pA, Bz-ATP and ATP, respectively). Longer application of ATP (1 mM for 30 s) produced larger, slowly developing, currents which reached a plateau after approximately 15–20 s and were reversible on washing. Under these conditions, in the presence of ATP, ethidium bromide uptake could be demonstrated. Further applictions of 1 mM ATP produced rapid currents of the same magnitude as those observed during the 30 s application. Subsequent determination of concentration-effect curves to Bz-ATP, ATP and 2-methylthio-ATP yielded EC₅₀ values of 58.3, 298 and 505 μM, respectively. These affects of ATP were antagonized by pyridoxal-phosphate-6-azophenyl- 2′, 4′-disulphonic acid (PPADS; 30 μM) but not suramin (100 μM).
4. In low divalent solution, repeated application of 1 mM ATP for 1 s produced successively larger currents which reached a plateau, after 8 applications, of 466% of the first application current. PPADS (30 μM) prevented this augmentation, while 5-(N,N-hexamethylene)-amiloride (HMA) (100 μM) accelerated it such that maximal augmentation was observed after only one application of ATP in the presence of HMA. At a bath temperature of 32°C, current augmentation also occurred in normal divalent cation containing solution.
5. These data demonstrate that mouse microglial NTW8 cells possess a purinoceptor with pharmacological characteristics resembling the P2X₇ receptor. We suggest that the current augmentation phenomenon observed reflects formation of the large cytolytic pore characteristic of this receptor. We have demonstrated that pore formation can occur under normal physiological conditions and can be modulated pharmacologically, both positively and negatively.

     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured.
2. ET-1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg.
3. The selective ET_B receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET_A receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET_B receptor antagonist, BQ788 (1 μM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively.
4. Exposure of tracheal segments to ET-1 (1–1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 μM) did not cause any significant NO release. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET-1.
5. These results demonstrate that in the isolated guinea-pig trachea activation of ET_B receptors results in a contractile response, whereas activation of ET_A receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

     

Pharmacological properties of P2X3-receptors present in neurones of the rat dorsal root ganglia

1. The electrophysiological actions of several agonists which may differentiate between P2X₁- and P2X₃-receptors were studied under concentration and voltage-clamp conditions in dissociated neurones of 1–4 day old rat dorsal root ganglia.
2. β,γ-Methylene-D-ATP (β,γ-me-D-ATP) (1–300 μM), diadenosine 5′,5′′′-P¹,P⁵-pentaphosphate (AP5A) (100 nM–300 μM), diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP4A) (300 nM–300 μM) and uridine 5′-triphosphate (UTP) (1 μM–1 mM) all activated concentration-dependent inward currents with a latency to onset of a few ms.
3. The concentration-response curves for β,γ-me-D-ATP and AP5A and ATP had similar maximum values, while that for AP4A had a lower maximum. The concentration-response curve to UTP was shallow and did not reach a maximum. β,γ-Methylene-L-ATP was virtually inactive. The rank order of agonist potency was ATP>AP5A≈amp;AP4A>β,γ-me-D-ATP>UTP>>β,γ-methylene-L-ATP.
4. The inward currents were inhibited by the P2-receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (10 μM). PPADS also inhibited responses to ATP (800 nM) and α,β-methylene ATP (2 μM) in a concentration-dependent manner.
5. This study shows that β,γ-me-D-ATP, AP5A, AP4A and UTP all act via a suramin- and PPADS-sensitive P2X-receptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The very low activity of β,γ-methylene-L-ATP suggests that the agonists were acting at the P2X₃-subtype to produce these effects.

     

Effects of antagonists at the human recombinant P2X7 receptor

1. We have used whole-cell patch clamping methods to examine the properties of the recombinant human P2X₇ (P2Z) receptor stably expressed in HEK-293 cells.
2. In an extracellular solution with lowered concentrations of divalent cations (zero Mg²⁺ and 0.5 mM Ca²⁺), both ATP and the nucleotide analogue, 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (Bz-ATP) evoked concentration-dependent whole-cell inward currents with maxima of 4658±671 and 5385±990 pA, respectively, at a holding potential of −90 mV. Current-voltage relationships determined using 100 μM Bz-ATP reversed at −2.7±3.1 mV, and did not display significant rectification.
3. Repeated applications of 300 μM Bz-ATP produced inward currents with similar rise-times (approx. 450 ms, 5–95% current development) but with progressively slower 95–5% decay times, with the eighth application of this agonist yielding a decay time of 197% of the first application.
4. Concentration-effect curves to ATP and Bz-ATP produced estimated EC₅₀ values of 780 and 52.4 μM, respectively. Consecutive concentration-effect curves to Bz-ATP produced curves with similar maxima and EC₅₀ values.
5. The non-selective P2 antagonists, pyridoxal-phosphate-6-azophenyl-, 2′,4′-disulphonic acid (PPADS) and suramin, both produced concentration-dependent increases in maximal inward currents to Bz-ATP, with IC₅₀ concentrations of approximately 1 μM and 70 μM, respectively. The profile of antagonism produced by PPADS was not that of a competitive antagonist.
6. The isoquinolene derivatives 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62) and calmidazolium both produced antagonism which was not competitive, with IC₅₀ concentrations of approximately 15 and 100 nM, respectively. HMA (5-(N,N-hexamethylene)- amiloride) was also an effective antagonist at a concentration of 10 μM. The group IIb metal, copper, also displayed antagonist properties at the human P2X₇ receptor, reducing the maximum response to Bz-ATP by about 50% at a concentration of 1 μM.
7. These data demonstrate that the human recombinant P2X₇ receptor displays functional behaviour which is similar to the recombinant rat P2X₇ receptor, but has a distinct pharmacological profile with respect to agonist and antagonist sensitivity.

     

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

1. Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination.
2. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]-tert-butyl bicyclophosphorothionate ([³⁵S]-TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.
3. The membranes of cells expressing β3 cDNAs bound [³⁵S]-TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels.
4. GABA (10 nM–100 μM) modulated [³⁵S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC₅₀ of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.
5. Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [³⁵S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.
6. Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.
7. Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μM). 5α-Pregnan-3α-ol-20-one (100 nM) enhanced propofol (100 μM)-evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α-pregnan-3α-ol-20-one (10 μM) caused only a modest facilitation (to 128±12% of control) of propofol (100 μM)-evoked currents mediated by β3 homomers.
8. Propofol (3–100 μM) activated α1β3 and β3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 μM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge'' current on cessation of propofol (300 μM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α-pregnan-3α-ol-20-one (10 μM) to cells expressing α1β3 receptors.
9. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

     

Effects of the cysteinyl leukotriene receptor antagonists pranlukast and zafirlukast on tracheal mucus secretion in ovalbumin-sensitized guinea-pigs in vitro

1. We investigated the inhibitory effects of the cysteinyl leukotriene (CysLT₁) receptor antagonists, pranlukast and zafirlukast, on ³⁵SO₄ labelled mucus output, in vitro, in guinea-pig trachea, induced by leukotriene D₄ (LTD₄) or by antigen challenge of sensitized animals. Agonists and antagonists were administered mucosally, except in selected comparative experiments where drugs were administered both mucosally and serosally to assess the influence of the epithelium on evoked-secretion.
2. LTD₄ increased ³⁵SO₄ output in a concentration-related manner with a maximal increase of 23 fold above controls at 100 μM and an approximate EC₅₀ of 2 μM. Combined mucosal and serosal addition of LTD₄ did not significantly affect the secretory response compared with mucosal addition alone. Neither LTC₄ nor LTE₄ (10 μM each) affected ³⁵SO₄ output. Pranlukast or zafirlukast significantly inhibited 10 μM LTD₄-evoked ³⁵SO₄ output in a concentration-dependent fashion, with maximal inhibitions of 83% at 10 μM pranlukast and 78% at 10 μM zafirlukast, and IC₅₀ values of 0.3 μM for pranlukast and 0.6 μM for zafirlukast. Combined mucosal and serosal administration of the antagonists (5 μM each) gave degrees of inhibition of mucosal-serosal 10 μM LTD₄-evoked ³⁵SO₄ output similar to those of the drugs given mucosally. Pranlukast (0.5 μM) caused a parallel rightward shift of the LTD₄ concentration-response curve with a pK_B of 7. Pranlukast did not inhibit ATP-induced ³⁵SO₄ output.
3. Ovalbumin (10–500 μg ml⁻¹) challenge of tracheae from guinea-pigs actively sensitized with ovalbumin caused a concentration-related increase in ³⁵SO₄ output with a maximal increase of 20 fold above vehicle controls at 200 μg ml⁻¹. The combination of the antihistamines pyrilamine and cimetidine (0.1 mM each) did not inhibit ovalbumin-induced ³⁵SO₄ output in sensitized guinea-pigs. Neither mucosal (10 μM or 100 μM) nor mucosal-serosal (100 μM) histamine had any significant effect on ³⁵SO₄ output.
4. Pranlukast or zafirlukast (5 μM each) significantly suppressed ovalbumin-induced secretion in tracheae from sensitized guinea-pigs by 70% and 65%, respectively.
5. We conclude that LTD₄ or ovalbumin challenge of sensitized animals provokes mucus secretion from guinea-pig trachea in vitro and this effect is inhibited by the CysLT₁ receptor antagonists pranlukast and zafirlukast. These antagonists may be beneficial in the treatment of allergic airway diseases in which mucus hypersecretion is a clinical symptom, for example asthma and allergic rhinitis.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.

     

Diazoxide- and leptin-activated KATP currents exhibit differential sensitivity to englitazone and ciclazindol in the rat CRI-G1 insulin-secreting cell line

1. The effects of the antidiabetic agent englitazone and the anorectic drug ciclazindol on ATP-sensitive K⁺ (K_ATP) channels activated by diazoxide and leptin were examined in the CRI-G1 insulin-secreting cell line using whole cell and single channel recording techniques.
2. In whole cell current clamp mode, the hyperglycaemic agent diazoxide (200 μM) and the ob gene product leptin (10 nM) hyperpolarised CRI-G1 cells by activation of K_ATP currents. K_ATP currents activated by either agent were inhibited by tolbutamide, with an IC₅₀ for leptin-activated currents of 9.0 μM.
3. Application of englitazone produced a concentration-dependent inhibition of K_ATP currents activated by diazoxide (200 μM) with an IC₅₀ value of 7.7 μM and a Hill coefficient of 0.87. In inside-out patches englitazone (30 μM) also inhibited K_ATP channel currents activated by diazoxide by 90.8±4.1%.
4. In contrast, englitazone (1–30 μM) failed to inhibit K_ATP channels activated by leptin, although higher concentrations (>30 μM) did inhibit leptin actions. The englitazone concentration inhibition curve in the presence of leptin resulted in an IC₅₀ value and Hill coefficient of 52 μM and 3.2, respectively. Similarly, in inside-out patches englitazone (30 μM) failed to inhibit the activity of K_ATP channels in the presence of leptin.
5. Ciclazindol also inhibited K_ATP currents activated by diazoxide (200 μM) in a concentration-dependent manner, with an IC₅₀ and Hill coefficient of 127 nM and 0.33, respectively. Furthermore, application of ciclazindol (1 μM) to the intracellular surface of inside-out patches inhibited K_ATP channel currents activated by diazoxide (200 μM) by 86.6±8.1%.
6. However, ciclazindol was much less effective at inhibiting K_ATP currents activated by leptin (10 nM). Ciclazindol (0.1–10 μM) had no effect on K_ATP currents activated by leptin, whereas higher concentrations (>10 μM) did cause inhibition with an IC₅₀ value of 40 μM and an associated Hill coefficient of 2.7. Similarly, ciclazindol (1 μM) had no significant effect on K_ATP channel activity following leptin addition in excised inside-out patches.
7. In conclusion, K_ATP currents activated by diazoxide and leptin show different sensitivity to englitazone and ciclazindol. This may be due to differences in the mechanism of activation of K_ATP channels by diazoxide and leptin.