Modulation of the delayed K+ current by histamine in guinea pig ventricular myocytes

Summary The effects of histamine on delayed K⁺ current (I_K) were investigated in patch-clamped single guinea pig ventricular myocytes. Histamine increased I_K with a maximal fractional response of 2.7 and a k_d of 9.4 × 10^–7 mol/l. At a concentration of 10^–8 mol/l, histamine did not increase I_K significantly, but increased I_Ca by 52% ± 12%. The voltage-dependence of I_K activation, the reversal potential and the time course of the I_K tail decay were not changed by histamine. Under pretreatment with 10^–4 mol/l of ranitidine, neither histamine (10^–6 mol/l) nor 2-pyridylethylamine (10^–4 mol/l) caused any sizable increase in I_K. When the cell was pretreated with a saturating dose of isoproterenol (10^–6 mol/l), histamine did not additively enhance I_K. The I_K enhancement by 3 × 10^–7 mol/l histamine was partially antagonized by concurrent exposure to 5 × 10^–6 mol/l carbachol. Whereas, use of a higher concentration of histamine (10^–6 mol/l) obscured the inhibitory effect of carbachol. It is concluded that histaminergic action of I_K is attributed exclusively to H₂ receptor-mediated reactions involving G_s protein and adenylate cyclase. Send offprint requests to Y. Habuchi at the above address     

Role of ATP-sensitive K+ channels in antinociception induced by R-PIA,an adenosine A1 receptor agonist

The influence of several K⁺ channel-acting drugs on antinociception induced by the adenosine A₁ receptor agonist (–)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA) was evaluated with a tail flick test in mice. The subcutaneous administration of R-PIA (0.5–8 mg/kg) induced a dose-dependent antinociceptive effect. The ATP-sensitive K⁺ (K_ATP) channel blocker gliquidone (2–8 g/mouse, i.c.v.) produced a dose-dependent displacement to the right of the R-PIA dose-response line, whereas the K_ATP channel opener cromakalim (32 g/mouse, i.c.v.) shifted it to the left. Several K_ATP channel blockers dose-dependently antagonized the antinociceptive effect of R-PIA, the order of potency being gliquidone > glipizide > glibenclamide (i.e., the same order of potency shown by these drugs in blocking K_ATP channels in neurons). In contrast, the K⁺ channel blockers 4-aminopyridine and tetraethylammonium did not antagonize the effect of R-PIA. These data suggest that antinociception produced by adenosine A₁ receptor agonists is mediated by the opening of ATP-sensitive K⁺ channels. The present results, together with those of previous studies, further support a role for K⁺ channel opening in the antinociceptive effect of agonists of receptors coupled to G_i/G_o proteins. Correspondence to: José M. Baeyens at the above address     

Inhibitors of ATP-sensitive potassium channels in guinea pig isolated ischemic hearts

During heart ischemia, ATP-sensitive potassium channels in the sarcolemmal membrane (sarcK_ATP) open and cause shortening of the action potential duration. This creates heterogeneity of repolarization, being responsible for the development of re-entry arrhythmias and sudden cardiac death. Therefore, the aim is to develop selective blockers of the cardiac sarcK_ATP channel. In the present study we established an in vitro model and classified 5 K_ATP channel inhibitors with respect to their potency and selectivity between cardiomyocytes and the coronary vasculature and compared the results with inhibition of Kir6.2/SUR2A channels expressed in HEK293 cells, recorded with the Rb⁺-efflux methods. We used Langendorff-perfused guinea pig hearts, where low-flow ischemia plus hypoxia was performed by reducing the coronary flow (CF) to 1.2 ml/min and by gassing the perfusion solution with N₂ instead of O₂. Throughout the experiment, the monophasic action potential duration at 90% repolarization (MAPD₉₀) was recorded. In separate experiments, high-flow hypoxia was produced by oxygen reduction in the perfusate from 95% to 20%, which caused an increase in the coronary flow. Under normoxic conditions, the substances glibenclamide, repaglinide, meglitinide, HMR 1402 and HMR 1098 (1 M each) reduced the CF by 34%, 38%, 19%, 12% and 5%, respectively. The hypoxia-induced increase in CF was inhibited by the compounds half-maximally at 25 nM, approximately 200 nM, 600 nM, approximately 9 M and >100 M, respectively. In control experiments after 5 min low-flow ischemia plus hypoxia, the MAPD₉₀ shortened from 121±2 to 99±2 ms (n=29). This shortening was half-maximally inhibited by the substances at concentrations of 95 nM, 74 nM, 400 nM, 110 nM and 550 nM, respectively. In HEK293 cells the Rb⁺-efflux through KIR6.2/SUR2A channels was inhibited by the compounds with IC₅₀ values of 21 nM, 67 nM, 205 nM, 60 nM and 181 nM, respectively. In summary, the present data demonstrate that the sulfonylurea glibenclamide, and the carbamoylbenzoic acid derivatives repaglinide and meglitinide are unselective blockers of K_ATP channels in cardiac cells and in the cardiac vascular system, whereas the sulfonylthioureas HMR 1402, and especially HMR 1098 selectively blocked the cardiac sarcK_ATP channel. Blockade of Kir6.2/SUR2A channels in HEK293 cells occurred with comparable efficacy as in the cardiac tissue, indicating that the expression system is suited for screening for novel inhibitors.     

Adenosine receptors mediate both contractile and relaxant effects of adenosine in main pulmonary artery of guinea pigs

In guinea pig main pulmonary artery precontracted with noradrenaline, adenosine exerted an initial phasic contraction followed by a tonic contraction and a slow relaxation. After selective blockade by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX: 10 nM) of A₁ receptors, adenosine only elicited a rapid relaxation. This initial response was characterized by use of adenosine (AR) and its analogues N⁶-cyclopentyl-adenosine (CPA), R-N⁶-phenyllsopropyladeno-sine (R-PIA), 2-chloroadenosine (CADO), 5-N-ethyl-carboxamidoadenosine(NECA), N⁶-2-(4-aminophenyl) ethyl adenosine (APNEA) and 2-p-((carboxyethyl)phenethylamino)-5-carboxamidoadenosine (CGS 21 680). The order of potency of the adenosine analogues for purine-induced phasic contraction was CPA > R-PIA > NECA = APNEA > AR > CGS 21 680 suggesting the involvement of activation of A₁ type adenosine receptors in the contraction phase. DPCPX antagonized the CPA-induced contraction with a pA₂ = 9.27 ± 0.26, but the Schild plot slope parameter was significantly lower than unity (0.58 ± 0.09). In contrast, in electrically driven guinea pig atrial myocardium (a tissue reported to possess A1 receptors), the DPCPX-CPA antagonism was purely competitive (pA₂ = 8.95 ± 0.06; slope = 0.93 ± 0.06). In the presence of 300 nM DPCPX, the rank order of potency for the purine-induced fast relaxation was NECA > CADO = AR > CGS 21 680 = R-PIA > CPA. The NECA- and adenosine-induced relaxation was influenced neither by 300 nM CP 66 713 (an antagonist at A_2a receptors), nor by endothelial removal and inhibition of nitric oxide synthase (100 M N^G-nitro-L-arginine: L-NOARG). The adenosine-induced relaxation was antagonized by 8-phenyltheophylline (8-PT), a potent A₁/A₂ antagonist. However, the rapid relaxation elicited by adenosine in the presence of 8-PT, was reversed and contraction developed. It is concluded that adenosine causes contraction via dual action on A₁ adenosine receptors and on xanthine-resistant sites. Our experiments with APNEA (a prototypic A₃ receptor agonist) did not support the suggestion that A₃ receptors are implicated in the xanthine-resistant component of adenosine-induced contraction, as DPCPX (300 nM) completely abolished and even reversed the APNEA-induced contraction. In addition, cromolyn (a mast cell stabilizing agent) did not influence the xanthine-resistant contraction induced by adenosine in the presence of DPCPX, 8-PT and dipyridamole (an adenosine uptake inhibitor). On the basis of the rank order of agonist potency, the receptors involved in the adenosine-induced rapid relaxation most likely is of the A_2b subtype. The opposing action of the xanthine-resistant contraction, however, did not allow a definitive pharmacological characterization of the receptor mediating relaxation.     

Enhancement of adenosine A1 receptor functions by benzoylthiophenes in guinea pig tissues in vitro

Previous reports on a series of benzoylthiophenes, including PD 81,723 {2-amino-4,5-dimethyl-3-(3-trifluoromethyl-benzoyl)thiophene}, have shown specific enhancement of agonist binding at the adenosine A₁ receptor. We have studied the effects of two substituted benzoylthiophenes, PD 78,416 {thieno[2,3-c]pyridine-6(5H)-carboxylic acid, 2-amino-3-benzoyl-4,7-dihydro-ethyl ester} and RS-74513-000 {2-amino-4-ethyl-5-methyl-3-(3-trifluoro-methyl-benzoyl) thiophene} on response elicited by adenosine A₁ receptors in isolated guinea pig left atrium and ileum.In the electrically paced left atrium, PD 78,416 antagonized negative inotropic effect elicited by the agonist CPA {N⁶-cyclopentyladenosine} with a pK_B value of 6.2 ± 0.2 (n = 4) . At a low concentration which had no antagonistic effect (0.1 M), PD 78,416 enhanced the effect of CPA. The concentration-response curve to CPA was shifted leftward by 5.1 fold (95% confidence limits 2.4–11.2). In field stimulated isolated ileum, PD 78,416 (0.1, 0.3, 1 M) did not enhance or antagonize effects of CPA. At concentrations above 1 M, PD 78,416 decreased electrically induced contraction. This effect was not sensitive to adenosine deaminase and was not antagonized by the A₁ antagonist CPX {8-cyclopentyl-1,3-dipropyl-xanthine} (1 M).Unlike PD 78,416, RS-74513-000 (0.01, 0.1, 1, 3, 10 M) did not antagonize or enhance effects of CPA in the left atrium. However, effects of CPA in ileum were enhanced by RS-74513-000 (1 and 3 M). Maximum enhancement was observed at 3 M; the concentration-response curve to CPA was shifted leftward by 3.2 fold (95% confidence limits 2.4–4.2). Higher concentrations of RS-74513-000 (10 and 30 M) decreased electrically induced contraction, this effect was not reversed by CPX. These findings confirmed that functional effects of A₁ adenosine receptor may be enhanced by substituted benzoylthiophenes in vitro. The differential effect of PD 78,416 and RS-74513-000 on cardiac and ileal A₁ receptors suggests that it may be possible to design selective enhancers for cardiac and neural functions.     

Some degree of overlap exists between the K+-channels opened by cromakalim and those opened by minoxidil sulphate in rat isolated aorta

Summary The effects of the K⁺ channel opening drugs minoxidil sulphate and cromakalim, on ⁴²K⁺ and ⁸⁶Rb⁺ efflux and on vasorelaxation in rat isolated aorta, were compared. In rat aortic rings precontracted with noradrenaline (100 nmol/l), minoxidil sulphate and cromakalim concentration-dependently inhibited induced tension by up to 90%, with pD₂ values of 7.35±0.1 and 7.17±0.1, respectively. Glibenclamide (300 nmol/l), produced 2200- and 19-fold rightward shifts in the concentration-relaxation curves to minoxidil sulphate and cromakalim, respectively, without an effect on the maximum relaxation.Both minoxidil sulphate and cromakalim increased the efflux of ⁴²K⁺ and ⁸⁶Rb⁺ from aorta in a concentration-dependent manner, with midpoints in the µmol/l range; the maximum efflux induced by minoxidil sulphate being approximately one tenth of that induced by cromakalim. The ratio of stimulated ⁸⁶Rb⁺/⁴²K⁺ efflux increased from 0.22 to 0.48 with increasing cromakalim concentrations, but was approximately constant (0.39) when the minoxidil sulphate concentration was varied. In the presence of minoxidil sulphate, the effects of cromakalim on ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited in a concentration-dependent manner, by up to 60%. In the continuing presence of cromakalim (300 nmol/l), minoxidil sulphate (10 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited by 45%, whereas conditioning with cromakalim (1 µmol/l) inhibited the ⁸⁶Rb⁺ efflux stimulated by additional superfusion of cromakalim (1 µmol/l) by 85%. Glibenclamide inhibited minoxidil sulphate (10 µmol/l)- and cromakalim (1 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux in a concentration-dependent manner with IC₅₀ values of approximately 80 nmol/l.In conclusion, the efflux data suggest that considerable overlap exists between the channels opened by minoxidil sulphate and those opened by cromakalim in rat aorta. Minoxidil sulphate has a weak efficacy as a K⁺ channel opener, and may act to open a homogeneous population of K⁺ channels. In contrast, the actions of cromakalim (1 µmol/l) are associated with large increases in tracer efflux, which are probably mediated via a heterogeneous population of K⁺ channels. However, only a small proprtion of this induced efflux appears to be required for relaxation. The differential inhibition by glibenclamide of the vasorelaxant effects of minoxidil sulphate and cromakalim may result from (a) the partial agonist properties of minoxidil sulphate in opening K⁺ channels and/or (b) additional mechanisms of vasorelaxation, which differ in their sensitivity to glibenclamide. Send offprint requests to U. Quasi at the above address     

Comparison of the effluxes of 42K+ and 86Rb+ elicited by cromakalim (BRL 34915) in tonic and phasic vascular tissue

Summary The cromakalim-induced effluxes of ⁴²K⁺ and ⁸⁶Rb⁺ were compared in rat aortic segments and in guinea-pig portal vein. In both vessels, low concentrations of cromakalim (0.1 M) increased the permeability to ⁸⁶Rb⁺ 3–4 times less than that to ⁴²K⁺; at 10 M the difference was about a factor of 1.3–2. In rat aorta, the threshold concentration of cromakalim for ⁴²K⁺ efflux was 0.03 M; with ⁸⁶Rb⁺ as the tracer ion it was 0.1 M. At similar concentrations, cromakalim relaxed the tension of aortic segments precontracted with 23 mM KCl (IC₅₀ = 0.06 ± 0.01 M). However, no concomitant increase in ⁴²K⁺ or ⁸⁶Rb⁺ efflux could be detected from this stimulated preparation at these concentrations. In guinea-pig portal vein, ⁴²K⁺ efflux measurements were performed in the presence and absence of the dihydropyridine Ca²⁺ entry blocker PN 200-110 (isradipine) yielding comparable results. In the presence of PN 200-110, where spontaneous activity and the K⁺ efflux associated with it were abolished, the threshold concentration of cromakalim for ⁴²K⁺ efflux was 0.02 M as compared to 0.06 M for ⁸⁶Rb⁺ efflux. In the absence of PN 200-110, spontaneous activity of the portal vein was inhibited by 70% and 90% at these concentrations. In double isotope experiments, the K⁺ channel inhibitor tetraethylammonium did not discriminate between the effluxes of ⁴²K⁺ and ⁸⁶Rb⁺ stimulated by cromakalim.It is concluded that in the two vascular tissues examined, cromakalim increased the permeability to ⁴²K⁺ more than to ⁸⁶Rb⁺, the difference being more marked at low cromakalim concentrations. The use of ⁴²K⁺ as the tracer ion narrows the apparent gap between the concentrations of cromakalim which elicit vasorelaxant effects and those which induce an observable increase in K⁺ permeability; however a significant difference persists.Part of the data was presented at the Winter Meeting of the British Pharmacological Society London 1988 [Br J Pharmacol 93 (1988) p 19] Send offprint requests to U. Quasi at the above address     

Quinidine inhibition of the muscarine receptor-activated K+ channel current in atrial cells of guinea pig

Summary Postsynaptic mechanisms underlying the anticholinergic effects of quinidine were examined in single atrial cells, using the tight-seal whole-cell recording technique. The solution in the glass pipettes contained guanosine-5triphosphate (GTP) or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolyzable GTP analogue). In both cases, acetylcholine (ACh), applied to the bath, induced a specific K⁺ current. In GTP-loaded cells, quinidine in the bath solution depressed the ACh-induced K⁺ current concentration-dependently. Atropine also blocked the K⁺ current. On the other hand, in GTP-S-loaded cells, the ACh-induced current was not blocked by atropine and persisted even when ACh was washed out from the bath, indicating that GTP-S causes uncoupling of the K⁺ channels from the muscarine receptors. Quinidine, however, did depress the increased K⁺ current concentration-dependently. The percent inhibition curves for quinidine to depress the K⁺ current were very similar between GTP-loaded and GTP-S-loaded cells. From these observations, we suggest that direct inhibition of the muscarine receptor-activated K⁺ channel current by quinidine, and not blockade of the muscarine receptor itself, is mainly responsible for the anticholinergic effects of the drug in atrial myocytes. Send offprint requests to Y. Kurachi at the above address     

The adenosine A1 receptor contributes to the stimulatory, but not the inhibitory effect of caffeine on locomotion: a study in mice lacking adenosine A1 and/or A2A receptors

Caffeine has biphasic effects on locomotion, and blockade of the adenosine A(2A) receptor (A2AR) is necessary for the stimulatory effect of low doses of caffeine, but not for the locomotor depressant effect observed at high doses. We wanted to elucidate the role of the adenosine A(1) receptor (A1R) in mediating the locomotor effects of increasing doses of caffeine using wild-type mice (A1R(WT)), mice heterozygous for (A1R(HET)), and mice lacking the adenosine A(1) receptor (A1R(KO)). Caffeine had the typical biphasic dose-effect relationship in all three genotypes, but the stimulatory action of caffeine was facilitated in the A1R(KO) mice. In order to investigate the interaction between blockade of A1Rs and A2ARs, mice lacking both receptors (A1R(KO)/A2AR(KO)) were tested. Regardless of A1R genotype, animals lacking A2AR were not stimulated by caffeine, whereas animals heterozygous for A2AR were. As expected, the A1R is not crucial for the stimulatory effect of caffeine, but seems to modulate the effect of caffeine exerted via A2AR blockade. Furthermore, these results suggest that the inhibitory effect of high doses of caffeine is due neither to blockade of the A1R, nor of the A2AR, and an effect independent of these adenosine receptors is likely.     

Cardiac glycosides: Correlations among Na+, K+-ATPase,sodium pump and contractility in the guinea pig heart

Summary Relationships among positive inotropic response to cardiac glycosides, Na⁺,K⁺-ATPase inhibition and monovalent cation pump activities were studied using paced Langendorff preparations of guinea-pig heart. Na⁺,K⁺-ATPase activity was estimated from the initial velocity of (³H)-ouabain binding in ventricular homogenates, and cation pump activity from ouabain-sensitive ⁸⁶Rb uptake of ventricular slices. These parameters were assayed in control, ouabain- or digitoxintreated hearts either at the time of inotropic response to the cardiac glycosides or during the course of drug washout. Development and loss of the inotropic response during ouabain or digitoxin perfusion and washout was accompanied by reduction and subsequent recovery of the initial ouabain binding velocity, respectively. If homogenates from glycoside-treated hearts were incubated at 37°C for 10 min during ouabain-binding studies, the levels of binding were not different from those of control hearts, indicating a rapid dissociation of the glycosides from cardiac Na⁺,K⁺-ATPase in this species. Despite differences in the time course of the loss of inotropic responses produced by ouabain or digitoxin, the relationship between Na⁺,K⁺-ATPase inhibition and inotropic responses were similar. Inotropic responses to digitoxin during perfusion, and subsequent los during washout, also were accompanied by a reduction and subsequent recovery of ⁸⁶Rb uptake. A correlation between inhibition of cation pump activity and positive inotropy has hitherto not been demonstrated. Thus, it appears that with cardiac glycosides, a relationship exists among contractility, cardiac Na⁺,K⁺-ATPase and monovalent cation pump activities.     

The modulation of Ca2+ and K+ channels but not changes in cAMP signaling contribute to the inhibition of glutamate release by cannabinoid receptors in cerebrocortical nerve terminals

While cannabinoid receptors activate multiple signaling pathways in the brain, it remains unclear what influence the inhibition of adenylylcyclase has on the inhibition of glutamate release. In cerebrocortical nerve terminals, the cannabinoid receptor agonist WIN55,212-2 reduced KCl-evoked glutamate release through a mechanism that restricted the rise of cytoplasmic free Ca²⁺, but not the changes in plasma membrane depolarization. These effects were consistent with the inhibition of Ca²⁺ channels. Furthermore, WIN55,212-2 reduced 4-aminopyridine (4AP) evoked glutamate release to a larger extent by modulating the behavior of both Ca²⁺ and K⁺-channels. The inhibition of 4AP-evoked release was associated with a decrease in cytoplasmic free Ca²⁺ and in plasma membrane depolarization that was reverted by the potassium channel blocker, tetraethylammonium. Interestingly, the reduction of KCl- and 4AP-evoked release by WIN55,212-2 was independent of adenylylcyclase activity and did not affect cAMP. Forskolin and the β-adrenergic receptor increase intrasynaptosomal cAMP and promote a PKA-dependent tetrodotoxin (TTX)-sensitive increase in the spontaneous release of glutamate. These two responses were reduced by WIN55,212-2. However, the glutamate release induced by Sp-8-Br-cAMPS, which directly activated PKA without affecting cAMP, was also similarly reduced by WIN55,212-2. Hence, we conclude that the inhibition of glutamate release by WIN55,212-2 is unrelated to changes in cAMP and that the inhibition of release that a decrease in cAMP might produce is occluded by the activation of additional pathways such as the inhibition of Ca²⁺ channels and/or the activation of K⁺-channels that strongly depress glutamate release.     

Comparative studies of the influence of various K+ concentration on the action of K-strophthidin, digitoxin and strophanthidin-3-bromoacetate on papillary muscle and on membrane-ATPSA of guinea pig hearts

The influence of increasing K⁺ concentrations (5, 16, and 50 mM) on the effects of different cardenolides — digitoxin (DIG), k-strophanthidin (STR) and strophanthidin-3-bromoacetate (SBA) — on the contractile force of isolated electrically stimulated papillary muscles and on the activity of the Na⁺, K⁺-activated ATPase of guinea pig hearts was studied under comparable experimental conditions.     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Characterization of the potency, selectivity, and pharmacokinetic profile for six adenosine A2A receptor antagonists

Antagonists of adenosine A_2A receptors (A_2A-antagonists) with different chemical structures have been developed by several pharmaceutical companies for the potential treatment of Parkinson’s disease. Pharmacological characterization of these antagonists was incomplete, and different assay conditions were used in different labs. Therefore, we characterized the potencies, selectivities, and pharmacokinetic profiles of six prototypical A_2A-antagonists. Displacements of [³H]MSX-2 and of [³H]CGS21680 binding to the human cloned and rat A_2A receptors were performed. The rank order of potency of antagonists to displace [³H]MSX-2 binding to the human A_2A was SCH58261 ≥ Biogen-34 ≥ Ver-6623 ≥ MSX-2 > KW-6002 > > DMPX. For the rat striatal A_2A, the order of potency was Biogen-34 ≥ SCH58261 ≥ Ver-6623 ≥ MSX-2 ≥ KW-6002 > > DMPX. SCH58261 was the most potent antagonist of the human A_2A with a K _i value of 4 nM, whereas Biogen-34 was the most potent antagonist of the rat A_2A with a K _i value of 1.2 nM. Similar results were obtained from cAMP assays. Selectivities of A_2A-antagonists were determined using radioligands [³H]DPCPX, [³H]ZM241385, and [¹²⁵I]-AB-MECA for A₁, A_2B, and A₃ receptors, respectively. KW-6002 and Biogen-34 exhibited the highest selectivity for A_2A vs A₁ (human and rat), respectively. The pharmacokinetic profiles of antagonists were evaluated in vivo in rats. DMPX and KW-6002 had the greatest oral bioavailability. In contrast, SCH58261, MSX-2, and Ver-6623 had low or poor oral bioavailability. In summary, SCH58261, Biogen-34, MSX-2, and Ver-6623 had high affinities for both human and rat A_2A receptors, with reasonable selectivity for A_2A over A₁ and A_2B receptors. They are suitable as A_2A-antagonists for in vitro pharmacological studies. Among the six A_2A-antagonists, KW-6002 is the best for use in in vivo animal studies, particularly for a CNS target, based on its bioavailability, half life, and brain penetration.     

Acetylcholine and adenosine activate the G protein-gated muscarinic K+ channel in ferret ventricular myocytes

The properties of the K⁺ channel activated by acetylcholine (ACh) and adenosine (Ado) were examined in single ferret ventricular myocytes using patch-clamp techniques. In the whole-cell configuration, ACh and Ado induced an inwardly rectifying K⁺ current and shortened the action potential duration. The effect of ACh was blocked by atropine, while the Ado effect was interrupted by 8-cyclopenty1,1,2-dipropyl xanthine, a specific Ado A₁ receptor antagonist. In cell-attached recordings, ACh and Ado added to the pipette solution activated a single population of inwardly rectifying K⁺ channels, distinct from the i _K1 channel. The channel had a slope conductance of 40 pS in symmetrical 150 mM K⁺ solutions and a mean open time of 0.8 ms. Excision of the patch into the inside-out patch configuration in guanosine triphosphate (GTP)-free solution abolished the channel activity. The channel was reversibly reactivated by adding GTP to the intracellular side of the patch. GTPS activated the channel irreversibly. When the inside-out patch was treated with the A protomer of pertussis toxin (PTX), intracellular GTP no longer activated the K⁺ channel. The results show that ferret ventricular myocytes possess a K⁺ channel activated by both muscarinic and Ado A1 receptors. Its electrophysiological properties and the gating by a PTX-sensitive G protein in a membrane-delimited fashion are identical with those of the muscarinic K⁺ channels in nodal and atrial tissues of other species. In conclusion, the G protein-gated muscarinic K⁺ channel is expressed in ferret ventricular myocardium and may underlie the direct negative inotropism of ACh and Ado in this tissue.     

Comparison of A1 adenosine receptors in brain from different species by radioligand binding and photoaffinity labelling

Summary Radioligand binding to A₁ adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1,3-[³H]dipropyl-xanthine ([³H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [³H]DPCPX binding showed that the most potent compounds were DPCPX with K _i values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with K _i values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radioligand 2-chloro-N ⁶-[³H]cyclopentyl-adenosine ([³H]CCPA) were less pronounced, ranging from a K _D value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [³H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoa-denosine (NECA) and S-N ⁶-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different K _D values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the classical A₁ receptor profile (R-PIA > -NECA > S-PIA) is not sufficient to speculate that A₁ receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences.     

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.     

Reduction of the equilibrium binding of cardiac glycosides and related compounds to Na+, K+-ATPase as a possible mechanism for the potassium-induced reversal of their toxicity

Summary The influence of potassium ions on the equilibrium state of the binding of cardiac glycosides and their derivatives to partially purified dog heart and rat brain enzyme preparations was studied in vitro. The addition of potassium to the incubation mixture containing enzyme preparation, ³H-ouabain, Na⁺, Mg²⁺ and ATP, at the time when the binding reaction is close to equilibrium, caused an immediate reduction of the bound drug concentration; the concentration apparently shifting toward a lower equilibrium state. The degree of the potassium-induced reduction in bound drug concentration was dependent on the potassium concentration and on the chemical structure of the compound. The binding of aglycones, pentacetyl-gitoxin and cassaine was affected to a greater extent than that of the glycosides. These data suggest that one of the mechanisms by which potassium antagonizes the toxic actions of digitalis on the heart is to reduce the drug binding to cardiac Na⁺, K⁺-ATPase.This work was supported by a U.S. Public Health Service Grant, HL-16052     

Assessment of the enantiomeric purity of R- and S-N6-phenylisopropyladenosine (PIA): Implications for adenosine receptor subclassification

A chiral column high-performance liquid chromatographic method was developed for the assessment of the enantiomeric purity of the stereoisomers of N⁶-phenylisopropyladenosine (PIA). The observed chiral purity of R-PIA was greater than 99.90%, whereas S-PIA was found to contain 4.4% of the R-enantiomer. In radioligand binding studies, the observed affinity of S-PIA for the adenosine A₁ receptor (IC₅₀ 240 nM) could entirely be attributed to its content of R-PIA (IC₅₀ 7.8 nM). Calculation of a theoretical IC₅₀ of pure S-PIA for the A2 receptor yielded a value of 6700 nM, which was 35-fold higher than for R-PIA (190 nM). Concludingly, the utilization of enantiomeric impure S-PIA in the definition of adenosine receptor subclasses is questionable. Correspondence to: R. Mathôt at the above address(–)-N⁶-(R-phenylisopropyl)adenosine is referred to in literature as L-PIA, I-PIA, (–)-PIA and R-PIA. The abbreviation R-PIA is commonly used     

Effects of U-37883A on intracellular Ca2+ -activated large-conductance K+ channels in pig proximal urethral myocytes

Kinetic studies of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydrochloride), a vascular ATP-sensitive K+ channel (KATP channel) blocker, were performed on pig urethral myocytes to investigate inhibitory effects on large-conductance intracellular Ca2+ -sensitive K+ channels (i.e., BKCa channels; 225 pS K+ channels) by use of single-channel recordings (outside-out and inside-out configuration). BKCa channels in pig urethral smooth muscles showed extracellular iberiotoxin (300 nM) sensitivity and voltage dependency. The alpha subunit of BKCa channel proteins was detected in the membrane fraction by use of Western blot technique. Application of U-37883A (> or =10 microM) reduced the activity of BKCa channels in a concentration-dependent manner, not only by decreasing mean openlife time but also by prolonging the mean closed time. These results shows that U-37883A affects channels other than the vascular KATP channel, and demonstrates how it inhibits the activities of BKCa channels in urethral smooth muscles.