Agonist-independent effect of an allosteric enhancer of the A1 adenosine receptor in CHO cells stably expressing the recombinant human A1 receptor

The allosteric enhancer PD 81,723, a 2-amino-3-benzoylthiophene derivative, has been shown to potentiate agonist binding to A1 adenosine receptors (A1AdoRs) and to enhance the functional effects of adenosine and adenosine analogs. The objective of this study was to determine whether the apparent agonist-independent effect of PD 81,723 observed in CHO cells stably expressing the recombinant human A1AdoR was due to the potentiation of the action of endogenous adenosine, to the presence of constitutive receptor activity and/or to the binding of PD 81,723 to the agonist binding site of the A1AdoR. The allosteric enhancer PD 81,723, the A1AdoR agonist (R)-N6-(2-phenylisopropyl)adenosine and adenosine all significantly inhibited forskolin-stimulated cAMP accumulation in intact cells and increased [35S]-5'-(gamma-thio)triphosphate binding to cell membranes. The effects of adenosine on cAMP formation and [35S]-5'-(gamma-thio)triphosphate binding were attenuated by adenosine deaminase, but the effects of PD 81,723 were not. In the presence of ADA, the A1AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine increased forskolin-stimulated cAMP accumulation in cells expressing the recombinant human A1AdoR but not in nontransfected CHO cells. In binding experiments, the agonist (R)-N6-(2-phenylisopropyl)adenosine, but not PD 81,723, significantly displaced the specific binding of the A1AdoR agonist [3H]-N6-cyclohexyladenosine and the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine. The results of this study demonstrate that in CHO cells stably expressing the recombinant human A1AdoR, the agonist-independent effect of PD 81,723 is not due to potentiation of the action of endogenous adenosine or mediated by the binding of the allosteric enhancer to the agonist binding site of the recombinant human A1AdoR. It is possible that these effects are due to potentiation of constitutive receptor activity by PD 81,723.     

Use of a fluorescent analog of CDP-DAG in human skin fibroblasts: characterization of metabolism, distribution, and application to studies of phosphatidylinositol turnover

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.     

Cholinergic short-term conditioning and activation of ATP-sensitive K+ current in cat atrial myocytes

In atrial myocytes, an initial exposure to acetylcholine (ACh1) exerts a short-term conditioning effect such that a second ACh exposure (ACh2) activates ATP-sensitive K+ current (IK,ATP). The purpose of the present study was to determine the mechanism underlying the short-term conditioning induced by ACh that results in subsequent ACh-induced activation of IK.ATP. Cat atrial myocytes were studied using a nystatin-perforated patch whole cell recording method. Changes in L-type Ca2+ current (ICa,L) amplitude were used as an index of relative changes in cyclic AMP (cAMP). The results show that when atrial myocytes are treated with two consecutive exposures to 10 microM ACh separated by a recovery interval, ACh2 activates a larger increase in potassium conductance (gK+) than ACh1. The additional ACh2-induced increase in gK+ is selectively blocked by 10 microM glibenclamide, identifying the current as IK,ATP. Moreover, ICa,L activated immediately after the withdrawal of ACh1 exhibited a transient increase in amplitude above control (+ 76%), consistent with rebound stimulation of cAMP. Rp-cAMPs (50 microM), a selective antagonist of cAMP-dependent protein kinase A, blocked the rebound stimulation of ICa,L and abolished ACh2-induced activation of IK,ATP. Thapsigargin (5 microM), an inhibitor of Ca2+ ATPase in the sarcoplasmic reticulum (SR), abolished ACh2-induced activation of IK,ATP without decreasing rebound stimulation of ICa,L. Rebound stimulation of ICa,L and ACh2-induced activation of IK,ATP both varied as a function of ACh1 duration. We conclude that withdrawal of an initial ACh exposure elicits a rebound cAMP-mediated stimulation of SR Ca2+ uptake. This mechanism induces a short-term conditioning in atrial myocytes such that a subsequent ACh exposure activates IK,ATP. The present results demonstrate novel cholinergic signaling mechanisms in the regulation of IK,ATP.     

Spectral filtering in neutron interferometry and wave-corpuscle dualism

1. Anti-acetylcholine effects of pilsicainide, flecainide, disopyramide and propafenone on the acetylcholine (ACh)-induced K+ current (IK.ACh) were examined in dissociated guinea-pig atrial myocytes under whole-cell voltage clamp by the use of the 'concentration-clamp' technique. 2. The IK.ACh was activated with a latency of about 100 ms after 1 microM ACh application and desensitized to a steady-state level. The latent period and the time to peak response were shortened with increasing ACh concentration. 3. The values of half-maximal inhibition (IC50) on the peak and steady state responses were 25 and 25 microM for pilsicainide, 1.7 and 2.0 microM for disopyramide, 19 and 2.0 microM for flecainide and 0.7 and 0.2 microM for propafenone, respectively. 4. Pilsicainide and disopyramide increased the latent period and the time to peak of IK.ACh in a concentration-dependent manner. Flecainide and propafenone did not change the latent period, but shortened the time to peak and hastened the decay of IK.ACh in a voltage-independent manner. 5. The results suggest that the mechanisms underlying the anti-acetylcholine effect of antiarrhythmic drugs are different among these drugs: i.e., pilsicainide and disopyramide mainly block the muscarinic ACh receptors while flecainide and propafenone inhibit the K+ channel itself as open channel blockers.     

Novel approach for enhancing atrioventricular nodal conduction delay mediated by endogenous adenosine

The 2-amino-3-benzoylthiophene derivative PD 81,723 potentiates the A1 receptor-mediated negative dromotropic effect of exogenous adenosine and adenosine receptor agonists in guinea pig isolated perfused and in situ hearts. The objective of this study was to determine whether PD 81,723 could amplify the cardiac actions of endogenous adenosine. Two approaches known to increase the myocardial interstitial concentration of adenosine--hypoxia, which increases the production of adenosine and the inhibition of adenosine kinase, which decreases its metabolism--were used to test this hypothesis. In guinea pig hearts in situ, PD 81,723 (2 mg/kg i.v.) potentiated the atrioventricular (AV) nodal conduction delay caused by hypoxemia (PaO2, 14 to 19 mm Hg). In guinea pig isolated hearts, PD 81,723 (5 mumol/L) increased by twofold the stimulus-to-His bundle (S-H) interval prolongations induced by both a 5-minute period of hypoxia (25% O2/70% N2/5% CO2) and the administration of the adenosine kinase inhibitor iodotubercidin (40 to 70 nmol/L) but had no effect on coronary conductance. Hypoxia and hypoxia plus PD 81,723 (5 mumol/L) caused equivalent increases in the concentration of adenosine in epicardial transudate, from 0.13 +/- 0.15 to 0.48 +/- 0.1 and 0.45 +/- 0.4 mumol/L, respectively. Similar to the allosteric enhancer, the nucleoside uptake blocker draflazine (0.1 mumol/L) also increased by twofold the S-H interval prolongation caused by hypoxia. In contrast to the allosteric enhancer, draflazine increased the concentration of adenosine in epicardial transudate during hypoxia from 0.48 +/- 0.15 to 1.5 +/- 0.4 mumol/L. Draflazine also increased coronary conductance by approximately twofold in guinea pig normoxic constant-fold perfused hearts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological mechanisms for the antiarrhythmic activities of naloxone on cardiac tissues

It has been reported that naloxone, an opioid antagonist, has antiarrhythmic activity in vivo. In Langendorff perfused rat hearts, we found that ischemia-reperfusion-induced ventricular tachyarrhythmia reverted to normal sinus rhythm after the treatment with naloxone (3 approximately 10 microM). The method of voltage and current clamp were used to study the underlying mechanism of its antiarrhythmic activity on isolated cardiac myocytes. In isolated rat ventricular and in guinea-pig and human atrial myocytes, naloxone prolonged the action potential duration reversibly. In rat ventricular myocytes, naloxone (1 approximately 30 microM) inhibited sodium current (I(Na)), transient outward potassium current (I(to)), and calcium current (I(Ca)). On the contrary, the addition of naloxone significantly increased inward rectifier potassium current (I(K1)). For the effect on I(Na), naloxone did not shift the inactivation curve of I(Na) but retarded the I(Na) recovery rate from inactivation state. Naloxone suppressed I(to) with a significant left-shift of the inactivation curve, however, the time course of I(to) recovery from inactivation was not affected. In guinea pig atrial myocytes, naloxone (10 microM) decreased the delayed rectifier K+ current (IK). These results show that naloxone exert various extent of inhibition on I(Na), I(to), IK and I(Ca). The prolongation of cardiac action potential is related to the inhibition of I(to) and IK. The antiarrhythmic activity of naloxone is more closely related to the inhibition of Na+ and K+ currents rather than the blockade of myocardial opioid receptors.     

RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of "regulators of G protein signaling" (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent "basal" GIRK current was suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of "slow" postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.     

ATP and adenosine inhibit transmitter release at the frog neuromuscular junction through distinct presynaptic receptors

1. The effects of exogenous ATP or adenosine on end-plate currents (e.p.cs; evoked by simultaneous action of a few hundred quanta of ACh) or on miniature e.p.cs (m.e.p.cs) were studied under voltage clamp conditions on frog sartorius muscle fibres. 2. ATP or adenosine (100 microM(-1) mM) reduced the e.p.c. amplitude but did not affect m.e.p.c. amplitude, decay time constant and voltage-dependence of m.e.p.c., suggesting that e.p.c. depression induced by these purines had presynaptic origin only. 3. The action of ATP, unlike that of adenosine, was prevented by the P2-purinoceptor antagonist suramin (100 microM). The stable ATP analogue alpha,beta-methylene ATP (100 microM), known to be desensitizing agent on P2X receptors, also abolished the depressant effect of ATP while sparing the action of adenosine. Concanavalin A, an inhibitor of ecto-5'-nucleotidase, did not affect the presynaptic action of exogenously applied ATP. 4. The presynaptic action of adenosine was prevented by theophylline (1 mM), a blocker of adenosine receptors, while the effect of ATP was not changed under these conditions. The selective blocker of A1 adenosine receptors, 8-cyclopentyl-1,3,dipropylxanthine (DPCPX; 0.1 microM), abolished the presynaptic action of adenosine but did not prevent the depressant effect of ATP. 5. The effects of ATP and adenosine (at nearly saturating concentration) were additive suggesting that these purines activated not only distinct receptors but also different intracellular signalling mechanisms. 6. In contrast to the hypothesis that at the neuromuscular junction ATP reduces transmitter release via enzymatic degradation to presynaptically active adenosine, our data suggest that ATP (through its own presynaptic receptors) directly inhibits ACh release. Thus, ATP and adenosine might be almost equipotent as endogenous prejunctional neuromodulators at the neuromuscular junction.     

cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes

EHNA (Erythro-9-[2-hydroxy-3-nonyl]adenine) is a wellknown inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent Ki value of approximately 1 microM and with negligible effects on Ca2+/calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low Km cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733- 738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca2+ current (ICa), we have examined the effect of EHNA on ICa in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 microM EHNA induced an increase in the amplitude of basal ICa ( approximately 80% at 1 microM) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on ICa was comparable in amplitude with the maximal effect of isoprenaline (1 microM), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2'-deoxycoformycin (1-30 microM), another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 microM) did not increase ICa. In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase ICa. However, under similar conditions, intracellular perfusion with 0.5 microM cGMP produced an 80% increase in ICa. As opposed to human cardiomyocytes, EHNA (1-10 microM) did not modify ICa in isolated rat ventricular and atrial myocytes. We conclude that basal ICa is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nucleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation.     

GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels

GIRK1 and GIRK4 subunits combine to form the heterotetrameric acetylcholine-activated potassium current (IKACh) channel in pacemaker cells of the heart. The channel is activated by direct binding of G-protein Gbetagamma subunits. The GIRK1 subunit is atypical in the GIRK family in having a unique ( approximately 125-amino acid) domain in its distal C terminus. GIRK1 cannot form functional channels by itself but must combine with another GIRK family member (GIRK2, GIRK3, or GIRK4), which are themselves capable of forming functional homotetramers. Here we show, using an extracellularly Flag-tagged GIRK1 subunit, that GIRK1 requires association with GIRK4 for cell surface localization. Furthermore, GIRK1 homomultimers reside in core-glycosylated and nonglycosylated states. Coexpression of GIRK4 caused the appearance of the mature glycosylated form of GIRK1. [35S]Methionine pulse-labeling experiments demonstrated that GIRK4 associates with GIRK1 either during or shortly after subunit synthesis. Mutant and chimeric channel subunits were utilized to identify domains responsible for GIRK1 localization. Truncation of the unique C-terminal domain of Delta374-501 resulted in an intracellular GIRK1 subunit that produced normal IKACh-like channels when coexpressed with GIRK4. Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the C terminus of GIRK4 localized to the cell surface. Deletion analysis of the GIRK4 C terminus identified a 25-amino acid region required for cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino acid region required for cell surface localization of GIRK4 homotetramers. GIRK1 appeared intracellular in atrial myocytes isolated from GIRK4 knockout mice and was not maturely glycosylated, supporting an essential role for GIRK4 in the processing and cell surface localization of IKACh in vivo.     

Properties of 3,4-diaminopyridine-evoked dopamine and acetylcholine release in rabbit caudate nucleus slices: involvement of facilitatory adenosine A2 receptors or nitric oxide?

The 3H-overflow from slices of the rabbit caudate nucleus preincubated with tritiated dopamine (DA), or choline, and then superfused and stimulated twice with 3,4-diaminopyridine (3,4-DAP; 25 microM, 1 min), was explored as an in vitro model for evoked release of DA, or acetylcholine (ACh), respectively. In both cases the 3,4-DAP-evoked 3H-overflow was tetrodotoxin-sensitive and Ca(2+)-dependent and hence most probably represents action potential-induced exocytotic release of DA or ACh, respectively. Using pairs of preferential agonists/antagonists it was shown, that evoked DA release was inhibited via presynaptic D2 autoreceptors (quinpirole/domperidone) and kappa-opioid receptors (U-50488H/norbinaltorphimine). No evidence was found for the presence of presynaptic adenosine A1 or A2 receptors on dopaminergic terminals. Moreover, 3,4-DAP-evoked DA release was unaffected by increased intracellular cyclic AMP levels or by drugs affecting the NO/guanylate cyclase pathway. In a similar manner it was shown that 3,4-DAP-evoked ACh release was inhibited via presynaptic muscarine autoreceptors (oxotremorine/atropine) and dopamine D2 heteroreceptors (quinpirole/domperidone). Again, no evidence for the involvement of the NO/guanylate cyclase system in the modulation of ACh release was found, whereas the presence of inhibitory adenosine A1 receptors, but not of facilitatory A2 receptors, could be clearly established. It is concluded, that 3,4-DAP-evoked 3H-overflow from rabbit caudate nucleus slices preincubated with [3H]DA or [3H]choline, represents a simple and useful in vitro model for action potential-induced DA or ACh release, respectively. Moreover, at least in this model or rabbit brain region, facilitatory adenosine A2 receptors and the NO/guanylate cyclase system seem not to be involved in the release of these transmitters.     

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.     

Modulation of [3H]acetylcholine release from cultured amacrine-like neurons by adenosine A1 receptors

We have investigated the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultured chick amacrine-like neurons. The release of [3H]ACh evoked by 50 mM KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca2+ channels. Ligand binding studies using [3H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca2+-dependent release of [3H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3H]ACh release may be due to a direct inhibition of N-type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Withdrawal of acetylcholine elicits Ca2+-induced delayed afterdepolarizations in cat atrial myocytes

BACKGROUND: Recent experiments in atrial myocytes indicate that withdrawal of cholinergic agonist can directly increase Ca2+ influx via L-type Ca2+ current and stimulate Ca2+ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca2+. Overload of cellular Ca2+ within the SR can initiate various types of atrial dysrhythmias. The present study was designed to determine whether withdrawal of acetylcholine (ACh) can elicit Ca2+-induced delayed afterdepolarizations (DADs) in atrial myocytes. METHODS AND RESULTS: A nystatin perforated-patch whole-cell method and fluorescence microscopy (indo 1) were used to measure electrical activities and intracellular free Ca2+ ([Ca2+]i), respectively. Withdrawal of ACh (1 micromol/L) increased action potential duration, shifted plateau voltage toward positive, and generated DADs that initiated spontaneous action potentials. Voltage-clamp analysis revealed that withdrawal of ACh elicited a rebound stimulation of L-type Ca2+ current (I(Ca,L)) (+45%) and Na/Ca exchange current (I(NaCa)) (+16%) and the appearance of transient inward current (I(ti)) and spontaneous [Ca2+]i transients. Each of these changes induced by withdrawal of ACh was abolished by Rp-cAMPs (50 to 100 micromol/L) or H-89 (2 micromol/L), inhibitors of cAMP-dependent protein kinase A. Ryanodine (1 micromol/L) abolished I(NaCa) and the appearance of I(ti) without decreasing the rebound stimulation of I(Ca,L) elicited by withdrawal of ACh. CONCLUSIONS: Withdrawal of ACh can elicit cAMP-mediated stimulation of Ca2+ influx via I(Ca,L) and uptake of SR Ca2+. As a result, cellular Ca2+ overload causes enhanced SR Ca2+ release and the initiation of DADs. These mechanisms may generate triggered and/or spontaneous atrial depolarizations elicited by withdrawal of vagal nerve activity.     

Activation of multiple sites by adenosine analogues in the rat isolated aorta

1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100 microM) E/[A] curves to NECA and CPA were right-shifted. However, no further shift of the CPA E/[A] curve was obtained when 8-SPT (50 microM) was administered concomitantly. The locations of curves to R-PIA and adenosine were unaffected by L-NAME (100 microM). 6. In the presence of PD 115,199 (0.1 microM) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 +/- 0.19). PD 115,199 (0.1 and 1 microM) gave smaller rightward shifts of E/[A] curves to R-PIA and CPA, but E/[A] curves to adenosine were not significantly shifted in the presence of PD 115,199 (0.1 or 1 microM). 7. The presence of ZM 241385 (3 nM-0.3 microM) caused parallel rightwad shifts of NECA E/[A] curves (pKB = 8.73 +/- 0.11). No significant shifts of E/[A] curves to adenosine, CPA or R-PIA were observed in the presence of 0.1 microM ZM 241385. 8. CGS 21680 (1 microM) elicited a relaxant response equivalent to approximately 40% of the NECA maximum response. In the presence of this concentration of CGS 21680, E/[A] curves to NECA were right-shifted in excess of 2-log units, whereas E/[A] curves to R-PIA were not significantly shifted. 9. BWA1433 (100 microM) caused a small but significant right-shift of the E/[A] curve to R-PIA yielding a pA2 estimate of 4.1 IB-MECA (N6-(3-iodo-benzyl)adenosine-5(1)-N-methyl uronamide) elicited relaxant responses which were resistant to blockade by 8-SPT (p[A]50 = 5.26 +/- 0.13). 10. The results suggest that whereas relaxations to NECA (10 nM-1 microM) are mediated via adenosine A2a receptors, which are located at least in part on the endothelium, R-PIA and CPA may activate A2b receptors on the endothelium and an additional, as yet undefined site, which is likely to be located on the smooth muscle and which is not susceptible to blockade by 8-SPT, PD 115,199 or ZM 241385. This site is unlikely to be an A3 receptor since the very small shift obtained in the presence of BWA1433 (100 microM), and the low potency of IB-MECA is not consistent with the affin     

Roles of calcium-activated and voltage-gated delayed rectifier potassium channels in endothelium-dependent vasorelaxation of the rabbit middle cerebral artery

1. The cellular mechanism(s) of action of endothelium-derived vasodilator substances in the rabbit middle cerebral artery (RMCA) were investigated. Specifically, the subtypes of potassium channels involved in the effects of endothelium-derived relaxing factors (EDRFs) in acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in this vessel were systematically compared. 2. In the endothelium-intact RMCA precontracted with histamine (3 microM), ACh induced a concentration-dependent vasorelaxation, which was sensitive to indomethacin (10 microM) or N(G)-nitro-L-arginine (L-NOARG; 100 microM); pD2 values 8.36 vs 7.40 and 6.38, P < 0.01 for both, n = 6 and abolished by a combination of both agents. ACh caused relaxation in the presence of high K+ PSS (40 mM KCl), which was not affected by indomethacin, but abolished by L-NOARG and a combination of indomethacin and L-NOARG. 3. In the presence of indomethacin, relaxation to ACh in the endothelium-intact RMCA precontracted with histamine was unaffected by either glibenclamide (10 microM), an ATP-sensitive K+ channel (K[ATP]) blocker, 4-aminopyridine (4-AP, 1 mM) or dendrotoxin (DTX, 0.1 microM), delayed rectifier K channel (Kv) blockers. However, relaxation responses to ACh were significantly inhibited by either LY83583 (10 microM) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 microM), guanylyl cyclase inhibitors, or charybdotoxin (CTX; 0.1 microM), iberiotoxin (ITX, 0.1 microM) and apamin (APA, 0.1 microM), large conductance Ca2+-activated K+ channels (BK[Ca]) blocker and small conductance Ca2+-activated K+ channel (SK[Ca]) blocker, respectively. 4. In the presence of L-NOARG, relaxation to ACh was unaffected by glibenclamide or the cytochrome P450 mono-oxygenase inhibitor, clotrimazole (1 microM), but was significantly inhibited by either 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, 10 microM) and 2',3'-dideoxyadenosine (2',3'-DDA, 30 microM), adenylyl cyclase inhibitors, or 4-AP, DTX, CTX, ITX and APA. 5. In the endothelium-denuded RMCA precontracted with histamine, authentic NO-induced relaxation was unaffected by glibenclamide, 4-AP and DTX, but significantly reduced by ODQ, ITX and APA. Authentic prostaglandin I2 (PGI2)-induced relaxation was unaffected by glibenclamide, but significantly reduced by 2',3'-DDA, 4-AP, DTX, ITX and APA. Forskolin-induced relaxation was significantly inhibited by high K+, CTX and 4-AP. 6. These results indicate that: (1) in the RMCA the EDRFs released by ACh are NO and a prostanoid (presumably PGI2), and there is no evidence for the release of a non-NO/PGI2 endothelium-derived hyperpolarizing factor (EDHF), (2) K(Ca) channels are involved in NO-mediated relaxation of the RMCA but both K(Ca) and Kv channels are involved in PGI2-mediated relaxation.     

Electrically evoked acetylcholine release from hippocampal slices is inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid antagonist, SR 141716A

This study examined the effect of the cannabinoid receptor agonist, WIN 55212-2, on the electrically evoked release of [14C]acetylcholine (ACh) from superfused brain slices from the hippocampus, a region with a high density of cannabinoid receptors. A comparison was also made with [14C]ACh release from the nucleus accumbens, which has relatively fewer cannabinoid receptors. In the hippocampal slices, WIN 55212-2 produced a dose-dependent inhibition of [14C]ACh release, with an EC50 of 0.03 microM and a maximal inhibition of 81% at 1 microM. In the nucleus accumbens slices, WIN 55212-2 produced a weak inhibition of [14C]ACh release, which did not quite reach statistical significance. The inhibition of electrically evoked hippocampal [14C]ACh release by WIN 55212-2 could be prevented by the cannabinoid receptor antagonist, SR 141716A (EC50, 0.3-1.0 microM). In addition to antagonizing the effects of WIN 55212-2, SR 141716A alone produced a 2-fold potentiation of the electrically stimulated [14C]ACh release in this region (EC50, 0.1-0.3 microM). By contrast, in nucleus accumbens slices, no potentiation of the stimulated release of [14C]ACh release by SR 141716A was observed. Basal [14C]ACh release was unaffected by WIN 55212-2 or SR 141716A in either area. These results suggest that cannabinoid receptor activation can produce a strong inhibition of ACh release in the hippocampus. Furthermore, the potentiation of ACh release in the hippocampus by SR 141716A alone suggests either that this compound is an inverse agonist at cannabinoid receptors or it is antagonizing the actions of an endogenous ligand acting on these receptors.     

Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus

The effects of cibenzoline on transmembrane action potentials were examined in right ventricular papillary muscles and in single ventricular myocytes isolated from guinea-pig hearts. In papillary muscles, cibenzoline > or = 3 microM caused a significant decrease in the maximum upstroke velocity (Vmax) of the action potential without affecting the action potential duration. The inhibition of Vmax was enhanced at higher stimulation frequencies. In the presence of cibenzoline, trains of stimuli at rates > or = 0.2 Hz led to a use-dependent inhibition of Vmax. The time constant for Vmax recovery (tauR) from the use-dependent block was 26.2 s. The use-dependent block of Vmax with cibenzoline was enhanced and tauR was shortened when the resting potential was depolarized by high (8, 10 mM) [K+]o. The curve relating membrane potential and Vmax in single myocytes was shifted by cibenzoline (10 microM) in a hyperpolarizing direction by 7.1 mV. In myocytes treated with cibenzoline (10 microM), a 10-ms conditioning clamp to 0 mV caused a significant decrease in Vmax of the subsequent test action potential; the Vmax inhibition was enhanced modestly in association with a prolongation of the 0 mV clamp pulse duration. In the presence of cibenzoline (3 microM), application of a train of depolarizing pulses (10 ms, 200 ms) to myocytes from the resting level (-80 mV) to 0 mV resulted in a progressive Vmax reduction in a pulse number-dependent manner. Unlike glibenclamide (30 microM), cibenzoline (10 microM) did not prevent the hypoxia-induced shortening of action potential duration in papillary muscles. These findings indicate that the onset and offset kinetics of use-dependent Na+ channel block by cibenzoline are slow. Given its state dependence, cibenzoline may be a blocker of activated Na+ channels. The inhibitory action of this compound on the ATP-sensitive K+ current (I(K), ATP) would be minimal or negligible at concentrations causing sufficient Na+ channel block.     

Comparative effects of glibenclamide, tedisamil, dofetilide, E-4031, and BRL-32872 on protein kinase A-activated chloride current in guinea pig ventricular myocytes

The modulation of the protein kinase A-activated chloride current (PKA-I[Cl]) may lead to modification of the cardiac action potential shape. The purpose of this study was to evaluate the effects of glibenclamide, tedisamil, dofetilide, E-4031, and BRL-32872 on the PKA-I(Cl). Experiments were conducted by using the patch-clamp technique in guinea pig ventricular myocytes. PKA-I(Cl) was activated by application of 1 microM isoproterenol and was inhibited by 1 microM propranolol, 10 microM acetylcholine, or 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS). The sulfonylurea receptor inhibitor, glibenclamide, inhibited PKA-I(Cl) at micromolar concentration. Among class III antiarrhythmic agents, tedisamil induced a dose-dependent inhibition of PKA-I(Cl) with a half effective concentration (EC50) of 7.15 microM (Hill coefficient, 0.54). This effect may contribute to action potential widening induced by tedisamil. In contrast, the selective inhibitors of the rapid component of the delayed rectifier K current (I[Kr]), dofetilide, and E-4031, as well as BRL-32872, that blocks I(Kr) and the L-type calcium current, did not significantly affect the amplitude of PKA-I(Cl), even at high concentrations (10-30 microM). These results demonstrate that compounds such as glibenclamide and tedisamil that are known to block the adenosine triphosphate (ATP)-sensitive K current also affect PKA-I(Cl). Furthermore it appears that blockade of PKA-I(Cl) is not a common feature for all class III antiarrhythmic agents.     

Endothelin-1 inhibits L-type Ca2+ current enhanced by isoprenaline in rat atrial myocytes

Endothelin-1 (ET-1) was shown to exert direct cardiac effects by complex signaling pathways and to interact with neurotransmitter regulation of cardiac activity. The effect of ET-1 was investigated on the beta-adrenergic stimulation of cardiac L-type Ca2+ current (ICaL) on isolated rat atrial myocytes by using the patch-clamp technique. ET-1 (5 x 10(-8) M) reversed the increase in ICaL induced by isoprenaline (10(-6) M) but had no effect on basal ICaL and on (-) Bay K 8644-increased ICaL (10(-6) M); so ET-1 might exert an effect only when the Ca2+ channels are phosphorylated. The antiadrenergic action of ET-1, blocked by BQ-123 (10(-6) M) and unaffected by IRL 1038 (3.5 x 10(-8) M) should be mediated by ET-A receptors. The inhibitory action of ET-1 was still observed when ICaL was previously increased by forskolin (3 x 10(-6) M), 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP; 200 microM), or cAMP (100 microM) in presence of isobutyl methyl xanthine (IBMX; 10(-6) M), suggesting that the antiadrenergic action of ET-1 on ICaL was exerted independent of the cAMP-dependent phosphorylation pathway. ET-1 is known to be an activator of phosphoinositide hydrolysis, resulting in an increased production of IP3 and diacylglycerol (DAG). A Ca(2+)-dependent inhibition of ICaL consequently to an elevation of the intracellular Ca2+ pool via IP3 might be excluded in the action of ET-1, because of the presence of EGTA in the intrapipette medium. ET-1 reversed the isoprenaline-induced increase in ICaL in the presence of protein kinase C inhibitor [PKC(19-31); 100 microM), making unlikely the involvement of a DAG-dependent activation of PKC. Therefore the antiadrenergic action of ET-1 might also be independent on the phosphoinositide pathway.