Involvement of adenosine in the effect of fluoxetine on isolated guinea-pig atria.

The effect of fluoxetine (FL) a selective serotonin reuptake inhibitor antidepressant was studied on the rate and force of contractions of isolated guinea-pig atria. FL (2-16 microg/ml) caused a decrease in the rate (13-45%) and contractile force (41-53%) of isolated guinea-pig atria in a dose-dependent manner. These negative inotropic and chronotropic effect of FL (4 microg/ml) were not prevented by atropine (1 microg/ml) and 3,7-dimethyl-1-propargylxanthine (DMPX; 1.5 microg/ml), an adenosine A(2) receptor antagonist, but 1,3-dipropargyl-8-cyclopentylxanthine (DPCPX;12 microg/ml), a specific adenosine A(1) receptor antagonist significantly blocked these effects (P < 0.001) and theophylline (30 microg/ml) a non- selective adenosine A(1)/A(2A) receptor antagonist also prevented the inotropic and chronotropic effects of FL. These results suggest that the negative chronotropic and inotropic effect of FL on isolated guinea-pig atria is probably mediated through an inhibition of the reuptake of adenosine or the A(1) receptor mechanism.     

Characterization of the 5-hydroxytryptamine receptor mediating the positive inotropic response in guinea-pig isolated left atria.

1. 5-Hydroxytryptamine (5-HT), in the presence of propranolol (1 microM), atropine (3 microM) and ketanserin (1 microM), induced a positive inotropic response of guinea-pig isolated electrically paced left atria (pEC50 = 7.52). The positive inotropic response was mimicked by alpha-methyl-5-HT (pEC50 = 7.26) and 5-carboxamidotryptamine (5-CT; pEC50 = 6.56) but not by sumatriptan or 1-(m-chlorophenyl) piperazine (m-CPP). 2. The 5-HT induced positive inotropic response was competitively antagonized by both mesulergine (pA2 = 7.68) and methiothepin (pA2 = 6.67). Methysergide was a surmountable antagonist at 3 nM producing a rightward shift in the 5-HT concentration-response curve giving an apparent pA2 = 9.2 with no significant reduction in the maximum. At higher concentrations, methysergide behaved as an insurmountable antagonist, significantly reducing the maximum response to 5-HT as well as producing rightward shifts in the 5-HT concentration-response curves. 3. The 5-HT-induced positive inotropic response was not antagonized by either tropisetron (10 microM) or yohimbine (10 microM). 4. The guinea-pig atrial 5-HT receptor does not satisfy the criteria for any of the currently recognised 5-HT receptor subtypes and appears to have some similarities to the atypical 5-HT receptors previously described in other peripheral tissues.     

Role of N-type calcium channels in autonomic neurotransmission in guinea-pig isolated left atria.

1. Calcium entry via neuronal calcium channels is essential for the process of neurotransmission. We investigated the calcium channel subtypes involved in the operation of cardiac autonomic neurotransmission by examining the effects of selective calcium channel blockers on the inotropic responses to electrical field stimulation (EFS) of driven (4 Hz) guinea-pig isolated left atria. In this tissue, a previous report (Hong & Chang, 1995) found no evidence for N-type channels involved in the vagal negative inotropic response and only weak involvement in sympathetic responses. 2. The effects of cumulative concentrations of the selective N-type calcium channel blocker, omega-conotoxin GVIA (GVIA; 0.1-10 nM) and the non-selective N-, P/Q-type calcium channel blocker, omega-conotoxin MVIIC (MVIIC; 0.01-10 nM) were examined on the positive (with atropine, 1 microM present) and negative (with propranolol, 1 microM and clonidine, 1 microM present) inotropic responses to EFS (eight trains, each train four pulses per punctate stimulus). 3. GVIA caused complete inhibition of both cardiac vagal and sympathetic inotropic responses to EFS. GVIA was equipotent at inhibiting positive (pIC50 9.29+/-0.08) and negative (pIC50 9.13+/-0.17) inotropic responses. MVIIC also mediated complete inhibition of inotropic responses to EFS and was 160 and 85 fold less potent than GVIA at inhibiting positive (pIC50 7.08+/-0.10) and negative (pIC50 7.20+/-0.14) inotropic responses, respectively. MVIIC was also equipotent at inhibiting both sympathetic and vagal responses. 4. Our data demonstrates that N-type calcium channels account for all the calcium current required for cardiac autonomic neurotransmission in the guinea-pig isolated left atrium.     

Interaction of amrinone with endogenous adenosine in guinea-pig atria.

In spontaneously beating atria from reserpine-treated guinea-pigs, amrinone (10 microM to 2 mM) induced a positive inotropic and chronotropic effect that was preceded by a transient reduction in contractile force and in frequency. Both the positive and negative effects were concentration-dependent. The inotropic action of amrinone was antagonized by low concentrations of 8-phenyltheophylline that compete with adenosine at R-receptors on plasma membrane without significantly influencing phosphodiesterase activity. Cumulative concentrations of amrinone (1 mM) antagonized the reduction of rate of contraction and amplitude induced by dipyridamole 1 microM in spontaneously beating atria and restored the maximum contractile effect reached in the absence of dipyridamole. In spontaneously beating preparations incubated in the presence of adenosine deaminase (1 u ml-1), amrinone lost its positive effects on the atria and only reduction of rate and contractile force was evident. Both effects were antagonized by scopolamine 1 mM thus indicating their cholinergic nature. Adenosine at 0.1 microM and 0.5 microM significantly inhibited the inotropic effect induced by amrinone (0.03 to 3 mM) and the concentration-effect curves of amrinone obtained in the absence and presence of adenosine clearly indicate a competitive antagonism between the two drugs. Thus the contractile activity of amrinone in spontaneously beating atria from reserpine-treated guinea-pigs originates from a displacement of adenosine from its R-receptor sites in the cardiac cell.     

Apparent affinity of some 8-phenyl-substituted xanthines at adenosine receptors in guinea-pig aorta and atria.

1 Some 8-phenyl-substituted, 1,3 dipropyl xanthines have previously been demonstrated to have a 20-400 fold greater affinity for A1 binding sites in rat CNS membranes than for A2 adenosine receptors in intact CNS cells from guinea-pigs. In the present study these compounds (1,3, dipropyl-8-phenylxanthine: DPPX; 1,3 dipropyl-8-(2 amino-4-chlorophenyl) xanthine: PACPX; 8-(4-(2-amino-ethyl)amino) carbonyl methyl oxyphenyl)-1,3-dipropylxanthine: XAC; and D-Lys-XAC) together with two that have not been reported to exhibit A1-receptor selectively (8-(p-sulphophenyl)theophylline: 8-PST; 8-(4-carboxy methyl oxyphenyl)-1,3-dipropylxanthine: XCC) have been evaluated as antagonists of the effects of 2-chloroadenosine in two isolated cardiovascular tissues. 2 The isolated tissues used were guinea-pig atria (bradycardic response) and aorta (relaxation), which are thought to possess A1 and A2 adenosine receptors, respectively. 3 All the xanthines antagonized responses evoked by 2-chloroadenosine in both tissues but did not affect responses evoked by acetylcholine (atria) or sodium nitrite (aorta). 4 The xanthines, 8-PST, XAC, D-Lys XAC, XCC and DPPX appeared to be competitive antagonists of the effects of 2-chloroadenosine, as Schild plot slopes did not differ significantly from unity. The 1,3-dipropyl substituted compounds had pA2 values from 6.5 to 7.4 and were more potent than the 1,3 dimethyl substituted 8-PST (pA2 4.9 to 5). 5 For individual xanthines, there was no difference between pA2 values obtained in the atria and in the aorta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of tachykinin receptors in isolated basilar arteries of guinea-pig.

In circular lengths cut from the basilar artery of guinea-pig (0.2-0.3 mm o.d.) relaxations induced by substance P and neurokinin A were highly susceptible to mechanical damage of the endothelium by rubbing. The precontraction induced by prostaglandin F2 alpha but not that of 124 mM potassium was reduced considerably by the rubbing procedure. Concentration-dependent relaxations were evoked by tachykinin agonists in the following order of potency: substance P = physalaemin greater than neurokinin A greater than eledoisin. Physalaemin was, however, a partial agonist, giving only half the maximum relaxation as compared to the other tachykinins. The two putative tachykinin receptor antagonists, spantide ([D-Arg1, D-Trp7,9, D-Leu11] substance P) and [D-Pro2, D-Trp7,9] substance P, shifted the concentration-dependent relaxations of substance P to the right in a parallel manner. Calculation of pA2 values and Schild plot analysis revealed pA2 values of 7.4-7.6 for spantide and 6.9-7.0 for [D-Pro2, D-Trp7,9] substance P, irrespective of whether substance P or neurokinin A was used as agonist. The pA2 values and the Schild plot analysis suggest a specific interaction between tachykinin agonists and antagonists that follow a simple bimolecular process. The results suggest the presence of tachykinin receptors of the 'SP-P' type in guinea-pig basilar arteries which, for induction of relaxation, involves the release of an endothelium-derived relaxing factor.     

Analysis of the atypical characteristics of adenosine receptors mediating negative inotropic and chronotropic responses of guinea-pig isolated atria and papillary muscles.

1. Adenosine receptor(s) mediating negative inotropy of paced left atria, isoprenaline-stimulated paced left atria and papillary muscles, and negative chronotropy of spontaneously beating right atria were characterized. 2. Isometric tension of guinea-pig isolated paced left atria and left ventricular papillary muscles and rate of contraction of spontaneously beating right atria were recorded. Papillary muscles were pre-stimulated with isoprenaline (1x10-8 M). Concentration-response curves (CRCs) for tension or rate reduction by N6-cyclopentyladenosine (CPA), the stereoisomers of N6-(2-phenylisopropyl)adenosine ((+)-PIA and (-)-PIA), 5'-(N-carboxamido)adenosine (NECA), N6-2-(4-aminophenyl)ethyladenosine (APNEA) and N6-(3-iodobenzyl)adenosine-5'-N-methyuromide (IB-MECA) revealed a potency order of CPA=(-)-PIA>NECA in right atria and papillary muscles, which is consistent with involvement of A1-receptors. The potency order in left atria was CPA=NECA>(-)-PIA>(+)-PIA>APNEA, which is not typical of A1 adenosine receptors. Weak activity of APNEA and IB-MECA discounts involvement of A3 receptors. 3. pA2 values for the antagonism of CPA by 8(p-sulfophenyl)theophylline (8-SPT) were calculated from Schild plots (log concentration-ratio against log 8-SPT concentration), the unity slopes of which indicated competitive antagonism. The pA2 value in the papillary muscles was significantly higher than for atrial preparations, indicating a possible difference in receptor characteristics between atrial and papillary muscle responses. 4.In left and right atria there was a limit to the displacement of the CPA CRCs at higher concentrations of 8-SPT. The 8-SPT-resistant component of the response is suggested to arise from duality of coupling of a common A1 receptor through either different G proteins or G protein subunits to independent transduction pathways. The results with papillary muscles can be explained by a typical A1 receptor coupled to a single transduction pathway.     

Characterization of opioid receptors modulating the function of capsaicin-sensitive neurons in guinea-pig atria

Transmural nerve stimulation of isolated atria, obtained from reserpine-pretreated guinea-pigs, in the presence of atropine and the β₁-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. [D-Ala², N-Me-Phe⁴, Gly⁵-ol] enkephalin (DAGO), [D-Ala², D-Leu⁵]enkephalin (DADLE), morphine and dynorphin dose dependently reduced the cardiac response to transmural nerve stimulation. The δ receptor selective agonist [D-Pen², D-Pen⁵]enkephalin (DPDPE), and the κ receptor agonist, U50488, were unable to modify the response. The inhibitory effect of all the active opioid agonists was antagonized by naloxone but not by the selective δ and κ opioid receptor antagonists, ICI 174.864 and MR 2266. These results suggest the presence on sensory nerve terminals of inhibitory opioid receptors belonging to the μ, but not to the δ and κ subtypes.     

Mechanism of inhibitory effect of citalopram on isolated guinea-pig atria in relation to adenosine receptor

The effect of citalopram (CTP), a selective serotonin reuptake inhibitor antidepressant was studied on the rate and force of contractions of isolated guinea-pig atria. CTP (2-32 microg/ml) caused a dose-dependent decrease in the contractile force (7%-62%) and in the rate of contractions (11%-72%). These negative inotropic and chronotropic effects of CTP (8 microg/ml) were not prevented by atropine (1 microg/ml) and 3,7 dimethyl-1-propargylxanthine (DMPX; 1.5 microg/ml), an adenosine A(2) receptor antagonist, but 1,3 dipropargyl-8-cyclopentylxanthine (DPCPX; 12 microg/ml), a specific adenosine A(1) receptor antagonist significantly blocked these effects (p < 0.001) and theophylline (30 microg/ml) a non-selective adenosine A(1)/A(2A) receptor antagonist also prevented the inotropic and chronotropic effects of CTP. These results suggest that the negative inotropic and chronotropic effect of CTP on isolated guinea-pig atria is probably mediated through an inhibition of the uptake of adenosine or the A(1) receptor mechanism.     

Characterization of adenosine receptors in isolated cerebral arteries of cat.

The effect of some adenosine analogues and xanthine derivatives were studied on isolated cerebral arteries from cats. The adenosine analogues caused an almost complete relaxation of cerebral arteries contracted by prostaglandin F2 alpha (PGF2 alpha, 30 microM). The order of potency was: 5-N-ethylcarboxamide adenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than L-N6-phenylisopropyl adenosine (L-PIA). The analogue D-PIA was very weak and its maximum effect was small. NECA and L-PIA enhanced [3H]-cyclic AMP accumulation in [3H]-adenine labelled feline pial vessels with similar absolute and relative potency to their relaxant effects. The relaxant effects of adenosine and of NECA were competitively antagonized by 8-phenyl-theophylline (pA2 = 6.5). The effect of theophylline and enprofylline could not be tested in higher concentrations than 30 or 10 microM because they affected the vessels directly. At these concentrations they were essentially inactive as adenosine antagonists. The non-xanthine phosphodiesterase inhibitor rolipram (0.1 and 100 microM) caused a slight but non-significant potentiation of the relaxant effect of adenosine. The results are compatible with the opinion that adenosine relaxes cerebral vessels by an action on adenosine A2-receptors. The effect may be linked to adenylate cyclase and can be antagonized by 8-phenyl-theophylline.     

Characterization of histamine receptors modulating inotropic and biochemical activities in rabbit left atria.

The experiments were performed to identify histamine H1- and H2-receptors in rabbit left atrium and to characterize the pharmacological properties mediated by the respective subtypes of histamine receptors. High-affinity saturable binding to the left atrial membranes was obtained for [3H]mepyramine, yielding a maximum binding capacity (Bmax) of 96 fmol/mg of protein and an equilibrium dissociation constant (KD) of 3.8 nM and also for [3H]tiotidine, yielding a Bmax of 126 fmol/mg of protein and a KD of 14.7 nM. In isolated left atrium, histamine produced a concentration-dependent positive inotropic effect, an effect which was competitively antagonized by cimetidine but not altered by chlorpheniramine. Schild analysis showed that the pA2 value for cimetidine was 6.55 and the slope was not significantly different from unity. An excellent correlation was found between the increase in force of contraction and cyclic AMP in the presence of histamine, suggesting that the positive inotropic effect of histamine in rabbit left atrium is dependent on an increased level of intracellular cyclic AMP through stimulation of histamine H2-receptors. Histamine also produced concentration-dependent stimulation of phosphoinositide hydrolysis as measured by [3H]inositol monophosphate accumulation. The phosphoinositide response to histamine was blocked by chlorpheniramine and mepyramine but not by cimetidine. The data indicate that histamine H1-receptors, in addition to histamine H2-receptors, are present in the rabbit left atrium. Although this tissue lacks an inotropic response to histamine H1-receptor stimulation, the histamine H1-receptors interact with histamine to mediate the stimulation of phosphoinositide hydrolysis.     

Papaverine enhances the effect of adenosine in guinea-pig atria

Summary Papaverine, while enhancing the force of contraction of guinea-pig atria, remarkably and dose-dependently enhanced the negative inotropic response of the atria to adenosine. It also enhanced the actions of ATP and other adenine nucleotides, but not those of 2-chloroadenosine and ACh. At similar concentrations, papaverine inhibited the uptake of adenosine by the atrial tissue during incubation with adenosine. Adenosine in the medium was degraded to inactive inosine during incubation with the atrial tissue, and papaverine reduced its degradation.The enhancing effect of papaverine on the action of adenosine on guinea-pig atria was like those of dipyridamole, 6-(2-hydroxy-5-nitrobenzyl)thioguanosine and cinepazide. The effect seemed to be due mainly to inhibition of adenosine uptake into the tissue. Inhibition of adenosine degradation may also have contributed to the action of papaverine, but this action was probably much less important than inhibition of adenosine uptake.     

Antagonist effects of seglitide (MK 678) at somatostatin receptors in guinea-pig isolated right atria.

Somatostatin (SS) exerts a negative inotropic effect in isolated atria. Here we report that in guinea-pig isolated right atria, seglitide, a potent cyclic hexapeptide somatostatin agonist, behaves as a competitive somatostatin receptor antagonist with pA2 values against SS14, SS25 and SS28, of 6.50 +/- 0.40, 6.24 +/- 0.08 and 6.09 +/- 0.06, respectively. Seglitide had little or no effect on the negative inotropic action of carbachol or N6-cyclohexyladenosine. Our findings indicate that the receptor-response coupling characteristics of guinea-pig atria are such that in this preparation seglitide has low intrinsic activity and behaves specifically as a somatostatin receptor antagonist.     

Characterization of adenosine receptors in the rat isolated aorta

• 1.1. Adenosine and its analogues relaxed the isolated rat aorta by an endothelium-dependent mechanism with an order of potency of 5′-N-ethylcarboxamidoadenosine (NECA) > 2-(p-(2-carboxyethyl)phenethylamino)-5′-N-ethylcarboxamidoadenosine (CGS 21680) > adenosine = N⁶-(2-(4-aminophenyl)ethyl)adenosine (APNEA = N⁶-cyclopentyladenosine (CPA) > 5′ - methylthioadenosine (MTA), although the maximal response achieved by CGS 21680 was less than that achieved by NECA.
• 2.2. Both 8-sulphophenyltheophylline (8-SPT) and MTA antagonized responses to the adenosine analogues, but there were some anomolous features of this antagonism and NECA was inhibited more powerfully than the other agonists. This suggests that as well as A_2a receptors mediating relaxation, the rat aorta may relax to adenosine analogues by other mechanisms.

     

Characterization of receptors on postganglionic cholinergic neurons in the guinea-pig isolated ileum.

Dopamine, apomorphine, noradrenaline and isoprenaline reduced the response of the isolated guinea-pig ileum to exogenous acetylcholine by a maximum of 40%. Propranolol reversed this inhibition whilst phentolamine and pimozide were ineffective, suggesting that the drugs were acting on a post-synaptic beta-adrenoceptor. The same agonists were more effective as inhibitors of the response to transmural electrical stimulation of the ileum, lower doses producing almost complete inhibition. This inhibition was partially antagonized by phentolamine, pimozide and propranolol. Clonidine proved to be the most potent inhibitor of the response to transmural electrical stimulation, whilst phenylephrine was ineffective. pA2 determinations showed that phentolamine was a potent antagonist of clonidine but a weak antagonist of apomorphine whilst for pimozide the opposite was true. The results suggest that there are two populations of prejunctional receptors on the cholinergic nerves innervating the smooth muscle of the guinea-pig ileum. One receptor is similar to a classical prejunctional alpha-adrenoceptor and the other resembles a central dopamine receptor.     

GABAA receptor-mediated positive inotropism in guinea-pig isolated left atria: evidence for the involvement of capsaicin-sensitive nerves.

1. Isolated left atria from reserpine-pretreated guinea-pigs, electrically driven (3 Hz) in the presence of atropine (1 microM), phentolamine (0.3 microM) and propranolol (1 microM), responded to a train of stimuli (10 Hz for 2.5s) with a delayed neurogenic positive inotropic response which was insensitive to hexamethonium (10 microM) but abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to calcitonin gene-related peptide (CGRP). 2. In these experimental conditions, gamma-aminobutyric acid (GABA) produced a concentration-related (10 microM-1 mM) positive inotropic response similar to that produced by electrical field stimulation. The effect of GABA was competitively antagonized by bicuculline methiodide (10 microM), a GABAA receptor antagonist. 3. The selective GABAA receptor agonists, muscimol and homotaurine mimicked the positive inotropic effect of GABA while baclofen, the selective GABAB receptor agonist, did not. 4. The action of GABA (1 mM) was abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to CGRP, while it was unaffected by hexamethonium. In contrast, the inotropic response to CGRP was unaffected by tetrodotoxin, omega-conotoxin, bicuculline methiodide, hexamethonium or in vitro capsaicin desensitization, but was abolished by CGRP desensitization. 5. In the spontaneously beating guinea-pig right atrium, GABA (1 microM) produced a small and transient positive chronotropic effect that was no longer observed after in vitro desensitization with capsaicin (1 microM). 6. In the guinea-pig isolated perfused heart from reserpine-pretreated animals (with atropine, phentolamine and propranolol in the perfusion medium), GABA (1 microM) produced a transient tachycardia and a small increase in coronary flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of dehydroemetine on isolated guinea-pig atria

1. The mechanisms of the inhibitory effect of dehydroemetine on the heart was investigated using spontaneously contracting isolated guinea-pig atria.

2. In normal Locke solution, dehydroemetine caused a reduction in the rate and amplitude of contraction of the atria. The effect was antagonized by adrenaline and augmented by acetylcholine.

3. Increasing the potassium concentration of the bathing medium increased the inhibitory effect of dehydroemetine on the atria, while decreasing the potassium concentration antagonized the effect of dehydroemetine.

4. The character of the inhibitory effect of dehydroemetine on the atria altered when the sodium concentration of the bathing solution was lowered to 75·6 mmol. In the normal sodium medium arrest of beat by dehydroemetine was gradual; in the low sodium medium, it was abrupt.

5. In the low, as in the normal sodium medium, the inhibitory effect of dehydroemetine was more marked the higher the potassium concentration of the bathing medium.

6. In both standard Locke solution and Locke solution containing 75·6 mmol sodium, the effect of dehydroemetine closely mimicked that obtained by adding excess potassium to the solution.

7. On the basis of the foregoing it was suggested that dehydroemetine acts by increasing the permeability of the myocardial cell membrane to potassium thereby leading to the accumulation of potassium in the extracellular space.

     

Acute desensitization of adenosine by adenosine infusion in isolated dog atria

We investigated the effects of a continuous infusion of adenosine on responses of sinus rate and developed tension to bolus injections of adenosine and acetylcholine in isolated, blood-perfused canine right atria. Each drug was directly injected into the sinus node artery of the isolated atrium. During adenosine infusions, the negative chronotropic and inotropic responses to bolus injections of adenosine were significantly inhibited in a dose-related manner, while the negative responses to administered acetylcholine were consistently potentiated. These results indicate that adenosine infusion has an acute desensitizing action on subsequent bolus adenosine, although the cholinergic action is enhanced by a continuous infusion of adenosine.