Activation of myocardial adenyl cyclase by histamine in guinea pig, cat, and human heart

Histamine has positive inotropic and chronotropic effects on the heart which are not abolished by beta adrenergic-blocking agents. Since the positive inotropic and chronotropic effects of other hormones on the heart are thought to be mediated by cyclic 3',5'-AMP, we examined the effect of histamine on adenyl cyclase in particulate preparations of guinea pig, cat, and human myocardium. Histamine at the peak of its dose-response curve, 3 x 10(-4)moles/liter, produced approximately a 300% increase in cyclic 3',5'-AMP accumulation in the guinea pig, 60% in the cat, and 90% in the human heart particles. Half-maximal activity for the histamine mediated activation of adenyl cyclase in the guinea pig was 9 x 10(-6)moles/liter, almost identical with that observed for norepinephrine in the same preparation. DL-Propranolol, 1 x 10(-5)moles/liter, did not abolish the activation of adenyl cyclase produced by histamine but did abolish the activation produced by norepinephrine. In contrast, diphenhydramine hydrochloride, Benadryl, 8 x 10(-5)moles/liter, abolished the activation of adenyl cyclase by histamine but not that produced by norepinephrine. These data suggest that there are at least two receptor sites in guinea pig heart mediating the activation of adenyl cyclase, one responsive to histamine, the other to norepinephrine. In addition, combined maximal doses of histamine and norepinephrine produced completely additive effects on the activation of adenyl cyclase, which suggests that at least two separate adenyl cyclase systems are present in the heart, each responsive to one of these hormones. However, definitive proof would require physical separation of the two enzymes.     

A1 and A2 adenosine receptors in rabbit cortical collecting tubule cells. Modulation of hormone-stimulated cAMP.

Adenosine analogs were used to investigate the cellular mechanisms by which adenosine may alter renal tubular function. Cultured rabbit cortical collecting tubule (RCCT) cells, isolated by immunodissection, were treated with 5'-N-ethylcarboxamideadenosine (NECA), N6-cyclohexyladenosine (CHA), and R-N6-phenylisopropyladenosine (PIA). All three analogs produced both dose-dependent inhibition and stimulation of RCCT cell cyclic AMP (cAMP) production. Stimulation of cAMP accumulation occurred at analog concentrations of 0.1 microM to 100 microM with the rank order of potency NECA greater than PIA greater than CHA. Inhibition occurred at concentrations of 1 nM to 1 microM with the rank order of potency CHA greater than PIA greater than NECA. These effects on cAMP production were inhibited by 1,3-diethyl-8-phenylxanthine and isobutylmethylxanthine. CHA (50 nM) blunted AVP- and isoproterenol-stimulated cAMP accumulation. This modulation of hormone-induced cAMP production was abolished by pretreatment of RCCT cells with pertussis toxin. Prostaglandin E2 production was unaffected by 0.1 mM CHA. These findings indicate the presence of both inhibitory (A1) and stimulatory (A2) receptors for adenosine in RCCT cells. Moreover, occupancy of the A1 receptor causes inhibition of both basal and hormone-stimulated cAMP formation through an action on the inhibitory guanine nucleotide-binding regulatory component, Ni, of the adenylate cyclase system.     

Influence of a phosphodiesterase inhibitor on the chronotropic effects of glucagon and norepinephrine in fetal mouse hearts

Fetal mouse hearts develop tachycardia in response both to norepinephrine and to glucagon, but although adenylate cyclase is stimulated and adenosine 3':5'-monophosphate (cyclic AMP) elevated by norepinephrine, no measurable changes are produced by glucagon. To test further the possible independence of glucagon chronotropy from the cyclic AMP system, the effects of a phosphodiesterase inhibitor were evaluated. The dose-response curve to norepinephrine was shifted to the left by the phosphodiesterase inhibitor 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Ro7-2956), but the dose-response curve to glucagon was unaltered. Thus, 10(-6) M norepinephrine produced an increase of 40 +/- 5 beats/min in hearts pretreated with Ro7-2956, as compared to an increase of 22 +/- 3 in control hearts (P less than .01). In contrast, 10(-6) M glucagon produced a rate increase of 25 +/- 4 beats/min in treated hearts vs. 26 +/- 4 beats/min in controls. These data are compatible with the hypothesis that adenylate cyclase and cyclic AMP are involved in the chronotropic response of the fetal mouse heart to norepinephrine but not to glucagon.     

Functional evidence for adenosine A2 receptor regulation of phosphatidylcholine secretion in cultured type II pneumocytes

We examined the effects of P1 purinoceptor agonists on secretion of phosphatidylcholine in primary cultures of rat type II pneumocytes. Adenosine and its nonmetabolizable analogs, 5'-N-ethylcarboxyamidoadenosine (NECA), N6-phenylisopropyladenosine (PIA) and 2-chloroadenosine, increased secretion, and this effect was dependent on concentration. At the highest concentration, all agonists stimulated phosphatidylcholine secretion approximately 2-fold. NECA, with an EC50 of 8.9 X 10(-8) M, was the most potent agonist. The order of potency was NECA greater than 2-chloroadenosine = L-PIA greater than or equal to adenosine greater than D-PIA. The stimulatory effect of 10(-6) M NECA was diminished by the P1 antagonists theophylline and 8-phenyltheophylline. The degree of stimulation by the adenosine analogs as well as its time course was the same as that induced by terbutaline. The effects of the adenosine analogs and of terbutaline were not additive, suggesting a similar mode of action for the beta adrenergic and purinoceptor agonists. Terbutaline and the adenosine analogs increased intracellular cyclic AMP levels. Again, NECA was the most potent adenosine analog. For NECA, L-PIA and adenosine, there was a significant correlation between effects on secretion and on cyclic AMP content. These data suggest that the effect of adenosine on phosphatidylcholine secretion in type II pneumocytes is mediated by the A2 subtype of the P1 purinoceptor.     

EFFECTS OF PURINES AND FORSKOLIN ON HAEMOLYSIS

Effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine on the antihaemolytic action of procaine, and the effect of ATP on the antihaemolytic actions of lidocaine, dibucaine, tetracaine, pentobarbitone, and chlorpromazine were investigated in rat erythrocytes. The effects of adenosine and its analogues D-N6-phenylisopropyladenosine (D-PIA), L-N6-phenylisopropyladenosine (L-PIA), N6-cyclohexyladenosine (CHA), 2-chloroadenosine, and N6-methyladenosine, as well as the effects of cyclic-AMP (cAMP), dibutyryl cAMP, and forskolin on haemolysis were also investigated in rat erythrocytes. ATP, ADP, and AMP, but not adenosine, antagonized in a concentration-dependent manner the antihaemolytic action of procaine, and ATP was ineffective against the antihaemolytic actions of lidocaine, dibucaine, tetracaine, pentobarbitone, and chlorpromazine. Adenosine and its analogues, but not N6-methyladenosine, protected erythrocytes against hypotonic haemolysis in a concentration-dependent manner. The order of potencies was: D-PIA greater than CHA greater than L-PIA greater than adenosine greater than 2-chloroadenosine. The effect of adenosine on haemolysis was not prevented by theophylline, 8-phenyltheophylline, or 8-sulfophenyltheophylline. Dibutyryl cAMP, a stable analogue of cAMP, and forskolin, a specific activator of adenylate cyclase, mimicked the antihaemolytic action of adenosine. cAMP was less efficient than adenosine. Adenine nucleotides antagonized the antihaemolytic action of procaine, probably due to calcium-chelating properties. In contrast, adenine nucleosides have antihaemolytic properties. The possibility that the antihaemolytic effects of these substances relate to cAMP and/or to lipophilicity is discussed.     

Cardiac effects of adenosine and adenosine analogs in guinea-pig atrial and ventricular preparations: evidence against a role of cyclic AMP and cyclic GMP

The effects of adenosine, the Ri site adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA), the Ra site agonist 5'-N-ethylcarboxamideadenosine (NECA) and the P site agonist 2',5'-dideoxyadenosine (DIDA) on force of contraction, cyclic AMP (cAMP) and cyclic GMP (cGMP) content and on transmembrane action potential were studied in isolated electrically driven left auricles and papillary muscles from guinea pigs. Furthermore, the effects on adenylate cyclase activity in a particulate membrane preparation were investigated. In the auricles, adenosine, PIA and NECA had negative inotropic effects which were accompanied by a shortening of the action potential. Theophylline antagonized these effects which are likely mediated by R site adenosine receptors. DIDA was ineffective. Except for a small positive inotropic effect of adenosine the analogs were ineffective in the papillary muscles. None of the mechanical effects was accompanied by a change in cAMP and cGMP content in the intact preparations. In the broken cell preparation PIA and NECA had no effect on adenylate cyclase activity. Adenosine and DIDA inhibited the enzyme. The latter effects can be classified as P site-mediated effects. In conclusion, distinct mechanical, i.e., negative inotropic effects of adenosine and its analogs in the heart are observed in auricular preparations only. These effects are unlikely to be related to the cAMP and/or cGMP system. Instead, they are probably due to a direct shortening of the action potential which, in turn, is conceivably due to an increase in K+ outward current and a secondary decrease in Ca++ inward current. This effect is apparently mediated by cardiac R site adenosine receptors which are not detectably coupled to adenylate cyclase.     

Adenosine and muscarinic cholinergic receptors attenuate cyclic AMP accumulation by different mechanisms in 1321N1 astrocytoma cells

An adenosine receptor has been characterized to unambiguously demonstrate that the inhibitory guanine nucleotide regulatory protein, Gi, of 1321N1 human astrocytoma cells is fully capable of functionally coupling to adenylate cyclase. Adenosine receptor agonists attenuated cyclic AMP accumulation by 35 to 75% with the order of potency of N6(R-phenylisopropyl)-adenosine greater than adenosine = 2-chloroadenosine greater than N6-methyladenosine = N6-benzyladenosine. 3-Isobutyl-1-methylxanthine competitively antagonized the effect of adenosine receptor agonists. Adenylate cyclase activity measured in cell-free preparations from 1321N1 cells was inhibited by N6(R-phenylisopropyl)-adenosine. Pretreatment of 1321N1 cells with pertussis toxin blocked both adenosine receptor-mediated inhibition of adenylate cyclase activity and attenuation of cyclic AMP accumulation. In contrast to the effects on responses to adenosine receptor agonists, 3-isobutyl-1-methylxanthine noncompetitively antagonized muscarinic receptor-mediated attenuation of cyclic AMP accumulation and pertussis toxin had no effect. These data are consistent with the ideas that Gi is fully functional in 1321N1 cells and links inhibitory adenosine receptors to adenylate cyclase, and that the muscarinic receptor of these cells couples to the phosphoinositide response system, but is incapable of functionally coupling through Gi to inhibit adenylate cyclase.     

Adenosine promotes neutrophil chemotaxis

We have previously (1-4) demonstrated that adenosine, by engaging specific receptors on the surface of neutrophils, inhibits generation of toxic oxygen metabolites by activated neutrophils and prevents these activated neutrophils from injuring endothelial cells. We now report the surprising observation that engagement of these same neutrophil adenosine receptors promotes chemotaxis to C5 fragments (as zymosan- activated plasma [ZAP]) or to the bacterial chemoattractant FMLP. When chemotaxis was studied in a modified Boyden chamber, physiologic concentrations of adenosine promoted chemotaxis by as much as 60%. Adenosine receptor analogues, 5'N-ethylcarboxamidoadenosine (NECA) and N6-phenylisopropyladenosine (PIA), also promoted chemotaxis; the order of agonist potency was consistent with that of an A2 adenosine receptor (NECA greater than PIA greater than or equal to adenosine). A potent antagonist at adenosine receptors, 8-p-sulfophenyltheophylline (10 microM), completely reversed NECA enhancement of chemotaxis but did not affect chemotaxis by itself. Neither NECA nor 2-chloroadenosine, a nonselective adenosine receptor agonist, alone was chemotactic or chemokinetic by checkerboard analysis. NECA also promoted chemotaxis quantitated by a different technique, chemotaxis under agarose, to the surrogate bacterial chemoattractant FMLP. These data suggest that engagement of adenosine A2 receptors uniquely modulates neutrophil function so as to promote migration of neutrophils to sites of tissue damage while preventing the neutrophils from injuring healthy tissues en route.     

Adenosine promotes histamine H1-mediated negative chronotropic and inotropic effects on human atrial myocardium

Because histamine and adenosine are coreleased from the ischemic heart, we investigated the effects of their interaction on human myocardium. Surgical specimens of human right atrium (i.e., pectinate muscles) responded to histamine with increases in spontaneous rate and contractile force. Adenosine, and the A1-selective adenosine agonist N6-cyclopentyladenosine (CPA), reduced the spontaneous rate and suppressed the positive chronotropic and inotropic effects of histamine. CPA was more potent than adenosine in slowing the spontaneous rate and in suppressing histamine's positive chronotropic effect, suggesting that the responses to CPA and adenosine are A1-mediated. CPA was also more potent than adenosine in attenuating histamine's positive inotropic effect on human ventricular papillary muscle. The adenosine-induced suppression of histamine's effects on pectinate muscles was mimicked by carbachol, which like adenosine is known to attenuate H2-mediated histamine-induced adenylate cyclase activation. The H1-selective histamine antagonist pyrilamine potentiated histamine's chronotropic and inotropic responses, and inhibited the attenuation of these responses by adenosine or carbachol. In contrast, pyrilamine failed to modify the adenosine-induced attenuation of the cardiac stimulatory effects of dimaprit, an H2-selective histamine agonist. Our data suggest that adenosine-induced suppression of histamine's positive chronotropic and inotropic effects on human myocardium results both from an A1-mediated attenuation of H2-stimulatory effects and from the uncovering of H1 negative chronotropic and inotropic effects. Thus, the results of the histamine-adenosine interaction may exceed the "retaliatory" purpose of adenosine release and uncover H1-mediated myocardial depression.     

Myocardial adenyl cyclase: activation by thyroid hormones and evidence for two adenyl cyclase systems

The mechanism responsible for the hyperdynamic circulatory state in hyperthyroidism has not been defined. Although certain cardiac manifestations resemble those caused by excessive adrenergic stimulation, recent evidence suggests that thyroid hormone exerts an effect on the heart that is independent of the adrenergic system. Since the inotropic and chronotropic effects of norepinephrine appear to be mediated by activation of adenyl cyclase, the possibility that thyroxine and triiodothyronine are also capable of activating adenyl cyclase was examined in the particulate fraction of cat heart homogenates.L-thyroxine and L-triiodothyronine increased the conversion of adenosine triphosphate-(32)P (ATP-(32)P) to cyclic 3',5'-adenosine monophosphate-(32)P (3',5'-AMP-(32)P) by 60 and 45% respectively (P < 0.01). A variety of compounds structurally related to the thyroid hormones, but devoid of thyromimetic activity did not activate adenyl cyclase: these included 3,5-diiodo-L-thyronine, L-thyronine, 3,5-diiodotyrosine, monoiodotyrosine, and tyrosine. D-thyroxine activated adenyl cyclase and half maximal activity was identical to that of the L-isomer. Although the beta adrenergic blocking agent propranolol abolished norepinephrine-induced activation of adenyl cyclase, it failed to alter activation caused by thyroxine. When maximal concentrations of L-thyroxine (5 x 10(-6) moles/liter) and norepinephrine (5 x 10(-5) moles/liter) were incubated together, an additive effect on cyclic 3',5'-AMP production resulted.THIS INVESTIGATION DEMONSTRATES: (a) thyroid hormone is capable of activating myocardial adenyl cyclase in vitro and (b) this effect is not mediated by the beta adrenergic receptor. Moreover, the additive effects of norepinephrine and thyroxine suggest that at least two separate adenyl cyclase systems are present in the heart, one responsive to norepinephrine, the other to thyroid hormone.These findings are compatible with the hypothesis that the cardiac manifestations of the hyperthyroid state may, in part, be caused by the direct activation of myocardial adenyl cyclase by thyroid hormone.     

Insurmountable beta receptor blockade by ICI 147,798 in rabbits

In rabbits, the characteristics of cardiac beta-1 receptor blockade produced by ICI 147,798, a novel beta receptor blocking agent with diuretic properties, were evaluated and compared with those of propranolol. In conscious rabbits, i.v. injections of 0.31, 1.0 and 3.1 mg/kg of ICI 147,798 and 1.0 mg/kg of propranolol caused significant bradycardia. ICI 147,798 produced a dose-dependent shift to the right of the dose-response (chronotropic) curve of isoproterenol with suppression of the maximal tachycardia, an effect characteristic of insurmountable beta receptor blockade. Propranolol also produced a shift to the right of the dose-response curve of isoproterenol without affecting the maximal tachycardia. ICI 147,798-induced antagonism was specific for beta adrenoceptors as it failed to modify the effects of acetylcholine, angiotensin II, phenylephrine, adenosine, histamine and prostaglandin E2 on mean arterial pressure and heart rate. In rabbits with prior autonomic blockade, ICI 147,798, like propranolol, failed to inhibit the positive chronotropic effects of theophylline which are mediated by postreceptor mechanisms. In reserpinized rabbits, ICI 147,798 was found to have no intrinsic sympathomimetic activity. Unlike the effects of propranolol, which were attenuated by first-pass through the hepatic vascular bed, the effects of ICI 147,798 were unaffected suggesting an absence of first-pass metabolism. The effects of propranolol (1.0 mg/kg i.v.) were not detectable at 24 hr after injection, whereas significant beta receptor blocking activity was still present at 24 hr after ICI 147,798 (1.0 mg/kg i.v.). The results suggest that ICI 147,798 is a specific, long-acting, insurmountable beta-1 receptor blocking agent without intrinsic sympathomimetic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand selectivity of dog coronary adenosine receptor resembles that of adenylate cyclase stimulatory (Ra) receptors

Adenosine receptors on the surface of coronary myocytes initiate coronary relaxation, but events subsequent to receptor occupancy are uncertain. We assayed the coronary vasoactivity of receptor-selective adenosine analogs in open chest dogs to test the hypothesis that the coronary adenosine receptor is an adenylate cyclase stimulatory (Ra) receptor. The potency order was: ethyl adenosine-5'-uronamide greater than cyclopropyl adenosine-5'-uronamide greater than 2-chloroadenosine greater than N6-[R(-)-1-phenyl-2-propyl] adenosine (R-PIA) greater than N6-cyclohexyladenosine greater than adenosine greater than S-PIA. Such a hierarchy of activity resembles that of the Ra receptors of thyroid and aortic myocyte adenylate cyclases and of tracheal smooth muscle relaxation. The potency order R-PIA greater than adenosine and the stereoselective coronary vasoactivity of R-PIA suggest that the coronary receptor may be a hybrid receptor which contains the "N6 site" typical of inhibitory (Ri) receptors in addition to the "5'-uronamide site' which expresses Ra activity.     

Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms.

The effects of adenosine deaminase and of pertussis toxin on hormonal regulation of lipolysis were investigated in isolated human fat cells. Adenosine deaminase (1.6 micrograms/ml) caused a two-to threefold increase in cyclic AMP, which was associated with an increase in glycerol release averaging 150-200% above basal levels. Clonidine, N6-phenylisopropyladenosine, prostaglandin E2, and insulin caused a dose-dependent inhibition of glycerol release in the presence of adenosine deaminase. Pretreatment of adipocytes with pertussis toxin (5 micrograms/ml) for 180 min resulted in a five- to sevenfold increase in cyclic AMP. Glycerol release was almost maximal and isoproterenol caused either no further increase or only a marginal additional increase of lipolysis after pretreatment with pertussis toxin, whereas cyclic AMP levels were 500 times higher than in controls. The effects of antilipolytic agents known to affect lipolysis by inhibition of adenylate cyclase activity, i.e., clonidine, N6-phenylisopropyladenosine, and prostaglandin E2, were impaired. In contrast, the antilipolytic action of insulin was preserved in adipocytes pretreated with pertussis toxin. As in controls, the peptide hormone had no detectable effect on cyclic AMP after pertussis toxin treatment. The findings support the view that the antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action. In addition, the results demonstrate that, upon relief of endogenous inhibition, human fat cell lipolysis proceeds at considerable (adenosine deaminase) or almost maximal (pertussis toxin) rates. A certain degree of inhibition, therefore, appears to be necessary for human fat cell lipolysis to be susceptible for hormonal activation.     

Adenosine attenuation of the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers

Adenosine is known to have effects on electrophysiologic parameters of the sinus node, AV node and atrium and to antagonize isoproterenol-induced increased inotropy in the ventricle. However, the effects on cardiac Purkinje fibers are not well established. Therefore, the purpose of the present experiments was to examine the effects of adenosine alone and adenosine on isoproterenol-treated canine cardiac Purkinje fibers. Microelectrode techniques were used to record transmembrane action potentials. Adenosine alone (10(-7) to 10(-4) M) produced no effects on action potential characteristics of paced fibers. Adenosine in concentrations of 10(-7), 10(-6), 10(-5) and 10(-4) M produced a 15, 24, 44* and 72*% attenuation of isoproterenol (10(-6) M)-induced action potential duration shortening, respectively (*P less than .001). In 7 of 7 fibers depolarized with 22 mM K+, adenosine (10(-4) M) ablated calcium-dependent action potentials restored with isoproterenol (10(-6) M). These effects were antagonized by theophylline (5 X 10(-5) M) and adenosine deaminase (1 U/ml). Action potential shortening due to superfusion of high calcium Tyrode's solution and calcium-dependent action potentials generated in Na+ free-high Ca++ Tyrode's solution were not antagonized by adenosine. Adenosine (10(-5) M) produced a negative chronotropic effect, increasing escape intervals from 2669 +/- 647 to 3702 +/- 717** msec in control fibers and from 1864 +/- 329 to 2658 +/- 399** msec in tyramine (10(-4) M)-treated fibers (**P less than .05), but failed to produce a negative chronotropic response in fibers pretreated with propranolol (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-level N-methyl-D-aspartate receptor activation provides a purinergic inhibitory threshold against further N-methyl-D-aspartate-mediated neurotransmission in the cortex.

N-methyl-D-aspartate (NMDA) is 33 times more potent at releasing adenosine than it is at releasing [3H]norepinephrine from slices of rat parietal cortex. Consequently, maximal adenosine release occurs at levels of NMDA receptor activation which release little norepinephrine. The potential modulatory role of the adenosine released during NMDA receptor activation on NMDA-evoked [3H]norepinephrine release was investigated. The A1-selective agonist R-(-)N6-(2-phenylisopropyl)adenosine (10 microM) decreased 100 microM NMDA-evoked [3H]norepinephrine release by 27%; this was reversed by the P1 antagonist 8-phenyltheophylline (8-PT, 10 microM), indicating that NMDA-evoked norepinephrine release from cortical slices is susceptible to purinergic modulation. On the other hand, 8-PT had no effect on [3H]norepinephrine release evoked by 100 microM NMDA, suggesting that endogenous adenosine, released during NMDA receptor activation, does not modulate [3H]norepinephrine release. However, [3H]norepinephrine release precedes adenosine release, so that the released adenosine may not be temporally available to modulate [3H]norepinephrine release. Pretreatment with a concentration of NMDA (10 microM) which releases substantial endogenous adenosine, but very little [3H]norepinephrine decreased subsequent 100 microM NMDA-evoked [3H]norepinephrine release by 52%. 8-PT partially reversed this inhibition, indicating that prereleased adenosine, acting at P1 purinoceptors, modulated subsequent NMDA-evoked [3H]norepinephrine release. These results suggest that adenosine, released during submaximal NMDA receptor activation, may provide an inhibitory threshold which must be overcome in order for other NMDA-mediated processes to proceed maximally.     

ICI 147,798: a slowly dissociable beta adrenoceptor antagonist that causes insurmountable beta-1 and surmountable beta-2 adrenoceptor antagonism in isolated tissues

ICI 147,798 has been shown to exhibit both diuretic and beta-antagonist properties in vivo. The present study investigated the nature and selectivity of the beta-antagonism in a variety of isolated tissues. ICI 147,798 produced a concentration-dependent suppression of the maximum chronotropic response of norepinephrine in guinea pig right atria (beta-1 adrenoceptor). ICI 147,798 caused a concentration-dependent shift to the right of the salbutamol concentration-response curve in the guinea pig trachea (beta-2 adrenoceptor), and Schild analysis suggested competitive inhibition. Propranolol produced parallel shifts to the right of the norepinephrine concentration-response curve in guinea pig right atria, except at relatively high concentrations. The inhibitory effects of propranolol in guinea pig right atria were reversed by greater than 95%, whereas the effects of ICI 147,798 were only slightly reversed after a 6-hr washout period. Preincubation of propranolol with ICI 147,798 in guinea pig right atria prevented completely the suppression of the norepinephrine maximum chronotropic response. Postincubation of propranolol with ICI 147,798 partially reversed the suppression of the maximum chronotropic response. ICI 147,798 had no effect on the maximum chronotropic responses of either histamine (H2-receptor) or forskolin (adenylate cyclase activation) in guinea pig right atria and had no effect on agonist responses in a variety of other receptor systems. The insurmountable beta-1 adrenoceptor antagonism was evaluated based on the assumptions of irreversible competitive antagonism, mixed competitive and noncompetitive antagonism and slowly dissociating competitive antagonism ("hemi-equilibrium" conditions). Concentration-dependent changes in norepinephrine KA values suggested the first three possibilities were unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine selectively attenuates H2- and beta-mediated cardiac responses to histamine and norepinephrine: an unmasking of H1- and alpha-mediated responses

Adenosine is known to attenuate the positive inotropic and chronotropic effects of norepinephrine and histamine by reducing cyclic AMP accumulation. We assessed whether adenosine, while inhibiting the cardiac responses mediated by beta and H2 receptors, leaves unmodified the responses mediated by alpha and H1 receptors. In isolated cardiac preparations from the guinea pig, adenosine antagonized the positive inotropic effect of histamine more than that of norepinephrine. This most likely occurred because, by attenuating H2 and beta responses, adenosine unmasked the H1-negative and alpha-1-positive components of the inotropic effects of histamine and norepinephrine. Consistent with this hypothesis, the pure H2 agonist impromidine appeared to be antagonized by adenosine less than histamine, and norepinephrine less than isoproterenol. In addition, adenosine antagonized the positive inotropic effect of norepinephrine in the presence of the alpha-1 blocker prazosin, whereas it did not affect the inotropic effect of phenylephrine. In the papillary muscle depolarized by 22 mM K+, adenosine antagonized the restoration of contractile responses induced by histamine or norepinephrine. This action of adenosine was reversed by the phosphodiesterase inhibitor papaverine and by the adenylate cyclase activator forskolin, suggesting that adenosine attenuates beta and H2 responses by suppressing the cyclic AMP-dependent facilitation of Ca++ influx promoted by the two amines. Our data indicate that adenosine selectively attenuates H2 and beta but not alpha and H1 responses. Thus, when catecholamines, histamine and adenosine are released together, as in myocardial ischemia, in addition to their individual effects, negative inotropism, decreased impulse conduction velocity and coronary constriction (i.e., H1- and alpha-mediated responses) may result from the adenosine-histamine-norepinephrine interaction.     

Relaxant effects of adenosine analogs on guinea pig trachea in vitro: xanthine-sensitive and xanthine-insensitive mechanisms

Adenosine analogs were tested for their ability to relax carbachol-contracted trachea in vitro. The rank order of potency was: 5'-N-ethylcarboxamidoadenosine (NECA) greater than 2-chloroadenosine (2-CIADO) greater than 5'-chloroadenosine = N6-R-1-phenyl-2-propyladenosine (R-PIA) greater than N6-cyclohexyladenosine greater than 2-phenylaminoadenosine (CV1808) greater than 5'-methylthioadenosine (MTA). The rank order of potency for NECA, 2-CIADO and R-PIA is characteristic of an A2 subtype of adenosine receptor. 8-Para-sulfophenyltheophylline (8-p-ST) and 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), were used to antagonize tracheal relaxation elicited by adenosine analogs. 8-p-ST antagonized the 2-CIADO, N6-cyclohexyladenosine, R-PIA and 5'-chloroadenosine responses, but had little or no effect on the CV1808 and MTA responses. 8-p-ST antagonized responses to NECA at concentrations of NECA up to approximately 30 microM, but had no effect on responses to higher concentrations of NECA. The differences in antagonist potency of 8-p-ST and the clear biphasic response of NECA are indicative of at least two mechanisms of adenosine analog action leading to tracheal relaxation. One mechanism is mediated through a xanthine-sensitive site, at which NECA acted in a potent manner, whereas the other mechanism or mechanisms are insensitive to blockade by xanthines and account for the effects of action of MTA and CV1808, as well as for NECA at high concentrations. The low potency of the A1-selective antagonist DPCPX indicates that the xanthine-sensitive site is an A2 type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine A1 and A2 receptors mediate presynaptic inhibition and postsynaptic excitation in guinea pig submucosal neurons

Intracellular recordings were made from guinea pig submucosal neurons in vitro. Adenosine, 2-[p-(carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS21680), 2-chloroadenosine (CADO), 5'-N-ethylcarboxamidoadenosine (NECA), R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), N6-cyclohexyladenosine (CHA) and 1-deaza-2-chloro-N6-cyclopentyladenosine (DCCPA) were applied by adding them to the superfusion solution. Adenosine (30 nM to 30 microM) depolarized S-type neurons neurons and this was mimicked by analogs with potency order: CGS21680 = NECA greater than R-PIA greater than CADO greater than adenosine greater than CHA much greater than DCCPA. 8-Cyclopentyltheophylline (CPT) blocked the depolarizing action of CADO or R-PIA; this antagonism was surmountable and the dissociation equilibrium constant (KD) estimated by the Schild method was 295 nM. Synaptic potentials were evoked by focal stimulation of nerve strands running between submucosal ganglia. The nicotinic excitatory postsynaptic potential was reduced by adenosine and analogs with potency order CHA = R-PIA greater than DCCPA = NECA = CADO greater than adenosine much greater than CGS21680; CPT competitively antagonized this effect of CADO or CHA with a KD of 13 nM. The noradrenergic inhibitory postsynaptic potential was also reduced; the potency order was R-PIA greater than CHA = CADO = NECA = DCCPA greater than adenosine much greater than CGS21680; the KD of CPT as an antagonist was 7 nM. It is concluded that adenosine directly depolarizes submucosal neurons by acting at an A2 receptor, and that it inhibits the release of acetylcholine (from intramural nerves) and noradrenaline (from sympathetic nerves) by acting at a presynaptic A1 receptor.     

Allosteric modulation and accelerated resensitization of human P2X(3) receptors by cibacron blue

The activity of ATP as a fast neurotransmitter is mediated by the P2X family of ligand-gated ion channels. P2X receptor subtypes are subject to functional modulation by a diverse set of factors, including pH, divalent cations, and temperature. The human P2X(3) (hP2X(3)) receptor subunit is expressed primarily in sensory ganglia where it exists as either a homomultimeric receptor or, in combination with P2X(2), as a heteromultimeric receptor. This article describes the allosteric modulatory effect of the putative P2X receptor antagonist cibacron blue on the activity of recombinant hP2X(3) receptors. In 1321N1 cells expressing the hP2X(3) receptor, cibacron blue mediated a 3- to 7-fold increase in both the magnitude and the potency of ATP-activated Ca(2+) influx and transmembrane currents. The half-maximal concentration of cibacron blue required to mediate maximal potentiation (EC(50) = 1.4 microM) was independent of the agonist used to activate the hP2X(3) receptor. The nonselective P2 receptor antagonist PPADS (pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid) caused a rightward shift of the cibacron blue concentration-effect curve, whereas increasing concentrations of cibacron blue attenuated PPADS antagonism. In addition to potentiating the effects of ATP at the hP2X(3) receptor, cibacron blue also produced a 6-fold increase in the rate of hP2X(3) receptor recovery from desensitization (from T(1/2) = 15.9 to 2.6 min), as evidenced by its ability to restore ATP responsiveness to acutely desensitized receptors. Consistent with the properties of other ligand-gated ion channels, these results suggest that hP2X(3) receptor activity can be allosterically modulated by a ligand distinct from the endogenous agonist.