Evidence against adenylate cyclase-coupled adenosine receptors in the guinea pig heart

(--)-N6-(Phenylisopropyl)adenosine (PIA, 10 microM) and 5'-N-(ethylcarboxamide)adenosine (NECA, 50 microM), potent agonists at the adenylate cyclase-coupled R-site adenosine receptor, were investigated for adenylate cyclase inhibition in a guinea pig ventricular membrane preparation by means of the dATP assay method. Neither compound influenced basal or isoproterenol-stimulated cyclase activity, irrespective of whether Na ions were present or not. These data suggest that R-site receptors may not be involved in the cardiac adenylate cyclase system of the guinea pig.     

Characterization of dopamine receptors mediating inhibition of adenylate cyclase activity in rat striatum

In the presence of SCH 23390, a potent blocker of D1 dopamine receptors, dopamine inhibits adenylate cyclase activity of synaptic plasma membranes isolated from rat striatum. Maximal inhibition corresponds to a 20-25% decrease of basal enzyme activity and is reached with 100 microM dopamine. The apparent IC50 of dopamine is 2.5 microM. The inhibitory effect of dopamine is mimicked by various dopamine receptor agonists with the following rank order of potency: (-)-propylnorapomorphine greater than or equal to bromocriptine greater than (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene = (-)-apomorphine greater than dopamine greater than LY 171555 greater than l-noradrenaline greater than l-phenylephrine. Clonidine and l-isoproterenol are inactive at 100 microM. Bromocriptine and LY 171555, two agents which stimulate selectively D2 receptors, inhibit striatal adenylate cyclase activity in the absence of SCH 23390. However, bromocriptine behaves like a partial agonist. A variety of neuroleptic drugs antagonize the dopamine inhibition with a rank order of potency which qualitatively correlates with their relative affinity for D2 receptors. l-Sulpiride (EC50 = 210 nM) and (+)-butaclamol (EC50 = 130 nM) are severalfold more potent than d-sulpiride (EC50 = 5 microM) and (-)-butaclamol (EC50 = 10 microM). The inhibitory effect of dopamine on striatal adenylate cyclase activity is dependent on the presence of GTP, with half-maximal inhibition occurring at 1 microM GTP. In the absence of SCH 23390, dopamine stimulates adenylate cyclase activity, reaching a maximum at 1 microM GTP. At higher concentrations of the nucleotide, the dopamine-stimulated enzyme activity decreases, and this decline is antagonized by the D2 receptor blocker l-sulpiride. Guanyl-5'-yl imidodiphosphate, a stable analogue of GTP, has a biphasic effect on the striatal adenylate cyclase activity, inhibiting at low concentration (from 1 to 100 nM) and stimulating at higher concentrations. Selective activation of D2 receptors by LY 171555 does not increase the extent of enzyme inhibition elicited by guanyl-5'-yl imidodiphosphate. Sodium chloride amplifies the inhibition of striatal adenylate cyclase activity by LY 171555 and reduces the potency of the D2 agonist by a factor of 4. The dopamine-inhibited enzyme activity is lost following intrastriatal injection of kainic acid. The results indicate that in rat striatum dopamine inhibits adenylate cyclase activity by acting on postsynaptic dopamine receptors with pharmacological properties of D2 type.     

A comparison between muscarinic receptor occupancy,adenylate cyclase inhibition,and inotropic response in human heart

Summary Binding to muscarinic receptors was compared with adenylate cyclase inhibition in membranes derived from human heart auricles, and with inhibition of the contraction of auricular muscle fibers.In the absence of GTP, agonists recognized two classes of receptors both of which bound antagonists with the same affinity. In the presence of GTP, both classes of receptors for agonists were converted into a single low affinity state.Carbachol and oxotremorine inhibited adenylate cyclase activity by 43%, pilocarpine being less efficient (–28%). The 3 agonists exerted similar inhibitory effects on the inotropic response, in 7 out of 9 preparations of electrically- and norepinephrine-stimulated fibers. Dose-effect curves suggested that spareness (or an amplification mechanism) was implicated in the occupancy of low affinity binding sites by carbachol and oxotremorine (but not by the partial agonist pilocarpine) and the resulting inhibition of both adenylate cyclase activity and contractile force.Abbreviations [³H] NMS, [N-methyl-³H] scopolamine methyl chloride - Gpp(NH)p guanosine 5-0-(2-3 imido) triphosphate - EGTA ethylene glycol bis (2-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Somatostatin receptors on cortical neurones and adenohypophysis: comparison between specific binding and adenylate cyclase inhibition

Primary cultures of mouse embryonic neurones from the cerebral cortex and rat pituitary membranes were used to identify and characterize further the somatostatin receptors coupled to an adenylate cyclase and to compare these receptors with specific binding sites for a non-reducible somatostatin analog. 125I-CGP 23996 on both tissues. 125I-CGP 23996 bound specifically to a single population of sites on cortical neurones and pituitary membranes, with a high affinity (Kd = 2.76 and 1.95 nM respectively). The rank order of potency of somatostatin-(1-14) and some analogs (somatostatin-28, [D-Trp8,D-Cys14]somatostatin-(1-14), native CGP) to displace 125I-CGP 23996 from its binding sites was similar on both tissues. Furthermore this rank order was also found identical for the inhibition of adenylate cyclase activity on cortical neuronal and pituitary membranes. Finally a good correlation was found between the order of potencies of somatostatin analogs evaluated from binding experiments and adenylate cyclase assays, suggesting the presence of the same receptor observed under two different affinity states. According to the classification of somatostatin receptors by Tran and his colleagues (1985) these results support the hypothesis that SSA is the somatostatin receptor coupled with an adenylate cyclase.     

Correlation between histamine-induced neuronal excitability and activation of adenylate cyclase in the guinea pig hippocampus

In a homogenate of guinea pig hippocampus histamine activated adenylate cyclase and in a hippocampal slice preparation it increased the firing rate of pyramidal cells in the CA3 region. Both activities were apparently mediated by H2 receptors. The concentration of histamine and of the H2 receptor agonist, impromidine, required to stimulate activity was similar in each test preparation with impromidine being about 100-fold more potent than histamine. Moreover, the H2 receptor antagonists, cimetidine and ICIA 5165, each reversed the activation by histamine of the two test preparations, with ICIA 5165 being about 100-fold more potent than cimetidine. Thus, there is a correlation between activation of cyclase and neuronal excitability induced by histamine. These observations support a large body of evidence suggesting that histamine is a neurotransmitter or modulator in the CNS.     

Pharmacological characterization of two 5-hydroxytryptamine receptors coupled to adenylate cyclase in guinea pig hippocampal membranes

Two 5-hydroxytryptamine (5-HT) receptors mediate stimulation of adenylate cyclase activity in membranes of adult guinea pig hippocampus. The two receptors were characterized with agonists and antagonists and with the aid of computerized curve-fitting procedures. Each receptor mediates about 50% of the maximal response to 5-HT. 5-HT is about 10-fold more potent in eliciting response through one cyclase-linked receptor (RH) than the other (RL). The concentrations of 5-HT that elicit half-maximal response through RH and RL are 43 +/- 6 nM and 414 +/- 53 nM, respectively. 5-Methoxytryptamine (5-MeOT) and 5-HT are approximately equipotent at each receptor. The agonists tryptamine and bufotenine are less potent than 5-HT at both receptors, and each is about 50-fold selective for RH. The two receptors are best discriminated by the agonists 5-carboxamidotryptamine (5-CONH2-T) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), both of which are selective for RH. 5-CONH2-T is about 7-fold more potent than 5-HT at RH. The rank order of agonist potencies at RH (5-CONH2-T greater than 8-OH-DPAT = 5-HT = 5-MeOT greater than bufotenine greater than tryptamine) differs from that at RL (5-HT = 5-MeOT greater than bufotenine greater than tryptamine = 5-CONH2-T greater than 8-OH-DPAT). Spiperone acts as a simple competitive antagonist at RH, with a dissociation constant of 20 nM, but it is at least 100-fold less potent as an antagonist at RL. The relatively low affinities of the selective 5-HT antagonists ketanserin and MDL 72222 for RH and RL indicate that neither receptor may be classified as the 5-HT2 or as the 5-HT3 (i.e., peripheral neuronal) type. The characteristics of RH suggest that it is a functional correlate of the 5-HT1A-binding site in brain. RL appears not to correspond to a known 5-HT-binding site, but it may be homologous to receptors that mediate 5-HT-stimulated adenylate cyclase activity in other systems such as infant rat colliculi. RH and RL may also mediate stimulation of adenylate cyclase activity by 5-HT in hippocampal membranes of adult rat.     

Comparison of the effects of histamine and tolazoline on adenylate cyclase activity from guinea pig heart.

Both histamine and tolazoline (2-benzyl-2-imidazoline) stimulated particulate fractions of adenylate cyclase from guinea pig myocardium. Tolazoline was one-tenth as potent, and about two-thirds as active at maximally effective levels, as was histamine. Enhancement of cyclase activity by tolazoline was additive with that by isoproterenol, and the histamine and tolazoline concentration-response curves were parallel, suggesting that tolazoline acted at the same site as histamine. At maximally effective concentrations, tolazoline did not affect ATPase or cyclic AMP phosphodiesterase activities associated with the cyclase preparations. The H1-receptor antagonist mepyramine, and the H2 antagonist, burimamide, blocked stimulation of cyclase by tolazoline at one-tenth the molarity of agonist. Both antagonists were less effective vs. histamine stimulation of heart cyclase in particulate fractions or whole homogenates, with mepyramine being generally more potent. It is suggested that the molecular basis of the stimulatory effect of tolazoline on cardiac tissue may be histaminergic stimulation of adenylate cyclase. Furthermore, the lack of potency of burimamide as a histamine antagonist and its lack of specificity compared to mepyramine, at the subcellular level, indicate that histamine-responsive adenylate cyclase from heart may not be a satisfactory molecular model for the H2 receptor pharmacology of histamine in cardiac tissue.     

Fentanyl isothiocyanate reveals the existence of physically associated mu- and delta-opioid receptors mediating inhibition of adenylate cyclase in rat neostriatum

Dopamine D-1 receptor-stimulated cyclic AMP efflux from superfused rat neostriatal slices was strongly inhibited by the delta-opioid receptor agonist, [D-Pen2, D-Pen5]enkephalin (DPDPE, 1 microM), and by the mu-opioid receptor agonist, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO, 1 microM). Naloxone (0.1 microM) fully antagonized the inhibitory effect of DAGO, leaving that of DPDPE virtually unchanged. Preincubation of the slices with the irreversible delta receptor ligand, fentanyl isothiocyanate (FIT, 1 microM) did not affect the inhibitory effect of DAGO, but prevented that of DPDPE. Naloxone no longer antagonized the inhibitory effect of DAGO when the delta receptors were selectively and irreversibly blocked by FIT. These data indicate that FIT and naloxone, acting on delta and mu receptors, respectively, may share a common binding site, suggesting the involvement of a functional mu, delta-opioid receptor-complex.     

5-Hydroxytryptamine 5-HT1B and 5-HT1D receptors mediating inhibition of adenylate cyclase activity

Summary 5-Hydroxytryptamine_1B (5-HT_1B) receptor mediated-inhibition of forskolin-stimulated adenylate cyclase activity in rat substantia nigra was characterized pharmacologically and compared to 5-HT_1D receptor mediated-inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra. Special attention was paid to the effects of drugs known to bind with high affinity to 5-HT_1B (pindolol, propranolol, cyanopindolol, SDZ 21-009, isamoltane) or 5-HT_1D recognition sites (yohimbine, rauwolscine).PEC₅₀ or pK _B values of a variety of 5-HT-receptor ligands (6 agonists including 5-HT, and 12 antagonists) for the inhibition of adenylate cyclase activity in rat substantia nigra, correlated significantly to the corresponding pK _D values at 5-HT_1B binding sites (r = 0.90, P = 0.0001). Amongst the ₂- and -adrenoceptor antagonists tested, none of the drugs expressed more than 35% of the intrinsic activity of 5-HT at 5-HT_1B receptors. When tested as antagonists, their pK _B values were in good agreement with their pK _D values for 5-HT_1B sites. By contrast, these drugs displayed marked intrinsic activity at 5-HT_1D receptors: their pEC₅₀ values were close to their pK _D values for 5-HT_1D sites and their effects could be potently antagonized by methiothepin. The rank orders of potency of the tested compounds at 5-HT_1B and 5-HT_1D were markedly different.The results strengthen the identity between 5-HT receptors mediating inhibition of adenylate cyclase activity in rat and calf substantia nigra and 5-HT_1B and 5-HT_1D binding sites, respectively. They underline the differences between these receptors in terms of intrinsic activities and potencies of drugs. Send offprint requests to: D. Hoyer at the above address     

Dynorphin-selective inhibition of adenylyl cyclase in guinea pig cerebellum membranes

Guinea pig cerebellum, which contains kappa opioid receptors uncontaminated by other opioid receptor types, was chosen to examine whether kappa receptors are coupled to adenylyl cyclase. Membranes were prepared from guinea pig cerebellum and pretreated at pH 4.5 to increase inhibitory activity, and adenylyl cyclase was assayed in the presence of dynorphin analogs as prototypical kappa agonists. Results showed that several dynorphin analogs inhibited adenylyl cyclase by 30-50%, whereas mu- and delta-preferring agonists had no effect. Dynorphin A and the kappa-selective compounds D-Pro10-dynorphin(1-11) and U-50,488H were the most potent agonists, with IC50 values of 0.03-0.05 microM, whereas other dynorphin gene products like dynorphin B and alpha-neo-endorphin were approximately 10-fold less potent. Like other Gi-coupled responses, dynorphin-inhibited adenylyl cyclase required GTP and sodium. Naloxone was a competitive antagonist for dynorphin-inhibited adenylyl cyclase, with 1 microM naloxone shifting the IC50 value of dynorphin A by 20-fold. The kappa-selective antagonist nor-binaltorphimine was even more potent, with 0.1 microM nor-binaltorphimine shifting the dynorphin IC50 value by 50-fold. These results suggest that dynorphin A and its analogs inhibit adenylyl cyclase by binding to a guanine nucleotide-binding protein-coupled opioid receptor whose pharmacological specificity matches those of kappa receptors.     

Development of pharmacological sensitivity to adenosine analogs in embryonic chick heart: role of A1 adenosine receptors and adenylyl cyclase inhibition

The developing chick heart was employed as a model system to explore temporal correlations between the onset of pharmacological sensitivity to adenosine analogs and the appearance of A1 adenosine receptors coupled to adenylyl cyclase. A characterization of the developmental profile for adenosine analog-induced negative chronotropic response revealed that isolated atria from 5- and 6-day embryos were unresponsive to adenosine analogs. The onset of pharmacological sensitivity occurred on embryonic day 7, as evidenced by a 27% reduction in atrial beating rate in the presence of 2-chloradenosine (2-CIA) (30 microM). The sensitivity of embryonic atria to 2-CIA increased continuously from day 7 to day 12 in ovo, when the atria became fully responsive to the negative chronotropic effect of this adenosine analog. In order to evaluate whether the developmental increase in pharmacological sensitivity to 2-CIA reflected changes in the number of A1 adenosine receptors, the ontogenesis of A1 adenosine receptors was assessed using the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine as a probe. Cardiac membranes from day 5 and day 6 embryos possessed approximately one third of the maximum number of A1 adenosine receptors expressed at later embryonic ages. Additionally, agonist/[3H] DPCPX competition curves revealed that the high affinity state receptors comprised a larger proportion of the total receptor population in membranes from day 6 as compared with day 12 embryos. These results suggest that there are pharmacologically inactive A1 receptors in hearts from day 5 and day 6 embryos. The developmental change in A1 receptor-mediated negative chronotropic response paralleled the increase in [3H]DPCPX binding sites from embryonic day 7 to day 10. Thus, a large fractional occupancy of A1 adenosine receptors is required to express negative chronotropy during this period of embryonic development. Studies of the sensitivity of adenylyl cyclase to inhibition by cyclopentyladenosine as a function of ontogenesis revealed that cyclopentyladenosine inhibited basal adenylyl cyclase activity to a similar maximal extent from embryonic day 5 through day 16. The efficacy of cyclopentyladenosine as an inhibitor of adenylyl cyclase activity was, therefore, stable during a developmental period when A1 receptor density increased approximately 3-fold. Hence, only a fraction of the A1 receptors present during embryogenesis need to be coupled to produce a maximum response with respect to adenylyl cyclase inhibition, which is an indication of the presence of spare receptors. receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of furosemide on parathyroid hormone stimulated guinea pig renal adenylate cyclase and thyrotrophin and fluoride stimulated human thyroid adenylate cyclase

The effect of furosemide 8 X 10(-4) mol/l an 8 X 10(-5) mol/l on parathyroid hormone stimulated adenylate cyclase was studied in renal tissue slices from guinea pigs. Furosemide caused a dose-dependent inhibition of the effect of parathyroid hormone on production of cyclic AMP, without having any significant effect on the basal cyclic AMP production. Furosemide in similar concentrations did not inhibit the stimulatory effect of thyrotrophin and fluoride in human thyroid homogenates suggesting that furosemide is not an universal inhibitor of adenylate cyclase and that the inhibition is not caused by a direct action of furosemide on the adenylate cyclase enzyme. Furosemide did not interfere with binding of cyclic AMP to cyclic AMP binding protein kinase from rabbit muscle. The results indicate that furosemide exerts an inhibitory influence either upon binding of parathyroid hormone to renal receptors or upon transmission of impulse from receptor to adenylate cyclase. The inhibitory influence of furosemide on parathyroid hormone action in kidney could explain the value of furosemide in the acute treatment of hypercalcaemia, but also suggest that chronic treatment with furosemide might interfere with normal calcium metabolism.     

Interaction of azelastine with adenosine receptors in guinea pig trachea

The effects of azelastine, 8-phenyltheophylline, NDGA, atropine and mepyramine on PIA-induced contraction and relaxation of isolated guinea pig tracheal chains were investigated. Atropine (1 nM) and mepyramine (1 microM) had no effect on PIA-induced relaxation whereas 8-phenyltheophylline (5 microM) caused strong inhibition of PIA-induced relaxation, indicating that the latter effect is mediated by stimulation of extracellular adenosine receptors. NDGA (0.5 microM) caused potentiation of PIA-induced relaxation. Azelastine (10 nM-1 microM) caused dose-dependent potentiation of PIA-induced relaxation. In another model for investigation of extracellular adenosine receptors, namely the negative inotropic effect in the electrically driven isolated guinea pig atrium, the action of PIA was fully reversed by the addition of 8-phenyltheophylline. In contrast, the negative inotropic effect of azelastine was not reversed by 8-phenyltheophylline, indicating that azelastine does not act on extracellular adenosine receptors. The negative inotropic effect of azelastine can be reversed by addition of calcium as for verapamil. It is concluded that the calcium-antagonistic and perhaps antiallergic properties of azelastine are responsible for the potentiation of extracellular adenosine receptor mediated relaxation by azelastine. Since asthmatics show increased hyperreagibility (bronchospasm) to inhalation of adenosine, the inhibition of PIA-induced contraction by azelastine indicates that the drug may be worthwhile in the treatment bronchial hyperreactivity in asthmatic patients.     

Selective blockade of dopamine D-1 receptors by SCH 23390 discloses striatal dopamine D-2 receptors mediating the inhibition of adenylate cyclase in rats

l-Glutamate but not methyl-D-aspartate (NMDA) or quisqualate (Quis) (10^?6 M) in vitro with or without preincubation increased significantly the K_D value of the [³H]N-propylnorapomorphine ([³H]NPA) binding sites by 21 and 36% respectively in striatal membranes of rat without influencing the striatal [³H]spiperone binding sites. The number of striatal [³H]NPA binding sites was not changed by l-glutamate (10^?6 and 10^?5 M) in vitro. There may thus exist interactions between striatal glutamate receptors — not related to excitatory amino-acid receptors of the NMDA or the QUIS type - and high affinity striatal DA receptors.     

Pharmacological characterization of the adenylate cyclase-coupled adenosine receptor in isolated guinea pig atrial myocytes

Although adenosine is known to activate K+ conduction in atrial tissue, there is still debate as to the involvement of cAMP-dependent mechanisms. In isolated adult guinea pig atrial myocytes, we demonstrate that the highly A1-selective adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine reduced basal cAMP levels by 30-40% in the absence and presence of the nonxanthine phosphodiesterase inhibitor Ro 20-1724. Isoprenaline caused a concentration-dependent increase in cAMP levels, which was more pronounced in the presence of the phosphodiesterase inhibitor. Several adenosine derivatives suppressed the isoprenaline-induced cAMP increase by approximately 80%. The rank order of potency was 2-chloro-N6-cyclopentyladenosine (IC50, 93 nM) greater than (R)-N6-phenylisopropyladenosine (IC50, 309 nM) greater than 5'-N-ethylcarboxamidoadenosine (IC50, 813 nM) much greater than (S)-N6-phenylisopropyladenosine (IC50, 26,300 nM). A similar but complete suppression of the isoprenaline-induced cAMP increase was produced by the muscarinic receptor agonist carbachol (IC50, 398 nM), which like adenosine is known to activate atrial K+ channels. The A1-adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine antagonized the effect of 2-chloro-N6-cyclopentyladenosine concentration-dependently, with a KB value of 9.6 nM. In atrial myocytes isolated from guinea pigs pretreated with pertussis toxin, the inhibitory effects of adenosine analogs on basal and isoprenaline-stimulated cAMP accumulation were markedly attenuated. It is concluded that the adenosine receptor in guinea pig atrial myocytes, which is known to be linked to K+ channels, is also coupled to adenylate cyclase via a pertussis toxin-sensitive guanine nucleotide-binding protein and shows the characteristics of the A1-adenosine receptor subtype.     

Halothane attenuation of muscarinic inhibition of adenylate cyclase in rat heart

Halothane stimulated basal adenylate cyclase activity in rat cardiac membranes. Maximal stimulation (54%) was obtained after equilibrating the membranes with 2% halothane. Halothane did not affect the fractional stimulation of adenylate cyclase activity produced by either forskolin or isoproterenol. However, halothane decreased carbamylcholine inhibition of adenylate cyclase activity stimulated by both forskolin and isoproterenol. Maximal depression of carbamylcholine inhibition of stimulated cyclase activity was obtained after equilibration with 1% halothane. These results are consistent with evidence from ligand binding studies and indicate that halothane disrupts muscarinic receptor-G-protein interactions.     

Inactivation of the guanine nucleotide regulatory site mediating inhibition of the adenylate cyclase in hamster adipocytes

Summary The influence of N-ethylmaleimide and trypsin was studied on stimulatory and inhibitory regulations of the hamster adipocyte adenylate cyclase. Treatment of intact adipocytes or adipocyte ghosts with N-ethylmaleimide decreased basal and forskolin-stimulated adenylate cyclase activities. In the pretreated membrane preparations, inhibition of the enzyme by GTP and by stable GTP analogues was abolished. Concomitantly, activation of the adenylate cyclase by NaCl and its inhibition by the antilipolytic agents, prostaglandin E₁ and nicotinic acid, were obliterated. In contrast, adenylate cyclase stimulation by ACTH and stable GTP analogues was not impaired but rather increased. Similarly, the NaCl-induced attenuation of the ACTH-stimulated enzyme activity was increased by the N-ethylmaleimide treatment. Limited proteolysis of hamster adipocyte ghosts with trypsin also obliterated GTP and prostaglandin E₁-induced inhibitions and NaCl-induced activation of the adenylate cyclase. In contrast, adenylate cyclase activity stimulated by isoproterenol was increased after trypsin treatment. The data suggest that the activity of the adenylate cyclase is regulated via two distinct guanine nucleotide sites and that treatment with N-ethylmaleimide and limited proteolysis with trypsin functionally eliminates the regulatory site mediating adenylate cyclase inhibition, leading to a state where the enzyme activity is regulated only via the stimulatory site. The differential effects of these treatments on NaCl-induced activation and attenuation of the adenylate cyclase suggest that sodium acts on both regulatory sites in an inhibitory manner, and that by the functional elimination of the inhibitory site, only the sodium-induced attenuation of the adenylate cyclase via the stimulatory site is observed.