Inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT receptor agonists

We measured the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig hippocampal membranes by 5-HT, 5-carboxamidotryptamine (CAT) and 8-hydroxy-2-(di-n-propylamino) tetralin (PAT). Low concentrations of these agonists inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent and saturable manner. The antagonist spiperone shifted the concentration-response curve to CAT to the right in a parallel manner. The EC50 values of CAT, PAT and 5-HT and the KB of spiperone suggest that this receptor may correspond to the 5-HT1A binding site.     

Partial and full dopamine D1 receptor agonists in mice and rats: relation between behavioural effects and stimulation of adenylate cyclase activity in vitro.

The dopamine (DA) D1 agonists, SK&F 83959, SK&F 75670, SK&F 38993, SK&F 81297 and SK&F 80723, had variable abilities to stimulate adenylate cyclase activity in rat striatal homogenates. Their efficacies, in relation to the effect of 100 microM DA were 0, 33, 69, 68 and 81%, respectively. In rats, all compounds induced (1) contralateral circling behaviour after unilateral 6-hydroxy-DA lesions, (2) ipsilateral circling behaviour after midbrain hemitransection after cotreatment with the D2 agonist quinpirole and (3) oral stereotypies after their combination with quinpirole. Maximum effects and rank order of potencies were similar in the three test models. In mice SK&F 83959, SK&F 75670 and SK&F 38393 inhibited methylphenidate-induced gnawing behaviour and induced no or only weak hypermotility. SK&F 81297 induced marked hypermotility which was partially inhibited by SK&F 83959 and SK&F 75670 and was completely blocked by the D1 antagonist, SCH 23390. It is concluded that no relation could be demonstrated between the efficacy to stimulate adenylate cyclase and to induce circling behaviours and stereotypies in rats. In contrast, a relation between biochemical and behavioural efficacies was found in the mouse models. The results suggest that different subtypes of D1 receptors mediate the behavioural effects reported in this study.     

Dopamine receptor agonists: selectivity and dopamine D1 receptor efficacy

Dopamine receptor selectivity was investigated for a number of dopamine receptor agonists. In vitro, the benzazepine derivatives, e.g., SKF 38393 and SKF 75670 as well as the isoquinoline derivatives, SKF 89626 and SKF 89615, were D1 receptor-selective. All other compounds like apomorphine, CY 208-243, 6,7-ADTN and 3-PPP were either D2-selective or did not discriminate between subtypes. In general, the same receptor profile seen in vitro was observed in vivo. The exceptions to this pattern were: compounds which did not cross the blood-brain barrier, like 6,7-ADTN and SKF 89626, and compounds which appeared nonselective in vitro but demonstrated D2 selectivity in vivo like apomorphine, CI 201-678 and CY 208-243. A number of compounds were characterized in detail with respect to a GTP-induced affinity shift in inhibition of [3H]SCH 23390 binding, and potency and efficacy in stimulating adenylate cyclase from rat striatum. Inhibition of specific [3H]SCH 23390 binding by these agonists in the absence of GTP occurred with Hill slopes below unity and could best be explained by a two-site model with a high (KH)- and low-affinity (KL) component. Inhibition of [3H]SCH 23390 binding in the presence of 15 microM GTP occurred with Hill slopes of unity. The KI values obtained in the presence of 15 microM GTP were similar to the KL values, the low-affinity component observed in the absence of GTP. The capability of the agonists to stimulate the adenylate cyclase was analyzed in relation to dopamine (efficacy = 100%). The efficacy of the benzazepine derivatives varied from 24 (SKF 75670) to 100% (SKF 83189), dependent on the substituents on the benzazepine core. The isoquinolines, SKF 89626 and SKF 89615 had full efficacy, whereas most other agonists tested appeared to have only partial efficacy. In summary, the present paper presents data on dopamine receptor selectivity and efficacy in stimulating adenylate cyclase for a number of dopaminergic agonists. These data may create a basis for selection of agonists in future characterizations of dopaminergic-mediated events.     

Cardiac adenylate cyclase. II. Structure-activity relationships for the activation of rat ventricle adenylate cyclase by beta-adrenoceptor agonists.

The ability of phenylethylamine derivatives to stimulate adenylate cyclase activity of a sarcolemma fraction of rat ventricular muscle has been studied. The structure-activity relationships for maximal activation of adenylate cyclase activity showed that a phenylethylamine derivative required two adjacent hydroxyl groups at the 3 and 4 positions of the phenyl ring, a hydroxyl group on the asymmetric β-carbon of the ethylamine side chain and an isopropyi group on the amine. The configuration of the hydroxyl group on the β-carbon was critical since the laevo isomer of this amine (isoprenaline) was highly active whilst the dextro isomer was almost inactive. The activation of adenylate cyclase activity by (?)-isoprenaline was blocked by ( ? )-propranolol but not (+)-propranolol or phentolamine. These structure-activity relationships are identical with those found for the actions of β-agonists and antagonists on cardiac muscle and therefore strengthen the hypothesis that the β-adreno-receptor is a component of adenylate cyclase in cardiac muscle. The order of potency of catecholamine derivatives to stimulate adenylate cyclase activity in intact cubes of rat ventricles was similar to that found in homogenates of ventricles, although the relative sensitivity of catecholamines in cubes was approximately 50 times higher. It was concluded that homogenization and preparation of sarcolemma membranes alters the sensitivity of adenylate cyclase to activation by catecholamines.     

Differential activation of G-proteins by mu-opioid receptor agonists

We investigated the ability of the activated mu-opioid receptor (MOR) to differentiate between myristoylated G(alphai1) and G(alphaoA) type G(alpha) proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each G(alpha) protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The G(alpha) subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified G(alpha) protein by CB(1) cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[(35)S]GTP(gamma)S exchange was then compared for G(alphai1) and G(alphaoA). Activation of MOR by DAMGO produced a high-affinity saturable interaction for G(alphaoA) (K(m)=20+/-1 nM) but a low-affinity interaction with G(alphai1) (K(m)=116+/-12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal G(alpha) activation among the agonists evaluated. Endomorphins 1 and 2, methadone and beta-endorphin activated both G(alpha) to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between G(alphai1) and G(alphaoA). Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two G(alpha). Differences in maximal activity and potency, for G(alphai1) versus G(alphaoA), are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects.     

Amelioration of ketamine-induced working memory deficits by dopamine D1 receptor agonists

Rationale  

Ketamine has been used in humans to model cardinal symptoms of schizophrenia, including working memory impairments and behavioral disorganization. Translational studies with ketamine in nonhuman primates promise to extend the neurobiological understanding of this model.     

Short- and long-term heterologous sensitization of adenylate cyclase by D4 dopamine receptors

The D₄ dopamine receptor, a member of the D₂-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D₄ receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D_4.2, D_4.4, or D_4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D₄ receptor variants. D₄ dopamine receptor-mediated sensitization was blocked by the D₄ antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for G_i/G_o proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D₄ receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of G_iα without altering the abundance of G_sα, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D₄ receptor may involve mechanisms that do not contribute to short-term sensitization. Received: 26 March 1998/Final version: 6 May 1998     

Identification of a D1 dopamine receptor, not linked to adenylate cyclase, on lactotroph cells.

1. We studied the lactotroph cells of the rat by both in vivo and in vitro pharmacological techniques for the presence of D1-receptors. Both approaches revealed the presence of D2-receptor, stimulated by quinpirole (resulting in an inhibition of prolactin secretion) and blocked by domperidone. 2. Administration of fenoldopam, the most selective D1-receptor agonist currently available, resulted in a dose-dependent decrease of prolactin secretion in vivo (after pretreatment with alpha-methyl-p-tyrosine) and in vitro (cultured pituitary cells). This increase was dose-dependently blocked by the selective D1-receptor antagonist, SCH 23390, and although the effect of fenoldopam was less than that obtained by D2-receptor stimulation, these data suggest that a D1-receptor also controls prolactin secretion. 3. In order to detect the location of these dopamine receptors, autoradiographic studies were performed by use of [3H]-SCH 23390 and [3H]-spiperone as markers for D1- and D2-receptors, respectively. Specific binding sites for [3H]-SCH 23390 were demonstrated. Fenoldopam dose-dependently reduced [3H]-SCH 23390 binding, but had no effect on [3H]-spiperone binding. Immunocytochemical labelling of prolactin cells after incubation with [3H]-SCH 23390 revealed that the granulae and hence, D1 binding sites were present on the lactotroph cells. 4. Radioligand binding studies performed on membranes from anterior pituitary cells revealed the presence of the D2-receptor (54 fmol mg-1 protein) with a Kd of 0.58 nM for [3H]-spiperone, but failed to detect D1-receptors. 5. Finally, we studied the effect of dopamine and of fenoldopam on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dopamine D1 and dopamine D2 receptor agonists on coronary and peripheral hemodynamics

This study evaluated the coronary dopamine receptors by using the dopamine D1 receptor agonist fenoldopam, dopamine D2 receptor agonist propylbutyldopamine, and their selective antagonists SCH23390 and domperidone. Left circumflex coronary flow (CF), coronary perfusion pressure at constant flow, left ventricular hemodynamics, and total peripheral vascular resistance (TPR) were measured in pentobarbital-anesthetized dogs at constant arterial pressures. At doses of 200, 500 and 5000 nM, both fenoldopam and propylbutyldopamine induced dose-related inotropic effects, as evidenced by maximal dp/dt and cardiac output, an increase in CF, decrease in coronary vascular resistance and a decrease in TPR. Fenoldopam was more potent in its cardiac and coronary effects while propylbutyldopamine was more potent peripherally. On the basis of dosage used, the positive inotropic effects of fenoldopam and propylbutyldopamine were much weaker than dopamine. After beta-receptor blockade, the inotropic and coronary effects of fenoldopam and propylbutyldopamine were extremely attenuated. Domperidone could largely antagonize the propylbutyldopamine-induced inotropic and coronary effects while SCH23390 showed no significant effect. In addition, under our experimental conditions, the fenoldopam- and propylbutyldopamine-induced decreases in TPR were markedly reduced by SCH23390 and domperidone, respectively. The results indicate that the coronary effects of fenoldopam and propylbutyldopamine result not from a primary coronary vasodilating action, but from vasodilation secondary to positive inotropic effects. Both dopamine D1 and dopamine D2 receptors are involved in the peripheral vascular hemodynamics.     

The enhancement of dopamine D1 receptor desensitization by adenosine A1 receptor activation

The present study was designed to examine the effects of adenosine A(1) receptor on dopamine D(1) receptor desensitization in a human embryonic kidney 293 cell line stably cotransfected with human adenosine A(1) receptor and dopamine D(1) receptor cDNAs (A(1)D(1) cells) by means of cAMP accumulation assay. Long-term exposure of A(1)D(1) cells to dopamine D(1) receptor agonist (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) caused a rapid desensitization of dopamine D(1) receptor. Coadministration of adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) potentiated the effect of SKF38393. This enhancement effect of CPA was blocked by adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) but not by pertussis toxin, indicating that this effect of CPA was mediated by adenosine A(1) receptor and was G(i) protein independent. Furthermore, the blockade of endogenous adenosine by adenosine deaminase or DPCPX attenuated dopamine D(1) receptor desensitization. Collectively, these results suggest that adenosine A(1) receptor plays an important role in the regulation of dopamine D(1) receptor by potentiating ligand-induced desensitization.     

Galphaq-coupled receptor signaling enhances adenylate cyclase type 6 activation

Calcium signaling robustly inhibits AC6 activity in membrane preparations and in intact cells via capacitative calcium entry (CCE). However, the release of intracellular calcium has not been demonstrated to robustly alter AC6 signaling and activation of Galpha(q)-coupled receptors in tissues that express AC6 enhances cyclic AMP accumulation. To specifically examine the ability of Galpha(q)-coupled receptors to modulate AC6 signaling in intact cells, we used stably transfected HEK-AC6 cells. We demonstrate that AC6 activation is potentiated by activation of endogenous muscarinic receptors expressed in HEK293 cells. Muscarinic receptor activation failed to potentiate the activation of the closely related AC5 isoform. Expression of recombinant Galpha(q)-coupled muscarinic or serotonin receptors, or constitutively active Galpha(q), also potentiated drug-stimulated cyclic AMP accumulation in HEK-AC6 cells. Muscarinic receptor-mediated potentiation of AC6 activation was not due to activation of PKC or modulation of Galpha(i/o)-mediated inhibition of AC6. We demonstrate that calcium chelation or inhibition of calmodulin attenuates the effect of carbachol on AC6 activation. These data support the hypothesis that Galpha(q)-coupled receptor-mediated calcium signaling potentiates AC6 activation in intact cells.     

Forskolin alters acetylcholine receptor gating by a mechanism independent of adenylate cyclase activation

The diterpene forskolin activates adenylate cyclase in a receptor-independent fashion and is commonly used to obtain a rapid elevation of intracellular cAMP levels. Application of 10-20 microM forskolin to Xenopus oocytes that express Torpedo nicotinic acetylcholine (ACh) receptors leads to an acceleration in the decay of ACh-elicited currents, which could be taken as evidence for modulation of ACh receptor gating by cAMP-dependent protein kinase. However, the effect is not mimicked by phosphodiesterase inhibitors or intracellular injection of a cAMP analog. In addition, 1,9-dideoxyforskolin, which is unable to activate adenylate cyclase, has a similar effect. Finally, the action of forskolin is rapidly reversible, with full onset and recovery occurring within the exchange time of the recording chamber. These results suggest that forskolin is a potent local anesthetic and that this property of this widely used compound must be taken into account when using it to study ion channel modulation.     

Effects of dopamine D1 and D2 receptor agonists and antagonists on bombesin-induced behaviors

Central administration of bombesin elicits excessive grooming and locomotor activity in rats. This grooming activity is one characterised by vigorous scratching of the face, nape and body flanks. Pretreatment with the D1 receptor antagonist SCH 23390 inhibited the expression of bombesin-induced activity with grooming being more inhibited than locomotion. Blockade of D2 receptors with eticlopride significantly attenuated the behavioral responses to bombesin. When SCH 23390 and eticlopride were administered concurrently, it was apparent that D1 blockade had a greater effect on grooming and D2 blockade a larger effect on locomotion. Stimulation of D1 receptors by SKF 38393 elicited non-stereotyped locomotor activity and a form of grooming behavior characterised by vigorous washing of the face and ventral body surfaces. Co-administration of bombesin and SKF 38393 resulted in a form of grooming which resembled that elicited by SKF 38393 alone. The specific D2 agonist PPHT elicited a form of locomotion characterised by a downward oriented head posture and slow ambulatory activity around the cage perimeter. Co-administration of PPHT and bombesin resulted in a complete suppression of bombesin-induced behaviors and was largely indistinguishable from activity observed under PPHT alone conditions. These data implicate both D1 and D2 receptor based mechanisms in the modulation/mediation of the behavioral effects of bombesin. Part of the bombesin-induced behavioral effects may be explained by (indirect) activation of (a) dopamine system(s).     

GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic cholinergic agonists

Summary In membrane-containing particles of rabbit myocardium, inhibition of adenylate cyclase (EC 4.6.1.1) by cholinergic agonists was studied. The cholinergic agonists, in the potency order oxotremorine > acetylcholine > carbachol, reduced basal adenylate cyclase activity with half-maximal effects at about 0.3, 1 and 3 M, respectively; maximal inhibition observed was about 40%. The enzyme inhibition occurred without apparent lag phase and was reversed by the muscarinic cholinergic antagonist, atropine, which finding indicates that muscarinic cholinergic receptors are involved in this process.As for stimulation of cardiac adenylate cyclase by the -adrenergic agonist, isoproterenol, the addition of GTP was necessary for maximal enzyme inhibition by the cholinergic agonists. The effects of GTP were half-maximal at about 0.2 and 1 M and maximal at about 3 and 30 M GTP for stimulation and inhibition, respectively. NaCl, which increased cardiac adenylate cyclase activity, facilitated the GTP-dependent cholinergic inhibition; the NaCl effect was maximal between 50 and 100 mM.In the presence of GTP, the cholinergic agonist, carbachol, not only reduced basal adenylate cyclase activity, but also inhibited adenylate cyclase stimulated by isoproterenol (100 M) or NaF (10 mM). In the presence of cholera toxin (40 g/ml), the GTP-induced activation of the enzyme was counteracted by carbachol. However, the stable GTP-analogues, guanylyl-5-imidodiphosphate and guanosine-5-O-(3-thiotriphosphate), which caused a persistent adenylate cyclase activation, reversed or prevented the carbachol-induced inhibition.The data indicate that cholinergic agonists inhibit cardiac adenylate cyclase by a process that requires (the hydrolyzable) GTP and involves sodium ions.Some of the data were presented in abstract form (Aktories and Jakobs, 1979)     

Synergistic behavioural effects of dopamine D1 and D2 receptor agonists are determined by circadian rhythms.

The effects of continuous subcutaneous infusions of rats for 336 h with vehicle, SKF 38393 (a dopamine D1 receptor agonist), (+)-4-propyl-9-hydroxynaphthoxazine (PHNO, a dopamine D2 receptor agonist) or both D1 and D2 receptor agonists, on locomotor activity were investigated. Rats were maintained under constant lighting conditions, either continuous dark (dark:dark) or continuous light (light:light), before and during drug treatments in order to determine the influence of free-running circadian rhythms on drug responses. The D2 receptor agonist initially increased locomotion in rats kept under dark:dark during both subjective night (period of maximum locomotion) and day (period of minimum locomotion), but had no effect in rats maintained in light:light throughout the 336 h of treatment. The motor stimulant effects of the D2 receptor agonist on rats kept in dark:dark increase during the course of treatment during subjective night (sensitization), but decreased during the rats' subjective day (tolerance). The D1 receptor agonist, SKF 38393, had no effect on its own regardless of the lighting conditions and the duration of treatment. However, the D1 receptor agonist interacted synergistically with the D2 receptor agonist in rats maintained under light:light, depending on the duration of treatment. Synergistic effects were also observed on initiation of treatment in rats under dark:dark but only during subjective day. Tolerance to the synergistic effects of the receptor agonists occurred as a function of treatment duration, but only during subjective day. The D1 receptor agonist blocked the effects of the D2 receptor agonist during the rats' subjective night after 100 h of treatment, but not after 25 or 325 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of kappa receptor agonists on D1 and D2 dopamine agonist and antagonist-induced behaviors

Striatal dynorphin-containing neurons receive dopaminergic inputs from the substantia nigra pars compacta and project primarily to the substantia nigra pars reticulata and entoped uncular nucleus. These neurons mainly express dopamine (DA) D₁ receptors and thus dynorphin system stimulation might be expected largely to influence D₁ receptor agonist or antagonist effects on motor function. It is well known the interaction existing between DA D₁ and D₂ drugs in the induction of behavioral effects. However, the effects of dynorphin on selective D₁ and D₂ DA agonist and antagonist-induced behaviors have not yet been investigated. Administration of the kappa agonists spiradoline (0.5, 1 and 5 mg/kg) or U50,488H (1, 10 and 25 mg/kg) decreased non-stereotyped grooming induced by the selective D₁ agonist SKF38393. This effect was inhibited by the non-selective opioid receptor antagonist naloxone (20 mg/kg) and by the selective kappa antagonist nor-binaltorphimine (nor-BNI, 20 mg/kg). Stereotypies induced by the selective D₂ agonist quinpirole were decreased by spiradoline (1 and 5 mg/kg) and by U50,488H (1, 10 and 25 mg/kg), while jerking movements of a type associated with increased D₂ receptor and decreased D₁ receptor stimulation emerged. Kappa agonist effects were inhibited by the prior administration of SKF38393 (10 mg/kg); these inhibitory effects were blocked by prior administration of the D₁ antagonist SCH23390 (5 mg/kg). Naloxone reversed the effects of both kappa agonists on quinpirole-induced stereotypies. Kappa agonists increased D₁ antagonist-induced catalepsy, but had no effect on D₂ antagonist-induced catalepsy. Naloxone and nor-BNI inhibited this effect. These results suggest that the motoric effects of D₁ receptor antagonists in part reflect stimulation of striatal dynorphin containing efferents.