Receptor-stimulated Adenylate Cyclase Research Articles

Increased Gsα within blood cell membrane lipid microdomains in some depressive disorders: An exploratory study

The stimulatory guanine nucleotide binding protein Gs couples many cellular receptors to adenylate cyclase, and the Gsα subunit activates all 9 isoforms of the adenylate cyclase catalytic unit to produce the enzyme product cyclicAMP or cAMP. In prefrontal cortex and cerebellum of unipolar depressive suicides, Rasenick and colleagues have found increased concentrations of Gsα in membrane lipid microdomains (Donati et al., 2008), where the ensconced Gsα is less likely to activate adenylate cyclase by receptor and postreceptor pathways (Allen et al., 2005, 2009).We report that a group of 7 depressed patients (DP-1) had (1) reduced activation of platelet receptor-stimulated adenylate cyclase by both prostaglandins E2 and D2 compared to controls, and (2) reduced postreceptor stimulation of adenylate cyclase by aluminum fluoride ion in both platelets and mononuclear leukocytes when compared to both another group of depressed patients (DP-2, n = 17) and to controls (n = 21). Our observations in the blood cells of the group DP-1 support the findings of Donati et al. (2008), and they reflect the importance of this interaction between the activated Gsα subunit and membrane lipid microdomains in the pathophysiology and treatment of some major depressive disorders.

Repeated quinpirole treatments produce neurochemical sensitization and associated behavioral changes in female hamsters

Repeated stimulation of dopaminergic pathways with dopamine receptor agonists can produce both neurochemical and behavioral sensitization. The present study was designed to examine whether repeated treatment with the D2-like dopamine receptor agonist, quinpirole, would produce neurochemical sensitization of D1 dopamine receptor-mediated processes and associated behavioral changes in female hamsters in a manner analogous to that previously used to sensitize heterologous dopamine signaling pathways in derived cell lines. Female hamsters received two injections of quinpirole (1.5 mg/kg) or saline each week for 7 weeks, during which time pouching behavior and body weight were monitored. Over the next 2 weeks, hamsters were tested for differences in prepulse inhibition of the acoustic startle response (PPI) and sexual behavior. Adenylate cyclase activation assays were then performed on dissected tissue from the nucleus accumbens and caudate-putamen. Repeated treatment with quinpirole increased pouching behavior and body weight and disrupted PPI. No changes in sexual activity in response to repeated quinpirole were found. Prior quinpirole treatment enhanced D1 dopamine receptor-stimulated adenylate cyclase activity in the caudate-putamen that was blocked by co-incubation with the D1 dopamine antagonist, SCH23390. These results show that repeated activation of D2-like receptors in vivo can produce changes in feeding behavior and sensory processing that is associated with sensitization of D1 dopamine receptor-mediated signaling in the caudate-putamen.

Prostaglandin E2 Protects Gastric Mucosal Cells from Apoptosis via EP2 and EP4 Receptor Activation

Prostaglandin E(2) (PGE(2)) has a strong protective effect on the gastric mucosa in vivo; however, the molecular mechanism of a direct cytoprotective effect of PGE(2) on gastric mucosal cells has yet to be elucidated. Although we reported previously that PGE(2) inhibited gastric irritant-induced apoptotic DNA fragmentation in primary cultures of guinea pig gastric mucosal cells, we show here that PGE(2) inhibits the ethanol-dependent release of cytochrome c from mitochondria. Of the four main subtypes of PGE(2) receptors, we also demonstrated, using subtype-specific agonists, that EP(2) and EP(4) receptors are involved in the PGE(2)-mediated protection of gastric mucosal cells from ethanol-induced apoptosis. Activation of EP(2) and EP(4) receptors is coupled with an increase in cAMP, for which a cAMP analogue was found here to inhibit the ethanol-induced apoptosis. The increase in cAMP is known to activate both protein kinase A (PKA) and phosphatidylinositol 3-kinase pathways. An inhibitor of PKA but not of phosphatidylinositol 3-kinase blocked the PGE(2)-mediated protection of cells from ethanol-induced apoptosis, suggesting that a PKA pathway is mainly responsible for the PGE(2)-mediated inhibition of apoptosis. Based on these results, we considered that PGE(2) inhibited gastric irritant-induced apoptosis in gastric mucosal cells via induction of an increase in cAMP and activation of PKA, and that this effect was involved in the PGE(2)-mediated protection of the gastric mucosa from gastric irritants in vivo.

Dissociable effects of the kappa-opioid receptor agonists bremazocine, U69593, and U50488H on locomotor activity and long-term behavioral sensitization induced by amphetamine and cocaine.

Mesolimbic dopaminergic neurotransmission plays a critical role in the locomotor effects of psychostimulant drugs, but a general involvement in the induction of long-term psychostimulant sensitization is questionable. By influencing dopaminergic neurotransmission, opioid drugs can alter the behavioral effects of psychostimulants. The effects of the kappa-opioid receptor agonists bremazocine, U69593, and U50488H on the locomotor stimulant and the long-term sensitizing effects of amphetamine and cocaine were investigated in rats. Unlike U69593 and U50488H, bremazocine is also an antagonist at mu- and delta-opioid receptors, as well as an agonist at a subtype of delta-opioid receptors inhibiting dopamine D1 receptor-stimulated adenylate cyclase. Bremazocine, U69593, and U50488H were administered prior to amphetamine and cocaine, and locomotor activity was measured. In separate studies, the opioids were co-administered with amphetamine and cocaine for 5 days, and locomotor sensitization was assessed 3 weeks post-treatment. Bremazocine and U69593 attenuated the psychomotor stimulant effects of amphetamine and cocaine. U50488H attenuated the locomotor effect of cocaine and biphasically affected amphetamine-induced locomotion, i.e., suppression followed by stimulation. Bremazocine prevented the development of amphetamine-induced but not cocaine-induced long-term sensitization. Neither U69593 nor U50448H affected the induction of long-term amphetamine or cocaine sensitization. In agreement with previous studies, the present data suggest that differential mechanisms underlie the acute stimulant versus the long-term sensitizing effects of psychostimulants, and the induction of long-term sensitization by amphetamine versus cocaine. Stimulation of kappa-opioid receptors does not seem to block the induction of long-term psychostimulant sensitization. Thus, bremazocine is likely to block the induction of amphetamine sensitization through a non-kappa-opioid receptor mechanism. We suggest that this effect of bremazocine is the result of its unique agonist action at a subtype of delta-opioid receptors, thereby acting as a functional dopamine D1 receptor antagonist. This would be consistent with the literature showing that the induction of long-term amphetamine sensitization depends on the activation of dopamine D1 receptors. In addition, the present data are in keeping with studies showing that dopamine neurotransmission is not critical for the induction of long-term cocaine sensitization.

Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1.

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioural and immune responses to stress, and has been implicated in the stress-like and other aversive consequences of drug abuse, such as withdrawal from alcohol. Two CRH receptors, Crhr1 and Crhr2, have been identified in the mouse. Crhr1 is highly expressed in the anterior pituitary, neocortex, hippocampus, amygdala and cerebellum, and activation of this receptor stimulates adenylate cyclase. Here we show that in mice lacking Crhr1, the medulla of the adrenal gland is atrophied and stress-induced release of adrenocorticotropic hormone (ACTH) and corticosterone is reduced. The homozygous mutants exhibit increased exploratory activity and reduced anxiety-related behaviour under both basal conditions and following alcohol withdrawal. Our results demonstrate a key role of the Crhr1 receptor in mediating the stress response and anxiety-related behaviour.

Early Emergence of Three Dopamine D1 Receptor Subtypes in Vertebrates: MOLECULAR PHYLOGENETIC, PHARMACOLOGICAL, AND FUNCTIONAL CRITERIA DEFINING D1A, D1B, AND D1C R/ECEPTORS IN EUROPEAN EELANGUILLA ANGUILLA

The existence of dopamine D1C and D1D receptors in Xenopus and chicken, respectively, challenged the established duality (D1A and D1B) of the dopamine D1 receptor class in vertebrates. To ascertain the molecular diversity of this gene family in early diverging vertebrates, we isolated four receptor-encoding sequences from the European eel Anguilla anguilla. Molecular phylogeny assigned two receptor sequences (D1A1 and D1A2) to the D1A subtype, and a third receptor to the D1B subtype. Additional sequence was orthologous to the Xenopus D1C receptor and to several other previously unclassified fish D1-like receptors. When expressed in COS-7 cells, eel D1A and D1B receptors display affinity profiles for dopaminergic ligands similar to those of other known vertebrate homologues. The D1C receptor exhibits pharmacological characteristics virtually identical to its Xenopus homologue. Functionally, while all eel D1 receptors stimulate adenylate cyclase, the eel D1B receptor exhibits greater constitutive activity than either D1A or D1C receptors. Semiquantitative reverse transcription-polymerase chain reaction reveals the differential distribution of D1A1, D1A2, D1B, and D1C receptor mRNA within the hypothalamic-pituitary axis of the eel brain. Taken together, these data suggest that the D1A, D1B, and D1C receptors arose prior to the evolutionary divergence of fish and tetrapods and exhibit molecular, pharmacological, and functional attributes that unambiguously allow for their classification as distinct D1 receptor subtypes in the vertebrate phylum.

CAMP-dependent regulation of Ca2+ channels expressed inXenopus oocytes

Rat forebrain- and heart-derived mRNA were used to express Ca2+ channels inXenopus oocytes to study their cAMP-dependent regulation. Forebrain and heart mRNA-directed Ca2+ channel currents (I Ba, 40 mM Ba2+ were used as a charge carrier) showed similar voltage dependence and macroscopic kinetics but different pharmacology, which allowed us to attribute them to N- and L-type, respectively. Brain mRNA-directedI Ba was insensitive to the dihydropyridine (DHP) antagonist nitrendipine and the agonist Bay K 8644, but could be inhibited by 70% by 1 μM of ω-conotoxin GVIA, whileI Ba directed by cardiac mRNA was extremely sensitive to DHP. Neither forebrain, nor heart mRNA-directedI Ba could be augmented by the external applications of the β-agonist isoproterenol (ISO, 10 μM), the adenylate cyclase (AC) activator forskolin (FSK, 10 μM), the phosphodiesterase inhibitor IBMX (200 μM), or their mixtures. “Cardiac”I Ba was also unresponsive to the external applications of a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (500 μM), as well as to the direct intracellular infusion of cAMP (300 μM). Blockade of cAMP-dependent phosphorylation pathway by intracellular perfusion of the oocytes with 200 μM Rp-cAMP plus 200 μM of a synthetic protein kinase A (PKA) inhibitor peptide also exerted no effect on the basal level ofI Ba, suggesting that the expressed Ca2+ channels are not fully phosphorylated in the resting state. Measurements of the concentration of cAMP in the control and heart mRNA-injected oocytes, using an enzyme-immunoassay system, showed that they display a similar basal cAMP concentration (2.0–2.5 μM); however, application of ISO + FSK increased the cAMP concentration 2- to 3-fold in mRNA-injected oocytes, but not in control oocytes. Thus, our data demonstrate that injection of rat cardiac mRNA intoXenopus oocytes results in the expression of receptor-stimulated AC and L-type Ca2+ channels, which do not respond to cAMP or PKA inhibitors. Unresponsiveness to cAMP-dependent regulation is not channel type-specific, since N-type Ca2+ channels expressed by means of forebrain mRNA are also insensitive to such regulation. Unresponsiveness of the channels to cAMP-mediated regulation is most probably due to lack/inaccessibility of PKA-dependent phosphorylation site(s), or loss of functional significance of phosphorylation.

Muscarinic m1 receptor-stimulated adenylate cyclase activity in Chinese hamster ovary cells is mediated by Gsα and is not a consequence of phosphoinositidase C activation

The mechanism underlying muscarinic m1 receptor-mediated increases in adenosine 3',5'-cyclic monophosphate (cAMP) was investigated in Chinese hamster ovary (CHO) cells expressing human recombinant m1 muscarinic receptors (CHO-ml cells). Stimulation of CHO-ml cells with carbachol resulted in marked accumulation of Ins(1,4,5)P3 and cAMP, in an atropine-sensitive manner, with EC50 values (log M) of -5.16 +/- 0.06 and -3.93 +/- 0.07 respectively. Basal and agonist-stimulated cAMP accumulation were unaffected by a 5 min pretreatment with l microM phorbol 12,13-dibutyrate and were not attenuated by pertussis toxin (100 ng/ml, 20h). Agonist-stimulated cAMP accumulation was also observed in CHO-ml cell membranes incubated in a buffer containing 100 nM free Ca2+. Guanosine 5'- [gamma-thio]triphosphate (10 microM) potentiated agonist-stimulated cAMP accumulation in CHO-ml cell membranes, implicating a G-protein involvement in this response. Co-incubation of carbachol with forskolin (10 microM) produced a greater than additive accumulation of cAMP in CHO-ml cells. Furthermore, a C-terminal-directed anti-Gs alpha serum attenuated both carbachol-stimulated (in CHO-ml cell membranes) and isoprenaline-stimulated (in CHO-beta 2 cell membranes) cAMP accumulation with a similar dose-dependency. These results suggest that muscarinic agonist-stimulated cAMP accumulation in CHO-ml cells occurs via activation of Gs alpha and not as a consequence of phosphoinositidase C activation.

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE 1 receptors and stimulatory G proteins

Chronic exposure of all- trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 μM; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 μM) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE, receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE 1 receptor number ( B max) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPγS (100 μM) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of G sα and S49 cyc − reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE 1 receptors and G s in situ by application of a C-terminal anti-G sα antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE 1 receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available G sα within the PGE 1 receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 μM)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE 1 receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.

Interaction of the antiarrhythmic agents SR 33589 and amiodarone with the beta-adrenoceptor and adenylate cyclase in rat heart.

1. The effects of SR 33589 and amiodarone on the cardiac beta-adrenoceptor were studied in vitro and after chronic treatment by means of [125I]-(-)-iodocyanopindolol ([125I]-(-)-CYP) binding and measurement of adenylate cyclase activity. 2. Binding of [125I]-(-)-CYP was inhibited in a dose-dependent manner by SR 33589 (IC50=1.8 +/- 0.4 microM, nH=0.93 +/- 0.06) and amiodarone (IC50=8.7 +/- 2.0 microM, nH=9.2 +/- 0.03). Saturation binding experiments indicated a non-competitive interaction such that SR 33589 (1 and 3 microM) and amiodarone (5 and 10 microM) reduced the Bmax of [125I]-(-)-CYP binding without any effect on the KD. Kinetic studies showed that the rate of association of [125I]-(-)-CYP was unchanged while the rate of dissociation was increased both in the presence of SR 33589 (10 microM) and amiodarone (30 microM).3. Under the same conditions, the receptor stimulated adenylate cyclase activity was inhibited in a dose-dependent, but non-competitive manner, by SR 33589 (isoprenaline-, glucagon- and secretin-stimulated enzyme inhibited 50% at 6.8 +/- 0.6 microM, 31 +/- 10 microM and 12 +/- 3 microM, respectively) while the basal, GTP- and GPP(NH)p-stimulated enzyme was inhibited by 5-10% and the NaF and forskolin-stimulated enzyme by 50% at 500 microM. Amiodarone exhibited a similar pattern of inhibition. 4. After chronic oral treatment (50, 100, 150 mg kg(-1) per day, 14 days), both SR 33589 and amiodarone produced a dose-dependent decrease in Bmax without any effect on KD as determined from [125I]-(-)-CYP saturation experiments and a decrease of the isoprenaline- and glucagon-stimulated adenylate cyclase activity without any effect on basal enzyme activity or activity when stimulated by agents acting directly on regulatory catalytic units. 5. Unlike amiodarone, SR 33589 does not contain iodine substituents. Plasma levels of T3, T4, and rT3 were changed after SR 33589 treatment except a decrease in T4 level at the highest dose whilst the T4 T3 ratio and the level of rT3 were dose-dependently increased by amiodarone treatment. 6. In vitro, SR 33589 and amiodarone were characterized as non-competitive beta-adrenoceptor antagonists. Chronic treatment led to a down-regulation of the beta-adrenoceptor; the down-regulation cannot be attributed to an indirect effect mediated by the thyroid hormones. To reconcile these opposing observations, we propose that SR 33589 and amiodarone interact with the beta-adrenoceptor at a site close to the intracellular loops which are involved in the coupling with Gs and contain the phosphorylable sites.

Detection of Adenylate Cyclase-Coupled Receptors in Xenopus Oocytes by Coexpression with Cystic Fibrosis Transmembrane Conductance Regulator

To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)-a cAMP-dependent Cl − channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl − current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP 2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists.

The Dopamine D1D Receptor: CLONING AND CHARACTERIZATION OF THREE PHARMACOLOGICALLY DISTINCT D1-LIKE RECEPTORS FROM Gallus domesticus

Three genomic clones encoding dopamine D1-like receptors were isolated from the avian species Gallus domesticus. Two of these genes encode proteins of 451 and 488 amino acids, which, based on deduced amino acid sequence identity and homology of exhibited pharmacological profiles, appear to be species homologs of mammalian and vertebrate D1/D1A and D5/D1B receptors, respectively. The third genomic clone, termed D1D, encodes a protein of 445 amino acids displaying a deduced amino acid sequence identity within putative transmembrane domains of 75% to mammalian D1/D1A and 77% to D5/D1B receptors with overall sequence homologies of only 49% and 46%, respectively. Membranes from COS-7 cells transfected with D1D DNA bound [3H]SCH-23390 in a saturable manner with high affinity (approximately 300 pM) and with a pharmacological profile clearly indicative of a dopamine D1-like receptor. The D1D receptor exhibited affinities for 6,7-dihydroxy-2-aminotetralin and dopamine 10-fold higher than D1/D1A receptors, characteristic of the D5/D1B receptor subfamily. In contrast, the D1D receptor bound dopaminergic agents, such as SKF-38393, apomorphine, pergolide, and lisuride, with affinities 10-fold higher than other cloned mammalian or vertebrate D1A/D1B receptor subtypes, while both clozapine and haloperidol displayed considerably lower affinity for the D1D receptor. Based on the low overall amino acid sequence identity (54%) and unique pharmacological profile, the avian dopamine D1D receptor does not appear to be a species homolog of the recently cloned vertebrate D1C receptor (Sugamori, K.S., Demchyshyn, L. L., Chung, M., and Niznik, H. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10536-10540). As with all cloned mammalian and vertebrate D1-like receptors, the D1D receptor stimulates adenylate cyclase activity in the presence of dopamine or SKF-82526. Northern blot analysis reveals the selective expression of both avian D1D and D1A receptor mRNAs only in brain with the D1B receptor more widely distributed and localized in tissues such as brain, kidney, and spleen. The isolation of four distinct vertebrate dopamine D1 receptor subtypes suggests the existence of additional mammalian D1 like receptor genes that may account for the observed pharmacological and biochemical multiplicity of dopamine D1-like receptor mediated events.

Repeated and chronic morphine administration causes differential long-lasting changes in dopaminergic neurotransmission in rat striatum without changing its δ- and κ-opioid receptor regulation

Repeated, once daily morphine treatment (14 days) as well as chronic morphine administration (6 days) caused a rebound reduction in the electrically evoked release of [ 3H]dopamine from superfused rat striatal slices 1 day after the last subcutaneous injection. Interestingly, whereas [ 3H]dopamine release remained significantly reduced for at least 3 weeks following morphine withdrawal in chronically treated (tolerant/dependent) rats, neurotransmitter release from dopaminergic nerve terminals gradually increased above control values following cessation of repeated morphine administration. Postsynaptically, dopamine D 1 receptor-stimulated adenylate cyclase appeared to be sensitized 1–3 days but was unchanged 3 weeks after chronic morphine treatment. In contrast, such an enhanced postsynaptic dopamine D 1 receptor efficacy did not occur 1–3 days following repeated morphine administration, but appeared to develop slowly resulting in a profound increase of dopamine-sensitive adenylate cyclase 3 weeks after the last injection. The inhibitory effect of dynorphin A-(1–13) on [ 3H]dopamine release, as well as that of [Met 5]enkephalin on dopamine D 1 receptor-stimulated adenylate cyclase appeared to be unchanged subsequent to repeated or chronic morphine treatment. These data indicate that, long after cessation of drug treatment, chronic morphine treatment causes a reduction whereas repeated morphine administration gradually induces an enhancement of opioid receptor-regulated dopaminergic neurotransmission due to local adaptive changes within the rat striatum. Such distinct long-lasting alterations of dopaminergic neurotransmission induced by different temporal patterns of morphine administration in projection areas of mesencephalic dopaminergic neurons may be related to the enduring effects of drug abuse such as behavioural sensitization and drug craving.

Adrenocorticotropin/α‐Melanocyte‐Stimulating Hormone (ACTH/MSH)‐Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor‐Coupled Mechanism

The regulation of adenylate cyclase activity by adrenocorticotropin/alpha-melanocyte-stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1-24), alpha-MSH (EC50 values 16 and 6 nM, respectively), and [Nle4,D-Phe7]alpha-MSH (EC50 value 1.6 nM), in the presence of forskolin (1 microM, optimal concentration). 1-9-Dideoxyforskolin did not augment the response of adenylate cyclase to ACTH-(1-24). Various peptide fragments were tested for their ability to enhance [3H]cyclic AMP production. [Nle4,D-Phe7]alpha-MSH increased [3H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1-24), ACTH-(1-16)-NH2, alpha-MSH, ACTH-(1-13)-NH2, [MetO4]alpha-MSH, [MetO2(4),D-Lys8,Phe9]ACTH-(4-9), ACTH-(7-16)-NH2, ACTH-(1-10), and ACTH-(11-24), in order of potency. This structure-activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1-24) stimulated adenylate cyclase activity in both striatal (maximal effect, approximately 20%) and septal slices (maximal effect, approximately 40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle4,D-Phe7]alpha-MSH on [3H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor-stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1-24) did not affect the dopamine D1 or D2 receptor-mediated modulation of adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Homologous desensitization of calcitonin receptors and calcitonin-dependent adenylate cyclase in T47D cells

Prolonged use of calcitonin (CT) in vivo leads to a loss of biological response (escape). To understand the molecular basis of this phenomenon, we examined desensitization of calcitonin receptors and down-regulation of adenylate cyclase response to CT in T47CD cells after a preincubation with CT. Preincubation with salmon or human CT (sCT, hCT) for 3 h led to a concentration-dependent loss of [125I]-sCT binding and a similar loss of adenylate cyclase response to a maximal stimulatory dose of sCT. At the same time there was an increased basal activity of the adenylate cyclase. After 24 h preincubation with sCT, basal cAMP levels fell considerably but not to basal levels. Time course experiments showed a delayed decline of maximally sCT-stimulated cAMP levels, which started after 1 h, while binding values declined over the first 60 min to one-third of the original values. Upon removal of CT from the medium, recovery of hormone binding occurred in parallel with the recovery of the adenylate cyclase response to sCT. T47D cells incubated with 25 mumol/l monensin, a lysosomal inhibitor, showed a persistent [125I]-sCT binding after removal of hormone, consistent with diminished intracellular receptor degradation. However, despite persistent binding to the cells, basal and stimulated cAMP levels dropped in the same manner as seen in controls. Our experiments support the view that tight binding of CT to its receptor stimulates adenylate cyclase in T47D cells, until receptors are removed by internalization. The physiological role of internalization of the CT receptor might be to end continuous stimulation of the adenylate cyclase, which occurs after binding.

Opioid receptors and inhibition of dopamine-sensitive adenylate cyclase in slices of rat brain regions receiving a dense dopaminergic input

In slices of rat nucleus accumbens, olfactory tubercle, frontal cortex and mediobasal hypothalamus exposed to dopamine (DA), the activation of DA D 1 receptors stimulated cyclic AMP (cAMP) formation whereas, in nucleus accumbens slices only, activation of D 2 receptors appeared to inhibit D 1 receptor-stimulated adenylate cyclase at the same time. Activation of μ-opioid receptors by [D-Ala 2,MePhe 4,Gly-ol 5]enkephalin (DAMGO; 1 μM),but not of δ-opioid receptors by 1 μM [D-Pen 2,D-Pen 5]enkephalin (DPDPE), inhibited (by 35–40%) DA-stimulated cAMP production in slices of nucleus accumbens and olfactory tubercle. When adenylate cyclase was stimulated by selective D 1 receptor activation, i.e. by DA in the presence of (−)-sulpiride, DPDPE reduced cAMP formation (by about 45%) in nucleus accumbens slices but not in slices of the other brain dregions. The κ-agonist, U 50,488, did not affect DA- or D 1 receptor-stimulated adenylate cyclase activity in any of the brain regions. Preincubation of nucleus accumbens slices with the irreversible δ-ligand, fentanyl isothiocyanate (FIT; 1 μM), not only antagonized the inhibitory effect of DPDPE but also prevented the antagonism by naloxone of the inhibitory effect of DAMGO. Therefore in nucleus accumbens opioids may inhibit DA-sensitive adenylate cyclase through activation of a μ/δ-opioid receptor complex, whereas in olfactory tubercle μ-receptors appear to mediate the inhibition of adenylate cyclase activity. Opioids do not seem to affect DA-stimulated cAMP formation in frontal cortex and mediobasal hypothalamus.

Modulation by GTP of basal and agonist-stimulated striatal adenylate cyclase activity following chronic blockade of D1 and D2 dopamine receptors: involvement of G proteins in the development of receptor supersensitivity.

Rats receiving injections of specific antagonists of dopamine receptors (SCH 23390 for D1, haloperidol for D2, and haloperidol+SCH 23390) once daily for 21 days develop a selective supersensitivity of the blocked receptors. To study the molecular correlates of these adaptive changes, we evaluated the involvement of GTP-binding proteins in the development of supersensitivity of dopamine receptors. By means of adenylate cyclase studies, we tested whether any of the treatments modified the functional response to GTP in striata dissected from control and treated rats. Our data show that the chronic blockade of D1 and/or D2 receptors potentiates both basal and dopamine receptor-stimulated adenylate cyclase activity in response to GTP. D1 receptor up-regulation correlates with an increased adenylate cyclase response to GTP, whereas D2 receptor up-regulation is accompanied by an enhanced GTP-induced inhibition of enzyme activity, in both basal and receptor-activated conditions. This potentiation does not seem to match the changes in mRNA content of Gs and Gi alpha subunits. Unexpectedly, however, a significant increase in Gi alpha subunit mRNA was found after the chronic blockade of D1 receptors; this result could be explained by cross-regulation between GTP-binding protein-mediated pathways. This cross-regulation could serve as a protective mechanism whereby cells exposing up-regulated receptors protect themselves from a condition of hyperactivity of the adenylate cyclase enzyme.

Cyclic AMP produces desensitization of prostacyclin and adenosine A 2 receptors in hybrid cell lines but does not affect G s function

Prostacyclin and adenosine A 2 receptors stimulate adenylate cyclase activity in the related somatic hybrid cell lines NG108-15 and NCB20. The role of cAMP in the desensitization of these receptors has been examined. Pretreatment for 17 h with forskolin or 8-bromo-cAMP had the same effect in both cell lines. There was no change in the response to sodium fluoride or forskolin, suggesting that the function of G s and adenylate cyclase were unaffected by increased levels of cAMP. Receptor responses were affected however; the maximum response to N-ethylcarboxamidoadenosine (an A 2 receptor agonist) was reduced by 30–40%, there was a small but consistent shift to the right of the dose-response curve for iloprost (a stable analogue of prostacyclin) and [ 3H]iloprost binding studies revealed a loss of prostacyclin receptors. However, the loss of receptor responsiveness was much smaller than that which occurs following pretreatment with prostacyclin or adenosine A 2 receptor agonists (Keen et al. (1989) Biochem. Pharmacol. 38, 3827–3833; Kelly et al. (1990) Br. J. Pharmacol. 99, 309–316) suggesting that cAMP may not play a major role in agonist mediated desensitization.

A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns.

Biogenic amines such as serotonin elicit or modulate a wide range of behaviours by interacting with multiple receptor subtypes. We have isolated cDNA clones encoding three distinct Drosophila serotonin receptors which belong to the G protein-coupled receptor family. When expressed in mammalian cells, these receptors activate different intracellular effector systems. The 5HT-dro1 receptor stimulates adenylate cyclase while the 5HT-dro2A and the 5HT-dro2B receptors inhibit adenylate cyclase and activate phospholipase C. Expression of all three receptors starts in late embryos and is restricted to distinct populations of cells in the central nervous system. The 5HT-dro2A receptor is predominantly expressed in midline motor neurons (VUM neurons) that innervate larval muscles thus suggesting a role for this receptor in motor control.

Differential effects of chronic agonist administration on μ-opioid receptor- and muscarinic receptor-regulated adenylate cyclase in rat striatal neurons

In cultured rat striatal neurons exposed to 10 μM morphine or oxotremorine for 24 hours, we observed an increased (about 30 %) dopamine D 1 receptor-stimulated cyclic AMP production, whereas no desensitization of μ-opioid receptor or muscarinic cholinergic receptor was found. However, whereas upregulation of dopamine D 1 receptor-stimulated adenylate cyclase activity upon 7 days morphine exposure was at least as pronounced as observed after 24 hours of exposure to the opioid, this adaptive phenomenon was virtually absent following one week of oxotremorine treatment. This reduced adenylate cyclase sensitization upon 7 days oxotremorine exposure appeared to coincide with desensitization of muscarinic cholinergic receptors. A possible role of the resistance of μ receptors to desensitization and the (resulting) upregulation of the neuronal adenylate cyclase system upon chronic receptor activation in the development of opiate tolerance and dependence is suggested.