Pharmacological modifications in dopaminergic neurotransmission affect the quinpirole-evoked suppression of serotonin N-acetyltransferase activity in chick retina: an impact on dopamine D4-like receptors

Dopamine (DA) plays an important role in the regulation of melatonin biosynthesis in retinas of several vertebrate species. In the retina of chick, the DA receptor controlling melatonin production represents a D4-like subtype. Stimulation of this receptor by quinpirole (QNP) results in a dose-dependent decline of the nighttime activity of serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthesis) and melatonin level of chick retina. The present study was undertaken to determine whether long-term treatment with antipsychotic drugs (clozapine-30 mg/kg, i.m.; sulpiride-100 mg/kg, i.m.; and raclopride-10 mg/kg, i.p., once daily for 21 days) and L-DOPA (80 mg/kg, i.p., once daily for 7 days) affects the response of the melatonin generating system of chick retina to the suppressive effect of QNP. Chronic administration to chicks of clozapine and sulpiride, but not raclopride, resulted in a markedly increased response of retinal NAT activity to the action of QNP. ED50 values for QNP were 3-times (clozapine) and 4-times (sulpiride) lower than those in the respective vehicle-treated control groups. On the other hand, QNP was significantly less potent in retinas of birds treated with L-DOPA than in control animals; the ED50 value for QNP was 3-times higher in birds injected with L-DOPA than in the vehicle-treated group. These results indicate that long-term treatment with clozapine, sulpiride and L-DOPA may modify the reactivity of D4-like DA receptors regulating NAT activity of chick retina. A possibility of modifications of circadian and electrophysiological processes within the eye following prolonged administration of DA-ergic drugs is discussed.     

Selective activation of Galphao by D2L dopamine receptors in NS20Y neuroblastoma cells

D2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2L receptors with the selective D2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Galphai1*, Galphai2*, Galphai3*, and Galphao*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPgammaS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Galpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein alpha subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2L cells with HSV-Galphao* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Galphai1*, HSV-Galphai2*, or HSV-Galphai3* failed to rescue either response. In summary, the current study provides strong evidence that the D2L dopamine receptor couples to Galphao in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2 receptor activation on adenylate cyclase activity.     

The Mel1a melatonin receptor is coupled to parallel signal transduction pathways

The recent cloning of a family of high affinity melatonin receptors has provided us with a unique opportunity to define the signal transduction pathways used by these receptors. We have studied signaling through the human Mel1a receptor subtype by stable expression of receptor complementary DNA in NIH 3T3 cells. Our data indicate that the human Mel1a receptor is coupled to inhibition of forskolin-stimulated cAMP accumulation by a pertussis toxin-sensitive G protein. Although melatonin alone is without effect on phosphoinositide hydrolysis, it potentiates the effects of PGF2 alpha stimulation on phospholipase C activation. Melatonin potentiates arachidonate release stimulated by PGF2 alpha and by ionomycin. The effects of melatonin on arachidonate release are sensitive to inhibition of protein kinase C. They are independent of the effects of melatonin on cAMP and do not appear to involve activation of mitogen-activated protein kinase. The effects of melatonin on both phosphoinositide hydrolysis and arachidonate release are sensitive to pertussis toxin treatment. Thus, we show that the melatonin signal is transduced by parallel pathways involving inhibition of adenylyl cyclase and potentiation of phospholipase activation.     

The pharmacology of adenylyl cyclase modulation by GABAB receptors in rat brain slices

GABAB receptor activation inhibits forskolin-stimulated adenylyl cyclase activity but augments noradrenaline-stimulated adenylyl cyclase activity. The present study investigated the pharmacology of these two GABAB receptor mediated responses. In a cross-chopped rat cortical slice preparation, it was confirmed that (-)baclofen inhibited forskolin-stimulated adenylyl cyclase activity and augmented noradrenaline-stimulated adenylyl cyclase activity. The potency of five further agonists was investigated (SKF97541, CGP47656, CGP44533, 3-APA and CGP44532). Of these agonists two compounds were significantly more potent as inhibitors of forskolin-stimulated adenylyl cyclase than as augmenters of noradrenaline-stimulated adenylyl cyclase activity, these were (-)baclofen (pEC50 = 6.07 +/- 0.29 and 5.04 +/- 0.17, respectively (p < 0.05)), and CGP47656 (pEC50 = 6.44 +/- 0.05 and 4.48 +/- 0.26, respectively (p < 0.05)). It is possible to explain this difference in potency by proposing that these compounds have low intrinsic efficacy, and the augmentation of noradrenaline-stimulated adenylyl cyclase has a low receptor reserve. In addition six antagonists (CGP49311A, CGP46381, CGP45024, CGP45397, CGP36742) were also tested for their ability to antagonize 10 microM (-)baclofen in these two assays. These antagonists ranged in potency as inhibitors of forskolin-stimulated adenylyl cyclase activity from CGP49311A (pEC50 = 5.45 +/- 0.30) to CGP36742 (pEC50 = 3.87 +/- 0.16). Each antagonist had similar potency in the two assays, suggesting that these two responses are mediated by pharmacologically similar receptors.     

Receptor subtype and density determine the coupling repertoire of the 5-HT2 receptor subfamily

The 5-Hydroxytryptamine (5-HT)2C receptor (originally known as the 5-HT1C receptor) is a member of the 5-HT2 subfamily of G protein coupled receptors, which is known to couple to phospholipase C. Within the 5-HT2 subfamily, only the 5-HT2C receptor also coupled to inhibition of forskolin-stimulated cAMP production when expressed at high density (12 pmol/mg membrane protein) in stably transformed AV12 cells. The 5-HT2C receptor coupled with high efficacy to both phospholipase C as measured by IP3 (inositol 1,4,5-trisphosphate) production and to inhibition of forskolin-stimulated cAMP production (EC50 = 2.98 nM +/- 0.9 and IC50 = 47.99 nM +/- 10.25 respectively). The 5-HT2A and 5-HT2B receptors, while coupling to phospholipase C with high affinity (EC50s of 19.24 nM +/- 6.44 and 1.24 nM +/- 0.136 respectively), did not decrease adenylyl cyclase activity. The 5-HT2C receptor actions in both systems showed the expected pharmacology for the 5-HT2C receptor, e.g., mesulergine antagonized the effects of 5-HT and spiperone did not. Preincubation of cells with PTX showed that the G protein coupling of the 5-HT2C receptor to phospholipase C is PTX insensitive, while the G protein coupling to inhibition of adenylyl cyclase is PTX sensitive, even to concentrations as low as 20 ng/ml of PTX. PTX pretreatment of the 5-HT2C bearing cells also unmasked a small stimulatory effect on adenylyl cyclase. When expressed at low density the 5-HT2C receptor potentiated forskolin-stimulated cAMP production by 2 fold while still maintaining its ability to enhance PI hydrolysis. A more modest potentiation of cAMP production was noted with low density expression of the 5-HT2B receptor. Thus the ability of the 5-HT2C receptor to interact with several effectors through at least two different G proteins is, in part, receptor subtype specific but also influenced by receptor density.     

Molecular analysis of Chs3p participation in chitin synthase III activity

Adenylyl cyclase superactivation, a phenomenon by which chronic activation of inhibitory Gi/o-coupled receptors leads to an increase in cAMP accumulation, is believed to play an important role as a compensatory response of the cAMP signaling system in the cell. However, to date, the mechanism by which adenylyl cyclase activity is regulated by chronic exposure to inhibitory agonists and the nature of the adenylyl cyclase isozymes participating in this process remain largely unknown. Here we show, using COS-7 cells transfected with the various AC isozymes, that acute activation of the D2 dopaminergic and m4 muscarinic receptors inhibited the activity of adenylyl cyclase isozymes I, V, VI, and VIII, whereas types II, IV, and VII were stimulated and type III was not affected. Conversely, chronic receptor activation led to superactivation of adenylyl cyclase types I, V, VI, and VIII and to a reduction in the activities of types II, IV, and VII. The activity of AC-III also was reduced. This pattern of inhibition/stimulation of the various adenylyl cyclase isozymes is similar to that we recently observed on acute and chronic activation of the mu-opioid receptor, suggesting that isozyme-specific adenylyl cyclase superactivation may represent a general means of cellular adaptation to the activation of inhibitory receptors and that the presence/absence and intensity of the adenylyl cyclase response in different brain areas (or cell types) could be explained by the expression of different adenylyl cyclase isozyme types in these areas.     

Sodium saccharin inhibits adenylyl cyclase activity in non-taste cells

We have studied the in vitro effect of sodium saccharin (NaSacch) on the rat adipocyte adenylyl cyclase complex. NaSacch (2.5-50 mM) inhibited significantly in a dose-dependent manner basal and isoproterenol-stimulated cAMP accumulation on isolated rat adipocytes. Similarly, NaSacch (2.5-50 mM) inhibited forskolin-stimulated adenylyl cyclase activity measured in the presence of Mg(2+)-ATP on adipocyte, astrocyte and thyrocyte membrane fractions. In contrast, NaSacch did not inhibit but slightly increased the forskolin-stimulated adenylyl cyclase activity measured in the presence of Mn(2+)-ATP and GDP beta S, a stable GDP analogue. The effect of NaSacch was not mediated through either the A1-adenosine receptor (A1R) or the alpha 2-adrenergic receptor (alpha 2AR). The inhibitory effect of NaSacch was additive to that of A1R agonist and was not blocked by the addition of the alpha 2AR antagonist RX 821002. Pretreatment of adipocytes with pertussis toxin slightly attenuated but did not abolish the inhibitory effect of NaSacch on forskolin-stimulated adenylyl cyclase activity on membrane fractions. These data suggest that the inhibitory effect of NaSacch on forskolin stimulated-adenylyl cyclase in adipocytes does not imply only Gi protein but also other direct or indirect inhibitory pathway(s) which remain to be determined.     

Characterization of the role of medial prefrontal cortex dopamine receptors in cocaine-induced locomotor activity.

Medial prefrontal cortex (mPFC) dopamine (DA) modulates the motor-stimulant response to cocaine. The present study examined the specific mPFC DA receptor subtypes that mediate this behavioral response. Intra-mPFC injection of the DA D?-like receptor agonist quinpirole blocked cocaine-induced motor activity, an effect that was prevented by coadministration of the D2 receptor antagonist sulpiride. Intra-mPFC injection of the selective D? receptor agonist PD 168,077 or the selective D? receptor agonist SKF 81297 did not alter the motor-stimulant response to cocaine. Finally, it was found that an intermediate dose of quinpirole, which only attenuated cocaine-induced motor activity, was not altered by SKF 81297 coadministration, suggesting a lack of synergy between mPFC D?, and D? receptors. These results suggest that D? receptor mechanisms in the mPFC are at least partly responsible for mediating the acute motor-stimulant effects of cocaine. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Dopamine modulates inwardly rectifying hyperpolarization-activated current (Ih) in cultured rat olfactory receptor neurons

The presence of dopamine receptors in olfactory receptor neurons (ORNs) suggests that odor sensitivity may be modulated by neurotransmitters at the level of primary sensory neurons. Using standard patch-clamp techniques on rat ORNs, we found that 1 microM dopamine, 500 microM SQ 22536 (SQ, an adenylyl cyclase inhibitor), 20 and 50 microM quinpirole (a selective dopamine D2 receptor agonist), and 1 mM adenosine 3', 5'-cyclic monophosphate (cAMP) modulate the hyperpolarization-activated current Ih. On hyperpolarizing from a holding potential of -58 mV, a small Cs+-sensitive inwardly rectifying current (Ih) was observed. Increases in extracellular K+ increased Ih amplitude without shifting its voltage dependence of activation, whereas increases in temperature produced an increase in Ih amplitude and a hyperpolarizing shift in the activation curve. Application of 1 microM dopamine reversibly shifted Ih activation to more negative potentials and decreased Ih current amplitudes. These effects were blocked by concomitant application of dopamine with sulpiride, a selective dopamine D2 receptor antagonist. The effects of dopamine were mimicked by quinpirole. Quinpirole (20 microM) decreased Ih current amplitude, but was without effect on Ih voltage dependence of activation. However, 50 microM quinpirole produced both a reduction of Ih peak currents and a hyperpolarizing shift in the activation curve for Ih. External application of the adenylyl cyclase inhibitor SQ 22536 produced a reversible decrease in peak currents but had no effect on Ih voltage dependence of activation, whereas internal application of cAMP shifted Ih activation to more depolarized potentials. Because Ih modulates cell excitability and spike frequency adaptation, our findings support a role for dopamine in modulating the sensitivity and output of rat ORNs to odorants.     

Gestational exposure to cocaine or pharmacologically related compounds: effects on behavior and striatal dopamine receptors

Gestational cocaine (COC) exposure has been reported to alter behavior and possibly dopamine (DA) receptors. In this paper, we further examined the effects of prenatal COC (40 mg/kg, s.c.) on DA receptor binding and the behavioral response to quinpirole, a DA D2 receptor agonist. In an attempt to elucidate possible mechanisms of such effects, we exposed pregnant dams to specific reuptake blockers; fluoxetine 12.5 mg/kg, a serotonin reuptake blocker; desipramine 10 mg/kg, a norepinephrine reuptake blocker; GBR-12909 10 mg/kg, a DA reuptake blocker; or to a local anesthetic, lidocaine 40 mg/kg. Drugs were administered once daily over gestational days 8-20. Control dams were injected with saline (SAL) or pair-fed to the COC group. Quinpirole challenge was performed in the offspring on post natal day 19. Two pups per litter were injected (s.c.) with 0.03 or 0.09 mg/kg quinpirole-HCl on post-natal day 19. The remaining pups in each litter were sacrificed for analysis of striatal DA receptors. Results showed that only COC exposure altered the behavioral response to the quinpirole challenge by increasing quinpirole-induced stereotypy and motor activity relative to SAL controls. DA receptor analysis showed no alteration in K(D) or B(MAX) for striatal D1 or D2 sites in any group. These results suggest that prenatal COC exposure produces alterations in function of the D2 receptor complex which are not reflected in K(D) or B(MAX) and that these effects are not fully mimicked by exposure to specific monoamine reuptake blockers or a local anesthetic.     

Stimulation of cAMP accumulation by the cloned Xenopus melatonin receptor through Gi and Gz proteins

The Xenopus melatonin receptor was expressed in human embryonic kidney 293 cells and assayed for cAMP accumulation. In transfected 293 cells expressing the melatonin receptor, melatonin dose-dependently inhibited the endogenous adenylyl cyclases. In contrast, melatonin stimulated the accumulation of cAMP in cells co-expressing the type II adenylyl cyclase. Both the inhibitory and stimulatory responses to melatonin were mediated via Gi-like proteins as they were blocked by pertussis toxin. Upon co-transfection with the alpha subunit of Gz, the ability of melatonin to regulate both type II and the endogenous adenylyl cyclases became refractory to pertussis toxin, indicating that the melatonin receptor can also couple to Gz. However, other pertussis toxin-insensitive G proteins such as Gq, G12 and G13 were unable to interact with the melatonin receptor.     

SH3 binding domains in the dopamine D4 receptor

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.     

Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol

Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs.     

Calcitriol attenuates the basal and vasoactive intestinal peptide-stimulated cAMP production in prolactin-secreting rat pituitary (GH4C1) cells

A clonal strain of prolactin-producing rat pituitary tumour cells (GH4C1 cells) was used to study the effect of calcitriol on cyclic adenosine monophosphate (cAMP) production. Calcitriol (10 nM) attenuated both the basal and vasoactive intestinal peptide (VIP)-stimulated cAMP production after 2 days' pretreatment of the cells. The effect was detectable at 1 nM and maximal at about 10 nM. Calcitriol was at least 100 times more potent than calcidiol and 24-hydroxycalcidiol. Calcitriol (10 nM, 4 days) did not affect the specific binding of 125I-VIP, but attenuated the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-stimulated (100 microM) adenylyl cyclase activity by 25%. Calcitriol (10 nM, 4 days) also attenuated both the Mn2+ (1 mM) and the forskolin-stimulated (10 microM) adenylyl cyclase activity by 43 and 41%, respectively. In conclusion, these data suggest that calcitriol attenuates the basal and VIP-stimulated cAMP production by inhibiting the catalytic subunit of the adenylyl cyclase as well as the amount of the G protein Gs alpha.     

Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes

Dopamine D2 receptor agonists are commonly used in the control of PRL-secreting adenomas, and the sensitivity of dopamine agonists during long term therapy is exquisite. However, the molecular mechanisms responsible for the maintenance of this cellular sensitivity to dopamine agonists remain poorly understood. In the present study, we examined the agonist-induced regulation of the human D2L receptor expressed to a specific activity of approximately 1 pmol receptor/mg protein in Sf9 insect cells. Treatment of D2L receptor-expressing cells with dopamine for up to 3 h resulted in no detectable change in the ligand-binding properties of the receptor and a approximately 120-fold reduction in the potency, but not the efficacy, of D2L receptors to mediate dopamine inhibition of forskolin-stimulated adenylyl cyclase activity. This resistance of the D2L receptor to agonist-induced desensitization was accompanied by a approximately 28% translocation of intracellular D2L receptors to the cell surface, as quantified by cellular fractionation and radioligand binding and visualized by whole cell immunocytochemical staining and confocal microscopy. Immunoblot analysis of the P2 membrane fraction revealed that surface D2L receptors comprised monomers and dimers. Treatment of D2L receptor-expressing cells with the protein synthesis inhibitor cycloheximide significantly reduced the basal expression level of receptors, but did not block the agonist-induced up-regulation of receptors. Longer periods of dopamine exposure for 24 h brought about a small increase in surface receptor density. However, when these studies were conducted in the presence of cycloheximide, receptor density was marginally reduced, suggesting that receptor synthesis accounts for the maintenance of cellular receptor density under these conditions. We conclude that the resistance of the D2L receptor-coupled adenylyl cyclase system to agonist-induced desensitization is attributed to the up-regulation of surface receptors after the translocation of existing intracellular receptors and de novo receptor synthesis.     

Active immunization using dendritic cells mixed with tumor cells inhibits the growth of primary breast cancer

The potential role of alpha2-adrenoceptors in modulating the activity of adenylyl cyclase in the rat striatum was examined. The selective alpha2-adrenoceptor agonist, UK14,304, produced a concentration-dependent inhibition of forskolin-stimulated accumulation of cAMP in striatal slices. The effect of UK14,304 was reversed by pre-incubation of striatal slices with the selective alpha2-adrenoceptor antagonist, RX821002. To determine whether alpha2C-adrenoceptors contribute to the alpha2-adrenoceptor-induced inhibition of forskolin-stimulated cAMP accumulation, an antisense oligodeoxynucleotide directed against alpha2C-adrenoceptor mRNA (alpha(2C)AS) or a random sequence (RS) was infused directly into the striatum. The ability of alpha(2C)AS to reduce the expression of alpha2C-adrenoceptors has been previously demonstrated. Alpha2C(AS) infusions did not reduce the ability of UK14,304 to inhibit forskolin-stimulated cAMP accumulation. Instead, alpha(2C)AS significantly enhanced forskolin-stimulated cAMP accumulation on the infusion side compared to the contralateral striatum. In contrast to the effects of alpha(2C)AS, infusions of RS had no effects on forskolin-stimulated cAMP accumulation or on the ability of UK14,304 to inhibit this effect. Incubation of striatal slices from untreated rats with RX821002 could mimic the ability of alpha(2C)AS infusion to enhance forskolin-stimulated cAMP accumulation, and did so in a concentration-dependent manner. Alpha2-adrenoceptors are negatively coupled to adenylyl cyclase in the rat striatum and alpha2C-adrenoceptors appear to be under tonic activation by an endogenous ligand in striatal slices.     

D4 dopamine receptor-mediated signaling events determined in transfected Chinese hamster ovary cells

A Chinese hamster ovary (CHO) cell line stably expressing a recombinant human D4 dopamine receptor made from a synthetic gene has been used to determine potential D4-mediated signaling events. We designed and synthesized a modified gene coding for a human D4 receptor with reduced G + C content but unaltered encoded amino acids. Stable expression of this gene was obtained in two cell lines, inducible expression in CHO lacI cells and constitutive expression in HEK293 cells. In CHO lacI cells induced to express D4 receptors but not in uninduced cells, dopamine and quinpirole inhibit forskolin-stimulated cAMP accumulation and potentiate ATP-stimulated [3H]arachidonic acid release through a mechanism that requires protein kinase C but is unaffected by membrane-soluble cAMP analogs. In addition, D4 receptor activation causes an increase in the rate of extracellular acidification measured by microphysiometry. This response is unaffected by protein kinase C down-regulation but is inhibited by removal of extracellular sodium and inhibitors of NaH-1 exchange, suggesting the involvement of a Na+/H+ exchanger. All responses are blocked by clozapine and are sensitive to pertussis toxin. D4 receptors, like other G(i)/G(o)-linked receptors, mediate multiple signaling events, and the pathways activated are similar to those used by D2 and D3 receptors expressed in similar cells.     

Identification and characterization of muscarinic receptors potentiating the stimulation of adenylyl cyclase activity by corticotropin-releasing hormone in membranes of rat frontal cortex

In membranes of the rat frontal cortex, acetylcholine (ACh) and other cholinergic agonists were found to potentiate the stimulation of adenylyl cyclase activity elicited by corticotropin-releasing hormone (CRH). Oxotremorine-M, carbachol and methacholine were as effective as ACh, whereas oxotremorine and arecoline were much less effective. The facilitating effect of Ach was potently blocked by the M1 antagonists R-trihexyphenidyl, telenzepine and pirenzepine and by the M3 antagonists hexahydro-sila-difenidol and p-fluorohexahydro-sila-difenidol, whereas the M2 and M4 antagonists himbacine, methoctramine, AF-DX 116 and AQ-RA 741 were less potent. The mamba venom toxin MT-1, which binds with high affinity to M1 receptors, was also a potent blocker. The pharmacological profile of the muscarinic potentiation of CRH receptor activity was markedly different from that displayed by the muscarinic inhibition of forskolin-stimulated adenylyl cyclase, which could be detected in the same membrane preparations. Moreover, the intracerebral injection of pertussis toxin impaired the muscarinic inhibition of cyclic AMP formation and reduced the Ach stimulation of [35S]GTPgammaS binding to membrane G proteins but failed to affect the facilitating effect on CRH receptor activity. The latter response was also insensitive to the phospholipase C inhibitor U-73122, the protein kinase inhibitor staurosporine and to the inhibitors of arachidonic acid metabolism indomethacin and nordihydroguaiaretic acid. These data demonstrate that in the rat frontal cortex, muscarinic receptors of the M1 subtype potentiate CRH transmission by interacting with pertussis toxin-insensitive G proteins.     

Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors

Eight dopamine receptor-like cDNA clones were isolated from the carp (Cyprinus carpio) retina and four dopamine receptor-like cDNA clones were isolated from the European eel (Anguilla anguilla) retina. These cDNA clones show high sequence and structural homology to the known dopamine receptor subtypes. The sequence similarity and phylogenetic analysis revealed that five subtypes (D1A3, D1A4, D1B, D1C and D1X) in the carp retina and four subtypes (D1A1, D1A2, D1B and D1C) in the eel retina are D1-like receptor subtypes, and three (D2, D4A and D4B) in the carp retina are D2-like receptor subtypes; no D2-like receptor was found in the eel. Carp D1A3 and D1A4, carp D4A and D4B, and eel D1A1 and D1A2 are highly homologous pairs of receptors which show significant, domain-specific differences to each other and to their species homologues. The structure of the third cytoplasmic loop in the carp D1X receptor was particularly different from the other D1-like receptors. The implications of these structural differences in terms of dopamine receptor activation and signalling are discussed. It is suggested that the known diverse physiological and pharmacological effects of dopamine on the retinal neurones are likely to be mediated through these multiple receptor subtypes which may be coupled to different signal transduction pathways.     

Differences in tryptophan binding to hepatic nuclei of NZBWF1 and Swiss mice: insight into mechanism of tryptophan''s effects

Catecholamine receptors of multiple classes have been shown to influence pineal melatonin synthesis in a species-specific manner. In these experiments, the effects of catecholamine receptor agonists on circadian melatonin rhythms of zebrafish (Danio rerio) pineal in vitro were examined. Cyclic application of adrenergic receptor agonists (norepinephrine, phenylephrine, clonidine, and isoproterenol) had no effect on zebrafish pineal melatonin release, nor on the circadian oscillator that regulates melatonin rhythms. Pineal melatonin release was partially suppressed by quinpirole, a D2 dopamine receptor agonist, but cyclic application of quinpirole did not reset the pineal circadian oscillator. Pineal melatonin release was unaffected by either dopamine or SKF38393, a D1 receptor agonist, suggesting that the effects of quinpirole were not mediated by dopamine receptors. The regulatory mechanisms underlying pineal melatonin rhythms appear to differ among teleosts.