Absence of dopamine sensitive adenylate cyclase in the A10 region, the origin of mesolimbic dopamine neurones.

Dopamine (DA) failed to stimulate the adenylate cyclase of the mesolimbic A10 DA nerve cell body area, in contrast to tis activating effect in the nigrostriatal A9 DA cell body area. The enzyme was stimulated by GMPPNP (a GTP analog) and NaF. This indicates the absence in the A 10 cell area of DA receptors with functional coupling on adenylate cyclase, in contrast to the A9 cell area where such DA receptors are believed to be located on afferent axon terminals.     

Melatonin receptors limit dopamine reuptake by regulating dopamine transporter cell-surface exposure

Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT₁ and MT₂ receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT₁ or MT₂ receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT₁ and MT₂) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.     

Extended and bent conformations of the mannose receptor family

In mammals, the mannose receptor family consists of four members, Endo180, DEC-205, phospholipase A₂ receptor and the mannose receptor. The extracellular domains of all these receptors contain a similar arrangement of domains in which an Nterminal cysteine-rich domain is followed by a single fibronectin type II domain and eight or ten C-type lectin-like domains. This review focuses on the threedimensional structure of the receptors in the mannose receptor family and its functional implication. Recent research has revealed that several members of this family can exist in at least two configurations: an extended conformation with the N-terminal cysteinerich domain pointing outwards from the cell membrane and a bent conformation where the N-terminal domains fold back to interact with C-type lectin-like domains at the middle of the structure. Conformational transitions between these two states seem to regulate the interaction of these receptors with ligands and their oligomerization. Received 25 October 2007; received after revision 23 November 2007; accepted 7 December 2007     

Natural peptide analgesics: the role of solution conformation

Endogenous opioids have been studied extensively since their discovery, in the hope of finding a perfect analgesic, devoid of the secondary effects of alkaloid opioids. However, the design of selective opioid agonists has proved very difficult. First, structural studies of peptides in general are hampered by their intrinsic flexibility. Second, the relationship between constitution and the so-called 'bioactive conformation' is far from obvious. Ideally, a direct structural study of the complex between a peptide and its receptor should answer both questions, but such a study is not possible, because opioid receptors are large membrane proteins, difficult to study by standard structural techniques. Thus, conformational studies of opioid peptides are still important for drug design and also for indirect receptor mapping. This review deals with conformational studies of natural opioid peptides in several solvents that mimic in part the different environments in which the peptides exert their action. None of the structural investigations yields a convincing bioactive conformation, but the global conformation of longer peptides in biomimetic environments can shed light on the interaction with receptors. Received 15 April 2001; received after revision 10 May 2001; accepted 11 May 2001     

Modulation of dopaminergic transmission by alpha-noradrenergic agonists and antagonists: Evidence for antidopaminergic properties of some alpha antagonists

Summary The effects on dopamine (DA) metabolism, on³H-spiperone binding and on amphetamine-induced stereotypies of a variety of drugs with different actions on alpha₁-and alpha₂-noradrenergic (NA) receptors have been investigated.The preferential alpha₂-antagonists yohimbine, rauwolscine, piperoxane and esproquin as well as the preferential alpha₁-antagonists corynanthine and WB4101 increased homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the rat striatum, mesolimbic area, and cortex. Prazosine and clonidine tended to reduce HVA and DOPAC. The preferential alpha₂-antagonists, tolazoline and RX-781094A, had no measurable effects on DA metabolism even at high doses.Those compounds which in comparable doses increased DA metabolism inhibited³H-spiperone binding in the hippocampus. The effects in the striatum and cortex were smaller and did not show a relation to those in hippocampus or on DA metabolism. Only the yohimbine alkaloids antagonized amphetamine-induced stereotypies.The results suggest that the effects on DA metabolism at least of yohimbine, rauwolscine, and corynanthine are related to their intrinsic antidopaminergic properties. The same might be true, although with a lesser degree of certainty, for piperoxane, esproquin, and WB4101.Since many of the tested compounds possessing alpha-antagonistic properties interacted with the DA system, a close molecular relationship between alpha-noradrenergic and DA receptors might be anticipated. The preference of these compounds for the hippocampal subtype of DA receptors might indicate a particular role of the latter in the regulation of DA metabolism. On the other hand, the antagonism against haloperidol's enhancing effect on DA metabolism by clonidine suggests a modulatory NA influence on DA transmission. The observation that clonidine reduced the effects of yohimbine and piperoxane to a lesser degree than that of haloperidol, is in agreement with this notion.Part of this work has been presented at the 13th Meeting of the Union of Swiss Societies of Experimental Biology, Lausanne, March 26/27. 1981 (for abstract see Waldmeier and Bischoff, 1981).     

Molecular and structural effects of inverse agonistic mutations on signaling of the thyrotropin receptor – a basally active GPCR

Several mutations that decrease the basal signaling activity of G-protein coupled receptors (GPCRs) with pathogenic implications are known. Here we study the molecular mechanisms responsible for this phenotype and investigate how basal and further activated receptor conformations are interrelated. In the basally active thyroid stimulating hormone receptor (TSHR) we combined spatially-distant mutations with opposing effects on basal activity in double-mutations and characterized mutant basal and TSH induced signaling. Mutations lowering basal activity always have a suppressive influence on TSH induced signaling and on constitutively activating mutations (CAMs). Our results suggest that the conformation of a basally ‘silenced’ GPCR might impair its intrinsic capacity for signaling compared to the wild-type. Striking differences in conformation and intramolecular interactions between TSHR models built using the crystal structures of inactive rhodopsin and partially active opsin help illuminate the molecular details underlying mutations decreasing basal activity. G. Kleinau, H. Jaeschke: These two authors contributed equally to this work. Received 31 July 2008; received after revision 12 September 2008; accepted 19 September 2008     

Effects of selective dopamine D1 and D2 antagonists on male rat sexual behavior

The effects of selective dopamine (DA) D1 and D2 antagonists on male rat sexual behavior were investigated. The D1 antagonist (+)SCH-23390, 25-100 micrograms kg-1 s.c. -20 min, and the D2 antagonist raclopride, 0.1-1.6 mg kg-1 s.c., -20 min, decreased both the number of mounts and intromissions preceding ejaculation. No statistically significant effects in the time up to ejaculation or in the time up to the first intromission were noted, whereas both compounds produced a statistically significant increase in the post-ejaculatory interval. The effect can generally be characterized as psychomotor inhibition, and no evidence was obtained for a specific role of DA D1 or D2 receptors in the mediation of male rat sexual behavior.     

Pimozide and pyrogen-induced fever in rabbits

Summary Pimozide, a selective blocker of DA receptors, partly inhibits but does not suppress either the DA-induced hyperthermia or the LPS-induced fever in rabbits. This suggests that a common DA-related mechanism could be at least partly involved in both responses.Acknowledgments. We wish to thank Laboratories Le Brun (Paris) for the generous supply of pimozide.     

Studies on GABA accumulation induced byγ-glutamyl-hydrazide in regions of rat brain following treatment with a tyrosine hydroxylase inhibitor and 6-hydroxy-dopamine

Summary Reduction of DA receptor activity via depletion of DA stores does not seem to influence GABA turnover in the forebrain and in the DA cell body rich region of the midbrain.This work was supported by a grant (MH25504-03) from the National Institute of Health, USA, and by Magn. Bergvalls Stiftelse and Funds from the Karolinska Institute, Stockholm, Sweden.     

Expression of membrane and nuclear melatonin receptor mRNA and protein in the mouse immune system

The neurohormone melatonin plays a fundamental role in neuroimmunomodulation of several mammalian species, including mice. This effect is supported by the existence of specific melatonin-binding sites in murine immunocompetent organs. Moreover, using melatonin receptor analogues, several effects of the neurohormone on mice physiology through its membrane and nuclear receptors have been described. The expression of these receptors has never been studied, despite indirect evidence showing the presence of melatonin receptor in the murine immune system. At present, the MT₁ and MT₂ membrane receptors, and nuclear receptors belonging to the RZR/ROR family have been related to the immunomodulator effect of melatonin. Here, we show the presence of membrane and nuclear melatonin-binding sites in mouse thymus and spleen, using the specific melatonin membrane (S 20098) and nuclear (CGP 52608) receptor agonist. To confirm the presence of melatonin receptors, we analyzed the presence of membrane and nuclear receptor mRNA and protein by RT-PCR, Southern blot, and Western blot. Thus, we show that MT₁ and ROR receptor mRNA and protein are expressed in both thymus and spleen, while MT₂ receptor mRNA is only detected in the thymus. This expression of melatonin receptors strongly supports the idea of an immunomodulatory role of melatonin through its receptors.Received 2 June 2003; received after revision 6 August 2003; accepted 14 August 2003     

P2Y<Subscript>2</Subscript> receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y₂ receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y₂ receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y₂ Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y₂ Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y₂ receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y₂ RGE mutant receptors. Consistent with the role of P2Y₂ receptors in vasodilation, receptor knockdown and overexpression of P2Y₂ RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y₂ receptor in shear stress-induced cytoskeletal alterations in HUVECs.     

The enteric neural receptor for 5-hydroxytryptamine

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Desensitization by human chorionic gonadotropin (hCG): demonstration of a negative control of gonadotropin receptors by hCG at the level of testicular Leydig cells]

A single injection of hCG (500 IU) to intact and hypophysectomized prepubertal rat increases plasma testosterone levels and decreases hCG receptors in the testicular Leydig cells for 4 to 5 days. In intact animals testosterone exhibit a rapid increase within hours and a delayed paradoxical increase between 60 and 96 hours. In hypophysectomized animals the first peak is not present. In both intact and hypophysectomized rats hCG receptors are almost undetectable between 10 and 96 hours following hCG injection. Since receptor occupancy cannot account for this phenomenon it is concluded that hCG is exerting a negative control on its own receptors.     

Interferon receptors and their role in interferon action

Interferon (IFN) proteins interact with cells through specific cell surface receptors, some of which have been purified and cloned. The alpha-IFNs and beta-IFN bind to a common receptor (type I), whereas gamma-IFN binds to a separate receptor (type II). Both types of high-affinity receptors have been demonstrated on a variety of receptors and the ways in which IFNs may affect cellular physiology and gene expression is discussed.     

Dual effect of cannabinoid CB<Subscript>1</Subscript> receptor stimulation on a vanilloid VR1 receptor-mediated response

Cannabinoid CB1 receptors and vanilloid VR1 receptors are co-localized to some extent in sensory neurons of the spinal cord and dorsal root ganglia. In this study, we over-expressed both receptor types in human embryonic kidney (HEK)-293 cells and investigated the effect of the CB1 agonist HU-210 on the VR1-mediated increase in intracellular Ca2+ ([Ca2+]i), a well-known response of the prototypical VR1 agonist capsaicin. After a 5-min pre-treatment, HU-210 (0.1 microM) significantly enhanced the effect of several concentrations of capsaicin on [Ca2+]i in HEK-293 cells over-expressing both rat CB1 and human VR1 (CB1-VR1-HEK cells), but not in cells over-expressing only human VR1 (VR1-HEK cells). This effect was blocked by the CB1 receptor antagonist SR141716A (0.5 microM), and by phosphoinositide-3-kinase and phospholipase C inhibitors. The endogenous agonist of CB1 and VR1 receptors, anandamide, was more efficacious in inducing a VR1-mediated stimulation of [Ca2+]i in CB1-VR1-HEK cells than in VR1-HEK cells, and part of its effect on the former cells was blocked by SR141716A (0.5 microM). Pre-treatment of CB1-VR1-HEK cells with forskolin, an adenylate cyclase activator, enhanced the capsaicin effect on [Ca2+]i. HU-210, which in the same cells inhibits forskolin-induced enhancement of cAMP levels, blocked the stimulatory effect of forskolin on capsaicin. Our data suggest that in cells co-expressing both CB1 and VR1 receptors, pre-treatment with CB1 agonists inhibits or stimulates VR1 gating by capsaicin depending on whether or not cAMP-mediated signalling has been concomitantly activated.     

The glycinergic inhibitory synapse

Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Research over the last 15 years has yielded new insights on glycine neurotransmission. Glycinergic synapses are now known not to be restricted to the spinal cord and the brainstem. Presynaptic machinery for glycine release and uptake, the structure and function of postsynaptic receptors and the factors (both pre- and postsynaptic) which control the strength of glycinergic inhibition have been extensively studied. It is now established that glycinergic synapses can be excitatory in the immature brain and that some inhibitory synapses can corelease γ-aminobutyric acid (GABA) and glycine. Moreover, the presence of glycine transporters on glial cells and the capacity of these cells to release glycine suggest that glycine may also act as a neuromodulator. Extensive molecular studies have revealed the presence of distinct subtypes of postsynaptic glycine receptors with different functional properties. Mechanisms of glycine receptors aggregation at postsynaptic sites during development are better understood and functional implications of variation in receptor number between postsynaptic sites are partly elucidated. Mutations of glycine receptor subunits have been shown to underly some human locomotor disorders, including the startle disease. Clearly, recent work on glycine receptor channels and the synapses at which they mediate inhibitory signalling in both young and adult animals necessitates an update of our vision of glycinergic inhibitory transmission. Received 8 September 2000; received after revision 1 December 2000; accepted 21 December 2000     

Serotonergic and cholinergic interaction in the regulation of pituitary-adrenal function in rats

It has been proposed that the central serotonergic inputs which modulate pituitary-adrenal secretion are mediated by cholinergic neurons. We have tested this hypothesis in intact rats. Male Sprague-Dawley rats were injected with cholinergic and serotonergic agents which enhanced transmitter function and with receptor blocking agents. Agents were injected, singly and in combination, into both unstressed and stressed animals. Since the response to cholinergic agents might be due to changes to vasopressin release, Brattleboro (vasopressin deficient) rats were also injected with cholinergic agents. The level of plasma corticosterone at 1-h post-injection was determined. Results indicate that the serotonin receptor blockade decreased the stimulatory, cholinergic effect of physostigmine. Cholinergic receptor blockers did not significantly reduce the corticosterone rise induced by 5-hydroxytryptophan. These results do not support the hypothesis of cholinergic mediation of serotonergic input. Nicotinic and muscarinic receptors appeared to exert opposing influences on the system. The nicotinic receptor antagonist was able to block the stimulatory effect of physostigmine. The muscarinic receptor antagonist significantly elevated plasma corticosterone levels. No differences were found in the effect of physostigmine on Brattleboro rats as compared to controls. These data are interpreted as suggesting that 1) the acetylcholine-induced stimulation of pituitary-adrenal function is mediated, in part, by serotonergic neurons; and 2) stimulation of nicotinic receptors is facilitatory whereas stimulation of muscarinic receptors is inhibitory to pituitary-adrenal function.