The Melanin-Concentrating Hormone (MCH) System Modulates Behaviors Associated with Psychiatric Disorders

Deficits in sensorimotor gating measured by prepulse inhibition (PPI) of the startle have been known as characteristics of patients with schizophrenia and related neuropsychiatric disorders. PPI disruption is thought to rely on the activity of the mesocorticolimbic dopaminergic system and is inhibited by most antipsychotic drugs. These drugs however act also at the nigrostriatal dopaminergic pathway and exert adverse locomotor responses. Finding a way to inhibit the mesocorticolimbic- without affecting the nigrostriatal-dopaminergic pathway may thus be beneficial to antipsychotic therapies. The melanin-concentrating hormone (MCH) system has been shown to modulate dopamine-related responses. Its receptor (MCH1R) is expressed at high levels in the mesocorticolimbic and not in the nigrostriatal dopaminergic pathways. Interestingly a genomic linkage study revealed significant associations between schizophrenia and markers located in the MCH1R gene locus. We hypothesize that the MCH system can selectively modulate the behavior associated with the mesocorticolimbic dopamine pathway. Using mice, we found that central administration of MCH potentiates apomorphine-induced PPI deficits. Using congenic rat lines that differ in their responses to PPI, we found that the rats that are susceptible to apomorphine (APO-SUS rats) and exhibit PPI deficits display higher MCH mRNA expression in the lateral hypothalamic region and that blocking the MCH system reverses their PPI deficits. On the other hand, in mice and rats, activation or inactivation of the MCH system does not affect stereotyped behaviors, dopamine-related responses that depend on the activity of the nigrostriatal pathway. Furthermore MCH does not affect dizocilpine-induced PPI deficit, a glutamate related response. Thus, our data present the MCH system as a regulator of sensorimotor gating, and provide a new rationale to understand the etiologies of schizophrenia and related psychiatric disorders

Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles

δ-Opioid receptors stimulate the metabolic sensor AMP-activated protein kinase through coincident signaling with G(q/11)-coupled receptors.

AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-d-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake

Stimulation of phosphoinositide hydrolysis by muscarinic receptor activation in the rat olfactory bulb.

The effect of muscarinic receptor activation on phosphoinositide hydrolysis in the rat olfactory bulb was investigated by determining either the inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) mass or the accumulation of [3H]inositol phosphates ([3H]InsPs). In miniprisms of rat olfactory bulb, carbachol produced an atropine-sensitive increase in Ins(1,4,5)P3 concentration. In a membrane preparation, the formation of Ins(1,4,5)P3 was stimulated by guanosine-5'-(3-O-thio) triphosphate (GTP gamma S), but not by carbachol. However, carbachol potentiated the GTP gamma S stimulation when the two agents were combined. In miniprisms prelabelled with [3H]myo-inositol, carbachol increased the accumulation of [3H]InsPs and this effect was significantly reduced by tissue treatment with either 1 microM phorbol 12-myristate 13-acetate or 1 mM dibutyryl cyclic AMP. Analysis of concentration-response curves indicated that carbachol (EC50 = 96 microM) and oxotremorine-M (EC50 = 8.2 microM) behaved like full agonists, whereas oxotremorine, BM5, arecoline and bethanechol were partial agonists. The carbachol stimulation of [3H]InsPs accumulation was counteracted with high affinity by the M1 antagonist pirenzepine (pA2 = 8.26), and less potently by the M3 antagonist para-fluorohexahydro-sila-difenidol (pA2 = 6.7) and the M2 antagonist AF-DX 116 (pA2 = 6.12). The biochemical and pharmacological properties of the muscarinic stimulation of phosphoinositide hydrolysis were compared with those displayed by the muscarinic stimulation of adenylate cyclase in the rat olfactory bulb

Synergistic interaction of muscarinic and opioid receptors with Gs-linked neurotransmitter receptors to stimulate adenylyl cyclase activity of rat olfactory bulb

We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor agonists stimulate adenylyl cyclase activity. In the present study we show that carbachol (CCh) and Leu-Enkephalin act synergistically with vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH), but not with l-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal adenylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect 125I-CRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5'-O-(3'-thiotriphosphate) for GTP allows the CRH stimulation, but abolishes the CCh enhancement of both basal and CRH-stimulated enzyme activities. Moreover, in vivo treatment of olfactory bulbs with pertussis toxin completely prevents the muscarinic and opioid effects. Thus, the synergistic interaction appears to result from opioid- and muscarinic-induced activation of a pertussis toxin-sensitive GTP-binding protein which may potentiate the adenylyl cyclase stimulation by the stimulatory GTP-binding protein activated by either VIP or CRH receptors

G protein-coupled corticotropin-releasing hormone receptors in rat retina

The presence of corticotropin-releasing hormone (CRH) receptors in rat retinal membranes was investigated by using [125I-Tyro]-ovine CRH ([125I]oCRH) as radioligand. The receptor binding was rapid, reversible, saturable and specific. The [125I]oCRH binding was completely displaced by different CRH-related peptides with a rank order of potency similar to that displayed in stimulating rat retinal adenylyl cyclase activity. Two populations of binding sites were detected: one with high affinity (Kd = 1.7 nM) and the other with low-affinity (Kd = 130 nM). The GTP analogue guanosine 5'-O-(3'-thiotriphosphate) reduced the high-affinity binding and increased the relative proportion of sites with low-affinity. Incubation of rat retinal membranes with the RM/1 antibody, which recognizes the carboxyl-terminus of the alpha subunit of the G protein Gs, prevented the CRH stimulation of adenylyl cyclase. In immunoblots, the RM/1 antibody recognized an immunoreactive protein band of 45 kDa and a protein with a similar electrophoretic mobility was ADP-ribosylated by cholera toxin. These data provide evidence for the presence of specific CRH receptors in rat retina and contribute to define the CRH signalling system in this tissue

PD 102807, a novel muscarinic M4 receptor antagonist, discriminates between striatal and cortical muscarinic receptors coupled to cyclic AMP

In membranes of Chinese hamster ovary cells expressing the cloned human M1-M4 muscarinic receptor subtypes, PD 102807, a novel M4 selective antagonist, was found to counteract the M4 receptor-induced stimulation of [35S]-GTPgammaS binding to membrane G proteins with a pK(B) of 7.40, a value which was 63-, 33- and 10-fold higher than those displayed at M1 (pK(B) = 5.60), M2 (pK(B) = 5.88) and M3 (pK(B) = 6.39) receptor subtypes, respectively. In rat striatal membranes, PD 102807 antagonized the muscarinic inhibition of dopamine (DA) D1 receptor-stimulated adenylyl cyclase with a pK(B) value of 7.36. In contrast, in membranes of rat frontal cortex, PD 102807 displayed lower potencies in antagonizing either the muscarinic facilitation of corticotropin releasing hormone (CRH)-stimulated adenylyl cyclase (pK(B) = 5.79) or inhibition of Ca2+/calmodulin (Ca2+/CaM)-stimulated enzyme activity (pK(B) = 5.95). In each response investigated, PD 102807 interacted with muscarinic receptors in a manner typical of a simple competitive antagonist. These data provide additional evidence that PD 102807 is a M4-receptor preferring antagonist and that this compound can discriminate the striatal muscarinic receptors inhibiting DA D1 receptor activity from the cortical receptors mediating the potentiation of CRH receptor signalling and the inhibition of Ca2+/CaM-stimulated adenylyl cyclase activity

Supersensitivity of striatal D2 dopamine receptors mediating inhibition of adenylate cyclase and stimulation of guanosine triphosphatase following chronic administration of haloperidol in mice

Adenylate cyclase and guanosine triphosphatase (GTPase) activities in response to dopamine (DA) were determined in membranes prepared from striata of mice treated with haloperidol for a period of 3 months. D1- and D2 receptor-mediated effects were investigated in the presence of 2 microM (-)-sulpiride and 0.1 microM SCH 23390, respectively. The drug treatment produced a 38% increase in the maximal inhibition of adenylate cyclase activity elicited by DA via D2 receptors. D1-mediated stimulation of adenylate cyclase was not affected. The enhanced D2 inhibition of adenylate cyclase was associated with a 45% increase in the stimulatory response of GTPase activity via D2 sites. These results indicate that D2 receptors linked to inhibition of adenylate cyclase and to stimulation of GTPase become supersensitive following in vivo chronic blockade of DA receptors