EXPRESSION OF RECEPTORS FOR MELANIN-CONCENTRATING HORMONE (MCH) IN DIFFERENT TISSUES AND CELL LINES

ABSTRACT

Melanin-concentrating hormone (MCH) is a potent orexigenic neuropeptide and a physiological antagonist of α-melanocyte-stimulating hormone (α-MSH) in the brain as well as at peripheral sites, including the pigmentary systems of specific vertebrates. Two receptor subtypes for MCH, MCH-R₁ and MCH-R₂, have been cloned, but other receptor subtypes are likely to exist. Based on our own data and the current literature, we have compared the expression of different receptors for MCH in various mammalian cell lines and tissues. Summarizing all data currently available, we conclude that the two cloned MCH receptors, MCH-R₁ and MCH-R₂, exhibit differences in their expression pattern, although MCH-R₁ is generally colocalized in all tissues where MCH-R₂ expression is found. It appears that MCH-R₁ is more abundant and has a wider distribution pattern than MCH-R₂. Other hypothetical MCH-R subtypes may be expressed in specific tissues, e.g., in the pigment cell system.     

Cellular models for the study of the pharmacology and signaling of melanin-concentrating hormone receptors

Cellular models for the study of the neuropeptide melanin-concentrating hormone (MCH) have become indispensable tools for pharmacological profiling and signaling analysis of MCH and its synthetic analogues. Although expression of MCH receptors is most abundant in the brain, MCH-R(1) is also found in different peripheral tissues. Therefore, not only cell lines derived from nervous tissue but also from peripheral tissues that naturally express MCH receptors have been used to study receptor signaling and regulation. For screening of novel compounds, however, heterologous expression of MCH-R(1) or MCH-R(2) genes in HEK293, Chinese hamster ovary, COS-7, or 3T3-L1 cells, or amplified MCH-R(1) expression/signaling in IRM23 cells transfected with the G(q) protein gene are the preferred tools because of more distinct pharmacological effects induced by MCH, which include inhibition of cAMP formation, stimulation of inositol triphosphate production, increase in intracellular free Ca(2+) and/or activation of mitogen-activated protein kinases. Most of the published data originate from this type of model system, whereas data based on studies with cell lines endogenously expressing MCH receptors are more limited. This review presents an update on the different cellular models currently used for the analysis of MCH receptor interaction and signaling.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

Distribution of serotonin (5-HT) and its receptors in the insect brain with focus on the mushroom bodies: lessons from Drosophila melanogaster and Apis mellifera

The biogenic amine serotonin (5-hydroxytryptamine, 5-HT) plays a key role in regulating and modulating various physiological and behavioral processes in both protostomes and deuterostomes. The specific functions of serotonin are mediated by its binding to and subsequent activation of membrane receptors. The vast majority of these receptors belong to the superfamily of G-protein-coupled receptors. We report here the in vivo expression pattern of a recently characterized 5-HT₁ receptor of the honeybee Apis mellifera (Am5-HT_1A) in the mushroom bodies. In addition, we summarize current knowledge on the distribution of serotonin and serotonin receptor subtypes in the brain and specifically in the mushroom bodies of the fruit fly Drosophila melanogaster and the honeybee. Functional studies in these two species have shown that serotonergic signaling participates in various behaviors including aggression, sleep, circadian rhythms, responses to visual stimuli, and associative learning. The molecular, pharmacological, and functional properties of identified 5-HT receptor subtypes from A. mellifera and D. melanogaster will also be summarized in this review.     

Pharmacological modulation of intracellular Ca2+ channels at the single-channel level

Synaptic signaling, memory formation, neuronal development, and neuronal pathology are strongly influenced by the properties of intracellular Ca²⁺ channels, ryanodine, and inositol 1, 4, 5 trisphosphate receptors. This review will focus on recently developed and discovered pharmacological tools to modulate these channel proteins at the single-channel level. It will allow the readers of Molecular Neurobiology to evaluate the current knowledge on the pharmacological modulation of intracellular Ca²⁺ channels and to direct future research efforts effectively using available experimental tools and concepts.     

Properties of receptors for neurotoxic phospholipases A2 in different tissues

A radioiodinated derivative of OS₂ (¹²⁵I–OS₂), a neurotoxic monochain phospholipase A₂ isolated from taipan venom, was previously found to bind to a specific brain membrane receptor with very high affinity.¹²⁵I–OS₂ is now used to identify the properties of neurotoxic phospholipase receptors in other tissues. Heart, skeletal muscle, kidney, lung, liver, pancreas, and smooth muscle membranes also contain high-affinity binding sites for toxic phospholipases A₂. In most tissues, two different types of receptor sites have been characterized for¹²⁵I–OS₂ with K_d1 and K_d2 values in the 1–5 pM and the 10–50 pM range respectively. Whereas all receptors are similar in the different tissues in terms of their affinity for¹²⁵I–OS₂, maximal binding site capacities were very different, varying from 1.4 pmol/mg of protein in brain to 0.01 pmol/mg of protein in pancreaas. In brain, heart, and skeletal muscle, receptor densities vary with in vivo development. Affinity labeling experiments have identified the subunit composition of OS₂ receptors and indicated that these receptors do not have identical structures in the different tissues. Binding competition studies with OS₂ and other toxic phospholipases showed tissue-dependent pharmacological profiles. All these results taken together suggest the existence of a family of receptors for neurotoxic phospholipases.The abbreviations used are PLA₂ phospholipase A₂ - DSS suberic acid bis-N-hydroxysuccinimide ester - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - OS₁ Oxyuranus scutellatus scutellatus toxin 1 - OS₂ Oxyuranus scutellatus scutellatus toxin 2 Special issue dedicated to Dr. Lawrence Austin.     

Role of endocannabinoids and endovanilloids in Ca signalling

Endocannabinoids and endovanilloids are, by definition, endogenous agonists at cannabinoid CB₁ or CB₂ receptors and transient receptor potential vanilloid-type-1 (TRPV1) channels, respectively. Due to the several ways through which cannabinoid receptors influence cytosolic Ca²⁺ concentrations, and to the fact that TRPV1 activation leads to the gating of cations, including Ca²⁺, both endocannabinoids and endovanilloids, taken separately, can strongly influence Ca²⁺ signalling. Moreover, CB₁/CB₂ receptors and TRPV1 channels are often expressed in the same or neighbouring cells, and this can lead to cross-talk between the two receptor types, which is further enriched by the fact that some endocannabinoids, like anandamide and N-arachidonoyldopamine, also activate TRPV1 channels. Finally, both endocannabinoids and endovanilloids also interact with non-cannabinoid, non-TRPV1 receptors and channels, and, although the full physiological relevance of such interactions is yet to be established, the “promiscuity” of these lipophilic molecules can increase even further the potential ways through which they affect Ca²⁺ signalling. Here we discuss the effects of endocannabinoids and endovanilloids on cytosolic Ca²⁺ concentrations and their potential biological consequences.     

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT₁R and CysLT₂R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-α), and leukotriene D₄ (LTD₄), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC₄ synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-α and LTD₄, to a lesser extent, up-regulate the CysLT₁R levels. Interestingly, TNF-α also reduced CysLT₂R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.     

Functional characterization of P2X3 receptors fused with fluorescent proteins

P2X receptor function in the CNS is poorly understood, and currently available data are partly inconsistent. In the presented study, we investigated P2X₃ receptors stably expressed in HEK293 cells. Non-stationary noise analysis of whole cell currents and rapid ATP application through flash photolysis allowed for assessing the single channel conductance (6.6?pS) and the fast activation kinetics of the receptor (20?ms). The characteristics of channel desensitization and pharmacological properties matched previous findings. The properties of wild type receptors were compared with P2X₃ constructs carrying a fluorescent tag (ECFP or DsRed₂) at the C-terminus. These fluorescently labeled subunits formed functional receptors, with neither the affinity of the ligand binding site nor channel properties (ion selectivity, gating kinetics, single channel conductance) differing from wild type. We conclude that both fusion proteins tested here are suitable for generating transgenic mice, which can be expected to promote understanding of the physiological role of P2X₃ receptors in CNS signaling.     

INTERACTION OF MELANIN-CONCENTRATING HORMONE (MCH), NEUROPEPTIDE E-I (NEI), NEUROPEPTIDE G-E (NGE), AND α-MSH WITH MELANOCORTIN AND MCH RECEPTORS ON MOUSE B16 MELANOMA CELLS

Melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between α-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-R_pc) and the different melanocortin (MC) receptors. Radioreceptor assays using [¹²⁵I]MCH, [¹²⁵I]α-MSH and [¹²⁵I]NEI as radioligands and bioassays were performed with MC1-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC₅₀s of α-MSH and NEI or NGE for [¹²⁵I]MCH displacement from mouse MCH-R_pc were 80-fold and, respectively, > 300-fold higher than that of MCH, and the IC₅₀s for MCH and NEI or NGE for [¹²⁵I]α-MSH displacement from mouse MC1-R were 50,000-fold and > 200,000-fold higher than that of α-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [¹²⁵I]α-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 μM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via G_s/olf or G_i/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gα_q activation in rat brain membranes was investigated. Agonist-induced Gα_q activation was assessed by increase in guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to Gα_q determined by [³⁵S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gα_q functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D₁-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D₂-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D₁-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT_2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT_2A receptor, but not by dopamine D₁-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D₁-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT_2A receptor has been mentioned.     

Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.     

Novel Cell Line Selectively Expressing Neuropeptide Y‐Y2 Receptors

Abstract

Neuropeptide Y (NPY) recognition by the human neuroblastoma cell lines SiMa, Kelly, SH‐SY5Y, CHP‐234, and MHH‐NB‐11 was analyzed in radioactive binding assays using tritiated NPY. For the cell lines CHP‐234 and MHH‐NB‐11 binding of [³H]propionyl‐NPY was observed with K_d‐values of 0.64 ± 0.07 nM and 0.53 ± 0.12 nM, respectively, determined by saturation analysis with non‐linear regression. The receptor subtype was determined by competition analysis using the subtype selective NPY analogues [Leu³¹, Pro³⁴]‐NPY (NPY‐Y₁, NPY‐Y₅), [Ahx^5‐24]‐NPY (NPY‐Y₂), [Ala³¹, Aib³²]‐NPY (NPY‐Y₅), NPY [3‐36] (NPY‐Y₂, NPY‐Y₅), and NPY [13‐36] (NPY‐Y₂). Both cell lines, CHP‐234 and MHH‐NB‐11, the latter one being characterized for NPY receptors for the first time, showed exclusive expression of NPY‐Y₂ receptors. In both cell lines binding of NPY induced signal transduction, which was monitored as reduction of forskolin‐induced cAMP production in an ELISA.     

Prostaglandin E2 suppresses β1-integrin expression via E-prostanoid receptor in human monocytes/macrophages

β₁-Integrins mediate cell attachment to different extracellular matrix proteins, intracellular proteins, and intercellular adhesions. Recently, it has been reported that prostaglandin E₂ (PGE₂) has anti-inflammatory properties such as inhibition of the expression of adhesion molecules or production of chemokines. However, the effect of PGE₂ on the expression of β₁-integrin remains unknown. In this study, we investigated the effects of PGE₂ on the expression of β₁-integrin in the human monocytic cell line THP-1 and in CD14⁺ monocytes/macrophages in human peripheral blood. For this, we examined the role of four subtypes of PGE₂ receptors and E-prostanoid (EP) receptors on PGE₂-mediated inhibition. We found that PGE₂ significantly inhibited the expression of β₁-integrin, mainly through EP₄ receptors in THP-1 cells and CD14⁺ monocytes/macrophages in human peripheral blood. We suggest that PGE₂ has anti-inflammatory effects, leading to the inhibited expression of β₁-integrin in human monocytes/macrophages, and that the EP₄ receptor may play an important role in PGE₂-mediated inhibition.     

GABA<Subscript>B</Subscript> Receptors in Neuroendocrine Regulation

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA_A and GABA_C (ionotropic receptors) and GABA_B (metabotropic receptor). Here we reviewed the participation of GABA_B receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA_B1 knock-out mice, that lack functional GABA_B receptors. Our general conclusion indicates that GABA_B receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA_B receptor activation, as demonstrated in the rat and also in the GABA_B1 knock-out mouse. In addition, hypothalamic and pituitary GABA_B receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA_B receptors depends on physiological conditions, being age and sex critical factors. These results indicate that patients receiving GABA_B agonists/antagonists should be monitored for possible endocrine side effects.     

METHIONINE PROXIMITY ASSAY,A NOVEL METHOD FOR EXPLORING PEPTIDE LIGAND–RECEPTOR INTERACTION

ABSTRACT

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,?p″-nitrobenzoylphenylalanine (NO₂Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT₁ and AT₂, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.     

β2‐Adrenoceptor Agonists Induce the Mammalian Clock Gene,hPer1, mRNA in Cultured Human Bronchial Epithelium Cells in vitro

The mammalian Per1 gene is one of the most important components of circadian clock function of the suprachiasmatic nucleus and peripheral tissues. We examined whether the β₂‐adrenoceptor agonists, procaterol and fenoterol, induce human Per1 mRNA expression in human bronchial epithelium. The in vitro stimulation of β₂‐adrenoceptor agonists in BEAS‐2B cells led to a remarkable increase in the level of hPer1 mRNA. Moreover, fenoterol or procaterol induced the phosphorylation of CREB in BEAS‐2B cells as verified by immunoblot analysis. β₂‐adrenoceptor agonists induced human Per1 mRNA expression by the signaling pathways of cAMP‐CREB in BEAS‐2B cells.