Inverse agonist and neutral antagonist actions of synthetic compounds at an insect 5-HT1 receptor

Abstract

Background and purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT(1) receptor of an insect model for neurobiology, physiology and pharmacology.

Experimental approach: A cDNA encoding for the Periplaneta americana 5-HT(1) receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum.

Key results: The P. americana 5-HT(1) receptor (Pea5-HT(1)) shares pronounced sequence and functional similarity with mammalian 5-HT(1) receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT(1) was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands.

Conclusions and implications: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT(1) receptor. The results presented here should facilitate further analyses of 5-HT(1) receptors in mediating central and peripheral effects of 5-HT in insects.

Figure 1

Amino acid sequence alignment of Pea5-HT₁ and orthologous receptors from Drosophila melanogaster (Dm5-HT_1A; accession no. CAA77570), Dm5-HT_1B (no. CAA77571), Papilio xuthus (Pxu5-HT₁, no. BAD72868) and Penaeus monodon (Pem5-HT₁, no. AAV48573). Identical residues (≥80%) between the receptors are shown as white letters against black, whereas conservatively substituted residues are shaded. Putative transmembrane domains (TM 1–TM 7) are indicated by grey bars. Potential N-glycosylation sites (+) and putative palmitoylation sites (*) of Pea5-HT₁ are labelled. Underlined letters represent the region within the third cytoplasmic loop from which the Pea5-HT₁-specific peptide antigen was derived. The amino acid position is indicated on the right.

Figure 2

Phylogenetic analysis of Pea5-HT₁ and various 5-HT receptors. Alignments were performed with BioEdit (version 7.0.5; Hall, 1999) using the core amino acid sequences lacking the variable regions of the amino and carboxyl terminus, and the third cytoplasmic loop. The genetic distance was calculated with MEGA4 (Tamura et al., 2007). The receptor sequences followed by their accession numbers are listed in the order illustrated: Panulirus interruptus (Pan5-HT₁, no. AY528822); Procambarus clarkii (Pro5-HT₁, no. ABX10973), Penaeus monodon (Pem5-HT₁, no. AAV48573), Periplaneta americana (Pea5-HT₁, no. FN298392), Drosophila melanogaster (Dm5-HT_1A, no. CAA77570), Dm5-HT_1B (no. CAA77571), Papilio xuthus (Pxu5-HT₁, no: BAD72868), Manduca sexta (Ms5-HT_1A, no. DQ840515), Bombyx mori (Bm5-HT, no. CAA64862), Ms5-HT_1B (no. DQ840516), Lymnaea stagnalis (Lym5-HT₁, no. L06803), human 5-HT_1A (no. NP_000515), human 5-HT_1B (no. NP_000854), human 5-HT_1D (no. NP_000855), human 5-HT_1E (no. NP_000856), human 5-HT_1F (no. NP_000857), human 5-HT₇ isoform a (no. NP_000863), human 5-HT₇ isoform d (no. NP_062873), human 5-HT₇ isoform b (no. NP_062874), Apis mellifera (Am5-HT₇, no. AM076717), Dm5-HT₇ (no. A38271), Aedes aegypti (Aae5-HT₇, no. AAG49292), Pan5-HT₂ (no. AY550910), Pro5-HT₂ (no. ABX10972), Dm5-HT₂ (no. CAA57429), Lym5-HT₂ (no. U50080), human 5-HT_2B (no. NP_000858), human 5-HT_2A (no. NP_000612), human 5-HT_2C (no. NP_000859), D. melanogaster ninaE-encoded rhodopsin 1 (DmninaE, no. NM_079683) and D. melanogaster FMRFamide receptor (DmFR, no. AAF47700). The numbers at the nodes of the branches represent the percentage bootstrap support for each branch. The scale bar allows conversion of branch lengths in the dendrogram to genetic distance between clades.

Figure 3

Tissue distribution of Pea5-ht1 mRNA. The 100 bp DNA ladder (marker) is shown on the left. Detection of PCR products amplified on total RNA isolated from brain, salivary glands, Malpighian tubules, midgut and muscle. Amplification failed when samples were digested with an RNase cocktail prior to RT-PCR (data not shown). The lower panel shows RT-PCR products amplified by using actin (accession no. AY116670)-specific primers as a control.

Figure 4

Western blot analysis with the anti-Pea5-HT₁ receptor antibody. Molecular weight marker in kDa. (A) Specificity of anti-Pea5-HT₁ receptor antibody tested on Periplaneta americana brain proteins. Lane 1: Western blot analysis with anti-Pea5-HT₁ antibody (1:20 000). A single band of ∼80 kDa was detected. Lane 2: Western blot analysis with anti-Pea5-HT₁ antibody (1:20 000) pre-absorbed with 15 µg·mL⁻¹ of the peptide used for immunization; no band was detected. (B) Western blot of membrane proteins (10 µg per lane) from brain tissue (b) and salivary glands (sg) treated without (–) or with (+) PNGase F. De-glycosylation resulted in a slight shift of the protein band from ∼80 to ∼75 kDa.

Figure 5

Immunohistochemical localization of Pea5-HT₁ receptors in brain sections. Vibratome sections of brains were double labelled with anti-5-HT (red) and anti-Pea5-HT₁ (green), and imaged by confocal microscopy. Each image shows the sum of multiple consecutive optical sections representing a total thickness of ∼20 µM. (A) Central part of an anterior frontal section of the cockroach brain (for schematic drawing, see C). (B) Central part of a more posterior frontal section of the cockroach brain. (C) Schematic drawings of sections of the cockroach brain. Grey boxes highlight the details displayed in A + B. Abbreviations: V, vertical lobe; AL, antennal lobe; Ca, calyx; Ch, chiasm region of nervi coporis cardiaci I; M, medial lobe; NCC I, nervus corporis carciaci I; PI, pars intercerebralis.

Figure 6

Modulation of intracellular cAMP levels in HEK 293 cells stably expressing the Pea5-HT₁ receptor and in non-transfected cells. The amount of cAMP is given as the percentage of the value obtained after incubation with 10 µM NKH-477 (100%), a water-soluble forskolin analogue. Error bars indicate SEM and are in some cases too small to be represented. The statistical analysis is based on a one-way anova followed by Dunnett's multiple comparison test; ***P < 0.0001. (A) Effect of NKH-477 and 5-HT (10 µM) on cAMP levels in non-transfected cells and in Pea5-HT₁-expressing cells. To determine the basal [cAMP]_i, cells were incubated with 100 µM IBMX only (basal). Data represent the mean ± SEM of 16 values from four experiments each performed in quadruplicate. Asterisks indicate statistically significant differences for drug versus NKH-477 (100%) for a given cell line. (B) Dose-dependent effect of 5-HT (10⁻⁹–3 × 10⁻⁵ M) on [cAMP]_i. Data represent the mean ± SEM of eight replicates from four experiments each performed in duplicate. (C) Effects of 5-HT receptor agonists (10 µM) on NKH-477-stimulated cAMP production in Pea5-HT₁-expressing cells. Data represent the mean ± SEM of 14 values from four experiments each performed in either triplicate or quadruplicate. Asterisks indicate statistically significant differences for drug versus NKH-477. (D) Effects of putative antagonists (10 µM) on 5-HT-mediated (500 nM) inhibition of NKH-477-stimulated cAMP production in Pea5-HT₁-expressing cells. Data represent the mean ± SEM of 16 values from four experiments each performed in quadruplicate. Asterisks indicate statistically significant differences for drug versus 5-HT.

Figure 7

Modulation of intracellular cAMP levels in HEK 293 cells stably expressing the Pea5-HT₁ receptor by WAY 100635 and methiothepin. (A) Dose-dependent effects of the neutral antagonist methiothepin and the inverse agonist WAY 100635 on NKH-477-stimulated cAMP production in the presence of 500 nM 5-HT. Data represent the mean ± SEM of eight replicates from two experiments representative of four similar experiments. (B) Dose-dependent effects of methiothepin and WAY 100635 on NKH-477-stimulated cAMP production in the absence of 5-HT. Data represent the mean ± SEM of eight replicates from two experiments representative of four similar experiments. (C) Inhibition of the effect of WAY 100635 (30 µM) on the NKH-477-stimulated cAMP production by methiothepin (10 µM). Data represent the mean ± SEM of eight replicates from two experiments representative of four similar experiments. Asterisks indicate statistically significant differences for drug versus NKH-477 (one-way anova followed by Dunnett's multiple comparison test; **P < 0.01).