Pharmacological characterization of the 5-HT1A receptor of Bombyx mori and its role in locomotion

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Abstract

Serotonin is involved in the regulation of many physiological and behavioral processes in vertebrates and invertebrates. The effects of serotonin are mediated through interactions of several 5-HT receptor types. The expression and pharmacological properties of 5-HT1 have received more attention than other serotonin receptors, but its functions at the individual level are little studied in arthropods. Silkworm, a Lepidoptera model, almost has no reports about serotonin receptors. To analyze the function of Bm5-HT1A receptor in vitro, the ORF of Bm5-HT1A was cloned into the pcDNA3.1 vector and expressed in HEK 293 cells. Serotonin activation of Bm5-HT1A-expressing cells decreased forskolin-stimulated cAMP synthesis and had the most potent effect compared to other biogenic amines. Serotonin reduced cAMP synthesis in a dose-dependent manner, and half-maximal activation (EC50) occurred at a concentration of 1.17 × 10−7 M (117 nM). The pharmacological analysis demonstrated that the rank potency of agonists was pimozide >8-OH-DPAT >5-MeOT ~ αm-5-HT, and antagonists was WAY-100635 > prazosin > SB-269970 > methiothepin at the Bm5-HT1A receptor. Injecting the antagonist of Bm5-HT1A receptor into larvae caused slow or weak motility, and adults lowered courtship vitality or moving speed. Injecting dsRNA of Bm5-HT1A into adults also dropped locomotivity in courtship. These results show that the Bm5-HT1A receptor is related to locomotor activity. This study provides the first information of serotonin receptor on pharmacological in silkworm and on individual functions in arthropods.

Introduction

Five-hydroxytryptamine (5-HT, serotonin) is an intracellular signaling molecule that regulates many major physiological and behavioral processes in animals (Dierick and Greenspan, 2007; Anstey et al., 2009; Ott et al., 2012). Serotonin effects are mediated through interactions with 5-HT receptors. Molecular cloning and gene expression techniques in vertebrates have characterized fourteen serotonin-receptor subtypes, grouped into seven classes (5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7) on the basis of their sequence similarities, gene organization, second messenger coupling pathways and pharmacological characteristics. Six of these belong to the G protein-coupled receptor superfamily; the class to which 5-HT3 belongs consists of ligand-gated cation channel receptors. The 5-HT1 and 5-HT5 receptors are coupled to the Gi/o proteins signaling pathway to inhibit adenylate cyclase activity when stimulated. The 5-HT2 receptors couple preferentially to Gq/11 proteins, which mediate the hydrolysis of inositol phosphates and lead to a subsequent increase in cytosolic Ca2+ levels. The 5-HT4, 5-HT6 and 5-HT7 receptors all couple preferentially with Gs proteins and promote cAMP production (Hoyer et al., 1994; Hannon and Hoyer, 2008; Nichols and Nichols, 2008).

Orthologous G protein-coupled serotonin receptors with conserved signaling pathways have been discovered in protostomes (Ranganathan et al., 2000; Carre-Pierrat et al., 2006; Sugamori et al., 1993; Gerhardt et al., 1996; Clark et al., 2004; Spitzer et al., 2008; Blenau and Baumann, 2001). The insect serotonin receptors can be classified based on mammalian counterparts 5-HT1-, 5-HT2- and 5-HT7-types (Troppmann et al., 2010); Dacks et al., 2013; Ono and Yoshikaw, 2004; Watanabe et al., 2011; Lee and Pietrantonio, 2003). Apis mellifera (Thamm et al., 2010; Schlenstedt et al., 2006; Thamm et al., 2013) as well as Drosophila melanogaster (Tierney, 2001; Hauser et al., 2006; Saudou et al., 1992; Witz et al., 1990; Colas et al., 1995) have five serotonin receptor subtypes (two 5-HT1, two 5-HT2 and 5-HT7) being characterized. However, only four serotonin receptors (two 5-HT1, 5-HT2, and 5-HT7) are predicted from the Tribolium castaneum genome, and two of these receptors (one 5-HT1 and 5-HT7) have been identified (Hauser et al., 2008; Vleugels et al., 2014; Vleugels et al., 2013). These indicate that current knowledge remains limited yet for serotonin receptors in insects. In Bombyx mori, this lepidopteran model, dopamine (Chen et al., 2017; Ohta et al., 2009), octopamine (Ohtani et al., 2010; Chen et al., 2010) and tyramine receptors (Huang et al., 2009) have been characterized extensively. But its serotonin receptors have rarely been researched, especially in cell pharmacology or individual function.

The 5-HT1 class is the largest subfamily of serotonin receptors and contains six distinct members (5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, 5-HT1E, and 5-HT1F) (Hannon and Hoyer, 2008; Nichols and Nichols, 2008; Gerhardt and Van, 1997). The 5-HT1A is an ancestral archetypical serotonin receptor, a major determinant of serotonergic cell activity and serotonin release because of its pre- and postsynaptic location in mammalian systems (Chalmers and Watson, 1991). In arthropods, 5-HT1 receives more attention than other serotonin receptors. The expression levels of 5-HT1 in the brain and in the eyestalk are up-regulated from serotonin injection in Metapenaeus ensis (Tiu et al., 2005). Both mRNA and the protein of the 5-HT1 receptor are found in the brain and salivary glands of Periplaneta americana (Troppmann et al., 2010). 5-HT1A and 5-HT1B are highly expressed in brain regions associated with learning and memory, such as the mushroom bodies in Drosophila melanogaster (Yuan et al., 2006; Johnson et al., 2011). The T. castaneum 5-HT1 transcript level in the brain was 3.5 times higher than in the optic lobes and was not detected in the salivary gland (Vleugels et al., 2013). The Ms5-HT1A receptors are likely to be expressed heterogeneously within the antennal lobe in Manduca sexta (Dacks et al., 2013). Simultaneously, functions in vitro and/or pharmacological profiles have been identified by the cells expressed 5-HT1A in bees (Thamm et al., 2010), cockroaches (Troppmann et al., 201), fruit flies (Saudou et al., 1992), red flour beetles (Vleugels et al., 2013), tobacco hornworms (Dacks et al., 2013), and cattle ticks (Spitzer et al., 2008). However, the study of 5-HT1A functions at the individual level (in vivo) is absent in arthropods. In this study, we analyzed the function of Bm5-HT1A receptor in HEK293 cells and its role on individual locomotivity by RNAi and injecting its antagonist.

Section snippets

Construction of expression vectors

The amplified products for the ORF of Bm5-HT1A were ligated into the EcoR I and Xho I sites of the expression vector pcDNA3.1 (+) to produce the recombinant pcDNA3.1 + Bm5-HT1A. The insertion was confirmed by DNA sequencing.

Transfections and cAMP assays

HEK-293 cells were grown in Dulbecco's modified Eagle's medium (D-MEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Invitrogen) at 37 °C with 5% CO2. Cells suspended in D-MEM containing 10% FBS were plated on 35-mm dishes for 1 day before transfection. The

Results

To determine the functional characteristics of Bm5-HT1A receptor in HEK 293 cells, we heterologously expressed the full-length of Bm5-HT1A in HEK 293 cells. Expression was confirmed by RT-PCR using total RNA extracted from transiently transfected cells (Supplementary Fig. S2). The control was an empty pcDNA3.1 vector under the same conditions. Since 5-HT1 receptors can drop cAMP levels which are low in normal cells by inhibiting adenylyl cyclase activity, 10 μM forskolin was applied to

Discussion

Activation of 5-HT1 receptor results in the inhibition of cAMP accumulation. This was demonstrated in D. melanogaster (Dm5-HT1A and Dm5-HT1B), T. castaneum (Tr5-HT1), M. sexta (Ms5HT1A and Ms5HT1B), P. americana (Pea5-HT1), and the crustaceans Panulirus interruptus (5-HT1αpan) and Procambarus clarkii (5-HT1αpro) (Saudou et al., 1992; Witz et al., 1990; Troppmann et al., 2010; Dacks et al., 2013; Vleugels et al., 2013; Thamm et al., 2010; Spitzer et al., 2008). In our study, the ORF of Bm5-HT1A

Conclusion

The function of silkworm serotonin receptors is first studied in vivo and in vitro. Our results may help to establish additional roles of this receptor subtypes in silkworm physiology and behavior.

Competing interests

The authors declare no competing or financial interests.

Funding

This work was supported by the Basic research and frontier exploration projects of Chongqing [grant numbers cstc2018jcyjAX0075]; and the Subsidy fund for the development of National Silk in Chongqing [grant number CQ2018JSCE05].

Acknowledgements

We would like to thank DengFeng Yan for his help in cell experiment and YaTeng Li for his help on English in manuscript.

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