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Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP3)/Ca2+ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT2 and 5-HT7 receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α-transfected mammalian cells with 5-HT elevates cytosolic [Ca2+] in a dose-dependent manner (EC50 = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP]i (EC50 = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT2α or Cv5-HT7 in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT7 receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT7 in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca2+- and cAMP-signalling cascades.
Background: Serotonin plays a pivotal role in regulating and modulating physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee (Apis mellifera), serotonin has been implicated in division of labor, visual processing, and learning processes. Here, we present the cloning, heterologous expression, and detailed functional and pharmacological characterization of two honeybee 5-HT2 receptors.
Methods: Honeybee 5-HT2 receptor cDNAs were amplified from brain cDNA. Recombinant cell lines were established constitutively expressing receptor variants. Pharmacological properties of the receptors were investigated by Ca2+ imaging experiments. Quantitative PCR was applied to explore the expression patterns of receptor mRNAs.
Results: The honeybee 5-HT2 receptor class consists of two subtypes, Am5-HT2α and Am5-HT2β. Each receptor gene also gives rise to alternatively spliced mRNAs that possibly code for truncated receptors. Only activation of the full-length receptors with serotonin caused an increase in the intracellular Ca2+ concentration. The effect was mimicked by the agonists 5-methoxytryptamine and 8-OH-DPAT at low micromolar concentrations. Receptor activities were blocked by established 5-HT receptor antagonists such as clozapine, methiothepin, or mianserin. High transcript numbers were detected in exocrine glands suggesting that 5-HT2 receptors participate in secretory processes in the honeybee.
Conclusions: This study marks the first molecular and pharmacological characterization of two 5-HT2 receptor subtypes in the same insect species. The results presented should facilitate further attempts to unravel central and peripheral effects of serotonin mediated by these receptors.