RESUMEN
Calcium acts as a second messenger in many cell types, including insect hemocytes. Intracellular calcium level has a definite role in innate and adaptive immune signaling. Biogenic amines such as octopamine (OA), tyramine (TA), dopamine (DA) and serotonin (5-HT) play various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs) that share a putative seven transmembrane domain structure. OA and 5-HT have been shown that can mediate insect hemocytic immune reactions to infections and invasions. Here, we showed that TA increase hemocyte spreading in the rice stem borer, Chilo suppressalis. Furthermore, we cloned a cDNA encoding a tyramine receptor type 2 from the hemocytes in the C. suppressalis, viz., CsTA2, which shares high sequence similarity to members of the invertebrate tyramine receptor family. The CsTA2 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with TA induced a dose-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in cells, with an EC50 value of 18.7±5.3 nM, whereas OA, DA, 5-HT and other potential agonists did not have this response. The mRNA is present in various tissues including nerve cord, hemocytes, fat body, midgut, Malpighian tubules, and epidermis in the larval stage. Western blot analysis and immunohistochemistry assay displayed that CsTA2 was detected and presented on hemocytes. We also showed that TA induced Ca(2+) release from the hemocytes of C. suppressalis.
Asunto(s)
Hemocitos/metabolismo , Mariposas Nocturnas/metabolismo , Receptores de Amina Biogénica/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Tiramina/farmacología , Animales , Calcio/fisiología , ADN Complementario/genética , Dopamina/farmacología , Células HEK293 , Humanos , Proteínas de Insectos/metabolismo , Larva/metabolismo , Mariposas Nocturnas/genética , Octopamina/farmacología , Receptores de Amina Biogénica/genética , Receptores Acoplados a Proteínas G/genética , Sistemas de Mensajero Secundario , Serotonina/farmacologíaRESUMEN
Octopamine receptors are attractive insecticide targets. To screen compounds acting at octopamine receptors simply and rapidly, we constructed a chemiluminescent reporter gene assay system that detects secreted placental alkaline phosphatase transcriptionally regulated by the cAMP response element for a silkworm octopamine receptor. This system proved useful in high-throughput screening to develop octopamine receptor-specific insecticides.
Asunto(s)
Fosfatasa Alcalina/genética , Evaluación Preclínica de Medicamentos/métodos , Genes Reporteros/genética , Insecticidas/farmacología , Placenta/enzimología , Receptores de Amina Biogénica/genética , Receptores de Amina Biogénica/metabolismo , Fosfatasa Alcalina/metabolismo , Femenino , Expresión Génica , Células HEK293 , Humanos , Embarazo , Receptores de Amina Biogénica/agonistas , Receptores de Amina Biogénica/antagonistas & inhibidoresRESUMEN
A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa(1)), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK-293 cells. OA above 1 microM led to an increase in intracellular cyclic AMP concentration ([cAMP](i)). The synthetic OA-receptor agonist demethylchlordimeform also elevated [cAMP](i) to the same maximal level (approximately 5-fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP](i) level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nM induced a transient or sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an alpha-adrenergic-like OA receptor of B. mori.
Asunto(s)
Bombyx/genética , Expresión Génica , Receptores Adrenérgicos/química , Receptores de Amina Biogénica/genética , Receptores de Amina Biogénica/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bombyx/química , Señalización del Calcio/efectos de los fármacos , Clonación Molecular , AMP Cíclico/biosíntesis , ADN Complementario/genética , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genoma de los Insectos/genética , Humanos , Datos de Secuencia Molecular , Octopamina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Amina Biogénica/biosíntesis , Receptores de Amina Biogénica/química , Tritio , Yohimbina/metabolismo , Yohimbina/farmacologíaRESUMEN
The LCR/MEL system (Locus Control Region/Murine Erythroleukaemia cells) was employed to express and characterize the Locusta migratoria tyramine receptor (TyrLoc), an insect G protein-coupled receptor. Functional agonist-dependent responses were recorded in stable, tyramine receptor expressing cell clones (MEL-TyrLoc). Tyramine elicited a dose-dependent increase of cytosolic Ca2+-ions and an attenuation of forskolin-induced cyclic adenosine monophosphate (AMP) production. Octopamine was shown to be a weak agonist for both responses. In addition, yohimbine proved to be a potent tyramine receptor antagonist. This study reports the first application of the LCR/MEL expression system in functional assays for G protein-coupled receptors and therefore expands the capabilities of this system by exploiting the functionality of the signal transduction pathways.