AmOct2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either - or -adrenergic-like octopamine receptors. Currently, one - and four -receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an 2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOct2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOct2R), phylogenetically groups in a clade closely related to human 2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOct2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOct2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked eciently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOct2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology

Elimination of a ligand gating site generates a supersensitive olfactory receptor

Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors

El Zonda, portavoz del espacio público sanjuanino

En este trabajo analizamos al semanario El Zonda (San Juan, 1839) como el medio representante de la esfera pública sanjuanina, surgida bajo los auspicios de un grupo de jóvenes intelectuales encabezados por Sarmiento, que intentaron constituirse en intermediarios válidos entre el poder provincial y la sociedad. Motivados por la convicción de que la educación y los periódicos eran los medios eficaces para promover el progreso local, auspiciaron valiosas iniciativas que impactaron de manera dispar en el espacio público cuyano, como la creación de la Sociedad Dramática y Filarmónica, la Sociedad Literaria y el Colegio de Pensionistas de Santa Rosa, además del semanario. Este periódico fue renovador en varios sentidos: presentó una nueva concepción estética periodística, al tiempo que se valió de novedosas estrategias comunicacionales para instalar un discurso “renovador” y captar nuevos lectores, incluyendo a las mujeres. Además asumió un perfil político-pedagógico, pero no partidario, abandonando todo fin económico. Inobjetablemente suscitó controversias no obstante su efímera existencia (sólo seis números) la que estuvo condicionada por la conjunción de varias razones: la exigüidad de suscripciones, que imposibilitaron su sostenimiento económico y, con el correr del tiempo, la disipación de la complacencia del poder político de turno, actitudes que podrían responder a las severas críticas a la sociedad local emitidas desde sus enunciados.Facultad de Periodismo y Comunicación Socia

PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana

The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP]i) whereas type 2 tyramine receptors can mediate Ca2+ signals or both Ca2+ signals and effects on [cAMP]i. Here; we report that the American cockroach (Periplaneta americana) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine

Full rescue of an inactive olfactory receptor mutant by elimination of an allosteric ligand-gating site

Ligand-gating has recently been proposed as a novel mechanism to regulate olfactory receptor sensitivity. TAAR13c, the zebrafish olfactory receptor activated by the death-associated odor cadaverine, appears to possess an allosteric binding site for cadaverine, which was assumed to block progress of the ligand towards the internal orthosteric binding-and-activation site. Here we have challenged the suggested gating mechanism by modeling the entry tunnel for the ligand as well as the ligand path inside the receptor. We report an entry tunnel, whose opening is blocked by occupation of the external binding site by cadaverine, confirming the hypothesized gating mechanism. A multistep docking algorithm suggested a plausible path for cadaverine from the allosteric to the orthosteric binding-and-activation site. Furthermore we have combined a gain-of-function gating site mutation and a loss-of-function internal binding site mutation in one recombinant receptor. This receptor had almost wildtype ligand affinities, consistent with modeling results that showed localized effects for each mutation. A novel mutation of the suggested gating site resulted in increased receptor ligand affinity. In summary both the experimental and the modeling results provide further evidence for the proposed gating mechanism, which surprisingly exhibits pronounced similarity to processes described for some metabotropic neurotransmitter receptors

Loss of HCN2 in Dorsal Hippocampus of Young Adult Mice Induces Specific Apoptosis of the CA1 Pyramidal Neuron Layer

Neurons inevitably rely on a proper repertoire and distribution of membrane-bound ion-conducting channels. Among these proteins, the family of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels possesses unique properties giving rise to the corresponding Ih-current that contributes to various aspects of neural signaling. In mammals, four genes (hcn1-4) encode subunits of HCN channels. These subunits can assemble as hetero- or homotetrameric ion-conducting channels. In order to elaborate on the specific role of the HCN2 subunit in shaping electrical properties of neurons, we applied an Adeno-associated virus (AAV)-mediated, RNAi-based knock-down strategy of hcn2 gene expression both in vitro and in vivo. Electrophysiological measurements showed that HCN2 subunit knock-down resulted in specific yet anticipated changes in Ih-current properties in primary hippocampal neurons and, in addition, corroborated that the HCN2 subunit participates in postsynaptic signal integration. To further address the role of the HCN2 subunit in vivo, we injected recombinant (r)AAVs into the dorsal hippocampus of young adult male mice. Behavioral and biochemical analyses were conducted to assess the contribution of HCN2-containing channels in shaping hippocampal network properties. Surprisingly, knock-down of hcn2 expression resulted in a severe degeneration of the CA1 pyramidal cell layer, which did not occur in mice injected with control rAAV constructs. This finding might pinpoint to a vital and yet unknown contribution of HCN2 channels in establishing or maintaining the proper function of CA1 pyramidal neurons of the dorsal hippocampus

AmOctα2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either α- or β-adrenergic-like octopamine receptors. Currently, one α- and four β-receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an α2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOctα2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOctα2R), phylogenetically groups in a clade closely related to human α2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOctα2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOctα2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked efficiently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOctα2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiolog

AmOct2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either - or -adrenergic-like octopamine receptors. Currently, one - and four -receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an 2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOct2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOct2R), phylogenetically groups in a clade closely related to human 2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOct2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOct2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked eciently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOct2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology

AmOct2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either - or -adrenergic-like octopamine receptors. Currently, one - and four -receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an 2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOct2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOct2R), phylogenetically groups in a clade closely related to human 2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOct2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOct2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked eciently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOct2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology

Dm5-HT2B_{2B}: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster

Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster, a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT1A, Dm5-HT1B, and Dm5-HT7 couple to cAMP signaling cascades, the Dm5-HT2A receptor leads to Ca2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT2B receptor. Knowledge about this receptor’s pharmacological properties is very limited. This is quite surprising because Dm5-HT2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT2B’s pharmacology, we evaluated the receptor’s response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT2B signaling in vitro and in vivo