THE ROLE OF CALCIUM IN THE MALPIGHIAN TUBULES OF THE KISSING BUG Rhodnius prolixus

Stimulation of urine production by the Malpighian (renal) tubules in Rhodnius prolixus is regulated by at least two diuretic hormones, CRF-related peptide and serotonin, that have traditionally been believed to function through the activation of cAMP-mediated intracellular second messenger pathways. In this study I demonstrate that serotonin stimulation triggered, in addition to cAMP, intracellular Ca2+ waves in the Malpighian tubule cells of R. prolixus. Treatment with the intracellular Ca2+ chelator BAPTA-AM blocked the intracellular Ca2+ waves and reduced serotonin-stimulated fluid secretion by 75%. This suggests a role for intracellular Ca2+ signaling in the excretory system of R. prolixus. Serotonin stimulated Malpighian tubules (MTs) exposed to Ca2+-free saline plus BAPTA-AM secreted an abnormal fluid, showing: increased K+ concentration, reduced Na+ concentration and lower pH. These results along with measurement of transepithelial potential (TEP) suggest that the basolateral Na+:K+:2Cl- cotransporter (NKCC) activity is reduced in tubule cells treated with BAPTA-AM, suggesting that Ca2+ is required to modulate the activity of the basolateral NKCC. Treatment with the non-hydrolysable cell-permeable cAMP analog, 8Br-cAMP, produced fluid with the same K+ and Na+ concentration and at the same secretion rate as serotonin-stimulated tubules. In addition, 8Br-cAMP triggered intracellular Ca2+ oscillations similar to those obtained with serotonin. 8Br-cAMP-stimulated tubules treated with BAPTA-AM decreased their fluid secretion by about 40% and increased Na+ concentration, similar to the effect observed on serotonin-stimulated tubules. Therefore, I conclude that the intracellular Ca2+ waves triggered by serotonin are mediated by cAMP. The role of inositol-3-phospate (InsP3) in Ca2+ release was tested by treating the tubules with the InsP3 receptor blocker xestospongin. The treatment decreased fluid secretion rate as well as the amplitude of Ca2+ waves in serotonin-stimulated tubules. These results suggest that serotonin activates the production of InsP3 and, most likely, diacylglycerol (DAG). Thus, I decided to test whether the protein kinase C (PKC) may be involved in serotonin-stimulated secretion. The PKC inhibitors chelerythrine and bisindolylmaleimide (BIM) decreased secretion fluid rate in serotonin-stimulated tubules by 50% and 70%, respectively. Fluid secreted by tubules treated with BIM showed no differences in K+ and Na+ concentrations compared to controls, however both ion fluxes decreased. The evidence suggests that PKC is involved in serotonin stimulated secretion; the mechanism is still not understood. Taken together, the results suggest that cAMP, Ca2+ and PLC-PKC pathway are involved in serotonin stimulated secretion. However cAMP stimulation is enough for maximal secretion rate. Therefore PLC-PKC must act downstream of cAMP. Based on those results we hypothesize that serotonin binds a GPCR, increasing cAMP by activation of an adenylate cyclase (AC). Subsequently, cAMP is somehow able to activate PLC, which finally produces Ca2+ release, PKC activation and NKCC upregulation

The neuropeptide CCHamide2 regulates diuresis in the Chagas disease vector Rhodnius prolixus

Given that hematophagous insects ingest large quantities of blood in a single meal, they must undergo a rapid post-prandial diuresis in order to maintain homeostasis. In the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae), the coordinated activity of the Malpighian tubules and anterior midgut maintains water and ion balance during the post-prandial diuresis. Three to four hours after the meal, the diuretic process finishes, and the animal enters an antidiuretic state to ensure water conservation until the next blood intake. The diuretic and antidiuretic processes are tightly regulated by serotonin and neuropeptides in this insect. In the present work, we report that the neuropeptide precursor CCHamide2 is involved in the regulation of the post-prandial diuresis in R. prolixus. Our results suggest a dual effect of RhoprCCHamide2 peptide, enhancing the serotonin-induced secretion by Malpighian tubules, and inhibiting serotonin-induced absorption across the anterior midgut. To our knowledge, this is the first report of a hormone presenting opposite effects in the two osmoregulatory organs (i.e. midgut and Malpighian tubules) in insects, probably reflecting the importance of a well-tuned diuretic process in hematophagous insects during different moments after the blood meal.Fil: Capriotti, Natalia. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Ianowski, Juan Pablo. University of Saskatchewan; CanadáFil: Gioino, Paula. University of Saskatchewan; CanadáFil: Ons, Sheila. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentin

The neuropeptide RhoprCCHamide2 inhibits serotonin-stimulated transcellular Na⁺ transport across the anterior midgut of the vector of Chagas disease, <i>Rhodnius prolixus</i>

Rhodnius prolixus is a blood-feeding insect vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. During each blood meal, the animals ingest large volumes of blood, that may be up to 12 times the unfed body mass. These blood meals impose a significant osmotic stress for the animals due to the hyposmotic condition of the ingested blood compared with the insect's hemolymph. Thus the insect undergoes a massive postprandial diuresis that allows for the excretion of the plasma fraction of the blood in less than two hours. Diuresis is performed by the excretory system, consisting of the Malpighian tubules and gut, under the control of diuretic and anti-diuretic factors. We investigated the ion transport machinery triggered by stimulation with the diuretic factor serotonin in the anterior midgut (i.e. crop) and the effect of the diuretic modulator RhoprCCHamide2. Ussing chamber assays revealed that serotonin-stimulated increase in transepithelial short-circuit current (Isc) was more sensitive to the blockage with amiloride than 5-N-ethyl-N-isopropyl amiloride (EIPA), suggesting the involvement of Na⁺ channels. Incubation in Na⁺-free, but not Cl⁻-free saline, blocked the effect of serotonin on Isc. Moreover, treatment with Na⁺-K⁺-2Cl⁻ cotransporter (NKCC) and Na⁺-Cl⁻ cotransporter (NCC) blockers had no effect on fluid secretion but was blocked by amiloride. Blockage of Na⁺/K⁺-ATPase with ouabain inhibited Isc but the H⁺-ATPase inhibitor bafilomycin had no effect. The neuropeptide RhoprCCHamide2 diminished serotonin-stimulated Isc across the crop. The results suggest that Na⁺ undergoes active transport via an apical amiloride-sensitive Na⁺ channel and a basolateral ouabain-sensitive Na⁺/K⁺-ATPase, while Cl⁻ is transported through a passive paracellular pathway.Centro Regional de Estudios Genómico

The neuropeptide CCHamide2 regulates diuresis in the Chagas disease vector <i>Rhodnius prolixus</i>

Given that hematophagous insects ingest large quantities of blood in a single meal, they must undergo a rapid post-prandial diuresis in order to maintain homeostasis. In the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae), the coordinated activity of the Malpighian tubules and anterior midgut maintains water and ion balance during the post-prandial diuresis. Three to four hours after the meal the diuretic process finishes, and the animal enters an antidiuretic state to ensure water conservation until the next blood intake. The diuretic and antidiuretic processes are tightly regulated by serotonin and neuropeptides in this insect. In the present work, we report that the neuropeptide precursor CCHamide 2 is involved in the regulation of the post-prandial diuresis in the kissing bug R. prolixus. Our results suggest a dual effect of RhoprCCHamide2 peptide, enhancing the serotonin-induced secretion by Malpighian tubules, and inhibiting serotonin-induced absorption across the anterior midgut. To our knowledge, this is the first report of a hormone presenting opposite effects in the two osmoregulatory organs (i.e. midgut and Malpighian tubules) in insects, probably reflecting the importance of a well-tuned diuretic process in hematophagous insects during different moments after the blood meal.Facultad de Ciencias ExactasCentro Regional de Estudios Genómico

The neuropeptide RhoprCCHamide2 inhibits serotonin-stimulated transcellular Na+ transport across the anterior midgut of the vector of Chagas disease, Rhodnius prolixus

Rhodnius prolixus is a blood-feeding insect vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. During each blood meal, the animals ingest large volumes of blood, that may be up to 12 times the unfed body mass. These blood meals impose a significant osmotic stress for the animals due to the hyposmotic condition of the ingested blood compared with the insect’s hemolymph. Thus the insect undergoes a massive postprandial diuresis that allows for the excretion of the plasma fraction of the blood in less than two hours. Diuresis is performed by the excretory system, consisting of the Malpighian tubules and gut, under the control of diuretic and antidiuretic factors. We investigated the ion transport machinery triggered by stimulation with the diuretic factor serotonin in the anterior midgut (i.e. crop) and the effect of the diuretic modulator RhoprCCHamide2. Ussing chamber assays revealed that serotonin-stimulated increase in transepithelial short-circuit current (Isc) was more sensitive to the blockage with amiloride than 5-N-ethyl-N-isopropyl amiloride (EIPA), suggesting the involvement of Na+ channels. Incubation in Na+-free, but not Cl−-free saline, blocked the effect of serotonin on Isc. Moreover, treatment with Na+–K+–2Cl− cotransporter (NKCC) and Na+–Cl− cotransporter (NCC) blockers had no effect on fluid secretion but was blocked by amiloride. Blockage of Na+/K+-ATPase with ouabain inhibited Isc but the H+-ATPase inhibitor bafilomycin had no effect. The neuropeptide RhoprCCHamide2 diminished serotonin-stimulated Isc across the crop. The results suggest that Na+ undergoes active transport via an apical amiloride-sensitive Na+ channel and a basolateral ouabain-sensitive Na+/K+-ATPase, while Cl− is transported through a passive paracellular pathway.Fil: Capriotti, Natalia. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Gioino, Paula. Saskatchewan University; CanadáFil: Ons, Sheila. Saskatchewan University; CanadáFil: Ianowski, Juan Pablo. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentin

Escribir las prácticas: una propuesta metodológica para investigaciones de Extensión o de Responsabilidad Social Universitaria

En la actualidad existe una significativa y variada producción bibliográfica sobre paradigmas, metodologías y técnicas de investigación, orientada primordialmente hacia investigaciones de corte académico y científico; mientras que, es relativamente escasa y nueva la producción y sistematización de abordajes destinados a guiar la labor de investigación en marcos de ecciones de extensión y/o responsabilidad social universitaria. Colaborando con la profundización de esta última línea, el objetivo principal de este artículo es iniciar una necesaria reflexión y sistematización de las acciones, saberes y prácticas que han guiado el trabajo enmarcado en un proyecto de responsabilidad social universitaria, a fin de bosquejar un esquema de abordaje para este tipo de intervenciones. Más que informar sobreel desarrollo del proyecto, el sentido de este escrito es marcadamente político: se trata, en otras palabras, de una propuesta para innovar en las formas de concebir y escribir las prácticas, tanto de la ciencia como de la comunidad y la política.Nowdays, there is a significant and varied bibliographic production on paradigms, methodologies, and techniques of research which is oriented primarily toward academic and scientific research; whereas, it is relatively scarce and new the production and systematization of approaches designed to guide the work of research into “extension” and/or “social responsibility” frameworks. Inside this last line, the main objective of this article is to start a necessary reflection and systematization of the actions, knowledge and practices that have guided the running of a project of social responsibility, in order to outline a scheme of approaching for this type of intervention. Rarher than reporting on the development of the project, the meaning of this writing is markedly political: it is, in other words, a proposal to innovate the ways of designing and writing practices, not only of the science, but also or politics and the community.Fil: Ciuffolini, María Alejandra. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de la Vega Avila Tulian, Candela. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Job, Sergio Fernando. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Ferrero, María Mercedes. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gallego, Ayelén Dominique. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Avalle, Gerardo. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hernández, Juliana. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ibáñez Mestres, Gonzalo. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Feldmann, Noelia. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Forclaz, Zarina. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Fernandez Gioino, Mauricio. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Gatica, Santiago. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Alonso Alasino, Ignacio. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Antonini, Tania. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Bordese, María Paula. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; ArgentinaFil: Guglielmetti, Lucía. Universidad Católica de Córdoba. Facultad de Ciencias Politicas y Relaciones Internacionales; Argentin