Isolation and functional characterization of calcitonin-like diuretic hormone receptors in Rhodnius prolixus.

Several families of diuretic hormones exist in insects, one of which is the calcitonin-like diuretic hormone (CT/DH) family. CT/DH mediates its effects by binding to family B G-protein coupled receptors (GPCRs). Here we isolate and functionally characterize two R. prolixusCT/DH receptor paralogs (Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2) using a novel heterologous assay utilizing a modified human embryonic kidney 293 (HEK293) cell line. Rhopr-CT/DH-R1 is orthologous to the previously characterized D. melanogasterCT/DH receptor (CG17415) while Rhopr-CT/DH-R2 is orthologous to the D. melanogaster receptor (CG4395), an orphan receptor whose ligand was unknown until now. We determine the cDNA sequences of three splice variants encoding Rhopr-CT/DH-R1 (Rhopr-CT/DH-R1-A, Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C) and two splice variants encoding Rhopr-CT/DH-R2 (Rhopr-CT/DH-R2-A and Rhopr-CT/DH-R2-B). Rhopr-CT/DH-R1-A and Rhopr-CT/DH-R2-A encode truncated receptors that lack six and seven of the characteristic seven transmembrane domains, respectively. Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C, which only differ by 2 amino acids in their C-terminal domain, can both be activated by Rhopr-CT/DH at equal sensitivities (EC50 = 200-300 nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC50 = 15 nM) compared to Rhopr-CT/DH-R1-B/C and also yields a much greater response (amplitude) in our heterologous assay. This is the first study to reveal that insects possess at least two CT/DH receptors, which may be functionally different. Quantitative PCR demonstrates that Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2 have distinct expression patterns, with both receptors expressed centrally and peripherally. Moreover, the expression analysis also identified novel target tissues for this neuropeptide, including testes, ovaries and prothoracic glands, suggesting a possible role for Rhopr-CT/DH in reproductive physiology and development.

Usefulness of procalcitonin clearance as a prognostic biomarker in septic shock. A prospective pilot study

Objective: To evaluate procalcitonin clearance as a prognostic biomarker in septic shock. Design: Prospective, observational pilot study. Setting: Intensive care unit. Patients: Patients admitted to the ICU due to septic shock and multiorgan dysfunction. Interventions: Serum concentrations of procalcitonin were determined within 12h of onset of septic shock and multiorgan dysfunction (coinciding with admission to the ICU), and the following extractions were obtained after 24, 48 and 72h in patients who survived. Data collected: Demographic data, Acute Physiology and Chronic Health Evaluation II score, and Sequential Organ Failure Assessment score, data on the primary focus of infection, and patient outcome (ICU mortality). Results: Procalcitonin clearance was higher in survivors than in non-survivors, with significant differences at 24h (73.9 [56.4-83.8]% vs 22.7 [-331-58.4], p<0.05) and 48h (81.6 [71.6-91.3]% vs -7.29 [-108.2-82.3], p<0.05). The area under the ROC curve was 0.74 (95%CI, 0.54-0.95, p<0.05) for procalcitonin clearance at 24h, and 0.86 (95%CI, 0.69-1.0, p<0.05) at 48h. Conclusions: ICU mortality was associated to sustained high procalcitonin levels, suggesting that procalcitonin clearance at 48h may be a valuable prognostic biomarker (AU)

Objetivo: Evaluar el aclaramiento de procalcitonina como biomarcador pronóstico del shock séptico. Diseño: Estudio piloto, observacional y prospectivo. Ámbito: Servicio de Medicina Intensiva. Pacientes: Enfermos ingresados en el Servicio de Medicina Intensiva por shock séptico y disfunción multiorgánica. Intervenciones: Determinación de las concentraciones séricas de procalcitonina en las primeras 12h de evolución del shock séptico (coincidiendo con el ingreso en el Servicio de Medicina Intensiva) y posteriormente a las 24 horas, 48 horas y a las 72 horas en los pacientes supervivientes. Variables recogidas: datos demográficos, score Acute Physiology and Chronic Health Evaluation II, score Sequential Organ Failure Assessment, datos relativos al foco de sepsis y al resultado del paciente (mortalidad en el Servicio de Medicina Intensiva). Resultados: El aclaramiento de procalcitonina fue mayor en los pacientes supervivientes respecto a los no supervivientes, con diferencias significativas a las 24 horas (73,9 [56,4-83,8]% vs 22,7 [-331-58,4], p<0,05) y las 48 horas (81,6 [71,6-91,3]% vs -7,29 [-108,2-82,3], p<0,05). El área por debajo de la curva ROC fue 0,74 (IC del 95%, 0,54 a 0,95, p<0,05) para el aclaramiento de procalcitonina a las 24 horas y 0,86 (IC del 95%, 0,69 a 1,0, p<0,05) para el aclaramiento de procalcitonina a las 48 horas. Conclusiones: La persistencia de concentraciones elevadas de procalcitonina se asoció a una mayor mortalidad. El aclaramiento de procalcitonina realizado a las 48h puede ser de utilidad como biomarcador pronóstico (AU)

Functional calcitonin gene-related peptide subtype 2 receptors in porcine coronary arteries are identified as calcitonin gene-related peptide subtype 1 receptors by radioligand binding and reverse transcription-polymerase chain reaction.

Calcitonin gene-related peptide (CGRP) receptors are classified into CGRP subtype 1 (CGRP(1)) and CGRP subtype 2 (CGRP(2)) based on the affinity of the antagonist, human alpha (halpha)-CGRP(8-37). halpha-CGRP(8-37) antagonizes CGRP(1) receptor-mediated responses with high affinity (K(B) < 100 nM) and antagonizes CGRP(2) receptor-mediated responses with low affinity (K(B) > 1 microM). CGRP(2) receptors have been previously reported to mediate relaxation of large porcine coronary arteries because this action is antagonized with low affinity by halpha-CGRP(8-37). In the present study, we used reverse transcription-polymerase chain reaction, radioligand binding, and values from our previously reported isolated tissue experiments to compare the CGRP receptor in porcine coronary arteries with the porcine CGRP(1) receptor stably expressed in human embryonic kidney (HEK) 293 cells. We identified calcitonin receptor-like receptor and receptor activity modifying protein 1 mRNA in coronary arteries. We also found that the ligand binding characteristics of the CGRP receptor in coronary arteries and the cloned CGRP(1) receptor were highly similar. K(I) values for halpha-CGRP(8-37) were 6.6 and 5.7 nM in porcine coronary arteries and the cloned CGRP(1) receptor, respectively. The affinities (K(B)) of halpha-CGRP(8-37) and five other antagonists were 22- to 707-fold lower in functional experiments measuring relaxation of coronary arteries than in radioligand binding experiments. Despite this difference in absolute affinity values, there was a high correlation of the rank order of affinity for the antagonists determined by the two methods. Thus halpha-CGRP(8-37) antagonizes CGRP-induced relaxation of porcine coronary arteries with low affinity at the CGRP(1) receptor. Taken together, these data do not support the existence of the CGRP(2) receptor.

Modulation of calcitonin binding by calcium: differential effects of divalent cations.

Binding of salmon calcitonin to bovine hypothalamic membranes is enhanced about 25% by calcium with a half-maximal effect at 15 mM calcium. In contrast, membranes prepared from a cell line expressing a recombinant human calcitonin receptor show no effect of calcium under similar conditions. The hypothalamic calcitonin receptor solubilized with CHAPS detergent retains an apparent Kd of 0.3 nM for salmon calcitonin; however, binding of calcitonin to the detergent-solubilized receptor complex can be inhibited by divalent cations in order of potency Mn > Ca approximately Sr approximately Mg >> NaCl with Mn and Ca having apparent Ki's of 5 mM and 20 mM respectively. Dixon and Scatchard plots of Mn and Ca inhibition of binding to the soluble receptor complex suggest a noncompetitive mechanism of inhibition. Calcium also inhibits calcitonin binding to a detergent-solubilized recombinant human calcitonin receptor. Inhibition of calcitonin binding is observed using two independent methods for determining soluble receptor-hormone complex and inhibition is reversed by EDTA.