ABSTRACT
OBJECTIVES@#To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions.@*METHODS@#HPMECs were randomly divided into an air group and a hyperoxia group (@*RESULTS@#Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB (@*CONCLUSIONS@#ERK1/2 and PKB may be the downstream targets of the ADM signaling pathway. ADM mediates the ERK/PKB signaling pathway by regulating CRLR/RAMP2 and participates in the protection of hyperoxia-induced lung injury.
Subject(s)
Humans , Adrenomedullin/genetics , Endothelial Cells , Hyperoxia/complications , Lung Injury , Receptor Activity-Modifying ProteinsABSTRACT
The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.
Subject(s)
Animals , Male , Adrenomedullin/pharmacology , Calcitonin Receptor-Like Protein/analysis , Muscle, Smooth/drug effects , Parasympatholytics/pharmacology , Penis/drug effects , Vasodilator Agents/pharmacology , /pharmacology , /analysis , Adrenomedullin/genetics , Adrenomedullin/metabolism , Blotting, Western , Calcitonin Receptor-Like Protein/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclooxygenase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Immunohistochemistry , Indazoles/pharmacology , Muscle Relaxation , Muscle, Smooth/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide/analysis , Nitric Oxide/analogs & derivatives , Penis/metabolism , Potassium Channels, Voltage-Gated/metabolism , Rats, Wistar , Real-Time Polymerase Chain Reaction , RNA, Messenger/metabolism , Receptor Activity-Modifying Protein 1/genetics , Receptor Activity-Modifying Protein 1/metabolism , /metabolism , /genetics , /metabolism , Receptors, Calcitonin Gene-Related Peptide/metabolismABSTRACT
Blood samples are used as a biological source to discover biomarkers of hematological and non-hematological disorders. The present study shows the impact of different experimental conditions associated with cell lysis buffer, TRI-reagent protocol and blood cell storage buffer and their correlation with the quantity, quality and Adrenomedullin gene expression levels of total RNA when RT-PCR technique is used. A leukocyte cell bank protocol is also proposed for further mRNA expression analysis using RNAlater as storage buffer. There is evidence that total RNA isolated from leukocyte concentrate stored for 1 month at -70 degrees C did not show significant differences concerning quality, purity and Adrenomedullin gene expression compared with the freshly processed leukocyte sample.