Seful in understating the regulation of sphinoglipid metabolism in the lung in general, and in the future design of therapies that target various ceramide species and metabolites. In the current study, we determined the expression of ceramide species and CerS in the lung and in principal alveolar cells, and examined the importance of CerS2 in lung function. We demonstrate that CerS2 is essential for proper lung sphingolipid homeostasis and airway function.from Bay Genomics. CerS2GT/+ (denoted in our manuscript CerS2/+) mice were intercrossed to generate CerS2GT/GT (denoted in our manuscript as CerS2-null) mice. CerS2-null mice were born in normal mendelian distribution. Experiments were performed in mice (both females and males) age 3 months, unless otherwise noted. To inhibit CerS pharmacologically, mice were administered fumonisin B1 (FB1) intraperitoneal (i.p.) (1.1 or 2.2 mg/kg [11]) daily, for 3 days.Lung Function MeasurementMice were anesthetized with ketamine followed by 5 isoflurane inhalation (5 ), and were intubated via a custom-made laryngoscope blade. Animals were mechanically ventilated with a rodent ventilator using room air, at a rate of 140 breaths per min, a tidal volume of 0.3 ml, and 5 cm H2O of positive end-expiratory pressure. Mice were placed on a heated (37uC) pad and pulmonary function tests were performed with the FlexiVent system (Scireq, Montreal, PQ, Canada).Materials and Methods Chemicals and ReagentsAll chemicals and reagents were from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.Cell CultureBeas2B cells, a transformed human bronchial cell line, were a kind gift from Dr.Phenanthriplatin Augustine Choi, Harvard University and were originally purchased from American Type Culture Collection (ATCC, Manassas, VA).Ramelteon They were used from passages 52. Primary human small airway epithelial cells (SAEC) and human lung microvascular endothelial cells were from Lonza, Walkersville, MD and were maintained up to passage 5 in complete culture medium consisting of EGM-2MV, supplemented with specific SingleQuotsH (Lonza, Walkersville, MD).PMID:24957087 Bronchoalveolar LavageBronchoalveolar lavage (BAL) fluid was collected by lavaging the lungs with three aliquots of 1 ml of saline. Samples were centrifuged (6 min, 5006g, 4uC). Cell pellets were collected in 1 ml of red blood cell lysis buffer and following washing, were resuspended in PBS and counted in a hemocytometer. Cytospin slides containing 10,000 cells were prepared using a 3-step stain set (Richard Allen Scientific). Slides were scored by an investigator blinded to the identity of the experimental groups.AnimalsAnimal studies were approved by the Institutional Animal Care and Use Committee at Indiana University (Indianapolis, IN) and at the Weizmann Institute of Science (Rehovot, Israel). CerS2 null mice were generated in our laboratory, as previously described [10], using CerS2 genetrap ES cells (Cers2Gt(S16-4B1)Sor) obtainedPLOS ONE | www.plosone.orgLung Tissue HarvestingFollowing euthanasia, lungs were flushed by perfusing 2610 ml of saline through the pulmonary circulation. The right bronchus was ligated and a pre-warmed solution of low melting point agarose (0.25 (v/v) in 10 (v/v) formalin/PBS) was slowlySphingolipid Homeostasis Impact on Airway Functionintroduced into the left lung under a constant pressure of 20 cm H2O. The lungs and the heart were dissected en block and cooled on ice for 50 min. The right lung was dissected and snap-frozen in liquid N2. The left lung was secti.
