Antigen stimulation of mast cells through high affinity receptors for IgE (FcepsilonRI) leads to Ca2+ mobilization and, as a consequence, the release of inflammatory mediators that are responsible for allergic diseases such as asthma. It is generally believed that inositol 1,4,5-trisphoshate (IP3) is responsible for mobilizing Ca2+ from the intracellular endoplasmic reticulum (ER) stores. Dr. Choi's laboratory has shown that the amount of IP3 produced by FcepsilonRI stimulation is not sufficient to cause Ca2+ mobilization through FcepsilonRI and sphingosine 1-phosphate (SIP), a product of sphingosine kinase (SK) may play a major role in Ca2+ mobilization through FcepsilonRI. Currently cytosolic form of SK has been cloned and in Dr. Choi's laboratory cloned a putative membrane-associated SK. The goal of this proposal is to test the hypothesis that the membrane-associated, not cytosolic, SK is responsible for Ca2+ mobilization through FcepsilonRI in mast cells. In these studies, a mast cell line, RBL-2H3 cells will be used as a model system as FcepsilonRI-mediated signaling has been extensively studied in this cell line. Strategies will be 1) To establish stable RBL-2H3 cell lines overexpressing the cytosolic or membrane-associated SK. 2) To determine which form of SK, cytosolic or membrane-associated, is responsible for S1P production in the endoplasmic reticulum (ER) membrane after FcepsilonRI clustering and Ca2+ mobilization. 3) To measure functional responses such as Ca2+ response, IP3 production and hexosaminidase release after FcepsilonRI clustering in the two cell lines.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31AI010492-01A1
Application #
6204675
Study Section
Special Emphasis Panel (ZRG1-IFCN-5 (02))
Program Officer
Hernandez, Milton J
Project Start
2000-08-01
Project End
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
1
Fiscal Year
2000
Total Cost
$26,029
Indirect Cost
Name
Meharry Medical College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Nashville
State
TN
Country
United States
Zip Code
37208