Cellular stimuli (e.g. hormones, transmitters, mitogens, chemotactic factors and other agonists) whose actions involve the mobilization of intracellular Ca2+ as a mediator of cellular responses stimulate the metabolism of membrane phosphoinositides. As a result, the membrane lipid phosphatidylinositol 4,5-bisphosphate is hydrolyzed to generate the product inositol 1,4,5-trisphosphate (IP3). IP3 has been proposed to be the second messenger for the release of intracellular stores of Ca2+. IP3 has been found to release Ca2+ in several types of permeabilized cells, and from isolated membrane fractions from a few cell types. Microinjection of IP3 into egg cells also releases internal Ca2+. The molecular site of action of IP3 is unknown, and the physiological role of IP3 in stimulus-response coupling is yet to be fully established. The available evidence indicates the existence of specific membrane IP3-binding sites and an IP3- activated Ca2+ channel. Our approach to this problem is to employ the powerful hybridoma technology for the production of monoclonal antibodies to identify and isolate the IP3- receptor/channel and to further investigate the physiological role of IP3 in cellular physiology. Our approach is to immunize mice with a purified platelet membrane fraction that releases Ca2+ in response to IP3, and thereby to obtain polyclonal and then monoclonal antibodies that effect the function of the IP3-receptor/channel. Aided by assays (ELISA) that detect antibodies to platelet membrane antigens, and a bioassay that reliably measures Ca2+ release by IP3, we have succeeded in cloning several hybridomas which produce monoclonal antibodies that can completely block the action of IP3. In this study our objectives are to produce and purify a variety of monoclonal antibodies that block the Ca2+-mobilizing action IP3, and to employ these antibodies to: (1) identify the molecular site of action of IP3 (i.e. the receptor/channel), (2) to isolate, purify and characterize the IP3-receptor/channel, of IP3- receptor/channel, and (3) to test the physiological significance of IP3 by studying responses to stimulation in intact cells (e.g. eggs, photoreceptor cells, etc.) that have been microinjected with monoclonal antibodies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL037422-08
Application #
2218457
Study Section
Special Emphasis Panel (NSS)
Project Start
1987-01-01
Project End
1996-12-31
Budget Start
1994-01-01
Budget End
1994-12-31
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Pharmacology
Type
Schools of Dentistry
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030