The Na/Ca exchanger couples the movement of Ca ions across the plasma membrane to the movement of Na ions in the opposite direction. It is the principal Ca efflux mechanism in cardiac myocytes and plays an important role in controlling the Ca content of myocardial cells and in regulating cardiac contractility. Little is known about the cellular mechanisms that regulate exchange activity. In this project, the general hypothesis to be investigated is that sphingolipids are physiological regulators of Na/Ca exchange activity and block the conformational transition associated with regulatory Ca activation of the exchanger. This hypothesis is based on preliminary results demonstrating that short chain ceramide analogs and sphingosine inhibit exchange activity in a manner that is specifically targeted to the exchanger's regulatory properties. Exchange activity will be measured using fluorescent cytosolic Ca indicators in transfected CHO cells expressing the cardiac Na/Ca exchanger and in neonatal rat cardiac myocytes. The project has 6 specific aims: (1) Initial characterizations of the effects of exogenous and endogenous ceramide or sphingosine on exchange activity in the transfected cells will be completed. (2) A new kinetic formulation of exchanger regulation, which specifically links the process termed """"""""Na-dependent inactivation"""""""" to changes in regulatory Ca-activation, will be developed. (3) The rate at which ceramide inhibition develops when cytosolic Ca is elevated or when internal Ca stores are filled will be measued in order to test the hypothesis that ceramide inhibition requires the formation of the Ca-depleted conformation of the exchanger. Other studies will test the prediction that ceramide does not inhibit the exchanger in the absence of Ca activation. (4) The hypothesis that ceramide inhibits exchange activity by perturbing lateral lipid subdomains such as lipid rafts and/or caveolae will be tested. (5) The studies in this aim will test the idea that pharmacological stabilization of the actin cytoskeleton locks the exchanger in an activated state, reducing its susceptibility to regulation by cytosolic Ca or inhibition by ceramide. (6) The physiological significance of ceramide inhibition of exchange activity will be assessed by measuring the influence of ceramide on (a) exchange activity, (b) the Ca-transient and (c) the amount of Ca in the sarcoplasmic reticulum in beating neonatal rat cardiac myocytes. Other studies will assess whether inhibition of exchange activity contributes to the cardiotoxic effects of TNFalpha.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL049932-12
Application #
6750640
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Przywara, Dennis
Project Start
1993-06-01
Project End
2005-12-31
Budget Start
2004-06-01
Budget End
2005-12-31
Support Year
12
Fiscal Year
2004
Total Cost
$314,000
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Pharmacology
Type
Schools of Medicine
DUNS #
623946217
City
Newark
State
NJ
Country
United States
Zip Code
07107
Condrescu, Madalina; Reeves, John P (2010) Inhibition of sodium-calcium exchange by KB-R7943: Dodecylamine and sphingosine in transfected Chinese hamster ovary cells. Cell Calcium 47:404-11
Turner, Jay D; Thomas, Andrew P; Reeves, John P et al. (2009) Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells. Cell Calcium 46:242-7
Chernysh, Olga; Condrescu, Madalina; Reeves, John P (2008) Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells. Am J Physiol Cell Physiol 295:C872-82
Reeves, John P; Abdellatif, Maha; Condrescu, Madalina (2008) The sodium-calcium exchanger is a mechanosensitive transporter. J Physiol 586:1549-63
Babich, Olga; Reeves, John; Shirokov, Roman (2007) Block of CaV1.2 channels by Gd3+ reveals preopening transitions in the selectivity filter. J Gen Physiol 129:461-75
Urbanczyk, Jason; Chernysh, Olga; Condrescu, Madalina et al. (2006) Sodium-calcium exchange does not require allosteric calcium activation at high cytosolic sodium concentrations. J Physiol 575:693-705
Condrescu, Madalina; Reeves, John P (2006) Actin-dependent regulation of the cardiac Na(+)/Ca(2+) exchanger. Am J Physiol Cell Physiol 290:C691-701
Hantash, Basil M; Thomas, Andrew P; Reeves, John P (2006) Regulation of the cardiac L-type calcium channel in L6 cells by arginine-vasopressin. Biochem J 400:411-9
Le, Hoa Dinh; Omelchenko, Alexander; Hryshko, Larry V et al. (2005) Allosteric activation of sodium-calcium exchange by picomolar concentrations of cadmium. J Physiol 563:105-17
Chernysh, Olga; Condrescu, Madalina; Reeves, John P (2004) Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger. Am J Physiol Cell Physiol 287:C797-806

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