This long term goal of our laboratories is to understand the kinetic mechanism that enable normal tissues to mount an appropriately timed and appropriately limited response to stress or crisis. Local release of adenine nucleotides and adenosine, and metabolism of these highly pharmacoactive compounds by cell surface enzymes are major components of the homeostatic response in many physiologic and pathophysiologic situations. We propose to evaluate the capacities of ventricular myocytes isolated from rabbit heart, and microvascular endothelial cells cultured from pig and rabbit heart, to produce extracellular adenosine and adenine nucleotides, to regulate extracellular nucleotide hydrolysis, and to respond to ATP, ADP, and adenosine.
The specific aims are: 1) Determine whether the metabolic and transport capabilities of each cell type sustain net adenosine efflux sufficient to account for the extracellular adenosine observed during coronary ischemia. 2) Determine whether hypoxia causes ATP release from myocytes and coronary microvascular endothelial cells. 3) Determine how the kinetic properties of the extracellular reaction sequence, ATP leads to ADP leads to AMP leads to adenosine, regulate the relative concentrations of these compounds at the surfaces of both cell types, and develop kinetic models which can predict the entire time course of the reaction sequence when a bolus of one or more of the components is introduced. 4) Examine cellular responses to ATP, ADP, and adenosine, and where possible, relate the rate and extent of these responses to the shifting extracellular agonist concentrations driven by release and extracellular metabolism. Stimulation (endothelial cells) and inhibition (myocytes) of cAMP synthesis and efflux will be used as cellular end-points for response to adenosine. Prostaglandin synthesis and changes in intracellular calcium concentration will be measured as indices of response to ATP and ADP. 5) Characterize the export of cAMP from each of the cell types, and determine the extent to which this process contributes to regulation of cellular cAMP turnover. Pharmacologically active concentrations of adenine nucleotides or adenosine occur extracellularly during neurotransmission, thrombus formation, and coronary ischemia. The experiments proposed here should provide important information for understanding the regulation of the rate and extent of coronary response during these events.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL031854-08
Application #
3343041
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-09-30
Project End
1994-04-30
Budget Start
1992-05-01
Budget End
1994-04-30
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Massachusetts Amherst
Department
Type
Schools of Arts and Sciences
DUNS #
153223151
City
Amherst
State
MA
Country
United States
Zip Code
01003
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Meghji, P; Pearson, J D; Slakey, L L (1995) Kinetics of extracellular ATP hydrolysis by microvascular endothelial cells from rat heart. Biochem J 308 ( Pt 3):725-31
Mahoney, M G; Slakey, L L; Hepler, P K et al. (1993) Independent modes of propagation of calcium waves in smooth muscle cells. J Cell Sci 104 ( Pt 4):1101-7
Meghji, P; Skladanowski, A C; Newby, A C et al. (1993) Effect of 5'-deoxy-5'-isobutylthioadenosine on formation and release of adenosine from neonatal and adult rat ventricular myocytes. Biochem J 291 ( Pt 3):833-9
Meghji, P; Pearson, J D; Slakey, L L (1992) Regulation of extracellular adenosine production by ectonucleotidases of adult rat ventricular myocytes. Am J Physiol 263:H40-7
Mahoney, M G; Randall, C J; Linderman, J J et al. (1992) Independent pathways regulate the cytosolic [Ca2+] initial transient and subsequent oscillations in individual cultured arterial smooth muscle cells responding to extracellular ATP. Mol Biol Cell 3:493-505
Schonherr, E; Jones, G A; Slakey, L L (1992) Gastric and salivary mucins inhibit angiotensin-converting enzyme. Inhibition is partly due to oligosaccharides. Biochem J 286 ( Pt 2):425-33
Xiong, Y M; Xu, S Z; Slakey, L L (1991) Modulation of response to adenosine in vascular smooth muscle cells cultured in defined medium. In Vitro Cell Dev Biol 27A:355-62
Fehr, T F; Dickinson, E S; Goldman, S J et al. (1990) Cyclic AMP efflux is regulated by occupancy of the adenosine receptor in pig aortic smooth muscle cells. J Biol Chem 265:10974-80
Slakey, L L; Gordon, E L; Pearson, J D (1990) A comparison of ectonucleotidase activities on vascular endothelial and smooth muscle cells. Ann N Y Acad Sci 603:366-78;discussion 378-9

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