A persistent increase in lung endothelial permeability to protein is a hallmark of the Adult Respiratory Distress Syndrome. Moreover, inflammatory mediator-initiated increase in the Area/Unit pathlength (Arho/Ax) of the Endothelial Junctional Space is the foundation of elevated solute permeability of the microvascular wall. The long term objectives of this research are to better understand the endothelial mechanisms that modulate pulmonary microvascular permeability to macromolecules. Intracellular level of cyclic nucleotide, adenosine 3',5' cyclic monophosphate (cAMP), or the activities of protein kinase C and A, and intracellular free Ca2+ concentration ([Ca2+]) will be specifically manipulated in vitro. The hypotheses to be tested are: 1) Agonist-induced changes of intracellular second messengers modulate endothelial morphology leading to the discrete Solute Permeability Mechanism: Restricted Diffusion or Convection and 2) Different mediator induced distinct cellular responses by discrete spatiotemporal alterations in [Ca2+] of cytoskeleton protein will be assessed in specific aims 1-3.
Specific aim 4 will test the Crone Hypothesis.
These aims will be tested using bovine lung endothelial cells from two distinct locations: large vessel (pulmonary artery endothelial cells, AEC) and macrovessel (MEC). Monolayers of AEC and MEC will be used as cellular models of the lung microvascular barrier to solutes. The following assays will be used: 1) The solute permeability mechanisms that define the barrier properties of endothelial monolayer will be measured for each experimental condition, 2) Endothelial surface area will be measured from differential interference contrast (DIC) digital images using Image-1 software (universal Imaging Corp), 3) Agonist-induced spatiotemporal alterations of intracellular [Ca2+] will be measured by fura-2 fluorescence digital images. 4) Agent-induced rearrangement of endothelial F-actin/myosin II will be measure by fluorescence digital images in living cells, 5) Electron microscopic analysis by rotary shadowing of the endothelial cytoskeleton will be performed, 6) Intracellular [cAMP] and [cGMP] will be measured by radioimmunoassay, 7) Protein kinase activities will be measured by P-labelled immunoaffinity protein SDS polyacrylamide gel electrophoresis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL048816-02
Application #
2224903
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1993-12-15
Project End
1998-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Arizona
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Mucha, David R; Myers, Carter L; Schaeffer Jr, Richard C (2003) Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase. Am J Physiol Heart Circ Physiol 284:H994-H1002
Schaeffer Jr, Richard C; Gratrix, Max L; Mucha, David R et al. (2002) The rat glomerular filtration barrier does not show negative charge selectivity. Microcirculation 9:329-42
Merkle, C J; Schuler, L A; Schaeffer Jr, R C et al. (2000) Structural and functional effects of high prolactin levels on injured endothelial cells: evidence for an endothelial prolactin receptor. Endocrine 13:37-46
Carbajal, J M; Gratrix, M L; Yu, C H et al. (2000) ROCK mediates thrombin's endothelial barrier dysfunction. Am J Physiol Cell Physiol 279:C195-204
Carbajal, J M; Schaeffer Jr, R C (1999) RhoA inactivation enhances endothelial barrier function. Am J Physiol 277:C955-64
Cohen, A W; Carbajal, J M; Schaeffer Jr, R C (1999) VEGF stimulates tyrosine phosphorylation of beta-catenin and small-pore endothelial barrier dysfunction. Am J Physiol 277:H2038-49
Carbajal, J M; Schaeffer Jr, R C (1998) H2O2 and genistein differentially modulate protein tyrosine phosphorylation, endothelial morphology, and monolayer barrier function. Biochem Biophys Res Commun 249:461-6
Diwan, A H; Honkanen, R E; Schaeffer Jr, R C et al. (1997) Inhibition of serine-threonine protein phosphatases decreases barrier function of rat pulmonary microvascular endothelial cells. J Cell Physiol 171:259-70
Schaeffer Jr, R C; Bitrick Jr, M S (1995) Effects of human alpha-thrombin and 8bromo-cAMP on large and microvessel endothelial monolayer equivalent ""pore"" radii. Microvasc Res 49:364-71