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Am J Physiol Cell Physiol (September 5, 2001). doi:10.1152/ajpcell.00256.2001
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Articles in PresS, published online ahead of print September 5, 2001
Am J Physiol Cell Physiol, 10.1152/ajpcell.00256.2001
Submitted on June 8, 2001
Accepted on August 24, 2001

Vascular Endothelial Cells Express Isoforms of Protein Kinase A Inhibitor

Hazel Lum1*, Zengping Hao1, Dave Gayle1, Priyadarsini Kumar2, Carolyn E Patterson3, and Michael D Uhler4

1 Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, IL, USA
2 Biological Chemistry, University of California, Davis, CA, USA
3 Medicine, Indiana University School of Medicine, Indianapolis, IN, USA
4 Biological Chemistry, University of Michigan, Ann Arbor, MI, USA

* To whom correspondence should be addressed. E-mail: hlum{at}rush.edu.

The expression and function of PKI (endogenous inhibitor of cAMP-dependent protein kinase) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAEC), foreskin microvessel (HMEC), and brain microvessel endothelial cells (HBMEC). RT-PCR using specific primers for human PKI{alpha}, human PKI{gamma}, and mouse PKIß sequences detected PKI{alpha} and PKI{gamma} mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI{alpha} and PKI{gamma} were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI{alpha} mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI{gamma} mRNA. Western blot analysis indicated that PKI{gamma} protein was detected in all three cell types; whereas PKI{alpha} was found in HBMEC. In summary, endothelial cells from three different vascular beds express PKI{alpha} and PKI{gamma}, which may be physiologically important in endothelial barrier function.




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