Related Experiment Videos
Vascular endothelial cells express isoforms of protein kinase A inhibitor
Hazel Lum1, Zengping Hao, Dave Gayle
1Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA. hlum@rush.edu
Abstract:
The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) 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 (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta 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 HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.
Insights
Endothelial cells express PKI alpha and PKI gamma, crucial for maintaining endothelial barrier function. These inhibitors regulate cellular responses to cAMP and thrombin, impacting vascular integrity.
Area of Science:
- Molecular Biology
- Cell Biology
- Vascular Biology
Background:
- The role of Protein Kinase Inhibitor (PKI) in endothelial cells remains largely uncharacterized.
- Endothelial barrier function is critical for vascular integrity and is modulated by various signaling pathways.
Purpose of the Study:
- To investigate the expression of endogenous PKI isoforms (PKI alpha and PKI gamma) in human endothelial cells.
- To determine the potential role of PKI in regulating endothelial barrier function.
Main Methods:
- Reverse Transcription Polymerase Chain Reaction (RT-PCR) to detect PKI mRNA.
- DNA sequencing and BLAST analysis for sequence verification.
- RNase protection assays to confirm mRNA integrity.
- Western blot analysis to detect PKI protein expression.
Main Results:
- PKI alpha and PKI gamma mRNA were detected in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs).
- PKI gamma protein was found in all three cell types, while PKI alpha protein was detected in HBMECs.
- Overexpression of PKI inhibited cAMP-mediated barrier increases and worsened thrombin-induced barrier decreases.
Conclusions:
- Human endothelial cells express PKI alpha and PKI gamma.
- These PKI isoforms are likely physiologically important in regulating endothelial barrier function.