Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells

Area of Science:

• Endothelial cell biology
• Biochemistry
• Cell signaling

Background:

• Prostacyclin (PGI2) is a key regulator of vascular function.
• The role of trypsin in endothelial cell signaling and PGI2 production is not fully understood.

Purpose of the Study:

• To investigate the correlation between trypsin binding, processing, and prostacyclin production in cultured adult bovine aortic endothelial (ABAE) cells.
• To characterize the cell-surface receptor involved in trypsin binding and its effect on PGI2 release.

Main Methods:

• Dose-response studies of PGI2 production and 125I-trypsin binding in ABAE cells.
• Characterization of trypsin receptor affinity (Kd) and density.
• Competition assays using unlabeled trypsin and thrombin.
• Comparison of trypsin internalization in ABAE cells versus human skin fibroblasts (HSF).

Main Results:

• ABAE cells produced PGI2 in a dose-dependent manner in response to trypsin, with saturation at 1 microgram/ml.
• 125I-trypsin binding exhibited dose-response kinetics and saturation, indicating specific high-affinity binding (Kd = 1.5 X 10(-8) M) to approximately 1.2 X 10(5) receptor sites per cell.
• Unlabeled trypsin competed for binding, while thrombin did not, suggesting a specific trypsin receptor.
• Minimal internalization and degradation of bound trypsin were observed in ABAE cells compared to HSF.

Conclusions:

• Trypsin stimulates prostacyclin production and release in ABAE cells.
• This stimulation is mediated by a specific cell-surface receptor for trypsin.
• The trypsin receptor is distinct from the thrombin receptor and shows limited internalization of the bound ligand.