Cyclic AMP increases bradykinin receptor binding affinity in human endothelial cells

Area of Science:

• Molecular pharmacology
• Endothelial cell biology
• Signal transduction

Background:

• Bradykinin receptors are present in human endothelial cells.
• The role of cyclic AMP (cAMP) in regulating bradykinin receptor function is not fully understood.

Purpose of the Study:

• To investigate the presence and regulation of bradykinin receptors in human endothelial cells.
• To determine if cyclic AMP influences bradykinin receptor binding characteristics.

Main Methods:

• Real-time PCR to detect bradykinin receptor mRNA.
• Immunoblotting to confirm B(2) receptor protein.
• Radioligand binding assays using (125)I-[Tyr(8)]-bradykinin to determine receptor affinity and capacity.
• Competition studies with B(1) and B(2) receptor antagonists.
• Cell stimulation with cAMP-elevating agents (dibutyryl-cAMP, cholera toxin, forskolin).

Main Results:

• Bradykinin B(1) and B(2) receptor mRNA and B(2) protein were detected in human endothelial cells.
• Binding assays revealed one class of binding sites with a dissociation constant of 352 ± 27 pM and maximal binding capacity of 39.3 ± 1.3 fmol/mg protein.
• Activation of adenylate cyclase by cAMP-elevating agents increased bradykinin binding by approximately 50%.
• Stimulation with dibutyryl-cAMP reduced the dissociation constant to 203 ± 18 pM, indicating increased B(1) receptor affinity, while maximal binding capacity remained unchanged.
• B(1) receptor antagonists competed binding similarly in stimulated and non-stimulated cells.

Conclusions:

• Human endothelial cells express bradykinin B(1) and B(2) receptors.
• Adenylate cyclase activators, through cyclic AMP signaling, enhance bradykinin B(1) receptor affinity, thereby increasing bradykinin binding without altering receptor capacity.
• These findings suggest a novel mechanism for regulating bradykinin signaling in endothelial cells.