Real-time evaluation of human secretin receptor activity using cytosensor microphysiometry

Area of Science:

• Pharmacology
• Cellular Biology
• Biochemistry

Background:

• The human secretin receptor is a G protein-coupled receptor.
• It is functionally linked to the cAMP second messenger system via adenylate cyclase stimulation.

Purpose of the Study:

• To functionally characterize the human secretin receptor.
• To evaluate its signal transduction pathway.
• To compare the sensitivity of cAMP accumulation and microphysiometry assays.

Main Methods:

• Subcloning of human secretin receptor cDNA into a mammalian expression vector.
• Expression in cultured Chinese Hamster Ovary (CHO) cells.
• Measurement of intracellular cAMP accumulation using radioimmunoassay (RIA).
• Measurement of extracellular acidification rate using Cytosensor microphysiometry.

Main Results:

• Human secretin and biotinylated human secretin were equipotent in both assays.
• Microphysiometry (EC50 = 0.1 nM) was more sensitive than cAMP assay (EC50 = 0.2-0.5 nM).
• Secretin-stimulated response involves cAMP, protein kinase A, and does not show robust homologous desensitization.

Conclusions:

• Microphysiometry is a more sensitive method for monitoring human secretin receptor activation.
• Protein kinase A is essential for the receptor's intracellular signaling.
• The human secretin receptor does not exhibit significant homologous desensitization upon repeated stimulation.