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

Abstract

Human secretin receptor is a G protein-coupled receptor that is functionally linked to the cAMP second messenger system by stimulation of adenylate cyclase. To functionally characterize the receptor and evaluate its signal transduction pathway, the full-length human secretin receptor cDNA was subcloned into the mammalian expression vector pRc/CMV and expressed in cultured CHO cells. Intracellular cAMP accumulation of the stably transfected cells was measured by a radioimmunoassay (RIA), while the extracellular acidification rate was measured by the Cytosensor microphysiometer. Human secretin and biotinylated human secretin were equipotent in both assays in a dose-dependent manner. The EC50 values of stimulating the intracellular cAMP accumulation and the extracellular acidification rate were 0.2-0.5 nM and 0.1 nM, respectively, indicating that microphysiometry is more sensitive than the cAMP assay in monitoring ligand stimulation of the human secretin receptor. The secretin-stimulated response could be mimicked by forskolin and augmented by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, indicating that the extracellular acidification response is positively correlated with intracellular cAMP level. The response could be abolished by the protein kinase A inhibitor H-89, suggesting that protein kinase A plays an essential role in the intracellular signaling of the receptor. Upon repeated stimulation by the ligand, the peak acidification responses did not change significantly at both physiological (0.03 nM and 3 nM) and pharmacological (0.3 microM) concentrations of human secretin, suggesting that the human secretin receptor did not exhibit robust homologous desensitization.

Functional Segregation of the Highly Conserved Basic Motifs within the Third Endoloop of the Human Secretin Receptor

“…Generally, the signal transduction mechanism of GPCRs involves ligandinduced changes that affect the conformation of the intracellular surface of the receptors and hence promote the coupling to G proteins (2)(3)(4)(5)(6). In the case of the secretin/VIP receptor subfamily, this stimulation process activates adenylate cyclase and eventually leads to the elevation of intracellular cAMP (7)(8)(9)(10)(11). Besides cAMP, other intracellular second messengers, such as Ca 2ϩ and inositol phosphates have also been reported (12)(13)(14).…”

A1 and A2A adenosine receptor modulation of α1‐adrenoceptor‐mediated contractility in human cultured prostatic stromal cells

“…To confirm the presence of functional adenosine receptor subtypes on HCPSC, we measured changes in pH i , using the fluorophore BCECF ( Rink et al , 1982 ), as a direct indicator of adenosine receptor activation. Microphysiometry studies have established that cellular proton efflux is modulated by various metabolic events, including the activation of G‐protein‐coupled receptors ( McConnell et al , 1992 ; Ikeda et al , 1999 ; Kobayashi et al , 2001 ) and subsequent effects on adenylyl cyclase activity ( Ng et al , 1999 ). This increase in proton efflux is a result of the breakdown of ATP to ADP with the subsequent release of P i and H + ( McConnell et al , 1992 ).…”

Algorithmically designed peptides ameliorate behavioral defects in animal model of ADHD by an allosteric mechanism

“…Protonic H + neutralizes the charge on the surface of the semiconductor, reducing the produced photocurrent at a rate linearly related to H + production. The applicability of this technique to systems with a variety of membrane receptor, signal transductive mechanisms is consistent with the findings that there may or may not be a relationship between positive EAR responses to peptides or dopamine in transfected cell systems and their native modulation via G-proteins and/or adenyl cyclase (Baumbach et al, 1998;Neve et al, 1992;Ng et al, 1999;Smalley et al, 1998). Time-dependent [H + ] production as EAR is integrated as a trapezoidal approximation of area under the curve, AUC.…”