Comparison of functional profiles at human recombinant somatostatin sst₂ receptor: simultaneous determination of intracellular Ca²⁺ and luciferase expression in CHO‐K1 cells

Abstract

1 Somatostatin (somatotropin release inhibiting factor; SRIF) acts via five G protein-coupled receptors (sst 1 -sst 5 ) that modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst 2 receptor stably expressed in CHO-K1 cells in a single experiment using a duplex assay for intracellular calcium and serum response element (SRE)-driven luciferase expression. 2 Intracellular calcium was measured using a fluorometric imaging plate reader II (FLIPR II). SRIF-14 rapidly and transiently increased intracellular calcium with a pEC 50 of 8.7470.03 (n ¼ 52). At 5 h after FLIPR II measurements, luciferase expression was determined. SRIF-14 concentrationdependently increased luciferase expression (pEC 50 ¼ 9.0670.03, n ¼ 52). 3 Natural and synthetic agonist/antagonist ligands for SRIF receptors were tested in the duplex assay. Correlation of agonist potencies and efficacies between the two responses were significant (r 2 ¼ 0.83 and 0.90, pEC 50 and E max , respectively). 4 Pertussis toxin pretreatment reduced SRIF-14/octreotide-mediated intracellular calcium increases by 45-47% and luciferase expression by 95-98%. 5 Thapsigargin pretreatment abolished the SRIF-14/octreotide-mediated intracellular calcium increase but had no effect on luciferase expression. 6 In conclusion, SRIF stimulates an increase in intracellular calcium and SRE-luciferase expression via human sst 2 receptors in CHO-K1 cells.

Inhibitory Effect of Somatostatin-14 on L-Type Voltage-Gated Calcium Channels in Cultured Cone Photoreceptors Requires Intracellular Calcium

“…We found that thapsigargin (10 M), an inhibitor of endoplasmic reticulum Ca 2ϩ ATPase, evoked a robust surge of intracellular Ca 2ϩ followed by its complete depletion, which provided evidence for the existence of intracellular calcium stores in chicken cone photoreceptors. Somatostatin has been shown to induce calcium release from intracellular calcium stores in different cell types such as pituitary cells and neuroblastoma ϫ glioma NG108-15 cells (Akbar et al 1994;Nunn et al 2004;Rhie et al 2003;Romoser et al 2001;Siehler et al 2005;Siffert et al 1995). Here, we showed that the inhibitory effect of SS14 on L-VGCCs was blocked by both the Ca 2ϩ chelator BAPTA-AM and thapsigargin.…”

“…Among the subtypes of SS receptors, sst2 is the most abundant in the chicken retina (Chen et al 2007) and is also expressed in salamander, rat, and human cone photoreceptors (Akopian et al 2000;Grigoryan et al 2003;Helboe et al 1997;Johnson et al 1999;Vasilaki et al 2001). In the retina, sst2 couples to both PTX-sensitive (Akopian et al 2000;Pavan et al 2004;Vasilaki et al 2003) and -insensitive G proteins (Chen et al 2007). The complex intracellular signaling elicited by SS not only relies on different SS receptors coupling to different G proteins, it also depends on the coupling between different SS receptors (Pavan et al 2004).…”

“…In the retina, sst2 couples to both PTX-sensitive (Akopian et al 2000;Pavan et al 2004;Vasilaki et al 2003) and -insensitive G proteins (Chen et al 2007). The complex intracellular signaling elicited by SS not only relies on different SS receptors coupling to different G proteins, it also depends on the coupling between different SS receptors (Pavan et al 2004). Interestingly, in the salamander retina, somatostatin elicits an increase of L-VGCC currents in cone photoreceptors but a decrease in rod photoreceptors (Akopian et al 2000).…”

“…In rod bipolar cell terminals, SS inhibits calcium influx through L-VGCCs (Johnson et al 2001). Interestingly, the content of SS is under circadian control in the rodent retina (Peinado et al 1990), and this rhythm correlates with the activities of ENSLI cells with a high sustained rate of activity in the dark and a low sustained rate of activity in the light (Morgan et al 1994). Hence we postulated that there could be a circadian phase-dependent effect of SS in the modulation of L-VGCCs in retinal photoreceptors.…”

Silencing of MUC8 by siRNA increases P2Y2-induced airway inflammation

“…G q ␣ activity is typically measured by assaying the concentrations of intracellular Ca 2ϩ or inositol 1,4,5-trisphosphate (IP 3 ), which are representative products of functional G q ␣. However, we employed an SRE-luc assay, which has been demonstrated to yield similar results to those obtained with Ca 2ϩ and IP 3 assays (17,20,21,23). After transfection with a construct driving the overexpression of P2Y 2 , cells were treated with either ATP or both ATP and IL-1␣.…”