Research Resource: Real-Time Analysis of Somatostatin and Dopamine Receptor Signaling in Pituitary Cells Using a Fluorescence-Based Membrane Potential Assay

Abstract

Stable somatostatin analogues and dopamine receptor agonists are the mainstay for the pharmacological treatment of functional pituitary adenomas; however, only a few cellular assays have been developed to detect receptor activation of novel compounds without disrupting cells to obtain the second messenger content. Here, we adapted a novel fluorescence-based membrane potential assay to characterize receptor signaling in a time-dependent manner. This minimally invasive technique provides a robust and reliable read-out for ligand-induced receptor activation in permanent and primary pituitary cells. The mouse corticotropic cell line AtT-20 endogenously expresses both the somatostatin receptors 2 (sst2) and 5 (sst5). Exposure of wild-type AtT-20 cells to the sst2- and sst5-selective agonists BIM-23120 and BIM-23268, respectively, promoted a pertussis toxin- and tertiapin-Q-sensitive reduction in fluorescent signal intensity, which is indicative of activation of G protein-coupled inwardly rectifying potassium (GIRK) channels. After heterologous expression, sst1, sst3, and sst4 receptors also coupled to GIRK channels in AtT-20 cells. Similar activation of GIRK channels by dopamine required overexpression of dopamine D2 receptors (D2Rs). Interestingly, the presence of D2Rs in AtT-20 cells strongly facilitated GIRK channel activation elicited by the sst2-D2 chimeric ligand BIM-23A760, suggesting a synergistic action of sst2 and D2Rs. Furthermore, stable somatostatin analogues produced strong responses in primary pituitary cultures from wild-type mice; however, in cultures from sst2 receptor-deficient mice, only pasireotide and somatoprim, but not octreotide, induced a reduction in fluorescent signal intensity, suggesting that octreotide mediates its pharmacological action primarily via the sst2 receptor.

Figure 1.. Somatostatin-induced changes in membrane potential in AtT-20 cells.

A, Fluorescent traces obtained after addition of SS-14 in a concentration range of 0.1pM to 10μM. Data were obtained using FlexStation3 running SoftMax Pro Microplate Data Acquisition and Analysis Software (Molecular Devices) by measuring a 60-second baseline of each well before agonist exposure. Results show a representative 1-day experiment where each curve represents a single well. Data are presented as relative decrease of fluorescence signal normalized to time of agonist exposure. B, Dose-response curve of SS-14 in AtT-20 cells. Dose-response curve was obtained with OriginPro using sigmoideal nonlinear fitting function of 3 independent experiments performed in triplicate (mean ± SD). Vehicle-induced changes in fluorescence signal were subtracted from signals obtained by agonist-containing solutions. Data are expressed as Δ F − F₀ (%).

Figure 2.. Characterization of somatostatin-induced membrane potential changes.

A, AtT-20 cells were either not exposed (−) or exposed (+) to 300-ng/mL PTX for 16 hours and subsequently stimulated with 1μM SS-14. B, AtT-20 cells were either not exposed (−) or exposed (+) to 500nM TPN-Q for 5 minutes and subsequently stimulated with 1μM SS-14 after baseline recording of 60 seconds. Shown are representative results of 3 independent experiments performed in quadruplicate (mean ± SD). Each point represents a relative change of fluorescence signal. Vehicle-induced changes in fluorescence signal (background) were subtracted from agonist-induced changes at a given concentration.

Figure 3.. Analysis of functional ssts in AtT-20 cells.

A, Agarose gel electrophoresis of the PCR products for mouse sst1, sst2, sst3, sst4, and sst5 receptors and GAPDH in AtT-20 cells. Reverse transcriptase was either not added (−), referring to lanes 1, 3, 5, 7, and 9, or added (+), referring to lanes 2, 4, 6, 8, and 10 to RNA isolates before converting into cDNA. The position of the PCR fragment is indicated by the DNA molecular weight marker (100–1000 bp). B, Dose-response curve for BIM-23120 (sst2 selective). AtT-20 cells were exposed to various concentrations of BIM-23120 ranging from 0.1pM to 1μM. Inset, Western blot analysis of sst2 in AtT-20 cells using the anti-sst2 antibody (UMB-1). The position of the molecular mass markers is indicated on the left (in kDa). C, Dose-response curve for BIM-23268 (sst5 selective). AtT-20 cells were exposed to various concentrations of BIM-23268 ranging from 1pM to 5μM. Inset, Western blot analysis of sst5 in AtT-20 cells using the anti-rsst5 antibody (6003). The position of the molecular mass markers is indicated on the left (in kDa). Dose-response curve was obtained with OriginPro using sigmoideal nonlinear fitting function of 4 independent experiments performed in duplicate (mean ± SEM). Vehicle-induced changes in fluorescence signal were subtracted from signals obtained by agonist-containing solutions. Shown are representative results from 1 of 3 independent experiments. Data are expressed as Δ F − F₀ (%).

Figure 4.. Response to sst subtype-selective agonists.

A, Wild-type AtT-20 were treated with BIM-23926 (sst1-selective). B, Wild-type AtT-20 were treated with BIM-23120 (sst2 selective). C, Wild-type AtT-20 were treated with L-796/778 (sst3 selective). D, AtT-20 cells stably transfected with hsst1 were treated with BIM-23926. E, AtT-20 cells stably transfected with hsst2 were treated with BIM-23120. F, AtT-20 cells stably transfected with hsst3 were treated with L-796/778. G, Wild-type AtT-20 cells were treated with L-803/087 (sst4 selective). H, Wild-type AtT-20 cells were treated with BIM-23268 (sst5 selective). I, Wild-type AtT-20 cells were treated with dopamine. J, AtT-20 cells stably transfected with hsst4 were treated with L-803/087. K, AtT-20 cells stably transfected with hsst5 were treated with BIM-23268 (sst5 selective). L, AtT-20 cells stably transfected with D2R were treated with dopamine. All agonist exposures were performed at a concentration of 1μM. Average responses of 5 independent experiments performed in triplicate (n = 5). Data of relative fluorescence signals shown were obtained by subtraction of vehicle-induced fluorescence changes from solutions containing agonists and expressed as F/F₀.

Figure 5.. Block with sst2-selective antagonist BIM-23627.

Primary agonist exposure was performed after recording baseline for 60 seconds. After 180 seconds, a secondary exposure was performed containing the sst2-selective antagonist BIM-23627 in a concentration of 10μM. Yielding a final molar agonist to antagonist ratio of 1:1. A, BIM-23120 (sst2-selective agonist). B, octreotide. C, somatoprim. D, BIM-23268 (sst5-selective agonist). E, pasireotide. F, SS-14. Shown are representative curves from 1 of 4 independent experiments performed in triplicate. Data for relative fluorescence signals shown were obtained by subtraction of vehicle-induced fluorescence changes from solutions containing agonist/antagonist and expressed as F/F₀.

Figure 6.. Activity of the sst2–D2R chimeric compound, BIM-23A760, in the presence and absence of D2R.

A, Wild-type AtT-20 cells were initially stimulated with 10μM BIM-23A760. In a secondary experiment, 10μM BIM-23627 was added to yield a 1 μM to 1 μM (1:1) ratio of agonist to antagonist. B, AtT-20 cells stably transfected with D2Rs were stimulated in the same manner as reported in A. C, AtT-20 cells either expressing D2Rs or not were exposed to BIM-23A760 in a concentration range of 10⁻⁵ M to 10⁻¹² M to yield a dose-response curve. Fitting was performed using a Levensberg-Marquadt Iteration algorithm and subsequent using a 4-parameter nonlinear regression for fitting concentration-response curves with OriginPro software. Each data point was determined in duplicates on 3 different occasions.

Figure 7.. Primary pituitary culture of wild-type and sst2−/− mice.

A, Membrane potential assay measurements in primary mouse pituitary cultures. Cells were either not exposed (−) or exposed (+) to 300-ng/mL PTX for 16 hours and subsequently stimulated with 10μM SS-14. Each trace represents data from 5 animals (each 3 males and 2 females) (mean ± SEM). Background fluorescence (F₀) of vehicle was subtracted from agonist-induced fluorescence signal (F) (F − F₀). B, left scale bar, Total pituitary cell extracts of wild-type mice were pretreated with 500nM TPN-Q for 5 minutes before agonist exposure with 10μM SS-14. Right scale bar, Total pituitary extracts of wild-type mice were pretreated with (+) 300-ng/mL PTX or left untreated (−) for 16 hours and stimulated with 10μM SS-14. C, Immunohistochemical staining for sst2 in mouse pituitary formalin-fixed and paraffin-embedded pituitary tissue using rabbit monoclonal anti-sst2 antibody UMB-1. D, Mouse pituitary extracts of wild-type (+/+) vs sst2 knockout (−/−) mice were exposed to 10μM solutions of SS-14, octreotide, pasireotide, or somatoprim. Statistical analysis was performed according to Mann-Whitney U test. Data are given as relative changes in fluorescence signal compared with vehicle-induced background signal determined as Δ relative fluorescence unit (RFU) (%).