pip2 sensor  (Addgene inc)

Journal: Molecular Pharmacology

Article Title: Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor

doi: 10.1124/mol.117.109678

Figure Lengend Snippet: Effect of pilocarpine on PIP2 hydrolysis. Cells were cotransfected with the PIP2 red biosensor and with the plasmids encoding M1R or M3R. Fluorescence was recorded in real time after cell stimulation with pilocarpine (Pilo) or CCh. (A) Representative images of CHO-K1 cells cotransfected with plasmids to overexpress M3R and the PIP2 sensor. The cells were grown on coverslips and imaged under fluorescence microscope in a flow chamber. (B) Traces show the average of fluorescence response from 20 to 30 HEK293T cells; red trace is response of M1R-expressing cells, green trace is M3R-expressing cells, and black is cells expressing only the PIP2 biosensor. Cells were challenged with the flow of solutions of 100 μM Pilo or CCh at the indicated times. (C) PIP2 responses from CHO-K1 cells transfected with M1R (red) or M3R (green). Cells were stimulated with 100 μM pilocarpine, Oxo-M, or CCh. (D) PIP2 sensor fluorescence recorded from M3R-overexpressing HEK293 cells were first challenged with 25 μM CCh in the presence of 300 μM pilocarpine (red and black horizontal bars) and then washed and stimulated again with 25 μM CCh (black bars). (E) Ca2+ responses from HEK293 cells expressing M3R (green) or control plasmid (black). Cells were stimulated with the mixture of pilocarpine and CCh or CCh alone, as in (D).

Article Snippet: We used a protein sensor that increases its fluorescence upon binding of PIP2 (Montana Molecular, Bozeman, MT).

Techniques: Fluorescence, Cell Stimulation, Microscopy, Expressing, Transfection, Control, Plasmid Preparation

Journal: Molecular Pharmacology

Article Title: Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor

doi: 10.1124/mol.117.109678

Figure Lengend Snippet: Ca2+ assay is more sensitive to agonist stimulation than is the PIP2 assay. CHO-K1 cells were transiently transfected with M1R- or M3R-encoding plasmids and analyzed for pilocarpine and CCh-stimulated Ca2+ increase and PIP2 hydrolysis. (A) M1R transfected cells were stimulated with indicated concentrations of CCh (black lines) or pilocarpine (red). Live-cell imaging of free Ca2+ (solid lines) or PIP2 responses (dashed lines) was performed as described in Materials and Methods. Data points denote the maximal amplitude of the response and expressed at the percentage of the maximal response (mean ± S.D.); n = 3 or more. (B) CHO-K1 cells were transfected to overexpress M3R and analyzed as in (A). (C) CHO-K1 cells were transfected with M3R, stimulated with 1 μM pilocarpine in the absence of extracellular Ca2+, and analyzed for their Ca2+ response. Data shown are representative of at least three such experiments done with independent transfections.

Article Snippet: We used a protein sensor that increases its fluorescence upon binding of PIP2 (Montana Molecular, Bozeman, MT).

Techniques: Transfection, Live Cell Imaging

Journal: Molecular Pharmacology

Article Title: Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor

doi: 10.1124/mol.117.109678

Figure Lengend Snippet: Analysis of Ca 2+ and PIP2 responses to CCh and pilocarpine in M1R- and M3R-expressing cells CHO-K1 or HEK293T cells were transfected with either M1R or M3R-expressing plasmids. Nontransfected HEK293 cells were used to study the endogenous M3R present in these cells. The EC 50 and E max were determined for pilocarpine (Pilo) and CCh in two functional assays: free Ca 2+ and PIP2 measurements. The experiments were performed as discussed in the text and in the Materials and Methods . Shown are means ± S.D. for the values determined in three to four independent experiments.

Article Snippet: We used a protein sensor that increases its fluorescence upon binding of PIP2 (Montana Molecular, Bozeman, MT).

Techniques: Transfection, Functional Assay

Journal: Molecular Pharmacology

Article Title: Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor

doi: 10.1124/mol.117.109678

Figure Lengend Snippet: Pilocarpine-stimulated Ca2+ and PIP2 signaling via endogenous and overexpressed M3R in HEK293 cells. HEK293 cells were transfected to overexpress M3R (red) or a control plasmid (lacZ, green), plated on glass coverslips, and stimulated with indicated concentrations of CCh. Ca2+ (solid line, filled circles) or PIP2 (dashed line, empty triangles) was measured in real time using a fluorescence microscope. Data points represent peak amplitude (mean ± S.D., n = 3) measured as average from 20 to 40 cells in a visual field.

Article Snippet: We used a protein sensor that increases its fluorescence upon binding of PIP2 (Montana Molecular, Bozeman, MT).

Techniques: Transfection, Control, Plasmid Preparation, Fluorescence, Microscopy

Journal: Molecular Pharmacology

Article Title: Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor

doi: 10.1124/mol.117.109678

Figure Lengend Snippet: Oxotremorine and cevimeline do not induce PIP2 hydrolysis via overexpressed M3R. (A) CHO-K1 cells were transfected to overexpress M3R and the PIP2 reporter, plated on glass coverslips, and stimulated with 100 μM of the indicated drugs. PIP2 responses (green lines) were measured in real time using a fluorescence microscope. The traces show an average of three experiments recording fluorescence from 20 to 30 cells for each compound. (B) Structures of the tested muscarinic agonists. ACh, acetylcholine; Pilo, pilocarpine; Oxo, oxotremorine.

Article Snippet: We used a protein sensor that increases its fluorescence upon binding of PIP2 (Montana Molecular, Bozeman, MT).

Techniques: Transfection, Fluorescence, Microscopy