Labeled peptides
SP labeled with Alexa 488 (S-13426), BODIPY Fl (S-13425), fluorescein (S-13424), Oregon Green 488 (S-13427) or tetramethylrhodamine (S-13428) on Lys3 were purchased from Molecular Probes (Eugene, OR), product numbers in parenthesis. The chemical formulae for the labeled compounds are shown in Fig. 1.
Spectra
Fluorophore spectral analysis was performed on a Perkin Elmer 512 Spectrofluorometer (Foster City, CA). Alexa 488-SP, BODIPY Fl-SP, fluorescein-SP, Oregon Green 488-SP and tetramethylrhodamine-SP were analyzed at a concentration of 10 nM, using PBS, pH 7.4, as a solvent.
Epifluorescence microscopy
CHO cells stably transfected with the cDNA of the rSPR were obtained from Dr. James Krause and maintained as previously described [18]. Transfected CHO cells were cultured overnight on 3-well HTC super cured glass slides (Cel-Line Associates, Inc., Newfield, NJ). The following day, the slides were rinsed with PBS. Two of the three wells from each slide were stained with 100 nM of one of the SP conjugates. The third well of each slide served as a negative control, in that the cells were incubated with PBS in the absence of a SP analog. Slides were incubated for 2 hrs at 4°C, to prevent internalization of the receptor and thus provide membrane staining. The cells were then rinsed and fixed with paraformaldehyde (2 %) (Fisher Scientific, Pittsburgh, PA) at 4°C for 20 min. Slides were viewed under a Nikon Eclipse E600FN fluorescent microscope (Fryer Company, Inc., Cincinnati, OH). The green fluorophores were excited using a FITC (fluorescein isothiocyanate) filter set (excitation 465-495 nm, dichroic 505LP, emission 515-555 nm). The red fluorophore was excited using a TRITC (tetramethylrhodamine isothiocyanate) filter set (excitation 528-553 nm, dichroic 565LP, emission 600-660 nm). Pictures were taken with a SPOT camera (Diagnostic Instruments, Inc., Sterling Heights, MI). All digital images of the green fluorophores were taken at a constant gain (16) and exposure time (0.7 s) to allow for direct comparison between cells and experiments. Pictures of tetramethylrhodamine-SP, the red fluorophore, were taken at a gain of 8 and a 0.4 s exposure time.
The specificity of the fluorescent staining to the rSPR was analyzed in two ways. First, incubating untransfected CHO cells with 100 nM of each analog assessed non-specific staining. Secondly, staining of rSPR-transfected CHO cells was performed in the presence of an excess of unlabeled SP. Transfected cells were pretreated with 1 μM unlabeled SP for 1 hr at 4°C. Then, the cells were rinsed and incubated with 1 μM unlabeled SP and 100 nM labeled SP for 1 hr at 4°C. The cells were rinsed, fixed and viewed as described above.
Image Analysis
Epifluorescence images from the wells containing only conjugated SP (100 nM) were analyzed using Scion Image (Scion Corporation, Frederick, MD). For each fluorophore, except tetramethylrhodamine, a line was drawn around the outside of the cell membrane of 10 cells (5 cells/experiment) to create a boundary; the area and pixel intensity were then measured within each boundary. Background, as determined by measuring the intensity and area of 10 control cells incubated with PBS in the absence of a fluorophore, was subtracted. Cell area was used to normalize between cells, thus the ratio of intensity to area was calculated for the treated and untreated cells.
Receptor binding
Radioligand competition binding assays utilizing CHO cells stably expressing the rSPR were performed with Bolton-Hunter [125I] SP (NEN, Boston, MA) at a concentration of 50 pM. Fluorescein-SP, Alexa 488-SP, BODIPY Fl-SP, fluorescein-SP, Oregon Green 488-SP or tetramethylrhodamine-SP was added at increasing concentrations, from 100 pM to 1 μM, to compete with the [125I] SP. In order to reach equilibrium, rSPR-expressing CHO cells (100,000 cells/well) were incubated in a pre-wetted Millipore Multiscreen 96 well BV filtration plate (France) with both radiolabeled and fluorescently labeled SP at 4°C for 2 hrs. Peptides and cells were prepared in Tris Buffered Saline Binding Buffer: 50 mM Tris, 120 mM NaCl, 0.2 mg/ml bacitracin, 20 μg/ml leupeptin, 20 μg/ml chymostatin, 0.1 % bovine serum albumin, pH 7.4. Following incubation, cells were filtered and washed using a Millipore vacuum manifold (France). Filters were punched and analyzed using a Packard Cobra II series auto-gamma counter (Meriden, CT) to determine counts per minute (cpm).
Non-specific binding was specified as the counts obtained with 1 μM unlabeled SP in the presence of radiolabeled SP. Statistical analysis showed that there was not a significant difference between the fluorescent conjugates and unlabeled SP when 1 μM of SP or its derivatives was present with 50 pM [125I] SP. In each experiment, specific binding was determined by subtracting nonspecific binding from the original cpm measurements. The data were normalized by calculating B/Bo for each fluorophore, where B is the cpm of [125I] SP specifically bound in the presence of non-radioactive SP and Bo is the cpm of [125I] SP specifically bound in the presence of 100 pM fluorescently labeled SP. The data were plotted as a function of competitor concentration vs. B/Bo. Sigmoidal (Hill, 3 parameter) regression was then performed on the data to determine the IC50, the concentration of fluorescent analog required to inhibit the binding of radiolabeled SP by 50 %, for each SP analog.
Calcium measurements
Dose-response curves for the ability of the SP analogs to produce Ca++elevations upon binding to the SPR were obtained as previously described [5]. Briefly, transfected CHO cells expressing the rSPR were cultured onto coverslips until ∼ 90% confluent. The coverslips were incubated in a Ca++-free solution containing fura-PE3 (AM) and pluronic acid (Texas Fluorescence Labs, Austin, TX) for 30 min., washed, then incubated in a Ca++-containing solution for 30 min. A CAF-110 Intracellular Ion Analyzer (Jasco Corp., Tokyo, Japan) was used to measure the fura- PE3 fluorescence emission. Cytosolic Ca++ concentrations were measured by taking the ratio of fluorescence emission at 510 nm by excitation at 340 and 380 nm. Dose-response curves were obtained by measuring the Ca++ responses produced by adding various concentrations (1 pM, 100 nM, 1 nM and 10 nM) of the labeled probes. Ionomycin (Sigma, St. Louis, MO) was added at a concentration of 10 μM at the end of each experiment to obtain the maximal Ca++ elevation for that coverslip. Ca++ responses were measured by subtracting the peak of each response from the baseline. Responses are expressed as a fraction of the ionomycin (maximum) response.
Two experiments were carried out to serve as negative controls. In the first control experiment, untransfected CHO cells were utilized to determine if any of the SP conjugates were eliciting Ca++ responses through a pathway independent of rSPR activation. Each of the SP isoforms was added to untransfected CHO cells at a concentration of 10 nM and the signals, if any, were recorded. If a SP conjugate caused an increase in Ca++ at 10 nM, then 0.1 and 1 nM concentrations for that fluorophore were also tested.
The second control experiment measured fluorescence produced by the SP analogs themselves, in the absence of cells. Each of the SP fluorophores was added to the Ca++ solution in the absence of cells at a concentration of 10 nM and increases in fluorescence, if any, were recorded. If a response was observed at a 10 nM concentration, then 0.1 and 1 nM concentrations were also tested for that SP analog. If a SP analog produced a Ca++ elevation in either control assay, then the average (n of 3) for the differences between the baseline and peak Ca++ signals for that concentration was calculated. The average was then subtracted from the Ca++ responses observed when that SP analog was added to transfected and untransfected CHO cells at that concentration.
Electrophysiology
Single neurons were dissociated from bullfrog sympathetic ganglia as described previously [19]. Briefly, ganglia were dissected, treated with enzymes and then stored in growth medium at 4°C for 1 to 3 days. The ganglia were triturated to release single neurons for daily use.
Whole cell recordings of isolated neurons were conducted at room temperature, ∼ 21°C. Drugs were applied by single cell superfusion [20] and the bath constantly perfused separately with extracellular solution. Whole cell recordings were made with electrodes with resistances of 0.25-1 MΩ when filled with intracellular solution.
The compositions of solutions for electrophysiology are shown in mM unless otherwise noted. Intracellular (or recording electrode) solution: KCl 120, MgCl2 2, HEPES 10, K4BAPTA 1, ATP 1.15, GTP 0.4, pH 6.8 (with KOH). Extracellular solution: NaCl 118, KCl 2.4, CaCl2 1.8, MgCl2 1.8, sodium pyruvate 5, glucose 5, HEPES 10, TTX 0.0003, pH 7.4 (with NaOH). Growth medium: NaCl 118, KCl 2.4, creatine 5.7, glucose 5, sodium pyruvate 5, 100× MEM vitamins 10 ml/L, penicillin 100 U/ml, streptomycin 100 μg/ml, 50× MEM essential amino acids 20 ml/L, 100× MEM non-essential amino acids 10 ml/L, HEPES 20, pH 7.4 (with NaOH).
IM was monitored by 500 ms pulses from a holding potential of -30 mV to -50 mV every 8 s. The recordings were filtered at 1 kHz and stored on magnetic tape. The IM relaxations were sampled on line at 2.4 kHz. IM was measured as the amplitude of the current tail following a voltage step from -50 mV to -30 mV [21]. To quantify the inhibition of IM, the current before drug and at the maximum inhibition during each response were measured and the percentage of inhibition of IM was calculated.
Statistical Analysis
The percent of ionomycin data from the Ca++ elevation experiments and the B/B0 data from the binding experiments were both subjected to two-way ANOVA analysis, with the independent variables being fluorescent conjugate and dose. One-way ANOVA analysis was used to describe the intensity to area measurements from the fluorescent images, except tetramethylrhodamine. These data, like the data obtained from the binding assay and Ca++ analysis, were analyzed for variance followed by the Student-Newman-Keuls test.
