Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation

Spectra properties of fluorescent analogs

The fluorescent properties of a fluorophore can change when the fluorophore is attached to another molecule, in this case a peptide. Furthermore, the conditions under which fluorescence is measured, such as pH, ionic strength and buffer composition, can affect fluorescence. The absorption and emission spectra of the fluorescent SP analogs were assessed to determine their fluorescence under experimental conditions used here. As shown in Fig. 2, the peak excitations of Alexa 488-SP, BODIPY Fl-SP, fluorescein-SP, Oregon Green 488-SP and tetramethylrhodamine-SP were 490 nm, 505 nm, 495 nm, 495 nm and 560 nm, respectively. The peaks of the emission curves were 515 nm, 510 nm, 520 nm, 520 nm and 575 nm, respectively.

Epifluorescence microscopy

Direct-labeling of the SPR with fluorescent ligands like those analyzed in this study would offer advantages over current methods of receptor labeling. Some of these advantages include the ability to label live cells and the ability to conduct receptor labeling without antibodies or radioactive ligands. Therefore, an important characteristic of the fluorescent SP analogs is their ability to bind to the SPR and to fluoresce under physiological conditions. In order to observe the direct-labeling of the SP conjugates to the SPR, CHO cells expressing the rat SPR (rSPR), were incubated with the SP analogs (100 nM) for 2 hrs at 4°C, to prevent internalization of the receptor, rinsed and fixed. Under these conditions, all of the labeled forms of SP, except for Alexa 488-SP, produced bright membrane staining (Fig. 3). Observation of transfected cells incubated with phosphate buffered saline (PBS) in the absence of labeled SP conjugate showed that the transfected cells had some intracellular auto-fluorescence (Fig. 3, F). The fluorophore-treated cells also show some dull intracellular fluorescence, similar to the auto-fluorescence seen in the unstained control cells. The intensity of the staining produced by the green fluorophores was compared by quantitative analysis. The intensities of the fluorescence after staining with BODIPY Fl-SP, fluorescein-SP and Oregon Green 488-SP were significantly greater (p < 0.0001) than the intensity of the fluorescence of cells treated with Alexa 488-SP. Tetramethylrhodamine-SP also produced intense membrane staining. The intensity of the staining with tetramethylrhodamine-SP could not be directly compared to the others due to its different excitation and emission wavelengths.

The specificity of the fluorescence staining was revealed by the finding that unlabeled SP effectively prevented the fluorescent staining of rSPR-transfected CHO cells. The fluorescence of these cells was similar to that of transfected cells incubated in PBS alone (Fig. 3, F) in that they possessed some intracellular auto-fluorescence but lacked membrane staining.

Application of the labeled analogs to untransfected CHO cells did not produce membrane staining (data not shown). Only intracellular auto-fluorescence, comparable to that observed with transfected cells incubated with PBS in the absence of labeled SP (Fig. 3, F), was seen in cells lacking the rSPR.

SP receptor binding

An important determinant of the usefulness of the SP conjugates is their ability to bind to and activate the SPR. Therefore, the ability of each of the fluorescent SP analogs to compete with [¹²⁵I] SP binding to the SPR was assessed using a heterologous expression system, rSPR-expressing CHO cells. The results are shown in Fig. 4. The concentration of fluorescent analog required to inhibit the binding of radiolabeled SP by 50 % (IC₅₀) was calculated from the binding studies. The rank order of potency for the competing peptides was: unlabeled SP (2.0 nM) > tetramethylrhodamine-SP (4.2 nM) > Oregon Green 488-SP (6.4 nM) > BODIPY-SP (18.0 nM) > fluorescein-SP (44.5 nM) >>> Alexa 488-SP. Alexa 488-SP (Fig. 4, A) did not compete with [¹²⁵I] SP for binding at the rSPR, therefore an IC₅₀ value could not be determined.

Two-way analysis of variance followed by Student-Newman-Keuls test showed that there was a dose-dependent effect (p < 0.001) on the binding of unlabeled SP and the SP analogs to the rSPR. Statistical analysis also demonstrated that the competition binding curves generated by the SP conjugates were significantly different than the unlabeled SP binding curve (p < 0.001). Further analysis showed that, with the exception of the binding curves produced by tetramethylrhodamine-SP and Oregon Green 488-SP, a significant difference was seen between the binding curves of the different SP conjugates (p < 0.05).

Calcium measurements

Activation of the SPR expressed in CHO cells results in an elevation in intracellular Ca⁺⁺ [7]. The biological activity of the SP conjugates was tested by examining their ability to elicit Ca⁺⁺ elevations. Dose-response curves for the labeled SP analogs and for unlabeled SP to produce an increase in intracellular Ca⁺⁺ in CHO cells transfected with the rSPR are shown in Fig. 5.

None of the labeled SP analogs produced Ca⁺⁺ elevations (n = 3) when added to untransfected CHO cells at a concentration of 10 nM (data not shown). When the fluorescent derivatives were added at a concentration of 100 nM they produced an increase in the fluorescence signal due to the inherent fluorescence of the probes. This could also be seen in the absence of cells. Due to this interference, the activity of the fluorescent probes to produce Ca⁺⁺ elevations could not be tested at concentrations higher than 10 nM.

Alexa 488-SP produced the most prominent increase in non-specific fluorescence in the absence of cells. When 1 nM or 10 nM Alexa 488-SP was added the fluorophore produced signals similar in intensity to those observed when this analog was added to transfected or untransfected CHO cells (data not shown). To determine if Alexa 488-SP could specifically produce a receptor-mediated Ca⁺⁺ response, the average of the fluorescence signal produced by Alexa 488-SP in the absence of cells was subtracted from the fluorescent signal obtained when Alexa 488-SP was added to SPR-expressing CHO cells. The results showed that Alexa 488-SP did not activate the SPR to produce a Ca⁺⁺ response. However, the other four SP analogs were able to elicit Ca⁺⁺ responses in rSPR-expressing CHO cells.

Statistical analysis, two-way analysis of variance followed by Student-Newman-Keuls test, demonstrated that unlabeled SP and the SP analogs, except Alexa 488-SP, elicited dose-dependent Ca⁺⁺ elevations (p < 0.001). Analysis also showed that there was not a significant difference (p > 0.05) in Ca⁺⁺ elevations generated by unlabeled SP or the various SP conjugates, with the exception of Alexa 488-SP which did not elicit a calcium response.

Effects of SP analogs on single neurons

The data thus far show that the labeled forms of SP, with the exception of Alexa 488-SP, can bind to and activate the receptor in a heterologous expression system. It is also of importance to determine the activity of the labeled ligands in a cell that endogenously expresses the SPR. The principal neurons of bullfrog sympathetic ganglia express a SPR that has been well characterized with regard to its structure and function. The bullfrog sympathetic ganglia SPR (bfSPR) has 69% homology to the rSPR at the amino acid level. Perhaps most importantly, nine of the ten residues that have been demonstrated to be important for SP binding to mammalian receptors are conserved in the bullfrog receptor. The effects of SP and other tachykinins at the bfSPR have been previously well characterized [8, 9, 5]. The primary biological response following activation of the ganglionic SPR is inhibition of the M-type K⁺ current, I_M.

SP produces a dose-dependent inhibition of I_M [9]. The responses of a given neuron to a given form of labeled SP have been compared to the responses of those same neurons to unlabeled SP at a concentration of 100 nM (Fig. 6). At this concentration SP typically elicits a 70 % depression of I_M. BODIPY Fl-SP and Oregon Green 488-SP produced an inhibition of I_M similar to that of unlabeled SP. Fluorescein-SP was variable in its ability to inhibit the current, being effective on only two of the four cells tested. At a concentration of 100 nM, neither Alexa 488-SP nor tetramethylrhodamine-SP inhibited I_M; thus they were further tested at a concentration of 1 μM. At this concentration tetramethylrhodamine-SP inhibited I_M by 57 ± 33 %, (n = 4), however Alexa 488-SP did not inhibit I_M. Dose-response curves for the analogs that inhibited I_M and for unlabeled SP are shown in the plots in Fig. 6. A dose-dependency was observed for the effects of BODIPY Fl-SP, fluorescein-SP and Oregon Green 488-SP.

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/B_o for each fluorophore, where B is the cpm of [¹²⁵I] SP specifically bound in the presence of non-radioactive SP and B_o 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/B_o. 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).

I_M 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 I_M relaxations were sampled on line at 2.4 kHz. I_M 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 I_M, the current before drug and at the maximum inhibition during each response were measured and the percentage of inhibition of I_M 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.