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

Abstract

BACKGROUND: Substance P (SP) is a peptide neurotransmitter found in central and peripheral nerves. SP is involved in the control of smooth muscle, inflammation and nociception. The amino acid sequence of SP is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. Five different forms of fluorescently labeled SP have recently been synthesized, in which Alexa 488, BODIPY Fl, fluorescein, Oregon Green 488 or tetramethylrhodamine has been covalently linked to SP at Lys3. Here, these novel analogs are characterized as to their ligand binding, receptor activation and fluorescence labeling properties. RESULTS: Competition binding studies, using radiolabeled [125I] SP, revealed that all of the labeled forms of SP, except for Alexa 488-SP, effectively competed with radiolabeled SP for binding at the rat SP receptor. With the exception of Alexa 488-SP, all of the SP analogs produced Ca++ elevations and fluorescence labeling of the SP receptor expressed in Chinese hamster ovary cells. In SP-responsive neurons, BODIPY Fl-SP and Oregon Green 488-SP were as effective as unlabeled SP in producing a reduction of the M-type K+ current. Fluorescein-SP produced variable results, while tetramethylrhodamine-SP was less potent and Alexa 488-SP was less effective on intact neurons. CONCLUSIONS: The above results show that fluorescent labeling of SP altered the biological activity and the binding properties of the parent peptide. Oregon Green 488 and BODIPY FL-SP are the most useful fluorophores for labeling SP without affecting its biological activity. Given these results, these probes can now be utilized in further investigations of the mechanisms of SPR function, including receptor localization, internalization and recycling.

Figure 1

Fluorescent SP analog structures. A) Alexa 488-SP, B) BODIPY Fl-SP, C) fluorescein-SP, D) Oregon Green 488-SP and E) tetramethylrhodamine-SP. The first 5 amino acids of SP (Arg¹-Pro²-Lys³-Pro⁴-Gln⁵) are shown, with each of the fluorophores conjugated to Lys³. R = Gln⁶-Phe⁷-Phe⁸-Gly⁹-Leu¹⁰-Met¹¹-NH₂. Alexa 488 is the most charged structure, BODIPY Fl is the smallest, and Alexa 488 and tetramethylrhodamine are the largest.

Figure 2

Spectral analysis. The excitation and emission spectra was determined for (A) Alexa 488-SP, (B) BODIPY Fl-SP, (C) fluorescein-SP, (D) Oregon Green 488-SP and (E) tetramethylrhodamine-SP. See results for peak data.

Figure 3

Fluorescence microscopy. CHO cells expressing the rSPR were stained with (A) Alexa 488-SP, (B) BODIPY Fl-SP, (C) fluorescein-SP, (D) Oregon Green 488-SP, (E) tetramethylrhodamine-SP and (F) PBS in the absence of a fluorophore. The intensity to area ratio of Alexa 488-SP was significantly less than the other green fluorophores. However a significant difference was not seen between BODIPY Fl-SP, fluorescein-SP or Oregon Green 488-SP.

Figure 4

Binding of conjugated and unconjugated SP to the rSPR. Competition binding of rSPR transfected CHO cells was performed with 50 pM [¹²⁵I] SP and increasing concentrations (1 pM, 100 pM, 1 nM, 100 nM and 1 μM) of each fluorescent SP analog. The calculated IC₅₀ values for BODIPY Fl-SP, fluorescein-SP, Oregon Green 488-SP, tetramethylrhodamine-SP and unconjugated SP are 18.0 nM, 44.5 nM, 6.39 nM, 4.20 nM and 1.98 nM, respectively. Note that Alexa 488-SP was unable to displace the radioisotope. Each data point represents an n of at least 4 from 2 or 3 separate experiments.

Figure 5

Calcium dose response data for the effects of conjugated and unconjugated SP at the rSPR expressed in CHO cells. Values are the peak Ca⁺⁺ elevation produced by each drug expressed as the % of the ionomycin response for that coverslip. ANOVA showed significant dose-dependency, but no difference between Ca⁺⁺ responses produced by the drugs was shown. Each data point represents the average of at least 5 experiments. Alexa 488-SP is not graphed since it did not elicit specific Ca⁺⁺ responses at the rSPR.

Figure 6

Top panel - Comparison of the effects of conjugated and unconjugated SP on single neurons. The bars show the percent inhibition of I_M produced by labeled or unlabeled SP at a concentration of 100 nM. Both drugs were applied to the same cell. Alexa 488-SP and tetramethylrhodamine-SP did not inhibit I_M, fluorescein-SP only inhibited the current 50% of the time, where as BODIPY Fl-SP and Oregon Green 488- SP produced inhibitions similar to unlabeled SP. Bottom panel - I_M inhibition dose response curve for labeled and unlabeled SP. Data is expressed as % I_M inhibition. Each point represents an average of 4 experiments, except fluorescein-SP, which is only an average of 2 experiments. A dose-dependency was observed for each drug that inhibited the I_M.