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

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.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.Substance P (SP) is a peptide neurotransmitter that has been shown to play a role in nociception, smooth muscle control, allergic responses, inflammation and glandular secretion [1]. The amino acid sequence of SP was determined in 1970 [2] after being isolated from mammalian gastrointestinal tract in 1931 [3]. SP acts as an agonist at the SP receptor (SPR), known as the neurokinin-1 receptor (NK1) in mammalian systems. SP activation of the SPR, a G-protein-coupled receptor, has a variety of effects in the nervous system including inhibition of the M-type K+ current (IM) [4]. The mechanistic properties of the SPR have been extensively studied in receptor-expression systems. When transfected into Chinese hamster ovary (CHO) cells, activation of the SPR results in an increase in intracellular Ca++ [5], accumulation of inositol phosphates and cAMP formation [6].The recent developmen