The neurokinin A receptor activates calcium and cAMP responses through distinct conformational states.

G protein-coupled receptors are thought to mediate agonist-evoked signal transduction by interconverting between discrete conformational states endowed with different pharmacological and functional properties. In order to address the question of multiple receptor states, we monitored rapid kinetics of fluorescent neurokinin A (NKA) binding to tachykinin NK2 receptors, in parallel with intracellular calcium, using rapid mixing equipment connected to real time fluorescence detection. Cyclic AMP accumulation responses were also monitored. The naturally truncated version of neurokinin A (NKA-(4-10)) binds to the receptor with a single rapid phase and evokes only calcium responses. In contrast, full-length NKA binding exhibits both a rapid phase that correlates with calcium responses and a slow phase that correlates with cAMP accumulation. Furthermore, activators (phorbol esters and forskolin) and inhibitors (Ro 31-8220 and H89) of protein kinase C or A, respectively, exhibit differential effects on NKA binding and associated responses; activated protein kinase C facilitates a switch between calcium and cAMP responses, whereas activation of protein kinase A diminishes cAMP responses. NK2 receptors thus adopt multiple activatable, active, and desensitized conformations with low, intermediate, or high affinities and with distinct signaling specificities.

Fluorescent siderophore-based chemosensors: iron(III) quantitative determinations.

A highly sensitive and selective method is described for a rapid and easy determination of iron(III). This procedure is based on fluorimetric detection combined with the attractive properties of siderophores and biomimetic ligands, which are strong and selective ferric chelators. Azotobactin delta, a bacterial fluorescent siderophore, three fluorescent derivatives of desferriferrioxamine B with a linear structure (NBD-, MA-, NCP-desferriferrioxamine B) and one tripodal biomimetic ligand of desferriferrichrome carrying an anthracenyl fluorescent probe were examined. A very efficient static quenching mechanism by iron was observed for all the ligands considered in this work. Our results identify azotobactin delta as the most promising chemosensor of ferric traces in water, more sensitive than the NBD-desferriferrioxamine B fluorescent ligand. Under more lipophilic conditions, the anthryl-desferriferrichrome biomimetic analogue showed similar analytical potential and was found to be more sensitive than the lipophilic MA- and NCP-desferriferrioxamine B. Their detection limits were respectively 0.5 ng mL-1 for azotobactin delta and 0.6 ng mL-1 for the anthryl tripodal chelator. The calibration curves were linear over the range 0-95 ng mL-1 and 0-180 ng mL-1. Various foreign cations have been examined and only copper(II) and aluminium(III) were shown to interfere when present in similar concentrations as iron(III). The developed procedure using fluorescent siderophores or biomimetic ligands of iron(III) may be applied (1) to monitor iron-(III)-dependent biological systems and (2) to determine iron(III) quantitatively in natural waters and in biological systems.