Light-Enhanced Fluorescence of Multi-Level Cavitands Possessing Pyridazine Upper rim.

Completely different fluorescence behaviour of cavitands based on a same calix[4]resorcinarene compound was observed. While the fluorescence intensity of the parent compound, tetramethyl-cavitand (1) slowly faded as a result of UV-light exposure, the emission of the three-level cavitand with pyridazine moieties at the upper rim (5a) was enhanced by the excitation in the UV-region. The structure of fluorescence emission (characterized by excitation-emission matrices) and the absorption of 5a remained unaltered. The analysis of fluorescence decay curves reveals the presence of two separated components assigned to two individual emitting species. The measured significant increase of the average lifetime and quantum yield is the consequence of the UV-light induced transition between the different states of 5a. These observations can be explained by the structural difference between 5a and 1. As a counterpart of the naked cavitand (1) with methyl substituents at the upper rim only, 5a has three additional moieties benzene, triazole and pyridazine levels. Computational studies proved the existence of two conformational isomers of 5a. Upon ultraviolet light excitation a "dark" to "light" conformational transition occurs between the two isomers. This hypothesis was confirmed by anisotropy decay measurements.

Evidence for the role of lipid rafts and sphingomyelin in Ca2+-gating of Transient Receptor Potential channels in trigeminal sensory neurons and peripheral nerve terminals.

Transient Receptor Potential (TRP) cation channels, such as TRP Vanilloid 1 and TRP Ankyrin repeat domain 1 (TRPV1 and TRPA1) are nocisensors playing important role to signal pain. Two "melastatin" TRP receptors, like TRPM8 and TRPM3 are also expressed in a subgroup of primary sensory neurons. These channels serve as thermosensors with unique thermal sensitivity ranges and are activated also by several exogenous and endogenous chemical ligands inducing conformational changes from various allosteric ("multisteric") sites. We analysed the role of plasma membrane microdomains of lipid rafts on isolated trigeminal (TRG) neurons and TRPV1-expressing CHO cell line by measuring agonist-induced Ca2+ transients with ratiometric technique. Stimulation-evoked calcitonin gene related peptide (CGRP) release from sensory nerve endings of the isolated rat trachea by radioimmunoassay was also measured. Lipid rafts were disrupted by cleaving sphingomyelin (SM) with sphingomyelinase (SMase), cholesterol depletion with methyl ß-cyclodextrin (MCD) and ganglioside breakdown with myriocin. It has been revealed that intracellular Ca2+ increase responses evoked by the TRPV1 agonist capsaicin, the TRPA1 agonsits allyl isothiocyanate (AITC) and formaldehyde as well as the TRPM8 activator icilin were inhibited after SMase, MCD and myriocin incubation but the response to the TRPM3 agonist pregnenolon sulphate was not altered. Extracellular SMase treatment did not influence the thapsigargin-evoked Ca2+-release from intracellular stores. Besides the cell bodies, SMase also inhibited capsaicin- or AITC-evoked CGRP release from peripheral sensory nerve terminals, this provides the first evidence for the importance of lipid raft integrity in TRPV1 and TRPA1 gating on capsaicin-sensitive nerve terminals. SM metabolites, ceramide and sphingosine, did not influence TRPA1 and TRPV1 activation on TRG neurons, TRPV1-expressing CHO cell line, and nerve terminals. We suggest, that the hydrophobic interactions between TRP receptors and membrane lipid raft interfaces modulate the opening properties of these channels and therefore, targeting this interaction might be a promising tool for drug developmental purposes.