Fast and slow interactions of n-alkanols with human 5-HT3A receptors: Implications for anesthetic mechanisms.

This is part of a continuing patch-clamp study exploring molecular actions of anesthetics and systematically varied related substances on 5-HT3A receptors as prototypes of ligand-gated ion channels. Specifically, n-alkanols, related to but simpler in structure than propofol, were studied to explore the complex actions of this leading intravenous anesthetic. Outside-out patches excised from HEK 293 cells heterologously expressing human 5-HT3A receptors were superfused with even-numbered n-alkanols (ethanol through n-tetradecanol) of different concentrations. Fast solution exchange for varying durations allowed separation of drug actions by their kinetics. Compared with propofol the electrophysiological responses to n-alkanols were not much simpler. n-Alkanols produced fast and slow inhibition or potentiation of current amplitudes, and acceleration of current rise and decay time constants, depending on exposure time, concentration, and chain-length of the drug. Inhibition dominated, characterized by fast and slow processes with time constants separated by two orders of magnitude which were similar for different n-alkanols and for propofol. Absolute interaction energies for ethanol to n-dodecanol (relative to xenon) ranged from -10.8 to -37.3kJmol(-1). No two n-alkanols act completely alike. Potency increases with chain length (until cutoff) mainly because of methylene groups interacting with protein sites rather than because of their tendency to escape from the aqueous phase. Similar wash-in time constants for n-alkanols and propofol suggest similar mechanisms, dominated by the kinetics of conformational state changes rather than by binding reactions.

Which agonist properties are important for the activation of 5-HT3A receptors?

PURPOSE: Why do anesthetics not activate excitatory ligand-gated ion channels such as 5-HT3 receptors in contrast to inhibitory ligand-gated ion channels? This study examines the actions of structural closely-related 5-HT derivatives and 5-HT constituent parts on 5-HT3A receptors with the aim of finding simpler if not minimal agonists and thus determining requirements for successful agonist action. EXPERIMENTAL APPROACH: Responses to 5-HT derivatives of human 5-HT3A receptors stably expressed in HEK 293 cells have been examined with the patch-clamp technique in the outside-out configuration combined with a fast solution exchange system. RESULTS: Phenol, pyrrole and alkyl amines, constituents of 5-HT, even at high concentrations, cannot activate 5-HT3A receptors but they can inhibit them. To date, tyramines are the smallest known agonists. However, an aromatic ring is not required for activation as acetylcholine is also an agonist of similar strength. CONCLUSION: Simultaneous interactions of adequate strength at two separate subsites within the 5-HT binding domain appear to be essential for successful agonist function. Anesthetics either fail to achieve this or the activation they produce is so weak that it is masked by a comparatively very strong inhibition.

Nuclear localization of insulin-like growth factor binding protein 3 in a lung cancer cell line.

Considerable evidence exists that lung cancer cell lines produce large amounts of insulin-like growth factor-binding proteins (IGFBPs). In addition, these cells are subject to an autocrine or paracrine growth control by insulin-like growth factors (IGFs). We now demonstrate by immunocytochemistry with IGFBP-3 antibodies that nuclei of a lung cancer cell line distinctly immunostain for IGFBP-3. This finding led us to investigate in more detail the localization of this protein that, to date, had only been known to occur extracellularly. Ligand blotting revealed that purified nuclear extracts contain a 43,000-Da IGFBP which can bind [I125]IGF-I. By Western blot this protein was identified as IGFBP-3. Thus, our data are consistent with the results of a previous structural study predicting a nuclear localization for IGFBP-3. Moreover, our findings raise the possibility that nuclear IGFBP-3 is functional and involved in the pathogenesis of lung cancer.