PubMed · 7260756
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B A Lowden. 1980-08-09. Error in glaucoma therapy.. https://pubmed.ncbi.nlm.nih.gov/7260756/
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The difference between infrared spectra of the nicotinic acetylcholine receptor (nAChR) recorded using the attenuated total reflectance technique in the presence and absence of carbamylcholine exhibits a complex pattern of positive and negative bands that provides a spectral map of the structural changes that occur in the nAChR upon agonist binding and subsequent desensitization. Two relatively intense bands are observed in the amide I region of the difference spectra recorded in 1H2O buffer near 1655 cm(-1) and 1620 cm(-1) that were previously interpreted in terms of either a net conversion of beta-sheet to alpha-helix or a reorientation of transmembrane alpha-helix accompanied by a change in structure of beta-sheet and/or turn [Baenziger, J. E., Miller, K. W., & Rothschild, K. J. (1993) Biochemistry 32, 5448-5454]. However, difference spectra recorded in 2H2O buffer reveal that these and other difference bands in the amide I region undergo downshifts in frequency upon peptide 1H/2H exchange that are much larger than the downshifts in frequency that are typically observed for the amide I vibrations of either alpha-helix or beta-sheet. Difference spectra recorded in 2H2O buffer within either minutes or hours of prior exposure of the nAChR to 2H2O exhibit the same amide I difference band shifts that are observed in difference spectra recorded after 3 days prior exposure of the nAChR to 2H2O. Most of the peptides that are involved in both ligand binding and the resting to desensitized conformational change and that give rise to bands in the difference spectra therefore exchange their hydrogens for deuterium on the seconds to minutes time scale. The frequencies of the difference bands, the magnitudes of the difference band shifts upon peptide 1H/2H exchange, and the rapidity of the hydrogen deuterium exchange kinetics of those structures that give rise to amide I bands in the difference spectra all suggest that the formation of a channel-inactive desensitized state results predominantly from a conformational change in solvent-accessible extramembranous regions of the polypeptide backbone as opposed to a large structural perturbation near the ion channel gate. A conformational change in the agonist binding site may be primarily responsible for channel inactivation upon desensitization.
We report that upon muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells, the extent of phosphoinositide-derived diacylglycerol (DG) conversion to phosphatidic acid (PA), operated by a DG kinase, is dependent on the potency of receptor stimulation and correlates with the reduction of phosphatidylinositol 4,5-bisphosphate mass. Evidence is provided that agonist-evoked Ca2+ mobilisation or protein kinase activation are not key events in triggering receptor-generated DG conversion to PA; furthermore, the phenomenon is compartmentalized, namely it occurs within a topologically restricted area that is poorly accessible to DG artificially generated by cell treatment with bacterial phosphatidylinositol-specific phospholipase C. Possible mechanisms driving regulation of the DG kinase operating in the transduction system investigated are discussed.
Oxidative stress appears to contribute to neuronal dysfunction in a number of neurodegenerative conditions, notably including Alzheimer's disease, in which cholinergic receptor-linked signal transduction activity is severely impaired. To test whether oxidative stress could contribute to deficits in cholinergic signaling, responses to carbachol were measured in human neuroblastoma SH-SY5Y cells exposed to H2O2. DNA binding activities of two transcription factors that are respondent to oxidative conditions, AP-1 and NF kappa B, were measured in nuclear extracts. H2O2 and carbachol individually induced dose- and time-dependent increases in AP-1 and NF kappa B. In contrast, when given together, H2O2 concentration dependently (30-300 microM) inhibited the increase after carbachol in AP-1. Carbachol's stimulation of NF kappa B was not inhibited except with a high concentration (300 microM) of H2O2, which was associated with impaired activation of protein kinase C. Lower concentrations of H2O2 (30-300 microM) inhibited carbachol-induced [3H]phosphoinositide hydrolysis, and this inhibition correlated (r = 0.95) with the inhibition of carbachol-induced AP-1. Activation [3H]phosphoinositide hydrolysis by the calcium ionophore ionomycin was unaffected by H2O2, indicating that phospholipase C and phosphoinositides were impervious to this treatment. In contrast, activation with NaF of G-proteins coupled to phospholipase C was concentration dependently inhibited by H2O2, indicating impaired G-protein function. These effects of H2O2 are similar to signaling impairments reported in Alzheimer's disease brain, which involve deficits in receptor- and G-protein-stimulated phosphoinositide hydrolysis, but not phospholipase C activity. Thus, these findings indicate that oxidative stress may contribute to impaired phosphoinositide signaling in neurological disorders in which oxidative stress occurs, and that oxidative stress can differentially influence transcription factors activated by cholinergic stimulation.