Laser intensity and wavelength dependence of Rose-Bengal-photosensitized inhibition of red blood cell acetylcholinesterase.

The intensity and wavelength-dependence of Rose-Bengal-mediated photoinhibition of red blood cell acetylcholinesterase has been studied. Irradiation of dye-membrane suspensions with 308 nm laser excitation resulted in enzyme inhibition almost 50% greater than that obtained with 514 nm laser excitation. Sodium azide and argon purging greatly decreased the photosensitized enzyme inhibition at both wavelengths. Although Rose Bengal photosensitized enzyme inhibition more efficiently upon excitation into Sn (308 nm) than into S1 (514 nm), Stern-Volmer analysis of sodium azide quenching data gave similar quenching efficiencies at both wavelengths. Irradiation of dye-membrane suspensions with increasing intensities (Nd:YAG, 532 nm, 40 ps pulse duration) resulted in a decrease in enzyme inhibition. Saturation of the Rose Bengal fluorescence intensity and light transmission occurred with nearly the same intensity-dependence, suggesting that ground-state depletion occurs at the higher intensities. Our results demonstrate that excitation of a sensitizer into higher-lying excited singlet states can result in enhanced sensitizing efficiency. However, attempts to populate such states in Rose Bengal by sequential two-photon absorption using high intensities resulted only in ground-state depletion.

Up-regulation of nicotinic acetylcholine receptors following chronic exposure of rats to mainstream cigarette smoke or alpha 4 beta 2 receptors to nicotine.

Smokers are reported to have a higher density of central nicotinic acetylcholine receptors (nAChRs) that non-smokers at autopsy. Whether this increased receptor density is a response to smoking or a result of genetic variability is not known. While sub-chronic treatment of rats and mice with nicotine results in upregulation of central nAChRs, changes in receptor density in response to cigarette smoke have not been studied previously. In this study, male Sprague-Dawley rats were exposed nose-only for 13 weeks to mainstream cigarette smoke followed by assessment of [3H]nicotine binding in five brain regions of smoke- and sham-exposed animals. In smoke-exposed animals, there was a significant increase in nAChR density in the cortex, striatum, and cerebellum (35, 25, and 31% increases, respectively), while there was no significant change in receptor density in the thalamus and hippocampus. Smoke exposure did not alter markedly the affinity of the receptor for nicotine in these brain regions. Furthermore, up-regulation of nAChRs did not alter the biphasic binding properties by which nicotine binds to its receptor. There were no changes in the association (fast phase) or isomerization (slow phase) rate constants, and the percent contribution of slow and fast phase binding to nAChRs was not altered in the up-regulated receptor population compared with control. Similar results were observed following chronic nicotine exposure of cultured cortical cells from fetal rat brain or cells transfected with the alpha 4 beta 2 nAChR subtype. These results show that the up-regulation following smoke exposure in the rat is phenomenologically similar to that observed in vitro. These data provide preliminary evidence for a relationship between cigarette smoking and nAChR up-regulation in vivo and suggest that similar mechanisms of upregulation may underlie chronic smoke exposure of live animals and nicotine exposure of artificially expressed alpha 4 beta 2 receptors in vitro.

Advances in the use of the fluorescent probe fura-2 for the estimation of intrasynaptosomal calcium.

Fura-2 has been used to measure intracellular Ca2+ with great success in a variety of cell and subcellular preparations, including synaptosomes. There is, however, a great deal of variability in the reported estimates of resting intrasynaptosomal Ca2+ ([Ca2+]i). Fura-2 AM is highly lipophilic and passes readily across the plasma membrane into the cytoplasm, where it is de-esterified and trapped. The lipophilicity of fura-2, however, promotes the formation of micelles in aqueous media, which may impede the passage of the probe across cell membranes. Our results suggest that some of the variability in the reported [Ca2+]i estimates may be related to fura-2 de-esterification and loading efficiencies. The use of the nonionic detergent pluronic F-127 is recommended to prevent the formation of fura-2 micelles. The use of a detergent is not always an acceptable practice, however, especially in studies in which detergent-lipid interactions may influence membrane parameters. We found that fatty acid free bovine serum albumin (BSA) (0.25%) greatly increases the intrasynaptosomal concentration of the probe, resulting in a significant increase in the signal-to-noise (S/N) ratio. The mechanism appears to be independent of effects of BSA on synaptosomal integrity and directly related to the prevention of fura-2 micelle formation, as evidenced by light spectroscopic scattering measurements. Thus, BSA appears to keep the probe in a form that crosses the synaptic plasma membrane more readily. The effectiveness of BSA in improving the loading of fura-2 into synaptosomes was comparable to the detergent pluronic F-127, making it possible to measure [Ca2+]i without compromising membrane integrity.

Membrane potential induced by external electric field pulses can be followed with a potentiometric dye.

A potential-sensitive dye was recently used to measure the spatial variation in the membrane potential induced by an externally applied electric field. In this work, we demonstrate that the time course of these induced potentials can also be followed. Two experimental systems were explored. Dye fluorescence from HeLa cells could be modulated by a train of field pulses; the relative fluorescence change measured with a lock-in amplifier was linear with the field and similar to the fluorescence responses obtained in the static measurements. A model membrane system consisting of a hemispherical bilayer allowed convenient measurement of the dye absorbance change as a function of the bathing solution conductivity. The charging time of the membrane was inversely related to the aqueous conductance as predicted by the theoretical solution to Laplace's equation.

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations.

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.