Steady-state catecholamine distribution in chromaffin granule preparations: a test of the pump-leak hypothesis of general anesthesia

The molecular mechanism of general anesthesia is not understood. Possible modes of action include binding at a protein site, such as a receptor or channel, or physical effects on membrane lipid properties. The pump-leak hypothesis suggests that anesthetics perturb the bilayer of synaptic vesicles, thereby increasing ionic permeability. This results in decay of proton gradients required for transport and accumulation of neurotransmitters. The subsequent loss of neurotransmitters from synaptic vesicles reduces the efficiency of synaptic transmission and results in the anesthetized state. We have determined the effects of general anesthetics on certain parameters of enzyme activity and membrane permeability relevant to the pump-leak hypothesis. We used chromaffin granules as a convenient model system and focused on clinically relevant anesthetic concentrations (ED50), quantitative measurements of permeability changes, and the kinetics of gradient decay. General anesthetics at ED50 have little or no effect on the proton-transport ATPase activity, but do cause modest increments in proton permeability that change the catecholamine distribution in actively pumping chromaffin granule preparations. We found that pH gradients do not collapse entirely under these conditions and that only a fraction of total catecholamine is lost from the chromaffin granules. When total collapse is induced by other means, efflux of catecholamines occurs with a half-time near 30 min. These results suggest that if the pump-leak hypothesis is valid, then very small losses of catecholamines must be sufficient to induce anesthesia. We conclude that the weight of evidence favors other mechanisms, notably direct binding of anesthetics to sensitive proteins.     

ACTION OF TRITON X-100 ON CHLOROPLAST MEMBRANES : Mechanisms of Structural and Functional Disruption

Addition of Triton X-100 to chloroplast suspensions to a final concentration of 100–200 µM causes an approximate tripling of chloroplast volume and complete inhibition of light-induced conformational changes, light-dependent hydrogen ion transport, and photophosphorylation. Electron microscopic studies show that chloroplasts treated in this manner manifest extensive swelling in the form of vesicles within their inner membrane structure. Triton was adsorbed to chloroplast membranes in a manner suggesting a partition between the membrane phase and the suspending medium, rather than a strong, irreversible binding. This adsorption results in the production of pores through which ions may freely pass, and it is suggested that the inhibition of conformational changes, hydrogen ion transport, and photophosphorylation by Triton is due to an inability of treated chloroplast membranes to maintain a light-dependent pH gradient. The observed swelling is due to water influx in response to a fixed, osmotically active species within the chloroplasts, after ionic equilibrium has occurred. This is supported by the fact that chloroplasts will shrink upon Triton addition if a nonpenetrating, osmotically active material such as dextran or polyvinylpyrrolidone is present externally in sufficient concentration (>0.1 mM) to offset the osmotic activity of the internal species.     

Endogenously produced CO2 induces stationary phase in tetrahymena cultures

Media concentration of total soluble CO₂ increases with culture age of Tetrahymena pyriformis. CO₂ is a weak acid and is capable of acidifying intracellular pH (pHi). Changes in pHi have been demonstrated to affect cell metabolism and growth in many systems. For these reasons, we investigated whether the concentrations of CO₂ produced in vitro were sufficient to affect cell proliferation and pHi in Tetrahymena. In this study, we used DMO to mimic the weak acid properties of CO₂. DMO is freely permeable to membranes in its uncharged form and has a pKa similar to that of CO₂/HCO. In addition, it has the advantages of being metabolically inert and non-volatile. At concentrations similar to endogenously produced CO₂, DMO acidifies pHi and arrests culture growth. In addition, procedures are described which decrease the media CO₂ concentrations in both growing and non-growing cultures. These conditions lead to increased maximum culture density at stationary phase. The data indicate that, under our conditions, accumulation of CO₂ in the culture leads to cessation of growth, probably through elimination of transmembrane pH gradients, which are necessary for regulation of metabolism and growth.     

15 Combinatorial chemistry in the prebiotic environment

The pathway leading to the origin of life presumably included a process by which polymers were synthesized abiotically from simpler compounds on the early Earth, then encapsulated to form protocells. Previous studies have reported that mineral surfaces can concentrate and organize activated mononucleotides, thereby promoting their polymerization into RNA-like molecules. However, a plausible prebiotic activation mechanism has not been established, and minerals cannot form cellular compartments. We are exploring ways in which nonactivated mononucleotides can undergo polymerization and encapsulation. We found that small yields of RNA-like molecules are synthesized by a condensation reaction when mixtures of amphiphilic lipids and mononucleotides are exposed to cycles of dehydration and rehydration. The lipids concentrate and organize the monomers within multilamellar liquid-crystalline matrices that self-assemble in the dry state. The chemical potential driving the polymerization reaction is supplied by the anhydrous conditions in which water becomes a leaving group, with heat providing activation energy. Significantly, the polymeric products are encapsulated in trillions of microscopic compartments upon rehydration. Each compartment is unique in its composition and contents, and can be considered to be an experiment in a natural version of combinatorial chemistry that would be ubiquitous in the prebiotic environment. A successful experiment would be a compartment that captured polymers capable of catalyzing their own replication. If this can be reproduced in the laboratory, it would represent a significant step toward understanding the origin of cellular life.     

Water transport by the bacterial channel alpha-hemolysin

This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels.     

Nanopores and nucleic acids: prospects for ultrarapid sequencing

DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.