The Effect of Inhalational Anaesthetics on the Swimming Velocity of Tetrahymena Pyriformis

0 Division of Computing and Statistics, Clinical Research Centre , Watford Road, Harrow, Middlesex HAi 2UJ, England 1 Division of Cell Pathology 2 Division of Anaesttiesia J. F. NUNN AND JEAN E. STURROCK E.J.WILLS AND JOAN E.RICHMOND AND C. K. McPHERSON The effect of 6 inhalational anaesthetics on speed of swimming (produced by ciliary movement) has been studied in Tetrahymena pyriformh. There was no evidence of stimulation at low dose levels and higher levels caused rapid, reversible, dose-dependent reduction in swimming velocity. The concentrations of anaesthetics which depressed motility by 50 % were of the same order as those required for anaesthesia in mammals, except in the case of cyclopropane, for which the required level was 4 times higher than the anaesthetic level. Correlation with lipid solubility was not as close as is the case for narcotic concentration. Oxygen consumption was reduced with increasing amounts of halothane in parallel with the reduction in swimming velocity. Halothane produced deciliation of Tetrahymena at about 10 times the anaesthetic dose for man; regrowth of cilia took place within 4 h of withdrawal of the drug. There were no changes in the ultrastructure of the cilia, basal bodies and associated microtubular systems at levels of halothane sufficient to stop cilial beat. At higher concentrations deciliation occurred immediately distal to the axosome and there was variable swelling of the mitochondria. THE OF INHALATIONAL OF TETRAHYMENA Inhalational anaesthetics have a wide range of biological actions, many of which do not appear to be concerned with the production of narcosis even though the mechanism of narcosis is still unexplained. Such actions are none the less important because they explain many of the side effects of anaesthetics and provide useful models for the elucidation of the interactions between these chemically inert agents and molecular receptor sites. Amongst the biological activities of anaesthetics may be included inhibition of certain enzymes (Ueda, 1965; White & Dundas, 1970; Brammall, Beard & Hulands, 1973), arrest of mitosis in metaphase (Ostergren, 1944; Nunn, Lovis & ffimball, 1971) and the depolymerization of certain labile microtubular systems (Allison et al. 1970). Depression of ciliary beat has long been recognized as a side effect of anaesthesia (Bernard, 1866; Hill, 1928) but, to the best of our knowledge, quantitative data are not available. For example, we do not know the magnitude of the effect of the partial pressures used in clinical anaesthesia, nor whether the effective pressures for different anaesthetics are related to their lipid solubility, as is the case for the production of narcosis (Miller, Paton & Smith, 1967). Chloral hydrate (Kennedy & Brittingham, 1968) and ethanol (Pitelka & Child, 1964) can produce deciliation in Paramecium caudatum but these drugs clearly show important differences from the inhalational anaesthetics. Quantitative data on the actions of inhalational anaesthetic agents are of particular interest in view of the uncertainty of their mode and molecular site of action. This group of drugs, of which xenon is a typical member, is unlikely to act by hydrogen or covalent bonding, or as a result of biotransformation. The relationship between narcotic potency and lipid solubility suggests action by solution in lipid or by hydrophobic interaction with non-polar areas of proteins (Schoenborn, Watson & Kendrew, 1965). In the studies to be described we have used the swimming speed of the ciliate protozoon Tetrahymena pyriformis to prepare dose-response curves for 6 anaesthetic agents. The effective pressure was then related to lipid solubility. Electron microscopy has been used to search for any morphological basis for the changes observed with the typical anaesthetic halothane. Tetrahymena pyriformis (strain S) was grown in axenic culture in 2 % proteose peptone. Observations of swimming speed were made after growth had proceeded to stationary phase. A hanging drop (containing 50-200 individuals) was suspended from a coverslip in an exposure chamber through which could be passed any required gas mixture. Exposures to 6 anaesthetics were made on 9-14 occasions for each agent at concentrations spanning the expected range for 25-75 % effect. A separate hanging drop was mounted for each experiment and controls were recorded before each treatment. For each anaesthetic the exposures were divided into 2 series run some weeks apart. For all the volatile anaesthetics studied, the dose-effect data could be satisfactorily pooled. Preparation of vapour concentrations Varying concentrations of methoxyflurane (2,2-dichloro-i,i-difiuoroethyl methyl ether), trichloroethylene, chloroform, halothane (2-bromo-2-chJoro-i,i,i-trifluoroethane) and diethyl ether were prepared by dilution of saturated vapour. The carrier gas in all cases was air but the oxygen concentration of the mixture was never less than 16 %. A preliminary study demonstrated that Tetrahymena will swim at normal speeds in a hanging droplet suspended in nitrogen for at least 20 min, and thus should not have been affected by the moderate reductions of oxygen tension encountered in this study. Cyclopropane was dispensed as a gas and mixed with oxygen to obtain the required concentrations. Vapour concentrations were determined by interference refractometry (Hulands & Nunn, 1970). All gas leaving the exposure chamber was passed through the refractometer (Riken Fine Optical Company, Model 27). The concentration of cyclopropane was determined by measuring the oxygen concentration of the mixture with a Servomex paramagnetic oxygen analyser (Model D C L 101). The exposure chamber consisted of a hollow brass cylinder, 3 mm in depth and 17 mm in internal diameter, fixed by epoxy resin to a microscope slide. The top of the chamber was sealed with a coverslip carrying the hanging drop, which had a mean volume of 0-02 ml. A drop of water in the bottom of the chamber maintained humidification. The inlet port was so shaped that the incoming gas caused swirling of the droplet, which assisted in mixing and equilibration. A gas flow of 130-160 ml/min was maintained for 10 min, after which time the slide was rotated and the inlet and outlet tubes quickly interchanged. The gas was now admitted for 5 min at 100 ml/min through the previous exit port, which was baffled, so that the droplet remained stationary during filming. At the conclusion of each run the temperature of the hanging drop was measured with a thermocouple and was found to range from 18 to 22 C. The time course of equilibration in this system was studied with diethyl ether which, because of its high solubility in water, would be expected to be the slowest-acting agent. Measurement of response Swimming velocity was expressed as the mean distance in micrometres travelled by 20 Tetrahymena in 1 s. Movement was recorded by microcinephotography (objective x 2 5 ; eyepiece x 8; 16 fra (...truncated)