THE EFFECT OF INHALATIONAL ANAESTHETICS ON THE SWIMMING ... · caudatum but these drugs clearly show important differences from the inhalational anaesthetics. Quantitative data on

J. Cell Sci. 15, 537-554 («974) 537Printed in Great Britain

THE EFFECT OF INHALATIONAL

ANAESTHETICS ON THE SWIMMING

VELOCITY OF TETRAHYMENA PYRIFORMIS

J. F. NUNN AND JEAN E. STURROCKDivision of Anaesttiesia,

E.J.WILLS AND JOAN E.RICHMONDDivision of Cell Pathology,

AND C. K. McPHERSONDivision of Computing and Statistics,Clinical Research Centre, Watford Road, Harrow, Middlesex HAi 2UJ, England

SUMMARYThe effect of 6 inhalational anaesthetics on speed of swimming (produced by ciliary move-

ment) has been studied in Tetrahymena pyriformh. There was no evidence of stimulation atlow dose levels and higher levels caused rapid, reversible, dose-dependent reduction inswimming 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 ofcyclopropane, 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 thereduction in swimming velocity. Halothane produced deciliation of Tetrahymena at about10 times the anaesthetic dose for man; regrowth of cilia took place within 4 h of withdrawalof the drug. There were no changes in the ultrastructure of the cilia, basal bodies andassociated microtubular systems at levels of halothane sufficient to stop cilial beat. At higherconcentrations deciliation occurred immediately distal to the axosome and there was variableswelling of the mitochondria.

INTRODUCTION

Inhalational anaesthetics have a wide range of biological actions, many of whichdo not appear to be concerned with the production of narcosis even though themechanism of narcosis is still unexplained. Such actions are none the less importantbecause they explain many of the side effects of anaesthetics and provide usefulmodels for the elucidation of the interactions between these chemically inert agentsand molecular receptor sites.

Amongst the biological activities of anaesthetics may be included inhibition ofcertain 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

538 J. F. Nunn and others

not available. For example, we do not know the magnitude of the effect of the partialpressures used in clinical anaesthesia, nor whether the effective pressures for differentanaesthetics are related to their lipid solubility, as is the case for the productionof narcosis (Miller, Paton & Smith, 1967). Chloral hydrate (Kennedy & Brittingham,1968) and ethanol (Pitelka & Child, 1964) can produce deciliation in Parameciumcaudatum but these drugs clearly show important differences from the inhalationalanaesthetics.

Quantitative data on the actions of inhalational anaesthetic agents are of particularinterest in view of the uncertainty of their mode and molecular site of action. Thisgroup of drugs, of which xenon is a typical member, is unlikely to act by hydrogenor covalent bonding, or as a result of biotransformation. The relationship betweennarcotic potency and lipid solubility suggests action by solution in lipid or byhydrophobic 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 ciliateprotozoon Tetrahymena pyriformis to prepare dose-response curves for 6 anaestheticagents. The effective pressure was then related to lipid solubility. Electron microscopyhas been used to search for any morphological basis for the changes observed withthe typical anaesthetic halothane.

METHODS

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 exposurechamber through which could be passed any required gas mixture. Exposures to 6 anaestheticswere made on 9-14 occasions for each agent at concentrations spanning the expected rangefor 25-75 % effect. A separate hanging drop was mounted for each experiment and controlswere recorded before each treatment. For each anaesthetic the exposures were divided into2 series run some weeks apart. For all the volatile anaesthetics studied, the dose-effect datacould 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 diethylether were prepared by dilution of saturated vapour. The carrier gas in all cases was air butthe oxygen concentration of the mixture was never less than 16 %. A preliminary studydemonstrated that Tetrahymena will swim at normal speeds in a hanging droplet suspendedin nitrogen for at least 20 min, and thus should not have been affected by the moderatereductions of oxygen tension encountered in this study. Cyclopropane was dispensed as a gasand 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 (RikenFine Optical Company, Model 27). The concentration of cyclopropane was determined bymeasuring the oxygen concentration of the mixture with a Servomex paramagnetic oxygenanalyser (Model DCL 101).

The exposure chamber consisted of a hollow brass cylinder, 3 mm in depth and 17 mm ininternal diameter, fixed by epoxy resin to a microscope slide. The top of the chamber wassealed 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

Anaesthetics and swimming velocity of Tetrahymena 539

so shaped that the incoming gas caused swirling of the droplet, which assisted in mixing andequilibration. A gas flow of 130-160 ml/min was maintained for 10 min, after which timethe slide was rotated and the inlet and outlet tubes quickly interchanged. The gas was nowadmitted for 5 min at 100 ml/min through the previous exit port, which was baffled, so thatthe droplet remained stationary during filming. At the conclusion of each run the temperatureof the hanging drop was measured with a thermocouple and was found to range from 18 to22 °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 20Tetrahymena in 1 s. Movement was recorded by microcinephotography (objective x 25; eye-piece x 8; 16 frames per second; bright-field illumination), and was analysed by single-frameprojection in a Specto Analysing projector. Magnification was directly determined by filminga 1-mm stage graticule. Individual Tetrahymena were randomly selected and the distancetravelled in i s was measured; those which did not move at all or moved in circles wereexcluded. The Tetrahymena in each drop served as their own controls, but since individualscould not be recognized there was no method of ensuring that the same creatures weremeasured during test and control periods. They were therefore treated as unpaired groups.

Calculation of drug effect

Drug effect was expressed as the ratio of the mean velocities of treated specimens to themean velocity for the same aliquot immediately before exposure to each concentration of eachanaesthetic. Satisfactorily linear relationships were obtained when this ratio was plotted ona probability scale against log dose (concentration of anaesthetic in the gas phase expressedas percentage of one atmosphere (I-OI x io6 N m~*)). However, an alternative transformationwhich also yields a linear plot is the logit of the response against log dose. This is very similarto the probit transformation but is more convenient for numerical processing of data. In thisapproach, response is expressed as logtp/q where p is the above ratio and q = i-p. Log.p/qwas regressed against log, dose using an ICL 1904 computer by the method of weightedleast squares. Thus the model used to explain the dose/response relationship was:

\og,p/q = a + b log, dose + e,

where a and b are constants to be fitted and e a random error term. Individual points wereweighted by a factor equal to the reciprocal of their variance (Finney, 1971). Since the coefficientof variation of swimming velocity was roughly constant in all experiments, the standard errorof p fell when the effect of the drug was increased. The weighting of points was thereforesubstantially increased at the higher dosage of each agent. The statistical analysis is discussedin detail elsewhere (McPherson, in preparation).

In comparing the response to different anaesthetics, it is useful to derive by interpolationthe concentration required for 50 % effect (p = q). This effective dose for 50 % responsecorresponds to the EDS0 used in quantal probit analysis. It should be stressed that thereis no intrinsic merit in the use of the concentration required for 50 % effect when comparingsensitivity to different agents, and the' ED,6' or' EDM' might be considered equally appropriate.The comparison of agents is valid whichever point is taken, provided that the log/dose-responsecurves are parallel. In the present study it will be seen that this is not always the case, althoughnone of the dose-response curves crosses another within the range of observations.

An automatic graph plotter was used to display for each anaesthetic the individual points,regression lines, and their confidence limits, both on logit/log co-ordinates (linear plots) andon linear co-ordinates (sigmoid curves). Student's t test was used to determine the significanceof the difference between slopes and EDt0 values. A x1 statistic was used to test the goodnessof fit of the model and in all agents this proved satisfactory.


Measurement of oxygen consumption

Oxygen consumption was determined by serial polarographic measurements of oxygentension of aliquots of cultures which had been exposed to various concentrations of halothaneand then sealed in a io-ml glass syringe maintained at 20 °C. For any concentration of halothanethe rate of fall of oxygen tension was constant between 200 and 20 mm Hg (268 and 268 xio3 N m"1). Partial pressures were converted to concentrations on the assumption thatsolubility of oxygen in the medium was the same as for water (0-031 ml/ml of water/atmosphere(I-OI x io6 N m~s)). Cells were killed with formalin and counted in a Sedgewick-Raftercounting chamber; results were calculated as rate of oxygen consumption per cell.

Preparation for electron microscopy

Halothane was vaporized in room air to give a series of concentrations from 10 to 8#o. %,checked by refractometry. The gas stream was then split and bubbled through 2 solutions.The first consisted of Tetrahymena in 2-5 ml culture medium, while the second comprisedthe same volume of 5 % glutaraldehyde in 0-2 M cacodylate buffer (pH 7-4). Both solutionswere at room temperature (20-23 °C). It had previously been demonstrated by ultravioletspectroscopy that water achieves saturation within 75 min of bubbling under similar conditions.Bubbling was therefore allowed to continue for 15 min, after which the 2 solutions wererapidly mixed and the Tetrahymena allowed to settle. It was established by light microscopythat bubbling with air caused no morphological change and did not interfere with swimmingvelocity. Controls treated in this manner were also fixed as described above.

The fixed Tetrahymena were examined by phase-contrast microscopy and also preparedfor electron microscopy as follows. After 1 h the cells were washed thrice in o-i M sodiumcacodylate/025 M sucrose solution, pH 74, postfixed for 1 h in 1 % osmium tetroxide inphosphate buffer (Millonig, 1961), then rinsed with distilled water and embedded in 2 %agar. Cut agar blocks were dehydrated in a graded series of ethanols and embedded inEpikote 812 (Luft, 1961). Thin sections were cut on a Sorvall MT-2 ultramicrotome, stainedwith alcoholic uranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963), andexamined in an AEI EM 6B electron microscope.

RESULTS

Swimming velocity

Exposure to all 6 anaesthetics resulted in a rapid reduction of swimming velocitythroughout the dose range employed for each agent. The rate of onset of action waslargely a function of the thickness of the droplet, and with a thin film was completein as little as 6 s. For a droplet of 0-02 ml, equilibration with diethyl ether, the agentwith the highest water solubility, was complete within 3 min (Fig. 1). Throughoutthe range of concentrations used in clinical practice the effect was rapidly reversedby withdrawal of the agent.

During the first minute of exposure to anaesthetic, Tetrahymena gathered in thecentre of the droplet as though trying to avoid the anaesthetic, which would, at thatstage, show a concentration gradient from the periphery to the centre. When equi-libration was complete, the organisms again became uniformly distributed butswimming was slower and more circuitous. Sometimes all forward movement ceased,but individuals continued to revolve slowly about their long axes. Slow reverseswimming was occasionally seen at higher concentrations of some agents, particularlycyclopropane. At a higher magnification single cilia could be seen moving slowlyand feebly.

Anaesthetics and swimming velocity of Tetrahymena

120 -

° 100

Fig. i. Time course of reduction of swimming velocity on exposure of a hangingdrop (002 ml) to IO % diethyl ether followed by recovery on exposure to air. Barsrepresent 2 standard errors of the means.

80

70

S 60

50

40

30

20

10

I

0-1 0 2 0 30-4 0-6 1 0 2 4 6 10 20 40 60 100

Anaesthetic concentration, % of one atmosphere

Fig. 2. Dose/response curves for 6 anaesthetic agents (log/logit coordinates).


There was no evidence of increased swimming velocity at the lower concentrationsof the agents, and the fastest rate observed was 87 % of control. Results were highlyreproducible for the 2 series of experiments carried out with the 5 volatile anaestheticagents. However, with cyclopropane, the iterative method for determination of theregression line failed to converge on the pooled data but produced satisfactory linesfor the 2 sets of data taken separately. Since there was no obvious reason for therejection of either set, we have included the results derived from both.

Table 1. Mean concentrations of the agents required for 50% reduction ofswimming velocity {expressed as % of atmosphere in the gas phase)

The lower section shows tests of significance of differencebetween values for pairs of agents (t values).

Trichloro-

Concentrationfor 50 % effect

95 % fiduciallimits forabove

ChloroformMethoxy-flurane

HalothaneDiethyl etherCyclopropane

ethylene

0 2 5

O-2O

0 3 0

S-S4II-O4

Chloro-form

0 3 6

0 2 90 4 1

—

5 1 1

18-74

—

Methoxy-flurane

0-47

0-40o-53

——

16-083i-99

—

Halothane

1-07

0-871 2 3

—.—

—

189277-84

Diethylether

4-33

3 1 0

5-34

——

——

40-27

Cyclopropane

Series

r6249

52-1468-57

——

——

Series

rr8063

71-18111-25

—

—

—

—

—

Dose/response curves are shown on log/logit co-ordinates in Fig. 2. Interpolatedvalues for 50% effective doses are shown in Table 1, together with t values for thedifferences in 50% effective doses for pairs of agents for which these values areclosest. The lowest t value is 5-11 and in each case p = o-ooi. The ranking order of50% effective doses therefore has a very high level of probability and the same istrue for the 75 % effective doses.

Deciliation

It was observed that after exposure to very high concentrations of halothane(more than 8-o %) no recovery occurred within the first few minutes of withdrawalof the anaesthetic. However, after 90 min some Tetraliymena were motile and themajority had regained a reasonable velocity within 4 h. Phase-contrast microscopyshowed that deciliation had occurred at concentrations above 8-o% and that thecilia had regrown when swimming eventually recommenced.


Oxygen consumption

Under control conditions, oxygen consumption approximated to 6 x io~9 ml/minper cell. Increasing halothane concentrations up to 4% produced an approximatelylinear decrease in oxygen consumption with 50% reduction occurring at about3 % halothane.

Electron microscopy

Control Tetrahymena and those exposed to halothane at concentrations of up to6-7% had essentially the same appearance (Figs. 4-10). The morphology of thenormal organism has been fully described by Allen (1967) and Hill (1972) and willnot be discussed in detail here. Survey views of control Tetrahymena are illustratedin Figs. 4 and 5. In the halothane-treated organisms, particular attention was paidto the structure of the somatic cilia and the microtubular systems of the kineties.As is apparent from the transverse sections shown in Figs. 6-8, the axonemalstructure of the cilia was unaffected, and there was no swelling. In particular itwas noted that the dynein arms and nexin connexions were all present after treatmentwith concentrations greatly in excess of those required to reduce swimming velocityby 90 %. Basal bodies were also normal and their associated microtubules, togetherwith the longitudinal microtubules, could all be readily identified (Figs. 9, 10).

On the other hand, Tetrahymena exposed to 8-9% halothane (Figs. 11-15) showedseveral abnormalities of which the most striking was the total loss of cilia, somaticand oral, from almost all organisms (Fig. 11). Separation of cilia occurred distal tothe axosome which could be seen above the terminal plate of the basal body in theremaining stump (Fig. 15). Cilia showing partial or complete degeneration of theirfibrils and other components were occasionally encountered (Figs. 13, 14) but in allcases the basal bodies with their associated microtubules and kinetodesmal fibresappeared intact (Fig. 12).

A further change apparent only in Tetraliymena treated with 8-9 % halothane wasvariable swelling of mitochondria. In such cases (Fig. n ) the matrix was ratified,but cristae and surface membranes generally appeared normal.

DISCUSSION

It is clear that the swimming speed of Tetrahymena pyriformis is very sensitiveto the action of inhalational anaesthetics. It is not possible at present to say whetherthe effect is due primarily to depression of rate or amplitude of cilial beat, or whetherit can be explained by lack of coordination of the pattern of beat. The techniqueswhich we have used do not permit visualization of the metachronal waves, andindividual cilia cannot be seen until movement is almost entirely arrested (at about4 times the concentration required for 50% effect). Under the latter circumstances,cilia were seen to be immobile or moving very feebly and slowly. However, at lowerconcentrations of anaesthetics (about twice that required for 50 % effect) there wassome evidence of lack of coordination, since Tetrahymena were occasionally seen

544 3- F- Nunn and otliers

rotating about their long axes without moving forwards or backwards. In all suchcases the rotational velocity was low and of the order of 0-5-1 rev/s.

Table 2 relates the concentration required for 50 % reduction of swimming speedto the mean concentration (in alveolar gas) which will prevent reaction to a painfulstimulus in dogs. There are few biological activities, other than the maintenanceof consciousness itself, which are equally sensitive to the action of anaesthetics. At

Table 2. Comparison of concentrations (as % in gas phase) of agents required foranaesthesia with concentrations required for 50% reduction of swimming velocity

MethoxyfluraneTrichloroethyleneChloroformHalothaneDiethyl etherCyclopropane

• Value interpolated from lipidEger et al. (1969).

Mean alveolarconcentration for

anaesthesiain dogs

0-230-24*0 7 70-873 0 0

17-5

solubility (Steward et al.

Concentrationrequired for 50 %

reduction ofswimming velocity

in Tetrahymena

0-470-250 3 61 07433

7156

1973), remaining values frorr

the concentration required for anaesthesia the only other effects which can beconsistently produced are the arrest of cell division, depolymerization of certainlabile microtubules and depression of bioluminescence. It seems unlikely that suchdiverse reactions are produced by anaesthetics acting on a common target site. Theinhibition of cell division in plants is mainly due to a colchicine-like effect on themitotic spindle (Levan & Ostergren, 1943) and there appears to be a similar actionon the microtubular array in the axonemes of Actinosphaerium nucleofilum (Allisonet al. 1970). The suppression of bacterial luminescence may be due to competitiveinhibition between the anaesthetic and the aliphatic aldehyde which is a substratein the reaction (G. D. Adey, B.Wardley-Smith& D. C. White, unpublished). However,the molecular, or indeed the cellular, target site for narcosis is unknown.

Our studies have not indicated the site of action of anaesthetics on cilial beat.One possibility would be the inhibition of ATP production in the cortical mito-chondria such as occurs in rat liver mitochondria, although at rather higher anaestheticconcentrations (Cohen & Marshall, 1968). However, the rapidity of action ofanaesthetics on cilial beat contrasts with the extreme slowness of response to hypoxiaand furthermore it is known that the enzymes of the Embden—Meyerhof pathwayare highly resistant to anaesthetics (Brammall et al. 1973). In anaesthetized mammalianbrain there is no reduction in concentration of ATP and other high-energy compounds(Nilsson & Seisjo, 1970).

Ion permeability or transport across the ciliary membrane might also be alteredby the action of anaesthetics, but studies of this phenomenon in the erythrocyte


membrane have shown significant changes only in response to concentrations ofanaesthetics about 10 times higher than those required for anaesthesia (Halsey, Smith& Wood, 1970). Furthermore, normal membrane integrity does not seem to beessential for ciliary beat. Provided that ATP is present, motility can be restoredto ciliates whose normal membrane semi-permeability and mechanisms for regulationof ion balance have been destroyed by saponin treatment (Pitelka & Child, 1964).

10000 r

uoo

4000

2000

1000

700

400

2. 200

DO

5- 100o60

40

20

10

Methoxyflurane

Trichloro-- ethylene

Chloroform

Halothane

Diethyrether

Cyclopropane

0-1 0-2 0-3 0-4 0-6 10 2 4 1050% Effective dose, % of one atmosphere

30 60 100

Fig. 3. Relationship between lipid solubility and concentration ofanaesthetic required for 50 % depression of swimming velocity.

It has been suggested that anaesthetics may form weak hydrophobic bonds withcertain proteins (Schoenborn et al. 1965; Balasubramanian & Wetlaufer, 1966).ATPase activity is presumed to be related to mechanochemical function (Gibbons& Rowe, 1965), and is located in the dynein arms (Gibbons, 1963). Thus it is possiblethat anaesthetics might bond directly with dynein, resulting in conformationalchange and loss of ATPase activity. However, no structural changes in dynein wereobserved by electron microscopy except at concentrations which caused degenerationof all cilial components.

Fig. 3 shows the relationship between lipid solubility and the concentrationrequired for production of 50% reduction of swimming velocity. Although there isan approximate overall relationship between potency and lipid solubility, 4 agents

35 C E L 35


deviate from the line of best fit by more than 2 standard errors, and the discrepanciesare highly significant. This is in contrast to narcosis, where the correlation betweenlipid solubility and effective narcotic concentration is extremely close (Eger, Lundgren,Miller & Stevens, 1969). This variation may indicate a difference between themolecular sites of action in the 2 cases.

The dispersion of axonemal microtubules seen in halothane-treated Actinosphaerium(Allison et al. 1970) was not observed here. It is hardly surprising that the transverseand posterior microtubules were unaltered, since these are thought merely to providestructural support for the cilium-basal body complex (see Allen, 1967). However, thebasal microtubules, which are more labile and may be involved in the coordinationof ciliary beat (Allen, 1967), were also apparently unaffected by halothane. Thus itwould appear that the effect of anaesthetics on the structural integrity of microtubulesis a variable one and may be unrelated to the production of narcosis.

Chloral hydrate and ethanol have been employed as deciliating agents in a numberof organisms (see review by Pitelka & Child, 1964). We have found that, with halo-thane, cilia were lost from Tetrahymena only at concentrations about ten times higherthan those required to produce anaesthesia in man. In all cases the site of detachmentwas distal to the axosome. Loss of somatic cilia at the same level in Parameciumcaudatum treated with chloral hydrate was also reported by Kennedy & Brittingham(1968), who suggested the possible importance of the axosome in cilial regrowth.However, the vesicular degeneration of cilial fibres observed by these authors wasnot seen in the present study.

We are indebted to Mr A. J. Jones for skilled technical assistance and Miss B. N. Dobsonfor typing the manuscript.

REFERENCES

AT.TF.NT, R. D. (1967). Fine structure, construction and possible functions of components ofthe cortex of Tetraliymena pyriformis. J. Protozool. 14, 553-565.

ALLISON, A. C , HULANDS, G. H., NUNN, J. F., KITCHING, J. A. & MACDONALD, A. C. (1970).

The effect of inhalational anaesthetics on the microtubular system in Actinosphaeriummicleofilum. J. Cell Sci. 7, 483-499.

BALASUBRAMANIAN, D. & WETLAUFER, D. B. (1966). Reversible alteration of the structure ofglobular proteins by anaesthetic agents. Proc. natn. Acad. Sci. U.S.A. 55, 762-765.

BERNARD, C. (1866). Ltfons sur les Propriitis des Tissus Vivants, p. 136. Paris: Bailliere.BRAMMALL, A., BEARD, D. & HULANDS, G. H. (1973). Effects of inhalational anaesthetic agents

on the enzyme glutamate dehydrogenase. Br. J. Anaesth. 45, 923-924.COHEN, P. J. & MARSHALL, B. E. (1968). Effects of halothane on respiratory control and

oxygen consumption of rat liver mitochondria. In Toxicity of Anaesthetics (ed. B. R. Fink),pp. 24—36. Baltimore: Williams and Wilkins.

EGER, E. I., LUNDGREN, C , MILLER, S. L. & STEVENS, W. C. (1969). Anaesthetic potencies ofsulphur hexafluoride, carbon tetrafluoride, chloroform and Ethrane in dogs. AnestJiesiology30, 120-135-

FINNEY, D. J. (1971). Statistical Method in Biological Assay. London: Griffin.GIBBONS, I. R. (1963). Studies on the protein components of cilia from Tetrahymena pyriformis.

Proc. natn. Acad. Sci. U.S.A. 50, 1002-1010.GIBBONS, I. R. & ROWE, A. J. (1965). Dynein: a protein with adenosine triphosphatase activity

from cilia. Science, N. Y. 149, 424-425.

Anaesthetics and svnmming velocity of Tetrahymena 547

HALSEY, M. J., SMITH, E. B. & WOOD, T. E. (1970). Effects of general anaesthetics on Na+transport in human red cells. Nature, Lond. 225, 1151-1152.

HILL, D. L. (1972). The Biochemistry and Physiology of Tetrahymena, pp. 3-12. New York:Academic Press.

HILL , L. (1928). The ciliary movement of the trachea studied in vitro: a measure of toxicity.Lancet 215, 802-805.

HULANDS, G. H. & NUNN, J. F. (1970). Portable interference refractometers in anaesthesia.Brit. J. Anaesth. 42, 1051-1059.

KENNEDY, J. R. & BRITTINCHAM, E. (1968). Fine structure changes during chloral hydratedeciliation of Paramecium caudatum. J. Ultrastruct. Res. 22, 530-545.

LEV AN, A. & OSTERGREN, G. (1943). The mechanism of c-mitotic action. Observations on thenaphthalene series. Hereditas 29, 381-443.

LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biochem.Cytol. 9, 409-414.

MILLER, K. W., PATON, W. D. M. & SMITH, E. B. (1967). The anaesthetic pressures ofcertain fluorine-containing gases. Br. J. Anaesth. 39, 910-918.

MILLONIG, G. (1961). Advantages of a phosphate buffer for OsO4 solutions in fixation.jf. appl. Physics 32, 1637.

NlLSSON, L. & SIESJO, B. K. (1970). The effect of anaesthetics upon labile phosphates andupon extra- and intracellular lactate, pyruvate and bicarbonate concentrations in the ratbrain. Acta physiol. scand. 80, 235-248.

NUNN, J. F., Lovis, J. D. & KIMBALL, K. L. (1971). Arrest of mitosis by halothane. Br. J.Anaesth. 43, 524-530.

OsTERGREN, C. (1944). Colchicine mitosis, chromosome contraction, narcosis and proteinchain folding. Hereditas 30, 420—467.

PITELKA, D. R. & CHILD, F. M. (1964). The locomotor apparatus of ciliates and flagellates;relations between structure and function. In Biochemistry and Physiology of Protozoa, vol. 3(ed. S. H. Hutner), pp. 131-198. New York and London: Academic Press.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.

SCHOENBORN, B. P., WATSON, H. C. & KENDREW, J. C. (1965). Binding of xenon to spermwhale myoglobin. Nature, Lond. 207, 28-30.

STEWARD, A., ALLOTT, P. R., COWLES, A. L. & MAPLESON, W. W. (1973). Solubility coefficientsfor inhaled anaesthetics for water, oil and biological media. Br. J. Anaesth. 45, 282-300.

UEDA, I. (1965). Effects of diethyl ether and halothane on firefly luciferin bioluminescence.Anesthesiology 26, 603-606.

WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals.J. biophys. biochem. Cytol. 4, 475-478.

WHITE, D. C. & DUNDAS, C. R. (1970). The effect of anaesthetics on emission of light byluminous bacteria. Nature, Lond. 226, 456-458.

(Received 21 December 1973)

35-2


Fig. 4. Control Tetrahymena pyriformis in approximately transverse section throughthe oral region showing oral cilia (oc) arising from basal bodies (arrows) in the oraldepression, and somatic cilia (sc) seen mainly in cross-section. Some of the somaticbasal bodies (bb) can be seen in the grooves between the ectoplasmic ridges. Mito-chondria (m) are compact and have a dense matrix, x 7500.Fig. 5. Tangential section through part of 2 kineties in a control Tetraliymena.Basal bodies (bb) with their associated kinetodesmal fibres (kd) and posterior micro-tubules (p) lie on the left. The cilia (c) arising in circumciliary depressions in theadjacent kinety have been sectioned at the level of the axosome (arrow). Transversemicrotubules (t) are present beneath the alveolate pellicle which shows irregularswelling, a variable artifact of fixation, x 38000.



Figs. 6-8. Transverse sections through somatic cilia of a control Tetrahymena(Fig. 6), and Tetrahymena after treatment with 2 % halothane (Fig. 7) and with6'7 % halothane (Fig. 8). The normal arrangement of 9 + 2 fibrils is present in all,and in favourably orientated cilia, the dynein arms (d) and nexin connexions (n)between the outer doublet fibrils can also be seen. X 100000.

Figs. 9, 10. Transverse sections through Tetrahymena showing the basal part oflongitudinally sectioned somatic cilia, x 76000.

Fig. 9. Control. The plane of section has passed through the anterior edge of thecilium and its basal body (bb), and one of the group of transverse microtubules (t).Basal microtubules (b) and longitudinal microtubules (/) are also present.

Fig. 10. After 2% halothane. The section is posterior to Fig. 9 and includes partof the electron-opaque core (o) which lies beneath the terminal plate (tp) of thebasal body (bb). One of the transverse microtubules (t), basal microtubules (b) andlongitudinal microtubules (/) are visible.


552 J- F. Nunn and others

Fig. I I . Approximate transverse section of Tetrahymena after treatment with 8 9 %halothane. Only the stumps of oral and somatic cilia remain attached to the basalbodies (arrows). Mitochondria (w) show various degrees of swelling and rarificationof their matrix (compare with Fig. 4). x 7500.

Fig. 12. Tangential section through part of a kinety of Tetrahymena after 8 9 %halothane. Basal bodies (bb) appear intact, and their kinetodesmal fibres (kd),posterior microtubules (p), transverse microtubules (t) and a basal microtubule (b)can be seen, depending on the level of the section, x 38000.

Fig. 13. Transverse section through a somatic cilium after treatment with 8 9 %halothane showing degeneration of subfibrils (arrows) or their complete loss (at x).x 100 000.


13

554 jf. F. Nunn and otliers

Fig. 14. Tetrahymena after 8 9 % halothane. A normal basal body (bb) has beensectioned at the level of the terminal plate. Kinetodesma] fibres (kd) and the parasomalsac (ps) are also present. There is advanced degeneration of all the internal componentsof the cilium (c) lying in the circumciliary depression at the top of the field, x 100 000.

Fig. 15. Transversely sectioned Tetrahymena after 8-9% halothane. The stumpof a somatic cilium with its axosome (a) can be seen above the terminal plate (tp).The basal body (bb), a transverse microtubule (t) and longitudinal microtubules (/)appear normal, x 76000.

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