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Permeability of the abdominal nerve cord of the American cockroach, Periplaneta Americana (L.) to aliphatic alcohols
-7. Insect Physiol., 1967, Vol. 13, pp. 691 to 698. Pergamon PLess Ltd.
Printed in Great Britain
PERMEABILITY OF THE ABDOMINAL NERVE CORD OF THE AMERICAN COCKROACH, PERIPLANETA AMERICANA (L.) TO ALIPHATIC ALCOHOLS* M. E. ELDEFRAWIT
and R. D. O’BRIEN
Section of Neurobiology and Behaviour, Cornell University, Ithaca, New York (Received 8 November 1966)
Abstract-The
rates of influx and efilux of C14- and HVabelled aliphatic alcohols in the abdominal nerve cord of the American cockroach were studied. Influx was biphasic and positively correlated with octanol/water partition coefficients, with a cut-off point at butanol. All alcohc$s, except methanol, established a higher concentration inside the nerve cord than outside within 50 min exposure. Influx rates of alcohols were lower than their analogous fatty acids, the difference being attributed to the absence of metabolism of the alcohols in the nerve cord. Compared to the analogous cations, the alcohols had higher influx rates, yet when comparing an alcohol with a cation with similar partition coefficient the infiux rates were similar. Charge therefore seems to lower influx because it increases the polarity. Efflux of the alcohols is characterized by a two-stage process with simultaneous rapid and slow phases. Based on the rates of the fast efflux, the nerve sheath appears to be equally permeable to alcohols and tetra-alkylammonium cations and 2 to 5 times less permeable to fatty acids. INTRODUCTION
THE movement of non-electrolytes in or out of the central nervous system (CNS) of insects has attracted very few investigators despite its paramount importance, particularly in relation to insecticidal action. Few data are available to show that non-ionizable organic molecules permeate the insect CNS freely. On the other hand, there are several reports to support this view in vertebrates (MARK et al., 1958; RALL et al., 1959; BRODIEet al., 1960; HANSSONand SCHMITERL~W,1961). The only direct measurement of penetration of non-electrolytes into the insect CNS was the work on the movement of glucose and sucrose into the abdominal nerve cord of the American cockroach (TREHERNE, 1960, 1962). O’BRIEN (1959) made indirect measurements of the penetration of the organophosphate tepp (diethyl phosphoric anhydride) into the intact and excised nerve cord of the American cockroach by measuring the resultant cholinesterase inhibition. Most of the studies on permeation into the insect CNS have been made with the inorganic ions Na f, Kf, and Ca2+ (TREHEFWE, 1961a, b, c, d, 1962) mainly because of their vital r61e in the propagation of the action potential. Though they are cations, they were found to permeate the abdominal nerve cord quite freely,
* This work was supported in part by NIH Grant GM 07804-06. t Permanent address, Faculty of Agriculture, University of Alexandria, Egypt, U.A.R. 691
692
M. E. ELDEFRAWI ANDR. D. O’BRIEN
and so did acetylcholine (in the absence of eserine) (TREHERNEand SMITH, 1965). Yet for unmetabolizable organic cations, ELDEFPZAWI and O’BRIEN (1967) found that increasing the liposolubility within a series of tetra-alkylammonium cations increased penetration into the cockroach abdominal nerve cord, and increasing the size decreased it. Increasing lipid solubility also increased penetration of fatty acids into the cockroach nerve cord (ELDEFRAWIand O’BRIEN, 1966). It was suggested that there exists a regulatory system that restricts influx of large, charged, unmetabolizable polar molecules in the American cockroach abdominal nerve cord (ELDEFRAWIand O’BRIEN, 1966) and the ganglia of the willow aphid, Tuberolachus salignus (Gmelin) (TOPPOZADAand O’BRIEN, 1967). It was of great interest to compare the above data with similar studies of organic non-electrolytes; a series of aliphatic alcohols was therefore chosen for the present study. The results of this work may help formulate a fuller understanding of the effects of polarity and charge upon influx into the insect CNS. MATERIALS
AND METHODS
Methyl alcohol-C14, specific activity 10 mc/mM, ethyl alcohol-Z-H3, specific activity 753 mc/mM, butyl alcohol-, hexyl alcohol-, and octyl alcohol-l&Y*, specific activity 1 mc/mM, were all available commercially. Stock solutions of the first two alcohols were made up in water, whereas those of the three higher alcohols were made in ethanol. Ringer solutions containing the different alcohols at molar concentration varying from 1 x 10m4to 2 x lOA were prepared. The final ethanol concentration was 2‘$, in the case of the three higher alcohols. The composition of the Ringer solution, and the nerve cord preparation and techniques for measuring influx and efflux are the same as described earlier (ELDEFRAWIand O’BRIEN, 1966). To investigate the degree of metabolism of octanol by the nerve cord tissue during the period of exposure, a nerve cord exposed to the alcohol for 1 hr was homogenized in acetone in a micro glass Bolab homogenizer (Bonus Labs, Reading, Mass, U.S.A.), then the homogenate was centrifuged and the supernatant chromatographed on Whatman No. 4 paper. A biphasic chromatographic system was employed using 25% fl-methoxy-propionitrile in acetone as a stationary phase and iso-octane saturated with /3-methoxypropionitrile as a moving phase. The paper strip was cut into Q in. sections after development and each section placed in a counting vial with 10 ml of BRAY’S (1960) dioxane cocktail and counted in a Tri-Carb liquid scintillation counter. RESULTS AND DISCUSSION InJEUX It is apparent that the influx of alcohols into the abdominal nerve cord of the cockroach is biphasic (Fig. l), as was found for fatty acids (ELDEFRAWIand O’BRIEN, 1966). With the exception of hexanol, it appears that the larger the molecular volume or liposolubility of the alcohol, the higher its influx. This is also apparent when the molar ratios at 30 min are used as an index of influx, and are plotted against the octanol/water partition coefficients of the alcohols (Fig. 2).
ALIPHATICALCOHOLS IN AMERICANCOCKROACH ARDOMINAZ. NERVECORD
693
The relationship ‘between influx and liposolubility is linear for the lower alcohols up to butanol, then there is a cut-off point, after which increase in liposolubility does not result in an increase in influx.
TIME
Similar cut-off points were found for the
(MINI
FIG. 1. The relation between time and influx of alcohols into the abdominal nerve cord: methanol ( e) ; ethanol ( 0) ; butanol ( A); hexanol ( 0) ; and octanol ( 0). Vertical lines represents the ranges of the triplicate determinations.
octanol 0
o
PARTITION
FIG. 2.
hexanol
C 0.
The relation between octanol/water partition coefficient and uptake of alcohols by the abdominal nerve cord.
influx of fatty acids into the cockroach abdominal nerve cord at octanoate, and in the aphid ganglia at valerate (TOPPOZADA and O’BRIEN, 1967). Such cut-off phenomena could have two possible explanations: the solubility of the high molecular weight materials might be less than the concentration which we attempted 45
M. E. ELDEFRAWIAND R. D. O’BRIEN
694
to obtain, i.e. the true concentration employed was less than it appeared.
This
explanation is unlikely because the cut-off point of the acids was different for the cockroach (ELDEFRAWI and O’BRIEN, 1966) and the aphid (TOPPOZADA and O’BRIEN, 1967). Alternatively,
the increase in liposolubility
as the series were
ascended may have been more than offset by a decrease in free diffusion rate caused by the large size. Comparing
the alcohols with the fatty acids (Table
l), it is apparent that the
rates of influx are much higher for each fatty acid than its analogous alcohol (1.3 x to 13 x for K,, and 1.4 x to 3 x for K,), even though the acids have about TABLE ~-INFLUX RATESINTO THEABDOMINALNERVECORD,AND OCTANOL/WATER PARTITION COEFFICIENTS, OF ALCOHOLS AND THEIRANALOGOUS ANIONSAND CATIONS
Compound
Octanol/water partition coefficient
k,min-l
*
k, min-l
Alcohols : CH,OH C2H,0H C&H,OH C,H,,CH C,H,,CH
0.15 0.4 4.6 51.0 560.0
0.010 0.032 0.054 0.050 0.070
0.003 0.018 0.032 0.019 0=035
Anions : t CH,COO C,H,COOC,H,,COOC,H,,COO-
0.19 2.0 22.5 245.0
0.04 0.30 OS6 0.70
0.027 0.074 0.136 0,295
-
0.013 0.006 0.009 0.015
Cations :$ GHaN+(CH& GH&+(CHa)a GH,,N+(CH& CaH,,N+(CHa)r
9.9 2.49 1.46 8.55
x x x x
10-4 IO+? lo+ 1O”2
*
* Rates calculated by the equation dC/dt = kc. i Values calculated from ELDEFRAWIand O’BRIEN (1966). $ Values from ELDEFRAWIand O’BRIEN (1967). half the partition coefficient values of their analogous alcohols. The higher influx rates of fatty acids are attributable mainly to their metabolism in the nerve tissue, which acts as a driving force to increase their influx. ELDEFRAWI and O’BRIEN (1966) found that octanoate was almost completely metabolized in the cockroach nerve cord after 90 min exposure. STEVENS and STETTLER (1967) also found that transport of fatty acids across the rumen epithelium increased from acetic to butyric, and so did the rate of metabolism of the acids in the rumen epithelium. In the case of octanol, no metabolism was detected even after 1 hr exposure, as shown
ALIPHATIC ALCOHOLS IN AMERICAN COCKROACH ABDOMINAL NRRVB CORD
695
in Fig. 3. Attempts were made to study the metabolism of butanol and ethanol by the same system, but preliminary experiments showed that all the parent material was lost by volatilization. However, it was shown that after incubation of the nerve tissue with these alcohols under the above conditions, no extractable, non-volatile metabolites could be detected. Another electrolyte, glucose, has an influx rate into the cockroach central nervous system of 0.036 min-l as calculated from TREHEFWE (1960), a value similar to the rate for butanol (Table l), despite the fact that glucose has much higher water solubility. Again, metabolism may be the determining factor, since TREHERNE(1960) found that glucose was metabolized rapidly by the cockroach nerve cord.
3-
2-
E 5 2 2 u
4-
@
(b)
:;:%:;:; ::::I$::
I
P.O.
S. F.
FIG. 3. Autoradiograrna of octanol-l-C14 (a) and the acetone extract of a nerve cord exposed to octanol-l-Cl4 for 1 hr (b). P.O. = point of origin; S.F. = solvent front.
Unlike the influx of alcohols and acids, that of the cations is monophasic and O’BRIEN 1967). When the monophasic influx rate is taken as $ and compared to the A8 values for alcohols, it is found that the analogous alcohols have higher rates (1.3-5.3 x ). However, one has to consider not only the presence of charge per se in the cation series but also its effect on liposolubility. If one compares a cation such as C,H1,N+(CH& (with a partition coefficient of O-086) and methanol (partition coefficient O*lS), it is found that the cation influxes at a (ELDEFRAWI
M. E.
696
ELDEFRAWI
AND R. D.
O’BRIEN
higher rate (Table 1). Therefore one may conclude that it is the effect of the charge in making the molecule more polar (water-soluble) rather than the presence of the charge per se that lowers its influx into the insect’s CNS.
Ejlux Efflux of the alcohols from a ligatured abdominal nerve cord is biphasic, with fast and slow components (Fig. 4). The size of the fast pool decreases as the alcohol increases in volume, while the opposite is true for the slow pool (Table 2).
FIG. 4. Efflux curves of alcohols from the abdominal nerve cord. Solid lines are experimental and broken lines are calculated by subtraction of the slow contribution from first part. Symbols as Fig. 1. TABLE
%--PROPERTIES
OF THE TWO POOL CH.ARACTRRISTICS OF THE BFFLUX OF ALCOHOLS FROM THE ABDOMINAL NERVE CORD
Slow pool
Fast pool t,,.5
k1 min-l
y0 size
to.5
k, min-l
0/0size
Per cent retained by nerve cord after 1 hr efflux
1.0 2.0 2.5 2.5 2-o
O-69 o-34 O-28 O-28 o-34
40 36 22 32 20
103 112 220 210 210
O-0067 O-0062 0*0031 0.0033 o-0033
60 64 78 68 80
39.7 37.6 68.0 57.3 70.3
Alcohol Methanol Ethanol Butanol Hexanol OctanoI.
Jz& 103 55 90 85 103
The rate constants of efflux from the two pools are higher for the lower alcohols. If one compares the percentage activity retained by nerve cord tissue after 1 hr of washing in Ringer solution, one finds that this proportion increases with increase
ALIPHATIC ALCOHOLS IN AMERICAN COCKROACH ABDOMINAL NERVE CORD
697
in molecular volume and thus with liposolubility (Table 2). It is suggested that this activity retained by the nerve cord is confined primarily to the intracellular components. Th e average sixes of the two pools in the case of the alcohols are similar to those of the fatty acids (ELDEFRAWIand O’BRIEN, 1966); the fast pool of the cations is larger and the slow pool is smaller (ELDEFRAWIand O’BRIEN, 1967). The fast efflux of alcohols is 2-6.5 x greater than that of glucose and 0.245 x that of fatty acids. This suggests that permeability of the nerve sheath to alcohols is lower than for glucose, higher than fatty acids, and similar to the tetra-alkylammonium cations. It is of great interest to note that the efflux of different ions and molecules from the insects CNS has always been found to be biphasic. Such curves were reported for glucose (TREHERNE,1960), Na+, Kf, Ca2+, Cl-, tritiated water, and sucrose (TREHERNE,1962), in the case of the cockroach nerve cord, and Na+, K+, Ca2+, Cl- from the nerve cord of the stick insect, Caruusius morosus (TREHERNE,1965). ELDEFRAWIand O’BRIEN (1966, 1967) found similar efflux curves for fatty acids, tetra-alkylammonium cations and acetylcholine in the cockroach abdominal nerve cord. It was suggested by TREHERNE(1961d) that the initial fast phase is attributable to a rapid diffusion from the extracellular spaces, and the slow phase to a slower extrusion from the cellular components of the CNS. This suggestion is supported by the fact that the biphasic nature of the efflux curve is unaffected by size, charge, or solubility as indicated by the data reviewed above. One may then expect that any compound would exhibit a biphasic efflux curve. However, the properties of the curve, such as sixes and rates of the two phases, are affected by the chemical nature of the compound and its physical properties. This observation is not incompatible with Treherne’s suggestion, for the partitioning of compounds between intra- and extra-cellular phases would undoubtedly differ. In conclusion, it appears that alcohols penetrate freely the abdominal nerve cord of the American cockroach, at rates comparable to those of glucose and Na+, but higher than their analogous tetra-alkylammonium cations. The higher the liposolubility of the alcohol, up to butanol, the higher its influx, the smaller is the size of the fast pool, and the higher is the percentage activity retained by the nerve cord after 1 hr of efflux. The effect of the regulatory mechanism, suggested to exist in the central nervous system of insects (ELDEFRAWIand O’BRIEN, 1967), is reflected in the influx of alcohols, by favouring smaller and less polar molecules.
REFERENCES ALBERTA. (1960) Selective Toxicity, p. 110. Wiley, New York. BRAYG. A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. 1,279-285. BRODIE B. B., KURZH., and SCHANKER L. S. (1960) The importance of dissociation constant and lipid-solubility in influencing J. Phunnac. exp. Ther. 130,20-25.
the passage of drugs into the cerebrospinal fluid.
ELDEFRAWM. E. and O’BRIENR. D. (1966) Permeability of the abdominal nerve cord of the American cockroach to fatty acids. J. Insect Physiol. 12,1133-1142.
698
M. E. ELDEFRAWIANDR. D. O’BRIEN
ELDEFRAWIM. E. and O’BRIEN R. D. (1967) Permeability of the abdominal nerve cord of the American cockroach, Periplaneta americana (L.) to quaternary ammonium salts. J. exp. Biol. In press. HANSSONE. and SCHMITERL~WC. G. (1961) A comparison of the distribution, excretion and metabolism of a tertiary (Promethazine) and a quaternary (Aprobit @) phenothiazine compound labelled with S35. Archs int. Pharmacodyn Ther. 131, 309-324. MARK L. C., BURNSJ. J., BRANDL., CAMPOMANES C. I., TROUSON., PAPPER E. M., and BRODIEB. B. (1958) The passage of thiobarbiturates and their oxygen analogs into brain. r. Pharmac. exp. Ther. 123, 70-73. O’BRIEN R. D. (1959) Effect of ionization upon penetration of organophosphates to the nerve cord of the cockroach. g. econ. Ent. 52, 812416. RALL D. P., STABENAUJ. R., and ZUBRODC. G. (1959) Distribution of drugs between blood and cerebrospinal fluid: general methodology and effect of pH gradients. J. Pharmac. exp. Ther. 125, 185-193. STEVENSC. E. and STETTLER B. K. (1967) Transport of fatty acid mixtures across rumen epithelium. Am. J. Physiol. In press. TOPPOZADAA. and O’BRIEN R. D. (1967) Permeability of the gangha of the wiIlow aphid, Tuberolachnus salignus (Gmelin) to organic ions. r. Iwect Physiol. To be published. TREHERNEJ. E. (1960) The nutrition of the central nervous system in the cockroach Periplaneta americana L. The exchange and metabolism of sugars. y. exp. Biol. 37,513-533. TREHERNEJ. E. (1961a) Sodium and potassium fluxes in the abdominal nerve cord of the cockroach Periplaneta americana L. J. exp. Biol. 38, 315-322. TREHJZRNEJ. E. (196lb) The movements of sodium ions in the isolated abdominal nerve cord of the cockroach Pe-riplaneta americana L. j? exp. Biol. 38, 629-636. TREHERNEJ. E. (196lc) The efflux of sodium ions from the last abdominal ganglion of the cockroach Periplaneta amevicana L. J. exp. Biol. 38, 729-736. TREHERNEJ. E. (196ld) The kinetics of sodium transfer in the central nervous system of the cockroach Periplaneta americana L. r. exp. Biol. 38, 737-746. TREHERNEJ. E. (1962) The distribution and exchange of some ions and molecules in the central nervous system of Pe-riplaneta americana L. r. exp. Biol. 39, 193-217. TRE~IERNEJ. E. (1965) The distribution and exchange of inorganic ions in the central nervous system of the stick insect Carausius morosus. J. exp. Biol. 42, 7-27. TREHERNEJ. E. and SMITH D. S. (1965) The penetration of acetylcholine into the central nervous tissues of an insect (Periplaneta americana L.). r. exp. Biol. 42, 13-21.
Printed in Great Britain
PERMEABILITY OF THE ABDOMINAL NERVE CORD OF THE AMERICAN COCKROACH, PERIPLANETA AMERICANA (L.) TO ALIPHATIC ALCOHOLS* M. E. ELDEFRAWIT
and R. D. O’BRIEN
Section of Neurobiology and Behaviour, Cornell University, Ithaca, New York (Received 8 November 1966)
Abstract-The
rates of influx and efilux of C14- and HVabelled aliphatic alcohols in the abdominal nerve cord of the American cockroach were studied. Influx was biphasic and positively correlated with octanol/water partition coefficients, with a cut-off point at butanol. All alcohc$s, except methanol, established a higher concentration inside the nerve cord than outside within 50 min exposure. Influx rates of alcohols were lower than their analogous fatty acids, the difference being attributed to the absence of metabolism of the alcohols in the nerve cord. Compared to the analogous cations, the alcohols had higher influx rates, yet when comparing an alcohol with a cation with similar partition coefficient the infiux rates were similar. Charge therefore seems to lower influx because it increases the polarity. Efflux of the alcohols is characterized by a two-stage process with simultaneous rapid and slow phases. Based on the rates of the fast efflux, the nerve sheath appears to be equally permeable to alcohols and tetra-alkylammonium cations and 2 to 5 times less permeable to fatty acids. INTRODUCTION
THE movement of non-electrolytes in or out of the central nervous system (CNS) of insects has attracted very few investigators despite its paramount importance, particularly in relation to insecticidal action. Few data are available to show that non-ionizable organic molecules permeate the insect CNS freely. On the other hand, there are several reports to support this view in vertebrates (MARK et al., 1958; RALL et al., 1959; BRODIEet al., 1960; HANSSONand SCHMITERL~W,1961). The only direct measurement of penetration of non-electrolytes into the insect CNS was the work on the movement of glucose and sucrose into the abdominal nerve cord of the American cockroach (TREHERNE, 1960, 1962). O’BRIEN (1959) made indirect measurements of the penetration of the organophosphate tepp (diethyl phosphoric anhydride) into the intact and excised nerve cord of the American cockroach by measuring the resultant cholinesterase inhibition. Most of the studies on permeation into the insect CNS have been made with the inorganic ions Na f, Kf, and Ca2+ (TREHEFWE, 1961a, b, c, d, 1962) mainly because of their vital r61e in the propagation of the action potential. Though they are cations, they were found to permeate the abdominal nerve cord quite freely,
* This work was supported in part by NIH Grant GM 07804-06. t Permanent address, Faculty of Agriculture, University of Alexandria, Egypt, U.A.R. 691
692
M. E. ELDEFRAWI ANDR. D. O’BRIEN
and so did acetylcholine (in the absence of eserine) (TREHERNEand SMITH, 1965). Yet for unmetabolizable organic cations, ELDEFPZAWI and O’BRIEN (1967) found that increasing the liposolubility within a series of tetra-alkylammonium cations increased penetration into the cockroach abdominal nerve cord, and increasing the size decreased it. Increasing lipid solubility also increased penetration of fatty acids into the cockroach nerve cord (ELDEFRAWIand O’BRIEN, 1966). It was suggested that there exists a regulatory system that restricts influx of large, charged, unmetabolizable polar molecules in the American cockroach abdominal nerve cord (ELDEFRAWIand O’BRIEN, 1966) and the ganglia of the willow aphid, Tuberolachus salignus (Gmelin) (TOPPOZADAand O’BRIEN, 1967). It was of great interest to compare the above data with similar studies of organic non-electrolytes; a series of aliphatic alcohols was therefore chosen for the present study. The results of this work may help formulate a fuller understanding of the effects of polarity and charge upon influx into the insect CNS. MATERIALS
AND METHODS
Methyl alcohol-C14, specific activity 10 mc/mM, ethyl alcohol-Z-H3, specific activity 753 mc/mM, butyl alcohol-, hexyl alcohol-, and octyl alcohol-l&Y*, specific activity 1 mc/mM, were all available commercially. Stock solutions of the first two alcohols were made up in water, whereas those of the three higher alcohols were made in ethanol. Ringer solutions containing the different alcohols at molar concentration varying from 1 x 10m4to 2 x lOA were prepared. The final ethanol concentration was 2‘$, in the case of the three higher alcohols. The composition of the Ringer solution, and the nerve cord preparation and techniques for measuring influx and efflux are the same as described earlier (ELDEFRAWIand O’BRIEN, 1966). To investigate the degree of metabolism of octanol by the nerve cord tissue during the period of exposure, a nerve cord exposed to the alcohol for 1 hr was homogenized in acetone in a micro glass Bolab homogenizer (Bonus Labs, Reading, Mass, U.S.A.), then the homogenate was centrifuged and the supernatant chromatographed on Whatman No. 4 paper. A biphasic chromatographic system was employed using 25% fl-methoxy-propionitrile in acetone as a stationary phase and iso-octane saturated with /3-methoxypropionitrile as a moving phase. The paper strip was cut into Q in. sections after development and each section placed in a counting vial with 10 ml of BRAY’S (1960) dioxane cocktail and counted in a Tri-Carb liquid scintillation counter. RESULTS AND DISCUSSION InJEUX It is apparent that the influx of alcohols into the abdominal nerve cord of the cockroach is biphasic (Fig. l), as was found for fatty acids (ELDEFRAWIand O’BRIEN, 1966). With the exception of hexanol, it appears that the larger the molecular volume or liposolubility of the alcohol, the higher its influx. This is also apparent when the molar ratios at 30 min are used as an index of influx, and are plotted against the octanol/water partition coefficients of the alcohols (Fig. 2).
ALIPHATICALCOHOLS IN AMERICANCOCKROACH ARDOMINAZ. NERVECORD
693
The relationship ‘between influx and liposolubility is linear for the lower alcohols up to butanol, then there is a cut-off point, after which increase in liposolubility does not result in an increase in influx.
TIME
Similar cut-off points were found for the
(MINI
FIG. 1. The relation between time and influx of alcohols into the abdominal nerve cord: methanol ( e) ; ethanol ( 0) ; butanol ( A); hexanol ( 0) ; and octanol ( 0). Vertical lines represents the ranges of the triplicate determinations.
octanol 0
o
PARTITION
FIG. 2.
hexanol
C 0.
The relation between octanol/water partition coefficient and uptake of alcohols by the abdominal nerve cord.
influx of fatty acids into the cockroach abdominal nerve cord at octanoate, and in the aphid ganglia at valerate (TOPPOZADA and O’BRIEN, 1967). Such cut-off phenomena could have two possible explanations: the solubility of the high molecular weight materials might be less than the concentration which we attempted 45
M. E. ELDEFRAWIAND R. D. O’BRIEN
694
to obtain, i.e. the true concentration employed was less than it appeared.
This
explanation is unlikely because the cut-off point of the acids was different for the cockroach (ELDEFRAWI and O’BRIEN, 1966) and the aphid (TOPPOZADA and O’BRIEN, 1967). Alternatively,
the increase in liposolubility
as the series were
ascended may have been more than offset by a decrease in free diffusion rate caused by the large size. Comparing
the alcohols with the fatty acids (Table
l), it is apparent that the
rates of influx are much higher for each fatty acid than its analogous alcohol (1.3 x to 13 x for K,, and 1.4 x to 3 x for K,), even though the acids have about TABLE ~-INFLUX RATESINTO THEABDOMINALNERVECORD,AND OCTANOL/WATER PARTITION COEFFICIENTS, OF ALCOHOLS AND THEIRANALOGOUS ANIONSAND CATIONS
Compound
Octanol/water partition coefficient
k,min-l
*
k, min-l
Alcohols : CH,OH C2H,0H C&H,OH C,H,,CH C,H,,CH
0.15 0.4 4.6 51.0 560.0
0.010 0.032 0.054 0.050 0.070
0.003 0.018 0.032 0.019 0=035
Anions : t CH,COO C,H,COOC,H,,COOC,H,,COO-
0.19 2.0 22.5 245.0
0.04 0.30 OS6 0.70
0.027 0.074 0.136 0,295
-
0.013 0.006 0.009 0.015
Cations :$ GHaN+(CH& GH&+(CHa)a GH,,N+(CH& CaH,,N+(CHa)r
9.9 2.49 1.46 8.55
x x x x
10-4 IO+? lo+ 1O”2
*
* Rates calculated by the equation dC/dt = kc. i Values calculated from ELDEFRAWIand O’BRIEN (1966). $ Values from ELDEFRAWIand O’BRIEN (1967). half the partition coefficient values of their analogous alcohols. The higher influx rates of fatty acids are attributable mainly to their metabolism in the nerve tissue, which acts as a driving force to increase their influx. ELDEFRAWI and O’BRIEN (1966) found that octanoate was almost completely metabolized in the cockroach nerve cord after 90 min exposure. STEVENS and STETTLER (1967) also found that transport of fatty acids across the rumen epithelium increased from acetic to butyric, and so did the rate of metabolism of the acids in the rumen epithelium. In the case of octanol, no metabolism was detected even after 1 hr exposure, as shown
ALIPHATIC ALCOHOLS IN AMERICAN COCKROACH ABDOMINAL NRRVB CORD
695
in Fig. 3. Attempts were made to study the metabolism of butanol and ethanol by the same system, but preliminary experiments showed that all the parent material was lost by volatilization. However, it was shown that after incubation of the nerve tissue with these alcohols under the above conditions, no extractable, non-volatile metabolites could be detected. Another electrolyte, glucose, has an influx rate into the cockroach central nervous system of 0.036 min-l as calculated from TREHEFWE (1960), a value similar to the rate for butanol (Table l), despite the fact that glucose has much higher water solubility. Again, metabolism may be the determining factor, since TREHERNE(1960) found that glucose was metabolized rapidly by the cockroach nerve cord.
3-
2-
E 5 2 2 u
4-
@
(b)
:;:%:;:; ::::I$::
I
P.O.
S. F.
FIG. 3. Autoradiograrna of octanol-l-C14 (a) and the acetone extract of a nerve cord exposed to octanol-l-Cl4 for 1 hr (b). P.O. = point of origin; S.F. = solvent front.
Unlike the influx of alcohols and acids, that of the cations is monophasic and O’BRIEN 1967). When the monophasic influx rate is taken as $ and compared to the A8 values for alcohols, it is found that the analogous alcohols have higher rates (1.3-5.3 x ). However, one has to consider not only the presence of charge per se in the cation series but also its effect on liposolubility. If one compares a cation such as C,H1,N+(CH& (with a partition coefficient of O-086) and methanol (partition coefficient O*lS), it is found that the cation influxes at a (ELDEFRAWI
M. E.
696
ELDEFRAWI
AND R. D.
O’BRIEN
higher rate (Table 1). Therefore one may conclude that it is the effect of the charge in making the molecule more polar (water-soluble) rather than the presence of the charge per se that lowers its influx into the insect’s CNS.
Ejlux Efflux of the alcohols from a ligatured abdominal nerve cord is biphasic, with fast and slow components (Fig. 4). The size of the fast pool decreases as the alcohol increases in volume, while the opposite is true for the slow pool (Table 2).
FIG. 4. Efflux curves of alcohols from the abdominal nerve cord. Solid lines are experimental and broken lines are calculated by subtraction of the slow contribution from first part. Symbols as Fig. 1. TABLE
%--PROPERTIES
OF THE TWO POOL CH.ARACTRRISTICS OF THE BFFLUX OF ALCOHOLS FROM THE ABDOMINAL NERVE CORD
Slow pool
Fast pool t,,.5
k1 min-l
y0 size
to.5
k, min-l
0/0size
Per cent retained by nerve cord after 1 hr efflux
1.0 2.0 2.5 2.5 2-o
O-69 o-34 O-28 O-28 o-34
40 36 22 32 20
103 112 220 210 210
O-0067 O-0062 0*0031 0.0033 o-0033
60 64 78 68 80
39.7 37.6 68.0 57.3 70.3
Alcohol Methanol Ethanol Butanol Hexanol OctanoI.
Jz& 103 55 90 85 103
The rate constants of efflux from the two pools are higher for the lower alcohols. If one compares the percentage activity retained by nerve cord tissue after 1 hr of washing in Ringer solution, one finds that this proportion increases with increase
ALIPHATIC ALCOHOLS IN AMERICAN COCKROACH ABDOMINAL NERVE CORD
697
in molecular volume and thus with liposolubility (Table 2). It is suggested that this activity retained by the nerve cord is confined primarily to the intracellular components. Th e average sixes of the two pools in the case of the alcohols are similar to those of the fatty acids (ELDEFRAWIand O’BRIEN, 1966); the fast pool of the cations is larger and the slow pool is smaller (ELDEFRAWIand O’BRIEN, 1967). The fast efflux of alcohols is 2-6.5 x greater than that of glucose and 0.245 x that of fatty acids. This suggests that permeability of the nerve sheath to alcohols is lower than for glucose, higher than fatty acids, and similar to the tetra-alkylammonium cations. It is of great interest to note that the efflux of different ions and molecules from the insects CNS has always been found to be biphasic. Such curves were reported for glucose (TREHERNE,1960), Na+, Kf, Ca2+, Cl-, tritiated water, and sucrose (TREHERNE,1962), in the case of the cockroach nerve cord, and Na+, K+, Ca2+, Cl- from the nerve cord of the stick insect, Caruusius morosus (TREHERNE,1965). ELDEFRAWIand O’BRIEN (1966, 1967) found similar efflux curves for fatty acids, tetra-alkylammonium cations and acetylcholine in the cockroach abdominal nerve cord. It was suggested by TREHERNE(1961d) that the initial fast phase is attributable to a rapid diffusion from the extracellular spaces, and the slow phase to a slower extrusion from the cellular components of the CNS. This suggestion is supported by the fact that the biphasic nature of the efflux curve is unaffected by size, charge, or solubility as indicated by the data reviewed above. One may then expect that any compound would exhibit a biphasic efflux curve. However, the properties of the curve, such as sixes and rates of the two phases, are affected by the chemical nature of the compound and its physical properties. This observation is not incompatible with Treherne’s suggestion, for the partitioning of compounds between intra- and extra-cellular phases would undoubtedly differ. In conclusion, it appears that alcohols penetrate freely the abdominal nerve cord of the American cockroach, at rates comparable to those of glucose and Na+, but higher than their analogous tetra-alkylammonium cations. The higher the liposolubility of the alcohol, up to butanol, the higher its influx, the smaller is the size of the fast pool, and the higher is the percentage activity retained by the nerve cord after 1 hr of efflux. The effect of the regulatory mechanism, suggested to exist in the central nervous system of insects (ELDEFRAWIand O’BRIEN, 1967), is reflected in the influx of alcohols, by favouring smaller and less polar molecules.
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ELDEFRAWM. E. and O’BRIENR. D. (1966) Permeability of the abdominal nerve cord of the American cockroach to fatty acids. J. Insect Physiol. 12,1133-1142.
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M. E. ELDEFRAWIANDR. D. O’BRIEN
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