Metabolism and significance of acetylcholine in the brain of the adult housefly, Musca domestica L.

J. Insect Physiol., 1966, Vol. 12, pp. 909 to 924. Pergamon Press Ltd.

Printed in Great Britain

METABOLISM AND SIGNIFICANCE OF ACETYLCHOLINE IN THE BRAIN OF THE ADULT HOUSEFLY, MUSCA DOMESTICA L. F. P. W. WINTERINGHAM Biochemistry Department,

Agricultural Research Council, Pest Infestation Laboratory, Slough, Buckinghamshire (Received

3 March

1966)

Abstract-Methods are described for the determination of [‘“Cl acetylcholine formed in the head tissues of the adult housefly in viva following [‘“Cl acetate injection. Acetylcholine estimated radiometrically and that assayed pharmacologically were in good agreement. This confirms that this ester largely, if not entirely, accounts for the pharmacological activity of head extracts as assayed with the frog rectus abdominis muscle preparation. The data indicated that the acetylcholine content of the brain is due to a steady state of synthesis and hydrolysis with a minimum turnover rate of 6.1 x lop5 pmoles acetylcholine/min/head. Turnover was apparently reduced in insects poisoned with acetylcholinesterase inhibitors. These and other observations are interpreted as showing that the ACh system of the brain is highly compartmentalized, synthesis from choline and intracellular acetate being controlled by the availability of choline arising from acetylcholinesterase action. No correlation was found between acetylcholine turnover in v,ivo and the incidence of light on the compound eye. Turnover was apparently unaffected by surgical elimination of possible nerve activity originating in the thoracic ganglion. It was, apparently, reduced during cyclopropane anaesthesia. Current problems of insect neurophysiology are critically discussed. INTRODUCTION

IN VERTEBRATES acetylcholine plays a vital role in preganglionic transmission of the autonomic nervous system, at the nerve endings of the parasympathetic system, skeletal neuromuscular junctions (KATZ, 1962; THESLEFFand QUASTEL, 1965 ; TRIGGLE, 1965), and in brain (GADDUM, 1963). Acetylcholine may also have a role in adrenergic transmission (BURN and RAND, 1965). NACHMANSOHN(1959) has postulated a role in general axonic transmission which, however, has been challenged or modified by others (EHRENPREIS,1964). Acetylcholine (ACh)* and the enzymes (CL4 and AChE)* which facilitate its synthesis and hydrolysis have been * Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase (E.C. 3.1.1.7); ChA, choline acetylase ( = acetyl coenzyme A: choline-0-acetyl transferase, E.C. 2.3.1.6); DFP, diisopropylphosphorofluoridate ; o-IPMC, o-isopropoxyphenyl-N-methylcarbamate ; TEPP, tetraethyl pyrophosphate; TMPP, tetramethyl pyrophosphate; ATP, adenosine triphosphate. 909

910

F. P. W. WINTERINGHAM

demonstrated in the tissues, and in nervous tissue especially, of many insect species yet the physiological role of ACh in insects remains to be demonstrated (COLHOUN, 1963). However, lipophilic inhibitors of AChE are toxic to insects and there is a good correlation between signs of poisoning and AChE inhibition estimated to have obtained in viva (MENGLE and CASIDA, 1958; SMALLMANand FISHER, 1958). Electrophysiological and histochemical studies have clearly indicated conductive nervous tissue as the site of action of anticholinergic drugs in insects (ROEDERet al., 1947; WINTON et al., 1958; MOLLOY, 1961). There is also some evidence that the ACh system may be involved in the mode of action of certain chlorohydrocarbon insecticides and in the mechanisms of resistance thereto (STERNBURGand HEWITT, 1962; WINTERINGHAM,1962a). These observations suggest that ACh plays a vital role in insect nervous function but not one necessarily entirely comparable with that in vertebrates. For example, there is some evidence that neuromuscular transmission in insects is not mediated by ACh (HARLOW, 1958; O’CONNOR et al., 1965). Possibly it is mediated by glutamic acid (KERKUTet al., 1965). There are high concentrations of ACh, AChE, and ChA in the head tissues of adult Diptera and the system is largely concentrated in the brain (LEWIS and SMALLMAN,1956; SMALLMAN,1956; MEHROTRA,1961; MOLLOY, 1961; ROTHSCHILD and HOWDEN,1961). The same components are present in vertebrate brain and the high concentrations of AChE in certain neurons suggest a cholinergic function (SHUTE and LEWIS, 1963; SALMOIRAGHIet al., 1965). Levels of ACh in the brains of mice or rats have been shown to depend upon physiological state. Thus, RICHTERand CROSSLAND(1949) reported significant increases in the ACh content of rat brain under anaesthesia and decreases in animals exhibiting emotional excitement or convulsions. KUROKAWAet al., (1963) have made related observations with mice. By means of the labelled pool technique following the injection of [2-l*C] acetate it is possible to estimate chemical levels of acetylcholine and its turnover in the intact insect in viva (WINTERINGHAM, 1956; 1962b; WINTERINGHAMand HARRISON,1956; 1961). This paper describes the application of these techniques to a study of the role and metabolism of ACh in the head tissues of the intact adult housefly, Musca domestica L. In particular an attempt has been made to measure turnover rate in viva in the belief that this parameter was more likely than chemical level to be related to physiological state.

MATERIALS

AND METHODS

Insects Adult female houseflies, Musca domestica L., were drawn within a 2 to 10 day period after emergence from a laboratory culture maintained at about 27°C. Average weight of the flies was 21 mg. For comparing the effects of different treatments ‘control’ and ‘treated’ groups of flies were always of the same age and batch.

METABOLISM

[2J4C]

AND SIGNIFICANCE

OF ACETYLCHOLINE

IN THE HOUSEFLY

911

Anhydrous sodium acetate

This was purchased from the Radiochemical Centre, Arnersham. An 0.4 M stock solution in glass-distilled water was prepared at a specific radioactivity of 2.5 me/m-mole and stored at - 15°C. A similar O-4 M reference solution was prepared at l/1000 of the specific radioactivity of the stock solution and used for reference counting (see p. 912). [Car60xy-1~C] acetylcholine chloride This was prepared as described by WINTERINGHAMand DISNEY (1964) and purchased from the Radiochemical Centre, Amersham. A 5 x 10~~ M stock solution in glass-distilled water was prepared at a specific radioactivity of 6.4 me/mmole and stored at - 15°C. Treatment of insects All experiments were conducted at 25 _t 2°C and batches of flies were conditioned for at least 1 hr at this temperature in normal laboratory lighting or in the dark before injecting them with [‘“Cl acetate. Insecticides in 2 ~1 acetone solution were applied to the dorsal surface of the thorax of flies immobilized with cyclopropane and held in fresh air without food or water for the required time (‘treated’ group). ‘Control’ groups were similarly treated with acetone only. Single flies of ‘treated’ or ‘control’ groups were then immobilized by exposure to cyclopropaneair mixture for 30 set and injected intrathoracically with 2.0 ~1 of stock solution of [2-r4C] sodium acetate equivalent to 0.8 ,umoles of salt and 2 PC of carbon-14. Flies which showed signs of injury, bleeding, etc. were rejected. Single injected insects were then held in small glass chambers in normal diffuse daylight, in the dark, or in a blue-filtered tungsten light flashing at an average frequency of about 3 c/s (i.e. equal dark or light periods of Q set) for a timed period of 1 to 120 min (‘metabolism period’). At the end of the metabolism period single flies were killed by exposing them to a jet of steam at 100°C for 30 sec. This rapidly inactivated the ACh enzyme system without loss of ACh from the head tissues. Entire flies were then dipped once in A.R. acetone to remove traces of condensed water, frozen in liquid nitrogen, and decapitated. Unthawed isolated heads in groups of 10 were homogenized for about 1 min at room temperature in a Potter-Elvehjem homogenizer containing 0.50 ml of 0.01 N HCl and 0.5 mg of non-radioactive acetylcholine bromide as carrier. The homogenate was centrifuged for 2 to 3 min at about 2500g. The clear supernatant was decanted off and stored at - 15°C pending analysis. Pharmacological assay of acetylcholine Acetylcholine in boiled housefly heads was assayed by means of the frog rectus abdominis muscle preparation exactly as described by LEWIS and SMALLMAN (1956), and in extracts prepared as above except for the omission of acetylcholine bromide carrier and the slight modifications tabulated (see p. 913).

912 Radiochromatographic

F. P. W. WINTERINGHAM assay of [‘“Cl acetylcholine formed in vivo

Labelled acetate injected into adult houseflies is very rapidly distributed throughout the tissues and metabolized, largely with the formation of [r4C] ketogenic amino acids (WINTERINGHAM,1956; PRICE, 1961). The [‘“Cl ACh formed in the head tissues was determined and separated from the large excess of other acidsoluble [r4C] metabolites by paper radiochromatographic analysis as follows. Aliquots (50 ~1) of extract were applied to l& in. strips of acid-washed Whatman No. 1 paper from an ‘Agla’ micrometer syringe and ascending chromatography allowed to proceed overnight at 27°C. in A.R. formic acid-acetone-water (14 : 60 : 26) mixture. The strip was dried in warm air and the carrier ACh zone (Rf about 0.95) located non-destructively by exposure to iodine vapour at about 40°C. The zone was reconcentrated on to a fresh strip of paper (WINTERINGHAM, 1953) and ascending chromatography repeated in A.R. formic acid-n-propanolwater (10 : 80 : 10) mixture. Carrier ACh was again located (rZ, about O-5), reconcentrated, and run overnight in n-propanol-water (90 : 10) mixture. The ACh was finally located (Rj about 0.3) and reconcentrated. A 14 in. x 1 cm section of the paper carrying the concentrated zone was cut out and suspended in 1.0 ml of 0.01 N HCI and briefly warmed to 100°C to dissolve the ACh. An ahquot of 0.50 ml was transferred to a 10 ml counting phial, 5-O ml of 7.5 per cent ‘Scinstant’ (Nuclear Enterprises (Great Britain) Ltd., NE-572) in dioxane added and carbon-14 activity assayed in the usual way at about 50 per cent counting sensitivity. Sensitivity was determined by the addition of 5.00 ~1 aliquots of [2-l*C] sodium acetate reference solution (see p. 911) as an internal standard and by recounting the samples. Two or more replicate chromatographic analyses were made of each extract. Replicates usually agreed to within the equivalent of 0.25 x lo-* pg ACh per head at the specific molar radioactivity of the injected acetate (see p. 914). Radiochemical purity and identity of the final [‘“Cl ACh fraction were indicated by exact co-chromatography with authentic ACh in the three separate solvent systems used and by the fact that mild alkaline hydrolysis of the extracts before paper chromatography eliminated carbon-14 of the final fraction. For this purpose O-02 ml of 1 N NaOH was added to O-2 ml of extract and the alkaline solution heated at 100°C for 5 min. The extract was reacidified by adding 0.03 ml of 1 N HCI and 62.5 ~1 aliquots applied to paper chromatograms to allow for dilution. Corrections were applied for losses of [‘*Cl ACh during chromatographic fractionation, concentration, etc. To determine this correction 5.00 ~1 aliquots of authentic p*C] ACh solution were added to initial chromatograms of hydrolysed extracts, and the [r4C]-activity finally recovered as the ACh fraction compared with that obtained by direct counting of 5-O ~1 aliquots under identical conditions. Recoveries varied from 76 to 93 per cent with a mean value of 82 per cent. A similar estimate of losses during paper chromatography by pharmacological assay of added and fractionated ACh indicated a mean recovery of 81 per cent. Although relatively tedious, paper chromatographic techniques were retained despite the advent of thin-layer chromatography since these studies were undertaken. Paper chromatograms had the important advantage of lending themselves to

METABOLISMAND SIGNIFICANCEOF ACETYLCHOLINEIN THE HOUSEFLY

913

automatic radiometric scanning. Typical radiochromatograms so obtained at the various stages of the ACh fractionation have been illustrated elsewhere (WINTERINGHAM, 1962b). Determination of acid-soluble [‘*Cl metabolites Five ~1 of extract were mixed with 5 ~1 of 0.5 N HCl on a small 1 cm glass square and dried with a jet of hot air to remove all free [W] acetate and excess HCl. The residue was dissolved in 0.5 ml of 0.01 N HCl and [l%] activity assayed as above. Determination of unmetabolized [““Cl acetate Five ~1 of extract were mixed with 5 ~1 of 0.05 N NaOH and dried as above. The residue was taken up in 0.5 ml of 0.01 N HCl and total [‘“Cl activity assayed as above. Free unmetabolized [‘“Cl acetate was obtained as the difference between total [‘“Cl activity and that determined as soluble metabolites above. RESULTS

Pharmacological assay of total ACh content of head tissues Results of the pharmacological assays for total ACh content of housefly heads are compared with those reported by LEWIS and SMALLMAN(1956) in Table 1. A lower ACh content was found in the present work than that reported originally by LEWIS and SMALLMAN(1956). Their result is probably more accurate since three separate extracts of 50 heads were used. The results of the present work were TABLE I-TOTAL

Group

ACh CONTENTOF HEADSOF ADULT HOUSEFLIES

Pretreatment of insects where different from that of LEWIS and SMALLMAN (1956)

Acetylcholine content (pmoles/head) 3.35 x 10-4

(1) (2) (3) (4) (5)

(6)

(7)

Steam-killed 30 set Steam-killed 60 set As in (3) ; duplicate As in (3) but insects first anaesthetized for 60 set in cyclopropane As in (2) but 0.8 pmoles NaOOC.CH, injected intrathoracically. Insects then held in dark for 20 min As in (6) but insects held in flashing blue light for 20 min

2.53 2.87 2.94 2.53 2.67

x x x x x

1O-4 1O-4 10-4 lo4 1O-4

Reference LEWIS and SMALLMAN (1956) Present work Present work Present work Present work Present work

2.94 x 10-t

Present work

2.87 x 1O-4

Present work

914

F. P. W. WINTERINGHAM

obtained on single extracts of 10 heads (see p. 911). There was no evidence of effects on total ACh content as a result of the special treatments employed for the present study. [‘“Cl Acetate metabolism in head tissues The overall metabolism of the injected [““Cl acetate and its persistence as free acetate are shown by the curves of Fig. 1. It will be seen that within 1 min of [‘“Cl acetate injection some 20 per cent of the injected acetate appeared within the head tissues as free acetate and that free [‘“Cl acetate was detectable up to 40 min. The curves also indicate an increase in the acid-soluble [‘“Cl fraction until a maximum was reached between 20 and 30 min. Whether the insects were held in the dark or in flashing light after the injection appeared to have no effect on the total metabolism or persistence of the [‘“Cl acetate of the head tissues.

Time min

.%ul CI

20

40

60

FIG. 1. Free [‘“Cl acetate and total acid-soluble [‘“Cl metabolites in head tissues of the adult housefly following intrathoracic injection of 0.8 pmoles of sodium [2-14C] acetate. Insects in darkness, 0 ; flashing blue light, A.

Incorporation of [‘“Cl acetate into the acetylcholine fraction of the head tissues in vivo The formation of [‘“Cl ACh in the head tissues of the intact insect as a function of time after injecting [2J4C] acetate is shown in Fig. 2. The data are expressed in pmole [‘“Cl ACh per head at the specific molar radioactivity of the injected acetate, i.e. assuming no isotopic dilution of the labelled precursors during the metabolism period. Most groups of insects were conditioned and held in normal laboratory lighting, but in some experiments (1 min and 5 min metabolism periods) the insects

were conditioned in total darkness and then held in darkness after the injections or exposed to flashing light. There was no evidence that the different conditions of lighting during the period of metabolism affected the rate of [‘“Cl ACh formation.

METABOLISM ANDSIGNIFICANCE OFACETYLCHOLINE IN THEHOUSEFLY

zu

n z -$ cl

5 3*0x10-4 .$ 0 .z

A

.t; Eu, =Li’

~.___‘____~___‘________________________

.z 24)*1l)-c

‘0’

f

915

.-g

1.0~10

-4

l 0 2cf

40 Time

60

120

min

FIG. 2. Incorporation of [14C] acetate into the ACh fraction of the head tissues of the adult housefly following intrathoracic injection of 0.8 pmoles of sodium [2J4C] acetate. The calculated curve (see Discussion) is shown as a broken line. Insects in normal light, 0 ; darkness, A ; flashing blue light, 0. EfJects of acetylcholinesterase inhibitors on [‘“Cl acetylcholine formation in vivo The effects of topically applied inhibitors of acetylcholinesterase on the formation of [‘“Cl ACh and upon the physiological state of the insects are compared in Table 2. The results are expressed as the ratio: pmole [“*Cl ACh formed in heads of the treated group/pmole [‘“Cl ACh formed in the control group. It will be seen that, whether phosphorylating or carbamylating agents, all the inhibitors caused a reduction of [14C] ACh synthesis, this reduction tending to be greatest when signs of poisoning suggested greatest inhibition of AChE in Go. It is interesting to note that these reductions of [‘“Cl ACh synthesis occurred under conditions which, if anything, were likely to have induced slight increases in the ACh content of the head tissues over the metabolism periods studied (cf. the data of LEWIS and FOWLER, 1956; SMALLMANand FISHER,1958; COLHOUN,1959). In separate experiments the oxygen consumption, respiratory quotient (R.Q.) and 14C02 output of DFP-poisoned flies (10 pg topically applied for 30 min prior to [‘“Cl acetate injection) were compared with those of control flies. Oxygen consumption was measured by means of the larger electrolytic respirometer described elsewhere (WINTERINGHAM,1959). Carbon dioxide and 14C02 absorbed in samples from the alkali well of the respirometer were determined in a combined manometric and counting unit also described earlier (WINTERINGHAM,1955). The results are summarized in Figs. 3 and 4. It will be seen that DFP treatment stimulated oxygen consumption, 14C0, output, and caused a slight progressive decrease in the acid-soluble [14C] metabolites of the whole insect, but had no significant effect on R.Q. over the 5 hr period (coefficient of variation associated with the R.Q. measurements was f 5.4 per cent). Although not shown in Fig. 3, DFP also had no significant effect on the proportions of acid-soluble [““Cl metabolites present

F. P. W. WINTERINGHAM

916

TABLE ~-EFFECTS OF TOPICALLYAPPLIEDAChE

Time allowed for Condition of insects [‘“Cl acetate metaat time of [‘“Cl bolism (lighting) acetate injection (mm)

Experiment No.

Treatment of insects before injection of [2-r4C] acetate

(1)

0.5 pg DFP. Held in normal light for 120 min 2.0 pg DFP. Held in normal light for 30 min 5.0 pg DFP. Held in normal light for 30 min 10.0 pg DFP. Held in normal light for 30 min As in (4) As in (4) 10 pg DFP. Held in darkness for 30-90 min As in (7) ; duplicate 10 pg TEPP. Held in normal light for 120 min 10 pg malathion. Held in normal light for 180 min 5 pg o-IPMC. Held in darkness for 30-90 min As in (11) ; duplicate

(2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

(12)

INHIBITORS [““Cl ACh of treated group [‘“Cl ACh of control group

Slight signs of excitability only Just knocked down

10 (normal)

0.72

10 (normal)

0.63

Prostrate

10 (normal)

0.27

Prostrate

10 (normal)

0.25

Prostrate Prostrate Prostrate

5 (normal) 30 (normal) 1 (flashing light)

0.24 0.15 0.17

Prostrate Just knocked down

1 (flashing light) 10 (normal)

0.22 0.40

Prostrate

10 (normal)

0.67

Prostrate

1 (darkness)

0.60

Prostrate

1 (darkness)

0.33

in the head tissues. These data demonstrate clearly that the effects of DFP on [‘“Cl ACh synthesis, and almost certainly the similar effects of the other AChE inhibitors, were not due to an overall inhibition of respiratory metabolism or of acetate oxidation.

1

I

I

I

I

10

20

30

40

50

Time of r”C]

acetate

‘/GY--60

metabolism

120 min

FIG. 3. Effects of 30 min exposure to 10 pg DFP applied topically on 14C02 expiration and formation of [‘“Cl acid-soluble metabolites. Expired 14C0, ratio, n ; acid-soluble-i4C ratio, 0.

METABOLISM

AND SIGNIFICANCE

OF ACETYLCHOLINE

IN THE HOUSEFLY

917

Effects of cutting of the central nerve cord between the thoracic ganglion and brain No correlation was found between [‘“Cl ACh synthesis and the incidence of visible light on the compound eyes (Fig. 2). It was possible that electrical activity and associated ACh turnover of the brain might be stimulated by activity of the thoracic ganglion. To test this possibility the [‘“Cl ACh synthesis was determined in insects in which the connexion between the two ganglionic regions had been severed. Single insects were pinned on their backs under a dissecting microscope while under cyclopropane anaesthesia. The prothoracic legs were amputated by cutting through the coxae near to the body. The fine point of a 1 mm scalpel blade was inserted through the one thoracic stump and a transverse cut made through the stergal sclerite between the two stumps. A fine, hook-shaped needle was then inserted through the incision and rotated to engage the central nerve cord anterior to the thoracic ganglion (cf. Fig. 1 of MOLLOY, 1961). The cord was gently raised, cut, and replaced. The wound was covered with molten paraffin wax (m.p. 54°C). This surgery resulted in loss of the proboscis extension and head movement responses to stimulation of the tarsal chemoreceptors with O-1 M sucrose (cf. SMYTH and ROYS, 1955). Ten live insects treated in this manner were collected and allowed to recover before injection. A control group received identical treatment except for severing the cord. [2-14C] Acetate was injected in the usual way and metabolism allowed to proceed for 5 min in the dark before killing, extraction, etc. [‘“Cl ACh recovered from the heads of treated and control groups was equivalent to 1.62 x 1O-4 and 1.47 x 1O-4 pmole respectively (cf. Fig. 2), this difference almost certainly not being significant. Isolation of the brain in this manner had not, apparently, reduced [‘“Cl ACh synthesis. Eflects of cyclopropane anaesthesia and/or dieldrin poisoning on [““Cl acetylcholine formation The effects of sustained cyclopropane anaesthesia on the [‘“Cl ACh formation in normal or in dieldrin-poisoned insects are shown in Table 3. In these experiments there was some variation in the acetate metabolism times but the average time of metabolism of 5 min was the same for the treated and control groups. It will be seen that anaesthesia alone reduced the [““Cl ACh formation whether the insects were normal (exp. 1) or at an advanced stage of dieldrin poisoning (exp. 3) at the time of acetate injection. Under the conditions of these experiments it was unlikely that cyclopropane had affected [““Cl ACh formation indirectly as a result of partial anoxia and a consequent fall in ATP and the ATP-dependent acetyl coenzyme-A synthetase activity. Levels of ATP in the head tissues appeared normal after comparable anaesthesia with either cyclopropane or 1,Zdichloroethane (WINTERINGHAM,1960). Dieldrin poisoning alone (exp. 2), apparently, had no effect but at the time of the acetate injections the insects had already passed through the earlier convulsions which are a typical sign of dieldrin poisoning in houseflies. The effects of dieldrin on oxygen consumption (see above) of non-anaesthetized insects were determined

918

F. P. W. WINTERINGHAM

TABLE 3-EFFRCTS

Experiment No.

OF CYCLOPROPANR ANAJXSTHESIAAND/OR DIELDRIN TREATMENT

Conditions and treatment of insects during [‘“Cl acetate metabolism period (5 min)

Treatment of insects prior to [i4C] acetate injections

-

(1)

[““Cl ACh of treated group [““Cl ACh of control group

Insects motionless as a result of sustained cyclopropane anaesduring metabolism thesia period

0.68

but showing No anaes-

0.99

knocked down but showing leg movements at time of the injection. Then motionless after injection as a result of sustained anaesthesia during metabolism period

0.73

(2)

0.37 pg dieldrin. Held 2-4 hr in normal light without food or water

All

(3)

As in (2)

All

knocked down leg movements. thesia

in a separate experiment and also shown in Fig. 4. It will be seen that at the time of the acetate injection (2-4 hr), oxygen consumption of the dieldrin-poisoned insects remained at a higher level than that of the control but the rate of [“*Cl ACh formation was apparently normal. Increased oxygen consumption per se was unlikely, therefore, to account for the reduced [‘“Cl ACh formation observed as a result of DFP treatment (see p. 915).

Time

hr

FIG. 4. Effects of exposure to 10 pg DFP or 0.37 pg dieldrin applied topically on oxygen consumption and effects of DFP on 5 hr respiratory quotient (R.Q.). Dieldrin control, 0 ; dieldrin-treated, A; DFP control, 0; DFP-treated, A.

METABOLISM

AND SIGNIFICANCE

OF ACETYLCHOLINE

IN THE HOUSEFLY

919

DISCUSSION

Turnover rate of brain acetylcholine in vivo The data of Fig. 1 indicate that after reaching a maximum after 20 min the total [‘“Cl acid-soluble fraction of the head tissues declined relatively slowly during the next 40 min. During this period this fraction consists largely of ketogenie [‘“Cl amino acids derived from the [‘“Cl acetyl-coenzyme A pool. The data of Fig. 1 and those of PRICE(1961) show that the free [‘“Cl acetate continued to fall off after 20 min in the head and total body tissues respectively so that after 1 hr free Ip-“C] acetate could not be detected. These observations imply that the level of free acetate was greatly in excess of the normal physiological level for at least 15 min after the [‘“Cl acetate injection. Therefore, an effectively constant specific radioactivity of the tissue acetate equal to that injected can also be assumed for the same period. The fact that the glycolytic supply of endogenous acetate was likely to be inhibited in the presence of an added excess of tissue acetate (WILLIAMSON, 1965) is further justification for the assumption. It follows that if the pharmacological activity of the head extracts be due entirely to the acetyl derivative of choline the maximum level of [““Cl ACh reached in 15 min or so (Fig. 2) should coincide with that determined pharmacologically. The mean level indicated by the data of LEWIS and SMALLMAN(1956) is shown as that portion of the dotted line parallel to the abscissa of Fig. 2 and demonstrates a remarkable agreement between the radiometric maximum and pharmacological level. The steady-state level determined pharmacologically in the present work was lower (cf. Table 1) than that indicated radiometrically (Fig. 2). Th is may have been due to a greater extraction efficiency for the [‘“Cl ACh assays owing to the presence of the ACh carrier during extraction. The virtually constant level of total ACh found in the head tissues of the adult housefly under a variety of conditions and the data of Fig. 2 imply a steady-state of synthesis and destruction in the brain, the brain (and optic lobes) being the principal sites of AChE activity of the head tissues (MOLLOY, 1961). If the specific radioactivity a of the [‘“Cl acetate precursor in vivo be constant until the [‘“Cl ACh has achieved its maximum and if x be the specific radioactivity of [‘“Cl ACh t min after injection it follows that dx/dt =K(a - x)/w,

(1)

where K is the rate of synthesis and equal rate of hydrolysis, and w the steadystate weight of total ACh per head. Since x = 0 when t = 0 integration gives: 1 -x/a

= e--ktf*.

(2)

This equation represents the steady-state labelled precursor-product relationship when the specific activity of the precursor remains constant (cf. ZILVERSMIT et al., 1943). The accumulation of [‘“Cl ACh as a function of time has been calculated from equation (2) and shown as the broken line in Fig. 2. For this purpose the value of 3.35 x 1O-4 pmole/head (LEWIS and SMALLMAN, 1956) was substituted

920

F. P. W. WINTERINGHAM

for w and Ktaken as the initial slope of the curve in Fig. 2 (6.1 x 1O-5 pmole/min/ head). Up to about 20 min the observed and calculated data almost certainly agree within experimental error. After 20 min the level of observed [““Cl ACh would be expected to fall off with the inevitable isotopic dilution of the [‘“Cl acetyl coenzyme A pool. On this basis the initial slope of the curve of Fig. 2 will represent the steady turnover rate of the total ACh of the active brain in vitro and is 6.1 x 1O-5 pmoles/ min/head. Differences in [‘“Cl ACh formed over periods of less than 10 min will then indicate differences in turnover rate. However, until some correlation can be unequivocally demonstrated in Z&J between the turnover rates estimated in this manner and nervous activity, the possibility of some rate-limiting step outside the synaptic region remains. In this event the turnover rate in the synaptic region might be faster than that estimated here and changes in turnover rate with physiological activity over and above the estimated minimum might remain undetected. E/ffects of acetylcholinesterase inhibitors The results of Table 2 demonstrate a marked reduction in apparent turnover rate under conditions of AChE inhibition. Yet these inhibitors have little effect on ACh synthesis in vitro and are, indeed, usually added to ensure accumulation of the ester by inhibiting the AChE normally present (e.g. see SMALLMAN, 1956). Similarly, these inhibitors were unlikely to have inhibited acetyl coenzyme A formation (see earlier). It follows that either ACh synthesis in vivo is tightly coupled to AChE activity as a hydrolytic enzyme or the formation of [‘“Cl ACh is a result of an AChE-dependent [‘“Cl acetate-ACh exchange reaction. The latter would require intimate contact between the total ACh and [““Cl acetate at the enzyme surface, which is incompatible with current views on the intracellular distribution of the components of the ACh system as studied in vertebrate brain (WHITTAKER, 1959; WHITTAKER and GRAY, 1959; WHITTAKER et al., 1964). The exchange reaction per se was also shown to be unlikely by the following experiment: Purified bovine erythrocyte acetylcholinesterase (E.C. 3.1.1.7) at a nominal concentration of 0.02 units/ml (I.U.B. definition) was incubated for 30 min at 37°C with initial concentrations of 1O-3 M ACh and 8 x 10m3 M sodium [2J4C] acetate (2.5 PC/m-mole) in buffer at pH 7.4. During this time when a little more than 50 per cent of the substrate was hydrolysed enzymically, samples were taken at 5 min intervals. Any [i4C] ACh formed during the incubation and, excess [‘“Cl acetate were separately assayed. At no stage could [““Cl ACh be detected. It is concluded, therefore, that in the synaptic region of the intact brain ACh synthesis and enzymic hydrolysis are tightly coupled so that synthesis proceeds only as one or both products of AChE activity become available. The ready access to the synaptic region of tissue acetate and its utilization by the ChA system indicated by the present studies point to the choline ion as the substrate through which synthesis and hydrolysis are linked. While rate of synthesis may be effectively controlled by local choline availability, the limitation on total ACh content is likely to be imposed structurally, e.g. by the capacity of the ‘synaptosome’ system

METABOLISM

AND SIGNIFICANCE

OF ACETYLCHOLIN-E

IN THE HOUSEFLY

921

described by WHITTAKERet al. (1964) for vertebrate brain. A highly compartmentalized ACh system in the synaptic region of the housefly brain can thus be visualized. This system contains all the components for limited storage, coupled synthesis and hydrolysis of ACh, and, probably, includes acetyl coenzyme A synthetase (E.C. 6.2.1.1.) to account for the rapid and direct use of acetate. SCHUBERTH(1965) h as reported the presence of this enzyme in nerve endings of rat brain. Despite the rapid turnover of ACh in erivo, the potential reservoirs of glycolytically formed acetate and choline as lecithin or phospholipid intermediates (cf. BRIDGESet al., 1965), there is not the rapid accumulation of ACh in viva to be expected as a result of AChE inhibition (LEWIS and FOWLER, 1956; SMALLMAN and FISHER, 1958; COLHOUN, 1959). This may now be seen as a blocking of the supply of free choline for synthesis and its compartmentalization. Further evidence of such compartmentalization is provided by the observations of BRIDGESet al. (1965), who found that when dietary choline of housefly larvae was replaced by a source of p-methylcholine the phosphatidylcholine content of the adult tissues was only 6 per cent of the normal level compared with an acetylcholine content of the head tissues equivalent to 30 per cent of the normal level. This suggests that choline of the central nervous system is protected against dietary deficiencies of choline and the corresponding changes in phospholipid choline. The relative insensitivity of insect central nerve to ionized forms of AChE inhibitors and to ACh (O’BRIEN, 1959; O’BRIEN and FISHER, 1958) has been ascribed to structural barriers such as the neural lamella although in the absence of the latter relatively high concentrations of ACh were still required to affect synaptic transmission in the last abdominal ganglion of the cockroach (TWAROG and ROEDER;1957). On the other hand, TREHERNEand SMITH (1965) found a rapid penetration of [‘“Cl ACh into the nerve cord of cockroach and concluded that the nerve sheath is not a significant barrier. The present studies have indicated a high degree of compartmentalization of ACh and of the factors which regulate its turnover in the synaptic region. It seems unlikely, therefore, that endogenous ACh or choline of the central nerve would achieve any pharmacologically significant equilibrium with the externally applied ions. Role of acetylcholine in insect brain function Despite the high concentration of cholinesterase in the region of the optic lobes (MOLLOY, 1961) and high concentrations of synaptic structures associated with optical reception (BULLOCKand HORRIDGE,1965), no correlation was found between overall ACh turnover and exposure of the compound eyes to light. LANGER(1962) has reported that exposure to light (254-436 rnp) of the compound eyes of isolated heads of CaZZiphora stimulated carbohydrate catabolism. However, the present studies have shown that exposure to AChE inhibitors under conditions likely to block central synaptic transmission reduced ACh turnover. Sustained cyclopropane anaesthesia also apparently reduced ACh turnover, but in these experiments possible effects on the total ACh content were not determined. However, in

F. P. W. WINTERINGHAM

922

vertebrates

brain anaesthesia has been found, if anything, to increase ACh content KUROKAWA et al., 1963). Of considerable interest in this context is the, report by STERNBURG and HEWITT

(RICHTER and CROSSLAND, 1949; (1962)

that central nerve AChE

subsequent

inhibition

of DDT-poisoned

by TMPP

and TEPP

cockroaches in viva while

was protected that

from

of unpoisoned

cockroaches was not. These workers rejected the possible protection of AChE in vivo by its own substrate. However, for the reasons discussed, while stimulated

central nerve activity would not be expected to cause a significant increase in total ACh it would be expected to increase turnover and, therefore, the effective concentration at the active sites of synaptic AChE. Recent studies of the kinetics of carbamate inhibition of AChE (WINTERINGHAM and FOWLER, in press) have shown that in the presence

of relatively

low concentrations

of ACh (1O-3 M) the enzyme

which does in fact occur in vivo, therefore indicates that the effective concentrations of ACh at the active sites of the enzyme are normally very low. Stimulated central activity may, accordingly, result in relatively large increases in effective substrate concentrations at the enzyme sites. This would not, however, be reflected in detectable changes in total ACh content of the central nervous system and may, indeed, only involve a small fraction of the total ACh. STERNBURG and HEWITT’S observations thus provide is largely protected.

indirect

evidence

as a result of DDT

The rapid inhibition

of increased

ACh

by carbamates,

turnover

during

insect

central

nerve

activity

poisoning.

Acknowledgements-The author is indebted to Mrs. ANN BROOKS,Miss M. A. MCKAY, Messrs. D. EVANS,K. S. FOWLER, A. HARRISON,and J. B. WALLER for assistance at various stages of the experimental work, and to Mr. S. E. LEWIS for advice on the pharmacological assay of acetylcholine.

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