Studies on action mechanisms of a possible false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium

Life Soiencea Vol. 14, pp . 1721-1733 Printed in U.S .A .

Pergamon Press

STUDIES ON ACTION MECHANISMS OF A POSSIBLE FALSE CHOLIMER6IC TRANSMITTER, (2-HYDROXYETHYL) METHYLDIETHYLAMMONIUM . Chung Y. Chiou Department of Pharmacology and Therapeutics University of Florida College of Medicine Gainesville, Florida 32610 (Received in final form 26 !larch 1974) SUMMARY (2-HydraX~ethyl) methyldiethylammoniun (DEC ; Diethylcholine) was found to inhibit cholinergic fibers slowly, both in skeletal muscle (ED5Q : 2 .25 x 10-5 M in chick biventer cervicis and 42 mg/kg in rat sciatic-gastrocnemius) and in smooth muscle preparations (EDSp: 7 .7 x 10 -4 M in transmurally stimulated guineapigi lean) without having any effect on dose-response curves of acetylcholine to contract chick biventer cervicis, frog rectus abdaminis and guinea-pig ileun, These results indicate that DEC acts at the prejunctional nerve fibers, but not at the postjunctional chollnergic receptor sites. DEC was acetylated efficiently both by choline acetyltransferase and by minced rat brain, suggesting that it can be acetylated to acetyl-DEC in the nerve ending . Acetyl-DEC was found to block acetylcholine actions competitively both in smooth and in skeletal muscle preparations (1 x 10-3 - 1 x 10 -2M) indicating that the acetylated product of DEC can serve as an antagonist at the cholinergic receptor site . It is therefore concluded that DEC is a false cholinergic transmitter . Attempts have been made to find an ideal false neurotransmitter in cholinergic systems, and triethylcholine (TEC) was once thought to be a promising one (2,4) .

However, since then TEC has been classified as

a hemicholinium-like agent which inhibits the synthesis of acetylcholine (ACh) (2,3), rather than as a false transmitter, because the acetylation of TEC by choline acetyltransferase (ChAc) could not be denonstrated (6,8) . In addition, acetyl-TEC is not an agonist nor an antagonist, and these activities would be essential in a false transmitter (1,9). 1721

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Paralyais of Cholinergic Neurone by DEC

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The possibility of using diethylcholine (DEC) as a false transmitter has been explored in this study because (a) DEC resembles choline more closely in molecular structure than does TEC; therefore, DEC could be acetylated more easily than TEC in the nerve ending ; and (b) acetyl-DEC is practically devoid of stimulant effects on the cholinergic receptor (g), which is one of the important requiranents for a false transmitter . Although the molecular strucutre of monoethylcholine is even closer to that of choline than is that of DEC, its activity as a false transmitter is ruled out because acetylmonoethylcholine is known to produce fairly strong cholinergic responses (g) . In this study the pharmacology and action mechanism of DEC have been investigated along with those of choline and TEC.

It is concluded from

the experimental data obtained that DEC is a false cholinergic transmitter . Methods Materials :

Diethylcholine iodide (hydroxyethyl methyldiethylammonium

iodide ; DEC) and acetyldiethylcholine iodide (acetoxyethylmethyldiethylammonium iodide ; acetyl-DEC) were synthesized in this laboratory by standard methods (g) .

The drugs used in this study included neostigmine

brromide, choline iodide, acetylcholine iodide (ACh), nicotine tartrate and acetyl-1-14 C-coenzyme A (acetyl-1- 14C-CoA, specific activity of 50 mCi/mM) (Int . Chem . b Nuclear Corp .) .

Choline acetyltransferase

(ChAc) was obtained from acetone powder of rat brain extracted with the method of Burgen et al . (4) . Rat blood pressure and sciatic nerve-9astrocnemius muscle preparation : Holtzman rats, weighing 300-400 g were anesthetized with 1 .6 g/kg of urethane .

The carotid artery was canulated for blood pressure determination

and the jugular vein for drug injection .

The sciatic nerve was exposed,

cut and stimulated with a pair of electrodes .

The parameters of interrupted

tetanic stimulation used were 250 Hz frequency, 0 .5 msec pulse width and a supramaximal voltage (4-8 volts) for 0.1 sec every 10 sec.

The gastroc-

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Paralysis of Cholinergic Neurons by DSC

1723

nemius muscle was freed from connective tissues and its tendon was tied with a thread which lead to the force transducer .

Drug solutions were

made with saline and injected into the rat with doses b.garithmically spaced .

The doses required to block 50% of the neuromuscular transmission

were determined with probit plot analysis (10) .

The hypotensive responses

of drugs were recorded and observed simultaneously, along with the neuromuscular blockade .

In order to study the choline reversal on neuromuscular

blockade induced by DEC, 10 mg/kg of choline was injected into the jugular vein 20 min after the injection of DEC. Superfused frog r~ectus abdominis muscle :

Rana

ip piers weighing 20-25 g

were decapitated and the rectus abdominis muscles were isolated (5) . Each muscle was mounted on a superfusion assembly and superfused with frog Ringer's solution (NaCI,

111 ; KC1, 1 .9 ; NaHC03, 4 .8 ; CaC1 2, 1 .5 ;

and glucose,ll .l mM) oxygenated with 95% 02-5% C02 at 25 °C .

The flow

rate of the superfusate was 3 to 4 ml/min and ACh solutions were injected into the streams of superfusate in volumes of not more than 0.1 ml .

The

isometric contractions of the muscle were measured with a force transducer and were recorded on a polygraph .

Dose-response curves of ACh were

constructed before and during superfusion of the muscle with various concentrations of DEC (1 x 10

4

- 1 x lÔ 2M) and acetyl-DEC

(1 x 10

-4

-

1 x 10 -2M) . Chick biventer cervicis nerve muscle preparation :

Chicks, 10 to 14 days

old, were killed with chloroform and the biventer cervicis muscles were dissected (7) .

For superfusion experiments, the muscle was mounted on a

superfusion assanbly and superfused with Tyrode's solution

(NaCI, 137;

KC1, 2 .7 ; CaC1 2 , 1 .35 ; MgC1 2, 1 .0 ; NaHC03 , 11 .9 ; NaH2P04, 0.36 ; dextrose, 11 .1 mM) oxygenated at 37 °C .

The same method described in the last section

was used to measure muscle contraction .

The dose-response curves were

constructed before and during superfusion of the muscle with various

Paralysis of Cholinergic Neurons by DSC

172 4

concentrations of DEC (1 x lÔ

4

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- 1 x lÔ 2M) and acetyl-DEC (1 x 10

4

-

1 x 10 2M) . For electrical stimulation the preparation was suspended in an organ bath of 40 ml capacity with a pair of platinum electrodes placed on the nerve tendon .

The nerve was stimulated with 250 Hz frequency, 0.5 cosec

pulse width and a supramaximal voltage for 0.1

sec every 10 sec .

Drug

solutions were added into the organ bath and the ~ of blockade of neuromuscular transmission was measured 60 min after the addition of drugs. ~50~s

were again determined with probat plot analysis (10) .

Guinea-aig ileum areaaration:

The terminal i11a were obtained from guinea-

pigs, weighing 300 to 400 g and were mounted on a superfusion assembly and superfused with oxygenated Tyrode's solution at 37°C.

The methods

used for measuring muscle contractions and for constructing dose-response curves before and during superfusion of the muscle with DEC (1 x 10

1 x 10 2M)

and acetyl-DEC (1 x

10

4

-4

-

- 1 x 10 2M) were the same as those

described in the section of the superfused frog rectus abdaminis muscle preparation . For transmural stimulation, the ileun was suspended in an organ bath of 40 ml capacity .

The lower end of the ileum was tied to a piece of

glass tubing frorom which an electrode protruded .

The upper end of the

ileum was attached to an isanetric force transducer with a coaxial circle of wire place at the upper end as the other electrode .

The intrinsic

nerves were stimulated with 10 Hz frequency, 0.5 cosec pulse width and a supramaximal

voltage for 1 sec every 10 sec.

This stimulation was known

to excite postganglionic parasympathetic fibers (11) .

The ~ of blockade

of the neuromuscular transmission and the ED 50 to inhibit neuromuscular transmission were determined as described in the last section . Acetvlation of DEC and its analogs:

Holtzman rat brain was used to make

acetone powder which was extracted with ice-cold extraction medium (NaCI, 100 mM ; disodium ethylenediamine tetraacetate, 1 mM ; cysteine,4 mM ; bovine

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Paralysie of Cholinergic Neurons by DBC

serun albuni n, 0 .05 ; and NaH2P04Na 2HP04, 10 mM at pH 7.0) (4) .

1725 The

acetone powder was extracted with the extraction medium (100 mg/4m1) for 20 min with constant stirring .

The extract was then centrifuged at 9,000 x

g at 4oC for 10 min and the supernatant fraction containing ChAc was retained . 0.05 M1 of enzyme extract was used in each incubation tube .

The

final volume of the incubation median was 0.2 ml with the following compositaon :

NaCI, 150mM ; neostigmine, 0 .05mM: substrate (DEC, TEC or choline),

5QuM ; acetyl CoA, 0 .6ni~i ; acetyl-1- 14C-CoA, 0.067mM and NaH2 P04Na 2HP0 4 (pH 7), 25mM (12) .

After 30 min incubation at 37 °C, the reaction was

stopped by adding 2 .8 ml of ice-cold water and immediately thereafter 500 mg of Bio-Rod A6 1 x 1 anion exchange resin to remove unreacted acetyl CoA.

After vigorous stirring for 30 sec, each incubation tube was

centrifuged for 5 min at 1,500 x g and 0.5 ml of the supernatant fraction was transferred to 10 ml of the scintillation solution (2,5-diphenyloxazole, 7g ; naphthalene, 100g and dioxane, 1,000 ml) and was counted 1n a Beckman liquid scintillation counter.

The remaining supernatant fluid was trans-

ferred to a clean centrifuge tube and 250 mg of Amberlite CG-120 cation exchange resin was added to trap choline derivatives .

After stirring and

centrifuging as above, 0.5 ml of the supernatant was again added to 10 ml of scintillation fluid for counting . The amount of acetylated product was calculated by subtracting the cpm obtained after both anionic and cationic exchange from the cpm obtained after anionic exchange alone.

10,000 cp~n is equivalent to 8.4 nmoles of

acetylated product synthesized/hr/mg acetone powder . Minced rat brain and its supernatant solution were also used instead of ChAc isolated from the acetone powder of rat brain to study the acetylation of DEC .

These experiments showed the uptake of DEC by the nerve ending,

and its acetylation at the intracellular site .

All experimental procedures

were the same as those described above : except that the isolated ChAc was

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Paralysie of Choliaergic Neurons by DEC

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replaced with minced rat brain, 10 mg wet weight in each incubation medium, or the supernatant solution of the minced brain tissue . Toxicity :

The LD 50 's of DEC, TEC and choline were determined with ICR

Charles River mice weighing 20-30g, according to Litchfield and Wilcoxon (10) .

The drugs were administered intraperitoneally with a microsyringe . Results

Effects of DEC on somatic fibers and the blood pressure :

DEC is able to

inhibit somatic fibers both in vivo (rat sciatic nerve gastrocnemius muscle preparation) and in vitro (chick biventer cervicis nerve muscle preparation) (Table 1) .

In in vitro experiments the neuromuscular transmission was

blocked slowly (40-60 min) after the administration of DEC and recovered slowly but steadily after washing .

In in vivo experiments, the ED 50 's of

DEC, TEC and choline to block sciatic nerve-gastrocnemius transmission were roughly the same (Table 1) .

The neuromuscular blockade induced by

DEC was reversed by choline indicating that choline and DEC are taken up by nerve endings through the same carrier system (Fig . 1) .

It is interesting

to note that the blood pressure was depressed by TEC and choline but not by DEC (Fig . 2) . Inhibition of parasympathetic fibers :

DEC was the most potent drug among

DEC, TEC and choline in blocking neurotransmission in transmurally stimulated guinea-pig ileum (Table 2) .

TEC was the poorest of all in inhibiting the

FIG. 1 Effects of choline (10 mg/kg) on the neurrorouscular blockade induced by diethylcholine (DEC, 50 mg/kg) in rat sciaticnerve-gastrocnemius muscle in vivo preparation . Time scale is one min each .

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172 7

Paralysie of Cholinergic Neurone by DEC TABLE 1 ED50's of Diethylcholine and its analogs to Block Neuranuscular Transmissions

rug

N

is iventer cervic s in vitro (M x 10 5)

1

Rat sciatic gastrocnaoius in vivo (m9/~

DEC

7

2.25(1 .18-4.28) a

6

42(21-84)

TEC

6

60(32-111)

6

28(18-44)

Choline

6

1280(711-2304)

5

46(19-115)

a95% confidence limits in parentheses

TABLE 2 ED~p 's of Diethylcholine and its Analogs for Blockade of Neurotransmission in :guinea-pig ileum is vitro ~~

U

Drug

N

DEC

5

7.70

TEC

4

>370

Choline

5

260

(M x 10 4)

TABLE 3 Acetylation of Diethylcholine and its Analogs by Rat Brain Choline Acetyltransferase Su strate

Choline

N

Re ative rate of acetylation (%)

Rate o

acety an on n mo es/ r/mg acetone ~owder) mean _ SE

14

100

5 .90±0.14

DEC

8

67

3 .92±0.07

TEC

8

24

1 .40+0.16

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Paralyaia of Cholinergio Neurone by DSC

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m x E E

FIG . 2 Effects of diethylcholine (DEC, 50 mg/kg), triethylcholine (TEC, 25 mg/kg) and choline (50 mg/kg) on the blood pressure of the rat . parasympathetic neuron and did not block 50% of the neurotransmission even at very high concentrations Acetylation of DEC by ChAc :

(37mM) .

DEC was acetylated efficiently both by isolated

ChAc (Table 3) and by minced rat brain (Table 4) .

DEC has been noted to

be acetylated as efficiently as choline with ChAc by Burgen et al . (4) . On the contrary, however,

same investigators failed to detect an appreciable

extent of DEC acetylation by ChAc (6,8) .

In this study, DEC was found to

acetylate only at a slightly lower rate than choline, but this was sufficient to inhibit the nervous tissues .

TEC was acetylated rather poorly compared

to choline and DEC (Tables 3 and 4) .

These latter results supported the

reports of Dauterniann and Mehrotra (6) and Hemsworth and Morris were ûnable to detect any acetylation of TEC .

(8) who

No ACh was synthesized

when supernatant solution of minced brain was used, indicating that no

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Paralysio of Choliaargio Netsrax~s by DSC

significant amount of ChAc was present in the extracellular fluid of the minced brain preparation . TABLE 4 Acetylation of Diethylcholine and its Analogs by minced rat brain

raté

Substrate

N

Choline

5

100

0 .89±0 .013a

DEC

5

61

0 .54±0 .008

TEC

5

39

0 .35±0 .005

Relative of acetylation (~)

Rate of acetylation (n moles/hr/mg minced brain) mean ± SE

a No ChAc activity was detected when supernatant solution of minced brain was used instead of minced brain itself . Effects

of

DEÇ and Açe~l- DEC on ACh responses :

DEC did not stimulate

smooth muscles nor skeletal muscles at the doses studied .

It was also

noted that acetyl-DEC stimulated the cholinergic receptors very poorly, which supported the finding of Hofton and Ing (9) .

Figs . 3, 4, and 5

show that DEC, up to 1 x lÔ 2M, had no effect on the dose-response curve of ACh for the contraction of guinea-pig ileum, frog rectus abdaninis and chick biventer cervicis, whereas acetyl-DEC shifted the dose-response curve of ACh to the right in a parallel fashion at concentrations at 1 x 10 3h1, 1 x lÔ 3M and 1 x 10 2M respectively .

These results indicate

that DEC itself is devoid of effect on the cholinergic receptor, while acetyl-DEC is a competitive antagonist at the cholinergic receptor site . Toxicity :

Table 5 shows the LD 50 's of the drugs studied .

Although the

LD~'s of DEC and TEC were fairly close, TEC killed mice within 60 min, whereas with DEC it took several hours for the mice to die .

No death

occurred after injection of choline at doses as high as 800 mg/kg .

1729

1730

Paralysis of Cholinergic Neurons by DEC

Vol . 14, No . 9

TABLE 5 LD 50 's of Diethylcholine and its Analogs in Mice

Drug

LD 50 (mg/kg)

DEC

69(52-92) a

several hours

TEC

79(62-101)

within 60 min

Choline

Duration between drug administration and death

~ 800

---

a95~ confidence limits in parentheses

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1

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1

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I

r

I

w

AChINQ)

Fis .

1

r

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Effects of diethylcholine (DEC) and acetyldiethylcholine (AcDEC) on the dose-response curves of acetylcholine (ACh) on the superNote that the dose-response curve of ACh fused guinea-pig ileum. was shifted to the right-hand side in a parallel fashion by acetyl-DEC (B), but not by DEC (A) . Each point is a mean of three (A) and four (B) values and bars represent standard errors .

s

1731

Paralysis of Cholinergic Neurons by DEC

Yol . 14, No . 9

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1

aa

I

w

I

a><

I

u

~

><

I

~~>,

I

us

I

u

I ><

u

I ><

I

r

ACh (mg)

FIG . 4 Effects of diethylcholine (DEC) and acetyldiethylcholine (AcDEC) on the dose-response curves of acetylcholine (ACh) on the superfused frog rectus abdaminis muscle . Note that similar results were obtained as in Fig . 1 . Each point is a mean of three (A) and four (B) values and bars represent standard errors .

" c " t- "

I

'-

Control DEC 1x10-4 M DEC 1%10-3 M DEC 1x10_2 M

ui

I

us

1

u

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I

I

>,

>I

" Control c Ac DEC " Ac DEC " Ac DEC

I

w

I

w

1x10 -4 M 1x10 -3 M 1x10_ 2 M

I

u

1

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I >t

I >t

L~J

r

ACA (mg )

FI~ . 5 Effects of diethylcholine (DEC) and acetyl~iethylcholine (AcDEC) on the dose-response curves of acetylcholine ACh) on the superfused chick biventer cervicis muscle . Note that similar results were obtained as in Fig . 1 . Each point is a mean of three (A) and four (B) values and bars represent standard errors .

r

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Pnralysis of Cholinargic N~urons by DBC

1732

Discussion A false cholinergic transmitter is defined as an agent which is acetylated in the nerve terminal and subsequently released as an acetylated form, similar to the acetylation of choline and its release as ACh, except that it interacts with cholinergic receptors without inducing cholinergic responses or muscle contractions (2) . In this study, DEC was found to inhibit somatic and parasympathetic neurons efficiently but slowly .

The slowness of onset of drug actions

indicates that DEC is not blocking the neuromuscular transmission at the receptor site ; because if that were the case, an abrupt onset of drug actions should occur.

The latter statement is further substantiated by

the fact that DEC concentrations as high as 1 x lÔ 2M did not affect the dose,~esponse curves of ACh in guinea-pig ileum, frog rectus abdaminis muscle and chick biventer cervicis muscle .

It is important to note that DEC was

acetylated efficiently by ChAc with acetyl-CoA, a crltical requirement for an agent to be a false cholinergic transmitter .

TEC was acetylated

very poorly, supporting the same observations obtained by others (6,8) . This is one of the major reasons why TEC was discarded as a false cholinergic transmitter . Holton and Ing (g) reported that acetyl-DEC produced very minute cholinergic actions in hypotensive actions of rats, hypottensive actions of atropinized rats, contractions of guinea-pig ileum, bradycardia and rRyocardium inhibition of frog heart and rabbit auricles and contractions of frog rectus abdaeinis muscles (1/400, 0, 1/300 that of ACh respectively) .

1/700, 1/1500, 1/600 and

These results clearly indicate that

acetyl-DEC is a poor stimulant of cholinergic receptors,

if any.

In addition,

acetyl-DEC was found to antagonize ACh responses competitively in guinea-pig ileum, frog rectus abdaninis muscle and chick blventer cervicis, indicating that acetyl-DEC does interact with the cholinergic receptor, and produces little effect of its own, just as in the case with a canpetitive antagonist .

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Paraly~i~ of Cholinergic N~uronr by DBC

1733

In conclusion, all experimental results obtained so far indicate that DEC fulfills the criteria required to be a false cholinergic transmitter . It is interesting to note that there is a major difference in pharniacological actions between DEC and TEC.

TEC produced hWpotenston

(1,2) which is due to ganglionic blockade (2) .

DEC on the other hand,

did not lower rat blood pressure, indicating that DEC serves as a false chollnergic transmitter without having appreciable side effects on the blood pressure . Acknowled9eoents The author is grateful to Mrs. Irene Schwartz, Miss Jan Rubin and Mr . Peter Rowell for their technical assistances and to Drs . Thomas Maren and Kenneth Leiburan for their comments on this manuscript .

This work

was supported in part by the National Institute of Neurological Diseases and Stroke (NS-09302-03) . References 1.

W.C . BOWMAN, B .A . HEMSWORTH, and M .J . RAND, Brit . J . Pharmacol . 19, 198-218 (1962) .

2.

W.C . BOWMAN and M.J . RAND, Brit . J- Pharmncol : 17, 176-195 (1961) .

3.

G. BULL and B.A . HEMSWORTH, Brit . Jam . Pharnmcol . 25, 228-233 (1965) .

4.

A.S .V . BURGEN, G . BURKE, and M .L . DESBARATS-SCHONBAI~I, Brit . J . Pharmaçol, 11, 308-312 (1956) .

5.

J .H . BURN, Practical Pharmacoloav , Blockwall Sci . Publ . Oxford, (1952) .

6.

W.C . DAUTERMANN and K.N . MEHROTRA, J. Neurochen. 10, 113-117 (1963) .

7.

B.L . GINSBORG and J . WARRINER, Brit . J. Pharmacol . ~, 410-411 (1960) .

8.

B.A . HEMSWORTH and D. MORRIS, J . Neurochen. 11, 793-803 (1964) .

9.

P. HOLTON and H .R . ING, Brit . J- Pharniacol . 4, 190-196 (1949) .

10 . J .T . LITCHFIELD, JR ., and F. WILCOXON, J. Pharmacol . EXp. Ther . 96, 99-113 (1949) . 11 . W.D .M . PATON, J . P

siol . 127, 40p-41p (1955) .

12 . B. K. SCHRIER and L. SHUSTER, J. Neurochen . 14, .977-985 (1967) .