Two kinds of cholinoreceptors on the non-visceral muscle of some echinodermata

('omp. Biochem. Physiol,, 1977. 1ol. 58C, PS- 1 to 12. Pergamon Press. Printed in Great Britain

TWO K I N D S OF C H O L I N O R E C E P T O R S O N THE N O N - V I S C E R A L MUSCLE OF SOME E C H I N O D E R M A T A S. A. SHELKOVNIKOV,L. A. STARSHINOVA AND E. V. ZEIMAL The Pharmacological Laboratory. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad, 194223, USSR

(Received 9 November 1976) Abstract - The pharynx protractor muscle of holothuria Cucumaria .japonica responds by contraction to both nicotinomimetics and muscarinomimetics. The assumption that it is due to the existence of the two kinds of cholinorecepto~'s on the muscle fibres has been tested. The following findings support the assumption: 1. Partial agonists-nicotinomimetics block the response to full agonists-nicotinomimetics, and do not block the response to muscarinomimetics. 2. Benzilylcholine mustards and dibenamine antagonize the action of muscarinomimetics selectively without changing response to nicotinomimetics. 3. N-cyclohexyl-N'-(fi-4-methylmorpholinium)-ethyl-carbodiimide blocks selectively the response to nicotinomimetics, that to muscarinomimetics being unaffected. The preliminary experiments on the dentis retractor muscle of the sea urchin Stron.qlyocentrotus intermedius suggest that there are two kinds of cholinoreceptors, nicotinic and muscarinic, on this muscle too.

INTRODUCTION Non-visceral muscle of some animals which belong to the phylum Echinodermata (holothuria, sea urchin) were shown to respond by contraction to both nicotinomimetics (N-mimetics) and muscarinomimetics (M-mimetics) (Itina. 1947; Bolt & Ewer, 1963: Michelson et al., 1965: Magazanik, 1965, 1968: Welsh, 1966; Mendes et aL, 1970; Lavrentieva, 1971), in contrast to Vertebrata skeletal muscles which are sensitive only to N-mimetics. The suggestion was made that the sensitivity both to N-mimetics and M-mimetics depends on the primitive, non-differentiated character of cholinoreceptors (ChR) of these animals (Ginetsinsky, 1947: Itina, 1947; Magazanik, 1965, 1968; Michelson et al., 1965; Mendes et al., 1970). The present paper examines an alternative suggestion: the existence of the two kinds of ChR, muscarinic and nicotinic, on the holothurian muscle fibers. MATERIALS AND METHODS The experiments were made on the island Putiatin near Vladivostok during July September, 1972 1975. Specimens of holothuria Cucumaria japonica were obtained by trawling and then stored in a stew plunged in the sea. The pharynx protractor muscle was excised and hung up in the aerated bath containing sea water (30 ml). The muscle contracted during preparation. To stretch it to its natural length, the muscle was allowed to relax under isotonic conditions with a load of 0.5 g. In 2 t{r the muscle reached a steady length (about 25 mm). Then the upper end of the muscle was attached to a transducer connected with the pen recorder, and contractions caused by pharmacological agents were recorded under isometric conditions. After the finishing of experiment the response to isotonic KC1 (0.5 M) was obtained. This response was taken to be 100% Drug solutions (<0.5ml) were injected into the bath with a syringe. The repetitive tests were made 20 min after the muscle had returned to the inital tension. For cholinomimetics the dose response curves were obtained, and the concentrations caused a contraction equal to 50°;0 of the

t-.B.P. 5S/IC

-A

maximal (ECs0) (see Fig. 1). For each cholinomimetic the value of the maximal response (as a percentage of the response to the isotonic KCl) was found as well. The chemical structure of the cholinomimetics used is shown in Table 1. When studying the potency of blocking agents they were added to the bath 1 hr before the testing of a cholinomimetic. This time was found to be sufficient to reach an equilibrium state: more long pretreatment did not enhance the blocking effect. After the equilibrium state had been reached, the dose-response curve for cholinomimetic was obtained (in the presence of a blocking agent). After washing out during 3(>60 min the initial sensitivity to cholinomimetic usually returned. By testing several concentrations of the cholinergic blocking agent the value A2 was obtained-the concentration of blocking agent causing two-fold shift of the dos~response curve for cholinomimetic (Shild, 1947). The chemical structures of the cholinergic blocking agents used are shown in Table 4. I

80x1°-7 z

'H" 6Pll

"6~!6~

4

.

e

/

p

j/

.6c

8.oxio

2~ ~ 8 0

~oc -

2C 6

8

Time, min

1(5e tO-" I0 -6 Carbaminoylcholine concentration, M

Fig. 1. Responses of the pharynx protractor muscle to the different concentrations of carbaminoy]choline (CCh). Left: isometric contractions caused by different concentrations of CCh (molar concentration is shown at each curve). Right: the dose-response curve for CCh (from the experiment shown on left). On the abscissa-~concentrations of CCh, on the ordinate--isometric responses (~ of the maximal contraction). Dashed line shows the finding of ECso value. ECs0 was found to be equal to 6.5 x 10 -8 M.

S. A. SHELKOVNIKOV,L. A. STARSHINOVAAND E. V. ZEIMAL Table 1. The action of cholinomimetics on the pharynx protractor muscle of Cucumaria ,japonica. Number of the experiments is given in the parentheses Drug

ECs0 M _+ S.E. O

(CH3) 3

--CH2CH2OCCH

3.1 x 10 6 + 0.4 × 10 -6 (10) *2.5 x 10 - s (3)

3-J

acetylcholine iodide O + ii' (CH 3)3N - - C H 2CH 2 0 C N H z' CI carbacholine chloride

6.1 x 10 - s _+ 0.8 x 10 -8 (10)

+

(CH3)3N--CH2CH2CH2CH2CH 3 •J pen tyltrimethylammonium iodide

8.0 x I0 -v (4)

O +

I~

(CH3)3N--CH2CH2COCH a •J analogue of acetylcholine with "'reversed" carboxylic group

3.0 x 10 - 7 + 0.5 × 10 . 7 (4)

(CH3)4I~" J -

3.8 x 10 5_+0.7 x 10 -5 (5)

tetramethylammonium iodide Muscarinomimetics (CH3) 3 N--CH2

CH3 .d7.3 x 10-v_+0.5 x 10 7(61)

methylfurmethide iodide H 0 I II CH2--C--O--C--CH3 N\~CHz--CH2 ~CH • C7H603 / CH2 --CH z aceclidine salicylate O

5.5 x 10 -7 + 0.6 × 10 -v (7)

+

2.0 x 10 -6 -I- 0.4 x 10 -6 (4)

C H 3 L o L C H 2 N (CH3)3 . j Dioxolane F-2268 iodide

O

~t

3~0 x 10-5_+0.7 x 10 5 (4)

(CH 3)3I~--CH 2 C H - - O C C H 3- C1

J

CH3 acetyl-fi-methylcholine chloride N / ~

1 \s/ / CH-

' H Br

3.0 x l0 -5 _+ 0.5 x 10 -5 (5)

C--o--ell

IJ 3 0 arecoline bromhydratc O

~N--CH2--C~C--CHz--N ~

Blocking effect (A 2 = 2 x 10-SM)

oxotremorine sesquioxolate Pilocarpine chlorhydrate

Without effect up to 1 x 10 3 M

Nicotinomimetics

.2 C4H606 CH 3

Nicotine bitartrate

1.5 x 10-°_+0.3 x 10 6(12) Maximal response is equal to 60 _+ 3% of that to KC1

Non-visceral muscle of some echinodermata Table 1. (Continuedt Drug

ECs0 M + S.E. O 6.0 × 10 5 +0.9 x 10 "~(3)

( C H 3 ) 3 N - - C H 2 C H 2 O C C H 2 C H 2CH3" CI-

Butyrylcholine chloride O

II

+ ( CH3)3 N--CH 2CH 2OCCH 2CH 3" C1

5.0 x 10 c~+ 1.0 x 10 6(4)

Propionylcholine chloride +N HI /

2.7 x 10 6 ± 0 . 4 x 10 6(3)

ICI--O--CH3 ,dO

Arecoline methiodide O

O 1.3

(CH3)3N--CH2CH2OC(CH2)6COCH2CH 2N(CH3)3 ' 2J

× l0 -6

~ 0.2 X 10 ~' (10)

Suberyldicholine diiodide (D-6) O

O

+ + ( C H s ) 3 N - - C H 2CH2CO(CH2)6OCCH2CH2N(CH3)3 - 2J

4.0 x 10 - 6 ± 0 . 9 x 10 ~'(6) Maximal response is equal to 5 0 ± 5 % of that toKC1

Hexatolinium diiodide * After treatment with anticholinesterase (armine 1 x 10 4M, 10 min)

Alkylating agents containing the /~-chloraethylamine group (dibenamine, A-I and A-5--for chemical structure see Table 5) were dissolved in sea water (pH 8.0) and left for 1 hr at room temperature (to form ethylenimmonium ion) before adding to the bath. After a definite time of contact with the muscle, the free molecules of ethylenimmonium ion were removed by washing out with the sodium thiosulphate 5 x 1 0 - 2 M over the period of 15min. After additional 45min of washing out with the sea water the dos~response curve for a cholinomimetic was obtained and compared with the initial dose response curve. Carbodiimide (for the chemical structure see Table 5) was allowed to be in contact with the muscle for l hr. After washing out with sea water for 1 hr the sensitivity of the muscle to the cholinomimetics was tested and compared with that before the treatment with carbodiimide. RESULTS AND DISCUSSION

The action of cholinomimetics The potency of different cholinomimetics on the

Cucumaria .japonica pharynx protractor muscle is shown in the Table 1. The most active proved to be carbacholine (CCh) (Fig. 1). Acetylcholine (ACh) was less p o t e n t - - p r o b a b l y due to its hydrolysis by cholinesterases (ChE) of the muscle. Since anticholinesterases themselves caused contractions of the holothurian pharynx protractor muscle (Magazanik, 1965) it was possible only in a few experiments to test the potency of A C h after C h E inhibition (with organop h o s p h o r o u s c o m p o u n d armine 1 x 1 0 - 4 M , for 10 rain). In these experiments ACh proved to be even more potent t h a n CCh. Analogue of ACh with "reversed" carboxylic group (non-hydrolysed) was active in concentrations one order of magnitude as great (providing that ACh hydrolysis was prevented

with anticholinesterases). This suggests that the "right" position of the components of the carboxylic group is important for interaction of ACh with C h R of h o l o t h u r i a n muscle. C o m p o u n d without carboxylic group, pentyltrimethylammonium, proved to be even less potent. These results, together with poor potency of t e t r a m e t h y l a m m o n i u m (able to interact only with anionic site of ChR) shows that the esterophilic site of ChR is i m p o r t a n t for interaction of ACh with holothurian muscle C h R as well as in the case of ChR of vertebrates. In accordance with previous studies on Cucumaria japonica (Lavrentieva, 1971) and Cucumaria frondosa (Michelson et al., 1965; Magazanik, 1968) it was found that pharynx protractor muscle responds by contractions not only to N-mimetics but also to M-mimetics. Table 2 shows the relative potencies of M-mimetics and N-mimetics on the h o l o t h u r i a n muscle as compared with their potency when acting on the typical M - C h R and N - C h R of vertebrates. Some of the M-mimetics studied (methylfurmethide (Mfm), F-2268, aceclidine) were found to be rather potent, their effective concentrations being comparable with those on the typical M-ChR. At the same time some other drugs which possess a high potency on the M - C h R of vertebrates proved to be weak or even ineffective on the h o l o t h u r i a n muscle. F o r example, mecholyl which is only twice as weak as CCh on the M - C h R of guinea-pig ileum was found to be weaker than CCh by a factor of 500 on the pharynx protractor muscle. Arecoline has also very feeble activity on the holothurian muscle. Oxotremorine and pilocarpine do not cause any contraction of the pharynx protractor muscle. W e were surprised to discover that oxotremorine had a blocking effect on holothurian muscle (Az = 2 x 10-5 M).

S. A. SHELKOVNIKOV,L. A. STARSHINOVAAND E. V. ZEIMAL Table 2. The relative potency of muscarinomimetics and nicotinomimetics on /he Cucumaria iaponica pharynx protractor muscle, flog rectus abdominis muscle and guinea-pig ileum

Equi-effective molar concentration

Cucumaria japonica pharynx protractor muscle

Drug Carbacholine Acetylcholine "'Reserved" acetylcholine Pentyltrimet hylammonium Tetramethylammonium Methylfurmethide Aceclidine Arecoline Acetyl-fl-methylcholine F-2268 Nicotine Suberyldicholine Hexatolinium Butyrylcholine Propionylcholine Arecoline methiodide

N-cholinoreceptors of the .~/-cholinoreceptors of frog rectus abdominis muscle the guinea-pig ileum

i

1

1

(6.1 x 10-SM) *0.4 0.5 13.0 623.0 Muscarinomimetics 12.0 9.0 500.0 5(~).0 32.0 Nicotinomimetics 24.0 21.0 66.0 1000.0 82.0 45.0

(5.0 x 10 ~'M) *0.4 t3.3

(1.0 x 10 VM) 1.4 3.0 {6.0 1000.0

t38.0 Without effect Without effect Without effect 8.0

0.4 37.0 100.0 2.0 0.1

§7.0 t0.04 tI.4 §0.6 IlO.1 §3.0

Without effect Without effect Without effect ij700.0 i;56.0 250.0

* After inhibition of cholinesterases + Danilov et al., 1974 ++Stephenson, 1956 § Michelson et al., I965 !I Curtis e¢ al., 1966. Thus, in spite of the high sensitivity of holothurian muscle to some M-mimetics, we c a n n o t say that its ChR are identical to vertebrate M - C h R in their sensitivity to M-mimetics. The same is likely to be true in respect to N-mimetics: ChR of h o l o t h u r i a n muscle differ from N - C h R of vertebrates in their sensitivity to N-mimetics. For example, butyrylcholine and propionylcholine whose potencies on vertebrate ChR are similar to that of ACh were found to be far less potent than ACh on the pharynx protractor muscle. 'The change in the sensitivity to N-mimetics and to M-minwtics after chronical denervation. One can imagine that the high sensitivity of the pharynx protractor muscle to b o t h N-mimetics and Mmimetics is due to the presence of some nervous cells whose ChR differ from those of the muscle. To elucidate this question, experiments with chronic denervation of the muscle were carried out. It was shown on Phascalosoma (Prosser & Melton, 19543 that when proboscis retractor muscles were excised and left in refrigerator for 5 7 days, not only nerve fibers degenerate, but also ganglion cells in ganglia attached to the muscle. At the same time the muscle

fibers remained in a good state. The authors concluded that "ganglion cells do not survive much better than do the nerve fibers when isolated from the animals". Having this in mind we can expect that on the Cucumaria muscle excised from the animal and kept in the refrigerator for several days, the nerve cells--if they do exist in this muscle--degenerate too. If the action of M-mimetics or N-mimetics is the result of the excitation of the nerve cells, we can expect these drugs to be ineffective on the denervated muscle. The ECso values for different cholinomimetics found on the fresh muscle and on the muscle kept for 7-10 days in the refrigerator are given in the Table 3. The Table shows that denervated muscles are as a rule as sensitive as normal muscles to both N-mimetics and M-mimetics. Sometimes an increase in the sensitivity to both kinds of cholinomimetics was observed, but never a decrease in sensitivity. The above suggestion that the action of one kind of cholinomimetics is directed not to the muscle C h R but is due to the excitation of the nerve cells seems to be unlikely in the light of these results.

Table 3. The effect of denervation on the potency of cholinornimetics on the pharynx protractor muscle of Cucumaria japonica, n = number of experiments ECso M ± S.E. Drug Nicotine Suberyldicholine Methylfurmethidc Carbacholine Tetramethylammonium

Normal muscle 1.5 ± 0.3 1.3 ± 0.2 7.3 _+ 0.5 6.1 ±0.8 3.8 +0.7

x lO-6(n = 12) x 10 6(n = 10) x lO-7(n = 70)

× 10 S(n = 10) x 10 5(n = 5)

Denervated muscle 1.0±0.2 8.0 ± 0.8 6.3 ± 0.6 7.4 ± 0.8 3.0 + 0.5

× x × × x

lO-6(n lO-V(n lO-V(n lO~8(n lO-S(n

= = = = -

3) 7) 15) 8} 5)

Non-visceral muscle of some echinodermata Table 4. Blocking potency of some cholinolytics against carbacholine on the pharynx protractor muscle of Cucumaria japonica, n = number of experiments Blocking potency (A2) M

Cucumaria japonica pharynx protractor muscle

Drug Atropine sulphate /--~ OH

C2H5

~C--C--O--CH2--CH2~I[ ' FtCL 0 C2H5

Frog rectus abdominis muscle

Muscarinolytics 6.0 x 10 5 + 0 . 8 x 10 s *7.7 x 10 5 (n=8) 1.2 × 10:4_+ 3 x 10 4 (n = 3)

*5.5 x 10 : s

Guinea pig ileum 1.0 x 10 9__ 0.2 x 10 -~ ( n = 10) 2.(1 x 10 '~ (n = 2)

Bcnactyzine +

~ / C - - ~--0 CH2-CH2-N(CH3' 3

2.5 x 10 ~ + 0.5 x 10 4 (n = 5)

7.0 x 10 lo + 0 . 8 × 10 to (,, = 10)

Benzilylcholine [Metacine) cH~

0 0 +~C--CH3 1.3 × 10 ~'± 1.1 × l0 ~' ~C--O--CHuCH2--N~CH3 ( n = 5) O "CL- H(~-cH3 CH3

t4.0 × 10 lo

Propanthelinium Nicotinolytics

Tubocurarine chloride

5.0 ± 1.6 x 10 6 (n=7)

6.0 +0.8 x 10 7 ( n = 5)

No blocking effect upto 1 × 10 4

O

[i + /C--O-CH2-CH2-N {C2H 5)3

(CHz)8 + \C--O --CH2~-CH2--N(C2 Hs) 3 .2J-

_. ~0 x 10 ~ +_ 2 . 4 x 10 ~ (n = 4)

+1.( / +

1.0 x 10 5 + 0.3 × 10 5 (n = 3)

No blocking effect upto 5 x 10 -4

x

10 e~

1.0 x 10 (n = 2)

7

Triethyl analogue of sebacoyldicholine

H5C2~.~1~'C2H5 ,jH~C2/'" ~ C z H 5

Tetraethylammonium * Michelson et al., 1965. t Barlow, 1968. ++Khromov-Borisov et al., 1975.

The potency q/ cholinergic blocking agents Agents known to block M-ChR and N-ChR reversibly. Table 4 s h o w s that C h R of the p h a r y n x protractor muscle can be b l o c k e d b o t h by the agents k n o w n to block selectively M - C h R (atropine, benactyzine, benzilylcholine, p r o p a n t h e l i n e ) and by t h o s e blocking selectively N - C h R of vertebrates {d-tubocurarine, triethyl analogue of sebacoyldicholine). Both kinds of drugs are, however, effective only at high concentrations. Nicotinic blocking agents are a little m o r e p o t e n t t h a n m u s c a r i n i c and yet the effective concentration of d - t u b o c u r a r i n e is one o r d e r of m a g n i t u d e as great on the h o l o t h u r i a n muscle as on the frog rectus a b d o m i n i s muscle. A t r o p i n e blocks C h R of the p h a r y n x p r o t r a c t o r muscle only at c o n c e n t r a t i o n s which are 5 orders of m a g n i t u d e as great as those b l o c k i n g M - C h R of vertebrates. Benactyzine and benzilylcholine p r o v e d to be even less potent. At the same time p r o p a n t h e l i n e which is only twice as strong as atropine o n the M - C h R o f the guinea-pig ileum (Barlow, 1968) p r o v e d to be 50 times as p o t e n t as atropine o n the h o l o t h u r i a n muscle. The above results as well as the d a t a o b t a i n e d when

studying the p o t e n c y o f c h o l i n o m i m e t i c s s h o w that C h R of h o l o t h u r i a n muscle differ b o t h from N - C h R and from M - C h R of vertebrates. It was s h o w n on the Cucumaria fi'ondosa p h a r y n x p r o t r a c t o r that the response to M - m i m e t i c agent M f m can be prevented by the same c o n c e n t r a t i o n s o f atropine or t u b o c u r a r i n e which are effective against ACh. It is these tindings that led to the c o n c l u s i o n that only one kind of a m b i v a l e n t C h R exists o n the H o l o t h u r i a muscle (Magazanik, 1965). W e have studied the blocking p o t e n c y o f the typical b l o c k a t o r s of N - C h R or M - C h R against N - m i m e t i c s or M - m i m e t i c s on the Cucumaria .japonica p h a r y n x p r o t r a c t o r muscle. In s o m e experiments the response to one c h o l i n o m i m e t i c agent was b l o c k e d to a greater extent than the response to another. F o r example, triethyl analogue of sebacoyldicholine in two experiments p r o v e d to be 5 6 times m o r e effective against nicotine t h a n against Mfm. At the same time a n o t h e r nicotinolytic, d-tubocnrarine, did not s h o w any selectivity in b l o c k i n g responses to N - m i m e t i c s as c o m p a r e d to M-mimetics. A t r o p i n e also b l o c k e d the response to M f m to the same extent as that to suberyldicholine (D-6) (Fig.

0

S . A . SHELKOVNIKOV, L. A. STARSHINOVA AND E. V. ZEIMAL ~O0 90-80 --

70--

so -

§

"4

@

.~'~

e"~

40

"c~ 30

~20 IO--

OLO-7

I

I

lO-S Concontral'ion

of

16 5 ogonist,

M

Fig. 2. The lack of a difference in the blocking effect of atropine against methylfurmethide (Mfm) and against suberyldicholine (D-6). The responses to Mfm (©, 0) and. to D-6 (~,A) show approximately the same shift towards greater concentrations in the presence of atropine 1 x 10 4 M. Control responses--open symbols, the responses in the presence of atropine closed symbols. 2). Thus we failed to find definitive regular differences between the blocking by nicotinolytics or by muscarinolytics of the responses to N-mimetics and to M-mimetics. These results would seem to justify against the existence of two kinds of ChR on the pharynx protractor muscle. However it is necessary to take into account that the muscarinolytics block ChR of the pharynx protractor muscle only at very high concentrations. For example, A2 for atropine is equal to 8 x 10 -5 M. At such concentrations atropine can also block the N-ChR of the frog rectus abdominis muscle (Table 4; see also Michelson et al., 1965). This suggests that atropine as well as other muscarinolytics used are probably unsuitable tools for differentiation of invertebrate ChR. It is more difficult to understand why tubocurarine does not block the response to N-mimetics stronger than that to Mfm if N-mimetics and M-mimetics act at the different points. It is interesting to note a rather high blocking potency of TEA on the pharynx protractor muscle. TEA is six times as strong as atropine and only twice as weak as tubocurarine. This emphasizes the important role of anionic site of ChR under investigation (as TEA is likely to react only with anionic site) and shows also that the action of tubocurarine on these ChR differs from that on the N-ChR of vertebrates (on vertebrate skeletal muscle ChR tubocurarine is much more active than TEA).

Potentiation of the responses to cholinomimetics caused by some cholinergic blocking agents. When studying the blocking potency of atropine, benzilylcholine (metacine) and propantheline it was found that these drugs at small concentrations increase muscle response to cholinomimetics instead of blocking it. For example, metacine ! x 10 6 M shifts the dose response curve for Mfm towards lower concentrations whereas its blocking activity appears only after its concentration is increased two orders of magnitude (Fig. 3). After washing out the sensitivity to cholinomime-

tics de&eased down to the initial level. Metacine, propantheline and atropine increase the sensitivity to cholinomimetics not more than 2-3 times. The increase ef metacine concentration from 1 x 1 0 - 6 M to 1 x 1 0 - 4 M does not lead to the further increase of sensitivity to cholinomimetic. The blocking effect appears from 1 x 10 -4 M. Propantheline potentiates the action of cholinomimetics at the concentration 1 x 1 0 - T M and at 1 x 1 0 - 6 M it shows a marked blocking effect. Potentiation of the effect of cholinomimetics by small concentrations of cholinergic blocking agents was earlier described in the literature (Liillman & FiSrster, 1953; Karassik. 1958, 1965; Hazard et al., 1959; Rozkova, 1968). Such effects of blocking agents is considered to be due to their influence on some regulating sites which are capable of changing the sensitivity of ChR to cholinomimetics (a kind of allosteric interaction).

The use of the pqrtial agonists to differentiate ChR of the pharynx protractor muscle. We tried to differentiate ChR of pharynx protractor muscle using partial agonsits as was done by Flacke & Yeoh (1968) on the dorsal leech muscle. The concentration of a partial agonist needed to produce the response which is maximal for the given partial agonist is considered to occupy all I00% of ChR even though this response is less than the maximal possible response of the muscle (Stephenson, 1956). If the effects of N-mimetics and M-mimetics are due to their action on the same kind of ChR, then any full agonist would not be able to increase the response produced by the partial agonist because all the receptors are already occupied by the partial agonist. If the full agonist can nevertheless increase the contraction caused by the maximal concentration of the partial agonist, it probably occurs on account of excitation of those ChR which cannot react with the partial agonist employed. Nicotine and hexatolinium proved to be partial agonists in our experiments on the pharynx protractor muscle. Mfm, aceclidine and mecholyl were

Mfm Mfm with metocine Bo

"

6(

Mfm with metocine IxlO3M (control)

/

/"

/,//I xld~M/

~. 3c

10-7

I

1

10-6 10-5 Concentrotion of egonist, M

Fig. 3. Potentiating and blocking effects of benzilylcholine (metacine). Dose response curves to Mfm in control (x) and in the presence of low (111)and high (e) metacine concentrations. Note that at the low concentration (1 × 10 - 6 M) metacine potentiates the response to Mfm (causing the shift of the dose-response curve for Mfm towards smaller concentrations) whereas at the high concentration (1 × 10 3 M) it shows the blocking effect (the shift of the dos~response curve for Mfm to the right).

Non-visceral muscle of some echinodermata

9C,o I8 -

F

f

(a}

70

o~ 6 0 -

/

/

A -D-~6 Mfm x IC)3M Ix I(DSM

~

<~ 50-E

40--

7

6o

~ 5e

I-

30--

30

2C--

2O

i x idSM ix idSM

iC C

D-6 IxIOSM I x167M3xI(~VM IxlO6M 3xl(D6 M

I00

f

90

(c)

8C

7C

ro~-e g 5o

Buch

H

~H

~H

~H

2 x 104M5x 107M 2x 106M Ix 1(~5M Ix 104M

f

A~

ix IO-4M ix IG4M

~ 4o I.--

3O 2O IC

D-6 ~N ~ N AN N IxI(~SM IxI(~TM 3xl(~'tM IxlO6M 3xlO6M employed as full agonists acting on M-ChR and D-6 or butyrylcholine--as full agonists acting on N-ChR. After the maximal response which can be produced by the partial agonist was reached, a full agonist-Nmimetic or a full agonist-M-mimetic was added to the bath at the concentration required to cause 100% response on the control muscle or even at greater concentration. It turned out that the full agonists-Nmimetics did not increase the muscle tension reached during the action of the partial agonists (nicotine and hexatolinium) whereas the full agonists-M-mimetics did increase muscle tension up to 100% (Fig. 4). These results ai'e in favour of the assumption that two kinds

Fig. 4. The differentiation of two kinds of ChR in the holothurian muscle using partial agonists. D-6 was used as full agonist-nicotinomimetic in (a) and (c) and butyrylcholine (BuCh) in (b) Nicotine (N) was used as partial agonist-nicotinomimetic agonist in (a) and (c) and hexatolinium (H) in (b). Note that with the increase of the concentration of N from 1 x 10 6 M t o 3 x 10 6M(a,c) aswell as with the increase of H concentration from 1 x 10 s M to 1 x 10 4M (b) the response does not increase: the value of the response which is maximal for N (or H) is reached. Full agonists-nicotinomimetics (D-6 in a and c. BuCh in b) fail to produce additional contraction in the presence of maximal concentration of the partial agonistsnicotinomimetics (N in a and c and H in b) whereas with the full agonists-muscarinomimetics Mfm (in a) or aceclidine (Ac in b) as well as with ACh (c) the maximal response can be reached. their structure have been used for investigation of pharynx protractor muscle ChR. Among them, alkylating agents and carbodiimide (for formulae see Table 5) e-bungarotoxin was shown to be ineffective in the blocking of holothurian muscle ChR (Magazanik et al., 1974).

Alkylating agents Agents containing the fl-chlorethylamine group in their molecule are known to undergo cyclization in solution forming ethylenimmonium ions (1) able to alkylate carboxylic groups of receptors (2) (see Gill & Rang, 1966):

R.\ N + / ~CH I z (1) Rr7" Rr/" ~(~H2 R . .-CHz 0 R~ iOi =R, 0 N I + -O--C--Rec-- N--CH2-CH~O--C--Rec ~N--CH2CH20H+-O-C-Rec ( 2 ) Rr'~ ~CH2 Rp~" slo~ R" R

~N--CH2CH2CL

H20==

of ChR exist in the holothurian muscle: one is capable of interacting with N-mimetics and another with M-mimetics. It is interesting to note that CCh and ACh at large concentrations can increase the muscle tension produced by the maximal concentration of the partial agonist-N-mimetic (similar to M-mimetics) i.e. ACh and CCh are likely to interact at large concentrations with M - C h R (Fig. 4c). Agents known to block ChR irreversibly. Some agents known to block ChR irreversibly by modifying

Dibenamine is known to block predominantly ~-adrenoreceptors. This agent blocks also M-ChR of vertebrates (Furchgott, 1954; Ariens, 1964; Takagi et al., 1965; Beddoe et al., 1971) and does not block N-ChR (see Table 5). Taking into account that a high affinity towards the given kind of receptors providing the reversible complex formation results in quick and selective alkylation of receptors the compounds A-I and A-5 synthesized on the base of the chemical structure of the potent reversible muscarinolytic agent benzilylcholine (Ioffe & Kuznetsov, 1964; Gill & Rang,

S. A. SHELKOVNIKOV,L. A. STARSHINOVAAND E. g. ZEIMAL

Table 5. The action of drugs blocking ChR irreversibly,, n - n u m b e r of experiments

C. japonica pharynx protractor muscle

Blocking agent C6HsCH2

Concentration (My and exposure 4 x 10 s 90' n = 13

CH2

\+/I CsHsCH2/N\c:H2

Dibenamine

CsH5

\

0 //

OH2 +/I

HOTCCOCH2CH2N r

1 x 10 .s 20' n = 5

Blocking of the responses to M-mimetics N-mimetics 20(_+4) times shift of the dose response curve without decrease of the maximal response 503/o (_+ 103'o) decrease of the maximal response

Concentrations (My and exposure needed to block N-ChR of M-ChR of frog rectus guinea-pig abdominis ileum muscle

No blocking effect

2xlO 10'

5

No blocking effect

5xlO 10'

"

No blocking effect up to 1 x 10 -3 30'

No blocking effect up to 1 x 10 4 30'

1 x 10 - s 10'

No blocking effect up to I x I0 3 30'

5 10 times shift of the doseresponse curve

No blocking effect up to 1 x 10 -~ 60'

1 x I0 ~ 60'

1×10 30'

3

A I

10 -5 15' n = 29

1 x C6He,

0

CH 2

\ I/ +/I HOTCCOCH2CH2N J

C,sH5 .

l

CH a

CICH2CH2

45% (+6~°J(,) decrease of the maximal response

A5 Hie\+ ~N~CH2 CH2N=C=N

Lo- @

10 -4" 60' n=4

1 x

Carbodiimide 5 X 10 4 60' 11=4

No blocking effect

1966) were used. A-1 a n d A-5 were s h o w n to be m o r e selective t o w a r d s M - C h R a n d to alkylate t h e m q u i c k e r a n d at s m a l l e r c o n c e n t r a t i o n s t h a n d i b e n a m i n e (Gill & R a n g , 1966; S h e l k o v n i k o v et al., 1974). O n t h e p h a r y n x p r o t r a c t o r muscle, a l k y l a t i n g a g e n t s u s e d were f o u n d to i n h i b i t the r e s p o n s e s to M-mimetic~Mfm, m e c h o l y l , aceclidine, F-2268. A t the s a m e t i m e t h e r e s p o n s e to N - m i m e t i c s (nicotine, D-6, b u t y r y l c h o l i n e a n d h e x a t o l i n i u m ) did n o t decrease after t h e t r e a t m e n t of t h e m u s c l e with alkylating a g e n t s at c o n c e n t r a t i o n s w h i c h block t h e r e s p o n s e to M - m i m e t i c s (Table 5). F o r e x a m p l e , Fig. 5 s h o w s t h a t alter t h e t r e a t m e n t of t h e m u s c l e with A-5 t h e r e s p o n s e to M f m r e a c h e d only 20% of m a x i m a l , w h e r e a s t h e r e s p o n s e to D - 6 w a s negative. T h e results s t r o n g l y s u g g e s t t h a t N - m i m e t i c s a n d M - m i m e t i c s interact with different k i n d s of C h R in t h e h o l o t h u r i a n muscle. D i b e n a m i n e p r o v e d to be less p o t e n t t h a n A-1 or A-5 (Table 5). It was also less selective t h a n A-I or A-5 t o w a r d s C h R . W h i l e A-1 a n d A-5 b l o c k e d res p o n s e s to M - m i m e t i c s w i t h o u t c h a n g i n g essentially the r e s p o n s e s of p h a r y n x p r o t r a c t o r m u s c l e to d o p a m i n e , d i b e n a m i n e inhibited t h e r e s p o n s e s to d o p a m i n e to a g r e a t e r e x t e n t t h a n t h e r e s p o n s e to M-mimetics.

T h e p r e l i m i n a r y e x p o s u r e of a l k y l a t i n g a g e n t to s o d i u m t h i o s u l p h a t e (5 × l0 -2 M. 10 m i n ) k n o w n to IOC- -

.2 5C

i-- 4c 3c _ o~- e, ~...~ °'"-"qu'

Ic

e / " I

io-r

io-6

Concentration of agonisi-, M Fig. 5. The differentiation of two kinds of ChR in the ho]othurian muscle using benziJylcholine mnstard. The responses to nicotinomimetic D-6 (A,A) do not change after treatment for 30rain with benzilylcholine mustard A-I (1 x 10 5 M) whereas those to muscarinomimetic Mfin (O,O) are greatly reduced.

Non-visceral muscle of some echinodermata I00 -90-8C--

Before A-5

70--

;Zi5

j~w"

6C

5C .2 4C

5C 2£ IC 10-~

I

I

I

i0-6

i0-5

10-4

Before A-5 +f~+

IOC

9C 8C

,,,,,/After A - 5 ( 3 min)

7C 6( 5C

+/~,,,/

4C

After 2 - ~

20 rain)

5( 2C

'o°1

I

[

I

l O-6

I 0 -5

10- 4

ConcenCrcrMon of rne'l'hylfurmethide,

M

Fig. 6. Blocking effect of different benzilylcholine mustard concentrations. Upper graph- the parallel shift of the dose-response curve for Mfm after the treatment of the muscle with A-5 1 x 10-6M during 90rain. Lower graph the decrease of the maximal response to Mfm due to treatment with A-5 1 x 10 -5 M. After treatment for 3rain 75"11 of the maximal response to Mfm can be reached; after treatment for 20 min only 400/,; of the maximal response can be achieved.

bind ethylenimmonium ions prevented the blocking of holothurian ChR. This means that the ethylenimmonium ion is responsible for alkylating ChR (as well as in the case of vertebrate M-ChR see Gill & Rang, 1966). Two effects can be distuinguished when studying the action of alkylating agents on the holothurian muscle. With small concentrations and long exposure a parallel shift of dose-response curves to greater concentrations of cholinomimetic occurs (Fig. 6A). At greater concentration of alkylating agent the main effect is the decrease of the maximal response. At a given concentration of alkylating agent the degree of the maximal response decrease depends on the duration of the treatment of the muscle with alkylating agent (Fig. 6B). These findings resemble those of Moran & Triggle (1970) obtained when studying the action of alkylating agents on M-ChR of guinea-pig ileum. The authors suggest that alkylating agents at low concentrations form a covalent bond, not with the same site of ChR with which cholinomimetics interact, but with some regulatory site. The reaction with the regulatory site is suggested to decrease the sensitivity of ChR towards cholinomimetics which results in a parallel shift of the dose response curve towards greater concentrations, whereas the maximal response can still be achieved provided the concentration of cholinomimetic is sufficiently raised. At higher concentrations, the alkylating agent is suggested to bind directly to anionic site of ChR, decreasing the total number of free receptors and, as a result, the value of the maximal response is decreased.

9

Such an explanation can probably be applied also to our results on the holothurian muscle. When washing out the alkylating agent the response of pharynx protractor muscle re-established at the same rate as that of guinea-pig ileum (Gill & Rang, 1966). After inhibiting the response to 50g{; of the maximal by A-l, A-5 or dibenamine, the return to the initial maximal response takes 10hr. This means that the alkylating agent liberates 5°',o of the occupied ChR every hour. This rate of ChR liberation is similar to that found on the M-ChR of guinea-pig ileum with these agents (Gill & Rang, 1966). This suggests that in pharynx protractor muscle the same chemical groups of ChR are alkylated as in the guinea-pig ileum--presumably some carboxylic groups. It is interesting to note that while A-I or A-5 are highly selective blockators of "muscarinic" ChR of holothurian muscle, their non-alkylating analogue, benzilylcholine (which is a very potent and very selective blockato,r of M-ChR of vertebrates) does not show either high affinity or high selectivity towards holothurian M-ChR, For example, A2 of benzilylcholine is equal to 3 x 10 4 M on holothurian muscle (cf. A2 = 1 × 10 9 M for M-ChR of guinea-pig ileum) and this drug is equally effective against M-mimetics and N-mimetics. This suggests that selectivity of A-I and A-5 towards "muscarinic" holothurian receptors is not connected with the complex formation stage but with the alkylation stage. It is possible that in "nicotinic" receptors of holothurian muscle, the chemical groups which these agents could alkylate are inaccessible for these alkylating agents.

Carbodiimide Carbodiimides are chemical reagents of the general formula R N - - C - - N R known to form covalent bonds with carboxylic and phosphate groups (Khorana, 1953). Some of them (for example l-ethyl-3/3-dimethylaminopropyl/carbodiimide) were used for the modification of ChR (Edwards et al., 1970). We have used another carbodiimide: N-cyclohexyl-N'-([#4-methylmorpholinium)ethyl-carbodiimide n-toluolsulphonate. In our experiments on N-ChR of the frog rectus I0O

o',Ob

9C

o,,v"j k'c -

#

70--

so

o~-

~7"

.,,'7" #Z

/ ~"

~- 5o 2d I0

-

Oj~

[

iO-S Concentration of agonist,

I

10 5 M

Fig. 7. Differentiation of two kinds o f ChR in the holothurian muscle using carbodiimide. The responses to M f m

do not change after the treatment with carbodiimide (Cd) 1 x 10 4M for 60rain whereas the dose response curve for D-6 is shifted towards the concentrations several times as great.

l0

S . A . SHELKOVNIKOV, L. A. STARSHINOVA AND E. V. ZEIMAL 100

6C

f,

"~ c

Ach

Mfm with÷ neostigmine// / / /

80

40 /

/

/

/

;/

/ /

I' I

/

+,,.1/

Mfm

Ach with //, neost igmine (control) / S +( c~o n t r o l ) I •

,

+

20 I i 0- 9

iO-a

I

I

I

I

I 0 -~

iO-e

i0 -5

i0 -4

Concentration

of

agonist,

M

Fig. 8. The increase in the sensitivity of the sea urchin dentis retractor muscle to ACh and in the presence of neostigmine. In the presence of neostigmine 2 x 10 *g/ml there is the the dose response curve for ACh towards the concentrations 3040 times as low and the the dose-response curve for Mfm towards the concentrations three orders of magnitude as low. curves--solid lines; curves in the presence of neostigmine broken lines. abdominis muscle, this carbodiimide irreversibly inhibited the response to ACh but it was without effect on the M-ChR of guinea pig ileum. Therefore we have used this agent for the differentiation of holothurian ChR. It was found that the treatment of the pharynx protractor muscle for an hour with carbodiimide l x 1 0 - 4 M irreversibly blocks the responses to N-mimetics (nicotine, butyrylcholine, D-6) but does not change the responses to Mfm (Fig. 7.). This suggests that carbodiimide can modify only "nicotinic" receptors of holothurian muscle (as well as N-ChR of frog muscle) whereas "muscarinic" receptors of this muscle [as well as M - C h R of guinea-pig ileum) are insensitive to carbodiimide.

Two kinds of ChR on the muscle of sea urchin Strongylocentrotus intermedius In the preliminary experiments it was found that the dentis retractor muscle of the sea urchin Strongylocentrotus intermedius responds by contraction both to N-mimetics (D-6, nicotine) and M-mimetics (Mfm) similar to the same muscle of the other sea urchins Strongylocentrotus droebachiensis (Magazanik, 1968), Echinometra lucunter and Echinus esculentus (Mendes et al., 1970). This muscle was found in our experiments to be much less sensitive to muscarinomimetic IOCF-

agent Mfm than the pharynx protractor muscle of C. japonica, EC50 being two orders of magnitude as great. Besides Mfm proved to be the partial agonist in most of experiments (the contraction did not reach the value which is maximal for the given muscle). We found that the sensitivity to Mfm was greatly enhanced in the presence of neostigmine. For example in the experiments shown in Fig. 8 there was a 2000-fold shift of the dose-response curve for Mfm in the presence of neostigmine 2 x 10 - 6 M whereas only a 34-fold shift of the dos~response curve for ACh occurred. This result was unexpected as Mfm has no esteric group and cannot be hydrolysed by cholinesterases. The response to nicotine was not changed in the presence of neostigmine. The response to Mfm can be selectively blocked by alkylating agent A-1 both in the absence and in the presence of neostigmine, the response to nicotine and ACh being unaffected (Fig. 9). These preliminary results suggest that on the retractor dentis muscle of Strongylocentrotus intermedius as well as on the Cucumaria japonica pharynx protractor muscle there exist two kinds of ChR.

The action of dopamine The pharynx protractor muscle of C. japonica was found to be sensitive not only to cholinomimetics but

N offer A-I ÷N \ f (control) /p \ o.,/

80--

.6O--

to Mfm shift of shift of Control

Z/Z

Mfm (control)

/

"c~ 4 0 -

I~

Ach After A - I /

20-

F Ach (control)

IO-7

/% ,

1(56 I (5~ Concentrotion of egonist~

Mfm offer A - I 164

Id ~

M

Fig. 9. Differentiation of the two kinds of ChR in the sea urchin dentis retractor muscle using benzilylcholine mustard A-I. The responses to ACh and to nicotine (N) do not change after treatment with A-I 4 x 10-5M for 30min, whereas those to Mfm disappeared.

Non-visceral muscle of some echinodermata also to dopamine. The contraction caused by dopamine developed slower than the response to cholinomimetics. ECso for dopamine (8.2 x 10 VM _+ 0.6 x 10 -7 M; n = 6) is close to that for Mfm. The response to dopamine can be blocked with haloperidol. For example, to obtain the same (50% of maximal) dopamine contraction in the presence of haloperidol 5 x 10 5 M the concentration of dopamine must be increased 12-30 times. The response to Mfm is not inhibited by haloperidol. Among the tested irreversible blocking agents dibenamine proved to be the most effective blockator of dopamine response whereas A-5 selectively inhibits the response to Mfm without affecting the response to dopamine. Dentis retractor muscle of S. intermedius did not respond to dopamine.

CONCLUSION The pharynx protractor muscle of Cucumaria japonica has a high sensitivity not only to N-mimetics (like other Deuterostomia somatic muscles) but also to M-mimetics. It was studied whether this is due to the possible ambivalence of holothurian ChR, i.e. their ability to react with both N-mimetics and M-mimetics, or to the existence of two different kinds of ChR, one of which is sensitive to N-mimetics and the other to M-mimetics. Several approaches were used to elucidate this question. First, we tried to make it clear whether or not the sensitivity to both N-mimetics and M-mimetics is connected with the presence of some nervous elements in the muscle. The experiments with chronic muscle denervation were made on the assumption that after denervation sensitivity only remains to those cholinomimetics which interact with the ChR of the muscle, whereas the sensitivity to cholinomimetics which are able to interact only with ChR of nervous elements will disappear. It proved to be that denervated muscle maintains the ability to respond to both N-mimetics and M-mimetics. This led us to conclude that the action of all the cholinomimetics studied is directed to the ChR of muscle fibers and not to the ChR of nervous elements. We tried to selectively block one or another kind of ChR using cholinolytics which are known to selectively block M-ChR or N - C h R of vertebrates. However, we could not differentiate holothurian receptors in this way. Both atropine and tubocurarine (as well as other drugs known to block M-ChR or N-ChR selectively) were effective only in high concentrations and were found to have the same blocking properties against M-mimetics and against N-mimetics. It is possible that the lack of drugs which would be really selective towards one or another kind of holothurian receptor should be responsible for this failure, c~-Bungarotoxins which are known to block irreversibly and selectively N-ChR of vertebrates, cannot be used for this purpose as they were found to be ineffective in blocking these receptors (Magazanik et al., 1974). Using partial agonists and drugs which irreversibly combine with ChR we have obtained the following data in favour of the existence of the two kinds of ChR in the Cucumaria .japonica pharynx protractor muscle: (1) partial, agonists of nicotinic type at the

11

concentrations at which they produced their maximal effect (and are considered to occupy 100,°o of ChR) block responses to full agonists-N-mimetics without decreasing the responses to full agonists-M-mimetics; (2) dibenamine and benzilylcholine mustards selectively block responses of holothurian muscle to M-mimetics without influence on the sensitivity to N-mimetics; (3) carbodiimide blocks responses of the pharynx protractor muscle only to N-mimetics without changing the responses to M-mimetics. The possibility of selectively blocking one or another kind of ChR suggests that both of them can act independently. This is another argument against the supposition that one kind of ChR belongs not to the muscle fibers but to the nervous elements of the muscle. One can suppose that "muscarinic" and "nicotinic" ChR belong to the different muscle fibers. In this case however not all the muscle fibers would participate in the response to N-mimetics or M-mimetics but only those having N-ChR or M-ChR respectively. Therefore neither N-mimetics nor M-mimetics could in this case produce the response as great as that caused by isotonic concentration of KC1 (all the muscle fibers participate in the response to KCI). In our experiments the values of the maximal responses to KCI, to Mfm and to D-6 were the same. This means that both N-mimetics and M-mimetics are able to act on all the muscle fibers, i.e. both N-ChR and M-ChR exist on every muscle fiber. When studying the action of alkylating agents we found that whereas the response to Mfm disappeared, the response to ACh or to carbacholine did not change (as well as the response to N-mimetics). On the other hand, carbodiimide inhibits not only the response to N-mimetics but the response to ACh as well. This suggests that natural transmitter ACh interacts predominantly with "nicotinic" ChR of the pharynx protractor muscle. The role of "muscarinic" ChR is not yet clear.

REFERENCES

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12

S.A. SHELKOVNIKOV,L. A. STARSHINOVAAND E. V. ZEIMAL

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