Amino acids and biogenic amines in smooth muscles and hemolymphs of different molluscs

Comp Blochem Ph~3ug. 1977 Iol 56C pp 19 to 24 Pergamon Press Printed In Great Britain

AMINO ACIDS A N D BIOGENIC AMINES IN SMOOTH MUSCLES A N D HEMOLYMPHS OF DIFFERENT MOLLUSCS D. VON WACHTENDONK AND M. KAPPLER Instltut fur Zoologle, TH Aachen, KopernikusstraBe 16, D 5100 Aachen, B R D

(Rece~ted 5 May 1976) Abstract--1 Microchromatography of dansyl compounds on 3 × 3 cm polyamide sheets was used to study the distribution of amino aods and blogenlc amines in smooth muscles and hemolymphs of the king crab and some molluscs 2 Using liquid scintillation counting, the amounts of glycine, glutamlc acid, 7-amino butyric acid and 5-hydroxytryptamlne was quantitatively determined 3 After stimulation of the penis retractor muscle (PRM), a smooth muscle in the mollusc Hehx pomatta, varmtaons of the concentrations of these putative neurotransmltters were quantitatively determined and compared with an untreated PRM 4 Only in the marine mollusc, Mytdus eduhs, large amounts of free amino acids and Iaurme were detected, all other tissues and hemolymphs of the other animals examined contained essentially less free amino acids 5 Some rare compounds could be Identified in a few tissues or hemolymphs sarcosine, ethanolamlne, putrescme and tryptamme The occurrence of free chohne has not been clearly indicated 6 The significance of these experiments has been briefly discussed

INTRODUCTION

scintillation counting of the radioactive labelled compounds.

Beside concern for protein synthesis, as osmoregulators and w~th 1on distribution across cellular memMATERIAL A N D M E T H O D S branes some amino acids show transmltter-hke Snarls (Helix pomatla~ and fresh water mussels (Anodonta properties, as more recent experiments have shown cygneal were collected locally, sea water mussels (Myttlus (Neuhoff et al, 1975, Denavlt-Saubi6 & Champagnat, eduhs) were bought from local fish traders, and the king 1975, Sonnhof et al., 1975) Some of these amino acids crabs (Ltmulus polyphemus) were kept in tanks of clrculatmay possibly be connected with the development of mg sea water until required a "catch-state" in smooth muscles of Invertebrates, Determination of 5-hydroxytryptamlne, glycme, glutaespecially molluscs While thxs "catch-mechanism" in mic acid and ),-amino butyric acid (GABA) was carried out accolding to the method of Neuhoff (1973j all its basics has not been fully explained as yet (Johansson, 1975), it is necessary for studying some (a) Preparation of the muscle tissue muscles--which are known to undergo a " c a t c h " ~ One-10 mg of freshly dissected muscle tissue (see Table not only in their physiological or pharmacologlcak 1) from each animal was homogenized in 10-100ld of but in their biochemical behavlour too 0 05 M sodlumbicarbonate, pH 10 0, to reach a final ratio The present paper shall point out the whole of the of 1 10 (w/v), and centrifuged for 30mm at 21,500g in avadable spectrum on solved amino acids, neuroa specially equipped Christ (Type Junior II KS) centrifuge transmitters and their metabohtes in smooth muscles Proteins were precipitated from each of the supernatant as well as hemolymphs of some chosen molluscs and solutions by the addition of the double vol (20-200/A) of cold acetone and the precipitation was completed by placgive information on possible similarities, which may give evidence for the origin of the "catch'-mechanlsm. ing the solutions in a freezer (-28~C) for 30rain Each of the solutions was allowed to thaw, again centrifuged The fast adductor muscle (FAM) of the fresh water at 21,500 g for 30 mln and 4 #1 respectively of the supernamussel Anodonta cyynea, the anterior byssus retractor rants were then transferred to clean mlcrotubes (about muscle (ABRM) of the marine mussel Myttlus eduhs 50/A content) and the penis retractor muscle (PRM) of the c o m m o n (b) Preparation oJ the hemolymphs snarl Hehx pomatza as well as their hemolymphs were chosen for these experiments, in addition the hemoTo 10 gl of each hemolymph, 10/A of 0 05 M sodlumbilymph of the king crab Ltmulus polyphemus was examcarbonate, pH 100, were added, well shaken and mixed lned. with 20 ltl cold acetone. After precipitation of the hemolymph proteins at -28°C in a freezer for 30 min, the soluUsing the dansyl technique in mlcroscale according tions were allowed to thaw and centrifuged at 21,500 0 to Neuhoff (1973), a registration of substances, which can react with dansyl chloride (1-dlmethylamlnonaph- for 30 man Four ~1 respectwely of the supernatants were then transferred to clean microtubes thalene-5-sulfonyl-chlorxde) because of their reactive amino- and phenolic hydroxyl groups, is possible to -(c) Dansvlatton procedure and chromatograph 3 the pmole-range by use of x4C-dansylchlorade; a Four #1 of the supernatants were mixed with the equal quantitative estimation can be achieved using liqmd vol of l"~C-dansylchloride m acetone (1 2 x 10-5mole/l, 19

20

D VON WACHTENDONK AND M KAPPLER Table 1

Group

Animal

Anisomyaria M),tdu~ eduhs (common edible sea mussed EulamelhAnodonta branchiata cygnea (Bivalvia) Gastropoda Helix pomatm (garden snail)

Animal Mytdus eduh~ 4nodonta c) 9nea Heh,¢ pomatm Merostomata LtmIdus (Xlphosura) Polyphemu~ (king crab)

Habitat marine

fresh water terrestrial

Habitat

Glycme content (all values in #g/g wet wt of tissue)

Glutamic acid content (pg/g wet wt)

GABA content (,ug/g wet wt)

5-hydroxytryptamme content (~tg/g wet wt)

1480_+65

906-+42

63_+21

43-+03

690_+30

869_+31

73_+05

15+_02

542-+35

492_+23

60-+12

32_+02

Tissue Anterior byssus retractor muscle (ABRMt Fast adductor muscle (FAM) Penis retractor muscle (PRM)

Tissue

Glycme content Glutamic aod GABA (all values content content 5-hydrox~tryptamme are pg/ml (pg/ml (pg/ml content hemolymph) hemolymph) hemolymph) (pg/ml hemolymph)

marine

hemolymph

155_+12

71_+11

45_+0.6

0

fresh water

hemolymph

25_+05

22_+02

21_+04

0

terrestrial

hemolymph

45 _+ 0.2

44 _+ 05

2.0 _+ 03

0

marine

hemolymph

33_+06

24_+03

39_+03

0

sp act 20 3 mC1/m-mole, Amersham Buchler, Braunschwelg, B R D ) m 50-pl capillaries, sealed and subsequently incubated m the dark for 30rain at 37'C After evaporation of the solvents the residues were again dissolved in 5 ,ul acetone/acetic acid (3 2, v/v), and 0 1 #1 of this mixture containing the dansylated compounds was then carefully apphed to a corner of a 3 x 3 cm polyamide sheet (Schleicher & Schull, F 1700 mlcropolyamlde) and developed m an ascending way Water/formic acid (100.3, v/v) was used for the first direction and benzene/acetic acid (9 1, v/v) for the second Standard amounts of glycine, glutamic acid, GABA resp 5-hydroxytryptamine was added to the extracts and chromatographed separately to render possible or to lighten their identification and determination Radloautographs of the microchromatograms from each sample were obtained in the dark by using a highly sensitive film (Agfa Gevaert, Dentus T 2), exposure time 7 days Development was achieved by use of Agfa Gevaert G 150 fired according to the direction of the manufacturer For quantitative measurements the fluorescent spots of glycine, glutamac acid, GABA and 5-hydroxytryptamine were marked with a soft pencil under u v light (254 nm), scraped off with a special razor-sphnter knife (Neuhoff, 1973), and transfered immediately into counting vials Each sample was suspended in 10ml of a scintillation liquid (4 g PPO and 01 g POPOP, both from Packard Instruments, dissolved in 1 l toluene for scintillation counting (Merck)), and counted in a Packard Trl-Carb Liquid Scantlllatmn Spectrometer, Model 2425

RESULTS R a & o a u t o g r a p h s illustrating the occurrence of I4C-dansylated substances are shown in Fig. 1, each p h o t o g r a p h as the original size of the mlcrochromatogram Table 2 lists qualltatwely all c o m p o u n d s m the pmole-range, which can react with 14C-dansylchlor1de With the aid of the autoradlography 31 substances could be ~dentlfied. some spots, which were detected m the tissues and hemolymphs respectively, still remain unknown. Glycme. glutamlc acid, G A B A and 5-hydroxytryptamme, which can act as neurotransm~tters, have been detected in all the muscle t~ssues and, beside 5-hydroxytryptamme, m all the hemolymphs too, as Fig 1 shows Using reaction curves, a quant~tatwe estlmat m n for these putatwe neurotransmltters was achieved by llqmd scintillation counting, for all these counts the background actwlty of 1 6 0 d l s / m l n has already been subtracted The quenching effect of the small amounts of polyamide layers is neghglble (Neuhoff & Wexse, 1970, unpublished results). Beside for 5-hydroxytryptamlne all other reaction curves were taken from Neuhoff (1973), Fig. 2 shows the corresponding curve for 5-hydroxytryptamme, which was obtained by reacting various amounts of 5-hydroxytryptamine with a constant a m o u n t of 14C-dansylchlonde.

Amino acids and biogenlc amines in molluscs

( a ) Anodonta ) hemolymph

( • ) Anodonta ) FAM

O

( b )Hehx ) hemolyrnph

( f ) Hehx) PRM

9F

tP

(

c

) Llmulus) hemolymph

21

Amounts of bls-dansyl-5-hydroxytryptamine can be calculated by dividing the dxs/mln of this bls-dansylated compound by 2, for 5-hydroxytryptamlne with its two reactive groups may also react with two moles of ~C-dansylchlorlde After the corrections and by use of the cahbratlon curves the resulting counts are converted into ,umoles of each of the examined compounds, Fable 1 summarizes these results. Muscle tissue from each of the species examined contained all the four substances being researched The ABRM of Mytflus contained the highest amounts of each amino acid and 5-hydroxytryptamine too Except in 5-hydroxytryptamlne and in glutamlc acid content the amounts of the other amino acids hardly differ in the examined muscles of both the other invertebrates Anodonta and Hehx Greater differences can be discovered in the hemolymphs than the smooth muscles of the examined invertebrates show, in addition to these invertebrates the hemolymph of the king crab L~mulus polyphemus was examined In all the body fluids GABA, glycine and glutamlc acid were detected 5-hydroxytryptamine could not be detected, but some of its metabolites, e g 5-hydroxytryptophane (spot 14), 5-hydroxylndole (spot 40) and tryptamine [spot 41).

( g ) MytHus) ABRM DISCUSSION

( d ) Myl"Hus) hemolymph

( h ) (l'l'C) --Dansylchlorlde impurlf'les ) treated hke tissue preparahons

45

46

47

0 0

0

042

48

0

49

o

044

O4a 0 41

40 0

39

0

34 0

0 38

031

O~' C)s3 oas

03~

2sO 2900g° 28 207

0 so

The method used in this study to detect and estimate amino acids and neurotransmltters is more senSltlve than other commonly used methods (e.g nlnhydrln, gas chromatography), for it allows in combination with 14C.labelled dansylchloride the demonstration of substances in the pmole-range. Evidence is given for the occurrence of taurme in the hemolymph of the king crab Ltmulus polyphemus, which was until now in doubt (Jeunlaux, 1971), taurlne was also found in the hemolymph of the snail Hehx pomat~a, which was not detected by Osborne (1972a) The effect of taurlne, however, is still doubtful "In small amounts taurlne can act as a hampering transmitter (Davlson & Kaczmarek, 1971), while larger amounts especially in marine invertebrates take active part in the osmotic equihbration between extracellular and Intracellular fluids (Pllgnm, 1953; Lange, 19631. Of the three examined molluscs only the marlne form, Mytflus, contains large amounts of taunne, the hemolymph of the king crab, Lmmlus, taken for comparison shows, in contrast, only a small taurine content. These differences may be explained by the form of the hemolymph circulation because, in contrast to Lunulus, Mytflus has an open hemolymph system While taurlne is in all tissues and hemolymphs in relatively large amounts, a possible use as a neurotransmitter can be excluded The osmoregulating function may be regarded as likely, although one

22

2

t

170 140015

016 06

70

S .4.o .6p~.._3 8c:~ -

..--,

02

13 10~12 Q 11

Fzg 1 Autoradtographs (top) of the different tzssues and hemolymphs in original size, after two d]menslonal mlcrochromatography of substances in muscle extracts or, m hemolymphs, having reacted with (14C)-labelled dansylcbloride Exposure hme 7 days Numbers m the corresponding map (bottom) refer to Table 2 for identification of the spots First d~rectmn 3°0 formac acid in water, second d]recnon benzene/acetic ac]d, 9 1 (v/v)

22

D

VON WACHTENDONK AND M

KAPPLER

Table 2

Myttlus

Spot No

Compound

1

Dans-taurme

2

Dansylchlorldeimpurities

3

Dans-argmln+N

4

Dans-hlstldin N-Dans-serotonln

5 6

N-Dans-tyrosln unknown

7

Dans-glutamlc

8

Dans-aspartlc

9

'-

acid acid

Anodonta

Hemolymph

ABRM

Hehx

Hemolymph

FAM

Hemolymph

L~mulus PRM

Hemolymph

+++

+++

++

++

++

+++

+

+++

+++

+

++

++

++

++

-

+

-

+

-

+

-

+ -

+ +

+

+

+ -

-

+

+ +

+ + +

+

+ +

+

+ +

+

+ +

+ +

+

+

+

+ +

+

unknown

-

+ +

-

+

nd

-

-

10

Dans-threonin (+Dansylchloride-impurities)

+

+

+

+

+

+

+

11

N-Dans-serln

+

+

+

+ +

+ +

+ +

+ +

12 13

Dans-asparagm Dans-glutamln

+ +

+ +

+ +

+ +

+

+ + +

+ +

14 15

Dans-5-hydroxytryptophan Dans-tryptophan

+

+ +

+ -

+ + + -

-

+

+ -

16 17

unknown unknown

-

+ +

-

+ + +

+ -

-

+ -

18 19 20

unknown unknown Dans-glycme

21

unknown

+

+

22

unknown

-

+

23 24

Dans-lysine Dans-ornithln

+ + + + + +

25 26

Bls-Dans-tyrosin Dans-alanin (+Dansyl-

27 28 29

Dans-methmmne Dans-phenylalanln Bls-Dans-hlstldln

30 31

Dans-leucine Dans-lsoleuclne

32

. +++

.

.

+ +++

++

+++

+ + ++

-

+

-

+

-

-

n d

-

-

-

+ +

+

+ + +

+ +

+ + + +

+ + +

+ + + + +

+ + + +

+ +

+ + +

+ + + +

+ + + + +

+ + + +

+ + + +

+ + +

+ +

+ + +

+ +

+ +

+ +

+ +

+ +

-

+ +

+ +

+ +

+ +

+

+

+

+ +

+

+

+

33

Dans-7-amlnobutyrIcacid Dans-vahne

+

+

-

+

+

+

+

34

Bls-Dans-serotonln

-

+

-

+

-

+

-

35 36

Dans-sarcosin Dans-ethanolamln

+ +

+

+ +

n d .

-

+

+ +

.

37

Dans-prohne

+

+ +

+

+

+

+ + +

+ +

38

unknown

+

+

-

-

-

+

+

39 40

Dans-putrescm Dans-5-hydroxylndol

+ +

+

-

+ + + +

+ +

+

-

41

Dans-tryptamln

-

-

-

+

+

-

-

42 43 44 45

unknown unknown unknown Dansylchloride-

+ -

+ -

+ + -

+ + +

+ + -

+ + -

+ + -

46 47 48

impurities unknown unknown Dansylchloride-

+ +

+ +

+ +

+ +

+ +

-

+ + +

49 50

impurities unknown Dans-choline

_

_

_

+ +

+

_

_

+++

.

.

+ +++

.

chloride-impurities)

+ + + + + + -

n d

(o)

hLgh c o n t e n t o f t h e r e s p e c t L v e c o m p o u n d medmm content of the respective compound low content of the respective compound not present n o t d e t e c t a b l e , c a 6 s e d by 1 4 C - d a n s y l c h l o r l d e i m p u r i t i e s

.

.

.

Amino acids and blogenlc amines m molluscs

/£/

E Q_

fg -

/~///// 4

C5_.+[mollL'] Fig 2 Reaction curve of 5-hydroxytryptamlne (N-dans-5hydroxytryptamme) log y = (6221 _+ 0 122) + (0945 + 0049) log x a = +0065

r = 0993

The sohd hne represents the theorehcal curve, the dashed line corresponds to the experimental curve

must note that the terrestrial Hehx as well as the Anodonta hwng in fresh water exhlNt similar amounts of taurme While all examined animal forms were caught or recewed in October and N o v e m b e r 1975 and immedmtely afterwards processed, changes due to the time of the year in amino acid and transmitter content should be considered (York & Twarog, 1973, Kappler & v o n Wachtendonk, 1975). Thus Osborne (1972a, b) in contrast to us found no taurlne m the hemolymph of Hehx, but the ammo acid methlonlne in his experiments Furthermore, four a d d m o n a l spots could be identified sarcosme (spot 35), ethanolamine (spot 36), putrescme (spot 39) and tryptamlne (spot 41), spot 50 is probably chohne. Similar results have been obtained by Hanley (1975) The occurrence of tryptamine m Anodonta and ItS role as neurotransmltter an early stages of ontogeny has been shown by L/lbos et al. (1964), it could be detected here in the fast adductor muscle (FAM) of Anodonta and in the h e m o l y m p h of Hehx As Table 1 shows, the a m o u n t of 5-hydroxytryptamine in all the examined muscle tissues is very low, compared with those of nervous tissue (Osborne,

23

1972b: Kappler & yon Wachtendonk, 1975), it ranges from 1.65 #g for the fast adductor muscle of Anodonta up to 4 3 ltg/g wet wt for the anterior byssus retractor muscle of Mytilus, the amounts of G A B A range from 60l~g (Hehx) up to 7 3 #g (Anodonta) per g wet wt of these tissues Also these values when compared to those of nervous tissues are low (Osborne, 1971). In all the hemolymphs no 5-hydroxytryptamine was detected but GABA was about one half of the a m o u n t determined for the smooth muscles Very great differences resulted by comparison of the glycane and glutamlc acid contents of the smooth muscles and the hemolymphs (see Table 1) Because of the large amounts of these amino acids it does not seem probable that they play a role in nervous transmission or muscle stimulation of the tissues examined The quahtatlve comparison of the chromatograms which was obtained from the muscle tissue of the different molluscs, yielded no corresponding facts, neither m the known amino acids nor in the unidentified spots, which could lead to the conclusion for the same course or a similar release of the "catch-state" Stimulation of smooth invertebrate muscles which are able to develop a "catch" led to changes in the amino acids and transmitter concentrations, so far as a reuptake or an enzymat~cal reduction of blogenic amines and amino acids is inhibited, as Table 3 indicates Similar results on nervous tissue were obtained by Neuhoff et al (1975) after a long term potentmtlon of monosynaptlc reflexes in the spinal cord of cats Further mvestigatlons will deal with this phenomenon, the hberatlon and increase of amino acids and transmitter values after stimulation of smooth muscles (yon W a c h t e n d o n k & Wabmtz, in preparatlon). Twarog (1966) considers the development of the "catch" p h e n o m e n o n through the interaction of 5-hydroxytryptamlne and Ca2+-lons on actm and myosin to be possible In summary, significant similarities in the examined smooth muscles were not found Electrophyslologlcal experiments in c o m b i n a t i o n with gaschromatographlcal analyses, especially relative to tertiary and quaternary a m m o n m m compounds like acetylchollne, should supply further Information on the chemical processes of the "'catch-mechamsm"

Acknowledgements--We are greatly indebted to Prof Dr V Neuhoff for having given us the possibility of learning the mlcrotechnlques in the Instltut fur Expenmentelle Medlzln, Gottlngen (BRD) Thanks are also due to Prof Dr H Stleve, Instttut fur Neuroblologle, Juhch (BRD), for preparing the hemolymph of the king crabs

Table 3

Tissue Pems retractor muscle

(Helix pomatta)

untreated treated*

Glyclne /~g/g wet wt

Glutamlc acid #g/g wet wt

GABA /~g/g wet wt

5-hydroxytryptamlne /~g/g wet wt

54 2 _+ 3 5 65 7 + 3.9

49 2 _+ 3 2 245.4 -I- 7 6

6 0 + 12 12 0 + 1 5

3 2 _+ 0 2 2 1 _+ 0 1

* Stimulated and treated with Lilly 110140 (5-hydroxytryptamlne re-uptake inhibition), for details see Wabnltz & yon Wachtendonk (1976) All values were achieved quantitatively by hqmd scintillation counting of the dansylated compounds, as described in Material and Methods, experiments were repeated six times

24

D VON WACHTENDONK AND M KAPPLER REFERENCES

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tlon of monosynaptlc reflexes in the cat spinal cord llI Neurochemtstrv 5, 254~269 OSBORNE N N (1971) Occurrence of GABA and taurme in the nervous systems of the dogfish and some invertebrates Comp Gen Pharmac 2. 433 438 OSBORNE N N (1972a) Serotonln, free amino acids and related substances occurring in the blood and nervous tissue of Helix aspersa (Gastropoda, Mollusca) Comp Gen Pharmac 3. 171 177 OSBORNE N N (1972b) Occurrence of glyclne and glutamlc acid in the nervous system of two fish species and some invertebrates Comp Blochem Phystol 43B, 579-585 PILGRIM R L C (1953) Osmotic relation in molluscan contractile tissues I Isolated ventrlal strip preparations from lamelhbranchs (Mvtdus eduh~ L. Ostrea eduhs L, Anodonta cygnea L ) J exp Btol 30, 297 317 SONNHOF U , GRAFE P . KRUMNIKL J, LINDER M & SCHINDLER L 0975) Inhibitory postsynaptlc actions of taurlne, GABA, and other amino acids on motoneurons of the isolated frog spinal cord Brain Re,s 100, 327 341 TWAROG B M (1966) Catch and the mechanisms of action of 5-hydroxytryptamlne on molluscan muscle A speculation Life Sct 5, 1201 1213 WABNHZ R & ",'ON WACHTENDONK D (1976) Evidence for serotomn (5-hydroxytryptamlne) as transmitter in the penis retractor muscle of Hehx pomatta L Experwntla 32, 707 709 YORK B & TWAROG B M (1973) Evidence for the release of serotonlne by relaxing nerves in molluscan muscle Comp Btochem Phys~ol 44A, 423-430