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Choline esterase activity in the tissues of the snail Helix Aspersa
Comp. Biochem. Physiol., 1969, Vol. 28, pp. 923 to 929. Pergamon Press. Printed in Great Britain
C H O L I N E ESTERASE A C T I V I T Y IN T H E T I S S U E S OF T H E SNAIL H E L I X A S P E R S A E. K O R N Department of Zoology, The University of Liverpool, England (Received 28 March 1968) A b s t r a c t - - 1 . The titrimetric method of Glick (1937) was used to measure
cholinesterase activity in various tissues of Helix aspersa. 2. Such activity was found in a variety of tissues and was particularly high in the heart. 3. The activity showed some of the properties of a true cholinesterase. INTRODUCTION THE GASTROPODheart is myogenic in nature (Needham, 1950), but its beat is regulated by extrinsic nerves (Carlson, 1905). The control is mainly exercised through the visceral nerve whose anatomy has been described (Jullien & Ripplinger, 1954) for Helix pomatia. Its course is similar in Helix aspersa. Stimulation of the visceral nerve may in different conditions cause acceleration or inhibition of the beat (Sz-R6sza & Per6nyi, 1966). Pharmacological studies have indicated the possibility that the neuromuscular transmitter substance involved in inhibition is acetylcholine, and in excitation is 5-hydroxytryptamine (Meng, 1960). Both these substances have been identified in the brain of H. aspersa (Kerkut & Cottrell, 1963). While both have been identified and shown to have pharmacological actions appropriate to the hypothesis of their role as mediators in a variety of molluscs (Bacq & Copp6e, 1937; Bacq, 1947; Welsh, 1954; Welsh & Moorhead, 1960), the evidence is extensive only in the case of the lameUibranch Venus mercenaria (Welsh & Taub, 1948; Florey & Florey, 1953; Welsh & Moorhead, 1959; Loveland, 1963). Anomalies in the response of mollusc hearts to acetylcholine (Pilgrim, 1954) have been used by some workers to argue against the hypothesis of cholinergy in the snail (Jullien et al., 1959). Since the presence of an enzyme system capable of inactivating acetylcholine is often used as one of the criteria of cholinergy (e.g. Bacq, 1947), it seemed valid to investigate this. The existence of such a system has been demonstrated in the blood of H. pomatia (Augustinsson, 1948; Bockendahl, 1962) and in the snail gut (Bockendahl & Mtiller, 1965). Moreover, histochemical tests have shown the presence of Gomori-positive substances in the nerve endings of H. aspersa (Sal~nki & Zsoltan-Nagy, 1965). The whole subject of transmitters in invertebrates has recently been reviewed fully (Cottrell & Laverack, 1968). 923
924
E. KORN
MATERIALS AND METHODS a. Titrimetric methods Glick's method for the estimation of esterase activity depends on the liberation of acid by the breakdown of choline esters. T h e p H is measured and maintained at a fixed point by the titration of a dilute solution of sodium hydroxide. T h e rate of addition of the alkali then represents the rate of breakdown of the choline ester, and the effect on this rate of the addition of tissue extracts provides a measure of the activity of the extracts (Glick, 1937). Standard solutions of acetylcholine chloride were made up in Meng's snail Ringer (1960) and stabilized by the addition of a few drops of HCI to bring them to a p H below 4. T h e y were stored at 0-10°C. Twenty-five-ml samples of this solution were pipetted into a 100-ml beaker and the p H measured continuously with a Pye p H meter. T h e solution was stirred continuously with a magnetic stirrer. When the temperature had risen to about 20°C, the p H was brought to 8 with 0"1 N NaOH. T h e time was then noted and as the p H dropped below 8, 0.005 N N a O H was run in from a burette, causing the p H to overshoot 8 slightly. It was then permitted to drop again and the operation repeated. Each time the p H passed 8"00, the time and the burette reading were noted. After a number of readings had been taken in this fashion, the tissue extract, or extract and inhibitor, were added, the p H adjusted to 8 once more, and a further series of readings taken. T h e p H of 8 was adopted as this optimized the difference between non-enzymatic hydrolysis (the rate of which rises sharply with solutions more alkaline than p H 8"5) and enzymatic hydrolysis, which is optimal at p H 9 (Glick, 1938). Care was taken to prevent the p H of the solution ever passing outside the range 7"9-8"1. Acetyl-, butyryl- and propionyl-cholines were used (all as the chloride salt). T h e titration of 1/zl of alkali corresponds to the hydrolysis of 0"9/~g of acetylcholine chloride. b. Preparation of extracts A number of snails (usually twenty-five) was rapidly dissected and the appropriate organs removed. T h e y were weighed on filter paper, scraped off into a small known quantity of cold Meng's Ringer, the filter paper reweighed, and the tissue then brought to a convenient concentration by the addition of further Ringer solution. T h e organs were then minced with fine scissors and crudely homogenized. T h e solution was allowed to stand for half an hour at 0-5°C. T h e supernatant was then decanted off from the tissue. Control experiments showed no measurably greater activity when the tissues were more fully homogenized, which suggests that we are here dealing with the lyozyme fraction (ErLnk5 et al., 1964). T h e extract could be stored frozen for upwards of 48 hr but was usually used immediately. RESULTS
I n t h e results, t h e q u a n t i t y o f e x t r a c t a d d e d is q u o t e d in g w e t wt. o f tissue. T h e v o l u m e a d d e d was n e v e r m o r e t h a n 1 ml. S i n c e t h e r e was s o m e v a r i a b i l i t y f r o m r u n to r u n in t h e rate o f n o n - e n z y m a t i c h y d r o l y s i s ( a l t h o u g h n o t w i t h i n t h e run), t h e p e r c e n t a g e a c c e l e r a t i o n c a u s e d b y a g i v e n e n z y m e p r e p a r a t i o n w a s u s e d for c o m p a r a t i v e p u r p o s e s r a t h e r t h a n t h e absolute rate of enzymatic hydrolysis. a. A l b u m e n g l a n d T h i s tissue h a d a l r e a d y b e e n r e p o r t e d as a r i c h s o u r c e o f esterase ( K e r k u t , p e r s o n a l c o m m u n i c a t i o n ) . R e s u l t s a r e s h o w n in T a b l e 1, rates b e i n g t a k e n f r o m t h e s l o p e values o f g r a p h s .
925
CHOLINE ESTERASE ACTIVITY IN THE TISSUES OF H E L I X ASPERSA
b. Comparison of the enzymatic activity of various tissues Although there was considerable variation in the activity of the extracts, those from the heart were more active than those from the brain and the albumen gland, which were roughly similar in activity. Rates of hydrolysis are shown in Table 2. The heart has the highest level of esterase activity/unit of weight. T A B L E 1 - - H Y D R O L Y S I S OF ACETYLCI-tOLINE BY EXTRACT OF
Non-enzymatic rate
Material added 100 mg albumen gland 100 mg albumen gland after 2 min boiling Ringer control
30 mg heart 50 mg brain 50 mg albumen gland
dspersa ALBUMEN GLAND
Rate after addition of extract
In/~l/sec of 0"005 N NaOH added
% Acceleration
0"52 0"46
0"70 0"48
35 4
0"63
0"67
6
T A B L E 2 - - H Y D R O L Y T I C ACTIVITY OF
Extract
H.
H. aspersa TISSUES
% Acceleration
Rate of enzymatic hydrolysis (#molesACh/g per hr)
49 54 43
317 97 72
Some activity was also found in the hepatopancreas but none in the foot musculature. Augustinsson (1948) found a high level of esterase activity in the hemocoelic fluid of H. poraatia. That the activity here measured is present in the tissues of the myocardium rather than in the hemocoelic fluid is shown by electrophoretic studies of esterases. The activity of the auricle was compared with that of the ventricle in another series of experiments. The auricles and the ventricle were separated by a transverse cut and ground separately. The auricle extracts showed a consistently higher activity/unit of weight than those of the ventricle. A single auricle, whose weight was approximately 3 mg, gave an acceleration of 30 per cent; a single ventricle (weight 15 mg) gave a 25 per cent acceleration. This suggests that the total hydrolytic activity of the heart is approximately equally divided between the two chambers of the heart, the auricle being some fivefold richer per unit of weight.
c. Enzyme inhibition Table 3 shows the abolition of enzymatic activity by treatment with 10 -4 eserine. The extract was incubated with the inhibitor for 50 rain at room temperature, and the experimental run alternated with runs of untreated preparation also maintained at room temperature. Eserine abolishes activity completely.
926
E. KORN
d. Relation of activity to substrate concentration For these runs the extract was prepared in a magnesium-rich Ringer (NaCI 5.9 g/l, MgC12 3-8 g/l), to maximize the activity at the lower substrate concentrations. Augustinsson (1948) has reported an activating effect of M g ~+ ions on cholinesterase. T h e r e was an approximately tenfold increase in the effectiveness of the extract on 10 -4 acetylcholine as c o m p a r e d with the m a g n e s i u m - p o o r extract. T A B L E 3 - - I N H I B I T I O N OF CHOLINESTERASE ACTIVITY BY ESERINE
Time from start of incubation (min)
Extract
% Acceleration
0 50 80
Normal Eserinized Normal
30 ( - 5) 19
T A B L E 4 - - - S u B S T R A T E CONCENTRATION AND CHOLINESTERASE ACTIVITY
% Acceleration Conc. substrate (ACh)
By 10 mg heart extract
By 100 mg albumen gland extract
1 0 -5
30 77 46 40
39 57 32 0
10 -4 10 -~ 10 -~
As T a b l e 4 shows, there is complete inhibition of albumen gland extract activity at high substrate concentration, but only partial inhibition of heart extract.
e. Substrate specificity of the extracts T a b l e 5 shows the comparative activities of different choline esters. T h e heart enzyme shows considerable specificity for the acetyl ester. T A B L E 5 - - H Y D R O L Y S I S OF VARIOUS CHOLINE ESTERS BY
Choline ester Acetylcholine Butyrylcholine Propionylcholine
H. aspersa
% Acceleration by 30 mg heart extract 59 ( - 5) 15
HEART EXTRACT
C H O L I N E ESTERASE ACTIVITY I N THE TISSUES OF H E L I X A S P E R S A
927
DISCUSSION Because of the method of preparation there is considerable variation between one extract and the next; for this reason, comparisons are avoided except as between runs made with samples of the same extract. Although quantitative values cannot be accorded much confidence, the data for one typical run of heart extract correspond to a rate of hydrolysis of acetylcholine of about 320/~moles/hr per g of tissue. This compares with the figure of 525/~moles/hr per ml found by Augustinsson (1948) for H. pomatia blood. The higher activity of the auricle is of some interest. The effect of acetylcholine on the heart is inhibitory (Kerkut & Cottrell, 1963) and it has been suggested that the mediator for the inhibitory fibres of the mixed visceral nerve is in fact acetylcholine. It has been suggested by Ripplinger (1957) that the innervation of the auricle (by the auricular branch of the visceral nerve) is exclusively inhibitory, while that of the ventricle contains both excitatory and inhibitory fibres. Something may also be surmised about the nature of the esterases involved. Specific acetylcholinesterases (true, type I, e-type cholinesterase) show a greater affinity for acetyl- than for butyryl- or propionylcholines, while in the case of the non-specific cholinesterases ("pseudo-", type II, s-type) activity increases with the length of the acyl chain (Gomori, 1952; Augustinsson & Nachmahnsohn, 1949). Moreover, for the former there is a distinct optimum concentration of substrate, whereas for the latter activity increases with increasing substrate concentration (Mendel et al., 1943). From the results shown in Table 5 it appears that the substrate specificity of the heart enzyme accords with that for the specific acetylcholinesterase. The activity-substrate curves for both albumen gland and heart extract show distinct maxima, though the shape of that for the heart might suggest a mixture of specific and non-specific enzymes. Augustinsson (1948) found the H. pomatia blood enzyme to be a specific acetylcholinesterase, while the dart sac contained unmixed non-specific cholinesterase. Using histochemical methods, Saklnki & Zsoltan-Nagy (1965) found in the neuropile of gastropods an enzyme which would hydrolyse acetyl- and butyrylthiocholines. If the snails were in the active state, enzymic activity was abolished by either eserine or DFP, but when the snails were taken in hibernation, the inhibitors were ineffective. They deduce from this that they are dealing with different fractions, in the former case a butyrylcholinesterase, in the latter an arylesterase. However, it is not clear how far it is permissible to assign to invertebrate enzymes names which refer to their substrate specificity, merely by the use of inhibitors whose specificity has been determined on vertebrate enzymes. The optimum substrate concentration for the heart enzyme is about 10 -3"'~. This is equivalent to a pSopt in molar terms of 2.8. Augustinsson found pSol, t for the H. pomatia blood enzyme to be 2.7 and Bockendahl about 2.5. The occurrence of a cholinesterase which appears to be specific acetylcholinesterase fulfils one of the conditions for cholinergy in this organ. It is not in itself valuable evidence for the hypothesis, since it occurs in so many other sites. No
928
E. Kom~
function has been suggested for the enzyme in the albumen gland; the role of this organ in the economy of the snail is not fully understood, but it appears to be concerned with the production of storage materials for the egg. SUMMARY 1. T h e addition of extracts of snail heart, brain and albumen gland increases the rate of hydrolysis of A C h solutions. T h e increase in the rate of hydrolysis is proportional to the amount of extract added. 2. T h e heart is m o r e active than any other tissue in terms of wet weight, the auricle being richer in enzyme than the ventricle. 3. T h e activity is abolished by boiling, by allowing the extract to stand or by treatment with 10 -4 eserine. 4. T h e heart enzyme is m o r e active on acetyl- than on butyryl- or propionylcholine. 5. As the concentration of the substrate is increased, the rate of hydrolysis increases to a m a x i m u m and then falls off. pSop ~ for the heart is about 2.8. 6. T h e results are consistent with the hypothesis of cholinergy in the cardioinhibitory fibres of the visceral nerve. REFERENCES AUGUSTINSSON K. B. (1948) Cholinesterases--a study in comparative enzymology. Acta physiol, scand. 15, Suppl. 52, 1-181. AUGUSTINSSON K. B. & NACHMAHNSOHND. (1949) Studies on cholinesterase--VI. The kinetics of the inhibition of acetyl cholinesterase, j~. biol. Chem. 179, 543-559. BACQZ. M. (1947) L'ac6tylcholine et l'adr6naline chez les Invert6br~s. Biol. Rev. 22, 73-91. BACQZ. M. & COPPICEG. (1937) R6action des Vers et des Mollusques h l'6s6rine. Existence des nerfs cholinergiques chez les Vers. Arch. int. Physiol. 45, 310-324. BOCKENDAHLH. (1962) Untersuchungen an der Acetylcholinesterase des Blutes von Helix pomatia. Hoppe-Seyler's Z. physiol. Chem. 328, 97-107. BOCK~NDAHLH. & MULLERT. M. (1965) Untersuchungen an der Acetylcholinesterase--VI. Das Vorkommen weiterer acetylcholinspaltender Esterasen in Helix pomatia. HoppeSeyler's Z. physiol. Chem. 343, 79-85. CARLSON A. J. (1905) Comparative physiology of the invertebrate heart--III. Physiology of the cardiac nerves in molluscs (continued). Am. J. Physiol. 14, 16-53. COTT~LL G. A. & LAW~U~CKM. S. (1968) Invertebrate pharmacology. A. Rev. Pharmac. 8, 273-298. ER;~qKb O., HXRK/~N~'~M., KOKKOJ. P. & R X I s ~ ' ~ L. (1964) Histochemical and starch gel electrophoretic characterization of desmo- and lyo-esterases in the sympathetic and spinal ganglia of the rat. J. Histochem. Cytochem. 12, 570-581. FLOREY E. & FLOREYE. (1953) ~ b e r die Bedeutung yon 5-Hydroxytryptamin als nerv6ser Aktionssubstanz bei Cephalopoden und dekapoden Crustaceen. Naturwlssenschaften 40, 413-414. GLICK D. (1937) Properties of cholinesterase in human serum. Biochem. jY. 31, 521-531. GLICK D. (1938) Studies of enzymatic histochemistry--XXV. A micromethod for the determination of cholinesterase and the activity-pH relationship of this enzyme, ft. gen. Physiol. 21, 289-297. GOMORI G. (1952) Microscopical Histochemistry. University of Chicago Press.
CHOLINE ESTERASE ACTIVITY IN THE TISSUES OF H E L I X / I S P E R S A
929
A., CARDOT J . , RIPPLINGER J. & JoLv M. (1959) Revue g6n6rale sur la r6gulation cardiaque chez les invert6br6s. Hypotheses r6centes. Annls scient. Univ. Besangon (2), Zool. Physiol. 12, 67-82. JULLIEN A. & RIPPLINGER J. (1954) Recherches sur la structure du nerf visc6ral de l'Escargot (Helix pomatia) et sur la distribution des fibres constitutives ~ l'int6rieur du myocarde. .4nnls scient. Univ. Besan~on (2), Zool. Physiol. 1, 71-77. KEaKUT G. A. & COTTm~LL G. (1963) Acetylcholine and 5-hydroxytryptamine in the snail brain. Comp. Biochem. Physiol. 8, 53-63. LOVELAND R. E. (1963) 5-Hydroxytryptamine, the probable mediator in the heart of Mercenaria (Venus) mercenaria. Comp. Biochem. Physiol. 9, 95-104. MENDEL B., MUNDELL D. B. & RUDNEY H. (1943) Studies on cholinesterase--III. Specific tests for true cholinesterase and pseudo-cholinesterase. Biochem. ft. 37, 473-476. MENC K. (1960) Untersuchungen zur Steuerung der Herzt~itigkeit bei Helix pomatia. Zool. fib. (Allg. Zool. Physiol. d. T.) 68, 539-566. NEEDHAM A. E. (1950) T h e neurogenic heart and ether anaesthesia. Nature, Lond. 166, 9-11. PILGRIM R. L. C. (1954) T h e action of acetylcholine on the hearts of lamellibranch molluscs. ft. Physiol. 125, 208-214. RIPPLINGEa J. (1957) Contribution h l'~tude de la physiologie du cceur et de son innervation extrins+que chez l'Escargot (Helix pomatia). Ann. scient. Univ. Besan~on (2), Zool. Physiol. 8, 3-179. SALANKI J. & ZSOLTAN-NAcY I. (1965) Histochemical investigations of cholinesterase in different molluscs with reference to functional conditions. Nature, Lond. 206, 842-843. Sz-R6szA K. & PEa~NYI L. (1966) Chemical identification of the excitatory substance released in Helix heart during stimulation of the extracardial nerve. Comp. Biochem. Physiol. 19, 105-113. WELSH J. H. (1954) Hydroxytryptamine: a neurohormone in the invertebrates. Fedn Proc. 13, 162-163. WELSH J. H. & MOOaHEAD M. (1959) T h e in vitro synthesis of 5-hydroxytryptamine from 5-hydroxytryptophan by nervous tissue of two species of mollusks. Gunma ft. reed. Sci. 8, 211-218. WELSH J. H. & MOOaHEAD M. (1960) T h e quantitative distribution of 5-hydroxytryptamine in the invertebrates, especially in their nervous systems, ft. Neurochem. 6, 146-149. WELSH J. H. & TAUB R. (1948) T h e action of choline and related compounds on the heart of Venus mercenaria. Biol. Bull., Woods Hole 95, 346-353.
JULLIEN
C H O L I N E ESTERASE A C T I V I T Y IN T H E T I S S U E S OF T H E SNAIL H E L I X A S P E R S A E. K O R N Department of Zoology, The University of Liverpool, England (Received 28 March 1968) A b s t r a c t - - 1 . The titrimetric method of Glick (1937) was used to measure
cholinesterase activity in various tissues of Helix aspersa. 2. Such activity was found in a variety of tissues and was particularly high in the heart. 3. The activity showed some of the properties of a true cholinesterase. INTRODUCTION THE GASTROPODheart is myogenic in nature (Needham, 1950), but its beat is regulated by extrinsic nerves (Carlson, 1905). The control is mainly exercised through the visceral nerve whose anatomy has been described (Jullien & Ripplinger, 1954) for Helix pomatia. Its course is similar in Helix aspersa. Stimulation of the visceral nerve may in different conditions cause acceleration or inhibition of the beat (Sz-R6sza & Per6nyi, 1966). Pharmacological studies have indicated the possibility that the neuromuscular transmitter substance involved in inhibition is acetylcholine, and in excitation is 5-hydroxytryptamine (Meng, 1960). Both these substances have been identified in the brain of H. aspersa (Kerkut & Cottrell, 1963). While both have been identified and shown to have pharmacological actions appropriate to the hypothesis of their role as mediators in a variety of molluscs (Bacq & Copp6e, 1937; Bacq, 1947; Welsh, 1954; Welsh & Moorhead, 1960), the evidence is extensive only in the case of the lameUibranch Venus mercenaria (Welsh & Taub, 1948; Florey & Florey, 1953; Welsh & Moorhead, 1959; Loveland, 1963). Anomalies in the response of mollusc hearts to acetylcholine (Pilgrim, 1954) have been used by some workers to argue against the hypothesis of cholinergy in the snail (Jullien et al., 1959). Since the presence of an enzyme system capable of inactivating acetylcholine is often used as one of the criteria of cholinergy (e.g. Bacq, 1947), it seemed valid to investigate this. The existence of such a system has been demonstrated in the blood of H. pomatia (Augustinsson, 1948; Bockendahl, 1962) and in the snail gut (Bockendahl & Mtiller, 1965). Moreover, histochemical tests have shown the presence of Gomori-positive substances in the nerve endings of H. aspersa (Sal~nki & Zsoltan-Nagy, 1965). The whole subject of transmitters in invertebrates has recently been reviewed fully (Cottrell & Laverack, 1968). 923
924
E. KORN
MATERIALS AND METHODS a. Titrimetric methods Glick's method for the estimation of esterase activity depends on the liberation of acid by the breakdown of choline esters. T h e p H is measured and maintained at a fixed point by the titration of a dilute solution of sodium hydroxide. T h e rate of addition of the alkali then represents the rate of breakdown of the choline ester, and the effect on this rate of the addition of tissue extracts provides a measure of the activity of the extracts (Glick, 1937). Standard solutions of acetylcholine chloride were made up in Meng's snail Ringer (1960) and stabilized by the addition of a few drops of HCI to bring them to a p H below 4. T h e y were stored at 0-10°C. Twenty-five-ml samples of this solution were pipetted into a 100-ml beaker and the p H measured continuously with a Pye p H meter. T h e solution was stirred continuously with a magnetic stirrer. When the temperature had risen to about 20°C, the p H was brought to 8 with 0"1 N NaOH. T h e time was then noted and as the p H dropped below 8, 0.005 N N a O H was run in from a burette, causing the p H to overshoot 8 slightly. It was then permitted to drop again and the operation repeated. Each time the p H passed 8"00, the time and the burette reading were noted. After a number of readings had been taken in this fashion, the tissue extract, or extract and inhibitor, were added, the p H adjusted to 8 once more, and a further series of readings taken. T h e p H of 8 was adopted as this optimized the difference between non-enzymatic hydrolysis (the rate of which rises sharply with solutions more alkaline than p H 8"5) and enzymatic hydrolysis, which is optimal at p H 9 (Glick, 1938). Care was taken to prevent the p H of the solution ever passing outside the range 7"9-8"1. Acetyl-, butyryl- and propionyl-cholines were used (all as the chloride salt). T h e titration of 1/zl of alkali corresponds to the hydrolysis of 0"9/~g of acetylcholine chloride. b. Preparation of extracts A number of snails (usually twenty-five) was rapidly dissected and the appropriate organs removed. T h e y were weighed on filter paper, scraped off into a small known quantity of cold Meng's Ringer, the filter paper reweighed, and the tissue then brought to a convenient concentration by the addition of further Ringer solution. T h e organs were then minced with fine scissors and crudely homogenized. T h e solution was allowed to stand for half an hour at 0-5°C. T h e supernatant was then decanted off from the tissue. Control experiments showed no measurably greater activity when the tissues were more fully homogenized, which suggests that we are here dealing with the lyozyme fraction (ErLnk5 et al., 1964). T h e extract could be stored frozen for upwards of 48 hr but was usually used immediately. RESULTS
I n t h e results, t h e q u a n t i t y o f e x t r a c t a d d e d is q u o t e d in g w e t wt. o f tissue. T h e v o l u m e a d d e d was n e v e r m o r e t h a n 1 ml. S i n c e t h e r e was s o m e v a r i a b i l i t y f r o m r u n to r u n in t h e rate o f n o n - e n z y m a t i c h y d r o l y s i s ( a l t h o u g h n o t w i t h i n t h e run), t h e p e r c e n t a g e a c c e l e r a t i o n c a u s e d b y a g i v e n e n z y m e p r e p a r a t i o n w a s u s e d for c o m p a r a t i v e p u r p o s e s r a t h e r t h a n t h e absolute rate of enzymatic hydrolysis. a. A l b u m e n g l a n d T h i s tissue h a d a l r e a d y b e e n r e p o r t e d as a r i c h s o u r c e o f esterase ( K e r k u t , p e r s o n a l c o m m u n i c a t i o n ) . R e s u l t s a r e s h o w n in T a b l e 1, rates b e i n g t a k e n f r o m t h e s l o p e values o f g r a p h s .
925
CHOLINE ESTERASE ACTIVITY IN THE TISSUES OF H E L I X ASPERSA
b. Comparison of the enzymatic activity of various tissues Although there was considerable variation in the activity of the extracts, those from the heart were more active than those from the brain and the albumen gland, which were roughly similar in activity. Rates of hydrolysis are shown in Table 2. The heart has the highest level of esterase activity/unit of weight. T A B L E 1 - - H Y D R O L Y S I S OF ACETYLCI-tOLINE BY EXTRACT OF
Non-enzymatic rate
Material added 100 mg albumen gland 100 mg albumen gland after 2 min boiling Ringer control
30 mg heart 50 mg brain 50 mg albumen gland
dspersa ALBUMEN GLAND
Rate after addition of extract
In/~l/sec of 0"005 N NaOH added
% Acceleration
0"52 0"46
0"70 0"48
35 4
0"63
0"67
6
T A B L E 2 - - H Y D R O L Y T I C ACTIVITY OF
Extract
H.
H. aspersa TISSUES
% Acceleration
Rate of enzymatic hydrolysis (#molesACh/g per hr)
49 54 43
317 97 72
Some activity was also found in the hepatopancreas but none in the foot musculature. Augustinsson (1948) found a high level of esterase activity in the hemocoelic fluid of H. poraatia. That the activity here measured is present in the tissues of the myocardium rather than in the hemocoelic fluid is shown by electrophoretic studies of esterases. The activity of the auricle was compared with that of the ventricle in another series of experiments. The auricles and the ventricle were separated by a transverse cut and ground separately. The auricle extracts showed a consistently higher activity/unit of weight than those of the ventricle. A single auricle, whose weight was approximately 3 mg, gave an acceleration of 30 per cent; a single ventricle (weight 15 mg) gave a 25 per cent acceleration. This suggests that the total hydrolytic activity of the heart is approximately equally divided between the two chambers of the heart, the auricle being some fivefold richer per unit of weight.
c. Enzyme inhibition Table 3 shows the abolition of enzymatic activity by treatment with 10 -4 eserine. The extract was incubated with the inhibitor for 50 rain at room temperature, and the experimental run alternated with runs of untreated preparation also maintained at room temperature. Eserine abolishes activity completely.
926
E. KORN
d. Relation of activity to substrate concentration For these runs the extract was prepared in a magnesium-rich Ringer (NaCI 5.9 g/l, MgC12 3-8 g/l), to maximize the activity at the lower substrate concentrations. Augustinsson (1948) has reported an activating effect of M g ~+ ions on cholinesterase. T h e r e was an approximately tenfold increase in the effectiveness of the extract on 10 -4 acetylcholine as c o m p a r e d with the m a g n e s i u m - p o o r extract. T A B L E 3 - - I N H I B I T I O N OF CHOLINESTERASE ACTIVITY BY ESERINE
Time from start of incubation (min)
Extract
% Acceleration
0 50 80
Normal Eserinized Normal
30 ( - 5) 19
T A B L E 4 - - - S u B S T R A T E CONCENTRATION AND CHOLINESTERASE ACTIVITY
% Acceleration Conc. substrate (ACh)
By 10 mg heart extract
By 100 mg albumen gland extract
1 0 -5
30 77 46 40
39 57 32 0
10 -4 10 -~ 10 -~
As T a b l e 4 shows, there is complete inhibition of albumen gland extract activity at high substrate concentration, but only partial inhibition of heart extract.
e. Substrate specificity of the extracts T a b l e 5 shows the comparative activities of different choline esters. T h e heart enzyme shows considerable specificity for the acetyl ester. T A B L E 5 - - H Y D R O L Y S I S OF VARIOUS CHOLINE ESTERS BY
Choline ester Acetylcholine Butyrylcholine Propionylcholine
H. aspersa
% Acceleration by 30 mg heart extract 59 ( - 5) 15
HEART EXTRACT
C H O L I N E ESTERASE ACTIVITY I N THE TISSUES OF H E L I X A S P E R S A
927
DISCUSSION Because of the method of preparation there is considerable variation between one extract and the next; for this reason, comparisons are avoided except as between runs made with samples of the same extract. Although quantitative values cannot be accorded much confidence, the data for one typical run of heart extract correspond to a rate of hydrolysis of acetylcholine of about 320/~moles/hr per g of tissue. This compares with the figure of 525/~moles/hr per ml found by Augustinsson (1948) for H. pomatia blood. The higher activity of the auricle is of some interest. The effect of acetylcholine on the heart is inhibitory (Kerkut & Cottrell, 1963) and it has been suggested that the mediator for the inhibitory fibres of the mixed visceral nerve is in fact acetylcholine. It has been suggested by Ripplinger (1957) that the innervation of the auricle (by the auricular branch of the visceral nerve) is exclusively inhibitory, while that of the ventricle contains both excitatory and inhibitory fibres. Something may also be surmised about the nature of the esterases involved. Specific acetylcholinesterases (true, type I, e-type cholinesterase) show a greater affinity for acetyl- than for butyryl- or propionylcholines, while in the case of the non-specific cholinesterases ("pseudo-", type II, s-type) activity increases with the length of the acyl chain (Gomori, 1952; Augustinsson & Nachmahnsohn, 1949). Moreover, for the former there is a distinct optimum concentration of substrate, whereas for the latter activity increases with increasing substrate concentration (Mendel et al., 1943). From the results shown in Table 5 it appears that the substrate specificity of the heart enzyme accords with that for the specific acetylcholinesterase. The activity-substrate curves for both albumen gland and heart extract show distinct maxima, though the shape of that for the heart might suggest a mixture of specific and non-specific enzymes. Augustinsson (1948) found the H. pomatia blood enzyme to be a specific acetylcholinesterase, while the dart sac contained unmixed non-specific cholinesterase. Using histochemical methods, Saklnki & Zsoltan-Nagy (1965) found in the neuropile of gastropods an enzyme which would hydrolyse acetyl- and butyrylthiocholines. If the snails were in the active state, enzymic activity was abolished by either eserine or DFP, but when the snails were taken in hibernation, the inhibitors were ineffective. They deduce from this that they are dealing with different fractions, in the former case a butyrylcholinesterase, in the latter an arylesterase. However, it is not clear how far it is permissible to assign to invertebrate enzymes names which refer to their substrate specificity, merely by the use of inhibitors whose specificity has been determined on vertebrate enzymes. The optimum substrate concentration for the heart enzyme is about 10 -3"'~. This is equivalent to a pSopt in molar terms of 2.8. Augustinsson found pSol, t for the H. pomatia blood enzyme to be 2.7 and Bockendahl about 2.5. The occurrence of a cholinesterase which appears to be specific acetylcholinesterase fulfils one of the conditions for cholinergy in this organ. It is not in itself valuable evidence for the hypothesis, since it occurs in so many other sites. No
928
E. Kom~
function has been suggested for the enzyme in the albumen gland; the role of this organ in the economy of the snail is not fully understood, but it appears to be concerned with the production of storage materials for the egg. SUMMARY 1. T h e addition of extracts of snail heart, brain and albumen gland increases the rate of hydrolysis of A C h solutions. T h e increase in the rate of hydrolysis is proportional to the amount of extract added. 2. T h e heart is m o r e active than any other tissue in terms of wet weight, the auricle being richer in enzyme than the ventricle. 3. T h e activity is abolished by boiling, by allowing the extract to stand or by treatment with 10 -4 eserine. 4. T h e heart enzyme is m o r e active on acetyl- than on butyryl- or propionylcholine. 5. As the concentration of the substrate is increased, the rate of hydrolysis increases to a m a x i m u m and then falls off. pSop ~ for the heart is about 2.8. 6. T h e results are consistent with the hypothesis of cholinergy in the cardioinhibitory fibres of the visceral nerve. REFERENCES AUGUSTINSSON K. B. (1948) Cholinesterases--a study in comparative enzymology. Acta physiol, scand. 15, Suppl. 52, 1-181. AUGUSTINSSON K. B. & NACHMAHNSOHND. (1949) Studies on cholinesterase--VI. The kinetics of the inhibition of acetyl cholinesterase, j~. biol. Chem. 179, 543-559. BACQZ. M. (1947) L'ac6tylcholine et l'adr6naline chez les Invert6br~s. Biol. Rev. 22, 73-91. BACQZ. M. & COPPICEG. (1937) R6action des Vers et des Mollusques h l'6s6rine. Existence des nerfs cholinergiques chez les Vers. Arch. int. Physiol. 45, 310-324. BOCKENDAHLH. (1962) Untersuchungen an der Acetylcholinesterase des Blutes von Helix pomatia. Hoppe-Seyler's Z. physiol. Chem. 328, 97-107. BOCK~NDAHLH. & MULLERT. M. (1965) Untersuchungen an der Acetylcholinesterase--VI. Das Vorkommen weiterer acetylcholinspaltender Esterasen in Helix pomatia. HoppeSeyler's Z. physiol. Chem. 343, 79-85. CARLSON A. J. (1905) Comparative physiology of the invertebrate heart--III. Physiology of the cardiac nerves in molluscs (continued). Am. J. Physiol. 14, 16-53. COTT~LL G. A. & LAW~U~CKM. S. (1968) Invertebrate pharmacology. A. Rev. Pharmac. 8, 273-298. ER;~qKb O., HXRK/~N~'~M., KOKKOJ. P. & R X I s ~ ' ~ L. (1964) Histochemical and starch gel electrophoretic characterization of desmo- and lyo-esterases in the sympathetic and spinal ganglia of the rat. J. Histochem. Cytochem. 12, 570-581. FLOREY E. & FLOREYE. (1953) ~ b e r die Bedeutung yon 5-Hydroxytryptamin als nerv6ser Aktionssubstanz bei Cephalopoden und dekapoden Crustaceen. Naturwlssenschaften 40, 413-414. GLICK D. (1937) Properties of cholinesterase in human serum. Biochem. jY. 31, 521-531. GLICK D. (1938) Studies of enzymatic histochemistry--XXV. A micromethod for the determination of cholinesterase and the activity-pH relationship of this enzyme, ft. gen. Physiol. 21, 289-297. GOMORI G. (1952) Microscopical Histochemistry. University of Chicago Press.
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A., CARDOT J . , RIPPLINGER J. & JoLv M. (1959) Revue g6n6rale sur la r6gulation cardiaque chez les invert6br6s. Hypotheses r6centes. Annls scient. Univ. Besangon (2), Zool. Physiol. 12, 67-82. JULLIEN A. & RIPPLINGER J. (1954) Recherches sur la structure du nerf visc6ral de l'Escargot (Helix pomatia) et sur la distribution des fibres constitutives ~ l'int6rieur du myocarde. .4nnls scient. Univ. Besan~on (2), Zool. Physiol. 1, 71-77. KEaKUT G. A. & COTTm~LL G. (1963) Acetylcholine and 5-hydroxytryptamine in the snail brain. Comp. Biochem. Physiol. 8, 53-63. LOVELAND R. E. (1963) 5-Hydroxytryptamine, the probable mediator in the heart of Mercenaria (Venus) mercenaria. Comp. Biochem. Physiol. 9, 95-104. MENDEL B., MUNDELL D. B. & RUDNEY H. (1943) Studies on cholinesterase--III. Specific tests for true cholinesterase and pseudo-cholinesterase. Biochem. ft. 37, 473-476. MENC K. (1960) Untersuchungen zur Steuerung der Herzt~itigkeit bei Helix pomatia. Zool. fib. (Allg. Zool. Physiol. d. T.) 68, 539-566. NEEDHAM A. E. (1950) T h e neurogenic heart and ether anaesthesia. Nature, Lond. 166, 9-11. PILGRIM R. L. C. (1954) T h e action of acetylcholine on the hearts of lamellibranch molluscs. ft. Physiol. 125, 208-214. RIPPLINGEa J. (1957) Contribution h l'~tude de la physiologie du cceur et de son innervation extrins+que chez l'Escargot (Helix pomatia). Ann. scient. Univ. Besan~on (2), Zool. Physiol. 8, 3-179. SALANKI J. & ZSOLTAN-NAcY I. (1965) Histochemical investigations of cholinesterase in different molluscs with reference to functional conditions. Nature, Lond. 206, 842-843. Sz-R6szA K. & PEa~NYI L. (1966) Chemical identification of the excitatory substance released in Helix heart during stimulation of the extracardial nerve. Comp. Biochem. Physiol. 19, 105-113. WELSH J. H. (1954) Hydroxytryptamine: a neurohormone in the invertebrates. Fedn Proc. 13, 162-163. WELSH J. H. & MOOaHEAD M. (1959) T h e in vitro synthesis of 5-hydroxytryptamine from 5-hydroxytryptophan by nervous tissue of two species of mollusks. Gunma ft. reed. Sci. 8, 211-218. WELSH J. H. & MOOaHEAD M. (1960) T h e quantitative distribution of 5-hydroxytryptamine in the invertebrates, especially in their nervous systems, ft. Neurochem. 6, 146-149. WELSH J. H. & TAUB R. (1948) T h e action of choline and related compounds on the heart of Venus mercenaria. Biol. Bull., Woods Hole 95, 346-353.
JULLIEN