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Control of the ciliary activity in the gills of Anodonta by acetylcholine
I52
Comp. gen. Pharma~.
CONTROL OF THE CILIARY ACTIVITY IN THE GILLS OF A.N'ODO.N'TA BY ACETYLCHOLINE K. Y. H. L A G E R S P E T Z , H I L L E V I
LJ~NSIMIES, HILPPA IMPIVAARA,
Am) K. S E N I U S Zoophysiological Laboratory, Department of Zoology, University of Turku, Finland
(Rerdwd 3 Nov., x969) ABSTRACT x. Acetylcholine (ACh) and eserine at low concentrations stimulated and at high concentrations inhibited the activity of the frontal cilia in the gills of the pelccypod Anodonta cygnea cdlatds. The effects of 5-HT were not definite. 2. ACh concentration and acetylcholinesterase activity in the gills of Anodonta were about to per cent of those found in Mytilus. The difference is apparently related with the relative number of ciliated cells in the gills. The 5-HT concentration was similar in both species. 3. It issuggested that the A C h systcm acts as a cellularcontrol mechanism of the ciliary activity,while 5-HT is involved in the higher level control by the branchial nerve.
BOLBmNO, Burn, a n d Shelley 0 9 5 3 ) a n d Milton (x969) have shown t h a t the choline a c e t y l a s e - a c e t y l c h o l i n e - acetylcholinesterase system is present in the ciliated gills o f the pelecypod Mytilus edul/s. This system is assumed to control the ciliary activity in the gills of Mytilus, a l t h o u g h 5 - H T (5" h y d r o x y t r y p t a m i n e , serotonin) has also been suggested to act as a local h o r m o n e controlling the beat of the cilia (Aiello, I957; Gosselin, x96I). Since knowledge a b o u t the control of ciliary activity in the gills o f the fresh water mussel, Anodonta, was needed for our studies on the temperature acclimation of ciliary activity, a n d nothing was k n o w n a b o u t the effects o f acetylcholine (ACh) on the ciliary activity in Anodonta, these effects were studied. Attempts were also m a d e to demonstrate the presence o f components o f the A C h system in the gills. As another possibility, ciliary control b y 5 - H T was also studied. MATERIALS AND M E T H O D S The test animals, their maintenance and handling, as well as the particle transport method used for the determination of the ciliary activity, were the same as described earlier (Lagerspetz and Dubitscher, 1966 ). However, the ciliary activity
was determined at I9-2x ° C. and in distilled water, with which the solutions were also made. The ciliary activity of excised gills was constant in these conditions for at least 04 hours. As the changes in/OH have a profound effect on the ciliary activity in Mflilus gills (Gray, x922, x928), the possible effects of variations in pH were studied in a preliminary series of experiments. The pH of the water was varied by additions of o.oI M HCI or o-oi M NaOH. A definite decrease in the ciliary activity was seen below pH 4 and above pH 8. In the experiments of Gray (t922) the ciliary activity in Mytilus already decreased below pH 7. Anodonta is in this respect much more tolerant to low pH. This is in accordance with the ecology of the species. As the pH of the solutions used varied only from pH 4"6 to pH 4"8, the effects of the solutions could not be due to differences in pH only. For the assays of ACh and acetylcholinesterase activity, respectively, the frog musculus rectus abdominis method (Burn, t952 ) and the method of Ellman, Courtney, Andres, and Featherstone (196x) were employed. Because of the low enzyme activity, homogenates of zoo mg. tissue per ml. were prepared. These were then centrifuged for io minutes at iI,65og in the cold, and the supernatant used for the measurement of the enzyme activity. The sediment showed no acetylcholinesterase activity. The 5-HT assays were performed using the method of Bogdanski, Pletscher, Brodie, and Udenfriend (I956) as modified by Kuntzman, Shore, Bogdauski, and Brodie (196I).
I97o, z, x52-I54
x53
C O N T R O L OF C I L I A R Y A C T I V I T Y
RESULTS T h e results of the experiments on the effects of A C h chloride and eserine (physostigmlne) sulphate on the ciliary activity are summarized in Table I and the course of two typical experiments is presented in Fig. i. A change of 4 per cent was calculated to represent a statistically significant response at P = o . o 5 level. ACh at low concentrations
cm/min
ACh content of 4 ~g. per g. in the gills of Mytilus. T h e acetylcholinesterase activity of the gill tissue at 320C. was an average of 3 t n M per g. per minute (4 determinations with animals kept previously at 22 ° C.). The activity is less than to per cent of that found by Bttlbring and others (I953) in Mytilus gills. The 5 - H T content of the gills in Anodonta was from o.x to o.6 ~tg. per g., an
4,8
• ACH
/
4.2
10 . 3
T/~ °E~10"2
3,6~ W~ W~ ~/
• Ac. !o' 0 |
Ik
10 -4
3,0 2,4
' 0
' 60
' 120
I 180
JI
, 240
3 0 0 rain
FIG. I.--The effect of acetylcholine (ACh) and eserlne (E) on the ciliary activity, measured as particle transport rate (cm. per minute) in the gills of Anodonta. Arrows indicate the transfer of gills to ACh and E solutions or to water (W) for the control measurements. (IO-Sto Io-6g. per ml.) stimulated the rate of the particle transport by frontal gill cilia, while ACh at high concentration (io -3 g. per ml.) had an inhibitory effect. Eserine had similar effects, a stimulating effect at Io-4g. per ml., and an inhibitory effect at xo-~ g. per ml. 5 - H T at concentrations from xo -~ to I o -s had no consistent effect, although the particle transport rate seemed to be more variable in 5 - H T solution than in water. The average content of A C h in the gills was o'44 pg. per g. (mean of 8 determinations). Biilbring and others (i953) found an
average of 0.36 lag. per g. (9 determinations). According to Aiello (I962), the 5 - H T content in the Mytilus gills varied between o.x and I.O I~g. per g. DISCUSSION T h e effects of ACh and the acetylcholinesterase inhibitor, eserine, on the ciliary activity in the gills of Anodonta are similar to those found by Bi~lbring and others (I953) in Nl.yti[~. This suggests that the ACh system in Anodonta also acts as a cellular mechanism controlling the ciliary activity in the gill
'54
LAGERSPETZ ET AL.
REFERENCES AmLLO, E. L. (1957), ' T h e influence of the branchial nerve and of five-hydroxytryptamine on the ciliary activity of Mytilus gill ', Biol. Bull. mar. biol. Lab., Woods Hole, xx3, 325 • -- - (I96O), ' Factors affecting ciliary activity on the gill of the mussel Mytilus edulis ', Table L--EFFECT ov ACETYLCHOLINEAND ESERINE ON THE CILIARYAcTivrrY (PARTICLETRANSPORT Physiol. Zo#l., 33, I2o-I35. - - - - (1962), ' Identification of the cilioexcitatory RATE' IN THE GILLSOF Anodonta substance present in the gill of the mussel Mytilus edulis ', 07. Cell comp. Physiol., 6% Suppl. STIMULATION (-~-) x , I7-2I. CONCENOR INHIBITION (--) No. oF BARRINOTON,E. J. W. (,967), Invertebrate Structure TRATION EXPERIMENTS (per cent of and Function, p. x86. London: Nelson. control) BOODANSKX,D. F., PLETSeHER, A., BRoDm, B. B., and UDENFRI~NV, S. (I956), ' Identification Acetylcholine and assay of serotonin in brain ', 07. Pharmac. 12 XO-S --I9. 9 exp. Ther., HT, 82-88. +x2.I I2 i0-5 BOLERrso, E., Bum~, J. H., and SHELLEY, H. IO-~ +17.3 (1953), ' Acetylcholine and ciliary movement in lO-S -~ I,~.I the gill plates of Mytilus edulis ', Proc. R. Soc. B, x4x, 445-466. Eserine BURN, J. H. (I952), Practical Pharmacology, pp. 1-4, iO-2 -27"3 Oxford: BlackweU. i0-3 + 5"2 ELL~a~a~, G. L., COURTNEV,K. D., ANDRES,V. J., i0-4 + I8.3 and F~.ATH~.~TONE,R. M. (I961), ' A new and rapid eolorimetric determination of acetylcholinesterase activity ', Biochem. Pharmac., 7, 88-95. aeetylcholinesterase activity in the gills of GOSSZLIN, R. E. (1961), ' T h e cilioexcitatory Anodonta a n d Mytilus. Since the incubation activity of serotonin ', 07. Cell comp. Physiol., temperature in the e n z y m e activity deter58, '7-25. minations o f Btilbring a n d others (I953) was GRAY, J. (i922), ' T h e mechanism of ciliary movement ', Proc. R. Soc. B, 93, I ° 4 - ' 3 L 37.5 ° C., i.e., 5.5 ° C. higher, the difference -- - (I928), Ciliary Movement, p. 8o. Cambridge: between the species is only to a small extent Cambridge University Press. due to the difference in the incubation KUNTZMAN,R., Saom~, P. A., BOODANSVa,D., and temperatures. O n the other hand, the BRODm, B. B. (196I), ' Microanalytical procedures for fluorometric assay of brain DOPAdifference is correlated with the relative 5HTP decarboxylase, norepinephrine and seroa m o u n t of ciliated epithelium in the gills tonin, and a detailed mapping of decarboxylase o f the two species, which is perhaps IO to activity in brain ', 07. aVeurochem., 6, 226--23~. ~o times greater in Mytffus than in Anodonta, LAOERSPETZ,K.Y.H., and Dtmrrscn~R, I. (z966), ' Temperature acclimation of the ciliary activity w h e n estimated f r o m the figures of the gill in the gills of Anodonta ", Comp. Biochem. Physiol., structure given by Barrington (I967). T h e '7, 665-67 I. 5 - H T content is similar in both species, a n d MILTON, A. S. (I959), ' Choline acetylase in the thus not correlated with the a m o u n t of gill plates of Mytilus edulis ', Proc. R. Soc. B, eiliated cells. These observations seem also xSO~ 24o--244 . to point out that the cellular control of ciliary activity is by the A C h system, while Key Word Index : Acetylcholine, 5-hydroxy5 - H T m i g h t act in a higher level control tryptamine, ciliary activity, gill tissue, acetylsystem involving the branchial nerve, as choline concentration, acetylcholinesterase activity, suggested by Aiello (196o). Anodonta eygnea eeUensis. epithelium or at least the activity of the frontal cilia. T h e effects o f 5 - H T are less definite. A n interesting observation is the large differences found in the A C h content a n d in the
Comp. gen. Pharma~.
CONTROL OF THE CILIARY ACTIVITY IN THE GILLS OF A.N'ODO.N'TA BY ACETYLCHOLINE K. Y. H. L A G E R S P E T Z , H I L L E V I
LJ~NSIMIES, HILPPA IMPIVAARA,
Am) K. S E N I U S Zoophysiological Laboratory, Department of Zoology, University of Turku, Finland
(Rerdwd 3 Nov., x969) ABSTRACT x. Acetylcholine (ACh) and eserine at low concentrations stimulated and at high concentrations inhibited the activity of the frontal cilia in the gills of the pelccypod Anodonta cygnea cdlatds. The effects of 5-HT were not definite. 2. ACh concentration and acetylcholinesterase activity in the gills of Anodonta were about to per cent of those found in Mytilus. The difference is apparently related with the relative number of ciliated cells in the gills. The 5-HT concentration was similar in both species. 3. It issuggested that the A C h systcm acts as a cellularcontrol mechanism of the ciliary activity,while 5-HT is involved in the higher level control by the branchial nerve.
BOLBmNO, Burn, a n d Shelley 0 9 5 3 ) a n d Milton (x969) have shown t h a t the choline a c e t y l a s e - a c e t y l c h o l i n e - acetylcholinesterase system is present in the ciliated gills o f the pelecypod Mytilus edul/s. This system is assumed to control the ciliary activity in the gills of Mytilus, a l t h o u g h 5 - H T (5" h y d r o x y t r y p t a m i n e , serotonin) has also been suggested to act as a local h o r m o n e controlling the beat of the cilia (Aiello, I957; Gosselin, x96I). Since knowledge a b o u t the control of ciliary activity in the gills o f the fresh water mussel, Anodonta, was needed for our studies on the temperature acclimation of ciliary activity, a n d nothing was k n o w n a b o u t the effects o f acetylcholine (ACh) on the ciliary activity in Anodonta, these effects were studied. Attempts were also m a d e to demonstrate the presence o f components o f the A C h system in the gills. As another possibility, ciliary control b y 5 - H T was also studied. MATERIALS AND M E T H O D S The test animals, their maintenance and handling, as well as the particle transport method used for the determination of the ciliary activity, were the same as described earlier (Lagerspetz and Dubitscher, 1966 ). However, the ciliary activity
was determined at I9-2x ° C. and in distilled water, with which the solutions were also made. The ciliary activity of excised gills was constant in these conditions for at least 04 hours. As the changes in/OH have a profound effect on the ciliary activity in Mflilus gills (Gray, x922, x928), the possible effects of variations in pH were studied in a preliminary series of experiments. The pH of the water was varied by additions of o.oI M HCI or o-oi M NaOH. A definite decrease in the ciliary activity was seen below pH 4 and above pH 8. In the experiments of Gray (t922) the ciliary activity in Mytilus already decreased below pH 7. Anodonta is in this respect much more tolerant to low pH. This is in accordance with the ecology of the species. As the pH of the solutions used varied only from pH 4"6 to pH 4"8, the effects of the solutions could not be due to differences in pH only. For the assays of ACh and acetylcholinesterase activity, respectively, the frog musculus rectus abdominis method (Burn, t952 ) and the method of Ellman, Courtney, Andres, and Featherstone (196x) were employed. Because of the low enzyme activity, homogenates of zoo mg. tissue per ml. were prepared. These were then centrifuged for io minutes at iI,65og in the cold, and the supernatant used for the measurement of the enzyme activity. The sediment showed no acetylcholinesterase activity. The 5-HT assays were performed using the method of Bogdanski, Pletscher, Brodie, and Udenfriend (I956) as modified by Kuntzman, Shore, Bogdauski, and Brodie (196I).
I97o, z, x52-I54
x53
C O N T R O L OF C I L I A R Y A C T I V I T Y
RESULTS T h e results of the experiments on the effects of A C h chloride and eserine (physostigmlne) sulphate on the ciliary activity are summarized in Table I and the course of two typical experiments is presented in Fig. i. A change of 4 per cent was calculated to represent a statistically significant response at P = o . o 5 level. ACh at low concentrations
cm/min
ACh content of 4 ~g. per g. in the gills of Mytilus. T h e acetylcholinesterase activity of the gill tissue at 320C. was an average of 3 t n M per g. per minute (4 determinations with animals kept previously at 22 ° C.). The activity is less than to per cent of that found by Bttlbring and others (I953) in Mytilus gills. The 5 - H T content of the gills in Anodonta was from o.x to o.6 ~tg. per g., an
4,8
• ACH
/
4.2
10 . 3
T/~ °E~10"2
3,6~ W~ W~ ~/
• Ac. !o' 0 |
Ik
10 -4
3,0 2,4
' 0
' 60
' 120
I 180
JI
, 240
3 0 0 rain
FIG. I.--The effect of acetylcholine (ACh) and eserlne (E) on the ciliary activity, measured as particle transport rate (cm. per minute) in the gills of Anodonta. Arrows indicate the transfer of gills to ACh and E solutions or to water (W) for the control measurements. (IO-Sto Io-6g. per ml.) stimulated the rate of the particle transport by frontal gill cilia, while ACh at high concentration (io -3 g. per ml.) had an inhibitory effect. Eserine had similar effects, a stimulating effect at Io-4g. per ml., and an inhibitory effect at xo-~ g. per ml. 5 - H T at concentrations from xo -~ to I o -s had no consistent effect, although the particle transport rate seemed to be more variable in 5 - H T solution than in water. The average content of A C h in the gills was o'44 pg. per g. (mean of 8 determinations). Biilbring and others (i953) found an
average of 0.36 lag. per g. (9 determinations). According to Aiello (I962), the 5 - H T content in the Mytilus gills varied between o.x and I.O I~g. per g. DISCUSSION T h e effects of ACh and the acetylcholinesterase inhibitor, eserine, on the ciliary activity in the gills of Anodonta are similar to those found by Bi~lbring and others (I953) in Nl.yti[~. This suggests that the ACh system in Anodonta also acts as a cellular mechanism controlling the ciliary activity in the gill
'54
LAGERSPETZ ET AL.
REFERENCES AmLLO, E. L. (1957), ' T h e influence of the branchial nerve and of five-hydroxytryptamine on the ciliary activity of Mytilus gill ', Biol. Bull. mar. biol. Lab., Woods Hole, xx3, 325 • -- - (I96O), ' Factors affecting ciliary activity on the gill of the mussel Mytilus edulis ', Table L--EFFECT ov ACETYLCHOLINEAND ESERINE ON THE CILIARYAcTivrrY (PARTICLETRANSPORT Physiol. Zo#l., 33, I2o-I35. - - - - (1962), ' Identification of the cilioexcitatory RATE' IN THE GILLSOF Anodonta substance present in the gill of the mussel Mytilus edulis ', 07. Cell comp. Physiol., 6% Suppl. STIMULATION (-~-) x , I7-2I. CONCENOR INHIBITION (--) No. oF BARRINOTON,E. J. W. (,967), Invertebrate Structure TRATION EXPERIMENTS (per cent of and Function, p. x86. London: Nelson. control) BOODANSKX,D. F., PLETSeHER, A., BRoDm, B. B., and UDENFRI~NV, S. (I956), ' Identification Acetylcholine and assay of serotonin in brain ', 07. Pharmac. 12 XO-S --I9. 9 exp. Ther., HT, 82-88. +x2.I I2 i0-5 BOLERrso, E., Bum~, J. H., and SHELLEY, H. IO-~ +17.3 (1953), ' Acetylcholine and ciliary movement in lO-S -~ I,~.I the gill plates of Mytilus edulis ', Proc. R. Soc. B, x4x, 445-466. Eserine BURN, J. H. (I952), Practical Pharmacology, pp. 1-4, iO-2 -27"3 Oxford: BlackweU. i0-3 + 5"2 ELL~a~a~, G. L., COURTNEV,K. D., ANDRES,V. J., i0-4 + I8.3 and F~.ATH~.~TONE,R. M. (I961), ' A new and rapid eolorimetric determination of acetylcholinesterase activity ', Biochem. Pharmac., 7, 88-95. aeetylcholinesterase activity in the gills of GOSSZLIN, R. E. (1961), ' T h e cilioexcitatory Anodonta a n d Mytilus. Since the incubation activity of serotonin ', 07. Cell comp. Physiol., temperature in the e n z y m e activity deter58, '7-25. minations o f Btilbring a n d others (I953) was GRAY, J. (i922), ' T h e mechanism of ciliary movement ', Proc. R. Soc. B, 93, I ° 4 - ' 3 L 37.5 ° C., i.e., 5.5 ° C. higher, the difference -- - (I928), Ciliary Movement, p. 8o. Cambridge: between the species is only to a small extent Cambridge University Press. due to the difference in the incubation KUNTZMAN,R., Saom~, P. A., BOODANSVa,D., and temperatures. O n the other hand, the BRODm, B. B. (196I), ' Microanalytical procedures for fluorometric assay of brain DOPAdifference is correlated with the relative 5HTP decarboxylase, norepinephrine and seroa m o u n t of ciliated epithelium in the gills tonin, and a detailed mapping of decarboxylase o f the two species, which is perhaps IO to activity in brain ', 07. aVeurochem., 6, 226--23~. ~o times greater in Mytffus than in Anodonta, LAOERSPETZ,K.Y.H., and Dtmrrscn~R, I. (z966), ' Temperature acclimation of the ciliary activity w h e n estimated f r o m the figures of the gill in the gills of Anodonta ", Comp. Biochem. Physiol., structure given by Barrington (I967). T h e '7, 665-67 I. 5 - H T content is similar in both species, a n d MILTON, A. S. (I959), ' Choline acetylase in the thus not correlated with the a m o u n t of gill plates of Mytilus edulis ', Proc. R. Soc. B, eiliated cells. These observations seem also xSO~ 24o--244 . to point out that the cellular control of ciliary activity is by the A C h system, while Key Word Index : Acetylcholine, 5-hydroxy5 - H T m i g h t act in a higher level control tryptamine, ciliary activity, gill tissue, acetylsystem involving the branchial nerve, as choline concentration, acetylcholinesterase activity, suggested by Aiello (196o). Anodonta eygnea eeUensis. epithelium or at least the activity of the frontal cilia. T h e effects o f 5 - H T are less definite. A n interesting observation is the large differences found in the A C h content a n d in the