- Email: [email protected]
Effects of dopamine on anaerobic metabolism and ciliary activity in bivalve gills
Comp. Gen. Pharrtu~., 1974, Vol. 5, PP. 51 to 59. Pergamon Press. Printed in Great Britain
51
EFFECTS OF D O P A M I N E O N ANAEROBIC METABOLISM AND CILIARY ACTIVITY IN BIVALVE GILLS C A R L J. M A L A N G A School of Pharmacy, West Virginia University Medical Center, Morgantown, West Virginia 26506, U.S.A.
(Received 19 March I973) ABSTRACT x. Manometric measurements show that I x xo-4 M dopamlne (DA) stimulates the rate of anaerobic glycolysis in the gills of Modiolus demissus, Mytilus edulis, and Modiolus modiolus. 2. The stimulation of glycolysis in M. demissus gill is mimicked, to a lesser extent, by L-dopa. 3" Dopamine, in concentrations from x × io -7 M to xo-4 M, inhibits lateral ciliary activity, measured stroboscopically, in M. dem/ssusgill. 4- Dopamine and e-dopa (x × xO-4 M) completely, but reversibly, stop metachronal w a v e activity in M. dem/ssu~. 5. Crawling rates of Modiolus and Mytilus gill pieces are stimulated by dopamine (i × io -~ M), apparently by stimulation of frontal ciliary activity. THE role of catecholamines in the control of ciliary activity in the bivalve gill has been shrouded with controversy. Bulbring, Burn, and Shelley (I 953) reported that epinephrine stimulated particle transport by the frontal cilia on the gill of Mytilus edulis and, to a lesser extent, stimulated lateral ciliary activity measured stroboscopically. Gosselin (i966), however, reported that epinephrine, norepinephrine, and dopamine had little or no effect on the lateral cilia of Mytilis gill. Even though epinephrine and norepinephrine stimulated anaerobic lactic acid production in the gill of Modiolus demissus, Moore and Gosselin (1962) felt that the action of these substances could not be considered of physiological significance because catecholamines Were not detected in their gill preparations. These observations are at variance with a number of recent reports. Paparo and Aiello (I97O) showed that dopamine and high= frequency electrical stimulations of the branchial nerve were cilio-inhibitory to the lateral cilia in Mytilus gill preparations. In a preliminary report, Malanga (I97i) stated that dopamine and L-dopa reversibly stopped the metachronal wave motion in normally active excised gills of Modiolus demissus.
The identification of endogenous dopamine b u t not norepinephrine, in gill extracts of three species of marine mussel (Malanga, Wenger, and Aiello, i972 ) provided additional evidence to suggest dopamine as a physiological regulator in the bivalve gill. According to that study endogenous dopamine levels could be increased over tenfold by incubating gills with L-dopa prior to extraction, thus suggesting a mechanism for enzymatic synthesis through decarboxylation of the traditional precursor. Recent reviews by Woodruff (197I) and Hornykiewicz (i971) strongly support the argument that the physiological significance of dopamine extends.far beyond its role as an immediate precursor in the biosynthesis of norepinephrine. Dopaminergic mechanisms have been demonstrated in a number of invertebrate systems as well as in mammals, and it has been stated that elucidation of the functions of dopamine in invertebrate nervous systems might enrich our knowledge of the functional role of dopamine and other biogenic amines in the mammalian nervous system (Hornykiewicz, 1971). The dependence of ciliary activity on energy metabolism and the stimulatory
Gomp. Gen. Pharmac.
MALANGA
52
effects of 5 - h y d r o x y t r y p t a m i n e , a p o t e n t cilio-excitatory n e u r o h u m o u r , o n gill m e t a bolism have been well d o c u m e n t e d (Aiello, I96O; Moore, M i l t o n , a n d Gosselin, I961 ; Moore a n d Gosselin, 1962 ; Usuki, 1962 ; M a l a n g a a n d Aiello, I97I). T h e present study investigates the effect of d o p a m i n e on glycolytic m e t a b o l i s m i n the gills of Modiolus demissus, Mytilus edulis, a n d Modiolus modiolus, a n d attempts to correlate a n y metabolic actions with those affecting the m e c h a n i c a l activity of gill cilia. MATERIALS AND M E T H O D S Specimens of Modiolus demissus, Mytilus edulis, and Modiolus modioluswere obtained commercially from Northeast Marine Specimen Co., Woods Hole, Massachusetts. Mussels 6-8 era. in length were used throughout this study and were mainrained for up to x month in a 3o-gal. marine aquarium. The aquarium contained artificial sea water (Instant Ocean, Aquariums Inc.) at pH 7"8±o-2, specific gravity I.o23±o'ooI at 15-I6 ° C. The water was constantly filtered and aerated by a forced-air gravel filter system. Prior to each experiment, animals were opened by slicing through the posterior and anterior adductor muscles; gills were excised and placed in Petri dishes containing artificial sea water (ASW) removed from the aquarium. Gills were transferred after 3° minutes to fresh ASW at room temperature and remained in this solution for at least x hour to allow for the removal of secreted mUCus.
All metabolic experiments were conducted according to standard manometric techniques (Umbreit, Burris, and Stauffer, x957) at 25°C. employing a Warburg constant-volume respirometer with calibrated flasks and manometers (Bronwill Scientific). Fifteen-ml. Warburg flasks with a single vented side-arm were routinely employed. The volume of fluid in the main compartment was 2"5 ml. The basal medium in all flasks was artificial sea water plus 25 x I o - 3 M sodium bicarbonate (ASWB). A gas mixture of 95 per cent N,/5 per cent CO2 was bubbled through the ASWB for 15 minutes immediately after preparation. The flasks were gassed with 95 per cent N!/5 per cent CO2 through a multiple outlet manifold for 2o-3o minutes prior to the start of manometric readings and were shaken at 12o c.p.m, throughout the experiment. Dopamine HC1 (Sigma Chemical Co.) and laevodopa (Hoffman La Roche Labs) were prepared as stock solutions, IO -8 M a n d 5 x i o -3 M respectively, in ASWB pins 1o mg. per cent sodium ascorbate to retard oxidation prior to gassing with N,/COI.
Doparnine or L-dopa solutions (0"25 ml.) were ti.'pped from side-arms into the main compartment at time-zero to yield final con.centrations of xxlo
-~Mor
5xxo-'M-
Each of the two gills from an animal was cut into 2 or 4 equal pieces. Each gill piece was gently drained and blotted on moistened filterpaper and wet weights were determined to the nearest mg. on an electronic balance (Mettler, H i o) before transferring the tissue to a Warburg flask. Tissue weights varied between Ioo and x5o mg. per flask. Thermobars were identical to control and drug-containing flasks minus tissue. Anaerobic determinations of metabolic rates were based indirectly on the rate of metabolic acid production as evidenced by the evolution of C O , from a bicarbonate-carbonic acid buffer under an atmosphere of 95 per cent N2/5 per cent CO2. Since CO2 evolution maintained a linear relationship with time during the measurement period, the best-fit line was determined by the method of least squares and the slope taken as the rate. The standard error of the mean slope was calculated, and the statistical significance of the difference between mean rates was determined by the t-test with probability values less than o-o5 taken to be significantly different. Results were expressed as ~tl. CO2 evolved per o-I g. wet weight per minute (qCO2). In experiments involving the study of ciliary activity demibranch gill pieces, 4-7 mm. wide, were employed, and all observations were conducted at 22-25°C. Ciliary responses were observed in two ways. Drug effects on lateral cilia were determined by their ability to stop metachronal wave activity in the normally active Modiolus gill or to stimulate the appearance of metachronal waves in the normally quiescent Mytilus gill. Demibranch pieces were trans%rred to small Petri dishes (6o× 15 ram.) containing 4.5ml. ASW plus xomg. per cent ascorbate (ASWA) and observed microscopically under IOOX magnification. Drugs were dissolved in ASWA as lO × stock solutions and o. 5 or o'55 ml. was added to give a final volume ofs.o or 5"55 ml. in each dish. Results were recorded as positive or negative with respect to metachronal wave motion in untreated controls and in the same demibranch piece after addition of drug or ASWA solution. To study the effects of increasing dopamine concentrations on lateral ciliary activity, stroboscopic synchronization of metachronal wave motion was performed. The stroboscopic light source was a Grass Photostimulator (Model PS22) with a pulse frequency adjustable from I to 6o flashes per second. The rate of beating of the lateral cilia was taken to be the stroboscopic flash frequency at which the metachronal wave appears to stop smoothly before changing direction. This method was first described by Gray (I93O) and has been repeatedly used by Aiello (196o , i97o ) and Gosselin ( I 9 6 I , 1966).
~974, 5
DOPAMINE IN BIVALVE GILLS
Gills used for these studies were excised as previously described but were not cut into pieces. Whole gills were pinned to two strips of waterproof aquarium sealer, which were applied and allowed to set approximately 2-3 cm. apart on the bottom o f a Petri dish. The dish contained 2o ml. ASWA at the start of each experiment to determine the control or basal rate of lateral ciliary activity. When control rates were determined ~o ml. of the ASWA were removed and replaced with 1o ml. o f a dopamine solution in ASWA to yield the final concentrations of x × 1o-'-I x 1o-4 34" in fivefold increments. Lateral beat frequencies for each preparation were determined by averaging the rates of Io randomly chosen gill filaments within two lateral fields under zoo x magnification. At the end of each experiment the I × xo-4 M dopamine solution was replaced by three 2o-ml. washes with ASWA to assure the reversibility of the dopamine effect. The other parameter used to determine drug effects on the activity of gill cilia was crawling rate. When placed in Petri dishes containing ASW, most demibranch gill pieces will crawl along the bottom of the dish in a straight line. This method, originally reported by Nomura and Tomita (i933) , has been used extensively in physiological studies on ciliary activity in oyster and mussel gill (Weller and Ronkln, i952 ; Usuki, 1962; Hoshi and Shimada, i964). The primary factor believed to be responsible for this crawling activity is the beating of the frontal cilia, although water currents created by the lateral cilia may also influence the crawling rate (Gosselin and O'Hara, 196 I). Demibranch pieces of approximately 5 × 1o ram. were placed in Petri dishes (95 ×2o mnh.) containing 3° ml. ASWA and allowed to crawl between glass slides glued to the bottom of the
Table L---EFFECT
MOLLUSC
dish 9 ram. apart with a mm. ruler placed beneath the crawl path. Each demibranch piece was allowed to crawl a total of 6o-I2O mm. during which the time required to crawl each Io-, 2o-, and 4o-mm. distance was recorded. All rates were calculated as mm. per second and the average crawl rate for each demibranch piece was recorded. Only those pieces which crawled in a consistantly straight line were used. The tissue was then carefully transferred to a similar Petri dish containing drug dissolved in 3° ml. of ASWA. After 2- 5 minutes, crawling rates were again determined as in the control dish. Mean crawl rates4S.E.M. for controls were then compared to drugtreated rates by means of the t-test. Probability values less than o'o 5 indicated significant differences between means. RESULTS W h e n gill pieces of the 3 species of m a r i n e mussels were i n c u b a t e d u n d e r a n a t m o s p h e r e of N f l C O 2 i n the presence of b i c a r b o n a t e as previously described, glycolytic m e t a b o l i s m was d e m o n s t r a t e d b y the e v o l u t i o n of C O o. T h e rate o f C O 2 e v o l u t i o n d u r i n g the 2 - h o u r i n c u b a t i o n p e r i o d was m a r k e d l y s t i m u l a t e d i n gills of all 3 species b y the a d d i t i o n of x × i o -4 M d o p a m i n e (Table I). Because of the g r e a t e r p e r c e n t a g e increase i n the gills of Modiolus demissus, this species was used to d e t e r m i n e the effect o f L-dopa o n a n a e r o b i c m e t a b o l i s m . Table II shows t h a t L-dopa does significantly increase the r a t e o f C O 2 e v o l u t i o n a t c o n c e n t r a t i o n s o f I × xo -4 M a n d 5 × 1o-4 M . T o d e t e r m i n e h o w closely
OF D O P A M I N E ON T H E R A T E OF A N A E R O B I C GLYCOLY$1B IN T H E GILLS OF BIVALVE M O L L U S C S
EXPERIMENTAL CONDITIONS
Modiolus demissus Endogenous control Mytilus edulis
53
JV
q C O 2 (-[-S.E.).
PERCENTAGE OF ENDOGENOUS CONTROL
+ d o p a m i n e I x Io -4 M
6 6
o-I93 (-4-0.007) 0"377 (+o'ox5)*
-I95
Endogenous control + d o p a m i n e I × io -4 M
6 6
o.I]8 (4-0.002) o.I75 (4-0.003)*
-148
6 6
o'128 4-(o'ooi) o-I85 (4-0.006)*
-145
Modiolus modiolus Endogenous control + d o p a m i n e I x IO-~ M
* Significantly different from control at o. I per cent level (P
Comp. Gem Pharmac.
MALANGA
54 Table//.--EFFECT
OF L-DOPA ON THE IN TIIE GILLS OF
EXPERIMENTAL CONDITIONS
JV
~ A T E OF ANAEROBIC GLYCOLYSIS
Modiolus demissus
qCOz (4-S.E.)
PERCENTAGE OF
ENDOGENOUS CONTROL
Endogenous control +L-dopa I X xo-' M
6 6
o" 157 (-t-o"oo3) o.2io (4.o.oo6)*
-I35
Endogenous control +L-dopa 5 x xo-* M
4 8
°"I35 (4.0"005) o.249 (4-0"008)*
-184
* Significantly greater than control at o. I per cent level (P
total C O s levels a c h i e v e d in 3 h o u r s closely a p p r o a c h e s , to w i t h i n 9o p e r cent, t h e d o p a m i n e - s t i m u l a t e d levels r e a c h e d after 2 hours. T h e d o t t e d line w i t h i n the histog r a m s o f the L - d o p a e x p e r i m e n t r e p r e s e n t the CO,. levels after 2 hours a n d f u r t h e r
/~1. COz evolved per I00 mg. per 2 hours 0 I
0 l
0 !
i'J i
I
Modiolus demissus control
Modiolus demissus4- dopamine I X 10`4 M
[41. COz evolved per I00 mg. per 3 hours -
~
0 I
0 I
w
~
I
I
0
I
Modlolus demlssus control Modiolus demissus -I- L-dopa
I )~ [ 0 -4 M
Modlolus demlssus + L-clopa
5 Y- 10-4 M
~. . . . i . . . . a
~--- j- .... ~
'
o~
i
I
[co
FIG. t.--Comparison of the effects of dopamine and L-dopa on anaerobic glycolysis in the gills of
Modiolus demissus. Two bars (top) represent total COs evolved per 1oo mg. gill wet weight in 2 hours 4- standard errors; three bars (bottom) represent total COs evolution after 3 hours. The dashed lines within the bars (bottom) indicate total COa evolution after 2 hours. The numbers at the top of each bar represent the numbers of gills used to obtain each mean value. Dopamine and L-dopa were added at time-zero.
1974, 5
DOPAMINE IN BIVALVE GILLS
suggests a l a g p e r i o d r e q u i r e d for the L - d o p a i n d u c e d effect to a p p r o a c h t h a t o f d o p a m i n e . W h e n gills o f Modiolus demissus a r e o b s e r v e d microscopically, prominent metachronal waves, i n d i c a t i n g l a t e r a l c i l i a r y activity, a r e visible on all f i l a m e n t s for days. D o p a m i n e c o n c e n t r a t i o n s f r o m 1o -7 M to IO - 4 M i n h i b i t t h e r a t e o f b e a t i n g o f the l a t e r a l cilia. T h e response is g r a d e d a n d r e a c h e s c o m p l e t e i n h i b i t i o n a t d o p a m i n e c o n c e n t r a t i o n s bet w e e n I o - S M a n d I o - 4 M (Fig. 2). T h e a c t u a l b e a t frequencies for c o n t r o l a n d d o p a m i n e i n h i b i t e d rates a r e listed in Table III. A l t h o u g h d o s e - r e s p o n s e curves w e r e n o t a t t e m p t e d for L - d o p a - t r e a t e d gills, l a t e r a l c i l i a r y a c t i v i t y in d e m i b r a n c h gill pieces o f M. demissus was c o m p l e t e l y s t o p p e d b y I o -4 M L - d o p a w i t h i n 5 minutes. T h e c i l i a r y s t o p p a g e seen a t IO -4 M d o p a m i n e was u s u a l l y elicited in less t h a n 2 m i n u t e s . T h e s e results r e p r e s e n t o b s e r v a t i o n s on d e m i b r a n c h gill pieces f r o m 5 a n i m a l s . Effects o f d o p a m i n e a n d L - d o p a o n l a t e r a l c i l i a r y a c t i v i t y in Mytilus edulis gill pieces
55
Table III.--EvFECT OF DOPAMINE ON LATERAL CILIARY ACTIVITY IN THE GILL OF Modiolus dem~Ju~ IN THE PRESENCE OF VARYING CONCEN-
TXAT~ONSOF DOPAMmE CrLIARY BEAT FREQUENCY
TREATMENT
At *
(c.p.s.±S.E.M.) Control in A S W A + D A [ × zo -~ M q - D A 5 × Io-7 M + D A x × i o -6 M + D A 5 x xo-6 M q-DA I x zo -s M + D A 5 × xo-S M + D A I × IO-4 M
z4.74- I-I 11.7q- I'3J" Io'I-FI'5
5 5 4
8-o+i.o
5
7"3+ z'o 5"94-0"6 1.7±I. 7 o.o
4 5 4 5
* At = Number of gill preparations (I gill preparation = the average of 1o gill filaments). t Not significantly less than control at 5 per cent level. Lateral ciliary beat frequency in cycles (or beats) per second -4- standard error of the mean was determined stroboscopically 5-xo minutes after exposure to each dopamine concentration. Only one out of four gill preparations showed activity at 5 × Io-6 M dopamine,
10C
8C o
8
6C
0 40
=
4C
n
2C
I
16-4
5×lO-S
I
IO-S
5×10 -6
I
10-6
I
5×10-7
I
10-7
Molar concentraUon of dopamtne FIo. 2.---Graded inhibitory effect ofdopamine on lateral ciliary activity in the gills ofModiolus demissus. Gill preparations were bathed in increasing dopamine concentrations from x x IO-7 M to x x IO-4 M, each exposure lasting approximately 15-2o minutes. The inhibition is represented as the percentage of control lateral ciliary beat frequency. Each point represents the mean-bS.E.M, of 4 or 5 gill preparations with each preparation representing the average of IO gill filaments. Only one out of four gills showed activity at 5 x xo -5 M dopamine.
Comp. Gen. Pharmac.
MALANGA
56
could not be ascertained, because metachronal waves stop in this species when the gill is excised and cut into pieces. Dopamine and L-dopa, therefore, had no apparent effect on lateral activity in excised Mytilus gill pieces. Dopamine lO -4 M did stop all metachronal wave activity in demibranch pieces of Modiolus modiolus gill (5 animals). The dopamine effect was always reversible. In an attempt to correlate the metabolic stimulation produced by dopamine with any possible stimulatory effect on ciliary activity, another group of gill cilia was studied. Crawling rates of gill pieces could be readily determined and the effect of dopamine on this parameter was ascertained. Dopamine markedly stimulates crawling by Modiolus demissus and Mytilus edulis gill pieces (Table IV). The inhibitory influence of dopamine on the lateral cilia of Modiolus demissus cannot be overlooked as a possible contributing factor in the stimulation of crawling in this species. However, the stimulatory effect of dopamine on the crawling of Mytilus gill pieces is more likely due to an excitatory response by the frontal cilia, believed to be responsible for crawling, since the laterals do not appear to be affected by dopamine. As shown in Table IV, the DA stimulation of crawling by Mytilus gill is reversible by
washing in ASWA and is reaffected by a second exposure to IDA. DISCUSSION T h e initial report by Paparo and Aiello (197o) that dopamine (DA) inhibited beat frequency of the lateral gill cilia in Mytilus edulis, and the report by M a l a n g a and others (i972) that dopamine is present in gill extracts of three species of marine mussels, including M. edulis, p r o m p t e d the investigation of the effects of DA on lateral ciliary activity of Modiolus and gill metabolism of the three species found to contain DA. Since serotonin stimulates lateral ciliary activity as well as anaerobic and aerobic metabolism (Aiello, I97o; Moore and others, 196I; Moore and Gosselin, I962; M a l a n g a and Aiello, I97I ), it was thought that D A might inhibit metabolism. T h e effect of increasing D A concentrations on lateral ciliary activity in Modiolus dem/ssus, with minor differences, appears comparable to the inhibitory effect reported by Paparo and Aiello (x97o) for M. edulis. Although their concentration-response curve ranges from o.ooI to I.O lag. per ml. (5 × I ° - 9 5 × I o - S M ) , only dopamine concentrations from o.o 5 to I.Olag. per ml. (2.5× IO-75 × I°-S M) significantly inhibited lateral
Table IV.--DoPAMINERGIC STIMULATION OF CRAWLING RATES OF BIVALVE GILL PmCES
SPECIES
TREATMENT
PERCENTAGE
RATE4-S.E.M.* CONTROL
dVt
P<
Modiolus demissus
Control in ASWA +DA xo-* M
o.355-4-o.o21 0.5864-0.026
-x65
20 20
-o'oot
Mytilus edulis
Control in ASWA + D A 1o-4 M Control in ASWA after wash +DA Io -4 M 2nd exposure
o.4o9 + o'o2 t o.6o8+o-o31 0.487+0.026 o.6x4+o.o3 x
-i5o -126
t8 18 15 I5
-o.ooi -0"005
* Rate in mm. per second. 1"jV = Number of gill pieces. Each gill piece or demibranch, approximately 5 x lO mm., was allowed to crawl a total of 6o-12o mm. with times recorded every xo mm. Six-twelve rates (mm. per second) were calculated for each gill piece, and these rates were averaged to determine the mean rate for each gill piece. One animal was used to provide about 4 gill pieces. The mean crawling rate -4- S.E.M. was then calculated for each treatment from the total number (W) of gill pieces. Probability values (P <) were determined from a Student t distribution comparing means-4-S.E.M, for each treatment.
Leech Ringer I
Hyposmotic Ringer '
!~:.:!lllll
lmin
f+If!t~i !:ll,~Ur~,..
.u~i~ .t ,liijjl ill j iJdHiUt i~.J~Jl lJl JlLiui,. --r
"7,-
I " 'T-
l
- r"
-T .... T
5 [ , t [1[ ,,II,INIIII~,~Ill IL~ldntl}~Jll ~lll~ i : r-
-I'-
!--T---,!
----
~,--~--
r-
' III, -,-~-
10 Jill ldlzLllllllJ,JiUJJLIIILIIIlLILH HIll.111111I,iili
20 ,--" .....................
T-
W.-,~" ' t
~
"T'*
iT-~F"
~
Ii[i
I --m~
I
60
75
¢,q I
!
5sec FIG. x . - - T i m e course ofchanges of the frequency of spontaneous spike activity in a hyposmotic m e d i u m (2 5 ~ of the NaCI was omitted).
Hyposmot ic Ringer
"'""iiJ[illJlJ I~_.~ - r - - r - T - - r 1
I
I
1
:
',.[ I,IILLJ[.t,I 1~[11[1.[[ 11 ,I1i1i 1',
"
II
3.... T - - r - 1
I
t
1
*
1
T
Adrenaline 3xlO%/ml 5
3mln
,lllJ,lllllllllllL[LllL[L[llltllliilJJJ ''~ Jh '~' . . . .
7
. . . .
I
. . . .
r~--~7:---'"7
. . . .
• .
.
.
.
.
.
10
.
.
.
.
~; ;l.lliiIl.l.[[,iI filL[Ii 1.1.[[1l.lJ,[l.11I.tLI.;.
.
Recovery
>
!
!
5sec Fro. o . - - T h e effect of adrenaline on the spontaneous spike activity of Retzius nerve cells located in the first and second free ganglion, which had been equilibrated in hyposmotic medium (2 5 ~ of the NaC1 was omitted) for 12o minutes.
Hyposmotic Ri nger
Adrenattne 3xlC)5/mt
5min
""ili Ht,IIh,tlJlLI[llllhllllil,. I
1
t
i I
m l l ill
-4 Propranolot lO/g mt ,++~,,tt!T11
,!tit,
1 ....
I+ t t i l t
ljij,ijttiliiJllJl IiIiL i,JJJ,JJJiJ+. 2O '''~
*''l
TIt'Pt
Plp~tI+
!'++rll
'
lJl.llj£Ji:lJiitJlhlJ!ililJ!jh~l!J!ltl 3O
jjJjJjJJJJJJJJJJJj Recovery
uililllillt.lUlil![llil[[!!!!illiilii, I E i
I
5 sec Fio. 3 . - - T h e effect of propranolol on the spontaneous spike activity induced by adrenaline. The cells were firstly equilibrated in hyposmotic medium and then treated with adrenaline, propranolol and finally washed again with hyposmotic solution.
I974, 5
DOP~LWS IN BIVALVE GILLS
beat frequency below control levels, with maximum inhibition of 3 beats per sec. (b.p.s.) at 5 × I ° - e M DA. In the present study with Modiolus, using an electronic, rather than mechanical, strobe light, beat frequencies below 5b.p.s. could not be accurately determined, but statistically significant inhibition was noted at 5 × I o -? M DA progressing to a reversible complete cessation of lateral activity at between 5 × I°-5 and I × IO-4 M DA. Metachronal wave activity in Modiolus modiolus also was stopped completely by I × io - 4 M DA. In Modiolus demissus 5 × 1°-e M DA produced a 5 ° per cent inhibition (I4.7-7. 3 b.p.s.), whereas Paparo and Aiello (197o) reported a 7° per cent inhibition in Mytilus gill at the same concentration with 5 ° per cent inhibition occurring at about 5 × I o -7 M DA. Although they did not report the effects of I × 10-5-1 × 10 -4 M DA, they did state that concentrations greater than I.O ~tg. per ml. (5 × I°-6 M) caused beat frequencies less than 3 b.p.s. Aside from the obvious differences in species, seasonal variation, and laboratories, two factors must be considered in an attempt to account for the tenfold difference in DA concentration required to produce 5 ° per cent inhibition of lateral ciliary activity in the two species of marine mussels. In the report by Paparo and Aiello (I97O) beat frequencies were measured 30 minutes after the addition of each DA concentration, whereas in the present study beat frequencies were recorded 5-IO minutes after each addition of DA. Also to be considered in comparing the lateral ciliary inhibition by DA in Mytilus and Modiolus is the control or basal rate of beating from which the DA inhibition is measured. T h e report by Paparo and Aiello shows the basal rate in Mytilus to be between 9 and iob.p.s.,. whereas the basal rate in Modiolus averaged I 4 . 7 + i . I b.p.s, in the present study. Although this difference is certainly within the range reported by previous workers using stroboscopic synchronization of bivalve gill lateral ciliary activity (Gosselin, 1966; Aiello, I97O), it has been often noted that the initial basal rate of the lateral cilia will influence
57
the magnitude of response to cilio-excitatory substances like serotonin. In the present study it was noted that lateral cilia with a higher basal rate of beating showed activity at 5 × I ° - 6 M DA and were completely stopped only at I x IO-* M DA, while gills with a slower basal rate were completely inhibited at 5 × 10 -5 M. In any event, data thus far presented strongly suggest that DA may be a cilio-inhibitory physiological regulator of lateral ciliary activity in the gills of Modiolus demissus and Mytilus edulis. T h e stimulation of anaerobic metabolism produced by dopamine and L-dopa in the gills of three bivalve species did not appear to correlate with the cilio-inhibitory effect on the lateral cilia. T h a t is, if ciliary activity is directly dependent on the rate of metabolism and A T P synthesis, one might have expected a decrease in the rate of anaerobic energy metabolism. Endogenous glycogen is the primary energy source in the bivalve gill and its rate of disappearance has been correlated with ciliary activity (Moore and Gosselin, I962 ; Usuki, 1962 ). Gosselin (I966) showed that glycogen disappearance was far greater under anaerobic conditions, and serotonin exerted a far greater effect on glycogen breakdown anaerobically than aerobically. Malanga and Aiello (i97i) then proved that serotonin does stimulate lateral cilia anaerobically, an effect believed to be linked to the stimulation of anaerobic metabolism, since the cilio-excitatory response could be blocked with the glycolytic inhibitors iodoacetate and fluoride. It is possible that the metabolic stimulation by dopamine is linked to an inhibitory hyperpolarization process in the lateral cilia involving the stimulation of a Na+/K + ATPase. However, m e m b r a n e potentials have yet to be measured in the lateral ciliated cells and it is unknown whether cilio-inhibition is accompanied by hyperpolarization. Preliminary experiments in this laboratory in an attempt to block the dopamine stimulation of metabolism with ouabain have proven unsuccessful. While studying the cilio-inhibitory effects of dopamine on lateral cilia in demibranch pieces, it was noted that the gill pieces
58
Comp. Gen. Pharmac.
MALANGA
crawled out of the microscope field more rapidly than usual. This observation led to investigation of the influence of dopamine on crawling rates, a parameter believed to be dependent on forces generated by the beating of the frontal cilia. As discussed under Materials and Methods, this technique has been fairly extensively used in physiological studies on ciliary activity. However, as in studies utilizing particle transport by bivalve gill cilia, many of the past crawling data are open to question since the report by Gosselin and O ' H a r a (I96I). They showed that drug effects on lateral ciliary activity can markedly influence particle transport or crawling by bivalve gills. For this reason, experiments involving the effect of DA on crawling of Modiolus gill pieces were duplicated with Mytilus gill. In Modiolus, the lateral cilioinhibition produced by DA might have contributed to the stimulation of crawling by these gill pieces; whereas, in Mytilus, the lateral cilia are quiescent in excised gill pieces so the stimulation of crawling by DA is more likely due to a stimulation of frontal ciliary activity. A future report from this laboratory will show that the stimulated crawling response to DA is truly a dopaminergic response, which is mimicked by epinine H_Br, blocked by ergonovine maleate, and not responsive to norepinephrine or apomorphine. Although the initial reports on the effects of DA in bivalve gills (Paparo and Aiello, 197o; Malanga, 197i ) and the present study point to inhibitory control of lateral cilia, the present report also suggests that DA may also exert an excitatory influence on cells other than the laterals. If dopamine does in fact stimulate frontal ciliary activity, as suggested by crawling data, this effect may be mediated through the stimulation of glycolysis in the frontal ciliated cells of the bivalve gill. The concept of a single neurotransmitter or neurohumour affecting two closely related cell types in opposite ways is not too difficult to appreciate if one considers the different physiological functions these cell types perform. The lateral cilia function in creating the inhalent and exhalent water currents
which pass through the animal and gills carrying life-supporting food and oxygen. The frontal cilia are involved with the transport of particulate matter trapped in a mucous sheet either towards the mouth for ingestion or towards rejection tracts to be expelled in the exhalent current. Although highly speculative, it is conceivable that DA release may be triggered during filtration or feeding for the facilitation of particle transport. A final consideration in an attempt to account for the stimulation of frontal ciliary activity by DA involves speculation on a possible composite D A / 5 H T receptor in the gill, analogous to that proposed by Woodruff (I97I) for the brain of Helix aspersa. Since Gosselin (I966) has stated that all ciliated cell types in the bivalve gills tested are stimulated by serotonin, it is possible that DA stimulates frontal cilia by combining with a serotonin receptor as a partial agonist. Preliminary data in the present study offer no solution to this problem. Work is currently in progress to uncover any possible relationship between serotonin and dopamine effects on frontal cilia. ACKNOWLEDGEMENTS
The author wishes to thank Dottle Davidson, Sandra Propst, and Terry Prettyman for their technical assistance, and acknowledges the advice and support of Dr. Edward Aiello and the late Dr. R. O. Bachmann.
REFERENCES A~LLO, E. 096o), 'Factors affecting ciliary
activity on the gill of the mussel Mytilus edulis ', Physiol. Zool., 33, 12o--135. AmLLO, E. (I97o), 'Nervous and chemical stimulation of gill cilia in bivalve molluscs ', Physiol. god., 43, 6o--70. BOLBRINO,E., BURN,J., and SHELLEY,H. (1953) , ' Acetylcholine and ciliary movement in the gill plates of Mytilus edulis ', Proc. R. Soc. B., x4x , 445-466.
GOSSELIN, R. E. (t96x), ' T h e cilioexcitatory activity of serotonin ', 07. Cell. Comp. Physiol., 58, x 7-26.
GOSSELIN, R. E. (I966), 'Physiologic regulators of ciliary motion ', Am. Rev. resp. Dis., 93, 4x-59 • GOSSELm, R. E., and O'HARA, G. (196I), ' A n unsuspected source of error in studies of particle transport by lamellibranch gill cilia ', o7. Cell. Comp. Physiol., 58, I-9.
1974, 5
D O P A M I N E IN B I V A L V E G I L L S
GRAY, J. (I93o), ' T h e mechanism of ciliary movement. VI. Photographic and stroboscopic analysis of ciliary movement ', Proc. R. Soc. B., xo7, 313-332. Ho~wIez, O. (x97z), ' Dopamine: its physiology, pharmacology and pathological neurochemistry ', in Biogeni¢ Amines and Physiological Membranes in Drug Therapy (ed. Biel and Abood), pp. I73-258. New York: Marcel Dekker. HosnI, T., and S n ~ A , T. (I964), ' Contributions to the marine biology from the J a p a n sea: physiology of ciliary movement. V I I I . Effect of azide, cyanide, rodoacetic acid and 2, 4dinitrophenol on ciliary activity and O2 consumption of the gill of Mytilu~ edulis ', Sci. Rep. Niigata Univ., Ser. D. x, 77-83. MALA~OA, C.J. (t971), ' Effects of dopamine and L-dopa on ciliary activity and anaerobic glycolysis in bivalve gills ', (abstr.) Am. Zool., xx, 661. MALANOA, C.J., and AmLLO, E. (I97I), ' Anaerobic cilio-excitation and metabolic stimulation by 5-hydroxytryptamine in bivalve gill ', Comp. Gen. Pharmac., 2, 456-468. MALANGA, C. J., WENOER, G., and AXELLO, E. (I972), 'Endogenous dopamine in bivalve gills ', Comp. Biochem. Physiol., 43, 825-83o. MooR~, K., and Goss~.Ln% R. (1962), ' Effects of 5-hydroxytryptamine on the anaerobic metabolism and phosphorylase activity of lamellibranch gill ',07. Pharmac. Exp. Ther., x38~ I45-I53.
59
MOORE, K., MILTON, A., and GOSSELIN,R. (196I), ' Effects of 5-hydroxytryptamine on the respiration of excised lamellibranch gill ', Br. 07. Pharma¢., x7, 078-285. NoMuRA, S., and TOMITA, G. (1933), ' A simple method for measuring the mechanical activity of cilia ', 07. Shanghai Sci. Inst., See. IV. x, 29-39PAPARO, A., and AmLLO, E. (I97o), 'Cilioinhibitory effects of branchial nerve stimulation in the mussel, Mytilus edulis', Comp. Gen. Pharmac., x, 241-25o. U~m~IT, W., BuRRts, R., and STAUFFER, J. (I957) , Manometric Techniques. Minneapolis: Burgess Publishing Co. Usvxi, I. (i962), ' E n e r g y source for the ciliary movement and the respiration and its metabolism in oyster gill ', Sci. Rep. Tohoku Univ., Ser. IV 28, 59-83. WELLER, H., and RONXXN, R. (I952), ' Effects of 2,4-dinitrophenol upon oxygen consumption and ciliary activity in the ctenidia of Mytilus ', Proc. Soc. Exp. Biol. Med., 8x, 65-66. WOODRUFF, G. (197I), ' D o p a m i n e receptors: a review ', Comp. Gen. Pharmac., 2, 439-455.
Key Word Index: Dopamine, Modiolus demissus, Mytilus edulis, Modiolus modiolus, bivalve gill, ciliary activity, anaerobic metabolism, lateral cilia, crawling rates, frontal cilia, marine mussels.
51
EFFECTS OF D O P A M I N E O N ANAEROBIC METABOLISM AND CILIARY ACTIVITY IN BIVALVE GILLS C A R L J. M A L A N G A School of Pharmacy, West Virginia University Medical Center, Morgantown, West Virginia 26506, U.S.A.
(Received 19 March I973) ABSTRACT x. Manometric measurements show that I x xo-4 M dopamlne (DA) stimulates the rate of anaerobic glycolysis in the gills of Modiolus demissus, Mytilus edulis, and Modiolus modiolus. 2. The stimulation of glycolysis in M. demissus gill is mimicked, to a lesser extent, by L-dopa. 3" Dopamine, in concentrations from x × io -7 M to xo-4 M, inhibits lateral ciliary activity, measured stroboscopically, in M. dem/ssusgill. 4- Dopamine and e-dopa (x × xO-4 M) completely, but reversibly, stop metachronal w a v e activity in M. dem/ssu~. 5. Crawling rates of Modiolus and Mytilus gill pieces are stimulated by dopamine (i × io -~ M), apparently by stimulation of frontal ciliary activity. THE role of catecholamines in the control of ciliary activity in the bivalve gill has been shrouded with controversy. Bulbring, Burn, and Shelley (I 953) reported that epinephrine stimulated particle transport by the frontal cilia on the gill of Mytilus edulis and, to a lesser extent, stimulated lateral ciliary activity measured stroboscopically. Gosselin (i966), however, reported that epinephrine, norepinephrine, and dopamine had little or no effect on the lateral cilia of Mytilis gill. Even though epinephrine and norepinephrine stimulated anaerobic lactic acid production in the gill of Modiolus demissus, Moore and Gosselin (1962) felt that the action of these substances could not be considered of physiological significance because catecholamines Were not detected in their gill preparations. These observations are at variance with a number of recent reports. Paparo and Aiello (I97O) showed that dopamine and high= frequency electrical stimulations of the branchial nerve were cilio-inhibitory to the lateral cilia in Mytilus gill preparations. In a preliminary report, Malanga (I97i) stated that dopamine and L-dopa reversibly stopped the metachronal wave motion in normally active excised gills of Modiolus demissus.
The identification of endogenous dopamine b u t not norepinephrine, in gill extracts of three species of marine mussel (Malanga, Wenger, and Aiello, i972 ) provided additional evidence to suggest dopamine as a physiological regulator in the bivalve gill. According to that study endogenous dopamine levels could be increased over tenfold by incubating gills with L-dopa prior to extraction, thus suggesting a mechanism for enzymatic synthesis through decarboxylation of the traditional precursor. Recent reviews by Woodruff (197I) and Hornykiewicz (i971) strongly support the argument that the physiological significance of dopamine extends.far beyond its role as an immediate precursor in the biosynthesis of norepinephrine. Dopaminergic mechanisms have been demonstrated in a number of invertebrate systems as well as in mammals, and it has been stated that elucidation of the functions of dopamine in invertebrate nervous systems might enrich our knowledge of the functional role of dopamine and other biogenic amines in the mammalian nervous system (Hornykiewicz, 1971). The dependence of ciliary activity on energy metabolism and the stimulatory
Gomp. Gen. Pharmac.
MALANGA
52
effects of 5 - h y d r o x y t r y p t a m i n e , a p o t e n t cilio-excitatory n e u r o h u m o u r , o n gill m e t a bolism have been well d o c u m e n t e d (Aiello, I96O; Moore, M i l t o n , a n d Gosselin, I961 ; Moore a n d Gosselin, 1962 ; Usuki, 1962 ; M a l a n g a a n d Aiello, I97I). T h e present study investigates the effect of d o p a m i n e on glycolytic m e t a b o l i s m i n the gills of Modiolus demissus, Mytilus edulis, a n d Modiolus modiolus, a n d attempts to correlate a n y metabolic actions with those affecting the m e c h a n i c a l activity of gill cilia. MATERIALS AND M E T H O D S Specimens of Modiolus demissus, Mytilus edulis, and Modiolus modioluswere obtained commercially from Northeast Marine Specimen Co., Woods Hole, Massachusetts. Mussels 6-8 era. in length were used throughout this study and were mainrained for up to x month in a 3o-gal. marine aquarium. The aquarium contained artificial sea water (Instant Ocean, Aquariums Inc.) at pH 7"8±o-2, specific gravity I.o23±o'ooI at 15-I6 ° C. The water was constantly filtered and aerated by a forced-air gravel filter system. Prior to each experiment, animals were opened by slicing through the posterior and anterior adductor muscles; gills were excised and placed in Petri dishes containing artificial sea water (ASW) removed from the aquarium. Gills were transferred after 3° minutes to fresh ASW at room temperature and remained in this solution for at least x hour to allow for the removal of secreted mUCus.
All metabolic experiments were conducted according to standard manometric techniques (Umbreit, Burris, and Stauffer, x957) at 25°C. employing a Warburg constant-volume respirometer with calibrated flasks and manometers (Bronwill Scientific). Fifteen-ml. Warburg flasks with a single vented side-arm were routinely employed. The volume of fluid in the main compartment was 2"5 ml. The basal medium in all flasks was artificial sea water plus 25 x I o - 3 M sodium bicarbonate (ASWB). A gas mixture of 95 per cent N,/5 per cent CO2 was bubbled through the ASWB for 15 minutes immediately after preparation. The flasks were gassed with 95 per cent N!/5 per cent CO2 through a multiple outlet manifold for 2o-3o minutes prior to the start of manometric readings and were shaken at 12o c.p.m, throughout the experiment. Dopamine HC1 (Sigma Chemical Co.) and laevodopa (Hoffman La Roche Labs) were prepared as stock solutions, IO -8 M a n d 5 x i o -3 M respectively, in ASWB pins 1o mg. per cent sodium ascorbate to retard oxidation prior to gassing with N,/COI.
Doparnine or L-dopa solutions (0"25 ml.) were ti.'pped from side-arms into the main compartment at time-zero to yield final con.centrations of xxlo
-~Mor
5xxo-'M-
Each of the two gills from an animal was cut into 2 or 4 equal pieces. Each gill piece was gently drained and blotted on moistened filterpaper and wet weights were determined to the nearest mg. on an electronic balance (Mettler, H i o) before transferring the tissue to a Warburg flask. Tissue weights varied between Ioo and x5o mg. per flask. Thermobars were identical to control and drug-containing flasks minus tissue. Anaerobic determinations of metabolic rates were based indirectly on the rate of metabolic acid production as evidenced by the evolution of C O , from a bicarbonate-carbonic acid buffer under an atmosphere of 95 per cent N2/5 per cent CO2. Since CO2 evolution maintained a linear relationship with time during the measurement period, the best-fit line was determined by the method of least squares and the slope taken as the rate. The standard error of the mean slope was calculated, and the statistical significance of the difference between mean rates was determined by the t-test with probability values less than o-o5 taken to be significantly different. Results were expressed as ~tl. CO2 evolved per o-I g. wet weight per minute (qCO2). In experiments involving the study of ciliary activity demibranch gill pieces, 4-7 mm. wide, were employed, and all observations were conducted at 22-25°C. Ciliary responses were observed in two ways. Drug effects on lateral cilia were determined by their ability to stop metachronal wave activity in the normally active Modiolus gill or to stimulate the appearance of metachronal waves in the normally quiescent Mytilus gill. Demibranch pieces were trans%rred to small Petri dishes (6o× 15 ram.) containing 4.5ml. ASW plus xomg. per cent ascorbate (ASWA) and observed microscopically under IOOX magnification. Drugs were dissolved in ASWA as lO × stock solutions and o. 5 or o'55 ml. was added to give a final volume ofs.o or 5"55 ml. in each dish. Results were recorded as positive or negative with respect to metachronal wave motion in untreated controls and in the same demibranch piece after addition of drug or ASWA solution. To study the effects of increasing dopamine concentrations on lateral ciliary activity, stroboscopic synchronization of metachronal wave motion was performed. The stroboscopic light source was a Grass Photostimulator (Model PS22) with a pulse frequency adjustable from I to 6o flashes per second. The rate of beating of the lateral cilia was taken to be the stroboscopic flash frequency at which the metachronal wave appears to stop smoothly before changing direction. This method was first described by Gray (I93O) and has been repeatedly used by Aiello (196o , i97o ) and Gosselin ( I 9 6 I , 1966).
~974, 5
DOPAMINE IN BIVALVE GILLS
Gills used for these studies were excised as previously described but were not cut into pieces. Whole gills were pinned to two strips of waterproof aquarium sealer, which were applied and allowed to set approximately 2-3 cm. apart on the bottom o f a Petri dish. The dish contained 2o ml. ASWA at the start of each experiment to determine the control or basal rate of lateral ciliary activity. When control rates were determined ~o ml. of the ASWA were removed and replaced with 1o ml. o f a dopamine solution in ASWA to yield the final concentrations of x × 1o-'-I x 1o-4 34" in fivefold increments. Lateral beat frequencies for each preparation were determined by averaging the rates of Io randomly chosen gill filaments within two lateral fields under zoo x magnification. At the end of each experiment the I × xo-4 M dopamine solution was replaced by three 2o-ml. washes with ASWA to assure the reversibility of the dopamine effect. The other parameter used to determine drug effects on the activity of gill cilia was crawling rate. When placed in Petri dishes containing ASW, most demibranch gill pieces will crawl along the bottom of the dish in a straight line. This method, originally reported by Nomura and Tomita (i933) , has been used extensively in physiological studies on ciliary activity in oyster and mussel gill (Weller and Ronkln, i952 ; Usuki, 1962; Hoshi and Shimada, i964). The primary factor believed to be responsible for this crawling activity is the beating of the frontal cilia, although water currents created by the lateral cilia may also influence the crawling rate (Gosselin and O'Hara, 196 I). Demibranch pieces of approximately 5 × 1o ram. were placed in Petri dishes (95 ×2o mnh.) containing 3° ml. ASWA and allowed to crawl between glass slides glued to the bottom of the
Table L---EFFECT
MOLLUSC
dish 9 ram. apart with a mm. ruler placed beneath the crawl path. Each demibranch piece was allowed to crawl a total of 6o-I2O mm. during which the time required to crawl each Io-, 2o-, and 4o-mm. distance was recorded. All rates were calculated as mm. per second and the average crawl rate for each demibranch piece was recorded. Only those pieces which crawled in a consistantly straight line were used. The tissue was then carefully transferred to a similar Petri dish containing drug dissolved in 3° ml. of ASWA. After 2- 5 minutes, crawling rates were again determined as in the control dish. Mean crawl rates4S.E.M. for controls were then compared to drugtreated rates by means of the t-test. Probability values less than o'o 5 indicated significant differences between means. RESULTS W h e n gill pieces of the 3 species of m a r i n e mussels were i n c u b a t e d u n d e r a n a t m o s p h e r e of N f l C O 2 i n the presence of b i c a r b o n a t e as previously described, glycolytic m e t a b o l i s m was d e m o n s t r a t e d b y the e v o l u t i o n of C O o. T h e rate o f C O 2 e v o l u t i o n d u r i n g the 2 - h o u r i n c u b a t i o n p e r i o d was m a r k e d l y s t i m u l a t e d i n gills of all 3 species b y the a d d i t i o n of x × i o -4 M d o p a m i n e (Table I). Because of the g r e a t e r p e r c e n t a g e increase i n the gills of Modiolus demissus, this species was used to d e t e r m i n e the effect o f L-dopa o n a n a e r o b i c m e t a b o l i s m . Table II shows t h a t L-dopa does significantly increase the r a t e o f C O 2 e v o l u t i o n a t c o n c e n t r a t i o n s o f I × xo -4 M a n d 5 × 1o-4 M . T o d e t e r m i n e h o w closely
OF D O P A M I N E ON T H E R A T E OF A N A E R O B I C GLYCOLY$1B IN T H E GILLS OF BIVALVE M O L L U S C S
EXPERIMENTAL CONDITIONS
Modiolus demissus Endogenous control Mytilus edulis
53
JV
q C O 2 (-[-S.E.).
PERCENTAGE OF ENDOGENOUS CONTROL
+ d o p a m i n e I x Io -4 M
6 6
o-I93 (-4-0.007) 0"377 (+o'ox5)*
-I95
Endogenous control + d o p a m i n e I × io -4 M
6 6
o.I]8 (4-0.002) o.I75 (4-0.003)*
-148
6 6
o'128 4-(o'ooi) o-I85 (4-0.006)*
-145
Modiolus modiolus Endogenous control + d o p a m i n e I x IO-~ M
* Significantly different from control at o. I per cent level (P
Comp. Gem Pharmac.
MALANGA
54 Table//.--EFFECT
OF L-DOPA ON THE IN TIIE GILLS OF
EXPERIMENTAL CONDITIONS
JV
~ A T E OF ANAEROBIC GLYCOLYSIS
Modiolus demissus
qCOz (4-S.E.)
PERCENTAGE OF
ENDOGENOUS CONTROL
Endogenous control +L-dopa I X xo-' M
6 6
o" 157 (-t-o"oo3) o.2io (4.o.oo6)*
-I35
Endogenous control +L-dopa 5 x xo-* M
4 8
°"I35 (4.0"005) o.249 (4-0"008)*
-184
* Significantly greater than control at o. I per cent level (P
total C O s levels a c h i e v e d in 3 h o u r s closely a p p r o a c h e s , to w i t h i n 9o p e r cent, t h e d o p a m i n e - s t i m u l a t e d levels r e a c h e d after 2 hours. T h e d o t t e d line w i t h i n the histog r a m s o f the L - d o p a e x p e r i m e n t r e p r e s e n t the CO,. levels after 2 hours a n d f u r t h e r
/~1. COz evolved per I00 mg. per 2 hours 0 I
0 l
0 !
i'J i
I
Modiolus demissus control
Modiolus demissus4- dopamine I X 10`4 M
[41. COz evolved per I00 mg. per 3 hours -
~
0 I
0 I
w
~
I
I
0
I
Modlolus demlssus control Modiolus demissus -I- L-dopa
I )~ [ 0 -4 M
Modlolus demlssus + L-clopa
5 Y- 10-4 M
~. . . . i . . . . a
~--- j- .... ~
'
o~
i
I
[co
FIG. t.--Comparison of the effects of dopamine and L-dopa on anaerobic glycolysis in the gills of
Modiolus demissus. Two bars (top) represent total COs evolved per 1oo mg. gill wet weight in 2 hours 4- standard errors; three bars (bottom) represent total COs evolution after 3 hours. The dashed lines within the bars (bottom) indicate total COa evolution after 2 hours. The numbers at the top of each bar represent the numbers of gills used to obtain each mean value. Dopamine and L-dopa were added at time-zero.
1974, 5
DOPAMINE IN BIVALVE GILLS
suggests a l a g p e r i o d r e q u i r e d for the L - d o p a i n d u c e d effect to a p p r o a c h t h a t o f d o p a m i n e . W h e n gills o f Modiolus demissus a r e o b s e r v e d microscopically, prominent metachronal waves, i n d i c a t i n g l a t e r a l c i l i a r y activity, a r e visible on all f i l a m e n t s for days. D o p a m i n e c o n c e n t r a t i o n s f r o m 1o -7 M to IO - 4 M i n h i b i t t h e r a t e o f b e a t i n g o f the l a t e r a l cilia. T h e response is g r a d e d a n d r e a c h e s c o m p l e t e i n h i b i t i o n a t d o p a m i n e c o n c e n t r a t i o n s bet w e e n I o - S M a n d I o - 4 M (Fig. 2). T h e a c t u a l b e a t frequencies for c o n t r o l a n d d o p a m i n e i n h i b i t e d rates a r e listed in Table III. A l t h o u g h d o s e - r e s p o n s e curves w e r e n o t a t t e m p t e d for L - d o p a - t r e a t e d gills, l a t e r a l c i l i a r y a c t i v i t y in d e m i b r a n c h gill pieces o f M. demissus was c o m p l e t e l y s t o p p e d b y I o -4 M L - d o p a w i t h i n 5 minutes. T h e c i l i a r y s t o p p a g e seen a t IO -4 M d o p a m i n e was u s u a l l y elicited in less t h a n 2 m i n u t e s . T h e s e results r e p r e s e n t o b s e r v a t i o n s on d e m i b r a n c h gill pieces f r o m 5 a n i m a l s . Effects o f d o p a m i n e a n d L - d o p a o n l a t e r a l c i l i a r y a c t i v i t y in Mytilus edulis gill pieces
55
Table III.--EvFECT OF DOPAMINE ON LATERAL CILIARY ACTIVITY IN THE GILL OF Modiolus dem~Ju~ IN THE PRESENCE OF VARYING CONCEN-
TXAT~ONSOF DOPAMmE CrLIARY BEAT FREQUENCY
TREATMENT
At *
(c.p.s.±S.E.M.) Control in A S W A + D A [ × zo -~ M q - D A 5 × Io-7 M + D A x × i o -6 M + D A 5 x xo-6 M q-DA I x zo -s M + D A 5 × xo-S M + D A I × IO-4 M
z4.74- I-I 11.7q- I'3J" Io'I-FI'5
5 5 4
8-o+i.o
5
7"3+ z'o 5"94-0"6 1.7±I. 7 o.o
4 5 4 5
* At = Number of gill preparations (I gill preparation = the average of 1o gill filaments). t Not significantly less than control at 5 per cent level. Lateral ciliary beat frequency in cycles (or beats) per second -4- standard error of the mean was determined stroboscopically 5-xo minutes after exposure to each dopamine concentration. Only one out of four gill preparations showed activity at 5 × Io-6 M dopamine,
10C
8C o
8
6C
0 40
=
4C
n
2C
I
16-4
5×lO-S
I
IO-S
5×10 -6
I
10-6
I
5×10-7
I
10-7
Molar concentraUon of dopamtne FIo. 2.---Graded inhibitory effect ofdopamine on lateral ciliary activity in the gills ofModiolus demissus. Gill preparations were bathed in increasing dopamine concentrations from x x IO-7 M to x x IO-4 M, each exposure lasting approximately 15-2o minutes. The inhibition is represented as the percentage of control lateral ciliary beat frequency. Each point represents the mean-bS.E.M, of 4 or 5 gill preparations with each preparation representing the average of IO gill filaments. Only one out of four gills showed activity at 5 x xo -5 M dopamine.
Comp. Gen. Pharmac.
MALANGA
56
could not be ascertained, because metachronal waves stop in this species when the gill is excised and cut into pieces. Dopamine and L-dopa, therefore, had no apparent effect on lateral activity in excised Mytilus gill pieces. Dopamine lO -4 M did stop all metachronal wave activity in demibranch pieces of Modiolus modiolus gill (5 animals). The dopamine effect was always reversible. In an attempt to correlate the metabolic stimulation produced by dopamine with any possible stimulatory effect on ciliary activity, another group of gill cilia was studied. Crawling rates of gill pieces could be readily determined and the effect of dopamine on this parameter was ascertained. Dopamine markedly stimulates crawling by Modiolus demissus and Mytilus edulis gill pieces (Table IV). The inhibitory influence of dopamine on the lateral cilia of Modiolus demissus cannot be overlooked as a possible contributing factor in the stimulation of crawling in this species. However, the stimulatory effect of dopamine on the crawling of Mytilus gill pieces is more likely due to an excitatory response by the frontal cilia, believed to be responsible for crawling, since the laterals do not appear to be affected by dopamine. As shown in Table IV, the DA stimulation of crawling by Mytilus gill is reversible by
washing in ASWA and is reaffected by a second exposure to IDA. DISCUSSION T h e initial report by Paparo and Aiello (197o) that dopamine (DA) inhibited beat frequency of the lateral gill cilia in Mytilus edulis, and the report by M a l a n g a and others (i972) that dopamine is present in gill extracts of three species of marine mussels, including M. edulis, p r o m p t e d the investigation of the effects of DA on lateral ciliary activity of Modiolus and gill metabolism of the three species found to contain DA. Since serotonin stimulates lateral ciliary activity as well as anaerobic and aerobic metabolism (Aiello, I97o; Moore and others, 196I; Moore and Gosselin, I962; M a l a n g a and Aiello, I97I ), it was thought that D A might inhibit metabolism. T h e effect of increasing D A concentrations on lateral ciliary activity in Modiolus dem/ssus, with minor differences, appears comparable to the inhibitory effect reported by Paparo and Aiello (x97o) for M. edulis. Although their concentration-response curve ranges from o.ooI to I.O lag. per ml. (5 × I ° - 9 5 × I o - S M ) , only dopamine concentrations from o.o 5 to I.Olag. per ml. (2.5× IO-75 × I°-S M) significantly inhibited lateral
Table IV.--DoPAMINERGIC STIMULATION OF CRAWLING RATES OF BIVALVE GILL PmCES
SPECIES
TREATMENT
PERCENTAGE
RATE4-S.E.M.* CONTROL
dVt
P<
Modiolus demissus
Control in ASWA +DA xo-* M
o.355-4-o.o21 0.5864-0.026
-x65
20 20
-o'oot
Mytilus edulis
Control in ASWA + D A 1o-4 M Control in ASWA after wash +DA Io -4 M 2nd exposure
o.4o9 + o'o2 t o.6o8+o-o31 0.487+0.026 o.6x4+o.o3 x
-i5o -126
t8 18 15 I5
-o.ooi -0"005
* Rate in mm. per second. 1"jV = Number of gill pieces. Each gill piece or demibranch, approximately 5 x lO mm., was allowed to crawl a total of 6o-12o mm. with times recorded every xo mm. Six-twelve rates (mm. per second) were calculated for each gill piece, and these rates were averaged to determine the mean rate for each gill piece. One animal was used to provide about 4 gill pieces. The mean crawling rate -4- S.E.M. was then calculated for each treatment from the total number (W) of gill pieces. Probability values (P <) were determined from a Student t distribution comparing means-4-S.E.M, for each treatment.
Leech Ringer I
Hyposmotic Ringer '
!~:.:!lllll
lmin
f+If!t~i !:ll,~Ur~,..
.u~i~ .t ,liijjl ill j iJdHiUt i~.J~Jl lJl JlLiui,. --r
"7,-
I " 'T-
l
- r"
-T .... T
5 [ , t [1[ ,,II,INIIII~,~Ill IL~ldntl}~Jll ~lll~ i : r-
-I'-
!--T---,!
----
~,--~--
r-
' III, -,-~-
10 Jill ldlzLllllllJ,JiUJJLIIILIIIlLILH HIll.111111I,iili
20 ,--" .....................
T-
W.-,~" ' t
~
"T'*
iT-~F"
~
Ii[i
I --m~
I
60
75
¢,q I
!
5sec FIG. x . - - T i m e course ofchanges of the frequency of spontaneous spike activity in a hyposmotic m e d i u m (2 5 ~ of the NaCI was omitted).
Hyposmot ic Ringer
"'""iiJ[illJlJ I~_.~ - r - - r - T - - r 1
I
I
1
:
',.[ I,IILLJ[.t,I 1~[11[1.[[ 11 ,I1i1i 1',
"
II
3.... T - - r - 1
I
t
1
*
1
T
Adrenaline 3xlO%/ml 5
3mln
,lllJ,lllllllllllL[LllL[L[llltllliilJJJ ''~ Jh '~' . . . .
7
. . . .
I
. . . .
r~--~7:---'"7
. . . .
• .
.
.
.
.
.
10
.
.
.
.
~; ;l.lliiIl.l.[[,iI filL[Ii 1.1.[[1l.lJ,[l.11I.tLI.;.
.
Recovery
>
!
!
5sec Fro. o . - - T h e effect of adrenaline on the spontaneous spike activity of Retzius nerve cells located in the first and second free ganglion, which had been equilibrated in hyposmotic medium (2 5 ~ of the NaC1 was omitted) for 12o minutes.
Hyposmotic Ri nger
Adrenattne 3xlC)5/mt
5min
""ili Ht,IIh,tlJlLI[llllhllllil,. I
1
t
i I
m l l ill
-4 Propranolot lO/g mt ,++~,,tt!T11
,!tit,
1 ....
I+ t t i l t
ljij,ijttiliiJllJl IiIiL i,JJJ,JJJiJ+. 2O '''~
*''l
TIt'Pt
Plp~tI+
!'++rll
'
lJl.llj£Ji:lJiitJlhlJ!ililJ!jh~l!J!ltl 3O
jjJjJjJJJJJJJJJJJj Recovery
uililllillt.lUlil![llil[[!!!!illiilii, I E i
I
5 sec Fio. 3 . - - T h e effect of propranolol on the spontaneous spike activity induced by adrenaline. The cells were firstly equilibrated in hyposmotic medium and then treated with adrenaline, propranolol and finally washed again with hyposmotic solution.
I974, 5
DOP~LWS IN BIVALVE GILLS
beat frequency below control levels, with maximum inhibition of 3 beats per sec. (b.p.s.) at 5 × I ° - e M DA. In the present study with Modiolus, using an electronic, rather than mechanical, strobe light, beat frequencies below 5b.p.s. could not be accurately determined, but statistically significant inhibition was noted at 5 × I o -? M DA progressing to a reversible complete cessation of lateral activity at between 5 × I°-5 and I × IO-4 M DA. Metachronal wave activity in Modiolus modiolus also was stopped completely by I × io - 4 M DA. In Modiolus demissus 5 × 1°-e M DA produced a 5 ° per cent inhibition (I4.7-7. 3 b.p.s.), whereas Paparo and Aiello (197o) reported a 7° per cent inhibition in Mytilus gill at the same concentration with 5 ° per cent inhibition occurring at about 5 × I o -7 M DA. Although they did not report the effects of I × 10-5-1 × 10 -4 M DA, they did state that concentrations greater than I.O ~tg. per ml. (5 × I°-6 M) caused beat frequencies less than 3 b.p.s. Aside from the obvious differences in species, seasonal variation, and laboratories, two factors must be considered in an attempt to account for the tenfold difference in DA concentration required to produce 5 ° per cent inhibition of lateral ciliary activity in the two species of marine mussels. In the report by Paparo and Aiello (I97O) beat frequencies were measured 30 minutes after the addition of each DA concentration, whereas in the present study beat frequencies were recorded 5-IO minutes after each addition of DA. Also to be considered in comparing the lateral ciliary inhibition by DA in Mytilus and Modiolus is the control or basal rate of beating from which the DA inhibition is measured. T h e report by Paparo and Aiello shows the basal rate in Mytilus to be between 9 and iob.p.s.,. whereas the basal rate in Modiolus averaged I 4 . 7 + i . I b.p.s, in the present study. Although this difference is certainly within the range reported by previous workers using stroboscopic synchronization of bivalve gill lateral ciliary activity (Gosselin, 1966; Aiello, I97O), it has been often noted that the initial basal rate of the lateral cilia will influence
57
the magnitude of response to cilio-excitatory substances like serotonin. In the present study it was noted that lateral cilia with a higher basal rate of beating showed activity at 5 × I ° - 6 M DA and were completely stopped only at I x IO-* M DA, while gills with a slower basal rate were completely inhibited at 5 × 10 -5 M. In any event, data thus far presented strongly suggest that DA may be a cilio-inhibitory physiological regulator of lateral ciliary activity in the gills of Modiolus demissus and Mytilus edulis. T h e stimulation of anaerobic metabolism produced by dopamine and L-dopa in the gills of three bivalve species did not appear to correlate with the cilio-inhibitory effect on the lateral cilia. T h a t is, if ciliary activity is directly dependent on the rate of metabolism and A T P synthesis, one might have expected a decrease in the rate of anaerobic energy metabolism. Endogenous glycogen is the primary energy source in the bivalve gill and its rate of disappearance has been correlated with ciliary activity (Moore and Gosselin, I962 ; Usuki, 1962 ). Gosselin (I966) showed that glycogen disappearance was far greater under anaerobic conditions, and serotonin exerted a far greater effect on glycogen breakdown anaerobically than aerobically. Malanga and Aiello (i97i) then proved that serotonin does stimulate lateral cilia anaerobically, an effect believed to be linked to the stimulation of anaerobic metabolism, since the cilio-excitatory response could be blocked with the glycolytic inhibitors iodoacetate and fluoride. It is possible that the metabolic stimulation by dopamine is linked to an inhibitory hyperpolarization process in the lateral cilia involving the stimulation of a Na+/K + ATPase. However, m e m b r a n e potentials have yet to be measured in the lateral ciliated cells and it is unknown whether cilio-inhibition is accompanied by hyperpolarization. Preliminary experiments in this laboratory in an attempt to block the dopamine stimulation of metabolism with ouabain have proven unsuccessful. While studying the cilio-inhibitory effects of dopamine on lateral cilia in demibranch pieces, it was noted that the gill pieces
58
Comp. Gen. Pharmac.
MALANGA
crawled out of the microscope field more rapidly than usual. This observation led to investigation of the influence of dopamine on crawling rates, a parameter believed to be dependent on forces generated by the beating of the frontal cilia. As discussed under Materials and Methods, this technique has been fairly extensively used in physiological studies on ciliary activity. However, as in studies utilizing particle transport by bivalve gill cilia, many of the past crawling data are open to question since the report by Gosselin and O ' H a r a (I96I). They showed that drug effects on lateral ciliary activity can markedly influence particle transport or crawling by bivalve gills. For this reason, experiments involving the effect of DA on crawling of Modiolus gill pieces were duplicated with Mytilus gill. In Modiolus, the lateral cilioinhibition produced by DA might have contributed to the stimulation of crawling by these gill pieces; whereas, in Mytilus, the lateral cilia are quiescent in excised gill pieces so the stimulation of crawling by DA is more likely due to a stimulation of frontal ciliary activity. A future report from this laboratory will show that the stimulated crawling response to DA is truly a dopaminergic response, which is mimicked by epinine H_Br, blocked by ergonovine maleate, and not responsive to norepinephrine or apomorphine. Although the initial reports on the effects of DA in bivalve gills (Paparo and Aiello, 197o; Malanga, 197i ) and the present study point to inhibitory control of lateral cilia, the present report also suggests that DA may also exert an excitatory influence on cells other than the laterals. If dopamine does in fact stimulate frontal ciliary activity, as suggested by crawling data, this effect may be mediated through the stimulation of glycolysis in the frontal ciliated cells of the bivalve gill. The concept of a single neurotransmitter or neurohumour affecting two closely related cell types in opposite ways is not too difficult to appreciate if one considers the different physiological functions these cell types perform. The lateral cilia function in creating the inhalent and exhalent water currents
which pass through the animal and gills carrying life-supporting food and oxygen. The frontal cilia are involved with the transport of particulate matter trapped in a mucous sheet either towards the mouth for ingestion or towards rejection tracts to be expelled in the exhalent current. Although highly speculative, it is conceivable that DA release may be triggered during filtration or feeding for the facilitation of particle transport. A final consideration in an attempt to account for the stimulation of frontal ciliary activity by DA involves speculation on a possible composite D A / 5 H T receptor in the gill, analogous to that proposed by Woodruff (I97I) for the brain of Helix aspersa. Since Gosselin (I966) has stated that all ciliated cell types in the bivalve gills tested are stimulated by serotonin, it is possible that DA stimulates frontal cilia by combining with a serotonin receptor as a partial agonist. Preliminary data in the present study offer no solution to this problem. Work is currently in progress to uncover any possible relationship between serotonin and dopamine effects on frontal cilia. ACKNOWLEDGEMENTS
The author wishes to thank Dottle Davidson, Sandra Propst, and Terry Prettyman for their technical assistance, and acknowledges the advice and support of Dr. Edward Aiello and the late Dr. R. O. Bachmann.
REFERENCES A~LLO, E. 096o), 'Factors affecting ciliary
activity on the gill of the mussel Mytilus edulis ', Physiol. Zool., 33, 12o--135. AmLLO, E. (I97o), 'Nervous and chemical stimulation of gill cilia in bivalve molluscs ', Physiol. god., 43, 6o--70. BOLBRINO,E., BURN,J., and SHELLEY,H. (1953) , ' Acetylcholine and ciliary movement in the gill plates of Mytilus edulis ', Proc. R. Soc. B., x4x , 445-466.
GOSSELIN, R. E. (t96x), ' T h e cilioexcitatory activity of serotonin ', 07. Cell. Comp. Physiol., 58, x 7-26.
GOSSELIN, R. E. (I966), 'Physiologic regulators of ciliary motion ', Am. Rev. resp. Dis., 93, 4x-59 • GOSSELm, R. E., and O'HARA, G. (196I), ' A n unsuspected source of error in studies of particle transport by lamellibranch gill cilia ', o7. Cell. Comp. Physiol., 58, I-9.
1974, 5
D O P A M I N E IN B I V A L V E G I L L S
GRAY, J. (I93o), ' T h e mechanism of ciliary movement. VI. Photographic and stroboscopic analysis of ciliary movement ', Proc. R. Soc. B., xo7, 313-332. Ho~wIez, O. (x97z), ' Dopamine: its physiology, pharmacology and pathological neurochemistry ', in Biogeni¢ Amines and Physiological Membranes in Drug Therapy (ed. Biel and Abood), pp. I73-258. New York: Marcel Dekker. HosnI, T., and S n ~ A , T. (I964), ' Contributions to the marine biology from the J a p a n sea: physiology of ciliary movement. V I I I . Effect of azide, cyanide, rodoacetic acid and 2, 4dinitrophenol on ciliary activity and O2 consumption of the gill of Mytilu~ edulis ', Sci. Rep. Niigata Univ., Ser. D. x, 77-83. MALA~OA, C.J. (t971), ' Effects of dopamine and L-dopa on ciliary activity and anaerobic glycolysis in bivalve gills ', (abstr.) Am. Zool., xx, 661. MALANOA, C.J., and AmLLO, E. (I97I), ' Anaerobic cilio-excitation and metabolic stimulation by 5-hydroxytryptamine in bivalve gill ', Comp. Gen. Pharmac., 2, 456-468. MALANGA, C. J., WENOER, G., and AXELLO, E. (I972), 'Endogenous dopamine in bivalve gills ', Comp. Biochem. Physiol., 43, 825-83o. MooR~, K., and Goss~.Ln% R. (1962), ' Effects of 5-hydroxytryptamine on the anaerobic metabolism and phosphorylase activity of lamellibranch gill ',07. Pharmac. Exp. Ther., x38~ I45-I53.
59
MOORE, K., MILTON, A., and GOSSELIN,R. (196I), ' Effects of 5-hydroxytryptamine on the respiration of excised lamellibranch gill ', Br. 07. Pharma¢., x7, 078-285. NoMuRA, S., and TOMITA, G. (1933), ' A simple method for measuring the mechanical activity of cilia ', 07. Shanghai Sci. Inst., See. IV. x, 29-39PAPARO, A., and AmLLO, E. (I97o), 'Cilioinhibitory effects of branchial nerve stimulation in the mussel, Mytilus edulis', Comp. Gen. Pharmac., x, 241-25o. U~m~IT, W., BuRRts, R., and STAUFFER, J. (I957) , Manometric Techniques. Minneapolis: Burgess Publishing Co. Usvxi, I. (i962), ' E n e r g y source for the ciliary movement and the respiration and its metabolism in oyster gill ', Sci. Rep. Tohoku Univ., Ser. IV 28, 59-83. WELLER, H., and RONXXN, R. (I952), ' Effects of 2,4-dinitrophenol upon oxygen consumption and ciliary activity in the ctenidia of Mytilus ', Proc. Soc. Exp. Biol. Med., 8x, 65-66. WOODRUFF, G. (197I), ' D o p a m i n e receptors: a review ', Comp. Gen. Pharmac., 2, 439-455.
Key Word Index: Dopamine, Modiolus demissus, Mytilus edulis, Modiolus modiolus, bivalve gill, ciliary activity, anaerobic metabolism, lateral cilia, crawling rates, frontal cilia, marine mussels.