Inactivation of the Ca2+-pump in the mussel, Mytilus edulis

Cm. Pkurmuc.. Vol. I I, pp. 403 to 406 0 Pergamon Press Ltd 1980. Printed m Great Britam

0306.3623/80/0701-0403102.00/0

INACTIVATION OF THE CA2+-PUMP MUSSEL, MYTILUS EDULIS

IN THE

ANTHONY PAPARO* and PETER SATlRt *Department of Anatomy/School of Medicine and Department of Zoology, Southern Illinois University, Carbondale, IL 62901, U.S.A. tDepartment of Anatomy/Albert Eistein College of Medicine, Yeshiva University, Bronx, New York 10461, U.S.A. (Received 12 September 1979) Abstract-l. Lateral ciliary activity was studied on intact and isolated gill cell preparations in the mussel, Mytilus edulis. 2. It has been previously demonstrated by the senior author that the lateral cilia are under the control of both excitatory and inhibitory axons present in the branchial nerve. Furthermore, it was shown that low frequency [5 Hz; neurotransmitter released is serotonin (5-HT)] and high frequency (50 HZ; neurotransmitter released is dopamine (DA)) nerve stimulation activated cilia-excitatory and/or cilioinhibitory axons. 3. This study confirms the above findings and in addition shows that perfusing intact and isolated gill cell preparations with salyrgan (Ca *+-ATPase poison) in the presence of external Ca*+ is also cilioinhibitory. Salyrgan potentiates the cilia-inhibition produced by high frequency nerve stimulation/DA. It antagonizes the cilia-excitation that occurred after low frequency nerve stimulation/5-HT. The addition of lanthanum to the salyrgan perfusate enhances the cilia-inhibitory response of the latter probably by more effectively stopping the Ca*+-pump. 4. It is postulated that salyrgan effects the Cazf -pump of lateral ceils by slowing or stopping (with addition of lanthanum) the pump so that external Ca *+ leaks across the cell membrane. A subsequent rise in internal Ca* + concentration produces lateral ciliary arrest.

INTRODUCTION Lateral cilia in the gill of Mytilus are under the control of the cilia-excitatory and cilia-inhibitory axons contained within the branchial nerve. Furthermore, electrical stimulation of the nerve at: (a) 5 pulses per set and exposure to serotonin (5HT) each accelerate ciliary beating, and (b) 50 pulses per set and exposure to dopamine (DA) each inhibit ciliary activity (Paparo & Aiello, 1970). Stimulation of the branchial nerve was postulated to mobilize Ca’+ within the gill (Paparo & Murphy, 1975). The important role of intracellular-Ca’+ controlling ciliary activity has been well documented for Paramecium (Naitoh & Kaneko, 1972), Crithidia (Holwill & McGregor, 1976). More recently Walter & Satir (1978) have shown that lateral ciliary activity are arrested within very narrow limits of 5 x lo-‘M to 8 x lo-‘M Ca’+. Ciliary arrest was also induced by perfusing gills with Ca*+ and Salyrgan [Ca ’ +-ATPase poison of sarcoplasmic reticulum (Satir, 19761-j. The purpose of this study is to elucidate the relationship of salyrgan/Ca2 + -pump/nerve stimulation in the elicitation of both cilia-excitation and cilio-inhibition of lateral activity. MATERIALS AND

METHODS

All experiments were performed on the mussel, Mytilus These animals were kept for 2 weeks in an Instant Ocean Aquarium (I = 18°C; pH = 7.5; density = 1.025) prior to experimentation.

edulis.

Preparation

of intact

gill and measurement

of ciliary

action

The posterior adductor muscle was cut and a visceral ganglion-branchial nerve-gill preparation was isolated. A field of view was chosen which contained from 10&200 gill filaments. The frequency (rate of ciliary beating) was esti403

mated by synchronizing the rate of flashing of a calibrated, stroboscopic light with the rate of beating of the lateral cilia. Measurements were made from dorsal to ventral border, and from left to right across the field, giving 12 sets of measurements. The average rate of beating varied from about 10 to 12 Hz, in Rila Instant Ocean Mix containing less than lo-* M. Ca’+. It was this calculated average that was assigned a percentage frequency (activity) value of 100 that was used as the zero-point for each experiment. Preparation of ciliary action

isolated

lateral

cells

and

measurement

of

Suspensions of lateral cells from the gill of Mytilus were obtained by placing the gill in a solution containing 15 x 10m3M sodium chloride, 50 x 10e6 M potassium chloride, and 40 x 10m3M N-hydroxyethylpiperazineN-2-ethane-sulfonic acid (HEPES). The PH of the medium was 7.4. The intact gill was left in a refrfigerator (13-16°C) overnight (12-15 hr) in the medium. It was then’removed and left at room temperature (21-23°C). After 5 hr. all cell exfoliation is complete. The motility of lOG200 lateral cells in this solution was noted in a field of view to determine percentage motility (activity). This procedure is a modification of that of Walter & Satir (1978). stimulation of branchial nerve and drug perfusion The branchial nerve was dissected and a concentric electrode was placed at a point midway between its emergence from the visceral ganglion and its passage into the dorsal axis of the gill. A stimulator supplied electrical pulses (0.1 V, 5 or 50 biphasic pulses set- ‘, 3.0 msec pulse duration) for the first 2 min. Drugs were continually added to the perfusion dish in sea water at 21°C. In the case of isolated preparations the desired concentration of drug was achieved by dilution with medium containing exfoliated cells. The following drugs were used: 5-hydroxvtryptamine (5-HT. serotonin): 3,4-dihydroxyphenylethylamine~ (DA, dopamine); calcium (Ca*‘); lanthanum (La3+); and o[(3-hydroxymercuri-2methoxypropyl) carbamyl] phenoryacetic acid (Salyrgan). Electrical

404

ANTHONYPAPAROand PETERSATIR I00

INTACT GILL

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CONTROL

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o ' ..... ,; ...... ,~,.o,.,.d..,,~% 5

10"3M

4 X 1~3M La3+ "

20

5 10 15 20 TIME (min) in 102M Ca2+

TIME (rain) in SALYRGAN

Fig. 1. The effect of salyrgan/Ca2÷ on the percentage activity of lateral cilia in intact and isolated gill cell preparations. The control curves labelled no Ca 2÷ represent measurements in solutions with no exogenous Ca 2+ and a probable free Ca :+ of less than 10 -a M. The inhibition of ciliary activity observed with salyrgan requires the presence of high exogenous Ca 2+ in the external medium for intact and isolated gill cell preparation. In addition, the presence of La 3 + in the isolated gill cell preparation significantly potentiates the effect of salyrgan. The latter observation is probably due to the interference with Ca z ÷ by La 3÷ in what must be a somewhat leaky cell system (Fig. 1). Salyrgan inhibits the cilio-excitatory response brought about by low frequency (5 Hz) electrical nerve stimulation. La 3÷ enhances this inhibition (Fig. 2). The longer the intact gill preparation is left in the salyrgan perfusate, the less of a chance low freINTACT GILL

t~

l

/

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50"~5 hz.l.10-3M/ SALYRGA + 3 01 4 ; 10"3M ~a 5

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~

SALYRGAN I 15

TIME (mln/

Fig. 2. The effect of low frequency (5 Hz) nerve stimulation/ salyrgan/La 3+ on percent lateral frequency changes in intact gill.

Fig. 4. The effect of high frequency (50 Hz) nerve stimulation/salyrgan/La3+ on percent lateral frequency changes in intact gill. quency nerve stimulation has to restore lateral activity. After 16 rain, low frequency nerve stimulation appears to have an inhibitory effect on activity of lateral cilia presumably by depolarizing the membrane with subsequent rise intracellular-Ca 2 + (Fig. 3). As anticipated, salyrgan potentiates the cilio-inhibitory response brought about by the high frequency (50 Hz) branchial nerve stimulation. Again, La 3+ enhances the effect of salyrgan by bringing about a more significant cilio-inhibition (Fig. 4). The cilio-excitatory neurotransmitter (5-HT) which is presumably released by low frequency nerve stimulation antagonizes the cilio-inhibitory effect of salyrgan with or without addition of La 3 +. 5-HT is a significant competitive antagonizer of salyrgan. A significant restoration of the cilio-excitatory response of 5-HT occurred even after 16min pre-treatment of intact gill preparations with salyrgan (Figs 5 and 6). Furthermore, if one confines the field of view to the crushed ends of gill filaments, where presumably 5-HT is being endogenously released, a significant degree of competitive antagonism of 5-HT can be visualized without the addition of exogenous 5-HT to the perfusate (Fig. 7). 5-HT does not completely reverse the effect of salyrgan in isolated lateral cell preparations. This presumably is due to the increased permeability of exposed cell membrane present in isolated cells in these preparations (Fig. 8). The cilio-inhibitory neruotransmitter (DA), presumably released by high frequency (50 Hz) nerve INTACT GILL

10"eM5HT

250,

200-

200 U

150,

.z.. 150

~

loo.

~ ~.

so.

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+

10"3M SALYRGAN

~ 100,

s

CONTROL 5O

0 5

10

15

20

25

30

TIME (min) in SALYRGAN (10"3M)

Fig. 3. The effect of pretreatment with satyrgan on the restoration of percent lateral frequency brought about by 5 Hz nerve stimulation in intact gill.

I 5

I 10

I I 15 20 TIME(rain}

I 25

"1 30

Fig. 5. The effect of 5-HT/salyrgan/La3+ on percentage lateral frequency changes in intact gill.

Inactivation of the Ca 2+-pump

405

INTACT GILL

~

250'~

ISOLATED LATERAL CELLS

9 100.~

CONTROL

200

:3

150"

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SALYRGAN

50-

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o

I 30

Fig. 6. The effect of pretreatment with salyrgan on the restoration of lateral frequency brought about by 5-HT in intact gill. stimulation, enhances the cilio-inhibition brought about by salyrgan in intact (Fig. 7) and isolated (Fig. 8) gill cell preparations. DISCUSSION

This study supports the hypothesis that Ca 2 + plays a critical role in the observed cilio-excitatory and cilio-inhibitory response produced by nerve stimulation. Indeed, Ca 2 ÷ also has been demonstrated to cause the release of endogenous neurotransmitters at chemical synapses (Blaustein et al., 1978) and action potentials in giant axons (Baker et al., 1971; Rasmussen, 1970, 1971). In metazoan cilia, increasing the cytoplasmic-Ca z+ often causes ciliary arrest (Meech, 1974a,b; Satir, 1975). Changes in intracellular free Ca 2÷ concentration is important in the control of many cellular processes (Romero & Wittman, 1971; Meech, 1972). Recently, Walter & Satir (1978) have shown that lateral cilia are particularly sensitive to changes of free intracellular C a 2÷ within narrow limits (5 × 10 - 7 M to 8 × 10 -7 M). Since the lateral cilia are responsible for the water currents over the gill (Field, 1922), a small change in intracellular-Ca2+ could signal a partial or complete shutdown of the filtering process in unfavorable conditions. This study further relates postulated Ca 2 ÷ flow across the membrane of the lateral cilia to neuro-regulatory processes. In particular to observation of activity after low frequency (5-HT release) and high frequency (DA INTACT GILL CONTROL

IO0l

50-

lO2M'~"q

o

I 5

~

I~ 5

e~ SALYRGAN

;~-~" ~ 10 15 TIME (rain)

I 20

Fig. 8. The effect of (5-HT and DA)/salyrgan on the percent lateral motility changes in isolated gill cell preparations. release) stimulation of the gill of Paparo & Aiello (1970). The release of endogenous neurotransmitters can presumably activate (5-HT; cilio-excitor) or inactivate (DA; cilio-inhibitor) a Ca 2+ ATPase pump described in the ciliary membrane (Satir, 1976). The cilio-inhibitory effect of salyrgan in this study is produced by poisoning Ca 2 + ATPase and therefore Ca 2 + transport out of the cilium since no arrest is observed if K + is substituted for Ca 2 + in intact or isolated gill cell preparation. Since the isolated ciliated gill cells show the same salyrgan-induced arrest as intact gill, the response can be completely explained by direct increase in cytoplasmic (Ca 2+) in ciliated tissue and not in underlying nonciliated tissue elements of gill. However, it would appear from this study that the Ca 2 +-pump is not completely shut down by salyrgan, since La 3+ enhances the observed cilio-inhibition caused by salyrgan alone. La 3+ presumably binds irreversibly in place of Ca 2 ÷ to Ca 2 ÷ channels within lateral cell membrane. Leaks across the cell membrane can be produced by micro-injury by laser irradiation (Motokawa & Satir, 1974) which can produce arrest in the presence of sufficient external Ca 2÷. Presumably, high frequency nervous stimulation mimics laser irradiation in that membrane depolarization is probably responsible for local rise in internal-Ca 2 +, either directly by an increase in Ca 2 ÷ permeability or as the initial part of the process that inactivates the Ca 2 ÷ pump. In conclusion, this study has demonstrated the important role of intracellular-Ca2+ in controlling lateral ciliary activity. Future research, in explaining ciliary behavior in general, would be to localize the Ca 2 + binding components within the ciliary axoneme. In this regard, it is noteworthy that calmodulin has recently been found in Paramecium cilia (Maihle et al., 1979). Acknowledoement--This work was supported, in part, by funds provided by the United States Department of Interior as authorized under the Water Resources Act of 1964, and by USPHS Grant HL 22560 which provided partial support while the (senior) author was on sabbatical leave at Dept. of Anatomy, Albert Einstein Coll. Med.

10-~ 5.r

REFERENCES

I E 10

:"~ 15

I 20

',"~25

TIME(mln)

Fig. 7. The effect of (5-HT and DA)/salyrgan on the percent lateral frequency changes in the intact gill.

BAKER P. F., HODGKIN A. L. & RlDGWAVW. B. (1974) Depolarization and calcium entry in ~quid giant axons. J. Physiol., Lond. 218, 709-755. BANCROFTH. (1957) Introduction to Biostatistics. Hoeber, New York.

406

ANTHONY PAPAROand PETER SATIR

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