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The effect of salyrgan and lanthanum on the calcium-pump in the oyster Crassostrea virginica
Camp. Biochem. Physiol.Vol. 93C, No. 1, pp. 11l-l 14, 1989 Pnnted m Great Britain
0306~4492/89$3.00 + 0.00 0 1989 Pergamon Press plc
THE EFFECT OF SALYRGAN AND LANTHANUM ON THE CALCIUM-PUMP IN THE OYSTER CRASSOSTREA VIRGINICA ANTHONY A. PAPARO Department of Anatomy, School of Medicine, Department of Zoology, College of Science. Southern Illinois University, Carbondale, IL, USA and Visiting Professor of Marine Biology, Dauphin Island Sea Lab, P.O. Box 369-370, Dauphin Island, AL, USA (Received 14 July 1988) Abstract-l.
Lateral ciliary activity was studied in whole ctenidial preparations in the oyster, Crassostrea
virginica.
2. It has been previously demonstrated by the author that the lateral cilia are under the control of axons present in the branchial nerve and that serotonin (S-HT) is the released neurocilioexcitatory neurotransmitter. 3. This study confirms the above findings and in addition demonstrates that perfusates containing salyrgan (calcium-ATPase poison) in the presence of external calcium, in the various test salinities, is cilioinhibitory. The latter effect was reduced in the presence of the calcium chelator, EGTA. 4. The addition of lanthanum to the salyrgan perfusate enhances the cilioinhibitory response of the latter, presumably by binding in an irreversible manner to calcium channels within the lateral ciliated cell membrane. This functions to more effectively stop the calcium-pump. 5. It is postulated that salyrgan brings about an increase in the intracellular calcium pool within the lateral ciliated cell by poisoning the calcium-pump. This is further exacerbated in the presence of lanthanum and in the absence of the neurocilioexcitatory transmitter in the cut branchial nerve preparations. The latter presumably functions as a calcium-pump activator.
INTRODUCTION
MATERIALS AND METHODS
The American oyster Crassostrea virginica is a osmoconforming species of bivalve euryhaline, mollusc. Oysters pump water, together with the suspended food particles, into their mantle cavities and through the ctenidia by the action of tracts of lateral cilia which beat with a coordinated, metachronal rhythm (Nelson, 1960; Galtsoff, 1964). This species is found in large subtidal beds in estuarine areas on the Atlantic and Gulf costs of North America, and the animals experience both short and long term fluctuations in salinity (Butler, 1954). The lateral cilia in the ctenidia of Crassostrea are under the control of the serotonergic (5-HT, serotonin) cilioexcitatory axons contained within the branchial nerve (Paparo and Dean, 1984). Stimulation of the branchial nerve is postulated to mobilize calcium within the ctenidium (Paparo and Murphy, 1975; Paparo and Dean, 1984). Walter and Satir (1978) have shown that lateral ciliary activity is arrested within very narrow limits of 5 x lo-‘M-8 x lo-’ M calcium. Ciliary arrest was also induced by perfusing ctenidia with calcium and sayragan (a calcium-ATPase poison of the sarcoplasmic reticulum; Satir, 1976). The purpose of this study is to elucidate the mechanism of action of salyrgan, lanthanum and serotonin during salinity changes which activates the calcium-pump stimulation across the lateral cell membrane.
Oysters (Crassostrea uirginica Gmelin) were collected from natural subtidal beds on the north side of Dauphin Island, Alabama. The oysters were placed in standing seawater aquaria with undergravel filters filled with Instant Ocean Artificial seawater at salinity of 10% at 2428°C for three to five days before use for ciliary activity measurements. Oysters 6-9cm in length were shucked by breaking the ligament with an oyster knife and cutting the adductor muscle at the points of insertion on the upper and lower valves. The whole tissue was then maintained in a tray of clean artificial seawater at 10% salinity and 24°C. The mantle was excised from one side to expose the ctenidia. For an experiment, the tissue was transferred to a Petri dish containing the particular medium at 24°C on an adjustable microscope stage. A field of view was chosen which contained from 100 to 200 ctenidial filaments; the frequency (rate of lateral ciliary beating) was estimated by synchronizing the rate of flashing of a calibrated, stroboscopic light with the rate of beating of lateral cilia. Measurements were made from dorsal to ventral border, and from left to right across the field to determine the average at the beginning of each experiment. This calculated average (which varied from 10 to 12 Hz in a drug-free seawater perfusate) was assigned a percentage beating rate value of 100 that was used as the zero-point for each experiment. Drugs were continually added to the perfusion dish in seawater at 24°C. The following drugs were used: 10e6 M S-hydroxytryptamine (S-HT, serotonin); lo-) M O-[( 3- hydroxymercuri-2-methoxypropyl)carbamyl]phenoxyacetic acid (salyrgan); IO-’ M lanthanum; IO-’ M ethylene glycol-bis(amino-ethylether)-N:N’ tetra-acetic acid (EGTA). 111
112
ANTHONYA.PAPARO
(w=W
31~
3NllWB
AWIll
113
Oyster calcium pump RESULTS
The 10%0 habitat oysters, seawater pretreated preparations, which were perfused with various test salinities showed a significant (P < 0.01) reduction in the level of ciliary activity (28% and 6%; intact and cut nerve preparations, respectively). Perfusing these preparations with salyrgan in the presence of lanthanum produced a complete shut down of all ciliary activity (see Fig. 1). When the preparations were pretreated with EGTA there was significantly less of a reduction in the activity level, down to 69% and 66% (intact and cut nerve preparations, respectively). Salyrgan reduced the level of activity to 47% and 13%, while the addition of lanthanum further reduced this activity to 25% and 13% (intact and cut nerve preparations) (see Fig. 2). S-HT pretreated preparations showed a reduction in the level of ciliary activity of 57% and 44%, respectively, when perfused with the highest test salinity of 3O’k There was a further reduction in the level of activity down to 41% and 13%; 16% and 0%
5-HT / SEA WATER
for salyrgan and salyrgan plus lanthanum tions, respectively (see Fig. 3).
prepara-
DISCUSSION
The lateral cilia are arranged in tracts running proximally to distally along the ctenidial filaments (Nelson, 1960; Ribelin and Collier, 1977). The beating of these cilia is coordinated and under the control of the branchial nerve (Galtsoff, 1964; Aiello, 1974). The effect of a change in an oyster’s environment, therefore, may involve the animal’s nervous system or the ciliated epithelium itself. The results of exposing oyster tissue to sudden salinity changes support our earlier findings (Dean and Paparo, 1983; Paparo and Dean, 1984): a change in salinity inhibited lateral ciliary activity, with the degree of inhibition directly proportional to the magnitude of salinity change. This study supports the hypothesis that calcium plays a critical role in the observed cilioexcitatory response of nerve stimulation, since by cutting the branchial nerve one
PRETREATMENT CONTROL
SALYRGAN
20%* 30%, lO%o 20%. 30%0
SALYRGAN
+ LANTHANUM 10 % 0
TIME
(min)
Fig. 3. 5-HT pretreated 10% habitat salinity ctenidial intact (solid lines) and cut branchial nerve (broken lines) preparations were subsequently perfused with: salinity changes (top); salinity changes plus salyrgan (middle); salinity changes, salyrgan plus lanthanum (bottom).
ANTHONYA. PAPARO
114
could alter the effect of changing the salinity. Indeed, calcium also has been demonstrated to cause the release of endogenous neurotransmitters at the 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 calcium often causes ciliary arrest (Meech, 1974a, b; Satir, 1975). Changes in intracellular free [calcium] is important in the control of many cellular processes (Romero and Wittman, 1971; Meech, 1972). In a study by Walter and Satir (1978), it was shown that lateral cilia are particularly sensitive to changes in free intracellular calcium within narrow limits (5 x lo-’ M-8 x lo-‘M). Since the lateral cilia are responsible for the water currents over the ctenidium, a small change in intracellular calcium could signal a partial or complete shutdown of the filtering process in unfavorable conditions. The release of endogenous neurotransmittors can presumably activate (5-HT, cilioexitor) a calciumATPase pump described in the ciliary membrane (Satir, 1976). The cilioinhibitory effect of salyrgan is produced by poisoning the calcium-pump and therefore the transport out of the cilium. It would appear from this study that the calcium-pump is not completely shutdown by salyrgan, since lanthanum enhances the observed cilioinhibitory effect of salyrgan alone. Lathanum presumably binds irreversibly in place of calcium to the calcium channels within the lateral membrane. Leaks across the cell
membrane can be produced by microinjury by laser irradiation (Motokawa and Satir, 1974), which can produce arrest in the presence of sufficient exogenous calcium. The remarkable feature of the ciliary behavior is the capacity of the lateral ciliary activity on the ctenidium to acclimate to the new environmental conditions and eventually maintain a constant and high rate of activity. This aspect still remains to be studied. REFERENCES
Aiello E. (1974) Control of ciliary activity in metazoa. In Cilia and Flagella (Edited by Sleigh M. ,A.), pp. 353-376. Academic Press, London. Baker P. F., Hodgkin A. L. and Ridgway W. B. (1971) Depolarization and calcium entry in squid giant axons. J. Physiol., Lond. 218, 709-755.
Blaustein M. P., Ratzlaff R. W. and Kendrick N. K. (1978) The regulation of intracellular calcium in presynaptic nerve terminals. Ann. N.Y. Acad. Sci. 387. 195-212. Butler P. A. (1954) Summary of our knowledge of the oyster in the Gulf of Mexico. In Gulf of Mexico: Its Origin, Waters and Marine Life (Edited by Galtsoff P. S.). Bull. U.S. Fish Wildl. Serv. 55, 479-489.
Dean R. C. and Paparo A. (1983) Effects of salinity and calcium concentrations on ctenidial ciliary activity in the oyster Crassostrea uirginica (Gmelin). Comp. Biochem. Physiol. 14, 587-594.
Galtsoff P. S. (1964) The American oyster Crassostrea virginica Gmelin. Bull. U.S. Fish Wildl. Serv. 64, 1480. Meech R. W. (1972) Intercellular calcium injection causes increased potassium conductance in Aplysia nerve cells. Comp. Biochem. Physzol. 42. 493-499.
Meech R. W. (1974a) The sensitivity of Helix aspera neurons to injected calcium ions. J. Physiol., Lond. 237, 259-277.
Meech R. W. (1974b) Calcium influx induces a post-tetanic hyperpolarization in Aplysia neurons. Comp. Biochem. Physiol. 48, 387-395.
Motokawa T. and Satir P. (1974) Laser-induced spreading arrest of Mytilus gill cilia. J. Cell Biol. 366, 377-391. Nelson T. C. (1960) The feeding mechanism of the oyster. II. On the gills and palps of Ostrea edulis, Crassostrea virginica and C. angulaya. J. Morph. 107, 163-203. Paparo A. and Murphy J. (1975) The effect of Ca on the rate of beating of lateral cilia in Mytilus edulis-II. A response. to electrical stimulation of the branchial nerve. Comp. Biochem. Physiol. 50, 15-19.
Paparo A. and Dean R. C. (1984) Activity of the lateral cilia of the oyster Crassostrea virginica Gmelin: response to changes in salinity and to changes in potassium and magnesium concentration. Mar. Behav. Physiol. 11, 111-130. Rasmussen H. (1970) Cell communication, calcium ion and cyclic adenosine monophosphate. Science, Wash. 70, 404412.
Rasmussen H. (1971) Ionic and hormonal control of calcium homeostatis. Am. J. Med. SO, 567-588. Ribelin B. W. and Collier A. (1977) Studies of the gill ciliation of the American oyster, Crassostrea virginica (Gmelin). J. Morph. 151, 439448. Romero P. J. and Whittan R. (1971)The control by internal calcium of membrane permeability to sodium and potassium. J. Physiol.,. Lond. 214, 481-507. Satir P. (1975) Ionophore-mediated entry induces mussel gill ciliary arrest. Science 190, 586588. Satir P. (1976) Local design of membranes in relation to cell function. Eur. Congr. Electron Microsc. Jerusalem 6, 414.
Walter M. F. and Satir P. (1978) Calcium control of ciliary arrest in mussel gill cells. J. Cell Biol. 79, 11&120.
0306~4492/89$3.00 + 0.00 0 1989 Pergamon Press plc
THE EFFECT OF SALYRGAN AND LANTHANUM ON THE CALCIUM-PUMP IN THE OYSTER CRASSOSTREA VIRGINICA ANTHONY A. PAPARO Department of Anatomy, School of Medicine, Department of Zoology, College of Science. Southern Illinois University, Carbondale, IL, USA and Visiting Professor of Marine Biology, Dauphin Island Sea Lab, P.O. Box 369-370, Dauphin Island, AL, USA (Received 14 July 1988) Abstract-l.
Lateral ciliary activity was studied in whole ctenidial preparations in the oyster, Crassostrea
virginica.
2. It has been previously demonstrated by the author that the lateral cilia are under the control of axons present in the branchial nerve and that serotonin (S-HT) is the released neurocilioexcitatory neurotransmitter. 3. This study confirms the above findings and in addition demonstrates that perfusates containing salyrgan (calcium-ATPase poison) in the presence of external calcium, in the various test salinities, is cilioinhibitory. The latter effect was reduced in the presence of the calcium chelator, EGTA. 4. The addition of lanthanum to the salyrgan perfusate enhances the cilioinhibitory response of the latter, presumably by binding in an irreversible manner to calcium channels within the lateral ciliated cell membrane. This functions to more effectively stop the calcium-pump. 5. It is postulated that salyrgan brings about an increase in the intracellular calcium pool within the lateral ciliated cell by poisoning the calcium-pump. This is further exacerbated in the presence of lanthanum and in the absence of the neurocilioexcitatory transmitter in the cut branchial nerve preparations. The latter presumably functions as a calcium-pump activator.
INTRODUCTION
MATERIALS AND METHODS
The American oyster Crassostrea virginica is a osmoconforming species of bivalve euryhaline, mollusc. Oysters pump water, together with the suspended food particles, into their mantle cavities and through the ctenidia by the action of tracts of lateral cilia which beat with a coordinated, metachronal rhythm (Nelson, 1960; Galtsoff, 1964). This species is found in large subtidal beds in estuarine areas on the Atlantic and Gulf costs of North America, and the animals experience both short and long term fluctuations in salinity (Butler, 1954). The lateral cilia in the ctenidia of Crassostrea are under the control of the serotonergic (5-HT, serotonin) cilioexcitatory axons contained within the branchial nerve (Paparo and Dean, 1984). Stimulation of the branchial nerve is postulated to mobilize calcium within the ctenidium (Paparo and Murphy, 1975; Paparo and Dean, 1984). Walter and Satir (1978) have shown that lateral ciliary activity is arrested within very narrow limits of 5 x lo-‘M-8 x lo-’ M calcium. Ciliary arrest was also induced by perfusing ctenidia with calcium and sayragan (a calcium-ATPase poison of the sarcoplasmic reticulum; Satir, 1976). The purpose of this study is to elucidate the mechanism of action of salyrgan, lanthanum and serotonin during salinity changes which activates the calcium-pump stimulation across the lateral cell membrane.
Oysters (Crassostrea uirginica Gmelin) were collected from natural subtidal beds on the north side of Dauphin Island, Alabama. The oysters were placed in standing seawater aquaria with undergravel filters filled with Instant Ocean Artificial seawater at salinity of 10% at 2428°C for three to five days before use for ciliary activity measurements. Oysters 6-9cm in length were shucked by breaking the ligament with an oyster knife and cutting the adductor muscle at the points of insertion on the upper and lower valves. The whole tissue was then maintained in a tray of clean artificial seawater at 10% salinity and 24°C. The mantle was excised from one side to expose the ctenidia. For an experiment, the tissue was transferred to a Petri dish containing the particular medium at 24°C on an adjustable microscope stage. A field of view was chosen which contained from 100 to 200 ctenidial filaments; the frequency (rate of lateral ciliary beating) was estimated by synchronizing the rate of flashing of a calibrated, stroboscopic light with the rate of beating of lateral cilia. Measurements were made from dorsal to ventral border, and from left to right across the field to determine the average at the beginning of each experiment. This calculated average (which varied from 10 to 12 Hz in a drug-free seawater perfusate) was assigned a percentage beating rate value of 100 that was used as the zero-point for each experiment. Drugs were continually added to the perfusion dish in seawater at 24°C. The following drugs were used: 10e6 M S-hydroxytryptamine (S-HT, serotonin); lo-) M O-[( 3- hydroxymercuri-2-methoxypropyl)carbamyl]phenoxyacetic acid (salyrgan); IO-’ M lanthanum; IO-’ M ethylene glycol-bis(amino-ethylether)-N:N’ tetra-acetic acid (EGTA). 111
112
ANTHONYA.PAPARO
(w=W
31~
3NllWB
AWIll
113
Oyster calcium pump RESULTS
The 10%0 habitat oysters, seawater pretreated preparations, which were perfused with various test salinities showed a significant (P < 0.01) reduction in the level of ciliary activity (28% and 6%; intact and cut nerve preparations, respectively). Perfusing these preparations with salyrgan in the presence of lanthanum produced a complete shut down of all ciliary activity (see Fig. 1). When the preparations were pretreated with EGTA there was significantly less of a reduction in the activity level, down to 69% and 66% (intact and cut nerve preparations, respectively). Salyrgan reduced the level of activity to 47% and 13%, while the addition of lanthanum further reduced this activity to 25% and 13% (intact and cut nerve preparations) (see Fig. 2). S-HT pretreated preparations showed a reduction in the level of ciliary activity of 57% and 44%, respectively, when perfused with the highest test salinity of 3O’k There was a further reduction in the level of activity down to 41% and 13%; 16% and 0%
5-HT / SEA WATER
for salyrgan and salyrgan plus lanthanum tions, respectively (see Fig. 3).
prepara-
DISCUSSION
The lateral cilia are arranged in tracts running proximally to distally along the ctenidial filaments (Nelson, 1960; Ribelin and Collier, 1977). The beating of these cilia is coordinated and under the control of the branchial nerve (Galtsoff, 1964; Aiello, 1974). The effect of a change in an oyster’s environment, therefore, may involve the animal’s nervous system or the ciliated epithelium itself. The results of exposing oyster tissue to sudden salinity changes support our earlier findings (Dean and Paparo, 1983; Paparo and Dean, 1984): a change in salinity inhibited lateral ciliary activity, with the degree of inhibition directly proportional to the magnitude of salinity change. This study supports the hypothesis that calcium plays a critical role in the observed cilioexcitatory response of nerve stimulation, since by cutting the branchial nerve one
PRETREATMENT CONTROL
SALYRGAN
20%* 30%, lO%o 20%. 30%0
SALYRGAN
+ LANTHANUM 10 % 0
TIME
(min)
Fig. 3. 5-HT pretreated 10% habitat salinity ctenidial intact (solid lines) and cut branchial nerve (broken lines) preparations were subsequently perfused with: salinity changes (top); salinity changes plus salyrgan (middle); salinity changes, salyrgan plus lanthanum (bottom).
ANTHONYA. PAPARO
114
could alter the effect of changing the salinity. Indeed, calcium also has been demonstrated to cause the release of endogenous neurotransmitters at the 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 calcium often causes ciliary arrest (Meech, 1974a, b; Satir, 1975). Changes in intracellular free [calcium] is important in the control of many cellular processes (Romero and Wittman, 1971; Meech, 1972). In a study by Walter and Satir (1978), it was shown that lateral cilia are particularly sensitive to changes in free intracellular calcium within narrow limits (5 x lo-’ M-8 x lo-‘M). Since the lateral cilia are responsible for the water currents over the ctenidium, a small change in intracellular calcium could signal a partial or complete shutdown of the filtering process in unfavorable conditions. The release of endogenous neurotransmittors can presumably activate (5-HT, cilioexitor) a calciumATPase pump described in the ciliary membrane (Satir, 1976). The cilioinhibitory effect of salyrgan is produced by poisoning the calcium-pump and therefore the transport out of the cilium. It would appear from this study that the calcium-pump is not completely shutdown by salyrgan, since lanthanum enhances the observed cilioinhibitory effect of salyrgan alone. Lathanum presumably binds irreversibly in place of calcium to the calcium channels within the lateral membrane. Leaks across the cell
membrane can be produced by microinjury by laser irradiation (Motokawa and Satir, 1974), which can produce arrest in the presence of sufficient exogenous calcium. The remarkable feature of the ciliary behavior is the capacity of the lateral ciliary activity on the ctenidium to acclimate to the new environmental conditions and eventually maintain a constant and high rate of activity. This aspect still remains to be studied. REFERENCES
Aiello E. (1974) Control of ciliary activity in metazoa. In Cilia and Flagella (Edited by Sleigh M. ,A.), pp. 353-376. Academic Press, London. Baker P. F., Hodgkin A. L. and Ridgway W. B. (1971) Depolarization and calcium entry in squid giant axons. J. Physiol., Lond. 218, 709-755.
Blaustein M. P., Ratzlaff R. W. and Kendrick N. K. (1978) The regulation of intracellular calcium in presynaptic nerve terminals. Ann. N.Y. Acad. Sci. 387. 195-212. Butler P. A. (1954) Summary of our knowledge of the oyster in the Gulf of Mexico. In Gulf of Mexico: Its Origin, Waters and Marine Life (Edited by Galtsoff P. S.). Bull. U.S. Fish Wildl. Serv. 55, 479-489.
Dean R. C. and Paparo A. (1983) Effects of salinity and calcium concentrations on ctenidial ciliary activity in the oyster Crassostrea uirginica (Gmelin). Comp. Biochem. Physiol. 14, 587-594.
Galtsoff P. S. (1964) The American oyster Crassostrea virginica Gmelin. Bull. U.S. Fish Wildl. Serv. 64, 1480. Meech R. W. (1972) Intercellular calcium injection causes increased potassium conductance in Aplysia nerve cells. Comp. Biochem. Physzol. 42. 493-499.
Meech R. W. (1974a) The sensitivity of Helix aspera neurons to injected calcium ions. J. Physiol., Lond. 237, 259-277.
Meech R. W. (1974b) Calcium influx induces a post-tetanic hyperpolarization in Aplysia neurons. Comp. Biochem. Physiol. 48, 387-395.
Motokawa T. and Satir P. (1974) Laser-induced spreading arrest of Mytilus gill cilia. J. Cell Biol. 366, 377-391. Nelson T. C. (1960) The feeding mechanism of the oyster. II. On the gills and palps of Ostrea edulis, Crassostrea virginica and C. angulaya. J. Morph. 107, 163-203. Paparo A. and Murphy J. (1975) The effect of Ca on the rate of beating of lateral cilia in Mytilus edulis-II. A response. to electrical stimulation of the branchial nerve. Comp. Biochem. Physiol. 50, 15-19.
Paparo A. and Dean R. C. (1984) Activity of the lateral cilia of the oyster Crassostrea virginica Gmelin: response to changes in salinity and to changes in potassium and magnesium concentration. Mar. Behav. Physiol. 11, 111-130. Rasmussen H. (1970) Cell communication, calcium ion and cyclic adenosine monophosphate. Science, Wash. 70, 404412.
Rasmussen H. (1971) Ionic and hormonal control of calcium homeostatis. Am. J. Med. SO, 567-588. Ribelin B. W. and Collier A. (1977) Studies of the gill ciliation of the American oyster, Crassostrea virginica (Gmelin). J. Morph. 151, 439448. Romero P. J. and Whittan R. (1971)The control by internal calcium of membrane permeability to sodium and potassium. J. Physiol.,. Lond. 214, 481-507. Satir P. (1975) Ionophore-mediated entry induces mussel gill ciliary arrest. Science 190, 586588. Satir P. (1976) Local design of membranes in relation to cell function. Eur. Congr. Electron Microsc. Jerusalem 6, 414.
Walter M. F. and Satir P. (1978) Calcium control of ciliary arrest in mussel gill cells. J. Cell Biol. 79, 11&120.