TOXICOLOGYANDAPPLIEDPHARMACOLOGY
SHORT Effect
of Cadmium
33,384-387(1975)
COMMUNICATION on Epithelial
Membranes’
Effect of Cadmiumon Epithelial Membranes.FLEISHER, L. N., YORIO, T. BENTLEY, P. J. (1975). Toxicol. Appl. Pharmacol. 33, 384-387. Cdz+ (10e3~1) on theexternal sideof the frog skin, increasedtheelectricalpotential difference (PD), short-circuit current (SCC), and resistanceacrossthis membrane.The effect wasirreversible. Cd2+(10e3M) on the serosalsideof the toad urinary bladder inhibited the SCC, but this was increasedby 10e3M and 10e4M at the mucosaor 10e4M at the serosa.The increasesin SCCwereinhibited by amiloride, indicating the effect wasdueto a stimulation of active transmural sodiumtransport. The increasedresistanceseen in the frog skin may reflect a decreasedpermeability to chloride. These resultsindicate that cadmiumcan influenceactive ion transport and the permeability of epithelial membranes. AND
The occurrence and possible roles of toxic amounts of cadmium in human diseasehas become increasingly apparent (Gieske and Foulkes, 1974; Hine et al., 1973). This metal, which is potentially volatile, is used in many modern industrial processes, including the making of certain alloys, plastics, pigments, and batteries (Bremner, 1974). Although the toxic effects of Cd 2+ have been known for about 100 years, the possible industrial hazards associated with its use have not been appreciated until recently. In Japan, the ingestion of Cd2+ from contaminated drinking water has been shown to result in an osteoporotic disease(“itai-itai”) (Friedberg et al., 1971)and it may also cause nephritis, hypertension, and damage to the lungs, testes, and liver. In the present study we describe the effects of cadmium on the electrical properties and ion permeability of the frog skin and toad urinary bladder in uitro. These preparations behave in many respectslike mammalian epithelia, especially the kidney, and they are widely used as models for studying the latter. METHODS The ventral skin from frogs (Ranapippiens) was usedto make diaphragm preparations in divided chambers bathed on each side with Ringer’s solution (Ussing and Zerahn, 1951). Toad (Bufo marinus) urinary bladder preparations were made as sacs(Bentley, 1958). Due to variability in the different batches of toads, animals from the same shipment were used for each group of experiments. The electrical potential difference (PD), short-circuit current (SCC), and electrical resistancewere measuredbetween the two sidesof each membrane. The PD was measured(with a voltmeter, Keithley 200b) through a pair of calomel cells which were connected to each side of the membrane by saturated KCI-agar bridges. The SCC was applied from an external battery through 1 Supported by NSF Grant GB-28543X (PJB), Mount Sinai Graduate School Fellowship (TY). Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
a CUNY 384
Research Assistantship (LNF), and a
SHORT
a similar pair of bridges and a pair of Ag-AgCl ampmeter. The statistical analysis was preformed between two sample means (Fisher, 1950). RESULTS
385
COMMUNICATION
AND
cells. The current was read with an using a Student’s t test for differences
DISCUSSION
When CdZ+ ( 10e3M) was placed in the external (but not the internal) solution bathing the frog skin, the SCC, PD, and resistance all increased (Table 1). These effects were maximal within 10 min and were not reversible when the Cd2+ was removed. Lower concentrations were without effect. TABLE
1
EFFECTSOF CdZ+ ONTHEELECTRICALPARAMETERSOFFROGSKIN (IN VITRO)~
AND TOAD BLADDER
see Cd”+ concentration and treatment
(El!, Initial
lo-min
exposure
-
(pA/3.8 cm2 skin or/100 mg bladder) Initial
IO-min exposure
Skin 10m3 M Outside (33) 10W3 M Inside (8)
29 + 2.4 26 k 2.8
58 k 4.4’ 27k3.1
89 5 7.6 86 k 8.5
Bladder 10mm3 M Mucosal(l5) 1O--3 M Serosal (8)
43 * 4.3 42 L- 5.2
52 Ifr 4.6 33 + 6.2
144 rf- 15.4 246 + 36.3
214 + 20.3” 161 + 11.8’
1O--j M Mucosal(5) 10-j M Serosal(7)
66 5- 7.8 57 f 7.6
89 + 8.2’ 62 + 9.5
83 5 10.2 255 + 27.7
20-min exposure 130 rt 17.0’ 402 * 40.0”
124? 91 *
9.0b 7.8
’ Results are expressed in means + SE of the number of experiments in parentheses. The electrical resistance was calculated (PD/SCC) in 15 skin preparations; it rose from 1.40 + 0.11 to 1.91 2 0.15 Kohms-cm2 when the CdZ+ was present in the outer solution (p i 0.01). b p < 0.01 for change from initial value. c p < 0.05 for change from initial value.
In toad bladders CdZ+ (10m3and 10-4~) also increased the KC when added to the mucosal bathing solution (Table 1). The PD was increased by 10e4hl Cd’+ (but not 10m3~) but, in contrast to the skin, the resistance did not increase. Lower concentrations of Cd2+ had no significant effects on the mucosal side of the bladder. When 10m3~ Cd2+ was placed in the serosal solution, both the PD and SCC declined, and after 25 min the SCC was only 14% of the initial value. This effect was not reversible. At 10m4~ on the serosal side, however, Ca2+ increased the SCC across the bladder, though the effect was somewhat slower and, like at the mucosa, took about 20 min to reach maximal levels. Cadmium at a concentration of 10e5~ on the bladder serosa had no effect. The SCC acrossthese preparations usually reflects active Na transport (Ussing and Zerahn, 1951; Leaf et al., 1958), but in order to further confirm that the observed increasesin SCC also reflected this, amiloride, a specific inhibitor of Na transport in
386
SHORT
COMMUNICATION
these membranes (Bentley, 1968; Ehlich and CrabbC, 1968) was added to the external solutions. This drug (~O+M) promptly reduced the SCC to near zero levels. Thus, the increase in SCC probably reflects higher rates of Na transport across the membranes. Such an increase in cation transport is not expected to be associated with the increased resistance that was observed in the frog skin (Table I) and may reflect an additional effect in decreasing anion, possibly Cl, permeability. When an impermeant anion SO:- was substituted for Cl- in the bathing media, Cd2+ produced a decline in the resistance (Kohms-cm2); from 4.2 f. 0.28 (14) to 3.2 ? 0.22 in 5 min (p < 0.05). This may reflect its action on Na transfer in the absence of an ability to alter Cl permeability. The present results show that Cd2+ can influence certain processes in epithelial membranes and depending on the surface exposed, can increase or decrease active transmural Na transport. The Cd2+ used acutely in these experiments is unbound to other molecules and the concentrations are higher than those that occur in vivo. However, exposure may be chronic in nature and long-term accumulation of the metal may occur. The stimulation of ion transport that we observed is reminiscent of the observation of Vander (1962) who showed that Cd2+ increased Na transport across the renal tubules of dogs. The sensitivity of these membranes to the actions of cadmium also may reflect its renal toxicity. The precise nature of the action of cadmium is uncertain at this time, but these membranes have a known sensitivity to SH reacting drugs, as well as ions that bind to Ca2+ sites (Rasmussen et al., 1960; Bentley, 1973; Wietzerbin et aE., 1974) and either of these could be involved. CdZ+ also combines with alkyl groups (Wakefield, 1971). Such in vitro preparations of epithelial membranes may be useful for studying the mechanismsinvolved in cadmium toxicity. REFERENCES BENTLEY,P. J. (1958).The effectsof neurohypophysialextracts on water transfer acrossthe wall of the isolatedurinary bladder of the toad Bufo marims. J. Endocrinol. 18,201-209. BENTLEY,P. J. (1968). Amiloride: A potent inhibitor of sodium transport acrossthe toad bladder.J. Physiol. 195, 317-330. BENTLEY,P. J. (1973). Someproperties of the hydro-osmotic vasopressin“effector”: interactions with N-ethylmaleimide.J. Endocrinol. 59,99-106. BREMNER, I. (1974).Heavy metal toxicities. Quart. Rev. Biophys. 7, 75-124. EHLICH, E. N. AND CRABBY, J. (1968). The mechanismof action of amipramizide.Pflugers Arch. 302,79-96.
FISHER,R. A. Statistical Methods for Research Workers, pp. 114-173.Oliver and Boyd Ltd., Edinburgh, England. FRIEDBERG, L., PISCATOR, M. ANDNORDBERG, G. (1971).Cadmium in the environment, pp. 11l-l 19. Chem.Rubber Publ. Co., Cleveland,Ohio. GIESKE,T. H. ANDFOULKES,E. C. (1974).Acute effects of cadmium on proximal tubular function in rabbits. Toxicol. Appl. Pharmacol. 27, 292. HINE, C. H., WRIGHT, J. AND GOODMAN,D. (1973). Tissuelevels of cadmium in different diseasestates.Toxicol. Appl. Pharmacol. 27,476-477. LEAF, A., ANDERSON, J. ANDPAGE,L. B. (1958).Active sodiumtransport by the isolatedtoad bladder.J. Gen. Physiol. 41, 657-668. RASMUSSEN, H., SCHWARTZ, I. L., SCHOESSLER, M. A. AND HOCHSTER, G. (1960). Studieson the mechanismof action of vasopressin.Proc. Nat. Acad. Sci. USA 46,1278-1287. USSING, H. H. AND ZERAHN, K. (1951). Active transport of sodiumasthe sourceof electric current in the short-circuited isolatedfrog skin. Acta Physiol. Stand. 23, 110-l 17.
SHORT
VANDER, A. 203, 1-5.
3x7
COMMUNICATION
J. (1962). Effect of cadmium on renal tubular sodium transport. Amer. J. Physiol.
B. J. (1971). Alkyl derivatives of the group II metals. In Adwrces in Inorganic (H. I. Emelus,and A. G. Sharpe,Eds.), Vol. 11,pp. 342425. Academic Press,New York. WIETZERBIN, J., LANGE, Y. AND GARY-B• BO, C. M. (1974).Lanthanuminhibition of the action of oxytocin on water transfer of the frog urinary bladder: effect on the serosaland apical membrane.J. hfemhr. Biol. 17, 2740. WAKEFIELD,
Chemistry and Radiochemistry
Departments oj’Pharmacology and Ophthalmology, The Mount Sinai School of Medicine oj’The City Unirersity of New York, New York 10029 Rewired Januarv 13,197s; accepted March 26,1975
L. N.
FLEISHER
T. YORK) P. J. BFN’rLt?
SHORT Effect
of Cadmium
33,384-387(1975)
COMMUNICATION on Epithelial
Membranes’
Effect of Cadmiumon Epithelial Membranes.FLEISHER, L. N., YORIO, T. BENTLEY, P. J. (1975). Toxicol. Appl. Pharmacol. 33, 384-387. Cdz+ (10e3~1) on theexternal sideof the frog skin, increasedtheelectricalpotential difference (PD), short-circuit current (SCC), and resistanceacrossthis membrane.The effect wasirreversible. Cd2+(10e3M) on the serosalsideof the toad urinary bladder inhibited the SCC, but this was increasedby 10e3M and 10e4M at the mucosaor 10e4M at the serosa.The increasesin SCCwereinhibited by amiloride, indicating the effect wasdueto a stimulation of active transmural sodiumtransport. The increasedresistanceseen in the frog skin may reflect a decreasedpermeability to chloride. These resultsindicate that cadmiumcan influenceactive ion transport and the permeability of epithelial membranes. AND
The occurrence and possible roles of toxic amounts of cadmium in human diseasehas become increasingly apparent (Gieske and Foulkes, 1974; Hine et al., 1973). This metal, which is potentially volatile, is used in many modern industrial processes, including the making of certain alloys, plastics, pigments, and batteries (Bremner, 1974). Although the toxic effects of Cd 2+ have been known for about 100 years, the possible industrial hazards associated with its use have not been appreciated until recently. In Japan, the ingestion of Cd2+ from contaminated drinking water has been shown to result in an osteoporotic disease(“itai-itai”) (Friedberg et al., 1971)and it may also cause nephritis, hypertension, and damage to the lungs, testes, and liver. In the present study we describe the effects of cadmium on the electrical properties and ion permeability of the frog skin and toad urinary bladder in uitro. These preparations behave in many respectslike mammalian epithelia, especially the kidney, and they are widely used as models for studying the latter. METHODS The ventral skin from frogs (Ranapippiens) was usedto make diaphragm preparations in divided chambers bathed on each side with Ringer’s solution (Ussing and Zerahn, 1951). Toad (Bufo marinus) urinary bladder preparations were made as sacs(Bentley, 1958). Due to variability in the different batches of toads, animals from the same shipment were used for each group of experiments. The electrical potential difference (PD), short-circuit current (SCC), and electrical resistancewere measuredbetween the two sidesof each membrane. The PD was measured(with a voltmeter, Keithley 200b) through a pair of calomel cells which were connected to each side of the membrane by saturated KCI-agar bridges. The SCC was applied from an external battery through 1 Supported by NSF Grant GB-28543X (PJB), Mount Sinai Graduate School Fellowship (TY). Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
a CUNY 384
Research Assistantship (LNF), and a
SHORT
a similar pair of bridges and a pair of Ag-AgCl ampmeter. The statistical analysis was preformed between two sample means (Fisher, 1950). RESULTS
385
COMMUNICATION
AND
cells. The current was read with an using a Student’s t test for differences
DISCUSSION
When CdZ+ ( 10e3M) was placed in the external (but not the internal) solution bathing the frog skin, the SCC, PD, and resistance all increased (Table 1). These effects were maximal within 10 min and were not reversible when the Cd2+ was removed. Lower concentrations were without effect. TABLE
1
EFFECTSOF CdZ+ ONTHEELECTRICALPARAMETERSOFFROGSKIN (IN VITRO)~
AND TOAD BLADDER
see Cd”+ concentration and treatment
(El!, Initial
lo-min
exposure
-
(pA/3.8 cm2 skin or/100 mg bladder) Initial
IO-min exposure
Skin 10m3 M Outside (33) 10W3 M Inside (8)
29 + 2.4 26 k 2.8
58 k 4.4’ 27k3.1
89 5 7.6 86 k 8.5
Bladder 10mm3 M Mucosal(l5) 1O--3 M Serosal (8)
43 * 4.3 42 L- 5.2
52 Ifr 4.6 33 + 6.2
144 rf- 15.4 246 + 36.3
214 + 20.3” 161 + 11.8’
1O--j M Mucosal(5) 10-j M Serosal(7)
66 5- 7.8 57 f 7.6
89 + 8.2’ 62 + 9.5
83 5 10.2 255 + 27.7
20-min exposure 130 rt 17.0’ 402 * 40.0”
124? 91 *
9.0b 7.8
’ Results are expressed in means + SE of the number of experiments in parentheses. The electrical resistance was calculated (PD/SCC) in 15 skin preparations; it rose from 1.40 + 0.11 to 1.91 2 0.15 Kohms-cm2 when the CdZ+ was present in the outer solution (p i 0.01). b p < 0.01 for change from initial value. c p < 0.05 for change from initial value.
In toad bladders CdZ+ (10m3and 10-4~) also increased the KC when added to the mucosal bathing solution (Table 1). The PD was increased by 10e4hl Cd’+ (but not 10m3~) but, in contrast to the skin, the resistance did not increase. Lower concentrations of Cd2+ had no significant effects on the mucosal side of the bladder. When 10m3~ Cd2+ was placed in the serosal solution, both the PD and SCC declined, and after 25 min the SCC was only 14% of the initial value. This effect was not reversible. At 10m4~ on the serosal side, however, Ca2+ increased the SCC across the bladder, though the effect was somewhat slower and, like at the mucosa, took about 20 min to reach maximal levels. Cadmium at a concentration of 10e5~ on the bladder serosa had no effect. The SCC acrossthese preparations usually reflects active Na transport (Ussing and Zerahn, 1951; Leaf et al., 1958), but in order to further confirm that the observed increasesin SCC also reflected this, amiloride, a specific inhibitor of Na transport in
386
SHORT
COMMUNICATION
these membranes (Bentley, 1968; Ehlich and CrabbC, 1968) was added to the external solutions. This drug (~O+M) promptly reduced the SCC to near zero levels. Thus, the increase in SCC probably reflects higher rates of Na transport across the membranes. Such an increase in cation transport is not expected to be associated with the increased resistance that was observed in the frog skin (Table I) and may reflect an additional effect in decreasing anion, possibly Cl, permeability. When an impermeant anion SO:- was substituted for Cl- in the bathing media, Cd2+ produced a decline in the resistance (Kohms-cm2); from 4.2 f. 0.28 (14) to 3.2 ? 0.22 in 5 min (p < 0.05). This may reflect its action on Na transfer in the absence of an ability to alter Cl permeability. The present results show that Cd2+ can influence certain processes in epithelial membranes and depending on the surface exposed, can increase or decrease active transmural Na transport. The Cd2+ used acutely in these experiments is unbound to other molecules and the concentrations are higher than those that occur in vivo. However, exposure may be chronic in nature and long-term accumulation of the metal may occur. The stimulation of ion transport that we observed is reminiscent of the observation of Vander (1962) who showed that Cd2+ increased Na transport across the renal tubules of dogs. The sensitivity of these membranes to the actions of cadmium also may reflect its renal toxicity. The precise nature of the action of cadmium is uncertain at this time, but these membranes have a known sensitivity to SH reacting drugs, as well as ions that bind to Ca2+ sites (Rasmussen et al., 1960; Bentley, 1973; Wietzerbin et aE., 1974) and either of these could be involved. CdZ+ also combines with alkyl groups (Wakefield, 1971). Such in vitro preparations of epithelial membranes may be useful for studying the mechanismsinvolved in cadmium toxicity. REFERENCES BENTLEY,P. J. (1958).The effectsof neurohypophysialextracts on water transfer acrossthe wall of the isolatedurinary bladder of the toad Bufo marims. J. Endocrinol. 18,201-209. BENTLEY,P. J. (1968). Amiloride: A potent inhibitor of sodium transport acrossthe toad bladder.J. Physiol. 195, 317-330. BENTLEY,P. J. (1973). Someproperties of the hydro-osmotic vasopressin“effector”: interactions with N-ethylmaleimide.J. Endocrinol. 59,99-106. BREMNER, I. (1974).Heavy metal toxicities. Quart. Rev. Biophys. 7, 75-124. EHLICH, E. N. AND CRABBY, J. (1968). The mechanismof action of amipramizide.Pflugers Arch. 302,79-96.
FISHER,R. A. Statistical Methods for Research Workers, pp. 114-173.Oliver and Boyd Ltd., Edinburgh, England. FRIEDBERG, L., PISCATOR, M. ANDNORDBERG, G. (1971).Cadmium in the environment, pp. 11l-l 19. Chem.Rubber Publ. Co., Cleveland,Ohio. GIESKE,T. H. ANDFOULKES,E. C. (1974).Acute effects of cadmium on proximal tubular function in rabbits. Toxicol. Appl. Pharmacol. 27, 292. HINE, C. H., WRIGHT, J. AND GOODMAN,D. (1973). Tissuelevels of cadmium in different diseasestates.Toxicol. Appl. Pharmacol. 27,476-477. LEAF, A., ANDERSON, J. ANDPAGE,L. B. (1958).Active sodiumtransport by the isolatedtoad bladder.J. Gen. Physiol. 41, 657-668. RASMUSSEN, H., SCHWARTZ, I. L., SCHOESSLER, M. A. AND HOCHSTER, G. (1960). Studieson the mechanismof action of vasopressin.Proc. Nat. Acad. Sci. USA 46,1278-1287. USSING, H. H. AND ZERAHN, K. (1951). Active transport of sodiumasthe sourceof electric current in the short-circuited isolatedfrog skin. Acta Physiol. Stand. 23, 110-l 17.
SHORT
VANDER, A. 203, 1-5.
3x7
COMMUNICATION
J. (1962). Effect of cadmium on renal tubular sodium transport. Amer. J. Physiol.
B. J. (1971). Alkyl derivatives of the group II metals. In Adwrces in Inorganic (H. I. Emelus,and A. G. Sharpe,Eds.), Vol. 11,pp. 342425. Academic Press,New York. WIETZERBIN, J., LANGE, Y. AND GARY-B• BO, C. M. (1974).Lanthanuminhibition of the action of oxytocin on water transfer of the frog urinary bladder: effect on the serosaland apical membrane.J. hfemhr. Biol. 17, 2740. WAKEFIELD,
Chemistry and Radiochemistry
Departments oj’Pharmacology and Ophthalmology, The Mount Sinai School of Medicine oj’The City Unirersity of New York, New York 10029 Rewired Januarv 13,197s; accepted March 26,1975
L. N.
FLEISHER
T. YORK) P. J. BFN’rLt?
