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Secretion of nitrate by rectal gland of squalus acanthias
03I%9629/93 $6.00 + 0.00
Camp. Biochem. Physiol. Vol. 104A,No. 2, pp. 255-259, 1993
0 1993Pergamon Press Ltd
Printed in Great Britain
SECRETION OF NITRATE BY RECTAL GLAND OF SQUALUS ACANTHIAS PATRICIO SILVA* and FRANKLIN H. EPSTEIN Departments of Medicine, New England Deaconess Hospital, Boston, MA 02215 and Beth Israel Hospital, Harvard Medical School, The Joslin Diabetes Center, The Harvard Center for the Study of Kidney Diseases, and The Mount Desert Island Biological Laboratory, Salsbury Cove, ME, U.S.A. (Received
19 May 1992; accepted 24 June 1992)
Rectal glands secrete nitrate at 30% of their capacity to secrete chloride. 2. Nitrate secretion is directly related to its concentration at constant chloride concentrations. 3. Chloride has a biphasic effect on nitrate secretion. 4. Hill coefficients at chloride < 100mM are eaual to 1. while at 100 mM indicate inhibition of nitrate by chloride. 6. Lineweaver-Burk plots at chloride < 100 indicate a single site, while at 100mM indicate inhibition of nitrate by chloride. 7. Bumetanide inhibits nitrate secretion. 8. The data suggest that nitrate interacts with one of the two chloride sites of the chloride transporter.
Abstract-l.
INTRODlJmION
The rectal gland of the spiny dogfish, Squafus acanthius, secretes chloride against an electrochemical gradient by what is termed a secondary active process. The energy for this transport process is provided by Na-K-ATPase located in the basolateral cell membrane of the cells lining the rectal gland tubules. Chloride enters the cell across the basolateral membrane via a neutral Na + : K + :2Cl- cotransporter and enters the lumen following an electrical gradient that favors its efflux across the luminal border (Ernst and Van Rossum, 1982; Eveloff et al., 1978; Silva and Myers, 1986; Silva et al., 1977). In ion substitution experiments performed to determine the stoichiometry of the chloride transport system (Silva et al., 1983; Solomon et al., 1977) we observed that when chloride was replaced by nitrate in the perfusate the rate of secretion of sodium was substantially larger than that of chloride, suggesting that sodium was being transported together with nitrate. In the present series of experiments we explored this question more completely using a direct measurement of nitrate concentration in rectal gland secretion. MATERIALS AND
METHODS
Dogfish of either sex were taken by gill nets or by hook and line from Frenchman Bay, Maine, and kept in marine live cars until used, usually within 3 days of capture. Dogfish were killed by segmental transection of the spinal cord. The rectal glands were removed via an abdominal incision. *Present address: New England Deaconess Hospital, 110 Francis St, Boston, MA 02215, U.S.A.
Rectal glands were perfused using a method previously described by Silva et al. (1990). The rectal gland artery, vein and duct were catheterized with PE90 tubing. The glands were placed in a glass perfusion chamber maintained at 15°C with running sea water. The glands were perfused by gravity at a pressure of 40 mm Hg. The composition of the basic perfusion media was (in mM): Na, 280; Cl, 290; K, 5; bicarbonate, 8; phosphate, 1; Ca, 2.5; Mg, 3; sulfate, 0.5; urea, 350, glucose 5, pH 7.6 when gassed with 99% 0,/l% CO,. The nitrate perfusion medium contained nitrate instead of chloride, otherwise it was the same as the basic medium. Whenever partial substitutions of chloride with nitrate were made the final osmolality of the medium was kept constant by the addition of gluconate. Each experimental protocol started and ended with three consecutive 10min collection periods of perfusion with a medium of normal chloride composition. Changes in the composition of the perfusate were made after three or more collection periods. All the perfusion media contained theophylline 2.5 x low4 M and dibutyryl cyclic AMP 5 x 10m5M to stimulate rectal gland secretion (Stoff et al., 1977). Rectal gland secretion was collected in 1.5 ml conical centrifuge tubes or calibrated glass pipettes when volume was less than 200 pl/lO min. Chloride was measured by amperometric titration using a Buchler-Cotlove chloridometer. Sodium and potassium were measured with an 1L 343 flame photometer. Nitrate was measured by an ion selective electrode (Radiometer, Copenhagen) in a 1:20 dilution of duct fluid. The electrode was calibrated against a series of nitrate solutions of the same electrolyte composition as that of the rectal gland secretion. The slope of the electrode was -50.1 + 0.72 mV per decade. Concentrations of chloride
255
PATRICIOSILVA and FRANKLINH. EPSTEIN
256
Log
nitrate c~ncsntration(mhi)
Fig. 1. Relationship between the concentration of nitrate in solution and the reading in millivolts of an electrode sensi-
tive to nitrate at concentrations of chloride of 0 and 50 mM. The slope of the electrode was - 50.1 + 0.72 mV per decade. Chloride at a concentration of 50 mEq/l interfered with the measurement of nitrate only when the latter was less than 5 mEq/l.
0 to 50 mEq/l in the calibration solution resulted in an underestimate of the concentration of nitrate only when the latter was less than 5 mEq/l (Fig. 1). Since the concentration of nitrate in the diluted sample of recta1 gland secretion in the experiments reported was always higher than 5 mEq/l, the possibility of chloride interference with the nitrate electrode was negligible. The concentration of nitrate in the recta1 gland secretion, measured directly by the nitrate electrode, correlated well with that calculated from the difference between the concentration of sodium and chloride (slope 0.99 + 0.0, P -C0.001). It is therefore possible to estimate nitrate concentration reasonably accurately in experiments on nitrate excretion in which nitrate cannot be measured directly. Results are expressed as pEq/hr/g wet wt. The values are mean f SEM. Statistical significance was determined using Student’s t-test, paired t-test or analysis of variance wherever applicable. ranging
chloride concentration in the perfusate progressively and reciprocally replaces nitrate, the rate of secretion of nitrate first increases, to reach a maximum at chloride concentrations in the perfusate between 30 and 60 mM. Subsequently, at concentrations of chloride above 60 mM, the rate of secretion of nitrate is progressively inhibited down to levels lower than those seen when chloride is absent. The observation that chloride can first stimulate and then inhibit nitrate transport suggests that nitrate might interact with the Na + : K + : 2Cl~ carrier. As chloride in the perfusate is increased the rate of secretion of chloride increases in parallel. The rate of secretion of sodium, which in the absence of chloride, is similar to that of nitrate, also rises in proportion to the concentration of chloride in the perfusate but reaches a maximum long before that of the chloride secreted. Nitrate secretion at constant chloride concentration
from
The nature of the interaction between nitrate and chloride was investigated in a series of experiments in which the secretion of nitrate was measured at different concentrations of chloride in the perfusate (25, 50 and 100 mM) and nitrate concentrations in the perfusate increasing from 10 to 190 mM. In these experiments, chloride concentration in the perfusate was held constant, and gluconate served as a substitute for nitrate, to bring total anion concentration in the perfusing solution to 290mEq/l. The results are summarized in Fig. 3. The secretion of nitrate increases as a function of the concentration of nitrate in the perfusate at all concentrations of chloride studied. As noted earlier (Fig. 2), the secretion of
RESULTS
Nitrate secretion When nitrate replaced all chloride in the perfusate, stimulated glands secreted nitrate at 535 +_73 pEq/ hr/g (N = 9), about 30% of the rate at which those same glands secreted chloride (1841 f 170 pEq/hr/g, N = 9) when shark Ringer solution containing chloride as the chief anion was used to perfuse the same glands. Sodium secretion when nitrate was the sole anion in the perfusate was 538 f 64, N = 9. These experiments indicate that the recta1 gland has the capacity to transport nitrate, though at a lower rate than chloride. Isotonic replacement of nitrate by chloride Figure 2 shows the rate of secretion of nitrate, chloride and sodium by recta1 glands as a function of the concentration of chloride in the perfusate. As
or 0
,
,
,
,
,
50
100
‘50
200
250
Chloride
concentration
(mM)
in pertusate
Fig. 2. Secretion of chloride, sodium and nitrate by isolated perfused rectal glands as a function of chloride in the perfusate. Chloride was replaced reciprocally by nitrate. This figure summarizes the results of 21 perfusions. Each point represents values observed in at least four perfusions. No single perfusion was carried over the entire range of chloride concentrations. As the concentration of chloride in the perfusate is progressively increased the rate of secretion of nitrate increases from 535 + 73 at OmM chloride to 920.5 k 102.4 at 30 mM chloride, P < 0.01, remaining unchanged up to a concentration of 60 mM chloride. As the chloride concentration continues to increase the rate of nitrate secretion falls to a low of 185.2 k 36.4 at 190 mM chloride, P < 0.01 vs 0 or 60 mM chloride.
Nitrate secretion by rectal gland
251
Table I. Kinetic parameters of the secretion of nitrate by isolated Derfused rectal alands
Chloride (mM)
l/Y “S l/S
v “S v/s
s/v vs s
K+~Eqlhrl8) 50
593f 194 887f 203
733* 103 982 k 81
624 + 198
279
+ 194
388 k 65
404+
@W 2s
32.4
+ 0.9
+ 8.3
36.6
+ 8.2
50
15.9 + 1.2
16.0:
2.6
22.0
16.8
100
13.0 + 2.7
16.3
+ 5.4
28.7
+ 16.2
100 &,
;;
0
31.9
991
+ 189 142
.,.,.,.,_,.,_, 0
2s
SO
Nitrate
75
I00
concentration
125
(mM)
,so
175
in perfusate
Fig. 3. Secretion of nitrate by isolated perfused rectal glands at constant concentrations of chloride in the perfusate. Balance anion was gluconate. There were four perfusions in each group. The secretion of nitrate increases as a function of the concentration of nitrate in the perfusate at all concentrations of chloride studied, P < 0.007 by two-way analysis of variance. The rate of nitrate secretion was significantly different among the three groups by two-way analysis of variance, P < 0.004. Values are mean f SEM, N = 4 for all groups.
nitrate was highest at a concentration of chloride in the perfusate of 50mM and lowest at 100mM. Lineweaver-Burk plots of the data shown in Fig. 3 were linear only for the experiments at 25 and 50 mM (Fig. 4). The plot at 100 mM showed a convex curve (insert in Fig. 4) indicating negative cooperativity, again suggesting that chloride, at concentrations greater than 50 mM, inhibits nitrate transport. The estimated maximal secretion (V,,,.,) and MichaelisMenten constant (I&,) values for nitrate obtained from these plots are shown in Table 1. Similar estimates for V,,, and K,,, were obtained from V versus V/S plots and S/V versus S plots and are also shown in Table 1. It should be noted that the values for Vmaxcalculated from these plots are consistent with those observed at high concentrations of nitrate in the perfusate in Fig. 3.
Using the V,,,,, observed at the highest nitrate concentration in each experiment, Hill plots were constructed. The Hill plots are shown in Fig. 5. The Hill coefficients for the 25 and 50 mM chloride concentrations were 0.83 +_0.15, r = 0.96 and 1.1 +_0.20, r = 0.95, respectively, not significantly different from 1, suggesting that nitrate interacts with its transport system at a single site. The Hill coefficient at 100 mM chloride was 0.39 f 0.23, r = 0.71, indicating negative cooperativity and consistent with the observation that chloride at concentrations greater than 50mM inhibits nitrate secretion. Effect of bumetanide on nitrate secretion
Bumetanide, that inhibits chloride secretion in transporting epithelia by interacting with the Na + : K + : 2Cl- cotransporter, at a concentration of
-1.5
f==-yq
1
IS
,,.
0
1
1OOmMChhxide
::::
0.0
0.5
Log 0
l/Nitrate
concentratbn
25mMChbfida
(mM)
Fig. 4. Lineweaver-Burk plots of the data shown in the previous figure. The plots were linear only for the experiments at 25 and 50mM. The plot at 1OOmM (insert) showed a convex curve indicating negative cooperativity, again suggesting that chloride, at concentrations greater than 50 mM, inhibits nitrate transport. Values are mean & SEM.
1.0
Nitrate
1.5
2.0
concentration
2,s
3.0
(mM)
Fig. 5. Hill plots of the secretion of nitrate shown in Fig. 3. The Hill coefficients are 0.83 kO.15, 1.10&0.20 and 0.39 + 0.23 for 25, SO and 100 mM chloride, respectively. The Hill plots were constructed using the V,, observed at the highest nitrate concentration in each experiment. The Hill coefficients for the 25 and 50mM chloride concentrations were not significantly different from 1, suggesting that nitrate interacts with its transport system at a single site. The Hill coefficient at 100 mM chloride was less than 1, indicating negative cooperativity and consistent with the observation that chloride at concentrations greater than 50 mM inhibits nitrate secretion. Values are mean f SEM.
PATRICIOSILVAand FRANKLINH. EPSTEIN
258
Control
Bumetanide
Fig. 6. Effect of bumetanide 10m6M on the secretion of nitrate by isolated perfused rectal glands. Bumetanide inhibited nitrate secretion by as much as 60% at a nitrate concentration of 100 mM. Values are mean + SEM, N = 21 for control and 11 for bumetanide, P i 0.001. 10e6 M inhibited the secretion of nitrate. Bumetanide inhibited nitrate secretion by 60% at a nitrate concentration of 100mM (Fig. 6). Bumetanide inhibited nitrate secretion to the same extent that it inhibits chloride secretion in this gland (Palfrey er al., 1984).
DISCUSSION The preceding results demonstrate that the rectal gland of the shark, a chloride transporting epithelium, can also transport nitrate. The transport of nitrate has been observed in a number of different biological systems. Nitrate is taken up by an active process and serves as an electron acceptor in bacteria, (Lara et nl., 1987; Miki and Lin, 1975; Noji and Taniguchi, 1987); it can substitute for chloride in the sheep red cell (Lauf, 1983); it can be transported together with sodium in rat proximal and distal colon (Wurmli et al., 1987); competes with chloride exchange in Ehrlich ascites tumor cells (Hoffmann et al., 1979; Levinson, 1985); and germane to the present report, it can substitute for chloride in the duck salt-gland, an epithelial organ that transports chloride by a mechanism similar to that of the rectal gland (Ernst and Van Rossum, 1982). In yet another epithelial structure that transports chloride via the Na + : K + : 2Cl- cotransporter, the thick ascending limb of mammalian kidneys, nitrate can partially support transport-dependent oxygen consumption (Silva et al., 1987). The capacity for the transport of nitrate by the stimulated rectal gland is substantial. The rate of transport when nitrate was the main anion in the perfusate was 500 pEq/hr/g, or approximately a third of that of chloride and several times greater than that of the unstimulated transport of chloride. This high rate of transport suggests that the rectal gland cells have a transport system that can accommodate nitrate. Thus far only one transport system capable of transporting anions has been identified in the rectal gland cell, the Na + : K + : 2Cl_ cotransporter, which
has two independent binding sites for chloride of different, high and low, affinities. Although the rectal gland is rich in carbonic anhydrase and an anion exchange mechanism may well be present, inhibitors of anion exchange such as SITS have no effect on chloride secretion by the rectal gland (Solomon et al., 1978). The effect of chloride on the transport of nitrate offers an insight into the nature of the nitrate transport system. Chloride has a dual effect on the secretion of nitrate. At concentrations up to 50mM (Fig. 3) chloride clearly enhances nitrate secretion. At higher concentrations, on the other hand, chloride inhibits nitrate secretion. At concentrations up to 50 mM, chloride increases both the maximal secretion rate (I’,,,,,) and the affinity of the transport system for nitrate (Table 1). Increasing the concentration of chloride in the perfusate above 50 to 60 mM (Figs 2 and 3) decreases the maximal secretion rate. These changes are statistically significant to P < 0.001 by two-way analysis of variance. (The effect of increasing the concentration of chloride on the affinity of the transport system for nitrate cannot be evaluated properly because of the curvilinear nature of the plots.) The K,,, for nitrate is approximately a third of the average Km for chloride of approximately 50, the latter calculated from kinetic analysis assuming two independent chloride sites (Silva and Myers, 1986). This observation coupled with the Hill coefficient of close to 1 suggests that nitrate can successfully compete with chloride for the low affinity chloride site. This may explain the stimulatory effect of chloride at concentrations below its average K,,, and its inhibitory effect above that level. Although the application of kinetic analysis to electrolyte transport in a complex system is by nature hazardous, we suggest that it may provide useful insights in this case. The observation that nitrate interacts with its transport system at only one site, coupled with the dual effect of chloride on its secretion, suggests that the interaction of nitrate is limited to one of the two chloride binding sites of the Na + : K + : 2Cl_ cotransporter, probably the low affinity binding site. When chloride is present at low concentrations, it occupies the high affinity binding site and increases the rate of operation of the carrier. At high concentrations it inhibits nitrate secretion by displacing it from the other binding site. The concept that nitrate is transported by the Na’ : K+ :2Cl- cotransporter is supported further by the observation that bumetanide, that binds and blocks this membrane transporter (Forbush et al., 1992; Palfrey et al., 1984), inhibits the transport of nitrate by 60%. The demonstration that nitrate can be transported by the Na+ :K+ :2Cl- cotransporter in rectal gland cells suggests that this mechanism may serve to move nitrate and perhaps other anions across plasma membranes of a variety of cells possessing the capacity for chloride cotransport.
Nitrate
secretion
Acknowledgements-This research was supported by grants provided by the National Institutes of Health, NI-HDK18078, NIHHL35998 and NIEHS I-P30-ES03828, the National Science Foundation NSF, DCB-850826. The technical help of Mesdames M. Silva, K. Spokes and M. Taylor and Messrs. P. Silva Jr. and J. P. Silva is gratefully acknowledged.
REFERENCES
Ernst S. A. and Van Rossum G. D. V. (1982) Ions and energy metabolism in duck salt-gland: possible role of furosemide-sensitive co-transport of sodium and chloride. J. Physiol. 325, 333-352. Eveloff J., Kinne R., Kinne S. E., Murer H., Silva P., Epstein F. H., Stoff J. and Kinter W. B. (1978) Coupled sodium and chloride transport into plasma membrane vesicles prepared from dogfish rectal gland. PjZgers Arch 378, 87-92. Forbush B., Payne J., Xu J., Lytle C., Benz E., Forbush J., Zhu T. T. and Jones G. (1992) Distribution of the Na-KC1 cotransporter in the spiny dogfish, Squalus acanrhias. Bull. Mt. Desert Is. Biol. Lab. 31, 82-83. Hoffmann E. K., Simonsen L. 0. and Sjohlm C. (1979) Membrane potential, chloride exchange, and chloride conductance in Ehrlich mouse ascites tumour cells. J. Physiol. (Lond.), 2%, 61-84. Lara C., Romero J. M. and Guerrero M. G. (1987) Regulated nitrate transport in the cyanobacterium Anacystis nidulans. J. Bacreriol. 169, 437&4378. Lauf P. K. (1983) Thiol-dependent passive K/Cl transport in sheep red ceils: I. Dependence on chloride and external ions. J. Membrane Biol. 73, 237-245. Levinson C. (1985) Sodium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells. J. Membrane Biol. 87, 121-130. Miki K. and Lin E. C. (1975) Electron transport chain from glycerol 3-phosphate to nitrate in Escherichia coli. J. Bact. 124. 1288-1294.
CBN.4) 104/2-F
by rectal gland
259
Noji S. and Taniguchi S. (1987) Molecular oxygen controls nitrate transport of Escherichia coli nitrate-respiring cells. J. biol. Chem. 262, 944-9443. Palfrey H. C., Silva P. and Epstein F. H. (1984) Sensitivity of CAMP-stimulated salt secretion in shark rectal gland to loop diuretics. Am. J. Physiol. 246, C242-246. Silva P., Koenig B., Lear S., Eveloff J. and Kinne R. (1987) Dibutyryl cyclic AMP inhibits transport dependent Qo, in cells isolated from the rabbit medullary ascending limb. PJliigers Arch 409, 74-80. Silva P., Myers M., Landsberg A., Silva P. J., Silva P. J., Silva M., Brown R. and Epstein F. H. (1983) Stoichiometry of sodium chloride transport by the shark rectal gland. Bull. M.D.I.B.L. 23, 47-50. Silva P. and Myers M. A. (1986) Stoichiometry of sodium chloride transport by rectal gland of Squulus acanthias. Am. J. Physiol. 250, F5169. Silva P., Solomon R. and Epstein F. H. (1990) Shark Rectal Gland. Academic Press, San Diego. Silva P., Stoff J., Field M., Fine L., Forrest J. N. and Epstein F. H. (1977) Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport. Am. J. Physiol. 233, F298-306. Solomon R., Silva P.. Epstein J., Taylor M., Stevens A., Stoff J. S. and Epstein F. H. (1978) Further studies on the mechanism of chloride transport in the rectal gland of Squalus acanthias. Bull. M.D.I.B.L. 18, 13-16. Solomon R. J., Silva P., Stevens A., Epstein J., Stoff J. S., Spokes K. and Epstein F. H. (1977) Mechanism of chloride transport in the rectal gland of Squalus acanthias: ionic selectivity. Bull. M.D.I.B.L. 17, 5943. Staff J. S., Silva P., Field M., Forrest J., Stevens A. and Epstein F. H. (1977) Cyclic AMP regulation of active chloride transport in the rectal gland of marine elasmobranchs. J. exp. Zool. 199, 443-448. Wurmli R., Wolffram S. and Scharrer E. (1987) Influence of nitrate and nitrite on electrolyte transport by the rat small and large intestine. Comp. Biochem. Physiol. @?A, 127-129.
Camp. Biochem. Physiol. Vol. 104A,No. 2, pp. 255-259, 1993
0 1993Pergamon Press Ltd
Printed in Great Britain
SECRETION OF NITRATE BY RECTAL GLAND OF SQUALUS ACANTHIAS PATRICIO SILVA* and FRANKLIN H. EPSTEIN Departments of Medicine, New England Deaconess Hospital, Boston, MA 02215 and Beth Israel Hospital, Harvard Medical School, The Joslin Diabetes Center, The Harvard Center for the Study of Kidney Diseases, and The Mount Desert Island Biological Laboratory, Salsbury Cove, ME, U.S.A. (Received
19 May 1992; accepted 24 June 1992)
Rectal glands secrete nitrate at 30% of their capacity to secrete chloride. 2. Nitrate secretion is directly related to its concentration at constant chloride concentrations. 3. Chloride has a biphasic effect on nitrate secretion. 4. Hill coefficients at chloride < 100mM are eaual to 1. while at 100 mM indicate inhibition of nitrate by chloride. 6. Lineweaver-Burk plots at chloride < 100 indicate a single site, while at 100mM indicate inhibition of nitrate by chloride. 7. Bumetanide inhibits nitrate secretion. 8. The data suggest that nitrate interacts with one of the two chloride sites of the chloride transporter.
Abstract-l.
INTRODlJmION
The rectal gland of the spiny dogfish, Squafus acanthius, secretes chloride against an electrochemical gradient by what is termed a secondary active process. The energy for this transport process is provided by Na-K-ATPase located in the basolateral cell membrane of the cells lining the rectal gland tubules. Chloride enters the cell across the basolateral membrane via a neutral Na + : K + :2Cl- cotransporter and enters the lumen following an electrical gradient that favors its efflux across the luminal border (Ernst and Van Rossum, 1982; Eveloff et al., 1978; Silva and Myers, 1986; Silva et al., 1977). In ion substitution experiments performed to determine the stoichiometry of the chloride transport system (Silva et al., 1983; Solomon et al., 1977) we observed that when chloride was replaced by nitrate in the perfusate the rate of secretion of sodium was substantially larger than that of chloride, suggesting that sodium was being transported together with nitrate. In the present series of experiments we explored this question more completely using a direct measurement of nitrate concentration in rectal gland secretion. MATERIALS AND
METHODS
Dogfish of either sex were taken by gill nets or by hook and line from Frenchman Bay, Maine, and kept in marine live cars until used, usually within 3 days of capture. Dogfish were killed by segmental transection of the spinal cord. The rectal glands were removed via an abdominal incision. *Present address: New England Deaconess Hospital, 110 Francis St, Boston, MA 02215, U.S.A.
Rectal glands were perfused using a method previously described by Silva et al. (1990). The rectal gland artery, vein and duct were catheterized with PE90 tubing. The glands were placed in a glass perfusion chamber maintained at 15°C with running sea water. The glands were perfused by gravity at a pressure of 40 mm Hg. The composition of the basic perfusion media was (in mM): Na, 280; Cl, 290; K, 5; bicarbonate, 8; phosphate, 1; Ca, 2.5; Mg, 3; sulfate, 0.5; urea, 350, glucose 5, pH 7.6 when gassed with 99% 0,/l% CO,. The nitrate perfusion medium contained nitrate instead of chloride, otherwise it was the same as the basic medium. Whenever partial substitutions of chloride with nitrate were made the final osmolality of the medium was kept constant by the addition of gluconate. Each experimental protocol started and ended with three consecutive 10min collection periods of perfusion with a medium of normal chloride composition. Changes in the composition of the perfusate were made after three or more collection periods. All the perfusion media contained theophylline 2.5 x low4 M and dibutyryl cyclic AMP 5 x 10m5M to stimulate rectal gland secretion (Stoff et al., 1977). Rectal gland secretion was collected in 1.5 ml conical centrifuge tubes or calibrated glass pipettes when volume was less than 200 pl/lO min. Chloride was measured by amperometric titration using a Buchler-Cotlove chloridometer. Sodium and potassium were measured with an 1L 343 flame photometer. Nitrate was measured by an ion selective electrode (Radiometer, Copenhagen) in a 1:20 dilution of duct fluid. The electrode was calibrated against a series of nitrate solutions of the same electrolyte composition as that of the rectal gland secretion. The slope of the electrode was -50.1 + 0.72 mV per decade. Concentrations of chloride
255
PATRICIOSILVA and FRANKLINH. EPSTEIN
256
Log
nitrate c~ncsntration(mhi)
Fig. 1. Relationship between the concentration of nitrate in solution and the reading in millivolts of an electrode sensi-
tive to nitrate at concentrations of chloride of 0 and 50 mM. The slope of the electrode was - 50.1 + 0.72 mV per decade. Chloride at a concentration of 50 mEq/l interfered with the measurement of nitrate only when the latter was less than 5 mEq/l.
0 to 50 mEq/l in the calibration solution resulted in an underestimate of the concentration of nitrate only when the latter was less than 5 mEq/l (Fig. 1). Since the concentration of nitrate in the diluted sample of recta1 gland secretion in the experiments reported was always higher than 5 mEq/l, the possibility of chloride interference with the nitrate electrode was negligible. The concentration of nitrate in the recta1 gland secretion, measured directly by the nitrate electrode, correlated well with that calculated from the difference between the concentration of sodium and chloride (slope 0.99 + 0.0, P -C0.001). It is therefore possible to estimate nitrate concentration reasonably accurately in experiments on nitrate excretion in which nitrate cannot be measured directly. Results are expressed as pEq/hr/g wet wt. The values are mean f SEM. Statistical significance was determined using Student’s t-test, paired t-test or analysis of variance wherever applicable. ranging
chloride concentration in the perfusate progressively and reciprocally replaces nitrate, the rate of secretion of nitrate first increases, to reach a maximum at chloride concentrations in the perfusate between 30 and 60 mM. Subsequently, at concentrations of chloride above 60 mM, the rate of secretion of nitrate is progressively inhibited down to levels lower than those seen when chloride is absent. The observation that chloride can first stimulate and then inhibit nitrate transport suggests that nitrate might interact with the Na + : K + : 2Cl~ carrier. As chloride in the perfusate is increased the rate of secretion of chloride increases in parallel. The rate of secretion of sodium, which in the absence of chloride, is similar to that of nitrate, also rises in proportion to the concentration of chloride in the perfusate but reaches a maximum long before that of the chloride secreted. Nitrate secretion at constant chloride concentration
from
The nature of the interaction between nitrate and chloride was investigated in a series of experiments in which the secretion of nitrate was measured at different concentrations of chloride in the perfusate (25, 50 and 100 mM) and nitrate concentrations in the perfusate increasing from 10 to 190 mM. In these experiments, chloride concentration in the perfusate was held constant, and gluconate served as a substitute for nitrate, to bring total anion concentration in the perfusing solution to 290mEq/l. The results are summarized in Fig. 3. The secretion of nitrate increases as a function of the concentration of nitrate in the perfusate at all concentrations of chloride studied. As noted earlier (Fig. 2), the secretion of
RESULTS
Nitrate secretion When nitrate replaced all chloride in the perfusate, stimulated glands secreted nitrate at 535 +_73 pEq/ hr/g (N = 9), about 30% of the rate at which those same glands secreted chloride (1841 f 170 pEq/hr/g, N = 9) when shark Ringer solution containing chloride as the chief anion was used to perfuse the same glands. Sodium secretion when nitrate was the sole anion in the perfusate was 538 f 64, N = 9. These experiments indicate that the recta1 gland has the capacity to transport nitrate, though at a lower rate than chloride. Isotonic replacement of nitrate by chloride Figure 2 shows the rate of secretion of nitrate, chloride and sodium by recta1 glands as a function of the concentration of chloride in the perfusate. As
or 0
,
,
,
,
,
50
100
‘50
200
250
Chloride
concentration
(mM)
in pertusate
Fig. 2. Secretion of chloride, sodium and nitrate by isolated perfused rectal glands as a function of chloride in the perfusate. Chloride was replaced reciprocally by nitrate. This figure summarizes the results of 21 perfusions. Each point represents values observed in at least four perfusions. No single perfusion was carried over the entire range of chloride concentrations. As the concentration of chloride in the perfusate is progressively increased the rate of secretion of nitrate increases from 535 + 73 at OmM chloride to 920.5 k 102.4 at 30 mM chloride, P < 0.01, remaining unchanged up to a concentration of 60 mM chloride. As the chloride concentration continues to increase the rate of nitrate secretion falls to a low of 185.2 k 36.4 at 190 mM chloride, P < 0.01 vs 0 or 60 mM chloride.
Nitrate secretion by rectal gland
251
Table I. Kinetic parameters of the secretion of nitrate by isolated Derfused rectal alands
Chloride (mM)
l/Y “S l/S
v “S v/s
s/v vs s
K+~Eqlhrl8) 50
593f 194 887f 203
733* 103 982 k 81
624 + 198
279
+ 194
388 k 65
404+
@W 2s
32.4
+ 0.9
+ 8.3
36.6
+ 8.2
50
15.9 + 1.2
16.0:
2.6
22.0
16.8
100
13.0 + 2.7
16.3
+ 5.4
28.7
+ 16.2
100 &,
;;
0
31.9
991
+ 189 142
.,.,.,.,_,.,_, 0
2s
SO
Nitrate
75
I00
concentration
125
(mM)
,so
175
in perfusate
Fig. 3. Secretion of nitrate by isolated perfused rectal glands at constant concentrations of chloride in the perfusate. Balance anion was gluconate. There were four perfusions in each group. The secretion of nitrate increases as a function of the concentration of nitrate in the perfusate at all concentrations of chloride studied, P < 0.007 by two-way analysis of variance. The rate of nitrate secretion was significantly different among the three groups by two-way analysis of variance, P < 0.004. Values are mean f SEM, N = 4 for all groups.
nitrate was highest at a concentration of chloride in the perfusate of 50mM and lowest at 100mM. Lineweaver-Burk plots of the data shown in Fig. 3 were linear only for the experiments at 25 and 50 mM (Fig. 4). The plot at 100 mM showed a convex curve (insert in Fig. 4) indicating negative cooperativity, again suggesting that chloride, at concentrations greater than 50 mM, inhibits nitrate transport. The estimated maximal secretion (V,,,.,) and MichaelisMenten constant (I&,) values for nitrate obtained from these plots are shown in Table 1. Similar estimates for V,,, and K,,, were obtained from V versus V/S plots and S/V versus S plots and are also shown in Table 1. It should be noted that the values for Vmaxcalculated from these plots are consistent with those observed at high concentrations of nitrate in the perfusate in Fig. 3.
Using the V,,,,, observed at the highest nitrate concentration in each experiment, Hill plots were constructed. The Hill plots are shown in Fig. 5. The Hill coefficients for the 25 and 50 mM chloride concentrations were 0.83 +_0.15, r = 0.96 and 1.1 +_0.20, r = 0.95, respectively, not significantly different from 1, suggesting that nitrate interacts with its transport system at a single site. The Hill coefficient at 100 mM chloride was 0.39 f 0.23, r = 0.71, indicating negative cooperativity and consistent with the observation that chloride at concentrations greater than 50mM inhibits nitrate secretion. Effect of bumetanide on nitrate secretion
Bumetanide, that inhibits chloride secretion in transporting epithelia by interacting with the Na + : K + : 2Cl- cotransporter, at a concentration of
-1.5
f==-yq
1
IS
,,.
0
1
1OOmMChhxide
::::
0.0
0.5
Log 0
l/Nitrate
concentratbn
25mMChbfida
(mM)
Fig. 4. Lineweaver-Burk plots of the data shown in the previous figure. The plots were linear only for the experiments at 25 and 50mM. The plot at 1OOmM (insert) showed a convex curve indicating negative cooperativity, again suggesting that chloride, at concentrations greater than 50 mM, inhibits nitrate transport. Values are mean & SEM.
1.0
Nitrate
1.5
2.0
concentration
2,s
3.0
(mM)
Fig. 5. Hill plots of the secretion of nitrate shown in Fig. 3. The Hill coefficients are 0.83 kO.15, 1.10&0.20 and 0.39 + 0.23 for 25, SO and 100 mM chloride, respectively. The Hill plots were constructed using the V,, observed at the highest nitrate concentration in each experiment. The Hill coefficients for the 25 and 50mM chloride concentrations were not significantly different from 1, suggesting that nitrate interacts with its transport system at a single site. The Hill coefficient at 100 mM chloride was less than 1, indicating negative cooperativity and consistent with the observation that chloride at concentrations greater than 50 mM inhibits nitrate secretion. Values are mean f SEM.
PATRICIOSILVAand FRANKLINH. EPSTEIN
258
Control
Bumetanide
Fig. 6. Effect of bumetanide 10m6M on the secretion of nitrate by isolated perfused rectal glands. Bumetanide inhibited nitrate secretion by as much as 60% at a nitrate concentration of 100 mM. Values are mean + SEM, N = 21 for control and 11 for bumetanide, P i 0.001. 10e6 M inhibited the secretion of nitrate. Bumetanide inhibited nitrate secretion by 60% at a nitrate concentration of 100mM (Fig. 6). Bumetanide inhibited nitrate secretion to the same extent that it inhibits chloride secretion in this gland (Palfrey er al., 1984).
DISCUSSION The preceding results demonstrate that the rectal gland of the shark, a chloride transporting epithelium, can also transport nitrate. The transport of nitrate has been observed in a number of different biological systems. Nitrate is taken up by an active process and serves as an electron acceptor in bacteria, (Lara et nl., 1987; Miki and Lin, 1975; Noji and Taniguchi, 1987); it can substitute for chloride in the sheep red cell (Lauf, 1983); it can be transported together with sodium in rat proximal and distal colon (Wurmli et al., 1987); competes with chloride exchange in Ehrlich ascites tumor cells (Hoffmann et al., 1979; Levinson, 1985); and germane to the present report, it can substitute for chloride in the duck salt-gland, an epithelial organ that transports chloride by a mechanism similar to that of the rectal gland (Ernst and Van Rossum, 1982). In yet another epithelial structure that transports chloride via the Na + : K + : 2Cl- cotransporter, the thick ascending limb of mammalian kidneys, nitrate can partially support transport-dependent oxygen consumption (Silva et al., 1987). The capacity for the transport of nitrate by the stimulated rectal gland is substantial. The rate of transport when nitrate was the main anion in the perfusate was 500 pEq/hr/g, or approximately a third of that of chloride and several times greater than that of the unstimulated transport of chloride. This high rate of transport suggests that the rectal gland cells have a transport system that can accommodate nitrate. Thus far only one transport system capable of transporting anions has been identified in the rectal gland cell, the Na + : K + : 2Cl_ cotransporter, which
has two independent binding sites for chloride of different, high and low, affinities. Although the rectal gland is rich in carbonic anhydrase and an anion exchange mechanism may well be present, inhibitors of anion exchange such as SITS have no effect on chloride secretion by the rectal gland (Solomon et al., 1978). The effect of chloride on the transport of nitrate offers an insight into the nature of the nitrate transport system. Chloride has a dual effect on the secretion of nitrate. At concentrations up to 50mM (Fig. 3) chloride clearly enhances nitrate secretion. At higher concentrations, on the other hand, chloride inhibits nitrate secretion. At concentrations up to 50 mM, chloride increases both the maximal secretion rate (I’,,,,,) and the affinity of the transport system for nitrate (Table 1). Increasing the concentration of chloride in the perfusate above 50 to 60 mM (Figs 2 and 3) decreases the maximal secretion rate. These changes are statistically significant to P < 0.001 by two-way analysis of variance. (The effect of increasing the concentration of chloride on the affinity of the transport system for nitrate cannot be evaluated properly because of the curvilinear nature of the plots.) The K,,, for nitrate is approximately a third of the average Km for chloride of approximately 50, the latter calculated from kinetic analysis assuming two independent chloride sites (Silva and Myers, 1986). This observation coupled with the Hill coefficient of close to 1 suggests that nitrate can successfully compete with chloride for the low affinity chloride site. This may explain the stimulatory effect of chloride at concentrations below its average K,,, and its inhibitory effect above that level. Although the application of kinetic analysis to electrolyte transport in a complex system is by nature hazardous, we suggest that it may provide useful insights in this case. The observation that nitrate interacts with its transport system at only one site, coupled with the dual effect of chloride on its secretion, suggests that the interaction of nitrate is limited to one of the two chloride binding sites of the Na + : K + : 2Cl_ cotransporter, probably the low affinity binding site. When chloride is present at low concentrations, it occupies the high affinity binding site and increases the rate of operation of the carrier. At high concentrations it inhibits nitrate secretion by displacing it from the other binding site. The concept that nitrate is transported by the Na’ : K+ :2Cl- cotransporter is supported further by the observation that bumetanide, that binds and blocks this membrane transporter (Forbush et al., 1992; Palfrey et al., 1984), inhibits the transport of nitrate by 60%. The demonstration that nitrate can be transported by the Na+ :K+ :2Cl- cotransporter in rectal gland cells suggests that this mechanism may serve to move nitrate and perhaps other anions across plasma membranes of a variety of cells possessing the capacity for chloride cotransport.
Nitrate
secretion
Acknowledgements-This research was supported by grants provided by the National Institutes of Health, NI-HDK18078, NIHHL35998 and NIEHS I-P30-ES03828, the National Science Foundation NSF, DCB-850826. The technical help of Mesdames M. Silva, K. Spokes and M. Taylor and Messrs. P. Silva Jr. and J. P. Silva is gratefully acknowledged.
REFERENCES
Ernst S. A. and Van Rossum G. D. V. (1982) Ions and energy metabolism in duck salt-gland: possible role of furosemide-sensitive co-transport of sodium and chloride. J. Physiol. 325, 333-352. Eveloff J., Kinne R., Kinne S. E., Murer H., Silva P., Epstein F. H., Stoff J. and Kinter W. B. (1978) Coupled sodium and chloride transport into plasma membrane vesicles prepared from dogfish rectal gland. PjZgers Arch 378, 87-92. Forbush B., Payne J., Xu J., Lytle C., Benz E., Forbush J., Zhu T. T. and Jones G. (1992) Distribution of the Na-KC1 cotransporter in the spiny dogfish, Squalus acanrhias. Bull. Mt. Desert Is. Biol. Lab. 31, 82-83. Hoffmann E. K., Simonsen L. 0. and Sjohlm C. (1979) Membrane potential, chloride exchange, and chloride conductance in Ehrlich mouse ascites tumour cells. J. Physiol. (Lond.), 2%, 61-84. Lara C., Romero J. M. and Guerrero M. G. (1987) Regulated nitrate transport in the cyanobacterium Anacystis nidulans. J. Bacreriol. 169, 437&4378. Lauf P. K. (1983) Thiol-dependent passive K/Cl transport in sheep red ceils: I. Dependence on chloride and external ions. J. Membrane Biol. 73, 237-245. Levinson C. (1985) Sodium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells. J. Membrane Biol. 87, 121-130. Miki K. and Lin E. C. (1975) Electron transport chain from glycerol 3-phosphate to nitrate in Escherichia coli. J. Bact. 124. 1288-1294.
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Noji S. and Taniguchi S. (1987) Molecular oxygen controls nitrate transport of Escherichia coli nitrate-respiring cells. J. biol. Chem. 262, 944-9443. Palfrey H. C., Silva P. and Epstein F. H. (1984) Sensitivity of CAMP-stimulated salt secretion in shark rectal gland to loop diuretics. Am. J. Physiol. 246, C242-246. Silva P., Koenig B., Lear S., Eveloff J. and Kinne R. (1987) Dibutyryl cyclic AMP inhibits transport dependent Qo, in cells isolated from the rabbit medullary ascending limb. PJliigers Arch 409, 74-80. Silva P., Myers M., Landsberg A., Silva P. J., Silva P. J., Silva M., Brown R. and Epstein F. H. (1983) Stoichiometry of sodium chloride transport by the shark rectal gland. Bull. M.D.I.B.L. 23, 47-50. Silva P. and Myers M. A. (1986) Stoichiometry of sodium chloride transport by rectal gland of Squulus acanthias. Am. J. Physiol. 250, F5169. Silva P., Solomon R. and Epstein F. H. (1990) Shark Rectal Gland. Academic Press, San Diego. Silva P., Stoff J., Field M., Fine L., Forrest J. N. and Epstein F. H. (1977) Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport. Am. J. Physiol. 233, F298-306. Solomon R., Silva P.. Epstein J., Taylor M., Stevens A., Stoff J. S. and Epstein F. H. (1978) Further studies on the mechanism of chloride transport in the rectal gland of Squalus acanthias. Bull. M.D.I.B.L. 18, 13-16. Solomon R. J., Silva P., Stevens A., Epstein J., Stoff J. S., Spokes K. and Epstein F. H. (1977) Mechanism of chloride transport in the rectal gland of Squalus acanthias: ionic selectivity. Bull. M.D.I.B.L. 17, 5943. Staff J. S., Silva P., Field M., Forrest J., Stevens A. and Epstein F. H. (1977) Cyclic AMP regulation of active chloride transport in the rectal gland of marine elasmobranchs. J. exp. Zool. 199, 443-448. Wurmli R., Wolffram S. and Scharrer E. (1987) Influence of nitrate and nitrite on electrolyte transport by the rat small and large intestine. Comp. Biochem. Physiol. @?A, 127-129.