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45Ca discrimination by the active shell gland of the fowl
Comp. Biochem. Physiol., 1973, Vol. 44A, pp. 201 to 205. Pergamon Press. Printed in Great Britain
8sSr/~SCa DISCRIMINATION BY THE ACTIVE SHELL GLAND OF THE FOWL K. S I M K I S S t, S. H U R W I T Z 2 and A. BAR S 1Department of Zoology and Comparative Physiology, Queen Mary College, London E.1. ; and 2Animal Science, Volcani Centre, P.O.B.6, Bet Dagan, Israel (Received 23 March 1972) A mixture of ssSr and 45Ca was injected into the veins of laying hens and the 8SSr/45Ca ratio of plasma, shell gland mucosa and eggsheU were measured. 2. There is a discrimination against strontium in the body and the 85Sr/ 45Ca ratio of the plasma rises progressively. 3. The discrimination against strontium is only 1"5 times that of calcium in the passage from plasma to eggshell 4. Calcium binding protein is present in the shell gland but its formation constant for calcium is about seven times that of strontium and it is not, therefore, considered to play a central part in the transfer of cations during eggshell calcification. Abstract--1.
INTRODUCTION THE MOVEMENTof calcium within and across cells has attracted increasing interest in the last decade. Various types of cells and their organelles have been studied but no generally accepted theory has yet been proposed. It is not surprising, therefore, that great interest was shown in the discovery by Wasserman & Taylor (1963) of a protein with a high calcium binding ability in the intestinal mucosa of chickens. T h e occurrence of this calcium binding protein (CaBP) both qualitatively and quantitatively correlated well with calcium transport under a wide variety of circumstances (Wasserman, 1968). As a result of this, it has been suggested that CaBP may be involved in some mechanism of calcium transport across cellular membranes. If this were the case, then any simple concept based upon such a principle would be expected to involve a high discrimination against strontium since Wasserman et al. (1968) obtained the following formation constants for CaBP Ca 2.6 × 105 M -1, Sr 3 . 9 × 1 0 4 M -t, Ba 5 . 8 x 1 0 8 M -1. The shell gland of the oviduct of the domestic fowl transports large quantities of calcium (2 g Ca in 20 hr) during eggshell formation. This tissue contains CaBP (Corradino et al., 1968) but the evidence to date: indicates that it shows very little Sr/Ca discrimination (Drori et al., 1964). 201
202
K. SIMKISS, S. HURWITZAND A. BAR
T h e following experiments were, therefore, undertaken to investigate the ssSr/ 45Ca discrimination of the shell gland and its possible relationship with CaBP.
MATERIALS AND METHODS A stock solution of S5Sr and 4zCa was prepared of approximately 30/zCi/ml of each isotope and the molar ratio of St/Ca was made up to unity by the addition of SrC12. Single doses of 1 ml were used for intravenous injections into three fowls (Nos. 43, 44 and 45) about 5 hr after oviposition when the egg could just be felt entering the shell gland. Blood samples were taken from the opposite wing vein 3"5, 5"25 and 9 hr after injection and the birds were then killed by dislocation of the neck. In two other birds (Nos. 37 and 38) the isotopes were injected 5 hr after the egg entered the shell gland and blood samples were taken at the corresponding times of 5"25 and 9 hr of calcification after which the birds were also killed. The eggshells were removed from the oviducts at the time of death, washed and dried. Samples of mucosa tissue were separated from the shell gland, carefully blotted and homogenized. The eggshells were ashed at 800°C for 3 hr and then dissolved in hydrochloric acid. Weighed samples of homogenized shell gland mucosa and of plasma were digested with nitric acid and hydrogen peroxide, brought to dryness and dissolved in hydrochloric acid. These were counted for 45Ca in a Tri-Carb liquid scintillation counter and for ssSr in an automatic gamma well counter. Corrections were made for szSr counts in the a~Ca channel. Other samples of the same shell gland homogenates were heated to 60°C for 5 min to give a crude supernatant preparation of CaBP. This was counted for 45Ca and s~Sr and also used for electrophoretic demonstration of CaBP. In another experiment plasma samples taken from a laying hen and from a cockerel were treated with the stock solution of ssSr and asCa isotopes and then used to prepare an ultrafiltrate through Visking tubing at various times up to 3 hr as previously described (Hurwitz & Bar, 1968). The ultrafiltrate was counted for ssSr and *sCa. Crude CaBP preparations of supernatants from intestinal and shell gland mucosa homogenates were subjected to equilibrium dialysis with the isotope mixture for 4 hr and the distribution of the two isotopes was measured on both sides of the membrane. RESULTS AND DISCUSSION T h e ssSr/aSCa ratio of isotopes in the injected dose was measured and a correction factor applied so that it could be regarded as unity throughout the experiments. T h e ratio of 858r/45Ca in the blood plasma was measured as a function of the time after the injection and is given in Table 1 together with the ratios found in the shell gland mucosa and the eggshell at the time of death. It is apparent that the S5Sr/45Ca ratio in the plasma increases with time suggesting a preferential removal of asCa from the pool. T h e shell gland mucosa has a low ratio indicating that it shows considerable discrimination against strontium in the uptake of isotopes while the eggshell itself contains almost equal quantities of radioactive material. I n order to make more detailed estimates of the discrimination shown between the isotopes it is necessary to estimate the ionic ratios of the material that is presented to the tissues during the period of the experiment. T h e ratios of the isotopes in the blood change with time, since the organs of the body continually discriminate against strontium, and in order to compensate for this the plasma ratios which were determined experimentally were weighted by the time between sampling and a
ssSr/45Sr DISCRIMINATION BY FOWL SHELL GLAND
203
TABLE 1--85Sr/45Ca RATIOS IN VARIOUS TISSUES EXPRESSED AS ~/o DOSE/g × 103 r
Plasma values (hr after start of calcification) No.
0
3"50
5"00
5"25
9"00
Shell gland mucosa
37 38 43 44 45
--1"0* 1"0" 1"0"
--1"324 1"387 1"174
1"0* 1"0" ----
1"173 1"120 1"537 1"600 1,251
1"737 1"554 1'730 1"996 1"400
0"493 0"556 0"445 0"428 0"397
Bird
Eggshell 1"064 0"954 0"919 0"963 0.901
**Note t h a t all data are corrected to give e q u i v a l e n t values of ssSr a n d 45Ca in t h e stock solution. T h e ratio (ssSr/4~Ca) tissue/(S6Sr/*SCa) dose is therefore always
unity for the initial injections into plasma. mean value was then calculated. These values are for total SSSr/45Ca in the plasma and in order to convert them to ionic ratios it is necessary to estimate the additional effects of binding by plasma proteins. This effect was evident from the in vitro plasma ultrafiltration results which showed a filtrate ratio of 1.1 : 1 for SSSr/45Ca, independent of the time of equilibration in these experiments. As the ultrafiltration sample initially had a uniform ratio of isotopes, the results indicate a selective binding of calcium by the plasma proteins. Weighted mean values of total plasma sSSr/45Ca ratios were therefore multiplied by 1.1 to give the estimated mean ionic ratios shown in Table 2. TABLE 2--O.R. SHELL/PLASMA CALCULATED FROM ESTIMATES OF IONIC s5Sr/'SCa RATIO IN PLASMA
Bird No. 37 38 43 44 45
Shell
Estimated ionic ssSr/45Ca (weighted mean plasma × ultrafiltrated discrimination)
O.R. shell/plasma
1.06 0.95 0.92 0.96 0-90
1.59 1.46 1.73 1.87 1.45
0.66 0.65 0.60 0.60 0.65
T h e discrimination ratio of a tissue between two isotopes is normally expressed in physiological studies as the observed ratio (O.R.) of sample/precursor. T h e O.R. shell/plasma is shown in Table 2 for the two groups of birds. T h e values obtained are remarkably consistent at about 0.6 even for different time periods and stages of calcification. This indicates that about six strontium atoms are deposited upon the shell for every ten calcium atoms when the two isotopes are presented equally to the shell gland. If one tries to analyse the phenomenon further, however, by
204
K.
SIMKISS,S.
HURWlTZ AND A. BAR
determining discrimination at the plasma/sheU gland and shell gland/eggshell regions then the results are more complex. Shell gland mucosa contains about 40 per cent extracellular fluid (Sirnkiss, 1969; Hurwitz & Cohen, unpublished data) and this fluid presumably has the same SaSr/45Ca ratio as plasma ultrafiltrates. The total tissue ratio can therefore be divided to give extraceUular and intracellular isotope ratios. On this basis the intracellular fluid of the mucosal cells has a snSr/ 45Ca ratio of approximately 0.35 to 0.25 giving a discrimination ratio of about 5 against strontium at the plasma/mucosa region and of about 3 against calcium at the mucosa/eggshell surface. A discrimination ratio of about 5 against strontium at the plasma/mucosa region is the sort of result that CaBP would produce although the ratio is rather small for the known formation constants. If one accepts this result at its face value, however, one is left with a physiologically unlikely phenomenon of a preferential secretion of strontium at the mucosa/eggshell surface and it is difficult to explain this in terms of removing bound ions from CaBP. We have ignored the effects of crystallization upon the discrimination against strontium at the site of eggshell formation but these would tend to increase the Sr/Ca discrimination factor. It appears more likely, therefore, that the intracellular ratios are of doubtful significance and merely reflect the accumulation of bound calcium in the mucosal cells. Evidence for such a slowly exchanging calcium fraction within the cells is obtained from the decrease in ssSr/45Ca ratios of mucosa with time (Table 1, compare birds 37 and 38 with 43, 44 and 45). Previous studies of SSSr/aSCa discrimination in the laying bird obtained values of 0-927 between blood and eggshell (Drori et aL, 1964). This result, however, was based purely upon analyses of incorporated dose into the shell and it is very close to our own value of 0.96 (Table 1). In isolation, however, this figure underestimates the discrimination since it overlooks the continual rise in ssSr/45Ca ratios in the plasma caused by the overall discrimination of the body against strontium. It is in fact likely that in the system being studied, the rise in the SSSr/45Ca ratio of the plasma is at least partly caused by the activity of the shell gland itself, so that birds 37 and 38 which were injected later in calcification show initially more rapid rises in plasma SSSr/aSCa ratios than birds number 43, 44 and 45 which were just starting to calcify (Table 1). This factor is, in fact, probably the main variable in calculating the mean plasma ratios of isotopes even on a time weighted basis. The mucosa which was studied in these experiments was shown to contain a strong band of CaBP when subjected to electrophoresis. Equilibrium dialysis of the same material indicated that there was good evidence for 4~Ca binding but no detectable binding of s5Sr in these samples. These results are in keeping with the known formation constants of CaBP but are not capable of explaining the poor O.R. plasma/shell found in these experiments. This suggests that CaBP does not seem to play a central role in the transfer of calcium across the shell gland since the protein has a ssSr/45Ca discrimination factor of about 7 whereas the plasma/eggshell system in vivo has an overall discrimination of only about 1.5 in favour of calcium.
a s S r / 4 ~ S r DISCRIMINATION BY F O W L SHELL GLAND
205
Acknowledgements--One of us (K. S.) would like to thank the Volcani Institute for an invitation to visit it during its 50th Anniversary Celebrations and for the use of its facilities during this work. T h e research was assisted by a grant for the visit from the Royal Society/ Israeli Exchange Programme. REFERENCES CORRADINO R. A., WASSERMANR. H., PUBLOS M. H. & CHANG S. I. (1968) Vitamin D~ induction of a calcium-binding protein in the uterus of the laying hen. Arch. Biochem. 125, 378-380. DRORI D., VOLCANI R., FEtCE T., SHALMONE. & PASSYN. (1964) Factors affecting the Ca 4~ and Sr a9 transfer in the laying hen. Poult. Sci. 43, 486-491. HURWITZ S. & BAR A. (1968) Calcium exchange in plasma of the fowl Biochem. biophys. Acta 156, 389-393. SIMKISS K. (1969) Intracellular p H during calcification. Biochem. ft. 111,647-652. WASSEaMAN R. H. (1968) Calcium transport by the intestine: a model and comment on Vitamin D action. Calc. Tiss. Res. 2, 301-333. WASSERMANR. H., CORRADINOR. A. & TAYLOR A. N. (1968) Vitamin D dependent calcium binding protein: purification and some properties, ft. biol. Chem. 243, 3978-3986. WASSERMANR. H. & TAYLOR A. N. (1963) Vitamin D inhibition of radiocalcium binding b y chick intestinal homogenates. Nature, Lond. 198, 30-32.
Key Word Index----ssSr; ~Ca; shell gland; calcium binding protein; Gallus domesticus.
8sSr/~SCa DISCRIMINATION BY THE ACTIVE SHELL GLAND OF THE FOWL K. S I M K I S S t, S. H U R W I T Z 2 and A. BAR S 1Department of Zoology and Comparative Physiology, Queen Mary College, London E.1. ; and 2Animal Science, Volcani Centre, P.O.B.6, Bet Dagan, Israel (Received 23 March 1972) A mixture of ssSr and 45Ca was injected into the veins of laying hens and the 8SSr/45Ca ratio of plasma, shell gland mucosa and eggsheU were measured. 2. There is a discrimination against strontium in the body and the 85Sr/ 45Ca ratio of the plasma rises progressively. 3. The discrimination against strontium is only 1"5 times that of calcium in the passage from plasma to eggshell 4. Calcium binding protein is present in the shell gland but its formation constant for calcium is about seven times that of strontium and it is not, therefore, considered to play a central part in the transfer of cations during eggshell calcification. Abstract--1.
INTRODUCTION THE MOVEMENTof calcium within and across cells has attracted increasing interest in the last decade. Various types of cells and their organelles have been studied but no generally accepted theory has yet been proposed. It is not surprising, therefore, that great interest was shown in the discovery by Wasserman & Taylor (1963) of a protein with a high calcium binding ability in the intestinal mucosa of chickens. T h e occurrence of this calcium binding protein (CaBP) both qualitatively and quantitatively correlated well with calcium transport under a wide variety of circumstances (Wasserman, 1968). As a result of this, it has been suggested that CaBP may be involved in some mechanism of calcium transport across cellular membranes. If this were the case, then any simple concept based upon such a principle would be expected to involve a high discrimination against strontium since Wasserman et al. (1968) obtained the following formation constants for CaBP Ca 2.6 × 105 M -1, Sr 3 . 9 × 1 0 4 M -t, Ba 5 . 8 x 1 0 8 M -1. The shell gland of the oviduct of the domestic fowl transports large quantities of calcium (2 g Ca in 20 hr) during eggshell formation. This tissue contains CaBP (Corradino et al., 1968) but the evidence to date: indicates that it shows very little Sr/Ca discrimination (Drori et al., 1964). 201
202
K. SIMKISS, S. HURWITZAND A. BAR
T h e following experiments were, therefore, undertaken to investigate the ssSr/ 45Ca discrimination of the shell gland and its possible relationship with CaBP.
MATERIALS AND METHODS A stock solution of S5Sr and 4zCa was prepared of approximately 30/zCi/ml of each isotope and the molar ratio of St/Ca was made up to unity by the addition of SrC12. Single doses of 1 ml were used for intravenous injections into three fowls (Nos. 43, 44 and 45) about 5 hr after oviposition when the egg could just be felt entering the shell gland. Blood samples were taken from the opposite wing vein 3"5, 5"25 and 9 hr after injection and the birds were then killed by dislocation of the neck. In two other birds (Nos. 37 and 38) the isotopes were injected 5 hr after the egg entered the shell gland and blood samples were taken at the corresponding times of 5"25 and 9 hr of calcification after which the birds were also killed. The eggshells were removed from the oviducts at the time of death, washed and dried. Samples of mucosa tissue were separated from the shell gland, carefully blotted and homogenized. The eggshells were ashed at 800°C for 3 hr and then dissolved in hydrochloric acid. Weighed samples of homogenized shell gland mucosa and of plasma were digested with nitric acid and hydrogen peroxide, brought to dryness and dissolved in hydrochloric acid. These were counted for 45Ca in a Tri-Carb liquid scintillation counter and for ssSr in an automatic gamma well counter. Corrections were made for szSr counts in the a~Ca channel. Other samples of the same shell gland homogenates were heated to 60°C for 5 min to give a crude supernatant preparation of CaBP. This was counted for 45Ca and s~Sr and also used for electrophoretic demonstration of CaBP. In another experiment plasma samples taken from a laying hen and from a cockerel were treated with the stock solution of ssSr and asCa isotopes and then used to prepare an ultrafiltrate through Visking tubing at various times up to 3 hr as previously described (Hurwitz & Bar, 1968). The ultrafiltrate was counted for ssSr and *sCa. Crude CaBP preparations of supernatants from intestinal and shell gland mucosa homogenates were subjected to equilibrium dialysis with the isotope mixture for 4 hr and the distribution of the two isotopes was measured on both sides of the membrane. RESULTS AND DISCUSSION T h e ssSr/aSCa ratio of isotopes in the injected dose was measured and a correction factor applied so that it could be regarded as unity throughout the experiments. T h e ratio of 858r/45Ca in the blood plasma was measured as a function of the time after the injection and is given in Table 1 together with the ratios found in the shell gland mucosa and the eggshell at the time of death. It is apparent that the S5Sr/45Ca ratio in the plasma increases with time suggesting a preferential removal of asCa from the pool. T h e shell gland mucosa has a low ratio indicating that it shows considerable discrimination against strontium in the uptake of isotopes while the eggshell itself contains almost equal quantities of radioactive material. I n order to make more detailed estimates of the discrimination shown between the isotopes it is necessary to estimate the ionic ratios of the material that is presented to the tissues during the period of the experiment. T h e ratios of the isotopes in the blood change with time, since the organs of the body continually discriminate against strontium, and in order to compensate for this the plasma ratios which were determined experimentally were weighted by the time between sampling and a
ssSr/45Sr DISCRIMINATION BY FOWL SHELL GLAND
203
TABLE 1--85Sr/45Ca RATIOS IN VARIOUS TISSUES EXPRESSED AS ~/o DOSE/g × 103 r
Plasma values (hr after start of calcification) No.
0
3"50
5"00
5"25
9"00
Shell gland mucosa
37 38 43 44 45
--1"0* 1"0" 1"0"
--1"324 1"387 1"174
1"0* 1"0" ----
1"173 1"120 1"537 1"600 1,251
1"737 1"554 1'730 1"996 1"400
0"493 0"556 0"445 0"428 0"397
Bird
Eggshell 1"064 0"954 0"919 0"963 0.901
**Note t h a t all data are corrected to give e q u i v a l e n t values of ssSr a n d 45Ca in t h e stock solution. T h e ratio (ssSr/4~Ca) tissue/(S6Sr/*SCa) dose is therefore always
unity for the initial injections into plasma. mean value was then calculated. These values are for total SSSr/45Ca in the plasma and in order to convert them to ionic ratios it is necessary to estimate the additional effects of binding by plasma proteins. This effect was evident from the in vitro plasma ultrafiltration results which showed a filtrate ratio of 1.1 : 1 for SSSr/45Ca, independent of the time of equilibration in these experiments. As the ultrafiltration sample initially had a uniform ratio of isotopes, the results indicate a selective binding of calcium by the plasma proteins. Weighted mean values of total plasma sSSr/45Ca ratios were therefore multiplied by 1.1 to give the estimated mean ionic ratios shown in Table 2. TABLE 2--O.R. SHELL/PLASMA CALCULATED FROM ESTIMATES OF IONIC s5Sr/'SCa RATIO IN PLASMA
Bird No. 37 38 43 44 45
Shell
Estimated ionic ssSr/45Ca (weighted mean plasma × ultrafiltrated discrimination)
O.R. shell/plasma
1.06 0.95 0.92 0.96 0-90
1.59 1.46 1.73 1.87 1.45
0.66 0.65 0.60 0.60 0.65
T h e discrimination ratio of a tissue between two isotopes is normally expressed in physiological studies as the observed ratio (O.R.) of sample/precursor. T h e O.R. shell/plasma is shown in Table 2 for the two groups of birds. T h e values obtained are remarkably consistent at about 0.6 even for different time periods and stages of calcification. This indicates that about six strontium atoms are deposited upon the shell for every ten calcium atoms when the two isotopes are presented equally to the shell gland. If one tries to analyse the phenomenon further, however, by
204
K.
SIMKISS,S.
HURWlTZ AND A. BAR
determining discrimination at the plasma/sheU gland and shell gland/eggshell regions then the results are more complex. Shell gland mucosa contains about 40 per cent extracellular fluid (Sirnkiss, 1969; Hurwitz & Cohen, unpublished data) and this fluid presumably has the same SaSr/45Ca ratio as plasma ultrafiltrates. The total tissue ratio can therefore be divided to give extraceUular and intracellular isotope ratios. On this basis the intracellular fluid of the mucosal cells has a snSr/ 45Ca ratio of approximately 0.35 to 0.25 giving a discrimination ratio of about 5 against strontium at the plasma/mucosa region and of about 3 against calcium at the mucosa/eggshell surface. A discrimination ratio of about 5 against strontium at the plasma/mucosa region is the sort of result that CaBP would produce although the ratio is rather small for the known formation constants. If one accepts this result at its face value, however, one is left with a physiologically unlikely phenomenon of a preferential secretion of strontium at the mucosa/eggshell surface and it is difficult to explain this in terms of removing bound ions from CaBP. We have ignored the effects of crystallization upon the discrimination against strontium at the site of eggshell formation but these would tend to increase the Sr/Ca discrimination factor. It appears more likely, therefore, that the intracellular ratios are of doubtful significance and merely reflect the accumulation of bound calcium in the mucosal cells. Evidence for such a slowly exchanging calcium fraction within the cells is obtained from the decrease in ssSr/45Ca ratios of mucosa with time (Table 1, compare birds 37 and 38 with 43, 44 and 45). Previous studies of SSSr/aSCa discrimination in the laying bird obtained values of 0-927 between blood and eggshell (Drori et aL, 1964). This result, however, was based purely upon analyses of incorporated dose into the shell and it is very close to our own value of 0.96 (Table 1). In isolation, however, this figure underestimates the discrimination since it overlooks the continual rise in ssSr/45Ca ratios in the plasma caused by the overall discrimination of the body against strontium. It is in fact likely that in the system being studied, the rise in the SSSr/45Ca ratio of the plasma is at least partly caused by the activity of the shell gland itself, so that birds 37 and 38 which were injected later in calcification show initially more rapid rises in plasma SSSr/aSCa ratios than birds number 43, 44 and 45 which were just starting to calcify (Table 1). This factor is, in fact, probably the main variable in calculating the mean plasma ratios of isotopes even on a time weighted basis. The mucosa which was studied in these experiments was shown to contain a strong band of CaBP when subjected to electrophoresis. Equilibrium dialysis of the same material indicated that there was good evidence for 4~Ca binding but no detectable binding of s5Sr in these samples. These results are in keeping with the known formation constants of CaBP but are not capable of explaining the poor O.R. plasma/shell found in these experiments. This suggests that CaBP does not seem to play a central role in the transfer of calcium across the shell gland since the protein has a ssSr/45Ca discrimination factor of about 7 whereas the plasma/eggshell system in vivo has an overall discrimination of only about 1.5 in favour of calcium.
a s S r / 4 ~ S r DISCRIMINATION BY F O W L SHELL GLAND
205
Acknowledgements--One of us (K. S.) would like to thank the Volcani Institute for an invitation to visit it during its 50th Anniversary Celebrations and for the use of its facilities during this work. T h e research was assisted by a grant for the visit from the Royal Society/ Israeli Exchange Programme. REFERENCES CORRADINO R. A., WASSERMANR. H., PUBLOS M. H. & CHANG S. I. (1968) Vitamin D~ induction of a calcium-binding protein in the uterus of the laying hen. Arch. Biochem. 125, 378-380. DRORI D., VOLCANI R., FEtCE T., SHALMONE. & PASSYN. (1964) Factors affecting the Ca 4~ and Sr a9 transfer in the laying hen. Poult. Sci. 43, 486-491. HURWITZ S. & BAR A. (1968) Calcium exchange in plasma of the fowl Biochem. biophys. Acta 156, 389-393. SIMKISS K. (1969) Intracellular p H during calcification. Biochem. ft. 111,647-652. WASSEaMAN R. H. (1968) Calcium transport by the intestine: a model and comment on Vitamin D action. Calc. Tiss. Res. 2, 301-333. WASSERMANR. H., CORRADINOR. A. & TAYLOR A. N. (1968) Vitamin D dependent calcium binding protein: purification and some properties, ft. biol. Chem. 243, 3978-3986. WASSERMANR. H. & TAYLOR A. N. (1963) Vitamin D inhibition of radiocalcium binding b y chick intestinal homogenates. Nature, Lond. 198, 30-32.
Key Word Index----ssSr; ~Ca; shell gland; calcium binding protein; Gallus domesticus.