Ca content of duck eggshell gland mucosa homogenate and the rate of Ca2+ binding to its subcellular fractions during and after the formation of the eggshell

Comp. Biochem. Physiol. Vol. 77B, No. 4, pp. 655-663, 1984 Printed in Great Britain

0305-0491/84 $3.00+ 0.00 (~ 1984PergamonPress Ltd

Ca C O N T E N T OF D U C K E G G S H E L L G L A N D M U C O S A H O M O G E N A T E A N D THE R A T E OF Ca 2+ B I N D I N G TO ITS S U B C E L L U L A R F R A C T I O N S D U R I N G A N D A F T E R THE F O R M A T I O N OF THE E G G S H E L L * C. E. LUNDHOLM Department of Pharmacology, Link6ping University, Sweden (Tel: 013-19 1040) (Received 13 September 1983) Abstract--1. From a homogenate of the eggshell gland mucosa of Indian runner ducks (Anas platy-

rhynchos var.), different particulate subfractions which bound Ca2+ in the presence of ATP were separated by differential centrifugation. 2. In one of these microsomal subfractions (FIII), which was rich in both cytochrome c oxidase and 5-nucleotidase and was the most active of all in binding Ca2+, the rate of Ca2+ binding was dependent on the functional state of the gland. 3. It was significantlyhigher in glands activelyforming an eggshell than when the shell had been formed. FIII may contain calcium-secreting granules of the eggshell gland.

INTRODUCTION

Some ecotoxicological agents such as chlorinated hydrocarbons, especially DDT and its metabolite DDE, and heavy metals such as organic mercuric compounds, are able to reduce the eggshell thickness in several species of birds (Cooke, 1973; Hayes, 1975; Ratcliffe, 1970). During an investigation of the mode of action o f p - p ' - D D E on eggshell thickness in ducks (Lundholm, 1982), the question arose how the secretion of Ca 2+ from the eggshell gland occurred and how it was regulated during the eggshell formation. Of special interest was the question of which subcellular structures are involved in the Ca 2+ translocation during Ca 2+ secretion, since this matter had previously received little attention (Hohman and Schraer, 1966). Most studies on the Ca secretion of the gland have been carried out on the intact organ in vivo or in vitro. During the formation of an eggshell in the hen, the gland is capable of secreting about 2 g of calcium or about 10% of the total body calcium in approx. 15 hr (Simkiss and Taylor, 1971). During this time the organism has to accomplish two functions--the first is to provide the plasma with sufficient calcium to support the calcium secretion of the gland, and the second to transport the calcium from the plasma to the lumen of the shell gland. With regard to the first function, oestrogen, probably in interaction with androgens, increases the plasma concentration of calcium in egg-laying birds and also the formation of medullary bone, which constitutes a reserve from which calcium is mobilized during the eggshell formation (Taylor et al., 1971). It has been observed that increased renal formation of the vitamin D metabolite 1~, 25-(OH)2 D 3 occurs at ovulation (Kenny, *Financial support was provided by the Swedish Natural Science Research Council and the Bofors NobelPharma C., Karlskoga, Sweden.

1976). This hormone increases the absorption of calcium in the intestine by stimulating calcium transport in the epithelial cells, probably by the synthesis of special proteins (De Luca, 1978). It also stimulates resorption of calcium from the bones (Tanaka and De Luca, 1971). Concerning the second function, the shell gland consists of the mucosal cells of the uterus. Of these cells the columnar cell layer, located directly on the basement membrane, is probably the main calciumsecreting part, whereas the tubular gland cells penetrating the muscular layer probably secrete NaHCO3 (Simkiss and Taylor, 1971; Mongrin and Carter, 1977). Several investigators (Hohman and Schraer, 1966; Gay and Schraer, 1971; Pearson et al., 1977; Talbot and Taylor, 1974; Eastin and Spaziani, 1978a,b) have studied the calcium secretion of the shell gland and the way in which the secretion of calcium is regulated. Most of the established observations have been summarized by Eastin and Spaziani (1978a,b). The secretion of calcium is an energydependent reaction (Ehrenspeck et al., 1967), but is not dependent on an electrical potential difference across the gland (Ehrenspeck et al., 1971). The calcium secretion occurs partly directly and partly from an exchange reaction between Ca 2+ and Na 2+ in the shell gland (Eastin and Spaziani, 1978a). The increase in Ca 2+ secretion during shell formation is not dependent on the innervation of the uterus but may be mediated by a humoral factor. The extension of the uterus by the presence of an egg in the oviduct contributes to the stimulation of the Ca :+ secretion but is not the main stimulus (Eastin and Spaziani, 1978a). The chick oviduct contains high affinity binding proteins for 1~, 25-(OH)2D3 (Coty, 1980) and the plasma concentration of this steroid shows a circadian change in the hen, which is synchronous with the eggshell formation (Abe et al., 1979). The production of this compound by the kidneys is reduced in

655

656

C . E . LUNDHOLM

old hens laying eggs with thin shells ( A b e et al., 1982), a n d it has been s u g g e s t e d that this h o r m o n e m a y be involved in the calcium t r a n s p o r t in the shell g l a n d (Bar a n d N o r m a n , 1981). M o r e o v e r , the renal biosynthesis o f 1~, 25-(OH)zD3 is s t i m u l a t e d by oestradiol in i m m a t u r e b i r d s (Baksi a n d K e n n y , 1976). W h e t h e r le, 25-(OH)zD 3 is the h u m o r a l f a c t o r that stimulates the calcium s e c r e t i o n by the g l a n d d u r i n g the f o r m a t i o n o f the shell, o r w h e t h e r it has a permissive a c t i o n for this factor is n o t yet k n o w n , however. T h e d o m e s t i c fowl is resistant to the eggshell t h i n n i n g effects o f D D E a n d D D T ( C o o k , 1973), b u t in the I n d i a n r u n n e r duck, D D E was especially c a p a b l e o f r e d u c i n g the thickness o f the eggshell ( L u n d h o l m , 1980). D u c k s o f this variety are g o o d egg-laying birds (Olsson, 1954) a n d lay their eggs at a m o r e regular time in the m o r n i n g t h a n the d o m e s t i c hen. In the p r e s e n t i n v e s t i g a t i o n the C a 2+ b i n d i n g to different subcellular fractions o f eggshell g l a n d m u cosal cells f r o m I n d i a n r u n n e r d u c k s was t h e r e f o r e c o m p a r e d at two different stages o f eggshell form a t i o n . T h e effect o f D D E a d m i n i s t e r e d in vivo on the Ca 2+ b i n d i n g to these s t r u c t u r e s was also s t u d i e d a n d is r e p o r t e d elsewhere [ L u n d h o l m , in press (a)].

MATERIAL

AND METHODS

Experiments in vivo One-year-old Indian runner ducks (Anas platyrhynchos var.) were bred from my own colony of ducks. The ducks were kept in an outdoor pen with free access to commercial chickenfood (Pullfor V/irp Hel E, 3°~ calcium) and water. The pens were connected to a house in which the ducks could get shelter. In the middle of April, when all hens laid eggs, one group, consisting of 12 hens and one male, were isolated in a pen. Eggs were collected daily. The length and breadth of the eggs were determined to the nearest 0.05 ram. A 5 mm hole was drilled at the equator of the egg and the content was removed by compressed air. The eggshell was washed with water and dried and the shell weight (shell + membrane) was determined to the nearest 0.1 mg with a sensitive laboratory balance. The eggshell index (EI) proposed by Ratcliffe (1970) was calculated as follows: shell weight (mg) El= shell length (mm) x breadth (ram) Determination o] Ca content of blood plasma, eggshell, uterine fluid and eggshell gland mucosa The ducks were killed by a sharp blow on the head and exsanguinated. They were killed either at 6-7 p.m. or at 8 a.m., i.e. 13-15 hr before or within 1 hr before egg-laying, which usually occurred between 7 and 9 a.m. Before killing, a check was made by palpitation that the eggshell gland (uterus) contained an egg. In the following, animals killed at 6-7 p.m. with a calcifying egg in the uterus are referred to as 'secreting' birds and those killed at 8 a.m. with a fully calcified egg, as 'non-secreting' birds. The latter expression may not be exactly true (Pearson et al., 1977, Eastin and Spaziani 1978a), but is convenient for the present purpose. Samples of arterial blood of about 5 ml were obtained at the time of slaughter. The samples were centrifuged at 750 x g for 15min and the plasma was frozen at -20:'C until analyzed. One hundred microliters of plasma were wet-ashed with 1 ml of HNO 3 (Suprapure Merck) for 24 hr. The samples were then diluted with 0.75~ EDTA and the Ca content was determined by atomic absorption spectrophotometry, with

a Pye Unicam SP90 apparatus as earlier described (Lundholm, 1982).

Eggshells About 100 mg of eggshell (shell + membrane) were wetashed in 1 ml of HNO 3 (Suprapure Merck) for 24 hr and samples were diluted with 0.75% EDTA and analyzed by atomic absorption spectrophotometry. Uterine fluid Uterine fluid was obtained from the eggshell gland mucosa at the time of slaughter and frozen at 2 0 C until analyzed. To 100 #1 of uterine fluid, 1 ml HNO 3 (Suprapure) was added and samples were diluted with 0.75°~, EDTA and analyzed by atomic absorption spectrophotometry. Eggshell gland mucosa The Ca content of the mucosa was determined as described by Lundholm (1982). Preparation q[ subcellular fractions The method described by Nilsson et al. (1978a) for intestinal smooth muscle was used with the following modifications: The eggshell gland was rapidly excised and placed in ice-cold 0.3 M sucrose. All subsequent operations were carried out with ice-cold solutions and instruments. The mucosal layer was scraped off from the muscular layer with a stainless steel knife. The mucosa (~4.5 g) was divided into three 1.5 g portions. Each portion was homogenized for 3 x 20 sec at 700 x rpm with a glass to glass homogenizer in 10 ml of a medium containing 0.3 M sucrose, 0.03 M Tris (pH 7.2) and 2raM ascorbic acid. After each homogenization the pestle and homogenizer were rinsed with 5 ml of the homogenization medium, resulting in a tenfold dilution of the tissue. The homogenate was first centrifuged at 750 × g for 15 rain to obtain the nuclear fraction. This fraction was washed once by resuspending the pellet in 5 ml of homogenization medium, and then recentrifuged. The supernatants were combined. The crude mitochondrial fraction was obtained by centrifuging the supernatant at 17,000 x g for 15min. This pellet was washed once with 2.5ml of homogenization medium. The combined supernatants were further centrifuged at 40,000 x g for 90 min to obtain a microsomal pellet. This pellet was suspended in 1.5ml of homogenization medium and homogenized by hand with a glass Teflon homogenizer. The microsomal suspension was layered on a discontinuous sucrose gradient with the following composition from the bottom to the top: 2.5 ml 55~(~ sucrose, 3 ml 45'~o sucrose, 3 ml 37~'/oand 3 ml 29°/~,sucrose. After centrifuging at t10,000 x g for 120min, four microsomal bands were seen at the boundaries of the sucrose layers and collected with the aid of a Pasteur pipette with a broad tip. The bands were designated (from top to bottom) FI, FII, FIII and FIV. In some experiments especially mentioned in the text, the crude mitochondrial pellet was resuspended in 2 ml of the homogenization medium and layered on the same discontinuous sucrose gradient as was used for preparation of the microsomal subfractions. After centrifugation at 10,000 x g for 20 min, four mitochondrial bands were obtained. That located between 29 and 37°,o sucrose (MII) had a somewhat higher relative activity of cytochrome c oxidase (6.8 _+ 1.0) and a lower relative activity of 5-nucleotidase (1.5 _+ 0.8) than the crude mitochondrial fraction (Fig. 4). The Ca 2~ binding of MII was still only partly inhibited (by about 755°) by sodium azide or oligomycin (see Results). Since the 'true' mitochondrial Ca 2+ binding still had to be established with the help of specific mitochondrial Ca 2~ -binding inhibitors. the 'crude' mitochondrial fraction was routinely used, however.

The fractions were analyzed for the marker enzyme cytochrome c oxidase by the method of Cooperstein and Lazarow ( 1951) for mitochondria, and with 5'-nucleotidase

Ca 2+ binding by duck eggshell gland mucosa by the method of Avruch and Wallach (1971) for plasma membrane. NADPH-cytochrome c reductase was determined as described by Avruch and Wallach (1971). Ca 2+ binding of eggshell gland mucosa fractions The Ca2+-binding studies of the whole eggshell gland mucosal homogenate and of the subcellular fractions prepared from it were performed as described by Lundholm (1982)• Ca binding took place at 37°C in an incubation medium containing 120mM KCI, 10mM NaC1, 0.02M histidine buffer (pH 7.2), 1 mM MgCI2, 1 mM ATP, 5 mM sodium oxalate and 10-4 M CaCI 2 labelled with 45Ca. To some of the incubation solutions 5 mM sodium azide or 10#g/ml of oligomycin was added to inhibit the Ca 2+ binding by the mitochondria (Lehninger et al., 1967, Brierley and Jung, 1981). The Ca binding was terminated by the addition of 500/~1 of 0.1 M KC1 and the sample was rapidly filtered through a 45 #m Millipore filter. The radioactivity retained on the filters was determined in a Packard Tri Carb liquid scintillation counter. Protein was measured as described by Lowry et al. (1951). RESULTS

Eggshell index and calcium content of fully developed and partially formed eggshells The rate of Ca 2+ secretion by the active eggshell gland in vivo varies in relation to the time of egglaying (Schraer and Schraer, 1965, Eastin and Spaziani, 1978a). In the hen a maximum rate was observed 18-13 hr before egg-layering. At this time the rate of secretion was 5-7 times higher than 2 hr before or after oviposition, when it was at its minimum (Eastin and Spaziani, 1978a). The ducks laid eggs between 7 and 9 a.m. The birds that were killed at 6-7 p.m. were classified as 'secreting', and those killed at 8 a.m. as 'non-secreting' birds although in the latter case some Ca 2+ secretion may have been present (see below). There may also have been some variation in Ca 2+ secretion according to the point in the secretory cycle at which the ducks secreting Ca 2+ were killed and the eggshell gland mucosa was taken for analysis. E l o f t h e eggs soon to be laid was 2.2 _+ 0.04 (range 1.88-2.37), whereas that of the NON-SECRETING SECRETING

i

PLASMA

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Fig. 1. Calcium content of the mucosa of the shell gland (mg/g dry weight) and blood plasma (mmol/l) of ducks with an egg with a mature shell ('non-secreting') or with an eggshell during formation in the shell gland ('secreting'). Mean _+ SEM, n = 5. Statistically significant differences are denoted: * = P < 0.05.

657

100

SECRETING

80

NON-SECRETING

60

==4o,~ H

i ~

20

0

~

6

26 Time (min)

Fig. 2. Time course of the binding of 45Ca2+ (#mol/g prot.) by the eggshell gland mucosa homogenate from secreting and non-secreting ducks. Mean _+ SEM, n = 5. *P < 0.05; *(*) = P < 0.02. eggs being formed was 0.67 _+ 0.05 (range 0.53-1.01). (In calculating E1 the eggshell membranes containing proteins were also included; their weight was 1-2% of the mature shell). The total calcium content of the fully formed eggshell determined by atomic spectrophotometry was 2.7 _+ 0.1 g. F r o m Elit was calculated that in the secreting ducks, i.e. those killed during the shell formation, about 30% of the total calcium content of the mature eggshell had been secreted.

Calcium content of the eggshell gland mucosa, uterine fluid and blood plasma in non-secreting ducks The calcium content was significantly lower, by 57% in the eggshell gland mucosa from secreting birds than in that from non-secreting ones (Fig. 1). The plasma calcium level was somewhat lower in secreting than in non-secreting ducks, but the difference was not significant (Fig. 1). Sufficient uterine fluid for determination of its calcium content was obtained in all secreting ducks but only in one of the non-secreting ones. The mean calcium content was 4.2 + 2.0 m M in the secreting ducks and 3.5 in the 'non-secreting' birds. This semi-quantitative test may indicate that it was more the amount of secreting fluid than its calcium content that was changed at the time of oviposition. Ca 2+ binding by a homogenate of eggshell gland mucosa from secreting and non-secreting ducks; effect of sodium azide The time curves of the binding of 45Ca2+ by the homogenate of eggshell gland mucosa from the two categories of ducks are presented in Fig. 2. The rate of binding was most rapid during the first 2.5 rain. After 5 min the binding increased linearly with time for at least 20 min. The binding reported in the following was mostly determined during the first 5 min. As seen in Fig. 2, the Ca 2+ binding was significantly higher at 2.5 and 5 min in the homogenate from secreting than from non-secreting ducks. It was probable that there was still a difference at least up to 20 rain, but that this did not reach a statistically significant level.

C. E. LUNDHOLM

658

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previous knowledge about the Ca:+-binding properties of the different subcellular fractions from other tissues.

Homogenate

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Fig. 3. Histogram of the total, sodium azide insensitive ('microsomal') or azide-sensitive ('mitochondrial') 45Ca2+ binding by the whole homogenate of eggshell glands from non-secreting or secreting ducks. Mean + SEM, n = 5; *=P<0.05: **-P<0.01. In an earlier investigation the Ca 2+ binding to the homogenate was found to be divided into a part inhibited by 5mM sodium azide and an azideinsensitive part (Lundholm, 1982). The azideinsensitive part was probably attributable to Ca binding to intact or fragments of mitochondria, which are sensitive to azide (Lehninger et al., 1967). This has also been demonstrated in tissues such as smooth muscle (Carsten, 1969) and fibroblasts (Moore and Pastan, 1978). As a mean of all experiments, 44~o of the Ca :+ binding was inhibited by sodium azide after 5 min, whereas the rest was insensitive. On comparison between secreting and non-secreting birds, the azidesensitive ('mitochondrial') binding was found to be almost similar in these two categories (Fig. 3). The azide-insensitive ('microsomal') Ca :+ binding was, on the other hand, significantly (P < 0.01) greater in the secreting (18.9 4- 1.8 x 10-gmole/mg protein/ 5 min) than in the non-secreting birds (11.1 4-0.5 x 10 9mole). It was therefore suspected that the higher rate of Ca 2+ binding to the whole homogenate in secreting than in the non-secreting birds was due to greater "microsomal' Ca: ~ binding in the former group. It was therefore of interest to separate the homogenate into its different subcellular fractions so that the Ca:* binding could be studied in the microsomal fractions without the help of inhibitors.

Subcellular fractions of the eggshell gland mucosa In order to ascertain in which subcellular fraction or fractions (plasma membrane, mitochondria, nuclear fraction, endoplasmatic reticulum and/or secretory granules) the observed changes in Ca :+ binding in the whole homogenate took place during the different phases of eggshell formation, these fractions were isolated and characterized, and their Ca2+-binding properties studied. Only partial separation of the eggshell gland mucosal cells into different fractions had been performed previously (Hohman and Schraer, 1966). The differentiation and characterization of the fractions were accomplished with the help of marker enzymes and on the basis of

Cytochrome c oxidase, 5-nucleotidase and NADPHcytochrome c reductase in the subcellular fractions As a marker for mitochondria and mitochondrial fragments, cytochrome c oxidase has been used in several tissues (Hogeboom et al., 1946, De Duve, 1972), including the eggshell gland mucosa (Hohman and Schraer, 1966). A marker for microsomes from the plasma membrane is 5-nucleotidase. NADPH-cytochrome c reductase has been reported to be located in the endoplasmic reticulum (Avruch and Wallach, 1971) and was therefore also determined in the fractions of the eggshell gland. Its activity was so low that it could not be used as a marker enzyme, however. The crude homogenate was separated into subfractions as described in Materials and Methods, and the relative activities of the enzymes in these fractions in relation to those in the whole homogenate are depicted in Fig. 4. The mitochondrial fraction showed, on the average, a four-times higher relative activity of cytochrome c oxidase than the whole homogenate. There was also a three times higher relative activity of 5-nucleotidase in this fraction, indicating a contamination with microsomal fragments. As reported in Methods, it was possible to purify the mitochondria to some degree by means of further gradient centrifugation, but for reasons mentioned there, this step was not used in this part of the investigation. The crude microsomal fraction had an approximately four times higher activity of 5-nucleotidase than the whole homogenate, but still contained cytochrome c oxidase (Fig. 4). This crude fraction was separated into four subfractions (FI FIV) after centrifugation on a discontinuous sucrose gradient. In FI the relative activity of 5-nucleotidase was about nine times higher than that in the whole homogenate, whereas the relative activity of cytochrome c oxidase was 0.8 times that in the whole homogenate (Fig. 4). Among the other subfractions, the relative activity of 5-nucleotidase decreased, whereas that of cytochrome c oxidase increased with increasing fraction numbers when compared with the values for FI. Ca :~ binding by the different suhfractions in nonsecreting ducks; effects of oxalate The time curve of the Ca 2+ binding by the crude fractions in a representative experiment is shown in Fig. 5A. The most marked difference was noted at 5 min, the crude microsomal fraction bound least, and the mitochondrial fraction most, Ca 2+/rag protein. At 20 min only the nuclear fraction showed a markedly lower binding capacity than the other fractions. When the crude microsomal fraction had been divided into subfractions by gradient centrifugation, a very pronounced difference in the rate of Ca :+ binding became apparent between them (Fig. 5B). FI and FII displayed relatively more rapid Ca 2+ binding during the first 5 min than later. The rate of binding was highest in FIII, where it was virtually constant during the whole 20min period. The rate of Ca 2+

Ca -,+ binding by duck eggshell gland mucosa cyt. C ox.

659

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N Mit.Mic.

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Fig. 4. Relative specific activity of cytochrome c oxidase and 5-nucleotidase in the crude fraction prepared from the shell gland mucosa homogenate--H; N, nuclear; Mit., mitochondrial and Mic., microsomal fraction. The microsomal subfractions are denoted FI--FIV. Mean _+SEM n = 4-6. The mean cytochrome c oxidase activity in the whole homogenate was 1.22_+ 0.11 (AE/mg protein/min) and the 5-nucleotidase activity 70.9 + 9 nmole adenosine/mg protein/60 min. binding during the first 5 min was about four times higher in FIII than in FI. The total 45Ca2+ binding after 20 min was eight times higher in FIII than in FI. After 5 min the Ca 2+ binding to FIII was also 2.5 times higher in FIII than in the mitochondrial fraction (Fig. 5A,B). Even in relation to the furtherpurified mitochondrial fraction (MII, see Materials and Methods), FIII had bound (as a mean of 9 experiments) 1.8 times more Ca 2+. The actual values in fractions from the same secreting ducks were 82.8 _+ 7.1 × 10 -9 mole/rag protein/5 rain in FIII and 47.3 + 6 . 6 x 10 9mole/rag protein/5min in the purified mitochondrial fraction MII. The difference was significant (P < 0.01). The very high rate and high Ca 2+ binding capacity in FIII could not therefore be explained as a consequence of contamination with mitochondria.

300

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The Ca2+-binding curves of FI] and FIV lay between those of FI and FIII (Fig. 5B). Since sodium oxalate stimulates the Ca 2+ uptake in vesicular particles but not that in membranous fragments (Hasselbach, 1974), the reason for the difference between FIII and FI may be that the former contained more vesicular structures than the latter. In the absence of oxalate the Ca 2+ binding during the first 5 min was reduced to about 50~ in FI and to about 35~o in FIII as compared with the controls (Fig. 6). Even in the absence of oxalate the total Ca 2+ binding was greater in FIII than in FI (difference 20.8 + 3.4 × 10 9 mole/mg protein/5 min; P < 0.01). The explanation for the difference between FI and FIII cannot therefore be solely that the microsomes had a membranous rather than a vesicular structure.

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Fig. 5. A. Time response of the binding of 4SCa2+ by a homogenate (H) of a shell gland mucosa and by the crude fractions prepared from it (Mic., microsomal, Mit., mitochondrial, N, nuclear). B. 4~Ca binding by the microsomal subfractions (FI-FIV) prepared from the crude microsomal fractions.

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Fig. 6. Effect of sodium azide (5 mM) (10#g/ml) or absence of oxalate on the of the microsornal subfractions FI and secreting ducks. Mean+_SEM, n = 5. *** = P < 0.001.

or oligomycin Ca2+ binding FIII of non** = P <0.01;

Calcium content of different subcellular fractions A factor that may have influenced the CaZ+-binding rate of the subcellular fractions was their initial calcium content. If the Ca 2+-binding sites were already almost saturated from the beginning or, in the case of the vesicular structures, if the initial intra-vesicular Ca 2+ concentration was high, the rate of Ca 2+ binding might have been lowered. The total initial calcium content was therefore determined by atomic absorption spectroscopy in the fractions isolated from secreting ducks. It was found (Table 1) that with the exception of the 'nuclear' fraction, which had a lower value, the calcium contents of the mitochondrial, FI and FIII fractions varied from 1.1-1.3x 10 4mole/g protein without any significant differences between the fractions. With regard to the value for the supernatant over the 40,000 x g fraction, this was not the 'cytoplasmic' concentration, since this supernatant still contained light or 'small' microsomes (Nilsson, 1978b). There was thus no marked differences in the initial calcium content between the subcellular fractions in which Ca 2+ binding was determined.

Effects of sodium azide, oligomycin and trifluoperazine on the Ca 2+ binding of the subcellular fractions After separation of the subcellular Ca 2+ binding fractions by differential and gradient centrifugation, the Ca 2+-binding properties of these fractions were also studied with the help of inhibitors of Ca 2+ binding. Sodium azide and oligomycin (Lehninger et al., 1967) are claimed to specifically inhibit the A T P Table 1. Calcium content ( m g / g prot.) of the different subcellular fractions of a h o m o g e n a t e from eggshell gland mucosa. Shell glands from secreting ducks. Means _+ SEM, n = 9 Total homogenate Nuclear fraction Mitochondrial fraction F I microsomal fraction F I I I microsomal fraction Supernatant 40,000 x g

0.98 1.93 5.21 4.29 5.63 0.33

+ 0.08 + 0.14 +_ 0.26 _+ 0.31 ± 0.96 -- 0.04

dependent Ca 2+ binding of the mitochondria but not that of the microsomes. Several other compounds also inhibit the respiratory-coupled Ca 2+ binding of the mitochondria (Brierly and Jung, 1981), an uptake mechanism that was of little or no importance under the present experimental conditions in vitro. Ca 2+ binding was thus not reduced by 1 mM KCN (data not given). Recently calmodulin has been suggested to be involved in the Ca 2+ binding to the plasma membrane, and the phentiazine derivative trifluoperazine in a concentration of < 5 0 p M is a rather specific calmodulin antagonist (Wets and Wallace, 1980). A study was therefore made of the way in which these three compounds, sodium azide, oligomycin and trifluoperazine, influenced the Ca 2+ binding of the complete homogenate and the different purified sub-cellular fractions. In preliminary experiments the lowest concentration of the compound to produce maximal inhibition was determined (data not given). These experiments, which were performed in eggshell gland mucosa of secreting ducks, are presented in Fig. 6. In the whole homogenate sodium azide and oligomycin reduced the Ca 2~ binding to 51 and 37'1% of the control binding, respectively. In the purified mitochondrial fraction (MII) the corresponding values were 28 and 23~. In FI sodium azide had, on the contrary, a stimulating action (118 + "/ whereas _ 3jo), oligomycin caused no or very little change in Ca 2+ binding (104 + 5~o). In FIII the actions of the two compounds were obviously different; azide did not change the Ca 2+ binding (101 + 6~o) of the control value, whereas oligomycin reduced it to 67 + 5')%. With regard to the effect of trifluoperazine, this compound almost completely abolished the Ca -`+ binding in the whole homogenate, to 3 + 1);; of that in the controls. In the purified subfractions the effect of the phentiazine derivative was weaker. The main effect was localized to the purified mitochondria (MII), where the binding amounted to 11 _+4.'<'~iof the controls. In FI and FIII the binding was reduced to 23 + 5~o and 67 + 5~o, respectively, of the control values. The action of trifluoperazine on the Ca 2+ binding to the eggshell gland mucosa is from a physiological point of view of interest since it may indicate that calmodulin is involved in the Ca 2+ -binding process and that Ca 2+, via calmodulin, controls its own binding in the mucosa. The drug did not differentiate specifically between the mitochondria and microsomal Ca 2+ binding, however. In this respect oligomycin and especially sodium azide were better chemical tools.

Differences between secreting and non-secreting ducks in the Ca 2+ binding to subcellulur [factions Jfom eggshell gland mucosa Mitochondrial Jraction. There was no significant difference between secreting and non-secreting birds in the rate of Ca 2+ binding to the crude mitochondrial fraction (Fig. 7), whereas such a difference was found for the whole homogenate (Fig. 2). When, by adding sodium azide, the Ca 2+ binding of the mitochondrial fraction was divided into an azideinsensitive and an azide-sensitive part, the former ('microsomal') binding was found to be significantly

Ca2+ binding by duck eggshell gland mucosa [~

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FI[

FII[

Fig. 7. A. Histogram of the Ca 2+ binding of the crude mitochondrial fraction of the shell gland homogenate from non-secreting and secreting ducks. The parts of the binding insensitive or sensitive to inhibition by sodium azide is shown too. B. Histogram of the Ca 2+ binding of the microsomal subfractions FI-FIII from secreting and non-secreting ducks. Mean -t- SEM, * = P < 0.05; *** = P < 0.01. greater in the secreting than in the non-secreting low, had a higher content of calcium in the eggshell birds. The azide-sensitive ('mitochondrial') binding gland mucosa than 'secreting' birds, which were was about 50~o greater in the non-secreting than in calcifying an egg and probably secreting Ca at a the secreting birds, but the difference was not higher rate. The rate of binding of 4~Ca2+ by a homogenate of significant (Fig. 7). Microsomal subfractions. In the microsomal sub- the eggshell gland--the mucosal cells--was, on the fraction FI there was no significant difference in Ca 2+ other hand, higher in the non-secreting than in the binding between secreting and non-secreting birds secreting birds (Fig. 2). When the Ca 2+ binding of the (Fig. 7B). FI, with its relatively low cytochrome c whole homogenate was divided into an azideoxidase activity but high 5-nucleotidase activity, and sensitive ('mitochondrial') and an azide-insensitive its insensitivity to the inhibitory effect of sodium ('microsomal') part, it was found that it was the azide and oligomycin, was probably a comparatively binding attributable to the microsomes that was pure microsomal preparation of the plasma mem- enhanced in the secreting ducks. Hohman and Schraer (1966) observed that the brane. In FII, with its somewhat higher relative activity of cytochrome c oxidase, the binding tended mitochondrial fraction accumulated more injected to be greater in the secreting than in the non-secreting 45Ca in vivo when the gland was resting than when it was secreting. A similar effect may have been present birds. The microsomal subfraction showing the most in this study, but this could not be verified statistically potent Ca 2+ binding was FIII. In secreting shell gland (Fig. 7). It has been suggested that the mitochondria it bound 80.1 _+4.6 x 10 -9mOle/mg protein/5 min act as a kind of buffer system in the regulation of and in non-secreting gland 59.4 + 7.1; the difference cytoplasmic Ca 2+ (Bygrave, 1977; Carafoli and was significant (Fig. 7). It is therefore probable that Crompton, 1978). It may therefore be speculated that a large part of the observed difference in the azide- under the experimental conditions of Hohman and insensitive binding to the whole homogenate (Fig. 3) Schraer (1966) the mitochondria bound an excess of between secreting and non-secreting ducks was attri- 45Ca that was not secreted. Mitochondrialfractions. The crude mitochondrial butable to FIII. fraction showed a four to five times higher relative activity of cytochrome c oxidase but also an approximately three times higher relative activity of DISCUSSION 5-nucleotidase than the whole homogenate. The proVariations in the Ca 2+ content of eggshell gland muportions of azide-insensitive and azide-sensitive Ca z+ cosa and in the Ca 2+ binding by the homogenate in binding were not very different in the mitochondrial relation to the time o f egg-laying fraction from those in the whole homogenate (Fig. 4); The Ca 2+ metabolism of the eggshell gland shows the purification of mitochondria was not complete, cyclic variations in relation to the time of egg-laying even though the fraction was rich in mitochondria. A (Schraer and Schraer, 1965; Eastin and Spaziani, further purification step was only partially successful. After addition of sodium azide or oligomycin, the 1978a). In the present experiments, the calcium content of 'mitochondrial' and 'microsomal' Ca 2+ binding could the shell gland mucosa varied in relation to the time be further dissociated. These inhibitors of mitoof egg-laying. 'Non-secreting' ducks, which had a chondrial Ca z+ binding reduced the Ca ~+ uptake to fully calcified egg in their uterus and in which the about 75~ of the total Ca 2+ binding of the purified secretion of Ca 2+ from the shell gland probably was mitochondrial fraction (MII).

662

C.E. LUNDHOLM

Microsomal.[?actions. The crude microsomal fraction was divided into four subfractions by centrifugation on discontinuous sucrose gradients. In the lightest of them (FI) the high relative activity of 5-nucleotidase (nine times higher than in the whole homogenate) and low relative activity of cytochrome c oxidase (0.8 times that in the whole homogenate) indicated that it contained an abundance of fragments of the plasma membrane but was relatively free from mitochondrial fragments. Azide or oligomycin did not decrease the Ca 2+ binding by FI, further supporting this assumption. In the absence of sodium oxalate the Ca 2+ binding after 5 min was reduced by about 50~o whereas the corresponding figure for FIII was 65~o, indicating that FI had a lower content of vesicular structures than FIII. This was supported by the observation that the Ca 2+ binding curve of FI declined with time and was less steep than that of FII[. Of the microsomal subfractions, FIII showed the most pronounced Ca 2+ binding. FIII was able to bind more than 300 nmole/mg protein after 20 min (Fig. 5), whereas FI only bound 40 nmole/mg during the same time. It is therefore quite probable that FIII contained calcium-binding and calcium-secreting granules of the eggshell gland mucosa. This is further strengthened by the finding that the Ca 2+ binding of FIII was significantly greater in the shell gland mucosa of 'secreting' than in 'non-secreting' ducks. In another investigation I also observed that in ducks treated in vivo with DDE until eggshell thinning had occurred this compound had reduced both the total calcium content of FIII and the rate of Ca 2+ binding of this subfraction in secreting birds in comparison with untreated controls. The calcium content of the uterine fluid was also reduced by DDE (Lundholm, in press, a). The Ca2+-binding properties of FIII therefore seemed to be of special interest and I have studied them in some other birds (Lundholm, in press, b). FIII isolated from secreting eggshell glands of the hen also bound large amounts of Ca 2+ (150 nmole/mg protein/5 min, i.e. almost twice the amount in the duck, of 80 nmole/mg protein/5 min). As in the duck (Fig. 5), FIII from the hen bound much more Ca 2+ than the purified mitochondrial fraction (MII) of the shell gland mucosa (36 nmole/mg protein/5 min). The high Ca 2+ binding by FIII was therefore not dependent on contamination with mitochondria. There was a difference, however, between the Ca 2+ binding of FIII of Indian runner ducks and the hen. In these experiments on ducks the Ca 2+ binding of FIII was not azide-sensitive but was reduced to about 70~ of the control value by oligomycin (Fig. 6), whereas in the hen about 50~o of the binding was inhibited by sodium azide and 90~ by oligomycin. These observations raise the question of the origin and composition of the Ca:+-binding particles in FIII and whether they are Ca2+-secreting granules of the shell gland. Johnston et al. (1963) and Aitken (1971), who studied this problem in the eggshell gland mucosa of the domestic fowl by electronmicroscopy observed in the columnar surface epithelium--the cells of which are probably those that secrete Ca 2~ (Mongin and Carter, 1977)~two kinds of cells with

different granulae. In one cell type with an apically located nucleus the granules were derived from the endoplasmic reticulum or the Golgi apparatus, whereas in the other cell type with a basally located nucleus the granules seemed to have been formed from modified mitochondria. These authors provided no conclusive evidence that these granules were involved in the secretion of Ca 2÷ by the eggshell gland, however. Talbot and Tyler (1974) reported that the content of organic matter varied in the different layers of the eggshell of the hen. This observation may indicate that the composition of the secretory granulae of the eggshell gland varies during the shell formation. SUMMARY The calcium content of and the rate of ATPdependent Ca2+-binding to a homogenate of the eggshell gland mucosa from Indian runner ducks (Anas platyrhynchos var.) with a calcifying egg Csecreting') ducks or a mature eggshell in the gland ('non-secreting') were studied. The calcium content was higher in the gland from non-secreting than from secreting ducks, but the rate of ATP-dependent Ca 2+ binding to the homogenate was lower in the former than in the latter birds. The particulate fraction of the homogenate was separated into subfractions by differential centrifugation. Neither in the fraction rich in the marker enzyme cytochrome c oxidase (mitochondria) nor in that (FI) rich in 5-nucleotidase (microsomes of the plasma membrane), did the rate of Ca 2+ binding differ significantly between secreting and nonsecreting ducks. In the microsomal subfraction (FIII) which was the most active of all in binding Ca 2+, the contents of both marker enzymes were 3~4 times higher than in the whole homogenate. The rate of Ca 2+ binding to FIII was higher in secreting than in non-secreting ducks and this subfraction may contain Ca 2+-secreting granules of the eggshell gland mucosa.

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