Eur. J. Biochem. 21 (1971) 433-437

Calcium and the Exportable Protein in Rat Parotid Gland Parallel Subcellular Distribution and Concomitant Secretion

David WALLACH and Michael SCHRAMM Department of Biological Chemistry, The Hebrew University of Jerusalem

(Received April 1/May 21, 1971)

Most of the calcium (> SOO/O) present in rat parotid gland slices was secreted concomitantly with the exportable protein. Detailed kinetics showed that secretion induced by catecholamines and by N 6 monobutyryl adenosine 3' : 5'-monophosphate proceeded a t an approximately con- stant ratio of calcium to amylase (60 nmoles calcium/mg exported protein). It was calculated that the calcium which is tightly bound to amylase could account for only l i O / o of the exported cal- cium.

Among the subcellular fractions of the gland, the secretory granules showed the highest calciumlprotein ratio which was identical with that found for the material secreted by the slices. Both amylase and calcium were sedimented in a crude granule fraction to the extent of 500/, of the total present in the homogenate of the gland.

Various treatment that caused release of amylase from the crude secretory granule fraction also caused release of calcium to a similar extent. The calcium in secretory granules was not readily exchangeable when the granules were suspended in a medium containing 45Ca. The possible role of calcium in the aggregation of the exportable proteins in the secretory granules is discussed in view of the information on the presence of relatively high concentrations of bivalent cations in some other gland systems.

In a previous communication it was reported that the rat parotid gland contains a high concentration of calcium which, upon homogenization of the tissue, causes extensive damage to the mitochondria [I]. It was also shown that the parotid gland contains a specific pump for calcium ions [ Z ] . Work by Dreis- bach, on the submandibular gland in vivo, showed that calcium is being effectively secreted after in- jection of the catecholamine isoprenaline [3,4]. With this information a t hand, an attempt was made to examine in detail the relation between protein se- cretion and calcium secretion in tissue slices and to establish the subcellular location of the calcium. The work leads to the conclusion that most of the calcium is accumulated in the secretory granules together with the exportable protein and that the calcium and the protein are secreted concomitantly.

MATERIALS AND METHODS Incorporation of 45Ca

into Rat Parotid Glands in vivo Albino rats (160-2OOg) were used. 45Ca (5 to

40 $3) in 0.5 ml saline, containing also 2 mM CaCl,, was injected intraperitoneally during slight ether

Unusual Abbreviation. Butyryl cyclic AMP, Ne-mono- butyryl adenosine 3': 6'-monophosphate.

anaesthesia. Since the animals eat a t night [5], the injection was given at 6 a.m., when lights were turned on. The dissection of the glands was done [6] either 6 or 28 h after the injection. Throughout that time, the rats were kept without food, receiving water ad lib.

Isolation of Subcellular Practions from Rat Parotid Glands

Glands were collected a t 37 "C in aerated Krebs Ringer bicarbonate (Ringer) medium [7] which con- tained no calcium. Homogenization and fractionation were done as described by Amsterdam et al. [8]. All homogenization media were adjusted to pH 7.4 with Na,CO, and contained 0.2 tJ.g/ml diphenyl para- phenylene diamine.

Parotid Slice System The glands were collected as above, sliced and in-

cubated as previously described [7]. Unless otherwise mentioned, calcium was omitted from the Ringer medium.

Anulyticul Nethods Amylase was determined according to Bernfeld [9].

Protein content was estimated with the Lowry reagent using bovine serum albumin as the standard [iO]. Calcium and magnesium content were determined

434 Concomitant Secretion of Calcium and Amylase Eur. J. Biochem.

after wet dashing with a perchloric-nitric acid mix- ture [ll] or after deproteinization with loo/,, cold trichloracetic acid. The recovery of calcium was found to be the same for both of these extraction procedures. The measurements were done with a Perkin-Elmer atomic absorption spectrophotometer, Model 303. To overcome interference by phosphate and to equalize enhancement effects by alkaline ions and low pH, all the samples contained 50mM KC1, 50mM NaC1, 1 I mM SrC1, and 0.6 M trichloroacetic acid. To remove traces of calcium, glassware was washed with concentrated nitric acid. Plasticware was immersed overnight in 50 mM Na-EDTA and was washed thor- oughly afterwards with glass distilled water.

45Ca was measured in a toluene-triton scintillation mixture, using a Packard scintillation counter.

Chemicals Epinephrine bitartrate was purchased from K and

Klab, (U.S.A.). Butyryl cyclic AMP was obtained from Boehringer Mannheim GmbH (Mannheim, Ger- many). 45CaC12 was purchased from New England Nuclear (U.S.A.).

RESULTS

Secretion of Calcium by Gland Slices Induction of amylase secretion by epinephrine

and butyryl cyclic AMP caused concomitant secretion of calcium (Fig. 1). The kinetics indicated that similar relative amounts of calcium and amylase were being secreted. At 90 min about 80°/, of both calcium and amylase, initially present in the slices, had been released into the medium. At this time, the media of the systems containing either epinephrine or buty- ryl cyclic AMP showed a calcium content of 58 to 60 nmoles/mg protein secreted.

To check kinetics of calcium secretion by a more sensitive and accurate procedure the glands were labeled in vivo with 45Ca and the secretion of the radio- active calcium was subsequently studied in the slice system. 45Ca secretion confirmed the pattern observed for total non-labeled calcium (compare Figs. 1 and 2). There is a small, but possibly significant difference in the initial rates of calcium and amylase secretion in the systems containing either butyryl cyclic AMP (Figs. 1 and2) or 2,4-dinitrophenol (Fig.2). Due to the limits of the accuracy of the methods, the results do not show the small fraction of non exportable Ca2+ which must be presumed to be present in the gland cells. It was not possible to obtain an accurate figure for calcium secretion when the Ringer medium con- tained added calcium. This was mainly because cal- cium carbonate precipitated on the slices. A rough estimate of calcium secretion induced by epinephrine in Ringer medium containing 1 mM calcium was, however, obtained by comparison with a control

'* t

" 20 40 60 80 100

Time (min)

Fig. 1. Secretion of amylase and calcium by gland slices. Glands were collected after 6 h of starvation, sliced and incubated in Ringer medium which did not contain calcium. At the indicated times, samples of 1 ml were drawn from the medium for calcium and amylase determination, and were replaced by 1 ml fresh Ringer medium. The slices in each system contained about 10000 units of amylase and about 1 pmole glandular calcium. Concentrations of reagents were as follows: epinephrine, 50 pM; butyryl cyclic AMP, 1 mM. ___- , calcium; ~ , amylase; 0, 0, epinephrine; 0, m,

butyryl cyclic AMP; A, A, control

l o o t

Time (min)

Fig. 2. Secretion of amylase and 45Ca by gland slices. The glands were collected 6 h after injection of 6 pCi of 45Ca to each rat. The incubation and sampling procedures were as described for Fig.1. The slices in each system contained about 10000 units of amylase and about 5000 counts/min 45Ca. Concentrations of reagents were as follows: Epinephrine, 50 @I; butyryl cyclic AMP, 1 mM; 2,4-dinitrophenol, 1 mM. ----, 45Ca; - , amylase; 0, 0, epinephrine; 0, m,

butyryl cyclic AMP; v, V, 2,4-dinitrophenol

Vol.21, No.3, 1971 D. WALLACH and M. SCHEtAMW 435

Table 1. Ca2+ and Mg2+ content in subcellular fractions of rat parotid gland and binding of added 45Ca during homogenization Experiment I-Glands were collected from 16 rats after 24 h starvation. Homogenization and fractionation were carried out at 4 "C in a 0.3 M sucrose medium. The total homogenate contained 360 mg protein, 125000 units amylase, 11 pmoles calcium and 16 pmoles magnesium. The amylase and calcium in the purified fractions shown below accounted for about 50°/, of the amount initially present in the homo- genate. Experiment 11-Glands from six rats were collected and fractionated as above. The homogenization medium contained 0.03 pCi 45Ca per ml a t a concentration of 1 FBI

Expt I Expt I1

W a bound Subcellular fraction Amylase Mg'+ Ca*+ during homo-

genization

Homogenate 350 45 30 5 500 Secretory

granules 710 25 60 700 Mitochondria 80 25 25 5 000 Microsomes 20 100 45 11 000 Postmicroso-

ma1 super- natant 350 50 25 7 000

slice system which did not receive hormone. The slices, after 90 min secretion, lost 78O/, of the amylase and about 60 ,Ilo ofthe calcium initially present in the slices.

Subcellular Distribution of Calcium in the Gland It is shown in Table 1 that the secretory granule

fraction contains the highest calcium concentration per mg protein. The ratio of 60 nmoles/mg protein in the secretroy granule fraction is the same as that found for the protein specifically secreted by the slices.

The secretory granules showed a negligible uptake of 45Ca when the latter was added to the homogeni- zation medium. This observation indicates that the high calcium content in this fraction is indeed intrinsic to the granules. It may be further concluded that the granule membrane is either quite impermeable to calcium or that the calcium inside is only sluggishly dissociable. Thus calcium would be packed into the granules only during the period of their formation.

The relatively high concentration of calcium in the microsomal fraction is probably due to absorption from the soluble fraction by the ribosomes. This as- sumption is supported by the finding that 45Ca added to the homogenate became effectively bound to the microsomes (Table 1). Their ability to bind large amounts of Mg2+ is well known [I21 (Table 1). It should be further noted that the calcium in the micro- soma1 fraction amounted to only go/, of the total calcium of the homogenate. The calcium content of the postmicrosomal fraction represented 30 O/,

Table 2. Distribution of amylase and calcium between a crude secretory granule fraction and the supernatant

Accumulation of 45Ca in vivo in the rat parotid gland, collec- tion of glands and homogenization were as described in Methods. Glands from 3 rats, containing about 10000 units of amylase and 10000 counts 45Ca/min were used for each fractionation. A crude secretory granule fraction was sedi- mented from the homogenate by centrifugation for 10 min a t 100Oxg and was washed once in the medium used for

homogenization

Relative amount Tempe- in the crude secretory

ratwe of granule fraction isolation

Time

injection after "Ca Isolation

medium

Amylase 4sCa

honrs "C of total

6 0.3 M sucrose 25 65 50 28 0.3 M sucrose 4 60 65 28 0.15 M KC1 25 40 50 6 0.15 M KCI 25 55 50 6 0.15 M KCl 4 45 45

Table 3. Release of 45Ca and amylase from secretory granules by various agents

Experiment I-A purified secretory granule fraction was isolated a t 4 "C in 0.3 M sucrose, 28 h after 45Ca injection. The fraction contained 1000 counts 45Ca/min and 2500 units of amylase. The granules were suspended in 1 m10.3 M sucrose to which 4 ml distilled water were added. After spinning for 10 min a t 1 4 0 0 ~ g, amylase and 45Ca were determined on the supernatant and on the pellet. Experiment 11-A crude secretory granule fraction was isolated a t 4°C in 0.15M KCl medium, 6 h after 45Ca injection as described in the legend of Table 2. Samples containing 350 units of amylase and 2000 counts 45Ca/min were added to 0.15 M KCl medium containing also 30 mM buffer or p-hydroxymercuribenzoate (PMB) as shown below. After 10 min incubation, release of 45Ca and amylase from the granules was measured as in

Expt 1

Release into snpernatant Experiment Treatment

Amylase "Ca

a/1. of total

I. Purified granule fraction hypotonic lysis 97 98

11. Crude granule pH 7.5, Tris 10 28 fraction pH 5.5, acetate 36 45

DH 4.5, acetate 85 91 Go addition 10 26 1 UM PMB 14 29

10'pM PMB 38 49

of the total calcium and is ascribed to the destruction of part of the secretory granules during homogeni- zation. This is strongly indicated by the finding that the ratio calcium/amylase in the supernatant is identical with this ratio in the secretory granules.

Further experiments were carried out to test whether indeed the high calcium content found in the secretory granule fraction is in the same granules as the amylase. It is shown in Tables 2 and 3 that various conditions for fractionation and treatment of the

436 Concomitant Secretion of Calcium and Amylase Eur. J. Biochem.

Fraction no.

Fig.3. Fractionation of the secretory granule bound calcium by gel filtration. A pursed granule fraction was isolated from 24 glands 6 h after injection of 40 pCi of 45Ca to each rat. The homogenization was done in 0.3 M sucrose medium at 4 O C . The granules were lysed by resuspension in 0.8 ml of 0.05 M cold Tris buffer (pH 7.4). The membranes were sedi- mented by centrifugation for 6min in a Beckman 152 centrifuge. A sample of 0.5 ml from the supernatant (con-

granule fraction did not cause a drastic change in the Calamylase ration in the fraction. The crude secretory granule fraction also contains the nuclei and cell debris. These contaminants, by absorbing calcium solubilized during homogenization might release it during subsequent incubation and cause the relatively small discrepancies between calcium release and amy- lase release shown in Table 3.

It is shown in Fig.3 that about 14O/, of the cal- cium in the secretory granules is eluted from a Se- phadex column in the void volume together with the protein. Most of the calcium emerging with the pro- tein is probably tightly bound to mamylase. Given that a-amylase represents 3001, of the total secretory granule protein [13], that it tightly binds one atom Ca per mole [14], that its mol. wt is 50000 [15] and that the Calprotein ratio of the secretory granules is 60 nmolelmg (Table l), it is calculated that 11 of the total calcium is bound to mamylase.

DISCUSSION

Calcium appears to fuliil several different functions in the cell of the exocrine parotid gland. A low con- centration in the medium is required for induction of enzyme secretion by epinephrine in slices [16]. Higher concentrations, equivalent to those normally present in blood (2 mM) must be added to the medium

5.0

--. 4.0 $

0 4 c

s 3.0 Cm e

8 - 2.0 5 E

c c

c 3 :: 1.0 5

e c

a

taining 1900 units of amylase and 17600 counts Wa/min) was applied to a Sephadex 0-25 column (1 x 15 cm) which had been equilibrated with 0.05 M Tris buffer (pH 7.4). The same buffer served for the elution at 4OC. The flow rate was 1.2 ml/min and fractions of 1 ml were collected. Protein concentration (A----A) in the eluant is given as absorbance at 280 nm. 6Ca content ( 0 ) and amylase activity (0) eluted in each fraction are expressed as percent of total input

bathing slices in order to produce K+ release and vac- uole formation by epinephrine [7].

As shown in the current work, there are large amounts of calcium inside the gland, segregated in the secretory granules andsecretedin conjunction with the exportable protein. Since this exportable calcium appears to be accumulated in membrane-bound struc- tures, it is not surprising that it cannot replace the requirement for added calcium to produce secretion as noted above.

Several approaches were used to establish whether indeed the calcium in the glands is intimately asso- ciated with the exportable protein. Kinetics studies showed a close parallelism between the secretion of amylase and calcium when induced either by epin- ephrine or by butyryl cyclic AMP. Subcellular frac- tionation showed that the highest concentration per mg protein, of both amylase and calcium, is found in the secretory granule fraction. Furthermore, the ratio nmoles Ca/mg protein in the secretory granule fraction was equivalent to that of the material se- creted by the slices. Additional experiments on the secretory granule fractions demonstrated parallel release of amylase and calcium under various condi- tions. Thus, the diverse experimental approaches all point to the same conclusion, that calcium in the pa- rotid gland is packaged and secreted with the export- able protein. In this respect, calcium H e r s markedly

Vo1.21, No.3, 1071 D. WALLACH and M. SCHRAMM 437

from K+, the latter being released by a mechanism which is independent of protein secretion [7].

It is not clear a t which stage of transport, calcium joins the exportable protein on its way from the ribo- somes to the secretory granules. A calcium pump has been demonstrated in smooth membrane vesicles of the rat parotid gland [ 2 ] . It is known that the secretory granule is formed in the area of the Golgi complex which is abundant in smooth membrane vesicles [17,6]. It is therefore, tempting to suggest that thc exportable protein is joined on its way by calcium somewhere after leaving the rough endo- plasmic reticulum and prior to formation of the mature secretory granule.

It is noteworthy that divalent metals are also present a t high concentration in a number of other gland systems, and in these, too, the metal is probably associated with the secretory material[3,18-22].More specific evidence for the presence of high metal concen- trations in secretory granules has been reported for chromaffin granules containing calcium [18,19] and for insulin granules containing zinc [20,21]. For the latter, there is some indication that the metal might serve to form a complex with the insulin [23,24]. Hokin's studies [25] on secretory granules of dog pancreas showed that the exportable proteins in the granules might exist as an insoluble aggregate. Taking these observations into account andincluding especial- ly the present findings on calcium in the parotid gland, we would like to propose that in several gland systems the metal serves in the aggregation of the exportable material.

This work was supported by a grant from the National Institutes of Health, U.S.A. (No. 5 RO1-BIO-AM-10451-06).

1.

2.

3.

REFERENCES Feinstein. H.. and Schramm, M.. Eur. J . Biochem. 13 . ,

(1970) 158.'

Acta, 203 (1970) 326.

953.

Selinger, Z., Naim, E., and Lasser, M., Biochim. Biophys.

Dreisbach, R. H., Proc. SOC. Exp. Bid. Med. 116 (1964)

4. Dreisbach, R. H., Proc. SOC. Exp. Biol. Med. 126 (1967) 279.

5. Sreebny, L. M., and Johnson, D. A., Arch. Oral Biol. 14 (1969) 397.

6. Amsterdam, A., Ohad, I., and Schramm, M., J . Cell Bid. 41 (1969) 753.

7. Batzri, S., Amsterdam, A., Selinger, Z., Ohad, I . , and Schramm, M., Proc. Nat. Acad. Sci. U. 8. A. 68 (1971) 121.

8. Amsterdam, A., Schramm, If., Ohad, I., Salomon, Y., and Selinger, Z., J . Cell Biol. 1971, in press.

9. Bernfeld, P., in Methods in Enzymology (edited by S. P. Colowick and N. 0. Kadan) . Academic Press. New York 1955, Vol. I, p. 146.

"

10. Lowrv. 0. H., Rosebroueh. N. J., Farr, A. L., and Ran- dafi,'R. J., J . Biol. C&m. 193 (1951) 265. '

11. Allan, J. E., Spectrochimica Acta, 17 (1961) 459. 12. Petermann, M. L., The Physical and Chemical Properties

of Ribosomes, Elsevier Publishing Company, Amster- dam 1964, p. 105.

13. Schramm, M., in Salivary Glands and their Secretion (edited bv L. M. Sreebnv and J. Mever). Pereamon " I, " Press, Ox2ord 1964, p. 3 i k

14. Stein, E. A.. Hsiu, J., and Fischer, E. H., Biochemistrv, 3 (1964) 56.

65 (1962) 200.

203 (1970) 335.

577.

don), 205 (1965) 42.

15. Loyter, A., and Schramm, M., Biochim. Biophys. Acta,

16. Selinger, Z., and Naim, E., Biochim. Biophys. Acta,

17. Jamieson, J. D., and Palade, G. E., J . Cell Biol. 34 (1967)

18. Borowitz, J. L., Fuwa, K., and Weiner, N., Nature (Lon-

19. Borowitz, J. L., Biochem. Pharmacol. 19 (1970) 2475. 20. Logothetopoulos, J., Kaneko, M., Wienshall, G. A., and

Best, C . H., in The Structure and Metabolism of the Pancreatic Islets (edited by S. E. Brolin, B. Hellman, and H. Knutson), Pergamon Press, Oxford 1964, p. 333.

21. Maske, H., Diabetes, 6 (1957) 335. 22. Mawson. C. A.. and Fischer. M. I.. Nature (London). 167

, I

(1951 j859. '

23. Howell, S. L.. Young. D. A.. and Lacv, P. E.. J . Cell " ,

Biol.'41 (1969) 167:'

Science (Washington), 116 (1952) 394. 24. Hallas-Mdler, K., Petersen, K., and Schlichtkrull, J.,

25. Hokin, L. E., Biochim. Biophys. Acta, 18 (1955) 379.

D. Wallach and M. Schramm Department of Biological Chemistry The Hebrew University of Jerusalem 20 Mamilla Road, Jerusalem, Israel