Archs oral Biol. Vol. 14, pp. 1395-1400, 1969. Pergmon Press. Printed in Great Britain.

RADIOAUTOGRAPHY OF Ca45 IN RAT SALIVARY GLANDS

R. H. DREISBACH and R. TAUGNER

Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305, U.S.A. and Physiologisches Institut, Heidelberg, Germany

Summary-Radioautographic visualization of Ca 45 in rat salivary glands indicated marked storage in submaxillary gland acinar cells. Acinar cells of the sublingual gland stored somewhat less, while acinar cells of the parotid and granular tubule cells of the submaxillary gland contained only slightly more Ca45 than connective tissue. The uptake of Ca45 in submaxillary and parotid gland acinar cells was greatly reduced 2 hr after isoproterenol. After chronic administration of isoproterenol, radioautographic visualization of CY5 localization indicated that the great increase in storage of calcium in the submaxillary gland produced by this drug probably resulted from an increase in the proportion of the gland made up by acinar cells.

INTRODUCTION

AN ELEVATED calcium level is found in salivary glands, ranging from 6 peq./g in rat

parotid (DREISBACH, 1964a) and 9 peq./g in human submaxillary gland (KRAINTZ, 1966) to 21 peq./g in rat submaxillary gland (DREISBACH, 1957) and 42 peq./g in cattle

submaxillary gland (KRAINTZ, 1966). Much of the calcium stored in rat submaxillary glands disappears on stimulation by pilocarpine (DREISBACH, 1959) and the amount disappearing from the gland can be correlated with an initial transient elevation in

saliva calcium. The loss of calcium from submaxillary and parotid glands is even

greater after isoproterenol administration and, here again, the calcium stored in the

gland is secreted into the saliva (DREISBACH, 1964a). After isoproterenol, the calcium

stored in the gland contributes to an initial transient elevation which can reach 72 peq./g of saliva (DREISBACH, 1967). Thus the calcium in the gland could be important in rats in maintaining a calcium concentration in saliva greater than can be maintained by the secretory process alone. Whether the same is true in human beings has not been tested.

Chronic administration of isoproterenol causes a marked increase in the size of submaxillary and sublingual glands in rats (SELYE, VEILLEUX and CANTIN, 1961; SCHNEYER, 1962; SEIFERT, 1962) and an increase in calcium content of the sub- maxillary gland (DREISBACH, 1964a).

The great diversity of cell types in salivary glands (POHTO, 1966; LEESON, 1967)

poses the question of the role each cell type plays in calcium metabolism in salivary glands. Previous studies have attempted to determine the calcium content of different salivary gland cells by histochemical tests (ROHDENBURG, and GEIGER, 1928) or by microincineration (H~BEL, 1954; SCH~~TZLE, 1962) but, since these studies utilized

1395

1396 R. H. DREISBACH AND R. TAUGNER

tissues fixed in aqueous media, the calcium or other mineral localization in the cells was probably altered post-mortem.

Although iodide localization in hamster salivary glands has been studied by radio- autography (MCGEE, MASON and DUGUID, 1967), no previous study of calcium localization in salivary glands by this method has been found. No references were found to the use of radioautography to study alterations in localization of a substance after acute stimulation of salivary glands but radioautography has been used to study the enlargement phenomenon after isoproterenol (SEIFERT, BACKMANN and PIEPER, 1966).

METHODS

Commercially obtained Wistar strain rats were fed a pelleted laboratory ration up to the time of the experiment. All injections were subcutaneous. Administration of isoproterenol for 1 week was given as previously described (DREISBACH, 1964a). For in vivo experiments, Ca45 (0.1-0.5 mCi) was injected 2 hr prior to removal of glands under ether anaesthesia. Glands were frozen in liquid N2 immediately after removal.

Frozen section radioautography was carried out by the method of TAUGNER (1966). Tissues were cut at 10 p in a cryostat at -15°C and alternate sections were used for histology or for radioautography on Kodak NTB or Ilford K2 glass plates (1 in. x 3 in.) having 10 TV thick emulsion. Sections for radioautography were applied to the emulsion by means of a fine camel hair brush where they adhered satisfactorily. Application of any adherent or solvent reduced the detiition of the Ca4’ localization. Sections for histology were fixed in Bouin’s fluid and stained with haematoxylin and eosin. The figures are a selection from experiments on 10 rats.

RESULTS

In vivo

(1) Adult rut. In the submaxillary gland, the Ca 45 localized much more intensely in acinar cells as compared to duct cells or cells of granular tubules (Figs. 1 and 2).

Uptake of Ca45 in sublingual gland acinar cells was distinctly less than that of acinar cells of the submaxillary gland (Figs. 1 and 2) but the uptake in sublingual acinar cells was greater than in submaxillary gland granular tubule cells. The uptake in duct cells of the sublingual gland appeared to be less than that in duct or granular tubule cells of the submaxillary gland. A faint lobulation could be seen in the radio- autograph of some areas of the sublingual gland corresponding to the structure of the gland. The Ca45 uptake as shown by radioautography of the sublingual gland reflected the calcium content which has been found by chemical analysis to be about 2/3 that of the submaxillary gland (DREISBACH, 1964a).

The parotid gland showed a distinctly different pattern of uptake of Ca45 from either submaxillary or sublingual glands (Fig. 3). There was only a slight difference between acinar cells, ducts and connective tissue. Occasional collecting ducts showed greater accumulation of the Ca45 whereas the smaller ducts were not distinguishable from acini. The lesser uptake of Ca4’ revealed by radioautography correlates well

RADIOAUTOGRAPHY OF cd5 IN RAT SALIVARY GLANDS 1397

with the calcium concentration which is only 6 peq./g wet weight for whole gland (DREISBACH, 1964a).

(2) Immature rut. A l-month-old male rat showed less uptake of Ca45 in the sub- lingual gland than in the submaxillary gland and only slight specificity of uptake in acinar cells of the submaxillary gland as compared to other cells in this gland (Figs. 4 and 5). Only slight differences in uptake in different cells of the sublingual gland could be seen. The calcium content of similar immature rats averaged 10.2 peq./g wet weight for the sublingual gland and 10.7 peq./g wet weight for the submaxillary gland.

(3) Acute isoproterenol. When 10 mg/kg of isoproterenol was given subcutaneously with Ca45 and glands were removed 2 hr later, marked changes occurred in the localization of the isotope in some glands but not in others. Figures 6 and 7 show the great reduction in uptake of Ca4’ which occurred in the submaxillary gland with almost complete disappearance of the differences in uptake between acinar cells and granular tubule cells. The sublingual gland was not affected by the isoproterenol and continued to show the same differentiation in uptake between acinar cells and ducts (Figs. 6 and 7) as was shown by the control untreated rat (Figs. 1 and 2). In the parotid gland, the slight specificity of uptake that was evident in the control rat disappeared completely and the connective tissue surrounding the gland showed greater uptake than the gland itself.

(4) Chronic isoproterenol. When the localization of Ca45 was studied 24 hr after the last of a series of injections of isoproterenol, the radioautograph (Fig. 9) showed that the proportion of cells in the submaxillary gland which stored Ca45 was increased as compared to the control (Fig. 2). Granular tubule cells were not apparent in the section and the acinar cells were greatly enlarged (Fig. 8).

The parotid had expanded so much that it was possible to cut a section which showed this gland as well as the sublingual and submaxillary glands (Figs. 8 and 9). The Ca45 uptake in the parotid gland at this time was minimal and was consistent with a calcium content of 3-4 peq./g wet weight found 24 hr after isoproterenol injections (DREISBACH, 1964a). Only a faint trace of parotid architecture can be seen in the radioautograph as compared to the other glands (Fig, 9).

In contrast to the submaxillary and parotid glands, the Ca45 uptake and histo- logical appearance of the sublingual gland did not change (Figs. 8 and 9).

DISCUSSION

If the radioautographic localization of Ca45 represents the concentration of Ca in the various cells of the gland, then the concentration of calcium in submaxillary gland acinar cells of the rat is remarkably high. The approximate concentration can be estimated from the proportion of the entire gland consisting of acinar cells. TAMARIN and SREEBNY (1965) have reported that acini make up 63 per cent of the intralobular tissue in adult rats. On the other hand, JACOBY and LEESON (1959) find that tubules in the fully developed gland take up half, or even more, of the total lobular space. Out- side the peripheral lobules, ducts make up a larger portion of the gland, reducing the fraction of the total that can consist of acinar cells. Since whole gland contains about 18 peq. of calcium per g of wet weight, and if acinar cells make up half or less of the

1398 R. H. DREISBACH AND R. TAUGNER

total gland, then the concentration of calcium in acinar cells could be more than 36 peq. of calcium per g. In contrast to adult rats, this storage mechanism for calcium in a l-month old rat was less well developed. At this age, acinar development is ahead of granular tubule development (JACOBY and LEESON, 1959).

The mechanism by which this high calcium level of the submaxillary gland is maintained is still unknown. It could be maintained by the presence of an intra- cellular binding substance (LEUNG and DRAUS, 1962; DREISBACH, 1962). Another possibility is that a specific mechanism transports the calcium across the cell mem- brane. The possibility has been described of a transport system that is speeded in both directions by sympathetic and parasympathetic drugs and is inhibited by metabolic inhibitors (DREISBACH, 1964b). Whether such a transport system plays a part in maintaining the elevated gland calcium has not been determined.

In almost all experiments, the relative calcium content indicated by radioauto- graphy 2 hr after the injection of Ca45 was similar to that found in previous experi- ments by chemical analysis. Only in the glands of immature rats was a difference apparent. In the sublingual and submaxillary glands of immature rats, the calcium content was about the same, IO.2 peq./g for the sublingual gland and 10.7 peq./g for the submaxillary gland. In contrast, a radioautograph showed distinctly less Ca45 in the sublingual gland than in the submaxillary gland. Whether this difference resulted from a slower exchange of Ca45 in the sublingual gland or from some other mechan- ism has not been determined.

Radioautographs made after acute isoproterenol reflect the fall of submaxillary gland calcium from 18 *2 to 3 * 1 peq./g wet weight and of parotid gland calcium from 6 -0 to 2 - 7 peq./g wet weight 2 hr after injection (DREISBACH, 1964a). Calcium content of the sublingual gland by analysis was unchanged after acute isoproterenol and the radioautograph also indicated no change from the untreated animal. After chronic isoproterenol, the submaxillary gland calcium concentration rose from 18 ~2 to 33 -7 peq./g wet weight 24 hr after the last of 10 daily injections, along with a 5-fold increase in wet weight. At this time the sublingual gland weight and calcium content were unchanged (DREISBACH, 1964a). The lack of change in weight of the sublingual gland after chronic administration of isproterenol agrees with the findings of SEIFERT (1962) and of SEIFERI et al. (1966) for the major sublingual gland but not with the findings of SCHNEYER (1962) who found 25 per cent enlargement of acinar cell size. The parotid gland also increased greatly in size after isoproterenol but the calcium content never increased above the control (DREISBACH, 1964a).

The radioautographs indicate that the increased concentration of calcium in the submaxillary gland after chronic isoproterenol administration could result at least partially from the greater proportion of the gland made up by acinar cells (SEIFERT, 1962; SCHNEYER, 1962). If the acinar cells make up 40 per cent of the total gland in untreated rats having gland calcium averaging 18 *2 peq./g, the proportion of acinar cells would need to be 74 per cent after isoproterenol to give a whole-gland calcium concentration of 33 -7 peq./g without any change in calcium content of acinar cells in relation to weight. The question of greater content of calcium per unit weight of acinar cells might be determinable by grain-counting techniques. However, this would

RADIOAUMGRAFWY OF Ca4’ IN RAT SALIVARY GLANDS 1399

require precise determination of the rate of exchange of the Ca45 into all of the different cell types and maintaining blood levels of Ca45 constant over a sufficiently long time to allow equilibration with intracelluar calcium to occur in all cell types.

The 2 hr period used in the present experiments for equilibrating extracellular Ca4s with intracellular calcium represents a compromise between the slow transfer of Ca45 into the gland and the rapidly falling plasma Ca4’ resulting from bone uptake. The half-time of equilibration of Ca4s in submaxillary gland is about 90 min in untreated rats (DREISBACH, 1961). Thus a longer time after injection of Ca4’ would allow more complete exchange with intracellular calcium. On the other hand, bone uptake of Ca4s removes the isotope from the blood at a rate sufficient to lower the blood Ca45 specific activity by half in the second hour after subcutaneous administra- tion. Thus, longer equilibration time does not help to increase the amount of isotope in the gland.

Acknowledgement-This investigation was supported by United States Public Health Service Research Grant DE-794 from the National Institute of Dental Research, Bethesda, Maryland and the Ministeriums fur wissenschaftliche Fors- chung, Bundesrepublic Deutschland.

R&m&-La mise en evidence radioautographique de Ca4’, dans les glandes salivaires de rats, indique une accumulation marquee dans les cellules acineuses de la glande sous- maxillaire. Les cellules acineuses de la glande sub-linguale en renferment moins, alors que les cellules acineuses de la parotide et les cellules granuleuses des canalicules de la glande sous-maxillaire ne contiennent que lkgerement plus de Ca4’ que le tissu conjonctif. Le marquage en Ca45 des cellules acineuses de la sous-maxillaire et de la parotide est nettement reduit 2 heures aprts administration d’isoproterenol. Apres administration prolongee d’isoproterenol. la mise en evidence radioautographique de la localisation du Ca4’ indique que l’augmentation importante du calcium au niveau de la glande sous- maxillaire, induite par ce medicament, est probablement IiCe a l’augmentation en cellules acineuses glandulaires.

Zusammenfassung-Radioautographische Untersuchungen iiber die Verteilung von Ca45 in den Speicheldrtisen der Ratte ergaben eine deutliche Speicherung in den Driisen- zellen der Glandulae submandibulares. Die Drtisenzellen der Glandulae sublinguales speicherten etwas weniger, wlhrend die Drtisenzellen der Glandulae parotides und die granulierten tubularen Zellen der Glandulae submandibulares kaum mehr Ca45 enthiel- ten als das Bindegewebe. Zwei Stunden nach der Applikation von Isoproterenol war die Aufnahme von Ca45 in den Drtisenzellen der Gil. submandibulares und parotides erheblich verringert. Nach fortdauemder Verabreichung von Isoproterenol fand sich stark vermehrte Speicherung von Ca45 . m den Gll. submandibulares. Dies ist mdglicher- weise auf eine Vergrogerung der Drtisenzellen zuriickzufiihren.

REFERENCES

DREISBACH, R. H. 1957. Accumulation of calcium 45 by salivary glands. Proc. sot. exp. Biol. Med. 96, 555-558.

DREISBACH, R. H. 1959. Secretion of calcium by rat submandibular gland. Am. J. Physiol 196, 645- 648.

DRESIBACH, R. H. 1961. Effect of pilocarpine on transfer of Ca45 and K42 to rat submaxillary gland and kidney. J. Pharmac. exp. Ther. 131, 257-260.

DREISBACH, R. H. 1962. Calcium binding by rat submaxillary glands in vitro. Biochem. J. 82,71-75. DREISBACH, R. H. 1964a. Effect of isoproterenol on calcium metabolism in rat salivary gland. Proc.

sot. exp. Biol. Med. 116,953-956.

1400 R. H. DREISBACH AND R. TAUGNER

DREISBACH, R. H. 1964b. Regulation of Ca45 transfer in rat salivary and lacrimal glands in vitro. Am. J. Physiol. 207, 1015-1020.

DREISBACH, R. H. 1967. Effect of isoproterenol on calcium, protein, and electrolytes of rat sub- maxillary gland. Proc. sot. exp. Biol. Med. 126, 279-281.

HIIBEL, E. 1954. Vergleichende und experimentele Untersuchungen tiber den Aschengehalt der Spei- cheldriisen. Z. f mikrosk.-anat. Forsch. 60,33-64.

JACOBY, F. and LEESON, C. R. 1959. The post-natal development of the rat submaxillary gland. J. Anat. 93,201-216.

KRAINTZ, L. 1966. Comparison of the calcium concentration of submaxillary salivary glands. Nature, Lond. 209, 215-216.

LEESON, C. R. 1967. Structure of salivary glands. In: Handbook of Physiology, Vol. 2 (Sec. 6), pp. 463-95.

LEUNG, S. W. and DRAUS, F. J. 1962. The calcium binding characteristics of a salivary gland mucoid. Archs oral Biol. 7,327-332.

MCGEE, J. O’D., MASON, D. K. and DUGUID, W. P. 1967. The site of iodide concentration in hamster salivary glands as demonstrated by autoradiography. Archs oral Biol. 12, 1189-1193.

POHTO, P. 1966. Catecholamine-induced salivary gland enlargement in rats. Acta odont. stand. 24, Suppl. 45, l-73.

ROHDENBURG, G. L. and GEIGER, J. 1928. The distribution and amount of calcium and of potassium in normal tissue of the mouse, Archs Path. 6,215-227.

SCHNEYER, C. A. 1962. Salivary gland changes after isoproterenol-induced enlargements. Am. J. Physiol. 203, 232-236.

SCH;~TZLE, W. 1962. Histochemie der Speicheldrtisen. Acta histochem. 13, 62-112. SEIFERT, G. 1962. Experimentelle Speicheldriisenvergrosserungen nach Einwirkung von Noradrenalin.

Beitr. path. Anat. 126, 321-251. SEIFERT, G., BACKMANN, R. and PIEP~~, A. 1966. Uber die RNS-Synthese und RNS-Migration bie

der experimentellen Sialadenose. Beitr. path. Anat. 134, 370-381. SELYE, H., VEILLEUX, R. and CANTIN, M. 1961. Excessive stimulation of salivary gland growth by

isoproterenol. Science, N. Y. 133, 44-45. TAMARIN, A. and SREEBNY, L. M. 1965. The rat submaxillary salivary gland. A correlative study by

light and electron microscopy. J. Morph. 117,295-356. TAUGNER, R. 1966. Renale “Stapelung” und Ausscheidung vonHgZo3-Mersayl bei der Ratte. Naunyn-

Schmeidebergs. Arch. exp. Path. Pharrnak. 255, 254-265.

RADIOAUTOGRAPHY OF Ca’” IN RAT SALIVARY GL4NDS

FIGS. I and 2. 210 g female rat. 0.5 mCi Ca4’. Radiographexposed for49 days. Sub-

lingual gland above, submaxillary gland below. 70

FIG. 3. Parotid from same rat as Fig. I. 70

FIGS. 4 and 5. 60 g male rat. 0.5 mCi Cad5. Sublingual gland above, submaxillary gland

below. Radiograph exposed for 13 days. 70

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R. H. DREISBACH AND R. TAUGNER

FIGS. 6 and 7. 160 gmale rat. 0.5 mCi Ca4” and 10 mg/kg isoproterenol simultaneously in different injection sites. Sublingual gland above, submaxillary gland below. Radio-

graph exposed for 98 days. \ 70

FIGS. 8 and 9. 200 g male rat given isoproterenol for I week. Last injection 24 hr prior to post-mortem. 0.5 mc Ca45. Parotid upper, sublingual gland right, submaxillary

gland lower. Radiograph exposed for 52 days. u 70

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