Diurnal variation in secretory components of the rat parotid gland

Archs oral Bid.

Vol. 14, pp. 397405,

DIURNAL

1969. Pergamon Press. Printed in Cit. Britain.

VARIATION IN SECRETORY COMPONENTS OF THE RAT PAROTID GLAND L. M. SREEBNY and D. A. JOHNSON

Departments

of Oral Biology and Pathology, University of Washington, Washington 98105, U.S.A.

Seattle,

Summary-The parotid gland in rats fed ad libitum exhibits a diurnal cyclic pattern which is correlated with food intake. This is characterized by a change in gland weight, and is accompanied histologically by corresponding changes in acinar diameter and granule content. The gland levels of three enzymes (amylase, deoxyribonuclease, and ribonuclease), comprising 55 per cent of the secretory protein, were minimal at the end of the eating cycle. These then progressively increased and the highest levels were observed just prior to eating. During the eating cycle, all three enzymes progressively decreased. The changes observed in gland protein were largely the result of cyclic variations in the levels of secretory protein. Preliminary experiments indicate that the non-protein gland weight change may also result from secretory compounds. It was concluded that the diurnal pattern of the rat parotid gland was due to storage and expulsion of secretory components located in the zymogen granules of acinar cells. INTRODUCTION

been noticed in this laboratory that the dry weight and the amylase activity of the parotid glands of rats was somewhat higher in the afternoon than in the morning. As rats are nocturnal, the majority of their food intake is at night. Since food intake is a known stimulus for salivation, we wondered if the increase in dry weight and amylase activity during the day reflected a cyclic pattern within the gland which was dependent on food intake. To test for such a diurnal variation within the gland, and for its dependency on food intake, the parotid glands of rats were examined at intervals of 4 hr over a 24 hr period. IT HAD

MATERIALS

AND

METHODS

Adult, male, Sprague-Dawley rats (Northwest Rodent Laboratories, Pullman, Washington) were used in these experiments. The animals were housed individually in cages in a room in which light was provided from 8 a.m. to 6 p.m. The animals were allowed to adjust for 2 to 3 days and then body weight and daily food intake for each animal was followed for an additional 4 days. The animals were divided into six groups of seven animals per group so that the mean body weight and mean food intake were the same for each group. An additional group of five animals of similar body weight and food intake was used to determine the food intake pattern during the 24 hr of the experiment. This was measured at intervals of 2 hr beginning at 5 a.m. on the morning of the experiment and ending at 5 a.m. on the following morning. 397

398

L. M. SREEBNY

AND

D. A. JOHNSON

At 5 a.m. the parotid glands of the first group of experimental animals were removed. At intervals of 4 hr thereafter, the parotid glands of another group of animals were removed. The glands of group six, the last group, were dissected at 1 a.m., 20 hr later. All the animals were sacrificed with ether; they were allowed to eat ad Iibitum until this time. One parotid gland of each animal was carefully dissected, weighed, frozen on dry ice, lyophilized and stored at -20°C. This gland was then finely minced and used for the biochemical assays. A portion of the opposite gland was excised, fixed immediately in Zenker’s solution (MCMANUS and MOWRY, 1960) and prepared for histologic evaluation. A portion of the lyophilized gland was homogenized in a teflon-glass homogenizer in the cold in a buffer containing 0 .Ol M NaCl and 0 -02 M phosphate (PH 6 -9). Proteins were determined on this homogenate. For enzyme analysis, the homogenate was centrifuged at 1OOOgin the cold for 5 min. Amylase was immediately determined on the supernatant and the remainder was frozen for deoxyribonuclease (DNase) and ribonuclease (RNase) assays: Extracts of the tissues for RNA and DNA determinations were prepared by a modification (SREEBNY, WANAMAKER and ROBINOVITCH, 1965) of the method of SCHNEIDER(1945). Each of the above determinations is expressed as mg per total gland. Proteins were determined by the method of LOWRYet al., (1951) with the modification described by BAILEY(1962). A human serum pool was used as the standard. Amylase was determined by the method of BERNFELD(1951) with the exception that the salt concentration of the buffer was modified to 0.01 M NaCl (FISCHERand STEIN, 1960). A glucose standard curve was used to express amylase activity. The glucose value was then multiplied by a factor of 1.259 to convert to mg of maltose (BRUNER,1964). The amylase activity, expressed as mg maltose equivalents formed in 3 min at 3o”C, was subsequently converted to mg of amylase by using the specific activity of purified amylase isolated from rat parotid saliva (2500 mg of maltose per mg of amylase protein, LOYTERand SCHRAMM,1962). The RNase activity was determined by incubating an appropriately diluted aliquot of a pooled sample in 1 per cent yeast RNA (Worthington Biochemicals) for 20 min at 37°C. The buffer in this assay was that of DICKMAN,AROSKARand KROPF (1956) and contained O-2 M NaCl and 0.05 M tris, pH 8.1 at 25°C. The reaction mixture also contained 0.001 per cent gelatin. The reaction was stopped by acid precipitation with 25 per cent perchloric acid containing 0.75 per cent uranyl acetate (KALNITSKY,HUMMELand DIERKS, 1959) and immediately placed on ice. Following centrifugation, a 1:30 dilution was made and the OD,,, material was determined in a Beckman DB spectrophotometer. A non-incubated (i.e. zero time) control tube was included for each sample. The standard was beef pancreatic ribonuclease (3X crystallized, Worthington Biochemicals). DNase was determined by the DNAmethyl green method of KURNICK(1950) as modified by SREEBNY,RUARKand TAMARIN(1967). The standard employed was bovine pancreatic DNase (IX crystallized, Worthington Biochemicals). DNA was determined by the diphenylamine method of DI~CHE(1955). Highly

DIURNAL

VARIATION

IN SECRETORY

COMPONENIS

OF THE RAT PAROTID

GLAND

399

polymerized DNA obtained from Worthington Biochemicals was used as the standard. RNA was determined on another aliquot of the same extraction by an orcinol method similar to that of MEJBAUM(1939). The concentrations of the reagents in the reaction mixture were 1 g% orcinol, 0.2 g% ferric alum and 22 per cent HCl. The boiling time was increased to 45 min. MILLER, GOLDERand MILLER (1951) have described the effect of these changes. In instances in which saliva was used, parotid saliva was collected by the method of ROBINOVITCH,SREEBNYand SMUCKLER(1968). The data were analyzed by the “F” test for analysis of variance. Data wherein P was less than or equal to 0.05 were considered significant. The values are expressed as the mean -& 1 standard error (S.E.). RESULTS The food intake (Fig. 1) was expressed as cumulative per cent. Between 6 p.m. and 3 a.m., the animals ate at a fairly constant rate of 8 per cent/hr. After 3 a.m. and until 8 a.m., the rate was somewhat less, but constant (ca. 4.5 per cent/hr). Between

Time

FIG. 1. Relation

of the parotid

of .day

gland wet and dry weight and food intake pattern to time of day.

8 a.m. and 6 p.m. only a minimal amount of food was eaten. The food intake over the 24 hr averaged 21.9 g. (This was 2.7 g less than the mean daily food intake noted for these animals during the previous three days.) The mean initial body weight for all groups was the same (322 f 5). During the day (5 a.m. to 5 p.m.) the body weight of the animals progressively decreased 14 g and then increased (Table 1). Unlike body weights, the wet and dry weights of the parotid gland increased progressively between 5 a.m. and 5 p.m. (Fig. 1) and then decreased. The wet weight increased 12 mg or 8 per cent, but the change was not significant. The water content of the gland did not change significantly during the day and varied between 142 A.O.B. 1414-E

L.M.

400

TABLE ~.PHYSICALAND

5 a.m. Body weight (g)

322+ 5

Parotid gland? wet wt. (mg)

194.5 4.5

9 a.m.

+

A. JOHNSON

SREEBNYANDD.

BIOCHEMICALDATA*

1 p.m.

3201 7

316+ 7

195.8 i 6-O

203.5 6.8

5 p.m. 308* 6

*

206.6 8.0

f

9 p.m.

1 a.m.

317% 7

318k 6

n.s.

198.8 f 8-O

IIS.

203.6 3.8

rt

. F-teat

50.671: I.45

53.32+ 1.89

59.14-c 2.78

58*87& 2.23

56.41 f 1.02

52*77& 1.90

0.05

DNA (mg)

1*04* 0.09

O-98+ 0.09

1.021 0.08

0.92% 0.04

1*08* 0.03

l-03* O-04

n.s.

RNA (mg)

4.44* 0.18

4*39+ 0.15

4.221 0.25

4.17% 0.21

4*29f 0.23

4*46& 0.24

n.s.

26.56& 0.98

29.02& l-14

31.37& 1.44

32*03& 0.98

29.71 f 0.77

28*06+ 0.96

O-05

5.87* 0.29

7.471 0.77

10.201 O-76

10*97& 0.52

8.63& 0.64

7*83* o-59

0.05

20*69* 0.84

21*55& 1.08

21.17& o-73

31.06* 0.75

20.23 + 0.84

ns.

dry wt. (mg)

protein (mg)

secretory

protein (mg)$

nonsecretory protein (mg)Q * t $ 9

21.081: 0.92

Each value represents the mean f 1S.E. Each value is the amount per total gland. Amylase x 3.053 (see discussion). Protein-secretory protein.

and 148 mg. The dry weight showed a sign&cant (P < 0.05) change during the day, increasing 8 a4 mg (17 per cent) between 5 a.m. and 1 to 5 p.m. Most of the changes in wet weight were a reflection of the changes in dry weight. The protein content of the gland (Table 1) showed a significant variation during the day (P < 0.05). The increase in gland protein between 5 a.m. and 5 p.m. was 5 a47 mg (21 per cent). The three secretory enzymes showed analogous changes during the day (Fig. 2). Amylase increased l-68 mg (86 per cent) between 5 a.m. and 5 p.m. The changes in amylase were significant (P < 0.01). During this same time interval, deoxyribonuclease increased 1.45 mg (124 per cent) and ribonuclease increased 5.52 pg (68 per cent). DNA per total gland (Table 1) remained constant (ca l-01 mg). RNA showed a slight but regular decrease during the day and a subsequent rise after 5 p.m. (Table 1). Qualitative evaluation of many histologic sections distinctly revealed that the acinar diameter increased between 5 a.m. and 5 p.m. (Fig. 3). There was also an apparent increase in the number of granules within each cell. Compared with 5 a.m., the nuclei of the cell at 5 p.m. were more irregular in shape and located more basally in the cell.

DIURNAL VARIATION IN SECRETORY COMPONENTS OF THE RAT PAROTID GLAND

01

5am

I

9am

1

lpm

I

5pm

Time of day

1

9pm

I

lam

401

I 5am

FIG.

2. Relation of parotid gland enzyme content to time of day. For amylase, each point represents the mean of individual determinations of seven animals; for DNase and RNase these represent the means of two determinations on pooled specimens. TABLE 2. ENZYME RAnos

Amylase : DNase 5 a.m. 9 a.m. 1 p.m. 5 p.m. 9 p.m. 1 a.m.

l-68 l-68 1.45 1.39 1.44 1.44

Amylase : RNase 239 265 300 265 245 231

DISCUSSION

A consistent observation in this study was the regularity of the diurnal pattern for parotid gland weight and individual secretory proteins. The curves for gland wet and dry weight, protein, and secretory enzymes had a low at 5 a.m. and a peak at 5 p.m. These highs and lows correlated with the changes in the eating cycle of the animals (Figs. 1 and 2). During periods of minimal food consumption (5 a.m. to 5 p.m.), the components increased; at the onset of the eating cycle (5 p.m.) each component declined. If food was withheld, this decline was not observed, and in fact, the gland weight and secretory proteins continued to increase (unpublished results). Histologic evidence showed an increase in the acinar diameter; biochemically there was no change in total DNA (Table 1). The increase in gland weight therefore

402

L. M. SREEBNY AND D. A. JOHNSON

was judged to be the result of acinar cell enlargement. DALY and MIRSKY(1952) also found no change in DNA during the secretory cycle of the rat pancreas. Sixty-six per cent of the gland dry weight increase resulted from an increase in protein. Increases in three secretory enzymes (amylase, DNase, and RNase) accounted for 55 per cent of the protein increase. The three secretory enzymes measured seemed to show the same cyclic pattern during the 24 hr. That the amount of increase for each was different was probably a reflection of differences in the rate of synthesis. But, if the pattern for each enzyme was indeed the same, the ratio of one enzyme to another should remain constant. The ratio of amylase: DNase was essentially constant (Table 2). Amylase:RNase showed the same degree of variability (ca 20 per cent) as amylase : DNase. However, unlike the amylase : DNase, the amylase : RNase showed a cyclic pattern with a low at 5 a.m. and high at 1 p.m. This pattern may be related to the effect of the ribonuclease inhibitor demonstrated in this gland (ROBINOVITCHet al., 1968). It has been shown in this laboratory that in parotid saliva, the ratio of amylase to protein is constant with up to 100 per cent increases in total gland amylase (SREEBNY and JOHNSON,1968). In this previous report the ratio was given as 0.5. This was based on using a factor of 1.9 to convert mg of glucose to the equivalent weight of maltose. BRUNERhas shown (1964) that the colour development with DNSA of 0 *794 mg of glucose is equal to that of 1 mg of maltose. Thus to obtain the equivalent weight of maltose in terms of equal colour, a factor of 1.259 is used. When this new factor is used, it is found that amylase comprises 32.8 per cent of the protein in saliva. Since the ratio of secretory enzymes to total protein in the gland and in the secretion is constant, and since the ratio of amylase to protein in the saliva is constant (O-328), the approximate amount of gland secretory protein can be calculated by multiplying the amylase value by a factor of 3 ~053. When the calculated values of secretory protein (Table 1) are subtracted from the total gland protein at each time interval, the remainder (“non-secretory protein”) is a constant. This further supports the use of this factor and indicates that the secretory proteins may be responsible for the cyclic changes in gland protein. Using the calculated value of secretory protein, amylase comprises 32.8 per cent, DNase 22 * 1 per cent, and RNase 0.1 per cent. Although essentially all of the protein increase (and thus 66 per cent of the gland dry weight increase) was due to secretory protein, the remaining 34 per cent of the dry weight increase has not been determined. It is assumed that this is also composed of secretory components and it is likely that a large fraction of this is mucoprotein. Preliminary experiments indicate that about 60 per cent of lyophilized cannulated parotid saliva is protein; thus the remaining 40 per cent consists of non-protein secretory components. MANDEL,THOMPSONand ELLISON(1964) reported that four carbohydates in human parotid saliva (hexose, hexosamine, fucose, and sialic acid) comprise, by weight, about 20 per cent that contributed by protein. PAS positive material indicative of protein-bound carbohydrates has been demonstrated in the secretory granules of the rat parotid gland (WATERHOUSE and WILLIAh$ 1967). During the 24 hr period, RNA showed a regular, though insignificant decrease from 1 a.m. to 5 p.m. The reason for this is unknown, but since RNase increases

DIURNAL

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IN SECRETORY

COMPONENTS

OF THE RAT

PAROTID

GLAND

403

as RNA decreases, the change in amount of RNA may be a reflection of some physiological process within the cell. It may, however, be an artifact resulting from RNase activity released during the processing of tissues. The changes in enzyme levels within the gland can be considered to represent the net effects of synthesis and expulsion. In the case of the ascending part of the curve, the enzyme levels increase since synthesis and storage predominate. After 5 p.m. expulsion plays the more prominent role and the enzyme levels decrease. With regard to the descending portion of the curve, irregularities in the pattern of decline were seen. The rapid decrease in enzyme levels after 5 p.m. was probably related to expulsion following gland stimulation (i.e. ingestion of food) and may also be the result of a depression in the rate of synthesis. FARBERand SIDRANSKY (1956) with the rat pancreas and GROMET-ELHANAN and WINNICK (1963) with the rat parotid showed a slight depression in the rate of amino acid incorporation during the early phase of secretion following gland stimulation. Similarly, in the mouse pancreatic system of MORRIS and DICKMAN(1960), the levels of incorporation of amylase and ribonuclease dropped significantly during the first hour after stimulation of secretion by pilocarpine administration. During the 24 hr period, the food intake for these animals was 2.7 g less than usual. Previous studies in this laboratory have demonstrated that with decreased food, the parotid gland becomes hypertrophic due to storage of secretory material (SREEBNYand JOHNSON,1968). This, along with a possible acceleration in synthesis which has been demonstrated l-4 hr after stimulation (MORRISand DICKMAN,1960; GROMFX-ELHANAN and WINNICK, 1963) may account in part for the plateau observed between 9 p.m. and 1 a.m. The drop seen in the graph between 1 a.m. and 5 a.m. is actually a “projected” drop since all graphs have been arbitrarily returned to their original 5 a.m. levels. Since food intake is less, it is quite probable that this starting level would not have been reached. Acknowledgeme&--This research project was supported by National Institutes of Health grants DE-02120 and DE-00102. R&m&-La glande parotide de rats, nourris % volont6, prksente une activitC cyclique diurne en rapport avec l’ingestion alimentaire. 11s’agit d’un changement pond&al de la glande, qui s’accompagne histologiquement d’une modification du diamktre des acini ainsi que du contenu en granules. Le contenu enzymatique en amylase, desoxyribonuclbse et ribonucl&ase, comportant 55 pour cent des protkines s&Sees, est le plus bas ii la fin du cycle alimentaire. Ces enzymes augmentent ensuite progressivement et les concentrations les plus BlewSeessont atteintes juste avant alimentation. Pendant le rapas, ces trois enzymes diminuent progressivement. Leschangementsprotbiques,observ~%danslaglande sont principalement li6s aux variations cycliques des proteines &r&es. Des essais prkliminaires indiquent que le changement de poids de la fraction glandulaire nonprotCique peut i3re lit A des phases de &r&ion. 11 semble que l’activite diurne de la glande parotide de rat soit li6e 51.la mise en rkserve et 21la s&%tion de composts entrant dans la constitution de granules de zymoghne des cellules acineuses. Zusammenfassung-Die Parotisdriisen von ad libirum gefiitterten Ratten zeigen einen von der Nahrungsaufnahme abhtingigen Tagesrhythmus. Dieser ist durch einen Wechsel des Driisengewichts charakterisiert und histologisch von entsprechenden Vertinderungen

L. M. SREEBNYAND D. A. JOHNSON

404

im Durchmesser der Azini und des Granulagenaltes begleitet. Die Konzentrationen der drei Enzyme Amylase, Desoxyribonuclease und Ribonuclease, die 55 Prozent des sekretorischen Eiweisses ausmachen, waren gegen Ende der FreBperiode niedrig. Sie stiegen dann progressiv an, und die hijchsten Konzentrationen wurden unmittelbar vor der Nahrungsaufnahme beobachtet. Wahrend der Nahrungsaufnahme fielen alle drei Enzyme fortlaufend ab. Die beobachteten Veranderungen des Driiseneiweisses waren vomehmlich das Ergebnis zyklischer Variationen im Gehalt an sekretorischem EiweiD. Vorllufige Untersuchungen deuten darauf hin, da13 die eiweil3unabhangigen Veranderungen des Driisengewichts ebenfalls von sekretorischen Komponenten abhangen. Es wird der Schlul3 gezogen, dag der Tagesrhythmus der Parotisdriise der Ratte auf eine Speicherung und Ausschiittung sekretorischer Komponenten zuriickzufiihren ist, die in den zymogenen Granula der Azinuszellen lokalisiert sind.

REFERENCES BAILEY,J. L. 1962. Miscellaneous analytical methods. In: Techniques in Protein Chemistry. Chap. 11, pp. 293-304, Elsevier, New York. BERNFELD,P. 1951. Enzymes of starch degradation and synthesis. In: Advances in EnzymoLogy (Edited by NORD, F. F.) Vol. 12, pp. 379-428, Interscience, New York. BRUNER,R. L. 1964. Determination of reducing value. 3, 5-dinitrosalicylic acid method. In: Methods of Carbohydrate Chemistry (Edited by WHISTLER,R. L.) Vol. 6, pp. 67-71, Academic Press, New York. DALY, M. M. and MIRSKY, A. E. 1952. Formation of protein in the pancreas. J. gen. Physiol. 36, M3-254.

DICKMAN,S. R., AROSKAR,J. P. and KROPF, R. B. 1956. Activation and inhibition of beef pancreas ribonuclease. Biochim. Biophys. Acta 21, 539-545. DISCHE,Z. 1955. Color reactions of nucleic acid components. In: Nucleic Acids (Edited by CHARGAFF, E. and DAVIDSON,J. N.) Chap. 9, pp. 285-305, Academic Press, New York. FARBER.E. and SIDRANSKY.H. 1956. Changes in Protein metabolism in the rat pancreas on stimulation. J. dial. Chem. 222,23?-248.

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_

FISCHER, E. H. and STEIN, E. A. 1960. a-amylases In: The Enzymes (Edited by BOYER,P., LARDY, H. and MYRBACK.K.) Ghan. 19. Vol. 4 (2nd ed.) Academic Press, New York. GROMET-ELHANAN, Z.‘and WINLICK; T. 1963. Microsomes as sites of a-amylase synthesis in the rat parotid gland. Biochim. Biophys. Acta 69, 85-96. KALNITSKY,G., HUMMEL,J. P. and DIERKS,C. 1959. Some factors which affect the enzymatic digestion of ribonucleic acid. J. biol. Chem. 234, 1512-I 516. KURNICK, N. B. 1950. The determination of desoxyribonuclease activity by methyl green; application to serum. Archs Biochem. 29, 41-53. LOWRY.0. H.. ROSEBROUGH. N. J.. FARR. A. L. and RANDALL.R. J. 1951. Protein measurement with thefolin dhenol reagent.‘J. bioi. Chem. 193,265-275. ’ LOOTER,A. and SCHRAMM,M. 1962. The glycogen-amylase complex as a means of obtaining highly purified a-amylases. Biochim. Biophys. Acta 65, 200-206. MANDEL,I. D., THOMPSON,JR., R. and ELLISON,S. A. 1964. The carbohydrate components of human submaxillary saliva. Archs oral Biol. 9, 601-609. MCMANUS, J. F. A. and MOWRY, R. W. 1960. Staining Methods, Histologic and Histochemical. p. 19, Hoeber, New York. MEIBALJM,W. 1939. Uber die Bestimmung Kleiner Pentosemengen, insbesondere in Derivaten der Adenylsaure. 2. Physioi. Chem. 258, 117-120. MILLER.G. L.. GOLDER. R. H. and MILLER.E. E. 1951. Determination of nentoses. effect of varving _ proportions of components of Bial’s cblor reagent. Analyt. Chem. 2j, 904-905. MORRIS, A. J. and DICKMAN, S. R. 1960. Biosynthesis of ribonuclease in mouse pancreas. J. biol. Chem. 235, 1404-1408.

ROBINO~ITCH,M. R., SREEBNY,L. M. and SMUCKLER,E. A. 1968. Ribonuclease and ribonuclease inhibitor of the rat parotid gland and its secretion. J. biol. Chem. 243, 3441-3446. SCHNEIDER,W. C. 1945. Phosphorus compounds in animal tissues. 1. Extraction and estimation of desoxypentose nucleic acid and of pentose nucleic acid. J. biol. Chem. 161,293-303. SREEBNY,L. M. and JOHNSON,D. A. 1968. Effect of food consistency and decreased food intake on rat parotid and pancreas. Am. J. Physiol. 215, 455-460.

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G. W. and TAMARIN, A. 1967. The distribution of deoxyribonuclease in the salivary glands and pancreas of various mammals. Archs oral Biol. 12,777-781. SREEBNY,L. M., WANAMAKER, B. B. and ROBINOVITCH, M. R. 1965. The DNase of the rat parotid gland. J. dent. Res. 44,463-466. WATERHOUSE, J. P. and WILLIAMS,R. A. D. 1967. Some carbohydrate-containing components of the parotid gland and pilocarpine-stimulated parotid saliva of the rat. Archs oral Biol. 12,99-108.

PLATE 1 OVERLEAF

DIURNAL

VARIATION

(4 FIG.

IN SECRETORY

COMPONENTS

OF THE RAT PAROTID

GLAND

(b)

3. Histology of the parotid gland: (a) = 5 a.m., (b) = 5 p.m. Haematoxylin eosin. x 1280.

and

PLATE

1

A.O.B. f.p. 406