Exocrine pancreatic secretion in the syrian golden hamster Mesocricetus auratus—I. Basic values

Camp. Biochem. Ph.wol., Vol. 66A, pp 473 to 477 0 Pergamon Press Ltd 1980 Prmted m Great Brawn

EXOCRINE SYRIAN

0300.9629/80,‘0701-0473802.00/O

PANCREATIC

SECRETION

IN THE

GOLDEN HAMSTER MESOCRICETUS AURATUS-I. BASIC VALUES

A. SCOTT HELGESON, PARVIZ POUR, TERENCE LAWSON and CARTER J. GRANDEAN Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha,

NB 68105 U.S.A.

(Receiced

4 July 1979)

Abstract-l. Pancreatic secretion was studied in starved and fed Syrian hamsters. 2. pH did not change over the 20-hr collection period. 3. Flow rate increased steadily through l&l2 hr at which time it remained constant or decreased slowly. 4. Protein concentration was higher in starved than in fed animals and decreased with time in both. 5. Ionic concentrations were seen to vary with K+ and Na+ remaining constant, while Ca’+ and relationship and their combined Mg ” decreased with time. HCO; and Cl- exhibited a complementary total decreased with time as did SOi- and PO:-. 6. DNA and protein synthesis were evaluated in pancreatic tissue.

INTRODUCTION

situations, will hopefully provide information about processes within pancreatic cells under various conditions. Webster and his group (Morrisset & Webster, 1970; Morrisset & Webster, 1972; Tucker & Webster, 1972; Webster et al., 1974) seem to feel this procedure possesses significance for understanding overall pancreatic function. Starved animals were examined to minimize the large individual differences between parameters measured, and hence a state of near synchronous metabolism might be achieved (Desnuelle et al., 1962).

Physiological studies of mammalian pancreas are extensive, and have included both human subjects and several species of laboratory animals (Bell et al., 1972; Bernacki er al., 1976; Hotz et al., 1975; Knoebel, 1971). For unknown reasons, the Syrian golden hamster has been excluded almost entirely from these studies, although this species has been used increasingly since 1941 in many biological research fields (Homburger, 1972). The lack of information on the physiology of the hamster pancreas is a great oversight, since this species is unique for studying pancreatic cancer induction (Pour et al., 1974). The alarmingly increased incidence of and mortality from pancreatic cancer in the United States makes elucidation of its etiology an urgent matter. Since induced tumors in hamsters resemble in many respects those in man (Pour et al., 1973, this experimental model promises an understanding of pancreatic carcinogenesis through collection of relevant data on physiological characteristics of the hamster pancreas. The findings could give insight into etiology and parameters for early detection and prevention of this disease. The present work concentrates on selected parameters of exocrine pancreatic function in starved and fed Syrian golden hamsters and is specifically conc8rned with exocrine secretion, including the rate, pH, protein and inorganic ion content of the flow, which is collected over 20 hr. A long collection period seemed essential to gain an overall view of the animals’ secretory patterns. The ions chosen for examination were potentially affected by changes in the state of ductular cells, and since the carcinogenic processes under our scrutiny have definite effects upon duct cell homeostasis, it is hoped there will be discernible differences in the way these ions are secreted under pathological, as opposed to non-pathological conditions. DNA and protein synthesis, while impractical as indicators of pancreatic dysfunction in most clinical

MATERIALS AND METHODS Outbred S-week-old Syrian golden hamsters from the Eppley Colony were used. They were housed in plastic cages (Macrolon) on San-i-ccl bedding in groups of five by sex and maintained under standard laboratory conditions (room temperature, 22 + 2°C; humidity, 55 k 5%; lightdark cycle, 12 hr-I 2 hr; 10 x air changes per hour). The animals were given Wayne pelleted diet (Allied Mills, Chicago, Ill.) and water ad lihitum, and weighed daily. At the time of use the average body weight for males was 130 g, while for females it was 125 g. Pancreatic juice was collected regularly in the two hamster groups at selected times after cannulation of the distal common pancreatic duct and ligation of the common bile duct (for detailed anatomy of the hamster pancreas, see Takahashi et al., 1977), after sodium pentobarbital anesthesia (75 mg/kg). One group of animals had free access to food and water, whereas in the second group animals were not allowed food for 20 hr prior to surgery. After surgery both groups had free access to water, but were denied food for the 20-hr collection period. Collections were started approximately 1 hr after surgery to reduce the influence of anesthesia on pancreas function (Peterson & Grossman, 1977). After 1 hr the hamsters were fully alert and active again. Secretions were collected in tared vials, weighed and converted to volumes using the average specifii gravity (1.056) determined in all samples. Collected material was maintained in an ice bath at 0°C throughout the time period and then frozen at - 10°C. At the end of each collection period, a new tared vial was set into place. Collection periods were as follows: Period I, I1 am-2p.m.; 473

474

A. SCOTT HELGESON et al

period 2, 2 pm-5 p.m.; period 3, 5 p.m.-9 p.m. and period 4, 9 p.m.-7 a.m. After 20 hr of collection, animals were sacrificed, their pancreas were excised and fixed in 10% buffered formalin and prepared for histology by conventional methods. Pancreatic juice smears were done during each collection period to evaluate the gross cellular content of pancreatic secretions. The quantity of cells was estimated by + (1-3 cells), + + (?-lo cells) and + + + (over 10 cells) per IO-power field. pH Values were measured after collected samples thawed. Total protein was determined by the method of Lowry et al., 1951). Protein and DNA synthesis was measured by intraperitoneal injections of 0.25 pCi/kg body wt [3H]phenylalanine or C3H]thymidine (New England Nuclear, Boston), to fed and fasted animals of both sexes. respectively. After 15 min the animals were decapitated and the pancreases removed. Pancreatic DNA and protein were isolated by the methods of Shibko et al. (1967). The 15-min incorporation period resulted in adequate uptake and yet was short enough not to complicate timed procedures planned for later. Aliquots of DNA or protein were dissolved in Aquasol (New England Nuclear, Boston, Mass.) in scintillation

vials and their radioactivity was determined by counting with a liquid scintillation counter (Beckman, LS 335). Ionic concentrations of potassium (K’), sodium (Nat). calcium (Ca”) and magnesium (Mg”) were quantitated by atomic absorption (Perkin Elmer 303, Perkin Elmer Co.). Chloride (Cl-) and bicarbonate (HCO,) were quantitated using an Oxford titrator and supplied reagent sets (Oxford Laboratories, Foster City, California). Phosphate (HPO:-) and sulfate (SO:-) were quantitated by two modifications of the method of Weissman & Pileggi (1974).

sodium was at twice the concentration in (S) as in (F). Calcium levels varied between 1.75-fold to 2.15-fold higher in (F) than (S), depending upon the collection period. Magnesium levels began at a 3.5-fold greater value in (F) than in (S), then dropped to l&fold greater value by period 4. Bicarbonate levels in (F) were 3 to 4-fold greater than in (S). while chloride levels were relatively constant between both groups, with an obvious reciprocity in the bicarbonate and chloride levels, as reported in the literature (Ribet et al., 1968; Rothman & Brooks, 1966; Sarles, 1977; Scratcherd & Case, 1973). Phosphate levels were generally the same, except for the decrease at period 2 in (F). Sulfate values, on the other hand were IO-fold greater in (F) than in (S). Although the results are only preliminary at this time, the rate of DNA and protein biosynthesis are basic parameters to which future results may be compared. Therefore, their syntheses are included to see what the basal levels are and if they were affected by availability of food. No difference was found for DNA synthesis between male (F) or (S) animals. The amount of incorporation was 18 + 9 dpm/pg DNA. The level of synthesis in both (F) and (S) female animals was higher than in males and was slightly starvation upon (49 f 30 vs depressed 38 f 36 dpm/pg DNA, respectively). Protein synthesis in both sexes was 50dpm/pg protein for (S) animals and 100 dpm/pg protein for (F) animals.

RESULTS

DISCUSSION

Tables 1 and 2 give results concerning pH, flow rate, protein concentration and excretion rate, and ionic concentration of secretions. Table 1 shows that pH was relatively constant, regardless of whether animals were recently fed. The fed animals had a consistent but slightly higher pH value. The greatest variation in flow rate from basal to peak was in starved animals that had 44% of the maximum. There was an obvious difference between protein concentration in fed (F) and starved (S) secretions, as starved was five-fold greater than fed. Total protein secreted per hour reflected this difference in concentration in a similar fashion. Potassium levels (see Table 2), although slightly higher in the fed animals, fluctuated similarly in all collection periods for both (F) and (S) animals, while

Table 1. Normal

values of pH*, flow rate* and protein* in fed (F) and starved male hamsters Flowt (~l/hr/lMg.b.w.) F

PH Period Period

1

Penod

2

Perwd

3

Period

4

Time

Sex

Hourst o-3 Hoursf 3-6 Hovrsf 6-10 Hoursf IO-20

F M F

M F M F M

The overall objective of these studies was to establish as many basic parameters of Syrian golden hamster pancreatic physiology, as was feasible. These values then would serve as the basis upon which future experiments would be compared. Rather wide variations were seen. However, these could be expected (Desnuelle et al., 1962) and do not detract from the overall objective. Previous studies on pancreatic secretory activity, whether or not juice was collected exactly by our methods, in man and in other animals have been of short duration (seldom longer than 6 hr per procedure) (Kanno et al., 1977; Lowry et al., 1951; Reber et al., 1977), which does not take into account the animal’s diurnal, nocturnal physiological variations. We chose an overall collection time of 20 hr to allow

F 9.24 9.24 9 33 9.11 9.35 9.27 9.46 9.25

* f + + + + * k

S 0 15 0.16 0.18 0.25 0.20 0.1 0.37 0.24

I

8.87 8.71 9.14 9.15 9.04 9.17 8.94 9.06

* * * i + * * f

0.51 0.15 0.37 0.46 0.64 0.30 0.35 0.56

83.00 79.00 98.M) 105.00 136.00 105.00 137.00 93.00

* + k f + * * +

50 45 36 45 32 20 44 19

Protein concentratwn (mg/ml) F s

S

58.5 f 645 * 78.1 * 90.0 * 137.0 k 165.0 + 113.0 A NO+36

(S) g-week-old

22 9.75 21 20.8 26.4 24 3 32 3

5.73 5.24 4.36 3.80 3.06 3.41 2 62 2.67

* * f + * i + +

Values represent mean k SD of a minimum of seven animals. t Computed on a basis of 1.056 specific gravity average for hamster $ Zero time corresponds to about 1 hr post surgery. 6 Average body weight at time of use was 3 130 g, P 125 g.

2.15 0.85 1.17 1.27 0 78 1.46 0.69 0.9

24.6 22.3 194 18.4 16.5 14.1 10.9 11.2

PVJlCl” (,zg/hr’lOOg.b.w.k F S

* 1.97 _I 4.53 * 4.5 _i 4 02 _e 2.1 i 544 * 4.9 * 4.33

473 414 427 398 413 359 360 249

*

pancreatic

female and

secretions.

* f f k i i k k

123 38 4: 56 25 29 30 17

1440 1460 1512.5 1660 2270 2330 1238 1010

* 220 + 291 k 464 + 398 i 276 _+ 660 + 793 + 780

Exocrine pancreatic secretionTable 2. Ionic concentration Time

Sex

K’

Nat

415

I

of pancreatic secretions in 8-week-old fed (F) and starved (S) hamsters caz *

Mg’ *

Cl

W-0;

HPO:

so:-

Fed: HOUIS 03 HOUR 3d HOUIS 6-10 HOUS l&20 srartd

F M F M F M F M

17.65 10.67 14.32 II.18 II.94 13.35 17.67 14.25

k 0.63 +T0.36 * 8.08 i 033 * 1.53 5 3 89 * 2.71 k 6 57

41.65 42.44 43.57 40.96 43.61 42.22 4.17 42.74

f 1.7 + 3 * 2.04 f 0.61 k 3.13 k 1.57 52.13 k 3.91

HOIXS o-3 H0UlS 3-6 HOUS 6.10 HOUCS 1O-20

F M F M F M F M

12.61 13.3 12.3 9.69 15.9 10.23 16.73 13.04

+ f * + + + f k

98.34 97 26 93.78 90 92.9 93 96 92.5 94.1

+ k k * + + + f

45 4.35 4.55 2.86 8.95 2.22 4.71 4.3

3.57 5.35 9.57 13.83 0.96 5.78 4.35 6.78

12.13 9.43 8.83 9.58 7.6 7.34 7.19 8 13

* * + k k + k +

195 1.55 0.75 I.8 18 1.4 2.45 2.25

14.6 17.19 10.53 8.47 798 6.42 5.68 62

7.64 8 54 4.24 3.7 3.14 3.29 3.29 3.94

+ i & * f k + *

I 75 1.3 I.15 I.13 1.5 I 51 1.15 I2

4 28 4.85 3.78 4.03 3.04 362 3.13 3 04

k k * f + k + k

2.71 8.64 3.04 1.07 1.9 1.65 1.48 1.56

+ 0.535 + 0.494 k 0.66 k 0 92 k 0.61 kO82 f 0.51 * 0.74

143.1 2M) 160.6 209.5 231.6 2293 175.4 197

* 100 * loo * 101.5 f 84.6 k 133 4 k 765 i 141.4 +_ 29 I

80 84 67 2 56 22 53.85 45.04 45.02 48 74 47 7

+ k f + k i + *

25.6 17.94 19.68 17.21 I7 83 I7 13.K 19 24

29.13 312 45 82 3704 51.66 51 87 54 5402

2 * + + + ? f f

144 1432 18.33 17.52 13.34 4 59 41.2 3.6

8444 74.5 64.61 60.16 514 51.11 55 I9 4394

f 294 i 20.6 k 1867 * 13.31 i- 1286 *Y I i 77 _c 173

1.0 * 0 13 0833 k 0.138 0.4 k 0 032 0.46 k 0.23 0667kOl53 0.671 k 0 13 0 473 * 0.025 0.46 * 0 071

0.07 0.06 0077 0064 0034 0 034 0.048 0.036

i k * f f f i i

0.014 0.029 0033 0.037 0014 0.02 0.014 0.0094

114*044 117*05 IO8 *035 117*0344 0.913 f 03R 0.83 + 0 37 0 7R + 0 26 04R + 030

0.0092 f 0.0104 i OOlc4 f OOOYK+ O.W63 f 00052 + 00123 f 00096 t

0 005 0004 0.005 0.005 0.0058 0.0037 00052 0073

Values represent mean + SD for two minimum of nine animals and are expressed in MEgIl.

for effects, if any, of the circadian patterns in physiological activity upon the process of pancreatic exocrine secretion. Flow rate (given for (F) and (S) males and females in Table 1) increased gradually in both starved and fed hamsters until late evening, whereupon it began to decrease slightly. The pH remained constant at or around 9.0, which is close to the pH reported for pancreatic secretions in other studies (Knoebel, 1971; Rawls et al., 1963). Protein concentration in (F) hamsters correlated with data in man and other animals, including other hamster species, dogs, and cats (Bernacki et al., 1976; Knoebel, 1971; Reber et al., 1977: Tucker & Webster, 1972). It is known that food deprivation in man may cause atrophy of the entire pancreas with a loss of protein and vacuolization of tissue (Veghelyi & Kemeny, 1962). Within 2 days, as much as 20”/:, of pancreatic weight is lost during food deprivation, we found considerably more cellular debris (e.g. actual broken cells and dead cells) in the secretions of (S) animals than in (F) animals. (We also found that the pancreas of fasted hamsters was histologically altered due to vacuolization and concluded the increased protein content of (S) secretion could well be entirely due to structural debris.) Considering the ionic patterns (Table 2), the same relationships were seen in similar studies. For example, calcium concentration in (F) hamsters and other mammals (Sarles, 1977) shares a direct relationship with protein concentration, and is similarly seen in the (S) hamsters. However, it is apparent that the ratio of calcium to protein is not constant, and the higher protein concentration in (S) animals without a proportionally high Ca2’ level can be partially explained by the morphological alterations in (S) hamsters. That is, the excess protein in (S) is not physiological but instead consists of cell structural debris which should not be associated with as great an amount of calcium. Despite the fact that calcium concentration is lower in (S) than in (F) hamsters, it is still parallel to protein concentration and leads us to conclude that the calcium-protein relationship in (S) still holds. Fluctuation of magnesium and potassium levels and their comparability in both (F) and (S) animals correlated with reported data in other species (Meldo-

lesi. 1976; Sarles, 1977; Sullivan rf al., 1974; White et al.. 1977), with an overall trend for potassium to remain at a semi-constant level. As with Ca2+, magnesium concentration in both groups decreased with time. The reason for constant sodium concentration in hamsters, as in other species (Sarles. 1977; Scratcherd & Case, 1973; Sullivan et al.. 1974; White et al., 1977) compared to potassium values, could be due to the different secretory mechanisms into the ductal system, i.e., active secretion of Na’ as opposed to simple diffusion of K’ from the extracellular space (Scratcherd & Case, 1973: Sullivan et al.. 1974). The overall high value of Nat in (S) hamsters might result from its increased release by or from degenerating pancreatic cells due to the animal’s state of starvation. This may also explain the difference between (S) and (F), with regard to bicarbonate concentration, since cells responsible for secretion of this important anion may have been functionally altered in (S) animals, The known reciprocal relationship (Ribet et al., 1968; Rothman & Brooks, 1966; White et al., 1977) between HCO, and Cl- was maintained in both groups. Since these two anions, considered the most significant in pancreatic juice, are secreted actively, their patterns could be a measure of the competence of ductular cells (Bell et al., 1972: Sullivan et uI.. 1974). Phosphate was higher in (S) and sulfate higher in (F) hamsters. However. in both groups there was a reciprocity between the two, although not as obvious as that between chloride and bicarbonate. The importance of the reciprocal relationship between sulfate and phosphate has yet to be discovered. An examination of pancreatic DNA synthesis showed no differences between (F) and (S) hamsters, but a large difference between males and females. Protein synthesis activity in (F) hamsters was twice that in (S) hamsters, a result which we would expect according to Veghelyi (Veghelyi & Kemeny, 1962) and Sarles (1977) who found that fasting does depress protein synthesis in the pancreas. Basically the following trends were observed for the pancreatic secretory physiology of the Syrian golden hamster: (1) pH remained constant at about 9.0 over the entire 20-hr collection period, regardless of food intake or lack thereof. (2) Flow rate increased steadily

A. Srorr HELGESON ri ~1,

476

for IO-12 hr, then remained constant or decreased slowly for (F) and (S) animals in the final 8 hr. (3) Protein concentration was significantly higher in (S) than in (Ff hamsters, yet followed the same pattern of decreasing with time in both groups and reaching its Iowest point between 9p.m. and 7 a.m. {4) K+ and Na+ ions remained at relatively constant concentrations in (F) and (S) groups, with K” fluctuating more. (5) In both groups, CaZi and MS’+ concentration decreased with time. (6) HCO; and Cl- exhibited a complementary relationship, with a tendency for the combined total of both to increase with time. (7) HPOi- and SOi- had a similar relationship to HCO; and Cl- ; however, the tendency was for the combined total of sulfate and phosphate to decrease with time. (8) There were no significant differences for any of the above measured parameters between male and female hamsters in both groups. (9) In comparing the data between (F) and (S) hamsters, a significantly higher value for protein, Nat and SO:- eoncentration and tower levels of Ca’*, Mg” * and HCO; were found in (S) hamsters. There is no doubt that investigations concerning pancreatic function are worthwhile and constantly generate more interest (Allen & White, 1975; Kanno & Yamm~moto, 1977; Reber er at., 1977; Sulivan ef al., 1974; White et al., 1977). By establjshing numerous basic parameters in hamster pancreatic physiology, we have initiated a program upon which future comparisons can be made. We are currently expanding the basic information while also investigating how the diseased pancreas affects these parameters.

SUMMARY

Pancreatic secretion was studied in starved and fed Syrian hamsters. Examination of the collected pancreatic secretions showed no sign;~~ant pH changes. A steadily increasing flowrate was seen for 10-12 hr, at which time Row remained constant or slowly decreased. While protein concentration was signifi~ntly higher in starved than in fed animals, it decreased with time and reached its lowest point between 12 and 20 hr after collection began. K* and Na+ remained at eons&m ~on~n~at~on in both groups, while CaZt and Mgz+ decreased with time. HCO; and Cl- exhibited a complementary relationship, with their combined total increasing with time. HPO:and SO:-, while showing a complementary relationship as well, had a combined total which decreased with time. Starved hamsters, as a goup, showed a sj~i~~tiy higher value for protein, Na* and SO:and lower levels of Ca 2+, Mg2’ and HCO;. DNA synthesis was measured in the pancreas of starved and fed hamster groups and no difference was seen between them. Analysis of protein synthesis determinations revealed that this activity in fasted pancreas was half that of non-fasted. Histologicaf abservations demonstrated that fasting is detr~menra~ to pancreatic structure. Acknowledgements-This study was supported by a grant from the National Pancreatic Cancer Project No. 5-R26-CA20198-01. The authors wish to thank Thorkatla

Donnelly for her surgical expertise, Lance Hines for technical aid and Mardelle Susman for her editorial assistance.

REFERENCES

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