Effects of Extracellular Calcium and Magnesium on ...

Effects of Extracellular Calcium and Magnesium on BileSalt-Stimulated Amylase Release from Rat Pancreatic Acini

Scand J Gastroenterol Downloaded from informahealthcare.com by University of Lund on 12/08/10 For personal use only.

R.-D. DUAN & C. ERLANSON-ALBERTSSON Dept. of Physiological Chemistry, University of Lund, Lund, Sweden

Duan R-D, Erlanson-Albertsson C. Effects of extracellular calcium and magnesium on bile-salt-stimulated amylase release from rat pancreatic acini. Scand J Gastroenterol 1986, 21, 1211-1216 The effects of extracellular calcium and magnesium on bile-salt-induced amylase release from rat pancreatic acini have been studied. The amylase releases caused by from 0.25 mM to 1.0 mM taurodeoxycholate (TDC) and by taurochenodeoxycholate (TCDC) at a concentration of more than 0.75mM were reduced by ethylenediaminetetraacetic acid (EDTA) and increased by verapamil. EDTA and verapamil had no significant effect on the taurocholate (TC) (1.0-5.0 mM)-stimulated amylase release. The inhibiting effect of EDTA began to appear already during the initial 5 min and was not parallel to any change of lactate dehydrogenase release. The TDCand TCDC-stimulated amylase release was strongly dependent on the concentrations of extracellular calcium and was only weakly dependent on extracellular magnesium. The TC-induced amylase release was slightly increased only at high concentrations of calcium and magnesium. It is suggested that the mechanism of dihydroxy bile-saltinduced amylase release from rat pancreatic acini is different from that of trihydroxy bile salt. The dihydroxy bile-salt-stimulated amylase release is dependent on extracellular calcium but does not seem to be related to the uptake of calcium by the acini. Key words: Amylase; bile salt; calcium; EDTA; lactic dehydrogenase: magnesium; rat pancreatic acini; verapamil Charlotte Erlanson-Alberrsson, M .D . , Dept. of Physiological Chemistry, Vniuersitv of Lund, Box 94, S-221 00 Lund. Sweden

Recent studies have indicated that bile salt, apart from its well-known detergent effect on cell membranes, can stimulate the secretion of water, electrolytes (1-S), and enzymes (6-from 8) isolated enterocytes and hepatocytes with a specific mechanism involving calcium transport (9-10) or a change in the cyclic AMP levels of the cells (1113). Our laboratory has reported that bile salt also has specific stimulatory effects on exocrine pancreatic acini at quite low concentrations M) without causing any damage to the cells (14). The mechanism of enzyme release from pancreatic acini by bile salts at these low concentrations is not known. The present work was designed to observe the influence of extracellular calcium and magnesium

on the bile-salt-stimulated amylase release. It was found that the effect of dihydroxy bile salt but not that of trihydroxy bile salt was calcium-dependent: the stimulation by calcium was not related in a simple manner to the uptake of calcium by the pancreatic acini.

MATERIALS AND METHODS Materials

White Sprague-Dawley rats (200-300 g) were from Anticimex, Sweden. Collagenase was obtained from Boehringer-Mannheim GmbH, FRG. Verapamil was obtained from Sigma Chemical Co., USA. The sources of other sub-

1212

R.-D.Duan & C. Erlanson-Albertsson

aliquots (0.5 ml) of acini were added to 2 ml incubation solution, and 0.5 ml of the suspension was taken and immediately centrifuged. Enzyme activities in these samples were taken as the zero Methods Preparation of pancreatic acini. The prepara- time value, which was subtracted from the values tion of pancreatic acini was in accordance with obtained after incubation. To assay total enzyme the methods of Williams et al. (15) with some activity, other aliquots (0.5 ml) of acini were modifications, as described previously (14). The added to 4.5 ml diluent solution (16) (for assay basic medium contained 103 mM NaCI, 4.78 mM amylase) or distilled water (for LDH), followed KC1, 1.16mM MgS04, l.lOmM KH2P04, by sonification by 15 sec in a B-12 sonifier with a 2.5 mg/ml D-glucose, 25 mM Hepes, 2% (v/v) probe-type sonicator. Enzyme activities in these amino acids mixture (50-fold concentrated), 1% samples were taken as total value. (v/v) vitamin mixture (100-fold concentrated), Amylase activity was assayed by the methods of and 1% (v/v) L-glutamine. The pH of the basic Ceska et al. (17,18), using the Phadebas reagent medium was 7.4. The dissociation solution con- containing blue starch polymer. Lactate dehydrotained 0.25-0.30 mg/ml collagenase, 2 mM genase (LDH) activity was assayed by the methCaCI2, and 0.1 mg/ml soybean trypsin inhibitor ods ol Amador et al. (19) and Richards et al. (STI). The standard incubation solutions con- (20), using the kit from Sigma Chemical Co. Entained 1.0 mM CaCI2, 0.5% bovine serum albu- zyme release was expressed as percentage of total min, and 0.1 mg/ml STI in basic medium. Acini activity that appeared in the medium during the obtained from one pancreas were suspended in incubation periods. The significance (P < 0.05) 20 ml incubation solution and preincubated for of the effects was determined with Student's t 60 min. test. Assay of enzymes. Unless otherwise stated, aliquots (0.5 ml) of acini were added to 2.0 ml RESULTS incubation solution containing the agents tested for 30min, 0.5 ml of the suspension was centri- Effects of ED TA on bile-salt-induced amylase fuged at 10,000rpm for 30sec, and the super- release from pancreatic acini Acini were preincubated in incubation solution natant was assayed for enzyme activities. Other


stances used in the preparation of pancreatic acini were the same as described previously (14).

1. Fig. 1 . The effect of EDTA (0.2 mM) on amylase release caused by TDC, TCDC, and TC. Acini were preincubated in Ca"-free incubation solution for 30 min and then added to standard or Ca+*-free EDTA-containing incubation solution, to incubate with bile salts for 30 min. Results are means of six separate experiments. Results are given for the standard incubation solution (M and )for Ca++free, EDTA-containing incubation solution (0-- -0).Vertical bars represent 21 SEM (as in the other figures) ('P < 0.01).


Calcium and Magnesium Effects on Amylase Release

without calcium for 30 min and was then transferred to standard incubation solution of Ca++free incubation solution containing 0.2 mM EDTA and incubated with different concentrations of sodium taurocholate (TC), taurochenodeoxycholate (TCDC), and taurodeoxycholate (TDC) for 30 min. The results are shown in Fig. 1. In the range of 0.25 to 1.O mM the TDCinduced amylase release in Ca++-free, EDTAcontaining incubation solution was less than that in the standard incubation solution. As for TCDC, the inhibition of amylase release by EDTA only appeared at a TCDC concentration greater than 0.75 mM. The amylase release stimulated by TC in both incubation solutions was not

BASAL

1213

significantly different at concentrations between 0.5 and 5.0mM. At 5.0mM concentration the ability of T C to stimulate amylase release was equal to that of TDC and TCDC. Time course of the effect of EDTA on amylase release caused by TDC and TCDC After preincubation in incubation solution without calcium, 0.1 ml of acini were added to 4 ml standard or calcium-free incubation solution containing 0.2 mM EDTA. Samples were taken at different intervals and amylase release assayed. The inhibition of amylase release by EDTA began to appear already during the initial 5 min (Fig. 2). The differences in the rate of amylase release between the calcium-containing and the EDTAcontaining incubation solutions were greater in the initial period (the slope being 5.0 and 3.0 for TDC, 7.1 and 4.0 for TCDC, respectively) and then became nearly parallel at 2 h (the slope being 0.34 and 0.26 for TDC, 0.43 and 0.33 for TCDC, respectively). Comparison of amylase and LDH release caused by bile salt and carbachol in standard and in Ca+ free ED TA-containing incubation solution The amylase and LDH releases caused by taurolithocholate (TLC) (0.5 mM), TDC (0.5 mM), TCDC (0.5 mM), tauroursodeoxycholate (TUDC) (1.0 mM), TC (2.5 mM), and carbachol (1 pM) in calcium-containing standard and in Ca++-free EDTA-containing incubation solution were measured (Fig. 3). In all cases the LDH releases in the EDTA-containing incubation solution were increased, but the change in amylase release varied with the type of bile salt used as stimulus. No significant correlation was present ( P > 0.5). + -

U-

o

1'5

io

60 Tlme (min)

120

Fig. 2. Thc. effect of EDTA (0.2 mM) on the time course of amylase release stimulated by TDC (0.75 mM) and TCDC (1 .0 mM). Acini were preincubated in Ca"-free incubation solution for 30 min. 1 ml of acini were added or to 4 ml Ca"-free EDTAto 4 ml standard (W) to incubate containing incubation solution (e--O), with TDC and TCDC. At different time intervals 0.5ml samples were taken, and amylase activity assayed. Results are means of four separate experiments.

Effect of different concentrations of Ca++ and M g + + on bile-salt-stimulated amylase release Acini were preincubated in calcium-free or magnesium-free incubation solutions for 30 min and was then added to incubation solutions containing different concentrations of calcium or magnesium and incubated with TDC (0.75 mM), TCDC (1.0 mM), or TC (2.5 mM) for 30 min. The effect of different concentrations of Ca++on


1214

R.-D. Duan & C. Erlanson-Albertsson

,-..

amylase release caused by bile salts is shown in Fig. 4. The amylase releases stimulated by TDC and TCDC increased significantlywith an increase in the concentration of calcium. There was no difference in amylase release caused by TC at concentrations of extracellular calcium between 0 and 1.0 mM. Above 1.0 mM extracellular calcium the TC-induced amylase release increased slightly, but the statistical significance ( P < 0.05) began to appear first at 2.0 mM concentration of calcium. The basal amylase release was unchanged by different concentrations of Ca+ . The effect of different concentrations of Mg++ on amylase release caused by TDC (0.75mM), TCDC (l.OmM), and TC (2.5mM) are shown in Fig. 5. The TCDC-, TDC-, and TC-induced amylase release increased slightly with an increase in the concentration of magnesium, but statistical significance ( P < 0.05) was present only at high concentrations. The basal amylase release was not significantly changed by magnesium. +

Control TOC

TCDC TUDC

TC

TLC

Carbachol

Fig. 3. The amylase and LDH release caused by TLC (0.5 mM). TDC (0.5 mM), TCDC (0.5 mM). TUDC (l.OmM), TC (1.0mM). and carbachol (lO-'M) in standard or in Ca**-free EDTA (0.2 mM)-containing incubation solution. Acini were preincubated in Ca++free incubation solution for 30 min, added to different incubation solutions containing the agents mentioned above. and incubated for 30 min. Results were means of four separate experiments ('P < 0.05).

//

3 0 501

**

Y

O

TCDC

Effect of verapamil on bile-salt-induced amylase release The pancreatic acini were preincubated in calcium-free incubation solution for 30 min, transferred to standard incubation solution with or

I

T DC TC

al 104

"

0

6.5

1:O

f.5

2.0

f5(rnM)

Ca**Concent rotion Fig. 4. Effect of different extracellular Ca++ concentrations on amylase release caused by TC (2.5 mM), TDC (0.75mM), and TCDC (1.0mM). Acini were preincubated in a Ca"-free incubation solution for 30 min, added to incubation solution containing different concentrations of Ca++,and incubated with bile salts for 30 min. The results are means of four separate experiments ('P < 0.05; * * P < 0.01).

I Mg*+ C o n c e n t r a t i o n Fig. 5 . Effect of different extracellular Mg++ concentrations on amylase release caused by TC (2.5 mM), TDC (0.75mM), and TCDC (1.0mM). Acini were preincubated in a Mg"-free incubation solution for 30 min, added to incubation solution containing different concentrations of Mg", and incubated with bile salts for 30 min. The results are means of four separate experiments ('P < 0.05).


Calcium and Magnesium Effects on Amylase Release

0

025 050 075

1 DC

TCDC

100

0

10

20

30

*O

1215

50

TC

Fig. 6 . Effect of verapamil(2 x M) on amylase release caused by TDC. TCDC. and TC. Acini were preincubated in a Ca' '-free incubation solution for 30 min. added to incubation solution with (C--O)or without (W) veraDamil. and incubated with bile salts for 30 min The' results are means of four separate experiments ( * P < 0.05).

without verapamil (2 x 10-'M), and incubated with different concentrations of TDC, TCDC, and T C for 30 min. The results are shown in Fig. 6. The amylase release stimulated by T D C at from 0.25 mM to 1.0 mM was increased, as was the amylase release stimulated by TCDC at a concentration above 0.75 mM. In contrast, the amylase release stimulated by TC at from 1.0 to 5.0mM was not significantly changed by verapamil. DISCUSSION The present work shows that the extracellular calcium influences the amylase release caused by bile salts. The effects of calcium varied with the different bile salts. In a calcium-free. EDTAcontaining medium, the amylase release caused by T D C (from 0.25 to 1.0 mM) and TCDC (above 0.75 mM) was lower than that in standard incubation solution (containing 1 mM Ca++),whereas that caused by T C (from 0.5 to 5.0 mM) was not significantly altered. The potency of the stimulated amylase release of the three bile salts was nearly the same. Although E D T A is a chelating agent both for calcium and magnesium, the effect of different concentrations of calcium and magnesium on bilesalt-stimulated amylase release showed that the amylase release caused by TDC and TCDC

increased with the concentration of extracellular calcium, whereas extracellular magnesium was without any effect. The inhibitory effect of E D T A observed in the above experiments was therefore mainly due to thexhelating of calcium. The basal amylase release in this study was not altered by removing the extracellular calcium, which is in contrast to other studies on dispersed fragments of rat pancreas (21-23). This may be due to different in vitro pancreatic preparations, as we are using acini instead of fragments. This is in agreement with the work of Gardner et al. (24). who also found no change in basal amylase release in pancreatic acini of the guinea pig by removing extracellular calcium. Why extracellular calcium is involved in the bile-salt-induced amylase release from pancreatic acini is not clear. The comparison of the change in amylase release with that in L D H release caused by bile salt in calcium-containing and in calcium-free, EDTA-containing solution (Fig. 1) excluded the possibility of damage to the acini, supporting the idea that bile salt may have a specific secretory mechanism on pancreatic acini (14). As far as we know, the effects of pancreatic secretagogues such as cholecystokinin, carbachol, vasoactive intestinal polypeptide, c-AMP, c-GMP, and A23187 on amylase release were reduced in a calcium-free medium (21,22,24,

R.-D. Duan & C . Erlanson-Albertsson


1216

25). Yet the effect of bile salt appears to be dif- REFERENCES ferent. This is because the effect of removing 1. Pope JL, Parkinson TM, Olsen JA. Biochim calcium on TDC- and TCDC-induced amylase Biophys Acta 1966, 130, 218-232 2. Binder HJ, Rawlins CL. J Clin Invest 1973, 72, release appeared already during the initial 5 min 1460-1466 and not after lOmin, as shown for other secre- 3. Bright-Asare P, Binder HJ. Gastroenterology 1973, 64, 81-88 tagogues (24). It may indicate that calcium is 4. Goerg KL, Gross M, Nell G, Kummel W, Schulz involved in a quite early step of the dihydroxy L. Arch Pharmacol 1980, 312, 91-97 bile-salt-stimulated amylase release. Although 5 . Goerg KL, Kummel W, Nell G. Digestion 1983,26, dihydroxy bile salts have some calcium ionophore 105-113 6. Bossmann B, Haschen KJ. J Clin Chem Clin Bioactivity in the intestinal brush border (9, lo), chem 1983, 21, 1-9 the increase in Ca++ uptake does not seem to 7. Billington D, Evans CE. Godfrey PP, Coleman R. be the mechanism for the dihydroxy bile-saltBiochem J 1980, 188. 321-327 induced amylase release from pancreatic acini, 8. Schmidt K, Schilmerich J , Ritter H , Schmitt I. Klin Wochenschr 1982, 60,237-242 since verapamil, a calcium channel blocker, did 9. DD, Forsyth GW. Membrane Biol 1982, not decrease the effect of TDC and TCDC. 70, 125-133 Rather, a stimulation was observed nn adding 10. Maeng DD, Forsyth GW. Digestion 1984,30. 138180 be that ver- 11. Binder HJ, Filburn C , \'olpe DT. Gastroenterology verapamil*The apamil blocks the calcium uptake rather than the 1975, 68, 503-508 calcium efflux, in this manner maintaining a high 12. Conley DRl Coyne MJ, Bonorris GG, Chung A, Schoenfield LJ. 1976, 21, 453-458 extracellular concentration of calcium and 13. ~ ~ ~TS, Phillips i ~ SF, ~ Dozois l l KR,~ Go vLw. increasing the TDC- and TCDC-induced amylase Gastroenterology 1978, 74, 11-15 14. Duan RD, Erlanson-Albertsson, C. Scand J Gastrorelease. enterol 1985. 20, 1239-1245 Calcium ions can bind to bile salt at con- 15, Williams IA, Korc M, Dormer KC. Am Physiol centrations above their critical micellar con1978. 235. E517-ES24 centration (27,28), and this binding is greater for 16. Peikin SR. Rottman AT, Batzri S, Gardner JD. Am J Physiol 1978. 235. E743-E749 the dihydroxy bile salt than for the trihydroxy bile 17. Ceska M, Birath K, Brown B. Clin Chim Acts 1979, salt (291. However, the concentrations of bile 26. 437-444 salts in this study were lower than the critical 18. Ceska M. Brown B. Birath K. Clin Chim Acta 1979, 26, 445-453 micellar concentration of the bile salts; such a 19, Amador E, LE, Wacker WF, Clin Chem calcium binding might therefore not be present 1963, 9, 391-399 20. Richards AH, Labisuki RM, Vanderlinde RE. Clin (28). Chem 1975, 21, 1018 ~~

~I

ACKNOWLEDGEMENTS Prof. Bengt Borgstrom is thanked for stimulating discussions, Ms Ruth Loven for typing the manuscript. and Mr. Arne Johansson for valuable support. This work was by a from the World Health Organization to R.-D. Duan and was also supported by grants from the Och University Of Lurid; Thorsten Segerfalks Stiftelse; and Albert

Received 23 May 1986 Accepted 19 June 1986

21. Robberecht P, Christophe I. Am J Physiol 1971, 220, 911-917 (London) 1973.235. 22. Cse KM, Clausen T. J Phvsiol . . 75-102 23. Heslers o, Fast D, Tenenhouse A, Biochim Biophys Acta 1972, 279, 561-572 24. Gardner JD, Costenheder CL, Uhlemann ER. Am J Physiol 1979, 236, E745-E762 25. Williams IA, Chandler D. Am J Physiol 1975,208, 1729-1732 26. Gardner JD, Walker MP, Rottman AJ. Am J Physiol 1980, 238, G451G466 27. Moore EW, Celic L, Ostrow JD. Gastroenterology 1982, 83, 1079-1089