Maturation of muscarinic agonist receptors in rat developing pancreas and its relation to maximal enzyme secretion

Life Sciences, Vol. 29, pp. 2771-2779 Printed in the U.S.A.

Pergamon Press

MATURATION OF MUSCARINIC AGONIST RECEPTORS IN RAT DEI~LOPING PANCREAS AND ITS RELATION TO MAXIMAL ENZYME SECRETION Yvan Dumont, Benoft CSt~, Louise Larose, Guy Poirier and Jean Morisset Centre de Recherches en Physiopathologie Digestive D~partement de Biologie, Facult~ des Sciences, Universit~ de Sherbrooke, Sherbrooke, P. Quebec, Canada, JIK 2RI (Received in final form November 5, 1981)

Sum~nary The true KDS of [3H](-) QNB binding to muscarinic receptors were found to be 4.13 and 6 . 4 3 x i 0 -II M in pancreas of 21 day fetal and adult rats. The competition curves of specific [3H](-) QNB bound by two antagonists have shown that the affinity of these drugs did not change with age with Hill coefficients near unity. However, with the agonist carbamylcholine as the competitive drug, a more flat curve was obtained with a Hill coefficient below unity. At least two populations of carbamylcholine binding sites were found with different KDS: a K H around 7 x 1 0 -7 M and a K L around 3 x 10 -5 M. These two populations were present during all the developmental periods studied. The ED50 of bethanechol stimulated amylase secretion did not change within the age limit studied (from Ii to 365 days). The high affinity sites for carbamylcholine would seem to be the receptor implicated in the physiological response of the pancreas. It is well established that pancreatic acinar cell secretion is modulated by the gastrointestinal hormones cholecystokinin and secretin and the parasympathetic nervous system. The modulation of the ecbolic secretion by the cholinergic muscarinic receptor was established on the basis that atropine can prevent cholinergic stimulation of pancreatic acinar cell depolarization (i), calcium efflux (2, 3), cyclic GMP accumulation (4) and enzyme secretion (5, 6). We have recently shown that [3H]-quinuclidinyl benzilate [3H]-QNB, a potent muscarinic antagonist, binds specifically to pancreas homogenates (7). We have also established that a relationship exists between the concentration of muscarinic receptors and the maximal secretory response of the pancreas to cholinergic agonist during the development of the rat, from 5 to 365 days of age (8). We have suggested from the receptor concentration measured by antagonist binding that all muscarinic receptors had to be occupied to elicit maximal secretory response of the tissue. However data derived from competition curves involving [3H]-antagonist/agonist for this pancreatic muscarinic receptor showed Hill coefficients constantly below unity, suggesting that the agonist was interacting with more than one class of receptor sites (9); such a phenomenon has also been reported in other systems (i0, ii). In the brain, a direct evaluation of muscarinic agonist binding sites with [3H]-cis-methyl-dioxalane has also shown two populations of muscarinic receptors (12). 0024-3205/81/262771-09502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

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In order to further characterize the pancreatic muscarinic receptor as a function of age, we have determined the true KDS of [3H](-) QNB for the muscarinic receptor and the affinity of agonist and antagonists to inhibit [3H](-) QNB binding and finally the affinity of bethanechol in stimulating amylase secretion. Our data suggest that the high affinity muscarinic agonist binding sites could be the population of receptors involved in the cholinergic physiological response of the exocrine pancreas. Materials and Methods Male Sprague-Dawley rats were used throughout these studies. In order to obtain 21 day fetal pancreas and those from rats aged 1 to 365 days, the procedure of Larose and Morisset (13) was followed. Before sacrifice, all animals were fasted overnight and killed by decapitation; their pancreas was then removed, placed in ice-cold 0.32 M sucrose until 1-2 g of tissue was obtained. They were then trimmed of fat tissue, lymphatic nodes and major blood vessels. The tissue was then homogenized in 0.32 M cold sucrose at low setting on a Polytron (PT 20, Brinkman Instruments) and the homogenate was filtered through four layers of cheese-cloth. Protein was measured according to Lowry et al. (14) using bovin serum albumin as a standard. DNA was extracted according to Mainz et al. (15) and determined according to Volkin and Cohn (16) with calf thymus DNA as the standard. Amylase activity was assayed according to Bernfeld (17); a unit of amylase activity is that liberating 1.0 ~mole of reducing groups calculated as maltose, per minute at 37°C. The ligand binding assay was performed according to a modification of Yamamura and Snyder's method (18) as reported by Larose et al. (7). Specific [3H] QNB binding was experimentally determined from the difference between [3H] QNB bound in the absence and presence of 1 ~M atropine sulfate. Incubations were carried out at 37oc in 25 ml of Na-K phosphate buffer 50 mM, pH 7.4, containing [3H](-) QNB at 10 -10 M and protein concentrations between 25 to 50 ~g/ml for 150 min (except for the association and dissociation studies of the [3HI(-) QNB receptor complex). The reaction was stopped by filtration through GF/B glass fibre filter followed by four rinses with 5 ml of ice-cold buffer. Samples were shaken overnight at room temperature in a scintillation cocktail solution BBS-3-toluene-Ready Solv-NA (i : 2: 5), and counted in a Beckman LS-7000 liquid scintillation spectrometer with an efficiency of 43%. Analyses of the data were performed according to the equations described by Fields et al. (19). The theoretical binding curves [3H] (-) QNB/carbamylcholine were fitted to the experimental data points by non linear least square regression analysis using a computer program. Individual data points were the mean of 3 to 6 observations in triplicate and were weighed according to the inverse of the standard error of the mean. The program provides estimates of the best-fit parameter values together with estimates of the SEM and confidence limits for each parameter. The latter estimates assume that each parameter makes an independent contribution to the residual sum of squares about the optimum solution. Incubation of pancreatic tissue for secretion studies was performed as previously described by Larese and Morisset (13). Amylase secretion was used to monitor pancreatic enzyme release under stimulation by bethanechol at different concentrations. [3H](-) QNB (30-43 Ci/mmol) was purchased from Amersham; GF/B glass fibre filters were obtained from Whatman; all other drugs and chemical products were from commercial sources.

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Results

The true dissociation constant (KD) was determined by the ratio between the rate constants of dissociation (k_l) and association (k+l) of the reeeptor-ligand complex; this method gives a K D v a l u e independent of the equilibrium and receptor concentration. As shown in Table I, the mean of 3 association and dissociation studies in 21 day fetal pancreas gives a k+l of 2 . 0 1 x 1 0 8 M -I min-i and a k_ 1 of 5 . 5 8 x 1 0 -3 min -I yielding a true K D (k_i/k+l) of 4 . 1 3 x i 0 -II M. In adult pancreas, the mean of 6 studies shows a k+ 1 of 0 . 8 4 x 1 0 8 M-I min-i and a k_ 1 of 5 . 4 0 x 1 0 -3 min -I yielding a true K D of 6 . 4 3 x i 0 -II M similar to that of fetal pancreas. The T½ of dissociation was 125 ± 8 min in the 21 day fetal pancreas and 128 ± 6 min in the 60 days old pancreas. TABLE 1 Determination of the True K D of Muscarinic Pancreatic Receptor in 21 Day Feotus and 60 Days Old Rat

DaysAge

k+ 1 M-I Min_ 1

k_ 1 Min_ 1

Foetus (3) (21)

2.01x108 ±0.80

5 . 5 8 x 1 0 -3 ± 0.26

4 . 1 3 x i 0 -II ± 1.22

0.84 x 108 ±0.18

5.40 x 10 -3 ±0.37

6.43 x i0 -II ± 0.78

60

(6)

KDM

The rate constant of association (k+l) and dissociation (k_l) of the complex [3H](-) QNB-receptor in function of time was determined by the equation presented by Fields et al. (19). The concentration of [3H](-) QNB used: i0 -I0 M, the times studied for the association: i0, 20, 30, 60, 90, 120, 180 and 240 min, and for the dissociation: i0, 20, 30, 40, 60, 90, 120 and 180 min. The dissociation was initiated after 60 min of association by the addition of atropine sulfate 10 -6 M. Numbers in parentheses represent the number of experiments. ~ Each value represents the mean ± SE. Student's t test d%d not show any difference between the foetus and the 60 days old rats. Competition studies were performed with two antagonists, atropine and QNB and one agonist, carbamylcholine. Both antagonists exhibit the same pattern of inhibition of specific [3H](-) QNB binding to fetal and post-natal pancreas (Fig. i). However, carbamylcholine exhibits a flatter competition curve than those of QNB or atropine. The Hill coefficient (NH) and the IC50 calculated from the antagonist curves are presented in Table 2. We can observe that the Hill coefficient stood near unity with the IC50 remaining the same with age. Concerning carbamylcholine, we found two populations of muscarinic agonist receptors with different KDS by analyzing the data of competition of [3H](-) QNB by carbamylcholine by a non linear regression. The KDS of the high affinity sites ranged between 2 . 2 5 x 1 0 -7 and i i . 0 x 1 0 -7 M while those of the low affinity sites stood between 1 . 4 5 x 1 0 -5 and 4 . 9 2 x 1 0 -5 M (Table 3). There are no significant differences between all ages studied in the KH, KL, the percent of low affinity sites and the Hill coefficients for carbamylcholine as evaluated by an analysis of variance.

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5O

,,=

0o0fB IJ-

-~

'f~o °°,A -12

10

LOGOFTHEDRUG 8

-6

-4

-2

FIG. i Typical experiment of inhibition of specific [3H](-) QNB binding to pancreas homogenate by (±) QNB (D), atropine sulphate (i) and carbamylcholine (o). A: fetal 21 days; B: ii days old; C: 60 days old pancreas. TABLE 2 The Concentration of Antagonist Inhibiting 50% of Specific [3H] QNB Bound and the Hill Coefficient of These Antagonists

Age

IC50 of (±) QNB a

NHb

IC50 of Atropine

NH

Foetal

(3)

1 . 9 9 x i 0 -I0 ± 0.81

0.98 ± 0.07

1 . 1 2 x l O -9 ± 0.65

1.02 ± 0.05

Ii

(4)

2 . 5 1 x i 0 -I0 ± 0.54

1.09 ± 0.04

1 . 7 8 x i 0 -9 ± 0.74

0.97 ± 0.03

60

(4)

1 . 7 7 x i 0 -I0 ± 0.67

1.03 ± 0.i0

i o 9 9 x i 0 -9 ± 0.39

1.04 ± 0.04

a. The IC50 represents the concentration of the drug inhibiting 50% of [3H] (-) QNB specifically bound at I0 -I0 M of [3H](-) QNB. bo N H is the Hill coefficient determined from the competition curves. Each value represents the m e a n ± S E . Numbers in parentheses indicate the number of experiments. No significant difference was observed between the three ages studied for the 3 parameters listed as determined by analysis of variance.

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TABLE 3 Competition of [3HI QNB Binding by Carbamylcholine

Age Days

as a Function of Age

K H x 10 -7 M b

K L X 10-5 M b

% of Low

NHC

6.06 ± 2.12

1.45 ± 0.48

50 ± 7

0.78±0.13

1 (3)

7.71± 2.18

4.93 ± 2.54

40 ± ii

0.85 ± 0.26

5 (3)

9.03 ± 3.03

2.70± 1.85

37± i0

0.46 ± 0 . i O

ii (4)

8.78± 3.82

3.39 ± 1.35

54± 8

0.61±0.06

19 (5)

7.78± 1.37

4.41± 1.54

37 ± 4

0.66 ± O . O 8

23 (3)

4.37 ± 1.36

2.11±0.34

60± 3

0.67 ±0.12

30 (3)

ii.00 ± 2.46

3.85 ±0.59

54 ± 3

0.72 ±,0.14

60 (5)

6.50 ± 1.29

2.45 ±0.37

61± 3

0.62 ±0~.16

365 (3)

2.25 ±0.75

4.78 ±1.20

55± 3

0.47 ±0.06

Foetal 21 (6) a After Birth

a. b. c.

Number of experiments Value of K high and K low given by the computer program corrected by the equation presented by Fields et al. (19). Hill coefficient calculated from the mean of the experimental data. Each value represents the mean ± SE. Analysis of variance did not show any difference within each column.

As shown in Table 4, secretion of amylase stimulated in vitro by bethanechol was also studied and the ED50 obtained from dose response curves did not change within the age limit studied. Discussion We have shown previously that homogenates from pancreas of foetus, young and older rats bind [3H](±) QNB with high affinity to a single population of muscarinic receptors (7,8). However, the KDS determined by the slope of all the Scatchard plots varied from 3 . 8 x i 0 -II M to 1 4 . 4 x i 0 - i i M (8). In order to test the validity of these variations, we have determined the true K D by measuring the rate constant of association (k+l) and dissociation (k_ l) at two different ages. Indeed, we know that the K D determined by a Scatchard plot is dependent on the concentration of muscarinic receptors (18,19), thus introducing possible experimental errors even though this relationship of K D to receptor concentration is not yet understood. In rat parotid gland for example, the K~s extrapolated as the concentration of receptors falls to zero was 4 . 0 x i 0 -~I M in the adult and 2 . 4 x i 0 -II M in the younger rats, giving values similar to those calculated from k-i/k+l ( 4 . 2 x i 0 -II M) in adult rats (20). These K D values observed in the rat parotid gland were very similar to those found by kinetic studies in the pancreas, that is 6 . 4 3 x i 0 -II M at 60 days of age and 4 . 1 3 x i 0 -II M at 21

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TABLE 4 The Affinity of Bethanechol in Stimulating Pancreatic Amylase Release as a Function of Age

Age Days

ED50 pM

Age Days

ED50 pM

ii

2.3

±0.28

23

4.13±0.72

13

3.7

±0.51

25

3.66 i 0.49

15

3.6

±0.69

27

3.80 ± 0.62

17

3 . 0 7 ± 0.71

60

4.53 ± 0.47

19

3.45 ± 0.59

365

5.30 ± 0.66

21

5.20 ± 0 . 4 6

The ED50 was determined from dose-response curves to bethanechol by measuring amylase secreted in vitro. Each dose-response curve was performed with the following concentrations of agonist: 5 x 1 0 -7 , 10 -6 , 5 x 1 0 -6 , 10 -5 , 5 x 1 0 -5 and 10 -4 M. Each value represents the mean ±SE of nine different curves with one piece of pancreas for each point on each curve. Analysis of variance showed no difference between the ED50 at all ages. days of foetal life. So it can be assumed that the variations observed previously in the K D app are probably related to the concentration of muscarinic receptors used in the assay (8). Competition curves of [3}{] QNB binding by two antagonists, QNB and atropine, gave the same IC50 for the 3 ages evaluated. The Hill coefficients were near unity for all the competition curves, being similar to those previously reported by Larose et al. (7) in adult pancreas. Those IC50 also agree well with the physiological potency of each drug in inhibiting amylase secretion stimulated by bethanechol 10 -5 M (6,7). This stability of the receptor affinity as a function of age was also observed in developing chick and avian hearts (21,22). Under our experimental conditions, we have also determined that the pancreatic receptor was stereoselective at all ages as 10 -8 M (+) benzetimide inhibited 85% of specific [3}{] QNB binding whereas at the same concentration (-) benzetimide was effective only at 10% (data not shown). Previous results (13) indicate that pancreas of 21 day foetus and newborn rats did not secrete enzymes in response to bethanechol. However, those tissues have muscarinic receptor populations similar to those of older rats (8). This dichotomy between the presence of muscarinic receptors and the unresponsiveness to cholinergic stimulation may result from a change of muscarinic agonist receptor affinity which could not be detected by a antagonist. The answer to this phenomenon does not seem to involve receptor affinity to the agonist since the ED50 of bethanechol stimulated amylase release in vitro did not change over the period of ii days to a year of age; moreover, the high and low affinity populations of receptor for

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carbamylcholine detected from [3H](-) QNB binding competition were identified at each developmental period studied without evident variation in their affinity. Only one report, in mouse hippocampus, has presented an increase in the affinity of the receptor for carbamylcholine with age, but the other regions of the brain (cortex, striatum and thalamus) present K H of 5 . 2 x 1 0 - 7 M and K L of 7 . 9 x 1 0 -5 M (23), values that are very similar to those reported in this study for the pancreas and also in rat brain (24). To explain this lack of pancreatic secretory response in early life, one would then expect that some biochemical reactions involved in the stimulus secretion coupling mature only after birth. In support of an immature limiting step posterior to the muscarinic receptor coupling, is the observation that exogenous dibutyryl cyclic AMP, 8 Br-cGMP or the calcium ionophore A-23187 can initiate enzyme secretion from foetal pancreas (25,26). We have recently established a correlation between the development of the pancreatic secretory response to a cholinergic stimulation and the total muscarinic receptor sites by using [3HI QNB as the ligand (8). This observation could suggest that all the QNB binding sites needed to be occupied for maximal enzyme secretion. However, all the competition curves of [3H] QNB by the agonist carbamylcholine gave Hill coefficients constantly below one in homogenates prepared from rats of all ages; this characteristic has also been reported in other organs of adult (i0,ii) and developing animals (21), suggesting that the agonist binds to more than one class of muscarinic receptors. This possibility has been more directly emphasized by direct evaluation of muscarinic agonist binding sites using [3H]-cis-methyldioxalane as marker in rat fore brain (12). In these studies, we have also found at least two populations of muscarinic binding sites for carbamylcholine. These two populations have the same developmental pattern since the percentage of low affinity sites for carbamylcholine was not significantly different b e t w e e n t h e different ages studied. Recently, studies performed on pancreatic dispersed acini (9) have shown that Hill coefficients calculated from secretion dose-response curves established with different agonists were near unity. These data then suggest that one population of muscarinic receptor could be implicated in enzyme secretion. It is believed that this population would involved the high affinity sites since the ED50 for the carbamylcholine-induced enzyme secretion was 4 x10-7 M, value close to the K H established for this same agonist (Table 3). It was also shown that full occupancy of the high affinity sites corresponded to maximal enzyme secretion (9). The low affinity class of receptors could be the same molecular unit but in a different state. This is suggested from studies looking at the effects of GppNHp on agonist affinity binding sites to B-adrenergic (27-30), ~-adrenergic (31), dopaminergic (32) and muscarinic (33-36) receptors. It is also possible that we are dealing with two different receptors with the low affinity class of still unknown physiological role. Further studies are thus needed to explain the possible role of the low affinity sites. Acknowledgements The authors wish to express their thanks to G. Chabot for her technical assistance and N. Vanasse for typing the manuscript. This work was supported by Grant MA-7320 from the Medical Research Council of Canada and Grant A0415 from NSERC of Canada.

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Re fe rences i. 2. 3. 4. 5. 6. 7.

8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

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L.B. ROSENBERG, W.R. ROESKE and H.I. YAMAMURA, Europ. J. Pharmacol. 56, 179-180 (1979) L.B. ROSENBERG, W.R. ROESKE and H.I. YAMAMURA, J. Biol. Chem. 255, 820823 (1980) F.J. EHLERT, H.I. YAMAMURA, D.J. TRIGGLE and W.R. ROESKE, Europ. J. Pharmacol. 61, 317-319 (1980)