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Comparative inhibitory effects of pirenzepine and atropine on cholinergic stimulation of exocrine and endocrine rat pancreas
GASTROENTEROLOGY
1985;89:408-14
Comparative Inhibitory Effects of Pirenzepine and Atropine on Cholinergic Stimulation of Exocrine and Endocrine Rat Pancreas MAKOTO OTSUKI, TAKAHIKO NAKAMURA, YOSHINORI OKABAYASHI, TORU OKA, MASATOSHI FUJII, and SHIGEAKI BABA Second Department of Internal Chuo-ku, Kobe 650, Japan
Medicine,
Kobe University
The effects of pirenzepine on carbamylcholine-stimulated exocrine and endocrine pancreatic functions were compared with those of atropine in both the isolated pancreatic acini and the isolated perfused pancreas of rats. In the isolated acini pirenzepine and atropine produced a concentration-dependent inhibition of amylase secretion initiated by carbamylcholine. This inhibition resulted in a rightward shift in the dose-response curve for carbamylcholine-stimulated amylase secretion without altering the maximal increase in amylase secretion. Pirenzepine was, however, -300 times less potent than atropine in inhibiting the stimulated amylase release. A similar difference in potency was observed with respect to carbamylcholine stimulation of pancreatic juice, amyiase, and insulin release from the isolated perfused pancreas. The maximal inhibitory concentration of pirenzepine on a maximal effective concentration of carbamylcholine for stimulating pancreatic exocrine secretion was 10 PM, whereas that of atropine was 30 nM. The present data define the pirenzepine receptors in the exocrine and endocrine pancreas as low-affinity-type receptors. Pirenzepine is a tricyclic, pyridobenzodiazepine derivative that has been shown to reduce gastric acid secretion (l-5). There is strong evidence that the mechanism of action of pirenzepine involves the blockade of muscarinic receptors (6-8). Its inhibitory effect on gastric acid secretion seems similar to those of classical anticholinergics and it is used in Received August 20, 1984. Accepted February 14, 1985. Address requests for reprints to: Dr. Makoto Otsuki, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. 0 1985 by the American Gastroenterological Association 0016-5085/85/$3.30
School
of Medicine,
Kusunoki-cho,
Europe and Japan to treat patients with peptic ulcer (3-5). Hammer et al. (6,7,9) have demonstrated that pirenzepine distinguishes subclasses of muscarinic receptors within one tissue and also binds to receptors of various tissues with a wide heterogeneity of affinities. In agreement with these in vitro studies, clinical experience has shown that pirenzepine has more selective antisecretory properties on gastric secretion, producing less side effects, than conventional anticholinergic drugs (8). Although it is accepted that pirenzepine reduces gastric acid secretion by an antimuscarinic mechanism, considerable controversy exists on the effects of pancreatic exocrine secretion (10-13). Several investigators observed the inhibitory effects of pirenzepine on pancreatic juice (12)and enzyme secretion (ll-13), whereas others failed to demonstrate the decrease of secretory volume (10,ll)and bicarbonate output (11,12). The effectiveness of pirenzepine on muscarinic receptors in the exocrine and endocrine pancreas is still unknown. The present study was therefore undertaken to further evaluate the antimuscarinic properties of pirenzepine on carbamylcholine-stimulated exocrine and endocrine functions and to define muscarinic receptors subtypes in the exocrine and endocrine pancreas in both the isolated pancreatic acini and the isolated perfused pancreas. Materials
and Methods
Materials The following materials were used in this study: carbamylcholine chloride (carbachol), atropine, and soybean trypsin inhibitor (type 1-S) from Sigma Chemical Co., Abbreviation
used in this paper:
IRI, immunoreactive
insulin.
August 1985
PIRENZIPINE AND PANCREATIC FUNCTION
St. Louis, MO.; chromatographically purified collagenase from Worthington Biochemicals, Freehold, N.J.; minimal Eagle’s medium amino acid supplement from Grand Island Biological, Grand Island, N.Y.; bovine plasma albumin (fraction V) from Miles Laboratories, Elkhart, Ind. and from Reheis, Chicago, Ill.; Dextran T-70 from Pharmacia Fine Chemicals, Uppsala, Sweden; Phadebas amylase test from Shionogi Pharmaceutical Co., Osaka, Japan, Pirenzepine was kindly provided by Nippon Boehringer Ingelheim Co., Kawanishi, Japan. Synthetic cholecystokinin-octapeptide was a gift from Dr. Miguel Ondetti of the Squibb Institute for Medical Research, Princeton, N.J.
Animals Male Wistar rats weighing -250 g were used throughout the experiment. The animals were kept at 23°C on a 12-h light-dark cycle and had free access to water and a standard laboratory diet.
Isolated Release
Pancreatic
Acini
and
AmyJase
The isolated pancreatic acini were prepared by the methods reported previously (13,14). In brief, the pancreas was injected with Krebs-Henseleit bicarbonate buffer containing minimal Eagle’s medium amino acid supplement, 40-45 U/ml purified collagenase, 2 mg/ml bovine plasma albumin, and 0.1 mg/ml soybean trypsin inhibitor. The injected pancreatic tissue was incubated at 37°C with shaking 120 times/min for 50 min, followed by mechanical disruption of the tissue by forceful pipetting through plastic pipettes. Acini were then purified by filtration through 150~pm nylon cloth and centrifugation through 4% bovine plasma albumin. Acini were preincubated for 60 min in N-2-hydroxyethylpiperazine-N’-2-ethane-sulfonic acid (HEPES)-buffered Ringer solution. This solution was similar to Krebs-Henseleit bicarbonate buffer but contained 10 mM HEPES (pH 7.4) as buffer and 0.5% bovine plasma albumin and was gassed with 100% 02, After preincubation, acini were centrifuged and resuspended in fresh HEPES-buffered Ringer solution at a density of 0.3-0.4 mg protein/ml. Aliquots (2 ml) were distributed into 25-ml polycarbonate Erlenmeyer flasks, secretagogues were added in the presence or absence of pirenzepine, and the flasks were incubated at 37°C with shaking at 60 times/min. Amylase release in response to various concentrations of carbamylcholine or cholecystokinin-octapeptide was determined using the procedure reported previously (14,15). In all experiments triplicate flasks were used to determine the amylase release stimulated by each concentration of secretagogue.
Isolated Perfused Pancreas and Exocrine and
Endocrine
Secretions
The isolated and perfused rat pancreas was prepared as previously reported (16). The perfusate was Krebs-Ringer bicarbonate solution containing 4.6% Dex-
409
tran T-70, 0.25% bovine plasma albumin, and 2.8 mM glucose. It was gassed with a 95% 02-5% COz mixture and adjusted to a pH of 7.4. The inlets of the vascular perfusion were the superior mesenteric and celiac arteries, and the outlet was the portal vein. The flow rate through the pancreas was kept constant at 2 mlimin. After a single passage through the pancreas, the complete effluent was collected in chilled tubes at l-min intervals for measurement of insulin concentration. To measure the pancreatic exocrine secretion, a calibrated capillary tube was attached to the free end of the pancreatic cannula, which was inserted into the distal end of the common duct at a point shortly before its entrance into the duodenum, and tied in place. The proximal end of the bile duct was ligated. The capillary tube was replaced every 10 min to measure the flow rate of the pancreatic juice. The sample of pancreatic juice was diluted with 5% bovine plasma albumin and stored at -20°C for subsequent assay. Experiments were performed after a 40-min equilibration period. The glucose concentration was then changed to 8.3 mM. Ten minutes later, carbamylcholine was added at a concentration of 1 PM for 40 or 60 min. Pirenzepine or atropine was superimposed for 20 min on carbamylcholine stimulation.
Assay Amylase activity in the incubation medium, sonicated acini, and the pancreatic juice was determined by a chromogenic method with the Phadebas amylase test (17). Amylase release from the isolated acini was expressed as the percentage of the total content of enzyme in the acini at the beginning of the incubation that was released, whereas the amylase release from the isolated perfused pancreas was expressed as Somogyi units per 10 min. Immunoreactive insulin (IRI) in the portal effluent was measured by polyethylene glycol radioimmunoassay (18) using crystalline rat insulin as a reference standard.
Statistics Results were expressed as mean f standard error (SE]. For a statistical evaluation of the difference between mean values of the various groups of experiments, the Wilcoxon rank sum test was used. For comparison of data from various levels within the individual experiments, Wilcoxon’s signed-rank test for paired samples was used.
Results The effect of pirenzepine and atropine was tested on a maximal effective concentration of carbamylcholine (1 PM) for stimulating amylase secretion from the isolated rat pancreatic acini (15). Pirenzepine and atropine produced a concentration-dependent inhibition of amylase secretion initiated by 1 PM carbamylcholine (Figure 1). From Figure 1 the IDea for atropine was estimated at 3.2 nM and for
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OTSUKI
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ET AL.
10-10 10-g 10-E 10-7 10-6 10-s Antagonist
Figure
GASTROENTEROLOGY
Concentration
10-d
(M)
I. Effects of pirenzepine and atropine on carbamylcholine-stimulated amylase release from the isolated pancreatic acini. The acini were incubated for 30 min at 37°C with various concentrations of pirenzepine or In each experiatropine plus 1 PM carbamylcholine. ment each value was determined in triplicate, and the results shown are mean * SE from four separate experiments.
pirenzepine at 890 nM. Pirenzepine was, therefore, about 300-fold less potent than atropine on a molar basis in inhibiting the carbamylcholine-stimulated amylase secretion. The secretory effect of 100 pM cholecystokinin-octapeptide (18.0% + 0.6% n = 4) was not impaired by 100 nM atropine (18.3% * 1.2%, n = 4) nor by 10 PM pirenzepine (18.5% ? 1.2%, n = 4). Neither atropine nor pirenzepine modified basal amylase release. In the acini incubated with increasing concentration of carbamylcholine, amylase secretion increased, became maximal with 1 PM carbamylcholine, and then decreased at carbamylcholine concentrations >l PM (Figure 2). Atropine and pirenzepine caused a rightward shift in the doseresponse curve for carbamylcholine-stimulated amylase secretion. The concentration of carbamylcholine required to induce a half-maximal release in the presence of 10 ~_LM pirenzepine was 12 PM, whereas that in the presence of 0.1 PM atropine was 36 PM. Thus, in this protocol pirenzepine was also 300-fold less potent than atropine. The inhibitory action of pirenzepine was further investigated in dynamic experiments with the isolated perfused rat pancreas. Coaddition of 10 PM pirenzepine and 1 PM carbamylcholine to the perfused pancreas for 20 min resulted in a complete inhibition of pancreatic juice and amylase secretion (Table 1). When pirenzepine was removed from the perfusate, the flow rate of pancreatic juice increased rapidly. In contrast to the flow of pancreatic juice,
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the recovery of amylase output after the removal of pirenzepine was slow. The amylase output in response to the carbamylcholine stimulation from the pirenzepine-pretreated pancreas was significantly lower than that with carbamylcholine stimulation alone in the corresponding period. When 1 PM carbamylcholine was infused in the presence of 8.3 mM glucose, two distinctly different phases of IRI release appeared. There was an early and rapid release of IRI that subsided within -2 min, followed by a late and slow release phase that continuously increased in rate until termination of the carbamylcholine infusion. Both phases of the carbamylcholine-induced IRI release were affected by an addition of pirenzepine. The cumulative output of IRI for the 20-min period during pirenzepine addition was significantly lower than that with carbamylcholine stimulation alone, but was similar to that with 8.3 mM glucose alone in the corresponding period (Table 1). After termination of the pirenzepine infusion, a biphasic IRI increase in response to the carbamylcholine stimulation was observed. The cumulative output of IRI for the 20-min period after the removal of pirenzepine was significantly lower than that with carbamylcholine stimulation alone in the corresponding period but was similar to that in the first 20 min. Addition of pirenzepine after a 20-min perfusion with 1 PM carbamylcholine produced a concentration-dependent inhibition of the stimulatory effects
20 r
I
0
1o-7 10-s 1o-5 10-d 10-j Carbamylcholine
Figure
10-2
(M)
2. Effects of pirenzepine and atropine on carbamylcholine-stimulated amylase release from the isolated pancreatic acini. The acini were incubated for 30 min at 37°C with various concentrations of carbamylcholine alone, carbamylcholine plus pirenzepine, or carbamylcholine plus atropine. In each experiment each value was determined in triplicate, and the results shown are mean 2 SE from four separate experiments.
August 1985
Table
PIRENZIPINE AND PANCREATIC FUNCTION
411
1. Cumulative Outputs of Pancreatic Juice, Amylase, and Immunoreactive Insulin During a N-Minute Perfusion With 1 PM Carbamylcholine With or Without an Addition of 10 PM Pirenzepine Time after the start of experiments Carbamylcholine k pirenzepine (21-40 min)
Carbamylcholine (41-60 min)
Pancreatic juice (~1/20 min) Carbamylcholine alone (5) Carbamylcholine plus pirenzepine
(5)
65.7 t 1.7 21.4 t 2.7”
54.1 2 1.5 69.9 k 2.6a
Amylase (SUIIO min) Carbamylcholine alone (5) Carbamylcholine plus pirenzepine
(5)
8906.1 2 803.4 1157.5 r 145.3”
7256.8 ? 649.4 4440.1 rfr 649.4”
1032.6 + 108.8 242.4 t 24.2a 214.7 t 33.1
1717.6 L 81.5 1056.8 ? 96.5” 307.3 c 49.9
IRI (ng/20 min) Carbamylcholine alone (5) Carbamylcholine plus pirenzepine Glucose 8.3 mM alone 141
(5)
Carbamy!choline was added from 20 to 80 min in the presence of 8.3 mM glucose and pirenzepine was superimposed on carbamylcholine stimulation for XI min from 20 to 40 min. Values are mean + SE of the number of experiments indicated in parentheses. Pancreatic juice, amylase, and immunoreactive insulin (IRI] secretions were measured simultaneously in the same experimental preparation. SU, Somogyi units. a Significant difference vs. carbamylcholine stimulation alone in the corresponding period (p < 0.05).
of carbamylcholine on the secretion of pancreatic juice and amylase (Table 2). Termination of 300 nM pirenzepine infusion resulted in an immediate increase of‘ both pancreatic juice flow and amyl&e output to a level comparable to that with carbamylcholine stimulation alone in the corresponding period. The recovery pf pancreatic exocrine responses after the removal of 10 PM pirenzepine, however, was slow. Both pancreatic juice and amylase outputs during the first 10 min after pirenzepine removal
Table
2. Cumulative Outputs of Pancreatic Carbamykholine With or Without
were less than those during the second lo-min period. Addition of 1 and 30 nM atropine to the carbamylcholine perfusion effectively suppressed pancreatic exocrine secretion to a level comparable to that found with 300 nM and JO cln/r pirenzepine infusion, respectively. Pirenzeping was, therefore, 300-fold less potent than atropine in inhibiting the carbamylcholine-stimulated pancreatic exocrine secretion in the isolated perfused pancreas. Although an immediate increase in pancreatic juice and amy-
Juice and Amylase During an Addition of Pirenzepine
a go-Minute or Atropine
Perfusion
With
1 FM
Time after the start of experiment 21-40 min Pancreatic juice (@20 min) Carbamylcholine alone (6) Carbamylcholine plus pirenzepine 300 nM (5) 10 I*M (5) Carbamylcholine plus atropine 1 nM (4) 30 nM (5) Amylase (SULW min) Carbamylcholine alone (6) Carbamylcholine plus pirenzepine 300 nM (5) 10 PM (51 Carbamylcholine plus atropine 1 nM (5) 30 nM (5)
41-60 min
61-80 min
64.6 + 2.9
54.8 k 3.2
43.3 -c 4.0
66.1 f 1.7 69.8 2 8.2
49.0 2 2.4 17.2 2 2.0a,b
52.8 2 7.5 49.6 -c 7.3
67.7 +- 3.7 67.3 ? 4.1
47.5 * 3.3 11.1 ? 2.4”,b
47.7 c 2.9 12 2 rt 3 0a.b
8511.3 -+ 671.8
7620.4 + 850.6
6029.5 -c 407.7
7442.2 + 212.9 8717.1 ” 828.2
3463.6 + 416.2” 1600.2 ? 141.3’,”
5687.8 k 722.3 4982.1 2 75.7O
7778.5 +- 403.6 7783.3 + 578.7
5135.9 2 703.2 746.1 k 44.7”.b
5572.9 k 450.9 601.8 + 32.7°,b
Carbamylcholine was added from 20 to 80 min and pirenzepine or atropine was superimposed on carbamylcholine stimulation for 20 min from 40 to 60 min. Values are mean + SE of the number of experiments indicated in parentheses. Outputs of pancreatic juice and amylase were determined in the same experimental preparation shown in Figure 3. SU, Somogyi units. a Significant difference vs. carbamylcholine stimulation alone in the corresponding period (p < 0.05). b Significant difference vs. lower concentration of the same antagonist in the corresponding period (p < 0.05).
412
GASTROENTEROLOGY
OTSUKI ET AL.
Atropine (3Onfd)
Pirenzeb-ie(lOM
Control
Vol. 89, No. 2
Glucose(mM)
F40. F ; 30 . v) 520. 3 a g
10 _ O-
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Time(min) Figure 3. Ability of 10 PM pirenzepine (middle) and 30 nM atropine (right) to inhibit stimulation of immunoreactive insulin (IRI) release from the isolated perfused pancreas caused by 1 yM carbamylcholine. Each value represents mean k SE of five separate experiments.
lase outputs was observed at the termination of 1 nM atropine infusion, the suppressive effect of 30 nM atropine on pancreatic exocrine secretion remained throughout the experiment, even after the termination of atropine infusion. Pirenzepine also produced a concentration-dependent inhibition of the stimulatory effect of carbamylcholine on IRI secretion, although a biphasic IRI response was observed even in the presence of pirenzepine (Figure 3, middle). When the addition of pirenzepine was terminated, two distinctly different phases of IRI release appeared, as observed during the initial 20-min period of carbamylcholine stimulation. In similar experiments, a comparable suppression of IRI release was observed when the infusion of 300 nM and 10 PM pirenzepine was replaced by 1 nM and 30 nM atropine, respectively (Figure 3,
Table
right). Although two distinctly different phases of IRI release appeared after atropine removal, the peak of an early and rapid release of IRI was significantly lower (Figure 3, right) than that obtained after pirenzepine removal (Figure 3, middle). In contrast to pirenzepine, the suppressive effect of atropine on carbamylcholine-stimulated IRI release remained even after the termination of atropine infusion. The cumulative output of IRI for the 20-min period during an addition of atropine to the carbamylcholine infusion was similar to that obtained during pirenzepine addition. Pirenzepine at a concentration of 300 nM and atropine at a concentration of 1 nM suppressed the secretory response of IRI to 43.4% and 48.9%, respectively, of that to carbamylcholine stimulation alone in the corresponding period (Table 3). However, the cumulative output of IRI for the 20-
3. Cumulative Output of lmmunoreactive Insulin During a 60-Minute With or Without an Addition of Pirenzepine or Atropine
Perfusion
With
1 PM Carbamylcholine
Time after the start of experiments
Control (4) Carbamylcholine alone (6) Carbamylcholine plus pirenzepine 300 nM (5) 10 FM (5) Carbamylcholine plus atropine 1 nM (4) 30 nM (5)
21-40 min
41-60 min
61-80 min
214.7 -e 33.1 1002.5 f 124.2
307.3 - 49.9 1665.3 2 174.8
416.4 t 81.7 2063.2 * 199.3
917.4 + 90.0 981.6 2 91.5
723.3 f 114.8” 676.7 2 120.9”
1184.0 k 216.0a 1043.5 t 134.3”
905.6 2 72.4 903.5 + 92.3
813.6 2 16.0” 660.3 2 58.4”,b
965.7 2 47.2” 865.7 2 96.2a,b
Carbamylcholine was added from 20 to 80 min in the presence of 8.3 mM glucose and pirenzepine or atropine was superimposed on carbamylcholine stimulation for 20 min from 40 to 60 min as shown in Figure 3. Values are mean f SE of the number of experiments indicated in parentheses (nanograms per 20 min). 0 Significant difference vs. carbamylcholine alone in the corresponding period (p < 0.05). b Significant difference VS. lower concentration cf the same antagonist in the corresponding period (p < 0.05).
August
1985
min period after the removal of atropine was significantly lower than that after pirenzepine removal in the corresponding period. The cumulative output of IRI for the 2%min period after the removal of pirenzepine was significantly lower than that with carbamylcholine stimulation alone in the corresponding period, but was similar to that in the first 20-min period with carbamylcholine stimulation alone.
Discussion The present study demonstrates that pirenzepine, a tricyclic pyridobenzodiazepine derivative, antagonizes the actions of carbamylcholine on the exocrine and endocrine pancreas. The results of the present study, therefore, are in agreement with previous reports that indicated a decrease in pancreatic juice (12) and enzyme output (11-13) after pirenzepine administration. Moreover, the present investigation extends these observations by demonstrating that the insulin release initiated by carbamylcholine is also significantly suppressed by pirenzepine and that the ability of pirenzepine to inhibit the action of carbamylcholine is reversible with restoration of full responsiveness to carbamylcholine. However, pirenzepine is about 300-fold less potent than atropine on a molar basis in inhibiting the stimulated pancreatic exocrine and endocrine responses. The fluid and enzyme secretion from the exocrine pancreas is controlled by the nerves as well as by the gastrointestinal hormones. Cholinergic mechanisms are involved in both direct stimulation of acinar cells (1920, and present study] and potentiation of the response to several pancreatic stimulants (20-22). In addition, the vagovagal cholinergic reflex mediating the early pancreatic enzyme response to intestinal stimulation with L-tryptophan and sodium oleate has been demonstrated (23). From these, it has generally been thought that cholinergic mechanisms are most important with regard to nervous control of pancreatic exocrine function. As is the case with atropine, pirenzepine acts as a competitive antagonist in the interaction of carbamylcholine with carbamylcholine (acetylcholine) receptors on acinar cells. Pirenzepine as well as atropine causes a rightward shift in the dose-response curve of carbamylcholine-stimulated amylase release but does not alter the maximal increase in amylase secretion caused by carbamylcholine. This antagonism is selective for carbamylcholine in that pirenzepine does not alter the actions of cholecystokinin, which has a mode of action similar to that of carbamylcholine but acts through different receptors (24). Pirenzepine appears to be bound to the receptors on acinar cells in a rapidly dissociating state, as
PIRENZIPINE AND PANCREATIC
FUNCTION
413
pirenzepine did not cause residual inhibition on carbamylcholine-stimulated pancreatic juice and amylase release. In contrast, atropine had a much longer duration of action than pirenzepine. The present study has clearly demonstrated that -the pancreas perfused with 30 nM atropine for 20 min did not respond to carbamylcholine even when atropine infusion was terminated. Atropine is known to inhibit the cholinergic-mediated enhancement of respiration in the pancreas (25). It is conceivable that pirenzepine, at a high concentration, also prevents the increase in respiration in the pancreas, as the peak response in the pancreas preperfused with 10 PM pirenzepine was attained during the second 10 min of stimulation after pirenzepine removal. Such an inhibitory effect on acinar cells might have some influence on amylase release in response to the subsequent stimulation in the isolated perfused pancreas, where infusion of pirenzepine is immediately followed by carbamylcholine stimulation without washing. It seems that the reversal of pancreatic juice flow and amylase secretion after the removal of pirenzepine infusion appears to be doseand time-dependent. The present findings of the stimulatory effect of carbamylcholine on insulin secretion and its inhibition by pirenzepine are consistent with results observed with vagal stimulation and atropine in the isolated, in situ cross-perfused pancreas (26), or with acetylcholine and atropine in both the isolated perfused pancreas (27) and the isolated islet system (28). Although pancreatic juice flow and amylase output were greatly attenuated by the addition of 10 PM pirenzepine, biphasic insulin secretion was observed even in the presence of pirenzepine when added after a 20-min perfusion with carbamylcholine. Moreover, pirenzepine pretreatment had no influence on carbamylcholine-stimulated insulin release determined during a subsequent stimulation. In contrast to pirenzepine, a high concentration of atropine produced a persistent inhibition of carbamylcholine-stimulated insulin release, as was the case for pancreatic exocrine secretion. The complete reversal of carbamylcholine-stimulated insulin release after the termination of the pirenzepine infusion and the incomplete suppression of carbamylcholine-stimulated insulin response by pirenzepine suggests a weaker effect of pirenzepine on the endocrine pancreas as compared with the exocrine pancress . Muscarinic receptors have been classified into two classes based on binding studies and pharmacologic responses in both isolated tissue and intact systems: (a) those with high affinity and (b) those with low affinity for pirenzepine (6,7,9). It has been demonstrated that pirenzepine is only 2-10 times less
414
OTSUKI ET AL.
potent than atropine in tissues with high-affinity muscarinic receptors in the stomach and peripheral ganglia, whereas it is -200 times less potent than atropine at its low-affinity receptors in the atria, ileum, and pancreas (1,2,6,7,9,13). Pirenzepine thus inhibits acid and pepsin secretion by blocking highaffinity muscarinic receptors with minimal interference of muscarinic receptors of the low-affinity type. In the present study, the concentration of pirenzepine required to inhibit the secretory response of pancreatic juice, amylase, and insulin to 1 PM carbamylcholine was -300 times higher than that of atropine. These results may define pirenzepine receptors in the exocrine and endocrine pancreas as low-affinity-type receptors similar to those of the low-affinity type present in the atria and the ileum (6,7). Accordingly, it seems unlikely that inhibitory doses of pirenzepine on acid secretion cause inhibitory effects on the exocrine and endocrine pancreas.
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pancreatic secretion in man. Arzneimittel-Forschung Drug Res 1977;27:684-7. 11. Kuntzen 0, Kaess H. Zur Wirkung von intravenos appliziertern Pirenzepin auf die exokrine Pankreasfunktion. In: Blum AL, Hammer R, eds. Die Behandlung des Ulcus pepticum mit Pirenzepin. Miinchen: Demeter, 1979:131-5. H. Exkretorische Pankreasfunktion nach 12. Huchzermeyer oraler Gabe von Pirenzepin und Atropin. In: Blum AL, Hammer R, eds. Die Behandlung des dlcus pepticum mit Pirenzepin. Miinchen: Demeter, 1979:136-8. 13. Dehaye JP, Winand J, Hermans L, Poloczek P, Christophe J. Inhibitory effects of pirenzepine on muscarinic stimulation of rat pancreas. Eur J Pharmacal 1983;92:259-64. 14. Williams JA, Korc M, Dormer RL. Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini. Am J Physidl 1978;235:E517-24. 15. Otsuki M, Williams JA. Effect of diabetes mellitus on the regulation of enzyme secretion by isolated rat pancreatic acini. J Clin Invest 1982;70:148-56. 16. Otsuki M, Sakamoto C, Yuu H, et al. Discrepancies between the doses of cholecystokininor caerulein-stimulating exocrine and endocrine responses in perfused isolated rat pancreas. J Clin Invest 1979;63:478-84. 17. Ceska M, Birath K, Brown B. A new and rapid method for the clinical determination of a-amylase activities in human serum and urine. Clin Chim Acta 1969;26:437-44. 18. Desbuquois B, Aurbach GD. Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassay. J Clin Endocrinol Metab 1971;33:732-8. 19. Williams JA. An in vitro evaluation of possible cholinergic and adrenergic receptors affecting pancreatic amylase secretion Proc Sot Exp Biol Med 1975;150:513-6. 20. Gardner JD, Jackson MJ. Regulation of amylase release from dispersed pancreatic acinar cells. J Physiol (Lond) 1977; 270:439-54. 21. Collen MJ, Sutliff VE, Pan G-Z, Gardner JD. Postreceptor modulation of action of VIP and secretin on pancreatic enzyme secretion by secretagogues that mobilize cellular calcium. Am J Physiol 1982;242:G423-8, 22. Beglinger C, Grossman MI, Solomon TE. Interaction between stimulants of exocrine pancreatic secretion in dogs. Am J Physiol 1984;246:G173-9. 23. Singer MV, Solomon TE, Wood J, Grossman MI. Latency of pancreatic enzyme response to intraduodenal stimulants. Am J Physiol 1980;238:G23-9. 24. Gardner JD. Regulation of pancreatic exocrine function in vitro: initial steps in the actions of secretagogues. Annu Rev Physiol 1979;41:550-6. 25. Morisset J, Webster PD: Effects of atropine on pigeon pancreas. Am J Physiol 1970;219:1286-91. 26. Bergman RN, Miller RE. Direct enhancement of insulin secretion by vagal stimulation of the isolated pancreas. Am J Physiol 1973;225:481-6. 27. Iversen J. Effect of acetylcholine on the secretion of glucagon and insulin from the isolated, perfused canine pancreas. Diabetes 1973;22:381-7. 28. Malaisse W, Malaisse-Lagae F, Wright PH, Ashmore J. Effects of adrenergic and cholinergic agents upon secretion of insulin in vitro. Endocrinology 1967;80:975-8.
1985;89:408-14
Comparative Inhibitory Effects of Pirenzepine and Atropine on Cholinergic Stimulation of Exocrine and Endocrine Rat Pancreas MAKOTO OTSUKI, TAKAHIKO NAKAMURA, YOSHINORI OKABAYASHI, TORU OKA, MASATOSHI FUJII, and SHIGEAKI BABA Second Department of Internal Chuo-ku, Kobe 650, Japan
Medicine,
Kobe University
The effects of pirenzepine on carbamylcholine-stimulated exocrine and endocrine pancreatic functions were compared with those of atropine in both the isolated pancreatic acini and the isolated perfused pancreas of rats. In the isolated acini pirenzepine and atropine produced a concentration-dependent inhibition of amylase secretion initiated by carbamylcholine. This inhibition resulted in a rightward shift in the dose-response curve for carbamylcholine-stimulated amylase secretion without altering the maximal increase in amylase secretion. Pirenzepine was, however, -300 times less potent than atropine in inhibiting the stimulated amylase release. A similar difference in potency was observed with respect to carbamylcholine stimulation of pancreatic juice, amyiase, and insulin release from the isolated perfused pancreas. The maximal inhibitory concentration of pirenzepine on a maximal effective concentration of carbamylcholine for stimulating pancreatic exocrine secretion was 10 PM, whereas that of atropine was 30 nM. The present data define the pirenzepine receptors in the exocrine and endocrine pancreas as low-affinity-type receptors. Pirenzepine is a tricyclic, pyridobenzodiazepine derivative that has been shown to reduce gastric acid secretion (l-5). There is strong evidence that the mechanism of action of pirenzepine involves the blockade of muscarinic receptors (6-8). Its inhibitory effect on gastric acid secretion seems similar to those of classical anticholinergics and it is used in Received August 20, 1984. Accepted February 14, 1985. Address requests for reprints to: Dr. Makoto Otsuki, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. 0 1985 by the American Gastroenterological Association 0016-5085/85/$3.30
School
of Medicine,
Kusunoki-cho,
Europe and Japan to treat patients with peptic ulcer (3-5). Hammer et al. (6,7,9) have demonstrated that pirenzepine distinguishes subclasses of muscarinic receptors within one tissue and also binds to receptors of various tissues with a wide heterogeneity of affinities. In agreement with these in vitro studies, clinical experience has shown that pirenzepine has more selective antisecretory properties on gastric secretion, producing less side effects, than conventional anticholinergic drugs (8). Although it is accepted that pirenzepine reduces gastric acid secretion by an antimuscarinic mechanism, considerable controversy exists on the effects of pancreatic exocrine secretion (10-13). Several investigators observed the inhibitory effects of pirenzepine on pancreatic juice (12)and enzyme secretion (ll-13), whereas others failed to demonstrate the decrease of secretory volume (10,ll)and bicarbonate output (11,12). The effectiveness of pirenzepine on muscarinic receptors in the exocrine and endocrine pancreas is still unknown. The present study was therefore undertaken to further evaluate the antimuscarinic properties of pirenzepine on carbamylcholine-stimulated exocrine and endocrine functions and to define muscarinic receptors subtypes in the exocrine and endocrine pancreas in both the isolated pancreatic acini and the isolated perfused pancreas. Materials
and Methods
Materials The following materials were used in this study: carbamylcholine chloride (carbachol), atropine, and soybean trypsin inhibitor (type 1-S) from Sigma Chemical Co., Abbreviation
used in this paper:
IRI, immunoreactive
insulin.
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PIRENZIPINE AND PANCREATIC FUNCTION
St. Louis, MO.; chromatographically purified collagenase from Worthington Biochemicals, Freehold, N.J.; minimal Eagle’s medium amino acid supplement from Grand Island Biological, Grand Island, N.Y.; bovine plasma albumin (fraction V) from Miles Laboratories, Elkhart, Ind. and from Reheis, Chicago, Ill.; Dextran T-70 from Pharmacia Fine Chemicals, Uppsala, Sweden; Phadebas amylase test from Shionogi Pharmaceutical Co., Osaka, Japan, Pirenzepine was kindly provided by Nippon Boehringer Ingelheim Co., Kawanishi, Japan. Synthetic cholecystokinin-octapeptide was a gift from Dr. Miguel Ondetti of the Squibb Institute for Medical Research, Princeton, N.J.
Animals Male Wistar rats weighing -250 g were used throughout the experiment. The animals were kept at 23°C on a 12-h light-dark cycle and had free access to water and a standard laboratory diet.
Isolated Release
Pancreatic
Acini
and
AmyJase
The isolated pancreatic acini were prepared by the methods reported previously (13,14). In brief, the pancreas was injected with Krebs-Henseleit bicarbonate buffer containing minimal Eagle’s medium amino acid supplement, 40-45 U/ml purified collagenase, 2 mg/ml bovine plasma albumin, and 0.1 mg/ml soybean trypsin inhibitor. The injected pancreatic tissue was incubated at 37°C with shaking 120 times/min for 50 min, followed by mechanical disruption of the tissue by forceful pipetting through plastic pipettes. Acini were then purified by filtration through 150~pm nylon cloth and centrifugation through 4% bovine plasma albumin. Acini were preincubated for 60 min in N-2-hydroxyethylpiperazine-N’-2-ethane-sulfonic acid (HEPES)-buffered Ringer solution. This solution was similar to Krebs-Henseleit bicarbonate buffer but contained 10 mM HEPES (pH 7.4) as buffer and 0.5% bovine plasma albumin and was gassed with 100% 02, After preincubation, acini were centrifuged and resuspended in fresh HEPES-buffered Ringer solution at a density of 0.3-0.4 mg protein/ml. Aliquots (2 ml) were distributed into 25-ml polycarbonate Erlenmeyer flasks, secretagogues were added in the presence or absence of pirenzepine, and the flasks were incubated at 37°C with shaking at 60 times/min. Amylase release in response to various concentrations of carbamylcholine or cholecystokinin-octapeptide was determined using the procedure reported previously (14,15). In all experiments triplicate flasks were used to determine the amylase release stimulated by each concentration of secretagogue.
Isolated Perfused Pancreas and Exocrine and
Endocrine
Secretions
The isolated and perfused rat pancreas was prepared as previously reported (16). The perfusate was Krebs-Ringer bicarbonate solution containing 4.6% Dex-
409
tran T-70, 0.25% bovine plasma albumin, and 2.8 mM glucose. It was gassed with a 95% 02-5% COz mixture and adjusted to a pH of 7.4. The inlets of the vascular perfusion were the superior mesenteric and celiac arteries, and the outlet was the portal vein. The flow rate through the pancreas was kept constant at 2 mlimin. After a single passage through the pancreas, the complete effluent was collected in chilled tubes at l-min intervals for measurement of insulin concentration. To measure the pancreatic exocrine secretion, a calibrated capillary tube was attached to the free end of the pancreatic cannula, which was inserted into the distal end of the common duct at a point shortly before its entrance into the duodenum, and tied in place. The proximal end of the bile duct was ligated. The capillary tube was replaced every 10 min to measure the flow rate of the pancreatic juice. The sample of pancreatic juice was diluted with 5% bovine plasma albumin and stored at -20°C for subsequent assay. Experiments were performed after a 40-min equilibration period. The glucose concentration was then changed to 8.3 mM. Ten minutes later, carbamylcholine was added at a concentration of 1 PM for 40 or 60 min. Pirenzepine or atropine was superimposed for 20 min on carbamylcholine stimulation.
Assay Amylase activity in the incubation medium, sonicated acini, and the pancreatic juice was determined by a chromogenic method with the Phadebas amylase test (17). Amylase release from the isolated acini was expressed as the percentage of the total content of enzyme in the acini at the beginning of the incubation that was released, whereas the amylase release from the isolated perfused pancreas was expressed as Somogyi units per 10 min. Immunoreactive insulin (IRI) in the portal effluent was measured by polyethylene glycol radioimmunoassay (18) using crystalline rat insulin as a reference standard.
Statistics Results were expressed as mean f standard error (SE]. For a statistical evaluation of the difference between mean values of the various groups of experiments, the Wilcoxon rank sum test was used. For comparison of data from various levels within the individual experiments, Wilcoxon’s signed-rank test for paired samples was used.
Results The effect of pirenzepine and atropine was tested on a maximal effective concentration of carbamylcholine (1 PM) for stimulating amylase secretion from the isolated rat pancreatic acini (15). Pirenzepine and atropine produced a concentration-dependent inhibition of amylase secretion initiated by 1 PM carbamylcholine (Figure 1). From Figure 1 the IDea for atropine was estimated at 3.2 nM and for
410
OTSUKI
0
ET AL.
10-10 10-g 10-E 10-7 10-6 10-s Antagonist
Figure
GASTROENTEROLOGY
Concentration
10-d
(M)
I. Effects of pirenzepine and atropine on carbamylcholine-stimulated amylase release from the isolated pancreatic acini. The acini were incubated for 30 min at 37°C with various concentrations of pirenzepine or In each experiatropine plus 1 PM carbamylcholine. ment each value was determined in triplicate, and the results shown are mean * SE from four separate experiments.
pirenzepine at 890 nM. Pirenzepine was, therefore, about 300-fold less potent than atropine on a molar basis in inhibiting the carbamylcholine-stimulated amylase secretion. The secretory effect of 100 pM cholecystokinin-octapeptide (18.0% + 0.6% n = 4) was not impaired by 100 nM atropine (18.3% * 1.2%, n = 4) nor by 10 PM pirenzepine (18.5% ? 1.2%, n = 4). Neither atropine nor pirenzepine modified basal amylase release. In the acini incubated with increasing concentration of carbamylcholine, amylase secretion increased, became maximal with 1 PM carbamylcholine, and then decreased at carbamylcholine concentrations >l PM (Figure 2). Atropine and pirenzepine caused a rightward shift in the doseresponse curve for carbamylcholine-stimulated amylase secretion. The concentration of carbamylcholine required to induce a half-maximal release in the presence of 10 ~_LM pirenzepine was 12 PM, whereas that in the presence of 0.1 PM atropine was 36 PM. Thus, in this protocol pirenzepine was also 300-fold less potent than atropine. The inhibitory action of pirenzepine was further investigated in dynamic experiments with the isolated perfused rat pancreas. Coaddition of 10 PM pirenzepine and 1 PM carbamylcholine to the perfused pancreas for 20 min resulted in a complete inhibition of pancreatic juice and amylase secretion (Table 1). When pirenzepine was removed from the perfusate, the flow rate of pancreatic juice increased rapidly. In contrast to the flow of pancreatic juice,
Vol.
89. No.
2
the recovery of amylase output after the removal of pirenzepine was slow. The amylase output in response to the carbamylcholine stimulation from the pirenzepine-pretreated pancreas was significantly lower than that with carbamylcholine stimulation alone in the corresponding period. When 1 PM carbamylcholine was infused in the presence of 8.3 mM glucose, two distinctly different phases of IRI release appeared. There was an early and rapid release of IRI that subsided within -2 min, followed by a late and slow release phase that continuously increased in rate until termination of the carbamylcholine infusion. Both phases of the carbamylcholine-induced IRI release were affected by an addition of pirenzepine. The cumulative output of IRI for the 20-min period during pirenzepine addition was significantly lower than that with carbamylcholine stimulation alone, but was similar to that with 8.3 mM glucose alone in the corresponding period (Table 1). After termination of the pirenzepine infusion, a biphasic IRI increase in response to the carbamylcholine stimulation was observed. The cumulative output of IRI for the 20-min period after the removal of pirenzepine was significantly lower than that with carbamylcholine stimulation alone in the corresponding period but was similar to that in the first 20 min. Addition of pirenzepine after a 20-min perfusion with 1 PM carbamylcholine produced a concentration-dependent inhibition of the stimulatory effects
20 r
I
0
1o-7 10-s 1o-5 10-d 10-j Carbamylcholine
Figure
10-2
(M)
2. Effects of pirenzepine and atropine on carbamylcholine-stimulated amylase release from the isolated pancreatic acini. The acini were incubated for 30 min at 37°C with various concentrations of carbamylcholine alone, carbamylcholine plus pirenzepine, or carbamylcholine plus atropine. In each experiment each value was determined in triplicate, and the results shown are mean 2 SE from four separate experiments.
August 1985
Table
PIRENZIPINE AND PANCREATIC FUNCTION
411
1. Cumulative Outputs of Pancreatic Juice, Amylase, and Immunoreactive Insulin During a N-Minute Perfusion With 1 PM Carbamylcholine With or Without an Addition of 10 PM Pirenzepine Time after the start of experiments Carbamylcholine k pirenzepine (21-40 min)
Carbamylcholine (41-60 min)
Pancreatic juice (~1/20 min) Carbamylcholine alone (5) Carbamylcholine plus pirenzepine
(5)
65.7 t 1.7 21.4 t 2.7”
54.1 2 1.5 69.9 k 2.6a
Amylase (SUIIO min) Carbamylcholine alone (5) Carbamylcholine plus pirenzepine
(5)
8906.1 2 803.4 1157.5 r 145.3”
7256.8 ? 649.4 4440.1 rfr 649.4”
1032.6 + 108.8 242.4 t 24.2a 214.7 t 33.1
1717.6 L 81.5 1056.8 ? 96.5” 307.3 c 49.9
IRI (ng/20 min) Carbamylcholine alone (5) Carbamylcholine plus pirenzepine Glucose 8.3 mM alone 141
(5)
Carbamy!choline was added from 20 to 80 min in the presence of 8.3 mM glucose and pirenzepine was superimposed on carbamylcholine stimulation for XI min from 20 to 40 min. Values are mean + SE of the number of experiments indicated in parentheses. Pancreatic juice, amylase, and immunoreactive insulin (IRI] secretions were measured simultaneously in the same experimental preparation. SU, Somogyi units. a Significant difference vs. carbamylcholine stimulation alone in the corresponding period (p < 0.05).
of carbamylcholine on the secretion of pancreatic juice and amylase (Table 2). Termination of 300 nM pirenzepine infusion resulted in an immediate increase of‘ both pancreatic juice flow and amyl&e output to a level comparable to that with carbamylcholine stimulation alone in the corresponding period. The recovery pf pancreatic exocrine responses after the removal of 10 PM pirenzepine, however, was slow. Both pancreatic juice and amylase outputs during the first 10 min after pirenzepine removal
Table
2. Cumulative Outputs of Pancreatic Carbamykholine With or Without
were less than those during the second lo-min period. Addition of 1 and 30 nM atropine to the carbamylcholine perfusion effectively suppressed pancreatic exocrine secretion to a level comparable to that found with 300 nM and JO cln/r pirenzepine infusion, respectively. Pirenzeping was, therefore, 300-fold less potent than atropine in inhibiting the carbamylcholine-stimulated pancreatic exocrine secretion in the isolated perfused pancreas. Although an immediate increase in pancreatic juice and amy-
Juice and Amylase During an Addition of Pirenzepine
a go-Minute or Atropine
Perfusion
With
1 FM
Time after the start of experiment 21-40 min Pancreatic juice (@20 min) Carbamylcholine alone (6) Carbamylcholine plus pirenzepine 300 nM (5) 10 I*M (5) Carbamylcholine plus atropine 1 nM (4) 30 nM (5) Amylase (SULW min) Carbamylcholine alone (6) Carbamylcholine plus pirenzepine 300 nM (5) 10 PM (51 Carbamylcholine plus atropine 1 nM (5) 30 nM (5)
41-60 min
61-80 min
64.6 + 2.9
54.8 k 3.2
43.3 -c 4.0
66.1 f 1.7 69.8 2 8.2
49.0 2 2.4 17.2 2 2.0a,b
52.8 2 7.5 49.6 -c 7.3
67.7 +- 3.7 67.3 ? 4.1
47.5 * 3.3 11.1 ? 2.4”,b
47.7 c 2.9 12 2 rt 3 0a.b
8511.3 -+ 671.8
7620.4 + 850.6
6029.5 -c 407.7
7442.2 + 212.9 8717.1 ” 828.2
3463.6 + 416.2” 1600.2 ? 141.3’,”
5687.8 k 722.3 4982.1 2 75.7O
7778.5 +- 403.6 7783.3 + 578.7
5135.9 2 703.2 746.1 k 44.7”.b
5572.9 k 450.9 601.8 + 32.7°,b
Carbamylcholine was added from 20 to 80 min and pirenzepine or atropine was superimposed on carbamylcholine stimulation for 20 min from 40 to 60 min. Values are mean + SE of the number of experiments indicated in parentheses. Outputs of pancreatic juice and amylase were determined in the same experimental preparation shown in Figure 3. SU, Somogyi units. a Significant difference vs. carbamylcholine stimulation alone in the corresponding period (p < 0.05). b Significant difference vs. lower concentration of the same antagonist in the corresponding period (p < 0.05).
412
GASTROENTEROLOGY
OTSUKI ET AL.
Atropine (3Onfd)
Pirenzeb-ie(lOM
Control
Vol. 89, No. 2
Glucose(mM)
F40. F ; 30 . v) 520. 3 a g
10 _ O-
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Time(min) Figure 3. Ability of 10 PM pirenzepine (middle) and 30 nM atropine (right) to inhibit stimulation of immunoreactive insulin (IRI) release from the isolated perfused pancreas caused by 1 yM carbamylcholine. Each value represents mean k SE of five separate experiments.
lase outputs was observed at the termination of 1 nM atropine infusion, the suppressive effect of 30 nM atropine on pancreatic exocrine secretion remained throughout the experiment, even after the termination of atropine infusion. Pirenzepine also produced a concentration-dependent inhibition of the stimulatory effect of carbamylcholine on IRI secretion, although a biphasic IRI response was observed even in the presence of pirenzepine (Figure 3, middle). When the addition of pirenzepine was terminated, two distinctly different phases of IRI release appeared, as observed during the initial 20-min period of carbamylcholine stimulation. In similar experiments, a comparable suppression of IRI release was observed when the infusion of 300 nM and 10 PM pirenzepine was replaced by 1 nM and 30 nM atropine, respectively (Figure 3,
Table
right). Although two distinctly different phases of IRI release appeared after atropine removal, the peak of an early and rapid release of IRI was significantly lower (Figure 3, right) than that obtained after pirenzepine removal (Figure 3, middle). In contrast to pirenzepine, the suppressive effect of atropine on carbamylcholine-stimulated IRI release remained even after the termination of atropine infusion. The cumulative output of IRI for the 20-min period during an addition of atropine to the carbamylcholine infusion was similar to that obtained during pirenzepine addition. Pirenzepine at a concentration of 300 nM and atropine at a concentration of 1 nM suppressed the secretory response of IRI to 43.4% and 48.9%, respectively, of that to carbamylcholine stimulation alone in the corresponding period (Table 3). However, the cumulative output of IRI for the 20-
3. Cumulative Output of lmmunoreactive Insulin During a 60-Minute With or Without an Addition of Pirenzepine or Atropine
Perfusion
With
1 PM Carbamylcholine
Time after the start of experiments
Control (4) Carbamylcholine alone (6) Carbamylcholine plus pirenzepine 300 nM (5) 10 FM (5) Carbamylcholine plus atropine 1 nM (4) 30 nM (5)
21-40 min
41-60 min
61-80 min
214.7 -e 33.1 1002.5 f 124.2
307.3 - 49.9 1665.3 2 174.8
416.4 t 81.7 2063.2 * 199.3
917.4 + 90.0 981.6 2 91.5
723.3 f 114.8” 676.7 2 120.9”
1184.0 k 216.0a 1043.5 t 134.3”
905.6 2 72.4 903.5 + 92.3
813.6 2 16.0” 660.3 2 58.4”,b
965.7 2 47.2” 865.7 2 96.2a,b
Carbamylcholine was added from 20 to 80 min in the presence of 8.3 mM glucose and pirenzepine or atropine was superimposed on carbamylcholine stimulation for 20 min from 40 to 60 min as shown in Figure 3. Values are mean f SE of the number of experiments indicated in parentheses (nanograms per 20 min). 0 Significant difference vs. carbamylcholine alone in the corresponding period (p < 0.05). b Significant difference VS. lower concentration cf the same antagonist in the corresponding period (p < 0.05).
August
1985
min period after the removal of atropine was significantly lower than that after pirenzepine removal in the corresponding period. The cumulative output of IRI for the 2%min period after the removal of pirenzepine was significantly lower than that with carbamylcholine stimulation alone in the corresponding period, but was similar to that in the first 20-min period with carbamylcholine stimulation alone.
Discussion The present study demonstrates that pirenzepine, a tricyclic pyridobenzodiazepine derivative, antagonizes the actions of carbamylcholine on the exocrine and endocrine pancreas. The results of the present study, therefore, are in agreement with previous reports that indicated a decrease in pancreatic juice (12) and enzyme output (11-13) after pirenzepine administration. Moreover, the present investigation extends these observations by demonstrating that the insulin release initiated by carbamylcholine is also significantly suppressed by pirenzepine and that the ability of pirenzepine to inhibit the action of carbamylcholine is reversible with restoration of full responsiveness to carbamylcholine. However, pirenzepine is about 300-fold less potent than atropine on a molar basis in inhibiting the stimulated pancreatic exocrine and endocrine responses. The fluid and enzyme secretion from the exocrine pancreas is controlled by the nerves as well as by the gastrointestinal hormones. Cholinergic mechanisms are involved in both direct stimulation of acinar cells (1920, and present study] and potentiation of the response to several pancreatic stimulants (20-22). In addition, the vagovagal cholinergic reflex mediating the early pancreatic enzyme response to intestinal stimulation with L-tryptophan and sodium oleate has been demonstrated (23). From these, it has generally been thought that cholinergic mechanisms are most important with regard to nervous control of pancreatic exocrine function. As is the case with atropine, pirenzepine acts as a competitive antagonist in the interaction of carbamylcholine with carbamylcholine (acetylcholine) receptors on acinar cells. Pirenzepine as well as atropine causes a rightward shift in the dose-response curve of carbamylcholine-stimulated amylase release but does not alter the maximal increase in amylase secretion caused by carbamylcholine. This antagonism is selective for carbamylcholine in that pirenzepine does not alter the actions of cholecystokinin, which has a mode of action similar to that of carbamylcholine but acts through different receptors (24). Pirenzepine appears to be bound to the receptors on acinar cells in a rapidly dissociating state, as
PIRENZIPINE AND PANCREATIC
FUNCTION
413
pirenzepine did not cause residual inhibition on carbamylcholine-stimulated pancreatic juice and amylase release. In contrast, atropine had a much longer duration of action than pirenzepine. The present study has clearly demonstrated that -the pancreas perfused with 30 nM atropine for 20 min did not respond to carbamylcholine even when atropine infusion was terminated. Atropine is known to inhibit the cholinergic-mediated enhancement of respiration in the pancreas (25). It is conceivable that pirenzepine, at a high concentration, also prevents the increase in respiration in the pancreas, as the peak response in the pancreas preperfused with 10 PM pirenzepine was attained during the second 10 min of stimulation after pirenzepine removal. Such an inhibitory effect on acinar cells might have some influence on amylase release in response to the subsequent stimulation in the isolated perfused pancreas, where infusion of pirenzepine is immediately followed by carbamylcholine stimulation without washing. It seems that the reversal of pancreatic juice flow and amylase secretion after the removal of pirenzepine infusion appears to be doseand time-dependent. The present findings of the stimulatory effect of carbamylcholine on insulin secretion and its inhibition by pirenzepine are consistent with results observed with vagal stimulation and atropine in the isolated, in situ cross-perfused pancreas (26), or with acetylcholine and atropine in both the isolated perfused pancreas (27) and the isolated islet system (28). Although pancreatic juice flow and amylase output were greatly attenuated by the addition of 10 PM pirenzepine, biphasic insulin secretion was observed even in the presence of pirenzepine when added after a 20-min perfusion with carbamylcholine. Moreover, pirenzepine pretreatment had no influence on carbamylcholine-stimulated insulin release determined during a subsequent stimulation. In contrast to pirenzepine, a high concentration of atropine produced a persistent inhibition of carbamylcholine-stimulated insulin release, as was the case for pancreatic exocrine secretion. The complete reversal of carbamylcholine-stimulated insulin release after the termination of the pirenzepine infusion and the incomplete suppression of carbamylcholine-stimulated insulin response by pirenzepine suggests a weaker effect of pirenzepine on the endocrine pancreas as compared with the exocrine pancress . Muscarinic receptors have been classified into two classes based on binding studies and pharmacologic responses in both isolated tissue and intact systems: (a) those with high affinity and (b) those with low affinity for pirenzepine (6,7,9). It has been demonstrated that pirenzepine is only 2-10 times less
414
OTSUKI ET AL.
potent than atropine in tissues with high-affinity muscarinic receptors in the stomach and peripheral ganglia, whereas it is -200 times less potent than atropine at its low-affinity receptors in the atria, ileum, and pancreas (1,2,6,7,9,13). Pirenzepine thus inhibits acid and pepsin secretion by blocking highaffinity muscarinic receptors with minimal interference of muscarinic receptors of the low-affinity type. In the present study, the concentration of pirenzepine required to inhibit the secretory response of pancreatic juice, amylase, and insulin to 1 PM carbamylcholine was -300 times higher than that of atropine. These results may define pirenzepine receptors in the exocrine and endocrine pancreas as low-affinity-type receptors similar to those of the low-affinity type present in the atria and the ileum (6,7). Accordingly, it seems unlikely that inhibitory doses of pirenzepine on acid secretion cause inhibitory effects on the exocrine and endocrine pancreas.
References 1. Kitagawa H, Kurahashi
2.
3.
4. 5
6
7
8.
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