J. Physiol. (1977), 264, pp. 819-835 With 8 text-figure8 Printed in Great Britain
819
SECRETION OF FLUID AND AMYLASE IN THE PERFUSED RAT PANCREAS
By 0. H. PETERSEN AND N. UEDA* From the Department of Physiology, The University, Dundee DD1 4HN
(Received 24 May 1976) SUMMARY
1. The isolated rat pancreas was perfused with physiological salt solutions of varying composition. Flow of pancreatic juice and output of amylase during rest and after stimulation with pure secretin, pure cholecystokinin-pancreozymin (CCK-PZ), caerulein or acetylcholine (ACh) were measured. 2. Basal fluid secretion was abolished replacing perfusion fluid Na+ or Cl- by Tris+ or S042- respectively. Readmission of Na+ or C1- caused a transient increase above the normal control level of both fluid and amylase output. Exposure to K+-free solution severely reduced fluid output and K+ readmission resulted in a transient increase in secretary rate. 3. Maximal stimulation with ACh (10-7 M), CCK-PZ (1I5 x 10-1o M) or caerulein (10-10 M) caused marked sustained fluid and amylase secretion. Maximal secretin stimulation (5.7 x 10-9 M) caused marked sustained fluid but only a small sustained amylase secretion following an initial transient. 4. Under continuous secretin stimulation, replacement of the C02/ HC03- buffered control fluid by a C02/HCO3--free Tris buffered solution caused a sharp decrease in pancreatic juice flow. In the absence of extracellular C02/HC03- secretin did not evoke fluid or enzyme secretion. In contrast the effects of ACh, CCK-PZ or caerulein were independent on
C02/HCO3-. Monobutyryl cyclic AMP (10-3 M) caused marked sustained fluid secretion and transient enzyme secretion. The effect was entirely dependent on the presence of C02/HC03- in the perfusion fluid. 5. Ouabain (104-10-3 M) markedly inhibited both secretin- and caerulein-evoked fluid secretion while caerulein-evoked amylase secretion was hardly affected. Similar findings were made with K+-free solution. * Wellcome Japanese Post-doctoral Research Fellow. Present address: the Second Department of Internal Medicine, Asahikawa University School of Medicine, Asahikawa, Japan.
0. H. PETERSEN AND N. UEDA 820 6. The effect of maximal secretin stimulation on amylase secretion was greatly augmented in the presence of a maximally stimulating concentration of caerulein. The effects on fluid secretion of secretin and caerulein were simply additive. The effects of secretin on both amylase and fluid secretion, in the presence of caerulein, were entirely dependent on the presence of C02/HC03- in the perfusion fluid. 7. We conclude that two different fluid secretion processes occur in the rat exocrine pancreas. One stimulated by ACh and CCK-PZ, that is independent of extracellular C02/HCO3- and another stimulated by secretin involving H+ or HC03- transport. Only the effects of secretin seem to be mediated by intracellular cyclic AMP. INTRODUCTION
It is well known that acetylcholine (ACh), cholecystokinin-pancreozymin (CCK-PZ) and gastrin, stimulants acting on the acinar cell membrane (Petersen & Ueda, 1975), evoke enzyme secretion while secretin, acting on duct cells (Schulz, Yamagata & Weske, 1969; Greenwell, 1975), evokes secretion of a bicarbonate-rich fluid. Dockray (1972) has shown that CCK-PZ evokes marked fluid secretion in the in vivo rat pancreas and Sewell & Young (1975) have shown that while the secretin-evoked rat pancreatic juice is rich in bicarbonate, COK-PZ evokes a secretion with a low bicarbonate concentration. The aim of the present work was to test in an isolated perfused rat pancreatic preparation (Ueda, 1975) the effect of the pure physiological pancreatic stimulants on both fluid and amylase secretion and to describe the ionic dependency of the effects of ACh, CCK-PZ, and secretin. METHODS An isolated perfused preparation of rat pancreas (Ueda, 1975) was used in all experiments. Male rats weighing 300-400 g were fasted overnight before experiments, but allowed free access to water. The rats were anaesthetized with urethane (1.5 g/kg i.P.). To perfuse the whole pancreas, both caeliac and superior mesenteric arteries were cannulated separately by polyethylene tubes (1 mm o.d.). The oxygenated Krebs-Henseleit perfusion fluid was infused through these tubes by means of a roller pump at a constant flow rate (0.8-0.9 ml./min each, total flow rate 1.6-1.8 ml./min) and a pressure of 35 mmHg. The venous effluent was drained through a portal vein cannula. The proximal end of the common duct was ligated and the distal end of the common duct was cannulated by a thin steel cannula having a small dead space. A calibrated silicone rubber tube (1 cm = 3.3 pl.) was attached to the free end of the steel cannula and replaced every 10 min. Stomach, spleen and intestine, except part of the duodenum, were freed from the pancreas after ligating all vascular branches not supplying the pancreas. The isolated pancreas was placed in a tissue bath. A physiological salt solution with the same composition as the
PERFUSED RAT PANCREAS
821
vascular perfusion fluid (but without Dextran), warmed to 370 C, flowed through the bath at a rate of 10 ml./min. The control solution had the following composition (mM): NaCl 103, KCl 4*7, CaCi2 2-56, MgCl, 1-15, NaHCO3 25, NaH2PO4 1-15, D-glucose 2-8, Na pyruvate 4 9, Na fumarate 2-7 and Na glutamate 4 9. Dextran (0 7 mM) (mol. wt. 70,000, Pharmacia, Sweden) was added to the solution that was gassed with 95%0 2 and 5% CO2. This solution was modified in several ways. To Ca2+-free solutions, 10 4 MEGTA (ethylene glycol-bis-(f-amino ethylether) N,N'-tetra acetic acid) (Sigma) was added. A bicarbonate-free and phosphate-free solution buffered by Tris (1-5 mM) with augmented NaCl concentration to maintain its osmolarity was frequently used. This solution was gassed with pure 0° and the pH was adjusted to 7-4. When Na-free solution was used, all NaCl was replaced by Tris Cl or LiCl and pyruvic acid, glutamic acid and fumaric acid were added instead of the Na salts, pH being adjusted by Tris (base) and HCl. In Cl--free solutions, SO42+ was added. Ca2+ concentration was augmented to 10 mM-CaSO4. (The high concentration of CaSO4 was used to compensate, partially, for the low calcium ion concentration that exists in the presence of sulphate; Hill & Howarth, 1957). The osmolality of the perfusion fluids (290+ 5 m-osmole/kg H2O) were frequently checked by a cryoscopic method. Measurement of amylase in the pancreatic juice was carried out by a modification of the method of Bernfeld (1955). One unit (u.) of amylase is defined as the amount of amylase which liberates 1 0 mg maltose from starch (Zulkofsky's soluble starch, Merck, West Germany) in 5 min at pH 7-1 and 370 C. Pure natural secretin and pure natural CCK-PZ from Karolinska Institute Stockholm, were used. Synthetic caerulein was a gift from Kyowa Hakko Kogyo Ltd (Tokyo, Japan). N6-mono-butyryl adenosine 3',5'-cyclic monophosphate and N2-monobutyryl guanosine 3',5'-cyclic monophosphate came from Sigma. RESULTS
The effect of ACh, CCK-PZ, caerulein and 8ecretin During perfusion with the standard Krebs-Henseleit solution in the absence of any applied stimuli a small basal secretion was always obtained (Table 1). Addition of an appropriate dose of any of the physiological stimulants, or an analogue of CCK-PZ such as caerulein, resulted in a large sustained increase in fluid secretion. In preliminary experiments the concentrations of hormones evoking maximal responses were established. These concentrations were then routinely used (Table 1). Dose-response curves for the effect of caerulein on amylase output from perfused rat pancreas have been reported and the maximally stimulating concentration of caerulein in the present work (10-10 M) agrees well with that previously found (0.8 x 10-10 M). (Ueda, 1974) As seen in Table 1 the effects of ACh, CCK-PZ and caerulein were very similar in causing a larger fluid secretion than that obtained following secretin stimulation and while the latter stimulant only evoked a small increase in amylase output all the other agents had a very marked stimulatory effect on enzyme secretion. Since pure CCK-PZ and caerulein had the same effect on fluid and enzyme
822
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823 PERFUSED RAT PANCREAS secretion and since it is known that caerulein has exactly the same effect on the pancreatic acinar cell membrane potential and resistance as CCK-PZ (N. Iwatsuki, K. Kato and A. Nishiyama, submitted for publication) it seemed reasonable to use caerulein for most of the experiments in this way also excluding the possible effect of trace impurities. 2.64 Secretin (57 x10-' M)
Cr. (10-I m)
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Time (hr) Fig. 1. The effect of secretin and caerulein (Cr.) on fluid secretion (open columns) and amylase secretion (stippled columns) in the presence and absence of CO21HC00- in the perfusion fluid.
The importance of C02/H003In six experiments the effect of taking away extracellular C02/HC03- on secretin- or caerulein-evoked secretion was tested. Fig. 1 shows that the responses evoked by secretin were rapidly abolished while caerulein was able to evoke the usual response with respect to both fluid and amylase secretion even after 30 min of perfusion with the C02/HCO3--free solution. The effect of removing perfusion fluid C02/HC03- was always fully reversible. Results similar to the one shown in Fig. 1 were obtained using pure COK-PZ (two experiments) and Aah (Table 1). The output of amylase during stimulation with ACh or caerulein was independent of
824 0. H. PETERSEN AND N. UEDA the presence of perfusion fluid C02/HCO3-, whereas the fluid secretion rate was slightly reduced during exposure to the C02/HCO3--free solution. This can at least in part be explained by a reduction of the basal secretion rate (Table 1).
The importance of Na+ and ClBasal secretion of both fluid and amylase was abolished following replacement of all perfusion fluid Na+ by Tris+ or Cl- by SO42+ (Fig. 2). Readmission of Na+ (in the presence of atropine to avoid any possible ACh release from nerve endings within the tissue) caused some stimulation of amylase secretion and a large increase in fluid secretion rate. Both C.
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819
SECRETION OF FLUID AND AMYLASE IN THE PERFUSED RAT PANCREAS
By 0. H. PETERSEN AND N. UEDA* From the Department of Physiology, The University, Dundee DD1 4HN
(Received 24 May 1976) SUMMARY
1. The isolated rat pancreas was perfused with physiological salt solutions of varying composition. Flow of pancreatic juice and output of amylase during rest and after stimulation with pure secretin, pure cholecystokinin-pancreozymin (CCK-PZ), caerulein or acetylcholine (ACh) were measured. 2. Basal fluid secretion was abolished replacing perfusion fluid Na+ or Cl- by Tris+ or S042- respectively. Readmission of Na+ or C1- caused a transient increase above the normal control level of both fluid and amylase output. Exposure to K+-free solution severely reduced fluid output and K+ readmission resulted in a transient increase in secretary rate. 3. Maximal stimulation with ACh (10-7 M), CCK-PZ (1I5 x 10-1o M) or caerulein (10-10 M) caused marked sustained fluid and amylase secretion. Maximal secretin stimulation (5.7 x 10-9 M) caused marked sustained fluid but only a small sustained amylase secretion following an initial transient. 4. Under continuous secretin stimulation, replacement of the C02/ HC03- buffered control fluid by a C02/HCO3--free Tris buffered solution caused a sharp decrease in pancreatic juice flow. In the absence of extracellular C02/HC03- secretin did not evoke fluid or enzyme secretion. In contrast the effects of ACh, CCK-PZ or caerulein were independent on
C02/HCO3-. Monobutyryl cyclic AMP (10-3 M) caused marked sustained fluid secretion and transient enzyme secretion. The effect was entirely dependent on the presence of C02/HC03- in the perfusion fluid. 5. Ouabain (104-10-3 M) markedly inhibited both secretin- and caerulein-evoked fluid secretion while caerulein-evoked amylase secretion was hardly affected. Similar findings were made with K+-free solution. * Wellcome Japanese Post-doctoral Research Fellow. Present address: the Second Department of Internal Medicine, Asahikawa University School of Medicine, Asahikawa, Japan.
0. H. PETERSEN AND N. UEDA 820 6. The effect of maximal secretin stimulation on amylase secretion was greatly augmented in the presence of a maximally stimulating concentration of caerulein. The effects on fluid secretion of secretin and caerulein were simply additive. The effects of secretin on both amylase and fluid secretion, in the presence of caerulein, were entirely dependent on the presence of C02/HC03- in the perfusion fluid. 7. We conclude that two different fluid secretion processes occur in the rat exocrine pancreas. One stimulated by ACh and CCK-PZ, that is independent of extracellular C02/HCO3- and another stimulated by secretin involving H+ or HC03- transport. Only the effects of secretin seem to be mediated by intracellular cyclic AMP. INTRODUCTION
It is well known that acetylcholine (ACh), cholecystokinin-pancreozymin (CCK-PZ) and gastrin, stimulants acting on the acinar cell membrane (Petersen & Ueda, 1975), evoke enzyme secretion while secretin, acting on duct cells (Schulz, Yamagata & Weske, 1969; Greenwell, 1975), evokes secretion of a bicarbonate-rich fluid. Dockray (1972) has shown that CCK-PZ evokes marked fluid secretion in the in vivo rat pancreas and Sewell & Young (1975) have shown that while the secretin-evoked rat pancreatic juice is rich in bicarbonate, COK-PZ evokes a secretion with a low bicarbonate concentration. The aim of the present work was to test in an isolated perfused rat pancreatic preparation (Ueda, 1975) the effect of the pure physiological pancreatic stimulants on both fluid and amylase secretion and to describe the ionic dependency of the effects of ACh, CCK-PZ, and secretin. METHODS An isolated perfused preparation of rat pancreas (Ueda, 1975) was used in all experiments. Male rats weighing 300-400 g were fasted overnight before experiments, but allowed free access to water. The rats were anaesthetized with urethane (1.5 g/kg i.P.). To perfuse the whole pancreas, both caeliac and superior mesenteric arteries were cannulated separately by polyethylene tubes (1 mm o.d.). The oxygenated Krebs-Henseleit perfusion fluid was infused through these tubes by means of a roller pump at a constant flow rate (0.8-0.9 ml./min each, total flow rate 1.6-1.8 ml./min) and a pressure of 35 mmHg. The venous effluent was drained through a portal vein cannula. The proximal end of the common duct was ligated and the distal end of the common duct was cannulated by a thin steel cannula having a small dead space. A calibrated silicone rubber tube (1 cm = 3.3 pl.) was attached to the free end of the steel cannula and replaced every 10 min. Stomach, spleen and intestine, except part of the duodenum, were freed from the pancreas after ligating all vascular branches not supplying the pancreas. The isolated pancreas was placed in a tissue bath. A physiological salt solution with the same composition as the
PERFUSED RAT PANCREAS
821
vascular perfusion fluid (but without Dextran), warmed to 370 C, flowed through the bath at a rate of 10 ml./min. The control solution had the following composition (mM): NaCl 103, KCl 4*7, CaCi2 2-56, MgCl, 1-15, NaHCO3 25, NaH2PO4 1-15, D-glucose 2-8, Na pyruvate 4 9, Na fumarate 2-7 and Na glutamate 4 9. Dextran (0 7 mM) (mol. wt. 70,000, Pharmacia, Sweden) was added to the solution that was gassed with 95%0 2 and 5% CO2. This solution was modified in several ways. To Ca2+-free solutions, 10 4 MEGTA (ethylene glycol-bis-(f-amino ethylether) N,N'-tetra acetic acid) (Sigma) was added. A bicarbonate-free and phosphate-free solution buffered by Tris (1-5 mM) with augmented NaCl concentration to maintain its osmolarity was frequently used. This solution was gassed with pure 0° and the pH was adjusted to 7-4. When Na-free solution was used, all NaCl was replaced by Tris Cl or LiCl and pyruvic acid, glutamic acid and fumaric acid were added instead of the Na salts, pH being adjusted by Tris (base) and HCl. In Cl--free solutions, SO42+ was added. Ca2+ concentration was augmented to 10 mM-CaSO4. (The high concentration of CaSO4 was used to compensate, partially, for the low calcium ion concentration that exists in the presence of sulphate; Hill & Howarth, 1957). The osmolality of the perfusion fluids (290+ 5 m-osmole/kg H2O) were frequently checked by a cryoscopic method. Measurement of amylase in the pancreatic juice was carried out by a modification of the method of Bernfeld (1955). One unit (u.) of amylase is defined as the amount of amylase which liberates 1 0 mg maltose from starch (Zulkofsky's soluble starch, Merck, West Germany) in 5 min at pH 7-1 and 370 C. Pure natural secretin and pure natural CCK-PZ from Karolinska Institute Stockholm, were used. Synthetic caerulein was a gift from Kyowa Hakko Kogyo Ltd (Tokyo, Japan). N6-mono-butyryl adenosine 3',5'-cyclic monophosphate and N2-monobutyryl guanosine 3',5'-cyclic monophosphate came from Sigma. RESULTS
The effect of ACh, CCK-PZ, caerulein and 8ecretin During perfusion with the standard Krebs-Henseleit solution in the absence of any applied stimuli a small basal secretion was always obtained (Table 1). Addition of an appropriate dose of any of the physiological stimulants, or an analogue of CCK-PZ such as caerulein, resulted in a large sustained increase in fluid secretion. In preliminary experiments the concentrations of hormones evoking maximal responses were established. These concentrations were then routinely used (Table 1). Dose-response curves for the effect of caerulein on amylase output from perfused rat pancreas have been reported and the maximally stimulating concentration of caerulein in the present work (10-10 M) agrees well with that previously found (0.8 x 10-10 M). (Ueda, 1974) As seen in Table 1 the effects of ACh, CCK-PZ and caerulein were very similar in causing a larger fluid secretion than that obtained following secretin stimulation and while the latter stimulant only evoked a small increase in amylase output all the other agents had a very marked stimulatory effect on enzyme secretion. Since pure CCK-PZ and caerulein had the same effect on fluid and enzyme
822
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823 PERFUSED RAT PANCREAS secretion and since it is known that caerulein has exactly the same effect on the pancreatic acinar cell membrane potential and resistance as CCK-PZ (N. Iwatsuki, K. Kato and A. Nishiyama, submitted for publication) it seemed reasonable to use caerulein for most of the experiments in this way also excluding the possible effect of trace impurities. 2.64 Secretin (57 x10-' M)
Cr. (10-I m)
1-98 *
COJ/HCO;
.
--
I
Trs
COJHCO,
Tris
-I
1232
~~~~~~~~~60 0
0.66:
40 E
I-
2~~~~~~~2
Time (hr) Fig. 1. The effect of secretin and caerulein (Cr.) on fluid secretion (open columns) and amylase secretion (stippled columns) in the presence and absence of CO21HC00- in the perfusion fluid.
The importance of C02/H003In six experiments the effect of taking away extracellular C02/HC03- on secretin- or caerulein-evoked secretion was tested. Fig. 1 shows that the responses evoked by secretin were rapidly abolished while caerulein was able to evoke the usual response with respect to both fluid and amylase secretion even after 30 min of perfusion with the C02/HCO3--free solution. The effect of removing perfusion fluid C02/HC03- was always fully reversible. Results similar to the one shown in Fig. 1 were obtained using pure COK-PZ (two experiments) and Aah (Table 1). The output of amylase during stimulation with ACh or caerulein was independent of
824 0. H. PETERSEN AND N. UEDA the presence of perfusion fluid C02/HCO3-, whereas the fluid secretion rate was slightly reduced during exposure to the C02/HCO3--free solution. This can at least in part be explained by a reduction of the basal secretion rate (Table 1).
The importance of Na+ and ClBasal secretion of both fluid and amylase was abolished following replacement of all perfusion fluid Na+ by Tris+ or Cl- by SO42+ (Fig. 2). Readmission of Na+ (in the presence of atropine to avoid any possible ACh release from nerve endings within the tissue) caused some stimulation of amylase secretion and a large increase in fluid secretion rate. Both C.
1.32
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