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Effects of phorbol esters and secretin on pancreatic juice secretion in the anaesthetized rat
Gen. Pharmac. Vol. 21, No. 4, pp. 465-469, 1990 Printed in Great Britain.All rights reserved
0306-3623/90$3.00+ 0.00 Copyright ~' 1990PergamonPress plc
EFFECTS OF PHORBOL ESTERS A N D SECRETIN ON PANCREATIC JUICE SECRETION IN THE ANAESTHETIZED RAT G. M. SALIDO,* L. P. F~Ncxs, ~P. J. CAMELLO,J. SINGH,! J. A. MADglD and J. A. PARIENTE Department of Physiology, Faculty of Veterinary Science, University of Extremadura, 10080-Caceres, Spain and tSchool of Applied Biology, Lancashire Polytechnic, Preston, England (Received 20 September 1989)
Al~lan~--I. An investigation was made of the effects of phorbol esters, i 2-O-tetradeconylphorbol acetate (TPA) and secretin on pancreatic juice secretion in the anaesthetized rat. TPA (10-t2-10-s mol/kg body wt) evoked marked dose-dependent increases in secretory rate and total protein output. 2. An inactive phorbol ester (4",-phorbol-12-13-didecanoate;4ctPDD) had no effect on the secretory rate but increased total protein output compared to saline control animals. 3. When TPA was administered in combination with the protein kinase C inhibitor, Polymyxin B (10-Smol/kg body wt) both secretory rate and protein output were significantly reduced (P <0.001) compared to TPA alone. 4. Secretin (50-1600 pmol/kg body wt) increased both pancreatic juice flow and total protein output in a dose-dependent manner. 5. Simultaneous administration of secretin (50-1600 pmol/kg body wt) and TPA (10-I° mol/kg body wt) resulted in a marked attenuation in the secretin-induced secretory rate while secretin-evoked protein output was unaffected. 6. The results indicate that protein kinase C activation is associated with pancreatic juice secretion and it may also modulate secretin-induced pancreatic juice flow in the anaesthetized rat.
INTRODUCTION Previous studies on exocrine pancreatic secretion using the in vitro pancreas have demonstrated the presence of two distinct stimulus-secretion coupling mechanisms (Gardner, 1979; Schulz and Stolze, 1980; Petersen, 1982). One pathway is activated by secretin, vasoactive intestinal polypeptide and cathecholamines resulting in the increase in endogenous adenosine-3,5-cyclic monophosphate (cyclicAMP) while the other pathway is stimulated by acetylcholine (ACh), cholecystokinin (CCK) and bombesin-like peptides and involves mobilization of cellular calcium (Ca ~÷) (Petersen and Iwatsuki, 1978; Gardner, 1979; Schulz and Stolze, 1980; Petersen, 1982; Case, 1984). More recently, some studies have shown that the phorboi esters can stimulate enzyme secretion in isolated pancreatic segments and acini without affecting either Ca 2+ mobilization or cyclicAMP metabolism (Argent et al., 1978; Gunther and Jamieson, 1979; De-Pont and Fleuren-Jakobs, 1984; Singh, 1985). This secretory action of TPA is associated with the activation of the phospholipid and Ca ~+-dependent enzyme protein kinase C (Nishizuka, 1984, 1988; Noguchi et al., 1985) as the selective inhibitor of protein kinase C, Polymyxin B can completely block the TPA-induced enzymatic secretion (Wrenn and Wooten, 1984). These recent experiments with TPA suggest the existence for a third
*To whom correspondence should be addressed.
stimulus-secretion coupling pathway controlling enzyme secretion in the isolated pancreatic segments and acini (De-Pont and Fleuren-Jakobs, 1984; Singh, 1988). From a functional point of view no evidence is hvailable as to whether TPA can evoke pancreatic juice secretion and, moreover, modify secretagogueinduced secretory responses in the in vivo pancreas. As such, this study was designed to investigate the effects of phorbol esters on the rate of pancreatic juice secretion and total protein output in the absence and presence of the protein kinase C inhibitor, Polymyxin B in the anaesthetized rat. Furthermore, it was also decided to examine any interactions between TPA and the gut hormone secretin on pancreatic juice secretion. A preliminary account of some aspects of this work was presented to the Physiological Society (Camello et al., 1989).
MATERIALS AND METHODS
Animals and surgical preparation All experiments were performed on overnight-fasted adult Wistar rats (200-450 g body wt) of either sex. The animals were anaesthetized by 0.5-0.75 g/kg i.p. urethane. After tracheal intubation, the fight carotid artery was cannulated to measure systemic blood pressure with a blood pressure transducer connected to a multichannel pen recorder. Drugs were administered via cannula inserted into the left jugular vein. After performing a laparotomy, the pylorus was ligated and the bile-pancreatic duct exposed and cannulated at its entrance to the duodenum for the collection of pancreatic juice. The hepatic end of the bile-pancreatic duct
465
G . M . SALIDOet al.
466 200
~
[] 180
-
•
Inactive phorbol ester Saline
;1,0 •
.G
120
m ~ :alO0
¢ ¢
// //
S ¢
/
/
/
10 -12
10 "11
10 "10
10 -9
30
60
90
120
10 -8 mol / kg 150 Time { min )
Fig. 1. Time-course effects of administering either saline or varying concentrations (10-~-'--10-s mol/kg body wt) phorbol esters on pancreatic juice flow in the anaesthetized rat preparation. Solid columns, saline; open columns, inactive phorbol ester, single bar columns, TPA and double bar columns, TPA plus 10 -8 mol/kg Polymyxin B (a protein kinase C, inhibitor).
was also cannulated and bile was reinfused into the duodenum. Throughout experiments the body temperature was kept between 31 and 33°C with a heating pad.
Experimental design After 45 min of flow stabilization, pancreatic juice was collected on ice for 210 min at 30 min intervals. Pancreatic juice collected between 0 and 30 min was taken as the resting value. Drug or saline administration started at the minute 30. In this study several different experiments were performed using saline, TPA (Sigma), inactive phorbol ester (Sigma), Polymyxin B (Sigma) and secretin (Sigma). All drugs were freshly prepared in 0.9% w/v sodium chloride solution. Firstly, control experiments (saline-treated animals) were performed to determine the effect of the time of cannulation on pancreatic juice secretion. In the second set of experiments the effects of TPA and an inactive phorbol ester (4ctPDD) on pancreatic juice output were determined in different animal preparations. The phorbol esters were administered by means of i.v. bolus injections at doses of 10-~2-10 -s mol/kg body wt. Thirdly, the protein kinase C inhibitor, Polymyxin B (10 -s mol/kg body wt) was injected i.v. as bolus in combination with varying doses (10-t:-I0 -s mol/kg body wt) of TPA. Fourthly, the effect of administering different doses of the gut hormone secretin on pancreatic juice secretion was also investigated in the absence and presence of TPA (10 -j° mol/kg body wt) in separate experiments. Secretin was injected in bolus form at doses of 50, 100, 200, 400, 800 and 1600 pmol/kg body wt.
Estimation offlow of pancreatic juice and total protein output Secretory rates were determined by weighing tared Eppendorph tubes, assuming a density of secretion equal to water. All samples for protein determination were frozen immediately following collection, stored overnight at -20°C and assayed the following day. Several determinations of protein content were made the same day as collection and compared to determinations after freezing and storing and no significant differences were observed. Total protein concentrations were estimated by the method of Bradford (1976) with bovine serum albumin (Sigma) used as the standard.
Data analysis The secretory rates of pancreatic juice were normalised to prestimulated levels and the percentage change was given. Results were analysed by using the Student's t-test for paired data and P < 0.05 was taken as significant. RESULTS
Unstimulated pancreatic secretion In anaesthetized rats the resting flow was 0.27 + 0.04/zl/min 3 0 m i n after the initial i.v. injection of saline compared to 0.33 + 0.05 ~l/min after 150 min. In contrast, protein output in pancreatic juice during the initial 3 0 m i n collection period was 3 . 9 6 + 1.28/~g/min compared to 3.01 + 0 . 3 0 / ~ g / m i n after 150 rain. These control experiments show that saline administration caused a small increase in the flow rate (Figs I and 3; solid columns) whereas protein output (Figs 2 and 4; solid columns) was reduced during the same period. Alternatively, these changes may be due to an alteration in the resting juice flow from the time of cannulation to the end of the experiment.
Effects o f phorbol esters on pancreatic secretion Intravenous bolus injection of T P A (10 - j z 10-8 moi/kg body wt) resulted in marked dose-dependent increases in both the rate of pancreatic juice flow (Fig. I; single bar columns) and total protein output (Fig. 2; single bar columns). These effects of T P A were significantly reduced (P < 0.001) when the protein kinase C inhibitor Polymyxin B (10-Smol/kg body wt) was injected in combination with different doses of T P A (Figs 1 and 2; double bar columns). In contrast, administration of the inactive phorbol ester (10-~2-10-Smol/kg body wt) had little or no effect on the secretory rate compared to control saline (Fig. 1; open columns). However, at high doses (10-9-10-Smol/kg body wt) the inactive phorbol ester evoked an increase in total protein output compared to saline control (Fig. 2; open columns).
Secretagogue-evoked pancreatic secretion 350
300 --
D •_~
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467
I
TPA 4- Polymyxin B TPA Inactive phorbol ester Saline
/ / /
2
~ ~ lSO -
/
10-12 30
10-11 60
' " /1
/.~ ix
10-10 90
10-9 120
/
/
10 .8 moI / kg 150 Time { rain )
Fig. 2. Time-course effects of administering either saline or varying concentrations (10-t:-I0-s mol/kg body wt) of phorbol esters on total protein output in pancreatic juice in the anaesthetized rat. Solid columns, saline; open columns, an inactive phorbo] ester; single bar column, TPA and double bar columns, TPA plus 10-s mol/kg Polymyxin B.
Effect o f secretin on pancreatic juice secretion in absence and presence o f TPA
Intravenous administration of increasing doses of secretin between 50 and 1600pmol/kg body wt induced dose-dependent increases in pancreatic juice flow compared to control saline (Fig. 3; open columns). In the combined presence of TPA (10-~o mol/kg), the secretin-evoked pancreatic juice flow was markedly reduced compared to secretin
alone but remained elevated about saline control (Fig. 3; single bar columns). The gut hormone had little or no effect on total protein output in pancreatic juice at a lower dose. However, administration of high doses resulted in marked increases in protein output compared to saline control (Fig. 4; open columns). When TPA was combined with secretin there was little or no effect on protein output compared to intravenous administration of secretin alone (Fig. 4; single bar columns).
320 300 " 280
[ ] Secretin + TPA [ ] Secretin •
Saline
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260
240
~i = o
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/~
o E160
A A
71
120 100 50 30
100 60
200 90
400 120
800 150
1600 pmol Secretin/Kg 180 Time ( rn~ )
Fig. 3. Time-course effects of administering saline (solid columns), secretin (open columns; 50-1600pmol/kg body wt) and secretin in combination with 10-t°mol/kg body wt TPA on rate of pancreatic juice flow in the anaesthetized rat.
G. M. SALIDOet al.
468
200 -- [] Secteti~+ TPA [] Secretin • Saline
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Fig. 4. Time-course effects of administering saline (solid columns), secretin (open columns, 50--1600 pmol/kg body wt) and secretin in combination with 10-z0mol/kg body wt TPA on total protein output in pancreatic juice. DISCUSSION Petersen and Grossman (1977) found that pancreatic secretion in rats may vary considerably with the conditions of the study and that in conscious rats pancreatic secretion of water, bicarbonate and protein was much greater than in anaesthetized rats, both basally and during stimulation by secretin and cholecystokinin. In our experimental conditions, i,e. anaesthetized rats with recirculation of bile into the duodenum but without recirculation of pancreatic juice, the basal protein output and the juice flow were similar to those obtained by others (Petersen and Grossman, 1977) in anaesthetized rats without recirculation of bile and pancreatic juice into the duodenum. In saline treated animals the pancreatic juice flow increases progressively as time passes reaching a magnitude of 20% at the end of the experimental period. Total protein output showed progressive and parallel reductions throughout the experimental period. The progressive increase of the juice flow together with the reduction of total protein output has also been observed in anaesthetized rabbit (De Dios et al., 1986; Pariente et al., 1989) and at the moment there is no convincing explanation for these effects. The non-recirculation of pancreatic juice under our experimental conditions does not seem to be the answer because recirculation suppresses pancreatic secretion (Petersen and Grossman, 1977). As compared to the saline-treated animals, bolus injections of increasing doses of TPA induced a dose-dependent increase in the secretory rate of pancreatic juice and total protein output. These effects were only partially reduced by Polymyxin B, an inhibitor of the protein kinase C. In contrast, Polymyxin B completely blocked the action of TPA in isolated rat pancreatic acini (Wrenn and Wooten, 1984). At present, there is no explanation for this
discrepancy. On the other hand the inactive phorbol ester (4a-phorbol-12,13-didecanoate) had no effect on the secretory rate but increased the total protein output in a similar manner to TPA plus Polymyxin B. These results indicate that protein kinase C activation is associated with rat pancreatic juice secretion. It is also worth noting that neither the phorbol esters nor Polymyxin B had any detectable effect on blood pressure during administration. Intravenous administration of anaesthetized rat preparation with increasing doses of secretin resulted in a gradual increase in juice flow but differential increases in protein output. Both effects of secretin have already been demonstrated in the pancreas of rats both in vivo (Petersen and Grossman, 1977) and in vitro intact preparation (Yoshida and Kanno, 1987). It is now well established that combination of two secretagogues acting via different stimulus-secretion coupling mechanisms can potentiate one another (Gardner, 1979; Schuitz and Stolze, 1980; Petersen, 1982; Case, 1984; De-Pont and Fieuren-Jakobs, 1984; Singh, 1985). This study investigates further the interaction between TPA and secretin on pancreatic juice secretion, especially since previous studies have demonstrated that TPA can potentiate the secretory responses evoked by ACh and noradrenaline as well as the responses induced by the Ca 2+ ionophore A23187 and dibutyryl cyclic AMP (De-Pont and Fleuren-Jakobs 1984; Singh, 1988; Francis and Singh, 1989). Surprisingly, our results show no potentiation between TPA and secretin on either pancreatic juice flow or protein output in the anaesthetized rat. At present we have no explanation for this observation. by Wellcome Trust and Spanish Ministry of Education and Science.
Acknowledgement--Supported
Secretagogue-evoked pancreatic secretion REFERENCES
Argent B. E., Case R. M. and Hirst F. C. (1978) The effect of phorbolmyristate acetate on amylase secretion and 4SCa2+ effiux from rat pancreas. J. Physiol. (Lond.)285, 53P. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Camello P. J., Francis L. P., Madrid J. A., Pariente J. A., Salido G. M. and Singh J. (1989) Phorbol ester stimulates juice secretion and inhibits secretin-evoked secretory response on the in vivo rat pancreas. J. Physiol. 4(18), 99P. Case R. M. (1984) The role of Ca 2+ stores in secretion. Cell Calcium 5, 89-11 I. De Dios i., Arranz A. and Lopez M. A. (1986) The effect of hypotherrnia on exocrine pancreatic secretion in rabbits. Comp. Biochem. Physiol. 83, 677-681. De-Pont J. J. H. A. M. and Fleuren-Jakobs A. M. M. (1984) Synergistic effect of A23187 and phorbol ester on amylase secretion from rabbit pancreatic acini. FEBS Lett. 170, 67-68. Francis L. P. and Singh J. (1989) Effects of acetylcholine, phorbol ester and diacylglycerol kinase inhibition on [~H]leucine-labelled protein secretion from isolated permeabilized rat pancreatic acini. J. Physiol. (Lond.) 410, 49P. Gardner J. D. (1979) Regulation of pancreatic exocrine function in vitro: initial steps in the actions of secretagogues. A. Rev. Physiol. 41, 55-66. Gunther G. R. and Jamieson J. D. (1979) Increased cyclic G M P does not correlate with protein discharge from pancreatic acinar cells. Nature 280, 318-320. Nishizuka Y. (1984) The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 3/Ig, 693-698.
469
Nishizuka Y. (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334, 661-665. Noguchi M., Adachi H., Gardner G. D. and Jensen R. T. (1985) Calcium-activated phospholipid dependent protein kinase in pancreatic acinar cells. Am. J. Physiol. 2415, G692-GT08. Pariente J. A., Madrid J. A. and Salido G. M. (1989) Histamine receptors in unstimulated pancreatic exocrine secretion of the rabbit. Agents Actions 28, 62-69. Petersen O. H. (1982) Stimulus-excitation coupling in plasma membranes of pancreatic acinar cells. Biochim. Biophys. Acta 694, 163-184. Petersen H. and Grossman M. I. (1977) Pancreatic exocrine secretion in anaesthetized and conscious rats. Am. J. Physiol. 233(6), E530-E536. Petersen O. H. and lwatsuki N. (1978) The role of calcium in pancreatic acinar cell stimulus-secretion coupling: an electrophysiological approach. Ann. N.Y. Acad. Sci. 307, 599-617. Schulz I. and Stolze H. H. (1980) The exocrine pancreas: the role of secretagogues, cyclic nucleotides and calcium in enzyme secretion. A. Rev. Physiol. 42, 127-156. Singh J. (1985) Phorbol ester (TPA) potentiates noradrenaline and acetylcholine-evoked amylase secretion in the rat pancreas. FEBS Lett. 180, 191-195. Singh J. (1988) Synergistic effects of secretagogues on amylase secretion in the rat pancreas: evidence of a third stimulus-secretion coupling pathway. In Gastrointestinal and Hepatic Secretions: Mechanism and Control. pp. 225-228. Univ. of Calgary Press, Calgary. Wrenn R. W. and Wooten M. W. (1984) Dual calciumdependent protein phosphorylation systems in pancreas and their differential regulation of Polymyxin B. Life Sci. 35, 267-276. Yoshida T. and Kanno T. (1987) The role of extra-cellular calcium in the secretory response of the exocrine pancreas to secretin and forskolin. Biomed. Res. g, 233-240.
0306-3623/90$3.00+ 0.00 Copyright ~' 1990PergamonPress plc
EFFECTS OF PHORBOL ESTERS A N D SECRETIN ON PANCREATIC JUICE SECRETION IN THE ANAESTHETIZED RAT G. M. SALIDO,* L. P. F~Ncxs, ~P. J. CAMELLO,J. SINGH,! J. A. MADglD and J. A. PARIENTE Department of Physiology, Faculty of Veterinary Science, University of Extremadura, 10080-Caceres, Spain and tSchool of Applied Biology, Lancashire Polytechnic, Preston, England (Received 20 September 1989)
Al~lan~--I. An investigation was made of the effects of phorbol esters, i 2-O-tetradeconylphorbol acetate (TPA) and secretin on pancreatic juice secretion in the anaesthetized rat. TPA (10-t2-10-s mol/kg body wt) evoked marked dose-dependent increases in secretory rate and total protein output. 2. An inactive phorbol ester (4",-phorbol-12-13-didecanoate;4ctPDD) had no effect on the secretory rate but increased total protein output compared to saline control animals. 3. When TPA was administered in combination with the protein kinase C inhibitor, Polymyxin B (10-Smol/kg body wt) both secretory rate and protein output were significantly reduced (P <0.001) compared to TPA alone. 4. Secretin (50-1600 pmol/kg body wt) increased both pancreatic juice flow and total protein output in a dose-dependent manner. 5. Simultaneous administration of secretin (50-1600 pmol/kg body wt) and TPA (10-I° mol/kg body wt) resulted in a marked attenuation in the secretin-induced secretory rate while secretin-evoked protein output was unaffected. 6. The results indicate that protein kinase C activation is associated with pancreatic juice secretion and it may also modulate secretin-induced pancreatic juice flow in the anaesthetized rat.
INTRODUCTION Previous studies on exocrine pancreatic secretion using the in vitro pancreas have demonstrated the presence of two distinct stimulus-secretion coupling mechanisms (Gardner, 1979; Schulz and Stolze, 1980; Petersen, 1982). One pathway is activated by secretin, vasoactive intestinal polypeptide and cathecholamines resulting in the increase in endogenous adenosine-3,5-cyclic monophosphate (cyclicAMP) while the other pathway is stimulated by acetylcholine (ACh), cholecystokinin (CCK) and bombesin-like peptides and involves mobilization of cellular calcium (Ca ~÷) (Petersen and Iwatsuki, 1978; Gardner, 1979; Schulz and Stolze, 1980; Petersen, 1982; Case, 1984). More recently, some studies have shown that the phorboi esters can stimulate enzyme secretion in isolated pancreatic segments and acini without affecting either Ca 2+ mobilization or cyclicAMP metabolism (Argent et al., 1978; Gunther and Jamieson, 1979; De-Pont and Fleuren-Jakobs, 1984; Singh, 1985). This secretory action of TPA is associated with the activation of the phospholipid and Ca ~+-dependent enzyme protein kinase C (Nishizuka, 1984, 1988; Noguchi et al., 1985) as the selective inhibitor of protein kinase C, Polymyxin B can completely block the TPA-induced enzymatic secretion (Wrenn and Wooten, 1984). These recent experiments with TPA suggest the existence for a third
*To whom correspondence should be addressed.
stimulus-secretion coupling pathway controlling enzyme secretion in the isolated pancreatic segments and acini (De-Pont and Fleuren-Jakobs, 1984; Singh, 1988). From a functional point of view no evidence is hvailable as to whether TPA can evoke pancreatic juice secretion and, moreover, modify secretagogueinduced secretory responses in the in vivo pancreas. As such, this study was designed to investigate the effects of phorbol esters on the rate of pancreatic juice secretion and total protein output in the absence and presence of the protein kinase C inhibitor, Polymyxin B in the anaesthetized rat. Furthermore, it was also decided to examine any interactions between TPA and the gut hormone secretin on pancreatic juice secretion. A preliminary account of some aspects of this work was presented to the Physiological Society (Camello et al., 1989).
MATERIALS AND METHODS
Animals and surgical preparation All experiments were performed on overnight-fasted adult Wistar rats (200-450 g body wt) of either sex. The animals were anaesthetized by 0.5-0.75 g/kg i.p. urethane. After tracheal intubation, the fight carotid artery was cannulated to measure systemic blood pressure with a blood pressure transducer connected to a multichannel pen recorder. Drugs were administered via cannula inserted into the left jugular vein. After performing a laparotomy, the pylorus was ligated and the bile-pancreatic duct exposed and cannulated at its entrance to the duodenum for the collection of pancreatic juice. The hepatic end of the bile-pancreatic duct
465
G . M . SALIDOet al.
466 200
~
[] 180
-
•
Inactive phorbol ester Saline
;1,0 •
.G
120
m ~ :alO0
¢ ¢
// //
S ¢
/
/
/
10 -12
10 "11
10 "10
10 -9
30
60
90
120
10 -8 mol / kg 150 Time { min )
Fig. 1. Time-course effects of administering either saline or varying concentrations (10-~-'--10-s mol/kg body wt) phorbol esters on pancreatic juice flow in the anaesthetized rat preparation. Solid columns, saline; open columns, inactive phorbol ester, single bar columns, TPA and double bar columns, TPA plus 10 -8 mol/kg Polymyxin B (a protein kinase C, inhibitor).
was also cannulated and bile was reinfused into the duodenum. Throughout experiments the body temperature was kept between 31 and 33°C with a heating pad.
Experimental design After 45 min of flow stabilization, pancreatic juice was collected on ice for 210 min at 30 min intervals. Pancreatic juice collected between 0 and 30 min was taken as the resting value. Drug or saline administration started at the minute 30. In this study several different experiments were performed using saline, TPA (Sigma), inactive phorbol ester (Sigma), Polymyxin B (Sigma) and secretin (Sigma). All drugs were freshly prepared in 0.9% w/v sodium chloride solution. Firstly, control experiments (saline-treated animals) were performed to determine the effect of the time of cannulation on pancreatic juice secretion. In the second set of experiments the effects of TPA and an inactive phorbol ester (4ctPDD) on pancreatic juice output were determined in different animal preparations. The phorbol esters were administered by means of i.v. bolus injections at doses of 10-~2-10 -s mol/kg body wt. Thirdly, the protein kinase C inhibitor, Polymyxin B (10 -s mol/kg body wt) was injected i.v. as bolus in combination with varying doses (10-t:-I0 -s mol/kg body wt) of TPA. Fourthly, the effect of administering different doses of the gut hormone secretin on pancreatic juice secretion was also investigated in the absence and presence of TPA (10 -j° mol/kg body wt) in separate experiments. Secretin was injected in bolus form at doses of 50, 100, 200, 400, 800 and 1600 pmol/kg body wt.
Estimation offlow of pancreatic juice and total protein output Secretory rates were determined by weighing tared Eppendorph tubes, assuming a density of secretion equal to water. All samples for protein determination were frozen immediately following collection, stored overnight at -20°C and assayed the following day. Several determinations of protein content were made the same day as collection and compared to determinations after freezing and storing and no significant differences were observed. Total protein concentrations were estimated by the method of Bradford (1976) with bovine serum albumin (Sigma) used as the standard.
Data analysis The secretory rates of pancreatic juice were normalised to prestimulated levels and the percentage change was given. Results were analysed by using the Student's t-test for paired data and P < 0.05 was taken as significant. RESULTS
Unstimulated pancreatic secretion In anaesthetized rats the resting flow was 0.27 + 0.04/zl/min 3 0 m i n after the initial i.v. injection of saline compared to 0.33 + 0.05 ~l/min after 150 min. In contrast, protein output in pancreatic juice during the initial 3 0 m i n collection period was 3 . 9 6 + 1.28/~g/min compared to 3.01 + 0 . 3 0 / ~ g / m i n after 150 rain. These control experiments show that saline administration caused a small increase in the flow rate (Figs I and 3; solid columns) whereas protein output (Figs 2 and 4; solid columns) was reduced during the same period. Alternatively, these changes may be due to an alteration in the resting juice flow from the time of cannulation to the end of the experiment.
Effects o f phorbol esters on pancreatic secretion Intravenous bolus injection of T P A (10 - j z 10-8 moi/kg body wt) resulted in marked dose-dependent increases in both the rate of pancreatic juice flow (Fig. I; single bar columns) and total protein output (Fig. 2; single bar columns). These effects of T P A were significantly reduced (P < 0.001) when the protein kinase C inhibitor Polymyxin B (10-Smol/kg body wt) was injected in combination with different doses of T P A (Figs 1 and 2; double bar columns). In contrast, administration of the inactive phorbol ester (10-~2-10-Smol/kg body wt) had little or no effect on the secretory rate compared to control saline (Fig. 1; open columns). However, at high doses (10-9-10-Smol/kg body wt) the inactive phorbol ester evoked an increase in total protein output compared to saline control (Fig. 2; open columns).
Secretagogue-evoked pancreatic secretion 350
300 --
D •_~
5Z [] i7 •
467
I
TPA 4- Polymyxin B TPA Inactive phorbol ester Saline
/ / /
2
~ ~ lSO -
/
10-12 30
10-11 60
' " /1
/.~ ix
10-10 90
10-9 120
/
/
10 .8 moI / kg 150 Time { rain )
Fig. 2. Time-course effects of administering either saline or varying concentrations (10-t:-I0-s mol/kg body wt) of phorbol esters on total protein output in pancreatic juice in the anaesthetized rat. Solid columns, saline; open columns, an inactive phorbo] ester; single bar column, TPA and double bar columns, TPA plus 10-s mol/kg Polymyxin B.
Effect o f secretin on pancreatic juice secretion in absence and presence o f TPA
Intravenous administration of increasing doses of secretin between 50 and 1600pmol/kg body wt induced dose-dependent increases in pancreatic juice flow compared to control saline (Fig. 3; open columns). In the combined presence of TPA (10-~o mol/kg), the secretin-evoked pancreatic juice flow was markedly reduced compared to secretin
alone but remained elevated about saline control (Fig. 3; single bar columns). The gut hormone had little or no effect on total protein output in pancreatic juice at a lower dose. However, administration of high doses resulted in marked increases in protein output compared to saline control (Fig. 4; open columns). When TPA was combined with secretin there was little or no effect on protein output compared to intravenous administration of secretin alone (Fig. 4; single bar columns).
320 300 " 280
[ ] Secretin + TPA [ ] Secretin •
Saline
1"
260
240
~i = o
220 _
=o ~ g'r
180
I
3" -
T
/
'
/~
o E160
A A
71
120 100 50 30
100 60
200 90
400 120
800 150
1600 pmol Secretin/Kg 180 Time ( rn~ )
Fig. 3. Time-course effects of administering saline (solid columns), secretin (open columns; 50-1600pmol/kg body wt) and secretin in combination with 10-t°mol/kg body wt TPA on rate of pancreatic juice flow in the anaesthetized rat.
G. M. SALIDOet al.
468
200 -- [] Secteti~+ TPA [] Secretin • Saline
~o2 8o
[ ~
ilil
f/
,0o i l /
50 30
l
/ 100 60
!
't!tiil!:
/ 200 90
120
150
)molSectetin/Ko 180 Time(nil}
Fig. 4. Time-course effects of administering saline (solid columns), secretin (open columns, 50--1600 pmol/kg body wt) and secretin in combination with 10-z0mol/kg body wt TPA on total protein output in pancreatic juice. DISCUSSION Petersen and Grossman (1977) found that pancreatic secretion in rats may vary considerably with the conditions of the study and that in conscious rats pancreatic secretion of water, bicarbonate and protein was much greater than in anaesthetized rats, both basally and during stimulation by secretin and cholecystokinin. In our experimental conditions, i,e. anaesthetized rats with recirculation of bile into the duodenum but without recirculation of pancreatic juice, the basal protein output and the juice flow were similar to those obtained by others (Petersen and Grossman, 1977) in anaesthetized rats without recirculation of bile and pancreatic juice into the duodenum. In saline treated animals the pancreatic juice flow increases progressively as time passes reaching a magnitude of 20% at the end of the experimental period. Total protein output showed progressive and parallel reductions throughout the experimental period. The progressive increase of the juice flow together with the reduction of total protein output has also been observed in anaesthetized rabbit (De Dios et al., 1986; Pariente et al., 1989) and at the moment there is no convincing explanation for these effects. The non-recirculation of pancreatic juice under our experimental conditions does not seem to be the answer because recirculation suppresses pancreatic secretion (Petersen and Grossman, 1977). As compared to the saline-treated animals, bolus injections of increasing doses of TPA induced a dose-dependent increase in the secretory rate of pancreatic juice and total protein output. These effects were only partially reduced by Polymyxin B, an inhibitor of the protein kinase C. In contrast, Polymyxin B completely blocked the action of TPA in isolated rat pancreatic acini (Wrenn and Wooten, 1984). At present, there is no explanation for this
discrepancy. On the other hand the inactive phorbol ester (4a-phorbol-12,13-didecanoate) had no effect on the secretory rate but increased the total protein output in a similar manner to TPA plus Polymyxin B. These results indicate that protein kinase C activation is associated with rat pancreatic juice secretion. It is also worth noting that neither the phorbol esters nor Polymyxin B had any detectable effect on blood pressure during administration. Intravenous administration of anaesthetized rat preparation with increasing doses of secretin resulted in a gradual increase in juice flow but differential increases in protein output. Both effects of secretin have already been demonstrated in the pancreas of rats both in vivo (Petersen and Grossman, 1977) and in vitro intact preparation (Yoshida and Kanno, 1987). It is now well established that combination of two secretagogues acting via different stimulus-secretion coupling mechanisms can potentiate one another (Gardner, 1979; Schuitz and Stolze, 1980; Petersen, 1982; Case, 1984; De-Pont and Fieuren-Jakobs, 1984; Singh, 1985). This study investigates further the interaction between TPA and secretin on pancreatic juice secretion, especially since previous studies have demonstrated that TPA can potentiate the secretory responses evoked by ACh and noradrenaline as well as the responses induced by the Ca 2+ ionophore A23187 and dibutyryl cyclic AMP (De-Pont and Fleuren-Jakobs 1984; Singh, 1988; Francis and Singh, 1989). Surprisingly, our results show no potentiation between TPA and secretin on either pancreatic juice flow or protein output in the anaesthetized rat. At present we have no explanation for this observation. by Wellcome Trust and Spanish Ministry of Education and Science.
Acknowledgement--Supported
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