169
Fundam CIin Pharmacol(l992) 6,169-176 0 Elsevier, Paris
CRL41405: a drug with a new pharmacological profile on pancreatic exocrine secretion in the rat C Nagain, J Chariot, C Vaille, C Roze
*
INSERM U2.39, Fnculte'X Bichat, I6 rue H tluchord, 7.5018 Paris, Frorice (Received 17 January 1992; accepted 6 April 1992)
Summary - The recently described compound CRLA1405 displays central effects suggesting possihle antidepressive and awakening properties. In order to further analyze the pliarmacology of t l U ~compound, its effects were studied on basal and stimulated pancreatic secretion in anaestlietized rats. CRL41405 alone (7-20 niglkg, sc) liad no effect on basal pancreatic secretion. Larger doses (67-200 mgkg) increased hasal secretion tlirougli the stiiiiulation of cliolinergic muscarinic receptors, the effect being antagonized by atropine. CRL41405 (20 niglkg) suppressed the 2-deoxyglucose-induced (hut not the acetylclioline-induced) stiniulation of pancreatic secretion through an alpha-:! adrenoceptor inluhitory nieclianism that was hlocked by idazoxan (0.3 mglkg, sc). In addition, a beta adrenoceptor mediated stimulation of sodium and bicarhonate excretion (blocked by propranolol) was evidenced when the alplia-2 inhibition was suppressed by idazoxan. Under alpha-:! adrenoceptor hlockade. water and electrolyte stimulation by CRLA1405 could he demonstrated on hasal. 2-deoxyglucose-induced and acetylclioline-induced pancreatic secretion. This original profile makes CRL41405 a unique drug in pancreatic pliarinacology. pancreatic secretion / nand /3 adrenoreptors / CRL41405 / rat model
Introduction
CRL4 1405 (a-hexamethyleneimino, p-anlino propiophenone dichlorhydrate, fig 1) is a recently synthesized compound (French patent No 88-01565) whose pharmacology has not as yet been described in detail. Unpublished studies indicate that CRLA1405 has central effects suggesting an antidepressive and awakening action in rats and mice (J Duteil, personal communication). These effects include: antagonism of aponiorphine-, reserpineand oxotremorine-induced hypothermia, decrease in comportmental despair in mice, antagonism of barbital-induced sleep, increase in spontaneous
* Correspondence and reprints
motility and in exploratory motility in mice. CRL41405 has few peripheral effects, displaying in large doses only a moderate alpha-adrenoceptor stimulation (mydriasis, antagonism of reserpine ptosis and oxotremorine-induced tremor, reduction of femoral blood flow after intra-arterial injection in dogs). Chronic administration of CRL41405 induced localized foci of cell vacuolization in the pancreas, and this histological finding prompted us to study the pancreatic effects of this drug. Analysing the action of CRLA1405 on basal and stimulated pancreatic secretion showed an original profile of adrenoceptor stimulation which is described here.
170
C Nagain
Fig 1. Formula of CRL41405.
Materials and methods Animals Male Wistar rats weighing 280-300 g were obtained from Iffa Credo, Les Oncins, L’Arbresle, France and were maintained on a standard laboratory pelleted chow (UAR 113, Alimentation Rationnelle, Villemoisson, Epinay-sur-Orge. France). Collection of puncrearic juice and experirrrentul sclrcdules The rats were deprived of food at 11 am, the day before the experiments, but were allowed free access to water. They were anaesthetized with ethylurethane the following morning (1.125 g k g , im) and an acute pancreatic fistula was prepared. Bile was diverted, and the purc pancreatic secretion was collected with a continuous dilution method (Rozk et al, 1975). The temperature was maintained at 38 f 05°C throughout the study. Sodium (flame photometer), bicarbonate (Chariot and RozC, 1976), and total protein (UV absorbance at 180 nm) were determined in 20-min samples of pancreatic juice, beginning 4 h after surgery. Since the concentration of sodium in the pancreatic juice is constant at about 145 mmoVl in this preparation, monitoring the sodium output is equivalent to measuring the vol of juice secreted (Rozk et al, 1975). The average of the two 20-min fractions collected between f = - 40 min and t = 0 was taken as the basal value. The effect of CRL41405 was first studied on basal secretion after the subcutaneous (sc) injection of different doses (7; 20; 67; 200 mgkg). The mechanism of action of CRL41405 on basal secretion was studied hy associating vagotomy. performed by sectioning both vagus nerve trunks in the neck, at the same time as fitting the pancreatic fistula, or atropine sulphate, administered 20 min before CRL41405 as a primer holus intravenous (iv) injection of 75 pg/kg. followed by a 3 h intravenous infusion of 75 p d k g h . This dose of atro-
ef nl
pine has been shown to be the smallest that can maximally inhibit basal pancreatic secretion in conscious rats (Chariot et al, 1987). The effect of CRL41405 was also measured on secretion stimulated by 2-deoxy-~-glucose(2DG) or acetylcholine. At time zero, an iv injection of 75 m g k g of 2DG was given. This dose has been shown to produce a central stimulation of pancreatic secretion which is about 50% maximal (Appia et al, 1984). Acetylcholine was iv infused as increasing doses (60, 180, 600 and 1 800 pgkgh. each dose infused for 40 min). CRL41405 was injected sc, 5 min before 2DG or before beginning acetylcholine infusion. In antagonist experiments (idazoxan, prazosin, propranolol, naloxone), the antagonist drug was injected sc, 5 min before CRL41405. The doses of antagonists were chosen as follows from results obtained in our laboratory o n pancreatic secretion in rats: idazoxan 0.3 mgkg sc, an alpha-2-blocking dose validated against clonidine (Chariot et al, 1988); prazosin 1 mgkg sc, the largest alpha-1-blocking dose tolerated in our conditions (Nagain ct al. 1991); propranolol 1 mg/kg sc. a beta-blocking dose validated against isoproterenol (RozC et al, 1976); naloxone 1 mg/kg sc. a dose blocking the pancreatic effect of 5 mgkg methadone (Nagain et (11. 1991).
2DG, acetylcholine. atropine and propranolol were purchased from Sigma Chemicals (St Louis, USA), nuloxone was purchased from Endo Laboratories Inc (New York. USA), CRL41405 was a gift from the Centre de Rccherches Lafon (Maisons-Alfort, France), idazoxan was a gift of Reckitt and Colman (Kingstonupon-Hull. IJK), prazosin was a gift from Pfizer (Brussels, Belgium). Sfci t i s firs
The results are given as the means f SEM (standard error of the mean) in groups of 5-10 rats. The response to stimulants (CRL41405. 2DG, acetylcholine) are exprcssed as thc anmunt of pancreatic secretion produced over the basal level (A). This response is shown either as the time-related variation in secretion, measured in ?O-nlin samples of pancreatic juice, or as the integrated response over the basal level, measured during thc 3 li following CRL41405 or 2DG injection (= cumulative 3 h response). Statistical differences were assessed by analysis of variance, followed when necessary by a
CRLA 1405 and pancreatic secretion
multigroup comparisnll test (SchellC’s test, Winer, 1971). A P value of c 0.05was considered significant.
Results
B ~ s t rpcmcrerrric l secretion Effect of CRLAIJOS on bnscrl secretion Sc injections of CRL41405 produced a dose-related increase in pancreatic secretion (figs 2 and 3 ) which was significant with the doses of 67 and 200 mgkg for sodiunl and bicarbonate output and with the dose of 67 mgkg for protein output (fig 3). ‘The CRL41405-induced stimulation was progressive and long lasting (more than 180 nlin) (fig 2). The peak response represented respectively about 30% (sodium and bicarbonate) and 10% (protein) of the maximal stimulation induced by cholecystokinin (CCK), as previously deternuned in the same experimental model (Nagain et nl, 1989). CRL41405 at the dose of 200 mgkg also produced systemic effects, including respiratory rhythm problems for 10 min after the injection, togcther with chromdacryorrhea. Cholinergic dependence of CRWI4OS stitliu/trtion of Orrsnl secretiorl In these experiments, atropine sulphare infusion or vagotomy was associated with a large dose of CRL41405 (200 nig/kg). The results of thcse prctreatments are shown in figure 4 as the integrated 3 h response over the basal level. Vagotomy did not change the bicarbonate and protein responses to CRL41405 but decreased by SO% (P c 0.05) the sodium response. Atropine reduced by 9 0 % the effect of CRL41405 on sodium and bicarbonate (P < 0.01) and by 60% (NS) the small effect of CRL41405 on protein. Atropine also abolished the systemic effects of CRL41405 on respiration and tears.
Effect of CRLAI4M on ZDG-stit~~ul~tetl secretion 2DG (75 m g k g ) induced a progressive (peak at
171
80 min) and long-lasting (more than 180 min) stimulation of pancreatic secretion. The peak response represented about 50% (sodium and bicarbonate) and 30% (protein) of the maximal response to CCK (Appia el al, 19844; Nagain el al, 1989). CRL41405 (20 mgkg, sc), injected 5 min before 2 DG, reduced the pancreatic response to 2DG.
7mglkg 20mglkg + 67mglkg 200mgkg -a-
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Fig 2. Tinie course of the stimulation of pancreatic secretion of sodiuni. hicarhonate and total protein in the pancreatic juice by increasing suhcutaneous doses of CRL41405. The results are expressed as variations (A) over the basal level, measured in 20-niin samples of pancreatic juice. M f SEM in groups of 6 nts per dose.
172
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expressed as the integrated 3 h response over the basal level (fig S), by 67% (P c 0.01) for sodium, by 62% (P < 0.01) for bicarbonate and by 74% (P < 0.01) for protein.
Mechanism of action of CRW1405 on 2DG-stinzulated pancreatic secretion The inhibition of the 2DG effect induced b y 20 mgkg of CRL41405 was not suppressed by the previous injection of naloxone (1 mgkg, sc), propranolol (1 mgkg,sc), or prazosin (2 mgkg,s ~ ) , but was markedly changed by idazoxan
a
.1 C
+At
+VX
CRL41405 Fig 4. Effect of atropine (75 p g k g + 75 pgkg.11, iv) and hilateral tmiical vagotomy on the pancreatic stinidation produced by 200 mgkg CRLA1405, sc. C = control group of vehicle treated rats. The results are expressed as the 3 h integrated response over the hasal level. M f SEM in groups of 6 n t s . * P < 0.05, ** P < 0.01 with respect to contml group (C).
(0.3 mgkg,sc) (fig 5). While idazoxan completely suppressed the CRL41405-induced inhibition of the 2DG effect on pancreatic secretion of protein, the combination of idazoxan and CRL41405 increased the hydroelectrolytic pancreatic response to 2DG. by 194% (P < 0.01) for sodium and 178% ( P < 0.01) for bicarbonate (fig 5).
CRL41405 and pancreatic secretion
Effect of CRU1405 on acetylcholine-stimulated pancreatic secretion
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Acetylcholine infused alone produced a doserelated increase in pancreatic secretion (fig 6). The addition of idazoxan alone or of CRL41405 alone (20 mgkg, sc) had little effect upon the acetylcholine stimulation. In contrast, the association of CRL41405 + idazoxan significantly increased ( P c 0.01) the sodium and bicarbonate response to all doses of acetylcholine but had very little effect on the protein response.
Effect of CRL41405 on brrsril secretion in the presence of itkrzoxon
CRL2DG
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+Nx+Pz +P +I +I+P 2DG+CRL41405
Fig 5. Effect of
CRL41405 (20 m a g , sc) on the pancreatic response to 2 deoxy-D-glucose (2DG; 75 rng/kg, iv). CRLA1405 was injected alone or associated with a pre-treatment by naloxone (Nx, 1 rngkg, sc), prazosin (Pz, 2 rngkg, sc), propranolol (P, 1 m g k g , sc), idazoxan (I, 0.3 nigkg, sc) or idazoxan + propranolol (I + P). The absence of effect of CRL41405 alone on basal secretion lias been shown for comparison (CRL).The results are expressed as the 3 ti integrated response over the basal level. M f SEM in groups of 5-10 rats. ** P < 0.01 with respect to group 2DG.
The previous results led us to investigate the effect of CRL41405 on basal pancreatic secretion in the presence of idazoxan (fig 7). Idazoxan did not change the effect of the smallest dose of CRL41405 (7 mgkg), but significantly increased the sodium ( P c 0.01) and bicarbonate ( P c 0.01) responses to 20 and 67 mg/kg of CRL41405, and the protein response ( P c 0.01) to 20 mgkg of CRL41405. The stimulation of s d i u m and bicarbonate pancreatic secretion produced by CRL41405 in the presence of idazoxan was powerful, since the peak secretion reached 50% (sodium) and 80% respectively (bicarbonate) of the maximal effect obtained with CCK. This effect peaked 40-60 min after the CRL41405 injection. In contrast, the protein stimulation produced by CRL41405 + idazoxan was progressive and late: it peaked only 160-180 min after the CRL41405 injection. The injection of propranolol (1 mgkg sc) 5 min before idazoxan totally reversed the increase of response to CRL41405 produced by idazoxan.
Discussion
The increase in sodium and bicarbonate response to 2DG produced by combining CRL41405 + idazoxan was abolished by the further addition of propranolol(1 mgkg, sc; fig 5).
CRL41405, sc administered in the rat, was able to modulate external pancreatic secretion through several mechanisms: both beta- (stimulatory) and
-
174
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Minutes Fig 6. Effect of CRL41405 (CRL, 20 mgkg, sc) alone or associated with idazoxan (I, 0.3 mgkg, sc) on pancreatic secretion stimulated by increasing doses of acetylcholine. The absence of effect of CRL alone on hasal secretion has been shown for comparison. The results are expressed as the secretion over the hasal level in 20-min samples. M f SEM in groups of 6 rats. ** P < 0.01 with respect to the acetylcholine group alone.
alpha-2 (inhibitory) adrenoceptor stimulation were evidenced at moderate doses, whereas large doses induced cholinergic activation. Basal pancreatic secretion was not changed by small and moderate doses of CRL41405 alone, because inhibition and stimulation mediated by
0
--+I
7
+I +I+P
20 CRL41405 (mg/kg sc)
+I +I+P
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Fig 7. Effect of CRL41405 associated with idazoxan (I, 0.3 nigkg) and propranolol (1 mgkg, sc) on basal pancreatic secretion. The results are expressed as the 3 11 integrated respouse over the basal level. M f SEM in groups of 5 to 6 rats. ** P c 0.01 with respect to group CRL41405 alone at the same dose.
adrenoceptors were balanced. In large doses, CRL41405 stimulated basal pancreatic secretion through the activation of a cholinergic mechanism.
CRL.41405 aiid pancreatic secrctioii
The efficacy of CRL41405 was however moderate, since the maximal secretion remained under 30% (sodium and bicarbonate) and 10% (protein) of the maximal CCK-induced stimulation nieasured in our laboratory in other experiments (Nagain et 01, 1989). Doses larger than 200 mgkg were not used. because of cholinergic systemic side-effects. The effects of 200 mg/kg of CRL41405 (pancreatic stimulation and systemic side-effects) were totally abolished by atropine infusion, thus demonstrating their muscarinic dependence. Vagotomy also moderately decreased the action of CRL41405 on sodium output, a finding which seems difficult to explain. This may be due to the fact that sodium output may to some extent be reduced by vagotoniy. When moderate doses of CRL41405 were injected after idazoxan, a clear stimulation of sodium and bicarbonate output was found. This stiniulation was already maximal after 20 nig/kg CRL41405, and seems to be related to a beta-adrenoceptor stimulation, since it was suppressed by propranolol. Similar beta-adrenoceptor related stimulation of water and electrolytes excretion has been documented following the infusion of several sympathetic amines (isoproterenol, epinephrine, norepinephrine, dopanline) in the same experimental model (Rozc? et cil, 1976; Chariot et crl, 1983). This stimulation appears to be related to a CAMP mediated event in pancreatic cells (Lingard and Young, 1983). CRL41405 was also able to inhibit pancreatic secretion stiniulated by 2DG. When 20 m g k g of CRL41405 (a dose which had no effcct on basal secretion) was administered, a significant inhibition of 2DG stimulation of all the variables of pancreatic secretion (sodium, bicarbonate and total protein) was found. This inhibition was likely to depend upon the central and/or peripheral inhibition of neurotransniitter release from vagal fibers, since the direct effect of acetylcholine on pancreatic cells was not clearly changed by CRL4140S. The CRL41405 inhibition of 2DG effect was not altered by naloxone, propranolol, or prazosin, suggesting that i t was independent from opiate
175
receptors, as well as from beta- and alpha-1 adrenoceptors. In contrast, the CRL41405 inhibition of 2DG-stimulated secretion was markedly altered by idazoxan, indicating that an alpha-2 adrenoceptor-mediated event was involved. Pancreatic inhibition by clonidine, guanabenz and other alpha-2 agonists has been documented previously (Rozc?et crl, 1981; Chariot et crl, 1988); i t involves both central and peripheral actions (Chariot er al, 1988). Whereas the 2DG-induced pancreatic protein stimulation recovered its normal value after idazoxan + CRL41405, the 2DG-induced sodium and bicarbonate stimulation was apparently potentiated by idazoxan + CRL41405. In fact, the betastimulating effect of CRL41405 was unveiled by alpha-2 adrenoceptor blockade, and the observed effect corresponded approximately to the addition of ~Iicidazoxan-unveiled effect of CRL41405 on basal secretion, to the effect of 2DG per se. A similar situation was observed in the experiments with acetylcholine. While CRL41405 alone, or idazoxan alone, did not clearly change the response of pancreatic cells to their direct stimulation by acetylcholine, the idazoxan-unveiled effect of CRL41405 added a constant amount of sodium and bicarbonate excretion above the dose-related response to acetylcholine, thus showing a leftwards shift of the acetylcholine dose-response curve. In conclusion, CRL41405 displayed a complex and original pattern of adrenoceptor-related effects on pancreatic secretion in rats. Under the appropriate conditions, we observed: i) an alpha-:! adrenoceptor-mediated inhibition of 2DG-stimulated secretion; ii) a bcta adrenoceptor-niediated stimulation of water and electrolyte secretion, that can add to the cfl'ect of 2DG or acetylcholine. Muscarinic stimulation may also be observed, but only after large doses of CRL41405.
Ackiiowledgmetits Financial support was provided in part by the Conseil Scicntil'ique of FacultC X Bichat and by the Association Charlcs Dohray.
176
C Nagain
References Appia F, De la Tour J. Chariot J, Roz6 C (1984) Action du 5-thio-glucose sur la sCcr6tion pancrkatique externe chez le rat. Comparaison avec le 2-dCsoxy-glucose. Gustrocnt4rol CIin Biol8,746-748 Chariot J, Appia F, Del Tacca M, Tsocas A, Rose C (1988) Central and peripheral inhibition of exocrine pancreatic secretion by alpha-2 adrenergic agonists in the rat. Plzurniacol RCS Coniniun 20, 707-717 Chariot J, De la Tour J, Vaille C, Roz6 C (1987) Comparative effects of pirenzepine and atropine on pancreatic secretion in conscious rats. Arch Int Pharniucodyn Tlicr 285, 158-165 Chariot J, Roz6 C (1976) DCterinination automatist% de tr8s faibles concentrations de bicarbonate. Application au suc pancrkatique. Ann Biol Cliri 34. 269-272 Chariot J, Roz6 C, De la Tour J, Souchard M, Vaille C (1983) Modulation of stimulated pancreatic secretion by sympathomimetic amines in the rat. Pharrtiacology 26,313-323 Lingard JM, Young JA (1983) PAdrenergic control of exocrine secretion by perfused rat pancreas in vitro. Am J Pliysiol245, G690-G696
el
ti/
Nagain C, Chariot J. Vaille C, Roz6 C (1991) Effect of modafinil on pancreatic exocrine secretion in the rat. A comparison with methadone. Eur J Plzurmacol201, 203-2 08 Nagain C, Mendre C, Rodriguez M, Bernard C. Martinez J, Rozk C (1989) External pancreatic secretion in the rat. Supra-maximal inhibition induced by the cholecystokinin octapeptide (CCK 26-33) and analogs altered on the 2 8 - 2 9 bond. Peptides 10, 104 1-1047 Roz6 C, Chariot J. Appia F, Pascaud X. Vaille C (1981) Clonidine inhibition of pancreatic secretion in rats: a possible central site of action. Eur J Pharntacol 76, 381-390 Rozd C, La Tour J de, Chariot J, Souchard M. Debray C (1975)Tcchnique d’Ctude de la s6crCtion pancrkatique externe chez le rat. Biol GastroentCrol (Paris) 8 . 19 1-295 Rozd C, La Tour J de, Chariot J. Souchard M. Vaille C, Dupont C, Jean E, Wepierre J (1976) Isoproterenolinduced pancreatic secretion in rats: a comparison with secretin. Biornedicinc 24. 410-417 Wincr BJ (1971) Statistical principles in experimental dcsign. Mc Craw IIiII, New York
Fundam CIin Pharmacol(l992) 6,169-176 0 Elsevier, Paris
CRL41405: a drug with a new pharmacological profile on pancreatic exocrine secretion in the rat C Nagain, J Chariot, C Vaille, C Roze
*
INSERM U2.39, Fnculte'X Bichat, I6 rue H tluchord, 7.5018 Paris, Frorice (Received 17 January 1992; accepted 6 April 1992)
Summary - The recently described compound CRLA1405 displays central effects suggesting possihle antidepressive and awakening properties. In order to further analyze the pliarmacology of t l U ~compound, its effects were studied on basal and stimulated pancreatic secretion in anaestlietized rats. CRL41405 alone (7-20 niglkg, sc) liad no effect on basal pancreatic secretion. Larger doses (67-200 mgkg) increased hasal secretion tlirougli the stiiiiulation of cliolinergic muscarinic receptors, the effect being antagonized by atropine. CRL41405 (20 niglkg) suppressed the 2-deoxyglucose-induced (hut not the acetylclioline-induced) stiniulation of pancreatic secretion through an alpha-:! adrenoceptor inluhitory nieclianism that was hlocked by idazoxan (0.3 mglkg, sc). In addition, a beta adrenoceptor mediated stimulation of sodium and bicarhonate excretion (blocked by propranolol) was evidenced when the alplia-2 inhibition was suppressed by idazoxan. Under alpha-:! adrenoceptor hlockade. water and electrolyte stimulation by CRLA1405 could he demonstrated on hasal. 2-deoxyglucose-induced and acetylclioline-induced pancreatic secretion. This original profile makes CRL41405 a unique drug in pancreatic pliarinacology. pancreatic secretion / nand /3 adrenoreptors / CRL41405 / rat model
Introduction
CRL4 1405 (a-hexamethyleneimino, p-anlino propiophenone dichlorhydrate, fig 1) is a recently synthesized compound (French patent No 88-01565) whose pharmacology has not as yet been described in detail. Unpublished studies indicate that CRLA1405 has central effects suggesting an antidepressive and awakening action in rats and mice (J Duteil, personal communication). These effects include: antagonism of aponiorphine-, reserpineand oxotremorine-induced hypothermia, decrease in comportmental despair in mice, antagonism of barbital-induced sleep, increase in spontaneous
* Correspondence and reprints
motility and in exploratory motility in mice. CRL41405 has few peripheral effects, displaying in large doses only a moderate alpha-adrenoceptor stimulation (mydriasis, antagonism of reserpine ptosis and oxotremorine-induced tremor, reduction of femoral blood flow after intra-arterial injection in dogs). Chronic administration of CRL41405 induced localized foci of cell vacuolization in the pancreas, and this histological finding prompted us to study the pancreatic effects of this drug. Analysing the action of CRLA1405 on basal and stimulated pancreatic secretion showed an original profile of adrenoceptor stimulation which is described here.
170
C Nagain
Fig 1. Formula of CRL41405.
Materials and methods Animals Male Wistar rats weighing 280-300 g were obtained from Iffa Credo, Les Oncins, L’Arbresle, France and were maintained on a standard laboratory pelleted chow (UAR 113, Alimentation Rationnelle, Villemoisson, Epinay-sur-Orge. France). Collection of puncrearic juice and experirrrentul sclrcdules The rats were deprived of food at 11 am, the day before the experiments, but were allowed free access to water. They were anaesthetized with ethylurethane the following morning (1.125 g k g , im) and an acute pancreatic fistula was prepared. Bile was diverted, and the purc pancreatic secretion was collected with a continuous dilution method (Rozk et al, 1975). The temperature was maintained at 38 f 05°C throughout the study. Sodium (flame photometer), bicarbonate (Chariot and RozC, 1976), and total protein (UV absorbance at 180 nm) were determined in 20-min samples of pancreatic juice, beginning 4 h after surgery. Since the concentration of sodium in the pancreatic juice is constant at about 145 mmoVl in this preparation, monitoring the sodium output is equivalent to measuring the vol of juice secreted (Rozk et al, 1975). The average of the two 20-min fractions collected between f = - 40 min and t = 0 was taken as the basal value. The effect of CRL41405 was first studied on basal secretion after the subcutaneous (sc) injection of different doses (7; 20; 67; 200 mgkg). The mechanism of action of CRL41405 on basal secretion was studied hy associating vagotomy. performed by sectioning both vagus nerve trunks in the neck, at the same time as fitting the pancreatic fistula, or atropine sulphate, administered 20 min before CRL41405 as a primer holus intravenous (iv) injection of 75 pg/kg. followed by a 3 h intravenous infusion of 75 p d k g h . This dose of atro-
ef nl
pine has been shown to be the smallest that can maximally inhibit basal pancreatic secretion in conscious rats (Chariot et al, 1987). The effect of CRL41405 was also measured on secretion stimulated by 2-deoxy-~-glucose(2DG) or acetylcholine. At time zero, an iv injection of 75 m g k g of 2DG was given. This dose has been shown to produce a central stimulation of pancreatic secretion which is about 50% maximal (Appia et al, 1984). Acetylcholine was iv infused as increasing doses (60, 180, 600 and 1 800 pgkgh. each dose infused for 40 min). CRL41405 was injected sc, 5 min before 2DG or before beginning acetylcholine infusion. In antagonist experiments (idazoxan, prazosin, propranolol, naloxone), the antagonist drug was injected sc, 5 min before CRL41405. The doses of antagonists were chosen as follows from results obtained in our laboratory o n pancreatic secretion in rats: idazoxan 0.3 mgkg sc, an alpha-2-blocking dose validated against clonidine (Chariot et al, 1988); prazosin 1 mgkg sc, the largest alpha-1-blocking dose tolerated in our conditions (Nagain ct al. 1991); propranolol 1 mg/kg sc. a beta-blocking dose validated against isoproterenol (RozC et al, 1976); naloxone 1 mg/kg sc. a dose blocking the pancreatic effect of 5 mgkg methadone (Nagain et (11. 1991).
2DG, acetylcholine. atropine and propranolol were purchased from Sigma Chemicals (St Louis, USA), nuloxone was purchased from Endo Laboratories Inc (New York. USA), CRL41405 was a gift from the Centre de Rccherches Lafon (Maisons-Alfort, France), idazoxan was a gift of Reckitt and Colman (Kingstonupon-Hull. IJK), prazosin was a gift from Pfizer (Brussels, Belgium). Sfci t i s firs
The results are given as the means f SEM (standard error of the mean) in groups of 5-10 rats. The response to stimulants (CRL41405. 2DG, acetylcholine) are exprcssed as thc anmunt of pancreatic secretion produced over the basal level (A). This response is shown either as the time-related variation in secretion, measured in ?O-nlin samples of pancreatic juice, or as the integrated response over the basal level, measured during thc 3 li following CRL41405 or 2DG injection (= cumulative 3 h response). Statistical differences were assessed by analysis of variance, followed when necessary by a
CRLA 1405 and pancreatic secretion
multigroup comparisnll test (SchellC’s test, Winer, 1971). A P value of c 0.05was considered significant.
Results
B ~ s t rpcmcrerrric l secretion Effect of CRLAIJOS on bnscrl secretion Sc injections of CRL41405 produced a dose-related increase in pancreatic secretion (figs 2 and 3 ) which was significant with the doses of 67 and 200 mgkg for sodiunl and bicarbonate output and with the dose of 67 mgkg for protein output (fig 3). ‘The CRL41405-induced stimulation was progressive and long lasting (more than 180 nlin) (fig 2). The peak response represented respectively about 30% (sodium and bicarbonate) and 10% (protein) of the maximal stimulation induced by cholecystokinin (CCK), as previously deternuned in the same experimental model (Nagain et nl, 1989). CRL41405 at the dose of 200 mgkg also produced systemic effects, including respiratory rhythm problems for 10 min after the injection, togcther with chromdacryorrhea. Cholinergic dependence of CRWI4OS stitliu/trtion of Orrsnl secretiorl In these experiments, atropine sulphare infusion or vagotomy was associated with a large dose of CRL41405 (200 nig/kg). The results of thcse prctreatments are shown in figure 4 as the integrated 3 h response over the basal level. Vagotomy did not change the bicarbonate and protein responses to CRL41405 but decreased by SO% (P c 0.05) the sodium response. Atropine reduced by 9 0 % the effect of CRL41405 on sodium and bicarbonate (P < 0.01) and by 60% (NS) the small effect of CRL41405 on protein. Atropine also abolished the systemic effects of CRL41405 on respiration and tears.
Effect of CRLAI4M on ZDG-stit~~ul~tetl secretion 2DG (75 m g k g ) induced a progressive (peak at
171
80 min) and long-lasting (more than 180 min) stimulation of pancreatic secretion. The peak response represented about 50% (sodium and bicarbonate) and 30% (protein) of the maximal response to CCK (Appia el al, 19844; Nagain el al, 1989). CRL41405 (20 mgkg, sc), injected 5 min before 2 DG, reduced the pancreatic response to 2DG.
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Fig 2. Tinie course of the stimulation of pancreatic secretion of sodiuni. hicarhonate and total protein in the pancreatic juice by increasing suhcutaneous doses of CRL41405. The results are expressed as variations (A) over the basal level, measured in 20-niin samples of pancreatic juice. M f SEM in groups of 6 nts per dose.
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expressed as the integrated 3 h response over the basal level (fig S), by 67% (P c 0.01) for sodium, by 62% (P < 0.01) for bicarbonate and by 74% (P < 0.01) for protein.
Mechanism of action of CRW1405 on 2DG-stinzulated pancreatic secretion The inhibition of the 2DG effect induced b y 20 mgkg of CRL41405 was not suppressed by the previous injection of naloxone (1 mgkg, sc), propranolol (1 mgkg,sc), or prazosin (2 mgkg,s ~ ) , but was markedly changed by idazoxan
a
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CRL41405 Fig 4. Effect of atropine (75 p g k g + 75 pgkg.11, iv) and hilateral tmiical vagotomy on the pancreatic stinidation produced by 200 mgkg CRLA1405, sc. C = control group of vehicle treated rats. The results are expressed as the 3 h integrated response over the hasal level. M f SEM in groups of 6 n t s . * P < 0.05, ** P < 0.01 with respect to contml group (C).
(0.3 mgkg,sc) (fig 5). While idazoxan completely suppressed the CRL41405-induced inhibition of the 2DG effect on pancreatic secretion of protein, the combination of idazoxan and CRL41405 increased the hydroelectrolytic pancreatic response to 2DG. by 194% (P < 0.01) for sodium and 178% ( P < 0.01) for bicarbonate (fig 5).
CRL41405 and pancreatic secretion
Effect of CRU1405 on acetylcholine-stimulated pancreatic secretion
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Acetylcholine infused alone produced a doserelated increase in pancreatic secretion (fig 6). The addition of idazoxan alone or of CRL41405 alone (20 mgkg, sc) had little effect upon the acetylcholine stimulation. In contrast, the association of CRL41405 + idazoxan significantly increased ( P c 0.01) the sodium and bicarbonate response to all doses of acetylcholine but had very little effect on the protein response.
Effect of CRL41405 on brrsril secretion in the presence of itkrzoxon
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Fig 5. Effect of
CRL41405 (20 m a g , sc) on the pancreatic response to 2 deoxy-D-glucose (2DG; 75 rng/kg, iv). CRLA1405 was injected alone or associated with a pre-treatment by naloxone (Nx, 1 rngkg, sc), prazosin (Pz, 2 rngkg, sc), propranolol (P, 1 m g k g , sc), idazoxan (I, 0.3 nigkg, sc) or idazoxan + propranolol (I + P). The absence of effect of CRL41405 alone on basal secretion lias been shown for comparison (CRL).The results are expressed as the 3 ti integrated response over the basal level. M f SEM in groups of 5-10 rats. ** P < 0.01 with respect to group 2DG.
The previous results led us to investigate the effect of CRL41405 on basal pancreatic secretion in the presence of idazoxan (fig 7). Idazoxan did not change the effect of the smallest dose of CRL41405 (7 mgkg), but significantly increased the sodium ( P c 0.01) and bicarbonate ( P c 0.01) responses to 20 and 67 mg/kg of CRL41405, and the protein response ( P c 0.01) to 20 mgkg of CRL41405. The stimulation of s d i u m and bicarbonate pancreatic secretion produced by CRL41405 in the presence of idazoxan was powerful, since the peak secretion reached 50% (sodium) and 80% respectively (bicarbonate) of the maximal effect obtained with CCK. This effect peaked 40-60 min after the CRL41405 injection. In contrast, the protein stimulation produced by CRL41405 + idazoxan was progressive and late: it peaked only 160-180 min after the CRL41405 injection. The injection of propranolol (1 mgkg sc) 5 min before idazoxan totally reversed the increase of response to CRL41405 produced by idazoxan.
Discussion
The increase in sodium and bicarbonate response to 2DG produced by combining CRL41405 + idazoxan was abolished by the further addition of propranolol(1 mgkg, sc; fig 5).
CRL41405, sc administered in the rat, was able to modulate external pancreatic secretion through several mechanisms: both beta- (stimulatory) and
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Minutes Fig 6. Effect of CRL41405 (CRL, 20 mgkg, sc) alone or associated with idazoxan (I, 0.3 mgkg, sc) on pancreatic secretion stimulated by increasing doses of acetylcholine. The absence of effect of CRL alone on hasal secretion has been shown for comparison. The results are expressed as the secretion over the hasal level in 20-min samples. M f SEM in groups of 6 rats. ** P < 0.01 with respect to the acetylcholine group alone.
alpha-2 (inhibitory) adrenoceptor stimulation were evidenced at moderate doses, whereas large doses induced cholinergic activation. Basal pancreatic secretion was not changed by small and moderate doses of CRL41405 alone, because inhibition and stimulation mediated by
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Fig 7. Effect of CRL41405 associated with idazoxan (I, 0.3 nigkg) and propranolol (1 mgkg, sc) on basal pancreatic secretion. The results are expressed as the 3 11 integrated respouse over the basal level. M f SEM in groups of 5 to 6 rats. ** P c 0.01 with respect to group CRL41405 alone at the same dose.
adrenoceptors were balanced. In large doses, CRL41405 stimulated basal pancreatic secretion through the activation of a cholinergic mechanism.
CRL.41405 aiid pancreatic secrctioii
The efficacy of CRL41405 was however moderate, since the maximal secretion remained under 30% (sodium and bicarbonate) and 10% (protein) of the maximal CCK-induced stimulation nieasured in our laboratory in other experiments (Nagain et 01, 1989). Doses larger than 200 mgkg were not used. because of cholinergic systemic side-effects. The effects of 200 mg/kg of CRL41405 (pancreatic stimulation and systemic side-effects) were totally abolished by atropine infusion, thus demonstrating their muscarinic dependence. Vagotomy also moderately decreased the action of CRL41405 on sodium output, a finding which seems difficult to explain. This may be due to the fact that sodium output may to some extent be reduced by vagotoniy. When moderate doses of CRL41405 were injected after idazoxan, a clear stimulation of sodium and bicarbonate output was found. This stiniulation was already maximal after 20 nig/kg CRL41405, and seems to be related to a beta-adrenoceptor stimulation, since it was suppressed by propranolol. Similar beta-adrenoceptor related stimulation of water and electrolytes excretion has been documented following the infusion of several sympathetic amines (isoproterenol, epinephrine, norepinephrine, dopanline) in the same experimental model (Rozc? et cil, 1976; Chariot et crl, 1983). This stimulation appears to be related to a CAMP mediated event in pancreatic cells (Lingard and Young, 1983). CRL41405 was also able to inhibit pancreatic secretion stiniulated by 2DG. When 20 m g k g of CRL41405 (a dose which had no effcct on basal secretion) was administered, a significant inhibition of 2DG stimulation of all the variables of pancreatic secretion (sodium, bicarbonate and total protein) was found. This inhibition was likely to depend upon the central and/or peripheral inhibition of neurotransniitter release from vagal fibers, since the direct effect of acetylcholine on pancreatic cells was not clearly changed by CRL4140S. The CRL41405 inhibition of 2DG effect was not altered by naloxone, propranolol, or prazosin, suggesting that i t was independent from opiate
175
receptors, as well as from beta- and alpha-1 adrenoceptors. In contrast, the CRL41405 inhibition of 2DG-stimulated secretion was markedly altered by idazoxan, indicating that an alpha-2 adrenoceptor-mediated event was involved. Pancreatic inhibition by clonidine, guanabenz and other alpha-2 agonists has been documented previously (Rozc?et crl, 1981; Chariot et crl, 1988); i t involves both central and peripheral actions (Chariot er al, 1988). Whereas the 2DG-induced pancreatic protein stimulation recovered its normal value after idazoxan + CRL41405, the 2DG-induced sodium and bicarbonate stimulation was apparently potentiated by idazoxan + CRL41405. In fact, the betastimulating effect of CRL41405 was unveiled by alpha-2 adrenoceptor blockade, and the observed effect corresponded approximately to the addition of ~Iicidazoxan-unveiled effect of CRL41405 on basal secretion, to the effect of 2DG per se. A similar situation was observed in the experiments with acetylcholine. While CRL41405 alone, or idazoxan alone, did not clearly change the response of pancreatic cells to their direct stimulation by acetylcholine, the idazoxan-unveiled effect of CRL41405 added a constant amount of sodium and bicarbonate excretion above the dose-related response to acetylcholine, thus showing a leftwards shift of the acetylcholine dose-response curve. In conclusion, CRL41405 displayed a complex and original pattern of adrenoceptor-related effects on pancreatic secretion in rats. Under the appropriate conditions, we observed: i) an alpha-:! adrenoceptor-mediated inhibition of 2DG-stimulated secretion; ii) a bcta adrenoceptor-niediated stimulation of water and electrolyte secretion, that can add to the cfl'ect of 2DG or acetylcholine. Muscarinic stimulation may also be observed, but only after large doses of CRL41405.
Ackiiowledgmetits Financial support was provided in part by the Conseil Scicntil'ique of FacultC X Bichat and by the Association Charlcs Dohray.
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C Nagain
References Appia F, De la Tour J. Chariot J, Roz6 C (1984) Action du 5-thio-glucose sur la sCcr6tion pancrkatique externe chez le rat. Comparaison avec le 2-dCsoxy-glucose. Gustrocnt4rol CIin Biol8,746-748 Chariot J, Appia F, Del Tacca M, Tsocas A, Rose C (1988) Central and peripheral inhibition of exocrine pancreatic secretion by alpha-2 adrenergic agonists in the rat. Plzurniacol RCS Coniniun 20, 707-717 Chariot J, De la Tour J, Vaille C, Roz6 C (1987) Comparative effects of pirenzepine and atropine on pancreatic secretion in conscious rats. Arch Int Pharniucodyn Tlicr 285, 158-165 Chariot J, Roz6 C (1976) DCterinination automatist% de tr8s faibles concentrations de bicarbonate. Application au suc pancrkatique. Ann Biol Cliri 34. 269-272 Chariot J, Roz6 C, De la Tour J, Souchard M, Vaille C (1983) Modulation of stimulated pancreatic secretion by sympathomimetic amines in the rat. Pharrtiacology 26,313-323 Lingard JM, Young JA (1983) PAdrenergic control of exocrine secretion by perfused rat pancreas in vitro. Am J Pliysiol245, G690-G696
el
ti/
Nagain C, Chariot J. Vaille C, Roz6 C (1991) Effect of modafinil on pancreatic exocrine secretion in the rat. A comparison with methadone. Eur J Plzurmacol201, 203-2 08 Nagain C, Mendre C, Rodriguez M, Bernard C. Martinez J, Rozk C (1989) External pancreatic secretion in the rat. Supra-maximal inhibition induced by the cholecystokinin octapeptide (CCK 26-33) and analogs altered on the 2 8 - 2 9 bond. Peptides 10, 104 1-1047 Roz6 C, Chariot J. Appia F, Pascaud X. Vaille C (1981) Clonidine inhibition of pancreatic secretion in rats: a possible central site of action. Eur J Pharntacol 76, 381-390 Rozd C, La Tour J de, Chariot J, Souchard M. Debray C (1975)Tcchnique d’Ctude de la s6crCtion pancrkatique externe chez le rat. Biol GastroentCrol (Paris) 8 . 19 1-295 Rozd C, La Tour J de, Chariot J. Souchard M. Vaille C, Dupont C, Jean E, Wepierre J (1976) Isoproterenolinduced pancreatic secretion in rats: a comparison with secretin. Biornedicinc 24. 410-417 Wincr BJ (1971) Statistical principles in experimental dcsign. Mc Craw IIiII, New York
