Nitric oxide modulates pancreatic basal secretion and response to cerulein in the rat: Effects in acute pancreatitis

GASTROENTEROLOGY1995;108:1855-1862

Nitric Oxide Modulates Pancreatic Basal Secretion and Response to Cerulein in the Rat: Effects in Acute Pancreatitis XAVIER MOLERO,* FRANCISCO GUARNER,* ANTONIO SALAS, t MARISABEL MOURELLE,* VALENTi PUIG,* and JUAN R. MALAGELADA* *Digestive System ResearchUnit, Hospital GeneralVail d'Hebron, AutonomousUniversityof Barcelona, Barcelona;and tDepartment of Pathology, Hospital Mutua de Terrassa,Terrassa, Spain

Background~Aims: Nitric oxide synthase activity is de-

tected in the pancreas, but the role of NO on pancreatic function has not been fully characterized. The aim of this study was to evaluate the role of NO in normal and diseased pancreatic function. Methods: Amylase and NO secretion were measured in vivo in rats and in vitro in dispersed acini, with and without NO synthesis blockade, by NG-nitro-L-arginine methyl ester (L-NAME). Rats were subjected to cerulein-induced pancreatitis, and the effects of L-NAME or NO donors were assessed. Results: L-NAME reduced amylase output to 60% of basal. This effect was reversed by L-arginine. The secretory response to optimal doses of cerulein induced a poor amylase secretion and a marked release of NO. High doses of cerulein in combination with L-NAME inhibited NO formation and amylase secretion. In dispersed acini, supramaximal cerulein concentrations induced NO release, but the amylase dose-response curve was not modified by NO inhibition. In acute pancreatitis, L-NAME increased amylasemia and tissue myeloperoxidase activities, whereas NO donors reduced amyiasemia, lipasemia, and the histological damage score. Conclusions: The L-arginine/NO pathway facilitates basal and stimulated pancreatic secretion in vivo. NO donor drugs may improve the course of acute pancreatitis. 'itric oxide is generated from L-arginine by an enzymatic pathway originally shown in vascular endothelial cells 1 but also present in a variety of cell types, including macrophages and neurons. 2 In the pancreas, N O synthase may play a physiological role in [[3 cells 3 and neurons. 4 N O is a molecule that diffuses easily across membranes, a property that enables it to function as an intracellular and intercellular messenger) It has been shown to be involved in vascular smooth muscle relaxation; inhibition of platelet aggregation; nonadrenergic, noncholinergic neurotransmission2; gallbladder relaxation6; osteoclastic inhibitionV; L-arginine-dependent insulin secretion3; and several leukocyte-dependent inflammatory processes. 8-1° The biological actions of N O are medi-

N

ated via activation of guanylate cyclase, but N O may also participate in some other 5'-cyclic guanosine monophosphate-independent functions. 7-9 Inhibition of endogenous N O production results in vasoconstriction. 11 In the pancreas, intracellular 5'-cyclic guanosine monophosphate concentration increases in response to certain secretagogues, such as cholecystokinin, .2 suggesting that this nucleotide participates in the regulation of acinar cell function. When guanylate cyclase is inhibited, calcium influx to the cell is diminished severely. I3 Nitroso compounds generate N O either spontaneously (sodium nitroprusside [SNP]) or enzymatically (glyceryl trinitrate) and, therefore, are considered N O donors. They certainly activate guanylate cyclase in the acinar cell, resulting in an increase of intracellular 5'-cyclic guanosine monophosphate concentration. 12'14 Previous studies in vitro showed that SNP slightly enhanced amylase secretion, although it had no effect on the cholecystokinin-stimulated response. 14 In the hemodynamically isolated dog pancreas, nitrites seem to stimulate pancreatic secretion. 15 The purpose of the present study was to investigate the role of endogenously generated N O on pancreatic enzyme secretion in the basal state and in response to hormonal stimulation. Because N O is a key factor in the regulation of vascular tone and because acute pancreatitis is associated with impaired vascular function, 16 we also investigated a potential role for N O in the development of acute pancreatitis in the cerulein-hyperstimulation model. Materials

and Methods

Materials Cerulein, N%nitro-L-arginine-methyl ester (L-NAME), L-arginine, o-dianisidine, reduced nicotinamide adenine dinuAbbreviations used in this paper: L-NAME,NGnitro-L-arginine-methyl ester; NADPH, reduced nicotinamideadenine dinucteotidephosphate; SNP, sodiumnitroprusside. © 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00

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MOLERO ET AL.

cleotide phosphate (NADPH), naphthylenediamine, trypan blue, hexadecyltrimethylammonium bromide, and collagenase type 11 were obtained from Sigma Chemical Co. (Alcobendas, Spain). Phenylephrine was purchased from Roig Farma (Barcelona, Spain). SNP was obtained from Merck (Darmstadt, Germany), and glyceryl trinitrate was obtained from Berenguer-Infale (Sant Just Desvern, Spain). Nitrate reductase, flavin adenine dinucleotide, lactate dehydrogenase, and pyruvate were obtained from Boehringer G m b H (Mannheim, Germany). All other reagents were of the highest purity commercially available.

Methods In vivo experiments. Control of exocrine pancreatic secretion. To monitor exocrine pancreatic secretion, we used a modi-

fication of the model described by Niederau et al. 17 Briefly, male Sprague-Dawley rats, weighing 2 0 0 - 3 0 0 g, were fasted 16 hours before surgery. Anesthesia was induced by intraperitoneal injection of 100 mg/kg of ketamine. A polyethylene cannula (PE-10; Clay Adams, Parsippany, NJ) was placed inside the left jugular vein for intravenous administration of drugs and for continuous saline perfusion at 3 mL/h, using a syringe pump. A heparinized (10 U/mL) polyethylene catheter (PE-50) was inserted into the carotid artery and connected to a pressure transducer for continuous monitoring of blood pressure. A laparotomy was then performed, and the pancreatobiliary duct was cannulated through its duodenal opening with polyethylene tubing (PE-10). Pancreaticobiliary secretion was allowed to drain and equilibrate for 30 minutes before the experiments. A new dose of 50 mg/kg of ketamine was administered intramuscularly 15 minutes before the onset of juice collection. Amylase output was calculated in periods of 10 minutes. The mean amylase output obtained from two 10-minute fractions after the equilibration time was used as a measure of basal output. Drugs were administered as an intravenous bolus injection at the end of the second basal period except for phenylephrine, which was administered as an intravenous perfusion. Secretions were collected in six separate 10-minute fractions in tared vessels. Fluid volumes were estimated by weight. Because pancreatic basal secretion tends to fluctuate spontaneously over time and because it shows marked interindividual variation even under similar experimental conditions, *a we decided to standardize our results on unstimulated basal secretion by expressing data as the percentage of variation in amylase output from basal (taken as 100%). Ketamine was used as the anesthetic of choice because it has no effect on mean mesenteric blood flow, 19 whereas urethane stimulates somatostatin release2° and would be unsuitable for the present study. Separate experiments were conducted to calculate the secretory response to (1) vehicle only (control), (2) L-NAME (30 mg/kg), (3) ].-NAME + t-arginine (100 mg/kg), and (4) phenylephrine (10 gg" kg - I " min-1). In a second series of experiments, we investigated the effects of inhibiting nitric oxide synthase on the pancreatic response to hormonal stimulation.

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Optimal (0.5 ~tg/kg) and supramaximal (10 btg/kg) bolus doses of cerulein were administered with or without L-NAME. Amylase output and N O formation (determined by the accumulation of nitrite and nitrate) were measured in pancreatic juice. Experiments were performed in 9 different rats for each experimental condition, and results are expressed as means _+ SE. Induction of acute pancreatitis, Experimental acute pancreatitis was achieved by administering a total of four intraperitoheal injections of cerulein at the dose of 20 gg/kg of body weight at 1-hour intervals as described by Tani et al., 21 with minor modifications. To evaluate the role of N O in the development of ceruleininduced acute pancreatitis, the N O synthase inhibitor I~NAME (30 mg/kg) was administered intraperitoneally with each cerulein injection. The N O donors SNP or glyceryl trinitrate (0.5 mg/kg in 0.5% bovine serum albumin and saline solution) were administered intraperitoneally at the beginning of the experiments and then 3 hours later. Blood was obtained by cardiac puncture for determination of amylase and lipase activities 9 hours after the first cerulein injection. Rats were then killed, and the pancreas was removed and trimmed of fat and ganglia. For histological examination, the pancreas was fixed in 10% formaldehyde for 24 hours, embedded in paraffin, and stained with H&E. The organs were examined blindly by the participating pathologist (A.S.), who graded the coded slides for each gland according to a histological score that evaluated edema, vacuolization, inflammation, and cellular necrosis based on modifications of pubiished s c o r e s . 22'23 Grading for edema was scaled as follows: 0, absent; 1, interlobular; 2, moderate interlobular and intra-acinar; and 3, severe interlobular and intraacinar edema. Necrosis (degranulation and fragmentation) and vacuolization were graded as follows: 0, absent or rare; l, < 3 0 % acinar cells; and 2, > 3 0 % acinar ceils. Inflammation refers to neutrophilic infiltration and was scored as follows: 0, absent; 1, perivascular infiltration; 2, moderate septal and intra-acinar infiltration; and 3, severe neutrophilic infiltration. Separate experiments were performed to quantify tissue edema and leukocyte infiltration. Fragments of excised pancreas were immediately wet weighed, desiccated at 160°C for 48 hours, and reweighed. Pancreatic water content was calculated as the percentage of total weight. 24 Additional fragments were frozen at - 8 0 ° C until tissue myeloperoxidase activity was determined. Tissue was processed according to the method described by Grisham et al., 25 with minor modifications. Briefly, three fragments of the gland ( ~ 1 0 0 mg each) were separately homogenized (100 mg/mL) in 50 mmol/L potassium phosphate buffer (pH 6), containing 0.01% soybean trypsin inhibitor and 0.1 mmol/L phenylmethylsulfonyl fluoride and using a motor-driven tissue homogenizer (Tissue Tearor 985370; Biospec Products Inc.). Homogenates were centrifuged at 12,000g for 10 minutes, and pellets were solubilized in 1 mL of the same buffer containing 0.5 % hexadecyltrimethylammonium bromide. Homogenates were then sonicated (three bursts of 10 seconds) and centrifuged at 12,000g for 10 minutes. Supernatants were assayed for myeloperoxidase activity.

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NITRIC OXIDE EFFECTS ON PANCREATIC FUNCTION

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Caerulein concentration (log M) Figure 1. Effect of NO synthase inhibition on amylase (curves) and NO2/N03 (bars) release by dispersed pancreatic acini. Acini from the

same gland were preincubated for 5 minutes with 10 mmol/L L-NAME (Q,[]) or vehicle ([3,l@) and then exposed to graded concentrations of cerulein for 30 minutes (n = 8). *P < 0.05 compared with basal concentration. **P < 0.05 compared with the response obtained at O, 0.1, and 0.3 nmol/L cerulein. +P < 0.05 compared with L-NAMEtreated acini.

In vitro experiments. Rats weighing 200 g were fasted for 16 hours and killed by cervical dislocation, and the pancreas was removed rapidly. Pancreatic acini were isolated by collagenase digestion of the gland, as described by Bruzzone et al. 26 Amylase and NOe/NO 3 release from acini were determined in the supernatant after incubation with graded concentrations of cerulein over a 30-minute period at 37°C. Acini were preincubated for 5 minutes with 10 mmol/L t - N A M E or vehicle at room temperature. To determine the viability of the preparation, dispersed acini were incubated with trypan blue (400 ~mol/L) for 5 minutes and washed, and the number of cells excluding the dye was recorded. Data, as shown in Figure 1, are expressed as the percentage of the total initial amylase content, as determined in the supernatant of an equivalent aliquot of acini sonicated in nanopure water. In each experiment, each amylase and nitrite concentration was determined in duplicate, and results are shown as mean + SE from eight separate experiments. Biochemical determinations. Amylase activity was determined by the O~-amylase EPS test (Boehringer GmbH) for BM/Hitachi system 717. Two hundred-microliter aliquots of 1:100 to 1:1000 dilutions of the sample were used in the assay. Lipase activity was determined by a turbidimetric method (Boehringer GmbH). Myeloperoxidase activity was measured in aliquors ofsolubi-

1857

lized pancreatic tissue. Fifty microliters was combined with 2.95 mL of 50 mmol/L phosphate buffer, p H 6, containing 0.5 % hexadecyltrimethylammonium bromide, 0.167 mg/mL 0-dianisidine hydrochloride, and 0.005% hydrogen peroxide. The change in absorbance at 460 nm was measured with a Shimadzu UV-160A spectrophotometer. One unit of myeloperoxidase activity was defined as the amount of enzyme reducing 1 ~mol of peroxide per minute at 25°C. Protein content was estimated by the BCA Protein Assay (Pierce Chemical Co., Rockford, IL). N O formation was measured by the accumulation of nitrite and nitrate after a previously described procedure with minor modifications.26 Briefly, nitrate was reduced to nitrite with 0.5 units of nitrate reductase in 500 btL of the sample in the presence of 50 btmol/ L N A D P H and 5 ~mol/L flavin adenine dinucleotide. The excess of N A D P H was oxidized in the presence of 0.2 mmol/ L pyruvate and 1 btg of lactate dehydrogenase. 27 Nitrite was determined by adding 1 mmol/L sulfanilic acid and 100 mmol/ L CtH. After 5 minutes of incubation, tubes were centrifuged at 10,000g, and the supernatant was transferred to a 1-mL microcubette. Absorbance was read at 548 nm in a Shimadzu UV-160 spectrophotometer before and 15 minutes after incubation with 1 mmol/L naphthylenediamine. Results were compared with a standard of NaNO2. All biochemical determinations were performed in duplicate. Statistical determinations. Data are expressed as means _+ SE. A two-tailed Student's t test for unpaired values was used for statistical comparison of mean values for amylase, lipase, percentage of pancreatic water content, tissue myeloperoxidase activity, and histological data. Nitrite concentrations were compared using a two-tailed Student's t test for paired values.

Results Effect of NO Synthase Blockade on Basal Pancreatic Secretion In vivo studies. I n h i b i t i o n of N O synthase activity by an intravenous bolus of 30 m g / k g of ]L-NAME increased arterial blood pressure by a factor of 1.8 and reduced basal amylase o u t p u t by half. This effect was reversed p a r t l y by 100 m g / k g of the natural substrate of the enzyme L-arginine (P < 0.01) (Figure 2). O~-Adrenergic agonists cause systemic vasoconstriction and reduce pancreatic blood flow. 28 Infusion of the syst e m i c vasopressor p h e n y l e p h r i n e (an O~l-adrenergic agonist), at doses that p r o d u c e d a sustained increase in blood pressure c o m p a r a b l e to t h a t p r o d u c e d by ]~-NAME, also reduced amylase o u t p u t to 57.3% + 5.5% of basal outp u t (P < 0.01 vs. basal) and r o u g h l y o u t l i n e d the LN A M E o u t p u t curve. In vitro studies. More than 9 8 % of dispersed acinat ceils excluded t r y p a n blue. Spontaneous amylase release and basal N O 2 / N O 3 concentration was not modified by L - N A M E t r e a t m e n t .

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GASTROENTEROLOGY Vol. 108, No. 6

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Figure 2. Effect of L-NAME on basal amylase output. Data are presented as the percentage of variation in amylase output from basal output in lO-minute periods in response to bolus doses of L-NAME (30 mg/kg, e), L-NAME plus L-arginine (100 mg/kg, 0), or vehicle (&). Each point is the mean _+ SE of five separate experiments for each experimental condition. The asterisks represent significant differences compared with controls and L-NAME plus L-arginine-treated groups (P < 0.05).

Effect of NO Synthase Blockade on Hormone-Stimulated Pancreatic Secretion In vivo studies. Amylase output was markedly stimulated by an optimal dose of cerulein (0.5 btg/kg). Administration of L-NAME inhibited the 30-minute amylase output response to this optimal dose (P < 0.05) (Figure 3). A supramaximal dose of cerulein (10 t.tg/kg) induced a very weak secretory response in 30 minutes, but when the dose was administered in combination with LNAME, pancreatic secretion was inhibited profoundly (Figure 3). The reduction in amylase secretion produced

by this bolus dose of L-NAME was reversible, returning to control levels by 60 minutes after the injection. NO2/NO3 output determined in biliopancreatic juice was very low and close to detection limits when optimal doses of cerulein were used. However, NOi/NO3 output increased sharply in response to supramaximal intravenous bolus doses of cerulein (Figure 3). This NO2/NO 3 production was significantly inhibited by L-NAME treatment. In vitro s t u d i e s . In dispersed pancreatic acini, cerulein-stimulated amylase secretion was not modified by treatment with L-NAME (Figure 1). However, in response to cerulein, acini released NO2/NO 3 in a concentration-dependent manner that was statistically different from basal output at 0.3 nmol/L and maximal at the highest cerulein concentration used (10 nmol/L). This NO2/NO3 release was inhibited by L-NAME treatment and showed a good correlation with the supramaximal secretory inhibition part of the cerulein dose-response curve (Figure 2). These results are in close agreement with the observed NO2/NO3 release in the in vivo experiments (Figure 3).

Effect of NO on Cerulein-lnduced Acute Pancreatitis Repeated intraperitoneal supramaximal doses of cerulein resulted in a sharp increase in serum amylase and lipase concentration, compared with the control group. LNAME significantly potentiated cerulein-induced hyper-

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Figure 3. Effect of NO synthase inhibition on cerulein-stimulated amylase and NO output. Amylase and NO2/N03 outputs were determined in response to (A) optimal (0.5 pg/kg, []) and (B) supramaximal (10 pg/kg, II) doses of cerulein. CR+NE bars represent amylase and NO2/N03 output in response to cerulein (CR) plus L-NAME (30 mg/kg) (NE). Data are cumulative for the first 30-minute period after the administration of cerulein (n = 9 for each experimental condition). *P < 0.05 compared with CR+NE bars. •P < 0.001 compared with N O j N 0 3 output in response to optimal doses of cerulein.

June 1995

NITRIC OXIDE EFFECTS ON PANCREATIC FUNCTION

1859

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Figure 4, (A) Serum amylase and (B) lipase activities in ceruleininduced acute pancreatitis. Rats received four intraperitoneal injections at 1-hour intervals of vehicle (control group), cerulein (20 ~tg/ kg), cerulein plus L-NAME (30 mg/kg), or cerulein plus SNP (0.5 mg/ kg) at 0 and 3 hours. Blood was withdrawn 9 hours after the initiation of the experiments. Each bar represents the mean + SE of eight experiments. Asterisks denote significant differences compared with cerulein-only group (P < 0,05).

amylasemia. On the contrary, the N O donor SNP reduced both serum amylase and lipase activity (P < 0.01 vs. cerulein alone) (Figure 4). At the low dose used in the experiments (0.5 mg/kg, intraperitoneally administered bolus), blood pressure decreased slightly (by > 2 0 % of its initial value) and quickly recovered. At the end of the experiments, all groups tested had similar concentrations of serum urea and creatinine (data not shown), indicating a preservation of renal function. Glyceryl trinitrate also reduced serum activities of amylase (16.9 + 1.8 UlmL vs. 26.6 + 3.6 U/mL in the cerulein-alone group; P < 0.01) and lipase (0.9 + 0.1 UImL vs. 3.6 + 0.5 U/mL in the cerulein-alone group; P < 0.001) to the same extent as SNP. Furthermore, SNP was able to decrease, slightly but significantly, the histological damage score from 7.4 _+ 0.2 for the cerulein-alone group to 6.1 ___ 0.2 for cerulein plus S N P treated rats (P < 0.05; n = 1). The quantification of pancreatic water content and the measurement of tissue myeloperoxidase activity were assessed to further characterize the slight improvement in the serum enzyme activities and in the pathological score. At the low doses used, SNP significantly reduced the amount of pancreatic water retention induced by cerulein (P < 0.05 vs. cerulein-alone group and vs. cerulein plus L-NAME groups) (Figure 5). However, SNP failed to reduce significantly tissue myeloperoxidase activity compared with the cerulein-alone group, but LNAME seemed to increase this marker for polymorphonuclear infiltration (Figure 6).

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In the present study, we investigated the role of N O in the regulation of pancreatic secretion in the basal state and in response to cerulein, both in vivo and in

CR + SNP

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Figure 5. Pancreatic edema expressed as the percentage of pancreatic water content in the animal groups shown in Figure 4. Values shown are means ± SE for 10 or more animals in each group. *P < 0.05 compared with cerulein-only and cerulein plus L-NAME groups. **P < 0.05 compared with all other groups. CR, cerulein.

vitro. We also analyzed the effect of N O in ceruleininduced acute pancreatitis in the rat. N O is generated from L-arginine by the action of N O synthase. N O is a potent stimulus for the activation of the enzyme guanylate cyclase, which converts guanosine triphosphate into 5'-cyclic guanosine monophosphate. 2 Because N O diffuses through membranes, ir can affect the cell in which N O has been synthesized, or it can affect neighboring cells. There is an endogenous basal N O production by the endothelium that depends on the activity of the constitutive main form of the enzyme N O synthase. This constitutive N O synthase generates N O in low quantities) Its blockade in vivo leads to vasoconstriction and hypertension.ll W e observed in vivo that endogenous N O production blockade by L-NAME inhibited basal secretion and that this effect was partly reversed by L-arginine. Phenyleph-

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Figure 6. Tissue myeloperoxidase activity in pancreas of rats treated in the same manner as described in the legend of Figure 4. Values are means _+ SE for 6 or more animals in each group. *P < 0.05 compared with values of cerulein-only and cerulein plus SNP groups. **P < 0.05 compared with all other groups, CR, cerulein.

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MOLERO ET AL.

rine, an O~-adrenergic agonist that reduces pancreatic blood flow, 28 increased blood pressure (reflecting vasoconstriction) and decreased amylase output to the same extent as L-NAME. This finding suggests that NO may play a permissive role in spontaneous pancreatic secretion, perhaps via the maintenance of adequate blood flow. When stimulated by an optimal dose of cerulein, the amylase output 30 minutes later was markedly enhanced, but this response did not correlate with a measurable increase in NO2/NO3 output. However, administration of L-NAME blunted the increase in enzymatic activity in pancreatic juice. Administration of high doses of cerulein results in smaller amylase secretion than optimal doses, both in vitro ~2and in vivo. .7 L-NAME treatment potentiated this trend in vivo. At these high doses, NO2/NO 3 output was markedly increased, and because NO2/NO~ formation could be inhibited by L-NAME, these results indicate an activation of NO synthase in response to high doses of cerulein. Thus, in vivo, inhibition of NO2/NO3 release by L-NAME in response to high doses of cerulein correlated with a further decrease in amylase output. Considering the in vivo and in vitro data together, we speculate that induction of NO synthase function at high doses of cerulein (10 btg/kg and 10 nmol/L) may be the consequence of an activation of pathways that operate during the "blocked secretory state" that is induced by high doses of cerulein. Indeed, L-NAME effectively inhibits NO2/NO3 release but fails to reverse the inhibited amylase secretion in dispersed acini. On the contrary, it further impairs enzyme output in vivo, perhaps because of the inhibition of both the constitutive and the inducible form of NO synthase. It is known that large doses of cerulein cause acute edematous pancreatitis in rats. 29 Because L-NAME enhanced supramaximal inhibition of pancreatic secretion, we reasoned that it may also facilitate the induction of pancreatitis by cerulein. In our pancreatitis model, LNAME in combination with cerulein resulted in a much higher serum amylase concentration than cerulein alone. L-NAME failed to increase serum lipase activity and failed to worsen tissue damage when compared with the control, but L-NAME enhanced tissue myeloperoxidase activity, a marker for neutrophil infiltration. However, at the low doses used in the experiments, SNP (an NO donor) reduced both serum amylase and lipase activities and also improved, slightly but significantly, the histological damage score. This indicates that the additional influx of NO was beneficial in the course of acute pancreatitis in this model. There are few reliable and accurate parameters to grade the severity of the acute edematous form of experimental

GASTROENTEROLOGY Vol. 108, No. 6

pancreatitis. For the parameters that we used, L-NAME did not clearly aggravate all of them. This could be explained on the basis of a common pathway of tissue damage for L-NAME and cerulein, so that further stimuli would not produce further damage. On the other hand, SNP reduced amylase and lipase concentrations as well as the histological damage score and tissue edema, suggesting that the addition of NO in low quantities might ameliorate acute pancreatitis or that cerulein counteracts endogenous NO effects to a certain extent. In fact, high doses of cerulein reduce pancreatic blood flow up to 50% 3o and vasoconstrictors have been shown to worsen experimental pancreatitis. 31 L-NAME was fully effective in inhibiting NO2/NO3 release, but under our experimental conditions, it failed to modify amylase secretion by dispersed acini under basal or stimulated conditions. It is possible that the effects observed in vivo are caused by endothelial or neural NO production (constitutive NO synthase) affecting the vascular bed, assuring an adequate blood perfusion and permeability to the whole gland. NO exerts its actions in a variety of cell types through both dependent and independent 5'-cyclic guanosine monophosphate mechanisms, v'8 We may consider three other NO targets that could have a protective role in acute pancreatitis. Increased microvascular permeability is believed to be implicated in the pathophysiology of acute pancreatitis, 32 to the point that some experimental therapeutic approaches have been aimed at protecting microvascular derangement. 33 NO has been shown to maintain vascular integrity in endotoxin-induced acute intestinal damage in the rat. 34 Accordingly, NO may protect the pancreas by preventing an alteration in the vascular permeability. Polymorphonuclear leukocyte infiltration of pancreatic tissue is an outstanding feature of virtually all models of acute pancreatitis. Leukocytes may initiate or potentiate cell damage through several mechanisms, including free oxygen radical and cytokine production. Impairment of endogenous NO generation enhances leukocyte adhesion to the endothelium and leukocyte emigration to the tissue, 5a° a pattern characteristic of acute inflammation. By keeping NO at optimal levels with SNP, leukocyte infiltration may be reduced in acute pancreatitis. NO is certainly generated in large quantities by activated macrophages and lymphocytes. This NO is believed to be responsible for the cytotoxicity of these cells) There is now a large body of evidence suggesting that low levels of NO (such as those generated by the constitutive form of NO synthase) may have beneficial or protective effects in a variety of systems, whereas large, uncontrolled NO production is associated with tissue damage. 5'35

June 1995

Finally, oxygen free radicals are generated in acute pancreatitis and may further exacerbate cellular injury. 36 The hypoxic tissue and leukocytes are the two main sources of these free radicals. Oxygen free radical scavengers, such as superoxide dismutase, reduce the severity of acute pancreatitis. 37 It has been shown that superoxide dismutase prolongs the effects of N O and protects NO, at least in part, by preventing the reaction of superoxide with NO. 8 Hence, it has been suggested that N O protection by superoxide dismutase is an important component of the ability of superoxide dismutase to prevent tissue injury) '38 Moreover, N O not only acts as a superoxide scavenger itself, but it also inhibits neutrophil superoxide anion production via direct effects on membrane components of the N A D P H oxidase. 9 W e can conclude from the present report that the Larginine/NO pathway acts as a modulator of basal and stimulated pancreatic secretion in vivo. Furthermore, N O donors may have the ability to improve the course of acute pancreatitis through a multifactorial mechanism. The specific function of N O synthase activity in acinar cells remains to be determined. During the review process of the present work, Konturek et al. 39 published an article describing the effects of N O in pancreatic secretion in dogs. Although using a different arginine analogue, their results are in accordance with our own observations in rats, suggesting that the effects of manipulating endogenous N O production on pancreatic secretion is not limited to a single mammalian species.

NITRIC OXIDE EFFECTS ON PANCREATIC FUNCTION 1861

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References 21. 1. Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1 9 8 8 ; 3 3 3 : 6 6 4 666. 2. Moncada S. The 1991 UIf yon Euler Lecture. The L-arginine:nitric oxide pathway. Acta Physiol Scand 1 9 9 2 ; 1 4 5 : 2 0 1 - 2 2 7 . 3. Schmidt HHH, Warner TD, Ishii K, Sheng H, Murad F. Insulin secretion from pancreatic B cells caused by barginine-derived nitrogen oxides. Science 1 9 9 2 ; 2 2 5 : 7 2 1 - 7 2 3 . 4. Shimosegawa T, Abe T, Satoh A, Asakura T, Yoshida K, Koizumi M, Toyota T. Histochemical demonstration of NADPH-diaphorase activity, a marker for nitric oxide synthase, in neurons of the rat pancreas. Neurosci Lett 1992; 1 4 8 : 6 7 - 7 0 . 5. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1 9 9 3 ; 3 2 9 : 2 0 0 2 - 2 0 1 2 . 6. Mourelle M, Guarner F, Molero X, Moncada S, Malagelada JR. Regulation of gallbladder motility by the arginine: nitric oxide pathway in the guinea pigs. Gut 1993; 3 4 : 9 1 1 - 9 1 5 . 7. Maclntyre I, Zaidi M, Alam ASMT, Datta HK, Moonga BS, Lidbury PS, Hecker M, Vane JR. Osteoclastic inhibition: an action of nitric oxide not mediated by cyclic GMP. Proc Natl Acad Sci USA 1991; 8 8 : 2 9 3 6 - 2 9 4 0 . 8. Murphy ME, Sies H. Reversible conversion of nitroxyl anion to nitric oxide by superoxide dismutase. Proc Natl Acad Sci USA 1991;88:10860-10864. 9. Clancy RM, Leszczynska-Piziak J, Abramsen SB. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide

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Received January 21, 1993. Accepted February 17, 1995. Address requests for reprints to: Xavier Molero, M.D., Digestive System Research Unit, Hospital General Vail d'Hebron, 08035 Barcelona, Spain. Fax: (34) 3-428-18-83. Supported in part by grant PM 92-0192 from the Ministerio de EducaciOn y Ciencia, Spain. Presented in part at the annual meeting of the American Gastroenterolog~j Association held in San Francisco, California, in May 1992. The authors thank Dr. Rosa M. Segura for performing lipase determinations and Drs. Mar|a Antol|n and Ana Garc|a for technical help.