Gut 1996; 39: 539-544

L-Arginine, nitric oxide, and intestinal secretion:studies in rat jejunum in vivo

F H Mourad, L J D O'Donnell, E A Andre, C P Bearcroft, R A Owen, M L Clark,M J G Farthing

Digestive DiseasesResearch Centre,Medical College ofSt Bartholomew'sHospital,Charterhouse Square,LondonF H MouradL J D O'DonnellE A AndreC P BearcroftR A OwenM L ClarkM J G FarthingCorrespondence to:ProfessorM J G Farthing,Digestive Diseases ResearchCentre, St Bartholomew'sand the Royal LondonSchool of Medicine andDentistry, Tumner Street,London El 2AD.

Accepted for publication30 May 1996

AbstractBackground-L-Arginine has been shownto induce fluid secretion in humanjejunum. Nitric oxide, a derivative ofL-arginne is thought to have an importantrole as an intestinal secretagogue.Aim-To determine the effect of L-arginine and the nitric oxide synthaseinhibitor, nitro L-arginine methyl ester(L-NAME), on fluid and electrolyte move-ment in rat jejunum.Methods-A 25 cm segment ofrat jejunumwas perfused in situ with iso-osmoticsolutions containing either (1) saline,(2) D-arginine 20, (3) L-arginine 20, (4)L-NAME 0-1, 1, or 20 mmol/l, or (5) acombination of L-arginine 20 and L-NAME 0.1, 1, or 20 mmol/l. In furthergroups the effect of a subcutaneousinjection of L-NAME 100 mglkg wasexamined in rats pretreated with either D-or L-arginine 500 mg/kg.Results-L-Arginine, unlike D-arginine,induced fluid secretion despite beingbetter absorbed (mean -7 3 v 17-0 ,l/min/g; p<001). L-NAME at 0.1 mmol/l hadno effect on basal fluid movement butreversed L-arginine induced secretion(7.8; p<005). L-NAME at 1 and 20 mmol/linduced fluid secretion (-15.4 and -28-4,respectively), which was enhanced by theaddition of L-argilnlne (-30.0 and -41-0,respectively; both p<005). A subcutan-eous injection of L-NAME resulted inmarked fluid secretion (-39.9) and histo-logical evidence of intestinal ischaemia.These changes were attenuated or re-versed by pretreatment with subcutan-eous L- but not D-arginine.Conclusions-L-Arginine induces intes-tinal fluid secretion through production ofnitric oxide. There is a delicate balancebetween the effect of nitric oxide as asecretagogue and its effect on maintainingblood flow and thus preventing intestinalischaemia.(Gut 1996; 39: 539-544)

Keywords: arginine, nitric oxide, L-NAME, intestinaltransport, bowel ischaemia.

Actively transported sugars and most aminoacids enhance small intestinal absorption ofwater and electrolytes.l` However, L-arginine(L-Arg), unlike other amino acids, has beenshown by us and others to induce water

secretion when perfused in human jejunum.' 5A satisfactory explanation has never been putforward to explain this phenomenon, thoughit has been suggested that L-Arg inducedsecretion by a local effect that could beinhibited by the calmodulin antagonist chlor-promazine.5Over the past few years L-Arg has been found

to be the precursor of the free radical nitricoxide (NO), which has an important role as amediator of neural, cardiovascular, andgastrointestinal function.`8 Nitric oxide isderived from the guanidino terminal of L-Argby the action of the stereospecific enzyme nitricoxide synthase (NOS) which, in the gut, ispresent in the myenteric plexus9 10 andsubmucosal arterioles and venules."' 12 Inaddition, it has been shown that NO could beproduced by enterocytes through both theconstitutive and the inducible NOS." In vitrostudies have unveiled a role for NO as asecretagogue in the ileum14 '5 and colon,16-18and in vivo studies have suggested a possiblerole for NO in the laxative action of castoroil,"9 magnesium sulphate,20 and in thepathophysiology of secretion induced byEscherichia coli heat stable enterotoxin.`5 Inaddition, NO is a vasodilator and its con-tinuous endogenous production is important inmaintaining gastric microcirculation andmucosal integrity.2' 22 However, the effect ofendogenous NO on small intestinal waterabsorption/secretion and mucosal integrity isunknown. The aim of this study was toexamine (a) the pathophysiology of L-Arginduced jejunal secretion and its relation withNO production, and (b) the structural andfunctional effects of NOS inhibition in ratjejunum by examining the water and elec-trolyte movement and the histological changesin the jejunum after administration of theNOS inhibitor nitro L-arginine methyl ester(L-NAME).

Methods

In vivo perfusion modelMale adult Wistar rats (180-220 g bodyweight) were fasted for 18 hours with freeaccess to water. The rats were anaesthetised byintraperitoneal injection of sodium pento-barbitone (60 mg/kg) and maintained through-out the experiment by interval intraperitonealinjections (1 5-30 mg/kg) as necessary. Theabdomen was opened through a midlineincision and a 25 cm segment of jejunum

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TABLE I Composition and osmolality of the different solutions perfused into ratjejunum

L-Arg L-NAME Na Cl Osmolality(mmol/l) (mmoUl) (mmot'l) (mmoZ/l) (mOsmol/kg)

Saline 158-2 156-5 300L-Arg 20 139-9 159-6 300D-Arg 20 139-5 160 300L-Arg+L-NAME 0-1 20 0.1 140 158 300L-Arg+L-NAME 1 20 1 140 159 300L-Arg+L-NAME 20 20 20 128 150-8 300L-NAME0 1 0.1 156 158 300L-NAME 1 1 154-5 159 300L-NAME 20 20 136-4 157 300

starting 5 cm distal to the ligament of Treitzwas isolated between two cannulae and theabdomen closed.23 The isolated segment was

perfused in situ with the test solution at a rateof 0.5 ml/min for 30 minutes to reach steadystate and then three effluent collections of 10minutes each were obtained for assessment ofwater and electrolyte movement. All solutionsused were rendered iso-osmotic (300 mOsmol/kg) by addition ofNaCl, adjusted to pH 7.2 bythe addition of 1 N HCI or 1 N NaOH, andcontained 4 ,uCi/l ['4C]polyethylene glycol4000 as a non-absorbable volume marker.Animals were kept at 37°C using a heat pad.At the end of the experiments, urine and bloodsamples were taken and the rats were killed byan overdose of pentobarbitone; the perfusedintestinal segment was removed, rinsed,blotted, and desiccated in an oven at 100°C toobtain dry weight. The samples of effluentwere analysed immediately or kept frozen at-20'C and analysed within two weeks. Steadystate condition was shown by less than 5%variation in water movement between con-secutive 10 minute collections, and the valueswere accepted only if recovery of radioactivePEG fell between 95 and 105%23 and no

radioactivity was detected in urine and bloodsamples. At the end of the experiments thegross appearance of the intestine was assessedby an independent observer for the presenceof ischaemia and then jejunal sections weretaken for histological examination by lightmicroscopy. Tissue sections were coded andexamined 'blind' by a histopathologist.

Effect ofNO precursor, L-Arg, on water andelectrolyte transport in ratjejunumIn three groups of animals the intestinalsegment was perfused with either iso-osmoticsaline or a solution containing L-Arg (20 mmol/1) or its inactive enantiomer D-Arg at the same

concentration. Effluent radioactivity, sodiumchloride, and Arg were measured and netwater, electrolyte, and Arg movement cal-culated accordingly.

In parallel groups of animals L-Arg or D-Argin doses of 100 and 500 mg/kg in 0.3 ml salinewas given subcutaneously and 35 minuteslater jejunal perfusion was performed with iso-osmotic saline.

Effect ofL-NAME on water and electrolytetransport in ratjejunumIntestinal perfusion was performed in sixgroups of animals with solutions containing the

NOS inhibitor, L-NAME at concentrations of0O1, 1, or 20 mmol/l with or without L-Arg 20mmolIl (Table I). The solutions were perfusedfor 30 minutes to reach steady state and thenthree collections of the effluent of 10 minuteseach were collected.To study the effect of systemic L-NAME,

three groups of animals were injected sub-cutaneously with either L-Arg or D-Arg 500mg/kg in 0.3 ml saline, or 0.3 ml saline alone,followed after 15 minutes by subcutaneousL-NAME 100 mg/kg in 0.3 ml saline. After afurther 20 minutes jejunal perfusion with aniso-osmotic saline solution was performed.

Analytical methods['4C]PEG concentrations in the effluent weremeasured in triplicate by liquid scintillationspectroscopy in LKB Wallac Ultra-beta 1210scintillation counter. Sodium and potassiumconcentrations were determined by flamephotometry (Instrument Laboratories 943),and chloride concentration by Coming 925chloride analyser (Coming, UK). Solutionosmolality was analysed using the vapourpressure technique with a Wescor 5500 osmo-meter. L-Arg and D-Arg concentrations in theeffluent were measured by the colorimetricmethod of Sakagushi as previously des-cribed.24 25 In brief, 0.5 ml of sample solutionand 0.5 ml of 20% potassium hydroxide weremixed in a test tube colled in an ice-water bath.Acetic anhydride (0.075 ml) was added andthe tube shaken vigorously in the ice. One hourlater 0.5 ml of 0.01% l-naphthol in 10%potassium hydroxide solution was addedfollowed by 0.2 ml of 0.06% hypobromitesolution. This was followed by 0.5 ml ofalcohol and one hour later a second additionof 0.2 ml of hypobromite solution was made.The optical density was measured at 520 nmand compared with that of known concen-trations of Arg. The net water, electrolyte, andArg movement was calculated and expressedrespectively in ,ul/min/g and ,moUmin/g of dryintestinal weight. Positive values denote ab-sorption and negative values denote secretion.

HistologyJejunal sections were stained with haema-toxylin and eosin and histology was evaluatedby a pathologist unaware of the experimentalprotocol from which the tissue originated.

MaterialsL-Arg, D-Arg, and L-NAME were obtainedfrom Sigma Chemical Company, UK. Radio-labelled polyethylene glycol ([`4C]PEG 4000)was obtained from Amersham Intemationaland all other chemicals were supplied byBritish Drug House (BDH Chemicals).

StatisticsResults are expressed as mean (SEM) in eachgroup of animals studied. Analysis of variance(ANOVA) was used for multiple comparisons

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c 30

E 20

10E cm

oI-'-

- A

I

T

Saline L-Arg DiArg(n = 24) (n = 41) (n = 8)

c0E._

EE(0±

Saline L-Arg DArg(n = 24) (n - 41) (n _ 8)

10 CI8 ~-

E 6> 5

E E 3-

OoZL2

Saline L-Arg D-Arg(n = 24) (n = 41) (n a 8)

Figure 1: Water (A), sodium (B) and chloride (C)movement in ratjejunum perfused with either saline, L-Arg20 mmol 1, or D-Arg 20 mmol/l. Values are expressed asmean (SEM). Positive values denote absorption andnegative values denote secretion. (A) ANOVA p<0 001;*p<0.00001 compared with saline andp<0 01 comparedwith D-Arg. (B) ANOVA p<0 01; *p<0.001 comparedwith saline andp<0 05 compared with D-ARG.(C) ANOVA p<0 05; *p<0.05 compared with saline.

and an unpaired Student's t test for individualcomparisons.

Results

Effect of the NO precursor, L-Arg on water andelectrolyte transportPerfusion of rat jejunum with an iso-osmoticsaline solution (Fig 1A) resulted in a mean(SEM) net water absorption of 18 3 (3 6),uA/min/g dry intestinal weight, whereas 20mmol/l of L-Arg caused water secretion (-7 3(2-6); p<00000l compared with saline). Thisphenomenon was not observed with D-Argwhere net water absorption (17-0 (9 0)) wasnot different from the saline control, butsignificantly different from the L-Arg (p<0 05)(Fig 1A). Likewise, sodium and chlorideabsorption were greater with saline solutionthan with L-Arg containing solution (Figs 1Band 1C). Paradoxically, D-Arg resulted in adecrease in chloride absorption than saline (Fig1C). Absorption of L-Arg was greater than itsD-enantiomer (9-8 (2 0) Fmolmin/g v 2-2(0-5); p<001).Subcutaneous administration of L-Arg or

D-Arg in a dose of 100 or 500 mg/kg had noeffect on water (Fig 2) and electrolyte move-ment (data not shown). All histological sec-tions obtained after either perfusion with Argor following subcutaneous administration ofArg were normal.

a 30E 25

20>.r 20 - T l5EE _-

Saline L-Arg L-Arg D-Arg D-Arg(n = 24) 100 500 100 500

(n = 8) (n = 7) (n = 8) (n = 8)

Figure 2: Water movement in ratjejunum perfused withiso-osmotic saline solution after a subcutaneous injection ofL-Arg or D-Arg at a dose of 100 and 500 mg/kg. Valuesare expressed as mean and SEM. Positive values denoteabsorption and negative values denote secretion.ANOVA NS.

0

E

E

0

A A+NO.1 A AA+N1 A A+ N20(n -41) (n= 9) (n=41) (n =10) (n.=41) (n313)

NO.1 NI N20(n = 8) (n 15) (n = 24)

Figure 3: Water movement in ratjejunum after perfusion with solutions containing L-Arg,L-NAME, or L-Arg L-NAME. Values are expressed as mean(SEM). Positive valuesdenote absorption and negative values denote secretion. (1) ANOVA p<0 05; *p<0.01compared with A. (2) ANOVA p<0-01; **p<0-004 compared with A andp<0-05compared with Nl. (3) ANOVA p<0 01; +p<0*003 and ++p<0 0001 compared with A,and ++p<0*05 compared with N20. (A=L-Arg 20 mmol/; NO1, Nl, N20=L-NAME at0-1, 1, and 20 mmol/l respectively).

Effect ofNOS inhibition with L-NAMEThe NOS inhibitor L-NAME perfused intra-luminally in a concentration of 0-1 mmolI hadno effect on water and electrolyte movement,but in higher doses of 1 and 20 mmolI it ledto water secretion (Fig 3) and to a markeddecrease or reversal of electrolyte absorption(Table II).

Perfusion with combinations of L-NAMEand L-Arg (Fig 3) showed that L-NAME at aconcentration of 0-1 mmolI reversed L-Arginduced secretion to absorption. At thehigher concentrations of L-NAME (1 and 20mmol/l), however, L-Arg induced secretion wasenhanced rather than reversed (Fig 3).

All intestinal segments perfused with L-NAME at 1 and 20 mmol/l with or withoutL-Arg appeared grossly dusky and ischaemicbut there were no obvious abnormalities onlight microscopy.A subcutaneous injection of L-NAME (100

mg/kg) 20 minutes before starting the jejunalperfusion caused severe intestinal secretion(-39 9 (5 4)) (Fig 4). Pretreatment with L-Arg(500 mg/kg) subcutaneously, partially butsignificantly attenuated the secretory response.D-Arg had no effect on L-NAME inducedintestinal secretion (Fig 4). Sodium andchloride movement paralleled that of water(Table II).

Intestinal segments perfused after L-NAMEinjection showed marked macroscopic changes;the intestine looked ischaemic with obviousareas of infarction. Microscopically, there weretypical histological changes of ischaemia with

I s *L

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TABLE II Sodium and chloride move,containing either L-Arg 20 mm0lii, L-1L-Arg 20 + L-NAME at the above diJ(SEM)

L-Arg

L-NAME 0 1 mmol/lL-Arg+L-NAME 0 1 mmol/l

L-NAME 1 mmolAIL-Arg+L-NAME 1 mmol/l

L-NAME 20 mmol/lL-Arg+L-NAME 20 mmolI

Comparison is made among groups. All*p<0Q005 and **p<0Q05 compared withtp<0-0005 and ttp<0-05 compared wittp<0005 compared with L-NAME 1 an§p<0-001 and §§p<0 05 compared withp<'0-003 compared with L-NAME 20 a

W

c

E-

E --

ment in ratjejunum perfused with solutions D-Arg caused water absorption despite beingNJAME 01, 1, or 20 mmol/; or a combination of less well absorbed. In addition, these studiesfferent concentrations. Values are expressed as mean lesscwe absoretI oni relatesindicate that secretion induced by L-Arg relates

to a local effect in the intestine, as parentalNa (,umo1Imin/g) Cl (/Lmol/min/g) L-Arg administration in high dose failed to0-8 (1)*t§ 3 9 (1)**t alter net water movement (Fig 2). Other

6 (1-2) 7-3 (1-3) investigators have shown that L-Arg and other4-1 (0-8) 5-3 (1) NO donors induce changes in short circuit023 (28)#t -2-42 (1-2) current in the rat and guinea pig ileum in

-57 (0 6) -1-97 (0-07) vitro,'4 15 but intraperitoneal injection of L-Arg-1-36 (1-4)§§ -4-59 (1-8) did not affect intestinal water movement in4-96 (1-2) -46 (2 3) rats,9 supporting a local effect of L-Arg on

intestinal mucosa. The effect of L-Arg andANOVA p<0-05. nitric oxide donors on intestinal water move-L-NAME 0-1 and L-Arg+L-NAME 0-1.:hL-Arg+L-NAME 1. ment could be due to the ability of NO toLd L-Arg+L-NAME 1. stimulate soluble guanylate cyclase resulting inL-Arg+L-NAME 20.nd L-Arg+L-NAME 20. the production of the second messenger cyclic

26GMP, a potent prosecretory agent.We have investigated whether L-Arg induces

10 secretion in the small bowel through formationo _ ___ of NO by examining the effect of NOS

-10 _ inhibitor L-NAME given intraluminally on-20 L-Arg induced secretion. L-NAME at a-30 - concentration of 0d mmolll reversed L-Arg-40 - induced secretion to absorption, indicating a-50 _ I role for NO in this process. Surprisingly,

L-NAME L-Arg + D-Arg + however, L-NAME at a concentration of 1 and(n 10) L-NAME i-NAME 20 mmol/l given alone caused secretion in the

rat jejunum and enhanced rather than reversede 4: Water movement in ratjejunum perfused with an water secretion induced by L-Arg, which maymotic saline solution after a subcutaneous injection of[ME 100 mg/kg alone, or after pretreatment with imply that these substances induce watertaneous L-Arg or D-Arg 500 mg/kg. Values are secretion by different mechanisms. The effectsssed as mean (SEM). Positive values denote of L-NAME at higher doses are more difficultption and negative values denote secretion. ANOVA91; *p<0.01 compared with L-NAME and with to explain. The effect ofL-NAME on itestial+ L-NAME. water transport in previous studies has been

conflicting. Miller et al found that adminis-tration of L-NAME orally to guinea pigs for

nucosal oedema and haemorrhages with seven days resulted in ileal water secretion.27degenerative changes of the surface Rolfe and Levin showed that L-NAME

helium at the tip of villi (Fig 5). All these administration in rats by the intraperitonealiges were prevented by pretreatment with route in a dose of 40 mg/kg reversed Escherichia.g (Fig 5B) but not D-Arg. coli heat stable toxin (Sta) induced secretion to

absorption, implying that NO is a secreta-gogue'5; however Schirgi-Degen and Beubler

cussion found that intravenous L-NAME adminis-no acids are actively transported in the tration led to net fluid secretion in rat jejunum[1 intestine and in general enhance water and enhanced Sta secretion, concluding thatelectrolyte absorption.' 3 4 However, the NO has a proabsorptive role in the intestine.28

Lsic amino acid L-Arg has been shown to In these studies intestinal ischaemia and his-ice water secretion in human jejunum.' 5 tology were not evaluated.ilarly, in our experiments we have shown Our studies suggest that there is a fineL-Arg induced water secretion when per- balance between the direct secretory effectd in rat jejunum (Fig 1). This phenomenon of NO on enterocytes or via prosecretoryereospecific and not osmotically driven as neurones and its role in maintaining intestinal

B

Figure 5: Histological sectionsfrom ratjejunum after a subcutaneous injection ofL-NAME 100 mg/kg (A) showingsubmucosal oedema and haemorrhages with early degenerative changes at the tip of villi or (B) after pretreatment withsubcutaneous L-Arg 500 mg/kg showing no histological abnormalities (original magnification X 100).

Figur'so-osxL-NAsubcu,expresabsorb

D-Arg

subnearlyepitichanL-Ar

DiscAmismalanddibainduSimithatfuseis Stt

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L-Arginine, nitric oxide, and intestinal secretion 543

blood flow. Nitric oxide could be involved inintestinal secretion either by acting directlyon the epithelium or indirectly stimulatingneuronal reflexes`5 or stimulating the release ofother agents from enteric nerves that canstimulate secretion. In addition, oxyradicalshave been shown to stimulate intestinal elec-trolyte transport in rabbit and rat intestine,29 30and therefore NO and other nitrogen oxidescould stimulate secretion by the nature of theirfree radical structure. L-NAME is absorbedfrom the small intestine and Gardiner et al havefound a 25% decrease in mesenteric blood flowin rats drinking a solution containing 0. 1 mg/ml L-NAME.3' We would expect the highestconcentration and the total amount of L-NAME administered in our experimentswould cause a significant fall in mesentericblood flow. With perfusions of L-NAME at 1and 20 mmol/l the intestine was clearlyischaemic, though not severely enough to causehistological changes. This may be due to thefact that histological changes usually appearwithin 30 minutes to one hour after totalcirculatory arrest to the intestine,32 33 whereasin the present experiments there was probablya decrease but not complete interruption ofintestinal blood flow. It is well known thatvasoconstrictor agents like vasopressin causewater and electrolyte secretion in the smallintestine.34 In addition Robinson et al haveshown that intestinal ischaemia causes netwater and electrolyte secretion secondary to aprofound inhibition of water and sodiumabsorption by villus enterocytes, whereas theintact crypt cells continue to secrete sodiumchloride and water.35 36 The concentration ofL-NAME required to reverse L-Arg inducedsecretion in our study was low compared withprevious studies, where the dose of L-Arg ableto counteract the L-NAME effect can bethreefold to 100-fold higher than that of theNOS inhibitor depending on tissues andspecies.37 This could be explained by the factthat L-NAME may be better absorbed thanL-Arg, or the concentration of NO needed toactivate secretion either directly or throughneuronal reflexes is much lower than that re-quired for vasodilatation. Although L-NAMEhas been found to have an antimuscariniceffect,38 it is unlikely that it has an importantrole in our experiments as the antimuscariniceffect is independent of NO production andthus it would not be antagonised by the NOprecursor L-Arg.

Systemic administration of L-NAME ledto severe intestinal secretion associated withmacroscopic and microscopic evidence ofintestinal ischaemia with some areas of infarc-tion. This is further evidence of the importanceof NO in maintaining mesenteric circulationand thus intestinal mucosal integrity. Bothintestinal ischaemia and intestinal secretionwere markedly attenuated by pretreatmentwith L-Arg but not D-Arg, confirming thespecificity of the effect. Our results are inaccordance with those of Schirgi-Degen andBeubler,28 in that L-NAME given parenterallyinduced secretion in rat jejunum; we disagree,however, with their conclusion that NO is

an absorbagogue as the L-NAME inducedsecretion in their experiments is most likely dueto decreased blood flow.

Nitric oxide exerts its biological effectslocally either as a paracrine mediator or as aneurotransmitter. We propose that within theintestine, inhibition of NO release in thevicinity of the vasculature causes ischaemia andconsequent intestinal secretion, and thatincreased NO production in the vicinity ofenterocytes also promotes intestinal secretion.The secretion induced by L-Arg perfusion isdue to increased NO production in the vicinityof enterocytes. The secretion produced by sub-cutaneous administration ofL-NAME is due tointestinal ischaemia. Secretion induced byperfusion with L-Arg is reversed by perfusionin combination with 0O1 mmolIl L-NAMEowing to inhibition of NO production in thevicinity of the enterocytes. However, perfusionwith higher concentrations of L-NAME para-doxically enhances L-Arg induced secretionby affecting the vasculature and producingischaemia and consequent secretion. Thusthere is a delicate balance between the effect ofNO and inhibitors of NO on enterocyte andvascular function.

In summary, L-Arg induces water andelectrolyte secretion in the rat small intestinethrough an NO mediated mechanism dueeither to a direct effect on enterocytes orthrough neuronal activation. Intraluminal andsystemic NOS inhibition leads to severeintestinal secretion by decreasing mesentericblood flow resulting in intestinal ischaemia.There seems to be a fine balance between theprosecretory effect of NO on enterocytes ormyenteric neurones and its role in maintainingblood flow and thus preventing intestinalischaemia.

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