European Journal of Pharmacology, 191 (1990) 303-309 303

Elsevier

UP 51610

vasoactive intestin
nhibitory transmiss

Chun Guang Li and Michael J. Rand

Department of Pharmacology, University of Melbourne, Melbourne, Victoria 3052, Australia

Received 4 September 1990. accepted 11 September 1990

The nitric oxide (NO) synthesis inhibitor NG-monomethyl L-arginine (L-NMMA) reduced NANC-mediated
relaxations of isolated strips of the rat gastric fundus elicited by low frequencies or shcrt periods of field stimulation,
but D-NMMA had no effect. The inhibitory effect of L-NMMA on NANC-mediated relaxations was partially
reversed by L-arginine but was not affected by D-arginine. A VIP antibody abolished the relaxant response to VIP
and reduced the responses to stimulation. Residual responses to stimulation in the presence of VIP antibody were
further reduced by L-NMMA. The tone of the fundus strip was slightly increased by L-NMMA and slightly reduced
by L-arginine. The relaxation produced by VIP was slightly reduced by L-NMMA and enhanced by L-arginine.
Relaxations produced by peptide histidine isoleucine, sodium nitroprusside or isoprenaline were not affected by
L-NMMA or L-arginine. The results suggest that NO as well as VIP is involved in NANC-mediated relaxations of the
rat gastric fundus.

Gastric fundus (rat); NG-Monomethyl-L-A&tine (L-NMMA); L-arginine; Nitric oxide; Non-adrenergic.

non-cholinergic transmission; Vasoactive intestinal polypeptide (VIP)

1. Introduction There is evidence indicating that nitric oxide

The gastric fundus has an inhibitory non-
adrenergic non-choline@ (NANC) innervation
and there is some evidence suggesting that the
NANC transmitter may be vasoactive intestinal
polypeptide (VIP) in the guinea-pig (Grider et al.,
1985), rat (Lefebvre, 1986; De Beurme and
Lefebvre, 1987; 1988; Kamata et al., 1988) and cat
(D’Amato et al., 1988). However, the incomplete
blockade of NANC-mediated relaxations by VIP
antiserum indicates that a non-VIP component
may be involved (De Beurme and Lefebvre, 1988;
D’Amato et al., 1988).
Correspondence to: C.G. Li. Department of Pharmacology,
University of Melbourne. Melbourne, Victoria 3052. Australia.
(NO) is a mediator of NANC-mediated relaxa-
tions of the anococcygeus muscle of the rat (Li
and Rand, 1989; Gillespie et al., 1989; Ramagopal
and Leighton, 1989) and mouse (Gibson et al.,
1990), and dog cerebral arteries (Toda and
Okamura, 1990) and duodenal muscle strips (Toda
et al., 1990). This is based on the findings that
NG-monomethyl L-arginine (L-NMMA), which
inhibits the synthesis of NO from L-arginine in
endothelial cells and thereby blocks endothelium-
dependent relaxations of vascular smooth muscle
(Rees et al., 1989) also blocks NANC-mediated
relaxations of the tissues mentioned above. There-
fore, we used L-NMMA to investigate the possi-
bility that NO might mediate the non-VIP compo-
nent of the NANC-mediated relaxation in the rat
isolated gastric fundus muscle. A preliminary

0014-2999/90/$03.50 Q 1990 Elsevier Science Publishers B.V. (Biomedical Division)

a~~~~~~t of this work has been communicated to NANC relaxation or relaxations induced by
lasian Society of Clinical and Experi- agonists, the incubation times for these agents
rm~co1ogists (Li and Rand, 1990). were lo-20 min. as specified in Results. Subse-
quent responses to field stimulation or agonists
were obtained in the presence of test drugs, or in
2. ateri met their absence to provide time-controlled observa-
tions.
ale Sprague-Dawley rats (200-300 g) were When VIP antibody was used, control re-
apitated and the stomach was removed. Longi- sponses to agonists and field stimulation were
ina muscle strips of the ventral part of the elicited. then the tissue was incubated with VIP
fundus approximately 15 mm long by 3 mm wide antibody for 60 min before any further observa-
were obtained by cutting parallel to the greater tions were made; thereafter VIP antibody was
cumature and set up in an 8 ml organ bath in replaced in the organ bath whenever the PSS was
physiological salt solution (PSS) at a resting ten- changed. The VIP antibody has been described by
sion of 1 g for isotonic recording. The composition Walsh and Wong (1987). The freeze-dried powder
of the PSS was (mM): NaCl 118; KC1 4.7: was dissolved in PSS at a concentration of 400
NaHCO, 25: MgSO, 0.45; KH,PO, 1.03; CaC12 pg/ml. equivalent to a serum dilution of 1 : 170.
2.5: D-( t- )-glucose 11.1; disodium edetate 0.067: Drugs used were: L- and D-arginine (Well-
ascorbic acid 0.14. The PSS was gassed with 5% come), atropine sulphate (Sigma), gtanethidine
CO, in 0, and maintained at 37” C. The PSS sulphate (Ciba), 5-hydroxytryptamine creatinine
contained atropine (3 FM) and guanethidine (5 sulphate (serotonin, Sigma), indomethacin (Merck
pM) to block cholinergic and adrenergic involve- Sharp & Dohme), isoprenaline hydrochloride
ment in responses to field stimulation of intramu- (Winthrop), and L- and D-No-monomethyl
ral nerves and serotonin (10 PM) to raise the tone arginine (L-NMMA. D-NMMA, Wellcome),
of the smooth muscle. In some experiments. in- peptide histidine isoleucine (porcine PHI fragment
domethacin (10 PM) was present in the PSS to l-27, Sigma), sodium nitroprusside (Sigma),
avoid the influence of endogenous prostaglandins. tetrodotoxin (Sigma), vasoactive intestinal peptide
The tissue was allowed to equilibrate for at least (VIP, human: Auspep, Australia), VIP antibody
40 min. with changes of the PSS every 10 min. 7913, freeze-dried powder (Cure Radioimmunoas-
before making experimental observations. say Laboratory, U.S.A.).
After the equilibration period, NANC-media- Quantitative data are expressed as means + S.E.
ted relaxations were elicited by stimulating the (n = number of observations). Differences be-
intramural nerves with square wave pulses of 1 ms tween means were compared by Student’s t-test,
duration and supramaximal field strength (17 and values of P < 0.05 were taken to indicate
V/cm) delivered from a Grass S88 stimulator statistical significance.
through parallel platinum wire electrodes on either
side of the strip: other stimulation parameters are
given in Results. When agonists producing relaxa-
tion (VIP. peptide histidine isoleucine (PHI), 3. Results
sodium nitroprusside and isoprenaline) were used,
successive responses to a single concentration of Transmural stimulation (l-5 Hz) in the pres-
each (contact time 5 min) were elicited in each ence of atropine and guanethidine to block re-
preparation. The concentrations of agonists pro- sponses to stimulation of cholinergic and adren-
duced submaximal responses in the range of those ergic nerves and serotonin to raise the tone pro-
elicited by field stimulation at frequencies be- duced relaxation of gastric fundus strips. The re-
tween 1 and 5 Hz (established in preliminary laxation was not affected by propranolol (1 PM)
experiments). When the effects of L-NMMA, L- or phentolamine (1 PM), but was abolished by
arginine or other enantiomers were tested on tetrodotoxin (1 p M).

6 (fig. 2). The relaxation produced by a higher con-
bp B
c3 IS0 st4P 64 PHI Ia VIP
Control I_-NMMA I_-ARG
90 270 jbM
Fig. 2. Effects of L-NMMA (30 and 90 pM). D-NMMA (90
FM). L-arginine (L-ARG, 90 and 270 pM) and D-arginine
(D-ARG, 90 pM) on NANC relaxations of rat gastric fundus
strips to field stimulation (1 Hz for 10 s) (A), relaxations
elicited by VIP (1 nM). PHI (30 nM). sodium nitroprusside
(SNP. 50 nM) and isoprenahne (ISO. 6 nM) (B). Relaxations in
each preparation before the addition of L- or D-NMMA or L-
or D-arginine was taken as 100%. Drugs were added 20 rnin
before obsemations were made. In drug combination experi-
.ments. following 20 min of exposure to L-NMMA. tissues were
exposed to L- or D-arginine for 20 min. All data are expressed
as means+ S.E.M. (n = 3-S). * indicates P -z 0.05 compared
with control. * * indicates P < 0.05 compared with L-NMMA
alone.
L-NMMA was partially reversed by repeated
washing with fresh PSS.
D-NMMA (90 PM) had no effect on NANC-
mediated relaxations (fig. 2). NANC-mediated re-
laxations were not significantly affected by L-
arginine (90 and 270 PM) or D-arginine (90 PM).
The reduction of stimulation-induced relaxations
at I Hz for 10 s produced by L-NMMA (30 PM)
was partially reversed by L-arginine (90 PM), but
was not affected by D-arginine (90 pMj (fig. 2).
Similarly, the reduction of stimulation-induced re-
laxations at 1 Hz for 60 s produced by L-NMMA
(90 FM) to 39.28 of control (see above) was
partially reversed by L-arginine, the relaxations
after exposure to L-arginine (270 PM) being 61.6
f 3.9% (n = 4) of control responses.
3.3. Effects of L-NMMA and L-arginine on agonist-
induced relaxations
The relaxation of the gastric fundus produced
by a low concentration of VIP (1 nM) was slightly
but significantly reduced by L-NMMA (90 PM)
centration of VIP (10 nM) was not significantly
affected by L-NMMA, being 97.8 + 3.4% (n = 3)
of control responses. L-Arginine (270 FM) slightly
enhanced the relaxation elicited by VIP (1 nM)
(fig. 2). Neither L-NMMA (90 PM) nor L-arginine
(270 PM) significantly affected the relaxations
elicited by PHI (30 nM), sodium nitroprusside (50
nM) or isoprenaline (6 nM) (fig. 2).
3.4. Effects of VIP antibody
Incubation of gastric fundus strips with VIP
antibody (400 pg/ml, equivalent to a serum dilu-
tion of 1: 170) abolished the relaxant action of
VIP (l-2 nM), but relaxations produced by isopre-
naline (6 nM) or sodium nitroprusside (50 nM)
were not significantly affected, being 104.5 f 6.9%
(n = 3) and 101.2 f 17.4% (n = 3), respectively, of
control response. Incubation with VIP antibody
reduced relaxations elicited by field stimulation
with lg0 s trains of pulses at 1 Hz to 6i.6 + 7.6%
(n = 6) of control responses. In other experiments
in the presence of indcmethacin (IO FLM), VIP
antibody reduced the relaxations to 72.9 + 4.2%
(n = 8) of control responses.
The relaxation elicited by stimulation at 5 Hz
for 180 s after incubation with VIP antibody was
not appreciably decreased in magnitude but re-
covered more rapidly than in the absence of VIP
antibody (fig. 3). Stimulation at 0.5 Hz did not
invariably produce relaxation. However, when a
relaxation was produced, it was appreciably af-
fected by VIP antibody (fig. 3); in three such
experiments, VIP antibody reduced the relaxations
to 80.4 f 6.3% (n = 3) of control responses.
The residual responses to field stimulation after
 1 mm[
1 VIP antibodv 7913
3L;;lin

Fig. 3. Effects of VIP antibody 7913 (400 ~g/ml, equivalent to a serum dilution of 1: 170) and L-NMMA (90 FM) on the
NANC-fiats relaxations of the rat gastric fundus elicited by 180 s trams of field stimulation, VIP and isoprenaline (ISO) in the
presence of guanethidine (5 CM), atropine (3 PM). serotonin (10 FM) and indomethacin (10 JIM). The tissue was incubated with VIP
antibody for 60 min before exposure to L-NMMA (90 CM) for 10 mm. Isotonic recordings were amplified Itfold; the vertical
calibration has been corrected for amplification.

incubation with VIP antibody were further re-
duced or abolished by L-NMMA (90 ,uM) (fig. 3).
When stimulation was at 0.5 Hz for 180 s, L-
NMMA abolished the residual responses. In the
presence of indomethacin (10 PM), as in fig. 3, the
residual responses to simulation at 1 and 5 Hz for
180 s after incubation with VIP antibody were
reduced by L-NMMA (90 PM) to 35.5 + 4.2%
(n = 4) and 76.9 f 6.1% (n = 51, respectively, of
the co~esponding control responses. In other ex-
periments in the absence of indomethacin, the
residual responses to stimulation at 1 and 5 Hz for
180 s were reduced by L-NMMA (90 PM) to
31.0f4.5% (n = 3) and 73.1+4.3% (n= 3}, re-
spectively, of the corresponding control responses.
4. Ikxussi~a
It has been suggested that VIP is a possible
candidate as a NANC i~bito~ transmitter to
the smooth muscle of the gastric fundus of the cat
(D’Amato et al., 1988) and rat (Lefebvre, 1986;
De Beurme and Lefebvre, 1987; 1988). In the
present study, incubation of rat gastric fundus
strips with a VIP antibody blocked VIP-induced
relaxations and reduced NANC-mediated relaxa-
tions elicited by long (180 s) trains of stimulation,
which supports the view that VIP is involved in
NANC transmission to smooth muscle in the rat
gastric fundus (De Beurme and Lefebvre, 1988).
However, a partial inhibition of the stimulation-
induced relaxations of rat gastric fundus strips by
VIP antibody indicates the probability that a non-
VIP component is also involved (De Bezttme and
Lefebvre, 1988).
L-NMMA, but not D-NMMA, abolished or
markedly reduced NANC-mediated relaxations of
rat gastric fundus strips elicited by short (10 s)
trains of field stimulation at 1 and 5 Hz or longer
(180 s) trains at 0.5 and 1 Hz. The inhibitory
effect of L-NMMA was partially reversed by L-
arginine but not by D-arginine. These
enantiomer-specific effects run parallel to those
observed on endothelium-dependent relaxations of
vascular smooth muscle (Rees et al.. 1989), and to
those observed on NANC-m~iated relaxations of
the rat anococcygeus muscle (Li and Rand, 1989),
and suggest that NO is involved in NANC trans-
mission in the rat gastric fundus: we refer to such
transmission processes as nitrergic.
The residual relaxations elicited by field stimu-
lation with 180 s trains at 0.5, 1 and 5 Hz after
incubation with the VIP antibody were further
reduced or abolished by L-NMMA, indicating
that the non-VIP component of NANC transmis-
sion is nitrergic. The possibility that VIP released
from the nerve terminals may stimulate the gener-
ation of NO can not be excluded, since L-NMMA
slightly decreased and L-arginine slightly in-
creased the relaxant action of a low concentration
of VIP, but L-NMMA produced a considerably
greater reduction of stimulation-induced than of
VIP-induced relaxations, and also reduced stimu-
lation-induced relaxations when the action of VIP
was blocked in the presence of VIP antibody.
 owever. the present experiments do not preclude does not appear to be a non-specific stimulant of
rhe .&b&t> there may be yet another compo- smooth muscle since it did not produce contrac-
nent. as the NANC relaxations are not compktely tions of rat isolated bladder and guinea-pig taenia
by a combination of VIP antibody and coli (Li and Rand, unpublished observations), and
it did not inhibit NANC mediated relaxations of
reduced by an alternative processing of the taenia coli and rat duodenum (Li and Rand,
ursor peptide and it has been sug- unpublished observations). The contractile effect
gested that it too may be involved in NANC- of L-NMMA suggests that there is a tonic produc-
mediated relaxations of the rat gastric fundus (De tion of NO in the rat gastric fundus, as already
Reurme and Lefebvre. 1988). In rat gastric fundus suggested for rat anococcygeus muscle (Li and
strips. PHI was less potent than VIP in producing Rand, 1989) and that the most likely sources of
a relaxant effect: nevertheless. it is possible that the NO is NANC nerve terminals. The L-
PHI could relax the gastric fundus if it were NMMA-induced contraction was not affected by
released from netve terminals. However, the possi- tetrodotoxin, so the NO was not released by
bility that PHI could stimulate the generation of spontaneously occurring propagated nerve im-
NO is unlikely since L-NMMA did not affect the pulses, but it is not unreasonable to assume that
relaxations elicited by PHI. there may be a spontaneous tetrodotoxin-insensi-
Prostaglandins may modulate NANC inhibi- tive release of the NANC transmitter, as there is
toty neurotransmission in the stomach (Gustafs- with the classical transmitters (Blaschke and
son and Delbro. 1988); however, in rat gastric ovnas, 1981).
fundus strips in the presence of indomethacin, the It has been reported that L-NMMA produced
effect of L-NMMA on the NANC relaxations was contractions of some vascular preparations
not changed. suggesting that endogenous pros- (Thomas et al., 1989). It ma.y be that L-NMMA
taglandins are not involved in the inhibitory ac- has both a specific action arising from inhibition
tion of L-NMMA on NANC transmission to of NO synthesis and a non-specific effect in rais-
smooth muscle of the gastric fttndtts. ing the tone of smooth muscle. However, NANC-
The simplest interpretation of the inhibitory mediated relaxations of the gastric fundus, like
effects of L-NMMA and VIP antibody on field that of the rat anococcygeus muscle, were in-
stimulation-induced relaxations of rat gastric hibited by L-NMMA whereas the relaxations pro-
funduz ;:+s is that there are (at least) two NANC duced by isoprenaline and sodium nitroprusside
mediators acting or; ?he smooth muscle effector were not affected. This rules out the possibility
cells: NO and VIP (possibly piti, PHI). The NO that the inhibition of NANC-mediated relaxations
component appears to function preferentially c.+n by L-NMMA is attributable to a nonspecific ef-
stimulation is with short trains or low frequencies feci.
of stimulation. There is at present no way of The small reiaxation of rat gastric fundus strips
determining whether the two mediators come from produced by exogenous L-argii<n~ suggests that
the satte or from different nerve terminals. How- the resting release of NO is not maxiuia!!y
ever, it is worth noting that in noradrenergic and activated. However, exogenous L-arginine had no
cholinergic neurones, noradrenaline and effect on stimulation-induced relaxations of rat
acetylcholine are preferentially released by low gastric fundus strips, or of dog cerebral arteries
frequency stimulation of short duration whereas and duodenal muscle strips (Toda and Okamura,
peptide cotransmitters are released by higher fre- 1990; Toda et al., 1990) whereas it has been
quencies and longer durations of stimulation found to augment NANC relaxations in the
(Bartfai et al., 1988). anococcygeus muscle of the rat (Li and Rand,
L-NMMA produced a small but clear contrac- 1989) and mouse (Gibson et al., 1990). The reason
tion of the rat gastric fundus strip, which resem- for the differences is not clear.
bled its effect on the rat anococcygeus muscle (Li The fact that the inhibitory effect of L-NMMA
and Rand, 1989; Gillespie et al., 1989). L-NMMA on NANC-mediated relaxations of gastric fundus
strips is only partially reversed by L-arginine or
by repeated washing with fresh PSS suggests that
L-NMMA is strongly bound to the NO-generating
enzymes. In contrast, the inhibitory effect of L-
NMMA on NANC-mediated relaxations of the
rat anococcygeus muscle was more readily re-
versed (Li and Rand, 1989). The reason for this is
not clear, but one possibility is that different
&-enzymes may be involved in generating NO in
these tissues.
This work was supported by a National Health and Medi-
cal Research Council Programme Grant. C.G. Li was in receipt
of a Melbourne University Postgraduate Scholarship. We are
indebted to Dr. S. Moncada, The Wellcome Research Labora-
tories, Beckenham, UK, for supplying samples of L-NMMA.
D-NMMA, L-arginine and D-arginine, and to Dr. J.H. Walsh,
Cure Radioimmunoassay Laboratory, Veteran’s Administra-
tion Center, Los Angeles, USA. for supplying VIP antibody
7913.
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