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Stimulation by carbachol of mucus gel thickness in rat stomach involves nitric oxide
ELSEVIER
European Journal of Pharmacology 263 (1994) 199-202
Short communication
Stimulation by carbachol of mucus gel thickness in rat stomach involves nitric oxide Kenneth J. Price a, Peter J. Hanson a9*,Brendan J.R. Whittle b a Pharmaceutical Sciences Institute, Aston Uniuersity, Aston Triangle, Birmingham, B4 7ET, UK b Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR33B.7, UK
Received 16 June 1994; revised MS received 20 July 1994; accepted 22 July 1994
Abstract Instillation administration dose-dependently L-NAME was did not reduce
of carbachol(150 pg/kg) into the gastric lumen in vivo increased the thickness of the mucus gel layer. Intravenous of the inhibitor of nitric oxide (NO) synthase, NG-nitro-L-arginine methyl ester (L-NAME, 0.4-5 mg/kg) reduced the stimulation by carbachol, the half-maximal inhibitory dose being 0.57 mg/kg. This effect of abolished by administration of L-arginine but not by o-arginine (100 mg/kg i.v.). By contrast L-NAME (5 mg/kg) the stimulatory effect of i&alumina1 16,16-dimethyl prostaglandin E, (50 pg/kg) on mucus gel thickness. These
results implicate NO in the cholinergic activation of gastric mucus secretion. Keywords: Nitric oxide (NO); Gastric mucosa; Mucus; Stomach
1. Introduction
Intragastric administration of nitric oxide (NO) donors in vivo increased the thickness of the layer of mucus overlying the gastric epithelium (Brown et al., 1992a). The mucus layer protects the mucosa from the damaging effects of acid and pepsin (Allen et al., 1993), and short-term changes in its thickness are used as an index of alterations in the rate of mucus secretion (Kerss et al., 1982). NO donors also enhanced the release of mucus from suspensions of rat gastric mucosal cells in vitro (Brown et al., 1993). Furthermore, the dibutyryl analogue of guanosine 3’,5’-cyclic monophosphate (cyclic GMP) stimulated mucus release both in vivo and in vitro (Brown et al., 1992a, 1993). The above results, when combined with the presence of Ca ‘+-dependent NO synthase in gastric mucosal epithelial cells (Brown et al., 1992b), suggest a signalling pathway leading from NO synthase activation and subsequent stimulation of guanylate cyclase by released NO to a cyclic GMP-dependent activation of mucus secretion. Muscarinic acetylcholine receptor agonists increase intracellular Ca2+ in gastric mucous * Corresponding 0733.
author. Tel. 021 359 3611 ext. 5330, fax 021 359
0014-2999/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZOO14-2999(94)00455-2
cells (Seidler and Pfeiffer, 1991), and might therefore activate NO synthase and trigger the proposed pathway leading to mucus secretion. Thus, the aim of the present work was to investigate whether NO was involved in the stimulatory effect of the muscarinic agonist carbachol on the thickness of the gastric mucus gel in vivo by using the specific inhibitor of NO synthase NG-nitro-L-arginine methyl ester (L-NAME). 2. Materials
and methods
2.1. General
Male Wistar rats (175-225 g), which had been deprived of food, but not water, for the previous 18 h, were anaesthetised with sodium pentobarbitone (60 mg/kg i.p.>. L-NAME (0.4-5 mg/kg) was administered as a bolus intravenous injection in sterile isotonic saline (1 ml/kg). Control animals received saline alone. The abdomen was opened 5 min after the intravenous injection by a midline incision and isotonic saline (5 ml/kg), or saline containing carbachol (150 pg/kg) or 16,16-dimethyl prostaglandin E, (50 pg/kg), was introduced into the gastric lumen through the wall of the forestomach by means of a syringe and a 26-gauge needle. Pilot, and previous experiments (McQueen et
200
I(J. Price et al. / European Journal of Pharmacology 263 (1994) 199-202
al., 1983)' showed that the above doses of carbachol and 16,16-dimethyl prostaglandin E 2 produced nearmaximal effects on the thickness of the mucus gel layer. In further experiments to evaluate the specificity of the action of L-NAME, L-arginine or o-arginine (100 m g / k g ) were injected intravenously 5 min before L-NAME and 10 min before instillation of carbachol into the stomach. The gastric mucosa was exposed to agents for 30 min, and the stomach was then removed from the animal and cooled to 4°C. A 'blister' of muscle was produced on the dorsal and ventral surfaces of the mid-corpus mucosa by injection of saline into the wall, and the muscle and then the mucosa were removed. Four sections, each 1 mm thick, were cut from the stomach as described previously (Kerss et al., 1982; Brown et al., 1992a). Sections were mounted transversely in isotonic saline and viewed between 0 and 30 min after sectioning under phase contrast by using an inverse microscope, during which time there was no change in the dimensions of the mucus layer (Kerss et al., 1982). The thickness of the mucus layer was determined by measurement with an eyepiece graticule, and with the observer unaware of the previous treatment of the rat. Mucus thickness was calculated as the arithmetic mean of at least 50 measurements per rat.
2.2. Materials L-NAME, D-arginine, L-arginine, carbachol and 16,16-dimethyI prostaglandin E 2 were purchased from Sigma Chemical Co. (Poole, UK). Rats were from Bantin and Kingman (Hull, UK).
2.3. Analysis of results Data are expressed as means + S.E.M. where (n) is the number of animals. Comparisons between measurements of mucus thickness made under two different conditions were performed by using a Mann-Whitney test, while the effects of more than two treatments were analysed by a Kruskal-Wallis test and a non-parametric equivalent of the Newman-Keuls test (Zar, 1984). Non-parametric tests were used because the distribution of measurements of mucus thickness for each animal may depart from normality (Kerss et al., 1982). P values less than 0.05 were considered as significant in all cases. The half-maximally effective dose of L-NAME was calculated by using the program BASEFIT (Barlow, 1983).
3. Results
The thickness of the mucus layer 30 min after administration of saline into the gastric lumen was 108 +
[--OmPCE~--] 220
t
~ 200
Carbachol
180
t
~ 160 ~ 140 ~
120 100
I
Basal
D-Arg
I
I
L-Arg
L-NAME L-NAME Fig. 1. Effect of pretreatment (5 min) with intravenous saline (clear bars) or NG-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg i.v., shaded bars) on the thickness of the mucus gel layer after intragastric instillation of isotonic saline (5 ml/kg), saline plus earbachol (150 /xg/kg) or saline plus 16,16-dimethyl prostaglandin E 2 (DmPGE 2, 50 ~g/kg). In some experiments (heavily shaded bars) Dor L-arginine (100 mg/kg) was administered intravenously 5 min before L-NAME. Results are means + S.E.M. from six rats for each treatment: *significant difference (P < 0.05) from basal, and * significant reduction from the response to the mucus secretagogue in the gastric lumen and saline administered intravenously.
7.5/~m (n = 6) which was not different (Mann-Whitney test) from the value of 113 + 11 /zm (n = 6) obtained with the lumen empty. Intragastric carbachol (150 /zg/kg) increased ( P < 0.05) the thickness of the mucus layer measured 30 min after administration (Fig. 1). Intravenous injection of L-NAME (0.4-5 mg/kg), 5 min before intragastric instillation of carbachol, dose-dependently reduced the stimulation of mucus thickness by carbachol, which was abolished ( P < 0.05) with the highest dose of L-NAME (Fig. 1 and 2). The dose of L-NAME causing half-maximal inhibition of carbachol-stimulated mucus release was calculated to be 0.57 mg/kg. Intravenous adminis170 ~
160 150
~
140
~
130
N 12o 110 100 0.5 1 5 L-NAME (mg/kg) Fig. 2. Dose-dependent effect of NG-nitro-L-arginine methyl ester (L-NAME, 0.4-5 mg/kg), administered intravenously as a bolus 5 min before intragastric instillation of carbachol (150 p,g/kg), on the thickness of the mucus gel layer determined 30 min after administration of carbachol. Results are means + S.E.M. from 4-6 rats for each dose. There was a significant effect of the dose of L-NAME on the thickness of the layer of mucus (P < 0.05, using a Kruskal-Wallis test).
KJ. Price et al. / European Journal of Pharmacology 263 (1994) 199-202
tration of L-arginine (100 m g / k g ) 5 min before LN A M E (5 m g / k g ) prevented the inhibitory action of L - N A M E on the response to intraluminal carbachol, but the equivalent dose of D-arginine had no such effect. Intravenous administration of L - N A M E (5 m g / k g ) or L-arginine (100 m g / k g ) , with m e a s u r e m e n t made 30 min after saline alone was introduced into the gastric lumen, gave values of 129 + 4 . 6 / z m (n = 5) and 128 + 9 . 7 / z m (n = 6) respectively for mucus thickness which were not significantly different (Kruskal-Wallis test) from the control result of 108 + 7.5/zm (n = 6). By contrast to results obtained with carbachol, LN A M E (5 m g / k g i.v.) did not inhibit the stimulation of mucus thickness by intraluminal administration of 16,16-dimethyl prostaglandin E 2 ( 5 0 / ~ g / k g ) , as shown in Fig. 1.
4. Discussion The present results demonstrate that the inhibitor of N O synthase, L-NAME, causes a dose-dependent inhibition of the stimulatory effect of carbachol on the thickness of the mucus layer in rat stomach. This action of L - N A M E appeared to involve inhibition of N O synthase since it was prevented by prior administration of L-arginine, the physiological substrate for the enzyme (Moncada et al., 1991), but not by the inactive isomer, D-arginine. Thus, although L - N A M E has been demonstrated to exert antagonist actions at muscarinic receptors in vitro, such actions were not prevented by concurrent administration of L-arginine (Buxton et al., 1993). Intravenous administration of L - N A M E causes a rise in blood pressure and a reduction in gastric mucosal blood flow ( T e p p e r m a n and Whittle, 1992). However, it is unlikely that such changes interfered nonspecifically with the maintenance of the mucus gel layer for the stimulatory effect of 16,16-dimethyl prostaglandin E 2 on mucus thickness was not reduced by L-NAME, nor did L - N A M E affect the dimensions of the mucus layer in the absence of secretagogues. L - N A M E (5 m g / k g i.v.) does not interfere directly with gastric acid secretion (Esplugues et al., 1993) which makes it unlikely that the effects of L - N A M E on mucus gel thickness seen in the present work were caused by changes in acid production. These present findings that imply an involvement of endogenous N O in cholinergic activation of mucus secretion are compatible with the observed stimulation of mucus secretion by exogenously generated N O (Brown et al., 1993a,b). Stimulation of the release of mucus by cholinergic activation of preparations of gastric mucosa in vitro is dependent upon intracellular Ca 2÷ (Seidler and Sewing, 1989). Cholinergic stimulation of the release of
201
Ca 2+ into the cytosol of epithelial cells (Seidler and Pfeiffer, 1991) may therefore initiate a pathway leading to mucus secretion by activating a Ca2+-calmodulin-de pendent form of N O synthase, known to be located in these cells (Brown et al., 1992b). By contrast with carbachol, stimulatory effects of prostaglandin E 2 on mucus secretion in vitro required neither intracellular nor extracellular Ca 2÷ (Seidler and Sewing, 1989), and indeed L - N A M E did not inhibit mucus secretion induced by the 16,16-dimethyl analogue of prostaglandin E 2 in the present study. The origin of the N O involved in mucus secretion stimulated by carbachol may also be from sites outside of the epithelial cell. Thus N O produced by cholinergic activation of N O synthase in endothelial cells (Moncada et al., 1991) or other ceils might be able to diffuse to mucous cells. In addition, a neuromodulator role of N O (Sanders and Ward, 1992), which may influence mucus secretion in vivo cannot be excluded. The present results using L - N A M E therefore indicate an involvement of N O in the pathway by which a muscarinic acetylcholine receptor agonist stimulates gastric mucus secretion, and hence constitutive N O synthase may be involved in the physiological regulation of mucus secretion.
Acknowledgement This work is supported by a project grant from the Medical Research Council UK.
References Allen, A., G. Flemstrom, A. Garner and E. Kivilaakso, 1993, Gastroduodenal mucosal protection, Physiol. Rev. 73, 823. Barlow, R.B., 1983, Biodata Handling with Microcomputers (Elsevier, Amsterdam) p. 163. Brown, J.F., P.J. Hanson and B.J.R. Whittle, 1992a, Nitric oxide donors increase mucus gel thickness in rat stomach, Eur. J. Pharmacol. 223, 103. Brown, J.F., B.L. Tepperman, P.J. Hanson, B.J.R. Whittle and S. Moncada, 1992b, Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa, Biochem. Biophys. Res. Commun. 184, 680. Brown, J.F., A.C. Keates, P.J. Hanson and B.J.R. Whittle, 1993, Nitric oxide generators and cGMP stimulate mucus secretion by rat gastric mucosal cells, Am. J. Physiol. 265, G418. Buxton, I.L.O., D.J. Cheek, D. Eckman, D.P. Westfall, K.M. Sanders and K.D. Keel, 1993, N~-Nitro L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists, Circ. Res. 72, 387. Esplugues, J.V., M.D. Barrachina, S. Calatayud, J.M. Pique and B.J.R. Whittle, 1993, Nitric oxide mediates the inhibition by interleukin-1/3 of pentagastrin-stimulated rat gastric acid secretion, Br. J. Pharmacol. 108, 9. Kerss, S., A. Allen and A. Garner, 1982, A simple method for measuring thickness of the mucus gel layer adherent to rat, frog and human gastric mucosa: influence of feeding prostaglandin, N-acetylcysteine and other agents, Clin. Sci. 63, 187.
202
K.J. Price et al. / European Journal of Pharmacology 263 (1994) 199-202
McQueen, S., D. Hutton, A. Allen and A. Garner, 1983, Gastric and duodenal surface mucus gel thickness in rat: effect of prostaglandins and damaging agents, Am. J. Physiol. 245, G388. Moncada, S., R.M.J. Palmer and E.A. Higgs, 1991, Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev. 43, 109. Sanders, K.M. and S.M. Ward, 1992, Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission, Am. J. Physiol. 262, G379. Seidler, U. and A. Pfeiffer, 1991, Inositol phosphate formation and
[Ca2+ ]i in secretagogue-stimulated rabbit gastric mucous cells, Am. J. Physiol. 260, G133. Seidler, U. and K.-Fr. Sewing, 1989, CaZ+-dependent and -independent secretagogue action on gastric mucus secretion in rabbit mucosal explants, Am. J. Physiol, 256, G739. Tepperman, B.L. and B.J.R. Whittle, 1992, Endogenous nitric oxide and sensory neuropeptides interact in the modulation of the rat gastric microcirculation, Br. J. Pharmacol. 105, 171. Zar, J.H., 1984, Biostatistical Analysis (Prentice-Hall, Englewood Cliffs, N J) p. 200.
European Journal of Pharmacology 263 (1994) 199-202
Short communication
Stimulation by carbachol of mucus gel thickness in rat stomach involves nitric oxide Kenneth J. Price a, Peter J. Hanson a9*,Brendan J.R. Whittle b a Pharmaceutical Sciences Institute, Aston Uniuersity, Aston Triangle, Birmingham, B4 7ET, UK b Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR33B.7, UK
Received 16 June 1994; revised MS received 20 July 1994; accepted 22 July 1994
Abstract Instillation administration dose-dependently L-NAME was did not reduce
of carbachol(150 pg/kg) into the gastric lumen in vivo increased the thickness of the mucus gel layer. Intravenous of the inhibitor of nitric oxide (NO) synthase, NG-nitro-L-arginine methyl ester (L-NAME, 0.4-5 mg/kg) reduced the stimulation by carbachol, the half-maximal inhibitory dose being 0.57 mg/kg. This effect of abolished by administration of L-arginine but not by o-arginine (100 mg/kg i.v.). By contrast L-NAME (5 mg/kg) the stimulatory effect of i&alumina1 16,16-dimethyl prostaglandin E, (50 pg/kg) on mucus gel thickness. These
results implicate NO in the cholinergic activation of gastric mucus secretion. Keywords: Nitric oxide (NO); Gastric mucosa; Mucus; Stomach
1. Introduction
Intragastric administration of nitric oxide (NO) donors in vivo increased the thickness of the layer of mucus overlying the gastric epithelium (Brown et al., 1992a). The mucus layer protects the mucosa from the damaging effects of acid and pepsin (Allen et al., 1993), and short-term changes in its thickness are used as an index of alterations in the rate of mucus secretion (Kerss et al., 1982). NO donors also enhanced the release of mucus from suspensions of rat gastric mucosal cells in vitro (Brown et al., 1993). Furthermore, the dibutyryl analogue of guanosine 3’,5’-cyclic monophosphate (cyclic GMP) stimulated mucus release both in vivo and in vitro (Brown et al., 1992a, 1993). The above results, when combined with the presence of Ca ‘+-dependent NO synthase in gastric mucosal epithelial cells (Brown et al., 1992b), suggest a signalling pathway leading from NO synthase activation and subsequent stimulation of guanylate cyclase by released NO to a cyclic GMP-dependent activation of mucus secretion. Muscarinic acetylcholine receptor agonists increase intracellular Ca2+ in gastric mucous * Corresponding 0733.
author. Tel. 021 359 3611 ext. 5330, fax 021 359
0014-2999/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZOO14-2999(94)00455-2
cells (Seidler and Pfeiffer, 1991), and might therefore activate NO synthase and trigger the proposed pathway leading to mucus secretion. Thus, the aim of the present work was to investigate whether NO was involved in the stimulatory effect of the muscarinic agonist carbachol on the thickness of the gastric mucus gel in vivo by using the specific inhibitor of NO synthase NG-nitro-L-arginine methyl ester (L-NAME). 2. Materials
and methods
2.1. General
Male Wistar rats (175-225 g), which had been deprived of food, but not water, for the previous 18 h, were anaesthetised with sodium pentobarbitone (60 mg/kg i.p.>. L-NAME (0.4-5 mg/kg) was administered as a bolus intravenous injection in sterile isotonic saline (1 ml/kg). Control animals received saline alone. The abdomen was opened 5 min after the intravenous injection by a midline incision and isotonic saline (5 ml/kg), or saline containing carbachol (150 pg/kg) or 16,16-dimethyl prostaglandin E, (50 pg/kg), was introduced into the gastric lumen through the wall of the forestomach by means of a syringe and a 26-gauge needle. Pilot, and previous experiments (McQueen et
200
I(J. Price et al. / European Journal of Pharmacology 263 (1994) 199-202
al., 1983)' showed that the above doses of carbachol and 16,16-dimethyl prostaglandin E 2 produced nearmaximal effects on the thickness of the mucus gel layer. In further experiments to evaluate the specificity of the action of L-NAME, L-arginine or o-arginine (100 m g / k g ) were injected intravenously 5 min before L-NAME and 10 min before instillation of carbachol into the stomach. The gastric mucosa was exposed to agents for 30 min, and the stomach was then removed from the animal and cooled to 4°C. A 'blister' of muscle was produced on the dorsal and ventral surfaces of the mid-corpus mucosa by injection of saline into the wall, and the muscle and then the mucosa were removed. Four sections, each 1 mm thick, were cut from the stomach as described previously (Kerss et al., 1982; Brown et al., 1992a). Sections were mounted transversely in isotonic saline and viewed between 0 and 30 min after sectioning under phase contrast by using an inverse microscope, during which time there was no change in the dimensions of the mucus layer (Kerss et al., 1982). The thickness of the mucus layer was determined by measurement with an eyepiece graticule, and with the observer unaware of the previous treatment of the rat. Mucus thickness was calculated as the arithmetic mean of at least 50 measurements per rat.
2.2. Materials L-NAME, D-arginine, L-arginine, carbachol and 16,16-dimethyI prostaglandin E 2 were purchased from Sigma Chemical Co. (Poole, UK). Rats were from Bantin and Kingman (Hull, UK).
2.3. Analysis of results Data are expressed as means + S.E.M. where (n) is the number of animals. Comparisons between measurements of mucus thickness made under two different conditions were performed by using a Mann-Whitney test, while the effects of more than two treatments were analysed by a Kruskal-Wallis test and a non-parametric equivalent of the Newman-Keuls test (Zar, 1984). Non-parametric tests were used because the distribution of measurements of mucus thickness for each animal may depart from normality (Kerss et al., 1982). P values less than 0.05 were considered as significant in all cases. The half-maximally effective dose of L-NAME was calculated by using the program BASEFIT (Barlow, 1983).
3. Results
The thickness of the mucus layer 30 min after administration of saline into the gastric lumen was 108 +
[--OmPCE~--] 220
t
~ 200
Carbachol
180
t
~ 160 ~ 140 ~
120 100
I
Basal
D-Arg
I
I
L-Arg
L-NAME L-NAME Fig. 1. Effect of pretreatment (5 min) with intravenous saline (clear bars) or NG-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg i.v., shaded bars) on the thickness of the mucus gel layer after intragastric instillation of isotonic saline (5 ml/kg), saline plus earbachol (150 /xg/kg) or saline plus 16,16-dimethyl prostaglandin E 2 (DmPGE 2, 50 ~g/kg). In some experiments (heavily shaded bars) Dor L-arginine (100 mg/kg) was administered intravenously 5 min before L-NAME. Results are means + S.E.M. from six rats for each treatment: *significant difference (P < 0.05) from basal, and * significant reduction from the response to the mucus secretagogue in the gastric lumen and saline administered intravenously.
7.5/~m (n = 6) which was not different (Mann-Whitney test) from the value of 113 + 11 /zm (n = 6) obtained with the lumen empty. Intragastric carbachol (150 /zg/kg) increased ( P < 0.05) the thickness of the mucus layer measured 30 min after administration (Fig. 1). Intravenous injection of L-NAME (0.4-5 mg/kg), 5 min before intragastric instillation of carbachol, dose-dependently reduced the stimulation of mucus thickness by carbachol, which was abolished ( P < 0.05) with the highest dose of L-NAME (Fig. 1 and 2). The dose of L-NAME causing half-maximal inhibition of carbachol-stimulated mucus release was calculated to be 0.57 mg/kg. Intravenous adminis170 ~
160 150
~
140
~
130
N 12o 110 100 0.5 1 5 L-NAME (mg/kg) Fig. 2. Dose-dependent effect of NG-nitro-L-arginine methyl ester (L-NAME, 0.4-5 mg/kg), administered intravenously as a bolus 5 min before intragastric instillation of carbachol (150 p,g/kg), on the thickness of the mucus gel layer determined 30 min after administration of carbachol. Results are means + S.E.M. from 4-6 rats for each dose. There was a significant effect of the dose of L-NAME on the thickness of the layer of mucus (P < 0.05, using a Kruskal-Wallis test).
KJ. Price et al. / European Journal of Pharmacology 263 (1994) 199-202
tration of L-arginine (100 m g / k g ) 5 min before LN A M E (5 m g / k g ) prevented the inhibitory action of L - N A M E on the response to intraluminal carbachol, but the equivalent dose of D-arginine had no such effect. Intravenous administration of L - N A M E (5 m g / k g ) or L-arginine (100 m g / k g ) , with m e a s u r e m e n t made 30 min after saline alone was introduced into the gastric lumen, gave values of 129 + 4 . 6 / z m (n = 5) and 128 + 9 . 7 / z m (n = 6) respectively for mucus thickness which were not significantly different (Kruskal-Wallis test) from the control result of 108 + 7.5/zm (n = 6). By contrast to results obtained with carbachol, LN A M E (5 m g / k g i.v.) did not inhibit the stimulation of mucus thickness by intraluminal administration of 16,16-dimethyl prostaglandin E 2 ( 5 0 / ~ g / k g ) , as shown in Fig. 1.
4. Discussion The present results demonstrate that the inhibitor of N O synthase, L-NAME, causes a dose-dependent inhibition of the stimulatory effect of carbachol on the thickness of the mucus layer in rat stomach. This action of L - N A M E appeared to involve inhibition of N O synthase since it was prevented by prior administration of L-arginine, the physiological substrate for the enzyme (Moncada et al., 1991), but not by the inactive isomer, D-arginine. Thus, although L - N A M E has been demonstrated to exert antagonist actions at muscarinic receptors in vitro, such actions were not prevented by concurrent administration of L-arginine (Buxton et al., 1993). Intravenous administration of L - N A M E causes a rise in blood pressure and a reduction in gastric mucosal blood flow ( T e p p e r m a n and Whittle, 1992). However, it is unlikely that such changes interfered nonspecifically with the maintenance of the mucus gel layer for the stimulatory effect of 16,16-dimethyl prostaglandin E 2 on mucus thickness was not reduced by L-NAME, nor did L - N A M E affect the dimensions of the mucus layer in the absence of secretagogues. L - N A M E (5 m g / k g i.v.) does not interfere directly with gastric acid secretion (Esplugues et al., 1993) which makes it unlikely that the effects of L - N A M E on mucus gel thickness seen in the present work were caused by changes in acid production. These present findings that imply an involvement of endogenous N O in cholinergic activation of mucus secretion are compatible with the observed stimulation of mucus secretion by exogenously generated N O (Brown et al., 1993a,b). Stimulation of the release of mucus by cholinergic activation of preparations of gastric mucosa in vitro is dependent upon intracellular Ca 2÷ (Seidler and Sewing, 1989). Cholinergic stimulation of the release of
201
Ca 2+ into the cytosol of epithelial cells (Seidler and Pfeiffer, 1991) may therefore initiate a pathway leading to mucus secretion by activating a Ca2+-calmodulin-de pendent form of N O synthase, known to be located in these cells (Brown et al., 1992b). By contrast with carbachol, stimulatory effects of prostaglandin E 2 on mucus secretion in vitro required neither intracellular nor extracellular Ca 2÷ (Seidler and Sewing, 1989), and indeed L - N A M E did not inhibit mucus secretion induced by the 16,16-dimethyl analogue of prostaglandin E 2 in the present study. The origin of the N O involved in mucus secretion stimulated by carbachol may also be from sites outside of the epithelial cell. Thus N O produced by cholinergic activation of N O synthase in endothelial cells (Moncada et al., 1991) or other ceils might be able to diffuse to mucous cells. In addition, a neuromodulator role of N O (Sanders and Ward, 1992), which may influence mucus secretion in vivo cannot be excluded. The present results using L - N A M E therefore indicate an involvement of N O in the pathway by which a muscarinic acetylcholine receptor agonist stimulates gastric mucus secretion, and hence constitutive N O synthase may be involved in the physiological regulation of mucus secretion.
Acknowledgement This work is supported by a project grant from the Medical Research Council UK.
References Allen, A., G. Flemstrom, A. Garner and E. Kivilaakso, 1993, Gastroduodenal mucosal protection, Physiol. Rev. 73, 823. Barlow, R.B., 1983, Biodata Handling with Microcomputers (Elsevier, Amsterdam) p. 163. Brown, J.F., P.J. Hanson and B.J.R. Whittle, 1992a, Nitric oxide donors increase mucus gel thickness in rat stomach, Eur. J. Pharmacol. 223, 103. Brown, J.F., B.L. Tepperman, P.J. Hanson, B.J.R. Whittle and S. Moncada, 1992b, Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa, Biochem. Biophys. Res. Commun. 184, 680. Brown, J.F., A.C. Keates, P.J. Hanson and B.J.R. Whittle, 1993, Nitric oxide generators and cGMP stimulate mucus secretion by rat gastric mucosal cells, Am. J. Physiol. 265, G418. Buxton, I.L.O., D.J. Cheek, D. Eckman, D.P. Westfall, K.M. Sanders and K.D. Keel, 1993, N~-Nitro L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists, Circ. Res. 72, 387. Esplugues, J.V., M.D. Barrachina, S. Calatayud, J.M. Pique and B.J.R. Whittle, 1993, Nitric oxide mediates the inhibition by interleukin-1/3 of pentagastrin-stimulated rat gastric acid secretion, Br. J. Pharmacol. 108, 9. Kerss, S., A. Allen and A. Garner, 1982, A simple method for measuring thickness of the mucus gel layer adherent to rat, frog and human gastric mucosa: influence of feeding prostaglandin, N-acetylcysteine and other agents, Clin. Sci. 63, 187.
202
K.J. Price et al. / European Journal of Pharmacology 263 (1994) 199-202
McQueen, S., D. Hutton, A. Allen and A. Garner, 1983, Gastric and duodenal surface mucus gel thickness in rat: effect of prostaglandins and damaging agents, Am. J. Physiol. 245, G388. Moncada, S., R.M.J. Palmer and E.A. Higgs, 1991, Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev. 43, 109. Sanders, K.M. and S.M. Ward, 1992, Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission, Am. J. Physiol. 262, G379. Seidler, U. and A. Pfeiffer, 1991, Inositol phosphate formation and
[Ca2+ ]i in secretagogue-stimulated rabbit gastric mucous cells, Am. J. Physiol. 260, G133. Seidler, U. and K.-Fr. Sewing, 1989, CaZ+-dependent and -independent secretagogue action on gastric mucus secretion in rabbit mucosal explants, Am. J. Physiol, 256, G739. Tepperman, B.L. and B.J.R. Whittle, 1992, Endogenous nitric oxide and sensory neuropeptides interact in the modulation of the rat gastric microcirculation, Br. J. Pharmacol. 105, 171. Zar, J.H., 1984, Biostatistical Analysis (Prentice-Hall, Englewood Cliffs, N J) p. 200.