The response of the blood proteins to ablation of the capsaicin-sensitive nerves

Effects of neurotoxic doses of capsaicin (150 mg/kg) on the protein content in electrophoretic fractions (PAAG) in the Wistar rat plasma were studied. In early period (7 days) after administration of capsaicin, an increase of the alpha1-, alpha2-globulins and a decrease of the albumin, gamma-globulins, were observed. After 14-30 days, increase of the albumin and decrease of the alpha1-, gammay-globulins were detected. The ablation of the capsaicin-sensitive nerves abrogated the changes of positive and negative acute phase reactants induced by zymosan and diminished the content of gamma-globulins.     

Effect of interleukin-1 on lymphocyte capacity of releasing factors affecting platelet adhesion and aggregation,blood coagulation and fibrinolysis

Effects of recombinant interleukin-1 (IL-1) involved an inhibition of adhesion and thrombocyte aggregation. In heparinised blood, the IL-1 activates production of protein C (PnC), alpha 2-macroglobulin (alpha 2-MG) and alpha 1-antitrypsin (alpha 1-AT) and facilitates formation of the fibrinolysis inhibitors. In fibrinolysed blood, the IL-1 suppresses production of PnC, alpha 2-MG, alpha 1-AT, and inhibits the fibrinolysis. In a blood clot, the IL-1 suppresses production of PnC, alpha 2-MG, alpha 1-AT and activates the fibrinolysis.     

The influence of sensory nerves and CGRP on the pancreatic regeneration after repeated episodes of acute pancreatitis in rats.

Stimulation of capsaicin sensitive nerves or administration of calcitonin gene-related peptide (CGRP) before induction of acute pancreatitis (AP) attenuates pancreatic damage, whereas CGRP administration after development of AP aggravates lesion of pancreatic tissue. The aim of this study was to determine the effect of prolonged activity of sensory nerves or CGRP administration on the pancreatic repair after repeated episodes of AP. Five episodes of acute caerulein-induced pancreatitis (10 microg/kg/h for 5 h s.c.) were performed at weekly intervals in rats receiving either vehicle or capsaicin at the sensory nerve stimulatory dose (0.5 mg/kg, 3 times daily), or CGRP (10 microg/kg, 3 times daily). Two weeks after the last induction of AP morphological signs of pancreatic damage, pancreatic blood flow (PBF), serum and pancreatic amylase activity, fecal chymotrypsin activity, pancreatic weight, pancreatic RNA and DNA content, as well as, serum interleukin-1beta (Il-1beta ) were assessed. Pancreata of animals receiving vehicle alone showed almost full recovery within two weeks after last episode of pancreatitis induction. In capsaicin-treated group of rats, we observed the increase in PBF by 44% and in serum Il-1beta concentration by 91%. The pancreatic amylase activity, fecal activity of chymotrypsin, pancreatic nucleic acids content and DNA synthesis were decreased. In rats treated with CGRP the alterations in PBF, serum Il-1beta concentration, as well as, in pancreatic and fecal activity of enzymes were similar to capsaicin treated group but less pronounced. We conclude that prolonged activity of capsaicin-sensitive sensory nerves and the presence of their main mediator-CGRP during pancreatic regeneration after AP leads to pancreatic functional insufficiency typical for chronic pancreatitis.     

Different susceptibility of elastase inhibitors to inactivation by proteinases from Staphylococcus aureus and Pseudomonas aeruginosa.

Neutrophil elastase is thought to contribute to the lung pathology in patients with cystic fibrosis (CF). Therefore, intrapulmonary application of elastase inhibitors might be beneficial for these patients. Inactivation of such inhibitors by bacterial proteinases, however, is an important consideration in this therapy. We studied the effects of Staphylococcus aureus proteinase (STAP) and Pseudomonas aeruginosa elastase (PsE) on native (alpha 1-AT) and recombinant (rAAT) alpha 1-antitrypsin, recombinant secretory leukocyte proteinase inhibitor (rSLPI) and the leech inhibitor eglin C. All inhibitors were inactivated by these bacterial proteinases showing pronounced differences in their susceptibilities to proteolytic cleavage. Comparing the turnover rate (mol of inhibitor inactivated by one mol bacterial proteinase/min), rAAT and alpha 1-AT were approximately 20,000-fold more susceptible to STAP than rSLPI and 50,000-fold more susceptible than eglin C. Pseudomonas aeruginosa elastase inactivated all inhibitors more rapidly than STAP. rAAT and alpha 1-AT were 13-fold and 17,000-fold more susceptible than rSLPI and eglin C, respectively. Incubation of the rAAT-elastase complex with equimolar amounts of STAP did not result in release of elastase activity. Upon simultaneous addition of STAP and leukocyte elastase to rAAT, there was undisturbed elastase inhibition indicating that complex formation with elastase proceeded at a faster rate than inactivation of rAAT by the bacterial proteinase. From these results of inactivation in vitro and considering the immunogenic potential of the inhibitors studied here, we conclude that rSLPI may be the appropriate choice for anti-elastase therapy in CF.     

Role of capsaicin-sensitive nerve fibers in uterine contractility in the rat

The possible participation of capsaicin-sensitive sensory nerves in the modulation of neurogenic contractions was studied in nonpregnant and term pregnant rat uteri. Neurogenic contractions were elicited by electric field stimulation (40 V, 1-70 Hz, 0.6 msec) in intact uteri and uteri that were previously exposed to capsaicin in vitro. In capsaicin pretreated preparations obtained both from nonpregnant and term pregnant rats, a dose-dependent increase in the amplitude of uterine contractions was detected. Prior systemic treatment of the rats with capsaicin (130 mg/kg, s.c.) abolished the effect of in vitro capsaicin administration on the amplitude of neurogenic contractions. Use of a specific antagonist of calcitonin gene-related peptide revealed that depletion of this peptide, which normally elicits uterine smooth muscle relaxation, may be responsible for the increased responsiveness of the uterus to low-frequency stimulation. Experiments on the localization of calcitonin gene-related peptide in uterine tissue specimens exposed to capsaicin revealed dose-dependent depletion of calcitonin-gene related peptide-immunoreactive nerves innervating blood vessels and the myometrium. The findings indicate that capsaicin-sensitive afferent nerves, by the release of sensory neuropeptides, significantly contribute to the modulation of uterine contractility both in nonpregnant and term pregnant rats. It is suggested that uterine sensory nerve activation may be part of a trigger mechanism leading to preterm contractions evoked by, for example, inflammation.     

Enkephalin-like immunofluorescence in nerves of the rat iris following systemic capsaicin injection

A network of nerve fibers with an enkephalin-like immunoreactivity was demonstrated in rat iris whole mounts. Systemic administration of capsaicin in doses which caused partial (5 mg/kg) or complete (50 mg/kg) disappearance of substance P-containing fibers in the iris did not cause degeneration of enkephalin-positive nerve fibers. The enkephalin-immunoreactive network seemed intact also after a capsaicin dose of 250 mg/kg. In fact, the fluorescence intensity of the nerve fibers showing enkephalin-immunoreactivity was often increased three days after a capsaicin injection in a dose of 50 mg/kg. The mechanism behind this effect of capsaicin remains to be elucidated, but could be due either to a direct effect on the enkephalin-positive nerves or involve the disappearance of substance P nerves and/or a simultaneous inflammatory response. However, an increased fluorescence intensity of the enkephalin-immunoreactive fibers was sometimes seen also without capsaicin treatment.     

Effect of electrical stimulation of the trigeminal nucleus caudatus on the blood microvessel permeability in the eye in normal and capsaicin-treated rats

Electrical stimulation of the trigeminal caudate nucleus increased permeability of the eye blood microvessels in rats. Ten days after capsaicin pre-treatment (total dose 150 mg/kg, s/c for 2 days: 20, 30, 50, 50 mg/kg) the electrical stimulation did not evoke any changes of the microvascular permeability in the eye. The data suggest that the effect of the stimulation is mediated through capsaicin-sensitive neurones of the trigeminal ganglion.     

Contribution of capsaicin-sensitive sensory neurons to stress-induced increases in gastric tissue levels of prostaglandins in rats

We examined whether capsaicin-sensitive sensory neurons might be involved in the increase in the gastric tissue level of prostaglandins, thereby contributing to the reduction of water immersion restraint stress (WIR)-induced gastric mucosal injury in rats. Gastric tissue levels of calcitonin gene-related peptide (CGRP), 6-keto-PGF1alpha, and PGE2 were transiently increased 30 min after WIR. These increases were significantly inhibited by subcutaneous injection of capsazepine (CPZ), a vanilloid receptor antagonist, and by functional denervation of capsaicin-sensitive sensory neurons induced by the administration of high-dose capsaicin. The administration of capsaicin (orally) and CGRP (intravenously) significantly enhanced the WIR-induced increases in the gastric tissue level of prostaglandins 30 min after WIR, whereas CGRP-(8-37), a CGRP receptor antagonist, significantly inhibited them. Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of nitric oxide (NO) synthase (NOS), and that with indomethacin inhibited the WIR-induced increases in gastric tissue levels of prostaglandins, whereas either pretreatment with aminoguanidine (AG), a selective inhibitor of the inducible form of NOS, or that with NS-398, a selective inhibitor of cyclooxygenase (COX)-2, did not affect them. CPZ, the functional denervation of capsaicin-sensitive sensory neurons, and CGRP-(8-37) significantly increased gastric MPO activity and exacerbated the WIR-induced gastric mucosal injury in rats subjected to 4-h WIR. The administration of capsaicin and CGRP significantly increased the gastric tissue levels of prostaglandins and inhibited both the WIR-induced increases in gastric MPO activity and gastric mucosal injury 8 h after WIR. These effects induced by capsaicin and CGRP were inhibited by pretreatment with L-NAME and indomethacin but not by pretreatment with AG and NS-398. These observations strongly suggest that capsaicin-sensitive sensory neurons might release CGRP, thereby increasing the gastric tissue levels of PGI2 and PGE2 by activating COX-1 through activation of the constitutive form of NOS in rats subjected to WIR. Such activation of capsaicin-sensitive sensory neurons might contribute to the reduction of WIR-induced gastric mucosal injury mainly by inhibiting neutrophil activation.     

Small doses of capsaicin given intragastrically inhibit gastric basal acid secretion in healthy human subjects.

Although the direct inhibitory effect of small dose of capsaicin on gastric secretory responses was proved in animal observations, the role of capsaicin-sensitive afferent nerves (CSAN) and the effect of capsaicin applied in small and high doses on gastric secretion in human has not been clarified yet. In this study we investigated the influence of different small doses (100-800 microg) of capsaicin given intragastrically through an orogastric tube on gastric basal secretory responses in 10 healthy human subjects. Gastric basal secretory responses (volume, H+-concentration, H+-output) were measured from the suctions of gastric juice for a 1-h period. It has been found that: a) capsaicin dose-dependently inhibited the volume and H+-output of gastric juice; b) ID50 was found to be about 400 microg for capsaicin on gastric acid secretion; c) the time interval for capsaicin-induced gastric inhibition existed for about 1 h indifferently from the higher dose (800 microg) of capsaicin given after. It has been concluded that the capsaicin (given in small doses) inhibits the gastric basal acid output via stimulation of the inhibition of capsaicin sensitive afferent nerves.     

Proteinase-proteinase inhibitor complex in rats under oxidative stress caused by administration of cobalt chloride

Mechanisms of proteinase-inhibitor proteinase system response was estimated following of cobalt chloride injection. The increase proteinase activity, which led to significant decrease of alpha-2-macroglobulin (alpha-2-MG) level was established that indicated to the removal of the proteinase in complex with alpha-2-MG from the organism. Increase of alpha-1-proteinase inhibitor (alpha-1-PI) trypsin-inhibitory activity in the kidneys testify about removal of oxidative alpha-1-PI.     

Interaction between human α2-macroglobulin and duodenase, a serine proteinase with dual specificity

Interaction between a serine proteinase from bovine duodenum and human serum alpha(2)-macroglobulin (alpha(2)-MG) was studied. alpha(2)-MG is established to be one of the most effective duodenase inhibitors. The enzyme is completely inhibited in less than 30 sec at equimolar ratio of the inhibitor and enzyme (concentration 2 x 10(-8) M). Under identical conditions, the rate of duodenase association with alpha(2)-MG is at least 2.5-fold higher than the rate of chymotrypsin association with this inhibitor. The interaction with duodenase results in proteolysis of the inhibitor subunit in the "bait region". Similarly to other proteases, duodenase in the complex with alpha(2)-MG retains the intact catalytic apparatus and ability to hydrolyze some small substrates. But the duodenase-inhibitor complex is fully inactive to proteins (bovine serum albumin). The stoichiometry of the enzyme interaction with the inhibitor is 2 : 1 (mol/mol). Based on the association rate constant and the termination time of the duodenase and alpha(2)-MG in vivo association, alpha(2)-MG is suggested to be a physiological regulator of the enzyme.     

Inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin A plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.     

alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors alpha1-proteinase inhibitor, alpha1-antichymotrypsin, and alpha2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.     

Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358

alpha 1-Antitrypsin (alpha 1-AT) was recently identified as a major physiologic plasma inhibitor of activated protein C. The reaction with activated protein C of recombinant alpha 1-AT containing amino acid substitutions at the reactive center was studied. The substitution of Arg358 for Met, as observed in a patient with a severe bleeding disorder with the mutant alpha 1-AT Pittsburgh, increased the association rate constant for activated protein C from 1.1 x 10(1) to 4.9 x 10(4) M-1 s-1. The association rate constant of activated protein C with protein C inhibitor, a native plasma serpin that contains Arg354 at the reactive site, is 6 x 10(3) M-1 s-1 in the absence of heparin. Plasma containing 4 microM [Arg358]alpha 1-AT inhibited activated protein C activity by greater than 95% in 15 s, and the inhibited activated protein C was shown by immunoblotting to exist as activated protein C-inhibitor complexes. In controls 50% loss of activated protein C activity in normal plasma occurred in 19 min. Double-substituted [Pro357,Met358]alpha 1-AT----[Ala357,Arg358]alpha 1-AT had similar reactivity toward activated protein C as the single-substituted [Arg358]alpha 1-AT. Thus, replacement of the reactive center Met358 of alpha 1-AT by Arg358, analogous to Arg354 of protein C inhibitor, results in an activated protein C inhibitor that is more potent than either of the native inhibitors. Comparison of the association rate constant of the [Arg358]alpha 1-AT for activated protein C to that for thrombin (4 x 10(4) versus 3 x 10(5) M-1 s-1) suggests that thrombin would be more effectively inhibited than activated protein C, thereby giving an explanation for bleeding rather than thrombosis in the alpha 1-AT Pittsburgh patient.     

An increase in the synthesis and release of calcitonin gene-related peptide in two-kidney,one-clip hypertensive rats

Previous investigations have indicated that capsaicin-sensitive sensory nerves play an important role in modulation of the peripheral resistance of the circulation system. In the present study, we examined the role of capsaicin-sensitive sensory nerves in two-kidney, one-clip (2K1C) renovascular hypertension rats. Systolic blood pressure (BP) was monitored by the tail-cuff method throughout the experiment. Concentrations of calcitonin gene-related peptide (CGRP) in the plasma, the level of CGRP mRNA in dorsal root ganglia (DRG) and the density of CGRP immunoreactive (CGRP-ir) fibers in mesenteric artery were measured. Blood pressure was significantly elevated at day 10 postoperation (BP was 143+/-10 and 114+/-7 mm Hg for 2K1C and Sham groups, respectively, p<0.05). Treatment with capsaicin, which selectively depletes neurotransmitters in sensory nerves, enhanced hypertensive responses to clipping (BP was 168+/-7 and 143+/-10 mm Hg at day 10 postoperation for Cap1+2K1C and 2K1C groups, respectively, p<0.05), and BP in the rats treated with a second injection of capsaicin was greater than that in the rats treated with a single injection of capsaicin (At day 30 postoperation, BP was 199+/-7 and 166+/-9 mm Hg for Cap2+2K1C and 2K1C groups, respectively, p<0.01; mean arterial pressure was 185.2+/-6.6 and 150.5+/-4.1 mm Hg for Cap2+2K1C and 2K1C groups, respectively, p<0.01). The expression of alpha-CGRP mRNA in DRG (122.87+/-3.67 arbitrary units, p<0.05), the level of CGRP in the plasma (75.40+/-4.99 pg/ml, p<0.01) and the density of CGRP-ir fibers in mesenteric artery (525.67+/-31.42 intersections, p<0.05) were significantly increased in 2K1C rats. Treatment with capsaicin, a single injection or a second injection, prevented the increased in the expression of CGRP mRNA in DRG. However, the decreased level of CGRP was only observed in the rats treated with a second capsaicin. These results suggest that in 2K1C hypertensive rats, the activity of capsaicin-sensitive sensory nerves is increased, which is playing a compensatory depressor role to partially counteract the increase in blood pressure, and that the cardiovascular actions of CGRP is mediated by the alpha-CGRP isoform.     

Characterization of a peptide released during the reaction of human alpha 1-antitrypsin and bovine alpha-chymotrypsin.

The release of a peptide (molecular weight: about 3,600) was observed during complex formation between human alpha 1-antitrypsin (alpha 1-AT) and bovine alpha-chymotrypsin, when monitored by gel-electrophoresis in the presence of sodium lauryl sulfate. Release of the peptide was proportional to the extent of complex formation. Peptides of the same molecular weight were also released during the complex formation of alpha 1-AT with bovine trypsin or porcine elastase. The peptide released from the complex with bovine alpha-chymotrypsin was composed of 32 amino acid residues, which did not correspond to the composition of any 32 amino acid segment in the bovine alpha-chymotrypsin sequence. The N- and C-terminal sequences of the peptide were determined to be H-(Ser)-Ile-Pro-Pro-Glu- and -Gln-Lys-OH, respectively. Though there was some uncertainty as to the N-terminal sequence, it is quite different from that of the original alpha-AT molecule, and showed a similarity to the sequences of the leaving group sides of the reactive sites in some legume proteinase inhibitors. The C-terminal 2 residues were identical with those of native alpha 1-AT. These results suggest that the peptide was released from the C-terminal region of alpha 1-AT uon interaction with alpha-chymotrypsin. It is tempting to suggest that alpha 1-AT inhibits a serine proteinase by the acyl enzyme mechanism at a residue adjacent to the amino group of the N-terminus of this peptide and that this peptide is liberated as a leaving group in the enzymic process.     

The role of inter-alpha-trypsin inhibitor and other proteinase inhibitors in the plasma clearance of neutrophil elastase and plasmin

The plasma clearance of neutrophil elastase, plasmin, and their complexes with human inter-alpha-trypsin inhibitor (I alpha I) was examined in mice, and the distribution of the proteinases among the plasma proteinase inhibitors was quantified in mixtures of purified inhibitors, in human or murine plasma, and in murine plasma following injection of purified proteins. The results demonstrate that I alpha I acts as a shuttle by transferring proteinases to other plasma proteinase inhibitors for clearance, and that I alpha I modulates the distribution of proteinase among inhibitors. The clearance of I alpha I-elastase involved transfer of proteinase to alpha 2-macroglobulin and alpha 1-proteinase inhibitor. The partition of elastase between these inhibitors was altered by I alpha I to favor formation of alpha 2-macroglobulin-elastase complexes. The clearance of I alpha I-plasmin involved transfer of plasmin to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Results of distribution studies suggest that plasmin binds to endothelium in vivo and reacts with I alpha I before transfer to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Evidence for this sequence of events includes observations that plasmin in complex with I alpha I cleared faster than free plasmin, that plasma obtained after injection of plasmin contained a complex identified as I alpha I-plasmin, and that a murine I alpha I-plasmin complex remained intact following injection into mice. Plasmin initially in complex with I alpha I more readily associated with alpha 2-plasmin inhibitor than did free plasmin.     

Disruption of the murine alpha1-antitrypsin/PI2 gene.

Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.     

Roles of capsaicin-sensitive sensory nerves, endogenous nitric oxide, sulfhydryls, and prostaglandins in gastroprotection by momordin Ic, an oleanolic acid oligoglycoside, on ethanol-induced gastric mucosal lesions in rats.

The roles of capsaicin-sensitive sensory nerves (CPSN), endogenous nitric oxide (NO), sulfhydryls (SHs), prostaglandins (PGs) in the gastroprotection by momordin Ic, an oleanolic acid oligoglycoside isolated from the fruit of Kochia scoparia (L.) SCHRAD., on ethanol-induced gastric mucosal lesions were investigated in rats. Momordin Ic (10 mg/kg, p.o.) potentially inhibited ethanol-induced gastric mucosal lesions. The effect of momordin Ic was markedly attenuated by the pretreatment with capsaicin (125 mg/kg in total, s.c., an ablater of CPSN), N(G)-nitro-L-arginine methyl ester (L-NAME, 70 mg/kg, i.p., an inhibitor of NO synthase), N-ethylmaleimide (NEM, 10 mg/kg, s.c., a blocker of SHs), or indomethacin (10 mg/kg, s.c., an inhibitor of PGs biosynthesis). The attenuation of L-NAME was abolished by L-arginine (300 mg/kg, i.v., a substrate of NO synthase), but not by D-arginine (300 mg/kg, i.v., the enatiomer of L-arginine). The effect of the combination of capsaicin with indomethacin, NEM, or L-NAME was not more potent than that of capsaicin alone. The combination of indomethacin and NEM, indomethacin and L-NAME, or indomethacin and NEM and L-NAME increased the attenuation of each alone. These results suggest that CPSN play an important role in the gastroprotection by momordin Ic on ethanol-induced gastric mucosal lesions, and endogenous PGs, NO, and SHs interactively participate, in rats.