- Email: [email protected]
Capsaicin-like activity of N-ethylmaleimide in rat stomach
Gen. Pharmac. Vol. 23, No. 1, pp. 39-41, 1992
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press pie
Printed in Great Britain. All fights reserved
CAPSAICIN-LIKE ACTIVITY OF N - E T H Y L M A L E I M I D E IN RAT STOMACH S. EVANGELISTA, 1 DANIELA RENZI, 2 PAOLA GUZZI, 2 C. SURRENTI,2 P. SANTICIOLI 3 a n d C. A. MAGGI 3
~Pharmacology Department, Malesii Pharmaceuticals, via Porpora 22, 50144 Firenze, Italy [Tel. 39-55-353221; Fax 39-55-361037] 2Gastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Firenze, Italy and 3Menarini Pharmaceuticals, Pharmacology Department, Firenze, Italy (Received 18 June 1991)
Abstract--1. N-Ethylmaleimide (NEM) and capsaicin induced a release of calcitonin gene-related peptide-like immunoreactivity (CGRP-Ii) in superfusates from rat gastric corpus. 2. The prior application of capsalcin completely blocked CGRP-Ii release by NEM while the exposure to NEM reduced the capsaicin-evoked CGRP-Ii outflow by about 51%. 3. These findings provide evidence that NEM might exert capsaicin-like activity in rat stomach and the release of CGRP-Ii from capsaicin-sensitive afferent fibres might influence the properties exerted by NEM in this tissue.
INTRODUCTION
Capsaicin is a selective neurotoxin which affects a certain population of primary afferents containing several neuropeptides. Among the latter, calcitonin gone-related peptide (CGRP) is considered a marker of the capsaicin-sensitive afferent innervation of the stomach (Green and Dockray, 1988). Release of neuropeptides from capsaicin-sensitive afferents is thought to be involved in some functions of the stomach such as motility and ulcer formation (Maggi and Meli, 1988). Thus, systemic pretreatment with capsaicin induced a worsening of gastric ulcers produced by several agents (Evangelista et al., 1988) probably by depleting the protective sensory neuropeptides from gastric afferents (Maggi and Meli, 1988) whereas release of peptides induced by acute capsaicin administration is protective in gastric ulcers (Holzer and Lippe, 1988; Renzi et al., 1988). N-Ethylmaleimide (NEM) has been extensively used as depletor of gastric non-protein sulfhydryl (SH) pool (Szabo et al., 1981) which is thought to be an important factor in gastric ulcerogenesis. Ethanolinduced gastric lesions have been associated with a rapid decrease in gastric SH (Szabo et al., 1981) and the systemic pretreatment with NEM, given i.p. or s.c., has been reported to reverse the antiulcer properties of some antiulcer compounds (Evangelista and Meli, 1988). Conversely, NEM, given by oral route, has been shown to afford protection against ethanolinduced lesions (Szabo et al., 1987). Here we present direct evidence that NEM possesses capsaicin-like activity on rat gastric sensory nerves.
bovine serum albumin and 30#M thiorphan. A 60min equilibration period was allowed to elapse before drug administration. Fractions (4 ml) were collected every 2 rain in tubes containing enough acetic acid to obtain a 2 N final concentration. At the end of the experiments the tissues were blotted and weighed. The superfusates were immediately freeze-dried and reconstituted with the assay buffer (0.1 M pH 7.4 phosphate buffer containing 0.9% NaC1, 0.01% NaN 3 and 0.1% bovine serum albumin) for radioimmunoassay of CGRP-li (Evangelista et al., 1989). Briefly, 100/~1 (10-1000fmol/ml) of alpha-rat CGRP standard or the sample was incubated with 100pl of an appropriate dilution of rabbit anti-human CGRP-Ii serum (1:400,000, Peninsula) for 48hr at 4°C. One hundred microliters (3000cpm) of [125I]iodohistidyl-human CGRP (Amersham) were added and further incubated for 48 hr at 4°C. The separation of free from bound antigen was achieved using second antibody immunopreeipitation. Coefficient of percentage variation was less than I0% for values between 20-300 fmol/ml. The lower detection limit was 2.5 fmol/tube. Crossreactivity of the antiserum was 100% for both rat and human, alpha and beta CGRP and less than 0.001% for salmon calcitonin. Solutions of I00 #M of NEM or 1 #M of capsaicin did not interfere with CGRP radio-immunoassay. Statistical analysis was performed by means of analysis of variance followed by multiple comparison procedures. RESULTS
Capsaicin (1 # M), the known selective stimulant of gastric sensory fibers, and to a lesser extent NEM (100#M) were able to release CGRP-li from rat stomach (Fig. 1). The prior exposure to NEM affected CGRP-Ii outflow induced by capsaicin (upper panel in Fig. 1). The total evoked release (TER, see Renzi et al., 1988) induced by capsaicin after a previous exposure to NEM (upper panel in Fig. 1; TER = 558 _+ 298) was significantly different (at P < 0.05) as compared to TER induced by the first exposure to capsaicin (lower panel in Fig. 1; T E R = 1141 _ 132).
MATERIALS AND METHODS
Male albino SD rats weighing 200-250g were used. Samples from rat stomach, taken from corpus region, were minced with forceps (total weight 300-400 ms) and placed in 2 ml organ baths, maintained at 37°C and superfused (2 ml/min) with oxygenated Krebs solution containing 0.1% 39
40
S. EVANGELISTAe t al.
2 min
I
|
700 -
600" A
$
41111. 45 rain
2O0 NEM 100 gM m m
!
0. nO 0
CAPS 1 gM O O
1000 900-
700 °
45 rain CAPS 1 gM
NEM 100 gM
Fig. 1. Release of CGRP-Ii by N-ethylmaleimide (NEM, 100/~M) or capsaicin (CAPS, 1/zM) in superfusates of rat gastric corpus. Shaded zones indicate the exposure to NEM or CAPS. A period of 45 min was allowed between the first and the second exposure to NEM or CAPS. Each value represents mean __+SE of 4 experiments, * and ** = <0.05 and <0.01 as compared to basal values.
As shown in the lower panel of Fig. 1, the previous exposure to capsaicin produced a marked desensitization and NEM was unable to induce a further release of CGRP-li from rat stomach. DISCUSSION Anatomical and neurochemical investigations have shown that, in the rat stomach, CGRP is entirely contained in capsaicin-sensitive primary afferents and is considered a selective marker for a sub-set of gastric afferents (Green and Dockray, 1988). The present findings show that NEM releases CGRP-li from the rat stomach and possesses capsaicin-like desensitizing properties. In experiments aiming to deplete SH pool, NEM is usually given systemically at a dose of 5-10 mg/kg, which itself produced a worsening of gastric ulcers if given by subcutaneous route (Evangelista and Meli, 1988). Conversely, intragastric pretreatment with NEM dose-dependently decreased the hemorrhagic erosions caused by concentrated solutions of ethanol (Szabo et al., 1987). Although the actual NEM concentrations which are reached at gastric level after systemic administration are not known, our results demonstrate that, in principle, NEM could stimulate first and then block, at least in part, the capsaicinsensitive afferents. It should be considered that oral capsaicin decreased the lesions induced by ethanol
(Holzer and Lippe, 1988) through the release of gastroprotective peptides (Lippe et al., 1989) contained in the afferent fibres, namely CGRP (Holzer et al., 1990), while high doses of capsaicin produced an aggravation of gastric lesions (Evangelista et al., 1988). In view of the above, we suggest that the capsaicinlike properties of NEM should be considered in any further discussion relative to this substance in the field of experimental ulcers. REFERENCES
Evangelista S. and Meli A. (1988) Sulfhydryls and gastric mucosa protection. Dig. Dis. Sci. 33, 1338-1339. Evangelista S., Maggi C. A., Giuliani S. and Meli A. (1988) Further studies on the role of the adrenals in the capsaicin-sensitive "gastric defence mechanism". Int. J. Tiss. Reac. 10, 253-255. Evangelista S., Renzi D., Mantellini P., Surrenti C. and Meli A. (1989) Duodenal ulcers are associated with a depletion of duodenal calcitonin gene-related peptidelike immunoreactivity in rats. Eur. J. Pharmac. 164, 389-391. Green T. and Dockray G. J. (1988) Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea pig. Neuroscience 25, 181-193. Holzer P. and Lippe I. Th. (1988) Stimulation of afferent nerve endings by intragastric capsaicin protects against ethanol-induced damage of gastric mu¢osa. Neuroscience 27, 981-987.
Capsaicin-like activity of NEM Holzer P., Peskar B. M., Peskar B. A. and Amann R. (1990) Release of calcitonin gene-related peptide induced by capsaicin in the vascularly perfused rat stomach. Neurosci. Lett. 108, 195-200. Lippe I. Th., Lorbach M. and Holzer P. (1989) Close arterial infusion of calcitonin gene-related peptide into the rat stomach inhibits aspirin- and ethanol-induced hemorrhagic damage. Reg. Peptides 26, 35-46. Maggi C. A. and Meli A. (1988) The sensory-efferent function of capsaicin-sensitive sensory neurons. Gen. Pharmac. 19, 1-43.
GP 23/|--D
41
Renzi D., Santicioli P., Maggi C. A., Surrenti C., PradeUes P. and Meli A. (1988) Capsaicin-induced release of substance P-like immunoreactivity from guinea pig stomach /n vitro and /n vivo. Neurosci. Lett. 92, 254-258. Szabo S., Trier J. S. and Frankel P. W. (1981) Sulfhydryi compounds may mediate gastric cytoprotection. Science 214, 200-202. Szabo S., Pihan G., Raza A., Mullen E. A. and Hauschka P. V. (1987) Multiple mechanisms of cell injury in the gastric mucosa. Fed. Proc. 46, 1152-1153.
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press pie
Printed in Great Britain. All fights reserved
CAPSAICIN-LIKE ACTIVITY OF N - E T H Y L M A L E I M I D E IN RAT STOMACH S. EVANGELISTA, 1 DANIELA RENZI, 2 PAOLA GUZZI, 2 C. SURRENTI,2 P. SANTICIOLI 3 a n d C. A. MAGGI 3
~Pharmacology Department, Malesii Pharmaceuticals, via Porpora 22, 50144 Firenze, Italy [Tel. 39-55-353221; Fax 39-55-361037] 2Gastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Firenze, Italy and 3Menarini Pharmaceuticals, Pharmacology Department, Firenze, Italy (Received 18 June 1991)
Abstract--1. N-Ethylmaleimide (NEM) and capsaicin induced a release of calcitonin gene-related peptide-like immunoreactivity (CGRP-Ii) in superfusates from rat gastric corpus. 2. The prior application of capsalcin completely blocked CGRP-Ii release by NEM while the exposure to NEM reduced the capsaicin-evoked CGRP-Ii outflow by about 51%. 3. These findings provide evidence that NEM might exert capsaicin-like activity in rat stomach and the release of CGRP-Ii from capsaicin-sensitive afferent fibres might influence the properties exerted by NEM in this tissue.
INTRODUCTION
Capsaicin is a selective neurotoxin which affects a certain population of primary afferents containing several neuropeptides. Among the latter, calcitonin gone-related peptide (CGRP) is considered a marker of the capsaicin-sensitive afferent innervation of the stomach (Green and Dockray, 1988). Release of neuropeptides from capsaicin-sensitive afferents is thought to be involved in some functions of the stomach such as motility and ulcer formation (Maggi and Meli, 1988). Thus, systemic pretreatment with capsaicin induced a worsening of gastric ulcers produced by several agents (Evangelista et al., 1988) probably by depleting the protective sensory neuropeptides from gastric afferents (Maggi and Meli, 1988) whereas release of peptides induced by acute capsaicin administration is protective in gastric ulcers (Holzer and Lippe, 1988; Renzi et al., 1988). N-Ethylmaleimide (NEM) has been extensively used as depletor of gastric non-protein sulfhydryl (SH) pool (Szabo et al., 1981) which is thought to be an important factor in gastric ulcerogenesis. Ethanolinduced gastric lesions have been associated with a rapid decrease in gastric SH (Szabo et al., 1981) and the systemic pretreatment with NEM, given i.p. or s.c., has been reported to reverse the antiulcer properties of some antiulcer compounds (Evangelista and Meli, 1988). Conversely, NEM, given by oral route, has been shown to afford protection against ethanolinduced lesions (Szabo et al., 1987). Here we present direct evidence that NEM possesses capsaicin-like activity on rat gastric sensory nerves.
bovine serum albumin and 30#M thiorphan. A 60min equilibration period was allowed to elapse before drug administration. Fractions (4 ml) were collected every 2 rain in tubes containing enough acetic acid to obtain a 2 N final concentration. At the end of the experiments the tissues were blotted and weighed. The superfusates were immediately freeze-dried and reconstituted with the assay buffer (0.1 M pH 7.4 phosphate buffer containing 0.9% NaC1, 0.01% NaN 3 and 0.1% bovine serum albumin) for radioimmunoassay of CGRP-li (Evangelista et al., 1989). Briefly, 100/~1 (10-1000fmol/ml) of alpha-rat CGRP standard or the sample was incubated with 100pl of an appropriate dilution of rabbit anti-human CGRP-Ii serum (1:400,000, Peninsula) for 48hr at 4°C. One hundred microliters (3000cpm) of [125I]iodohistidyl-human CGRP (Amersham) were added and further incubated for 48 hr at 4°C. The separation of free from bound antigen was achieved using second antibody immunopreeipitation. Coefficient of percentage variation was less than I0% for values between 20-300 fmol/ml. The lower detection limit was 2.5 fmol/tube. Crossreactivity of the antiserum was 100% for both rat and human, alpha and beta CGRP and less than 0.001% for salmon calcitonin. Solutions of I00 #M of NEM or 1 #M of capsaicin did not interfere with CGRP radio-immunoassay. Statistical analysis was performed by means of analysis of variance followed by multiple comparison procedures. RESULTS
Capsaicin (1 # M), the known selective stimulant of gastric sensory fibers, and to a lesser extent NEM (100#M) were able to release CGRP-li from rat stomach (Fig. 1). The prior exposure to NEM affected CGRP-Ii outflow induced by capsaicin (upper panel in Fig. 1). The total evoked release (TER, see Renzi et al., 1988) induced by capsaicin after a previous exposure to NEM (upper panel in Fig. 1; TER = 558 _+ 298) was significantly different (at P < 0.05) as compared to TER induced by the first exposure to capsaicin (lower panel in Fig. 1; T E R = 1141 _ 132).
MATERIALS AND METHODS
Male albino SD rats weighing 200-250g were used. Samples from rat stomach, taken from corpus region, were minced with forceps (total weight 300-400 ms) and placed in 2 ml organ baths, maintained at 37°C and superfused (2 ml/min) with oxygenated Krebs solution containing 0.1% 39
40
S. EVANGELISTAe t al.
2 min
I
|
700 -
600" A
$
41111. 45 rain
2O0 NEM 100 gM m m
!
0. nO 0
CAPS 1 gM O O
1000 900-
700 °
45 rain CAPS 1 gM
NEM 100 gM
Fig. 1. Release of CGRP-Ii by N-ethylmaleimide (NEM, 100/~M) or capsaicin (CAPS, 1/zM) in superfusates of rat gastric corpus. Shaded zones indicate the exposure to NEM or CAPS. A period of 45 min was allowed between the first and the second exposure to NEM or CAPS. Each value represents mean __+SE of 4 experiments, * and ** = <0.05 and <0.01 as compared to basal values.
As shown in the lower panel of Fig. 1, the previous exposure to capsaicin produced a marked desensitization and NEM was unable to induce a further release of CGRP-li from rat stomach. DISCUSSION Anatomical and neurochemical investigations have shown that, in the rat stomach, CGRP is entirely contained in capsaicin-sensitive primary afferents and is considered a selective marker for a sub-set of gastric afferents (Green and Dockray, 1988). The present findings show that NEM releases CGRP-li from the rat stomach and possesses capsaicin-like desensitizing properties. In experiments aiming to deplete SH pool, NEM is usually given systemically at a dose of 5-10 mg/kg, which itself produced a worsening of gastric ulcers if given by subcutaneous route (Evangelista and Meli, 1988). Conversely, intragastric pretreatment with NEM dose-dependently decreased the hemorrhagic erosions caused by concentrated solutions of ethanol (Szabo et al., 1987). Although the actual NEM concentrations which are reached at gastric level after systemic administration are not known, our results demonstrate that, in principle, NEM could stimulate first and then block, at least in part, the capsaicinsensitive afferents. It should be considered that oral capsaicin decreased the lesions induced by ethanol
(Holzer and Lippe, 1988) through the release of gastroprotective peptides (Lippe et al., 1989) contained in the afferent fibres, namely CGRP (Holzer et al., 1990), while high doses of capsaicin produced an aggravation of gastric lesions (Evangelista et al., 1988). In view of the above, we suggest that the capsaicinlike properties of NEM should be considered in any further discussion relative to this substance in the field of experimental ulcers. REFERENCES
Evangelista S. and Meli A. (1988) Sulfhydryls and gastric mucosa protection. Dig. Dis. Sci. 33, 1338-1339. Evangelista S., Maggi C. A., Giuliani S. and Meli A. (1988) Further studies on the role of the adrenals in the capsaicin-sensitive "gastric defence mechanism". Int. J. Tiss. Reac. 10, 253-255. Evangelista S., Renzi D., Mantellini P., Surrenti C. and Meli A. (1989) Duodenal ulcers are associated with a depletion of duodenal calcitonin gene-related peptidelike immunoreactivity in rats. Eur. J. Pharmac. 164, 389-391. Green T. and Dockray G. J. (1988) Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea pig. Neuroscience 25, 181-193. Holzer P. and Lippe I. Th. (1988) Stimulation of afferent nerve endings by intragastric capsaicin protects against ethanol-induced damage of gastric mu¢osa. Neuroscience 27, 981-987.
Capsaicin-like activity of NEM Holzer P., Peskar B. M., Peskar B. A. and Amann R. (1990) Release of calcitonin gene-related peptide induced by capsaicin in the vascularly perfused rat stomach. Neurosci. Lett. 108, 195-200. Lippe I. Th., Lorbach M. and Holzer P. (1989) Close arterial infusion of calcitonin gene-related peptide into the rat stomach inhibits aspirin- and ethanol-induced hemorrhagic damage. Reg. Peptides 26, 35-46. Maggi C. A. and Meli A. (1988) The sensory-efferent function of capsaicin-sensitive sensory neurons. Gen. Pharmac. 19, 1-43.
GP 23/|--D
41
Renzi D., Santicioli P., Maggi C. A., Surrenti C., PradeUes P. and Meli A. (1988) Capsaicin-induced release of substance P-like immunoreactivity from guinea pig stomach /n vitro and /n vivo. Neurosci. Lett. 92, 254-258. Szabo S., Trier J. S. and Frankel P. W. (1981) Sulfhydryi compounds may mediate gastric cytoprotection. Science 214, 200-202. Szabo S., Pihan G., Raza A., Mullen E. A. and Hauschka P. V. (1987) Multiple mechanisms of cell injury in the gastric mucosa. Fed. Proc. 46, 1152-1153.