Linkage between capsaicin-stimulated calcitonin peptide and somatostatin release in rat stomach TETUYA INUI, YOSHIKAZU KINOSHITA, TOSHIYUKI YAMATANI, AND TSUTOMU
AKINORI CHIBA
gene-related
YAMAGUCHI,
Third Division and Division of Gerontology, Department of Medicine, Kobe University School of Medicine, Kobe 650, Japan
INUI, TETUYA, CHI, TOSHIYUKI
YOSHIKAZU YAMATANI,
KINOSHITA, AKINORI YAMAGUAND TSUTOMU CHIBA. Linlzuge
between capsaicin-stimulated calcitonin gene-related peptide and somatostatin release in rat stomach. Am. J. Physiol. 261 (Gas-
trointest. Liver Physiol. 24): G770-G774, 1991.-Calcitonin gene-relatedpeptide (CGRP) is present in the stomach, and exogenousCGRP stimulates gastric somatostatin release.A study was undertaken to elucidate the functional linkage between CGRP and somatostatinin the stomach. Newborn Wistar rats were madeCGRP deficient by intraperitoneal injection of capsaicin 2 days after birth, and then 2.5 mo later, release of CGRP and somatostatinwasexaminedby vascularperfusion of the isolated stomach. In CGRP-deficient rats, neither the content nor basal secretion of gastric somatostatin differed from that in normal rats, and although none of several secretagoguesinduced CGRP secretion, the somatostatin response to glucagon was well preserved, indicating the presence of normally functioning D cells. On the other hand, arterial infusion of capsaicinsignificantly increasedthe releaseof not only CGRP but also somatostatinfrom the stomachof normal rats. In CGRP-deficient rats, however, capsaicin produced no corresponding effect. Finally, human CGRP-(E&37), a CGRPreceptor antagonist,completely inhibited the increaseof gastric somatostatin induced by both rat ar-CGRPand capsaicininfusion in normal rats. Thus the capsaicin-inducedincrease of somatostatin releaseappearsto be mediated by CGRP in the stomach. afferent neurons;acid secretion;calcitonin gene-relatedpeptide antagonist
more, Bunnett et al. (1) have also shown that CGRP stimulates the release of pro-somatostatin-derived peptides exclusively from the gastric fundus. These results raise the possibility that functional linkage exists between gastric CGRP and somatostatin. On the other hand, recent studies have also revealed that considerable amounts of CGRP in the gut including the stomach are localized to afferent neurons whose cell bodies appear to be located in the dorsal root ganglia (11, 16). Indeed, it has been reported that administration of the sensory neurotoxin capsaicin to neonatal rats caused almost complete loss of immunoreactive CGRP in the stomach, suggesting that capsaicin may be a useful tool for exploring the physiological significance of CGRPcontaining afferent neurons innervating the stomach (23, 26)
In the present study therefore, to elucidate the functional relationship between CGRP-containing neurons and somatostatin-producing D cells in the stomach, we depleted CGRP in the afferent neurons innervating the stomach by treating neonatal rats with capsaicin and investigated the release of these peptides from the isolated perfused stomach of these animals. In addition, to further clarify the role of gastric CGRP in somatostatin release, we eliminated the endogenous release of CGRP using human CGRP-(8-37), a specific antagonist of CGRP. MATERIALS
ESTABLISHED that the stomach contains a number of biologically active peptides such as somatostatin, gastrin, and gastrin-releasing peptide that are involved in the regulation of various gastric functions (10, 28). In addition to these peptides, recent studies have demonstrated that calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide encoded by alternative transcripts of the calcitonin gene (25), is present at high concentration in the stomach (22, 24) and may play important physiological roles, including inhibition of gastric acid secretion (17, 27). Previously, using not only isolated rat stomach (30) but also isolated canine fundic D cells (3), we demonstrated that exogenous CGRP stimulates gastric somatostatin release and suggested that inhibition of gastric acid secretion induced by CGRP in vivo (17, 27) may be mediated by local release of somatostatin within the stomach. FurtherIT IS WELL
G770
0193-1857/91
$1.50 Copyright
AND
METHODS
Chemicals and peptides. [Tyr’lrat c~-CGRP, rat CYCGRP, [Tyr’] somatostatin, somatostatin, and human glucagon were purchased from Peninsula Laboratories (Belmont, CA). Capsaicin was obtained from Sigma Chemical (St. Louis, MO). Human CGRP-I and human CGRP-(8-37) were synthesized using an automatic solid-phase synthesizer (430-A Peptide Synthesizer, Applied Biosystems). Animals. Newborn Wistar rats (Shizuoka Dobutsu, Shizuoka, Japan) were divided into two groups, and rats of one group were injected intraperitoneally with capsaitin (50 mg/kg in a solution of 10 pi/g body wt) 2 days after birth (capsaicin-treated rats). Littermate control rats each received an equal volume of vehicle, which consisted of 10% ethanol, 10% Tween 80, and 80% saline (control rats). The animals were kept in a temperaturecontrolled and air-conditioned room under artificial
0 1991 the American
Physiological
Society
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GASTRIC
G771
CGRP AND SOMATOSTATIN
lighting (lights on 0600-1800 h) and fed Oriental laboratory chow (Oriental Yeast, Tokyo) with tap water ad libitum. At 2.5 mo after the treatment, male rats weighing 300-350 g were used for the experiments. There was no significant difference in body weight between the two groups, and only one of 35 capsaicin-treated and one of 32 control rats had hemorrhagic gastritis; these rats were not used for subsequent experiments. Tissue extraction. After an overnight fast, the animals were killed by decapitation, and the stomach of each was quickly resected and divided into the cardia, fundus, antrum, and pylorus. The tissues were boiled in 0.1 N ‘ acetic acid for 15 min and homogenized with a Polytron tissue grinder. After chilling, the homogenate was centrifuged at 1,800 g for 30 min at 2”C, and the supernatant was lyophilized and stored at -20°C. The residues were dissolved in assay buffer and assayed for immunoreactive CGRP and somatostatin. Perfusion of isolated stomach. After an overnight fast, the animals were anesthetized with chloral hydrate (400 mg/kg ip), and the stomach of each was isolated by the method described previously (4). All the perfusions were performed with 4.6% dextran (mean mol wt 70,000)Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose (DKRBG) gassed with 95% 02-5% COn via the left gastric artery at a flow rate of 2 ml/min without recirculation at 37°C. After a prestimulation period (20 min) with DKRBG alone, each stimulant was infused for 10 min, and the gastric venous effluents were collected at l- or 2-min intervals in chilled tubes containing aprotinin (Bayer, Leverkusen, GFR) (50 U/ml). To remove the dextraa, two volumes of 98% acetone containing 0.35 N acetic acid were added to the samples followed by stirring and centrifugation at 1,800 g for 15 min. The supernatant was evaporated under air to remove the acetone and then lyophilized and stored at -20°C until assayed. Measurement of immunoreactive CGRP and somatostutin. Immunoreactive CGRP and somatostatin in both the tissue extracts and the perfusates were determined by specific radioimmunoassays, respectively, as described previously (4, 14). Specific antiserum to CGRP was produced in the rabbit by repeated immunization with synthetic rat a-CGRP (24). The minimum detectable quantity of the CGRP assay was 15 pg/ml, and mean inhibitory concentration value with rat a-CGRP was 1,080 pg/ml. Intra- and interassay variations were 4.8 and 8.0%, respectively (14). This CGRP antiserum cross-reacted with rat flCGRP lo6 times less potently than rat (Y-CGRP. The minimal detectable quantity of the somatostatin assay with antiserum RA-823 was 10 pg/ml. Intra- and interassay variations were 5.4% and 8.5%, respectively (4). Statistical analysis. Statistical analysis was done by Student’s t test when two means were compared.
1). In capsaicin-treated rats, furthermore, the basal secretion of CGRP from isolated perfused stomach was markedly reduced, whereas that of somatostatin was unchanged (Fig. 2). In addition to the reduction of basal secretion, none of several CGRP-secretagogues such as N6,2’-0-dibutyryladenosine 3’,5’-cyclic monophosphate (DBcAMP) (10e3 M) or theophylline (5 mM) (14) was able to stimulate CGRP secretion from the stomach of capsaicin-treated rats (data not shown), suggesting degeneration of CGRP-containing neurons by neonatal treatment with capsaicin. In contrast, glucagon ( 10e7 M), a representative stimulant of gastric somatostatin release
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FIG. 1. Effects of neonatal treatment with capsaicin on gastric somatostatin and calcitonin gene-related peptide (CGRP) contents in rats. Newborn rats were iniected intraperitoneally with capsaicin (50 mg/kg) 2 days after birth, and peptide-concentrations were measured 2.5 mo after treatment. Control animals received vehicle alone. Values are means * SE from 6 animals. * P < 0.01 vs. control.
300 I
2\ g 200 -
0’
J
, -10
I 10
1 0
I 20
Time (min)
RESULTS
Capsaicin treatment of newborn rats markedly decreased the content of CGRP in every part of the stomach. In contrast, the gastric somatostatin content was not influenced by neonatal treatment with capsaicin (Fig.
FIG. 2. Effects of acute administration of capsaicin on release of gastric CGRP and somatostatin from isolated perfused stomachs of capsaicin-treated (O-O) and control (O---O) rats. After preperfusion for 20 min, capsaicin (10m6M) was infused through gastric artery for 10 min. Each value represents mean + SE from 8 perfusions. *P < 0.01 vs. control.
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G772
GASTRIC
CGRP
AND
(4, 5), induced a significant increase of somatostatin secretion even in capsaicin-treated rats, the peak value of which (1,121 t 195 pg/ml) was not different from that in control rats (1,134 t 133 pg/ml) (Fig. 3). These data indicate that gastric D cell function was well preserved in capsaicin-treated rats, in contrast with CGRP-containing neurons. On the other hand, acute arterial infusion of capsaicin (10m6 M) into the isolated stomach evoked significant increase of not only CGRP but also somatostatin in control rats, in the release showing peak values of 334 t 28 and 650 t 91 pg/ml at 3 min after the start of capsaicin infusion, respectively (Fig. 2). In capsaicin-treated rats, by contrast, despite the presence of functional D cells, acute arterial infusion of capsaicin had no incremental effect on the release of either CGRP or somatostatin from the isolated stomach (Fig. 2). As reported previously (30), human CGRP-I ( 10B8 M) elicited a significant increase of somatostatin release from the isolated stomach of rats (Fig. 4). Recently, on the other hand, we have found that a COOH-terminal fragment of human CGRP, human CGRP-(8-37), is an antagonist of CGRP receptors on rat hepatocyte plasma membranes (7). Therefore, we examined the effects of human CGRP-(8-37) on CGRP-induced somatostatin release from rat stomach and found that human CGRP(8-37) ( 10W6 M) also acts as a specific antagonist of CGRP receptors on rat gastric D cells because it completely inhibited the increase of somatostatin release induced by CGRP (Fig. 4). As a next step therefore, to examine whether the increase of somatostatin release induced by capsaicin infusion in control rats was mediated by gastric CGRP, human CGRP-(8-37) was administered simultaneously with capsaicin. Interestingly, human CGRP-(8-37) ( 10D6 M) significantly inhibited the capsaicin ( 10e6 M) -induced increase of somatostatin secretion from the isolated stomach of control rats (Fig. 5) .
SOMATOSTATIN
* 0
,
I
-10
0
I
I
10
20
Time (min)
FIG. 4. Effect of human CGRP-(8-37) on of somatostatin release from isolated perfused After preperfusion for 20 min, human CGRP-I through gastric artery with (O-O) or without (8-37) (10s6 M) for 10 min. Values are means * P c 0.01 vs. human CGRP-I alone. 1 thCGRP
Time
(8-37)
CGRP-induced increase stomachs of control rats. (10s8 M) was infused (O---O) human CGRPt SE from 6 perfusions.
1
(mid
FIG. 5. Effect of human CGRP-(8-37) on capsaicin-induced increase of somatostatin release from isolated perfused stomachs of control rats. After preperfusion for 20 min, capsaicin (10m6 M) was infused through gastric artery with (O-O) or without (O---O) human CGRP-(8-37) (10m6 M) for 10 min. Values are means 2 SE from 6 perfusions. * P < 0.01 vs. capsaicin alone.
DISCUSSION 1200-
10
20
Time (min)
3. Effects of human glucagon on gastric somatostatin release isolated perfused stomachs of capsaicin-treated (O-O) and control (0- - -o) rats. After preperfusion for 20 min, human glucagon (low7 M) was infused through gastric artery for 10 min. Values are means t SE rats was signiffrom 6 perfusions-N&e of values in capsaicin-treated icantly different from those in control rats (P > 0.05). FIG.
from
Recent studies have demonstrated that CGRP-containing neurons are widely distributed in peripheral tissues (8, 22-24) and that these neurons are markedly depleted by neonatal treatment with the sensory neurotoxin capsaicin, suggesting that CGRP is predominantly present in sensory neurons (2, 18). Indeed, Sternini et al. (26) and Mulderry et al. (23) have reported that capsaicin treatment of newborn rats almost completely eliminated CGRP in the rat stomach, lending support to the idea that CGRP in the stomach is derived mainly from primary afferent neurons. In the present experiment, we also showed that neonatally administered capsaicin markedly decreased not only the content but also the basal secretion of immunoreactive CGRP from rat stomach, confirming the previous reports (11,23,26). In these animals, furthermore, neither DBcAMP nor theophylline, established secretagogues of gastric CGRP (14), was able to stimulate CGRP secretion. Because our antibody against CGRP recognized rat ,8-CGRP lo6
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GASTRIC
CGRP
AND
times less potently than rat wCGRP and rat wCGRP was used as a standard in our CGRP radioimmunoassay, the possibility remains that ,&CGRP was still present and released even in capsaicin-treated rats in this study and was not detected by our CGRP radioimmunoassay. This seems unlikely, however, because use of both specific and nonspecific radioimmunoassays for wCGRP by Mulderry et al. (23) revealed that wCGRP predominates in the rat stomach. Thus it appears that CGRP in the stomach is largely depleted in rats treated neonatally with capsaicin. In contrast to its effect on CGRP-containing neurons, neonatally administered capsaicin had no significant effect on gastric D cells. Indeed, neither the content nor the basal secretion of somatostatin was changed by neonatal treatment with capsaicin. Moreover, the gastric somatostatin response to glucagon in capsaicin-treated rats was quite similar to that in control animals, indicating that the function of somatostatin-producing cells was well preserved even in capsaicin-treated animals. These data are compatible with the known fact that most of the somatostatin in the stomach originates from D cells (29), which are present mainly within the epithelial layer of the fundic as well as antral mucosa and thus insensitive to capsaicin. One point of interest in our study was that acute administration of capsaicin caused simultaneous release of CGRP and somatostatin from the isolated rat stomach. Because various effects of acute capsaicin are considered to be due to the release of CGRP or substance P (20), and moveover, Maggi et al. (21) have reported the direct stimulation of CGRP secretion by capsaicin from isolated guinea pig gallbladders, it appeared that in the present study capsaicin acted directly on CGRP-containing afferent nerve fibers innervating the stomach to stimulate CGRP secretion. As for secretion of somatostatin from D cells, however, it is still unclear from these data whether capsaicin acts directly on D cells. In this regard, it is worth noting that in addition to CGRP release, the somatostatin response to acute capsaicin also disappeared in CGRP-depleted rats with functional D cells after neonatal treatment with capsaicin. More strikingly, the present study clearly demonstrated that a specific antagonist of CGRP receptors, human CGRP(8-37), markedly inhibited the capsaicin-induced increase of somatostatin secretion in control rats. Together with the previous finding by Bunnett et al. (1) that exogenous CGRP potently enhanced somatostatin release from the gastric fundus, all of these results strongly indicate that somatostatin secretion induced by acute administration of capsaicin is mediated by local relase of CGRP in the stomach. One finding that might contradict this hypothesis is that despite the decrease of both CGRP content and release induced by neonatal capsaicin, the neurotoxin had no effect on either the content or basal secretion of somatostatin. These results indicate that CGRP neurons in the stomach might not exert a tonic inhibitory action on D cells. Aiternatively, neonatal capsaicin may also have depleted the content of another peptide present in sensory neurons whose action is to decrease somatostatin secretion. Indeed, we have demonstrated previouslv that substance P, which is present
G773
SOMATOSTATIN
in the stomach (12) and known to be colocalized with CGRP in sensory neurons (II), exerts an inhibitory action on gastric somatostatin release from isolated rat stomach (6). It is generally accepted that CGRP in the stomach is present almost exclusively in afferent nerve fibers whose cell bodies may be located in the dorsal root ganglia (11, 23,26). Thus it is conceivable from our previous (14) and present studies that CGRP is released from the peripheral endings of afferent neurons in the stomach and may have certain physiological functions in the vicinity of the peripheral endings of afferent neurons. In support of this idea, Maggi et al. (21) have demonstrated the release of CGRP from sensory nerve endings in guinea pig gallbladder and suggested that released CGRP is involved in the desensitization of muscle contraction. Furthermore, CGRP in the heart has also been found to be released from capsaicin-sensitive nerves by environmental stimuli such as anoxia (E), as well as by bradykinin, an endogenous mediator of inflammation (9). Thus, as suggested by Maggi and Meli (20), all of these findings may reflect the sensory-efferent function of capsaicin-sensitive sensory neurons. Holtzer and Sametz (13) have recently reported that although functional ablation of afferent neurons by neonatally administered capsaicin enhances the formation of gastric mucosal lesions in response to indomethacin or ethanol, acute intragastric administration of capsaicin protects the gastric mucosa against aspirin. Moreover, Maggi et al. (19) have shown that exogenous administration of CGRP exerts anti-ulcer activity in rats. These results indicate that CGRP-containing afferent neurons in the stomach may play a role in protection of the gastric mucosa against damaging agents. CGRP is known to be a potent inhibitor of gastric acid secretion in vivo (17, 27), and it has been suggested by us and others that this inhibition by CGRP is mediated by gastric somatostatin (1, 30). Moreover, the present study demonstrated a functional link between CGRP-containing neurons and D cells in the stomach. Thus an interesting possibility yet to be explored is that CGRP may be released from the peripheral endings of afferent neurons on gastric irritation and exerts an inhibitory action against the formation of gastric mucosal lesions by inducing the release of somatostatin, thus playing a role in the short-circuit reflexes in localized areas of the stomach. We are grateful for the secretarial skills of Naomi Kabuchi. This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. Address for reprint requests: T. Chiba, Div. of Gerontology, Dept. of Medicine, Kobe Univ. School of Medicine, Kusunoki-cho, 7-5-1, Chuo-ku, Kobe 650, Japan. Received
29 January
1991; accepted
in final
form
25 June 1991.
REFERENCES 1. BUNNETT,
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GASTRIC
CGRP AND SOMATOSTATIN
M. A. GHATEI, G. TERENGHI, S. R. BLOOM, AND J. M. POLAK. Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: effect of capsaicin treatment and surgical denervations. Neuroscience 19: 605-627, 1986. 3. CHIBA, T., J. PARK, AND T. YAMADA. Calcitonin gene related peptide (CGRP) stimulates somatostatin secretion from isolate gastric D-cells via cyclic AMP dependent mechanisms (Abstract). Gastroenterology 96: A86, 1989. 4. CHIBA, T., Y. SEINO, Y. GOTO, S. KADOWAKI, T. TAMINATO, H. ABE, Y. KATO, S. MATSUKURA, M. NOZAWA, AND H. IMURA. Somatostatin release from isolated perfused rat stomach. Biochem.
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1093-1101,1989. 22. MULDERRY, P. K., M. A. GHATEI, A. E. BISHOP, M. POLAK, AND S. R. BLOOM. Distribution and
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AKINORI CHIBA
gene-related
YAMAGUCHI,
Third Division and Division of Gerontology, Department of Medicine, Kobe University School of Medicine, Kobe 650, Japan
INUI, TETUYA, CHI, TOSHIYUKI
YOSHIKAZU YAMATANI,
KINOSHITA, AKINORI YAMAGUAND TSUTOMU CHIBA. Linlzuge
between capsaicin-stimulated calcitonin gene-related peptide and somatostatin release in rat stomach. Am. J. Physiol. 261 (Gas-
trointest. Liver Physiol. 24): G770-G774, 1991.-Calcitonin gene-relatedpeptide (CGRP) is present in the stomach, and exogenousCGRP stimulates gastric somatostatin release.A study was undertaken to elucidate the functional linkage between CGRP and somatostatinin the stomach. Newborn Wistar rats were madeCGRP deficient by intraperitoneal injection of capsaicin 2 days after birth, and then 2.5 mo later, release of CGRP and somatostatinwasexaminedby vascularperfusion of the isolated stomach. In CGRP-deficient rats, neither the content nor basal secretion of gastric somatostatin differed from that in normal rats, and although none of several secretagoguesinduced CGRP secretion, the somatostatin response to glucagon was well preserved, indicating the presence of normally functioning D cells. On the other hand, arterial infusion of capsaicinsignificantly increasedthe releaseof not only CGRP but also somatostatinfrom the stomachof normal rats. In CGRP-deficient rats, however, capsaicin produced no corresponding effect. Finally, human CGRP-(E&37), a CGRPreceptor antagonist,completely inhibited the increaseof gastric somatostatin induced by both rat ar-CGRPand capsaicininfusion in normal rats. Thus the capsaicin-inducedincrease of somatostatin releaseappearsto be mediated by CGRP in the stomach. afferent neurons;acid secretion;calcitonin gene-relatedpeptide antagonist
more, Bunnett et al. (1) have also shown that CGRP stimulates the release of pro-somatostatin-derived peptides exclusively from the gastric fundus. These results raise the possibility that functional linkage exists between gastric CGRP and somatostatin. On the other hand, recent studies have also revealed that considerable amounts of CGRP in the gut including the stomach are localized to afferent neurons whose cell bodies appear to be located in the dorsal root ganglia (11, 16). Indeed, it has been reported that administration of the sensory neurotoxin capsaicin to neonatal rats caused almost complete loss of immunoreactive CGRP in the stomach, suggesting that capsaicin may be a useful tool for exploring the physiological significance of CGRPcontaining afferent neurons innervating the stomach (23, 26)
In the present study therefore, to elucidate the functional relationship between CGRP-containing neurons and somatostatin-producing D cells in the stomach, we depleted CGRP in the afferent neurons innervating the stomach by treating neonatal rats with capsaicin and investigated the release of these peptides from the isolated perfused stomach of these animals. In addition, to further clarify the role of gastric CGRP in somatostatin release, we eliminated the endogenous release of CGRP using human CGRP-(8-37), a specific antagonist of CGRP. MATERIALS
ESTABLISHED that the stomach contains a number of biologically active peptides such as somatostatin, gastrin, and gastrin-releasing peptide that are involved in the regulation of various gastric functions (10, 28). In addition to these peptides, recent studies have demonstrated that calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide encoded by alternative transcripts of the calcitonin gene (25), is present at high concentration in the stomach (22, 24) and may play important physiological roles, including inhibition of gastric acid secretion (17, 27). Previously, using not only isolated rat stomach (30) but also isolated canine fundic D cells (3), we demonstrated that exogenous CGRP stimulates gastric somatostatin release and suggested that inhibition of gastric acid secretion induced by CGRP in vivo (17, 27) may be mediated by local release of somatostatin within the stomach. FurtherIT IS WELL
G770
0193-1857/91
$1.50 Copyright
AND
METHODS
Chemicals and peptides. [Tyr’lrat c~-CGRP, rat CYCGRP, [Tyr’] somatostatin, somatostatin, and human glucagon were purchased from Peninsula Laboratories (Belmont, CA). Capsaicin was obtained from Sigma Chemical (St. Louis, MO). Human CGRP-I and human CGRP-(8-37) were synthesized using an automatic solid-phase synthesizer (430-A Peptide Synthesizer, Applied Biosystems). Animals. Newborn Wistar rats (Shizuoka Dobutsu, Shizuoka, Japan) were divided into two groups, and rats of one group were injected intraperitoneally with capsaitin (50 mg/kg in a solution of 10 pi/g body wt) 2 days after birth (capsaicin-treated rats). Littermate control rats each received an equal volume of vehicle, which consisted of 10% ethanol, 10% Tween 80, and 80% saline (control rats). The animals were kept in a temperaturecontrolled and air-conditioned room under artificial
0 1991 the American
Physiological
Society
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GASTRIC
G771
CGRP AND SOMATOSTATIN
lighting (lights on 0600-1800 h) and fed Oriental laboratory chow (Oriental Yeast, Tokyo) with tap water ad libitum. At 2.5 mo after the treatment, male rats weighing 300-350 g were used for the experiments. There was no significant difference in body weight between the two groups, and only one of 35 capsaicin-treated and one of 32 control rats had hemorrhagic gastritis; these rats were not used for subsequent experiments. Tissue extraction. After an overnight fast, the animals were killed by decapitation, and the stomach of each was quickly resected and divided into the cardia, fundus, antrum, and pylorus. The tissues were boiled in 0.1 N ‘ acetic acid for 15 min and homogenized with a Polytron tissue grinder. After chilling, the homogenate was centrifuged at 1,800 g for 30 min at 2”C, and the supernatant was lyophilized and stored at -20°C. The residues were dissolved in assay buffer and assayed for immunoreactive CGRP and somatostatin. Perfusion of isolated stomach. After an overnight fast, the animals were anesthetized with chloral hydrate (400 mg/kg ip), and the stomach of each was isolated by the method described previously (4). All the perfusions were performed with 4.6% dextran (mean mol wt 70,000)Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose (DKRBG) gassed with 95% 02-5% COn via the left gastric artery at a flow rate of 2 ml/min without recirculation at 37°C. After a prestimulation period (20 min) with DKRBG alone, each stimulant was infused for 10 min, and the gastric venous effluents were collected at l- or 2-min intervals in chilled tubes containing aprotinin (Bayer, Leverkusen, GFR) (50 U/ml). To remove the dextraa, two volumes of 98% acetone containing 0.35 N acetic acid were added to the samples followed by stirring and centrifugation at 1,800 g for 15 min. The supernatant was evaporated under air to remove the acetone and then lyophilized and stored at -20°C until assayed. Measurement of immunoreactive CGRP and somatostutin. Immunoreactive CGRP and somatostatin in both the tissue extracts and the perfusates were determined by specific radioimmunoassays, respectively, as described previously (4, 14). Specific antiserum to CGRP was produced in the rabbit by repeated immunization with synthetic rat a-CGRP (24). The minimum detectable quantity of the CGRP assay was 15 pg/ml, and mean inhibitory concentration value with rat a-CGRP was 1,080 pg/ml. Intra- and interassay variations were 4.8 and 8.0%, respectively (14). This CGRP antiserum cross-reacted with rat flCGRP lo6 times less potently than rat (Y-CGRP. The minimal detectable quantity of the somatostatin assay with antiserum RA-823 was 10 pg/ml. Intra- and interassay variations were 5.4% and 8.5%, respectively (4). Statistical analysis. Statistical analysis was done by Student’s t test when two means were compared.
1). In capsaicin-treated rats, furthermore, the basal secretion of CGRP from isolated perfused stomach was markedly reduced, whereas that of somatostatin was unchanged (Fig. 2). In addition to the reduction of basal secretion, none of several CGRP-secretagogues such as N6,2’-0-dibutyryladenosine 3’,5’-cyclic monophosphate (DBcAMP) (10e3 M) or theophylline (5 mM) (14) was able to stimulate CGRP secretion from the stomach of capsaicin-treated rats (data not shown), suggesting degeneration of CGRP-containing neurons by neonatal treatment with capsaicin. In contrast, glucagon ( 10e7 M), a representative stimulant of gastric somatostatin release
z
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FIG. 1. Effects of neonatal treatment with capsaicin on gastric somatostatin and calcitonin gene-related peptide (CGRP) contents in rats. Newborn rats were iniected intraperitoneally with capsaicin (50 mg/kg) 2 days after birth, and peptide-concentrations were measured 2.5 mo after treatment. Control animals received vehicle alone. Values are means * SE from 6 animals. * P < 0.01 vs. control.
300 I
2\ g 200 -
0’
J
, -10
I 10
1 0
I 20
Time (min)
RESULTS
Capsaicin treatment of newborn rats markedly decreased the content of CGRP in every part of the stomach. In contrast, the gastric somatostatin content was not influenced by neonatal treatment with capsaicin (Fig.
FIG. 2. Effects of acute administration of capsaicin on release of gastric CGRP and somatostatin from isolated perfused stomachs of capsaicin-treated (O-O) and control (O---O) rats. After preperfusion for 20 min, capsaicin (10m6M) was infused through gastric artery for 10 min. Each value represents mean + SE from 8 perfusions. *P < 0.01 vs. control.
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G772
GASTRIC
CGRP
AND
(4, 5), induced a significant increase of somatostatin secretion even in capsaicin-treated rats, the peak value of which (1,121 t 195 pg/ml) was not different from that in control rats (1,134 t 133 pg/ml) (Fig. 3). These data indicate that gastric D cell function was well preserved in capsaicin-treated rats, in contrast with CGRP-containing neurons. On the other hand, acute arterial infusion of capsaicin (10m6 M) into the isolated stomach evoked significant increase of not only CGRP but also somatostatin in control rats, in the release showing peak values of 334 t 28 and 650 t 91 pg/ml at 3 min after the start of capsaicin infusion, respectively (Fig. 2). In capsaicin-treated rats, by contrast, despite the presence of functional D cells, acute arterial infusion of capsaicin had no incremental effect on the release of either CGRP or somatostatin from the isolated stomach (Fig. 2). As reported previously (30), human CGRP-I ( 10B8 M) elicited a significant increase of somatostatin release from the isolated stomach of rats (Fig. 4). Recently, on the other hand, we have found that a COOH-terminal fragment of human CGRP, human CGRP-(8-37), is an antagonist of CGRP receptors on rat hepatocyte plasma membranes (7). Therefore, we examined the effects of human CGRP-(8-37) on CGRP-induced somatostatin release from rat stomach and found that human CGRP(8-37) ( 10W6 M) also acts as a specific antagonist of CGRP receptors on rat gastric D cells because it completely inhibited the increase of somatostatin release induced by CGRP (Fig. 4). As a next step therefore, to examine whether the increase of somatostatin release induced by capsaicin infusion in control rats was mediated by gastric CGRP, human CGRP-(8-37) was administered simultaneously with capsaicin. Interestingly, human CGRP-(8-37) ( 10D6 M) significantly inhibited the capsaicin ( 10e6 M) -induced increase of somatostatin secretion from the isolated stomach of control rats (Fig. 5) .
SOMATOSTATIN
* 0
,
I
-10
0
I
I
10
20
Time (min)
FIG. 4. Effect of human CGRP-(8-37) on of somatostatin release from isolated perfused After preperfusion for 20 min, human CGRP-I through gastric artery with (O-O) or without (8-37) (10s6 M) for 10 min. Values are means * P c 0.01 vs. human CGRP-I alone. 1 thCGRP
Time
(8-37)
CGRP-induced increase stomachs of control rats. (10s8 M) was infused (O---O) human CGRPt SE from 6 perfusions.
1
(mid
FIG. 5. Effect of human CGRP-(8-37) on capsaicin-induced increase of somatostatin release from isolated perfused stomachs of control rats. After preperfusion for 20 min, capsaicin (10m6 M) was infused through gastric artery with (O-O) or without (O---O) human CGRP-(8-37) (10m6 M) for 10 min. Values are means 2 SE from 6 perfusions. * P < 0.01 vs. capsaicin alone.
DISCUSSION 1200-
10
20
Time (min)
3. Effects of human glucagon on gastric somatostatin release isolated perfused stomachs of capsaicin-treated (O-O) and control (0- - -o) rats. After preperfusion for 20 min, human glucagon (low7 M) was infused through gastric artery for 10 min. Values are means t SE rats was signiffrom 6 perfusions-N&e of values in capsaicin-treated icantly different from those in control rats (P > 0.05). FIG.
from
Recent studies have demonstrated that CGRP-containing neurons are widely distributed in peripheral tissues (8, 22-24) and that these neurons are markedly depleted by neonatal treatment with the sensory neurotoxin capsaicin, suggesting that CGRP is predominantly present in sensory neurons (2, 18). Indeed, Sternini et al. (26) and Mulderry et al. (23) have reported that capsaicin treatment of newborn rats almost completely eliminated CGRP in the rat stomach, lending support to the idea that CGRP in the stomach is derived mainly from primary afferent neurons. In the present experiment, we also showed that neonatally administered capsaicin markedly decreased not only the content but also the basal secretion of immunoreactive CGRP from rat stomach, confirming the previous reports (11,23,26). In these animals, furthermore, neither DBcAMP nor theophylline, established secretagogues of gastric CGRP (14), was able to stimulate CGRP secretion. Because our antibody against CGRP recognized rat ,8-CGRP lo6
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GASTRIC
CGRP
AND
times less potently than rat wCGRP and rat wCGRP was used as a standard in our CGRP radioimmunoassay, the possibility remains that ,&CGRP was still present and released even in capsaicin-treated rats in this study and was not detected by our CGRP radioimmunoassay. This seems unlikely, however, because use of both specific and nonspecific radioimmunoassays for wCGRP by Mulderry et al. (23) revealed that wCGRP predominates in the rat stomach. Thus it appears that CGRP in the stomach is largely depleted in rats treated neonatally with capsaicin. In contrast to its effect on CGRP-containing neurons, neonatally administered capsaicin had no significant effect on gastric D cells. Indeed, neither the content nor the basal secretion of somatostatin was changed by neonatal treatment with capsaicin. Moreover, the gastric somatostatin response to glucagon in capsaicin-treated rats was quite similar to that in control animals, indicating that the function of somatostatin-producing cells was well preserved even in capsaicin-treated animals. These data are compatible with the known fact that most of the somatostatin in the stomach originates from D cells (29), which are present mainly within the epithelial layer of the fundic as well as antral mucosa and thus insensitive to capsaicin. One point of interest in our study was that acute administration of capsaicin caused simultaneous release of CGRP and somatostatin from the isolated rat stomach. Because various effects of acute capsaicin are considered to be due to the release of CGRP or substance P (20), and moveover, Maggi et al. (21) have reported the direct stimulation of CGRP secretion by capsaicin from isolated guinea pig gallbladders, it appeared that in the present study capsaicin acted directly on CGRP-containing afferent nerve fibers innervating the stomach to stimulate CGRP secretion. As for secretion of somatostatin from D cells, however, it is still unclear from these data whether capsaicin acts directly on D cells. In this regard, it is worth noting that in addition to CGRP release, the somatostatin response to acute capsaicin also disappeared in CGRP-depleted rats with functional D cells after neonatal treatment with capsaicin. More strikingly, the present study clearly demonstrated that a specific antagonist of CGRP receptors, human CGRP(8-37), markedly inhibited the capsaicin-induced increase of somatostatin secretion in control rats. Together with the previous finding by Bunnett et al. (1) that exogenous CGRP potently enhanced somatostatin release from the gastric fundus, all of these results strongly indicate that somatostatin secretion induced by acute administration of capsaicin is mediated by local relase of CGRP in the stomach. One finding that might contradict this hypothesis is that despite the decrease of both CGRP content and release induced by neonatal capsaicin, the neurotoxin had no effect on either the content or basal secretion of somatostatin. These results indicate that CGRP neurons in the stomach might not exert a tonic inhibitory action on D cells. Aiternatively, neonatal capsaicin may also have depleted the content of another peptide present in sensory neurons whose action is to decrease somatostatin secretion. Indeed, we have demonstrated previouslv that substance P, which is present
G773
SOMATOSTATIN
in the stomach (12) and known to be colocalized with CGRP in sensory neurons (II), exerts an inhibitory action on gastric somatostatin release from isolated rat stomach (6). It is generally accepted that CGRP in the stomach is present almost exclusively in afferent nerve fibers whose cell bodies may be located in the dorsal root ganglia (11, 23,26). Thus it is conceivable from our previous (14) and present studies that CGRP is released from the peripheral endings of afferent neurons in the stomach and may have certain physiological functions in the vicinity of the peripheral endings of afferent neurons. In support of this idea, Maggi et al. (21) have demonstrated the release of CGRP from sensory nerve endings in guinea pig gallbladder and suggested that released CGRP is involved in the desensitization of muscle contraction. Furthermore, CGRP in the heart has also been found to be released from capsaicin-sensitive nerves by environmental stimuli such as anoxia (E), as well as by bradykinin, an endogenous mediator of inflammation (9). Thus, as suggested by Maggi and Meli (20), all of these findings may reflect the sensory-efferent function of capsaicin-sensitive sensory neurons. Holtzer and Sametz (13) have recently reported that although functional ablation of afferent neurons by neonatally administered capsaicin enhances the formation of gastric mucosal lesions in response to indomethacin or ethanol, acute intragastric administration of capsaicin protects the gastric mucosa against aspirin. Moreover, Maggi et al. (19) have shown that exogenous administration of CGRP exerts anti-ulcer activity in rats. These results indicate that CGRP-containing afferent neurons in the stomach may play a role in protection of the gastric mucosa against damaging agents. CGRP is known to be a potent inhibitor of gastric acid secretion in vivo (17, 27), and it has been suggested by us and others that this inhibition by CGRP is mediated by gastric somatostatin (1, 30). Moreover, the present study demonstrated a functional link between CGRP-containing neurons and D cells in the stomach. Thus an interesting possibility yet to be explored is that CGRP may be released from the peripheral endings of afferent neurons on gastric irritation and exerts an inhibitory action against the formation of gastric mucosal lesions by inducing the release of somatostatin, thus playing a role in the short-circuit reflexes in localized areas of the stomach. We are grateful for the secretarial skills of Naomi Kabuchi. This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. Address for reprint requests: T. Chiba, Div. of Gerontology, Dept. of Medicine, Kobe Univ. School of Medicine, Kusunoki-cho, 7-5-1, Chuo-ku, Kobe 650, Japan. Received
29 January
1991; accepted
in final
form
25 June 1991.
REFERENCES 1. BUNNETT,
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GASTRIC
CGRP AND SOMATOSTATIN
M. A. GHATEI, G. TERENGHI, S. R. BLOOM, AND J. M. POLAK. Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: effect of capsaicin treatment and surgical denervations. Neuroscience 19: 605-627, 1986. 3. CHIBA, T., J. PARK, AND T. YAMADA. Calcitonin gene related peptide (CGRP) stimulates somatostatin secretion from isolate gastric D-cells via cyclic AMP dependent mechanisms (Abstract). Gastroenterology 96: A86, 1989. 4. CHIBA, T., Y. SEINO, Y. GOTO, S. KADOWAKI, T. TAMINATO, H. ABE, Y. KATO, S. MATSUKURA, M. NOZAWA, AND H. IMURA. Somatostatin release from isolated perfused rat stomach. Biochem.
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