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Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa 078-8510, Japan
Address all correspondence and requests for reprints to: Toshikatsu Okumura, M.D., Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa 078-8510, Japan. E-mail: okumurat{at}asahikawa-med.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical CRF9–41, completely blocked the central CART-induced
inhibition of gastric acid secretion. All these results suggest that
CART acts in the brain to inhibit gastric function via brain CRF
system. The vagal pathway and the prostaglandin system are not involved
in the acid inhibition. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With regard to the pathophysiological relevance of CART in the brain, Kristensen et al. (4) raised a hypothesis that CART is a new anorectic peptide because intracerebroventricular administration of CART (55–102) potently inhibits food intake and fasting reduces the expression of CART mRNA in the arcuate nucleus. Lambert et al. also showed that centrally injected CART peptide fragments significantly suppressed feeding (5). In fa/fa rats and ob/ob mice, the CART mRNA is virtually absent from the arcuate nucleus and in ob/ob mice the expression is increased upon leptin treatment (4). These results suggest that leptin-induced anorexia may be mediated by brain CART. Based upon these evidences, endogenous CART may be one of the regulatory peptides that play a physiological role in the control of feeding behavior.
However, we do not know yet at this moment a physiological relevance of CART in body functions other than the regulation of feeding behavior. CART neurons are distributed in the paraventricular hypothalamic nucleus, the dorsomedial hypothalamic nucleus, and the lateral hypothalamic nucleus (2, 3). These hypothalamic nuclei are involved in not only regulation of feeding but control of gut function (6). In the present study, we made a hypothesis that CART may act in the brain to regulate gastric functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemicals
We used CART (55–102) in this study according to the previous
study (4). It has been shown that CART (55–102) is involved in the
control of food intake because intracerebroventricular injection of the
CART (55–102) peptide potently inhibits food intake (4). The peptide
corresponding to CART (55–102) was identified in ovine hypothalamic
extracts during the isolation of somatostatin-like peptide (7). It is
therefore speculated that the C-terminal CART could be an endogenous
peptide that corresponds to a naturally occurring form of CART.
Synthetic rat CART (55–102) was purchased from Peptide Institute Inc., Osaka, Japan and was dissolved in normal saline
just before experiments. Indomethacin (Sigma, St. Louis,
MO) was dissolved in a vehicle (7.5% sodium bicarbonate solution). A
CRF antagonist, -helical CRF9–41, and pentagastrin was purchased
from Peninsula Laboratories, Inc. (St. Helens, UK) and was
dissolved in normal saline just before experiments.
Measurement of food intake
Evaluation of food consumption was done as described previously
(8). Each rat was transferred to a individual metabolic cage 1 day
before the experimental procedures and kept isolated throughout the
experiments. This experiment was performed in 15 animals deprived of
food for 24 h but with free access to water.
Twenty-four-hour-fasted rats received intracisternal injection of CART
or saline in a volume of 10 µl.
Intracisternal injection is defined as an administration of a solution
into the cisterna magna. Intracisternal injections were performed
acutely under short ether anesthesia (
2 min) by puncture of the
occipital membrane with a 10-µl-Hamilton microsyringe after rats were
mounted in a stereotaxic apparatus (David Kopf Instruments, Tijunga,
CA). The presence of cerebrospinal fluid in the Hamilton syringe upon
aspiration before injection insured correctness of needle placement
into the cisterna magna. Immediately after the intracisternal
injection, rats were returned to their cages, and preweighted rat chow
was given to each rat and the weight of uneaten food was measured every
1 h for 8 h.
Measurement of gastric secretion
Gastric secretion was measured using the pylorus-ligation method
as described previously (9). Rats received intracisternal injection of
1, 2, or 4 µg of CART or saline. Intracisternal injection was
performed as described above. Following the intracisternal injection
and ligation of the pylorus under brief ether anesthesia (5 min),
rats were returned to their cages. Four or 8 h after the
treatment, rats were killed by overdose of ether. The stomachs were
removed, and the gastric content were collected and centrifuged. The
volume of gastric secretion was measured and the amount of gastric acid
was determined by titration with 0.01 N NaOH to a pH of
7.0. The unit of gastric acid output is equivalent (Eq). Ip injection
of 4 µg of CART dissolved in 0.5 ml of saline was performed to
examine the possibility that centrally administered CART was acting
periphery by leaking into the circulation. As a control, normal saline
(0.5 ml) alone was injected ip under short ether anesthesia. In the
experiment to examine the time-course effect of CART on gastric acid
secretion, rats received intracisternal injection of 4 µg dose of
CART or saline, and gastric secretion was measured 4 or 8 h after
the injection.
To investigate the role of the vagus nerve in inhibition of gastric acid secretion by central CART, rats underwent bilateral gastric branch vagotomy as described previously (10). Briefly, under ether anesthesia (approximately 5 min), a ventral abdominal incision was made along the midline, and the liver was cranially and laterally retraced to expose the right and left gastric branches of the vagus nerve along the abdominal esophagus, which were identified under an operative microscope. Both nerves were separated and freed for a length of 3 mm from the esophagus at a site immediately rostral to the esophagogastric junction and then cut. CART in a dose of 2 µg or saline was administered intracisternally in vagotomized rats with or without ip injection of 75 µg/kg pentagastrin. Background stimulation with pentagastrin was used because gastric acid output in vagotomized rats is very low, which would mask the inhibitory action of CART. The dose of pentagastrin was according to a previous publication (10). Immediately after the treatments, the pylorus was ligated. These procedures including vagotomy were performed under ether anesthesia within 10 min. Two hours after the treatment, gastric contents were collected and gastric acid output was measured as described above.
To test the hypothesis that prostaglandins are involved in central CART-induced acid inhibition, we examined the effect of indomethacin, a prostaglandin synthesis inhibitor, on the acid inhibition by intracisternal injection of CART. Indomethacin at a dose of 10 mg/kg was administered sc 60 min before CART injection at a dose of 2 µg and pylorus ligation. The dose of indomethacin was selected as described previously (10). Four hours after the treatment, gastric contents were collected and gastric acid output was measured as described above.
The possibility that CRF, a neuropeptide that inhibits acid secretion
through the central action, may mediate the CART-induced acid
inhibition was evaluated. To address the problem, -helical CRF9–41,
an antagonist for CRF (11), was used to block the action of endogenous
CRF in the brain. After brief ether anesthesia, pylorus-ligated rats
received intracisternal administration of -helical CRF9–41 (10
µg/5 µl) or saline (5 µl) together with CART (2 µg/5 µl) or
saline (5 µl), and gastric acid secretion was measured 4 h after
the treatment as described above. The dose (10 µg) of -helical
CRF9–41 used in this study was selected according to a previous
publication (12).
Measurements of gastric emptying
Gastric emptying was measured using a modification of the phenol
red method which has been published previously (13). The test meal
consisted of a solution of 50 mg phenol red dissolved in 100 ml aqueous
methylcellulose (1.5%, wt/vol). This liquid meal was given by oral
intubation in an amount of 1.5 ml/rat under brief ether anesthesia.
Sixty minutes after the test meal, the animals were killed by overdose
of ether to collect the gastric contents to measure gastric emptying.
Under brief ether anesthesia, rats received intracisternal injection of
1, 2, or 4 µg of CART. Immediately after the injection of CART, the
test meal was administered into the stomach. Sixty minutes after the
test solution, the animals were killed and the stomach was removed to
measure the rate of gastric emptying. Four rats were killed immediately
after the meal intubation, the gastric content of phenol red contents
determined, and these calculations were used as zero emptying point. To
measure the gastric contents, the stomach was exposed by laparotomy,
quickly ligated at the pylorus and the esophago-gastric junction and
then removed. The stomach and its contents were homogenized in 40 ml of
0.1 M NaOH and phenol red content determined using methods
described elsewhere (13). Briefly, this assay involves precipitation of
proteins with 20% trichloroacetic acid, alkalization with 0.5
M NaOH, and a colorimetric assay at 560 nm. Gastric
emptying was calculated according to the following formula (PR, phenol
red):
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Statistical analysis
The results are expressed as mean ± SEM.
Statistical analysis was performed by ANOVA and subsequent Fisher’s
LSD test. P < 0.05 was considered statistically
significant.
Ethical considerations
Experiments were conducted in accordance with the Guide for the
Care and Use of Laboratory Animals published by the Public Health
Service. The approval of the Research and Development and Animal Care
committees at the Asahikawa Medical College was obtained for all
studies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, centrally administered CART in a dose of 1 µg significantly inhibited
food consumption for 3 h. Furthermore, 2 µg dose of CART
injected into the cisterna magna potently suppressed food intake up to
8 h after injection. The inhibitory action of CART on feeding was
dose dependent. These results confirmed that CART acts in the brain to
suppress food intake.
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illustrates the effects of
intracisternal injection of CART on gastric secretion in
pylorus-ligated conscious rats. A 1-µg dose of CART did not alter
gastric secretion. However, a 2- or 4-µg dose of CART significantly
reduced the volume of gastric juice and inhibited gastric acid output
(Fig. 2A). Volume of gastric juice and acid output were inhibited by
central CART in a parallel fashion. In contrast, ip administration of
CART in a dose of 4 µg failed to inhibit acid secretion (Table 1). Figure 2B illustrates the time-course
effects of intracisternal CART on gastric secretion. Gastric secretion
was still inhibited 8 h after the central injection of CART,
indicating that the acid inhibitory action of CART was
long-lasting.
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illustrated the
effect of vagotomy on the CART-induced inhibition of acid secretion.
Vagotomy by itself potently inhibited gastric acid secretion when
compared with vagus-intact animals as shown in Fig. 2
. In the
vagotomized rats, intracisternal injection of CART did not
significantly suppress acid secretion. The lack of inhibition of acid
secretion may be due to the low baseline of acid secretion in the
vagotomized animals. We therefore next increased the acid baseline by
ip administration of pentagastrin in vagotomized animals and examined
the effects of intracisternal injection of CART on gastrin-stimulated
gastric secretion. In this condition, intracisternal CART significantly
inhibited gastric acid secretion.
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illustrates the effect of indomethacin,
a cyclooxygenase inhibitor, on the CART-induced inhibition of gastric
acid. In saline-treated control rats, intracisternal injection of
CART in a dose of 2 µg significantly inhibited gastric acid
secretion. Similarly, intracisternal CART suppressed acid production in
rats pretreated with indomethacin.
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demonstrates the role of
endogenous brain CRF in the CART-induced acid inhibition. In
saline-treated control rats, intracisternal CART significantly
inhibited gastric acid secretion. -helical CRF9–41, an antagonist
for CRF, by itself did not change gastric acid secretion, being in
agreement with previous reports (12, 14). Injection of CART into the
cisterna magna failed to inhibit gastric acid secretion in rats treated
with -helical CRF9–41.
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shows the effects of
intracisternal administration of CART on gastric emptying. Centrally
injected CART dose-dependently delayed the rate of gastric emptying.
The significant inhibition of gastric emptying was obtained by a 2-µg
dose of CART. However, suppression of gastric emptying could not be
found after ip injection of CART (Table 2), indicating that CART acts centrally
in the brain to suppress gastric emptying.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The dose of CART to achieve a significant inhibition of gastric acid output and gastric emptying was 2 µg/rat. Almost same dose range of CART inhibited food consumption and gastric function. According to the previous findings, intracerevroventricular CART in doses of 1 and 2 µg/rat inhibited food consumption and anti-CART antibody increased feeding (4, 15). In addition, fasting down-regulated CART mRNA in the hypothalamus (4). These results strongly suggest that endogenous CART plays a role in the central control of feeding behavior. Considering this evidence, the present observation that the inhibition of gastric acid secretion and gastric emptying was observed by the same dose range of CART to inhibit feeding may suggest that suppression of gastric acid and gastric emptying might occur at a physiological concentration of CART. However, we cannot be sure that intracisternal injection of CART resulted in local brain concentrations that might normally be present in a rat.
Intracisternal injection of CART dose-dependently inhibited food intake and gastric emptying. In contrast, a dose-dependent action was not clearly found in the CART-induced acid inhibition. We do not know at this moment the reason why dose-response of gastric acid secretion was not clearly observed in rats treated with central injection of CART. The lack of dose-dependent inhibition of acid secretion by CART in the present study might be due to a relatively small number of animals or the time period tested. Otherwise, more than 4 µg dose of CART might be needed for further inhibition of acid secretion.
So far, a number of neuropeptides and chemicals have been examined to have a role in the central control of feeding behavior. It is of interest that majority of neuropeptides or chemicals that influence feeding also contribute to the central regulation of gastrointestinal function. These include CRF (16, 17), interleukin-1 ß (18, 19, 20), basic fibroblast growth factor (21, 22, 23), apolipoprotein A-IV (9, 10, 13, 24), and orexins (25, 26). The present observation that centrally administered CART inhibited not only food intake but also gastric acid and gastric emptying, furthermore supporting our hypothesis that a anorectic/orexigenic chemical may be involved in central control of gut function.
The present study clearly demonstrated that CART acts in the brain to delay gastric emptying. The delayed gastric emptying by CART might provide a mechanism by which CART has an anorectic action because appetite is likely to be lost when the stomach remains filled. Broberger et al. (27) have very recently demonstrated the presence of CART in the rat vagus nerve and nodosa ganglion (n.g.). This neuroanatomical evidence suggests that CART in the vagal afferent fibers may transmit visceral sensation such as gastric distention from the stomach to the brain stem. The present observation that CART may act in the brain to suppress gastric emptying indicates that CART acts as an efferent signal from the brain to the stomach. In this case, the source of CART may be in the hypothalamus. We cannot, however, exclude the possibility that CART originated in the n.g. may act in the brain to inhibit gastric function. In the latter case, CART may function as a chemical messenger to regulate gastric functions controlled by visceral sensation through the vagal afferent fibers from the stomach.
The vagus nerve is one of the final common pathways that transmit centrally integrated output from the brain to the stomach in the regulation of gastric acid secretion (6). We therefore examined a role of the vagal system in the inhibition of gastric acid secretion by central CART using surgically vagotomized rats. Gastrin-stimulated acid secretion in vagotomized rats is a different model when compared with the pylorus-ligation alone. However, there is no way other than the present procedures to examine a role of the vagus nerve in the central CART-induced acid inhibition. So far, a couple of studies have been done using a gastrin-stimulated gastric secretion model with pylorus-ligation in vagotomized rats to clarify that centrally applied chemicals inhibited gastric secretion in a vagal-dependent or independent manner (10, 28). We therefore examined the effect of intracisternal injection of CART on pentagastrin-stimulated gastric acid secretion in vagotomized rats. In the vagotomized animals, intracisternal CART did inhibit the gastrin-stimulated gastric acid secretion, suggesting that the vagus nerve dose not mediate the central CART-induced inhibition of gastric acid secretion.
It has been reported that central prostaglandin E2 is involved in the regulation of acid secretion. Centrally administered prostaglandin E2 inhibited gastric acid secretion (29, 30), and central (400 µg) or peripheral (10 mg/kg) administration of indomethacin reverses the inhibitory action of neuropeptides and cytokines on gastric secretion. These include dermorphin, calcitonin gene related peptide and interleukin-1 (19, 31). The dose of indomethacin (10 mg/kg) used in this study is known to suppress prostaglandin synthesis both in the brain and the stomach (29). Although it is not known whether central CART is involved in the metabolism of prostaglandins, we tested the hypothesis that prostaglandins are involved in the CART-induced inhibition of gastric acid secretion. The present finding that centrally injected CART significantly suppressed acid secretion in rats pretreated with indomethacin excludes the prostaglandin pathway in the mechanism of action of CART.
CRF in the brain plays a role in the regulation of gastric acid
secretion. For example, intracisternal injection of CRF inhibits
gastric acid secretion in rats (17). CRF injected into the
cerebrospinal fluid mimics the effects of various stressors on gastric
acid secretion in rats (32, 33). It is furthermore demonstrated that
the CRF antagonist, -helical CRF9–41, injected into the
cerebrospinal fluid prevents stress-induced alternation of gastric
secretion (14, 34). Thus, brain CRF plays a role in mediating
stress-induced alternation of gastric secretory function. Considering
these evidence, we tested a hypothesis that CRF in the brain may
mediate the CART-induced inhibition of gastric acid secretion. The
present result indicates that the CART-induced inhibition of gastric
acid secretion was completely blocked by -helical CRF9–41. This
evidence suggests that endogenous CRF in the brain mediates the
CART-induced acid inhibition. The CART-induced inhibition of acid
did not depend on the vagal system because of a persistence of acid
inhibition by central injection of CART in vagotomized animals as
demonstrated in this study. Previous studies demonstrated that CRF in
the brain exerts its inhibitory action on gastric secretion via a vagus
independent pathway (12, 35). This evidence may furthermore support our
hypothesis that CRF in the brain mediates the CART-induced acid
inhibition through a vagus-independent pathway.
Vrang et al. (15) have injected CART into the cerebrospinal fluid in rats and examined its effect on c-fos expression in the brain. According to their data, CART induced c-fos expression in several hypothalamic and brain stem structures. In the hypothalamus, high number of c-fos immunoreactive cells were observed in the paraventricular nucleus of the hypothalamus (PVN) and in the posterior part of the dorsomedial hypothalamic nucleus. It has been shown that CRF neurons are distributed mainly in the PVN (36, 37, 38). In fact, c-fos positive elements in the PVN seen after CART injection were observed in the area where most of the CRF containing neurons are located (15). This evidence led us to speculate that centrally administered CART activates neurons in the PVN that contain CRF. This speculation may explain the mechanism by which CART acts in the brain to inhibit acid secretion through endogenous CRF. In other words, we suggest that CART acts in the CRF-containing neurons in the PVN, and then released CRF inhibits gastric acid secretion through a vagus-independent mechanism.
In summary, the present study suggests for the first time that CART may act in the brain to inhibit not only feeding but gastric function such as gastric acid secretion and gastric emptying. Endogenous CRF in the brain may mediate the acid inhibition by CART.
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Footnotes |
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Received January 21, 2000.
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