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Endocrinology Vol. 138, No. 9 3749-3755
Copyright © 1997 by The Endocrine Society

ARTICLES

Central Action of Adrenomedullin to Inhibit Gastric Emptying in Rats1

V. MartÍnez, F. Cuttitta and Y. Taché

CURE: Digestive Diseases Research Center, West Los Angeles Veterans Administration Medical Center, Department of Medicine and Brain Research Institute, University of California School of Medicine, Los Angeles, California 90073; Biomarkers and Prevention Research Branch, Division of Cancer Prevention and Control, National Cancer Institute, National Institutes of Health (F.C.), Rockville, Maryland 20850-3300

Address all correspondence and requests for reprints to: Vicente Martínez, D.V.M., Ph.D., West Los Angeles Veterans Administration Medical Center, Building 115, Room 203, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: vmartine{at}ucla.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The central action of human adrenomedullin (AM) to influence gastric emptying and the peripheral mechanisms involved were studied in conscious rats. The 20-min rate of gastric emptying of a methylcellulose solution was assessed after intracisternal (ic) injection of AM or rat {alpha}-calcitonin gene-related peptide ({alpha}CGRP). AM and {alpha}CGRP dose-dependently inhibited gastric emptying with ic ED50 values of 120 and 100 pmol, respectively. Human proadrenomedullin N-terminal 20 peptide (150–600 pmol, ic) and AM (150 pmol, iv) had no effect. The inhibitory actions of AM and {alpha}CGRP (150 pmol, ic) were completely blocked by the CGRP antagonist, human CGRP-(8–37) injected ic at 30 µg, but not at 15 µg. The CRF antagonist, [D-Phe12,Nle21,38,C{alpha}MeLeu37]CRF-(12–41) (10 µg/rat) injected ic prevented ic rat/human CRF (150 pmol)-induced 53% inhibition of gastric emptying while not modifying the effect of AM. The action of AM (150 pmol, ic) was abolished by bilateral adrenalectomy or the ß-adrenergic blocker, propranolol (1 mg/kg, ip), but was not altered by indomethacin (5 mg/kg, ip) or subdiaphragmatic vagotomy. These results indicate that ic AM and {alpha}CGRP equipotently inhibit gastric emptying through mechanisms similarly antagonized by a high dose of CGRP-(8–37). The central AM action is mediated through adrenal-dependent, ß-adrenergic pathways independently from activation of central CRF receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADRENOMEDULLIN (AM) is a newly identified peptide originally isolated from a human pheochromocytoma by monitoring its stimulatory action on platelet cAMP production (1). Cloning from human, rat, and porcine complementary DNAs coding for prepro-AM showed that the structure of AM is highly homologous across species, with 52 amino acids in the human and pig and 50 residues in the rat (2). AM is considered to be a member of the calcitonin gene-related peptide (CGRP) superfamily, including CGRP, calcitonin, and amylin (3). These peptides have the 6- to 7-amino acid N-terminal ring structure in common and the amidated C-terminus as well as overlapping biological effects (2, 3). In particular, peripheral administrations of CGRP and AM have similar hypotensive actions due to the powerful vasodilation in several vascular beds in various species (2, 3, 4).

Several peptides influence gastric motor function upon administration into the brain through modulation of the autonomic nervous system activity to the gastrointestinal tract (5, 6). In particular, neuroanatomical and pharmacological studies using CRF receptor antagonists such as {alpha}-helical CRF-(9–41), [D-Phe12]CRF-(12–41), or astressin established the physiological relevance of brain CRF in mediating stress-induced gastric stasis (7, 8, 9). A few studies indicate that peptides related to the CGRP superfamily, namely {alpha}CGRP, calcitonin, and amylin, inhibit gastric emptying upon central injection in rats (10, 11, 12). The central action of AM to influence gut function has not yet been explored. However, AM messenger RNA is expressed in the rat brain (13), and AM immunoreactivity is localized in the hypothalamus and medulla oblongata (13, 14). Specific binding sites for AM are present in the hypothalamus and brainstem (15, 16). The AM receptor was recently cloned from the rat lung complementary DNA (17). Pharmacological characterization of rat AM and human CGRP receptors in transfected cells and various tissue membranes, including brain membranes, indicates that AM displays a highly specific recognition over CGRP at the AM receptor and a cross-reactivity with the CGRP1 receptor subtype (13, 15, 16, 17, 18, 19, 20). Taken together, these observations suggest a possible central action of AM on gastric motor function.

Subsequent analysis of the AM precursor revealed that proteolytic cleavage and amidation of pro-AM results, in addition to AM, in a unique 20-residue peptide, termed proadrenomedullin N-terminal 20 peptide [PAMP, or pro-AM-(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)] (21). PAMP immunoreactivity was distributed similarly to that of AM (22). Specific binding sites for PAMP have been identified recently in various rat tissues, including the brain, although in distinct locations from AM binding sites (23). PAMP is structurally unrelated to the CGRP superfamily, does not stimulate cAMP (23), and displays a transient hypotensive effect when injected peripherally related to the inhibition of sympathetic neural transmission at nerve endings (24, 25).

Therefore, the present study was designed to examine in conscious rats 1) whether the novel peptide AM, which is related to the CGRP superfamily, shares a similar central action as {alpha}CGRP to inhibit gastric emptying upon intracisternal (ic) injection; 2) to test the influence of ic injection of PAMP, the AM-gene related peptide, which is structurally unrelated to the CGRP superfamily; 3) to establish whether the inhibitory actions of {alpha}CGRP and AM are sensitive to the antagonistic effect of ic injection of CGRP-(8–37); 4) to assess whether AM action is secondary to activation of central CRF receptors; and 5) to define the neurohumoral pathways involved in AM action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague-Dawley rats (Harlan, San Diego, CA), weighing 240–280 g, were used. Animals were housed in group cages with free access to food (Purina rat chow, Ralston Purina, St. Louis, MO) and tap water and were maintained under controlled conditions of lighting (12-h light, 12-h dark cycle) and temperature (21–23 C). Animals were fasted, but had free access to water (or water containing 0.9% NaCl and glucose after adrenalectomy) for 18–20 h before gastric emptying studies. Experiments were performed between 1100–1500 h and were conducted under V.A. Animal Component of Research Protocol number 96-080-08.

Drugs and treatments
Human AM and human PAMP (Peptide Institute, Osaka, Japan) were dissolved in distilled water to a 1.35 x 10-4 M solution. Ten-microliter aliquots of the stock solution were stored at -70 C. Further dilutions were performed in 0.9% sterile saline (Sigma Chemical Co., St. Louis, MO) before use. Rat {alpha}CGRP and human/rat CRF (Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA) and the CGRP antagonist, human CGRP-(8–37) (University of Quebec, Montreal, Canada), were kept in powder form at -70 C and dissolved in sterile saline immediately before the experiments. The CRF antagonist, [D-Phe12]CRF-(12–41) ([D-Phe12,Nle21,38,C{alpha}MeLeu37]CRF-(12–41); The Salk Institute) was kept in powder form at -70 C and dissolved in distilled water (pH 7.0, warmed to 37 C) before use. Indomethacin (Sigma) was dissolved in 1% sodium bicarbonate; propranolol hydrochloride (Sigma) and phentolamine mesylate (Ciba Pharmaceutical Co., Summit, NJ) were dissolved in saline.

Intracisternal injections were performed acutely under short enflurane anesthesia (2–3 min; 5.5% vapor concentration in O2; Ethrane-Anaquest, Madison, WI) by puncture of the occipital membrane with a 50-µl Hamilton syringe (Hamilton, Reno, NV) in rats placed in ear bars of a stereotaxic equipment. The presence of cerebrospinal fluid in the Hamilton syringe upon aspiration before injection insured correctness of needle placement into the cisterna magna. After the injection, animals were returned to their home cages for the duration of the experiment. The total volume of ic injection was 10 µl either as a single injection or as two consecutive 5-µl injections. Intravenous injections were performed acutely under short enflurane anesthesia (5 min) by injection of 0.1 ml into the jugular vein. Intraperitoneal injections were performed in 0.5 ml. Unless otherwise stated, all doses represent dose per rat.

Measurement of gastric emptying
Gastric emptying was determined by the phenol red method, as previously described (8). A suspension of continuously stirred 1.5% methylcellulose (Sigma Chemical Co.) and phenol red (0.5%; Sigma Chemical Co.) was given intragastrically (1.5 ml) to conscious rats. After a 20-min period, rats were killed by CO2 inhalation. The abdominal cavity was opened, the gastroesophageal junction and the pylorus were clamped, and the stomach was removed, rinsed in 0.9% saline, and placed in 100 ml of 0.1 N NaOH and homogenized (Polytron, Brinkmann Instruments, Westbury, NY). The suspension was allowed to settle for 1 h at room temperature, and 5 ml of the supernatant were added to 0.5 ml of 20% trichloroacetic acid (wt/vol) and then centrifuged at 3000 rpm at 4 C for 20 min. The supernatant was mixed with 4 ml of 0.5 N NaOH, and the absorbance of the sample was read at 560 nm (Shimadzu UV-260, Cole Scientific, Moorpark, CA). Phenol red recovered from animals killed immediately after administration of the methylcellulose solution was used as the standard (0% emptying). The percent emptying during the 20-min period was calculated according to the following equation: gastric emptying (%) = 1 - (absorbance of test sample/absorbance of standard) x 100.

Experimental protocols
Effects of AM, {alpha}CGRP, or PAMP injected ic and AM injected iv.
Rats were injected ic with saline, AM (30, 75, or 150 pmol), {alpha}CGRP (30, 75, or 150 pmol), or PAMP (150, 300, or 600 pmol). Other groups were injected iv with either saline or AM (150 pmol). Ten minutes later, the 20-min rate of gastric emptying was measured.

Effects of ic CGRP antagonist or CRF antagonist.
Rats were injected ic with saline or CGRP-(8–37) (15 or 30 µg) and immediately thereafter, with saline, {alpha}CGRP (150 pmol), or AM (150 pmol). In another study, rats were injected ic with vehicle (distilled water, pH 7.0) or [D-Phe12]CRF-(12–41) (10 µg) and immediately thereafter with saline, CRF (150 pmol), or AM (150 pmol). Ten minutes after the ic injections, the 20-min rate of gastric emptying was measured.

Effects of indomethacin, vagotomy, adrenalectomy, and adrenergic blockade.
The following pretreatments were performed: indomethacin (5 mg/kg) or vehicle (1% sodium bicarbonate) injected ip 60 min before the experiments; propranolol (1 mg/kg), phentolamine (1 mg/kg), or saline injected ip 15 min before the experiments; subdiaphragmatic vagotomy (achieved by a circular seromuscular myotomy of the esophagus, about 2 cm from the gastroesophageal junction) or sham operation (laparotomy and manipulation of the stomach); and bilateral adrenalectomy or sham operation (laparotomy and manipulation of abdominal viscera). Subdiaphragmatic vagotomy and adrenalectomy were performed 48 and 24 h before the experiments, respectively, in fasted rats under ketamine hydrochloride (75 mg/kg, ip; Ketaset, Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (5 mg/kg, ip; Rompun, Mobay Co., Shawnee, KS) anesthesia. All pretreated groups were injected ic with either saline or AM (150 pmol), and 10 min later, the 20-min rate of gastric emptying was determined.

Statistical analysis
Results are expressed as the mean ± SE. Comparisons between groups were performed using one-way ANOVA followed by a Student-Newman-Keuls multiple comparison test. When the effects of two treatments and their reciprocal interaction was studied, data were analyzed by a two-way ANOVA with replication. When the two-way ANOVA revealed significant effects of the treatments, data were reanalyzed using one-way ANOVA followed by a Student-Newman-Keuls multiple comparison test to determine differences among groups. P < 0.05 was considered statistically significant. The mean effective dose (ED50), defined as the dose of peptide that induced a 50% inhibition of gastric emptying compared with the rate of emptying in vehicle-treated animals (taken as 0% inhibition), was determined by nonlinear regression (Prism, version 2.0, GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of AM, {alpha}CGRP, or PAMP injected ic and AM injected iv
In rats injected ic with saline, the 20-min rate of gastric emptying of a nonnutrient viscous solution was 59.8 ± 4.1%, as assessed during the 10- to 30-min period after the ic injection (n = 11; Fig. 1Go). AM or {alpha}CGRP injected ic induced a dose-dependent inhibition of gastric emptying, with ED50 values of 120 pmol (95% confidence interval, 86–167 pmol; r2 = 0.99) and 100 pmol (95% confidence interval, 65–155 pmol; r2 = 0.979), respectively (Fig. 1Go). AM and {alpha}CGRP injected ic at 150 pmol inhibited gastric emptying by 56–59% compared with that in the vehicle group. By contrast, AM (150 pmol) injected iv did not influence the rate of gastric emptying [vehicle, 44.8 ± 5.8% (n = 6); AM, 53.4 ± 8.6% (n = 6); P > 0.05; data not shown]. The ic injection of PAMP (150, 300, and 600 pmol) failed to produce significant changes in gastric emptying (Fig. 1Go).



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  		Figure 1. Effect of ic injection of {alpha}CGRP, AM, and PAMP on gastric emptying in conscious rats. Rats were injected ic (10 µl) with vehicle, rat {alpha}CGRP, human AM, or human PAMP, and 10 min later, the 20-min rate of gastric emptying of a nonnutrient solution was measured. Data are expressed as the mean ± SE of the rat number shown on the top of the columns. *, P < 0.05 vs. vehicle.

 
Effects of ic CGRP antagonist or CRF antagonist
{alpha}CGRP and AM injected ic at 150 pmol inhibited gastric emptying to 17.8 ± 5.5% and 18.6 ± 5.1%, respectively, compared with 60.8 ± 8.9% in ic vehicle-treated rats (Fig. 2Go). The CGRP antagonist, CGRP-(8–37) injected ic at a dose of 15 or 30 µg did not modify the basal rate of gastric emptying (Fig. 2Go). The CGRP antagonist injected ic at 15 µg induced a nonsignificant attenuation of {alpha}CGRP- and AM-induced inhibition of gastric emptying [CGRP-(8–37) plus CGRP, 28.5 ± 3.9% (n = 3); CGRP-(8–37) plus AM, 35.6 ± 8.0% (n = 3)], whereas at 30 µg, there was a complete reversal of the {alpha}CGRP and AM inhibitory effect [CGRP-(8–37) plus CGRP, 67.9 ± 12.1%; CGRP-(8–37) plus AM, 65.4 ± 12.2%; n = 4 for each group; P < 0.05; Fig. 2Go].



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  		Figure 2. Reversal of intracisternal AM- and {alpha}CGRP-induced inhibition of gastric emptying by ic injection of the CGRP-1 antagonist, CGRP-(8–37) in conscious rats. Human CGRP-(8–37) was injected ic (5 µl) immediately before ic injection of vehicle, rat {alpha}CGRP (150 pmol), or human AM (150 pmol); 10 min later, the 20-min rate of gastric emptying of a nonnutrient solution was measured. Data are expressed as the mean ± SE of the rat number shown on top of the columns. *, P < 0.05 vs. vehicle treatment or respective peptide plus CGRP-(8–37) (30 µg) [ANOVA, F(8,25) = 7.503; P < 0.0001].

 
CRF or AM injected ic at 150 pmol inhibited gastric emptying to 25.0 ± 6.9% and 17.3 ± 7.2%, respectively, compared with 53.2 ± 4.9% in ic vehicle-treated rats (Fig. 3Go). The ic injection of the CRF antagonist, [D-Phe12]CRF-(12–41) (10 µg), had no effect on the basal rate of gastric emptying by itself and blocked ic CRF (150 pmol)-induced delayed gastric emptying (Fig. 3Go). By contrast, ic injection of [D-Phe12]CRF-(12–41) (10 µg) did not modify the inhibitory action of ic AM ([D-Phe12]CRF-(12–41) plus AM, 20.9 ± 6.0%; n = 6; P > 0.05 vs. vehicle plus AM; Fig. 3Go).



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  		Figure 3. Influence of intracisternal injection of the CRF antagonist, [D-Phe12,Nle21,38,C{alpha}MeLeu37]CRF-(12–41), [D-Phe12]CRF-(12–41), on ic CRF- and AM-induced inhibition of gas-tric emptying in conscious rats. [D-Phe12]CRF-(12–41) was injected ic immediately before the ic injection (5 µl) of vehicle, rat/human CRF (150 pmol), or human AM (150 pmol); 10 min later, the 20-min rate of gastric emptying was determined. Data are expressed as the mean ± SE of the rat number shown on top of the columns. *, P < 0.05 vs. vehicle plus vehicle; [D-Phe12]CRF-(12–41) plus vehicle or CRF plus [D-Phe12]CRF-(12–41) [by ANOVA, F(5,20) = 6.278; P = 0.0012].

 
Effects of indomethacin, vagotomy, adrenalectomy, or adrenergic blockade
The delayed gastric emptying induced by AM (150 pmol, ic) was not modified by pretreatment with indomethacin (5 mg/kg, ip; -60 min). Indomethacin by itself did not influence the rate of gastric emptying (Table 1Go).


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  		Table 1. Effect of indomethacin and vagotomy on ic AM-induced inhibition of gastric emptying in conscious rats

 
Subdiaphragmatic vagotomy 48 h before the experiments significantly decreased the rate of gastric emptying in the ic vehicle control group (Table 1Go). The rate of gastric emptying after ic AM (150 pmol) was similar in vagotomized compared with sham-operated animals (Table 1Go). However, taking into consideration the differences in basal emptying, AM inhibited gastric emptying by 62.4 ± 3.3% and 46.2 ± 6.3% in sham and vagotomized animals, respectively; these results are not significantly different (P > 0.05).

Bilateral adrenalectomy 24 h before the experiments completely reversed the inhibitory effect of ic AM (150 pmol) compared with that in sham-operated animals (percent gastric emptying: adrenalectomy plus AM, 57.0 ± 6.6%; sham operation plus AM, 21.2 ± 2.2%; n = 4–5; P < 0.05). Adrenalectomy produced a small, but nonsignificant, rise in the rate of gastric emptying (Fig. 4Go).



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  		Figure 4. Reversal by adrenalectomy or ß-adrenergic blockade of ic AM-induced inhibition of gastric emptying in conscious rats. Bilateral adrenalectomy was performed 24 h before the experiments. Human AM (150 pmol) or vehicle was injected ic (10 µl), and 10 min later, the 20-min rate of gastric emptying was determined. ß-Adrenergic blockade was achieved by pretreatment with propranolol 15 min before the ic injection of AM (150 pmol). Data are expressed as the mean ± SE of the rat number shown on top of the columns. *, P < 0.05 vs. all other groups.

 
Blockade of ß-adrenergic receptors with propranolol (1 mg/kg, ip) completely prevented ic AM-induced inhibition of gastric emptying (percent gastric emptying: propranolol plus AM, 54.8 ± 1.1%; vehicle plus AM, 19.5 ± 4.5%; n = 5–6; P < 0.05; Fig. 4Go). Pretreatment with propranolol increased the rate of gastric emptying by 20% compared with that in vehicle-treated animals, although this value was not statistically significant (Fig. 4Go). Phentolamine (1 mg/kg, ip) by itself reduced the rate of gastric emptying to 24.3 ± 6.4 (n = 4; P < 0.05 vs. vehicle-treated animals). Under these conditions, the inhibitory effect of AM (150 pmol, ic) was not modified (vehicle plus AM, 19.5 ± 4.5; phentolamine plus AM, 21.9 ± 5.7; n = 4–5; P > 0.05).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results show that ic injection of human AM inhibits gastric emptying of a nonnutrient solution in conscious rats with an estimated ED50 of 120 pmol. Peptides injected into the cerebrospinal fluid can leak or be transported into the peripheral circulation (26). However, it is most likely that AM acts in the central nervous system, as the peptide injected iv at 150 pmol did not modify the rate of gastric emptying. These results provide the first demonstration that AM acts in the brain to influence gastric motor function.

These data also indicate that AM shares a similar central action to influence gastric function as other peptides of the CGRP superfamily. Rat calcitonin, rat {alpha}CGRP, and amylin administered into the lateral brain ventricle (icv) inhibit gastric emptying of a nonnutrient meal in conscious rats with ED50 values of 10, 100, and 600 pmol, respectively (11, 12). Likewise, in the present study, rat {alpha}CGRP injected into the cisterna magna delays gastric emptying, as previously reported (10). Based on the similar ED50 for AM (120 pmol) and {alpha}CGRP (100 pmol), both peptides appear to be equipotent to suppress gastric emptying upon ic injection. The lack of effect of human PAMP, the structurally unrelated product of the human AM gene (24), when injected ic at doses up to 600 pmol shows the peptide specificity of AM action. Although PAMP immunoreactivity and binding sites are present in the brain (23, 24), its central biological actions remain elusive.

CGRP-(8–37) injected ic blocked the inhibition of gastric emptying in response to ic AM. The dose at which the complete reversal was observed required a 60-fold molar excess of CGRP-(8–37) over that of AM, whereas a 30-fold excess was subthreshold. The recent pharmacological characterization of the cloned rat AM receptor in transfected cells demonstrates the highly specific recognition of AM over CGRP at the AM receptor and a weak antagonistic effect of AM by CGRP-(8–37) with a Ki of 10-6 M (17). Likewise, rat hypothalamic binding assays indicate that human AM (10-10-10-8 M), unlike rat {alpha}CGRP-(8–37) (10-6 M), competes for labeled AM binding sites (16). In addition to binding to its own receptor, AM cross-reacts with the cloned dog CGRP-1 receptor subtype (18, 19). In the rat hypothalamic binding assay, human AM competes for the labeled rat {alpha}CGRP-binding sites with an order of potency showing rat {alpha}CGRP > CGRP-(8–37) > human AM (13, 16). Therefore, based on the reversal by ic CGRP-(8–37), the present results would be consistent with an AM action exerted at CGRP-binding sites sensitive to competition by CGRP-(8–37). However, both rat {alpha}CGRP and human AM are equipotent to inhibit gastric emptying and similarly sensitive to the antagonistic action of CGRP-(8–37). Therefore, these observations are difficult to reconcile with previously established affinities of AM, CGRP, and CGRP-(8–37) on AM or CGRP-1 receptors (16, 27, 28). In addition, the ratio of {alpha}CGRP/CGRP-(8–37) at which the reversal is observed is higher than that reported for icv {alpha}CGRP to suppress food intake or induce pressor responses (1:1 or 1:17, respectively) (28, 29). Different antagonistic potencies of CGRP-(8–37) in various in vitro bioassays have been used to postulate the existence of multiple CGRP receptor subtypes (28). In particular, membrane studies in the rat nucleus accumbens revealed the existence of a unique class of sites possessing high affinity binding for CGRP, amylin, and salmon calcitonin, suggesting the existence of a CGRP receptor subtype 3 (30, 31). Based on these observations, it may be speculated that a receptor subtype(s) with similar high affinity for human AM and rat {alpha}CGRP is involved. The pharmacological characterization of AM/CGRP receptor subtypes involved would benefit from advances in the development of more specific antagonists and the cloning of CGRP receptor subtypes (32).

CGRP and CRF share a similar spectrum of central actions related to the inhibition of gastric acid secretion and food intake, protection against ethanol lesions, and increased sympathetic activity and mean arterial blood pressure (33, 34, 35, 36, 37). A physiological role of endogenous brain CRF to inhibit gastric emptying has been established under stress conditions in rats (7, 9). Neuroanatomical evidence also indicates that 35% of CRF neurons in specific brain areas receive direct input from CGRP nerve terminals (38), suggesting that activation of CGRP receptors may activate CRF neurons. However, ic injection of the CRF antagonist [D-Phe12]CRF-(12–41) did not modify the 50% inhibition of gastric emptying induced by ic AM while blocking ic CRF-induced similar inhibition of gastric emptying, as previously reported (8). These results indicate that gastric stasis induced by ic AM is not mediated by central CRF receptors and is not secondary to a nonspecific stress-related effect. Likewise, although AM induces a sustained hypertensive effect upon central injection, these alterations do not play a role as the ic doses inhibiting gastric emptying are lower than those producing vascular changes (39, 40). For example, in rats, icv injection of AM at doses up to 176 pmol failed to induce any change in mean arterial blood pressure or heart rate, while inhibiting water intake (40) and gastric emptying (present study).

Adrenalectomy completely abolished and vagotomy tended to attenuate, although not significantly, ic AM-induced gastric stasis. Likewise, {alpha}CGRP injected ic induced delayed gastric transit was completely reversed by adrenalectomy and slightly attenuated by vagotomy (10). Moreover, the central AM effect was blocked by propranolol, suggesting an action mediated through adrenergic ß-receptors. Similar ß-adrenergic mechanisms have been shown to mediate ic injection of {alpha}CGRP-induced decreased gastric motility (41). AM injected ic stimulates abdominal sympathetic nerve activity, and icv injection of {alpha}CGRP increases catecholamine release (29, 33, 39). Taken together, these findings indicate that AM injected ic inhibits gastric emptying through sympathetic adrenal-dependent ß-adrenoreceptors, as previously reported for ic injection of {alpha}CGRP (10, 41). By contrast, {alpha}CGRP injected icv was previously reported to inhibit gastric emptying through vagal-dependent pathways, whereas noradrenergic mechanisms were not involved (11). Such a difference may be related to different brain areas (hypothalamic vs. brainstem) at which {alpha}CGRP or related peptides act upon icv compared with ic injection (42). The exact brain sites involved in ic AM- and {alpha}CGRP-induced inhibition of gastric emptying need to be established. Possible responsive brainstem nuclei may include the area postrema/nucleus tractus solitarius and/or the trigeminal subnucleus caudalis based on the responsiveness of these sites to direct injection of AM and {alpha}CGRP (43, 44) and their projections to influence sympathetic outflow (43, 45, 46).

Unlike ic injection of other peptides, such as interleukin-1ß (47), AM action does not involve PG mechanisms. The vagal- and PG-independent mechanism of action of ic AM to inhibit gastric emptying contrasts with the cholinergic PG pathways mediating ic AM-induced gastric cytoprotection against ethanol lesions in rats (48). Interestingly, in these studies, the effect of ic AM was not blocked by ic CGRP-(8–37) at 30 µg, suggesting modulation by distinct receptors resulting in activation of different efferent pathways. Differences in the peripheral neurohumoral pathways mediating motor- vs. secretory-related events have been observed for several centrally acting neuropeptides (10, 36). In particular, upon ic injection of {alpha}CGRP, the gastric motor alterations, as described above, depend mainly on sympatho-adrenal components, whereas the secretory effects are not mediated by the sympathetic nervous system (10, 36).

CGRP-(8–37) injected ic at a dose that prevented AM- or {alpha}CGRP-induced delayed gastric emptying did not alter basal rate of gastric emptying in rats. Consistent with these findings, no agonist action of CGRP-(8–37) was reported upon icv injection at doses up to 80 µg (28). These results suggest that endogenous AM and {alpha}CGRP do not modulate the basal rate of gastric emptying in rats. Similar observations have been reported for other centrally acting peptides such as CRF, which does not play a role in basal but is involved in stress-related alterations of gastric emptying (7, 8, 9). Therefore, these data do not preclude a possible regulatory role of AM under pathophysiological conditions. The elevation of circulating AM and gene transcription by endotoxin (49) may be involved in endotoxin-induced gastric stasis (50), as AM influences neuronal activity in the area postrema (44), and humoral stimuli are conveyed via vascular channels to the area postrema and part of the nucleus tractus solitarius (44, 51).

In summary, our study demonstrates for the first time that human AM injected ic in picomole amounts, acts in the brain to inhibit gastric emptying. By contrast, human PAMP, the structurally unrelated product of the human AM gene, injected ic up to 4-fold higher doses had no effect. Compared with {alpha}CGRP, AM injected ic displayed a similar potency, sensitivity to the antagonistic action of CGRP-(8–37), and sympathetic mediation through adrenal-dependent ß-adrenergic mechanisms. The central action of AM is not secondary to activation of the CRF receptor-induced increase in sympathetic outflow. These results indicate that AM and {alpha}CGRP act via common mechanisms. The CGRP receptor subtypes involved in the central actions of AM and {alpha}CGRP to induce gastric stasis and the pathophysiological significance remain to be established.


   Acknowledgments
 
The authors thank Dr. Jean Rivier (The Salk Institute, La Jolla, CA) for the generous supply of rat CRF, rat {alpha}CGRP, and the CRF antagonist, and Dr. Serge St. Pierre (University of Quebec, Canada) for the generous supply of the CGRP antagonist. Mr. P. Kirsh is acknowledged for helping with the preparation of the manuscript.


   Footnotes
 
1 This work was supported by NIDDK Grants DK-33061 and DK-41301 (Center Grant, Animal Core) and NIMH Grant MH-00663. Back

Received March 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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