Gastric motor effects of peptide and non-peptide ghrelin agonists in mice in vivo and in vitro

Abstract

Background and aims: The gastroprokinetic activities of ghrelin, the natural ligand of the growth hormone secretagogue receptor (GHS-R), prompted us to compare the effect of ghrelin with that of synthetic peptide (growth hormone releasing peptide 6 (GHRP-6)) and non-peptide (capromorelin) GHS-R agonists both in vivo and in vitro.

Methods: In vivo, the dose dependent effects (1-150 nmol/kg) of ghrelin, GHRP-6, and capromorelin on gastric emptying were measured by the 14C octanoic breath test which was adapted for use in mice. The effect of atropine, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), or D-Lys3-GHRP-6 (GHS-R antagonist) on the gastroprokinetic effect of capromorelin was also investigated. In vitro, the effect of the GHS-R agonists (1 microM) on electrical field stimulation (EFS) induced responses was studied in fundic strips in the absence and presence of L-NAME.

Results: Ghrelin, GHRP-6, and capromorelin accelerated gastric emptying in an equipotent manner, with bell-shaped dose-response relationships. In the presence of atropine or l-NAME, which delayed gastric emptying, capromorelin failed to accelerate gastric emptying. D-Lys3-GHRP-6 also delayed gastric emptying but did not effectively block the action of the GHS-R agonists, but this may be related to interactions with other receptors. EFS of fundic strips caused frequency dependent relaxations that were not modified by the GHS-R agonists. L-NAME turned EFS induced relaxations into cholinergic contractions that were enhanced by ghrelin, GHRP-6, and capromorelin.

Conclusion: The 14C octanoic breath test is a valuable technique to evaluate drug induced effects on gastric emptying in mice. Peptide and non-peptide GHS-R agonists accelerate gastric emptying of solids in an equipotent manner through activation of GHS receptors, possibly located on local cholinergic enteric nerves.

Figure 1

Effect of ghrelin on gastric emptying in mice, as determined by the ¹⁴C octanoic breath test. Typical CO₂ excretion curves obtained after ingestion of a solid meal enriched with ¹⁴C octanoic acid, 30 minutes after intraperitoneal injection of ghrelin (75 nmol/kg) or 0.9% NaCl in the same mouse.

Figure 2

Intraindividual variations in gastric emptying parameters (T_1/2 and T_lag) in mice. Mice were injected with 0.9% NaCl (0.2 ml intraperitoneally) on five different experimental days (days 0, 3, 15, 25, and 35), 30 minutes before the start of the ¹⁴C octanoic breath test, and the effect on T_1/2 and T_lag was determined. Values are mean (SEM) of 12 mice. No significant differences were found.

Figure 3

Interindividual variations in gastric emptying parameters (T_1/2 and T_lag) in mice. T_1/2 and T_lag were determined from the CO₂ excretion curves in 36 mice after intraperitoneal injection of 0.9% NaCl 30 minutes before the start of the ¹⁴C octanoic breath test. Between mice the coefficient of variation (CV) was 32% for T_1/2 and 41% for T_lag, with means of 77 minutes and 39 minutes, respectively.

Figure 4

Effects of bethanechol, atropine, and N^G-nitro-L-arginine methyl ester hydrochloride (L-NAME) on gastric emptying parameters in mice. Mice underwent three consecutive breath tests consisting of subcutaneous injection of 0.9% NaCl followed by bethanechol (20 mg/kg), atropine (1 mg/kg), or L-NAME (50 mg/kg) and then 0.9% NaCl again. The effect on T_1/2 (A) and T_lag (B) was calculated from the CO₂ excretion curves of the control and drug treated mice. Results are mean (SEM) of 6–12 mice. *p<0.05, **p<0.01 versus saline treatment.

Figure 5

Dose dependent effects of ghrelin, growth hormone releasing peptide 6 (GHRP-6), and capromorelin on gastric emptying in mice. Mice were injected with increasing doses of ghrelin (1–150 nmol/kg), GHRP-6 (6–150 nmol/kg), or capromorelin (6–150 nmol/kg) 30 minutes before ingestion of a meal enriched with ¹⁴C octanoic acid and the effects on gastric emptying parameters, T_1/2 (A) and T_lag (B), were determined. Results are expressed as per cent decrease in time compared with injection of saline given before and after injection of the ghrelin agonists. Results are mean (SEM) of at least 12 mice. *p<0.05, **p<0.01, ***p<0.001 indicate significant decreases compared with saline treatment.

Figure 6

Effects of atropine (A) and N^G-nitro-L-arginine methyl ester hydrochloride (L-NAME) (B) on the gastroprokinetic actions of capromorelin in mice. Mice were pretreated with atropine (1 mg/kg) or L-NAME (50 mg/kg) 15 minutes before administration of capromorelin (75 nmol/kg) and the effect was compared with that of treatment with capromorelin and atropine or L-NAME alone in the same mice. Results are mean (SEM) of at least 12 mice.

Figure 7

Effects of D-Lys³-growth hormone releasing peptide 6 (GHRP-6) on gastric emptying and on the gastroprokinetic action of capromorelin in mice. The CO₂ excretion curves were obtained after ingestion of a solid meal enriched with ¹⁴C octanoic acid, 30 minutes after intraperitoneal injection of NaCl or capromorelin. To evaluate the effect of D-Lys³-GHRP-6, mice were pretreated for 15 minutes with D-Lys³-GHRP-6 (5.6 µmol/kg) before administration of capromorelin or saline. All experiments were performed in the same mice. CO₂ excretion curves are mean results of six mice.

Figure 8

Representative tracings of mechanical responses of mice fundic strips elicited by electrical field stimulation (0.5–32 Hz) under different conditions: (A) control; (B) in the presence of N^G-nitro-L-arginine methyl ester hydrochloride (L-NAME 300 μM); (C) L-NAME plus atropine (5 μM); and (D) L-NAME plus atropine plus tetrodotoxin (1 μM).

Figure 9

Effects of ghrelin (A), growth hormone releasing peptide 6 (GHRP-6) (B), and capromorelin (C) on electrical field stimulation induced mechanical responses of mice fundic strips. Muscle strips were electrically stimulated (0.5–32 Hz) preceding and during incubation with N^G-nitro-l-arginine methyl ester hydrochloride (L-NAME 300 µM) in the absence or presence of ghrelin (1 µM), GHRP-6 (1 µM), or capromorelin (1 µM), and the tension of the responses was measured. Results are mean (SEM) of five strip preparations from different mice. *p<0.05, **p<0.01 versus the frequency spectrum in the presence of L-NAME in the same strip preparation.