# Activation of protease-activated receptor-2 increases in vitro jejunal muscle responses to cholinergic and tachykinergic stimulation in rats

Nadège Chambert, Rafael Garcia Villar, Nicolas Cenac, Bruno Guiard, Jean

Fioramonti, Lionel Bueno

► To cite this version:

Nadège Chambert, Rafael Garcia Villar, Nicolas Cenac, Bruno Guiard, Jean Fioramonti, et al. Activation of protease-activated receptor-2 increases in vitro jejunal muscle responses to cholinergic and tachykinergic stimulation in rats. 18. International Symposium on Neurogastroenterology and Motility, Sep 2001, Madison, United States. 2001, Neurogastroenterol Mot 13 (4 Suppl.). [hal-01600694](https://hal.science/hal-01600694)

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Abstracts of papers accepted for presentation
at the XVIII International Symposium on
Neurogastroenterology and Motility

16–20 September, Madison, WI, USA
higher ($P < 0.05$) than in the control (49.3 ± 5.3%). There were no differences in the dye recovery from proximal, mid or distal small intestine between MI and control rats, respectively. 27.5 ± 5.2 vs. 31.8 ± 5.5%, 17.2 ± 5.6 vs. 15.8 ± 7.1% and 1.65 ± 0.6 vs. 3.2 ± 3.8%. Central venous pressure in MI rats (3.8 ± 0.5 cmH2O) was higher ($P < 0.05$) than in the control group (0.3 ± 0.5 cmH2O). There were no differences between the infarcted and control rats either in arterial pressure (95 ± 7.5 vs. 94.7 ± 8.6 mmHg) or heart rate values (429.6 ± 31.8 vs. 417 ± 32.8 bpm). The results show that acute MI delays gastric emptying of liquid in awake rats.

### ACKNOWLEDGMENTS

Financial support from CNPq and FUNCAP.

### REFERENCE

1 *Circulation* 1981; 63: 960.

### Interleukin (IL)-1β-induced damage to intracellular LOS Ca²⁺ stores in acute experimental oesophagitis (AO) in the cat

W. CAO, L. CHENG, K. M. HARNETT, J. BEHAR & P. BIANCANI

R.I. Hospital and Brown University, Providence RI

HCl-induced experimental acute oesophagitis (AO) reduces *in vivo* lower oesophageal (LOS) resting pressure, *in vitro* tone, and contraction of LOS smooth muscle cells in response to IP3 and thapsigargin, suggesting that AO affects releasable intracellular Ca²⁺ stores. IL-1β reduces LOS tone and mimics the changes induced by oesophagitis. We therefore measured cytosolic Ca²⁺ levels in response to a maximally effective dose (Emax) of ACh in Fura 2-loaded LOS cells from normal [N] and oesophagitis [E] animals and after 2 h exposure to 100 U mL⁻¹ IL-1β (IL). In Fura 2-loaded N cells Emax ACh caused contraction and increased cytosolic Ca²⁺ levels. In N cells incubated in normal Ca²⁺ medium cytosolic Ca²⁺ increased from 93 ± 8 to 537 ± 54 nM [n = 8]. In Ca²⁺-free medium, cytosolic Ca²⁺ increased from 59 ± 7 to 483 ± 57 nM [n = 5]. The amplitude of the Ca²⁺ increase was not different in normal [444 nM] and in Ca²⁺-free medium [424 nM], confirming that LOS contraction induced by Emax ACh depends on intracellular Ca²⁺ release. In E cells Emax ACh increased cytosolic Ca²⁺ by 259 nM [from 43 ± 6 to 287 ± 28 nM, n = 7] in normal Ca²⁺ medium. The cytosolic Ca²⁺ increase was reduced more than 50% ($P < 0.05$) to 103 nM [from 44 ± 3 to 147 ± 51 nM, n = 9] in Ca²⁺-free medium, confirming that releasable Ca²⁺ stores are reduced by oesophagitis. In IL cells the response to Emax ACh was similar to that in E cells, as cytosolic Ca²⁺ increased by 297 nM [from 70 ± 10 to 367 ± 49 nM, n = 9] in normal Ca²⁺ and by 144 nM [from 44 ± 3 to 188 ± 95 nM, n = 5] in Ca²⁺-free medium. In addition, IL-1β significantly increased ($P < 0.05$) the production of hydrogen peroxide [H₂O₂]. Exposure of N cells to H₂O₂ produced results similar to those observed in E cells and in IL cells. These data suggest that IL-1β may contribute to the damage to LOS intracellular Ca²⁺ stores observed in acute experimental oesophagitis, possibly by increasing production of H₂O₂.

### ACKNOWLEDGMENT

Supported by NIH RO1-DK57030.

### Activation of protease-activated receptor-2 increases *in vitro* jejunal muscle responses to cholinergic and tachykinergic stimulation in rats

N. CHAMBERT, R. GARCIA-VILLAR, N. CENAC, B. GUIARD, J. FIORAMONTI

& L. BUENO

INRA Neuro-Gastroenterology and Nutrition Unit, Toulouse, France

Proteinase-activated receptors-2 (PAR-2) have been identified throughout the gastrointestinal tract. Activated by cleavage of a tethered peptide sequence by trypsin or mast cell tryptase, PAR-2 have been assigned a role in neuro-immune modulation. The PAR-2-activating synthetic peptide SLIGRL mimics the action of the endogenous protease in rats. The present study was aimed at investigating whether activation of jejunal PAR-2 may lead to an alteration of *in vitro* contractility responses to cholinergic and tachykinergic stimulation in rats. Methods. Ten male Wistar rats (200–250 g) were used for this purpose. Eight full-thickness strips of jejunum were collected from each rat and placed in the direction of longitudinal

fibres in an 8-chamber superfusion system (Krebs, 37 °C, 2 mL min⁻¹) for *in vitro* contractility studies (isometric system). Four strips were used to investigate the contractile responses to porcine trypsin (1, 4 and 10 µM), SLIGRL (10, 40, 100 and 400 µM), and the inactive reverse peptide LSIGRL (100 and 400 µM). The other four strips were used to construct concentration-response curves to carbachol (1 nM to 0.1 mM) and neurokinin A (NKA; 0.1 nM to 0.1 µM) before and after application of either porcine trypsin (4 µM), SLIGRL (400 µM), or LSIGRL (400 µM). For both neurotransmitters, potency was expressed as the EC₅₀ (confidence interval 95%; nM) and maximal contractile effect as the Emax (in percentage of the response induced by 60 nM KCl in the same strip). Emax values are means ± SEM (n = 10) *Results*. At the concentrations used both SLIGRL and LSIGRL did not induce any contractile response of jejunal strips. In contrast, trypsin (4 and 10 µM) produced a transient biphasic response: short inhibition followed by concentration-related contraction. Carbachol and NKA induced concentration-related contractile responses in controls and treated strips: EC50s which, overall, ranged from 130 to 220 nM for carbachol and 67–93 nM for NKA, were not different between all groups ($P > 0.05$); in contrast Emax were significantly enhanced in SLIGRL-treated strips (269 ± 19%, $P < 0.001$ and 242 ± 20%, $P < 0.001$ for carbachol and NKA, respectively) and trypsin-treated strips (236 ± 16%, $P < 0.01$ and 268 ± 18%, $P < 0.001$, respectively) but not in LSIGRL-treated strips ($P > 0.05$), compared to controls (196 ± 8% and 178 ± 13%, respectively). Conclusion. This study shows that *in vitro* activation of PAR-2 receptors by SLIGRL or trypsin enhances intestinal muscle responsiveness to cholinergic and tachykinergic stimulation. These data bring support for a role of PAR-2 located at the digestive tract in the pathophysiological regulation of intestinal contractility.

### Intracisternal ghrelin stimulates gastric contractility via vagal pathway

C.-Y. CHEN, M. MILLION, D. ADELSON, S. ST-PIERRE* & Y. TACHÉ

Division of Digestive Diseases, Department of Medicine, UCLA and CURE, VAGLAHS, Los Angeles, California

*PQ Canada

**Background.** Ghrelin (Ghr), a novel 28-amino-acid acylated peptide, recently isolated from rat and human stomach, is identified as the endogenous ligand for the growth hormone secretogogue receptor.¹ It is well known that many gastrointestinal peptides participate in the regulation of gut functions. Intravenous administration of Ghr stimulates gastric acid secretion (GAS) and gastric motility in rats.² In addition to their peripheral location, Ghr-producing neurones are also present in the hypothalamus. Intracerebroventricular injection of Ghr increases feeding, body weight gain in rats,³ and promotes appetite in mice.⁴ Taken together, Ghr has central and as yet undefined peripheral physiological functions. However, the effects of central Ghr on gastric secretory and motor functions are still unknown. Aim. To investigate the impacts of central administration of Ghr on GAS and gastric contractility (GC) in urethane-anesthetized rats. Methods. GAS was measured by the flush technique through double lumen gastric cannula, and GC was assessed by acutely implanted strain gauge sutured to serosal side of gastric corpus to monitor contractions of circular muscles in 24 h fasted male SD rats (250–300 g). I.c. injection was done through acutely implanted i.c. catheter. Intravenous 0.9% saline (1.2 mL h⁻¹) was infused through catheter in external jugular vein to keep the blood pressure stable, whereas body temperature was maintained at 36–37 °C. I.c. Ghr at 0.5, 1, and 5 µg 5 µL⁻¹ rat⁻¹ or i.c. vehicle (saline, 5 µL) was given. Collection of GAS and recording of GC were performed 30 min before and 90 min after ic injection. Results I.c. Ghr at 5 µg rat⁻¹ significantly stimulated the low basal GC, as shown by the increase in area under curve (AUC) of contractions, number of spikes, and contractile spike amplitude, compared with i.c. vehicle (Table 1). Truncal vagotomy (TV) blocked the stimulatory effect of i.c. Ghr on GC. There was a tendency to increase GAS after i.c. injection of Ghr, however, but it did not reach statistical significance. Conclusion. I.c Ghr at 5 µg rat⁻¹ induces selective stimulation of GC via vagal pathway activation, while GAS is not altered.