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1 The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44- and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. 2 To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3 Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4 We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Go6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Go6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (ɛ, δ), but not conventional or atypical PKCs. Further analyses suggest that PKCɛ is required for the activation of ERK1/2. 5 Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC- and PKCɛ-dependent pathway. British Journal of Pharmacology (2006) 148, 350–365. doi:10.1038/sj.bjp.0706727

In cells overexpressing active MEKK1 to enhance c-Jun trans-activation, expression of rat cholecystokinin 1 receptor increased the activity of c-Jun while in the same experimental conditions overexpression of mouse cholecystokinin 1 receptor repressed it. This differential trans-activation is specific, since it was not observed for either the other overexpressed kinases (MEK, PKA) or for other transcription factors (ATF2, ELK-1, CREB). This differential behaviour was also detected in a human colon adenocarcinoma cell-line naturally producing high levels of endogenous MEKK1. This differential behaviour between the two receptors on the MEKK1-induced c-Jun trans-activation was independent of the activation state of JNK, of the phosphorylation level of c-Jun and of its ability to bind its specific DNA responsive elements. Two amino acids (Val43 and Phe50 in the mouse cholecystokinin 1 receptor, replaced by Leu43 and Ileu50 in the rat cholecystokinin 1 receptor) localized in the first transmembrane domain were found to play a crucial role in this differential behaviour. MEKK1 probably activates a transcriptional partner of c-Jun whose activity is maintained or increased in the presence of the rat cholecystokinin 1 receptor but repressed in the presence of the mouse cholecystokinin 1 receptor. British Journal of Pharmacology (2006) 147, 951–958. doi:10.1038/sj.bjp.0706690

The aim of this study was to analyse the antisecretory mechanism of L-365,260 in vitro in isolated rabbit gastric glands. We showed that compound L-365,260, described as a non-peptide specific competitive CCK-B receptor antagonist, was able to dose-dependently inhibit [14C]-aminopyrine accumulation induced by histamine (10−4 M), carbachol (5×10−5 M), 3-isobutyl-1-methyl-xanthine (IBMX) (5×10−6 M) and forskolin (5×10−7 M) with similar IC50 values respectively of 1.1±0.6×10−7 M, 1.9±1.2×10−7 M, 4.2±2.0×10−7 M and 4.0±2.8×10−7 M. We showed that L-365,260 acted beyond receptor activation and production of intracellular second messengers and that it had no action on the H+/K+-ATPase. We found that L-365,260 inhibited cyclic AMP-induced [14C]-aminopyrine accumulation in digitonin-permeabilized rabbit gastric glands, suggesting that this compound acted, at least in part, as an inhibitor of the cyclic AMP-dependent protein kinase (PKA) pathway. Keywords: L-365,260; isolated rabbit gastric glands; [14C]-aminopyrine accumulation; H+/K+-ATPase; protein kinase inhibitor Introduction Acetylcholine, gastrin and histamine are the three most important mediators that stimulate gastric acid secretion by interacting with specific receptors located at the basolateral membrane of the parietal cells. Acetylcholine interacts with a muscarinic M3 receptor (Kajimura et al., 1992), gastrin with a CCK-B/gastrin receptor (Kopin et al., 1992) and histamine with a histamine H2 receptor (Black et al., 1972). It has been established that the gastric histamine H2 receptor is coupled to the formation of cyclic AMP (Chew et al., 1980). Activation of the parietal cell M3 cholinoceptor or the CCK-B/gastrin receptor leads to an elevation of [Ca2+]i (Chew et al., 1992). The observation that cholinergic or gastrin stimulation of acid secretion is potentiated by histamine (Berglindh et al., 1976; Soll, 1978; Chew & Hersey, 1982; Oiry et al., 1995) suggests that cyclic AMP and [Ca2+]i interact at some level. Whatever the complexity of the mechanism of acid secretion, the binding of these ligands to the receptors on the basolateral membrane of the parietal cell generates changes in second messengers and activation of protein kinase cascades that lead to final activation of the H+/K+-adenosine triphosphatase (H+/K+-ATPase) (EC 3.6.1.36) (Sachs et al., 1976; Forte & Soll, 1989). One role of cyclic AMP is the activation of cyclic AMP-dependent protein kinase (PKA) (Chew, 1985). PKA has specific functional targets and also initiates a phosphorylation cascade through activation of other downstream protein kinases the actions of which cause proton pump activation and cytoskeletal rearrangements which are characteristic of the stimulated parietal cell. It has been suggested that Ca2+/calmodulin-dependent kinase may play an important regulatory role in the modulation of cytoskeletal structure and function. Recently, Tsunoda et al. (1992) reported that Ca2+/calmodulin-dependent kinase II (CaMK II) mediates cholinergic-stimulated parietal cell secretion. In resting parietal cells, the H+/K+-ATPase is sequestered in the cytoplasmic membrane compartments of low K+ permeability: the tubulovesicles. Stimulation of the parietal cells causes a cytoskeletal reorganization and a fusion of the tubulovesicles with the apical plasma membrane, transferring the proton pump to that surface (Forte & Soll, 1989). The H+/K+-ATPase constitutes the final step common to each secretagogue in the production of acid by parietal cells and so provides a target for inhibitors used therapeutically as anti-ulcer agents such as omeprazole (Fellenius et al., 1981). In a previous study in which we compared the acid secretion induced by the C-terminal octapeptide of cholecystokinin (CCK-8) and [Leu11]gastrin(5–17) in isolated rabbit gastric glands (Oiry et al., 1995), we confirmed the ability of histamine to potentiate the action of CCK-8 (or [Leu11]gastrin(5–17)). To better understand the cooperativity between CCK and histamine, we tested the potency of various classes of CCK receptor antagonists (L-365,260; PD-135,158; YM-022; JMV-180 and L-364,718) as inhibitors of histamine-induced [14C]-aminopyrine accumulation. We found that PD-135,158; YM-022; JMV-180 and L-364,718 had no effect on histamine-induced [14C]-aminopyrine accumulation. Surprisingly, only compound L-365,260 appeared active and even more potent than cimetidine to inhibit histamine-induced [14C]-aminopyrine accumulation. Compound L-365,260, already described in the literature as a competitive and specific CCK-B/gastrin receptor antagonist (Lotti & Chang, 1989), presented a different and specific behaviour compared to other CCK-B/gastrin antagonists (PD-135,158, YM-022 and JMV-180). It has been mentioned that in the rat in vivo, low doses of L-365,260 inhibited pentagastrin-stimulated secretion (Hirst et al., 1991), whereas higher doses inhibited basal-, histamine- and carbachol-stimulated secretion (Hirst et al., 1991; Nishida et al., 1992; Pendley et al., 1993). In order to provide a better understanding of the role of L-365,260 in acid secretion in vitro, we decided to investigate further its antisecretory mechanism in isolated rabbit gastric glands.

1. The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44- and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. 2. To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3. Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4. We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Gö6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Gö6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (epsilon, delta), but not conventional or atypical PKCs. Further analyses suggest that PKCepsilon is required for the activation of ERK1/2. 5. Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC- and PKCepsilon-dependent pathway.