Growth hormone secretagogue receptors in rat and human gastrointestinal tract and the effects of ghrelin.

The peptide hormone ghrelin is known to be present within stomach and, to a lesser extent, elsewhere in gut. Although reports suggest that gastric function may be modulated by ghrelin acting via the vagus nerve, the gastrointestinal distribution and functions of its receptor, the growth hormone secretagogue receptor (GHS-R), are not clear and may show signs of species-dependency. This study sought to determine the cellular localisation and distribution of GHS-R-immunoreactivity (-Ir) using immunofluorescent histochemistry and explore the function of ghrelin in both human and rat isolated gastric and/or colonic circular muscle preparations in which nerve-mediated responses were evoked by electrical field stimulation. The expression of GHS-R-Ir differed to a greater extent between species than between gut regions of the same species. Both the human and rat gastric and colonic preparations (n=3 each) expressed GHS-R-Ir within neuronal cell bodies and fibres, cells associated with gastric glands and putative entero-endocrine and/or mast cells. Smooth muscle cells and epithelia were devoid of GHS-R-Ir and only rat preparations expressed GHS-R-Ir on nerve fibres associated with the muscle layers. GHS-R-Ir was fully competed in all cases in pre-adsorption studies and antiserum specificity was confirmed using a cell line transiently expressing the rat GHS-R. In rat isolated forestomach circular muscle, ghrelin 0.1-10 microM had no effect on smooth muscle tension but concentration-dependently facilitated the amplitude of contractions evoked by excitatory nerve stimulation (n=4-7; P<0.05 for each concentration versus vehicle; n=18). When examined under similar conditions, in both rat distal colon (n=4-6, P>0.05 each) and human ascending (n=3) and sigmoid (n=1) colon, these concentrations of ghrelin were without effect (P>0.05 each). The data suggest that ghrelin has the potential to profoundly affect gastrointestinal functions in both species and at least one of these functions is to exert a gastric prokinetic activity. Moreover, we suggest that this activity of ghrelin is mediated via the enteric nervous system, in addition to known vagus nerve-dependent mechanisms.

GABA(B) receptor function in the ileum and urinary bladder of wildtype and GABA(B1) subunit null mice.

1. GABA(B1) receptor subunit knockout mice were generated and the effects of the GABA(B) receptor agonist, baclofen, were evaluated within the peripheral nervous system (PNS) of wildtype (+/+), heterozygote (+/-) and knockout (-/-) animals. For this purpose, neuronally-mediated responses were evoked in both the isolated ileum and urinary bladder, using selective electrical field stimulation (EFS). 2. In ileum resected from 4-8-week-old-mice, low frequencies of EFS (0.5 Hz) evoked irregular muscle contractions which were prevented by atropine 1 microM and reduced by baclofen (33.4 +/- 5.6%, 100 microm). The latter effect was antagonized by the GABA(B) receptor antagonist CGP54626 0.2 microm. Baclofen 100 microm did not affect contractions of similar amplitude induced by carbachol, indicating that the ability of baclofen to inhibit cholinergic function in mouse ileum may be due to an action at prejunctional GABA(B) receptors. 3. To avoid the development of grand mal seizure by GABA(B1) (-/-) mice, a behaviour observed when the mice were greater than 3 weeks old, it was necessary to study the effects of this knockout in 1-3-week-old-animals. However, at this age, EFS at 0.5 Hz did not evoke robust muscle contractions. Consequently we used EFS at 5 Hz, which did evoke cholinergically mediated contractions, found to be of similar amplitude in (+/+) and (+/-) mice, of both 1-3 weeks and 4-8 weeks of age. At this frequency of EFS, baclofen reduced the amplitude of the evoked contractions [n = 6 (+/+) and n = 5 (+/-), IC50 19.2 +/- 4.8 microm) and this effect was greatly reduced in the presence of CGP54626 0.2 microm. 4. In urinary bladder from 1-3-week-old-mice, using higher frequencies of EFS to evoke clear, nerve-mediated contractions (10 Hz), baclofen 10-300 microm concentration-dependently inhibited contractions in (+/+) mice (IC50 9.6 +/- 3.8 microm). This effect was inhibited by CGP54626 (0.2 microm, 46.2 +/- 13.6% inhibition, 300 microm baclofen n = 7) a concentration which, by itself, had no effect on the EFS-evoked contractions. 5. The effects of baclofen in both ileum and urinary bladder were absent in the GABA(B1) receptor subunit (-/-) mice; however, responses to EFS were unaffected in (-/-) when compared to the (+/+) mice. 6. Our data suggest that, as in the central nervous system (CNS), the GABA(B1) receptor subunit is an essential requirement for GABA(B) receptor function in the enteric and PNS. As such, these data do not provide a structural explanation for the existence of putative subtypes of GABA(B) receptor, suggested by studies such as those in which different rank-orders of GABA(B) agonist affinity have been reported in different tissues.