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ML Baudet and S Harvey

Although avian and mammalian species differ significantly in their regulation of GH secretion, preliminary studies have demonstrated in vivo GH responses to ghrelin in chickens, as in mammals. However, the relative potency of ghrelin as a GH-releasing hormone (GHRH) in birds is uncertain, as is its site of action.The intravenous administration of human ghrelin to immature chickens promptly increased the circulating GH concentration (within 10 min), although this was transitory and was only maintained for 20 min. This GH response was dose-related with an EC50 of approximately 3.0 microg/kg, comparable with the reported potency of human GHRH in chickens. When incubated with dispersed pituitary cells, human ghrelin induced dose-dependent GH release over a range of 10(-6) to 10(-9) M, with an EC50 of 7.0 x 10(-8) M, comparable with that induced by human GHRH (EC50 6.0 x 10(-8) M), although it was less effective at doses of 10(-6) to 10(-8) M. This was due to direct effects on pituitary somatotrophs, since human ghrelin increased GH release (determined by the reverse hemolytic plaque assay) from individual pituitary cells. The incubation of these cells with human ghrelin induced a dose-dependent increase in the numbers of somatotrophs secreting GH and in the amount of GH released by each cell. In summary, these results demonstrated that ghrelin is a dose-related GH-releasing factor in chickens with a potency comparable with that induced by human GHRH. The GH-releasing action of ghrelin is due, at least in part, to stimulatory actions on the numbers of somatotrophs induced to release GH and upon the amount of GH released from individual somatotrophs.

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AE Murphy, H Peek, ML Baudet, and S Harvey

GH has previously been shown to be present in peripheral extrapituitary tIssues of chick embryos, but the cellular distribution of GH immunoreactivity is still uncertain because of differing immunohistochemical findings. The possibility that this uncertainty reflects differences in fixation of the embryonic tIssues was assessed by comparing GH immunoreactivity in tIssues fixed in 4% (w/v) paraformaldehyde or Carnoy's fluid (60% ethanol (v/v); 30% chloroform (v/v); 10% acetic acid (v/v)). A widespread distribution of GH immunoreactivity was seen in paraformaldehyde-fixed tIssues, although it was particularly intense in the spinal cord, dorsal and ventral root ganglia, notochord, myotome, epidermis, crop, heart, lung and humerus. In marked contrast, GH immunoreactivity in embryonic tIssues fixed with Carnoy's was more discrete and mainly restricted to marginal and mantle layers of the spinal cord, spinal nerves, the ventral root ganglia and the extensor nerve of the anterior limb bud. Since these are neural derivatives, Carnoy's fixation appears to preferentially result in neural GH staining, whereas GH staining in neural and non-neural tIssues is seen after paraformaldehyde fixation. Carnoy's, because it is a precipitive fixative, may only fix large GH moieties, whereas GH in peripheral tIssues includes numerous molecular variants, many of which are of relatively small size. Paraformaldehyde, because it is a cross-linking fixative, preferentially fixes peptides and small proteins, and it may therefore fix more GH moieties than Carnoy's fluid. Carnoy's fixation appears to underestimate GH immunoreactivity in immunohistochemical studies on the cellular distribution of GH-like proteins in embryonic chicks.