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In this study, we established a radioimmunoassay (RIA) specific for ghrelin from the bullfrog Rana catesbeiana using a novel antibody raised against the C-terminal amino acid sequence of bullfrog ghrelin [13-28]. We also examined the distribution of ghrelin-producing cells in the stomachs of bullfrogs using this antibody and a cRNA probe specific for the bullfrog ghrelin gene. Ghrelin levels in plasma and stomach extracts were approximately 150 fmol/ml and 83-135 fmol/mg wet tissue, respectively. Reverse-phase high performance liquid chromatographic analysis, combined with bullfrog ghrelin RIA, revealed that ghrelin immunoreactivity in the stomach was composed of non-acylated ghrelin (des-acyl ghrelin) and several acylated forms of ghrelin bearing different fatty acid modifications, which could induce increases in intracellular Ca2+ in cells expressing the rat GH secretagogue receptor. In the stomach, the major storage form was acylated ghrelin. In bullfrog plasma, however, the majority of ghrelin immunoreactivity was des-acyl ghrelin and C-terminal fragments of frog ghrelin. Acylated ghrelin forms comprised only minor peaks. Ghrelin-immunopositive and ghrelin mRNA-expressing cells were observed within the mucosal layer of the stomach. Following starvation, significant increases in plasma ghrelin levels and stomach ghrelin mRNA levels were observed as early as 10 days after starvation. These results indicate that ghrelin is present in the stomach and plasma of the bullfrog, which can be detected with our novel antibody. Interestingly, the primary storage form of ghrelin in the stomach differed from the circulating form dominating in the plasma. Furthermore, increases in ghrelin levels in plasma and mRNA levels in the stomach after starvation suggest the possible involvement of ghrelin in energy homeostasis in the bullfrog.

A bullfrog (Rana catesbeiana) thyroid-stimulating hormone (TSH) beta-subunit (TSHbeta) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHbeta cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHbeta corresponded to those positively stained with an antiserum against human (h) TSHbeta. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75+/-0.07ng TSH/100microl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose-response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHbeta coupled with the alpha-subunit by an immunoprecipitation experiment using the TSHbeta antiserum and an alpha-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro. The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.

We have identified the amphibian ghrelin from the stomach of the bullfrog. We also examined growth hormone (GH)-releasing activity of this novel peptide in both the rat and bullfrog. The three forms of ghrelin identified, each comprised of 27 or 28 amino acids, possessed 29% sequence identity to the mammalian ghrelins. A unique threonine at amino acid position 3 (Thr(3)) in bullfrog ghrelin differs from the serine present in the mammalian ghrelins; this Thr(3) is acylated by either n-octanoic or n-decanoic acid. The frog ghrelin-28 has a complete structure of GLT (O-n-octanoyl)FLSPADMQKIAERQSQNKLRHGNM; the structure of frog ghrelin-27 was determined to be GLT(O-n-octanoyl)FLSPADMQKIAERQSQNKLRHGN; frog ghelin-27-C10 possessed a structure of GLT(O-n-decanoyl)FLSPADMQKIAERQSQNKLRHGN. Northern blot analysis demonstrated that ghrelin mRNA is predominantly expressed in the stomach. Low levels of gene expression were observed in the heart, lung, small intestine, gall bladder, pancreas, and testes, as revealed by reverse transcription polymerase chain reaction analysis. Bullfrog ghrelin stimulated the secretion of both GH and prolactin in dispersed bullfrog pituitary cells with potency 2-3 orders of magnitude greater than that of rat ghrelin. Bullfrog ghrelin, however, was only minimally effective in elevating plasma GH levels following intravenous injection into rats. These results indicate that although the regulatory mechanism of ghrelin to induce GH secretion is evolutionary conserved, the structural changes in the different ghrelins result in species-specific receptor binding.

The coexistence of prolactin (PRL) and growth hormone (GH) was previously demonstrated in newly hatched bullfrog (Rana catesbeiana) tadpoles, whereas in adult bullfrogs, there were no cells containing both PRL and GH. However, a cell blot assay with enzymatically dispersed adult pituitary cells demonstrated the existence of cells secreting both PRL and GH. The number of cells secreting both PRL and GH was reduced by a protein synthesis inhibitor, cycloheximide, but not by an RNA synthesis inhibitor, actinomycin D. In situ hybridization and immunostaining of intact pituitary glands revealed the existence of GH mRNA in some of the PRL-immunoreactive cells and of PRL mRNA in some of the GH-immunoreactive cells. We propose that dispersion of the pituitary cells triggered the translation of GH mRNA in the PRL cells and/or of PRL mRNA in the GH cells.