UBIQUITOUS DISTRIBUTION OF GROWTH-HORMONE RECEPTORS AND OR BINDING-PROTEINS IN ADENOHYPOPHYSEAL TISSUE

Liver GH receptor (GHR)-like mRNA has been shown to be widely distributed throughout rat and rabbit pituitary glands. In the present study RNA extracted from rabbit anterior pituitary glands was reverse transcribed, and the cDNA amplified by the polymerase chain reaction (PCR) in the presence of 3'- and 5'-flanking oligonucleotide primers for the extracellular and transmembrane domains of the rabbit GHR. A 499-basepair (bp) fragment was generated, identical in size to that in rabbit liver, kidney, and adipose tissue. Digestion of this fragment with a restriction endonuclease (SalI) produced moieties of 280 and 219 bp, as observed for the amplified cDNA fragments from liver, kidney, and adipose tissue. In situ hybridization of a cRNA probe for the rabbit GHR with cryostat sections of the anterior pituitary gland was demonstrated. Specific hybridization occurred throughout the adenohypophysis and was present in somatotroph and nonsomatotroph cells, identified by hybridization of the same tissue sections with a complementary riboprobe for rat GH mRNA. Electron microscopy and immunogold staining, using monoclonal antibodies against the extracellular domain of the rat (MAb 263) or rabbit (MAb 7) GHR, demonstrated the presence of the receptor or binding protein throughout rat and rabbit anterior pituitary gland. Immunostaining occurred in somatotroph and nonsomatotroph cells and was widely distributed throughout the intracellular and nuclear compartments. In GH-secreting cells gold particles were specifically accumulated in the secretory granules and heterochromatin, but were also present in mitochondria, Golgi, endoplasmic reticulum, cytoplasm, nucleoplasm, and cellular and nuclear membranes. A similar distribution of GHR immunoreactivity was observed within rat and rabbit hepatocytes. Specific binding sites for radiolabeled ovine GH to cytosolic fractions and to crude solubilized preparations of plasma and nuclear membranes of the rat anterior pituitary gland were also demonstrated. The binding of the tracer to these sites was inhibited by prior exposure to MAb 263, which binds to epitopes in the ligand-binding domain of the rat GHR. These results provide evidence of 1) the expression and translation of the GHR gene in rat and rabbit adenohypophysis in 2) GH and non-GH-secreting cells, and 3) the presence of GH receptors/binding proteins throughout the intracellular and nuclear compartments of these cells.