Ultrastructural distribution of growth hormone (GH) mRNA and GH intron I sequences in rat pituitary gland: effects of GH releasing factor and somatostatin.

We have measured the distribution of growth hormone (GH) mRNA or intron I sequences by in situ hybridization on ultrathin frozen sections of pituitaries removed from rats injected with saline, GH-releasing factor (GRF) or somatostatin. A 4-fold increase in labeling of the anterior lobe was observed after GRF, no changes with somatostatin. After ultrastructural in situ hybridization, labeling with the GH cDNA probe was specific to somatotrophs. Two populations of cells containing few or many secretory granules were labeled mainly in the cytoplasm or in both cytoplasm and nucleus. Some cells showed labeling at the perinuclear membrane. Hybridization with the GH intron I probe showed the same cell specificity with silver grains mainly located in the nucleus. After GRF, sequences hybridizing to growth hormone cDNA were increased mainly in the nucleus of somatotrophs when compared to mock-injected rats, indirectly suggesting an increase in the transcriptional activity of the growth hormone gene. After somatostatin, the density of labeling in the nucleus was increased suggesting that somatostatin may prevent the export of growth hormone mRNA molecules from the nucleus to the cytoplasm.

Immunocytochemical localization, binding, and effects of atrial natriuretic peptide in rat adipocytes.

The metabolic effects of atrial natriuretic peptide (ANP) have not been widely investigated. Since adipocyte cells represent a model system extensively used to examine the metabolic actions of many peptide hormones, we sought to establish whether ANP could bind to adipocyte membranes, alter cyclic nucleotide metabolism, and affect spontaneous or hormone-stimulated lipolysis. Using in vitro autoradiographic techniques, radiolabelled ANP was found to bind specifically to mammary gland fat cells. Additionally, endogenous ANP-like immunoreactivity could be localized in the plasma membrane compartment and cytoplasmic matrix of fat cells, but not in fat vacuoles. [125I]ANP bound to single high affinity sites (Kd = 0.72 nM) in fat cell membranes. The binding was rapid (equilibrium within 1 min at 25 degrees C) and specific. The atrial peptide was capable of stimulating a time- and concentration-dependent increase in cGMP accumulation in isolated adipocytes, but had no effect on spontaneous or stimulated [-)-isoproterenol, ACTH, forskolin) cAMP formation. ANP did not alter the increase in glycerol production stimulated by l-epinephrine in isolated fat cells. While i.v. infusion of ANP stimulated a marked increase in circulating levels of cGMP, the atrial peptide did not alter plasma triglyceride levels. These data demonstrate the presence of specific ANP binding sites on adipocyte membranes and internalization of ANP-associated immunoreactivity. These receptors are biochemically functional given the ability of ANP to augment cGMP formation. The peptide, however, does not exert an action on adipocyte lipolysis. Adipocytes, therefore, represent an ANP target tissue in which the physiological action of the peptide is yet to be defined.

Synthesis, internalization, and localization of atrial natriuretic peptide in rat adrenal medulla.

Some, though not all studies, have indicated that atrial natriuretic peptide (ANP) can bind to adrenal medullary cells. ANP-like immunoreactivity (ANP-LI) has also been identified in catecholamine-secreting cells. Together, these findings suggest that ANP may be taken up and/or synthesized in the adrenal medulla. The present study was designed to ascertain, by in situ hybridization, whether adrenal chromaffin cells could synthesize ANP, to define by an in vivo ultrastructural autoradiographic approach, whether ANP could, in fact, bind to rat adrenal medulla cells, to determine whether there was a cellular [noradrenaline (NA) vs. adrenaline (A)] selectivity in the binding process, and to establish whether extracellular [125I]ANP could be internalized by these cells. The cellular and subcellular distribution of endogenous ANP-LI was also investigated in both cell types by cryoultramicrotomy and immunocytochemical approaches. The in situ hybridization studies indicate the presence of mRNA to ANP in about 15% of adrenal medullary cells. Intravenous injection of [125I]ANP resulted in a 3-fold, preferential and specific radiolabeling of A-as compared to NA-containing cells. In A-containing cells, plasma membranes were significantly labeled 2 and 5 min post injection; cytoplasmic matrix, mitochondria, and secretory granules throughout the time course studied (1-30 min post injection). Lysosomes, rough endoplasmic reticulum, Golgi apparatus, and nuclei were not labeled. ANP-LI was identified in both NA- and A-containing cells; in the former, it was almost exclusively localized in secretory vesicles, in the latter it was detected in plasma membranes, cytoplasmic matrix, nuclear euchromatin, some mitochondria and relatively fewer granules than in NA-containing cells. The findings suggest that ANP may be synthesized primarily in NA-containing cells and that A-containing cells primarily bind and internalize the extracellular (endogenous or exogenous) atrial peptide. The data suggest that ANP secreted by adrenal medullary chromaffin cells may have distal paracrine actions or interactions with coreleased catecholamines and neuropeptides. Binding and internalization may reflect an action of ANP on the secretory function of A-containing cells.

Ontogeny of gonadotropic and thyrotropic cells in fetal mouse anterior pituitary. Comparison between two species C57 BL6 and Balb/C.

The fetal period constitutes a determinant stage in the ontogenesis of the hypothalamo-hypophysial-gonadal system. This work mainly concerns gonadotropic and thyrotropic functions and compares their different aspects in two strains of fetal mice. Balb/c and C57 BL6 fetal mice were studied at 16, 17, 18 and 19 days of gestation. The appearance and distribution of immunoreactive gonadotropic and thyrotropic cells of the anterior pituitary were observed by immunocytology using an indirect method with anti-porcine luteinizing hormone beta serum, anti-bovine thyrotrope hormone serum after saturation with bovine luteinizing hormone and anti-rat luteinizing hormone serum. In the two strains and in both sexes, LH gonadotropes appeared at 17 days of gestation and preferentially localized in the ventral part of the anterior lobe; a similar distribution was noted at 18 days and there was an increase in the number and staining intensity of labeled cells. By 19 days of gestation the gonadotrophs seemed more numerous, more generally distributed throughout the gland and often abutted to sinusoidal capillaries. An account of immunoreactive cells with anti-porcine luteinizing hormone serum and statistical evaluation of the results performed by variance analysis showed significant differences between the two strains. LH gonadotropic cells were always more numerous at each day of gestation in Balb/c fetuses especially in female fetuses. The possibility of a different evolution and/or differentiation for this cell population is discussed. Comparison of gonadotropic function between rat fetuses, mouse fetuses and human fetuses lead us to conclude that mouse fetuses appeared as an experimental model more closely related to human fetuses.

In situ hybridization to rat brain and pituitary gland of growth hormone cDNA.

Using in situ hybridization, we have investigated the presence of mRNA coding for growth hormone (GH) in the rat brain. Using 32P-labeled GH cDNA as a probe, the pituitary gland showed hybridization in unfixed sections. Using 3H-labeled GH cDNA hybridized to fixed sections, only cells in the anterior pituitary were labeled in good agreement with the localization of somatotropes. In the brain, wide zones were labeled with 32P-GH cDNA: the caudate putamen, the striatum, the ventral thalamus, the formatio reticularis and the basal cortex. With the 3H GH-cDNA probe, more discrete regions of the brain showed hybridization: the basal cortex, the outside part of the hippocampus and the caudate putamen.

Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat. A quantitative autoradiographic study after cryoultramicrotomy.

To identify anterior pituitary cell types containing GnRH binding sites and to study the internalization process of this peptide by target cells under physiological conditions, autoradiography was performed on rat anterior pituitaries removed at specific time intervals (2-60 min) after intravenous injection of mono-radioiodinated 125I-GnRH into intact males. At electron-microscopic level, gonadotrophs and lactotrophs appeared to contain silver grains. Concomitant administration of an excess of unlabeled GnRH with the radioiodinated hormone prevented this localization indicating the specificity of the reaction. The time-course study in gonadotrophs showed that 2 min after injection silver grains could be found over the plasma membrane, secretory granules and nuclear membrane. Similar results were observed 5 and 15 min after injection. Extensive label was observed over the nucleus and nuclear membrane 15 to 60 min after injection. The injection of a radioiodinated GnRH agonist [D-Trp6, Pro9 (Net), DesGly10]-GnRH produced comparable results. In contrast, the injection of 125I-[D-pGlu1, D-Phe2, Trp3,6]-GnRH, an antagonist of GnRH, produced positive labeling only at the plasma membrane without internalization. These results indicate that, after binding with receptors on the plasma membrane, GnRH is rapidly internalized, accumulating in secretory granules, and localizing over the nuclear membrane and later, in the nucleus. Association of radioactivity with secretory granules could be related to a specific action of GnRH at this level or to receptor recycling, and presence of label in the nucleus may be related to stimulation of neosynthesis of LH and GnRH receptors.