Secretion of melanocyte-stimulating hormone and adrenocorticotropin from transplanted pituitary pars intermedia in stressed and nonstressed rats

Rats bearing kidney grafts of the pituitary pars intermedia were divided into three groups: unstressed, acutely stressed, and chronically stressed. Corresponding sham-operated rats were used for comparisons. Twenty days after grafting, the rats were sacrificed and alpha-melanocyte-stimulating hormone (alpha-MSH), adrenocorticotropin (ACTH), and corticosterone were estimated in plasma. The adrenal/body weight ratio and DNA content of the glands were also investigated. The following results were obtained: MSH was found not to be increased in unstressed rats, but it was in grafted animals subjected to acute and chronic swimming stress. ACTH and corticosterone rose in all three groups. Adrenal/body weight ratio and DNA content increased only in grafted chronically stressed rats. Moreover, plasma corticosterone was found higher in grafted hypophysectomized rats than in non-grafted hypophysectomized animals. Administration of ergocryptine to nonstressed grafted rats induced a decrease in the blood content of ACTH and MSH, indicating that the grafts were the source of a part of the circulating ACTH. On the other hand, the fall in MSH levels could show the effect of the drug upon the pars intermedia. Comparison of the ratios of both hormones released in incubations showed that grafts secreted more ACTH than MSH; on the other hand, when intact neurointermediate lobes were incubated, MSH predominated over ACTH. For the first time it is demonstrated that the pars intermedia can secrete ACTH in vivo. Nevertheless, the ability to secrete this hormone is not a property of normal intact pars intermedia, but it manifests in the transplantations probably due to the overactivity of light cells induced by chronic stoppage of dopaminergic inhibition.     

Thyrotropin-releasing hormone does not alter the release of melanocyte-stimulating hormone or adrenocorticotropic hormone from the rat pars intermedia.

Thyrotropin-releasing hormone (TRH) has been reported to stimulate the release of melanocyte-stimulating hormone (MSH) from the pars intermedia (PI) of Rana esculenta. To test its effect on the rat PI, acutely dispersed rat PI cells, as well as whole nervosa-intermedias (NI), were incubated with synthetic TRH, from 10(--10)--10(--4)M. TRH did not alter the release of either MSH or adrenocorticotropic hormone (ACTH).     

GABAergic regulation of melanocyte-stimulating hormone secretion from the pars intermedia of Xenopus laevis: immunocytochemical and physiological evidence

alpha-MSH secretion from the amphibian pars intermedia is under inhibitory hypothalamic control, and the catecholamine dopamine is thought to be the physiological MSH release-inhibiting factor. In the present study we evaluated the possible role of the neurotransmitter gamma-aminobutyric acid (GABA) in the regulation of the pars intermedia of Xenopus laevis. Immunocytochemical staining with antibodies to glutamic acid decarboxylase showed the presence of a rich GABAergic network in the intermediate lobe of the pituitary gland. Administration of GABA to superfused neurointermediate lobes caused a rapid and dose-dependent inhibition of basal release of MSH and immunoreactive endorphin. Pulse-chase experiments revealed that GABA gave a coordinate inhibition of the release of all peptides derived from proopiomelanocortin. In vivo administration of GABA resulted in almost complete pigment aggregation in dermal melanophores of both adults and larvae. Altogether, our results indicate that GABA is a physiologically important factor for regulation of the pars intermedia in Xenopus laevis.     

Serum melanocyte-stimulating hormone levels and pituitary melanocyte-stimulating hormone content during pseudopregnancy in the rat.

Pseudopregnancy (PSP) was induced in rats by vaginal stimulation and the levels of MSH in serum and pituitary were examined. One hour after vaginal stimulation, the serum MSH level was increased, and a cyclical variation was observed by the second day of PSP. There were two surges of about 50 pg/ml, one in the morning and the other at night. On the sixth day of PSP, the pattern of serum levels still showed two peaks, but while the nocturnal peak remained at 50 pg/ml, the diurnal value was halved. The pituitary MSH content also underwent cyclical variations; the lowest levels coincided with the peak levels in serum. These observations raise the possibility that MSH is involved in PSP.     

Stereological analysis of the effects of 6-hydroxydopamine on the ultrastructure of the melanocyte-stimulating hormone cell of the pars intermedia of the pituitary of Xenopus laevis.

It has been confirmed that intracisternal administration of the sympatholytic compound 6-hydroxydopamine (6-OHDA) to the toad Xenopus brings about degenerative changes in the appearance of the catecholaminergic innervation of the pars intermedia of the pituitary. This observation has been extended by stereological analysis of the ultrastructural composition of the presumed melanocyte-stimulating hormone (MSH) cells of the pars intermedia, which reveals that following the administration of this compound there is an increase in the amount of rough endoplasmic reticulum and a decrease in the numbers of secretory granules. These changes are consistent with increased secretory activity and are considered to reflect the removal of an inhibitory catecholaminergic influence from the MSH cells. 6-OHDA also brings about a prolonged darkening of the animals, but it is suggested that this cannot be due entirely to increased circulating levels of MSH in view of the finding that 6-OHDA has a pituitary-independent effect on pigmentation, as demonstrated by the administration of 6-OHDA to hypophysectomised animals when a transitory darkening occurs.     

In vitro biosynthesis of beta-endorphin, gamma-lipoprotein, and beta-lipotropin by the pars intermedia of beef pituitary glands.

Labeled amino acids were incorporated with proteins during incubations of isolated cells from the pars intermedia of beef pituitary glands. After 3 hr of incubation with methionine and [3H]lysine, approximately equivalent amounts of labeled gamma-lipotropin and beta-endorphins were isolated. They were found to be major synthesis products of the pars intermedia. In contrast, very little labeled beta-lipotropin was recovered. Another major synthesis product of the pars intermedia was also purified. Its partial amino acid sequence and molecular weight were determined and it was concluded that this peptide cannot be identified as any known pituitary hormone or protein fragment.     

Immunoreactive pit-1 protein in hyperplastic pars intermedia induced by calcitonin of the rat pituitary gland

To elucidate the effects of synthetic salmon calcitonin (sCT) on the cells in the rat pituitary gland, we histopathologically and immunohistochemically examined the early changes after 4 or 13 weeks treatment with sCT 120 IU/kg. Focal proliferative lesions of the anterior pituitary glands were consistently found after treatment with sCT for 13 weeks. Histologically, the cells with the focal proliferative lesions were classified into the following three groups: 1) enlarged basophilic cell focus, 2) vacuolated cell focus and 3) chromophobe cell focus. These focal proliferative lesions had positive staining only for the alpha-subunit and failed to show Pit-1 protein immunoreactivity. The sCT treatment also increased the thickness of the pars intermedia. Hypertrophy of the pars intermediate cells was characteristically seen. Furthermore, Pit-1 protein immunoreactivity was clearly detected in the nuclei of the hyperplastic pars intermediate cells. All pars intermediate cells were equally stained by alpha- or beta-MSH and beta-endorphin in both vehicle- and sCT-treatment. No difference was seen. These findings strongly suggest a very close relationship between Pit-1 protein immunoreactivity and cellular proliferation induced by sCT.     

A kininogenase resembling glandular kallikrein in the rat pituitary pars intermedia

The purpose of this study was to determine the distribution and characteristics of kinin-generating proteases (kininogenases) in rat pituitary tissue. Male rat pituitaries were dissected into their various lobes, sonicated in saline containing 12 mM deoxycholic acid, and assayed for protease activity at pH 8.5 using kininogen and chromogenic peptide substrates; kinin generation was measured by RIA. The rat pituitary was found to contain kininogenase activity highly concentrated in the pars intermedia; this activity was strongly inhibited by aprotinin and was resistant to soybean trypsin inhibitor. Antiserum against rat urinary kallikrein also inhibited the intermediate pituitary kininogenase. The kininogenase product was identified as bradykinin by reversed-phase high performance liquid chromatography. Homogenization of neurointermediate pituitaries in 0.25 M sucrose buffer (pH 7.5) followed by differential centrifugation (1,000 X g, 5 min; 10,000 X g, 20 min; 105,000 X g, 70 min) demonstrated that most of the kininogenase activity was in the 10,000 X g pellet. Kinin generation by neurointermediate pituitary extracts had a pH optimum of 8.0, and such extracts also hydrolyzed chromogenic peptide substrates for kallikreins. In addition, neurointermediate pituitary extracts were found to contain a distinct protease which hydrolyzed a chromogenic peptide substrate for thrombin. The intermediate pituitary kininogenase resembles glandular kallikrein and possibly may participate in prohormone processing.     

ACTH production by the pars intermedia of the rainbow trout pituitary.

During in vitro culture, the pars intermedia of the rainbow trout pituitary synthesises and releases far greater amounts of immunologically and biologically active adrenocorticotrophin (ACTH) than does the pars distalis. This effect is inhibited by cycloheximide. Radio-immunossay of culture media for α-Melanocyte-stimulating hormone (α-MSH) and ACTH demonstrate the secretion also of trout α-MSH and a high molecular weight protein with ACTH immunoreactivity. The findings support the concept that ACTH and α-MSH may be derived from a common high molecular weight protein precursor. Some possible physiological implications of ACTH secretion by the pars intermedia are discussed.     

Concomitant synthesis of beta-endorphin and alpha-melanotropin from two forms of pro-opiomelanocortin in the rat pars intermedia.

In the pars intermedia of rat pituitary glands, two forms of a common precursor for corticotropin (ACTH) and beta-lipotropin with apparent molecular weights of 34,000 and 36,000 were resolved by sodium dodecyl sulfate/acrylamide gradient slab gel electrophoresis. High-performance liquid chromatographic analysis of [35S]methionine-labeled tryptic fragments of the two forms of the precursor revealed that both contained copies of ACTH-(1-8) and beta-lipotropin-(61-69) sequences. When biosynthetic studies were performed in the presence of tunicamycin, the 34,000- and 36,000-dalton forms were replaced by a peptide with an apparent molecular weight of 32,000. It was therefore concluded that the 34,000- and 36,000-dalton forms of the precursor represent two glycoprotein variants of similar polypeptides, differing in the number of asparagine-linked carbohydrate moieties. During pulse-chase incubations with [35S]methionine, the precursor forms were cleaved into two major groups of labeled products: (i) beta-endorphin and (ii) a mixture of ACTH fragments closely related to alpha-melanotropin. No ACTH-(1-39) was found at the end of a 2-hr chase period, suggesting that ACTH is not a significant hormone product of the rat pars intermedia.     

High-resolution radioautographic localization of beta-adrenergic receptors in the pars intermedia of the rabbit pituitary

In order to study the cellular and subcellular distribution of beta-adrenergic receptors in the pars intermedia of the rabbit pituitary, we have developed a technique for the high-resolution radioautographic localization of beta-adrenergic receptors, using [125]-cyanopindolol as the ligand. The most suitable fixative was the McLean fixative to which 0.1% glutaraldehyde was added. In semithin sections, specific labeling was localized over all the secretory cells. At the ultrastructural structural level, plasma membrane, nuclear envelope, and mitochondria appeared to be significantly labeled. These observations are consistent with previous results indicating the presence of beta-adrenergic receptors at the level of the plasma membrane. On the other hand, the exact role of intracellular receptors remains to be clarified. Moreover, this technique which allows the localization of beta-adrenergic receptors at the ultrastructural level should be very useful to study the distribution of these receptors in other tissues, especially the central nervous system.     

Biosynthesis of beta-endorphin from beta-lipotropin and a larger molecular weight precursor in rat pars intermedia.

When isolated rat pars intermedia cells were incubated for 10 min with radioactive amino acids, one major labeled protein with a molecular weight of 30,000 +/- 1500 was extracted. This protein was shown to contain in its sequence the antigenic determinants for corticotropin and beta-melanotropin by immunoprecipitation. When the radioactivity incorporated into this large molecular weight protein during the first 10 min was chased by a further incubation in presence of an excess of unlabeled amino acid, the initial protein was degraded into several smaller peptides including beta-endorphin and beta-lipotropin. Another 18,000-dalton peptide was also observed and was tentatively identified as a large molecular form of corticotropin. From the kinetics of the maturation of the initial precursor, it is concluded that the initial cleavage of the 30,000-dalton peptide gives rise to beta-lipotropin and the 18,000-dalton form of corticotropin. beta-Lipotropin is subsequently cleaved to form beta-endorphin.