Extraction and characterization of a cytochemically assayable Na+/K+-ATPase inhibitor/glucose-6-phosphate dehydrogenase stimulator in the hypothalamus and plasma of man and the rat

ABSTRACT Some physicochemical properties of partially purified hypothalamic material from the spontaneously hypertensive rat, and of plasma from man and the rat, have been characterized using a validated cytochemical bioassay which measures the ability of biological fluids to stimulate fresh guinea-pig kidney glucose-6-phosphate dehydrogenase (G6PD) after 2 min of exposure to the test substance, as an indication of their ability to inhibit Na+/K+ adenosine triphosphatase (Na+/K+-ATPase) after 4–6 min of exposure. The G6PD-stimulating activity of both hypothalamic extract and plasma is soluble in water and insoluble in chloroform. During electrophoresis the activity from both sites appears in the same fractions and travels considerably further than lysine. After high-pressure liquid chromatography the activity of hypothalamic extract appears in a discreet fraction which does not absorb u.v. light. The activity of both the hypothalamic extract and plasma survives boiling and acid hydrolysis, but is substantially inhibited by prior incubation with digoxin antibody. From ultrafiltration studies, the substance responsible for the ability to stimulate G6PD appears to have a molecular weight of less than 500. The G6PD-stimulating activity of hypothalamic extracts was destroyed by ashing and by base hydrolysis. The ability of plasma of high activity to stimulate G6PD is considerably increased by incubating at 37 °C for 15 min and destroyed by incubation for 45 min. It is concluded that these and several other previously noted similarities suggest that the cytochemically assayable Na+/K+-ATPase-inhibiting/G6PD-stimulating activity in the plasma and hypothalamus may be due to the same ouabain-like substance. J. Endocr. (1987) 112, 299–303

Different mechanisms controlling FSH and LH release in Japanese quail (Coturnix coturnix japonica): evidence for an inherently spontaneous release and production of FSH

ABSTRACT Plasma concentration of FSH and LH and their content in the adenohypophyses of Japanese quail were estimated by homologous radioimmunoassays based on chicken hormones. The results consistently showed that the plasma concentration of FSH was slightly higher than that of LH, although the FSH content in the adenohypophysis was much lower than that of LH in immature, mature and acutely photostimulated quail. These results prompted experiments in vitro to determine whether the difference was intrinsic. Adenohypophyses were collected and each cut mid-sagittally into two halves; one half was incubated for 20 h with or without chicken hypothalamic extract, and the medium was changed every 1 or 2 h to estimate the FSH and LH released. The other half served to estimate the initial content of FSH and LH in the adenohypophysis. The amount of FSH released was three times more than the original content even when adenohypophyses were incubated without hypothalamic extract, and the residual FSH content in the pituitary after 20 h of incubation was equal to the initial FSH content. In contrast, under the same incubation conditions, little LH was released and the residual LH content was decreased. When hypothalamic extract was added to the medium, LH release was enhanced sixfold compared with the control, whereas the increase in FSH was less than twofold. Spontaneous FSH release was markedly decreased in a Ca2+-deficient incubation medium, suggesting that the release was not due to leakage but to an active secretion mechanism. We therefore suggest, for the first time, that the release and production of FSH are largely autonomous, whereas release and production of LH are rigidly controlled and regulated by the releasing hormone in avian species. J. Endocr. (1986) 108, 239–245

Mechanisms of release of prolactin from fowl anterior pituitary glands incubated in vitro: effects of calcium and cyclic adenosine monophosphate

ABSTRACT Fowl anterior pituitary glands were bisected and each half was pretreated in either Medium 199 or medium containing EGTA to deplete endogenous calcium (Ca2+) stores, after which they were incubated in Medium 199, or Ca2+-free medium, containing prolactin release-stimulating agents and verapamil, a Ca2+ channel blocker. High K+ concentrations, hypothalamic extract, synthetic thyrotrophin-releasing hormone (TRH) and dibutyryl cyclic AMP (dbcAMP) all stimulated release of prolactin from control (non EGTA-treated) hemianterior pituitary glands. The effects of TRH and dbcAMP were not additive, but the response to submaximal concentrations of TRH was augmented by theophylline, a phosphodiesterase inhibitor. Reduction of Ca2+ availability with EGTA or verapamil reduced basal release of prolactin, prevented the prolactin-stimulating effects of high K+ concentrations and TRH, and markedly attenuated responses to hypothalamic extract and dbcAMP, EGTA being more effective than verapamil. Increasing the Ca2+ concentration of the medium did not augment basal or stimulated release of prolactin. These results suggest that both Ca2+ and cyclic AMP may act as intracellular mediators in the release of prolactin. Both basal and stimulated release of prolactin depend upon the presence of Ca2+. Although influx from the medium may be the major source of Ca2+, endogenous stores of Ca2+, perhaps mobilized by dbcAMP, may be able to maintain some release of prolactin. The prolactin-stimulating effects of TRH may be mediated by cyclic AMP. J. Endocr. (1985) 105, 183–188

Effects of synthetic mammalian thyrotrophin releasing hormone, somatostatin and dopamine on the secretion of prolactin and growth hormone from amphibian and reptilian pituitary glands incubated in vitro

ABSTRACT Pituitary glands of grassfrog (Rana pipiens), bullfrog (Rana catesbeiana), clawed toad (Xenopus laevis) and two species of terrapin (Chrysemys picta and Pseudemys scripta) were incubated in medium containing hypothalamic extract (HE), thyrotrophin releasing hormone (TRH), somatostatin, dopamine, or combinations of these treatments. Prolactin and GH concentrations in the medium were determined by densitometry after polyacrylamide-gel electrophoretic separation. Hypothalamic extract stimulated secretion of both hormones in all species tested. Thyrotrophin releasing hormone stimulated secretion of prolactin and GH, showing a biphasic pattern of response. Dopamine had little effect alone, but inhibited HE-and TRH-stimulated release of prolactin, but not GH, in both amphibia and reptiles. Somatostatin by itself had no apparent effect on release of hormones, but it inhibited HE- and TRH-stimulated release of GH from both amphibian and reptilian pituitary glands. These results indicate that factors affecting mammals and birds also interact in the regulation of secretion of prolactin and GH in lower vertebrate species. J. Endocr. (1984) 102, 175–180