Gy. Rappay

Effect of electrical stimulation of the neurohypophysis on ACTH release in rats with hypothalamic lesions.

Electrical stimulation of the neural lobe (NL) of the pituitary induces a rise in plasma corticosterone indicating the release of adrenocorticotropin (ACTH) in rats pretreated with dexamethasone, morphine and pentobarbitone. 7-8 days after placing an anterolateral cut around the medial basal hypothalamus (MBH), the rats failed to respond with ACTH release to electrical stimulation of the NL; the number of nerve fibers and terminals in the NL decreased to less than 5% of the normal; and ACTH releasing activity of acid extracts of the NL was undetectable using both in vivo and in vitro tests, which are insensitive to vasopressin. After lesions of the paraventricular nuclei the stimulation of the NL elecited a rise of plasma corticosterone that was significantly less than that in the controls. These results suggest that the NL of the pituitary contains electrically excitable fibers capable of releasing corticotropin-releasing factor (CRF; distinct from vasopressin) and that these fibers probably originate from outside the MBH, with a portion of them coming from or through the paraventricular nuclei or their immediate vincinity.

Inhibition of growth hormone and prolactin secretion by a serine proteinase inhibitor

The action of the tripeptide aldehyde t-butyloxycarbonyl-DPhe-Pro-Arg-H (boc-fPR-H), belonging to a family of serine proteinase inhibitors, on the release of immunoreactive prolactin (iPRL) and growth hormone (iGH) has been studied. In rat anterior pituitary cell cultures and pituitary quarters 1 mM boc-fPR-H inhibited basal iPRL and iGH release. Thyroliberin-induced iPRL release by cultured cells was also markedly inhibited with a concomitant accumulation of intra-cellular iPRL. During the short- and long-term exposure of cells to boc-fPR-H there no changes in total cell protein contents and in activities of some lysosomal marker enzymes. A wide scale of unchanged parameters characteristic for cellular metabolism indicated that the tripeptide aldehyde has no cytotoxic effect. The marked inhibition of basal as well as stimulated hormone release in the presence of the enzyme inhibitor might suggest that at least a portion of the hormones is released via a proteolytic enzyme-dependent process.

Intracellular distribution of arylsulphatase activity in adrenal cortical cells of the rat

SummaryArylsulphatase (B) activity has been demonstrated by means of an electronmicroscopic cytochemical method (substrate: p-nitrocatechol sulphate; capturing ion: barium, pH 5.5) in the lysosomes, in many Golgi elements, and occasionally in the endoplasmic reticulum of rat adrenal cortical cells.

Lysosomal enzyme activities in hypo- and hypersecretory anterior pituitary cells

SummaryThe involvement of lysosomes in ACTH and prolactin secretion was studied. Lysosomes were visualized in the anterior pituitary by their non-specific esterase (gold thioacetic acid technique) or acid phosphatase (Gomori technique) activity. Corticotrophs and mammotrophs were identified by postembedding immunocytochemistry for their respective hormones. Corticotrophs were rendered hypersecretory by bilateral adrenalectomy (7 or 12 days prior to examination), hyposecretory by dexamethasone administration. Prolactin secretion was enhanced by 17-beta-estradiol, prolactin release was inhibited by bromoergocriptine administration. Long-term hypersecretion of ACTH was accompanied by the presence of numerous autophagic vacuoles often containing secretory granules in the corticotrophs. Lysosomal enzyme-containing tubules and small lysosomes were abundant in the cytoplasm near the cell membrane, among the mature secretory granules. Feed-back inhibition of ACTH release by dexamethasone resulted in the extension of enzyme-containing tubules, continuous with cisternae and small lysosomes anywhere in the cytoplasm and in the appearance of numerous crinophagic vacuoles. A higher frequency of tubular lysosomes was described at the periphery of mammotrophs stimulated by 17-beta-estradiol. Bromoergocriptine caused a high incidence of characteristic crinophagic vacuoles in the prolactin cells. The concept of crinophagy has been extended to the corticotrophs. Morphological phenomena were attributed to the traffic and increased turnover of membranes, ligands and cytoplasmic organelles during stimulated secretion.

Microspectrophotometric data on the kinetics of acid phosphatase activity in cells removed from the peritoneal cavity of the rat

SynopsisThe extinctions (optical densities) of cells incubated for acid phosphatase in a histochemical azo-coupling procedure (naphthol AS-BI prosphate-hexazotized pararosaniline) have been measured microspectrophotometrically as a function of the incubation time and substrate concentration. A microcuvette was designed for the incubation. The amount of the reaction product in the cells at 23±1°C was found to be proportional to the incubation time for at least 40 min. Lineweaver-Burk plots were linear for some cells while in others nonlinear plots suggested the presence of two or more enzymes. This suggestion was supported by results obtained from polyacrylamide gel electrophoresis experiments.

Is calcium or cyclic AMP involved in the inhibitory effect on pituitary hormone secretion of the tripeptide aldehyde proteinase inhibitors

The mechanism by which tripeptide aldehyde proteinase inhibitors decrease prolactin (PRL) and growth hormone (GH) secretion was studied. Agents known to modify the intracellular levels of cyclic adenosine monophosphate (cAMP) or cytosolic free calcium were used in monolayer cultures of the rat anterior pituitary gland. The phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), 8-bromo-cAMP and forskolin all stimulated PRL release. Boc-D-Phe-Pro-arginal (Boc-DPPA) at 1 mmol/l concentration was a potent inhibitor of basal PRL release and significantly decreased the effect of 8-Br-cAMP, forskolin or IBMX (0.5 mmol/l). Forskolin (1 mumol/l) stimulated ACTH, PRL and GH release and all these effects were decreased by 100 mumol/l of Boc-D-Phe-Phe-lysinal (Boc-DPPL). Neither tripeptide aldehyde affected the forskolin-induced rise in intracellular cAMP. Growth hormone releasing factor (hpGRF, 1 nmol/l) stimulated both GH release and intracellular cAMP generation; Boc-DPPL (100 mumol/l) significantly decreased stimulated GH release without affecting cAMP accumulation. Increasing medium K+ to 10 times normal level stimulated PRL release presumably by enhancing Ca2+ entry into the cells and 1 mmol/l Boc-DPPA decreased high potassium-stimulated PRL release. The ionophore A-23187 stimulated PRL release at 10 mumol/l but not at 1 mumol/l. At 1 mumol/l A-23187 prevented the Boc-DPPA-induced inhibition of PRL release. These findings suggest that the tripeptide aldehyde proteinase inhibitors inhibit PRL and GH release at a site beyond cAMP formation.

Lysosomes in anterior pituitary corticotrophs of the rat

SummaryE600 resistent non-specific esterase activity or acid phosphatase activity were localized in corticotrophic cells identified by postembedding immunocytochemistry (PAP or protein A-immunogold techniques). The lysosomal system of this cell type consists of dense bodies, of a population of small lysosomes mostly situated at the cell periphery in the vicinity of secretory granules as well as of tubular structures. These latter were located either in the central part of the cytoplasm and probably belonged to the Golgi apparatus or at the cell periphery, partly in the extensions. Small lysosomes occurred to be in continuity with enzyme-containing tubules. In a few structures lysosomal enzyme activity and ACTH immunoreactivity overlapped. Some autophagic vacuoles seemed to contain secretory granule matrix. It is suggested that the concept of crinophagy can be extended to the corticotrophs, though the lysosomal system may be involved in the specific function of this cell type by other mechanisms as well.

Corticoliberin (CRF) activity in selected hypothalamic regions in rats with anterolateral hypothalamic cuts

Abstract The hypothalamic path of the corticoliberin (CRF) containing fibers was investigated in male rats with an anterolateral cut around the medial basal hypothalamus (MBH). Frozen samples of different brain regions were taken from cryostat sections and they were extracted and bioassayed using immunoreactive ACTH release from cell cultures of anterior pituitary as an index of CRF activity. Three days after placing an anterolateral cut there was no change in CRF activity of tissue surrounding a thalamic cut, but a significant rise was observed in the strip of tissue surrounding the hypothalamic cut at the level of the retrochiasmatic area. Seven days after the operation CRF activity decreased in the samples of the stalk-median eminence as well as the arcuate nucleus but it was maintained in the samples taken from that portion of the lateral retrochiasmatic area, which was outside the hypothalamic cut. We suggest that CRF containing fibers traverse the basallateral part of the retrochiasmatic area and the arcuate-ventro-medial region of the MBH en route to the stalk-median eminence and neural lobe. Within 3 days following partial isolation of the MBH neurosecretory material including CRF accumulates near the hypothalamic cut by redistribution in the damaged neurons, while by 7 days after transection the distal part of the axons in the MBH degenerates and is removed.

Microspectrophotometry of mitochondrial cytochrome P-450 in single adrenal cells.

SynopsisThe cytochrome P-450 is known to be a key enzymic component in steroid hydroxylation. Biochemically it is localized in adrenal cells, both in the mitochondrial and in the microsomal fractions. Owing to its characteristic light absorptivity, attempts were made at its localization and measurement in a microspectrophotometric system. Specific difference spectra were obtained in the cytoplasm of some cells from an adrenal cell suspension before and after saturation with carbon monoxide. Scanning at 450 nm showed slender absorption maxima after CO saturation randomly distributed in the cytoplasm. These may be attributed to mitochondria or larger cytoplasmic functional units including mitochondria.

Hydrolytic enzyme activity of rat thymic cells grown in vitro

SummaryAn electron-microscopic study of the fine-structural localization of acid phosphatase, non-specific esterase and aryl sulphatase activity in cultured rat thymus tissue showed the existance of three different types of cell, two of which conformed to the epithelial reticular cells and the macrophages present in vivo. The origin of the third is unknown. In all three types, and even within the very same cell, the lysosomes differ in activity. This finding argues in favour of the heterogeneity in the lysosomes of thymic cells.