Content of beta-LPH and its fragments (including endorphins) in anterior and intermediate lobes of the bovine pituitary gland

Radioimmunoassays (RIAs) specific for β-LPH_1–47, β-endorphin, α-MSH and β-MSH have been used to identify immunoreactive components in acid extracts from anterior and intermediate lobes of bovine pituitary gland after separation by chromatography on Sephadex G-50. When components in extracts of both lobes, eluting at the same position, were measured with the β-endorphin and β-LPH_1–47 RIA systems, marked quantitative differences were seen. The main components reacting with the β-LPH_1–47 system in anterior pituitary extract co-migrated with β-LPH and γ-LPH while in the intermediate lobe, the main immunoreactive component eluted at a position slightly later than β-endorphin. When the β-endorphin RIA system was used, relatively low amounts of immunoreactive material co-migrating with β-endorphin were seen in the anterior lobe extract while a highly predominant peak eluting at a position slightly later than β-endorphin was observed in intermediate lobe extract. Some β-MSH was seen in the intermediate lobe. These date indicate that the processing of β-LPH is markedly different in the anterior and intermediate bovine pituitary lobes: β-endorphin immunoreactive material predominates in the intermediate lobe whereas β-LPH and γ-LPH predominate in the anterior lobe.     

Delta-sleep-inducing peptide (DSIP) inhibited CRF-induced ACTH secretion from rat anterior pituitary gland in vitro

Delta-sleep-inducing peptide (DSIP, 10(-9) - 10(-7) M) significantly inhibited the CRF-induced ACTH release from rat anterior pituitary quarters in vitro. 10(-8) M DSIP showed the most prominent inhibition. DSIP (10(-8) M) also inhibited the CRF-activated cAMP levels in anterior pituitary tissue. DSIP did not influence basal ACTH or cAMP levels. Prostaglandin E2 (PGE2)-release from anterior pituitary quarters was not changed by DSIP. From these results, we conclude that DSIP inhibits CRF-induced ACTH release at the pituitary level through the inhibition of the cAMP system in corticotrophs. The involvement of PGE2 in this phenomenon is unlikely.     

Endorphins are located in the intermediate and anterior lobes of the pituitary gland, not in the neurohypophysis.

Immunocytofluorescence techniques with well characterized anti-sera to α-endorphin and β-endorphin show presence of these two peptides in all cellular elements of the pars intermedia of the rat hypophysis, and in discrete cells of the pars distalis (adenohypophysis) at the complete exclusion of the neurohypophysis (pars nervosa, posterior lobe).     

Estrogen regulation of the rat anterior pituitary gland proteome

Estrogen is known to affect the regulation of all six of the established anterior pituitary gland (AP) hormones, but little is known of the specifics of its regulation of the AP hormones, their isoforms, and nonhormonal AP proteins. We used difference gel electrophoresis in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting to quantify the effects of estrogen on the AP-soluble protein fraction in rats. Two-month-old rats were ovariectomized and used at 6 months of age. They were injected subcutaneously with sesame oil vehicle or 50 mug estradiol valerate in vehicle and studied 48 hrs later, approximately 3 hrs before the time of the anticipated onset of the estrogen-induced surges of gonadotropins in blood. The APs were pooled, and the soluble protein fraction was examined in replicate analyses. After DeCyder software analysis, we identified 26 protein spots that had a 1.5-fold or greater average increase in the experimental group relative to the controls. Nineteen showed a 1.5-fold or greater decrease. Estrogen increased levels of the more acidic isoforms of growth hormone and prolactin and of proteins involved in protein synthesis, folding, and secretion (e.g., eukaryotic translation elongation factor 2, ERp57, ERp29, Hsc70-ps1, calreticulin, coatomer delta subunit, and secretogranin II) and of some metabolic enzymes (e.g., arginosuccinate synthetase, enolase 1, creatine kinase B, phosphoglycerate mutase, malate dehydrogenase, pyruvate kinase, and aldolase A). The majority of the downregulated proteins were involved in RNA or DNA interactions (e.g., five heterogeneous nuclear ribonucleoproteins, DEAD-box proteins 17 and 48, ssDNA binding protein PUR-alpha, PTB-associated splicing factor, and Pigpen protein), but isovaleryl coenzyme A dehydrogenase, mitochondrial aldehyde dehydrogenase, stathmin 1, vinculin, radixin, and secretogranin III were also reduced. Our results indicate that estrogen acts in vivo within 48 hrs to modulate levels of a significant number of AP proteins.     

Dynamics and regulation of TSH secretion by superfused anterior pituitary cells

Superfused dispersed cells respond rapidly to 2- to 10-min pulses of TRH (10(-10) to 10(-7) M) in a dose-dependent manner. The effects of decreasing the stimulus duration can be overcome by a proportional increase in concentration of TRH. A TRH stimulus of 10 min or greater duration results in a sharp peak in TSH secretion followed by a lower plateau. Somatostatin (10(-8) M inhibits the response to TRH (t X 10(-9) M). T3 (2.0 microgram/dl) inhibits TRH-induced TSH secretion by superfused pituitary fragments, but not by dispersed cells. Corticosterone (50 microgram/dl), however, inhibits crude CRF-induced ACTH secretion by such cells.     

Cholinesterase in the posterior and intermediate lobes of the pituitary

Summary Posterior and intermediate lobes of pituitary glands of cat, rabbit, beef, and rat were examined histochemically for specific (AChE) and non-specific (BuChE) cholinesterase by light and electron microscopy. Acetylthiocholine was utilized in conjunction with ethopropazine to demonstrate AChE, and butyrylthiocholine with BW 284C51 to demonstrate BuChE. Glandular cells of the intermediate lobe of cat, rabbit and rat contained variable amounts of AChE, whereas those of beef contained BuChE. In the posterior pituitary, AChE was detected in the cat, BuChE in the beef and rat, and both AChE and BuChE in the rabbit. In the posterior lobe of all species examined, cholinesterase, whether true or pseudo enzyme, as the case may be, was localized to certain pituicytes and pituicyte-neuron junctions. These histochemical studies failed to identify cholinergic neurons in the posterior pituitary. Large blood vessels of the pituitary were innervated apparently by adrenergic nerves only. Speculations on the role of pituicyte cholinesterase in posterior pituitary secretion are presented.Supported by the Medical Research Council of Canada.Medical Research Associate of the MRC of Canada.     

ACTH in bovine pituitary intraglandular colloid, the holocrine secretion of intermediate lobe cells

The presence of bovine pituitary intermediate lobe peptides in intraglandular colloid, the holocrine secretion of intermediate lobe cells, is explored by ELISA. Intraglandular colloid collected immediately after sacrificing the animal, is placed in phosphate buffered saline, pH 7.6. This material is homogenized, centrifuged to remove extraneous tissue, lyophilized and stored at -20 degrees C. ACTH in intraglandular colloid is measured by competitive ELISA. Human ACTH (1-24) is used in the preparation of the solid phase antigen and as the standard for competition. The antibody is rabbit anti-human ACTH (1-24), and the alkaline phosphatase conjugate is goat anti-rabbit IgG with p-nitrophenyl phosphate as substrate. It is concluded that ACTH is present in bovine pituitary intraglandular colloid of intermediate lobe origin and that the colloid may serve as a transport medium for intermediate lobe materials.     

Existence of a common precursor to ACTH and endorphin in the anterior and intermediate lobes of the rat pituaitary

Extracts of rat anterior and intermediate-posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for immunoactive ACTH and endorphin. In both lobes the major forms of immunoactive ACTH have apparent molecular weights of 31,000 (31K), 20–21K, 14K, and 4.5K, and the major forms of immunoactive endorphin have apparent molecular weights of 31K (coincident with the peak of immunoactive ACTH), 13K (a βLPH-like peptide), and 3.5K (a β-endorphin-like peptide). However, the quantitative distribution of immunoactivity among the various forms differs greatly between the lobes. Assays using an extreme COOH-terminal ACTH antiserum indicate that the 31K ACTH/endorphin molecule in rat antierior and intermediate pituitary is similar to the pro-ACTH/endorphin molecule from mouse pituitary tumor cells. A radioimmunoassay that is specific for the NH₂-terminal non-ACTH, nonendorphin segment (referred to as 16K fragment) of the mouse pro-ACTH/endorphin molecule was used to assay extracts of rat pituitary. In addition to detecting material at 31K and 20–21K, the 16K fragment radioimmunoassay detects significant amounts of cross-reactive material with an apparent molecular weight of 16K in extracts of both lobes. This result also suggests that the structure and processing of the rat 31K ACTH/endorphin molecule is similar to that of mouse tumor cell pro-ACTH/endorphin. Cell suspensions were prepared from the anterior and intermediate lobes of the rat pituitary and maintained in culture for a 24-h period. The isolated cells from both lobes incorporate [³H] phenylalanine into immunoprecipitable ACTH- and endorphin-containing molecules. By sequential immunoprecipitation with ACTH and endorphin antisera, it is possible to demonstrate directly that a single molecule (31K ACTH/endorphin) has antigenic determinants for both ACTH and endorphin. Significant amounts of 31K ACTH/endorphin are released into the culture medium by isolated anterior lobe and intermediate lobe cells. The isolated intermediate lobe cells synthesize and secrete relatively large amounts of a β-endorphin-like molecule; the isolated anterior lobe cells secrete significant amounts of both a βLPH-like molecule and a β-endorphin like molecule. These same quantitative differences between anterior and intermediate lobe tissue were observed in immunoassays of extracts of the separated lobes and probably reflect differences in the processing of the common precursor. The isolated anterior lobe cells can be stimulated to release increased amounts of immunoprecipitable ACTH and endorphin by incubation with a cyclic AMP analog and a phosphodiesterase inhibitor.     

Electron microscopic histochemistry of cholinesterase in the posterior,intermediate, and anterior lobes of the rat pituitary

Summary The three lobes of the pituitary gland of the rat were examined histochemically for specific (AChE) and non-specific (BuChE) cholinesterase at the light and electron microscopic levels. Acetylthiocholine was utilized in conjunction with ethopropazine to demonstrate AChE, and butyrylthiocholine with BW284C51 to demonstrate BuChE. Using the histochemical method of Lewis and Shute, only BuChE was detected in the posterior pituitary by both light and electron microscopy; the enzyme was localized to certain pituicytes, including the endoplasmic and nuclear membranes of these cells and the pituicyte-neurosecretory neuron junctions. Endothelial cells of the posterior pituitary were also BuChE-positive. In the intermediate lobe, AChE was localized to the polygonal glandular cells, whereas BuChE was localized to cells of the interlobular septa and to elongated, densely staining cells which penetrate the lobules. In the anterior lobe, cells were positive for AChE, whereas follicular cells were positive for BuChE.Supported by the Medical Research Council of Canada.Medical Research Associate of the MRC of Canada.The authors wish to acknowledge the technical assistance of Mrs. Maria Prasher.