The subsynaptosomal distribution and release of [3H]acetylcholine synthesized by rat cerebral cortical synaptosomes

Synaptosomes were prepared from rat cerebral cortex and incubated in [³H]choline for periods ranging from 1 to 90 min. The [³H]ACh synthesized during this period was found only in the cytoplasm and in a membrane-associated fraction. A negligible amount of the newly formed [³H]ACh was recovered in the vesicular fraction despite concerted efforts to protect a hypothetical population of labile vesicles. The specific activity of the membrane-associated component, accounting for 21% of the total [³H]ACh, was by far the highest. This membrane-associated fraction was not released by hypotonic shock or homogenization and apparently was not in association with the monodisperse synaptic vesicles. The [³H]ACh was released in a calcium dependent manner. This investigation has determined that the ACh synthesized by synaptosomes is localized in only two fractions, cytoplasmic and membrane-associated; that this newly synthesized ACh can be released from synaptosomes by a process consistent with physiological release; and that at least part of the ACh released was originally present in the cytoplasm.     

Binding of a tritiated thyrotropin-releasing factor to a prolactin secreting clonal cell line (GH3)

The GH3 pituitary clonal cell line, which secretes prolactin and is responsive to thyrotropin-releasing factor, TRF, has been used to study the binding of a strongly labelled ³H-TRF (60 Ci/mM). This binding is time-dependent. It increases linearly in the first 15 min of incubation, thereafter reaching a plateau up to 60 min, independent of the dose (from 0.27 to 27 nM). This kinetic is similar to that of the increase of prolactin release induced by TRF. The specificity has been tested by comparing the affinity of GH3 pituitary cells with those of 3T3 fibroblasts and C6 glial cells and by competition experiment with unlabelled TRF and other peptides.After a constant period of incubation (30 min), the amount of bound molecules is dose-dependent. The two different rates of augmentation with increasing doses for low (1.35 to 155 nM) and high (155 to 1 080 nM) concentrations of ³H-TRF, suggest the involvement of at least two components in the³H-TRF binding to GH3 cells. Moreover, the presence of radioactive material within the cells is revealed by an autoradiographic study performed after a 30 min incubation with ³H-TRF.     

Culture requirements for optimal expression of 1α,25-dihydroxyvitamin D3-enhanced thyrotropin secretion

Summary An effect of the hormone, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on hormone secretion by normal rat pituitary cells was investigated in vitro. Based on previous findings using GH₄C₁ cells, dispersed anterior pituitary cell cultures were prepared and maintained in serum-free conditions for up to 6 d. Under these circumstances, there was no effect of 1,25(OH)₂D₃ to alter medium or cell-associated levels of thyrotropin (TSH), prolactin (PRL), or growth hormone (GH). Cultures maintained under these conditions had lower medium and cell-associated hormone levels and lesser responses to agonists than cultures maintained in serum-supplemented medium. In the presence of 10% charcoal-treated fetal bovine serum, treatment with 10⁻⁸ M 1,25(OH)₂D₃ for 24 h selectively increased TRH (10⁻¹⁰ to 10⁻⁷ M)-induced TSH secretion (P<0.001), with maximal enhancement observed at 10⁻⁹ M TSH-releasing hormone (TRH). Enhancement of TSH secretion by 1,25(OH)₂D₃ was detected after 15 min exposure to TRH. There was no effect on agonist-induced PRL or GH secretion or on cell-associated hormone levels. The effect was evident after 24 h treatment with 1,25(OH)₂D₃, and decreased thereafter. Several other steroid hormones had no effect on 10⁻⁹ M TRH-induced TSH secretion. These data contrast with the effect of 1,25(OH)₂D₃ in GH cells. They suggest that 1,25(OH)₂D₃ may act selectively in the normal pituitary to modulate TSH secretion.     

Cholecystokinin octapeptide releases growth hormone from the pituitary in vitro

Cholecystokinin-octapeptide (CCK-8)(10^?6 to 10^?8M) produced a marked increase in growth hormone (GH) release from incubated rat anterior pituitary quarters and from cultured GH₃ pituitary tumor cells. Although several CCK-8 analogues also caused GH release, bombesin, secretin and pancreatic polypeptide had no effect on GH secretion $\text{in vitro}$. In the GH₃ cell line, CCK-8 (10^?7M) reversed the inhibitory effect of somatostatin (10^?5M) on GH release. As CCK immunoreactivity has been demonstrated to be present in the hypothalamus, these results suggest that CCK-8 may be a physiologically important growth hormone releasing factor.     

Studies on growth hormone secretion VI. Effects of dibutyryl cyclic AMP,prostaglandin E1, and indomethacin on growth and hormone secretion by rat pituitary tumor cells in culture

The growth of rat pituitary tumor cells (GH₁ line) maintained in monolayer culture was inhibited by dibutyryl cyclic AMP in a dose-related fashion. Neither PGE₁ (2.8 × 10^?5M) nor indomethacin (2.8 × 10^?6M) had any significant effect on cell proliferation. Release of GH into the culture medium was stimulated by the cyclic AMP derivative but not by PGE₁ or indomethacin. In short term experiments (15 min.) both in intact monolayers and in trypsin-treated cells incubated in suspension, PGE₁ caused a 2–10 fold increase in cyclic AMP levels. This response, however, appeared to be of short duration reaching a maximum in 10 minutes. It is suggested that, at least in this line of pituitary tumor cells, PGE₁ does not mimic the effect of cyclic AMP, for it probably cannot sustain the elevated intracellular levels of this nucleotide which seem to be necessary for growth inhibition and enhanced GH secretion.     

Prostaglandin E2 (PGE2)-induced growth hormone (GH) release: Effect of intrahypothalamic and intrapituitary implants

Overiectomized rats were unilaterally implanted with a 23-gauge stainless steel cannula in different hypothalamic areas or in the pituitary gland and subsequently were treated with estrogen (sc, 10 μg estradiol benzoate, Eb). Two days after the estrogen injection, an inner cannula containing PGE₂ or PHF_2α at its tip was inserted into the cannula. Other animals were implanted with empty inner cannula. Plasma GH concentrations were measured by RIA in blood samples drawn from the jugular vein while the animals were lightly etherized before (−2) and at 20, 40, 60 and 120 min following the implantation. Plasma GH levels in control animals bearing an empty cannula in the body of the arcuate nucleus-median eminence region (BARH-ME) were signifantly depressed by the ether stress. The implantation of PGF_2α in this area was completely ineffective in preventing ether stress-induced decline in plasma GH. By contrast, PGE₂ implanted in BARH-ME or the post-chiasmatic region of the hypothalamus (HARH-ME) elevated plasma GH 20 min following its implantation and partially prevented the subsequent decrease in GH levels induced by ether stress. PGE₂ implants located in several other hypothalamic areas failed to induce GH release or to prevent the decline in GH levels induced by ether stress. However, PGE₂ implanted in the pituitary gland elicited a marked increase in plasma GH at 20 min and completely prevented the subsequent ether stress-induced decline in GH levels.The results suggest that PGE₂ can act at both hypothalamic (ARH-ME) and pituitary levels to stimulate GH release. At the hypothalamus, PGE₂ may inhibit GH-inhibiting factor (GIF) release or induce release of GH releasing factor (GHRF).     

Control of the production of two protein hormones by rat pituitary cells in culture

Summary A clonal strain of rat pituitary tumor cells (GH₃) has continued to produce the pituitary protein hormones growth hormone (GH) and prolactin during 5 years of continued growth in monolayer culture. Studies of the effects of external stimuli have indicated that, in spite of the physical similarity of these protein hormones (each is a single polypeptide of molecular weight ≈23,000), their production is controlled by different mechanisms. Addition of hydrocortisone (HC) (3×10⁻⁶ m) to the growth medium leads, after a lag of 12 to 24 hr, to an increased relative rate (rate in experimental cells divided by rate in control cells) of GH production. The relative rate reaches a maximum of 5 to 8 at 30 to 100 hr. Stimulation by HC of GH production is observed in cells growing in either the stationary or the exponential phase of growth. Indirect estimates indicate that, in exponentially growing cells, GH represents about 2% and 14% of the total protein synthesized by control and fully stimulated cells, respectively. Maintenance of the stimulated state requires HC. HC decreases both the growth rate of GH₃ cells and their incorporation of amino acids into acid-insoluble material. At the same time that HC stimulates GH production it decreases the relative rate of prolactin production to about 0.2 to 0.3. On the other hand, addition of acid extracts of bovine hypothalamus, cerebral cortex, kidney, or liver (0.3 to 1.0 mg of protein per ml) to the medium leads to an increase of the relative rate of prolactin production to 6 to 9, while decreasing the relative rate of GH production to about 0.5. Chromatographic fractionation of simple extracts of bovine liver has yielded a macromolecular, heatlabile fraction exhibiting these effects at a concentration as low as 20 μg per ml. GH₃ cells which have been adapted to growth in suspension culture produce both GH and prolactin. HC is observed to stimulate GH production and suppress prolactin production by cells growing in this state, without affecting the growth rate of the cells. This investigation was supported in part by Research Grant AM 11011 from the National Institute of Arthritis and Metabolic Diseases.     

Thyroid hormone and apotransferrin regulation of growth hormone secretion by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium

Summary Growth hormone (GH) production by GH₁ rat pituitary tumor cells in iron restricted serum-free defined medium requires apotransferrin (apoTf) and triiodothyronine (T₃). As measured by radioimmunoassay, apoTf plus T₃ induced GH levels 2 to 4-fold above controls. Deletion of either apoTf or T₃ arrested GH secretion. ApoTf/T₃ defined medium regulated GH production as effectively as whole serum. Because glucocorticoids enhance GH secretion in serum containing cultures, the effects of dexamethasone were evaluated in apoTf/T₃ defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoTf/T₃ defined medium. Even under these conditions, the response to dexamethasone remained T₃ dependent. These observations indicate that a yet to be characterized serum factor(s), other than apoTf, regulates the reponse to the steroid hormone. This is the first report of thyroid hormone regulation of GH secretion by rat pituitary tumor cells under completely serum-free chemically defined conditions.     

Permissive Effect of Insulin-like Growth Factor I (IGF-I) on Gonadotropin Releasing-hormone Action on In Vitro Growth Hormone Release,in Rainbow Trout (Oncorhynchus mykiss)

The aim of the work was to study the influence of insulin-like growth factor I (IGF-I) on GnRH-induced GH release by cultured pituitary cells of normally growing rainbow trout (Oncorhynchus mykiss), collected at different stages of gametogenesis. When pituitary cells were pre-incubated with human IGF-I (10⁻⁸ M) for 48 hours they became responsive to sGnRH (10⁻⁸ to 10⁻⁶ M) in the subsequent 24-hour incubation period, depending on the sexual stage, while not IGF-I pre-incubated cells were always non-responsive to GnRH. The permissive effect of IGF-I was detected in immature fish or those at the beginning of the gametogenesis, but not in mature fish. IGF-I inhibition of GH release during the preincubation period varies also with the sexual stage and is greater in immature than in mature fish. The permissive effect of IGF-I seems specific to somatotropes since IGF-I does not modify GnRH action on GtH₂ release. This work suggests that GnRH action on GH release can vary for a particular fish species depending on the physiological status.     

Specificity of the stimulatory effect of prostaglandins on hormone release in rat anterior pituitary cells in culture

Specificity of the effect of prostaglandins (PGs) on hormone release by the anterior pituitary gland was studied using cells in primary culture. Growth hormone (GH) release is stimulated by all eight PGs studied, PGE₁ and E₂ being 1000-fold more potent than the corresponding PGFs. The release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) remains unchanged upon addition of PGEs. While the basal release of thyrotropin (TSH) is only slightly stimulated by concentrations of PGEs above 10⁻⁶M, an important potentiation of the stimulatory effect of thyrotropin-releasing hormone on TSH release is observed. The release of GH, TSH and LH is stimulated equally well by PGAs and PGBs at concentrations higher than 10⁻⁶M, 3 × 10⁻⁶M, and 10⁻⁵M, respectively. PGFs do not affect the release of any of the measured pituitary hormones at concentrations below 10⁻⁴M. The stimulation of GH release by PGE₂ can be inhibited by the PG antagonist 7-oxa-13-prostynoic acid, a half-maximal inhibition being found at a concentration of 4 × 10⁻⁵M of the antagonist in the presence of 10⁻⁶M PGE₂. In the presence of somatostatin (10⁻⁸M), the inhibition of GH release cannot be reversed by PGE₂ at concentrations up to 10⁻⁴M. 8-bromo-cyclic AMP-induced GH release is additive with that produced by PGE₂.The present data show that 1) of the five pituitary hormones measured, only GH release is stimulated by prostaglandins at relatively low concentrations, 2) the PGE-induced GH release can be competitively inhibited by 7-oxa-13-prostynoic acid, 3) the inhibition of GH release by somatostatin cannot be reversed by PGE₂ and 4) the PGEs increase the responsiveness of the thyrotrophs to TRH.     

Pituitary localization of 3H-spiroperidol by an Uptake/Storage Mechanism?

The lack of a pituitary imaging agent combined with the considerable clinical value for such an agent prompted an examination of ³H-spiroperidol (³HSp). Spiroperidol was selected for initial evaluation based on its high affinity for D₂ receptors which are known to be present in the pituitary. A time course study of ³HSp concentration in rat pituitary and other tissues was conducted. Pituitary activity levels were found to be constant from 5 min to 4 h and were about 8 times levels in corpus striatum at 1 h. Blocking studies with (+)-butaclamol and with unlabelled spiroperidol suggested the existence of both a D₂ receptor mediated binding localization and a second uptake which is postulated to be an internalization process. Further studies involving ultracentrifugation of pituitary homogenates resulted in evidence for association of ³HSp with dense subcellular particles. ³HSp thus appears to be internalized by pituitary cells.     

Effect of prostaglandin F2α on anterior pituitary function in man

Five healthy adult men received iv PGF_2α at dosages of 0.05, 0.20 and 2.0 μg/kg/min for 30 min. There were no significant changes in serum FSH, LH or TSH levels. Serum GH and cortisol levels were slightly increased at the highest dosage. These responses were associated with, and presumably a result of, stressful side effects. Thus, PGF_2α cannot be used as a provocative test of pituitary hormone reserve.Prostaglandins (PG's) have recently been implicated in the release of a number of hormones from the anterior pituitary gland. The stimulation of GH release by PG's of the E series from incubated rat pituitary slices has been demonstrated. $\text{In vivo}$ stimulation by PGE₁ of ACTH in rats and of GH release in man has also been shown.The present study was undertaken in order to examine the efficacy of iv administration of PGF_2α as a provocative test of anterior pituitary hormone reserve in man. The responses in circulating levels of gonadotropins, TSH, GH, and cortisol (as an index of ACTH) were measured.     

Antagonism of prostaglandin-promoted pituitary cyclic amp accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid

The studies reported here confirm the previously observed potent stimulus to growth hormone (GH) secretion by prostaglandin E₁ (PGE₁). Proportional increments in GH secretion were observed following $\text{in vitro}$ addition of PGE₁ over a concentration range of 10^?7 to 10^?5 M. Growth hormone secretion could not be further stimulated by higher concentrations of prostaglandin. Prostaglandin E₁ also increased cyclic AMP concentration in the pituitary explants in a proportional fashion, which correlated closely with its potency as a growth hormone secretogogue. In order to define more precisely the mechanism by which prostaglandin acts, the effects of prostaglandin antagonist, 7-oxa-13-prostynoic acid, on GH secretion and cyclic AMP accumulation were investigated. Addition of the antagonist alone had no consistent effects on GH secretion or cyclic AMP levels in the pituitary. However, the antagonist significantly reduced the stimulation of hormone release and cyclic AMP accumulation found following addition of PGE₁. Increasing the concentration of antagonist further diminished prostaglandin stimulated hormone release and nucleotide accumulation. The antagonist failed to block the stimulatory effects of theophylline and dibutyryl cyclic AMP on GH release, indicating that the inhibition observed occurred prior to intracellular accumulation of the cyclic nucleotide. These results are consistent with the hypothesis that a prostaglandin receptor on the pituitary somatotrope is linked to the adenyl cyclase-cyclic AMP system.     

Cloning and differential expression of the growth hormone in Pampus argenteus

The growth hormone (GH) is a pluripotent hormone produced by the pituitary in vertebrates. It plays important roles in the growth, development, and metabolism of vertebrates.We cloned GH cDNA sequence of Pampus argenteus (GenBank: KT257176). Multi‐sequence analysis revealed P. argenteus GH cDNA contained four conservative cysteine residues positions (Cys⁶⁹, Cys¹⁷⁷, Cys¹⁹⁴, and Cys²⁰²) and shared more than 51.5% identity with homologues from other reported bony fish GHs, except that of Lepisosteus osseus. We used semi‐quantitative RT‐PCR and quantitative real‐time PCR to detect GH expression in 10 tissues and GH expression levels in the pituitary at six different growth stages, and also detected GH content in serum at different growth stages . qPCR showed that GH mRNA was detected in the liver, muscle, kidney, intestine, pituitary, olfactory bulb, stomach, heart, gill, and ovary. The highest level of P. argenteus GH mRNA was observed in the pituitary (P < 0.01, n = 3). At different growth stages, P. argenteus GH expression first increased, decreased, and increased again. GH gene expression levels and the variations of serum GH levels of P. argenteus were consistent with the growth rate and associated with the sexual maturity. In addition, in situ hybridization was used to locate the GH expression in pituitary. In situ hybridization showed that the GH‐positive cells were round, oval, or irregular and often gathered into groups or presented branches along the nerve fibers.     

Synthesis and biological activity of four gamma-melanotropin peptides derived from the cryuptic region of the adrenocorticotropin/beta-lipotropin precursor.

A third melanotropin coding fragment named γ-MSH was discovered by Nakanishi et al (Nature $\text{278}$, 423–427 (1979)) in the cryptic region outside the portion coding for ACTH and β-LPH in the ACTH/β-LPH precursor mRNA isolated from the intermediate lobe of bovine pituitary. Four possible γ-MSH peptides derived from this coding fragment were synthesized by solid-phase methodology and their bioactivity determined in an $\text{in vitro}$ MSH assay as well as the anterior pituitary primary culture assay. Relative to α-MSH, the melanotropic activities of Ac-γ₁-MSH, γ₁-MSH, γ₂-MSH and γ₃-MSH are 7.3 × 10^?4, 3.3 × 10^?5, 1.4 × 10^?4 and 4.6 × 10^?7 respectively. None of these γ-MSH peptides releases LH, FSH, PRL, GH and TSH in the pituitary culture medium at a dose as high as 100 ng per dish.     

ANALYSIS OF THE PREFERENTIAL RELEASE OF NEWLY SYNTHESIZED ACETYLCHOLINE BY CORTICAL SLICES FROM RAT BRAIN WITH THE AID OF TWO DIFFERENT LABELLED PRECURSORS

—The release of newly synthesized acetylcholine (ACh) by cortical slices from rat brain in the presence of 25 mm -KCl was studied. The slices were incubated for 5 min in a medium containing both [2-¹⁴C]pyruvate and choline labelled with 3 deuterium atoms (choline-d₃) in order to label at the same time the acetyl moiety and the choline moiety of ACh. The non-labelled ACh and the ACh-d₃ were measured by pyrolysis-gas chromatography/mass spectrometry, and the [^I4C]ACh by liquid scintillation counting. It was found that the newly formed [⁴C]ACh as well as the newly formed ACh-d₃ had a more than 2.5 times greater probability of being released than the preformed non-labelled ACh. These findings strongly suggest that it is not simply the ACh synthesized immediately inside the nerve ending membrane from incoming undiluted labelled choline, which is preferentially released, but that all newly formed ACh has a greater probability of being released than preformed ACh. No preferential release of newly formed ACh was observed when the incubation medium contained 5.6 mm -pyruvate instead of 10 mm -glucose + 0.6 mm -pyruvate. The cause of this difference remains unexplained.     

Zinc retention differs between primary and transformed cells in response to zinc deprivation

Previous studies in our laboratory have demonstrated that reducing the availability of zinc with the extracellular metal chelator DTPA (diethylenetriaminepentaacetate) enhances, rather than inhibits, the thyroid hormone induction of growth hormone mRNA in GH3 rat anterior pituitary tumor cells. To understand the actions of the chelator on cellular zinc status, we observed the effects of DTPA on ⁶⁵Zn uptake and retention. DTPA reduced the uptake of ⁶⁵Zn by GH3 cells from the medium, but when GH3 cells were prelabeled with ⁶⁵Zn, it resulted in greater retention of the isotope. In primary hepatocytes, DTPA both reduced the uptake of ⁶⁵Zn from the medium and increased efflux from prelabeled cells. To investigate this difference, we studied the effects of DTPA on radioactive zinc flux in the H4IIE (rat hepatoma), MCF-7 (human breast cancer) and Hs578Bst (nontransformed human mammary) cell lines and in rat primary anterior pituitary cells. DTPA reduced the uptake of ⁶⁵Zn in all cell lines examined. DTPA increased the retention of ⁶⁵Zn in prelabeled H4IIE, MCF-7 and Hs578Bst cells but reduced it in primary pituitary cells. Time course experiments showed that ⁶⁵Zn efflux is shut down rapidly by DTPA in transformed cells, whereas the chelator causes greater efflux from primary hepatocytes over the first 6 h. Experiments with ¹⁴C-labeled DTPA confirmed that this chelator does not cross cell membranes, showing that it operates entirely within the medium. Expression of ZnT-1, the efflux transporter, was not affected by DTPA in H4IIE cells. Thus, zinc deprivation enhanced zinc retention in established cell lines but increased efflux from primary cells, perhaps reflecting differing requirements for this mineral.     

Poly(A)-protein interactions and transport of mRNA in isolated nuclei.

RNA-protein complexes (RNP), formed in a cell-free system using nuclei from GH cells, prelabelled, with [³H] leucine,were isolated from nucleoplasm and from postnuclear supernatant (PNS). Radioactive proteins, associated with newly synthesized HnRNP and mRNP-like material in the PNS were examined. On SDS gels, the [³H] protein pattern of poly(A)HnRNP was found to be more complex than that of PNS poly(A)-RNP. Only three radioactive proteins (78K, 100K and 120K) were associated with the poly(A) segments of cell-free formed HnRNP and PNS poly(A)-RNP. Cordycepintriphosphate and α-amanitin reduced the transport of cell-free formed poly(A) RNP associated [³H] proteins to the extent of 52% and 46% respectively. It may be concluded that the poly(A) segment of newly synthesized mRNA-like material is directly or indirectly (by providing binding sites for specific proteins) responsible for the transport of poly(A) containing mRNA.     

Thyroliberin is rapidly transferred to the nucleus of GH3 pituitary cells at both 4°C and 37°C

The time-course of thyroliberin transfer to the nucleus of GH3/B6 rat pituitary prolactin cells was studied by both autoradiography and cell fractionation of intact cells exposed to [³H]thyroliberin at 4°C or 37°C. It was previously shown that thyroliberin is not degraded in these conditions. It is found by autoradiography that [³H]-thyroliberin is transferred to the nucleus of GH3/B6 cells within 5 min at least at both 37° C and 4°C. Consistent results are obtained by fractionation of cells exposed to [³H]thyroliberin at 37°C. However after binding at 4°C 50% of the cell radioactivity is extractible by glutaraldehyde and after fractionation the isolated nuclei retain only 1–1.5% of the cell radioactivity. This suggests the existence of both tightly bound and loosely bound internalized thyroliberin molecules.