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The parathyroid hormone-related peptide (PTHrP) gene has been reported to be subject to a wide variety of physiological and pharmacological controls. Two distinct patterns of PTHrP mRNA response have been recognized, one characterized by a prolonged or plateau response lasting many hours to days and the second characterized by rapid induction-deinduction kinetics and lasting 1 to several hours. The kinetics of the second pattern are similar to those displayed by primary response genes like nuclear protooncogenes, cytokines, and growth factors. In GH4C1 rat pituitary cells, 17 beta-estradiol induced a rapid and transient increase in PTHrP mRNA expression, with a peak response at 1-2 h. This response appeared to be due to a rapid and transient burst in gene transcription, which by runoff analysis was maximal at 20-40 min and declined thereafter. PTHrP mRNA half-life was 30 min in these cells and was unaltered by estradiol. Cycloheximide did not block the 17 beta-estradiol-induced response but rather prolonged it, and runoff analysis revealed that this effect was due to a prolongation or persistence of PTHrP gene transcription. These findings suggest that the transient nature of the native response reflects the effects of an estrogen-inducible repressor. All of these features are characteristic of a prototypical primary response gene.

Myoblasts transfected with HB10D insulin secrete more hormone than those transfected with wild-type insulin, as published previously, indicating that production of wild-type insulin is not efficient in these cells. The ability of non-beta-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH(4)C(1) cells stably transfected with proinsulin, two thirds of (35)S-proinsulin was degraded within 3 h of synthesis, whereas (35)S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of (35)S-proinsulin was degraded within 3 h after synthesis, whereas (35)S-GH was stable. In transiently transfected fibroblast COS cells, (35)S-proinsulin was stable for longer, but less than 10% was secreted 8 h after synthesis. Proinsulin formed a concentrated patch detected by immunofluorescence in transfected cells that did not colocalize with calreticulin or BiP, markers for the endoplasmic reticulum, but did colocalize with membrin, a marker for the cis-medial Golgi complex. Proinsulin formed a Lubrol-insoluble aggregate within 30 min after synthesis in non-beta-cells but not in INS-1E cells, a beta-cell line that normally produces insulin. More than 45% of (35)S-HB10D proinsulin was secreted from COS cells 3 h after synthesis, and this mutant formed less Lubrol-insoluble aggregate in the cells than did wild-type hormone. These results indicate that proinsulin production from these non-beta-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of beta-cells may prevent aggregation of proinsulin to allow efficient production.

Treatment of rat pituitary GH4C1 cells with estradiol, insulin, and epidermal growth factor induces secretory granule accumulation, PRL storage, and stabilization of ICA512, a membrane protein associated with secretory granules. In these investigations we found that the same treatment induced accumulation over 2-fold of other proteins in the secretory pathway, including synaptosomal-associated protein of 25 kDa (SNAP-25), synaptotagmin III, synaptobrevin, synaptophysin, and cyclophilin B, and did not affect accumulation of others, including synaptotagmin I, calnexin, and glucose-regulated protein 94. The induction of proteins was not a coordinate event, because epidermal growth factor alone maximally stimulated SNAP-25 accumulation, but not that of synaptotagmin III. Induction of SNAP-25 accumulation occurred without an increase in its synthesis, and induction of cyclophilin B occurred without an increase in its messenger RNA accumulation, suggesting that accumulation may be caused by stabilization of the proteins. SNAP-25 immunofluorescence was located in the cytoplasm and on the plasma membrane and sometimes was heavily concentrated in protrusions from the cell surface, especially in hormone-treated cells. Frequenin immunofluorescence was also sometimes concentrated in intense patches, but did not colocalize with SNAP-25. Growth hormone and prolactin immunofluorescence was not found in the protrusions and sometimes did not colocalize with each other when they were present in the same cell. Hormone treatment of GH4C1 cells therefore induces accumulation of specific proteins in all parts of the secretory pathway and causes morphological changes in addition to accumulating secretory granules.

We examined the biological activity and immunological reactivity of four mutants of human PRL. Two were mutants that changed the ability of human PRL to inhibit rat PRL storage when transfected into a rat pituitary cell line:mutations S34A and N31T. Two mutations were in regions of PRL that are highly conserved. One, des(3-11)-PRL, removed the N-terminal cystine loop that most PRLs, except those from certain fish, have, and no GHs have. The other, S90A, mutated a serine that is present in all PRLs but those from some fish and in all GHs. The immunological properties of des(3-11)-PRL were reduced 10-fold compared to those of wildtype human PRL in a RIA using NIH antihuman PRL-3, AFP C11580; the others were similar to those of wild-type PRL. The biological activity of des(3-11)-PRL was the most affected; activity was reduced about 8-fold compared to that of wild-type PRL in the Nb2 cell assay. The activities of the others were similar to that of the wild type. Serine 90 may be partially buried by loops connecting the alpha-helixes. The mutation of serine 90 did not affect the stability of the molecule in vitro, determined by comparing the red shift in tryptophan fluorescence that occurs with increasing concentrations of urea in S90A and wild-type PRL. The activity of S34A and N31T mutations indicates there is no correlation between biological activity and ability to affect storage. The N-terminal cystine loop may be conserved because it is needed for biological activity, but the conservation of serine 90 in GH and PRL must be determined by other properties, such as spacial requirements.

GH4C1 cells, rat pituitary tumor cells, produce PRL, but store little relative to normal lactotrophs; treating cells with estradiol, insulin, and epidermal growth factor increases both PRL storage and accumulation of dense core granules. Normal lactotrophs contain several differently charged forms of PRL. We investigated whether inducing PRL storage in GH4C1 cells altered the production of these forms. Four forms of PRL that differed by charge were found by immunoblots of two-dimensional gels of extracts of female rat pituitary glands and of secretory granules isolated from the glands. Four forms were also secreted by GH4C1 cells. The relative abundance of the four forms in the medium of GH4C1 cells, determined by [35S]amino acid incorporation for 24h, was 1.2 +/- 0.58%, 91.3 +/- 1.09%, 6.3 +/- 0.72%, and 1.2 +/- 0.39% of total PRL (mean +/- SEM), from the most basic to the most acidic, respectively. Treatment with 1 nM estradiol, 300 nM insulin, and 10 nM epidermal growth factor did not significantly change the relative abundance of the forms. All four forms also were found in GH4C1 cells after 2 h of incubation with [35S]amino acids, although no incorporation of 32PO4 was detectable over the same incubation time. We conclude that GH4C1 cells produce four forms of PRL that differ by charge, as normal lactotrophs do. The increase in storage of PRL caused by insulin, estrogen, and epidermal growth factor does not result in or is caused by increased secretion of a specific form.

PRL release from rat lactotrophs in response to TRH is Ca2+ dependent. TRH-induced PRL release is inhibited either after repeated pulses of TRH or in the presence of dopamine. TRH, however, generates increases in intracellular Ca2+ concentrations ([Ca2+]i) in both conditions. Calcium/calmodulin-dependent protein kinase-II (CaM kinase-II) is a ubiquitous enzyme implicated in secretion. To determine whether down-regulation of CaM kinase-II activity caused the lack of responsiveness to increases in [Ca2+]i, we measured the generation of calcium/calmodulin-independent kinase activity. Anterior pituitary cells contain a 50-kilodalton form of CaM kinase-II, determined by immunoblot, and the enzyme is in lactotrophs, determined by immunocytochemistry. TRH rapidly and transiently increased calcium/calmodulin-independent kinase activity; the increase was maximal by 15 sec and returned to basal by 2 min. When TRH pulses (1 microM, 15 sec) were applied every 10 min, each pulse caused an increase in calcium/calmodulin-independent kinase activity of similar magnitude, and the activity returned to basal values between pulses. Pretreatment of cells with dopamine (1 microM; 30 min) inhibited PRL release, but did not prevent the increase in calcium/calmodulin-independent kinase activity. These results indicate that TRH still activates CaM kinase-II when PRL release is inhibited. Dopamine and repeated pulses of TRH must inhibit PRL release at a site after the TRH-induced increase in [Ca2+]i and at a site other than CaM kinase-II.

There are three steps in the formation of secretory granules: aggregation of proteins to form the dense cores of granules, accumulation of appropriate membrane proteins necessary for function of the granules, and removal of extraneous membrane and inappropriate proteins by small vesicles. Formation of protein aggregates may be the initial step in this process, which is not well understood. Assays of aggregation of human prolactin and growth hormone in neuroendocrine cells indicate that acidic intracellular compartments are necessary, and Zn2+ and Cu2+ may facilitate aggregation through low affinity binding sites. There is more than one way to make proteins aggregate in solution; precipitates of human prolactin formed in "crowded" conditions most closely resemble what is likely to occur in cells. Understanding the properties of aggregates formed in cells may be important, as there are several examples of granules with different contents that function differently; human R183H-growth hormone, a mutant that causes autosomal dominant isolated growth hormone deficiency, also appears to be an example. Recognition of surface motifs on aggregates of proteins may be important to localize correctly membrane proteins necessary for function, an explanation for the means by which granule content may influence function.

Rat prolactin in the dense cores of secretory granules of the pituitary gland is a Lubrol-insoluble aggregate. In GH(4)C(1) cells, newly synthesized rat prolactin and growth hormone were soluble, but after 30 min about 40% converted to a Lubrol-insoluble form. Transport from the endoplasmic reticulum is necessary for conversion to Lubrol insolubility, since incubating cells with brefeldin A or at 15 degrees C reduced formation of insoluble rat (35)S-prolactin. Formation of Lubrol-insoluble aggregates has protein and cell specificity; newly synthesized human growth hormone expressed in AtT20 cells underwent a 40% conversion to Lubrol insolubility with time, but albumin did not, and human growth hormone expressed in COS cells underwent less than 10% conversion to Lubrol insolubility. del32-46 growth hormone, a naturally occurring form of growth hormone, and P89L growth hormone underwent conversion, although they were secreted more slowly, indicating that there is some tolerance in structural requirements for aggregation. An intracellular compartment with an acidic pH is not necessary for conversion to Lubrol insolubility, because incubation with chloroquine or bafilomycin slowed, but did not prevent, the conversion. GH(4)C(1) cells treated with estradiol, insulin, and epidermal growth factor accumulate more secretory granules and store more prolactin, but not more growth hormone, than untreated cells; Lubrol-insoluble aggregates of prolactin and growth hormone formed to the same extent in hormone-treated or untreated GH(4)C(1) cells, but prolactin was retained longer in hormone-treated cells. These findings indicate that aggregation alone is not sufficient to cause retention of secretory granule proteins, and there is an additional selective process.

Human PRL binds Zn2+, but the function of the binding is not known. We investigated the effect on PRL production in pituitary cells by obtaining clones of GH4C1 cells stably transfected with human H27A-PRL, a mutant that does not bind Zn2+. Unexpectedly, clones transfected with the mutant human PRL made little rat PRL. Untransfected GH4C1 cells made between 0.5 to 10 μg rat PRL/105 cells in 24 h. Clones transfected with vector alone (four of four), wild type human PRL (six of six), or with human K69A-PRL (two of two) made amounts of rat PRL in the same range. Clones transfected with human H27A-PRL (five of five) made 0.003–0.1 μg rat PRL/105 cells in 24 h, and the production of rat PRL mRNA was reduced. Human H27A-PRL was not efficiently secreted; 20–40% newly synthesized H27A-PRL was degraded by 60 min, and there was usually a delay in release of newly synthesized H27A-PRL. Reduction of rat PRL production is not mediated through the PRL receptor, because no sequences for the receptor in GH4C1 cells were detected by RT-PCR. Proteins involved in folding, such as BiP, were not specifically elevated in the H27A-PRL clones. In transient transfections, in which cells have not undergone selection, we found no evidence for disulfide-bonded aggregates of the mutant protein. The results indicate that Zn2+ binding stabilizes PRL in the secretory pathway; the instablility of the mutant protein may trigger effects that suppress rat PRL production directly or that indirectly result in selection of clones with low rat PRL production.

GH4C1 cells increase storage of rat PRL and accumulation of dense core secretory granules when they are treated with 300 nM insulin, 1 nM estradiol, and 10 nM epidermal growth factor. In the experiments reported here, intracellular rat PRL increased from 4% of the total amount produced in 24 h in control cultures to 15% in treated cultures. When GH4C1 cells were transfected with DNA sequences so that they coexpressed human PRL, the storage of rat PRL was no longer induced by hormone treatment; transfected clones contained less than 5% of the total produced in 24 h in both control and treated cultures. The sum of rat and human PRL produced by these clones was not more than rat PRL produced by the untransfected cells, so the storage capacity of the cells was not exceeded. The transfected clones made between 1 to 40 times more rat than human PRL. Release of human and rat PRL was stimulated by depolarizing the cells, indicating both still were in a regulated pathway, even though storage could not be induced. Mutations of human PRL with threonine substituted for asn31 or alanine substituted for ser34 did not block induction of rat PRL storage when coexpressed in GH4C1 clones. We conclude the ability to increase rat PRL storage is a process with marked specificity because it is inhibited by relatively low amounts of human PRL and inhibition requires asn31 and ser34 in human PRL. Such specificity is consistent with a receptor-mediated mechanism that concentrates PRL into dense cores of secretory granules.

The dihydropyridine Ca2+ channel activator BAY K 8644 (1 microM) stimulated basal prolactin secretion from perifused primary cultures of anterior pituitary cells and potentiated the stimulation of prolactin secretion by 1 microM thyrotropin-releasing hormone (TRH) 5-fold over 30 min. This potentiation was mimicked by other dihydropyridine agonists CGP 28392 and (+)-SDZ 202-791 and by (-)-BAY K 8644 (1 microM), but not by (+)-BAY K 8644. The Ca2+ channel antagonist nimodipine, at a concentration sufficient to block BAY K 8644-stimulated 45Ca2+ uptake in GH4C1 anterior pituitary tumor cells, decreased basal prolactin secretion and blocked the enhancement of basal and TRH-stimulated secretion by BAY K 8644. These results suggest that dihydropyridine agonists potentiate TRH-induced secretion through interaction with known stereospecific sites on Ca2+ channels. In GH4C1 cells, BAY K 8644 alone did not affect inositol polyphosphate accumulation, but potentiated TRH-stimulated accumulation of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. Accumulation of the Ca(2+)-mobilizing isomer inositol 1,4,5-trisphosphate was not potentiated, suggesting that potentiation of TRH-stimulated hormone secretion by BAY K 8644 does not result from synergistic stimulation of phospholipase C, but may correlate with enhanced inositol trisphosphate-3-kinase activity.

Rhodamine 123 is a lipophilic cationic fluorescent dye that localizes in mitochondria. We found that 17 beta-estradiol changes the ability of GH4C1 cells, clonal rat pituitary tumor cells, to retain rhodamine 123. Cells incubated with 10 micrograms/ml rhodamine 123 for 30 min at 37 C took up about equal amounts of rhodamine 123, as determined by fluorescence microscopy, regardless of whether they had been treated with estradiol. After three 5-min washes at 37 C, cells treated with 1 nM estradiol for 7 days before incubation with rhodamine 123 had lost more fluorescence than untreated cells. We further characterized the effect by flow cytometry. The difference in fluorescence between control and treated cells ranged from 50- to 500-fold. The effect of estradiol was maximal at 10(-10) M and took a week to develop fully. The effect is specific for estradiol, because estradiol and diethylstilbestrol reduced retention of rhodamine 123 fluorescence at 10(-10) M, but the same concentrations of dihydrotestosterone, progesterone, dexamethasone, and cholesterol did not. To test if the effect on rhodamine 123 fluorescence was caused by activation of the multidrug resistance transport system, we examined the effect of estradiol on the retention of daunomycin, a known substrate of the transport system. Estradiol treatment caused a 3-fold decrease in daunomycin fluorescence. We isolated clones resistant to estradiol-induced loss of rhodamine 123 fluorescence by flow cytometry and found that two clones still showed an estradiol-induced decrease in daunomycin fluorescence equivalent to that of the parent line.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine inhibits prolactin production from cell cultures of rat pituitary glands with a half-maximal effect at about 0.2 μM, and from GH-cells, clonal strains of rat pituitary cells, with a half-maximal effect at about 1 μM. The inhibition ranges between 80 and 40 % of control values. Inhibition is detectable at 2 hours, and continues for days in the presence of the anticholinesterase, eserine. Muscarinic agonists mimic the cholinergic inhibition and nicotinic agonists do not. The inhibition is blocked by atropine, a muscarinic antagonist, and not by hexamethonium, a nicotinic antagonist.

Thyrotropin-releasing hormone (TRH) has 3 effects on clonal strains of rat pituitary cells in culture (GH-cells). Two long-term effects of TRH on GH-cells, which are measurable after 3 h or longer, have been previously reported; these are an increase in prolactin synthesis and a decrease in growth hormone production. We report here that TRH also stimulates the rapid release of stored intracellular prolactin. We have investigated the role of cyclic AMP as a possible mediator of the effects of TRH on GH-cells. Cyclic AMP concentrations are higher in cells treated with TRH compared with paired controls; a maximum difference of greater than 150% of control values is detected at 15 min if the incubation is performed in serum-free medium in the presence of 1 mM theophylline. The concentration of TRH required to give half-maximum increases in both prolactin release and cyclic AMP accumulation is 0.3 nM; half-maximal increases in prolactin synthesis occur at 3 nM TRH. Exogenous cyclic AMP (1 mM) causes only a slight increase in prolactin release; 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (1 mM) do not cause significant release. Phosphodiesterase inhibitors (0.3 mM theophylline, 0.03 mM isobutyl-methylxanthine) increase prolactin release but their effects on hormone synthesis are more complicated. Isobutylmethylxanthine, 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (0.4 MM) increase prolactin synthesis, but do not significantly affect growth hormone synthesis. Theophylline increases the synthesis of both hormones. Dibutyryl cyclic AMP (0.5 mM or more) increases prolactin release and both growth hormone and prolactin synthesis, but equivalent amounts of sodium butyrate have the same effects. We conclude that in GH-cells under carefully defined experimental conditions: 1) TRH causes an increase in intracellular cyclic AMP concentrations; 2) the increase in endogenous cyclic AMP and the effects of phosphodiesterase inhibitors are consistent with a model with cyclic AMP as a mediator of the effects of TRH on prolactin release; however, they do not prove this model, because the interpretation of these results depends on assumptions which may not all be valid; and 3) none of the analogs of cyclic AMP or the phosphodiesterase inhibitors tested mimic the decrease in growth hormone production caused by TRH.

We have examined the effects of cholera toxin, a specific probe for processes which are caused by an increase in cAMP, on clonal strains of rat pituitary cells (GH cells) in culture. We found that 5 ng/ml cholera toxin increased the amount of intracellular cAMP after a lag period of 30-60 min. After a similar lag period, cholera toxin increased the release of PRL into the medium by 50% and caused a decrease in intracellular PRL to 60% of control values at 90 min. PRL synthesis was increased to 100% above control after 1 week of treatment, and GH synthesis was elevated to 280% above control. TRH caused an increase in PRL release and synthesis but a decrease of GH synthesis. Depending on the dose, TRH prevented or reduced the cholera toxin-induced rise in GH synthesis. We conclude that the decrease in GH synthesis caused by TRH is not likely to be mediated through an increase of intracellular cAMP.

The gene for subunit II of cytochrome oxidase is part of the mitochondrial genome. 17 beta-Estradiol, 1 nM, increased the levels of cytochrome oxidase II mRNA in the GH4C1 pituitary tumor cell line; the increases ranged from 3- to 16-fold over controls in different experiments. Insulin, 300 nM, estradiol, 1 nM, and epidermal growth factor, 10 nM, together caused a larger increase in cytochrome oxidase II mRNA accumulation than did estradiol alone. The dose-response relationship for the induction of cytochrome oxidase II mRNA by estradiol was similar to that for PRL mRNA; maximal induction occurred at about 10(-9) M. This concentration is 10-fold greater than that required for maximal stimulation of cell proliferation and of 1C28, another estrogen-inducible mRNA, indicating that the increase in cytochrome oxidase II mRNA is not a result of increasing the growth rate of the cells. The increase in cytochrome oxidase II mRNA was not caused by an increase in the number of copies of the cytochrome oxidase II gene. Estradiol therefore must induce in the mitochondria an increase in transcription or a decrease in degradation of cytochrome oxidase II mRNA.

Bromocriptine, a dopamine agonist, inhibited secretion of PRL and did not affect GH release from rat anterior pituitary cells in culture. The reversal of this inhibition of PRL release by butaclamol, a dopamine antagonist, was stereospecific; 10 nM d-butaclamol completely reversed the inhibition caused by 10 nM bromocriptine, while l-butaclamol had no effect at concentrations up to 10 microM. However, both enantiomers at 10 microM inhibited PRL release to 30% and GH release to 91% of control values. Two other dopamine antagonists also inhibited hormone release. Haloperidol (10 microM) inhibited PRL release to 23% of control values and did not affect GH release; 3.3 microM pimozide inhibited PRL and GH release to 18% and 38% of control values, respectively. These data indicate that, the inhibition of PRL by antipsychotic drugs is not mediated through the dopamine receptor.

To investigate the relationship of changes in cytosolic free calcium concentrations [( Ca2+]c) caused by TRH to changes in PRL secretion, we simultaneously monitored PRL release and [Ca2+]c, using the fluorescent Ca2+ indicator indo-1, in freshly isolated perifused cells from rat anterior pituitary glands. We found that a 30-sec pulse of 100 nM TRH triggered a transient spike of [Ca2+]c, but prolonged PRL release for up to 30 min; continuous administration of TRH caused a sustained elevation in [Ca2+]c, but the same pattern and amount of PRL release as that caused by the pulse of TRH. PRL secretion was refractory to further pulses of TRH given at 10-min intervals for 40 min, but did respond to a second pulse of TRH given 40 min after the first pulse with no intervening pulses. Pulses of TRH given every 10 min still triggered spikes of [Ca2+]c of the same magnitude as the first pulse, indicating that the cause of the refractory state must occur at a post-receptor step that is after the mobilization of [Ca2+]c. A 30-sec pulse of a high concentration of KCl caused a transient spike of [Ca2+]c and transient, not prolonged, release. Additional pulses of KCl cause progressively less PRL release, although the magnitude of the spikes in [Ca2+]c did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate whether dopaminergic reduction of PRL secretion is caused by an inhibition of release of Ca2+ from intracellular stores, we used a perifusion system to monitor simultaneously changes in intracellular Ca2+ concentrations ([Ca2+]c) and changes in PRL secretion from rat anterior pituitary cells, and from enriched populations of lactotrophs. We eliminated influx of extracellular Ca2+ by using medium with no added Ca2+ and 0.2 mM EGTA, conditions which abolished the increase in [Ca2+]c caused by 56 mM KCl. In this low Ca2+-containing medium, 100 nM TRH induced a burst of [Ca2+]c; the magnitude of the peak was the same whether the cells were perifused with low Ca2+-containing medium for 2 or 20 min before adding TRH, indicating the source of intracellular Ca2+ was stable under these conditions. After 2 min in this medium, TRH was still able to stimulate almost as much PRL release as in medium containing 1.8 mM Ca2+, and 1 microM dopamine inhibited this release, but did not affect the magnitude of the TRH-induced increase in [Ca2+]c. Preincubation for 5 min with dopamine did not affect the ability of a 30-sec incubation with TRH to stimulate the accumulation of inositol phosphate, inositol bisphosphate, and inositol trisphosphate, either in medium containing 1.8 mM Ca2+, or in low Ca2+-containing medium. Preincubation with dopamine for 5 min had no effect on TRH-induced mobilization of intracellular calcium. A source of Ca2+ is needed to refill internal Ca2+-stores discharged by TRH, and as dopamine lowered [Ca2+]c which might fill these stores, we tested to see if dopamine prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel PTH-like peptide has recently been purified and cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. We surveyed the expression of mRNAs encoding this peptide in normal tissues by Northern analysis. One or more low abundance hybridizing transcripts was identified in poly(A)+ RNA prepared from human keratinocytes, thyroid, bone marrow, and fibroblasts, from bovine hypothalamus, pituitary, parathyroid, adrenal cortex, and adrenal medulla, and from rat brain, stomach mucosa, and fetal but not adult liver. One or more hybridizing transcripts was also identified in poly(A)+ RNA prepared from a number of established lines, including rat pituitary (GH4), rat pheochromocytoma (PC 12), human osteosarcoma (TE-85), and human medullary carcinoma (TT) cells. Northern analysis of mRNAs from abnormal human parathyroid tissue revealed an overexpression of transcripts for the PTH-like peptide which appeared to be specific for adenomatous or autonomous glands. These findings suggest that the PTH-like peptide is expressed in a number of endocrine and nonendocrine tissues, that it is developmentally expressed in at least one tissue (fetal liver), and that the regulation of its expression is abnormal in human parathyroid adenomas.

Storage forms of PRL were studied in control and cysteamine-treated cultures of estradiol-induced tumors in Fischer 344 rats and in secretory granules isolated from these tumors to further investigate the mechanism of action of cysteamine on PRL. The two major bands visible when protein is stained after electrophoresis of isolated granules migrate to the position of PRL and GH monomers. Electrophoresis under reducing conditions changes the position, but does not noticeably increase the amount of each band. [3H]PRL in cells labeled for 8 h with [3H]leucine also exists predominantly as monomer. Immunoreactivity of PRL in cell lysates or isolated granules is not affected by incubation with reducing agents beta-mercaptoethanol or glutathione at concentrations up to 5 mM, but cysteamine decreases PRL immunoreactivity in isolated granules at concentrations of 3 mM and higher. Electrophoresis of isolated granules after incubation with 25 mM cysteamine for 1 h demonstrates that cysteamine converts PRL to the reduced form. After 4 h, or after dilution of the granules before solubilization, the amount of reduced monomer is decreased, and larger molecular weight species appear. The reduced monomer can be recovered by electrophoresis under reducing conditions. The fully immunoreactive form can be recovered by incubation for 1 h with dithiothreitol at concentrations of 0.3 mM-3 mM. These data indicate that: PRL exists predominantly in monomeric form in the rat pituitary gland, and cysteamine reduces PRL, and formation of disulfide-linked aggregates of PRL occurs subsequently under some conditions.

The antiestrogens Ly-117018 and tamoxifen increased prolactin production about 2-fold in primary cultures of male rat anterior pituitary cells. The dose-response relationship was biphasic; 10−10 M Ly-117018 and 10−7 M tamoxifen caused maximal stimulation, but higher concentrations caused no stimulation and completely antagonized the 5-fold stimulation caused by estrogen. The calmodulin antagonists, trifluoperazine, pimozide and W7 also prevented estrogen induction of prolactin production. Increasing concentrations of estradiol reversed inhibition by tamoxifen but not by pimozide. These results indicate that, in normal pituitary cells, estrogen antagonists may behave as partial agonists at low concentrations and as full antagonists at higher concentrations (tamoxifen, prolactin, calmodulin, 17βestradiol).

Dopaminergic inhibition of PRL release stimulated by agents that affect cytosolic Ca2+ concentrations, C-kinase activity, and cAMP levels was studied in perifused rat anterior pituitary cells cultured on cytodex beads. We used A23187 (20 microM) to increase intracellular Ca2+, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 50 nM) to stimulate C-kinase, forskolin (10 microM) to increase intracellular cAMP, and 8-bromo-cAMP to mimic cAMP. Dopamine (10 microM) inhibited PRL release to 20-60% of the basal release within 10 min. After 30 min of preincubation with dopamine, the absolute amount of release stimulated by 100 nM TRH was strongly inhibited, although the pattern of release, a quick burst followed by sustained release at a lower rate, was the same in the presence or absence of dopamine. A23187 (20 microM) caused a rapid burst of PRL release that subsided within 10 min, and TPA (50 nM) caused a sustained release that began within 4 min and continued for at least 30 min. TPA and A23187 combined caused a rapid burst of release followed by a sustained phase of release similar to that caused by TRH. Preincubation with dopamine inhibited the absolute amount of PRL release caused by A23187 alone, TPA alone, or the two combined, although, as with TRH, the pattern of release remained the same. Forskolin (1 or 10 microM) or 8-bromo-cAMP (3 mM) induced a 1.5- to 2-fold increase in PRL release, and this was completely prevented by dopamine. Preincubation with both dopamine and 8-bromo-cAMP or forskolin restored the amount of release stimulated by TPA alone or TPA and A23187 in the presence of dopamine to the level of release stimulated by these agents in the absence of dopamine. Therefore, activating either the cAMP messenger system or the Ca2+ system alone will not abolish dopaminergic inhibition, but activating the two together will. These results suggest that dopamine blocks release by inhibiting both adenylate cyclase and a step in the Ca2+ messenger system.

Cyclo(Histidyl-Proline) is a metabolite of thyrotropin-releasing hormone. It has been suggested that this peptide plays a role in regulating prolactin secretion in GH cells. An investigation of the effect of cyclo(His-Pro) on GH cells indicated that it does not affect basal prolactin release or accumulation or the levels stimulated by TRH. cAMP levels in GH cells are elevated by TRH or VIP, but not influenced by cyclo(His-Pro). cGMP levels in GH cells are not affected by either TRH or cyclo(His-Pro). While there is specific binding of TRH to receptors in GH cells, no such receptors for cyclo(His-Pro) are detectable. It is suggested that GH cells are unresponsive to cyclo(His-Pro).

The hormones bombesin and thyrotropin-releasing hormone (TRH) stimulated formation of inositol-monophosphate, bisphosphate, trisphosphate and tetrakisphosphate with parallel time courses in GH 4 C 1 cells, while a more polar inositol polyphosphate peak, consisting of inositol-pentakisphsophate and perhaps also inositol-hexakisphosphate, was unaffected by either hormone. Although bombesin and TRH had similar potencies in stimulating inositol trisphosphate production (K m = 30 nM and 40 nM, respectively), TRH was significantly more efficacious than bombesin. Maximal stimulation of inositol-1,4,5-trisphosphate formation by TRH was not further increased by addition of a maximally effective dose of bombesin, suggesting that the two hormones act through stimulation of a common pool of phospholipase C, and this enzyme pool can be fully stimulated by TRH, alone.

In isolated rat anterior pituitary cells, KN-62 (10 microM), an isoquinoline sulfonamide inhibitor of calcium/calmodulin-dependent protein kinase II, inhibited high KCl(50 milliM)-stimulated prolactin secretion almost completely, with an IC50 of 95 nM KN-62 inhibited TRH-induced prolactin secretion less effectively. KN-04, a compound that is over 100-fold less active in inhibiting purified calcium/calmodulin-dependent protein kinase II, also inhibited high KCl-stimulated prolactin secretion with an IC50 of 500 nM. KN-62 and KN-04 (10 microM) both inhibited high KCl-stimulated increases in intracellular Ca2+ concentrations. We conclude that KN-62 and KN-04 inhibit activation of voltage-dependent calcium channels in anterior pituitary cells either directly or indirectly.

Spiperone, a dopamine antagonist widely used as a specific ligand for dopamine and serotonin receptors, is actively accumulated into the F4C1 strain of rat pituitary tumor cells. The accumulation of 10 nM [3H]spiperone was linear for 3 min and reached a steady state after 10 min. Spiperone accumulation was reduced 50% by preincubation with 5 microM reserpine, an inhibitor of biogenic amine transport into secretory granules, and was also blocked by monensin and ammonium chloride, both of which increase the pH of intracellular storage organelles. Uptake was not affected by replacing sodium in the buffer with lithium at equimolar concentrations. Spiperone at 1 microM inhibited by over 50% serotonin transport into membrane vesicles isolated from platelet dense granules; this concentration inhibited the Na+-dependent plasma membrane transport system less than 10%. The data indicate spiperone specifically interacts with the secretory granule amine transport system and suggest that this transport system is found in the F4C1 pituitary cell strain as well as in platelets and neurons. The data also suggest that experiments utilizing spiperone to measure dopamine and serotonin receptors be interpreted with caution.

17 beta-Estradiol increases the growth rate of GH4C1 cells with a half-maximally effective concentration (EC50) that is about 10-fold less than the EC50 for the stimulation of PRL production. We have examined the effects of five other estrogens: estriol, estrone, 17 alpha-estradiol, and the metabolism-resistant analogs ethynyl estradiol and moxestrol. All were full agonists for both effects, and all were more potent for the stimulation of cell growth than for stimulation of PRL production. The order of analog potency for both biological effects was the same as the order of potency for inhibiting saturable [3H]estradiol binding to intact cells. Therefore, both biological effects appear to be mediated through the same receptor, and metabolism of 17 beta-estradiol is unlikely to account for the difference in the concentrations required to elicit the two effects. We selected two estrogen-responsive clones from a cDNA library made from GH4C1 cells. The clones were chosen because they were induced at the estrogen concentrations that stimulate growth. Estradiol caused maximal stimulation of the mRNAs corresponding to the two recombinant clones at 10(-10) M, a concentration over 10-fold lower than that required for maximal stimulation of PRL mRNA. These data indicate that a difference in sensitivity to estrogen occurs at the level of mRNA accumulation as well as at the level of the biological responses.

GH4C1 cells are a rat pituitary tumor cell strain that secretes PRL and GH but contains almost no secretory granules. Treatment of GH4C1 cells with a combination of estradiol (1 nM), insulin (300 nM), and epidermal growth factor (10 nM) increased the cellular content of PRL by more than 30-fold above control levels but only increased PRL accumulation in the medium 6-fold. To determine whether the increase in intracellular PRL was accompanied by an increase in secretory granules, we compared the numbers of granules in ultrathin sections from untreated GH4C1 cells and from cells treated with the combined hormone regimen and found a nearly 50-fold increase in granule number. Only 75% of the granules stained for PRL by the protein-A gold technique; the other 25% stained for neither PRL nor GH. The occasional granules found in untreated GH4C1 cells stained for PRL. The data demonstrate that the number of granules in GH4C1 cells can be regulated by hormone treatment and that the increase in intracellular PRL is found in storage granules.

The 5-fluoroimidazole analogue of thyrotropin-releasing hormone, obtained by total synthesis from 5-fluoro-L-histidine, neither binds to rat pituitary cells nor stimulates release of prolactin from them. Levine-Pinto et. al. reported an agonist, which was generated during presumptive photofluorination of the hormone and which they believed to be the 5-fluoro analogue. In addition to the striking contrast in biological activities, the chemical properties of the agonist differ markedly from those of our peptide and are inconsistent with expectation for the fluoroimidazole moiety. Despite its inactivity in pituitary functions, the authentic 5-fluoro analogue mimics the natural hormone with respect to cardiovascular responses in the central nervous system.

The mechanism by which cysteamine reduces the PRL content of pituitary cells was studied in primary cultures of estradiol-induced pituitary tumors in Fischer 344 rats. The PRL content of these cells was effectively decreased by cysteamine, with an IC50 of 0.2 mM. Cells previously labeled with [3H]leucine were exposed to cysteamine (0.25 mM), and the intracellular content of [3H]PRL was measured by immunological or nonimmunological means, that is by immunoprecipitation and electrophoresis or by electrophoresis alone. The intracellular concentration of immunoreactive [3H]PRL was reduced by 53% by cysteamine, whereas [3H]PRL quantified by electrophoresis alone was not significantly affected. Our data indicate that cysteamine reduces the PRL content of pituitary tumor cells by causing the loss of its immunoreactivity.

Measurements of concentrations of cytosolic free Ca2+ ([Ca2+]c) in individual cells has frequently demonstrated periodic transients in [Ca2+]c rather than sustained elevated levels. To determine in anterior pituitary cells if such short and repetitive [Ca2+]c transients stimulated prolactin release, we used a perifusion system with cells loaded with the fluorescent Ca2+-indicator, indo-1. A one second pulse of 100 mM KCl caused an increase in [Ca2+]c with a half peak width of about 18 seconds and an almost coincident increase in prolactin secretion. Subsequent pulses of KCl each caused increases in [Ca2+]c and prolactin release that were the same as the first, up to a pulse frequency of one every two minutes. Increasing the frequency to 1 pulse every minute or 1 pulse every 30 seconds, however, resulted in a serial decline in the amount of prolactin released by each pulse even though each pulse caused a similar peak Ca2+ response. These findings demonstrate that cells become adapted to transient increases in [Ca2+]c of the same magnitude so that they no longer release prolactin if the increases in [Ca2+]c occur frequently enough. Cells may use frequency-encoded Ca2+ signals to stimulate release of prolactin at low frequency to prevent the adaptation that occurs at higher frequencies.

We have used primary cultures of male rat anterior pituitary cells to characterize two effects of pretreatment with 17β-estradiol: 1) the ability to overcome the inhibition of PRL accumulation in the medium (production) by dihydroergocryptine, a dopamine agonist, and 2) the stimulation of PRL production. Medium containing untreated serum contained 20 pm estradiol (E2). In this medium, incubation with dihydroergocryptine for 12 or 24 h caused half-maximal inhibition of PRL production at 150 pm; maximal inhibition reduced values to 32% of the control value. Pretreatment with 50 nm E2 for 5 days decreased the maximal inhibition of PRL production caused by dihydroergocryptine to 65% of control values, with little or no effect on the concentration of ergot which caused half-maximal inhibition. In addition, pretreatment with 50 nm E2 did not increase basal PRL production. Medium containing charcoaltreated serum was calculated to contain less than 2 pm E2. In this medium, dihydroergocryptine caused half-maximal ...

The effects of 12- O -tetradecanoylphorbol-13-acetate (TPA, an activator of C-kinase), the cation ionophore A23187, forskolin (an activator of adenylate cyclase) and thyrotropin-releasing hormone (TRH) on prolactin release from anterior pituitary cells in primary culture were investigated and compared to the effects of these same agents on prolactin release from GH 4 C 1 cells. In both GH 4 C 1 cells and primary pituitary cultures, 100 nM TRH increased prolactin release 3- to 5-fold within 4 min after the stimulation started. This peak response was followed by a fall to a sustained increased rate of release approximately 1.5-fold above the basal rate. The decline after the early peak was slower in primary cultures than in GH 4 C 1 cells. Addition of 20 μM A23187 to primary cultures caused a rapid 2- to 4-fold increase in release that fell to basal values within 12 min after the stimulation started. In GH 4 C 1 cells, A23187 caused a rise in prolactin release of less than 2-fold that was sustained longer than the rise seen in primary cultures. Perifusion of either type of cells with 50 nM TPA caused a rapid 2- to 2.5-fold increase in release that also was sustained for 30 min or more in both types of cells. Perifusion with combined TPA and A23187 caused a 3- to 5-fold increase in rate of release from each cell type that declined rapidly to a 2-fold sustained release in primary cultures, and that declined more slowly in GH 4 C 1 cells. Forskolin, l μM, had only a small effect by itself, but potentiated the effect of TPA or combined TPA and A23187 in both types of cells. Forskolin did not potentiate the effect of TRH in primary cultures. Although there are differences in responses of the 2 types of cells, the overall patterns of response were similar. The data are consistent with a model in which 2 parts of the Ca 2+ messenger system participate in the action of TRH: an increase in cytosolic Ca 2+ and an activation of protein kinase C.

A naturally occurring variant of human (h)GH, designated 20K, is equipotent with hGH(22K) in stimulating growth in hypophysectomized rats. However, despite high growth-promoting activity in vivo, 20K is a poor inhibitor of 125I-hGH(22K) binding to GH receptors in radioreceptor assays. In order to resolve the differences between bioassay and radioreceptor assay data, we have examined the binding of 20K to rat and rabbit liver GH receptors. Our data indicate that the GH receptor in rat liver binds 20K with an affinity only slightly lower than that for hGH(22K), in accordance with bioassay data. In the rabbit, however, 20K is bound with high affinity by only a small subset of the GH receptors which bind hGH(22K) with high affinity. The GH receptors which bind 20K appear to belong to the same of subset of receptors which bind rat and rabbit GH with high affinity.

We investigated the role of Ca++ in cAMP-stimulated PRL release from 1) primary cultures of male rat pituitary glands, 2) primary cultures of estrogen-induced pituitary tumors from Fischer rats, and 3) the pituitary tumor cell line GH4C1. Forskolin, an activator of adenylate cyclase, increased intracellular cAMP concentrations in GH4C1 cells at least 20-fold within 15 min. This increase occurred in the presence or absence of added extracellular Ca++ or in the presence of D600 or Co++. Forskolin increased PRL release from the three types of cells. The three systems differed in the Ca++ sensitivity of forskolin-induced release, but showed little difference in the Ca++ sensitivity of K+-induced release. This was shown in two ways. The cells were incubated either 1) in a medium without added Ca++ or 2) in the presence of a Ca++ channel inhibitor, D600. In normal cells, K+- and forskolin-induced release were equally inhibited when extracellular Ca++ was removed or D600 was added. In GH4C1 cells, Ca++ removal or D600 addition (100 microM) completely blocked K+-induced release, but had little effect on forskolin-induced release. The response of Fischer tumor cells was intermediate between those of normal and GH4C1 cells. 45Ca++ uptake by GH4C1 cells was not affected by forskolin, whereas the release of 45Ca++ from preloaded cells was increased slightly only 30 min after the addition of forskolin in three of four experiments. The difference in Ca++ requirements between normal and GH4C1 cells for forskolin stimulation may be due to the release of cellular Ca++ stores by cAMP. These stores may not be as large in normal cells as they are in GH4C1 cells, and therefore the requirement for extracellular Ca++ occurs. Alternatively, GH4C1 cells may release PRL by a mechanism different from that which normal cells use.

The effects of TRH and a methylated analog [pyroglutamyl-N3im-methyl-histidyl-proline-amide (MeTRH)] on the release of PRL and TSH were studied in dispersed primary cell cultures of rat pituitary glands. Both short term effects (occurring within 2–6 h) and long term effects (occurring within 3 days) were examined. Dose-response curves for TRH and MeTRH were indistinguishable for the short term release of PRL, with half-maximal values ranging from 2–5 nm for both compounds in different experiments. However, MeTRH was more active than TRH in releasing TSH; MeTRH released TSH with a half-maximal concentration of < 0.5 nm, whereas with TRH, half-maximal stimulation occurred at 3 nm. In long term studies, TRH increased both TSH and PRL accumulation in the medium, with half-maximal activity at 5 nm. Under these conditions, MeTRH was more active with both hormones; a halfmaximal TSH increase occurred at 0.4 nm, and a half-maximal PRL increase occurred at about 0.8 nm. Incubation with TRH for 3 days increased [3H...

We have investigated the effects of veratridine, a Na+ channel activator, and ouabain, an inhibitor of Na+-K+-ATPase, on short term (1-h) PRL release from primary cultures of rat anterior pituitary cells and from the rat anterior pituitary cell line GH4C1 in culture. Both compounds should increase intracellular Na+. Veratridine (20-500 microM) and ouabain (0.1-3 mM) stimulated PRL release from normal cells. The stimulation was inhibited by the omission of Ca++ from the release buffer or by preincubation with the calcium channel blocker D600 (20-500 microM), suggesting a role for Ca++ in the action of these compounds. Ouabain (1 mM), but not veratridine (200 microM), stimulated PRL release from GH4C1 cells, an effect that was also inhibited by calcium channel blockers. In the presence of the dopaminergic agonist bromocriptine (30 nM), the amount of stimulated release by veratridine (200 microM) and ouabain (1 mM) was reduced by 50%. The veratridine effect was only partially inhibited by preincubation of the cells with the Na+ channel blocker tetrodotoxin (1 and 10 microM), but the effect was inhibited completely when Na+ in the buffer was replaced by choline, suggesting that the action of veratridine requires extracellular Na+. The results of this study indicate that 1) ouabain- and veratridine-stimulated PRL release are largely dependent on Ca++; 2) veratridine appears to act through a tetrodotoxin-insensitive mechanism; and 3) stimulation of PRL release by these compounds is similar to that by 50 mM KCl and cAMP in its sensitivity to bromocriptine.

Dispersed cells from anterior pituitary glands of male rats were cultured embedded in collagen gels or on a plastic surface. After 6 or more days in culture, cells in collagen produced more prolactin than those on plastic. The cultures in collagen had fewer fibroblasts than those on plastic. Many cells cultured in collagen developed processes, and a few cells in every culture had long extended processes that sometimes branched and resembled those of neurons. About 60% of the cells in culture including cells with well developed processes bound the monoclonal antibody A2B5, an antibody that binds to neuronal cells in culture. Fibroblasts did not have detectable binding of A2B5. Some cells with short processes reacted with antiserum to prolactin or ACTH, but the cells with neuronlike processes did not. Collagen gels may provide a matrix in which cells from the anterior pituitary gland can differentiate into neuronlike cells.

We purified lactotrophs from pituitary tumors induced by estrogen in ovariectomized female Fischer 344 rats from 80% of the population before to more than 90% after purification through a continuous Percoll density gradient. The percentage of lactotrophs was evaluated by immunofluorescence. The patterns of PRL release stimulated by 100 nM TRH, 20 microM A23187 (a Ca++ ionophore), 50 nM 12-O-tetradecanoyl-phorbol-13-acetate (a C-kinase activator), or combinations of these agents, or inhibited by 10 microM dopamine were similar in perifused primary cultures of tumor lactotrophs to patterns in cultures of anterior pituitary cells from female retired breeders used previously. In particular, dopamine completely inhibited the release stimulated by forskolin. Intracellular cAMP concentrations and PRL accumulation in the medium were measured in monolayer cultures of purified tumor lactotrophs. In 9 separate experiments, forskolin (10 microM) increased intracellular cAMP concentrations more than 60-fold above control after 30 min of incubation. Preincubation (30 min) with dopamine (10 microM) reduced the cAMP accumulation caused by forskolin, but levels were still at least 20-fold above basal levels in most experiments. PRL release was stimulated 2-fold with forskolin alone, but there was no stimulation of PRL release by forskolin in the presence of dopamine even though cAMP levels were elevated above basal. Therefore, a decrease in cAMP levels is not necessary to inhibit PRL release, and dopamine must have a mechanism for inhibiting PRL release in addition to inhibiting adenylate cyclase.