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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.

Estimates of the frequency of GH deficiency range from 1:4,000 to 1:10,000. Most cases are sporadic and presumed to be secondary to a wide variety of aetiologies. However, in families with consanguinity, or when a second case occurs in the same family, a genetic cause may be suspected. Given that the patient is isolated GH deficient four distinct familial types of isolated GH deficiencies (IGHD) are well-differentiated on the basis of inheritance, hormonal deficiencies as well as molecular analyses. Two forms are autosomal recessively (IGHD type IA and IB), one is autosomal dominantly (IGHD type II) and one X-linked inherited. In this review, we focus on the secretory pathway of peptide hormones in general and on the possible mechanisms causing IGHD type II in detail. Most interestingly, in IGHD type II the apparently same phenotype of IGHD is caused by distinct GH-1 gene alterations leading to different blockades within the secretory pathway. Furthermore, this type of IGHD, in addition to some other specific GH-1 gene mutations, provides the most important opportunity to shed light on cell-biological mechanisms far beyond its pure description at the DNA/RNA level.

Soluble protein hormones are concentrated and stored in secretory granules The cisternal maturation model for transport of proteins through the Golgi complex allows a major role for formation of reversible aggregates as a means of both concentrating and sorting hormones, since soluble proteins will be removed in small vesicles, leaving behind the aggregated hormones. The storage of secretory granule proteins, however, is more selective than would be expected if passive aggregation were the only process involved. Aggregation of hormones in the secretory pathway may not be completely passive, but may be controlled by the cells. In addition to aggregation, other layers of sorting must exist, because there is selective retention of proteins after aggregation or packaging into granules.

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.

Familial isolated GH deficiency type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32–71-GH, a protein that cannot fold normally. The mechanism by which this mutant suppresses the secretion of wild-type GH encoded by the normal allele is not known. Coexpression of del32–71-GH with wild-type human GH in transient transfections of the neuroendocrine cell lines GH4C1 and AtT20 suppressed accumulation of wild-type GH. The suppression of wild-type GH accumulation by del32–71-GH was a posttranslational effect on wild-type GH caused by decreased stability, rather than decreased synthesis, of wild-type GH. Coexpression of del32–71-GH with human PRL did not suppress accumulation of PRL, indicating that there was not a general suppression of secretory pathway function. Accumulation of del32–71-GH protein was not necessary for the suppression of wild-type GH, because del32–71-GH did not accumulate in the neuroendocrine cell line...

I. Introduction: Concentrating Protein Hormones in Secretory Granules II. A Well Characterized Sorting Mechanism: Lysosomal Hydrolases A. Sorting is linked to transport B. Specific transport is associated with specific vesicles III. Sorting of Soluble Transported Proteins in the Endoplasmic Reticulum A. Sorting in yeast B. Sorting in mammalian cells C. Implications for sorting into granules IV. Two Models for the Functioning of the Golgi Complex A. Vesicular transport of proteins vs. maturation of stacks B. Implications for sorting into granules V. Models for Formation of Secretory Granules A. Active budding off of secretory granules vs. active budding off of everything else B. Implications for sorting into granules VI. Hormone Aggregates in Cells A. Evidence for their existence B. Properties of hormone aggregates in cells VII. Hormone Aggregates in Solution VIII. Does Aggregation Cause Sorting in Cells? A. Ways to measure sorting B. Does sorting in cells correlate with aggregation in solution? IX. What K...

The sections in this article are: 1 Intracellular Transport in Vesicles 2 Membrane Fusion and Exocytosis 2.1 Investigations in Synaptic Vesicles 2.2 Investigations in Yeast 2.3 Reconstituted Systems of Transport 2.4 Convergence of Separate Approaches 2.5 Calcium Dependence of Stimulated Neurotransmitter Release 2.6 Docking Vesicles to the Correct Membrane 2.7 Other Fusion Mechanisms 2.8 Evidence for SNAP-NSF-Mediated Docking/Fusion With Dense Core Granules 2.9 Exocytosis in Endocrine Cells 3 Membrane Budding and Endocytosis 3.1 Budding Involving Clathrin 3.2 Budding Involving COPI/ARF 3.3 Endocytosis in Endocrine Cells 3.4 Formation of Secretory Granules in Endocrine Cells

Treatment with 1 nM estradiol, 300 nM insulin, and 5 nM epidermal growth factor induces secretory granule accumulation and prolactin storage in GH4C1 rat pituitary tumor cells. The same triple treatment induced more than 6-fold accumulation of both the precursor (100 kDa, pro-ICA512) and the mature forms (60-70 kDa, ICA512 transmembrane fragment) of ICA512, a receptor protein tyrosine phosphatase-like protein that is preferentially localized in secretory granule membranes. Accumulation of ICA512 resembles that of prolactin storage, for the combination of all three, estradiol, insulin, and epidermal growth factor, gave the greatest induction, which was maximal at 4 days. This effect was specific, as the levels of the small GTP-binding protein Rab3, which is also associated with secretory granule membranes, were unaffected by the triple hormone/growth factor treatment. Increased transcription and translation of ICA512 could only partially account for its 6-fold accumulation, as ICA512 messenger RNA and ICA512 synthesis levels were 1.8 +/- 0.35- and 1.6 +/- 0.17-fold in triple treated GH4C1 cells compared with those in untreated cells, respectively. Pulse-chase procedures showed that pro-ICA512 was more stable in treated cells. These results indicate that the enlargement of the secretory granule compartment results in the stabilization of ICA512 and raise the possibility that trafficking of secretory granules may affect ICA512's function and vice versa.

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.

Prolactin and GH form reversible aggregates in the trans-Golgi lumen that become the dense cores of secretory granules. Aggregation is an economical means of sorting, because self-association removes the hormones from other possible pathways. Secretory granules containing different aggregates show different behavior, such as the reduction in stimulated release of granules containing R183H-GH compared with release of those containing wild-type hormone. Aggregates may facilitate localization of membrane proteins necessary for transport and exocytosis of secretory granules, and therefore understanding their properties is important. Three types of self-association have been characterized: dimers of human GH that form with Zn2+, low-affinity self-association of human prolactin caused by acidic pH and Zn2+ with macromolecular crowding, and amyloid fibers of prolactin. The best candidate for the form in most granules may be low-affinity self-association because it occurs rapidly at Zn2+ concentrations that are likely to be in granules and reverses rapidly in neutral pH. Amyloid may form in older granules. Determining differences between aggregates of wild type and those of R183H-GH should help to understand why granules containing the mutant behave differently from those containing wild-type hormone. If reversible aggregation of other hormones, including those that are proteolytically processed, is the crucial act in forming granules, rather than use of a sorting signal, then prohormones should form reversible aggregates in solution in conditions that resemble those of the trans-Golgi lumen, including macromolecular crowding.

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.

The treatment of GH4C1 cells, a prolactin-producing rat anterior pituitary cell line, with estradiol (1 nM), insulin (300 nM) and epidermal growth factor (10 nM) has been previously shown to substantially increase both the intracellular level of prolactin, as well as the number of secretory granules. In this study, we examined the effect of this treatment on levels of carboxypeptidase E (CPE), a prohormone-processing enzyme. GH4C1 cells contain CPE mRNA and enzymatic activity. The secretion of both prolactin and CPE activity from GH4C1 cells is stimulated 10-fold by 50 mM KCl and 2- to 3-fold by 100 nM thyrotropin-releasing hormone, suggesting that these two proteins are contained in secretory granules. Treatment of GH4C1 cells with estradiol, insulin, and epidermal growth factor causes an increase in the intracellular level of CPE to approximately 2-fold of control values. This change is much smaller than the change in the level of prolactin: intracellular prolactin is increased 140-fold by the treatment. Kinetic analysis of the CPE activity indicates that the treatment does not alter the Km of substrate hydrolysis, with the change in activity the result of an increase in apparent Vmax. Northern blot analysis indicates that the level of CPE mRNA is not influenced (less than 10%) by the treatment, whereas the level of prolactin mRNA is increased 9-fold. These results indicate that CPE is not coordinately regulated with prolactin in the GH4C1 cell line, although some regulation of CPE activity does occur.

A prominent feature of neuroendocrine cells is the large dense-core vesicles that contain concentrated stores of protein hormones. Keeping stores of hormones in these vesicles, commonly known as secretory granules, means that large amounts of protein hormones can be rapidly released when needed, so the delay required to synthesize, fold, process, and transport hormones through the secretory pathway is avoided. The release of hormones is triggered by an increase in the concentration of cytosolic Ca2+, which causes the membranes of the secretory granule to fuse with the plasma membrane, releasing the contents into the extracellular space. All vesicular traffic in cells occurs by membrane fusion; the membrane fusion of these other vesicles differs from the membrane fusion that leads to regulated protein hormone release from secretory granules, because other vesicles fuse constitutively and the fusion of these vesicles is not stimulated by increases in Ca2+ in the cytosol. In the last decade of the 20th century, definite progress was made in understanding the mechanisms by which the release of protein hormones occurs; much of the progress came about because most membrane fusion processes have characteristics in common, regardless of whether they occur in yeast or human.

The subcellular structure of peptide hormone-producing cells includes the nucleus, cytoplasm, mitochondria, lysosomes, and plasma membrane, which are present in all cells. The parts of the secretory pathway present in all cells, the endoplasmic reticulum and Golgi complex, are more predominant in peptide hormone-secreting cells, and in these cells there are specialized subcellular components called secretory granules that store hormones until they are needed.

In neuroendocrine cells, protein hormones are stored in a concentrated form in secretory granules, so that large amounts may be rapidly released when needed. The process of concentration is effective; prolactin is 200 times more concentrated in secretory granules than it is in the endoplasmic reticulum. In addition to being concentrated, protein hormones are segregated from other soluble proteins in the secretory granules, such as lysosomal enzymes. The mechanisms by which this segregation occurs are beginning to be elucidated.

Neuroendocrine cells and other secretory cell types are able to store secretory proteins in a concentrated form for extended periods until the release of large quantities of protein is triggered. The proteins are stored in dense core secretory granules. The dense cores of these granules are made up of large, insoluble aggregates that form by self-association. These aggregates solubilise rapidly into monomeric proteins in their native conformations when released from the cells by exocytosis of secretory granules. Formation of aggregates is an early event in secretory granule formation in at least some cell types. The function of secretory granules containing protein aggregates varies, depending upon the contents. This may occur because recognition of an aspect, such as a surface motif, of the aggregate facilitates correct assembly of the membrane proteins necessary for transport and exocytosis of the granules. Understanding the principles necessary for aggregation of protein hormones may help in the formulation of proteins for clinical use. Formation of aggregates of human prolactin has been investigated both in cells and in solution. In cells, the aggregation of human prolactin requires a mildly acidic pH, and is slowed in the presence of a membrane-permeable chelator of zinc. In solution, the aggregation of human prolactin at mildly acidic pH and physiological concentrations of Zn(2+) resembles that which occurs in cells if the reaction is performed with macromolecular crowding, which will mimic the conditions in cells. The factors causing protein aggregation and the extent to which aggregation plays a role in secretory granule formation are likely to vary with the protein and cell type. Further understanding of the principles involved in forming these aggregates that readily disassociate may enhance the ability to formulate protein preparations. Knowledge of the exact residues involved in the protein : protein interfaces in the aggregates of secretory granule proteins may lead to the ability to use small molecules to interfere with self-association and to regulate the storage of secretory granule proteins.

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.

In some individuals with autosomal dominant isolated growth hormone deficiency, one copy of growth hormone lacks amino acids 32-71 and is severely misfolded. We transfected COS7 cells with either wild-type human growth hormone or Delta 32-71 growth hormone and investigated subcellular localization of growth hormone and other proteins. Delta 32-71 growth hormone was retained in the endoplasmic reticulum, whereas wild-type hormone accumulated in the Golgi apparatus. When cells transfected with wild-type or Delta 32-71 growth hormone were dually stained for growth hormone and the Golgi markers beta-COP, membrin or 58K, wild-type growth hormone was colocalized with the Golgi markers, but beta-COP, membrin and 58K immunoreactivity was highly dispersed or undetectable in cells expressing Delta 32-71 growth hormone. Examination of alpha-tubulin immunostaining showed that the cytoplasmic microtubular arrangement was normal in cells expressing wild-type growth hormone, but microtubule-organizing centers were absent in nearly all cells expressing Delta 32-71 growth hormone. To determine whether Delta 32-71 growth hormone would alter trafficking of a plasma membrane protein, we cotransfected the cells with the thyrotropin-releasing hormone (TRH) receptor and either wild-type or Delta 32-71 growth hormone. Cells expressing Delta 32-71 growth hormone, unlike those expressing wild-type growth hormone, failed to show normal TRH receptor localization or binding. Expression of Delta 32-71 growth hormone also disrupted the trafficking of two secretory proteins, prolactin and secreted alkaline phosphatase. Delta 32-71 growth hormone only weakly elicited the unfolded protein response as indicated by induction of BiP mRNA. Pharmacological induction of the unfolded protein response partially prevented deletion mutant-induced Golgi fragmentation and partially restored normal TRH receptor trafficking. The ability of some misfolded proteins to block endoplasmic reticulum-to-Golgi traffic may explain their toxic effects on host cells and suggests possible strategies for therapeutic interventions.

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.

Protein folding and transport in the secretory pathway of cells is a controlled process, facilitated by chaperones. Proteins that do not fold well elicit several different programmed responses from the cells. A comparison of mutants of growth hormone that result in growth hormone deficiency suggests that cells do not respond in the same way to all growth hormone mutants that cannot fold, because some mutants are dominant and some are recessive. Causes for autosomal dominant hormone deficiencies include accumulation of toxic or dysfunctional forms, competition for chaperones important for folding or transport, induction of protein degradation in the endoplasmic reticulum, or long-term responses of the cells to synthesis of proteins that do not fold that decrease hormone synthesis or cell viability.

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.

The ability of protein hormones to self-associate is likely to play an important role in concentrating hormones into secretory granules; therefore, the aggregation properties of human PRL and H27A mutant were investigated. Human PRL bound (65)Zn++; the Scatchard analysis was convex up, and limited by the solubility of PRL, but at least 0.7 mole Zn++ bound per mole of PRL. Binding of (65)Zn++ to H27A-PRL was greatly reduced. The biological activity in an Nb2 cell assay and the circular dichroism spectrum of wild type and H27A-PRL were similar, indicating the H27A mutant folded correctly, and the binding of Zn++ to the high affinity site is not essential for biological activity. Dynamic light scattering measurements indicated 10 and 20 mu M Zn++ caused some aggregation of both wild type and H27A-PRL. Sedimentation equilibrium analysis indicated that PRL is primarily a monomer in the absence of Zn++ and that there is increasing self-association in the presence of 5 and 10 mu M Zn++. The mutant H27A exhibited a greater tendency to aggregate without changing detectably the mode of association. Although human PRL binds Zn++ as human GH does, it differs in that the ability to bind Zn++ and to self-associate were decoupled in PRL. Human PRL must have two types of interactions with Zn++; one is binding to a site involving histidine 27, and the other is weaker interactions that induce self-association of PRL.

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.

Publisher Summary This chapter discusses the 3-acetyl pyridine model of Parkinsonism. The degeneration of the nigrostriatal dopamine neurons is the primary morphological substrate of idiopathic Parkinson's disease (PD). The administration of 3-acetylpyridine (3-AP) results in the near complete degeneration of the climbing fiber innervation of the cerebellum with a concomitant loss of the inferior olivary (IO) neurons that give rise to the climbing fibers. The 3-AP also induces the degeneration of substantia nigra (SN) neurons. The 3-acetylpyridine administration to susceptible species results in the slow degeneration of the nigrostriatal dopamine system. Thus, 3-AP-induced neurotoxicity may be a useful animal model in studies of Parkinsonism. In particular, this pyridine toxin provides a means whereby the orderly progression of compensatory mechanisms that result from partial striatal DA depletion can be studied. Alterations in mitochondrial transcription may be one of these compensatory responses, and thereby contribute to the sustained function of the system under duress, yet paradoxically herald the ultimate degeneration of the nigrostriatal neurons.

Rhodamine 123 is a mitochondrial dye that is retained for prolonged periods by carcinoma cells. While investigating causes of retention of this dye, we found that 10 microM progesterone caused a rapid stimulation of efflux of rhodamine 123 within 15 min from KB V20C cells, which overexpress the multidrug resistance pump. Progesterone did not stimulate efflux from KB cells that do not overexpress the pump, and verapamil blocked rhodamine 123 efflux in the presence or absence of progesterone, indicating that rhodamine 123 is removed from KB V20C cells by the multidrug resistance pump. Progesterone, however, is unlikely to stimulate rhodamine 123 efflux by simply increasing pump activity for two reasons: (1) progesterone inhibited the efflux of daunomycin from KB V20C cells, so it did not stimulate efflux of all drugs, and (2) progesterone inhibited efflux of rhodamine 123 from L1210/VMDRC cells and had little effect on Adr MCF7 cells; both overexpress the multidrug resistance pump. In the experiments with KB V20C cells, progesterone was the most active steroid tested. At 10 microM, progesterone caused a 70-fold stimulation, desoxycorticosterone, testosterone, promegestone and estradiol about 20-fold, and others had little or no effect. Progesterone may act by a non-genomic mechanism to decrease intracellular binding of rhodamine 123, making the dye accessible to the multidrug resistance pump.

Many membrane receptors have truncated soluble forms that circulate in the blood, and a protein in serum with characteristics of the extracellular domain of the human prolactin receptor has recently been identified. Because the extracellular domain of the prolactin receptor binds human growth hormone, does the prolactin-binding protein in serum affect the amount of bound circulating growth hormone? A more general question is whether this serum protein has a minor or a major effect on the biological activities of growth hormone and prolactin.

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.

PRL storage in GH4C1 cells, rat pituitary tumor cells, can be induced by treatment with a combination of estradiol, epidermal growth factor, and bovine insulin. This increase in storage is characterized by a preferential increase in intracellular PRL compared with secreted PRL and a 50-fold increase in the number of secretory granules. Treatment with the combined hormones stimulates PRL synthesis approximately 6-fold, but this effect is not sufficient to increase PRL storage, because epidermal growth factor alone increases PRL synthesis to the same extent without affecting storage. The cDNA for human proinsulin down-stream of the RSV-LTR promoter was transfected into GH4C1 cells to determine whether storage of another protein known to be targeted to the regulated pathway would also increase with hormone treatment. Proinsulin and PRL release were stimulated over the same time course and to the same peak height, compared to basal release, by both KCl and TRH, indicating that proinsulin is targeted to the regulated pathway in GH4C1 cells. There was little intracellular processing of proinsulin to insulin. Proinsulin synthesis increased 3.9-fold with the combined treatment, assessed by accumulation of proinsulin immunoreactivity in the medium and increases at the mRNA level. Treatment with the combined hormones did not cause the preferential increase in intracellular proinsulin that occurred with PRL; the increase in intracellular proinsulin could be accounted for primarily by effects on synthesis. These results suggest that storage of the two hormones can be differentially regulated.

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)

Established clonal strains of rat pituitary cells, GH-cells, responded prior to 1974 to 10−11 to 10−8M 17β-estradiol by increasing prolactin synthesis 2-fold and decreasing the production of growth hormone to between 20 and 70% of control values. In experiments in 1975-76, these effects of estradiol were no longer statistically significant. Unexpectedly, the estrogen antagonists enclomiphene, CI-628, tamoxifen, and Lilly 88751 caused increases in both prolactin and growth hormone synthesis at concentrations of about 10−7 to 10−6M after one week of treatment. These increases were prevented by 10−8M 17β-estradiol. Prolactin synthesis remained elevated for at least 11 days after removal of enclomiphene from the culture medium but, in the presence of estradiol, synthesis approached control levels by 11 days. In GH-cells, compounds which are estrogen antagonists in other systems mimic the previously observed effect of estradiol on prolactin synthesis, but have an effect opposite to that of estradiol on growth ...

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.

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.

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.

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.

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.

Synthesis of prolactin was measured in a clonal strain of functional pituitary cells in culture by incubating the cells with [3H]leucine; labeled prolactin was separated from the other labeled proteins by specific immunoprecipitation followed by dissociation with sodium dodecyl sulfate and disc gel electrophoresis. Accumulation of labeled prolactin in the cells was linear for at least 1 hour; labeled prolactin was released into the culture medium after 1 hour. Treatment with 28 nm thyrotropin-releasing hormone for 3 days increased prolactin accumulation in the medium 2.5-fold, and the increased synthesis of prolactin completely accounted for the increased accumulation. Treatment with 5 x 10-6 m hydrocortisone for 7 days decreased prolactin accumulation in the medium 2- to 5-fold, and the decrease in synthesis completely accounted for the decreased accumulation. Therefore the long term (3 to 7 days) effects on these cells of thyrotropinreleasing hormone and hydrocortisone result only in a change in the synthesis of prolactin and not in its rate of degradation. Pulse-chase experiments, using 10 µg per ml of cycloheximide or 10 mm leucine to block further incorporation of [3H]leucine into prolactin, show that there is no detectable degradation of prolactin under the usual conditions of culture. Incubation with 0.1 mm leucine does not prevent incorporation of radioactive leucine into prolactin and results in a 1.7-fold increase in labeled prolactin within 4 hours after the start of the chase. This result suggests there are two intracellular pools of leucine in GH cells, one of which does not appear to equilibrate readily with the medium.

Estrogen causes mammotroph and pituitary tumor growth i n vivo; we therefore studied its effect on growth of GH cells, a clonal line of rat pituitary tumor cells. Medium supplemented with gelding serum enabled us to detect effects of estradiol which are masked by the higher estrogen concentrations of commercially available horse serum. GH cells were treated with estradiol for 7 days. Cell number was affected in a biphasic fashion: estradiol at 10-11M and 10-11M increased cell number 6- to 13-fold. Concentrations between 3×10-11M and 10-9 M caused a dose-dependent decrease from the maximal cell number. The half maximal concentration for this inhibitory effect was about 10>-10M. Prolactin Production was stimulated 3-to 11-fold by estradiol, also with a half-maximal concentration of 10-10

The effects of 12-0-tetradecanoyl-phorbol-13-acetate (TPA), A23187, forskolin and thyrotropin-releasing hormone (TRH) on prolactin release from GH4C1 cells were compared. TPA caused a 2-fold release, maximum after 6 or more min, that was sustained for 30 min or more. A23187 caused only a small and variable response that peaked within 4 to 6 min. Combination of TPA and A23187 caused a rapid 3- to 5-fold increase in release that declined slowly. TRH increased prolactin release 3- to 5-fold, reaching a maximum within 4 min, followed by sustained release at lower rates. Forskolin had little effect by itself, but potentiated release caused either by combined TPA and A23187, or by TRH. These data are consistent with a model in which two branches of the Ca2+ messenger system participate in the action of TRH, a calmodulin branch and a C-kinase branch that interact to cause large amounts of sustained release. Forskolin, by regulating the cyclic AMP content of the cell determines the set point around which the Ca2+ messenger system operates.

A wheat germ embryo extract was used to translate cytoplasmic RNA isolated from rat pituitary tumor cells (GH-cells). This RNA directed the synthesis of a radioactive product which was precipitated with antiserum specific for rat prolactin. The molecular weight of this immunoprecipitated product was 24,500 as determined by electrophoresis in denaturing gels. Prolactin secreted by intact GH-cells had a molecular weight identical to standard pituitary prolactin, reported to be about 22,500. Our finding that a larger form of prolactin is made by the wheat germ system is similar to results recently described by Maurer, Stone and Gorski (J. Biol. Chem., in press). Thyrotropin-releasing hormone (TRH) stimulates prolactin synthesis in GH-cells, and cytoplasmic RNA isolated from cells treated with TRH directed the synthesis in wheat germ extracts of larger amounts of prolactin than RNA isolated from control cells. The increase in translatable cytoplasmic mRNA for prolactin corresponded to the increase in prolactin synthesis which suggests that the increase in prolactin synthesis in TRH-treated cells is a result of the accumulation of cytoplasmic mRNA for prolactin.

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.