Prolactin storage in a clonal strain of rat pituitary tumor cells is cell-cycle dependent.

GH4C1 cells (CH cells) are a clonal strain of rat pituitary tumor cells which secrete prolactin. We measured intracellular prolactin at different stages of the cell cycle using flow microfluorometry. Prolactin was stained by an indirect immunocytochemical technique using fluorescein isothiocyanate (FITC)-conjugated antiserum, and DNA was stained simultaneously with propidium iodide. We found that prolactin storage in GH cells was cell-cycle dependent; prolactin storage increased as cells passed from G1 to S to G2 + M. We have shown previously that insulin and 17 beta-estradiol act synergistically to increase intracellular prolactin three- to sevenfold and slow the rate of cell growth to approximately 70% of control cells. In this study we observed that insulin and estradiol increased prolactin storage at each stage of the cell cycle but did not affect the cell-cycle distribution of the population even though cell growth was slowed. We conclude that insulin and estradiol did not increase prolactin storage by affecting the cell-cycle distribution of the population.

Protein hormone storage in secretory granules: mechanisms for concentration and sorting.

Recent findings in cell biology have demonstrated there are several kinds of active sorting from the trans-Golgi network in all cells. The presence of several sorting pathways, using more than one sorting signal, in neuroendocrine cells means that mutations that direct a hormone to a constitutive pathway instead of a regulated one may not simply be interpreted as a signal for sorting to a regulated pathway. The use of three-dimensional electron microscopy of lactotrophs and the possibility that the trans-Golgi network is consumed during sorting has suggested a major role for hormone aggregation, not only as a sorting mechanism, but also as a mechanism for granule formation, in that other transport vesicles may bud from the trans-Golgi network, leaving the aggregated protein as the dense core granule. If aggregation plays such a role, it is unclear how it works in cases where the prohormone must be processed one or more times; does a porous aggregate or colloid form? Obtaining information about the kinds of aggregates that occur in cells is difficult, because at this time there is not a definitive way of knowing whether an aggregate that occurs in solution also occurs in cells. Although secretory granule proteins tend to aggregate relatively easily in solution, the separate treatment of different secretory granule proteins in the same cell make it unlikely that aggregation is a purely passive process, but suggests that the process of aggregation of each hormone is actively controlled in cells. Even if the ability to aggregate accounts for most of the sorting of cargo-secretory granule proteins into granules, other sorting must still occur to get correct membrane proteins necessary for transport and exocytosis into secretory granule membranes. Possible recognition sites for these secretory granule membrane proteins include the cargo itself in an aggregated form, membrane lipids in some unrecognized way, or the proteins and factors that specifically control aggregation of the cargo.

KN-62, a calcium/calmodulin-dependent protein kinase II inhibitor, inhibits high potassium-stimulated prolactin secretion and intracellular calcium increases in anterior pituitary cells.

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.

A serum prolactin-binding protein: implications for growth hormone.

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.

Inhibition of rat prolactin (PRL) storage by coexpression of human 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.

Estrogen induces accumulation of the mitochondrial ribonucleic acid for subunit II of cytochrome oxidase in pituitary tumor cells.

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.

Dopamine has no effect on thyrotropin-releasing hormone mobilization of calcium from intracellular stores in rat anterior pituitary cells.

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)

Ca2+ transients induced by thyrotropin-releasing hormone rapidly lose their ability to cause release of prolactin.

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)

Properties of human prolactin (PRL) and H27A-PRL, a mutant that does not bind Zn++.

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.

Estradiol decreases retention of rhodamine 123 fluorescence in GH4C1 pituitary tumor cells.

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)

Prolactin and insulin are targeted to the regulated pathway in GH4C1 cells, but their storage is differentially regulated.

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.

Inefficient secretion of human H27A-prolactin, a mutant that does not bind Zn2+.

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 microg rat PRL/10(5) 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 microg rat PRL/10(5) 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.

Expression of messenger ribonucleic acids encoding a parathyroid hormone-like peptide in normal human and animal tissues with abnormal expression in human parathyroid adenomas.

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.

Veratridine and ouabain stimulate calcium-dependent prolactin release.

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.

Depletion of pituitary prolactin by cysteamine is due to loss of immunological activity.

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.

Antipsychotic drugs inhibit prolactin release from rat anterior pituitary cells in culture by a mechanism not involving the dopamine receptor.

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.

The role of adenosine 3',5'-monophosphate in dopaminergic inhibition of prolactin release in anterior pituitary cells.

The relationship between cAMP stimulation and dopaminergic inhibition of PRL release was studied in primary cultures of rat anterior pituitary cells. Bromocriptine, a dopaminergic agonist, and cholera enterotoxin, isobutylmethylxanthine, theophylline, and 8-bromo-cAMP, agents which mimic cAMP action or cause increases in cAMP, were used. Short term PRL release (that which occurs within 1 or 2 h) was stimulated 1.5- to 2-fold by all of the cAMP agents. Bromocriptine (5 nM) decreased basal release and completely abolished any short term stimulation above basal caused by cholera enterotoxin and 8-bromo-cAMP. Isobutylmethylxanthine and theophylline did cause some stimulation of PRL release above basal, but the magnitude of the increase was half that in the absence of bromocriptine. The short term release stimulated by 8-bromo-cAMP was dependent on extracellular calcium. PRL accumulation in the medium for 1-3 days was increased by all of the cAMP agents. A long term increase caused by these agents was also observed in the presence of bromocriptine. The magnitude of the increase in release above basal was the same with and without bromocriptine, but the total PRL in the medium was always less in the presence of bromocriptine, since basal release was reduced. These results show that the short term inhibition of PRL release by bromocriptine cannot be completely reversed by agents which increase cAMP.

Insulin and 17 beta-estradiol increase the intracellular prolactin content of GH4C1 cells.

We investigated the effects of insulin and 17 beta-estradiol in PRL-producing rat pituitary tumor cells (GH cells) in culture. Incubation with 1.8 X 10(-7) M insulin and 1 X 10(-9) M estradiol always increased intracellular PRL significantly to a mean of 290% of control levels. Insulin or estradiol alone caused smaller increases in intracellular PRL that were significant in five out of six experiments with insulin and three out of five experiments with estradiol, producing average increases of 150% and 160% of controls, respectively. Combined treatment with insulin and estradiol increased PRL secreted into the medium to 160% of controls, which was smaller than the effect on intracellular PRL. The effects of insulin and estradiol were detectable after 2-4 days of treatment and were maximal between 6-8 days. The effects were dose dependent; the half-maximal dose of insulin was about 2 X 10(-8) M and of estradiol was about 5 X 10(-11) M. Treatment with both insulin and estradiol increased cellular uptake of [3H]leucine by 45% compared to controls, incorporation of [3H]leucine into PRL by 65%, and total protein by 50%. The increased incorporation into PRL may represent an increase in PRL synthesis, but it is not enough to account for the increased intracellular hormone. Insulin and estradiol did not change the time required for most newly synthesized PRL to be processed and released from the cells, which was 60 min. Therefore, the amount of intracellular PRL was not increased as a result of an increased intracellular transit time. We conclude that insulin and estradiol increase the amount of intracellular PRL as a result of either decreasing the rate of degradation or increasing the cells' capacity to store PRL.

Stimulation of the adenosine 3',5'-monophosphate and the Ca2+ messenger systems together reverse dopaminergic inhibition of prolactin release.

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.

Lack of correlation of distribution of prolactin (PRL) charge isoforms with induction of PRL storage.

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.