Effects of pretreatment with tetradecanoyl phorbol acetate on regulation of growth hormone and prolactin secretion from ovine anterior pituitary cells

Tetradecanoyl phorbol acetate (TPA) stimulates growth hormone (GH) and prolactin secretion from ovine anterior pituitary cells. Pretreatment of the cells with TPA abolishes this effect, presumably due to down-regulation of protein kinase C. Such pretreatment did not alter effects of thyrotropin-releasing hormone or dopamine on prolactin secretion, suggesting no involvement of protein kinase C. Pretreatment with TPA attenuated actions of GH-releasing hormone on GH release (but not actions on cyclic AMP levels), possibly due to depletion of cellular stores of GH. Such pretreatment also attenuated inhibition of GH release by somatostatin, possibly due to phosphorylation of receptors or associated proteins by protein kinase C.     

Phosphorylation of prolactin

Rat prolactin exhibits microheterogeneity when examined in electrophoretic systems, running as three isoforms having the same molecular weight but different net charges (prolactins 1, 2, and 3 with isoform 3 being the most acidic). As there is precedent for the phosphorylation of a pituitary hormone and phosphorylation is a common cause of microheterogeneity, we examined the possibility that rat prolactin existed in differentially phosphorylated forms. The investigation included examinations of rat prolactin phosphorylation both in vitro and in vivo. For the in vitro studies, purified rat prolactin was incubated with [gamma-32P]ATP and low levels of each of five purified protein kinases. Phosphorylated rat prolactin was identified by autoradiography of silver-stained one- and two-dimensional gels. For the in vivo studies, rat anterior pituitary cells in primary culture were incubated in the presence of H3 32PO4 for 2 or 12 h, after which time the proteins were extracted from the cells, cold acetone-precipitated, or immunoprecipitated and run on two-dimensional gels. We report the in vitro phosphorylation of rat prolactin by cAMP-dependent protein kinase, casein kinase I, protease-activated kinase I, and the calcium/phospholipid-dependent kinase, that phosphorylation with these kinases results in phosphate incorporation only into isoforms 2 and 3, and the phosphorylation of prolactin in rat pituitary cells in primary culture.     

''Cross-talk'' between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells.

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the -subunits of the stimulatory and inhibitory G-proteins of AC (G_s and G_i−2) in cultured prolactin-producing rat pituitary adenoma cells (GH₃ cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G_s levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G_s levels by more than 50% at all time-points. G_i−2 levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G_s protein -subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.     

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Adenosine 3'',5''-cyclic monophosphate-dependent release of prolactin from GH3 pituitary tumour cells. A quantitative analysis.

The involvement of cyclic AMP in mediating regulatory peptide-controlled prolactin release from GH3 pituitary tumour cells was investigated. Cholera toxin and forskolin elicited concentration-dependent increases in both GH3 cell cyclic AMP content and prolactin release. The maximum rise in prolactin release with these agents was 2-fold over basal. 8-Bromo-cyclic AMP produced a similar stimulation of prolactin release. The phosphodiesterase inhibitor isobutylmethylxanthine also produced an increase in prolactin release and GH3 cell cyclic AMP content. However, the magnitude of the stimulated prolactin release exceeded that obtained with any other agent. Thyrotropin-releasing hormone (thyroliberin) and vasoactive intestinal polypeptide produced a concentration-dependent rise in both cell cyclic AMP content and prolactin release. However, only vasoactive intestinal polypeptide elicited an increase in cell cyclic AMP content at concentrations relevant to the stimulation of prolactin release. Vasoactive intestinal polypeptide and thyrotropin-releasing hormone, when used in combination, were additive with respect to prolactin release. Vasoactive intestinal polypeptide and forskolin, at concentrations that were maximal upon prolactin release, were, when used in combination, synergistic upon GH3 cell cyclic AMP content but were not additive upon prolactin release. In conclusion the evidence supports a role for cyclic AMP in the mediation of vasoactive intestinal polypeptide- but not thyrotropin-releasing hormone-stimulated prolactin release from GH3 cells. A quantitative analysis indicates that a 50-100% rise in cyclic AMP suffices to stimulate cyclic AMP-dependent prolactin release fully.     

Transforming growth factor-beta and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system

Incubations of rat anterior pituitary cells with transforming growth factor (TGF)-beta 1 for 48 hr suppressed the secretion of basal prolactin (PRL) in a dose-dependent manner (ED50, 100 pg/ml). Activin, a gonadal hormone processing cysteine distribution similar to TGF beta, also suppressed basal PRL secretion, but it was less effective (ED50, 4 mg/ml). Treatment with TGF beta 1 significantly suppressed basal PRL secretion from the pituitary after 24 hr and up to 72 hr of incubation. TGF beta 1 also inhibited thyrotropin-releasing hormone-mediated PRL secretion and activin inhibited thyrotropin-releasing hormone-mediated PRL secretion slightly, but significantly. In addition, we also measured the secretion of growth hormone by cultured pituitary cells treated with TGF beta 1 or activin for 24 to 72 hr. TGF beta 1 and activin showed an opposite effect on growth hormone secretion; TGF beta stimulated and activin inhibited basal secretion of growth hormone. These results suggest that TGF beta 1 is a potent inhibitor of basal secretion of PRL by the pituitary, and both TGF beta 1 and activin play a multifunctional role in basal secretion of pituitary hormones.     

Protein phosphorylation in response to the tumor promoter TPA is dependent on the state of differentiation of muscle cells

We have shown previously (A. Sobel and A. H. Tashjian, Jr. (1983). J. Biol. Chem. 258, 10,312-10,324;A. Sobel and M.C. Boutterin (1985). Neurochem. Int. 7, 995-1006) that, in the pituitary-derived GH4C1 cells, thyrotropin-releasing hormone or the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) stimulates the phosphorylation of two sets of cytoplasmic proteins related to the regulation of prolactin synthesis and release, respectively. Interestingly, phosphoproteins with identical electrophoretic migration properties on two-dimensional gels were detected in cultured neonate or adult mouse muscle cells and in the L6 and C2 myogenic cell lines. In addition TPA, which is known to have many actions on muscle cell functions, proliferation, and differentiation, stimulated the phosphorylation of these same proteins in myoblasts in culture. After fusion of the proliferating myoblasts into differentiated myotubes, this TPA-induced stimulation was strongly reduced in normal muscle cell cultures where some mononucleate muscle and non-muscle cells remained present. It was totally abolished in the homogeneous L6 and C2 cell lines. These observations suggest that the same phosphoproteins may be related to the intracellular mechanisms involved in the transduction of extracellular regulatory signals in such distinct differentiated environments as those of pituitary and muscle cells. In muscle cells themselves, the regulation of the phosphorylation of these proteins is function of the cell's state of differentiation.     

Epidermal Growth Factor Promotes Uncoupling from Adenylyl Cyclase of the Rat D2S Receptor Expressed in GH4C1 Cells

Abstract: In anterior pituitary cells or when transfected into host cell lines, the D₂ dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH-3 pituitary tumor cell line, which lacks functional D₂ receptors, responds to epidermal growth factor (EGF) by expressing a D₂ receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in α subunit of the G protein G₁₃. In this study we have investigated the effects of EGF on the transduction mechanisms of D₂ receptors in GH4C1 cells transfected and permanently overexpressing the rat short D₂ receptor. Activation of D₂ receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP-dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D₂ receptor to inhibit cyclic AMP-dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of G₁₃ protein. These results suggest that the transmembrane signaling specificity of G protein-coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.     

DNA polymerase delta mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH₁ some hours after phytohemagglutinin stimulation than if they are irradiated in G₀ before stimulation. Post-treatment of lymphocytes irradiated in G₀ with the DNA polymerase inhibitor aphidicolin, which is effective against both pol and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G₁ four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol ineffective in producing increased yields in either G₀ or G₁ lymphocytes, we conclude that much of the G₀ to G₁ increase in yields is mediated by pol δ.     

Analogs of thyrotropin-releasing hormone using an aminoglycine-based template

Novel thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂) analogs, made by solid phase, were derived from the general scaffold pGlu-(D/L)Agl(X)-Pro-NH₂ where Agl = aminoglycine. Analogs ranged from X being a proton to an acylating agent derived from substituted (aromatic heterocyclic rings) formic or acetic acids or an aminotriazolyl moiety (3′-amino-1H-1′,2′,4′-triazolyl) built on N of aminoglycine or N^β of ,β-diaminoproprionic acid (Dpr). X was expected to mimic the electronic and structural characteristics of the imidazole ring of histidine. Analogs were purified by HPLC, characterized by mass spectrometry and isolated as either diastereoisomeric mixtures or pure isomers. Analogs, tested for their binding affinity to mouse pituitary TRH receptors, have apparent equilibrium inhibitory constants >1 μM.     

Palladium complexes with PCP ligands.: Preparation and structural studies of two forms of [(PCP)Pd(OH2)]BF4

Abstraction of chloride from the Pd complex {[η³-2,6-(^tBu₂PCH₂)₂C₆H₃)]PdCl with AgBF₄ in THF gives {[η³-2,6-(^tBu₂PCH₂)₂C₆H₃)]Pd(THF)}⁺BF₄ ⁻. Attemped crystallization of this THF complex produced the aqua complex {[η³-2,6-(^tBu₂PCH₂)₂C₆H₃)]Pd(OH₂)}⁺BF₄ ⁻. Crystal structures of two crystalline forms of this compound are reported. In {[η³-2,6-(^tBu₂PCH₂)₂C₆H₃)]Pd(OH₂)}⁺BF₄ ⁻·THF, one hydrogen of the water is hydrogen bonded to the oxygen of the THF, and the other hydrogen is hydrogen bonded to an F of the BF₄ ⁻ anion. Another crystalline form has no THF, but has both of the hydrogens of water hydrogen bonded to different BF₄ ⁻ anions, such that two different BF₄ ⁻ anions bridge two {[η³-2,6-(^tBu₂PCH₂)₂C₆H₃)]Pd(OH₂)}⁺ cations. A crystal structure is also reported for the palladium chloride complex [η³-2,6-(^tBu₂PCH₂)₂C₆H₃)]PdCl.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the in vitro release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all in vitro experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Establishment in culture of a multihormone-secreting cell strain derived from the MtT/F4 rat pituitary tumor.

A clonal cell strain F4C1 has been established from the transplantable rat pituitary tumor MtT/F4 and has been maintained in continuous culture for two years. The cells grow with a population doubling time of 48 hours; the karyotype with a modal number of 39 chromosomes includes a pair of large metacentric marker chromosomes. F4C1 cells in culture produce growth hormone and prolactin but not adrenocorticotropin in contrast to the MtT/F4 tumor which secretes all three hormones in the host rat. The cloned cells lack specific receptors for thyrotropin-releasing hormone and do not respond to this agent with increased prolactin or decreased growth hormone production. Treatment with hydrocortisone results in a small increase in growth hormone and a small decrease in prolactin production. Tumors generated in rats from injected F4C1 cells secrete prolactin and growth hormone but not adrenocorticotropin. The results suggest that growth hormone and prolactin are produced by a single cell type in the MtT/F4 tumor.     

Synthesis and characterization of organomolybdenum and organotungsten halides containing the η-pentabenzylcyyclopentadienyl ligand η-Bz5C5M(CO)3X (M = Mo, W; X = Cl, Br, I). The X-ray molecular structure of η-Bz5C5Mo(CO)3I

An improved synthetic procedure for pentabenzylcyclopentadiene Bz₅C₅H was developed. Six new organomolybdenum and organotungsten halides η⁵-Bz₅C₅M(CO)₃X(M = Mo, W; X = Cl, Br, I) were syntesized through the reaction of η⁵-Bz₅C₅M(CO)₃Li (derived from Bz₅C₅H, n-BuLi and M(CO)₆) with PCl₃, PBr₃ or I₂ and characterized by elemental analysis, IR and ¹H NMR spectroscopy. The structure of η⁵-Bz₅C₅Mo(CO)₃I was determined by single-crystal X-ray diffraction techniques. It crystallized in the monoclinic space groupp P2/c with cell parameters a = 13.294(4), B = 15.147(4), C = 19.027(3) Å, β = 108.32(2)°, V = 3637(2) ų, Z = 4 and D_x = 1.50 g cm⁻³. The final R value was 0.035 for 4564 observed reflections.     

Hetero-polynuclear complexes containing bridging diphenylphosphino-alkenes and -alkynes. The crystal structure of an Rh2{μ−ν:ν−P(Ph2) (CH2)3C≡CR}Co2 complex

The phosphinoalkenes Ph₂P(CH₂)_nCH=CH₂ (n= 1, 2, 3) and phosphinoalkynes Ph₂P(CH₂)_n C≡CR (R = H, N = 2, 3; R = CH₃, N = 1) have been prepared and reacted with the dirhodium complex (η−C₅H₅)₂Rh₂(μ−CO) (μ−η²−CF₃C₂CF₃). Six new complexes of the type (ν−C₅H₅)₂(Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃)L, where L is a P-coordinated phosphinoalkene, or phosphinoalkyne have been isolated and fully characterized; the carbonyl and phosphine ligands are predominantly trans on the Rh---Rh bond, but there is spectroscopic evidence that a small amount of the cis-isomer is formed also. Treatment of the dirhodium-phosphinoalkene complexes with (η−CH₃C₅H₄)Mn(CO)₂thf resulted in coordination of the manganese to the alkene function. The Rh₂---Mn complex [(η−C₅H₅)₂Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃) {Ph₂P(CH₂)₃CH=CH₂} (η−CH₃C₅H₄)Mn(CO)₂] was fully characterized. Simi treatment of the dirhodium-phosphinoalkyne complexes with Co₂(CO)₈ resulted in the coordination of Co₂(CO)₆ to the alkyne function. The Rh₂---Co₂ complex [(η−C₅H₅)₂Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃) {Ph₂PCH₂C≡CCH₃}Co₂(CO)₂], C₃₇H₂₅Co₂F₆O₇PRh₂, was fully characteriz spectroscopically, and the molecular structure of this complex was determined by a single crystal X-ray diffraction study. It is triclinic, space group (C_i¹, No. 2) with a = 18.454(6), B = 11.418(3), C = 10.124(3) Å, = 112.16(2), β = 102.34(3), γ = 91.62(3)°, Z = 2. Conventional R on |F| was 0.052 fo observed (I > 3σ(I)) reflections. The Rh₂ and Co₂ parts of the molecule are distinct, the carbonyl and phosphine are mutually trans on the Rh---Rh bond, and the orientations of the alkynes are parallel for Rh₂ and perpendicular for Co₂. Attempts to induce Rh₂Co₂ cluster formation were unsuccessful.     

Endogenous thyrotropin-releasing hormone in the anterior pituitary: sites of activity as identified by immunocytochemical staining.

Thyrotropin-releasing hormone (TRH) immunoreactivity was localized in the rat anterior pituitary with rabbit anti-TRH sera and the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique. Stain was present in secretory granules of cells possessing morphological characteristics of thyrotropes, gonadotropes and lactotropes. Antibody absorption studies with anti-TRH sera absorbed with TRH, 3 diastereoisomeric analogues of TRH, gonadotropin-releasing hormone (GnRH), bovine serum albumin, thyrotropin, prolactin, adrenocorticotropin, luteinizing hormone, follicle stimulating hormone were performed to determine the specificity of the staining reaction. Only absorption with TRH resulted in a significant reduction in staining intensity. In vitro experiments were then begun with hemipituitaries to ascertain if intrapituitary TRH might originate by sequestration of exogenous, plasma membrane bound TRH or by de novo synthesis. The results suggest that anterior pituitary TRH is of endogenous origin.     

Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis

The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T₃) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T₃ response requires both on-going mRNA and protein synthesis. In addition, the effects of T₃ were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T₃. Furthermore, the inhibition of SF-1 expression by DAX-1 markedly abolished T₃-mediated StAR expression in a time frame, which was consistent with decreased steroid biosynthesis. Specific involvement of SF-1 was further confirmed by assessing the 5′-flanking region of the mouse StAR gene, which identified a region between −254 and −110 bp that was essential for T₃ function. Importantly, it was found that the SF-1 binding site at position −135 bp of the 5′-flanking region was greatly involved in T₃-mediated reporter activity. Electrophoretic mobility shift assays (EMSA) also demonstrated involvement of SF-1 in T₃ function. The relevance of T₃-mediated LHR function was investigated in mice rendered hypo-and hyperthyroid, which accounted for up-regulation in the former and down-regulation in the latter group, respectively. These findings demonstrate a key role of thyroid hormone in maintaining mouse Leydig cell function, where thyroid hormone and StAR protein coordinately regulate steroid hormone biosynthesis.     

Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells. Differential effects of hydrocortisone and tissue extracts

Several established clonal strains of rat pituitary cells which produce growth hormone in culture have been shown to secrete a second protein hormone, prolactin. Prolactin was measured immunologically in culture medium and within cells by complement fixation. Rates of prolactin production varied from 6.6 to 12 µg/mg cell protein per 24 hr in four different cell strains. In these cultures ratios of production of prolactin to growth hormone varied from 1.0 to 4.1. A fifth clonal strain produced growth hormone but no detectable prolactin. Intracellular prolactin was equivalent to the amount secreted into medium in a period of about 1–2 hr. Both cycloheximide and puromycin suppressed prolactin production by at least 94%. Hydrocortisone (3 x 10^-6 M), which stimulated the production of growth hormone 4- to 8-fold in most of the cell strains, reduced the rate of prolactin production to less than 25% of that in control cultures. Conversely, addition of simple acid extracts of several tissues, including hypothalamus, to the medium of all strains increased the rate of production of prolactin six to nine times and decreased growth hormone production by about 50%. We conclude that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.