Progesterone and 17 beta-estradiol acutely stimulate nitric oxide synthase activity in rat aorta and inhibit platelet aggregation

The rapid non-genomic stimulatory action of progesterone (Pg) and estradiol (E2) on nitric oxide synthase (NOS) activity of endothelium intact aortic rings and its effect on platelet aggregation was investigated. First we measured the effect of the hormones on platelet aggregation when added to rat aortic strips (RAS) incubated in a PRP. RAS induced an antiaggregatory activity, which was enhanced by the presence of the hormones. The inhibitory action induced by the hormones was evoked in a dose dependent manner (10 pM-100 nM). These effects are specific for progesterone and 17-beta-estradiol, since either testosterone and 17-alpha-estradiol were devoid of activity. The hormones induced rapid responses, producing significant inhibition within 1 to 5 minutes of hormonal exposure. The addition of 10(-5) M L-NAME suppressed the antiaggregatory effect of 1 nM E2 or 10 nM Pg. Furthermore, we specifically quantified the NO generation by the 3H-citrulline technique. 10(-8) M E2 induced 2-fold increase of RAS citrulline production, while the increment induced by 10(-7) M Pg was 55% over control. Preincubation with 10(-5) M L-NAME completely suppressed the stimulatory action of 10(-9) M E2 or 10(-8) M Pg, confirming that the antiaggregatory factor released from the aortic tissue was NO. Preincubation with cycloheximide did not block the increment in NO induced by the hormones. In conclusion the present study provides for the first time evidence of acute, non-genomic effects of Pg on rat aorta NOS activity and platelet aggregation in coincidence with the results obtained with estradiol treatment.     

Stimulation of ascites tumor RNA polymerase II by protein kinase.

The activity of purified RNA polymerase II from Novikoff ascites tumor cells is stimulated 5-7-fold by a purified protein factor. This protein factor, designated HLF2, has extensive protein kinase activity and catalyzed the incorporation of gamma-32G from ATP into protein under normal RNA polymerase assay conditions. Protein phosphorylation is totally dependent on the presence of HLF2 and is stimulated 2-3-fold by the presence of highly purified RNA polymerase II. The purification procedure developed for the isolation of the polymerase stimulatory factor resulted in a 4000-fold purification of a protein kinase. Chromatography on carboxymethylcellulose, phosphocellulose, and Sephadex G-100 did not resolve polymerase stimulatory activity from protein kinase activity. Adenylimidodiphosphate (AMP-PNP), an inhibitor of protein kinases, inhibited the stimulatory activity of purified factor by 80%. The heat denaturation profile of protein kinase was paralleled by the loss of polymerase stimulatory activity. Concentrations of (NH4)2SO4 which are known to inhibit polymerase stimulation (Lee and Dahmus, 1973) also inhibit protein kinase activity. The protein kinase activity associated with stimulatory factor catalyzes the phosphorylation of basic proteins such as protamine or histone. The protein kinase is not stimulated by cyclic 3', 5'-AMP or -GMP over a concentration range of 10(-6)-10(-4)M. Furthermore, protein kinase activity is not inhibited by either the regulatory subunit of rabbit muscle protein kinase or by the heat-stable inhibitor of cyclic 3', 5'-AMP-dependent protein kinases. Protein kinase activity is stimulated by KCl or NH4Cl and is inhibited by MnCl2. The apparent Km values, determined in the presence of 4 mM Mg2+, are 0.02 mM for ATP, and 4.1 mM for GTP.     

Inhibitory effects of the antiprogestin, RU 486, on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs

The effects of RU 486 on the modulation of LH release by progesterone were investigated in cultured anterior pituitary cells from ovariectomized adult female rats. The inhibitory effect of progesterone on LH secretion was demonstrable in estrogen-treated pituitary cells, in which addition of 10(-6) M progesterone to cells cultured in the presence of 10(-9) M estradiol for 52 h reduced the LH response to GnRH (10(-11) to 10(-7) M). When RU 486 was superimposed upon such combined treatment with estradiol and progesterone, the suppressive effect of progesterone on GnRH-induced LH release was completely abolished. The converse (facilitatory) effect of progesterone on LH secretion was observed in pituitary cells pretreated with 10(-9) M estradiol for 48 h and then with 10(-6) M progesterone for 4 h. When RU 486 was added together with progesterone during the 4 h treatment period, the facilitatory effect of progesterone was blocked and LH release fell to below the corresponding control value. The direct effect of RU 486 on LH secretion in the absence of exogenous progesterone was evaluated in cells cultured in the absence or presence of 10(-9) M estradiol and then treated for 4 to 24 h with increasing concentrations of RU 486 (10(-12) to 10(-5) M) and stimulated with GnRH (10(-9) M) during the last 3 h of incubation. In estrogen-deficient cultures, 4 h exposure to RU 486 concentrations of 10(-6) M and above decreased the LH response to GnRH by up to 50%. In cultures pretreated with 10(-9) M estradiol, GnRH-stimulated LH responses was inhibited by much lower RU 486 concentrations, of 10(-9) M and above. After 24 h of incubation the effects of RU 486 were similar in control and estradiol-pretreated pituitary cell cultures. Thus, RU 486 alone has a significant inhibitory effect on LH secretion that is enhanced in the presence of estrogen. The antiprogestin is also a potent antagonist of both the inhibitory and the facilitatory actions of progesterone upon pituitary gonadotropin release in vitro.     

Effect of insulin-like growth factor-1 on steroidogenesis in cultured carp ovarian follicles: interactions with estradiol

The biological action of insulin like growth factor-1 (IGF-1) on follicular steroidogenesis during follicular development in common carp was examined. Studies were carried out by culturing small (1-2 mm diam.) and large (> 2 mm diam.) follicles. IGF-1 (0.3-100 ng/ml) had no effect on progesterone accumulation or aromatase activity during 48 hr culture of small follicles. Progesterone accumulation by large follicles was also unaffected by IGF-1 over the same period, although aromatase activity was stimulated in a dose dependent manner (8-fold increase over basal levels with a maximum stimulatory dose of 30 ng IGF-1/ml). In contrast, small and large follicles responded to IGF-1 in terms of both progesterone accumulation and aromatase activity after longer periods of culture (4 days for progesterone and 6 days for aromatase). Concurrent treatment of small follicles with estradiol (10(-7) M) enhanced the action of IGF-1 on both indices of steroidogenesis and advanced the time at which IGF-1 stimulated activity was first detectable. The effect of estradiol on follicular IGF-1 responsiveness were independent of cell number. In summary, these results demonstrate varied actions of IGF-1 carp ovarian follicular steroidogenesis in vitro. The results indicate that carp follicles acquire responsiveness to IGF-1 in terms of aromatase activity during follicular development in vivo and that estradiol can induce the response in vitro. The results also suggest that estrogen and progesterone biosynthesis by cultured carp ovarian follicles is differentially regulated by IGF-1. Together, these results provide new insights into the biological actions of IGF-1 in fish ovary.     

Comparison of the effect of cortisol on aromatase activity and androgen metabolism in two human fibroblast cell lines derived from the same individual

The effect of preincubation with cortisol on estrogen and androgen metabolism was investigated in human fibroblast monolayers grown from biopsies of genital and non-genital skin of the same person. The activity in the cells of aromatase, 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase and 3 alpha-hydroxysteroid oxidoreductase was investigated by isolating estrone, estradiol, estriol, dihydrotestosterone, androstanedione, androsterone, 3 alpha-androstanediol, testosterone and androstenedione after incubation of the cells with [14C]testosterone or [14C]androstenedione. For experiments with 14C-labeled substrate the cells were incubated in medium, charcoal stripped of steroids without Phenol Red. Preincubation from 6 to 36 h with cortisol in concentrations of 10(-8) - 10(-6) M showed maximal stimulation of aromatase activity after 12 h preincubation with cortisol in concentrations of 0.5-1.0 x 10(-6) M in both cell lines. When preincubation with cortisol was omitted no estrogen synthesis was detected. The formation of androgen was not altered after preincubation with cortisol. Pronounced differences were found in estrogen and in androgen metabolism in the two cell lines suggesting a local regulation of the hormonal environment. The aromatase activity, which is low in many tissues could be stimulated by cortisol without altering the androgen metabolism was found to be a suitable system for investigations of the cellular interconversion of androgens and estrogens and for investigations of the in vitro regulation of the enzymes involved.     

Cyclin-dependent kinase (cdk) inhibitors/cdk4/cdk2 complexes in early stages of mouse mammary preneoplasia.

The level of circulating ovarian hormones (estrogen and progesterone) alone or in combination with pituitary hormones have a potent mitogenic impact in the normal mammary gland, and they also play a pivotal role in the development and progression of mammary carcinoma. The differential effects of hormones on the molecular components of cyclin-dependent kinase (cdk) complexes in mammary epithelium of the hormone-dependent ductal outgrowth line, EL11, and the hormone-independent alveolar outgrowth line, TM2L, were the focus of this study. The two outgrowth lines, which represent early stages in mammary hyperplasia, were compared with normal mammary gland at different hormonal conditions: control, hormone stimulated by pituitary isograft, and hormone depleted by ovariectomy. Hormonal stimulation by a pituitary isograft resulted in DNA synthesis and lobuloalveolar development of normal mammary ducts, DNA synthesis but no lobuloalveolar development in the EL11 ductal outgrowth, and no changes either in DNA synthesis or in lobuloalveolar morphology in the TM2L outgrowth. The levels of cdk4- and cyclin D1-associated kinase activities were correlated with cell proliferation in only the alveolar phenotypes (i.e., in only hormonally stimulated normal virgin gland and in alveolar mammary outgrowth), whereas cyclin D2-dependent kinase activity was correlated with cell proliferation in only the alveolar preneoplasia. p16(INK4a) and p21(Cip1) protein levels were decreased at the earliest stages of preneoplasia, i.e., at immortalization, and were independent from changes in cyclin D1, which occurred later in preneoplasia. Although all cdk inhibitors changed in concordance with hormonal status reflected by proliferation levels, p27(Kip1) was the only cdk inhibitor that was up-regulated at the earliest stages of preneoplasia and may have a unique role in blocking alveolar differentiation in response to the loss of one or more of the cell cycle-negative regulators. We hypothesize that up-regulation of p27(Kip1) prevents immortalized ductal outgrowths (EL11) from progressing to the neoplastic state, even under hormonal stimulation.     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

Zinc chloride stimulates DNA synthesis of mouse embryonic stem cells: involvement of PI3K/Akt, MAPKs, and mTOR

Although zinc is one of the most important trace elements in the body, the mechanisms underlying zinc-induced cell proliferation have yet to be unraveled. Thus, we investigated the effect of zinc chloride (ZnCl(2)) on mouse embryonic stem (ES) cell proliferation and related signaling pathways. ZnCl(2) (40 microM) significantly increased [(3)H]-thymidine incorporation after 12 h of treatment. At moderate concentrations (> or =4 microM), ZnCl(2) increased cell cycle regulatory protein levels, [(3)H]-thymidine incorporation, and total cell numbers, but higher doses of ZnCl(2) (> or =200 microM) blocked this proliferative effect. ZnCl(2) induced the phosphorylation of Akt, c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), p44/42 MAPKs, and mammalian target of rapamycin (mTOR) in a time-dependent manner. Pretreatment of LY 294002 (a PI3K inhibitor, 10(-6) M), wortmannin (a PI3K inhibitor, 10(-7) M), or an Akt inhibitor (10(-5) M), which inhibited the activation of JNK/SAPK and p44/42 MAPKs, blocked the ZnCl(2)-induced expression of cyclins and cyclin-dependent kinases (CDKs). Furthermore, pretreatment with PD 98059 (a p44/42 inhibitor, 10(-5) M) or SP 600125 (a JNK inhibitor, 10(-6) M) inhibited ZnCl(2)-induced activation of mTOR, p70S6K, and 4E-BP1. In addition, rapamycin (an mTOR inhibitor, 10(-8) M) blocked the ZnCl(2)-induced increase in [(3)H]-thymidine incorporation and cell cycle regulatory protein expression. In conclusion, ZnCl(2) stimulated ES cell proliferation through the PI3K/Akt, p44/42 MAPKs, JNK/SAPK, and mTOR signal pathways.     

Estrogen regulation of progestin biosynthetic enzymes in cultured rat granulosa cells

The mechanism by which estrogens enhance gonadotropin-stimulated ovarian progestin production was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) in cultured rat granulosa cells. Cells from immature hypophysectomized, estrogen-treated rats were cultured for 3 days with follicle-stimulating hormone (FSH) and/or estrogens. Pregnenolone production was measured in the presence of cyanoketone which inhibits 3 beta-HSD activity. Activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. Some cells were also primed with FSH to induce luteinizing hormone (LH) receptors for studies on the effects of estrogens on LH-modulated parameters. Pregnenolone production by cultured granulosa cells was stimulated by FSH, while treatment with diethylstilbestrol (DES) or estradiol further enhanced the gonadotropin action. Treatment with FSH increased 3 beta-HSD activity. Similarly, concomitant treatment with DES further enhanced 3 beta-HSD activity in a dose-dependent manner with an apparent ED50 of 10(-8) M. Also, treatment with estrogens alone increased 3 beta-HSD activity. The increases in enzyme activity induced by estrogen alone or in combination with FSH were not associated with changes in the apparent Km values. FSH also stimulated 20 alpha-HSD activity by 2-fold in these cells, while concomitant treatment with DES did not affect the FSH action. In FSH-primed cells, LH stimulated pregnenolone production while the LH action was enhanced by concomitant treatment with the estrogens. Likewise, LH stimulated the activity of 3 beta-HSD, while concomitant DES treatment further augmented LH action. LH did not stimulate 20 alpha-HSD activity when added alone or in combination with DES. Thus, the estrogen enhancement of the gonadotropin-stimulated progesterone production in cultured rat granulosa cells is associated with increases in pregnenolone biosynthesis and the activity of the 3 beta-HSD enzyme, without affecting the 20 alpha-HSD activity.     

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeability properties of the Bufo bufo bladder as affected by isoprenaline and vasopressin

Isoprenaline, a beta adrenergic agonist, strongly increases both transepithelial fluxes across the urinary bladder of Bufo bufo; this effect is dose dependent, 10(-6)M being necessary for the maximal action. This effect is less selective than that of vasopressin: the ratio J urea/J thiourea is 3.8 under isoprenaline and 30.4 under vasopressin treatment. Both hormones differently affect the permeability of a mainly liposoluble molecule, i.e. antipyrine: vasopressin increases antipyrine permeability, while isoprenaline decreases it. Moreover diethylpyrocarbonate treatment of the luminal membrane strongly inhibits vasopressin effect on urea permeability leaving unmodified that of isoprenaline. However, the actions of both hormones are not additive. These results allows to assume that the tissue has a feedback mechanism which inhibits other hormonal action while the bladder is stimulated by a particular hormone.     

The effect of estradiol on the activity of cAMP-dependent protein kinase in cells of estradiol-dependent tumors of the rat mammary gland. The role of a thermally stable protein inhibitor of cAMP-dependent protein kinase

Stimulation of rat mammary tumour growth by estradiol is due to the activation of the adenylate cyclase system and cAMP-dependent protein kinases. A single administration of estradiol to ovariectomized rats causes a rise in the cAMP-dependent protein kinase activity in cell nuclei within the first 4-6 hours after injection. This effect is probably due to the translocation of enzymes into nuclei and an increase of their synthesis. The high level of the cAMP-dependent protein kinase activity in cell nuclei was observed in actively growing intact mammary tumours, in contrast to regressing ones in ovariectomized animals. This phenomenon can be accounted for by the decrease in the content of a thermostable protein inhibitor of cAMP-dependent protein kinases rather than by the high level of cAMP.     

Characteristics of different cytoplasmic and nuclear estrogen receptors appearing with continuous hormonal exposure

Biochemical properties of cytosol estrogen receptor (ERC) and nuclear estrogen receptor (ERN) from rat uteri continuously exposed in vivo to 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2) for 6 h have been studied on the basis of immunological recognition and chromatographic elution patterns. Overall concentrations of ERC and ERN did not change during this time period when receptor-saturating concentrations of [3H]E2 were maintained (Jakesz, R., Kasid, A., and Lippman, M. E. (1983) J. Biol. Chem. 258, 11798-11806); however, biochemical characteristics were different in ERC and ERN after short or long term hormonal exposure. When ERC from rats treated with estradiol for 30 min was applied to HAP or DEAE columns, two different ER binding components were seen. DNA binding in a cell-free system revealed that these binding components represented an activated and a nonactivated ERC population. After long term hormonal exposure (6 h), only one component of ERC with low DNA binding could be shown despite the preservation of an equivalent quantity of cytoplasmic binding activity. This binder does not react with a monoclonal antibody directed against extranuclear estrogen receptor species. These data suggest disappearance of the activated ERC population, with appearance of a new, immunologically nonrecognizable ERC species with 6 h of continuous hormonal exposure. Elution profiles of ERN on HAP chromatography reveal 2 different binding components at 30 min and at 6 h of continuous [3H]E2 exposure. There is an increase of the population eluted at higher molarity after 6 h of in vivo treatment. This later eluting binding component is the major DNA binder in vitro. ERN from both time points are recognized immunologically by monoclonal antibody. After reaction with the antibody, the sedimentation coefficient shifted to 8-9 S on sucrose gradients, but the previously described faster sedimentation of ERN extracted 6 h after injection persisted. We conclude that ER in both cellular compartments undergoes time-dependent alterations, which may be involved in the initiation of hormone action.     

Beta-adrenergic and muscarinic modulation of human embryonic stem cell-derived cardiomyocytes.

BACKGROUND: Embryonic stem cells provide the most promising tool for cell replacement therapy including transplantation of human embryonic stem (hES) cell- derived cardiomyocytes in the infarcted area of the heart. Here we provide data for differentiation of cardiomyocytes from hES cells and firstly describe their hormonal modulation. METHODS: Using Micro-Electrode Arrays as a novel electrical mapping technique of beating cardiomyocyte clusters within whole hES cell aggregates, we were able to measure the field potential generation and morphology changes during hormonal modulation. RESULTS: We found that isoproterenol provokes, similar to the mouse ES cell system, a strong positive chronotropic effect with an EC50 of around 10(-8) M. Moreover, isoproterenol stimulated with a higher EC50 value the slow field potential amplitude, FP(slow), indicating a stimulation of Ca2+ channels in ventricular-like ES cell-derived cardiomyocytes which is shown to be clearly independent from frequency modulation. In contrast, carbachol (10 microM) produced a transient negative chronotropic effect but had no effect on FP(slow). CONCLUSION: The Micro-Electrode system allows measurement of ionic channel modulation and chronotropic responsiveness in a pharmacological screening setup. Moreover, all our data indicate that cardiomyocytes derived from human embryonic stem cells exhibit a physiological response to the major hormones of the vegetative nervous system and might therefore serve as an ideal candidate for the use in cell replacement strategies.     

Thyroid hormonal activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A

The thyroid hormonal-disrupting activity of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) was examined and compared with that of bisphenol A, a typical estrogenic xenobiotic. TBBPA and TCBPA, halogenated derivatives of bisphenol A, markedly inhibited the binding of triiodothyronine (T(3); 1 x 10(-10) M) to thyroid hormone receptor in the concentration range of 1 x 10(-6) to 1 x 10(-4) M, but bisphenol A did not. The thyroid hormonal activity of TBBPA and TCBPA was also examined using rat pituitary cell line GH3 cells, which grow and release growth hormone (GH) depending on thyroid hormone. TBBPA and TCBPA enhanced the proliferation of GH3 cells and stimulated their production of GH in the concentration range of 1 x 10(-6) to 1 x 10(-4) M, while bisphenol A was inactive. TBBPA, TCBPA, and bisphenol A did not show antagonistic action, i.e., these compounds did not inhibit the hormonal activity of T(3) to induce growth and GH production of GH3 cells. TBBPA and TCBPA, as well as bisphenol A, enhanced the proliferation of MtT/E-2 cells, whose growth is estrogen-dependent. These results suggest that TBBPA and TCBPA act as thyroid hormone agonists, as well as estrogens.     

Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal

Treatment of adipocytes with okadaic acid (a specific inhibitor of type 1 and 2a protein phosphatases) resulted in a rapid 8-10-fold stimulation of cell extract myelin basic protein (MBP) kinase activity (t1/2 = 10 min) and kinase activity toward a synthetic peptide RRLSSLRA (S6 peptide) (t1/2 = 5 min). Insulin brought about a smaller stimulation of these two activities (t1/2 = 2.5 min). MBP kinase activity from cells treated with okadaic acid or insulin was resolved by anion exchange chromatography into two well defined peaks; S6 peptide kinase activity was less well resolved. The two partially purified MBP kinases were inactivated by the protein tyrosine phosphatase CD45 or by protein phosphatase 2a (PP-2a). In contrast, partially purified S6 peptide kinase activity was inactivated only by PP-2a or protein phosphatase 1 (PP-1). Furthermore, a 38-kDa protein which co-eluted with one peak of MBP kinase and a 42-kDa protein which co-eluted with the other peak of MBP kinase were phosphorylated on tyrosine after treatment with okadaic acid. These findings illustrate several important points concerning regulation of MBP and S6 peptide kinases. First, these protein kinases are regulated by phosphorylation, and, second, in the absence of hormonal stimuli their activities are strongly suppressed by protein phosphatases. Lastly, the increased tyrosine phosphorylation accompanying the activation of MBP kinases following okadaic acid treatment suggests a role for PP-2a in events that are mediated by tyrosine phosphorylation.     

Evidence for the presence of protein kinases which stimulate phosphorylation of c-erb A protein in rat kidney nuclei.

Protein kinases were separated from rat kidney nuclear extract by hydroxylapatite column chromatography. Five (I-V) different protein kinases were isolated when histone was used as a substrate. Two (I and III) of them stimulated phosphorylation of c-erb A-beta protein (50 kDa) expressed in Escherichia coli. The c-erb A product has an activity of high affinity T3 binding. One (I) of the kinases was dependent on cyclic adenosine 3',5'-monophosphate (cyclic AMP). The other kinase (III) was not dependent on cyclic nucleotides. The latter kinase was eluted from hydroxylapatite column with 0.05 M PO4 at pH 7.4. The sedimentation coefficient(s) estimated by continuous sucrose density gradient centrifugation was approximately 6.0 Km values for ATP were estimated by double reciprocal analyses, which gave 110.0 microM in the protein kinase I (in the presence of 10(-6) M cyclic AMP) and 25 microM in the protein kinase III, respectively. The data showed that 1.0 mol phosphate was incorporated into 80 mol of c-erb A protein (50 kDa) either in the presence of protein kinase I (with 10(-6) M cyclic AMP) or in the presence of protein kinase III. These results suggested that there are protein kinases for c-erb A protein, whose functional properties are similar to those of nuclear T3 receptor, in rat kidney nuclei.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.