Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequenc of biochemical events associated with the action of follicle-stimulating hormone (FSH) in the testis has been investigated using a Sertoli cell-enriched testis model system. The Sertoli cell-encriched testis, created by irradiation of male rats in utero, is devoid of germinal elements but contains a normal complement of supportive Sertoli cells. Comparison of the Sertoli cell-enriched testis with normal testis, demonstrates that the two types of testes contain equal numbers of FSH specific receptors, judged by the binding of labeled hormone. In addition, FSH over a concentration range from 6 X 10(-11) to 6 X 10(-9)M will stimulate the production of adenosine 3',5' monophosphate (cAMP) in the Sertoli cell-enriched testis in a manner indistinguishable from that of the normal testis. Incubation of Sertoli cell enriched testis also results in the activation of soluble cAMP-dependent protein kinase. This response to FSH is dependent upon the age of the animal and disappears at about 32 days of age. While sensitivity to the hormone can still be detected in mature Sertoli cell-enriched animals by the addition of the phosphodiesterase inhibitor 1-methyl-3-isobutyl-xanthine, no detectable increase in phosphodiesterase activity is apparent after 30 days of age. Injection of FSH into Sertoli cell-enriched animals results in an increase in total testicular protein synthesis as well as in the production of the Sertoli cell-specific protein, androgen-binding protein within 30 minutes. Furthermore, while hypophysectomy of Sertoli cell-enriched animals result in a decline of the testicular concentration of androgen-binding protein, the injection of FSH will stimulate and maintain the levels of androgen-binding protein in such animals. These results demonstrate that the Sertoli cell-enriched testis is capable of carrying out the sequence of biochemical events previously described for FSH in the normal testis and therefore, suggest that the Sertoli cell is the primary target cell for FSH action.
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PMID:Biochemical actions of follice-stimulating hormone in the sertoli cell of the rat testis. 17 98

Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and beta-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5'-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 microM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was cotransfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either gonadotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.
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PMID:Specific inhibition of protein kinase A in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter. 190 60

Ovarian follicles of Xenopus laevis frogs consist of a single large oocyte surrounded by follicle cells attached to the oocyte by gap junctions. Adenosine has been found to activate an outward K+ current in follicles. This response is reduced by microinjection of protein kinase inhibitor (PKI), suggesting that adenosine 3',5'-cyclic monophosphate (cAMP) mediates the response. To investigate this further, we verified previous studies that indicate that several methods of elevating cAMP in follicles activate hyperpolarizing outward currents. The potency of two adenosine analogues to hyperpolarize follicles, 5'-N-ethylcarboxamidoadenosine (NECA) greater than cyclopentyladenosine, is indicative of A2 receptors that are characteristically coupled to adenylyl cyclase. We also report for the first time that another stimulator of adenylyl cyclase, follicle-stimulating hormone (FSH), also induces a hyperpolarizing current in follicles which is carried by K+ and attenuated by injection of PKI. We used a novel procedure to completely remove follicle cells from oocytes. Intact follicles, but not oocytes completely stripped of follicle cells, hyperpolarized in response to FSH, NECA, dibutyryl cAMP, microinjected cAMP, and forskolin, but not to dideoxyforskolin (which does not activate adenylyl cyclase). Injection of the catalytic subunit of cAMP-dependent protein kinase (which is too large to traverse gap junctions) into oocytes of intact follicles failed to activate a K+ current. These data suggest that FSH and adenosine hyperpolarize follicles by stimulating adenylyl cyclase and that cAMP-dependent protein kinase must be activated on both sides of follicle cell-oocyte gap junctions to elicit a hyperpolarizing K+ current.
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PMID:Xenopus oocyte K+ current. I. FSH and adenosine stimulate follicle cell-dependent currents. 212 36

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.
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PMID:Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells. 242 18

Hypoxanthine and adenosine are present in preparations of mouse ovarian follicular fluid, and these purines maintain mouse oocytes in meiotic arrest in vitro (Eppig et al.: Biology of Reproduction 33:1041-1049. 1985). The first hypothesis tested in this study is that purines which maintain meiotic arrest act by maintaining meiosis-arresting levels of cyclic adenosine monophosphate (cAMP) in the oocyte. Oocyte-cumulus cell complexes were incubated in control medium (no added purines), or medium containing 0.75 mM adenosine, 4 mM hypoxanthine, or both for 3 hr and the percentage of the oocytes that underwent germinal vesicle breakdown (GVB) and the cAMP content of the intact complexes and the oocytes were determined. Adenosine alone had little inhibitory effect on GVB at this time point but sustained higher levels of cAMP in the oocytes. Hypoxanthine maintained 80% of cumulus cell-enclosed oocytes in meiotic arrest and also sustained higher cAMP levels in the oocytes. The addition of adenosine to hypoxanthine-containing medium increased the percentage of oocytes maintained in meiotic arrest, and increased the amount of cAMP in the oocytes above that maintained by either hypoxanthine or adenosine alone. Neither hypoxanthine, adenosine, nor hypoxanthine plus adenosine altered the cAMP content of intact complexes when assayed after 3 hr culture. Microinjection of an inhibitor of the catalytic subunit of cAMP-dependent protein kinase induced GVB in denuded oocytes cultured in medium containing hypoxanthine. This purine, therefore, maintained meiotic arrest by sustaining elevated cAMP levels within the oocytes. The second hypothesis tested in this study is that purines maintain meiosis-arresting levels of cAMP, at least in part, by inhibiting cAMP phosphodiesterase activity. In descending order of potency, 3-isobutyl-1-methylxanthine (IBMX), guanosine, hypoxanthine, adenosine, and xanthosine inhibited cAMP phosphodiesterase in oocyte lysates. Moreover, like the potent phosphodiesterase inhibitor IBMX, hypoxanthine augmented the meiotic arrest and cAMP accumulation mediated by follicle-stimulating hormone (FSH) in intact complexes. Therefore, inhibition of oocyte phosphodiesterase appears to be one mechanism by which the purines could maintain meiosis-arresting levels of cAMP.
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PMID:Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity. 247 69

The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium-dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2-dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP-dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C.
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PMID:Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C. 300 7

These studies were undertaken to determine the molecular events by which estradiol and follicle-stimulating hormone (FSH) stimulate in ovarian granulosa cells the increase in the content of one of the regulatory subunits of cAMP-dependent protein kinase type II, RII51 (Mr = 51,000), and its electrophoretic variants RII51.5 (Mr = 51,500) and RII52 (Mr = 52,000). To analyze the de novo synthesis of RII51/52, granulosa cells were cultured (10(6) cells/ml) for 0, 12, 24, or 48 h with estradiol (10 nM) +/- FSH (12.5, 25, and 50 ng/ml), 8-bromo-cAMP (0.25-3 mM), or forskolin (0.5-100 microM) and then pulse-labeled with [35S]methionine (300 microCi/ml; 4 h). Labeled RII51, present either in urea extracts of total cellular protein or after partial purification from a soluble cell extract by cAMP-Sepharose chromatography, was quantitated by autoradiography of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and by excision of the silver-stained spots of the RII51 variants from the gels and counting. Synthesis of RII51 and its electrophoretic variants was low in cells cultured with estradiol alone for 48 h, whereas it was increased 4-5-fold in cells cultured with estradiol and FSH. Changes in the synthesis of actin were minor throughout the culture period regardless of hormone treatment. Pulse-chase experiments using [35S]methionine provided evidence that the isoelectric variants RII51.5 and RII52 may be derived from RII51 by post-translation modification, such as phosphorylation. Labelling with [32P]orthophosphate showed that RII52 contained more radioactivity than RII51.5 and RII51. Northern and filter hybridization assays demonstrated a 6-10-fold dose- and time-dependent increase in the amount of RII51 mRNA in granulosa cells exposed to estradiol and FSH or estradiol and forskolin compared to those cultured with estradiol alone. In vitro translation of poly(A)+ mRNA of granulosa cells from estradiol- and FSH-treated hypophysectomized rats also demonstrated an increase in the content of translatable RII51 mRNA. These studies indicate that in cultured rat granulosa cells the synthesis of RII51 and the content of its mRNA are selectively increased by estradiol and cAMP in a time- and dose-dependent manner. Based on these observations, RII51 appears to be a useful marker to determine the molecular (genomic?) sites of estradiol and FSH action in differentiating rat granulosa cells.
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PMID:Hormonal regulation of the synthesis and mRNA content of the regulatory subunit of cyclic AMP-dependent protein kinase type II in cultured rat ovarian granulosa cells. 303 88

Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.
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PMID:Hormonal regulation of nuclear cyclic AMP-dependent protein kinase subunit levels in rat ovaries. 313 55

The regulatory subunit (R-II) of cAMP-dependent protein kinase type II is induced in rat ovarian granulosa cells by the synergistic actions of estradiol and follicle-stimulating hormone. The R-II from rat ovaries was compared with R-II from rat heart, rat brain, bovine heart, and bovine brain using immunological methods, 8-N3[32P]cAMP photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three isoforms of R-II were identified in rat ovarian cell extract (R-II54 Mr = 54,000, R-II52 Mr = 52,000, R-II51 Mr = 51,000), two isoforms of R-II in rat brain cell extract (Mr = 54,000, Mr = 52,000), and one isoform of R-II in rat heart cell extract (Mr = 54,000). Rat ovarian R-II54, heart R-II, and brain R-II (Mr = 54,000) were recognized by antiserum against rat heart R-II, whereas rat ovarian R-II52/R-II51 and rat brain R-II (Mr = 52,000) were not. In contrast, an antiserum raised against bovine heart R-II recognized all three isoforms of ovarian R-II as well as the lower molecular weight form of rat brain R-II. Ovarian types R-II52 and R-II51 but not R-II54 were increased selectively in granulosa cells by estradiol and follicle-stimulating hormone. In addition: 1) ovarian R-II52/51 subunits were purified to homogeneity and shown to recombine with C subunit from bovine heart to form a cAMP-dependent protein kinase; 2) pure R-II52/51 were not interconvertible to a higher molecular weight form by C subunit-dependent phosphorylation; 3) pure rat heart R-II (Mr = 54,000) and ovarian R-II52/51 exhibited distinct differences based on one- and two-dimensional peptide mapping; and 4) by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis pure R-II52/51 were resolved as three (rather than two) isoelectric variants which were clearly different from pure rat heart R-II54. Thus, the hormone-regulated form of R-II in rat ovarian granulosa cells appears to represent a gene product distinct from R-II54 in rat heart.
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PMID:Purification and characterization of hormone-regulated isoforms of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovaries. 393 66

The accumulation of two polypeptides, SCm1 and SCm2, in the medium of Sertoli cell cultures is enhanced by follicle-stimulating hormone (FSH) but is unaffected by either the cAMP analog, N6,O2'-dibutyrl cAMP or luteinizing hormone. The assigned molecular weights of SCm1 and SCm2 differ from those of androgen-binding protein subunits or any other previously identified Sertoli cell secretory product. Incubation of Sertoli cell cultures with either FSH or N6,O2'-dibutyryl cAMP also stimulates the incorporation of [35S]methionine into two intracellular polypeptides, SCc1 and SCc2. In addition, the phosphorylation of three intracellular polypeptides, SCc3, SCc4, and SCc5, is intensified when Sertoli cell cultures are treated with either FSH or N6,O2'-dibutyryl cAMP. Based on these results and on previous work, we conclude that (i) SCm1 and SCm2 may, like androgen-binding protein, be secreted by Sertoli cells and function extracellularly while SCc1 and SCc2 are involved in FSH-dependent intracellular activity; (ii) SCc3, SCc4, and SCc5 are possible substrates for FSH-stimulated, cAMP-dependent protein kinase activity; and (iii) SCc5 is an isoelectric variant of vimentin-type intermediate filament protein presumably involved in FSH- and N6,O2'-dibutyryl cAMP-induced Sertoli cell shape changes.
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PMID:Hormonal regulation of protein synthesis, secretion, and phosphorylation in cultured rat Sertoli cells. 629 7


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