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Marine Science Institute, University of Texas at Austin, 750 Channelview Drive, Port Aransas, Texas 78373, USA
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Marine Science Institute, University of Texas at Austin, 750 Channelview Drive, Port Aransas, Texas 78373, USA
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Marine Science Institute, University of Texas at Austin, 750 Channelview Drive, Port Aransas, Texas 78373, USA
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Marine Science Institute, University of Texas at Austin, 750 Channelview Drive, Port Aransas, Texas 78373, USA
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that mPRα acts as an intermediary in MIS induction of oocyte maturation in teleost fish. Recent results in human myometrial cells have shown that both mPRα and mPRβ are involved in downregulation of adenylyl cyclase activity through a pertussis toxin
Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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Department of Physiology, University of La Laguna, La Laguna, Spain
Department of Comparative Pathology, University of Córdoba, Córdoba, Spain
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from TX-treated OVX rats as well as TX-cultured anterior pituitary cells from OVX rats, this study investigated the action of E 2 on: (1) LHRH self-priming; (2) the potentiating effect of P 4 and adenylyl cyclase activator forskolin (FSK) on LHRH
Department of Cell Biology, Physiology and Immunology, University of Córdoba, Cordoba, Spain
Hospital Universitario Reina Sofia (HURS), Cordoba, Spain
CIBER de la Fisiopatología de la Obesidad y Nutrición (CIBERobn), Madrid, Spain
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inhibitors to various intracellular signaling pathways previously shown to be important in the actions of SST (adenylyl cyclase (AC), protein kinase A (PKA), phospholipase C (PLC), protein kinase C (PKC), extracellular Ca 2+ L-type channels, intracellular Ca
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Abstract
The rabbit corpus luteum possesses LH receptors that are coupled to adenylyl cyclase, but paradoxically it does not require LH as a luteotrophic factor for the maintenance of progesterone secretion. This suggests that rabbit luteal cells may not respond physiologically to LH. Therefore, the present study was undertaken to investigate the responsiveness of the rabbit corpus luteum of pseudopregnancy to human chorionic gonadotrophin (hCG) which acts on the same receptor as LH. Pseudopregnancy was induced by injection of 40 IU pregnant mare serum gonadotrophin followed 50 h later by an injection of 40 IU hCG (day 0). On days 7 and 11 of pseudopregnancy, corpora lutea were obtained and incubated for 2 or 5 h in the presence of either 0·1 or 1 μg/ml hCG or 1 mm monobutyryl cyclic AMP (bcAMP). Neither hCG nor bcAMP stimulated progesterone production by the isolated corpus luteum, despite a sustained high rate of progesterone production by the tissue throughout the incubation period. By contrast, Graafian follicles removed from the same ovaries and incubated under the same conditions responded both to hCG and bcAMP with large increases in progesterone production. To determine whether the cyclic AMP content of the corpus luteum was altered by in vitro exposure to hCG, day 7 and day 11 corpora lutea were incubated for 5 or 15 min with various concentrations of hCG, and cyclic AMP in the tissue was then measured. Even at the highest concentration of hCG tested (10 μg/ml), the cyclic AMP content of the corpus luteum was unaltered. Given this result, the acute effects of various concentrations of hCG on the adenylyl cyclase activity of homogenates of day 11 corpora lutea were examined. Consistent with previous reports of others, adenylyl cyclase activity was stimulated, but only at a high concentration of hCG (1 μg/ml), and the degree of stimulation of the enzyme (∼75%) was quite modest. By contrast, the adenylyl cyclase activity of homogenates of rabbit Graafian follicles was stimulated by even the lowest concentration of hCG tested (0·01 μg/ml). Thus, the adenylyl cyclase of the rabbit follicle is much more sensitive to hCG stimulation than the luteal form of the enzyme. Given the poor responsiveness of luteal adenylyl cyclase to hCG, the possibility was considered that cyclic AMP production in response to hCG might be obscured by luteal cell phosphodiesterase. When day 11 corpora lutea were incubated with hCG in the presence of the phosphodiesterase inhibitor, isobutyl methylxanthine (5 mm), there was a marked increase in the cyclic AMP content of the tissue. Despite this large increase in endogenous cyclic AMP, progesterone production by the corpora lutea was again unaffected. Thus, the rabbit corpus luteum is insensitive to stimulation in at least two major respects. The adenylyl cyclase coupled to the LH receptor is resistant to stimulation by LH (hCG), and steroidogenesis, as reflected by progesterone production in vitro, is not stimulated acutely by cyclic AMP. These results suggest that progesterone biosynthesis in the rabbit corpus luteum is not a regulated process, but rather a process that becomes autonomous as a result of the differentiation of granulosa cells into luteal cells. Therefore, the role of a luteotrophin, such as oestrogen in the rabbit, is to maintain the health and viability of the luteal cells, which have, as an intrinsic property, the capacity to produce progesterone at a high rate.
Journal of Endocrinology (1994) 143, 423–431
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Abstract
The functional capacity of the recombinant human FSH (hFSH) receptor was tested on the basis of gonadotrophin stimulation of cyclic AMP (cAMP) production by transient transfections of 293 cells and stable transfections of Chinese hamster ovary (CHO) cells. A CHO cell line expressed with the hFSH receptor cDNA covering the entire amino acid coding region revealed the presence of FSH binding site (K d 6·2 × 10−10 m) on the plasma membrane. Treatment of transfected cells with hFSH induced dose-dependent increases in intracellular cAMP production. These results indicate that the hFSH receptor functionally couples with endogenous adenylyl cyclase. Although rat FSH also induced dose-dependent increases in cAMP production, bovine FSH was effective only at high doses and human chorionic gonadotropin did not alter cAMP levels compared with control values.
Northern blot analysis with a cRNA probe derived from hFSH receptor cDNA indicated the presence of two common FSH receptor mRNA transcripts (2·4 and 4·1 kb) in RNA prepared from a human ovary and transfected cell lines.
Preincubation of CHO cells expressing a functional hFSH receptor (CHO-FSHR) with FSH for 16 h decreased the subsequent cAMP production resulting from a 30-min pulse of FSH stimulation. These results indicate that desensitization of the adenylyl cyclase response to FSH stimulation occurs in CHO-FSHR cells. This cell line therefore provides a tool with which to pursue detailed studies on the molecular basis of FSH-induced desensitization.
Journal of Endocrinology (1994) 141, 369–375
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ABSTRACT
The follicles of the ovary in the domestic hen are arranged in a hierarchy. Responsiveness of the adenylyl cyclase enzyme system of the granulosa cells to FSH decreases as follicles proceed towards ovulation. To test the hypothesis that this decline in FSH responsiveness could be the result of a decrease in FSH receptor numbers, an FSH receptor assay was characterized for chicken granulosa cells and used to measure receptor number and affinity of the largest (F1), third largest (F3) and fifth largest (F5) follicles removed 18 h before ovulation. The numbers of binding sites for F1, F3 and F5 follicles (n = 4) were 0·22 ± 0·05, 0·5 ± 0·14 and 1·22 ± 0·27 pmol hormone bound/mg protein respectively, and were significantly (P < 0·001) different among follicles. The apparent association constants for the F1, F3 and F5 follicles were not different and had a value of 23·4 ± 4·9 litres/nmol (n = 12). Our results indicate that FSH receptor numbers decrease in granulosa cells without a change in affinity as follicles approach ovulation. The decrease in FSH receptor numbers is associated with the reported decline in FSH-stimulated steroidogenesis and adenylyl cyclase activity which occurs during follicular maturation.
J. Endocr. (1987) 115, 303–310
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Abstract
Treatment of ovine pars tuberalis (oPT) cultures with forskolin activates adenylyl cyclase, leading to increased levels of cyclic AMP, activation of protein kinase A, phosphorylation of the calcium/cyclic AMP response-element binding protein and the increased synthesis and secretion of several proteins. Simultaneous treatment with melatonin inhibits or reverses these effects of forskolin. In the neonatal rat pituitary, the inhibitory effects of melatonin are mediated by changes in membrane potential.
This study therefore investigated whether the inhibitory action of melatonin in oPT cultures is also dependent on the modulation of plasma membrane potential. Treatment of cultures with the ionophore valinomycin selectively permeabilised the cell plasma membrane to potassium, thereby causing membrane hyperpolarisation. In cultures of oPT, valinomycin inhibited in a concentration-dependent manner (maximal effect 2 μm) the stimulatory action of forskolin (1 μm) on intracellular levels of cyclic AMP, indicating that the activity of adenylyl cyclase in this tissue is sensitive to hyperpolarisation of the plasma membrane. However, increasing the extracellular concentration of potassium from 5 mm to 100 mm, which would depolarise the plasma membrane, had no effect on the inhibitory action of melatonin (1 μm) in forskolin-stimulated cultures. This indicated that melatonin could be effective in cells with sustained depolarisation. To test directly whether integrity of the plasma membrane is essential for melatonin to inhibit adenylyl cyclase, cultures were treated with the cholesterol-chelating agent saponin (50 μg/ml). Saponin increased cellular permeability to trypan blue and enhanced the release of the cytoplasmic enzyme lactate dehydrogenase to the extracellular medium, demonstrating that cell plasma membranes had been permeabilised, thereby abolishing membrane polarity. In cultures pretreated with saponin there was a tendency for levels of cyclic AMP to be reduced. However, permeabilisation did not block the forskolin-stimulated increases in cyclic AMP levels nor did it alter the ability of melatonin to inhibit the production of cyclic AMP in forskolin-stimulated cultures.
This study demonstrated that, while it is possible to inhibit the stimulatory actions of forskolin in the oPT by increasing the permeability of cells to potassium and thereby hyperpolarising them, melatonin is able to inhibit cyclic AMP in permeabilised cells and so can act independently of changes in membrane potential.
Journal of Endocrinology (1995) 145, 471–478
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Prostaglandin E(2) (PGE(2)) potently activated glycogenolysis and gluconeogenesis in isolated rockfish (Sebastes caurinus) hepatocytes. The average degree of activation for glycogenolysis was 6.4+/-0.67-fold (mean+/-S.E.M.; n=37), and could be as much as 19-fold. Analysis of dose-concentration relationships between glycogenolytic actions and PGE(2) concentrations yielded an EC(50) around 120 nM in hepatocyte suspensions and 2 nM for hepatocytes immobilized on perifusion columns. For the activation of gluconeogenesis (1.74+/-0.14-fold; n=10), the EC(50) for suspensions was 60 nM. Intracellular targets for PGE(2) actions are adenylyl cyclase, protein kinase A and glycogen phosphorylase. Concentrations of cAMP increased with increasing concentrations of PGE(2), and peaked within 2 min of hormone application. In the presence of the phosphodiesterase inhibitor, isobutyl-3-methylxanthine, peak height was increased and peak duration extended. The protein kinase A inhibitor, Rp-cAMPS, counteracted the activation of glycogenolysis by PGE(2), implying that the adenylyl cyclase/protein kinase A pathway is the most important, if not exclusive, route of message transduction. PGE(2) activated plasma membrane adenylyl cyclase and hepatocyte glycogen phosphorylase in a dose-dependent manner. The effects were specific for PGE(2); smaller degrees of activation of glycogenolysis were noted for PGE(1), 11-deoxy PGE(1), 19-R-hydroxy-PGE(2), and prostaglandins of the A, B and Falpha-series. The selective EP(2)-receptor agonist, butaprost, was as effective as PGE(2), suggesting that rockfish liver contains prostaglandin receptors pharmacologically related to the EP(2) receptors of non-hepatic tissues of mammals. Rockfish hepatocytes quickly degraded added PGE(2) (t((1/2))=17-26 min). A similar ability to degrade PGE(2) has been noted in catfish (Ameiurus nebulosus) hepatocytes, but no glycogenolytic or gluconeogenic actions of the hormone are noted for this species. We conclude that PGE(2) is an important metabolic hormone in fish liver, with cAMP-mediated actions on glycogen and glucose metabolism, and probably other pathways regulated by cAMP and protein kinase A. The constant presence of EP(2)-like receptors is a unique feature of the fish liver, with interesting implications for function and evolution of prostaglandin receptors in vertebrates.
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SOCS (suppressor of cytokine signaling)-3 has recently been shown to be an insulin- and tumor necrosis factor (TNF)-alpha-induced negative regulator of insulin signaling. To further clarify a potential involvement of SOCS-3 in the development of insulin resistance, we measured differentiation-dependent SOCS-3 mRNA expression in 3T3-L1 adipocytes and studied its regulation by various hormones known to impair insulin signaling using quantitative real-time RT-PCR. There was a differentiation-dependent downregulation of SOCS-3 mRNA by 50% over the 9 day adipocyte differentiation course. Interestingly, besides insulin and TNF-alpha, chronic treatment of differentiated 3T3-L1 cells with 10 microM isoproterenol for 16 h stimulated SOCS-3 gene expression by about 3.5-fold. Furthermore, isoproterenol stimulated SOCS-3 mRNA expression in a dose-dependent manner with significant activation detectable at concentrations as low as 10 nM isoproterenol. Moreover, a strong 27- and 47-fold activation of SOCS-3 mRNA expression could be seen after 1 h of isoproterenol and GH treatment respectively. The stimulatory effect of isoproterenol could be almost completely reversed by pretreatment of 3T3-L1 cells with the beta-adrenergic antagonist propranolol. Finally, isoproterenol's action could be mimicked by stimulation of G(S)-proteins with cholera toxin and of adenylyl cyclase with forskolin and dibutyryl cAMP. Taken together, our results demonstrate a differentiation-dependent downregulation of SOCS-3 in adipocytes and suggest that SOCS-3 gene expression is stimulated by beta-adrenergic agents via activation of a G(S)-protein-adenylyl cyclase-dependent pathway. As SOCS-3 is a novel inhibitor of insulin signaling, the data support a possible role of this protein as a selectively regulated mediator of catecholamine-induced insulin resistance.
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TSH-treated pig thyroid cells reorganize into follicle-like structures and exhibit differentiated functions. TSH also induces a phosphotyrosine phosphatase (PTPase) activity evaluated by phosphorylated substrate hydrolysis. Incubation of thyrocytes with various concentrations of 8-bromo-cyclic AMP or forskolin induces an increase of PTPase activity in a dose-dependent manner. During the culture period, adenylyl cyclase sensitivity, protein binding iodine and PTPase activity progressively increase from the first to the fourth day of the culture. Chronic treatment with phorbol 12-myristate 13-acetate (PMA) significantly inhibits PTPase activity during the first 24 h following PMA addition. GF 109203X, a specific inhibitor of protein kinase C, abolishes the inhibitory effect of PMA. Electrophoresis of membrane extracts allowed us to demonstrate a phosphatase activity at 111 kDa (p111). Vanadate inhibits this activity, indicating that p111 is a PTPase. This p111 is significantly reduced in PMA-treated cells. These data suggest that PTPase activity evidenced at 111 kDa is correlated with a differentiated state of primary cultured pig thyroid cells induced by TSH.
