The role of ghrelin and some intracellular mechanisms in controlling the secretory activity of chicken ovarian cells

The general aim of these in-vitro experiments was to determine whether ghrelin controls the secretory activity of chicken ovarian cells and whether its action is mediated by TK-, MAPK-, CDK- or PKA-dependent intracellular mechanisms. We postulated that particular protein kinases could be considered as mediators of ghrelin action (a) if they are controlled by ghrelin, and (b) if blockers of these kinases modify the action of ghrelin. In our in-vitro experiments we investigated whether ghrelin altered the accumulation of TK, MAPK, CDK and PKA in chicken ovarian cells and whether ghrelin, with or without blockers of MAPK, CDK and PKA, affected the secretion of progesterone (P4), testosterone (T), estradiol (E2) or arginine-vasotocin (AVT). In the first series of experiments, the influence of a ghrelin 1-18 analogue (1, 10 or 100 ng/mL) was studied on the expression of TK, MAPK and PKA in cultured chicken ovarian granulosa cells. The percentage of cells containing TK/phosphotyrosine MAPK/ERK1, 2 and PKA was determined using immunocytochemistry. Ghrelin increased the expression of both TK and MAPK. The low concentration of ghrelin (1 ng/mL) increased the accumulation of PKA in ovarian cells whilst the high concentration (100 ng/mL) decreased it. The 10 ng/mL concentration had no effect. In the second series of experiments, the effects of the ghrelin analogue combined with an MAPK blocker (PD98059; 100 ng/mL), a CDK blocker (olomoucine; 1 microg/mL), or a PKA blocker (KT5720; 100 ng/mL), were tested for their effects on the secretion of hormones by cultured fragments of chicken ovarian follicular wall. P4, T, E2 and AVT secretions were measured using RIA and EIA. Ghrelin increased T and decreased E2, but did not affect P4 or AVT secretion. The PKA blocker promoted P4 secretion and suppressed E2 and AVT, but did not affect T secretion. It prevented or even reversed the effect of ghrelin on T and E2, but did not modify its effect on AVT secretion. The MAPK blocker enhanced P4 and T and reduced AVT, but did not affect E2 secretion. It was able to prevent or reverse the effect of ghrelin on T and E, and it induced a stimulatory effect of ghrelin on AVT secretion. The CDK blocker reduced the secretion of AVT, but had no effect on steroid hormone secretion. It induced the stimulatory influence of ghrelin on the secretion of P4 and AVT, but did not modify the effect of ghrelin on other hormones. These observations clearly demonstrate that ghrelin is a potent regulator of the secretory activity of ovarian cells and of TK, MAPK and PKA. Furthermore, they suggest that MAPK-, CDK- and PKA-dependent intracellular mechanisms are involved in the control of ovarian secretion and that they mediate the effects of ghrelin on these processes.     

Comparison of effects of protein kinase A, mitogen-activated protein kinase, and cyclin-dependent kinase blockers on rabbit ovarian granulosa cell functions

The aim of the present study was to define the role of protein kinase A (PKA)-, mitogen-activated protein kinase (MAPK)-, and cyclin-dependent kinase (CDK)-dependent pathways in the control of ovarian cell functions. The effects of PKA, MAPK, and CDK blockers (KT 5720, PD 98059, and olomoucine, respectively), given at doses of 0.001-10.0 μg/ml medium on functions of cultured rabbit granulosa cells were examined. Expression of PKA, MAPK/ERK1,2, secretory activity (IGF-I output), and proliferation (proliferating cell nuclear antigen, PCNA) in these cells were determined by RIA, immunocytochemistry and Western blotting. A PKA inhibitor, KT 5720 suppressed the expression of PKA and MAPK/ERK1,2, the IGF-I release, and the ratio of PCNA-positive cells in granulosa cells. A MAPK blocker, PD 98059 reduced the expression of MAPK/ERK1,2 (but not PKA), the IGF-I release, and percentage of PCNA-positive cells. A CDK blocker, olomoucine, increased the PKA expression, decreased the expression of MAPK/ERK1,2 and PCNA, but did not affect the IGF-I release. These observations confirm the involvement of PKs in control of basic ovarian functions and demonstrate the involvement of PKA in stimulation of ovarian cell proliferation and MAPK (but not CDK) and in promotion of ovarian IGF-I release. Different activity and specificity of the PKA, MAPK, and CDK blockers in their effects on PCNA and IGF-I suggests different biological role of these PKs in control of proliferative and secretory functions of rabbit ovarian cells.     

Action of protein kinase A regulators on secretory activity of porcine granulosa cells in vitro

To understand the role of protein kinase A (PKA) in the control of ovarian secretory activity, we examined effects of stimulators (db-cAMP, 6-Phe-cAMP, Sp-cDBIMPS) or inhibitors (Rp-cAMPS, KT5720) of PKA on the release of insulin-like growth factor I (IGF-I), progesterone (P) and estradiol (E) by cultured porcine granulosa cells using RIA. All the PKA stimulators db-cAMP (10-10000 ng/ml), 6-Phe-cAMP (10-10000 pmol) or Sp-cDBIMPS (1-10000 pmol) increased IGF-I almost at all doses tested. P release was stimulated by db-cAMP (at doses 100-10000 ng/ml), Sp-cDBIMPS (at 10-1000 pmol) and 6-Phe-cAMP (at 1000 and 10000 pmol). The release of E was stimulated by Sp-cDBIMPS (1-100 pmol), db-cAMP (1000 and 10000 ng/ml) and 6-Phe-cAMP (1000 and 10000 pmol). Since Sp-cDBIMPS, which activates preferentially PKA isozyme type II, showed stimulating effects at doses lower than those of 6-Phe-cAMP, a preferential activator of both, type I and II of PKA, it is assumed that PKA type II is more important for the control of ovarian steroidogenesis than type I. A PKA inhibitor Rp-cAMPS inhibited release of IGF-I (10000 pmol), P (1000 pmol) and E (1000 and 10000 pmol), whereas Rp-cAMPS, at doses higher than 1000 pmol, tended to reverse this inhibitory effect. Other PKA inhibitor KT5720 suppressed P (at 10-1000 ng/ml), but not IGF-I or E release.The stimulation of growth factor and sex steroid release by PKA activators, and suppression of the secretion some of these substances by PKA inhibitors may indicate the implication of PKA (probably site B) in up- and down-regulation of ovarian IGF-I and steroid release.     

Involvement of obestatin,cyclin-dependent kinase and protein kinase C in control of feline ovarian cell viability and hormones release

The aim of our in vitro study was to understand the role of obestatin, cyclin-dependent kinase (CDK) and protein kinase C (PKC) in the control of basic feline ovarian cell functions (viability, ovarian hormones release), as well as the role of protein kinases in mediating the effect of obestatin on these processes. For this purpose, we analyzed the effect of obestatin (0, 10 and 100 ng/mL) alone or in combination with CDK blocker olomoucine (100 ng/mL) or PKC blocker calphostin-c (100 ng/mL) on cultured feline ovarian fragments or granulosa cells. The release of progesterone (P4), testosterone (T) and estradiol (E2) by isolated ovarian follicular fragments were evaluated by ELISA. Granulosa cell viability was analysed using the Trypan blue exclusion test. It was observed that the addition of obestatin alone significantly increased the granulosa cell viability (at dose 100 ng/mL), promoted the release of P4 (at all doses added) and IGF-I (at dose 100 ng/mL) but decreased T (at all doses added). E2 output was below the detection limit in all groups. The addition of either olomoucine or calphostin-c reduced cell viability, P4, T and IGF-I release. Both olomoucine and caplhostin-c inverted the stimulatory effect of obestatin on granulosa cell viability and were able to prevent stimulatory action of obestatin on ovarian cell viability and on hormone and growth factor release and change it to an inhibitory action. These observations show that obestatin can directly regulate (mostly promote) basal feline ovarian cell functions (hormone release and viability). The inhibitory action of CDK and PKC blockers on these functions suggests, that both CDK and PKC can be promoters of ovarian cell viability and steroidogenesis in cats. Furthermore, the ability of both CDK and PKC to prevent olomoucine action demonstrates that obestatin action on the feline ovary could be mediated by these kinases.     

The role of IGF-I, cAMP/protein kinase A and MAP-kinase in the control of steroid secretion, cyclic nucleotide production, granulosa cell proliferation and preimplantation embryo development in rabbits

The aim of this study was to investigate the actions of insulin-like growth factor I (IGF-I) on the secretory and proliferative functions of rabbit ovarian cells and on early embryogenesis. It was found that addition of IGF-I at a lower concentration (1 ng/ml) stimulated progesterone secretion by cultured rabbit granulosa cells, whilst higher concentrations of IGF-I (10, 100 ng/ml) were inhibitory. IGF-I had no effect on estradiol secretion. Cyclic AMP secretion was slightly increased after addition of IGF-I at 10 ng/ml, but not by higher concentrations. Cyclic GMP secretion was stimulated by IGF-I at 100 ng/ml only. A blocker of protein kinase A, Rp-cAMPS, did not alter progesterone and estradiol secretion but did prevent the action of IGF-I on progesterone secretion. An immunocytochemical study demonstrated that IGF-I significantly increased the proportion of proliferating cell nuclear antigen-positive (PCNA-positive) cells. Rp-cAMP did not change cell proliferation but partially prevented the proliferation-stimulating effect of IGF-I. IGF-I (100 ng/ml) significantly increased the proportion of divided zygotes and the number of embryos reaching the morula/blastocyst stage. Blockers of PKA, Rp-cAMPS and KT5720, reversed the effects of IGF-I on zygote cleavage and embryo development. Addition of IGF-I (100 ng/ml) significantly increased MAPK within the cells (proportion showing immunoreactivity to ERK-1 and ERK-3 antibodies and intensity of a 42 kDa band related to ERK-2). Rp-cAMPS suppressed the basal ERK-2 immunoreactivity but not that of ERK-1 or ERK-3. It completely inhibited the IGF-I-induced activation of ERK-3 but not that of ERK-1 or ERK-2. This in vitro study demonstrates that IGF-I is a potent stimulator of ovarian secretion, proliferation and embryogenesis in rabbit. Its effects are mediated by cAMP/PKA- and, probably by, MAPK-dependent intracellular mechanisms.     

Evidence that growth factors IGF-I, IGF-II and EGF can stimulate nuclear maturation of porcine oocytes via intracellular protein kinase A

The aim of our in vitro experiments was to study the role of growth factors and protein kinase A (PKA)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. Oocytes were cultured with or without growth factors (IGF-I, IGF-II, EGF; 10 ng x mL(-1) medium) and inhibitors of PKA (Rp-cAMPS or KT5720; 100 ng x mL(-1)). Stages of meiosis were determined from the structure of chromosomes after staining with Giemza. Intracellular levels of PKA were evaluated immunocytochemically using primary antisera against the PKA regulatory and catalytic subunits and by Western immunoblotting using primary antiserum against the PKA catalytic subunit. It was found that after 24 h culture the majority of oocytes had resumed nuclear maturation (they were at a stage of meiosis after diplotene) and that after 48 h culture the majority of cells had completed maturation (they had reached metaphase II of meiosis). Addition of IGF-I, IGF-II or EGF, or a combination of IGF-I and EGF, significantly increased the proportion of oocytes which resumed and completed meiosis. Immunocytochemistry demonstrated a significant increase in the proportion of cells containing catalytic and, in some cases, the regulatory subunits of PKA after addition of IGF-I, IGF-II and EGF. Immunoblotting showed the presence of 2 forms of the PKA catalytic subunit within the oocytes (MW approximately 52 and 40 kD). EGF, but not IGF-I or IGF-II, increased the content of both isoforms. Inhibitors of PKA, when given alone, did not substantially influence the proportion of oocytes which resumed or completed meiosis. However, Rp-cAMPS and KT5720 both prevented the stimulatory effects of IGF-I, IGF-II and EGF on the resumption and completion of oocyte maturation. The present observations suggest (1) that IGF-I, IGF-II and EGF are potent stimulators of both resumption and completion of porcine oocyte nuclear maturation, (2) that PKA is present in oocytes, and (3) that PKA-dependent intracellular mechanisms can mediate the action of growth factors on porcine oocytes.     

The cyclic AMP-dependent protein kinase A pathway is involved in progesterone effects on calcitonin secretion from TT cells

It is well known that gonadal steroid hormones influence the level of plasma calcitonin (CT), but the mechanism by which progesterone affects CT secretion is not clear. Immortalized TT cells are a reliable model system for studying the endocrine function of human parafollicular cells. In the present study, the effects of progesterone on CT secretion were examined in TT cells. TT cells were incubated in medium containing vehicle (DMSO), progesterone or BSA-progesterone for 60 or 150 min, and then the levels of CT in the medium, progesterone receptors, cAMP accumulation and CT mRNA expression were measured. To study the correlation between progesterone effects and the cAMP-dependent protein kinase A (PKA) pathway, cell lysates or cells in 24-well plates were treated with either vehicle or progesterone plus RU486, SQ22536, KT5720, or 3-isobutyl-1-methylxanthine. Then, adenylyl cyclase and protein kinase A (PKA) activities were measured in the cell lysates, and the CT levels were measured in the medium from the 24-well plate. The activated cAMP response element binding protein (P-CREB) was also measured by immunofluorescence. Administration of 1 microM progesterone or 500 nM BSA-progesterone increased the secretion of CT by 381% and 100%, respectively. Progesterone receptors A and B were downregulated by progesterone treatment. The cAMP concentration, adenylyl cyclase and PKA activity, CT mRNA expression, and nuclear P-CREB concentrations all showed an increase after progesterone treatment. RU486, SQ22536 and KT5720 inhibited the progesterone-stimulated effects. These results suggest that a cAMP-dependent PKA pathway is involved in progesterone-stimulated effects on CT secretion from TT cells.     

Leptin potentiates antiproliferative action of cAMP elevation via protein kinase A down‐regulation in breast cancer cells

Previously, we have shown that leptin potentiates the antiproliferative action of cAMP elevating agents in breast cancer cells and that the protein kinase A (PKA) inhibitor KT‐5720 prevented the antiproliferative effects induced by the leptin plus cAMP elevation. The present experiments were designed to gain a better understanding about the PKA role in the antitumor interaction between leptin and cAMP elevating agents and on the underlying signaling pathways. Here we show that exposure of MDA‐MB‐231 breast cancer cells to leptin resulted in a strong phosphorylation of both ERK1/2 and STAT3. Interestingly, intracellular cAMP elevation upon forskolin pretreatment completely abrogated both ERK1/2 and STAT3 phosphorylation in response to leptin and was accompanied by a consistent CREB phosphorylation. Notably, leptin plus forskolin cotreatments resulted in a strong decrease of both PKA regulatory RIα and catalytic subunits protein levels. Importantly, pretreatment with the PKA inhibitor KT‐5720 blocked the forskolin‐induced CREB phosphorylation and prevented both the inhibition by forskolin of leptin‐induced ERK1/2 and STAT3 phosphorylation and the PKA subunits down‐regulation induced by the combination of leptin and forskolin. Altogether, our results indicate that leptin‐dependent signaling pathways are influenced by cAMP elevation and identify PKA as relevantly involved in the pharmacological antitumor interaction between leptin and cAMP elevating drugs in MDA‐MB‐231 cells. We propose a molecular model by which PKA confers its effects. Potential therapeutic applications by our data will be discussed. J. Cell. Physiol. 225: 801–809, 2010. © 2010 Wiley‐Liss, Inc.     

Rapid effects of estrogen to modulate G protein-coupled receptors via activation of protein kinase A and protein kinase C pathways.

17Beta-estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic neurons. We have used intracellular recordings from female guinea pig hypothalamic slices to characterize the receptor and intracellular pathway(s) mediating E2's rapid effects. E2 acts stereospecifically with physiologically relevant concentration-dependence (EC50 = 8 nM) to cause a fourfold reduction in the potency of the mu-opioid agonist (D-Ala2-N-Me-Phe4-Gly5-ol)-enkephalin and the GABA(B) agonist baclofen to activate an inwardly rectifying K+ conductance in hypothalamic neurons. Both the nonsteroidal estrogen diethylstilbestrol and the anti-estrogen ICI 164,384 blocked E2 actions to uncouple mu-opioid receptors. Using a pharmacological Schild analysis, we found that ICI 164,384 competed for this E2 receptor with a Ke of approximately 0.3 nM. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a rapid, nongenomic mechanism of E2 action. The effects of E2 were mimicked by the bath application of the protein kinase A (PKA) activators, forskolin and Sp-cAMP, and the protein kinase C (PKC) activator phorbol-12,13-dibutyrate. Furthermore, the selective PKA antagonists Rp-cAMP and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. In addition, the actions of E2 were blocked by the selective PKC inhibitor Calphostin C. Therefore, it appears that E2 can activate both PKA and PKC to cause a heterologous desensitization of both mu-opioid and GABA(B) receptors, which has the potential to alter synaptic transmission in many regions of the CNS.     

Some endocrine traits of transgenic rabbits. II. Changes in hormone secretion and response of isolated ovarian tissue to FSH and ghrelin

In the present in vitro experiments we examined FSH- and ghrelin-induced changes in ovarian hormone secretion by transgenic rabbits. Fragments of ovaries isolated from adult transgenic (carrying mammary gland-specific mWAP-hFVIII gene) and non-transgenic rabbits from the same litter were cultured with and without FSH or ghrelin (both at 0, 1, 10 or 100 ng/ml medium). The secretion of progesterone (P4), estradiol (E2) and insulin-like growth factor I (IGF-I) was assessed by RIA. It was observed that ovaries isolated from transgenic rabbits secreted much less P4, E2 and IGF-I than the ovaries of non-transgenic animals. In control animals FSH reduced E2 (at doses 1-100 ng/ml medium) and IGF-I (at 1-100 ng/ml), but not P4 secretion, whereas ghrelin promoted P4 (at 1 ng/ml) and IGF-I (at 100 ng/ml), but not E2 output. In transgenic animals, the effects were reversed: FSH had a stimulatory effect on E2 (at 100 ng/ml) and ghrelin had an inhibitory effect on P4 (at 10 ng/ml). No differences in the pattern of influence of FSH on P4 and IGF-I and of ghrelin on E2 and IGF-I were found between control and transgenic animals. The present observations suggest that 1) both FSH and ghrelin are involved in rabbit ovarian hormone secretion, 2) transgenesis in rabbits is associated with a reduction in ovarian secretory activity, and 3) transgenesis can affect the response of ovarian cells to hormonal regulators.     

Peptidoglycan-induced IL-6 production in RAW 264.7 macrophages is mediated by cyclooxygenase-2, PGE2/PGE4 receptors, protein kinase A, I kappa B kinase, and NF-kappa B

In this study, we investigated the signaling pathway involved in IL-6 production caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium, Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused concentration- and time-dependent increases in IL-6, PGE(2), and cAMP production. PGN-mediated IL-6 production was inhibited by a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), a PGE(2) (EP2) antagonist (AH6809), a PGE(4) (EP4) antagonist (AH23848), and a protein kinase A (PKA) inhibitor (KT5720), but not by a nonselective NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester). Furthermore, PGE(2), an EP2 agonist (butaprost), an EP2/PGE(3) (EP3)/EP4 agonist (misoprostol), and misoprostol in the presence of AH6809 all induced IL-6 production, whereas an EP1/EP3 agonist (sulprostone) did not. PGN caused time-dependent activations of IkappaB kinase alphabeta (IKKdbeta) and p65 phosphorylation at Ser(276), and these effects were inhibited by NS398 and KT5720. Both PGE(2) and 8-bromo-cAMP also caused IKKdbeta kinase alphabeta phosphorylation. PGN resulted in two waves of the formation of NF-kappaB-specific DNA-protein complexes. The first wave of NF-kappaB activation occurred at 10-60 min of treatment, whereas the later wave occurred at 2-12 h of treatment. The PGN-induced increase in kappaB luciferase activity was inhibited by NS398, AH6809, AH23848, KT5720, a protein kinase C inhibitor (Ro31-8220), and a p38 MAPK inhibitor (SB203580). These results suggest that PGN-induced IL-6 production involves COX-2-generated PGE(2), activation of the EP2 and EP4 receptors, cAMP formation, and the activation of PKA, protein kinase C, p38 MAPK, IKKdbeta, kinase alphabeta, p65 phosphorylation, and NF-kappaB. However, PGN-induced NO release is not involved in the signaling pathway of PGN-induced IL-6 production.     

Adenosine A2A receptor facilitates calcium-dependent protein secretion through the activation of protein kinase A and phosphatidylinositol-3 kinase in PC12 cells

Adenosine modulates a variety of cellular functions including calcium-dependent exocytosis. Activation of adenosine A(2A) receptor (A(2A)-R) facilitates neurotransmitter release in some cell types, although the underlying mechanisms are not fully understood. In this study, we found that treatment of PC12 cells with the A(2A)-R agonist CGS21680 promotes calcium-evoked secretion of the fusion protein between neuropeptide Y and modified yellow fluorescence protein (NPY-Venus). CGS21680 treatment of PC12 cells transiently increased the phosphorylation of p38 and JNK MAP kinases and Akt, as well as that of ATF2 and CREB, reaching maximal levels at around 10-15 min of CGS21680 treatment. Importantly, pretreatment of PC12 cells with the PI3K inhibitor LY294002, together with the protein kinase A (PKA) inhibitor KT5720, significantly inhibited CGS21680 enhancement of calcium-dependent NPY-Venus release. Moreover, expression of a dominant-negative form of Akt and the PKA inhibitory polypeptide protein kinase inhibitor (PKI) co-operatively inhibited the facilitating effect of CGS21680 on secretion of NPY-Venus. These data suggest that the PI3K-Akt and PKA pathways play a critical role in A(2A)-R-mediated facilitation of calcium-dependent secretion. We also found that CGS21680 treatment promoted recruitment of the NPY-Venus-containing vesicles to the proximity of the plasma membrane at around 10-15 min of CGS21680 treatment, which may in part account for the facilitated secretion by A(2A)-R activation.     

Effect of cGMP analogues and protein kinase G blocker on secretory activity, apoptosis and the cAMP/protein kinase A system in porcine ovarian granulosa cells in vitro

The aim of the present study was to examine the role of cGMP-dependent intracellular mechanisms in control of ovarian functions. In the first series of experiments we studied the effects of the cGMP analogues 8-pCPT-cGMP (0.001-100 nM), Rp-8-pCPT-cGMPS (0. 01-100 nM), Rp-8-Br-cGMPS (0.01-100 nM), and Rp-8-Br-PET-cGMPS (0.01-100 nM) on the release of progesterone, insulin-like growth factor I (IGF-I) and oxytocin by cultured porcine granulosa cells. In a second series of experiments, the effects of Rp-8-Br-PET-cGMPS (50 nM) and KT5822 (100 ng/ml), specific inhibitor of cGMP-dependent protein kinase (PKG), on cAMP, PKA, oxytocin and the occurrence of apoptosis in cultured cells were compared. The release of hormones and IGF-I into the culture medium was evaluated using a RIA, while the percentage of cells containing visible oxytocin, cAMP, as well as the regulatory and catalytic subunits of PKA was assessed using immunocytochemistry. Occurrence of apoptosis in these cells was detected using the TUNEL method. The stimulatory (8-pCPT-cGMP and Rp-8-pCPT-cGMPS), inhibitory (Rp-8-Br-cGMPS) and biphasic (Rp-8-Br-PET-cGMPS) effect of cGMP analogues on progesterone release was observed. All cGMP analogues used suppressed IGF-I release. All cGMP analogues decreased oxytocin release, but 8-pCPT-cGMP and Rp-8-Br-cGMPS, when given at low doses (0.01-0.1 and 1-10 nM, respectively) stimulated oxytocin output. Both, Rp-8-Br-PET-cGMPS and KT5822 increased the rate of incidence of apoptosis and percentage of cells containing immunoreactive cAMP. Both Rp-8-Br-PET-cGMPS and KT5822 decreased the proportion of cells containing immunoreactive oxytocin and regulatory subunit of PAK KT5822, but not Rp-8-Br-PET-cGMPS, increased the number of cells containing catalytic subunit of PKA. The present observations suggest the involvement of cGMP and PKG in control of the production of steroid, nonapeptide hormone, growth factor, cAMP and cAMP-dependent PKA, as well as the induction of apoptosis in porcine ovarian cells.     

Histamine inhibits atrial myocytic ANP release via H2 receptor-cAMP-protein kinase signaling

Changes in cyclic nucleotide production and atrial dynamics have been known to modulate atrial natriuretic peptide (ANP) release. Although cardiac atrium expresses histamine receptors and contains histamine, the role of histamine in the regulation of ANP release has to be defined. The purpose of the present study was to define the effect of histamine on the regulation of ANP release in perfused beating rabbit atria. Histamine decreased ANP release concomitantly with increases in cAMP efflux and atrial dynamics in a concentration-dependent manner. Histamine-induced decrease in ANP release was a function of an increase in cAMP production. Blockade of histamine H2 receptor with cimetidine but not of H1 receptor with triprolidine abolished the responses of histamine. Cell-permeable cAMP analog, 8-Br-cAMP, mimicked the effects of histamine, and the responses were dose-dependent and blocked by a protein kinase A (PKA)-selective inhibitor, KT5720. Nifedipine failed to modulate histamine-induced decrease in ANP release. Protein kinase nonselective inhibitor staurosporine blocked histamine-induced changes in a concentration-dependent manner. KT5720 and RP-adenosine 3',5'-cyclic monophosphorothioate, another PKA-selective inhibitor, attenuated histamine-induced changes. These results suggest that histamine decreases atrial ANP release by H2 receptor-cAMP signaling via PKA-dependent and -independent pathways.     

Effects of insulin-like growth factor-II on proliferation and differentiation of ovarian granulosa cells.

The effects of insulin-like growth factor-II (IGF-II) on the proliferation and differentiation of ovarian granulosa cells were studied in cultured human and porcine granulosa cells. IGF-II significantly increased basal progesterone secretion in granulosa cells at concentrations of 1-100 ng/ml. A stimulatory effect was also observed in gonadotropin-stimulated porcine granulosa cells treated with IGF-II. The secretion of estradiol by basal and gonadotropin-stimulated porcine granulosa cells was also significantly increased by adding IGF-II. IGF-II led to dose-dependent increases in [3H]thymidine incorporation into DNA and in the number of granulosa cells. To further characterize the cellular mechanisms underlying the stimulatory effects of IGF-II on the proliferation and differentiation of granulosa cells, we investigated the intermediary roles of cyclic AMP and intracellular Ca2+ concentration ([Ca2+]i). Treatment with 100 ng/ml IGF-II produced a significant increase in the basal accumulation of cyclic AMP in porcine granulosa cells. However, no change of [Ca2+]i by IGF-II was noted. IGF-II produced effects in accumulation that were similar to those of IGF-I. Our findings suggest that IGF-II may be a general stimulator in the proliferation and differentiation of granulosa cells, and that cyclic AMP may be a second messenger for the effects of IGF-II in ovarian granulosa cells.     

Steroid production in vitro by granulosa, theca, and luteal cells from goat ovaries

Basal progesterone (P4) production by isolated goat ovarian cells in vitro was in the order corpus luteum (CL) greater than granulosa (G) greater than theca (TH), while estradiol (E2) production was in the order TH greater than G greater than CL. In G cells, various concentrations (0.01 to 100 micrograms/ml) of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) increased P4 and E2 secretion. Testosterone (T, 10(-9) to 10(-5) M) produced dose-dependent increases in P4 and E2 secretion. Testosterone and LH together had an additive effect on E2 secretion. The combined effect of the lower (less than 10(-6) M) concentrations of T and LH on P4 production was marginally higher than either agent alone, but the increase was statistically insignificant; at higher concentrations of T (10(-6) and 10(-5) M) in combination with LH, P4 secretion was similar to that with LH alone, but was significantly (p less than 0.01 and less than 0.001, respectively) less compared to that with T alone. Follicle-stimulating hormone and T together produced a synergistic effect on E2 and an additive effect on P4 production. In TH cells, a dose-dependent increase in P4 and E2 production was observed with LH and hCG, but the effect of FSH was not significant. Testosterone produced a dose-dependent increase in P4 and E2 secretion. Testosterone and LH together induced higher steroid production than either agent alone. However, the increase was not statistically significant compared to T alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase A and regulation of neonatal Nav1.5 expression in human breast cancer cells: Activity-dependent positive feedback and cellular migration

Voltage-gated Na⁺ channels (VGSCs) are expressed in excitable cells (e.g. neurons and muscles), as well as in some classically ‘non-excitable’ cells (e.g. fibroblasts), and in carcinomas. In general, functional expression of VGSCs in plasma membrane (PM) is hierarchical and dynamic. Previously, we have shown that an activity-dependent positive feedback mechanism involving cAMP-dependent protein kinase A (PKA) plays a significant role in upregulation of VGSCs in strongly metastatic rat prostate cancer Mat-LyLu cells expressing Nav1.7. Here, we investigated the possible role of PKA in VGSC regulation and its functional consequences in strongly metastatic human breast cancer (BCa) MDA-MB-231 cells, where the neonatal splice form of Nav1.5 (nNav1.5) is the predominant VGSC present. Treatment with the PKA activator forskolin for 24 h increased mRNA and PM protein levels of nNav1.5, without changing the total VGSC protein level. Opposite effects were obtained by application of the PKA inhibitor KT5720 or the highly specific VGSC blocker tetrodotoxin (TTX), the latter implying activity-dependent upregulation. We tested the possibility, therefore, that the activity dependence of VGSC (nNav1.5) expression involved PKA. Indeed, TTX pretreatment reduced the level of phosphorylated PKA and eliminated basal and PKA-stimulated cellular migration. These data suggested that activity-dependent positive feedback mediated by PKA plays an important role in the functional expression of nNav1.5 in BCa, and in turn, this enhances the cells’ metastatic potential.     

Phagocytosis by Lymnaea stagnalis haemocytes: a potential role for phosphatidylinositol 3-kinase but not protein kinase A

The molecular events that regulate phagocytosis, an important innate immune response, in invertebrate defence cells (haemocytes) are poorly understood. Lymnaea stagnalis haemocytes were used as a model to elucidate the role of cell signalling pathways in phagocytosis by molluscan defence cells. The phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, significantly impaired haemocyte phagocytic activity in a dose-responsive manner with 10 microM LY294002 reducing internalization of fluorescent-conjugated Escherichia coli by 62% (P < or = 0.001). In contrast, the protein kinase A (PKA) inhibitor KT5720 was without effect. Therefore, PI3-K, but not PKA, appears to control phagocytosis by haemocytes in these gastropod molluscs.     

PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior

The laterocapsular division of the central nucleus of the amygdala (CeLC) has emerged as an important site of pain-related plasticity and pain modulation. Glutamate and neuropeptide receptors in the CeLC contribute to synaptic and behavioral changes in the arthritis pain model, but the intracellular signaling pathways remain to be determined. This study addressed the role of PKA, PKC, and ERK in the CeLC. Adult male Sprague-Dawley rats were used in all experiments. Whole-cell patch-clamp recordings of CeLC neurons were made in brain slices from normal rats and from rats with a kaolin/carrageenan-induced monoarthritis in the knee (6 h postinduction). Membrane-permeable inhibitors of PKA (KT5720, 1 μM; cAMPS-Rp, 10 μM) and ERK (U0126, 1 μM) activation inhibited synaptic plasticity in slices from arthritic rats but had no effect on normal transmission in control slices. A PKC inhibitor (GF109203x, 1 μM) and an inactive structural analogue of U0126 (U0124, 1 μM) had no effect. The NMDA receptor-mediated synaptic component was inhibited by KT5720 or U0126; their combined application had additive effects. U0126 did not inhibit synaptic facilitation by forskolin-induced PKA-activation. Administration of KT5720 (100 μM, concentration in microdialysis probe) or U0126 (100 μM) into the CeLC, but not striatum (placement control), inhibited audible and ultrasonic vocalizations and spinal reflexes of arthritic rats but had no effect in normal animals. GF109203x (100 μM) and U0124 (100 μM) did not affect pain behavior. The data suggest that in the amygdala PKA and ERK, but not PKC, contribute to pain-related synaptic facilitation and behavior by increasing NMDA receptor function through independent signaling pathways.