cAMP Effector System Research Articles

The interaction between protein kinase A and progesterone on basal and inflammation-induced myometrial oxytocin receptor expression.

Our previous work has shown myometrial PKA activity declines in term and twin-preterm labour in association with an increase in the expression of the oxytocin receptor (OTR). Here we investigate the action of cAMP/PKA in basal conditions, with the addition of progesterone (P4) and/or IL-1β to understand how cAMP/PKA acts to maintain pregnancy and whether the combination of cAMP and P4 would be a viable therapeutic combination for the prevention of preterm labour (PTL). Further, given that we have previously found that cAMP enhances P4 action we wanted to test the hypothesis that changes in the cAMP effector system are responsible for the functional withdrawal of myometrial P4 action. Myometrial cells were grown from biopsies obtained from women at the time of elective Caesarean section before the onset of labour. The addition of forskolin, an adenylyl cyclase activator, repressed basal OTR mRNA levels at all doses and P4 only enhanced this effect at its highest dose. Forskolin repressed the IL-1β-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1β-activation and nuclear transfer of NFκB and AP-1. P4 had similar effects and the combination P4 and forskolin had greater effects on OTR and NFκB than forskolin alone. While PKA knockdown had no effect on the ability of P4 to repress IL-1β-induced OTR expression it reversed the repressive effect of the combination of P4 and forskolin and resulted in a greater increase than observed with IL-1β alone. These studies suggest that cAMP acts via PKA to repress inflammation-driven OTR expression, but that when PKA activity is reduced, the combination of cAMP and P4 actually enhances the OTR response to inflammation, promoting the onset of labour and suggesting that changes in the cAMP effector system can induce a functional P4 withdrawal.

Changes in cAMP effector predominance are associated with increased oxytocin receptor expression in twin but not infection-associated or idiopathic preterm labour

We previously reported that at term pregnancy, a decline in myometrial protein kinase A (PKA) activity leads to an exchange protein activated by cyclic AMP (Epac1)-dependent increase in oxytocin receptor (OTR) expression, promoting the onset of labour. Here, we studied the changes in the cyclic adenosine monophosphate (cAMP) effector system present in different phenotypes of preterm labour (PTL). Myometrial biopsies obtained from women with phenotypically distinct forms of PTL and the levels of PKA and OTR were examined.Although we found similar changes in the cAMP effector pathway in all forms of PTL, only in the case of twin PTL (T-PTL) was myometrial OTR levels increased in association with these results. Although there were several changes in the mRNA levels of components of the cAMP synthetic pathway, the total myometrial cAMP levels did not change with the onset of any subtype of PTL. With regards to the expression of cAMP-responsive genes, we found that the mRNA levels of 4 of the 5 cAMP-down-regulated genes were increased in T-PTL, similar to our findings in term labour.These data signify that although changes in the cAMP effector system were common to all forms of PTL, only in T-PTL were OTR levels increased. Similarly, the mRNA levels of cAMP-repressed genes were only increased in T-PTL supporting the concept that the decline in PKA levels influences myometrial function driving the onset of T-PTL.

Role of calcium in the stimulation of ecdysteroidogenesis by recombinant prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori

The aim of this study was to clarify whether Ca 2+ plays a role in the mechanism by which recombinant prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by the prothoracic glands of the silkworm, Bombyx mori. Omission of Ca 2+ from the incubation medium, or inclusion of a Ca 2+ channel blocker (verapamil or lanthanum) did not affect basal ecdysteroid secretion, but greatly inhibited the stimulation of ecdysteroidogenesis by PTTH. The addition of a Ca 2+ ionophore (ionomycin or A23187) stimulated ecdysteroid secretion in a dose-dependent manner. Furthermore, thapsigargin, a potent Ca 2+-ATPase inhibitor, enhanced ecdysteroid secretion. In contrast, thimerosal, a thiol-oxidizing reagent, inhibited prothoracic gland activity. The mechanism of Ca 2+ action was examined further in relation to the cAMP effector system. Ca 2+ deprivation greatly suppressed the accumulation of cAMP in prothoracic glands treated with PTTH but had no effect on basal levels. Omission of Ca 2+ from the incubation medium, or inclusion of a Ca 2+ channel blocker (verapamil or lanthanum) did not affect the stimulation of ecdysteroid secretion in response to dibutyryl cAMP or 1-methyl-3-isobutylxanthine. The present study suggests that specific regulatory actions of Ca 2+ in prothoracic gland cells are exerted prior to cAMP generation.

Effect of Ionizing Radiation on In Vivo Striatal Release of Dopamine in the Rat

The time-course effect of ionizing radiation on the levels of basal and KCl-stimulated striatal release of dopamine (DA) was examined in vivo using microdialysis techniques. The basal level of extracellular DA in sham-irradiated controls was 0.172 +/- 0.042 pmol/sample (n = 9), and it increased 7.1-fold after the stimulation by 30 mM KCl (20microliters). However, the release of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), two metabolites of DA, was reduced significantly by 30 mM KCl (P < 0.05). In the presence of 10 microM forskolin, an activator of adenylate cyclase, a second stimulation by 30 mM KCl increased the release of DA 6.9-fold. Radiation exposure, at a dose of 10 Gy at 10 Gy/min, had no significant effect on the levels of either basal or KCl-stimulated release of DA or on the release of DOPAC and HVA. Striatal DA release increased in response to two consecutive challenges of KCl. However, the release of DA in response to the second challenge of KCl was significantly smaller than that after the first challenge (543 +/- 110% compared to 794 +/- 164%, P < 0.05; Student's paired t test). Pretreatment with 10 muM forskolin, which by itself had no significant effect on the level of basal release of DA, prevented the decreased response of DA to the second challenge of KCl. Our results suggest that radiation exposure at the dose we used has no significant effect on the level of the basal release of DA or the release of DA stimulated by 30mM KCl in the rat striatum, and that a reduced release of DA in response to repeated KCl stimulation might involve the cAMP effector system.

Influence of kaurenol on basal, LH- and forskolin-stimulated progesterone and cAMP production in avian granulosa cells

The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 μM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10μM), kaurenol (10–100 μM) dose-depend ently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenol's stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system.

Transforming growth factor beta regulates the inhibitory actions of epidermal growth factor during granulosa cell differentiation.

The effects of transforming growth factor beta (TGF-beta) on epidermal growth factor (EGF) receptor content and EGF action were studied in cultured granulosa cells from immature diethylstilbestrol-implanted rats. During follicle-stimulating hormone (FSH)-induced differentiation in vitro, EGF receptors increased by 20-fold as measured by the binding of 125I-EGF to the intact cells. Addition of TGF-beta during the 48-h culture period amplified the stimulatory effects of FSH on EGF receptors up to 2-fold, with ED50 and maximal concentrations of 2.5 and 8 pM, respectively. Also TGF-beta alone in amounts from 1.6 to 16 pM increased EGF receptor content 4-fold. The stimulatory effects of TGF-beta were due to increased numbers of EGF receptors/cell, since the growth factor had no effect on the Kd (3-5 X 10(-11) M) of the high-affinity EGF binding site. TGF-beta action was observed within 20 h of granulosa cell culture and was maximal by 48 h of a 96-h culture. The stimulatory actions of TGF-beta in gonadotropin-induced cells were exerted through the cAMP effector system of the granulosa cell, since the growth factor also amplified the induction of EGF receptors by cholera toxin, forskolin, and 8-bromo-cAMP. The augmentation of EGF receptors by TGF-beta resulted in a parallel 2-fold increase in the inhibitory effects of EGF on FSH-induced cAMP production and luteinizing hormone receptor expression during granulosa cell development. TGF-beta did not increase granulosa cell numbers during culture although it elevated [3H]thymidine incorporation into DNA by 2-fold over that of FSH-treated cells. These results indicate that TGF-beta regulates the effects of both FSH and EGF during granulosa cell differentiation and provides evidence that ovarian function may be controlled by the combined actions of gonadotropins and multiple growth factors.

Mechanisms by which calcium ions regulate the steroidogenic actions of luteinizing hormone in isolated ovarian cells in vitro.

Incubation of swine granulosa cells in chemically defined medium selectively deficient in calcium ions markedly impaired progesterone production in response to submaximal and maximally stimulating concentrations of LH. Accumulation of progesterone in response to LH was reduced significantly in both cells and medium, without a discernible shift in the time-course of progestin production. The reduction in progesterone accumulation could not be accounted by increased formation of the catabolite 20 alpha-hydroxypregn-4-en-3-one. In addition, progesterone secretion basally or in response to exogenously supplied pregnenolone was not altered in calcium-deficient incubations. Administration of verapamil or diltiazem, organic inhibitors of net transmembrane calcium uptake, also suppressed LH-stimulated progesterone production. Conversely, micromolar concentrations of the divalent cation ionophore A23187 significantly enhanced the stimulatory effects of LH. The mechanisms of calcium action were examined further in relation to the cAMP effector system. Calcium deprivation significantly suppressed the dose-dependent accumulation of cAMP in granulosa cells treated with LH but had no effect on basal levels. Omission of calcium ions from the extracellular medium also markedly impaired production of progesterone in response to 8-bromo-cAMP, cholera toxin, or 3-isobutyl-l-methylxanthine. The present studies suggest that calcium ions significantly modulate LH-stimulated progesterone biosynthesis in isolated ovarian cells in vitro. Specific regulatory actions of calcium ions in granulosa cells may be exerted at several levels, including LH-stimulated cAMP accumulation and at intracellular loci distal to the actual generation of cAMP.