Induction of type 2 deiodinase activity by cyclic guanosine 3',5'-monophosphate in cultured rat glial cells

We investigated the effects of cyclic guanosine 3',5'-monophosphate (cGMP) on type 2 iodothyronine deiodinase (D2) in cultured rat glial cells. Rat glial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum. When cells were cultured in the presence of 8-bromo cGMP (8-Br cGMP), an analogue of cGMP, D2 activity was increased in a time- and concentration-dependent manner. Lineweaver-Burk plots revealed that the stimulation of D2 activity by 8-Br cGMP (10(-3) M) was associated with fivefold increase in maximum velocity but without a significant change in Michaelis-Menten constant, suggesting that cGMP increases D2 activity via new enzyme synthesis. Both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are well known to increase the intracellular cGMP level via their guanylate cyclase-linked receptors in rat glial cells. In the present study, ANP (10(-6) M) and CNP (10(-6) M) significantly increased the D2 activity in rat glial cells (1.9-fold [ANP] or 2.3-fold [CNP] compared with control activity, respectively). Northern blot analysis demonstrated that D2 mRNA level increased in the presence of 8-Br cGMP (10(-3) M), and reached a plateau (six-fold) after 4 hours of incubation. The increment of D2 mRNA level by 8-Br cGMP was comparable with the increase of the D2 activity by this agent. Our data suggest that cGMP induces rat D2 activity, at least in part, at the pretranslational level, and that ANP and CNP increase D2 activity most likely via their guanylate cyclase-linked receptors in rat glial cells.     

Urinary cyclic adenosine 3',5'-monophosphate response in McCune-Albright syndrome: clinical evidence for altered renal adenylate cyclase activity

The recent finding of an activating mutation in the Gs alpha protein, the protein that couples receptors to stimulation of adenylate cyclase, from endocrine and nonendocrine tissues of patients with McCune-Albright syndrome (MAS) suggests that alterations in adenylate cyclase activity may account for the clinical abnormalities in these patients. Many patients with MAS have hypophosphatemia. This may result from the presence of the activating Gs alpha mutation in proximal renal tubules or the elaboration of a phosphaturic factor from fibrous dysplasia. We, therefore, sought to characterize renal cAMP generation and phosphate handling in MAS patients. Intravenous infusion of PTH is a classic clinical test used to evaluate hormonal responsiveness of renal proximal tubule adenylate cyclase and examine PTH-dependent phosphate clearance. We performed PTH infusion in 6 MAS patients, 10 normal subjects, and 6 patients with pseudohypoparathyroidism (PHP). The basal urinary cAMP (UcAMP) level in the MAS group [5.5 +/- 2.6 nmol/dL glomerular filtration (GF)] was elevated (P < 0.05) compared to those in both normal subjects (3.2 +/- 1.2 nmol/dL GF) and patients with PHP (1.9 +/- 0.6 nmol/dL GF). However, PTH-stimulated peak UcAMP (15.0 +/- 7.0 nmol/dL GF) and the peak/basal UcAMP ratio (3.1 +/- 1.7) in MAS were significantly lower than the respective values in normal subjects (30.8 +/- 16.9 nmol/dL GF and 9.3 +/- 2.9; P < 0.05 for both) and were statistically similar to the blunted levels in PHP (respectively, 3.1 +/- 1.5 nmol/dL GF and 2.0 +/- 1.7). By contrast, the PTH-induced phosphaturic response in MAS patients was similar to that in the normal subjects. Our study provides clinical evidence that MAS patients have altered renal adenylate cyclase activity, manifested by an elevated basal UcAMP, but a blunted UcAMP response to PTH stimulation. These observations are presumably due to a mutation in the Gs alpha protein in the renal tubules. Despite the blunted UcAMP excretion, the phosphaturic response to PTH in MAS patients is intact.     

Elevation of cyclic adenosine 3',5'-monophosphate potentiates activation of mitogen-activated protein kinase by growth factors in LNCaP prostate cancer cells

OBJECTIVE: Proinflammatory cytokines are involved in the pathogenesis of acute pancreatitis. The value of serum levels of tumor necrosis factor-alpha, interleukin-1-beta, interleukin-6, and interleukin-8 in predicting the outcome of acute pancreatitis was evaluated. METHODS: In 50 patients with acute pancreatitis, the serum concentrations of tumor necrosis factor-alpha, interleukin-1-beta, interleukin-6, interleukin-8, and C-reactive protein were determined on days 1, 2, 3, 4, and 7 after admission. Acute Physiology and Chronic Health Evaluation (APACHE II) scores were recorded on days 1, 2, and 3. RESULTS: Serum concentrations of interleukin-1-beta, interleukin-6, interleukin-8, and C-reactive protein on days 1-7 were significantly higher in patients with severe pancreatitis than in patients with mild pancreatitis. Patients with severe attacks had significantly elevated serum tumor necrosis factor-alpha concentrations on days 1-3 compared with those with mild attacks, but not on days 4 and 7. The median peak value of tumor necrosis factor-alpha, interleukin-1-beta, interleukin-6, and interleukin-8 was reached on day 1, in contrast to the median peak of C-reactive protein, which was reached on day 2. Using cutoff levels of 12 pg/ml for tumor necrosis factor-alpha, 1 pg/ml for interleukin-1-beta, 400 pg/ml for interleukin-6, 100 pg/ml for interleukin-8, 12 mg/dl for C-reactive protein, and 10 for the Acute Physiology and Chronic Health Evaluation (APACHE II) score, the accuracy rates for detecting severe pancreatitis were 72%, 82%, 88%, 74%, 80%, and 72%, respectively, on day 1 and 78%, 74%, 80%, 76%, 80%, and 78%, respectively, on day 2. CONCLUSION: Among the proinflammatory cytokines, interleukin-6 is the most useful parameter for early prediction of the prognosis of acute pancreatitis.     

Interleukin-6 production by rat granulosa cells in vitro: effects of cytokines, follicle-stimulating hormone, and cyclic 3',5'-adenosine monophosphate

In the present study we examined the influence of FSH as well as a number of well-established cytokines on interleukin (IL)-6 by rat granulosa cells in culture. Increasing concentrations of FSH, IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS) were incubated for 48 h with undifferentiated granulosa cells obtained from diethylstilbestrol-primed immature rats. The results demonstrate that FSH, IL-1 alpha, IL-1 beta, and LPS, but not TNF alpha, caused significant concentration-dependent increases in IL-6 release. We also examined the effects of dibutyryl-cAMP, forskolin, and 3-isobutyl-1-methyl-xanthine (IBMX) on IL-6 release by granulosa cells. Each of these agents caused a significant concentration-dependent increase in IL-6 production by granulosa cells in either the absence or presence of FSH. Taken together, these results show that the granulosa cell is not only a likely source of IL-6 but that the release of IL-6 can be regulated. Moreover, evidence suggests that cAMP may serve as a second messenger for the stimulated secretion of IL-6 by undifferentiated granulosa cells.     

Identification of cis-acting elements in the SUC2 promoter of Saccharomyces cerevisiae required for activation of transcription

We analyzed the effects of site-directed mutations in the SUC2 promoter of Saccharomyces cerevisiae. Analyses were performed in wild-type as well as mig1 and tup1 mutant strains after the promoter mutants were reintroduced into the native SUC2 locus on the left arm of chromosome IX. Mutation of the two GC boxes revealed that these elements play two distinct roles: they are, as expected, required for Mig1-mediated repression but they are also necessary for activation of the SUC2 promoter in response to glucose limitation. The individual GC boxes are functionally redundant with regard to Mig1-mediated repression, however, only the upstream GC box is essential for high level expression of SUC2. Microccocal nuclease sensitivity of the SUC2 promoter in derepressed cells was reduced in the GC box mutant promoters, particularly in the vicinity of the TATA box. The difference in nuclease sensitivity between wild-type and GC box mutant promoters was not evident in tup1- cells. The formation of nuclease-resistant chromatin does not require the GC boxes, indicating that other cis-acting elements can serve to recruit the Ssn6-Tup1 co-repressor complex to the SUC2 promoter.     

Synergistic antiproliferative actions of cyclic adenosine 3',5'-monophosphate, interleukin-1beta, and activators of Ca2+/calmodulin-dependent protein kinase in primary hepatocytes

cAMP and Ca2+ acted together with the acute phase cytokine interleukin-1beta (IL-1beta) to inhibit hepatocyte DNA replication. At sub-basal activity of cAMP-dependent protein kinase (PKA), neither IL-1beta nor the Ca2+-elevating hormone vasopressin affected hepatocyte proliferation. Basal level of PKA activity permitted IL-1beta action. Increased PKA activity also permitted vasopressin action and sensitized further towards IL-1beta, which acted at 10-50 pM concentrations. Vasopressin acted via Ca2+/calmodulin-dependent protein kinase II (CaMKII), and its action was mimicked by the serine/threonine phosphatase inhibitor microcystin, which activates CaMKII. Inhibitors (KN93 and KT5926) of CaMKII selectively counteracted the effects of vasopressin and microcystin on hepatocyte proliferation at concentrations similar to those required to inhibit CaMKII in vitro. Two-dimensional gel electrophoresis of 32P-prelabeled hepatocytes revealed a common set of proteins phosphorylated in response to vasopressin and microcystin. Their phosphorylation was counteracted by CaMKII inhibitor (KT5926). Phosphorylation of the CaMKII substrate phenylalanine hydroxylase (PAH; EC 1.14.16.1) was used as an endogenous marker of CaMKII activation. It was found that treatment of the cells with vasopressin or microcystin increased the phosphorylation of PAH, and that the vasopressin-induced PAH phosphorylation was inhibited by KT5926. In conclusion, the Ca2+-elevating hormone vasopressin potentiated the antiproliferative effects of cAMP and IL-1beta through CaMKII activation.     

Evidence for a role for the cyclic adenosine 3',5'-monophosphate/protein kinase-A pathway in regulation of the gonadotropin subunit messenger ribonucleic acids

cAMP regulation of gonadotropin secretion and subunit mRNA levels was studied in pituitary cells perifused with pulses of GnRH. Pituitary cells from 7-week-old male rats castrated at 5 weeks of age were stimulated hourly for 9-24 h with 1-min pulses of GnRH, the adenylate cyclase activator forskolin, the cell-permeable cAMP analog 8-bromo-cAMP (8Br-cAMP), or control medium. Cells were also treated with the nonsteroidal antiinflammatory drug flufenamic acid, which reduces pituitary cAMP levels. During perifusion, the effluent was collected in 10-min fractions for FSH and LH assay. At the completion of perifusion, total RNA was extracted, and gonadotropin subunit mRNA levels were quantitated by Northern analysis. Continuous administration of flufenamic acid gradually reduced the amplitude of GnRH-stimulated FSH and LH pulses to nadir values of 40 +/- 4.7% and 62 +/- 12% of the control value, respectively. Flufenamic acid decreased (P < 0.05) FSH beta and alpha-subunit mRNA levels and blocked the effect of GnRH to lengthen LH beta mRNA. Pulses of forskolin or 8Br-cAMP released LH and FSH, and continuous forskolin or 8Br-cAMP potentiated the gonadotropin stimulatory effect of GnRH. Forskolin or 8Br-cAMP increased (P < 0.05) FSH beta mRNA and alpha-subunit mRNA levels when administered in pulses, but not when administered continuously, and lengthened LH beta mRNA. The Nal-Glu GnRH antagonist blocked the effects of GnRH pulses, but not the effects of 8Br-cAMP or forskolin. In conclusion, lowering intracellular cAMP levels with flufenamic acid attenuated GnRH-stimulated gonadotropin secretion, decreased alpha-subunit and FSH beta mRNA levels, and blocked the effect of GnRH to lengthen LH beta mRNA, whereas 8Br-cAMP or forskolin produced the opposite effect. These data extend previous results which suggested that cAMP modulates gonadotropin secretion and indicate that the cAMP/A-kinase pathway regulates each of the gonadotropin subunit mRNAs.     

Mediation by epidermal growth factor of the estradiol-induced increase in cyclic guanosine 3',5'-monophosphate content in the rat uterus

Estrogen treatment of immature or ovariectomized mature rats induces an increase in uterine cGMP content, with a peak 2-3 h after hormone administration. This response to estrogenic action also develops in vitro, in incubated uterine horns, thus excluding the intervention of another organ. Its function is still unknown. We show here that treatment of incubated uterine horns from immature or mature rats with 8 nM epidermal growth factor (EGF), exactly mimicked the effect of 1 nM estradiol on cGMP levels. The estradiol-induced increase in uterine cGMP was canceled in the presence of the phosphotyrosine kinase inhibitor genistein. Like the cGMP response to EGF, the estradiol-induced increase in uterine cGMP was completely suppressed in the presence of an antimouse EGF antibody. On the other hand, whereas the induction of cGMP accumulation by estradiol in vivo or in vitro was suppressed by prior treatment of the animals with the pure antiestrogen ICI 164,384, such pretreatment had no effect on the EGF-induced increase in uterine cGMP content. Together, these data support the concept that the uterine cGMP response to estrogens is entirely due to auto/paracrine mediation by the EGF-EGF receptor system. Considering reports from the literature showing that EGF can directly induce the phosphorylated active form of the estrogen receptor, we speculate that this might implicate its action on cGMP, with the latter then intervening as cofactor of the involved phosphokinase(s).     

Receptor cross-talk can optimize assays for autoantibodies to the thyrotropin receptor: effect of phenylisopropyladenosine on adenosine 3',5'-monophosphate and inositol phosphate levels in rat FRTL-5 thyroid cells

Immunoglobulins (IgG) from patients with Graves' disease increase inositol phosphate (IP) as well as cAMP production in rat thyroid FRTL-5 cells; IgGs from normal control subjects do not. Graves' IgG-and TSH-induced IP formation is inhibited by blocking TSH receptor (TSHR) antibodies from hypothyroid patients with primary myxedema, as is the cAMP response; this suggests that the Graves' IgG are acting through the TSHR to induce both the cAMP and phosphatidyl-inositol 4,5-biphosphate signal cascades in FRTL-5 thyroid cells as in cells with recombinant TSHR. Optimal conditions for measuring the Graves' IgG-induced IP increase include a NaCl-free Hanks' Balanced Salt Solution (HBSS) buffer system and a P1 purinergic receptor agonist; the action of each is additive. Optimization by NaCl-free HBSS is similar to that observed in cAMP assays and is specific for TSH or Graves' IgG; thus, NaCl-free HBSS did not affect ATP-induced, and actually inhibited norepinephrine-induced, IP production in FRTL-5 cells. The P1 purinergic receptor agonist acts via receptor cross-talk, which also allows further optimization of cAMP assays. Thus, adenosine deaminase improves Graves' IgG-induced cAMP production by removing adenosine from the medium. Although NaCl-free HBSS improved TSH- or Graves' IgG-induced IP and cAMP production in cells with recombinant TSHR; the modulatory action of phenylisopropyladenosine was lost.     

Synergistic interaction between paclitaxel and 8-chloro-adenosine 3',5'-monophosphate in human ovarian carcinoma cell lines

Taxol is a unique anticancer agent that is used in treatment of advanced ovarian cancer. Taxol exposure results in the polymerization and stabilization of the microtubule skeleton of eukaryotic cells, hence blocking replication and intracellular motility. 8-Chloro-adenosine 3',5'-monophosphate (8-Cl-cAMP) is a cAMP analogue, currently in Phase II clinical trials, that displays growth inhibition at micromolar concentrations. The aim of this study was to assess the nature of the interaction between 8-Cl-cAMP and paclitaxel using the combination index (CI) method of Chou and Talalay, which uses the median-effect analysis. Two ovarian cancer cell lines, A2780 and OAW42, which differ in sensitivity to both drugs, were tested using the fixed-ratio design using various scheduling regimens. Concurrent exposure of both drugs resulted in highly synergistic interactions in both cell lines. CIs (mean +/- SE) with this schedule were 0.182 +/- 0.016, 0.315 +/- 0.32, and 0.618 +/- 0.637 at 20, 50, and 80% cell kill, respectively, in A2780 cells and 0.001 +/- 0.0009, 0.016 +/- 0.0075, and 0.184 +/- 0.168 at 20, 50, and 80% cell kill, respectively, in OAW42 cells. In both cell lines, synergy was effective over a 4-fold log range of concentration for either drug. Sequencing with paclitaxel for 24 h prior to 8-Cl-cAMP was the most effective regimen; it resulted in consistently low CIs of up to the 90% cell kill level for both cell lines. Exposure to 8-Cl-cAMP prior to paclitaxel was the least effective regimen. In conclusion, the combination of paclitaxel and 8-Cl-cAMP is highly synergistic in ovarian carcinoma cell lines, suggesting that 8-Cl-cAMP may stimulate the antitumor effect of the taxanes.