Effects of a complex mixture of persistent organic pollutants (POPs) on steroidogenesis in H295R cells under 10 μM forskolin stimulation - results from a pilot study
Kareem Eldin Mohammed AhmedHåvard G. FrøysaOdd André KarlsenKarin ZimmerHanne Friis BerntsenSteven VerhaegenErik RopstadRalf KellmannAnders Goksøyr
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Abstract This study describes the utilization of an LC-MS/MS based H295R assay to assess an environmentally relevant mixture of persistent organic pollutants (POPs). H295R cells were exposed to the POP mixture in two conditions stimulated with 10 μM forskolin and unstimulated. Most importantly, the unstimulated cells responded to the low concentration of the mixture with a significant down-regulation of dehydroepiandrosterone (DHEA). This response was not observed in forskolin-stimulated cells. In stimulated H295R cells, exposure to the highest concentration showed a trend towards induced production of mineralocorticoids and glucocorticoids, although this was not significant. On the other hand, in the same exposure concentration and condition, estrogen and androgen production tended to be down-regulated. In addition to these patterns of responses being different in the stimulated vs unstimulated condition, four steroids were not detectable in the unstimulated condition.We have examined the roles that cyclic AMP and protein synthesis play in the development of refractoriness in C6-2B rat glioma cells using the diterpene, forskolin, a general activator of cyclic AMP-generating systems. Forskolin-stimulated cyclic AMP accumulation peaked at 30 min and declined thereafter to 10% of peak levels by 3 hr despite the continued presence of sufficient forskolin to produce 98% of the control response when the incubation medium was transferred to naive cells. C6-2B cells treated for 3 hr with forskolin were refractory to a subsequent challenge with forskolin or isoproterenol. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) increased the degree of refractoriness developed after forskolin treatment. In the presence of IBMX, the induction of refractoriness by forskolin and forskolin-stimulated cyclic AMP accumulation were similarly dependent on forskolin concentration. Pre-treatment with isoproterenol or the cyclic AMP analogue, dibutyryl cyclic AMP, induced refractoriness to forskolin. When C6-2B cells were pre-treated with forskolin plus the protein synthesis inhibitor, cycloheximide, the development of refractoriness to forskolin or isoproterenol was attenuated. Cycloheximide prevented isoproterenol- or dibutyryl cyclic AMP-induced refractoriness to forskolin. These data provide further evidence that the onset of the refractory state in C6-2B cells is mediated by cyclic AMP and is a protein synthesis-requiring process.
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Abstract Dehydroepiandrosterone (DHEA), an adrenal androgen precursor, can be metabolized in target tissues into active sex steroids. It has been proposed that DHEA supplementation might result in restoration of physiological local sex steroid levels, but knowledge on the effect of DHEA treatment on local sex steroid levels in multiple tissues is lacking. To determine the effects of DHEA on tissue-specific levels of sex steroids, we treated orchiectomized (ORX) male mice with DHEA for 3 weeks and compared them with vehicle-treated ORX mice and gonadal intact mice. Intra-tissue levels of sex steroids were analyzed in reproductive organs (seminal vesicles, prostate, m. levator ani), major body compartments (white adipose tissue, skeletal muscle, and brain), adrenals, liver, and serum using a sensitive and validated gas chromatography–mass spectrometry method. DHEA treatment restored levels of both testosterone (T) and dihydrotestosterone (DHT) to approximately physiological levels in male reproductive organs. In contrast, this treatment did not increase DHT levels in skeletal muscle or brain. In the liver, DHEA treatment substantially increased levels of T (at least 4-fold) and DHT (+536%, P < 0.01) compared with vehicle-treated ORX mice. In conclusion, we provide a comprehensive map of the effect of DHEA treatment on intra-tissue sex steroid levels in ORX mice with a restoration of physiological levels of androgens in male reproductive organs while DHT levels were not restored in the skeletal muscle or brain. This, and the unexpected supraphysiological androgen levels in the liver, may be a cause for concern considering the uncontrolled use of DHEA.
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To visualize the adenylate cyclase (AC)-related second messenger system, [11C]forskolin, [11C]1-acetyl-7-deacetylforskolin, [11C]1,9-dideoxyforskolin and [11C]1-deoxyforskolin were synthesized by acetylation of the respective deacetyl-precursors using [11C]acetylchloride and dimethylaminopyridine. The radiochemical yield of [11C]forskolin, [11C]1-acetyl-7-deacetylforskolin, [11C]1,9-dideoxyforskolin and [11C]1-deoxyforskolin calculated from trapped [11C]CO2 were 5%, 10%, 15% and 18%, respectively. Since the 1- and 9-OH groups on the forskolin structure are critical for specific binding to AC (active type), we considered [11C]1-acetyl-7-deacetylforskolin, [11C]1,9-dideoxyforskolin and [11C]1-deoxyforskolin to be nonspecific forskolin analogs. A comparative study of [11C]forskolin and its analogs on the n-octanol/phosphate buffer (pH 7.4) partition ratio showed that [11C]1-acetyl-7-deacetylforskolin has similar physical properties to [11C]forskolin. In the mouse heart, kidneys, liver and lungs, more [11C]forskolin accumulated than [11C]1-acetyl-7-deacetylforskolin. Moreover, simultaneous [11C]forskolin with forskolin (10 micrograms) administration reduced the accumulation of [11C]forskolin particularly in the heart to the level of [11C]1-acetyl-7-deacetylforskolin. These results indicate that [11C]forskolin would be a useful imaging agent for the AC-related second messenger system.
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アデニールサイクラーゼ活性化作用をもつforskolinの心不全に対する作用検討の一環として,我々の開発した左心室心筋内プロテアーゼ注入による心筋破壊,輸液,メソキサミン投与による低拍出性末梢血管抵抗増加型イヌうっ血性心不全モデルを用いて,forskolin静注の効果を検討した.forskolin5.0 μg/kg静脈内投与により,大動脈血流量は0.50→0.72Z/min(平均値,N=7),総末梢血管抵抗19,980→10,390 dynesec/cm5,平均左房圧17.5→7.9 mmHg,左室拡張終期圧22.8→16.8 mmHgと変化した.体血圧は102.5→77.7 mmHgと減少し,心拍数は92→122 b/minと増加した.Vmaxは2.32→2.82 l/secと増加し,拡張期時定数Tは90.7→59.2msecと減少した.forskolinの増量により,総末梢血管抵抗,平均左房圧,左室拡張終期圧は用量依存性に減少した.以上のことから,forskolinは血管拡張作用と心筋収縮能,弛緩能の増加によりうっ血性心不全を改善する方向に作働するものと考えられる.
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Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage‐sensitive Ca 2+ channels (VSCCs) in a cyclic AMP (cAMP)‐independent manner in PC12 cells. Ca 2+ influx induced by membrane depolarization with 70 m M K + was inhibited when cells were preincubated with 10 µ M forskolin. Almost maximum inhibitory effect on Ca 2+ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca 2+ influx was not affected by the presence of 2′,5′‐dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin‐induced cAMP production. 1,9‐Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca 2+ influx induced by 70 m M K + without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.
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To explore the role of protein kinase A (PKA) in regulating tau phosphorylation and spatial memory, we injected forskolin, an activator of PKA, at different concentrations into the rat brains. We found that forskolin at concentrations up to 80 μM enh
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Phosphodiesterase inhibitor
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