Recent advances in biosynthesis of forskolin / 中国中药杂志

Forskolin is a complex labdane plant diterpenoid, which has been used in the treatment of a variety of diseases based on its activity as an activator of adenosine monophosphate(cAMP) cyclase. Natural forskolin exists only in the cork layer of the root of Coleus forskohlii. Due to the complexity of the extraction and chemical synthesis processes, the yield and purity of forskolin cannot meet commercial requirements. In recent years, with the rapid development of synthetic biology and the analysis and interpretation of many diterpene biosynthetic pathways, a new approach has been provided for the green production of forskolin. In this paper, the structure, activity, biosynthetic pathway and the heterologous biosynthesis of forskolin were reviewed. The problems and solutions in the heterologous biosynthesis of forskolin were also discussed and summarized, which will provide references for the construction of high-yielding forskolin engineering strains.

Forskolin inhibited the secretion of interleukin－1β in activated human THP－1 macrophages / 中华实用儿科临床杂志

Objective To investigate the effects of Forskolin on the activation of nucleotide－binding oligome-rization domain like receptor family,pyrin domain－containing 3(NLRP－3)inflammasome and the secretion of inter-leukin－1β(IL－1β)in activated human THP－1 macrophages,which can provide evidence for clinical treatment of inflammatory diseases in children.Methods Human monocyte cell line THP－1 cells were induced to differentiate into macrophages by Phorbol－12－myristate－13－acetate(PMA),and Forskolin(10,50,100 μmol/L)stimulated activa-ted macrophages by nigericin.The mRNA of the inflammasome markers NLRP－3,IL－1β and Caspase－1 were detec-ted by real－time quantitative polymerase chain reaction(qPCR)method.The protein of NLRP －3,pro －IL －1β, pro－Caspase－1 and Caspase－1 were detected by Western blot.The secretion of inflammatory cytokines IL－1β was detected by enzyme－linked immuno－sorbent assay(ELISA).Results Nigericin activated the cells and the mRNA expression of NLRP－3,IL－1β and Caspase－1 increased by 7.59 times(P＜0.001),579.10 times(P＜0.001)and 3. 59 times (P ＜0. 001 ),compared to non － activated cells;Forskolin had no effect on the mRNA expression of NLRP－3,Caspase－1 and IL－1β on activated THP－1 macrophages(P＞0.05).Western blot showed that Forskolin also had no effect on the protein expression of pro－Caspase－1 and pro－IL－1β in activated THP－1 macrophages (P＞0. 05),but the expression of NLRP －3 and Caspase －1 decreased significantly (P ＜0.001). The results of ELISA showed that the IL －1β secretion increased from basal 584. 0 nmol/L to activated 2 695. 6 nmol/L (t ＝16.031 1,P＜0. 001)on THP －1 macrophages by nigericin,but Forskolin(10,50,100 μmol/L)reduced it to 1 858. 4 nmol/L(t＝5.365 5,P ＜0. 001),1 467.9 nmol/L(t ＝8.047 5,P ＜0.001)and 1 246.7 nmol/L(t ＝10.199 0,P＜0.001)on the activated THP－1 macrophages.Conclusions Forskolin can not affect the expression of pro－cytokines at the gene transcription and protein translation levels in activated THP－1 cells,but inhibit the secre-tion of inflammatory cytokines IL－1β,so as to inhibit inflammatory response,which can be used to treat pediatric in-flammatory diseases caused by IL－1β.

Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification / Acúmulo de lipídios intracitoplasmáticos, desenvolvimento e criotolerância de embriões bovinos produzidos in vitro e tratados com diferentes concentrações de forskolin antes da vitrificação

The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5µM (Forsk 2.5 group), 5.0µM (Forsk 5.0 group) or 10.0µM (Forsk 10.0 group). Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri-Ingá® procedure. Although there were no significant differences (P>0.05) in the blastocyst rates between the Control group (44.9%) and the other treatments, the embryo production was lower (P<0.05) in Forsk 10.0 group (38.8%) compared to Forsk 2.5 (50.5%) and Forsk 5.0 (54.7%) groups. The intracytoplasmic lipid content (expressed in arbitrary units of pixels) in blastocysts from the Control group (1.00±0.03) was similar (P>0.05) to that found in Forsk 2.5 (0.92±0.03) and Forsk 10.0 groups (1.06±0.03) groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04) was lower than in the Control group (P<0.05). Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05) in Forsk 5.0 group (72.2%) compared to the Control group (46.2%). In conclusion, pre-treatment with forskolin at a concentration of 5.0 µM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.(AU)

Os embriões foram produzidos a partir de ovários obtidos em abatedouro e foram alocados em quatro grupos experimentais. Nos grupos tratados, o forskolin foi adicionado ao meio de cultivo in vitro no dia 6 do cultivo e os embriões foram incubados durante 24 horas com uma das seguintes concentrações: 2,5µM (grupo Forsk 2,5), 5,0µM (grupo Forsk 5,0) ou 10,0µM (grupo Forsk 10,0). Os embriões do grupo controle foram cultivados na ausência de forskolin. No dia 7 do cultivo, os blastocistos expandidos foram corados com o corante lipofílico Sudan Black B para a determinação do teor de lípidos intracitoplasmáticos ou foram criopreservados através do protocolo Vitri-Ingá®. Não foi observada diferença significativa (P>0,05) na taxa de produção de blastocistos entre o grupo Controle (44,9%) e os demais tratamentos, todavia observou-se menor produção de embriões (P<0,05) no grupo Forsk 10,0 (38,8%) em comparação com os grupos Forsk 2,5 (50,5%) e Forsk 5,0 (54,7%). A quantidade de lipídeos intracitoplasmáticos do grupo Controle (1,00±0,03) foi semelhante (P>0,05) a dos grupos Forsk 2,5 (0,92±0,03) e Forsk 10,0 (1,06±0,03); no entanto, o acúmulo de lípidos nos blastocistos do grupo Forsk 5.0 (0,82 ± 0,04) foi menor do que no grupo controle (P<0,05). A partir destes resultados, o grupo Forsk 5,0 foi testado quanto à criotolerância e foi observado que a taxa de re-expansão da blastocele 24 horas após o aquecimento foi maior (P<0,05) no grupo Forsk 5,0 (72,2%) quando comparado ao grupo Controle (46,2%). Em conclusão, o pré-tratamento com forskolin na concentração de 5,0 µM durante 24 horas antes da vitrificação foi eficiente para promover a redução da quantidade de lipídeos intracitoplasmáticos e, consequentemente, melhorou a criotolerância de embriões bovinos produzidos in vitro.(AU)

The Labdane Ent-3-Acetoxy-Labda-8(17), 13-Dien-15-Oic Decreases Blood Pressure In Hypertensive Rats / O Labdano Ácido Ent-3-Acetóxi-Labda-8(17), 13-Dieno-15-Óico Reduz Pressão Arterial Em Ratos Hipertensos

Abstract Background: Labdane-type diterpenes induce lower blood pressure via relaxation of vascular smooth muscle; however, there are no studies describing the effects of labdanes in hypertensive rats. Objective: The present study was designed to investigate the cardiovascular actions of the labdane-type diterpene ent-3-acetoxy-labda-8(17), 13-dien-15-oic acid (labda-15-oic acid) in two-kidney 1 clip (2K-1C) renal hypertension. Methods: Vascular reactivity experiments were performed in aortic rings isolated from 2K-1C and normotensive (2K) male Wistar rats. Nitrate/nitrite (NOx) measurement was performed in aortas by colorimetric assay. Blood pressure measurements were performed in conscious rats. Results: Labda-15-oic acid (0.1-300 µmol/l) and forskolin (0.1 nmol/l - 1 µmol/l) relaxed endothelium-intact and endothelium-denuded aortas from both 2K-1C and 2K rats. Labda-15-oic acid was more effective at inducing relaxation in endothelium-intact aortas from 2K pre-contracted with phenylephrine when compared to the endothelium-denuded ones. Forskolin was more potent than labda-15-oic acid at inducing vascular relaxation in arteries from both 2K and 2K-1C rats. Labda-15-oic acid-induced increase in NOx levels was lower in arteries from 2K-1C rats when compared to 2K rats. Intravenous administration of labda-15-oic acid (0.3-3 mg/kg) or forskolin (0.1-1 mg/kg) induced hypotension in conscious 2K-1C and 2K rats. Conclusion: The present findings show that labda-15-oic acid induces vascular relaxation and hypotension in hypertensive rats.

Resumo Fundamento: Diterpenos do tipo labdano induzem uma queda da pressão arterial por meio do relaxamento do músculo liso vascular; todavia, não há estudos que descrevam os efeitos de labdanos em ratos hipertensos. Objetivo: O presente estudo foi desenvolvido para investigar as ações cardiovasculares do labdano ácido ent-3-acetóxi-labda-8(17),13-dieno-15-óico (labda-15-óico) na hipertensão renal dois rins-1 clipe (2R-1C). Métodos: Foram feitos experimentos de reatividade vascular em anéis aórticos isolados de ratos machos 2R-1C e normotensos (2R). A medição de Nitrato/Nitrito (NOx) foi feita nas aortas por meio de ensaio colorimétrico. As medidas de pressão arterial foram feitas em ratos conscientes. Resultados: O ácido labda-15-óico (0,1 - 300 µmol/l) e a forscolina (0,1 nmol/l - 1 µmol/l) relaxaram as aortas com endotélio intacto e as aortas sem endotélio dos ratos 2R-1C e 2R. O labda-15-óico mostrou-se mais eficaz na indução do relaxamento em aortas com endotélio intacto de 2R pré-contraídas com fenilefrina em comparação àquelas sem endotélio. A forscolina mostrou-se mais potente do que o ácido labda-15-óico na indução do relaxamento vascular nas artérias tanto de ratos 2R-1C quanto de ratos 2R. O aumento dos níveis de NOx induzido pelo ácido labda-15-óico foi menor nas artérias de ratos 2R-1C em comparação a ratos 2R. A administração intravenosa de ácido labda-15-óico (0,3-3 mg/kg) ou forscolina (0,1-1 mg/kg) induziu hipertensão em ratos 2R-1C e 2R conscientes. Conclusão: Os presentes resultados mostram que o labda-15-óico induz relaxamento vascular e hipotensão em ratos hipertensos.

Experimental study on forskolin combined with bortezomib inducing apoptosis in bortezomib-resistant multiple myeloma cells / 中国癌症杂志

Background and purpose:Although bortezomib has become one of the major therapeutic agents against newly diagnosed or relapsed multiple myeloma (MM), there are some patients who become resistant to bor-tezomib and then relapse, emerging as a major obstacle to long-term survival of MM patients. It has been found that elevation of intracellular cyclic adenosine monophosphate (cAMP) levels could induce cell cycle arrest and apoptosis in MM cells,which has become an interesting approach to MM therapy. This study aimed to investigate possible effects of forskolin combined with bortezomib on bortezomib-resistant myeloma cells and further explore its mechanisms. Methods:The bortezomib-resistant MM cell lines H929-R and primary cells from patients who do not respond to bortezomib were used asin vitro models. The inlfuences of bortezomib and/or forskolin on MM cells were evaluated through cellular morphology, changes of cell distribution and apoptotic rate. Meanwhile, lfow cytometry analysis was used to detect mitochondrial transmembrane potential (ΔΨm) and the expression levels of apoptosis regulators in these cells before and after the treatment were detected by Western blot.Results:Bortezomib (20 nmol/L) synergized with forskolin (50nmol/L) to induce apoptosis of H929-R cells and bortezomib-resistant primary cells. In addition, borte-zomib synergized with forskolin to induce collapse of mitochondrial transmembrane and facilitate the degradation of anti-apoptosis proteins including Bcl-2 and Mcl-1.Conclusion:Bortezomib could synergize with forskolin to induce apoptosis in bortezomib-resistant MM cells.

Effect of forskolin on corticosterone secretion in Y1 mouse adrenocortical tumor cells / 中国药理学通报

Aim To study the effect of forskolin on cor-ticosterone secretion in mouse adrenocortical tumor cells. Methods Y1 cells were treated with 1μmol· L-1 or 10μmol·L-1 forskolin for 15 min to 24 h. Y1 cells growth morphology was observed, cell culture su-pernatants were collected and corticosterone was tested by ELISA kit. The cells total RNA was extracted using TRIzol kit and was reversely transcribed to obtain the cDNA, then was amplificated by real time quantitative PCR. The cells were lysed with RIPA lysis buffer and protein expressions were carried out by Western blot. Results Y1 cells morphology changed from flat adher-ent to shrink and spherical growth after 1μmol · L-1 forskolin treatment for 3 h. Compared with the control group, corticosterone levels were increased significantly by 1μmol · L-1 forskolin treatment for 24 h ( P <0. 01), then, forskolin significantly enhanced steroid ogenic acute regulatory protein ( Star) mRNA and pro-tein expression ( P <0. 01 ) , moreover, the steroido-genic enzymes such as Cyp11 a1 and Cyp11 b1 mRNA expressions were also up-regulated significantly by fors-kolin ( P <0. 01 ) . Additionally, Star mRNA expres-sion was increased significantly in a time-dependent re-sponse after 10μmol·L-1 forskolin treatment from 1 h to 12 h (P<0. 01). Furthermore, Nr4a1 and Nr4a2 mRNA expressions were up-regulated significantly after 10μmol · L-1 forskolin treatment from 15 min and reached the top at 1 h ( P<0. 01 ) . However, forsko-lin showed no effect on Mc2 r and Nr5 a1 mRNA expres-sions. Conclusion Y1 mouse adrenocortical tumor cells have a strong response to adenylate cyclase ago-nists, then, forskolin can be used to glucocorticoid se-cretion inducer reagent.

The Strategies for Production of Forskolin vis-a-vis Protection Against Soil Borne Diseases of the Potential Herb Coleus forskohlii Briq.

Coleus forskohlii is an important plant in Indian Ayurvedic medicine. It is the only source for forskolin among the plant kingdom. Forskolin has a unique property of activating almost all hormone sensitive adenylate cyclase enzymes in a biological system. This review article has highlighted on research developments of C. forskohlii for the production and to enhance the production of forskolin by employing various strategies, and also to protect the most potential herb against the soil borne wilt disease, causing a serious threat towards its propagation and cultivation by using effective integrated disease management technology.

Role of cGMP and cAMP in the hemodynamic response to intrathecal sildenafil administration

INTRODUCTION: Results from our laboratory have demonstrated that intracerebroventricular administration of sildenafil to conscious rats promoted a noticeable increase in both lumbar sympathetic activity and heart rate, with no change in the mean arterial pressure. The intracerebroventricular administration of sildenafil may have produced the hemodynamic effects by activating sympathetic preganglionic neurons in the supraspinal regions and spinal cord. It is well documented that sildenafil increases intracellular cGMP levels by inhibiting phosphodiesterase type 5 and increases cAMP levels by inhibiting other phosphodiesterases. OBJECTIVE: To examine and compare, in conscious rats, the hemodynamic response following the intrathecal administration of sildenafil, 8-bromo-cGMP (an analog of cGMP), forskolin (an activator of adenylate cyclase), or dibutyryl-cAMP (an analog of cAMP) in order to elucidate the possible role of the sympathetic preganglionic neurons in the observed hemodynamic response. RESULTS: The hemodynamic responses observed following intrathecal administration of the studied drugs demonstrated the following: 1) sildenafil increased the mean arterial pressure and heart rate in a dose-dependent manner, 2) increasing doses of 8-bromo-cGMP did not alter the mean arterial pressure and heart rate, 3) forskolin did not affect the mean arterial pressure but did increase the heart rate and 4) dibutyryl-cAMP increased the mean arterial pressure and heart rate, similar to the effect observed following the intrathecal injection of the highest dose of sildenafil. CONCLUSION: Overall, the findings of the current study suggest that the cardiovascular response following the intrathecal administration of sildenafil to conscious rats involves the inhibition of phosphodiesterases other than phosphodiesterase type 5 that increase the cAMP level and the activation of sympathetic preganglionic neurons.

Regulation of synthesis function of porcine ovarian granulose cells by mitogen-activated protein kinase signaling pathway / 中华妇产科杂志

Objective To investigate the role of mitogen-activated protein kinase(MAPK)signaling pathway regulating synthesis function of steroid hormone of porcine ovarian granulose cells.Methods Porcine ovarian granulose cells were cultured in medium added with dimethyl sulfoxide(DMSO)at concentration of 50μmol/L or with MAPK inhibitor PD98059 (PD) for 48 hours.Then,granulose cells in DMSO medium were added with activator of adenylate cyclase(20 μmoL/L)or blank agent for next 48 hours incubation,which were defined as observation group 1 and control group 1,similarly,granulose cells in PD medium were also added with activator of adenylate cyclase(20 μmol/L)or blank agent for next 48 hours,which were defined as observation group 2 and control group 2.The level of estradiol(E_2),progesterone (P)and testosterone (T) were detected by chemoluminescence.The mRNA expression of 17alpha-hydroxylase/17,20-lyase (CYP17) and aromatase P450 (P450 arom) were assessed by RT-PCR.Results (1)Hormone expression:the level of T,P and E_2 were(29.5±2.5)nmol/L,(80±5)nmol/L,(49±4) pmol/L in control group 1 and(42.3±3.4)nmol/L,(170±15)nmol/L,(75±6)pmol/L in control group 2,which showed significant difference(P<0.05).In the mean time,the level of T, P and E_2 were (106.2±7.6)nmol/L,(210±16)nmol/L,(130±11)pmol/L in observation group 2 and(47.2±3.5)nmol/L,(130±6)nmol/L,(81±6)pmol/L in observation group 1,which also reach statistical difference(P<0.05).(2)The expression of enzyme:the expression of CYP17 mRNA was inereased by 50% and P450 arom mRNA was decreased by 20% between control group 1 and 2. However, the mRNA expression of P450 arom and CYPI7 were upregulated remarkably, especially, the expression of CYP17 mRNA were increased by 125%. Conclusion MAPK signaling pathway plays an inhibitory role in regulating synthesis of steroid hormone of ovarian granulose cell.

Relaxation Patterns of Human Gastric Corporal Smooth Muscle by Cyclic Nucleotides Producing Agents

To elucidate the mechanism of cyclic nucleotides, such as adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5' -cyclic monophosphate (cGMP), in the regulation of human gastric motility, we examined the effects of forskolin (FSK), isoproterenol (ISO) and sodium nitroprusside (SNP) on the spontaneous, high K+ and acetylcholine (ACh)-induced contractions of corporal circular smooth muscle in human stomach. Gastric circular smooth muscle showed regular spontaneous contraction, and FSK, ISO and SNP inhibited its phasic contraction and basal tone in a concentration-dependent manner. High K+ (50 mM) produced sustained tonic contraction, and ACh (10 micrometer) produced initial transient contraction followed by later sustained tonic contraction with superimposed phasic contractions. FSK, ISO and SNP inhibited high K+-induced tonic contraction and also ACh-induced phasic and tonic contraction in a reversible manner. Nifedipine (1 micrometer), inhibitor of voltage-dependent L-type calcium current (VDCC(L)), almost abolished ACh-induced phasic contractions. These findings suggest that FSK, ISO and SNP, which are known cyclic nucleotide stimulators, inhibit smooth muscle contraction in human stomach partly via inhibition of VDCCL.

Forskolin Enhances Synaptic Transmission in Rat Dorsal Striatum through NMDA Receptors and PKA in Different Phases

The effect of forskolin on corticostriatal synaptic transmission was examined by recording excitatory postsynaptic currents (EPSCs) in rat brain slices using the whole-cell voltage-clamp technique. Forskolin produced a dose-dependent increase of corticostriatal EPSCs (1, 3, 10, and 30micrometer) immediately after its treatment, and the increase at 10 and 30micrometer was maintained even after its washout. When the brain slices were pre-treated with (DL)-2-amino-5-phosphonovaleric acid (AP-V, 100micrometer), an NMDA receptor antagonist, the acute effect of forskolin (10micrometer) was blocked. However, after washout of forskolin, an increase of corticostriatal EPSCs was still observed even in the presence of AP-V. When KT 5720 (5micrometer), a protein kinase A (PKA) inhibitor, was applied through the patch pipette, forskolin (10micrometer) increased corticostriatal EPSCs, but this increase was not maintained. When forskolin was applied together with AP-V and KT 5720, both the increase and maintenance of the corticostriatal EPSCs were blocked. These results suggest that forskolin activates both NMDA receptors and PKA, however, in a different manner.

PKA-Mediated Regulation of B/K Gene Transcription in PC12 Cells

B/K protein is a novel protein containing double C2-like domains. We examined the specific signaling pathway that regulates the transcription of B/K in PC12 cells. When the cells were treated with forskolin (50microM), B/K mRNA and protein levels were time-dependently decreased, reaching the lowest level at 3 or 4 hr, and thereafter returning to the control level. Chemicals such as dibutyryl-cAMP, cell- permeable cyclic AMP (cAMP) analogue and CGS21680, adenosine receptor A2A agonist, also repressed the B/K transcription. However, 1, 9-dideoxyforskolin did not show inhibitory effect on B/K transcription, suggesting direct involvement of cAMP in the forskolin-induced inhibition of B/K transcription. Effect of forskolin, dibutyryl cAMP and CGS21680 was significantly reduced in PKA-deficient PC12 cell line (PC12-123.7). One cAMP-response element (CRE) -like sequence (B/K CLS) was found in the promoter region of B/K DNA, and electrophoretic mobility shift assay indicated its binding to CREM and CREB. Forskolin significantly suppressed the promoter activity in CHO-K1 cells transfected with the constructs containing B/K CLS, but not with the construct in which B/K CLS was mutated (AC: TG). Taken together, we suggest that the transcription of B/K gene in PC12 cells may be regulated by PKA-dependent mechanism.

Forskolin promotes astroglial differentiation of human central neurocytoma cells

Human central neurocytoma is a kind of the brain tumors that are usually found in anterior part of the lateral ventricles. In this study, we established conditions that allowed proliferation of neurocytoma cells culture and analyzed characteristics of neurocytoma cells in vitro. For in vitro, a condition that used for culturing neural stem cells and contained basic fibroblast growth factor (bFGF) provided high proliferation. RT-PCR analaysis showed that nestin was found in neurocytoma cells, indicating that the neurocytomas possess neural stem cell properties. Interestingly, treatment of neurocytoma cells with forskolin increased expression of glial fibrillary acidic protein with a concomitant decrease in the nestin expression. Forskolin also induced morphological changes of neurocytoma cells to adopt an astrocyte-like phenotype. The results suggest that neurocyotma cells may have properties of multipotent neural stem cells.

Modulation of Pituitary Somatostatin Receptor Subtype (sst1-5) mRNA Levels by Growth Hormone (GH) -Releasing Hormone in Purified Somatotropes

We have previously reported that expression of the somatostatin receptor subtypes, sst1-5, is differentially regulated by growth hormone (GH) -releasing hormone (GHRH) and forskolin (FSK), in vitro. GHRH binds to membrane receptors selectively located on pituitary somatotropes, activates adenylyl cyclase (AC) and increases sst1 and sst2 and decreases sst5 mRNA levels, without significantly altering the expression of sst3 and sst4. In contrast FSK directly activates AC in all pituitary cell types and increases sst1 and sst2 mRNA levels and decreases sst3, sst4 and sst5 expression. Two explanations could account for these differential effects: 1) GHRH inhibits sst3 and sst4 expression in somatotropes, but this inhibitory effect is masked by expression of these receptors in unresponsive pituitary cell types, and 2) FSK inhibits sst3 and sst4 expression levels in pituitary cell types other than somatotropes. To differentiate between these two possibilities, somatotropes were sequentially labeled with monkey anti-rat GH antiserum, biotinylated goat anti-human IgG, and streptavidin-PE and subsequently purified by fluorescent-activated cell sorting (FACS). The resultant cell population consisted of 95% somatotropes, as determined by GH immunohistochemistry using a primary GH antiserum different from that used for FACS sorting. Purified somatotropes were cultured for 3 days and treated for 4 h with vehicle, GHRH (10 nM) or FSK (10micrometer). Total RNA was isolated by column extraction and specific receptor mRNA levels were determined by semi-quantitative multiplex RT-PCR. Under basal conditions, the relative expression levels of the various somatostatin receptor subtypes were sst2> sst5> sst3=sst1> sst4. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst3, sst4 and sst5 mRNA levels in purified somatotropes, comparable to the effects of FSK on purified somatotropes and mixed pituitary cell cultures. Taken together, these results demonstrate that GHRH acutely modulates the expression of all somatostatin receptor subtypes within GH-producing cells and its actions are likely mediated by activation of AC.

Adenylate cyclase stimulation and ocular hypertension inhibition by forskolin analogs / 中华实验眼科杂志

ObjectiveForskolin （FSK） analogs，isoforskolin （isoF），deacetylforskolin（deaF），and 1-acetylforskolin（1-aF），extracted from Coleus forskohlii native to Yunnan，were assayed for their adenylate cyclase stimulating activities in vitro and for effects of two analogs on ocular hypertension （OHT） in water-loaded rabbits．MethodsAdenylate cyclase stimulation was determined by protein-binding method of radioimmunoassay，and intraocular pressure was monitored by pneumatonometer．ResultsIt showed that isoforskolin and forskolin stimulated adenylate cyclase in vitro with almost equal activity，deacetylforskolin with milder activity，and 1-acetylforskolin with little activity in vitro．1％ deaF and 1-aF suppressed rabbit OHT induced by water-loading for at least 3h，with the maximal inhibitory rates of 6.0，10.9% respectively．ConclusionThis study suggests that two foskolin analogs （isoforskolin，deacetylforskolin） possess adenylate cyclase stimulation activities in vitro；deacetylforskolin and 1-acetylforskolin suppress OHT induced by water-loading in rabbits．

Human mesenchymal stem cells differentiate into neuron-like cells by increase in intracellular cyclic AMP / 中国病理生理杂志

AIM: To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells by the increase in intracellular cyclic AMP. METHODS: hMSC were separated from human marrow with Ficoll-Paque reagent and expanded in culture medium. hMSC were induced to differentiate into neurons with Forskolin and 3-isobutyl-1-methyl-xanthine (IBMX). Neuron-specific enolase(NSE), neurofilament(NF), glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry. RESULTS: hMSC were expanded as undifferentiated cells in culture for more than 10 passages. Forskolin/IBMX can induce hMSC to exhibit a neuronal phenotype, expressing NSE and NF-M in 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP. CONCLUSION: hMSC can be induced to differentiate into neurons by increase in the intracellular cAMP.

Cholera toxin mediated regulation of the expression of Gq alpha and G11 alpha GTP binding proteins

Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.

Effects of cAMP forskolin and cyanoketone on in vitro oocyte maturation in the catfish, Clarias batrachus.

This study investigated the interactive effects of cyanoketone (CK), an inhibitor of 3β-hydroxysteroid dehydrogenase on the effects of cAMP and forskolin (FK) on oocyte maturation in Clarias batrachus using an in vitro incubation technique. When the oocytes were incubated in the presence of 1 μg/ml 17α, 20β-dihydroxy-4-pregnen-3-one[l7α, 20β-DP, the maturation-inducing steroid (MIS) of this species] for 6h, they matured [85·3 + 1·36% germinal vesicle breakdown (GVBD)] normally after additional incubation for 20-30 h in plain medium. On the other hand, exposure to 1·0 and 8 0 mM of cAMP after MIS stimulation caused significant inhibition of GVBD but lower concentrations (0·1 and 0·5 mM) of cAMP were noninhibitory. However, when the oocytes were preincubated for 1 h with 1 μg/mI CK, a significant inhibition in the percentage of GVBD was recorded including the lower concentrations of c AMP. FK, an activator of adenylate cyclase, could significantly induce GVBD at all of its concentrations (0·1, 0·5, 1·0 and 10·0 μM) in a dose- and time-dependent manner. However, when the oocytes were exposed to 1 μg/ml CK for 1 h, prior to FK stimulation, a complete inhibition of GVBD occurred but when CK treatment was given after the FK stimulation, only a partial inhibition of maturation was observed. Taken together, these data indirectly suggest that FK induces catfish oocyte maturation probably by stimulating follicular production of Δ4 steroid ( 17α,20 β-DP)through an adenylate cyclase-c AMP-mediated pathway, a mechanism identical to the gonadotropin-induced oocyte maturation.

Interaction of forskolin with the effect of N-6-cyclopentyladenosine on norepinephrine release in rat hippocampus

As it has been reported that the depolarization-induced norepinephrine (NE) release is modulated by activation of presynaptic A-1-adenosine heteroreceptor and various lines of evidence indicate the involvement of adenylate cyclase system in A-1-adenosine post-receptor mechanism in hippocampus, it was attempted to delineate the role of adenylate cyclase system in the A-1-receptor-mediated control of NE release in this study. Slices from rat hippocampus were equilibrated with (3H)-NE and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 V cm-1, 2 ms, rectangular pulses). The influence of various agents on the evoked tritium-outflow was investigated. N-6-Cyclopentyladenosine (CPA), a specific A-1-adenosine receptor agonist, in concentrations ranging from 0.1 to 10 micrometer decreased the (3H)-NE release in a dose-dependent manner without any change of basal rate of release. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 micrometer), a selective A-1-receptor antagonist, inhibited the CPA effect. The responses to N-ethylmaleimide (3 & 10 micrometer), a SH-alkylating agent of G-protein, were characterized by increments of the evoked NE-release and the CPA effects were completely abolished by NEM pretreatment. Forskolin, a specific adenylate cyclase activator, in concentrations ranging from 0.1 to 30 micrometer increased the evoked and basal rate of NE release in a dose-dependent manner and the CPA effects were inhibited by forskolin pretreatment. Rolipram (1 & 10 micrometer), a phosphodiesterase inhibitor, did not affect the evoked NE release, but reduced the CPA effect. And 8-bromo-cAMP (100 & 300 micrometer), a membrane permeable cAMP analogue, inhibited the CPA effect significantly. These results suggest that the A-1-adenosine heteroreceptor plays an important role in NE-release via nucleotide-binding protein G-i in the rat hippocampus and that the adenylate cyclase system might be participated in this process.

Expression of signal transducing G proteins in human melanoma cell lines

Some malignant melanoma cells regress spontaneously by terminal differentiation, and understanding the mechanisms of this spontaneous regression can contribute to the development of a new therapy not only for melanoma but also for other cancers. The signal transducing G protein is one component of the signaling pathways for the differentiation-inducing molecules such as alpha-melanocyte-stimulating hormone (alpha-MSH) and cAMP. To investigate the role of G proteins in the differentiation process, we analyzed the expression of various G proteins by quantitative Western blot and cAMP response in human malignant melanoma cell lines. SK-MEL-3 cells expressed the largest amount of stimulatory G protein alpha subunit (G(s) alpha) and the largest amount of inhibitory G protein alpha subunit (G(i) alpha) was expressed in Malme-3M cells among the 4 melanoma cell lines analyzed in this experiment. The SK-MEL-28 cells exhibited largest amount of alpha subunit of G(q) and the beta subunits. The cAMP formation by forskolin stimulation was largest in the Malme-3M. The amount of cAMP formation did not show any correlation with the expression of G(s) alpha nor that of G(i) alpha. The population doubling time was longest in Malme-3M cells. In this experiment, we found that the melanoma cells vary widely both in the expression of various G proteins and in cAMP production depending on the cell lines.