Mountjoy KG, Robbins LS, Mortrud MT, Cone RD. The cloning of a family of genes that encode the melanocortin receptors. inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 steroid hydroxylase mRNA expression were potently inhibited by diphenyl-butylpiperidine T-type Ca2+ antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Cav3.2 Ca2+ current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Cav3.2 Ca2+ channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism. for 10 min at 4C. An aliquot (60 l) of the supernatant was reserved for estimation of total Rap1. The remaining supernatant was mixed with glutathione-agarose and incubated for 1 h at 4C. Samples were then centrifuged at 10,000 for 30 s at 4C, washed three times with lysis buffer, suspended in 40 l of 2 Lammeli buffer containing 10 mM DTT, and separated by 8C16% SDS-PAGE gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membrane, incubated with polyclonal anti-Rap1 antibodies (Millipore), and visualized by enhanced chemilluminescence (Pierce of Thermo Fisher Scientific, Rockford, IL). [3H]thymidine incorporation. For determination of DNA synthesis, the procedure used by Hornsby and Gill (25) was followed with some modifications. Briefly, 0.5 104 AZF cells were plated in triplicate in 35-mm fibronectin-treated dishes. Medium was changed after 6 h, and cells were incubated either without (control) or with agents. After 20 h, 10 Ci of [3H]thymidine (10 l of 1 1 mCi/ml stock solution) was added to cultures, and incubation continued for 4 h. Medium was then removed, cells were washed twice with ice-cold PBS, and 1 ml of a 1% aqueous solution of Triton X-100 was added. Cells were incubated at room temperature with this solution for 5 min, after which the entire contents of the plate were transferred to 10 ml of absolute ethanol, and insoluble material was collected on 2.4-cm glass fiber filters (GF/A; Whatman) with suction. The filters were washed twice with 10 ml of absolute ethanol and then transferred to 10 ml of Scintiverse BD scintillation cocktail and counted using a Beckman Coulter LS 6500 scintillation counter. A-kinase assay. PKA activity was measured with a SignaTECT cAMP-dependent protein kinase assay kit (Promega, Madison, WI) in which PKA-dependent phosphorylation of biotinylated peptides can be quantitatively measured as a function of PKA activity. AZF cells were plated on 60-mm fibronectin-treated dishes in DMEM-F-12+ at a density of 3C4 106 cells/dish. After 24 h, the medium was replaced with either control medium (DMEM-F-12+) or the same medium containing ACTH (1C24) or 8PT-Ade. At the end of the incubation period, cells were washed four times with ice-cold PBS and suspended in cold extraction buffer [25 mM TrisHCl, pH 7.4, 0.5 mM EGTA, 10 mM -mercaptoethanol, 0.5 mM Pefabloc-SC (Roche Applied Science, Indianapolis, IN)] and protease inhibitors with EDTA [Complete Mini protease inhibitor cocktail tablet (Roche Applied Science), 1/10 ml lysis solution]. Lysates were homogenized using a cold Dounce homogenizer and then centrifuged AMD3100 (Plerixafor) for 5 min at 4C, 14,000 < 0.0001 vs. respective control without cAMP compound using Student's and < 0.0001 vs. control without 8CPT-2-OMe-cAMP using Student's and shows that neither 8MeOPT-2-OMe-cAMP (100 M) nor.Bornstein SR, Engeland WC, Ehrhart-Bornstein M, Herman JP. Dissociation of ACTH and glucocorticoids. potently inhibited by diphenyl-butylpiperidine T-type Ca2+ antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Cav3.2 Ca2+ current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Cav3.2 Ca2+ channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism. for 10 min at 4C. An aliquot (60 l) of the supernatant was reserved for estimation of total Rap1. The remaining supernatant was mixed with glutathione-agarose and incubated for 1 h at 4C. Samples were then centrifuged at 10,000 for 30 s at 4C, washed three times with lysis buffer, suspended in 40 l of 2 Lammeli buffer containing 10 mM DTT, and separated by 8C16% SDS-PAGE gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membrane, incubated with polyclonal anti-Rap1 antibodies (Millipore), and visualized by enhanced chemilluminescence (Pierce of Thermo Fisher Scientific, Rockford, IL). [3H]thymidine incorporation. For determination of DNA synthesis, the procedure used by Hornsby and Gill (25) was followed with some modifications. Briefly, 0.5 104 AZF cells were plated in triplicate in 35-mm fibronectin-treated dishes. Medium was changed after 6 h, and cells were incubated either without (control) or with agents. After 20 h, 10 Ci of [3H]thymidine (10 l of 1 1 mCi/ml stock solution) was added to cultures, and incubation continued for 4 h. Medium was then removed, cells had been washed double with ice-cold PBS, and 1 ml of the 1% aqueous alternative of Triton X-100 was added. Cells had been incubated at area heat range with this alternative for 5 min, and the entire items from the dish had been used in 10 ml of overall ethanol, and insoluble materials was gathered on 2.4-cm glass fiber filters (GF/A; Whatman) with suction. The filter systems had been washed double with 10 ml of overall ethanol and used in 10 ml of Scintiverse BD scintillation cocktail and counted utilizing a Beckman Coulter LS 6500 scintillation counter. A-kinase assay. PKA activity was assessed using a SignaTECT cAMP-dependent proteins kinase assay AMD3100 (Plerixafor) package (Promega, Madison, WI) where PKA-dependent phosphorylation of biotinylated peptides could be quantitatively assessed being a function of PKA activity. AZF cells had been plated on 60-mm fibronectin-treated meals in DMEM-F-12+ at a thickness of 3C4 106 cells/dish. After 24 h, the moderate was changed with either control moderate (DMEM-F-12+) or the same moderate filled with ACTH (1C24) or 8PT-Ade. By the end from the incubation period, cells had been washed four situations with ice-cold PBS and suspended in frosty removal buffer [25 mM TrisHCl, pH 7.4, 0.5 mM EGTA, 10 mM -mercaptoethanol, 0.5 mM Pefabloc-SC (Roche Applied Research, Indianapolis, IN)] and protease inhibitors with EDTA [Complete Mini protease inhibitor cocktail tablet (Roche Applied Research), 1/10 ml lysis solution]. Lysates had been homogenized utilizing a frosty Dounce homogenizer and centrifuged for 5 min at 4C, 14,000 < 0.0001 vs. particular control without cAMP substance using Student's and < 0.0001 vs. control without 8CPT-2-OMe-cAMP using Student's and implies that neither 8MeOPT-2-OMe-cAMP (100 M) nor 8HPT-2-OMe-cAMP (100 M) elevated cortisol creation by AZF cells.In various other experiments, we discovered that, at concentrations from 10 to 100 M, 8MeOPT-Ade activated well-correlated, concentration-dependent increases in cortisol synthesis and CYP17 gene expression (Fig. in conjunction with 8Br-cAMP. On the other hand, no additivity was noticed for these three substances when found in mixture with ACTH. 8PT-adenine didn't activate PKA or inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 steroid hydroxylase mRNA appearance had been potently inhibited by diphenyl-butylpiperidine T-type Ca2+ antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the appearance of both CACNA1H mRNA and linked Cav3.2 Ca2+ current. These outcomes indicate that 8-chloro (however, not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are changed into a dynamic metabolite, 8CPT-adenine, that induces the appearance of genes coding for steroidogenic proteins in bovine AZF cells. Various other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that will require the appearance of useful Cav3.2 Ca2+ stations. These phenylthio-adenine substances and ACTH may induce cortisol synthesis through the same cAMP-independent system. for 10 min at 4C. An aliquot (60 l) from the supernatant was reserved for estimation of total Rap1. The rest of the supernatant was blended with glutathione-agarose and incubated for 1 h at 4C. Examples had been after that centrifuged at 10,000 for 30 s at 4C, cleaned 3 x with lysis buffer, suspended in 40 l of 2 Lammeli buffer filled with 10 mM DTT, and separated by 8C16% SDS-PAGE gel electrophoresis. Protein had been used in polyvinylidene fluoride membrane, incubated with polyclonal anti-Rap1 antibodies (Millipore), and visualized by improved chemilluminescence (Pierce of Thermo Fisher Scientific, Rockford, IL). [3H]thymidine incorporation. For perseverance of DNA synthesis, the task utilized by Hornsby and Gill (25) was implemented with some adjustments. Quickly, 0.5 104 AZF cells were plated in triplicate in 35-mm fibronectin-treated dishes. Moderate was transformed after 6 h, and cells had been incubated either without (control) AMD3100 (Plerixafor) or with realtors. After 20 h, 10 Ci of [3H]thymidine (10 l of just one 1 mCi/ml share alternative) was put into civilizations, and incubation continuing for 4 h. Moderate was then taken out, cells had been washed double with ice-cold PBS, and 1 ml of the 1% aqueous alternative of Triton X-100 was added. Cells had been incubated at area heat range with this alternative for 5 min, and the entire items from the dish had been used in 10 ml of overall ethanol, and insoluble materials was gathered on 2.4-cm glass fiber filters (GF/A; Whatman) with suction. The filter systems had been washed double with 10 ml of overall ethanol and used in 10 ml of Scintiverse BD scintillation cocktail and counted utilizing a Beckman Coulter LS 6500 scintillation counter. A-kinase assay. PKA activity was assessed using a SignaTECT cAMP-dependent proteins kinase assay package (Promega, Madison, WI) where PKA-dependent phosphorylation of biotinylated peptides could be quantitatively assessed being a function of PKA activity. AZF cells had been plated on 60-mm fibronectin-treated meals in DMEM-F-12+ at a thickness of 3C4 106 cells/dish. After 24 h, the moderate was changed with either control moderate (DMEM-F-12+) or the same moderate filled with ACTH (1C24) or 8PT-Ade. By the end from the incubation period, cells had been washed four situations with ice-cold PBS and suspended in frosty removal buffer [25 mM TrisHCl, pH 7.4, 0.5 mM EGTA, 10 mM -mercaptoethanol, 0.5 mM Pefabloc-SC (Roche Applied Research, Indianapolis, IN)] and protease inhibitors with EDTA [Complete Mini protease inhibitor cocktail tablet (Roche Applied Research), 1/10 ml lysis solution]. Lysates had been homogenized utilizing a frosty Dounce homogenizer and centrifuged for 5 min at 4C, 14,000 < 0.0001 vs. particular control without cAMP substance using Student's and < 0.0001 vs. control without 8CPT-2-OMe-cAMP using Student's and implies that neither 8MeOPT-2-OMe-cAMP (100 M) nor 8HPT-2-OMe-cAMP (100 M) elevated cortisol creation by AZF cells sometimes 48 h. On the other hand, by 48 h, 8MeOPT-Ade (50 M) activated a 12-fold upsurge in cortisol synthesis. In other experiments, we found that, at concentrations from 10 to 100 M, 8MeOPT-Ade stimulated well-correlated, concentration-dependent increases in cortisol synthesis and CYP17 gene expression (Fig. 4, and < 0.001; **< 0.0001 vs. respective control using Student's test. The robust stimulation of cortisol synthesis and CYP17 mRNA expression by 8MeOPT-Ade shows that 8-substituted adenine derivatives other than 8CPT-Ade activate the steroidogenic pathway. They also indicate that 8MeOPT-2-OMe-cAMP is usually ineffective because it is usually not converted to an active metabolite. 8PT-Ade stimulates cortisol synthesis and the expression of CYP11a and CYP17 mRNAs. Results obtained in this study have shown that this 8PT-adenine derivatives 8CPT-Ade and 8MeOPT-Ade both markedly stimulate the expression of a panel of genes coding for steroidogenic proteins, leading to pronounced increases in cortisol synthesis. In other experiments, we found that 8PT-Ade itself stimulated cortisol secretion and the expression of steroid hydroxlyases, with a temporal pattern, potency, and effectiveness similar to the other two PT-adenine derivatives. At concentrations from 1 to 50 M, 8PT-Ade.Bird IM, Mason JI, Rainey WE. Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 Rabbit polyclonal to SRF.This gene encodes a ubiquitous nuclear protein that stimulates both cell proliferation and differentiation.It is a member of the MADS (MCM1, Agamous, Deficiens, and SRF) box superfamily of transcription factors. in H295R human adrenocortical cells. Ca2+ current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Cav3.2 Ca2+ channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism. for 10 min at 4C. An aliquot (60 l) of the supernatant was reserved for estimation of total Rap1. The remaining supernatant was mixed with glutathione-agarose and incubated for 1 h at 4C. Samples were then centrifuged at 10,000 for 30 s at 4C, washed three times with lysis buffer, suspended in 40 l of 2 Lammeli buffer made up of 10 mM DTT, and separated by 8C16% SDS-PAGE gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membrane, incubated with polyclonal anti-Rap1 antibodies (Millipore), and visualized by enhanced chemilluminescence (Pierce of Thermo Fisher Scientific, Rockford, IL). [3H]thymidine incorporation. For determination of DNA synthesis, the procedure used by Hornsby and Gill (25) was followed with some modifications. Briefly, 0.5 104 AZF cells were plated in triplicate in 35-mm fibronectin-treated dishes. Medium was changed after 6 h, and cells were incubated either without (control) or with brokers. After 20 h, 10 Ci of [3H]thymidine (10 l of 1 1 mCi/ml stock answer) was added to cultures, and incubation continued for 4 h. Medium was then removed, cells were washed AMD3100 (Plerixafor) twice with ice-cold PBS, and 1 ml of a 1% aqueous answer of Triton X-100 was added. Cells were incubated at room heat with this answer for 5 min, after which the entire contents of the plate were transferred to 10 ml of absolute ethanol, and insoluble material was collected on 2.4-cm glass fiber filters (GF/A; Whatman) with suction. The filters were washed twice with 10 ml of absolute ethanol and then transferred to 10 ml of Scintiverse BD scintillation cocktail and counted using a Beckman Coulter LS 6500 scintillation counter. A-kinase assay. PKA activity was measured with a SignaTECT cAMP-dependent protein kinase assay kit (Promega, Madison, WI) in which PKA-dependent phosphorylation of biotinylated peptides can be quantitatively measured as a function of PKA activity. AZF cells were plated on 60-mm fibronectin-treated dishes in DMEM-F-12+ at a density of 3C4 106 cells/dish. After 24 h, the medium was replaced with either control medium (DMEM-F-12+) or the same medium made up of ACTH (1C24) or 8PT-Ade. At the end of the incubation period, cells were washed four occasions with ice-cold PBS and suspended in cold extraction buffer [25 mM TrisHCl, pH 7.4, 0.5 mM EGTA, 10 mM -mercaptoethanol, 0.5 mM Pefabloc-SC (Roche Applied Science, Indianapolis, IN)] and protease inhibitors with EDTA [Complete Mini protease inhibitor cocktail tablet (Roche Applied Science), 1/10 ml lysis solution]. Lysates were homogenized using a cold Dounce homogenizer and then centrifuged for 5 min at 4C, 14,000 < 0.0001 vs. respective control without cAMP compound using Student's and < 0.0001 vs. control without 8CPT-2-OMe-cAMP using Student's and shows that neither 8MeOPT-2-OMe-cAMP (100 M) nor.In: Williams Textbook of Endocrinology. Philadelphia, PA: W. antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Cav3.2 Ca2+ current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Cav3.2 Ca2+ channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism. for 10 min at 4C. An aliquot (60 l) of the supernatant was reserved for estimation of total Rap1. The remaining supernatant was mixed with glutathione-agarose and incubated for 1 h at 4C. Samples were then centrifuged at 10,000 for 30 s at 4C, washed three times with lysis buffer, suspended in 40 l of 2 Lammeli buffer made up of 10 mM DTT, and separated by 8C16% SDS-PAGE gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membrane, incubated with polyclonal anti-Rap1 antibodies (Millipore), and visualized by enhanced chemilluminescence (Pierce of Thermo Fisher Scientific, Rockford, IL). [3H]thymidine incorporation. For determination of DNA synthesis, the procedure used by Hornsby and Gill (25) was followed with some modifications. Briefly, 0.5 104 AZF cells were plated in triplicate in 35-mm fibronectin-treated dishes. Medium was changed after 6 h, and cells were incubated either without (control) or with brokers. After 20 h, 10 Ci of [3H]thymidine (10 l of 1 1 mCi/ml stock answer) was added to cultures, and incubation continued for 4 h. Medium was then removed, cells were washed twice with ice-cold PBS, and 1 ml of a 1% aqueous answer of Triton X-100 was added. Cells were incubated at room heat with this answer for 5 min, after which the entire contents from the dish had been used in 10 ml of total ethanol, and insoluble materials was gathered on 2.4-cm glass fiber filters (GF/A; Whatman) with suction. The filter systems had been washed double with 10 ml of total ethanol and used in 10 ml of Scintiverse BD scintillation cocktail and counted utilizing a Beckman Coulter LS AMD3100 (Plerixafor) 6500 scintillation counter. A-kinase assay. PKA activity was assessed having a SignaTECT cAMP-dependent proteins kinase assay package (Promega, Madison, WI) where PKA-dependent phosphorylation of biotinylated peptides could be quantitatively assessed like a function of PKA activity. AZF cells had been plated on 60-mm fibronectin-treated meals in DMEM-F-12+ at a denseness of 3C4 106 cells/dish. After 24 h, the moderate was changed with either control moderate (DMEM-F-12+) or the same moderate including ACTH (1C24) or 8PT-Ade. By the end from the incubation period, cells had been washed four instances with ice-cold PBS and suspended in cool removal buffer [25 mM TrisHCl, pH 7.4, 0.5 mM EGTA, 10 mM -mercaptoethanol, 0.5 mM Pefabloc-SC (Roche Applied Technology, Indianapolis, IN)] and protease inhibitors with EDTA [Complete Mini protease inhibitor cocktail tablet (Roche Applied Technology), 1/10 ml lysis solution]. Lysates had been homogenized utilizing a cool Dounce homogenizer and centrifuged for 5 min at 4C, 14,000 < 0.0001 vs. particular control without cAMP substance using Student's and < 0.0001 vs. control without 8CPT-2-OMe-cAMP using Student's and demonstrates neither 8MeOPT-2-OMe-cAMP (100 M) nor 8HPT-2-OMe-cAMP (100 M) improved cortisol creation by AZF cells sometimes 48 h. On the other hand, by 48 h, 8MeOPT-Ade (50 M) activated a 12-fold upsurge in cortisol synthesis. In additional experiments, we discovered that, at concentrations from 10 to 100 M, 8MeOPT-Ade activated well-correlated, concentration-dependent raises in cortisol synthesis and CYP17 gene manifestation (Fig. 4, and < 0.001; **< 0.0001 vs. particular control using Student's check. The robust excitement of cortisol synthesis and CYP17 mRNA manifestation by 8MeOPT-Ade demonstrates 8-substituted adenine derivatives apart from 8CPT-Ade.