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Expression of arachidonic acid-metabolizing cytochrome P450s in human megakaryocytic Dami cells.

Jarrar YB, Shin JG, Lee SJ - In Vitro Cell. Dev. Biol. Anim. (2013)

Bottom Line: Ethoxyresorufin-O-deethylase (EROD) activity was observed in Dami cells, and its activity was significantly decreased after treatment with the P450 inhibitor SKF-525A when compared to the control groups (60% reduction, P < 0.001).Furthermore, 15 ARA metabolites, including 8,9-EET, 14,15-EET, and 20-HETE, were detected by LC-MS/MS in ARA-treated Dami cells, and their levels were decreased with the treatment of the SKF-525A.The present data suggest the possibility that the P450s play a role in the metabolism of ARA and other CYP-related substrates in human megakaryocytes and that P450 expression in megakaryocytic cell lines may predict their existences in platelets with functional activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Inje University, 633-165 Gaegum-dong, Busanjin-gu, Busan, South Korea.

ABSTRACT
Cytochrome P450s (P450s) are involved in the metabolism of arachidonic acid (ARA), and ARA metabolites are associated with various cellular signaling pathways, such as blood hemostasis and inflammation. The present study demonstrates the expression of ARA-metabolizing P450s in the human megakaryocytic Dami cells using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunublotting analysis followed by activity assays using ARA as a substrate. In addition to the previously identified CYP5A1, both protein and mRNAs of CYP1A1, 2U1, and 2J2 bands were detected. Ethoxyresorufin-O-deethylase (EROD) activity was observed in Dami cells, and its activity was significantly decreased after treatment with the P450 inhibitor SKF-525A when compared to the control groups (60% reduction, P < 0.001). CYP1A1 protein expression in Dami cells was induced by 3-methylenecholantheren. This increase in CYP1A1 protein level was correlated with enhanced EROD activity (fourfold increase vs. the control), as well as with increased metabolites, such as 20-hydroxyeicosatrienoic acid (20-HETE), 14, 15-EET (14-,15-epoxyeicosatrienoic acid), and 14, 15-dihydroxyeicosatrienoic acid (14, 15-DHET). The expression of soluble epoxide hydrolase, an enzyme responsible for the synthesis of DHETs from EETs, was confirmed by RT-PCR. Furthermore, 15 ARA metabolites, including 8,9-EET, 14,15-EET, and 20-HETE, were detected by LC-MS/MS in ARA-treated Dami cells, and their levels were decreased with the treatment of the SKF-525A. The present data suggest the possibility that the P450s play a role in the metabolism of ARA and other CYP-related substrates in human megakaryocytes and that P450 expression in megakaryocytic cell lines may predict their existences in platelets with functional activities.

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EROD assays in Dami cells. The intact cells were incubated with 2 μg of 7-ER in a TN assay buffer for 20 min. The amount of resorufin formed was measured at excitation/emission wavelengths of 545/575 nm by comparison with a standard curve of known concentrations of resorufin. Further details are in the “Materials and Methods” section. EROD activity was expressed as nanomoles per minute per milligram of protein. Statistically significant differences as compared with the control group are indicated as ***P < 0.001, based on a two-tailed Student’s t test. Data are presented as the mean ± S.D. of reactions performed in triplicate.
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Fig2: EROD assays in Dami cells. The intact cells were incubated with 2 μg of 7-ER in a TN assay buffer for 20 min. The amount of resorufin formed was measured at excitation/emission wavelengths of 545/575 nm by comparison with a standard curve of known concentrations of resorufin. Further details are in the “Materials and Methods” section. EROD activity was expressed as nanomoles per minute per milligram of protein. Statistically significant differences as compared with the control group are indicated as ***P < 0.001, based on a two-tailed Student’s t test. Data are presented as the mean ± S.D. of reactions performed in triplicate.

Mentions: EROD assays of CYP1A1 were performed with Dami cells. The activity of resorufin formation was 112 ± 10 nmol/min/mg protein. To confirm the activity of P450, 40 μM of the P450 inhibitor SKF-525A was added to the reactions and compared with the non-SKF-525A-treated samples. Figure 2 shows that EROD activity decreased significantly with the addition of SKF-525A to Dami cells (60%, P < 0.001) when compared to the control groups. Because CYP1A1 (this study) and the nuclear Aryl hydrocarbon receptor (AhR) (Lindsey and Papoutsakis 2011) are both expressed in megakaryocytes, we investigated the inducibility of CYP1A1 in Dami cells using 3-MC which is an AhR agonist, after confirming the expression of AhR in Dami cells (Fig. 3A). Immunoblot analysis showed that the CYP1A1 protein level increased in Dami cells after 3-MC treatment (Fig. 3B), and that this increased protein level correlated with the increase in EROD activity (fourfold, P < 0.001) as compared with the DMSO-treated groups (Fig. 3C).Figure 2.


Expression of arachidonic acid-metabolizing cytochrome P450s in human megakaryocytic Dami cells.

Jarrar YB, Shin JG, Lee SJ - In Vitro Cell. Dev. Biol. Anim. (2013)

EROD assays in Dami cells. The intact cells were incubated with 2 μg of 7-ER in a TN assay buffer for 20 min. The amount of resorufin formed was measured at excitation/emission wavelengths of 545/575 nm by comparison with a standard curve of known concentrations of resorufin. Further details are in the “Materials and Methods” section. EROD activity was expressed as nanomoles per minute per milligram of protein. Statistically significant differences as compared with the control group are indicated as ***P < 0.001, based on a two-tailed Student’s t test. Data are presented as the mean ± S.D. of reactions performed in triplicate.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3713264&req=5

Fig2: EROD assays in Dami cells. The intact cells were incubated with 2 μg of 7-ER in a TN assay buffer for 20 min. The amount of resorufin formed was measured at excitation/emission wavelengths of 545/575 nm by comparison with a standard curve of known concentrations of resorufin. Further details are in the “Materials and Methods” section. EROD activity was expressed as nanomoles per minute per milligram of protein. Statistically significant differences as compared with the control group are indicated as ***P < 0.001, based on a two-tailed Student’s t test. Data are presented as the mean ± S.D. of reactions performed in triplicate.
Mentions: EROD assays of CYP1A1 were performed with Dami cells. The activity of resorufin formation was 112 ± 10 nmol/min/mg protein. To confirm the activity of P450, 40 μM of the P450 inhibitor SKF-525A was added to the reactions and compared with the non-SKF-525A-treated samples. Figure 2 shows that EROD activity decreased significantly with the addition of SKF-525A to Dami cells (60%, P < 0.001) when compared to the control groups. Because CYP1A1 (this study) and the nuclear Aryl hydrocarbon receptor (AhR) (Lindsey and Papoutsakis 2011) are both expressed in megakaryocytes, we investigated the inducibility of CYP1A1 in Dami cells using 3-MC which is an AhR agonist, after confirming the expression of AhR in Dami cells (Fig. 3A). Immunoblot analysis showed that the CYP1A1 protein level increased in Dami cells after 3-MC treatment (Fig. 3B), and that this increased protein level correlated with the increase in EROD activity (fourfold, P < 0.001) as compared with the DMSO-treated groups (Fig. 3C).Figure 2.

Bottom Line: Ethoxyresorufin-O-deethylase (EROD) activity was observed in Dami cells, and its activity was significantly decreased after treatment with the P450 inhibitor SKF-525A when compared to the control groups (60% reduction, P < 0.001).Furthermore, 15 ARA metabolites, including 8,9-EET, 14,15-EET, and 20-HETE, were detected by LC-MS/MS in ARA-treated Dami cells, and their levels were decreased with the treatment of the SKF-525A.The present data suggest the possibility that the P450s play a role in the metabolism of ARA and other CYP-related substrates in human megakaryocytes and that P450 expression in megakaryocytic cell lines may predict their existences in platelets with functional activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Inje University, 633-165 Gaegum-dong, Busanjin-gu, Busan, South Korea.

ABSTRACT
Cytochrome P450s (P450s) are involved in the metabolism of arachidonic acid (ARA), and ARA metabolites are associated with various cellular signaling pathways, such as blood hemostasis and inflammation. The present study demonstrates the expression of ARA-metabolizing P450s in the human megakaryocytic Dami cells using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunublotting analysis followed by activity assays using ARA as a substrate. In addition to the previously identified CYP5A1, both protein and mRNAs of CYP1A1, 2U1, and 2J2 bands were detected. Ethoxyresorufin-O-deethylase (EROD) activity was observed in Dami cells, and its activity was significantly decreased after treatment with the P450 inhibitor SKF-525A when compared to the control groups (60% reduction, P < 0.001). CYP1A1 protein expression in Dami cells was induced by 3-methylenecholantheren. This increase in CYP1A1 protein level was correlated with enhanced EROD activity (fourfold increase vs. the control), as well as with increased metabolites, such as 20-hydroxyeicosatrienoic acid (20-HETE), 14, 15-EET (14-,15-epoxyeicosatrienoic acid), and 14, 15-dihydroxyeicosatrienoic acid (14, 15-DHET). The expression of soluble epoxide hydrolase, an enzyme responsible for the synthesis of DHETs from EETs, was confirmed by RT-PCR. Furthermore, 15 ARA metabolites, including 8,9-EET, 14,15-EET, and 20-HETE, were detected by LC-MS/MS in ARA-treated Dami cells, and their levels were decreased with the treatment of the SKF-525A. The present data suggest the possibility that the P450s play a role in the metabolism of ARA and other CYP-related substrates in human megakaryocytes and that P450 expression in megakaryocytic cell lines may predict their existences in platelets with functional activities.

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