4 RESULTS
4.2 INHIBITION OF CORTISOL RELEASE FROM NCI-H295R CELLS
4.2 INHIBITION OF CORTISOL RELEASE FROM NCI-H295R CELLS
The involvement of OATs in cortisol release has been tested so far in bovine and rat, but not in human adrenals. We investigated the inhibition of cortisol release from NCI-H295R cells by probenecid, a potent inhibitor of OAT family, and by PAH and glutarate, model substrates of OAT1. The cells were pre-stimulated for 24 h with 10 µM forskolin. After washing the cells with warm PBS, cells were incubated with medium containing 10 µM forskolin with or without inhibitory substances (probenecid or PAH or glutarate). At the end of 24 h incubation period, medium samples were collected to determine the inhibitory effect of the substances on the cortisol release.
The rate of cortisol release in response to forskolin and the inhibition by different concentrations of test substances varied considerably among cultures. However, an inhibition of secretion was always visible.
The involvement of OATs in cortisol release has been tested so far in bovine and rat, but not in human adrenals. We investigated the inhibition of cortisol release from NCI-H295R cells by probenecid, a potent inhibitor of OAT family, and by PAH and glutarate, model substrates of OAT1. The cells were pre-stimulated for 24 h with 10 µM forskolin. After washing the cells with warm PBS, cells were incubated with medium containing 10 µM forskolin with or without inhibitory substances (probenecid or PAH or glutarate). At the end of 24 h incubation period, medium samples were collected to determine the inhibitory effect of the substances on the cortisol release.
The rate of cortisol release in response to forskolin and the inhibition by different concentrations of test substances varied considerably among cultures. However, an inhibition of secretion was always visible.
4.2.1 Inhibition of cortisol release by probenecid in NCI-H295R cells
4.2.1 Inhibition of cortisol release by probenecid in NCI-H295R cells
Probenecid inhibits the transport activity through OATs. In the presence of 0.25 mM Probenecid inhibits the transport activity through OATs. In the presence of 0.25 mM
Figure 4.3 Concentration-dependent inhibition of cortisol release by probenecid in forskolin pre-stimulated NCI-H295R cells. The cells were incubated for 24 h with 10 µM forskolin, washed, and incubated again with medium containing 10 µM forskolin, in the presence or absence of different concentrations of probenecid. The cortisol content of the medium was determined and normalized to the protein concentration. Data points are the means ± SEM of values from four different experiments. Each column is calculated as a percentage of cortisol release in forskolin stimulated cells not exposed to probenecid. The significance was performed by student’s t-test against control cells (***, P < 0.0001).
Figure 4.3 Concentration-dependent inhibition of cortisol release by probenecid in forskolin pre-stimulated NCI-H295R cells. The cells were incubated for 24 h with 10 µM forskolin, washed, and incubated again with medium containing 10 µM forskolin, in the presence or absence of different concentrations of probenecid. The cortisol content of the medium was determined and normalized to the protein concentration. Data points are the means ± SEM of values from four different experiments. Each column is calculated as a percentage of cortisol release in forskolin stimulated cells not exposed to probenecid. The significance was performed by student’s t-test against control cells (***, P < 0.0001).
probenecid, the cortisol release was 97.0 ± 5.5%, while 0.5 mM and 1 mM probenecid significantly inhibited the cortisol secretion to 64.0 ± 2.4% (P < 0.0001), and 37.1 ± 1.8% (P < 0.0001), respectively, compared to the forskolin stimulated control cells not exposed to probenecid (100 ± 2.6%) as shown in figure 4.3.
probenecid, the cortisol release was 97.0 ± 5.5%, while 0.5 mM and 1 mM probenecid significantly inhibited the cortisol secretion to 64.0 ± 2.4% (P < 0.0001), and 37.1 ± 1.8% (P < 0.0001), respectively, compared to the forskolin stimulated control cells not exposed to probenecid (100 ± 2.6%) as shown in figure 4.3.
4.2.2 Inhibition of cortisol release by PAH in NCI-H295R cells 4.2.2 Inhibition of cortisol release by PAH in NCI-H295R cells
PAH is a model substrate of several OATs. Inhibition of cortisol release by PAH was not effective at 0.01 mM concentration, showing 100.5 ± 2.6% as compared to forskolin stimulated control NCI-H295R cells (100 ± 2.6%). A significant reduction of cortisol secretion was observed by 0.1 mM, 1 mM, and 5 mM PAH concentrations, lowering the cortisol secretion to 72.4 ± 5.1% (P < 0.0001), 67.2 ± 6.1% (P < 0.0001), and 36.0 ± 4.3% (P < 0.0001), respectively, as compared to control 100% ± 2.6%(Figure 4.4).
PAH is a model substrate of several OATs. Inhibition of cortisol release by PAH was not effective at 0.01 mM concentration, showing 100.5 ± 2.6% as compared to forskolin stimulated control NCI-H295R cells (100 ± 2.6%). A significant reduction of cortisol secretion was observed by 0.1 mM, 1 mM, and 5 mM PAH concentrations, lowering the cortisol secretion to 72.4 ± 5.1% (P < 0.0001), 67.2 ± 6.1% (P < 0.0001), and 36.0 ± 4.3% (P < 0.0001), respectively, as compared to control 100% ± 2.6%
(Figure 4.4).
Figure 4.4 Concentration-dependent inhibition of cortisol release by PAH in forskolin pre-stimulated NCI-H295R cells. The cells were incubated for 24 h with 10 µM forskolin, washed, and incubated again with medium containing 10 µM forskolin, in the presence or absence of different concentrations of PAH. The cortisol content of the medium was determined and normalized to the protein concentration. Each column is calculated as a percentage value of cortisol release in forskolin stimulated cells not exposed to PAH. Data points are the means ± SEM of values from four different experiments. The significance was performed by student’s t-test against control cells (***, P < 0.0001).
Figure 4.4 Concentration-dependent inhibition of cortisol release by PAH in forskolin pre-stimulated NCI-H295R cells. The cells were incubated for 24 h with 10 µM forskolin, washed, and incubated again with medium containing 10 µM forskolin, in the presence or absence of different concentrations of PAH. The cortisol content of the medium was determined and normalized to the protein concentration. Each column is calculated as a percentage value of cortisol release in forskolin stimulated cells not exposed to PAH. Data points are the means ± SEM of values from four different experiments. The significance was performed by student’s t-test against control cells (***, P < 0.0001).
4.2.3 Inhibition of cortisol release by glutarate in NCI-H295R cells 4.2.3 Inhibition of cortisol release by glutarate in NCI-H295R cells
OAT1 and OAT3 work in the exchanger mode (Bakhiya et al. 2003; Burckhardt et al.2003). Both take up OA in exchange of dicarboxylates especially α-ketoglutarate. We checked the effect of glutarate on the cortisol secretion from the cells. At a concentration of 250 µM glutarate inhibits 31% of the cortisol release from NCI-H295R cells i.e., the total uptake was reduced to 68.9 ± 5.6% (P < 0.001) as compared to the forskolin stimulated control (100% ± 2.6%)
OAT1 and OAT3 work in the exchanger mode (Bakhiya et al. 2003; Burckhardt et al.
2003). Both take up OA in exchange of dicarboxylates especially α-ketoglutarate. We checked the effect of glutarate on the cortisol secretion from the cells. At a concentration of 250 µM glutarate inhibits 31% of the cortisol release from NCI-H295R cells i.e., the total uptake was reduced to 68.9 ± 5.6% (P < 0.001) as compared to the forskolin stimulated control (100% ± 2.6%)
Figure 4.5 Inhibition of cortisol release by glutarate in forskolin pre-stimulated NCI-H295R cells. The cells were incubated for 24 h with 10 µM forskolin, washed, and incubated again with medium containing 10 µM forskolin, in the presence or absence of different concentrations of glutarate. The cortisol content of the medium was determined by RIA and normalized to the protein concentration. Each column is calculated as a percentage value of cortisol release in forskolin stimulated cells not exposed to glutarate. Data points are the means ± SEM of values from three different experiments. The significance was performed by student’s t-test against control cells (**, P < 0.001).
Figure 4.5 Inhibition of cortisol release by glutarate in forskolin pre-stimulated NCI-H295R cells. The cells were incubated for 24 h with 10 µM forskolin, washed, and incubated again with medium containing 10 µM forskolin, in the presence or absence of different concentrations of glutarate. The cortisol content of the medium was determined by RIA and normalized to the protein concentration. Each column is calculated as a percentage value of cortisol release in forskolin stimulated cells not exposed to glutarate. Data points are the means ± SEM of values from three different experiments. The significance was performed by student’s t-test against control cells (**, P < 0.001).
4.2.4 Inhibition of cortisol release by cimetidine in NCI-H295R cells
4.2.4 Inhibition of cortisol release by cimetidine in NCI-H295R cells
The presence of 0.05 mM cimetidine in the culture medium did not significantly reduce cortisol release from NCI-H295R cells (Figure 4.6). The inhibition of cortisol release by 0.1 mM and 0.5 mM was 8.7 ± 5.9% (P < 0.001) and 56.2 ± 4.6% (P <
0.0001), respectively, as compared to forskolin stimulated control cells not exposed to cimetidine (100%).
The presence of 0.05 mM cimetidine in the culture medium did not significantly reduce cortisol release from NCI-H295R cells (Figure 4.6). The inhibition of cortisol release by 0.1 mM and 0.5 mM was 8.7 ± 5.9% (P < 0.001) and 56.2 ± 4.6% (P <
0.0001), respectively, as compared to forskolin stimulated control cells not exposed to cimetidine (100%).
Figure 4.6 Concentration-dependent inhibition of cortisol release from forskolin pre-stimulated NCI-H295R cells by cimetidine. The cells were incubated for 24 h with 10 µM forskolin, washed, and incubated again with medium containing 10 µM forskolin, in the presence or absence of different concentrations of cimetidine. The cortisol content of the medium was determined and normalized to the protein concentration. Each column is calculated as a percentage value of cortisol release in forskolin stimulated cells not exposed to cimetidine. Data points are the means ± SEM of values from two different experiments. The significance was performed by student’s t-test against control cells (**, P < 0.005; ***, P <
0.0001).
Figure 4.6 Concentration-dependent inhibition of cortisol release from forskolin pre-stimulated NCI-H295R cells by cimetidine. The cells were incubated for 24 h with 10 µM forskolin, washed, and incubated again with medium containing 10 µM forskolin, in the presence or absence of different concentrations of cimetidine. The cortisol content of the medium was determined and normalized to the protein concentration. Each column is calculated as a percentage value of cortisol release in forskolin stimulated cells not exposed to cimetidine. Data points are the means ± SEM of values from two different experiments. The significance was performed by student’s t-test against control cells (**, P < 0.005; ***, P <
0.0001).