Figure 1.
Changes in D2 receptor protein levels in lactotropic cells expressing D2S receptor isoform (D2S), D2L receptor isoform (D2L) or undetectable D2 receptors (V) and in enriched lactotropes (E–LT) following treatment with ethanol for a period of 24 h.
The level of D2 receptor protein in all cells was measured by using 25 mg of cellular proteins of each cell in Western blots. Actin was used as control housekeeping protein. In each column of the figure, representative blots were shown on the top and mean ± SEM values of D2 receptor protein and actin ratios were presented as histograms on the bottom. N = 4. *,***, P<0.05, P<0.001, respectively as compared to control (C)-treated group within a cell type. a, P<0.05, significantly different from the rest of the groups.
Figure 2.
Effect of ethanol on cellular levels of PRL (A–D), PRL gene transcription (E–H) and cell proliferation as measured by [3H]thymidine incorporation (I–L) in enriched lactotropes (E-LT; A, E, I) or lactotropic cells expressing D2S receptor isoform (D2S; B, F, J), D2L receptor isoform (D2L; C, G, K) or undetectable D2 receptors (V; D, H, L) cells following treatment with different doses of ethanol for a period of 24 h.
PRL gene transcription was measured by luciferase reporter activity. Cellular levels of PRL weremeasured by ELISA. Lactotropic cell proliferation was determined by measuring the [3H]thymidine incorporation into cells. Data are mean ± SEM values from 3–4 independent experiments. *, **,***, P<0.05, P<0.01, P<0.001, respectively as compared to “0”- dose- (vehicle)-treated group. a, P<0.05, significantly different from the “12.5 mM”- dose- treated group. bP<0.05, significantly different from the “25 mM”- dose- treated group.
Figure 3.
Effects of ethanol on cellular levels of G proteins (Gs, Gq11 and Gi3) in D2S (A, D, G), D2L (B, E, H) and V (C, F, I) cells following treatment with different doses of ethanol (0–50 mM) for a period of 24 h.
Quantitation of G protein levels was done using Western Blot and actin was used as a control housekeeping protein. Data were presented in gel blots as well as the ratio of the protein/actin values as histograms. Data are mean ± SEM values of 4 cultures. *, **,***, P<0.05, P<0.01, P<0.001, respectively as compared to “0”- dose- (vehicle)-treated group. a, P<0.05, significantly different from the “12.5 mM”- dose- treated group.
Figure 4.
Effects of dopamine and pertussis toxin on ethanol modulated G proteins expression in D2S (A, D, G), D2L (B, E, H) and V (C, F, I) cells.
Cells were treated with ethanol (EtOH; 50 mM) alone or with dopamine (DA; 5 µM) or PTX (100 ng/ml) for a period of 24 h. Quantitation of G protein levels was done using Western Blot and actin was used as a control housekeeping protein. Data were presented in gel blots as well as the ratio of the protein/actin values as histograms. Data are mean ± SEM values of 4 cultures. *, **,***, P<0.05, P<0.01, P<0.001, respectively as compared to the control (C) group. a, P<0.05, significantly different from PTX alone-treated group.
Figure 5.
Effects of dopamine and pertussis toxin on ethanol modulated cellular levels of PRL and cell proliferation in D2S (A, D, G), D2L (B, E, H) and V cells (C, F, I).
Cells were treated with the dopamine (DA) or pertussis toxin (PTX) as described in the legend of Fig. 4. Cellular levels of PRL were determined by Western blots (A–C) or by ELISA (D–F). Cell proliferation studies were conducted by measuring the [3H]thymidine incorporation into cells (G–I). N = 4–9. *, **,***, P<0.05, P<0.01, P<0.001, respectively as compared to the vehicle-treated group (C). a, P<0.05, significantly different from the EtOH only treatment.
Figure 6.
Effect of knock-down of Gi3 proteins by siRNA on ethanol modulated cellular levels of G proteins and PRL in D2S (A, C, E, G) and D2L cells (B, D, F, H).
Cells treated with control or 50 mM ethanol (EtOH) were co-incubated with or without siRNA for Gi3 for 24 h. Cellular levels of Gi3 (A, B), Gs (C, D) and PRL (E–F) were determined by Western blots and Actin as a control housekeeping protein. Cellular levels of PRL were verified by ELISA (G, H). N = 4–6. ***, P<0.001, *, P<0.05, significantly different from the vehicle-treated group (Cont). a, P<0.001, significantly different from siRNA only treatment. b, P<0.001, significantly different from ethanol only treatment.
Figure 7.
Effect of knock-down of Gs proteins by siRNA on ethanol modulated cellular levels of G proteins and PRL in D2S (A, C, E, G) and D2L cells (B, D, F, H).
Cells treated with control or 50 mM ethanol (EtOH) were co-incubated with or without siRNA for Gs for 24 h. Cellular levels of Gs (A, B), Gi3 (C, D) and PRL (E, F) were determined by Western blots and Actin as a control housekeeping protein. Cellular levels of PRL were verified by ELISA (G, H). N = 4–6. ***, P<0.001, *, P<0.05, significantly different from the vehicle-treated group (Cont). a, P<0.001, significantly different from the siRNA only treatment. b, P<0.001, significantly different from ethanol only treatment.
Figure 8.
A diagram summarizing the postulated role of D2 receptor signaling in mediation of ethanol action on PRL production and lactotropic cell proliferation.
It is hypothesized that ethanol action on PRL production and cell proliferation possibly involves the downregulation of the D2S-coupled-PTX-sensitive Gi3 protein that activates Gs protein-mediated signaling to stimulate PRL production and cell proliferation in lactotropes. Furthermore, by increasing the production of D2L, ethanol may decouple the D2 receptor influence on Gi3 and Gs interaction and thereby reduce the dopaminergic inhibitory control over PRL production and cell proliferation.