Gi protein-mediated inhibition of hormone secretion in pituitary prolactinomas by somatostatin 5 activation
Objective To explore the potential mechanism of somatostatin receptor 5(SSTR5)activation in inhibiting hormone secretion in pituitary prolactinoma through in vitro experiments.Methods We utilized our developed SSTR5 overexpressing GH3 cell line as an in vitro cell model for pituitary pro-lactinoma.Cells were stimulated with the SSTR5 agonist BIM23052 in combination with inhibitors of down-stream G protein pathways.The activity of the Prl-luc promoter was assessed using a luciferase reporter gene assay,while the concentration of prolactin(PRL)in the cell supernatant was measured using an en-zyme linked immunosorbent assay(ELISA)kit.Statistical analysis between groups was performed using Student's t-test and one-way ANOVA.Results The activity of Prl promoter in GH3SSTR5 cells after pre-treatment with the Gi protein inhibitor pertussis toxin,altered from the control group[Ctrl(Control)group(100.00±6.82)％vs.BIM(BIM23052)group(69.08±7.09)％,t=5.43,P＜0.01]to the interven-tion group[Ctrl group(107.86±12.40)％vs.BIM group(115.51±11.76)％,t=0.76,P＞0.05];the concentration change of PRL in cell supernatant shifted from[Ctrl group(100.00±12.38)％vs.BIM group(70.60±9.12)％,t=3.31,P＜0.05]to[Ctrl group(91.31±9.81)％vs.BIM group(89.91± 9.48)％,t=0.18,P＞0.05]after pertussis toxin pretreatment.These results indicated that pertussis tox-in can eliminate the effect of BIM23052 on Prl promoter activity.Overexpression of beta-ARK activated G beta-gamma subunit,and Prl promoter activity changed from blank transfection group[Ctrl group(100.00±3.27)％vs.BIM group(64.70±2.26)％,t=15.38,P＜0.01]to beta-ARK overexpression group[Ctrl group(96.91±5.36)％vs.BIM group(70.12±6.82)％,t=5.35,P＜0.05],and beta-ARK overexpression did not affect the inhibitory effect of BIM23052 on Prl promoter activity.Pretreatment of GH3SSTR5 cells with the cGMP analogue Rp8-pCPT-cGMPS,the PKG inhibitor KT5823 and PKG inhibi-tor,the PKC broad-spectrum inhibitor Staurosporin,and Gö6983 did not affect the inhibitory effect of BIM23052 on Prl promoter activity,in which the cGMP analogue Rp8-pCPT-cGMPS{ Prl promoter activity altered from the control group[Ctrl group(100.00±3.97)％vs.BIM group(52.98±5.16)％,t=12.5,P＜0.01]to intervention group[Ctrl group(95.99±5.38)％vs.BIM group(60.23±2.63)％,t=10.35,P＜0.01]};PKG inhibitor KT5823 {Prl promoter activity changed from the control group[Ctrl group(100.00±2.50)％vs.BIM group(72.64±0.45)％,t=18.66,P＜0.01]to the intervention group[Ctrl group(93.13±9.31)％vs.BIM group(53.54±11.50)％,t=4.30,P＜0.05]};PKG in-hibitor {control group[Ctrl group(100.00±7.55)％vs.BIM group(64.07±3.49)％,t=7.48,P＜0.01]to intervention group[Ctrl group(96.27±4.89)％vs.BIM group(74.17±1.16)％,t=7.62,P＜0.01]};Staurosporin {different concentration gradients of 0,5,50,100 nmol/L,Prl promoter activity ranged from Ctrl group[(100.00±5.07)％,(102.03±1.08)％,(131.85±4.33)％,(109.62± 7.98)％]to BIM group[(68.35±4.63)％,(74.24±9.64)％,(90.18±8.87)％,(85.24±6.63)％,P＜0.01]};Go6983 {control group[Ctrl group(100.00±7.81)％vs.BIM group(66.09±7.66)％,t=4.79,P＜0.05]to the intervention group[Ctrl group(92.37±5.78)％vs.BIM group(77.31± 1.54)％,t=3.43,P＜0.05]},indicating that PKG inhibitors and PKC inhibitors did not influence the inhibitory effect of BIM23052 on Prl promoter activity.Conclusion The inhibitory effect of SSTR5 activa-tion on PRL synthesis is mediated through Gi alpha,rather than through alternative pathways involving PKG or PKC.
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