IA-2 modulates dopamine secretion in PC12 cells

Abstract

The secretion of the hormone insulin from beta cells is modulated by the expression of the dense core vesicle transmembrane protein IA-2. Since IA-2 is found in neuroendocrine cells throughout the body, the present experiments were initiated to determine whether the expression of IA-2 also modulates the secretion of neurotransmitters. Using the dopamine-secreting pheochromocytoma cell line PC12, we found that the overexpressions of IA-2 increased the cellular content and secretion of dopamine, whereas the knockdown of IA-2 by siRNA decreased the cellular content and secretion of dopamine. Neither the overexpression nor knockdown of IA-2 influenced the uptake of [H(3)]dopamine by PC12 cells, but did influence the amount of [H(3)]dopamine secreted. Overexpression of IA-2 also increased the level of the dense core vesicle-associated proteins Rab3A, IA-2beta and secretogranin II, whereas the knockdown of IA-2 decreased the level of these proteins. We conclude that the expression of IA-2 profoundly influences the function of dense core vesicles and has a broad modulating effect on the cellular content and secretion of both hormones and neurotransmitters.

Fig. 1

IA-2 overexpression in PC12 cells. (a) Western blot showing overexpression of IA-2. (b) Dopamine content as determined by ELISA in mock-transfected PC12 cells (PC12/mock) and IA-2-transfected PC12 cells (PC12/IA-2). (c) Effect of low (LK) and high (HK) concentrations of K⁺ plus PMA on dopamine secretion in PC12/mock cells and PC12/IA-2 cells. The values are the mean ± SEM of three independent experiments (*, P < 0.05; **, P < 0.01).

Fig. 2

Fraction of dopamine secreted from cells stimulated with low K⁺, high K⁺ or high K⁺ plus PMA. (a) Mock or IA-2-transfected PC12 cells; (b) IA-2-transfected PC12 cells treated with control siRNA or IA-2 siRNA; (c) PC12 cells treated with control siRNA or IA-2 siRNA.

Fig. 3

Treatment of PC12/IA-2 cells and PC12 cells with IA-2 siRNA. Treatment of IA-2 overexpressing PC12 cells (PC12/IA-2) with IA-2 siRNA showing: (a) IA-2 expression by Western blot; (b) dopamine content; and (c) LK, HK and HK plus PMA-induced dopamine secretion. Treatment of PC12 cells with IA-2 siRNA showing: (d) IA-2 expression by Western blot; (e) dopamine content; and (f) LK, HK and HK plus PMA-induced dopamine secretion. The values are the mean ± SEM of three independent experiments (*, P < 0.05; **, P < 0.01).

Fig. 4

[H³]dopamine uptake and secretion by K⁺ stimulation. (a) [H³]dopamine uptake and (c) [H³]dopamine secretion by HK plus PMA as determined by measuring the radioactivity in the supernatant of mock-transfected PC12 cells (PC12/mock) as compared to IA-2-transfected PC12 cells (PC12/IA-2). (b) [H³]dopamine uptake and (d) [H³]dopamine secretion by HK plus PMA as determined by measuring the radioactivity in the supernatant of PC12 cells treated with IA-2 siRNA or control siRNA. The values are the mean ± SEM of triplicate wells in a single experiment, but are representative of three independent experiments (**, P < 0.01).

Fig. 5

Expression of DCV-associate proteins as determined by Western blot. (a) Transfection of PC12 cells with IA-2 (PC12/IA-2) increases the expression of the DCV-associated proteins Rab3, IA-2β, and secretogranin II (SgII). (b) Treatment of nontransfected PC12 cells with IA-2 siRNA decreased the expression of the DCV-associated proteins Rab3, IA-2β and SgII. Tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC) showed no change.