Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Abstract

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [(3)H]NE uptake capacity, an effect characterized by a robust decrease in the V(max) of the transport of [(3)H]NE. As expected, reserpine did not displace the binding of [(3)H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [(3)H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [(3)H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca(2+)/Ca(2+)-calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [(3)H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, alpha-methyl-p-tyrosine, increased [(3)H]NE uptake and eliminated the inhibitory effects of reserpine on [(3)H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca(2+)-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.

Figure 1

Time course of the effect of reserpine exposure on [³H]NE uptake in PC12 cells. Cells were exposed to reserpine (50 nM) and uptake was initiated after washing at different time points following exposures, as indicated under the bars. Asterisks indicates statistical significance (n=3 experiments performed on separate days; p < 0.01), comparing data to control values generated without reserpine exposure.

Figure 2

Effect of reserpine exposure on [³H]NE transport kinetics in PC12 cells. Data from control (empty symbols, no treatment) and reserpine exposure (filled symbols) conditions were analyzed by non-linear regression analyses (n=3 experiments performed on separate days). Cells were treated with 50 nM reserpine for 30 min prior to measurement of [³H]NE uptake. Reserpine exposure produced a robust decrease in transport capacity (V_max) with a modest decrease in K_m (see Results).

Figure 3

Effect of Ca²⁺ on the reserpine-induced decrease in [³H]NE uptake in PC12 cells. Ca²⁺-chelating agents (BAPTA/AM and BAPTA) were also present during reserpine (Res) treatment. Cells were treated with 50 nM reserpine for 30 min prior to measurement of [³H]NE uptake. Asterisks above bars indicate significant differences as compared to control values (measured in the absence of drugs), and specific comparisons between groups <that were significant> are noted above bars by lined demarcations ( n=2 experiments performed on separate days, with differences between the two experiments varying <5%).

Figure 4

Effect of CaMK activity on reserpine (Res) exposure-induced decrease in [³H]NE uptake in PC12 cells. Cells were pretreated with 10 μM KN93 or 10 μM KN92 (inactive analog) for 30 min prior to reserpine treatment and these compounds were also present during reserpine treatment. Cells were treated with 50 nM reserpine for 30 min prior to measurement of [³H]NE uptake. Asterisks above bars indicate significant differences as compared to control values (measured in the absence of drugs), and specific comparisons between groups <that were significant> are noted above bars by lined demarcations ( n=2 experiments performed on separate days, with differences between the two experiments varying <10%).

Figure 5

Effect of cAMP/PKA activity on reserpine (Res) exposure-induced decrease in [³H]NE uptake in PC12 cells. PC12 cells were treated with 2 mM 8-bromo-cAMP, or 100 nM staurosporine (stauro = staurosporine) prior to reserpine treatment. cAMP/PKA-regulating agents were also present during reserpine treatment. Cells were treated with 50 nM for 30 min prior to measurement of [³H]NE uptake. Asterisks above bars indicate significant differences as compared to control values (measured in the absence of drugs), and specific comparisons between groups <that were significant> are noted above bars by lined demarcations (n=3 experiments performed on separate days).

Figure 6

Effect of PKC activity on the reserpine (Res) exposure-induced decrease in [³H]NE uptake in PC12 cells. PC12 cells were treated with 50 nM PMA or 150 nM staurosporine (stauro = staurosporine) prior to reserpine exposure. PKC-regulating agents were also present during reserpine exposure. Cells were treated with 50 nM for 30 min prior to measurement of [³H]NE uptake. Asterisks above bars indicate significant differences as compared to control values (measured in the absence of drugs), and specific comparisons between groups <that were significant> are noted above bars by lined demarcations (n=3 experiments performed on separate days).

Figure 7

Effect of cGMP/PKG activity on reserpine (Res) exposure-induced decrease in [³H]NE uptake in PC12 cells. Cells were pretreated with 2 mM 8-Br-cGMP (8-bromo-cGMP), or 1 μM KT5823 (KT = KT5823) for 30 min prior to reserpine treatment. PKG-regulating agents were also present during reserpine treatment. Cells were treated with 50 nM reserpine for 30 min prior to measurement of [³H]NE uptake. Asterisks above bars indicate significant differences as compared to control values (measured in the absence of drugs), and specific comparisons between groups <that were significant> are noted above bars by lined demarcations (n=3 experiments performed on separate days).

Figure 8

Non-linear regression analysis of [³H]nisoxetine binding to whole PC12 cells following 30 min exposure to reserpine. Filled circles with a dashed non-linear regression line represent reserpine exposure and empty circles with a solid non-linear regression line represent control conditions (no reserpine). Cells were exposed to 50 nM reserpine prior to initiating the binding assay. B_max and K_D values of [³H]nisoxetine binding to surface NET are presented in Table 1 (n=3 experiments performed on separate days).

Figure 9

Effect of reserpine on [³H]NE uptake in three cell lines with varying amounts of catecholamine storage vesicles (PC12, high levels; SK-N-SH, low levels; 293-hNET, no storage vesicles). Cells were exposed to reserpine for 30 min exposure prior to measurement of [³H]NE uptake (n=3 experiments performed on separate days).

Figure 10

Effect of reserpine exposure on [³H]NE release from PC12 cells (A) and SK-N-SH cells (B). PC12 and SK-N-SH cells were treated with 50 nM and 5 μM reserpine, respectively, followed by the measurement of [³H]NE release after different time intervals. Asterisks above bars indicate significant differences as compared to control values (measured in the absence of reserpine; * P<0.05, **P<0.01; n=2 experiments performed on separate days, with differences between the two experiments varying <10%).

Figure 11

Effect of AMPT treatment on [³H]NE uptake in PC12 cells. PC12 cells were treated with AMPT or vehicle for 24 h, and then exposed to reserpine (50 nM) or vehicle 30 min prior to measuring [³H]NE uptake. Data were calculated as a percentage of control values determined with vehicle exposures (n=8; experiments performed on 3 separate days). Asterisks indicate significant differences from control values and lines above bars denote significant differences between designated groups.