Neurite Outgrowth Stimulated by Oxytocin Is Modulated by Inhibition of the Calcium Voltage-Gated Channels

Abstract

Neuropeptide oxytocin contributes to the regulation of the neuron differentiation and cell morphology. However, the precise mechanisms are not yet fully understood. Oxytocin receptor function and its coupling to calcium entry are obvious objects of interest in relation to the neuron morphology. Postsynaptic scaffolding proteins including SHANK proteins interact with other synaptic molecules and change dendritic morphology. SH-SY5Y neuroblastoma cell line represents a useful neurobiological in vitro model to study the short-term oxytocin effects on neurite outgrowth and underlying mechanisms. In the present study, we show that oxytocin induces an increase in the intracellular calcium in SH-SY5Y cells. Specificity of the calcium influx was verified by blockade of the oxytocin receptors with oxytocin receptor antagonist L-371,257. Neurite outgrowth stimulated by oxytocin was inhibited by specific voltage-gated calcium channel blockers. The exposure of SH-SY5Y cells to oxytocin resulted in a significant increase in the gene expression of SHANK1 and SHANK3 proteins. Overall, the present data indicate that oxytocin may contribute to the regulation of scaffolding proteins expression known to be associated with clusters of calcium channels at the cell membrane. It appears that oxytocin stimulated neurite outgrowth is, at least, in part dependent on the voltage-gated calcium channels.

Keywords: Calcium; Development; Neurite outgrowth; Oxytocin receptor; SHANK.

Fig. 1

Changes of intracellular calcium after stimulation with 1 µM oxytocin in SH-SY5Y cells incubated with or without oxytocin receptor antagonist L-371,257 (1 µM). After the dye incubation, intracellular calcium was measured in cells by plate reader. Cells were excited with wavelengths of 340 and 380 nm, and the ratio of fluorescence emitted at 505 nm was analyzed after subtraction of background fluorescence levels. Baseline fluorescence was recorded for 210 s followed by stimulation of calcium influx by addition of 1 μM oxytocin. Data are represented as mean of 5 independent measurements

Fig. 2

Changes of intracellular calcium after stimulation with 1 µM oxytocin in SH-SY5Y cells incubated with or without calcium channels inhibitors or BAPTA/AM. After the dye incubation, intracellular calcium was measured in cells by plate reader. Cells were excited with wavelengths of 340 and 380 nm, and the ratio of fluorescence emitted at 505 nm was analyzed after subtraction of background fluorescence levels. Cells were incubated with calcium channel inhibitors (Table 1) for 2 h before calcium measurement. Baseline fluorescence was recorded for 210 s followed by stimulation of calcium influx by addition of 1 μM oxytocin. Data are represented as mean of 5 independent measurements

Fig. 3

Changes of neurite length in SH-SY5Y cells incubated with 1 µM oxytocin and in combination with different inhibitors of calcium channels or BAPTA/AM. Neurite outgrowth was determined by manually tracing the length of the longest neurite per cell (using Image J software) after incubation with 1 μM oxytocin (OXT), 10 μM isradipine, 10 μM nifedipine, 2 μM mibefradil, 100 nM ω-agatoxin, and 10 μM BAPTA/AM for 48 h. Length of the cell neurite was evaluated randomly by three independent members of the research team. Data were pooled and statistically evaluated. Means are represented on bars ± SEM (n = 44–105 cells). Significantly different values are marked with *p < 0.05, ***p < 0.001 compared with control and ^### p < 0.001 compared with OXT group

Fig. 4

Changes in gene expression of SHANK1 protein in SH-SY5Y cells incubated in the presence of 1 µM oxytocin and/or 1 µM oxytocin receptor antagonist L-371,257. Graphs show relative mRNA expression measured by qPCR, normalized to GAPDH and calculated by 2^−ΔΔCt Livak method (Livak and Schmittgen 2001). Means are represented on bars ± SEM (n = 5). Significantly different values are marked with **p < 0.01 compared with control group

Fig. 5

Changes in gene expression of SHANK2 protein in SH-SY5Y cells incubated in the presence of 1 µM oxytocin and/or 1 µM oxytocin receptor antagonist L-371,257. Graphs show relative mRNA expression measured by qPCR, normalized to GAPDH and calculated by 2^−ΔΔCt Livak method (Livak and Schmittgen 2001). Means are represented on bars ± SEM (n = 5). Significantly different values are marked with *p < 0.05 or **p < 0.01 compared with control group

Fig. 6

Changes in gene expression of SHANK3 protein in SH-SY5Y cells incubated in the presence of 1 µM oxytocin and/or 1 µM oxytocin receptor antagonist L-371,257. Graphs show relative mRNA expression measured by qPCR, normalized to GAPDH and calculated by 2^−ΔΔCt Livak method (Livak and Schmittgen 2001). Means are represented on bars ± SEM (n = 5). Significantly different values are marked with **p < 0.01 compared with control group