Activation of ryanodine receptors induces calcium influx in a neuroblastoma cell line lacking calcium influx factor activity

Abstract

We have further characterized the Ca2+ signalling properties of the NG115-401L (or 401L) neuroblastoma cell line, which has served as an important cell line for investigating SOC (store-operated channel) influx pathways. These cells possess an unusual Ca2+ signalling phenotype characterized by the absence of Ca2+ influx when Ca2+ stores are depleted by inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). Previous studies found that Ca2+-store depletion does not produce a CIF (Ca2+ influx factor) activity in 401L cells. These observations have prompted the question whether 401L cells possess the signalling machinery that permits non-voltage-gated Ca2+ influx to occur. We tested the hypothesis that ryanodine-sensitive Ca2+ pools and activation of RyRs (ryanodine receptors) constitute a signalling pathway capable of inducing Ca2+ influx in 401L cells. We found that 401L cells express mRNA for RyR1 and RyR2 and that RyR activators induced Ca2+ release. Activation of RyRs robustly couples with Ca2+ influx responses in 401L cells, in sharp contrast with absence of Ca2+ influx when cells are treated with SERCA inhibitors. Thus it is clear that 401L cells, despite lacking depletion-induced Ca2+ influx pathways, express the functional components of a Ca2+ influx pathway under the control of RyR function. These findings further support the importance of the 401L cell line as an important cell phenotype for deciphering Ca2+ influx regulation.

Figure 1. Ca²⁺ influx is induced by ryanodine but not by CPA in NG115-401L cells

(A) The addition of CPA (50 μM) in the presence of extracellular Ca²⁺ (1.8 mM) increased the F₃₄₀/F₃₈₀ fluorescence ratio, but failed to activate Ca²⁺ influx when extracellular Ca²⁺ levels were increased to 10 mM. (B) 401L cells were incubated in a Ca²⁺-free medium (0 Ca²⁺ HBSS) and stimulated with ryanodine (1 μM). After the decay of the ryanodine response, Ca²⁺ (1 mM) was added back to the cells and responses were tested for sensitivity to Ni²⁺ (1 mM). (C) Cells stimulated as in (B) were tested for sensitivity to EGTA (5 mM) to determine their dependence on extracellular Ca²⁺. (D) Ba²⁺ (1 mM) was added to 401L cells stimulated with ryanodine (1 μM) in Ca²⁺-free HBSS. Arrows indicate the approximate time points of addition of the various agents. Bars indicate different treatment methods for bivalent ion exposure.

Figure 2. Common pharmacological activators of RyRs induce Ca²⁺ influx in NG115-401L cells

(A) 401L cells were treated with PCB95 (10 μM) in Ca²⁺-free HBSS, followed by challenge with Ca²⁺ (1 mM) and exposure to Ni²⁺ (1 mM). (B) In a Ca²⁺-containing (1.8 mM) medium, CMC (250 μM; solid trace) induced [Ca²⁺]_i release and activated sustained Ca²⁺ entry responses. The addition of high Ca²⁺ concentrations (10 mM) further increased the Ca²⁺ influx. CMC-induced Ca²⁺ influx was inhibited by La³⁺ (100 μM). In a Ca²⁺-free medium, CMC (250 μM, broken trace) induced a transient F₃₄₀/F₃₈₀ fluorescence peak. Restoration of external Ca²⁺ (1.2 mM) induced a Ca²⁺ influx response that was completely inhibited by Ni²⁺ (1 mM) treatment. (C) The RyR activator caffeine (40 mM) induced Ca²⁺ release in 401L cells in the presence of extracellular Ca²⁺ (1.8 mM). Caffeine treatment resulted in Ca²⁺ influx responses when high Ca²⁺ (10 mM) was added to the cells. Caffeine-induced Ca²⁺ influx responses were inhibited by Zn²⁺ (1 mM). Arrows indicate the approximate time points of addition of the various agents. Bars indicate different treatment methods for bivalent ion exposure.

Figure 3. Relationship of the RyR-gated and thapsigargin-sensitive Ca²⁺ pools in NG115-401L cells

(A) In a Ca²⁺-free medium (0 Ca²⁺ HBSS), addition of thapsigargin (TG, 1 μM) induced a [Ca²⁺]_i transient that rapidly decayed to pre-stimulus levels. Subsequent addition of CMC (500 μM) failed to stimulate an increase in [Ca²⁺]_i. (B) Response of 401L cells to ryanodine (1 μM) after stimulation with TG (1 μM) in Ca²⁺-free media. (C) The addition of ryanodine (1 μM) to 401L cells in a Ca²⁺-free medium induced a [Ca²⁺]_i transient reflecting release from intracellular Ca²⁺ stores. Subsequent addition of TG (1 μM) failed to cause an increase in [Ca²⁺]_i. (D) As in (A, B), addition of TG (1 μM) in a Ca²⁺-free medium elicited a [Ca²⁺]_i transient that returned to pre-stimulus levels. Subsequent addition of CMC (500 μM) failed to induce a response. The subsequent addition of ionomycin (2 μM) induced an increase of [Ca²⁺]_i in 401L cells. Arrows indicate the approximate time points of addition of the various agents.

Figure 4. ATP induces Ca²⁺ influx in NG 115-401L cells

(A) In a Ca²⁺-containing (1.8 mM) medium ATP (100 μM) mobilized Ca²⁺ from intracellular stores. Further addition of high Ca²⁺ concentrations (5 mM) to the medium increased the [Ca²⁺]_i responses. The ATP-induced Ca²⁺ influx responses were inhibited by Ni²⁺ (1 mM). (B) In a Ca²⁺-free medium, thapsigargin (TG, 1 μM) increased [Ca²⁺]_i. Subsequent addition of ATP (100 μM) failed to increase [Ca²⁺]_i. Restoration of Ca²⁺ (1.2 mM) to the cells induced Ca²⁺ influx responses. (C) Similar results were obtained by reversing the order of addition in (B); TG (1 μM) failed to increase [Ca²⁺]_i following Ca²⁺ release induced by ATP (100 μM). Adding back Ca²⁺ (1.2 mM) stimulated Ca²⁺ influx in the 401L cells. Arrows indicate the approximate time points of addition of the various agents.