Complex relationship between Ins(1,4,5)P3 accumulation and Ca2+ -signalling in a human neuroblastoma reveled by cellular differentiation

Abstract

1. Differentiation of SH-SY5Y neuroblastoma cells induces morphological and biochemical changes consistent with a more neuronal phenotype. These cells may therefore provide a model for studying phenomena such as signal transduction in a neuronal context whilst retaining the advantages of a homogenous cell population expressing a well characterized array of G-protein coupled receptors. 2. This study examined the effects of differentiating SH-SY5Y cells on muscarinic- and bradykinin-receptor-mediated phosphoinositide and Ca2+ signalling. Retinoic acid (10 microM, 6 days) along with a lowered serum concentration produced phenotypic changes consistent with differentiation including reduced proliferation and increased neurite outgrowth. 3. Differentiation increased the magnitude and potency of rapid Ins(1,4,5)P3 responses to a full muscarinic receptor agonist. Bradykinin receptor-mediated Ins(1,4,5)P3 signalling was also potentiated following differentiation. Determination of agonist-evoked accumulation of [3H]-inositol phosphates under lithium-block demonstrated these changes reflected enhanced phospholipase C activity which is consistent with observed increases in the expression of muscarinic and bradykinin receptors. 4. Despite the marked alterations in Ins(1,4,5)P3 signalling following differentiation, elevations of intracellular [Ca2+] were totally unaltered. Thus, in SH-SY5Y cells, the relationship between the elevations of Ins(1,4,5)P3 and intracellular [Ca2+] is agonist dependent and affected by the state of differentiation. This demonstrates that mechanisms other than the measured increase in Ins(1,4,5)P3 regulate the elevation of intracellular [Ca2+].

Figure 1

Time courses of agonist-mediated Ins(1,4,5)P₃ accumulation. Adherent populations of SH-SY5Y or SH-SY5Y_RA6 cells were challenged with 1 mM methacholine or 10 μM bradykinin for the indicated times. Data are means±s.e.mean, n=3.

Figure 2

Concentration-response relationships of agonist-mediated Ins(1,4,5)P₃ accumulation. Methacholine-mediated peak changes in Ins(1,4,5)P₃ mass in adherent SH-SY5Y or SH-SY5Y_RA6 cells. Data are mean±s.e.mean, n=3. EC₅₀ values (log₁₀ M, mean±s.e.mean) for methacholine were −4.96±0.03 (11 μM) and −5.36±0.07 (4.4 μM) in SH-SY5Y and SH-SY5Y_RA6 cells respectively (P<0.01).

Figure 3

Time courses of agonist-mediated accumulation of [³H]-InsPs in cells in which inositol monophosphatase activity was blocked with 10 mM Li⁺. (a) SH-SY5Y or SH-SY5Y_RA6 cells were challenged with 1 mM methacholine for the indicated times. (b) SH-SY5Y or SH-SY5Y_RA6 cells were challenged with 10 μM bradykinin for the indicated times. The lack of effect of buffer addition alone is also shown. All data are means±s.e.mean, n=3.

Figure 4

Time courses of agonist-mediated elevations of [Ca²⁺]_i. [Ca²⁺]_i was determined in adherent populations of fura-2 loaded SH-SY5Y cells during challenge with either 1 mM methacholine or 1 μM bradykinin added at 48 s and not removed. Data are representative of n=3–6 for each agonist.

Figure 5

Concentration-response relationships for agonist-mediated peak elevations of [Ca²⁺]_i. (a) Peak [Ca²⁺]_i responses to methacholine in populations of SH-SY5Y or SH-SY5Y_RA6 cells. (b) Peak [Ca²⁺]_i responses to bradykinin in SH-SY5Y or SH-SY5Y_RA6 cells. Data are mean±s.e.mean, n=3–7 for methacholine-mediated responses and n=3 for bradykinin-mediated responses. EC₅₀ values (log₁₀ M, mean±s.e.mean) were unaffected by retinoic acid treatment (−5.59±0.19 (2.6 μM) and −5.94±0.3 (1.1 μM) for methacholine and −7.65±0.2 (22 nM) and −7.81±0.2 (15 nM) for bradykinin in control and retinoic acid treated cells respectively.

Figure 6

Lack of effect of ryanodine on methacholine-mediated elevation of [Ca²⁺]_i. (a) Pre-incubation of SH-SY5Y cells with 10 μM ryanodine had no effect on the subsequent response to a sub-maximal concentration (2 μM) of methacholine. (b) Challenge of cells with a maximal concentration of methacholine (1 mM) in the presence of ryanodine (10 μM) had no effect on the subsequent response to a sub-maximal concentration of methacholine. All data are representative of a total of three experiments on single cells showing similar results.

Figure 7

Relationship between peak Ins(1,4,5)P₃ accumulation and maximal [Ca²⁺]_i elevation. Data represent the peak Ins(1,4,5)P₃ level and the associated elevation of [Ca²⁺]_i following challenge with a range of either methacholine or bradykinin concentrations in adherent SH-SY5Y and SH-SY5Y_RA6 cells. Data points represent mean values (n=3) for peak Ins(1,4,5)P₃ mass accumulations and means±s.e.mean, n=3–9 for peak [Ca²⁺]_i elevations. Curves were generated using a second order polynomial equation.