FIGURE 3.

A, HaCaT keratinocytes show strong repetitive calcium signals to histamine (n = 215; 100 μm, 10 s) and LPA (n = 165; 200 nm, 10 s) stimulation. ATP (100 μm) application serves as a positive control for cell viability. B, in monoculture, trigeminal neurons fail to react to histamine (n = 85) or LPA (n = 590) stimulation. C, trigeminal neurons react in a co-culture approach subsequent to keratinocytes. D, repetitive LPA application induced calcium signals in keratinocytes but failed to induce signal transmission to trigeminal neurons. E, quantification of the co-culture results. The number of responding cells at histamine stimulation was significantly increased in the co-cultures. In contrast, LPA application did not lead to a significant increase in the co-cultures (histamine: TrigMono n = 85 neurons in 3 monocultures, TrigCoCu n = 29 neurons in 7 co-cultures; histamine: TrigMono n = 590 neurons in 10 monocultures, TrigCoCu n = 95 neurons in 13 co-cultures). F, quantitative analysis of Ca²⁺ signals of trigeminal neurons in co-cultures induced by Javanol® (1 mm; n = 25 neurons in 7 co-cultures) or histamine (100 μm; n = 29 neurons in 7 co-cultures) stimulation. Shown are mean amplitudes normalized to high potassium (K⁺; 45 mm). ***, p < 0.001; Fisher's exact test; ns, not significant. Error bars, S.E.