FIGURE 5.

A, the ATP receptor blocker PPADS significantly inhibited the reactivity of trigeminal neurons in co-cultures. B, quantitative analysis of the responses of trigeminal neurons in co-cultures (TrigCoCu) and co-cultures in the presence of PPADS (TrigCoCu PPADS). PPADS lead to a decrease in the activity of trigeminal neurons upon Javanol® stimulation (Sandalore®: TrigCoCu n = 66 neurons in 23 co-cultures, TrigCoCu PPADS n = 88 in 17 co-cultures; Javanol®: TrigCoCu n = 25 neurons in 7 co-cultures, TrigCoCu PPADS n = 81 in 11 co-cultures). The ATP receptor blocker PPADS (200 μm) had no influence on reactivity (C) or calcium amplitudes (D) of trigeminal neurons in histamine (100 μm)-stimulated co-cultures (TrigCoCu, n = 29 neurons in 7 co-cultures) or histamine-stimulated co-cultures in the presence of PPADS (TrigCoCu PPADS, n = 89 neurons in 10 co-cultures). Amplitude levels were normalized to high potassium (K⁺; 45 mm). E, pannexin 1 inhibitor probenecid (10 μm) leads to a decrease in the activity of trigeminal neurons upon Javanol® stimulation (Javanol®: TrigCoCu n = 49 neurons in 5 co-cultures, TrigCoCu PPADS n = 47 in 6 co-cultures). F, quantification of Javanol®- and ATP-induced Ca²⁺ responses of HaCaT cells in co-cultures in the absence (n = 54 cells/72 cells) or presence of PPADS (n = 45 cells) or probenecid (n = 67 cells). Both inhibitors had no influence on reactivity of HaCaT cells in co-cultures. Shown are mean amplitudes of Ca²⁺ responses at repetitive applications of Javanol® (1–3; 1 mm) and ATP (100 μm, positive control). Amplitude levels were normalized to control measurements. G, quantification of high potassium (K⁺; 45 mm)-induced Ca²⁺ responses of trigeminal neurons in co-cultures in the absence (n = 43 neurons/48 neurons) or presence of PPADS (n = 43 neurons) or probenecid (n = 48 neurons). Amplitude levels were normalized to control measurements. Both inhibitors had no influence on reactivity of trigeminal neurons in co-cultures. In all measurements, PPADS (200 μm) or probenecid (10 μm) application was initiated 90 s before and continued through the Javanol® or K⁺ stimulation. *, p < 0.01; **, p < 0.01; ***, p < 0.001; ns, not significant; Fisher's exact test (B–E) or Student's t test (F and G).