Article Figures & Data
Figures
- Fig. 1.
Effect of 4-AP on [Ca2+]i. Near-confluent cultured type I astrocytes were loaded with fura-2. Cells were perfused with increasing 4-AP concentrations applied at the times indicated by thetop broken line arrows and washed at the times indicated by the solid line arrows below the trace. Increasing concentrations of 4-AP caused proportional [Ca2+]i elevation that reached an apparent plateau at 20 mm 4-AP, as shown in theinset. Removal of 4-AP caused a prompt return to baseline [Ca2+]i. The EC50 of 4-AP was ∼10 mm.
- Fig. 2.
Characterization of 4-AP-induced calcium response in type I astrocytes. A, Treatment with 10 mm4-AP caused a slow-onset long-lasting [Ca2+]i elevation, which was promptly reversed by washout. B, When extracellular calcium was removed, 4-AP was still able to increase [Ca2+]i. However, the shape of the response was modified to a transient peak with a quick return to baseline. C, The effect of 4-AP was tested in the absence of extracellular calcium and after depletion of intracellular calcium stores with thapsigargin (Thap). D, A 10 mm concentration of 4-AP was applied to astrocytes in calcium-free KRB. Subsequently calcium was reintroduced, and [Ca2+]i was monitored. Perfusion of the testing substances is indicated by the horizontal bars.
- Fig. 3.
Characterization of 4-AP-induced [Ca2+]i rise in cultured cortical neurons. A, A 10 mm concentration of 4-AP was applied to cortical neurons plated on glass coverslips. The compound caused a slow-onset long-lasting [Ca2+]i elevation. The [Ca2+]i elevation was promptly reversed by washing out the compound. B, When extracellular calcium was removed, 4-AP (10 mm) was still able to increase [Ca2+]i. However, the response was transient, and [Ca2+]ireturned promptly to baseline values. C, ICS were depleted in the absence of extracellular calcium with thapsigargin and CCE after reintroduction of calcium was measured. D, ICS were depleted with 4-AP in calcium-free medium. The neurons were subsequently exposed to thapsigargin, and 60 sec later calcium was reintroduced in the extracellular solution to elicit CCE. Perfusion of the testing substances is indicated by the horizontal bars.
- Fig. 4.
Effect of 4-AP on InsPt accumulation in astrocytes and neurons. A, 4-AP causes a concentration-dependent elevation of InsPt accumulation in type I astrocytes. Compared with the stimulation obtained with a 10 μm ATP (EC50, 30 μm), 10 mm 4-AP (EC50) was 50% less powerful (inset). B, 4-AP increased InsPtproduction in cortical neurons. Basal values are indicated by theopen triangle and the dashed line. *p ≤ 0.05 versus basal value.
- Fig. 5.
Effect of 4-AP on neurotransmitter-evoked calcium transients in astrocytes. Astrocytes were challenged with 10 μm ATP (A) or 100 nmbradykinin (C). The calcium response to both agonists was characterized by a fast and sharp [Ca2+]i elevation and a rapid return to a much lower but prolonged [Ca2+]ivalue. Astrocytes pre-exposed to 10 mm 4-AP and challenged with ATP (B) or bradykinin (D) showed a long-lasting large [Ca2+]ielevation. E and F show statistical analysis of the data extrapolated from the experiments in A andC 2 sec after agonist stimulation, and in B andD 2 sec before 4-AP washout. *p ≤ 0.05 versus value in control cells. Perfusion of the testing substances is indicated by the horizontal bars.
- Fig. 6.
Effect of 4-AP on CCE triggered by agonist-induced ICS depletion in astrocytes. ICS were depleted with ATP (A) or bradykinin in the absence of extracellular calcium. ICS emptying was controlled with a second ATP (A) or bradykinin (C) stimulation. When calcium was reintroduced, a weak [Ca2+]i elevation was generated because of CCE activation. B, ICS were depleted with 10 μm ATP in the presence of 4-AP. When calcium was reintroduced, [Ca2+]i elevation was very high and lasted until 4-AP was removed from the cells. The same effect was recorded when stores were depleted with bradykinin in the presence of 4-AP (D). E and Fshow the statistical validation of the data presented inA–D, respectively. Values were extrapolated from the experiments 2 sec before removal of 4-AP. *p value ≤ 0.05 versus control cells. Perfusion of the testing substances is indicated by the horizontal bars.
- Fig. 7.
4-AP potentiates thapsigargin-induced CCE in astrocytes. A, Calcium stores were depleted with a maximal concentration of thapsigargin (10 μm).B, Thapsigargin exposure in cells pretreated with 4-AP resulted in a large increase of CCE. C, Statistical validation of the data presented in A and B. Peak values were analyzed. *p value ≤ 0.05 versus control cells. Perfusion of the testing substances is indicated by thehorizontal bars.
- Fig. 8.
4-AP potentiates CCE in L6 cells.A, Application of 10 μm thapsigargin in calcium-free KRB caused an elevation of [Ca2+]i, (see insetto A). Reintroduction of calcium in the extracellular buffer was followed by CCE. The effect of thapsigargin alone it is highlighted in the inset. B, In the presence of 10 mm 4-AP, CCE was increased approximately ninefold.C, Statistical validation of the data extracted from control at peak and from 4-AP-exposed cells 2 sec before 4-AP washout. *p value ≤ 0.05 versus control cells. Perfusion of the testing substances is indicated by the horizontal bars.
- Fig. 9.
4-AP inhibits voltage-gated K+ currents in astrocytes. A, Astrocytes were voltage-clamped at a membrane voltage of −60 mV. Fifty-millisecond-long pulses were delivered at increasing voltages from −80 mV up to +50 mV, at 10 mV intervals. The I–Vprofile was generated with a positive current that was recorded up to ∼2 nA amplitude. The steady-state current was measured 40 msec after the start of the pulse. B, Example traces recorded in astrocytes before, after exposure to 10 mm 4-AP and after washout. C, 4-AP application reversibly blocked the late component (40 msec), whereas the early, fast inactivating component did not recover from blockage after 10 min washout (open barsare the normalized control current; black bars represent the currents after 4-AP application; hatched bars represent the amplitude of the current after 10 min washout). The fast component was calculated as the difference between the current at 5 and 40 msec after the beginning of the pulse. D displays the inhibitory effect of 20 mm TEA on potassium current in type I astrocytes. Control current was ∼1.38 + 0.63 pA and used as 100% in theopen bar. D, The effect of 20 mm TEA is represented by the black bar. Cells were washed out, and the recovery of K+ current is summarized in thehatched bar.
- Fig. 10.
Effect of DTx on CCE in astrocytes. We tested whether voltage-gated K+ channel blocker α-dendrotoxin potentiatied CCE evoked by ATP-induced ICS depletion.A, Response to 10 μm ATP in control cells.B, Cells were challenged with 10 μm ATP after a 3 min exposure to 100 nm DTx. C andD were the same experiments as shown in A andB, respectively, and they were performed in the absence of extracellular calcium. Calcium was reintroduced in the perfusion buffer, and CCE was monitored. Perfusion of the testing substances is indicated by the horizontal bar.
