Two different ionotropic receptors are activated by ATP in rat microglia

Abstract

1. Our aim was to assess whether ATP-induced inward currents in microglia are due to a single or more than one purinergic receptor. The ATP dose-response curve showed two components, whose presence might be due to the activation of high and low affinity receptors. 2. The P2Z/P2X7 specific receptor agonist benzoylbenzoyl-ATP (Bz-ATP) and some P2 receptor agonists were tested. The rank order of potency was Bz-ATP >> ATP = 2-methylthio-ATP (2-MeSATP) > alpha, beta-methylene ATP (alpha,beta-meATP) >= ADP. beta, gamma-MethyleneATP (beta,gamma-meATP), UTP and adenosine were ineffective. 3. The non-specific P2 receptor antagonist suramin antagonized by 92 +/- 2 % the inward current induced by 100 microM ATP, and by 51 +/- 8 and 68 +/- 6 % those induced by 3 mM ATP and 100 microM Bz-ATP, respectively. The P2Z/P2X7 antagonist oxidized ATP (oATP) almost abolished the inward current induced by 3 mM ATP or Bz-ATP, but was ineffective against 100 microM ATP. 4. Inward currents induced by low ATP concentrations (<= 100 microM) were generally followed by an almost complete and irreversible desensitization, while those elicited by ATP >= 1 mM showed only a partial decline. Interestingly, the inward current induced by 100 microM 2-MeSATP showed a large desensitization, while that induced by Bz-ATP did not. 5. In voltage-ramp experiments, the 100 microM ATP-induced current exhibited a slight inward rectification more visible at negative potentials, while the 3 mM ATP-induced current did not. 6. ATP induced a fast and large increase in [Ca2+] that promptly recovered in the continuous presence of low ATP doses, but did not recover in high ATP doses. As with desensitization, the response to Bz-ATP mimicked that of high doses of ATP. 7. When Ca2+ mobilization due to P2Y receptors was blocked by thapsigargin-induced Ca2+ depletion or by pertussis toxin treatment, 10 microM ATP was still able to induce a Ca2+ transient, which represented the contribution of the Ca2+ influx induced by P2X receptors 8. In conclusion, the inward currents and a fraction of the Ca2+ transients induced by ATP in microglia are due to at least two ATP-sensitive receptor channel types, whose different properties and sensitivity to ATP may be associated with different functional roles.

Figure 1. The dose-response curve of the ATP-induced inward current does not show a simple sigmoidal shape and shifts leftward in the absence of divalent cations

Dose-response curves of the ATP-induced inward current were obtained from peak current amplitudes recorded in the presence (○; n = 9-149) and absence (□; n = 3-8) of divalent cations. The inset shows an enlarged portion of the dose-response curve at a range of low ATP concentrations. In the presence of divalent cations the dose-response curve has a composite shape and in their absence the currents are larger and the dose-response curve shifts to the left.

Figure 2. Rank order of potency of P2 receptor agonists

The graph shows the mean amplitude of inward currents induced by different P2 receptor agonists at a concentration of 100 μm. For the computation of means only one agonist was tested in each cell. Other agonists (β,γ-meATP, UTP and adenosine) were unable to induce an inward current (data not shown).

Figure 3. Antagonistic effect of oATP and suramin on the inward current induced by ATP agonists

A, mean amplitude of inward currents recorded from untreated cells or cells treated with oATP for 2 h before and during recording with 300 μm oATP. Note the marked antagonistic effect of oATP on the currents induced by 3 mM ATP and 100 μm Bz-ATP and the lack of effect on the current induced by 100 μm ATP. B, decrease in the inward current due to 5 min application of 300 μm suramin. The antagonist was applied 5 min before and during recording of inward currents induced by ATP (100 μm or 3 mM) or 100 μm Bz-ATP. The inward current induced by 100 μm ATP was more sensitive to the antagonism by suramin. Three pairs of superimposed currents obtained before and in the presence of suramin are shown. Calibration bars: horizontal, 2 s; vertical, 50, 200 and 450 pA, for left to right traces, respectively.

Figure 4. Low ATP concentrations induce inward currents showing a more pronounced desensitization compared to high ATP concentrations

A shows typical inward currents induced by 100 μm ATP (left) and 3 mM ATP (right). Note the different profiles of desensitization obtained with the two concentrations. The continuous line superimposed on the decaying phase of the current traces is the best fit of the desensitization to a mono-exponential equation. Calibration bars: horizontal, 10 s; vertical, 100 pA and 400 pA, for left to right traces, respectively. B, mean percentage of desensitization of inward currents is plotted versus the ATP concentration. Note that the percentage of desensitization is significantly lower at high ATP concentrations (≥ 1 mM) compared to low ATP concentrations. C, distribution of the percentage of desensitization in cells treated with 100 μm ATP (left) and 3 mM ATP (right). With 100 μm ATP, two sub-populations of cells can be detected, with the majority of the cells showing a high percentage of desensitization.

Figure 5. P2X agonists induce inward currents showing a more pronounced desensitization than those induced by the P2Z/P2X7 agonist Bz-ATP

A, typical inward currents induced by ATP (upper trace) and 2-MeSATP (lower trace), both at a concentration of 100 μm. Note the similarity of the current desensitization. Calibration bars: horizontal, 10 s; vertical, 50 pA and 20 pA for upper and lower traces, respectively. B, typical inward currents induced by 3 mM ATP (upper trace) and 100 μm Bz-ATP (middle trace). Note the less pronounced desensitization compared to that obtained with 100 μm ATP or 2-MeSATP. The lower trace was obtained by applying 3 mM ATP to a cell treated with the P2Z/P2X7 antagonist oATP. Note that even if 3 mM ATP was used, the desensitization resembled that obtained with lower concentrations of the agonist. Calibration bars: horizontal, 10 s; vertical, 100, 100 and 40 pA for upper, middle and lower traces, respectively. C, the graph shows the mean percentages of desensitization of inward currents induced by 100 μm and 3 mM ATP in untreated and oATP-treated cells. The percentage of desensitization does not seem to vary after oATP treatment. D, distributions of the percentage of desensitization induced by 3 mM ATP in untreated (left) and oATP-treated (right) cells. Treatment with oATP revealed a sub-population of cells showing a high percentage of desensitization.

Figure 6. Inward currents induced by P2X receptor activation do not recover from desensitization

A, a 30 s application of an agonist was followed by a second application of the same agonist after a 2 min interval. The superimposed current traces were obtained using 100 μm ATP (left), 100 μm 2-MeSATP (middle) and 3 mM ATP (right; the latter in an oATP-treated cell). Note the lack of recovery from desensitization in all the cases shown. Calibration bars: horizontal, 10 s; vertical, 50, 30 and 30 pA, for left, middle and right traces, respectively. B, 100 μm ATP was applied for 20 s in order to caused full desensitization of the inward current. Then 100 μm 2-MeSATP (left trace) or 3 mM ATP (right trace) was applied after a 5 s interval. 2-MeSATP was almost ineffective due to the desensitization induced by the previous application of ATP, while 3 mM ATP caused a large current. Calibration bars: horizontal, 10 s; vertical, 50 and 100 pA for left and right traces, respectively.

Figure 7. Only inward currents induced by low ATP concentrations show inward rectification

A, voltage ramps from -100 to +50 mV (1 mV ms⁻¹) were applied 30 s before, 1 s after (at the current peak) and 30 s after (when the ATP-induced current reached steady state) the start of the application of 100 μm ATP. The current recorded in control was subtracted from the peak and steady-state currents and the resulting traces are shown in the graph as ATP-induced peak (P) and steady-state (SS) currents, respectively. Note the inward rectification of the peak current, and the reversal potential close to 0 mV. B, the current trace represents the pure 3 mM ATP-induced current at the peak (as in panel A). Note the lack of rectification in the voltage range from -70 to +50 mV and the reversal potential close to 0 mV.

Figure 8. ATP-induced depolarization: correlation with the inward current desensitization

A and C, depolarization induced by 20 s applications of 10 μm ATP (A) and 3 mM ATP (C) were recorded under current clamp. Note the decay of the depolarization in the presence of 10 μm ATP compared to the long-lasting depolarization obtained with 3 mM ATP. B and D, a 20 s applications of 10 μm ATP (B) or 3 mM ATP (D) was followed, after a 2 min interval, by a second application of the same concentration of the agonist. The second application of the low dose of ATP had little effect compared to that of the high dose.

Figure 10. Contributions of Ca²⁺ influx and Ca²⁺ release to the Ca²⁺ transient induced by P2 receptor activation

A, the effect of 10 μm ATP was measured in Ca²⁺-free solution and in the presence of Ca²⁺ in the same set of cells (average of 16 cells). B, microglial cells were bathed for 10 min in a solution containing 50 ng ml⁻¹ thapsigargin in order to empty endoplasmic reticulum Ca²⁺ stores. The complete depletion of Ca²⁺ stores was assumed when 100 μm UTP was unable to provoke Ca²⁺ mobilization. Even in this condition, 10 μm ATP induced a Ca²⁺ transient (average of 7 cells). C, microglial cultures were treated overnight with 10 ng ml⁻¹ pertussis toxin. When cells were challenged with 10 μm ATP in a Ca²⁺-free solution they were unable to respond with a Ca²⁺ increase, while in a normal Ca²⁺-containing solution they retained the ability to respond to ATP (average of 16 cells).

Figure 9. Rise of intracellular Ca²⁺ induced by ATP and ATP analogues

The agonists, each at the concentration of 100 μm, were applied for 30 s to the area containing the cells selected for recording. The traces represent the average response to the agonist calculated in populations comprising 7-21 cells. The traces shown were obtained from the first application of the test solution, each on a different set of cells.

Figure 11. ATP-induced Ca²⁺ rise: correlation with desensitization of the inward current

A and C, the rise of intracellular Ca²⁺ was induced by 30 s applications of 10 μm ATP (A) and 3 mM ATP (C). Note the different time course of the Ca²⁺ transients. B and D, a series of 30 s pulses of 10 μm ATP (B) and 3 mM ATP (D) was applied to the area containing the cells chosen for recording. The amplitude of the Ca²⁺ transients induced by 10 μm ATP decreased, while those induced by 3 mM ATP remained constant.