Effects of antagonists at the human recombinant P2X7 receptor |
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Authors: | I P Chessell A D Michel P P A Humphrey |
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Institution: | 1.Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ |
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Abstract: | - We have used whole-cell patch clamping methods to examine the properties of the recombinant human P2X7 (P2Z) receptor stably expressed in HEK-293 cells.
- In an extracellular solution with lowered concentrations of divalent cations (zero Mg2+ and 0.5 mM Ca2+), both ATP and the nucleotide analogue, 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (Bz-ATP) evoked concentration-dependent whole-cell inward currents with maxima of 4658±671 and 5385±990 pA, respectively, at a holding potential of −90 mV. Current-voltage relationships determined using 100 μM Bz-ATP reversed at −2.7±3.1 mV, and did not display significant rectification.
- Repeated applications of 300 μM Bz-ATP produced inward currents with similar rise-times (approx. 450 ms, 5–95% current development) but with progressively slower 95–5% decay times, with the eighth application of this agonist yielding a decay time of 197% of the first application.
- Concentration-effect curves to ATP and Bz-ATP produced estimated EC50 values of 780 and 52.4 μM, respectively. Consecutive concentration-effect curves to Bz-ATP produced curves with similar maxima and EC50 values.
- The non-selective P2 antagonists, pyridoxal-phosphate-6-azophenyl-, 2′,4′-disulphonic acid (PPADS) and suramin, both produced concentration-dependent increases in maximal inward currents to Bz-ATP, with IC50 concentrations of approximately 1 μM and 70 μM, respectively. The profile of antagonism produced by PPADS was not that of a competitive antagonist.
- The isoquinolene derivatives 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62) and calmidazolium both produced antagonism which was not competitive, with IC50 concentrations of approximately 15 and 100 nM, respectively. HMA (5-(N,N-hexamethylene)- amiloride) was also an effective antagonist at a concentration of 10 μM. The group IIb metal, copper, also displayed antagonist properties at the human P2X7 receptor, reducing the maximum response to Bz-ATP by about 50% at a concentration of 1 μM.
- These data demonstrate that the human recombinant P2X7 receptor displays functional behaviour which is similar to the recombinant rat P2X7 receptor, but has a distinct pharmacological profile with respect to agonist and antagonist sensitivity.
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Keywords: | Human P2X7 receptors suramin ATP antagonist pore KN-62 |
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