Identification of P2X7 (P2Z) receptor in N18TG-2 cells and NG108-15 cells.
ATP activates a nonselective cation current by stimulating the P2Z receptor in NG108-15 cells--a hybrid cell line of the mouse neuroblastoma N18TG-2 cells and the rat glioma C6Bu-1 cells. Recently, the P2X7 receptor was cloned from the rat brain and was found to have electrophysiological properties similar to those of the P2Z receptor. We examined the expression of P2X7 receptor mRNA in NG108-15 cells as well as in their parent cell lines, N18TG-2 and C6Bu-1 cells, by reverse transcription-polymerase chain reaction (RT-PCR). The cDNA templates from these cell lines were amplified with primers specific to the P2X7 receptor sequence. Positive signals were detected in the RT-PCR products from NG108-15 and N18TG-2 cells but not from C6Bu-1 cells. We next examined the effect of ATP on the membrane current in N18TG-2 cells and C6Bu-1 cells by whole-cell voltage clamp. In N18TG-2 cells, ATP induced a sustained current with a reversal potential of 9.3 +/- 1.2 mV (n = 22) in a concentration-dependent manner with an EC50 of 1.76 +/- 0.18 mM (n = 36). In contrast, ATP (1 mM) did not induce any current in C6Bu-1 cells. The ATP-induced current in N18TG-2 cells resembled that in NG108-15 cells in the following points: (a) The currents did not desensitize significantly. (b) EC50 values of ATP are of millimolar order. (c) Benzoylbenzoyl-ATP was a more potent agonist than ATP. (d) The current was larger in methanesulfonate than in Cl- external solution. (e) The current was larger at lower external Mg2+ concentrations. These results suggest that the hybrid NG108-15 cells possess a P2X7 receptor like the P2Z receptor and that the ability of expressing this channel originates from N18TG-2 cells but not from C6Bu-1 cells.
Kaiho
H
H
Department of Pharmacology, Fukushima Medical College, Japan.
Matsuoka
I
I
Kimura
J
J
Nakanishi
H
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002412:Cations / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005910:Glioma; D006822:Hybrid Cells / Q000737:chemistry* / Q000502:physiology; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics* / Q000378:metabolism; D058486:Receptors, Purinergic P2X7
D000345:Affinity Labels; D002412:Cations; D002712:Chlorides; C546469:P2rx7 protein, mouse; C546470:P2rx7 protein, rat; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium