P2X(4) purinoceptors mediate an ATP-activated, non-selective cation current in rabbit osteoclasts.
Extracellular nucleotides act as signaling molecules in numerous tissues. In bone, nucleotides stimulate osteoclast formation and activity; however, the receptors and signaling mechanisms underlying these effects have yet to be identified. To identify specific P2X purinoceptor subtypes in osteoclasts, degenerate oligonucleotide primers were used to PCR-amplify DNA fragments from a rabbit osteoclast cDNA library. A 372-base-pair fragment was obtained that encoded an amino acid sequence with 88% identity to the rat P2X(4) purinoceptor. The presence of P2X(4) mRNA in purified osteoclasts was confirmed by reverse transcription-PCR. Endogenous purinoceptors were functionally characterized in isolated rabbit osteoclasts by patch-clamp recording in whole-cell configuration. At negative membrane potentials, application of ATP or ADP rapidly activated an inward current followed by an outward current. In contrast, UTP or ADPbetaS elicited only an outward current, due to activation of a Ca(2+)-dependent K(+) conductance. The initial inward current was non-selective for cations and inactivated during agonist application. Furthermore, the inward current was insensitive to suramin and Cibacron blue, and was potentiated by Zn(2+). These characteristics are consistent with properties of P2X(4) purinoceptors. Activation of P2X(4) purinoceptors leads to cation influx and depolarization. Nucleotides, released at sites of trauma or inflammation, may act through these receptors on osteoclasts to stimulate bone resorption.
Naemsch
L N
LN
Department of Physiology, Division of Oral Biology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1.
Weidema
A F
AF
Sims
S M
SM
Underhill
T M
TM
Dixon
S J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D017124:Conserved Sequence; D017931:DNA Primers; D015723:Gene Library; D007473:Ion Channels / Q000502:physiology*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000502:physiology; D010010:Osteoclasts / Q000166:cytology / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011817:Rabbits; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D058848:Receptors, Purinergic P2X4; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D007473:Ion Channels; D009479:Neuropeptides; C548228:P2rx4 protein, rat; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate