Adenosine-5'-O-(3-thiotriphosphate) binding to human neutrophils. Evidence for a common nucleotide receptor.
Human polymorphonuclear neutrophils (PMN) respond to ATP with an elevation in intracellular calcium and a marked enhancement of O2-production in response to stimulation by the chemotactic peptide N'-formyl-Met-Leu-Phe (FMLP). These pertussis toxin-sensitive pathways appear to be mediated by a nucleotide receptor(s) on the surface of human PMN. In the current study, we have examined the binding to intact human PMN of the ATP analog, adenosine 5'-O-(3-thio[35S] triphosphate) [( 35S]ATP gamma S). On the basis of Scatchard analysis, the binding of [35S]ATP gamma S involves at least two sites, one of high and one of low affinity. In the presence of sodium thiophosphate, a compound which did not affect intracellular increases in calcium induced by ATP or N'-formyl-Met-Leu-Phe, a significant fraction of the [35S]ATP gamma S binding was eliminated. This reduction involved both high and low affinity binding of [35S]ATP gamma S and was related to a reduction in numbers of binding sites. The Kd values for the high affinity binding site were unaffected by the presence of sodium thiophosphate, although the low affinity Kd values were numerically increased by 2-fold. In the presence of thiophosphate, [35S]ATP gamma S binding was specific, saturable, and reversible, and was related to a single class of high affinity (Kd = 36 +/- 19 nM) binding sites (184 +/- 144 sites/cell), together with a second class of low affinity (Kd = 1110 +/- 503 nM) binding sites (13,562 +/- 6,851 sites/cells). Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block [35S]ATP gamma S binding to PMN, revealed a rank order of ATP gamma S greater than ATP greater than 2-MeS-ATP = 8-Bromo ATP greater than ADP = ITP greater than AMP-PCP = GTP much greater than CTP. A comparison between the ability of nucleotides to compete with [35S]ATP gamma S binding and their ability to induce a biologic response (elevation of intracellular calcium) revealed a close correlation (r2 = 0.83). These findings support the possibility of a common nucleotide PMN receptor functionally linked to a cellular response which involves increases in intracellular calcium.
Yu
G H
GH
Department of Pathology, University of Michigan Medical School, Ann Arbor.
Tarapchak
S J
SJ
Walker
B A
BA
Ward
P A
PA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*
D009711:Nucleotides; D010710:Phosphates; D011956:Receptors, Cell Surface; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D002118:Calcium; C035638:thiophosphoric acid