Inositol phosphates modulate human red blood cell Ca 2+—adenosine triphosphatase activity in vitro by a guanine nucleotide regulatory protein

Abstract

d- myo-inositol 1,4,5-trisphosphate [lns(1,4,5)P 3) inhibits human red blood cell (RBC) Ca 2+-stimulable, Mg 2+-dependent adenosine triphosphatase (Ca 2+-ATPase) activity in vitro. Because we have previously shown that adrenergic receptors exist on the human mature RBC membrane and can modulate Ca 2+-ATPase activity, we examined the possibility that a guanine nucleotide regulatory protein (G protein) mediated the lns(1,4,5)P 3 effect. Guanosine 5′- O-(3-thiotrisphosphate) (GTP γyS) 10 −4 mol/L also inhibited RBC Ca 2+-ATPase activity. Pertussis toxin 200 ng/mL blocked the effects of both lns(1,4,5)P 3 and GTP γS on Ca 2+-ATPase activity. In separate studies, pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation was shown to occur in RBC membranes under conditions in which measurements of Ca 2+-ATPase activity were performed. When lns(1,4,5)P 3 10 −7 mol/L and GTP γS 10 −6 mol/L were added to membranes concurrently, their inhibitory actions on the enzyme were additive. At greater concentrations of lns(1,4,5)P 3 (10 −6 to 10 −5 mol/L) and GTP γS (10 −4 mol/L), the inositol phosphate reversed the inhibitory effect of GTP γS. These observations indicate that the novel effect of lns(1,4,5)P 3 on the activity of a plasma membrane Ca 2+-ATPase depends at least in part on the action of a pertussis toxin-susceptible G protein.