Abstract
The regulation of Ca2+ transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca2+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca2+] to a steady state of around 0.2 microM. A vesicular ATP-dependent and vanadate-sensitive non-mitochondrial pool maintained this low [Ca2+] level. In the absence of ATP, a higher Ca2+ steady state of 0.6 microM was seen, exhibiting the characteristics of a mitochondrial Ca2+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca2+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca2+] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca2+] to the basal level. The action of inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a few cell types was studied. IP3 released (detectable within 2 s) Ca2+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IP3 additions. Under experimental conditions in which the ATP-dependent Ca2+ influx was blocked, the addition of IP3 resulted in a very large Ca2+ release from nonmitochondrial pool. The results strongly suggest that IP3 is a second messenger mediating intracellular Ca2+ mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways for Ca2+ transport, a Ca2+ ATPase (the influx component) and an IP3-sensitive efflux component activated during Ca2+ mobilization.
Highlights
From the Slnstitut de Biochimie Clinique,University of Geneva, Sentier de la Roseraie, 1211 Geneva 4,Switzerland and the SDiuisionof Infectious Diseases, University Cantonal Hospital, CH-12114,Switzerland
A MgATP-dependent Ca2+pump has been observed in the neutrophil plasma membrane [4,5],little isknown about theintracellular structures that participate in the regulation of the cytosolic Ca2+ homeostasis
The results strongly suggest that IPS is a second messenger mediating intracellular Ca2+mobilizationin human neutrophils
Summary
Isolation and Permeabilization of Human Neutrophils-Neutrophils were isolated from fresh blood samples (usually 96 ml) obtained from healthy volunteers. Ca2+Transport by Permeabilized Neutrophils warming at 37 “C for 5 min, a 100-fold concentrated solution of digitonin dissolved in water was added a t a final concentration of 10 p ~Th. e incubation was continued at 37 “Cfor 8min, and thereaction was stopped by the addition of a 5-fold excess volume of the same ice-cold buffer. Under these conditions and as shown for other cells [25,26,27,28], the neutrophil plasma membrane was selectivelypermeabilized.
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