Studies on stimulus-response coupling in human neutrophils

Human blood neutrophils suspended in Na + -free, high-K + , phosphate-buffered solution exhibit respiratory and secretory responses to N -formylmethionylleucylphenylalanine (fMet-Leu-Phe) much higher than those suspended in phosphate-buffered solution containing physiological concentration of K + and Na + . The differences between the responses are very marked at low doses of fMet-Leu-Phe (10 −9 , 10 −8 M), progressively decrease at higher doses, and disappear at the maximal stimulatory concentration of the peptide (10 −6 M). The higher responses of human neutrophils to fMet-Leu-Phe are not dependent on the membrane depolarization, that occurs when the cells are suspended in high-K + buffered solution, but on the absence, or on the low concentration, of Na + in the suspending medium. In fact: (i) the higher respiratory and secretory responses progressively decrease by substituting K + with Na + in the suspending solution, without change of the state of depolarization; (ii) the replacement of extracellular Na + with choline ions does not affect the transmembrane potential of neutrophils but induces higher respiratory and secretory responses to fMet-Leu-Phe; (iii) the membrane depolarization induced by gramicidin and by ouabain does not result in a higher respiratory response to chemotactic peptide. These results indicate that in human neutrophils Na + plays a regulative role in the stimulation of the respiratory burst and in the secretion induced by the chemotactic peptide. This regulation does not influence the maximal responses, but the threshold of the responses. K + is also involved at least in the respiratory response, since the effect of the absence of Na + is potentiated when the concentration of K + of the suspending solution is high. Furthermore, the finding that a very high respiratory burst and the secretion of β-glucuronidase and vitamin B-12-binding protein can be induced by fMet-Leu-Phe in human neutrophils in the absence of external Na + indicates that the entry of this cation and the consequent decrease in transmembrane potential are not necessary events for the activation of respiration and secretion by the peptide. The mechanism underlying the effect of the modification of ionic composition of the external medium is discussed in terms of the molecular events triggered by the stimulus at the level of the plasma membrane and of the recognition phenomena at the cell surface, that are common steps for the induction of the respiratory and secretory responses in neutrophils.