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Desensitization of calcium mobilization and cell function in human neutrophils.

ABSTRACT: Neutrophils pretreated with the chemoattractant formylmethionyl-leucyl-phenylalanine become unresponsive when re-exposed to the same ligand, a process termed desensitization. We have examined whether desensitization of transduction (Ca2+ mobilization) or of other cell functions (superoxide generation, enzyme release, or aggregation) occurs synchronously. Simultaneous studies of Ca2+ mobilization and aggregation by using Fura-2-loaded cells indicate that, under conditions where the aggregation response is abolished, most of the Ca2+ mobilization is unaltered. Further studies were then carried out to ascertain whether desensitization of Ca2+ mobilization could in fact be induced. Desensitization was observed, and was dependent on the number of exposures of the cells to the ligand, the concentration of the ligand, and whether the ligand was left in the medium or was removed. The pattern of resensitization was dependent on the experimental design. Under conditions where ligand was continuously present, no recovery of the Ca2+-mobilization response was seen with subsequent challenges. In contrast, on removal of ligand, this response showed partial recovery. Whereas complete desensitization of aggregation was noted, enzyme release showed a markedly lesser degree of desensitization and required more frequent exposures to the ligand before it was observed. Little or no desensitization of superoxide generation was observed regardless of the conditions utilized. Studies using phorbol myristate acetate as the ligand showed that Ca2+ mobilization and aggregation could be simultaneously inhibited. Our results suggest that discrete mechanisms of desensitization are possible in human neutrophils, and that desensitization of one particular function (aggregation) does not imply concomitant desensitization of other functions.

SUBMITTER: Lee GH

PROVIDER: S-EPMC1133243 | biostudies-other | 1989 Aug

REPOSITORIES: biostudies-other

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Project description:Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis.

Project description:BackgroundAntimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.Methodology/principal findingsPMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity.Conclusions/significanceFor the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.

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Desensitization of calcium mobilization and cell function in human neutrophils.

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