Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A2

Background: Antimicrobial 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 Findings: PMNs 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 CaMbinding 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/Significance: For 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

Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs.

Endothelium-derived nitric oxide (EDNO) plays a pivotal role in regulating pulmonary circulation. To determine whether there is a heterogeneity in EDNO-mediated responses of different sized pulmonary vessels, we studied small and large isolated pulmonary arteries of newborn lambs (diameter, 0.4-0.7 and 1.5-2.5 mm, respectively). The isometric tension of vessel rings were recorded while suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O2-5% CO2, 37 degrees C). In vessels preconstricted with norepinephrine, acetylcholine and bradykinin induced a greater relaxation of small pulmonary arteries than of large pulmonary arteries. Acetylcholine, bradykinin, and nitric oxide also induced a greater increase in cGMP content in small arteries than in large ones. The responses to acetylcholine and bradykinin were endothelium-dependent and inhibited by nitro-L-arginine, an inhibitor of nitric oxide synthase. In vessels without endothelium, the response to nitric oxide was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. The activity of soluble guanylyl cyclase of small arteries was greater than that of large arteries under basal conditions and after stimulation with S-nitroso-N-acetylpenicillamine, a nitric oxide donor. These results demonstrate that heterogeneity exists in EDNO-mediated relaxation of small and large pulmonary arteries in newborn lambs. A difference in the soluble guanylate cyclase activity of vascular smooth muscle may have contributed to this phenomenon