The Mechanism of Ciliary Stimulation by Acetylcholine: Roles of Calcium, PKA, and PKG

Stimulation of ciliary cells through muscarinic receptors leads to a strong biphasic enhancement of ciliary beat frequency (CBF). The main goal of this work is to delineate the chain of molecular events that lead to the enhancement of CBF induced by acetylcholine (ACh). Here we show that the Ca2+, cGMP, and cAMP signaling pathways are intimately interconnected in the process of cholinergic ciliary stimulation. ACh induces profound time-dependent increase in cGMP and cAMP concentrations mediated by the calcium–calmodulin complex. The initial strong CBF enhancement in response to ACh is mainly governed by PKG and elevated calcium. The second phase of CBF enhancement induced by ACh, a stable moderately elevated CBF, is mainly regulated by PKA in a Ca2+-independent manner. Inhibition of either guanylate cyclase or of PKG partially attenuates the response to ACh of [Ca2+]i, but completely abolishes the response of CBF. Inhibition of PKA moderately attenuates and significantly shortens the responses to ACh of both [Ca2+]i and CBF. In addition, PKA facilitates the elevation in [Ca2+]i and cGMP levels induced by ACh, whereas an unimpeded PKG activity is essential for CBF enhancement mediated by either Ca2+ or PKA

The Natural Cytotoxicity Receptor 1 Contribution to Early Clearance of Streptococcus pneumoniae and to Natural Killer-Macrophage Cross Talk

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC

A Missense Variation in <i>PHACTR2</i> Associates with Impaired Actin Dynamics, Dilated Cardiomyopathy, and Left Ventricular Non-Compaction in Humans

Dilated cardiomyopathy (DCM) with left ventricular non-compaction (LVNC) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure, and excessive risk of sudden cardiac death. Using whole-exome sequencing to investigate a possible genetic cause of DCM with LVNC in a consanguineous child, a homozygous nucleotide change c.1532G>A causing p.Arg511His in PHACTR2 was found. The missense change can affect the binding of PHACTR2 to actin by eliminating the hydrogen bonds between them. The amino acid change does not change PHACTR2 localization to the cytoplasm. The patient’s fibroblasts showed a decreased globular to fibrillary actin ratio compared to the control fibroblasts. The re-polymerization of fibrillary actin after treatment with cytochalasin D, which disrupts the actin filaments, was slower in the patient’s fibroblasts. Finally, the patient’s fibroblasts bridged a scar gap slower than the control fibroblasts because of slower and indirect movement. This is the first report of a human variation in this PHACTR family member. The knock-out mouse model presented no significant phenotype. Our data underscore the importance of PHACTR2 in regulating the monomeric actin pool, the kinetics of actin polymerization, and cell movement, emphasizing the importance of actin regulation for the normal function of the human heart

IL-1alpha and IL-1beta recruit different myeloid cells and promote different stages of sterile inflammation.

Item does not contain fulltextThe immune system has evolved to protect the host from invading pathogens and to maintain tissue homeostasis. Although the inflammatory process involving pathogens is well documented, the intrinsic compounds that initiate sterile inflammation and how its progression is mediated are still not clear. Because tissue injury is usually associated with ischemia and the accompanied hypoxia, the microenvironment of various pathologies involves anaerobic metabolites and products of necrotic cells. In the current study, we assessed in a comparative manner the role of IL-1alpha and IL-1beta in the initiation and propagation of sterile inflammation induced by products of hypoxic cells. We found that following hypoxia, the precursor form of IL-1alpha, and not IL-1beta, is upregulated and subsequently released from dying cells. Using an inflammation-monitoring system consisting of Matrigel mixed with supernatants of hypoxic cells, we noted accumulation of IL-1alpha in the initial phase, which correlated with the infiltration of neutrophils, and the expression of IL-1beta correlated with later migration of macrophages. In addition, we were able to show that IL-1 molecules from cells transfected with either precursor IL-1alpha or mature IL-1beta can recruit neutrophils or macrophages, respectively. Taken together, these data suggest that IL-1alpha, released from dying cells, initiates sterile inflammation by inducing recruitment of neutrophils, whereas IL-1beta promotes the recruitment and retention of macrophages. Overall, our data provide new insight into the biology of IL-1 molecules as well as on the regulation of sterile inflammation