The Type 2 Ryanodine Receptor of Neurosecretory PC12 Cells Is Activated by Cyclic ADP-ribose ROLE OF THE NITRIC OXIDE/cGMP PATHWAY

Of two neurosecretory PC12 cell clones that respond to NO donors and 8-bromo-cGMP with similar increases in cADP-ribose and that possess molecularly similar Ca2+ stores, only one (clone 16A) expresses the type 2 ryanodine receptor, whereas the other (clone 27) is devoid of ryanodine receptors. In PC12-16A cells, activation of the NO/cGMP pathway induced slow [Ca2+]i responses, sustained by release from Ca2+ stores. In contrast, PC12-27 cells were insensitive to NO donors. Likewise, in PC12-16A cells preincubated with NO donors, Ca2+ stores were partially depleted, as revealed by a test with thapsigargin, whereas those in clone 27 were unchanged. The NO-induced Ca2+ release was increased synergistically by caffeine, and the corresponding store depletion was magnified by ryanodine. The specificity for the NO/cGMP pathway was confirmed by the effects of two blockers of cGMP-dependent protein kinase I, while the role of cADP-ribose was demonstrated by the effects of its antagonist, 8-amino-cADP-ribose, administered to permeabilized cells. These results demonstrate in neurosecretory cells a ryanodine receptor activation pathway similar to that known in sea urchin oocytes. The signaling events described here could be of great physiological importance, especially in the nervous system

Nitric oxide action on growth factor-elicited signals. Phosphoinositide hydrolysis and [Ca2+]i responses are negatively modulated via a cGMP-dependent protein kinase I pathway.

Abstract The role of nitric oxide (NO) in the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and intracellular Ca2+ release responses induced by epidermal, platelet-derived, and fibroblast growth factors was investigated in three cell lines, a clone of NIH-3T3 fibroblasts overexpressing epidermal growth factor receptors and the tumoral epithelial cells A431 and KB. In all three cell types, pretreatment with NO donors decreased growth factor-induced PIP2 and Ca2+ responses, whereas pretreatment with NO synthase inhibitors increased them. The Ca2+-dependent PIP2 hydroysis induced by micromolar concentrations of the Ca2+ ionophore, ionomycin, was also modulated negatively and positively by NO donors and synthase inhibitors, respectively. In contrast, the Ca2+ content of the intracellular stores was unaffected by the various pretreatments employed. NO donors and synthase inhibitors induced an increase and decrease, respectively, of the intracellular cGMP formation in all three cell lines investigated. All of the effects of the NO donors were mimicked by 8-bromo-cGMP administration and abolished by pretreatment with the specific blocker of the cGMP-dependent protein kinase I, KT5823, which by itself mimicked the effects of the synthase inhibitors. Together with previous observations on G protein-coupled receptors, the present results demonstrate that PIP2 hydrolysis and Ca2+ release occur under the feedback control of NO, independently of the phospholipase C (β, γ, or δ type) involved and of the mechanism of activation. Such a control, which appears to be effected by the cGMP-dependent protein kinase I acting at the level of the phospholipases C themselves, might ultimately contribute to the inhibitory role of NO on growth previously observed with various cell types

Autocrine Nitric Oxide Modulates CD95-induced Apoptosis in γδ T Lymphocytes

Gammadelta T lymphocytes play an important early role in the defense against pathogens. Their function is terminated by acquisition of susceptibility to CD95-triggered apoptosis. Here we show that the regulation of this process depends on the activity of the endothelial NO synthase expressed by gammadelta T lymphocytes, which is modulated in an activation-dependent way. The effects of nitric oxide thus generated, mediated via cGMP generation, are exerted at at least two sites along the CD95 signaling cascade: one at, or upstream, and the other downstream of ceramide generation. At either site, nitric oxide/cGMP action is sufficient for protection from apoptosis. The effect of NO is selective for apoptosis induced by CD95 cross-linking, since it does not affect apoptotic program triggered by other stimuli. The evidence here reported demonstrates a new physiological role for nitric oxide, acting as a survival factor for T lymphocytes

The p75NTR-induced Apoptotic Program Develops through a Ceramide-Caspase Pathway Negatively Regulated by Nitric Oxide

SK-N-BE neuroblastoma cell clones transfected with p75(NTR) and lacking Trk neurotrophin receptors, previously reported to undergo extensive spontaneous apoptosis and to be protected by nerve growth factor (NGF) (Bunone, G., Mariotti, A., Compagni, A., Morandi, E., and Della Valle, G. (1997) Oncogene 14, 1463-1470), are shown to exhibit (i) increased levels of the pro-apoptotic lipid metabolite ceramide and (ii) high activity of caspases, the proteases of the cell death cascade. In the p75(NTR)-expressing cells, these parameters were partially normalized by prolonged NGF treatment, which, in addition, decreased apoptosis, similar to caspase blockers. Conversely, exogenous ceramide increased caspase activity and apoptosis in both wild-type and p75(NTR)-expressing cells. A new p75(NTR)-expressing clone characterized by low spontaneous apoptosis exhibited high endogenous ceramide and low caspase levels. A marked difference between the apoptotic and resistant clones concerned the very low and high activities of nitric-oxide (NO) synthase, respectively. Protection from apoptosis by NO was confirmed by results with the NO donor S-nitrosoacetylpenicillamine and the NO-trapping agent hemoglobin. We conclude that the p75(NTR) receptor, while free of NGF, triggers a cascade leading to apoptosis; the cascade includes generation of ceramide and increased caspase activity; and the protective role of NO occurs at step(s) in between the latter events

The nitric oxide-donor molsidomine modulates the innate inflammatory response in a mouse model of muscular dystrophy

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Macrophage plasticity in skeletal muscle repair

Macrophages are one of the first barriers of host defence against pathogens. Beyond their role in innate immunity, macrophages play increasingly defined roles in orchestrating the healing of various injured tissues. Perturbations of macrophage function and/or activation may result in impaired regeneration and fibrosis deposition as described in several chronic pathological diseases. Heterogeneity and plasticity have been demonstrated to be hallmarks of macrophages. In response to environmental cues they display a proinflammatory (M1) or an alternative anti-inflammatory (M2) phenotype. A lot of evidence demonstrated that after acute injury M1 macrophages infiltrate early to promote the clearance of necrotic debris, whereas M2 macrophages appear later to sustain tissue healing. Whether the sequential presence of two different macrophage populations results from a dynamic shift in macrophage polarization or from the recruitment of new circulating monocytes is a subject of ongoing debate. In this paper, we discuss the current available information about the role that different phenotypes of macrophages plays after injury and during the remodelling phase in different tissue types, with particular attention to the skeletal muscle

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell–derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration

Nitric Oxide Confers Therapeutic Activity to Dendritic Cells in a Mouse Model of Melanoma

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs

Ozonated autohemotherapy: protection of kidneys from ischemia in rats subjected to unilateral nephrectomy

<p>Abstract</p> <p>Background</p> <p>Ozonated autohemotherapy (OA) has been previously successfully used in the treatment of patients affected by peripheral occlusive arterial disease. OA consists of an intrafemoral reinfusion of autologous blood previously exposed to a mixture of oxygen/ozone (O₂/O₃). This study analyzes the effects of OA in protecting rat kidney from ischemia and ischemia/reperfusion damage.</p> <p>Methods</p> <p>We performed OA 30 min before the induction of 60 min renal ischemia or at the induction of 60 min postischemic reperfusion in rats subjected to unilateral nephrectomy. In addition, to evidence the possible protection induced by O₂/O₃on endothelial functions, the present study analyzes the in vitro effects of O₂/O₃on oxygen consumption by human umbilical vein endothelial cells (HUVEC).</p> <p>Results</p> <p>1) OA preserves rat kidney functions and architecture, as demonstrated by the improved levels of serum creatinine and blood urea nitrogen and by histology; 2) such protection does not correlate with the increase of plasmatic nitric oxide, but is compatible with a focal renal increase of renal βNADPH-diaphorase; 3) treatment of HUVEC with O₂/O₃significantly increases both the rate of oxygen consumption and the mitochondrial activity assessed by confocal microscopy.</p> <p>Conclusion</p> <p>The preservation of the mitochondrial activity of endothelium could in vivo limit the endothelial dysfunction provoked by the Isc or Isc/R processes.</p

Protective effect of EDTA preadministration on renal ischemia

BACKGROUND: Chelation therapy with sodium edetate (EDTA) improved renal function and slowed the progression of renal insufficiency in patients subjected to lead intoxication. This study was performed to identify the underlying mechanism of the ability of EDTA treatment to protect kidneys from damage. METHODS: The effects of EDTA administration were studied in a rat model of acute renal failure induced by 60 minutes ischemia followed or not by 60 minutes reperfusion. Renal ischemic damage was evaluated by histological studies and by functional studies, namely serum creatinine and blood urea nitrogen levels. Treatment with EDTA was performed 30 minutes before the induction of ischemia. Polymorphonuclear cell (PMN) adhesion capability, plasmatic nitric oxide (NO) levels and endothelial NO synthase (eNOS) renal expression were studied as well as the EDTA protection from the TNFα-induced vascular leakage in the kidneys. Data was compared by two-way analysis of variance followed by a post hoc test. RESULTS: EDTA administration resulted in the preservation of both functional and histological parameters of rat kidneys. PMN obtained from peripheral blood of EDTA-treated ischemized rats, displayed a significant reduction in the expression of the adhesion molecule Mac-1 with respect to controls. NO was significantly increased by EDTA administration and eNOS expression was higher and more diffuse in kidneys of rats treated with EDTA than in the controls. Finally, EDTA administration was able to prevent in vivo the TNFα-induced vascular leakage in the kidneys. CONCLUSION: This data provides evidence that EDTA treatment is able to protect rat kidneys from ischemic damage possibly through the stimulation of NO production