Extracellular Calcium Stores Research Articles

CML8 and GAD4 function in (Z)–3–hexenol–mediated defense by regulating γ–aminobutyric acid accumulation in Arabidopsis

(Z)–3–hexenol, a small gaseous molecule, is produced in plants under biotic stress and induces defense responses in neighboring plants. However, little is known about how (Z)–3–hexenol induces plant defense–related signaling. In this study, we uncovered how (Z)–3–hexenol treatment enhances plant resistance to insect attacks by increasing γ–aminobutyric acid (GABA) contents in Arabidopsis leaves. First, (Z)–3–hexenol increases the intracellular content of calcium as secondary messenger in Arabidopsis leaf mesophyll cells. Both intracellular and extracellular calcium stores regulate changes in calcium content. Then, CML8 and GAD4 transmit calcium signaling to affect (Z)–3–hexenol induced GABA content and plant resistance. Herein, CML8 interaction with GAD4 was examined via yeast two–hybrid assays, firefly luciferase complementation imaging, and GST pull–down assays. These results indicate that (Z)–3–hexenol treatment increased the GABA contents in Arabidopsis leaves based on CML8 and GAD4, thus increasing plant resistance to the insect Plutella xylostella. This study revealed the mechanism of activating plant insect defense induced by (Z)–3–hexenol, which guides the study of volatiles as biological pest control.

Sarm1 activation produces cADPR to increase intra-axonal Ca++ and promote axon degeneration in PIPN.

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).

Activation of Sarm1 Produces cADPR to Increase Intra-Axonal Calcium and Promote Axon Degeneration in CIPN

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a “dying-back” axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating CIPN.

Effect of reperfusion on vascular smooth muscle reactivity in three contraction models

BackgroundIschemia and reperfusion remain inseparable elements of numerous medical procedures such as by-pass surgery, organ transplantation or other cardiology and intervention radiology. The contraction of the smooth muscle of the vessel is considered to be one of the basic components leading to impaired perfusion, an increase in the oxygen deficit of the endothelium of the vessel, and subsequently also to tissues vascularized by the vessel.Main aim of this study was to evaluate the effect of ischemia and reperfusion on vascular smooth muscle cells stimulated pharmacologically with mastoparan-7 (direct G-protein activator) in comparison to stimulation of G-protein coupled receptor agonist - phenylephrine, and direct calcium channel activator - Bay K8644. Material and methodsExperiments were performed on isolated and perfused tail artery of Wistar rats. Contraction force in our model was measured by increased level of perfusion pressure with a constant flow. ResultsConcentration-response curves obtained for phenylephrine, mastoparan-7 and Bay K8644 presented a sigmoidal relation. Ischemia induced hyporreactivity of vessels, whereas during reperfusion the significant time related hyperreactivity for phenylephrine and mastoparan-7 only but not for Bay K8644. These reactions were secondary to the modulation of calcium influx from intra- and extracellular calcium stores. ConclusionsResults of our experiments suggest that mastoparan-7 significantly induces contraction of vascular smooth muscle cells not only for controls but in the presence of ischemia and reperfusion too. Potential therapeutic applications of the observed reactions are important. They may include regenerative processes within the nervous system, studies on the improvement of blood flow within the microcirculation, or antimicrobial activity. Modulation of the G protein-phospholipase C response may also be an interesting point of action of future drugs modifying the response to stimulation during ischemia in particular, such activities could take place during the transport of organs for transplantation.

Ultracytochemical visualization of calcium distribution in heart cells and erythrocytes of zebrafish Danio rerio

Detection of patterns of subcellular calcium distribution in the cardiovascular system can contribute to understanding its role in cardiac and blood function. The present study localized calcium in heart atrium, ventricle, and bulbus arteriosus as well as in erythrocytes of zebrafish Danio rerio using an oxalate-pyroantimonate technique combined with transmission electron microscopy. Intracellular calcium stores were detected in caveolae, mitochondria, and the nuclei of several zebrafish cardiac cell types. Melanin pigmentation containing calcium stores was detected in the pericardial cavity. Melanin might be an extracellular source of calcium for heart beating and/or a lubricant to prevent friction during beating process. Calcium deposits were also detected in the plasma membrane, cytoplasm and nucleus of erythrocytes as well as in blood plasma. Possible exchange of calcium between erythrocytes and blood plasma was observed. Interactions of such calcium stores and possible contribution of extracellular calcium stores such as melanin pigmentation to supply calcium for vital functions of heart cells should be addressed in future studies.

2,4,6-Trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide increases calcium influx in lipopolisaccharide-pre-treated arteries.

It has been demonstrated that 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide (m-3M3FBS) activates phospholipase C (PLC) and stimulates apoptosis in smooth muscle cells, which may increase vascular reactivity. The primary aim of the present study was to evaluate the physiological effects of the direct stimulation of PLC by m-3M3FBS on vascular smooth muscle reactivity in arteries pre-treated with lipopolysaccharides (LPS) as a model of septic shock. Experiments were performed on isolated and perfused tail arteries of Wistar rats. The contraction force in the model was measured by assessing increases in perfusion pressure at a constant flow. Parameters describing the concentration-response curves (CRCs) obtained for phenylephrine and arginine-vasopressin in the presence of LPS confirmed a decrease in vessels reactivity. In comparison with the controls, m-3M3FBS treatment caused a significant increase in LPS-untreated as well as pre-treated arteries. Furthermore, in the presence of m-3M3FBS, calcium influx from intra- as well as extracellular calcium stores was significantly higher for LPS-untreated and pre-treated arteries. The results of the present study suggested that m-3M3FBS significantly increased the reactivity of vascular smooth muscle cells pre-treated with LPS by increasing the calcium influx from intra- and extracellular calcium stores. Further studies investigating this mechanism are required to evaluate whether this pathway may be a potential therapeutic strategy to treat sepsis.

Effect of 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzene-sulfonamide on calcium influx in three contraction models.

2,4,6-Trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide (m-3M3FBS) activates phospholipase C and stimulates apoptosis; however, in smooth muscle cells it may increase the perfusion pressure. The main aim of the present study was to evaluate the physiological effect of direct stimulation of phospholipase C on vascular smooth muscle reactivity using three contraction models. Experiments were performed on the isolated and perfused tail artery of Wistar rats. The contraction force in the present model was measured by an increased level of perfusion pressure with a constant flow. Concentration-response curves (CRCs) obtained for phenylephrine, arg-vasopressin, mastoparan-7 and Bay K8644 presented a sigmoidal association. In comparison to the control curves, CRCs in the presence of m-3M3FBS were significantly shifted to the left except for Bay K8644. Analyses of calcium influx suggest that in the presence of m-3M3FBS the calcium influx from intra- and extracellular calcium stores was significantly higher. The results of the present experiments suggest that m-3M3FBS significantly increases the reactivity of vascular smooth muscle stimulated with metabotropic receptors or G-protein by an increase in calcium influx from intra- and extracellular calcium stores. The current knowledge regarding the apoptotic pathway shows the significance of calcium ions involved in this process, thus, m-3M3FBS may induce apoptosis by an increase of cytoplasmic calcium concentration; however, simultaneously, the use of this mechanism in therapy must be preceded by a molecular modification that eliminates a possible vasoconstriction effect.

Cyclosporine-A, but not tacrolimus significantly increases reactivity of vascular smooth muscle cells.

Application of cyclosporine-A (CsA) or tacrolimus is associated with numerous side effects. One of the main reasons for restricting usage of CsA is hypertension. In tacrolimus treated subjects the frequency of these phenomena is significantly lower. The known molecular mechanism of action of tacrolimus and cyclosporine-A seems to be the same, thus we decided to compare modulatory effect of drugs on vascular smooth muscle contractility. Experiments were performed on isolated and perfused tail artery of Wistar rats. Contraction force was measured by increased degree of perfusion pressure with a constant flow rate. Concentration-response curves for agonist in the presence CsA were significantly shifted to the left with increase in maximal responses. This effect was due to increased calcium influx from extracellular calcium stores whereas there were no significant changes in calcium influx in the presence of tacrolimus; concentration-response curve was comparable to controls. Our results strongly support the idea that main difference between effects on smooth muscle contractility of calcineurin-dependent immunosuppressants: CsA and tacrolimus is related to the different level of extracellular calcium influx to the cytoplasm. The elucidation of these mechanisms may permit the identification of new therapeutic strategies against CsA-induced hypertension.

Effect of phospholipase C inhibition on vascular smooth muscle contraction

Phospholipase C is the key enzyme responsible for the hydrolysis of membrane phospholipids phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to two intracellular transmitters - diacylglycerol (DAG) and inositol triphosphate (IP 3 ). PLC activation of smooth muscle leads to an increase in the concentration of IP 3 and DAG which initiates the increase in the concentration of calcium ions into the cytoplasm as a result of the flow from the intracellular and then from extracellular pool. Main aim of this study was to evaluate the effect of PLC inhibitor U-73122 on vascular smooth muscle contraction stimulated pharmacologically with phenylephrine (PHE, α 1 -adrenoceptor agonist) and arg-vasopressin (AVP, vasopressin receptor agonist). Material and methods. Experiments were performed on isolated and perfused tail artery of Wistar rats. Contraction force in our model was measured by increased level of perfusion pressure with a constant flow. Results. Reactivity of vascular smooth muscle cells to PHE (10 -9 - 10 -3 M/L), and AVP (10 -10 – 10 -4 M/L), in the control group and in the presence of U-73122 – phospholipase C inhibitor at concentrations 1, 3 and 10 μM/l was analyzed. EC 50 values calculated for PHE and AVP were 7.52 (±0.97) x10 -8 M/L and 1.84 (±0.6) x10 -8 M/L, respectively. The concentration-response curves obtained for PHE and AVP in the presence of U-73122 were shifted to the rightward with a decrease in maximal responses. In the presence of U-73122 the significant and dose dependent reduction in perfusion pressure was found for both agonists for calcium influx from intracellular calcium stores and from extracellular space. Conclusion. Results of our experiments suggest that in the presence of phospholipase C inhibitors, contraction induced by G-protein coupled receptors activation may by effectively inhibited by reduction in calcium influx from intra and extracellular calcium stores.

Modulatory effect of sodium nitroprusside and 8Br-cGMP on mastoparan-7 induced contraction

I n t r o d u c t i o n . Mastoparan-7 activates G-protein and stimulates apoptosis, but in smooth muscle cells leads to increase in perfusion pressure. Main a i m of this study was to evaluate the modulatory effect of 8Br-cGMP and sodium nitroprusside on vascular smooth muscle contraction induced by direct stimulation of G-protein with mastoparan-7. Ma t e r i a l a n d m e t h o d s . Experiments were performed on isolated and perfused tail artery of Wistar rats. Contraction force in our model was measured by increased level of perfusion pressure with a constant flow. R e s u l t s . Concentration-response curves obtained for mastoparan-7 presented an sigmoidal relation. In presence of SNP and 8Br-cGMP the significant and dose dependent leftward shift with significant reduction in maximal responses was present. Moreover, analyzing function of calcium stores the significant inhibition of influx from both, intra- and extracellular calcium stores was present. C o n c l u s i o n . Results of our experiments suggest that contraction induced by direct activation of G-protein with mastoparan-7 may by effectively inhibited in the presence of donors of nitric oxide such as sodium nitroprusside and in the presence of 8Br-cGMP.

Human Amniotic Fluid Stem Cells Seeded in Fibroin Scaffold Produce In Vivo Mineralized Matrix

This study investigated the potential of amniotic fluid stem cells (AFSCs) to synthesize mineralized extracellular matrix (ECM) within different porous scaffolds of collagen, poly-D,L-lactic acid (PDLLA), and silk fibroin. The AFSCs were initially differentiated by using an osteogenic medium in two-dimensional culture, and expression of specific bone proteins and the physiologic mineral production by the AFSCs were analyzed. In particular, during differentiation process, AFSCs expressed proteins like Runt-related transcription factor 2 (Runx2), Osterix, Osteopontin, and Osteocalcin with a sequential expression, analogous to those occurring during osteoblast differentiation, and produced extracellular calcium stores. AFSCs were then cultured on three-dimensional (3D) scaffolds and evaluated for their ability to differentiate into osteoblastic cells in vivo. Stem cells were cultured in vitro for 1 week in collagen, fibroin, and PDLLA scaffolds. The effect of predifferentiation of the stem cells in scaffolds on the subsequent bone formation in vivo was determined in a rat subcutaneous model. With the addition of a third dimension, osteogenic differentiation and mineralized ECM production by AFSCs were significantly higher. This study demonstrated the strong potential of AFSCs to produce 3D mineralized bioengineered constructs in vivo and suggests that fibroin may be an effective scaffold material for functional repair of critical size bone defects.

Role of intra- and extracellular calcium stores in mesothelial cell response to histamine

Ca2+ may play an important role in mesothelial cellular responses because Ca2+ acts as an intracellular messenger in a wide variety of cellular responses in different tissues. The present study was designed to clarify the mechanisms of cytosolic Ca2+ mobilization in the mesothelial cells. Rat pleural and pericardial mesothelial cells were maintained in vitro, and the Ca2+ movement was evaluated using fura 2. Histamine (30 microM to 10 mM) induced a biphasic elevation of intracellular levels of Ca2+ concentration ([Ca2+]i) that consisted of a rapid initial transient elevation followed by a sustained elevation. Neither removal of extracellular Ca2+ nor inhibition of Ca2+ influx by 1 microM nifedipine affected the histamine-induced initial transient elevation of [Ca2+]i in mesothelial cells. Nifedipine did not block histamine-induced sustained elevation of [Ca2+]i. KCl (25 and 50 mM) elicited a biphasic elevation of [Ca2+]i. However, this KCl-induced elevation of [Ca2+]i was abolished by nifedipine treatment. Ryanodine (10 microM) induced a transient elevation of [Ca2+]i in Ca(2+)-free solution. The histamine-induced elevation of [Ca2+]i was completely blocked by H1-receptor antagonists. These results suggest that the mesothelial cells have three pathways to increase [Ca2+]i: release from intracellular Ca2+ stores, Ca2+ influx through L-type voltage-dependent Ca2+ channels, and receptor-operated Ca2+ channels.