ATP triggers a robust intracellular [Ca2+ ]-mediated signalling pathway in human synovial fibroblasts.

NEW FINDINGS: What is the central question of this study? What are the main [Ca2+ ]i signalling pathways activated by ATP in human synovial fibroblasts? What is the main finding and its importance? In human synovial fibroblasts ATP acts through a linked G-protein (Gq ) and phospholipase C signalling mechanism to produce IP3 , which then markedly enhances release of Ca2+ from the endoplasmic reticulum. These results provide new information for the detection of early pathophysiology of arthritis. ABSTRACT: In human articular joints, synovial fibroblasts (HSFs) have essential physiological functions that include synthesis and secretion of components of the extracellular matrix and essential articular joint lubricants, as well as release of paracrine substances such as ATP. Although the molecular and cellular processes that lead to a rheumatoid arthritis (RA) phenotype are not fully understood, HSF cells exhibit significant changes during this disease progression. The effects of ATP on HSFs were studied by monitoring changes in intracellular Ca2+ ([Ca2+ ]i ), and measuring electrophysiological properties. ATP application to HSF cell populations that had been enzymatically released from 2-D cell culture revealed that ATP (10-100 µm), or its analogues UTP or ADP, consistently produced a large transient increase in [Ca2+ ]i . These changes (i) were initiated by activation of the P2 Y purinergic receptor family, (ii) required Gq -mediated signal transduction, (iii) did not involve a transmembrane Ca2+ influx, but instead (iv) arose almost entirely from activation of endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (IP3 ) receptors that triggered Ca2+ release from the ER. Corresponding single cell electrophysiological studies revealed that these ATP effects (i) were insensitive to [Ca2+ ]o removal, (ii) involved an IP3 -mediated intracellular Ca2+ release process, and (iii) strongly turned on Ca2+ -activated K+ current(s) that significantly hyperpolarized these cells. Application of histamine produced very similar effects in these HSF cells. Since ATP is a known paracrine agonist and histamine is released early in the inflammatory response, these findings may contribute to identification of early steps/defects in the initiation and progression of RA.

Resistance of cotton towards Xanthomonas campestris pv. malvacearum.

Interactions between Gossypium spp. and the bacterial pathogen Xanthomonas campestris pv. malvacearum are understood in the context of the gene-for-gene concept. Reviewed here are the genetic basis for cotton resistance, with reference to resistance genes, resistance gene analogs, and bacterial avirulence genes, together with the physiological mechanisms involved in the hypersensitive response to the pathogen, including production of signaling hormones, synthesis of antimicrobial molecules and alteration of host cell structures. This host-pathogen interaction represents the most complex resistance gene/avr gene system yet known and is one of the few in which phytoalexins are known to be specifically localized in HR cells at anti-microbial concentrations.

Lipid peroxidation in cotton: Xanthomonas interactions and the role of lipoxygenases during the hypersensitive reaction.

Lipid peroxidation, often associated with hypersensitive cell death, may be initiated either by active oxygen species (AOS) or lipoxygenases (LOX). Here we report a detailed analysis of this oxidative process in both incompatible and compatible interactions between the cotton cultivar Reba B50 and Xanthomonas campestris pv. malvacearum (Xcm). The hypersensitive reaction (HR) was characterized by a massive production of polyunsaturated fatty acid (PUFA) hydroperoxides together with typical tissue dehydration. Among these, isomers peroxidized on carbon 9, largely predominant, were chiral, showing an excess in the S enantiomer. The HR process was accompanied by an increase in 9S-LOX activity and preceded by transcription of a LOX gene (GhKLox1). These results showed that: (i) AOS produced during the oxidative burst were not involved in PUFA peroxidation during HR; and (ii) as previously described in elicited leaves of tobacco, the massive enzymatic lipid peroxidation was closely associated with hypersensitive cell death. During disease development in this cotton cultivar, the 9-lipoxygenation of PUFAs was late, weak, preceded by a faint accumulation of GhKLox1 transcripts, and associated with chlorosis but not with necrosis. Consequently, the main difference between incompatible and compatible interactions was in the precocity and intensity of the oxidative process, rather than in its nature. These data provide the evidence for a correlation between lipid peroxidation and hypersensitive cell death induced by pathogens.

Salicylic acid mediated by the oxidative burst is a key molecule in local and systemic responses of cotton challenged by an avirulent race of Xanthomonas campestris pv malvacearum.

We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O(2)(.-)), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H(2)O(2) infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2. 5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O(2)(.-)-generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H(2)O(2) is required for local and systemic accumulation of SA, which may locally control the generation of O(2)(.-). Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.