In addition to the traditional renin-angiotensin system, a great deal of evidence favors the existence of numerous independent tissue-specific renin-angiotensin systems. We report that mast cells are an additional source of renin and constitute a unique extrarenal renin-angiotensin system. We use renin-specific antibodies to demonstrate that cardiac mast cells contain renin. Extending this observation to the human mast cell line HMC-1, we show that these mast cells also express renin. The HMC-1 renin RT-PCR product is 100% homologous to Homo sapiens renin. HMC-1 cells also contain renin protein, as demonstrated both by immunoblot and immunocytochemical analyses. Renin released from HMC-1 cells is active; furthermore, HMC-1 cells are able to synthesize renin. It is known that, in the heart, mast cells are found in the interstitium in close proximity to nerves and myocytes, which both express angiotensin II receptors. Inasmuch as myocardial interstitium contains angiotensinogen and angiotensinconverting enzyme, and because we were able to detect renin only in mast cells, we postulate that the release of renin from cardiac mast cells is the pivotal event triggering local formation of angiotensin II. Because of the ubiquity of mast cells, our results represent a unique paradigm for understanding local renin-angiotensin systems, not just in the heart, but in all tissues. Our findings provide a rationale for targeting mast cells in conjunction with renin-angiotensin system inhibitors in the management of angiotensin II-related dysfunctions.T raditionally the renin-angiotensin system (RAS) has been viewed as a circulating axis, whereby renin is released into the circulation from the kidneys in response to decreased renal perfusion pressure, decreased delivery of NaCl at the macula densa, and͞or increased renal sympathetic nerve activity (1). The rate-limiting step in the formation of angiotensin II (ANG II) is the proteolytic action of renin, which cleaves angiotensinogen (Aogen) to the intermediate angiotensin I (ANG I). ANG I is then converted to ANG II by angiotensin-converting enzyme (ACE) at the endothelial surface (2, 3).In addition to this conventional pathway, many tissues, including heart and brain, are thought to be capable of local ANG II production via tissue-specific RAS (4, 5). Although Aogen, ANG I, and ACE, have been demonstrated in various organs, the presence of renin of extrarenal origin has been more difficult to prove and remains controversial. In this investigation, experiments were designed to determine whether renin could be detected in native tissue other than kidney. Because ANG II plays such a crucial role in cardiovascular disease, we focused our efforts on heart tissue, which we screened for renin by using an established polyclonal anti-renin Ab made to recombinant human renin (6). We found that cardiac mast cells were immunopositive for renin. In addition, we used a cultured mast cell line to extrapolate our observations from fixed heart slices to living cells.Our findings indicate that ma...

A B S T R A C TBackground: PLCg enzymes are key nodes in cellular signal transduction and their mutated and rare variants have been recently implicated in development of a range of diseases with unmet need including cancer, complex immune disorders, inflammation and neurodegenerative diseases. However, molecular nature of activation and the impact and dysregulation mechanisms by mutations, remain unclear; both are critically dependent on comprehensive characterization of the intact PLCg enzymes. Methods: For structural studies we applied cryo-EM, cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry. In parallel, we compiled mutations linked to main pathologies, established their distribution and assessed their impact in cells and in vitro. Findings: We define structure of a complex containing an intact, autoinhibited PLCg1 and the intracellular part of FGFR1 and show that the interaction is centred on the nSH2 domain of PLCg1. We define the architecture of PLCg1 where an autoinhibitory interface involves the cSH2, spPH, TIM-barrel and C2 domains; this relative orientation occludes PLCg1 access to its substrate. Based on this framework and functional characterization, the mechanism leading to an increase in PLCg1 activity for the largest group of mutations is consistent with the major, direct impact on the autoinhibitory interface. Interpretation: We reveal features of PLCg enzymes that are important for determining their activation status. Targeting such features, as an alternative to targeting the PLC active site that has so far not been achieved for any PLC, could provide new routes for clinical interventions related to various pathologies driven by PLCg deregulation. Fund: CR UK, MRC and AstaZeneca.

Askwith T, Zeng W, Eggo MC, Stevens MJ. Oxidative stress and dysregulation of the taurine transporter in high-glucose-exposed human Schwann cells: implications for pathogenesis of diabetic neuropathy. Am J Physiol Endocrinol Metab 297: E620 -E628, 2009. First published July 14, 2009; doi:10.1152/ajpendo.00287.2009.-In human Schwann cells, the role of taurine in regulating glucose-induced changes in antioxidant defense systems has been examined. Treatment with high glucose for 7 days induced reactive oxygen species, increased 4-hydroxynoneal adducts (20 Ϯ 5%, P Ͻ 0.05) and poly-(ADP-ribosyl)ated proteins (40 Ϯ 13%, P Ͻ 0.05). Increases in these markers of oxidative stress were reversed by simultaneous incubation in 0.25 mM taurine. Both high glucose and taurine independently increased superoxide dismutase and catalase activity and decreased glutathione levels, but their effects were not additive. Glucose reduced taurine transporter (TauT) mRNA and protein in a dose-dependent manner with maximal decreases of 66 Ϯ 6 and 63 Ϯ 12%, respectively (P Ͻ 0.05 both). The Vmax for taurine uptake was decreased in 30 mM glucose from 61 Ϯ 5 to 42 Ϯ 3 pmol ⅐ min Ϫ1 ⅐ mg protein Ϫ1(P Ͻ 0.001). Glucose-induced TauT downregulation could be reversed by inhibition of aldose reductase, a pathway that depletes NADPH and increases osmotic stress and protein glycation. TauT protein was increased more than threefold, and the Vmax for taurine uptake doubled (P Ͻ 0.05 both) by prooxidants. TauT downregulation was reversed both by treatment with the antioxidant ␣-lipoic acid, which increased TauT mRNA by 60% and Vmax by 50% (P Ͻ 0.05 both), and by the aldose reductase inhibitor sorbinil, which increased TauT mRNA 380% and Vmax by 98% (P Ͻ 0.01 both). These data highlight the potential therapeutic benefits of taurine supplementation in diabetic complications and provide mechanisms whereby taurine restoration could be achieved in order to prevent or reverse diabetic complications.THE RELATIONSHIP OF HYPERGLYCEMIA to the microvascular complications of diabetes is well established (13a, 76a, 76b), but the mechanisms contributing to the development and progression of complications are not well understood. Recently, increased oxidative/nitrosative stress has been implicated as a key pathogenetic pathway (9,18,62,78). Increased oxidative stress has been identified in the nerve (4, 24, 68), eye (54), vasculature (31), kidney (3, 32), and heart (29) in diabetic rodent models and in diabetic patients (23,25,43,44). Antioxidant approaches have been shown to prevent or reverse complications in experimental diabetes (50). Glucose-driven oxidative stress occurs due to an imbalance in the production of reactive oxygen species (ROS) in concert with impaired antioxidant defense. Excess ROS production can have many downstream pathogenic effects within the cell, including protein nitrosylation, formation of poly(ADP-ribosyl)ated (PAR) protein polymers, and apoptosis (22,35,49,51,56,63). Flux through the enzyme aldose reductase (AR) pathway at key sites for di...

DN is a very common and disabling complication that currently has no effective treatments other than diabetes control. The pathogenesis of DPN is complex and multi-factorial. Several disease-modifying and symptomatic treatments are currently under development. Oxidative and nitrosative stress have been identified as key pathogenic factors in the development of DPN and new treatments target these pathways and/or their downstream consequences. Gene therapy and growth factors have also emerged as potential new therapies that target particular cellular compartments as opposed to being delivered systemically. The recognition of the difficulty in reversing established DN has focused efforts on slowing its progression.