Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release

Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation.Transient receptor potential (TRP) channels represent a large and diverse family of nonselective cation channels that respond to a wide range of chemical and physical stimuli and biophysical properties (1). TRP cation channel subfamily M member 3 (TRPM3), a calcium-permeable nonselective cation channel (2), is a typical example of a polymodally gated TRP channel, in that it can be activated by ligands, such as pregnenolone sulfate (PS) and nifedipine, as well as by heat and membrane depolarization (3, 4). Interestingly, recent evidence indicates that combined stimulation with PS and clotrimazole (Clt) leads to the activation of two distinct permeation pathways in TRPM3: the central pore, which is Ca²⁺-permeable and carries an outwardly rectifying current, and an alternative ion permeation pathway that mediates an inwardly rectifying monovalent cation current (5).TRPM3 is highly expressed in somatosensory neurons, where it plays decisive roles in the nocifensive response to PS and heat, as well as in the development of heat hyperalgesia during inflammation (3, 6). In these neurons, TRPM3 is frequently coexpressed with TRPA1 and TRPV1, two TRP channels that have emerged as key regulators of neurogenic inflammation by triggering neuropeptide release from sensory nerve endings (7, 8). Whether activation of TRPM3 can also initiate the release of neuropeptides, such as substance P or calcitonin gene-related peptide (CGRP), which elicit vasodilation, vascular leakage, and other responses in peripheral cell types, is unclear, however. In addition, TRPM3 is expressed in pancreatic beta cells, where it is involved in controlling insulin release (4), as well as in various tissues, including brain, pituitary gland, eye, kidney, and adipose tissue (reviewed in ref. 9). The physiological roles of TRPM3 in these tissues remain only poorly understood, owing in part to the lack of potent and specific pharmacologic tools to modulate its action in vitro and in vivo.Here we describe the identification and characterization of a TRPM3 agonist, CIM0216, with a potency that greatly exceeds that of currently used agonists. This compound has the unique property to open both ion permeation pathways of TRPM3 without the requirement of other channel modulators. We further demonstrate that CIM0216 acts in a TRPM3-dependent manner to induce pain and evoke neuropeptide release from sensory nerve terminals in the skin, and also to release insulin from pancreatic islets. Collectively, these findings provide a novel powerful tool for use in further studies of the physiological functions of TRPM3, and identify TRPM3 as a novel player in neurogenic inflammation.     

Convergence of ion channel genome content in early animal evolution

Multicellularity has evolved multiple times, but animals are the only multicellular lineage with nervous systems. This fact implies that the origin of nervous systems was an unlikely event, yet recent comparisons among extant taxa suggest that animal nervous systems may have evolved multiple times independently. Here, we use ancestral gene content reconstruction to track the timing of gene family expansions for the major families of ion-channel proteins that drive nervous system function. We find that animals with nervous systems have broadly similar complements of ion-channel types but that these complements likely evolved independently. We also find that ion-channel gene family evolution has included large loss events, two of which were immediately followed by rounds of duplication. Ctenophores, cnidarians, and bilaterians underwent independent bouts of gene expansion in channel families involved in synaptic transmission and action potential shaping. We suggest that expansions of these family types may represent a genomic signature of expanding nervous system complexity. Ancestral nodes in which nervous systems are currently hypothesized to have originated did not experience large expansions, making it difficult to distinguish among competing hypotheses of nervous system origins and suggesting that the origin of nerves was not attended by an immediate burst of complexity. Rather, the evolution of nervous system complexity appears to resemble a slow fuse in stem animals followed by many independent bouts of gene gain and loss.Animal nervous systems are complex cellular networks that encode internal states and behavioral output. They achieve this complexity primarily in two ways. First, nervous systems encode information in a wiring scheme whose connections differ in strength and sign (excitatory or inhibitory). The strengths can often change in an activity-dependent fashion (1). Second, nervous systems have a dynamic neural code made up of all-or-none action potentials and subtler graded potentials (2). The shape, timing, and duration of evoked electrical potentials vary greatly among—and even within—neurons and can also be activity-dependent. These two types of complex signaling, respectively, among and within cells are the fundamental work of nervous systems (1), and they are made possible by the great variety of ion channel proteins expressed in neurons.Recent studies have found that most ion channels and proteins involved in the formation of synapses are ancient, having originated long before the advent of nervous systems or even of animal multicellularity (3–7). However, the nature of the first animals and of the cells from which nervous systems evolved are not well understood, although many theories exist (8–11), and little is known about the genomic events that facilitated the rise of complex nervous systems. New information about animal phylogeny has demanded a return to these old questions concerning the nature of the first animals and the evolutionary history of nervous systems (12–15).This new information concerns the placement of the ctenophores, or comb jellies. Recent studies place ctenophores as the sister group to all other metazoans, a surprising finding given that ctenophores are complex predators with fairly sophisticated nervous systems (15). In contrast, sponges, which traditionally were considered to be the sister group of the remaining animals (16), and placozoans do not have nervous systems (but see refs. 17 and 18). Recent genomic analyses have found that ctenophores are lacking many nervous system and muscle-associated genes, suggesting independent origins of these structures in ctenophores (15, 19, 20). Conversely, the genomic presence and expression of certain developmental genes involved in nervous system differentiation (13, 14) and genes expressed in the synapse (13, 21) indicate deep similarities between ctenophore nervous systems and others. These findings have revived the debate about whether animal nervous systems have one or more origins. Although it is clear that there has been some degree of homoplasy and/or secondary simplifications or losses, the nature and timing of these events remains debatable (refs. 14, 20, and 21 are excellent reviews on this subject).Many studies have addressed the origin of animal nervous systems by using comparative physiological, developmental, or morphological evidence (22–24). We used a different technique: ancestral gene content reconstruction. This approach has been used to explore the origin of multicellularity (25), the evolution of prokaryotic metabolism (26), and the expansion of G-protein–coupled receptors in animals (27). Gene duplication has long been known to be a major source of novelty and complexity (28), and many of the families we analyzed play few known roles outside of nervous systems. We therefore hypothesized that the elaboration of nervous systems coincided with an expansion of the ion-channel families that are expressed there. We used two methods (27, 29) to reconstruct the ancestral copy number for a variety of ion channel families and tracked the evolution of gene duplications across the animal and fungal tree. The evolution of some of these families have been studied by other groups (15, 27, 30–32), but here we combine current methods of ancestral genome content reconstruction with dense sampling of early branching species and gene families to search for patterns of gene duplication that might illuminate the early history of nervous systems.     

通道下经椎间孔微创腰椎融合联合经皮椎弓根内固定治疗腰椎间盘突出伴腰椎不稳

目的：探讨通道下经椎间孔微创腰椎融合（TLIF）联合经皮椎弓根内固定治疗腰椎间盘突出伴腰椎不稳的临床效果。方法对82例腰椎间盘突出伴腰椎不稳患者采用通道下微创 TLIF 联合经皮椎弓根内固定治疗。结果所有患者均获随访,时间4～36个月。患者术后腰腿痛症状均明显缓解或消失,JOA 评分术前为（8．52±1．2）分,术后末次随访时为（26．32±1．6）分,差异有统计学意义（P ＜0．05）;其中优68例,良10例,中4例。植骨融合率95．12％。结论通道下微创 TLIF 联合经皮椎弓根内固定治疗腰椎间盘突出伴腰椎不稳具有创伤小、并发症少等优点,临床疗效满意。     

Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis

Transient receptor potential canonical (TRPC) Ca²⁺-permeant channels, especially TRPC3, are increasingly implicated in cardiorenal diseases. We studied the possible role of fibroblast TRPC3 in the development of renal fibrosis. In vitro, a macromolecular complex formed by TRPC1/TRPC3/TRPC6 existed in isolated cultured rat renal fibroblasts. However, specific blockade of TRPC3 with the pharmacologic inhibitor pyr3 was sufficient to inhibit both angiotensin II- and 1-oleoyl-2-acetyl-sn-glycerol–induced Ca²⁺ entry in these cells, which was detected by fura-2 Ca²⁺ imaging. TRPC3 blockade or Ca²⁺ removal inhibited fibroblast proliferation and myofibroblast differentiation by suppressing the phosphorylation of extracellular signal-regulated kinase (ERK1/2). In addition, pyr3 inhibited fibrosis and inflammation-associated markers in a noncytotoxic manner. Furthermore, TRPC3 knockdown by siRNA confirmed these pharmacologic findings. In adult male Wistar rats or wild-type mice subjected to unilateral ureteral obstruction, TRPC3 expression increased in the fibroblasts of obstructed kidneys and was associated with increased Ca²⁺ entry, ERK1/2 phosphorylation, and fibroblast proliferation. Both TRPC3 blockade in rats and TRPC3 knockout in mice inhibited ERK1/2 phosphorylation and fibroblast activation as well as myofibroblast differentiation and extracellular matrix remodeling in obstructed kidneys, thus ameliorating tubulointerstitial damage and renal fibrosis. In conclusion, TRPC3 channels are present in renal fibroblasts and control fibroblast proliferation, differentiation, and activation through Ca²⁺-mediated ERK signaling. TRPC3 channels might constitute important therapeutic targets for improving renal remodeling in kidney disease.     

Store–Operated Ca2+ Channels in Mesangial Cells Inhibit Matrix Protein Expression

Accumulation of extracellular matrix derived from glomerular mesangial cells is an early feature of diabetic nephropathy. Ca²⁺ signals mediated by store–operated Ca²⁺ channels regulate protein production in a variety of cell types. The aim of this study was to determine the effect of store–operated Ca²⁺ channels in mesangial cells on extracellular matrix protein expression. In cultured human mesangial cells, activation of store–operated Ca²⁺ channels by thapsigargin significantly decreased fibronectin protein expression and collagen IV mRNA expression in a dose-dependent manner. Conversely, inhibition of the channels by 2-aminoethyl diphenylborinate significantly increased the expression of fibronectin and collagen IV. Similarly, overexpression of stromal interacting molecule 1 reduced, but knockdown of calcium release–activated calcium channel protein 1 (Orai1) increased fibronectin protein expression. Furthermore, 2-aminoethyl diphenylborinate significantly augmented angiotensin II–induced fibronectin protein expression, whereas thapsigargin abrogated high glucose– and TGF-β1–stimulated matrix protein expression. In vivo knockdown of Orai1 in mesangial cells of mice using a targeted nanoparticle siRNA delivery system resulted in increased expression of glomerular fibronectin and collagen IV, and mice showed significant mesangial expansion compared with controls. Similarly, in vivo knockdown of stromal interacting molecule 1 in mesangial cells by recombinant adeno–associated virus–encoded shRNA markedly increased collagen IV protein expression in renal cortex and caused mesangial expansion in rats. These results suggest that store–operated Ca²⁺ channels in mesangial cells negatively regulate extracellular matrix protein expression in the kidney, which may serve as an endogenous renoprotective mechanism in diabetes.     

Transient receptor potential channel modulators as pharmacological treatments for lower urinary tract symptoms (LUTS): myth or reality?

Transient receptor potential (TRP) channels belong to the most intensely pursued drug targets of the last decade. These ion channels are considered promising targets for the treatment of pain, hypersensitivity disorders and lower urinary tract symptoms (LUTS). The aim of the present review is to discuss to what extent TRP channels have adhered to their promise as new pharmacological targets in the lower urinary tract (LUT) and to outline the challenges that lie ahead.

• TRP vanilloid 1 (TRPV1) agonists have proven their efficacy in the treatment of neurogenic detrusor overactivity (DO), albeit at the expense of prolonged adverse effects as pelvic ‘burning’ pain, sensory urgency and haematuria.
• TRPV1 antagonists have been very successful in preclinical studies to treat pain and DO. However, clinical trials with the first generation TRPV1 antagonists were terminated early due to hyperthermia, a serious, on‐target, side‐effect.
• TRP vanilloid 4 (TRPV4), TRP ankyrin 1 (TRPA1) and TRP melastatin 8 (TRPM8) have important sensory functions in the LUT. Antagonists of these channels have shown their potential in pre‐clinical studies of LUT dysfunction and are awaiting clinical validation.

     

Association of hospital construction with the development of healthcare associated environmental mold infections (HAEMI) in pediatric patients with leukemia

Background

Healthcare associated mold infections (HAEMI) increase morbidity and mortality in children with leukemia. Excavation adjacent to Children's Medical Center Dallas (CMCD) April 2006–February 2007 provided an opportunity to determine if excavation adjacent to a hospital building is associated with increased risk of developing HAEMI in children receiving intensive chemotherapy for acute leukemia.

Methods

Children who began receiving intensive chemotherapy for acute leukemia at CMCD from 2004 to 2008 were identified (n = 275). Exposures to the CMCD campus during intensive chemotherapy and duration of neutropenia per exposure were recorded. Proven, probable, or possible invasive fungal disease (IFD) was classified using EORTC/MSG guidelines. Institutional guidelines categorized mold infections as definite or possible HAEMI. A bivariate time‐to‐event model compared the association of excavation with HAEMI and yeast infections, controlling for neutropenia.

Results

There were 7,454 CMCD exposures, 1,007 (13.5%) during excavation. Of 50 cases of IFD, 31 were HAEMI. By time‐to‐event analysis exposure to the CMCD campus during the excavation period was significantly associated with HAEMI (HR = 2.8, P = 0.01) but not yeast infections (HR = 0.75, P = 0.75). Neutropenia was significantly associated with both HAEMI and yeast infections (P < 0.001). Voriconazole prophylaxis did not prevent HAEMI in 42% of the 14 patients with AML who had been receiving this agent.

Conclusion

This study is the first to demonstrate an association between exposure to hospital construction that includes excavation and HAEMI in pediatric oncology patients. Since neutropenic patients need protection from aerosolized fungal spores during visits to expanding medical centers, preventive strategies with adherence monitoring need additional study. Pediatr Blood Cancer 2014;61:276–280. © 2013 Wiley Periodicals, Inc.     

浅谈仲景辛凉解表法

辛凉解表法虽首创于刘完素,完善于温病,但却溯源于仲景,仲景四方大青龙汤、越婢汤、麻杏石甘汤、白虎汤完整的体现了辛凉解表的思想。表热的根本在于卫气的阻滞,解表法最在意使卫气恢复流通,辛温、辛凉解表均不离辛者,以辛味发散或透达,邪气可随之出表,若单着眼于出汗者,已然失其本意。辛热开通气机,本不在发汗,汗液乃是随同郁热的开通一同从腠理而出,不同于西药单纯以发汗带出热量,而郁热并未解除。所以,纵使辛温解表,热如麻黄、桂枝也非为单纯发汗而设,乃取其辛散开通之意,汗液自蒸蒸而协同邪热一并而出。     

石决明散加减方治疗肝经积热型前葡萄膜炎的临床研究

目的观察中药石决明散加减方治疗肝经积热型前葡萄膜炎的临床效果。方法前葡萄膜炎患者60例随机分为两组各30例,对照组采用眼局部治疗,治疗组在对照组的基础上加用中药石决明散加减方中医辨证治疗,对比分析两组治疗效果。结果总有效率:治疗组为86.7%,对照组为76.7%,两组比较差异有统计学意义(P0.05);两组在随访中,复发率治疗组为10.0%,对照组为26.7%,差异有统计学意义(P0.05);中西医结合治疗其疗效优于对照组。结论中药石决明散加减方配合眼局部治疗可以缩短病程,提高疗效,减少复发。     

通督热针法与捻转补法治疗早期强直性脊柱炎对比研究

目的比较通督热针法与捻转补法治疗早期强直性脊柱炎的临床效果。方法选取在我院就诊的早期强直性脊柱炎患者76例,随机分为两组各38例。对照组实施捻转补法治疗,观察组采用通督热针法治疗,比较两组巴氏强直性脊柱炎疾病活动指数(BASDAI)、巴氏强直性脊柱炎功能指数(BASFI)评分及疗效。结果治疗前,两组患者BASDAI和BASFI评分比较,差异不显著(P0.05);治疗后,观察组BASDAI和BASFI评分均明显低于对照组,差异显著(P0.05);观察组治疗效果较对照组明显提高,差异显著(P0.05)。结论与捻转补法治疗比较,通督热针法可提高早期强直性脊柱炎患者治疗效果,改善患者症状。