Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels

Background and purpose:

ClC-K kidney Cl⁻ channels are important for renal and inner ear transepithelial Cl⁻ transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above ∼1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA.

Experimental approach:

We used site-directed mutagenesis and two-electrode voltage clamp analysis of wild-type and mutant channels expressed in Xenopus oocytes. Guided by the crystal structure of a bacterial CLC homolog, we screened 97 ClC-Ka mutations for alterations of NFA effects.

Key results:

Mutations of five residues significantly reduced the potentiating effect of NFA. Two of these (G167A and F213A) drastically altered general gating properties and are unlikely to be involved in NFA binding. The three remaining mutants (L155A, G345S and A349E) severely impaired or abolished NFA potentiation.

Conclusions and implications:

The three key residues identified (L155, G345, A349) are localized in two different protein regions that, based on the crystal structure of bacterial CLC homologs, are expected to be exposed to the extracellular side of the channel, relatively close to each other, and are thus good candidates for being part of the potentiating NFA binding site. Alternatively, the protein region identified mediates conformational changes following NFA binding. Our results are an important step towards the development of ClC-Ka activators for treating Bartter syndrome types III and IV with residual channel activity.     

Manipulation of liver regeneration with macrophages to influence the hepatic progenitor cell niche

Insufficient regeneration of the adult liver is believed to cause failure to recover from severe liver disease. An undifferentiated cell population with stem-cell-like qualities known as hepatic progenitor cells (HPCs) is hypothesised to have a central role in regeneration of the adult liver during massive or chronic liver disease. Stem cells in other organ systems are believed to reside in a specialised microenvironment or niche that supports their maintenance and function. The existence of a hepatic stem cell niche might provide a means of therapeutically manipulating endogenous HPCs in vivo as a regenerative therapy.To investigate the physiological potential of HPCs to regenerate the mammalian liver, we have established a novel model of hepatocellular injury and HPC activation using genetic manipulation of hepatocytes. After hepatocyte senescence and death in this model (AhCre Mdm2^flox), HPCs expand and bring about the complete regeneration of the liver parenchyma.We demonstrate that a stereotypical niche, consisting partly of macrophages, exists in both animal models and correlating human disease. Using cell tracking, we show active recruitment of extrahepatic macrophages into this niche during injury. In health, intravenous injection of macrophages results in macrophage engraftment to the liver niche, with subsequent HPC activation and changes to liver structure and function.Within the niche, macrophages use paracrine signalling to control both HPC proliferation and cell fate via TWEAK (tumour-necrosis-factor-like weak inducer of apoptosis) and the Wnt signalling pathway, respectively. After hepatocellular injury, macrophages ingest hepatocyte debris, and release Wnt which promotes HPC differentiation into hepatocytes. TWEAK is vital for HPC proliferation in the AhCre Mdm2^flox model of regeneration. Here, the absence of TWEAK signalling results in liver failure and mortality.This work has demonstrated for the first time the ability of a solid organ to fully regenerate in the adult mammal from progenitor cells, and additionally highlights mechanisms by which this process can be modulated by either small molecule or cell therapy.FundingUniversity of Edinburgh.     

4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4

Background and Purpose

The Ca²⁺-permeable cation channel TRPV4 is activated by mechanical disturbance of the cell membrane and is implicated in mechanical hyperalgesia. Nerve growth factor (NGF) is increased during inflammation and causes mechanical hyperalgesia. 4α-phorbol 12,13-didecanoate (4αPDD) has been described as a selective TRPV4 agonist. We investigated NGF-induced hyperalgesia in TRPV4 wild-type (+/+) and knockout (–/–) mice, and the increases in [Ca²⁺]_i produced by 4αPDD in cultured mouse dorsal root ganglia neurons following exposure to NGF.

Experimental Approach

Withdrawal thresholds to heat, von Frey hairs and pressure were measured in mice before and after systemic administration of NGF. Changes in intracellular Ca²⁺ concentration were measured by ratiometric imaging with Fura-2 in cultured DRG and trigeminal ganglia (TG) neurons during perfusion of TRPV4 agonists.

Key Results

Administration of NGF caused a significant sensitization to heat and von Frey stimuli in TRPV4 +/+ and –/– mice, but only TRPV4 +/+ mice showed sensitization to noxious pressure. 4αPDD stimulated a dose-dependent increase in [Ca²⁺]_i in neurons from +/+ and –/– mice, with the proportion of responding neurons and magnitude of increase unaffected by the genotype. In contrast, the selective TRPV4 agonist GSK1016790A failed to stimulate an increase in intracellular Ca²⁺ in cultured neurons. Responses to 4αPDD were unaffected by pretreatment with NGF.

Conclusions and Implications

TRPV4 contributes to mechanosensation in vivo, but there is little evidence for functional TRPV4 in cultured DRG and TG neurons. We conclude that 4αPDD activates these neurons independently of TRPV4, so it is not appropriate to refer to 4αPDD as a selective TRPV4 agonist.     

Herbicide Phosphinothricin Causes Direct Stimulation Hormesis

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤ 50 μM and growth inhibition at higher concentrations. The growth stimulation at low PPT concentrations is a result of activation of chloroplastic isoform GS2, while the growth suppression is caused by inhibition of both cytosolic GS1 and GS2 at higher PPT concentrations. Since the results are obtained in cell-free system (e.g. protein extracts), to which the principles of homeostasis are not applicable, this PPT effect is an unambiguous example of direct stimulation hormesis. A detailed molecular mechanism of concentration-dependent interaction of both PPT and a related GS inhibitor, methionine sulfoximine, with GS holoenzymes is proposed. The mechanism is in concurrence with all experimental and literature data.     

Distress due to unwanted side-effects of prostate cancer treatment is related to impaired well-being (quality of life)

Based on traditional quality of life scales, it has been suggested that known side-effects of prostate cancer treatment do not influence the quality of life. The present authors have developed an alternative approach to quality of life assessment applying epidemiological methods. Using a self administered questionnaire, we investigated 431 prostate cancer patients and an age stratified sample of 435 randomly selected men. Patients reporting any level of distress due to waning sexual functions (66%) or urine or bowel symptoms (38%), reported a lower psychological well-being compared to patients not reporting these symptoms or patients not distressed by their symptoms. Our results stress that an intact sexual and urinary and bowel functions are important for the quality of life among elderly men with or without prostate cancer.     

Combination of ECMO and cytokine adsorption therapy for severe sepsis with cardiogenic shock and ARDS due to Panton–Valentine leukocidin—positive <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> pneumonia and H1N1

Sepsis-induced cardiogenic shock in combination with severe acute respiratory failure represents a life-threatening combination that is often refractory to the conventional methods of treatment. We describe the case of a 33-year-old patient who developed acute cardiovascular collapse and ARDS secondary to superinfection of Panton–Valentine leukocidin—positive Staphylococcus aureus and H1N1 pneumonia who underwent successful combination therapy for severe sepsis-related cardiomyopathy and respiratory failure using extracorporeal membrane oxygenation and cytokine adsorption therapy.     

Strain‐dependent induction of allergic rhinitis without adjuvant in mice

BACKGROUND: To date, no murine models have been reported to show the induction of both antigen-specific IgE and nasal eosinophilia, two of the major hallmarks of allergic rhinitis, after local sensitization in the absence of adjuvants, a phenomenon which reflects natural exposure. In this report, we attempted to establish a murine model representing an initiation of allergic rhinitis. METHODS: BALB/c, CBA/J, and C57BL/6 mice were sensitized intranasally to Schistosoma mansoni egg antigen (SEA) solely. After repeated sensitization, serum Ab titers, nasal eosinophilia, and cytokine production by nasal lymphocytes were determined. RESULTS: BALB/c mice produced SEA-specific IgE after repeated sensitization. High-dose sensitization to SEA induced IgE production in CBA/J mice, while C57BL/6 mice did not show the production throughout the period observed, suggesting that IgE production was regulated genetically. BALB/c mice also exhibited nasal eosinophilia after the nasal challenge. In addition, nasal lymphocytes sensitized with SEA intranasally produced significant amount of IL-5 in vitro. CONCLUSIONS: These results suggest that intranasal sensitization with SEA in the absence of adjuvants induces a Th2 immune reaction, reflecting the hallmarks of the initiation of allergic rhinitis both in vivo and in vitro, which is genetically regulated.     

胃肠胰胰岛淀粉样多肽的定位和表达

胰岛淀粉样多肽(islet armyloid polypeptide,IAPP)是1986年瑞典学者Westermarket al[1,2 ]从胰岛素瘤患者的瘤组织,糖尿病猫及Ⅱ型糖尿病患者胰岛淀粉样沉积物中分离出来的一种多肽,几乎在同时,英国生物化学家Cooper et al[3,4]也从Ⅱ型糖尿病患者的胰岛淀粉样沉积物中分离出该肽.IAPP又称为amylin.对IAPP的分子结构、基因表达和生理作用等已有许多报道[5].近年来,在IAPP定位、表达及胃肠胰IAPP免疫反应(immunoreactive,IR)细胞定位、发生、发育方面的研究报道,为探讨IAPP的生理作用及疾病状态下的改变,提供了形态学依据,现综述如下.