Chloride, glutathiones, and insect-derived elicitors introduced into the xylem trigger electrical signaling.

Ricca assays allow the direct introduction of compounds extracted from plants or the organisms that attack them into the leaf vasculature. Using chromatographic fractionation of Arabidopsis (Arabidopsis thaliana) leaf extracts, we found glutamate was the most active low mass elicitor of membrane depolarization. However, other known elicitors of membrane depolarization are generated in the wound response. These include unstable aglycones generated by glucosinolate (GSL) breakdown. None of the aglycone-derived GSL-breakdown products, including nitriles and isothiocyanates, that we tested using Ricca assays triggered electrical activity. Instead, we found that glutathione and the GSL-derived compound sulforaphane glutathione triggered membrane depolarizations. These findings identify a potential link between GSL breakdown and glutathione in the generation of membrane depolarizing signals. Noting that the chromatographic fractionation of plant extracts can dilute or exchange ions, we found that Cl- caused glutamate receptor-like3.3-dependent membrane depolarizations. In summary, we show that, in addition to glutamate, glutathione derivatives as well as chloride ions will need to be considered as potential elicitors of wound-response membrane potential change. Finally, by introducing aphid (Brevicoryne brassicae) extracts or the flagellin-derived peptide flg22 into the leaf vasculature we extend the use of Ricca assays for the exploration of insect/plant and bacteria/plant interactions.

Dietary Potassium Supplementation Reduces Chronic Kidney Lesions Independent of Blood Pressure in Deoxycorticosterone-Acetate and High Sodium Chloride-Treated Mice.

We have previously shown that an excess of deoxycorticosterone acetate and high sodium chloride intake (DOCA/salt) in one-renin gene mice induces a high urinary Na/K ratio, hypokalemia, and cardiac and renal hypertrophy in the absence of hypertension. Dietary potassium supplementation prevents DOCA/salt-induced pathological processes. In the present study, we further study whether DOCA/salt-treated mice progressively develop chronic inflammation and fibrosis in the kidney and whether dietary potassium supplementation can reduce the DOCA/salt-induced renal pathological process. Results showed that (1) long-term DOCA/salt-treated one-renin gene mice developed severe kidney injuries including tubular/vascular hypertrophy, mesangial/interstitial/perivascular fibrosis, inflammation (lymphocyte's immigration), proteinuria, and high serum creatinine in the absence of hypertension; (2) there were over-expressed mRNAs of plasminogen activator inhibitor-1 (PAI-1), fibronectin, collagen type I and III, interferon-inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP1), transforming growth factor-ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), osteopontin, Nuclear factor kappa B (NF-κB)/P65, and intercellular adhesion molecule (ICAM)-1; and (3) dietary potassium supplementation normalized urinary Na/K ratio, hypokalemia, proteinuria, and serum creatinine, reduced renal hypertrophy, inflammations, and fibrosis, and down-regulated mRNA expression of fibronectin, Col-I and III, TGF-ß, TNF-α, osteopontin, and ICAM without changes in the blood pressure. The results provide new evidence that potassium and sodium may modulate proinflammatory and fibrotic genes, leading to chronic renal lesions independent of blood pressure.

Molecular basis for selective uptake and elimination of organic anions in the kidney by OAT1.

In mammals, the kidney plays an essential role in maintaining blood homeostasis through the selective uptake, retention or elimination of toxins, drugs and metabolites. Organic anion transporters (OATs) are responsible for the recognition of metabolites and toxins in the nephron and their eventual urinary excretion. Inhibition of OATs is used therapeutically to improve drug efficacy and reduce nephrotoxicity. The founding member of the renal organic anion transporter family, OAT1 (also known as SLC22A6), uses the export of α-ketoglutarate (α-KG), a key intermediate in the Krebs cycle, to drive selective transport and is allosterically regulated by intracellular chloride. However, the mechanisms linking metabolite cycling, drug transport and intracellular chloride remain obscure. Here, we present cryogenic-electron microscopy structures of OAT1 bound to α-KG, the antiviral tenofovir and clinical inhibitor probenecid, used in the treatment of Gout. Complementary in vivo cellular assays explain the molecular basis for α-KG driven drug elimination and the allosteric regulation of organic anion transport in the kidney by chloride.

Lysosomal chloride transporter CLH-6 protects lysosome membrane integrity via cathepsin activation.

Lysosomal integrity is vital for cell homeostasis, but the underlying mechanisms are poorly understood. Here, we identify CLH-6, the C. elegans ortholog of the lysosomal Cl-/H+ antiporter ClC-7, as an important factor for protecting lysosomal integrity. Loss of CLH-6 affects lysosomal degradation, causing cargo accumulation and membrane rupture. Reducing cargo delivery or increasing CPL-1/cathepsin L or CPR-2/cathepsin B expression suppresses these lysosomal defects. Inactivation of CPL-1 or CPR-2, like CLH-6 inactivation, affects cargo digestion and causes lysosomal membrane rupture. Thus, loss of CLH-6 impairs cargo degradation, leading to membrane damage of lysosomes. In clh-6(lf) mutants, lysosomes are acidified as in wild type but contain lower chloride levels, and cathepsin B and L activities are significantly reduced. Cl- binds to CPL-1 and CPR-2 in vitro, and Cl- supplementation increases lysosomal cathepsin B and L activities. Altogether, these findings suggest that CLH-6 maintains the luminal chloride levels required for cathepsin activity, thus facilitating substrate digestion to protect lysosomal membrane integrity.

Effects of Various Copper Sources and Concentrations on Performance, Skeletal Growth, and Mineral Content of Excreta in Broiler Chickens.

The experiment was designed to study the effect of supplemental sources and concentrations of copper on the performance and development and mineralization of tibia bones in broiler chickens. A 42-day feeding experiment was conducted utilising three copper sources, including copper sulphate (CuS), copper chloride (CuCl), and copper propionate (CuP), each with four different concentrations, i.e. 8, 100, 150, and 200 mg/kg. The body weight gain with 200 mg Cu/kg food was noticeably higher during the first 4-6 weeks of age. Due to the interaction between Cu sources and levels, there was no significant change in the body weight gained. The feed intake during various growing phases did differ significantly neither the main effect nor the interaction between different copper sources and levels. A CuP-supplemented diet (200 mg/kg food) considerably (P ≤ 0.05) improved the feed conversion ratio between 4-6 and 0-6 weeks. At the end of the experiment, a total of 72 tibia bones, i.e. six for each treatment were collected. A metabolic trial was conducted to look into mineral retention in broiler chickens on the final 3 days of the trial (40-42 days). Increased tibia bone zinc (Zn) levels were seen with the addition of 8 mg Cu/kg of Cu chloride, 100 mg Cu/kg of Cu propionate, 8 mg Cu/kg of Cu sulphate, and 8 mg/kg of Cu propionate to the diet. At higher levels of Cu (150 and 200 mg/kg diet), there was a significantly (P ≤ 0.01) reduced tibia Zn content. Cu sulphate treatment group had higher (P ≤ 0.01) tibia Cu content (8 mg Cu/kg diet). Cu sulphate supplemented diet had a greater excreta Zn content (P ≤ 0.01) than Cu chloride supplemented diet, and Cu propionate supplemented diet had the lowest excreta Zn content. Excreta with a higher Fe concentration were found in diets supplemented with copper sulphate and copper chloride (P ≤ 0.05) than in diets supplied with copper propionate. Thus, it may be concluded that feeding dietary Cu concentrations up to 200 mg Cu/kg diet, regardless of the different sources, had no negative effects on bone morphometry and mineralization parameters with the exception of a decrease in the tibia's zinc content.

Zerumbone, a major sesquiterpene from Syringa pinnatifolia Hemsl., exerts the sedative effect by regulating GABAergic nervous system.

ETHNOPHARMACOLOGICAL RELEVANCE: Zerumbone (ZER) is a humulane sesquiterpenoid isolated from Syringa pinnatifolia Hemsl. (SP), its content accounts for 64.7% of volatile oil and 0.86% of total ethanol extract (TEE), representing one of characteristic ingredient of SP. As a representative Mongolian medicine with anti-"Khii", anti-asthma, and clearing-heat effects, SP has been used for the treatment of cardiovascular diseases, upset, insomnia, and other symptoms. AIM OF STUDY: Previous results showed that TEE has sedative effect, but the pharmacological substances and its sedative mechanism remains unclear. This study aims to determine whether ZER, as one of major and characteristic sesquiterpenoids of SP, contributes to the sedative effect of SP and its underlying mechanism. MATERIALS AND METHODS: Locomotor activity and threshold dose of pentobarbital sodium sleep experiments were used to evaluate the sedative effects in mice. ELISA assay was used to examine the level of GABA/Glu ratio in rats hippocampus, cortex and hypothalamus tissue. The binding ability of ZER with glutamic acid decarboxylase 67 (GAD67) and Gephyrin protein were predicted by molecular docking. Western blot and Immunohistochemistry assay were used to determine the expression of GABAergic nerve system related proteins (GAD67, Gephyrin) in rat's hypothalamus. ZER was co-administrated with flumazenil and bicuculline (GABAA antagonist) to determine whether it acts on GABAA receptor. Furthermore, MQAE assay was used to test the effect of ZER on the chloride ion concentration in cerebellar granule cells. RESULTS: Current data demonstrated that ZER dose-dependently (5-20 mg/kg) reduces the locomotor activity and sleep latency of mice, and extend sleeping time of mice. The results of ELISA showed that ZER increases the level of GABA/Glu in rats brain tissue, in particular in hypothalamus. Molecular docking results revealed that ZER has a strong affinity to GAD67 and Gephyrin protein. The Western blot and Immunohistochemistry data indicated that ZER up-regulates the expression of GAD67 and Gephyrin protein in rat's hypothalamus. Antagonism test results demonstrated that flumazenil and bicuculline reverse the effect of ZER on threshold dose of pentobarbital sodium sleep experiments. In addition, ZER also could dose-dependently (5-20 µM) increase the chloride ion concentration in cerebellar granule cell, suggesting that ZER induces the opening of chloride channel, exerts central inhibitory effect. CONCLUSION: ZER has a significant sedative effect in mice and rat, and the effect is associated with GABAergic nervous system. The present results suggest that ZER, as one of the major bioactive ingredients of SP, contributes to the sedative effect and provide substantial evidence for its traditional use of anti-"Khii" in clinic of Syringa pinnatifolia.

The therapeutic potential of berberine chloride against SARM1-dependent axon degeneration in acrylamide-induced neuropathy.

Chronic acrylamide (ACR) intoxication causes typical pathology of axon degeneration. Moreover, sterile-α and toll/interleukin 1 receptor motif-containing protein 1 (SARM1), the central executioner of the programmed axonal destruction process under various insults, is up-regulated in ACR neuropathy. However, it remains unclear whether inhibitors targeting SARM1 are effective or not. Among all the pharmacological antagonists, berberine chloride (BBE), a natural phytochemical and the first identified non-competitive inhibitor of SARM1, attracts tremendous attention. Here, we observed the protection of 100 µM BBE against ACR-induced neurites injury (2 mM ACR, 24 hr) in vitro, and further evaluated the neuroprotective effect of BBE (100 mg/kg p.o. three times a week for 4 weeks) in ACR-intoxicated rats (40 mg/kg i.p. three times a week for 4 weeks). The expression of SARM1 was also detected. BBE intervention significantly inhibited the overexpression of SARM1, ameliorated axonal degeneration, alleviated pathological changes in the sciatic nerve and spinal cord, and improved neurobehavioral symptoms in ACR-poisoned rats. Thus, BBE exhibits a strong neuroprotective effect against the SARM1-dependent axon destruction in ACR neuropathy. Meanwhile, our study underscores the need for appropriate inhibitor selection in diverse situations that would benefit from blocking the SARM1-dependent axonal destruction pathway.

Electroacupuncture attenuates ischemic injury after stroke and promotes angiogenesis via activation of EPO mediated Src and VEGF signaling pathways.

Although electroacupuncture (EA) has been shown to be effective in the treatment of stroke, its mechanisms of action remain undefined. This study explored the therapeutic effects of EA in rats with cerebral ischemia-reperfusion injury (CIRI) and evaluated its possible mechanisms in promoting angiogenesis. To evaluate the effect of EA, we used 2, 3, 5-Triphenyl-2H-Tetrazolium Chloride (TTC) staining and behavior score to calculate the cerebral infarct volume and neurological deficit score after CIRI. Western blot (WB) analysis was employed to evaluate the expression of cluster of differentiation 34 (CD34), erythropoietin (EPO), vascular endothelial growth factor (VEGF) and phospho-Src (p-Src) in the brain of the rats with CIRI. On the other hand, we established an oxygen-glucose deprivation/reoxygenation (OGD/R) injury model using brain microvascular endothelial cells (BMECs), and analyzed cell viability and expression of VEGF or p-Src using cell counting kit-8 (CCK-8) and WB, respectively. Our data showed that EA at the GV26 acupoint could significantly promote the expression of CD34, EPO, VEGF and p-Src in CIRI rats. Our CCK-8 results demonstrated that intervention with recombinant EPO and VEGF proteins remarkably improved the viability of BMECs after OGD/R, while a Src inhibitor, PP1, reversed this phenotype. The WB results showed that the recombinant EPO protein increased the expression of VEGF and p-Src, which was significantly inhibited by PP1. Taken together, our findings showed that EA at the GV26 acupoint can significantly attenuate ischemic injury after stroke and promote angiogenesis via activation of EPO-mediated Src and VEGF signaling pathways. Besides, the upregulation of VEGF may also be associated with the activation of Src by EPO.

Differential sensitivity of metabolic pathways in sugar beet roots to combined salt, heat, and light stress.

Plants in nature commonly encounter combined stress scenarios. The response to combined stressors is often unpredictable from the response to single stresses. To address stress interference in roots, we applied salinity, heat, and high light to hydroponically grown sugar beet. Two main patterns of metabolomic acclimation were apparent. High salt of 300 mM NaCl considerably lowered metabolite amounts, for example, those of most amino acids, γ-amino butyric acid (GABA), and glucose. Very few metabolites revealed the opposite trend with increased contents at high salts, mostly organic acids such as citric acid and isocitric acid, but also tryptophan, tyrosine, and the compatible solute proline. High temperature (31°C vs. 21°C) also frequently lowered root metabolite pools. The individual effects of salinity and heat were superimposed under combined stress. Under high light and high salt conditions, there was a significant decline in root chloride, mannitol, ribulose 5-P, cysteine, and l-aspartate contents. The results reveal the complex interaction pattern of environmental parameters and urge researchers to elaborate in much more detail and width on combinatorial stress effects to bridge work under controlled growth conditions to growth in nature, and also to better understand acclimation to the consequences of climate change.

Unveiling unstable non-acid incidence in Holstein cows fed with corn silage or sugarcane.

We aimed to evaluate the incidence of unstable non-acid milk (UNAM) in cows fed either sugarcane or corn silage. Second, we aimed to evaluate the effect of daily variation (d 1 to 4) and alcohol grades (72, 78, and 80%) on UNAM incidence. The experiment was conducted as a split-plot crossover design, with 2 periods and 2 roughage types (sugarcane or corn silage). Thirteen multiparous Holstein cows with an average of 281 ± 29 d in milk were randomly distributed into 2 diets. Individual blood (analysis of total proteins, albumin, urea, calcium, phosphorus, magnesium, iron, chloride, glucose, and lactate) and milk samples (analysis of protein, fat, lactose and total solids, somatic cell count, and characterization of the protein profile) were collected during the last 4 d of each period. For UNAM identification, the alcohol test was conducted in milk samples at 4°C; specifically, if the sample presented the formation of clots, this would be noted as positive for UNAM. In addition, the Dornic acidity analysis was performed in the same samples to evaluate the true milk acidity. The use of sugarcane and higher degrees of alcohol were associated with increased UNAM. We observed no daily variation in UNAM. Nevertheless, we found no roughage type effect on the variables most commonly associated with UNAM, such as changes in salts in the casein micelle and, consequently, the zeta potential and the κ-casein (CN) fraction. The Pearson correlation analysis showed that the zeta potential and the concentrations of αS2-CN, blood ionic calcium, lactate, and glucose increased as the incidence of UNAM increased, showing a positive correlation among these variables. In contrast, the concentrations of lactose, phosphorus, and potassium decreased as UNAM increased, presenting a negative correlation. This study brought important discoveries to unveil why cows manifest UNAM. For instance, higher alcohol grades and cows fed with sugarcane had increased the incidence of UNAM. Additionally, animals with a higher incidence of UNAM (sugarcane-fed cows) were related to increased ionic calcium and glucose and changes in milk protein profile, with lower levels of BSA, ß-CN, and α-lactalbumin and greater αS1-CN content, all of which were correlated with UNAM. Nonetheless, this trial also provides evidence for the need for further studies to better understand the physiological mechanisms that directly affect the stability of milk protein.

The power of a novel combined anticancer therapy: challenge and opportunity of micotherapy in the treatment of Glioblastoma Multiforme.

Glioblastoma (GBM) is the most common and mortal primary brain tumor in human. After standard therapies, that include surgical resection followed by radiotherapy and chemotherapy, it is difficult to completely remove the tumor and the development of relapses and resistance is almost inevitable. The chemotherapy now available also show important side effects, to overcame those limitation, new platinum-based drugs are being synthetized, Pt(IV)Ac-POA, (OC-6-44)-acetate-diamine-chloride(2-(2-propynyl)octanoato)platinum(IV), a prodrug having an Histone-3-DeAcetylase-Inhibitor as axial ligands, is one of them. Moreover, new compounds of plant origin are increasingly seen as potential sources of benefits in oncological treatments. The aim of the study is to investigate the possible contribution of micotherapy in the fight against GBM, its role in the metabolism of reactive oxygen species (ROS) and its synergic effect with a new platinum-based compound, Pt(IV)Ac-POA, on human glioblastoma U251 cells. Through cytofluorimetric and immunofluorescence analysis, the ability of the micotherapy in study to regulate the cell cycle was assessed, and its importance in controlling the cellular redox state was also revealed, opening to the possibility of a new therapy in which micotherapy can support the activity of new chemotherapy while reducing its side effects controlling inflammatory conditions in the microenvironment. Additionally, the combined therapy appeared able to induce regulated form of necrosis, such as ferroptosis, and to hinder the establishment of resistance mechanisms.

Alterations in Intestinal Brush Border Membrane Functionality and Bacterial Populations Following Intra-Amniotic Administration (Gallus gallus) of Nicotinamide Riboside and Its Derivatives.

Nicotinamide riboside (NR) acts as a nicotinamide adenine dinucleotide (NAD+) precursor where NR supplementation has previously been shown to be beneficial. Thus, we synthesized and characterized nicotinamide riboside tributyrate chloride (NRTBCl, water-soluble) and nicotinamide riboside trioleate chloride (NRTOCl, oil-soluble) as two new ester derivatives of nicotinamide riboside chloride (NRCl). NRCl and its derivatives were assessed in vivo, via intra-amniotic administration (Gallus gallus), with the following treatment groups: (1) non-injected (control); and injection of (2) deionized H2O (control); (3) NRCl (30 mg/mL dose); (4) NRTBCl (30 mg/mL dose); and (5) NRTOCl (30 mg/mL dose). Post-intervention, the effects on physiological markers associated with brush border membrane morphology, intestinal bacterial populations, and duodenal gene expression of key proteins were investigated. Although no significant changes were observed in average body weights, NRTBCl exposure increased average cecum weight. NR treatment significantly increased Clostridium and NRCl treatment resulted in increased populations of Bifidobacterium, Lactobacillus, and E. coli. Duodenal gene expression analysis revealed that NRCl, NRTBCl, and NRTOCl treatments upregulated the expression of ZnT1, MUC2, and IL6 compared to the controls, suggesting alterations in brush border membrane functionality. The administration of NRCl and its derivatives appears to trigger increased expression of brush border membrane digestive proteins, with added effects on the composition and function of cecal microbial populations. Additional research is now warranted to further elucidate the effects on inflammatory biomarkers and observe changes in the specific intestinal bacterial populations post introduction of NR and its derivatives.

Cucumis sativus extract elicits chloride secretion by stimulation of the intestinal TMEM16A ion channel.

CONTEXT: Cucumber (Cucumis sativus Linn. [Cucurbitaceae]) is widely known for its purgative, antidiabetic, antioxidant, and anticancer therapeutic potential. However, its effect on gastrointestinal (GI) disease is unrecognised. OBJECTIVE: This study investigated the effect of C. sativus fruit extract (CCE) on intestinal chloride secretion, motility, and motor function, and the role of TMEM16A chloride channels. MATERIALS AND METHODS: CCE extracts were obtained from commercially available cucumber. Active fractions were then purified by HPLC and analysed by high resolution mass spectrometry. The effect of CCE on intestinal chloride secretion was investigated in human colonic T84 cells, ex vivo mouse intestinal tissue using an Ussing chamber, and the two-electrode voltage-clamp technique to record calcium sensitive TMEM16A chloride currents in Xenopus laevis oocytes. In vivo, intestinal motility was investigated using the loperamide-induced C57BL/6 constipation mouse model. Ex vivo contractility of mouse colonic smooth muscles was assessed by isometric force measurements. RESULTS: CCE increased the short-circuit current (ΔIsc 34.47 ± µA/cm2) and apical membrane chloride conductance (ΔICl 95 ± 8.1 µA/cm2) in intestinal epithelial cells. The effect was dose-dependent, with an EC50 value of 0.06 µg/mL. CCE stimulated the endogenous TMEM16A-induced Cl- current in Xenopus laevis oocytes. Moreover, CCE increased the contractility of smooth muscle in mouse colonic tissue and enhanced small bowel transit in CCE treated mice compared to loperamide controls. Mass spectrometry suggested a cucurbitacin-like analogue with a mass of 512.07 g/mol underlying the bioactivity of CCE. CONCLUSION: A cucurbitacin-like analog present in CCE activates TMEM16A channels, which may have therapeutic potential in cystic fibrosis and intestinal hypodynamic disorders.

ß-eudesmol but not atractylodin exerts an inhibitory effect on CFTR-mediated chloride transport in human intestinal epithelial cells.

Oriental herbal medicine with the two bioactive constituents, ß-eudesmol (BE) and atractylodin (AT), has been used as a remedy for gastrointestinal disorders. There was no scientific evidence reporting their antidiarrheal effect and underpinning mechanisms. Therefore, we aimed to investigate the anti-secretory activity of these two compounds in vitro. The inhibitory effect of BE and AT on cAMP-induced Cl- secretion was evaluated by Ussing chamber in human intestinal epithelial (T84) cells. Short-circuit current (ISC) and apical Cl- current (ICl-) were measured after adding indirect and direct cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activator. MTT assay was used to determine cellular cytotoxicity. Protein-ligand interaction was investigated by in silico molecular docking analysis. BE, but not AT concentration-dependently (IC50 of ~1.05 µM) reduced cAMP-mediated, CFTRinh-172 inhibitable Cl- secretion as determined by transepithelial ISC across a monolayer of T84 cells. Potency of CFTR-mediated ICl- inhibition by BE did not change with the use of different CFTR activators suggesting a direct blockage of the channel active site(s). Pretreatment with BE completely prevented cAMP-induced ICl-. Furthermore, BE at concentrations up to 200 µM (24 h) had no effect on T84 cell viability. In silico studies indicated that BE could best dock onto dephosphorylated structure of CFTR at ATP-binding pockets in nucleotide-binding domain (NBD) 2 region. These findings provide the first evidence for the anti-secretory effect of BE involving inhibition of CFTR function. BE represents a promising candidate for the therapeutic or prophylactic intervention of diarrhea resulted from intestinal hypersecretion of Cl.

Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

Tracing oral Na+ and K+ in sweat during exercise and recovery in horses.

NEW FINDINGS: What is the central question of this study? What are the mechanisms by which equine sweat glands transport sodium, potassium and water into sweat? What is the main finding and its importance? The flux of sodium into sweat does not have an active transport component, the flux of potassium into sweat is partially dependent on an active transport mechanism, and there is no evidence for paracellular transport. ABSTRACT: In two series of experiments, this study used radioactive sodium (Na+ ) and potassium (K+ ) to trace the net flux, and calculate the unidirectional fluxes, of these ions from extracellular fluid into sweat of horses during exercise and recovery. The effect of an oral electrolyte supplement (PNW) on the sweating responses and ion fluxes was also examined. Compared to 8 litres of water (controls), provision of 8 litres of PNW resulted in significantly increased sweating duration (P < 0.001). Two hours before exercise, 99 Tc-labelled diethylene-triamine-pentaacetate (DTPA) was administered i.v. to determine if there was paracellular flux of this molecule in sweat glands during the period of sweating. One hour before beginning moderate-intensity exercise, horses were nasogastrically administered either 24 Na (1-3 litres) or 42 K (8 litres) with water (control) or an electrolyte supplement. Both radiotracers appeared in sweat within 10 min of exercise onset, and the sweat specific activity of both ions increased during exercise (P < 0.001), approaching plasma specific activities. There was no appearance of 99 Tc-DTPA in sweat. The activities of 24 Na and 42 K, together with the concentrations Na+ , K+ and Cl- , argued against significant paracellular flux of these ions into the lumen of sweat glands. The flux analysis for 24 Na indicated a small intracellular pool within sweat gland cells, and no evidence for an active transport component. The flux analysis for 42 K indicated a relatively large intracellular equilibration pool within sweat gland cells, with evidence for an active transport component. The results are discussed with respect to the current understanding of sweat gland epithelial cell ion transport mechanisms at both the basal and the apical membranes. It appears likely that the majority of ions appearing in sweat pass through sweat gland epithelial cells by transcellular mechanisms that include ion transporting pathways as well as apical vesicular exocytosis.

Synthesis and Structure-activity Relationship of Aminoarylthiazole Derivatives as Potential Potentiators of the Chloride Transport Defect in Cystic Fibrosis.

BACKGROUND: Cystic fibrosis (CF) is the autosomal recessive disorder most common in Caucasian populations. It is caused by mutations in the cystic fibrosis transmembrane regulator protein (CFTR). CFTR is predominantly expressed at the apical plasma membranes of the epithelial cells lining several organs, and functions as a cAMP-regulated chloride/bicarbonate channel. To address the underlying causes of cystic fibrosis, two biomolecular activities are required, namely correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. OBJECTIVE: In our previous data, we demonstrated that some aminoarylthiazoles (AATs) have peculiar activity acting as correctors and as potentiator-like molecules. Curiously, a compound called 1 has been shown to be markedly active as a potentiator. Now, we have further modified its scaffold at different portions, for the identification of molecules with improved potency and effectiveness on mutant CFTR. METHODS: Starting from this active compound, we synthesized a small library trying to improve the activity as potentiators. To extrapolate the contribution of a particular structural portion to bioactivity, we selectively modified one portion at a time. RESULTS: Our study has provided a structure-activity relationship (SAR) on AATs and led to the identification of some compounds, with a particular ability to act as CFTR potentiators. CONCLUSION: Two compounds 2 and 13 appear to be promising molecules and could be used for the future development of potentiators of the chloride transport defect in cystic fibrosis.

Regulation of renal pendrin activity by aldosterone.

PURPOSE OF REVIEW: Pendrin resides on the luminal membrane of type B intercalated cells in the renal collecting tubule system mediating the absorption of chloride in exchange for bicarbonate. In mice or humans lacking pendrin, blood pressure is lower, and pendrin knockout mice are resistant to aldosterone-induced hypertension. Here we discuss recent findings on the regulation of pendrin. RECENT FINDINGS: Pendrin activity is stimulated during alkalosis partly mediated by secretin. Also, angiotensin II and aldosterone stimulate pendrin activity requiring the mineralocorticoid receptor in intercalated cells. Angiotensin II induces dephosphorylation of the mineralocorticoid receptor rendering the receptor susceptible for aldosterone binding. In the absence of the mineralocorticoid receptor in intercalated cells, angiotensin II does not stimulate pendrin. The effect of aldosterone on pendrin expression is in part mediated by the development of hypokalemic alkalosis and blunted by K-supplements or amiloride. Part of the blood pressure-increasing effect of pendrin is also mediated by its stimulatory effect on the epithelial Na-channel in neighbouring principal cells. SUMMARY: These findings identify pendrin as a critical regulator of renal salt handling and blood pressure along with acid--base balance. A regulatory network of hormones fine-tuning activity is emerging. Drugs blocking pendrin are being developed.

Dietary Chloride Deficiency Syndrome: Pathophysiology, History, and Systematic Literature Review.

Metabolic alkalosis may develop as a consequence of urinary chloride (and sodium) wasting, excessive loss of salt in the sweat, or intestinal chloride wasting, among other causes. There is also a likely underrecognized association between poor salt intake and the mentioned electrolyte and acid-base abnormality. In patients with excessive loss of salt in the sweat or poor salt intake, the maintenance of metabolic alkalosis is crucially modulated by the chloride-bicarbonate exchanger pendrin located on the renal tubular membrane of type B intercalated cells. In the late 1970s, recommendations were made to decrease the salt content of foods as part of an effort to minimize the tendency towards systemic hypertension. Hence, the baby food industry decided to remove added salt from formula milk. Some weeks later, approximately 200 infants (fed exclusively with formula milks with a chloride content of only 2-4 mmol/L), were admitted with failure to thrive, constipation, food refusal, muscular weakness, and delayed psychomotor development. The laboratory work-up disclosed metabolic alkalosis, hypokalemia, hypochloremia, and a reduced urinary chloride excretion. In all cases, both the clinical and the laboratory features remitted in ≤7 days when the infants were fed on formula milk with a normal chloride content. Since 1982, 13 further publications reported additional cases of dietary chloride depletion. It is therefore concluded that the dietary intake of chloride, which was previously considered a "mendicant" ion, plays a crucial role in acid-base and salt balance.

Salt and water balance after sweat loss: A study of Bikram yoga.

Bikram yoga is practiced in a room heated to 105°F with 40% humidity for 90 min. During the class a large volume of water and electrolytes are lost in the sweat, specifically, sodium is lost, the main cation of the extracellular fluid. There is little known about the volume of sweat and the amount of sodium lost in sweat during Bikram yoga or the optimum quantity of fluid required to replace these losses. The participants who took part in this small feasibility study were five females with a mean age of 47.4 ± 4.7 years and 2.6 ± 1.6 years of experience at Bikram yoga. The total body weight, water consumed, serum sodium concentration, serum osmolality, and serum aldosterone levels were all measured before and after a Bikram yoga practice. Sweat sodium chloride concentration and osmolality were measured at the end of the practice. The mean estimated sweat loss was 1.54 ± 0.65 L, while the amount of water consumed during Bikram yoga was 0.38 ± 0.22 L. Even though only 25% of the sweat loss was replenished with water intake during the Bikram yoga class, we did not observe a change in serum sodium levels or serum osmolality. The sweat contained 82 ± 16 mmol/L of sodium chloride for an estimated total of 6.8 ± 2.1 g of sodium chloride lost in the sweat. The serum aldosterone increased 3.5-fold from before to after Bikram yoga. There was a decrease in the extracellular body fluid compartment of 9.7%. Sweat loss in Bikram yoga predominately produced a volume depletion rather than the dehydration of body fluids. The sweating-stimulated rise in serum aldosterone levels will lead to increased sodium reabsorption from the kidney tubules and restore the extracellular fluid volume over the next 24 hr.