Permeabilization via the P2X7 purinoreceptor reveals the presence of a Ca2+ -activated Cl- conductance in the apical membrane of murine tracheal epithelial cells

Calcium-activated Cl- secretion is an important modulator of regulated ion transport in murine airway epithelium and is mediated by an unidentified Ca2+-stimulated Cl- channel. We have transfected immortalized murine tracheal epithelial cells with the cDNA encoding the permeabilizing P2X7 purinoreceptor (P2X7-R) to selectively permeabilize the basolateral membrane and thereby isolate the apical membrane Ca2+-activated Cl- current. In P2X7-R-permeabilized cells, we have demonstrated that UTP stimulates a Cl- current across the apical membrane of CF and normal murine tracheal epithelial cells. The magnitude of the UTP-stimulated current was significantly greater in CF than in normal cells. Ion substitution studies demonstrated that the current exhibited a permselectivity sequence of Cl- > I- > Br- > gluconate-. We have also determined a rank order of potency for putative Cl- channel blockers: niflumic acid ≥ 5-nitro-2-(3-phenylpropylamino)benzoic acid > 4,4'-diisothiocyanostilbene-2,2'-disulfonate > glybenclamide >> diphenlyamine-2-carboxylate, tamoxifen, and p-tetra-sulfonato-tetra-methoxy-calix[4]arene. Complete characterization of this current and the corresponding single channel properties could lead to the development of a new therapy to correct the defective airway surface liquid in cystic fibrosis patients

Probing the Role of Melanocortin Type 1 Receptor Agonists in Diverse Immunological Diseases

Background: The melanocortin α-melanocyte stimulating hormone (α-MSH), an endogenous peptide with high affinity for the melanocortin 1 receptor (MC1r), has demonstrated prevention and reversal of intestinal and ocular inflammation in animal models. Preclinical studies were performed to determine whether two MC1r receptor agonists, PL-8177 and PL-8331, exhibit actions and efficacy similar to α-MSH in preventing and reversing intestinal and ocular inflammation.Methods: Both PL-8177 and PL-8331 were assessed in a Eurofins LeadProfilingScreen selectivity panel including 72 in vitro assays. PL-8177 and PL-8331 were evaluated in an in vitro assay using human whole blood stimulated by lipopolysaccharide to determine inhibition of tumor necrosis factor alpha (TNF-α); for comparison, adrenocorticotropic hormone (ACTH) and α-MSH were used as positive controls. PL-8177, dosed at 0.5, 1.5, and 5.0 μg, was assessed in a cannulated rat model of dinitrobenzene sulfonic acid (DNBS)-induced bowel inflammation versus vehicle and oral sulfasalazine. PL-8177 was also dosed at 0.3 mg/kg/mouse injected intraperitoneally versus untreated controls and α-MSH treatment in mice with experimental autoimmune uveitis (EAU). PL-8331 at 3 doses, 3 times daily, was evaluated in a murine model of scopolamine-induced dry eye disease (SiccaSystemTM model), versus twice-daily Restasis® and Xiidra®.Results: Both PL-8177 and PL-8331 demonstrated no significant activity at the 1 μm concentration in any of the 72 in vitro assays. PL-8177 and PL-8331 inhibited lipopolysaccharide-induced TNF-α to a similar degree as ACTH and α-MSH. In the DNBS rat model of bowel inflammation, PL-8177 was significantly superior to untreated controls at all 3 doses (P < 0.05) in reducing bowel inflammation parameters, with effects similar to sulfasalazine. In the murine EAU model, PL-8177 significantly reduced retinal inflammation scores versus untreated controls (P = 0.0001) over 3–5 weeks, and to a similar degree as α-MSH. In the murine scopolamine-induced model of dry eye disease, PL-8331 reduced corneal fluorescein staining scores at all doses, significantly (P = 0.02) for the highest dose (1 × 10-5 mg⋅mL-1), and similarly to Restasis®; Xiidra® demonstrated no effect.Conclusion: The MC1r receptor agonists PL-8177 and PL-8331 exhibited actions similar to those of α-MSH in preventing and reversing intestinal and ocular inflammation in preclinical disease models

High-Precision U-Pb Zircon Age Calibration of the Global Carboniferous Time Scale and Milankovitch Band Cyclicity in the Donets Basin, Eastern Ukraine

High-precision ID-TIMS U-Pb zircon ages for 12 interstratified tuffs and tonsteins are used to radiometrically calibrate the detailed lithostratigraphic, cyclostratigraphic, and biostratigraphic framework of the Carboniferous Donets Basin of eastern Europe. Chemical abrasion of zircons, use of the internationally calibrated EARTHTIME mixed U-Pb isotope dilution tracer, and improved mass spectrometry guided by detailed error analysis have resulted in an age resolution o

On the biostratigraphy and cyclostratigraphy of the Moscovian Stage in the type area

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Expression of nucleotide-regulated Cl^- currents in CF and normal mouse tracheal epithelial cell lines

The dominant route for Cl−secretion in mouse tracheal epithelium is via Cl−channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(−/−)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/−)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10−4M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current ( Isc) and resistance of both cell lines, with larger increases in Iscbeing elicited by ionomycin (10−6M). Both cell lines expressed P2Y2receptors and responded to the purinergic agonists ATP, UTP, and 5′-adenylylimidodiphosphate (10−4M) with an increase in Isc. This response could be inhibited by DIDS and was abolished in the presence of Cl−-free Ringer solution. Reducing the mucosal Cl−concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(−/−) cells. Pretreatment of these cells with thapsigargin caused a direct increase in Iscand inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl−currents and may prove valuable tools in studying the properties of this pathway.</jats:p

«Health school» for arterial hypertension patients: district out-patient clinic experience

Aim. To analyze effectiveness of “Health School” (First Out-Patient Department, Yekaterinburg Central City Hospital No. 6) activity in arterial hypertension (AH) patients. Material and methods. The study included 120 patients (85 females), attending School sessions. Attendees’ questionnaire survey was performed at first and sixth sessions. Target blood pressure (BP) level achievement was assessed by office BP measurement and diary BP registration. Six-month results after stopping the program were also registered. Results. Among all participants, there were 70.8% females; 67 individuals under 60 were still working (58.3%), 41 had Stage I AH (34%). By the program’s end, 30 patients (25%) achieved target BP levels; in 42 individuals (35%), BP decreased by 30% from the baseline level. Six months later, 67 patients (55.8%) maintained target BP level. Increase in combination therapy usage, by the end of the program, was associated with increased administration of diuretics and angiotensin II receptor antagonists. Conclusion. Optimal program outlay included interactive lectures and individual consultation sessions, that gave a chance to reach wide audience of AH patients and achieve real-life positive results in their treatment