Evidence for NHE3-mediated Na transport in sheep and bovine forestomach

Na absorption across the cornified, multilayered, and squamous rumen epithelium is mediated by electrogenic amiloride-insensitive transport and by electroneutral Na transport. High concentrations of amiloride (>100 μM) inhibit Na transport, indicating Na+/H+ exchange (NHE) activity. The underlying NHE isoform for transepithelial Na absorption was characterized by mucosal application of the specific inhibitor HOE642 for NHE1 and S3226 for NHE3 in Ussing chamber studies with isolated epithelia from bovine and sheep forestomach. S3226 (1 μM; NHE3 inhibitor) abolished electroneutral Na transport under control conditions and also the short-chain fatty acid-induced increase of Na transport via NHE. However, HOE642 (30 μM; NHE1 inhibitor) did not change Na transport rates. NHE3 was immunohistochemically localized in membranes of the upper layers toward the lumen. Expression of NHE1 and NHE3 has been previously demonstrated by RT-PCR, and earlier experiments with isolated rumen epithelial cells have shown the activity of both NHE1 and NHE3. Obviously, both isoforms are involved in the regulation of intracellular pH, pHi. However, transepithelial Na transport is only mediated by apical uptake via NHE3 in connection with extrusion of Na by the basolaterally located Na-K-ATPase. The missing involvement of NHE1 in transepithelial Na transport suggests that the proposed “job sharing” in epithelia between these two isoforms probably also applies to forestomach epithelia: NHE3 for transepithelial transport and NHE1 for, among others, pHi and volume regulation

Sheep rumen and omasum primary cultures and source epithelia: barrier function aligns with expression of tight junction proteins

SUMMARYThe forestomachs of cows and sheep have historically served as important models for the study of epithelial transport. Thus, the ruminal epithelium was among the first tissues in which absorption of chloride against an electrochemical gradient was observed, requiring a tight paracellular barrier to prevent back-leakage. However, little is known about ruminal barrier function, despite the considerable implications for ruminant health. The tight junction proteins of the omasum have never been investigated, and no cell culture model exists. We present a new method for the isolation of cells from forestomach epithelia. Protein expression of cells and source tissues of sheep were studied using western blot, PCR and confocal laser scanning microscopy. Cultured cells were characterized by transepithelial resistance (TER) measurements and patch clamping. Cells developed TER values of 729±134 Ω cm2 (rumen) and 1522±126 Ω cm2 (omasum). Both primary cells and source epithelia of rumen and omasum expressed cytokeratin, occludin and claudins 1, 4 and 7 (but not claudins 2, 3, 5, 8 and 10), consistent with the observed paracellular sealing properties. Staining for claudin-1 reached the stratum basale. The full mRNA coding sequence of claudins 1, 4 and 7 (sheep) was obtained. Patch-clamp analyses of isolated cells proved expression of an anion conductance with a permeability sequence of gluconate&amp;lt;acetate&amp;lt;chloride. This is in accordance with a model that ruminal and omasal transport of anions such as chloride and acetate has to occur via a transcellular route and involves channel-mediated basolateral efflux, driven by Na+/K+-ATPase.</jats:p

Carbonic anhydrase influences asymmetric sodium and acetate transport across omasum of sheep

Objective Omasum is an important site for the absorption of short chain fatty acids. The major route for the transport of acetate is via sodium hydrogen exchanger (NHE). However, a discrepancy in the symmetry of sodium and acetate transport has been previously reported, the mechanism of which is unclear. In this study, we investigated the possible role of carbonic anhydrase (CA) for this asymmetry. Methods Omasal tissues were isolated from healthy sheep (N = 3) and divided into four groups; pH 7.4 and 6.4 alone and in combination with Ethoxzolamide. Electrophysiological measurements were made using Ussing chamber and the electrical measurements were made using computer controlled voltage clamp apparatus. Effect(s) of CA inhibitor on acetate and sodium transport flux rate of Na22 and 14C-acetate was measured in three different flux time periods. Data were presented as mean±standard deviation and level of significance was ascertained at p≤0.05. Results Mucosal to serosal flux of Na (JmsNa) was greater than mucosal to serosal flux of acetate (JmsAc) when the pH was decreased from 7.4 to 6.4. However, the addition of CA inhibitor almost completely abolished this discrepancy (JmsNa ≈ JmsAc). Conclusion The results of the present study suggest that the additional protons required to drive the NHE were provided by the CA enzyme in the isolated omasal epithelium. The findings of this study also suggest that the functions of CA may be exploited for better absorption in omasum

Clinical and electrophysiological efficacy of leaf extract of Gingko biloba L (Ginkgoaceae) in subjects with diabetic sensorimotor polyneuropathy

Purpose: To investigate the health benefits of Gingko biloba L. leave extract EGb 761 in patients with diabetic sensorimotor polyneuropathy (DSP).Method: In a randomized, double-blind, placebo trial, patients (n = 156) received Gingko biloba (120 mg/day) or placebo for 6 months. Efficacy of Gingko biloba extract EGb 761 was assessed on the reduction in the scores of “Short-Form McGill Pain Questionnaire” (SF-MPQ) and improvements in latency, amplitude and conduction velocity of four sensory (ulnar, median, peroneal and sural) and four motor (ulnar, median, peroneal and tibial) nerves.Results: Gingko biloba significantly improved the mean scores of sensory, affective, visual analogue score (VAS) and present pain intensity (PPI) of SF-MPQ on 3 and 6 month post-treatment. At the start of experiment in the Gingko biloba-treated group, scores for sensory, affective, VAS and PPI domains were 12.90 ± 7.21, 4.40 ± 1.79, 4.38 ± 1.69 and 3.24 ± 0.78 respectively. After 6-months treatment with Gingko biloba, scores for sensory, affective, VAS and PPI domains were reduced (p &lt; 0.01) to 7.24 ± 4.91, 3.11 ± 1.81, 3.37 ± 1.35 and 2.45 ± 0.65 respectively. Contrarily, in the placebo-administered group, sensory domain was (p &lt; 0.01) deteriorated after 6 month (4.70 ± 1.87) compared to the baseline (4.04 ± 1.67). Gingko biloba extract EGb 761 failed to influence the electrophysiological measures when compared with the placebo group. The common adverse effects observed with Gingko biloba were somnolence and dryness of mouth compared with the placebo-treated patientsConclusion: Gingko biloba extract EGb 761 seems to be an effective and safe drug for reducing symptoms of DSP without any apparent effect on the electrophysiological attributes of sensorimotor nerves.Keywords: Diabetic polyneuropathy, Diabetes mellitus, Electrophysiological parameters, Gingko biloba, Sensorimotor nerves, Short-Form McGill Pain Questionnair

Modulation des Na- und Acetat-Transports durch Heat Shock Proteine im Pansenepithel des Schafes

It has been known from previous studies that the rumen epithelium can adapt, both morphologically and functionally, to changes in diet. This process may take days or weeks however, a daily variation in ruminal pH, predominantly after meals, has also been documented particularly in case of SARA that causes a low ruminal pH (<5.5) for a period of 3-4 hours per day. This diurnal variation in rumen pH and SCFA concentration puts the rumen epithelium in a stressful scenario and demands a rescue mechanism from these short term challenges. It was therefore, the intention of this study to investigate the protective role of heat shock proteins and NHE in the rumen epithelium exposed on the luminal side to changes of pH, SCFA concentration, temperature or osmotic pressure. Using conventional Ussing chamber technique, in vitro, Na and acetate transport across the rumen epithelium were carried out to evaluate these protective effects whereas, concentrate fed animals with ruminal fermentation pattern resembling SARA were used to study the Hsp70 expression in vivo. The following results were obtained: 1\. Characterization of electroneutral Na transport via NHE clearly showed that the isoform NHE3 is mediating electroneutral Na transport. Application of the specific NHE3 blocker S3226 reduced Jms Na and Jnet, which was not different from Isc, indicating that S3226 abolished electroneutral Na transport. 2\. Incubation of isolated rumen epithelium in the Ussing chamber simulating the conditions of SARA i.e. low mucosal pH (6.4-6.0) and/or high SCFA (70-80mmol·l-1) concentrations, increasing temperature or osmotic pressure induced an over- expression of Hsp70. This expression was reduced when the stress conditions exceeded beyond a certain limit. For instance, an increase in temperature (>44°C), SCFA concentration on mucosal buffer (>100mmol·l-1) and decrease in pH on mucosal side (<5.5) reduced Hsp70 expression. 3\. Hsp70 expression could be reduced using a non specific (cycloheximide) or specific inhibitor (triptolide) thus allowing further studies in the presence or absence of Hsp70. Cycloheximide was most effective in a concentration of 3.0 mmol·l-1 while Triptolide was used in a concentration of 100 nmol·l-1. The use of these inhibitors did not affect the Isc or Gt and Na or acetate flux rates under control conditions. 4\. Electroneutral Na as well as acetate transport were significantly higher in the tissues after an over expression of Hsp70. This increase of flux rates was significantly reduced by treatment with cycloheximide or triptolide. 5\. Induction of Hsp expression by low rumen pH and an increase of temperature was confirmed in vivo by feeding concentrates. The tissues of these sheep exhibited higher Na transport rates. It is concluded that the diurnal variation of fermentation pattern are inducing mechanisms which help to protect the tissue against theses challenges. Hps 70 is obviously an integral part of this acute adaptation and enhances NHE3 activity. Extrusion of protons taken up at increasing rates at higher SCFA concentrations and low pH must be considered as a first line of protection against SARA conditions of the rumen fluid. Although the exact mechanism between Hsp expression and NHE3 activity is not clear, Hsp is obviously a protective mechanism in the rumen epithelium too.Zahlreiche Studien belegen, dass das Pansenepithel sich sowohl in funktioneller, als auch in morphologischer Hinsicht an Veränderungen in der Fütterung anpassen kann. Dieser Prozess kann Tage oder Wochen dauern; jedoch werden auch kurzfristige tägliche Schwankungen im pH des Pansens beobachtet, welcher insbesondere nach den Mahlzeiten und im Falle von SARA für 3-4 Stunden auf sehr niedrige Werte (<5.5) absinken kann. Diese tägliche Variation im pH Wert und in der Fettsäurekonzentration im Pansen stellt eine starke Belastung für das Pansenepithel dar und protektive Mechanismen sind erforderlich, um diesen akuten Belastungsschüben zu begegnen. Es war daher die Absicht dieser Studie, die Schutzfunktion eines Heat Shock Proteins (Hsp70) und des NHEs an Epithelien des Pansens zu untersuchen, welche auf der mukosalen Seite Veränderungen im pH, in der Konzentration kurzkettiger Fettsäuren, in der Temperatur oder dem osmotischen Druck ausgesetzt worden waren. Um die protektive Funktion dieser Proteine zu untersuchen, wurde der Transport von Na und Azetat über das Pansenepithel in konventionellen Untersuchungen in der Ussingkammer in vitro gemessen. Begleitend wurde an Tieren, bei welchen durch Konzentratfütterung im Pansen SARA-ähnliche Fermentationsbedingungen induziert worden waren, die in vivo Expression von Hsp70 untersucht. Die folgenden Ergebnisse wurden erhalten: 1\. Die Charakterisierung des elektroneutralen Natriumtransportes durch NHE zeigte klar, dass die Isoform NHE3 den elektroneutralen Natriumtransport vermittelt. Applikation des spezifischen NHE3 Blockers S3226 reduzierte Jms Na und Jnet, welche sich danach nicht signifikant vom gleichbleibenden Isc unterschieden. Dieses weist darauf hin, dass S3226 den elektroneutralen Natriumtransport hemmt. 2\. Die Inkubation von isoliertem Pansenepithel in der Ussingkammer unter SARA ähnlichen Bedingungen mit niedrigem pH (6.4-6.0) und/oder hoher Konzentration kurzkettiger Fettsäuren (70-80 mmol·l-1), ansteigender Temperatur oder Zunahme des osmotischen Druckes induzierte eine Überexpression von Hsp70. Diese Expression wurde reduziert wenn die Stressoren ein bestimmtes Maß überschritten. So kam es z.B. bei einer Zunahme der Temperatur >44ºC, einer Konzentration von kurzkettigen Fettsäuren >100 mM und einem Abfall des pHs auf der mukosalen Seite >5.5 zu einem Abfall der Hsp70 Expression. 3\. Die Expression von Hsp70 konnte durch einen nicht-spezifischen (Cycloheximid) oder spezifischen (Triptolid) Inhibitor abgesenkt werden, wodurch weitere Untersuchungen in der Anwesenheit und Abwesenheit von Hsp70 ermöglicht wurden. Cycloheximid war am effektivsten in einer Konzentration von 3.0 mmol·l-1, während Triptolid in einer Konzentration von 100 nmol·l-1 eingesetzt wurde. Unter Kontrollbedingungen hatte der Gebrauch dieser Inhibitoren weder auf den Kurzschlussstrom (Isc) noch auf die Leitfähigkeit (Gt), die Na- oder die Azetatfluxe einen Einfluss. 4\. Der elektroneutrale Transport von Na und der Transport von Azetat waren signifikant höher nach Überexpression von Hsp70. Diese Zunahme der Fluxraten wurde durch die Behandlung mit Cyclohexamid oder Triptolid signifikant reduziert. 5\. Die Induktion von Hsp70 Expression in vivo durch einen niedrigen pH des Pansens und durch eine Zunahme der Temperatur wurde durch die Untersuchung von Geweben von Schafen mit unterschiedlicher Fütterung bestätigt. Epithelien von Schafen, die eine hohe Kraftfutterration erhalten hatten, zeigten auch höhere Na Transportraten. Diese Ergebnisse führen zu der Schlussfolgerung, dass die tägliche Variation des Fermentationsmusters Mechanismen induziert, die das Gewebe vor der damit verbundenen Belastung schützt. Die Induktion von Hsp70 ist offenbar ein integraler Bestandteil dieser akuten Anpassungsreaktion und erhöht die Aktivität des NHE. Die Ausschleusung von Protonen, die mit zunehmender Fettsäurekonzentration und sinkendem pH vermehrt aufgenommen werden, muss als eine erste Schutzmaßnahme gegen die bei SARA vorherrschenden Bedingungen in der Pansenflüssigkeit gelten. Obwohl die genaue Natur des Zusammenhangs zwischen Hsp Expression und NHE3 Aktivitität nicht klar ist, stellen Hsp offenbar einen protektiven Mechanismus im Pansenepithel dar

Determining the true prevalence rate of Hepatitis-C in Pakistan

BackgroundHepatitis-C is a serious public health concern in Pakistan, with an average prevalence rate of 8.7 per cent, has been reported.AimsThe objectives of this study are multifold: to comprehensively review the published reports, determine positive predictive value (PPV) of Hepatitis-C using Bayes’ Theorem and to perform meta-analysis using both raw prevalence rates and PPV rates.Methods Using available search engines and databases, a comprehensive literature search was performed on Hepatitis-C in Pakistan. Positive predictive values for the reported prevalence rates were calculated using Bayes’ Theorem. Results Average PPV value found to be 33 per cent of the prevalence rate indicating that even after high prevalence reports, the chance of actual disease is still low to moderate, necessitating further confirmatory diagnosis. This discrepancy may be attributed to several factors, including the highly varying rates of prevalence; and test sensitivity and test specificity.ConclusionRandom samples from various geographical locations and population segments are required to determine a realistic Hepatitis-C prevalence in Pakistan; in the absence of such representative samples, novel analytical and modeling approaches may offer a viable alternative to determine the actual prevalence rate and help devise better public health policy to combat the disease

Determining the true prevalence rate of Hepatitis-C in Pakistan

Background Hepatitis-C is a serious public health concern in Pakistan, with an average prevalence rate of 8.7 per cent, has been reported. Aims The objectives of this study are multifold: to comprehensively review the published reports, determine positive predictive value (PPV) of Hepatitis-C using Bayes’ Theorem and to perform meta-analysis using both raw prevalence rates and PPV rates. Methods Using available search engines and databases, a comprehensive literature search was performed on Hepatitis-C in Pakistan. Positive predictive values for the reported prevalence rates were calculated using Bayes’ Theorem. Results Average PPV value found to be 33 per cent of the prevalence rate indicating that even after high prevalence reports, the chance of actual disease is still low to moderate, necessitating further confirmatory diagnosis. This discrepancy may be attributed to several factors, including the highly varying rates of prevalence; and test sensitivity and test specificity. Conclusion Random samples from various geographical locations and population segments are required to determine a realistic Hepatitis-C prevalence in Pakistan; in the absence of such representative samples, novel analytical and modeling approaches may offer a viable alternative to determine the actual prevalence rate and help devise better public health policy to combat the diseas

Pattern of varicocele vein blood gases in patients undergoing microsurgical Varicocelectomy

Abstract Background Varicocele is known to be associated with infertility and sperm disorders. The exact cause of this ailment is not fully understood. There are limited numbers of studies where venous blood gases (VBGs) of varicocele veins were determined with conflicting results. Therefore, we have investigated the pattern of VBGs in both internal spermatic and external spermatic varicocele veins and correlation with semen quality parameters in infertile individuals who underwent left microsurgical varicocelectomy. Methods Patients (n = 27) undergoing left microsurgical varicocelectomy at a tertiary care hospital, were included in the study. Before surgery, semen parameters and scrotal color Doppler ultrasonography was performed. During surgery, blood sample was drawn from varicocele veins (internal spermatic and external spermatic veins) and a peripheral arm vein of the same patient as a control. The VBGs of all veins under study were estimated and compared with each other. The VBGs were also correlated with various semen quality parameters. Data, expressed as Mean ± SD, regarding VBGs in three veins were analyzed using one-way ANOVA. The correlation between VBGs and semen quality parameters was determined using Pearson’s correlation. Differences were considered significant at p < 0.05. Results The pH was found to be higher (p < 0.01) in the internal spermatic vein compared with the external spermatic and the peripheral veins. Partial pressure of oxygen (pO2) and oxygen saturation (sO2) were higher (p < 0.01) in the internal spermatic vein compared with the peripheral vein. However, concentration of bicarbonate (HCO3) was lower (p < 0.01) in both veins compared with the peripheral vein. Partial pressure of carbon dioxide (pCO2) was also lower (p < 0.01) in the varicocele veins compared with the control vein. Conclusion The internal spermatic veins had higher pH and oxygen tension, but lower HCO3 and pCO2 levels compared with the control peripheral veins. External spermatic veins had lower pCO2 and HCO3 but other VBGs were similar to the peripheral veins. The shift of VBGs of internal spermatic vein toward arterial blood pattern may be a missing link to understand the pathophysiology of varicocele

Effect of Bacillus cereus and Phytase on the Expression of Musculoskeletal Strength and Gut Health in Japanese Quail (Coturnix japonica)

We conducted a 28-day trial to evaluate the influence of Bacillus cereus and phytase supplementation on the expression of musculoskeletal strength and intestinal histological features in Japanese quail. Two-hundred day-old quail chicks were divided into four groups with five replicates (n=10): the first group served as a control and was fed only a basal diet (BD); the second group was fed BD + 0.1% B. cereus; the third group was fed BD + 0.01% microbial phytase; and the fourth group was fed BD + 0.01% microbial phytase + 0.1% B. cereus. Compared to the control, individual and combined supplementation of probiotic and phytase increased (P&lt;0.05) the tibial weight, length, outside diameter, and weight of bone ash, but decreased (P&lt;0.05) the weight-to-length index. The water-holding capacity, fiber diameter, fiber cross-sectional area, number of fibers per unit area, fascicle diameter, and fascicle cross-sectional area increased significantly (P&lt;0.05) in birds fed on the combination of phytase and B. cereus. The villus height, width, depth, height-to-crypt depth, and surface area increased significantly (P&lt;0.05) in the B. cereus and phytase groups on an individual basis. The strength of the musculoskeletal system was fully expressed when B. cereus and phytase were given synergistically. However, the histological features of the intestines improved in birds fed on an individual basis