Animal Science

1 period. Two vitamin-trace mineral premixes containing either metal specific amino acid complexes or inorganic trace mineral sources were formulated to contain 200% of NRC (1988) sow requirements for iodine, copper, zinc, manganese, and iron. A separate vitamin premix containing no trace minerals served as the control. Premixes were stored in an environmentallycontrolled feed storage room and samples were collected every month to determine vitamin activity. Minimal monthly vitamin stock losses (0-1%) were observed for all vitamins except

This study used a total of 2020 Colombian forage resources of three families (Grass forages, legume forages, and other forage plants) to develop near infrared spectroscopy calibrations for predicting the nutritional value. Spectra were collected at 2 nm increments using a scanning visible/near infrared spectrometer. The reference data used for each forage were crude protein, crude ash, neutral detergent fiber, acid detergent fiber, acid detergent lignin, measured according to the Association of Official Analytical Chemists. Two chemometric tools for developing near infrared spectroscopy prediction models were compared: the GLOBAL modified partial least squares, and the calibration strategy known as LOCAL. The LOCAL procedure is designed to select, from a large database, samples with spectra resembling the sample being analyzed. Selected samples were used as calibration sets after one-tenth of the samples were selected for validation from each database. Predictions of nutrition indicators in validation samples using generic and specific calibrations were compared with both GLOBAL and LOCAL procedures. For all predicted forages, LOCAL resulted in a significant improvement in both standard error of prediction and bias values compared with GLOBAL. Determination coefficient values (r 2 ) also improved using the LOCAL algorithm, exceeding 0.9 for most forage sets. LOCAL calibration was then used with only one database (n 2020) comprising all the forage samples and SEP and r 2 were similar to those obtained in the three databases using LOCAL algorithm. Therefore, LOCAL can accurately predict the composition of different forages using only one database, and could offer a practical way to develop robust equations taking into account the biodiversity of Colombian forages.

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y1, P2Y2, P2Y4, and P2Y6 receptors. RT-PCR experiments also revealed expression of A2b adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current ( Isc) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na+ channel (ENaC) α-, β-, and γ-subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na+ transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation ...

lyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na ϩ transport and, in particular, the role of amiloride-sensitive Na ϩ channels in the apical membrane and the Na ϩ-K ϩ-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl Ϫ and K ϩ channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl Ϫ and K ϩ channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.

Non-technical summary The epithelial cells lining the ducts of the human mammary gland are responsible for modifying sodium and potassium concentrations in milk by actively absorbing sodium from and secreting potassium into the ductal fluid. In the present study we show that adenosine triphosphate (ATP) and uridine triphosphate (UTP) can stimulate sodium absorption and potassium secretion by a mechanism that involves increasing intracellular calcium and activation of calcium-dependent potassium channels. We discovered that addition of ATP or UTP to the luminal surface stimulates potassium secretion, whereas addition of the same concentrations to the epithelial surface normally exposed to the blood produces an increase in sodium absorption. These results provide a better understanding of the signalling mechanisms that control the concentrations of sodium and potassium present in milk.

Aeroallergens produced by Alternaria alternata can elicit life-threatening exacerbations of asthma in patients sensitized to this fungus. In this study, the effect of Alternaria on ion transport mechanisms underlying mucociliary clearance and airway epithelial barrier function was investigated in human airway epithelial cells. Apical exposure to Alternaria induced an increase in anion secretion that was inhibited by blockers of CFTR and Ca(2+)-activated Cl(-) channels. Stimulation of anion secretion was dependent on Ca(2+) uptake from the apical solution. Alternaria exposure also produced an increase in reactive oxygen species (ROS) that was blocked by pretreatment with the oxidant scavenger glutathione (GSH). GSH and the NADPH oxidase inhibitor/complex 1 electron transport inhibitor diphenylene iodonium chloride (DPI) blocked ATP release and the increase in intracellular [Ca(2+)] evoked by Alternaria Alternaria also decreased transepithelial resistance and a portion of this affect ...

Previous studies in our laboratory have shown that Na absorption across the porcine endometrium is stimulated by PGF 2␣ and cAMP-dependent activation of a barium-sensitive K channel located in the basolateral membrane of surface epithelial cells. In this study, we identify and characterize this basolateral, bariumsensitive K conductance. Porcine uterine tissues were mounted in Ussing chambers and bathed with KMeSO 4 Ringer solution. Amphotericin B (70 M) was added to the luminal solution to permeabilize the apical membrane and determine the current-voltage relationship of the basolateral K conductance after activation by 100 M CPT-cAMP. An inwardly rectifying current was identified which possessed a reversal potential of −53 mV when standard Ringer solution was used to bathe the serosal surface. The K:Na selectivity ratio was calculated to be 12:1. Administration of 5 mM barium to the serosal solution completely inhibited the current activated by cAMP under these conditions. In addition to these experiments, amphotericin-perforated whole cell patch clamp recordings were obtained from primary cultures of porcine surface endometrial cells. The isolated cells displayed an inwardly rectifying current under basal conditions. This current was significantly stimulated by CPT-cAMP and blocked by barium. These results together with our previous studies demonstrate that cAMP increases Na absorption in porcine endometrial epithelial cells by activating an inwardly rectifying K channel present in the basolateral membrane. Similar patch clamp experiments were conducted using cells from a human endometrial epithelial cell line, RL95-2. An inwardly rectifying current was also identified in these cells which possessed a reversal potential of −56 mV when the cells were bathed in standard Ringer solution. This current was blocked by barium as well as cesium. However, the current from the human cells did not appear to be activated by cAMP, indicating that distinct subtypes of inwardly rectifying K channels are present in endometrial epithelial cells from different species.

SUMMARY The uterine (shell gland) epithelium from the domestic chicken was mounted in Ussing chambers, bathed in symmetric avian saline solution on both apical and basolateral aspects and voltage clamped at 0 mV. The epithelium exhibited a basal short circuit current (Isc) that was partially inhibited by the epithelial Na+ channel (ENaC) blockers, amiloride and benzamil (IC50 values of 0.8 and 0.12 μmol l-1,respectively). Inhibition of basal Na+ absorption by 10 μmol l-1 amiloride was confirmed by measurements of transepithelial Na+ and Cl- fluxes, where inhibition of the apical-to-basolateral and net Na+ flux occurred, but no significant effects on Cl- fluxes were detected. The amiloride-insensitive portion of the basal Isc was both Cl- and HCO3- dependent and was inhibited by the Cl-channel blocker, diphenyl-2-carboxylate (DPC; 100 μmol l-1). Stimulation with 8-(4-chlorophenylthio)-cyclic 3′-5′, adenosine monophosphate (8-cpt cAMP) produced a sustained increase in Isc that was dep...

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increases sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or nontargeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels...

Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl− secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current ( Isc: thrombin, 21 ± 2 μA; SLIGRL, 83 ± 22 μA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca2+-chelating agent BAPTA-AM (50 μM) abolished the increase in Isc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 μM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated Isc was observed. In addition, basolateral treatment with the PGE2 receptor antagonist AH-6809 (25 μM) significantly inhibited the eff...

Whole cell perforated patch-clamp experiments were performed with adult rat alveolar epithelial cells. The holding potential was −60 mV, and depolarizing voltage steps activated voltage-gated K+ (Kv) channels. The voltage-activated currents exhibited a mean reversal potential of −32 mV. Complete activation was achieved at −10 mV. The currents exhibited slow inactivation, with significant variability in the time course between cells. Tail current analysis revealed cell-to-cell variability in K+ selectivity, suggesting contributions of multiple Kv α-subunits to the whole cell current. The Kv channels also displayed steady-state inactivation when the membrane potential was held at depolarized voltages with a window current between −30 and 5 mV. Analysis of RNA isolated from these cells by RT-PCR revealed the presence of eight Kv α-subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three β-subunits (Kvβ1.1, Kvβ2.1, and Kvβ3.1), and two K+ channel interacting protein ...

Kv4/K channel-interacting protein (KChIP) potassium channels are a major class of rapidly inactivating K channels in brain and heart. Considering the importance of alternative splicing to the quantitative features of KChIP gating modulation, a previously uncharacterized splice form of KChIP1 was functionally characterized. The KChIP1b splice variant differs from the previously characterized KChIP1a splice form by the inclusion of a novel amino-terminal region that is encoded by an alternative exon that is conserved in mouse, rat, and human genes. The expression of KChIP1b mRNA was high in brain but undetectable in heart or liver by RT-PCR. In cerebellar tissue, KChIP1b and KChIP1a transcripts were expressed at nearly equal levels. Coexpression of KChIP1b or KChIP1a with Kv4.2 channels in oocytes slowed K current decay and destabilized open-inactivated channel gating. Like other KChIP subunits, KChIP1b increased Kv4.2 current amplitude and KChIP1b also shifted Kv4.2 conductance-volta...

Mycobacterium avium subsp. paratuberculosis from infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km 2 containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS 900 PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests for M. avium...

Large conductance voltage-and Ca 2+-activated K + channels are important in regulating detrusor smooth muscle (DSM) function. Here, we examined systematically how the BK channel pharmacological activation modulates DSM contractility. NS1619, a potent BK channel activator, was utilized as a pharmacological tool to investigate the effect of BK channel activation on rat DSM contractility. Isometric tension recordings of DSM strips isolated from rat urinary bladder were performed systematically under various experimental conditions. NS1619 (30 μM) substantially diminished DSM spontaneous contraction amplitude, muscle force integral, frequency, duration and muscle tone. This effect was blocked by iberiotoxin, a BK channel selective inhibitor. NS1619 inhibited the phasic and tonic contractions in DSM strips precontracted with either the cholinergic agonist, carbachol (0.1 μM), or the depolarizing agent, KCl (20 mM and 60 mM KCl). In the presence of elevated KCl, the inhibitory effect of NS1619 was significantly reduced, indicating that BK channel activation is the underlying mechanism of NS1619 action. BK channel activation with NS1619 dramatically decreased the amplitude of electrical field stimulation (EFS)-induced contractions under a range of stimulation frequencies (0.5-50 Hz). In the presence of specific neurotransmitter inhibitors, BK channel activation with NS1619 significantly decreased both cholinergic and purinergic components of EFS-induced contractions. We conclude that BK channel activation with NS1619 significantly inhibited spontaneous, pharmacologically induced and nerve-evoked DSM contractions. Targeting the BK channel with selective openers may offer a unique opportunity to control DSM contractile activity, including pathophysiological conditions such as overactive bladder and detrusor overactivity, regardless of the underlying cause.

The functional properties of the G protein-coupled P2Y 1 receptor were investigated in Xenopus oocytes. Incubation of oocytes expressing either the human or turkey P2Y 1 receptor with adenine nucleotide agonists resulted in an increase in Cl ؊ current and activation of a novel cation current with an inwardly rectifying current-voltage relationship. Activation of either the human P2Y 2 (P 2U-purinergic) or M1 muscarinic receptor expressed in oocytes resulted in an increase in Cl ؊ current similar to that observed in P2Y 1 receptor-expressing oocytes but had no effect on cation current. P2 receptor agonists stimulated both the cation current and Cl ؊ current in P2Y 1 receptor-expressing oocytes with EC 50 values and an order of potency (2-methylthioadenosine diphosphate > 2-methylthioadenosine triphosphate (2MeSATP) > ATP > UTP) that were similar to those previously observed for activation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human or turkey P2Y 1 receptor. The P2Y receptor antagonists suramin and pyridoxal phosphate 6-azophenyl-2-4-disulfonic acid both shifted to the right the concentration-response relationship for 2Me-SATP for stimulation of oocyte currents. Although injection of oocytes with either GDP␤S (guanyl-5-yl thiophosphate) or GTP␥S (guanosine 5-3-O-(thio)triphosphate) resulted in loss of adenine nucleotide-promoted Cl ؊ channel activation, neither guanine nucleotide altered the 2MeSATPstimulated cation current. These data are consistent with the view that activation of the novel cation current by the P2Y 1 receptor does not involve a G protein. Tail current analysis of the novel P2Y 1 receptor-associated cation conductance revealed that the open channel current-voltage relationship was outwardly rectifying with a reversal potential of ؊38 mV for the turkey P2Y 1 receptor and ؊36 mV for the human P2Y 1 receptor. Replacement of Na ؉ with K ؉ ions in the bathing solution produced a shift in reversal potential to near zero mV, but significant outward rectification remained. The cation current was not permeable to either Ca 2؉ or Ba 2؉ and exhibited steady-state inactivation at holding potentials below ؊60 mV. These results indicate that the P2Y 1 receptor exhibits both metabotropic properties and a novel G protein-independent ionotropic response when expressed in Xenopus oocytes.

The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time-dependent alkalinization of the cytoplasm from an initial pH of 7.1±0.04 to 7.5±0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca2+]i). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca2+]i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na+-H+ exchange, proton channel or plasma membrane H+-ATPase activities. Measurements of intragranule pH with a cell permeant pH i...

Eosinophils play a primary role in the pathophysiology of asthma. In the lung, the activation state of the in®ltrating eosinophils determines the extent of tissue damage. Interleukin-5 (IL-5) and leukotriene B 4 (LTB 4) are important signaling molecules involved in eosinophil recruitment and activation. However, the physiological processes that regulate these activation events are largely unknown. In this study we have examined the mechanisms of human eosinophil NADPH oxidase regulation by IL-5, LTB 4 , and phorbol ester (PMA). These stimuli activate a Zn 2-sensitive plasma membrane proton channel, and treatment of eosinophils with Zn 2 blocks superoxide production. We have demonstrated that eosinophil intracellular pH is not altered by IL-5 activation of NADPH oxidase. Additionally, PKCd inhibitors block PMA, IL-5 and LTB 4 mediated superoxide formation. Interestingly, the PKCd-selective inhibitor, rottlerin, does not block proton channel activation by PMA indicating that the oxidase and the proton conductance are regulated at distinct phosphorylation sites. IL-5 and LTB 4 , but not PMA, stimulated superoxide production is also blocked by inhibitors of PI 3-kinase indicating that activation of this enzyme is an upstream event common to both receptor signaling pathways. Our results indicate that the G-protein-coupled LTB 4 receptor and the IL-5 cytokine receptor converge on a common signaling pathway involving PI 3-kinase and PKCd to regulate NADPH oxidase activity in human eosinophils.

The objective of this study was to investigate the mechanism of uridine 5′-triphosphate (UTP)-dependent inhibition of Na+absorption in porcine endometrial epithelial cells. Acute stimulation with UTP (5 μM) produced inhibition of sodium absorption and stimulation of chloride secretion. Experiments using basolateral membrane–permeabilized cell monolayers demonstrated a reduction in benzamil-sensitive Na+conductance in the apical membrane after UTP stimulation. The UTP-dependent inhibition of sodium transport could be mimicked by PMA (1 μM). Several PKC inhibitors, including GF109203X and Gö6983 (both nonselective PKC inhibitors) and rottlerin (a PKCδ selective inhibitor), were shown to prevent the UTP-dependent decrease in benzamil-sensitive current. The PKCα-selective inhibitors, Gö6976 and PKC inhibitor 20–28, produced a partial inhibition of the UTP effect on benzamil-sensitive Isc. Inhibition of the benzamil-sensitive Isc by UTP was observed in the presence of BAPTA-AM (50 μM), c...