Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons.

Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on alpha-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.     

Mechanisms of net chloride secretion during rotavirus diarrhea in young rabbits: do intestinal villi secrete chloride?

Rotaviral diarrheal illness is one of the most common infectious diseases in children worldwide, but our understanding of its pathophysiology is limited. This study examines whether the enhanced net chloride secretion during rotavirus infection in young rabbits may occur as a result of hypersecretion in crypt cells that would exceed the substantial Cl(-) reabsorption observed in villi. By using a rapid filtration technique, we evaluated transport of (36)Cl and D-(14)C glucose across brush border membrane (BBM) vesicles purified from villus tip and crypt cells isolated in parallel from the entire small intestine. Rotavirus infection impaired SGLT1-mediated Na(+)-D-glucose symport activity in both villus and crypt cell BBM, hence contributing to the massive water loss along the cryptvillus axis. In the same BBM preparations, rotavirus failed to stimulate the Cl(-) transport activities (Cl(-)/H(+) symport, Cl(-)/anion exchange and voltage-activated Cl(-) conductance) at the crypt level, but not at the villus level, questioning, therefore, the origin of net chloride secretion. We propose that the chloride carrier might function in both normal (absorption) and reversed (secretion) modes in villi, depending on the direction of the chloride electrochemical gradient resulting from rotavirus infection, agreeing with our results that rotavirus accelerated both Cl(-) influx and Cl(-) efflux rates across villi BBM.     

Phosphatidylethanolamine-esterified Eicosanoids in the Mouse: TISSUE LOCALIZATION AND INFLAMMATION-DEPENDENT FORMATION IN Th-2 DISEASE

In this study, murine peritoneal macrophages from naïve lavage were found to generate four phospholipids that contain 12-hydroxyeicosatetraenoic acid (12-HETE). They comprise three plasmalogen and one diacyl phosphatidylethanolamines (PEs) (16:0p, 18:1p, 18:0p, and 18:0a at sn-1) and are absent in macrophages from 12/15-lipoxygenase (12/15-LOX)-deficient mice. They are generated acutely in response to calcium mobilization, are primarily cell-associated, and are detected on the outside of the plasma membrane. Levels of 12-HETE-PEs in naïve lavage are in a similar range to those of free 12-HETE (5.5 ± 0.2 ng or 18.5 ± 1.03 ng/lavage for esterified versus free, respectively). In healthy mice, 12/15-LOX-derived 12-HETE-PEs are found in the peritoneal cavity, peritoneal membrane, lymph node, and intestine, with a similar distribution to 12/15-LOX-derived 12-HETE. In vivo generation of 12-HETE-PEs occurs in a Th2-dependent model of murine lung inflammation associated with interleukin-4/interleukin-13 expression. In contrast, in Toll receptor-dependent peritonitis mediated either by live bacteria or bacterial products, 12-HETE-PEs are rapidly cleared during the acute phase then reappear during resolution. The human homolog, 18:0a/15-HETE-PE inhibited human monocyte generation of cytokines in response to lipopolysaccharide. In summary, a new family of lipid mediators generated by murine macrophages during Th2 inflammation are identified and structurally characterized. The studies suggest a new paradigm for lipids generated by 12/15-LOX in inflammation involving formation of esterified eicosanoids.12/15-Lipoxygenase (12/15-LOX)² belongs to a family of lipid-peroxidizing enzymes that catalyze the oxygenation of polyunsaturated fatty acids to their corresponding hydroperoxy derivatives (1). They are best known for generation of free acid eicosanoids, comprising positional isomers of hydroperoxyeicosatetraenoic acid, which are subsequently converted into secondary products, including hydroxyeicosatetraenoic acid (HETE). The human homolog, 15-LOX1, is the most highly induced gene product in response to IL-4/IL-13 suggesting a potential role in Th2-driven immune responses such as autoimmune disease and allergy (2). Indeed, two recent studies indicate that mice deficient in 12/15-LOX are protected against Th2-dependent lung allergic disease (3, 4).Deficiency of 12/15-LOX in peritoneal macrophages (MΦ) alters their in vitro phenotype resulting in decreased IL-4 induction of scavenger receptor CD36, decreased stimulation of IL-12 synthesis by LPS and attenuated phagocytosis of apoptotic cells (5–7). However, the identities of the LOX products that regulate these processes are not clear, because several known products are unable to bypass the requirement for enzyme expression (7–10). Collectively, the studies infer the involvement of further uncharacterized 12/15-LOX products and indicate that the identification of novel lipids derived from this pathway is important.We recently reported that 15-LOX1 could generate 15-HETE-PE in response to calcium ionophore (11). In this study, we characterize generation of similar lipids by murine 12/15-LOX in vitro and in vivo. These new studies extend the previous findings to temporal generation of these lipids in immunologically distinct models of inflammation, as well as identifying potential biological mechanisms of action.     

Listeriolysin O-induced stimulation of mucin exocytosis in polarized intestinal mucin-secreting cells: evidence for toxin recognition of membrane-associated lipids and subsequent toxin internalization through caveolae

Lysteriolysin O (LLO) induces a microtubule-dependent activation of mucin exocytosis in the human mucin-secreting HT29-MTX. Cholesterol inhibits the LLO-induced mucin exocytosis, whereas the oxidized form of cholesterol had no inhibitory effect. LLO-induced mucin exocytosis inhibited by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. Inhibition of cholesterol synthesis in HT29-MTX cells results in a decrease in the LLO-induced mucin exocytosis. Other lipids such as gangliosides are able to inhibit the LLO-induced mucin exocytosis, suggesting that the binding of the toxin occurs at a multiplicity of membrane-associated lipids acting as receptors. Incubation of the toxin with lipids such as cholesterol or gangliosides does not decrease binding of LLO to target membranes. The present work also provides evidence that the LLO-induced mucin exocytosis develops independently of the pore-forming activity of the toxin. Finally, we demonstrated that the toxin associates with detergent-insoluble glycolipid microdomains (DIGs) containing VIP/21 caveolin, allowing internalization of the toxin and subsequent activation of the mucin exocytosis.     

Antagonistic activity of Lactobacillus acidophilus LB against intracellular Salmonella enterica serovar Typhimurium infecting human enterocyte-like Caco-2/TC-7 cells

To gain further insight into the mechanism by which lactobacilli develop antimicrobial activity, we have examined how Lactobacillus acidophilus LB inhibits the promoted cellular injuries and intracellular lifestyle of Salmonella enterica serovar Typhimurium SL1344 infecting the cultured, fully differentiated human intestinal cell line Caco-2/TC-7. We showed that the spent culture supernatant of strain LB (LB-SCS) decreases the number of apical serovar Typhimurium-induced F-actin rearrangements in infected cells. LB-SCS treatment efficiently decreased transcellular passage of S. enterica serovar Typhimurium. Moreover, LB-SCS treatment inhibited intracellular growth of serovar Typhimurium, since treated intracellular bacteria displayed a small, rounded morphology resembling that of resting bacteria. We also showed that LB-SCS treatment inhibits adhesion-dependent serovar Typhimurium-induced interleukin-8 production.     

Enhancement of TWIK-related Acid-sensitive Potassium Channel 3 (TASK3) Two-pore Domain Potassium Channel Activity by Tumor Necrosis Factor α

TASK3 two-pore domain potassium (K2P) channels are responsible for native leak K channels in many cell types which regulate cell resting membrane potential and excitability. In addition, TASK3 channels contribute to the regulation of cellular potassium homeostasis. Because TASK3 channels are important for cell viability, having putative roles in both neuronal apoptosis and oncogenesis, we sought to determine their behavior under inflammatory conditions by investigating the effect of TNFα on TASK3 channel current. TASK3 channels were expressed in tsA-201 cells, and the current through them was measured using whole cell voltage clamp recordings. We show that THP-1 human myeloid leukemia monocytes, co-cultured with hTASK3-transfected tsA-201 cells, can be activated by the specific Toll-like receptor 7/8 activator, R848, to release TNFα that subsequently enhances hTASK3 current. Both hTASK3 and mTASK3 channel activity is increased by incubation with recombinant TNFα (10 ng/ml for 2–15 h), but other K2P channels (hTASK1, hTASK2, hTREK1, and hTRESK) are unaffected. This enhancement by TNFα is not due to alterations in levels of channel expression at the membrane but rather to an alteration in channel gating. The enhancement by TNFα can be blocked by extracellular acidification but persists for mutated TASK3 (H98A) channels that are no longer acid-sensitive even in an acidic extracellular environment. TNFα action on TASK3 channels is mediated through the intracellular C terminus of the channel. Furthermore, it occurs through the ASK1 pathway and is JNK- and p38-dependent. In combination, TNFα activation and TASK3 channel activity can promote cellular apoptosis.     

The influence of body size on the diving behaviour and physiology of the bimodally respiring turtle, Elseya albagula

In aquatic vertebrates that acquire oxygen aerially dive duration scales positively with body mass, i.e. larger animals can dive for longer periods, however in bimodally respiring animals the relationship between dive duration and body mass is unclear. In this study we investigated the relationships between body size, aquatic respiration, and dive duration in the bimodally respiring turtle, Elseya albagula. Under normoxic conditions, dive duration was found to be independent of body mass. The dive durations of smaller turtles were equivalent to that of larger individuals despite their relatively smaller oxygen stores and higher mass specific metabolic rates. Smaller turtles were able to increase their dive duration through the use of aquatic respiration. Smaller turtles had a relatively higher cloacal bursae surface area than larger turtles, which allowed them to extract a relatively larger amount of oxygen from the water. By removing the ability to respire aquatically (hypoxic conditions), the dive duration of the smaller turtles significantly decreased restoring the normal positive relationship between body size and dive duration that is seen in other air-breathing vertebrates.     

A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes

The aim of this study was to assess inosine triphosphate (ITPase) expression in the different leukocyte populations present in peripheral blood samples of a nonimmune compromised control group. For this purpose, a multiparameter flow cytometric assay was developed and performed to study ITPase expression in peripheral leukocyte subpopulations of healthy volunteers (n = 20). Qualitative ITPase expression was assessed by determining the percentage of ITPase-positive cells. Quantitative data were obtained by measuring the median fluorescent intensity (MFI). Subcellular localization of ITPase was analyzed using immunocytochemistry. Immunocytochemistry showed that ITPase is present in all leukocytes and localized intracellular. Based on this finding, a multiparameter flow cytometric assay was developed using a Fix & Perm strategy. Qualitative and quantitative ITPase expression remained stable (variation, <10%) for at least 48 h after blood sampling. MFI values showed that activated monocytes contained significantly more ITPase when compared to the total monocyte fraction (P < 0.0001), which subsequently had a higher amount of expression than granulocytes (P < 0.0001). In addition, the phagocyte subpopulations ([activated] monocytes and granulocytes) contained significantly higher levels of ITPase when compared to lymphocytes (P < 0.0001). Within the lymphocyte fraction, it appeared that T-helper cells contained significantly higher ITPase levels when compared to cytotoxic T cells, B lymphocytes, and natural killer cells (P < 0.0001). Our study is the first which describes a flow cytometry assay to analyze ITPase expression in leukocytes qualitatively as well as quantitatively and visualizes the intracellular localization of ITPase in leukocytes. ? 2012 International Society for Advancement of Cytometry.