Involvement of tropomyosin in the sensitivity of Na+ + K+ ATPase to ouabain

The Na⁺/K⁺ ATPase sensitivity to ouabain was shown to be increased by 300 to 1000-fold after treatment of the plasma membrane by EDTA. Addition of proteins detached from the plasma membrane with Ca²⁺ ions to EDTA treated membranes reconstituted the original Na⁺/K⁺ ATPase resistance to ouabain inhibition. Tropomyosin with Ca²⁺ ions (not with Mg²⁺ ions) induced the same effect. When suboptimal doses of tropomyosin were used for such a reconstitution, the dose-response curve indicated a full reconstitution of a given percentage of enzyme molecules. This observation led us to assume a direct or indirect effect of tropomyosin on Na⁺/K⁺ ATPase functions.     

Inhibition of alpha-receptor-induced Ca2+ release and Ca2+ influx by Mn2+ and La3+

The influence of Mn2+ and La3+ on alpha-receptor-stimulated Ca2+ movements was examined in arterial smooth muscle of the rabbit aorta. Both cations cause an inhibition of phenylephrine (PE) contractile response which exhibits a different pattern at low and high cation concentrations. At 0.1-1.0 mM inhibition by Mn2+ and La3+ was predominately due to a reduction in Ca2+ influx reflected as inhibition of the slow phase of contraction and reduction in PE-stimulated 45Ca uptake. PE log dose-response curves were shifted to the right in a non-parallel manner by 1 mM Mn2+ such that responses to lower PE concentrations were more inhibited. However, in the presence of 10 mM Mn2+ PE responses are equally inhibited at all PE levels. At 10 mM both Mn2+ and La3+ also inhibited PE-stimulated Ca2+ release resulting in a reduction in both the rapid phase of contraction and in the magnitude of PE stimulation of 45Ca efflux. The effects of Nm2+ (1 or 10 mM) on contraction and 45Ca efflux were rapidly reversible, while the effect of La3+ was not. Inhibition of Ca2+ release by 10 mM Mn2+ and La3+ was not caused by displacement of releasable Ca2+, but appeared to reflect their occupation of a superficially located receptor modulating site. The inhibition of Ca2+ influx by lower concentrations of Mn2+ may illustrate the functional consequence of configurational changes in the alpha 2-form of the receptor which have been recently described at lower concentrations of divalent cations.     

Studies on the stable inhibition of Na+ + K+-ATPase by cassaine.

Exposure of rat brain Na+ + K+-ATPase (ATP phosphohydrolase E.C. 3.6.1.3) to concentrations of cassaine greater than 1 x 10(-4) M resulted in a poorly reversible inhibition of this enzyme. Inhibition did not require the presence of ATP and developed rapidly, but the final amount of inhibition observed was independent of time. The amount of inhibition observed at a given concentration of cassaine was reduced by increasing the concentration of membranes in the system. The inhibition of Na+ + K+-ATPase activity was associated with equivalent inhibition of the phosphorylation and (3H)-ouabain binding reactions of this enzyme, while the uninhibited enzyme was apparently kinetically normal. Concentrations of cassaine which produced this stable inhibition of Na+ + K+-ATPase had no effect on the Mg2+-activated ATPase or the NADH cytochrome-c-reductase activities of crude rat brain microsomal preparations. Cassaine inhibited the cholinesterase activity of rat brain microsomes with a Ki of about 5 x 10(-5) M, but his inhibition was fully reversible. The poorly reversible inhibitory actions of cassaine, thus, appeared specific for Na+ + K+-ATPase. Because this stable pattern of inhibition of the Na+ + K+-ATPase by cassaine required drug concentrations at least one hundred-fold greater than those which produce positive inotropic effects, it appears unlikely that this pattern of Na+ + K+-ATPase inhibition is involved in the cardiotonic actions of this drug.     

Vascular activation of K+ channels and Na+-K+ ATPase activity of estrogen-deficient female rats

The goal of the present study was to evaluate vascular potassium channels and Na⁺-K⁺-ATPase activity in estrogen deficient female rats. Female rats that underwent ovariectomy were assigned to receive daily treatment with placebo (OVX) or estrogen replacement (OVX + E2, 1 mg/kg, once a week, i.m.). Aortic rings were used to examine the involvement of K⁺ channels and Na⁺-K⁺-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 μM) and K⁺ channels blockers: tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 mM), charybdotoxin (ChTX, 0.1 mM) and iberiotoxin plus apamin. When aortic rings were pre-contracted with KCl (60 mM) or pre-incubated with TEA (5 mM), 4-aminopyridine (4-AP, 5 mM) and iberiotoxin (IbTX, 30 nM) plus apamin (0.5 μM), the ACh-induced relaxation was less effective in the ovariectomized group. Additionally, 4-AP and IbTX decreased the relaxation by sodium nitroprusside in all groups but this reduction was greater in the ovariectomized group. Estrogen deficiency also increased aortic functional Na⁺-K⁺ ATPase activity evaluated by K⁺-induced relaxation. L-NAME or endothelium removal were not able to block the increase in aortic functional Na⁺-K⁺ ATPase activity, however, TEA (5 mM) restored this increase to the control level. We also found that estrogen deficiency increased superoxide anion production and reduced nitric oxide release in aortic ring from ovariectomized animals. In summary, our results emphasize that the process underlying ACh-induced relaxation is preserved in ovariectomized animals due to the activation of K⁺ channels and increased Na⁺-K⁺ ATPase activity.     

Abacavir + dolutegravir + lamivudine for the treatment of HIV

Introduction: Since the last revision of both European and American guidelines (EACS and DHHS), new data from clinical trials and cohort studies, as well as experience in clinical practice, have prompted significant changes to the list of recommended/preferred options for the treatment of HIV infected patients, highlighted the role of INSTI-based regimens. Dolutegravir (DTG) in combination with abacavir/lamivudine (ABC/3TC) is one of these preferred regimens in multiple clinical scenarios, including treatment-naive and treatment-experienced patients.

Areas covered: In this article we describe the coformulation of ABC/3TC/DTG in a fixed-dose combination (FDC) approved in September 2014 for the treatment of HIV infection. We focused our research on the efficacy and safety data resulting from phase 2 and 3 clinical study, particularly on the results of both SPRING (1 and 2) and SINGLE studies.

Expert opinion: Triple combination therapy with ABC/3TC/DTG should be considered among the initial options for treatment-naive patients, being effective, well tolerated, with a high genetic barrier to resistance along with a convenient once-daily administration.

In treatment-experienced patients the single-tablet regimen (STR) based on ABC/3TC/DTG could be used as simplification strategy in subjects with sustained viral suppression, as the high genetic barrier of DTG should ensure a safe switch from both NNRTI or PI based regimens.     

Sodium fluoride produces a K+ efflux by increasing intracellular Ca2+ through Na+-Ca2+ exchange

Acute fluoride intoxication increases intracellular calcium (Cai), manifested by increased twitch tension in cardiac muscle, and by potassium efflux (mediated by Ca2+-dependent K+ channels) in fluoridated erythrocytes. Fluoride, like isoproterenol, stimulates adenylate cyclase, and could increase Cai via the effects of cAMP on Ca2+ channels. However, while the inotropic effects of fluoride mimicked isoproterenol in rat atria, their effects on the time course of isometric contraction were quite different. In addition, acetylcholine negated isoproterenol's effect on twitch tension but did not modulate the effects of fluoride. Further, the Ca2+ channel antagonist verapamil had no effect on fluoride-stimulated K+ efflux from erythrocytes. Fluoride also inhibits Na+-K+ ATPase, and increases intracellular Na+, so could increase Cai via Na+-Ca2+ exchange. Lanthanum, which blocks Na+-Ca2+ exchange, blocks fluoride-induced K+ efflux in erythrocytes. We conclude that the effects of fluoride on adenylate cyclase are not important in intact tissue, and that inhibition of Na+-K+ ATPase and subsequent Na2+-Ca2+ exchange may be the mechanism of increased Cai in acute fluoride toxicity.     

Effect of vanadium in the +5, +4 and +3 oxidation states on cardiac force of contraction, adenylate cyclase and (Na+ + K+)-ATPase activity

The influence of vanadium in the nominally +5 (NH4VO3; referred to as V5+), +4 (C10H14O5V and VOSO4; V4+) and +3 oxidation states (VCl3; V3+) on cardiac force of contraction, adenylate cyclase and (Na+ + K+)-ATPase activity was investigated in order to determine which form of vanadium mediates the cardiac effects. V5+, V4+ and V3+ (300 microM each) increased the force of contraction of isolated electrically driven cat papillary muscles by about 100%. In the presence of the reducing agent ascorbic acid (5 mM) none of the three compounds led to any distinct increase in force of contraction. On the particulate adenylate cyclase preparation from feline right ventricles only V5+ stimulated the enzyme activity by about 100%, whereas V4+ and V3+ were ineffective. In the presence of 5 mM ascorbic acid all three compounds were ineffective. In contrast, in the presence of the oxidizing agent diamide (azodicarboxylic acid-bis-dimethylamide; 1 mM) all three compounds became stimulatory. On the isolated (Na+ + K+)-ATPase V5+ (500 microM) alone reduced the basal activity by about 95%. In the presence of ascorbic acid the inhibitory effect of V5+ was greatly diminished. Similar results were obtained with V4+, V3+ (100 microM) alone inhibited (Na+ + K+)-ATPase activity only by about 40%. In the presence of ascorbic acid V3+ was ineffective. From the results it is concluded that positive inotropism, stimulation of adenylate cyclase and inhibition of (Na+ + K+)-ATPase by vanadium compounds likewise result from an action of vanadium in the +5 oxidation state.     

Targeting CD4+CD25+FoxP3+ regulatory T-cells for the augmentation of cancer immunotherapy

CD4+CD25+FoxP3+ T-regulatory (Treg) cells are vital to the maintenance of peripheral self tolerance and are implicated in tolerance to foreign antigens. Increasing evidence shows that Treg cells may also play an important role in immune evasion mechanisms employed by cancer. Treg cells are actively recruited and induced by tumors to block innate and adaptive immune priming, effector function and memory response, which can inhibit the efficacy of therapeutic cancer vaccines. As such, modulation of Treg cell function in cancer has been studied using various approaches, with encouraging preclinical and clinical findings. However, controlled and effective modulation of Treg cell function for cancer therapeutics will be contingent on a better understanding of the molecular basis of Treg cell interaction with tumor cells and ensuing immunosuppressive mechanisms.     

Generation of CD2+CD8+ NK Cells from c-kit+ Bone Marrow Cells in Porcine

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-kit⁺ bone marrow cells (c-kit⁺ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-kit⁺ BM cells that give rise to CD2⁺CD8⁺ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-kit⁺ BM cells than those of controls. And, we compared the ability of the cytotoxicity of CD2⁺CD8⁺ NK cells differentiated by cytokines from c-kit⁺ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100:1 for 4 h once a week. In results, CD2⁺CD8⁺ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-kit⁺ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-kit⁺ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.     

Interaction of clonidine with human placental Na+ -H+ exchanger

The effect of clonidine, an alpha 2-adrenergic receptor agonist, on the Na+ -H+ exchanger in human placental brush-border membrane vesicles was examined. The exchanger was inhibited by clonidine. The inhibition was freely reversible, and the apparent inhibition constant for the process was 250 microM. The nature of inhibition was found to be competitive with respect to Na+. The Dixon plot (1/v versus clonidine concentration) was linear (r2 = 0.998), indicating the interaction of the drug with a single site on the exchanger protein. Similar kinetic analyses with amiloride, a potassium-sparing diuretic, and cimetidine, a histamine type II receptor antagonist, revealed that these drugs also inhibited the Na+ -H+ exchanger by interacting with a single site on the protein. The presence of clonidine increased the intercepts without affecting the slopes of the l/v versus amiloride concentration and the l/v versus cimetidine concentration plots. These results demonstrate that all three drugs, amiloride, cimetidine and clonidine, interact with the human placental Na+-H+ exchanger at a single site in a mutually exclusive manner, and the site of interaction is identical with the Na+-binding site on the external surface of the exchanger protein.     

IL-17+ Foxp3+ T 细胞的研究进展

近来有文献报道复杂的细胞因子环境下, 调节性 T 细胞（regulatory T cells, Tregs）可以转变为表型和功能上酷似 Th17 的新的 T 细胞亚群, 即白细胞介素 （IL） -17 + Foxp3+T 细胞。IL-17 + Foxp3+T 细胞分泌 IL-17 并表达维甲酸受体相关孤儿受体γt（RORγt）, 在免疫系统中表现出双重特性。IL-17 + Foxp3 +T 细胞的发现为理解 Tregs 和 Th17的关系提供了新的思路。本文重点介绍了 IL-17 + Foxp3 +T 细胞的表型特征、 分化来源和多效性功能, 并进一步总结了炎症性疾病和肿瘤微环境中 IL-17+ Foxp3+T 细胞的作用。