Enhancement of Ca2+-dependent outward current in sheep bladder myocytes by evans blue dye.

Whole-cell and inside-out patch-clamp techniques were used to assess the action of a well-known dye, Evans blue, on membrane currents in bladder isolated smooth muscle cells from sheep. In whole cells Evans blue dose-dependently increased the outward current by up to fivefold. In contrast, Evans blue had no effect on inward Ca2+ current. The effect on outward current was abolished or reduced if the cells were bathed in Ca2+-free solution, iberiotoxin (5 x 10(-8) M), or charybdotoxin (5 x 10(-8) M), but was unaffected by externally applied caffeine (5 mM) or in cells exposed to heparin (1 mg/ml) via the patch pipette. In inside-out patches bathed in a Ca2+ concentration of 5 x 10(-7) M, Evans blue (10(-4) M) increased the open probability of large-conductance (298-pS) Ca2+-dependent K+ channels (BK channels), shifting the half maximal-activation voltage by -70 mV. We conclude that Evans blue dye acts as an opener of BK channels.

Ca2+ current and Ca(2+)-activated chloride current in isolated smooth muscle cells of the sheep urethra.

1. Isolated sheep urethral cells were studied using the perforated patch clamp technique (T = 37 degrees C). Depolarizing steps ranging from -40 to -10 mV evoked an inward current that peaked within 10 ms and a slower inward current. Stepping back to the holding potential of -80 mV evoked large inward tail currents. All three currents were abolished by nifedipine (1 microM). Substitution of external Ca2+ with Ba2+ resulted in potentiation of the fast inward current and blockade of the slow current and tails. 2. Changing the chloride equilibrium potential (ECl) from 0 to +27 mV shifted the reversal potential of the tail currents from 1 +/- 1 to 27 +/- 1 mV (number of cells, n = 5). Chloride channel blockers, niflumic acid (10 microM) and anthracene-9-carboxylic acid (9AC, 1 mM), reduced the slow current and tails suggesting that these were Ca(2+)-activated Cl- currents, ICl(Ca). 4. Caffeine (10 mM) induced currents that reversed at ECl and were blocked by niflumic acid (10 microM). 5. In current clamp mode, some cells developed spontaneous transient depolarizations (STDs) and action potentials. Short exposure to nifedipine blocked the action potentials and unmasked STDs. In contrast, 9AC and niflumic acid reduced the amplitude of the STDs and blocked the action potentials. 6. In conclusion, these cells have both L-type ICa and ICl(Ca). The former appears to be responsible for the upstroke of the action potential, while the latter may act as a pacemaker current.

Nitric Oxide-Mediated Signaling in the Bystander Response of Individually Targeted Glioma Cells.

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. In this study, human glioblastoma T98G cell nuclei were individually irradiated with an exact number of helium ions using a single-cell microbeam. It was found that when only 1 cell in a population of approximately 1200 cells was targeted, with one or five ions, cellular damage measured as induced micronuclei was increased by 20%. When a fraction from 1% to 20% of cells were individually targeted, the micronuclei yield in the population greatly exceeded that predicted on the basis of the micronuclei yield when all of the cells were targeted assuming no bystander effect was occurring. However when 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide (NO)-specific scavenger was present in the culture medium, the micronuclei yields reduced to the predicted values, which indicates that NO contributes to the bystander effect. By using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), NO was detected in situ, and it was found that NO-induced fluorescence intensity in the irradiated population where 1% of cell nuclei were individually targeted with a single helium ion was increased by 1.13 +/- 0.02-fold (P <0.005) relative to control with approximately 40% of the cells showing increased NO levels. Moreover, the medium harvested from helium ion-targeted cells showed a cytotoxic effect by inducing micronuclei in unirradiated T98G cells, and this bystander response was also inhibited by c-PTIO treatment. The induction of micronuclei in the population could also be decreased by c-PTIO treatment when 100% of cells were individually targeted by one or two helium ions, indicating a complex interaction of direct irradiation and bystander signals.

The myeloproliferative disorder-associated JAK2 V617F mutant escapes negative regulation by suppressor of cytokine signaling 3

The somatic JAK2 valine-to-phenylalanine (V617F) mutation has been detected in up to 90% of patients with polycythemia and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia and idiopathic myelofibrosis. Suppressor of cytokine signaling 3 (SOCS3) is known to be a strong negative regulator of erythropoietin (EPO) signaling through interaction with both the EPO receptor (EPOR) and JAK2. We report here that JAK2 V617F cannot be regulated and that its activation is actually potentiated in the presence of SOCS3. Instead of acting as a suppressor, SOCS3 enhanced the proliferation of cells expressing both JAK2 V617F and EPOR. Additionally, although SOCS1 and SOCS2 are degraded in the presence of JAK2 V617F, turnover of SOCS3 is inhibited by the JAK2 mutant kinase and this correlated with marked tyrosine phosphorylation of SOCS3 protein. We also observed constitutive tyrosine phosphorylation of SOCS3 in peripheral blood mononuclear cells (PBMCs) derived from patients homozygous for the JAK2 V617F mutant. These findings suggest that the JAK2 V617F has overcome normal SOCS regulation by hyperphosphorylating SOCS3, rendering it unable to inhibit the mutant kinase. Thus, JAK2 V617F may even exploit SOCS3 to potentiate its myeloproliferative capacity.