Zinc pyrithione activates K+ channels and hyperpolarizes the membrane of rat pulmonary artery smooth muscle cells

Figures

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  Fig 1. ZnPy relaxes pulmonary arteries. A. Record of tension developed in response to 1μM phenylephrine (PE), followed by cumulative addition of ZnPy (1x10-7 - 1x10-4 M). B. ZnPy concentration-response curve. Points and bars represent mean ± S.E.M. Curve is best fit to the Hill equation with IC50 = 4.3μM, minimum residual tension = 18.3% and Hill slope = 1.3. C. Record of tension developed in response to the cumulative addition of 1μM XE991, 10μM XE991 then 10μM ZnPy.

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  Fig 2. Properties of membrane hyperpolarisation induced by ZnPy in PASMC. A-B Original records of PASMC membrane potential before and after applying 10μM ZnPy (ZP) in the absence (A) and presence (B) of 10μM glibencamide, with and without the addition of 1μM XE991. C. Mean (±SEM) membrane potentials measured before and after applying ZnPy in the absence (n = 14) or presence (n = 12) of glibenclamide or glibenclamide plus XE991 (n = 12). C = control, W = wash. *p<0.05, **p<0.01, ***p<0.001 by repeated measures ANOVA with Tukey’s multiple comparisons test (glibenclamide present) or paired t-test (glibenclamide absent). D-F. Records of membrane potential during responses to ZnPy before and after exposing cells to 10mM TEA (D), 1μM paxilline (E) or 50nM iberiotoxin (F). G. Mean (±SEM) membrane potentials measured from the recordings in D-F. N = 10 (TEA), 15 (paxilline) and 12 (iberiotoxin). **p<0.01, ***p<0.001 by repeated measures ANOVA with Tukey’s multiple comparisons test.

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  Fig 3. ZnPy potentiates voltage-gated K+ current in PASMCs. A. K+ currents activated by 300ms steps to between -70 and 60 mV, applied from a holding potential of -80 mV at 5s intervals. Records under control conditions (left) and in the presence of 10μM ZnPy (right). Voltage protocol above control records. B. Current density plotted as a function of test potential before (control) and after applying 10μM ZnPy; mean±SEM of 3 cells. The difference between measurements in control conditions and after exposure to ZnPy is shown to indicate the currents induced by ZnPy. Inset shows the averaged difference currents at test potentials from -60 to -20 mV, obtained by digitally subtracting the control records from those during exposure to ZnPy. Broken line indicates zero current.

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  Fig 4. Properties of the ZnPy-induced current. A,C Examples of currents activated by a voltage step from -80 to 40 mV under control conditions, in the presence of 10μM ZnPy and after washing. Voltage protocol above traces. B. Difference current generated from the traces in A by digital subtraction of the control record from the record obtained in the presence of ZnPy. The best fit exponential function (red) with τ = 19.6ms is superimposed on the activation phase. D. Difference current generated from the traces in C, with the exponential function that best fit the activation time course (red) superimposed on the trace: two components were required (τ1 = 13.2ms, τ2 = 101ms), the faster component contributing 50.6% of the current. E. Plot of current amplitude against time with ZnPy applied at the times indicated by the bars.

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  Fig 5. XE991 reduces the ZnPy-induced current. A. Records of current activated by a voltage step from -80 to 40mV before and after exposure to 10μM ZnPy, under control conditions or in the presence of 10μM XE991. Each trace is the average of 3–4 consecutive records. B. Currents activated by ZnPy: traces are the average of records from 7 cells with bars indicating SEM. The ZnPy-induced current was obtained by subtracting control records from those in the presence of ZnPy (unblocked), or records in the presence of XE991 from those in the presence of XE991 plus ZnPy. The XE991-sensitive component was obtained by subtracting the ZnPy-induced current recorded in the presence of XE991 from that in its absence. The activation phase of the XE991-sensitive current was best fit by an exponential function (red), superimposed on the trace, with τ = 17.8ms. C. Plot of current amplitude against time, with ZnPy and XE991 applied as indictaed by the bars. Letters indicate amplitudes of traces in panel A.

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  Fig 6. Effects of BKCa channel blockers on the ZnPy-induced current. Upper traces show currents activated by a voltage step from -80 to 40mV before and after exposure to 10μM ZnPy, under control conditions or in the presence of 10mM TEA (A), 1μM paxilline (Pax, B) or 50nM iberiotoxin (IbTx, C). Each trace is the average of records from 6 (TEA and iberiotoxin) or 8 (paxilline) cells. Lower panels show the ZnPy-induced currents, obtained by digitally subtracting records in the absence of ZnPy from those in its presence, either in control conditions (unblocked) or after exposure to a BKCa blocker. The iberiotoxin-sensitive component in C, shown as the difference between the unblocked and iberiotoxin-inhibited currents, is superimposed by a double exponential function (red) that best fits the activation time course, with τ1 = 6.2ms and τ2 = 48ms: the fast component contributed 75.3% of the current.

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  Fig 7. Effect of ZnPy on the residual current recorded at 0 mV. Typical traces showing the current remaining after clamping a cell at 0 mV for ≥5 min, followed by the application of ZnPy (10μM) on its own or in the presence of 10mM TEA (A), 1μM paxilline (B) or 10μM XE991 (C). Drugs applied as indicated by the bars.