20-Hydroxyeicosatetraenoic acid contributes to the inhibition of K+ channel activity and vasoconstrictor response to angiotensin II in rat renal microvessels.

The present study examined whether 20-hydroxyeicosatetraenoic acid (HETE) contributes to the vasoconstrictor effect of angiotensin II (ANG II) in renal microvessels by preventing activation of the large conductance Ca(2+)-activated K(+) channel (KCa) in vascular smooth muscle (VSM) cells. ANG II increased the production of 20-HETE in rat renal microvessels. This response was attenuated by the 20-HETE synthesis inhibitors, 17-ODYA and HET0016, a phospholipase A2 inhibitor AACOF3, and the AT1 receptor blocker, Losartan, but not by the AT2 receptor blocker, PD123319. ANG II (10(-11) to 10(-6) M) dose-dependently decreased the diameter of renal microvessels by 41 ± 5%. This effect was blocked by 17-ODYA. ANG II (10(-7) M) did not alter KCa channel activity recorded from cell-attached patches on renal VSM cells under control conditions. However, it did reduce the NPo of the KCa channel by 93.4 ± 3.1% after the channels were activated by increasing intracellular calcium levels with ionomycin. The inhibitory effect of ANG II on KCa channel activity in the presence of ionomycin was attenuated by 17-ODYA, AACOF3, and the phospholipase C (PLC) inhibitor U-73122. ANG II induced a peak followed by a steady-state increase in intracellular calcium concentration in renal VSM cells. 17-ODYA (10(-5) M) had no effect on the peak response, but it blocked the steady-state increase. These results indicate that ANG II stimulates the formation of 20-HETE in rat renal microvessels via the AT1 receptor activation and that 20-HETE contributes to the vasoconstrictor response to ANG II by blocking activation of KCa channel and facilitating calcium entry

Effect of 17-ODYA on the inhibitory action of ANG II on K_Ca channel activity in renal VSM cells.

<p>Upper panel presents a representative tracing K_Ca channel activity in the cell-attached mode before and after addition of 17-ODYA (10^-7 M) and 17-ODYA plus ANG II to the bath. K_Ca channel currents were recorded with 140 mM KCl in the pipette at the resting membrane potential (pipette potential = 0 mV) and the cells were bathed in normal physiological salt solution in the presence of ionomycin. The middle panel summarizes the patch clamp recording mode. Lower panel summarizes the effects of 17-ODYA and 17-ODYA and ANG II on the open probability of the K_Ca channel in renal VSM cells (n = 4 cells from 4 rats). * indicates a significant difference versus the corresponding control value.</p

Effect of blockade of PLC and PLA₂ on the inhibitory action of ANG II on K_Ca channel activity in renal VSM cells.

<p>The upper panel presents representative tracings of single K_Ca channel currents recorded at the resting membrane potential (pipette potential = 0 mV) in the cell-attached mode before and after addition of PLC inhibitor U-73122 (10^-5 M) and U-73122 plus ANG II (10^-7 M) to the bath (left). The effects of the PLA₂ inhibitor, AACOF₃ (2 x 10^-5 M) and AACOF₃ plus ANGII (10^-7 M) are presented in the right panel. K_Ca channel currents were recorded with 140 mM KCl in the pipette at the resting membrane potential (pipette potential = 0 mV) and the cells were bathed in normal physiological salt solution in the presence of ionomycin. The middle panel summarizes the patch clamp recording mode. K_Ca channel currents were recorded as described in Figure 8. The lower left panel summarizes the effect of U-73122 and ANG II on the open probability of the K_Ca channel in renal VSM cells while the lower right panel depicts the effects of AACOF₃ on the response to ANGII. * indicates a significant difference versus control and # indicates a significant difference from the corresponding value recorded after PLC activity was inhibited with U-73122 or PLA2 activity was inhibited with AACOF3. </p

Effect of ANG II on the production of 20-HETE in renal microvessels.

<p>Comparison of the production of 20-HETE in renal microvessels treated with vehicle, ANG II and ANG II plus inhibitors of the synthesis of 20-HETE, 17-ODYA (10^-5 M) and HET0016 (10^-6 M), a phospholipase A₂ inhibitor, AACOF₃ (2 X 10^-5 M), a phospholipase C inhibitor, of U-73122 (10^-5 M), and the AT₁ receptor blocker, Losartan (10^-6 M). Numbers in the bars indicate the number of vessel preparations studied. # indicates a significant difference from the corresponding value in vessels treated with vehicle. * indicates a significant difference from the corresponding value in vessels treated with ANG II (10^-7 M).</p

Effect of Losartan on the inhibitory action of ANG II on K_Ca channel activity in renal VSM cells.

<p>The upper panel presents a representative current traces recording of the effects of Losartan and ANG II on the K_Ca channel activity recorded in the presence of ionomycin. K_Ca channel currents were recorded with 140 mM KCl in the pipette at the resting membrane potential (pipette potential = 0 mV) and the cells were bathed in normal physiological salt solution in the presence of ionomycin. The lower panel summarizes the effects of Losartan and ANG II on K_Ca channel activity recorded in the cell attached mode in the presence of ionomycin in the bath. * indicates a significant difference from the control value recorded from the same cells. # indicates a significant difference from the corresponding value recorded in the presence of ionomycin.</p

Effects of ANG II on [Ca²⁺]_i in renal VSM cells before and after 17-ODYA.

<p>The upper panel presents representative tracings of the effect of ANG II on [Ca²⁺]_i in renal VSM before and after blockade of the synthesis of 20-HETE with 17-ODYA (10^-5 M). The lower panel presents the peak and steady state [Ca²⁺]_i responses to ANG II measured before and after addition of 17-ODYA to the bath. Mean values <u>+</u> SE recorded from 25-30 cells (5-8 cells per experiment) isolated from 6 different renal VSM isolations are presented. * indicates a significant difference between ANG II and ANG II plus 17-ODYA treated vessels.</p

Effect of ANG II on intracellular calcium response in renal VSM cells.

<p>The upper panel presents a representative tracing of a [Ca²⁺]_i response of a single renal microvessel VSM cell to ANG II (10^-7 M) bathed in PSS before and after addition of ionomycin (10^-6 M). ANG II elicited a rise in [Ca²⁺]_i in renal VSM cells under control conditions. Addition of ionomycin raised baseline [Ca²⁺]_i and under these conditions ANG II had no additional effect on [ Ca²⁺]_i. The lower left panel summarizes the effects of on peak and plateau [Ca²⁺]_i responses to ANG II (10^-7 M) in the renal VSM cells. The right panel presents the [Ca²⁺]_i response to ANG II after addition of ionomycin. * indicates a significant rise in [Ca²⁺]_i over baseline. Mean values <u>+</u> SE recorded from 45-70 cells (5-10 cells per experiment) isolated from 9 different renal VSM cell isolation are presented. </p

Effects of 20-HETE on vasoconstrictor responses to ANG II in renal microvessels.

<p>Concentration response curves for the vasoconstrictor response to ANG II (10^-11 to 10^-6 M) in pressurized renal microvessels with the endothelium removed (N=5) are presented before and after addition of 17-ODYA (10^-5 M). 17-ODYA an inhibitor of the formation of 20-HETE had no significant effect on the EC50 but it reduced the maximal response to ANG II by 50%. # indicates a significant difference from the value at the lowest concentration of ANG II concentration (10^-11 M). * indicates a significant difference from the corresponding value prior to treatment of 17-ODYA (10^-5 M). </p