Superoxide differentially controls pulmonary and systemic vascular tone through multiple signalling pathways

Abstract

Aims: the aim of this study was to determine the relative importance of Ca(2+) sensitization, ion channels, and intracellular Ca(2+) ([Ca(2+)](i)) in the mixed constrictor/relaxation actions of superoxide anion on systemic and pulmonary arteries.

Methods and results: pulmonary and mesenteric arteries were obtained from rat. Superoxide was generated in arteries and cells with 6-anilino-5,8-quinolinequinone (LY83583). Following pre-constriction with U46619, 10 μmol/L LY83583 caused constriction in pulmonary and relaxation in mesenteric arteries. Both constrictor and relaxant actions of LY83583 were inhibited by superoxide dismutase and catalase. LY83583 caused Rho-kinase-dependent constriction in α-toxin-permeabilized pulmonary but not mesenteric arteries. Phosphorylation of myosin phosphatase-targeting subunit-1 (MYPT-1; as determined by western blot), was enhanced by LY83583 in pulmonary artery only. However, in both artery types, changes in tension were closely correlated with changes in phosphorylation of the 20 kDa myosin light chain as well as changes in [Ca(2+)](i) (as measured with Fura PE-3), with LY83583 causing increases in pulmonary and decreases in mesenteric arteries. When U46619 was replaced by 30 mmol/L K(+), all changes in [Ca(2+)](i) were abolished and LY83583 constricted both artery types. The K(V) channel inhibitor 4-aminopyridine abolished the LY83583-induced relaxation in mesenteric artery without affecting constriction in pulmonary artery. However, LY83583 caused a similar hyperpolarizing shift in the steady-state activation of K(V) current in isolated smooth muscle cells of both artery types.

Conclusions: superoxide only causes Rho-kinase-dependent Ca(2+) sensitization in pulmonary artery, resulting in constriction, and whilst it opens K(V) channels in both artery types, this only results in relaxation in mesenteric.

Figure 1

Vascular effects of LY83583 are via generation of reactive oxygen species. (A, bar chart) Effects of LY83583 (1 μmol/L: 1-LY, 10 μmol/L: 10-LY) on L-012 luminescence, taken as a measure of reactive oxygen species production in mesenteric artery (MA, n = 9–10), and pulmonary artery (PA, n = 6–21). *P < 0.05 for LY83583 vs. control (artery without LY83583). ^†P < 0.05 for dicoumarol (Dic, 10 μmol/L) vs. 10 μmol/L LY83583. (B) Measurement of tension (mN) in PA (left) and MA (right) pre-constricted for 10–15 min with U46619 (range: 100–200 nmol/L) and 1 mmol/L l-NAME before application of 10 μmol/L LY83583 for a further 15 min alone (PA n = 4, MA n = 12), or in the presence of catalase (200 U/mL, PA n = 4, MA n = 7) or superoxide dismutase (SOD) and catalase (200 U/mL each, PA n = 4, MA n = 9). Bar charts: Constriction in PA (left) and MA (right) measured at 15 min before and after application of LY83583, normalized to constriction induced by 80 mmol/L K⁺. Asterisk denotes significant constriction (PA) or relaxation (MA) in response to LY83583 (P < 0.01).

Figure 2

Effects of LY83583 on Ca²⁺-sensitization and phosphorylation of MYPT-1 and MLC₂₀. (A) Measurement of Ca²⁺-induced tension (mN) in α-toxin permeabilized PA (pCa 6.9, left) and MA (pCa 6.4, right). 15 min applications of 10 μmol/L LY83583 caused further constriction in PA (representative of 24 ± 5% increase, n = 8), but had no effect in MA (representative of n = 11). In both arteries these constrictions were relaxed by the Rho-kinase inhibitor Y27632 (10 μmol/L) (PA 81.6 ± 3.3%, n = 8 and MA 69.2 ± 2.9%, n = 11). (B) Example western blots and summary bar charts showing effects of 1 and 10 μmol/L LY83583 (1 LY or 10 LY) on MYPT-1 and MLC₂₀ phosphorylation in non-permeabilized PA (left) and MA (right), in the presence of 100 nmol/L U46619 and 1 mmol/L l-NAME (U46/LN). Ratio of phospho/total for each blot was calculated and effects of treatments expressed as a percentage change over control (untreated artery). Asterisk denotes significant enhancement vs. control (P < 0.05, PA n = 10–12, MA n = 12). Dagger denotes significant increase (PA) or decrease (MA) in response to LY83583 compared with U46/LN alone (P < 0.05).

Figure 3

Effects of LY83583 on relationship between constriction and [Ca²⁺]_i with U46619 pre-constriction. Example traces: Simultaneous measurement of tension (mN) and [Ca²⁺]_i (R_340/380) in PA (A) and MA (B). Following pre-constriction with 100 nmol/L U46619 and 1 mmol/L l-NAME, LY83583 was added cumulatively at 10 min intervals, as indicated. Note: constriction and [Ca²⁺]_i responses returned to the pre-constriction level when LY83583 was washed out in the continued presence of U46619/l-NAME. Summary charts: effects of LY83583 on [Ca²⁺]_i (open symbols) and constriction (closed symbols), normalized to responses induced by 80 mmol/L K⁺, in PA (A, n = 4–5) and MA (B, n = 4). Note: charts do not show effects of washout. Asterisk denotes significant enhancement or reduction of tension or [Ca²⁺]_i compared with the zero time-point (P < 0.05).

Figure 4

Effects of LY83583 on relationship between constriction and [Ca²⁺]_i with sub-maximal K⁺ pre-constriction. Example traces: Simultaneous measurement of tension (mN) and [Ca²⁺]_i (R_340/380) in PA (A) and MA (B). Following pre-constriction with 30 mmol/L KCl and 1 mmol/L l-NAME, LY83583 was added cumulatively at 10 min intervals, as indicated. Note: constriction and [Ca²⁺]_i responses returned to the pre-constriction level when LY83583 was washed out in the continued presence of U46619/l-NAME. Summary charts: effects of LY83583 on [Ca²⁺]_i (open symbols) and constriction (closed symbols), normalized to responses induced by 80 mmol/L K⁺ in PA (A, n = 4–6) and MA (B, n = 4–5). Note: charts do not show effects of washout. Asterisk denotes significant enhancement of tension or [Ca²⁺]_i compared with the zero time-point (P < 0.01).

Figure 5

Effects of K⁺ channel blockers on LY83583-induced constriction and relaxation. Measurements of tension (mN) in PA (A) and MA (B) pre-constricted for 10–15 min with U46619 (range: 10–100 nmol/L) and 1 mmol/L l-NAME. Example traces show effects of 10 μmol/L LY83583 in the absence (control) and presence of BK_Ca channel blocker paxilline, (1 μmol/L) or K_V channel blocker 4-aminopyridine (4-AP, 5 mmol/L). As shown in summary bar charts, significant enhancement of constriction caused by LY83583 in PA (A) was unaffected by either paxilline or 4-AP (*P < 0.05 vs. U46/LN alone, n = 4–6). In contrast, significant relaxation caused by LY83583 in MA (B) was only delayed by paxilline but abolished by 4-AP (*P < 0.01 vs. U46/LN alone, n = 6–12).

Figure 6

Effects of LY83583 on steady-state activation of voltage-gated K⁺ currents. Voltage protocols: cells serially subjected to a range of preconditioning pulses between −60 and +60 mV, each followed by a short test pulse to −20 mV (A, PASMC) or 0 mV (B, MASMC). Representative traces: tail currents evoked at relevant test potential before (control) and during application of LY83583 (10 μmol/L for 10 min). Note: different test potentials were chosen for ease of measurement of much smaller currents in MASMC. Arrows indicate point at which measurements were taken. Main summary plots: Tail current measurements from PASMC (A, n = 5) and MASMC (B, n = 6), normalized to maximum current amplitude, plotted against conditioning potential and fitted by Boltzmann non-linear regression. Inset bar-charts: V_A calculated from Boltzmann fit in PASMC (A, *P < 0.01 vs. control) and MASMC (B, *P < 0.01 vs. control). Note: all currents recorded in the presence of 1 μmol/L paxilline and 10 μmol/L glibenclamide to block BK_Ca and K_ATP channels.