SAH-induced MMP activation and K V current suppression is mediated via both ROS-dependent and ROS-independent mechanisms

Abstract

Voltage-gated potassium (K V) channels regulate cerebral artery tone and have been implicated in subarachnoid hemorrhage (SAH)-induced pathologies. Here, we examined whether matrix metalloprotease (MMP) activation contributes to SAH-induced K V current suppression and cerebral artery constriction via activation of epidermal growth factor receptors (EGFRs). Using patch clamp electrophysiology, we observed that K V currents were selectively decreased in cerebral artery myocytes isolated from SAH model rabbits. Consistent with involvement of enhanced MMP and EGFR activity in SAH-induced K V current suppression, we found that: (1) oxyhemoglobin (OxyHb) and/or the exogenous EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), failed to induce further K V current suppression after SAH and (2) gelatin zymography detected significantly higher MMP-2 activity after SAH. The removal of reactive oxygen species (ROS) by combined treatment with superoxide dismutase (SOD) and catalase partially inhibited OxyHb-induced K V current suppression. However, these agents had little effect on OxyHb-induced MMP-2 activation. Interestingly, in the presence of a broad-spectrum MMP inhibitor (GM6001), OxyHb failed to cause K V current suppression. These data suggest that OxyHb suppresses K V currents through both ROS-dependent and ROS-independent pathways involving MMP activation. The ROS-independent pathway involves activation of MMP-2, whereas the ROS-dependent pathway involves activation of a second unidentified MMP or ADAM (a disintegrin and metalloprotease domain).

Figure 1. OxyHb and HB-EGF suppressed K_V currents of cerebral artery myocytes from control, but not SAH model animals

a) Example of whole-cell K⁺ currents before and after 10 minute application of OxyHb (10 μM) to cerebral artery myocytes isolated from a control and SAH model animal. b) Summary of 4-AP-sensitive K_V currents obtained from control (n = 6) and SAH model (n= 4) animals. c) Examples of whole cell K⁺ currents before and after 10 minute application of HB-EGF (30 ng/ml) to cerebral artery myocytes isolated from control and SAH model animals. d) Summary data demonstrating that OxyHb and HB-EGF significantly suppressed K_V currents in cerebral artery myocytes from control, but not SAH model animals. OxyHb treatment; control: n = 7, SAH: n = 4, HB-EGF treatment; control: n = 6, SAH: n = 5. ** P < 0.01 vs control, unpaired students t-test.

Figure 2. MMP-2 activity is enhanced in cerebral arteries from SAH model animals

a) An example of gelatin zymography demonstrating activity of commercially purified MMP-2 and cerebral artery homogenate from a control animal. B) Gelatin zymography demonstrating enhanced MMP-2 activity in homogenates obtained from SAH model animals. c) Summary data showing significantly greater MMP-2 activity in cerebral artery homogenates from SAH model animals compared to cerebral artery homogenates from un-operated control animals. (n = 8 for each) d) Summary data demonstrating that MMP-2 mRNA levels are similar in cerebral artery homogenates obtained from control and SAH model animals (n = 7 for each). ** P < 0.01 vs control, unpaired students t-test.

Figure 3. ROS-dependent and ROS-independent MMP activation and K_V current suppression caused by exogenous OxyHb

a) Representative K_V current recordings demonstrating the ability of the free radical scavengers superoxide dismutase (SOD) and catalase or the MMP inhibitor, GM6001 to reduce OxyHb-induced K_V current suppression. b) Summary data demonstrating that GM6001 caused a greater inhibition of OxyHb-induced K_V current suppression than SOD/catalase. * P < 0.05; ** P < 0.01 SOD/catalase and GM6001 on OxyHb-induced K_V suppression (n = 7); § P < 0.05 GM6001 (n = 5) vs. SOD/catalase treatmtent on OxyHb-induced K_V suppression (n = 5). ANOVA followed by Tukey test. c) Representative gel and summary of zymography data demonstrating that SOD/catalase treatment did not prevent OxyHb-induced MMP-2 activation. * P < 0.05; ** P < 0.01 versus control. (Control: n = 8, OxyHb: n = 8, SOD/cat + OxyHb: n = 4) ANOVA followed by Tukey test.