(A–B) Rings were incubated with Krebs containing normal glucose (NG, 6mM) and/or Krebs containing PMA (0.1 μM) for 3 h before concentration response curve (CRC) to ACh was determined. Treatment of aortic rings with PMA for 3 h caused a significant reduction in maximal relaxation to ACh in females. (C–F) Rings from female rats were incubated with Krebs containing NG or Krebs containing LY379196 (1 μM), Fasudil (1μM), Ro-32-0432 (9 nM), or Rottlerin (6 μM) for 20 min or Krebs containing LY379196, Fasudil, Ro-32-0432 or Rottlerin and PMA (0.1 μM) for 3 h before the CRC to ACh was determined. Treatment of aorta with LY379196, but not Fasudil, Ro-32-0432 or Rottlerin prior to PMA restored the abnormal ACh relaxation of aorta. Data are presented as the mean ± S.EM. (*P < 0.05 paired t-test).