Delayed arteriolar relaxation after prolonged agonist exposure: functional remodeling involving tyrosine phosphorylation

1 Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia; and 2 Department of Medical Physiology, College of Medicine, Texas A&M University, College Station, Texas 77843 Submitted 13 November 2002 ; accepted in final form 23 April 2003 Although arteriolar contraction is dependent on Ca 2 + -induced myosin phosphorylation, other mechanisms including Ca 2 + sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization may contribute to contractile events. We hypothesized that if arteriolar smooth muscle exhibits time-dependent behavior this may be manifested in differences in relaxation after short- and long-term exposure to contractile agonists. Studies were conducted in isolated arterioles pressurized to 70 mmHg. In initial experiments ( n = 10), rate of relaxation was measured after acute (5 min) or prolonged (4 h) exposure to 5 µM norepinephrine (NE). Prolonged exposure to NE resulted in significantly ( P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of vessels exposed to NE for 4 h was observed after superfusion with 0 mM Ca 2 + buffer, indicating that the alteration in relaxation was reversible and Ca 2 + dependent. A similarly impaired dilation was not observed with 4-h exposure to KCl (75 mM). To determine mechanisms contributing to the effects of prolonged NE exposure, studies were performed in the presence of the microtubule depolymerizing agent demecolcine (10 µM) or a series of tyrosine phosphorylation inhibitors. Although demecolcine caused significant vasoconstriction ( P < 0.05) and potentiated NE vasoconstriction, it did not prevent the effect of long-term NE exposure on relaxation. Genistein, although having no effect on acute NE-induced contraction, concentration-dependently inhibited prolonged NE constriction. Similarly, Src (PP1) and p42/44 MAP kinase (PD-98059) inhibitors prevented maintenance of long-term NE contraction. The data indicate that prolonged exposure to NE induces biochemical alterations that impair relaxation after removal of the agonist. The contractile effects are Ca 2 + dependent and involve tyrosine phosphorylation but do not appear to involve the polymerization state of the microtubule network. myogenic tone; calcium ion; cytoskeleton Address for reprint requests and other correspondence: M. A. Hill, Microvascular Biology Group, School of Medical Science, RMIT Univ., Bundoora, Victoria 3083, Aust