Pulmonary Pharmacology & Therapeutics (1998) 11, 205–208 Article No. pu980139 PULMONARY PHARMACOLOGY & THERAPEUTICS Endothelin-1 Activates MAP Kinases and c-Jun in Pulmonary Artery Smooth Muscle I. A. Yamboliev*, A. Hruby, W. T. Gerthoffer Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557-0046, USA SUMMARY: We tested the coupling of endothelin receptors to mitogen-activated protein kinases (MAPK) and nuclear factor c-Jun in intact canine pulmonary artery smooth muscle. Muscle rings denuded of endothelium were stimulated with 10 -7  ET-1 and frozen during contraction. An ‘in-gel’ kinase assay with myelin basic protein as substrate revealed protein kinase activities at 98, 75, 55, 50, 44 and 40 kDa. Erk1 and Erk2 MAPK were activated by ET-1 to 5.4±0.97 and 4.03±1.54 times basal activity at 10 min. Using phospho-specific antibodies, we found increased threonine/tyrosine phosphorylation of p38 and JNK1 MAPK to 2.04±0.47 and 2.56±0.72 times basal. ET-1 increased the phosphorylation level of nuclear factor c-Jun with a time-course closely matching the activation of JNK1 and p38 MAPK. Therefore, endothelin receptors initiate intracellular signals leading to activation of Erk, p38 and JNK1 MAPK pathways and ultimately to nuclear targets. The activation of JNK1 MAPK seems closely related to the phosphorylation of nuclear transcription factor c-Jun.  1998 Academic Press KEY WORDS: ET-1, c-Jun, MAP kinase, Pulmonary artery, Smooth muscle. MATERIALS AND METHODS INTRODUCTION Methods The endothelin family consists of three closely related peptides (ET-1, ET-2 and ET-3) which activate at least Second and third branches of pulmonary artery were two G-protein-coupled receptors (ET A and ET B ). ET- dissected from adult mongrel dogs of either sex killed 1 is a powerful vasoconstrictor in isolated blood with pentobarbital sodium (45 mg/kg iv). Muscle rings vessels. Muscle contraction is mediated by a number of (2–3 mm) were equilibrated in physiological salt so- intracellular signal transduction molecules including lution and reproducible contractile responses obtained phospholipases C, D and A 2 , phosphatidylinositol 3- to potassium depolarizing solution. 7 The rings were kinase (PI-3K), protein kinase C (PKC) and focal then stimulated with 100 n ET-1 for times ranging adhesion kinase (FAK). 1,2 In addition, endothelins from 2–60 min, frozen in ice-cold 5 m NaF/acetone induce sustained activation of Erk MAPK. 3 It has been solution (-80°C), homogenized, and centrifuged at proposed that endothelin-induced MAPK activation 10 000 g, for 10 min. Protein concentration of the super- might be important for cell cycle entry and progression natants was assayed using the Bicinchoninic acid through the cell cycle, 4,5 or for induction of cell mot- method. 7 Erk MAPK activity was measured by an ‘in- ility. 6 In the present study we tested the effect of gel’ kinase assaying employing myelin basic protein ET-1 on activation of Erk, JNK1 and p38 MAPK, as as substrate as previously described. 7 Dual threonine/ well as the phosphorylation of c-Jun in intact smooth tyrosine phosphorylation of p38 and JNK1 MAPK, muscle strips of canine pulmonary artery. and serine phosphorylation of c-Jun was determined by immunoblotting. 8 Phosphoproteins in kinase assay gels were detected and quantitated using a BioRad Model 525 Molecular Imager (Hercules, California, * Author for correspondence. USA) to assess activation of Erk MAPK. Densitometry 1094–5539/98/020205+04 $30.00/0  1998 Academic Press 205