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MAPK and PKC activity are not required for H(2)O(2)-induced arterial muscle contraction
N J Pelaez1, S L Osterhaus, A S Mak
1Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120, USA.
Abstract:
H(2)O(2)-induced pulmonary arterial smooth muscle (PASM) contractions are independent of Ca(2+) and myosin light chain phosphorylation. The purpose of this study was to determine whether mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1 and ERK2, or protein kinase C (PKC) activation is required for H(2)O(2)-induced contraction. Porcine PASM strips were stimulated with 1 mM H(2)O(2), 120 mM KCl, or 10 microM phorbol myristic acetate and freeze clamped at various times during the contractions. Changes in relative amounts of tyrosine/threonine phosphorylated MAPK compared with total MAPK were measured. MAPK tyrosine phosphorylation levels increased in correlation with tension development. However, 50 microM PD-98059, a MAPK/ERK kinase-MAPK kinase blocker, reduced MAPK phosphorylation below resting levels, even though the magnitude of the isometric tension development was unaltered. Freeze-clamped PASM strips were placed in a PKC activity assay buffer containing (32)P and CaCl(2) to measure the total myelin basic protein phosphorylation. The data show that: 1) the time courses of PKC activity and force produced in response to H(2)O(2) do not correlate, and 2) MAPK activation may be a concurrent event with, or a consequence of, tension development in response to a variety of agonists but is not responsible for contractions to H(2)O(2), high K(+), or phorbol esters.
Insights
Hydrogen peroxide (H2O2) causes pulmonary arterial smooth muscle (PASM) contractions independently of calcium. Mitogen-activated protein kinase (MAPK) activation is not required for these H2O2-induced PASM contractions.
Area of Science:
- Physiology
- Biochemistry
- Pharmacology
Background:
- Pulmonary arterial smooth muscle (PASM) contractions are crucial for regulating vascular tone.
- Hydrogen peroxide (H2O2)-induced PASM contractions are known to be independent of intracellular calcium (Ca2+) and myosin light chain phosphorylation.
- The precise signaling pathways mediating H2O2-induced PASM contractions remain incompletely understood.
Purpose of the Study:
- To investigate the potential involvement of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in H2O2-induced PASM contractions.
- To determine if MAPK or PKC activation is a prerequisite for the development of force in PASM stimulated by H2O2.
Main Methods:
- Porcine PASM strips were utilized for mechanical tension measurements.
- Stimulation was performed using H2O2, high potassium (KCl), or phorbol myristic acetate (PMA).
- MAPK phosphorylation and PKC activity were assessed at various time points during contraction using biochemical assays and Western blotting.
Main Results:
- MAPK tyrosine phosphorylation levels increased in parallel with tension development.
- Inhibition of MAPK activation with PD-98059 did not alter the magnitude of H2O2-induced isometric tension.
- PKC activity did not correlate with the time course of force generation in response to H2O2.
Conclusions:
- MAPK activation is not essential for H2O2-induced PASM contractions.
- MAPK activation may be a downstream event or a consequence of contraction induced by various agonists, rather than a primary driver for H2O2-induced contractions.
- These findings suggest alternative signaling mechanisms are responsible for H2O2-mediated PASM contraction.