Protein phosphorylation changes in bovine carotid artery smooth muscle during contraction and relaxation

The protein phosphorylation changes associated with the contraction and relaxation of bovine carotid artery smooth muscle were studied using two-dimensional gel electrophoresis of labeled phosphoproteins. Muscle was stimulated with histamine, angiotensin II, 12-deoxyphorbol 13-isobutyrate (DPB) or high extracellular K+. Histamine induced a rapid and sustained contraction which was associated with an early (2 min) phosphorylation of 20 kDa myosin light chain (MLC) and two cytosolic proteins, Nos. 1 and 2, and with the late (60 min) phosphorylation of MLC, two isoelectric variants of desmin and ten other cytosolic proteins. Additionally, there was a decrease in the extent of phosphorylation of two cytosolic proteins, Nos. 9 and 10. Angiotensin II induced a rapid but transient contraction which was associated with the same early (2 min) phosphorylation changes, but with none of the late (60 min) changes. Elevation of the extracellular K+ concentration to 110 mM led to a sustained contraction which was associated with the phosphorylation of MLC and proteins Nos. 1 and 2 at both 2 and 60 min, but none of the other late phase phosphoproteins were seen. Addition of DPB, an activator of protein kinase C, induced a slowly developing but sustained contractile response which was associated with none of the early (5 min) phosphorylation changes. However, nearly all of late (60 min) protein phosphorylation changes were the same as those seen after histamine action. Addition of forskolin to either control or histamine-treated muscle led to an increase in the phosphorylation of three cytosolic proteins (Nos. 3, 8 and 13), and in the histamine-contracted muscle the dephosphorylation of MLC and proteins Nos. 4, 9, 10, 15 and 16. Similarly, forskolin induced a relaxation of DPB-treated muscle and the dephosphorylation of proteins Nos. 4, 9, 10, 15 and 16. These results suggest that there are two pathways by which histamine activates contraction: a Ca2+-calmodulin pathway which initiates the response, and a protein kinase C pathway which, along with the Ca2+-calmodulin pathway, sustains contraction.