Seong-Chun Kwon

Role of protein kinase C- or RhoA-induced Ca2+ sensitization in stretch-induced myogenic tone

OBJECTIVE It has been suggested that Ca(2+) sensitization mechanisms might contribute to myogenic tone. However, specific mechanisms have yet to be fully identified. Therefore, we investigated the role of protein kinase C (PKC)- or RhoA-induced Ca(2+) sensitization in myogenic tone of the rabbit basilar vessel. METHODS Myogenic tone was developed by stretch of rabbit basilar artery. Fura-2 Ca(2+) signals, contractile responses, PKC immunoblots, translocation of PKC and RhoA, and phosphorylation of myosin light chains were measured. RESULTS Stretch of the resting vessel evoked a myogenic contraction and an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) only in the presence of extracellular Ca(2+). Stretch evoked greater contraction than high K(+) at a given [Ca(2+)](i). The stretch-induced increase in [Ca(2+)](i) and contractile force were inhibited by treatment of the tissue with nifedipine, a blocker of voltage-dependent Ca(2+) channel, but not with gadolinium, a blocker of stretch-activated cation channels. The PKC inhibitors, H-7 and calphostin C, and a RhoA-activated protein kinase (ROK) inhibitor, Y-27632, inhibited the stretch-induced myogenic tone without changing [Ca(2+)](i). Immunoblotting using isoform-specific antibodies showed the presence of PKCalpha and PKCepsilon in the rabbit basilar artery. PKCalpha, but not PKCepsilon, and RhoA were translocated from the cytosol to the cell membrane by stretch. Phosphorylation of the myosin light chains was increased by stretch and the increased phosphorylation was blocked by treatment of the tissue with H-7 and Y-27632, respectively. CONCLUSIONS Our results are consistent with important roles for PKC and RhoA in the generation of myogenic tone. Furthermore, enhanced phosphorylation of the myosin light chains by activation of PKCalpha and/or RhoA may be key mechanisms for the Ca(2+) sensitization associated myogenic tone in basilar vessels.

Inhibitory mechanism of xestospongin‐C on contraction and ion channels in the intestinal smooth muscle

Xestospongin‐C isolated from a marine sponge, Xestospongia sp., has recently been shown to be a membrane‐permeable IP3 receptor inhibitor. In this study we examined the effects of this compound on smooth muscle from guinea‐pig ileum. In guinea‐pig ileum permeabilized with α‐toxin, xestospongin‐C (3 μM) inhibited contractions induced by Ca2+ mobilized from sarcoplasmic reticulum (SR) with IP3 or carbachol with GTP, but not with caffeine. In intact smooth muscle tissue, xestospongin‐C (3–10 μM) inhibited carbachol‐ and high‐K+‐induced increases in [Ca2+]i and contractions at sustained phase. It also inhibited voltage‐dependent inward Ba2+ currents in a concentration‐dependent manner with an IC50 of 0.63 μM. Xestospongin‐C (3–10 μM) had no effect on carbachol‐induced inward Ca2+ currents via non‐selective cation channels; but it did reduce voltage‐dependent K+ currents in a concentration‐dependent manner with an IC50 of 0.13 μM. These results suggest that xestospongin‐C inhibits the IP3 receptor but not the ryanodine receptor in smooth muscle SR membrane. In intact smooth muscle cells, however, xestospongin‐C appears to inhibit voltage‐dependent Ca2+ and K+ currents at a concentration range similar to that at which it inhibits the IP3 receptor. Xestospongin‐C is a selective blocker of the IP3 receptor in permeabilised cells but not in cells with intact plasma membrane.

Tyrosine kinase participates in vasoconstriction through a Ca2+- and myosin light chain phosphorylation-independent pathway

This study was undertaken to determine the role of tyrosine kinase on intracellular Ca2+ ([Ca2+]i), myosin light chain (MLC) phosphorylation, and contraction caused by norepinephrine (NE) in rat aorta. NE induced a sustained contraction with an increase of [Ca2+]i. On the other hand, NE increased the phosphorylation of the 20 kDa MLC transiently. Pretreatment with genistein and tyrphostin 25, tyrosine kinase inhibitors, significantly inhibited NE‐induced contraction, but did not affect the increase of [Ca2+]i and MLC phosphorylation. These results suggest that tyrosine kinase may regulate the NE‐mediated contraction without altering [Ca2+]i and MLC phosphorylation in rat aorta.