Soichi Miwa (1), Yasushi Iwamuro (2), Xiao-Feng Zhang (1), Taijiro Enoki
(3), Yasuo Okamoto (1),Makoto Okazawa (1) and Tomoh Masaki (4)
Departments of Departments of (1) Pharmacology, (2) Neurosurgery
and (3) Anesthesiology, Kyoto University Faculty of Medicine, Kyoto 606
- 8501, Japan
(4) National Cardiovascular Center Reseach Institute, 5 - 7 - 1 Fujishirodai,
Suita, Osaka 565 - 8565, Japan
Abstract: The contraction of the rat aorta induced by endothelin-1
(ET-1) requires entry of extracellur Ca2+, but involvement of
voltage-operated Ca2+ channel is minor. Using whole-cell recordings
of patch-clamp and monitoring of the intracellular free Ca2+
concentration ([Ca2+]i), we characterized Ca2+
entry channels in A7r5 cells activated by ET-1. ET-1 activates three types
of voltage-independent Ca2+ entry channels: two types of Ca2+-permeable
nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated
Ca2+ channel (SOCC). Furthermore, it was found that these channels
can be pharmacologically discriminated using Ca2+ channel blockers
such as SK&F 96365 and LOE 908. NSCC-1 is resistant to SK&F 96365,
but sensitive to LOE 908, whereas NSCC-2 is sensitive to both SK&F 96365
and LOE 908. SOCC is sensitive to SK&F 96365, but resistant to LOE 908.
Using these channel blockers, we analyzed Ca2+ entry channels
involved in the ET-1-induced contractions of rat thoracic aorta and increases
in [Ca2+]i of single smooth muscle cells. The responses
to lower concentrations of ET-1 (</= 0.1 nM) were abolished by either
SK&F 96365 or LOE 908 alone. In contrast, the responses to higher concentrations
of ET-1 (>/=1 nM) were suppressed by SK&F 96365 or LOE 908 to about
10% and 35% of controls, respectively, and abolished by combined treatment
with SK&F 96365 and LOE 908. These results show that the responses of
rat aorta to lower concentrations of ET-1 involve only one Ca2+
channel that is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas
those to higher concentrations of ET-1 involve NSCC-1, NSCC-2 and SOCC,
contributing 10%, 55% and 35%, respectively, to total Ca2+ entry.
Keywords: Endothelin, Vascular smooth muscle cell, Ca2+ channel,
LOE 908, SK&F 96365