Ca2+ images and K+ current during depolarization in smooth muscle cells of the guinea-pig vas deferens and urinary bladder.
1. Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo-3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea-pig vas deferens or urinary bladder. 2. Images obtained every 8 ms, during stepping from -60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 micrometer diameter) 'hot spots', over 95 % of which were located within 1.5 micrometer of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places. 3. Cd2+ or verapamil abolished both hot spots and Ca2+-activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+-induced Ca2+ release (CICR). 4. Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to -20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up-stroke of an evoked action potential under current clamp. 5. It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+-dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.
Imaizumi
Y
Y
Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan. yimaizum@phar.nagoya-cu.ac.jp
Torii
Y
Y
Ohi
Y
Y
Nagano
N
N
Atsuki
K
K
Yamamura
H
H
Muraki
K
K
Watanabe
M
M
Bolton
T B
TB
D000200:Action Potentials / Q000187:drug effects; D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004558:Electric Stimulation; D004594:Electrophysiology; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000819:agonists / Q000378:metabolism*; D001743:Urinary Bladder / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014649:Vas Deferens / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014966:Xanthenes
D000814:Aniline Compounds; D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium