Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum.
1. To characterize increases in cytosolic free Ca2+ concentration ([Ca2+]i) associated with discharge of action potentials, membrane potential and [Ca2+]i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques. 2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca2+]i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca2+]i transients with an almost constant amplitude. 3. Nicardipine (1 microM) and La3+ (10 microM), blockers of voltage-dependent Ca2+ channels (VDCCs), abolished both the action potential discharge and the [Ca2+]i transient. 4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca2+-activated K+ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca2+]i transients. 5. Thapsigargin (1 microM), an inhibitor of SR Ca2+-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca2+]i transients without affecting action potentials. In addition. in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca2+ current but exerted a similar effect on its concomitant rise in [Ca2+]i to that on the action potential-triggered [Ca2+]i transient. 6. Similar action potential-triggered [Ca2+]i transients were induced by brief exposures to high-K+ solution. They were not decreased, but rather increased, after depletion of intracellular Ca2+ stores by a combination of ryanodine (30 microM) and caffeine (10 mM) through an open-lock of Ca2+-induced Ca2+ release (CICR)-related channels. 7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca2+]i. Intracellular Ca2+ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca2+]i transients by replenishing cytosolic Ca2+. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca2+]i transient.
Kohda
M
M
Department of Veterinary Science, Faculty of Agriculture, Gifu University, Japan.
Komori
S
S
Unno
T
T
Ohashi
H
H
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D018999:Charybdotoxin / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D005470:Fluorometry; D016257:Fura-2; D006168:Guinea Pigs; D007082:Ileum / Q000378:metabolism* / Q000502:physiology; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009130:Muscle, Smooth / Q000378:metabolism* / Q000502:physiology; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D012433:Ryanodine / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D002121:Calcium Channel Blockers; D026902:Potassium Channel Blockers; D018999:Charybdotoxin; D012433:Ryanodine; D002110:Caffeine; D019284:Thapsigargin; D002118:Calcium; D016257:Fura-2