Zusammenfassung
Ca(2+) influx via Ca(2+) current (I(Ca)) during the action potential
(AP) was determined at 25 degrees C and 35 degrees C in isolated
rabbit ventricular myocytes using AP clamp. Contaminating currents
through Na(+) and K(+) channels were eliminated by using Na(+)- and
K(+)-free solutions, respectively. DIDS (0.2 mmol/L) was used to
block Ca(2+)-activated chloride current (I(Cl(Ca))). When the sarcoplasmic
reticulum (SR) was depleted of Ca(2+) by preexposure to 10 mmol/L
caffeine, total Ca(2+) entry via I(Ca) during the AP was approximately
12 micromol/L cytosol (at both 25 degrees C and 35 degrees C). Similar
Ca(2+) influx at 35 degrees C and 25 degrees C resulted from a combination
of higher and faster peak I(Ca), offset by more rapid I(Ca) inactivation
at 35 degrees C. During repeated AP clamps, the SR gradually fills
with Ca(2+), and consequent SR Ca(2+) release accelerates I(Ca) inactivation
during the AP. During APs and contractions in steady state, total
Ca(2+) influx via I(Ca) was reduced by approximately 50\% but was
again unaltered by temperature (5.6+/-0.2 micromol/L cytosol at 25
degrees C, 6.0+/-0.2 micromol/L cytosol at 35 degrees C). Thus, SR
Ca(2+) release is responsible for sufficient I(Ca) inactivation to
cut total Ca(2+) influx in half. However, because of the kinetic
differences in I(Ca), the amount of Ca(2+) influx during the first
10 ms, which presumably triggers SR Ca(2+) release, is much greater
at 35 degrees C. I(Ca) during a first pulse, given just after the
SR was emptied with caffeine, was subtracted from I(Ca) during each
of 9 subsequent pulses, which loaded the SR. These difference currents
reflect I(Ca) inactivation due to SR Ca(2+) release and thus indicate
the time course of local Ca(2+) in the subsarcolemmal space near
Ca(2+) channels produced by SR Ca(2+) release (eg, maximal at 20
ms after the AP activation at 35 degrees C). Furthermore, the rate
of change of this difference current may reflect the rate of SR Ca(2+)
release as sensed by L-type Ca(2+) channels. These results suggest
that peak SR Ca(2+) release occurs within 2.5 or 5 ms of AP upstroke
at 35 degrees C and 25 degrees C, respectively. I(Cl(Ca)) might also
indicate local Ca(2+), and at 35 degrees C in the absence of DIDS
(when I(Cl(Ca)) is prominent), peak I(Cl(Ca)) also occurred at a
time comparable to the peak I(Ca) difference current. We conclude
that SR Ca(2+) release decreases the Ca(2+) influx during the AP
by approximately 50\% (at both 25 degrees C and 35 degrees C) and
that changes in I(Ca) (and I(Cl(Ca))), which depend on SR Ca(2+)
release, provide information about local subsarcolemmal Ca(2+).
Nutzer