Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Abstract

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.

Figure 2

Evoked Ca²⁺ sparks and Ca²⁺ wave propagation depends on the magnitude and duration of I_Ca. Short clamp steps do not produce Ca²⁺ sparks, whereas lengthening the current duration results first in delayed Ca²⁺ sparks and then in Ca²⁺ wave propagation. (Top) Confocal line-scan images from a myocyte scanned along a single line at 4.16-ms intervals. The horizontal bar above each line-scan image indicates the period of the depolarizing pulse to −30 mV (A) or to −10 mV (B). The vertical bar indicates the diode flash used to synchronize optical and electrical data. (Bottom) The currents and voltage protocol are shown for each line-scan image in an expanded time scale. Note activation of Ca²⁺ sparks after termination of the depolarizing pulse. (C) The time course of a single Ca²⁺ spark (from 30-ms clamp step in A). The broken line shows a single exponential fit to the Ca²⁺ spark decay; τ = 59 ms.

Figure 1

Ryanodine receptors mediate calcium-induced calcium release and Ca²⁺ wave propagation in single urinary bladder myocytes. (Top) Selected x–y confocal images showing Ca²⁺ sparks and propagated Ca²⁺ waves from a series of images acquired every 8.3 ms after step depolarization of a fluo-4–loaded, voltage-clamped bladder myocyte to activate I_Ca. Relative fluorescence intensities are indicated by the color bar. (Bottom) Simultaneously recorded relative fluorescence profile from images as shown above and membrane current during a step depolarization to −30 mV (A) and to −10 mV (B and C). The slow tail current after Ca²⁺ release reflects activation of the Ca²⁺-sensitive chloride current. Numbers correspond to individual images shown at top. (C) The cell was dialyzed with 2 mg/ml heparin. The profile was obtained by averaging pixels within a 10 × 10 pixel (2.52 × 2.48 μm) box (outlined in the first image, top), placed over the area of Ca²⁺ spark initiation. Note that the change in relative fluorescence is delayed at −30 mV compared with that at −10 mV. A movie of Fig. 1 A showing images and current at 8.3 ms intervals is available at http://www.jgp.org/cgi/content/full/115/5/653/DC1.

Figure 4

Activation of a Ca²⁺ spark is dependent upon Ca²⁺ flux. (A) The relationship between the probability of Ca²⁺ spark occurrence and the Ca²⁺ flux in the associated current is shown. I_Ca from voltage-clamp steps was integrated and binned, and the probability of a Ca²⁺ spark occurring in each bin was determined. The solid line is a fit of the data to a generalized Boltzmann equation with parameters of 50% probability at 4.0 fmol of calcium, and a slope of 2.7. (B) Latencies for Ca²⁺ sparks obtained in voltage-clamp steps to −30 or −10 mV. The latency to Ca²⁺ spark after activation of I_Ca was determined from the first voltage-clamp step in experiments similar to Fig. 2; depolarization durations were 6 and 30 ms for voltage-clamp steps to −10 and −30 mV, respectively. Latencies were 32.0 ± 13.5 (n = 5) and 12.5 ± 2.7 (n = 10) at membrane potentials of −30 and −10 mV, respectively.

Figure 3

Ca²⁺ sparks and Ca²⁺ wave propagation are specified by the net flux of Ca²⁺, not the amplitude of I_Ca. (A, top) Line-scan image obtained during tail-current protocol. Depolarization (100 mV) period is indicated by the horizontal line, diode flash by the vertical line. (Middle) Voltage protocol and tail currents in time register with scan. (Bottom) Magnitude of Ca²⁺ flux (J Ca²⁺) during each repolarizing pulse. A propagated Ca²⁺ wave is triggered with maximum J Ca²⁺ and minimum I_Ca amplitude. (B) Fluorescence profile measured over a 1-μm area from line-scan images recorded as in A under control conditions and after exposure to caffeine (10 mM). Difference profile indicates the magnitude of Ca²⁺ release upon repolarization to −10 mV. Arrows indicate the point of repolarization. (C) I_Ca tail currents during repolarization to −10 mV before and during caffeine corresponding to the arrows in B.

Figure 5

Increased effective distance between L-type Ca²⁺ channels and ryanodine receptors in smooth muscle relative to cardiac muscle. Under conditions of high calcium buffering capacity, I_Ca fails to induce Ca²⁺ sparks in smooth muscle cells, whereas Ca²⁺ sparks are abundant in ventricular myocytes. (Top) Confocal x–y images from a series obtained at 8.3-ms intervals from smooth muscle (rabbit urinary bladder, A) and cardiac muscle (rat ventricle, B) cells dialyzed with 17 mM EGTA and 3 mM fluo-4. The scale bars are the same for A and B. (Bottom) Voltage-clamp protocol and corresponding Cd²⁺-sensitive membrane currents; numbered bars indicate time of corresponding images above.

Figure 6

Calcium-induced calcium release is loosely coupled to depolarizing stimuli. Low frequency depolarizing stimuli do not produce calcium release with every depolarization. Rather, a sufficient number of low frequency depolarizations result in nonpropagated Ca²⁺ sparks (local signal), and higher frequency stimuli produce repeated Ca²⁺ sparks that are propagated as Ca²⁺ waves (local and global signals). (A) Confocal x–y images of a smooth muscle cell during depolarizing stimuli applied at 2 s, and then at 0.1-s intervals. Images were obtained at the positions indicated below. (B) Profiles from images as above obtained every 16.7 ms. The global signal (above) shows the average relative fluorescence of the entire cell, and the local signal is from a 10 × 10 pixel region at the point of Ca²⁺ spark initiation. The inset shows the absence of Ca²⁺ sparks in a cell stimulated at 0.1-s intervals, after exposure to 10 μM ryanodine.