Ca2+ -induced Ca2+ release through localized Ca2+ uncaging in smooth muscle

Abstract

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).

Figure 1.

TPFP of caged Ca²⁺ in smooth muscle cells. Multiphoton uncaging of Ca²⁺ in smooth muscle. Cells (A–D) or tissues (E and F) were loaded with Fluo4 for the measurement of [Ca²⁺]_i and exposed to 730-nm excitation at the laser power (LP) shown under control conditions or after loading with caged Ca²⁺ (D-EDTA). At excitation power >5 mW, photodamage-induced Ca²⁺ release was observed in single cells in the absence of TPFP (no caged Ca²⁺) but was not observed <5 mW (C). Ca²⁺ release was only observed in cells and tissues loaded with D-EDTA (compare D with C and F with E) and was evoked in single cells at laser strengths below the threshold for photodamage (compare D with C). Profiles at right show fluorescence values from entire image series at the site of photolysis. Arrows indicate point of laser flash. Excitation powers shown were measured at the objective.

Figure 2.

Ca²⁺ waves triggered by localized Ca²⁺ uncaging. (A) Typical response to 88-ms two-photon excitation (4.7 mW) shows that two initial stimuli do not result in an observed release of Ca²⁺ (i.e., photoreleased Ca²⁺ is below the threshold of detection). A third pulse at the same location results in a localized release that propagates partially throughout the cell. Three pulses were within 15 s. (B) More slowly propagating asynchronous waves were occasionally observed with longer laser pulses or multiple short pulses. In the experiment shown, a single, longer pulse activated a slowly propagating Ca²⁺ wave at the site of laser focus after ∼200 ms. Note the slow time course of propagation and decay of the Ca²⁺ transient. (A and B) Bars, 10 μm. (C) Schematic showing areas in which TPFP activated Ca²⁺ release in single cells after a single 88-ms pulse. CICR was almost twofold more likely at the cell periphery. c, center; oc, off center; a, cell end.

Figure 3.

Activation of Ca²⁺ sparks by TPFP. (A) Sequential images obtained at 22-ms intervals before, during, and after TPFP (indicated by red bar). To show the relationship between the laser pulse focus location and the Ca²⁺ spark, the stimulation point is shown by the white circle on the image containing the scale bar. Note that the spark occurs at the site of focus of the uncaging pulse within 20 ms of the onset of the flash. (B) A similar experiment as in A, but demonstrating the activation of a Ca²⁺ spark ∼6 μm from the laser focus with a delay of ∼180 ms. Images taken from a series obtained at 22-ms intervals. Sequential images are shown starting with the third image, which also shows the point of laser focus (white circle). (A and B) Bars, 10 μm. (C) Line scan experiment demonstrating repeated activation of Ca²⁺ sparks with typical kinetics and spread at the site of laser focus. Ca²⁺ sparks were repeatedly triggered at the same site by a single photolysis stimulus (note first and second pulses). Inset shows last induced sparks at higher magnification and the relationship to the site of laser photolysis (white circle). Note the activation of separate Ca²⁺ sparks at ∼5 μm from the point of laser focus.

Figure 4.

Ryanodine and InsP₃Rs can mediate TPFP-induced CICR. (A) Incubation of cells in 10 μM ryanodine did not prevent cells from releasing Ca²⁺, whereas combined exposure to 30 μM ryanodine and xestospongin C completely eliminated Ca²⁺ release. Arrows indicate laser flashes. (B) Summary data indicating the propability of release in each condition. Note that the number of exposures to TPFP required to induce release was significantly higher in the presence of ryanodine. Error bars represent SD. *, P < 0.05; **, P < 0.01.

Figure 5.

Altered properties of Ca²⁺ release by ryanodine and InsP₃Rs. (A) Examples of TPFP experiments in tissues in the three conditions. Traces at right indicate fluorescence at the laser focus site throughout continuous experiments. Note the slow rise time in the presence of ryanodine. (B) Summary data from all similar experiments. Rise time and propagation were significantly slower in the presence of ryanodine, whereas xestospongin C had no significant effect. **, P < 0.01. Error bars represent SD.

Figure 6.

Increased delay to release by InsP₃Rs. (A) Examples of line scan experiments obtained during TPFP. Plots below indicate continuous fluorescence at the peak of release for each condition. Ryanodine markedly increased the delay between initiation of the laser uncaging flash and initial Ca²⁺ release. Laser excitation pulse was 100 ms in all experiments. As observed in xy experiments, rise time was markedly slowed in the presence of ryanodine. (B) Summary data from a series of line scan experiments indicates significant increase in delay in the presence of ryanodine. **, P < 0.01. Error bars represent SD.

Figure 7.

FKBP12.6 inactivation alters TPFP Ca²⁺ release. (A) Line scan experiments indicate a decrease in delay from initiation of the photolysis pulse to the observation of Ca²⁺ release. (B) Sequential xy images also indicate earlier release in FKBP12.6-null myocytes. Note different time scales. (C) Summary data indicate the marked change in kinetics of Ca²⁺ release in FKBP12.6-null tissues. *, P < 0.05; **, P < 0.01. Error bars represent SD.