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J Gen Physiol. May 1, 2000; 115(5): 653–662.
PMCID: PMC2217224

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 Ca2+ channels. In heart cells, a tight coupling between the gating of single L-type Ca2+ channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca2+ channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca2+ sparks and propagated Ca2+ waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca2+ channels. L-type Ca2+ channels can open without triggering Ca2+ sparks and triggered Ca2+ sparks are often observed after channel closure. CICR is a function of the net flux of Ca2+ ions into the cytosol, rather than the single channel amplitude of L-type Ca2+ channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca2+ channels are loosely coupled to RYR through an increase in global [Ca2+] due to an increase in the effective distance between L-type Ca2+ channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.

Keywords: calcium-induced calcium release, smooth muscle, Ca2+ sparks, excitation–contraction coupling, action potential signaling

INTRODUCTION

In striated muscle excitation–contraction (E-C) coupling is initiated by the gating of sarcolemmal L-type Ca2+ channels, which trigger the release of calcium from ryanodine receptors (RYRs) on the sarcoplasmic reticulum (Endo 1977; Fabiato 1983; Nabauer et al. 1989; Tanabe et al. 1990; McPherson and Campbell 1993). While the mechanism of coupling between L-type Ca2+ channels and RYRs is different in skeletal and cardiac myocytes, in both cell types local interactions between these proteins underlie calcium release. In skeletal myocytes, calcium entry is not required for calcium release (Armstrong et al. 1972), but gating of the L-type Ca2+ channel appears to be physically coupled to RYR opening (Tanabe et al. 1990; Nakai et al. 1998). Calcium entry through L-type Ca2+ channels triggers calcium-induced calcium release (CICR) in heart cells (Fabiato 1985; Nabauer et al. 1989), resulting in localized calcium release, termed Ca2+ sparks (Cheng et al. 1993; Cannell et al. 1995; Lopez-Lopez et al. 1995). This coupling process involves a local increase in [Ca2+]i in the microdomain of the L-type Ca2+ channel, which is sensed by the RYR, resulting in RYR gating, and several lines of evidence indicate that the opening of a single L-type Ca2+ channel triggers a Ca2+ spark in an obligatory fashion (Niggli and Lederer 1990; Cannell et al. 1995; Lopez-Lopez et al. 1995; Santana et al. 1996; Lipp and Niggli 1996; Collier et al. 1999). This tight coupling between gating of the voltage-dependent sarcolemmal channel and the sarcoplasmic reticular release channel underlies the full mobilization of Ca2+ that occurs in cardiac myocytes with each action potential.

RYRs are widely expressed in nonsarcomeric (smooth) muscle, neurons, and nonexcitable cells, although their role in calcium release and cellular signaling is poorly understood. In smooth muscle, RYRs are expressed on the sarcoplasmic reticulum (Carrington et al. 1995; Lesh et al. 1998) and triggered Ca2+ release has been inferred from measurements of calcium-activated membrane currents and spatially averaged [Ca2+]i (Zholos et al. 1992; Ganitkevich and Isenberg 1992, Ganitkevich and Isenberg 1995), but little direct evidence of CICR exists and the function of RYRs in E-C coupling is poorly understood. Spontaneous Ca2+ sparks have been reported in smooth muscle (Nelson et al. 1995; Mironneau et al. 1996; Gordienko et al. 1998), and recent experiments combining confocal microscopy and patch-clamp techniques have demonstrated localized calcium release during depolarizing voltage-clamp steps (Arnaudeau et al. 1997; Imaizumi et al. 1998), further supporting the existence of CICR in smooth muscle. Here we show that the L-type Ca2+ current (ICa) evokes CICR in single urinary bladder myocytes and establish the relationship between L-type Ca2+ channel opening and RYR-mediated calcium release in smooth muscle.

MATERIALS AND METHODS

Cell Isolation

Male New Zealand White rabbits were anesthetized (50 mg/kg ketamine, 5 mg/kg xylazine IM) and killed (100 mg/kg pentobarbital i.v.) in accordance with an approved laboratory animal protocol. The urinary bladder was removed, dissected in ice-cold oxygenated physiological salt solution, minced, and suspended in modified collagenase type II (Worthington Biochemical), 1 mg/ml protease type XIV and 5 mg/ml bovine serum albumin (Sigma-Aldrich) at 37°C for 35–40 min. Digested tissue was triturated with a wide-bore Pasteur pipette and passed through a 125-μm nylon mesh; cells were concentrated by low speed centrifugation, washed with fresh medium, resuspended, and stored at 4°C.

Electrophysiology

Single myocytes were placed in a chamber mounted on an inverted Nikon TE300 microscope (Nikon) and whole-cell recordings made as previously described (Wang and Kotlikoff 1997). In most experiments, pipettes were filled with (mM): 127 cesium glutamate, 10 HEPES (cesium-salt), 19 TEACl, 0.1 Tris-GTP, 1 Mg-ATP, 5 Tris2-creatine phosphate, and 0.1 fluo-4 (pentapotassium salt) at pH 7.2. Heparin (2–5 mg/ml) was added to the internal solution in some experiments. In experiments where the buffering capacity of the internal solution was increased, pipettes were filled with (mM): 110 CsCl, 5 HEPES (cesium-salt), 17 EGTA, 0.1 Tris-GTP, 1 Mg-ATP, 5 Tris2-creatine phosphate, and 3 fluo-4 (pentapotassium salt), with intracellular Ca2+ concentration adjusted to 100 nM at pH 7.2. The external solution contained (mM): 137 NaCl, 5 CsCl, 2 CaCl2, 1 NaH2PO4, 1.2 MgCl2, 10 HEPES, and 5 glucose at pH 7.4. Caffeine (10 mM) was applied to cells using a picospritzer (General Valve Corp.). Voltage-pulse protocols and the resulting membrane currents were acquired and analyzed using pCLAMP software (Axon Instruments, Inc.). Difference current was obtained either by linear leak subtraction or by blocking L-type Ca2+ current by substituting CaCl2 with 500 μM CdCl2. In experiments using ryanodine, cells were pre-incubated with the drug for 1 h at room temperature and 10 μM ryanodine was added to the bath solution.

Cells were field stimulated using a Grass S88 pulse stimulator (Grass Medical Instruments) connected to platinum wires placed in the recording chamber. The stimulus amplitude and duration were 70 V and 10 ms, respectively. Cells were incubated with 10 μM fluo-4 methoxymethylester (Molecular Probes, Inc.) and 0.02% pluronic acid in the dark for 20 min at room temperature, washed with fresh medium, and allowed to de-esterify for 40 min.

Recording and Measurement of Fluorescence

Fluo-4 fluorescence was excited with 488 nm light emitted from a Krypton/Argon laser and measured with a high speed laser scanning confocal head (Noran Oz), using a plan-apo, 60× water-immersion objective lens (1.2 NA; Nikon) and Intervision software on an Indy workstation (Silicon Graphics Inc.). x–y images were collected every 8.3 ms (256 × 240 pixels), and x–t images were obtained with line scans at 4.16-ms intervals for 2.13 s (512 × 480 pixels). Pixel size in the x axis was equal to 0.252 μm and in the y axis to 0.248 μm. To synchronize current and fluorescence measurements, a light emitting diode was placed in the path of the photomultiplier detector and switched on for 2 ms, 10 ms before the start of the voltage step. Images were analyzed using either Intervision software (Silicon Graphics Inc.) or a custom written analysis program using Interactive Data Language software (Research Systems Inc.), kindly provided by Drs. Mark Nelson and Adrian Bonev (Dept. of Pharamacology, University of Vermont, Burlington, VT). In all x–y images, a mean background fluorescence value was determined and subtracted from each pixel, and the images were smoothed using a 3 × 3 pixel median filter. Mean baseline fluorescence intensity (Fo) of a cell was obtained by averaging the first six to eight images that did not exhibit transient rises in intracellular Ca2+. Profiles of line-scan images were obtained over a 1-μm region and Fo was obtained by averaging the fluorescence of 30 pixels before a depolarizing step. Ratios of images (F/Fo) and profiles were constructed to reflect changes in fluorescence intensity over time. The previously described pseudo-ratio equation (Cheng et al. 1993) was used to estimate [Ca2+]i, using a Kd value for fluo-4 of 345 nM and assuming basal [Ca2+]i to be 100 nM. The Ca2+ spark criteria were a localized increase in fluorescence (F/Fo) ≥ 1.5, occurring in 20–30 ms, with a decay time of 50–80 ms. Ca2+ spark latencies were calculated as the time from the start of the voltage pulse to the point at which the fluorescence exceeded 5% of Fo, and were calculated for Ca2+ sparks occurring within the first voltage-clamp step of an experiment, so as not to bias results by an increase in [Ca2+]i resulting from preceding voltage-clamp steps. Ca2+ spark probability was calculated in the following manner. Voltage-clamp steps were analyzed to determine whether or not a Ca2+ spark occurred during a specific clamp step. Currents associated with each step were integrated to determine net Ca2+ flux, the fluxes were binned, and the probability was calculated by dividing the number of experiments in which a Ca2+ spark was evoked by the total number of experiments in the bin. Thus, the probability of a Ca2+ spark occurring in voltage-clamp steps after clamp steps in which no Ca2+ spark occurred is likely somewhat higher due to the accumulation of Ca2+ from previous steps. These probabilities were fit to a Boltzmann equation of the form:

equation M1

where J is the Ca2+ flux, J50 is the flux at which there is a 50% probability of evoking a Ca2+ spark, and k is the slope factor of the relationship. All statistical data are presented as mean ± SEM.

Online Supplemental Material

A movie depicting the entire experiment shown in Fig. 1 A is provided. The movie was constructed by superimposing the current and voltage traces up to the end of each image on the confocal images acquired at an interval of 8.3 ms; the contrast of the unratioed grey scale confocal images was adjusted to maximize the range between background and peak fluorescence (Adobe Photoshop). The stacked TIF files were converted to lower resolution JPG files and exported as a movie at 24 fps (Adobe Premier). Higher resolution, ratioed images at the beginning, middle, and end of this movie are shown in Fig. 1 A. This video can be found at http://www.jgp.org/cgi/content/full/115/5/653/DC1.

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 ...

RESULTS

The L-type Ca2+ Channel Current Triggers CICR

Depolarizing voltage-clamp steps activating ICa in single urinary bladder myocytes triggered one or several Ca2+ sparks and subsequent propagated Ca2+ waves (Fig. 1). Images acquired at 8.3-ms intervals showed that release began as elementary events at one or several foci, as previously reported (Imaizumi et al. 1998), and progressively expanded to propagated Ca2+ waves. The velocity of propagation of the Ca2+ wave was 94 ± 15 μm/s (n = 4), similar to Ca2+ wave velocity described in cardiac myocytes (Wussling et al. 1997), but substantially faster than Ca2+ waves propagated by InsP3 receptors in vascular cells (Bezprozvanny 1994).

Depolarizations activating smaller currents usually triggered a single Ca2+ spark and propagated Ca2+ wave, whereas larger currents initiated Ca2+ sparks from several sites that propagated and fused. The temporal relationship between ICa and Ca2+ sparks varied with the magnitude of the current, but Ca2+ sparks always occurred with a delay after current activation. In some cases, Ca2+ sparks were observed only after ICa was almost completely inactivated (Fig. 1 A). In separate experiments, Ca2+ sparks and Ca2+ waves were not altered by the dialysis of heparin (Fig. 1 C; n = 4), but were abolished by application of caffeine (10 mM; see Fig. 3; n = 9), incubation with ryanodine (10 μM; see Fig. 5; n = 11), or block of ICa with CdCl2 (500 μM; not shown; n = 9). The magnitude and kinetics of Ca2+ sparks triggered by ICa was similar to previously reported values for spontaneous Ca2+ sparks in smooth muscle. The mean rise time of triggered release events was 26.6 ± 1.6 ms, peak F/Fo = 1.9 ± 0.1, and the half time of decay of isolated (nonpropagated) Ca2+ sparks was 62 ± 16 ms (n = 5), which is similar to previous reports using similar methods (Perez et al. 1999). Thus, Ca2+ sparks and subsequent Ca2+ wave propagation triggered by the voltage-dependent calcium current is due to activation of RYRs by L-type Ca2+ channels.

Figure 3
Ca2+ sparks and Ca2+ wave propagation are specified by the net flux of Ca2+, not the amplitude of ICa. (A, top) Line-scan image obtained during tail-current protocol. Depolarization (100 mV) period is indicated by the horizontal line, diode flash by the ...
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, ICa fails to induce Ca2+ sparks in smooth muscle cells, whereas Ca2+ sparks ...

Relationship between ICa and CICR: Loose Coupling

The number of Ca2+ sparks triggered by ICa and the latency between the onset of the current and the appearance of Ca2+ sparks is in sharp contrast to CICR observed in heart cells, in which the latency of the release events (Wier et al. 1994; Cannell et al. 1995; Lopez-Lopez et al. 1995; Collier et al. 1999) closely follows the gating properties of individual L-type Ca2+ channels. The small number of Ca2+ spark sites evoked by ICa and the delay between L-type Ca2+ channel and RYR channel opening suggested a fundamentally different coupling process in smooth muscle. We hypothesized that, rather than sensing the local elevation of [Ca2+]i in the vicinity of the Ca2+ channel, smooth muscle RYRs were not sensitive to the opening of individual channels, but required a global rise in [Ca2+]i. To test this hypothesis, we first sought to determine whether activation of Ca2+ channels always lead to initiation of a Ca2+ spark.

As shown in Fig. 2, depolarizing voltage-clamp steps of short duration that initiated a calcium current, but little net calcium flux (J Ca2+), did not trigger Ca2+ sparks. When the duration of ICa was progressively increased, Ca2+ sparks were observed that occurred well after termination of the depolarizing step and did not propagate. Further lengthening the duration of ICa resulted in Ca2+ sparks that occurred closer to the period of current flow and finally in Ca2+ wave propagation. Activation of ICa without Ca2+ release does not occur in cardiac myocytes; rather, evidence suggests that the opening of a single L-type Ca2+ channel activates a Ca2+ spark (Santana et al. 1996; Collier et al. 1999). This observation and the demonstration of triggered Ca2+ sparks after current cessation indicate that L-type Ca2+ channels are loosely coupled to RYR channels. That is, L-type Ca2+ channels can open without initiating Ca2+ sparks in smooth muscle, and the probability of Ca2+ sparks occurring after activation of ICa is a function of current duration and magnitude (see below). It is unlikely that Ca2+ sparks during short ICa were missed and that late-occurring Ca2+ sparks were spontaneous events unrelated to ICa since: (a) measurements were made in line-scan mode using an extended slit width (z resolution at half max = 2.5 μM) to minimize the possibility of missed events; (b) spontaneous events were uncommon in the absence of ICa, but were always observed if J Ca2+ was sufficient; (c) the latency of late Ca2+ sparks decreased as J Ca2+ increased (Fig. 2); and (d) propagated Ca2+ waves, which were never observed spontaneously, often occurred after the termination of ICa.

Figure 2
Evoked Ca2+ sparks and Ca2+ wave propagation depends on the magnitude and duration of ICa. 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) ...

CICR Is a Function of the Magnitude of Ca2+ Influx, Not the Amplitude of ICa

As a further test of whether CICR in smooth muscle requires an increase in global [Ca2+]i or results from the local response of RYR to the opening of single L-type Ca2+ channels, we designed experiments to maximize J Ca2+ under conditions of low single-channel amplitude, and conversely to maximize the single channel current amplitude under conditions in which J Ca2+ is low. As shown in Fig. 3 A, bladder myocytes were depolarized to 100 mV for 100 ms to open L-type Ca2+ channels (without Ca2+ ion permeation), and then to varied potentials to systematically alter the magnitude and duration of the Ca2+ tail current. At more negative voltages (−70 mV) the magnitude of the instantaneous current (and the underlying single channel events) is relatively large (~0.3 pA; Rubart et al. 1996), but the current deactivation is rapid, resulting in little J Ca2+. Ca2+ sparks were never observed at clamp steps to −70 mV, indicating that brief channel openings of relatively large single channel amplitude do not trigger Ca2+ sparks. Conversely, when cells were stepped to −10 mV, where the single channel current amplitude is approximately threefold lower, but J Ca2+ is much larger due to a longer mean channel open time, CICR was routinely observed (n = 6). Thus, smooth muscle RYRs do not sense [Ca2+]i in the microdomain of the L-type Ca2+ channel, since large single channel events (which produce the highest local [Ca2+]i) do not evoke CICR in the absence of sufficient J Ca2+. Rather, CICR occurs at low single channel amplitude if the net J Ca2+ is sufficient. Moreover, the J Ca2+ requirements for propagated Ca2+ waves are incrementally greater than that required to achieve discrete Ca2+ sparks. The estimated global [Ca2+]i achieved immediately before spark propagation was ~230 nM (F/Fo = 1.33 ± 0.09, n = 8). After depletion of sarcoplasmic reticulum (SR) Ca2+ stores with caffeine (10 mM), tail current protocols did not result in Ca2+ sparks or propagated Ca2+ waves. As shown in Fig. 3 B, profiles from line-scan experiments obtained before and after caffeine exposure indicated that after SR depletion the tail protocol resulted in only a small rise in [Ca2+]i, relative to that observed in control steps, despite the fact the equivalent ICa obtained in both conditions (Fig. 3 C). In the experiment shown, the initial repolarization to −10 mV resulted in a rapid increase in local [Ca2+]i to greater than threefold baseline, whereas after caffeine application the increase was much smaller and slower.

The relationship between J Ca2+ and Ca2+ spark probability was examined quantitatively in voltage-clamp experiments. J Ca2+ was calculated from the integrated ICa in experiments such as that shown in Fig. 2, and the probability of a given J Ca2+ evoking a Ca2+ spark was determined. As shown in Fig. 4 A, the probability of an evoked Ca2+ spark increased sharply with J Ca2+. Fitting a generalized Boltzmann equation to the data, we determined that the flux at which the probability of evoking a Ca2+ spark was 50% occurred with a J Ca2+ of 4.0 fmol of Ca2+. We also examined the latency to Ca2+ spark in experiments at −30 and −10 mV (Fig. 4 B). Latencies were 32.0 ± 13.5 (n = 5) and 12.5 ± 2.7 (n = 10) in steps to −30 and −10 mV, respectively, significantly longer than observed in cardiac myocytes (<2 ms; Cannell et al. 1995). The voltage dependence of the latency is also consistent with a coupling mechanism related to net Ca2+ flux. That is, since the integral of ICa (and thus the flux of Ca2+ ions) increases more rapidly with time at −10 mV than at −30 mV (due mainly to the faster rate of current activation at −10 mV), the same Ca2+ flux is achieved in shorter time.

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. ICa from voltage-clamp steps was integrated and binned, and the probability ...

Uncoupling of CICR by Chelation of Cytosolic Ca2+

Loose coupling between L-type Ca2+ channels and RYR could result from an increase in the effective distance between these proteins, or could indicate a decreased affinity of the ryanodine receptor for Ca2+ ions. The spatial separation between a single L-type Ca2+ channel and RYR in cardiac cells has been estimated to be <100 nm, based on the fact that high concentrations of mobile Ca2+ buffers such as EGTA do not disrupt CICR (Collier and Berlin 1999), using models of radial diffusion of Ca2+ in a concentric shell (Klingauf and Neher 1997). To examine the effective distance between L-type Ca2+ channels and RYR, we sought to determine whether CICR is disrupted in smooth muscle cells in the presence of high concentrations of EGTA, and compared this result with experiments in heart cells recorded under identical conditions. As shown in Fig. 5, CICR was completely eliminated in smooth muscle cells dialyzed with 17 mM EGTA and 3 mM Fluo 4 ([Ca2+]i clamped at 100 nM; n = 6), whereas CICR was not affected in rat heart cells in equivalent protocols. Thus, Ca2+ ions entering through L-type Ca2+ channels at a distance >100 nm from RYRs are required for CICR in smooth muscle. We further investigated whether RYR are able to sense local Ca2+ entry by performing experiments in which we clamped [Ca2+]i at 250 and 500 nM, still maintaining 20 mM mobile Ca2+ buffer. We reasoned that under conditions of increased global [Ca2+]i, CICR might be triggered by a small additional increase in Ca2+ from near (<100 nm) L-type Ca2+ channels. However, CICR was not triggered in these experiments, suggesting that the functional distance between the L-type Ca2+ channel and RYR is substantially greater than in sarcomeric muscle, despite light microscopic evidence of a close association between calcium channels and RYRs in bladder smooth muscle (Carrington et al. 1995).

Link between Action Potential Discharge and CICR

To determine the relationship between action potential discharge and CICR under relatively physiological conditions, we examined CICR in fluo-4AM–loaded myocytes stimulated at varying frequencies. Rapid acquisition of confocal images during depolarizing stimuli indicated that Ca2+ release does not occur with each depolarization (Fig. 6). Rather, local Ca2+ sparks and propagated Ca2+ waves depend on action potential frequency, revealing complex signal integration at the level of calcium release. Thus, at low stimulation frequencies (0.5 Hz), nonpropagated Ca2+ sparks were observed only after accumulation of sufficient depolarizing stimuli (n = 4), and CICR took the form of discrete Ca2+ sparks. At higher frequency stimulation (10 Hz), similar to the frequency of spontaneous action potentials reported in guinea-pig bladder myocytes (Klockner and Isenberg 1985), Ca2+ sparks were propagated as Ca2+ waves and were repeatedly triggered, often from the same Ca2+ release site (n = 6). The frequency of initiation of the propagated Ca2+ waves was substantially lower than the stimulation frequency, resulting in an effective low-pass filter of high-frequency electrical signals.

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 ...

DISCUSSION

In sarcomeric myocytes, tight coupling exists between gating of the L-type Ca2+ channel and RYR such that essentially every L-type Ca2+ channel gating event results in the opening of one or more RYR. This coupling derives either from a physical interaction between the proteins in skeletal myocytes (Tanabe et al. 1990; Nakai et al. 1998), or in cardiac myocytes from an interaction between the Ca2+ ions permeating the L-type Ca2+ channel and subsequently gating RYR. The latter coupling process appears to involve a local sensing of permeating Ca2+ ions within the microdomain of a single L-type Ca2+ channel, such that opening of a single L-type Ca2+ channel is sufficient to activate a Ca2+ spark, since: (a) the occurrence of Ca2+ sparks is stochastic with a voltage sensitivity equivalent to the gating behavior of the L-type Ca2+ channel (Cannell et al. 1995; Collier et al. 1999); (b) the latency to occurrence of a Ca2+ spark after depolarization is equivalent to the latency to opening of an individual L-type Ca2+ channel (Lopez-Lopez et al. 1995; Santana et al. 1996; Collier et al. 1999); and (c) the coupling process is not disrupted in the presence of high concentrations of mobile Ca2+ buffer (Collier and Berlin 1999).

Despite the broad expression of L-type Ca2+ channels and RYR in many cell types, the existence and nature of CICR in nonsarcomeric cells, in which the distribution of L-type Ca2+ channels and RYR differs substantially from an orderly dyadic pattern, is not well established. In smooth muscle, evidence for CICR has been inferred from caffeine- and ryanodine-sensitive Cai transients evoked upon ICa activation (Zholos et al. 1992; Ganitkevich and Isenberg 1992, Ganitkevich and Isenberg 1995). More recently, confocal line-scan images acquired during flash photolysis of caged Ca2+ or peak ICa activation gave rise to localized increases in Ca2+ (Arnaudeau et al. 1997) and 2-D confocal images acquired during step depolarizations demonstrated areas of increased fluorescence intensity or “hot spots” (Imaizumi et al. 1998). Direct examination of CICR and the mechanism underlying the coupling between L-type Ca2+ channels and RyR is lacking, however.

Using both 2-D and line-scan confocal modes, we examined CICR as a function of the amplitude and duration of ICa, and provide direct visualization of CICR in x–y images obtained every 8.3 ms. A prominent feature of Ca2+ sparks activated by ICa is the very low number of evoked Ca2+ sparks relative to that seen during depolarization of cardiac myocytes (Fig. 1 and Fig. 2). While the frequency of Ca2+ sparks may be a function of SR loading and modulatory factors (Porter et al. 1998), the number of sparks observed after activation of ICa is dramatically lower than observed in cardiac cells and the ability to evoke Ca2+ release and Ca2+ waves with caffeine application suggests that the low efficiency of CICR coupling cannot be explained by poorly loaded SR. Visualization of individual Ca2+ sparks in heart cells requires that the amplitude of ICa be reduced (Cannell et al. 1995; Lopez-Lopez et al. 1995), while they were readily observed in bladder myocytes during voltage-clamp steps to activate ICa. Moreover, in smooth muscle, individual Ca2+ sparks spread in the form of a propagated Ca2+ wave, whereas in cardiac myocytes depolarization appears to result in CICR from each dyad, with little required propagation. Our data indicate that both the initial Ca2+ spark and the subsequent propagation occurs through the gating of RYR, since both were eliminated in the presence of ryanodine, and neither were affected by dialysis with heparin (Fig. 1).

A second major feature of CICR in smooth muscle relates to the nature of the coupling between the channels. Rather than every opening of L-type Ca2+ channels activating a Ca2+ spark, Ca2+ spark activation in smooth muscle cells was only observed when ICa was of sufficient magnitude or duration (Fig. 2 and Fig. 3). We term this relationship “loose coupling” since it differs dramatically from the obligate tight coupling that exists in heart cells. From experiments such as that shown in Fig. 2 and Fig. 3, it is clear that the opening of hundreds of L-type Ca2+ channels may not be sufficient to activate a Ca2+ spark if channel openings are not of sufficient duration. Experiments specifically designed to maximize single-channel amplitude and open-state probability, but minimize calcium flux, indicated that brief channel openings of maximal amplitude failed to activate Ca2+ sparks, whereas increasing the net Ca2+ flux at a lower single channel amplitude activated CICR. Thus, in smooth muscle, sufficient aggregate L-type Ca2+ channel activity is required to produce CICR in the form of discrete Ca2+ sparks, and further Ca2+ flux and increased global [Ca2+]i produces CICR in the form of propagated Ca2+ waves (Fig. 2 add 3). Taken together, these data indicate that RYRs appear to be coupled to L-type Ca2+ channels through a rise in global [Ca2+]i, rather than local elevations near the channel. This finding was further supported by the disruption of coupling by high concentrations of mobile Ca2+ buffer, conditions that do not affect the coupling between L-type Ca2+ channels and RYR in cardiac myocytes (Fig. 5). While these data could be explained by an increase in the spacing distance between the sarcolemmal and sarcoplasmic reticulum Ca2+ channels (L-type and RYR), it is also possible that the relatively few sites at which Ca2+ sparks are repeatedly observed (Imaizumi et al. 1998; Gordienko et al. 1998) represent a concentration of RYR sufficient to generate a resolvable Ca2+ spark, and that close connections exist between L-type Ca2+ channels and individual RYR, as has been reported (Carrington et al. 1995), but that these do not occur in the density required to generate a resolvable Ca2+ spark.

What then is the likely physiological relevance of loose coupling? In skeletal and cardiac myocytes, each action potential results in a twitch response that derives from RYR-mediated calcium release, triggered by local signals in the microdomain of the L-type Ca2+ channels. Thus, every neural signal evoking a postsynaptic action potential is obligatorily linked to a mechanical response. Moreover, in addition to tight coupling, the signal gain is quite high, since each channel opening results in a Ca2+ spark (activation of several RYRs), the duration of which is longer than the L-type Ca2+ channel opening (Cannell et al. 1995). We show here that in smooth muscle each action potential is not necessarily linked to CICR (Fig. 6), due to a coupling process that requires a sufficient rise in global [Ca2+]i. The uncoupling of Ca2+ release from the action potential introduces signal processing elements into the contractile response of smooth muscle. Features of “loose coupling” system are low gain (multiple L-type Ca2+ channels must open to produce Ca2+ sparks), discriminated responses (release takes the form of local Ca2+ sparks or globally propagated Ca2+ waves), and a marked lengthening of signal duration (Ca2+ waves last far longer than the action potential). Slight variations in this low gain, integrating system, such as a decrease in L-type Ca2+ channel density, likely underlies the fact that ICa does not appear to produce appreciable Ca2+ release in some smooth muscle cells, despite the presence of functional RYRs (Fleischmann et al. 1996; Kamishima and McCarron 1996). The dependence of Ca2+ release during E-C coupling on global increases in [Ca2+]i contrasts with the local signaling that underlies relaxation mediated by spontaneous Ca2+ release and the activation of sarcolemmal potassium channels (Nelson et al. 1995), providing a further example of a way in which local and global [Ca2+]i signals can be exploited to provide flexible cellular responses (Berridge 1997).

In summary, in the present study, we provide direct evidence of RYR-mediated Ca2+ release evoked by the L-type calcium current (CICR) in smooth muscle, demonstrate that the trigger stimulus for the Ca2+ release process is a global rather than local rise in [Ca2+]i, and show that this results in a functional uncoupling of a single action potential from Ca2+ release in smooth muscle cells. “Loose coupling” between L-type Ca2+ channels and RYR allows a functional uncoupling of the action potential and calcium release and provides a mechanism by which neural signals encoded at higher frequencies are transferred to slower mechanical responses.

Supplemental Material

Supplemental Material Index:

Acknowledgments

We thank Drs. Clara Franzini-Armstrong, W.K. Chandler, and Joshua R. Berlin for helpful comments, and Mr. Mario Brenes for technical support.

Supported by National Institutes of Health (NIH) grants HL45239 and DK52620 (to M.I. Kotlikoff). M.L. Collier is a NIH postdoctoral fellow (T32-DK07708-06).

Footnotes

The online version of this article contains supplemental material.

Abbreviations used in this paper: CICR, calcium-induced calcium release; E-C, excitation–contraction; RYR, ryanodine receptor; SR, sarcoplasmic reticulum.

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