Correspondence to: M.I. Kotlikoff, Department of Animal Biology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6046. Fax:215-573-6810 E-mail:mik{at}vet.upenn.edu.
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Abstract |
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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.
Key Words: calcium-induced calcium release, smooth muscle, Ca2+ sparks, excitationcontraction coupling, action potential signaling
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INTRODUCTION |
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In striated muscle excitationcontraction (E-C)1 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 (
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 (
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MATERIALS AND METHODS |
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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 3540 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 (
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, 60x water-immersion objective lens (1.2 NA; Nikon) and Intervision software on an Indy workstation (Silicon Graphics Inc.). xy images were collected every 8.3 ms (256 x 240 pixels), and xt images were obtained with line scans at 4.16-ms intervals for 2.13 s (512 x 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 xy images, a mean background fluorescence value was determined and subtracted from each pixel, and the images were smoothed using a 3 x 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 ( 1.5, occurring in 2030 ms, with a decay time of 5080 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:
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.
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RESULTS |
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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 (
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 (
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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 (
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 (
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;
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;
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 (
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-4AMloaded 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 (
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DISCUSSION |
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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 (
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 (
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 xy 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 (
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 (
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 (
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.
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: CICR, calcium-induced calcium release; E-C, excitationcontraction; RYR, ryanodine receptor; SR, sarcoplasmic reticulum.
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Acknowledgements |
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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).
Submitted: 30 August 1999
Revised: 28 March 2000
Accepted: 28 March 2000
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