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Originally published In Press as doi:10.1074/jbc.M708923200 on December 20, 2007

J. Biol. Chem., Vol. 283, Issue 11, 7206-7218, March 14, 2008
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A Single Luminally Continuous Sarcoplasmic Reticulum with Apparently Separate Ca2+ Stores in Smooth Muscle*

John G. McCarron1 and Marnie L. Olson

From the Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, Scotland, United Kingdom

Received for publication, October 30, 2007 , and in revised form, December 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Whether or not the sarcoplasmic reticulum (SR) is a continuous, interconnected network surrounding a single lumen or comprises multiple, separate Ca2+ pools was investigated in voltage-clamped single smooth muscle cells using local photolysis of caged compounds and Ca2+ imaging. The entire SR could be depleted or refilled from one small site via either inositol 1,4,5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) suggesting the SR is luminally continuous and that Ca2+ may diffuse freely throughout. Notwithstanding, regulation of the opening of RyR and IP3R, by the [Ca2+] within the SR, may create several apparent SR elements with various receptor arrangements. IP3R and RyR may appear to exist entirely on a single store, and there may seem to be additional SR elements that express either only RyR or only IP3R. The various SR receptor arrangements and apparently separate Ca2+ storage elements exist in a single luminally continuous SR entity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In smooth muscle, the endoplasmic reticulum (ER)2 exists as an extensive membrane network of tubules, flattened cisternae, and vesicles (1, 2) to comprise rough (RER, which carries ribosomes) and smooth (SER) reticula. RER predominates around the nucleus and is scattered throughout the cytoplasm and appears to be in luminal continuity with the SER (1, 3, 4). SER is found mainly near the cell surface to run in close apposition to the plasma membrane. In various tissues, changes in ER structure may occur at different developmental stages and when the cells experience prolonged activation (reviewed in Ref. 5). These changes may give the impression of a labile (pleiomorphic) ER network capable of modifications in structure and, consequently, function.

The Ca2+ storage element of the ER, the sarcoplasmic reticulum (SR), may be a specialized region of the SER that is endowed with those proteins responsible for the uptake (Ca2+ pumps), storage (calsequestrin), and release ryanodine receptor ((RyR) and inositol 1,4,5-trisphosphate receptor (IP3R)) of Ca2+ (4, 6). The SR, by regulating the cytoplasmic Ca2+ concentration ([Ca2+]c), plays a central role in Ca2+ signaling to contribute to activities such as gene expression, growth, contraction, and cell death (reviewed in Refs. 7-10). In determining the qualitative nature of the biological response, the structural arrangement of the SR through the cell may be instrumental in enabling different functions to occur and proceed from various regions of the cell. For example, the SR, as a series of multiple, separate Ca2+-storage elements, each with their own limited measure of Ca2+, may explain localized all-or-none responses in restricted regions of the cell, the graded release of Ca2+ from the SR in the "quantal" release process and the various sensitivities to IP3 measured throughout the cell (11-15). Yet, whether or not the SR exists as multiple separate Ca2+ storage units or as a single luminally continuous entity in which Ca2+ diffuses freely throughout, is unclear.

Evidence for the existence of multiple Ca2+ storage units comes from the heterogeneity of the SR in its Ca2+ handling capacity in smooth muscle (1, 4, 16). The SR near the center of the cells may be depleted less readily of its Ca2+ than that of peripherally located SR (1) and x-ray microprobe analysis revealed Ca2+-rich spots in the central and peripheral SR in portal vein and taenia coli (17, 18). Calsequestrin was not uniformly distributed through the SR network but clustered preferentially at sites most often within the peripheral SR (4). These calsequestrin-rich elements of the SR were also enriched with IP3R (4). This evidence provides a morphological basis for the existence of various arrangements of Ca2+ release channels.

The SR Ca2+ stores have been classified on the basis of the arrangement of IP3R and RyR and conflicting evidence exists regarding their number and receptor arrangement. To determine the organization of the SR Ca2+ stores, in native cells, the predominant experimental approach relies on functional data. The SR is depleted via one receptor (e.g. RyR) and whether or not Ca2+ is available to be released via the other receptor (e.g. IP3R) then determined. If exhaustion of the response arising from one receptor leaves the response of the other receptor largely unaffected, the two channels are suggested to be localized on two or more stores. Taking this experimental approach, a single store that contained both RyR and IP3R, has been proposed to exist based on the observation that depletion of the SR Ca2+ store by caffeine (which activates RyR) prevented IP3-mediated Ca2+ release (19-22). However, this result only indicates that IP3R and RyR access a common Ca2+ source. Multiple stores, each containing both receptors, may exist and such an arrangement would be fully compatible with the experimental observation. Indeed, in other studies, there may be separate stores for each receptor, i.e. one series of stores that express only RyR, another only IP3R. This view was derived from the observation that depletion of the RyR-sensitive store did not abolish agonist-evoked IP3-mediated Ca2+release and vice versa (23). Other more elaborate arrangements of SR Ca2+ stores are also proposed in various cell types (e.g. taenia coli, pulmonary artery, myometrium). One store may express RyR and IP3R and other stores, in the same cell, only IP3R (24-26). This conclusion followed the finding that depletion of the IP3-sensitive store abolished caffeine-evoked Ca2+ release, whereas depletion of the caffeine-sensitive store did not abolish Ca2+ release via IP3R. In yet other cell types (mesenteric artery, colonic smooth muscle) one store may express both RyR and IP3R, whereas a second, in the same cell, only RyR (27, 28).

Further evidence for the existence of separate stores has come from studies on the response of each of the receptors to inhibitors of the SR Ca2+ pump. Ca2+ pump inhibitors, e.g. thapsigargin and cyclopiazonic acid, deplete SR Ca2+ stores. If both receptors exist on a single store, Ca2+ release from each receptor should be equally sensitive to the inhibitors. Differences in the sensitivity of caffeine and IP3-mediated Ca2+ release to pump inhibitors has been interpreted as evidence of the existence of separate stores for each receptor (22, 29, 30). For example, the ryanodine/caffeine-sensitive store was not sensitive to thapsigargin or cyclopiazonic acid, whereas the IP3-sensitive Ca2+ store was depleted by the Ca2+ pump inhibitors in arterial myocytes (22, 30).

The structural organization of the store is thus often inferred from the functional consequences of store activation with differences in IP3R and RyR sensitivity to pharmacological agents (e.g. ryanodine and thapsigargin) that deplete the SR used widely to separate the store into subcompartments. However, the relationship between the sensitivity of the SR to pharmacological activators and inhibitors of Ca2+ release and whether or not subcompartments exist is unclear. Regions of various sensitivities to inhibitors and activators may also arise from higher densities of channels, pumps, or luminal Ca2+-binding proteins in different areas of the SR without a requirement for the existence of subcompartments (31).

The persistent uncertainty of the structure of the SR has prompted the present study in intact single smooth muscle cells. The results show that the SR is a single luminally continuous entity that contains both IP3R and RyR and in which Ca2+ may diffuse freely throughout. However, partial depletion of the SR may result in the appearance of multiple stores for each receptor. This situation arises by the regulation of the opening of IP3R and RyR by the [Ca2+] within the SR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Male guinea pigs (500-700 g) were humanely killed by cervical dislocation followed by immediate exsanguination in accordance with the guidelines of the Animal (Scientific Procedures) Act UK 1986. A segment of intact distal colon (~5 cm) was transferred to oxygenated (95% O2, 5%CO2) physiological saline solution composed of (mM): NaCl, 118.4; NaHCO3, 25; KCl, 4.7; NaH2PO4, 1.13; MgCl2, 1.3; CaCl2, 2.7; and glucose, 11 (pH 7.4). Following removal of the mucosa from the tissue, single smooth muscle cells, largely from circular muscle, were enzymatically dissociated (32). Cells were stored at 4 °C and used within 24 h of cell isolation. All experiments were carried out at room temperature (20 ± 2 °C).

Electrophysiology—Membrane currents were measured using conventional tight seal whole cell recording methods. The extracellular solution contained (mM): sodium glutamate, 80; NaCl, 40; tetraethylammonium chloride, 20; MgCl2, 1.1; CaCl2, 3; Hepes, 10; and glucose, 30 (pH 7.4 with NaOH). The pipette solution contained (mM): (Cs)2SO4, 85; CsCl, 20; MgCl2, 1; Hepes, 30; MgATP, 3; pyruvic acid, 2.5; malic acid, 2.5; NaH2PO4, 1; creatine phosphate, 5; guanosine phosphate, 0.5; and caged IP3 trisodium salt, 0.025. Whole cell currents were measured using an Axopatch 200B (Axon Instruments, Union City, CA), low-pass filtered at 500 Hz (8-pole bessel filter; Frequency Devices, Haverhill, MA), digitally sampled at 1.5 kHz using a Digidata interface and pClamp (version 8; Axon Instruments) and stored for analysis. In some experiments a second patch clamp electrode with modified extracellular solution (which contained 30 or 50 mM Ca2+) was used. The electrode was sealed "on-cell" in a "giga-seal" configuration to provide a localized Ca2+ source to the cell. Here, currents were measured using a second Axopatch 200B (Axon Instruments) and low-pass filtered at 500 Hz. The potential of the second on-cell electrode was 0 mV so that the entire cell, including the on "cell patch," was maintained at membrane potential set by the whole cell electrode, i.e. patch transmembrane potential = Vwhole cell electrode - Vpatch electrode.

Imaging—Cells were loaded with fluo-3 acetoxymethylester (AM) (10 µM) and wortmanin (10 µM; to prevent contraction) for at least 20 min prior to the beginning of the experiment. Two dimensional [Ca2+]c images were obtained using a wide-field digital imaging system (33). Single cells were illuminated at 488 nm (bandpass 14 nm) from a monochromator (Polychrome IV, T.I.L.L. Photonics, Martinsried, Germany) and imaged through an oil immersion objective (x40 UV 1.3 NA; Nikon UK, Surrey, United Kingdom). Excitation light was passed via a fiber optic guide through a 485-bandpass (15 nm) filter and a field stop diaphragm and reflected off a 505-nm long-pass dichroic mirror. Emitted light was guided through a 535-nm barrier filter (bandpass 45 nm) to an intensified, cooled, frame transfer CCD camera (Pentamax Gen IV, Roper Scientific, Trenton, NJ) operating in "virtual chip" mode with program WinView32 (Roper Scientific, Trenton, NJ). Full frame images (150 x 150 pixels), with a pixel size of 563 nm at the cell, were acquired at 20 or 100 frames s-1. Alternatively, to attain longer periods of data acquisition, the software program Metafluor (Molecular Devices Ltd., Wokingham, England) was used. Here the sampling was ~10 frames s-1 during, and 1 frame s-1 between [Ca2+]c transients. Ca2+ imaging data were recorded on a personal computer. Electrophysiological measurements and imaging data were synchronized by recording, on pClamp, a transistor logic output from the CCD camera, which reported its readout status, together with the electrophysiological information.

In those control experiments that involved the use BODIPY fluorescent ryanodine, [Ca2+]c was measured using fura-2 in a microfluorimeter system (34). Fura-2 was required in these experiments to avoid the overlap in fluorescence emitted from BODIPY and fluo-3. Briefly, [Ca2+]c was measured using membrane-impermeable fura-2 (potassium salt) introduced into the cell from the patch pipette. Fluorescence measurements were made using a microfluorimeter consisting of an inverted fluorescence microscope (Nikon diaphot and a photomultiplier tube with a bi-alkali photocathode). The excitation wavelengths (340 and 380 nm, 7 nm bandpass) were provided by a PTI deltascan (Photon Technology International Inc., East Sheen, London, UK). [Ca2+]c measurements were made at a frequency of 10 Hz. The excitation light passed through a 425-nm short pass filter and a field stop diaphram used to reduce background fluorescence. A 400-nm long pass dichroic mirror reflected the excitation wavelengths onto the cell. A 570-nm short pass dichroic mirror passed the emission light through a 505-nm barrier filter onto the photomultiplier for photon counting. Longer wavelengths from bright field illumination with a 610-nm Shott glass filter were reflected onto a CCD camera (Sony model XC-75) mounted onto the viewing port of the deltascan allowing the cell to be monitored during the course of the experiments.


Figure 1
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  		FIGURE 1.
Ca2+ release evoked by IP3 and caffeine. At -70 mV, locally photolyzed IP3 ({uparrow}, C) in a 10-µm diameter region, (bright spot in A left-hand panel; see also whole cell patch electrode (left side)) increased [Ca2+]c (B and C) in a single myocyte. Hydrostatic application of caffeine (10 mM; E, bottom panel) also increased [Ca2+]c (D and E). The [Ca2+]c images (B and D) are derived from the time points indicated by the corresponding numbers in C and E. [Ca2+]c changes in B and D are represented by color: blue, low, and red/white, high [Ca2+]c. The [Ca2+]c measurements (C and E) are derived from the area within the white square in A (right-hand panel).

 
In other experiments, the diffusion of ryanodine through the cell was tracked from the movement of tetramethylrhodamine ethyl ester perchlorate (TMRE) (1 µM). TMRE was applied to the cell together with ryanodine in a patch electrode sealed on the cell. TMRE fluorescence was measured together with that of the [Ca2+]c indicator fluo-4. Fluo-4 was used in these experiments because of the slight shift in its excitation spectrum when compared with that of fluo-3. This shift allowed a greater separation of the excitation wavelengths to be used for fluo-4 and TMRE (35). TMRE has a comparable molecular mass (514.96 Da) to ryanodine (493.55 Da) and so presumably each share similar diffusion characteristics. Excitation light for TMRE and fluo-4 (560 and 475 nm, respectively) passed through a dual bandpass (483/553 nm) excitation filter (bandpass 15 and 20 nm, respectively) reflected off a dual long pass dichroic mirror (transmissive in the ranges 505-540 and 577-640 nm). Emitted light passed through a dual bandpass 518/594-nm barrier filter (bandpass 25 and 18 nm, respectively) before reaching the CCD camera (35). Optical filters were purchased from Chroma (Rockingham, VT).

Localized Flash Photolysis—The output of a xenon flashlamp (Rapp Optoelecktronic, Hamburg, Germany), used to uncage IP3, was passed though a UG-5 filter to select ultraviolet light, focused, and merged into the excitation light path via a fiber optic bundle and long pass dichroic mirror at the lens part of the microscope epi-illumination attachment. The fiber optics diameter together with the lens magnification determined the area (spot size ~ 10 µm) of IP3 photolysis (33). The concentration of IP3 was altered to produce maximal and submaximal responses by changing the output of the xenon flashlamp. The output was reduced from ~38 mJ (maximal) to ~23 mJ (submaximal). In some figures the flash artifact has been blanked for clarity.

Data Analysis—Images were analyzed using the program Metamorph 5.1 or 6.2r5 (Molecular Devices Ltd.). Fluorescence signals were expressed as ratios (F/F0 or {Delta}F/F0) of fluorescence counts (F) relative to base-line (control) values (taken as 1) before stimulation (F0). Original fluorescence recordings were not filtered, smoothed, or averaged.

Summarized results are expressed as mean ± S.E. of n cells. A paired or unpaired Student's t test was applied to the raw data as appropriate; p < 0.05 was considered significant.

Drugs and Chemicals—Drugs were applied either by hydrostatic pressure ejection or addition to the extracellular solution as stated in the text. Concentrations in the text refer to the salts where appropriate. Fluo-3 and fluo-4 AM esters and TMRE were purchased from Invitrogen and caged Ins(1,4,5)P3-trisodium salt from SiChem GmbH (Bremen, Germany). Ryanodine was purchased from Alexis Biochemicals (Lausen, Switzerland). All other reagents were purchased from Sigma.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Flash photolysis of caged IP3 ({uparrow}) and hydrostatic application of caffeine (10 mM) each increased [Ca2+]c (Fig. 1) in a voltage-clamped (membrane potential, -70 mV) single colonic smooth muscle cell by activating IP3R and RyR, respectively. The relationship between the two types of receptors on one or more SR Ca2+ stores was studied by first depleting the ryanodine-sensitive store of Ca2+ and the Ca2+ rise evoked by IP3 examined subsequently. In this tissue, ryanodine (50 µM) depletes the SR of Ca2+ via RyR after the channel has been activated, e.g. by caffeine (27, 36, 37). If the receptors existed on a single structure to access a common Ca2+ store, depletion of the store via RyR should inhibit the Ca2+ rise mediated by IP3R.


Figure 2
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  		FIGURE 2.
Depleting RyR store abolishes the [Ca2+]C rise evoked by IP3. At -70 mV, caffeine (D) and IP3 (C,{uparrow}; bright spot in A, center panel; see also whole cell patch electrode (left side) and puffer pipette (right side)) evoked increases in [Ca2+]c in a voltage-clamped single myocyte (A and C). IP3-evoked Ca2+ increases (evoked every ~60 s) were not significantly reduced by ryanodine (50 µM; C, open bar above the trace). Activation of RyR by caffeine (10 mM, by pressure ejection, D), in the continued presence of ryanodine, initially increased [Ca2+]c (A and C). A second application of caffeine (D) to the same cell, however, some 90 s later, generated little increase in [Ca2+]c presumably because of SR store depletion; the effects of ryanodine on RyR require prior channel activation. The IP3 response was also subsequently inhibited ({uparrow}; A and C). Because the IP3-evoked Ca2+ transient was not blocked by ryanodine alone (only after RyR activation with caffeine), IP3-mediated Ca2+ release did not activate RyR. IP3R and RyR may share a common Ca2+ store; this is depleted of Ca2+ by ryanodine, after activation of RyR by caffeine, to reduce the Ca2+ available for IP3-mediated Ca2+ release to occur. The [Ca2+]c images (A, right and left panels) are derived from the time points indicated by the corresponding numbers in B. [Ca2+]c changes in A are represented by color: blue, low, and red/white, high [Ca2+]c.

 
At -70 mV, IP3 evoked approximately reproducible increases in [Ca2+]c (1.1 ± 0.4 {Delta}F/F0; n = 6; Fig. 2) as did caffeine (1.3 ± 0.5 {Delta}F/F0; Fig. 2). In ryanodine (50 µM) IP3-evoked Ca2+ transients remained at 97 ± 16% of their control value (measured after four IP3-evoked Ca2+ transients); IP3-mediated Ca2+ release does not activate CICR at RyR in this tissue (36, 37). Caffeine-evoked Ca2+ transients, on the other hand, were inhibited to 10 ± 4% of controls by ryanodine (50 µM; 0.11 ± 0.04 {Delta}F/F0) after two caffeine applications. Significantly, after inhibition of the caffeine-evoked Ca2+ transient, the next IP3-evoked Ca2+ transient was reduced to 8 ± 3% of control values (0.13 ± 3 {Delta}F/F0; n = 6; Fig. 2). These results are compatible with IP3R existing on the same Ca2+ store as RyR; depleting the SR store via RyR reduces the Ca2+ available to be released via IP3R (27, 36, 37). However, whereas the result suggests that IP3R accesses the same Ca2+ store as does RyR, it does not distinguish between the existence of single or multiple SR store elements each with both IP3R and RyR.

To examine whether or not the IP3-sensitive store formed a continuum on the luminal aspect of the SR throughout the cell or existed as multiple separate elements there, one small (10 µm) area of the SR was depleted of Ca2+ by the repeated localized photolysis of caged IP3 in a Ca2+-free solution and the consequence of this depletion on the amount of Ca2+ available for release from the remainder of the SR measured (Fig. 3). If the SR comprised separate elements, depleting one small area of the SR should have little effect on the total amount of Ca2+ available for release elsewhere in the SR. In the event depletion at one site also depleted the entire SR of Ca2+, suggesting that the IP3-sensitive Ca2+ store indeed existed as a single luminally continuous entity through which Ca2+ could diffuse freely (Fig. 3). Thus photolyzing caged IP3 at a 10-µm site at one end of the cell (Fig. 3, photolysis site 1) increased [Ca2+]c as revealed by the increase in the F/F0 ratio (Fig. 3; {Delta}IF/F0 1.96 ± 0.64; n = 6; p < 0.05). Subsequent (~90 s later) photolysis of caged IP3 at a 10-µm site at the other end of the same cell (Fig. 3, photolysis site 2) also evoked reproducible increases in [Ca2+]c ({Delta}F/F0 1.54 ± 0.39; n = 6). Repeated application of IP3 at photolysis site 2 in a Ca2+-free solution depleted the SR of Ca2+ as revealed by the decrease in the [Ca2+]c transient measured at site 2 ({Delta}F/F0 0.15 ± 0.08; n = 6). Significantly, when the IP3-evoked Ca2+ transient had been reduced at photolysis site 2, the Ca2+ increase evoked by IP3 at photolysis site 1 was also reduced ({Delta}F/F0 0.1 ± 0.09; n = 6). Thus, releasing Ca2+ at one site of the SR reduced the amount of ion available for release from other distant sites. As a control, at the end of the experiment, Ca2+ was restored to the bathing solution and, under these conditions, IP3-evoked Ca2+ transients recovered ({Delta}F/F0 1.49 ± 0.54; n = 6).


Figure 3
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  		FIGURE 3.
Depletion of the IP3-sensitive Ca2+ store in a localized area depletes the entire store of Ca2+. At -70 mV, locally photolyzed IP3 ({uparrow}, B) in a 10-µm diameter region, (photolysis site 1; bright spot in A, left-hand panel; see also patch electrode, left side) evoked Ca2+ transients (B). Results from photolysis site 1 are indicated by the magenta bar below the [Ca2+]c trace in B. When repositioned to photolysis site 2 (A, right-hand panel) subsequent photolysis ~90 s later produced a [Ca2+]c increase (B). Photolysis site 2 is indicated by the green line below the [Ca2+]c trace (B). In a Ca2+-free solution (containing EGTA (1 mM) and MgCl2 (3 mM); blue bar above the [Ca2+] trace) the [Ca2+c]c increase evoked by IP3 at photolysis site 2 declined in amplitude as the store was depleted of Ca2+ (B); IP3 was photolyzed every ~60 s. When the store content had been substantially reduced at photolysis site 2 (A) (as revealed by the smaller Ca2+ transients; B, the photolysis spot was repositioned (~90 s) and IP3 liberated at site 1 (A). Again, as at photolysis site 2, the response was now almost abolished compared with control. On restoring external Ca2+ (B, right-hand side) the Ca2+ increase evoked by IP3 at photolysis site 1 was restored toward control values after ~180 s. These results suggest that the SR is luminally continuous and within the SR Ca2+ is freely diffusible. [Ca2+]c measurements were made from a 5-µm diameter circle at the photolysis site. Thus when photolysis occurred at photolysis site 1 for [Ca2+]c, measurements were made from a 5-µm diameter circle at the photolysis site 1. When photolysis occurred at photolysis site 2, [Ca2+]c measurements were made from a 5-µm diameter circle at the photolysis site 2. C, the rise in [Ca2+]c plotted against distance from the photolysis site. Local photolyzed IP3 ({uparrow}) at photolysis site 1 increased [Ca2+]c (right-hand panel) that was maximal at, and decreased with each 10-µm increment away from, the release site (right panel). Region 1 (black circle) is the photolysis site. No increase in [Ca2+]c occurred beyond region 7. The Ca2+ traces (right-hand panel) were derived from the 5-µm circled colored regions shown in the left-hand panel. Colors in the circle regions in C (left-hand panel) correspond to those colors in the Ca2+ traces. Regions of photolysis sites are illustrated as black and white circles on the cell (C, left-hand panel).

 
Diffusion of some IP3 to the end of the cell distant from the site of photolysis is unlikely to explain the depletion of the SR at the second site. The concentration of IP3 will decrease substantially with diffusion from the release site (into the larger volume of the cell) and by metabolism of the inositide. The peak [Ca2+]c rise decreased steeply with distance from the release site (Fig. 3C) and no increase could be measured at regions further than 60 µm from the release site. The latter value suggests that relatively little IP3 reached the second site that, in this cell, was 158 µm away. Moreover, the [Ca2+]c increase through the cell includes Ca2+, which had simply diffused from the release site.

Nor is the decrease in IP3-evoked Ca2+ release a consequence of the Ca2+-free solution alone. Following a 7-min incubation in Ca2+-free solution (containing 3 mM MgCl2 and 1 mM EGTA), without exposure to IP3, the first Ca2+ transient was 96 ± 7% of control (n = 3). The transient subsequently declined to 3 ± 1% (n = 3) of control on repetitive IP3 release. These results suggest that localized photolysis of caged IP3, in a small region of the cell, had depleted the entire SR of Ca2+.

To confirm that the SR store was luminally continuous, the possibility that the entire SR could be refilled from one small site was investigated. Following depletion of the SR, by repeatedly applying IP3 to one small area of the cell in a Ca2+-free bathing solution, Ca2+ was readmitted but made available to only one site on the SR distant to the site of depletion (Fig. 4). The Ca2+ in a Ca2+-containing on cell patch electrode was the only available source of the ion for the entire cell. Under these conditions, the entire SR was found to have been replenished with Ca2+. This included the site previously depleted of Ca2+ despite the cell not having received any direct influx of the ion there (Fig. 4). Thus, in controls, the Ca2+ rise evoked by photolysis of caged IP3 was 1.1 ± 0.09 ({Delta}F/F0; n = 7). The [Ca2+]c rise decreased to 2 ± 1% ({Delta}F/F0 0.02 ± 0.01; n = 7) of control values in a Ca2+-free bathing solution. When the Ca2+ (30 or 50 mM) containing electrode was sealed onto the cell distant (92 ± 14 µm; n = 7) from the site of photolysis, IP3-mediated Ca2+ release was restored after ~3 min to 56 ± 17% of controls ({Delta}F/F0 0.35 ± 0.13; n = 7). Together these experiments demonstrate the entire IP3-sensitive store can be depleted and replenished from a single site; the IP3-sensitive store is a single, luminally continuous entity throughout which Ca2+ can diffuse freely and all IP3R are apparently present on a store that contains RyR.


Figure 4
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  		FIGURE 4.
Ca2+ can move through the SR to replenish a site previously depleted of the ion. At -70 mV, locally photolyzed IP3 ({uparrow}, C) in a 10-µm diameter region (bright spot in A, left-hand panel; see also whole cell patch electrode (left side)) increased [Ca2+]c (B and C). The [Ca2+]c images (B) are derived from the time points indicated by the corresponding numbers in C. [Ca2+]c changes in B are represented by color: blue, low, and red, high [Ca2+]c. A second photolysis of IP3 ~60 s later at the same site (C) generated an approximately comparable [Ca2+]c increase. In a Ca2+-free solution (containing 1 mM EGTA and 3 mM MgCl2; blue bar above the trace) the [Ca2+]c increase evoked by IP3 declined and was abolished as the store became depleted of Ca2+. When the Ca2+-containing electrode was subsequently sealed onto the cell (A, right-hand panel; C, red bar) the Ca2+ increase to IP3 at the photolysis region (A) was subsequently restored partially (C). The position of the region of [Ca2+]c measurement is shown as a white circle in A, center panel, and the time between each cell activation was ~60 s, except when the Ca2+-containing electrode was onto the cell, took ~180 s.

 
The corollary to this view, i.e. that all RyR were present on the store that contains IP3R, was next examined by depleting the IP3-sensitive store of Ca2+ and then determining the ability of caffeine to activate the RyR and evoke a Ca2+ transient. Because refilling of the IP3-sensitive store is dependent on external Ca2+ (38) the response to IP3 was reduced to 6 ± 1% of control in a Ca2+-free bathing solution (from {Delta}F/F0 1.0 ± 0.2 to 0.06 ± 0.2; n = 5; p < 0.05; Vm =-70 mV). However, after the almost complete loss of the IP3-evoked transient, caffeine evoked a Ca2+ transient that averaged 48 ± 9% of control values (0.48 ± 0.09 {Delta}F/F0 compared with 1.02 ± 0.17 {Delta}F/F0 prior to depletion of the IP3-sensitive store; n = 5; p < 0.05; Fig. 5).

There are at least two interpretations of the latter experiment. First, there are Ca2+ stores that contain only RyR (27, 28) and so are resistant to depletion by activating IP3R. Or, second, there is a single store with both receptors present and the activity of IP3R is controlled by the [Ca2+] available within the SR. As SR [Ca2+] falls, IP3R stops responding to IP3 before RyR does to caffeine.

As a first step in distinguishing between these possibilities, whether or not the RyR store was luminally continuous throughout the cell was determined. Were the RyR containing SR to comprise separate elements, depletion of one aspect of the store should not affect the Ca2+ available to be released in another area of the SR. In the event, the entire ryanodine-sensitive store was found to be luminally continuous and Ca2+ diffused freely throughout. Ryanodine (50 µM) depletes the SR of Ca2+ in this tissue (27, 36) and so was used to deplete the RyR store in a restricted region by applying it to one small site on the cell (Fig. 6). However, caffeine-evoked [Ca2+]c increases decreased uniformly throughout the whole cell, which includes those regions not exposed to ryanodine. Ryanodine was localized to one small part of the cell by sealing an on-cell patch containing the drug to one end of the cell in a giga-seal configuration. The position of ryanodine as it diffused through the cell was tracked by measuring the diffusion of fluorescence from TMRE, which was also present in the patch electrode. Ryanodine and TMRE share comparable molecular weights and so, presumably, diffusion characteristics (Fig. 6).

In these experiments, in control, caffeine applied to the whole cell evoked approximately reproducible increases in [Ca2+]c (Fig. 6). Ryanodine (50 µM) and TMRE (1 µM) were next applied to a small region of the cell via a patch electrode sealed on-cell (Fig. 6). Caffeine was again applied repeatedly to the whole cell to determine the Ca2+ available in the SR throughout the cell. As ryanodine diffused into the cell, caffeine-evoked Ca2+ transients decreased uniformly throughout the cell, which included those regions that had not been exposed to ryanodine (Fig. 6). Similar results were obtained from six separate cells. In the absence of TMRE, caffeine-evoked Ca2+ transients decreased with a similar time course when a pipette containing ryanodine (50 µm) was sealed on the cell (n = 3). Thus depletion of the SR by ryanodine at one site depleted the entire SR of Ca2+ suggesting that the ryanodine-sensitive Ca2+ store existed as a single luminally continuous structure through which Ca2+ could diffuse freely.


Figure 5
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  		FIGURE 5.
Depleting the IP3-sensitive store does not abolish the response to RyR activation. At -70 mV, hydrostatic application of caffeine (10 mM; D) and locally photolyzed IP3 ({uparrow}, C) in a 10-µm diameter region, (bright spot in A, center panel; see also whole cell patch electrode (left side)) increased [Ca2+]c (A and C). The [Ca2+]c images (A) are derived from the time points indicated by the corresponding numbers in B. [Ca2+]c changes in A are represented by color: blue, low, and red, high [Ca2+]c.IP3 ({uparrow}) and caffeine each evoked increases in [Ca2+]c (A and C). In a Ca2+-free solution (containing 1 mM EGTA and 3 mM MgCl2; blue bar above the trace) the IP3-evoked Ca2+ transient decrease as the store was depleted of Ca2+. Following depletion of the IP3-sensitive store, caffeine (D) evoked a Ca2+ transient that averaged 48 ± 9% of controls. The [Ca2+]c measurements (C) were derived from a 4-µm square centered in the photolysis region and the time between each cell activation was ~60 s.

 
The use of a separate fluorophore to estimate ryanodine diffusion through the cell was required because fluorescently labeled (BODIPY) ryanodine was largely ineffective in modulating caffeine-evoked Ca2+ rises when applied to the whole cell in control experiments. In these experiments, caffeine-evoked [Ca2+]c rises in the presence of fluorescent ryanodine (50 µM) that remained at 88 ± 5% of control values (Fig. 7; n = 3). Because fluorescent ryanodine did not inhibit caffeine-evoked Ca2+ increases (see also Ref. 39) it was unsuitable for use in the present experiments.

Together, these experiments (Figs. 3, 4, and 6) establish that the IP3- and caffeine-sensitive store is luminally continuous throughout the cell. Yet, depletion of the SR via IP3R reduced caffeine-evoked Ca2+ increases by only ~50%, which suggests that at least two separate stores exist. This paradox may arise from the regulation of IP3-mediated Ca2+ release by the [Ca2+] within the SR. To test this possibility and to determine whether the IP3-sensitive store stops responding to IP3 before the SR is depleted of Ca2+, the store was "depleted" in a Ca2+-free solution by repetitively applying IP3 at a concentration that produced a submaximal response. Depletion of the SR was evidenced by the reduced peak amplitude of the Ca2+ transient (Fig. 8A). After the SR had been apparently depleted in this way, a concentration of IP3 that produced a maximal response was applied and a substantial release of Ca2+ occurred (Fig. 8A). Here, the maximum increase in [Ca2+]c evoked by IP3 was 1.49 ± 0.41 ({Delta}F/F0; n = 6). The photolysis lamp energy was reduced to produce a submaximal increase (0.9 ± 0.32 {Delta}F/F0), 61 ± 8% of the maximum. In a Ca2+-free solution, repetitive submaximal [IP3] depleted the SR and the magnitude of the [Ca2+]c increase declined to 2.0 ± 0.8% (0.02 ± 0.01 {Delta}F/F0) of its control submaximal value (n = 6). After the SR was depleted in this way the lamp energy was returned to that which produced the maximum response and a [Ca2+]c increase occurred (0.49 ± 0.16 {Delta}F/F0), which was 35 ± 7% of maximum control (n = 7; Fig. 8A). This result demonstrated that the IP3-sensitive store contained significant residual quantities of Ca2+ even when apparently depleted. The lack of available Ca2+ within the SR may not therefore account for the inability of the lower [IP3] to evoke Ca2+ release.

RyR may also be regulated by the [Ca2+] within the SR (40-42). Support for this proposal, in the present cell type, is found in the observation that caffeine (1 mM) in the presence of ryanodine (50 µM) did not deplete the SR of Ca2+ despite the almost complete loss of a [Ca2+]c increase. The SR retained significant reserves of Ca2+ that could be liberated by a higher (10 mM) caffeine concentration (Fig. 8B). In these experiments, the increase in [Ca2+]c evoked by a submaximal concentration of caffeine (1 mM) was 50 ± 13% (0.4 ± 0.14 {Delta}F/F0; n = 4) of that of a higher concentration of caffeine (10 mM) (0.8 ± 0.16 {Delta}F/F0; n = 4). In ryanodine (50 µM), repetitive caffeine (1 mM) depleted the SR and the magnitude of the [Ca2+]c rise was reduced to 6 ± 3% of its control submaximal value (n = 4). When depleted in this way, caffeine (10 mM) evoked a [Ca2+]c increase (0.37 ± 0.16 {Delta}F/F0) that was 42 ± 10% of the control (n = 4; Fig. 8B). This result also demonstrated that the lack of available SR Ca2+ did not account for the inability of caffeine to evoke Ca2+ release.


Figure 6
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  		FIGURE 6.
Depletion of the RyR-containing store at one site depleted the entire store. Caffeine (F) applied to a single cell in controls (A, left-hand panel) evoked approximately reproducible [Ca2+]c increases (D, left-hand panel). [Ca2+]c increases are shown from six separate regions separated by 20 µm. After a patch pipette containing ryanodine (50 µM) and TMRE (1 µM) was sealed onto the cell (in a giga-seal configuration; A, middle panel) the caffeine-evoked Ca2+ transients declined (D, middle panel). The diffusion of ryanodine through the cell was monitored from the progression of TMRE fluorescence (B and E) in six regions of the cell (A, right-hand panel). Region 1 (black line, D) is the site of the ryanodine-containing electrode and region 6 (magenta line) furthest away from the electrode. The images (B) are derived from the time points indicated by the corresponding numerals in C.Ca2+ transients in all regions decreased comparably after the ryanodine-containing pipette was sealed onto the cell that include those regions not exposed to ryanodine as determined from the diffusion of TMRE (B and E). The breaks in the records last ~90 s in which new files were established. Measurements were derived from 1-pixel wide lines across the cell (A, right-hand panel) shown at 2-pixel width to facilitate visualization.

 
The latter results (Fig. 8) raise the possibility that activation of IP3R and RyR by concentrations of IP3 and caffeine that each produce comparable [Ca2+]c rises may not be equally effective in overcoming regulation of the receptors by SR luminal [Ca2+]. As the latter declines, regulation of RyR and IP3R by luminal [Ca2+] may terminate Ca2+ release from only one receptor type; such a result could be misinterpreted as evidence for the existence of multiple Ca2+ stores.

To test this possibility, the RyR store was depleted using caffeine (2 mM) and the ability of IP3 (125 µM) to evoke a response tested. Earlier experiments (Fig. 5; see also Refs. 27 and 28) demonstrated a significant residual response to caffeine when the SR was apparently depleted fully via IP3R. Here, at -70 mV, caffeine (2 mM) and IP3 (125 µM) evoked approximately reproducible increases in [Ca2+]c (1.3 ± 0.2 and 1.6 ± 0.3 {Delta}F/F0, respectively; n = 3; Fig. 9A). IP3-evoked Ca2+ transients were inhibited to 3 ± 3% of controls in a Ca2+-free bath solution (to 0.05 ± 0.01 {Delta}F/F0). Significantly, and in contrast to the results of Fig. 5, after inhibition of the IP3-evoked Ca2+ transient, the caffeine-evoked Ca2+ transient was now reduced to 1.9 ± 1% of control values (0.02 ± 0.01 {Delta}F/F0; Fig. 9A). The return of extracellular Ca2+ resulted in the restoration of the responses to IP3 and caffeine and the end of the experiment (Fig. 9A).


Figure 7
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  		FIGURE 7.
Lack of effect of BODIPY fluorescent ryanodine on caffeine-evoked [Ca2+]c rises. Caffeine (10 mM; B) evoked approximately reproducible rises in [Ca2+]c as indicated by the rise in the fura-2 fluorescence ratio (A). Fluorescent (BODIPY) ryanodine (unfilled bar) slowly reduced the caffeine-evoked [Ca2+]c rises by about 20% of their control values. The modest reduction, by fluorescent ryanodine, contrasts with the abolition of caffeine-evoked [Ca2+]c increases by the non-fluorescent form of ryanodine (e.g. Fig. 2). Because fluorescent ryanodine is relatively ineffective in reducing caffeine-evoked [Ca2+]c rises, it is unsuitable for use in the present study as a means to measure the diffusion of ryanodine through the cell and functional consequences.

 


Figure 8
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  		FIGURE 8.
Neither submaximal [IP3] nor [caffeine] deplete the SR store of Ca2+. A, at -70 mV high [IP3](pink, photolysed using a high lamp intensity;{uparrow}) increased [Ca2+]c. A lower [IP3](light blue,{uparrow}) evoked a submaximal [Ca2+]c increase. In a Ca2+-free bath solution (containing 1 mM EGTA and 3 mM MgCl2; dark blue bar) these increases declined, then disappeared. The absence of a response to [IP3] was not due to depletion of the store. Increasing [IP3] (pink, right side;{uparrow}) evoked further Ca2+ release. A mechanism, other than depletion of the store of Ca2+, e.g. "luminal" regulation of IP3R, may have accounted for the loss of response to IP3. The time between each IP3 challenge was ~60 s. B, caffeine (10 mM (iii)) indicated by pink (i) evoked approximately reproducible increases in [Ca2+]c (i). Caffeine (1 mM (ii)) indicated by light blue (i) evoked submaximal [Ca2+]c increases. In ryanodine (50 µM; for the duration of the unfilled bar) these increases declined to 12% of their control value (i). However, after the substantial reduction in response to submaximal caffeine (1 mM (ii)), caffeine (10 mM (iii)) evoked a [Ca2+]c rise of 77% of its control value. The break in the record is ~90 s in which a new data recording file was established.

 
Altering the concentration of IP3 and caffeine could also generate the appearance of a store that only contains IP3R (24, 26). Here the store was depleted of Ca2+ via RyR by repeatedly applying caffeine (1 mM) in the presence of ryanodine (50 µM; Fig. 9B). After the Ca2+ increase to caffeine was inhibited, IP3 evoked a substantial rise in [Ca2+]c (Fig. 9B). Thus in ryanodine (50 µM) the [Ca2+]c increase to caffeine (1 mM) was reduced to 13 ± 6% of controls (from 0.62 ± 0.16 {Delta}F/F0 to 0.08 ± 0.04 {Delta}F/F0; n = 6; Vm =-70 mV). However, after the inhibition of the caffeine-evoked transient, IP3 (250 µM) evoked a Ca2+ transient that averaged 84 ± 13% of control values (1.3 ± 0.31 {Delta}F/F0 compared with 1.57 ± 0.3 {Delta}F/F0 prior to depletion of the caffeine-sensitive store; n = 6; Fig. 5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the native cell type, methodologies for studying SR Ca2+ store subcompartments are limited and their existence is often inferred from the functional consequences of store activation. Multiple SR subcompartments are proposed to exist when there are differences in the sensitivity of IP3R and RyR to pharmacological agents (e.g. ryanodine and thapsigargin). The present results show the SR may exist as a single luminally continuous entity and Ca2+ is in free diffusional equilibrium throughout, in single colonic smooth muscle cells. Notwithstanding the luminal continuity, there are differences in the sensitivity of IP3R and RyR to pharmacological agents. "Depletion" of the SR via one receptor (either IP3R or RyR) may not abolish the Ca2+ release occurring via the other receptor and multiple stores with various receptor arrangements may appear to exist. The sensitivity differences, however, arise because the opening of RyR and IP3R is regulated by the agonist concentration acting on the cytoplasmic aspect of the receptors and the [Ca2+] within the SR (luminal [Ca2+]). As the latter declines, the receptors' sensitivity to their cytoplasmic ligands is reduced and one receptor may stop responding while Ca2+ release at the other is maintained. The control of the activity of IP3R and RyR by luminal [Ca2+] may generate functionally separate store responses that, hitherto, may have been interpreted as evidence for the existence of multiple structurally separate stores. These "functionally separate" stores exist on a single luminally continuous SR entity.


Figure 9
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  		FIGURE 9.
Various apparent SR receptor arrangements. A, caffeine (2 mM (ii)) and IP3 (125 µM;{uparrow}) each evoked approximately reproducible increases in [Ca2+]c. Removal of external Ca2+ (and addition of 1 mM EGTA and 3 mM MgCl2; blue bar) reduced the IP3-evoked Ca2+ transient. Following depletion of the IP3-sensitive store, the caffeine (ii)-evoked [Ca2+]c transient (i) was inhibited (in contrast to the results in Fig. 5). Reintroduction of Ca2+ (red bar) restored the IP3- and caffeine-evoked Ca2+ transients toward control values. B, caffeine (1 mM (ii)) evoked approximately reproducible increases in [Ca2+]c (i). IP3 (250µM;{uparrow}) also increased [Ca2+]c (i). Ryanodine (50 µM, open bar) inhibited caffeine-evoked [Ca2+]c increases by depletion of the SR. After the apparent depletion of caffeine-sensitive Ca2+ store, IP3-evoked a substantial [Ca2+]c increase (in contrast to the results in Fig. 2).

 
The conclusion, that the SR is a luminally continuous entity in which Ca2+ could diffuse freely throughout, was derived from the findings that the entire IP3-sensitive store could be depleted and refilled from one small site. When the SR was depleted by repetitively applying IP3 to one small site, diffusion of some IP3 to the end of the cell distant from the site of photolysis to deplete the SR is unlikely to explain the present findings. At the photolysis site, a large concentration of IP3 is released. This concentration would decrease substantially with diffusion from the release site and by metabolism of the inositide. Indeed, no detectable release of Ca2+ occurred within 65 µm of the second site. In these circumstances, it seems unlikely that any small concentration of IP3 that reached the second photolysis site, but failed to evoke any detectable release of Ca2+, could evoke a depletion of the SR that was comparable with that which occurred at photolysis site 1 where IP3 was released.

Depletion of the RyR-sensitive store at one site also depleted the entire store. This result suggests again a single luminally continuous store exists. The RyR containing store was depleted by attaching a pipette containing ryanodine to one end of the cell while caffeine was applied to the entire cell. Were the RyR containing SR to comprise separate elements, depletion of one aspect of the store should not affect the Ca2+ available to be released in another area of the SR. In the event, caffeine-evoked Ca2+ transients decreased uniformly throughout the cell. This result suggests that ryanodine, acting at one part of the cell, had decreased [Ca2+] uniformly throughout the SR that includes regions that had not been exposed to the drug. The position of ryanodine through the cell was determined by measuring the diffusion of a fluorescent indicator (TMRE), which was separate from ryanodine. The use of a separate fluorophore was required because fluorescent ryanodine, often used to localize ryanodine receptors, was essentially ineffective in altering a caffeine-evoked Ca2+ release. TMRE has a comparable molecular weight and so presumably diffusion characteristics, as ryanodine. In the absence of TMRE, ryanodine applied via a patch pipette inhibited Ca2+ release throughout the cell in a way that was similar in time course and extent as occurred in the presence of TMRE.

Our explanation for the appearance of multiple stores assumes that IP3R and RyR are regulated by luminal Ca2+. Evidence that this may be so comes from the observation that Ca2+ release evoked by low [IP3] or [caffeine], in a Ca2+-free medium or ryanodine, respectively, ceases as the SR Ca2+ content declines even when this store retains a significant residual quantity of Ca2+. The residual Ca2+ could be released when the concentration of IP3 or caffeine was increased. Thus the lack of available Ca2+ within the SR did not explain the inability of caffeine or IP3 to release the ion, and luminal regulation of the activity of the receptor is a more likely explanation for the results. This property of luminal regulation of the receptors may explain the appearance of multiple stores when pharmacological agents and functional data are used to separate store subcompartments. For example, after the SR had been apparently depleted of Ca2+ by IP3 (at a concentration which produced a maximal response) a substantial response to caffeine persisted (Fig. 5). This result is often interpreted (27, 28) as there being a store that only contains RyR and so is resistant to depletion by IP3. Conversely, after depletion of the SR with caffeine, a substantial response to IP3 could still be evoked (Fig. 9A). This result is often interpreted as there being a separate store that only contains IP3R (24, 26) and so is resistant to depletion by caffeine. However, each of the apparently separate stores for IP3R and RyR disappeared when the concentration of agonist (either caffeine or IP3) used to deplete the SR of Ca2+ was increased. This latter result suggests that the apparently separate stores may arise from partial depletion of the SR of Ca2+ and luminal regulation of the receptors. Thus depletion of the store via one receptor may, or may not, exhaust the response from the other receptor depending on: 1) the concentrations of the various agonists and position of the response from each receptor on its concentration-response relationship and 2) the sensitivity of the receptors to the luminal [Ca2+] in the regulation of their activity. Differences in the position of the agonist on its concentration-response curve, even when both responses are maximal, or in the ability of luminal Ca2+ to regulate the activity of the receptors, may lead to the appearance of multiple, separate, stores when this type of store depletion protocol is used to study store subcompartments, even when none exist.

In other native cell types a single luminally continuous store was also proposed to exist (reviewed in Ref. 43). In pancreatic acinar cells (44) the Ca2+ store in the apical region was refilled with Ca2+ originating from a pipette attached to the opposite side of the cell on the basolateral membrane. Thus Ca2+ can freely diffuse through the store (44). Direct monitoring of Ca2+ diffusion within the SR showed the ion to be in free equilibrium throughout, supporting the proposal of a single continuous lumen, in pancreatic acinar cells (45). In ventricular myocytes, photobleaching SR located fluo-5N at one end of the cell resulted in a decline in fluorescence in the unbleached region and an accompanying fluorescence recovery in the bleached region as dye re-equilibrated in the SR. These results suggest the SR is a luminally continuous structure in ventricular myocytes (46). A lipophilic fluorescent indicator injected into the soma of rat Purkinje neurons in cerebellar slices labeled the entire network extending right throughout the dendrites (47). This result suggests a continuous ER membrane structure exists. ER continuity also exists between soma and dendrites in rat midbrain (48). Dendritic portions of the ER could be replenished by Ca2+ moving from the soma via a luminally continuous ER (48).

The question of luminal continuity or discontinuity has also been addressed in various cultured cell types (HeLa, rat basophilic leukemia, and Chinese hamster ovary) by photobleaching a Ca2+ store-located green fluorescent protein (GFP) (49, 50). Prolonged GFP photobleaching in a small restricted region of the cell resulted in the disappearance of fluorescence throughout store. Thus GFP could move freely around the store to be eventually photobleached. After brief periods of photobleaching there followed a rapid restoration of fluorescence by the diffusion of GFP from sites neighboring the photobleached region (49, 51). These results suggest that the entire store was luminally continuous and GFP able to move freely throughout.

On the other hand, a single membrane structure throughout the SR network and Ca2+ being in free diffusional equilibrium throughout is not universally observed (52, 53). Whereas, morphologically, the SR appears as an interconnected network of tubules (29) it may adopt different configurations within the cell and components may even detach and reattach, so influencing the pattern and distribution of Ca2+ release channels (54). In Purkinje neurons, for example, IP3R-expressing regions may separate from other internal store elements (55). ER compartments that accumulate and release Ca2+ exist that are luminally discontinuous from the bulk of the ER in cultured hippocampal dendrites (56). Indeed, different Ca2+ concentrations have been measured in various regions of the SR using recombinant aequorin (49), electron microscopic determination of Ca2+ content (57), or fluorescent indicators loaded into the cell (29) suggesting that discontinuities may exist within the structures surrounding the lumen itself.

The metabolic status, life cycle stage, or prior experimental conditions of the cell may influence the appearance of subcompartments. In Chinese hamster ovary cells (UT-1 cells) overexpression of 3-hydroxy-methylglutaryl coenzyme A, an enzyme that regulates cholesterol biosynthesis, resulted in the development of additional stacks of ER that appeared to be in direct luminal continuity with the remainder of the ER (58). The stacks disappeared when 3-hydroxy-methylglutaryl coenzyme A was returned to control levels (59). In rat basophilic leukemia cells (51) the ER was a single luminally continuous structure but increases in [Ca2+]c, which persist for at least 10 min, led to the ER breaking into subcompartments. ER structural changes are also associated with fertilization and mitosis (60). Fertilization leads to a structural reorganization of the ER measured as a slowing of the diffusion of membrane probes and luminal proteins, in sea urchin eggs (61, 62) and, in mitosis, significant ER changes also occur that include the structure itself fragmenting into subcompartments (63, 64). Thus the metabolic status of the cell or certain life cycle stages may determine whether or not the store exists as a luminally continuous entity or is divided into subcompartments. Other structures within the cell such as Golgi, mitochondria, granules, and nucleus may also contribute to Ca2+ storage (31, 65-68) and generate subregions that appear to have various Ca2+ concentrations, especially when lipophilic Ca2+ indicators are used to image the distribution of [Ca2+] through the cell.

The structural arrangement of the SR as a series of multiple, separate, Ca2+-storage elements has been proposed to explain various physiological responses that include localized all-or-none responses, the graded release of Ca2+ from the SR and the various sensitivities to IP3 measured throughout the cell (11-15). The luminal continuity of the SR, reported in the present study, suggests the SR may generate each of these responses in the absence of subcompartments, in colonic myocytes at least. Other regulatory mechanisms that include the control of Ca2+ release by the luminal [Ca2+], clustering of release channels, Ca2+ storage proteins, or enzymes that metabolize IP3 to certain regions of the SR and a local positive feedback via CICR may combine to generate a diversity of Ca2+ release events and enable various physiological responses to proceed without a requirement for the existence of multiple Ca2+ storage elements (15, 69-73).

The present results, in colonic smooth muscle cells, suggest the IP3R and RyR containing SR is a single element in which Ca2+ may diffuse freely throughout. Regulation of the opening of IP3R and RyR by the [Ca2+] within the SR may generate the appearance of functionally separate stores when the SR is depleted by pharmacological agents; these functionally separate stores exist on a single luminally continuous SR entity.


   FOOTNOTES
 
* This work was supported by Wellcome Trust Grant 078054/Z/05/Z and British Heart Foundation Grant PG/06/016. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 44-141-548-4419; Fax: 44-141-552-2562; E-mail: john.mccarron{at}strath.ac.uk.

2 The abbreviations used are: ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; [Ca2+]c, cytosolic Ca2+ concentration; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor(s); RyR, ryanodine receptor(s); F/F0, the ratio of fluorescence counts relative to base-line counts before stimulation (F0); {Delta}F/F0, the magnitude of the change in F/F0; TMRE, tetramethylrhodamine ethyl ester perchlorate; GFP, green fluorescent protein; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum. Back



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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