A Single Luminally Continuous Sarcoplasmic Reticulum with Apparently Separate Ca2+ Stores in Smooth Muscle*

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.

Whether or not the sarcoplasmic reticulum (SR) is a continuous, interconnected network surrounding a single lumen or comprises multiple, separate Ca 2؉ pools was investigated in voltage-clamped single smooth muscle cells using local photolysis of caged compounds and Ca 2؉ imaging. The entire SR could be depleted or refilled from one small site via either inositol 1,4,5-trisphosphate receptors (IP 3 R) or ryanodine receptors (RyR) suggesting the SR is luminally continuous and that Ca 2؉ may diffuse freely throughout. Notwithstanding, regulation of the opening of RyR and IP 3 R, by the [Ca 2؉ ] within the SR, may create several apparent SR elements with various receptor arrangements. IP 3 R 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 IP 3 R. The various SR receptor arrangements and apparently separate Ca 2؉ storage elements exist in a single luminally continuous SR entity.
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 Ca 2ϩ 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 (Ca 2ϩ pumps), storage (calsequestrin), and release ryanodine receptor ((RyR) and inositol 1,4,5-trisphosphate receptor (IP 3 R)) of Ca 2ϩ (4,6). The SR, by regulating the cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] c ), plays a central role in Ca 2ϩ signaling to contribute to activities such as gene expression, growth, contraction, and cell death (reviewed in Refs. [7][8][9][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 Ca 2ϩ -storage elements, each with their own limited measure of Ca 2ϩ , may explain localized all-or-none responses in restricted regions of the cell, the graded release of Ca 2ϩ from the SR in the "quantal" release process and the various sensitivities to IP 3 measured throughout the cell (11)(12)(13)(14)(15)). Yet, whether or not the SR exists as multiple separate Ca 2ϩ storage units or as a single luminally continuous entity in which Ca 2ϩ diffuses freely throughout, is unclear.
Evidence for the existence of multiple Ca 2ϩ storage units comes from the heterogeneity of the SR in its Ca 2ϩ handling capacity in smooth muscle (1,4,16). The SR near the center of the cells may be depleted less readily of its Ca 2ϩ than that of peripherally located SR (1) and x-ray microprobe analysis revealed Ca 2ϩ -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 IP 3 R (4). This evidence provides a morphological basis for the existence of various arrangements of Ca 2ϩ release channels.
The SR Ca 2ϩ stores have been classified on the basis of the arrangement of IP 3 R and RyR and conflicting evidence exists regarding their number and receptor arrangement. To determine the organization of the SR Ca 2ϩ 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 Ca 2ϩ is available to be released via the other receptor (e.g. IP 3 R) 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 IP 3 R, has been proposed to exist based on the observation that depletion of the SR Ca 2ϩ store by caffeine (which activates RyR) prevented IP 3mediated Ca 2ϩ release (19 -22). However, this result only indicates that IP 3 R and RyR access a common Ca 2ϩ source. Multiple stores, each containing both receptors, may exist and such an arrangement would be fully compatible with the experimen-tal 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 IP 3 R. This view was derived from the observation that depletion of the RyR-sensitive store did not abolish agonist-evoked IP 3 -mediated Ca 2ϩ release and vice versa (23). Other more elaborate arrangements of SR Ca 2ϩ stores are also proposed in various cell types (e.g. taenia coli, pulmonary artery, myometrium). One store may express RyR and IP 3 R and other stores, in the same cell, only IP 3 R (24 -26). This conclusion followed the finding that depletion of the IP 3sensitive store abolished caffeine-evoked Ca 2ϩ release, whereas depletion of the caffeine-sensitive store did not abolish Ca 2ϩ release via IP 3 R. In yet other cell types (mesenteric artery, colonic smooth muscle) one store may express both RyR and IP 3 R, 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 Ca 2ϩ pump. Ca 2ϩ pump inhibitors, e.g. thapsigargin and cyclopiazonic acid, deplete SR Ca 2ϩ stores. If both receptors exist on a single store, Ca 2ϩ release from each receptor should be equally sensitive to the inhibitors. Differences in the sensitivity of caffeine and IP 3 -mediated Ca 2ϩ 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 IP 3sensitive Ca 2ϩ store was depleted by the Ca 2ϩ 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 IP 3 R 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 Ca 2ϩ 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 Ca 2ϩ -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 IP 3 R and RyR and in which Ca 2ϩ 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 IP 3 R and RyR by the [Ca 2ϩ ] within the SR.
Imaging-Cells were loaded with fluo-3 acetoxymethylester (AM) (10 M) and wortmanin (10 M; to prevent contraction) for at least 20 min prior to the beginning of the experiment. Two dimensional [Ca 2ϩ ] c images were obtained using a widefield 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 (ϫ40 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 ϫ 150 pixels), with a pixel size of 563 nm at the cell, were acquired at 20 or 100 frames s Ϫ1 . Alternatively, to attain longer periods of data acquisition, the software program Metafluor (Molecular Devices Ltd., Wokingham, England) was used. Here the sampling was ϳ10 frames s Ϫ1 during, and 1 frame s Ϫ1 between [Ca 2ϩ ] c transients. Ca 2ϩ 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, [Ca 2ϩ ] 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, [Ca 2ϩ ] c was measured using mem-brane-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). [Ca 2ϩ ] 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.
In other experiments, the diffusion of ryanodine through the cell was tracked from the movement of tetramethylrhodamine ethyl ester perchlorate (TMRE) (1 M). 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 [Ca 2ϩ ] 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 IP 3 , 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 ϳ 10 m) of IP 3 photolysis (33). The concentration of IP 3 was altered to produce maximal and submaximal responses by changing the output of the xenon flashlamp. The output was reduced from ϳ38 mJ (maximal) to ϳ23 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/F 0 or ⌬F/F 0 ) of fluorescence counts (F) relative to base-line (control) values (taken as 1) before stimulation (F 0 ). Original fluorescence recordings were not filtered, smoothed, or averaged.
Summarized results are expressed as mean Ϯ S.E. of n cells. A paired or unpaired Student's t test was applied to the raw data as appropriate; p Ͻ 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)P 3 -trisodium salt from SiChem GmbH (Bremen, Germany). Ryanodine was purchased from Alexis Biochemicals (Lausen, Switzerland). All other reagents were purchased from Sigma.

RESULTS
Flash photolysis of caged IP 3 (1) and hydrostatic application of caffeine (10 mM) each increased [Ca 2ϩ ] c (Fig. 1) in a voltage- clamped (membrane potential, Ϫ70 mV) single colonic smooth muscle cell by activating IP 3 R and RyR, respectively. The relationship between the two types of receptors on one or more SR Ca 2ϩ stores was studied by first depleting the ryanodine-sensitive store of Ca 2ϩ and the Ca 2ϩ rise evoked by IP 3 examined subsequently. In this tissue, ryanodine (50 M) depletes the SR of Ca 2ϩ 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 Ca 2ϩ store, depletion of the store via RyR should inhibit the Ca 2ϩ rise mediated by IP 3 Fig. 2). In ryanodine (50 M) IP 3evoked Ca 2ϩ transients remained at 97 Ϯ 16% of their control value (measured after four IP 3 -evoked Ca 2ϩ transients); IP 3 -mediated Ca 2ϩ release does not activate CICR at RyR in this tissue (36,37). Caffeine-evoked Ca 2ϩ transients, on the other hand, were inhibited to 10 Ϯ 4% of controls by ryanodine (50 M; 0.11 Ϯ 0.04 ⌬F/F 0 ) after two caffeine applications. Significantly, after inhibition of the caffeine-evoked Ca 2ϩ transient, the next IP 3 -evoked Ca 2ϩ transient was reduced to 8 Ϯ 3% of control values (0.13 Ϯ 3 ⌬F/F 0 ; n ϭ 6; Fig. 2). These results are compatible with IP 3 R existing on the same Ca 2ϩ store as RyR; depleting the SR store via RyR reduces the Ca 2ϩ available to be released via IP 3 R (27,36,37). However, whereas the result suggests that IP 3 R accesses the same Ca 2ϩ store as does RyR, it does not distinguish between the existence of single or multiple SR store elements each with both IP 3 R and RyR.
To examine whether or not the IP 3 -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 m) area of the SR was depleted of Ca 2ϩ by the repeated localized photolysis of caged IP 3 in a Ca 2ϩ -free solution and the consequence of this depletion on the amount of Ca 2ϩ 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 Ca 2ϩ available for release elsewhere in the SR. In the event depletion at one site also depleted the entire SR of Ca 2ϩ , suggesting that the IP 3 -sensitive Ca 2ϩ store indeed existed as a single luminally continuous entity through which Ca 2ϩ could diffuse freely (Fig.  3). Thus photolyzing caged IP 3 at a 10-m site at one end of the cell (Fig. 3, photolysis site 1) increased [Ca 2ϩ ] c as revealed by the increase in the F/F 0 ratio ( Fig. 3; ⌬IF/F 0 1.96 Ϯ 0.64; n ϭ 6; p Ͻ 0.05). Subsequent (ϳ90 s later) photolysis of caged IP 3 at a 10-m site at the other end of the same cell (  reduced (⌬F/F 0 0.1 Ϯ 0.09; n ϭ 6). Thus, releasing Ca 2ϩ 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, Ca 2ϩ was restored to the bathing solution and, under these conditions, IP 3 -evoked Ca 2ϩ transients recovered (⌬F/F 0 1.49 Ϯ 0.54; n ϭ 6). Diffusion of some IP 3 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 IP 3 will decrease substantially with diffusion from the release site (into the larger volume of the cell) and by metabolism of the inositide. The peak [Ca 2ϩ ] c rise decreased steeply with distance from the release site (Fig.  3C) and no increase could be measured at regions further than 60 m from the release site. The latter value suggests that relatively little IP 3 reached the second site that, in this cell, was 158 m away. Moreover, the [Ca 2ϩ ] c increase through the cell includes Ca 2ϩ , which had simply diffused from the release site.
Nor is the decrease in IP 3 -evoked Ca 2ϩ release a consequence of the Ca 2ϩ -free solution alone. Following a 7-min incubation in Ca 2ϩ -free solution (containing 3 mM MgCl 2 and 1 mM EGTA), without exposure to IP 3 , the first Ca 2ϩ transient was 96 Ϯ 7% of control (n ϭ 3). The transient subsequently declined to 3 Ϯ 1% (n ϭ 3) of control on repetitive IP 3 release. These results suggest that localized photolysis of caged IP 3 , in a small region of the cell, had depleted the entire SR of Ca 2ϩ .
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 IP 3 to one small area of the cell in a Ca 2ϩ -free bathing solution, Ca 2ϩ was readmitted but made available to only one site on the SR distant to the site of depletion (Fig. 4). The Ca 2ϩ in a Ca 2ϩ -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 Ca 2ϩ . This included the site previously depleted of Ca 2ϩ despite the cell not having received any direct influx of the ion there (Fig. 4). Thus, in controls, the Ca 2ϩ rise evoked by photolysis of caged IP 3 was 1.1 Ϯ 0.09 (⌬F/F 0 ; n ϭ 7). The [Ca 2ϩ ] c rise decreased to 2 Ϯ 1% (⌬F/F 0 0.02 Ϯ 0.01; n ϭ 7) of control values in a Ca 2ϩ -free bathing solution. When the Ca 2ϩ (30 or 50 mM) containing electrode was sealed onto the cell distant (92 Ϯ 14 m; n ϭ 7) from the site of photolysis, IP 3 -mediated Ca 2ϩ release was restored after ϳ3 min to 56 Ϯ 17% of controls (⌬F/F 0 0.35 Ϯ 0.13; n ϭ 7). Together these experiments demonstrate the entire IP 3 -sensitive store can be depleted and replenished from a single site; the IP 3 -sensitive store is a single,  luminally continuous entity throughout which Ca 2ϩ can diffuse freely and all IP 3 R are apparently present on a store that contains RyR. The corollary to this view, i.e. that all RyR were present on the store that contains IP 3 R, was next examined by depleting the IP 3 -sensitive store of Ca 2ϩ and then determining the ability of caffeine to activate the RyR and evoke a Ca 2ϩ transient. Because refilling of the IP 3 -sensitive store is dependent on external Ca 2ϩ (38) the response to IP 3 was reduced to 6 Ϯ 1% of control in a Ca 2ϩ -free bathing solution (from ⌬F/F 0 1.0 Ϯ 0.2 to 0.06 Ϯ 0.2; n ϭ 5; p Ͻ 0.05; V m ϭ Ϫ70 mV). However, after the almost complete loss of the IP 3 -evoked transient, caffeine evoked a Ca 2ϩ transient that averaged 48 Ϯ 9% of control values (0.48 Ϯ 0.09 ⌬F/F 0 compared with 1.02 Ϯ 0.17 ⌬F/F 0 prior to depletion of the IP 3 -sensitive store; n ϭ 5; p Ͻ 0.05; Fig. 5).
There are at least two interpretations of the latter experiment. First, there are Ca 2ϩ stores that contain only RyR (27,28) and so are resistant to depletion by activating IP 3 R. Or, second, there is a single store with both receptors present and the activity of IP 3 R is controlled by the [Ca 2ϩ ] available within the SR. As SR [Ca 2ϩ ] falls, IP 3 R stops responding to IP 3 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 Ca 2ϩ 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 Ca 2ϩ diffused freely throughout. Ryanodine (50 M) depletes the SR of Ca 2ϩ 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 [Ca 2ϩ ] 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 oncell 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 [Ca 2ϩ ] c (Fig. 6). Ryanodine (50 M) and TMRE (1 M) 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 Ca 2ϩ available in the SR throughout the cell. As ryanodine diffused into the cell, caffeine-evoked  Ca 2ϩ 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 Ca 2ϩ transients decreased with a similar time course when a pipette containing ryanodine (50 m) was sealed on the cell (n ϭ 3). Thus depletion of the SR by ryanodine at one site depleted the entire SR of Ca 2ϩ suggesting that the ryanodine-sensitive Ca 2ϩ store existed as a single luminally continuous structure through which Ca 2ϩ could diffuse freely.
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 Ca 2ϩ rises when applied to the whole cell in control experiments. In these experiments, caffeine-evoked [Ca 2ϩ ] c rises in the presence of fluorescent ryanodine (50 M) that remained at 88 Ϯ 5% of control values ( Fig. 7; n ϭ 3). Because fluorescent ryanodine did not inhibit caffeine-evoked Ca 2ϩ 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 IP 3 -and caffeine-sensitive store is luminally continuous throughout the cell. Yet, depletion of the SR via IP 3 R reduced caffeine-evoked Ca 2ϩ increases by only ϳ50%, which suggests that at least two separate stores exist. This paradox may arise from the regulation of IP 3 -mediated Ca 2ϩ release by the [Ca 2ϩ ] within the SR. To test this possibility and to determine whether the IP 3 -sensitive store stops responding to IP 3 before the SR is depleted of Ca 2ϩ , the store was "depleted" in a Ca 2ϩ -free solu-tion by repetitively applying IP 3 at a concentration that produced a submaximal response. Depletion of the SR was evidenced by the reduced peak amplitude of the Ca 2ϩ transient (Fig. 8A). After the SR had been apparently depleted in this way, a concentration of IP 3 that produced a maximal response was applied and a substantial release of Ca 2ϩ occurred (Fig. 8A). Here, the maximum increase in [Ca 2ϩ ] c evoked by IP 3 was 1.49 Ϯ 0.41 (⌬F/F 0 ; n ϭ 6). The photolysis lamp energy was reduced to produce a submaximal increase (0.9 Ϯ 0.32 ⌬F/F 0 ), 61 Ϯ 8% of the maximum. In a Ca 2ϩ -free solution, repetitive submaximal [IP 3 ] depleted the SR and the magnitude of the [Ca 2ϩ ] c increase declined to 2.0 Ϯ 0.8% (0.02 Ϯ 0.01 ⌬F/F 0 ) of its control submaximal value (n ϭ 6). After the SR was depleted in this way the lamp energy was returned to that which produced the maximum response and a [Ca 2ϩ ] c increase occurred (0.49 Ϯ 0.16 ⌬F/F 0 ), which was 35 Ϯ 7% of maximum control (n ϭ 7; Fig. 8A). This result demonstrated that the IP 3 -sensitive store contained significant residual quantities of Ca 2ϩ even when apparently depleted. The lack of available Ca 2ϩ within the SR may not therefore account for the inability of the lower [IP 3 ] to evoke Ca 2ϩ release.
RyR may also be regulated by the [Ca 2ϩ ] 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 M) did not deplete the SR of Ca 2ϩ despite the almost complete loss of a [Ca 2ϩ ] c increase. The SR retained significant reserves of Ca 2ϩ that could be liberated by a higher (10 mM) caffeine concentration (Fig. 8B). In these experiments, the increase in [Ca 2ϩ ] c evoked by a submaximal concentration of caffeine (1 mM) was 50 Ϯ 13% (0.4 Ϯ 0.14 ⌬F/F 0 ; n ϭ 4) of that of a higher concentration of caffeine (10 mM) (0.8 Ϯ 0.16 ⌬F/F 0 ; n ϭ 4). In ryanodine (50 M), repetitive caffeine (1 mM) depleted the SR and the magnitude of the [Ca 2ϩ ] c rise was reduced to 6 Ϯ 3% of its control submaximal value (n ϭ 4). When depleted in this way, caffeine (10 mM) evoked a [Ca 2ϩ ] c increase (0.37 Ϯ 0.16 ⌬F/F 0 ) that was 42 Ϯ 10% of the control (n ϭ 4; Fig. 8B). This result also demonstrated that the lack of available SR Ca 2ϩ did not account for the inability of caffeine to evoke Ca 2ϩ release.
The latter results (Fig. 8) raise the possibility that activation of IP 3 R and RyR by concentrations of IP 3 and caffeine that each produce comparable [Ca 2ϩ ] c rises may not be equally effective in overcoming regulation of the receptors by SR luminal [Ca 2ϩ ]. As the latter declines, regulation of RyR and IP 3 R by luminal [Ca 2ϩ ] may terminate Ca 2ϩ release from only one receptor type; such a result could be misinterpreted as evidence for the existence of multiple Ca 2ϩ stores.
To test this possibility, the RyR store was depleted using caffeine (2 mM) and the ability of IP 3 (125 M) 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 IP 3 R. Here, at Ϫ70 mV, caffeine (2 mM) and IP 3  control values (0.02 Ϯ 0.01 ⌬F/F 0 ; Fig. 9A). The return of extracellular Ca 2ϩ resulted in the restoration of the responses to IP 3 and caffeine and the end of the experiment (Fig. 9A).
Altering the concentration of IP 3 and caffeine could also generate the appearance of a store that only contains IP 3 R (24,26). Here the store was depleted of Ca 2ϩ via RyR by repeatedly applying caffeine (1 mM) in the presence of ryanodine (50 M ;  Fig. 9B). After the Ca 2ϩ increase to caffeine was inhibited, IP 3 evoked a substantial rise in [Ca 2ϩ ] c (Fig. 9B). Thus in ryanodine (50 M) the [Ca 2ϩ ] c increase to caffeine (1 mM) was reduced to 13 Ϯ 6% of controls (from 0.62 Ϯ 0.16 ⌬F/F 0 to 0.08 Ϯ 0.04 ⌬F/F 0 ; n ϭ 6; V m ϭ Ϫ70 mV). However, after the inhibition of the caffeine-evoked transient, IP 3 (250 M) evoked a Ca 2ϩ transient that averaged 84 Ϯ 13% of control values (1.3 Ϯ 0.31 ⌬F/F 0 compared with 1.57 Ϯ 0.3 ⌬F/F 0 prior to depletion of the caffeine-sensitive store; n ϭ 6; Fig. 5).

DISCUSSION
In the native cell type, methodologies for studying SR Ca 2ϩ 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 IP 3 R 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 Ca 2ϩ is in free diffusional equilibrium throughout, in single colonic smooth muscle cells. Notwithstanding the luminal continuity, there are differences in the sensitivity of IP 3 R and RyR to pharmacological agents. "Deple-   The conclusion, that the SR is a luminally continuous entity in which Ca 2ϩ could diffuse freely throughout, was derived from the findings that the entire IP 3 -sensitive store could be depleted and refilled from one small site. When the SR was depleted by repetitively applying IP 3 to one small site, diffusion of some IP 3 to the end of the cell distant from the site of pho-tolysis to deplete the SR is unlikely to explain the present findings. At the photolysis site, a large concentration of IP 3 is released. This concentration would decrease substantially with diffusion from the release site and by metabolism of the inositide. Indeed, no detectable release of Ca 2ϩ occurred within 65 m of the second site. In these circumstances, it seems unlikely that any small concentration of IP 3 that reached the second photolysis site, but failed to evoke any detectable release of Ca 2ϩ , could evoke a depletion of the SR that was comparable with that which occurred at photolysis site 1 where IP 3 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 Ca 2ϩ available to be released in another area of the SR. In the event, caffeine-evoked Ca 2ϩ transients decreased uniformly throughout the cell. This result suggests that ryanodine, acting at one part of the cell, had decreased [Ca 2ϩ ] 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 caffeineevoked Ca 2ϩ 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 Ca 2ϩ 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 IP 3 R and RyR are regulated by luminal Ca 2ϩ . Evidence that this may be so comes from the observation that Ca 2ϩ release evoked by low [IP 3 ] or [caffeine], in a Ca 2ϩfree medium or ryanodine, respectively, ceases as the SR Ca 2ϩ content declines even when this store retains a significant residual quantity of Ca 2ϩ . The residual Ca 2ϩ could be released when the concentration of IP 3 or caffeine was increased. Thus the lack of available Ca 2ϩ within the SR did not explain the inability of caffeine or IP 3 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 Ca 2ϩ by IP 3 (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 IP 3 . Conversely, after depletion of the SR with caffeine, a substantial response to IP 3 could still be evoked (Fig. 9A). This result is often interpreted as there being a separate store that only contains IP 3 R (24, 26) and so is resistant to depletion by caffeine. However, each of the apparently separate stores for IP 3 R and RyR disappeared when the concentration of agonist (either caffeine or IP 3 ) used to deplete the SR of Ca 2ϩ was increased. This latter result suggests that the apparently separate stores may arise from partial depletion of the SR of Ca 2ϩ 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 [Ca 2ϩ ] 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 Ca 2ϩ 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 Ca 2ϩ store in the apical region was refilled with Ca 2ϩ originating from a pipette attached to the opposite side of the cell on the basolateral membrane. Thus Ca 2ϩ can freely diffuse through the store (44). Direct monitoring of Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ release channels (54). In Purkinje neurons, for example, IP 3 R-expressing regions may separate from other internal store elements (55). ER compartments that accumulate and release Ca 2ϩ exist that are luminally discontinuous from the bulk of the ER in cultured hippocampal dendrites (56). Indeed, different Ca 2ϩ concentrations have been measured in various regions of the SR using recombinant aequorin (49), electron microscopic determination of Ca 2ϩ 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