Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

Robert Janiak, Sean M. Wilson, Stephen Montague, and Joseph R. Hume

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557


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To examine the nature of inositol 1,4,5-trisphosphate (IP3)-sensitive and ryanodine (Ryn)-sensitive Ca2+ stores in isolated canine pulmonary arterial smooth cells (PASMC), agonist-induced changes in global intracellular Ca2+ concentration ([Ca2+]i) were measured using fura 2-AM fluorescence. Properties of elementary local Ca2+ release events were characterized using fluo 3-AM or fluo 4-AM, in combination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca2+ stores with Ryn (300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-induced intracellular Ca2+ transients but had little or no effect on the initial IP3-mediated intracellular Ca2+ transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP3-induced intracellular Ca2+ transients but failed to attenuate the initial Caf-induced intracellular Ca2+ transient. These results suggest that in canine PASMC, IP3-, and Ryn-sensitive Ca2+ stores are organized into spatially distinct compartments while similar experiments in canine renal arterial smooth muscle cells (RASMC) reveal that these Ca2+ stores are spatially conjoined. In PASMC, spontaneous local intracellular Ca2+ transients sensitive to modulation by Caf and Ryn were detected, exhibiting spatial-temporal characteristics similar to those previously described for "Ca2+ sparks" in cardiac and other types of smooth muscle cells. After depletion of Ryn-sensitive Ca2+ stores, ANG II (8 nM) induced slow, sustained [Ca2+]i increases originating at sites near the cell surface, which were abolished by depleting IP3 stores. Discrete quantal-like events expected due to the coordinated opening of IP3 receptor clusters ("Ca2+ puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca2+ stores and elementary Ca2+ release events in isolated PASMC.

pulmonary artery; smooth muscle cells; sarcoplasmic reticulum; intracellular calcium; inositol 1,4,5-trisphosphate; calcium ion sparks


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THE IMPORTANCE OF RELEASE of Ca2+ from intracellular sarcoplasmic reticulum (SR) stores in smooth muscle cells (SMC) is well recognized (18, 39; see Ref. 21 for review). Ca2+ release can occur through a Ca2+-induced Ca2+-release (CICR) mechanism or through an inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release mechanism. Ryanodine receptors are key elements of CICR and can be selectively activated not only by Ca2+ but also by caffeine (50). Pharmacological studies have demonstrated that Ca2+ can be released from IP3-sensitive Ca2+ stores by a variety of different agonists including phenylephrine (PE), norepinephrine, serotonin, and ANG II (23). There is increasing evidence for expression of multiple isoforms of both ryanodine (31) and IP3 receptors (7, 28), suggesting that a complex variety of proteins may control intracellular Ca2+ release events involved in electromechanical and pharmacomechanical coupling in smooth muscle.

In some vascular SMC, functional studies suggest that caffeine- and ryanodine-sensitive Ca2+ release and IP3-evoked Ca2+ release may involve partially or completely overlapping Ca2+ stores. In rat portal vein SMC, norepinephrine and ATP appear to release Ca2+ from the same functional store, which contains both IP3 and ryanodine receptors (32). In contrast, in other types of vascular SMC, IP3-sensitive and ryanodine-sensitive SR Ca2+ stores may be organized into spatially separate compartments. In cultured rat aortic cells, IP3-sensitive Ca2+ stores were depleted by thapsigargin or cyclopiazonic acid (CPA), specific inhibitors of the SR Ca2+-ATPase (10, 41), but caffeine-sensitive Ca2+ stores were unaffected, suggesting that caffeine-sensitive stores may have a thapsigargin- and CPA-insensitive Ca2+-ATPase (43). Conflicting results have been reported in rat mesenteric arterial SMC. Thapsigargin was reported to block both norepinephrine- and caffeine-induced increases in intracellular Ca2+ concentration ([Ca2+]i) (2); however, a later study provided evidence that IP3- and ryanodine-sensitive SR Ca2+ stores are differentially affected by thapsigargin and CPA (11).

Presently, little is known about the nature of the intracellular Ca2+ store(s) in pulmonary arterial SMC (PASMC), even though the unique contractile response of this tissue to hypoxia may at least partially involve intracellular release of Ca2+ (see Ref. 45 for review). While there is considerable evidence for the existence of both IP3-sensitive and ryanodine-sensitive Ca2+ stores in PASMC (15, 16, 24, 46), whether these stores functionally or spatially overlap or represent separate spatially discrete Ca2+ pools is not known. Using isometric tension measurements in canine pulmonary arterial rings, we previously provided evidence that IP3-sensitive and ryanodine-sensitive Ca2+ stores in this tissue may be independent (19). However, contractile measurements in intact arterial smooth muscle strips are complicated by the presence of endothelial cells and by the inability to distinguish agonist-induced changes in [Ca2+]i from possible changes in Ca2+ sensitivity of contractile proteins. The first objective of this study was to directly measure agonist-induced changes in global [Ca2+]i in single canine PASMC to determine whether IP3-sensitive and ryanodine-sensitive Ca2+ stores are spatially independent or coupled. To this end, direct measurements of [Ca2+]i in single canine PASMC were performed using a conventional digital imaging microscope in cells loaded with the Ca2+ fluorescent dye, fura 2-AM. Similar experiments were performed on canine renal arterial SMC (RASMC) to compare the functional characteristics of intracellular Ca2+ stores in PASMC to those in cells from a different vascular arterial bed. A second objective of this study was to identify the elementary Ca2+ release events associated with IP3 and ryanodine receptors in PASMC. Spontaneous and agonist-induced localized Ca2+ release events were measured with confocal laser scanning microscopy using the Ca2+ indicators fluo 3-AM or fluo 4-AM.


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Cell isolation. Mongrel dogs of either sex were euthanized with pentobarbital sodium (45 mg/kg iv) and ketamine (15 mg/kg iv). The lungs and heart were excised en block, while the kidneys were excised separately. The third and fourth branches of pulmonary and renal arteries were dissected at 5°C to decrease cellular metabolic activity. The main pulmonary and renal arteries were flushed with physiological saline solution (PSS) containing (in mM) 125 NaCl, 5.36 KCl, 0.336 Na2HPO4, 0.44 K2HPO4, 11 HEPES, 1.2 MgCl2, 0.05 CaCl2, 10 glucose, 2.9 sucrose, pH 7.4 (adjusted with Tris), osmolarity 300 mosM (adjusted with sucrose). The PSS solution was continuously bubbled with 100% O2 during dissections. Arteries were cleaned of connective tissue, cut into small pieces, and placed in a tube containing fresh PSS. Tissue was immediately digested or cold stored in the refrigerator (5°C) up to 24 h. To disperse cells, tissue was placed in Ca2+-free PSS containing enzymes. Pulmonary and renal tissues were digested differently. Pulmonary arterial tissue was incubated with a solution containing (in mg/6 ml) 2 collagenase type XI, 0.1 elastase type IV, and 2 bovine serum albumin (fat free) for 16-18 h at 5°C. The tissue was next washed a few times with 5°C Ca2+-free PSS solution and then triturated with a fire-polished Pasteur pipette. Renal arterial tissue was incubated with PSS solution containing (in mg/2.50 ml) 5 collagenase XI, 0.4 elastase type IV, and 2 bovine serum albumin (fat free) for 18-23 min at 34°C. The tissue was then washed several times in warm (34°C) Ca2+-free PSS and subsequently triturated with fire-polished Pasteur pipettes. Tissue was cold stored until digested (0-24 h). After digestion, the resulting dispersed PASMC or RASMC were cold stored at 4°C for up to 8 h until experiments were performed. Only cells with normal morphology (i.e., elongated) were studied. Generally, cells that were used on day 2 were less responsive than cells used within the first 24 h; however, the effects of store depletion on their responses to agonists were the same.

Global [Ca2+]i measurements. Cytosolic Ca2+ was measured in SMC loaded with the ratiometric Ca2+ dye fura 2-AM (Molecular Probes, Eugene, OR) using a dual-excitation digital Ca2+ imaging system (IonOptix, Milton, MA) equipped with an intensified charge-coupled device (CCD). The imaging system was mounted on an inverted microscope (Nikon) outfitted with ×40 and ×100 [numerical aperture (NA) 1.3; Nikon, Melville, NY] oil immersion objectives. Fura 2-AM was dissolved in DMSO and added from a 1 mM stock to the cell suspension at a final concentration of 8 µM. Cells were loaded with fura 2-AM for 15 min at 34°C and an additional 20 min at room temperature in the dark. Cells were then washed for 30 min with normal Tyrode solution to allow for dye esterification. Cells were illuminated with a xenon arc lamp at 340 ± 15 and 380 ± 12 nm (Omega Optical, Brattleboro, VT), and emitted light was collected from regions that encompassed single cells with a CCD at 510 nm (Nikon). If cells contracted, the experiment was paused and the regions of interest resized. In most experiments, images were acquired at 1 Hz and stored on either compact disk or magnetic media for later analysis. The amplitudes of intracellular Ca2+ transients are expressed as the change in fluorescence ratio during caffeine, ANG II, and PE exposure from baseline measured in each cell in the presence of normal Tyrode solution alone. Background fluorescence was collected automatically and subtracted from the acquired fluorescence video images during each experiment. [Ca2+]i was estimated from the relation [Ca2+]i = Kd × (Sf2/Sb2) × (R - Rmin)/(Rmax - R) (14), where Kd is the dissociation constant and the values of Sf2, Sb2, Rmin, and Rmax were determined from an in situ calibration of fura 2 that was loaded into PASMC as described (R, fluorescence ratio; Rmin, minimum ratio; Rmax, maximum ratio). The Kd for fura 2 was assumed to be 224 nM. Specifically, following fura 2-AM loading, cells were exposed to either 3 µM 8-Br-A-23187 or 2.5 µM ionomycin while mitochondrial function was inhibited with 5 µM carbonyl cyanide m-chlorophenyl hydrazone. To determine Rmax, the Tyrode solution contained 3 mM Ca2+, and to determine Rmin, the solution contained 10 mM EGTA and no added Ca2+. Most bathing solutions used for calibrations contained 5 mM 2,3-butanedione monoxime to inhibit SMC contraction.

In most experiments, cells were perfused with a standard Tyrode solution of the following composition (in mM): 126 NaCl, 5.4 KCl, 0.3 NaH2PO4, 10 HEPES, 1 MgCl2, 2 CaCl2, 10 glucose, and 2.9 sucrose, pH 7.4 (adjusted with NaOH), 300 mosM (adjusted with sucrose). The rate of perfusion was ~2 ml/min, and intracellular Ca2+ transients were elicited by brief (~30 s) exposures to agonists (as illustrated by arrows in Figs. 1-4). Exposure times to ryanodine or CPA were longer in duration and are indicated by bars in the individual figures or in the figure legends.


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Fig. 1.   Effects of cyclopiazonic acid (CPA; 10 µM) on the amplitude of global intracellular Ca2+ transients induced by ANG II (1 µM) or caffeine (CAF; 10 mM) in pulmonary arterial smooth muscle cells (PASMC). A: representative intracellular Ca2+ transients elicited by brief caffeine or ANG II exposures in the absence or presence of CPA. B: summary of changes in intracellular Ca2+ transients elicited by the first (1), second (2), and/or third (3) brief ANG II or caffeine exposures in the presence of CPA. Each column represents mean ± SE (n = 4) change in estimated [Ca2+]i. Comparisons to that observed in the absence of CPA treatment were made. *P < 0.05.



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Fig. 2.   Effects of ryanodine (RYN; 300 µM) on the amplitude of global intracellular Ca2+ transients induced by ANG II (1 µM) or caffeine (10 mM) in PASMC. A: representative intracellular Ca2+ transients elicited by brief caffeine or ANG II exposures in the absence or presence of ryanodine. B: summary of changes in intracellular Ca2+ transients elicited by the first (1), second (2), and/or third (3) brief ANG II or caffeine exposures in the presence of ryanodine. Each column represents mean ± SE (n = 6) change in estimated [Ca2+]i. Comparisons to that observed in the absence of ryanodine treatment were made. *P < 0.05; **P < 0.01.



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Fig. 3.   Effects of CPA (10 µM) on the amplitude of global intracellular Ca2+ transients induced by phenylephrine (PE; 1 µM) or caffeine (10 mM) in renal arterial smooth muscle cells (RASMC). A: representative intracellular Ca2+ transients elicited by brief caffeine or PE exposures in the absence or presence of CPA. B: summary of changes in intracellular Ca2+ transients elicited by the first (1), second (2), and/or third (3) brief PE or caffeine exposures in the presence of CPA. Each column represents mean ± SE (n = 5) change in estimated [Ca2+]i. Comparisons to that observed in the absence of CPA treatment were made. *P < 0.05.



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Fig. 4.   Effects of ryanodine (300 µM) on the amplitude of global intracellular Ca2+ transients induced by PE (1 µM) or caffeine (10 mM) in RASMC. A: representative intracellular Ca2+ transients elicited by brief caffeine or PE exposures in the absence and presence of ryanodine. B: summary of changes in intracellular Ca2+ transients elicited by the first (1) and/or second (2) brief caffeine or PE exposures in the presence of ryanodine. Each column represents mean ± SE (n = 5) change in estimated [Ca2+]i. Comparisons to that observed in the absence of ryanodine treatment were made. *P < 0.05.

Local [Ca2+]i measurements and confocal microscopy. Confocal images of SMC were obtained using an Odyssey XL laser scanning imaging system (Noran Instruments, Middleton, WI) interfaced with an Indy workstation (Silicon Graphics, Mountainview, CA) and Intervision software. The confocal system was mounted to a Nikon diaphot microscope equipped with a ×60 water immersion objective lens (NA 1.2; Nikon). Local intracellular Ca2+ transients were observed in cells loaded with either the Ca2+ indicators fluo 3-AM or fluo 4-AM (Molecular Probes). Fluo 3- or fluo 4-AM was dissolved in DMSO and added from a 1 mM stock solution to the cell suspension to make a final concentration of 7 µM. Cells were loaded for 15-20 min at room temperature in the dark and then washed with normal Tyrode solution for 25 min to allow for deesterification. Cells were illuminated with a krypton-argon laser at 488 nm, and emitted light was collected with the confocal photomultiplier tube at wavelengths >515 nm. In some experiments, 128 × 96 µm (640 × 480 pixels) images were acquired every 33.3 ms (30 Hz; see Fig. 5); however, to improve temporal resolution, most experiments acquired 64 × 48 µm (320 × 240 pixels) images every 8.33 ms (120 Hz) continuously for 10 s. Experimental data were stored on compact discs for later analysis. Original images for the displayed figures were resized and cropped to illustrate regions of interest corresponding to frequent discharge sites.


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Fig. 5.   Spontaneous local intracellular Ca2+ transients (Ca2+ sparks) and their spatial-temporal characteristics in PASMC. A: series of 6 whole cell images acquired at a rate of 30 Hz, illustrating the initiation and spread of a spontaneous Ca2+ spark in a PASMC. The first image (0.0000 s) shows a full-frame image of the cell immediately before Ca2+ spark initiation (calibration bar = 10 µm) while the second image shows the initiation of a local intracellular Ca2+ transient, 0.0333 s later (red arrow). Subsequent images illustrate the spread and decay of the local intracellular Ca2+ transient with time. B: spatial-temporal characteristics of the local intracellular Ca2+ transients for each image shown in A. The F/Fo intensity was measured along a line (illustrated as red line in 0.0999 s image in A) through and perpendicular to the center of the spark's point source using Odyssey XL software. The spatial distributions of Ca2+ were fit with Gaussian curves (red lines), relative to the original initiation point of the spark. The full width at half-maximal intensity for the Ca2+ spark shown at time 0.0333 s was 1.95 µm.

Image data were analyzed using a custom program (kindly provided by Drs. M. Nelson and A. Bonev, University of Vermont, Burlington, VT) written in IDL 5.1 (Research Systems, Boulder, CO). Baseline fluorescence (Fo) was determined by averaging 20 images where no basal Ca2+ increases were observed. Ratio images were then constructed and analyzed for areas where F/ Fo increased rapidly. The average F/Fo fluorescence from 2.25 × 2.25 µm areas were plotted vs. time and analyzed for fluorescence increases using Origin software (Microcal Software, Northhampton, MA).

Chemicals and drugs. Ryanodine was obtained from Agrisystem International (Windgap, PA), and all others chemicals were purchased from Sigma (St. Louis, MO).

Statistical analysis.. All data are presented as means ± SE. Statistical difference in the same groups was determined by a paired Student's t-test (see Figs. 1-4) or by one-way ANOVA (Student-Newman-Keuls; see Figs. 6 and 7); a value of P < 0.05 was accepted as statistically significant. The n values reported usually reflect the total number of cells tested. For each type of experiment performed, cells isolated from multiple dogs were tested. For the experiments shown in Figs. 1-4, cells studied were isolated from a total of 9 dogs; for the experiments shown in Figs. 5-7, cells studied were isolated from a total of 13 dogs.


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Fig. 6.   Effects of caffeine (100 µM) and ryanodine (10 µM) on frequency of spontaneous Ca2+ sparks in PASMC. Four representative images recorded at a frequency of 120 Hz during a 10-s recording period in control conditions in a single cell (A) and in another cell during exposure to 100 µM caffeine (B). Ca2+ spark activity was continuously monitored in all 3 experimental conditions by measuring the average fractional increase in fluorescence (F/Fo) for the entire 10-s recording periods in 3 selected areas of the cells, indicated by the superimposed colored boxes of 2.25 × 2.25 µm. C: cumulative data on mean ± SE Ca2+ spark frequency (sparks · cell-1 · min-1) under control conditions (compiled from a total of 101 sparks, recorded from 28 cells, with 1-4 sites/cell), during brief exposure to caffeine alone (compiled from a total of 116 sparks, recorded from 11 cells, with 1-4 sites/cell), and during brief exposure to caffeine in the continuous presence of ryanodine (compiled from a total of 6 cells in which no sparks were detected). *P < 0.05. Since in these experiments only a subvolume of each cell was monitored for spark appearance, the frequencies reported underestimate the true spark frequency occurring in the entire cell.



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Fig. 7.   ANG II-induced changes in local [Ca2+]i in PASMC in the absence of Ca2+ sparks. Top: 4 representative images recorded at a frequency of 120 Hz during a 10-s recording period; Bottom: average fractional increase in fluorescence (F/Fo) for the entire 10-s recording period in 3 selected cell areas, indicated by the superimposed colored boxes of 2.25 × 2.25 µm, during brief 100 µM caffeine and >5 min 10 µM ryanodine exposure (two adjoining cells; A) and during exposure to 8 nM ANG II while being continuously exposed to ryanodine (10 µM) to eliminate spontaneous Ca2+ sparks (B). C: cumulative data on mean ± SE peak change in fractional increase in fluorescence (F/Fo) observed under control conditions (n = 13 cells), following brief caffeine and sustained ryanodine exposure (n = 10 cells), during exposure to ANG II in the continued presence of ryanodine (n = 14 cells), and finally during exposure to ANG II in PASMC continuously exposed to 10 µM ryanodine and 10 µM CPA (n = 8 cells). *P < 0.05. In these experiments, the peak fractional increase in fluorescence (F/Fo) during the 10-s recording period was measured in 3 selected areas and averaged in each cell.


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Independent IP3- and ryanodine-sensitive Ca2+ stores in canine PASMC. Brief applications of caffeine (10 mM) and ANG II (1 µM) to fura 2-loaded PASMC were used to evoke Ca2+ release from ryanodine- and IP3-sensitive Ca2+ stores, respectively. These brief caffeine or ANG II exposures elicited a transient rise in global [Ca2+]i, as evidenced by increases in the fura 2 fluorescence ratio (Fig. 1A). The magnitude and size of the caffeine- and ANG II-induced intracellular Ca2+ transients were generally reproducible and remained stable during multiple exposures to either agonist in control solutions (data not shown). After washout of caffeine and ANG II, exposure of cells to the selective SR Ca2+-ATPase inhibitor, CPA (10 µM) (29), caused a small increase in the resting fura 2 fluorescence ratio. A brief application of ANG II, in the continued presence of CPA, evoked a Ca2+ transient that appeared to have a larger amplitude (fluorescence ratio) than the control ANG II response observed in this cell. However, the mean ANG II-induced Ca2+ transient (fluorescence ratio) in the presence of CPA was similar in amplitude to that of the control ANG II response (Fig. 1B, n = 4). Subsequent ANG II exposures, in the continued presence of CPA, elicited significantly smaller Ca2+ transients (P < 0.05), suggesting that Ca2+ was depleted from the IP3-sensitive Ca2+ store. The abolition of ANG II-induced intracellular Ca2+ transients under these conditions did not affect the subsequent initial release of Ca2+ by caffeine. However, a second caffeine exposure under these conditions induced a smaller Ca2+ transient compared with control while the Ca2+ transient elicited by a third caffeine exposure was barely detectable. These data strongly suggest that the caffeine-induced Ca2+ release in PASMC is mediated through an intracellular Ca2+ store, which is spatially distinct from IP3-sensitive Ca2+ stores. The decrease in the amplitude of subsequent intracellular Ca2+ transients induced by repetitive caffeine applications in the presence of CPA suggests that ryanodine-sensitive Ca2+ stores do not reload effectively in the presence of CPA.

Corollary experiments were then performed to test whether depletion of the ryanodine-sensitive Ca2+ store in PASMC would affect ANG II-induced IP3-sensitive Ca2+ store release. A relatively high ryanodine (300 µM) concentration was used because it irreversibly inhibits ryanodine-sensitive Ca2+ release channels and effectively depletes ryanodine-sensitive Ca2+ stores when the channels open (26). Figure 2, A and B, shows that ryanodine exposure did not affect resting fura 2 fluorescence or the initial caffeine-induced intracellular Ca2+ transient, but virtually abolished all subsequent intracellular Ca2+ transients in response to caffeine. This is consistent with preferential binding of ryanodine to Ca2+ release channels in the activated state (40). This abolition of caffeine-induced intracellular Ca2+ transients by continuous exposure of the cell to ryanodine did not affect the amplitude of the subsequent initial ANG II-induced intracellular Ca2+ transient. The ability of ANG II to elicit intracellular Ca2+ transients following elimination of the caffeine-induced Ca2+ release is consistent with the interpretation that IP3- and ryanodine-sensitive Ca2+ stores are spatially distinct in PASMC. However, functional interactions between these appear to occur, since the amplitude of the Ca2+ transients elicited by successive ANG II exposures were significantly reduced in ryanodine (Fig. 2B), suggesting that refilling of the ANG II-releasable store may be dependent on normally functioning ryanodine-sensitive Ca2+ stores.

Coupled IP3- and ryanodine-sensitive Ca2+ stores in canine RASMC. Similar experiments were also performed on canine RASMC to compare the functional characteristics of intracellular Ca2+ stores in PASMC to SMC in a different vascular bed. The Ca2+ release responses observed in RASMC differed significantly from those of PASMC. In RASMC, the alpha -adrenergic agonist PE (1 µM) was used to release Ca2+ from IP3-sensitive Ca2+ stores (19). Figure 3, A and B, shows that an initial PE exposure elicited a large Ca2+ release in CPA-pretreated RASM. However, all subsequent PE exposures failed to cause much Ca2+ release, indicating that CPA promotes IP3-sensitive Ca2+ store depletion, similar to the ANG II- and CPA-induced emptying of IP3-sensitive Ca2+ stores in PASMC. In contrast to PASMC, in RASMC this CPA-induced IP3-sensitive Ca2+ store depletion eliminated all subsequent caffeine-induced Ca2+ transients. These results strongly suggest that IP3- and ryanodine-sensitive Ca2+ stores spatially overlap in RASMC.

Corollary experiments were then performed to test whether or not depletion of the ryanodine-sensitive Ca2+ store would also eliminate PE-induced Ca2+ transients in RASMC, which is expected if IP3- and ryanodine-sensitive Ca2+ stores are spatially conjoined. An initial caffeine exposure caused a substantial cytosolic Ca2+ increase in RASMC pretreated with 300 µM ryanodine, but Ca2+ transients in response to subsequent caffeine exposures were significantly reduced, consistent with depletion of the ryanodine-sensitive Ca2+ stores (Fig. 4, A and B). This maneuver also eliminated all Ca2+ transients in response to subsequent PE exposures, consistent with the hypothesis that IP3- and ryanodine-sensitive Ca2+ stores are spatially conjoined in RASMC. It is noteworthy that the initial caffeine-induced Ca2+ transient in the presence of 300 µM ryanodine failed to completely decline in some cells (Fig. 4A), and the fura 2 ratio remained elevated following washout of caffeine. This incomplete relaxation of the caffeine-induced Ca2+ transient may be due to ryanodine locking Ca2+ release channels into a subconductance state, causing slow sustained SR Ca2+ release, or, alternatively, it may be due to activation of a capacitative Ca2+ entry pathway in these cells (36, 44). Further experiments are required to test these possibilities.

Caffeine and ANG II cause differential subcellular Ca2+ release events in canine PASMC. Spontaneous local Ca2+ release events (Ca2+ sparks) were detected in fluo 3-loaded PASMC by measuring rapid local changes in [Ca2+]i using a real time laser scanning confocal microscope imaging system (see METHODS). Figure 5A shows six full-frame video images of a PASMC acquired at a frequency of 30 Hz, illustrating an example of spontaneous release of Ca2+ from a subcellular location in a PASMC. Spontaneous Ca2+ spark events were usually located near the cell edges and not in the cell center, suggesting that these Ca2+ release sites are located near the plasma membrane. Figure 5B shows the spatial-temporal characteristics of the spark shown in Fig. 5A. The spatial distributions of the fractional increase in fluorescence intensity were fit with Gaussian curves (red), relative to the initiation point. The full width at half-maximal (FWHM) intensity for the spark shown at time 0.0333 s was 1.95 µm. The average FWHM for sparks, which could clearly be attributed to initiation from a single point source, was 1.60 ± 0.2 µm (n = 3).

To test whether spontaneous Ca2+ spark events in PASMC are associated with ryanodine-sensitive Ca2+ release channels, the effects of caffeine and ryanodine on Ca2+ spark frequency were examined. In these experiments, video images of PASMC were acquired at a frequency of 120 Hz to improve temporal resolution. Figure 6A shows four example images recorded under control conditions during a 10-s recording period. Ca2+ spark activity was continuously monitored by measuring the average fractional increase in fluorescence (F/Fo) in selected areas of the cell, indicated by the superimposed colored boxes. Under control conditions, average basal spark frequency at room temperature was 21.6 ± 2.3 sparks · cell-1 · min-1 (Fig. 6C). The peak F/Fo was 1.0 ± 0.05, and the mean half time of decay of F/Fo was 50 ± 5.0 ms (compiled from a total of 42 sparks, recorded from 16 cells at 21 individual sites, with 1-3 sites/cell). In cells treated with caffeine (100 µM), average spark frequency increased to 62.3 ± 6.6 sparks · cell-1 · min-1. The peak F/Fo in caffeine-treated cells was 0.82 ± 0.05 and the mean half time of decay of F/Fo was 52 ± 4 ms (compiled from a total of 30 sparks, recorded from 5 cells at 10 individual sites, with 1-4 sites/cell); neither the amplitude nor the decay times were statistically different from control (P = 0.12 and 0.31, respectively). Ten micromolar ryanodine exposures for >15 min [with one brief (30-s) 100 µM caffeine exposure] nearly abolished all basal spark activity (see also Fig. 7A). These results verify that spontaneous Ca2+ spark events in PASMC are associated with ryanodine-sensitive Ca2+ release channels.

To release IP3-sensitive Ca2+ stores locally and in isolation, PASMC were first exposed to ryanodine (10 µM) for >5 min [with one brief (30-s) 100 µM caffeine exposure], which eliminated basal Ca2+ spark activity (Fig. 7A). Cells were then exposed to a low concentration of ANG II (8 nM) to release IP3-sensitive Ca2+ stores. Discrete quantal-like events associated with ANG II-induced Ca2+ release from IP3-sensitive stores were rarely observed in PASMC (Fig. 7B). Instead, ANG II appeared to induce slow, sustained, [Ca2+]i increases originating from multiple sites near the cell membrane. These effects were quantified by measuring the peak fractional increase in fluorescence (F/Fo) in selected areas of the cell under the various experimental conditions (Fig. 7C). These slow, ANG II-induced sustained increases in [Ca2+]i can be attributed to Ca2+ release from IP3-sensitive stores since 1) Ca2+ sparks associated with ryanodine-sensitive Ca2+ release channels were eliminated by depletion of ryanodine-sensitive Ca2+ stores, and 2) the ANG II-induced Ca2+ increase was nearly abolished by CPA pretreatment. The occasional quantal-like events observed at some sites (Fig. 7B, yellow trace) following ANG II exposure can probably be attributed to CICR from a few residual incompletely emptied ryanodine-sensitive Ca2+stores, since the spatial-temporal characteristics of these events resemble those of basally active Ca2+ sparks.

Organization of intracellular Ca2+ stores in PASMC and RASMC. In an earlier study, isometric tension measurements in canine pulmonary arterial rings suggested that IP3-sensitive and ryanodine-sensitive Ca2+ stores were spatially independent (19). Depletion of SR Ca2+ stores with ryanodine, which eliminated caffeine-induced contractions, had little or no effect on IP3-induced contractions. Thapsigargin or CPA, which abolished IP3-induced contractions, failed to attenuate caffeine-induced contractions. In the present study, using similar pharmacological agents, additional evidence for spatially distinct Ca2+ stores was obtained from measurements of global changes in [Ca2+]i in isolated single PASMC. In contrast, similar experiments in RASMC reveal that these Ca2+ stores appeared to be spatially conjoined.

The conclusion that intracellular Ca2+ stores may be organized differently in PASMC and RASMC is based primarily on functional studies using pharmacological tools to induce Ca2+ release from or to effect Ca2+ depletion of specific Ca2+ stores. Thus the validity of this conclusion is dependent on the specificity of the pharmacological agents used. Ryanodine is a highly selective agent; at low doses it can lock Ca2+ release channels into a subconductance state, eventually leading to Ca2+ depletion of the caffeine-sensitive SR, while at high doses it completely inhibits channel openings (37, 40). Ryanodine does not directly affect sarcolemmal Ca2+ channels (1), the plasma membrane ATPase (40), or IP3 receptors (18). CPA is a mycotoxin, which specifically inhibits the sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs) (10, 23) and thus blocks refilling of intracellular Ca2+ stores, leading to store depletion. PE and ANG II are well-established pharmacological tools that specifically cause rapid IP3 formation and Ca2+ release from IP3-sensitive stores. Although these agents are known to exert a number of other actions in smooth muscle, including modulation of sarcolemmal ion channels and intracellular signaling pathways (21), it is presumed that the ANG II- and PE-induced intracellular Ca2+ transients that were observed in both PASMC and RASMC can be attributed primarily to Ca2+ release from intracellular stores, since CPA consistently reduced these intracellular Ca2+ transients.

Data supporting either homogeneity or heterogeneity of intracellular Ca2+ stores involving the use of blockers of SERCAs in conjunction with activators of IP3 and ryanodine receptors have been obtained previously in a variety of different cell types (35). In vascular SMC, the best-characterized examples in which IP3-sensitive and ryanodine-sensitive SR Ca2+ stores appear to be organized into spatially separate compartments is cultured rat aortic cells (43) and cultured mesenteric arterial cells (11), although the latter result is controversial. In these studies, IP3- and ryanodine-sensitive SR Ca2+ stores appeared to be differentially depleted by thapsigargin and CPA, suggesting that caffeine-sensitive stores may have a thapsigargin- and CPA-insensitive Ca2+-ATPase. In contrast, our data in PASMC, while providing evidence for the existence of spatially separate IP3- and ryanodine-sensitive SR Ca2+ stores, suggest that refilling of both stores can be prevented by CPA. Thus, following depletion of the IP3-sensitive SR Ca2+ store (CPA + repeated ANG II exposures), the first caffeine response was unaffected by CPA (confirming the independence of the two stores), but subsequent caffeine responses were reduced and eventually eliminated, suggesting that CPA does in fact prevent reloading of the caffeine-sensitive store as well. These data suggest that the Ca2+-sequestration mechanisms of both the IP3- and caffeine-sensitive stores are sensitive to CPA and that differential store depletion can be effected by specific agonist-induced store emptying while blocking store repletion. In RASMC, where the stores appear to be conjoined, store emptying by either PE or caffeine, while blocking store repletion, is sufficient to cause depletion of the common Ca2+ pool. It should be noted that in all of our experiments, with relatively short exposure times to CPA, store depletion required initial agonist exposure, suggesting that the rate of Ca2+ leak from either store alone was insufficient to induce store depletion. It is possible that, with longer exposure times to CPA, heterogeneity in the rate of Ca2+ leak from the two stores (35) may give rise to an apparent differential sensitivity of IP3-sensitive Ca2+ stores and caffeine-sensitive Ca2+ stores to CPA or agonists.

It is not presently known whether the heterogeneous organization of IP3-sensitive Ca2+ stores and caffeine-sensitive Ca2+ stores that we observe in canine PASMC may be a common characteristic of PASMC. There is some evidence for a similar organization of intracellular Ca2+ stores in rat PASMC. Endothelin-induced [Ca2+]i oscillations mediated by Ca2+ release from IP3-sensitive Ca2+ stores were reported to be insensitive to tetracaine and ruthenium red, both inhibitors of CICR (17). It has also been reported that serotonin-induced intracellular Ca2+ transients, which are inhibited by CPA or nitric oxide pretreatment, are largely unaffected by caffeine (47). In contrast, in chemically skinned rat pulmonary arterial strips, thapsigargin and CPA were found to inhibit both norepinephrine- and caffeine-induced contractions (12).

However, functional studies that employ blockers of SERCAs in conjunction with activators of IP3 and ryanodine receptors can provide only limited information related to the actual morphological organization of intracellular Ca2+ stores. Morphological studies employing immunofluorescence confocal and immunoelectron microscopy to examine the relationship between IP3 and ryanodine receptor isoforms and SERCA isoforms in canine PASMC, and PASMC in other species and locations along the pulmonary arterial tree, are needed to substantiate conclusions drawn from these functional studies. It will be particularly interesting to examine the relationship of the two distinct functional intracellular Ca2+ stores described here to earlier reports documenting the existence of spatially resolvable central and peripheral (junctional) SR components in smooth muscle (39), including pulmonary artery (22). In smooth muscle, intracellular Ca2+ stores have been classified into at least three different subtypes: Salpha (containing both IP3 and ryanodine receptors) and Sbeta (containing only IP3 receptors) subtypes (18) and an Sgamma subtype (containing only ryanodine receptors) (3), the relative distribution of which varies considerably between different species and types of SMC (see Ref. 21). Within such a classification scheme, our data would be most compatible with the existence of Sbeta (agonist sensitive) and Sgamma as the major Ca2+ store subtypes in canine PASMC and Salpha as the major store subtype in canine RASMC.

Elementary Ca2+ release events in PASMC. Local intracellular Ca2+ transients due to the spontaneous opening of ryanodine receptors (Ca2+ sparks) have been previously characterized in SMC isolated from rat cerebral arteries (30), rat portal vein (27), guinea pig trachea (48), toad stomach (49), and guinea pig ileum (13). Depending on the functional organization of ryanodine-sensitive SR Ca2+ stores and their spatial relationship to sarcolemmal ion channels in different SMC, Ca2+ sparks are believed to regulate resting membrane potential through activation of Ca2+-dependent K+ channels, Ca2+-dependent Cl- channels, and nonselective cation channels (27, 34, 48, 49). Although Ca2+ sparks were originally believed to be generated by the opening of a single ryanodine receptor (8), it now seems clear that in most cells sparks are probably generated by a cluster of ryanodine receptors acting in concert (6, 38).

Local intracellular Ca2+ transients due to the spontaneous opening of ryanodine receptors have not been previously characterized in PASMC. Spontaneous local intracellular Ca2+ transients were observed in canine PASMC with spatial-temporal characteristics (FWHM, peak F/Fo, and mean half time of F/Fo decay) similar to those previously described for Ca2+ sparks in cardiac (6, 8) and SMC (27, 30, 48) using the Ca2+ indicators fluo 3-AM or fluo 4-AM in combination with confocal laser scanning microscopy. These events are attributable to the opening of ryanodine receptors since caffeine increased their frequency and ryanodine nearly eliminated them.

A characterization of the properties of elementary release events associated with IP3 receptor activation in canine PASMC was also attempted. Since ryanodine- and IP3-sensitive Ca2+ stores in these cells are spatially independent, it was anticipated that local intracellular Ca2+ transients, because of the opening of IP3 receptors, could be studied in isolation when ryanodine-sensitive Ca2+ stores were depleted. Under these conditions, low ANG II concentration exposures induced slow, sustained, local [Ca2+]i increases at multiple sites near the cell membrane. Discrete quantal-like events were not typically observed in marked contrast to the IP3-induced quantal-like, Ca2+ puffs, which have been characterized in Xenopus oocytes (33) and HeLa cells (5) and attributed to the coordinated opening of clusters of IP3 receptor/channels in the endoplasmic reticulum (33). In these and other cells, a hierarchy of IP3-evoked Ca2+ events, ranging from elementary Ca2+ blips to intermediary Ca2+ puffs, to global Ca2+ waves, has been proposed (25). More recently, an examination of the properties of a large population of IP3 Ca2+ release sites in HeLa cells suggests that IP3-evoked Ca2+ events, rather than behaving as discrete events, comprise a continuum of amplitudes and lifetimes (42). This is consistent with clusters of IP3 receptors containing variable numbers of channels and variable recruitment of channels within a cluster. Few studies to date have examined elementary events associated with IP3-evoked Ca2+ release in vascular SMC. However, the IP3-induced slow, sustained, increases in local [Ca2+]i that were observed in PASMC are similar to those previously described in rat portal vein cells (4), where IP3-induced quantal-like events also were not observed. One possible explanation for the differences observed in vascular SMC, compared with oocytes and HeLa cells, is that IP3 receptors may be organized differently, such that multiple IP3 channels may not be able to coordinate into functional clusters in vascular SMC. Future molecular and morphological studies are required to establish the actual organization of IP3 receptors, their relationship to one another and to ryanodine receptors, and the role that mitochondria (9) may play in regulation of [Ca2+]i in PASMC.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-49254.


    FOOTNOTES

Preliminary results of this study have been published in abstract form (20).

Present addresses: R. Janiak, ICON Clinical Research, Heinrich-Hertz-Strasse 26, D-63225 Langen, Germany; S. Montague, Smooth Muscle Research Group, Department of Physiology, Medical Biology Centre, The Queen's University of Belfast, 97 Lisburn Rd., Belfast BT9 7BL, UK.

Address for reprint requests and other correspondence: J. R. Hume, Dept. of Physiology and Cell Biology/351, Univ. of Nevada, School of Medicine, Reno, NV 89557 (E-mail: joeh{at}med.unr.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 December 1999; accepted in final form 22 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
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