Contribution of Ryanodine Receptor Subtype 3 to Ca2+ Responses in Ca2+-overloaded Cultured Rat Portal Vein Myocytes*

Jean MironneauDagger, Frédéric Coussin, Loice H. Jeyakumar§, Sidney Fleischer§, Chantal Mironneau, and Nathalie Macrez

From the Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, Bordeaux Cedex 33076, France and the § Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235

Received for publication, July 7, 2000, and in revised form, December 11, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca2+ release during Ca2+ sparks and global Ca2+ responses, evoked by activation of voltage-gated Ca2+ channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca2+ loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca2+ responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca2+ measurements revealed that the number of cells showing spontaneous Ca2+ sparks was strongly increased by superfusing the vascular myocytes in 10 mM Ca2+-containing solution. These Ca2+ sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca2+ responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca2+ responses in Ca2+-overloaded myocytes. Ca2+ transients evoked by photolysis of caged Ca2+ with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca2+]i sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca2+ loading, the RYR3 becomes activable by caffeine and local increases in [Ca2+]i.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Since the description of a Ca2+-induced Ca2+ release mechanism in skeletal muscle (1), the function of ryanodine receptor channels (RYRs)1 have been widely studied in both skeletal and cardiac muscles (2-7) and more recently in smooth muscle (8, 9). After the cloning and sequencing of three genes encoding different RYR subtypes, the localization and role of each RYR subtype in Ca2+ signaling have begun to be studied. Of the three RYRs, RYR subtype 3 (RYR3) is the most widely expressed (10), whereas RYR1 and RYR2 are mainly found in skeletal and cardiac muscles, respectively (11, 12). However, in arterial and venous smooth muscles, the three RYR subtypes have been identified (13, 14), but their role in Ca2+ release is still unclear.

The regulation of the different RYR subtypes has been extensively studied using single channel recordings in lipid bilayers. The single channel properties of RYR subtypes are rather similar, i.e. they form a large conductance channel permeable to monovalent and divalent cations, which can be activated by ATP, caffeine, and submicromolar concentrations of Ca2+; inhibited by Mg2+, ruthenium red, and millimolar concentrations of Ca2+; and modulated by ryanodine (15-17). However, differences in responses to cyclic ADP-ribose and caffeine as well as in sensitivities to Ca2+ activation and Ca2+ inactivation have been reported between RYR1 and RYR3 and between RYR3s cloned from skeletal and smooth muscles (17-20). It has been proposed that these differences may result from the expression of different splice variants of RYR3 (21).

The physiological contribution of the different RYR subtypes to Ca2+ signaling has been addressed first by using either RYR1 and RYR2 knockout mice (12, 22) or RYR3 knockout mice (23-25). In RYR1-null myotubes in culture, the Ca2+ release from the sarcoplasmic reticulum (SR) in response to increases in cytosolic Ca2+ concentration ([Ca2+]i) or caffeine is strongly reduced, but a similar decrease in caffeine sensitivity is also observed in RYR3-null neonatal myocytes, suggesting a possible co-contribution of each RYR subtype to Ca2+ signaling, at least at some stages of myogenesis. Accordingly, it has been recently proposed that, in embryonic skeletal muscle, both RYR1 and RYR3 may co-contribute to Ca2+ release during Ca2+ sparks (25). In addition, Ca2+ sparks produced independently in RYR1- or RYR3-null cells reveal similar spatio-temporal parameters (26). Another approach using antisense oligonucleotides, that specifically targeted each one of the RYR subtypes, has been used to determine which RYR subtypes are responsible for Ca2+ sparks and global Ca2+ responses in smooth muscle cells (14). It appears that both RYR1 and RYR2 are required for Ca2+ release during Ca2+ sparks and Ca2+ waves induced by activation of L-type Ca2+ currents and that RYR3 does not contribute to these Ca2+ signals.

In smooth muscle cells, Ca2+ sparks are observed spontaneously or in response to Ca2+ influx through L-type Ca2+ channels (8, 9, 27, 28), and their localization corresponds to coupling areas between the plasma membrane and the SR (28, 29). In rat portal vein myocytes, spatial and temporal recruitment of Ca2+ sparks results in propagating Ca2+ waves, which trigger cell contraction (30).

In the present study, we investigated the effects of elevating the extracellular Ca2+ concentration ([Ca2+]o) on both Ca2+ sparks and global Ca2+ responses induced by Ca2+, caffeine, and phenylephrine. Under conditions of increased SR Ca2+ loading, we provide the first evidence that the RYR3, which becomes activable by caffeine and localized increases in [Ca2+]i, is responsible for the increased global Ca2+ responses. We also found that spontaneous Ca2+ sparks are highly abundant but remain dependent on activation of only RYR1 and RYR2.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Preparation-- Rats (160-180 g) were killed by cervical dislocation. The portal vein was cut into several pieces and incubated for 10 min in low Ca2+ (40 µM) physiological solution, and then 0.8 mg/ml collagenase (EC 3.4.24.3), 0.20 mg/ml Pronase E (EC 3.4.24.31), and 1 mg/ml bovine serum albumin were added at 37 °C for 20 min. After this time, the solution was removed, and pieces of portal vein were incubated again in a fresh enzyme solution at 37 °C for 20 min. Tissues were placed in a enzyme-free solution and triturated using fire-polished Pasteur pipette to release cells. Cells were seeded at a density of 103 cells/mm2 on glass slides imprinted with squares for localization of injected cells. Cells were maintained in short term primary culture in medium M199 containing 2% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, 20 units/ml penicillin, and 20 µg/ml streptomycin; they were kept in an incubator gassed with 95% air and 5% CO2 at 37 °C. The myocytes were cultured in this medium for 4 days. The normal physiological solution contained 130 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 1.7 mM CaCl2, 11 mM glucose, and 10 mM HEPES (pH 7.4, with NaOH).

Microinjection of Oligonucleotides-- Phosphorothioate antisense oligonucleotides (denoted with the prefix "as") used in the present study were designed on the known cloned RYR sequences deposited in the GenBankTM sequence data base with Lasergene software (DNASTAR, Madison, WI). Sequences of all three RYR cDNAs were aligned with each other, and specific antisense oligonucleotide sequences were chosen in region of the cDNA of interest, completely different from the sequences of the two other RYR subtypes. Then antisense and scrambled sequences displaying putative binding to any other mammalian sequences deposited in GenBankTM were discarded. Oligonucleotides were injected into the nuclei of myocytes by a manual injection system (Eppendorf, Hamburg, Germany). Intranuclear oligonucleotide injection with Femtotips II (Eppendorf) was performed as previously described (14). The myocytes were then cultured for 3-4 days in culture medium, and the glass slides were transferred into the perfusion chamber for physiological experiments. The sequences of as1RYR1 and as2RYR1 are AGCGTGTGCAGCAGGCTCA and GCAATCCGCTCCCGCCCA, corresponding to nucleotides 325-343 and 584-601, respectively, of RYR1 cDNA deposited in GenBankTM (accession number X83932); those of as1RYR2 and as2RYR2 are GTGTCCTCACAGAAGTT and TGAAATCTAGTGCAGCCT, corresponding to nucleotides 137-153 and 1587-1604, respectively, of RYR2 cDNA (accession number X83933); and those of as1RYR3 and as2RYR3 are AAGTCAAGGGCATTTTTG and ACTTAGCCATGACACCAG, corresponding to nucleotides 502-519 and 557-574, respectively, of RYR3 cDNA (accession number X83934). In some control experiments, myocytes were injected with the following scrambled oligonucleotides: CACGCCTACGCACCTCCG, corresponding to a scrambled sequence of as2RYR1 (nucleotides 584-601 of RYR1 cDNA); AGTCGTACATGACTCGTA, corresponding to a scrambled sequence of as2RYR2 (nucleotides 1587-1604 of RYR2 cDNA); and CAGCACTATCAGTACGAC, corresponding to a scrambled sequence of as2RYR3 (nucleotides 557-574 of RYR3 cDNA).

Cytosolic Ca2+ Measurements-- In most experiments, cells were loaded by incubation in physiological solution containing 4 µM fluo 3-acetoxymethylester (fluo 3-AM) for 1 h at room temperature. These cells were washed and allowed to cleave the dye to the active fluo 3 compound for at least 30 min. Images were acquired using the line scan mode of a confocal Bio-Rad MRC1000 microscope connected to a Nikon Diaphot microscope. Excitation light was delivered by a 25-milliwatt argon ion laser (Ion Laser Technology, Salt Lake City, UT) through a Nikon Plan Apo × 60, 1.4 NA objective lens. Fluo 3 was excited at 488 nm, and emitted fluorescence was filtered and measured at 540 ± 30 nm. At the setting used to detect fluo 3 fluorescence, the resolution of the microscope was near 0.4 × 0.4 × 1.5 µm (x, y, and z axis). Images were acquired in the line scan mode at a rate of 6 ms/scan. Scanned lines were plotted vertically, and each line was added to the right of the preceding line to form the line scan image. In these images, time increased from the left to the right, and position along the scanned line was given by vertical displacement. Fluorescence signals are expressed as pixel per pixel fluorescence ratios (F/Fo), where F is the fluorescence during a response and Fo is the rest level fluorescence of the same pixel. Image processing and analysis were performed by using COMOS, TCSM, and MPL 1000 software (Bio-Rad).

In other experiments, cells were loaded by incubation in physiological solution containing 1 µM indo 1-AM for 30 min. [Ca2+]i measurements were estimated from the 405-/480-nm fluorescence ratio, as previously reported (31). The minimum and maximum fluorescence (Rmin and Rmax, respectively) values were determined in vivo, in the absence of Ca2+ and at saturating Ca2+, in cells superfused in 1.7 and 10 mM [Ca2+]o.

Caffeine and phenylephrine were applied by pressure ejection from a glass pipette for the period indicated on the records. All experiments were carried out at 26 ± 1 °C.

Flash Photolysis-- Caged Ca2+, 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA, tetra(acetoxymethylester) (DMNP-EDTA, AM) at 15 µM, was added to the bathing solution and maintained in the presence of cells for 1 h in an incubator at 37 °C. Photolysis was produced by a 1-ms pulse from a xenon flash lamp (Hi-Tech Scientific, Salisbury, United Kingdom) focused to a ~2-mm diameter spot around the cell. Light was band pass-filtered with a UG11 glass between 300 and 350 mm. Flash intensity could be adjusted by varying the capacitor-charging voltage between 0 and 380 V, which corresponded to a change in the energy input into the flash lamp from 0 to 240 J. On flash photolysis, Ca2+ was released within 2 ms, and the small percentage of conversion of the caged compound (~10%) allowed us to apply repetitive pulses without altering the Ca2+ responses and the reserve of caged Ca2+.

RYR Labeling-- Freshly dissociated and cultured myocytes (3 days after injection) were immunostained as previously described (30). Briefly, myocytes were incubated in the presence of anti-RYR3-specific antibody (20) (at 1:100 dilution) for 20 h at 4 °C and with the secondary antibody (donkey anti-rabbit IgG conjugated to fluorescein isothiocyanate, diluted at 1:200) for 3 h at 20 °C. Thereafter, cells were mounted in Vectashield. Images of the stained cells were obtained with the Bio-Rad confocal microscope. Control cells and injected cells on the same glass slide were compared with each other by keeping acquisition parameters constant (gray values, exposure time, aperture). Fluorescent labeling was estimated by gray level analysis using MPL software and expressed in arbitrary units of fluorescence.

Chemicals and Drugs-- Collagenase was obtained from Worthington. Fluo 3-AM and DMNP-EDTA, AM were from Molecular Probes (Leiden, The Netherlands). Caffeine was from Merck. Indo 1-AM, ryanodine, and cyclopiazonic acid were from Calbiochem. Medium M199 was from ICN (Costa Mesa, CA). Fetal calf serum was from Bio Media (Boussens, France). Streptomycin, penicillin, glutamine, and pyruvate were from Life Technologies, Inc. All primers and phosphorothioate antisense oligonucleotides were synthesized and purchased from Eurogentec (Seraing, Belgium). All other chemicals were from Sigma. The rabbit anti-RYR3-specific antibody was directed against the deduced amino acid sequence, 4326-4336 (11 amino acids), of rabbit RYR3 (20).

Data Analysis-- Data are expressed as means ± S.E.; n represents the number of tested cells. Significance was tested by means of Student's test. p values < 0.05 were considered as significant.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of High Ca2+-containing Solution on the Caffeine-induced Ca2+ Responses in Vascular Myocytes-- The effects of increasing the external Ca2+ concentration ([Ca2+]o) in rat portal vein myocytes were studied in a series of experiments, in which caffeine (10 mM) was applied through a micropipette positioned near the surface of the cells and the resulting [Ca2+]i changes were measured either in the entire cytosol (indo 1 experiments) or in a single line repeatedly scanned through the confocal cell section (fluo 3 experiments). In indo 1-loaded cells, the peak fluorescence (Delta  ratio 405/480 nm) as well as the total fluorescence measured during 10-s applications of caffeine (Delta  ratio × 10 s) increased in a time-dependent manner and reached a steady-state value within 1 h in 10 mM Ca2+-containing solution. In 1.7 mM [Ca2+]o, the values for the peak and total fluorescence were 0.69 ± 0.03 and 119.2 ± 7.6, respectively (n = 46). In 10 mM [Ca2+]o, these values increased to 0.98 ± 0.03 (n = 79) and 272.2 ± 26.1 (n = 46), respectively, indicating a 40% increase in caffeine response amplitude and a 230% increase in SR Ca2+ release. In contrast, the basal [Ca2+]i was not significantly increased (from 55 ± 8 nM in 1.7 mM [Ca2+]o to 64 ± 10 nM in 10 mM [Ca2+]o, n = 46). In fluo 3-loaded cells (Fig. 1), the amplitude of the caffeine-induced Ca2+ waves, measured from a 2-µm region of the line scan image, also increased from 2.01 ± 0.10 (Delta F/Fo, n = 32) in 1.7 mM [Ca2+]o to 2.66 ± 0.08 (n = 45) in 10 mM [Ca2+]o, indicating a 30% increase in Ca2+ response amplitude (Fig. 1B). Furthermore, the upstroke velocity of the caffeine-induced Ca2+ response, corresponding to the initiation site of the response, was enhanced from 8.57 ± 0.71 (Delta F/Fo·s-1, n = 32) in 1.7 mM [Ca2+]o to 33.57 ± 4.27 (n = 45) in 10 mM [Ca2+]o, indicating a 350% increase in Ca2+ release velocity (Fig. 1B). Taken together, these results suggest that the SR Ca2+ content of vascular myocytes is increased by sustained elevation in extracellular [Ca2+] and that both amplitude and upstroke velocity of the Ca2+ responses can be used as significant parameters to study the cellular mechanisms involved during increased SR Ca2+ loading.



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Fig. 1.   Effects of increased [Ca2+]o on caffeine-induced [Ca2+]i responses in rat portal vein myocytes. A, line scan images of fluorescence changes induced by 10 mM caffeine in 1.7 mM Ca2+-containing solution (a) and 1 h after increasing [Ca2+]o to 10 mM (b). Traces show (from top to bottom) caffeine application, line scan image, and averaged fluorescence from 2-µm regions (indicated by the small vertical bars) of the line scan image. B, compiled data showing the effects of 10 mM [Ca2+]o on both amplitude and upstroke velocity of caffeine-induced Ca2+ responses. Caffeine was applied at 10 mM. Bars show means ± S.E. in 1.7 mM [Ca2+]o (open bars) and 10 mM [Ca2+]o (filled bars), with the number of cells tested indicated in parentheses. Cells were obtained from five different batches. star , values significantly different from those obtained in 1.7 mM [Ca2+]o. Myocytes were loaded with fluo 3-AM.

To assess the role of the SR Ca2+ loading in the generation of large and fast Ca2+ responses to caffeine, the effects of 10 µM cyclopiazonic acid were first investigated on the caffeine-induced Ca2+ responses. Inhibition of the Ca2+ uptake capacity of the intracellular store by cyclopiazonic acid resulted in a small elevation of the basal [Ca2+]i and the suppression of the caffeine-induced Ca2+ response in the continuous presence of cyclopiazonic acid for 5 min (n = 6). In a second set of experiments, caffeine (10 mM) was applied in Ca2+-free, 0.5 mM EGTA-containing solution for 10 s (a time sufficient to remove voltage-dependent Ca2+ current) on myocytes superfused either in 1.7 mM [Ca2+]o or 10 mM [Ca2+]o. After 10 s in Ca2+-free solution, the amplitude of the caffeine-induced Ca2+ responses (measured with indo 1) was 0.55 ± 0.03 (Delta  ratio, n = 26) in myocytes pretreated with 1.7 mM [Ca2+]o and 0.79 ± 0.07 (n = 26) in myocytes pretreated with 10 mM [Ca2+]o, indicating an increase in Ca2+ response amplitude similar to that obtained in Ca2+-containing solutions (about 40%). Taken together, these results suggest that the increased accumulation of Ca2+ in the SR is responsible for the large and fast caffeine-induced Ca2+ responses under conditions of increased [Ca2+]o.

Antisense Oligonucleotide Strategy-- As recently published (14), we designed antisense oligonucleotides specifically targeting each RYR subtype mRNA. For each RYR subtype, two antisense sequences were chosen, one targeting the region of the mRNA amplified in PCR experiments (named as2RYR) and the other one (named as1RYR) designed to hybridize the mRNA outside the amplified fragment but close to the start codon. The time course of antisense oligonucleotide efficiency was determined by checking the ability of a mixture of as1RYR1 + as1RYR2 + as1RYR3 (10 µM each) to inhibit the Ca2+ waves induced by 10 mM caffeine in isolated myocytes superfused in 10 mM [Ca2+]o for 1 h. The Ca2+ responses were strongly inhibited 3 days after nuclear injection of the antisense oligonucleotides (83 ± 5%, n = 30); recovery began the fourth day with an inhibition of 48 ± 5% (n = 25). Nonspecific effects of antisense oligonucleotides were detected only at concentrations higher than 50 µM (for example, inhibition of RYR2 protein expression by 50 µM anti-Galpha o antisense oligonucleotide).

Immunodetection of RYR3 with an anti-RYR3-specific antibody (20) revealed a homogeneous distribution of fluorescence in cell sections from freshly isolated and cultured myocytes (Fig. 2, A and B). In cells injected with as1RYR3, the immunostaining was very weak (Fig. 2C), whereas it was not significantly changed in cells injected with as1RYR1 + 2 (Fig. 2, E and F). These results indicate that RYR3 is expressed in rat portal vein myocytes and can be selectively inhibited by asRYR3 without variation in the expression of the other RYR subtypes; they are in good agreement with previous data using BODIPY®-labeled ryanodine staining, which showed that inhibition of each one of the three RYR subtypes decreased by approximately one-third the specific fluorescence (14).



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Fig. 2.   Efficiency of RYR antisense oligonucleotides. Typical labeling of RYR3 is obtained with an anti-RYR3 specific antibody (20) in freshly isolated cells (A), in cells cultured for 4 days in control conditions (B), and after nuclear injection of as1RYR3 alone (C) or a mixture of as1RYR1 + 2 (E). D, scanning transmission image of the cell shown in C. F, compiled data expressed in arbitrary units (AU) of anti-RYR3 specific antibody fluorescence measured in control and injected cells. Nonspecific fluorescence was estimated in the presence of 2 µg/ml antigen peptide (hatched bar). Open bars (noninjected control cells) and filled bars (cells injected with asRYRs) show means ± S.E., with the number of cells tested indicated in parentheses. Cells were obtained from three different batches. star , values significantly different from those obtained in noninjected cells. Cells were immunostained 3 days after nuclear injection of antisense oligonucleotides. N, nucleus.

RYR Subtypes Involved in Ca2+ Sparks under Increased SR Ca2+ Loading-- We have previously reported that, in 1.7 mM [Ca2+]o, both RYR1 and RYR2 are required for Ca2+ release during Ca2+ sparks evoked by activation of voltage-gated Ca2+ channels (14). Since elevation of luminal [Ca2+] has been suggested to increase the activity of RYRs (32), we studied the parameters of spontaneous Ca2+ sparks in Ca2+-overloaded cells. In control cells, superfused in 1.7 mM [Ca2+]o, spontaneous Ca2+ sparks were rarely detected, in less than 25% of cells tested (7/32 cells), and the number of initiation sites per line scan image was 1.1 ± 0.1 (n = 7). In 10 mM [Ca2+]o, spontaneous Ca2+ sparks were detected in about 80% of cells tested (116/140 cells; Fig. 3A), and the number of initiation sites per line scan image was 2.17 ± 0.21 (n = 44, Fig. 3C). In contrast, the spatio-temporal parameters of Ca2+ sparks were not significantly different in 1.7 mM [Ca2+]o and 10 mM [Ca2+]o (Table I).



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Fig. 3.   Effects of RYR antisense oligonucleotides on the spontaneous Ca2+ sparks in 10 mM [Ca2+]o. A, percentage of cells showing spontaneous Ca2+ sparks in 1.7 mM [Ca2+]o and in 10 mM [Ca2+]o either in noninjected cells (open bars) or in cells injected with 10 µM asRYR1, 10 µM asRYR2, a mixture of as1RYR1 + 2, or a mixture of as1RYR1 + 2 + 3 at 10 µM each (filled bars). B, percentage of cells with spontaneous Ca2+ sparks in 10 mM [Ca2+]o, in noninjected cells (open bars) and in cells injected with 10 µM as1RYR3 or as2RYR3 (filled bars). C, number of initiation sites triggering spontaneous Ca2+ sparks per line scan image in 10 mM [Ca2+]o, in noninjected cells (open bars) and in cells injected with 10 µM as1RYR3 or as2RYR3 (filled bars). Bars show means ± S.E. in noninjected control cells (open bars) or in cells injected with asRYRs (filled bars), with the number of cells tested indicated in parentheses. Cells were obtained from five different batches. star , values significantly different from those obtained in noninjected cells in 10 mM [Ca2+]o. Myocytes were loaded with fluo 3-AM.


                              
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Table I
Effects of RYR3 antisense oligonucleotides on the parameters of Ca2+ sparks
Data are means ± S.E., with n indicating the number of cells tested in each conditions. FTHM, full time at half-maximal amplitude; FWHM, full width at half-maximal amplitude.

When cells were injected with asRYR1, asRYR2, or asRYR1 + 2, the number of cells with spontaneous Ca2+ sparks was strongly decreased (Fig. 3A). In contrast, the number of cells with spontaneous Ca2+ sparks and the number of initiation sites per line scan image were not significantly affected in cells injected with asRYR3 (Fig. 3, B and C). These results suggest that under both normal (14) and increased SR Ca2+ content conditions, Ca2+ sparks are due to activation of both RYR1 and RYR2 and that RYR3 does not contribute to triggering Ca2+ sparks. In addition, the spatio-temporal parameters of Ca2+ sparks in 10 mM [Ca2+]o were not significantly different in noninjected cells and in cells injected with asRYR3 (Table I). Taken together, these results suggest that increased SR Ca2+ loading potentiates the activity of Ca2+ release units formed by RYR1 and RYR2, leading to an increase in Ca2+ spark frequency without alterations of the spatio-temporal parameters.

RYR Subtypes Involved in Caffeine-induced Ca2+ Responses under Increased SR Ca2+ Loading-- We have previously shown that in 1.7 mM [Ca2+]o, caffeine triggers Ca2+ waves by activating both RYR1 and RYR2 (14). Consequently, inhibition of RYR1 or RYR2 by treatment with antisense oligonucleotides partly inhibited the caffeine-induced Ca2+ responses, whereas inhibition of RYR3 was ineffective (14). Using the same anti-RYR antisense oligonucleotides, we determined the role of each RYR subtype in the generation of the caffeine-induced Ca2+ responses under conditions of increased SR Ca2+ loading. Cells injected with asRYR1 or asRYR2 evoked Ca2+ responses to 10 mM caffeine that were similar to those elicited in control cells superfused in 10 mM [Ca2+]o (Fig. 4A). However, injection of both as1RYR1 + as1RYR2 and injection of asRYR3 alone significantly decreased the caffeine-induced Ca2+ responses by 55 and 35%, respectively (Fig. 4, A and B). Maximal inhibition (85%) was obtained in cells injected with a mixture of as1RYR1 + 2 + 3 (Fig. 4A). Scrambled RYR3 antisense oligonucleotides did not affect significantly the Ca2+ responses evoked by caffeine (Fig. 4B). It is noteworthy that the upstroke velocities of the Ca2+ responses in as1RYR1- and as1RYR2-injected cells were not significantly different from that measured in control cells, whereas that in as1RYR3-injected cells was decreased by 65% (Fig. 4C). Although injection of as1RYR1 + 2 decreased the upstroke velocity of the caffeine-induced Ca2+ response, this inhibition was significantly less than that obtained in as1RYR3-injected cells (Fig. 4C). These results indicate that the RYR3 becomes activable by caffeine in cells preloaded with high [Ca2+]o. Since inhibition of RYR1 or RYR2 can be compensated by activation of RYR3, these results suggest that the biophysical properties of the RYR3 may be different from those of RYR1 and RYR2.



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Fig. 4.   Effects of RYR antisense oligonucleotides on caffeine-induced Ca2+ responses in 10 mM [Ca2+]o. Caffeine was applied at 10 mM. A and B, effects on the amplitude of caffeine-induced Ca2+ responses. Bars show means ± S.E. in noninjected control cells (open bars) and in cells injected with 10 µM asRYR1 or asRYR2 or with a mixture of as1RYR1 + 2 or as1RYR1 + 2 + 3 at 10 µM each (A) or with 10 µM asRYR3 or scrambled (Scr) asRYR3 (filled bars) (B). C, effects on the upstroke velocity of caffeine-induced Ca2+ responses. Bars show means ± S.E. in noninjected control cells (open bars) or in cells injected with 10 µM as1RYR1, as1RYR2, as1RYR3, or a mixture of as1RYR1 + 2 (filled bars). The number of cells tested is indicated in parentheses. Cells were obtained from six different batches. star , values significantly different from those obtained in noninjected cells. star  star , value in as1RYR1 + 2-injected cells significantly different from that in as1RYR3-injected cells. Myocytes were loaded with fluo 3-AM.

RYR Subtypes Involved in [Ca2+]i Jump- and Phenylephrine-induced Ca2+ Responses-- To determine the effects of a direct increase in [Ca2+]i on activation of the RYR subtypes, flash photolysis of DMNP-EDTA was used to instantaneously elevate the [Ca2+]i, as previously reported (28). As illustrated in Figs. 5 (A and B) and 6A, both amplitude and full time at half-maximal amplitude (FTHM) of the Ca2+ responses evoked by a flash pulse of 66 J were increased significantly in 10 mM Ca2+-pretreated cells when compared with control cells superfused in 1.7 mM [Ca2+]o. The full time at half-maximal amplitude increased from 0.41 ± 0.02 s (n = 21) in 1.7 mM [Ca2+]o to 0.56 ± 0.03 s (n = 27) in 10 mM [Ca2+]o. The increase in [Ca2+]i due to Ca2+ release from the caged molecule in response to a 66-J flash pulse was estimated in cells pretreated with 100 µM ryanodine for 20 min to block all of the RYRs (Fig. 6A). In cells injected with as2RYR3, the amplitude of the Ca2+ responses evoked by flash photolysis of caged Ca2+ in 10 mM [Ca2+]o was significantly decreased (Figs. 5C and 6B) and became similar to that obtained in control cells superfused in 1.7 mM [Ca2+]o (Figs. 5A and 6A). In contrast, inhibition of RYR1 or RYR2 by treatment with antisense oligonucleotides had no significant effects on the Ca2+ responses evoked by flash photolysis of caged Ca2+ in 10 mM [Ca2+]o (Fig. 6B). However, injection of as2RYR1 + 2 significantly attenuated the Ca2+-induced Ca2+ responses (Fig. 6B). To evaluate the Ca2+ sensitivity of the RYR subtypes to [Ca2+]i, the amplitude of the Ca2+ transients obtained from the entire line scan images was plotted as a function of flash intensity in control cells and after inhibition of the RYR subtypes in 10 mM [Ca2+]o. As expected, the Ca2+-induced increase in [Ca2+]i was reduced by treatment with as2RYR3 and as2RYR1 + 2 at all of the flash intensities tested (Fig. 7A). Maximal inhibition was obtained in cells pretreated with 100 µM ryanodine for 20 min (Fig. 7A). It was noted that the curve obtained in 1.7 mM [Ca2+]o was similar to that obtained in 10 mM [Ca2+]o after inhibition of RYR3s (Fig. 7A). The Ca2+ sensitivity of RYR subtypes was examined by plotting the ratio between the peak Ca2+ transients and the maximal Ca2+ transient, at different flash intensities in cells superfused in 10 mM [Ca2+]o, before and after inhibition of RYR1 + 2 or RYR3 alone (Fig. 7B). The curves were practically superimposed, suggesting that the Ca2+ sensitivities of the three RYR subtypes to [Ca2+]i could be similar. These results show that in Ca2+-overloaded cells, the RYR3 is also activable by local increases in [Ca2+]i.



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Fig. 5.   Effects of RYR3 antisense oligonucleotides on Ca2+ responses evoked by flash photolysis of caged Ca2+ in 10 mM [Ca2+]o. Shown are typical Ca2+ transients evoked by UV flashes of 66 J in control cells in 1.7 mM [Ca2+]o (A) or 10 mM [Ca2+]o (B) and in a cell injected with 10 µM as2RYR3 and superfused in 10 mM [Ca2+]o (C). Traces show (from top to bottom) line scan fluorescence image and averaged fluorescence from a 2-µm region (indicated by the small vertical bar) of the line scan image. Myocytes were loaded with fluo 3-AM and caged Ca2+ (DMNP-EDTA, AM).



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Fig. 6.   Compiled data showing the effect of RYR antisense oligonucleotides on the amplitude of Ca2+ responses evoked by flash photolysis (66 J) of caged Ca2+. A, bars show means ± S.E. in 1.7 mM [Ca2+]o and in 10 [Ca2+]o in noninjected control cells in the absence (open bars) or in the presence of 100 µM ryanodine (filled bar). B, bars show means ± S.E. in 10 mM [Ca2+]o in noninjected cells (open bars) and in cells injected with 10 µM as2RYR3, as2RYR1, as2RYR2, and a mixture of as2RYR1 + 2 (filled bars). Cells were obtained from four different cell batches. star , value in 10 mM [Ca2+]o significantly different from that in 1.7 mM [Ca2+]o. star  star , values significantly different from those obtained in noninjected cells in 10 mM [Ca2+]o. Cells were obtained from five different batches. Myocytes were loaded with fluo 3-AM and caged Ca2+ (DMNP-EDTA, AM).



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Fig. 7.   Ca2+ sensitivity of RYR subtypes in 10 mM [Ca2+]o. A, peak Ca2+ transients induced by flash-photolytic Ca2+ jumps were measured from the entire line scan image and plotted against U.V. flashes of increasing energy, in noninjected control cells in the absence () or in the presence of 100 µM ryanodine for 20 min (black-triangle), and in cells injected with as2RYR3 () or as2RYR1 + 2 (open circle ). black-square, value obtained in 1.7 mM [Ca2+]o in noninjected cells. Data are means ± S.E. for 10-12 cells obtained from five different batches. B, normalized curves obtained by plotting Delta (F/Fo)/max Delta (F/Fo) against flash intensity in noninjected control cells () and in cells injected with as2RYR3 () or as2RYR1 + 2 (open circle ). Fitted curves were obtained with the Boltzmann equation. Myocytes were loaded with fluo 3-AM and DMNP-EDTA, AM.

We have reported previously that norepinephrine may activate RYRs following Ca2+ release via inositol 1,4,5-trisphosphate-gated Ca2+ channels (30). In Ca2+-overloaded cells, both amplitude and upstroke velocity of the Ca2+ waves induced by 10 µM phenylephrine (alpha 1-adrenergic agonist) were increased (Fig. 8A). The mean upstroke velocity and amplitude of the phenylephrine-induced Ca2+ responses increased from 13.6 ± 2.4 (Delta F/Fo·s-1, n = 14) to 27.9 ± 4.1 (n = 10) and from 1.90 ± 0.14 (Delta F/Fo, n = 14) to 2.34 ± 0.08 (n = 20), respectively, in 1.7 mM [Ca2+]o and in 10 mM [Ca2+]o (Fig. 8B). In 1.7 mM [Ca2+]o, inhibition of the RYR3 subtype by treatment with antisense oligonucleotides had no significant effect on the amplitude and upstroke velocity of phenylephrine-induced Ca2+ waves (Fig. 8B). In contrast, in 10 mM [Ca2+]o, inhibition of RYR3 reduced both the amplitude and the upstroke velocity of the phenylephrine-induced Ca2+ waves (Fig. 8B). Interestingly, the amplitude and upstroke velocity of the phenylephrine-induced Ca2+ responses in RYR3-deficient cells pretreated in 10 mM [Ca2+]o were similar to those obtained in control cells superfused in 1.7 mM [Ca2+]o (Fig. 8B). Taken together, these results suggest that the RYR3 is responsible for the enhancement of the phenylephrine-induced Ca2+ wave in Ca2+-overloaded cells.



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Fig. 8.   Effects of RYR3 antisense oligonucleotides on Ca2+ responses induced by phenylephrine in 10 mM [Ca2+]o. A, typical Ca2+ waves induced by 10 µM phenylephrine in 1.7 mM [Ca2+]o (a) or 1 h after increasing [Ca2+]o to 10 mM (b). Traces show (from top to bottom) phenylephrine application and averaged fluorescence from a 2-µm region of the line scan image. B, compiled data showing the effects of as1RYR3 oligonucleotide on the amplitude and upstroke velocity of phenylephrine-induced Ca2+ responses. Phenylephrine was applied at 10 µM. Bars show means ± S.E. in 1.7 mM [Ca2+]o and in 10 mM [Ca2+]o, either in noninjected cells (open bars) or in cells injected with 10 µM as1RYR3 (filled bars). The number of cells tested is indicated in parentheses. Cells were obtained from four different batches. star , values significantly different from those obtained in noninjected cells in 10 mM [Ca2+]o. Myocytes were loaded with fluo 3-AM.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Growing evidence suggests that the activity of RYRs of the SR can be influenced by luminal Ca2+ (32-34), but the contribution of the different RYR subtypes in increased Ca2+ release has never been investigated. Based on the antisense oligonucleotide strategy, the present study shows that, in rat portal vein myocytes, the RYR3 becomes activable under conditions of increased SR Ca2+ loading and is responsible for the increase in Ca2+ release during caffeine- and neuromediator-induced global Ca2+ responses. We confirm the contribution of both RYR1 and RYR2 but not of RYR3 to Ca2+ sparks in Ca2+-overloaded myocytes. Immunologic detection of RYR3s revealed that these receptors were present in freshly dissociated and cultured portal vein myocytes. Since the cellular distribution of RYR3 was homogeneous in the cell sections, this observation supports the idea that RYR3s do not constitute Ca2+ release units.

Elevation of extracellular [Ca2+] created the conditions for an increased SR Ca2+ loading, as previously reported in cardiac myocytes (35, 36), except that a pretreatment of 1 h in 10 mM [Ca2+]o was needed for vascular myocytes to reach a steady state. We found that inhibition of Ca2+ accumulation into the SR by cyclopiazonic acid completely suppressed the caffeine-induced Ca2+ responses, whereas removal of external Ca2+ for 10 s had no effect on the increased caffeine-induced Ca2+ responses in 10 mM [Ca2+]o, indicating that they were strictly dependent on Ca2+ release from the SR.

Under the conditions of increased SR Ca2+ loading, the frequency of spontaneous Ca2+ sparks was notably increased. The number of vascular myocytes showing spontaneous Ca2+ sparks was enhanced from less than 25% of cells tested to more than 80%, and the number of initiation sites per line scan image was higher than 2. A similar increase in Ca2+ spark frequency has been reported after a SR Ca2+ overloading in ventricular myocytes (37). Other experiments performed in stomach smooth muscle cells have shown that the Ca2+ spark frequency is dependent on the luminal Ca2+ concentration (38), but the RYR subtypes involved in this modulation have not been identified. Inhibition of either RYR1 or RYR2 by treatment with antisense oligonucleotides strongly reduced the number of vascular myocytes with spontaneous Ca2+ sparks, whereas inhibition of RYR3 was ineffective. Interestingly, inhibition of both RYR1 and RYR2 was not additive compared with inhibition of one RYR subtype, in accordance with our previous data showing that both RYR1 and RYR2 are required for activation of Ca2+ sparks under conditions of normal Ca2+ loading (14). The fact that spots of fluorescence were not detected in cells stained with an anti-RYR3-specific antibody (20) after inhibition of both RYR1 and RYR2 by treatment with antisense oligonucleotides supports the idea that RYR3s do not form clustered units and, therefore, could not give rise to Ca2+ sparks. This is in contrast with recent data showing that Ca2+ sparks with nearly identical properties were obtained in both RYR1- and RYR3-null embryonic skeletal cells (26). Moreover, the characteristics of Ca2+ sparks (i.e. the mean amplitude, time-to-peak, full time at half-maximal amplitude, and full width at half-maximal amplitude) (Table I), measured in noninjected cells and in cells injected with asRYR3, were not significantly different, supporting the idea that only RYR1 and RYR2 are engaged in Ca2+ spark activity in vascular myocytes. Several possibilities can be proposed to explain how increased luminal Ca2+ concentration regulates RYRs activity and enhances the number of spontaneous Ca2+ sparks. First, increased luminal [Ca2+] may exert an allosteric modulation of RYRs involving a luminal regulatory site, as previously suggested in cardiac myocytes (32). Second, it may increase the conductance as well as the open probability of RYR1 and RYR2. Our observations that the Ca2+ spark parameters were not modified in 10 mM [Ca2+]o and the data obtained from single channel experiments showing that increased luminal [Ca2+] enhanced the number of openings rather than their duration (32) do not support this proposal. Third, it may directly activate silent Ca2+ release units. This possibility would be consistent with previous observations that increased luminal [Ca2+] activated only RYRs affected by other modulators such as ATP (33). At the present time, these three possibilities cannot be clearly distinguished.

The sensitivity of RYR subtypes to [Ca2+]i jumps was evaluated by releasing Ca2+ by flash photolysis of caged Ca2+. The absence of shift for the normalized curves obtained in 10 mM [Ca2+]o, in control cells and in asRYR1 + 2-injected or asRYR3-injected cells, suggests that the three RYR subtypes could have a similar sensitivity to activation by local increases in [Ca2+]i. We have previously estimated that the threshold Ca2+ level for activation of Ca2+ sparks was ~95 nM, a value that is not significantly different from the threshold for activation of the purified native RYR1 from rabbit skeletal muscle and of the RYR3 from rabbit uterus in HEK293 cells (18). However, other authors found a less sensitivity for Ca2+ ions of the RYR3 from skeletal muscle (17, 19, 20). This difference might be related to tissue-specific alternative splicing of RYR3, since rat portal vein myocytes expressed predominantly RYR3-I mRNA2 in contrast to mouse skeletal muscle, which expressed only RYR3-II mRNA (21). Although activation of RYR3s does not give rise to Ca2+ sparks in vascular myocytes, our results obtained with flash photolysis of caged Ca2+ and phenylephrine applications indicate that local increases in [Ca2+]i induced experimentally or in response to activation of inositol 1,4,5-trisphosphate-gated channels are sufficiently high to activate RYR3s under conditions of increased SR Ca2+ loading. Therefore, our results support the idea that RYR3 needs a previous increase in luminal [Ca2+] to be activable by local increases in [Ca2+]i. Furthermore, when cells were injected with either asRYR1 or asRYR2, both caffeine- and Ca2+-induced Ca2+ responses were not significantly affected, suggesting that activation of RYR3s might compensate for the inhibition of RYR1s or RYR2s. This observation is in good agreement with the fact that the single channel properties of the RYR3 differ from those of RYR1. For example, the maximal open probability of the RYR3 activated by Ca2+ alone is close to unity, whereas that of the RYR1 is about 0.2-0.5, and the RYR3 is about 10 times less sensitive to inactivation by high Ca2+ concentrations than the RYR1 (17, 18). Recent data have confirmed that the recombinant RYR3, expressed in HEK293 cells, is sensitive to nanomolar [Ca2+] in the presence of 1 mM ATP and does not present inactivation at high Ca2+ concentration (41). Our observations that the upstroke velocity of the caffeine-induced Ca2+ response in asRYR1 + 2 is higher than that obtained in asRYR3 is in accordance with the high open probability of RYR3 compared with that of the other RYR subtypes.

Functional cells regulate their Ca2+ homeostasis, in part, by Ca2+ uptake into the SR and Ca2+ efflux to the extracellular space through specific Ca2+-ATPases, suggesting that localized and transient Ca2+ overloads of the SR may be of physiological relevance. Recently, potentiation of excitation-contraction coupling in cardiac myocytes has been correlated with an increase in free SR Ca2+ content (42). Since both RYR1 and RYR2 once activated are precluded from rapid reactivation as a result of RYR adaptation (40) or inactivation by Ca2+ ions (39), it can be postulated that the RYR3, which has been reported to be resistant to high Ca2+ concentrations (17, 18, 41), may maintain Ca2+ release when the other RYR subtypes are closed.

In conclusion, these results show that, in vascular myocytes, RYR3 can be activated by caffeine and local increases in [Ca2+]i, under conditions of increased SR Ca2+ loading.


    ACKNOWLEDGEMENT

We thank N. Biendon for secretarial assistance.


    FOOTNOTES

* This work was supported by grants from Centre National de la Recherche Scientifique, Centre National des Etudes Spatiales, Région Aquitaine, Pôle Médicament-Santé and Association Française contre les Myopathies, France.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.

Dagger To whom correspondence should be addressed. Tel.: 33 5 57 57 12 31; Fax: 33 5 57 57 12 27; E-mail: jean.mironneau@umr5017.u-bordeaux2.fr.

Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M005994200

2 J. L. Morel, C. Le Sénéchal, and J. Mironneau, unpublished data.


    ABBREVIATIONS

The abbreviations used are: RYR, ryanodine receptor; SR, sarcoplasmic reticulum; DMNP-EDTA, AM, 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA, tetra(acetoxymethylester); fluo 3-AM, fluo 3- acetoxymethylester.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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