Contribution of ryanodine receptor subtype 3 to ca2+ responses in Ca2+-overloaded cultured rat portal vein myocytes.

Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca(2+) loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca(2+) 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 Ca(2+) measurements revealed that the number of cells showing spontaneous Ca(2+) sparks was strongly increased by superfusing the vascular myocytes in 10 mm Ca(2+)-containing solution. These Ca(2+) 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 Ca(2+) responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca(2+) responses in Ca(2+)-overloaded myocytes. Ca(2+) transients evoked by photolysis of caged Ca(2+) with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca(2+)](i) sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca(2+) loading, the RYR3 becomes activable by caffeine and local increases in [Ca(2+)](i).

Since the description of a Ca 2ϩ -induced Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ ; inhibited by Mg 2ϩ , ruthenium red, and millimolar concentrations of Ca 2ϩ ; and modulated by ryanodine (15)(16)(17). However, differences in responses to cyclic ADP-ribose and caffeine as well as in sensitivities to Ca 2ϩ activation and Ca 2ϩ inactivation have been reported between RYR1 and RYR3 and between RYR3s cloned from skeletal and smooth muscles (17)(18)(19)(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 Ca 2ϩ signaling has been addressed first by using either RYR1 and RYR2 knockout mice (12,22) or RYR3 knockout mice (23)(24)(25). In RYR1-null myotubes in culture, the Ca 2ϩ release from the sarcoplasmic reticulum (SR) in response to increases in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] 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 Ca 2ϩ 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 Ca 2ϩ release during Ca 2ϩ sparks (25). In addition, Ca 2ϩ 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 Ca 2ϩ sparks and global Ca 2ϩ responses in smooth muscle cells (14). It appears that both RYR1 and RYR2 are required for Ca 2ϩ release during Ca 2ϩ sparks and Ca 2ϩ waves induced by activation of L-type Ca 2ϩ currents and that RYR3 does not contribute to these Ca 2ϩ signals.
In smooth muscle cells, Ca 2ϩ sparks are observed spontaneously or in response to Ca 2ϩ influx through L-type Ca 2ϩ 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 Ca 2ϩ sparks results in propagating Ca 2ϩ waves, which trigger cell contraction (30).
In the present study, we investigated the effects of elevating the extracellular Ca 2ϩ concentration ([Ca 2ϩ ] o ) on both Ca 2ϩ sparks and global Ca 2ϩ responses induced by Ca 2ϩ , caffeine, and phenylephrine. Under conditions of increased SR Ca 2ϩ loading, we provide the first evidence that the RYR3, which becomes activable by caffeine and localized increases in [Ca 2ϩ ] i , is responsible for the increased global Ca 2ϩ responses. We also found that spontaneous Ca 2ϩ sparks are highly abundant but remain dependent on activation of only RYR1 and RYR2.

EXPERIMENTAL PROCEDURES
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 Ca 2ϩ (40 M) 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 10 3 cells/mm 2 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 g/ml streptomycin; they were kept in an incubator gassed with 95% air and 5% CO 2 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 MgCl 2 , 1.7 mM CaCl 2 , 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 GenBank™ 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 GenBank™ 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 GCAATCCGCTC-CCGCCCA, corresponding to nucleotides 325-343 and 584 -601, respectively, of RYR1 cDNA deposited in GenBank TM (accession number X83932); those of as1RYR2 and as2RYR2 are GTGTCCTCACA-GAAGTT and TGAAATCTAGTGCAGCCT, corresponding to nucleotides 137-153 and 1587-1604, respectively, of RYR2 cDNA (accession number X83933); and those of as1RYR3 and as2RYR3 are AAGT-CAAGGGCATTTTTG 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: CACGCCTACG-CACCTCCG, 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 Ca 2ϩ Measurements-In most experiments, cells were loaded by incubation in physiological solution containing 4 M 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 ϫ 60, 1.4 NA objective lens. Fluo 3 was excited at 488 nm, and emitted fluorescence was filtered and measured at 540 Ϯ 30 nm. At the setting used to detect fluo 3 fluorescence, the resolution of the microscope was near 0.4 ϫ 0.4 ϫ 1.5 m (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/F o ), where F is the fluorescence during a response and F o 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 M indo 1-AM for 30 min. [Ca 2ϩ ] i measurements were estimated from the 405-/480-nm fluorescence ratio, as previously reported (31). The minimum and maximum fluorescence (R min and R max , respectively) values were determined in vivo, in the absence of Ca 2ϩ and at saturating Ca 2ϩ , in cells superfused in 1.7 and 10 mM [Ca 2ϩ ] 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 Ϯ 1°C.
Flash Photolysis-Caged Ca 2ϩ , 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA, tetra(acetoxymethylester) (DMNP-EDTA, AM) at 15 M, 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 ϳ2-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, Ca 2ϩ was released within 2 ms, and the small percentage of conversion of the caged compound (ϳ10%) allowed us to apply repetitive pulses without altering the Ca 2ϩ responses and the reserve of caged Ca 2ϩ .
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 Ϯ S.E.; n represents the number of tested cells. Significance was tested by means of Student's test. p values Ͻ 0.05 were considered as significant.  (Fig. 1B). Taken together, these results suggest that the SR Ca 2ϩ content of vascular myocytes is increased by sustained elevation in extracellular [Ca 2ϩ ] and that both amplitude and upstroke velocity of the Ca 2ϩ responses can be used as significant parameters to study the cellular mechanisms involved during increased SR Ca 2ϩ loading.

Effects of High Ca 2ϩ -containing Solution on the Caffeineinduced Ca 2ϩ Responses in Vascular
To assess the role of the SR Ca 2ϩ loading in the generation of large and fast Ca 2ϩ responses to caffeine, the effects of 10 M cyclopiazonic acid were first investigated on the caffeine-induced Ca 2ϩ responses. Inhibition of the Ca 2ϩ uptake capacity of the intracellular store by cyclopiazonic acid resulted in a small elevation of the basal [Ca 2ϩ ] i and the suppression of the caffeine-induced Ca 2ϩ response in the continuous presence of cyclopiazonic acid for 5 min (n ϭ 6). In a second set of experiments, caffeine (10 mM) was applied in Ca 2ϩ -free, 0.5 mM EGTA-containing solution for 10 s (a time sufficient to remove voltage-dependent Ca 2ϩ current) on myocytes superfused either in 1 (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 ϩ as1RYR2 ϩ as1RYR3 (10 M each) to inhibit the Ca 2ϩ waves induced by 10 mM caffeine in isolated myocytes superfused in 10 mM [Ca 2ϩ ] o for 1 h. The Ca 2ϩ responses were strongly inhibited 3 days after nuclear injection of the antisense oligonucleotides (83 Ϯ 5%, n ϭ 30); recovery began the fourth day with an inhibition of 48 Ϯ 5% (n ϭ 25). Nonspecific effects of antisense oligonucleotides were detected only at concentrations higher than 50 M (for example, inhibition of RYR2 protein expression by 50 M anti-G␣ 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 ϩ 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).  (14). Since elevation of luminal [Ca 2ϩ ] has been suggested to increase the activity of RYRs (32), we studied the parameters of spontaneous Ca 2ϩ sparks in Ca 2ϩ -overloaded cells. In control cells, superfused in 1.7 mM [Ca 2ϩ ] o , spontaneous Ca 2ϩ 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 Ϯ 0.1 (n ϭ 7). In 10 mM [Ca 2ϩ ] o , spontane-  (Table  I).

RYR Subtypes Involved in
When cells were injected with asRYR1, asRYR2, or asRYR1 ϩ 2, the number of cells with spontaneous Ca 2ϩ sparks was strongly decreased (Fig. 3A). In contrast, the number of cells with spontaneous Ca 2ϩ 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 Ca 2ϩ content conditions, Ca 2ϩ sparks are due to activation of both RYR1 and RYR2 and that RYR3 does not contribute to triggering Ca 2ϩ sparks. In addition, the spatio-temporal parameters of Ca 2ϩ sparks in 10 mM [Ca 2ϩ ] o were not significantly different in noninjected cells and in cells injected with asRYR3 (Table I). Taken together, these results suggest that increased SR Ca 2ϩ loading potentiates the activity of Ca 2ϩ release units formed by RYR1 and RYR2, leading to an increase in Ca 2ϩ spark frequency without alterations of the spatio-temporal parameters.  (14). Consequently, inhibition of RYR1 or RYR2 by treatment with antisense oligonucleotides partly inhibited the caffeine-induced Ca 2ϩ 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 Ca 2ϩ responses under conditions of increased SR Ca 2ϩ loading. Cells injected with asRYR1 or asRYR2 evoked Ca 2ϩ responses to 10 mM caffeine that were similar to those elicited in control cells superfused in 10 mM [Ca 2ϩ ] o (Fig. 4A). However, injection of both as1RYR1 ϩ as1RYR2 and injection of asRYR3 alone significantly decreased the caffeine-induced Ca 2ϩ responses by 55 and 35%, respectively (Fig. 4, A and B). Maximal inhibition (85%) was obtained in cells injected with a mixture of as1RYR1 ϩ 2 ϩ 3 (Fig. 4A). Scrambled RYR3 antisense oligonucleotides did not affect significantly the Ca 2ϩ responses evoked by caffeine (Fig. 4B). It is noteworthy that the upstroke velocities of the Ca 2ϩ 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 ϩ 2 decreased the upstroke velocity of the caffeine-induced Ca 2ϩ response, this inhibition was significantly less than that obtained in as1RYR3-injected cells (Fig. 4C) (28). As illustrated in Figs. 5 (A and B) (Fig. 6B). However, injection of as2RYR1 ϩ 2 significantly attenuated the Ca 2ϩ -induced Ca 2ϩ responses (Fig. 6B). To evaluate the Ca 2ϩ sensitivity of the RYR subtypes to [Ca 2ϩ ] i , the amplitude of the Ca 2ϩ 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 [Ca 2ϩ ] o . As expected, the Ca 2ϩinduced increase in [Ca 2ϩ ] i was reduced by treatment with as2RYR3 and as2RYR1 ϩ 2 at all of the flash intensities tested (Fig. 7A). Maximal inhibition was obtained in cells pretreated with 100 M ryanodine for 20 min (Fig. 7A). It was noted that the curve obtained in 1.7 mM [Ca 2ϩ ] o was similar to that obtained in 10 mM [Ca 2ϩ ] o after inhibition of RYR3s (Fig. 7A). The Ca 2ϩ sensitivity of RYR subtypes was examined by plotting the ratio between the peak Ca 2ϩ transients and the maximal Ca 2ϩ transient, at different flash intensities in cells superfused in 10 mM [Ca 2ϩ ] o , before and after inhibition of RYR1 ϩ 2 or RYR3 alone (Fig. 7B). The curves were practically superimposed, suggesting that the Ca 2ϩ sensitivities of the three RYR subtypes to [Ca 2ϩ ] i could be similar. These results show that in Ca 2ϩ -overloaded cells, the RYR3 is also activable by local increases in [Ca 2ϩ ] i .

RYR Subtypes Involved in Caffeine-induced
We have reported previously that norepinephrine may activate RYRs following Ca 2ϩ release via inositol 1,4,5-trisphosphate-gated Ca 2ϩ channels (30). In Ca 2ϩ -overloaded cells, both amplitude and upstroke velocity of the Ca 2ϩ waves induced by 10 M phenylephrine (␣ 1 -adrenergic agonist) were increased (Fig. 8A). The mean upstroke velocity and amplitude of the phenylephrine-induced Ca 2ϩ responses increased from 13.   rine-induced Ca 2ϩ waves (Fig. 8B). Interestingly, the amplitude and upstroke velocity of the phenylephrine-induced Ca 2ϩ responses in RYR3-deficient cells pretreated in 10 mM [Ca 2ϩ ] o were similar to those obtained in control cells superfused in 1.7 mM [Ca 2ϩ ] o (Fig. 8B). Taken together, these results suggest that the RYR3 is responsible for the enhancement of the phenylephrine-induced Ca 2ϩ wave in Ca 2ϩ -overloaded cells. DISCUSSION Growing evidence suggests that the activity of RYRs of the SR can be influenced by luminal Ca 2ϩ (32)(33)(34), but the contribution of the different RYR subtypes in increased Ca 2ϩ 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 Ca 2ϩ loading and is responsible for the increase in Ca 2ϩ release during caffeine-and neuromediator-induced global Ca 2ϩ responses. We confirm the contribution of both RYR1 and RYR2 but not of RYR3 to Ca 2ϩ sparks in Ca 2ϩoverloaded 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 Ca 2ϩ release units.
Elevation of extracellular [Ca 2ϩ ] created the conditions for an increased SR Ca 2ϩ loading, as previously reported in cardiac myocytes (35,36), except that a pretreatment of 1 h in 10 mM [Ca 2ϩ ] o was needed for vascular myocytes to reach a steady state. We found that inhibition of Ca 2ϩ accumulation into the SR by cyclopiazonic acid completely suppressed the caffeineinduced Ca 2ϩ responses, whereas removal of external Ca 2ϩ for 10 s had no effect on the increased caffeine-induced Ca 2ϩ responses in 10 mM [Ca 2ϩ ] o , indicating that they were strictly dependent on Ca 2ϩ release from the SR.
Under the conditions of increased SR Ca 2ϩ loading, the frequency of spontaneous Ca 2ϩ sparks was notably increased. The number of vascular myocytes showing spontaneous Ca 2ϩ 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 Ca 2ϩ spark frequency has been reported after a SR Ca 2ϩ overloading in ventricular myocytes (37). Other experiments performed in stomach smooth muscle cells have shown that the Ca 2ϩ spark frequency is dependent on the luminal Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ sparks under conditions of normal Ca 2ϩ 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 Ca 2ϩ sparks. This is in contrast with recent data showing that Ca 2ϩ sparks with nearly identical properties were obtained in both RYR1-and RYR3-null embryonic skeletal cells (26). Moreover, the characteristics of Ca 2ϩ 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 Ca 2ϩ spark activity in vascular myocytes. Several possibilities can be proposed to explain how increased luminal Ca 2ϩ concentration regulates RYRs activity and enhances the number of spontaneous Ca 2ϩ sparks. First, increased luminal [Ca 2ϩ ] may exert an allosteric modulation of RYRs involving a luminal regulatory site, as previously suggested in cardiac myocytes (32 We have previously estimated that the threshold Ca 2ϩ level for activation of Ca 2ϩ sparks was ϳ95 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 Ca 2ϩ 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 mRNA 2 in contrast to mouse skeletal muscle, which expressed only RYR3-II mRNA (21). Although activation of RYR3s does not give rise to Ca 2ϩ sparks in vascular myocytes, our results obtained with flash photolysis of caged Ca 2ϩ and phenylephrine applications indicate that local increases in [Ca 2ϩ ] i induced experimentally or in response to activation of inositol 2 J. L. Morel, C. Le Sénéchal, and J. Mironneau, unpublished data. 1,4,5-trisphosphate-gated channels are sufficiently high to activate RYR3s under conditions of increased SR Ca 2ϩ loading. Therefore, our results support the idea that RYR3 needs a previous increase in luminal [Ca 2ϩ ] to be activable by local increases in [Ca 2ϩ ] i . Furthermore, when cells were injected with either asRYR1 or asRYR2, both caffeine-and Ca 2ϩ -induced Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ concentrations than the RYR1 (17,18). Recent data have confirmed that the recombinant RYR3, expressed in HEK293 cells, is sensitive to nanomolar [Ca 2ϩ ] in the presence of 1 mM ATP and does not present inactivation at high Ca 2ϩ concentration (41). Our observations that the upstroke velocity of the caffeine-induced Ca 2ϩ response in asRYR1 ϩ 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 Ca 2ϩ homeostasis, in part, by Ca 2ϩ uptake into the SR and Ca 2ϩ efflux to the extracellular space through specific Ca 2ϩ -ATPases, suggesting that localized and transient Ca 2ϩ 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 Ca 2ϩ content (42). Since both RYR1 and RYR2 once activated are precluded from rapid reactivation as a result of RYR adaptation (40) or inactivation by Ca 2ϩ ions (39), it can be postulated that the RYR3, which has been reported to be resistant to high Ca 2ϩ concentrations (17,18,41), may maintain Ca 2ϩ 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 [Ca 2ϩ ] i , under conditions of increased SR Ca 2ϩ loading.