In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline– and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3−/−) mice, hypoxia-induced, but not submaximal noradrenaline–induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3−/− mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline– and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline–induced Ca2+ and contractile responses in PASMCs.

Introduction

Ca2+ release from the sarcoplasmic/endoplasmic reticulum (SR) through the intracellular Ca2+ channels inositol 1,4,5-triphosphate receptors (IP3Rs) and RyRs is critical for numerous cellular responses. The functional redundancy of these channels substantially complicates the understanding of the physiological role of RyRs in vascular smooth muscle cells (SMCs). We have recently shown that activation of RyRs with caffeine induces Ca2+ release and prevents subsequent neurotransmitter-induced responses, and vice versa, in freshly isolated rat pulmonary artery SMCs (Wang et al., 2003), indicating that RyRs and IP3Rs may be functionally colocalized in the same SR. Similar functional colocalization of both receptors in the same SR has also been found in cultured rat pulmonary artery smooth muscle cells (PASMCs) (Zhang et al., 2003) and other types of vascular myocytes (Leijten and van Breemen, 1984; Amedee et al., 1990; Pacaud and Loirand, 1995; Boittin et al., 1999; Janiak et al., 2001). These findings, together with the fact that Ca2+ activates RyRs and induces Ca2+ release from the SR, a process termed as Ca2+-induced Ca2+ release (CICR), raise a possibility that Ca2+ released from IP3Rs may open the neighboring RyRs and then induce further Ca2+ release, contributing to neurotransmitter-induced Ca2+ and contractile responses in PASMCs. In support of this hypothesis, a recent study performed in cultured PASMCs has shown that photorelease of caged IP3 can stimulate RyR-mediated Ca2+ sparks (Zhang et al., 2003). However, it should be noted that cell culture may significantly affect expression levels and functions of RyRs in vascular SMCs (Masuo et al., 1991; Cortes et al., 1997; Vallot et al., 2000; Thorne and Paul, 2003).

It is well known that hypoxia exposure induces pulmonary, but not systemic (mesenteric and cerebral) artery vasoconstriction (Madden et al., 1992; Vadula et al., 1993; Yuan et al., 1993; Wang et al., 2003). This unique hypoxia-induced pulmonary vasoconstriction serves as an important physiological process for maintaining arterial oxygenation. We and other investigators have recently shown that the depletion of SR Ca2+ with caffeine (through activation of RyRs) reduces or abolishes hypoxia-induced increases in [Ca2+]i and contraction in PASMCs (Salvaterra and Goldman, 1993; Post et al., 1995; Jabr et al., 1997; Dipp et al., 2001; Wang et al., 2003). Similarly, ryanodine, an agent that binds with high affinity to RyRs, largely or completely inhibits hypoxic responses (Vadula et al., 1993; Jabr et al., 1997; Dipp et al., 2001; Morio and McMurtry, 2002). These data suggest that RyRs possibly play an important role in hypoxic responses in PASMCs.

Three subtypes of RyRs (RyR1, RyR2, and RyR3) are expressed in mammalian cells, each encoded by distinct genes. However, studies on mRNA expression of RyR subtypes in cultured vascular SMCs or vascular tissues have yielded conflicting results, showing expression of RyR1, RyR2, and RyR3 all (Neylon et al., 1995; Coussin et al., 2000; Lohn et al., 2001), abundant RyR3, little RyR2, and no RyR1 (Ledbetter et al., 1994; Vallot et al., 2000), as well as RyR1 only (Vallot et al., 2000). These conflicting data may occur due to the changes in RyR expression patterns and levels during cell culture or due to the contamination with other types of cells in experiments using vascular tissues (Vallot et al., 2000). Nonetheless, little is known about expression patterns and levels of RyR subtypes in PASMCs.

Using the antisense oligonucleotide technique to selectively suppress the expression of RyR subtypes, Coussin et al. (2000) have shown that RyR1 and RyR2, but not RyR3, participate in Ca2+ release following activation of voltage-dependent Ca2+ currents (ICa) in cultured portal vein SMCs. However, studies using freshly isolated portal vein and coronary artery myocytes indicate that ICa fails to induce Ca2+ release (Ganitkevich and Isenberg, 1995; Kamishima and McCarron, 1996). It has been reported that caffeine- and noradrenaline-induced arterial muscle contractions are not significantly changed in RyR3 knockout (RyR3−/−) mice (Takeshima et al., 1996). In contrast, a recent report has shown that the frequency of Ca2+ sparks and STOCs (spontaneous transient outward currents) are increased in RyR3−/− mouse cerebral artery myocytes (Lohn et al., 2001). These discrepancies possibly reflect differential expression patterns and levels of RyR subtypes in systemic artery SMCs, in addition to different experimental conditions. Moreover, physiological functions of RyR subtypes in PASMCs are largely unknown.

In the present study, we first sought to determine physiological functions of RyRs in freshly isolated PASMCs by examining whether these Ca2+ release channels were involved in noradrenaline- and hypoxia-induced increases in [Ca2+]i and contraction using several structurally different RyR blockers. Next we examined mRNA and protein expression patterns and levels of RyR subtypes in freshly isolated PASMCs using real-time quantitative RT-PCR and immunofluorescence staining. Finally, we examined whether RyR3 gene knockout affected noradrenaline- and hypoxia-induced Ca2+ and contractile responses in freshly isolated PASMCs. Our data indicate that RyRs may amplify neurotransmitter-induced Ca2+ release and contraction in PASMCs, which likely occurs through a local IP3R–RyR interaction. These Ca2+ release channels also play an important role in hypoxia-induced Ca2+ release and contraction in PASMCs. All three subtypes of RyRs are expressed in PASMCs, and the amount of mRNA transcript is equivalent. RyR3 gene knockout significantly inhibits hypoxia-, but not noradrenaline-induced Ca2+ release and contraction in PASMCs, suggesting a unique function for this RyR subtype.

Materials And Methods

Cell Preparation

Freshly isolated cells were obtained from rat resistance (external diameter < 300 μm) pulmonary arteries, since these regional cells show stronger responses to hypoxia (Madden et al., 1985, 1992; Archer et al., 1996). The procedure for cell isolation was similar to that described previously (Wang et al., 2003). Under approved animal care and use protocols, Sprague-Dawley rats were killed by an intraperitoneal injection of sodium pentobarbital (150 mg/kg). Resistance pulmonary arteries were removed and cleaned of the connective tissue and endothelium. Small pieces (1 × 10 mm) of the arteries were incubated in nominally Ca2+-free physiological saline solution (PSS) containing papain (Sigma-Aldrich) and dithioerythritol at 37°C for 20 min, then in nominally Ca2+-free PSS containing type H and II collagenase (Sigma-Aldrich) and 100 μM Ca2+ for 10–15 min (37°C), and finally in ice cold nominally Ca2+-free PSS for 15 min. Single cells were harvested by gentle trituration, and then stored on ice for use up to 8 h. PSS contained (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 10 HEPES, 1.8 CaCl2, and 10 glucose (pH 7.4).

RyR3 knockout (RyR3−/−) mice were obtained as described previously (Bertocchini et al., 1997), and maintained as heterozygotes, which were continually backcrossed to generate RyR3−/− and wild-type (RyR3+/+) mice. Freshly isolated resistance (third and smaller branches) PASMCs from RyR3−/− and RyR3+/+ (same age and sex) mice were prepared using the same procedure as described above.

Cultured human PASMCs were purchased from Cambrex and cultured in smooth muscle growth medium in a humidified atmosphere of 5% CO2 in air at 37°C. The medium contained 5% FBS, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin. Cells at the fifth and sixth passage were used for experiments.

Measurements of Whole-cell [Ca2+]i

Measurements of whole-cell [Ca2+]i were made by a dual excitation wavelength fluorescence method as described previously (Wang et al., 2003), using the IonOptix fluorescence photometric system (Milton). PASMCs were loaded with 4 μM fura-2/AM for 20 min. The dye was excited at 340- and 380-nm wavelengths (Xenon 75 W arc lamp). The emission fluorescence at 510 nm was detected by a photomultiplier tube. Photo bleaching was minimized by using neutral density filters and by shuttering excitation light between sampling periods. Background fluorescence was determined by removing the cell from the field after the experiment.

Membrane Current Recording

Whole-cell membrane currents were measured using nystatin-perforated or standard patch clamp techniques (Wang et al., 2003) with a patch clamp amplifier (EPC-9; Heka Electronics). For the perforated patch clamp experiments, patch pipettes filled with intracellular solution had a resistance of 2–3 MΩ. Nystatin was included in the pipette solution at a final concentration of 250 μg/ml. When electrical access was detected, cells were clamped at a holding potential of −55 mV. Membrane capacitance and series resistance were continuously monitored and compensated, and experiments initiated following a decrease in the access resistance to <40 MΩ. In some experiments, the standard whole-cell technique was used to dialyze the tested reagents into the cell. In this case, the resistance of the patch pipettes was 1–2 MΩ. The pipette solution contained (in mM) 125 CsCl, 5 NaCl, 1.2 MgCl2, 3 EGTA, 1 CaCl2, and 10 HEPES (pH 7.3) for perforated patch clamp experiments, and 125 CsCl (or KCl), 5 NaCl, 1.2 MgCl2, 3 ATP-Mg, and 10 HEPES (pH 7.3) for standard whole-cell recordings.

Reverse Transcriptase (RT) PCR

The expression of RyR mRNAs in cultured human PASMCs was examined using RT-PCR. Total RNAs were isolated using Absolutely RNA Miniprep Kit (Stratagene). One-step RT-PCR was performed using SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen) according to the manufacturer's instructions, supplemented with the following forward and reverse oligonucleotide primers: 5′-GGCTGGTATATGGTGATGTC-3′ and 5′-TCGCTCTTGTTGTAGAACTTGCGG-3′for human RyR1 gene, 5′-ACAGCATGGCCCTTTACAAC-3′ and 5′-TTGGCTTTCTCTTTGGCTGT-3′ for human RyR2 gene, and 5′-CTGTCATCTGTAACTCACAA-3′ and 5′-GTGTCAAAGTAGTCATTGCCAA-3′for human RyR3 gene. The amplified products were electrophoresed on an agarose gel, stained with ethidium bromide, and visualized by UV illumination.

For real-time quantitative RT-PCR experiments, freshly isolated rat resistance PASMCs were used to yield total RNAs to avoid the contamination of neurons, endothelial and other types of cells. Isolated cells were collected using a Burleigh PCS-5300 manipulator under the help of a Nikon inverted microscope. Total RNAs were obtained using Absolutely RNA Nanoprep Kit (Stratagene). The first-strand cDNAs were synthesized from RNAs using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). The resultant cDNAs were amplified by specific target gene forward and reverse primers: 5′-TCTTCCCTGCTGGAGACTGT-3′ and 5′-TGGGAGAAGGCACTTGAGG-3′ for rat RyR1 gene, 5′-ACAACCCAAATGCTGGTCTC-3′ and 5′-TCCGGTTCAGACTTGGTTTC-3′ for rat RyR2 gene, and 5′-CTGGCCATCATTCAAGGTCT-3′ and 5′-GTCTCCATGTCTTCCCGTA-3′ for rat RyR3 gene, with the iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc.) using an iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, Inc.). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. To quantify the target gene mRNA levels, known RyR1, RyR2, RyR3, and GADPH DNAs were used for constructing standard curves. Known DNAs and unknown sample cDNAs at series dilutions (1:10) were simultaneously amplified. Real-time PCR was run for one cycle at 95°C for 3 min followed immediately by 40 cycles at 95°C for 20 s, 58°C (to be varied with different primers) for 20 s, and 72°C for 20 s. The fluorescence was measured after each of the repetitive cycles. A melting point dissociation curve generated by the instrument was used to confirm that only a single product was present. To validate the specificity, no reverse transcriptase and no template control experiments were performed to confirm no fluorescence resulting from either genomic DNA contamination or PCR step. The PCR amplification products were also verified by electrophoresis and sequencing analysis. The absolute expression levels of subtype RyR mRNAs in cells were calculated from standard curves of known DNAs. The absolute mRNA expression levels of target genes were also normalized to levels of GADPH mRNAs to yield the relative levels of target genes.

Immunofluorescence Staining

The experimental protocol was similar to that described previously (Zhang et al., 2001). In brief, freshly isolated rat PASMCs were fixed in 4% paraformaldehyde in PSS for 15 min at room temperature, incubated with 0.2% Triton X-100 in PSS for 30 min, and then blocked for 1 h with 2.5% BSA PSS. After that, cells were incubated with specific primary antibody at 4°C overnight, followed by Alexa488- or Alexa594-conjugated anti-mouse or anti-rabbit antibody (1:750 dilution) (according to the host species of primary antibody) for 2 h. Immunofluorescence staining was examined using Zeiss LSM510 laser scanning confocal microscope. The z interval was adjusted to 1 μm to obtain sufficient fluorescence signals. Alexa488 and Alexa594 were excited at 488 and 543 nm using a krypton-argon laser, and fluorescence was detected using 505 and 585-nm bandpass filters, respectively.

Smooth Muscle Tension Measurements

The third branches of rat or mouse pulmonary arteries were sectioned into segments 3 mm in length, and placed in 2-ml tissue baths (Radnoti). One end of the muscle strip was fixed to a small clip, and the other end connected to a highly sensitive force transducer (Harvard Apparatus). The bath solution contained (in mM): 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose (pH 7.4), aerated with 20% O2, 5% CO2, and 75%N2, and warmed at 35°C. The strips were set at a resting tone of 250 mg. Contractile force was recorded using a PowerLab/4SP recording system (AD Instruments). In experiments examining noradrenaline-induced muscle contraction, concentration–response curves were constructed by fitting the mean values of data with Origin Version 7 software (OriginLab Corporation), using the nonlinear Boltzmann equation: y = A2 + (A1 − A2)/(1 + exp((x − x50)/dx)), where A1 was minimal noradrenaline-induced contraction, A2 maximal contraction, x the logarithm of noradrenaline concentration, x50 the logarithm of concentration for half-maximal contraction (EC50), and dx the slope factor of the curve.

Hypoxia

In experiments using freshly isolated PASMCs, hypoxia was achieved by switching a perfusing solution from a bath solution equilibrated with 20% O2, 5% CO2, and balance with N2 (normoxic) to a solution equilibrated with 1% O2, 5% CO2, and balance with N2 mixtures (hypoxic). For muscle strip experiments, hypoxia was achieved by switching the bath solution gas mixtures from 20% O2, 5% CO2, and 75% N2 to 1% O2, 5% CO2, and 94% N2. The oxygen tension of the solution was continuously monitored by means of an oxygen electrode (OXEL-1, WPI). The bath Po2 was ≥140 and 10–20 Torr in the normoxic and hypoxic solution, respectively (Wang et al., 2000).

Reagents and Antibodies

Alexa488- and Alexa594-conjugated anti-mouse and anti-rabbit antibodies, as well as fura-2/AM were obtained from Molecular Probes; IP3 and ruthenium red from Calbiochem; anti-RyR1 antibody from Upstate Biotechnology; anti-actin (smooth muscle) antibody, anti-myosin (smooth) antibody, caffeine, chloro-m-cresol, dantrolene, noradrenaline, nystatin, ryanodine, and tetracaine from Sigma-Aldrich. Anti-RyR2 and anti-RyR3 antibodies were provided by A.F. Lai (University of Wales, Cardiff, UK). The isoform-specific RyR antibodies were tested for confirmation of their specificity.

Data Analysis

All values were expressed as means ± SEM of n samples investigated. Student's t test was used to determine the significance of differences between two observations, whereas one-way ANOVA was used for multiple comparisons. A P value of <0.05 was considered to be significant.

Results

Ryanodine Receptors and Inositol Triphosphate Receptors Functionally Overlap in PASMCs

Previous studies have shown that activation of RyRs with caffeine, similar to activation of IP3Rs with noradrenaline, induces Ca2+ release from the SR in freshly isolated canine and rat PASMCs (Janiak et al., 2001; Wang et al., 2003). Here we sought to compare the extent of Ca2+ release following maximal activation of RyRs and IP3Rs. Freshly isolated rat PASMCs were loaded with fura-2/AM and voltage clamped at −55 mV using the perforated patch clamp technique. Cs+ patch pipette solution was used to block outward K+ currents. Under these conditions, application of caffeine (10 mM) induced an increase in [Ca2+]i and an inward Ca2+-activated Cl current (ICl(Ca)) (Wang et al., 1997b, 2003). A typical example of these experiments is shown in Fig. 1 A. In a total of 16 cells tested, the mean increase in [Ca2+]i was 702 ± 27 nM (from a resting level of 112 ± 17 to 815 ± 29 nM), and the current had a mean amplitude of 581 ± 42 pA (Fig. 1 B). Stimulation of adrenergic receptors with noradrenaline (300 μM) to activate IP3Rs induced Ca2+ and current responses with similar amplitudes to caffeine-induced responses (Fig. 1 A). The mean increase in [Ca2+]i following application of noradrenaline was 674 ± 43 nM, and mean amplitude of ICl(Ca) was 613 ± 33 pA (n = 17) (Fig. 1 B). Thus, maximal activation of RyRs with caffeine and IP3Rs with noradrenaline induces equivalent Ca2+ release and associated ICl(Ca), which is consistent with previous findings (Janiak et al., 2001; Wang et al., 2003) and suggests that RyRs are highly expressed in PASMCs.

Our recent study has shown that prior application of noradrenaline blocks subsequent caffeine-induced Ca2+ release in freshly isolated rat PASMCs, and vice versa, indicating that both RyRs and IP3Rs may functionally overlap (Wang et al., 2003). To further test this view, we examined the effect of IP3 on caffeine-induced Ca2+ release in freshly isolated rat PASMCs. In these experiments, voltage-clamped (−55 mV) myocytes were first dialyzed with IP3 to induce Ca2+ release through IP3Rs selectively, and then exposed to caffeine. As shown in Fig. 2 A, dialysis of IP3 induced a Ca2+-activated Cl current (Ca2+ release). The mean amplitude of the currents was 507 ± 52 pA (n = 5). However, subsequent application of caffeine failed to induce any response in all five cells tested. In converse experiments, cells were first exposed to caffeine and then dialyzed with IP3. Caffeine-induced Ca2+ release was assessed by examining the activity of single Ca2+-activated K+ (KCa) channels under cell-attached patch clamp conditions, using K+ patch pipette solution. Application of caffeine (10 mM) caused a burst increase in activity of single KCa channels in a cell (Fig. 2 B). The mean current amplitude in a total of five cells was 23 ± 4 pA. Following the caffeine response, the cell membrane was ruptured to obtain the whole-cell patch clamp configuration, and IP3 (100 μM) was dialyzed into the cell through the patch pipette. However, dialysis of IP3 failed to induce any current in the continued presence of caffeine. These results are consistent with our previous findings (Wang et al., 2003), further indicating that both RyRs and IP3Rs functionally overlap in rat PASMCs.

It has been reported that caffeine may inhibit IP3-induced Ca2+-activated Cl currents (Ca2+ release) in Xenopus oocytes (Parker and Ivorra, 1991), cerebellum IP3Rs reconstituted in planar lipid bilayers (Bezprozvanny et al., 1994), and IP3-induced Ca2+ release in permeabilized A7r5 cells in the absence of ATP (Missiaen et al., 1994). To exclude the possibility that IP3 activity was blocked by caffeine, we examined the effect of chloro-m-cresol (CMC), another RyR activator without reported actions on IP3Rs, on noradrenaline-induced increase in [Ca2+]i. Application of CMC (1 mM) induced an increase in [Ca2+]i in freshly isolated rat PASMCs. The mean increase in [Ca2+]i by CMC was 755 ± 67 nM (n = 6). In the continued presence of CMC, however, application of noradrenaline (300 μM) no longer evoked Ca2+ release in six cells tested. On the other hand, application of noradrenaline induced an increase in [Ca2+]i with a mean amplitude of 686 ± 70 nM, and prevented subsequent CMC-induced responses in seven cells tested.

It is known that cell culture may result in significant changes in RyR expression levels and patterns in vascular SMCs, as caffeine fails to induce Ca2+ release (Masuo et al., 1991; Cortes et al., 1997; Vallot et al., 2000). Thus, we sought to utilize the cultured human PASMCs as an alternative approach to examine if caffeine could inhibit neurotransmitter-induced Ca2+ release. Application of caffeine (10 mM) could not evoke an increase in [Ca2+]i in a cultured human PASMC, although subsequent application of ATP (100 μM) induced a large response in the same cell (Fig. 3 A). Similar observations were obtained from six other cells. Moreover, CMC (1 mM) was also unable to induce any Ca2+ release (n = 6). We then examined the expression of RyRs in cultured human PASMCs using RT-PCR. As shown in Fig. 3 B, the PCR products for RyR1, RyR2, and RyR3 genes with the predicted sizes of 224, 265, and 431 bp were found. These data indicate that cultured human PASMCs lack responses to RyR agonists, although they express RyR mRNAs, which are consistent with previous reports in cultured rat aortic SMCs (Masuo et al., 1991; Cortes et al., 1997; Vallot et al., 2000).

Using these cultured human myocytes, we examined the effect of caffeine on neurotransmitter-induced Ca2+ release. As an example shown in Fig. 3 C, application of ATP (10 μM) induced similar increases in [Ca2+]i in a cell before and after exposure of caffeine (10 mM) for 5 min. The mean increases in [Ca2+]i before and after treatment of caffeine were 378 ± 29 and 398 ± 13 nM, respectively (P > 0.05, n = 5). Similarly, ATP-induced increase in [Ca2+]i in the absence of extracellular Ca2+ (nominally Ca2+-free bath solution containing 1 mM EGTA) to prevent Ca2+ influx was not affected by treatment with caffeine (10 mM) for 5 min (unpublished data). These results suggest that caffeine has no inhibitory effect on ATP-mediated Ca2+ release through IP3Rs. Taken together, we believe that the failure of noradrenaline or IP3 to induce an effect in the presence of caffeine is not due to the direct inhibition of IP3-mediated Ca2+ release. Rather, these data further support the view that prior application of caffeine to activate RyRs may deplete SR Ca2+, and then block subsequent IP3-induced Ca2+ release. Thus, RyR- and IP3-sensitive Ca2+ stores appear to overlap functionally in rat PASMCs.

Ryanodine Receptors Amplify Submaximal Neurotransmitter–induced Ca2+ Release and Contraction in PASMCs

The functional colocalization of RyRs and IP3Rs in the SR, together with the known function of RyRs in mediating Ca2+-induced Ca2+ release in SMCs (Imaizumi et al., 1998; Collier et al., 2000), led us to hypothesize that Ca2+ released from IP3Rs during stimulation of neurotransmitter receptors might activate neighboring RyRs, contributing to neurotransmitter-induced responses. To test this hypothesis, we first examined the effect of the RyR antagonist ruthenium red on noradrenaline-induced increase in [Ca2+]i in freshly isolated rat PASMCs. As shown in Fig. 4 A, repeated application of noradrenaline (10 μM) induced consistent increases in [Ca2+]i in a control cell. Similar observations were obtained from eight other cells (Fig. 4 C). In contrast, noradrenaline induced a smaller increase in [Ca2+]i in a cell after treatment with ruthenium red (50 μM) for 5 min (Fig. 4 B). The average increases in [Ca2+]i following noradrenaline (10 μM) exposure were 436 ± 44 nM before ruthenium red treatment and 141 ± 17 nM after ruthenium red treatment (P < 0.05, n = 10) (Fig. 4 C). Similarly, pretreatment with the structurally different RyR antagonists ryanodine (100 μM) and tetracaine (10 μM) also significantly inhibited submaximal noradrenaline (10 μM)–induced increases in [Ca2+]i (Fig. 4 C). However, ruthenium red and tetracaine, unlike ryanodine, did not significantly affect maximal noradrenaline (300 μM)–induced Ca2+ release (Fig. 4 D). Thus, RyRs may amplify Ca2+ release following submaximal stimulation of adrenergic receptors with noradrenaline in PASMCs, which occurs through the local IP3R–RyR interaction (Ca2+-induced Ca2+ release mechanism).

We next examined the effects of RyR antagonists on noradrenaline-induced muscle contraction in rat pulmonary artery strips. As shown in Fig. 5, in the presence of ruthenium red (50 μM), ryanodine (100 μM), tetracaine (10 μM), or dantrolene (10 μM) for 10 min to block RyRs, concentration–response curves for noradrenaline-induced muscle contraction were shifted to the right. Ruthenium red, tetracaine, and dantrolene did not affect noradrenaline-induced maximal responses, while ryanodine reduced the maximum contraction. These data further suggest that Ca2+ release following submaximal stimulation of neurotransmitter receptors is able to open the neighboring RyRs, which causes further Ca2+ release from the SR and then amplifies neurotransmitter-induced increase in [Ca2+]i and associated contraction in PASMCs.

Since RyR antagonists have been reported to inhibit IP3Rs directly or indirectly (Vites and Pappano, 1992; Yamazawa et al., 1992; Iino et al., 1994; Tovey et al., 2000; Zimmermann, 2000; Flynn et al., 2001), we sought to examine the specificity of the RyR antagonists on neurotransmitter-induced Ca2+ release in cultured human PASMCs, since these cells lack functional expression of RyRs (Fig. 3). Application of ATP induced similar increases in [Ca2+]i in a control cell and a cell pretreated with ruthenium red (50 μM) for 5 min (Fig. 6 A). The mean increases in [Ca2+]i by ATP (10 μM) were 522 ± 72 in six control cells and 452 ± 48 nM in six cells pretreated with ruthenium red, respectively (P > 0.05). Similarly, ATP (100 μM)-induced increases in [Ca2+]i were not affected by ruthenium red (50 μM) (Fig. 6 B). Moreover, treatment with ryanodine (100 μM) and tetracaine (10 μM) for 5 min had no effect on ATP-induced increases in [Ca2+]i (Fig. 6 B). These results indicate that the RyR antagonists block neurotransmitter-induced Ca2+ release and contraction in PASMCs by specifically inhibiting RyRs, rather than by affecting IP3Rs.

Ryanodine Receptors Mediate Hypoxia-induced Ca2+ Release and Contraction in PASMCs

It has been reported that pretreatment with caffeine and ryanodine can inhibit hypoxic increase in [Ca2+]i in PASMCs (Salvaterra and Goldman, 1993; Vadula et al., 1993; Cornfield et al., 1994; Wang et al., 2003) and hypoxic vasoconstriction in isolated pulmonary artery strips and lungs (Jabr et al., 1997; Dipp et al., 2001; Morio and McMurtry, 2002). Considering that the inhibitory effects of these compounds could occur due to the depletion of SR Ca2+, we sought to use ruthenium red and tetracaine, the RyR antagonists that do not open the channels and deplete SR Ca2+, to examine hypoxic increase in [Ca2+]i in freshly isolated rat PASMCs. Fig. 7 A shows examples of hypoxia-induced increase in [Ca2+]i in a control cell and a cell pretreated with ruthenium red (50 μM) for 5 min. Ruthenium red significantly inhibited hypoxia-induced response. As summarized in Fig. 7 B, hypoxia-induced increases in [Ca2+]i were 442 ± 19 nM in control cells and 188 ± 12 nM in ruthenium red–treated cells (n = 8, P < 0.05). Similarly, pretreatment with ryanodine (100 μM) and tetracaine (10 μM) for 5 min to block RyRs also inhibited hypoxia-induced increases in [Ca2+]i (Fig. 7 B). Therefore, hypoxia may result in the opening of RyRs and subsequent Ca2+ release in PASMCs.

In the next experiments, we investigated the effect of RyR antagonists on hypoxic vasoconstriction in freshly isolated rat pulmonary artery strips. In control muscle strips, repeated hypoxic exposure induced consistent vasoconstrictions (Fig. 8). However, in muscle strips pretreated with ruthenium red (50 μM) for 10 min, hypoxic pulmonary vasoconstriction was markedly inhibited. In a total of seven identical experiments, hypoxic vasoconstrictions were decreased from 170 ± 14 mg before treatment with ruthenium red to 98 ± 5 mg after treatment with ruthenium red (P < 0.05) (Fig. 8 C). Similarly, ryanodine (100 μM) and tetracaine (10 μM) also significantly blocked hypoxic vasoconstrictions. Taken together, these data suggest that RyRs serve as an important target for hypoxia, by which these Ca2+ release channels are opened during hypoxic stimulation, contributing to hypoxic increase in [Ca2+]i and contraction in PASMCs.

All Three Subtypes of Ryanodine Receptors Are Expressed in PASMCs

To further determine expression patterns and levels of RyR subtype expression in PASMCs, we examined the expression of RyR1, RyR2, and RyR3 mRNAs in freshly isolated rat myocytes using real-time quantitative RT-PCR. Freshly isolated cells were used to avoid potential contaminations with neuronal, endothelial, and other types of cells as well as to avoid significant changes in expression patterns and levels of RyRs during cell culture. To verify that the elongated cells freshly isolated from pulmonary arteries were SMCs, we examined the expression of smooth muscle actin and myosin in these cells using immunofluorescence staining. As expected, both smooth muscle–specific markers were expressed in these cells (Fig. 9 A). Using these cells (PASMCs) as a source of mRNAs, RyR1, RyR2, and RyR3 were amplified by PCR, indicating the presence of all three RyR subtype mRNAs in PASMCs. In addition, absolute mRNA expression levels of RyR subtypes, which were calculated from standard curves of known DNAs, were similar. Similarly, there were no differences between relative mRNA expression levels of all three RyR subtypes, which were generated by normalizing their mRNA levels to GADPH mRNA levels (n = 8, Fig. 9 B). The amplification products of RyR subtypes were further examined by gel electrophoresis. Predicted sizes of the RyR1, RyR2, and RyR3 PCR products were 93, 130, and 83 bp, respectively (Fig. 9 C). Moreover, sequencing analysis confirmed that the PCR products matched the known RyR1, RyR2, and RyR3 mRNA sequences. To complement mRNA expression patterns of RyR subtypes, we studied the expression of RyR1, RyR2, and RyR3 proteins in freshly isolated PASMCs using immunofluorescence staining. As examples show in Fig. 9 D, all RyR1, RyR2, and RyR3 proteins were detected. Similar observations were made in three or four separate experiments. These data are consistent with mRNA expression in PASMCs.

Type-3 Ryanodine Receptors Mediate Hypoxia-induced, but not Noradrenaline-induced, Ca2+ Release and Contraction in PASMCs

Because RyR3 is expressed in PASMCs, we sought to examine if neurotransmitter-induced Ca2+ release was affected in PASMCs from RyR3−/− mice. Fig. 10 A shows an example of these experiments, in which noradrenaline (10 μM) induced similar increases in [Ca2+]i in wild-type (RyR+/+) and RyR3−/− mouse PASMCs. The mean increases in [Ca2+]i were 358 ± 24 nM in RyR3+/+ PASMCs (n = 8) and 331 ± 17 nM in RyR3−/− PASMCs (n = 9) (Fig. 10 B). Similarly, concentration–response curves for noradrenaline-induced muscle contractions were not altered in pulmonary artery strips from RyR3−/− mice (Fig. 10 C).

Next we determined if hypoxia-induced increases in [Ca2+]i were altered in RyR3−/− mouse PASMCs. As examples show in Fig. 11 A, hypoxia-induced increase in [Ca2+]i was significantly smaller in RyR3−/− than that in RyR3+/+ PASMCs. The mean hypoxia-induced increases in [Ca2+]i were decreased from 379 ± 25 nM in wild-type cells (n = 12) to 200 ± 11 nM in RyR3−/− cells (n = 19, P < 0.05) (Fig. 11 B). To test the potential role of other RyR subtypes in hypoxic response, we studied the effect of the RyR antagonists ruthenium red and tetracaine on hypoxic increase in [Ca2+]i in freshly isolated PASMCs from RyR3−/− mice. Pretreatment with ruthenium red (50 μM) for 5 min significantly inhibited hypoxic increase in [Ca2+]i in RyR3−/− PASMCs. The mean hypoxic increases in [Ca2+]i in RyR3−/− cells un-pretreated (n = 30) and pretreated with ruthenium red (n = 24) were 220 ± 10 and 101 ± 11 nM, respectively (P < 0.05). Similarly, pretreatment with tetracaine (10 μM) for 5 min could also inhibit hypoxic response in RyR3−/− PASMCs (Fig. 11 C). Consistent with the reduced hypoxic increase in [Ca2+]i in RyR3−/− PASMCs, hypoxic pulmonary vasoconstriction in RyR3−/− mice was significantly inhibited. As summarized in Fig. 11 D, hypoxia-induced muscle tension was 25 ± 3 mg in RyR3−/− pulmonary artery strips (n = 8) and 37 ± 4 mg in RyR3+/+ pulmonary artery strips (n = 7) (P < 0.05). Collectively, these results indicate that RyR3 mediates hypoxia-, but not noradrenaline-induced Ca2+ release in pulmonary artery myocytes. Moreover, RyR1 and/or RyR2 may also play a role in hypoxic response in PASMCs.

Discussion

We and other investigators have recently demonstrated that prior application of caffeine to deplete SR Ca2+ by opening RyRs prevents subsequent neurotransmitter-induced Ca2+ release through IP3Rs, and vice versa, in freshly isolated and cultured rat PASMCs (Wang et al., 2003; Zhang et al., 2003), suggesting that RyRs and IP3Rs are functionally coupled in the SR. Consistent with this view, this study has demonstrated that dialysis of IP3 to directly activate IP3Rs induces Ca2+ release (Ca2+-activated Cl currents) and blocks subsequent caffeine responses in freshly isolated rat PASMCs (Fig. 2 A). Conversely, prior application of caffeine to induce Ca2+ release abolishes subsequent IP3-induced responses (Fig. 2 B).

It has been previously noted that caffeine inhibits IP3-induced Ca2+-activated Cl currents (Ca2+ release) in Xenopus oocytes, cerebellar IP3Rs reconstituted in planar lipid bilayers, and IP3-induced Ca2+ release in permeabilized A7r5 cells in the absence of ATP by affecting the binding of IP3 to IP3Rs (Parker and Ivorra, 1991; Bezprozvanny et al., 1994; Missiaen et al., 1994), although other investigators have shown that caffeine does not have an effect on the binding of IP3 to its receptor (Brown et al., 1992; Toescu et al., 1992; McNulty and Taylor, 1993). To determine whether the failure of noradrenaline or IP3 to induce Ca2+ release following prior application of caffeine simply reflects an inhibitory effect on IP3Rs, we examined the effect of CMC, another RyR activator that has not been reported to inhibit IP3Rs, on noradrenaline-induced Ca2+ release in freshly isolated rat PASMCs. Similar to caffeine, CMC also induces a large increase in [Ca2+]i and blocks subsequent noradrenaline-induced Ca2+ release. As another approach to examine the specificity of caffeine as a selective agonist of RyRs in PASMCs, we performed experiments in cultured human PASMCs, since cell culture may result in significant changes in RyR expression levels and patterns in vascular SMCs, in which caffeine is ineffective (Masuo et al., 1991; Cortes et al., 1997; Vallot et al., 2000). Consistent with these data, we have found that the RyR agonists caffeine and CMC both fail to induce Ca2+ release, although RyR1, RyR2, and RyR3 mRNAs are found in cultured human PASMCs. In addition, caffeine does not inhibit ATP-induced increases in [Ca2+]i in these cultured cells in the presence and absence of extracellular Ca2+. These findings, together with the fact that application of ATP to stimulate purinergic receptors can induce Ca2+ release through IP3Rs, although also trigger Ca2+ influx through nonselective cation channels in a variety of cells including vascular SMCs (Tawada et al., 1987; Lagaud et al., 1996; North and Barnard, 1997; Szado et al., 2003), indicate that caffeine has no inhibitory effect on IP3-mediated Ca2+ release. Taken together, our data indicate that the failure of noradrenaline and IP3 to induce Ca2+ release in freshly isolated rat PASMCs in the presence of caffeine is not due to the inhibition of IP3Rs, but rather due to prior depletion by caffeine of SR Ca2+ stores, where RyRs and IP3Rs functionally overlap. In support of this view, direct measurements of SR Ca2+ have shown that caffeine and muscarinic receptor agonists cause identical patterns of SR Ca2+ depletion in gastric SMCs (White and McGeown, 2002). The reasons for the different effects of caffeine on IP3-induced Ca2+ release reported by different investigators are unknown, but they may be related to different experimental conditions. For example, caffeine significantly inhibits IP3-induced Ca2+ release in the absence of ATP, but not in the presence of 1.5 and 5 mM ATP (McNulty and Taylor, 1993; Missiaen et al., 1994). In addition, different cell types may show different responses to caffeine. It is known that Xenopus oocytes only express type-1 IP3Rs (Parys et al., 1992), whereas vascular myocytes (e.g., PASMCs) express all three subtypes of IP3Rs (type 1, 2, and 3 IP3Rs) (Zheng et al., 2004). Thus, the differences in expression patterns and levels of IP3R subtypes may also contribute to differential effects of caffeine on IP3-induced Ca2+ release. This may be the case in some smooth muscle cells and species. In agreement with this view, a well-designed study using canine PASMCs has shown the functionally distinct IP3- and ryanodine-sensitive Ca2+ stores (Janiak et al., 2001). Similar observation has been also observed in rabbit and guinea pig colonic SMCs (Young et al., 1999; Flynn et al., 2001).

The functional roles of RyRs have not been well established in vascular SMCs. In this study, we have found that full activation of RyRs with caffeine and IP3Rs with noradrenaline induce equivalent Ca2+ release in rat PASMCs (Fig. 1), suggesting that RyRs are highly expressed. Prior application of the structurally different RyR antagonists ruthenium red, ryanodine, and tetracaine can significantly inhibit submaximal noradrenaline–induced increases in [Ca2+]i. Consistent with these results, these RyR antagonists shift concentration–response curves for noradrenaline-induced contraction to the right. These results suggest that Ca2+ released from IP3Rs following submaximal agonist stimulation may open neighboring RyRs and then induce further Ca2+ release from the SR (CICR), which amplifies agonist-induced increase in [Ca2+]i and contraction in PASMCs. Since it has been reported that RyR antagonists may inhibit IP3Rs directly or indirectly (Vites and Pappano, 1992; Yamazawa et al., 1992; Iino et al., 1994; Tovey et al., 2000; Zimmermann, 2000; Flynn et al., 2001), one might argue that the inhibition of IP3Rs could account for the effects of the RyR antagonists on noradrenaline-induced Ca2+ release and contraction in PASMCs. However, our data and previous reports indicate that this may not be the case in pulmonary artery (vascular) SMCS. First, none of the RyR antagonists ruthenium red, tetracaine, and dantrolene affect maximal noradrenaline–induced increase in [Ca2+]i and cell contraction (Figs. 4 and 5). Second, ruthenium red, ryanodine, and tetracaine do not block ATP (10 and 100 μM)-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+ in cultured human PASMCs that lack functional expression of RyRs (Fig. 6). Third, recent studies have shown that dialysis of RyR antibody blocks Ca2+ release following stimulation of α-adrenergic receptors with noradrenaline in cultured portal vein SMCs (Boittin et al., 1999), and the IP3R antagonists xestospongin C and 2-aminoethoxydiphenyl inhibit RyR-generated Ca2+ sparks in portal vein myocytes (Gordienko and Bolton, 2002). Finally, photorelease of caged IP3 can induce Ca2+ sparks in cultured PASMCs (Zhang et al., 2003). Taken together, the RyR antagonists may block neurotransmitter-induced Ca2+ release and contraction in PASMCs by specifically inhibiting RyRs, rather than by affecting IP3Rs.

It is known that ryanodine concentrations <10 μM activate RyRs, whereas concentrations >10 μM block RyRs in isolated SR preparations (Meissner, 1986). We have found that ryanodine at 100 μM, unlike the other RyR antagonists ruthenium red, tetracaine, and dantrolene, inhibits maximal noradrenaline–induced Ca2+ release and associated contraction in PASMCs (Figs. 5 and 6). These results suggest that following application of ryanodine (100 μM), this agent may initially activate RyRs possibly due to the low concentration, causing partial depletion of SR Ca2+.

Several studies have shown that ryanodine inhibits hypoxia-induced increases in [Ca2+]i in cultured PASMCs and hypoxic pulmonary vasoconstriction in isolated pulmonary artery and lung preparations (Vadula et al., 1993; Cornfield et al., 1994; Jabr et al., 1997; Morio and McMurtry, 2002), indicating that RyRs potentially play an important role in hypoxic responses. Considering that ryanodine possibly affects the SR Ca2+ (Figs. 5 and 6), we sought to examine the effects of ruthenium red and tetracaine (the RyR antagonists without affecting the SR Ca2+) on hypoxic responses. As shown in Fig. 7, prior application of ruthenium red and tetracaine, similar to ryanodine, also inhibit hypoxia-induced increases in [Ca2+]i in PASMCs. Consistent with these results, hypoxic pulmonary vasoconstriction is also significantly inhibited in pulmonary artery strips pretreated with ruthenium red, tetracaine, and ryanodine. Therefore, RyRs may be important targets for hypoxia, by which Ca2+ release from the SR contributes to hypoxic increase in [Ca2+]i and contraction in PASMCs.

Several studies using RT-PCR have reported that all three RyR subtype mRNAs are expressed in aorta, cerebral, and mesenteric arteries as well as portal vein (Neylon et al., 1995; Coussin et al., 2000; Lohn et al., 2001). In contrast, other groups have shown that abundant RyR3, little RyR2, and no RyR1 mRNAs are present in aorta (Ledbetter et al., 1994; Vallot et al., 2000). Moreover, a recent study has reported that RyR1 mRNA is present in aorta with endothelium, but not in the tissue free of endothelium (Vallot et al., 2000). These conflicting data in systemic artery SMCs possibly occur due to changes in expression patterns and levels of RyR subtype mRNAs resulting from cell culture and/or due to the contamination with nonmuscle cells in experiments using vascular tissues (Vallot et al., 2000). Furthermore, there have been no reports aimed at examining mRNA expression of RyR subtypes in pulmonary artery myocytes. Therefore, we sought to examine mRNA expression of RyR subtypes in freshly isolated rat PASMCs using real-time quantitative RT-PCR. As shown in Fig. 9 B, all three RyR subtype mRNAs are expressed in freshly isolated rat PASMCs with similar expression levels. Consistent with mRNA expression, immunofluorescence staining experiments using specific anti-RyR1, anti-RyR2, and anti-RyR3 antibodies indicate that all three subtype RyR proteins are expressed in PASMCs (Fig. 9 D). In support of our findings, Zhang et al. (2003) have recently shown that RyR1 and RyR2 proteins are detected in cultured pulmonary artery myocytes by immunofluorescence staining, although a nonspecific anti-RyR2 antibody was used and RyR3 protein expression was not examined in their study.

RyR3−/− mice have apparently normal growth and reproduction, but show a reduction in neonatal skeletal muscle contraction, an increase in locomotor activity, as well as an impairment of hippocampal synaptic plasticity and spatial learning (Takeshima et al., 1996; Bertocchini et al., 1997; Balschun et al., 1999). A study using cerebral artery myocytes from RyR3−/− mice has revealed that the frequency of Ca2+ sparks and STOCs (spontaneous transient inward currents) are increased (Lohn et al., 2001), whereas another report has shown that caffeine- and noradrenaline-induced muscle contractions are not significantly changed in RyR3−/− mouse aortic arteries (Takeshima et al., 1996). In this study, we have found that submaximal noradrenaline–induced increases in [Ca2+]i are similar in PASMCs from RyR3−/− and RyR3+/+ mice. In parallel, concentration–response curves for noradrenaline-induced muscle contraction are unaffected in pulmonary artery strips from RyR3−/− mice (Fig. 10 C). In contrast, hypoxia-induced increase in [Ca2+]i is significantly smaller in PASMCs from RyR3−/− mice than from RyR3+/+ mice. Similarly, hypoxic pulmonary vasoconstriction is also significantly inhibited in RyR3−/− mice (Fig. 11). It is evident that RyR3 plays an important role in hypoxia-induced, but not neurotransmitter-induced, Ca2+ release and contraction in PASMCs. This fact, together with previous findings that hypoxic increase in [Ca2+]i and vasoconstriction are unique cellular responses in pulmonary, but not systemic (mesenteric and cerebral), arteries (Madden et al., 1992; Vadula et al., 1993; Yuan et al., 1993; Wang et al., 2003), suggests that distinct patterns and/or levels of RyR3 expression between PASMCs and cells from the systemic circulation might account for the known differences in the response to hypoxia in these different arterial beds, although further experiments are required to confirm this view.

Our data indicate that the RyR antagonists ruthenium red and tetracaine can further, but not completely block hypoxic increase in [Ca2+]i in RyR3−/− mouse PASMCs (Fig. 11C). This provides evidence that other RyR subtypes (RyR1 and/or RyR2) as well as additional mechanisms may also be involved in hypoxic responses in PASMCs. In reality, it is well established that hypoxia can block voltage-dependent K+ channels (Post et al., 1992, 1995; Archer et al., 1993, 1998; Yuan et al., 1993; Wang et al., 1997a; Hulme et al., 1999), inhibit Ca2+/Na+ exchanger (Wang et al., 2000), and activate capacitative Ca2+ entry in PASMCs (Fantozzi et al., 2003; Lin et al., 2004; Ng et al., 2005). The blockade of voltage-dependent K+ channels by hypoxia depolarizes cell membrane, which may presumably activate voltage-dependent Ca2+ channels and then cause Ca2+ influx across cell membrane. The hypoxic inhibition of Ca2+/Na+ exchanger can prevent intracellular Ca2+ removal, contributing to hypoxic increase in [Ca2+]i. Thus, it is apparent that multiple pathways of Ca2+ release, entry, and removal are likely to be responsible for hypoxic increase in [Ca2+]i and associated contraction in PASMCs.

Acknowledgments

This work was supported by National Institutes of Health grants HL049426 (H.A. Singer), HL045239 (M.I. Kotlikoff), HL064043, and HL075190, and the American Heart Association (0256269T and 0340160N) (Y.-X. Wang).

Olaf S. Andersen served as editor.

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Abbreviations used in this paper: CMC, chloro-m-cresol; IP3R, inositol 1,4,5-triphosphate receptor; PASMC, pulmonary artery smooth muscle cell; PSS, physiological saline solution; RT-PCR, reverse transcriptase PCR; SMC, smooth muscle cell.