Thrombin-mediated increases in cytosolic [Ca²⁺] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Abstract

Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt). In this study, we compared the effect of thrombin on Ca²⁺ signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca²⁺]_cyt in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca²⁺]_cyt in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca²⁺ induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca²⁺ release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca²⁺ transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca²⁺. Ca²⁺ oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca²⁺ influx and Ca²⁺ reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca²⁺ influx, intracellular Ca²⁺ release, and reuptake underlie the Ca²⁺ transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.

thrombin, a major effector protease of the coagulation cascade, is best known for its role in the conversion of fibrinogen into fibrin. Thrombin can operate as an inflammatory agent by stimulating endothelial cells (EC) to produce and/or secrete chemotactic factors by promoting monocyte recruitment and by increasing endothelial permeability (8, 9). At the same time, thrombin regulates the inflammation processes by blocking aggregation of platelets, adhesion of monocytes to endothelium, and by releasing nitric oxide (NO) from EC (35).

In the lung, thrombin has a number of effects not limited to coagulation, which may play significant roles in the development of lung injury and pulmonary hypertension. Of particular interest are its actions related to chronic thromboembolic pulmonary hypertension where it plays inherent roles in the formation of the persistent thrombus and where it may endure for prolonged periods of time in the pulmonary artery. Increased luminal native α-thrombin was shown to increase the clearance of albumin across the pulmonary endothelial monolayer causing morphological changes and resulting in pulmonary artery endothelial cell (PAEC) contraction and formation of intercellular gaps (14). In ensuing studies, PAEC contraction and disruption of the endothelial barrier by thrombin has been attributed to 1) increased Ca²⁺ influx and/or release (15, 18, 26, 27, 38), 2) cytoskeletal rearrangements (3, 6, 41), 3) altered adenylate cyclase function (11), 4) activation of canonical transient receptor potential channels (TRPC) (15, 22, 30, 33, 36, 37), and 5) mitochondrial membrane potential loss (5). Thrombin also acts as a mitogen, promoting the proliferation of fibroblasts (4, 17, 34) or airway smooth muscle cells (20). Ca²⁺ signaling is therefore central to the function of thrombin; increased Ca²⁺ influx and/or release from intracellular stores, such as endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), is known to modulate the structure and contractility of pulmonary endothelial and smooth muscle cells (22, 32, 33). Intercellular gaps forming due to PAEC contraction could then expose pulmonary artery smooth muscle cells (PASMC) to thrombin. Activation of protease-activated receptors (PAR) on PASMC will then have vasoactive and mitogenic effects.

In the current study, we compared the effect of thrombin on Ca²⁺ signaling in human PAEC and PASMC. Our results show that thrombin-mediated intracellular Ca²⁺ mobilization involves different stores in PAEC and PASMC. Importantly, we identify a novel thrombin-releasable store in PAEC that is insensitive to cyclopiazonic acid (CPA), caffeine, and ryanodine, which may be crucial in understanding the pathogenesis of diseases such as chronic thromboembolic pulmonary hypertension (CTEPH), where pulmonary arteries are particularly exposed to thrombin.

MATERIALS AND METHODS

Cell preparation and culture.

Human PASMC and PAEC from normal subjects purchased from Lonza were used for experiments. Cryopreserved cells were plated onto cover slips or Petri dishes and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C in smooth muscle growth medium (SMGM; Lonza) for PASMC and in endothelium growth medium (EGM; Lonza) for PAEC. SMGM was composed of smooth muscle basal medium supplemented with 5% FBS, human epidermal growth factor, human fibroblast growth factor, and insulin. EGM was composed of endothelium basal medium supplemented with 2% FBS, human epidermal growth factor, human fibroblast growth factor, and insulin. PASMC and PAEC were used for experiments between passages 2 and 6. The morphology of the cells was examined using phase contrast microscopy.

Measurement of [Ca²⁺]_cyt.

Cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) was measured in single human PASMC and PAEC using ratiometric Ca²⁺ label fura 2-AM. Cells grown on cover slips were loaded with fura 2-AM (4 μM) in the dark at room temperature for 30 min before being transferred to a perfusion chamber on the Nikon TMS microscope stage. Loaded cells were superfused with modified Krebs solution (MKS) for 30 min at 32°C to remove residual extracellular dye and to allow intracellular esterases to cleave cytosolic fura 2-AM into active fura 2. The MKS contained (in mM) 138 NaCl, 4.7 KCl, 1.2 NaH₂PO₄, 1.2 MgSO₄, 10 HEPES, 1.8 CaCl₂, and 10 glucose, pH 7.4. In Ca²⁺-free MKS, CaCl₂ was replaced by equimolar MgCl₂.

Fura 2 fluorescence (excited by a xenon lamp at 510 nm and emission recorded at 340 and 380 nm) was collected using a ×40 Nikon UV-Fluor objective and a charge-coupled device camera. The fluorescence signals emitted from the cells were captured every 2 s using InCytIM2 software (Intracellular Imaging). [Ca²⁺]_cyt was measured as the ratio of fura 2 fluorescence emission at 340 nm and 380 nm (F₃₄₀/F₃₈₀) normalized to baseline (F₀) and is reported as F/F₀.

RT-PCR.

Total RNA was extracted from human PASMC and PAEC following cell lysis with TRIzol (Invitrogen). Addition of chloroform followed by centrifugation was used to separate the aqueous phase containing the RNA; RNA was then precipitated using isopropyl alcohol. RNA concentration and quality were determined from OD260–280 measurements. SuperScript First-Strand Synthesis System (Invitrogen) was used to synthesize cDNA. RNA (2 μg) was first treated with DNase I and the reaction stopped with 10% 25 mM EDTA (65°C 10 min). The reverse transcription (RT) reaction consisted of three phases: 1) 10 μl DNase-treated RNA, oligo(dT) dNTP (1 μl, 10 mM) at 65°C for 5 min; 2) first-strand buffer (4 μl, 5×), 0.1 M DTT (2 μl), RNaseOUT heated to 42°C for 2 min; and 3) SuperScript II reverse transcriptase added to the samples for 50 min at 42°C and 15 min at 70°C. For negative controls, DNase-RNase-free H₂O was added to the RT reaction instead of SuperScript II.

Platinum PCR SuperMix (Invitrogen) was used to perform all PCR reactions. The mixture contained 22 U/ml complexed recombinant Taq DNA polymerase with Platinum Taq antibody, 22 mM Tris·HCl (pH 8.4), 55 mM KCl, 1.65 mM MgCl₂, 220 μM dNTP, and stabilizers. Total volume of each reaction tube was 25 μl. PCR primer sequences are included in Table 1. The specificity of the sense and antisense oligonucleotides were examined using the NCBI-BLAST program. PCR was performed according to the following sequence: 5 min at 95°C followed by 35°C cycles of denaturation at 95°C for 30 s, primer annealing at 55 for 30 s, and primer extension at 72°C for 20 s and ending with 72°C for 7 min. PCR products were electrophoresed through 1.2% agarose gels containing 10 μM Gelstar Nucleic Acid stain (Lonza) and were visualized by ultraviolet illumination. A positive control GAPDH was used to semiquantify the RT-PCR products. The net intensity values of cDNA bands was measured by Image J software, and the PCR products were normalized to the GAPDH product from the same cDNA sample and PCR reaction and run on the same gel.

Table 1.

Primer sequences used for RT-PCR

Gene Name (Accession No.)	Product Size, bp	Sequence (Sense/Antisense)	Location, nt
PAR-1 (NM_001992)	708	5′-tgtgaactgatcatgtttatg-3′/	2542–2562
		5′-ttcgtaagataagagatatgt-3′	3249–3229
PAR-2 (NM_00542)	428	5′-gtgagaggctgactttctc-3′/	55–73
		5′-agggtgcttcttcttagttc-3′	482–462
PAR-3 (NM_004101)	599	5′-gaaagccctcatctttgcag-3′/	188–207
		5′-aggtgaaaggatggacgatg-3′	786–766
PAR-4 (NM_003950)	244	5′-ggcaacctctatggtgccta-3′/	1121–1140
		5′-ttcgacccagtacagccttc-3′	1364–1345
GAPDH (NM_002046)	243	5′-gacaacgaatttggctacagc-3′/	1045–1065
		5′-gatggtacatgacaaggtgc-3′	1287–1268

Chemicals.

Human thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). All other chemicals were purchased from Sigma (St. Louis, MO) and prepared as stock solutions in their respective solvents. CPA was dissolved in DMSO to make a stock solution of 10 mM. Aliquots of the stock solution were then diluted to the final concentration and used the day of each experiment. pH values were readjusted to 7.4 after the addition of drugs to the Ca²⁺-free solution.

Statistics and data analysis.

Data are expressed as means ± SE; n represents the number of cells. Statistical analysis was performed using paired Student's t-test. Differences were considered to be significant at P < 0.05.

RESULTS

Comparable PAR expression in PASMC and PAEC.

PASMC and PAEC from normal subjects (Fig. 1A) were first evaluated for expression of PAR subtypes. The mRNA expression for all PAR subtypes (PAR 1–4) was present in both PASMC (Fig. 1B) and PAEC (Fig. 1C). Semiquantitative analysis, when expression was normalized to GAPDH intensity (Fig. 1D), indicated no statistically significant differences in receptor subtype expression between the two cell lines. Indeed, PAR-2 and PAR-3 expression levels were higher in PASMC, and PAR-4 expression was higher in PAEC. Overall, these data indicate that the mRNA expression level of all PAR is comparable between PASMC and PAEC.

Fig. 1.

The mRNA expression levels of protease-activated receptors (PAR) are comparable in human pulmonary artery smooth muscle cells (PASMC) and pulmonary artery endothelial cells (PAEC). A: phase contrast image of cultured human PASMC (left) and PAEC (right). B and C: RT-PCR amplified products for PAR-1, PAR-2, PAR-3, PAR-4, and GAPDH in PASMC. M, 1,000-bp DNA ladder. RT-PCR-amplified products of GAPDH in the presence (+RT) or absence (−RT) of transcriptase are shown as controls. D: summarized data (n = 3–5) showing the mRNA levels of PAR 1–4, normalized to GAPDH, in PASMC (black bars) and PAEC (gray bars).

Thrombin causes kinetically different [Ca²⁺]_cyt transients in PAEC and PASMC.

We directly compared the thrombin-induced increases in [Ca²⁺]_cyt in PASMC and PAEC. Application of thrombin (10 nM) caused large transient increases in [Ca²⁺]_cyt in both cell types (Fig. 2A), but the transients differed with respect to both their amplitude and duration (Fig. 2B). Peak amplitude of the Ca²⁺ transient was significantly greater in PASMC (2.72 ± 0.11 F/F₀, n = 11) than in PAEC (2.28 ± 0.12 F/F₀, n = 19; P < 0.05). Thrombin-induced Ca²⁺ transients also decayed faster in PASMC than in PAEC. Fitting of the recovery from the peak response to the baseline with an exponential decay function indicated that the duration of the Ca²⁺ transients was 162.4 ± 15.2 s for PASMC (n = 11) and 458.3 ± 31.2 s for PAEC (n = 18; P < 0.001). These results suggest a differential regulation of the release and reuptake of Ca²⁺ mechanisms in PAEC and PASMC.

Fig. 2.

Different kinetics of thrombin-induced transient cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) increases in PASMC and PAEC. A: representative data depict the transient increase in [Ca²⁺]_cyt induced by 10 nM thrombin in PASMC (top) and PAEC (bottom); [Ca²⁺]_cyt elevations are described as the F/F₀ ratio of fura 2 fluorescence. B: summarized data showing the amplitude (left) and duration (right) of thrombin-induced [Ca²⁺]_cyt transients in PASMC (black bars, n = 11) and PAEC (gray bars, n = 18–19). *P < 0.05, ***P < 0.001 vs. PASMC.

We also calculated the area under the curve (AUC) for the [Ca²⁺]_cyt transients in human PASMC and PAEC. AUC was equal to 214.9 ± 26.3 for PAEC and 77.1 ± 10.7 for PASMC (P < 0.0001). The values of AUC indicated that there was substantially more thrombin-induced Ca²⁺ release in PAEC than in PASMC, reflecting the slower kinetics of the recovery phase of the [Ca²⁺]_cyt transients in PAEC that we have described.

Differential role of CPA-sensitive Ca²⁺ stores in thrombin-induced Ca²⁺ release.

As shown in Fig. 2, the thrombin-mediated increase in [Ca²⁺]_cyt in PAEC has a much slower kinetic of “recovery” (reuptake or extrusion from the cytoplasm) to basal [Ca²⁺]_cyt than observed in PASMC. In PASMC, the more transient nature of the change in [Ca²⁺]_cyt suggests that thrombin-mediated Ca²⁺ release and influx take place rapidly and are likely to overlap. To determine if the differential kinetics are due to the relative contribution of different intracellular Ca²⁺ stores, experiments were designed to determine the importance of ER/SR Ca²⁺ stores to the thrombin-induced increase in [Ca²⁺]_cyt. Figure 3A shows data from PASMC and PAEC exposed to thrombin in the presence of 10 μM CPA, an inhibitor of the SR/ER Ca²⁺-ATPase pump that depletes Ca²⁺ stored in the IP₃-sensitive SR/ER.

Fig. 3.

Cyclopiazonic acid (CPA) treatment significantly inhibited thrombin-induced Ca²⁺ release in PASMC, but not in PAEC. A: representative traces of [Ca²⁺]_cyt changes (denoted by fluorescence ratio, F/F₀) in PASMC (top) and PAEC (bottom) treated with CPA (10 μM) and thrombin (10 nM) in the presence and absence (0Ca) of extracellular Ca²⁺. B: summarized data showing the resting [Ca²⁺]_cyt [basal fluorescence intensity (a), the amplitude of CPA-induced Ca²⁺ release (b), the amplitude of thrombin-induced Ca²⁺ release (c), the ratio of thrombin- and CPA-induced Ca²⁺ release (d), and the peak (e) and plateau (f) amplitude of CCE in PASMC (black bars; n = 15) and PAEC (gray bars; n = 16)]. C: time course of the decay phase of CPA-induced transient increase in [Ca²⁺]_cyt in PASMC and PAEC superfused with Ca²⁺-free solution. **P < 0.01, ***P < 0.001.

Detailed analysis of these experiments showed that resting [Ca²⁺]_cyt was significantly lower in PASMC than in PAEC (P < 0.001) (Fig. 3Ba). In both cell types, CPA caused a transient [Ca²⁺]_cyt increase in the absence of extracellular Ca²⁺ (Fig. 3, A and Bb), but its amplitude was significantly greater in PASMC (1.08 ± 0.09 F/F₀) than in PAEC (0.42 ± 0.04; P < 0.001). Furthermore, the CPA-induced Ca²⁺ transient showed different kinetics in PASMC and PAEC (Fig. 3C), reflecting those observed in response to thrombin in Fig. 2. Subsequent exposure to thrombin in Ca²⁺-free+CPA solution caused a substantial rapid Ca²⁺ release in all PAEC, whereas only a very small proportion of PASMC showed a brief, low amplitude response to thrombin (Fig. 3Bc, P < 0.01). When the thrombin response is expressed as a ratio relative to the CPA-induced Ca²⁺ release, a significantly higher thrombin-mediated CPA-insensitive Ca²⁺ release is observed in PAEC (Fig. 3Bd, P < 0.001).

Upon reintroduction of Ca²⁺ to the perfusate in the presence of CPA, a biphasic capacitative Ca²⁺ entry (CCE) was observed. The peak and plateau amplitudes of this CCE were significantly greater in PASMC than in PAEC (Fig. 3, Be and Bf; P < 0.001). The data suggests that, in addition to causing Ca²⁺ influx through plasma membrane channels, thrombin-induced increases in [Ca²⁺]_cyt occur by promoting Ca²⁺ release from different intracellular stores in PASMC and PAEC. To confirm that the thrombin-mediated signaling pathway, via G protein-coupled receptors (GPCR), was not uncoupled and that the PAR were not desensitized, the experiment in PASMC was repeated in the presence of extracellular Ca²⁺ (Fig. 4). As expected, with constant extracellular Ca²⁺ present, the CPA-mediated Ca²⁺ transient reached steady state; addition of thrombin consistently caused a significant Ca²⁺ transient, indicating that in the presence of extracellular Ca²⁺, thrombin still evokes an increase in [Ca²⁺]_cyt and that CPA does not disassociate signaling pathways or desensitize receptors.

Fig. 4.

Thrombin signaling pathway remains intact after CPA pretreatment in human PASMC. A: representative trace (F/F₀) of thrombin-mediated calcium release after CPA pretreatment in the presence of extracellular Ca²⁺. B: averaged data from 39 cells (means ± SE) indicating the increase in [Ca²⁺]_cyt in PASMC after CPA and thrombin treatment in the presence of extracellular Ca²⁺ expressed as the F₃₄₀/F₃₈₀ ratio. Basal, the basal level of [Ca²⁺]_cyt; CPA-Peak and CPA-Plateau, the amplitude of CPA-induced increases in [Ca²⁺]_cyt (peak and plateau); Thrombin, the thrombin-induced increase in [Ca²⁺]_cyt (on top of CPA-induced increase in [Ca²⁺]_cyt). ***P < 0.001 vs. Basal or CPA-Plateau bar.

Caffeine- and ryanodine-sensitive stores are not thrombin-releasable stores in PAEC.

As shown in Figs. 2 and 3, thrombin clearly caused Ca²⁺ release in PAEC from an intracellular store that could not be depleted by CPA. We verified whether ryanodine-sensitive stores were modulated by thrombin in PAEC. Pretreatment of PAEC with a cocktail containing CPA (10 μM), caffeine (1 mM), and ryanodine (10 μM) caused a Ca²⁺ transient in Ca²⁺-free solutions (Fig. 5). Depletion of ryanodine- and caffeine-sensitive stores had no effect on the thrombin-induced increase in [Ca²⁺]_cyt. These results suggest that thrombin stimulates Ca²⁺ mobilization from an intracellular store that is not sensitive to caffeine, ryanodine, or CPA.

Fig. 5.

Treatment with CPA, caffeine, and ryanodine fails to inhibit thrombin-induced Ca²⁺ release in PAEC. A: representative trace (denoted by the fluorescence ratio, F/F₀) indicating thrombin (10 nM)-mediated Ca²⁺ release after pretreatment with CPA, caffeine (Caf), and ryanodine (Ry). B: averaged data (means ± SE) indicating the increase in [Ca²⁺]_cyt in PAEC pretreated with a combination of CPA (10 μM), caffeine (1 mM), and ryanodine (10 μM) in the absence of extracellular Ca²⁺. Basal, the basal level of [Ca²⁺]_cyt; CPA-Peak and CPA-End, the amplitude of [Ca²⁺]_cyt increases at the beginning (CPA-Peak) of application of CPA, Caf, and Ry and at the time right before application of thrombin (CPA-End); Thrombin, the thrombin-induced increase in [Ca²⁺]_cyt (on top of CPA/Caf/Ry-induced increase in [Ca²⁺]_cyt). ***P < 0.001 vs. Basal or CPA-End bar.

Thrombin causes rhythmic [Ca²⁺]_cyt oscillations in PASMC.

In human PASMC, low-concentration thrombin (10 nM) induced [Ca²⁺]_cyt oscillations with different frequency and amplitudes (Fig. 6). Removal of extracellular Ca²⁺ abolished thrombin-induced Ca²⁺ oscillations; this effect was reversible, i.e., restoration of extracellular Ca²⁺ restored thrombin-induced Ca²⁺ oscillations (Fig. 7, A and B). Upon removal of Ca²⁺, thrombin often triggered a single Ca²⁺ transient whose amplitude was comparable to that of oscillations observed in Ca²⁺-containing perfusate (Fig. 7C). Finally, CPA effectively blocked thrombin-induced Ca²⁺ oscillations (Fig. 8), again indicating that CPA-sensitive intracellular stores are also thrombin-sensitive stores in human PASMC.

Fig. 6.

Thrombin induces rhythmic Ca²⁺ oscillations in PASMC. The representative traces depict the Ca²⁺ oscillations induced by 10 nM thrombin in 3 separate PASMC, shown as higher (top, 0.0055 Hz), medium (middle, 0.0036 Hz), and lower (bottom, 0.00033 Hz) frequency.

Fig. 7.

Thrombin-induced Ca²⁺ oscillations require Ca²⁺ influx in PASMC. A: sample traces showing Ca²⁺ oscillations induced by thrombin (10 nM) in the presence or absence (0Ca) of external Ca²⁺ in the perfusate. B and C: bar graphs depict oscillation frequency (B) and oscillation amplitude (C) in PASMC (n = 8) exposed to thrombin before (white bars) and during (black bars) removal of external Ca²⁺, as well as upon return of Ca²⁺ to the perfusate (gray bars). ***P < 0.001 vs. thrombin.

Fig. 8.

CPA reversibly attenuates thrombin-induced Ca²⁺ oscillations in PASMC superfused with Ca²⁺-containing solution. Left: representative record showing 100 nM thrombin-induced [Ca²⁺]_cyt changes (denoted by the fluorescence ratio, R₃₄₀/R₃₈₀) in PASMC before, during, and after treatment with 5 μM CPA. Right: individual traces show the effect of 5 μM CPA on the frequency and waveform of Ca²⁺ oscillations induced by 100 nM thrombin.

Together, the results suggest that thrombin-induced activation of PAR, in addition to calcium influx mechanisms, increases [Ca²⁺]_cyt via Ca²⁺ release from CPA-sensitive SR/ER. The IP₃-mediated store depletion then causes CCE via store-operated channels (SOC); however, the incoming Ca²⁺ is probably directly or rapidly sequestered into the SR/ER to refill Ca²⁺ into these intracellular stores. In other words, the thrombin-induced Ca²⁺ oscillations depend on the capacity or levels of Ca²⁺ in the SR/ER, whereas the store-operated Ca²⁺ influx plays an important role in refilling the stores so that a “normal” Ca²⁺ oscillation can occur.

DISCUSSION

In the lung, the formation of fibrotic clots due to the persistence of pulmonary embolisms can lead to vascular injury, and, particularly, to disruption of the endothelial barrier. Such occluding clot regions, typical of chronic thromboembolic pulmonary hypertension, are sites that can accumulate thrombin as well as vasoactive and mitogenic agents produced by circulating platelets or by endothelial cells themselves. In the endothelial monolayer, changes in microvascular permeability or vascular leakage are major downstream effects of altered [Ca²⁺]_cyt. In vascular smooth muscle cells, increased [Ca²⁺]_cyt can translate into contraction and/or proliferation. Thrombin is known to induce vascular injury predominantly via effects on endothelial cells leading to endothelial barrier dysfunction due to mobilization of Ca²⁺ and rearrangement of the cytoskeleton (12). In the current study, we evaluated the impact of thrombin on Ca²⁺ signaling in human PASMC and PAEC.

Many of the cellular effects of thrombin are consistent with a primary role in vessel wound healing and revascularization (10) and involve a multitude of cell types (i.e., platelets, endothelium, smooth muscle cells, neutrophils, leukocytes, neurons, glial cells) in the systemic response to vascular damage. Thrombin receptors, known as PAR, are members of the G protein superfamily of seven-transmembrane domain receptors. Through binding to PAR expressed on smooth muscle cells (PAR 1–4), thrombin has been shown to modulate Ca²⁺ signaling, in turn increasing cell proliferation and migration (2). We found that the mRNA expression of all four thrombin-specific PAR subtypes was similar in human PASMC and PAEC (Fig. 1), although there was a tendency for PASMC to express more PAR-2 and PAR-3 than PAEC, whereas PAEC expressed more PAR-4 than did PASMC. PAR-1 function has been linked to regulation of endothelial barrier function (19, 37). PAR-2 is also expressed in vascular tissue and highly vascularized organs, where it is purported to play a role in regulating vascular tone (endothelium-dependent relaxation) and the chronic response to vessel inflammation and wound healing (21). PAR-3, expressed in many cell types, may be involved in the vascular inflammatory response (29) and smooth muscle cell proliferation (7). Platelet PAR-3 deficiency can also protect against thrombosis in mice (41). Some have even suggested that PAR-3 may act as a cofactor for PAR-4 activation by thrombin, rather than as a functional receptor (28, 41). PAR-4 itself may have an important role in regulating vascular tone (16) and endothelial cell proliferation (13). We previously reported that mRNA expression of PAR-1 and PAR-3 was increased in PASMC from idiopathic pulmonary arterial hypertension (IPAH) patients compared with PASMC from normal subjects and normotensive patients (31). These results suggest that thrombin signaling, at least in PASMC, may be involved in triggering pulmonary vasoconstriction and vascular remodeling in IPAH patients.

In the current study, we showed that thrombin causes significant increases in [Ca²⁺]_cyt in both PASMC and PAEC, which could be attributed to both Ca²⁺ influx and Ca²⁺ release from the sarcoplasmic/endoplasmic reticulum. Indeed, thrombin has previously been shown to stimulate Ca²⁺ influx mechanisms, particularly via the canonical transient receptor potential channels, TRPC1 and 6 (1, 33). Such thrombin-mediated TRPC-dependent elevations in [Ca²⁺]_cyt represent an important mechanism of increased intracellular Ca²⁺. In addition, we have shown here that thrombin has specific effects on Ca²⁺ release from intracellular stores that appear to be different in PASMC and PAEC. These complementary studies into the regulation of thrombin-mediated elevations of [Ca²⁺]_cyt may indicate an interaction of thrombin-sensitive stores with either different TRPCs or the same TRPCs activated via different mechanisms in the different cell types.

Our findings indicate a substantial thrombin-dependent release of Ca²⁺ from intracellular stores in both PASMC and PAEC; however, in PASMC this release is entirely CPA sensitive, indicating predominant Ca²⁺ release from IP₃- and CPA-sensitive stores in the SR. Thrombin was able to still initiate a rise in intracellular Ca²⁺ in the presence of extracellular Ca²⁺ after store depletion by CPA, confirming that the GPCR-mediated signaling pathways were still intact and functional in these cells.

While CPA- and IP₃-sensitive stores appeared to play major roles in the [Ca²⁺]_cyt response to thrombin in PASMC, it was apparent that PAEC possessed another source of Ca²⁺. In PAEC, a significant thrombin-mediated Ca²⁺ release was still present even after pretreatment with a cocktail of CPA, caffeine, and ryanodine, implicating a substantial Ca²⁺ release from a different intracellular store. These data show that the thrombin-mediated Ca²⁺ release in PAEC may involve sources in addition to an IP₃-sensitive ER that can be depleted by CPA: for example, 1) ER stores that cannot be depleted by CPA, and 2) non-ER-based stores. Although we have yet to identify the source, this finding is similar to one we previously described in human PAEC treated with histamine where pretreatment with caffeine, ryanodine, bafilomycin, and FCCP did not abolish histamine-induced Ca²⁺ transients in PAEC (23). While the ER/SR represent the largest intracellular store in most excitable and nonexcitable cells (including PASMC and PAEC), mitochondria, Golgi apparatus, lysosomes, peroxisomes, and endosomes all represent other potential Ca²⁺ sources (25). We did observe equivalent results in response to thrombin using a similar cocktail (i.e., caffeine, ryanodine, bafilomycin, and FCCP) (Agange and Yuan, unpublished observations). The presence of this additional source of intracellular Ca²⁺ specific to PAEC and stimulated by two different agonists, histamine and thrombin, may have a significant role in vascular injury in both inflammatory and thromboembolic pulmonary vascular diseases. The activation of such stores by distinct GPCR signaling pathways (histamine G_q coupled, PAR either G_αi, G_α12/13, or G_q coupled) therefore indicate that this Ca²⁺ release from a novel intracellular store in PAEC may be important in understanding mechanisms of endothelial dysfunction in pulmonary vascular disease. The data in the current manuscript are supportive of previously published work from our laboratory describing a similar “histamine-releasable store” specific to PAEC. We are continuing to determine the precise nature of this store and to ascertain if these thrombin- and histamine-inducible stores are indeed activated via the same specific mechanism in PAEC. Thrombin and histamine have been shown to contribute in different manners to vascular endothelial barrier dysfunction, histamine in a physiological Ca²⁺-calmodulin dependent pathway and thrombin by a Ca²⁺ sensitization, potentially involving protein tyrosine phosphorylation and RhoA activation (39, 40).

Whole cell Ca²⁺ oscillations are generally associated with rhythmic cell contraction and vasomotion (24). In our experiments, we observed Ca²⁺ oscillations in response to thrombin in several PASMC studied (Figs. 6–8). The oscillations varied dramatically in frequency, but not significantly in amplitude. Oscillations could be abolished by removal of extracellular Ca²⁺ and by treatment with CPA, indicating that their underlying mechanism involved primarily Ca²⁺ influx from the extracellular space, and that their occurrence is highly regulated by reuptake of Ca²⁺ into the ER/SR by CPA-sensitive Ca²⁺-ATPase pumps. The putative link between Ca²⁺ influx and ER/SR refilling also indicates that store-operated Ca²⁺ influx via TRPC is involved. As the oscillations were observed in so few PASMC, and not in PAEC, the physiological role of the Ca²⁺ oscillations is unclear at this time.

Together, our results suggest that the mechanisms underlying thrombin-mediated calcium release are different in PASMC and PAEC. Thrombin causes Ca²⁺ release from CPA-sensitive IP₃-modulated stores and Ca²⁺ influx from the extracellular space potentially via store-operated Ca²⁺ channels in PASMC; however, in PAEC, the thrombin-mediated Ca²⁺ release is predominantly via intracellular stores insensitive to CPA, caffeine, and ryanodine. Finally, we demonstrate that only PASMC exhibit Ca²⁺ oscillations in response to thrombin, ostensibly due to release-reuptake cycling within the SR. The CPA-, caffeine-, and ryanodine-insensitive Ca²⁺ release in the PAEC may contribute substantially to thrombin-mediated vascular injury and plays a significant role in endothelial barrier dysfunction in pulmonary diseases such as CTEPH.

GRANTS

This work is supported in part by grants from National Heart, Lung, and Blood Institute Grants HL-54043, HL-064945, and HL-66012.