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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

Division of Pulmonary and Critical Care Medicine, Department of Medicine, and Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California

Submitted 17 May 2005 ; accepted in final form 8 September 2005

	ABSTRACT

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Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca²⁺ release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) were observed in the absence of extracellular Ca²⁺. Capacitative Ca²⁺ entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca²⁺ stores with 10 µM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca²⁺ influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca²⁺ release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca²⁺-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca²⁺]_cyt. In PAEC bathed in Ca²⁺-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca²⁺]_cyt but did not affect initial transient increase in [Ca²⁺]_cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca²⁺]_cyt increases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca²⁺ stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca²⁺ release.

intracellular calcium stores; oscillations; pulmonary hypertension

Histamine exerts a vasocontrictive effect, presumably via activation of H₁ receptors, but also exerts a vasodilatory effect on pulmonary arterial muscle preparations (9, 39). Whereas the dual effects of histamine on vessel caliber of human pulmonary arteries have been known for some time, the Ca²⁺ signals that underlie stimulation of PASMC and PAEC with histamine have not been extensively examined. Previous studies have shown that histamine evokes oscillatory membrane currents via release of Ca²⁺ from internal stores in freshly dissociated rabbit PASMC (64) and induces Ca²⁺ release and influx in human umbilical vein endothelial cells (37) and PAEC (47, 60). PAEC also show an increase in [Ca²⁺]_cyt in response to hypercapnia, where Ca²⁺ is released from thapsigargin (TG)-insensitive stores (38). A relatively higher dose of histamine elicits heparin-inhibitable Ca²⁺ release (concurrent with significant phosphatidylinositol hydrolysis) in human PAEC (60). The nature (and identity) of the inositol 1,4,5-trisphosphate (IP₃)-sensitive stores involved in the response to histamine, however, was not closely examined in these previous reports.

Sustained pulmonary vasoconstriction and excessive pulmonary arterial medial hypertrophy significantly contribute to the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with idiopathic pulmonary arterial hypertension (46). Agonist-mediated PASMC contraction and mitogen-mediated cell proliferation play important roles in triggering pulmonary vasoconstriction and stimulating pulmonary vascular remodeling. The agonist- or mitogen-mediated rises in [Ca²⁺]_cyt serve as important stimuli for cell contraction and motility, gene expression, cell cycle progression, and cell growth (6, 17, 20, 21, 52). Increased resting [Ca²⁺]_cyt and enhanced Ca²⁺ influx have been demonstrated to cause contraction and stimulate proliferation in PASMC and to increase AP-1 DNA binding activity and stimulate gene expression of growth factors in PAEC (13). Interactions between PASMC and PAEC have been implicated in the development of pulmonary arterial hypertension (24).

In this study, we examined and compared the histamine-mediated regulation of [Ca²⁺]_cyt in human PASMC and PAEC. Our results indicate that similar pathways of Ca²⁺ influx, such as capacitative Ca²⁺ entry (CCE), can be activated by Ca²⁺ release from internal stores or by depletion of Ca²⁺ from intracellular stores in both cell types. In PASMC, the intracellular stores involved in regulating CCE are also the major stores responsible for histamine-mediated Ca²⁺ mobilization. In PAEC, however, the internal stores involved in triggering CCE contribute only partially to histamine-mediated initial transient Ca²⁺ mobilization but play a critical role in histamine-mediated oscillatory [Ca²⁺]_cyt changes in the absence of extracellular Ca²⁺. More important, our results indicate that human PAEC may contain a hitherto undescribed histamine-releasable store that is insensitive to cyclopiazonic acid (CPA), TG, caffeine, ryanodine, FCCP, and bafilomycin and is minimally present in human PASMC.

	MATERIALS AND METHODS

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Cell preparation and culture. Human PASMC and PAEC from normal subjects (Cambrex, Walkersville, MD) were used in the experiments. PASMC were derived from intrapulmonary arteries of three individuals who were 8 (male Caucasian), 15 (male Caucasian), and 26 (female) years old, respectively, whereas PAEC were derived mainly from extrapulmonary arteries (including main pulmonary artery and left and right branches of main pulmonary artery) from two individuals ages 18 (male Caucasian) and 24 (female) years. Both PASMC and PAEC were cryopreserved at passage 3, replated onto flasks to amplify cell number for two or three passages, and then used for the proposed experiments for two or three passages. In other words, PASMC and PAEC were used for the experiments at the fifth to ninth passages.

The cells were plated onto coverslips or petri dishes and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C in smooth muscle growth medium (SmGM; Cambrex) for PASMC and in endothelium growth medium (EGM; Cambrex) for PAEC. SmGM was composed of smooth muscle basal medium (SmBM) supplemented with 5% FBS, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 µg/ml insulin. EGM was composed of endothelium basal medium (EBM) supplemented with 2% FBS, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 µg/ml insulin. Cells were subcultured or plated onto 25-mm coverslips with trypsin-EDTA buffer when 70–90% confluence was achieved. In some experiments, PAEC were growth arrested by incubation in EBM for 24 h. There was no significant morphological difference between proliferating (cultured in EGM) and growth-arrested (cultured in EBM for 24 h) cells. Our previous studies (unpublished observations) showed that most cells (>90%) cultured in the basal medium (without serum and growth factors) are in G₀/G₁ phases of the cell cycle, whereas >66% of cells cultured in the growth medium (with serum and growth factors) are in S/G₂/M phases.

Measurement of [Ca²⁺]_cyt. Human PASMC and PAEC on coverslips were placed in a recording cell chamber on the stage of an inverted microscope (Nikon). [Ca²⁺]_cyt in single PASMC and PAEC was measured with the Ca²⁺-sensitive fluorescent indicator fluo-4 AM. Cells were loaded with fluo-4 at room temperature for 15 min using a physiological salt solution (PSS) containing 10 µM fluo-4 AM, 1.5% DMSO (vol/vol), and 0.03% cremophor EL (vol/vol). The fluo-4-loaded cells were then washed with PSS for 15 min to remove excess extracellular dye and to allow intracellular esterases to cleave cytosolic fluo-4 AM into active fluo-4. The PSS contained (in mM) 140 NaCl, 5 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 HEPES, and 5.6 glucose (pH 7.4 at 22°C). In Ca²⁺-free PSS, 1–2 mM EGTA was added to chelate residual Ca²⁺. In nominally Ca²⁺-free PSS, CaCl₂ was omitted and no EGTA was added.

Excitation (488 nm) was provided by a xenon arc lamp. Fluorescence emission (>510 nm) was collected with a x40 Nikon Plan Fluor objective (0.75 numerical aperture) and a charge-coupled device (CCD) camera (Stanford Photonics). "Ca²⁺ images" or fluorescence images based on the fluorescence signals emitted from the cells were acquired at 1 Hz and stored on a Macintosh computer for later analysis. Although we may refer to the figures as Ca²⁺ images, all figures are simply Ca²⁺-dependent fluo-4 fluorescence images.

Mn²⁺ quench experiments. Human PASMC and PAEC were incubated in control PSS containing 10 µM fura-2 AM and 1.5% DMSO for 30 min. Cells were then washed for 15 min with PSS to allow cleavage of the loaded fura-2 AM to active fura-2. Excitation of fura-2 to yield Ca²⁺-insensitive signals was selected with a narrow-bandwidth excitation filter (360 nm, bandwidth 10 nm). To maximize signal-to-noise ratio, the excitation was left on for 920 ms and the CCD camera was configured to collect photons throughout this period. Fluorescence emission (510 nm) was collected at 1 Hz. To evaluate Mn²⁺ entry triggered by store depletion, cells were first incubated in Ca²⁺-free medium for 5 min and then bathed in Ca²⁺-free medium (with 1–2 mM EGTA) containing 10 µM CPA for 15 min. After incubation with CPA, the solution was then switched to nominally Ca²⁺-free solution containing 1 mM Mn²⁺. The narrow-bandwidth excitation filter ensured that the signals collected were not affected by calcium ions. The Mn²⁺ quench experiments were designed to be Ca²⁺ insensitive. The excitation wavelength for this protocol was 360 nm, which is a Ca²⁺-insensitive wavelength for fura-2. Therefore, fura-2 fluorescence should not change (or should change very little) with increases in [Ca²⁺]_cyt. Indeed, we did not observe an increase in fluorescence signals (at 360-nm wavelength) in the absence of extracellular Mn²⁺ during application of histamine to cells (both PASMC and PAEC), thus validating that the signal we detected with 360-nm excitation was not artificially altered by changes in intracellular Ca²⁺ concentration.

Measurement of area of cells. The area of PASMC and PAEC was determined using ImageJ software (version 1.32j; National Institutes of Health, Bethesda, MD). Briefly, the outlines of the cells were traced manually, and the corresponding areas of cells were calculated by the software and recorded in the computer for further analysis. Values of the area were then reported as a normalized value, A/A₀, where A is the cell area after exposure to histamine and A₀ is the area of the same cell before exposure to histamine. The percent change of cell area [(A/A₀) x 100] was used to indicate histamine-mediated changes in cell area and size.

Chemicals. All chemicals were purchased from Sigma (St. Louis, MO) and prepared as stock solutions in the appropriate solvent. Histamine was prepared as an aqueous stock solution and then diluted to the final concentrations with the appropriate saline solution. CPA and TG were dissolved in DMSO to make a stock solution of 10 mM. Aliquots of the stock solution were then diluted into the appropriate solutions to their final concentration on the day of use. Solutions’ pH values were measured after addition of the drugs and readjusted to 7.4 when necessary.

A cell-permeant analog of 3,5-bis(trifluoromethyl)pyrazole (BTP), or N-(4-(3,5-bis[(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (Calbiochem), that specifically inhibits store-operated Ca²⁺ channels with an IC₅₀ of 10 nM (23, 26, 69) was dissolved in DMSO to make a stock solution of 10 mM, which was then diluted to the final concentration in bath solutions on the day of use. A specific inhibitor of PLC, 1-(6-[([17]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122; Sigma), was used in some experiments to examine the involvement of PLC in histamine-mediated increases in [Ca²⁺]_cyt. U-73122 was dissolved in DMSO (50%) and ethanol (50%) to make a stock solution of 25 mM; aliquots were then diluted to the final concentration in bath solutions on the day of use (48, 68).

Statistics and data analysis. Regions of interest (ROI) were chosen in each cell to extract fluorescence values. The fluorescence (F) data were then normalized relative to the basal fluorescence value (F₀) of each individual ROI to yield the normalized value F/F₀. Results are summarized as means ± SE of n cells obtained from multiple coverslips. Each experiment was performed at least three times. Statistical significance was tested with paired and unpaired Student’s t-tests as well as one-way ANOVA, with a P value <0.05 taken as significant.

	RESULTS

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Histamine-mediated increases in [Ca²⁺]_cyt are comparable in human PASMC and PAEC in absence of extracellular Ca²⁺. Extracellular application of 12.5 µM histamine did not change the cell shape or size (Fig. 1, A and B) but significantly increased [Ca²⁺]_cyt in a similar fashion in PASMC and PAEC bathed in Ca²⁺-free solution (Fig. 1C), although these two cell types are morphologically different (Fig. 1A). The histamine-mediated increases in [Ca²⁺]_cyt were composed of an initial rapid transient rise followed by either a sustained or an oscillatory increase due to Ca²⁺ release and Ca²⁺ release-resequestration cycling (Fig. 1C). The oscillatory patterns of histamine-induced [Ca²⁺]_cyt responses were composed of traveling waves that were not always evident at a 1-Hz sampling rate.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Characteristics of histamine-mediated Ca2+ release in human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC). A: contrasting morphology typical of human PASMC and PAEC in which cytosolic Ca2+ concentration ([Ca2+]cyt) was measured before (control, left) and during (Hist, right) application of histamine (12.5 µM). B: summary data (means ± SE) showing % changes in area of PASMC (n = 27 cells) and PAEC (n = 42 cells) after application of histamine. Cell area was determined from images like those in A. Values are reported as the normalized value A/A0, where A is area during exposure to histamine and A0 is cell area in basal condition. C: representative records of [Ca2+]cyt changes [denoted by fluorescence value normalized to basal fluorescence value (F/F0)] in PASMC (left) and PAEC (right) before, during, and after application of 12.5 µM histamine in Ca2+-free (0Ca, with 2 mM EGTA) solution. Each trace in C represents F/F0 changes in an area of interest located in the cytosol of a single cell. D: amplitude distributions of the histamine-mediated initial peak [Ca2+]cyt transients in PASMC (top) and PAEC (bottom). Mean fluorescence ratios (F/F0) are comparable between PASMC (2.48 ± 0.08, n = 35 cells) and PAEC (2.48 ± 0.04, n = 111 cells). E: frequency distribution of the histamine-induced oscillatory changes in [Ca2+]cyt in PASMC (top) and PAEC (bottom): 48% of PASMC (17 of 35) and 40% of PAEC (44 of 111) exhibited oscillatory changes in [Ca2+]cyt upon stimulation with histamine.

In vascular smooth muscle cells, there are at least two functionally distinct receptor-mediated Ca²⁺ pools in the SR: an IP₃-releasable store that is sensitive to CPA and TG and a ryanodine-sensitive store that is sensitive to caffeine (8, 14, 58). The next set of experiments was designed to define 1) whether the Ca²⁺-storing capacity, or the concentration of releasable Ca²⁺, in the SR or endoplasmic reticulum (ER) ([Ca²⁺]_SR/ER) is different between PASMC and PAEC; 2) which intracellular stores, i.e., the CPA- or caffeine-sensitive stores, are responsible for histamine-mediated [Ca²⁺]_cyt increases in PASMC and PAEC; and 3) whether histamine-mediated Ca²⁺ release is derived from different internal stores in PASMC and PAEC.

Passive depletion of intracellularly stored Ca²⁺ with CPA causes CCE in PASMC and PAEC. Treatment of human PASMC and PAEC with 10 µM CPA, a sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor (49) that passively depletes intracellularly stored Ca²⁺ (potentially in the CPA-sensitive or IP₃-sensitive SR/ER), induced a relatively slow increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺ (Fig. 2A). Prolonged or long-term blockade of SERCA with CPA in the absence of extracellular Ca²⁺, by promoting Ca²⁺ leakage (from the SR/ER to the cytosol) and by inhibiting Ca²⁺ sequestration (from the cytosol to the SR/ER), would deplete Ca²⁺ from the SR/ER (or IP₃-sensitive stores). Emptying or reduction of Ca²⁺ from the SR/ER can then activate store-operated Ca²⁺ channels (SOC) and induce CCE (41–43). Indeed, after 10- to 15-min treatment of PASMC and PAEC with 10 µM CPA in Ca²⁺-free solution, restoration or reintroduction of extracellular Ca²⁺ induced a rise in [Ca²⁺]_cyt apparently due to CCE (Fig. 2, A and B). The results clearly indicate that CCE can be induced successfully in both cell types by a 15-min incubation with CPA.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Cyclopiazonic acid (CPA)-mediated passive store depletion induces capacitative Ca2+ entry (CCE) in human PASMC and PAEC. A: representative records (a) of [Ca2+]cyt changes (denoted by F/F0) in PASMC (left) and PAEC (right) before, during, and after extracellular application of 10 µM CPA in the absence (0Ca) or presence of extracellular Ca2+. Summarized data (b; means ± SE) show the time course of CPA-mediated changes in [Ca2+]cyt in PASMC (n = 36 cells) and PAEC (n = 23 cells). Reintroduction of extracellular Ca2+ 15 min after CPA treatment increased [Ca2+]cyt, apparently as a result of CCE (right). The records of CCE-mediated [Ca2+]cyt changes in the absence of extracellular Ca2+ (left) and the records of CCE-mediated [Ca2+]cyt changes after restoration of extracellular Ca2+ (right) are from different cells. B: representative records (a) and summarized data (b; means ± SE) showing Mn2+ quench of fura-2 fluorescence signals at 360 nm in PASMC (n = 24 cells) and PAEC (n = 16). The cells were pretreated with CPA (10 µM) for 15 min before addition of Mn2+. C: summarized data (means ± SE) showing the time course of CCE-induced increases in [Ca2+]cyt (denoted by fluo-4 fluorescence signals, F/F0) in PASMC (a; n = 43) and PAEC (b; n = 59) in the absence (Cont) or presence (BTP2) of 1 µM BTP2. The control curves were constructed from the data presented in Ab (right).

The initial rise in [Ca²⁺]_cyt induced by CPA (due to Ca²⁺ leakage from SR/ER to cytosol) in the absence of extracellular Ca²⁺ was kinetically different (P < 0.05) between PASMC and PAEC (Fig. 2Ab, left), whereas the [Ca²⁺]_cyt increase due to store depletion-mediated CCE was virtually similar in PASMC and PAEC (Fig. 2Ab, right, B, and C). The quenching experiments (Fig. 2B) further indicate that CPA-mediated CCE is not different between PASMC and PAEC.

As shown in a separate set of Mn²⁺ quench experiments, the CPA-mediated Ca²⁺ influx was partially inhibited by nifedipine (1 µM) in PASMC (by 39%; Fig. 3A) but not in PAEC (Fig. 3B). In PASMC, CCE may shift the resting membrane potential to a less negative level and activate nifedipine-sensitive, voltage-dependent Ca²⁺ channels, contributing to the sustained Ca²⁺ influx. However, in PAEC, nifedipine had no effect on the CPA-mediated CCE because these cells do not express voltage-dependent Ca²⁺ channels.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Effect of nifedipine on CPA-mediated Mn2+ entry in human PASMC and PAEC. Left: background photobleaching rate (in absence of extracellular Mn2+) associated with the experiments for each cell type [PASMC (A) and PAEC (B)]. Right: summarized data (means ± SE) showing Mn2+ quench of fura-2 fluorescence signals in PASMC (A) and PAEC (B) in the absence (CPA; n = 25 for PASMC and n = 59 for PAEC) or presence (CPA+Nif; n = 40 for PASMC and n = 137 for PAEC) of 1 µM nifedipine. The cells were pretreated with 10 µM CPA for 15 min before addition of Mn2+. Gray lines denote extrapolation of the data points (0–35 s) before extracellular Mn2+ was applied to cells (time of Mn2+ application is indicated by vertical dashed lines).

Differential contribution of CPA-sensitive stores to histamine-mediated Ca²⁺ release in PASMC and PAEC. Treatment of PASMC and PAEC with CPA (10 µM for 15 min) depletes intracellularly stored Ca²⁺ in the CPA-sensitive stores (e.g., IP₃-sensitive SR/ER). In PASMC bathed in Ca²⁺-free solution, passive depletion of Ca²⁺ from the CPA-sensitive stores, in addition to causing CCE (see Figs. 2 and 3), almost abolished the histamine-mediated transient increase in [Ca²⁺]_cyt (Fig. 4A, left, and B), whereas in PAEC, CPA treatment did not eliminate the histamine-induced rise in [Ca²⁺]_cyt (Fig. 4A, right, and B). Only a small proportion of PASMC showed brief, low-amplitude, transient responses to application of histamine after depletion of intracellular stores by CPA, indicating that internal stores in PASMC were largely depleted by CPA. However, all PAEC (51 of 51 cells examined) treated with CPA still showed a robust transient Ca²⁺ release in response to application of histamine. Maximal mean fluorescence observed was 1.68 ± 0.03 (n = 51 cells) in PAEC treated with CPA (Fig. 4B). These observations indicate that 15-min CPA treatment could largely deplete intracellular stores that are responsible for histamine-induced Ca²⁺ release in PASMC but could not deplete the histamine-releasable intracellular stores in PAEC. These data further suggest that, in addition to inducing Ca²⁺ influx through Ca²⁺-permeable channels in the plasma membrane, histamine-mediated increases in [Ca²⁺]_cyt occur by promoting Ca²⁺ release from different intracellular stores in PASMC and PAEC.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effects of pretreatment with CPA on histamine-mediated Ca2+ release in human PASMC and PAEC. A: representative traces of histamine-stimulated changes in [Ca2+]cyt (denoted by F/F0) in PASMC (left) and PAEC (right) after incubation in Ca2+-free (0Ca) solution containing 10 µM CPA. A small proportion of PASMC demonstrated low amplitude and transient responses, whereas a majority of the cells showed no response to histamine. All PAEC, in contrast, showed a robust transient response to stimulation with histamine. B and C: summarized data (means ± SE) showing the time course (B) and averaged maximal values (C) of histamine-mediated increases in [Ca2+]cyt (F/F0) in PASMC (n = 36) and PAEC (n = 51) after treatment with 10 µM CPA in 0Ca solution. ***P < 0.001 vs. PASMC. D: comparison of the amplitude (left) and kinetics (right) of histamine-induced increases in [Ca2+]cyt (denoted by F/F0) in PAEC treated with (CPA, n = 111) or without (Cont; curve constructed from data shown in Fig. 1B) 10 µM CPA. Data shown at right are normalized to the maximal level of changes in [Ca2+]cyt; the maximum F/F0 value was set at 100% according to the formula [(F/F0) – 1]/(Fmax – 1). Histamine-mediated increases in [Ca2+]cyt were lower in amplitude and faster in decay after treatment with CPA.

Caffeine-sensitive stores are not histamine-releasable stores in PASMC and PAEC. In human PASMC, short-term (<5 min) treatment with 1 mM caffeine did not induce an obvious increase in [Ca²⁺]_cyt and had no effect on histamine-mediated increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺. Long-term (15 min) pretreatment with caffeine slightly (but insignificantly) increased the histamine-induced [Ca²⁺]_cyt release (Fig. 5A). The amplitude of histamine-induced initial peak [Ca²⁺]_cyt transient in PASMC treated with 1 mM caffeine for 15 min (F/F₀, 3.22 ± 0.22; n = 14 cells from 6 experiments) was slightly but statistically significantly (P < 0.05) higher than in control PASMC (F/F₀, 2.48 ± 0.08; n = 35 cells from 4 experiments) and PASMC treated with 1 mM caffeine for 5 min (F/F₀, 2.51 ± 0.16; n = 23 cells from 5 experiments) (Fig. 5B). The percentage of cells showing oscillatory responses to histamine was fairly similar in control (29%) and caffeine-treated (25% at 5 min and 50% at 15 min) cells. The percentage of cells showing oscillations from previous experiments in Ca²⁺-free solution was 49% (see Fig. 1C), which was similar to that in cells treated with caffeine for 15 min (50%). These results indicate that most of the Ca²⁺ in human PASMC is stored in the CPA-sensitive stores but that very little is stored in "caffeine-sensitive stores." The Ca²⁺ released from caffeine-sensitive stores can actually be sequestered by CPA-sensitive SERCA to the CPA-sensitive stores in vascular smooth muscle cells (14–16, 58); this is probably why histamine-mediated rise in [Ca²⁺]_cyt due to Ca²⁺ release from intracellular stores is slightly enhanced in PASMC treated with caffeine (Fig. 5A, right, and B).

View larger version (27K): [in this window] [in a new window]   Fig. 5. Effect of pretreatment with caffeine on histamine-mediated Ca2+ release in human PASMC. A: summarized data (means ± SE) showing the time course of [Ca2+]cyt changes in response to extracellular application of 1 mM caffeine (Caf, in Ca2+-free solution, n = 15; left) and to histamine (right) in control cells and cells pretreated with 1 mM caffeine for 5 or 15 min (n = 14–24). B: summarized data (means ± SE) showing the amplitude of histamine-mediated initial peak increase in [Ca2+]cyt. *P < 0.05 vs. 0 and 5 min. C: % of cells in which oscillatory [Ca2+]cyt increases were observed in response to histamine in control cells and cells treated with caffeine (5 or 15 min).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Effect of pretreatment with caffeine on histamine-mediated Ca2+ release in human PAEC. Representative traces (A) and summarized data (B; means ± SE) of [Ca2+]cyt changes (denoted by F/F0) in response to 1 mM caffeine in the Ca2+-free (0Ca) solution (left; n = 93) and to 12.5 µM histamine in cells incubated in Caf-containing 0Ca solution (right; for 15 min, n = 77). Application of caffeine alone did not evoke robust increases in [Ca2+]cyt (only 6 of 93 cells showed very small and brief transients in response to caffeine). Histamine-mediated oscillatory changes in [Ca2+]cyt could still be observed in some cells in the presence of caffeine (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effects of pretreatment with thapsigargin (TG) on histamine-mediated Ca2+ mobilization in human PAEC. A: representative traces of [Ca2+]cyt changes in PAEC in response to application of 1 µM TG (left) and to application of 12.5 µM histamine (after 15 min of exposure to TG; right) under Ca2+-free conditions. B: summarized data (means ± SE) of [Ca2+]cyt changes in PAEC (denoted by F/F0) in response to 1 µM TG in Ca2+-free (0Ca) solution (left; n = 76) and to 12.5 µM histamine in cells incubated in TG-containing 0Ca solution (right; for 15 min, n = 80).

View larger version (29K): [in this window] [in a new window]   Fig. 8. Effects of pretreatment with CPA, caffeine, FCCP, and bafilomycin on histamine-mediated Ca2+ mobilization in human PAEC. Representative traces (A) and summarized data (B; means ± SE) of [Ca2+]cyt changes (denoted by F/F0) in response to extracellular application of 12.5 µM histamine in Ca2+-free (0Ca) solution in PAEC pretreated with 10 µM CPA alone for 30 min (a; n = 107), CPA + 1 mM caffeine (b; n = 114), CPA + 5 µM FCCP (c; n = 71), or CPA + 0.5 µM bafilomycin (d; n = 80). Pretreatment with CPA alone, CPA + caffeine, CPA + FCCP, or CPA + bafilomycin did not significantly affect histamine-mediated increases in [Ca2+]cyt in PAEC superfused with Ca2+-free solution.

To examine whether mitochondria and lysosomes are potential histamine-releasable Ca²⁺ stores in PAEC, we investigated the effect of histamine on cells treated with FCCP, a mitochondrial uncoupler that depletes Ca²⁺ from mitochondria (12), and bafilomycin, a vacuolar proton pump inhibitor (or vacuolar H⁺-ATPase) that depletes Ca²⁺ from lysosome-related organelles in various cell types (29). Bath application of 5 µM FCCP to PAEC that had previously been incubated in 10 µM CPA (15 min) evoked a small, detectable increase in [Ca²⁺]_cyt (data not shown). A robust, brief transient could still be evoked by histamine in PAEC even after treatment (9 min) with combined CPA and FCCP (Fig. 8, Ac and Bc). The amplitudes of histamine-induced [Ca²⁺]_cyt transients in PAEC treated with CPA (30 min), CPA + caffeine, and CPA + FCCP were all comparable (Fig. 8, Ba–Bc). Furthermore, combined treatment with CPA + bafilomycin (0.5 µM for 15 min) also failed to eliminate histamine-induced [Ca²⁺]_cyt transients in PAEC (Fig. 8, Ad and Bd). These results suggest that the histamine-induced transient Ca²⁺ release is neither from CPA- and caffeine-sensitive SR/ER nor from mitochondria or lysosome-like organelles.

To further confirm that a ryanodine-sensitive store is not involved in the residual response to histamine, we examined whether histamine was able to induce Ca²⁺ release from intracellular stores in PAEC treated with combined CPA, caffeine, and ryanodine. As shown in Fig. 9A, histamine still caused a significant increase in [Ca²⁺]_cyt in PAEC treated for 15 min with CPA (10 µM), caffeine (1 mM), and ryanodine (100 µM) in the absence of extracellular Ca²⁺. Because a high concentration of ryanodine efficiently blocks Ca²⁺ release through ryanodine receptors, these experiments provide further evidence that there is a unique histamine-releasable store in human PAEC, which is neither the CPA- or TG-sensitive store nor the ryanodine- or caffeine-sensitive store. Although this unique store was insensitive to CPA, TG, caffeine, ryanodine, FCCP, and bafilomycin, our study indicated that the histamine-induced Ca²⁺ release from this store still depended on activation of PLC. Inhibition of PLC with U-73122 (50 µM) in the presence of CPA (10 µM) abolished the histamine-mediated Ca²⁺ release in PAEC (Fig. 9B). These data suggest that the histamine-releasable (CPA and TG insensitive) store is functionally coupled to PLC; receptor-mediated activation of PLC is required to trigger Ca²⁺ release from this store.

View larger version (26K): [in this window] [in a new window]   Fig. 9. Effect of pretreatment with the combination of CPA, caffeine, and ryanodine, or pretreatment with U-73122, on histamine-mediated Ca2+ mobilization in human PAEC. A: representative traces (a) and summary data (b; means ± SE) of [Ca2+]cyt changes in PAEC in response to application of CPA (10 µM), caffeine (Caf, 1 mM), and ryanodine (Ry, 100 µM) (left; n = 38) and to application of 12.5 µM histamine (after 15 min of exposure to CPA + Caf + Ry; right, n = 42), under Ca2+-free conditions. B: representative traces (a) and summary data (b; means ± SE) of [Ca2+]cyt changes in PAEC in response to application of histamine (after 15 min of exposure to 10 µM CPA and 50 µM U-73122). Data (n = 20) in b show abolition of the response to histamine in PAEC that were pretreated with CPA + U-73122.

View larger version (19K): [in this window] [in a new window]   Fig. 10. Effect of pretreatment with CPA on histamine-mediated Ca2+ mobilization in growth-arrested human PAEC. Representative traces (A) and summarized data (B; means ± SE) of [Ca2+]cyt changes in PAEC (precultured in endothelium basal medium for 24 h) in response to application of 10 µM CPA (left; n = 74) and to application of 12.5 µM histamine (after 15 min of exposure to CPA; right, n = 69), under Ca2+-free conditions. Almost all (66 of 69) growth-arrested cells treated with CPA showed a response to bath application of histamine similar to that of proliferating PAEC cultured in endothelium growth medium.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

CPA-sensitive Ca²⁺ stores contribute to histamine-mediated Ca²⁺ release. On activation of respective receptors by agonists or ligands (e.g., histamine), Ca²⁺ release may be derived from one or more types of intracellular Ca²⁺ stores including the SR/ER, acidic stores, Golgi apparatus, and mitochondria. Ca²⁺ can be released via action of IP₃ on IP₃ receptors (IP₃R) located on the internal store membrane and/or via Ca²⁺-induced Ca²⁺ release from stores that contain ryanodine receptors. The SR/ER Ca²⁺ stores can be further subdivided into spatially and functionally distinct subcompartments that can unload and refill separately from each other. CPA- and IP₃- as well as caffeine- and ryanodine-sensitive stores have been well demonstrated in vascular smooth muscle cells, neurons, and astrocytes (14–16, 58).

Our results from the present study indicate that the histamine-mediated Ca²⁺ release in human PASMC is derived mainly from CPA- and IP₃-sensitive stores because incubation of cells in 10 µM CPA was able to eliminate the response to stimulation with histamine. In PAEC, a major proportion of Ca²⁺ is also released from CPA- and IP₃-sensitive stores in response to histamine stimulation, because treatment of PAEC with CPA significantly attenuated the histamine-mediated rise in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺. In addition, preincubation of PAEC with CPA also altered the kinetics of the histamine-mediated rise in [Ca²⁺]_cyt; the CPA treatment eliminated histamine-induced [Ca²⁺]_cyt oscillations. These observations suggest that CPA- and IP₃-sensitive stores play a significant role in histamine-stimulated increases in [Ca²⁺]_cyt in both PASMC and PAEC.

Another significant difference between PASMC and PAEC shown in our study was that histamine-mediated oscillation frequency was greater in PAEC than in PASMC (Fig. 1E). Ca²⁺ oscillations are regulated by 1) the balance between Ca²⁺ release from intracellular stores through Ca²⁺ release channels and Ca²⁺ sequestration back into the stores via Ca²⁺-Mg²⁺-ATPase in the SR/ER (SERCA), 2) the balance between Ca²⁺ influx through Ca²⁺ channels and Ca²⁺ extrusion via Ca²⁺-Mg²⁺-ATPase in the plasma membrane, and 3) the subcellular organization of the Ca²⁺ release and sequestration system (18, 65). Because PAEC are morphologically smaller than PASMC and tend to have a much smaller cytosol area or store-to-surface distance than PASMC, the relatively higher frequency of Ca²⁺ oscillations in PAEC might be due to 1) a shorter distance between the SR/ER (or Ca²⁺ release channels and SERCA) and the plasma membrane (or Ca²⁺-permeable channels and Ca²⁺ pumps), 2) a smaller subplasmalemmal area, and 3) different organization of Ca²⁺ release and uptake systems. It is unclear whether the activation threshold of Ca²⁺ channels and Ca²⁺ pumps in the SR and plasma membrane differs between human PASMC and PAEC.

Depletion of CPA-sensitive Ca²⁺ stores is sufficient to induce CCE in PASMC and PAEC. Maintaining a high concentration (or capacity) of Ca²⁺ in the SR/ER ([Ca²⁺]_SR/ER) is not only important in the signal transduction process required for cell contraction, migration, and gene expression but also necessary for appropriate lipid and protein synthesis in the SR/ER. Depletion of Ca²⁺ from intracellular stores (e.g., the SR/ER) as a result of activation of IP₃ receptors or inhibition of SERCA has been demonstrated to mediate CCE by opening SOC in many cell types. CCE plays a significant role in maintaining the filling state of SR/ER (41). The intracellular Ca²⁺ store that contributes to regulating SOC activity or controlling CCE can be depleted actively by opening Ca²⁺ release channels or passively by blocking Ca²⁺-Mg²⁺-ATPase in the SR/ER. Our results from this study indicate that emptying or reduction of IP₃-sensitive stores by CPA is sufficient to induce CCE in both human PASMC and PAEC. The CPA-mediated CCE in PASMC and PAEC is sensitive to the SOC blocker BTP2 (23, 26, 69). At present, the exact mechanisms involved in the induction of CCE are still unclear. Several models have been described: 1) a chemical coupling mechanism in which a soluble messenger released from intracellular stores serves as a trigger to open plasmalemmal SOC and induce CCE, 2) a conformational coupling mechanism in which the direct or indirect (via cytoskeleton) interaction between SOC in the plasma membrane and IP₃R in the SR mediates opening of SOC and induction of CCE, and 3) a physical coupling mechanism in which the SR is physically, yet indirectly, tethered to the sarcolemmal SOC by one or more scaffolding proteins (40, 42, 45, 61).

Histamine-mediated Ca²⁺ release derives from multiple internal stores in PAEC. A significant difference exists between PASMC and PAEC with regard to the stores involved in the response to stimulation with histamine. In PASMC, pretreatment with CPA, which depletes CPA- or IP₃-sensitive stores, almost abolished histamine-mediated increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺. In PAEC, pretreatment with CPA did not eliminate histamine-mediated increase in [Ca²⁺]_cyt due to Ca²⁺ mobilization, although CPA treatment affected the kinetics of histamine-mediated Ca²⁺ release. These data indicate that the CPA-sensitive Ca²⁺ stores are not the only stores that are responsible for histamine-mediated Ca²⁺ release in PAEC, although the level of Ca²⁺ in these stores contributes to the regulation of CCE and activity of SOC.

In addition to the SR/ER, the membranes of other cytoplasmic organelles, such as the mitochondrion, Golgi apparatus, lysosome, peroxisome, and endosome, also contain transport proteins that are responsible for the import and export of specific metabolites and ions (e.g., Ca²⁺, Na⁺, and K⁺). In eukaryotic cells, approximately half the total area of membrane encloses the SR/ER, whereas 15% of the total cell volume is occupied by the SR/ER. Therefore, the SR/ER is undoubtedly the most predominant intracellular Ca²⁺ store in mammalian and human cells. Nevertheless, in many cell types, the mitochondrion, Golgi apparatus, lysosome, and endosome have also been shown to function as intracellular Ca²⁺ stores (36). Because the CPA-sensitive SR/ER is not the only intracellular organelle to store agonist-releasable Ca²⁺ in PAEC, it is important to define other potential stores that are responsible for histamine-mediated Ca²⁺ release.

Pretreatment of PAEC with the combination of CPA (10 µM) + caffeine (1 mM) + ryanodine (100 µM), CPA + FCCP (5 µM), or CPA + bafilomycin A1 (0.5 µM) (10) all failed to eliminate histamine-mediated increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺. In PASMC bathed in Ca²⁺-free solutions, however, pretreatment with effective doses of CPA (5–10 µM) alone almost abolished histamine-mediated increase in [Ca²⁺]_cyt. These results suggest that PAEC contain an alternate source of histamine-releasable Ca²⁺ that is minimally present in PASMC. This internal Ca²⁺ store is insensitive to CPA (or TG) and caffeine (or ryanodine), so it is not the CPA- and IP₃-sensitive SR/ER or caffeine- and ryanodine-sensitive SR/ER.

An intimate relationship exists between the SR/ER and mitochondria. Ca²⁺ released from the SR/ER can be taken up by neighboring mitochondria and result in a transient increase in mitochondrial [Ca²⁺]. The continuous flux of Ca²⁺ into and out of mitochondria, which occurs during stimulation with histamine of some cell types, is then linked to the refilling of nearby SR/ER stores (3, 33, 35). The inability of CPA and FCCP (a proton ionophore that dissipates the mitochondrial membrane potential and depletes Ca²⁺ from mitochondria) to eliminate histamine-mediated Ca²⁺ release, however, suggests that mitochondria do not directly contribute to the histamine-induced elevation of [Ca²⁺]_cyt in PAEC.

The Ca²⁺ pools (or the SR/ER) that are resistant to SERCA inhibitors (e.g., CPA, TG) have been shown in some mammalian cells (56, 63). The bafilomycin-sensitive V-type ATPases (10) that are associated with acidic Ca²⁺ storage compartments may also exist in the SR/ER and other unknown internal stores. However, the inability of CPA and bafilomycin to eliminate histamine-mediated Ca²⁺ mobilization indicates that the internal stores utilizing bafilomycin-sensitive V-type ATPases do not directly contribute to histamine-mediated Ca²⁺ release in human PAEC.

Together, the results from this study indicate that human PAEC possess a unique internal Ca²⁺ store that is minimally present in PASMC. The identity of this PAEC-specific internal Ca²⁺ store is presently unknown; whether it is the Golgi apparatus requires further investigation. Identification of this novel Ca²⁺ pool in human PAEC and its possible contribution to normal or pathological cellular processes represents a new dimension in our understanding of Ca²⁺ homeostasis and its effects on pulmonary vascular endothelial function. Although it seems not to be coupled to CCE, this unique internal Ca²⁺ store (insensitive to CPA, TG, caffeine, ryanodine, FCCP, and bafilomycin) has sufficient capacity to store and release Ca²⁺ to mediate histamine-induced functional effect on PAEC. This unique store may serve as a "reserved" resource for agonist- and receptor-mediated increases in [Ca²⁺]_cyt, especially when PAEC are exposed to multiple agonists and growth factors. Amplitude and frequency modulation of intracellular Ca²⁺ signals play important roles in regulating cell contraction, mobility, proliferation, differentiation, and apoptosis; this unique Ca²⁺ store in human PAEC may greatly contribute to the agonist (or histamine)-mediated activation of cytoplasmic enzymes, such as nitric oxide synthase, adenylyl cyclases, and calmodulin kinases, and of transcription factors, such as c-Jun, c-Fos, and cAMP response element binding protein (5, 7, 54).

In summary, a rise in [Ca²⁺]_cyt in PASMC serves as a major trigger for pulmonary vasoconstriction, whereas an increase in [Ca²⁺]_cyt in PAEC, in addition to inducing cell mobility and migration, may also be an important stimulus for activating enzymes that produce endothelium-derived relaxing factors (e.g., nitric oxide). Our data from this study demonstrate the following. 1) PASMC and PAEC share the same intracellular stores and mechanisms for triggering CCE but use different intracellular stores and mechanisms for agonist (e.g., histamine)-mediated Ca²⁺ mobilization. 2) Both PASMC and PAEC are able to generate oscillations in the absence of extracellular Ca²⁺; the Ca²⁺ oscillation in PASMC seems to be due mainly to release-uptake cycling through the same store (i.e., CPA-sensitive store), whereas the Ca²⁺ oscillation in PAEC is caused predominantly by release-uptake cycling among different stores. 3) Multiple intracellular Ca²⁺ stores in PAEC ensure agonist-mediated Ca²⁺ release or signaling; a unique internal store that is not required for triggering CCE but partially involved in agonist-induced Ca²⁺ release exists in PAEC. 4) The major intracellular stores in PASMC are CPA-sensitive stores that not only are responsible for agonist-mediated Ca²⁺ mobilization but also contribute to controlling the activity of SOC and regulation of CCE.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

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

	ACKNOWLEDGMENTS

 
We thank Dr. C. V. Remillard for assistance in preparation of the manuscript and A. Nicholson for assistance in preparation of human vascular smooth muscle and endothelial cells.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. X.-J. Yuan, Division of Pulmonary and Critical Care Medicine, Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Drive, MC 0725, La Jolla, CA 92093-0725 (e-mail: xiyuan{at}ucsd.edu)

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

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