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Am J Physiol Lung Cell Mol Physiol 293: L1135-L1142, 2007. First published August 10, 2007; doi:10.1152/ajplung.00432.2006
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Reduced store-operated Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats

Michael L. Paffett, Jay S. Naik, Thomas C. Resta, and Benjimen R. Walker

Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico

Submitted 1 November 2006 ; accepted in final form 6 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic hypoxia (CH)-induced pulmonary hypertension may influence basal endothelial cell (EC) intracellular Ca2+ concentration ([Ca2+]i). We hypothesized that CH decreases EC [Ca2+]i associated with membrane depolarization and reduced Ca2+ entry. To test this hypothesis, we assessed 1) basal endothelial Ca2+ in pressurized pulmonary arteries and freshly isolated ECs, 2) EC membrane potential (Em), 3) store-operated Ca2+ current (ISOC), and 4) store-operated Ca2+ (SOC) entry in arteries from control and CH rats. We found that basal EC Ca2+ was significantly lower in pressurized pulmonary arteries and freshly isolated ECs from CH rats compared with controls. Similarly, ECs in intact arteries from CH rats were depolarized compared with controls, although no differences were observed between groups in isolated cells. ISOC activation by 1 µM thapsigargin displayed diminished inward current and a reversal potential closer to 0 mV in cells from CH rats compared with controls. In addition, SOC entry determined by fura 2 fluorescence and Mn2+ quenching revealed a parallel reduction in Ca2+ entry following CH. We conclude that differences in the magnitude of SOC entry exist between freshly dispersed ECs from CH and control rats and correlates with the decrease in basal EC [Ca2+]i. In contrast, basal EC Ca2+ influx is unaffected and membrane depolarization is limited to intact arteries, suggesting that Em may not play a major role in determining basal EC [Ca2+]i following CH.

pulmonary hypertension; electrochemical gradient and calcium entry

THE PULMONARY CIRCULATION is characterized by a low-pressure, low-resistance profile with little to no vascular tone in nonpathological conditions. Chronic exposure to hypoxia leads to sustained pulmonary hypertension (32) and altered endothelial function (3). However, the mechanisms that influence endothelial Ca2+ homeostasis within the pulmonary circulation following chronic hypoxia (CH) are largely unknown. Endothelial intracellular Ca2+ concentration ([Ca2+]i) is essential for the synthesis and release of vasoactive compounds that modulate vascular reactivity (14, 47) and the regulation of paracellular fluid flux (30, 44). With the exception of the pulmonary microvasculature (43, 45), voltage-gated Ca2+ channels have scarcely been observed in pulmonary arteriolar endothelium, and plasmalemmal Ca2+ entry through other entry pathways is largely dependent on membrane potential (Em; see Refs. 17 and 42). In addition, the formation of nitric oxide (NO) and prostacyclin are largely dependent on agonist-induced hyperpolarization and Ca2+ influx (21). Nonselective cation channels and store-operated Ca2+ (SOC) entry are the best-characterized endothelial Ca2+ entry pathways (18, 44, 46); however, limited information exists regarding their functional roles in determining endothelial [Ca2+]i in the pulmonary vasculature, especially in the hypertensive setting. Although a variety of physiological stimuli such as shear stress, cytokines, and growth factors have been shown to affect endothelial cell (EC) Ca2+ entry (22, 26, 31), the contribution of CH-induced pulmonary hypertension to EC Ca2+ homeostasis has been minimally investigated.

Controversy exists regarding the effect of hypoxia per se on pulmonary artery EC Ca2+ homeostasis. Hampl et al. (13) found that the initial transient increase in [Ca2+]i elicited by acute hypoxia in bovine pulmonary artery ECs was presumably from enhanced intracellular store release, although a subsequent depolarization resulting in a concomitant decrease in [Ca2+]i has also been observed (40). Furthermore, others (2) have found a role for the Na+/Ca2+ exchanger (NCX) operating in reverse mode resulting in an influx of Ca2+ in human umbilical vein ECs following acute hypoxia. Conversely, Murata et al. (28) showed decreased carbachol-induced relaxation in pulmonary arteries from CH hypertensive rats that was associated with a blunted sustained rise in EC [Ca2+]i indicative of reduced Ca2+ influx. More recently, the effects of CH have been studied in cultured human pulmonary artery ECs in which basal [Ca2+]i and SOC entry were enhanced (11). This finding is one of the first correlating enhanced SOC entry with increased basal [Ca2+]i. However, it is unknown if this effect of CH on endothelial SOC entry and basal [Ca2+]i observed in cultured cells occurs in the intact vasculature during CH-induced pulmonary hypertension, where factors such as altered shear stress and humoral influences could affect endothelial function. Indeed, observations of diminished pulmonary NO production following CH despite increased expression of endothelial NO synthase (eNOS; see Ref. 23) may be due to alterations in more than one determinant of NO synthesis of which basal EC [Ca2+]i could be reduced rather than increased following CH in the intact vasculature.

In the present study, we examined EC [Ca2+]i homeostasis in intact arteries and freshly dispersed cells from control and CH-induced pulmonary hypertensive rats. We employed electrophysiology and fluorescence techniques to test the hypothesis that CH-induced pulmonary hypertension is associated with a decrease in basal endothelial [Ca2+]i in which membrane depolarization and reduced SOC entry pathways may contribute to changes in endothelial Ca2+ homeostasis.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Exposure of rats to CH. Male Sprague-Dawley rats (200–250 g; Harlan Industries) were used for all studies. Rats exposed to CH were placed in a hypobaric chamber with barometric pressure maintained at ~380 torr for 4 wk. Age-matched control rats were housed in similar cages under ambient barometric pressure (~630 torr). The hypobaric chamber was opened 3 times/wk to provide fresh rat chow, water, and clean bedding. All animals were maintained on a 12:12-h light-dark cycle.

Cannulation of small pulmonary arteries. Rats were euthanized with pentobarbital sodium (32.5 mg ip), and the heart and lungs were exposed by midline thoracotomy. The left lung was rapidly excised and placed in ice-cold physiological saline solution (PSS) containing the following (in mM): 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose. The PSS was aerated with 10% O2-6% CO2-balance N2 gas mixture throughout the course of the dissection, cannulation, and experiments. The left lung was pinned out in a Silastic-coated dissection dish filled with ice-cold PSS. Intrapulmonary arteries [3rd- and 4th-order, 200–400 µm inner diameter (ID)] were dissected from the cranial most region of the left lung, and the parenchymal lung tissue was removed before transferring to an arteriograph (CH-1; Living Systems). Next, the proximal end of the artery was cannulated and secured with a single strand of silk suture. The vessel lumen was gently flushed to remove any blood before to cannulation of the distal end. The arteriograph was transferred to an inverted microscope (TS-100F; Nikon), and the preparation was superfused with PSS equilibrated with a normoxic gas mixture at 37°C for 30 min before experimentation. PO2, PCO2, and pH of the superfusate were periodically assessed by a blood gas analyzer (Radiometer).

Measurement of EC [Ca2+]i in pressurized small pulmonary arteries. EC [Ca2+]i of pressurized pulmonary arteries was assessed using the ratiometric Ca2+-sensitive fluorescent indicator fura 2-AM (Invitrogen). Selective endothelial loading has been described previously (5, 19, 25) and consisted of luminal perfusion of a 3 µM fura 2 solution mixed with 0.5 vol of 20% pluronic acid (0.05%) in PSS via a pressure servocontrolled peristaltic pump (Living Systems). The Ca2+-sensitive dye was loaded at ~23°C for 3 min at a pressure of 5 mmHg and subsequently washed for 10 min at 37°C. Following some experiments, the endothelium was disrupted with moose main fiber to assess the degree of fluorescence loss indicating selective endothelial loading. Ratiometric changes in EC [Ca2+]i at luminal pressures of 5, 12, and 35 mmHg were determined by alternating a xenon arc lamp light source between 340 and 380 nm bandpass filters at 1 Hz (Ionoptix Hyperswitch) and collecting the interleaved fura 2 fluorescent emissions at 510 nm with a photomultiplier tube. Off-line ratiometric analysis was performed with Ion Wizard 5.0 software (Ionoptix).

Endothelial identification and isolation. Small pulmonary arteries were isolated in ice-cold HEPES buffered PSS (HBSS) solution. The HBSS contained the following (in mM): 150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose, titrated to pH 7.4 with NaOH. Intrapulmonary arteries (3rd- and 4th-order, 200–400 µm ID) were dissected from the cranial most region of the left lung and carefully cleaned of surrounding lung parenchyma. Arteries were cut longitudinally and incubated for 1 h in basal endothelial growth medium with 10% BSA containing 10 µg/ml of acetylated low-density lipoprotein label with a fluorescent probe, 1,1' -dioctadecyl-3,3,3',3' -tetramethyl-indocarbocyanine perchlorate (Ac-LDL-DiI) at 37°C. Following the labeling protocol, adapted from Hogg et al. (15), pulmonary arteries were treated with 0.2 mg/ml dithiothreitol and 0.2 mg/ml papain in HBSS for 45 min at 37°C. Vessels were carefully removed from the digestion solution and placed in 1 ml of HBSS containing 2 mg/ml BSA. Single pulmonary ECs and endothelial sheets were then released by gentle trituration with a small-bore fire-polished Pasteur pipette and stored at 4°C. One to two drops of the cell suspension were placed on a poly-L-lysine-coated glass cover slip mounted on an inverted fluorescence microscope for 30 min before superfusion. Pulmonary ECs were identified by the selective uptake of fluorescently labeled probe Ac-LDL-DiI using a rhodamine filter before each electrophysiology experiment.

Measurement of EC Em. EC Em was recorded from 3rd- and 4th-order pulmonary arteries using sharp electrodes. Artery strips were secured in a 35-mm culture dish with the luminal surface exposed. The preparation was superfused (2 ml/min) with a HBSS recording solution warmed to 37°C. Sharp electrodes (60–80 M{Omega}), initially backfilled with Lucifer yellow (16.6 mg/ml in 1 M LiCl) followed by 1 M KCl, permitted post hoc epifluorescence dye identification of ECs vs. inadvertent impalement of vascular smooth muscle (VSM) cells by the distinct cellular morphological and dye transfer characteristics of each cell type previously described (8). Alternatively, freshly dispersed ECs obtained from small pulmonary arteries described above were superfused with HBSS under constant flow (2 ml/min) at room temperature (~23°C). Em of isolated EC sheets was recorded by sharp electrode using a Neuroprobe amplifier (A-M Systems). Analog output was low pass filtered at 1 kHz and visualized and analyzed by a Windows-based oscilloscope (Axoscope; Axon Instruments).

Measurement of isolated EC store-operated Ca2+ current. Freshly dispersed ECs obtained from small pulmonary arteries described above were superfused under constant flow (2 ml/min) at room temperature (~23°C) in an extracellular solution containing (in mM): 120 tetraethylammonium aspartate, 5 calcium aspartate, 5 CaCl2, 10 HEPES, and 0.5 3,4-diaminopyridine, titrated to pH 7.4 with methane sulfonic acid. Currents were recorded in the conventional whole cell patch-clamp configuration from electrically isolated single ECs in voltage-clamp mode. Store-operated Ca2+ current (ISOC) was measured in freshly dispersed cells in which the intracellular recording solution was composed of the following (in mM): 130 N-methyl- D-glucamine, 10 HEPES, 1.15 EGTA, 1 Ca(OH)2, 2 Mg2+-ATP, 1 N-phenylanthranilic acid, and 0.1 5-nitro-2(3-phenylpropylamino benzoic acid), titrated to pH 7.2 with methane sulfonic acid. The osmolarity of all solutions was adjusted to 290–300 mosmol/lH2O with sucrose. Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) was inhibited with the inclusion of thapsigargin (TG) to the intracellular solution (1 µM) to elicit ISOC in response to the voltage-step protocol. In a parallel set of experiments, vehicle time controls in the absence of TG were also performed. Whole cell ISOC was measured in response to voltage steps applied from –100 to +60 mV in 20-mV increments with a duration of 200 ms and 2-s intervals. A holding potential of 0 mV between each voltage step was chosen to inactivate L-type voltage-gated Ca2+ channels that may be present. This voltage stimulus was performed after obtaining stable whole cell configuration and every 5 min thereafter while monitoring whole cell current at a holding potential of –50 mV. Voltage ramps were applied in a similar fashion and randomly interleaved over an identical range of potentials at a rate of 0.71 V/s. Biophysical criteria for successful recordings were as follows: 1) seal resistance ≥1 G{Omega}, 2) series resistance ≤35 M{Omega}, and 3) a rightward shift of the Erev near + 40 mV following ISOC activation.

Measurement of EC SOC entry. In separate experiments, freshly dispersed ECs were seeded on cover slips for a minimum of 30 min before fura 2 loading. Cells were loaded with 3 µM fura 2 for 15 min at ~23°C and washed in HBSS for 15 min at 37°C. Basal [Ca2+]i was measured under Ca2+-replete conditions at 37°C (in mM): 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with 8 M NaOH), followed by a switch to Ca2+-free superfusate in which CaCl2 was substituted with equimolar MgCl2, and 0.1 mM EGTA was added to chelate residual Ca2+. Intracellular stores were depleted with 10 µM cyclopiazonic acid (CPA) or 1 µM TG under Ca2+-free conditions to determine the magnitude of intracellular Ca2+ release. Next, cells were returned to Ca2+-containing HBSS in the continued presence of the SERCA inhibitors to measure SOC entry. Ratiometric changes in EC [Ca2+]i from peak SOC entry relative to baseline [Ca2+]i were performed to determine the magnitude of SOC entry. Additionally, Mn2+ quenching of fura 2 fluorescence was determined in both freshly isolated ECs and the endothelium in intact pressurized arteries recently described by Gokina and Goecks (12). These experiments were conducted at the estimated in vivo pulmonary artery pressures of 12 and 35 mmHg for control and CH rats, respectively. Each preparation was excited at the isobestic wavelength (360 nm), and emission wavelength recorded at 510 nm. SOC entry was quantified by the rate of quenching of fura 2 fluorescence with Mn2+ following the addition of 500 µM MnCl2 in a Ca2+-free HBSS or PSS (without EGTA) in store-depleted cells and endothelium in pressurized arteries, respectively. In an attempt to limit the effects of the SERCA inhibiton to the endothelium, the intact vessel preparation consisted of luminal administration of CPA for 15 min before and during the application of Mn2+. To correlate endothelial SOC entry with basal Ca2+ entry, parallel experiments utilizing this technique in isolated cells and pressurized arteries were performed in the absence of CPA to determine basal EC influx.

Calculations and statistics. All data are expressed as means ± SE. Values of n refer to the number of animals in each group for intact vessel experiments and number of cells for experiments utilizing freshly dispersed cells. The Student's t-test or ANOVA was used where appropriate for all comparisons between control and CH groups. If differences were detected by ANOVA, individual groups were compared with the Student-Newman-Keul's test. A probability of ≤0.05 was accepted as statistically significant for all comparisons.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of CH on basal EC [Ca2+]i. Basal EC [Ca2+]i was determined by values of the ratio of fluorescence at 340 to 380 nm in small pulmonary arteries (Fig. 1). Basal EC [Ca2+]i was significantly lower in small arteries from CH rats compared with controls at all of the selected pressures. Additionally, as pressure increased from 12 to 35 mmHg, basal endothelial Ca2+ decreased in control arteries. Ratiometric measurements of EC Ca2+ were similarly reduced (0.944 ± 0.091 vs. 0.629 ± 0.051) in freshly isolated ECs from CH vs. controls, respectively.


Figure 1
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  		Fig. 1. Basal endothelial cell (EC) Ca2+ is independent of pressure in chronic hypoxia (CH) arteries (n = 4) and reduced compared with controls (n = 4). Summary data for ratiometric values for EC Ca2+ in small arteries pressurized to 5, 12, and 35 mmHg. P ≤ 0.05 vs. respective control (*) and vs. control value at 5 and 12 mmHg (**). Data are means ± SE.

 
Effect of CH on pulmonary EC Em. Figure 2 shows EC Em data from intact arteries and acutely isolated pulmonary artery ECs from control and CH rats. Raw tracings of EC Em recordings from intact arteries are presented in Fig. 2A. Pulmonary EC Em in intact arteries from CH rats were more depolarized than controls (Fig. 2, A and B). In contrast, Em from isolated pulmonary artery ECs was not significantly different between groups (Fig. 2B).


Figure 2
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  		Fig. 2. Endothelial membrane potential (Em) is depolarized in intact pulmonary arteries from CH rats. A: raw sharp electrode tracing of endothelial Em recordings from intact arteries. B: summary data for intact arteries and freshly isolated endothelial preparations. Multiple recordings (4–10) ≥45 s from each vessel are considered a single observation (n). Intact arteries: control (n = 4) and CH (n = 7); freshly isolated: control (n = 4) and CH (n = 6). *P ≤ 0.05 vs. control. Data are means ± SE.

 
Effect of CH on pulmonary EC ISOC. TG and CPA-induced SOC entry in freshly isolated pulmonary artery ECs are shown in Figs. 3 and 4. The time course of ISOC activation is illustrated in Fig. 3A and is consistent with previous characterization (4). Whole cell ISOC was subsequently blocked in each group by 20 µM La3+ (Fig. 3A). Steady-state current was also examined in response to a voltage-step protocol before and after the activation of ISOC in freshly isolated cells (Fig. 3B). Time-dependent activation of whole cell ISOC was absent and appeared to be nonrectifying. Time controls in which TG was not present in the pipette did not show the characteristic inward ISOC following dialysis of the pipette solution. Current-voltage relationships generated from the voltage-step protocol revealed a significant reduction in the inward current at potentials from –100 to 0 mV and a leftward shift in the reversal potential (Erev) in ECs from CH rats compared with controls (Fig. 3C).


Figure 3
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  		Fig. 3. Freshly isolated ECs from CH hypertensive pulmonary arteries exhibit a reduced inward Ca2+ current. A: raw current tracing depicting the TG-induced time course of activation of store-operated Ca2+ current (ISOC) and La3+ sensitivity of the current. B: whole cell currents were measured in response to voltage-step protocol (inset) and were performed in series corresponding to A (from left to right) subsequent to patch rupture, following stable ISOC activation, and after application of 20 µM La3+. Time controls (vehicle) were performed, and time-dependent activation of ISOC was not observed. C: current-voltage relationship derived from voltage-step and ramp (inset) protocols in each group; control (n = 5) vs. CH (n = 6). *P ≤ 0.05 vs. control. Data are means ± SE.

 

Figure 4
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  		Fig. 4. Cyclopiazonic acid (CPA)-induced store-operated Ca2+ (SOC) entry is diminished in freshly isolated endothelial sheets (40–100 cells/sheet) from CH pulmonary arteries. A: raw tracings showing change in the ratio of fluorescence at 340 to 380 nm (F340/F380) upon reapplication of extracellular Ca2+ following store depletion (control vs. CH ± La3+). B: summary data illustrating the change in fura 2 ratio for CPA-induced SOC entry following the readdition of Ca2+ between control and CH ECs ± La3+ (n = 6/group). P ≤ 0.05 vs. control (*) and vs. CH (**). Data are means ± SE.

 
Effect of CH on pulmonary EC SOC entry and basal Ca2+ influx. In addition to measuring whole cell ISOC, SOC entry was assessed by reapplication of extracellular Ca2+ (1.8 mM) to fura 2-loaded ECs following store depletion with 10 µM CPA. Figure 4A illustrates the CPA-induced SOC entry protocol in freshly isolated ECs from small pulmonary arteries from control and CH rats in the presence or absence of the trivalent blocker La3+. The magnitude of SOC entry following passive store depletion was significantly less in EC sheets from CH rats compared with controls, and these responses were effectively inhibited by 20 µM La3+ (Fig. 4B). TG (1 µM) was also used to deplete intracellular Ca2+ stores in parallel studies, and the normalized change in fura 2 ratio subsequent to the readdition of Ca2+ was similarly reduced (0.278 ± 0.124 vs. 0.407 ± 0.053) in ECs from CH vs. controls, respectively. Furthermore, differences in SOC entry between groups were independent of the magnitude of store release subsequent to SERCA inhibition (Fig. 4A) in which no significant differences were found between changes in peak amplitude of the fura 2 ratio from CH (0.564 ± 0.032) and control (0.482 ± 0.040) ECs, respectively. To correlate the observation that diminished SOC entry contributes to reduced basal Ca2+, we assessed basal Ca2+ influx in isolated cells and intact pressurized arteries. Therefore, we estimated basal endothelial Ca2+ influx and SOC entry using the Mn2+ quenching technique. Basal endothelial Ca2+ influx was not reduced following CH compared with control in either preparation (Fig. 5, A and C); however, SOC entry determined by the rate of Mn2+ quenching of fura 2 fluorescence, expressed as percent per minute during the linear component of the response, was similarly reduced in intact pressurized pulmonary arteries following CH (4.152 ± 0.973) compared with controls (6.152 ± 0.598). This observation is consistent with our initial findings in isolated ECs from CH hypertensive rats, which displayed a reduced rate of Mn2+ quenching compared with controls (5.613 ± 0.650 vs. 9.674 ± 1.538). Data were also presented as percent change of fluorescence (360 nm) from baseline, as shown in Fig. 5, B and D.


Figure 5
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  		Fig. 5. Basal endothelial Ca2+ influx is unaltered in freshly isolated ECs and pressurized arteries following CH, although SOC entry determined by Mn2+ quenching of fura 2 fluorescence is reduced. A and B: changes in endothelial F360 fura 2 fluorescence (% of baseline) following the addition of Mn2+ in Ca2+-free HEPES buffered physiological saline solution (HBSS) solution in the presence or absence of CPA (10 µM) to isolated cells from control and CH rats. C and D: arteries from control and CH rats were pressurized to 12 and 35 mmHg, respectively, and changes in endothelial F360 fura 2 fluorescence following the addition of Mn2+ in Ca2+-free PSS solution in the presence or absence of CPA (10 µM) are shown. *P ≤ 0.05 vs. control. Data are means ± SE.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of this study are 1) basal EC [Ca2+]i in pressurized small pulmonary arteries and freshly isolated cells are reduced following CH, 2) ECs in small pulmonary arteries isolated from CH rats are depolarized compared with controls; however, freshly isolated EC Em does not differ between groups, 3) EC ISOC and SOC entry are diminished following CH compared with controls, and 4) basal Ca2+ influx is unchanged following CH despite a decrease in SOC entry . Our findings suggest that a depolarized Em and reduced SOC entry may only partially or indirectly contribute to altered endothelial Ca2+ homeostasis. Other mechanisms may account for the decrease in basal endothelial [Ca2+]i following CH-induced pulmonary hypertension.

The present study is the first to investigate the effects of CH-induced pulmonary hypertension on basal EC [Ca2+]i using pressurized, intact small intrapulmonary arteries. Our results suggest that CH alters EC Ca2+ homeostasis, resulting in reduced basal [Ca2+]i in the intact vasculature. Similarly, we observed a reduced basal [Ca2+]i in freshly isolated ECs from CH hypertensive arteries. In contrast to our findings, several studies using cultured ECs demonstrate increased [Ca2+]i in response to either acute or CH. For example, Hampl et al. (13) observed an immediate and transient increase in [Ca2+]i in cultured bovine pulmonary artery ECs exposed acutely to hypoxia that was likely due to Ca2+ release from intracellular stores. In a similar preparation, Berna et al. (2) found increased Na+-glucose cotransport in response to acute hypoxia, leading to elevated EC [Ca2+]i presumably by the NCX operating in reverse mode. Interestingly, others (40) have shown an opposite effect of acute hypoxia to reduce EC [Ca2+]i through membrane depolarization and altered Ca2+ influx. Whereas the above studies examined the effect of acute hypoxia on EC [Ca2+]i, Fantozzi et al. (11) more recently demonstrated that CH (72-h exposure to normobaric hypoxia) results in increased basal [Ca2+]i in cultured human pulmonary artery ECs. Discrepancies between these findings in cultured ECs and those of the present study may be due to our examination of ECs from intact arteries of animals exposed to CH where not only hypoxia but also altered mechanical forces resulting from hypertension and vascular remodeling may contribute to changes in the phenotype of the endothelium. Therefore, we felt it necessary to assess basal EC Ca2+ corresponding to luminal pressures that correspond to arterial normotensive (12 mmHg) and hypertensive (35 mmHg) states within the lung. Ratiometric values for basal EC Ca2+ appear to be offset to a lower set point at each pressure tested following CH. Interestingly, the transition from 12 to 35 mmHg in control arteries reduced basal EC Ca2+, suggesting acute pressure increases may diminish EC Ca2+ levels.

We also show that the endothelium in intact small pulmonary arteries from CH pulmonary hypertensive rats is depolarized compared with controls. Given the importance of Em for Ca2+ influx across the plasma membrane in ECs (20), this factor may contribute to the observed reduction of basal [Ca2+]i following CH exposure. However, the cause of this EC depolarization is not clear. Interestingly, Sadanaga et al. (35) have demonstrated that systemically hypertensive rats exhibit a reduction in whole cell voltage-gated potassium (Kv) currents in freshly dissociated ECs, leading to membrane depolarization, presumably by a decrease in Kv1.5 isoform expression. However, in the present study, we did not observe differences in Em between groups following cell dissociation (Fig. 2B, left), suggesting that CH-induced depolarization is not due to altered ionic conductances inherent to the endothelium.

An alternative interpretation for the observed EC depolarization in intact vessels from CH rats (Fig. 2B, right) is that CH enhanced heterocellular communication between the VSM and endothelium whereby VSM Em drives endothelial Em through low-resistance pathways, such as myoendothelial gap junctions. Although this interpretation is speculative, several others have previously shown that VSM Em is more depolarized following CH relative to controls (29, 38, 39), and this depolarization persists upon cell isolation. Myoendothelial communication has been extensively documented in a variety of systemic vascular beds (7, 9), but there is little direct evidence for this phenomenon in the pulmonary circulation. Tracey and coworkers (41) observed that gap junction blockade inhibited endothelium-dependent vasorelaxation of bovine pulmonary arteries to bradykinin following NOS blockade, suggesting a role of gap junctions in conducted hyperpolarization between endothelium and VSM in this vascular bed. Furthermore, Seiden et al. (37) demonstrated that a prolonged VSM membrane depolarization by high extracellular K+ concentration is associated with attenuated endothelium-dependent pulmonary vasodilation, suggesting that VSM-mediated depolarization influences EC Em via a number of mechanisms, including the possibility of low-resistance heterocellular pathways. However, the reduction in driving force (~12 mV) that we observed in CH intact arteries compared with controls is modest and may be insufficient to significantly lower basal [Ca2+]i. Additionally, we found EC Em was normalized following enzymatic dispersal despite the reduced basal [Ca2+]i (Figs. 1 and 2B), suggesting that modest changes in Em do not contribute strongly to reduced [Ca2+]i, and other mechanisms may be affording a greater influence on basal EC homeostasis.

In addition to Em, endothelial SOC entry represents a primary mode of Ca2+ influx in which the response to passive or active store depletion initiates a slowly developing and sustained rise in [Ca2+]i (4). Our present findings indicate that a La3+-sensitive SOC entry determined by whole cell patch clamp and fura 2 fluorescent techniques is reduced in freshly isolated endothelium from pulmonary hypertensive rats, which may contribute to decreased basal EC [Ca2+]i. The magnitude of the currents elicited by passive store depletion in our preparation across both groups is larger than some reports of endothelial ISOC (4) and lymphocytic Ca2+ release-activated Ca2+ currents (10). However, the magnitude of current densities activated by TG (24) and F-actin disruption (30) in ECs isolated from pulmonary arteries compare closely to our results. Although the majority of the currents in the present study did not display strong inward rectification, a small percentage did show a slight nonohmic feature that was not evident in the mean data. Furthermore, the Erev (+24 mV) that we obtained from control cells suggests a concomitant activation of nonselective cation channels by TG that has been reported by Zhang et al. (48) and may contribute to the overall departure from the classical ISOC current-voltage characteristics. In addition to the reduced inward Ca2+ current in the CH group, it also shows a modest left-ward shift in the Erev closer to zero, which may be due to a loss in the partial Ca2+ selectivity of this current seen in controls.

Interestingly, recent findings have correlated basal EC [Ca2+]i with SOC entry (11) in cultured human pulmonary artery ECs. In contrast to our results, these investigators demonstrated that CH enhanced endothelial SOC entry through an activator protein-1-sensitive mechanism that resulted in increased transient receptor potential channel C4 isoform expression and basal EC [Ca2+]i. Furthermore, they observed enhanced CPA-mediated store release following CH (72 h) in cultured ECs compared with control. Contradictory to these results, we found no difference in the magnitude of store release between groups. Discrepancies between the findings of Fantozzi et al. (11) and our results could be attributed to either the duration or the nature of hypoxic stimulus. Because our experiments utilized acutely isolated ECs from CH-induced hypertensive pulmonary arteries rather than cultured cell lines, the hypertension associated with CH could be responsible for decreased SOC entry. Interestingly, our results are consistent with earlier findings of diminished agonist-induced Ca2+ entry in main pulmonary arteries from CH hypertensive rats (28), suggesting that CH-induced pulmonary hypertension impairs endothelial Ca2+ entry. One possible explanation for these findings is that, following CH, SOC entry may become less selective for Ca2+ as illustrated by the slight leftward shift in the Erev in Fig. 3C. Although we found differences in the magnitude of SOC entry elicited by passive store depletion with CPA (Figs. 4 and 5), we were unable to detect any significant differences between basal Ca2+ influx using the Mn2+ technique in isolated cells or pressurized arteries (Fig. 5, A and C). It appears that the decrease in SOC entry following CH does not contribute to a decrease in basal endothelial Ca2+ influx, and alterations in other Ca2+-handling mechanisms may contribute to the reduced basal Ca2+ observed in isolated pressured arteries and freshly isolated cells. These results suggests that a more complex mechanism for store-dependent Ca2+ entry indirectly regulates endothelial Ca2+ homeostasis or this reduction may be a downstream effect of a different mechanism(s) that results in reduced basal endothelial Ca2+ following CH. Moreover, the lack of any difference in basal Ca2+ influx between groups may be due to the absence of any circulating humoral factors/endogenous ligands that may contribute to some level of tonic SOC entry activation in the in vivo setting. Although SOC entry is reduced following CH, it cannot entirely account for the reduction in basal endothelial Ca2+ by virtue that basal Ca2+ influx does not appear to be altered following CH under these experimental conditions.

Alterations in EC Ca2+ homeostasis following CH-induced pulmonary hypertension have important implications for endothelial function and vascular control in this setting. For example, basal synthesis of NO by the Ca2+/calmodulin-sensitive enzyme eNOS may be altered. Although it has been established that eNOS expression is elevated in arterial segments of the pulmonary circulation following CH (33) and agonist-induced endothelium-derived NO-dependent dilations are similarly enhanced in isolated-perfused lungs from CH rats (34), contrasting evidence of attenuated endothelium-dependent relaxation to acetylcholine and endothelin-1 (1, 6) also exists. Furthermore, it remains controversial whether elevated eNOS expression produces a corresponding increase in basal NO synthesis following CH. Several investigators have shown that agonist-dependent (16) and -independent (27) lung NO production is elevated following CH. However, Sato et al. (36) observed that lung NO production was not enhanced in CH rats maintained under hypoxic conditions, suggesting that NO production may be limited by reduced oxygen tension in the CH lung. Although it appears that PO2 can influence NO production, it has been observed that abnormal interactions between eNOS and the regulatory proteins caveolin-1 and heat-shock protein 90 may also decrease agonist-induced NO production in pulmonary arteries following CH (28). In addition, Millatt et al. (23) provided evidence that the incidence of pulmonary hypertension following CH correlates with levels of the endogenous NOS inhibitor asymmetric dimethylarginine. Indeed, many variables including EC [Ca2+]i that are affected by CH may influence NO production in this setting.

In conclusion, we have demonstrated that basal endothelial [Ca2+]i is reduced in small isolated, pressurized pulmonary arteries and freshly isolated ECs following CH compared with controls. Furthermore, reductions in EC SOC entry do not appear to correlate with basal endothelial Ca2+ influx, and modest membrane depolarization may not play a critical role in determining basal Ca2+ following CH. However, other Ca2+-handling mechanisms might contribute to altered endothelial function observed in this clinically relevant setting.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grants HL-07736 (M. L. Paffett), HL-58124 and HL-63207 (B. R. Walker), and HL-77876 (T. C. Resta).


   ACKNOWLEDGMENTS
 
We thank Dr. Karen Sweazea and Jessica Snow for comments on the manuscript and Minerva Murphy for assistance with the hypobaric facility.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. L. Paffett, Vascular Physiology Group, Dept. of Cell Biology and Physiology, 1 Univ. of New Mexico MSC08 4750, Albuquerque, NM 87131 (e-mail: mpaffett{at}salud.unm.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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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