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(Circulation Research. 2003;93:346.)
© 2003 American Heart Association, Inc.

Cellular Biology

Ca_v3.1 (_1G) T-Type Ca²⁺ Channels Mediate Vaso-Occlusion of Sickled Erythrocytes in Lung Microcirculation

Songwei Wu, Johnson Haynes, Jr, James T. Taylor, Boniface O. Obiako, James R. Stubbs, Ming Li, Troy Stevens

From the Departments of Pharmacology (S.W., T.S.), Medicine (J.H.), and Pathology (J.R.S.), the Center for Lung Biology (S.W., J.H., T.S.), and the Comprehensive Sickle Cell Center (J.H., B.O.O.), University of South Alabama College of Medicine, Mobile, Ala, and the Department of Pharmacology (J.T.T., M.L.), Tulane University School of Medicine, New Orleans, La.

Correspondence to Songwei Wu, MD, Center for Lung Biology and Department of Pharmacology, MSB 3370, University of South Alabama College of Medicine, Mobile, AL 36688. E-mail swu{at}jaguar1.usouthal.edu

	Abstract

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In the present study, we demonstrate that lung microvascular endothelial cells express a Ca_v3.1 (_1G) T-type voltage-gated Ca²⁺ channel, whereas lung macrovascular endothelial cells do not express voltage-gated Ca²⁺ channels. Voltage-dependent activation indicates that the Ca_v3.1 T-type Ca²⁺ current is shifted to a positive potential, at which maximum current activation is -10 mV; voltage-dependent conductance and inactivation properties suggest a "window current" in the range of -60 to -30 mV. Thrombin-induced transitions in membrane potential activate the Ca_v3.1 channel, resulting in a physiologically relevant rise in cytosolic Ca²⁺. Furthermore, activation of the Ca_v3.1 channel induces a procoagulant endothelial phenotype; eg, channel inhibition attenuates increased retention of sickled erythrocytes in the inflamed pulmonary circulation. We conclude that activation of the Ca_v3.1 channels selectively induces phenotypic changes in microvascular endothelial cells that mediate vaso-occlusion by sickled erythrocytes in the inflamed lung microcirculation.

Key Words: endothelial cells • store-operated Ca²⁺ entry • P-selectin • von Willebrand factor • coagulation

	Introduction

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Sickle cell anemia patients exhibit a chronic state of inflammation that increases endothelial cell expression of surface adhesive proteins.¹ Adherence of sickled erythrocytes to abnormally adhesive lung microvascular endothelium initiates vaso-occlusion and acute chest syndrome.^1–6 Endothelial cell adhesion to sickled erythrocytes is mediated through vascular cell adhesion molecule 1, _vß₃ integrins, glycoproteins Ib, IX, and V, and CD36.⁷ Such cell-cell interactions are promoted by the regulated secretion of von Willebrand factor (vWf) and lipid procoagulants from the endothelium.⁷ Collectively, these processes provide a means by which endothelial cells rapidly and selectively alter the microenvironment of individual vascular beds and modulate the interrelated processes of coagulation, fibrinolysis, inflammation, and vaso-occlusion.

Although intracellular events that transduce this abnormally adhesive endothelial cell surface are incompletely understood, the importance of elevated cytosolic Ca²⁺ ([Ca²⁺]_i) has been clearly established.^8–12 Thrombin and other G_q-linked neurohumoral inflammatory agonists increase endothelial cell [Ca²⁺]_i,¹³ sufficient to cause rapid vWf secretion and P-selectin upregulation.^8,9,14–17 However, specific Ca²⁺ entry pathways that mediate these effects are poorly understood, particularly in microvascular endothelial cells obtained from the prominent site of vaso-occlusion^18,19; indeed, we have previously shown that G_q-linked agonists activate distinct Ca²⁺ signaling pathways in lung microvascular and macrovascular endothelium.^20–22 Therefore, our present challenge is to identify a channel(s) that provides the Ca²⁺ source needed to promote coagulation and vaso-occlusion.

Voltage-gated Ca²⁺ channels trigger vesicular translocation in excitable cells, although endothelial cells are nonexcitable and are generally believed to lack the expression of voltage-gated ion channels.²³ This perception may be partly due to the preponderance of studies that have used endothelial cells derived from conduit vessels; the expression of voltage-gated Ca²⁺ channels has recently been characterized in both brain and adrenal capillary endothelium.^24,25 The present study indicates that lung microvascular endothelial cells express a T-type voltage-gated Ca²⁺ channel, whereas lung macrovascular (eg, pulmonary artery) endothelial cells do not express voltage-gated Ca²⁺ channels. Importantly, G_q-linked agonists cause an initial transient hyperpolarization and, subsequently, a large sustained depolarization in lung endothelial cells.^26–28 Likewise, loss of shear stress and generation of reactive oxygen species during ischemia induce lung endothelial cell membrane depolarization.^29,30 These changes in membrane potential could activate T-type Ca²⁺ channels. We hypothesized that Ca²⁺ entry through T-type Ca²⁺ channels in lung microvascular endothelium is an important amplification step that promotes the local retention of sickled erythrocytes leading to vaso-occlusion.

	Materials and Methods

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Cell Culture
Rat pulmonary artery endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs) were isolated, cultured, and purity-verified using a method previously described.²²

Patch-Clamp Electrophysiology
Whole-cell macroscopic currents were recorded using an EPC-9 amplifier (HEKA Elektronik). Data were acquired with Pulse/PulseFit software (HEKA) and filtered at 2.9 kHz. Voltage-dependent currents were corrected for linear leak and residual capacitance using an online P/n subtraction paradigm. (The definition for the so-called P/n leak correction protocol is that in a voltage range, where voltage-dependent channels are not active, a scaled-down version of the pulse protocol is applied n times, and resulting current is averaged, scaled, and subtracted from that elicited by the main test pulse.) Extracellular (bath) solution contained (mmol/L) CaCl₂ 10, tetraethylammonium chloride 110, CsCl 10, and HEPES 10 (pH 7.4, adjusted with tetraethylammonium hydroxide). Intracellular (pipette) solution contained (mmol/L) N-methyl-D-glucamine 130, EGTA 10, BAPTA 5, HEPES 10, MgCl₂ 6, CaCl₂ 4, and Mg-ATP 2 (pH 7.2, adjusted with methane sulfonic acid). All solutions were adjusted to 290 to 300 mOsm with sucrose. Various drug dilutions were applied to cells by a gravity-driven perfusion device, operated by a perfusion valve controller (model VC-6, Warner Instrument).

Molecular Biology
Total RNA was extracted with RNA Stat-60 (Tel-Test "B") from cells grown to 100% confluence (10⁷ cells) in 75-cm² tissue culture flasks. First-strand synthesis was performed with reverse transcriptase and oligo(dT) primer (Invitrogen) on 1 µg DNase I-treated total RNA. Polymerase chain reaction (PCR) was then performed with the following sets of primers: Ca_v3.1, 5'-CATTTGCTGTGCC-TTCTTCA-3' (sense) and 5'-ATCCACACCCACAGCATCCAG-3' (antisense); Ca_v3.2, 5'-TGAATTCACGCAGGACGT-3' (sense) and 5'-CAGCTGTGCACATGATGA-3' (antisense). PCR products were ligated into TA cloning vector pCR2.1 (Invitrogen) and transformed into chemically competent Escherichia coli. Positive clones (verified by PCR analysis) were selected and grown in Luria-Bertani broth (Sigma) with kanamycin (50 µg/mL) for 18 to 20 hours at 37°C. Plasmids were isolated by the QIAprep Spinprep system (QIAGEN) and submitted to the Biopolymer Laboratory at the University of South Alabama for automated fluorescence sequence analysis (AB373XL DNA stretch sequencer). Sequencing of both strands using double-stranded plasmids as templates and universal primers confirmed the product accuracy. Nucleotide and amino acid alignments were achieved with BLAST (National Center for Biotechnology Information, Bethesda, Md) and DNASIS v2.0 (Hitachi Software Engineering America) programs.

Cytosolic Ca²⁺ Measurements
[Ca²⁺]_i was estimated in confluent PAECs and PMVECs using fura 2-AM (Molecular Probes). Calculations of free [Ca²⁺]_i are routinely made using modifications of the formula described by Grynkiewicz et al³¹ in 1985.

Blood Collection and Preparations: Erythrocyte Isolation, ⁵¹Cr Labeling, and Neutrophil Isolation
Blood collection and blood cell preparations were performed as described previously.³² The activation of polymorphonuclear cells (PMNs) was achieved in the test tube by the addition of phorbol myristate acetate (20 ng/mL) to PMNs suspended in 1 mL HBSS.

Isolated Perfused Lung and Erythrocyte Retention Assay
Isolated perfused lung and erythrocyte retention assays were performed as described by Haynes et al.³² In the study using mibefradil (Hoffmann-La Roche), pimozide, or flunarizine, lungs were incubated with mibefradil (20 µmol/L), pimozide (10 µmol/L), and flunarizine (10 µmol/L) for 30 minutes and then perfused to eliminate contact of the drug with circulating cells. Activated PMNs were subsequently added to the perfusate reservoir (200 000/mL). Lungs were perfused in a recirculating fashion for 30 minutes, and the sickled erythrocyte retention/adherence was determined as previously described.³²

Data Analysis
Numerical data are reported as mean±SEM. A value of P<0.05 was considered significant.

	Results

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PMVECs Possess a T-Type Ca²⁺ Channel
Using a conventional whole-cell configuration of patch-clamp recordings, we examined functional expression of T-type Ca²⁺ channels in cultured rat PMVECs and PAECs. With Ca²⁺ (10 mmol/L) as a charge carrier, we recorded that membrane depolarization of PMVECs produced transient robust Ca²⁺ currents that were insensitive to high tetrodotoxin concentrations (data not shown). The current exhibited a low threshold of activation and inactivated quickly during depolarization voltage steps (Figure 1A). Analysis of current-voltage (I-V) relationships in PMVECs (Figure 1B) revealed that these currents were consistently activated at -60 mV; maximum current activation was observed at -10 mV (Figure 1B). Currents were typical of T-type Ca²⁺ channels. Despite repeated efforts, we did not observe a voltage-dependent Ca²⁺ current in PAECs (Figures 1A and 1B).

View larger version (19K): [in this window] [in a new window]   Figure 1. T-type Ca2+ currents in PMVECs. Ca2+ (10 mmol/L) was used as a charge carrier. A, Currents evoked in PMVECs by increasing depolarization from -70 to 60 mV with 10-mV increments from a holding potential of -90 mV. Arrow indicates peak current recorded at test pulse to -10 mV. Note criss-crossing pattern of current traces.37 No currents were evoked in PAECs using the same protocol. B, Averaged I-V relationship of peak currents obtained from 55 PMVECs (solid squares) and 20 PAECs (open squares).

Molecular Characterization of PMVEC T-Type Voltage-Gated Ca²⁺ Channels
We next examined whether the T-type Ca²⁺ channel expressed in PMVECs was encoded by one of three known members of the T-channel family identified in neurons, cardiomyocytes, and pancreatic ß-cells, namely, Ca_v3.1 (_1G), Ca_v3.2 (_1H), and Ca_v3.3 (_1I), respectively.^33–36 Among the three subtypes of T-type Ca²⁺ channels, Ca_v3.3 has been described only in neurons, and it possesses a slower voltage-dependent activation and inactivation compared with other T-channel types³⁵ and with the PMVEC current (Figure 1A). Therefore, it is likely that the PMVEC current is mediated by either Ca_v3.1, Ca_v3.2, or both channels. Total RNA isolated from PMVECs and PAECs was amplified using sequence-specific primers designed to target Ca_v3.1 and Ca_v3.2 pore regions, respectively. A single Ca_v3.1 product of expected size was selectively amplified from PMVEC RNA (Figure 2A). Sequence analysis revealed that the cloned product was 100% homologous to the previously reported rat Ca_v3.1 subunit (Figure 2B). These data reveal that Ca_v3.1 subunits form functional T-type Ca²⁺ channels in PMVECs. Reverse transcription (RT)-PCR analysis using sequence-specific primers targeting _1B (Ca_v2.2, N-type), _1C (Ca_v1.2), _1D (Ca_v1.3), _1F (Ca_v1.4), and _1S (Ca_v1.1) (L-type) did not yield any product in PMVECs (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. Presence of T-type Ca2+ channel Cav3.1 subunit in PMVECs. A, RT-PCR cloning of T-type Ca2+ channel was performed using total RNA from PMVECs and PAECs. Primers were designed for RT-PCR to amplify the pore region of the cytoplasmic segment spanning domain III of Cav3.1 and Cav3.2. B, Deduced amino acid sequencing of the PCR product identified it as the Cav3.1 subunit of T-type Ca2+ channel, demonstrating that PMVECs express the T-type Ca2+ channel Cav3.1 subunit, resembling that formed in pancreatic ß cells.36

Biophysical Characterization of PMVEC T-Type Ca²⁺ Channels
We characterized the gating properties of PMVEC Ca_v3.1 channels. Voltage dependence of channel activation was obtained from the above I-V relationships by calculating conductance from the equation G_T=I_T/(V-V_rev), where I_T is the current amplitude, V is the membrane potential, and V_rev is the reversal potential for Ca²⁺ (a value of 70 mV was used in our calculations). Normalized values of channel conductance were plotted as a function of the membrane potential (Figure 3A). The steady-state inactivation properties were characterized by applying a standard double-pulse protocol (ie, a 1500-ms prepulse from -80 to -20 mV with 5-mV increments, followed by a 100-ms test pulse at -20 mV). The currents were measured at -20 mV after varying 1500-ms prepulse potentials (Figure 3B). Peak currents were normalized to maximum current and then plotted as a function of the prepulse potential (Figure 3C). Both activation and inactivation were fitted with a Boltzmann equation of the form G/G_max or I/I_max=1/{1+exp[(V-V_1/2)/k]}, where V is the membrane potential (for activation) or prepulse potential (for inactivation), V_1/2 is the voltage at which activation or inactivation is half maximal, and k is the slope factor. For activation and inactivation, the calculated V_1/2 values were -28.4 and -51.4 mV, respectively, and k values were 7.084 and -5.556, respectively. Superimposition of activation and inactivation curves predicted a "window current" in the range of -60 to -30 mV (Figure 3C). To confirm that such a window-current range of voltages occurs in physiological conditions, the voltage dependence of the channel activation was determined for a second time in 2 mmol/L extracellular Ca²⁺ by measuring T tail-current amplitudes at various depolarizing pulses (Figure 3E) that fully activate Ca_v3.1 currents, with duration of each pulse adjusted to the average time-to-peak current values (Figure 3D). Normalized tail-current amplitudes were plotted as a function of the membrane potential and fitted using the Boltzmann equation with V_1/2=-30.8 mV, virtually identical to the activation curve obtained in 10 mmol/L Ca²⁺ (Figure 3F).

View larger version (23K): [in this window] [in a new window]   Figure 3. Activation and inactivation properties of the T-type Ca2+ currents in PMVECs. A, B, and C, Ca2+ (10 mmol/L) was used as a charge carrier. A, Normalized values of channel conductance were plotted as a function of the membrane potential (n=46). The smooth curve corresponds to the best fit obtained using the described Boltzmann equation G/Gmax=1/{1+exp[(V-V1/2)/k]}. B, Steady-state inactivation was estimated from measurement of the current amplitude at -20 mV after a 1500-ms predepolarization pulse of increasing amplitude (-80 to -20 mV, 5-mV increment). C, Normalized steady-state inactivation curve (n=12) was best fitted with the Boltzmann equation I/Imax=1/{1+exp[(V-V1/2)/k]}. The dashed curve represents the activation curve from panel A. Thus, a steady-state T current, referred to as a window current, is predicted by the overlap of the voltage range for activation and inactivation, -60 to -30 mV in PMVECs. D and E, Determination of the steady-state activation was based on the measurement of tail-current amplitudes. Physiological Ca2+ (2 mmol/L) was used as a charge carrier. Currents were evoked by depolarizations from -70 to 80 mV, followed by a 20-ms repolarization to the potential of -120 mV. For each depolarization, the tail-current amplitude was measured for pulse durations adjusted from the membrane potential/time-to-peak plot shown in panel D. Panel E shows representative fully activated PMVEC T-type Ca2+ current tail traces at various depolarizing pulses. F, Normalized steady-state activation obtained from the protocol described in panel A is shown (closed circles, n=6). The smooth curve corresponds to the best fit obtained using the Boltzmann equation, with I/Imax being a function of the test potential (V) according to the equation I/Imax=1/{1+exp[(V-V1/2)/k]}, and half-activation potential (V1/2) of -30.8 mV and a slope (k) of 13.2. The dashed and dash-dotted curves represent activation and inactivation curves, respectively, from panels A and C (10 mmol/L Ca2+). Thus, a predicted window-current range of voltages near -60 to -30 mV in physiological conditions has been confirmed in PMVECs.

We characterized deactivation properties of PMVEC Ca_v3.1 channels from tail-current analysis (Figures 4A and 4B). Ca_v3.1 currents were activated by a transition from a holding potential of -90 to -10 mV for 7 ms, and a family of tail currents was recorded for subsequent repolarization from -120 to -20 mV. Tail currents were fitted with a single exponential. The rate of deactivation, which reflects the rate of channel closing after removal of membrane depolarization, was measured as the rate of decay of tail currents at different repolarization potentials, named deactivation time constants (), and plotted as a function of membrane repolarization potential. The tail-current kinetics were slower when membrane repolarization potential was more positive. Deactivation time constants increased by e-fold per 31.3 mV of repolarization, reflecting strong voltage dependence of the deactivation process for Ca_v3.1 currents (Figure 4B).³⁷

View larger version (15K): [in this window] [in a new window]   Figure 4. Deactivation properties of the T-type Ca2+ currents in PMVECs. A, Representative PMVEC T-type Ca2+ current tail traces. Currents were evoked using the following voltage protocol: a 7-ms step to -10 mV, followed by repolarization to potentials from -120 to -20 mV with 10-mV increment. B, Plot of mean deactivation time constant () as a function of membrane repolarization potentials (n=6). Smooth curve represents exponential function of repolarization potential (e-fold per 31.3 mV).

Pharmacological Characterization of PMVEC T-Type Ca²⁺ Channels
We examined sensitivity of PMVEC T-type Ca²⁺ channels to inhibition by Ni²⁺ and four molecules (mibefradil, kurtoxin, pimozide, and flunarizine, which are all considered T-type channel blockers) (Figure 5). We used a T-tail current to measure peak Ca²⁺ current amplitude and to obtain the precise concentration of Ni²⁺ required to inhibit the PMVEC current. Ni²⁺ weakly inhibited the PMVEC T current (IC₅₀ 380.25 µmol/L), characteristic of the Ca_v3.1 and not Ca_v3.2 subtype of T channels.³⁸ Despite the insensitivity of the endogenous PMVEC Ca_v3.1 channel to Ni²⁺ blockade, we found inhibition using mibefradil, a benzimidazolyl-substituted tetraline derivative, considered one of the most potent T-type channel blockers,³⁹ in good agreement with previous studies on recombinant Ca_v3.1 T-type channels.^40,41 In similar experiments, we also observed inhibition of PMVEC T currents by kurtoxin (Peptides International, Inc), pimozide, and flunarizine. Kurtoxin is a newly identified scorpion toxin that binds to the Ca_v3.1 channel with high affinity and distinguishes between Ca_v3.1 and other types of voltage-gated Ca²⁺ channels, including Ca_v2.1 (_1A, P/Q-type), Ca_v2.2 (_1B, N-type), Ca_v1.2 (_1C, L-type), and Ca_v2.2 (_1E, R-type),⁴² whereas a diphenylbutylpiperidine antipsychotic drug, pimozide, and a diphenyldiperazine derivative, flunarizine, have both been shown to be potent T-type channel blockers.⁴³

View larger version (16K): [in this window] [in a new window]   Figure 5. Pharmacological profile of PMVEC T-type Ca2+ channels. Dose-effect curves for Ni2+ (open squares, n=5), mibefradil (solid squares, n=6), kurtoxin (Peptides International) (solid circles, n=3), pimozide (open circles, n=3), and flunarizine (open inverted triangles, n=3) indicate IC50 values of 380.25 µmol/L (Ni2+), 1.22 µmol/L (mibefradil), 0.11 µmol/L (kurtoxin), and 0.74 µmol/L (pimozide). The effect of increasing concentrations of Ni2+ and pimozide on the peak T-tail Ca2+ current amplitude is illustrated in the left panel. The currents were evoked by a 10-ms depolarization at 0 mV (Ni2+, mibefradil) or a 5-ms depolarization at 10 mV (kurtoxin, pimozide, and flunarizine) from a holding potential of -90 mV, and the tail current was recorded for subsequent repolarization at -100 mV (Ni2+) or -120 mV (pimozide). Current traces from bottom to top correspond to control, 0.001, 0.01, 0.1, 0.5, 1, 5, and 10 (in mmol/L Ni2+) and 0.001, 0.01, 0.1, 0.5, 1, 5, and 10 (in µmol/L pimozide), respectively.

T-Type Ca²⁺ Channel Contributes to Thrombin or Thapsigargin-Induced Ca²⁺ Entry in PMVECs
Physiological activation of T-type Ca²⁺ channels occurs when the membrane potential shifts into the window-current range, which is -60 to -30 mV in PMVECs. Importantly, G_q-linked neurohumoral inflammatory agonists cause an initial transient hyperpolarization and, subsequently, a large sustained depolarization in lung endothelial cells.^26–28 This subsequent depolarizing current is of sufficient magnitude to shift the membrane potential to the T-channel window current range of voltages, suggesting that thrombin and other G_q-linked inflammatory agonists would activate the PMVEC T channel.

Prior studies in nonexcitable cells, such as endothelia, have established that the principal mode of Ca²⁺ entry across the plasmalemma is through so-called store-operated Ca²⁺ entry channels.²³ We have recently examined store-operated Ca²⁺ entry pathways in PMVECs and PAECs and have observed that the magnitudes of Ca²⁺ release and entry are not associated between these cell types.²² Therefore, it is conceivable that PMVECs possess novel Ca²⁺ entry mechanisms that are distinct from store-operated Ca²⁺ entry pathways. To evaluate the contribution of Ca_v3.1 T currents to agonist-induced rises in [Ca²⁺]_i, we inhibited the Ca_v3.1 channels using mibefradil (10 µmol/L, IC₁₀₀) and then applied thrombin (10 U/mL, G_q-linked agonist) or thapsigargin (1 µmol/L, direct activation of store-operated Ca²⁺ entry). Mibefradil reduced the thrombin- and thapsigargin-induced increase in [Ca²⁺]_i (Figures 6A and 6B), indicating that activation of store-operated Ca²⁺ entry also results in Ca²⁺ permeation through Ca_v3.1 channels in PMVECs. To ensure that these results were due to the activation of T channels after a physiological transition into the window-current range of voltages and not due to nonspecific actions of mibefradil, studies were repeated in low extracellular Ca²⁺. This protocol is commonly used to examine the magnitude of Ca²⁺ release and to discriminate between Ca²⁺ release and Ca²⁺ entry components of the global [Ca²⁺]_i response. As seen in Figure 6C, mibefradil (IC₁₀₀) did not reduce thapsigargin-stimulated Ca²⁺ release in PMVECs (Figure 6C), indicating that T-channel inhibition does not disrupt store-operated Ca²⁺ entry pathways. Furthermore, mibefradil did not decrease the thrombin-induced Ca²⁺ response in PAECs (Figure 6D), which lack expression of T channels. Thus, G_q-linked agonists directly activate Ca²⁺ entry through Ca_v3.1 channels in PMVECs.

View larger version (21K): [in this window] [in a new window]   Figure 6. Mibefradil inhibits the thrombin- and thapsigargin (TG)-induced rise in PMVEC [Ca2+]i. A and B, [Ca2+]i responses to thrombin (10 U/mL) and TG (1 µmol/L) in PMVECs with (n=5 and 5, respectively) or without 10-minute pretreatment of mibefradil (10 µmol/L) (n=12 and 5, respectively), measured in the buffer containing 2 mmol/L extracellular Ca2+. Mibefradil dramatically reduced the sustained thrombin-and TG-induced increase in [Ca2+]i (P<0.05 for each). C, [Ca2+]i responses to TG (1 µmol/L) in PMVECs with (n=5) or without 10-minute pretreatment of mibefradil (10 µmol/L) (n=5), measured in the buffer containing 100 nmol/L extracellular Ca2+. Mibefradil-treated PMVECs exhibited no evidence of a decrease in Ca2+ release (P=NS). D, [Ca2+]i responses to thrombin (10 U/mL) in PAECs with (n=3) or without 10-minute pretreatment of mibefradil (10 µmol/L) (n=3), measured in the buffer containing 2 mmol/L extracellular Ca2+. Mibefradil had no effect on PAEC [Ca2+]i responses to thrombin (P=NS). Units on the y-axis scale represent the ratio of Ca2+ bound (340-nm) to unbound (380-nm) wavelengths for fura 2 fluorescence. Traces in dashed lines indicate the ratio in mibefradil-pretreated cells, and traces in solid lines indicate the ratio in control cells.

Ca_v3.1 T-Type Ca²⁺ Channels Contribute to Sickled Erythrocyte Retention in Lung Microcirculation
Recently, Haynes et al³² developed a lung inflammation model to investigate sickled erythrocyte retention in the intact pulmonary circulation. They found that activated PMNs promoted sickled erythrocyte retention in an isolated perfused lung preparation. The combination of platelet activating factor and leukotriene B₄ increased sickled erythrocyte retention, and a 5-lipoxygenase inhibitor (zileuton) attenuated sickled erythrocyte retention. Their findings suggest that neurohumoral inflammatory mediators promote the adhesion of sickled erythrocytes to endothelial cells.

Many inflammatory mediators released from PMNs stimulate receptor-G_q-linked pathways and may therefore consolidate to activate T channels within the lung microvasculature necessary for vaso-occlusion. Using the model developed by Haynes et al,³² we examined whether Ca_v3.1 channels contribute to sickled erythrocyte retention. Sickled erythrocytes (5.8±0.1x10⁸/g) were retained in the inflamed lung circulation (n=12). Lung pretreatment with mibefradil (20 µmol/L) for 30 minutes completely abolished the effect of activated PMNs on sickled erythrocyte retention (Figure 7). Similarly, pimozide and flunarizine abolished the PMN-induced lung sickle cell retention. These findings indicate that vaso-occlusion proceeds in the inflamed lung via a mechanism that involves the T-type Ca²⁺ channel.

View larger version (17K): [in this window] [in a new window]   Figure 7. Inhibition of T-type Ca2+ channels attenuates the increased retention of sickled erythrocytes in lungs perfused with activated PMNs. Groups are as follows: n=12 for activated PMNs (control), n=6 for activated PMNs+mibefradil (20 µmol/L), and n=3 for activated PMNs+pimozide (10 µmol/L) and for activated PMNs+flunarizine (10 µmol/L). P<0.001, *P<0.0001, and *P<0.0001 for activated PMNs+mibefradil, activated PMNs+pimozide, and activated PMNs+flunarizine vs control, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Developmental studies indicate that lung conduit vessels arise from angiogenesis, whereas the microcirculation arises from vasculogenesis,⁴⁴ suggesting that macrovascular and microvascular endothelial cells arise from embryologically distinct origins. Such heterogeneity has been illustrated in Ca²⁺-dependent signal transduction pathways.^20,22 In the present study, we demonstrate the selective expression of Ca_v3.1 T-type voltage-gated Ca²⁺ channels in lung microvascular endothelial cells. Functional expression of Ca_v3.1 is based on the following observations: (1) T-type Ca²⁺ currents are present in PMVECs and not PAECs; (2) PMVEC currents are poorly sensitive to Ni²⁺ blockade (IC₅₀ 380.25 µmol/L), which discriminates Ca_v3.1 among three T-type Ca²⁺ channel isoforms; and (3) only Ca_v3.1 is expressed in PMVECs. The present study is the first demonstration that pulmonary endothelial cells (specifically microvascular endothelial cells) possess a T-type voltage-gated Ca²⁺ channel.

An important property of T-type Ca²⁺ channels is that they display window currents. That is, there is a voltage range at which channels do not completely inactivate; therefore, they conduct Ca²⁺. This property is commonly defined by the presence of an overlap region between the steady-state inactivation and activation curves. T-type window currents play an important role in regulating a variety of cellular functions, including neuronal excitability,⁴⁵ ß-cell secretion,⁴⁶ and myoblast terminal differentiation.⁴⁷ We examined voltage-dependent activation and inactivation kinetics of the PMVEC T-type Ca²⁺ channels. Superimposition of voltage-dependent activation and inactivation curves of T-type Ca²⁺ currents reveals a window current in the range of -60 to -30 mV. This PMVEC T-type window-current region is expanded compared with that of native Ca_v3.1 channels expressed in HEK-293 cells (-65 to -55 mV)⁴⁸ and with that of endogenous Ca_v3.1 channels in cardiac myocytes (-65 to -45 mV).⁴⁹ This expanded T-type window current is due to a right-shifted steady-state inactivation curve, which suggests that more channels are available to open. The expanded window current together with the right-shifted I-V relationship appears to represent a unique physiological relevance of Ca_v3.1 channels in PMVECs. The T-type window range is very close to the resting membrane potential of many endothelial cells (-70 to -60 mV or -40 to -10 mV). Thus, small depolarizing currents shift the membrane potential into the window-current range of voltages, which is important to sustain physiologically relevant Ca²⁺ entry.

In endothelial cells, the [Ca²⁺]_i response to G_q-linked agonists is broadly characterized by Ca²⁺ release from intracellular stores and Ca²⁺ entry across the cell membrane after Ca²⁺ store depletion, which are due to the activation of store-operated and receptor-operated Ca²⁺ channels, which are thought to represent the principal Ca²⁺ entry pathways.⁵⁰ However, data shown in the present study resolve another mechanism of Ca²⁺ entry, eg, Ca²⁺ entry through Ca_v3.1 T-type Ca²⁺ channels within the window-current range of voltages. Indeed, mibefradil dramatically reduced thrombin- and thapsigargin-stimulated Ca²⁺ entry in PMVECs without affecting Ca²⁺ release. We exclude other "nonspecific" effects of mibefradil that could occur at its IC₁₀₀ dose, because mibefradil is without effect in PAECs, which possess store-operated Ca²⁺ entry pathways but do not possess T channels. Thus, thrombin depolarizes the PMVEC membrane potential into a window-current range, which is sufficient to activate Ca_v3.1 channels.

In our isolated perfused lung model, inhibition of T-type Ca²⁺ channels completely abolished the increased retention of sickled erythrocytes in the inflamed pulmonary circulation. These findings suggest that platelet-activating factor, leukotriene B₄, and likely other inflammatory agonists consolidate their PMVEC actions by signaling through Ca_v3.1 channels. Thus, Ca²⁺-dependent phenotypic changes in microvascular endothelial cells, ie, stimulated secretion of multimeric vWf and upregulation of adhesion molecules, mediate vaso-occlusion of sickled erythrocytes in the lung microcirculation through Ca_v3.1 channels.

Taken together, our observations support a novel potential therapeutic strategy using selective T-type Ca²⁺ channel antagonists for the prevention of vaso-occlusive crisis in sickle cell anemia or even for the treatment of thrombotic disorders. Although current antithrombotic therapy involves the use of thrombolytic, antiplatelet, and anticoagulant agents, none of these drugs targets endothelial cells with the procoagulant phenotypic changes necessary for triggering the extrinsic pathway of coagulation. Thus, a specific T-type Ca²⁺ channel antagonist may provide broadly ranging anticoagulant activity and have therapeutic utility in microvascular thromboembolic disorders of various etiologies in the cardiovascular, pulmonary, and hematological systems.

	Acknowledgments

 
This study was supported by NIH Grants HL-56056 and HL-66299 (to Dr Stevens), DK50151 (to Dr Li), and American Heart Association National Center Scientist Development Grant No. 0235137N (to Dr Wu). We thank Judy Creighton and Tray Hamil for their excellent technical assistance with cell culture experiments.

	Footnotes

 
Original received May 28, 2003; revision received July 9, 2003; accepted July 9, 2003.

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