L-Type Calcium Channel Blockers Exert an Antiinflammatory Effect by Suppressing Expression of Plasminogen Receptors on Macrophages
L-type Ca2+ channel (LTCC) blockers, represented by amlodipine and verapamil, are widely used antihypertensive drugs that also have antiinflammatory activities. Plasminogen (Plg) is an important mediator of macrophage recruitment, and this role depends on its interaction with Plg receptors (Plg-Rs). Plg-Rs include histone 2B, α-enolase, annexin 2, and p11, all proteins which lack signal sequences for cell surface export. When human or murine monocytoid cells were induced to differentiate into macrophages, their Plg binding and Plg-R expression increased by 4-fold. These changes were suppressed by pretreatment with verapamil and amlodipine. Expression of the Cav1.2 LTCC pore subunit was induced in differentiated macrophages, and siRNA against this subunit suppressed the upregulation of Plg binding and Plg-Rs. In vivo, amlodipine and verapamil suppressed peritoneal macrophage recruitment in response to thioglycollate by >60% at doses that did not affect blood pressure. In drug-treated animals, macrophages migrated into but not through the peritoneal membrane tissue and showed reduced surface expression of Plg-Rs. These findings demonstrate that Plg-R expression on macrophages is dependent on Cav1.2 LTCC subunit expression. Suppression of Plg-Rs may contribute to the antiinflammatory effects of the widely used LTCC blockers.
Involvement of plasminogen (Plg) and its active enzyme plasmin (Plm) in macrophage recruitment has been documented in a number of inflammatory models in Plg−/− mice.1–4 When bound to cell surfaces, Plg/Plm facilitates macrophage migration across adhesive substrates by direct degradation of extracellular matrix (ECM) proteins and by activating matrix metalloproteinase. Plg receptors (Plg-Rs) are abundant on the surface of macrophages and are heterogeneous but are usually characterized by the presence of a C-terminal lysine, which interacts with the lysine-binding sites of Plg.5,6 The major Plg-Rs expressed on macrophages are α-enolase, histone (H)2B, annexin 2, and p11.7–10 We and other have demonstrated that inflammation induces increased Plg binding to mouse macrophages and differentiated human macrophages.11–16 The mechanisms that promote upregulation of Plg-Rs during monocyte maturation are poorly understood because none of the major Plg-Rs has a signal sequence for export through the “conventional” endoplasmic reticulum (ER)/Golgi pathway.
In excitable cells, L-type Ca2+ channels (LTCCs) are voltage-dependent Ca2+ channels composed of a pore-forming α1 subunit (Cav1.1, Cav1.2, Cav1.3, or Cav1.4) and several associated auxiliary subunits (α2-δ, -β, -γ). Cav1.2 pore subunits have also been detected in cells of leukocyte lineage.17–19 Besides lowering blood pressure, LTCC blockers have antiinflammatory effects. Amlodipine, a long-acting dihydropyridine, limits the progression of arteriosclerosis and decreases cardiovascular events.20,21 Verapamil, a phenylalkylamine, also has antiinflammatory properties.22 In search of a mechanism regulating exteriorization of Plg-Rs during monocyte to macrophage differentiation, we now report a profound effect of amlodipine and verapamil on exteriorization of H2B and other Plg-Rs in vitro. We further demonstrate that these drugs suppress macrophage recruitment and Plg-R expression on these cells in vivo.
Materials and Methods
An expanded Material and Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Human monocytoid THP-1 cells were stimulated to differentiate with 250 U/mL interferon (IFN)γ (eBioscience) plus 100 nmol/L VD3 (Calbiochem) for 0 to 2 days in complete growth medium under conditions similar to those described by Kim et al.12
Fluorescence-Activated Cell Sorting
Rabbit polyclonal anti-α1 Cav1.2 (Alomone) was used to detect LTCC on various cell types. Phycoerythrin-conjugated anti-CD14 (eBioscience) was used to measure monocyte differentiation to macrophages.
Plg binding was assessed as described previously.15
Cell Surface Biotinylation and Western Blotting
Cell surface biotinylation on THP-1 cells followed by Western blot was performed as previously described.15
Cav1.2 subunit of LTCC was knocked down by small interfering (si)RNA designed to target Cav1.2 subunit (SiCav1.2, Dharmacon) using the nucleofection protocol described by Amaxa. Control siRNA (SiControl) was a scrambled sequence and was provided by Ambion.
RNA Isolation and RT-PCR
RNA isolation and RT-PCR were performed using RNeasy Mini Kits (Qiagen) and OneStep RT-PCR kit (Qiagen), respectively. The primers used to amplify Cav11.2 transcripts have been described previously.18
Intracellular Ca2+ Measurements
Fura-2–loaded THP-1 cells (Fura-2 from Invitrogen) were stimulated with 1 μmol/L fMLP (Sigma), and Ca2+ mobilization was recorded using a fluorimeter (Photon Technology International) with dual excitation at 340 and 380 nm.
In Vitro Matrigel Invasion
In vitro Matrigel assay was performed as previously described.15
In Vivo Experiments
Amlodipine, verapamil, or their vehicle controls were administered via subcutaneously implanted osmotic minipump, 0.25 μL/h (Alzet, Durect). Peritoneal inflammation was induced by IP administration of thioglycollate (TG). Cells within the peritoneal lavage, collected 3 days after TG, were counted and peritoneal membrane (PM) that lines the pancreas was isolated for further study.23
Anti-Mac3 (BD Biosciences) and biotinylated anti-rat antibody (mouse adsorbed, Vector) were used to stain sections of PM and developed with Vectastain avidin–biotin complex (ABC) reagent (Vector) using DAB as a substrate and counterstained with hematoxylin.
Rabbit polyclonal anti-peptide antibodies against H2B, α-enolase, annexin 2, p11, or nonimmune rabbit IgG were reacted with 8-μm sections, followed by Alexa-488–labeled anti-rabbit IgG (Invitrogen).
Values are expressed as means±SD; probability values are based on paired Student’s t tests. Results with P<0.05 were considered significantly different.
Monocyte Differentiation Induces Plg Binding
To investigate the molecular pathway(s) by which cell surface expression of Plg-Rs is modulated during monocyte activation, an in vitro model, THP-1 differentiation to macrophages, was implemented. When THP-1 cells were treated with IFNγ+VD3, expression of the macrophage differentiation marker CD14 was substantially enhanced on day 1 and further increased on day 2 (Figure 1A). As measured by FACS, Alexa-488–labeled Plg binding increased 4-fold at day 2 (Figure 1A). In parallel, cell surface expression of the major Plg-Rs on these cells, annexin 2, α-enolase, H2B, and p11, increased by 3- to 4-fold (Figure 1B, left, and Figure 1C), as measured by Western blots with specific antibodies to each protein.15 CD14 expression was also increased at the cell surface and in whole cell lysates (Figure 1B and 1C). Consistent with a previous report,14 cellular annexin 2 expression increased with differentiation, both at the protein and mRNA levels (Figure 1B and 1C). However, increased cell surface expression of α-enolase, H2B, and p11 occurred in the absence of detectable changes in total cellular expression at either the protein or mRNA levels, suggesting that changes in cell surface expression were independent of new protein synthesis (Figure 1B, right, and Figure 1C; Online Figure I). Similar results were demonstrated recently for α-enolase,16 using lipopolysaccharide to induce its cell surface expression. Together, these observations suggest changes in protein trafficking.
LTCC Inhibitors Regulate Surface Expression of Plg-Rs
THP-1 cells pretreated with Brefeldin A, which blocks transport of protein from the ER to the Golgi, reduced Plg binding by <25% (Figure 2A; P=0.02). Pathways involved in “nonconventional” protein secretion can include the ABC1 transporter.24 However, glyburide, an ABC1 transporter inhibitor, did not reduce Plg binding significantly (Figure 2A). Endolysosomal recycling (interleukin-1β) and Na+/K+-ATPase (fibroblast growth factor-2) have also been implicated as mediators of nonconventional protein surface expression.25,26 However, methylamine, which blocks endosomal recycling25 and ouabain, a Na+/K+-ATPase inhibitor, had no effect on differentiation-induced Plg binding to THP-1 cells (Figure 2B). Increased [Ca2+]in is a primary cellular response associated with monocyte differentiation.27 Pretreatment of THP-1 cells with BAPTA-AM, a cell-permeant calcium chelator, blocked the IFNγ+VD3-induced increase in Plg binding by 98% (Figure 2B). Consistent with a role for [Ca2+]in in modulating Plg-R expression, ionomycin, a Ca2+ ionophore, enhanced Plg binding to the IFNγ+VD3 treated THP-1 cells by 34±9.8% (P=0.01).
To further investigate the role of [Ca2+]in in regulation of Plg binding, the effects of selective Ca2+ channel inhibitors were tested. Pretreatment of THP-1 cells with ω-agatoxin and ω-conotoxin, which block P-type and N-type Ca2+ channels, respectively, had no effect on Plg binding (Figure 2B). However, 2 unrelated LTCC blockers, verapamil and amlodipine, effectively suppressed differentiation-induced Plg binding (Figure 2B). The concentration of these LTCC blockers (10 μmol/L) was selected based on a previous study on their effects on adhesion of THP-1 cells.17 LTCC blockers can also attenuate the synthesis of superoxide anions.28 However, pretreatment of cells with tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidininoxyl), a stable membrane-permeable superoxide dismutase mimetic, had only a modest effect (26%; P=0.06) on the increase in Plg binding induced by differentiation (Figure 2B). Activation of LTCC is associated with cAMP generation,29 and IFNα mediates α-enolase cell surface expression via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)1/2 activation.30 Pretreatment of cells with the adenylate cyclase inhibitor SQ22536 or the ERK1/2 phosphorylation inhibitor PD98059 had no effect on differentiation-induced Plg binding (Online Figure II, C), even though PD98059 blocked differentiation-induced cAMP generation and SQ22536 blocked ERK1/2 phosphorylation in the cells (Online Figure II, A and B).
Presence of Functional LTCCs in Monocytes and Macrophages
Three independent approaches were used to evaluate the presence and function of LTCC in the differentiated monocytoid cells. First, RT-PCR was performed on RNA isolated from THP-1 cells, human blood monocyte–derived macrophages (HBMMΦ), and TG-induced mouse peritoneal macrophages (TGMPMΦ). Using primers18 amplifying the IVS4-IVS6 domain of the Cav1.2 subunit of LTCC, an intense band of the predicted size (350 kb) was amplified in each cell type (Figure 3A). Human aortic smooth muscle cells (HASMCs), used as a positive control,31 also yielded a similar product. All of these cells also expressed LTCC β1 and β2 subunits (RT-PCR; data not shown). Second, using FACS, an anti-Cav1.2 antibody against a Cav1.2 intracellular sequence epitope reacted well with permeabilized HASMCs and THP-1, TGMPMΦ, and HMDMΦ cells. This signal was substantially reduced by exposure to the immunizing peptide (Figure 3B). Additionally, Cav1.2 expression was enhanced on treatment of THP-1 cells with IFNγ+VD3 for 0 to 2 days (Online Figure III, A). Third, Ca2+ mobilization in response to fMLP increased with differentiation (Online Figure III, B). This Ca2+ entry was blunted by amlodipine or verapamil, implicating LTCC in the [Ca2+]in response (Figure 3C). However, [Ca2+]in was unchanged when cells were treated with 50 mmol/L KCl to induce depolarization (data not shown), suggesting that the LTCC present in THP-1 cells are not voltage-gated.
To eliminate the nonspecific actions of LTCC blockers, THP-1 cells were nucleofected with siRNA against Cav1.2 (SiCav1.2) or a control siRNA (SiControl) and then stimulated with IFNγ+VD3. The upregulation of Cav1.2 induced by differentiation was suppressed by SiCav1.2, but not by SiControl (Online Figure IV). Moreover, differentiation-induced Plg binding was reduced by ≈90% (Figure 5A) by SiCav1.2 compared to SiControl. Interference with Cav1.2 expression also suppressed the [Ca2+]in response induced by fLMP in differentiated THP-1 cells (Figure 5C).
Involvement of LTCCs and [Ca2+]in in Upregulation of Plg-Rs on Stimulated THP-1 Cells
We sought to determine whether the dependence of Plg binding on LTCC and their function was attributable to suppression of one or more Plg-Rs. When THP-1 cells were pretreated with BAPTA-AM and then stimulated with IFNγ+VD3, BAPTA-AM prevented the cell surface upregulation of each of the 4 Plg-Rs (Figure 4A, left and right), without changing whole cell content of these proteins, ie, trafficking of these Plg-Rs is Ca2+-dependent. In THP-1 cells pretreated with amlodipine or verapamil and then induced to differentiate, induction of each of the 4 Plg-Rs was reduced by the lower concentration (5 μmol/L) and was almost fully blocked by their higher concentration (10 μmol/L), with no effect on whole cell levels of these proteins (Figure 4B, top and bottom). Similarly, SiCav1.2 but not SiControl suppressed surface expression without affecting whole cell levels of the Plg-Rs (Figure 5B).
LTCCs Blockers Impair Macrophage Recruitment In Vivo by Suppressing Plg-Rs
LTCC inhibitors are known to have antiinflammatory effects,20–22,28 and we sought to determine whether suppression of Plg-R expression might underlie this activity. To test this possibility, we turned to a widely used inflammatory model. Verapamil and amlodipine were administered to mice via subcutaneously implanted mini-pumps beginning 2 days before thioglycollate (TG)-induced peritonitis, which involves blood monocyte differentiation into macrophages and their recruitment to the site of inflammation.3,32 Amlodipine inhibited macrophage recruitment by 54% (P=0.004) at 1 mg/kg per day and by 87% (P=9.22×10−7) at 3 mg/kg per day compared to its vehicle control (Figure 6A). With verapamil, inhibition at the lower (1 mg/kg per day) and higher doses (3 mg/kg per day) was 82% (P=1.24×10−6) and 64% (P=0.001), respectively. The differences in recruitment at the high and low dose of verapamil were not significant (P=0.11). The vehicle control for each drug was not different from the recruitment observed in untreated mice. Even though these drugs are antihypertensive, hypotensive effects were only detected at the higher doses of both drugs (Table). At the lower doses at which both amlodipine and verapamil suppressed macrophage recruitment by 54 and 80%, respectively, systolic blood pressure was unaffected (Table). Similar effects of these doses of LTCC blockers on blood pressure were observed by Kataoka et al.33 These results suggest that the drugs were used in a biologically relevant range.
The reduction in macrophage recruitment in response to TG in mice deficient in Plg3 arises from their accumulation in the membrane (PM) of the peritoneal cavity.23 If the decrease of macrophage recruitment by the antihypertensive drugs was associated with modulation of Plg binding and Plg-R expression, the antiinflammatory effects of amlodipine and verapamil also might arise from the accumulation of macrophages in the PM. Hematoxylin/eosin staining demonstrated a large accumulation of cells in the PM of mice treated at the lower, nonhypertensive dose of amlodipine and verapamil (Figure 6B). The accumulated cells in the PM of the drug-treated mice were identified as macrophages based on Mac3 staining (Figure 6C): the abundance of Mac3-positive cells was 15±2.5% with amlodipine and 27±5.3% with verapamil versus 3.3±0.4% (P=0.001) and 3.4±0.4% (P=0.001), respectively, in PM of vehicle-treated mice. Thus, the accumulation was enhanced ≈7-fold by verapamil and ≈5-fold with amlodipine (Figure 6D).
To determine whether the accumulation of macrophages in the PM of animals on the antihypertensive drugs was attributable to effects on Plg-R expression, we examined the expression of the 4 Plg-Rs of interest on the surface of macrophages arrested in the PM of the drug-treated mice compared to accumulated macrophages of Plg−/− mice. Confocal analyses of PM sections revealed H2B, α-enolase, annexin 2, and p11 on the surface (cells were not permeabilized) of arrested macrophages (Figure 7A) in Plg−/− mice. H2B was also present in the matrix of the PM in the Plg−/− mice. After verapamil treatment, α-enolase levels were unaffected on the macrophages in the PM of the Plg+/+ mice compared to Plg−/− mice (Figure 6A and 6B). However, cell surface distribution of annexin 2 and H2B were drastically reduced in the drug-treated Plg+/+ versus the Plg−/− mice (Figure 7A and 7B). p11 cell surface distribution was also reduced in drug-treated macrophages compared to Plg−/− macrophages (Figure 7A and 7B). These results suggest that LTCC blockers are antiinflammatory, and this phenomenon occurs in concert with reductions of Plg-Rs on the surface of macrophages.
Receptors for Plg play an important role in regulating inflammatory cell recruitment.15,16,23,34 In the present study, we have sought to address the longstanding dilemma as to how Plg-Rs are regulated on inflammatory cells, using THP-1 differentiation to macrophages to drive surface expression of Plg-Rs. Stimulation of THP-1 cells with IFNγ+VD3 led to α4-fold increase in Plg binding, consistent with a previous report.13 Concomitant with this increase, stimulation of the cells for 24 to 48 hours led to increased cell surface expression of the Plg-Rs. For H2B, α-enolase, and p11, the increase in cell surface localization occurred without detectable increases in total cellular protein and mRNA levels. In contrast, cellular protein expression, mRNA levels, and cell surface expression of annexin 2 were all increased with differentiation of the THP-1 cells, consistent with a previous report.14
Several inhibitors of known nonconventional pathways of protein export were tested, using glyburide (to block the ABC1 transporter), ouabain (Na+/K+ ATPase), and methylamine (endosomal pathway). All of these inhibitors had minimal effects on Plg binding and Plg-R expression. In contrast, the intracellular [Ca2+]in chelator, BAPTA-AM, suppressed the increment in Plg binding induced by IFNγ+VD3 by 98% and completely blocked the increased expression of the 4 Plg-Rs assessed. To further dissect the role of [Ca2+]in in regulation of Plg-Rs, we tested the effects of verapamil and amlodipine, 2 distinct LTCC inhibitors. Both LTCC blockers completely suppressed the upregulation of Plg-Rs by IFNγ+VD3. In contrast, ionomycin, which increases [Ca2+]in, led to a significant increase in Plg binding; its modest effect may be attributable to repression of H2B gene expression by ionomycin.35
In support of the pharmacological data implicating the LTCC and cytosolic calcium in regulation of Plg-R membrane localization, we found that THP-1 cells, as well as mouse and human macrophages, express the LTCC Cav1.2. To our knowledge, this is the first study to document the presence of Cav1.2 transcript and protein in human and mouse macrophages. Differentiation of monocytes to macrophages was associated with increased Cav1.2 expression and a concomitant increase in fMLP-stimulated Ca2+ mobilization (measured with fura2). This response was blocked by amlodipine, verapamil, and siRNA against Cav1.2. In macrophages, the LTCC may be non–voltage-gated, because addition of KCl did not induce intracellular Ca2+ mobilization. This speculation is consistent with the nature of LTCC in other nonexcitable cells, such as T and B lymphocytes,18,19 and likely explains our inability to measure voltage-gated calcium channel activity using whole cell patch clamp (data not shown).
That knockdown of SiCav1.2 with siRNA showed the same inhibitory effects as verapamil and amlodipine on Plg-R cell surface expression suggests that the drug effects are attributable to their interactions with LTCCs. SiCav1.2 inhibited upregulation of Plg-R cell surface expression, Plg binding, and Plg-mediated Matrigel invasion (Online Figure V, A and B). Anti-H2B Fab fragments, which block Plg binding to H2B on the cell surface,15 reduced Plm-mediated THP-1 cell Matrigel invasion by 45% compared to nonimmune rabbit IgG Fab (Online Figure V, B), but there was no additional effect of anti-H2B Fab on the SiCav1.2-treated cells (Online Figure V, B), showing that the suppressive effect of SiCav1.2 on Plm-mediated THP-1 Matrigel invasion was indeed attributable to reduction of one or more candidate Plg-Rs.
Previous work by Brown et al36 showed that verapamil reduced tumor necrosis factor-α mRNA expression in response to ultrafine particles in rat alveolar macrophages, but we did not observe changes in whole cell protein or mRNA expression of Plg-Rs in THP-1 cells by either verapamil or amlodipine. Studies of intracellular signaling have implicated adenylase cyclase and ERK/MAPK as pathways dependent on LTCC, including in IFNα-mediated enolase cell surface expression. However, in our study, inhibitors of these events failed to block upregulation of Plg-Rs. The details of the pathway(s) by which LTCC and intracellular Ca2+ modulate Plg-R cell surface localization remains to be elucidated in future studies.
Macrophage recruitment in a TG-induced mouse peritonitis model is reduced by >60% in Plg-deficient mice3 and is associated with a large accumulation of these cells in the PM.23 In our study, we showed that both amlodipine and verapamil led to a marked suppression (82% to 87%) of macrophage emigration into the peritoneal cavity. The antiinflammatory effects of amlodipine and verapamil have been demonstrated in previous studies in other models.20–22,28,33 As in Plg−/− mice, suppression of migration was associated with accumulation of macrophages in the PM of the verapamil and amlodipine treated mice. In comparing accumulated macrophages in mice treated with LTCC blockers and Plg deficiency, the drugs reduced cell surface expression of H2B and annexin 2 markedly. Cell surface localization of p11 was also reduced, but not as extensively, and α-enolase expression was unaltered. The availability of low levels of Plg-Rs may explain why the drugs were slightly less effective than absence of Plg in suppressing macrophage recruitment. In addition to cell surface localization, H2B protein was found in the ECM in the Plg−/− mice. Heparan sulfate proteoglycans are known to bind the N termini of histones,37 and released histones have been shown to localize in the ECM.38 Matrix bound H2B may provide another mechanism for localizing Plg, enhancing its activation to Plm and, thereby, facilitating inflammatory cell migration.
In summary, our study identifies a pivotal role of increased [Ca2+]in in the upregulation of cell surface expression of Plg-Rs on monocyte differentiation into macrophages. We further show that verapamil and amlodipine, 2 commonly used LTCC blockers, suppress surface expression of Plg-Rs. The influence of these drugs on cell surface expression of Plg-Rs has been demonstrated both in vitro and in vivo. This previously unrecognized activity of these widely used drugs may contribute to their beneficial antiinflammatory effects.
We gratefully acknowledge Cleveland Clinic coworkers Christina Gaughan and Rui Chen for assistance in BP and [Ca2+]in measurement, respectively. We thank Dr Jane Hoover-Plow and laboratory members, Dr Maradumane Mohan for valuable discussion and technical help with in vitro experiments, and Drs Judy Drazba and John Peterson for help with microscopy studies.
Sources of Funding
This work was supported by NIH grant HL17964 (to E.F.P.) and American Heart Association Fellowship 0825638D (to R.D.).
Original received October 20, 2008; resubmission received May 4, 2009; accepted June 1, 2009.
Ploplis VA, French EL, Carmeliet P, Collen D, Plow EF. Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice. Blood. 1998; 91: 2005–2009.
Plow EF, Herren T, Redlitz A, Miles LA, Hoover-Plow JL. The cell biology of the plasminogen system. FASEB J. 1995; 9: 939–945.
Hajjar KA. The endothelial cell tissue plasminogen activator receptor. Specific interaction with plasminogen. J Biol Chem. 1991; 266: 21962–21970.
MacLeod TJ, Kwon M, Filipenko NR, Waisman DM. Phospholipid-associated annexin A2–S100A10 heterotetramer and its subunits: characterization of the interaction with tissue plasminogen activator, plasminogen, and plasmin. J Biol Chem. 2003; 278: 25577–25584.
Felez J, Miles LA, Plescia J, Plow EF. Regulation of plasminogen receptor expression on human monocytes and monocytoid cell lines. J Cell Biol. 1990; 111: 1673–1683.
Kim S-O, Plow EF, Miles LA. Regulation of plasminogen receptor expression on monocytoid cells by β1-integrin dependent cellular adherence to extracellular matrix proteins. J Biol Chem. 1996; 271: 23761–23767.
Brownstein C, Deora AB, Jacovina AT, Weintraub R, Gertler M, Faisal Khan KM, Falcone DJ, Hajjar KA. Annexin II mediates plasminogen-dependent matrix invasion by human monocytes: enhanced expression by macrophages. Blood. 2004; 103: 317–324.
Das R, Burke T, Plow EF. Histone H2B as a functionally important plasminogen receptor on macrophages. Blood. 2007; 110: 3763–3772.
Wygrecka M, Marsh LM, Morty RE, Henneke I, Guenther A, Lohmeyer J, Markart P, Preissner KT. Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung. Blood. 2009; 113: 5588–5598.
Yu T, Morita I, Shimokado K, Iwai T, Yoshida M. Amlodipine modulates THP-1 cell adhesion to vascular endothelium via inhibition of protein kinase C signal transduction. Hypertension. 2003; 42: 329–334.
Stokes L, Gordon J, Grafton G. Non-voltage-gated L-type Ca2+ channels in human T cells: pharmacology and molecular characterization of the major alpha pore-forming and auxiliary beta-subunits. J Biol Chem. 2004; 279: 19566–19573.
Pitt B, Byington RP, Furberg CD, Hunninghake DB, Mancini GB, Miller ME, Riley W. Effect of amlodipine on the progression of atherosclerosis and the occurrence of clinical events. PREVENT Investigators. Circulation. 2000; 102: 1503–1510.
Chandra M, Shirani J, Shtutin V, Weiss LM, Factor SM, Petkova SB, Rojkind M, Dominguez-Rosales JA, Jelicks LA, Morris SA, Wittner M, Tanowitz HB. Cardioprotective effects of verapamil on myocardial structure and function in a murine model of chronic Trypanosoma cruzi infection Brazil Strain): an echocardiographic study. Int J Parasitol. 2002; 32: 207–215.
Hamon Y, Luciani MF, Becq F, Verrier B, Rubartelli A, Chimini G. Interleukin-1beta secretion is impaired by inhibitors of the Atp binding cassette transporter, ABC1. Blood. 1997; 90: 2911–2915.
Andrei C, Dazzi C, Lotti L, Torrisi MR, Chimini G, Rubartelli A. The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles. Mol Biol Cell. 1999; 10: 1463–1475.
Kockx M, Guo DL, Huby T, Lesnik P, Kay J, Sabaretnam T, Jary E, Hill M, Gaus K, Chapman J, Stow JL, Jessup W, Kritharides L. Secretion of apolipoprotein E from macrophages occurs via a protein kinase A and calcium-dependent pathway along the microtubule network. Circ Res. 2007; 101: 607–616.
Gollasch M, Haase H, Ried C, Lindschau C, Morano I, Luft FC, Haller H. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J. 1998; 12: 593–601.
Kataoka C, Egashira K, Ishibashi M, Inoue S, Ni W, Hiasa K, Kitamoto S, Usui M, Takeshita A. Novel anti-inflammatory actions of amlodipine in a rat model of arteriosclerosis induced by long-term inhibition of nitric oxide synthesis. Am J Physiol Heart Circ Physiol. 2004; 286: H768–H774.
Kozian D, Proulle V, Nitsche A, Galitzine M, Martinez MC, Schumann B, Meyer D, Herrmann M, Freyssinet JM, Kerbiriou-Nabias D. Identification of genes involved in Ca2+ ionophore A23187-mediated apoptosis and demonstration of a high susceptibility for transcriptional repression of cell cycle genes in B lymphoblasts from a patient with Scott syndrome. BMC Genomics. 2005; 6: 146.
Brown DM, Donaldson K, Borm PJ, Schins RP, Dehnhardt M, Gilmour P, Jimenez LA, Stone V. Calcium and ROS-mediated activation of transcription factors and TNF-alpha cytokine gene expression in macrophages exposed to ultrafine particles. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L344–L353.
Watson K, Gooderham NJ, Davies DS, Edwards RJ. Nucleosomes bind to cell surface proteoglycans. J Biol Chem. 1999; 274: 21707–21713.
Henriquez JP, Casar JC, Fuentealba L, Carey DJ, Brandan E. Extracellular matrix histone H1 binds to perlecan, is present in regenerating skeletal muscle and stimulates myoblast proliferation. J Cell Sci. 2002; 115: 2041–2051.