Melatonin inhibits voltage-gated potassium KV4.2 channels and negatively regulates melatonin secretion in rat pineal glands.

Melatonin is synthesized in and secreted from the pineal glands, and regulates circadian rhythms. Although melatonin has been reported to modulate the activity of ion channels in several tissues, its effects on pineal ion channels remain unclear. In the present study, the effects of melatonin on voltage-gated K+ (KV) channels, which play a role in regulating the resting membrane potential, were examined in rat pinealocytes. The application of melatonin reduced pineal KV currents in a concentration-dependent manner (IC50=309 mM). An expression analysis revealed that KV4.2 channels were highly expressed in rat pineal glands. Melatonin-sensitive currents were abolished by the small interfering RNA knockdown of KV4.2 channels in rat pinealocytes. In human embryonic kidney 293 (HEK293) cells expressing KV4.2 channels, melatonin decreased outward currents (IC50=479 mM). Inhibitory effects were mediated by a shift in voltage dependence from steady-state inactivation to a hyperpolarizing direction. This inhibition was observed even in the presence of 100 nM luzindole, an antagonist of melatonin receptors. Melatonin also blocked the activity of KV4.3, KV1.1, and KV1.5 channels in reconstituted HEK293 cells. The application of 1 mM melatonin caused membrane depolarization in rat pinealocytes. Furthermore, KV4.2 channel inhibition by 5 mM 4-aminopyridine attenuated melatonin secretion induced by 1 mM noradrenaline in rat pineal glands. These results strongly suggest that melatonin directly inhibited KV4.2 channels and caused membrane depolarization in pinealocytes, resulting in a decrease in melatonin secretion through parasympathetic signaling pathway. This mechanism may function as a negative-feedback mechanism of melatonin secretion in pineal glands.

Corosolic acid ameliorates vascular remodeling in pulmonary arterial hypertension via the downregulation of STAT3 signaling.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that is characterized by vascular remodeling of the pulmonary artery. PAH remodeling is primarily caused by the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). Therefore, an inhibitory mechanism is expected as a target for the treatment of PAH. Corosolic acid (CRA) is a pentacyclic triterpenoid extracted from the leaves of Banaba (Lagerstroemia speciosa) that exerts anti-diabetic, anti-inflammatory, and anti-tumor effects. In the present study, the effects of CRA on PAH remodeling were examined using PASMCs from idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline (MCT)-induced pulmonary hypertensive (PH) rats. CRA inhibited the excessive proliferation of IPAH-PASMCs in a concentration-dependent manner (IC50 = 14.1 µM). It also reduced the migration of IPAH-PASMCs. The CRA treatment downregulated the expression of signal transducer and activator of transcription 3 (STAT3) in IPAH-PASMCs. In MCT-PH rats, the administration of CRA (1 mg/kg/day) attenuated increases in right ventricular systolic pressure, pulmonary vascular remodeling, and right ventricular hypertrophy. CRA also decreased the expression of STAT3 in pulmonary arterial smooth muscles from MCT-PH rats. In conclusion, the anti-proliferative and anti-migratory effects of CRA in PASMCs ameliorated PAH remodeling by downregulating STAT3 signaling pathways.

Pimaric acid reduces vasoconstriction via BKCa channel activation and VDCC inhibition in rat pulmonary arterial smooth muscles.

Pulmonary vessels play a pivotal role in oxygen circulation. We previously demonstrated that pimaric acid (PiMA) activated large-conductance Ca2+-activated K+ (BKCa) channels and inhibited voltage-dependent Ca2+ channels (VDCCs). In the present study, PiMA attenuated vasoconstriction induced by high K+ or endothelin-1 in rat pulmonary arterial smooth muscles (PASMs). PiMA also reduced high K+-induced cytosolic [Ca2+] increase in PASM cells. PiMA increased BKCa currents and decreased VDCC currents. BKCa channels and VDCCs were formed by the α/ß1 and α1C/α1D/ß2/ß3 subunits, respectively. These results indicate that PiMA induces vasorelaxation through the dual effects of BKCa channel activation and VDCC inhibition in PASMs.

SKF96365 activates calcium-sensing receptors in pulmonary arterial smooth muscle cells.

In pulmonary arterial smooth muscle cells (PASMCs), an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt) is involved in many physiological processes such as cell contraction and proliferation. However, chronic [Ca2+]cyt increases cause pulmonary vasoconstriction and vascular remodeling, resulting in pulmonary arterial hypertension (PAH). Therefore, [Ca2+]cyt signaling plays a substantial role in the regulation of physiological and pathological functions in PASMCs. In the present study, the effects of SKF96365 on [Ca2+]cyt were examined in PASMCs from normal subjects and idiopathic pulmonary arterial hypertension (IPAH) patients. SKF96365 is widely used as a blocker of non-selective cation channels. SKF96365 did not affect the resting [Ca2+]cyt in normal-PASMCs. However, SKF96365 increased [Ca2+]cyt in IPAH-PASMCs in a concentration-dependent manner (EC50 = 18 µM). The expression of Ca2+-sensing receptors (CaSRs) was higher in IPAH-PASMCs than in normal-PASMCs. The SKF96365-induced [Ca2+]cyt increase was inhibited by CaSR antagonists, NPS2143 and Calhex 231. The CaSR-mediated [Ca2+]cyt increase was facilitated by SKF96365 and the activation was blocked by NPS2143 or Calhex 231. In addition, the SKF96365-induced [Ca2+]cyt increase was reduced by siRNA knockdown of CaSRs. Taken together, SKF96365 activates CaSRs in IPAH-PASMCs and promotes [Ca2+]cyt signaling.

Upregulated ClC3 Channels/Transporters Elicit Swelling-Activated Cl- Currents and Induce Excessive Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling of the pulmonary artery, which is mainly attributed to the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) comprising the medial layer of pulmonary arteries. The activity of ion channels associated with cytosolic Ca2+ signaling regulates the pathogenesis of PAH. Limited information is currently available on the role of Cl- channels in PASMCs. Therefore, the functional expression of ClC3 channels/transporters was herein investigated in the PASMCs of normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). Expression analyses revealed the upregulated expression of ClC3 channels/transporters at the mRNA and protein levels in IPAH-PASMCs. Hypoosmotic perfusion (230 mOsm) evoked swelling-activated Cl- currents (ICl-swell) in normal-PASMCs, whereas 100 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) exerted the opposite effects. The small interfering RNA (siRNA) knockdown of ClC3 did not affect ICl-swell. On the other hand, ICl-swell was larger in IPAH-PASMCs and inhibited by DIDS and the siRNA knockdown of ClC3. IPAH-PASMCs grew more than normal-PASMCs. The growth of IPAH-PASMCs was suppressed by niflumic acid and DIDS, but not by 9-anthracenecarboxylic acid or T16Ainh-A01. The siRNA knockdown of ClC3 also inhibited the proliferation of IPAH-PASMCs. Collectively, the present results indicate that upregulated ClC3 channels/transporters are involved in ICl-swell and the excessive proliferation of IPAH-PASMCs, thereby contributing to the pathogenesis of PAH. Therefore, ClC3 channels/transporters have potential as a target of therapeutic drugs for the treatment of PAH.

The Rho kinase 2 (ROCK2)-specific inhibitor KD025 ameliorates the development of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that is characterized by the irreversible remodeling of the pulmonary artery. Although several PAH drugs have been developed, additional drugs are needed. Rho kinases (ROCKs) are involved in the pathogenesis of PAH, and thus, their inhibitors may prevent the development of PAH. However, the therapeutic benefits of ROCK isoform-specific inhibitors for PAH remain largely unknown. The in vitro and in vivo effects of the ROCK2-specific inhibitor, KD025, were examined herein using pulmonary arterial smooth muscle cells (PASMCs) from idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline (MCT)-induced pulmonary hypertensive (PH) rats. The expression of ROCK1 was similar between normal- and IPAH-PASMCs, whereas that of ROCK2 was markedly higher in IPAH-PASMCs than in normal-PASMCs. KD025 inhibited the accelerated proliferation of IPAH-PASMCs in a concentration-dependent manner (IC50 = 289 nM). Accelerated proliferation was also reduced by the siRNA knockdown of ROCK2. In MCT-PH rats, the expression of ROCK2 was up-regulated in PASMCs. Elevated right ventricular systolic pressure in MCT-PH rats was attenuated by KD025 (1 mg/kg/day). These results strongly suggest that enhanced ROCK2 signaling is involved in the pathogenic mechanism underlying the development of PAH, including accelerated PASMC proliferation and vascular remodeling in patients with PAH. Therefore, ROCK2 may be a novel therapeutic target for the treatment of PAH.

Comparative analysis of age in monocrotaline-induced pulmonary hypertensive rats.

Pulmonary arterial hypertension (PAH) is a rare, progressive, and fatal cardiovascular/lung disease. The incidence rate is affected by age. Monocrotaline (MCT, 60 mg/kg)-treated rats are widely used as an experimental PAH model. Here, we found that young rats died at a mean of 23.4 days after MCT injection, whereas adult rats survived for over 42 days. However, young (7-week-old) and adult (20-week-old) MCT-treated rats developed PAH, and had upregulated Ca2+-sensing receptor and transient receptor potential canonical subfamily 6 channel expression in pulmonary arteries. The present study provides novel information for elucidating the mechanism underlying the age difference in PAH patients.

Eicosapentaenoic acid ameliorates pulmonary hypertension via inhibition of tyrosine kinase Fyn.

Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial vasoconstriction and remodeling. Src family tyrosine kinases, including Fyn, play critical roles in vascular remodeling via the inhibition of STAT3 signaling. EPA is known to inhibit Fyn kinase activity. This study investigated the therapeutic potential and underlying mechanisms of EPA and its metabolite, resolvin E1 (RvE1), to treat PAH using monocrotaline-induced PAH model rats (MCT-PAH), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs). Administration of EPA 1 and 2 weeks after MCT injection both ameliorated right ventricular hypertrophy, remodeling and dysfunction, and medial wall thickening of the pulmonary arteries and prolonged survival in MCT-PAH rats. EPA attenuated the enhanced contractile response to 5-hydroxytryptamine in isolated pulmonary arteries of MCT-PAH rats. Mechanistically, the treatment with EPA and RvE1 or the introduction of dominant-negative Fyn prevented TGF-ß2-induced endothelial-to-mesenchymal transition and IL-6-induced phosphorylation of STAT3 in cultured HPAECs. EPA and RvE1 suppressed Src family kinases' activity as evaluated by their phosphorylation status in cultured HPAECs and HPASMCs. EPA and RvE1 suppressed vasocontraction of rat and human PA. Furthermore, EPA and RvE1 inhibited the enhanced proliferation and activity of Src family kinases in HPASMCs derived from patients with idiopathic PAH. EPA ameliorated PAH's pathophysiology by mitigating vascular remodeling and vasoconstriction, probably inhibiting Src family kinases, especially Fyn. Thus, EPA is considered a potent therapeutic agent for the treatment of PAH.

MicroRNA-mediated downregulation of K+ channels in pulmonary arterial hypertension.

Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel ß1 subunit (BKCaß1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaß1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

Platelet-derived growth factor up-regulates Ca2+-sensing receptors in idiopathic pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease associated with remodeling of the pulmonary artery. We previously reported that the Ca2+-sensing receptor (CaSR) is up-regulated in pulmonary arterial smooth muscle cells (PASMCs) from patients with idiopathic PAH (IPAH) and contributes to enhanced Ca2+ responses and excessive cell proliferation. However, the mechanisms underlying the up-regulation of CaSR have not yet been elucidated. We herein examined involvement of platelet-derived growth factor (PDGF) on CaSR expression, Ca2+ responses, and proliferation in PASMCs. The expression of PDGF receptors was higher in PASMCs from patients with IPAH than in PASMCs from normal subjects. In addition, PDGF-induced activation of PDGF receptors and their downstream molecules [ERK1/2, p38, protein kinase B, and signal transducer and activator of transcription (STAT) 1/3] were sustained longer in PASMCs from patients with IPAH. The PDGF-induced CaSR up-regulation was attenuated by small interfering RNA knockdown of PDGF receptors and STAT1/3, and by the treatment with imatinib. In monocrotaline-induced pulmonary hypertensive rats, the up-regulation of CaSR was reduced by imatinib. The combination of NPS2143 and imatinib additively inhibited the development of pulmonary hypertension. These results suggest that enhanced PDGF signaling is involved in CaSR up-regulation, leading to excessive PASMC proliferation and vascular remodeling in patients with IPAH. The linkage between CaSR and PDGF signals is a novel pathophysiological mechanism contributing to the development of PAH.-Yamamura, A., Nayeem, M. J., Al Mamun, A., Takahashi, R., Hayashi, H., Sato, M. Platelet-derived growth factor up-regulates Ca2+-sensing receptors in idiopathic pulmonary arterial hypertension.

Calcilytics inhibit the proliferation and migration of human prostate cancer PC-3 cells.

The carcinogenesis and development of prostate cancer are mediated by enhanced Ca2+ signaling. In the present study, the pharmacological profile of the Ca2+-sensing receptor (CaSR) antagonists (calcilytics) was examined in human prostate cancer PC-3 cells. NPS2143 and Calhex 231 blocked extracellular Ca2+-induced increases in cytosolic [Ca2+]. NPS2143 and Calhex 231 inhibited cell proliferation (IC50 = 7.4 and 10.3 µM, respectively) and migration. The exposure to NPS2143 or Calhex 231 down-regulated CaSR protein expression. These results demonstrated that calcilytics inhibited cell proliferation/migration and down-regulated CaSR expression in human prostate cancer cells, suggesting their potential as novel therapeutic drugs for prostate cancer.

Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and Is a Therapeutic Target in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is a cytozyme that regulates intracellular nicotinamide adenine dinucleotide levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation, and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling and that inhibition of NAMPT could attenuate pulmonary hypertension. METHODS: Plasma, mRNA, and protein levels of NAMPT were measured in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension and in the lungs of rodent models of pulmonary hypertension. Nampt+/- mice were exposed to 10% hypoxia and room air for 4 weeks, and the preventive and therapeutic effects of NAMPT inhibition were tested in the monocrotaline and Sugen hypoxia models of pulmonary hypertension. The effects of NAMPT activity on proliferation, migration, apoptosis, and calcium signaling were tested in human pulmonary artery smooth muscle cells. RESULTS: Plasma and mRNA and protein levels of NAMPT were increased in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension, as well as in lungs of rodent models of pulmonary hypertension. Nampt+/- mice were protected from hypoxia-mediated pulmonary hypertension. NAMPT activity promoted human pulmonary artery smooth muscle cell proliferation via a paracrine effect. In addition, recombinant NAMPT stimulated human pulmonary artery smooth muscle cell proliferation via enhancement of store-operated calcium entry by enhancing expression of Orai2 and STIM2. Last, inhibition of NAMPT activity attenuated monocrotaline and Sugen hypoxia-induced pulmonary hypertension in rats. CONCLUSIONS: Our data provide evidence that NAMPT plays a role in pulmonary vascular remodeling and that its inhibition could be a potential therapeutic target for pulmonary arterial hypertension.

Capsaicin-induced Ca2+ signaling is enhanced via upregulated TRPV1 channels in pulmonary artery smooth muscle cells from patients with idiopathic PAH.

Capsaicin is an active component of chili pepper and a pain relief drug. Capsaicin can activate transient receptor potential vanilloid 1 (TRPV1) channels to increase cytosolic Ca2+ concentration ([Ca2+]cyt). A rise in [Ca2+]cyt in pulmonary artery smooth muscle cells (PASMCs) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. In this study, we observed that a capsaicin-induced increase in [Ca2+]cyt was significantly enhanced in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with normal PASMCs from healthy donors. In addition, the protein expression level of TRPV1 in IPAH PASMCs was greater than in normal PASMCs. Increasing the temperature from 23 to 43°C, or decreasing the extracellular pH value from 7.4 to 5.9 enhanced capsaicin-induced increases in [Ca2+]cyt; the acidity (pH 5.9)- and heat (43°C)-mediated enhancement of capsaicin-induced [Ca2+]cyt increases were greater in IPAH PASMCs than in normal PASMCs. Decreasing the extracellular osmotic pressure from 310 to 200 mOsmol/l also increased [Ca2+]cyt, and the hypo-osmolarity-induced rise in [Ca2+]cyt was greater in IPAH PASMCs than in healthy PASMCs. Inhibition of TRPV1 (with 5'-IRTX or capsazepine) or knockdown of TRPV1 (with short hairpin RNA) attenuated capsaicin-, acidity-, and osmotic stretch-mediated [Ca2+]cyt increases in IPAH PASMCs. Capsaicin induced phosphorylation of CREB by raising [Ca2+]cyt, and capsaicin-induced CREB phosphorylation were significantly enhanced in IPAH PASMCs compared with normal PASMCs. Pharmacological inhibition and knockdown of TRPV1 attenuated IPAH PASMC proliferation. Taken together, the capsaicin-mediated [Ca2+]cyt increase due to upregulated TRPV1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PASMC proliferation in patients with IPAH.

Pathogenic role of calcium-sensing receptors in the development and progression of pulmonary hypertension.

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca(2+) influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate [Ca(2+)]cyt in PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca(2+)-induced [Ca(2+)]cyt increase in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca(2+)-induced [Ca(2+)]cyt increase in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice (casr(-/-)) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.

Notch Activation of Ca(2+) Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca(2+) entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6(-/-) mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca(2+) concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.

Dihydropyridine Ca(2+) channel blockers increase cytosolic [Ca(2+)] by activating Ca(2+)-sensing receptors in pulmonary arterial smooth muscle cells.

RATIONALE: An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH). OBJECTIVE: Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC. METHODS AND RESULTS: The nifedipine-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt). CONCLUSIONS: The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.

Activation of Notch signaling by short-term treatment with Jagged-1 enhances store-operated Ca(2+) entry in human pulmonary arterial smooth muscle cells.

Notch signaling plays a critical role in controlling proliferation and differentiation of pulmonary arterial smooth muscle cells (PASMC). Upregulated Notch ligands and Notch3 receptors in PASMC have been reported to promote the development of pulmonary vascular remodeling in patients with pulmonary arterial hypertension (PAH) and in animals with experimental pulmonary hypertension. Activation of Notch receptors by their ligands leads to the cleavage of the Notch intracellular domain (NICD) to the cytosol by γ-secretase; NICD then translocates into the nucleus to regulate gene transcription. In this study, we examined whether short-term activation of Notch functionally regulates store-operated Ca(2+) entry (SOCE) in human PASMC. Treatment of PASMC with the active fragment of human Jagged-1 protein (Jag-1) for 15-60 min significantly increased the amplitude of SOCE induced by passive deletion of Ca(2+) from the intracellular stores, the sarcoplasmic reticulum (SR). The Jag-1-induced enhancement of SOCE was time dependent: the amplitude was maximized at 30 min of treatment with Jag-1, which was closely correlated with the time course of Jag-1-mediated increase in NICD protein level. The scrambled peptide of Jag-1 active fragment had no effect on SOCE. Inhibition of γ-secretase by N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) significantly attenuated the Jag-1-induced augmentation of SOCE. In addition to the short-term effect, prolonged treatment of PASMC with Jag-1 for 48 h also markedly enhanced the amplitude of SOCE. These data demonstrate that short-term activation of Notch signaling enhances SOCE in PASMC; the NICD-mediated functional interaction with store-operated Ca(2+) channels (SOC) may be involved in the Jag-1-mediated enhancement of SOCE in human PASMC.

Flow shear stress enhances intracellular Ca2+ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension.

An increase in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for pulmonary arterial medial hypertrophy in patients with idiopathic pulmonary arterial hypertension (IPAH). Vascular smooth muscle cells (SMC) sense the blood flow shear stress through interstitial fluid driven by pressure or direct exposure to blood flow in case of endothelial injury. Mechanical stimulus can increase [Ca(2+)]cyt. Here we report that flow shear stress raised [Ca(2+)]cyt in PASMC, while the shear stress-mediated rise in [Ca(2+)]cyt and the protein expression level of TRPM7 and TRPV4 channels were significantly greater in IPAH-PASMC than in normal PASMC. Blockade of TRPM7 by 2-APB or TRPV4 by Ruthenium red inhibited shear stress-induced rise in [Ca(2+)]cyt in normal and IPAH-PASMC, while activation of TRPM7 by bradykinin or TRPV4 by 4αPDD induced greater increase in [Ca(2+)]cyt in IPAH-PASMC than in normal PASMC. The bradykinin-mediated activation of TRPM7 also led to a greater increase in [Mg(2+)]cyt in IPAH-PASMC than in normal PASMC. Knockdown of TRPM7 and TRPV4 by siRNA significantly attenuated the shear stress-mediated [Ca(2+)]cyt increases in normal and IPAH-PASMC. In conclusion, upregulated mechanosensitive channels (e.g., TRPM7, TRPV4, TRPC6) contribute to the enhanced [Ca(2+)]cyt increase induced by shear stress in PASMC from IPAH patients. Blockade of the mechanosensitive cation channels may represent a novel therapeutic approach for relieving elevated [Ca(2+)]cyt in PASMC and thereby inhibiting sustained pulmonary vasoconstriction and pulmonary vascular remodeling in patients with IPAH.

Enhanced Ca(2+)-sensing receptor function in idiopathic pulmonary arterial hypertension.

RATIONALE: A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca(2+)](cyt) and enhanced Ca(2+) influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). OBJECTIVE: We examined whether the extracellular Ca(2+)-sensing receptor (CaSR) is involved in the enhanced Ca(2+) influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. METHODS AND RESULTS: In normal PASMC superfused with Ca(2+)-free solution, addition of 2.2 mmol/L Ca(2+) to the perfusate had little effect on [Ca(2+)](cyt). In IPAH-PASMC, however, restoration of extracellular Ca(2+) induced a significant increase in [Ca(2+)](cyt). Extracellular application of spermine also markedly raised [Ca(2+)](cyt) in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca(2+)-induced [Ca(2+)](cyt) rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca(2+)-mediated [Ca(2+)](cyt) increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. CONCLUSIONS: The extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca(2+) influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.

Protective effects of fluvoxamine against ischemia/reperfusion injury in isolated, perfused guinea-pig hearts.

Serotonin (5-hydroxytryptamine; 5-HT) is known to be activated during ischemia-reperfusion and triggers contractile dysfunction and pathological apoptosis. Here, the beneficial effects of the selective serotonin reuptake inhibitor (SSRI) fluvoxamine was demonstrated on ischemia-reperfusion injury in guinea-pig hearts perfused using the Langendorff technique. The recovery (%) of left ventricular developed pressure (LVDP) by fluvoxamine (5×10(-8) M) was 95.4% (control: 32%), which was consistent with the inhibition of mitochondrial Ca(2+)([Ca(2+)]m) uptake induced by changes in the Ca(2+) content and acidification of the perfusate, and similar to reperfusion following global ischemia in Langendorff-perfused hearts. Fluvoxamine inhibited the increase in [Ca(2+)]m induced by changes in the Ca(2+) content of the perfusate in perfused preparations of mitochondria, which was similar to the results obtained with the mitochondrial permeability transition pore (MPTP) opener atractyroside. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were significantly less in fluvoxamine-treated hearts than in control hearts, with decreases in caspase-3 activity. These results suggest that SSRI inhibits opening of the MPTP by preventing [Ca(2+)]m overload-induced apoptosis related to the endogenous accumulation of 5-HT in ischemia-reperfusion hearts.