Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH_i), typically due to activation of Na⁺/H⁺ exchange (NHE). In the systemic vasculature, ET-1 increases pH_i, Na⁺/H⁺ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH_i and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH_i and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH_i and NHE activity induced by increasing concentrations of ET-1 (10⁻¹⁰ to 10⁻⁸ M). We found that application of exogenous ET-1 increased pH_i and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH_i or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH_i.     

Proteomic Analysis Reveals that Proteasome Subunit Beta 6 Is Involved in Hypoxia-Induced Pulmonary Vascular Remodeling in Rats

Background

Chronic hypoxia (CH) is known to be one of the major causes of pulmonary hypertension (PH), which is characterized by sustained elevation of pulmonary vascular resistance resulting from vascular remodeling. In this study, we investigated whether the ubiquitin proteasome system (UPS) was involved in the mechanism of hypoxia-induced pulmonary vascular remodeling. We isolated the distal pulmonary artery (PA) from a previously defined chronic hypoxic pulmonary hypertension (CHPH) rat model, performed proteomic analyses in search of differentially expressed proteins belonging to the UPS, and subsequently identified their roles in arterial remodeling.

Results

Twenty-two proteins were differently expressed between the CH and normoxic group. Among them, the expression of proteasome subunit beta (PSMB) 1 and PSMB6 increased after CH exposure. Given that PSMB1 is a well-known structural subunit and PSMB6 is a functional subunit, we sought to assess whether PSMB6 could be related to the multiple functional changes during the CHPH process. We confirmed the proteomic results by real-time PCR and Western blot. With the increase in quantity of the active subunit, proteasome activity in both cultured pulmonary artery smooth muscle cells (PASMCs) and isolated PA from the hypoxic group increased. An MTT assay revealed that the proteasome inhibitor MG132 was able to attenuate the hypoxia-induced proliferation of PASMC in a dose-dependent manner. Knockdown of PSMB6 using siRNA also prevented hypoxia-induced proliferation.

Conclusion

The present study revealed the association between increased PSMB6 and CHPH. CH up-regulated proteasome activity and the proliferation of PASMCs, which may have been related to increased PSMB6 expression and the subsequently enhanced functional catalytic sites of the proteasome. These results suggested an essential role of the proteasome during CHPH development, a novel finding requiring further study.     

AQP1促进肺动脉平滑肌细胞的增殖与迁移（应作者要求撤稿）

该文应作者要求已撤稿。肺动脉平滑肌细胞（PASMCs）的迁移和增殖是肺动脉重塑进而造成肺动脉高压的主要病理基础。水通道蛋白1（AQP1）具有促进上皮细胞、内皮细胞迁移的作用,但机制不清。由于AQP1也表达于血管平滑肌细胞,推测AQP1可能参与缺氧诱导的PASMCs增殖及迁移。通过PCR和免疫印迹分析,检测AQP的表达以及缺氧对AQP表达水平的影响,并通过细胞迁移以及增殖实验观察AQP1在缺氧诱导的PASMCs迁移与增殖中的作用。AQP1在PASMCs和主动脉平滑肌细胞（AoSMCs）均表达,但缺氧只增加PASMCs中AQP1的表达,以及促进PASMCs的迁移与增殖。敲除AQP1可抑制PASMCs的增殖以及缺氧诱导的细胞增殖和迁移。过表达AQP1促进PASMCs的增殖和迁移。缺氧促进β联蛋白在PASMCs内的表达。敲除β联蛋白后,抑制AdAQP1所介导的PASMCs迁移与增殖。这些结果表明,缺氧可促进AQP1在肺动脉内的表达,AQP1可通过β联蛋白对PASMCs的增殖和迁移进行调节。     

Hypoxia promotes rabbit pulmonary artery smooth muscle cells proliferation through a 15-LOX-2 product 15(S)-hydroxyeicosatetraenoic acid

The initial event of hypoxic pulmonary hypertension is acute hypoxic pulmonary vasoconstriction followed by remodeling of pulmonary arteries. Although 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] is found to be able to induce hypoxic pulmonary vasoconstriction, role of 15(S)-HETE in pulmonary artery smooth muscle cells (PASMCs) proliferation has been studied less. We sought evidence for a role of 15(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that hypoxia enhances 15-lipoxygenase-2 (15-LOX-2) expression and stimulates cultured rabbit PASMCs proliferation. 15(S)-HETE at concentration 0.1 μM stimulated proliferation of PASMCs and induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 15(S)-HETE-stimulated PASMC proliferation was blocked by the MEK inhibitors PD-98059. Hypoxia (3% O(2))-stimulated PASMC proliferation was blocked by U0126, a MEK inhibitor, as well as by NDGA and CDC, inhibitors of 15-LOX, but not by the p38 MAPK inhibitor SB-202190. We conclude that 15-LOX-2 and its product, 15(S)-HETE, are important intermediates in hypoxia-induced rabbit PASMC proliferation and may participate in hypoxia-induced pulmonary hypertension.     

Partial HIF-1alpha deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia

Chronic hypoxia depolarizes and reduces K+ current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1alpha [Hif1a(+/-)] and from their wild-type [Hif1a(+/+)] littermates after 3 wk in air or 10% O2. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, K+ current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/-) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/-) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/-) mice, no reduction in K+ current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1alpha is sufficient to impair hypoxia-induced depolarization, reduction of K+ current density, and PASMC hypertrophy.     

15-oxo-Eicosatetraenoic acid prevents serum deprivation-induced apoptosis of pulmonary arterial smooth muscle cells by activating pro-survival pathway

Pulmonary arterial hypertension (PAH) is a progressive condition in which remodeling of the pulmonary vasculature plays an important role. The vascular remodeling involves pulmonary arterial smooth muscle cell (PASMC) proliferation and apoptosis, which is affected by several arachidonic acid metabolites. 15-oxo-Eicosatetraenoic acid (15-oxo-ETE) is one of the metabolites. However, the biological role of 15-oxo-ETE in PASMCs remains unknown. Here we show evidence for the modulation of PASMC apoptosis by 15-oxo-ETE. We found that 15-oxo-ETE increased rat and human PASMC viability. Consistently, 15-oxo-ETE attenuated nuclear fragmentation and DNA strand breaks, decreased caspase-3 activity, reduced mitochondrial depolarization, and increased Bcl-2 expression. Interestingly, the anti-apoptotic effect of 15-oxo-ETE was lost when the Akt intracellular signaling pathway was blocked. Taken together, we have established that 15-oxo-ETE protects PASMCs against apoptosis through the Akt pathway. These results suggest that 15-oxo-ETE seems to be a potential agent for PAH controls by preventing unwanted PASMC death.     

LncRNA NRON alleviates atrial fibrosis via promoting NFATc3 phosphorylation

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a dominant role in the development of pulmonary arterial hypertension (PAH). Some studies and our previous work found that disturbance of fatty acid metabolism existed in PAH. However, the mechanistic link between fatty acid catabolism and cell proliferation remains elusive. Here, we identified an essential role and signal pathway for the key rate-limiting enzyme of mitochondrial fatty acid β-oxidation, carnitine palmitoyltransferase (CPT) 1, in regulating PASMC proliferation in PAH. We found that CPT1 was highly expressed in rat lungs and pulmonary arteries in monocrotaline-induced PAH, accompanied by decreased adenosine triphosphate (ATP) production and downregulation of the AMPK-p53-p21 pathway. Platelet-derived growth factor (PDGF)-BB upregulated the expression of CPT1 in a dose- and time-dependent manner. PASMC proliferation and ATP production induced by PDGF-BB were partly reversed by the CPT1 inhibitor etomoxir (ETO). The overexpression of CPT1 in PASMCs also promoted proliferation and ATP production and subsequently inhibited the phosphorylation of AMPK, p53, as well as p21 in PASMCs. Furthermore, AMPK was activated by ETO, which increased the expression of p53 and p21, and the proportion of cells in the cell cycle G₂/M phase in response to PDGF-BB stimulation in PASMCs. Our work reveals a novel mechanism of CPT1 regulating PASMC proliferation in PAH, and regulation of CPT1 may be a potential target for therapeutic intervention in PAH.

     

Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells

Pulmonary vascular medial hypertrophy in primary pulmonary hypertension (PPH) is mainly caused by increased proliferation and decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Mutations of the bone morphogenetic protein (BMP) receptor type II (BMP-RII) gene have been implicated in patients with familial and sporadic PPH. The objective of this study was to elucidate the apoptotic effects of BMPs on normal human PASMCs and to examine whether BMP-induced effects are altered in PASMCs from PPH patients. Using RT-PCR, we detected six isoforms of BMPs (BMP-1 through -6) and three subunits of BMP receptors (BMP-RIa, -RIb, and -RII) in PASMCs. Treatment of normal PASMCs with BMP-2 or -7 (100-200 nM, 24-48 h) markedly increased the percentage of cells undergoing apoptosis. The BMP-2-mediated apoptosis in normal PASMCs was associated with a transient activation or phosphorylation of Smad1 and a marked downregulation of the antiapoptotic protein Bcl-2. In PASMCs from PPH patients, the BMP-2- or BMP-7-induced apoptosis was significantly inhibited compared with PASMCs from patients with secondary pulmonary hypertension. These results suggest that the antiproliferative effect of BMPs is partially due to induction of PASMC apoptosis, which serves as a critical mechanism to maintain normal cell number in the pulmonary vasculature. Inhibition of BMP-induced PASMC apoptosis in PPH patients may play an important role in the development of pulmonary vascular medial hypertrophy in these patients.     

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.     

Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels

Activity of voltage-gated potassium (Kv) channels controls membrane potential, which subsequently regulates cytoplasmic free calcium concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMCs). Acute hypoxia inhibits Kv channel function in PASMCs, inducing membrane depolarization and a rise in [Ca²⁺ ]_cyt that triggers vasoconstriction. Prolonged hypoxia inhibits expression of Kv channels and reduces Kv channel currents in PASMCs. The consequent membrane depolarization raises [Ca²⁺]_cyt, thus stimulating PASMC proliferation. The present review discusses recent evidence for the involvement of Kv channels in initiation of hypoxic pulmonary vasoconstriction and in chronic hypoxia-induced pulmonary hypertension.     

Inhibition of Excessive Cell Proliferation by Calcilytics in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca²⁺ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca²⁺-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca²⁺ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC₅₀ = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC₅₀ = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC₅₀ = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.     

Pulmonary vascular response to normoxia and K(Ca) channel activity is developmentally regulated

To address developmental regulation of pulmonary vascular O(2) sensing, we tested the hypotheses that 1) fetal but not adult pulmonary artery smooth muscle cells (PASMCs) can directly sense an acute increase in O(2), 2) Ca2+-sensitive K(+) (K(Ca)) channel activity decreases with maturation, and 3) PASMC K(Ca) channel expression decreases with maturation. We used fluorescence microscopy to confirm that fetal but not adult PASMCs are able to sense an acute increase in O(2) tension. Acute normoxia induced a 22 +/- 2% decrease in cytosolic Ca2+ concentration ([Ca2+](i)) in fetal PASMCs and no change in ([Ca2+](i)) in adult PASMCs (P < 0.01). The effects of K(+) channel antagonists were studied on fetal and adult PASMC ([Ca2+](i)). Iberiotoxin (10(-9) M) caused PASMC ([Ca2+](i)) to increase by 694 +/- 22% in the fetus and caused no change in adult PASMCs. K(Ca) channel expression and mRNA levels in distal pulmonary arteries from fetal and adult sheep were examined. Both K(Ca) channel protein and mRNA expression in the distal pulmonary vasculature decreased with maturation. We conclude that maturation-dependent changes in PASMC O(2) sensing render the fetal PASMCs uniquely sensitive to an acute increase in O(2) tension at a biologically critical time point.     

Expression and activity of cAMP phosphodiesterase isoforms in pulmonary artery smooth muscle cells from patients with pulmonary hypertension: role for PDE1

Pulmonary hypertension (PHT) is associated with increased vascular resistance due to sustained contraction and enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC); the abnormal tone and remodeling in the pulmonary vasculature may relate, at least in part, to decreased cyclic nucleotide levels. Cyclic nucleotide phosphodiesterases (PDEs), of which 11 families have been identified, catalyze the hydrolysis of cAMP and cGMP. We tested the hypothesis that PASMC isolated from patients with PHT, either idiopathic pulmonary arterial hypertension (IPAH) or secondary pulmonary hypertension (SPH), have increased expression and activity of PDE isoforms that reduce the responsiveness of agents that raise cellular cAMP. Real-time PCR and immunoblotting demonstrated that the expression of PDE1A, PDE1C, PDE3B, and PDE5A was enhanced in PASMC from both IPAH and SPH patients compared with control PASMC. Consistent with this enhanced expression of PDEs, agonist-stimulated cAMP levels were significantly reduced in IPAH and SPH PASMC unless a PDE inhibitor was present. The use of specific PDE inhibitors revealed that an increase in PDE1 and PDE3 activity largely accounted for reduced agonist-induced cAMP levels and increased proliferation in IPAH and SPH PASMC. Treatment with PDE1C-targeted small interference RNA enhanced cAMP accumulation and inhibited cellular proliferation to a greater extent in PHT PASMC than controls. The results imply that an increase in PDE isoforms, in particular PDE1C, contributes to decreased cAMP and increased proliferation of PASMC in patients with PHT. PDE1 isoforms may provide novel targets for the treatment of both primary and secondary forms of the disease.     

Over-expression of PKGIα inhibits hypoxia-induced proliferation, Akt activation, and phenotype modulation of human PASMCs: the role of phenotype modulation of PASMCs in pulmonary vascular remodeling

The proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a role in pulmonary vascular remodeling (PVR). Recently, it was shown that vascular smooth muscular cell phenotype modulation is important for their proliferation in other diseases. However, little is known about the role of human PASMC phenotype modulation in the proliferation induced by hypoxia and its molecular mechanism during PVR. In this study, we found using primary cultured human PASMCs that hypoxia suppressed the expression of endogenous PKGIα, which was reversed by transfection with a recombinant adenovirus containing the full-length cDNA of PKGIα (Ad-PKGIα). Ad-PKGIα transfection significantly attenuated the hypoxia-induced downregulation of the expression of smooth muscle α-actin (SM-α-actin), myosin heavy chain (MHC) and calponin in PASMCs, indicating that hypoxia-induced phenotype modulation was blocked. Furthermore, flow cytometry and ³H-TdR incorporation demonstrated that hypoxia-induced PASMC proliferation was suppressed by upregulation of PKGIα. These results suggest that enhanced PKGIα expression inhibited hypoxia-induced PASMC phenotype modulation and that it could reverse the proliferation of PASMCs significantly. Moreover, our previous work has demonstrated that Akt protein is activated in the process of hypoxia-induced proliferation of human PASMCs. Interestingly, we found that Akt was not activated by hypoxia when PASMC phenotype modulation was blocked by Ad-PKGIα. This result suggests that blocking phenotype modulation might be a key up-stream regulatory target.     

Kinetic dissection of two distinct proton binding sites in Na+/H+ exchangers by measurement of reverse mode reaction

We examined the effect of intracellular acidification on the reverse mode of Na+/H+ exchange by measuring 22Na+ efflux from 22Na+-loaded PS120 cells expressing the Na+/H+ exchanger (NHE) isoforms NHE1, NHE2, and NHE3. The 5-(N-ethyl-N-isopropyl)amiloride (EIPA)- or amiloride-sensitive fraction of 22Na+ efflux was dramatically accelerated by cytosolic acidification as opposed to thermodynamic prediction, supporting the concept that these NHE isoforms are activated by protonation of an internal binding site(s) distinct from the H+ transport site. Intracellular pH (pHi) dependence of 22 Na+ efflux roughly exhibited a bell-shaped profile; mild acidification from pHi 7.5 to 7 dramatically accelerated 22Na+ efflux, whereas acidification from pHi 6.6 gradually decreased it. Alkalinization above pHi 7.5 completely suppressed EIPA-sensitive 22Na+ efflux. Cell ATP depletion and mutation of NHE1 at Arg440 (R440D) caused a large acidic shift of the pHi profile for 22Na+ efflux, whereas mutation at Gly455 (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na+/H+ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.     

PVT1促进PAH大鼠肺动脉平滑肌细胞增殖和迁移的作用机制研究

摘要 目的：研究浆细胞瘤多样异位基因1（PVT1）在肺动脉高压（PAH）大鼠中对于肺动脉平滑肌细胞(PASMCs)增殖和迁移的作用及其可能的机制。方法：将16只成年雄性SD大鼠随机分为肺动脉高压组（PAH组）和对照组,每组8只大鼠。PAH组大鼠通过单次项背部皮下注射MCT溶液造模,对照组大鼠给予单次项背部皮下注射等量生理盐水。通过胸右心室穿刺法测量右心室压力。取各组大鼠肺组织,并进行原代肺动脉平滑肌细胞分离培养。通过RT-qPCR和Western blot检测PVT1及Fxr1在PAH组织及PASMCs中的表达水平;通过HE染色评估PAH组织的血管壁形态;免疫荧光法检测HPASMC的纯度;CCK-8法和伤口愈合迁移实验检测PASMCs增殖和迁移情况。结果：与对照组相比,PAH组大鼠肺组织血管壁厚度偏厚、肺动脉压显著升高（P<0.05）。PVT1在PAH组大鼠的PAH组织和PASMCs中的表达水平显著上调（P<0.05）,且其表达与肺动脉压呈正相关。与对照组相比,转染sh-PVT1的PASMCs显示出较低的细胞活力,同时转染sh-PVT1有效敲低了PASMCs中PVT1的表达水平（P<0.01）。与对照组相比,PVT1的敲低抑制了PASMCs迁移能力（P<0.01）。在转染pcDNA-PVT1的PASMCs中发现较高的增殖能力,PVT1的过表达促进了PASMCs迁移能力（P<0.01）。与对照组相比,Fxr1在PAH模型组的PAH组织和PASMCs中的表达水平显著上调（P<0.01）。结论：PVT1通过调节Fxr1的表达促进PASMCs的增殖和迁移,PVT1可能是PAH诊断和预测指标。     

Intracellular pH regulation by Na⁺/H⁺ exchanger-1 (NHE1) is required for growth factor-induced mammary branching morphogenesis

Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H+exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H+) across plasma membranes in exchange for extracellular sodium ions (Na+). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 μM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα>PI3K>NHE1>pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis.     

Atrial natriuretic peptide-dependent modulation of hypoxia-induced pulmonary vascular remodeling

Hypoxic stress upsets the balance in the normal relationships between mitogenic and growth inhibiting pathways in lung, resulting in pulmonary vascular remodeling characterized by hyperplasia of pulmonary arterial smooth muscle cells (PASMCs) and fibroblasts and enhanced deposition of extracellular matrix. Atrial natriuretic peptide (ANP) reduces pulmonary vascular resistance and attenuates hypoxia-induced pulmonary hypertension in vivo and PASMC proliferation and collagen synthesis in vitro. The current study utilized an ANP null mouse model (Nppa-/-) to test the hypothesis that ANP modulates the pulmonary vascular and alveolar remodeling response to normobaric hypoxic stress. Nine-10 wk old male ANP null (Nppa-/-) and wild type nontransgenic (NTG) mice were exposed to chronic hypoxia (10% O(2), 1 atm) or air for 6 wks. Measurement: pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial and alveolar remodeling were assessed. Hypoxia-induced pulmonary arterial hypertrophy and muscularization were significantly increased in Nppa-/- mice compared to NTG controls. Furthermore, the stimulatory effects of hypoxia on alveolar myofibroblast transformation (8.2 and 5.4 fold increases in Nppa-/- and NTG mice, respectively) and expression of extracellular matrix molecule (including osteopontin [OPN] and periostin [PN]) mRNA in whole lung were exaggerated in Nppa-/- mice compared to NTG controls. Combined with our previous finding that ANP signaling attenuates transforming growth factor (TGF)-beta-induced expression of OPN and PN in isolated PASMCs, the current study supports the hypothesis that endogenous ANP plays an important anti-fibrogenic role in the pulmonary vascular adaptation to chronic hypoxia.     

Sildenafil alters calcium signaling and vascular tone in pulmonary arteries from chronically hypoxic rats

Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been recently proposed as a therapeutic tool to treat or prevent pulmonary artery hypertension (PAHT). We thus studied the effect of sildenafil on both the calcium signaling of isolated pulmonary artery smooth muscle cells (PASMCs) and the reactivity of pulmonary artery (PA) obtained from chronic hypoxia (CH)-induced pulmonary hypertensive rats compared with control (normoxic) rats. CH rats were maintained in an hypobaric chamber (50.5 kPa) for 3 wk leading to full development of PAHT. Intracellular calcium concentration ([Ca2+]i) was measured in PASMCs loaded with the calcium fluorophore indo 1. Unlike in control rats, sildenafil (10-100 nM) decreased the resting [Ca2+]i value in PASMCs obtained from CH rats. In PASMCs from both control and CH rats, sildenafil concentration dependently inhibited the [Ca2+]i response induced by G-coupled membrane receptor agonists such as angiotensin II and phenylephrine but had no effect on the amplitude of the [Ca2+]i response induced by caffeine. Sildenafil (0.1 nM-1 microM) concentration dependently reduced basal PA tone that is present in CH rats and relaxed PA rings precontracted with phenylephrine in both control and CH rats. These data show that sildenafil is a potent pulmonary artery relaxant in CH rats and that it normalizes CH-induced increases in resting [Ca2+]i and basal tone. Consequently, pharmacological inhibition of sildenafil-sensitive PDE5 downregulates the Ca2+ signaling pathway involved in this model of pulmonary hypertension.