Pulmonary angiogram (left) and cast (right) of a segment of arterial tree in human lung (A), and the proposed three schemes describing this complex structure by the Weibel model (B), the Strahler model (C), and the diameter‐defined Strahler's system (D).

Electric circuit of model cell membrane and intracellular ion homeostasis. Single‐section (A) and parallel‐conductance (B) model of cell membrane. For the parallel‐conductance model, one assumes independent conductance channels for K⁺, Na⁺, and Cl⁻. (C) Schematic diagram showing major ion channels and transporters that determine membrane potential as well as electrical and chemical gradients for K⁺, Na⁺, Ca²⁺, and Cl⁻ in pulmonary artery smooth muscle cells (PASMC). Resting membrane potential (E_m) for a PASMC is −40 to −60 mV. E_K, E_Na, E_Ca, and E_Cl represent the equilibrium potentials for K⁺, Na⁺, Ca²⁺, and Cl⁻, respectively. The size of the arrows indicates the driving force for the given ions. Inset, a diagram showing the channel protein embedded in the lipid membrane.

Regulation of intracellular [Ca²⁺] (A) and the major causes of membrane depolarization (B) in pulmonary artery smooth muscle cells. SERCA, Ca²⁺‐Mg²⁺ ATPase (Ca²⁺ pump) in the SR/ER membrane; PM, Ca²⁺‐Mg²⁺ ATPase (Ca²⁺ pump) in the plasma membrane; NCX, Na⁺‐Ca²⁺ exchanger; RyR, ryanodine receptor; IP₃R, inositol 1,4,5‐trisphosphate (IP₃) receptor; E_m, membrane potential.

Topological structure of the K_v channel α‐subunit (A) and voltage‐dependent Ca²⁺ channels (VDCC) (B). (A) A planar representation of a K_v channel α‐subunit (a) and its interaction with other α‐subunits (b) and the β‐subunits (c) via the N‐terminal T1 domain. The graphs in b and c show a 4α‐tetramer and an 4α/4β‐octamers, respectively. The pore of the 4α‐tetramer is shown in b. (B) A planar representation of a VDCC subunit. Inset: diagrammatical representation of a VDCC channel in the plasma membrane with associated subunits. The pore region (P) forming between the S5 and S6 subunits is indicated in red.

A rise in [Ca²⁺]_cyt is a major trigger for pulmonary vasoconstriction. When [Ca²⁺]_cyt rises in pulmonary artery smooth muscle cells, Ca²⁺ binds to calmodulin (CaM), which causes contraction by activating (or phosphorylating) myosin light chain kinase (MLCK) and indirectly causes the inhibition of myosin light chain phosphatase (MLCP) via Rho kinase (ROK). Activation of G protein‐coupled receptors (GPCR) can also cause Ca²⁺‐independent contraction (or Ca²⁺ sensitization) through the RhoA/ROK pathway.

A rise in [Ca²⁺]_cyt is an important stimulus of pulmonary artery smooth muscle cells proliferation. In addition to activating MLCK and causing contraction, cytosolic Ca²⁺ also activates CaM kinase (CaMK) and mitogen‐activated protein kinase (MAPK), as well as various transcription factors (e.g., NFAT, CREB, AP‐1 family members, and NF‐κB), to facilitate cell passage through the cell cycle and cause cell proliferation. The arrows in the inset indicate the Ca/CaM‐sensitive steps in the cell cycle. MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; PVR, pulmonary vascular resistance; PAP, pulmonary arterial pressure; SR/ER, sarcoplasmic (or endoplasmic) reticulum.

Structure of transient receptor potential channel (TRPC). (A) Planar views of the TRPC1, TRPV1, and TRPM1 channel subunits. (B) Representative traces reflecting changes in [Ca²⁺]_cyt (left panel) recorded in pulmonary artery smooth muscle cells (PASMC) before and during application of 10 μM cyclopiazonic acid (CPA), a SERCA inhibitor that depletes intracellular Ca²⁺ stores. 0 Ca²⁺, Ca²⁺‐free solution. The rise in [Ca²⁺]_cyt due to store‐operated Ca²⁺ entry (SOCE) or capacitative Ca²⁺ entry (CCE) is indicated in the shadow area. The right panel shows representative whole‐cell currents recorded in human PASMC cells before (−CPA) and after (+CPA) depletion of intracelluarly stored Ca²⁺ from the SR using 10 μM CPA.

Oligomerization and translocation of STIM to SR/ER‐plasma membrane junctions are the major mechanism involved in SOCE through Orai tetramer‐formed SOC. (A) The Ca²⁺ sensor EF‐hand domain in the N terminus of STIM has bound Ca²⁺ (red circles) when the SR/ER Ca²⁺ store is filled with Ca²⁺ (up to 1 mM). Depletion of Ca²⁺ from the SR/ER causes Ca²⁺ to unbind from the low‐affinity EF‐hand of STIM, which subsequently leads to STIM oligomerization and translocation to the SR/ER‐plasma membrane junctions. (B) STIM accumulation in the vicinity of Orai channel dimmers induces Orai channels to cluster in the adjacent plasma membrane. The C‐terminal effector domain of STIM causes Orai channels to open by direct binding of the distal coiled‐coil domain, and triggers Ca²⁺ entry. Two STIMs can activate a single SOC formed by an Orai tetramer; channel activation of Orai may involve a preliminary step of assembling Orai dimmers into a functional tetramer.

Functional coupling of receptors,  transient receptor potential channel (TRPC) and NCX in caveolae in pulmonary artery smooth muscle cells (PASMC). (A) Electron microscopy graphs showing the structure of plasma membrane in PASMC isolated from normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). IPAH PASMC have increased flask‐like invaginations of the plasma membrane consistent with the morphology of caveolae (indicated by arrows) and as determined by the number of caveoli per membrane length. (B) and (C) Schematic diagram depicting the caveolin‐1 binding domains of TRPC1 (B) and the potential mechanisms (C) that are involved in ligand‐mediated regulation of intracellular [Ca²⁺]_cyt through G protein‐coupled receptors (GPCR), TRPC, and Na⁺/Ca²⁺ exchangers (NCX) in caveolae. CBM, cav1‐binding motif; PBD, protein 4‐binding domain; CSD, caveolin‐scaffolding domain; PM, the plasma membrane; ER/SR, endoplasmic reticulum and sarcoplasmic reticulum; G, G protein; IP₃, inositol 1,4,5‐trisphosphate; IP₃R, IP₃ receptor; SERCA, SR/ER Ca²⁺‐Mg²⁺ ATPase; Cav‐1, caveolin‐1.

Structure of K⁺ channels. Planar membrane topologies of single K⁺ channel subunits for voltage‐gated K⁺ (K_v) channels (A), Ca²⁺‐activated (BK_Ca) K⁺ channels (B), inward rectifier K⁺ (K_IR) channels (C), and two‐pore domain (K_2P) K⁺ channels (D), respectively. The pore‐forming loop is indicated and the voltage sensor in the transmembrane (TM) domain 4 for K_v and BK_Ca channels. Membrane topology of a K_2P channel subunit featuring two pore regions, P1 and P2 and four TM‐spanning domains, M1 to 4 and cytoplasmic N‐ and C‐ termini (D). The ATP‐sensitive K⁺ (K_ATP) channels are heterooctamers formed by the pore‐forming K_IR subunits and the regulatory subunits, SUR (e.g., Kir6.1/SUR2B and Kir6.2/SUR2B) (as shown in C).

Structure of voltage‐gated Na⁺ channels. (A) Planar schematic diagram showing structural arrangement of Na⁺ channel α and β₁/β₂ subunits. (B) Representative currents showing inward Na⁺ currents (I_Na) in human pulmonary artery smooth muscle cells (PASMC), elicited by depolarizing the cell from a holding potential of −70 mV to a series of test potentials ranging from −80 and +80 mV in 20 mV increments. The inset indicates the summarized I‐V relationship curve for I_Na. (C) Representative I_Na at 0 mV from PASMC before, during and after extracellular application of 1 μM tetrodotoxin.

Ca²⁺‐activated Cl⁻ currents (I_ClCa) in pulmonary artery smooth muscle cells (PASMC). (A) A representative family of whole‐cell I_ClCa in PASMC elicited by a series of test potentials from −60 to +60 mV in 20‐mV increments (from a holding potential of −70 mV). The cells are superfused with Modified Kreb's solution (MKS) including 1.8 mM Ca²⁺. (B) Representative traces of I_ClCa recorded in PASMC superfused with MKS (Control and Recovery) and Ca²⁺‐free MKS with 10 mM BaCl₂ (0Ca‐10Ba). Replacement of extracellular Ca²⁺ with Ba²⁺ significantly enhanced inward cation current, but abolished the outward Cl⁻ currents and the inward tail currents, which were carried by Cl⁻ influx and efflux, respectively, through Cl_Ca channels. (C) The superimposed record of whole‐cell I_ClCa in PASMC before (Control) and during extracellular application of niflumic acid.

Gap junction (GJ) and GJ channels in vascular smooth muscle and endothelial cells. The changes in membrane potential (E_m) and [Ca²⁺]_cyt due to Ca²⁺ influx or release in one smooth muscle cell (SMC) can be communicated to an adjacent SMC through GJ channels and to an endothelial cell (EC) through GJ channels in the myoendothelial junction (MEJ). Ca²⁺ and electrical signals can also go through GJ channels between ECs. In addition to Ca²⁺ and other cations/anions, GJ channels also allow small molecules to go through, for example, IP₃, DAG, and other second messengers in the cytosol. ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; δE_m, changes in E_m (i.e., membrane depolarization, hyperpolarization, or repolarization); ROC, receptor‐operated Ca²⁺ channels.

Formation of gap junction (GJ) channels and topology of the connexin (Cx). (A) A schematic diagram showing how the hemichannels in the membrane of one cell interact with the hemichannels in another cell to form GJ channels. Not all hemichannels form GJ channels; the hemichannels can also function as a non‐selective cation/anion channels. (B) The Cx is a membrane protein with four transmembrane domains and cytoplasmic N‐ and C‐termini. There are multiple cysteine residues in the extracellular E1 and E2 segments that make the channel sensitive to the regulation of oxidation and reduction.

Inhibition of K⁺ channels causes membrane depolarization and causes pulmonary vasoconstriction. (A) Close of K⁺ channels in pulmonary artery smooth muscle cells (PASMC) causes membrane depolarization, which subsequently opens VDCC, enhances Ca²⁺ influx, increases [Ca²⁺]_cyt, and induces pulmonary vasoconstriction. Opening of K⁺ channels, on the other hand, causes membrane hyperpolarization (close to the K⁺ equilibrium potential), decreases VDCC activity and causes pulmonary vasodilation. (B) Representative records of whole‐cell K⁺ currents (a), membrane potential (E_m, b) and [Ca²⁺]_cyt (c) in PASMC before (control), during (4‐AP) and after (wash) extracellular application of 5 mM 4‐amynopyridine (4‐AP). A representative record of tension measurement in an isolated pulmonary arterial ring before, during, and after 4‐AP treatment is shown in d.

Activity of K⁺ channel is involved in apoptotic volume decrease (AVD) and apoptosis. (A) Diagram showing the chronological order of morphological and biochemical changes during apoptotic stimulation. Activation of K⁺ channels leads to a loss of intracellular K⁺, which relieves the inhibitory effect of K⁺ on cytoplasmic caspases and nucleases, increases caspase‐mediated cleavage, and ultimately induces nuclear breakage and apoptosis. Activation of K⁺ channels also accelerates AVD, which further facilitates the process leading to apoptosis. (B) Changes in cell morphology before (Control) and during treatment with apoptosis inducers. Exposure of cells to pro‐apoptotic triggers activates K⁺ (and Cl⁻) channels, induces AVD, and ultimately causes DNA fragmentation and nuclear breakage. The image on the right showing DAPI‐stained nuclei in pulmonary artery smooth muscle cells treated with staurosporine; the cells undergoing apoptosis (showing significant nuclear breakage) are indicated by arrows. The AVD due to K⁺ (Cl⁻ and H₂O) efflux occurs prior to nuclear condensation and DNA fragmentation.

Two pathways of apoptosis. When death receptors (DR) are activated (e.g., by Fas ligand), cleavage of procaspase 8 (and/or 10) to active caspase 8 is an important initial step to induce apoptosis. Cytochrome c (Cyt c), which can be released from the mitochondria to the cytosol when cells are exposed to UV light or when mitochondrial membrane potential (δΨ_m) is depolarized, activates cytoplasmic caspase 9. Active caspases 8 and 9 then activate caspases 3/6/7 and cause DNA fragmentation and nuclear breakage, and eventually cell death. Cytosolic cytochrome c also activates K_v channels, induces K⁺ loss, decreases cytosolic [K⁺], which increases caspase activity and accelerates AVD, and ultimately promote apoptosis 229.

Cellular mechanisms by which acute hypoxia causes pulmonary vasoconstriction. (A) Multiple pathways are involved in hypoxia‐induced increase in [Ca²⁺]_cyt in pulmonary artery smooth muscle cells (PASMC). Acute hypoxia can enhance Ca²⁺ influx through receptor‐operated Ca²⁺ channels (ROC) by activating phospholipase C (PLC) and diacylglycerol (DAG), through voltage‐dependent Ca²⁺ channels (VDCC) by membrane depolarization due to inhibition of K⁺ (K_v and K_2P) channels and activation of Ca²⁺‐activated Cl⁻ (Cl_Ca) channels, and through store‐operated Ca²⁺ channels (SOC) by active depletion of Ca²⁺ from the SR/ER. Acute hypoxia can also enhance Ca²⁺ release from the SR/ER, lysosome and mitochondrial by activating IP₃ receptors (IP₃R) and ryanodine (RyR) receptors, producing pyridine nucleotides (cyclic adenosine diphosphate‐ribose, cADPR and nicotinic acid adenine dinucleotide phosphate, NAADP), and inhibiting electron transportation chain (ETC), respectively. Store depletion may also increase Na⁺ influx through SOC and increases cytosolic [Na⁺], which would subsequently stimulate inward Ca²⁺ transportation via the reverse mode of Na⁺/Ca²⁺ exchange (NCX). (B) Representative records of whole‐cell K_v currents (I_K(V)), membrane potential (E_m), and [Ca²⁺]_cyt in PASMC before (normoxia), during (hypoxia) and after (recovery) exposure to hypoxia. (C) Acute hypoxia decreases I_K(V) only in KCNA5‐transfected PASMC, but not in mesenteric artery smooth muscle cells (MASMC) transfected with KCNA5 gene.

Cellular mechanisms by which chronic hypoxia causes pulmonary vascular remodeling and vasoconstriction. Chronic exposure to hypoxia downregulates K_v and K_2P channels and upregulates Ca²⁺‐activated Cl⁻ (Cl_Ca) channels in pulmonary artery smooth muscle cells (PASMC). The resultant membrane depolarization opens voltage‐dependent Ca²⁺ channels (VDCC) and increases [Ca²⁺]_cyt. Chronic hypoxia also upregulates VDCC (α_1C), transient receptor potential channel (TRPC1/3/4/6), and Orai2/STIM2 proteins in PASMC. The resultant augmentation of Ca²⁺ influx through these upregulated Ca²⁺ channels further increase [Ca²⁺]_cyt and ultimately causes pulmonary vasoconstriction and vascular remodeling. Downregulation of K⁺ channels also contributes to pulmonary vascular remodeling by inhibiting AVD and apoptosis in PASMC.

Schematic diagram showing the causes of elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH, A) and the angiographic and histological features of the pulmonary vasculature in IPAH patients (B). The angiogram show significant “loss” of small vessels in the IPAH patient (B, upper panels), which is mainly due to sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling characterized by medial and intimal hypertrophy, neointimal proliferation, and obliteration of small pulmonary arteries and arterioles (B, lower panels). EVG, elastic Van Gieson staining.

Patterns of vascular remodeling (A) and common histological changes in the pulmonary vasculature in patients with pulmonary hypertension (B). (A) The remodeling of a vessel to a larger lumen with the same wall thickness is termed “outward hypertrophic” (or eccentric hypertrophy), since cross‐sectional area is increased. Conversely, vessel narrowing with increased wall thickness occurs in chronic hypertension and may be “inward eutrophic” (e.g., smaller lumen with a somewhat thicker wall, but the same cross‐sectional area) or “inward hypertrophic” or “concentric hypertrophy” (i.e., smaller lumen with sufficient wall thickening to increase cross‐sectional area). A common assumption is that changes in cross‐sectional area indicate changes in wall mass (as implied by the terms “hypotrophic” or hypertrophic”). This, of course, is only correct if vessel length is not altered 210. (B) In the lung, pulmonary vascular remodeling consists in concentric hypertrophy, eccentric hypertrophy (associated with adventitial lesion and hypertrophy and extracellular matrix lesions), and obliteration of small vessels due to intimal lesions, plexiform lesions and in situ thrombosis. Concentric hypertrophy, in situ thrombosis and obliteration increase pulmonary vascular resistance (PVR) by narrowing the affected arterial lumen, while eccentric hypertrophy contributes to increasing PVR by increasing the wall stiffness or decreasing the vascular wall compliance (which is one of the major causes for reduced distention and recruitment in pulmonary circulation).

Schematic diagram showing the pathogenic role of dysfunctional K_v channels in the development of pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). Decreased K⁺ channel function and expression not only stimulate pulmonary artery smooth muscle cells (PASMC) proliferation by increasing [Ca²⁺]_cyt, but also inhibits PASMC apoptosis by attenuating apoptotic volume decrease (AVD) and decreasing cytoplasmic caspase activity. The increased proliferation and inhibited apoptosis in PASMC may play an important role in initiation and/or progression of pulmonary vascular remodeling. VDCC, voltage‐dependent Ca²⁺ channels.

Bone morphogenetic proteins (BMP) enhance whole‐cell K_v currents (I_K(V)) in pulmonary artery smooth muscle cells (PASMC). (A) Schematic diagram depicting the proposed role of BMP‐mediated regulation of K_v channel expression and function in human PASMC. There are two types of BMP receptors (BMP‐RI and BMP‐RII) which dimerize with one another and form a BMP ligand‐receptor complex. The activated BMP‐RI phosphorylates and activates the receptor‐activated Smads (R‐Smad) which then form dimerized complexes with Co‐Smads and enter the nucleus. The R‐Smad/co‐Smad interact with DNA in the nucleus and regulate the transcription of various target genes whose primers contain the Smad binding sequence (5′‐AGAC‐3′). In the nucleus, Smad‐1 (a R‐Smad) and Smad‐4 (a co‐Smad) in association with different corepressors appear to be involved in downregulating the expression of Bcl‐2, an antiapoptotic protein that blocks the release of cytochrome c from the mitochondrial intermembrane space to the cytosol. Bcl‐2 also downregulates the mRNA expression and inhibits the function of sarcolemmal K⁺ channels in PASMC. (B) Representative K_v currents (left panels), following 300 ms step depolarization at potentials ranging between −60 and +80 mV from a holding potential of −70 mV, and summarized current‐voltage (I‐V) curves (right panel) in PASMC treated with (BMP‐2) or without (Control) 200 nM BMP‐2 for 24 h. (C) Representative single‐channel K⁺ currents in control and BMP‐2‐treated PASMC (left panels). Bar graph depicts averaged open‐state probability (NP_o) of single‐channel K⁺ currents at +60 mV in control and BMP‐2‐treated PASMC. **P < 0.01 versus Control.

Voltage‐dependent and ‐independent regulation of [Ca²⁺]_cyt in pulmonary artery smooth muscle cells (PASMC). Decreased K_v channel activity causes membrane depolarization which subsequently opens voltage‐dependent Ca²⁺ channels (VDCC), increases Ca²⁺ influx, and raises [Ca²⁺]_cyt. Upon activation of membrane receptors including G protein‐coupled receptors (GPCR) and receptor tyrosine kinases (RTK) by ligands, store‐operated Ca²⁺ channels (SOC) are activated by store depletion as a result of IP₃‐mediated Ca²⁺ mobilization from the sarcoplasmic reticulum (SR), while receptor‐operated Ca²⁺ channels (ROC) are activated by diacylglycerol (DAG). Increased [Ca²⁺]_cyt is a major trigger for PASMC contraction, proliferation and migration.

Pathogenic role of transient receptor potential channel 6 (TRPC6) in the development of idiopathic pulmonary arterial hypertension (IPAH). (A) Proposed cellular mechanisms involved in excessive increase in [Ca²⁺]_cyt in pulmonary artery smooth muscle cells (PASMC) isolated from patients with IPAH. Upregulated TRPC subunits (e.g., TRPC3/6), due to genetic mutations and/or environmental stimulation, lead to the increased number of receptor‐operated (ROC) and store‐operated (SOC) Ca²⁺ channels in the plasma membrane, which enhances receptor‐operated (ROCE) and store‐operated (SOCE) Ca²⁺ entry and increases [Ca²⁺]_cyt in PASMC. Opening of SOC and ROC, potentially formed by TRPC subunits, not only causes Ca²⁺ influx, but also Na⁺ influx. The increased cytoplasmic [Na⁺] in the close proximity to Na⁺/Ca²⁺ exchangers (NCX) activates the reverse mode of NCX and promotes inward Ca²⁺ transport. NCX1 is also upregulated in IPAH PASMC in comparison to normal PASMC. (B) and (C) Representative currents (B), elicited by a ramp depolarization from −100 to +100 mV, and [Ca²⁺]_cyt (C) before and during application of the membrane permeable diacylglycerol analogue, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), in PASMC from normal subjects and IPAH patients.