p75 neurotrophin receptor regulates agonist-induced pulmonary vasoconstriction

Abstract

A member of the TNF receptor family, the p75 neurotrophin receptor (p75^NTR) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of p75^NTR in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of p75^NTR in mouse pulmonary arteries and the putative role of p75^NTR in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and p75^NTR knockout (p75^−/−) mice. Our data indicated that p75^NTR is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery rings from p75^−/− mice exhibited significantly elevated active tension due to endothelin-1-mediated Ca²⁺ influx. Furthermore, the contraction due to capacitative Ca²⁺ entry (CCE) in response to phenylephrine-mediated active depletion of intracellular Ca²⁺ stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and p75^−/− rings. Active tension induced by serotonin, U-46619 (a thromboxane A₂ analog), thrombin, 4-aminopyridine (a K⁺ channel blocker), and high extracellular K⁺ in p75^−/− rings was similar to that in WT rings. Deletion of p75^NTR did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact p75^NTR signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion.

neurotrophins are unique growth factors that interact with two separate types of transmembrane receptors: tropomyosin receptor kinases (Trk) and the p75 neurotrophin receptor (p75^NTR), with the latter being a member of the TNF superfamily of membrane receptors (38). A wealth of in vitro experiments has shown that neurotrophins and pro-neurotrophins can induce apoptosis via p75^NTR (15). In vivo, p75^NTR expression and induction are associated with increased apoptosis in vascular smooth muscle cells (SMCs) from atherosclerotic lesions (15, 41) as well as in promoting apoptosis in oligodendrocytes and neurons in injury models in the nervous system (1, 2, 26). Conversely, Chu et al. (9) demonstrated that p75^NTR is essential for neuronal survival, suggesting that its true role in regulating cell growth is unclear (for a review, see Ref. 7). p75^NTR expression may also play an important role in cell differentiation, particularly as it relates to recovery from tissue injury. Passino et al. (27) demonstrated that depletion of p75^NTR exacerbated liver pathology and prevented liver repair by attenuating the differentiation of hepatic stellate cells to myofibroblasts. Similarly, Cosgaya et al. (10) showed that p75^NTR regulates the differentiation of Schwann cells to a myelinating phenotype. p75^NTR is upregulated in glial cells in multiple sclerosis plaques (11), in neurons undergoing zinc-triggered stroke (26), spinal cord injury (2, 37), and sciatic nerve injury (36). Since its expression does not always correlate with apoptosis in vivo, its biological role in disease pathogenesis is not fully understood.

A recent study (33) has demonstrated a novel biological role for p75^NTR as a regulator of fibrin deposition after nerve injury and in a model of lung fibrosis. The expression of p75^NTR in nonneuronal cells, such as endothelial cells (ECs), SMCs, and hepatic stellate cells, has also led to a link between p75^NTR upregulation and pathologies such as atherosclerosis (41), melanoma formation (13), lung inflammation (30), and liver disease (27); all of these disease are associated with defects in fibrin degradation. Chronic thromboembolic pulmonary hypertension (CTEPH) is another disease whose main etiology is decreased fibrinolysis and pulmonary vasculopathy. Morris et al. (23) provided evidence to suggest that the resistance of CTEPH patient fibrin to plasmin-mediated lysis may be due to alterations in fibrinogen. However, previous studies of fibrinolysis also suggested that an upregulation of tissue-type plasminogen activator and type 1 plasminogen activator inhibitor (PAI-1) may also contribute to the deranged fibrinolysis in CTEPH patients (25). The expression of PAI-1 itself is dependent on the expression and function of p75^NTR; p75^NTR increases PAI-1 (33), but only after lung injury (in this case, triggered by LPS treatment). Therefore, it is possible that the increased PAI-1 in CTEPH patients is due to enhanced p75^NTR activity following the vascular injury that initiated formation of the fibrotic clot.

In the human lung, neurotrophins and their receptors (high-affinity Trk and low-affinity p75) have been detected in various cell types, including epithelia, airway and vascular smooth muscle, alveoli, and neurons (32). Interestingly, p75^NTR is detectable only in ganglionic neurons and pulmonary artery (PA) SMCs (PASMCs) in human lungs (32). Rat PASMCs also exhibit similar specific immunoreactivity for p75^NTR (31). Furthermore, expression of all neurotrophin receptors, including p75^NTR, is enhanced in smooth muscle (both bronchial and PASMCs) from spontaneously hypertensive rats compared with normotensive animals (31). Pulmonary vasoconstriction, such as that observed in pulmonary hypertensive subjects, is due to contraction of PASMCs within the vascular wall. Based on these findings, we hypothesized that p75^NTR may have a novel role in regulating PA vasoreactivity. In the present study, we evaluated the agonist-mediated vasoconstriction and nitric oxide-mediated vasodilation of pulmonary arteries isolated from wild-type (WT) and p75^NTR knockout (p75^−/−) mice. Our findings suggested that p75^NTR deletion selectively augments the development of active tension of the PA in response to agonist-induced capacitative Ca²⁺ entry (CCE) and to endothelin-1 (ET-1). Vasoreactivity to other vasoactive compounds such as serotonin (5-HT), U-46619 (U4; a thromboxane A₂ analog), 4-aminopyridine (4-AP; a K⁺ channel blocker), and thrombin appeared not to be altered by p75^NTR deletion.

MATERIALS AND METHODS

Animals and isolation of PA rings.

Male 5- to 8-wk-old p75^−/− mice (19) were from a C57Bl/6 background and purchased from The Jackson Laboratory. Balb-c mice and C57Bl/6J mice were used as controls. Use of mice for the experiments presented in this study was approved by the Institutional Animal Care and Use Committee of the University of California-San Diego. After mice had been decapitated using a procedure approved by the Institutional Animal Care and Use Committee, the lungs were quickly removed from the mouse. The right and left branches of the main PA and the first order of intrapulmonary arteries were isolated within 45 min of tissue retrieval. Adipose and connective tissues were carefully removed, and the remaining arteries were cut into 1- to 2-mm-long rings.

Tension measurements.

Two tungsten hooks (75 μm diameter) were carefully inserted through the lumen of PA rings. One hook was mounted on the bottom of a perfusion chamber, and the other hook was connected to an isometric force transducer (Harvard Apparatus). Isometric tension was continuously monitored and acquired using DATAQ software (DATAQ Instruments). Resting passive tension was set at 300 mg (43), and rings were equilibrated for 1 h at resting tension and then challenged three times with 40 mM K⁺ perfusate to obtain a stable contractile response. The optimal resting (or basal) tension (300 mg) was determined by our previous study (43) showing that increasing the basal tension from 100 to 300 mg significantly augmented the 40 mM K⁺-induced active tension in isolated mouse PA rings. To compare active tensions in different arterial rings, the ability to normalize active tension to vessel weight or length is an important factor in interpreting the results. The PA rings we used in this study were too small for us to obtain an accurate weight, so the agonist-induced pulmonary vasoconstriction was compared in different arterial rings based on the amplitude of active tension (determined by the difference between the total tension and basal tension).

Isolated PA rings were superfused with modified Krebs solution (MKS; at 37°C) consisting of (in mM) 138 NaCl, 1.8 CaCl₂, 4.7 KCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5 HEPES, and 10 glucose (pH 7.4). For Ca²⁺-free solution (0Ca-MKS), CaCl₂ was replaced by equimolar MgCl₂, and 1 mM EGTA was added to chelate residual Ca²⁺. In the high-K⁺ solution, NaCl was replaced by equimolar KCl to maintain osmolarity. The active tension induced by agonists was normalized by the basal tension and expressed as the net increase in tension (in mg).

For small vessels like the mouse PA rings used in this study, it is difficult to insert hooks into the lumen without damaging ECs. Our previous histological data showed that ECs were present in the PA rings after contraction experiments (43). Perfusion of bradykinin, an endothelium-dependent vasodilator, caused a marked relaxation in mouse PA rings contracted by 40 mM K⁺ (43). These data indicated that small vessels can be mounted with a functional endothelium. In this study, however, endothelial function (e.g., bradykinin-mediated vasodilation) was not determined for each vessel. The comparison of agonist-induced pulmonary vasoconstriction and vasodilation between WT and p75^−/− mice might be affected by the potential damage of endothelial function in the isolated vessels.

Histological preparation.

After the contraction experiment, PA rings were fixed overnight in 10% neutral buffered formalin. The formalin-fixed rings were serially cut for microscopic examination. The vessel tissues were processed and embedded in paraffin blocks in an automatic tissue processor (Sakura Tissue-Tek VIP, Sakura Finetek). Paraffin-embedded tissues were cut into 5-μm-thick sections for staining with either lectin (endothelium specific) or α-actin (smooth muscle specific).

p75^NTR immunohistochemistry.

Isolated PA rings were embedded into OCT, frozen, sectioned on a cryostat, and placed onto glass slides. Tissue sections were then fixed in methanol for 7 min at −20°C. After being washed in PBS (3 washes for 5 min each), sections were blocked in 3% BSA in PBS plus 0.1% Triton for 30 min, followed by three 5-min washes in PBS. Antibodies against p75^NTR (goat anti-p75^NTR, 1:10 dilution, Santa Cruz Biotechnology), CD31 (rat polyclonal, 1:10 dilution, BD Pharmingen), and desmin (goat polyclonal, 1:10 dilution, Santa Cruz Biotechnology) were diluted in 1% BSA in PBS plus 0.1% Triton and incubated overnight at 4°C. Primary antibodies were washed off with PBS (3 washes for 5 min each), and secondary antibodies (donkey anti-goat FITC, 1:200 dilution; donkey anti-rabbit Cy3, 1:200 dilution; and anti-mouse aminomethyl-coumarin, 1:200 dilution) were incubated for 30 min at room temperature. Tissue sections were washed in PBS (3 times for 5 min each) and mounted with Slowfade (Molecular Probes). Images were acquired on an Axioplan II epifluorescence microscope (Carl Zeiss MicroImaging) using dry Plan-Neofluar lenses using ×10 [0.3 numerical aperture (NA)], ×20 (0.5 NA), or ×40 (0.75 NA) objectives equipped with an Axiocam HRc digital camera and the Axiovision image-analysis system.

Smooth muscle α-actin staining and immunofluorescence.

Frozen lung tissue sections prepared from WT and p75^−/− mice were thawed, fixed in 4% paraformaldehyde-PBS for 10 min, and then washed in PBS. Sections were incubated 1 h at room temperature in a blocking solution composed of PBS supplemented with 2% BSA, 0.1% Triton X-100, and 2% normal serum (Santa Cruz Biotechnology). Sections were incubated overnight at 4°C with anti-smooth muscle α-actin monoclonal antibody (1:400 dilution, Sigma) diluted in blocking solution. Sections were washed three times (10 min each) in PBS and incubated 1 h at room temperature with FITC-conjugated secondary antibodies diluted in blocker. Sections were incubated in 4′,6-diamidino-2-phenylindole (DAPI; 5 μM) staining solution for 5 min and washed in PBS before mounted in anti-fade mounting solution. Fluorescence images were taken from a DeltaVision RT Deconvolution Microscope System using a ×10 objective. The number of vessels per field was obtained by averaging the numbers of vessels from 8 to 10 images.

RT-PCR.

Total RNA was extracted from brains, hearts, and lungs of WT and p75^−/− mice with TRIzol reagent (Invitrogen). RNA (1 μg) was first treated with DNase I (Invitrogen) before being transcribed to cDNA with Superscript II reverse transcriptase (Invitrogen). Canonical transient receptor potential (TRPC1-7) channel cDNA was amplified by PCR on a Bio-Rad thermal cycler using Platinum Blue PCR SuperMix (Invitrogen). The total volume of each reaction tube was 25 μl. PCR primer sequences are shown in Table 1. Specificities of sense and antisense oligonucleotides were examined using the National Center for Biotechnology Information BLAST program. PCR products were separated by agarose gel electrophoresis. β-Actin was used as an internal positive control for semiquantification. The net intensity values of cDNA bands was measured by Image J software, and PCR products were normalized to the GAPDH product from the same cDNA sample PCR and run on the same gel.

Table 1.

Oligonucleotide sequences of the primers used for RT-PCR

Gene Name	GenBank Accession No.	Size, bp	Sequence	Location, nt	Chromosome
TRPC1	NM_011643	146	Sense: 5′-AGCCTCTTGACAAACGAGGA-3′	1397–1542	9 E4
			Antisense: 5′-ACCTGACATCTGTCCGAACC-3′
TRPC2	NM_011644	201	Sense: 5′-GTGTGGTAAGCCCAAACAAC-3′	912–1112	7 F1
			Antisense: 5′-GGTGCTGTCTTTGTCTTGTC-3′
TRPC3	NM_019510	240	Sense: 5′-CCACTCAAGGTCTAGGATCA-3′	1001–1240	3 B
			Antisense: 5′-CCATTCAGAATGGCTTCCAC-3′
TRPC4	NM_016984	294	Sense: 5′-CTCTGGAGGAAGCTGAGATT-3′	338–631	3 D
			Antisense: 5′-CCAAGATGATGGGTGTGATG-3′
TRPC5	NM_009428	225	Sense: 5′-GCTGTGGAACTTCTGCTTAG-3′	734–958	X F2
			Antisense: 5′-CGAACTGGATACACACTCCA-3′
TRPC6	NM_013838	234	Sense: 5′-GAATGCAGCCAGAAGCAGAA-3′	1056–1289	9 A1
			Antisense: 5′-GTCCAAGAGACCAACAACGA-3′
TRPC7	NM_012035	229	Sense: 5′-GGACAAGCCTGCGTATTCTA-3′	69–297	13 B2
			Antisense: 5′-GTCTTGGATTCCTCCAGCAT-3′
β-Actin	NM_007393	90	Sense: 5′-ACGGCCAGGTCATCACTATT-3′/	810–899	5 G2
			Antisense: 5′-TGCCACAGGATTCCATACC-3′

GenBank accession numbers are accession numbers in GenBank for the sequence used in designing the primer. TRPC, canonical transient receptor potential Ca²⁺ channels.

Chemicals.

All drugs were from Sigma unless otherwise indicated. 4-AP, phentolamine (Phen), sodium nitroprusside (SNP; Fluka), and 5-HT were dissolved directly into MKS on the day of use. U4 was prepared as a stock in ethanol. Phenylephrine (PE) and ET-1 were prepared as concentrated stock solutions in distilled water. Cyclopiazonic acid (CPA) was dissolved in DMSO to make a stock solution of 50–100 mM. Bovine thrombin was prepared as a 1,000 U/ml stock in saline. All stock solutions were aliquoted and kept frozen at −20°C until use. On the day of experiments, aliquots of the stock solutions were dissolved in MKS to the proper concentrations. The pH values of all solutions were measured after the addition of the drugs and readjusted to 7.4. DAPI was prepared as a 10 mM stock solution in an antibody buffer containing 500 mM NaCl, 20 μM NaN₃, 10 μM MgCl₂, and 20 μM Tris·HCl (pH 7.4); it was diluted (1:100) in PBS before use.

Statistics.

The composite data are expressed as means ± SE. Statistical analysis was performed using paired or unpaired Student's t-tests or ANOVA and post hoc tests (Student-Newman-Keuls) where appropriate. Differences were considered to be significant at P < 0.05.

RESULTS

Expression of p75^NTR in PA rings.

Isolated PA rings from WT mice were stained with a p75 antibody, desmin, and CD31 to identify p75^NTR expression in SMCs (desmin marked) and ECs (CD31 marked) within the vessel wall. As shown in Fig. 1, p75^NTR was detected not only in the smooth muscle (Fig. 1, A, B, and D) but also in the endothelium (Fig. 1, A–C). Double staining for p75^NTR and desmin, and p75^NTR and CD31, demonstrated the colocalization of p75^NTR in both ECs (Fig. 1E) and SMCs (Fig. 1F), respectively. This was confirmed by triple staining with p75^NTR, desmin, and CD31 (Fig. 1E). Double staining for desmin and CD31 showed little colocalization, as expected (Fig. 1G).

Fig. 1.

Expression of the p75 neurotrophin receptor (p75^NTR) in the pulmonary artery (PA). A: immunohistochemical analysis of p75^NTR expression (green) in isolated mouse PA rings using CD31 (blue) and desmin (red) as markers of endothelial cells and smooth muscle cells, respectively. Inset, triple staining revealed areas of colocalization between p75^NTR and CD31 (yellow arrowhead) and p75^NTR and desmin (red arrowheads). The box outlines the area magnified further in B–G. B–D: higher-magnification images of p75^NTR staining alone (B; green staining), CD31 alone (C; blue staining), and desmin alone (D; red staining). E and F: double staining for p75^NTR and CD31 again showed colocalization (E; greenish-blue staining), as did double staining for p75^NTR and desmin (F; light brown staining). G: double staining for desmin and CD31 showed little colocalization.

Mouse lung p75^NTR expression.

Lung sections obtained from WT and p75^−/− mice were fixed and mounted. Immunohistochemical staining with a p75 antibody showed its expression in the airways, lung parenchyma, and pulmonary vasculature in WT mice but not in p75^−/− mice (Fig. 2A). The expression of p75^NTR seemed to be concentrated in SMCs lining the airway (Fig. 2A, top images; magnification: ×2.5) and PAs (Fig. 2A, bottom inset; magnification: ×20). Nuclear staining with DAPI was consistent in WT and p75^−/− sections.

Fig. 2.

Active tension induced by active store depletion-induced capacitative Ca²⁺ entry (CCE) is enhanced by p75^NTR deletion. A: fixed and mounted lungs slices from wild-type (WT; left) and p75^NTR knockout (p75^−/−; right) mice were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and p75^NTR antibody (red). Insets (magnified boxes from top images) show p75^NTR staining in the wall of PAs of WT animals only. B: representative traces showing tension generated by exposures of WT and p75^−/− PA rings to 40 mM K⁺ (40K). C: summary of active tension induced by 40K in WT (n = 8) and p75^−/− (n = 8) PA rings. D: representative tension traces of rings exposed to phenylephrine (PE) and phentolamine (Phen) in the absence or presence of extracellular Ca²⁺ to stimulate CCE (shaded areas). E: summarized data describing active tension in WT (n = 8) and p75^−/− (n = 8) PA rings induced by Ca²⁺ release (PE, 0Ca) from the sarcoplasmic reticulum (SR; left), by CCE (PE + Phen + Ca; middle), and by receptor-operated Ca²⁺ influx (PE + Ca, no Phen; right) according to the protocol shown in D. ROC, receptor-operated Ca²⁺ channel. *P < 0.05 vs. WT.

p75^NTR deletion does not significantly affect high K⁺-mediated active tension.

Increasing the extracellular K⁺ concentration from 4.7 to 40 mM shifts the K⁺ equilibrium potential and causes membrane depolarization. The high K⁺-induced vasoconstriction is primarily due to opening of voltage-dependent Ca²⁺ channels (VDCCs) on the surface membrane of vascular SMCs. Removal or chelation of extracellular Ca²⁺ almost abolished the high K⁺-induced vasoconstriction, whereas blockade of VDCCs with nifedipine (or verapamil) significantly inhibited high-K⁺-mediated vasoconstriction, in isolated mouse PA rings (43). We verified whether p75^NTR deletion altered the contraction of isolated mouse PAs. A challenge with 40 mM K⁺-MKS resulted in membrane depolarization and generation of tension due to Ca²⁺ influx through VDCCs in the plasma membrane of PASMCs. The active tension induced by 40 mM K was slightly greater in PAs from p75^−/− mice compared with rings from WT mice, but the difference was not statistically significant (Fig. 2, B and C; P = 0.12, n = 8). These results suggest that pulmonary vasoconstriction via Ca²⁺ influx through VDCCs is not significantly different in isolated PAs between WT and p75^−/− mice.

p75^NTR deletion significantly enhances the pulmonary vasoconstriction due to active store depletion-induced CCE.

Agonist-induced pulmonary vasoconstriction is usually initiated by a transient rise in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) due to Ca²⁺ mobilization (or release) from intracellular stores, such as the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). Subsequent depletion of Ca²⁺ from intracellular stores (e.g., the SR) opens store-operated Ca²⁺ channels (SOCs) and further increases [Ca²⁺]_cyt by promoting Ca²⁺ influx through SOCs. Store depletion-mediated Ca²⁺ influx is also referred to as CCE. In addition, agonist-mediated increases in [Ca²⁺]_cyt include Ca²⁺ entry through receptor-operated Ca²⁺ channels (ROCs) activated by diacylglycerol and PKC. Therefore, agonist-induced smooth muscle contraction is primarily caused by a rise in [Ca²⁺]_cyt due to Ca²⁺ release, CCE through SOCs, and Ca²⁺ influx via ROCs, although membrane depolarization and Ca²⁺ influx through VDCCs are also involved in agonist-induced vasoconstriction.

The next set of experiments was designed to examine whether CCE-induced contraction was affected by p75^NTR. As shown in Fig. 2D, left, application of the α₁-adrenergic agonist PE to PA rings in the absence of extracellular Ca²⁺ resulted in a small transient contraction, which was due obviously to Ca²⁺ release from intracellular stores. After the intracellular Ca²⁺ store has been depleted, the α-adrenergic antagonist Phen was applied to PA rings (for ∼10 min) to block α-receptors and return the level of intracellular second messengers (e.g., diacylglycerol) to the baseline level. The reintroduction of extracellular Ca²⁺ (1.8 mM) in the presence of PE and Phen caused a sustained elevation in active tension, which was apparently caused by CCE (Fig. 2D, shaded area). The amplitudes of Ca²⁺ release-mediated PA contraction were comparable in WT and p75^−/− mice (Fig. 2, D and E, left), whereas the amplitudes of CCE-induced PA contraction were significantly greater in PA rings from p75^−/− mice than in rings from WT mice (Fig. 2, D and E, middle). Removal of Phen from the media (in the continued presence of PE and Ca²⁺) resulted in a large contraction, which was believed to be due to Ca²⁺ influx through ROCs (and VDCCs). The amplitudes of ROC-mediated contraction were, however, not significantly different between PA rings from WT and p75^−/− mice (Fig. 2, D and E, right).

These observations suggest that p75^NTR activation regulates agonist-mediated pulmonary vasoconstriction by selectively inhibiting CCE-mediated contraction. p75^NTR, however, appeared to have no significant effect on PE-induced pulmonary vasoconstriction due to Ca²⁺ influx via ROCs and VDCCs (see Fig. 2B).

p75^NTR deletion fails to affect pulmonary vasoconstriction due to passive store depletion-induced CCE.

CCE, such as that observed using PE (Fig. 2), occurs due to depletion of Ca²⁺ from the SR. There are two ways to deplete the SR Ca²⁺ stores. In the case of PE, the SR was depleted by the downstream production of inositol 1,4,5-trisphosphate (IP₃), which causes Ca²⁺ release through IP₃ receptors on the SR membrane. This process is also referred to as active depletion of Ca²⁺ from the SR. In addition, the SR can also be depleted by blocking Ca²⁺ sequestration or uptake from the cytosol to the SR. CPA is a reversible inhibitor of the SR Ca²⁺ pump [sarco(endo)plasmic reticulum Ca²⁺-ATPase] that “passively” depletes Ca²⁺ from intracellular stores in the absence of extracellular Ca²⁺ (34). As shown in Fig. 3, CPA induced a small contraction in the absence of extracellular Ca²⁺ due to Ca²⁺ leakage from the SR to the cytosol (Fig. 3A). After intracellular stores had been depleted, restoration of extracellular Ca²⁺ caused a large contraction due to CCE. However, the contractions caused by the passive store deletion-mediated CCE were not significantly different between PA rings from WT and p75^−/− mice (Fig. 3, A and B). These results indicate that, in contrast to CCE induced by active store depletion (e.g., by PE), p75^NTR deletion does not affect contraction mediated by CCE due to passive store depletion. Our next experiments examined the possibility that p75^NTR can modulate CCE triggered by mechanisms other than SR Ca²⁺ depletion.

Fig. 3.

Active tension induced by passive store depletion-induced CCE is not affected by p75^NTR deletion. A: representative tension tracings from WT and p75^−/− PA rings exposed to cyclopiazonic acid (CPA) in the absence and presence of extracellular Ca²⁺, with the latter used to stimulate CCE (shaded region) as a result of passive Ca²⁺ leakage from the SR to the cytosol. B: summarized data (means ± SE) showing active tension induced by 40K and CPA-induced CCE in PA rings from WT (n = 8) and p75^−/− (n = 6) mice.

p75^NTR is involved in regulating ET-1-mediated pulmonary vasoconstriction.

ET-1 is a potent vasoconstrictor that exerts its contractile action on PA rings by activating ET type A and B receptors (8, 21). As shown in Fig. 4, the application of 0.1 μM ET-1 caused a transient contraction in PA rings isolated from WT and p75^−/− mice (Fig. 4A). However, the amplitude of ET-1-induced active tension in PA rings isolated from p75^−/− mice was much greater than in rings from WT mice (Fig. 4, A–C). In addition to the amplitude of the transient contraction, the kinetics of ET-1-mediated contraction in PA rings from p75^−/− mice seemed to be different from those in rings from WT mice. The ET-1-induced PA contraction in p75^−/− mice contained an initial transient contraction followed by a sustained contraction before ET-1 was washed out, whereas the ET-1 induced contraction in WT mouse PA rings only had a transient contraction (Fig. 4A). These results suggest that p75^NTR is involved in regulating ET-1-induced PA contraction; the activation (or presence) of p75^NTR inhibits ET-1-mediated pulmonary vasoconstriction.

Fig. 4.

Endothelin-1 (ET-1)-induced pulmonary vasoconstriction is enhanced by p75^−/− deletion. A: representative tracings from WT and p75^−/− PA rings exposed to 0.1 μM ET-1 in the presence of extracellular Ca²⁺. B and C: summarized data (means ± SE) showing absolute tension before (Cont), during (ET-1), and after (Wash) application of 0.1 μM ET-1 (B) and ET-1-mediated active tension (C) in PA rings from WT (n = 8) and p75^−/− (n = 8) mice. *P < 0.05 vs. WT; **P < 0.01 vs. Cont.

Effect of p75^NTR on pulmonary vasoconstriction induced by 5-HT, a thromboxane analog, and 4-AP.

In addition, we examined the effects of a variety of vasoactive stimulants that target membrane receptors and/or ion channels. Stimulation with 5 μM 5-HT, 10 μM U4 (a thromboxane A₂ analog), and 5 mM 4-AP (a K⁺ channel inhibitor) all caused the contraction of PA rings from WT and p75^−/− mice (Fig. 5). The amplitudes of active tension and time to peak contraction kinetics did not appear to be affected by the p75^NTR deletion.

Fig. 5.

Active tension induced by serotonin (5-HT), thromboxane A2 analog U-46619 (U4), and 4-aminopyridine (4-AP) is not affected by p75^NTR deletion. A–C: representative tracings (a), summarized absolute tension (b), and summarized active tension (c) in WT and p75^−/− PA rings exposed to 5 μM 5-HT (A; n = 8), 10 μM U4 (B; n = 8), and 5 mM 4-AP (C; n = 8) in the presence of extracellular Ca²⁺. **P < 0.01 and ***P < 0.001 vs. Cont.

In related experiments, the extracellular application of a high concentration (10 U/ml) of thrombin elicited reversible transient contractions of WT and p75^−/− PA rings that were not apparent at a lower concentration (0.1 U/ml; Fig. 6, A and B). The active tension generated by 10 U/ml thrombin in PA rings, although slightly large, was not statistically different in p75^−/− (P = 0.11) mice compared with WT controls (Fig. 6C). Our findings suggest that normal p75^NTR activity may differentially regulate vasoconstrictor signaling and, more specifically, that intact p75^NTR signaling may be involved in suppressing ET-1-mediated vasoconstriction while having little effect on pulmonary vasoconstriction induced by 5-HT and U4.

Fig. 6.

Thrombin (Thromb) dose dependently enhances PA contraction. A and B: representative (left) and summarized (right) tension measurements from WT and p75^−/− rings exposed to 0.1 (A) or 10 U/ml (B) Thromb in the presence of external Ca²⁺. C: active tension induced by 0.1 and 10 U/ml Thromb in WT (n = 8) and p75^−/− (n = 8) PA rings. ***P < 0.001 vs. Cont.

p75^NTR deletion does not alter SNP-induced vasodilation.

Previous experiments have focused primarily on excitation-contraction coupling using vasoconstrictors. We next tested whether vasodilation in response to the nitric oxide donor SNP was altered by p75^NTR deletion. PA rings were precontracted by an exposure to 25 mM K⁺ and then exposed to increasing doses of SNP (1 pM–10 μM). In PA rings from both WT and p75^−/− mice, SNP caused progressive dose-dependent relaxation (Fig. 7, A and B). However, based on the results shown in Fig. 7C, which depicts the percent active tension generated by 25 mM K⁺ as a function of SNP contraction, SNP-induced pulmonary vasodilation in PA rings from p75^−/− mice was not significantly different from that in rings from WT mice. These data suggest that p75^NTR deletion does not alter the vasodilatory response to SNP.

Fig. 7.

Nitric oxide-mediated pulmonary vasodilation is not affected by p75^NTR deletion. A and B: representative tracings from WT (A) and p75^−/− (B) PA rings precontracted with 25 mM K⁺ (25K) and exposed to incremental concentrations of the nitric oxide donor sodium nitroprusside (SNP; 1 pM–10 μM, as indicated by arrows). C: summarized data from WT (n = 5) and p75^−/− (n = 5) PA rings treated with SNP. Data are presented as percentages of active tension produced by 25K.

p75^NTR deletion alters TRPC expression.

CCE function is linked to the expression of TRPC channels. Having observed a change in CCE in PA rings isolated from p75^−/− mice, we examined and compared TRPC expression in tissues isolated from WT and p75^−/− mice. Extracts from the brain, heart, and lungs from WT and p75^−/− mice were evaluated for total mRNA expression of seven TRPC isoforms. Brain tissues from WT and p75^−/− animals exhibited similar levels (not statistically different) of all TRPC subunits (Fig. 8A). TRPC1 was more highly expressed in the p75^−/− heart, but the increase was not statistically significant compared with WT (Fig. 8B); only TRPC1–3 and TRPC6 isoforms were detected in the heart. In the lungs, both TRPC1 and TRPC2 were more highly expressed (P < 0.05 and P < 0.01, respectively) in p75^−/− tissues (Fig. 8C). TRPC3 and TRPC6 expression were also higher, but not significantly, in p75^−/− lungs. The data suggest that p75^NTR may suppress the expression of TRPC subunits underlying CCE and receptor-operated Ca²⁺ entry in pulmonary tissues.

Fig. 8.

Differential canonical transient receptor potential (TRPC) mRNA expression in WT and p75^−/− tissues. A–C: bar graphs depicting TRPC expression (relative to β-actin controls) for all TRPC isoforms in whole cDNA extracts from the brain (A), heart (B), and lung (C) from WT (n = 3) and p75^−/− (n = 2) mice. *P < 0.05 and **P < 0.01 vs WT.

Pulmonary vascular remodeling in p75^−/− mice.

Three main factors contribute to the development of pulmonary hypertension: enhanced and sustained vasoconstriction, vascular remodeling (in the form of intimal and medial hypertrophy), and in situ thrombosis, all of which can ultimately lead to the narrowing and/or obliteration of the vascular lumen and increased pulmonary vascular resistance. Having ascertained the increased contractility of PA rings from p75^−/− animals, we investigated whether pulmonary vascular remodeling might also be evident in the knockout animals. Figure 9A shows smooth muscle α-actin in lung cross sections from WT and p75^−/− mice. Increased wall thickness was evident in p75^−/− sections. In some segments, obliteration of the vascular lumen was well under way, suggesting that these animals likely developed pulmonary hypertension. We quantified the remodeling by evaluating the wall thickness and diameter of vessels in lung cross sections. In p75^−/− mice, the PA wall was thickened (P < 0.001) and the external diameter was decreased compared with WT mice (Fig. 9B). The ratio of thickness to diameter was also significantly increased in p75^−/− mice. We also verified the extent of vascularization in the lungs. Figure 9C shows the numbers of vessels counted per field (at ×10 magnification) in WT and p75^−/− lung cross sections; vascular pruning was evident in the lungs of p75^−/− mice. Taken together, these observations suggest that p75^−/− mice exhibit histological and pathological changes consistent with pulmonary hypertension.

Fig. 9.

Pulmonary vascular remodeling is evident in p75^−/− animals. A: smooth muscle α-actin staining of 5 different PA cross sections from both WT and p75^−/− mouse lungs. B: bar graphs showing pulmonary vascular remodeling in terms of wall thickness (left), external arterial diameter (middle), or a ratio of the two parameters (right). C: pulmonary neovascularization shown as the number of vessels per field (×10). Data in B and C are presented for WT and p75^−/− mice (n = 5 for each). ***P < 0.001 vs WT.

DISCUSSION

Excitation-contraction coupling in vascular smooth muscle is primarily initiated by an increase in [Ca²⁺]_cyt in vascular SMCs. A rise in [Ca²⁺]_cyt in PASMCs is a major trigger for pulmonary vasoconstriction and thus an important intracellular signaling process for excitation-contraction coupling in PAs. Smooth muscle contraction and relaxation can be regulated by changes in membrane potential in SMCs. When the SMC membrane is depolarized (e.g., with high-K⁺ perfusate or by blockade of K⁺ channels in the plasma membrane), Ca²⁺ influx through VDCCs is a major recourse for the elevated [Ca²⁺]_cyt and serves as an important mechanism for SMC contraction.

Smooth muscle contraction and relaxation can also be regulated by mechanisms independent of changes in membrane potential. The elevated [Ca²⁺]_cyt in SMCs, upon activation of membrane receptors, is mainly caused by Ca²⁺ influx through ROCs and/or SOCs and by Ca²⁺ release from intracellular Ca²⁺ stores, such as the SR or ER. However, many contractile agonists cause vasoconstriction by a complex mechanism involving Ca²⁺ release from different intracellular stores and Ca²⁺ influx through different channels (e.g., ROCs, SOCs, and VDCCs).

As shown in this study, pulmonary vasoconstriction induced by membrane depolarization, as a result of high-K⁺-mediated shift of the K⁺ equilibrium potential or 4-AP-mediated inhibition of voltage-gated K⁺ channels on the surface membrane of PASMCs, was not significantly different in PA rings from WT and p75^−/− mice (Figs. 2, B and C, and 5C). These results suggest that p75^NTR is probably not involved in membrane depolarization-induced Ca²⁺ influx through VDCCs in PASMCs.

The amplitude of active tension induced by 5-HT (Fig. 5A), the thromboxane A₂ analog (U4; Fig. 5B), and thrombin (Fig. 6) appeared to be comparable in PA rings from WT and p75^−/− mice. However, ET-1-induced active tension in PA rings from p75^−/− mice was significantly greater than in rings from WT mice (Fig. 4). These results indicate that that p75^NTR and/or its downstream signaling cascades selectively regulate ET-1-induced pulmonary vasoconstriction. It is still unclear how p75^NTR interferes with ET-1 and endothelin receptor signaling in PASMCs to inhibit PA smooth muscle contraction. p75^NTR is known to function as a coreceptor for several receptors, such as TrkA (3), NogoR (40), and PAR3 (6), and regulate their downstream signaling. One of the possibilities is that p75^NTR selectively interacts with the ET-1 signaling pathway to mediate Ca²⁺ mobilization (or depletion) from intracellular stores (i.e., IP₃-sensitive SR).

Pulmonary vasoconstriction induced by active store depletion-mediated CCE, induced by the α-receptor agonist PE, was significantly enhanced by p75^NTR depletion (Fig. 2, D and E), whereas smooth muscle contraction induced by passive store depletion-induced Ca²⁺ influx through SOCs was comparable in PA rings from WT and p75^−/− mice (Fig. 3). Taken together, these data suggest that p75^NTR may constitutively inhibit the production of IP₃ or activation of IP₃ receptors on the SR membrane. The inhibitory effect of p75^NTR seems to be selective because it only affects ET-1- and PE-induced pulmonary vasoconstriction.

p75^NTR has been primarily characterized in the regulation of cellular differentiation and/or survival in the nervous system. Recent studies have identified novel biological roles of p75^NTR outside of the nervous system in the regulation of lung fibrosis (33) and inflammation (31) and liver regeneration (27). Few studies have evaluated its importance in smooth muscle physiology, and none have evaluated its putative role in regulating muscular contraction. Ricci et al. (32) demonstrated the expression of p75^NTR mRNA in human PASMCs. We confirmed the predominant expression of p75^NTR in PASMCs in our murine model. Whole animal knockout of p75^NTR abolished its expression in lung arteries. However, membrane depolarization-induced vasoconstriction (i.e., by 40 mM K and 4-AP) was not significantly altered by p75^NTR deletion, unlike agonist-induced contraction.

Colocalization of p75^NTR with CD31 also suggested the expression of p75^NTR in ECs from WT animals. This is a novel observation, as very few studies have evaluated its importance in vascular ECs. In a recent study, Caporali et al. (5) demonstrated that gene transfer-induced expression of p75^NTR impairs the survival, proliferation, migration, and adhesion capacities of cultured ECs and endothelial progenitor cells. Intramuscular p75^NTR delivery also impaired angiogenesis and neovascularization in human umbilical vein ECs. p75^NTR in cultured porcine aortic ECs has also been associated with EC migration and angiogenesis (29). In an experimental autoimmune encephalomyelitis model (murine model of multiple sclerosis), p75 expression was linked to altered cellular infiltration and interaction of activated ECs with cells of the immune system (18); a similar role may be involved in brain injury and regulation of the blood-brain barrier (24). In our experiments, the endothelium was intact in the PA rings, and no work was conducted on endothelium-denuded vessels, so we cannot evaluate the potential impact of endothelial p75^NTR expression of pulmonary vascular function. However, judging from the excessive wall hypertrophy and vascular lumen obliteration we observed in p75^−/− tissues (Fig. 9), we can hypothesize that the decreased p75^NTR expression may somehow have contributed to either 1) increased EC proliferation or 2) increased PASMC proliferation due to infiltration of mitogenic and proliferative factors from the vascular lumen subsequent to impairment of the endothelial barrier. Whether this is truly the case remains to be investigated.

p75^NTR can interact directly with phosphodiesterase 4A to target cAMP degradation to the plasma membrane (33). In the lung, cAMP downregulation by p75^NTR results in decreased proteolytic activity of tissue plasminogen activator and increased pulmonary fibrosis. As it relates to vascular tone, cAMP is an intracellular second messenger that usually causes pulmonary vasodilation (12, 28); therefore, cAMP downregulation can lead to increased vasoconstriction. However, ET-1-induced pulmonary vasoconstriction was significantly enhanced in PA rings from p75^−/− mice; that is, p75^NTR exerts an inhibitory effect on ET-1-mediated PA contraction. These observations suggest that p75^NTR-modulated cAMP production and/or degradation is not involved in controlling ET-1- and PE-induced pulmonary vasoconstriction. In addition, p75^NTR deletion did not have a significant effect on SNP-induced pulmonary vasodilation, further indicating that p75^NTR-mediated changes in intracellular second messengers (e.g., cAMP and cGMP) may not directly affect agonist-induced vasoconstriction and vasodilation.

We and others have previously demonstrated that CCE can modulate pulmonary vascular tone in rat PAs (14, 17, 22, 35, 39, 42). CCE is often associated with store depletion-induced Ca²⁺ influx via SOCs (4). We showed that CCE induced by CPA (i.e., by passive store depletion) was not altered by p75^NTR deletion. However, agonist (e.g., PE)-induced CCE, or CCE induced by active store depletion, was significantly enhanced by p75^NTR deletion, although even this response varied with the nature of the agonist. Active tension induced by PE and ET-1 was significantly enhanced in p75^−/− mouse PAs, whereas tension generated by 5-HT, U4, 4-AP, and thrombin remained unaffected. ET-1 is the most potent vasoconstrictor of PAs. The indication that its vasoactive functions are suppressed by p75^NTR expression suggests that p75^NTR may be an important regulator not only of pulmonary fibrosis (33) but also of pulmonary vascular tone.

GRANTS

This work was supported in part by National Institutes of Health (NIH) Predoctoral Fellowship F31-NS-060478 (to B. D. Sachs) and by NIH Grants NS51470 (to K. Akassoglou) and HL-064945, HL-054043, and HL-066012 (to J. X.-J. Yuan).