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Keywords:

  • drug delivery;
  • endothelin;
  • endothelin receptors;
  • G-protein coupled receptor;
  • peptide;
  • signal transduction and modulators (activation/inhibition)

Abstract

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

TAT (a 13-mer derived from the HIV-1 Tat protein)-linked cell-permeable peptides deliver plasma membrane impermeable cargos into the cell. We investigated the effect of a TAT-linked intracellular third loop of the endothelin-1 type B receptor on endothelin-1 activation of ERK. The effect of this peptide on ERK activation was determined in ETB receptor cDNA-transfected Chinese hamster ovary cells and in ETA- and ETB-expressing human pulmonary artery smooth muscle cells obtained from a normal and a bone morphogenetic protein-2 receptor, exon 1–8 deletion subject, with pulmonary hypertension. In the Chinese hamster ovary cells the peptide, at optimum 10 μm concentration, suppressed endothelin-1 activation. In the normal human pulmonary artery smooth muscle cells, the peptide marginally enhanced endothelin-1 activation of ERK. However, it markedly enhanced the endothelin-1 activation of ERK in the bone morphogenetic protein-2 receptor human pulmonary artery smooth muscle cells. While the effective concentration for endothelin-1 activation of ERK remained unchanged in the bone morphogenetic protein-2 receptor human pulmonary artery smooth muscle cells, the number of ETB receptors declined by 2/3. These data point to the intracellular third loop peptide as having variable receptor interactive effects with both signal repressive and enhancing capabilities. Peptides that can alter endothelin-1 signal capabilities are potentially important in the study and treatment of pulmonary hypertension.

Cell-permeable peptides (CPP) such as the 13-mer (TAT) derived from the HIV transactivating regulatory protein are able to rapidly cross the plasma membrane via direct hydrophobic penetration or via endocytosis (1–5). These peptides, consisting of short strongly cationic sequences, can be covalently linked to a variety of molecules to facilitate their crossing the hydrophobic plasma membrane barrier. Once in the cytoplasm, the CPP have the potential to regulate specific receptor-generated signals. In fact, peptides that mirror intracellular motif sequences of G-protein coupled receptors (GPCR) are now being used to regulate receptor-initiated signaling (6–8). Other CPP cargos are also being translocated into cells and tissues (9–15). With regard to vascular function, angiotensin signaling through the angiotensin II type 1 (AT1) receptor has been previously shown to be suppressed with specific motif mimicking CPP (16).

Endothelin-1 (ET-1) is an important vasoactive effector associated with vascular constriction and pulmonary/cardiac/vascular diseases (17,18). ET-1 receptor blockers, particularly blockers of both the type A receptor (ETA) and type B (ETB) receptors, are commonly used in the treatment of pulmonary hypertension. This treatment has met with mixed success. One shortcoming for this treatment is the global effect of the blockers on all the ET-1 signals regardless of their beneficial or harmful effects on vascular physiology. For example, while the ETA receptor is a strong contributor toward vasoconstriction, the ETB receptor may be contributing to vasodilation. The ETB, but not the ETA receptor, has been linked to the production of NO, a vasodilator (19). The ETB receptor is also involved in prostacyclin production (20). Promoting vasodilation and limiting smooth muscle cell proliferation are the hallmarks for the treatment of pulmonary arterial hypertension (PAH). Thus, targeted specific regulation of the ET-1 receptor signal transduction to block harmful signal cascades, while promoting beneficial cascades, will prove important in the treatment of ET-1-associated diseases such as PAH.

In this communication, we report on the actions of a TAT-linked third intracellular loop (IC3) region of the ETB receptor as it functions in Chinese hamster ovary (CHO) cells expressing the human ETB receptor and human smooth muscle cell populations derived from the pulmonary arteries of transplanted lungs expressing both the ETA and ETB receptors: one population from a normal lung and one from a subject with PAH. The objective of this study was to determine whether these type of peptides can be used to alter ET-1 signaling and therefore potentially reverse the adverse effects of pulmonary hypertension.

Methods and Materials

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Cell culture

Chinese hamster ovary, cells were cultured in F-12 growth media plus 10% fetal bovine serum (FBS) in P100 cell culture plates at 37 °C and 5% CO2. Stock cultures were passaged at 85–100% confluency at 1–10 dilutions. Cells were plated in 6-well plates for Western blot experiments. Human pulmonary artery smooth muscle cells (hPASMC), including those from the bone morphogenetic protein-2 receptor (BMPR2) mutated subject with pulmonary hypertension, were derived as described by Comhair et al. (21) and the cells were kindly gifted to us. They were maintained at less than passage 10 in DMEM/F12 15 mm Hepes from Invitrogen (Carlsbad, CA, USA) and 10% FBS from Lonza (Basel, Switzerland).

Stable overexpression of ETB receptor

cDNA encoding the human ETB receptor in pcDNA 3.1 was obtained from the Missouri S&T cDNA Resource Center (Rolla, MO, USA). The construct was cut with BamHI and the ends blunted. Then, the cDNA was excised with XhoI and cloned into pcMIN which had been cut with XbaI and blunted followed by digestion with XhoI. The ETB receptor cDNA in pcMIN was transfected into CHO cells and stable transfectants selected with 500 μg/mL geneticin. pcMIN is a bicistronic vector with the CMV promoter driving the expression of first the receptor gene and then the antibiotic resistance gene so that all surviving cells expressed the receptor (22,23).

CPP peptides

The CPP, TAT plus the intracellular third loop of the human ETB receptor (TAT/IC3B), or the third loop of the human ETA receptor (TAT/IC3A) was synthesized by 21st Century Biochemicals, Marlboro, MA, using Fmoc/t-Bu solid-phase peptide chemistry. The crude products were purified to homogeneity by preparative high-performance liquid chromatography and converted to an acetate salt to avoid the exposure of cells to trifluoroacetate. The final peptides were characterized by analytical high-performance liquid chromatography (purity >95%), nanospray mass spectrometry, and the sequence confirmed via collision-induced fragmentation. To follow the uptake of the TAT/IC3, a 5-FAM (5-fluorescein coupled) fluorophore was linked to the N-terminal of the TAT/IC3B peptide (5-FAM-Ahx- GRKKRRQRRRPP-RKKSGMQIALNDHLKQ-amide). The synthesis was carried out by Drs Mierke and Rupasinghe at Dartmouth University. The peptides were dissolved in endotoxin-free water to obtain 5 mm stock solutions.

Determination of peptide cell penetration

5-FAM tagged TAT/IC3B peptide (10 μm) in serum-free medium was added to wild-type CHO cultures grown in 6-well plates. After 30 min at 37 °C, to cleave adhering CPP from the cell membranes and to detach the cells from the wells, the cells were washed twice with PBS and then trypsinized and resuspended in PBS as a single cell suspension. The single cell suspension was transferred into fluorescence activated cell sorting (FACS) tubes at a concentration of 1.5 × 106 cells/mL. Cells were analyzed by FACS on a FacsCalibur (BectonDickinson, Franklin Lakes, NJ, USA) within 1 h after trypsinization. A total of 8000 gated cells per sample were counted. Data were analyzed using FlowJo software version 7.6.5 for Microsoft (TreeStar, San Carlos, CA, USA). As controls, cells were incubated with peptide-free medium. In addition to flow cytometry, laser scanning confocal microscopy was performed on a Zeiss (Thornwood, NY, USA) Axiovert 200M LSM. Briefly, wild-type CHO cells were grown sparse on glass coverslips inside p35 Petri dishes in growth medium. Before loading on the microscope, cells were washed with serum-free medium and resuspended in HEPES buffered saline. Then, 10 μm of 5-FAM tagged TAT/IC3B peptide was added immediately and viewed on the microscope.

Endothelin-1 binding

Confluent cells in 24-well plates were preincubated in ice-cold binding buffer (140 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 0.8 mm MgSO4, 5 mm glucose, 25 mm HEPES, 0.1% BSA, pH 7.4) for 15 min. 125I labeled ET-1 was mixed with cold ET-1 to obtain a concentration of 0.25 nm. Cells were incubated with the labeled ET-1 for total binding and with the labeled ET-1 plus 1 μm cold ET-1 for non-specific binding. After incubation on ice for 2 h, the cells were washed three times with 1-mL binding buffer per well, solubilized with 0.2% SDS, and radioactivity determined with a gamma counter (Packard). Preliminary experiments with CHO cells expressing only ETA receptors showed that BQ123 at 1 μm completely blocked ET-1 binding. Binding of ET-1 to CHO cells expressing only the ETB receptor was not affected by BQ123. For experiments with hPASMC that express both the ETA and ETB receptors, we bound ET-1 in the presence of BQ123 to estimate the number of ETB receptors. ETA receptor numbers were calculated by subtracting ETB receptor numbers from the total receptor numbers.

Western blotting

ETB receptor cDNA-transfected or wild-type CHO cells were grown to 100% confluency in 6-well plates and washed two times with PBS. Serum-free F-12 media (1 mL) was added to each well. TAT linked to peptide or TAT alone was preincubated at a final concentration of 10 μm for 30 min at 37 °C and 5% CO2. ET-1 was used at a final optimal concentration of 5 nm. Plates were again incubated at 37 °C and 5% CO2 for 5 min, placed on ice, and washed two times with ice-cold PBS, followed by the addition of 100 μL RIPA buffer plus Roche Complete protease inhibitor (Roche Applied Science, Indianapolis, IN, USA). Cells were scraped from the wells and cell lysates were pipetted into 1.5-mL Eppendorf tubes, vortexed, and incubated at 4 °C for 15 min, followed by centrifugation at 13 000 g for 20 min at 4 °C. Supernatants were transferred to new Eppendorf tubes and frozen at −20 °C until use. Protein concentrations were determined by BCA method. SDS-PAGE was run with 4% stacking gels and 10% separating gels. SDS-PAGE separations were transferred on nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) at 100 V and 4 °C for 1 h. Membranes were washed three times for about 5 min with TBS-T (20 mm Tris, 150 mm NaCl, 0.1% Tween 20, pH 7.6), blocked at room T for 1 h with 5% powdered milk in TBS-T, washed again three times, for 5 min, and incubated in 1° rabbit antibody (Ab) for pERK1/2 and GAPDH or alpha-tubulin (Cell Signaling Technologies, Beverly, MA, USA) at 4 °C overnight in 1:1000 Ab dilutions plus 5% BSA in TBS-T. Blots were washed again in TBS-T followed by 1-hour incubation in the corresponding anti-rabbit 2° Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:10 000 in 5% powdered milk TBS-T. Blots were then washed three times in TTBS-T, and developed in ECL detection solution from Amersham (GE Healthcare Biosciences, Pittsburgh, PA, USA) for 1 min before exposure to Kodak BioMax XAR film. The image was then analyzed using the NIH ImageJ (http://imagej.nih.gov/ij/) image analysis software to determine the intensity of each band. Relative ERK activity was determined by taking the band intensity for normalized ERK divided by the band intensity for normalized GAPDH or alpha-tubulin.

Statistical analysis

Statistical evaluation of the data was carried out using the Student’s t-test. Probability values <0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Expression of ETB receptors in stably transfected CHO cells

Chinese hamster ovary cells have been reported not to express the ET-1 receptors (24,25). Indeed, results shown in Figure 1A substantiate that ET-1 failed to activate ERK in wild-type CHO cells. When the CHO cells were exposed to medium containing 10% FBS, a marked activation of ERK ensued. Stable transfection of the wild-type CHO cells with the ETB receptor cDNA resulted in CHO cells with ET-1 driven increase in pERK (Figure 1B). As further illustrated in Figure 1B, at 2.5 nm ET-1, very little activation of ERK took place. However, starting at 5 nm ET-1, strong activation occurred. This activation peaked at approximately 10 nm ET-1. ERK activation maximized at 5-min postexposure to ET-1 (data not shown).

image

Figure 1.  (A) ERK phosphorylation in wild-type Chinese hamster ovary (CHO). ERK phosphorylation was measured in response to serum-free medium alone (control), 5 nm ET-1, 10% FBS, 10 μm TAT/IC3B, or 10 μm TAT/IC3B and 5 nm ET-1. Blot is representative of three separate experiments. (B) Concentration curve of ERK activation. ETB-transfected CHO cells were stimulated with increasing concentrations of ET-1 in serum-free medium. Both Western blots were probed with pERK and alpha-tubulin. Bar graph represents the average of triplicate samples ± SD from three representative experiments.

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Linking of the ETB IC3 to TAT CPP, its homology, and cellular entry

As sketched in Figure 2A, the N-terminus of the ETB receptor IC3 region was covalently linked to the C-terminus of the TAT/IC3B peptide. The IC3 of the ETB receptor showed some homology with the endothelin-1 ETA receptor IC3 region, particularly at the C-terminus. There was no homology with either the bradykinin B2 or the angiotensin AT1 or AT2 IC3 regions (Figure 2B).

image

Figure 2.  (A) Illustration of the TAT/IC3B cell-permeable peptide for ETB receptor and its positioning in the cell membrane. (B) Amino acid sequence alignment of the IC3 loop for human ETB, ETA, AT1 (angiotensin II type 1 receptor), AT2 (angiotensin II type 2 receptor), and BK2 (bradykinin receptor B2). The portion of the ETB used in our TAT/IC3B peptide is shown in bold. (C) Flow cytometry analysis of FAM-TAT/IC3B crossing into wild-type Chinese hamster ovary (CHO) cells. CHO cells that were incubated for 30 min with 10 μm 5-FAM-labeled TAT/IC3B peptide (solid line) or peptide-free medium (gray filling under the dashed curve). Cells were trypsinized and resuspended in PBS as single cell suspensions before flow cytometry. (D) Laser scanning confocal microscopic image of wild-type CHO cells incubated with FAM-TAT/IC3B. Cells were rinsed with serum-free medium and suspended in HEPES buffer. Then, 10 μm of FAM-TAT/IC3B was added. Left panel shows fluorescence only and the right panel shows cells without fluorescence.

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To illustrate cell entry, a fluorescently labeled TAT/IC3B peptide was synthesized. As demonstrated in Figure 2C, FACS analysis showed that the TAT/IC3B conjugated to 5-FAM crossed into the wild-type CHO cells within 30 min of exposure. This confirms results obtained by others that TAT complexes cross freely into cells (1–3,26–28). Laser confocal analysis of this fluorescent entry was then performed to obtain evidence as to the manner of the cellular incursion. As shown in Figure 2D, cellular fluorescence was not found in distinct pockets but was instead dispersed throughout the cytoplasm (29). It is interesting to note that the CPP complex did not cross into the nucleus.

Effect of the TAT/IC3B on ET-1 activation of ERK

The ET-1-induced ERK activation was then tested in the presence of increasing concentrations of the TAT/IC3B peptide. Optimal effective concentration of the TAT/IC3B to inhibit ET-1-caused ERK activation is illustrated in Figure 3. The addition of the peptide at 2.5 and 5 μm had no discernible effect on ERK activation. However, starting at 10 μm, the TAT/IC3B peptide clearly inhibited ET-1 induction of pERK. This inhibition remained steady to 50 μm TAT/IC3B concentrations. All experiments following this observation were carried out at the lower, 10 μm TAT/IC3B, to limit anomalous TAT effects. At the 10 μm concentration TAT alone or TAT/IC3B alone, in the absence of ET-1, had no effect on ERK activation (data not shown). The peptide clearly inhibited ET-1-promoted ERK activation in the CHO cells expressing ETB receptors.

image

Figure 3.  Concentration-dependent effect of TAT/IC3B on ET-1-induced ERK activation in Chinese hamster ovary (CHO) cells expressing ETB receptors. ETB-expressing CHO cells in 6-well plates were grown to confluence and incubated with increasing concentrations of TAT/IC3B in serum-free medium, followed by stimulation with 5 nm ET-1. Western blot analysis of ETB-expressing CHO cell lysates was used to show that ERK activation in response to ET1 (top) is optimal at 5 μm TAT/IC3B peptide, relative to alpha-tubulin (bottom). Data represent the average of triplicate samples ± SD from a representative experiment of at least three experiments. *p < 0.05 compared with control.

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ETA and ETB receptor integrity and number in BMPR2 hPASMC compared to normal hPASMC

Human pulmonary artery smooth muscle cells express both ETA and ETB receptors. To substantiate that the effectiveness of these receptors did not change in the BMPR2 hPASMC, both the normal and hPASMC were tested for their response to ET-1 (Figure S1). Because these cells contain both ETA and ETB receptors, the effective ET-1 concentrations were determined in the presence of BQ123, an ETA antagonist, and BQ788, an ETB antagonist. Indeed, the BMPR2 hPASMC continued to respond at very similar cET-1 concentrations as the normal hPASMC with optimum for ETA at 0.3 nm ET-1 and for ETB at 0.7 nm optimum.

However, when receptor number was determined in both cell populations, it became clear that both the ETA and ETB number diminished in the BMPR2 cells. This is illustrated in Figure 4. The binding of 125I-ET-1 to ETB and ETA in the hPASMC was determined in the presence and absence of BQ123 which blocks the binding of ET-1 to ETA completely (data not shown). The binding of ET-1 to the BMPR2 hPASMC was low, 40% relative to that found in the normal smooth muscle cells. While the ETA receptor number was also reduced in the BMPR2 hPASMC, their number/cell still remained greater than the ETBR/cell in the normal hPASMC.

image

Figure 4.  The binding of ETA and ETB receptors in Chinese hamster ovary (CHO) and human pulmonary artery smooth muscle cells (hPASMC). Confluent cells were bound with 125I labeled ET-1 (0.25 nm) as described in the Methods and Materials section. Non-specific binding was determined in the presence of 1 μm cold ET-1. For smooth muscle cells, ETB receptor binding was determined in the presence of 1 μm BQ123. ETA receptor binding was calculated from total specific binding minus total specific binding in the presence of BQ123.

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ERK activation in hPASMC and effect of TAT/IC3B

Figure 5A illustrates ERK activation in response to ET-1 in normal and the BMPR2 mutation-derived hPASMC. The activation of ERK by ET-1 shifted in the BMPR2 mutation-derived cells. In the normal cells, ET-1 signaled primarily through the ETB receptor, whereas in the BMPR2 mutation-derived cells, ERK activation was primarily ETA receptor routed. This was demonstrated with use of ET-1 receptor binding inhibitors. Smooth muscle cells (normal and BMPR2 mutation derived) exposed to BQ123, an ETA blocker, and BQ788, an ETB blocker, showed that in the normal cells, ET-1-induced ERK activation occurred through the ETB receptor. Blocking of the ETB receptor reduced inhibited ERK activation below the control levels. In the BMPR2 cells, the ET-1 activation was blocked by the ETA blocker BQ123. The blocking of ETA action reduced pERK levels below control. In this case, the ETB receptor had no action on ERK.

image

Figure 5.  (A) ERK activation in normal and BMPR2 human pulmonary artery smooth muscle cells (hPASMC) and the effects of ET-1 receptor inhibitors (BQ123 or BQ788) on ET-1-induced ERK activation. hPASMC were grown to confluency in 6-well plates and incubated with 1 μm BQ123 or 0.5 μm BQ788 in serum-free medium followed by 5 nm ET-1 stimulation. ERK activation was determined by Western blot analysis. (B) The effects of TAT/IC3B on ERK activation in normal hPASMCs and BMPR2 PASMCs. The hPASMC were grown to confluency in 6-well plates and incubated with TAT/IC3B or TAT/IC3A in serum-free medium followed by 5 nm ET-1 stimulation. ERK activation was determined by Western blot analysis. Data represent the average of triplicate samples ± SD from a representative experiment of at least three experiments. *p < 0.05 compared with ET-1 only treated sample.

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The effect of the TAT/IC3B peptide on ET-1 ERK activation was then examined in normal and the BMPR2 hPASMC as illustrated in Figure 5B. TAT/IC3B in combination with ET-1 increased pERK slightly in the normal hPASMC cells. Surprisingly in the BMPR2 cells, the presence of the TAT/IC3B enhanced the ET-1 activation of ERK markedly. In comparison, TAT/IC3A, the IC3 sequence of ETA, had only a negligible effect.

Discussion

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Cell-permeable peptides composed of specific receptor motif sequences present promising tools to regulate receptor action both at the investigative and clinical levels (30–32). These CPP may be of particular interest with respect to the actions of angiotensin and ET-1 which have central roles in the physiology of the heart, lung, and vascular function and regulate vasodilation, fibrosis, and cell proliferation. Regulating specific physiologically deleterious and beneficial signal transductions by these receptors would prove of important vascular benefit. However at this time, regulation of signals engendered by these effectors is only at a very rudimentary stage. Many signals promoted by the AT1, as well as ETB receptors, are necessary and beneficial and should not be blocked, but actually promoted. Others are harmful and lead to hypertension or unwanted cell proliferation. We formerly duplicated the IC2 sequence of the angiotensin AT1 receptor and linked it to a TAT CPP. The TAT/IC2 peptide penetrated into the cytosol rapidly. At 10 μm, we successfully inhibited angiotensin activation of PI turnover, Ca2+ influx, and the activation of Akt. These actions were based on the interference of the AT1 receptor to signals transmitted by the IC2 of the WT AT1 receptor linked to TAT (17).

Here, we examined the action of a CPP carrying a motif mimicking the IC3 of the ETB receptor. We focused on the ETB receptor because, unlike the ETA receptor it has the potential to promote the production of NO and prostacyclin, effectors participating in vasodilation and proliferative arrest (19,20). Thus, with specific regulation of its signal cascading ability, it has the potential to prove beneficial in diseases such as PAH. Confocal microscopy using 5-FAM-labeled TAT/IC3B showed that the cellular incursion of this CPP was not taking place via endocytosis but through direct penetration. The TAT/IC3B proved evenly dispersed throughout the cytoplasm with no distinct vesicular compartmentalization (29). Interestingly, the CPP failed to penetrate the nucleus. At this time, we have no explanation for this occurrence.

We chose the IC3 of the receptor because this region contains motifs for the activation of ERK signaling in vasoactive GPCR (33,34). The sequence was attached to the TAT peptide. We were interested to determine whether the motif mimicking CPP acted just to suppress receptor signaling by the ETB receptor system or may also be capable of enhancing receptor signals. In this regard, it is interesting to note that a peptide based on the C-terminus of the rat angiotensin receptor (AT1AR) was shown to rapidly cross into live cells and alone, in absence of ligand stimulation, promotes blood vessel contraction (16). The peptide interacted with the same selectivity toward G-protein subtypes as agonist-activated AT1AR. For example, inhibition of phospholipase C abolished its contractile action. Another peptide derived from human glucagon-like peptide receptor induced insulin release from isolated pancreatic islets. The mechanism was again found to be shared with the original glucagon receptor. Thus, a receptor motif emulating peptide can either act alone or in conjunction with the receptor as it is activated. In our case, the TAT/IC3B peptide showed no action by itself, only in concert with exposure of the cells to ET-1.

It is premature to put forward a mechanistic explanation for the actions of the TAT/IC3B. Further studies are clearly necessary to determine the mechanism(s) bringing about the differential effect of the CPP which is taking place in the ET-1/ETB system. Possible mechanisms can only be considered at this time. Clearly, the CHO cells expressed only the ETB receptor while the hPASMC express both the ETA and ETB receptors. Heteromerization of the ETA/ETB receptors has been reported (35–38). The effect of the peptide on homologous dimers (ETB/ETB) in the CHO cells may be different from effect in cells containing ETB/ETA dimers as taking place in the human smooth muscle cells. Furthermore, while the ETB and ETA receptors maintained their integrity in the BMPR2 hPASMC, the number of ETB receptors/cell diminished critically relative to normal hPASMC. This was manifested by the principal ERK activator becoming the ETA receptor in the BMPR2 hPASMC. While the TAT/IC3A peptide had little effect, and its points of homology with the TAT/IC3B proven unimportant, the presence of the TAT/IC3B could be acting to replenish this ETB motif in the ETA/ETB complex in the BMPR2 cells.

Thus, our results suggest that CPP can be used to either promote or inhibit receptor signal transduction.

This finding is important because of the ultimate potential to inhibit deleterious signals while allowing for positive signals to not only continue but be enhanced. With regard to pulmonary hypertension, the ETB receptor is regarded as, at least in part, contributing beneficially and is related to smooth muscle dilation as opposed to ETA receptor-related constriction. Thus, enhancement of selected ETB receptor signals by CPP could prove to be a positive contribution toward combating this disease.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

This work was supported in part by NIH/NHLBI grant R01-HL-025776. C.O.S was supported by NIH/NIA training grantT32-AG-000115 and J.L.W. was supported by NIH/NHLBI postdoctoral research supplement grant R01-HL-025776-25S1. We are thankful for the assistance of Albert Lee and Dr. Vickery Trinkaus-Randall in the BUSM Confocal Facility.

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  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Figure S1. Western blot analysis of hPASMC ET-1 titration with ETA (1 μ<SMALLCAPS>M</SMALLCAPS> BQ123) or ETB (0.5 μ<SMALLCAPS>M</SMALLCAPS> BQ788) inhibitors.

FilenameFormatSizeDescription
CBDD_1405_sm_FigS1.tif1951KSupporting info item

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