**2. Materials and methods**

#### **2.1. Materials**

of cyclic nucleotides in the normoxic newborn pulmonary circulation assist in the downregulation of postnatal PAFR-mediated responses and that under hypoxic conditions, increasing the levels of cyclic nucleotides will abrogate PAF-mediated vasoconstriction thereby ameliorating PAF-induced persistent pulmonary hyperten‐

Platelet-activating factor (PAF) is an endogenous phospholipid which evokes a wide range of biological activities, such as vasoconstriction and systemic hypotension [1], mainly under pathophysiological conditions. The discovery of PAF, its cellular origin, and biological actions were first reported by Benveniste and Associates [2, 3]. Following these reports, investigations of the physiological effects of PAF involved its roles in fetal lung maturation and lung function [4-9], and its role in reproduction where it is involved in implantation of embryos, among other effects [10, 11]. PAF produces a myriad pathological effects in vivo, including platelet aggre‐ gation [12-14], mediation of immune response and bronchoconstriction [15, 16], and smooth muscle contraction [17-21], which hinges on its role as an inflammatory mediator and vaso‐ constrictor [17]. In the fetus, PAF plays an important physiological role in maintaining a high level of vasomotor tone in the pulmonary circulation [22]. Therefore, the high PAF receptor (PAFR) binding in fetal lamb lungs supports the existence of a high level of pulmonary vasomotor tone in utero [23]. On the other hand, in lungs of the newborn lamb, PAFR binding and receptor mRNA expression are low, suggesting a down regulation of PAFR-mediated

PAF acts by binding to its Gq protein isoform of G protein-coupled receptors, which is a seven transmembrane receptor [24]. Activation of G protein-coupled receptors by an agonist results in activation of signal transduction pathways [25], which may involve recruitment of intra‐ cellular second messengers such as cAMP, cGMP, inositol 1,4,5-triphosphate (IP3), and calcium [26, 27]. cAMP and cGMP act via their endogenous receptors, cAMP-dependent protein kinase (cAMP/PKA) and cGMP-dependent protein kinase (cGMP/PKG), respectively, to elicit relaxation of smooth muscle, and cAMP and cGMP mediate relaxation of pulmonary vessels, but cGMP has been shown to be more effective than cAMP in producing relaxation of perinatal ovine pulmonary vessels [28, 29]. Acute hypoxia upregulates PAFR-mediated intracellular signaling in fetal ovine pulmonary vascular smooth muscle [30]. Chronic hypoxia in the perinatal period may result in abnormal upregulation of PAFR protein expression, PAFR binding, and PAFR-mediated cell signaling, leading to increased pulmonary vasomotor tone and vascular remodeling, a key event in the onset of clinical disorders such as persistent

We are interested in understanding the mechanisms of pulmonary vascular relaxation at birth. Our primary hypothesis is that with oxygenation at birth and the increased production of

**Keywords:** Pulmonary artery, PAFR binding, cyclic nucleotides, siRNA

sion of the newborn.

**1. Introduction**

460 Muscle Cell and Tissue

effects in vivo [22, 23].

pulmonary hypertension of the newborn (PPHN) [31].

The study was approved by the Institutional Animal Care and Use Committee of Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Pregnant ewes (146-148 d gestation, term being 150 d) were purchased from Nebekar Farms (Santa Monica, CA). Authentic standards of PAF (C16-PAF) as well as 8-Br-cAMP, Rp-cAMPS, 8-Br-cGMP, Rp-8 pCPT-cGMPS were purchased from Biomol, Plymouth Meeting, PA. Radiolabeled PAF standards and substrates: hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, 1-O-[ace‐ tyl-3 H-(N)]-, (3 H-acetyl-C16-PAF), 21.5 Ci/mmol (370 GBq/mmol), and 3 H-thymidine were purchased from Perkin Elmer Life Sciences (Boston, MA). siRNA to PKA-Cα and its control were purchased from Cell Signaling Technologies (Carlsbad, CA). Phenylmethysulfonyl fluoride, leupeptin, pepstatin, as well as bovine serum albumin, were purchased from Sigma Chemical Company (St. Louis, MO). Antibody to PKA and PKG were purchased from Cell Signaling, while PAFR antibody was purchased from Cayman Chemical Company (Ann Arbor, MI). Studies were done with freshly made reagents. Ecolite(+) liquid scintillation cocktail was purchased from MP Biochemicals (Irvine, CA).

### **2.2. Methods**

Arterial intrapulmonary vessels were isolated from freshly killed term fetal lambs and then smooth muscle cells were harvested from the excised arteries and veins under sterile condi‐ tions as previously reported [20, 32]. Cells were used at the 3rd to 10th passage. Cell phenotype did not change from 1st to 10th passage as determined by the expression of α-smooth muscle actin and myosin light-chain kinase proteins.

#### **2.3. Study conditions**

Studies were done with adherent cells in normoxia and in hypoxia.

*Normoxia:* Cells were studied in humidified incubator at 37 °C aerated with 5% CO2 in air.

*Hypoxia:* An incubator set at 37 °C was first equilibrated for at least 1 h with a gas mixture of 2% O2, 10% CO2, and balance N2 to maintain incubator culture media pO2 < 40 Torr, and to mimic fetal lung environment, and monitored with TED 60T percent oxygen sensor, Teledyne Analytical Instruments (City of Industry, CA). Cells were then placed in this equilibrated incubator and continuously aerated with the hypoxia gas mixture throughout the duration of the study.

#### **2.4. Study of PAFR binding**

### *2.4.1. General protocol*

Receptor-binding assays were performed during hypoxia and normoxia as we previously reported [30]. Briefly, after incubation in normoxia or hypoxia, unbound 3 H-PAF was washed off with ice-cold phosphate buffer saline, and then incubated on ice for 30 to 45 min in saline/ EDTA mixture containing 154 mM saline and 5 mM EDTA. Receptor bound 3 H-PAF was extracted on Whatman GF/C membrane filters using inline vacuum system. Then culture flasks or dishes were washed with calcium-free 0.25% bovine serum albumin-containing Tyrodes buffer, pH 6.4. Cell-bound PAF radioactivity was quantified by scintillation spectrometry (Beckman Instruments, Fullerton, CA). In studies probing the interaction of PAF with its receptors in the presence of other agonist or antagonists, cells were pre-incubated with the agent before the addition of 3 H-PAF, and then incubated further according to the specific experimental protocol.

#### *2.4.2. Specific protocols*

*Effect of cAMP/cGMP on PAFR binding:* Cells were pre-incubated for 30 min, in normoxia or hypoxia, with buffer for controls or 10 µM each of cell permeable cyclic nucleotide analogs, 8- Br-cAMP or 8-Br-cGMP; the cAMP/PKA receptor inhibitor Rp-cAMPS or the cGMP/PKG receptor inhibitor Rp-8-pCPT-cGMPS. Then 1.0nM 3 H-PAF was added and incubated for 30 min more.

*Effect of cAMP/cGMP on PAFR protein expression:* Cells were pre-incubated for 3 hr min, in hypoxia or normoxia, with buffer for controls or 10 µM each of cell-permeable cyclic nucleotide analogs, 8-Br-cAMP or 8-Br-cGMP. Membrane proteins were isolated from cultured cells and probed for PAFR protein expression by Western blotting.

*Proliferation assay:* Sub-confluent cells were serum starved by culturing in 0.1% fetal bovine serum (FBS) for 72 hr, then cells were cultured in 10% FBS with or without the test agents in the presence of 5µCi/well of 3 H-thymidine and incubated for 24 hr more in the oxygen condition [32]. Cell DNA that were labeled with 3 H-thymidine were extracted with 0.5N NaOH and quantified on LKB 6500 scintillation counter (Beckman Coulter, Fullerton, CA).

*Transient cell transfection:* Cells were seeded in 6-well culture plates at 5 × 104 cells per well in an antibiotic-free growth media and allowed to stabilize for 24 h. Then they were treated with 1.5µg/ml of each plasmid in lipofectamine transfection reagent with 50nM of PKA-Cα siRNA according to the vendor's protocol (Cell Signaling) and incubated for 48 hr after which the transfection medium was replaced with fresh 10% FBS culture medium, which was also used to study cell proliferation or to study PAFR binding. Transfection efficiency was between 25% and 35% within 24 hr of transfection as judged by the pGFP fluorescence [32]. The proliferative phenotype of transfected cells was compared to that of untransfected cells. In studying cell proliferation or PAFR binding, transfected cells were incubated for 24 hr for proliferation study or 30 min for PAFR binding, with and without 10nM PAF in 10% FBS. Control for cell proliferation is 10% FBS alone while cells transfected with scrambled siRNA (sham siRNA) were used as control for PKA-Cα siRNA effect.

#### **2.5. Western blotting**

mimic fetal lung environment, and monitored with TED 60T percent oxygen sensor, Teledyne Analytical Instruments (City of Industry, CA). Cells were then placed in this equilibrated incubator and continuously aerated with the hypoxia gas mixture throughout the duration of

Receptor-binding assays were performed during hypoxia and normoxia as we previously

off with ice-cold phosphate buffer saline, and then incubated on ice for 30 to 45 min in saline/

extracted on Whatman GF/C membrane filters using inline vacuum system. Then culture flasks or dishes were washed with calcium-free 0.25% bovine serum albumin-containing Tyrodes buffer, pH 6.4. Cell-bound PAF radioactivity was quantified by scintillation spectrometry (Beckman Instruments, Fullerton, CA). In studies probing the interaction of PAF with its receptors in the presence of other agonist or antagonists, cells were pre-incubated with the

*Effect of cAMP/cGMP on PAFR binding:* Cells were pre-incubated for 30 min, in normoxia or hypoxia, with buffer for controls or 10 µM each of cell permeable cyclic nucleotide analogs, 8- Br-cAMP or 8-Br-cGMP; the cAMP/PKA receptor inhibitor Rp-cAMPS or the cGMP/PKG

*Effect of cAMP/cGMP on PAFR protein expression:* Cells were pre-incubated for 3 hr min, in hypoxia or normoxia, with buffer for controls or 10 µM each of cell-permeable cyclic nucleotide analogs, 8-Br-cAMP or 8-Br-cGMP. Membrane proteins were isolated from cultured cells and

*Proliferation assay:* Sub-confluent cells were serum starved by culturing in 0.1% fetal bovine serum (FBS) for 72 hr, then cells were cultured in 10% FBS with or without the test agents in

*Transient cell transfection:* Cells were seeded in 6-well culture plates at 5 × 104 cells per well in an antibiotic-free growth media and allowed to stabilize for 24 h. Then they were treated with 1.5µg/ml of each plasmid in lipofectamine transfection reagent with 50nM of PKA-Cα siRNA according to the vendor's protocol (Cell Signaling) and incubated for 48 hr after which the transfection medium was replaced with fresh 10% FBS culture medium, which was also used to study cell proliferation or to study PAFR binding. Transfection efficiency was between 25%

and quantified on LKB 6500 scintillation counter (Beckman Coulter, Fullerton, CA).

H-PAF, and then incubated further according to the specific

H-thymidine and incubated for 24 hr more in the oxygen

H-PAF was added and incubated for 30

H-thymidine were extracted with 0.5N NaOH

H-PAF was washed

H-PAF was

reported [30]. Briefly, after incubation in normoxia or hypoxia, unbound 3

EDTA mixture containing 154 mM saline and 5 mM EDTA. Receptor bound 3

the study.

462 Muscle Cell and Tissue

**2.4. Study of PAFR binding**

agent before the addition of 3

the presence of 5µCi/well of 3

receptor inhibitor Rp-8-pCPT-cGMPS. Then 1.0nM 3

probed for PAFR protein expression by Western blotting.

condition [32]. Cell DNA that were labeled with 3

experimental protocol.

*2.4.2. Specific protocols*

min more.

*2.4.1. General protocol*

*Preparation of proteins for Western analysis:* Proteins were prepared from stimulated and unstimulated cells that were studied in normoxia or hypoxia according to our previous reports. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Western blotting for PAFR quantification following our published methods [20, 30, 32, 33]. After x-ray exposure, band corresponding to PAFR protein was quantified and normalized to β-actin density.

#### **2.6. Data analysis**

All numerical data are mean ± SEM. In all instances where radioisotope was used, background radioactivity was subtracted before quantifying radioactivity. Data were analyzed with twotailed *t*-test followed with ANOVA (GraphPad Prism 6, San Diego, CA). Results were consid‐ ered significant at *p* < 0.05.
