**3. Results**

Figure 1 shows the effect of 8-Br-cAMP and 8-Br-cGMP on PAF binding to its receptors in pulmonary arterial smooth muscle cells (PASMC) (fmol/106 cells). For both 8-Br-cAMP and 8- Br-cGMP, data are mean ± SEM, *n* = 6; \**p* < 0.05, different from normoxia; + *p* < 0.05, different from PAF alone in normoxia or hypoxia. Cells treated with 10nM PAF alone, binding in normoxia was 9.7 ± 1.0, which increased to 17.2 ± 1.0 in hypoxia. Pretreatment of cells with 10µM of 8-Br-cAMP decreased PAF by 31% in normoxia and by 29% in hypoxia, respectively. Inhibition of endogenous cAMP effect with 1µM of the cAMP/PKA receptor inhibitor, RpcAMPS in normoxia or hypoxia increased PAF binding by 65% compared to effect of PAF alone in normoxia and by 35% compared to effect of PAF alone in hypoxia. Also, pretreatment of cells with 10µM Br-cGMP decreased PAF binding by 38% and 40% in normoxia and hypoxia, respectively, compared to the effect of PAF alone. Inhibition of endogenous cGMP activity with the cGMP/PKG receptor antagonist Rp-8-pCPT-cGMPS (Rp-pCPT) increased PAF binding by 20% compared to PAF alone in normoxia, and by 30% in comparison to binding by PAF alone in hypoxia. In general, hypoxia increased PAFR binding; exogenous cAMP or cGMP attenuated PAFR binding while inhibition of endogenous cAMP or cGMP activity with specific cAMP/PKA or cGMP/PKG receptor antagonists restored PAFR binding to levels higher than effect of PAF alone.

#### Hanouni et al

**Figure 1.** Effect of cAMP and cGMP on PAF receptor binding FPASMC. Data are means ± SEM, n = 6. Cells were preincubated for 30 min in normoxia or hypoxia with buffer alone, or 10µM of 8-Br-cAMP or 10µM of 8-Br-cGMP then 10nM PAF was added and incubated for 30 min more as described in the methods section. Hyoxia increased PAF re‐ ceptor binding and both 8-Br-cAMP and 8-Br.cGMP inhibited PAF receptor binding in normoxia and hypoxia. The specific inhibitors of PKA and PKG signaling; Rp-cAMPs and Rp-8-pCPT-cGMP reversed inhibitory effects of 8-BrcAMP and 8-cGMP. \*p <0.05, different from PAF normoxia; +p <0.05, different from PAF alone in normoxia or hypoxia.

Effect of cyclic nucleotides cAMP and cGMP on PAF stimulation of fetal pulmonary arterial smooth muscle cells (FPASMC) growth (3 H-thymidine DPM × 103 ) is shown in Figure 2. For both 8-Br-cAMP and 8-Br-cGMP, data are mean ± SEM, *n* = 6, \**p* < 0.05, different from normoxia; + *p* < 0.05, different from PAF alone in normoxia; # *p* < 0.05, different from PAF alone in normoxia or hypoxia. Treatment of cells with 10nM of PAF in normoxia caused a 15% increase in cell proliferation compared to effect of FBS control (55.9 ± 2.2). In hypoxia, PAF increased cell proliferation by 25% compared to effect of FBS control in hypoxia (70.5 ± 8.4). Incubation with 10µM 8-Br-cAMP in normoxia or hypoxia produced no difference in cell proliferation com‐ pared to PAF effect in hypoxia (Figure 2a). However, co-incubation of cells with 10nM PAF in the presence of cAMP significantly inhibited cell proliferation below effect of FBS control or 10nM PAF in normoxia and hypoxia. With cGMP (Figure 2b), treatment of cells with 10µM 8- Br-cGMP in normoxia or hypoxia decreased cell proliferation by 40% in normoxia compared to FBS control, and by 48% in compared to effect of 10nM PAF in normoxia. On the other hand, 8-Br-cGMP significantly increased cell proliferation compared to the effect of FBS control and 10nM PAF in hypoxia. Co-incubation of cells with PAF and 8-Br-cGMP in normoxia decreased cell proliferation by 77% in compared to FBS control and by 80% compared to PAF alone. Coincubation with 8-Br-cGMP and PAF in hypoxia produced no significant difference in cell growth compared to effects of FBS and 10nM PAF in hypoxia. Thus, cAMP and cGMP produced different effects of PAF stimulation of FPASMC growth during normoxia and hypoxia.

Figure 3 shows representative Western blots of effect of cAMP and cGMP on PAFR protein expression. For both 8-Br-cAMP and 8-Br-cGMP, data are mean ± SEM, *n* = 3. The statistics are as shown in the figures. The figures are not meant to be a cross-comparison of the effects of 8- Br-cAMP and 8-Br-cGMP on PAFR protein expression, but rather to compare effect of each cyclic nucleotide on PAFR protein expression. However, the figures show that hypoxia increased PAFR protein expression and in both cases, cAMP (Figure 3a) and cGMP (Figure 3b) decreased PAFR protein expression compared to control conditions.

#### Hanouni et al

Hanouni et al

464 Muscle Cell and Tissue

+

hypoxia.

smooth muscle cells (FPASMC) growth (3

*p* < 0.05, different from PAF alone in normoxia; #

**Figure 1.** Effect of cAMP and cGMP on PAF receptor binding FPASMC. Data are means ± SEM, n = 6. Cells were preincubated for 30 min in normoxia or hypoxia with buffer alone, or 10µM of 8-Br-cAMP or 10µM of 8-Br-cGMP then 10nM PAF was added and incubated for 30 min more as described in the methods section. Hyoxia increased PAF re‐ ceptor binding and both 8-Br-cAMP and 8-Br.cGMP inhibited PAF receptor binding in normoxia and hypoxia. The specific inhibitors of PKA and PKG signaling; Rp-cAMPs and Rp-8-pCPT-cGMP reversed inhibitory effects of 8-BrcAMP and 8-cGMP. \*p <0.05, different from PAF normoxia; +p <0.05, different from PAF alone in normoxia or hypoxia.

Effect of cyclic nucleotides cAMP and cGMP on PAF stimulation of fetal pulmonary arterial

both 8-Br-cAMP and 8-Br-cGMP, data are mean ± SEM, *n* = 6, \**p* < 0.05, different from normoxia;

or hypoxia. Treatment of cells with 10nM of PAF in normoxia caused a 15% increase in cell proliferation compared to effect of FBS control (55.9 ± 2.2). In hypoxia, PAF increased cell proliferation by 25% compared to effect of FBS control in hypoxia (70.5 ± 8.4). Incubation with 10µM 8-Br-cAMP in normoxia or hypoxia produced no difference in cell proliferation com‐ pared to PAF effect in hypoxia (Figure 2a). However, co-incubation of cells with 10nM PAF in the presence of cAMP significantly inhibited cell proliferation below effect of FBS control or 10nM PAF in normoxia and hypoxia. With cGMP (Figure 2b), treatment of cells with 10µM 8- Br-cGMP in normoxia or hypoxia decreased cell proliferation by 40% in normoxia compared to FBS control, and by 48% in compared to effect of 10nM PAF in normoxia. On the other hand, 8-Br-cGMP significantly increased cell proliferation compared to the effect of FBS control and 10nM PAF in hypoxia. Co-incubation of cells with PAF and 8-Br-cGMP in normoxia decreased cell proliferation by 77% in compared to FBS control and by 80% compared to PAF alone. Coincubation with 8-Br-cGMP and PAF in hypoxia produced no significant difference in cell growth compared to effects of FBS and 10nM PAF in hypoxia. Thus, cAMP and cGMP produced different effects of PAF stimulation of FPASMC growth during normoxia and

H-thymidine DPM × 103

) is shown in Figure 2. For

*p* < 0.05, different from PAF alone in normoxia

**Figure 2.** Effect of cAMP and cAMP on PAF stimulation of FPASMC proliferation. Data are means ± SEM, n = 6. Cells were pre-incubated for 30 min with buffer alone, or with 10 µM 8-Br-cAMP, or 10µM of 8-Br-cGMP, then PAF was added and incubated for 24 hr more as described in methods section. PAF increased cell proliferation in normoxia and hypoxia. Effect of 8-Br-cAMP on cell proliferation in normoxia or hypoxia, co-incubation of 8-Br-cAMP and PAF de‐ creased PAF-induced cell proliferation. 8-Br-cGMP alone decreased cell proliferation in normoxia, and co-incubation with PAF further decreased PAF stimulation of cell proliferation. \*p <0.05, different from normoxia; +p <0.05, different from effect of 10% FBS; #p <0.05, different from PAF effect.

We then investigated the effect of PKA siRNA PAFR binding and PAF stimulation of FPASMC proliferation in normoxia only. For both binding and proliferation, data are mean ± SEM, *n* = 6; \**p* < 0.05, different from 10% FBS control, + *p* < 0.05, different from PAF alone, # *p* < 0.05, different from 10% FBS control, PAF alone, and 8-Br-cAMP (+cAMP). In Figure 4a, 8-Br-cAMP decreased PAFR binding by 26% (PAF alone control, 10.2 fmol/106 cells). Pretreatment of cells with 50nM of the PKA-Cα siRNA increased PAFR binding by 47% compared to effect of PAF alone (control) and by 52% compared to effect of cAMP alone. The effect of sham PKA-Cα siRNA (sham) was not different from PAF alone. In Figure 4b, PAF alone stimulated greater cell proliferation (3 H-thymidine DPM × 103 ) by 45% compared to 10% FBS control (101.2 ± 5.3). Effect of 8-Br-cAMP on cell proliferation was 120.3 ± 7.8 which is 19% lower than effect of 10% FBS control and 20% lower than effect of 10nM PAF alone. As with PAFR binding, PKA-Cα increased cell proliferation and reversed the effect of cAMP on PAF binding. PAFR binding produced a 2-fold increase in cell proliferation compared to 10% FBS control and 37% increase compared to effect of 10nM PAF. The effect of the sham siRNA control was not different from effect of 10% FBS control.

#### Hanouni et al

**Figure 3.** Effects of cAMP and cGMP on PAF receptor protein expression in normoxia and hypoxia. Data are means ±SEM, n= 3. Cells were incubated in for 24 hr in normoxia or hypoxia with buffer alone, or with 10µM of 8-Br-cAMP, figure 3a, or with 10µM 8-Br-cGMP, figure 3b. PAF receptor (PAFR) expression was measureb by Western blotting and normalized to expression of beta-actin standard. Rp-8-pCPT-cGMPS, then 5nM PAF was added as needed and incu‐ bated for 20 min more. Treatment of cell with 8-Br-cAMP or 8-Br-cGMP surpressed PAFR protein expression in nor‐ moxia and hypoxia. \*p <0.05, different from control; +p <0.05, different from normoxia.

**Figure 4.** Effects of cAMP and and siRNA to PKA-cα on PAFR binding and PAF stimulation of cell proliferation. Data are means ± SEM, n= 5. Studies were done as described in methods section. PAFR binding (figure 4a) and cell prolifera‐ tion were determined. PAFR binding was attenuated by 8-Br-cAMP, but siRNA to PKA-cα increased PAFR binding. Effect of sham siRNA was not different from PAF alone. In figure 4b, 8-Br-cAMP decreased cell proliferation compared to effect of PAF alone. siRNA to PKA cα increased cell proliferation. Effect of sham siRNA was not different from 10% FBS or 8-br-cAMP. \*p <0.05, different from PAF alone of 10% FBS control; +p <0.05, different from PAF alone; #p <0.05, different from all other condition.
