Alteration of vascular reactivity in heart failure: role of phosphodiesterases 3 and 4

Abstract

Background and purpose: This study examined the role of the main vascular cAMP-hydrolysing phosphodiesterases (cAMP-PDE) in the regulation of basal vascular tone and relaxation of rat aorta mediated by β-adrenoceptors, following heart failure (HF).

Experimental approach: Twenty-two weeks after proximal aortic stenosis, to induce HF, or SHAM surgery in rats, we evaluated the expression, activity and function of cAMP-PDE in the descending thoracic aorta.

Key results: HF rat aortas exhibited signs of endothelial dysfunction, with alterations of the NO pathway, and alteration of PDE3 and PDE4 subtype expression, without changing total aortic cAMP-hydrolytic activity and PDE1, PDE3 and PDE4 activities. Vascular reactivity experiments using PDE inhibitors showed that PDE3 and PDE4 controlled the level of PGF2α -stimulated contraction in SHAM aorta. PDE3 function was partially inhibited by endothelial NO, whereas PDE4 function required a functional endothelium and was under the negative control of PDE3. In HF, PDE3 function was preserved, but its regulation by endothelial NO was altered. PDE4 function was abolished and restored by PDE3 inhibition. In PGF2α -precontracted arteries, β-adrenoceptor stimulation-induced relaxation in SHAM aorta, which was abolished in the absence of functional endothelium, as well as in HF aortas, but restored after PDE3 inhibition in all unresponsive arteries.

Conclusions and implications: Our study underlines the key role of the endothelium in controlling the contribution of smooth muscle PDE to contractile function. In HF, endothelial dysfunction had a major effect on PDE3 function and PDE3 inhibition restored a functional relaxation to β-adrenoceptor stimulation.

Figure 1

cAMP-PDE activity in aorta with or without functional endothelium, isolated from SHAM and HF rats. (A) Total cAMP-PDE activity was determined in the presence of 1 μM [³H]-cAMP in lysates of SHAM and HF endothelium-intact (ENDO+) or endothelium-denuded (ENDO−) aortas. (B) cAMP-PDE activity pattern was determined in the absence (vehicle) or presence of a selective PDE family inhibitor (PDE1: 10 μM MIMX; PDE2: 100 nM BAY; PDE3: 1 μM cilostamide (Cil); PDE4: 10 μM Ro) or a non-selective PDE inhibitor (IBMX: 1 mM). Results are expressed in % of cAMP-PDE activity measured in the absence of inhibitors. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, significantly different from vehicle group. ^#P < 0.05, significant effect of endothelial removal.

Figure 2

Expression of PDE3 and PDE4 proteins (A–D) and mRNAs (E) in aorta isolated from SHAM and HF rats. (A–C) Representative Western blot images showing PDE3A, PDE3B, PDE4B and β-actin expression in aorta from SHAM and HF rats. (D) Corresponding graph showing relative expression level of PDE3A, PDE3B and PDE4B proteins. Results are expressed in % of the mean expression level in SHAM group. Data are means ± SEM of 9–12 SHAM and 9–11 HF rats, detected in three to four independent immunoblots. (E) PDE3A, PDE3B, PDE4B and PDE4D mRNA expression in aorta from SHAM and HF rats. Results are expressed in % of the mean expression level in SHAM group. Data are means ± SEM of six SHAM and six HF rats. *P < 0.05, significant effect of HF.

Figure 3

Evaluation of vascular reactivity in aorta isolated from SHAM and HF rats with or without functional endothelium. (A) Contractile response to a depolarizing solution of 60 mM KCl in SHAM and HF endothelium-intact (ENDO+) or endothelium-denuded (ENDO−) rat aortas. (B) CRCs to PGF_2α (1 nM to 30 μM) in SHAM and HF ENDO+ and ENDO− aortas. (C): Relaxant-response curves to carbachol (1 nM to 10 μM) on PGF_2α-precontracted ENDO+ aortas isolated from SHAM and HF rats. Data are means ± SEM. ^##P < 0.01, ^###P < 0.001, significant effect of endothelial removal; ^$$P < 0.01, ^$$$P < 0.001, significant effect of HF.

Figure 4

Effect of PDE3 or PDE4 inhibition on PGF_2α-induced contraction in aorta isolated from SHAM and HF rats with or without functional endothelium. CRCs to PGF_2α (1 nM to 30 μM) were performed in SHAM (A and C) and HF (B and D) endothelium-intact (ENDO+) or endothelium-denuded (ENDO−) aortas, in the absence (control) or presence of selective PDE inhibitors: (A and B) 1 μM Cil for PDE3; (C and D) 10 μM Ro for PDE4. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, significant effect of PDE inhibitor in each group.

Figure 5

Effect of L-NAME, Cil and endothelium removal on PDE3 or PDE4 inhibition-induced relaxant response in precontracted aorta isolated from SHAM and HF rats. (A–D) CRCs to Cil (1 nM to 30 μM; A and B) or Ro (1 nM to 30 μM; C and D) were performed in endothelium-intact arteries (ENDO+) pretreated in the absence (control) or presence of the NOS inhibitor (300 μM L-NAME) and in endothelium-denuded arteries (ENDO−/control) isolated from SHAM (A and C) and HF (B and D) rats and precontracted with PGF_2α. (E–F) CRCs to Ro (1 nM to 30 μM) were performed either in endothelium-intact (ENDO+) or endothelium-denuded (ENDO−) arteries isolated from SHAM (E) and HF (F) rats, and pretreated in the absence (control) or presence of 1 μM Cil. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, significant effect of L-NAME or PDE inhibitor; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, significant effect of endothelial removal.

Figure 6

Effect of L-NAME and endothelium removal on relaxation to β-adrenoceptor stimulation in precontracted aorta isolated from SHAM and HF rats. CRCs to isoprenaline (0.01 nM to 30 μM) were performed in endothelium-intact arteries (ENDO+) pretreated in the absence (control) or presence of the NOS inhibitor (300 μM L-NAME) and in endothelium-denuded arteries (ENDO−/control) isolated from SHAM (A) and HF (B) rats and precontracted with PGF_2α. Data are means ± SEM. ***P < 0.001, significant effect of L-NAME. ^###P < 0.001, significant effect of endothelial removal.

Figure 7

Effect of PDE3 or PDE4 inhibition on relaxant response to β-adrenoceptor stimulation in precontracted aorta isolated from SHAM and HF rats. CRCs to isoprenaline (0.01 nM to 30 μM) were performed in endothelium-intact (A and B), in endothelium-denuded (C and D) or in L-NAME-pretreated (E and F) aortas isolated from SHAM (A, C, E) or HF (B, D, F) rats pretreated in the absence (control) or presence of the PDE3 inhibitor (1 μM Cil), or the PDE4 inhibitor (10 μM Ro). Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, significant effect of PDE inhibitor.

Figure 8

Effect of PDE3 or PDE4 inhibition on cAMP and cGMP levels in aorta isolated from SHAM and HF rats. Cyclic AMP (A) and cGMP (B) levels were determined in lysates of SHAM and HF endothelium-intact (ENDO+) or endothelium-denuded (ENDO−) rings pretreated in the absence (vehicle) or presence of the PDE3 inhibitor (1 μM Cil) or the PDE4 inhibitor (10 μM Ro). Results are expressed in % of mean cyclic nucleotide levels measured in the absence of inhibitors. Data are means ± SEM of n rings from different animals. **P < 0.01, significantly different from vehicle group. ^#P < 0.05, significant effect of endothelial removal.