Expression, activity, and function of phosphodiesterases in the mature and immature ductus arteriosus

Abstract

A patent ductus arteriosus is due in large part to increased sensitivity of the premature ductus to PGE2. After PGE2 stimulation, cAMP concentrations are higher in the immature than in the mature ductus. cAMP concentrations depend on the rates of adenyl cyclase production and phosphodiesterase (PDE)-mediated degradation. We used ductus from immature (n = 25) and mature (n = 21) fetal sheep to investigate whether a developmental increase in PDE activity could explain the diminished cAMP accumulation that follows PGE2 stimulation in the mature ductus. With advancing gestation, mRNA expression of the smooth muscle PDE isoforms (PDE1A, 1B, 1C, 3A, 3B, 4D, and 5A) increased in the ductus as did their hydrolytic activities. Selective inhibitors of PDE1, PDE3, and PDE4 relaxed the mature and immature ductus in the presence of inhibitors of prostaglandin and nitric oxide production. The mature ductus required higher concentrations of each of the PDE inhibitors to inhibit its tension to the same extent as in the immature ductus. There were no developmental changes in PDE expression in the fetal aorta. In conclusion, we observed a developmental increase in cAMP and cGMP PDE activity that contributes to the decreased sensitivity of the late-gestation ductus arteriosus to vasodilators like PGE2.

Figure 1

Real Time PCR measurements of PDE isoforms (PDE1A, 1B, 3A, 3B, 4D, 5A) are greater in the mature (■) than in the immature ductus (□) arteriosus. ΔCT(MDH-gene) represents the difference in CT between the expression of the housekeeping gene MDH and the gene of interest. Each unit of ΔCT(MDH-gene) represents a 2-fold increase in a gene’s mRNA. The more negative the ΔCT(MDH-gene), the fewer the number of starting copies of a gene (mRNA). N= number of separate animals used (immature = 7; mature = 7). (=): p<0.05, immature compared with mature. (*): p<0.05, mature ductus compared with mature aorta.

Figure 2

cAMP and cGMP PDE activities are greater in the mature (■) than in the immature ductus (□) arteriosus. Ductus homogenates were incubated in assay buffer containing either ³H-cAMP (panels A, B, and C) or ³H-cGMP (panels D and E). PDE activity was measured either in the absence (basal) or presence of selective PDE inhibitors (cilostamide 1×10⁻⁶ M, for PDE3; rolipram 5×10⁻⁶ M, for PDE4; and MY5445 5×10⁻⁶ M, for PDE5); or activators (CaCl₂/calmodulin (4×10⁻³ M)/10 units, respectively), for PDE1). We determined the PDE activity for each PDE family (Δ) by subtracting the basal activity from the activity in the presence of inhibitor or activator. Basal¹: no EGTA was added to the assay buffer in CaCl₂/calmodulin experiments (see Methods). N= number of separate animals used (immature = 5; mature = 5). (*): p<0.05, immature compared with mature.

Figure 3

Higher concentrations of PDE1, 3 and 4 inhibitors are required to inhibit isometric tension in the mature ductus arteriosus (■) than in the immature ductus (○). Ductus rings were precontracted with 30% oxygen, indomethacin and L-NAME (see Methods). Dose responses for 8-MM-IBMX, cilostamide, milrinone, rolipram, and MY5445 were studied. Tension is expressed as a percentage of baseline Net Tension (see Methods). (*): p<0.05, EC50 (immature) < EC50 (mature). n= number of separate animals used. 8-MM -IBMX: EC50 (immature) = 0.4±0.4 × 10⁻⁴M (n= 6); EC50 (mature) = 1.5±0.7 × 10⁻⁴M (n= 5), p<0.05. Cilostamide: EC50 (immature) = 2.9±0.8 × 10⁻⁷M (n= 4); EC50 (mature) = 8.4±1.5 × 10⁻⁷M (n= 4), p<0.05. Milrinone: EC50 (immature) = 0.7±0.1 × 10⁻⁶M (n= 6); EC50 (mature) = 9.4±2.2 × 10⁻⁶M (n= 7), p<0.05. Rolipram: EC50 (immature) = 0.7±0.1 × 10⁻⁴M (n= 6); EC50 (mature) = 1.3±0.1 × 10⁻⁴M (n= 7), p<0.05. MY5445: EC50 (immature) = not measurable (n= 4); EC50 (mature) = not measurable (n= 4).