Abstract
At birth, release of endogenous vasodilators such as nitric oxide and prostacyclin facilitate pulmonary vasodilation via the cyclic nucleotides, cGMP and cAMP. Interaction of cyclic nucleotides and platelet-activating factor (PAF)-mediated responses in pulmonary vascular smooth muscle is not known. We studied the effects of cGMP and cAMP on PAF-mediated responses in ovine fetal intrapulmonary venous smooth muscle cells. Studies were done in hypoxia or normoxia with buffer with 8-Br-cGMP (BGMP) and 8-Br-cAMP (BAMP), as well as cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) inhibitors. All groups were treated with 1 nM PAF and incubated for 30 min for the binding assay or 20 min for measurement of inositol 1,4,5-phosphate (IP3) production. BGMP and BAMP decreased PAF binding in normoxia by 63 and 14%, respectively. Incubations with the PKG inhibitor Rp-8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate sodium and the PKA inhibitor Rp-adenosine-3′,5′-cyclic monophosphorothioate abrogated the inhibitory effects of BGMP and BAMP. PAF-stimulated IP3 production was 8565 ± 314 dpm/106 cells in hypoxia and 5418 ± 118 dpm/106 cells in normoxia, a 40% decrease. BGMP attenuated PAF-stimulated IP3 production by 67 and 37% in hypoxia and normoxia, respectively; the value for BAMP was 44% under both conditions. Pretreatment with PKG or PKA inhibitor abrogated BGMP and BAMP inhibition of IP3 release. PAF receptor (PAFr) protein expression decreased in normoxia, but pretreatment with 10 nM PAF up-regulated PAFr expression. Pretreatment with PAF decreased expression and activities of PKG or PKA proteins in normoxia and hypoxia. Our data demonstrate the existence of cGMP/cAMP-PAF cross-talk in pulmonary vascular smooth muscle cells, which may be one mechanism by which PAFr-mediated vasoconstriction is down-regulated at birth.
Footnotes
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This work was supported in part by Grant HL-077819 from the National Heart Lung and Blood Institute and Grant IR25 GM56902 from the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, MD.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.111914.
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ABBREVIATIONS: PAF, platelet-activating factor; PAFr, PAF receptor; IP3, inositol 1,4,5-triphosphate; PKG, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; PPHN, persistent pulmonary hypertension of the newborn; SMC-PV, intrapulmonary venous smooth muscle cells; 8-Br-cGMP, 8-bromoguanosine-3′,5′-cyclic monophosphate; Rp-8-pCPT-cGMPS, Rp-8-(4-Chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate sodium; 8-Br-cAMP, 8-bromoadenosine-3′,5′-cyclic monophosphate; Rp-cAMPS, Rp-adenosine-3′,5′-cyclic monophosphorothioate; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; PBS, phosphate-buffered saline; IBMX, 3-isobutyl-1-methyl-xanthine; SNP, sodium nitroprusside; PAGE, polyacrylamide gel electrophoresis, NO, nitric oxide.
- Received August 2, 2006.
- Accepted November 2, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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