Background-Mutations in the type II receptor for bone morphogenetic protein (BMPR-II), a receptor member of the transforming growth factor-␤ (TGF-␤) superfamily, underlie many cases of familial and sporadic primary pulmonary hypertension (PPH). We postulated that pulmonary artery smooth muscle cells (PASMCs) from patients with PPH might demonstrate abnormal growth responses to TGF-␤ superfamily members. Methods and Results-For studies of 3 H-thymidine incorporation or cell proliferation, PASMCs (passages 4 to 8) were derived from main pulmonary arteries. In control cells, 24-hour incubation with TGF-␤ 1 (10 ng/mL) or bone morphogenetic protein (BMP)-2, -4, and -7 (100 ng/mL) inhibited basal and serum-stimulated 3 H-thymidine incorporation, and TGF-␤ 1 and BMPs inhibited the proliferation of serum-stimulated PASMCs. In contrast, TGF-␤ 1 stimulated 3 H-thymidine incorporation (200%; PϽ0.001) and cell proliferation in PASMCs from PPH but not from patients with secondary pulmonary hypertension. In addition, BMPs failed to suppress DNA synthesis and proliferation in PASMCs from PPH patients. Reverse transcription-polymerase chain reaction of PASMC mRNA detected transcripts for type I (TGF-␤RI, Alk-1, ActRI, and BMPRIB) and type II (TGF-␤RII, BMPR-II, ActRII, ActRIIB) receptors. Receptor binding and cross-linking studies with 125 I-TGF-␤ 1 confirmed that the abnormal responses in PPH cells were not due to differences in TGF-␤ receptor binding. Mutation analysis of PASMC DNA failed to detect mutations in TGF-␤RII and Alk-1 but confirmed the presence of a mutation in BMPR-II in 1 of 5 PPH isolates. Conclusions-We

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1 8-Iso prostaglandin F 2a ) is one of a series of prostanoids formed independently of the cyclo-oxygenase pathway. It has been shown to be upregulated in many conditions of oxidant stress where its formation is induced by free radical-catalysed actions on arachidonic acid. As 8-iso PGF 2a is formed in vivo in diseases in which oxidant stress is high such as septic shock, we have assessed the relative potency and e cacy of this compound in pulmonary arteries from control and lipopolysaccharide (LPS)-treated rats. 2 Several studies have characterized the contractile actions of 8-iso PGF 2a on various smooth muscle preparations, but its potential dilator actions have not been addressed. Thus these studies examined both the contractile and dilator actions of 8-iso PGF 2a in rat pulmonary artery rings. The thromboxane mimetic U46619, PGE 2 sodium nitroprusside (SNP) and acetyl choline (ACh) were used for comparison. Each prostanoid had to be dissolved in ethanol to a maximum concentration of 1610 72 M. At high concentrations, ethanol directly contracted pulmonary vessels. We were therefore limited by the actions of the vehicle such that we were unable to add prostanoids at concentrations higher than 1610 74 M. In some cases this meant that maximum responses were not achieved and in these cases the E max and pD 2 values are apparent estimates. 3 The following rank order of potency was obtained from contractile studies; U4661948-iso PGF 2a 4PGE 2 , each prostanoid producing concentration-dependent contractions (10 710 ± 3610 74 M, 10 79 ± 10 74 M, 10 78 ± 10 74 M, respectively). As has been shown previously for other smooth muscle preparations, the thromboxane receptor (TP) antagonist ICI 192605, (1610 76 , 1610 75 and 1610 74 M), inhibited the contractions of 8-iso PGF 2a in a concentration-dependent fashion. 4 The nitric oxide synthase inhibitor, N G -nitro-L-arginine methyl ester (L-NAME; 1610 74 M), enhanced the contractile function of both 8-iso PGF 2a and PGE 2 , but had no e ect on that caused by U46619. Similarly, L-NAME inhibited the dilator function of all agents tested except the exogenous nitric oxide (NO) donor SNP, indicating that PGE 2 and 8-iso PGF 2a like ACh, act through the release of NO. The speci®city of the e ects of L-NAME were con®rmed in studies with the inactive enantiomer D-NAME (1610 74 M), which did not a ect the contractile or the dilator actions of 8-iso PGF 2a . Furthermore, ICI 192605 enhanced the dilator actions of 8-iso PGF 2a , suggesting that the dilator component of 8-iso PGF 2a was achieved via activation of a non-TP receptor. 5 Isoprostanes may modulate vascular tone by a direct action on TP receptors to cause contraction and via a distinct receptor leading to the release of NO to cause dilatation.

Long-term infusion of prostacyclin, or its analogs, is an effective treatment for severe pulmonary arterial hypertension. However, dose escalation is often required to maintain efficacy. The aim of this study was to investigate the mechanisms of prostacyclin receptor desensitization using the prostacyclin analog cicaprost in rat pulmonary artery smooth muscle cells (PASMCs). Desensitization of the cAMP response occurred in 63 nM cicaprost after a 6-h preincubation with agonist. This desensitization was reversed 12 h after agonist removal, and resensitization was inhibited by 10 microg/ml of cycloheximide. Desensitization was heterologous since desensitization to other G(s)alpha-adenylyl cyclase (AC)-coupled agonists, isoproterenol (1 microM), adrenomedullin (100 nM), or bradykinin (1 microM), was also reduced by preincubation with cicaprost. The reduced cAMP response to prolonged cicaprost exposure appeared to be due to inhibition of AC activity since the responses to the directly acting AC agonist forskolin (3 microM) and the selective AC5 activator NKH-477 were similarly reduced. Expression of AC2 and AC5/6 protein levels transiently decreased after 1 h of cicaprost exposure. The PKA inhibitor H-89 (1 microM) added 1 h before cicaprost preincubation (6 h, 63 nM) completely reversed cicaprost-induced desensitization, whereas the PKC inhibitor bisindolylmaleimide (100 nM) was only partly effective. Desensitization was not prevented by the G(i) inhibitor pertussis toxin. In conclusion, chronic treatment of PASMCs with cicaprost induced heterologous, reversible desensitization by inhibition of AC activity. Our data suggest that heterologous G(s)alpha desensitization by cicaprost is mediated predominantly by a PKA-inhibitable isoform of AC, most likely AC5/6.