Introduction: Persistent pulmonary hypertension of the newborn (PPHN) is associated with an elevated thromboxane to prostacyclin ratio, pulmonary artery (PA) hyperreactivity and hypersensitivity. Thromboxane receptor (TP), coupling with G-protein Gαq causes pulmonary vasoconstriction; whereas prostacyclin receptor (IP), coupling with Gαs, causes vasodilation and TP phosphorylation via adenylyl cyclase (AC)-cAMP-protein kinase A (PKA), desensitizes TP. Both TP phosphorylation and Gαq palmitoylation play major roles in regulation of signaling through the TP-Gαq complex. We hypothesized that increased Gαq palmitoylation and decreased AC activity could cause hypoxic TP hyperresponsiveness. We studied the impact of hypoxia on selected post-translational modifications of the receptor-G-protein complex, determining TP vasoconstriction: Gαq palmitoylation, TP phosphorylation and upstream AC activity. Methods: Force responses to thromboxane mimetic U46619, palmitoylation inhibition by 2-bromopalmitate (2-BP) and AC activation (forskolin) were studied by myography in hypoxic PPHN and control newborn swine pulmonary artery. Ca2+ mobilization was studied by fluorescent calcium indicators fura-2AM in pulmonary myocytes (PASMC), and fluo-4NW in HEK293 cells. Effects of hypoxia on Gαq palmitoylation were studied by metabolic labeling. Gαq cysteines and TP serines were mutated to determine sites of post-translational modifications. Protein expression and receptor-G-protein coupling were studied by Western blot and co-immunoprecipitation. PKA activity was assayed; and AC activity quantified. Results: Hypoxia increases Gαq palmitoylation, without increasing total palmitate uptake. Palmitoylation inhibition decreases U46619-stimulated force generation as well as Ca2+ mobilization in PPHN PA rings and hypoxic PASMC. Mutation of palmitoylable cysteine and palmitoylation inhibition proportionately decrease U46619-mediated Ca2+ mobilization in HEK293 cells. TP serine phosphorylation is decreased by hypoxia due to decreased PKA activity; this causes TP hypersensitivity and hyper-reactivity. Serine 324 of TPα is the target of PKA-mediated desensitization. AC activator-induced relaxation is reduced in PPHN PA. Basal and receptor-stimulated AC activity are decreased in hypoxic PASMC. Decreased AC activity is not due to decreased AC expression, ATP availability nor increased Gαi activation. Conclusion: Increased Gαq palmitoylation plays a role in TPα hyper-responsiveness in hypoxic PPHN. Hypoxia also reduces responses to agents acting through AC, unleashing TP-mediated vasoconstriction. Reactivation of pulmonary AC might be useful therapeutically to promote vasodilation and TP desensitization. / October 2016

Introduktion Selektiva serotoninåterupptagshämmare (SSRI) är idag de mest använda läkemedlen för behandling av depression, både i Sverige och runt om i världen. Studier som gjorts pekar dock på att användning av dessa preparat under graviditet kan medföra vissa risker för fostret. Ett samband har setts mellan moderns exponering för SSRI och ökad risk för persistent pulmonell hypertension hos nyfödda (PPHN). Det är en ovanlig men mycket allvarlig kardiovaskulär sjukdom, där omkring 10-20 % av de barn som drabbas avlider. Trots detta är SSRI ett förstahandsval vid behandling av gravida, då obehandlad depression anses innebära en större risk för såväl kvinna som foster. Syfte Att studera sambandet mellan SSRI och PPHN, och uppskatta hur stor risk som behandling under graviditet innebär. Dessutom undersöks skillnader, vad gäller risk, mellan specifika preparat. Metod Den här litteraturstudien omfattar sju originalstudier där risken för PPHN vid behandling med SSRI har studerats. Sex kohortstudier och en fallkontrollstudie har inkluderats, där fyra av dessa undersökt om tidpunkten för själva exponeringen har någon betydelse. Artikelsökningar har gjorts i databasen PubMed. Resultat Fem av sju studier visade på en signifikant ökad risk för PPHN vid SSRI-behandling under sen graviditet, medan två studier inte fann något samband. Den relativa risken varierade mellan 1.12 - 6.1 i de studier där ett samband påvisats. För behandling under tidig graviditet visade två av fyra studier på ett signifikant samband, med en relativ risk på 2.3 resp. 2.4. Inga stora skillnader mellan specifika preparat kunde påvisas, dock sågs en något högre risk vid behandling med paroxetin, jämfört med övriga SSRI. Diskussion I flera av studierna finns många felkällor, där störfaktorer och bias bidrar till osäkerhet. Studierna har stor variation vad gäller inklusionskriterer, antal deltagare och hantering av eventuella störfaktorer, vilket gör resultaten mer eller mindre tillförlitliga. Slutsats Behandling med SSRI under graviditetens andra hälft tycks vara förknippat med en ökad risk för PPHN. Den låga relativa risken och det faktum att sjukdomen är mycket ovanlig innebär dock att den absoluta risken är väldigt låg. Ett eventuellt samband vid behandling under tidig graviditet kan inte uteslutas. Preparaten sertralin och citolapram är att föredra framför paroxetin.

INTRODUCTION: Persistent Pulmonary Hypertension of the Newborn (PPHN) is defined as the failure of normal circulatory relaxation in the lungs at birth. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. Actin polymerization (APM), regulated by Rho GTPases, stabilizes force generation. We studied basal and thromboxane (TP)-induced APM and contraction in normoxic and hypoxic PA myocytes and rings. We also examined the downstream signaling pathways regulating hypoxia and TP-induced APM, and the role that actin plays in TP receptor internalization. METHODS: Smooth muscle myocytes from 2nd to 6th generation PAs of newborn piglets were cultured and exposed to hypoxia (10% O2) or normoxia (21% O2) for 72 hrs, then challenged with 10-6M TP-agonist U46619. APM was quantified by laser-scanning cytometry and stress fiber isolation. Downstream signaling pathways of TP receptor were studied by immunoprecipitation, Rhotekin-RBD and PAK-PBD affinity precipitation, Western blot, immunofluoresence and ELISA. Isometric force to serial concentrations of U46619 was measured in resistant PAs from PPHN and 3-day control swine. RESULTS: Hypoxia induced 2-fold APM via alpha- and gamma-actin isoforms, which contributed to increase U46619-induced contraction. Hypoxia decreased TP association with G12/13 in favor of Gαq. Basal RhoA and Cdc42 activity increased in hypoxia, while Rac activity decreased. U46619-challenge did not further alter RhoA activity in hypoxic cells, but increased Cdc42 and Rac activity. Hypoxia increased phosphorylation of LIMK and PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia, but increased after U46619 exposure. Jasplakinolide significantly stabilized gamma filaments, increasing force generation; cytochalasin D depolymerized all actin isoforms, which attenuated contractile force. Both actin-modifying agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. CONCLUSIONS: PA myocytes exhibit marked RhoA- and Rac-dependent APM in hypoxia. The additional APM response to U46619 challenge is independent of RhoA, but requires Cdc42 signaling. Hypoxia induces APM in PA myocytes, particularly causing an increase in filamentous alpha- and gamma-actin that contributes to increased U46619-induced force generation, a characteristic of PPHN. Dynamic actin also facilitates internalization of the TP receptor. Determining the mechanism that controls TP-mediated APM maybe beneficial as a potential target for PPHN.