Activation of neutral sphingomyelinase is involved in acute hypoxic pulmonary vasoconstriction

Abstract

The mechanisms involved in hypoxic pulmonary vasoconstriction (HPV) are not yet fully defined. The aim of the study was to determine the role of protein kinase C ζ (PKCζ) and neutral sphingomyelinase (nSMase) in HPV. Ceramide content was measured by immunocytochemistry and voltage-gated potassium channel (K_V) currents were recorded by the patch clamp technique in isolated rat pulmonary artery smooth muscle cells (PASMC). Contractile responses were analysed in rat pulmonary arteries mounted in a wire myograph. Pulmonary pressure was recorded in anesthetized open-chest rats. Protein and mRNA expression were measured by western blot and RT–PCR, respectively. We found that hypoxia increased ceramide content in PASMC which was abrogated by inhibition of nSMase, but not acid sphingomyelinase (aSMase). The hypoxia-induced vasoconstrictor response in isolated pulmonary arteries and the inhibition of K_V currents were strongly reduced by inhibition of PKCζ or nSMase but not aSMase. The nSMase inhibitor GW4869 prevented HPV in vivo. The vasoconstrictor response to hypoxia was mimicked by exogenous addition of bacterial Smase and ceramide. nSMase2 mRNA expression was ∼10-fold higher in pulmonary compared with mesenteric arteries. In mesenteric arteries, hypoxia failed to increase ceramide but exogenous SMase induced a contractile response. nSMase-derived ceramide production and the activation of PKCζ are early and necessary events in the signalling cascade of acute HPV.