Ceramide inhibits K_v currents and contributes to TP-receptor-induced vasoconstriction in rat and human pulmonary arteries

Abstract

Neutral sphingomyelinase (nSMase)-derived ceramide has been proposed as a mediator of hypoxic pulmonary vasoconstriction (HPV), a specific response of the pulmonary circulation. Voltage-gated K(+) (K(v)) channels are modulated by numerous vasoactive factors, including hypoxia, and their inhibition has been involved in HPV. Herein, we have analyzed the effects of ceramide on K(v) currents and contractility in rat pulmonary arteries (PA) and in mesenteric arteries (MA). The ceramide analog C6-ceramide inhibited K(v) currents in PA smooth muscle cells (PASMC). Similar effects were obtained after the addition of bacterial sphingomyelinase (SMase), indicating a role for endogenous ceramide in K(v) channel regulation. K(v) current was reduced by stromatoxin and diphenylphosphine oxide-1 (DPO-1), selective inhibitors of K(v)2.1 and K(v)1.5 channels, respectively. The inhibitory effect of ceramide was still present in the presence of stromatoxin or DPO-1, suggesting that this sphingolipid inhibited both components of the native K(v) current. Accordingly, ceramide inhibited K(v)1.5 and K(v)2.1 channels expressed in Ltk(-) cells. Ceramide-induced effects were reduced in human embryonic kidney 293 cells expressing K(v)1.5 channels but not the regulatory subunit K(v)β2.1. The nSMase inhibitor GW4869 reduced the thromboxane-endoperoxide receptor agonist U46619-induced, but not endothelin-1-induced pulmonary vasoconstriction that was partly restored after addition of exogenous ceramide. The PKC-ζ pseudosubstrate inhibitor (PKCζ-PI) inhibited the K(v) inhibitory and contractile effects of ceramide. In MA ceramide had no effect on K(v) currents and GW4869 did not affect U46619-induced contraction. The effects of SMase were also observed in human PA. These results suggest that ceramide represents a crucial signaling mediator in the pulmonary vasculature.

Changes in the Cerebrospinal Fluid Ceramide Profile After Subarachnoid Hemorrhage

“…Ceramide, in particular, has been shown to reduce the diameter of small bovine coronary arteries in a dosedependent manner and to mediate vasoconstriction in rat and human pulmonary arteries. 19,20 In addition, in a canine animal model, Zheng et al 21 showed that both ceramide analogs and neutral-sphingomyelinase cause vasoconstriction of cerebral arterial rings and increase intracellular Ca 2ϩ concentration in cerebral vascular smooth muscle cells. Mechanistically, ceramide regulates the activity of enzymes that control vasomotor response such as endothelial-and inducible-nitric oxide synthase.…”

Oxygen sensing and signal transduction in hypoxic pulmonary vasoconstriction

“…Recently, PKCξ-dependent NADPH oxidase activation has been suggested to be induced by increased ceramide levels due to mitochondrial-dependent activation of neutral sphingomyelinase (nSMase) [122][123][124][125]. However, inhibition of the nSMase also decreased U46619-induced vasoconstriction in pulmonary arteries [122]. In line with the notion that non-phagocytic NADPH oxidases may transfer hypoxic responses, genetic studies showed that deletion of the gp91 subunit of the phagocytic NADPH oxidase did not affect HPV [126].…”

“…In this regard, it was suggested that ROS release from mitochondria activates PKCε, which in turn activates NADPH oxidase to enhance ROS production and leads to a calcium increase [121]. Recently, PKCξ-dependent NADPH oxidase activation has been suggested to be induced by increased ceramide levels due to mitochondrial-dependent activation of neutral sphingomyelinase (nSMase) [122][123][124][125]. However, inhibition of the nSMase also decreased U46619-induced vasoconstriction in pulmonary arteries [122].…”

CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

“…nSMase is activated upon oxidative stress (20), possibly via arachidonic acid liberation by phospholipase A 2 (21), which all have been linked to HPV (1). In addition, ceramide accumulates in PASMCs upon hypoxia (22), mediates caveolar TRPC6 translocation in lung endothelial cells (23), and contributes to constriction of pulmonary artery rings (19,24). Although ceramide may thus potentially act as a direct mediator of HPV, it may also serve as substrate for the formation of other, bioactive sphingolipids, most notably sphingosine-1-phosphate (S1P).…”