Sphingosine kinase 1 (Sk1) is an important regulator of myogenic tone and smooth muscle cell Ca2+ sensitivity. Phosphorylation of Sk1 at serine 225, which results in its translocation to the plasma membrane, is required for full enzymatic activation. To investigate the functional role of Sk1 phosphorylation and localization, isolated resistance arteries from the hamster gacilis muscle were transfected with GFP or various mutants of Sk1. Elevation of transmural pressure stimulated the translocation of Sk1, but not a non-phosphorylatable mutant of Sk1 (SkS225A). Compared to GFP controls, expression of SkS225A reduced resting and myogenic tone, resting Ca2+, pressure-induced Ca2+ elevations and Ca2+ sensitivity. Inhibition of ERK1/2 signalling (PD98059), which putatively phosphorylates Sk1, had similar effects on resting and myogenic tone. In an attempt to rescue the effects of the S225A mutation, we employed a membrane anchored version of the mutant. Surprisingly, only resting Ca2+ and pressure-induced Ca2+ elevations were restored. Interestingly, expression of a membrane anchored wild-type Sk1 enzyme also reduced resting tone, myogenic vasoconstriction and Ca2+ sensitivity compared to GFP controls, but to a lesser extent than the anchored S225A mutant. We conclude that phosphorylation-dependent translocation of Sk1 to the plasma membrane is critical for its regulatory function in vascular smooth muscle cells. This process can not be imitated by forced localization of Sk1 with a membrane anchor.

The spiral modiolar artery (SMA) is the exclusive supplier of blood to the inner ear, and thus constriction of this artery can cause ischemia-related inner ear pathologies. We have previously shown that exogenous sphingosine-1-phosphate (S1P) induces strong vasoconstriction of the SMA. We proposed: (i) that endogenous S1P generated by the sphingosine kinase 1 (Sk1) plays a key role in SMA tone regulation and (ii) that TNFα(i.e., a relevant pathological mediator) is a potent activator of Sk1. Gerbil SMA were isolated, cannulated (25mmHg transmural pressure) and incubated with plasmids encoding GFP, GFP-labelled wild-type Sk1 (Sk1-GFP) or a catalytically inactive mutant of Sk1 (Sk1G82D) under organ culture conditions for 19-21h. Expression of GFP did not affect vasoconstriction induced by exogenous S1P (EC50=115nM, n=5) or the relationship between diameter and Ca2+i. Expression of Sk1G82D (n=7) significantly shifted the diameter/Ca2+i relationship to the right. In contrast, incubation with 1nM TNFα (2h, n=6) left-shifted the curve, an effect that was prevented by expression of Sk1G82D (n=7) or pretreatment with the TNFα inhibitor Ethanercept (1μg/ml, n=4). TNFα also induced a translocation of Sk1-GFP from the cytosolic to the plasma membrane compartment, indicating activation of Sk1. We conclude that endogenous Sk1 is a physiological regulator of SMA tone, acting primarily as a modulator of contractile apparatus Ca2+ sensitivity. This mechanism can be targeted by TNFα, possibly linking several inner ear pathologies to dysfunction of the microcirculation.