Differential in vitro interactions of the Janus kinase inhibitor ruxolitinib with human SLC drug transporters.

Interactions of the Janus kinase (JAK) inhibitor ruxolitinib with solute carriers (SLCs) remain incompletely characterised. The present study was therefore designed to investigate this issue.The interactions of ruxolitinib with SLCs were analysed using transporter-overexpressing human embryonic kidney HEK293 cells. Substrate accumulation was detected by spectrofluorimetry, liquid chromatography coupled to tandem mass spectrometry or scintillation counting.Ruxolitinib was found to potently inhibit the activities of organic anion transporter 3 (OAT3), organic cation transporter 2 (OCT2), multidrug and toxin extrusion 1 (MATE1) and MATE2-K (half maximal inhibitory concentration (IC50) < 10 µM). It blocked OAT1, OAT4, OATP1B1, OATP1B3, OATP2B1 and OCT3, but in a weaker manner (IC50 > 10 µM), whereas OCT1 was not impacted. No time-dependent inhibition was highlighted. When applying the US Food and Drug Administration (FDA) criteria for transporters-related drug-drug interaction risk, OCT2 and MATE2-K, unlike MATE1 and OAT3, were predicted to be in vivo inhibited by ruxolitinib. Cellular uptake studies additionally indicated that ruxolitinib is a substrate for MATE1 and MATE2-K, but not for OAT3 and OCT2.Ruxolitinib in vitro blocked activities of most of SLC transporters. Only OCT2 and MATE-2K may be however clinically inhibited by the JAK inhibitor, with the caution for OCT2 that in vitro inhibition data were generated with an FDA-non recommended fluorescent substrate. Ruxolitinib MATEs-mediated transport may additionally deserve attention for its possible pharmacological consequences in MATE-positive cells.

Mitochondrial HSP90 Accumulation Promotes Vascular Remodeling in Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and limited therapeutic options. Although the mechanisms contributing to vascular remodeling in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs), have led to the emergence of the cancer-like concept. The molecular chaperone HSP90 (heat shock protein 90) is directly associated with malignant growth and proliferation under stress conditions. In addition to being highly expressed in the cytosol, HSP90 exists in a subcellular pool compartmentalized in the mitochondria (mtHSP90) of tumor cells, but not in normal cells, where it promotes cell survival. OBJECTIVES: We hypothesized that mtHSP90 in PAH-PASMCs represents a protective mechanism against stress, promoting their proliferation and resistance to apoptosis. METHODS: Expression and localization of HSP90 were analyzed by Western blot, immunofluorescence, and immunogold electron microscopy. In vitro, effects of mtHSP90 inhibition on mitochondrial DNA integrity, bioenergetics, cell proliferation and resistance to apoptosis were assessed. In vivo, the therapeutic potential of Gamitrinib, a mitochondria-targeted HSP90 inhibitor, was tested in fawn-hooded and monocrotaline rats. MEASUREMENTS AND MAIN RESULTS: We demonstrated that, in response to stress, HSP90 preferentially accumulates in PAH-PASMC mitochondria (dual immunostaining, immunoblot, and immunogold electron microscopy) to ensure cell survival by preserving mitochondrial DNA integrity and bioenergetic functions. Whereas cytosolic HSP90 inhibition displays a lack of absolute specificity for PAH-PASMCs, Gamitrinib decreased mitochondrial DNA content and repair capacity and bioenergetic functions, thus repressing PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In vivo, Gamitrinib improves PAH in two experimental rat models (monocrotaline and fawn-hooded rat). CONCLUSIONS: Our data show for the first time that accumulation of mtHSP90 is a feature of PAH-PASMCs and a key regulator of mitochondrial homeostasis contributing to vascular remodeling in PAH.

Effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells.

AIMS: The requirement of endothelial NO synthase (NOS3) calcium to produce NO is well described, although the effect of NO on intracellular calcium levels [Ca(2+)](i) is still confusing. Therefore, NO and [Ca(2+)](i) cross-talk were studied in parallel in endothelial cells possessing a functional or a dysfunctional NO pathway. METHODS AND RESULTS: Dysfunctional porcine endothelial cells were obtained either in vitro by successive passages or in vivo from regenerated endothelium 1 month after coronary angioplasty. Activity of NOS3 was characterized by conversion of arginine to citrulline, BH(4) intracellular availability, cGMP, and superoxide anion production. Imaging of the Ca(2+) indicator FURA 2-AM was recorded and sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) pump activity was analysed by (45)Ca(2+) uptake into cells. In endothelial cells with a functional NO pathway, NOS3 inhibition increased [Ca(2+)](i) and, conversely, an NO donor decreased it. In aged cells with an uncoupled NOS3 as shown by the reduced BH(4) level, the increase in superoxide anion and the lower production of cGMP and the decrease in NO bioavailability were linearly correlated with the increase in basal [Ca(2+)](i). Moreover, when stimulated by bradykinin, the calcium response was reduced while its decay was slowed down. These effects on the calcium signalling were abolished in calcium-free buffer and were similarly induced by SERCA inhibitors. In aged cells, NO improved the reduced SERCA activity and tended to normalize the agonist calcium response. CONCLUSION: In control endothelial cells, NO exerts a negative feedback on cytosolic Ca(2+) homeostasis. In aged cells, uncoupled NOS3 produced NO that was insufficient to control the [Ca(2+)](i). Consequently, under resting conditions, SERCA activity decreased and [Ca(2+)](i) increased. These alterations were reversible as exogenous NO, in a cGMP-independent way, refilled intracellular calcium stores, reduced calcium influx, and improved the agonist-evoked calcium response. Therefore, prevention of the decrease in NO in dysfunctional endothelium would normalize the calcium-dependent functions.

Effects of benazepril, an angiotensin-converting enzyme inhibitor, combined with CGS 35066, a selective endothelin-converting enzyme inhibitor, on arterial blood pressure in normotensive and spontaneously hypertensive rats.

Continuous intra-arterial administration of a selective endothelin-converting enzyme (ECE) inhibitor CGS 35066 at a dose of 30 mg/kg decreased the mean arterial blood pressure (MABP) in conscious unrestrained normotensive rats and spontaneously hypertensive rats (SHRs). At that dose, the magnitude of the antihypertensive effects was greater in SHRs than in normotensive rats. Additional administration of an angiotensin-converting enzyme (ACE) inhibitor benazapril (lotensin) further reduced MABP in normotensive rats and completely blocked hypertension in SHRs. However, when the selective ECE inhibitor was subsequently removed, blood pressure was less inhibited in normotenive rats whereas it remained strongly inhibited in SHRs by the ACE inhibitor alone. These results imply that simultaneous treatment with benazepril and CGS 35066 gave additive antihypertensive effects in normotensive rats but not in SHRs, when both compounds were administered at a dose of 30 mg/kg. Our results suggest that: (i) the endothelin (ET) system together with the renin-angiotensin system contribute to the maintenance of blood pressure in normal healthy rats; (ii) while an ECE inhibitor acts as an antihypertensive agent on its own, the sole efficacy of ACE inhibitor at that dose is sufficient to block MABP without the participation of the ET system in SHR.