No abstract

Calcium transients. GCaMP6f EHTs were analyzed using standard methods (see Supplemental Methods), and parameters were calculated in pClamp. GCaMP6f cells were obtained courtesy of the Conklin laboratory (Gladstone Institutes,

Pharmacological openers of adenosine triphosphate-sensitive K + (K ATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema limit their clinical usefulness. It is presumed that the arterial dilation induced by K ATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, K ATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic K ATP channel opener cromakalim (0.01-3 µmol/L) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01-100 µmol/L) had similar effects at clinically relevant plasma concentrations. High speed in-vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01-10 µmol/L) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time PCR and flow cytometry identified the abundant K ATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory SUR2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K + currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. Significance Statement Potassium channel openers (KCOs) are potent antihypertensive medications, but off-target effects including severe peripheral edema limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the K ATP channels in lymphatic muscle cells may be unique from their counterparts in This article has not been copyedited and formatted. The final version may differ from this version.

Sandhoff disease, one of several related lysosomal storage disorders, results from the build up of N-acetyl-containing glycosphingolipids in the brain and is caused by mutations in the genes encoding the hexosaminidase beta-subunit. Affected individuals undergo progressive neurodegeneration in response to the glycosphingolipid storage. (1)H magnetic resonance spectra of perchloric acid extracts of Sandhoff mouse brain exhibited several resonances ca 2.07 ppm that were not present in the corresponding spectra from extracts of wild-type mouse brain. High-performance liquid chromatography and mass spectrometry of the Sandhoff extracts post-MRS identified the presence of N-acetylhexosamine-containing oligosaccharides, which are the likely cause of the additional MRS resonances. MRS of intact brain tissue with magic angle spinning also showed additional resonances at ca 2.07 ppm in the Sandhoff case. These resonances appeared to increase with disease progression and probably arise, for the most part, from the stored glycosphingolipids, which are absent in the aqueous extracts. Hence in vivo MRS may be a useful tool for detecting early-stage Sandhoff disease and response to treatment.