Rosiglitazone improves aortic arginine transport, through inhibition of PKCα, in uremic rats

Peroxisome proliferator-activated receptor (PPAR) agonists were shown to inhibit atherosclerosis through augmentation of endothelial nitric oxide synthase (eNOS) activity. In addition, rosiglitazone exerts a beneficial effect in chronic renal failure (CRF). Since l-arginine transport by CAT-1 (the specific arginine transporter for eNOS) is inhibited in uremia, we aimed to explore the effect of rosiglitazone on arginine transport in CRF. Arginine uptake by aortic rings was studied in control animals, rats, 6 wk following 5/6 nephrectomy (CRF) and rats with CRF treated with rosiglitazone. The decrease of arginine transport in CRF was prevented by rosiglitazone. Immunobloting revealed that CAT-1 protein was decreased in CRF but remained unchanged following rosiglitazone administration. Protein content of the membrane fraction of PKCα and phosphorylated CAT-1 increased significantly in CRF, effects that were prevented by rosiglitazone. PKCα phosphorylation was unchanged but significantly attenuated by rosiglitazone in CRF. Ex vivo administration of phorbol-12-myristate-13-acetate to rosiglitazone-treated CRF rats significantly attenuated the effect of rosiglitazone on arginine uptake. The decrease in cGMP response to carbamyl-choline (eNOS agonist) was significantly attenuated by rosiglitazone in CRF. Western blotting and immunohistochemistry analysis revealed that protein nitration was intensified in the endothelium of CRF rats and this was attenuated by rosiglitazone. In conclusion, rosiglitazone prevents the decrease in arginine uptake in CRF through both depletion and inactivation of PKCα. These findings are associated with restoration of eNO generation and attenuation of protein nitration and therefore may serve as a novel mechanism to explain the beneficial effects of rosiglitazone on endothelial function in uremia.

Ca2+-Induced Ca2+ Release in the Pancreatic β-Cell: Direct Evidence of Endoplasmic Reticulum Ca2+ Release

The role of the Ca2+-induced Ca2+ release channel (ryanodine receptor) in MIN6 pancreatic β-cells was investigated. An endoplasmic reticulum (ER)-targeted “cameleon” was used to report lumenal free Ca2+. Depolarization of MIN6 cells with KCl led to release of Ca2+ from the ER. This ER Ca2+ release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca2+ channel antagonists and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca2+ was also inhibited by ryanodine, which did not alter voltage-gated Ca2+ channel activation. Both ER and cytoplasmic Ca2+ changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca2+ but no detectable change in ER Ca2+. Carbamyl choline caused ER Ca2+ release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca2+-induced Ca2+ release augments cytoplasmic Ca2+ signals in pancreatic β-cells.

Inhibition of dihydropyridine-sensitive calcium entry in hypoxic relaxation of airway smooth muscle

Hypoxia dilates airways in vivo and reduces active tension of airway smooth muscle in vitro. To determine whether hypoxia impairs Ca2+ entry through voltage-dependent channels (VDC), we tested the ability of dihydropyridines to modulate hypoxia-induced relaxation of KCl- and carbamyl choline (carbachol)-contracted porcine bronchi. Carbachol- or KCl-contracted bronchial rings were exposed to progressive hypoxia in the presence or absence of 1 microM BAY K 8644 (an L-type-channel agonist). In separate experiments, rings were contracted with carbachol or KCl, treated with nifedipine (a VDC antagonist), and finally exposed to hypoxia. BAY K 8644 prevented hypoxia-induced relaxation in KCl-contracted bronchi. Nifedipine (10(-5) M) totally relaxed KCl- contracted bronchi. Carbachol-contracted bronchi were only partially relaxed by nifedipine but were completely relaxed when the O2 concentration of the gas was reduced from 95 to 0%. These data indicate that hypoxia can reduce airway smooth muscle tone by limiting entry of Ca2+ through a dihydropyridine-sensitive pathway, but that other mechanisms also contribute to hypoxia-induced relaxation of carbachol-contracted bronchi.

Analysis of Ca 2+ fluxes and Ca 2+ pools in pancreatic acini

45 Ca 2+ movements have been analysed in dispersed acini prepared from rat pancreas in a quasi-steady state for 45 Ca 2+ . Carbamyl choline (carbachol; Cch) caused a quick 45 Ca 2+ release that was followed by a slower 45 Ca 2+ ‘reuptake’. Subsequent addition of atropine resulted in a further transient increase in cellular 45 Ca 2+ . The data suggest the presence of a Cch-sensitive ‘trigger’ pool, which could be refilled by the antagonist, and one or more intracellular ‘storage’ pools. Intracellular Ca 2+ sequestration was studied in isolated acini pretreated with saponin to disrupt their plasma membranes. In the presence of 45 Ca 2+ (1 µM), addition of ATP at 5 mM caused a rapid increase in 45 Ca 2+ uptake exceeding the control by fivefold. Maximal ATP-promoted Ca 2+ uptake was obtained at 10 µM Ca 2+ (half-maximal at 0.32 µM Ca 2+ ). In the presence of mitochondrial inhibitors it was 0.1 µM (half-maximal at 0.014 µM). 45 Ca 2+ release could still be induced by Cch but the subsequent reuptake was missing. The latter was restored by ATP and atropine caused further 45 Ca 2+ uptake. Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca 2+ , oxalate and ATP which were absent in intact cells or cells pretreated with A23187. The data suggest the presence of a plasma membrane-bound Cch-sensitive ‘trigger’ Ca 2+ pool and ATP-dependent Ca 2+ storage systems in mitochondria and rough endoplasmic reticulum of pancreatic acini. It is assumed that Ca 2+ is taken up into these pools after secretagogue-induced Ca 2+ release.