Background:
Reactive oxygen species (ROS) and calcium ions (Ca2+) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca2+ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca2+ signaling.
Methods:
Vascular smooth muscle cells were primary cultured from rat aorta. Ca2+ and ROS were monitored mainly using fura-2 and HyPer family probes. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques.
Results:
Ang II–induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca2+, and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca2+ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-G
βγ
-PI3K
γ
(phosphoinositide 3-kinase
γ
) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-Gα
q/11
-PLC (phospholipase C)-PKC
β
(protein kinase C
β
) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice.
Conclusions:
NOX1 mediates Ca2+ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.
Nanoxel-PM
TM
(Nanoxel) is a docetaxel-loaded methoxy-poly(ethylene glycol)-
block
-poly(D,L-lactide) (mPEG-PDLLA). This newly developed and marketed nanoformulation exhibits an improved pharmacokinetic profile, efficacy, and safety. Although the safety of Nanoxel to docetaxel as well as its bioequivalence must be clinically confirmed, all biological activities have not been examined in
in vitro
or
in vivo
studies. Here, the toxicity in a cultured cell system and the effects on blood cells were tested with Nanoxel and docetaxel. The
in vitro
cytotoxicity of Nanoxel was found to be comparable to or slightly lower than that of docetaxel depending on the concentrations tested or the cell types. Neither docetaxel nor Nanoxel induced erythrocytes hemolysis and produced reactive oxygen species up to 100 μM. However, Nanoxel was able to enhance the aggregatory response of platelets to collagen, whereas docetaxel attenuated such aggregation in a range of 50–100 μM, while thrombin-induced aggregation was not affected by either of them. Docetaxel or Nanoxel did not alter basal level of Ca
2+
and 5-hydroxytryptamine-evoked Ca
2+
transient in vascular smooth muscle cells. These results suggest that the mPEG-PDLLA micellar formulation alters the toxicological properties of docetaxel, and that extra cautions are needed when evaluating the safety of nanomedicine.
Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a widely distributed natural flavonol. It interacts with albumin, and thereby generates a fluorescence signal quantitatively. Based on such optical characteristics, we postulated that fisetin was applicable to the quantitation of albumin as an indicator. To establish the fisetin-based albumin assay, we examined the optical properties of fisetin and fisetin–albumin complex. The assay conditions were fine-tuned to fit for the actual concentration of serum albumin and to generate an optimal signal with a high signal-to-background ratio. The reaction between fisetin and albumin was linear in a wide range of concentrations. Non-protein serum components did not interfere with the reaction. The reactivity of fisetin was apparently specific for albumin among serum proteins. Both plasma and serum were compatible with the assay. The samples could be stored in a refrigerator or a freezer without the loss of reactivity toward fisetin. The generation and decay rates of the signal were acceptable for manual handling. The recovery of fortified albumin in serum was confirmed and the assay was validated with human sera. Fisetin-based albumin assay is suitable for clinical laboratory testing, considering the simple and short procedure, high specificity and sensitivity, linearity over a wide range of albumin concentrations, and, presumably, potential automatability.
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