Biomedical subjects

Zhenhua Huang

Publications and source records attributed to Zhenhua Huang.

6 recordsLinked to original sources

PubMed2007

Activation of ERK, JNK, Akt, and G-protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK-293 cells.

Bradykinin (BK) and angiotensin II (AngII) often have opposite roles in cardiovascular diseases. Our aim here was to construct hybrid receptors which bind AngII but signal as BK. Various sequences of the intracellular face of the AngII type I receptor, AT1R, were replaced with corresponding sequences from the bradykinin B2 receptor (BKB2R). The hybrids demonstrated a number of signaling characteristics of the BKB2R. For example, the hybrids demonstrated BK as opposed to AngII like phosphorylation of Akt and JNK. The hybrids containing the BKB2R intracellular loop 2 (IC2) displayed minimal G-protein, Galphai/Galphaq, linked signaling. Computer based molecular models suggested that Ser-Met-Gly from the IC2 of the BKB2R is detrimental for the Galphai/Galphaq coupled functions of this hybrid. The return of Lys-Ser-Arg of the AT1R to this hybrid led to almost full recovery of Galphai and Galphaq activation. The design and production of AT1/BKB2 hybrid receptors is a potential approach in the treatment of hypertension related diseases where the presence of AngII, its AT1 receptor and the consequent signal transduction has proven detrimental.

Amino Acid Sequence↗

PubMed2006

Modulation by bradykinin of angiotensin type 1 receptor-evoked RhoA activation of connective tissue growth factor expression in human lung fibroblasts.

The mechanisms regulating the opposing physiological actions of bradykinin (BK) and angiotensin II (AngII) are not well understood. Here we investigate signaling interactions between these two effectors. Connective tissue growth factor (CTGF) expression in IMR-90, human lung fibroblasts, is used as the endpoint target. In these cells the BK B2 receptor (BKB2R) is expressed constitutively, while no binding of AngII is detected. An inducible expression system is used to insert AngII receptor 1 (AT1R) and to obtain a signal level in response to AngII at the magnitude of BK. AngII and BK activate G protein-coupled targets, arachidonate release from cellular phospholipid stores, and intracellular phosphatidylinositol turnover equally. Both activate ERK, JNK, and p38 equally. However, AngII activates, whereas BK inactivates, RhoA. AngII induces a rapid (1 h) CTGF mRNA expression. RhoA siRNA and RhoA activation inhibitor, Y-27632, markedly reduce the AngII effect. Simultaneous treatment with BK and AngII attenuates the AT1R action. Additionally, BK in the absence of AngII lowers CTGF mRNA expression below basal levels over a span of 4 h. An AT1R/BKB2R chimera lacking heterotrimeric G protein coupling continues to activate MAP kinases to the same extent as wild-type (WT) AT1R and BKB2R. However, the increase of CTGF mRNA expression by this mutant is low, almost identical with that obtained by the simultaneous treatment of the WT AT1R-expressing cells with BK and AngII. In this context the chimeric receptor displays the characteristics of both receptors. These data demonstrate that, in human lung fibroblasts, BK modulates the action of AngII through the small G protein RhoA, but in a Galphai/Galphaq-independent manner.

Amino Acid Sequence↗

PubMed2005

The change of intracellular pH is involved in the cisplatin-resistance of human lung adenocarcinoma A549/DDP cells.

We had reported that the intracellular pH (pHi) of human lung adenocarcinoma A549 cells, which is sensitive to cisplatin, was more acidic than that of cisplatin-resistant A549/DDP cells. The correlation between the change of the pHi and cisplatin-resistance of A549/DDP cells was further studied by altering pHi in consequence of the change of CO2 concentration of the incubator. The pHi alterations of the cells were monitored by using the fluorescence probe of BCECF-AM. The results indicated that the pHi was more alkaline at lower CO2 concentration (2% CO2 in the incubator) and more acidic at higher CO2 concentration (8% CO2 in the incubator) for both A549 and A549/DDP cells compared with those of both A549 and A549/DDP cells cultured at 5% CO2 as the normal condition. Accumulation of bodipy-cisplatin, a fluorescence probe used for drug resistance assays, in A549 cells incubated at 2%, 5%, and 8% CO2 was increased 8.4%, 17.4%, and 23.5% compared to A549/DDP cells, respectively. Intracellular sequestration and distribution of bodipy-cisplatin imaged by laser scanning confocal microscopy indicated that bodipy-cisplatin was more encapsulated in acidic compartments of A549/DDP cells as shown with acridine orange, a dye that specifically labels acidic organelles in the cells. These results can be further confirmed in liposome systems with different pH gradients. It is proposed from the above results that the change of pHi in especially more acidic compartments in A549/DDP cells involves their cisplatin resistance.

Adenocarcinoma↗

PubMed2004

Fluorescent modified phosphatidylcholine floppase activity of reconstituted multidrug resistance-associated protein MRP1.

Multidrug resistance-associated protein (MRP1) may function as a floppase in human red blood cells to translocate phosphatidylserine and/or phosphatidylcholine from inner membrane leaflet to outer leaflet. Here we report that the purified and reconstituted MRP1 protein into asolectin proteoliposomes is mainly in an inside-out configuration and possesses the ability to flop a fluorescent labeled phosphatidylcholine (NBD-PC) from outer leaflet (protoplasmic) to inner leaflet (extracytoplasmic). The reconstituted MRP1 protein retains endogenous ATPase activity. ATP hydrolysis is required for the flopping since removal of ATP and/or Mg2+ inhibits the translocation of NBD-PC. Further evidence to support this conclusion is that the translocation of NBD-PC is inhibited by vanadate, which traps ATP hydrolysis product ADP in the nucleotide binding domains. In addition, the translocation of NBD-PC by proteoliposomes containing MRP1 protein is in a glutathione-dependent manner, similar to the process of translocating anticancer drugs such as daunorubicin. verapamil, vincristine, vinblastine, doxorubicin and oxidized glutathione partially inhibited the translocation of NBD-PC, whereas MK 571, an inhibitor of MRP1 protein, inhibited the translocation almost completely. Taken together, the purified and reconstituted MRP1 protein possesses the ability to flop NBD-PC from outer to inner leaflet of the proteoliposomes.

4-Chloro-7-nitrobenzofurazan↗

PubMed2004

The relationship between MRP1 activities and its NBD conformational changes.

MIANS, a sulfhydryl-reactive fluorescence, was used to label the cysteines of MRP1 (multidrug resistance protein), and the results indicated that an increase in fluorescence intensity and a large emission blue shift took place after two Cys residues of MRP1 reacted with MIANS, which demonstrated that labeled Cys residues in MRP1 reside in a relatively hydrophobic environment. The experimental results obtained from fluorescence resonance energy transfer further uncover that two Cys residues of MRP1 modified by MIANS located in the vicinity of its NBDs, of which one lies close to NBD1, and the other near NBD2. ATP, ADP and anticancer drugs can all reduce the rate of reaction of MRP1 with MIANS. The collisional quenchers, acrylamide, l-, and Cs+ were used to assess local environments of MIANS bound to MRP1 and the results showed that the region around the MIANS-labeled cysteine is positively charged. Both MIANS and NEM, which are sulfhydryl-reactive reagents, inhibited MRP1 ATPase activity, whereas anticancer drugs activated it. These results demonstrated that all nucleotides and drugs could induce changes in conformation of the NBDs in MRP1. Nucleotides can bind directly to NBDs, but drugs may react first with TMDs, which in turn alters the accessibility of the two Cys residues bound by MIANS and affects MRP1 ATPase activity, which is coupled with the transport of its substrates. Taken together, the above experimental results provide direct evidence for further study on the coupling of translocation of the transported species to hydrolysis of ATP in MRP1.

Adenosine Diphosphate↗

PubMed2003

NMR studies of the relationship between the changes of membrane lipids and the cisplatin-resistance of A549/DDP cells.

Changes of membrane lipids in cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells during the apoptotic process induced by a clinical dose of cisplatin (30 &mgr;M) were detected by 1H and 31P-NMR spectroscopy and by membrane fluidity measurement. The apoptotic phenotypes of the two cell lines were monitored with flow cytometry. The assays of apoptosis showed that significant apoptotic characteristics of the A549 cells were induced when the cells were cultured for 24 hours after treatment with cisplatin, while no apoptotic characteristic could be detected for the resistant A549/DDP cells even after 48 hours. The results of 1H-NMR spectroscopy demonstrated that the CH2/CH3 and Glu/Ct ratios of the membrane of A549 cells increased significantly, but those in A549/DDP cell membranes decreased. In addition, the Chol/CH3 and Eth/Ct ratios decreased for the former but increased for the latter cells under the same conditions. 31P-NMR spectroscopy indicated levels of phosphomonoesters (PME) and ATP decreased in A549 but increased in A549/DDP cells after being treated with cisplatin. These results were supported with the data obtained from 1H-NMR measurements. The results clearly indicated that components and properties of membrane phospholipids of the two cell lines were significantly different during the apoptotic process when they were treated with a clinical dose of cisplatin. Plasma membrane fluidity changes during cisplatin treatment as detected with the fluorescence probe TMA-DPH also indicate marked difference between the two cell lines. We provided evidence that there are significant differences in plasma membrane changes during treatment of cisplatin sensitive A549 and resistant A549/DDP cells.

Journal Article↗