Angiotensin II (Ang II) is a potent vasopressor peptide that interacts with 2 major receptor isoforms -AT1 and AT2. Although blood pressure is increased in AT2 knockout mice, the underlying mechanisms remain undefined because of the low levels of expression of AT2 in the vasculature. Here we overexpressed AT2 in vascular smooth muscle (VSM) cells in transgenic (TG) mice. Aortic AT1 was not affected by overexpression of AT2. Chronic infusion of Ang II into AT2-TG mice completely abolished the AT1-mediated pressor effect, which was blocked by inhibitors of bradykinin type 2 receptor (icatibant) and nitric oxide (NO) synthase (L-NAME). Aortic explants from TG mice showed greatly increased cGMP production and diminished Ang II-induced vascular constriction. Removal of endothelium or treatment with icatibant and L-NAME abolished these AT2-mediated effects. AT2 blocked the amiloride-sensitive Na + /H + exchanger, promoting intracellular acidosis in VSM cells and activating kininogenases. The resulting enhancement of aortic kinin formation in TG mice was not affected by removal of endothelium. Our results suggest that AT2 in aortic VSM cells stimulates the production of bradykinin, which stimulates the NO/cGMP system in a paracrine manner to promote vasodilation. Selective stimulation of AT2 in the presence of AT1 antagonists is predicted to have a beneficial clinical effect in controlling blood pressure.

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein-coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II-induced ERK activation. Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca2+ ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II-treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II-induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-beta receptor was not phosphorylated on Ang II stimulation, and Ang II-induced c-jun gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II-induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca2+/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.

Morphological changes of liposomes caused by interactions between liposomal membranes and talin, a cytoskeletal submembranous protein, were studied by direct, real-time observation by using high-intensity dark-field microscopy. Surprisingly, when talin was added to a liposome solution, liposomes opened stable holes and were transformed into cup-shaped liposomes. The holes became larger with increasing talin concentration, and finally the cup-shaped liposomes were transformed into lipid bilayer sheets. These morphological changes were reversed by protein dilution, i.e., the sheets could be transformed back into closed spherical liposomes. We demonstrated that talin was localized mainly along the membrane verges, presumably avoiding exposure of its hydrophobic portion at the edge of the lipid bilayer. This is the first demonstration that a lipid bilayer can stably maintain a free verge in aqueous solution. This finding refutes the established dogma that all lipid bilayer membranes inevitably form closed vesicles and suggests that talin is a useful tool for manipulating liposomes.Phospholipids spontaneously assemble into bilayer membranes in aqueous solution and necessarily form liposomes, which are closed-membrane vesicles (1). Liposomes often have been studied as simplified models of biological membranes (2-5) and are now used as such in a number of applications from pharmacology to bioengineering (6), for example, as carriers of DNA vectors or anticancer drugs for internal deliveries. However, studies of interaction mechanisms between liposome membranes and biological components, such as DNA or protein, are now still in progress (5,7,8), and the dynamic behavior of such complexes in solution has remained unclear. Therefore, real-time approaches by using optical microscopy to study the dynamic behavior of liposomes resulting from interactions between liposomal membranes and biological elements are very important.Liposomes can be visualized with several types of optical microscopes. In this study, we used high-intensity dark-field microscopy (9-11), because dark-field microscopy is the best way to visualize the intact three-dimensional morphology and the dynamic behavior of individual lamellar liposomes in solution, and only this type of microscopy provides real-time, high-contrast images. In practice, other types of high-contrast microscopes, such as phase contrast or differential interference, still yield poor contrast for individual lamellar liposomes.In this study, we investigated morphological changes of liposomes caused by talin. Talin is an actin-binding, peripheralmembrane protein that localizes at focal contacts in cells and that links actin filaments with plasma membranes through integrin (12-15). It has also been reported that talin can bind to membranes directly and can promote actin polymerization (16-18). MATERIALS AND METHODSPreparation and Observation of Liposomes. Liposomes were prepared as described previously (9-11). Lipid films were generated by dissolving phospholipids in a chloroform͞...

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Effects of moderate nutrient enrichment (NO { 3 : ,5 mmol L 21 , PO 3{ 4 : ,0.3 mmol L 21 ) on two carbon (C) fixation rates (photosynthesis and calcification) of the zooxanthellate coral Acropora pulchra were investigated under laboratory conditions. The coral branches were incubated in the nutrient condition for three different periods (0, 5, 10 d) to observe changes in tissue biomass and zooxanthellate chlorophyll a (Chl a) concentration. Next, the incubated corals were simultaneously transferred to nutrient-depleted seawater containing 13 C-labeled dissolved inorganic carbon to assay net photosynthesis and calcification rates. Chl a concentration per unit surface area increased 2.6-fold for the 10-d enrichment, and net photosynthetic rates were also stimulated up to a similar level (2.8-fold). Tissue biomass of the host coral and zooxanthellae was approximately doubled during the period. On the other hand, calcification rates only increased 1.3-fold, suggesting that even moderate nutrient loading resulted in one-sided enhancement of the algal photosynthetic activity. The measured C fixation ratios of organic C : skeletal C were higher than the structural ratios, and the inconsistency became greater as Chl a concentration increased. The increased photosynthetic products could be excessively stored in the organic tissue and/or released into the ambient seawater.

Symbioses between microalgae and animal hosts have the advantage of acquiring and sharing autotrophically produced organic carbon (C) as their energy source. However, the stoichiometry and turnover rates of biological elements in symbioses are not fully understood because of complicated metabolic interactions. We report the first comprehensive and simultaneous measurement of C and nitrogen (N) flows through coral-dinoflagellate symbiosis by using the unique approach of dual-isotope labeling with C andN, in situ chasing, and isotope-mixing models. The coral autotrophy occurred with much lower C:N ratios than previously thought, and the autotrophically produced N-rich organic matter was efficiently transferred to the animal host through two different pathways. In contrast to the dynamic N cycles within the symbiosis, the N uptake from the ambient seawater was extremely limited, which enabled the coral symbiosis to sustain N with a long turnover time (1 year). These findings suggest that coral endosymbionts are not under N limitation but are actively producing organic N and driving microscale N cycles in the reef ecosystem. The present techniques could be applied to further quantify the C and N cycles in other symbiotic interactions and reveal their ecological advantages.

All studies analyzing the localization of angiotensin II (Ang II) receptors in the human kidney have been performed at the protein level using ¹²⁵I-Ang II as a probe. In this study, cellular localizations of Ang II type l (AT1-R) and type 2 (AT2-R) receptor mRNAs in the adult human renal cortex were examined for the first time using in situ hybridization, and their expression patterns determined by RNase protection assay were compared with those in other human tissues. In the human renal cortex obtained from tumor-free portions in renal cell carcinoma, AT1-R mRNA levels were about 8- to 10-fold higher than AT2-R mRNA levels. Human liver and aorta predominantly expressed AT1-R mRNA, while human right atrium contained both AT1-R and AT2-R mRNAs. Ligand-binding assays revealed that the total Ang II receptor number in the human renal cortex was 16.0 ± 3.3 fmol/mg protein, similar to that in liver (17.7 ± 5.8) but significantly higher than in right atrium (11.6 ± 3.2) and aorta (5.6 ± 2.7). Relative distribution ratios of AT1-R and AT2-R numbers in the renal cortex and right atrium were 82/17 and 56/42%, respectively. In situ hybridization study indicated that strongest AT1-R mRNA signals were located in interlobular arteries and tubulointerstitial fibrous regions surrounding interlobular arteries and glomeruli, followed in decreasing order by glomeruli and cortical tubules. Expression of AT2-R mRNA was highly localized in interlobular arteries. Cells present in tubulointerstitial regions were positive for vimentin and collagen type 1, indicating that the majority of the cells present in the regions are fibroblasts. Presence of strong AT1-R mRNA signals in the tubulointerstitial fibrous regions surrounding arteries and glomeruli and the expression of AT2-R mRNA in the interlobular artery were the first evidence, suggesting a pharmacological framework for the differential effects of Ang II receptor subtype mediated renal function in the adult human kidney.

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