Biomedical subjects

Romer Gonzalez-Villalobos

Publications and source records attributed to Romer Gonzalez-Villalobos.

3 recordsLinked to original sources

PubMed2005

Megalin binds and internalizes angiotensin-(1-7).

Megalin is a multiligand receptor heavily involved in protein endocytosis. We recently demonstrated that megalin binds and mediates internalization of ANG II. Although there is a strong structural resemblance between ANG II and ANG-(1-7), their physiological actions and their affinity for the angiotensin type 1 receptor (AT(1)R) are dissimilar. Therefore, the hypothesis of the present work was to test whether megalin binds and internalizes ANG-(1-7). The uptake of ANG-(1-7) was determined by exposure of confluent monolayers of BN/MSV cells (a model representative of the yolk sac epithelium) to fluorescently labeled ANG-(1-7) (100 nM) and measurement of the amount of cell-associated fluorescence after 4 h by flow cytometry. Anti-megalin antisera and an AT(1)R blocker (olmesartan) were used to interfere with uptake via megalin and the AT(1)R, respectively. ANG-(1-7) uptake was prevented by anti-megalin antisera (63%) to a higher degree than olmesartan (13%) (P < 0.001). In analysis by flow cytometry of binding experiments performed in brush-border membrane vesicles isolated from kidneys of CD-1 mice, anti-megalin antisera interfered with ANG-(1-7) binding more strongly than olmesartan (P < 0.05 against positive control). Interactions of megalin with ANG-(1-7) at a molecular level were studied by surface plasmon resonance, demonstrating that ANG-(1-7) binds megalin dose and time dependently and with an affinity similar to ANG II. These results show that the scavenger receptor megalin binds and internalizes ANG-(1-7).

Angiotensin I↗

PubMed2005

Calcium and chloride channel activation by angiotensin II-AT1 receptors in preglomerular vascular smooth muscle cells.

The pathways responsible for the rapid and sustained increases in [Ca(2+)](i) following activation of ANG II receptors (AT(1)) in renal vascular smooth muscle cells were evaluated using fluorescence microscopy. Resting intracellular calcium concentration [Ca(2+)](i) averaged 75 +/- 9 nM. The response to ANG II (100 nM) was characterized by a rapid initial increase of [Ca(2+)](i) by 74 +/- 6 nM (n = 35) followed by a decrease to a sustained level of 12 +/- 2 nM above baseline. The average time from peak to 50% reduction from the peak value (50% time point) was 32 +/- 4 s. AT(1) receptor blockade with 1 microM candesartan (n = 5) prevented the responses to ANG II. In nominally calcium-free conditions (n = 8), the peak increase in [Ca(2+)](i) averaged 42 +/- 7 nM but the sustained phase was absent and the 50% time point was reduced to 11 +/- 4 s. L-type calcium channel blockade with diltiazem reduced the peak [Ca(2+)](i) to 24 +/- 8 nM and the sustained level to 4 +/- 2 nM (n = 10). In cells preincubated in low Cl(-) (3.0 mM), the peak response to ANG II was suppressed as was the sustained response. Blockade of chloride channels with DIDS eliminated both the peak and sustained responses (n = 11); chloride channel blockade with DPC (n = 17) suppressed the peak increase in [Ca(2+)](i) to 18 +/- 5 and also prevented the sustained response. IP3 receptor blockade by 10 microM TMB-8 (n = 6) reduced the peak to 22 +/- 8 and prevented the sustained response. Exposure to 10 microM TMB-8 in the presence of Ca(2+)-free medium prevented the ANG II response (n = 9). In the presence of 100 microM DPC and 10 microM TMB-8 (n = 7), the ANG II response was also prevented. Thus the rapid initial increase in [Ca(2+)](i) is due not only to release from intracellular stores, but also to Ca(2+) influx from the extracellular fluid. Although Ca(2+) entry via L-type calcium channels is responsible for the major portion of the sustained response, other entry pathways participate. The finding that chloride channel blockers markedly attenuate both rapid and sustained responses indicates that chloride channel activation contributes to, rather than being the consequence of, the initial rapid response.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2004

Megalin binds and internalizes angiotensin II.

Megalin is an abundant membrane protein heavily involved in receptor-mediated endocytosis. The major functions of megalin in vivo remain incompletely defined as megalin typically faces specialized milieus such as glomerular filtrate, airways, epididymal fluid, thyroid colloid, and yolk sac fluid, which lack many of its known ligands. In the course of studies on ANG II internalization, we were surprised when only part of the uptake of labeled ANG II into immortalized yolk sac cells (BN-16 cells) was blocked by specific peptide inhibitors and direct competitors of the angiotensin type 1 receptor. This led us to test if megalin was a receptor for ANG II. Four lines of direct evidence demonstrate that megalin and, to a lesser extent, its chaperone protein cubilin are receptors for ANG II. First, in BN-16 cells anti-megalin and anti-cubilin antisera interfere with ANG II uptake. Second, also in BN-16 cells, pure ANG II competes for uptake of a known megalin ligand. Third, in proximal tubule cell brush-border membrane vesicles extracted from mice, anti-megalin antisera interfere with ANG II binding. Fourth, purified megalin binds ANG II directly in surface plasmon resonance experiments. The finding that megalin is a receptor for ANG II suggests a major new function for the megalin pathway in vivo. These results also indicate that ANG II internalization in some tissues is megalin dependent and that megalin may play a role in regulating proximal tubule ANG II levels.

Angiotensin II↗