Caveolae act as signalling platforms serving as concentrating points for numerous signalling molecules, as well as regulating flux through many distinct signalling cascades. RhoA proteins have been identified as potential actors in the pathophysiology of the cardiovascular system. We used sucrose gradient fractionation and immunoblotting to determine caveolin-1 and RhoA presence in the kidney cortex of streptozotocin-induced T1 diabetes rats (4-week duration), and of diabetic rats treated with angiotensin receptor blocker losartan (4 weeks, 20 mg/kg/day) to retard renal hypertension. Positive RhoA/caveolin-1 co-immunoprecipitation result was detected in the caveolar fraction that corresponded to the light-scattering band obtained from diabetic rats, compared to negative co-immunoprecipitation result in the caveolar fraction obtained from control rats. The detection of RhoA protein in the caveolar fractions and the prospective RhoA/caveolin-1 association can be used to examine the role of these signalling reactions in the pathophysiology of microvascular complications in type 1 diabetes
- MeSH
- antagonisté receptorů pro angiotenzin terapeutické užití MeSH
- diabetes mellitus 1. typu farmakoterapie metabolismus MeSH
- experimentální diabetes mellitus farmakoterapie metabolismus MeSH
- imunoprecipitace MeSH
- kaveolin 1 metabolismus MeSH
- kaveoly metabolismus MeSH
- krysa rodu rattus MeSH
- losartan terapeutické užití MeSH
- náhodné rozdělení MeSH
- potkani Wistar MeSH
- rhoA protein vázající GTP metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Caveolin-1 (CAV-1) is the main structural component of caveolae, acting as a modulator of signal transduction. CAV-1 might be involved in the pathophysiology of microvascular complications in Type 1 diabetes (DM). We sought to determine whether fractionation on sucrose gradient (SF), a method routinely utilized for isolation of caveolar fractions in homogenous cell lines, is applicable for CAV-1-related studies in tissues with multiple cell types, such as the normal rat kidney cortex (C). Using this method, we also determined whether streptozotocininduced DM in rats (4-week duration) leads to changes in renal subcellular targeting of CAV-1, and evaluated the effects of tight metabolic control (insulin, 12 IU/day) and angiotensin receptor blocker, losartan (4 weeks, 20 mg/kg/day). Immunoblotting of individual fractions obtained from C revealed CAV-1 expression in fractions 4-6 that corresponded to light scattering band that typically forms after separating cellular fractions on SF. These fractions were considered to be caveolar fractions. In C, CAV-1 was also detectable in fractions 8-10. These and all other fractions except caveolar fractions were considered to be noncaveolar fractions. A ratio of caveolar/non-caveolar expression of CAV-1 (CNCR) was computed for each renal cortex allowing comparisons of CAV-1 subcellular distribution in C and DM rats, and effects of treatments. Using this approach, DM was characterized by marked increases in CNCR as compared to C (5.54±1.56 vs. 2.65±1.33, p<0.05) that were reduced by treatment with insulin (0.78±0.24, p<0.01 vs. DM) or losartan (0.84±0.06, p<0.01 vs. DM). In summary, analysis of CAV-1 following the SF of renal cortex detected similar distribution of the protein as in homogenous cell lines, DM-induced changes in CAV-1 targeting, and the effects of pharmacological treatments. This suggests applicability of SF in studies focusing on CAV-1 targeting in organs with various cell lines in vivo.
- MeSH
- diabetes mellitus 1. typu metabolismus MeSH
- experimentální diabetes mellitus MeSH
- financování organizované MeSH
- imunohistochemie MeSH
- kaveolin 1 metabolismus MeSH
- kaveoly metabolismus MeSH
- krysa rodu rattus MeSH
- ledviny metabolismus MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
