The G protein signaling cascade is a key player in cell signaling. Cascade activation leads to a redistribution of its members in various cellular compartments. These changes are likely related to the "second wave" of signaling from endosomes. Here, we set out to determine whether Gs signaling cascade members expressed at very low levels exhibit altered mobility and localize in clathrin-coated structures (CCSs) or caveolae upon activation by β2 -adrenergic receptors (β2 AR). Activated β2 AR showed decreased mobility and sustained accumulation in CCSs but not in caveolae. Arrestin 3 translocated to the plasma membrane after β2 AR activation and showed very low mobility and pronounced accumulation in CCSs. In contrast, Gαs and Gγ2 exhibited a modest reduction in mobility but no detectable accumulation in or exclusion from CCSs or caveolae. The effector adenylyl cyclase 5 (AC5) showed a slight mobility increase upon β2 AR stimulation, no redistribution to CCSs, and weak activation-independent accumulation in caveolae. Our findings show an overall decrease in the mobility of most activated Gs signaling cascade members and confirm that β2 AR and arrestin 3 accumulate in CCSs, while Gαs , Gγ2 and AC5 can transiently enter CCSs and caveolae but do not accumulate in and are not excluded from these domains.
Detection of patterns of subcellular calcium distribution in the cardiovascular system can contribute to understanding its role in cardiac and blood function. The present study localized calcium in heart atrium, ventricle, and bulbus arteriosus as well as in erythrocytes of zebrafish Danio rerio using an oxalate-pyroantimonate technique combined with transmission electron microscopy. Intracellular calcium stores were detected in caveolae, mitochondria, and the nuclei of several zebrafish cardiac cell types. Melanin pigmentation containing calcium stores was detected in the pericardial cavity. Melanin might be an extracellular source of calcium for heart beating and/or a lubricant to prevent friction during beating process. Calcium deposits were also detected in the plasma membrane, cytoplasm and nucleus of erythrocytes as well as in blood plasma. Possible exchange of calcium between erythrocytes and blood plasma was observed. Interactions of such calcium stores and possible contribution of extracellular calcium stores such as melanin pigmentation to supply calcium for vital functions of heart cells should be addressed in future studies.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčné jádro metabolismus MeSH
- dánio pruhované fyziologie MeSH
- erytrocyty metabolismus MeSH
- kardiomyocyty metabolismus MeSH
- kaveoly metabolismus MeSH
- melaniny metabolismus MeSH
- mitochondrie metabolismus MeSH
- srdeční komory cytologie MeSH
- srdeční síně cytologie MeSH
- transmisní elektronová mikroskopie MeSH
- vápník analýza MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- cévní endotel růst a vývoj účinky léků MeSH
- kaveoly MeSH
- lidé MeSH
- nádory * krevní zásobení terapie MeSH
- nanočástice * škodlivé účinky využití MeSH
- nosiče léků MeSH
- systémy cílené aplikace léků * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- novinové články MeSH
Airway smooth muscle (ASM) membrane depolarization through KCl opens L-type voltage dependent Ca2+ channels (Ca(v)1.2); its opening was considered the cause of KCl contraction. This substance is used to bypass intracellular second messenger pathways. It is now clear that KCl also activates RhoA/Rho kinase (ROCK) pathway. ROCK isoforms are characterized as ROCK1 and ROCK2. Because ROCK1 seems the most abundant isotype in lung, we studied its participation in KCl stimulated bovine ASM. With methyl-beta-cyclodextrin (MbetaCD) we disrupted caveolae, a membrane compartment considered as the RhoA/ROCK assembly site, and found that KCl contraction was reduced to the same extent (~26%) as Y-27632 (ROCK inhibitor) treated tissues. We confirmed that KCl induces ROCK activation and this effect was annulled by Y-27632 or MbetaCD. In isolated plasmalemma, ROCK1 was localized in non-caveolar membrane fractions in Western blots from control tissues, but it transferred to caveolae in samples from tissues stimulated with KCl. Ca(v)1.2 was found at the non-caveolar membrane fractions in control and MbetaCD treated tissues. In MbetaCD treated tissues stimulated with KCl, contraction was abolished by nifedipine; only the response to Ca(v)1.2 opening remained as the ROCK component disappeared. Our results show that, in ASM, the KCl contraction involves the translocation of ROCK1 from non-caveolar to caveolar regions and that the proper physiological response depends on this translocation.
- MeSH
- centrálně působící myorelaxancia farmakologie MeSH
- chlorid draselný farmakologie MeSH
- hladké svalstvo účinky léků metabolismus MeSH
- kaveoly účinky léků metabolismus MeSH
- kinázy asociované s rho metabolismus MeSH
- orgánové kultury - kultivační techniky MeSH
- skot MeSH
- svalová kontrakce účinky léků fyziologie MeSH
- trachea účinky léků metabolismus MeSH
- transport proteinů účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
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
- MeSH
- beta-1-adrenergní receptory fyziologie MeSH
- finanční podpora výzkumu jako téma MeSH
- heterotrimerní G-proteiny agonisté chemie metabolismus MeSH
- kaveoly chemie MeSH
- signální transdukce MeSH
- sodíko-draslíková ATPasa fyziologie chemie MeSH
- srdeční glykosidy farmakologie MeSH
- zpětná vazba fyziologická fyziologie MeSH
- Publikační typ
- přehledy MeSH
