Biomedical subjects

Annarita Graziani

Publications and source records attributed to Annarita Graziani.

5 recordsLinked to original sources

PubMed2006

Cellular cholesterol controls TRPC3 function: evidence from a novel dominant-negative knockdown strategy.

TRPC3 (canonical transient receptor potential protein 3) has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. In the present study, we investigated the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibited a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contained a haemagglutinin epitope tag in the second extracellular loop, conferred antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation was found to increase surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. We suggest the cholesterol content of the plasma membrane as a determinant of cellular TRPC3 activity and provide evidence for cholesterol dependence of TRPC3 surface expression.

Calcium Signaling↗

PubMed2006

TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells.

Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.

Animals↗

PubMed2006

TRPC4 expression determines sensitivity of the platelet-type capacitative Ca2+ entry channel to intracellular alkalosis.

The present study was designed to analyze the molecular basis of the intracellular pH-dependent capacitative Ca2+ entry (CCE) of human platelets and megakaryocytic cells, specifically to test the hypothesis that members of the classical transient receptor potential (TRPC) protein family are involved in the CCE pathway that is promoted by intracellular alkalosis. Human platelets as well as the tested megakaryocytic cell lines (CMK cells, MEG-01 cells) and HEK293 cells displayed thapsigargin-induced CCE and responded to monensin with comparable elevation in intracellular pH. Promotion of CCE by monensin-induced intracellular alkalosis, however, was profound in mature platelets, moderate in CMK cells and lacking in MEG-01 cells as well as in HEK293 cells. Analysis of the TRPC expression pattern by immunoblotting revealed that mature platelets and CMK cells express TRPC4 along with TRPC1 and TRPC3, while TRPC4 is lacking in MEG-01 cells. HEK293 cells displayed CCE characteristics as well as lack of TRPC4 expression similar to MEG-01 cells. Over-expression of TRPC4 in HEK293 cells was found to result in a gain of pH-sensitivity of CCE with clearly detectable promotion of CCE in response to monensin. These results suggest that platelet CCE channel complexes contain TRPC4 as a molecular component that determines sensitivity of CCE to intracellular alkalosis.

Alkalosis↗

PubMed2004

Cholesterol- and caveolin-rich membrane domains are essential for phospholipase A2-dependent EDHF formation.

OBJECTIVE: Cholesterol-rich membrane domains, which contain the scaffold protein caveolin-1 (Cav-1) (caveolae), represent an important structural element involved in endothelial signal transduction. The present study was designed to investigate the role of these signaling platforms in the generation of endothelial-derived hyperpolarizing factor (EDHF). METHODS: Caveolae were disrupted by cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD 10 mM). MbetaCD-induced modulation of non-nitric oxide-/non-prostanoid-dependent (EDHF)-mediated vasorelaxation was studied in pig coronary arteries. Effects of MbetaCD on endothelial Ca(2+) signaling and phospholipase A(2) (cPLA(2)) activity were determined using fura-2 imaging and measurement of [(3)H]-arachidonate mobilization in cultured pig aortic endothelial cells (PAEC). Cellular localization of caveolin-1 and phospholipase A(2) was investigated by cell fractionation, and interaction of cPLA(2) with caveolin-1 was tested by immunoprecipitation experiments. RESULTS: MbetaCD inhibited EDHF-mediated relaxations of pig coronary arteries induced by bradykinin (100 nM) or ionomycin (300 nM) but not relaxations induced by the NO donor DEA/NO (1 microM). Exposure of arteries to cholesterol-saturated MbetaCD failed to affect EDHF-mediated relaxations. Cholesterol depletion with MbetaCD did not affect bradykinin or ionomycin-induced Ca(2+) signaling in pig aortic endothelial cells, but was associated with enhanced basal and reduced Ca(2+)-dependent release of arachidonic acid (AA). Cell fractionation experiments indicated targeting of cPLA(2) to low density, caveolin-1 rich membranes and immunoprecipitation experiments demonstrated association of phospholipase A(2) with the scaffold protein of caveolae, caveolin-1. Cholesterol depletion with MbetaCD did not disrupt the interaction between cPLA(2) and caveolin-1 but prevented targeting of cPLA(2) to low density membranes. Exogenous supplementation of arachidonic acid after cholesterol depletion partially restored EHDF responses in pig coronary arteries. CONCLUSION: The integrity of caveolin-1-containing membrane microdomains is prerequisite for arachidonic acid recruitment and EDHF signaling in porcine arteries.

Animals↗

PubMed2004

Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger.

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) entry but not TRPC3-mediated Na(+) currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na(+) after Na(+) loading with monensin resulted in significant rises in intracellular free Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca(2+) signaling.

Anti-Arrhythmia Agents↗