Tissue specific membrane association of α1T, a truncated form of the α1 subunit of the Na pump

AbstractWe have assessed the Na pump α-subunit isoform content utilizing site directed antibodies in two vascular smooth muscle (VSM) preparations known to contain functional Na pump sites, VSM microsomal fractions (Na+,K+-ATPase) and intact primary confluent cells (ouabain inhibited 86Rb uptake). A comparison of isoform content was made with kidney microsomes. Both VSM and kidney microsomes contained a full length α1 subunit (~100 kDa) as well as a truncated subunit, α1T (~66 kDa). SDS treatment of VSM microsomes effected an increase in Na+,K+-ATPase and a retention of α1T. SDS treated kidney microsomes retained the α1 isoform and Na+,K+-ATPase. Confluent VSM cells showed no detectable α1T, only α1T. In the absence of detectable full length α1, the α1T protein may represent a functional Na pump component in canine VSM

Activation of RelA homodimers by tumour necrosis factor α: a possible transcriptional activator in human vascular endothelial cells

In vascular endothelial cells, cytokines induce genes that are expressed in inflammatory lesions partly through the activation of transcription factor NF-κB (nuclear factor-κB). Among the members of the NF-κB/rel protein family, homodimers of the RelA subunit of NF-κB can also function as strong transactivators when expressed in cells. However, the functional role of endogenous RelA homodimers has not been clearly elucidated. We investigated whether RelA homodimers are induced in cytokine-treated vascular endothelial cells. Gel mobility-shift and supershift assays revealed that a cytokine TNFα (tumour necrosis factor α) activated both NF-κB1/RelA heterodimers and RelA homodimers that bound to a canonical κB sequence, IgκB (immunoglobulin κB), in SV40 (simian virus 40) immortalized HMEC-1 (human dermal microvascular endothelial cell line 1). In HMEC-1 and HUVEC (human umbilical-vein endothelial cells), TNFα also induced RelA homodimers that bound to the sequence 65-2κB, which specifically binds to RelA homodimers but not to NF-κB1/RelA heterodimers in vitro. Deoxycholic acid, a detergent that can dissociate the NF-κB–IκB complex (where IκB stands for inhibitory κB), induced the binding of the RelA homodimers to 65-2κB from the cytosolic fraction of resting HMEC-1. Furthermore, TNFα induced the transcriptional activity of a reporter gene that was driven by 65-2κB in HMEC-1. These results suggest that in addition to NF-κB1/RelA heterodimers, TNFα also induces RelA homodimers that are functionally active. Thus RelA homodimers may actively participate in cytokine regulation of gene expression in human vascular endothelial cells