Estrogen (E2) protects against cardiovascular diseases, including coronary artery disease and stroke, in part, by regulating vascular endothelial functions through the mediation of its receptors. Two subtypes of estrogen receptor (ER), ERa and ERb, have been identified. It is generally accepted that the ER is located either predominately or exclusively in the nucleus in both ligand-free and ligand-bound states.

A growing body of evidence suggests that E2 can also evoke rapid, transcription-independent responses in the vascular endothelium. These responses are receptor mediated since they can be blocked by a specific ERa antagonist. Membrane impeded ligands, such as bovine serum albumin (BSA)-conjugated E2, can also elicit similar effects in these cells, leading to the proposal that a membrane form of the ER acts as the mediator. In addition recent work has shown, using cellular fractionation and immunoblots, that only those ERa which are associated with the caveolae can rapidly activate the endothelial nitric oxide synthase.

We have examined the three dimensional (3D) distribution of ERa in rat coronary and cerebral artery endothelia. To map the position of ERs in these cells, fixed en face vascular tissues were dual labeled with site-directed ERa antibodies and the nuclear stain DAPI, or with ERa and caveolin-1 antibodies.

A novel ERa isoform
Western blots of rat septal or basilar lysate, ovarian or uterine homogensate, and recombinant human ER proteins show a 55kDa band. This band is unlikely to be any previously reported isoform because the N-terminal,hinge and C-terminal epitopes are intact.

Distribution of N-terminal anti-ERa
Distribution of N-terminal anti-ERa (ER21) in septal artery endothelial cell shows the classic nuclear distribution.

Distribution of hinge anti-ERa
The distribution of hinge anti-ERa (SRA-1010) and C-terminal anti-ERa (below) is primarily extra-nuclear in these cells. This distribution was cell-dependent: An extranuclear distribution was found in human ovarian surface epithelial cells whereas in rat smooth muscle myometrial cells its distribution was intra-nuclear

Distribution of C-Terminal anti-ERa
The distribution of C-Terminal anti-ERa (SRA-1000) in septal artery endothelia is almost identical to that of the hinge antibody. This antibody also produced extranuclear labeling in human OSE cells.

Colocalisation between ERa and Caveolin-1
Caveolin-1 localises in the caveolae and marks the exterior of the endothelial cell. The stereo pair of a cell labeled with SRA-1010 for ERa and caveolin-1 shows that many (35%) of the estrogen receptors external to the nucleus colocalise with the caveolae. These receptors are potential membrane estrogen receptors.

Little or no colocalisation between ERa and ERb
The stereo pair of a cell labeled with ER21 for ERa and CO1531 for ERb shows few colocalised voxels (white), indicating that in these cells there is little chance of heterodimerisation.

Similar results to the above were obtained from endothelial cells from rat basilar arteries.

More detailed information can be found in:
Dan P, Cheung JC, Scriven DRL, and Moore ED
Epitope Dependent Localization of Estrogen Receptor a, but not b, in En Face Arterial Endothelium. Am J Physiol Heart Circ Physiol

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