Cell-specific expression and function of IKCa channels in cerebral endothelial cells but not smooth muscle cells: Relevance to the mechanism of EDHF-mediated dilation

Background

We recently hypothesized that EDHF-mediated dilations in cerebral arteries and arterioles involves hyperpolarization of the endothelium by activation of intermediate-conductance Ca2+-sensitive K channels (IKCa) as a critical initiating step. Whole vessel studies are consistent with this hypothesis in the rat middle cerebral artery (MCA). However, the specific cell type(s) that express functionally relevant IKCa channels remains to be demonstrated. Here we provide direct evidence for the cell-specific location of IKCa channels within rat cerebral arteries.

Methods

Freshly dispersed smooth muscle or endothelial cells (10 to 20 cells) were harvested and evaluated by RT-PCR. Two rounds of PCR with nested primers were used to determine cell-specific expression of eNOS, SM22α, GAPDH, and IKCa. The initial assignment of cell type was based upon morphological criteria at the time of cell harvesting. Confirmation of this assignment was based upon the expression (or lack of expression) of cell specific markers. Protein was evaluated by Western from whole vessel preparations and by immunofluorescence in freshly dispersed cells. KCa channel function (IKCa and BKCa) was evaluated by whole-cell patch clamp in freshly dispersed endothelial and smooth muscle cells. Cultured cells were not used in these studies given that KCa channel expression has been shown to be significantly altered by cell culture.

Results

In general, cells that were initially identified as endothelial expressed eNOS but not SM22α, whereas the reverse was true for cells initially identified as smooth muscle. Results were discarded from samples in which GAPDH was not expressed. IKCa message was found in endothelial cells but not in smooth muscle cells. Western analysis demonstrated IKCa immunoreactivity at the predicted size from MCA protein extracts. The cell-specific origin of the immunoreactivity was assessed in freshly dispersed MCA cells by immunofluorescence. Anti-IKCa immunofluorescence appeared on clusters of small rounded cells that were identified as endothelium in separate experiments. Whole-cell voltage clamp recordings from endothelial cells demonstrated Ca2+-sensitive outward current that was blocked by TRAM-34, a selective IKCa channel blocker. Smooth muscle cells did not demonstrate TRAM-34 sensitive current, although they did possess significant current that was sensitive to BKCa channel blockers, iberiotoxin (IbTx 0.1 ìM) or tetraethylammonium (TEA 2 mM).

Conclusions

We conclude that IKCa channels are present and functionally relevant in the endothelium (but not smooth muscle) within cerebral arteries. This cell-specific location of IKCa channels is consistent with the hypothesis that activation of endothelial IKCa channels is 1) capable of hyperpolarizing the endothelium and 2) critical to the mechanism of EDHF-dependent dilation of cerebral arteries.