Molecular investigations of BKCa channels and the modulatory β-subunits in porcine basilar and middle cerebral arteries
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Large conductance calcium-activated potassium (BKCa) channels are fundamental in the regulation of cerebral vascular basal tone. We investigated the expression of the mRNA transcripts for the BKCa channel and its modulatory β-subunits (β1–β4) in porcine basilar and middle cerebral arteries using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Western blotting was used to detect immunoreactivity for the porcine BKCa channel α-subunit and β-subunit proteins. The BKCa channel α-subunit RNA and protein distribution patterns were visualized using in situ hybridization and immunofluorescence studies, respectively. The study verified that the BKCa channel α-subunit is located to smooth muscle cells of porcine basilar and middle cerebral arteries. The mRNA transcript for β1-, β2- and β4-subunit were shown by RT-PCR in porcine basilar and middle cerebral arteries. However, at the protein level, only, the β1-subunit protein was found by western blotting.
KeywordsBKCa channels Porcine cerebral arteries RT-PCR Western blotting In situ hybridization Immunofluorescence
The BKCa antibody was kindly provided by Hans-Günther Knaus, Innsbruck, Austria. We thank Dorthe Meinertz and Birgit Poulsen for their technical assistance. This study was supported by the Lundbeck Foundation as part of the Lundbeck Foundation Center for Neurovascular Signaling (LUCENS), Danish Research Council, Region of Copenhagen, Kong Christian den tiende Foundation, Aase og Ejnar Danielsens Foundation, Foundation of 17.12.1981, Illum Foundation, Augustinus Foundation, Lippmann Foundation, Foundation of Lægevidenskabens Fremme, Brødrene Hartmanns Foundation, Dansk Horton Hovedpineforenings legat, Harboefoundation, Familien Hede Nielsens Foundation and Toyota-Fonden, Denmark.
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