Pharmacological and molecular comparison of K_ATP channels in rat basilar and middle cerebral arteries

Abstract

ATP-sensitive potassium (K_ATP) channels play an important role in the regulation of cerebral vascular tone. In vitro studies using synthetic K_ATP channel openers suggest that the pharmacological profiles differ between rat basilar arteries and rat middle cerebral arteries.

To address this issue, we studied the possible involvement of endothelial K_ATP channels by pressurized arteriography after luminal administration of synthetic K_ATP channel openers to rat basilar and middle cerebral arteries. Furthermore, we examined the mRNA and protein expression profile of K_ATP channels to rat basilar and middle cerebral arteries using quantitative real-time PCR (Polymerase Chain Reaction) and Western blotting, respectively. In the perfusion system, we found no significant responses after luminal application of three K_ATP channel openers to rat basilar and middle cerebral arteries. In contrast, abluminal application caused a concentration-dependent dilatation of both arteries, that was more potent in basilar than in middle cerebral arteries. Quantitative real-time PCR detected the presence of mRNA transcripts of the K_ATP channel subunits Kir6.1, Kir6.2, SUR1 and SUR2B, while SUR2A mRNA was barely detected in both rat basilar and middle cerebral arteries. Of the five mRNAs, the expression levels of Kir6.1 and SUR2B transcripts were predominant in both rat basilar and middle cerebral arteries. Western blotting detected the presence of Kir6.1, Kir6.2, SUR1 and SUR2B proteins in both arteries. Densitometric measurements of the Western blot signals further showed higher expression levels of Kir6.1 and SUR2B proteins in rat middle cerebral arteries than was found in rat basilar arteries. In conclusion, our in vitro pharmacological studies showed no evidence for functional endothelial K_ATP channels in either artery. Furthermore, the results indicate that Kir6.1/SUR2B is the major K_ATP channel complex in rat basilar and middle cerebral arteries.

Introduction

ATP-sensitive potassium (K_ATP) channels were first discovered in cardiac myocytes (Noma, 1983) and later found in many other tissues like pancreatic β-cells (Ashcroft et al., 1984), skeletal muscles (Spruce et al., 1985), neurons (Ashford et al., 1988), vascular smooth muscle cells (Standen et al., 1989) including cerebral blood vessels (Kleppisch and Nelson, 1995).

K_ATP channels couple intracellular metabolic changes to the electrical activity of the plasma membrane. Furthermore, the channels respond to multiple endogenous vasodilators (Nelson and Brayden, 1993) and synthetic compounds (Faraci and Sobey, 1998).

K_ATP channels are tetrameric ion channel complexes assembled by two subunits: a subunit of the inward-rectifying K⁺ channel family (Kir6.x) and a sulfonylurea receptor (SUR) subunit of the ATP-binding cassette (ABC) protein superfamily (Aguilar-Bryan and Bryan, 1999, Aguilar-Bryan et al., 1998, Clement et al., 1997, Seino and Miki, 2003).

The Kir6.x subunits are the pore-forming structures through which K⁺ ions transverse the membrane, while the SUR subunits confer different drug sensitivities to the channel complex.

Two genes each code for the two known Kir6.x subfamily members (Kir6.1 and Kir6.2) (Inagaki et al., 1995, Sakura et al., 1995) and for the two SUR members (SUR1 and SUR2) (Aguilar-Bryan et al., 1995, Inagaki et al., 1996). Alternative splicing of SUR2 generates at least two functionally relevant splice variants (SUR2A and SUR2B) with distinct pharmacological profiles (Inagaki et al., 1996, Isomoto et al., 1996, Yokoshiki et al., 1998).

K_ATP channels play an important role in the regulation of cerebral vascular tone (Faraci and Sobey, 1996, Faraci and Sobey, 1998, Kitazono et al., 1995). K_ATP channel openers and blockers have been shown to exert pharmacological action in cerebral arteries (Nelson and Quayle, 1995). Activators of K_ATP channels produce hyperpolarization and relaxation of cerebral arteries (Faraci and Sobey, 1998). Activity of these channels is inhibited by sulfonylureas like glibenclamide (Kleppisch and Nelson, 1995).

In previous in vitro pharmacological and conventional mRNA expression experiments, we suggested that the rat basilar and middle cerebral arteries were likely to be composed of Kir6.1 co-associated with SUR2B (Jansen-Olesen et al., 2005). Furthermore, we suggested that endothelial K_ATP channels may be involved in K_ATP channel opener-induced relaxation of basilar arteries, but not middle cerebral arteries. We hypothesize that rat basilar and middle cerebral arteries predominantly consist of the Kir6.1/SUR2B combination of K_ATP channels and that functional K_ATP channels exist in the endothelium of rat basilar arteries. To further study these hypotheses, we have used a perfusion model, that allow us to apply the compounds locally either luminally or abluminally. In addition, we have compared the mRNA expression profile of the K_ATP channel subunits in the two cerebral arteries by high-sensitive real-time PCR (Polymerase Chain Reaction) quantification. Finally, the expression of K_ATP channel subunits was analyzed by Western blotting to assess the expression profile at the protein level.