Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: possible role in glucose sensing

Abstract

ATP-sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose-sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT-1 and GLUT-3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia-like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT-1 and GLUT-3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT-1 and GLUT-3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes.

Fig. 1

Protein and mRNA levels of K(ATP) channel subunits expressed in human chondrocytes. A: Protein content of each Kir6 and SUR subunit in whole cell extracts from human chondrocytes exposed to high glucose (HG) (30 mM) for 18 h or left untreated (control). The protein content of each subunit was evaluated by Western blotting and normalized to the respective β-tubulin band. B: Western blot images representative of the protein content of each Kir6 and SUR subunit expressed in human chondrocytes according to OA grade, as defined in Materials and Methods Section. MW, molecular weight protein marker. C: mRNA content of each Kir6 and SUR subunit in human chondrocytes exposed to high glucose (HG) (30 mM) for 18 h or left untreated (control). The mRNA content of each subunit was evaluated by qRT-PCR and normalized to the respective 18S rRNA content.

Fig. 2

In situ immunofluorescence staining of K(ATP) channel subunits in normal (OA grades 0–1) human cartilage. Each section was stained with an antibody specific for the subunit (green) identified in the upper right corner of the representative image and counterstained with DAPI (blue) to allow visualization of nuclei. Negative controls (column on the right) were obtained by omitting the primary antibody for each subunit. Cartilage sections were viewed and images captured at 100× and 400× (insets) magnifications.

Fig. 3

Regulation of total GLUT-1 and GLUT-3 protein content by glibenclamide in chondrocytes from different OA grades. A: Relative GLUT-1 protein levels normalized to the respective β-tubulin band, in whole cell extracts from human chondrocytes of OA grades 0–1 (n = 7), 2–3 (n = 7), and 4 (n = 6) treated with the indicated concentrations of glibenclamide for 18 h. B: Relative GLUT-3 protein levels normalized to the respective β-tubulin band, in whole cell extracts from human chondrocytes of OA grades 0–1 (n = 3), 2–3 (n = 5), and 4 (n = 6) treated with the indicated concentrations of glibenclamide for 18 h. Results are expressed in percentage relative to control untreated cells of the same OA grade ***P < 0.005; **P < 0.01; *P < 0.05 relative to the respective control cells. MW, molecular weight protein marker. C: GLUT-1 and -3 mRNA levels in chondrocytes grades 0–1 treated with 10 nM glibenclamide for the time periods indicated, relative to untreated chondrocytes of the same OA grade.