Metabolic organization in vascular smooth muscle: distribution and localization of caveolin-1 and phosphofructokinase

Abstract

We have shown that a compartmentation of glycolysis and gluconeogenesis exists in vascular smooth muscle (VSM) and that an intact plasma membrane is essential for compartmentation. Previously, we observed that disruption of the caveolae inhibited glycolysis but stimulated gluconeogenesis, suggesting a link between caveolae and glycolysis. We hypothesized that glycolytic enzymes specifically localize to caveolae. We used confocal microscopy to determine the localization of caveolin-1 (CAV-1) and phosphofructokinase (PFK) in freshly isolated VSM cells and cultured A7r5 cells. Freshly isolated cells exhibited a peripheral (membrane) localization of CAV-1 with 85.3% overlap with PFK. However, only 59.9% of PFK was localized with CAV-1, indicating a wider distribution of PFK than CAV-1. A7r5 cells exhibited compartmentation of glycolysis and gluconeogenesis and displayed two apparent phenotypes distinguishable by shape (spindle and ovoid shaped). In both phenotypes, CAV-1 fluorescence overlapped with PFK fluorescence (83.1 and 81.5%, respectively). However, the overlap of PFK with CAV-1 was lower in the ovoid-shaped (35.9%) than the spindle-shaped cells (53.7%). There was also a progressive shift in pattern of colocalization from primarily the membrane in spindle-shaped cells (both freshly isolated and cultured cells) to primarily the cytoplasm in ovoid-shaped cells. Overall, cellular colocalization of PFK with CAV-1 was significant in all cell types (0.68 ≥ R2 ≤ 0.77). Coimmunoprecipitation of PFK with CAV-1 further validated the possible interaction between the proteins. We conclude that a similar distribution of one pool of PFK with CAV-1 contributes to the compartmentation of glycolysis from gluconeogenesis.

Expression of caveolin‐1 in lymphocytes induces caveolae formation and recruitment of phosphofructokinase to the plasma membrane

“…The demonstration that CAV-1 functions as a scaffolding protein for the membrane recruitment of PFK is a very important step in elucidating the physical basis for glycolytic-membrane interactions and their role in the organization of carbohydrate metabolism as observed in our previous studies on VSM (59). Moreover, our studies are consistent with studies that have demonstrated that CAV-1 coimmunoprecipitates with PFK-M from 293T cells transiently overexpressing V5-tagged PFK-M (34).…”

“…In vascular smooth muscle, a compartmentation of glycolytic metabolism has been well established, and the role of caveolae in the compartmentation has been proposed (1). We have found previously that PFK appeared to colocalize with a fairly ubiquitous plasma membrane protein named caveolin-1 (CAV-1), consistent with a role for CAV-1 as an anchor for glycolysis to the plasma membrane (59). However, colocalization with confocal microscopy simply implies that two proteins are in close proximity.…”

Role of caveolae in ouabain-induced proliferation of cultured vascular smooth muscle cells of the synthetic phenotype

“…These recent studies have suggested that the mechanism of ouabain activation occurred through protein-protein interactions that had been previously shown for cardiac muscle and kidney epithelial cells (17,27,47). However, the cascade activates an hypertrophic pathway in cardiomyocytes and a proliferative pathway in the kidney cells.…”

“…The compound ␤-methyl-cyclodextrin has been shown to deplete membrane cholesterol (14,47). Thus if treatment of VSMCs would prevent the specific ouabain activation of ERK1/2, this would certainly lend credence to the suggestion that ouabain is activating the cascade via an interaction with a specific population of ␣ 1 -subunits.…”

Role for SUR2A in Coupling Cardiac K_ATP Channels to Caveolin-3

“…K ATP channel microenvironment has been considered to be different from "bulk" cytosol as these channels exist in multimolecular complexes with [34][35][36][37][38] and are functionally regulated by many metabolic enzymes [39,40]. Glycolytic enzymes are specifically localized to caveolae [41]. It's been suggested that subsarcolemmal ATP may play an important role in activation of K ATP channels at relative high intracellular ATP [42].…”