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Christopher D Hardin

Publications and source records attributed to Christopher D Hardin.

7 recordsLinked to original sources
PubMed2006

Overexpression of caveolin-1 results in increased plasma membrane targeting of glycolytic enzymes: the structural basis for a membrane associated metabolic compartment.

Although membrane-associated glycolysis has been observed in a variety of cell types, the mechanism of localization of glycolytic enzymes to the plasma membrane is not known. We hypothesized that caveolin-1 (CAV-1) serves as a scaffolding protein for glycolytic enzymes and may play a role in the organization of cell metabolism. To test this hypothesis, we over-expressed CAV-1 in cultured A7r5 (rat aorta vascular smooth muscle; VSM) cells. Confocal immunofluorescence microscopy was used to study the distribution of phosphofructokinase (PFK) and CAV-1 in the transfected cells. Areas of interest (AOI) were analyzed in a central Z-plane across the cell transversing the perinuclear region. To quantify any shift in PFK localization resulting from CAV-1 over-expression, we calculated a periphery to center (PC) index by taking the average of the two outer AOIs from each membrane region and dividing by the central one or two AOIs. We found the PC index to be 1.92 +/- 0.57 (mean +/- SEM, N = 8) for transfected cells and 0.59 +/- 0.05 (mean +/- SEM, N = 11) for control cells. Colocalization analysis demonstrated that the percentage of PFK associated with CAV-1 increased in transfected cells compared to control cells. The localization of aldolase (ALD) was also shifted towards the plasma membrane (and colocalized with PFK) in CAV-1 over-expressing cells. These results demonstrate that CAV-1 creates binding sites for PFK and ALD that may be of higher affinity than those binding sites localized in the cytoplasm. We conclude that CAV-1 functions as a scaffolding protein for PFK, ALD and perhaps other glycolytic enzymes, either through direct interaction or accessory proteins, thus contributing to compartmented metabolism in vascular smooth muscle.

Animals↗
PubMed2006

Caveolins in vascular smooth muscle: form organizing function.

Caveolae are becoming increasingly recognized as an important organizational structure for a variety of signal and energy-transducing systems in vascular smooth muscle (VSM). In this review, we discuss the emerging role of the caveolins in organizing and modulating the basic functions of smooth muscle: contraction, growth/proliferation, and the energetic support systems that support these functions. With clear alterations in cell metabolism and function in VSM with altered caveolin-1 (Cav-1) protein expression and with cardiovascular abnormalities associated with Cav-1 null mice, the caveolin family of proteins may play an important role in the function and dysfunction of VSM.

Animals↗
PubMed2005

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

Compartmentation of carbohydrate metabolism has been shown in a wide range of tissues including reports of one compartment of glycolysis associated with the plasma membrane of cells. However, only in the erythrocyte has the physical basis for plasma membrane-associated glycolytic pathway been established. We have previously found that phosphofructokinase (PFK) appeared to colocalize with the fairly ubiquitous plasma membrane protein caveolin-1 (CAV-1), consistent with a role for CAV-1 as an anchor for glycolysis to the plasma membrane. To test the hypothesis that CAV-1 functions as a scaffolding protein for PFK, we transfected human lymphocytes (a cell without CAV-1 expression) with human CAV-1 cDNA. We demonstrate that expression of CAV-1 in lymphocytes results in the formation of caveolae at the plasma membrane and affects the subcellular localization of PFK by recruiting PFK to the plasma membrane. Targeting of PFK by CAV-1 also was validated by the significant colocalization between the proteins after transfection, which resulted in a correlation of 0.97 +/- 0.004 between the two fluorophores. This finding is significant in as much as it illustrates the CAV-1 feasibility of generating binding sites for glycolytic enzymes on the plasma membrane. We therefore conclude that CAV-1 functions as a scaffolding protein for PFK and that this may contribute to the elucidation of the basis for carbohydrate compartmentation to the plasma membrane in a wide variety of cell types.

Caveolae↗
PubMed2004

Caveolin-1 functions as a scaffolding protein for phosphofructokinase in the metabolic organization of vascular smooth muscle.

Using confocal microscopy, we have demonstrated a similar distribution of phosphofructokinase (PFK) with caveolin-1 (CAV-1) mainly at the periphery (membrane) in freshly isolated vascular smooth muscle (VSM) cells and in cultured A7r5 VSM cells. Co-immunoprecipitation analysis validated the interaction between the proteins. To further test the hypothesis that PFK and CAV-1 are colocalized, we used small interfering RNA (siRNA) to downregulate CAV-1 expression and disrupt the protein-protein interactions between PFK and CAV-1. Transfection of cultured A7r5 cells with CAV-1 siRNA resulted in a decreased level of immunoreactive CAV-1 and a consequent shift in the distribution of PFK with less localization of PFK to the periphery of the cells and increased immunoreactivity at the perinuclear region as compared to control. Analysis of the average PFK intensity across cultured A7r5 cells demonstrated a higher central:peripheral intensity ratio (CPI ratio) in siRNA-treated cells than in the control. These results validate the possible role of CAV-1 as a scaffolding protein for PFK as evidenced by the significant redistribution of PFK after CAV-1 downregulation. We therefore conclude that CAV-1 may function as a scaffolding protein for PFK and that this contributes to the compartmentation of glycolysis from other metabolic pathways in VSM.

Animals↗
PubMed2003

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

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 > or = R2 < or = 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.

Animals↗
PubMed2003

Mitochondrial oxidative substrate selection in porcine bladder smooth muscle.

PURPOSE: Alterations in bladder smooth muscle (BSM) metabolism due to alterations in plasma lipid levels may be important with the increasingly high fat diets eaten by most Americans. To determine the susceptibility of BSM to lipotoxicity we examined the normal pattern of mitochondrial substrate selection in BSM and the ability of BSM to respond to changes in metabolic substrate provision. MATERIALS AND METHODS: BSM strips were incubated in 5 mM 1-13C-glucose and 0 to 5 mM 1,2-13C-acetate. The pattern of substrate use measured by 13C-nuclear magnetic resonance using BSM extracts. BSM was also cultured for 4 days to elicit changes in cell phenotype. RESULTS: At physiological levels of glucose and acetate about 50% of the substrate used by mitochondria was glucose. When acetate concentration was changed from physiological levels (0.1 mM) to pathophysiological levels (0.5 mM), BSM was able to increase the use of acetate, while sparing the use of glucose and intracellular substrates, likely lipids. Above 0.5 mM acetate BSM was unable to further use acetate. With increasing acetate use anaplerosis increased, consistent with a depletion of tricarboxylic acid cycle intermediates. After 4 days of organ culture BSM mitochondria used significantly more unlabeled intracellular substrates and less 13C labeled glucose than control bladder, consistent with metabolic adaptation to increase lipid use, such as what occurs with hyperlipidemia. CONCLUSIONS: We conclude that BSM has modest plasticity of the pattern of mitochondrial substrate selection and excess lipid provision may be able to induce lipotoxicity in BSM, resulting in impaired detrusor function.

Acetates↗