The role of inhibition of the
CPT enzymes responsible for accumulation of long chain acylcarnitines (LCAC) during
hypoxia in the proximal tubule has not been previously examined. We have characterized
CPT enzyme activities in mitochondrial fractions of rabbit proximal tubules.
Malonyl CoA-sensitive
CPT I activity (1.1 +/- 0.3 nmol/min/mg
protein), and
detergent-solubilized,
malonyl CoA-insensitive
CPT II activity (2.3 +/- 0.4 nmol/min/mg
protein) were readily detected in proximal tubule mitochondrial fractions. Subjecting rabbit proximal tubules to various periods of
hypoxia did not significantly change mitochondrial
CPT I or
CPT II activities. Thirty minutes of
hypoxia resulted in an increase in
lysophospholipid mass from 440 +/- 105 to 720 +/- 93 pmol/mg
protein, N = 5, LCAC mass from 79 +/- 11 to 618 +/- 34 pmol/mg
protein, N = 5, and
lactate dehydrogenase (LDH) release from 9 +/- 1% to 46 +/- 3%, N = 8. Pretreatment of proximal tubules with two different
CPT inhibitors,
glybenclamide (Glyb) 400 microM and
oxfenicine (Oxfe) 1 mM, resulted in reduction in the magnitude of
hypoxia-induced
lysophospholipid formation 490 +/- 160 (Glyb), 342 +/- 150 pmol/mg
protein (Oxfe), N = 4,
hypoxia-induced LCAC formation 295 +/- 27 (Glyb), 128 +/- 16 pmol/mg
protein (Oxfe). N = 5, and LDH release 25 +/- 1% (Glyb) and 19 +/- 2% (Oxfe), N = 8. The protective effect of
CPT inhibition was also associated with increased production of
lactate suggesting the modulation of a substrate-mediated metabolic switch. Immunoblots demonstrated that
hypoxia caused a time dependent hydrolysis of
fodrin-alpha subunit and that
CPT inhibition protected against
hypoxia-induced
fodrin proteolysis. These data suggest a unifying hypothesis that links
phospholipase A2 (PLA2) activation, and
hypoxia-mediated
fodrin proteolysis to the proximal tubule mitochondrial
CPT system. I propose that
CPT inhibition may represent a novel mechanism to ameliorate proximal tubule cell death during
hypoxia.