The mechanism by which cyclopiazonic acid potentiates accumulation of tetraphenylphosphonium in cultured renal epithelial cells

Cyclopiazonic acid (CPA), a fungal metabolite produced by Aspergillus and Penicillium, potentiated the accumulation of the quaternary cation tetraphenylphosphonium (TPP+) in cultured pig renal epithelial cells. This is the first report of a natural product mediating the tight and apparently nonsaturable binding of a membrane potential probe to subcellular compartments. The potentiated TPP+ accumulation was dose dependent, nonsaturable, and not a result of hyperpolarization across the plasma membrane. Cyclopiazonic acid-potentiated accumulation was completely inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP). Dinitrophenol (DNP), tetrahexylammonium (THA), and n-ethylmaleimide (NEM) were also effective inhibitors of CPA-potentiated TPP+ accumulation. Although CPA-potentiated TPP+ uptake appeared to be energy dependent, TPP+ efflux (in the presence of CCCP) from CPA-treated cells was incomplete and most of the TPP+ accumulated in the presence of CPA was tightly bound. Dicyclohexylcarbodiimide (DCC), verapamil, and monensin also stimulated TPP+ accumulation, but the TPP+ which accumulated in the presence of these compounds was not tightly bound. As with controls, fractionation of cells which had accumulated TPP+ in the presence of DCC, verapamil, or monensin always resulted in near complete recovery (greater than 93%) of the TPP+ in the cytosolic fraction, whereas with CPA, greater than 88% of the TPP+ was recovered noncovalently bound in the plasma membrane and mitochondrial fractions. These results are consistent with the hypothesis that CPA-potentiated TPP+ accumulation is a result of potentiated partitioning of TPP+ into the plasma membranes and mitochondria of LLC-PK1 cells.