Decreases in cAMP phosphodiesterase activity in hepatocytes cultured with herbimycin A due to cellular microtubule polymerization related to inhibition of tyrosine phosphorylation of α-tubulin

Abstract

The increase in cellular cAMP concentration during 10-min incubation of rat hepatocytes with glucagon or forskolin was enhanced markedly when the hepatocytes had been cultured for several hours with herbimycin A. This effect of herbimycin was accompanied by inhibition of tyrosine- phosphorylation of cellular proteins including α-tubulin, antagonized by coaddition of Na3VO4 plus H2O2, which also antagonized the herbimycin- induced tyrosine phosphorylation, and overcome by the addition to the 10-min incubation medium of a certain inhibitor of cAMP phosphodiesterase (PDE), which caused a huge accumulation of cAMP. The effective PDE inhibitors were 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and 4-(3- butyloxy-4-methoxyphenyl)-2-imidazolidinone (Ro-20-1724, a PDE4 inhibitor), in addition to 3-isobutyl-1-methylxanthine (a nonselective inhibitor). Rapid breakdown of the once-accumulated CAMP in cultured hepatocytes during the subsequent incubation without PDE inhibitors was progressively prevented when the concentration of herbimycin was increased from 0.3 to 10 μM during prior culture. This effect of herbimycin to inhibit PDE activity in intact cells was abolished by coaddition of a microtubule-disrupting agent, either colchicine or vinblastine, into the culture, but remained unchanged if the vinblastine-containing medium was further supplemented with taxol, a microtubule-stabilizing agent, which by itself mimicked the effect of herbimycin. None of these agents, which thus affected PDE activity in intact cells, inhibited the PDE activity assayable in the cell lysates. The taxol- like and vinblastine-suppressible action of herbimycin to stimulate microtubular assembly was antagonized by Na3VO4/H2O2, as confirmed by confocal microscopic images of the cells stained with fluorescein-bound anti- (α-tubulin). Thus, 4-h culture of hepatocytes with herbimycin inhibits phosphorylation of the C-terminal tyrosine residue of α-tubulin, thereby stimulating formation of a microtubular network which is responsible for the inhibition of PDE4 in the intact cells by an unknown mechanism.