A NOVEL TECHNIQUE FOR VISUALIZING THE INTRACELLULAR-LOCALIZATION AND DISTRIBUTION OF TRANSPORTED POLYAMINES IN CULTURED PULMONARY-ARTERY SMOOTH-MUSCLE CELLS

The use of a combination of monofluorescein adducts of spermidine (FL- SPD) and spermine (FL-SPM) with confocal laser scanning microscopy (CL SM) provides a useful means for monitoring the fate and lime-dependent changes in the distribution of transported polyamines within living c ells. Polyamine-fluorescein adducts were synthesized from fluorescein isothiocyanate and the appropriate polyamine. Monofluorescein polyamin e adducts (ratio 1:1) were isolated using thin layer chromatography, a nd the structure and molecular weight of the monofluorescein polyamine adducts were confirmed using NMR and mass spectroscopy, respectively. The covalent linkage of the fluorescent adduct moiety to SPD and SPM did not influence their rate of uptake by bovine pulmonary artery smoo th muscle cells (PASMC). Similar to C-14-SPD and C-14-SPM, the rate of uptake of C-14-FL-SPD and C-14-FL-SPM in PASMC was temperature-depend ent. Treatment for 24 h with difluoromethylornithine (DFMO), a selecti ve blocker of the enzyme ornithine decarboxylase and an inducer of the polyamine transport system, significantly increased the cellular upta ke of C-14-FL-SPD and C-14-FL-SPM compared to that of control cells. W hen compared to control cells, treatment of PASMC with the pyrrolizidi ne alkaloid monocrotaline for 24 h also significantly increased the ce llular uptake of C-14-FL-SPD and C-14-FL-SPM. On the other hand, 24 h treatment of PASMC with a polymer of SPM, a selective blocker of the p olyamine transport system, or with free spermine, markedly reduced the cellular accumulation of C-14-FL-SPD and C-14-FL-SPM. After a 20-min treatment of PASMC with FL-SPD or FL-SPM, CLSM revealed that adduct fl uorescence was localized in the cytoplasm of living cells. Treatment w ith DFMO increased the cytoplasmic accumulation of both FL-SPD and FL- SPM. In addition, the fluorescence observed in the cytoplasm of chines e hamster ovary cells (CHO) was significantly higher than that detecte d in the cytoplasm of their polyamine transport deficient variants (CH OMGBG). The results of this study provide the first evidence of the ut ility of a novel method for visualizing the uptake, distribution, and cellular localization of transported polyamines in viable cultured mam malian cells. (C) 1998 Elsevier Science B.V. All rights reserved.