zy-17617b and Choriocarcinoma

The JAR human placental choriocarcinoma cells express the amino acid transport system L. The activity of this system is Na(+)-independent and is stimulated by acidic extracellular pH. Treatment of cells with the calmodulin antagonist CGS 9343B results in a marked stimulation of the system L activity. At a CGS 9343B concentration of 50 microM, the stimulation of activity measured at pH 7.5 is about 75-100%. This effect is not blocked by cycloheximide, actinomycin D, colchicine or cytochalasin D suggesting that the stimulation is not due to de novo synthesis of the carrier protein or recruitment of the carrier protein from an intracellular pool. The stimulatory effect of CGS 9343B is reproducible with other calmodulin antagonists. Treatment with CGS 9343B significantly modifies pH sensitivity of the system. The stimulatory effect of H+ is markedly reduced in treated cells compared to control cells. The stimulation of activity at pH 5.5 vs. pH 7.5 is 55% in control cells but only 8% in treated cells. Similarly, the stimulatory effect of CGS 9343B is reduced by H+. The stimulation of activity seen with 50 microM CGS 9343B is 80% at pH 8.0, but only 26% at pH 5.5. In addition, H+ and CGS 9343B affect the kinetic parameters of system L in a similar manner, the stimulation in both cases being primarily due to an increase in the maximal velocity. The apparent competitive nature between the effects of H+ and CGS 9343B is also observed with other calmodulin antagonists. These results show that the transport function and pH sensitivity of the amino acid transport system L in placental choriocarcinoma cells are modulated by calmodulin by processes which do not involve de novo synthesis nor recruitment of the carrier protein.

Topics: Alanine; Amino Acids; Benzimidazoles; Biological Transport; Calmodulin; Choriocarcinoma; Humans; Hydrogen-Ion Concentration; Kinetics; Leucine; Taurine; Tumor Cells, Cultured

We investigated the involvement of calmodulin-dependent cellular processes in the regulation of the human serotonin transporter in placental choriocarcinoma cells. Treatment of JAR and BeWo cells with the selective calmodulin antagonist 1,3-dihydro-1-[1-((4-methyl-4H, 6H-pyrrolo[1,2-a][4,1]-benzoxapin-4-yl)-methyl)-4-piperindinyl+ ++]- 2H-benzimidazol-2-one (CGS93-43B (CGS)) for 1 h decreased the imipramine-sensitive serotonin transport activity markedly. The inhibitory effect was specific and was reproducible with other calmodulin antagonists. The basal serotonin transport activity as well as the activity that was stimulated by cholera toxin were inhibited to a similar extent. The CGS-induced inhibition was accompanied by a decrease in the maximal velocity and in the affinities of the transporter for Na+ and Cl- and an increase in the affinity for serotonin. There was, however, no change in the Na+/Cl-/serotonin stoichiometry. The inhibition of the transport activity induced by the treatment of intact cells with CGS was observable in plasma membrane vesicles isolated from these cells. Treatment with CGS had no effect on steady state levels of the serotonin transporter mRNAs nor on the transporter density in the plasma membrane. We conclude that the serotonin transporter is regulated by calmodulin-dependent processes in human placental choriocarcinoma cells involving posttranslational modification, most likely phosphorylation/dephosphorylation, of the transporter protein.

Topics: Alanine; Benzimidazoles; Biological Transport; Calmodulin; Carrier Proteins; Cell Line; Cell Membrane; Choriocarcinoma; Cocaine; Female; Gene Expression; Humans; Iodine Radioisotopes; Kinetics; Leucine; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Placenta; RNA, Messenger; Serotonin; Serotonin Plasma Membrane Transport Proteins; Transcription, Genetic; Tumor Cells, Cultured; Uterine Neoplasms