Abstract
The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5-fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation.
Highlights
From the DeDartmentsof tBiochemistrv and Molecular Biology and §Physiology and Endocrinology, MedicCaolllege of Georgia, Augusta, Geo’rgia 30912-2100
Very little information mimic the action of cholera toxin, elicitinga 1.6-2.5- is available concerning whether or not hormones and/or inswTafoimhgiltdenihdisfesti5t,icim0amanuuPtlplalMyartitooobNtnyer-yi(noes2ffi-mfktaehimucneitlatnoassofneeerectoohihntuooyhsllneii)birnn-aict5out-trrobia.xasntioTsinqophiunsoeirantoenoeffoltrfaftieghancccoeehttnsoiciuvlezeliletrflydaso.n-tttirrhnaaacnttehslpelhuoalrrsaeitnregdrus.ilecaTcatoiotoendnodutmohrfaektstnphsoerewontglseeeeidrrnsgoketmointnohaidensuereltCartaiesn(sCtohpn-eoklyriftnueaanrssceiit)ninisocgiunlnelvtousoftrluevthddeeyd toxin on serotonin transport is specific because, under endothelial cells [18].Even though isolated plasma membrane similar conditions, cholera toxin inhibits 3-0-methyl- vesicles offer a number of advantages to characterize certain D-glUCOSe transportand does not influence taurine aspects of the transporter such as ion requirement, kinetics, transport in this cell line
We provide evidence here which shows for the first time that theserotonin transporter activity is under CAMP-dependent regulation in thiscell line
Summary
Depending upon the individual experiment, but in most cases it was 16 h. Even though the cells were preincubated with choline chloride medium for 45 min prior to initiation of serotonin uptake, it is possible that these conditions were not enough obligatory dependence on C1- for its maximal activity. After the preincubation the buffer of serotonin into the cells from choline chloride-containing medium did not alter the concentrations of the intracellular K+ and extracellular C1-, these ions might have significantly stimulated the uptake of serotonin in the presence of traces of extracellular Na+. Incubation was carriedout for 5 min after which the cells were washed four times with the imipramine-containing buffer, and serotonin transport was determined. Anion specificity for serotonin transport in human placental choriocarcinoma cells regulation of the serotonin transporter in the JAR cell line can be studied by investigating the effect of cholera toxin on Transport buffers were prepared by substituting 140 mM NaCl with an equal concentration of the Na+ salts of differentanions.
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