ABSTRACT
Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.
Subject(s)
Glioma/metabolism , Receptors, Serotonin/metabolism , Signal Transduction/physiology , ADP-ribosyl Cyclase/antagonists & inhibitors , ADP-ribosyl Cyclase/metabolism , Adenylyl Cyclase Inhibitors , Animals , Calcium/metabolism , Cyclic AMP/metabolism , GTP-Binding Proteins/metabolism , Gene Expression , Heparin/pharmacology , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Patch-Clamp Techniques , Potassium Channel Blockers/pharmacology , Rats , Receptors, Serotonin/genetics , Second Messenger Systems/physiology , Serotonin/pharmacology , Signal Transduction/drug effects , Tumor Cells, Cultured/drug effectsABSTRACT
The screening and quantitation of methamphetamine (MP) in urine using dansyl chloride (DNC) as the derivatization reagent were studied. Urinary MP derivatized with DNC could be detected by visual observation of the fluorescence in a solid-phase extraction column such as a Sep-Pak C18 cartridge to which the whole reaction solution was applied. The DNC-derivatized MP was eluted from the cartridge and then identified and quantitated by gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). In the GC-MS analysis with the MS detector in the electron-impact mode, DNC-derivatized MP and amphetamine (AP), exhibited diagnostic molecular ion peaks. The intensities of the molecular ions were 15% (DNC-MP) and 35% (DNC-AP) of the base peak (a fragment ion because of the loss of dimethylnaphthalene from M+), demonstrating that this method of derivatization has a major advantage for confirming APs by GC-MS. MP derivatized with DNC could be determined by HPLC with ultraviolet detection. Because a good correlation (r = 0.95) between the GC-MS and HPLC method for urinary MP was confirmed, both HPLC and GC-MS appear to be useful tools for determining urinary MP. The intensity of the cartridge fluorescence due to DNC-derivatized MP was approximately related to the urinary content of MP determined by HPLC or GC-MS, although a false positive in the visual fluorescence was observed in some urinary specimens from healthy volunteers. From these results, screening and confirmation/determination following DNC derivatization is proposed as a suitable method for the analysis of MP.
