P.1.049 - Messenger RNA for platelet 5HT transporter protein: Studies in rats with constitutionally altered serotonin homeostasis

Abstract

Serotonin (5-hydroxytryptamine, 5HT) transporters occupy central position in the process of fast elimination of neurotransmitter from the synaptic cleft and thus, are directly involved in the regulation of the 5HT neuronal activity. They are also regarded as targets of pharmacodynamic action of antidepressant drugs such as tryciclic antidepressants or the novel class of selective 5HT reuptake inhibitors, currently used in the treatment of depression, obsessive-compulsive disorder, panic attacks, anxiety, eating disorders, aggressive behavior and substance abuse. Blood platelets express structurally identical transporter protein on their membranes and are used as accessible peripheral models for studies of the 5HT transport mechanism in basic neurobiology as well as in biological psychiatry (Jernej B et al., 1996). We have recently introduced a method permitting ex vivo monitoring of transmembrane serotonin transport kinetics in rat platelets (Jernej B et al., 1994). By selective breeding for high and low platelet serotonin levels and respective activities of platelet serotonin transporter, two sublines (high and low) of Wistar-derived rats with constitutional differences in these traits were obtained. Here we report on our studies of gene expression for 5HT transmembrane transporter protein on platelets of these animals with genetically altered serotonin homeostasis. After measuring the activity of platelet serotonin transporter, animals with extreme Vmax values were selected from each subline. Blood samples were taken from 3-5 animals pertaining to the same subline and, after centrifuging, the obtained platelet rich plasma (PRP) samples were pooled together. By the use of acid guanidinium thiocyanate-phenol-chloroform extraction, ≈ 15 μ g of total platelet RNA was isolated from a typical starting sample of 16 ml of pooled PRP (9 × 109 platelets) (Hranilovi D. et al., 1995). Northern blot analysis, using 32P-labeled serotonin transporter cDNA as a probe, identified ≈ 3.3 kb long serotonin transporter mRNA, which could be quantified densitometrically. By the use of the same procedure, ≈ 1 kb long mRNA for cyclophilin was also identified and it was used to control for variations in the amount of RNA loaded and for further quantification of serotonin transporter mRNA. Values of platelet serotonin transporter activity (Vmax - reflecting the number of transporter protein molecules) and transporter mRNA levels, measured in PRP pools of selectively bred animals, showed analogous differences between sublines. If values of Vmax and transporter mRNA from the high-subline's rats are referred to as 100%, animals from the low subline demonstrated values of respective parameters in the range of 43-63%. It could be assumed that changes in transcription rate of serotonin transporter gene have occurred in megakaryocytes of these animals, indicating possible mutations in regulatory region of the gene.