Serotonin uptake via plasma membrane monoamine transporter during myocardial ischemia-reperfusion in the rat heart in vivo

Abstract

Serotonin (5-HT) accumulates in the heart during myocardial ischemia and induces deleterious effects on the cardiomyocytes through receptor-dependent and monoamine oxidase-dependent pathways. We aimed to clarify the involvement of extra-neuronal monoamine transporters in the clearance of 5-HT during ischemia and reperfusion in the heart. Using a microdialysis technique in the anesthetized Wistar rat heart, we monitored myocardial interstitial 5-HT and 5-hydroxyindole acetic acid (5-HIAA) concentration by means of electro-chemical detection coupled with high-performance liquid chromatography (HPLC-ECD). Effects of inhibitors of the plasma membrane monoamine transporter (PMAT) and the organic cation transporter 3 (OCT3) (decynium-22 and corticosterone) on the 5-HT and 5-HIAA concentrations during baseline, coronary occlusion, and reperfusion were investigated. Basal dialysate 5-HT concentration were increased by local administration of decynium-22, but not by corticosterone. Addition of fluoxetine, a serotonin transporter (SERT) inhibitor further increased the 5-HT concentration upon during administration of decynium-22. Decynium-22 elevated the background level of 5-HT during coronary occlusion and maintained 5-HT concentration at a high level during reperfusion. Production of 5-HIAA in the early reperfusion was significantly suppressed by decynium-22. These results indicate that PMAT and SERT independently regulate basal level of interstitial 5-HT, and PMAT plays a more important role in the clearance of 5-HT during reperfusion. These data suggest the involvement of PMAT in the monoamine oxidase-dependent deleterious pathway in the heart.

Keywords: 5-HT; in vivo cardiac microdialysis; ischemia reperfusion injury; organic cation transporter; plasma membrane monoamine transporter; serotonin transporter.

Figure 1

Schematic illustration of the cardiac microdialysis. A dialysis probe was implanted transversely into the lateral wall of the left ventricle. Interstitial 5‐HT and 5‐HIAA diffuse into perfusate across the dialysis membrane, and pharmaceutical agents diffuse into the interstitial space according to the concentration gradient. Dialysate from the myocardial interstitium was sampled and then analyzed using HPLC‐ECD. A silk suture was passed around the coronary artery and occluded as necessary

Figure 2

Time course of the experimental protocols. Protocol 1) The implanted probe was perfused with Ringer's solution containing various concentrations of decynium‐22 or corticosterone. Fluoxetine was co‐administered with the highest concentration of decynium‐22 or corticosterone. An aliquot of dialysate was sampled at baseline and 45–60 min into each perfusion period for 15 min. Protocol 2 and 3) Aliquots of dialysate were sampled before, during, and after the coronary occlusion as shown in the timeline. In the protocol 3, the perfusate was changed to the solution containing 100 µM decynium‐22 at 60 min before the baseline sampling

Figure 3

Effects of local administration of decynium‐22 or corticosterone on the baseline dialysate 5‐HT concentration (n = 3). Decynium‐22 significantly increased 5‐HT concentration at 100 µM (a), meanwhile corticosterone had no effect on 5‐HT concentration (b). Addition of fluoxetine in the perfusate further increased the 5‐HT concentration in both decynium‐22 and corticosterone treatment (a and b). Data are shown in individual data points and mean ± SE. *p < .05, versus baseline (−/−). One‐way repeated ANOVA followed by Bonferroni's post‐test. #p < .05, versus (100 µM decynium‐22 or 1 mM corticosterone alone). One‐way repeated ANOVA followed by Bonferroni's post‐test

Figure 4

Effect of decynium‐22 on dialysate 5‐HT and 5‐HIAA concentration during coronary occlusion and reperfusion. In control group (n = 7), 5‐HT increased during coronary occlusion and then immediately decreased after reperfusion, while 5‐HIAA kept the baseline level during coronary occlusion and abruptly increased after reperfusion. In decynium‐22 group (n = 7), background level of 5‐HT rose at baseline and during coronary occlusion and did not return to the baseline level after reperfusion. At baseline and during coronary occlusion 5‐HIAA level was not affected by decynium‐22, however, the increase in 5‐HIAA in the early reperfusion was significantly suppressed. Data are presented as box‐whisker plots. The horizontal line in the box indicates the median, and the dot in the box indicates the mean. #p < .05, versus Control. Two‐way repeated ANOVA followed by Bonferroni's post‐test

Figure 5

Putative pathway involving in the 5‐HT metabolism during ischemia and reperfusion in the heart. In a baseline condition (left), PMAT takes up 5‐HT into cells including cardiomyocytes, endothelial cells, and fibroblast cells to maintain background level of interstitial 5‐HT lower. During ischemia (middle), PMAT keeps the 5‐HT uptake rate while interstitial 5‐HT increases. In spite of the increase in intracellular 5‐HT, interstitial 5‐HIAA does not change due to reduced 5‐HT degradation in the cells. During reperfusion (right), high‐capacity transporter PMAT contributes to the clearance of the increased interstitial 5‐HT, meanwhile reoxygenation may accelerate 5‐HT degradation, therefore, intracellular 5‐HT is metabolized to 5‐HIAA