The supernatant was then centrifuged at 175 0 x g for 20 min and

The supernatant was then centrifuged at 175 0 x g for 20 min and the resulting pellet resuspended in 2.5 ml of calcium free Krebs buffer fmM: 119.5 NaCI. 3.3 KCI, 1.2 KHzPO 1.2 MgSO 11 glucose, 0.03 EDTA, 2 EGTA, 25 NaHCO 0.6 ascorbic acid . The buffer was gassed continuously with 95 0, S C02. After 5 min preincubation at 37 C, DA loaded synaptosomes. This effect was not mimicked by the non specific 5 HT agonist, d LSD, suggesting that surface located 5 HT receptors are not involved. Furthermore, 5 HT induced release was not inhibited by the 5 W, antagonists, MDL 72222 or GR 38032. This latter result contrasts with the findings of Blandina et al who concluded that 5 HT, receptors were involved in stimulating the basal release of DA since the effect of S HIT was mimicked by the 5 HT, agonist 2 methyld HT and the increased basal release evoked by both 5 HT and 2 methyld HT could be competitively blocked by the S HT, antagonist ICS 205 930. As reported by Nurse et al 5 HT enhanced release was prevented by the DA uptake blocker, nomifensine, but not by the 5 HT specific uptake blocker, imipramine.
Cocaine, which blocks both DA and 5 HT uptake, also potently antagonized S HT induced release. These results suggest that the DA uptake carrier, which is known to be capable of 5 HT transport is necessary for the 5 HT enhancement of tritium efflux. There are several ways to account for this observation. One possibility is that 5 HT enhances DA efflux by a process of facilitated exchange diffusion, SB-742457 selleckchem similar to that proposed to account for the amine releasing action of amphetamine and tyramine . Thus, the inward transport of 5 HT by the uptake carrier would make more carrier sites available on the inside of the membrane for the outward transport of cytoplasmic DA, leading to an increased basal efflux of this amine. Furthermore, an increase in the cytoplasmic sodium concentration as a result of the co transport of Na? with .5 HT would also increase inhibitor chemical structure carrier availability for the outward transport of DA.
It is also possible that if the uptake of 5 HT is sufficiently vigorous, the Na? co transported with the 5 HT could depolarize the terminal to the level needed for neurotransmitter release. This explanaiion PI3K Inhibitor can be excluded though since the 5 HT enhanced DA efflux was observed in calcium free saline. Another way 5 HT could enhance tritium efflux is by a reserpine like action, in which 5 HT, after entering dopaminergic terminals, would cause the depletion of vesicular DA stores. By analogy with the action of rcserpine, an enhancement of tritium efflw by such a mechanism would result in the release of label predominafery in the form of DA metabolites, rather than as DA itself.

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