Yukako Yamazaki

Effects of various dopamine uptake inhibitors on striatal extracellular dopamine levels and behaviours in rats

In vivo central effects of some dopamine uptake inhibitors were evaluated in both brain microdialysis and behavioural studies in rats, and compared with their in vitro affinities to dopamine uptake sites. IC50 values of GBR12909 (1-[2- bis(4-fluorophenyl)methoxy]ethyl]-4-(3- phenylpropyl)piperazine), diclofensine, mazindol, amfonelic acid and nomifensine for inhibiting 1 nM [3H]GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) binding to rat striatal membrane were 7.0, 36, 81, 187 and 290 nM, respectively. In the brain microdialysis study, dopamine levels in the striatal dialysates were increased to 16.3- (GBR12909), 14.1- (nomifensine), 4.8- (diclofensine) and 1.9-fold (amfonelic acid) the respective basal levels 40-60 min after i.p. administration (0.1 mmol/kg) and thereafter decreased slowly but remained at the elevated levels for a further 3 h, while mazindol gradually increased dopamine levels though less pronouncedly than others (1.7-fold 200 min after administration). Remarkable and comparable stereotyped behaviours (licking and forepaw treading) were continuously observed at least for 3 h after administration of GBR12909, nomifensine and amfonelic acid, while stereotypies induced by diclofensine and mazindol were moderate and marginal, respectively. In vivo potencies of dopamine uptake inhibitors to increase the extracellular dopamine levels in the striatum tended to correlate with their in vitro affinities to dopamine uptake sites except in the case of nomifensine, and correlated significantly with their potencies to induce stereotyped behaviours except in the case of amfonelic acid. Based on these findings, pharmacological characteristics of these dopamine uptake inhibitors are discussed.

Ca2+‐Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin‐Dependent Kinases

Abstract: We studied effects of Ca2+ in the incubation medium on [3H]dopamine ([3H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca2+ (0–30 min) and Ca2+ concentration (0–10 mM) in Krebs‐Ringer medium affected [3H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 mM Ca2+ after a 10‐min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in Vmax of the [3H]DA uptake process. On the other hand, [3H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 mM Ca2+. The Ca2+‐dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca2+‐free medium following preincubation with 1 mM Ca2+. Protein kinase C inhibitors did not affect apparently Ca2+‐dependent enhancement of the uptake, whereas 1‐[N,O‐bis(1,5‐isoquinolinesulfonyl)‐N‐methyl‐l‐tyrosyl]‐4‐phenylpiperazine (KN‐62; a Ca2+/calmodulin‐dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN‐62 and wortmannin appeared to be additive. N‐(6‐Aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide hydrochloride (W‐7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca2+‐dependent enhancement of [3H]DA uptake is mediated by activation of calmodulin‐dependent protein kinases.

Possible involvement of calmodulin-dependent kinases in Ca2+-dependent enhancement of [3H]5-hydroxytryptamine uptake in rat cortex

Effects of Ca2+ on [3H]5-hydroxytryptamine (5-HT) uptake into rat cortical synaptosomes were studied. The uptake was enhanced in the presence of Ca2+ in Krebs-Ringer medium and the uptake at 0.3-5 mM Ca2+ was 2.4-2.7 times greater than that observed in the absence of Ca2+. The maximal increase at the concentration of 1 mM Ca2+ was achieved after 2 min preincubation. Ca(2+)-dependent enhancement of the [3H]5-HT uptake reflected an increase in Vmax of the uptake process. However, Kd and Bmax values for [3H]paroxetine were not significantly changed in the presence of 1 mM Ca2+ compared with Ca(2+)-free condition. On the other hand, uptake was still enhanced after synaptosomes were washed with Ca(2+)-free after preincubation with 1 mM Ca2+. Staurosporine (a protein kinase C inhibitor) and wortmannin (a myosin light chain kinase inhibitor) did not affect Ca(2+)-dependent enhancement of the uptake, whereas 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazin e (KN-62, an inhibitor of Ca2+ /calmodulin-dependent kinase II) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a calmodulin antagonist) significantly reduced it. Moreover, L-type, but not P- or N-type, voltage-dependent Ca(2+)-channel blockers suppressed enhancement of the uptake. These results indicate that Ca(2+)-dependent enhancement of [3H]5-HT uptake is mediated by activation of calmodulin-dependent protein kinases, suggesting a possibility of calmodulin-dependent regulation of in vivo 5-HT uptake.

Effects of imipramine and sertraline on protein kinase activity in rat frontal cortex.

Three-week administration of sertraline or imipramine to rats (10 mg/kg, intraperitoneally, twice a day) increased ex vivo cyclic AMP-dependent protein kinase activity in the soluble but not in the particulate fraction of the frontal cortex. However, cyclic AMP-dependent protein kinase activity was not affected in either fraction of the parietotemporal cortex and hippocampus. Neither antidepressant altered protein kinase C activity in the soluble and particulate fractions or Ca2+/calmodulin-dependent protein kinase II activity in the frontal cortex. Therefore, sertraline and imipramine both selectively enhance cyclic AMP-dependent protein kinase activity in the frontal cortex. This enhancement might be involved in their biochemical mechanisms.

Role of calcium ions in dopamine release induced by sodium cyanide perfusion in rat striatum

We have previously reported a transient and remarkable increase in dopamine (DA) release in the rat striatum during application of 2 mM sodium cyanide (NaCN) through a brain microdialysis membrane. In the present study we examined the involvement of extracellular Ca2+ in this response. Rats were divided into 4 groups. In the NaCN group a microdialysis probe inserted into the striatum was perfused with Ringers solution containing 2 mM NaCN for 60 min. The Ca2+ free + NaCN group was subjected to perfusion with NaCN dissolved in Ca2+ free Ringers solution, and the CdCl2 + NaCN group with the same plus 0.3 mM CdCl2 (a non-specific Ca2+ channel blocker). In the NaCN and Ca2+ free + NaCN groups DA levels in the dialysates increased to 36- and 44-fold of the control level, respectively, while this was suppressed to only a 16-fold increase in the CdCl2 + NaCN group. In response to a 100 mM KCl perfusion given 3 hr later DA levels were increased (22-fold) in the control group. On the other hand this response was inhibited in the NaCN group (3-fold), but not in the other two groups. An in vitro study with striatal slices showed a gradual increase in intracellular Ca2+ during incubation with 2 mM NaCN. These results suggest that excessive influx of extracellular Ca2+ during NaCN perfusion may contribute partly to the increase in the extracellular DA level in the striatum, and also to the suppression of a DA increase in response to high K+ stimulation observed 3 hr later.

Behavioral stimulation without alteration of β and 5-HT receptors and adenylate cyclase activity in rat brain after chronic sertraline administration

Abstract Effects of chronic treatment with selective 5-HT reuptake inhibitors (SSRIs) on the monoaminergic functions have not been much investigated in compared with tricyclic antidepressants. Therefore, we compared the effects of 3-week treatment with sertraline, a potent SSRI, to those of imipramine (10 mg/kg, IP, twice a day), on monoamine receptors and adenylate cyclase (AC) activity in rat brain. Two-week treatment with both sertraline and imipramine reduced immobility in the water wheel test to the comparable extent. Sertraline treatment did not affect Kd and Bmax of [3H]CGP12177 and [3H]ketanserin bindings or cAMP accumulation by norepinephrine, isoproterenol, 5’-guanylylimidodiphosphate [Gpp(NH)p] and forskolin in the cortical membrane compared with vehicle-treated rats. On the other hand, imipramine treatment decreased Bmax of both bindings and norepinephrine- or isoproterenol-stimulated cAMP accumulation. Treatment with either antidepressant induced no apparent changes in [3H]8-OH-DPAT [2-(N, N-dipropylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene] binding in the hippocampal membrane. These results suggested that chronic treatment of sertraline induced little effect on monoamine receptors and AC activity in the brain and that the alteration of these functions may not be primarily involved in antidepressive effects of antidepressants, at least of SSRIs.

Effect of local cyanide perfusion on rat striatal extracellular dopamine and its metabolites as studied by in vivo brain microdialysis

To investigate in vivo effects of energy failure on functions of dopaminergic neurons, we administered 0 (control), 0.2, 1 or 2 mM sodium cyanide (NaCN) dissolved in Ringers solution for 60 min into the rat striatum through a brain microdialysis membrane. During NaCN perfusion, a transient and concentration-dependent increase in dopamine (DA) levels in the dialysate was observed. The maximum DA level during 2 mM NaCN perfusion was found to be 63-fold higher than the control levels. Leves of 3,4-dihydroxyphenylacetic acid and homovanillic acid were continuously lowered during and after NaCN perfusion. These data suggest that suppression of ATP production by NaCN induces an abrupt and remarkable increase in dopamine release from the nerve terminal in the striatum.