Showing papers by "Masato Inazu published in 2002"

Expression and functional characterization of the extraneuronal monoamine transporter in normal human astrocytes

Abstract: In this study we examined the functional expression of the extraneuronal monoamine transporter (EMT) in normal human astrocytes (NHA). RT-PCR with EMT-specific primers demonstrated the presence of EMT mRNA in NHA. The RT-PCR products were subjected to restriction-site analysis using three different enzymes (HinfI, SacI and BclI). The restriction patterns with the three enzymes were identical and were exactly as expected from the known restriction map of human EMT cDNA. DNA sequencing was performed for the RT-PCR products from NHA. Sequence analysis demonstrated that the sequences of RT-PCR products were identical to that of EMT. The extract of NHA was immunoblotted with anti-EMT polyclonal antibody raised against EMT polypeptides. Western blotting indicated that anti-EMT polyclonal antibody recognized a band of 63 kDa. Immunocytochemical staining using anti-EMT polyclonal antibody in NHA revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or Golgi membranes, showed a considerable level of immunoreactivity. We examined the time course of temperature-dependent [3H]MPP+ uptake in NHA for 60 min. Temperature-dependent [3H]MPP+ uptake increased in a time-dependent manner for the initial 45 min and almost reached a plateau level (8.70 ± 0.59 pmol/mg protein) at 60 min. In the presence of 3 µm decynium22 (D22) (the most potent EMT inhibitor), temperature-dependent [3H]MPP+ uptake was strongly reduced by 61% (3.39 ± 0.76 pmol/mg protein at 60 min). D22-sensitive [3H]MPP+ uptake was saturable over a MPP+ concentration of 6.25–200 µm. Km for this process was 78.01 ± 7.64 µm and Vmax was 295.4 ± 12.8 pmol/mg protein/min. D22-sensitive [3H]MPP+ uptake was reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding Ba2+ to the uptake buffer. These results provide evidence that the MPP+ transport activity in NHA is potential-sensitive. Moreover, D22-sensitive [3H]MPP+ uptake was independent of extracellular Na+. D22-sensitive [3H]MPP+ uptake was inhibited by D22, various organic cations, steroids and monoamine neurotransmitters. Our results showed that the EMT is functionally expressed in NHA and may also play a key role in the disposition of cationic drugs, neurosteroids, the neurotoxin MPP+ and monoamine neurotransmitters in the brain.

Astroglial dopamine transport is mediated by norepinephrine transporter.

Abstract: The aim of this study was to clarify the characteristics of the dopamine (DA) transport mechanism in cultured rat cortical astrocytes. Reverse transcription-polymerase chain reaction (RT-PCR) with DA transporter (DAT)-, norepinephrine (NE) transporter (NET)- and organic cation transporter 3 (OCT3)-specific primers demonstrated that rat cortical astrocytes and frontal cortex expressed DAT, NET and OCT3 mRNA. Specific [3H]DA and [3H]NE uptake were each partly inhibited by 1 µM decynium 22, an extraneuronal monoamine transporter (EMT) and OCT inhibitor. The selective NE uptake inhibitor nisoxetine (0.1 µM) and the tricyclic antidepressant desipramine (1 µM) very potently inhibited the specific uptake of both [3H]DA and [3H]NE in astrocytes. In contrast, 0.1 µM GBR-12935, a selective and potent DA uptake inhibitor, had no inhibitory activity on the uptake of either compound. These results suggest that cortical astrocytes regulate extracellular DA and NE concentrations through the uptake of DA and NE by the glial NET but not by DAT. Furthermore, an uptake2 mechanism contributes to DA uptake in cortical astrocytes.

Functional expression of the norepinephrine transporter in cultured rat astrocytes.

Abstract: We assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [3H]NE uptake increased in a time-dependent manner, and this uptake involves temperature- and Na+-sensitive mechanisms. The Na+-dependent [3H]NE uptake was saturable, and the Km for the process was 539.3 +/- 55.4 nm and the Vmax was 1.41 +/- 0.03 pmol/mg protein/min. Ouabain, a Na+-K+ ATPase inhibitor, significantly inhibited Na+-dependent [3H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na+-dependent [3H]NE uptake. On the other hand, GBR-12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT-PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti-NET polyclonal antibody recognized a major band of 80 kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re-uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.

Novel Diphenylalkyl Piperazine Derivatives with Dual Calcium Antagonistic and Antioxidative Activities.

Abstract: Two types of novel diphenylalkyl piperazine derivatives containing the thio or aminopropanol moiety substituted by phenyl or benzyl group were synthesized, and evaluated for their calcium antagonistic and antioxidative activities. These compounds showed apparent inhibitions against KCl-induced contractions in isolated rat aorta. Among them, phenylamino compound 9 and benzylamino compound 13 also possessed potent inhibitory activities against auto-oxidative lipid peroxidations in canine brain homogenates. Two representative compounds 3a and 9 were evaluated for their inhibitory activities against KCl-induced contractions in isolated canine arteries (basilar, coronary, mesenteric, and renal). Both compounds showed the most potent inhibitions to basilar artery.

Anti-atherosclerotic effects of tamoxifen in cholesterol-fed ovariectomized rabbits

Abstract: It has been indicated that the anti-estrogen agent, tamoxifen, developed for the treatment of breast cancer, may act on the vascular system as an estrogen agonist. However, to our knowledge few reports suggest that tamoxifen exerts anti-atherogenic actions. In the present study, we evaluated the anti-atherosclerotic effects of tamoxifen in ovariectomized cholesterol-fed rabbits. Ovariectomized rabbits were fed a 1% cholesterol diet and divided into 4 groups: control group (C, n=5); estrogen treatment (E, n=6); low-dose tamoxifen treatment (0.5 mg/kg) (LT, n=6); and high-dose tamoxifen (1.0 mg/kg) (HT, n=7). After 6 weeks, both Oil red O-positive areas on the intimal surfaces of aortae and the ratios of intimal to medial areas (I/M ratios) measured from cross-sections of aortae were significantly lower in groups E, LT and HT compared with group C. Although there were no significant differences in serum NOx (NO2 and NO3) levels among the 4 groups, NOx levels were slightly higher in groups E, LT and HT than group C. Acetylcholine (ACh) was administered to all animals, and the responses of ear arteriole diameters were compared among the 4 groups. While ear arteriole diameters were significantly decreased in group C, no significant changes were observed in groups E, LT or HT following ACh administration. Ratios of ear arteriole diameters after to before ACh administration were significantly greater in groups E, LT and HT compared to group C. These findings suggest that tamoxifen exerts anti-atherosclerotic effects, and that these effects are attributed to the maintenance of vascular endothelial function.