Pharmacological characterization of dopamine transport in cultured rat astrocytes.

The effects of GBR-12909 (selective DA uptake inhibitor), zimelidine (selective 5-HT uptake inhibitor) and nisoxetine (selective NE uptake inhibitor) on the uptake of 30 nM [3H]DA into cultured rat astrocytes were examined. [3H]DA uptake was inhibited by approximately 50% by GBR-12909 or zimelidine in a concentration-dependent manner (100 nM to approximately 10 microM). Furthermore, the inhibition curves of GBR-12909 were biphasic, and uptake was completely inhibited by a high concentration of GBR-12909 (100 microM). [3H]DA uptake was also inhibited by approximately 50% by nisoxetine in a concentration-dependent manner (0.1 to approximately 100 nM), and nisoxetine was more potent than GBR-12909 or zimelidine. The inhibitory potencies were in the order nisoxetine > GBR-12909 > zimelidine. The uptake of [3H]DA under Na+-free conditions was approximately 50% of that under normal conditions. Thus, DA was taken up by both Na+-dependent and Na+-independent mechanisms. Nisoxetine (100 nM), zimelidine (100 microM) and GBR-12909 (10 microM) inhibited [3H]DA uptake into astrocytes only in the presence of Na+. On the other hand, this uptake was completely inhibited by a high concentration of GBR-12909 (100 microM) in the absence of Na+. The present data suggest that the Na+-dependent uptake of [3H]DA in cultured rat astrocytes may occur in the NE uptake system. Furthermore, astrocytes express the extraneuronal monoamine transporter (uptake2), which is an Na+-independent system, and this transporter is involved in the inactivation of centrally released DA.     

Evidence for nonclassical alpha adrenoceptor blockade by prazosin in isolated rabbit blood vessels

In isolated rabbit ear artery, prazosin shifted the concentration-response curve for norepinephrine (NE) to the right and increased the slope of the curve in the ED₅₀ region. Prazosin caused a smaller shift to the right of the concentration-response curve for NE in rabbit small saphenous vein and had no effect on the slope in the ED₅₀ region. The prazosin-induced increase in the slope of the NE concentration-response curve did not occur in the ear artery when neuronal and extraneuronal uptake were inhibited by desipramine (DMI) and deoxycorticosterone acetate (DOCA). Prazosin concentration ratios were significantly larger in the ear artery than in the small saphenous vein at all prazosin concentrations in the presence or absence of DMI and DOCA. Significant differences were found between prazosin dissociation constants (K_B) determined at three concentrations of prazosin in ear artery. No such differences were found between phentolamine dissociation constants determined in this vessel. Prazosin dissociation constants determined in small saphenous vein were higher than those determined in ear artery. These findings suggest that prazosin behaves as a noncompetitive antagonist in ear artery and that there are differences between the alpha adrenoceptors of the ear artery and those of the small saphenous vein.     

In vitro effect of phencyclidine and other psychomotor stimulants on serotonin uptake in human platelets

Phencylidine, ketamine and fluoxetine inhibited serotonin (5-HT) uptake in a non-competitive manner in human blood platelets whereas d- and 1-amphetamine produced a competitive inhibition of 5-HT uptake. Phencyclidine (IC₅₀, 2.5 μM) was one-hundredth as potent as fluoxetine (IC₅₀, 22 νM) but ten times more potent than ketamine (IC₅₀, 25 μM) and d-amphetamine (IC₅₀, 24 μM) and three times more potent than 1-amphetamine (IC₅₀, 80 μM) in inhibition of 5-HT uptake by human blood platelets. The possibility that inhibition of 5-HT may contribute to some of the proposed serotonergic effects of psychomotor stimulants is discussed.     

Changes in rat brain monoamine turnover following chronic antidepressant administration

Changes in rat brain monoamine turnover were studied following the chronic administration of five agents which markedly differ in their patterns of monoamine uptake inhibition. Compounds (10 mg/kg, i.p.) were injected once daily for 14 days and experiments undertaken 24 h after the last injection. Chronic administration of desipramine or mianserin elevated brain MOPEG-SO₄ content and the α-MT-induced reduction in brain NA levels was enhanced by chronic desipramine. either antidepressant altered turnover of brain DA or 5-HT. Steady state levels of brain 5-HIAA or striatal levels of DOPAC or HVA were also unchanged. Chronically administered Org 6582, a selective inhibitor of 5-HT uptake, decreased basal and attenuated the probenecid-induced increase iin brain 5-HIAA levels. Chronic Org 6582 had no effect on NA or DA turnover and on the levels of MOPEG-SO₄, DOPAC or HVA. Neither maprotiline nor chlorimipramine altered turnover of NA, DA or 5-HT or levels of metabolites. Thus, in contrast to the acute situation, chronically administered desipramine increases rat brain NA turnover. Conversely, acute and chronic Org 6582 administration yield similar findings, viz. a decrease in turnover. These observations suggest that rat brain 5-HT systems are more resistant than NA systems to adaptive changes following a prolonged inhibition of monoamine uptake.     

Selective inhibition of serotonin uptake by trazodone,a new antidepressant agent

The inhibitory effect of trazodone, a non tricyclic antidepressant, on 5-HT and catecholamine uptake into the synaptosomal preparation from the rat brain was compared with that of chlorimipramine. The inhibition of 5-HT uptake by trazodone is competitive with a K_i of 1.6 × 10^?6 M. Trazodone inhibits ³H-5-HT, ³H-NE and ³H-DA uptake with an IC₅₀ of 1.4 × 10^?6, 3.1 × 10^?4 and 5.2 × 10^?4 M, respectively. Therefore trazodone is 220 and 370 times more potent in inhibiting 5-HT than NE and DA uptake, respectively. The respective IC₅₀ values of chlorimipramine were 0.9 × 10^?7, 3.6 × 10^?6 and 4.0 × 10^?6 M for ³H-5-HT, ³H-NE and ³H-DA.     

Neurotransmitter-induced changes in arachidonate incorporation into rat brain phospholipids: pharmacological manipulations of receptors,presynaptic uptake,mao activity and deacylation-reacylation processes

The incorporation of [³H]arachidonic acid ([³H]AA) into phospholipids (PL) of rat brain, was studied in cerebral cortex slices in the presence and absence of norepinephrine (NE), serotonin (5-HT) and carbamylcholine (CCH). Both NE and 5-HT produced a concentration-dependent effect of stimulating [³H]AA incorporation into phosphatidylinositol (PI) while attenuating incorporation into other PL. Addition of CCH had no apparent effect. The β-adrenergic agonist, isoproterenol, had an effect similar to that seen with equimolar concentrations of NE, whereas the α₁ agonist, phenylephrine, or the α₂ agonist, clonidine, did not produce significant changes. However, application of the NE-receptor blockers, propranolol or prazosin, in the presence of NE, did not modify the NE-induced effects. Similarly, the 5-HT-receptor blockers, methysergide or ketanserine, failed to modify the 5-HT-induced effects, indicating that the neurotransmitter-produced changes may not be receptor mediated. Manipulations of the NE or 5-HT reuptake systems by imipramine (IMI) or desipramine (DMI) had a small additive effect on the neurotransmitter-produced changes in [³H]AA incorporation, suggesting that a functional presynaptic reuptake system is not required for the NE or 5-HT-produced effects. The possibility that the NE or 5-HT effects involve the oxidative metabolism of the monoamines by MAO was also investigated. The MAO inhibitors tranylcypromine and pargyline had no appreciable effect on the neurotransmitter-induced changes in [³H]AA incorporation whereas clorgyline clearly reduced the increase in [³H]AA incorporation into PI seen in the presence of NE or 5-HT, but this clorgyline effect may not be related to its activity as MAO inhibitor. The phospholipase A₂ inhibitor mepacrine had no significant effect on the NE-produced increase in [³H]AA incorporation into PI, but it antagonized the NE-produced decrease in [³H]AA incorporation into PC. Delta-9-Tetrahydrocannabinol, which acts as acyltransferase inhibitor, antagonized the NE-produced increase in [³H]AA incorporation into PI without appreciably influencing the NE-produced decrease in [³H]AA incorporation into PC. These findings suggest that the neurotransmitter-produced increase in [³H]AA incorporation into PI is mediated by stimulation of a specific lyso-PI arachidonyl transferase. The neurotransmitter effects on arachidonate incorporation may have physiological significance in view of the importance of processes of deacylation and reacylation of membrane PL in regulating the function of neuronal membranes.     

Noncompetitive Amine Uptake Inhibition by the New Antidepressant Pridefine

Abstract: Pridefine (AHR-1118) is a pyrrolidine derivative with clinically established antidepressant efficacy. Previous work from this laboratory indicates that pridefine is a reuptake blocker of catecholamines and serotonin with weak releasing activity. This study characterized the mode of amine uptake inhibition by pridefine as noncompetitive. The uptake experiments were performed utilizing ouabain instead of zero-degree controls to differentiate between the passive and active components of uptake. Furthermore, the passive component was resolved into diffusion and binding of substrate. Correction was made for the effects of ouabain on binding. Kinetic constants determined from Lineweaver-Burk plots were: K_m= 3 × 10^?7 M for NE, K_m= 9 × 10^?8 M for DA, and K_m= 3 × 10^?8 M for 5-HT. Dixon analyses of uptake at various pridefine concentrations indicated noncompetitive inhibition with K_i= 2.5 × 10^?6 M for NE uptake, K_i= 2.0 × 10^?6 M for DA uptake, and K_i= 1 × 10^?5 M for 5-HT uptake. These constants compare well with IC₅₀ values for the same transmitters: NE, IC₅₀= 2.4 × 10^?6 M; DA, IC₅₀= 2.8 × 10^?6 M; 5-HT, IC₅₀= 1.0 × 10^?5 M. The in vitro results indicate that pridefine is relatively specific as a catecholamine uptake blocker. It differs from tricyclic antidepressants which are reportedly competitive inhibitors of monoamine uptake. The possible mechanisms by which pridefine acts as a noncompetitive inhibitor are discussed.     

Interactions between different antidepressants and morphine alter gastrointestinal transit in mice

To determine different serotoninergic antidepressants' effects on the gastrointestinal (GI) inhibiting effect induced by morphine, mice were pretreated with mianserin (a tetracyclic antidepressant with multiple 5-HT receptor subtypes interactions) and with fluoxetine (a selective 5-HT reuptake inhibitor). Mianserin alone, produced gastrointestinal inhibition in a dosedependent manner. Naloxone did not reverse this inhibiting effect, indicating that different mechanism of action are involved in morphine- and mianserin-induced inhibition of the gastrointestinal transit. Fluoxetine injected alone produced an increased propulsive motility of the GI transit. This effect was not reversed by naloxone. Fluoxetine did not reduce significantly mianserin-induced inhibition of GI transit. Fluoxetine also mildly reversed morphine-induced gastrointestinal inhibition, suggesting some degree of involvement of the opiates through the serotoninergic system.     

Effects of acidic fibroblast growth factor and epidermal growth factor on subconfluent fetal rat calvaria cell cultures: DNA synthesis and alkaline phosphatase activity

The effects of acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) were examined in subconfluent fetal rat calvaria cell cultures, in the presence of 2% serum. Maximal effect of aFGF and EGF on DNA synthesis measured by [³H]thymidine incorporation was observed after 18 h. aFGF stimulated DNA synthesis by 3.5-fold with an ED₅₀ of 0.75 ng/ml while a 2.3-fold EGF stimulation was recorded with an ED₅₀ of 0.067 ng/ml. 5-Bromo-2-deoxyuridine staining showed a higher stimulation of proliferation in the scattered cells than in the cell clusters. An 18 h aFGF or EGF treatment decreased alkaline phosphatase (ALP) activity by 40 and 23%, respectively, as compared with control cultures. This inhibition was more pronounced after 48 h in the presence of the effectors but no modification of the ALP electrophoretic mobility was observed. These data suggest that aFGF is a less potent mitogen than EGF and a higher inhibitor of ALP activity in fetal rat calvaria cell culture.     

Inhibition of nucleic acid synthesis in leukemia 1210 cells by antimetabolites of coenzyme Q10

Thirteen diversified antimetabolites of coenzyme Q₁₀ which have antitumor activity $\text{in vivo}$ were tested for inhibition of uptake of tritiated thymidine and uridine into DNA and RNA, respectively, of L1210 cells grown in tissue culture. Eight of these antimetabolites have inhibitory activities of the same order of magnitude as the used anticancer drugs, rubidazone and ellipticine. 5-ω-Phenylpropylmercapto-2,3-dimethoxy-1,4-benzoquinone was particularly potent to inhibit nucleic acid synthesis; ED₅₀ for DNA = 2.1 μM and ED₅₀ for RNA = 4.0 μM.     

Local Influence of Endogenous Norepinephrine on Extracellular Dopamine in Rat Medial Prefrontal Cortex

Abstract: Noradrenergic and dopaminergic projections converge in the medial prefrontal cortex and there is evidence of an interaction between dopamine (DA) and norepinephrine (NE) terminals in this region. We have examined the influence of drugs known to alter extracellular NE on extracellular NE and DA in medial prefrontal cortex using in vivo microdialysis. Local application of the NE uptake inhibitor desipramine (1.0 µM) delivered through a microdialysis probe increased extracellular DA (+149%) as well as NE (+201%) in medial prefrontal cortex. Furthermore, desipramine potentiated the tail shock-induced increase in both extracellular DA (stress alone, +64%; stress + desipramine, +584%) and NE (stress alone, +55%; stress + desipramine, +443%). In contrast, local application of desipramine did not affect extracellular DA in striatum, indicating that this drug does not influence DA efflux directly. Local application of the α₂-adrenoceptor antagonist idazoxan (0.1 or 5.0 mM) increased extracellular NE and DA in medial prefrontal cortex. Conversely, the α₂-adrenoceptor agonist clonidine (0.2 mg/kg; i.p.) decreased extracellular NE and DA in medial prefrontal cortex. These results support the hypothesis that NE terminals in medial prefrontal cortex regulate extracellular DA in this region. This regulation may be achieved by mechanisms involving an action of NE on receptors that regulate DA release (heteroreceptor regulation) and/or transport of DA into noradrenergic terminals (heterotransporter regulation).     

Determination of the reversible uptake of 3H-serotonin in synaptosomes: an adequate evaluation of the contribution of nonspecific uptake

Serotonin (5-HT) uptake in synaptosomes was studied at 0 degree C (1), at 37 degrees C in the presence of 100 microM imipramine (IIa) or 100 microM zimelidine (IIb), in the absence of Na+ ions (III) in the incubation medium. A significant decrease (P less than 0.01) of the uptake rate has been found (III greater than IIa, b greater than I). Nonspecific uptake inhibition was thought to be the cause of these differences. Determination of specific uptake, using control III, has shown Na+-dependent transport of 5-HT only on the one type of carrier (Km = 174 + 24 nM). It is concluded that determination of 5-HT nonspecific uptake, using control III, has permitted a more exact evaluation of specific uptake parameters, than determination, using controls I and IIa, b.     

Cardiac sympathetic neuroprotective effect of desipramine in tachycardia-induced cardiomyopathy

Cardiac sympathetic transmitter stores are reduced in the failing heart. In this study, we proposed to investigate whether the reduction of cardiac sympathetic neurotransmitters was associated with increased interstitial norepinephrine (NE) and reactive oxygen species in congestive heart failure (CHF), using a microdialysis technique and salicylate to detect .OH generation. Rabbits with and without rapid ventricular pacing (340 beats/min) were randomized to receive desipramine (10 mg/day) or placebo for 8 wk. Rapid pacing produced left ventricular dilation and systolic dysfunction. The failing myocardium also showed reduced tissue contents of NE and tyrosine hydroxylase protein and activity. In contrast, myocardial interstitial NE was increased in CHF (0.89 +/- 0.11 ng/ml) compared with the sham-operated animals (0.26 +/- 0.03 ng/ml). In addition, cardiac oxidative stress was increased in CHF animals as measured by myocardial interstitial .OH radical, tissue oxidized glutathione, and oxidized mitochondrial DNA. Desipramine treatment produced significant NE uptake inhibition as evidence by an exaggerated pressor response and a greater increase of myocardial interstitial NE in response to intravenous NE infusion but no significant effects on cardiac function or hemodynamics in sham-operated or CHF animals. However, desipramine treatment attenuated the reductions of tissue NE and tyrosine hydroxylase protein and activity in CHF. Desipramine also prevented the reduction of tyrosine hydroxylase produced by NE in PC12 cells. Thus the reduction of cardiac sympathetic neurotransmitters is related to the increased interstitial NE and tissue oxidative stress in CHF. Also, normal neuronal uptake of NE is required for NE or its oxidized metabolites to exert their neurotoxic effects.     

Comparison of naloxonazine and beta-funaltrexamine antagonism of mu 1 and mu 2 opioid actions.

beta-Funaltrexamine (beta-FNA) irreversibly blocks morphine analgesia, lethality and its inhibition of gastrointestinal transit, confirming that these actions involve mu receptors. In dose-response studies, beta-FNA antagonized all the actions with similar potencies (ID50 values of 12.1, 11.3 and 12.3 mg/kg, respectively). beta-FNA also reduced intra-cerebroventricular and intrathecal DAMGO analgesia equally well (ID50 values of 6.09 and 7.7 mg/kg, respectively). Naloxanazine blocked systemic morphine analgesia (ID50 value 9.5 mg/kg) and supraspinal DAMGO analgesia (ID50 value 6.1 mg/kg) as potently as beta-FNA. However, against spinal DAMGO analgesia, morphine's inhibition of gastro-intestinal transit or lethality, naloxonazine (ID50 values 38.8, 40.7 and 40.9 mg/kg, respectively) was significantly less active than beta-FNA (p less than 0.05). beta-FNA remains a valuable tool in the classification of mu opioid actions. Within the mu category, actions can be defined as either mu 1 (naloxonazine-sensitive) or mu 2 (naloxonazine-insensitive).     

Synthesis and antinociceptive effects of endomorphin-1 analogs with C-terminal linked by oligoarginine

Endomorphins (EMs) cannot be delivered into the central nervous system (CNS) in sufficient quantity to elicit antinociception when given systemically because they are severely restricted by the blood-brain barrier (BBB). In the present study, we investigated herein a series of EM-1 analogs with C-terminal linked by oligoarginine in order to improve the brain delivery and antinociception after systemic administration. Indeed, all these analogs decreased the opioid receptor affinity and in vitro pharmacological activity. Moreover, analogs 4, 7-9 produced a less potent antinociceptive activity after intracerebroventricular (i.c.v.) administration, with the ED₅₀ values about 11- to 13-fold lower potencies than that of EM-1. Nevertheless, our results revealed that EM-1 failed to induce any significant antinociception at a dose of 50 μmol/kg after subcutaneous (s.c.) administration, whereas equimolar dose of these four analogs produced a little low but significant antinociceptive effects. Naloxone (10 nmol/kg, i.c.v.) significantly blocked the antinociceptive effects, indicating an opioid and central mechanism. These results demonstrated that C-terminal of EM-1 linked to oligoarginine improved the brain delivery, eliciting potent antinociception following peripheral administration.     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

Differential antagonism of endomorphin-1 and endomorphin-2 supraspinal antinociception by naloxonazine and 3-methylnaltrexone

To determine if different subtypes of mu-opioid receptors were involved in antinociception induced by endomorphin-1 and endomorphin-2, the effect of pretreatment with various mu-opioid receptor antagonists beta-funaltrexamine, naloxonazine and 3-methylnaltrexone on the inhibition of the paw-withdrawal induced by endomorphin-1 and endomorphin-2 given intracerebroventricularly (i.c.v.) were studied in ddY male mice. The inhibition of the paw-withdrawal induced by i.c.v. administration of endomorphin-1, endomorphin-2 or DAMGO was completely blocked by the pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine (40 mg/kg), indicating that the antinociception induced by all these peptides are mediated by the stimulation of mu-opioid receptors. However, naloxonazine, a mu1-opioid receptor antagonist pretreated s.c. for 24h was more effective in blocking the antinociception induced by endomorphin-2, than by endomorphin-1 or DAMGO given i.c.v. Pretreatment with a selective morphine-6 beta-glucuronide blocker 3-methylnaltrexone 0.25mg/kg given s.c. for 25 min or co-administration of 3-methylnaltrexone 2.5 ng given i.c.v. effectively attenuated the antinociception induced by endomorphin-2 given i.c.v. and co-administration of 3-methylnaltrexone shifted the dose-response curves for endomorphin-2 induced antinociception to the right by 4-fold. The administration of 3-methylnaltrexone did not affect the antinociception induced by endomorphin-1 or DAMGO given i.c.v. Our results indicate that the antinociception induced by endomorphin-2 is mediated by the stimulation of subtypes of mu-opioid receptor, which is different from that of mu-opioid receptor subtype stimulation by endomorphin-1 and DAMGO.     

Regional Differences in 5-Hydroxytryptamine and Catecholamine Uptake in Primary Astrocyte Cultures

The uptake of 3H-labelled 5-hydroxytryptamine (5-HT, serotonin) norepinephrine ([3H]NE), and 3,4-dihydroxyphenylethylamine ([ 3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+-dependent uptake showed marked regional differences. For [3H]5-HT the magnitude of uptake was corpus striatum greater than or equal to cerebral cortex greater than hippocampus, whereas for [3H]NE the order was hippocampus greater than corpus striatum greater than cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+-dependent uptake. [3H]5-HT uptake was specifically inhibited by 10(-7) M fluoxetine whereas [3H]NE uptake was preferentially inhibited by 10(-7) M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.