Down-Regulation of Norepinephrine Transporters on PC12 Cells by Transporter Inhibitors

Abstract: To investigate the regulation of norepinephrine transporters (NETs) in vitro, we measured the binding of the NET-selective ligand [³H]nisoxetine in homogenates of PC12 cells after exposure of intact cells to the NET inhibitor desipramine (DMI). A 3-day exposure of PC12 cells to DMI robustly reduced the B _max, but not the K _D, of [³H]nisoxetine binding to NETs. Reduction of the binding of [³H]nisoxetine was dependent on both the concentration of DMI and the time of exposure to DMI. Reduction of [³H]nisoxetine binding to NETs produced by a 1-day exposure to DMI reverted to preexposure levels 48 h after cessation of DMI exposure. Similar down-regulation of NETs was found when PC12 cells were exposed to another NET-selective drug, nisoxetine, which is structurally unrelated to DMI. In contrast, exposure of cells to the serotonin transporter-selective drug citalopram, or the NET substrate norepinephrine, had no effects on the binding of [³H]nisoxetine to NETs. The down-regulation of NETs was paralleled by a DMI-induced reduction in the uptake of [³H]norepinephrine in intact PC12 cells. It can be inferred from these data that inhibitors of the NET can down-regulate NETs directly, and do so in the absence of changes in the synaptic concentration of norepinephrine.     

Down-Regulation of the Human Norepinephrine Transporter in Intact 293-hNET Cells Exposed to Desipramine

Abstract: The effects of continuous exposure of cultured cells expressing the human norepinephrine transporter (hNET) to the hNET inhibitor desipramine on hNET expression and function were studied. Exposure of HEK-293 cells transfected stably with the hNET cDNA (293-hNET cells) to desipramine for 3 days reduced the specific binding of [³H]nisoxetine in membrane homogenates in a concentration-dependent manner. The magnitude of the reductions in [³H]nisoxetine binding to hNET was dependent on the length of time of the exposure to desipramine, reaching 77% after a 21-day exposure. The reduction of [³H]nisoxetine binding returned to control levels within 72 h after a 3-day exposure to desipramine. Reductions in [³H]nisoxetine binding to hNET were accompanied by time-dependent and exposure concentration-dependent reductions in hNET protein levels as determined by western blotting. Similar to binding, hNET protein levels returned to control levels 72 h after cessation of desipramine exposure. Northern blotting indicated that exposure of 293-hNET cells to desipramine did not significantly alter hNET mRNA levels. Uptake of [³H]norepinephrine by 293-hNET cells was markedly reduced after a 3-day exposure to desipramine. However, desipramine exposure had no effect on uptake of [³H]glutamate or [³H]-alanine. The present findings imply that down-regulation of the hNET in 293-hNET cells induced by desipramine results from a selective reduction in hNET protein levels, presumably a consequence of either a reduction in the translation of hNET mRNA or from an enhanced degradation of hNET protein.     

The expression of dopamine beta-hydroxylase, tyrosine hydroxylase, and Phox2 transcription factors in sympathetic neurons: evidence for common regulation during noradrenergic induction and diverging regulation later in development

During differentiation of sympathetic neurons in chick embryos, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) mRNAs become detectable during the same developmental period and are both induced by BMP 4. Later during sympathetic ganglion development, DBH is detectable in TH-positive and -negative cells. Moreover, BMPs reduce DBH mRNA in cultures of sympathetic neurons while leaving TH unaffected. The data provide evidence for a common regulation of TH and DBH early during sympathetic neuron differentiation and indicate that BMPs promote their initial expression but not the maintenance during later development. The time course of Phox2a and 2b expression suggests an evolutionary conserved role in noradrenergic induction. In addition, Phox2a, Phox2b, and c-ret may be involved in the differentiation of cholinergic sympathetic neurons.     

INCREASE IN THE RELATIVE RATE OF SYNTHESIS OF DOPAMINE-β-HYDROXYLASE IN IN THE NUCLEUS LOCUS COERULEUS ELICITED BY RESERPINE

Abstract— Three days following a single injection of reserpine (10 mg/kg, i.p.) the activity and amount of dopamine-β-hydroxylase (DBH) are increased nearly 2-fold in the noradrenergic cell bodies of the nucleus locus coeruleus of rat. To determine if this increased accumulation of DBH is due to an increased rate of enzyme synthesis, [³H]amino acids were infused into the IVth ventricle of reserpine-and saline-injected rats. This method was 35 times more effective than intracisternal infusion and 600 times more effective than intravenous infusion. DBH protein was isolated from the locus coeruleus by immunoprecipitation and SDS-electrophoresis. These steps proved crucial for the complete isolation of DBH from other labelled proteins. Indeed, only 10–15% of the immunoprecipitate was finally identified as labelled DBH protein. The rate of incorporation of [³H]leucine into DBH protein of locus coeruleus was increased to 181%, of control following reserpine, whereas that into TCA-precipitable protein was unchanged. A similar result was obtained using [³H]lysine. In contrast, the apparent half-life of the enzyme did not change following reserpine. The relative rate of synthesis of DBH ([³H]DBH/³H-total protein), denoting selectivity of response, was increased in the locus coeruleus of reserpine-treated rats to 154% of control ( P < 0.01). These findings indicate that increased synthesis accounts for the observed increase in DBH protein in the locus coeruleus following reserpine administration.     

Cyclothiazide Selectively Potentiates AMPA- and Kainate-Induced [3H]Norepinephrine Release from Rat Hippocampal Slices

Abstract: Activation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of ionotropic glutamate receptors has been shown to result in a rapid desensitization of the receptor in the presence of certain agonists. One effect of AMPA receptor desensitization in the hippocampus may be to decrease the efficacy of AMPA receptor agonists at stimulating the release of norepinephrine from noradrenergic terminals. Recently, cyclothiazide was reported to inhibit AMPA receptor desensitization by acting at a distinct site on AMPA receptors. We have examined the effect of cyclothiazide on AMPA- and kainate (KA)-induced norepinephrine release from rat hippocampal slices to determine whether cyclothiazide would increase the efficacy of AMPA-induced [³H]norepinephrine release by inhibiting AMPA receptor desensitization. Cyclothiazide was observed to potentiate markedly both AMPA- and KA-induced [³H]norepinephrine release. This potentiation is selective for AMPA/KA receptors as cyclothiazide did not potentiate N -methyl- d -aspartate-induced [³H]norepinephrine release or release induced by the nonspecific depolarizing agents veratridine and 4-aminopyridine. These results demonstrate that AMPA receptor-mediated modulation of [³H]norepinephrine release from rat brain slices is a useful approach to studying the cyclothiazide modulatory site on the AMPA receptor complex.     

Exposure to an enriched environment selectively increases the functional response of the pre-synaptic NMDA receptors which modulate noradrenaline release in mouse hippocampus

We evaluated the impact of environmental training on the functions of pre-synaptic glutamatergic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and nicotinic receptors expressed by hippocampal noradrenergic nerve terminals. Synaptosomes isolated from the hippocampi of mice housed in enriched (EE) or standard (SE) environment were labeled with [³H]noradrenaline ([³H]NA) and tritium release was monitored during exposure in superfusion to NMDA, AMPA, epibatidine or high K⁺. NMDA -evoked [³H]NA release from EE hippocampal synaptosomes was significantly higher than that from SE synaptosomes, while the [³H]NA overflow elicited by 100 μM AMPA, 1 μM epibatidine or (9, 15, 25 mM) KCl was unchanged. In EE mice, the apparent affinity of NMDA or glycine was unmodified, while the efficacy was significantly augmented. Sensitivity to non-selective or subtype-selective NMDA receptor antagonists (MK-801, ifenprodil and Zn²⁺ ions) was not modified in EE. Finally, the analysis of NMDA receptor subunit mRNA expression in noradrenergic cell bodies of the locus coeruleus showed that NR1, NR2A, NR2B and NR2D subunits were unchanged, while NR2C decreased significantly in EE mice as compared to SE mice. Functional up-regulation of the pre-synaptic NMDA receptors modulating NA release might contribute to the improved learning and memory found in animals exposed to an EE.     

IN VIVO RELEASE OF ENDOGENOUSLY SYNTHESIZED CATECHOLAMINES FROM THE CAT BRAIN EVOKED BY ELECTRICAL STIMULATION AND BY d-AMPHETAMINE

Abstract— The cerebral ventricles of spinal-sectioned cats were perfused with artificial cerebrospinal fluid after the intraventricular administration of [³H]DOPA or [³H]tyrosine. Endogenously synthesized [³H]dopamine or [³H]norepinephrine were identified in the perfusate. Electrical stimulation of catecholaminergic nerve tracts in the hypothalamus increased the efflux of both catecholamines. The addition of d -amphetamine to the perfusing cerebrospinal fluid caused a large increase in [³H]dopamine and a small increase in [³H]norepinephrine appearing in the perfusate. Most of the endogenously synthesized [³H]catecholamines detected in the perfusate following stimuli originated from structures bordering the lateral cerebral ventricle. Thus, norepinephrine and dopamine can be synthesized in and released from catecholaminergic nerve terminals in structures bordering the cerebral ventricles.     

Opioid Effects on [3H]Norepinephrine Release from Dissociated Embryonic Locus Coeruleus Cell Cultures

Abstract: The acute and chronic effects of opioid exposure on [³H]norepinephrine ([³H]NE) release were examined in cell cultures of embryonic rat locus coeruleus (LC). Initial morphological and biochemical characterization of the cultures indicated that the cells exhibited properties similar to those observed in situ. Specific [³H]NE uptake was saturable with a K _m value of 222 ± 52 n M . [³H]NE accumulated by LC cells was released in response to 20 m M K⁺ stimulation, in a calcium-dependent manner. Both components of neurotransmitter release, spontaneous and K⁺ evoked, were significantly inhibited by β-endorphin, with the latter being maintained in the presence of tetrodotoxin. The pharmacology of the opioid effect was consistent with that of μ-receptor activation. The effect of chronic exposure to the μ-selective agonist fentanyl (1 μ M ) was examined following 4 days of drug treatment. Although there was no significant effect of fentanyl on K⁺-evoked [³H]NE release, these cells were tolerant to the acute inhibitory effect of β-endorphin. These results indicate that this is an appropriate system for examining the effects of acute and chronic opioid treatment on noradrenergic cells in vitro. In addition, this system may be useful as a CNS model for examining mechanisms that underlie tolerance and dependence following chronic opioid exposure.     

THE EFFECT OF PHOSPHOLIPASE C, PHOSPHOLIPASE A2 AND NEURAMINIDASE ON THE UPTAKE OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN

Abstract— The uptake of [³H]norepinephrine ([³H]NE) and [³H]serotonin ([³H]5-HT) by rat brain synaptosomes is reduced as a result of pretreatment of the synaptosomes with phospholipase C (EC 3.1.4.3) or phospholipase A₂ (EC 3.1.1.4). This effect is not due to inhibition of the Na⁺-K⁺-ATPase but rather is caused by hydrolysis of neuronal membrane phospholipids, mainly phosphatidylcholine, which seem to be important to the uptake.     

Endogenous Noradrenaline Activates α2-Adrenoceptors on Serotonergic Nerve Endings in Human and Rat Neocortex

Abstract: Slices from human neocortex preincubated with [³H]serotonin ([³H]5-HT) were superfused and stimulated electrically to investigate whether the α₂-adrenoceptors on serotonergic terminals can be stimulated by endogenous noradrenaline (NA) released from neighboring noradrenergic fibers. The stimulation-evoked ³H overflow, representing action potential-induced, exocytotic release of 5-HT, was depressed by the NA uptake blocker (+)-oxaprotiline. Rauwolscine (a mixed α₂-adrenoceptor antagonist/5-HT autoreceptor agonist) or phentolamine [a combined α- adrenoceptor/5-HT autoreceptor antagonist; the latter drug in the presence of (+)-oxaprotiline] enhanced the release when the 5-HT autoreceptors had previously been blocked by metitepine. Under hypothermia the release of 5-HT was found to be decreased and that of NA to be increased; under these conditions idazoxan (an α₂-adrenoceptor antagonist) enhanced the release of 5-HT. In neocortex slices from rats (+)-oxaprotiline similarly depressed the release of 5-HT (measured with the same methods) as in human tissue. When rats were pretreated with 6-hydroxydopamine, the inhibitory effect of exogenous NA on 5-HT release was increased, and in slices from rats pretreated with desipramine, it was decreased. In conclusion, α₂-heteroreceptors can be activated by endogenous NA released from neighboring noradrenergic fibers. Because regulatory processes analogous to those in rats probably occur in humans as well, an up- or down-regulation of α₂- heteroreceptors in depressed patients with a (pathological) decrease or a (therapeutic) enhancement of the noradrenergic neurotransmission may also be assumed to occur.