Functional expression of the norepinephrine transporter in cultured rat astrocytes

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.     

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [³H]NE uptake capacity, an effect characterized by a robust decrease in the V_max of the transport of [³H]NE. As expected, reserpine did not displace the binding of [³H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [³H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [³H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca²⁺/Ca²⁺–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [³H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [³H]NE uptake and eliminated the inhibitory effects of reserpine on [³H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca²⁺-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.     

Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.     

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.     

Chronic depolarization stimulates norepinephrine transporter expression via catecholamines

Chronic depolarization increases norepinephrine (NE) uptake and expression of the norepinephrine transporter (NET) in sympathetic neurons, but the mechanisms are unknown. Depolarization of sympathetic neurons stimulates catecholamine synthesis, and several studies suggest that NET can be regulated by catecholamines. It is not clear if the depolarization-induced increase in NET is because of nerve activity per se, or is secondary to elevated catecholamines. To determine if induction of NET mRNA was a result of increased catecholamines, we used pharmacological manipulations to (i) inhibit tyrosine hydroxylase activity in neurons depolarized with 30 mm KCl, thereby preventing increased catecholamines, or (ii) stimulate tyrosine hydroxylase activity in the absence of depolarization. Inhibiting the depolarization-induced increase in catecholamines prevented the up-regulation of NET mRNA, but did not block the increase in tyrosine hydroxylase (TH) mRNA. Furthermore, stimulating catecholamine production in the absence of depolarization elevated NE uptake, NET protein, and NET mRNA in sympathetic neurons. Similarly, elevating endogenous catecholamines in SK-N-BE2M17 neuroblastoma cells increased NE uptake and NET expression. These data suggest that chronic depolarization of sympathetic neurons induces NET expression through increasing catecholamines, and that M17 neuroblastoma cells provide a model system in which to investigate catechol regulation of NET expression.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Activation of glutamate receptors promotes a calcium-dependent and transporter-mediated release of purines in cultured avian retinal cells: possible involvement of calcium/calmodulin-dependent protein kinase II

Calcium-dependent release of purines was previously demonstrated in cultures of chick retinal cells stimulated with high potassium concentrations but there is no evidence for an exocytotic mechanism of adenosine release from presynaptic terminals. Here we show that activation of NMDA or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate glutamate ionotropic receptors promotes a two- to three-fold increase in the release of purines from these cultures. Approximately 96% of intracellular radioactivity is found as nucleotides after incubation with [(3)H]adenosine, but more than 85% of glutamate-stimulated released material is found as inosine (60%), hypoxanthine (19.9%) and adenosine (7.8%). The release is prevented by removal of extracellular calcium, by the transporter blocker nitrobenzylthioinosine, or inhibitors of calcium/calmodulin-dependent protein kinase II (CAMK II). The uptake of [(3)H]adenosine, but not of [(3)H]GABA or [(3)H]choline, is also blocked by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62), N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl)phenyl-N-[2-hydroxiethyl]-4-methoxybenzenesulfonamide (KN93) or the myristoylated autocamtide-2-related inhibitory peptide, suggesting that the enzyme modulates the nucleoside transporter. The distribution of intracellular purines was not affected by KN62. These results indicate that activation of glutamate receptors triggers the release of purines from retinal cells by a mechanism involving calcium influx, CAMK II and the nitrobenzylthioinosine-sensitive nucleoside transporter. The regulation of adenosine release by glutamate receptors and CAMK II could have important consequences in the presynaptic control of glutamate release.     

Transport of dopamine and levodopa and their interaction in COS-7 cells heterologously expressing monoamine neurotransmitter transporters and in monoaminergic cell lines PC12 and SK-N-SH

The present study investigated the effects of levodopa, a precursor of dopamine (DA) therapeutically used for the treatment of Parkinson's disease, on DA transport in the two different systems, COS-7 cells heterologously expressing rat monoamine transporter cDNA and in monoaminergic cell lines PC12 and SK-N-SH. Levodopa enhanced uptake of [3H]DA and [3H]norepinephrine (NE) but not [3H]serotonin in the transfected COS-7 cells in a concentration-dependent manner. On the other hand, in PC12 and SK-N-SH cells where NET is functionally expressed, levodopa enhanced [3H]DA and [3H]NE uptake at low concentrations and inhibited the uptake at higher concentrations. The effects of levodopa on catecholamine transporters in the opposite direction suggest a different mechanism at the intra- and extracellular sites in a levodopa transport-dependent and independent manner.     

Functional importance of the C-terminus of the human norepinephrine transporter

Three C-terminal variants of the human norepinephrine transporter (hNET) are known: the wild-type hNET in which exon 14 encodes the last seven amino acids and two variants with either three or 18 amino acids encoded by an alternatively spliced exon 15. In transfected HEK293 cells we compared by means of [(3)H]norepinephrine ([(3)H]NE) uptake and [(3)H]nisoxetine ([(3)H]NIS) binding the functional properties of the wild-type hNET with those of the more abundant long splice variant containing exon 15 (hNET-Ex15L) and of two artificial hNET mutants lacking either the last three (hNET-Ex14-4) or all seven (hNET-Ex14-0) C-terminal amino acids of exon 14. No differences among the NET isoforms were observed concerning the K(m) for uptake of NE and the K(D) for binding of NIS. However, compared with the wild-type hNET, the three isoforms (hNET-Ex15L, hNET-Ex14-4 and hNET-Ex14-0) showed a pronounced decrease in V(max) of [(3)H]NE uptake and B(max) of [(3)H]NIS binding which correlated with strongly reduced surface expression of the transporter isoforms. The decrease in surface expression of the hNET isoforms is probably a consequence of the lack of the three amino acids leucine, alanine and isoleucine at the C-terminal end which may represent a motif facilitating cell surface expression of the hNET. Expression of hNET-Ex15L exerted a dominant negative effect on plasma membrane expression of the wild-type hNET and thus may represent a novel mechanism for regulation of noradrenergic neurotransmission.     

Presynaptic Mechanisms Underlying the γ-Aminobutyric Acid-Evoked Receptor-Independent Release of [3H]Norepinephrine in Rat Hippocampus

The effects of gamma-aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA-induced [3H]NE release was counteracted by the GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA-induced release of [3H]NE was Ca2+-dependent and tetrodotoxin-sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.     

Dual phases of functional change in norepinephrine transporter in cultured bovine adrenal medullary cells by long-term treatment with clozapine

The effects of long-term treatment with clozapine, a prototype of atypical antipsychotic drugs, on the functional activity, synthesis and mRNA of norepinephrine (NE) transporter were examined in bovine adrenal medullary cells in culture. Treatment of cells with clozapine at 0.1-3.0 microM concentrations produced dual phases of changes in [(3)H]NE uptake, i.e. the first phase showed a decrease in [(3)H]NE uptake at 2-48 h, and the following phase showed an increase in uptake at 72-168 h. Treatment with clozapine for 6 h decreased V(max) to 40% of the control without changing the K(m) value for [(3)H]NE uptake. However, treatment with clozapine for 96 h increased V(max) by 56% over the control without a change in K(m). Scatchard plot analysis of [(3)H]desipramine (DMI) binding to membranes isolated from cells treated with clozapine for 6 h revealed a decrease in B(max) without any change in K(d); in contrast, treatment with clozapine for 96 h caused an increase in B(max) without any change in K(d). Both actinomycin D and cycloheximide, which are inhibitors of protein synthesis, suppressed the clozapine (96 h)-induced increase in [(3)H]NE uptake. Treatment of cells with clozapine for 12-96 h increased the level of NE transporter mRNA in a concentration-dependent manner (0.3-3.0 microM). These findings suggest that treatment of cells with clozapine results in the down-regulation and subsequent up-regulation of NE transporter. The latter change may be caused by the synthesis of new proteins of NE transporter via an increase in its mRNA.     

The inhibition of iodide uptake in the thyroid gland by the fluorescent dye, ANS.

A sensitive norepinephrine assay has been used to measure the release of endogenous norepinephrine from an $\text{in vitro}$ preparation of rat hypothalamus. The addition of KCl to the preparation was found to consistently stimulate the efflux of norepinephrine. This norepinephrine outflow was shown to be due to actual release of NE as opposed to inhibition of NE uptake. KCl-stimulated release was found to be temperature and Ca⁺⁺ dependent.     

Modulation of GABA-augmented norepinephrine release in female rat brain slices by opioids and adenosine

GABA_A receptor activation augments electrically-stimulated release of norepinephrine (NE) from rat brain slices. Because this effect is not observed in synaptoneurosomes, GABA probably acts on inhibitory interneurons to disinhibit NE release. To determine whether opioids or adenosine influence GABA-augmented NE release, hypothalamic and cortical slices from female rats were superfused with GABA or vehicle in the presence and absence of 10 M morphine or 100 M adenosine. GABA augments [³H]NE release in the cortex and hypothalamus. Morphine alone has no effect on [³H]NE release, but attenuates GABA augmentation of [³H]NE release in both brain regions. Adenosine alone modestly inhibits [³H]NE release in the cortex, but not in the hypothalamus. Adenosine inhibits GABA-augmented [³H]NE release in both brain regions. The general protein kinase inhibitor H-7, augments [³H]NE release in both brain regions and may have additive effects with GABA in cortical slices. These results implicate opioid and adenosine interneurons and possibly protein kinases in regulating GABAergic influences on NE transmission.     

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.     

Depolarizing stimuli and neurotransmitters utilize separate pathways to activate protein kinase C in sympathetic neurons.

Several types of extracellular signals affect the function of peripheral neurons. Depolarizing stimuli cause sudden increases in permeability to various ions leading to propagation of nerve impulses and release of transmitter substances. Neurons also receive external signals via neurotransmitter receptors located on the membrane. Different types of receptors present on sympathetic neurons are believed to modulate stimulation-evoked release of norepinephrine. We have investigated the effects of depolarizing stimuli and neurotransmitters on different signaling pathways in homogeneous cultures of chick sympathetic neurons. Depolarizing stimuli (35 mM KCl; electrical stimulation, 1 Hz for 5 min) and neurotransmitters (acetylcholine and 5-hydroxytrypatmine) enhanced membrane binding of protein kinase C by 2-5-fold. 35 mM KCl increased formation of 1,2-diacylglycerol and hydrolysis of [3H]phosphatidycholine without affecting [3H] phosphoinositide hydrolysis. Neurotransmitters increased [3H]inositol phosphates and 1,2-diacylglycerol without affecting the hydrolysis of [3H]phosphatidylcholine. 5-Hydroxytryptamine and acetylcholine (muscarinic component) did not increase Ca2+ concentration in the Indo-1-loaded neuronal cell body or the growth cone, but 35 mM KCl and electrical stimulation caused a marked increase in Ca2+ concentration in both regions of sympathetic neurons. We believe this to be the first demonstration of these two types of signalling mechanisms co-existing in sympathetic neurons; depolarization activate the phosphatidylcholine pathway and neurotransmitters activate the phosphatidylinositol pathway. The importance of two pathways in controlling neuronal Ca2+ concentration and the release of transmitter is discussed.     

Ca2+ Mobilized by Caffeine from the Inositol 1,4,5-Trisphosphate-Insensitive Pool of Ca2+ in Somatic Regions of Sympathetic Neurons Does Not Evoke [3H]Norepinephrine Release

The effects of electrical stimulation, muscarinic and serotonergic agonists, and caffeine on [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) content, intracellular free Ca2+ concentration ([Ca2+]i), and release of [3H]norepinephrine ([3H]NE) were studied in cultured sympathetic neurons. Neuronal cell body [Ca2+]i was unaffected by muscarinic or serotonergic receptor stimulation, which significantly increased [3H]Ins(1,4,5)P3 content. Stimulation at 2 Hz and caffeine had no effect on [3H]Ins(1,4,5)P3, but caused greater than two-fold increase in [Ca2+]i. Only 2-Hz stimulation released [3H]NE. Caffeine had no effect on the release. When [Ca2+]i was measured in growth cones, only electrical stimulation produced an increase in [Ca2+]i. The other agents had no effect on Ca2+ at the terminal regions of the neurons. We conclude that Ins(1,4,5)P3-insensitive, but caffeine-sensitive Ca2+ stores in sympathetic neurons are located only in the cell body and are not coupled to [3H]NE release.     

Volatile anesthetics stimulate the phorbol ester evoked neurotransmitter release from PC12 cells through an increase of the cytoplasmic Ca2+ ion concentration

PC12 cells preloaded with [3H]norepinephrine release this neurotransmitter at a slow rate (basal release). This rate is increased by the addition of phorbol myristate acetate (PMA), but not by a biologically inactive phorbol ester. This effect most likely is mediated by protein kinase C, since desensitization of this kinase abolished the stimulation of the neurotransmitter release by PMA. Unexpectedly, clinical concentrations of the volatile anesthetics halothane, enflurane, isoflurane and methoxyflurane stimulated the PMA evoked neurotransmitter release in good correlation with their anesthetic potency. Since the volatile anesthetics increased the cytoplasmic Ca2+ concentration of the PC12 cells in a dose dependent manner it seems very likely that the effect of the anesthetics on the PMA-evoked neurotransmitter release is mediated by this rise in Ca2+ concentration.     

Neuroendocrine properties of intrinsic cardiac adrenergic cells in fetal rat heart

Intrinsic cardiac adrenergic (ICA) cells in developing rat heart constitute a novel adrenergic signaling system involved in cardiac regulation. Regulatory mechanisms of ICA cells remain to be defined. Immunohistochemical study of fetal rat hearts demonstrated ICA cells with catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). The mRNA of TH and PNMP was also detected in fetal rat hearts before sympathetic innervation. Immunoreactivity of norepinephrine transporter (NET) was localized to ICA cells in rat heart tissue and primary cell culture. For the functional study, the activity of intracellular Ca2+ concentration ([Ca2+]i) transients was quantified by a ratio fluorescent spectrometer in cultured ICA cells and myocytes. ICA cells generated spontaneous [Ca2+]i transients that were eliminated by tetrodotoxin or Ca(2+)-free solutions and showed greatly reduced amplitude with the addition of L-type Ca2+ channel blocker nifedipine. [3H]norepinephrine studies demonstrate release and uptake of norepinephrine. Functional interaction between catecholamines produced by the ICA cells and cocultured myocytes was evident by the effect of the beta-adrenergic blocker atenolol eliciting a dose-dependent reduction in the amplitude and frequency of [Ca2+]i transients of beating myocytes. Hypoxia inhibited [Ca2+]i transient activity of ICA cells, which subsequently produced a reoxygenation-mediated rebound augmentation of [Ca2+]i transients. We conclude that ICA cells are capable of catecholamine synthesis, release, and uptake. They generate spontaneous [Ca2+]i transient activity that can be regulated by oxygen tension. ICA cells may provide an alternative adrenergic supply to maintain cardiac contractile and pacemaker function at rest and during stress in the absence of sympathetic innervation.     

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content

The cardiac neuronal norepinephrine (NE) transporter (NET) in sympathetic neurons is responsible for uptake of released NE from the neuroeffector junction. The purpose of this study was to assess the chamber distribution of cardiac NET protein measured using [(3)H]nisoxetine binding in rat heart membranes and to correlate NE content to NET amount. In whole mounts of atria, NET was colocalized in nerve fibers with tyrosine hydroxylase (TH) immunoreactivity. NE content expressed as micrograms NE per gram tissue was lowest in the ventricles; however, NET binding was significantly higher in the left ventricle than the right ventricle and atria (P < 0.05), resulting in a significant negative correlation (r(2) = 0.922; P < 0.05) of NET to NE content. The neurotoxin 6-hydroxydopamine, an NET substrate, reduced NE content more in the ventricles than the atria, demonstrating functional significance of high ventricular NET binding. In summary, there is a ventricular predominance of NET binding that corresponds to a high NE reuptake capacity in the ventricles, yet negatively correlates to tissue NE content.     

Halothane Increases Non-vesicular [<Superscript>3</Superscript>H]dopamine Release from Brain Cortical Slices

Experimental data suggest that halothane anesthesia is associated with significant changes in dopamine (DA) concentration in some brain regions but the mechanism of this effect is not well known. Rat brain cortical slices were labeled with [³H]DA to further characterize the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [³H]DA that was dependent on incubation time and anesthetic concentration (0.012, 0.024, 0.048, 0.072 and 0.096 mM). This effect was independent of extracellular or intracellular calcium. In addition, [³H]DA release evoked by halothane was not affected by TTX (blocker of voltage-dependent Na⁺ channels) or reserpine (a blocker of vesicular monoamine transporter). These data suggest that [³H]DA release induced by halothane is non-vesicular and would be mediated by the dopamine transporter (DAT) and norepinephrine transporter (NET). GBR 12909 and nomifensine, inhibitors of DAT, decreased the release of [³H]DA evoked by halothane. Nisoxetine, a blocker of NET, reduced the release of [³H]DA induced by halothane. In addition, GBR 12909, nisoxetine and, halothane decrease the uptake of [³H]DA into rat brain cortical slices. A decrease on halothane-induced release of [³H]DA was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium, which are known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na⁺/K⁺ ATPase pump inhibitor, which induces DA release through reverse transport, decreased [³H]DA release induced by halothane. It is suggested that halothane increases [³H]DA release in brain cortical slices that is mediated by DAT and NET present in the plasma membrane.