Neuronal Norepinephrine Transporter Research Articles

Cardiac Uptake of the Adrenergic Imaging Agent meta-Iodobenzylguanidine (mIBG) Is Mediated by Organic Cation Transporter 3 (Oct3).

Heart failure (HF) is a chronic disease affecting 1%-2% of the global population.123I-labeled meta-iodobenzylguanidine (mIBG) is US Food and Drug Administration-approved for cardiac imaging and prognosis risk assessment in patients with HF. As a norepinephrine analog, mIBG is believed to be transported into adrenergic nerve terminals by the neuronal norepinephrine transporter (NET) and hence image sympathetic innervation of the myocardium. We previously showed that mIBG is an excellent substrate of organic cation transporter 3 (OCT3), an extraneuronal transporter expressed in cardiomyocytes. Here, we evaluated the in vivo impact of Oct3 on mIBG disposition and tissue distribution using Oct3 knockout mice. Oct3 +/+ and Oct3 -/- mice were administered with mIBG intravenously, and mIBG plasma pharmacokinetics and tissue exposures were determined. In Oct3 +/+ mice, mIBG exhibited extensive accumulation in multiple tissues (heart, salivary gland, liver, and adrenal gland). No difference was observed in overall plasma exposure between Oct3 +/+ and Oct3 -/- mice. Strikingly, cardiac mIBG was depleted in Oct3 -/- mice, resulting in 83% reduction in overall cardiac exposure (AUC0-24 h: 12.7 vs. 2.1 μg × h/g). mIBG tissue exposure (AUC0-24 h) was also reduced by 66%, 36%, and 31% in skeletal muscle, salivary gland, and lung, respectively, in Oct3 -/- mice. Our data demonstrated that Oct3 is the primary transporter responsible for cardiac mIBG uptake in vivo and suggested that cardiac mIBG imaging mainly measures OCT3 activity in cardiomyocytes but not NET-mediated uptake in adrenergic nerve endings. Our findings challenge the current paradigm in interpreting cardiac mIBG imaging results and suggest OCT3 as a potential genetic risk marker for HF prognosis. SIGNIFICANCE STATEMENT: 123I-labeled meta-iodobenzylguanidine is used for cardiac imaging and risk assessment in heart failure patients. Contrary to the current belief that meta-iodobenzylguanidine (mIBG) tracks cardiac sympathetic innervation due to its uptake by the neuronal norepinephrine transporter, the authors demonstrated that cardiac mIBG uptake is mediated by the extraneuronal transporter Oct3. Their findings warrant a re-evaluation of the scientific rationale behind cardiac mIBG scan and further suggest organic cation transporter 3 as a risk factor for disease progression in heart failure patients.

Preliminary Evaluation of 18F-Labeled Benzylguanidine Analogs as NET Tracers for Myocardial Infarction Diagnosis.

Heart failure (HF) remains a major cause of late morbidity and mortality after myocardial infarction (MI). To date, no clinically established 18F-labeled sympathetic nerve PET tracers for monitoring myocardial infarction are available. Therefore, in this study, we synthesized a series of 18F-labeled benzyl guanidine analogs and evaluated their efficacy as cardiac neuronal norepinephrine transporter (NET) tracers for myocardial imaging. We also investigated the preliminary diagnostic capabilities of these tracers in myocardial infarction animal models, as well as the structure-activity relationship of these tracers. Three benzyl guanidine-NET tracers, including [18F]1, [18F]2, and [18F]3, were synthesized and evaluated in vivo as PET tracers in a myocardial infarction mouse model. [18F]LMI1195 was used as a positive control for the tracers. H&E staining of the isolated myocardial infarction heart tissue sections was performed to verify the efficacy of the selected PET tracer. Our data show that [18F]3 had a moderate decay corrected labeling yield (~10%) and high radiochemical purity (>95%) compared to other tracers. The uptake of [18F]3 in normal mouse hearts was 1.7±0.1%ID/cc at 1 h post-injection (p. i.), while it was 2.4±0.1, 2.6±0.9, and 2.1±0.4%ID/cc in the MI mouse hearts at 1, 2, and 3 days after surgery, respectively. Compared with [18F]LMI1195, [18F]3 had a better myocardial imaging effect in terms of the contrast between normal and MI hearts. The area of myocardial infarction shown by PET imaging corresponded well with the infarcted tissue demonstrated by H&E staining. With an obvious cardiac uptake contrast between normal mice and the myocardial infarction mouse model, [18F]3 appears to be a potential tool in the diagnosis of myocardial infarction. Therefore, it is necessary to conduct further structural modification studies on the chemical structure of [18F]3 to improve its in vivo stability and diagnostic detection ability to achieve reliable and practical imaging effects.

Inhibition of the norepinephrine transporter rescues vascular hyporeactivity to catecholamine in obstructive jaundice

In patients with obstructive jaundice, the cardiovascular system exhibits hypotension and vascular hyporeactivity. Most norepinephrine is taken up through the neuronal norepinephrine transporter (NET), which is implicated in cardiovascular diseases. A previous study demonstrated that pharmacological NET inhibition could increase resting blood pressure. However, the role of NETs in vascular hyporeactivity induced by obstructive jaundice is poorly understood. This study used the NET inhibitor nisoxetine and a rat model of bile duct ligation (BDL) to investigate whether NET is associated with BDL-induced vascular hyporeactivity. Rats were injected with nisoxetine via the tail vein for 7 consecutive days after BDL. Samples of the superior cervical sympathetic ganglion (SCG) and thoracic aortic rings were processed for investigations. Our results showed that NET expression in the SCG was significantly increased after BDL. Nisoxetine prevented the augmentation of NET expression, increased α1-adrenoceptor activation, and enhanced the weakened contractile responses of thoracic aortic rings after BDL. Our study demonstrates that nisoxetine plays a protective role in BDL-induced vascular hyporeactivity through increased α1-adrenoceptor activation in rats.

Depletion of cardiac catecholamine stores impairs cardiac norepinephrine re-uptake by downregulation of the norepinephrine transporter.

In heart failure (HF), a disturbed cardiac norepinephrine (NE) homeostasis is characterized by depleted cardiac NE stores, impairment of the cardiac NE re-uptake by the neuronal norepinephrine transporter (NET) and enhanced cardiac NE net release. Reduced cardiac NE content appears to be caused by enhanced cardiac NE net release from sympathetic neurons in HF, triggered by neurohumoral activation. However, it remains unclear whether reduced NE itself has an impact on cardiac NE re-uptake, independent of neurohumoral activation. Here, we evaluated whether depletion of cardiac NE stores alone can regulate cardiac NE re-uptake. Treatment of Wistar rats with reserpine (5 mg/kg/d) for one (1d) or five days (5d) resulted in markedly reduced cardiac NE content, comparable to NE stores in experimental HF due to pressure overload. In order to assess cardiac NE re-uptake, the specific cardiac [3H]-NE uptake via the NET in a Langendorff preparation was measured. Reserpine treatment led to decreased NE re-uptake at 1d and 5d compared to saline treatment. Expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of the NE synthesis, was elevated in left stellate ganglia after reserpine. Mechanistically, measurement of NET mRNA expression in left stellate ganglia and myocardial NET density revealed a post-transcriptional downregulation of the NET by reserpine. In summary, present data demonstrate that depletion of cardiac NE stores alone is sufficient to impair cardiac NE re-uptake via downregulation of the NET, independent of systemic neurohumoral activation. Knowledge about the regulation of the cardiac NE homeostasis may offer novel therapeutic strategies in HF.

Changes in sympathetic neurovascular function following spinal cord injury

The effects of spinal cord injury (SCI) on sympathetic neurovascular transmission have generally been ignored. This review describes changes in sympathetic nerve-mediated activation of arterial vessels to which ongoing sympathetic activity has been reduced or silenced following spinal cord transection in rats. In all vessels studied in rats, SCI markedly enhanced their contractile responses to nerve activity. However, the mechanisms that augment neurovascular transmission differ between the rat tail artery and mesenteric artery. In tail artery, the enhancement of neurovascular transmission cannot be attributed to changes in sensitivity of the vascular muscle to α1- or α2-adrenoceptor agonists. Instead the contribution of L-type Ca2+ channels to activation of the smooth muscle by nerve-released noradrenaline is greatly increased following SCI. By contrast, mesenteric arteries from SCI rats had increased sensitivity to phenylephrine but not to methoxamine. While both phenylephrine and methoxamine are α1-adrenoceptor agonists, only phenylephrine is a substrate for the neuronal noradrenaline transporter. Therefore the selective increase in sensitivity to phenylephrine suggests that the activity of the neuronal noradrenaline transporter is reduced. While present evidence suggests that sympathetic vasoconstrictor neurons do not contribute to the normal regulation of peripheral resistance below a complete SCI in humans, the available evidence does indicate that these experimental findings in animals are likely to apply after SCI in humans and contribute to autonomic dysreflexia.

Depression‐like Symptoms are Associated with Dysbalance in Vascular Norepinephrine Uptake. This is Worsening by Antidepressants and Increases the Risk for Hypertension.

Antidepressants affecting norepinephrine (NE) reuptake are known to increase the risk for hypertension. Anhedonia ‐ a decreased ability to experience pleasures ‐ is a central symptom in depression and can be evoked in preclinical models exploring the role of the stressful environment in mediating affective signs and autonomic function.Some rats exposed to Chronic Mild Stress (CMS) developed depression‐like symptoms (i.e. anhedonia, measured by sucrose intake) while others were resilient to 8 wks of CMS. Normal blood pressure and decreased cardiac function suggested increased peripheral resistance in rats with depression‐like symptoms. Chronic treatment with escitalopram (SSRI) did not affect blood pressure but normalized behavior in ~50% of treated rats, e.g. responders.Resistance (small mesenteric and femoral) arteries from CMS and non‐stressed rats responded similarly to NE in vitro. Inhibition of neuronal reuptake with cocaine increased NE sensitivity more in rats with depression‐like symptoms than in resilient and non‐stressed groups. Corticosterone‐sensitive extra‐neuronal catecholamine uptake was diminished in rats exposed to CMS. These changes were associated with upregulation of the neuronal NE transporter and reduced expression of the extra‐neuronal transporter (OCT‐2) in arteries from 'depressed' rats. Escitalopram further upregulated neuronal NE reuptake in all groups.Our results indicate that depression‐like symptoms are associated with changes in NE uptake in the vascular wall and could explain pro‐hypertensive action of some antidepressants.

Regulation of the norepinephrine transporter by endothelins: a potential therapeutic target.

Neuronal norepinephrine (NE) uptake is a crucial step in noradrenergic neurotransmission that regulates NE concentration in the synaptic cleft. It is a key mechanism mediated by the NE transporter (NET) which takes the neurotransmitter into the presynaptic neuron terminal or the adrenal medulla chromaffin cell. The activity of NET is short and long terms modulated by phosphorylation mediated by protein kinases A, C, and G and calcium-calmodulin-dependent protein kinase, whereas the transporter availability at the cell surface is regulated by glycosylation. Several neuropeptides like angiotensins II, III, and 1-7, bradykinin, natriuretic peptides, as well as endothelins (ETs) regulate a wide variety of biological effects, including noradrenergic transmission and in particular neuronal NE uptake. Diverse reports, including studies from our laboratory, show that ETs differentially modulate the activity and expression of NET not only in normal conditions but also in diverse cardiovascular diseases such as congestive heart failure and hypertension. Current literature supports a key role for the interaction between ETs and NE in maintaining neurotransmission homeostasis and further suggests that this interaction may represent a potential therapeutic target for various diseases, particularly hypertension.

Aldosterone augments Na+-induced reduction of cardiac norepinephrine reuptake.

Impairment of the cardiac norepinephrine (NE) reuptake by the neuronal NE transporter contributes to enhanced cardiac NE net release in congestive heart failure. Elevated plasma levels of aldosterone (AL) promote sympathetic overstimulation in failing hearts by unclear mechanisms. Our aim was to evaluate if elevated AL and/or alterations in Na(+) intake regulate cardiac NE reuptake. To test the effects of AL and Na(+) on cardiac NE reuptake, Wistar rats were fed a normal-salt (NS) diet (0.2% NaCl), a low-salt (LS) diet (0.015% NaCl), or a high-salt (HS) diet (8% NaCl). Another group of animals received AL infusion alone (0.75 μg/h) or AL infusion plus HS diet. Specific cardiac [(3)H]NE uptake via the NE transporter in a Langendorff preparation and AL plasma levels were measured at different time points between 5 and 42 days of treatment. To compare these findings from healthy animals with a disease model, Dahl salt-sensitive rats were investigated as a model of congestive heart failure with endogenously elevated AL. In summary, neither exogenous nor endogenous elevations of AL alone were sufficient to reduce cardiac NE reuptake. Only the HS diet induced a reduction of NE reuptake by 26%; additional infusion of AL augmented this effect to a further reduction of NE reuptake by 36%. In concordance, Dahl salt-sensitive rats treated with a HS diet displayed elevated AL and a marked reduction of NE reuptake. We conclude that exogenous or endogenous AL elevations alone do not reduce cardiac NE reuptake, but AL serves as an additional factor that negatively regulates cardiac NE reuptake in concert with HS intake.

Differential expression of tyrosine hydroxylase and transporters in the right and left stellate ganglion of socially isolated rats

Chronic isolation stress of adult rat males acted increasing gene expression of tyrosine hydroxylase (TH) and neuronal norepinephrine transporter (NET) in the right stellate ganglia, while vesicular monoamine transporter 2 (VMAT2) level remained unchanged. The stress decreased protein level of TH, as well as mRNA levels for NET and VMAT2 in the left stellate ganglia, but expressed no effect on protein levels of these two transporters. These results demonstrate asymmetry in noradrenergic genes in the right and left stellate ganglia during stress and provide molecular evidence to help explain the difference in response to the stress.

Regional changes in cardiac and stellate ganglion norepinephrine transporter in DOCA–salt hypertension

Uptake of norepinephrine via the neuronal norepinephrine transporter is reduced in the heart during deoxycorticosterone (DOCA)-salt hypertension. We hypothesized that this was due to reduced norepinephrine transporter mRNA and/or protein expression in the stellate ganglia and heart. After 4 weeks of DOCA-salt treatment there was no change in norepinephrine transporter mRNA in either the right or the left stellate ganglia from hypertensive rats (n=5-7, p>0.05). Norepinephrine transporter immunoreactivity in the left stellate ganglion was significantly increased (n=4, p<0.05) while the right stellate ganglion was unchanged (n=4, p>0.05). Whole heart norepinephrine content was significantly reduced in DOCA rats consistent with reduced uptake function; however, when norepinephrine was assessed by chamber, a significant decrease was noted only in the right atrium and right ventricle (n=6, p<0.05). Cardiac norepinephrine transport binding by chamber revealed that it was only reduced in the left atrium (n=5-7, p>0.05). Therefore, 1) contrary to our hypothesis reduced reuptake in the hypertensive heart is not exclusively due to an overall reduction in norepinephrine transporter mRNA or protein in the stellate ganglion or heart, and 2) norepinephrine transporter regulation occurs regionally in the heart and stellate ganglion in the hypertensive rat heart.

Do cannabinoids exhibit a tyramine-like effect?

The major constituent of the cannabis plant, Δ(9)-tetrahydrocannabinol, has stimulatory and depressant effects on cardiovascular functions. There is evidence from an in vivo study on the urethane-anaesthetized rat that part of the stimulatory effects is related to a tyramine-like activity. In the present study, we examined whether Δ(9)-tetrahydrocannabinol induces carrier-mediated noradrenaline release in vitro. The study was extended to another phytocannabinoid, cannabidiol, to the synthetic cannabinoids CP 55,940 and WIN 55,212-2 and to the endocannabinoids anandamide and 2-arachidonoyl glycerol. Tissue pieces of the renal cortex from the mouse and the rat were preincubated with (3)H-noradrenaline and superfused. The effect of the cannabinoids on basal (3)H-noradrenaline release was studied. Tyramine served as a positive control. In the mouse kidney, basal (3)H-noradrenaline release was increased by tyramine 0.1, 1 and 10 μM by 39, 91 and 212 %, respectively, and, in the rat kidney, (3)H-noradrenaline release was increased by tyramine 10 μM by 158 %. All effects were abolished by desipramine 1 μM, an inhibitor of the neuronal noradrenaline transporter. The cannabinoids at 0.1, 1 and 10 μM (CP 55,940 at 0.1, 1 and 3.2 μM) did not affect (3)H-noradrenaline release in the mouse kidney. The highest concentration of the cannabinoids (10 μM and in the case of CP 55,940 3.2 μM) also failed to affect (3)H-noradrenaline release in the rat kidney. In conclusion, the cannabinoids Δ(9)-tetrahydrocannabinol, cannabidiol, CP 55,940, WIN 55,212-2, anandamide and 2-arachidonoyl glycerol do not possess a tyramine-like effect on noradrenaline release.

Cardiac syncope due to pain

Neurocardiogenic syncope comprises situations triggered by neurological reflexes resulting in abnormal responses of the neurocardiovascular system that cause loss of consciousness. A vast number of clinical conditions may cause this disorder including pain, defecation, micturition, swallowing, cough, sudden fear or excitement, exercise, and long-time standing. Treatment options for syncope prevention are not satisfactory. Several agents were used for pharmacological treatment without success. Selective inhibitors of neuronal norepinephrine transporter (NET) like duloxetine may play a role in neurally mediated syncope by increasing synaptic norepinephrine levels. Therefore, we report the effect of duloxetine in a patient with pain-induced syncope resistant to standard regimens.

Norepinephrine transporter function and human cardiovascular disease

Approximately 80-90% of the norepinephrine released in the brain or in peripheral tissues is taken up again through the neuronal norepinephrine transporter (NET). Pharmacological studies with NET inhibitors showed that NET has opposing effects on cardiovascular sympathetic regulation in the brain and in the periphery. Furthermore, NET is involved in the distribution of sympathetic activity between vasculature, heart, and kidney. Genetic NET dysfunction is a rare cause of the postural tachycardia syndrome. The condition is characterized by excessive adrenergic stimulation of the heart, particularly with standing. Conversely, NET inhibition may be beneficial in hypoadrenergic states, such as central autonomic failure or neurally mediated syncope, which results from acute sympathetic withdrawal. Biochemical studies suggested reduced NET function in some patients with essential hypertension. Furthermore, cardiac NET function appears to be reduced in common heart diseases, such as congestive heart failure, ischemic heart disease, and stress-induced cardiomyopathy. Whether NET dysfunction is a consequence or cause of progressive heart disease in human subjects requires further study. However, studies with the nonselective NET inhibitor sibutramine suggest that reduced NET function could have an adverse effect on the cardiovascular system. Given the widespread use of medications inhibiting NET, the issue deserves more attention.

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.

Evaluation of LMI1195, a Novel 18 F-Labeled Cardiac Neuronal PET Imaging Agent, in Cells and Animal Models

Heart failure has been associated with impaired cardiac sympathetic neuronal function. Cardiac imaging with radiolabeled agents that are substrates for the neuronal norepinephrine transporter (NET) has demonstrated the potential to identify individuals at risk of cardiac events. N-[3-Bromo-4-(3-[18F]fluoro-propoxy)-benzyl]-guanidine (LMI1195) is a newly developed 18F-labeled NET substrate designed to allow cardiac neuronal imaging with the high sensitivity, resolution, and quantification afforded by positron emission tomography (PET). LMI1195 was evaluated in comparison with norepinephrine (NE) in vitro and 123I-meta-iodobenzylguanidine (MIBG) in vivo. The affinity (Ki) of LMI1195 for NET was 5.16 ± 2.83 μmol/L, similar to that of NE (3.36 ± 2.77 μmol/L) in a cell membrane-binding assay. Similarly, LMI1195 uptake kinetics examined in a human neuroblastoma cell line had Km and Vmax values of 1.44 ± 0.76 μmol/L and 6.05 ± 3.09 pmol/million cells per minute, comparable to NE (2.01 ± 0.85 μmol/L and 6.23 ± 1.52 pmol/million cells per minute). In rats, LMI1195 heart uptake at 15 and 60 minutes after intravenous administration was 2.36 ± 0.38% and 2.16 ± 0.38% injected dose per gram of tissue (%ID/g), similar to 123I-MIBG (2.14 ± 0.30 and 2.19 ± 0.27%ID/g). However, the heart to liver and lung uptake ratios were significantly higher for LMI1195 than for 123I-MIBG. In rabbits, desipramine (1 mg/kg), a selective NET inhibitor, blocked LMI1195 heart uptake by 82%, which was more effective than 123I-MIBG (53%), at 1 hour after dosing. Sympathetic denervation with 6-hydroxydopamine, a neurotoxin, resulted in a marked (79%) decrease in LMI1195 heart uptake. Cardiac PET imaging with LMI1195 in rats, rabbits, and nonhuman primates revealed clear myocardium with low radioactivity levels in the blood, lung, and liver. Imaging in rabbits pretreated with desipramine showed reduced heart radioactivity levels in a dose-dependent manner. Additionally, imaging in sympathetically denervated rabbits resulted in low cardiac image intensity with LMI1195 but normal perfusion images with flurpiridaz F 18, a PET myocardial perfusion imaging agent. In nonhuman primates pretreated with desipramine (0.5 mg/kg), imaging with LMI1195 showed a 66% decrease in myocardial uptake. In a rat model of heart failure, the LMI1195 cardiac uptake decreased as heart failure progressed. LMI1195 is a novel (18)F imaging agent retained in the heart through the NET and allowing evaluation of the cardiac sympathetic neuronal function by PET imaging.

Endothelin-1 and -3 modulate the neuronal norepinephrine transporter through multiple signalling pathways in the rat posterior hypothalamus

We have previously reported that endothelin-1 and -3 modulate different steps of noradrenergic transmission in the hypothalamus. We showed that endothelins modify neuronal norepinephrine transport activity through the regulation of the kinetic constant and internalization. In the present work we sought to define the endothelin receptors and intracellular mechanisms involved in the down-regulation of neuronal norepinephrine uptake induced by endothelin-1 and -3 in the rat posterior hypothalamic region. Results showed that endothelin-1 reduced norepinephrine uptake through ET B receptors, whereas endothelin-3 through a non-conventional or atypical endothelin receptor. In both cases, the effect on norepinephrine uptake was coupled to protein kinase A and C as well as nitric oxide pathways. However, neither protein kinase G nor intracellular or extracellular calcium and calcium/calmodulin-dependent protein kinase II were involved. In addition, the same intracellular mechanisms participated in the reduction of nisoxetine binding (norepinephrine transporter internalization index) induced by both endothelins. Present findings reveal the underlying mechanisms involved in the regulation of the neuronal norepinephrine transporter by endothelins and further support the role of these peptides in the modulation of noradrenergic transmission at the presynaptic nerve endings in the posterior hypothalamus.

Intracellular signaling mechanisms underlying the activity and internalization of the neuronal norepinephrine transporter by endothelin‐3 in the rat posterior hypothalamus

Brain norepinephrine (NE) is involved in relevant physiological functions regulated by the hypothalamus. We previously reported that endothelins (ET) participate in the short term regulation of NE uptake in the anterior and posterior hypothalamus by changing the kinetic and internalization of the neuronal NE transporter (NET). In the present work we studied the mechanisms involved in the regulation of NET by ET‐3 in the rat posterior hypothalamic region (PHR). Experiments were performed in vitro and neuronal NE uptake and nisoxetine binding were used as indexes of activity and internalization of NET, respectively. Results showed that ET‐3 reduced neuronal NE uptake through the activation of a non conventional ET receptor involving PKC, PKA and PKG pathways. Furthermore ET‐3 enhanced NET internalization. However incubation with inhibitors of PKC (GF‐109203x), PKA (H‐89) or PKG (KT5823) inhibited ET‐3 response on NET internalization. Present findings suggest that the effect of ET‐3 on NET in the rat PHR is mediated by a non‐conventional ET receptor coupled to various intracellular pathways including PKC, PKA and PKG.(UBACyT B079, CONICET‐PIP 5929, ANPCyT PICT‐38333)

Regulation of the neuronal norepinephrine transporter by endothelin-1 and -3 in the rat anterior and posterior hypothalamus

We previously reported that endothelin-1 and endothelin-3 modulate norepinephrine neuronal release and tyrosine hydroxylase activity and expression in the hypothalamus. In the present study we sought to establish the role of endothelin-1 and -3 in the regulation of norepinephrine uptake in the anterior and posterior hypothalamus. Results showed that in the anterior hypothalamus endothelin-3 increased neuronal norepinephrine uptake whereas endothelin-1 decreased it. Conversely, in the posterior hypothalamic region both endothelins diminished the neuronal uptake of the amine. Endothelins response was concentration dependent and maintained at all studied times. Endothelins also modified the kinetic and internalization of the NE neuronal transporter. In the anterior hypothalamic region endothelin-3 increased the V max and the B max whereas endothelin-1 decreased them. However, in the posterior hypothalamic region both endothelins diminished the V max as well as B max. Neither endothelin-1 nor endothelin-3 modified neuronal norepinephrine transporter K d in the studied hypothalamic regions. These findings support that in the posterior hypothalamic region both endothelins diminished neuronal norepinephrine transporter activity by reducing the amine transporter expression on the plasmatic membrane. Conversely, in the anterior hypothalamic region endothelin-3 enhanced neuronal norepinephrine transporter activity by increasing the expression of the transporter on the presynaptic membrane, whereas endothelin-1 induced the opposite effect. Present results permit us to conclude that both endothelins play an important role in the regulation of norepinephrine neurotransmission at the presynaptic nerve endings in the hypothalamus.

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.

Regulation and Role of the Presynaptic and Myocardial Na+/H+ Exchanger NHE1: Effects on the Sympathetic Nervous System in Heart Failure

In acute myocardial ischemia and in chronic heart failure, sympathetic activation with excessive norepinephrine (NE) release from and reduced NE reuptake into sympathetic nerve endings is a prominent cause of arrhythmias and cardiac dysfunction. The Na(+)/H(+) exchanger NHE1 is the predominant isoform in the heart. It contributes to cellular acid-base balance, and electrolyte, and volume homeostasis, and is activated in response to intracellular acidosis and/or activation of guanine nucleotide binding (G) protein-coupled receptors. NHE1 mediates its signaling via protein kinases A (PKA) or C (PKC). In cardiomyocytes, NHE1 is restricted to specialized membrane domains, where it regulates the activity of pH-sensitive proteins and modulates the driving force of the Na(+)/Ca(2+) exchanger. During acute ischemia/reperfusion and in heart failure the activity/amount of NHE1 is increased, leading to intracellular Ca(2+) overload and promoting structural (apoptosis, hypertrophy) and functional (arrhythmias, hypercontraction) myocardial damage. In sympathetic nerve endings, increased NHE1 activity results in the accumulation of axoplasmic Na(+) that diminishes the inward and/or favors the outward transport of NE via the neuronal norepinephrine transporter (NET). The increased NE levels within the nerve-muscle junction facilitate the sustained stimulation of myocardial alpha- and beta-adrenoceptors (ARs), which in turn aggravate the increases in myocardial NHE1 activity and the associated deleterious effects. Furthermore, the responsiveness of the beta-AR declines overtime, which results in further release of NE, initiating a vicious cycle. Accordingly, NHE1 is a potential candidate for targeted intervention to suppress this feedback loop.