Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter

HEK 293 cells stably expressing the human serotonin transporter (hSERT) were grown on coverslips, preincubated with [(3)H]5-hydroxytryptamine (5-HT), and superfused. Substrates of the hSERT [e.g., p-chloroamphetamine (PCA)], increased the basal efflux of [(3)H]5-HT in a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine) also raised [(3)H]5-HT efflux, reaching approximately one-third of the maximal effect of the hSERT substrates. In uptake experiments, both groups of substances inhibited [(3)H]5-HT uptake. Using the low-affinity substrate [(3)H]N-methyl-4-phenylpyridinium (MPP(+)) to label the cells in superfusion experiments, reuptake inhibitors failed to enhance efflux. Similar results were obtained using human placental choriocarcinoma (JAR) cells that constitutively express the hSERT at a low level. By contrast, PCA raised [(3)H]MPP(+) efflux in both types of cells, and its effect was inhibited by paroxetine. The addition of the Na(+),K(+)-ATPase inhibitor ouabain (100 microM) to the superfusion buffer enhanced basal efflux of [(3)H]5-HT-loaded hSERT cells by approximately 2-fold; the effect of PCA (10 microM) was strongly augmented by ouabain, whereas the effect of imipramine was not. The Na(+)/H(+) ionophore monensin (10 microM) also augmented the effect of PCA on efflux of [(3)H]5-HT as well as on efflux of [(3)H]MPP(+). In [(3)H]5-HT-labeled cells, the combination of imipramine and monensin raised [(3)H]5-HT efflux to a greater extent than either of the two substances alone. In [(3)H]MPP(+)-labeled cells, imipramine had no effect on its own and fully reversed the effect of monensin. The results suggest that the [(3)H]5-HT efflux caused by uptake inhibitors is entirely due to interrupted high-affinity reuptake, which is ongoing even under superfusion conditions.     

Reboxetine: functional inhibition of monoamine transporters and nicotinic acetylcholine receptors

The present study determined whether repeated administration of the antidepressant and selective norepinephrine (NE) uptake inhibitor reboxetine resulted in an adaptive modification of the function of the NE transporters (NETs), serotonin (5-HT) transporters, or dopamine (DA) transporters. Because antidepressants may be effective tobacco smoking cessation agents and because antidepressants have recently been shown to interact with nicotinic acetylcholine receptors (nAChRs), the interaction of reboxetine with nAChRs was also evaluated. Repeated administration of reboxetine (10 mg/kg i.p., twice daily for 14 days) did not alter the potency or selectivity of reboxetine inhibition of [(3)H]NE, [(3)H]DA, or [(3)H]5-HT uptake into striatal or hippocampal synaptosomes (IC(50) values = 8.5 nM, 89 microM, and 6.9 microM, respectively). In a separate series of experiments, reboxetine did not inhibit (K(i) > 1 microM) [(3)H]methyllycaconitine, [(3)H]cytisine, or [(3)H]epibatidine binding to rat whole brain membranes. However, at concentrations that did not exhibit intrinsic activity, reboxetine potently inhibited (IC(50) value = 7.29 nM) nicotine-evoked [(3)H]NE overflow from superfused hippocampal slices via a noncompetitive mechanism. In the latter experiments, the involvement of NET was eliminated by inclusion of desipramine (10 microM) in the superfusion buffer. Reboxetine also inhibited (IC(50) value = 650 nM) nicotine-evoked (86)Rb(+) efflux at reboxetine concentrations that did not exhibit intrinsic activity in this assay. Thus, in addition to inhibition of NET function, reboxetine inhibits nAChR function, suggesting that it may have potential as a smoking cessation agent.     

Histamine H(3)-receptors: a new frontier in myocardial ischemia

In protracted myocardial ischemia, sympathetic nerve endings undergo ATP depletion, hypoxia and pH(i) reduction. Consequently, norepinephrine (NE) accumulates in the axoplasm, because it is no longer stored in synaptic vesicles, and intraneuronal Na(+) concentration increases, as the Na(+)/H(+) exchanger (NHE) is activated. This forces the reversal of the Na(+)- and Cl(-)-dependent NE transporter, triggering a massive carrier-mediated release of NE and thus, arrhythmias. Indeed, NE overflow in myocardial ischemia directly correlates with the severity of arrhythmias. Histamine H(3)-receptors (H(3)R) have been identified as inhibitory heteroreceptors in adrenergic nerve endings of the heart. In addition to inhibiting NE exocytosis from sympathetic nerve endings, selective H(3)R agonists attenuate carrier-mediated release of NE in both animal and human models of protracted myocardial ischemia. Whereas H(3)R-mediated attenuation of exocytotic NE release involves an inhibition of N-type Ca(2+)-channels, H(3)R-mediated reduction of carrier-mediated NE release is associated with diminished NHE activity. In addition to inhibiting NE release, H(3)R stimulation significantly attenuates the incidence and duration of ventricular fibrillation. Although other presynaptic receptors also modulate NE release from sympathetic nerve endings, H(3)R stimulation reduces both exocytotic and carrier-mediated NE release, whereas alpha(2)-adrenoceptor agonists attenuate NE exocytosis but enhance carrier-mediated NE release. Furthermore, unlike adenosine A(1)-receptors, whose activation reduces both exocytotic and carrier-mediated NE release, H(3)R stimulation is devoid of negative chronotropic and dromotropic effects (i.e., sinoatrial and atrioventricular nodal functions are unaffected). Because excess NE release can trigger severe arrhythmias and sudden cardiac death, negative modulation of NE release by H(3)R agonists may offer a novel therapeutic approach to myocardial ischemia.     

Multiple cellular mechanisms mediate the effect of lobeline on the release of norepinephrine

The complex effect of lobeline on [(3)H]norepinephrine ([(3)H]NE) release was investigated in this study. Lobeline-induced release of [(3)H]NE from the vas deferens was strictly concentration-dependent. In contrast, electrical stimulation-evoked release was characterized by diverse effects of lobeline depending on the concentration used: at lower concentration (10 microM), it increased the release and at high concentration (100 and 300 microM), the evoked release of [(3)H]NE was abolished. The effect of lobeline on the basal release was [Ca(2+)]-independent, insensitive to mecamylamine, a nicotinic acetylcholine receptor antagonist, and to desipramine, a noradrenaline uptake inhibitor. However, lobeline-induced release was temperature-dependent: at low temperature (12 degrees C), at which the membrane carrier proteins are inhibited, lobeline failed to increase the basal release. Lobeline dose dependently inhibited the uptake of [(3)H]NE into rat hippocampal synaptic vesicles and purified synaptosomes with IC(50) values of 1.19 +/- 0.11 and 6.53 +/- 1.37 microM, respectively. Lobeline also inhibited Ca(2+) influx induced by KCl depolarization in sympathetic neurons measured with the Fura-2 technique. In addition, phenylephrine, an alpha(1)-adrenoceptor agonist, contracted the smooth muscle of the vas deferens and enhanced stimulation-evoked contraction. Both effects were inhibited by lobeline. Our results can be best explained as a reversal of the monoamine uptake by lobeline that is facilitated by the increased intracellular NE level after lobeline blocks vesicular uptake. At high concentrations, lobeline acts as a nonselective Ca(2+) channel antagonist blocking pre- and postjunctional Ca(2+) channels serving as a counterbalance for the multiple transmitter releasing actions.     

Na(+)-dependent transporters mediate HCO(3)(-) salvage across the luminal membrane of the main pancreatic duct

To study the roles of Na(+)-dependent H(+) transporters, we characterized H(+) efflux mechanisms in the pancreatic duct in wild-type, NHE2(-/-), and NHE3(-/-) mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na(+)-dependent H(+) efflux in the microperfused duct was inhibited by 1.5 microM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 microM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na(+)-dependent H(+) efflux by approximately 45%. Notably, the remaining luminal Na(+)-dependent H(+) efflux in ducts from NHE3(-/-) mice was inhibited by 50 microM HOE 694. Hence, approximately 55% of luminal H(+) efflux (or HCO(3)(-) influx) in the pancreatic duct is mediated by a novel, HOE 694-sensitive, Na(+)-dependent mechanism. H(+) transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na(+)-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na(+) and HCO(3)(-) to produce a pancreatic juice that is poor in HCO(3)(-) and rich in Cl(-) during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO(3)(-)-rich pancreatic juice.     

Bupropion inhibits nicotine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine and from rat hippocampal slices preloaded with [(3)H]norepinephrine

Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent.     

6-NO(2)-norepinephrine increases norepinephrine release and inhibits norepinephrine uptake in rat spinal synaptosomes

Nitric oxide has been shown to react under physiologic conditions with norepinephrine (NE) to produce 6-nitro-norepinephrine (6-NO(2)-NE), a compound that enhances NE release in the brain. Previous studies suggest that 6-NO(2)-NE is formed in the spinal cord and stimulates spinal NE release to produce analgesia. The purpose of the current studies was to examine the mechanisms by which 6-NO(2)-NE stimulates NE release in the spinal cord. Crude synaptosomes were prepared from spinal cords of male Sprague-Dawley rats and loaded with [(3)H]NE. Incubation of synaptosomes with 6-NO(2)-NE resulted in a release of NE, with a threshold of 1 microM 6-NO(2)-NE and a maximum effect of 30% fractional release. NE transporter inhibitors desipramine and nomifensine blocked NE release from 6-NO(2)-NE, and desipramine exhibited an IC(50) of 9.6 microM. NE release from 6-NO(2)-NE was dependent on external Na(+), but not Ca(2+) or the activity of guanylate cyclase. 6-NO(2)-NE also blocked uptake of [(3)H]NE into synaptosomes, with an IC(50) of 8.3 microM. These data are consistent with a direct action of 6-NO(2)-NE on noradrenergic terminals in the spinal cord to release NE. This action is independent of guanylate cyclase activation, and most likely shares a common mechanism with classic monoamine releasers such as amphetamine that cause direct release of NE from vesicles into the nerve terminal cytoplasm, leading to extracellular release by reverse transport.     

A kinetic study of the ouabain-induced efflux of norepinephrine from the dog saphenous vein

Dog saphenous vein strips were incubated with [3H]norepinephrine ([3H]NE), 1.4 microM, after inhibition of the NE-metabolizing enzymes and extraneuronal uptake, and superfused for up to 290 min. From the 70th min onwards the strips were exposed to 10 microM ouabain, some of them being subject to electrical stimulation from the 140th min onwards. Other strips were exposed to either 1, 10 or 100 microM ouabain from the 70th min onwards. The spontaneous efflux of [3H]NE had a long half-time (156 min), and over 90% of the [3H]NE accumulated did not participate in efflux ("bound fraction"). Ouabain, 10 microM, induced a pronounced increase of the rate of efflux of [3H]NE, which was delayed in its onset and reached a maximum at t = 135 min of superfusion. Increasing the concentration of ouabain decreased both the delay to the beginning of the overflow and the time to maximum efflux and increased the maximum rate of efflux. In Ca(++)-free medium (during the superfusion period), the maximum rate of efflux was lower than in Ca(++)-containing medium, but was attained earlier. The bound fraction amounted to 22% when the efflux was induced by 10 microM ouabain in Ca(++)-containing medium, a value unnaffected by electrical stimulation but reduced markedly by omitting calcium. The results support the view that the efflux of [3H]NE induced by ouabain is delayed and that it is both carrier-mediated and due to exocytosis.     

Characterization of the inhibitory effect of some antidepressant drugs on the outward transport of norepinephrine in the ischemic myocardium

The noradrenergic amine carrier has an important role in mediating the local release of norepinephrine (NE) during myocardial ischemia. The effects of the antidepressant or putative antidepressant drugs desipramine, 2-hydroxy desipramine, nisoxetine, (S)-oxaprotiline and (R)-oxaprotiline with respect to this release mechanism were investigated in the isolated rat heart submitted to total stop-flow ischemia and subsequent reperfusion. During the reperfusion phase a massive efflux of NE was observed, which was reduced in a concentration-dependent manner in the presence of the antidepressant drugs. The potency (pIC50) of the antidepressants in inhibiting the ischemia-induced NE release varied between 9.16 (desipramine) and 6.17 [(R)-oxaprotiline]. On the other hand, lidocaine (10 microM) or clonidine (1 microM) did not reduce the magnitude of the NE efflux. No attenuation in NE efflux could be detected when desipramine (0.1 microM) was present only during the reperfusion period. During repeated periods (3 x 20 min) of ischemia, desipramine (0.1 microM) reduced markedly the loss of NE. In another experiment in the isolated guinea pig heart, desipramine (0.1 microM) was also found to attenuate the ischemia-induced mobilization of NE to the same significant extent as in the isolated perfused rat heart. Chronic treatment (3 weeks) of rats with desipramine as well as acute administration of desipramine to rats (2 hr before the initiation of perfusion) caused a pronounced reduction in the ischemia-induced release of NE in the isolated perfused heart. In conclusion, these experiments strongly suggest that a major part of the ischemia-induced release of NE is mediated by the amine carrier associated with the noradrenergic nerve terminal membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulus-dependent modulation of [(3)H]norepinephrine release from rat neocortical slices by gabapentin and pregabalin

Gabapentin (GBP; Neurontin) has proven efficacy in several neurological and psychiatric disorders yet its mechanism of action remains elusive. This drug, and the related compounds pregabalin [PGB; CI-1008, S-(+)-3-isobutylgaba] and its enantiomer R-(-)-3-isobutylgaba, were tested in an in vitro superfusion model of stimulation-evoked neurotransmitter release using rat neocortical slices prelabeled with [(3)H]norepinephrine ([(3)H]NE). The variables addressed were stimulus type (i.e., electrical, K(+), veratridine) and intensity, concentration dependence, onset and reversibility of action, and commonality of mechanism. Both GBP and PGB inhibited electrically and K(+)-evoked [(3)H]NE release, but not that induced by veratridine. Inhibition by these drugs was most pronounced with the K(+) stimulus, allowing determination of concentration-effect relationships (viz., 25 mM K(+) stimulus: GBP IC(50) = 8.9 microM, PGB IC(50) = 11.8 microM). R-(-)-3-Isobutylgaba was less effective than PGB to decrease stimulation-evoked [(3)H]NE release. Other experiments with GBP demonstrated the dependence of [(3)H]NE release inhibition on optimal stimulus intensity. The inhibitory effect of GBP increased with longer slice exposure time before stimulation, and reversed upon washout. Combination experiments with GBP and PGB indicated a similar mechanism of action to inhibit K(+)-evoked [(3)H]NE release. GBP and PGB are concluded to act in a comparable, if not identical, manner to preferentially attenuate [(3)H]NE release evoked by stimuli effecting mild and prolonged depolarizations. This type of modulation of neurotransmitter release may be integral to the clinical pharmacology of these drugs.     

Structure-activity relationship between guanidine alkyl derivatives and norepinephrine release: site(s) and mechanism(s) of action

The effect of guanidine alkyl derivatives on the evoked release of [3H]norepinephrine [( 3H]NE) from spleen strips was examined. Guanidine, methyl guanidine and N,N-dimethyl guanidine all enhanced the field-stimulated release of [3H]NE 2- to 3-fold, whereas N,N'-dimethyl guanidine and propyl guanidine were without effect. The latter compound blocked the stimulatory effect of an equimolor concentration (4 mM) of guanidine. Guanidine enhanced moderately the field-stimulated release of [3H]NE from spleen strips pretreated with phenoxybenzamine. The efflux of [3H]NE from spleen slices induced by calcium ionophore A-23187 was not altered by guanidine incubation. The effect of guanidine on intracellular calcium movement was also tested by monitoring the effect of the drug on evoked secretion of ATP from human platelets. Guanidine did not modify this release. It is concluded that guanidine and its active structural derivatives augment [3H]NE release by increasing the influx of calcium through the voltage-sensitive calcium channels, but not by the mobilization of intracellular calcium pools. The biochemical basis for the action of the guanidinium cation is discussed.     

N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors regulating hippocampal norepinephrine release. II. Evidence for functional cooperation and for coexistence on the same axon terminal.

The possible interactions between activation of N-methyl-D-aspartic acid (NMDA) receptors and non-NMDA receptors regulating the release of [3H]norepinephrine [( 3H]NE) have been investigated in superfused synaptosomes from rat hippocampus. NMDA--at a concentration (100 microM) which, in a medium containing 1.2 mM Mg++ ions, did not evoke [3H]NE release--acquired releasing activity in the presence of equimolar concentrations of quisqualic acid (QA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. The [3H] NE release evoked by NMDA plus QA in the presence of Mg++ ions was Ca(++)-dependent, partly tetrodotoxin-sensitive, inhibited by clonidine but insensitive to desipramine. The NMDA receptor antagonists D-2-amino-5-phosphonopentanoic acid (D-AP5) and (+)-5-methyl-10,11-dihydro-5-H-dibenzo[a,d]cycloepten-5,10-imine (MK-801) antagonized the NMDA-induced [3H]NE release in Mg(++)-free medium; the IC50 values amounted, respectively, to 81.4 microM and to 1.11 microM. When NMDA was tested in the presence of QA and Mg++ ions, the affinity of D-AP5 was enormously increased (IC50 = 40 nM; i.e., more than 6 orders of magnitude); the affinity of MK-801 was found to be augmented by 350-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the human norepinephrine transporter by cocaine and amphetamine

Certain antidepressant and psychostimulant drugs block the uptake of norepinephrine from the synaptic cleft by inhibiting norepinephrine transporter (NET) function. The effects of chronic occupation of the NET by these drugs on NET expression are poorly understood. We previously described down-regulation of the NET in cultured cells after continuous exposure to the tricyclic antidepressant desipramine. Here, the effects of structurally unrelated NET ligands, cocaine and amphetamine, on levels of NET and on NET function in HEK-293 cells transfected with human NET cDNA were investigated. All drug exposures were followed by incubation in drug-free media before harvesting and assays. Exposure of intact cells to cocaine for 3 days did not significantly affect the B(max) or K(D) of [(3)H]nisoxetine binding to NET in membrane homogenates, and did not alter levels of NET immunoreactivity or NET mRNA. In contrast, incubation of cells with amphetamine significantly reduced [(3)H]nisoxetine binding to NET and levels of NET immunoreactivity in a time-dependent manner, although levels of NET mRNA appeared to be unaffected. Exposures to cocaine or amphetamine resulted in significant reductions of [(3)H]norepinephrine uptake, although the magnitude of the reduction produced by amphetamine was much greater than cocaine. [(3)H]Nisoxetine binding to NET and NET protein levels were also reduced by exposure of cells to high concentrations of norepinephrine, although norepinephrine exposures were accompanied by changes indicative of cellular toxicity. Cocaine and amphetamine have distinctly different effects on NET expression after continuous exposure. The ability of only certain drugs to down-regulate the NET may provide clues to the unique therapeutic effects of antidepressants that are NET ligands.     

Modulatory role of alpha adrenoceptors on the release of [3H]norepinephrine elicited by preganglionic stimulation of the cat superior cervical ganglion

In the isolated cat superior cervical ganglion labeled in vitro with [3H]norepinephrine ([3H]NE), the overflow of radioactivity evoked by preganglionic stimulation at 10 Hz (80 V, 2 msec duration for 5 min) was reduced to 50% of control values by the alpha adrenoceptor agonists clonidine (0.001 microM) and methoxamine (12.0 microM). The alpha adrenoceptor antagonist phenoxybenzamine (2.9 microM) produced a 2-fold increase in the overflow of [3H]NE elicited by nerve stimulation. Preincubation with drugs that reduce the neuronal uptake of norepinephrine in the isolated ganglion (8.8 microM cocaine and 0.33 microM desipramine) did not modify the release of [3H]NE by preganglionic stimulation. However, a higher concentration of desipramine (3.3 microM) produced a 4-fold increase in the overflow of tritium evoked by stimulation. As this concentration of desipramine produced a shift to the right in the concentration-response curve to methoxamine in the isolated nictitating membrane of the cat, the conclusion is drawn that a feedback mechanism mediated through presynaptic alpha adrenoceptors regulates the release of [3H]NE induced by preganglionic stimulation of the cat superior cervical ganglion. In addition, it is suggested that regulatory mechanisms for norepinephrine release by nerve stimulation are not restricted to nerve terminals but are also present in dendrites of the postganglionic adrenergic neurons.     

Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule

The proximal convoluted tubule (PCT) reabsorbs most of the filtered bicarbonate. Proton secretion is believed to be mediated predominantly by an apical membrane Na(+)/H(+) exchanger (NHE). Several NHE isoforms have been cloned, but only NHE3 and NHE2 are known to be present on the apical membrane of the PCT. Here we examined apical membrane PCT sodium-dependent proton secretion of wild-type (NHE3(+/+)/NHE2(+/+)), NHE3(-/-), NHE2(-/-), and double-knockout NHE3(-/-)/NHE2(-/-) mice to determine their relative contribution to luminal proton secretion. NHE2(-/-) and wild-type mice had comparable rates of sodium-dependent proton secretion. Sodium-dependent proton secretion in NHE3(-/-) mice was approximately 50% that of wild-type mice. The residual sodium-dependent proton secretion was inhibited by 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Luminal sodium-dependent proton secretion was the same in NHE3(-/-)/NHE2(-/-) as in NHE3(-/-) mice. These data point to a previously unrecognized Na(+)-dependent EIPA-sensitive proton secretory mechanism in the proximal tubule that may play an important role in acid-base homeostasis.     

Spermidine reverses arcaine's inhibition of N-methyl-D-aspartate-induced hippocampal [3H]norepinephrine release

The inhibition of N-methyl-D-aspartate (NMDA)-induced [3H]norepinephrine ([3HNE) release by a putrescine analog was studied. We report that arcaine, diguanidinobutane, a putative competitive polyamine antagonist, completely and noncompetitively antagonized NMDA-induced [3H]NE release from rat hippocampal minces with an IC50 value of 102 microM. Arcaine did not alter kainate- or potassium-induced [3H]NE release suggesting a specific effect on NMDA-mediated responses. Spermidine did not alter NMDA-induced [3H]NE release, nor did it reverse the effect of arcaine when introduced in a normal physiologic superfusion buffer. However, spermidine reversed the effect of arcaine when superfusing with buffer that contained 5% (v/v) of the organic solvent dimethylsulfoxide. This finding suggests that the polyamine site may be located at the intracellular surface of the cell membrane. Our results provide the first evidence for polyamine modulation of the NMDA receptor ionophore complex in a functional physiologic system.     

Angiotensin-converting enzyme-independent angiotensin formation in a human model of myocardial ischemia: modulation of norepinephrine release by angiotensin type 1 and angiotensin type 2 receptors

Angiotensin II (Ang II) promotes norepinephrine (NE) release from cardiac sympathetic nerve endings. We assessed in a human model in vitro whether locally formed Ang II contributes to NE release in myocardial ischemia. Surgical specimens of human right atrium were incubated in anoxic conditions. After 70 min of anoxia, NE release (carrier-mediated; caused by NE transporter reversal) was 8-fold greater than normoxic release. Angiotensin-converting enzyme inhibition with enalaprilat failed to reduce anoxic NE release. In contrast, prevention of chymase-dependent Ang II formation with chymostatin, Bowman-Birk inhibitor, or alpha(1)-antitrypsin significantly inhibited anoxic, but not exocytotic, NE release. Two mast-cell stabilizers, cromolyn and lodoxamide, markedly reduced NE release, implicating cardiac mast cells as a major source of chymase. Angiotensin type 1 receptor (AT(1)R) blockade with EXP3174 inhibited NE release, whereas angiotensin type 2 receptor (AT(2)R) blockade with PD123319 did not. Interestingly, PD123319 reversed the inhibitory effect of EXP3174. Furthermore, synergisms were uncovered between EXP3174 and an AT(2)R agonist, and between EXP3174 and a Na(+)/H(+) exchanger inhibitor. Thus, angiotensin-converting enzyme-independent Ang II formation via chymase is important for carrier-mediated ischemic NE release in the human heart. Locally generated Ang II promotes NE release by acting predominantly at AT(1)Rs, which are likely coupled to the Na(+)/H(+) exchanger. Effects of Ang II at AT(2)Rs, seemingly opposite to those resulting from AT(1)R activation, are uncovered when AT(1)Rs are blocked. Because NE release is associated with coronary vasoconstriction and arrhythmias, and mast-cell density and chymase content increase in the ischemic heart, the notion that chymase-generated Ang II plays a major role in carrier-mediated NE release may have important clinical implications.     

The selective serotonin reuptake inhibitor citalopram induces the storage of serotonin in catecholaminergic terminals

We investigated whether selective inhibition of serotonin (5-hydroxytryptamine; 5-HT) transporter with citalopram leads to accumulation of 5-HT in catecholaminergic neurons. In the rabbit olfactory tubercle, citalopram (1-10 microM) inhibited [(3)H]5-HT uptake; however, the maximal degree of inhibition achieved was 70%. Addition of nomifensine (1-10 microM) was required for complete inhibition of [(3)H]5-HT uptake. In slices labeled with 0.1 microM [(3)H]5-HT, cold 5-HT (0.03-1 microM) induced a large increase in the efflux (release) of stored [(3)H]5-HT, an effect blocked by coperfusion with 1 microM citalopram. Similar concentrations (0.03-1 microM) of norepinephrine (NE) or dopamine (DA) failed to release [(3)H]5-HT. When labeling with 0.1 microM [(3)H]5-HT was carried out in the presence of citalopram, 1) low concentrations of 5-HT failed to release [(3)H]5-HT; 2) DA and NE were more potent and effective in releasing [(3)H]5-HT than in control slices; 3) coperfusion of NE, DA, or 5-HT with citalopram enhanced the release of [(3)H]5-HT induced by the catecholamines but not by 5-HT; and 4) coperfusion of NE or DA with nomifensine antagonized NE- and DA-evoked [(3)H]5-HT release, with a greater effect on NE than on DA. These results suggest that in the rabbit olfactory tubercle, where there is coexistence of 5-HT, NE, and DA neurons, inhibition of the 5-HT transporter led to accumulation of 5-HT in catecholaminergic terminals. Thus, during treatment with selective serotonin uptake inhibitors (SSRIs), 5-HT may be stored in catecholaminergic neurons acting as a false neurotransmitter and/or affecting the disposition of DA and/or NE. Transmitter relocation may be involved in the antidepressant action of SSRIs.     

Differential effects of short and prolonged exposure to carvedilol on voltage-dependent Na(+) channels in cultured bovine adrenal medullary cells

We examined the effects of short and prolonged exposure to carvedilol, an antihypertensive and beta-adrenoceptor blocking drug, on voltage-dependent Na(+) channels in cultured bovine adrenal medullary cells. Carvedilol (1-100 microM) reduced (22)Na(+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels. Carvedilol also suppressed veratridine-induced (45)Ca(2+) influx and catecholamine secretion in a concentration-dependent manner similar to that of (22)Na(+) influx. Prolonged exposure of the cells to 10 microM carvedilol increased [(3)H]saxitoxin ([(3)H]STX) binding, which reached a plateau at 12 h and was still observed at 48 to 72 h. Scatchard analysis of [(3)H]STX binding revealed that carvedilol increased the B(max) value (control, 14.9 +/- 0.9 fmol/10(6) cells; carvedilol, 23.8 +/- 1.2 fmol/10(6) cells) (n = 3, P < 0.05) without altering the K(d) value, suggesting a rise in the number of cell surface Na(+) channels. The increase in [(3)H]STX binding by carvedilol was prevented by cycloheximide, an inhibitor of protein synthesis, whereas carvedilol changed neither alpha- nor beta(1)-subunit mRNA levels of Na(+) channels. The carvedilol-induced increase of [(3)H]STX binding was abolished by brefeldin A and H-89, inhibitors of intracellular vesicular trafficking of proteins from the trans-Golgi network and of cyclic AMP-dependent protein kinase (protein kinase A), respectively. The present findings suggest that short-term treatment with carvedilol reduces the activity of Na(+) channels, whereas prolonged exposure to carvedilol up-regulates cell surface Na(+) channels. This may add new pharmacological effects of carvedilol to our understanding in the treatment of heart failure and hypertension.