Research ArticleNEUROPHARMACOLOGY

Multiple Cellular Mechanisms Mediate the Effect of Lobeline on the Release of Norepinephrine

Ernô Sántha, Beáta Sperlágh, Tibor Zelles, Gabriella Zsilla, Péter T. Tóth, Balázs Lendvai, Mária Baranyi and E. Sylvester Vizi
Journal of Pharmacology and Experimental Therapeutics July 2000, 294 (1) 302-307;

Abstract

The complex effect of lobeline on [3H]norepinephrine ([3H]NE) release was investigated in this study. Lobeline-induced release of [3H]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 μM), it increased the release and at high concentration (100 and 300 μM), the evoked release of [3H]NE was abolished. The effect of lobeline on the basal release was [Ca2+]-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°C), at which the membrane carrier proteins are inhibited, lobeline failed to increase the basal release. Lobeline dose dependently inhibited the uptake of [3H]NE into rat hippocampal synaptic vesicles and purified synaptosomes with IC50 values of 1.19 ± 0.11 and 6.53 ± 1.37 μM, respectively. Lobeline also inhibited Ca2+ influx induced by KCl depolarization in sympathetic neurons measured with the Fura-2 technique. In addition, phenylephrine, an α1-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 Ca2+ channel antagonist blocking pre- and postjunctional Ca2+ channels serving as a counterbalance for the multiple transmitter releasing actions.

Although lobeline was originally considered as a pure nicotinic agonist, recent observations suggest that its pharmacological action is more complex than was thought previously. In our earlier study, it was found that the inhibition of carrier-mediated processes possibly underlies the loss of serotonin-releasing effects of lobeline at 7°C in hippocampal slices. Therefore, it has been proposed that lobeline acts by reversal of the monoamine uptake (Lendvai et al., 1996). Teng et al. (1997)observed that lobeline increased dopamine (DA) release via inhibition of vesicular DA uptake, which leads to a depletion of vesicular DA content and to an increase in cytosolic DA level. Furthermore, lobeline was found to be 28-fold more potent to inhibit vesicular [3H]DA uptake than to release [3H]DA from rat striatum (Teng et al., 1998). In contrast to plasma membrane carriers, low temperature does not influence the exocytotic type of transmitter release (for review, seeVizi, 1998). Therefore, using low temperature, the two primary types of transmitter release can be separated.

However, the release process from a nerve terminal is also regulated by several factors, perhaps fluctuation of resting cytoplasmic Ca2+ concentration being most important (Zucker, 1999). Voltage-dependent Ca2+ channel (VDCC) activity regulates transmitter release and significantly contributes to the operation of intracellular [Ca2+] stores. The finding that lobeline (10–300 μM) antagonized the high voltage-activated calcium current in a dose-dependent manner in rat sympathetic neurons suggests that lobeline can cause direct inhibition of VDCCs (Toth and Vizi, 1998). None of these effects fit well to the classic view of nicotinic receptor function, which is thought to mediate Na+, K+, and Ca2+ fluxes on activation. Nevertheless, there is evidence indicating that lobeline is a true nicotinic receptor ligand: 1) because it is able to displace [3H]nicotine binding from central nicotinic receptors with high affinity (Yamada et al., 1985; Lippiello and Fernandes, 1986; Benerjee and Abood, 1989;Broussolle et al., 1989); 2) it has a positive effect on learning and memory (Decker et al., 1993); and 3) it causes tachycardia, hypertension (Teng et al., 1997), and hyperalgesia (Hamann and Martin, 1994), in line with nicotinic receptor-mediated actions.

In this study, we made an attempt to understand further the complex effect of lobeline on transmitter release using different model systems. It was demonstrated that lobeline can induce norepinephrine (NE) release from peripheral tissues using several distinct mechanisms, including the reversal of membrane carrier and the inhibition of vesicular uptake. In addition, these effects are also accompanied by a “brake” mechanism, the inhibition of VDCCs.

Experimental Procedures

Release of Tritiated NE from Guinea Pig Vas Deferens.

The experiments were performed on male guinea pigs (body weight, 200–300 g). The vasa deferentia were excised and incubated for 40 min at 37°C in 1 ml of Krebs' solution containing 1-[7,8-3H]NE (10 μCi/ml, 36.0 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL). Experiments were performed at 37°C in modified Krebs' solution 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 0.03 Na2EDTA, ascorbic acid, 0.3, andd-glucose 12.5, which was continuously saturated with carbogen gas (95% O2 + 5% CO2). After incubation the vasa were transferred to thermoregulated (37°C) organ baths (internal volume, 3 ml). In some experiments, the bath temperature was reduced to 12°C using FRIGOMIX R (B. Braun Biotech International, Allentown, PA) from the sixth fraction (18th min after starting the collection) until the end of the experiment. The preparation was perfused at a rate of 1 ml/min. After 60 min of preperfusion, the outflow was collected in 3-ml (3-min) fractions for an additional 60 min. Tissues were stimulated at 60 V, 8 Hz, 1-ms impulse duration for 1 min (480 pulses), via platinum electrodes using a Grass S88 stimulator during the 3rd and 15th collection periods (S1 and S2). Mecamylamine and desipramine (DMI) were added at the 6th fraction to the perfusion fluid. Lobeline was perfused from the 8th collection period until the end of the experiment. At the end of the perfusion period, the tissue was removed from the organ bath and suspended in 500 μl of 10% trichloroacetic acid. To determine radioactivity released from the tissue, aliquots (0.5 ml) of the perfusate samples were assayed, and a 100-μl aliquot was assayed for tissue radioactivity. Radioactivity was determined in a liquid scintillation counter (Packard 1900; Packard, Meriden, CT). The outflow of tritium was expressed as fractional release (FR), i.e., as the percentage of the amount of radioactivity in the tissue at the time of the release. To calculate electrical field stimulation-induced overflow, the mean of the basal release determined before and after the stimulation was subtracted from the total efflux of radioactivity from the tissue in response to electrical stimulation. The effects of drugs on electrical stimulation-induced outflow were expressed by the calculated ratio of FR S2 over FR S1(FRS2/FRS1). The drug effect on baseline release of [3H]NE was calculated as a ratio of the averages of the FR values of the 13th and 14th fractions (FRR2) and the 1st and the 2nd fractions (FRR1). The mechanical response of the vas deferens smooth muscle was simultaneously recorded with a force displacement transducer and a potentiometric recorder (Omniscribe, Gistel, Belgium) under a resting tension of 1 g.

Hippocampal Synaptic Vesicle Preparation.

The hippocampi of male rats (180–200 g) were dissected out on ice, and the preparation of vesicles was performed according to Erickson et al. (1990). Tissues were pooled and homogenized in 0.32 M sucrose (1:10 g/ml) with a Teflon pestle, and the homogenate was centrifuged at 2000g for 10 min. The supernatant was centrifuged at 10,000g for 30 min at 4°C, and the pellet (buffy coat) was gently suspended in 2 ml of 0.32 M ice-cold sucrose and subjected to osmotic shock with distilled water and homogenized as above. After keeping the preparation on ice for 15 min, the osmolarity was restored by adding of 0.25 M potassium HEPES (pH 6.5) and 1 M potassium tartrate (pH 7.4) in 1/10 volume and centrifuged at 20,000g for 20 min. The supernatant was centrifuged (55,000g for 60 min). The resulting pellet was discarded, and the vesicles were sedimented at 100,000g for 60 min.

Measurement of [3H]NE Uptake into Synaptic Vesicles.

The pellet was suspended in assay buffer (pH 7.4) containing 25 mM HEPES, 100 mM potassium tartrate, 0.1 mM EDTA, 0.05 mM EGTA, 0.5 mM ascorbic acid, and 2 mM magnesium-ATP. In a final volume of 250 μl of assay buffer, an aliquot of the pellet suspension (0.1–0.2 mg of protein) was incubated with 5 nM [3H]NE and with lobeline (0.001–100 μM) for 10 min at 37°C. Uptake was terminated by the addition of 3 ml of ice-cold assay buffer containing 2 mM magnesium sulfate (washing buffer). Samples were filtered with a Brandel cell harvester using GF/B filters soaked in 0.1% polyethylenimine. Filters were washed three times with washing buffer, and the radioactivity trapped on the filters was counted in a Packard scintillation counter. Nonspecific uptake was determined by incubation of the samples at 0°C. The protein content of the preparation was measured by the method of Lowry et al. (1951)using CuEDTA.

Preparation of Synaptosomes from Hippocampus.

Purified synaptosomes were prepared according to the method of Dodd et al. (1981). Hippocampi of male rats (160–180 g) were homogenized in 0.32 M sucrose (1:10 g/ml) by Teflon pestle and centrifuged at 1000g (ω2t = 1.57 × 108 rad2/s). The supernatant was layered onto 1/2 volume of 1.2 M sucrose and centrifuged at 220,000g(ω2t = 1.6 × 1010 rad2/s). The gradient interface was carefully collected, diluted with 0.32 M sucrose, and layered onto 1/2 volume of 0.8 M sucrose and centrifuged as described above. The pellet containing synaptosomes was used for uptake experiments.

Measurement of [3H]NE Uptake into Hippocampal Synaptosomes.

Synaptosomal pellet was suspended in carbogenated Krebs' assay buffer containing 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 10 mM d-glucose, 25 mM NaHCO3, 0.3 mM ascorbic acid, and 0.01 mM pargyline (pH 7.4). Aliquots of synaptosomal suspension were preincubated with lobeline (0.001–100 μM) for 5 min at 37°C in a final volume of 1 ml of assay solution. After preincubation, [3H]NE (5 nM) was added to the tubes and incubation was continued for 5 min. Uptake was terminated by adding 3 ml of assay buffer. Samples were filtered through GF/B filters by using a Brandel cell harvester. Filters were washed with Krebs' solution. Nonspecific uptake was determined by incubation of the samples at 0°C.

Measurement of Intracellular Calcium.

Intracellular calcium measurements were performed on single sympathetic neuronal cells prepared from the superior cervical ganglion of 1- to 3-day-old Wistar rat pups as described by Toth and Miller (1995). The cells, plated on 22-mm diameter coverslips, were loaded with 3 μM Fura-2/AM [(Teflabs, Austin, TX) dissolved in dimethyl sulfoxide containing 20% w/v Pluronic F-127 (Molecular Probes Inc., Eugene, OR)] for 60 min in the dark at room temperature. Cells were then rinsed three times and incubated further for 30 min to complete hydrolysis of acetoxymethyl ester groups. Dye loading, de-esterification, and the entire experiment were performed in a HEPES-buffered salt solution, pH 7.4, composed of 154 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM d-glucose, and 10 mM HEPES. After de-esterification, the coverslips were mounted on a metal recording chamber (Intracell Ltd., Herts, UK) and placed on the stage of a Nikon Diaphot inverted epifluorescence microscope. A Photon Technology International (South Brunswick, NJ) microfluorimetric system was used for the ratio measurements. Fura-2 was excited with light from a 75-W xenon arc lamp, alternately at 340 and 380 nm with a bandwidth of 4 nm through a Nikon Fluor ×100 oil immersion objective. Fluorescence intensity at 510 ± 20 nm was measured by means of a photomultiplier tube. Measurements were limited to a field of view slightly larger than the cells, with a rectangular diaphragm. Background correction was performed before the fluorescence ratio was calculated. Background light levels were determined on a cell-free area. Intracellular calcium levels were expressed as the ratio of fluorescence intensities at 340 to 380 nm excitations. The metal recording chamber contained 1 ml of buffer, and the cells were superfused at a rate of 3 ml/min. Depolarization-induced calcium influx was produced by changing the perfusion solution for 20 s from low K+ (5 mM) to high K+ (50 mM) with K+ exchanged for Na+ reciprocally.

HPLC Analysis.

The concentrations of [3H]NE and its3H-metabolites of the guinea pig vas deferens superfusate fluid were determined using a Gilson HPLC system with electrochemical and liquid scintillation detection. For the separation, Nucleosil 3 C-18 analytical column (150 × 4.0 mm) was used. The mobile phase was 50 mM sodium phosphate, 25 mM citric acid, pH 3.6, 0.25 mM EDTA, 0.75 mM octane sulfonic acid sodium salt, and 5% acetonitrile:methanole (3.5:1.0). The perfusion fluid was centrifuged, and a 750-μl aliquot was mixed with 25 μl of 2.0 μM concentrations of unlabeled standards (dihydroxyphenyl ethylglycol, NE, epinephrine, normetanephrine). The sample was enriched on a guard column in stripping mode. The flow rate was 1.0 ml/min. In each 1-min sample of the effluent, the radioactivity was determined by liquid scintillation counting. The recovery of radiolabeled activity was 91.74 ± 3.74% (n = 28).

Statistics.

The statistical significance of the results was determined with one-way ANOVA followed by Tukey's test;P < .05 was considered significant. All data are expressed as mean ± S.E.

Drugs.

1-[7,8-3H]NE was purchased from Amersham Pharmacia Biotech, lobeline HCl from Sigma Chemical Co. (St. Louis, MO), phenylephrine was from Serva feinbiochemica (Heidelberg/New York), mecamylamine from Erypharm Italiana, DMI HCl from Research Biochemicals International (Natick, MA), and tetrabenazine (TB) from Fluka AG (Buchs, Switzerland). Drugs were prepared on the day of use.

Results

Effect of Lobeline on the Release of [3H]NE from the Vas Deferens.

After 60 min of loading with [3H]NE, followed by a 60-min washout, the tissue uptake was 2312.4 ± 74.7 kBq/g (n = 40). The tritium outflow at rest was 0.228 ± 0.008% of the total content of radioactivity during a 3-min collection period (n = 40). The electrical field stimulation-evoked release of [3H]NE from vasa deferentia was 0.776 ± 0.043% (n = 36). When the stimulation was repeated at the 42nd min, the FRS2/FRS1 ratio was 0.858 ± 0.046 (n = 4) in the control experiment. By the end of the experiment, 0.10 ± 0.003% (n = 4) of the total radioactivity taken up was released. Lobeline (10–300 μM), added 24 min after the beginning of the sample collection period, increased the resting outflow of [3H]NE from vas deferens in a concentration-dependent manner. To examine whether the radioactivity of the samples represents the release of [3H]NE and to further analyze the effect of lobeline, HPLC combined with electrochemical detection separation and subsequent radiochemical detection were performed. The [3H]NE/[3H]3,4-dihydroxyphenylethylene glycol ([3H]DOPEG) ratio was 0.88 ± 0.17 (n = 4) at rest. Electrical field stimulation increased the release of tritiated NE (Fig.1) with a [3H]NE/[3H]DOPEG ratio of 4.26 ± 2.45 (n = 4). Lobeline caused a large tritiated NE release (Fig. 1); however, the [3H]NE/[3H]DOPEG ratio (0.50 ± 0.01; n = 4) was smaller than in the case of baseline outflow or electrical stimulation that indicates the cytoplasmic origin of the lobeline-induced release. Because the release of radioactivity faithfully follows the release of [3H]NE, in the following, we will use the term [3H]NE release.

Figure 1
Figure 1

The released total radioactivity by lobeline (line) indicates the increase of [3H]NE (black bars) and its primary metabolite [3H]DOPEG (gray bars) release from the vas deferens after electrical stimulation (S1) and administration of lobeline. [3H]NE and [3H]DOPEG content of the release samples are determined using HPLC separation combined with radiochemical detection and expressed as disintegrations per minute in samples. Note that lobeline (100 μM) evokes a [3H]NE release comparable in magnitude with the one induced by electrical field stimulation (60 V, 8 Hz, for 1 min). The release of metabolites also increases as a sign of elevated cytoplasmic metabolism. Data represent mean ± S.E. (n = 4 in all groups).

When the temperature of the perfusion fluid was reduced to 12°C, the basal release decreased, whereas the electrical stimulation-evoked release was identical with that obtained at 37°C (Fig.2A), indicating that exocytotic release was not affected by low temperature. In contrast, the effect of lobeline on basal release of [3H]NE was completely inhibited at 12°C (Fig. 2). The nicotinic receptor antagonist mecamylamine (10 μM) did not prevent the effect of lobeline (10 μM) on the basal release of [3H]NE (FRR2/FRR1 was 1.93 ± 0.29; n = 4). Similarly, the monoamine uptake inhibitor DMI (30 μM) failed to alter the effect of lobeline (10 μM) on the basal release of [3H]NE (Fig. 2B). Omitting Ca2+ ions from the perfusion fluid in the presence of EGTA (1 mM) abolished the stimulation-evoked release of [3H]NE by the first electrical stimulation. On the other hand, the effect of lobeline on the resting release of [3H]NE was not changed significantly in Ca2+-free medium (Fig. 2B).

Figure 2
Figure 2

Lobeline reverses the uptake carrier to release [3H]NE. A, time course of the effect of low temperature on the 100-μM lobeline-induced release. Electrical field stimulation (arrowheads) and lobeline induce release of [3H]NE from vas deferens. The large [3H]NE outflow by the application of lobeline at 37°C is completely abolished by lowering the temperature of the perfusate to 12°C. In sharp contrast, the release attributable to electrical stimulation is unaffected. The effect of lobeline on the [3H]NE release is expressed as FR. B, quantification of the effect of various treatment suggests the uptake reversal as basic releasing mechanism. Numbers below the bars represent the used concentrations of lobeline (μM). The ratio of FRR2/FRR1, as a measure for transmitter releasing action of lobeline, is not changed by DMI treatment or in Ca2+-free medium, but low temperature (12°C) abolished the [3H]NE release by lobeline in all concentration used. Results represent mean ± S.E. (n = 4 in all groups) (***P < .001 versus lobeline alone).

Effect of Lobeline on the Uptake of [3H]NE.

The specific uptake of [3H]NE was determined by subtracting the uptake value at 0°C from the total value at 37°C. At a nonsaturating concentration of [3H]NE (5 × 10−9 M), the uptake into rat hippocampal vesicles was 16.61 ± 2.10 fmol/mg of protein/10 min. Lobeline concentration dependently inhibited the uptake of [3H]NE into vesicles with an IC50 value of 1.19 ± 0.11 μM (Fig.3). The specific uptake of [3H]NE into purified rat hippocampal synaptosomes was 387 ± 24.4 fmol/mg of protein/5 min using 5 × 10−9 M NE concentration. Lobeline inhibited the uptake of [3H]NE with an IC50 value of 6.53 ± 1.37 μM (Fig. 3). Furthermore, lobeline (100 μM) was able to decrease significantly the [3H]NE uptake when it was added to the solution during the loading period in guinea pig vas deferens. The tissue uptake of [3H]NE was 2609.5 ± 187.5 kBq/g in the control experiment and 1378.8 ± 56.1 kBq/g with lobeline (P < .001; n = 4). TB, a specific high-affinity inhibitor of the vesicular monoamine transporter in 1 μM concentration, inhibited the uptake of3[H]NE in synaptosomes by 69.2 ± 0.7%. Additional inhibition was not possible to obtain even using 10 μM concentration of the drug (71.1 ± 3.1%), indicating that inhibition of the vesicular uptake of 3[H]NE was complete. Using 10 μM TB plus 5, 10, and 100 μM lobeline, the inhibition was increased further (78.8 ± 2.8%, 88.9 ± 2.8%, P ≥ .01, and 93.9 ± 1.1%, P ≥ .01).

Figure 3
Figure 3

Lobeline inhibits [3H]NE uptake into vesicles and synaptosomes in rat hippocampus. Curves represent averages of two independent experiments. Each concentration was run with three parallels. IC50 values were determined using PRISM 2 (GraphPad Software Inc., San Diego, CA) program using nonlinear regression.

Effect of Lobeline on Prejunctional VDCCs.

When lobeline was applied at 10 μM, it significantly increased the electrical stimulation-evoked release. In contrast, at 100 μM and higher concentrations, it depressed the evoked release of [3H]NE, i.e., the FRS2/FRS1 ratio was 0.01 ± 0.01 in the presence of 100 μM lobeline (Fig.4B). Low temperature did not influence the inhibitory effect of lobeline on stimulation-evoked release.

Figure 4
Figure 4

Lobeline inhibits pre- and postjunctional VDCCs. A, effect of lobeline on high K+ depolarization-induced [Ca2+]i increase using single-cell fluorescence measurements of rat sympathetic ganglion cells. Twenty-second perfusions of 50 mM KCl elicited [Ca2+]i transients. Lobeline (300 μM) inhibited the depolarization-induced [Ca2+]iincrease (horizontal bar). B, the ratio of releases induced by the second and first electrical stimulations (FRS2/FRS1) increases when lobeline is used in 10 μM, showing a facilitation of VDCCs. FRS2/FRS1 decreases at a higher concentration of lobeline, suggesting an evolving inhibition on prejunctional VDCCs. C, effect of lobeline on contraction of guinea pig vas deferens. Phenylephrine (100 μM) potentiated the stimulation-indicated contraction and the induced spontaneous contractions (C/a). When phenylephrine (100 μM) and lobeline (300 μM) were applied together, the effect of phenylephrine on the postjunctional site was blocked (C/b). (*P < .05; ***P < .001 versus control).

Effect of Lobeline on Ca2+-Influx.

Perfusion of the sympathetic neuronal cells with 50 mM K+increased the intracellular Ca2+ concentration ([Ca2+]i) in a repeatable manner (Fig. 4A) by producing depolarization-induced Ca2+ influx (Thayer et al., 1988). Lobeline (300 μM), in the highest concentration used in the release experiments and which caused nearly maximal inhibition of Ca2+channels (Toth and Vizi, 1998), inhibited the K+depolarization-evoked [Ca2+]i increase in Fura-2 loaded cells. The effect of lobeline was reversible (Fig. 4A).

Effect of Lobeline on Mechanical Responses of Vas Deferens.

The effect of lobeline on the contraction of the vas deferens produced by field stimulation and phenylephrine was also examined. Electrical field stimulation at 4 Hz and 480 shocks resulted in a biphasic response that is known to be the result of the cotransmitter action of ATP and NE (Vizi et al., 1992). The electrical stimulation-induced response was completely prevented by lobeline. Phenylephrine, an α1-agonist, increased smooth muscle tone and spontaneous activity and increased biphasic response to field stimulation (Fig. 4C/a). When lobeline (300 μM) was added to the perfusion solution 6 min before phenylephrine (100 μM), the α1-agonist was unable to induce contraction or to enhance the contraction evoked by electrical stimulation. These findings indicate that lobeline prevented the direct effect of phenylephrine on the smooth muscle, i.e., on postjunctional α1-receptors (Fig. 4C/b).

Discussion

In this study, we have shown various aspects of pre- and postjunctional effects of lobeline to the release of [3H]NE. Although previous studies have suggested that the uptake of NE (Lindmar and Loffelholz, 1972) and carrier-mediated neurotransmitter release (Vizi et al., 1985, 1986) are temperature-dependent, it has just recently been recognized that 12°C is the cut-off point at which exocytotic and carrier-mediated release can be separated (Vizi, 1998; Vizi and Sperlagh, 1999). In agreement with this theory, our experiments showed that electrical stimulation-evoked release of [3H]NE, known to be the result of vesicular exocytosis, remained unaffected at 12°C. In contrast, the direct transmitter-releasing effect of lobeline was low temperature-sensitive. Therefore, it is reasonable to suggest that the action of lobeline involves a carrier-mediated outward transport of [3H]NE by a reversal of the flow of carrier-mediated monoamine uptake in the presynaptic membrane. Simple inhibition of the plasma membrane carrier by lobeline can be excluded because low temperature would then further increase the release or, at least, cause no change.

In agreement with the findings of Teng et al. (1997, 1998) on dopaminergic neurons, our data also suggest that lobeline inhibits vesicular catecholamine uptake that most likely results in a cytoplasmic NE accumulation in the nerve terminal. Our results that IC50 values for vesicular and synaptosomal [3H]NE uptake are in the same order of magnitude suggest that the inhibitory effect of lobeline on synaptosomal uptake is primarily attributable to the vesicular inhibition because the synaptosomal preparation includes synaptic vesicles, and the effect of lobeline on uptake must include it. However, the finding that lobeline has a surplus inhibitory effect on NE uptake after TB treatment needs explanation. When the direction of the NE transport has already been changed by lobeline, it is assumed that the transporter is not ready to bind the released transmitters on the extracellular side. This should result in an apparent inhibition of the synaptosomal uptake flow after the vesicles have been depleted by TB. There are drugs such as tyramine (Driessen et al., 1996) and histamine (Boudreau and Vohra, 1991) that can enter the cell using the membrane uptake carrier to evoke NE release. The uptake of tyramine and histamine can be blocked by DMI. The fact, that the monoamine uptake blocker DMI did not influence the effect of lobeline clearly shows that lobeline operates the carrier in a mode that is different from the one sensitive to DMI. As a highly lipophilic compound, lobeline can pass the membrane via passive diffusion. The failure of DMI to inhibit the effect of lobeline not only shows the route used by lobeline to enter the cell, but also that the carrier cannot be blocked by DMI when NE transport is reversed by lobeline. Although cytoplasmic NE is available for the monoaminooxidase enzyme, a significant amount of [3H]NE must have escaped from intracellular metabolism, because not only the metabolites but also NE itself considerably contribute to the released radioactivity in response to lobeline (Fig. 1). Therefore, the cellular-releasing effect of lobeline is based on multiple sites: first, it increases the releasable pool of NE in the cytoplasm; second, lobeline enhances the release from the cytoplasmic transmitter pool by the reversal of the plasma membrane uptake (Fig.5).

Figure 5
Figure 5

Schematic drawing of the concept of how lobeline can induce release in multiple ways. The effect of lobeline on the uptake carrier and vesicular uptake corroborate each other. Blockade of the vesicular uptake would increase cytoplasmic NE, whereas uptake reversal in plasma membrane releases NE from this transmitter pool. In contrast to these enhancing effects, lobeline could also inhibit VDCCs at a higher concentration. In addition, lobeline can also prevent the effect of the released transmitter on the postjunctional site by inhibiting the smooth muscle VDCCs. Based on literature data, the putative binding of lobeline on nAChRs is also included.

The question arises whether lobeline acted on the nicotinic acetylcholine receptor(s) (nAChRs). It is well known that stimulation of nAChR causes relatively high calcium influx (Patrick et al., 1993;Seguela et al., 1993). The nAChR-induced release of NE from the hippocampus is [Ca2+]o-dependent (Vizi et al., 1995). Lobeline is able to bind to the central nicotinic receptors with very high affinity (Yamada et al., 1985; Lippiello and Fernandes, 1986; Benerjee and Abood, 1989; Broussolle et al., 1989). On the other hand, lobeline-induced release was independent on the extracellular Ca2+ level in our experiments, and the effect of lobeline was insensitive to mecamylamine, a nicotinic receptor antagonist. Similarly, lobeline-induced outflow has been shown to be mecamylamine-insensitive in the hippocampus (J. P. Kiss, K. Windisch, and E. S. Vizi, unpublished data) and striatal slices (Teng et al., 1997). The participation of the mecamylamine-insensitive α7-nAChR in the effect of lobeline also seems unlikely, because lobeline acts as an antagonist on human α7-nAChR expressed inXenopus oocytes (Briggs and McKenna, 1998). In sharp contrast, NE release, induced by other nicotinic agonists such as 1,1-dimethyl-4-phenylpiperazinium and nicotine, can be reversed by nicotinic antagonists in the vas deferens (Todorov et al., 1991) and hippocampus (Kiss et al., 1997; Sershen et al., 1997). Therefore, it appears that lobeline does not behave as a classic nicotinic agonist on the NE release in the vas deferens.

Different types of Ca2+ channels take part in the releasing process in sympathetic neurons of vas deferens that involves N-, P-, and Q-type VDDCs (Waterman, 1997). In our experiments, high concentrations (100 and 300 μM) of lobeline inhibited [3H]NE release evoked by electrical stimulation. Lobeline inhibited Ca2+ influx induced by KCl in cultured sympathetic neurons measured by the Fura-2 technique, indicating that lobeline could inhibit the somatic VDCCs. Our results support the recent observations that lobeline (10–300 μM) antagonized the high voltage-activated calcium current in a dose-dependent manner using whole-cell patch clamp in rat sympathetic neurons (Toth and Vizi, 1998). The inhibition of these channels by lobeline can stop release-activating mechanisms and appears as an opposing effect of the releasing action of lobeline. One advantage of the vas deferens preparation is the possibility of the simultaneous recording of smooth muscle contractions and the release of [3H]NE that shows pre- and postjunctional effects at the same time. Electrical nerve stimulation results in biphasic contraction mediated by the cotransmitter action of ATP and NE, released from sympathetic nerve terminals (Kasakov et al., 1988;Vizi et al., 1992) and acting on postjunctional α1-adrenoceptors and P2xpurinoceptors (Burnstock, 1990). Lobeline abolished the smooth muscle contraction evoked by electrical field stimulation consistently with its suggested presynaptic VDCC inhibitory effect. Because muscle contraction by the α1-adrenoceptor agonist phenylephrine, which acts on the postjunctional site, can also be prevented by lobeline, it seems reasonable to conclude that lobeline inhibits both pre- and postjunctional VDCCs.

Together, the results of our experiments show that two primary routes converge after the application of lobeline to release NE from the cytoplasm, including a vesicular uptake inhibition and a reversal of the plasma membrane carrier. At a higher concentration range (100–300 μM), blockade of the pre- and postsynaptic VDCCs contributes to the complex action of lobeline.

Footnotes

  • Send reprint requests to: Dr. E. Sylvester Vizi, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Szigony u. 43., H-1450 P.O. Box 67, Hungary. E-mail: esvizi{at}koki.hu

  • ↵1 This study was supported in part by the Hungarian Research Fund (OTKA) (T022450, T029859), Hungarian Medical Research Foundation (194/96, 195/96, 53/98), and a Philip Morris research grant.

  • Abbreviations:
    DA
    dopamine
    VDCC
    voltage-dependent Ca2+ channel
    NE
    norepinephrine
    FR
    fractional release
    TB
    tetrabenazine
    nAChR(s)
    nicotinic acetylcholine receptor(s)
    [Ca2+]i
    intracellular calcium concentration, DMI, desipramine
    DOPEG
    3,4-dihydroxyphenylethylene glycol
    • Received December 10, 1999.
    • Accepted April 3, 2000.

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Research ArticleNEUROPHARMACOLOGY

Multiple Cellular Mechanisms Mediate the Effect of Lobeline on the Release of Norepinephrine

Ernô Sántha, Beáta Sperlágh, Tibor Zelles, Gabriella Zsilla, Péter T. Tóth, Balázs Lendvai, Mária Baranyi and E. Sylvester Vizi
Journal of Pharmacology and Experimental Therapeutics July 1, 2000, 294 (1) 302-307;
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