(Hypertension. 1996;28:414-420.)
© 1996 American Heart Association, Inc.
Articles |
Vesicular Monoamine Transport Inhibitors
Novel Action at Calcium Channels to Prevent Catecholamine Secretion
the Department of Medicine and Center for Molecular Genetics, University of California, and Department of Veterans Affairs Medical Center, San Diego, Calif.
Correspondence to Daniel T. O'Connor, MD, Department of Medicine (9111H), University of California, San Diego, 3350 La Jolla Village Dr, San Diego, CA 92161. E-mail doconnor@ucsd.edu.
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Abstract |
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Vesicular monoamine transport (VMAT) inhibitors, such as reserpine and tetrabenazine, impair vesicular catecholamine storage in chromaffin cells and sympathetic neurons, thereby lowering blood pressure. Here we describe a novel action of VMAT inhibitors—blockade of L-type voltage-gated calcium channels—that may also influence catecholamine release from both PC12 rat pheochromocytoma cells and bovine adrenal chromaffin cells. When given alone, VMAT inhibitors acutely release catecholamines from chromaffin cells in a dose-dependent fashion. However, VMAT inhibitors block catecholamine secretion stimulated by either nicotinic cholinergic agonists or cell membrane depolarization, each of which rely on the opening of L-type channels; the inhibition was more potent after long-term exposure to VMAT inhibitors (IC50 <100 nmol/L). Reserpine blocked nicotinic-stimulated catecholamine release from neurite-bearing PC12 cells. Reserpine also antagonized catecholamine release triggered by combined membrane depolarization and the dihydropyridine L-type channel agonist Bay K8644, and reserpine blocked cellular uptake of extracellular 45Ca2+ in response to nicotine. Taken together, these results indicate that VMAT inhibitors are also antagonists at L-type voltage-gated calcium channels. Classic L-type channel antagonists (verapamil or nifedipine) also exhibited the reciprocal actions; acutely, they released norepinephrine from chromaffin cells, and chronically, they depleted cellular catecholamine stores, albeit with inferior molar potency to reserpine (IC50 <1 nmol/L). We conclude that VMAT inhibitors and L-type calcium channel antagonists exert reciprocal inhibitory actions on each other's more classic pharmacological targets. Furthermore, these novel actions are seen at concentrations of these compounds frequently taken to be specific in vitro and likely to occur during antihypertensive treatment in vivo.
Key Words: reserpine • tetrabenazine • catecholamines • calcium channels • calcium antagonists • dihydropyridines • PC12 cells
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Introduction |
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Reserpine has been used for decades as an antihypertensive agent.1 2 3 4 5 6 7 Its mechanism of action is usually characterized as catecholamine depletion from sympathetic nerve endings and chromaffin cells, leading to diminished catecholamine release during sympathoadrenal secretion.8 9 10 11 12 Reserpine and its congeners (such as tetrabenazine) cause catecholamine depletion through competitive inhibition of catecholamine transport from cytosol into storage vesicles, acting as antagonists at the vesicular monoamine transporter (VMAT) in the membrane of the storage organelle.13 14 Acute catecholamine release is followed by chronic inhibition of catecholamine secretion as a result of diminished releasable vesicular catecholamine stores.8 9 10 11
While studying the catecholamine-depleting effects of VMAT inhibitors in chromaffin cells, we noticed an unexpected and apparently paradoxical action of the compounds: inhibition of secretagogue-stimulated catecholamine release, reminiscent of the action of calcium channel antagonists.15 16 17 18 19 20 We characterized this effect further and found that VMAT inhibitors block such catecholamine release by acting as noncompetitive antagonists at cell surface L-type voltage-gated calcium channels. To generalize these findings, we studied two VMAT inhibitors (reserpine and tetrabenazine) in two cell systems (PC12 rat pheochromocytoma cells and bovine adrenal chromaffin cells) as well as in neurite-bearing PC12 cells. This action of reserpine and the VMAT inhibitors has implications for their antihypertensive mechanisms.
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Cell Culture
Rat pheochromocytoma PC12 cells21 (passage 8; obtained from Dr David Schubert, Salk Institute, La Jolla, Calif) were grown at 37°C/6% CO2 in 10-cm plates or six-well plates and Dulbecco's modified Eagle's high-glucose medium supplemented with 5% fetal bovine serum, 10% horse serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Bovine adrenal chromaffin cells were prepared and grown in primary culture as previously described.22
Secretagogue-Mediated Norepinephrine Release
Norepinephrine secretion was monitored as previously described.23 PC12 cells were plated on poly-D-lysine–coated polystyrene dishes (Falcon Labware), labeled for 3 hours with 1 µCi L-[3H]norepinephrine (71.7 Ci/mmol, DuPont-NEN) in 1 mL of PC12 growth medium, washed twice with release buffer ([mmol/L] NaCl 150, KCl 5, CaCl2 2, HEPES 10, pH 7), and incubated at 37°C for 30 minutes in release buffer with or without secretagogues. Secretagogues included nicotine (60 µmol/L), cell membrane depolarization (55 mmol/L KCl), and the L-type calcium channel agonist Bay K8644 (1 µmol/L). The release buffer for experiments involving KCl as secretagogue had NaCl reduced to 100 mmol/L for maintenance of tonicity. After 30 minutes, secretion was terminated by aspiration of the release buffer and lysing of cells into 150 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L HEPES (pH 7), and 0.1% (vol/vol) Triton X-100. Release buffer and cell lysates were assayed for L-[3H]norepinephrine by liquid scintillation counting, and results were expressed as percent secreted (Amount Released/[Amount Released+Amount in Cell Lysate]x100).
Vesicular Norepinephrine Uptake and Storage
PC12 cells were seeded and labeled with L-[3H]norepinephrine as described above. Ascorbic acid (0.1 mmol/L) was added to the medium each day to prevent extracellular catecholamine oxidation. After 2 days of treatment with reserpine (versus control), medium and cell lysates were prepared for norepinephrine assay by liquid scintillation counting. Data were expressed as percent uptake (Amount in Cell Lysate/[Amount in Release Medium+Amount in Cell Lysate]x100).
45Ca2+ Uptake
Cells were seeded onto poly-D-lysine–coated six-well polystyrene culture dishes 2 days before assay. Cells were rinsed with 1 mL of release buffer and preincubated with 1 mL of release buffer for 30 minutes at 37°C. Then the cells were incubated for 5 minutes at 37°C in 1 mL of release buffer (without calcium) containing 2 µCi of 45Ca2+ (14.95 mCi/mg, DuPont-NEN). The drugs tested were present in the release buffer during the 5-minute incubation period. Calcium uptake was stopped simultaneously in all six wells by inversion of the plate, so all wells were decanted simultaneously, followed promptly by addition of ice-cold release buffer containing 2 mL of 1 mmol/L LaCl3 for termination of further uptake of extracellular labeled calcium.24 The culture dishes were then rinsed twice with ice-cold release buffer. One milliliter of cell lysis buffer was added to cells in each well and collected for liquid scintillation counting. The data were expressed as counts per minute per well.
Differentiation of PC12 Cells With Nerve Growth Factor
PC12 cells were split to 50% confluence and then treated with nerve growth factor (2.5S form, Boehringer Mannheim, 100 ng/mL). The medium was changed every other day, with nerve growth factor added to the new medium. After 5 days of treatment, the neurite-bearing cells were used for secretion studies as described before.
Radioligand Binding
Binding to L- or N-type voltage-gated calcium channels was tested through the National Institute of Mental Health (NIMH)/NOVASCREEN Psychotherapeutic Drug Discovery and Development Program (Contract No. N01NIMH-20003; Novascreen, Baltimore, Md). Reserpine was dissolved in neat dimethyl sulfoxide, which was diluted to a final concentration of 0.4% or less in the binding assays.
Nitrendipine competitive binding assays25 (for L-type channels) were performed in 0.8 vol of receptor preparation from rat brain cortical membranes, 0.1 vol radioligand ([3H]nitrendipine [70 to 87 Ci/mmol; final concentration, 0.2 nmol/L]), and 0.1 vol of the test compound (reserpine)/cold ligand (nonspecific binding determinant)/4% dimethyl sulfoxide (total binding determinant). Binding reactions were carried out for 60 minutes at 25°C in 50 mmol/L Tris-HCl, pH 7.7. Reactions were terminated by rapid vacuum filtration onto glass fiber filters (Whatman) followed by rapid washing with cold buffer. Radioactivity trapped onto the filters was determined by liquid scintillation (3H) counting and compared with control values for ascertainment of any interactions of reserpine with the nitrendipine binding sites. Specific binding (determined by displacement with 1 µmol/L nifedipine) represented 85% of total binding. In this assay, the Bmax was 166 fmol/mg tissue wet weight, with a Kd of 0.2 nmol/L. Inhibition constants (Ki) (nanomoles per liter) for reference compounds were nifedipine, 0.89; saxitoxin, 13.8, 
-conotoxin, >10 000, and apamin, >10 000.
-Conotoxin competitive binding assays26 (for N-type channels) were performed in 0.8 vol of receptor preparations from rat brain cortical membranes, 0.1 vol of radioligand (125I–-conotoxin GVIA [2000 Ci/mmol; final concentration, 10 pmol/L]), and 0.1 vol of the test compound (reserpine)/cold ligand (nonspecific binding determinant)/4% dimethyl sulfoxide (total binding determinant). Binding reactions were carried out for 30 minutes at 25°C in 50 mmol/L HEPES, pH 7.4, containing 0.2% bovine serum albumin. Reactions were terminated as noted above, and radioactivity trapped onto the filters was determined by gamma spectrometry (125I) and compared with control values for ascertainment of any interactions of reserpine with the -conotoxin binding site. Specific binding (determined by displacement with 100 nmol/L -conotoxin GVIA) represented 75% of total binding. In this assay, the Bmax was 23.0 fmol/mg tissue wet wt, with a Kd of 0.01 nmol/L. Ki values (nanomoles per liter) for reference compounds were -conotoxin GVIA, 0.06; and verapamil, nitrendipine, and diltiazem, all >100 000. Ki values were derived by the Cheung-Prusoff equation: 
Statistics
Results are shown as mean±SE. Group values were compared by ANOVA or t test, as appropriate.
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Results |
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Acute (30-minute) exposure to reserpine released prestored L-[3H]norepinephrine from both PC12 (rat pheochromocytoma) and bovine chromaffin cells in a reserpine dose–dependent fashion (Fig 1
). Tetrabenazine also acutely released norepinephrine, although with less potency at higher doses (0.1 to 10 µmol/L; data not shown).
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When administered chronically (for 28 hours), the catecholamine-releasing action of reserpine on PC12 cells was even more prominent than its acute effects, especially at the lower (1 to 100 nmol/L) doses tested (Fig 2).
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In the setting of nicotinic-stimulated catecholamine release from chromaffin cells, reserpine had very different effects on secretion, depending on its dose and the type of chromaffin cell studied (Fig 3). Nicotine (60 µmol/L) typically provoked net secretion of approximately 25% of cell total stores of [3H]norepinephrine during a 30-minute incubation. At a high dose (10 µmol/L), reserpine inhibited nicotinic-stimulated catecholamine release in both PC12 and bovine chromaffin cells. At lower doses (1 to 100 nmol/L), reserpine added to nicotinic release from PC12 although not from bovine chromaffin cells. In this same setting of nicotinic-stimulated release, tetrabenazine produced dose-dependent (0.001 to 10 µmol/L) inhibition of release in both chromaffin cell types (data not shown).
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The effects of reserpine on nicotinic-stimulated catecholamine release from PC12 cells depended on the duration of reserpine exposure (Fig 4). After prolonged (28-hour) preexposure of PC12 cells, reserpine caused only dose-dependent inhibition (rather than any stimulation at any dose) of nicotinic-triggered catecholamine release, with a half-maximal inhibitory concentration of less than 100 nmol/L reserpine; this contrasted with the effect of acute (coexposure with nicotine) reserpine to augment nicotinic-stimulated catecholamine secretion at low doses (1 to 100 nmol/L reserpine) (Figs 3 and 4
).
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The inhibition of nicotinic-stimulated catecholamine secretion by reserpine was shared by the L-type voltage-gated calcium channel antagonists nifedipine and verapamil (Figs 5 and 6). In PC12 cells, the calcium channel antagonist nifedipine (IC50~0.088 µmol/L) was a far more effective antagonist than either verapamil (IC50~5.39 µmol/L) or reserpine (IC50~5.98 µmol/L). The three antagonists exhibited dose-dependent inhibition, over the dose range from 1 nmol/L to 10 µmol/L, in bovine chromaffin cells; in the bovine chromaffin cell system, the relative potencies of the three inhibitors were nifedipine (IC50~0.0008 µmol/L)>verapamil (IC50~0.033 µmol/L)>reserpine (IC50~2.65 µmol/L).
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All three inhibitors (reserpine, nifedipine, and verapamil) also antagonized norepinephrine release triggered by cell membrane depolarization with 55 mmol/L KCl (Fig 7), especially at a high dose (10 µmol/L).
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In nerve growth factor–differentiated (neurite-bearing) PC12 cells, actions of reserpine were similar to those in undifferentiated PC12 cells: In the absence of nicotine, 10 µmol/L reserpine acutely released catecholamines (net release, 4.25±0.5% of cell stores over 30 minutes); 60 µmol/L nicotine alone released 40.7±0.5% of cell stores, and the addition of 10 µmol/L reserpine to nicotine was inhibitory (reducing secretion to 29.8±0.8% of cell stores). Neurite-bearing PC12 cells displayed more nicotine-induced norepinephrine secretion (net secretion of 40.7% of cell total norepinephrine stores in response to nicotine) than did untreated PC12 cells (net secretion of ~25% of cell total norepinephrine stores in response to nicotine). When nerve growth factor–differentiated PC12 cells were depolarized with 55 mmol/L KCl (Fig 8), both reserpine and nifedipine inhibited membrane depolarization–induced secretion; by contrast, the N-type calcium channel–specific antagonist -conotoxin was without effect.
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We used the dihydropyridine Bay K8644, an agonist (channel opener) at L-type voltage-gated calcium channels, to augment the catecholamine secretory response to membrane depolarization in PC12 cells (augmented by ~37% at 1 µmol/L Bay K8644; Fig 9). In this experiment, each inhibitor (reserpine, verapamil, and nifedipine; each at 10 µmol/L) antagonized the dose-dependent augmentation by Bay K8644 of norepinephrine release; for verapamil and reserpine, the antagonism was most apparent at lower doses of Bay K8644 (1 to 10 nmol/L), whereas the dihydropyridine nifedipine was an effective antagonist even at the highest Bay K8644 concentration (1 µmol/L).
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All three antagonists (reserpine, verapamil, and nifedipine; each at 10 µmol/L) blocked nicotine-stimulated 45Ca2+ uptake by PC12 cells (Fig 10).
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Reserpine did not displace the binding of either -conotoxin to brain membrane N-type calcium channels (only 1.5% inhibition of 125I–-conotoxin binding by 10 µmol/L reserpine) or nitrendipine to brain membrane L-type calcium channels (no inhibition of [3H]nitrendipine binding by 10 µmol/L reserpine).
Acute (30-minute) exposure of [3H]norepinephrine-loaded PC12 cells to not only reserpine but also verapamil and nifedipine released norepinephrine stores in a dose-dependent fashion, with a rank order of potency of reserpine>verapamil>nifedipine (Fig 11).
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Chronic (48-hour) exposure of PC12 cells to reserpine, verapamil, or nifedipine also depleted cellular norepinephrine stores in a dose-dependent fashion (Fig 12). In this setting, reserpine was clearly more potent than verapamil and nifedipine, especially at lower doses (1 to 100 nmol/L).
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Discussion |
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Taken together, the studies on secretory inhibition (Figs 3 and 4
) by VMAT inhibitors suggest that the inhibitory mechanism is blockade of L-type voltage-gated calcium channels. Nicotinic cholinergic agonists (eg, nicotine [Figs 3 and 4]) as well as membrane depolarization (by 55 mmol/L KCl [Fig 7]) each require calcium entry through such channels in triggering catecholamine secretion from chromaffin cells.23 27 28 29 Like the L-type calcium channel antagonists verapamil and nifedipine, reserpine and tetrabenazine inhibited norepinephrine secretion as induced by both nicotine (Figs 3 through 6) and membrane depolarization (Fig 7). Since secretion of catecholamines in response to such stimulation requires extracellular calcium influx,27 we examined 45Ca2+ uptake and found substantial blockade of nicotinic-stimulated 45Ca2+ uptake by not only nifedipine and verapamil but also reserpine. Finally, we found that reserpine, as well as established calcium channel antagonists, inhibited secretory stimulation by the L-type channel dihydropyridine agonist Bay K8644 (Fig 9).
Previous studies indicate that reserpine may have calcium channel blocker–like activity in vascular,30 intestinal,30 and uterine31 smooth muscle. Our studies were done on isolated chromaffin cells in culture. Is L-type calcium channel inhibition by reserpine likely to occur in vivo, perhaps during antihypertensive treatment? Peak plasma reserpine concentrations during human antihypertensive therapy are in the range of 0.4 to 0.6 ng/mL, or approximately 0.7 to 1.0 nmol/L,32 and reserpine is further concentrated in adrenergic tissues.33 Since we found dose-dependent inhibition of nicotinic-stimulated catecholamine release at reserpine concentrations down to 1 nmol/L (Figs 3 and 4), blockade of L-type channels by reserpine may well occur during human antihypertensive therapy.
Since reserpine does not displace [3H]nitrendipine from L-type calcium channels, it must interact with a site other than that on the 
-subunit of the channel occupied by dihydropyridines.34 The L-type channel is a multimeric structure,35 raising the additional possibility that other subunits of the channel might be targets for novel antagonists such as the VMAT inhibitors.
We obtained no evidence that VMAT inhibitors interact with N-type calcium channels; for example, reserpine did not displace 125I–-conotoxin from N-type calcium channels. However, L-type channel antagonists effectively blocked catecholamine secretion in these chromaffin cell studies (Fig 8), and even neurite-bearing (nerve growth factor–differentiated) PC12 cells did not exhibit inhibition of membrane depolarization–stimulated catecholamine secretion by the N-type–selective channel antagonist -conotoxin (Fig 8). Hence, the chromaffin cell secretory systems we studied may not be informative for actions on N-type channels.
If VMAT inhibitors exhibit L-type calcium channel blockade, is the converse also true? That is, do L-type calcium channel antagonists inhibit catecholamine vesicular storage? Acutely, both reserpine and L-type channel antagonists released norepinephrine from its vesicular stores in chromaffin cells (Fig 11); chronically, each agent depleted PC12 cells of stored norepinephrine, although reserpine had clearly superior molar potency for this effect (Fig 12). Terland et al20 also reported that calcium channel antagonists (including verapamil and the dihydropyridines amlodipine and nicardipine) diminished catecholamine storage in chromaffin granules by inhibiting development of the granule transmembrane proton gradient; however, the concentrations required in those studies (IC50 values ~12 to 150 µmol/L) were substantially higher than those required for the effects we report (Figs 11 and 12): We found dose-dependent acute catecholamine release (Fig 11) and chronic catecholamine depletion (Fig 12) by verapamil and nifedipine in the range of 1 nmol/L to 10 µmol/L. During oral administration to humans,36 peak plasma concentrations of verapamil are approximately 300 ng/mL (or ~0.7 µmol/L), and those of nifedipine are approximately 175 ng/mL (or ~0.5 µmol/L). Thus, the catecholamine storage–depleting actions of calcium channel antagonists may well occur during antihypertensive treatment in humans. Indeed, during antihypertensive treatment in humans,37 we found that the plasma concentration of norepinephrine rose acutely after felodipine and declined after chronic verapamil, actions consistent with vesicular catecholamine depletion and reminiscent of the time course of plasma norepinephrine changes seen during acute versus chronic reserpine treatment of human hypertension.12
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Acknowledgments |
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This study was supported by the Department of Veterans Affairs, the National Institutes of Health, and the American Heart Association. We gratefully acknowledge the NIMH/NOVASCREEN Psychotherapeutic Drug Discovery and Development Program (Contract No. N01NIMH20003) for in vitro receptor binding assays.
Received March 27, 1996; first decision April 19, 1996; accepted May 21, 1996.
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 S. K. Mahata, M. Mahata, A. R. Wakade, and D. T. O’Connor Primary Structure and Function of the Catecholamine Release Inhibitory Peptide Catestatin (Chromogranin A344-364): Identification of Amino Acid Residues Crucial for Activity Mol. Endocrinol., October 1, 2000; 14(10): 1525 - 1535. [Abstract] [Full Text]
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M. L. Mundorf, K. P. Troyer, S. E. Hochstetler, J. A. Near, and R. M. Wightman Vesicular Ca2+ Participates in the Catalysis of Exocytosis J. Biol. Chem., March 24, 2000; 275(13): 9136 - 9142. [Abstract] [Full Text] [PDF]
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 L. Taupenot, M. Mahata, S. K. Mahata, and D. T. O’Connor Time-Dependent Effects of the Neuropeptide PACAP on Catecholamine Secretion : Stimulation and Desensitization Hypertension, November 1, 1999; 34(5): 1152 - 1162. [Abstract] [Full Text] [PDF]
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M. Mahata, S. K. Mahata, R. J. Parmer, and D. T. O'Connor Proadrenomedullin N-Terminal 20 Peptide : Minimal Active Region to Regulate Nicotinic Receptors Hypertension, November 1, 1998; 32(5): 907 - 916. [Abstract] [Full Text] [PDF]
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C. V. Taylor, L. Taupenot, S. K. Mahata, M. Mahata, H. Wu, S. Yasothornsrikul, T. Toneff, C. Caporale, Q. Jiang, R. J. Parmer, et al. Formation of the Catecholamine Release-inhibitory Peptide Catestatin from Chromogranin A. DETERMINATION OF PROTEOLYTIC CLEAVAGE SITES IN HORMONE STORAGE GRANULES J. Biol. Chem., July 21, 2000; 275(30): 22905 - 22915. [Abstract] [Full Text] [PDF]
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