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(Circulation. 2000;101:403.)
© 2000 American Heart Association, Inc.

Clinical Investigation and Reports

N-Type Calcium Channels Control Sympathetic Neurotransmission in Human Heart Atrium

G. J. Molderings, MD; J. Likungu, MD; M. Göthert, MD

From the Institute of Pharmacology and Toxicology, University of Bonn (G.J.M., M.G.) and the Clinic of Cardiovascular Surgery (J.L.), University of Bonn, Bonn, Germany.

Correspondence to G.J. Molderings, MD, Institute of Pharmacology and Toxicology, University of Bonn, Reuterstraße 2B, D-53113 Bonn, Germany. E-mail molderings{at}uni-bonn.de

	Abstract

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Background—Because knowledge about the type of calcium channels involved in action potential–induced norepinephrine release from the human peripheral sympathetic nervous system is sparse, we investigated which types of calcium channels are functionally important in the sympathetic nerves of human cardiac tissue.

Methods and Results—In superfused segments of human right atrial appendages, the type of calcium channels that control [³H]norepinephrine release evoked by transmural electrical stimulation was determined. [³H]norepinephrine release was almost abolished by 0.2 µmol/L -conotoxin GVIA (a selective blocker of N-type channels) but was not modified by 0.1 µmol/L -agatoxin IVA (a selective blocker of P- and Q-type channels). Mibefradil (a T-type and N-type calcium channel blocker) at concentrations of 0.3 to 3 µmol/L reduced the evoked tritium overflow in a frequency- and calcium-dependent manner, whereas 0.1 to 10 µmol/L amlodipine, diltiazem, and verapamil (selective blockers of L-type channels) were ineffective.

Conclusions—Norepinephrine release from cardiac sympathetic nerves is triggered by Ca²⁺ influx via N-type but not L- and P/Q-type calcium channels. The inhibitory effect of mibefradil on norepinephrine release at clinically relevant concentrations is probably due to its blocking action on N-type Ca²⁺ channels. This property of mibefradil is unique among the calcium channel blockers that have been or still are therapeutically applied and may considerably contribute to its slight negative chronotropic effect in vivo.

Key Words: atrium • calcium channels • norepinephrine • mibefradil

	Introduction

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The depolarization-induced norepinephrine release from central and peripheral norepinephrine neurons occurs by exocytosis triggered by an influx of extracellular calcium via voltage-dependent calcium channels. The latter are activated when the surface membrane is depolarized by an action potential. At present, 6 classes of voltage-dependent transmembrane calcium channels have been identified on the basis of functional properties such as voltage- and time-dependent kinetics, single-channel conductance, pharmacology, and cellular distribution (for review, see Reference 1¹ ): long-lasting conductance or L-type channels (inhibited by verapamil, diltiazem, and dihydropyridines); neural or N-type channels (blocked by -conotoxin GVIA); transient or T-type channels (blocked by mibefradil with moderate selectivity over N-type channels^{2 3} ); P- and Q-type channels (blocked by -agatoxin IVA); and R-type channels (blocked by Ni²⁺). N-, P-, Q-, and R-type calcium channels have been identified in central neurons. L- and T-type calcium channels are abundant in the periphery, particularly in the cardiovascular system.

In the peripheral sympathetic nervous system of humans, little is known about the type of calcium channels involved in depolarization-induced norepinephrine release. Therefore, the aim of the present study was to examine which types of calcium channels are functionally important in the sympathetic nerve terminals of human cardiac tissue. This question was also addressed in the context of the possibility that the favorable bradycardic influence of the novel calcium channel blocker mibefradil^{4 5} might be due to an inhibition of norepinephrine release by inhibiting calcium influx into the cardiac sympathetic nerve terminals.⁶ The purpose of the present experiments with mibefradil was to provide evidence for the suggestion that mibefradil inhibits norepinephrine release by blocking calcium channels in the postganglionic sympathetic axon terminals.

Preliminary accounts on the present data have been given at the 9th International Symposium on Vascular Neuroeffector Mechanisms.⁷

	Methods

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Segments of macroscopically normal human right atrial appendages were obtained from normotensive 30- to 75-year-old male or female patients undergoing open-heart surgery. After the atrial appendages were removed (via a routine procedure for cannulation of the right atria), they were immediately transferred into fresh, cold physiological salt solution (for composition, see below) and stored at 4°C until the beginning of the experiment. The patients were not treated with adrenoceptor agonists or antagonists or with drugs that influence the storage or release of norepinephrine. After premedication with pethidine and promethazine, the patients were anesthetized (both induction and maintenance) with flunitrazepam and fentanyl. During maintenance of anesthesia, they were ventilated with mixtures of oxygen and air. Pancuronium was administered for neuromuscular blockade. The study was approved by the local ethics committee.

The experiments on strips (3x15 mm) of the atrial appendages began, as a rule, within 20 hours (or sooner, in a few cases) after removal; preliminary experiments, started 2 or 20 hours after removal, had revealed that norepinephrine release and its drug-induced modification did not differ. The strips were incubated for 60 minutes in 1.5 mL of physiological salt solution (37°C) containing (-)-[ring-2,5,6-³H]-norepinephrine 0.2 µmol/L (specific activity 42 Ci/mmol). Subsequently, they were mounted vertically in an organ bath (tension adjusted to 2.0 g) between 2 parallel platinum electrodes and superfused with [³H]norepinephrine-free physiological salt solution (37°C) at a rate of 2 mL/min. The composition of the solution was (in mmol/L): NaCl 118, NaH₂PO₄ 1.2, NaHCO₃ 25, KCl 4.7, CaCl₂ 1.6 (unless stated otherwise; in a few experiments, 3.2 or 4.8 mmol/L), MgSO₄ 1.2, glucose 11.0, ascorbic acid 0.3, Na₂ EDTA 0.03 (aerated with 95% O₂ and 5% CO₂). Throughout superfusion, this solution contained desipramine 0.6 µmol/L and corticosterone 40 µmol/L for blockade of the neuronal and extraneuronal uptake of norepinephrine, respectively.

The superfusate was collected in 4-minute fractions. Five (or in a few experiments, 3 or 4) periods of transmural electrical stimulation (0.66, 2 [standard stimulation parameter], 6, or 10 Hz; 360 rectangular impulses of 200 mA and 0.3 ms) were applied to each strip after 94 (S₁; conditioning stimulus), 126 (S₂), 158 (S₃), 190 (S₄), and 222 (S₅) minutes of superfusion. At the end of superfusion, the strips were solubilized with Soluene (Packard), and the radioactivity in the superfusate samples and tissues was determined by liquid scintillation counting. Separate control experiments were performed for each series of experiments with the drugs.

Tritium efflux was calculated as the fraction of tritium present in the strip at the onset of the respective collection period. Basal tritium efflux was determined in the superfusate collection periods immediately before S₂, S₃, S₄, or S₅ (ie, t₂, t₃, t₄, and t₅). Stimulation-evoked tritium overflow was calculated by subtraction of the basal efflux from the total efflux during the 16 minutes subsequent to the onset of stimulation; basal efflux decreased linearly from the collection period before to that 16 to 20 minutes after onset of stimulation. Evoked tritium overflow was calculated as a percentage of tissue tritium at the onset of stimulation, and the ratios of the overflow evoked by S₃, S₄, or S₅ over that evoked by S₂ (overflow evoked by S₂ through S₅ was less variable than that evoked by S₁) were determined.

Results are given as mean±SEM of data obtained in tissue strips from n different hearts (where n is the number of experiments). Student’s t tests for unpaired data were used for comparison of the mean values. As an estimate of drug potency, IC₂₅ values (negative logarithm of the concentrations producing 25% inhibition of evoked tritium overflow; for terminology, see Reference 8⁸ ) were determined by interpolation from the nearest points of the concentration-response curves.

Drugs used were (-)-[ring-2,5,6-³H]-norepinephrine (specific activity 42 Ci/mmol; New England Nuclear); desipramine hydrochloride (Ciba-Geigy); corticosterone, -conotoxin GVIA, -agatoxin IVA, diltiazem, verapamil, and amlodipine (Sigma); and mibefradil dihydrochloride (a gift of Hoffmann-La Roche, Grenzach-Wyhlen, Germany). Drugs were dissolved in saline or water with 1 exception: corticosterone was dissolved in 1,2-propandiol, and the stock solution was further diluted with saline. All experiments with verapamil and amlodipine were performed under exclusion of light.

	Results

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Basal Efflux and Electrically Evoked Tritium Overflow in Control Experiments
When no test drug was administered before, during, and after S₁ through S₅, basal tritium efflux during t₂ from segments of atrial appendages preincubated with [³H]norepinephrine was 4.55±0.54 nCi/min (in a representative series of control experiments; n=8; 2 Hz, 1.6 mmol/L calcium), which corresponds to a fractional rate of efflux of 0.00054±0.00002 min^-1. Basal efflux decreased with time, as reflected by t_n/t₂ ratios that declined from t₃/t₂ (0.94±0.02) to t₅/t₂ (0.87±0.02). Neither changes in Ca²⁺ concentration nor application of the test drugs in the concentration range tested modified basal tritium efflux.

In control experiments in the absence of the test drugs, transmural electrical stimulation elicited an increase in tritium overflow above basal efflux (Table). At all frequencies of stimulation and calcium concentrations, the evoked overflow decreased from S₂ to S₅, as reflected by S_n/S₂ ratios that declined from S₃/S₂ to S₅/S₂ (Table).

View this table: [in this window] [in a new window]   Table 1. Control Values for Stimulation-Evoked Tritium Overflow

Effects of Calcium Channel Antagonists
-Conotoxin GVIA 0.2 µmol/L nearly abolished the electrically evoked (2 Hz, 3 minutes, 360 impulses) [³H] overflow (Figure 1), whereas -agatoxin IVA 0.1 µmol/L was without effect (Figure 1). The L-type calcium channel blockers amlodipine, diltiazem (Figure 2), and verapamil (Figure 3A) failed to modify the evoked [³H] overflow. Verapamil also did not change the electrically evoked [³H] overflow at the increased stimulation frequencies of 6 and 10 Hz (360 impulses; Figure 3A).

View larger version (43K): [in this window] [in a new window]   Figure 1. Effects of -conotoxin GVIA and -agatoxin IVA on electrically evoked (2 Hz) tritium overflow from segments of human atrial appendages. Ordinate: ratios S3/S2 expressed as percentages of those in the corresponding drug-free control experiments. Drugs were present from 12 minutes before until 20 minutes after onset of S3. Mean±SEM of 3 to 6 experiments. ***P<0.001 (compared with corresponding control experiments).

View larger version (15K): [in this window] [in a new window]   Figure 2. Effect of amlodipine and diltiazem on electrically evoked tritium overflow from superfused segments of human atrial appendages preincubated with [3H]norepinephrine. Five periods of transmural electrical stimulation (2 Hz, 360 impulses; S1-S5) were applied. Amlodipine () and diltiazem () were present at increasing concentrations from 12 minutes before until 20 minutes after onset of S3, S4, and S5. Ordinate: ratios Sn/S2 expressed as percentages of those in corresponding drug-free control experiments. Mean±SEM of 7 to 9 experiments.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of verapamil (A) and mibefradil (B) on electrically evoked tritium overflow from superfused segments of human atrial appendages preincubated with [3H]norepinephrine at different frequencies of stimulation. Five periods of transmural electrical stimulation (S1 through S5, 360 impulses) were applied. Verapamil and mibefradil were present at increasing concentrations from 12 minutes before until 20 minutes after onset of S3, S4, and S5. •, 0.66 Hz; , 2 Hz; , 6 Hz; and , 10 Hz. Ordinates: ratios Sn/S2 expressed as percentages of those in corresponding drug-free control experiments. Mean±SEM of 5 to 10 experiments. *P<0.05, **P<0.02, ***P<0.001 (compared with corresponding control experiments); +P<0.05, +++P<0.001 (compared with corresponding drug concentration in experiments performed at 2 Hz). In the case of experiments with individual verapamil concentrations, data obtained at various stimulation frequencies did not significantly differ from each other.

Under the standard experimental conditions (2 Hz, 360 impulses; 1.6 mmol/L Ca²⁺), the T- and N-type calcium channel blocker mibefradil concentration-dependently inhibited the electrically evoked [³H] overflow with a pIC₂₅ value of 6.64 (Figure 4, open circles). The concentration-response curve of mibefradil was shifted right when the Ca²⁺ concentration was increased to 3.2 mmol/L (Figure 4, closed circles; pIC₂₅ 5.95). An additional increase in Ca²⁺ concentration to 4.8 mmol/L did not induce a further rightward shift of the concentration-response curve compared with that at 3.2 mmol/L (Figure 4, closed squares; pIC₂₅ 6.09). A rightward shift of the concentration-response curve for mibefradil was also observed when the preparations were stimulated electrically for 1 minute with an increased frequency of 6 Hz (360 impulses; Figure 3B, closed squares; pIC₂₅ 6.00). In contrast, lowering of the stimulation frequency to 0.66 Hz (9 minutes, 360 impulses) tended to shift the concentration-response curve for mibefradil to the left (Figure 3B, closed circles; pIC₂₅ 6.71).

View larger version (18K): [in this window] [in a new window]   Figure 4. Effect of mibefradil on electrically evoked tritium overflow from superfused segments of human atrial appendages preincubated with [3H]norepinephrine at 3 different calcium concentrations in superfusion solution. Five periods of transmural electrical stimulation (2 Hz, 360 impulses; S1 through S5) were applied. Mibefradil was present at increasing concentrations from 12 minutes before until 20 minutes after onset of S3, S4, and S5. , 1.6 mmol/L calcium; •, 3.2 mmol/L calcium; and , 4.8 mmol/L calcium. Ordinate: ratios Sn/S2 expressed as percentages of those in corresponding drug-free control experiments. Mean±SEM of 6 to 11 experiments. **P<0.02, ***P<0.001 (compared with corresponding control experiments); +P<0.05 (compared with corresponding drug concentration in experiments performed at 1.6 mmol/L calcium concentration in superfusion solution).

	Discussion

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The aim of the present study was to characterize the calcium channels that are involved in the control of sympathetic transmission in human heart atrium and to extend our findings previously obtained in the same preparation with mibefradil.⁶ The electrically evoked tritium overflow reflects quasi-physiological, Ca²⁺-dependent release of labeled and unlabeled norepinephrine from the postganglionic sympathetic axon terminals.⁹ This view is supported by our previous findings¹⁰ in human atrial appendages that the electrically evoked tritium overflow is abolished by tetrodotoxin, as well as in the absence of Ca²⁺ in the superfusion fluid. On the basis of these considerations, the evoked tritium overflow may be denoted as norepinephrine release.

N-type calcium channels have been shown to be predominant in controlling neurotransmitter release in sympathetically innervated preparations from animal species in vitro^{11 12 13} (for brief review, see Reference 7⁷ ) and in vivo.^{14 15} In agreement with those findings, norepinephrine release from the sympathetic nerves of the human heart was prevented by the highly selective N-type channel blocker -conotoxin GVIA (present study). In contrast, the P- and Q-type calcium channel blocker -agatoxin IVA was without influence on the electrically evoked norepinephrine release. Hence, the ability of -conotoxin GVIA to almost abolish the evoked norepinephrine release, whereas -agatoxin IVA was without effect, indicates that Ca²⁺ influx into the human cardiac sympathetic axon terminals occurs via N-type Ca²⁺ channels, whereas P- and Q-type channels are not involved.¹⁶

Mibefradil mimicked the inhibitory effect of -conotoxin GVIA on norepinephrine release. This inhibitory effect of mibefradil was dependent on the extracellular Ca²⁺ concentration. An increase in extracellular Ca²⁺ concentration from 1.8 to 3.6 mmol/L, leading to an elevated intraneuronal Ca²⁺ availability for stimulus-release coupling by an increased influx,¹⁷ reduced the potency of mibefradil in inhibiting evoked norepinephrine release. This finding is compatible with a quasi-competitive type of inhibition, ie, at a certain state of blockade of a given number of calcium channels, more Ca²⁺ ions can permeate the channel if the extracellular Ca²⁺ concentration is increased; conversely, a higher concentration of a calcium channel blocker is necessary at increased extracellular Ca²⁺ concentration to produce a certain degree of inhibition of Ca²⁺ influx. This interpretation is not ruled out by the finding that a further increase in Ca²⁺ concentration above 3.2 mmol/L to 4.8 mmol/L did not produce an additional reduction in the inhibitory potency of mibefradil: beyond a saturation level of extracellular Ca²⁺ concentration at which the maximum amount of Ca²⁺ already penetrates the channel at a given level of blockade (in the present experiments, this level appeared to be 3.2 mmol/L Ca²⁺), an increase in extracellular Ca²⁺ concentration will not lead to an additional increase in intraneuronal Ca²⁺ availability for stimulus-release coupling. Moreover, to maintain the level of channel blockade, it will not be necessary to increase the amount of the calcium channel blocker. Similarly, an increase in stimulation frequency, which also leads to an increase in intraneuronal Ca²⁺ concentration (see References 9 and 18^{9 18} ), also reduced the potency of mibefradil to inhibit evoked norepinephrine release, whereas a decrease in stimulation frequency tended to increase its potency. Because mibefradil can potently block neuronal N-type calcium channels in addition to T-type channels,^{2 3} the ability of mibefradil to mimic the effect of -conotoxin GVIA in human atrial appendages strongly suggests that the inhibitory effect of mibefradil on norepinephrine release from the cardiac sympathetic nerves is due to blockade of N-type Ca²⁺ channels.

Verapamil, diltiazem, and the dihydropyridine derivatives amlodipine and, as shown previously, nifedipine,⁶ which block the L-type calcium channel, did not inhibit the evoked norepinephrine release in human atrial appendages at concentrations up to 1 and 10 µmol/L, respectively. These results obtained at the standard stimulation frequency of 2 Hz suggest that L-type channels are not involved in Ca²⁺ influx into human cardiac sympathetic nerves during depolarization. Whether or not verapamil, diltiazem, and the dihydropyridines are capable of interacting with presynaptic calcium channels at clinically relevant concentrations is still a matter of debate. High-affinity binding of dihydropyridines to nervous tissue has been demonstrated, and it has been proposed that these binding sites are L-type calcium channels.¹⁹ However, in functional studies in which an inhibition of evoked norepinephrine release was observed by dihydropyridines and verapamil, it only occurred either at high stimulation frequencies (10 Hz) or at drug concentrations 1 µmol/L.^{19 20 21 22 23} In the present study, even 1 µmol/L verapamil failed to inhibit evoked norepinephrine release at stimulation frequencies of up to 10 Hz. Because the upper limit of the clinically relevant plasma concentration of the L-type calcium channel blockers is 0.1 µmol/L,²⁴ it is rather unlikely that an inhibition of norepinephrine release will occur with these drugs in vivo at therapeutic doses. However, combined L- and N-type calcium channel blockers would be suitable to inhibit norepinephrine release. Such drugs may be assumed to be clinically beneficial, because they would counteract the reflex tachycardia in response to blood pressure reduction.

Taken together, in human heart, norepinephrine release from sympathetic nerves is triggered by Ca²⁺ influx via N-type but not L-type calcium channels. The inhibitory effect of clinically relevant concentrations of mibefradil (which has been applied therapeutically until recently) on norepinephrine release is probably due to its blocking effect on N-type calcium channels. This unique property of mibefradil may contribute to the slight negative chronotropic effect of the drug in vivo. In the Mortality Assessment in Congestive Heart Failure (MACH)-1 trial, mibefradil did not result in clinical benefits in patients with cardiac failure and showed a trend toward an increased mortality; this may be assumed to be due to an interaction with T-type calcium channels in the conduction system and/or to drug interactions.²⁵ In the meantime, mibefradil has been withdrawn from the market because of its complex pharmacokinetic interactions. Nevertheless, it might serve as a leading compound to design future calcium channel antagonists devoid of its unfavorable pharmacokinetic interactions but sharing its beneficial properties. In particular, drugs that block both the vascular L-type and N-type calcium channels and that do not substantially pass the blood/brain barrier should lead to vasodilation without direct negative inotropism, and they should not induce sympathetic reflex activation but may cause mild bradycardia. Such drugs may represent progress in the treatment of cardiovascular diseases such as hypertension and coronary heart disease.

	Acknowledgments

 
This study was supported by a grant of the Deutsche Forschungsgemeinschaft. The technical assistance of D. Funccius and M. Hartwig is gratefully acknowledged.

Received June 16, 1999; revision received August 18, 1999; accepted August 26, 1999.

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