Heinrich Heine University of Diisseldorf. Frank Weinsberg (~). Ulf Bickmeyer 9 Herbert Wiegand. Medical Institute of Environmental Hygiene at the Heinrich ...
Arch Toxicol (1995) 69:191-196
9 Springer-Verlag 1995
Frank Weinsberg- Ulf Bickmeyer 9Herbert Wiegand
Effects of inorganic mercury (Hg2+) on calcium channel currents and catecholamine release from bovine chromaffin cells
Received: 7 June 1994/Accepted: 31 August 1994
Abstract The effects of inorganic mercury (Hg 2§ on calcium channel currents and the potassium-evoked catecholamine release of bovine chromaffin cells in culture were examined. The effects of cadmium (Cd 2 +), known to block calcium channels and reduce catecholamine release of chromaffin cells, were studied for comparison. Calcium channel currents were recorded in the whole-cell configuration of the patch-clamp technique. Hg 2+ is a potent calcium channel blocker in bovine chromaffin cells. The ICso value is about 3 pM, the Hill slope 1.46. In a concentration of 100 pM, Hg 2 + blocked the currents completely; 100 pM Cd 2 + had the same effect. Potassium-evoked catecholamine release from chromaffin cells was measured at different timepoints with high-performance-liquid-chromatography (HPLC) under control conditions and in the presence of different Hg 2+ concentrations. Low Hg 2+ concentrations (0.l and 1 ~tM) did not affect the amount of the catecholamines epinephrine (E) and norepinephrine (NE) which was released. Under identical conditions 1 ~tM Cd 2+ also had no effect on release. With 10 ~tM Hg 2 + there was a time-dependent increase in the potassium-evoked catecholamine release (by 27% after 8 min). The E/NE ratio was not altered, suggesting that the release of both hormones was increased similarly. In contrast to this, the release was slightly reduced with 10 pM Cd 2+. In the presence of 100 laM Hg 2+, there was a reduction of the release during an early phase, followed by an increase. The reduction is most probably due to the fast and effective calcium channel block by Hg 2+ in this high concentration. The calcium channel block by 100 pM Cd 2+ also reduced the release Parts of this work is included in the thesis of Frank Weinsberg at the Heinrich Heine University of Diisseldorf. Frank Weinsberg ( ~ ) . Ulf Bickmeyer 9Herbert Wiegand Medical Institute of Environmental Hygiene at the Heinrich Heine University, Dfsseldorf, Auf'm Hennekamp 50, 40225 Diisseldorf, Germany
significantly. Catecholamine release of bovine chromaffin cells is driven into two opposite directions by Hg 2 +. On the one hand, a calcium channel block reduces the release, while on the other hand effects occur which can increase the release. Both tendencies occur simultaneously, but have different concentration- and timedependencies; therefore one can overcome the other under specific conditions. The catecholamine output at a given timepoint reflects the "sum" of these different effects.
Key words Inorganic mercury Bovine chromaffin cells. Calcium channels Catecholamine release. Cadmium
Introduction In addition to toxic effects on the kidneys and the gastrointestinal tract, inorganic mercury induces neurological symptoms such as behavioural changes, tremor and erethism. The reasons for these clinical effects are not fully understood, but many studies have been performed in vitro to elucidate those effects. It is known that inorganic mercury compounds can increase the spontaneous and evoked transmitter release in various preparations (for review see Minnema and Cooper 1989). Several mechanisms have been discussed to cause these effects, for example alterations of intracellular calcium homeostasis (Binah et al. 1978), calcium mimetic effects (Hare et al. 1990) or inhibition of the Na-K-ATPase (Magour et al. 1987). Another effect of Hg 2+ ions is a block of voltageactivated calcium channels. This block was suggested because potassium-evoked 4SCa2+ uptake into rat brain synaptosomes (Nachshen 1984) and into isolated nerve terminals (Hewett and Atchison 1992) is inhibited in the presence of Hg 2+ ions. Patch-clamp recordings on Aplysia neurons and rat dorsal root ganglion
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(DRG) cells showed the block of voltage-activated calcium channels by Hg2 + directly (Pekel et al. 1993). Cultured bovine chromaffin cells (Livett 1984) are a well established model for studies on the secretion of transmitters. The cells release the catecholamines epinephrine, norepinephrine and dopamine. In bovine chromaffin cells blockage of voltage-activated calcium channels (by cadmium for instance) leads to an inhibition of the calcium-dependent, potassium-evoked catecholamine release (e.g. Holz et al. 1982). Therefore Hg 2§ might be able to drive potassiumevoked catecholamine release in two opposite directions. On the one hand, a possible increase in release may take place, on the other hand, there may be inhibition of the release by a calcium channel block. The aim of this study was to understand more about the concentration- and the time-dependency of these opposing Hg 2§ effects. For comparison, we examined effects of Cd 2 +, known to block calcium channels (e.g. Fox et al. 1987) and to reduce catecholamine release of bovine chromaffin cells (e.g. Holz et al. 1982).
Catecholamine release experiments Cells were held in the culture medium described above for 6 days. The following procedures were then performed: (a) Washing: the medium was removed and the wells were washed twice with a buffer solution containing: 125.6 mM NaCI; 4.8 mM KC1, 2.2mM CaC12*2H20; 2 5 m M HEPES; 1.8mM MgC12; 5.6 mM glucose. (b) Stimulation: the wash solution was removed and exchanged against fresh buffer (of the same composition) containing Hg 2§ (as HgC12), Cd 2+ (as CdCI2) or no metal (controls). The cells were subsequently stimulated with 50 mM potassium for 1 or 8 rain. After that time the buffer was removed and its catecholamine content determined with H P L C (Miiller and Unsicker 1981).
Catecholamine determination with H P L C Ascorbic acid (1.2 mM) was added to the buffer samples to protect the catecholamines from oxidation and the samples were then treated as described previously (Bickmeyer et al. 1994). Dihydrobenzoylamine (DHBA) was added as a standard (concentration: 170 ng/ml). The H P L C detects each catecholamine as a distinct peak. The unknown amounts of epinephrine, norepinephrine and dopamine were compared to the known amount of DHBA by comparing the areas under the peaks.
Materials and methods Culture method Primary cultures of bovine chromaffin cells were prepared by the method of Marxen et al. (1989) and kept in culture as previously described (Weinsberg et al. 1994). Cells were cultured in 35 mm culture dishes at a density of 500000/mlper dish for the electrophysiological experiments or 24-well plates at a density of 500 000/ml per well for the catecholamine release experiments. Both dishes and wells were coated with rat tail collagen.
Electrophysiological experiments The patch-clamp recordings (Hamill et al. 1981) were performed with the EPC-7 patch-clamp amplifier (List Electronics) and the data were analyzed using the computer program CED Electrophysiology package V5.5 (Cambridge Electronic Design). All recordings were carried out at room temperature between day 1 and day 3 in culture. Every culture dish contained three or four cover slips. For the experiments, one cover slip was transferred into another dish containing 2 m l of the following bath solution: 135mM tetraethylammonium (TEA)-CI; 10 mM N-(2-hydroxyethyl)-piperazineN-2-ethanesulphonic acid (HEPES); 1.2 mM MgC12; 10 mM BaCI2; 2 ~tM tetrodotoxin (TTX); pH was adjusted to 7.2 with TEA-OH. Barium currents through calcium channels were recorded with firepolished patch pipettes with 2-7 Mf~ resistance. The pipette solution was: 135 mM CsCI; 10mM HEPES; 10mM ethyleneglycolbis(2aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA); 2 mM MgCI2; 4 mM Na-ATP; pH was adjusted to 7.2 with TEA-OH. Calcium channel currents were evoked every 30 s with 100 ms voltage steps to 0 mV from a holding potential of - 70 mV. Under these conditions, current amplitude normally increased after the establishment of the whole-cell configuration and reached a constant level within a few minutes. From that point, stable calcium channel currents could be recorded without "run-down" for up to 45 min in the best cases. Hg 2 § (in the form of HgCI2) was applied after currents had stabilized by adding 1 ml bath solution supplemented to 3 times the desired Hg 2§ concentration to the bath, which contained 2 ml bath solution.
Epinephrine- and norepinephrine-secreting chromaffin cells Chromaffin cells consist of two distinct cell types, one synthesizing and secreting mainly epinephrine (E), the other mainly norepinephrine (NE). The fraction of these cell types is similar to the fractions of E and NE in the total catecholamine content of a culture (e.g. Moro et al. 1991). The total catecholamine content of different cell preparations was in average 67 +__11% E, 3 1 _ 10% NE and 2 + 2 % dopamine (cells from nine different cell preparations were lyzed with 0.5% Triton X-100 and the catecholamine content measured with HPLC, values are mean _ SD). In 8 min of depolarization with 50 mM potassium, the cells usually release around 10% of their total catecholamine content. The main part of the release happens during the first minute (Fig. 1, compare also Table 1). As previously shown (Rubin and Miele 1968; Marley and Livett 1987), there is preferential
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t(min) Fig. l Time course of the potassium-evoked catecholamine release (50 mM potassium, 8 rain) of a typical cell preparation of bovine chromaffin cells under control conditions. Values are mean ___SD, n = 3. The absolute amounts of the released catecholamines could be different in different cell preparations. Therefore the amount after 8 min was defined as 100% to compare the results from different cell preparations
193 Table 1 Amount of released catecholamines (sum of released E and NE) and E/NE ratio after 1 min and after 8 min of potassium depolarization under control conditions and in the presence of different Hg 2 + concentrations. The amount was measured and normalized to the amount after 8 rain under control conditions (which was defined as 100%)
Amount
E/NE
1 min Control 0.1 gM 1 ~tM 10 gM 100 gM
66 64 63 71 51
1.27 1.18 1.18 1.21 1.34
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100 97 103 127 117
0.90 0.92 0.93 0.90 1.21
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Fig. 2 Concentration-effect curve for the inhibition of calcium channel currents of bovine chromaflin cells by Hg 2§ Currents were evoked with 100 ms voltage steps to 0 mV from a holding potential of - 70 mV every 30 s. Current amplitude was measured before and 8 min after Hg 2§ application (with 50 and 100 ~tM the amplitude was measured after two minutes; after that time the block was complete in every experiment). Values are mean + SD the numbers above the error bars indicate the numbers of distinct experiments at each concentration. The ICs0 value was calculated as 3 pM and the Hill slope as 1.46 (computer program "Inplot")
NE release during potassium depolarizations (compare E/NE ratio in Table 1). "4-
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Results The effects of inorganic mercury (Hg 2 +) on voltageactivated calcium channel currents and the calciumdependent, potassium-evoked catecholamine release of bovine chromaffin cells in culture were examined. For comparison, we examined effects of cadmium, which blocks voltage-activated calcium channels and reduces catecholamine release of chromaffin cells.
Effects of Hg 2+
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Hg 2+ is a potent calcium channel blocker in bovine chromaffin ceils. The block is concentration dependent, with an ICs0 value of about 3 gM. The Hill slope is 1.46 (Fig. 2). The peak of the current-voltage relationship (I/V curve) was usually shifted by Hg 2§ to more positive potentials. Original current traces and an I/V curve from an experiment with 5 gM are shown in Fig. 3. The block was faster with higher concentrations: 100 gM Hg 2+ usually gave a complete block in not more than 90 s. The time course of experiments with different Hg 2+ concentrations is shown in Fig. 4. In one cell there was a slight recovery from the block upon washing, but usually the block was irreversible. In rat D R G neurons Hg 2+ not only blocks calcium channels, but also induces leak currents (Pekel et al. 1993),
Fig. 3 A Original current trace showing calcium channel currents of a chromaffin cell under control conditions and 8 min after application of 5 gM Hg 2+. B Current-voltage relationship of the same cell before (diamonds) and after treatment (circles). In both A and B no leak correction was made
possibly by activating a slow inward membrane current (Arakawa et al. 1991). In two of six cells tested with 1 0 p M Hg 2+ some leak currents appeared after 6-7 rain, but usually such currents were not found with Hg a+ concentrations of 10ktM or less. With higher
194
cium channels (Fox et al. 1987). Calcium channel currents of chromaffin cells were blocked completely by 100 pM Cd 2 + (not shown).
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t(min) Fig. 4 Time course of the calcium channel block by different Hg 2 + concentrations in typical experiments. Hg 2+ was added directly after the voltage step at time zero. Triangles 0.5 p M Hg 2 +, diamonds 10 gM, circles 100 p M
concentrations (50 or 100 gM) usually an increase in the leak current occurred after about 1-2 min.
Potassium-evoked catecholamine release The "amount of released catecholamines" is defined as the sum of released epinephrine (E) and norepinephrine (NE). Due to the small amounts, dopamine was not included in the analysis. Since E- and NE-containing chromaffin cells might be affected differently, the E/NE ratio ( = amount of released E/amount of released NE) was also analyzed. The amount of released catecholamines and the E/NE ratio were determined after 1 min and after 8 min of potassium depolarization in the presence of different Hg 2+ concentrations and compared to controls (Table 1). Concentrations of 0.1, 1 and 10 gM had no effect during the first minute. With 100 laM there was a significant reduction of the catecholamine amount (Table 1), but no effect on the E/NE ratio. The inhibition of the release was therefore similar in both E- and NE-chromaffin cells. After 8 min there was no alteration of the amount or the E/NE ratio with 0.1 and 1 IxM Hg 2 +. These concentrations do not affect potassium-evoked catecholamine release of bovine chromaffin cells in our experimental conditions. With 10 gM the catecholamine amount was increased by 27%, while the E/NE ratio was not altered compared to controls, indicating that both E and N E release increased similarly. In the presence of 100 laM Hg 2+ the catecholamine amount was also increased, but not so much as with 10 laM (by 17%). In contrast to controls, the E/NE ratio was increased significantly.
Effects of C d 2 + Calcium channel currents For comparison we examined the effects of cadmium (Cd 2 +), which is known to block voltage-activated cal-
Catecholamine release (50 m M potassium, 8 min) was affected by Cd2 + in a concentration-dependent manner. 1 pM Cd 2+ had no effect on the release. With 10 gM Cd 2§ there was a slight, but not significant reduction; the E/NE ratio was not affected. With 100 ~tM the release was reduced to 53 __ 14% of controls (n = 20, mean ___SD). The E/NE ratio was slightly, but not significantly, increased with 100 pM Cd 2 +. Discussion Hg 2+ is a potent calcium channel blocker in bovine chromaffin cells. Our results with regard to the ICs0 value and the Hill slope are similar to those reported in a recent paper by Pekel et al. (1993) for rat D R G neurons. In their paper an effective calcium channel block by such low Hg 2+ concentrations had been reported for the first time. The shift of the current-voltage relationship and the irreversibility of the block is also similar in chromaffin cells and D R G neurons. However, one difference occurred. The increase in the leak current reported to appear in rat D R G neurons with concentrations above 2 gM (Pekel et al. 1993) was not seen in most of the experiments with bovine chromaffin cells, even with 10 gM Hg 2+. In higher concentrations (50 or 100 gM) usually an increase in the leak current occurred, which might correspond to that described in D R G neurons. In this respect, bovine chromaffin cells are possibly less sensitive to Hg 2+ than rat D R G neurons, while the calcium channel block seems to be similar in both cell types. The potassium-evoked catecholamine release of chromaffin cells is calcium dependent (e.g. Holz et al. 1982). Since the release is strongest during this early period, a calcium channel block must be strong and must develop fast to produce a significant reduction of the release. 10 gM Hg z+ (and also 10 ~M Cd 2+) did not reduce the release significantly during the first minute, the development of the calcium channel block seems to be too slow with this concentration. It should also be noted that the time course of the development of the calcium channel block might differ during electrophysiological experiments from that during the release experiments because of the different experimental conditions. M O W i t h 100 gM Hg 2+ there was indeed a significant reduction of the release during the first minute, most probably due to the fast and effective calcium channel block. The calcium channel block is only one aspect of the effects of Hg 2 + on catecholamine release of chromaffin
195
cells. There are other, probably intracellular effects which increase the release. At the neuromuscular junction, Miyamoto (1983) reported that sodium and calcium channel blockers prevented the effects of Hg 24. He suggested that Hg 2+ acts intracellularly, following entry into the cells through sodium and calcium channels. In hepatocytes Hg uptake is not affected by calcium channel blockers, suggesting that Hg does not enter these cells through calcium channels (Blazka and Shaik 1991). Hg 2+ was shown to alter intracellular calcium homeostasis (Binah et al. 1978) and to have calcium mimetic effects (Hare et al. 1990). We found increased catecholamine release after 8 min, but not after 1 min of depolarization in the presence of 10 gM Hg 2 4. The increase obviously takes some time to develop, what fits in with idea that Hg 2§ first enters the cells and then increases release via intracellular effects. Under the experimental conditions used here, the increased release seems to be exocytotic and not due to non-specific cell damage. The membrane integrity as tested with trypan blue exclusion was apparently not impaired with 10 gM Hg 2+ (the number of cells incorporating the dye was slightly, but not obviously, increased). The electrophysiological experiments provide further evidence for this idea, because we found no obvious increase of leak currents with 10 gM Hg 2 + (or less). The situation is somewhat different with 100 laM Hg 2+. The early reduction of the release suggests that during the first minute 100/aM Hg 2+ does not destroy tile cells, because in this case one would expect an increase of catecholamines. Subsequently the catecholamine output was increased, but not so much as with 10 gM Hg 2+. In nearly every cell the membrane integrity (as tested with trypan blue exclusion) was impaired. However, despite this impairment, 100 gM Hg2 + did not induce a massive cateeholamine increase. As described above (Materials and methods), the cells release, under control conditions, usually around 10% of their total catecholamine content during 8 min of depolarization with 50 m M potassium. In the presence of 100 p.M Hg2+ the catecholamine amount was increased by a factor of 1.17 (Table I), or in other words from around 10% of the total catecholamine content to around 12%. This does not suggest a massive leak of catecholamines as a consequence of nonspecific cell damage (at least during these 8 min). Non-specific actions of Hg z+ on membrane permeability, which may not necessarily be linked to transmitter release, have been reported previously, but with lower concentrations (Hare at al. 1990). However, it cannot be excluded, that the catecholamine increase in the presence of 100 laM Hg z+ is related to nonspecifc toxic effects at such a high concentration. This may also hold for the increase of E/qqE in the presence of 100 taM Hg 2+ . Since this increase occurred only with 100 gM Hg 24 a preferential action of Hg 2 + on E- or NE-secreting chromaffin cells cannot be verified on the basis of these data.
We conclude that H g 2+ drives potassium-evoked catecholamine release of bovine chromaffin cells in two opposite directions during acute exposure. It reduces the release by a calcium channel block and increases the release by other, probably intracellular, effects. Both these mechanisms occur simultaneously, but under specific conditions one can overcome the other, because the effects have different concentrationand time-dependencies. The catecholamine output at a given timepoint reflects the "sum" of these different effects [a similar concept has been proposed for mercury effects on isolated myocardial tissue by Halbach (1990)]. Nachshen (1984) examined the Hg 2§ block of 4SCa2 + influx into rat brain synaptosomes during depolarizations and found ICso values of 120 gM (for a fast inactivating component) and 20 gM (for a slow inactivating component) after a 10 s Hg 2 + incubation. Hewett and Atchison (1992) found similar values (155 ~tM and 49 laM, respectively) in isolated rat nerve terminals. Our results and those from Pekel et al. (1993), show that Hg 2§ ions can block vertebrate calcium channels much more effectively during longer incubations. Some differences have also been reported regarding the effects of Hg 2 + on transmitter release. At neuromuscular junctions, for instance, the evoked transmitter release is firstly increased (and later reduced) by Hg 2 + (Manalis and Cooper 1975; Binah et al. 1978), while the potassium-evoked [3H]-dopamine release from mouse striatal slices is decreased by Hg 2+ (McKay et al., 1986). The concept of simultaneously occurring opposing Hg 2+ effects on transmitter release with different concentration- and time-dependencies might help to explain some of these different results. Acknowledgements We thank Erika Miiller an Karl-Heinz Redelings for excellent technical assistance.
References Arakawa O, Nakahiro M, Narahashi T (1991) Mercury modulation of GABA-activated chloride channels and non-specific cation channels in rat dorsal root ganglion neurons. Brain Res 551: 58-63 Bickmeyer U, Weinsberg F, Wiegand H (1994) Effects of deltamethrin on catecholamine secretion of bovine chromaffin ceils. Arch Toxicol 68:532-534 Binah O, Meiri U, Rahamimoff H (1978)The effects of HgC12 and mersalyl on mechanisms regulating intracellular calcium and transmitter release. Eur J Pharmacol 51:453-457 Blazka ME, Shaik ZA (1991) Differences in cadmium and mercury uptake by hepatocytes: role of calcium channels. Toxicol Appl Pharmacol 110:355-363 Fox AP, Nowicky MC, Tsien RW (1987) Kinetic and pharmacological properties distinguish three types of calcium currents in chick sensory neurons. J Physiol 394:149-172 Hamill OP, Marty A, Neher E, Sakmann B, Sigworth F (1981) Improved patch-clamp techniques for high-resolution current recording from cell and cell-free patches. Pfliiger's Arch 391: 85-100
196 Halbach S (1990) Mercury compounds: lipophilicityand toxic effects on isolated myocardial tissue. Arch Toxicol 64:315-319 Hare MF, Rezazadeh SM, Cooper GP, Minnema DJ, Michaelson IA (1990) Effects of inorganic mercury on 3H dopamine release and calcium homeostasis in rat strial synaptosoms. Toxicol Appl Pharmacol 102:316-330 Hewett SJ, Atchison WD (1992) Effects of charge and lipophilicity on mercurial-induced reduction of 45Ca2§ uptake in isolated nerve terminals of the rat. Toxicol Appl Pharmacol 113:267-273 H olz RW, Senter RA, Frye RA (1982) Relationship between calcium uptake and catecholamine secretion in primary dissociated cultures of adrenal medulla. J Neurochem 39:635-646 Livett BG (1984) Adrenal medullary chromaffin cells in vitro. Physiol Rev 64:1103-1161 Magour S, Maser H, Greim H (1987) The effect of mercuric chloride and methyl mercury on brain microsomal Na +K +-ATPase after partial delipidization with lubrol. Pharmacol Toxicol 60: 184--186 M analis RS, Cooper GP (1975) Evoked transmitter release increased by inorganic mercury at the frog neuromuscularjunction. Nature 257:690--691 Marley PD, Livett BG (1987) Differences between the mechanisms of adrenaline and noradrenaline secretion from isolated, bovine, adrenal chromaffin cells. Neurosci Lett 77:81-86 Marxen P, Fuhrmann U, Bigalke H (1989) Gangliosides mediate inhibitory effects of tetanus and botulinum A neurotoxins in chromaffin cells. Toxicon 27:849-859 McKay SJ, Reynolds JN, Racz WJ (1986) Effects of mercury compounds on the spontaneous and potassium-evoked release of
[3H]-dopamine from mouse striatal slices. Can J Physiol Pharmacol 64:1507-1514 Minnema DJ, Cooper GP (1989) Assessment of the effects of lead and mercury in vitro on neurotransmitter release. In: Foulkes EC, (ed) Biological effects of heavy metals. CRC Press, Boca Raton, pp 18 57 Miyamoto MD (1983) Hg 2+ causes neurotoxicity at an intracellular site following entry through Na and Ca channels. Brain Res 267: 375-379 Moro MA, Garcia AG, Langley OK (1991) Characterization of two chromaffin cell populations isolated from bovine adrenal medulla. J Neurochem 57:363-369 Miiller TH, Unsicker K (1981) High-performance liquid chromatography with electrochemical detection as a highly efficient tool for studying catecholaminergic systems. I. Quantification of noradrenaline, adrenaline and dopamine in cultured adrenal medullary cells. J Neurosci Methods 4:39-52 Nachshen D (1984) Selectivity of the calcium binding site in synaptosome Ca channels. Inhibition of Ca influx by multivalent metal cations. J Gen Physiol 83:941 967 Pekel M, Platt B, Biisselberg D (1993) Mercury (Hg 2§ decreases voltage-gated calcium channel currents in rat DRG and Aplysia neurons. Brain Res 632:121-126 Rubin RP, Miele E (1968) A study of the differential secretion of epinephrine and norpepinephrine from the perfused cat adrenal gland. J Pharmacol Exp Ther 164:115-121 Weinsberg F, Biekmeyer U, Wiegand H (1994) Effects of tetrandrine on calcium channel currents of bovine chromaffin cells. Neuropharmacology 33:885-890
