Skip to main content
PMC home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1999 Oct 15;520(Pt 2):503–512. doi: 10.1111/j.1469-7793.1999.00503.x

Role of calcium channels in catecholamine secretion in the rat adrenal gland

Takahiro Nagayama *, Takayuki Matsumoto *, Fumiyo Kuwakubo *, Yasuo Fukushima *, Makoto Yoshida *, Mizue Suzuki-Kusaba *, Hiroaki Hisa *, Tomohiko Kimura *, Susumu Satoh *
PMCID: PMC2269601  PMID: 10523418

Abstract

  1. We elucidated the contribution of voltage-dependent Ca2+ channels to cholinergic control of catecholamine secretion in the isolated perfused rat adrenal gland.

  2. Nifedipine (0.3–3 μm) inhibited increases in noradrenaline output induced by transmural electrical stimulation (1–10 Hz) and acetylcholine (6–200 μm), whereas it only slightly inhibited the adrenaline output responses. Nifedipine also inhibited the catecholamine output response induced by 1,1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 μm) but not by methacholine (10–300 μm).

  3. ω-Conotoxin MVIIC (10–1000 nm) inhibited the catecholamine output responses induced by electrical stimulation but not by acetylcholine, DMPP and methacholine.

  4. ω-Conotoxin GVIA (50–500 nm) had no inhibitory effect on the catecholamine output responses.

  5. These results suggest that L-type Ca2+ channels are responsible for adrenal catecholamine secretion mediated by nicotinic receptors but not by muscarinic receptors, and that their contribution to noradrenaline secretion may be greater than that to adrenaline secretion. P/Q-type Ca2+ channels may control the secretion at a presynaptic site.

Adrenal medullary chromaffin cells secrete catecholamines in response to nicotinic agonists (Douglas & Rubin, 1961; Wakade, 1981; Amy & Kirshner, 1982). Activation of the nicotinic receptors opens non-selective cation channels (Zhou & Neher, 1993) to depolarize the membrane of chromaffin cells (Douglas et al. 1967; Biales et al. 1976; Kilpatrick, 1984). This depolarization opens voltage-dependent Na+ and Ca2+ channels, and the subsequent elevation of intracellular free Ca2+ is an essential step in the exocytotic secretion of adrenal catecholamines (Cheek et al. 1989; Kim & Westhead, 1989).

Several types of voltage-dependent Ca2+ channels are present on adrenal chromaffin cells, but the role of each type in the catecholamine secretion process remains controversial. Cat chromaffin cells possess L- and N-type voltage-dependent Ca2+ channels which each carry 50 % of the Ca2+ current (Albillos et al. 1994), but the L-type Ca2+ channels dominate the exocytotic process (Lopez et al. 1994a). Bovine chromaffin cells possess not only L- (Artalejo et al. 1991) and N-type voltage-dependent Ca2+ channels (Hans et al. 1990; Artalejo et al. 1992) but also P- (Mintz et al. 1992; Gandía et al. 1994) and Q-type voltage-dependent Ca2+ channels (Lopez et al. 1994b), and the L- and Q-type Ca2+ channels dominate the exocytotic process (Lomax et al. 1997). Rat chromaffin cells possess L-, N-, P- and Q-type voltage-dependent Ca2+ channels (Gandía et al. 1995). Both L- and N-type Ca2+ currents have been shown to be recruited during exocytosis from rat chromaffin cells (Kim et al. 1995). Thus not all of the voltage-dependent Ca2+ channels present in chromaffin cells may contribute to the secretion of catecholamines.

Adrenal catecholamine secretion is also mediated by muscarinic receptors in various species (Douglas & Poisner, 1965; Harish et al. 1987; Nakazato et al. 1988; Kimura et al. 1992). Concerning the role of voltage-dependent Ca2+ channels in the muscarinic receptor-mediated secretion of catecholamines, observations obtained with L-type voltage-dependent Ca2+ channel blockers in in vitro studies remain controversial. Verapamil, an L-type voltage-dependent Ca2+ channel blocker, does not affect muscarine-induced catecholamine secretion from perfused rat adrenal gland (Harish et al. 1987). In contrast, isradipine, another L-type voltage-dependent Ca2+ channel blocker, inhibits the methacholine-induced catecholamine secretion from cat chromaffin cells (Uceda et al. 1992). On the other hand, little is known about the involvement of N-type voltage-dependent Ca2+ channels in the muscarinic receptor-mediated secretion of catecholamines (Uceda et al. 1994).

In the present study, we investigated the effects of nifedipine, an L-type voltage-dependent Ca2+ channel blocker, ω-conotoxin GVIA, an N-type voltage-dependent Ca2+ channel blocker, and ω-conotoxin MVIIC, a P/Q-type voltage-dependent Ca2+ channel blocker, on the secretion of adrenaline and noradrenaline from the isolated perfused rat adrenal gland in response to transmural electrical stimulation, acetylcholine, the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP) and the muscarinic agonist methacholine to elucidate the functional role of voltage-dependent Ca2+ channels in controlling the adrenal secretion of adrenaline and noradrenaline. The effects of hexamethonium, a nicotinic antagonist, and atropine, a muscarinic antagonist, on catecholamine secretion induced by transmural electrical stimulation and acetylcholine were also examined.

METHODS

Animal preparation

All procedures for handling animals were approved by the Animal Experimentation Committee of Tohoku University Graduate School of Pharmaceutical Sciences. Male Wistar rats, weighing 250-350 g, were housed at 21-24°C and maintained on a standard diet and water ad libitum. Rats were anaesthetized with sodium pentobarbital (50 mg kg−1, i.p.). The left adrenal gland was exposed by a mid-line incision of the abdomen. The stomach, intestines and parts of the liver were not removed, but were pushed over to the right side and covered with saline-soaked gauze pads to obtain enough working space for tying blood vessels and for cannulation. A polyethylene cannula, used for perfusion of the adrenal gland, was inserted into the adrenal vein through the renal vein after all the branches of the adrenal vein, the renal artery and the renal vein were ligated. Then the adrenal gland, along with the tied blood vessels and the cannula, was carefully removed from the animal and a small slit was made into the adrenal cortex just opposite the entrance of the adrenal vein. Perfusion of the adrenal gland was started to ensure that no leak was present and the perfusate escaped only from the slit of the adrenal gland. The adrenal gland was placed on a bipolar platinum electrode used for transmural electrical stimulation. The adrenal gland together with the electrode was placed in a water-jacketed chamber, the temperature of which was maintained at 37°C with a thermostatically controlled water circulator (NTT-1200, EYELA, Tokyo, Japan). After extraction of the adrenal gland, the animal was killed by exsanguination.

Perfusion of the adrenal gland

The adrenal gland was perfused by means of a peristaltic pump (MP-3A, EYELA) at a rate of 0.2 ml min−1. The perfusion was carried out with Krebs-Henseleit solution of the following composition (mm): NaCl, 118; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.6; KH2PO4, 1.2; NaHCO3, 24.9; and glucose, 11.1. Krebs-Henseleit solution was maintained at 37°C by the thermostat bath, and bubbled with a mixture of 95 % O2 and 5 % CO2. Perfusate samples were collected in chilled tubes containing 50 μl of 0.1 M perchloric acid, to prevent oxidation of catecholamines. Before starting a experiment, the adrenal gland was initially perfused for 60 min with Krebs-Henseleit solution.

Transmural electrical stimulation

Transmural electrical stimulation (duration, 1 ms; supramaximal voltage, 50 V) was applied by a bipolar platinum electrode with an electronic stimulator (SEN-3301, Nihon Kohden, Tokyo, Japan) and an isolation unit (SS-302J, Nihon Kohden). Stimulus frequency was raised stepwise from 1 to 2, 5 and 10 Hz at 5 min intervals, stimulation at each frequency being applied for 40 s.

Administration of cholinergic agonists

Different concentrations of acetylcholine, DMPP and methacholine solution were administered into the perfusion stream through a branching polyethylene catheter. These drugs were infused by using a microsyringe pump (CMA/200, Bioanalytical Systems, West Lafayette, IN, USA). The stimulus dose was raised stepwise at 5 min intervals, stimulation at each concentration being applied for 40 s.

Experimental protocol

The rats were divided into 20 groups. In group 1 (n= 11), the effects of repeated transmural electrical stimulation on increases in catecholamine (adrenaline and noradrenaline) output were examined without drug treatment. A set of transmural electrical stimulation was repeated four times at 30 min intervals. In group 2 (n= 10), the effects of repeated application of acetylcholine on increases in catecholamine output were examined without drug treatment. A set of acetylcholine infusion (6, 20, 60 and 200 μm) was repeated four times at 30 min intervals. In groups 3 (n= 16) and 4 (n= 15), the effects of repeated application of DMPP (5, 10, 20 and 40 μm) and methacholine (10, 30, 100 and 300 μm) on increases in catecholamine output were examined, respectively, with the same protocol as used in group 2. In group 5 (n= 6), the effects of hexamethonium on the transmural electrical stimulation-induced increases in catecholamine output were examined. The first set of transmural electrical stimulation was regarded as a control (1st trial). Perfusion with 10, 30 and 100 μm hexamethonium-containing Krebs-Henseleit solution was started 10 min before the start of the second, third and fourth trials, respectively. In group 6 (n= 6), the effects of hexamethonium on the acetylcholine-induced increases in catecholamine output were examined. The first set of acetylcholine infusion was regarded as a control (1st trial). Perfusion with 10, 30 and 100 μm hexamethonium-containing Krebs-Henseleit solution was started 10 min before the start of the second, third and fourth trials, respectively. The effects of atropine (0.3, 1 and 3 μm) on increases in catecholamine output induced by transmural electrical stimulation (group 7; n= 6) and acetylcholine (group 8; n= 8) were examined with the same protocol as used in the hexamethonium experiments. The effects of nifedipine (0.3, 1 and 3 μm) on increases in catecholamine output induced by transmural electrical stimulation (group 9; n= 11), acetylcholine (group 10; n= 11), DMPP (group 11; n= 7) and methacholine (group 12; n= 8) were examined with the same protocol as described above. The effects of ω-conotoxin GVIA (5, 50 and 500 nm) on increases in catecholamine output induced by transmural electrical stimulation (group 13; n= 8), acetylcholine (group 14; n= 10), DMPP (group 15; n= 12) and methacholine (group 16; n= 10) were examined with the same protocol. The effects of ω-conotoxin MVIIC (10, 100 and 1000 nm) on increases in catecholamine output induced by transmural electrical stimulation (group 17; n= 8), acetylcholine (group 18; n= 8), DMPP (group 19; n= 8) and methacholine (group 20; n= 6) were also examined.

Perfusate sampling

Perfusate was sampled before and during transmural electrical stimulation or infusion of the cholinergic agonists to determine catecholamine output. Sampling during the basal state was performed for 60 s just before stimulation. In preliminary experiments, it was found that the stimuli-induced catecholamine responses returned to the prestimulation level within about 20 s after stopping the transmural electrical stimulation or the agonist infusion. Thus, sampling during transmural electrical stimulation at each frequency or during infusion of the cholinergic agonist at each concentration was performed for 60 s.

Determination of adrenal catecholamine output

Catecholamines in perfusate samples were measured by high-performance liquid chromatography with electrochemical detection (LC-4C, Bioanalytical Systems), as described previously (Kimura et al. 1988). Adrenaline and noradrenaline output (ng min−1) were calculated by multiplying perfusate catecholamine concentration (ng ml−1) by perfusion rate (0.2 ml min−1). The basal catecholamine output was determined from samples collected just before transmural electrical stimulation and infusion of the cholinergic agonists. The stimuli-induced increases in catecholamine output were calculated by subtracting basal catecholamine output from that obtained during the stimulus state.

Analysis of data

The results are expressed as means ±s.e.m. throughout the study. Two-factor analysis of variance (ANOVA) was used for statistical analysis of the data. When ANOVA showed a statistical difference, Dunnett's test was used to determine the significance level. P values less than 0.05 were considered to be statistically significant. Significance of differences was determined at the highest stimulation frequency or cholinergic agonist concentration.

Drugs

The drugs used were hexamethonium chloride, atropine sulfate, nifedipine, acetylcholine chloride, DMPP iodide, acetyl-β-methylcholine (methacholine) chloride (all from Sigma), ω-conotoxin GVIA and ω-conotoxin MVIIC (Peptide Institute, Osaka, Japan). Nifedipine was dissolved in ethanol and diluted to the required concentrations with Krebs-Henseleit solution under dim light immediately before use. Other drugs were dissolved in Krebs-Henseleit solution.

RESULTS

Increases in catecholamine output in response to transmural electrical stimulation, acetylcholine, DMPP and methacholine

Basal adrenaline and noradrenaline output from the adrenal gland at 60 min after initial perfusion was 20.1 ± 1.4 (n= 185) and 3.1 ± 0.2 ng min−1 (n= 185), respectively, in all groups. There were no differences in these basal values among the experimental groups. Transmural electrical stimulation (1-10 Hz) or infusion of acetylcholine (6-200 μm), DMPP (5-40 μm) and methacholine (10-300 μm) into the adrenal gland produced frequency- or concentration-dependent increases in adrenaline and noradrenaline output (Table 1 and Table 2). The increases in catecholamine output induced by these stimuli did not vary during the time course of the experiment (1st-4th trials, Table 1 and Table 2). Ratios of adrenaline to noradrenaline for increases in catecholamine output induced by transmural electrical stimulation (10 Hz), acetylcholine (200 μm), DMPP (40 μm) and methacholine (300 μm) during the control stimulation period were 3.9 ± 0.2 (n= 50), 4.7 ± 0.2 (n= 53), 4.1 ± 0.2 (n= 43) and 14.9 ± 1.5 (n= 39), respectively. The ratio obtained with methacholine was greater than those obtained with other stimuli.

Table 1.

Adrenal catecholamine output from perfused rat adrenal glands in response to transmural electrical stimulation (ES) and acetylcholine (ACh)

Adrenaline output (ng min−1)
1st trial 2nd trial 3rd trial 4th trial
Group 1(n= 11)
 ES 1 Hz 14.0 ± 2.7 9.9 ± 2.2 9.7 ± 1.6 8.2 ± 1.9
 ES 2 Hz 34.4 ± 7.1 37.9 ± 8.5 30.8 ± 7.9 27.6 ± 6.3
 ES 5 Hz 107.1 ± 20.2 117.3 ± 21.9 106.6 ± 22.3 101.7 ± 23.0
 ES 10 Hz 195.2 ± 31.9 211.7 ± 41.6 197.1 ± 36.4 191.3 ± 42.9
Group 2 (n= 10)
 ACh 6 μm 17.7 ± 5.3 13.8 ± 5.2 9.5 ± 4.0 6.6 ± 3.0
 ACh 20 μm 64.4 ± 12.7 53.6 ± 16.3 47.2 ± 13.9 38.6 ± 11.0
 ACh 60 μm 127.3 ± 27.5 123.2 ± 28.2 116.3 ± 22.0 97.7 ± 20.3
 ACh 200 μm 195.9 ± 40.8 217.5 ± 41.5 207.1 ± 33.3 200.0 ± 35.6
Noradrenaline output (ng min−1)
1st trial 2nd trial 3rd trial 4th trial
Group 1(n= 11)
 ES 1 Hz 1.4 ± 0.3 1.3 ± 0.2 0.9 ± 0.2 0.8 ± 0.2
 ES 2 Hz 4.9 ± 1.1 5.6 ± 1.1 4.9 ± 1.2 3.8 ± 0.9
 ES 5 Hz 22.5 ± 3.6 24.8 ± 4.1 22.1 ± 4.1 19.0 ± 3.8
 ES 10 Hz 50.4 ± 8.7 53.0 ± 10.0 49.9 ± 8.8 46.6 ± 9.3
Group 2 (n= 10)
 ACh 6 μm 1.8 ± 0.4 1.5 ± 0.6 1.0 ± 0.4 0.5 ± 0.3
 ACh 20 μm 8.1 ± 1.4 6.6 ± 1.6 5.4 ± 1.3 4.9 ± 1.1
 ACh 60 μm 22.2 ± 4.1 21.2 ± 3.8 19.0 ± 2.8 15.8 ± 2.7
 ACh 200 μm 56.8 ± 10.3 65.6 ± 10.5 58.9 ± 8.6 56.3 ± 9.4
Open in a new tab

Values are means ± s.e.m.; n= number of rats per group. There were no significant differences (P > 0.05) between the values during the 1st trial and those during the 2nd, 3rd or 4th trials.

Table 2.

Adrenal catecholamine output from perfused rat adrenal glands in response to DMPP and methacholine (MCh)

Adrenaline output (ng min−1)
1st trial 2nd trial 3rd trial 4th trial
Group 3 (n= 16)
 DMPP 5 μm 13.4 ± 3.1 13.7 ± 3.6 15.0 ± 4.8 18.2 ± 3.9
 DMPP 10 μm 55.1 ± 7.7 62.9 ± 10.4 59.4 ± 9.7 58.6 ± 8.7
 DMPP 20 μm 143.3 ± 17.4 150.7 ± 17.1 137.4 ± 14.2 140.9 ± 12.3
 DMPP 40 μm 192.9 ± 20.7 198.7 ± 22.1 188.3 ± 16.9 192.8 ± 19.9
Group 4 (n= 15)
 MCh 10 μm 22.6 ± 5.0 15.6 ± 3.9 11.8 ± 3.4 9.0 ± 3.3
 MCh 30 μm 46.3 ± 9.7 53.2 ± 9.6 46.3 ± 7.6 36.6 ± 7.1
 MCh 100 μm 89.7 ± 15.5 105.5 ± 16.9 98.3 ± 15.5 91.3 ± 13.8
 MCh 300 μm 130.9 ± 21.1 150.2 ± 21.9 133.2 ± 22.1 150.6 ± 19.5
Noradrenaline output (ng min−1)
1st trial 2nd trial 3rd trial 4th trial
Group 3 (n= 16)
 DMPP 5 μm 1.6 ± 0.3 1.8 ± 0.4 1.5 ± 0.4 2.0 ± 0.4
 DMPP 10 μm 8.2 ± 1.2 10.0 ± 1.7 9.0 ± 1.3 8.7 ± 1.6
 DMPP 20 μm 27.8 ± 3.6 30.0 ± 3.6 26.8 ± 3.4 25.4 ± 3.0
 DMPP 40 μm 55.5 ± 6.7 56.6 ± 7.9 54.6 ± 5.8 51.3 ± 5.2
Group 4 (n= 15)
 MCh 10 μm 0.7 ± 0.2 0.2 ± 0.2 0.1 ± 0.1 0.2 ± 0.2
 MCh 30 μm 1.7 ± 0.5 2.1 ± 0.4 1.6 ± 0.4 1.5 ± 0.4
 MCh 100 μm 5.2 ± 1.1 6.5 ± 1.2 6.4 ± 1.1 5.5 ± 1.1
 MCh 300 μm 12.5 ± 2.0 14.8 ± 2.3 13.1 ± 2.3 15.7 ± 2.0
Open in a new tab

Values are means ± s.e.m.; n= number of rats per group. There were no significant differences (P > 0.05) between the values during the 1st trial and those during the 2nd, 3rd or 4th trials.

Effects of hexamethonium and atropine on the transmural electrical stimulation- and acetylcholine-induced increases in catecholamine output

Hexamethonium (10, 30 and 100 μm) significantly inhibited the transmural electrical stimulation- and acetylcholine-induced increases in adrenaline and noradrenaline output (Fig. 1). The inhibitory effect of hexamethonium on the acetylcholine-induced increase in noradrenaline output was greater than that for adrenaline output. Atropine (0.3, 1 and 3 μm) significantly inhibited the acetylcholine-induced increases in adrenaline and noradrenaline output (Fig. 2), but it caused only a slight inhibition of the transmural electrical stimulation-induced catecholamine secretion at the highest concentration (3 μm). Neither hexamethonium nor atropine affected basal adrenaline and noradrenaline output (data not shown).

Figure 1. Effects of hexamethonium on adrenaline and noradrenaline output from perfused rat adrenal glands in response to transmural electrical stimulation and acetylcholine.

Figure 1

Open in a new tab

Transmural electrical stimulation (ES; A) or acetylcholine infusion (ACh; B) was applied to the adrenal gland in the absence (control, •) or presence of hexamethonium (10 μm, ^; 30 μm, ▵; 100 μm, □). Symbols and vertical bars represent means ±s.e.m. ** P < 0.01 compared with corresponding control response obtained before hexamethonium treatment. Significance of differences was determined at 10 Hz transmural electrical stimulation or with 200 mm acetylcholine.

Figure 2. Effects of atropine on adrenaline and noradrenaline output from perfused rat adrenal glands in response to transmural electrical stimulation and acetylcholine.

Figure 2

Open in a new tab

Transmural electrical stimulation (ES; A) or acetylcholine infusion (ACh; B) was applied to the adrenal gland in the absence (control, •) or presence of atropine (0.3 μm, ^; 1 μm, ▵; 3 μm, □). Symbols and vertical bars represent means ±s.e.m. * P < 0.05, ** P < 0.01 compared with corresponding control response obtained before atropine treatment.

Effects of nifedipine on the transmural electrical stimulation-, acetylcholine-, DMPP- and methacholine-induced increases in catecholamine output

Nifedipine (0.3, 1 and 3 μm) significantly inhibited the transmural electrical stimulation- and acetylcholine-induced increases in noradrenaline output, and tended to inhibit the adrenaline response but the effect was not statistically significant (Fig. 3). The DMPP-induced increases in adrenaline and noradrenaline output were inhibited by nifedipine (Fig. 4A). In contrast, the methacholine-induced increases in adrenaline and noradrenaline output were not affected by even the highest dose (3 μm) of nifedipine (Fig. 4B). Basal adrenaline and noradrenaline output were not affected by nifedipine (data not shown).

Figure 3. Effects of nifedipine on adrenaline and noradrenaline output from perfused rat adrenal glands in response to transmural electrical stimulation and acetylcholine.

Figure 3

Open in a new tab

Transmural electrical stimulation (ES; A) or acetylcholine infusion (ACh; B) was applied to the adrenal gland in the absence (control, •) or presence of nifedipine (0.3 μm, ^; 1 μm, ▵; 3 μm, □). Symbols and vertical bars represent means ±s.e.m. * P < 0.05, ** P < 0.01 compared with corresponding control response obtained before nifedipine treatment.

Figure 4. Effects of nifedipine on adrenaline and noradrenaline output from perfused rat adrenal glands in response to DMPP and methacholine.

Figure 4

Open in a new tab

DMPP (A) or methacholine (MCh; B) infusion was applied to the adrenal gland in the absence (control, •) or presence of nifedipine (0.3 μm, ^; 1 μm, ▵; 3 μm, □). Symbols and vertical bars represent means ±s.e.m.** P < 0.01 compared with corresponding control response obtained before nifedipine treatment.

Effects of ω-conotoxin GVIA

ω-Conotoxin GVIA (5, 50 and 500 nm) did not affect the transmural electrical stimulation-, acetylcholine-, DMPP- or methacholine-induced increases in adrenaline and noradrenaline output. The 10 Hz transmural electrical stimulation-, 200 μm acetylcholine-, 40 μm DMPP- and 300 μm methacholine-induced increases in adrenaline output were 206 ± 36 (n= 8), 234 ± 47 (n= 10), 218 ± 39 (n= 12) and 135 ± 17 ng min−1 (n= 10) during the control period and 199 ± 21, 238 ± 45, 272 ± 53 and 149 ± 25 ng min−1 during treatment with 500 nmω-conotoxin GVIA, respectively, and the increases in noradrenaline output were 52 ± 7 (n= 8), 55 ± 13 (n= 10), 57 ± 5 (n= 12) and 11 ± 1 ng min−1 (n= 10) during the control period and 45 ± 5, 49 ± 12, 56 ± 8 and 11 ± 1 ng min−1 during treatment with 500 nmω-conotoxin GVIA, respectively. Basal adrenaline and noradrenaline output were not affected by ω-conotoxin GVIA (data not shown).

Effects of ω-conotoxin MVIIC

ω-Conotoxin MVIIC (10, 100 and 1000 nm) inhibited increases in adrenaline and noradrenaline output induced by transmural electrical stimulation but not by acetylcholine (Fig. 5). The DMPP- and methacholine-induced increases in adrenaline and noradrenaline output were not affected by even the highest concentration (1000 nm) of ω-conotoxin MVIIC. The 40 μm DMPP- and 300 μm methacholine-induced increases in adrenaline output were 198 ± 23 (n= 8) and 125 ± 23 ng min−1 (n= 6) during the control period and 179 ± 21 and 99 ± 19 ng min−1 during treatment with 1000 nmω-conotoxin MVIIC, respectively, and the increases in noradrenaline output were 52 ± 8 (n= 8) and 12 ± 2 ng min−1 (n= 6) during the control period and 41 ± 5 and 10 ± 2 ng min−1 during treatment with 1000 nmω-conotoxin MVIIC, respectively. Basal adrenaline and noradrenaline output were not affected by ω-conotoxin MVIIC (data not shown).

Figure 5. Effects of ω-conotoxin MVIIC on adrenaline and noradrenaline output from perfused rat adrenal glands in response to transmural electrical stimulation and acetylcholine.

Figure 5

Open in a new tab

Transmural electrical stimulation (ES; A) or acetylcholine infusion (ACh; B) was applied to the adrenal gland in the absence (control, •) or presence of ω-conotoxin MVIIC (10 nm, ^; 100 nm, ▵; 1000 nm, □). Symbols and vertical bars represent means ±s.e.m.**P < 0.01 compared with corresponding control response obtained before ω-conotoxin MVIIC treatment.

DISCUSSION

The transmural electrical stimulation-induced increases in adrenaline and noradrenaline output were strongly inhibited by hexamethonium, whereas these catecholamine output responses were less sensitive to atropine. These results indicate that transmural electrical stimulation-evoked secretion of catecholamines is predominantly mediated by nicotinic receptors. These findings are consistent with previous observations in the perfused rat adrenal gland (Wakade & Wakade, 1983). The acetylcholine-induced increases in adrenaline and noradrenaline output were also inhibited by hexamethonium and atropine, indicating that exogenously applied acetylcholine causes secretion of adrenaline and noradrenaline mediated by nicotinic and muscarinic receptors. The inhibition by atropine of adrenaline secretion was almost to the same degree as the inhibition of noradrenaline secretion, but the inhibition by hexamethonium of noradrenaline secretion was greater than that of adrenaline secretion. It is well known that adrenaline and noradrenaline are present in separate adrenal chromaffin cells (Hillarp & Hökfelt, 1953). Therefore, our results suggest that noradrenaline secretion from noradrenaline-containing cells in response to acetylcholine may be predominantly mediated by nicotinic receptors, and muscarinic receptors may play a minor role in noradrenaline secretion in the rat adrenal gland (see Fig. 6). This is supported by the observation in the present study that methacholine, a muscarinic agonist, produced a smaller secretion of noradrenaline than of adrenaline, and thus the ratio of adrenaline to noradrenaline in the catecholamine response to methacholine was 3 to 4 times greater than the ratio with other stimuli.

Figure 6. Schematic diagram of the cholinergic nerve ending-chromaffin cell synapse in the adrenal gland of rat.

Figure 6

Open in a new tab

This diagram demonstrates possible localizations of voltage-dependent Ca2+ channels and cholinergic receptors mediating adrenal catecholamine secretion. Adrenaline-containing cells possess synaptic nicotinic receptors, extrasynaptic nicotinic and muscarinic receptors, and L-type voltage-dependent Ca2+ channels close to the extrasynaptic nicotinic receptors. Noradrenaline-containing cells possess L-type voltage-dependent Ca2+ channels close to synaptic nicotinic receptors. P/Q-type voltage-dependent Ca2+ channels are located on cholinergic nerve endings.

Although hexamethonium strongly inhibited the transmural electrical stimulation-induced increase in adrenaline output, it only slightly inhibited the acetylcholine-induced increase in adrenaline output. These results indicate that the contribution of nicotinic receptors to adrenaline secretion differs between the endogenous and exogenous acetylcholine-induced responses. This difference may be explained by assuming a differential distribution of nicotinic and muscarinic receptors on the adrenaline-containing cell membrane; nicotinic receptors are located predominantly in synaptic zones, whereas muscarinic receptors are found predominantly in extrasynaptic regions (see Fig. 6). Endogenous acetylcholine released from nerve terminals would mainly activate intrasynaptic nicotinic receptors, because rapid destruction by acetylcholinesterase of released acetylcholine would restrict its diffusion into extrasynaptic regions. Exogenous acetylcholine delivered through the perfusate could diffuse into both synaptic zones and extrasynaptic regions and would activate nicotinic and muscarinic receptors.

Nifedipine, an L-type voltage-dependent Ca2+ channel blocker, had no effect on the methacholine-induced increases in adrenaline and noradrenaline output. This result agrees with the observation that verapamil, an L-type voltage-dependent Ca2+ channel blocker, does not affect muscarine-induced catecholamine secretion from perfused rat adrenal gland (Harish et al. 1987). This finding and those of the present study suggest that the influx of extracellular Ca2+ through L-type voltage-dependent Ca2+ channels is not involved in the muscarinic receptor-mediated pathway. The activation of muscarinic receptors has been suggested to promote Ca2+ release from intracellular storage sites and cause the secretion of catecholamines from the adrenal gland of cat (Yamada et al. 1988) and guinea-pig (Nakazato et al. 1988). It has been reported that extracellular Ca2+ deprivation does not affect muscarine-evoked catecholamine secretion from perfused rat adrenal gland (Harish et al. 1987). Therefore, the muscarinic receptor-mediated secretion of catecholamines in rat adrenal chromaffin cells may exclusively depend on the elevation of intracellular free Ca2+ mobilized from intracellular storage sites.

Nifedipine inhibited the increases in noradrenaline output induced by transmural electrical stimulation, acetylcholine and the nicotinic agonist DMPP. The inhibitory effects of nifedipine on the transmural electrical stimulation- and acetylcholine-induced secretion of noradrenaline can be explained by its blocking action on the nicotinic receptor-mediated pathway. L-type voltage-dependent Ca2+ channels may thus contribute to the secretion of noradrenaline mediated by nicotinic receptors. Nifedipine also inhibited the DMPP-induced increase in adrenaline output, suggesting that L-type voltage-dependent Ca2+ channels also contribute to nicotinic receptor-mediated adrenaline secretion. On the other hand, the inhibition by nifedipine of the transmural electrical stimulation- and acetylcholine-induced increases in adrenaline output was only slight, and this effect was not statistically significant. It was reported that furnidipine, an L-type voltage-dependent Ca2+ channel blocker, inhibits the K+-induced secretion of noradrenaline more than that of adrenaline in bovine chromaffin cells (Lomax et al. 1997). These findings, including our results, suggest that the contribution of L-type voltage-dependent Ca2+ channels to noradrenaline secretion is greater than that to adrenaline secretion. Our results obtained using hexamethonium suggest that the contribution of nicotinic receptors to acetylcholine-induced adrenaline secretion is relatively small compared with that to noradrenaline secretion. This explains the weak inhibitory effect of nifedipine on acetylcholine-induced adrenaline secretion. Here, the question arises as to why nifedipine influences the nicotinic receptor-mediated secretion of adrenaline differently; a potent inhibition in the case of DMPP and a weak inhibition in the case of transmural electrical stimulation. This difference might be explained by assuming a differential distribution of L-type voltage-dependent Ca2+ channels on the adrenaline-containing cell membrane in synaptic zones and extrasynaptic regions. If L-type voltage-dependent Ca2+ channels are primarily concentrated in extrasynaptic regions but less so in synaptic zones, they could affect the depolarization during activation of extrasynaptic nicotinic receptors but hardly affect the depolarization during activation of intrasynaptic nicotinic receptors (see Fig. 6). It was reported that the L-type voltage-dependent Ca2+ channel blocker isradipine partially inhibits electrical stimulation- and acetylcholine-induced catecholamine secretion, but potently inhibits nicotine- and K+-induced secretion in the perfused rat adrenal gland (López et al. 1992). This observation is consistent with our results in the present study.

Electrophysiological studies have suggested that in rat chromaffin cells L-type voltage-dependent Ca2+ channels predominate but other subtypes of voltage-dependent Ca2+ channels such as N-, P- and Q-type are also present (Gandía et al. 1995). N-type Ca2+ current has been shown to be recruited during exocytosis from rat chromaffin cells (Kim et al. 1995), but there has been no direct evidence for the participation of N-type voltage-dependent Ca2+ channels in catecholamine secretion. In the present study, ω-conotoxin GVIA, an N-type voltage-dependent Ca2+ channel blocker, did not affect the secretion of adrenaline and noradrenaline in response to transmural electrical stimulation, acetylcholine, DMPP or methacholine. Also, ω-conotoxin GVIA did not affect the transmural electrical stimulation-induced secretion of adrenaline and noradrenaline during treatment with nifedipine (data not shown). The failure of ω-conotoxin GVIA to affect the catecholamine secretion responses may not be due to a poor tissue penetration ability or insufficient concentration, because a submicromolar concentration (10 nm) of ω-conotoxin GVIA has been reported to reduce nerve stimulation-induced catecholamine secretion by 75 % in perfused bovine adrenal gland under experimental conditions similar to those used here (O'Farrell et al. 1997). In the present study, we used a wide range of concentrations (5-500 nm) of ω-conotoxin GVIA. Thus, our results suggest that N-type voltage-dependent Ca2+ channels have no role in the secretion of catecholamines from rat adrenal gland.

ω-Conotoxin MVIIC, a P/Q-type voltage-dependent Ca2+ channel blocker, inhibited increases in adrenaline and noradrenaline output induced by transmural electrical stimulation but not the increases induced by acetylcholine, DMPP and methacholine. The differential effects of ω-conotoxin MVIIC on secretion between endogenous and exogenous acetylcholine may be explained by assuming that the distribution of P/Q-type voltage-dependent Ca2+ channels is limited to presynaptic sites. If P/Q-type voltage-dependent Ca2+ channels are primarily located on presynaptic nerve endings but not on postsynaptic chromaffin cells, they could contribute to the release of acetylcholine due to transmural electrical stimulation but would not contribute to the secretion of catecholamines in response to cholinergic agonists (see Fig. 6).

In conclusion, our results suggest that L-type voltage-dependent Ca2+ channels located on rat adrenal medullary cells are responsible for the process of adrenal catecholamine secretion mediated by nicotinic receptors but not by muscarinic receptors, and that their contribution to noradrenaline secretion may be greater than that to adrenaline secretion. N-type voltage-dependent Ca2+ channels may not contribute to catecholamine secretion. P/Q-type Ca2+ channels may control the secretion at presynaptic sites.

Acknowledgments

This work was supported in part by Research Fellowships of Japan Society for the Promotion of Science for Young Scientists and by grant no. 10877371 for Scientific Research from The Ministry of Education, Science and Culture, Japan.

References

  1. Albillos A, Artalejo AR, López MG, Gandía L, García AG, Carbone E. Calcium channel subtypes in cat chromaffin cells. The Journal of Physiology. 1994;477:197–213. doi: 10.1113/jphysiol.1994.sp020184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amy C, Kirshner N. 22Sodium uptake and catecholamine secretion by primary cultures of adrenal medulla cell. Journal of Neurochemistry. 1982;39:132–142. doi: 10.1111/j.1471-4159.1982.tb04711.x. [DOI] [PubMed] [Google Scholar]
  3. Artalejo CR, Dahmer MK, Perlman RL, Fox AP. Two types of Ca2+ currents are found in bovine chromaffin cells: facilitation is due to the recruitment of one type. The Journal of Physiology. 1991;432:681–707. doi: 10.1113/jphysiol.1991.sp018406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Artalejo CR, Perlman RL, Fox AP. ω-Conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the ‘classic’ N type. Neuron. 1992;8:85–95. doi: 10.1016/0896-6273(92)90110-y. [DOI] [PubMed] [Google Scholar]
  5. Biales B, Dichter M, Tischler A. Electrical excitability of cultured adrenal chromaffin cells. The Journal of Physiology. 1976;262:743–753. doi: 10.1113/jphysiol.1976.sp011618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheek TR, Jackson TR, O'Sullivan AJ, Moreton RB, Berridge MJ, Burgoyne RD. Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of Fura-2 in cocultured cells. Journal of Cell Biology. 1989;109:1219–1227. doi: 10.1083/jcb.109.3.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Douglas WW, Kanno T, Sampson SP. Influence of the ionic environment on the membrane potential of adrenal chromaffin cells and on the depolarizing effect of acetylcholine. The Journal of Physiology. 1967;191:107–121. doi: 10.1113/jphysiol.1967.sp008239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Douglas WW, Poisner AM. Preferential release of adrenaline from the adrenal medulla by muscarine and pilocarpine. Nature. 1965;208:1102–1103. doi: 10.1038/2081102a0. [DOI] [PubMed] [Google Scholar]
  9. Douglas WW, Rubin RP. The role of calcium in the secretory response of the adrenal medulla to acetylcholine. The Journal of Physiology. 1961;159:40–57. doi: 10.1113/jphysiol.1961.sp006791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gandía L, Albillos A, García AG. Bovine chromaffin cells possess FTX-sensitive calcium channels. Biochemical and Biophysical Research Communications. 1994;194:671–676. doi: 10.1006/bbrc.1993.1874. [DOI] [PubMed] [Google Scholar]
  11. Gandía L, Borges R, Albillos A, García AG. Multiple calcium channel subtypes in isolated rat chromaffin cells. Pflügers Archiv. 1995;430:55–63. doi: 10.1007/BF00373839. [DOI] [PubMed] [Google Scholar]
  12. Hans M, Illes P, Takeda K. The blocking effects of ω-conotoxin on Ca current in bovine chromaffin cells. Neuroscience Letters. 1990;114:63–68. doi: 10.1016/0304-3940(90)90429-d. [DOI] [PubMed] [Google Scholar]
  13. Harish OE, Kao L-S, Raffaniello R, Wakade AR, Schneider AS. Calcium dependence of muscarinic receptor-mediated catecholamine secretion from the perfused rat adrenal medulla. Journal of Neurochemistry. 1987;48:1730–1735. doi: 10.1111/j.1471-4159.1987.tb05730.x. [DOI] [PubMed] [Google Scholar]
  14. Hillarp NA, Hökfelt B. Evidence of adrenaline and noradrenaline in separate adrenal medullary cells. Acta Physiologica Scandinavica. 1953;30:55–68. doi: 10.1111/j.1748-1716.1954.tb01074.x. [DOI] [PubMed] [Google Scholar]
  15. Kilpatrick DL. Ion channels and membrane potential in stimulus-secretion coupling in adrenal paraneurons. Canadian The Journal of Physiology and Pharmacology. 1984;62:477–483. doi: 10.1139/y84-077. [DOI] [PubMed] [Google Scholar]
  16. Kim K-T, Westhead EW. Cellular responses to calcium from extracellular and intracellular sources are different as shown by simultaneous measurements of cytosolic calcium and secretion from bovine chromaffin cells. Proceedings of the National Academy of Sciences of the USA. 1989;86:9881–9885. doi: 10.1073/pnas.86.24.9881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim SJ, Lim W, Kim J. Contribution of L- and N-type calcium currents to exocytosis in rat adrenal medullary chromaffin cells. Brain Research. 1995;675:289–296. doi: 10.1016/0006-8993(95)00085-5. [DOI] [PubMed] [Google Scholar]
  18. Kimura T, Katoh M, Satoh S. Inhibition by opioid agonists and enhancement by antagonists of the release of catecholamines from the dog adrenal gland in response to splanchnic nerve stimulation: evidence for the functional role of opioid receptors. Journal of Pharmacology and Experimental Therapeutics. 1988;244:1098–1102. [PubMed] [Google Scholar]
  19. Kimura T, Shimamura T, Satoh S. Effects of pirenzepine and hexamethonium on adrenal catecholamine release in responses to endogenous and exogenous acetylcholine in anesthetized dogs. Journal of Cardiovascular Pharmacology. 1992;20:870–874. doi: 10.1097/00005344-199212000-00004. [DOI] [PubMed] [Google Scholar]
  20. Lomax RB, Michelrna P, Núñez L, García-Sancho J, García AG, Montiel C. Different contributions of L- and Q-type Ca2+ channels to Ca2+ signals and secretion in chromaffin cell subtypes. American Journal of Physiology. 1997;272:C476–484. doi: 10.1152/ajpcell.1997.272.2.C476. [DOI] [PubMed] [Google Scholar]
  21. López MG, Albillos A, de la Fuente MT, Borges R, Gandía L, Carbone E, García AG, Artalejo AR. Localized L-type calcium channels control exocytosis in cat chromaffin cells. Pflügers Archiv. 1994a;427:348–354. doi: 10.1007/BF00374544. [DOI] [PubMed] [Google Scholar]
  22. López MG, Shukla A, García AG, Wakade AR. A dihydropyridine-resistant component in the rat adrenal secretory response to splanchnic nerve stimulation. Journal of Neurochemistry. 1992;58:2139–2144. doi: 10.1111/j.1471-4159.1992.tb10956.x. [DOI] [PubMed] [Google Scholar]
  23. López MG, Villarroya M, Lara B, Sierra RM, Albillos A, García AG, Gandía L. Q- and L-type Ca2+ channels dominate the control of secretion in bovine chromaffin cells. FEBS Letters. 1994b;349:331–337. doi: 10.1016/0014-5793(94)00696-2. [DOI] [PubMed] [Google Scholar]
  24. Mintz IM, Venema VJ, Swiderek KM, Lee TD, Bean BP, Adams ME. P-type calcium channels blocked by the spider toxin ω-agatoxin-IVA. Nature. 1992;355:827–829. doi: 10.1038/355827a0. [DOI] [PubMed] [Google Scholar]
  25. Nakazato Y, Ohga A, Oleshansky M, Tomita U, Yamada Y. Voltage-independent catecholamine release mediated by the activation of muscarinic receptors in guinea-pig adrenal glands. British Journal of Pharmacology. 1988;93:101–109. doi: 10.1111/j.1476-5381.1988.tb11410.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. O'Farrell M, Ziogas J, Marley PD. Effects of N- and L-type calcium channel antagonists and (±)-Bay K8644 on nerve-induced catecholamine secretion from bovine perfused adrenal glands. British Journal of Pharmacology. 1997;121:381–388. doi: 10.1038/sj.bjp.0701131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Uceda G, Artalejo AR, de la Fuente MT, López MG, Albillos A, Michelena P, García AG, Montiel C. Modulation by L-type Ca2+ channels and apamin-sensitive K+ channels of muscarinic responses in cat chromaffin cells. American Journal of Physiology. 1994;266:C1432–1439. doi: 10.1152/ajpcell.1994.266.5.C1432. [DOI] [PubMed] [Google Scholar]
  28. Uceda G, Artalejo AR, López MG, Abad F, Neher E, García AG. Ca2+-activated K+ channels modulate muscarinic secretion in cat chromaffin cells. The Journal of Physiology. 1992;454:213–230. doi: 10.1113/jphysiol.1992.sp019261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wakade AR. Studies on secretion of catecholamines evoked by acetylcholine or transmural stimulation of the rat adrenal gland. The Journal of Physiology. 1981;313:463–480. doi: 10.1113/jphysiol.1981.sp013676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wakade AR, Wakade TD. Contribution of nicotinic and muscarinic receptors in the secretion of catecholamines evoked by endogenous and exogenous acetylcholine. Neuroscience. 1983;10:973–978. doi: 10.1016/0306-4522(83)90235-x. [DOI] [PubMed] [Google Scholar]
  31. Yamada Y, Teraoka H, Nakazato Y, Ohga A. Intracellular Ca2+ antagonist TMB-8 blocks catecholamine secretion evoked by caffeine and acetylcholine from perfused cat adrenal glands in the absence of extracellular Ca2+ Neuroscience Letters. 1988;90:338–342. doi: 10.1016/0304-3940(88)90212-1. [DOI] [PubMed] [Google Scholar]
  32. Zhou Z, Neher E. Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells. Pflügers Archiv. 1993;425:511–517. doi: 10.1007/BF00374879. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES