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Mechanism of adrenal catecholamine release by divalent mercury
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
28,82-87
(19%)
Mechanism of Adrenal Catecholamine Release by Divalent Mercury J. L. BOROWITZ Department of Pharmacology and Toxicology, Purdue University, West Lafayette, Indiana 47907 Received June 19,1973; accepted September 27,1973
Mechanism of Adrenal Catecholamine Release by Divalent Mercury. J. L. (1974). Toxicol. Appl. Pharmacol. 28,82-87. The decline of mercury-induced catecholamine release from isolated perfused bovine adrenals is biphasic when mercury concentrations of 9 x 1O-5M or above are used. After initiation ofcatecholaminerelease by 3 x 1O-5M mercury, the response declinesin a monophasic manner which corresponds to the first phase of decline seen after exposure of adrenals to higher mercury concentrations. Neither phase is altered by 5 x 10m3M magnesium, but the second phase is eliminated when calcium is omitted from the medium. 45Ca washout from labeled adrenals is decreased by 2 x 10e4 M Hgz+ but is not affected by 3 x 10-5~ HgZ+. Acetylcholine and mercury interact approximately in an adBOROWITZ,
ditive manner in releasing adrenal catechoIamines. The initial phase of mer-
cury-induced adrenal catecholamine release may be due to amine displacement by the mercury ion. The secondary phase of adrenal catecholamine releaseprobably involves alteration of membrane structures by mercury. Divalent mercury is the most potent of the ionsof subgroup IIB of the periodic table in releasing adrenal catecholamines (Hart, 1972). Also, the duration of the effect of mercury on the adrenal medulla is much more prolonged than that of cadmium or barium (Hart, 1972). These results prompted a more thorough study of the effect of Hg2+ on the adrenal medulla. METHODS Bovine adrenals were obtained at a local slaughterhouse, placed on ice during transport, and used about 1 hr postmortem. The glands were perfused through the adrenal vein at a rate of 10 ml/min with aerated Tris-buffered Locke’s solution at room temperature (24°C) as previously described (Borowitz, 1971). All agonists were given in a volume of 10 ml. Catecholamine release was measured calorimetrically (von Euler and Hamberg, 1949). Resting secretion averaged 20 + 2 pg/min (SE) in 30 representative glands. In the concentration-response series, divalent mercury dissolved in the Locke’s solution was infused in successively increasing concentrations in volumes of 10 ml. Each gland was exposed to only 1 concentration-response treatment. In other experiments involving only 1 concentration of mercury, exposure to Hg2+ was not repeated. To test the effect of Hgz+ on 45Ca washout from bovine adrenals, glands were labeled with 45Ca (1 pCi/ml, 10 ml). Extracellular 45Ca was then washed out for 18 min Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
82
MERCURY
AND
ADRENAL
MEDULLA
83
with 45Ca-free Locke’s solution before exposure to mercury. Aliquots of the perfusates (0.1-0.5 ml) were counted in a scintillation counter with an efficiency of 90 %. The counting cocktail consisted of 0.6% PPO, 0.01% POPOP in a 1: 1 mixture of toluene and 2ethoxyethanol; 15 ml of cocktail and 0.5 ml of water including the sample were used. High concentrations of mercury (1 x 10e3 M and above) catalyzed oxidation of catecholamines in the perfusates. This slightly elevated the standard curve (5 % at 250 pg/ sample). Therefore, catecholamine release was estimated according to standard curves prepared in the presence of mercury when high concentrations of mercury were used in the experiments. RESULTS Figure 1 shows that adrenal catecholamine release in response to Hg2+ (3 x 10V5 M, 10 ml) declined in a linear manner on a semilog plot. Higher concentrations of mercury induced adrenal catecholamine release which declined in 2 phases on a semilog plot, an initial rapid phase and a slower secondary phase. “Half-lives” for the decline of cate800 600
. 3xt0-4M~Hg,n=5 A Z~iO-~ti-Hg, n-17 l ‘9x10-5M Hg, n=lO o 3x10-5f&Hg, n=9
400 6 ‘z
200
D .P ;i z T Bcl
100 80 60 40
.E ‘D i k 6 y z .-%’ ; 0 $ s
20
10 8
6 4
2
1 0.
2
‘4 6 Minutes dfter
‘.8 10 exposure to Hg
12
14
FIG. 1. Adrenal catecholamine release by various concentrations of mercury. The indicated concentrations of mercury acetate were infused for 1 min in lO-ml volumes and perfusate was collected at I-min intervals thereafter. Release during infusion of mercury is shown at zero time. Release during the subceeding minute intervals is plotted at half-minute marks. Means + SE are given.
cholamines in the perfusates were evaluated graphically (Borowitz, 1971) in a manner similar to that used to estimate radioactive decay. The initial phase of the decline of catecholamine release had a half-life of about 2.4 min. This half-life is approximately the same as that for the monophasic decline of catecholamine in adrenal perfusates after treatment with 3 x 10e5 M mercury. The second phase of decline of catecholamine in the perfusates, seen after treatment with concentrations of 9 x 10m5M mercury and above,
84
BOROWITZ
had a half-life of 6.2 min. The slopes of the 2 phases of decline after exposure to 2 x 10e4 Hgz+ were significantly different (p < 0.005) by comparison of regression coefficients. Because of the well known effects of Hg*+ on thiol groups, the effect of I-cysteine on the adrenal response to mercury was tested. In 4 glands 10V3 M cysteine perfused 5 min before and during stimulation with Hg*+ (2 x 10V4 M) completely blocked catecholamine release. Figure 2 shows that the initial response to mercury was slightly enhanced (p < 0.05 at 3,4, and 5 min after Hg2+; the data were analyzed using Student’s t test) in a calciumfree medium. The second phase of the decline of catecholamine in the perfusates, however, was inhibited by this treatment. M
free media,
n=8
joo 80 60
10
I 2
I 4 Minutes
I 6
.B
I 10
I 12
1 4 14
after mercury stimulation
FIG. 2. Effect of a calcium-free medium on mercury-induced adrenal catecholamine release. Glands were exposed to a calcium-free medium for 20 min prior to exposure to mercury. Data from Fig. 1 are included for comparison. Means + SE are given.
45Calcium washout curves (Fig. 3) show that 2 x 10m4 M mercury decreased 45Ca washout from labeled adrenal glands. The lower concentration of Hg*+ (3 x 10m5 M), however, had no apparent effect on 45Ca washout. Despite the involvement of calcium (Figs. 2 and 3) in the effect of mercury on the adrenal medulla, magnesium (5 x 10m3~) failed to alter the pattern of catecholamine release in response to Hg*+ (2 x 10M4M, 2 glands) except for a slight inhibition initially. Figure 4 shows that acetylcholine and 3 x lo-’ M Hg*+ act approximately in an additive manner to release catecholamines from the adrenal medulla. Acetylcholine alone (100 pg/ml, 10 ml) gave a peak catecholamine release of 87 f 17.3 pg/min (SE; n = 12) with a half-life of 1.5 min. This half-life of the response to acetylcholine alone is comparable to that of 1.8 min seen during the initial phase of the response to a mixture of acetylcholine and Hg*+ 3 x 10e5 M. When the glands were exposed to acetylcholine and a higher concentration of Hg *+ , the effect of acetylcholine was evident initially, but overall the shape of the catecholamine washout curve closely resembled that of Hg*+ alone.
MERCURY
FIG.
AND
ADRENAL
85
MEDULLA
3. Effect of mercury on 45Ca washout from bovine adrenal glands.
0
2
4 Minutes
6 after
8
10
12
14
stimulation
FIG. 4. Interaction between acetylcholine and mercury in adrenal medulla. Acetylcholine, 100 pug free base/ml (total 1 mg), was included in the lo-ml mercury solutions so that the glands were exposed to both agonists simultaneously. Data from Fig. 1 are shown for comparison. Means + SE are given. The response to the combination of acetylcholine and the low dose of Hgz+ was significantly different (p < 0.05) from the response to Hg2+ alone through the tirst 3 min. Acetylcholine added to 2 x lo-” M Hg*+ gave a response significantly (p < 0.05) higher at 0,2.5,3.5,4.5, and 5.5 min than the response to Hgz+ alone.
86
BOROWITZ
DISCUSSION
Concentrations of divalent mercury above 9 x 10e5 M induce adrenal catecholamine release which declines in a biphasic manner. Two different mechanisms appear to be involved in the response, each accounting for one of the phases of decline. Initially, mercury-induced adrenal catecholamine release very likely involves the direct action of mercury on catecholamine stores since it is independent of extracellular calcium and not blocked by magnesium. Adrenal catecholamine release by acetylcholine is not altered by 3 x 10m5M mercury. The decline of catecholamine in the perfusate initially follows a pattern similar to that seen when acetylcholine alone is used as the stimulus. Therefore, short term exposure to 3 x 10e5 M mercury has little effect on physiological adrenal catecholamine release mechanisms. Divalent mercury apparently traps 45Ca in bovine adrenals yet catecholamine release by Hg’+ is little affected by magnesium which is known to block calcium-mediated release (Rubin et al., 1967; Rahwan et al., 1973). Thus any calcium trapped in adrenal cells by HgZf is nonfunctional in terms of catecholamine secretion. Mercury binds to erythrocyte membranes and produces a decrease in red cell osmotic fragility (Weed et al., 1962). In higher doses, mercury increases erythrocyte fragility. The membrane effects of mercury in adrenal medulla may result in the fixation of calcium to binding sites on various membranes of the cell including those of the endoplasmic reticulum. The observed decrease in 45Ca efflux caused by 2 x 10m4M Hg2+ may therefore be a reflection of the effect of mercury on the conformation of adrenal cellular membranous elements. Although adrenal calcium efflux diminishes during Hg2+ treatment, the permeability of membranes generally increases after exposure to Hg’+. For example Mn’+, phosphate, and various anions penetrate membranes of yeast cells more easily after mercury treatment (Passow and Rothstein, 1960). The general increase in membrane permeability caused by mercury may contribute to its ability to release adrenal catecholamines. It is suggested that the second phase of the decline of catecholamine noted in adrenal perfusates involves altered membrane integrity. ACKNOWLEDGMENTS The able technical assistanceof Mrs. Ivna Shanbaky is acknowledged. This work was supported by NIH Grant No. AM 16153 from the Institute of Arthritis and Metabolic Diseases. REFERENCES J. L. (1971). Duration of catecholamine release from bovine adrenal medulla. Amer. J. Physiol. 220, 1194-l 198. HART, D. T. (1972). Catecholamine release from the bovine adrenal gland by cadmium and related divalent metal ions. Thesis, Purdue University Libraries. PASSOW, H. AND ROTHSTEIN, A. (1960). The binding of mercury by the yeast cell in relation to changes in permeability. J. Gen. Phys. 43, 621-633. RAHWAN, R. G., B~ROWTIZ, J. L. AND MIYA, T. S. (1973). The role of intracellular calcium in catecholamine secretion from the bovine adrenal medulla. J. Phurmucol. Exp. Ther. 184, 106-118. BOROWITZ,
MERCURY
AND
ADRENAL
MEDULLA
87
R. P., FEINSTEIN, M. G., JAANLJS, S. D. AND PAIMRE, M. (1967). Inhibition of catecholamine secretion and calcium exchange in cat adrenal glands by tetracaine and magnesium
RUBIN,
J. Pharmacol.
Exp. Ther. 155,463-471.
HAMBERG, U. (1949). Calorimetric determination of adrenalin and noradrenalin. Acta Physiol. Stand. 19, 74-84. WEED, R., EBER, J. AND ROTHSTEIN, A. (1962). Interaction of mercury with human erythrocytes. J. Gen. Phys. 45, 395-410. VON
EULER,
U.
S. AND
AND
APPLIED
PHARMACOLOGY
28,82-87
(19%)
Mechanism of Adrenal Catecholamine Release by Divalent Mercury J. L. BOROWITZ Department of Pharmacology and Toxicology, Purdue University, West Lafayette, Indiana 47907 Received June 19,1973; accepted September 27,1973
Mechanism of Adrenal Catecholamine Release by Divalent Mercury. J. L. (1974). Toxicol. Appl. Pharmacol. 28,82-87. The decline of mercury-induced catecholamine release from isolated perfused bovine adrenals is biphasic when mercury concentrations of 9 x 1O-5M or above are used. After initiation ofcatecholaminerelease by 3 x 1O-5M mercury, the response declinesin a monophasic manner which corresponds to the first phase of decline seen after exposure of adrenals to higher mercury concentrations. Neither phase is altered by 5 x 10m3M magnesium, but the second phase is eliminated when calcium is omitted from the medium. 45Ca washout from labeled adrenals is decreased by 2 x 10e4 M Hgz+ but is not affected by 3 x 10-5~ HgZ+. Acetylcholine and mercury interact approximately in an adBOROWITZ,
ditive manner in releasing adrenal catechoIamines. The initial phase of mer-
cury-induced adrenal catecholamine release may be due to amine displacement by the mercury ion. The secondary phase of adrenal catecholamine releaseprobably involves alteration of membrane structures by mercury. Divalent mercury is the most potent of the ionsof subgroup IIB of the periodic table in releasing adrenal catecholamines (Hart, 1972). Also, the duration of the effect of mercury on the adrenal medulla is much more prolonged than that of cadmium or barium (Hart, 1972). These results prompted a more thorough study of the effect of Hg2+ on the adrenal medulla. METHODS Bovine adrenals were obtained at a local slaughterhouse, placed on ice during transport, and used about 1 hr postmortem. The glands were perfused through the adrenal vein at a rate of 10 ml/min with aerated Tris-buffered Locke’s solution at room temperature (24°C) as previously described (Borowitz, 1971). All agonists were given in a volume of 10 ml. Catecholamine release was measured calorimetrically (von Euler and Hamberg, 1949). Resting secretion averaged 20 + 2 pg/min (SE) in 30 representative glands. In the concentration-response series, divalent mercury dissolved in the Locke’s solution was infused in successively increasing concentrations in volumes of 10 ml. Each gland was exposed to only 1 concentration-response treatment. In other experiments involving only 1 concentration of mercury, exposure to Hg2+ was not repeated. To test the effect of Hgz+ on 45Ca washout from bovine adrenals, glands were labeled with 45Ca (1 pCi/ml, 10 ml). Extracellular 45Ca was then washed out for 18 min Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
82
MERCURY
AND
ADRENAL
MEDULLA
83
with 45Ca-free Locke’s solution before exposure to mercury. Aliquots of the perfusates (0.1-0.5 ml) were counted in a scintillation counter with an efficiency of 90 %. The counting cocktail consisted of 0.6% PPO, 0.01% POPOP in a 1: 1 mixture of toluene and 2ethoxyethanol; 15 ml of cocktail and 0.5 ml of water including the sample were used. High concentrations of mercury (1 x 10e3 M and above) catalyzed oxidation of catecholamines in the perfusates. This slightly elevated the standard curve (5 % at 250 pg/ sample). Therefore, catecholamine release was estimated according to standard curves prepared in the presence of mercury when high concentrations of mercury were used in the experiments. RESULTS Figure 1 shows that adrenal catecholamine release in response to Hg2+ (3 x 10V5 M, 10 ml) declined in a linear manner on a semilog plot. Higher concentrations of mercury induced adrenal catecholamine release which declined in 2 phases on a semilog plot, an initial rapid phase and a slower secondary phase. “Half-lives” for the decline of cate800 600
. 3xt0-4M~Hg,n=5 A Z~iO-~ti-Hg, n-17 l ‘9x10-5M Hg, n=lO o 3x10-5f&Hg, n=9
400 6 ‘z
200
D .P ;i z T Bcl
100 80 60 40
.E ‘D i k 6 y z .-%’ ; 0 $ s
20
10 8
6 4
2
1 0.
2
‘4 6 Minutes dfter
‘.8 10 exposure to Hg
12
14
FIG. 1. Adrenal catecholamine release by various concentrations of mercury. The indicated concentrations of mercury acetate were infused for 1 min in lO-ml volumes and perfusate was collected at I-min intervals thereafter. Release during infusion of mercury is shown at zero time. Release during the subceeding minute intervals is plotted at half-minute marks. Means + SE are given.
cholamines in the perfusates were evaluated graphically (Borowitz, 1971) in a manner similar to that used to estimate radioactive decay. The initial phase of the decline of catecholamine release had a half-life of about 2.4 min. This half-life is approximately the same as that for the monophasic decline of catecholamine in adrenal perfusates after treatment with 3 x 10e5 M mercury. The second phase of decline of catecholamine in the perfusates, seen after treatment with concentrations of 9 x 10m5M mercury and above,
84
BOROWITZ
had a half-life of 6.2 min. The slopes of the 2 phases of decline after exposure to 2 x 10e4 Hgz+ were significantly different (p < 0.005) by comparison of regression coefficients. Because of the well known effects of Hg*+ on thiol groups, the effect of I-cysteine on the adrenal response to mercury was tested. In 4 glands 10V3 M cysteine perfused 5 min before and during stimulation with Hg*+ (2 x 10V4 M) completely blocked catecholamine release. Figure 2 shows that the initial response to mercury was slightly enhanced (p < 0.05 at 3,4, and 5 min after Hg2+; the data were analyzed using Student’s t test) in a calciumfree medium. The second phase of the decline of catecholamine in the perfusates, however, was inhibited by this treatment. M
free media,
n=8
joo 80 60
10
I 2
I 4 Minutes
I 6
.B
I 10
I 12
1 4 14
after mercury stimulation
FIG. 2. Effect of a calcium-free medium on mercury-induced adrenal catecholamine release. Glands were exposed to a calcium-free medium for 20 min prior to exposure to mercury. Data from Fig. 1 are included for comparison. Means + SE are given.
45Calcium washout curves (Fig. 3) show that 2 x 10m4 M mercury decreased 45Ca washout from labeled adrenal glands. The lower concentration of Hg*+ (3 x 10m5 M), however, had no apparent effect on 45Ca washout. Despite the involvement of calcium (Figs. 2 and 3) in the effect of mercury on the adrenal medulla, magnesium (5 x 10m3~) failed to alter the pattern of catecholamine release in response to Hg*+ (2 x 10M4M, 2 glands) except for a slight inhibition initially. Figure 4 shows that acetylcholine and 3 x lo-’ M Hg*+ act approximately in an additive manner to release catecholamines from the adrenal medulla. Acetylcholine alone (100 pg/ml, 10 ml) gave a peak catecholamine release of 87 f 17.3 pg/min (SE; n = 12) with a half-life of 1.5 min. This half-life of the response to acetylcholine alone is comparable to that of 1.8 min seen during the initial phase of the response to a mixture of acetylcholine and Hg*+ 3 x 10e5 M. When the glands were exposed to acetylcholine and a higher concentration of Hg *+ , the effect of acetylcholine was evident initially, but overall the shape of the catecholamine washout curve closely resembled that of Hg*+ alone.
MERCURY
FIG.
AND
ADRENAL
85
MEDULLA
3. Effect of mercury on 45Ca washout from bovine adrenal glands.
0
2
4 Minutes
6 after
8
10
12
14
stimulation
FIG. 4. Interaction between acetylcholine and mercury in adrenal medulla. Acetylcholine, 100 pug free base/ml (total 1 mg), was included in the lo-ml mercury solutions so that the glands were exposed to both agonists simultaneously. Data from Fig. 1 are shown for comparison. Means + SE are given. The response to the combination of acetylcholine and the low dose of Hgz+ was significantly different (p < 0.05) from the response to Hg2+ alone through the tirst 3 min. Acetylcholine added to 2 x lo-” M Hg*+ gave a response significantly (p < 0.05) higher at 0,2.5,3.5,4.5, and 5.5 min than the response to Hgz+ alone.
86
BOROWITZ
DISCUSSION
Concentrations of divalent mercury above 9 x 10e5 M induce adrenal catecholamine release which declines in a biphasic manner. Two different mechanisms appear to be involved in the response, each accounting for one of the phases of decline. Initially, mercury-induced adrenal catecholamine release very likely involves the direct action of mercury on catecholamine stores since it is independent of extracellular calcium and not blocked by magnesium. Adrenal catecholamine release by acetylcholine is not altered by 3 x 10m5M mercury. The decline of catecholamine in the perfusate initially follows a pattern similar to that seen when acetylcholine alone is used as the stimulus. Therefore, short term exposure to 3 x 10e5 M mercury has little effect on physiological adrenal catecholamine release mechanisms. Divalent mercury apparently traps 45Ca in bovine adrenals yet catecholamine release by Hg’+ is little affected by magnesium which is known to block calcium-mediated release (Rubin et al., 1967; Rahwan et al., 1973). Thus any calcium trapped in adrenal cells by HgZf is nonfunctional in terms of catecholamine secretion. Mercury binds to erythrocyte membranes and produces a decrease in red cell osmotic fragility (Weed et al., 1962). In higher doses, mercury increases erythrocyte fragility. The membrane effects of mercury in adrenal medulla may result in the fixation of calcium to binding sites on various membranes of the cell including those of the endoplasmic reticulum. The observed decrease in 45Ca efflux caused by 2 x 10m4M Hg2+ may therefore be a reflection of the effect of mercury on the conformation of adrenal cellular membranous elements. Although adrenal calcium efflux diminishes during Hg2+ treatment, the permeability of membranes generally increases after exposure to Hg’+. For example Mn’+, phosphate, and various anions penetrate membranes of yeast cells more easily after mercury treatment (Passow and Rothstein, 1960). The general increase in membrane permeability caused by mercury may contribute to its ability to release adrenal catecholamines. It is suggested that the second phase of the decline of catecholamine noted in adrenal perfusates involves altered membrane integrity. ACKNOWLEDGMENTS The able technical assistanceof Mrs. Ivna Shanbaky is acknowledged. This work was supported by NIH Grant No. AM 16153 from the Institute of Arthritis and Metabolic Diseases. REFERENCES J. L. (1971). Duration of catecholamine release from bovine adrenal medulla. Amer. J. Physiol. 220, 1194-l 198. HART, D. T. (1972). Catecholamine release from the bovine adrenal gland by cadmium and related divalent metal ions. Thesis, Purdue University Libraries. PASSOW, H. AND ROTHSTEIN, A. (1960). The binding of mercury by the yeast cell in relation to changes in permeability. J. Gen. Phys. 43, 621-633. RAHWAN, R. G., B~ROWTIZ, J. L. AND MIYA, T. S. (1973). The role of intracellular calcium in catecholamine secretion from the bovine adrenal medulla. J. Phurmucol. Exp. Ther. 184, 106-118. BOROWITZ,
MERCURY
AND
ADRENAL
MEDULLA
87
R. P., FEINSTEIN, M. G., JAANLJS, S. D. AND PAIMRE, M. (1967). Inhibition of catecholamine secretion and calcium exchange in cat adrenal glands by tetracaine and magnesium
RUBIN,
J. Pharmacol.
Exp. Ther. 155,463-471.
HAMBERG, U. (1949). Calorimetric determination of adrenalin and noradrenalin. Acta Physiol. Stand. 19, 74-84. WEED, R., EBER, J. AND ROTHSTEIN, A. (1962). Interaction of mercury with human erythrocytes. J. Gen. Phys. 45, 395-410. VON
EULER,
U.
S. AND