AMERICAN JOURNAL OF PHYSIOLOGY VoI. 229, No. 1, July 1975. Printed in U.S.A.
Catecholamine
release: mechanism
mercury-induced
vascular
smooth
of muscle
HAROLD S. SOLOMON AND NORMAN K. HOLLENBERG Departments of Medicine and Radiolog?, Peter Bent Brigham Hos@lal and Harvard Boston, Massachusetis 02 1 I.5
SOLOMON,
HAROLD
S.,
AND
NORMAN
amhe mechanism of mercury-induced contraction. Am. J Physiol. 229(l): release
l
K.
HOLLENBERG. vascular
CatechdSmou
th
muscle
8-12. 1975.-The mechanism by which mercuric ion (I-IgClz) induces contraction of vascular smooth muscle was defined in the kidney of anesthetized dogs and in rabbit aortic strips. In vivo, HgCl2 injected into the renal artery induced a dose-related reduction in renal blood flow (electromagnetic flowmeter) and glomerular filtration rate (creatinine clearance). An intra-arterial infusion of phenoxybenzamine (PO@ significantly reduced the vascular response to HgCl2 (P < 0.001). I n vitro, alpha-adrenergic blockade with phentolamine and POB prevented mercury-induced contraction, whereas agents that block serotonin, histamine, acetylcholine, and angiotensin did not do so. Norepinephrine receptor LLprotection” from phenoxybenzamine blockade sustained the response to HgC12. Reserpine pretreatment produced a parallel reduction in the response of the aorta to tyramine and mercury. The results are consistent with an indirect action of mercuric ion via release of endogenous catecholamines. trace metals; blood pressure; vascular reactivity; acute renal failure; renal blood flow; alpha-adrenergic angiotensin antagonists
nephrotoxins; blockade;
SMOOTH MUSCLE IXspOIISe to bichloride of is well documented (4, 13-16, 18, 19). Despite the number of studies performed to explore how mercury induces a vascular response, the mechanism has remained obscure. During the course of an investigation designed to explore the effects of nephrotoxins on vascular smooth muscle, we observed a profound effect of an alpha-adrenergic blocking agent on the response of the rabbit aorta to bichloride of mercury in vitro. This study was performed, therefore, to explore in detail the role of catecholamines in the vascular response to mercury. Because of the prominent action of mercury on renal function (4, 18, 19) and a well-documented role of the renal vasculature in the pathogenesis of acute renal failure, studies were also performed in vivo on the canine renal vasculature. A
VASCULAR
mercury
METHODS
In vitro. The in vitro studies were performed in the isolated rabbit aortic strip (lo), based on the original description of Furchgott and Bhadrakon (7). In brief four strips from each rabbit aorta were mounted with 4 g of tension in muscle chambers with a IO-ml working volume containing
contraction
Medical
School,
a modified Krebs-bicarbonate medium. The solution was maintained at 37 & 0.5”C and aerated constantly with a gas mixture containing 95 % 0 2 - 5 $6 COZ, which maintained bath pH at 7.4. Tsotonic contractions were monitored with a Harvard Instruments force transducer and recorded on a Harvard Instruments recorder. The tissues were allowed to equilibrate for 60 min prior to drug administration. The bath arrangement allowed continuous removal of solute solutions and agents at appropriate intervals. The volume used for the wash was at least 10 times the bath volume, and relaxation of the smooth muscle preparations after drug removal was passive. All studies reported below were performed in at least eight rabbit aortic strips derived from at least four animals. In each study at least one strip was used as a control to assess the stability of that preparation. In 8 experiments on 32 strips, dose-response relationships were defined for norepinephrine bitartrate (Levophed, Winthrop Laboratories), serotonin hydrogen oxalate (Calbiochern), angiotensin II amide (Hypertensin-CIBA), and HgClz (Sigma Chemical Co.). Doses ranged from 1 to 1,000 rig/ml for norepinephrine, serotonin, and angiotensin, calculated as the salt, and up to 10 pg/ml for ‘mercury. The term mercuric ion and the concentration administered are used for convenience in the following presentation, despite the fact that the concentration of ionic Hg+f in the Krebs solution must have been reduced by excess chloride ion, which binds the metal in complexes (14). While the free concentration was considerably lower, it was probably a consistent function of the administered dose. In another series of experiments, pharmacologic blockade was induced with one of the following agents; phentolamine mesylate (Regitine; Ciba Pharmaceutical CO.); phenoxybenzamine hydrochloride (Dibenzyline; Smith Kline & French); atropine sulfate (Sigma Chemical CO.); cyproheptadine hydrochloride (Periactin; Merck Sharp & Dohme); methysergide maleate (Sansert; Sandoz Pharmaceuticals); diphenhydramine hydrochloride (Benadryl; Parke, Davis 8r. Co.); [Sari, Al$]angiotensin II (Norwich Pharmaceutical Co.). The adequacy of blockade was assessed for specific agonists (norepinephrine, serotonin, histamine, angiotensin 11) in each experiment, as appropriate. Antagonists were used in a dose range of 0.1-100 pg/ml, and the agonists were assessed over a full dose-response range. Reserpine (Serpasil, Ciba Pharmaceutical Co.) in a
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
VASCULAR
Sh3OOTH
MUSCLE
CATECHOLAMINE
RELEASE
dose of 5 mg/kg was administered intraperitoneally at 24-h intervals for 2 or 3 days prior to study in five rabbits. The adequacy of catecholamine depletion in the reserpine pretreated rabbit aorta was assessed by challenge with tyramine (100 pg/ml) in vitro (11). In eight strips serial tyramine doses were given until tachyphylaxis to tyramine developed. In another series of experiments the specificity of the protection provided by phenoxybenzamine was assessed. The approach suggested by Furchgott (6) involving Yeceptor protection” was used. In these experiments norepinephrine (100 rig/ml) was added to the bath 3 min prior to the administration of phenoxybenzamine (30 pg/ml). After 5 min of contact with phenoxybenzamine, the tissues were rinsed free of both agents. In each protection experiment another tissue strip was exposed to an identical phenoxybenzamine concentration for an identical interval in the absence of norepinephrine. Similar experiments were also performed with serotonin, acetylcholine, and histamine as the agonists. ~JZviva. The in vivo experiments were performed in nine adult mongrel dogs, anesthetized with sodium pentobarbital, 30 mg/kg, and maintained with 50-mg supplemental doses as required. The preparation in this laboratory has been described in detail (2). Intubation with a cuffed endotracheal tube allowed control of ventilation, as required, with a Harvard respirator. A nasogastric tube was placed in the stomach for the administration of a tap water load of 5 ml/kg. Both ureters were catheterized with PE200 polyethylene tubing, and serial ZO- to 30-min. urine samples were collected for the measurement of volume and creatinine excretion. Serum creatinine was also measured to allow the calculation of endogenous creatinine clearance. A red Kifa catheter was placed into the renal artery via the femoral artery under Auoroscopic control for the intra-arterial administration of the agents. A Statham electromagnetic Aowmeter probe, 3,O-4.0 mm in diameter,
PHENTOLAMINE
1 Fq/ml
+
I
NOREPINEPHRINE
1
(ng/mJ)
FIG. 1. Effect of alpha-adrenergic blockade mine (POB) and phentoIamine in rabbit aortic reduction in responses to mercuric ion (10 pg/ml) foIlowing exposure to alpha-adrenergic blocking
1
l-lq ++
I
with phenoxybenzastrips. Note striking and norepinephrine agents.
9 was placed around the renal artery through a retroperitoneal flank approach. Arterial blood pressure was monitored through the Kifa catheter with a Statham P23 DC pressure transducer. Pressure, renal blood flow, and heart rate were recorded on a Grass polygraph. Following a period of 30-60 min to allow the stabilization of the animal after the preparation and for control urine collections, HgClz was administered as a bolus through the renal arterial catheter. The agent was diluted so that the volume injected was less than 3 ml, and following injection the catheter was flushed with 2 ml of 0.9 % heparinized saline+ The HgCl2 was given at 15-min intervals in the following log-dose increments; O,Ol, 0.03, 0.1, 0.3, and 0.7 to a cumulative 1.14-mg/kg dose. In five experiments alpha-adrenergic blockade was obtained prior to HgC12 administration by infusing phenoxybenzamine, 1-3 mg/kg, into the renal artery over 15 min. The adequacy of blockade was tested by injecting norepinephrine, 2.5 pug, into the renal artery before and after This dose of norepinephrine, phenoxybenzamine infusion. which normally reduces renal blood flow transiently to zero, was blocked by phenoxybenzamine in all experiments. After adequate blockade was achieved, usually 20-30 min later, HgCl2 was administered as described above. RESULTS
The contractile response of the rabbit aorta to mercuric ion was similar to that described earlier by Perry et al. (14). The contraction was characteristically slow; several minutes were required to reach a maximum, as opposed to the seconds required for the response to norepinephrine and angiotensin. Tracings of typical responses are shown in Fig. 1. The threshold for response was approximately 0.3 pg/ml, with a maximum at about 10 pg/ml. Metal-induced contraction was followed by spontaneous relaxation of the strip, frequently to its original length, following which it was unresponsive to Hg++, norepinephrine, and angiotensin II, as described (14). A mercury dose of 10 pg/ml was used in all of the following experiments. An excellent correlation was found between the contractile response to a 0. l-&ml norepinephrine dose (x) and to the mercuric ion at 10 pg/ml (y = 0.86x - 11; r = 0.89; F = 28.3; E’ < 0.001). A representative response to mercury is shown in Fig. 1. A correlation between the contractile response to mercury, conversely, could not be defined for angiotensin or serotonin. Alpha-adrenergic blockade with phentolamine and phenoxybenzamine prevented the response to mercuric ion Representative tracings are shown in Fig. 1. Doses of either blocking agent too small to induce alpha-adrenergic blockade (less than 0.1 pg/ml) also failed to alter the response to mercuric ion. Even large doses of the other blocking agents, cyproheptadine, methysergide, atropine, diphenhydramine, and [Sari , Ala 8] angiotensin II, failed to alter the response to mercuric ion, despite concentrations which shifted the responsiveness to serotonin, acetylcholine, histamine, or angiotensin 11 1,OOO-fold. Protection of the norepinephrine receptor against the effect of phenoxybenzamine also sustained the response to mercuric ion. A representative series of tracings is shown in
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H. S. SOLOMON
10
I
NOREPINEPHRINE
(nghl)’
I
Hg ++
I
FIG. 2. Protection of norepinephrine receptors from blockade induced by phenoxybenzamine (POB) was induced by superimposing phenoxybenzamine dose on norepinephrine-treated strip* Note that dose of POB was larger than that used earlier (compare with Fig. I), but a much smaller degree of bIockade of norepinephrine ensues and there is a well-sustained response to mercuric ion (10 pg/ml),
Fig. 2. Similar protection experiments with histamine and acetylcholine failed to protect the aortic strip from the effects of mercuric ion. Note that a phenoxybenzamine concentration 3 times higher than that which totally obliterated the response to Hg++ (Fig. 1) did not block Hg++-induced contraction when the norepinephrine receptors were “protected.” Reserpine pretreatment had a variable influence on the aortic strip response to mercuric ion. When there was a residual response to tyramine in the reserpine-treated strip, the response to mercuric ion was also sustained (Fig-. 3). In the aortic strip resistant to the effect of tyramine, the response to mercury was blunted or absent. When aortic strips taken from reserpine-pretreated rabbits were challenged with tyramine serially until total tachyphylaxis to tyramine was achieved, the strips were also unresponsive to mercury (Fig. 3). In vivo, HgCl2 produced a dose-related reduction in renal blood flow (Figs. 4 and 5). The threshold dose for both blood flow and creatinine clearance was 0.01 mg/kg into the renal artery with an ED-50, the dose that induced a 50 % reduction, between 0.03 and 0.10 “g/kg. A dose of 1.O mg/kg produced an 80 % reduction in both renal blood flow and glomerular filtration rate. Phenoxybenzamine pretreatment produced a significant reduction in the responses to norepinephrine and HgClz in vivo (Figs 4 and 5). The ED-50 for renal blood flow shifted from 0.06 to 028 mg/kg, approximately a fourfold shift (P < 0.01). In the case of glomerular filtration the ED-50 shifted from 0,02 to 0.21 mg/kg, approximately a lo-fold shift (P < 0.055)+ HgClz broke through the blockade, however, so that a 70 % reduction in both renal blood flow and glomerular filtration rate occurred with the 0.7mg/kg dose.
AND
N. K. HOLLENBERG
intrinsic responsiveness of the aortic strips to norepinephrine and to the mercuric ion. Second, two active alphaadrenergic blocking agents, phenoxybenzamine and phentolamine, blocked responses to mercuric ion in doses that blocked norepinephrine. Both agents, the first a beta haloalkylamine and the second an imidazoline derivative, also block a wide variety of receptors, including those for histamine, serotonin, and acetylcholine at somewhat higher doses (12). Specific antagonists to these agonists, however, did not block the response to mercuric ion. Third, it was possible to enhance the specificity of blockade with phenoxybenzamine by inducing specific “protection” (6) of the norepinephrine receptors : such protection also prevented phenoxybenzamine-induced blockade of the response to mercuric ion. Protection of the other receptors, on the other hand, did not prevent a response to mercuric ion. Fourth, the experiments with reserpine and with tyramine tachyphylaxis provided further support for the role of catecholamines in the sequence and also provided insight into the mechanism by which mercuric ion acted on a catecholamine receptor, It is apparent from this series of experiments that mercuric ion does not act directly on the alpha-adrenergic receptor, but rather does so indirectly by causing the release of endogenous catecholamines. When endogenous catecholamine stores were depleted by pretreatment with reserpine, as indicated by failure to respond to the indirect agent, tyramine (lo), the response to mercuric ion disappeared. The fact that the reserpine pretreatment produced a variable residual response to tyramine perhaps provides insight into the differences in this study and that reported by Perry et al. (14) earlier. They failed to demonstrate an efiect of reserpine on mercuric ion-induced contraction of rabbit aortic strips, but they did not assess the adequacy of catecholamine depletion and they administered only a single 5-mg dose to the rabbits. It seems likely that residual catecholamines accounted for this failure to demonstrate an effect of reserpine, since we pretreated for 2 or 3 days with the same dose and still found residual catecholamines.
0t
02
-4
4
08
05 4
4
4
nfv +
4
#
+
IJ-
4
4
DXSCUSSION Several lines of evidence defined in this study suggest that mercury-induced vascular smooth muscle contraction occurred via activation of norepinephrine receptors and was probably due to endogenous catecholamine release. First, there was a high degree of correlation between the
I
TYRAMINE
(IOOpg/ml)
1 ’
Hg++
’
’SEROTONIN (lOpg/ml)
FIG. 3, Jn strips taken from reserpine-pretreated rabbits, induction of tachyphylaxis to tyramine (100 pg/ml) obliterated response to mercuric ion (10 pg/mI). Note that these strips remained responsive . to serotonin.
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
’
SMOOTH
VASCULAR RENAL FLOW (ml/min)
MUSCLE
CATECHOLAMINE
11
RELEASE
4002oo-
OBP
FIG. 4. Tracing of renal hemodvnamic and blood”pressure responses ;o graded doses of H&12. Note total obliteration of renal vascular response to a large dose of norepinephrine following phenoxybenzamine administration.
zoo-
hmHg)
m
IOO- -.-
W
-_1_~
82-85
97x
OTIME
(min)
O-3
3OM
1
NE 2.5pg T POB ( 3mg/kg
33 65-68
t NE
2.5pg
t
Hg O.ly/kg
t
Hg O.Sy/kg
Hg
.I00 O.?y/
kg
id.)
Moreover, tachyphylaxis to tyramine made the strips unresponsive to mercuric ion. Many of the studies done in vitro were, for technical reasons, extremely difficult to perform in vivo. To the extent that the observations could be made in vivo, with the intraarterial administration of phenoxybenzamine, the results parallel those in vitro. Phenoxybenzamine, in a dose that induces striking reduction in the response to 2.5 pg of norepinephrine, also blunted the vascular response to mercuric ion. Larger doses of mercuric ion, however, overcame this effect of phenoxybenzamine. The dose-response curve for phenoxybenzamine plateaus at about 10 mg/kg (8). Such large doses, however, have prominent additional cardiovascular effects including alteration in arterial pressure, and so these doses were not used in the study. We cannot be certain, therefore, whether the residual vascular response to larger doses of mercury despite phenoxybenzamine reflects a) the inadequacy of alpha-adrenergic blockade, b) an additional, direct effect of the mercuric ion on the vascular smooth muscle, or c) the induction of some secondary effect-perhaps activation of the renin-angiotensin system secondary to a tubular action of mercury (17) This question is of some importance in view of the striking capacity of the mercuric ion to induce acute renal failure and the present interest in the role of vasoactive agents in the pathogenesis of that syndrome (9) + Sherwood et al. (18) demonstrated a similar blood flow reduction with mercury injected into the renal artery, a response that they attributed to mechanical occlusion of small renal vessels induced by capillary cell swelling, an explanation similar to that provided for the “no-reflow” phenomenon which follows prolonged renal ischemia (5). Sherwood and Lavender’s (18) interpretation was based on the response to mannitol, which transiently increased renal blood flow following mercury, a response that they attributed to the osmotic effect of mannitol on the renal capillaries Such a phenomenon may contribute to the renal vascular response to larger doses of mercury, which alphaadrenergic blockade did not prevent. The mechanism by which the mercuric ion results in catecholamine release was not defined in this study. There is excellent evidence that the mercuric ion, probably through binding with critical sulfhydryl groups, induces disruption of cell membrane, for example, in red cells (3, 20). Perry’s observation that the contractile apparatus became un-
O-
20 -
40
-
60
-
80
-
0.01
0.03
HgC12 FIG. 5. Rena1 vascular response chloride injected into renal artery. by phenoxybenzamine is significant
0.10
hg/kg
0.30
I.0
id
to cumulative Reduction (P < 0.01).
doses of mercuric of response induced
responsive not only to additional mercuric ion, but also to norepinephrine-an observation confirmed in this study-is consistent with the disruption of the entire machinery. Another observation made in this study, lowever, makes such a gross action of the mercuric ion extremely unlikely: the contracti!e response to serotonin was maintained. Thus, if the response to mercuric ion reflects an influence on the cell membrane, it is much more likely to be focal and specific, involving the loci which include the receptors for angiotensin and norepinephrine, rather than total, nonspecific disruption of the membrane. In conclusion, we have demonstrated in vitro and in the canine renal vasculature a dose-related response to mercuric ion which is due to catecholamine release. The mechanism by which the mercuric ion induces catecholamine release remains poorly defined (l), but it is unlikely on the basis of our observations that this reflects simple, nonspecific disruption of the cell membrane. It is a pleasure to acknowledge the assistance provided in various parts of this study by Ms. K. Henricks, B. Nevins, I. Dowgialo, E. Gonski, and Mr. R. Johnson.
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12
H. S. SOLOMON
This investigation was supported by National Institutes of Health Grants GM-19674 and HE-l 1668 and Army Research and Development Command DAMD 17 74 4023. In conducting the research described in this report, the investigators
adhered prepared Council,
to the “Guide for Laboratory by the National Academy
Received
for publication
1 July
AND N. K. HOLLENBERG Animal Facilities of Sciences-National
and Care” Research
1974.
REFERENCES J, 1;. Mechanism of adrenal catecholamine release by 1. BOROWITZ, divalent mercury. Toxicol, A#. Pharmacol. 28 : 82-87, 1974. W* J. H., N. K. HOLLENBERG, AND H. L. ABRAMS. 2. CALDICOTT, Characteristic response of renal vascular bed to contrast media: evidence for vasoconstriction induced by renin-angiotensin system. Invest. Radial. 5 : 539-547, 1970, J. W. The pharmacology of mercury compounds. 3. CLARKSON, Ann. Rev. Pharmacol, 12 : 375-406, 1972. W. J,, AND D. E, OKEN. Renal micropuncture study 4. FLANIGAN, of the development of anuria in the rat with mercury-induced acute renal failure. J. Clin. Invest. 44 : 449-457, 1965. 5. FLORES, J., D. R. DIBONA, C. H. BECK, AND A. LEAF. The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute. J. Clin. Invest, 5 1: 118-126, 1972. 6. FURCHGOTT, R. F. Dibenamine blockade in strips of rabbit aorta and its use in differentiating receptors. J. Pharmacol. Exptl. Therap. 111: 265-284, 1954. 7. FURCHGOTT, R. F., AND S. BHADRAKON. Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs. J. Pharmacol. ExptL Therap. 108: 129-144, 1953. 8. HOLLENBERG, N. K. The Role of the Sympathetic Nervous System in the Develofment of Decompensation During Hemorrhagic Shock (PhD Thesis). Winnipeg: Univ, of Manitoba, 1965. 9. HOLLENBERG, N. K., D. F. ADAMS, D. E, OKEN, H. 3;. ABRAMS, AND J+ P. MERRILL. Acute renal failure due to nephrotoxins: renal hemodynamic and angiographic studies in man. lvew Engl. J. Med. 282 : 1329-1334, 1970. 10. HOLLENBERG, N. K., L. R. BARBERO, AND K. P. J. HXNRICHS. Angiotensin tachyphylaxis and vascular angiotensinase activity. Experientia 27: 1032-1034, 1971. Influence of 11. KRZANOWSKI, J. J., JR., AND R. A. WOODBURY. with dibenamine in aortic tyramine on receptor interaction strips from reserpine-treated rabbits, J. Pharmacol. Exptl. Therap. 154 : 472-480, 1966.
12. 13.
14.
15,
16.
NICKERSON, M., AND N. K. HOLLENBERG. Blockade of alphaadrenergic receptors. In: Physiol. Pharmacology, edited by W. S. Root and F. G. Hofmann. New York: Academic, 1967, p. 243-306. PERRY, H. M., JR., AND A. YU.NICE. Acute pressor effects of intraarterial cadmium and mercuric ions in anesthetized rats. Proc. SOL Exptl. Biol. Med. 120 : 805-808, 1965. PERRY, H, M,, JR., E. SCHOEPFLE, AND J. BOURGOIGNIE. In vitro production and inhibition of aortic vasoconstriction by mercuric, cadmium, and other metal ions, hoc, SOL Exfltl. Biol. Med. 124: 485-490, 1967. PERRY, H. M., JR., M. ERLANGER, A. YUNICE, AND E. F. PERRY. Mechanisms of the acute hypertensive effect of intra-arterial cadmium and mercury in anesthetized rats. J. Lab. Clin. Med. 70: 963-972, 1967. PERRY, H, hi., JR., M. ERLANGER, A. YUNICE, E. SCHOEPFLE, AND E. F. PERRY. Hypertension and tissue levels following intravenous cadmium, mercury, and zinc. Am. J. Phys, 2 15 : 755-761,
1970. 17. SCHNERMANN, J., W. NAGEL, AND K. THURAU. Die frtihdistale natriumkonzentration in rattennieren nach renaler ischgmie und hgmorrhagischer hypotension : Ein beitrag zur pathogenese der postisch%mischen und posth%morrhZgischen filtraterniedrigung. P@egers A&z. 287 : 296-3 10, 1966. 18, SHERWOOD, T., J. P. LAVENDER, AND S. B. RUSSELL. Mercuryinduced renal vascular shut-down : observations in experimental acute renal failure. European J. C&n. Invest. 4 : 1, 1974. 19. SOLOMON, H. S., AND N. K. HOLLENBERG. Renal corticaf blood flow in mercury-induced canine acute renal failure, Proc, Intern. Gongr.
Nephrol.,
5th,
Mexico
City,
1972,
20. VERITY, M. A. Mercury-induced renal necrosis based upon heavy metal-membrane J. 83: 573-574, 1972.
p. 68.
necrosis: a model of cell interaction. Am. Heart
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
Catecholamine
release: mechanism
mercury-induced
vascular
smooth
of muscle
HAROLD S. SOLOMON AND NORMAN K. HOLLENBERG Departments of Medicine and Radiolog?, Peter Bent Brigham Hos@lal and Harvard Boston, Massachusetis 02 1 I.5
SOLOMON,
HAROLD
S.,
AND
NORMAN
amhe mechanism of mercury-induced contraction. Am. J Physiol. 229(l): release
l
K.
HOLLENBERG. vascular
CatechdSmou
th
muscle
8-12. 1975.-The mechanism by which mercuric ion (I-IgClz) induces contraction of vascular smooth muscle was defined in the kidney of anesthetized dogs and in rabbit aortic strips. In vivo, HgCl2 injected into the renal artery induced a dose-related reduction in renal blood flow (electromagnetic flowmeter) and glomerular filtration rate (creatinine clearance). An intra-arterial infusion of phenoxybenzamine (PO@ significantly reduced the vascular response to HgCl2 (P < 0.001). I n vitro, alpha-adrenergic blockade with phentolamine and POB prevented mercury-induced contraction, whereas agents that block serotonin, histamine, acetylcholine, and angiotensin did not do so. Norepinephrine receptor LLprotection” from phenoxybenzamine blockade sustained the response to HgC12. Reserpine pretreatment produced a parallel reduction in the response of the aorta to tyramine and mercury. The results are consistent with an indirect action of mercuric ion via release of endogenous catecholamines. trace metals; blood pressure; vascular reactivity; acute renal failure; renal blood flow; alpha-adrenergic angiotensin antagonists
nephrotoxins; blockade;
SMOOTH MUSCLE IXspOIISe to bichloride of is well documented (4, 13-16, 18, 19). Despite the number of studies performed to explore how mercury induces a vascular response, the mechanism has remained obscure. During the course of an investigation designed to explore the effects of nephrotoxins on vascular smooth muscle, we observed a profound effect of an alpha-adrenergic blocking agent on the response of the rabbit aorta to bichloride of mercury in vitro. This study was performed, therefore, to explore in detail the role of catecholamines in the vascular response to mercury. Because of the prominent action of mercury on renal function (4, 18, 19) and a well-documented role of the renal vasculature in the pathogenesis of acute renal failure, studies were also performed in vivo on the canine renal vasculature. A
VASCULAR
mercury
METHODS
In vitro. The in vitro studies were performed in the isolated rabbit aortic strip (lo), based on the original description of Furchgott and Bhadrakon (7). In brief four strips from each rabbit aorta were mounted with 4 g of tension in muscle chambers with a IO-ml working volume containing
contraction
Medical
School,
a modified Krebs-bicarbonate medium. The solution was maintained at 37 & 0.5”C and aerated constantly with a gas mixture containing 95 % 0 2 - 5 $6 COZ, which maintained bath pH at 7.4. Tsotonic contractions were monitored with a Harvard Instruments force transducer and recorded on a Harvard Instruments recorder. The tissues were allowed to equilibrate for 60 min prior to drug administration. The bath arrangement allowed continuous removal of solute solutions and agents at appropriate intervals. The volume used for the wash was at least 10 times the bath volume, and relaxation of the smooth muscle preparations after drug removal was passive. All studies reported below were performed in at least eight rabbit aortic strips derived from at least four animals. In each study at least one strip was used as a control to assess the stability of that preparation. In 8 experiments on 32 strips, dose-response relationships were defined for norepinephrine bitartrate (Levophed, Winthrop Laboratories), serotonin hydrogen oxalate (Calbiochern), angiotensin II amide (Hypertensin-CIBA), and HgClz (Sigma Chemical Co.). Doses ranged from 1 to 1,000 rig/ml for norepinephrine, serotonin, and angiotensin, calculated as the salt, and up to 10 pg/ml for ‘mercury. The term mercuric ion and the concentration administered are used for convenience in the following presentation, despite the fact that the concentration of ionic Hg+f in the Krebs solution must have been reduced by excess chloride ion, which binds the metal in complexes (14). While the free concentration was considerably lower, it was probably a consistent function of the administered dose. In another series of experiments, pharmacologic blockade was induced with one of the following agents; phentolamine mesylate (Regitine; Ciba Pharmaceutical CO.); phenoxybenzamine hydrochloride (Dibenzyline; Smith Kline & French); atropine sulfate (Sigma Chemical CO.); cyproheptadine hydrochloride (Periactin; Merck Sharp & Dohme); methysergide maleate (Sansert; Sandoz Pharmaceuticals); diphenhydramine hydrochloride (Benadryl; Parke, Davis 8r. Co.); [Sari, Al$]angiotensin II (Norwich Pharmaceutical Co.). The adequacy of blockade was assessed for specific agonists (norepinephrine, serotonin, histamine, angiotensin 11) in each experiment, as appropriate. Antagonists were used in a dose range of 0.1-100 pg/ml, and the agonists were assessed over a full dose-response range. Reserpine (Serpasil, Ciba Pharmaceutical Co.) in a
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
VASCULAR
Sh3OOTH
MUSCLE
CATECHOLAMINE
RELEASE
dose of 5 mg/kg was administered intraperitoneally at 24-h intervals for 2 or 3 days prior to study in five rabbits. The adequacy of catecholamine depletion in the reserpine pretreated rabbit aorta was assessed by challenge with tyramine (100 pg/ml) in vitro (11). In eight strips serial tyramine doses were given until tachyphylaxis to tyramine developed. In another series of experiments the specificity of the protection provided by phenoxybenzamine was assessed. The approach suggested by Furchgott (6) involving Yeceptor protection” was used. In these experiments norepinephrine (100 rig/ml) was added to the bath 3 min prior to the administration of phenoxybenzamine (30 pg/ml). After 5 min of contact with phenoxybenzamine, the tissues were rinsed free of both agents. In each protection experiment another tissue strip was exposed to an identical phenoxybenzamine concentration for an identical interval in the absence of norepinephrine. Similar experiments were also performed with serotonin, acetylcholine, and histamine as the agonists. ~JZviva. The in vivo experiments were performed in nine adult mongrel dogs, anesthetized with sodium pentobarbital, 30 mg/kg, and maintained with 50-mg supplemental doses as required. The preparation in this laboratory has been described in detail (2). Intubation with a cuffed endotracheal tube allowed control of ventilation, as required, with a Harvard respirator. A nasogastric tube was placed in the stomach for the administration of a tap water load of 5 ml/kg. Both ureters were catheterized with PE200 polyethylene tubing, and serial ZO- to 30-min. urine samples were collected for the measurement of volume and creatinine excretion. Serum creatinine was also measured to allow the calculation of endogenous creatinine clearance. A red Kifa catheter was placed into the renal artery via the femoral artery under Auoroscopic control for the intra-arterial administration of the agents. A Statham electromagnetic Aowmeter probe, 3,O-4.0 mm in diameter,
PHENTOLAMINE
1 Fq/ml
+
I
NOREPINEPHRINE
1
(ng/mJ)
FIG. 1. Effect of alpha-adrenergic blockade mine (POB) and phentoIamine in rabbit aortic reduction in responses to mercuric ion (10 pg/ml) foIlowing exposure to alpha-adrenergic blocking
1
l-lq ++
I
with phenoxybenzastrips. Note striking and norepinephrine agents.
9 was placed around the renal artery through a retroperitoneal flank approach. Arterial blood pressure was monitored through the Kifa catheter with a Statham P23 DC pressure transducer. Pressure, renal blood flow, and heart rate were recorded on a Grass polygraph. Following a period of 30-60 min to allow the stabilization of the animal after the preparation and for control urine collections, HgClz was administered as a bolus through the renal arterial catheter. The agent was diluted so that the volume injected was less than 3 ml, and following injection the catheter was flushed with 2 ml of 0.9 % heparinized saline+ The HgCl2 was given at 15-min intervals in the following log-dose increments; O,Ol, 0.03, 0.1, 0.3, and 0.7 to a cumulative 1.14-mg/kg dose. In five experiments alpha-adrenergic blockade was obtained prior to HgC12 administration by infusing phenoxybenzamine, 1-3 mg/kg, into the renal artery over 15 min. The adequacy of blockade was tested by injecting norepinephrine, 2.5 pug, into the renal artery before and after This dose of norepinephrine, phenoxybenzamine infusion. which normally reduces renal blood flow transiently to zero, was blocked by phenoxybenzamine in all experiments. After adequate blockade was achieved, usually 20-30 min later, HgCl2 was administered as described above. RESULTS
The contractile response of the rabbit aorta to mercuric ion was similar to that described earlier by Perry et al. (14). The contraction was characteristically slow; several minutes were required to reach a maximum, as opposed to the seconds required for the response to norepinephrine and angiotensin. Tracings of typical responses are shown in Fig. 1. The threshold for response was approximately 0.3 pg/ml, with a maximum at about 10 pg/ml. Metal-induced contraction was followed by spontaneous relaxation of the strip, frequently to its original length, following which it was unresponsive to Hg++, norepinephrine, and angiotensin II, as described (14). A mercury dose of 10 pg/ml was used in all of the following experiments. An excellent correlation was found between the contractile response to a 0. l-&ml norepinephrine dose (x) and to the mercuric ion at 10 pg/ml (y = 0.86x - 11; r = 0.89; F = 28.3; E’ < 0.001). A representative response to mercury is shown in Fig. 1. A correlation between the contractile response to mercury, conversely, could not be defined for angiotensin or serotonin. Alpha-adrenergic blockade with phentolamine and phenoxybenzamine prevented the response to mercuric ion Representative tracings are shown in Fig. 1. Doses of either blocking agent too small to induce alpha-adrenergic blockade (less than 0.1 pg/ml) also failed to alter the response to mercuric ion. Even large doses of the other blocking agents, cyproheptadine, methysergide, atropine, diphenhydramine, and [Sari , Ala 8] angiotensin II, failed to alter the response to mercuric ion, despite concentrations which shifted the responsiveness to serotonin, acetylcholine, histamine, or angiotensin 11 1,OOO-fold. Protection of the norepinephrine receptor against the effect of phenoxybenzamine also sustained the response to mercuric ion. A representative series of tracings is shown in
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H. S. SOLOMON
10
I
NOREPINEPHRINE
(nghl)’
I
Hg ++
I
FIG. 2. Protection of norepinephrine receptors from blockade induced by phenoxybenzamine (POB) was induced by superimposing phenoxybenzamine dose on norepinephrine-treated strip* Note that dose of POB was larger than that used earlier (compare with Fig. I), but a much smaller degree of bIockade of norepinephrine ensues and there is a well-sustained response to mercuric ion (10 pg/ml),
Fig. 2. Similar protection experiments with histamine and acetylcholine failed to protect the aortic strip from the effects of mercuric ion. Note that a phenoxybenzamine concentration 3 times higher than that which totally obliterated the response to Hg++ (Fig. 1) did not block Hg++-induced contraction when the norepinephrine receptors were “protected.” Reserpine pretreatment had a variable influence on the aortic strip response to mercuric ion. When there was a residual response to tyramine in the reserpine-treated strip, the response to mercuric ion was also sustained (Fig-. 3). In the aortic strip resistant to the effect of tyramine, the response to mercury was blunted or absent. When aortic strips taken from reserpine-pretreated rabbits were challenged with tyramine serially until total tachyphylaxis to tyramine was achieved, the strips were also unresponsive to mercury (Fig. 3). In vivo, HgCl2 produced a dose-related reduction in renal blood flow (Figs. 4 and 5). The threshold dose for both blood flow and creatinine clearance was 0.01 mg/kg into the renal artery with an ED-50, the dose that induced a 50 % reduction, between 0.03 and 0.10 “g/kg. A dose of 1.O mg/kg produced an 80 % reduction in both renal blood flow and glomerular filtration rate. Phenoxybenzamine pretreatment produced a significant reduction in the responses to norepinephrine and HgClz in vivo (Figs 4 and 5). The ED-50 for renal blood flow shifted from 0.06 to 028 mg/kg, approximately a fourfold shift (P < 0.01). In the case of glomerular filtration the ED-50 shifted from 0,02 to 0.21 mg/kg, approximately a lo-fold shift (P < 0.055)+ HgClz broke through the blockade, however, so that a 70 % reduction in both renal blood flow and glomerular filtration rate occurred with the 0.7mg/kg dose.
AND
N. K. HOLLENBERG
intrinsic responsiveness of the aortic strips to norepinephrine and to the mercuric ion. Second, two active alphaadrenergic blocking agents, phenoxybenzamine and phentolamine, blocked responses to mercuric ion in doses that blocked norepinephrine. Both agents, the first a beta haloalkylamine and the second an imidazoline derivative, also block a wide variety of receptors, including those for histamine, serotonin, and acetylcholine at somewhat higher doses (12). Specific antagonists to these agonists, however, did not block the response to mercuric ion. Third, it was possible to enhance the specificity of blockade with phenoxybenzamine by inducing specific “protection” (6) of the norepinephrine receptors : such protection also prevented phenoxybenzamine-induced blockade of the response to mercuric ion. Protection of the other receptors, on the other hand, did not prevent a response to mercuric ion. Fourth, the experiments with reserpine and with tyramine tachyphylaxis provided further support for the role of catecholamines in the sequence and also provided insight into the mechanism by which mercuric ion acted on a catecholamine receptor, It is apparent from this series of experiments that mercuric ion does not act directly on the alpha-adrenergic receptor, but rather does so indirectly by causing the release of endogenous catecholamines. When endogenous catecholamine stores were depleted by pretreatment with reserpine, as indicated by failure to respond to the indirect agent, tyramine (lo), the response to mercuric ion disappeared. The fact that the reserpine pretreatment produced a variable residual response to tyramine perhaps provides insight into the differences in this study and that reported by Perry et al. (14) earlier. They failed to demonstrate an efiect of reserpine on mercuric ion-induced contraction of rabbit aortic strips, but they did not assess the adequacy of catecholamine depletion and they administered only a single 5-mg dose to the rabbits. It seems likely that residual catecholamines accounted for this failure to demonstrate an effect of reserpine, since we pretreated for 2 or 3 days with the same dose and still found residual catecholamines.
0t
02
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4
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05 4
4
4
nfv +
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+
IJ-
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4
DXSCUSSION Several lines of evidence defined in this study suggest that mercury-induced vascular smooth muscle contraction occurred via activation of norepinephrine receptors and was probably due to endogenous catecholamine release. First, there was a high degree of correlation between the
I
TYRAMINE
(IOOpg/ml)
1 ’
Hg++
’
’SEROTONIN (lOpg/ml)
FIG. 3, Jn strips taken from reserpine-pretreated rabbits, induction of tachyphylaxis to tyramine (100 pg/ml) obliterated response to mercuric ion (10 pg/mI). Note that these strips remained responsive . to serotonin.
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’
SMOOTH
VASCULAR RENAL FLOW (ml/min)
MUSCLE
CATECHOLAMINE
11
RELEASE
4002oo-
OBP
FIG. 4. Tracing of renal hemodvnamic and blood”pressure responses ;o graded doses of H&12. Note total obliteration of renal vascular response to a large dose of norepinephrine following phenoxybenzamine administration.
zoo-
hmHg)
m
IOO- -.-
W
-_1_~
82-85
97x
OTIME
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3OM
1
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33 65-68
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2.5pg
t
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t
Hg O.Sy/kg
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Moreover, tachyphylaxis to tyramine made the strips unresponsive to mercuric ion. Many of the studies done in vitro were, for technical reasons, extremely difficult to perform in vivo. To the extent that the observations could be made in vivo, with the intraarterial administration of phenoxybenzamine, the results parallel those in vitro. Phenoxybenzamine, in a dose that induces striking reduction in the response to 2.5 pg of norepinephrine, also blunted the vascular response to mercuric ion. Larger doses of mercuric ion, however, overcame this effect of phenoxybenzamine. The dose-response curve for phenoxybenzamine plateaus at about 10 mg/kg (8). Such large doses, however, have prominent additional cardiovascular effects including alteration in arterial pressure, and so these doses were not used in the study. We cannot be certain, therefore, whether the residual vascular response to larger doses of mercury despite phenoxybenzamine reflects a) the inadequacy of alpha-adrenergic blockade, b) an additional, direct effect of the mercuric ion on the vascular smooth muscle, or c) the induction of some secondary effect-perhaps activation of the renin-angiotensin system secondary to a tubular action of mercury (17) This question is of some importance in view of the striking capacity of the mercuric ion to induce acute renal failure and the present interest in the role of vasoactive agents in the pathogenesis of that syndrome (9) + Sherwood et al. (18) demonstrated a similar blood flow reduction with mercury injected into the renal artery, a response that they attributed to mechanical occlusion of small renal vessels induced by capillary cell swelling, an explanation similar to that provided for the “no-reflow” phenomenon which follows prolonged renal ischemia (5). Sherwood and Lavender’s (18) interpretation was based on the response to mannitol, which transiently increased renal blood flow following mercury, a response that they attributed to the osmotic effect of mannitol on the renal capillaries Such a phenomenon may contribute to the renal vascular response to larger doses of mercury, which alphaadrenergic blockade did not prevent. The mechanism by which the mercuric ion results in catecholamine release was not defined in this study. There is excellent evidence that the mercuric ion, probably through binding with critical sulfhydryl groups, induces disruption of cell membrane, for example, in red cells (3, 20). Perry’s observation that the contractile apparatus became un-
O-
20 -
40
-
60
-
80
-
0.01
0.03
HgC12 FIG. 5. Rena1 vascular response chloride injected into renal artery. by phenoxybenzamine is significant
0.10
hg/kg
0.30
I.0
id
to cumulative Reduction (P < 0.01).
doses of mercuric of response induced
responsive not only to additional mercuric ion, but also to norepinephrine-an observation confirmed in this study-is consistent with the disruption of the entire machinery. Another observation made in this study, lowever, makes such a gross action of the mercuric ion extremely unlikely: the contracti!e response to serotonin was maintained. Thus, if the response to mercuric ion reflects an influence on the cell membrane, it is much more likely to be focal and specific, involving the loci which include the receptors for angiotensin and norepinephrine, rather than total, nonspecific disruption of the membrane. In conclusion, we have demonstrated in vitro and in the canine renal vasculature a dose-related response to mercuric ion which is due to catecholamine release. The mechanism by which the mercuric ion induces catecholamine release remains poorly defined (l), but it is unlikely on the basis of our observations that this reflects simple, nonspecific disruption of the cell membrane. It is a pleasure to acknowledge the assistance provided in various parts of this study by Ms. K. Henricks, B. Nevins, I. Dowgialo, E. Gonski, and Mr. R. Johnson.
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12
H. S. SOLOMON
This investigation was supported by National Institutes of Health Grants GM-19674 and HE-l 1668 and Army Research and Development Command DAMD 17 74 4023. In conducting the research described in this report, the investigators
adhered prepared Council,
to the “Guide for Laboratory by the National Academy
Received
for publication
1 July
AND N. K. HOLLENBERG Animal Facilities of Sciences-National
and Care” Research
1974.
REFERENCES J, 1;. Mechanism of adrenal catecholamine release by 1. BOROWITZ, divalent mercury. Toxicol, A#. Pharmacol. 28 : 82-87, 1974. W* J. H., N. K. HOLLENBERG, AND H. L. ABRAMS. 2. CALDICOTT, Characteristic response of renal vascular bed to contrast media: evidence for vasoconstriction induced by renin-angiotensin system. Invest. Radial. 5 : 539-547, 1970, J. W. The pharmacology of mercury compounds. 3. CLARKSON, Ann. Rev. Pharmacol, 12 : 375-406, 1972. W. J,, AND D. E, OKEN. Renal micropuncture study 4. FLANIGAN, of the development of anuria in the rat with mercury-induced acute renal failure. J. Clin. Invest. 44 : 449-457, 1965. 5. FLORES, J., D. R. DIBONA, C. H. BECK, AND A. LEAF. The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute. J. Clin. Invest, 5 1: 118-126, 1972. 6. FURCHGOTT, R. F. Dibenamine blockade in strips of rabbit aorta and its use in differentiating receptors. J. Pharmacol. Exptl. Therap. 111: 265-284, 1954. 7. FURCHGOTT, R. F., AND S. BHADRAKON. Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs. J. Pharmacol. ExptL Therap. 108: 129-144, 1953. 8. HOLLENBERG, N. K. The Role of the Sympathetic Nervous System in the Develofment of Decompensation During Hemorrhagic Shock (PhD Thesis). Winnipeg: Univ, of Manitoba, 1965. 9. HOLLENBERG, N. K., D. F. ADAMS, D. E, OKEN, H. 3;. ABRAMS, AND J+ P. MERRILL. Acute renal failure due to nephrotoxins: renal hemodynamic and angiographic studies in man. lvew Engl. J. Med. 282 : 1329-1334, 1970. 10. HOLLENBERG, N. K., L. R. BARBERO, AND K. P. J. HXNRICHS. Angiotensin tachyphylaxis and vascular angiotensinase activity. Experientia 27: 1032-1034, 1971. Influence of 11. KRZANOWSKI, J. J., JR., AND R. A. WOODBURY. with dibenamine in aortic tyramine on receptor interaction strips from reserpine-treated rabbits, J. Pharmacol. Exptl. Therap. 154 : 472-480, 1966.
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NICKERSON, M., AND N. K. HOLLENBERG. Blockade of alphaadrenergic receptors. In: Physiol. Pharmacology, edited by W. S. Root and F. G. Hofmann. New York: Academic, 1967, p. 243-306. PERRY, H. M., JR., AND A. YU.NICE. Acute pressor effects of intraarterial cadmium and mercuric ions in anesthetized rats. Proc. SOL Exptl. Biol. Med. 120 : 805-808, 1965. PERRY, H, M,, JR., E. SCHOEPFLE, AND J. BOURGOIGNIE. In vitro production and inhibition of aortic vasoconstriction by mercuric, cadmium, and other metal ions, hoc, SOL Exfltl. Biol. Med. 124: 485-490, 1967. PERRY, H. M., JR., M. ERLANGER, A. YUNICE, AND E. F. PERRY. Mechanisms of the acute hypertensive effect of intra-arterial cadmium and mercury in anesthetized rats. J. Lab. Clin. Med. 70: 963-972, 1967. PERRY, H, hi., JR., M. ERLANGER, A. YUNICE, E. SCHOEPFLE, AND E. F. PERRY. Hypertension and tissue levels following intravenous cadmium, mercury, and zinc. Am. J. Phys, 2 15 : 755-761,
1970. 17. SCHNERMANN, J., W. NAGEL, AND K. THURAU. Die frtihdistale natriumkonzentration in rattennieren nach renaler ischgmie und hgmorrhagischer hypotension : Ein beitrag zur pathogenese der postisch%mischen und posth%morrhZgischen filtraterniedrigung. P@egers A&z. 287 : 296-3 10, 1966. 18, SHERWOOD, T., J. P. LAVENDER, AND S. B. RUSSELL. Mercuryinduced renal vascular shut-down : observations in experimental acute renal failure. European J. C&n. Invest. 4 : 1, 1974. 19. SOLOMON, H. S., AND N. K. HOLLENBERG. Renal corticaf blood flow in mercury-induced canine acute renal failure, Proc, Intern. Gongr.
Nephrol.,
5th,
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City,
1972,
20. VERITY, M. A. Mercury-induced renal necrosis based upon heavy metal-membrane J. 83: 573-574, 1972.
p. 68.
necrosis: a model of cell interaction. Am. Heart
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