Sensitization to the Generalized Shwartzman Reaction by Catechol-O-Methyltransferase Inhibitors Jean-Gilles Latour, PhD, and Claudette Leger-Gauthier, MS
The generalized Shwartzman reaction (GSR) was produced by a single injection of endotoxin in male rats pretreated with catechol-o-methyltransferase (COMT) inhibitors (tropolone, pyrogallol). Such a result was not obtained with inhibitors (pargyline, phenelzine, isocarboxazide) of the monoamine oxidase (MAO). The inhibitors of the COMT were found to enhance the action of endotoxin on the coagulation system such as evidenced by the increased consumptions of Hageman factor, fibrinogen, and platelets. Tropolone-treated rabbits did not require exogenous stimulation of a-adrenergic receptor sites by norepinephrine to localize thrombi in the glomerular capillaries when Hageman factor was activated by ellagic acid and fibrinolysis inhibited by f-aminocaproic acid. It is concluded that interference with the degradation of circulating catecholamines results in sensitization to the generalized Shwartzman reaction. (Am J Pathol 92:377-388, 1978)
THE GENEERALIZED SHWARTFZMAN REACTION is the end result of a complex interplay between coagulation, fibrinolvsis, and vasomotor reactions subsequent to the entrance of bacterial endotoxin in the circulation.' The phenomenon is characterized bv glomerular capillary thrombosis leading to bilateral renal cortical necrosis. Stimulation of the a-adrenergic receptors has been identified as an essential component of the reaction.2 Blockade of the a-receptor sites was showrn to prevent the phenomenon induced in the pregnant rat;3'4 rabbits made tolerant to large doses of epinephrine do not develop the reaction.5 On the other hand, norepinephrine localizes thrombi in the glomerular capillaries of rabbits injected with thrombin 6 and precipitates circulating soluble fibrin monomers.7 The involvement of the adrenal response to endotoxin in the pathogenesis of the Shwartzman reaction has been demonstrated. Adrenalectomized rabbits are protected from the reaction, whereas administration of norepinephrine or glucocorticoids to these animals restores their susceptibilitv to the Shwartzman reaction.8 Finallv, the glucocorticoids were From the Laboratory of Experimental Pathology. Institut de Cardiologie de Montreal. and the Department of Pathology. Universit6 de M1ontr6al. Quebec. Canada Supported by Grant MA-4478 from the Medical Research Council of Canada and by the JeanLouis LUvesque Foundation Dr Latour is 'Chercheur Boursier' of the Conseil de la recherche en sante de Quebec. mrs Le'ger-Gauthier is a Fellow- of the Canadian Heart Foundation Accepted for publication April 3. 1978 Address reprint requests to Dr J. G Latour. Institut de Cardiologie de MIontr&al. 5000 Belanger Street East. M1ontr6al. Quebec. Canada HIT IC8 0002-9440/78/081 0-0377$01.00 377
378
LATOUR AND LEGER-GAUTHIER
American Journal of Pathology
show-n to sensitize to the Shwartzman phenomenon through an action mediated by the a-adrenergic receptor sites.9 The ability of the glucocorticoids to enhance the vascular reactivity to catecholamines is well known; among the several mechanisms suspected is the reported inhibition of catechol-o-methvltransferase (COMT)."0 Therefore, know-n inhibitors of COMT and monoamine oxidase (MAO) ,were investigated as sensitizing agents for the generalized Shwartzman reaction. Our results showr that inhibitors of COMT prepare the normal rat and rabbit for the Shwartzman phenomenon by promoting a-adrenergic stimulation. Materals and Methods Animals
Holtzman male rats (.375 to 400 g) and New Zealand rabbits (1.8 to 2.2 kg) were used. They %vere fed standard pellets and given water ad libitum. W7ith the rats under ether anesthesia. blood samples were collected from a jugular vein into plastic syninges containing the appropriate anticoagulant.
Infusions Slow intravenous infusions lasting 4.3 hours were performed in rabbits (Experiment 3) under pentobarbital anesthesia (30 mg 'kg). A catheter introduced into the superior vena cava bv ay of the right ear vein was connected to a sigmamotor infusion pump and used for ellagic acid. The other drugs were given by use of a Sage syringe pump via an infusion set (Butterflv 2.3, Abbott Laboratories, Chicago, Ill.) inserted in the marginal vein of the left ear.
Drugs Endotoxin
Salmonella typhosa 0901 endotoxin boivin ty-pe (Difco Laboratories, Detroit) was dissolved in physiologic sterile saline and injected into a jugular vein at the dosage of 1 mg'kg 2 hours after tropolone or distilled water, 15 minutes after pyrogallol, and 1 hour after the last administration of the NMAO inhibitors (Experiments 1, 3, and 4). MAO Inhibitors
Pargyline chlorhydrate (Abbott Laboratories, Montreal), phenelzine sulfate (Wamer Chilcott, Toronto), and isocarboxazide (Hoffmann-La Roche Ltee, Vaudreuil, Quebec) were obtained commercially. They were suspended in distilled water and fed to the rats on .3 successive days bv way of a stomach tube at the dosages of 40 mg, 20 mg, and 25 mg/kg, respectively. COMT Inhibitors
PN-rogallol (Pvrogallic acid. Fisher Scientific, Montreal) and tropolone (Aldrich Chemicals Co.. Montreal) were used as catechol-o-methvltransferase inhibitors.11-13 They were dissoved in sterile distilled water and injected subcutaneously in rats at the respective doses of 500 mg /kg and 3M0 mg,/kg. In the rabbit experiment (Experiment 5), tropolone
Vol. 92, No. 2 August 1978
GENERAUZED SHWARTZMAN REACTION
379
was prepared in 5% dextrose-saline and the pH of the solution was adjusted to 7.4 with IN NaOH. The drug was given intravenously at the dosage of 1 mg,/kg,/min. Blagic Acid
Ellagic acid (K and K Laboratories Inc., Plainview, N.Y.) was stirred in physiologic saline and infused at the dosage and rate of 12.5 mg '50 ml 'kg, 'hr (Experiment 5). Norepinephrine
Levarterenol bitartrate (Levophed, Winthrop Laboratories, New York) was diluted with 5% dextrose-saline and given at the rate of 10 ml/kg,/hr. The drug was infused at the dose of 1.25 qg 'kg 'min for 3 hours, then at 2 jig /kg/min for the next one-half hour, and finally at 2.5 ug /kg /min for the last hour (Experiment 5). E-Aminocaproic Acid (EACA)
Amicar (Lederle Laboratories. Pearl River, N.Y.) was diluted with 5% dextrose-saline such that 5 mg "kg 'min was infused in a volume of 5 ml /kg,/hr (Experiment 5). Blood Tests Plasma recalcification times and cephalin clotting times were performed on citrated plasma as already described.1' A Bell and Alton 15 rabbit brain cephalin extract was used as a platelet substitute in the latter test. Dilute blood clot lysis times were estimated with the use of 0.12 MI sodium acetate buffer pH 74.1617 The platelet counts were performed with a Coulter Electronic particle counter as reported elsewhere in detail.1' The hematocrits were measured in heparinized capillanr tubes, and Hageman factor was assayed on Factor-XIIdeficient plasma." Clottable fibrinogen was estimated according to the original method of Ratnoff and Menzie.19 Histlogy At the end of the experiments (4 hours after the injection of endotoxin in Experiments 1, .3, and 4 and after the 4.5-hour infusion period in Experiment 5), the rats were killed with chloroform and the rabbits were killed with an overdose of pentobarbital. An autopsy was performed on each animal, and the kidneys were fixed in 10% neutral formalin. Sections of both kidnevs were stained with phosphotungstic-acid hematoxylin (PTAH) and examined for the presence of fibrin thrombi.
Results COMT and MAO Inhibitors and Sensitizati (Experiment 1)
of the Rat to the Generalized Shwtzman Reaction
As indicated in Table 1, none of the rats given only endotoxin (Group 1) showed glomerular thrombosis. On the contrarv, endotoxin elicited glomerular thrombosis in 59.4% (P < 0.001 vs Groups 1 and 2) and in 64.7% (P < 0.001 vs Groups 1 and 4) of the rats pretreated, respectivelv, with tropolone (Group 3) and pvrogallol (Group 5). Tropolone (Group 2) and pyrogallol (Group 4) alone without endotoxin did not elicit glomerular thombosis. Endotoxin failed to induce the Shwartzman reaction in pargyline- (Group 6) and phenelzine- (Group 7) pretreated rats. However, a
American Journal of Pathology
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380
Table 1-COMT and MAO Inhibitors and Sensitization of the Male Rat to the Generalized Shwartzman Reaction
Group
Treatment
Total No. of animals
1 2 3 4 5 6 7 8
Distilled water + endotoxin Tropolone + saline Tropolone + endotoxin Pyrogallol + saline Pyrogallol + endotoxin Pargyline + endotoxin Phenelzine + endotoxin Isocarboxazide + endotoxin
39 35 32 20 17 16 17 20
Percent with glomerular thrombosis 0.0 0.0 59.40.0 64.7t 0.0 0.0
10.01t
P < 0.001 vs Groups 1 and 2. t P < 0.001 vs Groups 1 and 4. t P < 0.05 vs Group 1.
slight (10%) but significant incidence (P < 0.05) of glomerular thrombosis was observed with isocarboxazide (Group 8). Glomerular thrombosis elicited by endotoxin in tropolone- and pvrogallol-pretreated animals was not generalized to all glomeruli. The lesions were disseminated in both kidneys, but groups of 5 to 10 occluded glomeruli were often surrounded bv manv which had remained normal. The severity of the reaction is illustrated in Figure 1, which shows the observed typical glomerular lesion. Effects of COMT hhibitors In Vivo on (Experiment 2)
Bband Spoteous Fibtyss
In comparison to the cephalin times and recalcification times of the control rats given water, the cephalin times and recalcification times of the animals injected with tropolone were shorter bv, respectivelv, 25% (P < 0.001) and 3057c (P < 0.001) after 4 hours (Text-figure 1). Similar changes were observed 2¼4 hours after pyrogallol with respective reductions of 34% (P < 0.001) and 40%E (P < 0.001) of the cephalin and recalcification clotting times. However, neither tropolone nor pyrogallol modified the clot lvsis times. Platelet and Fb*ogen CosLwnpons in Rats Pretreated Wrth COMT hIhibitors and Given an Intravous Ircuon of Endotoxin (Experimet 3)
The blood platelet count dropped bv 15% (P < 0.01) and 26% (P < 0.001), respectivelv, 2 and 4 hours after an injection of endotoxin in control rats given onlv water subcutaneouslv (Text-figure 2). However, the platelet count was reduced bv 38% (P < 0.001) and 27% (P < 0.001), respectivelv, 4 hours after endotoxin in tropolone- and pyrogallol-pre-
GENERALIZED SHWARTZMAN REACTION
Vol. 92, No. 2 August 1978 CrEPIAUIN TIMF7
381
CaOT LYS TIUME
EECALAICATION Ttuc WBirL
0.01
-
0.001 t
TEXT-FIGURE 1-Effects of COMIT inhibitors on blood coagulability and spontaneous fibrinolysis in the rat. Results: Mean (12 rats per group) ± SE. Dotted columns, water: lined columns, tropolone; solid columns. psTogallol.
treated rats. In these groups there was no significant change after 2 hours. Tropolone did not produce by itself anv change in the circulating platelet count for the duration of the experiment, but pvrogallol administration was followed bv a significant (P < 0.001) increase (28%) in the platelet count after 4 hours. Fibrinogen was reduced bv 27% (P < 0.001) 4 hours after the injection of endotoxin in control rats. However, the drop was 48% (P < 0.001) and 38% (P < 0.001), respectively, in the animals pretreated with tropolone and pyrogallol. Two hours after administration of tropolone and pyrogallol, a 15% (P < 0.001) reduction in clottable fibrinogen was noted in both groups. However, normal or near normal values were measured after 4 to 6 hours in the groups given tropolone or pyrogallol and injected with saline instead of endotoxin. A gradual decrease in hematocrit, as a consequence of successive blood removals, reached 18% (P < 0.001) at the end of the experiment in the control animals given onlv water as a pretreatment and saline in place of endotoxin. This response was quite similar to that observed (22%, P < 0.001) in the rats given endotoxin. However, the changes were not as pronounced when tropolone and pyrogallol were given as a pretreatment. The drops were, respectivelv, 10% (P < 0.001) and 1% (not significant) in the rats given endotoxin and 6 % (P < 0.001) and 16% (P < 0.001) in those given saline in association with tropolone and pyrogallol. Hageman Factor Consumpftion i Rats Prstrated With COMT Inhibitors and Given Injection of Edotoxn (Experimnt 4)
an Intavenous
Three hours after the injection of physiologic saline in rats pretreated with water, tropolone, and pvrogallol, Hageman factor activitv was, respectivelv, 101.2 ± 1.5%, 97.3 ± 2.8%, and 104.2 ± 2% of the preexperimental (100%) values. However, the injection of endotoxin was
382
LATOUR AND
LeGER-GAUTHIER
American Journal of Pathology
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GENERALIZED SHWARIZMAN REACTIUN . .
Vol. 92, No. 2 August 1978
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100 TEAT-FIGURE 3-Consumption in Hageman factor following endotoxin in rats pretreated with CO\1T inhibitors. Pre-ex-
perimental values were set arbias the 100%l reference. The *-alues w-ere corrected for changes in plasma volume using the hematocrit as the index. Resuits: Mean (10 rats per group) i SE. Dotted columns, before endotoxin: solid columns. 3 hours after endotoxin.
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followed by a decrease of 38% (P < 0.001), 68% (P < 0.001), and 76% (P < 0.001), respectively, in the groups given water, tropolone, and pvrogallol as pretreatment (Text-figure 3). The consumptions in Hageman factor in these last two groups were significantlv (P < 0.001) more pronounced than those obtained in the controls given water as pretreatment. Incidence of Giomerular Thrombosis When Tropolone is Substited for Noreprephnee, EACA, or Elagic Acid i the Syntetc Shwartzman Reaction (Experiment 5)
As indicated in Table 2, the combined infusion of ellagic acid, norepinephrine, and EACA according to the original protocol of McKay et al 2 for production of the so-called svnthetic Shwartzman reaction resulted in glomerular thrombosis in 14 of the 16 rabbits treated (Group 1). Deletion of norepinephrine (Group 2) resulted in an incidence of onlv 1 of 34 rabbits. In this animal, only a few tiny fibrin thrombi were evident. Table 2-Incidence of Glomerular Thrombosis When Tropolone is Substituted for Norepinephrine, EACA, or Ellagic Acid in the Synthetic Shwartzman Reaction
Percent with Group
Treatment
No. of rabbits
1 2 3 4 5
Ellagic acid + norepinephrine + EACA Ellagic acid + saline + EACA Ellagic acid +norepinephrine + tropolone Ellagic acid + tropolone + EACA Tropolone + norepinephrine + EACA
34 22 23 20
*P < 0.001 vs Groups 2 to 5. < 0.01 vs Group 4.
tP
16
glomerular thrombosis 87.5* 2.9 0.0
26.0t 0.0
384
LATOUR AND
LUGER-GAUTHIER
American Joumal of Pathology
When tropolone was substituted for norepinephrine (Group 4), 6 of the 23 rabbits (P < 0.001 vs Groups 2, 3, and 5) showed glomerular thrombosis. In contrast to this, when tropolone was given as a replacement for EACA (Group 3) or ellagic acid (Group 5), none of the animals demonstrated fibrin deposition in their glomerular capillaries. Discussion The results of this investigation indicate that agents such as tropolone and pyrogallol which interfere with COMT sensitize the normal animal to the generalized Shwartzman reaction (Table 1). It is demonstrated that the mechanism involved corresponds to an enhancement of the a-adrenergic response to endotoxin. The evidence is that tropolone can only be substituted with success for norepinephrine for production of the svnthetic Shwartzman reaction (Table 2). The additional observation that inhibitors of the MAO are not effective in preparing the animal for the Shwartzman phenomenon implies that the circulating catecholamines are those involved in the reaction. This conclusion is based on the respective activities and distributions of the two enzymes in the organism, the COMT being primarily implicated in degradation of blood catecholamines and MAO acting predominantly on tissue catecholamines.20 These results are in close continuity with our previous observation that adrenalectomy prevents the Shwartzman reaction5 and that the adrenal response to endotoxin is essential for production of the phenomenon. These results provide additional evidence that sensitization to the GSR by corticosteroids is mediated via the a-adrenergic receptor sites 9 and possibly, at least in part, through an inhibition of the COMT. Therefore, any drug capable of interfering with the removal of the circulating catecholamines such as occurs in enzymatic degradation or tissue uptake or any condition that may promote overproduction or release of catecholamines is susceptible to prepare for the GSR by providing the essential a-adrenergic component of the reaction. The role of the catecholamines in the production of the generalized Shwartzman reaction is complex. Administered in vivo, these hormones provoke the activation of Hageman factor 15 and the precipitation of soluble fibrin monomers.7 They promote or induce platelet aggregation 21 and induce vasomotor reactions in the kidneys, which localize fibrin deposition in the glomeruli when intravascular coagulation is triggered by an intravenous injection of thrombin." The serotonin derived from the platelets during the course of intravascular coagulation, and which is
Vol. 92, No. 2 August 1978
GENERAI 17FD SHWARTZMAN REACTION
385
catabolized by MAO, has only a slight and secondary effect on production of these vasomotor reactions and production of the GSR.4 In our study, administration of tropolone or pyrogallol resulted in hypercoagulabiity, but Hageman factor and platelets were not consumed nor was fibrinolysis activated. However, on injection of endotoxin, the two drugs promoted the consumption of Hageman factor, platelets, and fibrinogen and the production of the generalized Shwartzman reaction. Whether the catecholamines released by endotoxin and protected from degradation by COMT inhibition are responsible for overconsumption of these blood coagulation parameters is not known, but, as already stressed,'6 a close correlation seems to exist between the two phenomena. Nevertheless, besides its effects on the coagulation processes, a-adrenergic stimulation was found to be essential for precipitation of the phenomenon and localization of thrombosis in the glomerular capillaries.4'6 In summary, several workers have identified the implication of catecholamines in the course of disseminated intravascular coagulation induced by endotoxin. Infusion of catecholamines under various experimental conditions was found to be essential- for production of the generalized Shwartzman reaction,"2'8 and pharmacologic agents interfering with the a-adrenergic receptors were shown to prevent the phenomenon." The present results with COMT inhibitors give proof that the massive release of catecholamines needed for production of the generalized Shwartzman reaction is achieved in vivo in the normal animal on injection of a single dose of endotoxin and that conditions or drugs which prepare or sensitize for the Shwartzman reaction may do so by interfering either with production, release, or neutralization of blood catecholamines. References 1. McKay DG, Latour JG. Lopez AM: Production of the generalized Shwartzman reaction by activated Hagemena factor and a-adrenergic stimulation. Thromb Diath Haemorrh 26:71-76, 1971 2. McKay DG, Muller-Berghaus G, Cruze V: Activation of Hageman factor by ellagic acid and the generalized Shwartzman reaction. Am J Pathol 54:393-420, 1969 3. Muller-Berghaus G, McKay DG: Prevention of the generalized Shwartzman reaction in pregnant rats by a-adrenergic blocking agents. Lab Invest 17:276-280, 1967 4. Latour JG, lkger-Gauthier C, Groulx C: Prevention of the generalized Shwartzman reaction by bradykinin and prostaglandins. Thromb Haemostas 38:224, 1977 5. Hall DL, Broom JS, Brunson JG: Effects of epinephrine tolerance on the Shwartzman phenomenon. Am J Pathol 44:431-440, 1964 6. Latour JG, lkger C: Production of glomerular capillary thrombosis by thrombin and vasoactive hormones. Blood Vessel Interactions: Systems in Special Tissues. First World Congress for Microcirculation, Toronto, Canada, 1975. Edited by J Grayson, W Zingg. New York, Plenum Publishing Corp., 1976 (Abstr)
386
LATOUR AND LEGER-GAUTHIER
American Journal of Pathology
7. Miiller-Berghaus G, Mann B: Precipitation of ancrod-induced soluble fibrin by aprotinin and norepinephrine. Thromb Res 2:305-322, 1973 8. Latour JG, McKav DG: Requirement of the adrenal glands for provocation of the generalized Shwartzrnan reaction. Lab Invest 22:281-285, 1970 9. Latour JG, Prejean JB, Margaretten W: Corticosteroids and the generalized Shwartzman reaction: Mechanisms of sensitization in the rabbit. Am J Pathol 65:189-202, 1971 10. Kalsner S: Mechanism of hvdrocortisone potentiation of responses to epinephrine and norepinephrine in rabbit aorta. Circ Res 24:383-395, 1969 11. Belleau B, Burba J: Occupancv of adrenergic receptors and inhibition of catecholo-methyl transferase by tropolones. J Med Chem 6:755-759, 1963 12. Mavrides C, Missala K, D'Iorio A: The effect of 4-methvl-tropolone on the metabolism of adrenaline. Can J Biochem Physiol 41:1581-1587, 1963 13. Hattori K, Matsuura M, Fujiwara M, Shimamoto K: Inhibition of catechol-omethyltransferase by hydroxybenzenes and related compounds. Jpn J Pharmcol 19:282-286, 1969 14. Latour JG, Leger C: Prevention by glucocorticoids of disseminated intravascular coagulation induced by endotoxin: Mechanisms. J Lab Clin Med 85:934-949, 1975 15. Bell WN, Alton HG: A brain extract as a substitute for platelet suspensions in the thromboplastin generation test. Nature 174:880-881, 1954 16. Gallimore MJ: Effects of diluents on blood clot lvsis. J Clin Pathol 20:234-238. 1967 17. Marchandise B, Latour JG, Bourassa MG, Beaulac P, Yu SK: Platelet hvperreactivity to fhrombin in patients with myocardial infarction and normal or slightly abnormal coronary arteries. Clin Invest Med (In press) 18. McKay DG, Latour JG, Parrish MH: Activation of Hageman factor by a-adrenergic stimulation. Thromb Diath Haemorrh 23:417-422, 1970 19. Ratnoff OD, Menzie C: A new method for the determination of fibrinogen in small samples of plasma. J Lab Clin Med 37:316-320, 1951 20. PevTin L, Dalmaz Y: La secretion et l'inactivation peripheriques des catecholamines (adrenaline, noradre'naline, dopamine). J Phvsiol (Paris) 70:353-433, 1975 21. Yu SK, Latour JG: Potentiation bv a and inhibition by 0-adrenergic stimulations of rat platelet aggregation: A comparative study with human and rabbit platelets. Thromb Haemostas 37:413-422, 1977
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The generalized Shwartzman reaction (GSR) was produced by a single injection of endotoxin in male rats pretreated with catechol-o-methyltransferase (COMT) inhibitors (tropolone, pyrogallol). Such a result was not obtained with inhibitors (pargyline, phenelzine, isocarboxazide) of the monoamine oxidase (MAO). The inhibitors of the COMT were found to enhance the action of endotoxin on the coagulation system such as evidenced by the increased consumptions of Hageman factor, fibrinogen, and platelets. Tropolone-treated rabbits did not require exogenous stimulation of a-adrenergic receptor sites by norepinephrine to localize thrombi in the glomerular capillaries when Hageman factor was activated by ellagic acid and fibrinolysis inhibited by f-aminocaproic acid. It is concluded that interference with the degradation of circulating catecholamines results in sensitization to the generalized Shwartzman reaction. (Am J Pathol 92:377-388, 1978)
THE GENEERALIZED SHWARTFZMAN REACTION is the end result of a complex interplay between coagulation, fibrinolvsis, and vasomotor reactions subsequent to the entrance of bacterial endotoxin in the circulation.' The phenomenon is characterized bv glomerular capillary thrombosis leading to bilateral renal cortical necrosis. Stimulation of the a-adrenergic receptors has been identified as an essential component of the reaction.2 Blockade of the a-receptor sites was showrn to prevent the phenomenon induced in the pregnant rat;3'4 rabbits made tolerant to large doses of epinephrine do not develop the reaction.5 On the other hand, norepinephrine localizes thrombi in the glomerular capillaries of rabbits injected with thrombin 6 and precipitates circulating soluble fibrin monomers.7 The involvement of the adrenal response to endotoxin in the pathogenesis of the Shwartzman reaction has been demonstrated. Adrenalectomized rabbits are protected from the reaction, whereas administration of norepinephrine or glucocorticoids to these animals restores their susceptibilitv to the Shwartzman reaction.8 Finallv, the glucocorticoids were From the Laboratory of Experimental Pathology. Institut de Cardiologie de Montreal. and the Department of Pathology. Universit6 de M1ontr6al. Quebec. Canada Supported by Grant MA-4478 from the Medical Research Council of Canada and by the JeanLouis LUvesque Foundation Dr Latour is 'Chercheur Boursier' of the Conseil de la recherche en sante de Quebec. mrs Le'ger-Gauthier is a Fellow- of the Canadian Heart Foundation Accepted for publication April 3. 1978 Address reprint requests to Dr J. G Latour. Institut de Cardiologie de MIontr&al. 5000 Belanger Street East. M1ontr6al. Quebec. Canada HIT IC8 0002-9440/78/081 0-0377$01.00 377
378
LATOUR AND LEGER-GAUTHIER
American Journal of Pathology
show-n to sensitize to the Shwartzman phenomenon through an action mediated by the a-adrenergic receptor sites.9 The ability of the glucocorticoids to enhance the vascular reactivity to catecholamines is well known; among the several mechanisms suspected is the reported inhibition of catechol-o-methvltransferase (COMT)."0 Therefore, know-n inhibitors of COMT and monoamine oxidase (MAO) ,were investigated as sensitizing agents for the generalized Shwartzman reaction. Our results showr that inhibitors of COMT prepare the normal rat and rabbit for the Shwartzman phenomenon by promoting a-adrenergic stimulation. Materals and Methods Animals
Holtzman male rats (.375 to 400 g) and New Zealand rabbits (1.8 to 2.2 kg) were used. They %vere fed standard pellets and given water ad libitum. W7ith the rats under ether anesthesia. blood samples were collected from a jugular vein into plastic syninges containing the appropriate anticoagulant.
Infusions Slow intravenous infusions lasting 4.3 hours were performed in rabbits (Experiment 3) under pentobarbital anesthesia (30 mg 'kg). A catheter introduced into the superior vena cava bv ay of the right ear vein was connected to a sigmamotor infusion pump and used for ellagic acid. The other drugs were given by use of a Sage syringe pump via an infusion set (Butterflv 2.3, Abbott Laboratories, Chicago, Ill.) inserted in the marginal vein of the left ear.
Drugs Endotoxin
Salmonella typhosa 0901 endotoxin boivin ty-pe (Difco Laboratories, Detroit) was dissolved in physiologic sterile saline and injected into a jugular vein at the dosage of 1 mg'kg 2 hours after tropolone or distilled water, 15 minutes after pyrogallol, and 1 hour after the last administration of the NMAO inhibitors (Experiments 1, 3, and 4). MAO Inhibitors
Pargyline chlorhydrate (Abbott Laboratories, Montreal), phenelzine sulfate (Wamer Chilcott, Toronto), and isocarboxazide (Hoffmann-La Roche Ltee, Vaudreuil, Quebec) were obtained commercially. They were suspended in distilled water and fed to the rats on .3 successive days bv way of a stomach tube at the dosages of 40 mg, 20 mg, and 25 mg/kg, respectively. COMT Inhibitors
PN-rogallol (Pvrogallic acid. Fisher Scientific, Montreal) and tropolone (Aldrich Chemicals Co.. Montreal) were used as catechol-o-methvltransferase inhibitors.11-13 They were dissoved in sterile distilled water and injected subcutaneously in rats at the respective doses of 500 mg /kg and 3M0 mg,/kg. In the rabbit experiment (Experiment 5), tropolone
Vol. 92, No. 2 August 1978
GENERAUZED SHWARTZMAN REACTION
379
was prepared in 5% dextrose-saline and the pH of the solution was adjusted to 7.4 with IN NaOH. The drug was given intravenously at the dosage of 1 mg,/kg,/min. Blagic Acid
Ellagic acid (K and K Laboratories Inc., Plainview, N.Y.) was stirred in physiologic saline and infused at the dosage and rate of 12.5 mg '50 ml 'kg, 'hr (Experiment 5). Norepinephrine
Levarterenol bitartrate (Levophed, Winthrop Laboratories, New York) was diluted with 5% dextrose-saline and given at the rate of 10 ml/kg,/hr. The drug was infused at the dose of 1.25 qg 'kg 'min for 3 hours, then at 2 jig /kg/min for the next one-half hour, and finally at 2.5 ug /kg /min for the last hour (Experiment 5). E-Aminocaproic Acid (EACA)
Amicar (Lederle Laboratories. Pearl River, N.Y.) was diluted with 5% dextrose-saline such that 5 mg "kg 'min was infused in a volume of 5 ml /kg,/hr (Experiment 5). Blood Tests Plasma recalcification times and cephalin clotting times were performed on citrated plasma as already described.1' A Bell and Alton 15 rabbit brain cephalin extract was used as a platelet substitute in the latter test. Dilute blood clot lysis times were estimated with the use of 0.12 MI sodium acetate buffer pH 74.1617 The platelet counts were performed with a Coulter Electronic particle counter as reported elsewhere in detail.1' The hematocrits were measured in heparinized capillanr tubes, and Hageman factor was assayed on Factor-XIIdeficient plasma." Clottable fibrinogen was estimated according to the original method of Ratnoff and Menzie.19 Histlogy At the end of the experiments (4 hours after the injection of endotoxin in Experiments 1, .3, and 4 and after the 4.5-hour infusion period in Experiment 5), the rats were killed with chloroform and the rabbits were killed with an overdose of pentobarbital. An autopsy was performed on each animal, and the kidneys were fixed in 10% neutral formalin. Sections of both kidnevs were stained with phosphotungstic-acid hematoxylin (PTAH) and examined for the presence of fibrin thrombi.
Results COMT and MAO Inhibitors and Sensitizati (Experiment 1)
of the Rat to the Generalized Shwtzman Reaction
As indicated in Table 1, none of the rats given only endotoxin (Group 1) showed glomerular thrombosis. On the contrarv, endotoxin elicited glomerular thrombosis in 59.4% (P < 0.001 vs Groups 1 and 2) and in 64.7% (P < 0.001 vs Groups 1 and 4) of the rats pretreated, respectivelv, with tropolone (Group 3) and pvrogallol (Group 5). Tropolone (Group 2) and pyrogallol (Group 4) alone without endotoxin did not elicit glomerular thombosis. Endotoxin failed to induce the Shwartzman reaction in pargyline- (Group 6) and phenelzine- (Group 7) pretreated rats. However, a
American Journal of Pathology
LATOUR AND L-GER-GAUTHIER
380
Table 1-COMT and MAO Inhibitors and Sensitization of the Male Rat to the Generalized Shwartzman Reaction
Group
Treatment
Total No. of animals
1 2 3 4 5 6 7 8
Distilled water + endotoxin Tropolone + saline Tropolone + endotoxin Pyrogallol + saline Pyrogallol + endotoxin Pargyline + endotoxin Phenelzine + endotoxin Isocarboxazide + endotoxin
39 35 32 20 17 16 17 20
Percent with glomerular thrombosis 0.0 0.0 59.40.0 64.7t 0.0 0.0
10.01t
P < 0.001 vs Groups 1 and 2. t P < 0.001 vs Groups 1 and 4. t P < 0.05 vs Group 1.
slight (10%) but significant incidence (P < 0.05) of glomerular thrombosis was observed with isocarboxazide (Group 8). Glomerular thrombosis elicited by endotoxin in tropolone- and pvrogallol-pretreated animals was not generalized to all glomeruli. The lesions were disseminated in both kidneys, but groups of 5 to 10 occluded glomeruli were often surrounded bv manv which had remained normal. The severity of the reaction is illustrated in Figure 1, which shows the observed typical glomerular lesion. Effects of COMT hhibitors In Vivo on (Experiment 2)
Bband Spoteous Fibtyss
In comparison to the cephalin times and recalcification times of the control rats given water, the cephalin times and recalcification times of the animals injected with tropolone were shorter bv, respectivelv, 25% (P < 0.001) and 3057c (P < 0.001) after 4 hours (Text-figure 1). Similar changes were observed 2¼4 hours after pyrogallol with respective reductions of 34% (P < 0.001) and 40%E (P < 0.001) of the cephalin and recalcification clotting times. However, neither tropolone nor pyrogallol modified the clot lvsis times. Platelet and Fb*ogen CosLwnpons in Rats Pretreated Wrth COMT hIhibitors and Given an Intravous Ircuon of Endotoxin (Experimet 3)
The blood platelet count dropped bv 15% (P < 0.01) and 26% (P < 0.001), respectivelv, 2 and 4 hours after an injection of endotoxin in control rats given onlv water subcutaneouslv (Text-figure 2). However, the platelet count was reduced bv 38% (P < 0.001) and 27% (P < 0.001), respectivelv, 4 hours after endotoxin in tropolone- and pyrogallol-pre-
GENERALIZED SHWARTZMAN REACTION
Vol. 92, No. 2 August 1978 CrEPIAUIN TIMF7
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TEXT-FIGURE 1-Effects of COMIT inhibitors on blood coagulability and spontaneous fibrinolysis in the rat. Results: Mean (12 rats per group) ± SE. Dotted columns, water: lined columns, tropolone; solid columns. psTogallol.
treated rats. In these groups there was no significant change after 2 hours. Tropolone did not produce by itself anv change in the circulating platelet count for the duration of the experiment, but pvrogallol administration was followed bv a significant (P < 0.001) increase (28%) in the platelet count after 4 hours. Fibrinogen was reduced bv 27% (P < 0.001) 4 hours after the injection of endotoxin in control rats. However, the drop was 48% (P < 0.001) and 38% (P < 0.001), respectively, in the animals pretreated with tropolone and pyrogallol. Two hours after administration of tropolone and pyrogallol, a 15% (P < 0.001) reduction in clottable fibrinogen was noted in both groups. However, normal or near normal values were measured after 4 to 6 hours in the groups given tropolone or pyrogallol and injected with saline instead of endotoxin. A gradual decrease in hematocrit, as a consequence of successive blood removals, reached 18% (P < 0.001) at the end of the experiment in the control animals given onlv water as a pretreatment and saline in place of endotoxin. This response was quite similar to that observed (22%, P < 0.001) in the rats given endotoxin. However, the changes were not as pronounced when tropolone and pyrogallol were given as a pretreatment. The drops were, respectivelv, 10% (P < 0.001) and 1% (not significant) in the rats given endotoxin and 6 % (P < 0.001) and 16% (P < 0.001) in those given saline in association with tropolone and pyrogallol. Hageman Factor Consumpftion i Rats Prstrated With COMT Inhibitors and Given Injection of Edotoxn (Experimnt 4)
an Intavenous
Three hours after the injection of physiologic saline in rats pretreated with water, tropolone, and pvrogallol, Hageman factor activitv was, respectivelv, 101.2 ± 1.5%, 97.3 ± 2.8%, and 104.2 ± 2% of the preexperimental (100%) values. However, the injection of endotoxin was
382
LATOUR AND
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American Journal of Pathology
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Vol. 92, No. 2 August 1978
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perimental values were set arbias the 100%l reference. The *-alues w-ere corrected for changes in plasma volume using the hematocrit as the index. Resuits: Mean (10 rats per group) i SE. Dotted columns, before endotoxin: solid columns. 3 hours after endotoxin.
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followed by a decrease of 38% (P < 0.001), 68% (P < 0.001), and 76% (P < 0.001), respectively, in the groups given water, tropolone, and pvrogallol as pretreatment (Text-figure 3). The consumptions in Hageman factor in these last two groups were significantlv (P < 0.001) more pronounced than those obtained in the controls given water as pretreatment. Incidence of Giomerular Thrombosis When Tropolone is Substited for Noreprephnee, EACA, or Elagic Acid i the Syntetc Shwartzman Reaction (Experiment 5)
As indicated in Table 2, the combined infusion of ellagic acid, norepinephrine, and EACA according to the original protocol of McKay et al 2 for production of the so-called svnthetic Shwartzman reaction resulted in glomerular thrombosis in 14 of the 16 rabbits treated (Group 1). Deletion of norepinephrine (Group 2) resulted in an incidence of onlv 1 of 34 rabbits. In this animal, only a few tiny fibrin thrombi were evident. Table 2-Incidence of Glomerular Thrombosis When Tropolone is Substituted for Norepinephrine, EACA, or Ellagic Acid in the Synthetic Shwartzman Reaction
Percent with Group
Treatment
No. of rabbits
1 2 3 4 5
Ellagic acid + norepinephrine + EACA Ellagic acid + saline + EACA Ellagic acid +norepinephrine + tropolone Ellagic acid + tropolone + EACA Tropolone + norepinephrine + EACA
34 22 23 20
*P < 0.001 vs Groups 2 to 5. < 0.01 vs Group 4.
tP
16
glomerular thrombosis 87.5* 2.9 0.0
26.0t 0.0
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American Joumal of Pathology
When tropolone was substituted for norepinephrine (Group 4), 6 of the 23 rabbits (P < 0.001 vs Groups 2, 3, and 5) showed glomerular thrombosis. In contrast to this, when tropolone was given as a replacement for EACA (Group 3) or ellagic acid (Group 5), none of the animals demonstrated fibrin deposition in their glomerular capillaries. Discussion The results of this investigation indicate that agents such as tropolone and pyrogallol which interfere with COMT sensitize the normal animal to the generalized Shwartzman reaction (Table 1). It is demonstrated that the mechanism involved corresponds to an enhancement of the a-adrenergic response to endotoxin. The evidence is that tropolone can only be substituted with success for norepinephrine for production of the svnthetic Shwartzman reaction (Table 2). The additional observation that inhibitors of the MAO are not effective in preparing the animal for the Shwartzman phenomenon implies that the circulating catecholamines are those involved in the reaction. This conclusion is based on the respective activities and distributions of the two enzymes in the organism, the COMT being primarily implicated in degradation of blood catecholamines and MAO acting predominantly on tissue catecholamines.20 These results are in close continuity with our previous observation that adrenalectomy prevents the Shwartzman reaction5 and that the adrenal response to endotoxin is essential for production of the phenomenon. These results provide additional evidence that sensitization to the GSR by corticosteroids is mediated via the a-adrenergic receptor sites 9 and possibly, at least in part, through an inhibition of the COMT. Therefore, any drug capable of interfering with the removal of the circulating catecholamines such as occurs in enzymatic degradation or tissue uptake or any condition that may promote overproduction or release of catecholamines is susceptible to prepare for the GSR by providing the essential a-adrenergic component of the reaction. The role of the catecholamines in the production of the generalized Shwartzman reaction is complex. Administered in vivo, these hormones provoke the activation of Hageman factor 15 and the precipitation of soluble fibrin monomers.7 They promote or induce platelet aggregation 21 and induce vasomotor reactions in the kidneys, which localize fibrin deposition in the glomeruli when intravascular coagulation is triggered by an intravenous injection of thrombin." The serotonin derived from the platelets during the course of intravascular coagulation, and which is
Vol. 92, No. 2 August 1978
GENERAI 17FD SHWARTZMAN REACTION
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catabolized by MAO, has only a slight and secondary effect on production of these vasomotor reactions and production of the GSR.4 In our study, administration of tropolone or pyrogallol resulted in hypercoagulabiity, but Hageman factor and platelets were not consumed nor was fibrinolysis activated. However, on injection of endotoxin, the two drugs promoted the consumption of Hageman factor, platelets, and fibrinogen and the production of the generalized Shwartzman reaction. Whether the catecholamines released by endotoxin and protected from degradation by COMT inhibition are responsible for overconsumption of these blood coagulation parameters is not known, but, as already stressed,'6 a close correlation seems to exist between the two phenomena. Nevertheless, besides its effects on the coagulation processes, a-adrenergic stimulation was found to be essential for precipitation of the phenomenon and localization of thrombosis in the glomerular capillaries.4'6 In summary, several workers have identified the implication of catecholamines in the course of disseminated intravascular coagulation induced by endotoxin. Infusion of catecholamines under various experimental conditions was found to be essential- for production of the generalized Shwartzman reaction,"2'8 and pharmacologic agents interfering with the a-adrenergic receptors were shown to prevent the phenomenon." The present results with COMT inhibitors give proof that the massive release of catecholamines needed for production of the generalized Shwartzman reaction is achieved in vivo in the normal animal on injection of a single dose of endotoxin and that conditions or drugs which prepare or sensitize for the Shwartzman reaction may do so by interfering either with production, release, or neutralization of blood catecholamines. References 1. McKay DG, Latour JG. Lopez AM: Production of the generalized Shwartzman reaction by activated Hagemena factor and a-adrenergic stimulation. Thromb Diath Haemorrh 26:71-76, 1971 2. McKay DG, Muller-Berghaus G, Cruze V: Activation of Hageman factor by ellagic acid and the generalized Shwartzman reaction. Am J Pathol 54:393-420, 1969 3. Muller-Berghaus G, McKay DG: Prevention of the generalized Shwartzman reaction in pregnant rats by a-adrenergic blocking agents. Lab Invest 17:276-280, 1967 4. Latour JG, lkger-Gauthier C, Groulx C: Prevention of the generalized Shwartzman reaction by bradykinin and prostaglandins. Thromb Haemostas 38:224, 1977 5. Hall DL, Broom JS, Brunson JG: Effects of epinephrine tolerance on the Shwartzman phenomenon. Am J Pathol 44:431-440, 1964 6. Latour JG, lkger C: Production of glomerular capillary thrombosis by thrombin and vasoactive hormones. Blood Vessel Interactions: Systems in Special Tissues. First World Congress for Microcirculation, Toronto, Canada, 1975. Edited by J Grayson, W Zingg. New York, Plenum Publishing Corp., 1976 (Abstr)
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7. Miiller-Berghaus G, Mann B: Precipitation of ancrod-induced soluble fibrin by aprotinin and norepinephrine. Thromb Res 2:305-322, 1973 8. Latour JG, McKav DG: Requirement of the adrenal glands for provocation of the generalized Shwartzrnan reaction. Lab Invest 22:281-285, 1970 9. Latour JG, Prejean JB, Margaretten W: Corticosteroids and the generalized Shwartzman reaction: Mechanisms of sensitization in the rabbit. Am J Pathol 65:189-202, 1971 10. Kalsner S: Mechanism of hvdrocortisone potentiation of responses to epinephrine and norepinephrine in rabbit aorta. Circ Res 24:383-395, 1969 11. Belleau B, Burba J: Occupancv of adrenergic receptors and inhibition of catecholo-methyl transferase by tropolones. J Med Chem 6:755-759, 1963 12. Mavrides C, Missala K, D'Iorio A: The effect of 4-methvl-tropolone on the metabolism of adrenaline. Can J Biochem Physiol 41:1581-1587, 1963 13. Hattori K, Matsuura M, Fujiwara M, Shimamoto K: Inhibition of catechol-omethyltransferase by hydroxybenzenes and related compounds. Jpn J Pharmcol 19:282-286, 1969 14. Latour JG, Leger C: Prevention by glucocorticoids of disseminated intravascular coagulation induced by endotoxin: Mechanisms. J Lab Clin Med 85:934-949, 1975 15. Bell WN, Alton HG: A brain extract as a substitute for platelet suspensions in the thromboplastin generation test. Nature 174:880-881, 1954 16. Gallimore MJ: Effects of diluents on blood clot lvsis. J Clin Pathol 20:234-238. 1967 17. Marchandise B, Latour JG, Bourassa MG, Beaulac P, Yu SK: Platelet hvperreactivity to fhrombin in patients with myocardial infarction and normal or slightly abnormal coronary arteries. Clin Invest Med (In press) 18. McKay DG, Latour JG, Parrish MH: Activation of Hageman factor by a-adrenergic stimulation. Thromb Diath Haemorrh 23:417-422, 1970 19. Ratnoff OD, Menzie C: A new method for the determination of fibrinogen in small samples of plasma. J Lab Clin Med 37:316-320, 1951 20. PevTin L, Dalmaz Y: La secretion et l'inactivation peripheriques des catecholamines (adrenaline, noradre'naline, dopamine). J Phvsiol (Paris) 70:353-433, 1975 21. Yu SK, Latour JG: Potentiation bv a and inhibition by 0-adrenergic stimulations of rat platelet aggregation: A comparative study with human and rabbit platelets. Thromb Haemostas 37:413-422, 1977
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