hyperthermia in only one of two studies. This reduction was (Received 15 July 1975)

Transcription

1 J. Phy8iol. (1976), 257, pp With 8 text-figurem Printed in Great Britain ANTAGONISM BY ANTIPYRETICS OF THE HYPERTHERMIC EFFECT OF A PROSTAGLANDIN PRECURSOR, SODIUM ARACHIDONATE, IN THE CAT BY WESLEY G. CLARK AND H. RICK CUMBY From the Department of Pharmacology, Southwestern Medical School, University of Texas Health Science Center at Dallas, Dallas, Texas 75235, U.S.A. (Received 15 July 1975) SUMMARY 1. Injection of sodium arachidonate (1-4 fg) into lateral cerebral ventricles of unanaesthetized cats caused shivering and rapid development of dose-related hyperthermic responses. Unless arachidonate is hyperthermogenic per se, this indicates that in vivo formation of prostaglandins, or perhaps an endoperoxide intermediate, can cause hyperthermia. 2. Tolerance gradually developed when arachidonate was administered repeatedly at intervals of 1-7 days. Examination of the brains of several tolerant animals revealed in each case marked enlargement of the lateral ventricles which apparently accounted for the diminished response to arachidonate. 3. Sodium salicylate (4, 16 mg/kg, I.v.) antagonized arachidonate but only after a 3-4 hr latent period. 4. Paracetamol (1, 4 mg/kg, i.v.) reduced the hyperthermic effect of arachidonate but a dose of 4 mg/kg antagonized centrally administered bacterial endotoxin more effectively than it did arachidonate. 5. Indomethacin (4 /tg/kg, i.v.) significantly reduced arachidonateinduced hyperthermia in only one of two studies. This reduction was comparable to the hypothermic effect of indomethacin in afebrile animals and was attributed to a non-specific action on thermoregulatory function rather than to inhibition of prostaglandin synthesis. Indomethacin antagonized endotoxin and leucocytic pyrogen to a greater degree than it did arachidonate. 6. Comparison of the relative effectiveness of the antipyretics in blocking hyperthermic responses to pyrogens and to sodium arachidonate indicates that, if prostaglandins do mediate pyrogen-induced fever, these antipyretics exert their primary action at a step before prostaglandin synthesis.

2 582 WESLEY G. CLARK AND H. RICK CUMBY INTRODUCTION Inhibition of prostaglandin synthesis has been proposed as the mechanism of action of antipyretics (Vane, 1971). It is not yet certain that prostaglandins mediate pyrogen-induced fevers. Most, if not all, of the evidence for such mediation (Coceani, 1974; Veale & Cooper, 1974) is also compatible with production of hyperthermia by prostaglandins and pyrogens by initially independent pathways which subsequently converge. There have been reports that pyrogens can evoke fever in animals which do not respond with hyperthermia to prostaglandins (Baird, Hales & Lang, 1974; Pittman, Veale & Cooper, 1975; Veale & Cooper, 1975) and that prostaglandin E1 injections into the medulla oblongata of the rat caused hypothermia (Lipton, Welch & Clark, 1973). Even if prostaglandins are necessary for pyrogen-induced fever, the present evidence that direct prostaglandin synthetase inhibition is the mechanism by which antipyretics act is circumstantial, based primarily on in vitro studies (Flower & Vane, 1972) and on determinations of changes in concentrations of prostaglandinlike materials in c.s.f. during fever (Feldberg & Gupta, 1973; Feldberg, Gupta, Milton & Wendlandt, 1973; Philipp-Dormston & Siegert, 1974). In fact, an increase in c.s.f. prostaglandin levels is apparently not necessary for pyrogen-induced fever (Cranston, Hellon & Mitchell, 1975a), and in any case it is not known if concentrations of prostaglandins sufficient to cause hyperthermia are attained in brain regions involved in thermoregulation. Splawinski, Reichenberg, Vetulani, Marchaj & Kaluia (1974) reported that intraventricular administration of arachidonic acid, the precursor of prostaglandin E2, to the rat evoked hyperthermia and that aspirin blocked this effect. We have evaluated the hyperthermic effect of intraventricular injection of sodium arachidonate in the cat. From prior studies with leucocytic pyrogen in our laboratory, an estimate was made of equi-antipyretic doses of sodium salicylate, paracetamol and indomethacin. The effectiveness of these doses and multiples of each antipyretic in antagonizing arachidonate was determined. The ability of paracetamol and indomethacin to reduce responses to arachidonate was also compared with their effect against pyrogens. The results indicate that inhibition of prostaglandin synthetase cannot account entirely for the antipyretic activity of these agents. METHODS Twenty-five cats weighing kg were used. Procedures for care and feeding of the animals, for recording body temperature chronically from the retroperitoneal space, for implanting i.v. catheters or lateral cerebral ventricular cannulas, for sterilization of glassware and for otherwise avoiding contamination by pyrogens

3 ARACHIDONATE HYPERTHERMIA AND ANTIPYRETICS 583 have been described previously (McCarthy & Borison, 1966; Clark & Moyer, 1972). Environmental temperature was maintained at C. Sodium arachidonate and bacterial endotoxin were injected intraventricularly in 1 ml. -9 % NaCl solution. Ventricular cannulas were flushed with -2 ml. NaCl solution between tests. Antipyretics were given I.v. and were flushed in with 1- ml. NaCl solution. Except for experiments in which leucocytic pyrogen was infused or injected intraventricularly and studies of tolerance development to arachidonate, cross-over experimental designs were used in which five to eight cats received each of the tests in randomly determined order at intervals of at least 48 hr. A Sage Model 351 pump was used for infusions. The brains of some cats tolerant to 2-8 lug sodium arachidonate were removed and preserved in 1 % formalin. Body temperature at the time of antipyretic or vehicle administration was used as the base line for determination of temperature changes in those experiments in which antipyretics were given during hyperthermic responses. In all other experiments, the average of temperature readings, 15 and 3 min before the initial drug injection was used as base line. Unless otherwise specified, hyperthermogenic agenfs were injected at 1. a.m. + 5 min. Deviations of body temperature from base line were tabulated at 15 min intervals, and changes in temperature were quantified as a 'thermal response index' (TRI), one unit of which is equivalent to a 1 C change lasting for 1 hr (Clark & Cumby, 1975). Unless otherwise indicated, TRIs were determined from the time of the final injection for the number of hours indicated by a subscript. Results were analysed by the Wilcoxon matched-pairs signed-ranks test (Siegel, 1956). Materials. Stock solutions of 1-3 mg/ml. sodium arachidonate (Nu-Chek-Prep, Inc., Elysian, Minnesota) in NaCl solution were kept frozen at -9 C. These were thawed as necessary to obtain portions for injections and then refrozen. There was no appreciable loss of potency in samples stored up to 3 months. Stock solutions of indomethacin (Merck, Sharp and Dohme, 2-8,ug/ml.) in 95 % ethanol, of sodium salicylate (1 mg/ml.) and Salmonella typhosa endotoxin (Difco, 2-,ug/ml.) in NaCl solution and of leucocytic pyrogen were stored at 4 C. The procedure for preparation of leucocytic pyrogen has been described (Clark & Cumby, 1975). Paracetamol (2 mg/ml.) in 95% ethanol was kept at room temperature. RESULTS Hyperthermic responses to various doses of sodium arachidonate. Intraventricular administration to cats which had not previously received sodium arachidonate caused shivering and rapid development of large and prolonged hyperthermic responses (Fig. 1, Table 1 a). Saline injection in the same animals was without appreciable effect. When five cats were given a series of injections of various doses of sodium arachidonate, the mean responses were dose-related (Table 1 b). 'Tolerance' to arachidonate. If arachidonate was administered repeatedly at intervals of 1-7 days, a diminution of its hyperthermic activity occurred. This is indicated by the data in Table 1. The cats receiving sodium arachidonate for the first time responded as well to 2,ug as the cats in the crossover study did to 4,ug. An example of complete tolerance development is shown in Fig. 2. The difference in response to any two consecutive

4 584 WESLEY G. CLARK AND H. RICK CUMBY injections was small. Examination of the brains of seven tolerant cats revealed marked enlargement of the lateral ventricles. The apparent tolerance to arachidonate was probably due primarily to a progressive dilution of the injected material by increasing volumes of c.s.f. Inhibition ofarachidonate-induced hyperthermia. Since tolerance developed Sodium arachidonate (2 ug in 1 ml.) 2 Saline solution U. E, -I I U,_J <EV.J Time after injection (hr) Fig. 1. Mean hyperthermic responses of eight cats to initial intraventricular injections of sodium arachidonate and NaCl solution. TABLE 1. Development of hyperthermia after lateral cerebral ventricular injection of sodium arachidonate. Results are expressed as mean values with the range in parentheses Maximum increase Dose No. of (,ug) cats TR12 (A' C x hr) in body temperature ( C) (a)* (-4-2 to 6.2) (.1 to 1-) (23-8 to 43.4) (1-4 to 2 8) (b)t (-2-2 to 28-2) (1.1 to 2.2) (7.1 to 32-4) (1.5 to 2-4) (17-4 to 36-1) (1.7 to 2-9) * Cats which had not previously received sodium arachidonate. t Crosts-over stidy,

5 ARACHIDONATE HYPERTHERMIA AND ANTIPYRETICS 585 slowly, with only small differences between consecutive injections, it was possible to use a cross-over design to compare the effects of an antipyretic and vehicle on the response to arachidonate. From previous studies with leucocytic pyrogen (Clark & Moyer, 1972; Clark & Cumby, 1975), it was estimated that 4 mg/kg, 1 mg/kg and 1 jtg/kg were approximately equi-antipyretic i.v. doses of sodium salicylate, paracetamol and indomethacin respectively. The ability of these doses and fourfold multiples 3 E6z2 -$ U ' Time after injection (hr) Fig. 2. Development of tolerance to 4,tg sodium arachidonate in a cat given a series of twelve injections over a period of 23 days. The numbers in the circles indicate the position of each response in the sequence. of each to inhibit arachidonate-induced hyperthermia was compared to vehicle alone in paired cross-over studies (Table 2). The dose of sodium arachidonate given to any animal was the same for both tests in a specific cross-over, but when necessary the dose was increased from 2 to 4-8 /tg for a subsequent study to maintain good responses in spite of slowly developing tolerance. The order of administration of antipyretic and vehicle, given 2 hr after arachidonate injection, was randomized so that four animals received antipyretic on the first test and four on the second. Sodium salicylate produced prolonged reductions in the hyperthermic response to arachidonate (Fig. 3) which were delayed for 3-4 hr after antipyretic injection. Salicylate injection was actually followed, during the first hour or so, by a small but statistically significant increase in contrast to the vehicle.

6 586 WESLEY G. CLARK AND H. RICK CUMBY Sodium salicylate A\~~~~~(16 mg/kg) -1 w. -2 O _} 9\ Sodium salicylate.' \5- ~~~~~~(4 mg/kg) Time after injection of antipyretic or vehicle (hr) Fig. 3. Inhibition of arachidonate-induced hyperthermia by sodium salicylate. The continuous lines indicate the mean change in response of eight cats to sodium arachidonate after injection of vehicle. The dashed lines indicate the change after injection of antipyretic. Paracetamol Paracetamol E(4 rk(1 mg/kg) U~~~~~~~m/g E -o -D c._ C U Al II Indomethacin 1 Indor I ~~~~tu4 ag/kg) (1 Fig Time after injection of antipyretic or vehicle (hr) 8 1 Inhibition of arachidonate-induced hyperthermia by paracetamol and indomethacin. See legend of Fig. 3 for details.

7 ARACHIDONATE HYPERTHERMIA AND ANTIPYRETICS 587 Q ; o SV V I] C] V W -O 3 3 V V/ 1 A C] _ 3 3 V V/ ; xr-i _ Q Q CO C] CO 6 1 to CIT m CZ Cm 1 C; -C CO ] ce o- C) Ce IC ei N -IPI: O~_6t o O ss 5 o 6 c C O _ s I; I; CI) - 1 C O jz j C] j ~ 6 _ 3 3;-: - 3 _ C) " ce C); O d-q -q -4 Ol 1 1 CC CC' -) C> C? }4.1,-- OCI) C) o o -_)o "t zz.- _- _- 't t 3 o 1: <: C) ~. > * _ >~) ~~~~._* ~~~C._ o C) -+- Cl t -C

8 588 WESLEY G. CLARK AND H. RICK CUMBY Paracetamol likewise inhibited the response to arachidonate (Fig. 4). When seven of the cats given 4 mg/kg were retested in an afebrile state with the same dose of paracetamol or with vehicle, the mean maximum hypothermic effect was only.3 C, and the mean difference between control and treated TRI5 values was 4 units. The 1,g/kg dose of indomethacin did not significantly reduce the response to arachidonate while the larger dose did (Fig. 4), particularly over the period from 2-5 hr after injection. However, the reduction.by 4,tg/kg was not much greater than the hypothermic effect ofindomethacin reported previously (Clark & Cumby, 1975) and re-examined below (Fig. 6, Table 4). 5 Paracetamol Indomethacin U 54 A _oo~co ' '-I5. Q~~~~~~~~~~. ~ 4. 2 po * C Time after injection of antipyretic or vehicle (hr) Fig. 5. Comparison of changes in hyperthermic responses to arachidonate and endotoxin after administration of paracetamol (4 mg/kg) or indomethacin (4 fig/kg). Mean responses of eight cats. -, pyrogen + vehicle; pyrogen + antipyretic; -, arachidonate + vehicle; - - -, arachidonate + antipyretic. Comparison of the ability of antipyretics to antagonize centrally injected sodium arachidonate and bacterial endotoxin. Although 4 mg/kg paracetamol antagonized arachidonate in the experiments reported above, the magnitude of the reduction in temperature was less than that reported previously (Clark, 197) when comparable doses were given during fever after intraventricular injection of endotoxin. Accordingly, antagonism of arachidonate and endotoxin by paracetamol was compared (Fig. 5, Table 3a). Because hyperthermia develops more slowly after intraventricular endotoxin than after arachidonate injection, endotoxin was given at 1 a.m. and the arachidonate at noon. The antipyretic or vehicle was injected at 2 p.m. at which time mean elevations of body temperature were

9 ARACHIDONATE HYPERTHERMIA AND ANTIPYRETICS 589 over 2 C. Although responses to both arachidonate and endotoxin were significantly reduced by paracetamol, endotoxin was antagonized to a considerably greater degree. In the first three cats listed (Table 3a), paracetamol had a minimal effect on the response to arachidonate but still antagonized endotoxin markedly. (b) Mean TABLE 3. Antagonism of sodium arachidonate and endotoxin by antipyretics in individual cats Indomethacin (4,ag/kg) Mean TRI5 (A' C x hr) f -A- --- Sodium arachidonate Antipyretic Control Antipyretic A (a) Paracetamol -1v5-1@ (4 mg/kg) *2 4* * @3-1@ * * - 3-8* 1i7.6-3* * * P < 5 v8. control * *2 1. Endotoxin Control Antipyretic * *2-3*4 2* -3* '6-3* *8-7 *3-5-8 *9-4-7** *2 2* *3 -*3-8 -1'8 1* *4-3 1*8 - -8** **P <-.1. A 6* * * X * *6 U -._ Co = C. -1 L I I I I I I Time after injection (hr) Fig. 6. Hypothermic effect of indomethacin. Mean responses of seven afebrile cats to injection of ethanol vehicle (continuous line) or indomethacin (4,ugfkg, dashed line).

10 59 WESLEY G. CLARK AND H. RICK CUMBY TABLE 4. Hypothermia and reduction of hyperthermic responses to sodium arachidonate produced by indomethacin (4 jug/kg). Results are expressed as mean values in seven cats with the range in parentheses (a) Other treatment None (b) Sodium arachidonate (c) Leucocytic pyrogen TRI5 (A C x hr) C A e Control Indomethacin A (1.4 to -5.2) - -8 (1-4 to -38) 2-6 (7.1 to -.8) 2 _ (-I- to -5 ) -1-9 (1.7 to -6.4) -1-9 ( 3 to -36) P 1- > > < -2 U' I- EU. E o 4. D. V C U I W I I V % r - %. - - _.f -1 I I 2 4 Time after injection of leukocytic pyrogen (hr) Fig. 7. Antagonism of leucocytic pyrogen by indomethacin pre-treatment. Mean responses of seven cats to pyrogen given I.v. as a single bolus 3 min after ethanol vehicle (continuous line) or indomethacin (4,ug/kg, dashed line).

11 ARACHIDONATE HYPERTHERMIA AND ANTIPYRETICS 591 When a similar study was performed with indomethacin, 4,ug/kg, endotoxin was significantly antagonized while arachidonate was not (Fig. 5, Table 3b) in contrast to the result of the previous cross-over (Fig. 4, Table 2). When seven of the same cats were given indomethacin or vehicle alone, the magnitude of the hypothermic response (Fig. 6, Table 4a) was similar to the degree of antagonism of arachidonate (Table 4b). -5 r -.Ir. E la. C -.5 S- -1*F C U ' -2* I I I I I I I I I Time after injection of indomethacin or vehicle (hr) Fig. 8. Indomethacin antagonism of hyperthermia produced by arachidonate or by prolonged i.v. infusion of leucocytic pyrogen in an individual cat. See Fig. 5 for key. The rate of pyrogen infusion was constant (14 #Il./min) from hr until the infusion was stopped, indicated by the arrows. Antagonism of leucocytic pyrogen by indomethacin. The antipyretic effect of indomethacin given 3 min before leucocytic pyrogen was also examined in the same seven cats as above (Fig. 7, Table 4c). The response to the pyrogen was essentially abolished in contrast to the small change in the response to arachidonate. To determine if such inhibition occurred only when indomethacin was given prior to pyrogen, the same dose of indomethacin (4,ug/kg) was given during fevers produced by intraventricular injection

12 592 WESLEY G. CLARK AND H. RICK CUMBY or by i.v. infusion of leucocytic pyrogen in four sets of experiments. One example is illustrated in Fig. 8. In each case indomethacin caused a marked reduction in the response to pyrogen. The decrease in response to arachidonate was slower and much less. DISCUSSION The results of this study in the cat clearly show that, as in the rat (Splawinski et al. 1974), intraventricular injection of prostaglandin precursors can raise body temperature, presumably by conversion to prostaglandins (Ziel & Krupp, 1975), and that antipyretics can reduce the hyperthermia. However, there were a number of indications that the pyrogenic activity of agents such as leucocytic pyrogen or bacterial endotoxin is more readily inhibited by antipyretics than is the hyperthermic activity of arachidonate. Salicylates given i.v. shortly before leucocytic pyrogen inhibit development of fever (Clark & Moyer, 1972; Lin & Chai, 1972). Even when salicylate was administered during an infusion of leucocytic pyrogen, the antipyretic effect began within 1 min after injection (Adler, Rawlins, Rosendorff & Cranston, 1969; Cranston, Luff, Rawlins & Rosendorff, 197). Furthermore, i.v. administration of salicylates during fever evoked by central administration of leucocytic pyrogen has been shown to cause defervescence within 3-6 min (Cranston, Hellon, Luff, Rawlins & Rosendorff, 197; Chai, Lin, Chen & Wang, 1971; Lin & Chai, 1972). Yet sodium salicylate caused no reduction in the response to arachidonate during the first 3-4 hr after injection in the cat. Hence salicylate antagonizes leucocytic pyrogen at a time when it was ineffective in blocking arachidonate. Splawinski et al. (1974) did report a significant reduction in the hyperthermic response to arachidonic acid within 1 hr after intragastric administration of aspirin, 15 mg/kg, to rats. However, this was at least partially due to a hypothermic action that salicylates have in the rat (Balint & Thuranszky, 1964; Bizzi, Garattini & Veneroni, 1965; Satinoff, 1972; Polk & Lipton, 1975). Salicylates do not cause hypothermia in cats (Clark, 197), even in a cold environment (Cranston, Hellon & Mitchell, 1975 b). Unlike sodium salicylate, paracetamol began to reduce arachidonateinduced hyperthermia shortly after injection. However, paracetamol antagonized centrally injected endotoxin to a greater extent. In fact, in some animals no appreciable change in the response to arachidonate was produced by paracetamol even though it greatly inhibited endotoxininduced hyperthermia. If responses to both endotoxin and arachidonate are mediated by prostaglandins and if paracetamol acts solely to inhibit prostaglandin synthetase, endotoxin should possibly have been more

13 ARACHIDONATE HYPERTHERMIA AND ANTIPYRETICS 593 difficult than arachidonate to antagonize since endotoxin caused larger and more sustained elevations of body temperature. Yet the opposite was true. The low dose of indomethacin did not block sodium arachidonate while the larger dose significantly inhibited arachidonate in only one of two studies. Indomethacin, like paracetamol, reduced the response to endotoxin to a greater degree than that to arachidonate. Inhibition of endotoxin was statistically significant and clearly greater than the hypothermic effect of indomethacin. The maximum mean reduction of the response to arachidonate at any specific time was.5 C and at 1 hr after indomethacin injection was only.2 C. Yet when indomethacin, 4 fig/kg, was given 3 min before leucocytic pyrogen (Fig. 7; Clark & Cumby, 1975), the maximum fever, which would have developed about 1 hr after indomethacin injection, was abolished. Pretreatment with the same dose of indomethacin had previously been shown to delay and reduce fever production by intraventricular injection of leucocytic pyrogen (Clark & Cumby, 1975). Such degrees of inhibition of leucocytic pyrogen do not require that the animals be pretreated with indomethacin. When given during high fevers produced by central injection or i.v. infusion of leucocytic pyrogen, indomethacin still exerted a powerful antipyretic effect which was considerably greater than its ability to antagonize sodium arachidonate. The reduction in arachidonate-induced hyperthermia was relatively small even when significant statistically and was comparable to the hypothermic effect of the same dose in afebrile animals. Milton (1973) has reported a slight reduction in the hyperthermic response to prostaglandin E1 after indomethacin administration which he attributed to an effect on heat loss mechanisms. Our results strongly suggest that the change in response to arachidonate after indomethacin was due to a nonspecific action rather than the result of a specific inhibition of prostaglandin synthetase. Thus, while the present study does provide indirect evidence that paracetamol and sodium salicylate in the doses used inhibit the conversion of arachidonate to prostaglandin E2 in vivo, the ability of these antipyretics and indomethacin to antagonize pyrogens to a greater extent than or during different time periods from arachidonate does not support the hypothesis that antipyretics reduce. fever by inhibiting prostaglandin synthetase. Baird et at. (1974) mention that the echidna, which responds to intraventricular injection of prostaglandins of the E series with hypothermia, nevertheless develops febrile responses to bacterial endotoxin and that paracetamol can prevent the fever. If prostaglandins are necessary for fever in less primitive animals such as the cat, antipyretics would decrease prostaglandin synthesis indirectly by inhibiting an earlier step 22 PHY 257

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