Prostaglandins are not stored in body tissue, but are synthesized on. fever which was inhibited by indomethacin, but not by PGE

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1 J. Physiol. (1977), 267, pp With 8 text-figure8 Printed in Great Britain EFFECTS OF PROSTAGLANDIN ANTAGONISM ON SODIUM ARACHIDONATE FEVER IN RABBITS BY HELEN LABURN, D. MITCHELL AND C. ROSENDORFF From the Department of Physiology, University of the Witwatersrand Medical School, Johannesburg 21, South Africa (Received 2 December 1976) SUMMARY 1. Sodium arachidonate, the prostaglandin precursor substance, when injected intraventricularly into rabbits, results in dose-dependent hyperthermia, which is rapid in onset and of several hours duration. 2. Arachidonate fever was inhibited by intraventricular injection of indomethacin, but not by the simultaneous intraventricular injection of either of the two prostaglandin antagonists SC 1922 or HR Both antagonists effectively inhibited the fever induced by the intraventricular injection of an equipotent dose of PGE1. 4. Our results show that a derivative of arachidonic acid other than prostaglandin is pyrogenic. INTRODUCTION Cranston, Duff, Hellon, Mitchell & Townsend (1976) reported recently that the simultaneous injection of antagonists of prostaglandins of the E series (PGE) has no effect on the latency, rise time or amplitude of the fever produced by the intraventricular injection of leucocyte pyrogen in rabbits. They interpreted their observations to mean that PGE is not an essential intermediate in the neurochemical mechanisms responsible for pyrogen fever. Their findings are in direct conflict with a body of evidence implicating PGE in just such a role (see Feldberg, 1975). In particular, if PGE is not an essential intermediate in fever, then the attractive hypothesis (Vane, 1973), that the aspirin-like drugs (including indomethacin and paracetamol) owe their antipyretic action to their ability to inhibit PG synthesis, cannot be valid. Prostaglandins are not stored in body tissue, but are synthesized on demand from the fatty acid precursor arachidonic acid. The present paper concerns the effect on body temperature of intraventricular injections of sodium arachidonate in rabbits. Sodium arachidonate induced a dosedependent fever which was inhibited by indomethacin, but not by PGE

2 56 H. LABURN AND OTHERS antagonists. The results suggest that a derivative of arachidonic acid other than prostaglandin is also pyrogenic. The existence of such a substance would reconcile the observations of Cranston et al. (1976) with the evidence attributing prostaglandin an essential role in fever. METHODS Thirty New Zealand White rabbits weighing between 2- and 3-5 kg were used. At least 1 week before the experiments were conducted, a Perspex headplate was fixed to the rabbit's skull under general anaesthesia. The construction and positioning of the headplate allowed the introduction of fine cannulae (o.d. = 4 mm) into a lateral cerebral ventricle (Monnier & Gangloff, 1961), through which intraventricular microinjections could be made. The injections consisted of various doses of sodium arachidonate, of arachidonate together with indomethacin and of arachidonate together with prostaglandin antagonists. The appropriate control injections, including those of PGE1, were also made. The pyrogenic effect of arachidonic acid was investigated using doses of 6-32 nmol/sodium arachidonate (Sigma) dissolved in 25 /zl. sterile distilled water. These and all subsequent injections were flushed through the injection cannulae using 25 1zl. sterile rabbit artificial c.s.f. (Cameron & Semple, 1968) and were passed through a -22 /sm Millipore filter before injection. Each dose was injected into six animals. In all subsequent experiments a single intermediate dose (25 nmol) of sodium arachidonate was employed. One series of experiments concerned the action of indomethacin on the pyrogenic effects of sodium arachidonate. In this series the actions were compared of (a) 28 nmol indomethacin (Sigma) dissolved on 2 pl. dimethyl sulphoxide (DMSO), (b) 25 nmol sodium arachidonate in 25 dzl. distilled water and (c) the solutions of indomethacin and arachidonate combined. A second series ofexperiments concerned the action of the prostaglandin antagonist 1 -acetyl-2-(8-chloro-1, 1l-dihydrobenz (b,f) (1,4)oxazepine hydrazine-1-carbonyl) (SC 1922, Searle). Here the action of 25 nmol sodium arachidonate dissolved in 25 #1. distilled water was compared with that of 25 nmole sodium arachidonate dissolved in 25,u1. water plus 15 #zmol SC 1922 dissolved in 2 Iud. DMSO. Because SC 1922 precipitates when in contact with an aqueous solution, a small air bubble (about 3 /d.) was drawn into the injection cannula to separate the SC 1922 solution from aqueous solutions. SC 1922 appears to precipitate in the cerebral ventricles following contact with cerebrospinal fluid and it is present in the ventricular system for several weeks after injection (Cranston et al. 1976). The experiments using SC 1922 were, therefore, the last experiments performed on an animal and no animal received more than one such treatment. The efficacy of SC 1922 in blocking prostaglandin fever was established by substituting for the sodium arachidonate solution, a solution of PGE1 (Upjohn), 4 nmol in 15,1. distilled water, to which 2,1. DMSO had been added. This dose of PGE1 was found in pilot experiments to give the same fever as the 25 nmol dose of sodium arachidonate. In the third series of experiments, the action of the structurally different prostaglandin antagonist 8-ethoxycarbonyl-1,1 1-dihydro-A-prostaglandin (HR 546, Hoechst) was investigated. The series was identical in design to the second series except that 44 nmol of HR 546 dissolved in 2 p1. ethanol was substituted for the SC 1922 dissolved in DMSO. Sodium arachidonate and SC 1922 were dissolved in the appropriate solvents immediately before use. Solutions of PGE, were made up from a stock solution of 25 pzmol/ml. in ethanol stored at -4 C. The HR 546 was supplied from the manufacturers in an ethanol solution.

3 SODIUM ARACHIDONATE FEVER All experiments were conducted at a room temperature of 2-23 C. The rabbits were conscious and restrained in conventional stocks throughout the experimental period. Body temperature was measured using an indwelling thermistor probe (YSI) inserted into the rabbit's rectum, the output of the thermistor bridge being continuously recorded on a chart recorder (Kipp BD 5) E.8 U 6 C tv U-r Time after injection (min) Fig. 1. The effects on rectal temperature of intraventricular injection of various doses of sodium arachidonate. O 6 nmol; *, 13 nmol; [1, 25 nmol; * 32 nmol; *, control injections of sterile distilled water. Each point represents the mean change in rectal temperature from pre-injection values (zero on temperature scale and corresponding line at C) for six rabbits.

4 562 H. LABURN AND OTHERS RESULTS Effects of sodium arachidonate Fig. 1 shows the effects on rectal temperature of intraventricular injections of sterile distilled water, and of doses of sodium arachidonate between 6 and 32 nmol, each result being the mean of observations on six animals. The increase in rectal temperature following sodium arachidonate injection was rapid in onset and of several hours duration. I.8 I ' Dose (nmol) Fig. 2. Mean elevations in rectal temperature, as measured 2 min after injection, induced by the various doses of sodium arachidonate, and by control injections of sterile distilled water (at zero dose). Each point represents the mean + S.E. of six observations. Abscissa, dose of sodium arachidonate (nmol). A dose of 6 nmol arachidonate resulted in a small increase in rectal temperature which resembled that of control injections of sterile water, particularly in the latter part of the experimental period. Doses of 13, 25 and 32 nmol produced mean elevations in rectal temperature which were dose-dependent and significantly different from the control responses (P < -2, Student's t test). The peak rises in rectal temperature occurred approximately 3 hr after injection. Fig. 2 shows the mean elevations in

5 SODIUM ARACHIDONATE FEVER 563 rectal temperature + S.E. 2 min after injection of the various doses of sodium arachidonate and of sterile distilled water. Effects of indomethacin Fig. 3 shows that injection of indomethacin together with 25 nmol sodium arachidonate resulted in a significant reduction of the hyperthermia following arachidonate alone. Injection of indomethacin alone L- 4 U bo U Time after injection (min), Fig. 3. Changes in rectal temperature of rabbits induced by intraventrioular injection of sodium arachidonate (@), sodium arachidonate + indomethacin () and of indomethacin alone ( A). Each point represents the mean of six observations. At 3min intervals the mean + S.E. is shown. Zero on temperature scale and corresponding line at C represent temperature at times of injection (zero on abscissa). resulted in small deviations of rectal temperature which were never significantly different from zero (Fig. 3). Mean rectal temperature values after injection of arachidonate alone and after injection of arachidonate plus indomethacin were significantly different after 2 min and for the subsequent 14 min (P < 2).

6 564 H. LABURN AND OTHERS Ejects of SC 1922 Fig. 4 shows the mean effects on rectal temperature of injection of PGE, alone, and of injection of SC 1922 together with PGE1. PGE1 caused a rise in rectal temperature which was rapid in onset and which lasted several hours. This fever could be abolished by simultaneous administration of SC The changes in rectal temperature following PGE1 + SC 1922 never differed significantly from zero. 1*4 1. E U Time after injection (min) Fig. 4. Changes in rectal temperature of rabbits induced by intraventricular injection of PGE1 (O), and of PGE1 + SC 1922 (). Additional details as in Fig. 3. In contrast, the same dose of SC 1922 injected together with sodium arachidonate affected an arachidonate hyperthermia equivalent in magnitude to that following PGE1, only in the first 3 min after injection (Fig. 5). Thereafter, the rise in rectal temperature following arachidonate + SC 1922 injection was not significantly different from, and resembled closely that induced by arachidonate alone.

7 SODIUM ARACHIDONATE FEVER 565 Effects of HR 546 HR 546 when injected together with PGE1 significantly reduced the fever induced by PGE1 alone. Fig. 6 shows the mean rectal temperature responses to PGE1, and to injection of PGE1 together with HR 546. The mean rectal temperature values following PGE1 + HR 546 injection differ from those following PGE1 alone 4 min after injection and for the subsequent 1 min of the experiment. 1*4 U 124 $5 1..W E U.6-2 ( g Time after injection (min) Fig. 5. Changes in rectal temperature of rabbits induced by intraventricular injection of sodium arachidonate () and of sodium arachidonate + SC 1922 (). Additional details as in Fig. 3. The effect of HR 546 on the hyperthermia induced by sodium arachidonate is shown in Fig. 7. The mean response to arachidonate injection together with HR 546, and that of arachidonate alone, are significantly different only in the first 4 min after injection. Thereafter a gradual long-lasting hyperthermia ensued, whether or not the HR 546 was present.

8 566 H. LABURN AND OTHERS DISCUSSION Two important results emerge from our observations. The first is that arachidonic acid, or at least its sodium salt, produces a dose-dependent fever when injected intraventricularly in rabbits. The second is that arachidonate fever, like leucocyte pyrogen fever in rabbits (Cranston et al. 1976), is not blocked by antagonists of prostaglandins W 86.4 C Time after injection (min) I I _ I I I I I Fig. 6. Changes in rectal temperature of rabbits induced by intraventricular injection of PGE1 () and of PGE1 + HR 546 (). Additional details as in Fig. 3. Arachidonate fever Sodium arachidonate injected into the cerebral ventricles results in a fever which is dose-dependent and rapid in onset. Clark & Cumby (1976) recently described similar results following arachidonate injection in cats, and Splawinski, Reichenberg, Vetulani, Marchaj & Kaluza (1974) have shown that sodium arachidonate is pyrogenic in rats. Our results suggest that the hyperthermic effects of arachidonic acid are due to its derivatives and not to the precursor substance itself. Addition of indomethacin to the arachidonate injectate reduced significantly and almost to zero the rise in rectal temperature induced by arachidonate alone. The inhibition of arachidonate fever by indomethacin does not persist for the entire duration of the experimental period, presumably because indomethacin is meta-

9 SODIUM ARACHIDONATE FEVER 567 bolized more rapidly than sodium arachidonate. In addition it seems likely that the active, pyrogenic derivatives of arachidonate are evolved continuously and over a long period of time, allowing the hyperthermic effects of precursor injection to persist for several hours a1l I- 1 a- 8 E U *S *4 (U Time after injection (min) Fig. 7. Changes in rectal temperature of rabbits induced by intraventricular injection of sodium arachidonate () and of sodium arachidonate + HR 546 (). Additional details as in Fig. 3. Clark & Cumby (1976) were unable to inhibit the fever induced by intraventricular arachidonate injection by intravenous administration of indomethacin. We also injected a low dose of indomethacin by this route and found it ineffective in attenuating the effects of sodium arachidonate (unpublished observations). Splawinski et al. (1974) were, however, able to reduce the pyrogenic effects of sodium arachidonate in rats after pretreatment with aspirin. Arachidonic acid and prostaglandin antagonism Our experiments have shown that derivatives of arachidonic acid are pyrogenic. One such derivative is PGE, which when injected alone into the cerebral ventricles or anterior hypothalamus (Stitt, 1973) results in fever, and which can be detected in elevated concentrations of the c.s.f.

10 568 H. LABURN AND OTHERS of febrile animals (Dey, Feldberg, Gupta, Milton & Wendlandt, 1974). However, SC 1922, which is a specific antagonist of PGE (Sanner, 1974) and which completely blocks PGE1 fever, leaves unaffected, except for the first few minutes, an equipotent fever resulting from sodium arachidonate injection. Thus PGE is not the only pyrogenic derivative of arachidonic acid. The structurally different prostaglandin antagonist HR 546 has similar effects to the SC 1922, but it is not as good an antagonist of PGE, as Cranston et al. (1976) also found. Fig. 8. Diagrammatic representation of the formation of degradation products of arachidonic acid (from Hamberg & Samuelsson, 1975). Arrows indicate position of action of indomethacin and of antagonists of prostaglandins. What then is the alternative pyrogenic derivative of arachidonic acid? Fig. 8 shows that the prostaglandins are not the only degradation products of this fatty acid, and that prostaglandin endoperoxide or the thromboxanes could be responsible for the arachidonate-induced fever during prostaglandin blockade. Recently Hamberg, Svensson & Samuelsson (1975) suggested that the roles previously ascribed to prostaglandins in platelet aggregation and vascular smooth muscle contraction, should, in fact, be attributed to prostaglandin endoperoxide, or more probably to the thromboxanes.

11 SODIUM ARACHIDONATE FEVER 569 Neurochemitdry of fever Our results suggest that the break-down of arachidonic acid produces at least two pyrogenic derivatives, one of which is prostaglandin. Prostaglandin may be the faster acting derivative, because arachidonate fever is depressed by prostaglandin antagonists for the first few minutes (Figs. 5, 7). The existence of two pyrogenic derivatives of arachidonic acid may explain the results of Cranston et al. (1976). If leucocyte pyrogen releases both derivatives from arachidonic acid then the presence of a prostaglandin antagonist would not necessarily affect leucocyte pyrogen fever. An alternative explanation of the results of Cranston et al. (1976) is that leucocyte pyrogen acts directly on the anterior hypothalamus. Indomethacin, together with the other aspirin-like drugs prevents the oxidation of arachidonic acid to prostaglandin endoperoxide (Needleman, Moncada, Bunting, Vane, Hamberg & Samuelsson, 1976). These drugs, therefore, prevent the formation of all the pyrogenic derivatives of arachidonic acid. If the antipyretic action of the aspirin-like drugs is concerned with the arachidonic acid pathway, then they act to inhibit arachidonic acid break-down rather than to inhibit prostaglandin synthesis. We are grateful to the Upjohn Company for the supplies of PGE1, to Searle Laboratories for the SC 1922 and to Hoechst AG for the HR 546. We also wish to thank Lorna Kerr for technical assistance, and the South African Medical Research Council for supporting this work. REFERENCES CAMERON, I. R. & SEmPTLE, S. J. G. (1968). The central respiratory stimulant action of salicylates. Clin. Sci. 35, CLARK, W. G. & CuMBY, H. R. (1976). Antagonism by antipyretics of the hyperthermic effect of a prostaglandin precursor, sodium arachidonate in the cat. J. Phy8iol. 257, CRANSTON, W. I., DUFF, G. W., HELLON, R. F., MITCHELL, D. & TowNsEND, Y. (1976). Evidence that brain prostaglandin synthesis is not essential in fever. J. Physiol. 259, DEY, P. K., FELDBERG, W., GUPTA, K. P., MILTON, A. S. & WENDLANDT, S. (1974). Further studies on the role of prostaglandin in fever. J. Physiol. 241, FELDBERG, W. (1975). Body temperature changes and fever: changes in our views during the last decade. Proc. R. Soc. B 191, HAMBERG, M. & SAMUELSSON, B. (1974). Prostaglandin endoperoxides. VII. Novel transformations of arachidonic acid in guinea pig lung. Biochem. biophy8. Res. Commun. 61, HAMBERG, M., SVENSSON, J. & SAMUELSSON, B. (1975). Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc. natn. Acad. Sci. U.S.A. 72, MONNIER, M. & GANGLOFF, H. (1961). Atla8 for Stereotaxic Re8earch on the Conscious Rabbit. London: Elsevier.

12 57 H. LABURN AND OTHERS NEEDLEMAN, P., MONCADA, S., BUNTING, S., VANE, J. R., HAMBERG, M. & SAMUEL- SSON, B. (1976). Identification of an enzyme in platelet microsomes which generates thromboxane A2 from prostaglandin endoperoxides. Nature, Lond. 261, SANNER, J. H. (1974). Substances that inhibit the actions of prostaglandins. Arch8 intern. Med. 133, SPLAWINSKI, J. A., REICHENBERG, K., VETULANI, J., MARCHAJ, J. & KALUZA, J. (1974). Hyperthermic effect of intraventricular injections of arachidonic acid and prostaglandin E2 in the rat. Pol. J. Pharmacot. Pharm. 26, STITT, J. T. (1973). Prostaglandin E4 fever induced in rabbits. J. Phy8iol. 232, VANE, J. R. (1973). Inhibition of prostaglandin biosynthesis as the mechanism of action of aspirinlike drugs. Adv. Biosci. 9,