PfliJgersArchiv

Pfl/igers Arch. 374, 15-21 (1978)

Etropean Journal of Physiology 9 by Springer-Verlag 1978

Hyperthermic Effects of Arachidonic Acid, Prostaglandins E2 and in Rats JACEK A. SPLAWIi~SKI 1, ZBIGNIEW GORKA z, ELZBIETA ZACNY 2, and BARBARA WOJTASZEK 1 1 Department of Pharmacology, Copernicus Medical Academy, 16 Grzeg6rzecka Street, 31-531 Krak6w 2 Institute of Pharmacology, Polish Academy of Sciences, 8 Smetna Street, 31-344 Krak6w, Poland

Abstract. The aim of the present study was to investigate the possibility that endotoxin fever in rats is mediated by arachidonic acid (AA) which in turn is converted to the active metabolites such as prostaglandin (PG) Ea, PGF2~, thromboxane A2 (TxA2), or prostacyclin (PG12). Evidence is presented indicating that PGE2 induces fever (not hyperthermia) by acting on the anterior hypothalamic preoptic area. Conversely, both PGF2~ and AA produce mutually similar hyperthermia and there is no correlation between their microinjection sites in the diencephalon and the observed hyperthermic response. In addition, evidence is presented suggesting that involvement of other metabolites of AA, namely TxA2 and PGIz in the mediation of endotoxin fever in rats seems unlikely. Only PGE2-induced fever is significantly similar, consistent with the parameters of this study, to endotoxin-induced fever in rats. AA-induced hyperthermia is probably brought about by increased levels of PGF2~ or both PGF2~ and PGE2 in the hypothalamus following AA injection. It seems highly unlikely that endotoxin produces fever in rats through the increased availability of free AA or through the activation of the PG endoperoxide synthetase in the hypothalamus. The mechanism by which endotoxin may increase PGE2 levels in the rat hypothalamus remains unknown. Key words." Arachidonic acid - Prostaglandins Thromboxane A2 - Prostacyclin - Fever.

INTRODUCTION Convincing evidence has accumulated over the last few years to the effect that a metabolite of arachidonic acid (AA), i.e. prostagtandin (PG) E2, mediates fever

produced by endotoxin (for ref. see Milton, 1976). The first step in the metabolism of AA is the cyclooxygenation of AA to cyclic endoperoxides (PGG2 and PGH2) and this step is inhibited by aspirin-like drugs. The unstable PGG2 and PGH2 are either isomerised to PGE2, reduced to PGF2~ or converted to thromboxane A2 (TxA2) or to prostacyclin (PGI2) by specific enzymes (Pace-Asciak, 1977). The yield of the particular end-product depends on the biochemical activity of the given tissue (Pace-Asciak and Rangaraj, 1977). Bovine seminal vesicle microsomes convert AA mainly to PGE2 and PGF2~ (Flower and Vane, 1974), while blood platelet microsomes convert AA almost exclusively to TxA2 (Samuelsson, 1976). Recent work of Cranston et al. (1976) and Laburn et al. (1977) indicate that in rabbits TxA2, and not PGE2, may mediate fever induced by pyrogens. There are no intracellular stores for PGs (Piper and Vane, 1971) and the disposal of TxA2 is probably achieved by a spontaneous breakdown since the tt/2 of this compound is about 30 s (Samuelsson, 1976). It therefore seems that the levels of TxA2, as well as PGs, are regulated in vivo by the availability of PGG2 and PGH2 and by the biochemical activity of a given tissue. The concentration of PGG2 and PGH2 in tissues depends on the availability of AA since the release of this precursor is probably the rate limiting step in PG biosynthesis (Flower and Blackwell, 1976). On the basis of the above data, it would seem possible that endotoxin increases the availability of AA in the hypothalamus of the rat. If that is the case, the administration of AA should mimic the endotoxin-induced pyretic effect in rats. In fact, an intraventricular injection of AA raises the body temperature of rats, cats, and rabbits (Sptawifiski et al., 1974; Clark and Cumby, 1976; Laburn et al., 1977) but this could represent hyperthermia and not fever. There-

0031-6768/78/0374/0015/$1.40

16

fore, in the present study the mechanisms by which AA, PGE2, and PGF2= increase body temperature of rats were compared. To further ascertain which metabolite of AA is the ultimate mediator of its effect on the rat body temperature, the conversion of AA by the hypothalamic homogenate and microsomes was studied in vitro. The obtained results do not confirm the assumption that AA in rats mediates pyretic effect of endotoxin.

Pfliigers Arch. 374 (1978)

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METHODS Cannulas were implanted stereotaxically into the left lateral ventricle or into various brain structures of male Wistar rats ( 2 8 0 350 g) according to the coordinates of Skinner (1971). The details of the surgical procedure, the injection technique, the method of carbon dioxide production measurement and the verification procedure were described previously (Sptawifiski et al., 1977). The coordinates of the identified sites are shown in Figure 1. The oesophageal (T~s) and skin (T~) temperature measurements were performed as previously described (Sptawiflski et al., 1977), however, one additional measurement was performed 15min following intracerebral administration of the drugs. The following thermoregulatory parameters were calculated : A T max. (the maximum change from the initial temperature), TRI (thermal response index, the area under the temperature curve, one unit equivalent to a change of I~ lasting for 1 h) and RTC (rate of temperature change, calculated by dividing A T max. by time in hours it took to occur). The experiments were carried out at an ambient temperature of 22 • 1~ The respiratory frequency was measured by counting the flank movements. All solutions were prepared in heat-sterilized glassware (150~ for 12 h) while polyethylene tubes and cannulas were stored in 70 ~ ethanol to insure aseptic conditions. Reagents used to prepare the salts of injected drugs were heated at 150~ for 12 h. PGF2~ tromethamine salt was used. The storage and the preparation of sodium salts of PGEz and AA was described previously (Splawiflski et al., 1973). The pH of solutions of PGE2, PGF2~, and AA was adjusted to 7 . 5 - 8.0 before injection. To estimate the capacity of the rat hypotbalami to generate various metabolites of AA, the hypot.halami of rats were isolated and homogenized (4~ for 2 rain in Krebs bicarbonate buffer (pH 7.4) 1:4 (w/v). The protein content in the homogenate was determined by the method of Gornall et al. (1949). PGE2 and PGF2~ were extracted from the homogenate by diethyl ether, separated by thin layer chromatography and estimated using bioassay (see below). To estimate TxA2 production, the homogenate of the hypothalami was incubated with ice-cold diethyl ether to remove lipids which interfere with TxAz bioassay. Other details of TxA2 generation and bioassay are presented elswhere (Gryglewski, 1977). To estimate PGI2 production, microsomes were prepared from the hypothalamic homogenate. PGI2 production was measured according to the method of Gryglewski et al. (1976). The substrates used for TxA2 and PGI2 generation were: crude ether extract of PG endoperoxides (Gryglewski, 1977) and AAsodium salt. PGE2 and PGF2~ were differentially bioassayed from TxA2, using the superfusion technique of Vane with subsequent modifications (for ref. see Gryglewski, 1977). The following detector organs were superfused in a cascade with Krebs bicarbonate buffer (at 37 ~C and 5 ml/min): a rat stomach strip, a rat colon, and a strip of mesenteric artery from the rabbit. Statistical evaluation of results was performed as previously described (Sptawifiski et al., 1977).

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Fig. 1. Top: Anatomical map of the rat brain made according to Skinner's (1971) atlas. A parasagittal p l a n e - c a . 0.8 mm from m i d - l i n e - o f explored sites. 1 dorsomedial; 2 anterior; 3 anterior preoptic; 4 posterior (A 4.25); 5 posterior (A 4.75); 6 ventromedial (A 5.25); 7 ventromedial (A 5.75) hypothalamic nuclei. Abbreviations: A anterior; H horizontal; Ca commissura anterior; F fornix; CO chiasma opticum. Bottom: Relationship between RTCe~ and the distance & t h e microinjection site from the frontal bregma plane (A 6.25). (O), PGE2 (0.2 Ixg); ( x ), PGF2~ (1.0 ~tg); (9 arachidonic acid (AA) (10.0 btg). The regression lines were calculated by the method of least squares. The equations for the regression lines and the correlation coefficients (r) are given in the text

RESULTS

Anatomical Mapping of Sites Sensitive to PGE2, PGF2~, and AA Thirty seven microinjections of PGE2 (0.2 btg) in 37 rats given into various hypothalamic nuclei were analysed after histological verification. The RTC~ values declined as the horizontal distance between the'bregma frontal plane and the microinjection site increased: y = 4.36 + 1.72x, r (correlation coefficient) = 0.62, P < 0.001 (Fig. 1). Similarly, there was an inverse relationship between the A Tes max. and TRies values and the distance from the bregma frontal plane: y = 1.73 + 0.7x (r = 0.57, P < 0.001)

J. A. Sp~awifiski et al. : Hyperthermic Effects of Arachidonic Acid, Prostaglandins E2 and F~

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Table 1. Mean values ofRTCo~(A To~max/h),TRIo~(A T~xh),andA T~ max(~ PGF2~,andarachidonic acid (AA) injected (10 ~tl) into the lateral brain ventricle (IVC) or microinjected (0.5 gl) into the anterior preoptic hypothalamic (AH/PO) or anterior hypothalamic (AH) nuclei. Doses are expressed in gg (of free acid) per rat. Significance (t test) was assessed by comparison with appropriate (see Methods) control. The AH/PO was the most sensitive nucleus to PGE2. The AH was the most sensitive nucleus to PGF2~ and AA. Asterisks: *** P < 0.001; ** P < 0.01; * P < 0.05 Compound

Route

Dose (~tg)

N

RTCo~ + S.E.M.

TRIo~ + S.E.M.

A To~max. • S.E.M.

PGE2

IVC

0.02 0.2 2.0

11 5 5

1.95 • 0.29*** 3.60 • 0.84*** 4.60 • 0.17"**

0.73 _+ 0.15'* 0.70 • 0.19" 2.17 • 0.35***

0.72 _+ 0.06*** 1.08 • 0.14"* 2.30 _+ 0.08***

PGF~

IVC

0.2 2.0 20.0

15 14 ~5

1.51 _+ 0.22*** 3.03 + 0.41"** 4.05 • 0.42***

1.30 • 0.32 1.40 + 0.40 5.15 _+ 0.53***

0.83 +_ 0.09* 1.61 • 0.19"** 2.69 • 0.15"**

AA

IVC

10.0 100.0

10 17

1.25 _+ 0.32** 1.33 • 0.22***

2.24 • 0.58*** 2.51 • 0.46***

1.17 + 0.15"** 1.30 • 0.16"**

PGEz

AH/PO

0.02 0.2 2.0

2 4 2

3.8 6.55 • 0.39*** 6.8

1.49 2.26 _+ 0.95* 3.4

1.5 2.18 • 0.47** 3.4

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AH

0.5 L0

5 8

3.04 + 0.69*** 3.37 • 0.68***

1.53 • 0.24* 4.08 _+ 0.66***

1.36 • 0.14"* 2.05 _+ 0.28***

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and y = 1.73 + 0.84x (r = 0.52, P < 0.001), respectively. Following 29 microinjections o f PGF2~ (1.0 gg) into various h y p o t h a l a m i c nuclei (29 rats), there was no correlation between RTCes values and the distance of the microinjected site f r o m the bregma plane: y = 2.60 + 0.Tx, r = 0.32, P > 0.05 (Fig. 1). F o r A Tes max. and TRies this correlation existed but was hardly significant r = 0.4 (P ~ 0.05) and r = 0.39 (P ~ 0.05), respectively. Following 25 microinjections o f A A (10.0 lag) into various h y p o t h a l a m i c nuclei (25 rats), no correlation was observed between RTC~s values and the distance o f the microinjected site f r o m the b r e g m a plane: y = 2 . 4 3 + 0 . 3 2 x , r=0.17, P>0.1 (Fig. l). There was also no correlation between the TRies or A Tes max. values and the distance of the microinjected site f r o m the bregma plane. The changes in A Tes max. p r o d u c e d by microinjections of appropriate control solutions into the various h y p o t h a l a m i c nuclei did not differ significantly f r o m pre-injection mean values of the b o d y temperature.

Dose-Response Relationship 1. Injections into the Cerebral Lateral Ventricle. The threshold doses of PGE2 and PGF2~ elevating Tes of rats are between 0 . 0 0 2 - 0 . 0 2 tag and 0 . 0 2 - 0 . 2 lag per rat, respectively. As m a y be seen in Table 1, the dose-response curves for PGE2 and PGF2~ are parallel

when RTCes is measured. These curves are parallel only at high dose ranges when A Te~ max. is measured. Dose-response curves calculated for TRies are not parallel at all. The threshold dose o f A A , which elevates Tes o f rats is between 1 . 0 - 1 0 . 0 lag per rat. T h e doseresponse (RTCes, TRies and A Tes max.) curves are flat in c o m p a r i s o n to those constructed for PGE2 and PGF2= (Table 1). 2. Microinjections into the H y p o t h a l a m i c Nuclei. PGE2 and PGF2~ were microinjected into those h y p o thalamic nuclei which showed the maximal sensitivity to either c o m p o u n d . The recorded changes o f the t h e r m o r e g u t a t o r y parameters (RTCes, TRL~, and A Tes max.) are higher and r o u g h l y parallel to those observed after injections o f c o r r e s p o n d i n g doses o f either P G into the cerebral ventricle. However, in contrast to intraventricular injections, only changes in TRies and A Tes max. are dose-dependent while the changes in RTCes, except for the smallest dose of PGE2, are not. Microinjections o f 10.0 lag o f A A into the A H (the h y p o t h a l a m i c nucleus m o s t sensitive to A A ) induce a rise in Tes with TRies and A T~s max. values c o m p a r a b l e to those observed after injection o f the same dose of A A into the cerebral ventricle (Table 1).

Vegetative and Behavioural Reactions Following Administration of PGE2, PGFz~, or AA into the Anterior Hypothalamus. The rise in Tes induced by PGE2 microinjeeted into the A H is a c c o m p a n i e d by a sig-

18

Pfliigers Arch. 374 (1978)

Table 2. Mean values of TRI~ (A T~x0.Sh) and A T~max. (~ of conscious rats observed during 30 rain following microinjections (0.5 gl) of PGE2, PGF2,, and arachidonic acid (AA) into the anterior hypothalamic nucleus. Doses are expressed in ~tg (of free acid) per rat. Significance (t-test)was assessed by comparison with appropriate (see Methods) controls: * P < 0.05 Compound

Dose (gg)

N

PGE2 PGF2~ AA

0.2 1.0 10.0

12 8 8

TRI~ _+ S.E.M.

--0.15 _ 0.06* 0.08 _+ 0.09 0.14 + 0.09

A T~ max. -F S.E.M, --0.58 _+ 0.24* 0.18 -t- 0.29 0.37 _+ 0:32

La~j

or AA display piloerection. The respiratory frequency following microinjections of PGE2, PGF2~, or AA does not change significantly.

The Synthesizing Capacity of the Rat Hypothalamus to Produce PGE2, PGF2~, TxA2, and PGI2. Homogenisation of the hypothalami (at 4 ~C), performed without addition of exogenous substrate, generates 17.7 pg of PGE2 and 35.6 pg of PGF2~ per mg of protein. The addition of AA substantially increases biosynthesis of PGs by homogenates (Fig. 2). However, in the presence of AA or a mixture of crude PGGz and PGH2 (see Methods), only traces of T x A 2 - i n 2 out of 8 experiments- were detected. When hypothalamic microsomes are incubated with AA or a mixture of crude PGG2 and PGHz no PGI2 is generated as judged by the absence of the anti-aggregatory properties of the incubation mixture (3 experiments).

q ~,,,j DISCUSSION PBE=

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HYPO. AA TIME

-

-

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- .(~ .05 .0S 25 - 25 25 40 10

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Fig. 2. Bioassay of prostaglandin (PG) E2-1ike and thromboxane A2 (TxAz)-like substances generated by the hypothaiamic homogenate (HYPO.). PGE2 relaxes mesenteric strip and contracts rat stomach. U-46619, synthetic PG endoperoxide analog, contracts mesenteric strip and is used as reference standard for TxA2. The detector tissues by the use of antagonists are selectively sensitive to PGs and TxA2. The presence of indomethacin (10 -6 g/ml) in the buffer excludes possibility that the response of tissues to arachidonic acid (AA) is due to conversion of AA into PGs or TxA2 within the tissues. At arrows 50 gl aliquots of components of incubation mixture (HYPO. or AA) or aliquots of incubation mixture (carried out on ice for 10, 40, and 70 s) were superfused over the bioassayed tissues, x Incubation was carried out at 37~

nificant fall of the tail skin temperature whereas, after PGF2~ or AA, a rise in T~ is observed (Table 2). Carbon dioxide production was measured before and after microinjections of PGE2 (0.2 ~tg), PGF2, (0.5 gg) or AA (10.0 gg) into the AH. PGE2 does not significantly increase CO2 production whereas PGF2~ and AA significantly (P < 0.02 and P < 0.05, respectively) increase CO2 production by 0 . 5 ~ and 0.9~, respectively, in comparison to the control values. The rise in Te~ induced by PGE2, PGF2,, or AA is accompanied by drowsiness. Following microinjections, rats assume a hunch-back posture in the corner of the plastic cage. Rats injected with PGE2, PGF2,,

The Mechanism of Action of PGE2. Microinjections of PGE2 into the AH/PO or AH of rats at an ambient temperature of 22 ~C induces a rise in Tes accompanied by the fall of the tail skin temperature (TJ. No increase in CO2 production is observed providing that the RQ remains constant during the experiment. It seems therefore that at the ambient temperature of 22~ the heat is gained through the tail vessels vasoconstriction resulting in a fall of Ts. Similarly, during fever induced by PGE1 in rabbits and monkeys at an ambient temperature of 2 2 - 24 ~C heat is gained mainly by peripheral vasoconstriction (Stitt, 1973; Crawshaw and Stitt, 1975). PGE2 in rats acts in a manner similar to that of endotoxin (Sptawifiski et al., 1977), i.e. fall in Ts, lack of stimulation of CO2 production, and lack of compensatory reactions (leading to heat loss) were observed during Tes rise in both cases. Therefore, a safe assumption is that the observed rise in the rat To~ following PGE2 injection is a regulated one and thus represents fever. As in the case ofPGEt (Veale and Whishaw, 1976; Williams et al., 1977) the area between the anterior commissure and the optic chiasm is apparently one of the ultimate sites of action of PGE2 in rats. The rise in Te~ following injection of PGE2 into this area corresponds to the highest RTCe~ values, the highest peak, and the largest TRI~ value measured. A significant correlation observed between RTCos, TRies, A T~ max. and the distance o f the injection site from the AH/PO supports this conchasion. Thus, the rise in To~caused by microinjections of PGE2 in other than AH/PO areas is probably due to a diffusion or trans-

J. A. Sptawiflskiet at.: HyperthermicEffec*sof ArachidonicAcid. ProstaglandinsEz mad Fz= port of PGE2 to the anterior centre of maximum se~nsitivity. A similar site of action is found in rats for the pyretic effect of E. coli endotoxi~ administered intracerebrarly (Splawifiski et al., t977). The onIy difference between the mode of action of PGE2 and that of endotoxin is the time of onset of fever. The value of RTC~s for 0.5 gg of endotoxin injected into the AH/PO is 0.73 ~C/h (Sptawifiski et al.. 1977) while 20 ng of PGE2 introduced into the same area yields a value of 3.8 ~C/h. Thus, threshold dose of PGE2 acts about five times faster then threshold dose of endotoxin. This is in agreement with the theory that PGE2 mediates endotoxin fever. However, recent experiments (see below) indicate t h a t - a t least in rabbits- PGEa may not be involved in mediation of pyrogen fever_

The Mechan&m of Action of AA and PGF2~. Cranston et al. (1976) have found that PGE2-induced fever in rabbits can be blocked without affecting the pyretic response to leucocyte pyrogen. Recently Laburn et al. (1977) reported that SC-19220, an antagonist of PGE2-induced fever in rabbits, did not block the long-term rise of the rabbit body temperature caused by AA which was attenuated by indomethacim These authors ascribed the effect of AA to one of its metahotile, other than ?GEz. probabIy TxA> A straightforward implication is that AA through the thromboxane pathway mediates fever induced by pyrogens. This hypothesis was tested in rats by studying the mechanism of a rise in body temperature induced by AA. It has already been shown that the increment of rise after an intraventricular injection of AA is decreased by aspirin (Sptawifiski et al., 1974) and that endotoxin-induced fever in rats is also reduced by aspirin (Sptawiflski et al., 1977). If endotoxin activity to produce fever in rats is due to the release of AA (which in turn is converted into TxA2) then the mechanism of the rise in rats Tos following AA administration should be similar to the mechanism of endotoxin fever. However. results obtained in the present study indicate that this in not the case. Firstly, the vegetative reactions following AA are not related to a rise in the body temperature or rats. AA significantly stimulates CO2 production. On the other hand, the rise in T~, following AA administration into the AH is accompanied by a rise in T~, indicating vasodi[ation of the tail vessels. These facts, therefore, suggest that AA induces hyperthermia, not fever. Conversely. endotoxin (Sp~awiflski et al., 1977), like PGEz. does not stimulate CO2 production and it induces tail vessels vasoconstriction. !~asoregulation of tail vessels represents a major thermoregulatory mechanism in the rat ,(Rand et at., 1965).

19

The vasodilation of the rat tail vessels following AA injection most probably represents a compensatory reaction. Secondly, in contrast to the response to endotoxin (Sptawihski et al., I977) or POEz, fo]Iowing AA injection there is no correlation between the hyperthermic response and the distance of the injection site from the bregma plane, This suggest thai the AH/PO or AH are not the primary sites of AA action. Thirdly, in vitro data indicate that the end-products of AA metabolism by the hypothalamic homogenate are mainly PGF2= and PGE2. Detection of TxA2 in only 2 out of 8 experiments in the present study indicates that TxA2 is not the main metabolite of AA in the hypothalamic tissue_ However, these experiments suggest that the hypothalamic tissue possesses a small, but definite, capability to produce TxA2. Similar results were reported by Wolfe et al. (1976b) for the rat cortex, although these authors detected more TxA2 than PGs. The question as to which end-product of AA is a main metabolite in vivo in the rat hypothalamus remains to be answered since addition of cofactors or the substrate load may redirect the AA metabolism in those tissues which produce a mixture of metabolites. For example, ram seminal vesicles convert AA mainly to PG[z. however, increasing substrate cortcentration increases the productien of PGEz. especially in the presence of reduced glutathione (Cottee el al., 1977). Fourthly, Clark and Cumby (1976) have reported that antipyretics are more effective against endotoxin fever than against the AA-induced rise in the cat body temperature. The antagonistic effect of aspirin against AAinduced hyperthermia in rats (Splawifiski et al., 1974) results, at least partially, from the inhibition of the conversion of AA into an active metabolite. The possibility that PGE2, TxA2, or PGI2 are active metabolites of the AA effect in rats seems to be very unlikely on the basis of the presented evidence. PGF2~ was shown to induce a rise in the rat body temperature after intraventricular administration (Feldberg and Saxena, 1975; G6rka et al., 1976). The present study confirms these results and suggests that PGF2~, like AA. induces hyperthermia rather than fever. This conclusion is based on the observation that vegetative reactions are not integrated to raise the rat body temperature, i.e. an increase in CO2 production and the tail vessels vasodilation are observed (see above under AA). As in the case of AA. the AH/PO or AH apparentIy are ~ot the principle sites of PGFz~ action. Interestingly, the AH is the most sensitive area explored to PGF2= and AA as well. These findings therefore suggest that the active metabolite of AA action in rats is PGF2=.

20 However, this conclusion does not agree with the dose-response relationship data. The dose-response curves for A A and PGF2~, after their intraventricular administration, are not parallel. This may exclude a c o m m o n mechanism of action or may indicate that A A is transported from the ventricle by a different mechanism than PGF2~. The latter possibility is suggested by the fact that the magnitude of the hyperthermic response to A A and its duration do not change when, instead of intraventricular injection, the same dose of A A is given into the AH. It is also possible that the hyperthermic action of A A in rats is mediated by not one but several metabolites: PGF2~ and PGE2. In the present study it is shown that hypothalamic tissue produces PGF2~ and PGE2. It is also possible that the increased concentration of PGF2~, or both PGF2~ and PGE2, in the rat hypothalamus (if it takes place after administration of AA) is not due to the conversion of AA into these metabolites but to the inhibiting effect of A A on 15-hydroxy-PG-dehydrogenase (Marrazzi and Matschinsky, 1972), an enzyme involved in the catabolism of PGE2 and PGF2~. In fact, body temperature is raised when polyphloretin phosphate, an inhibitor of this enzyme, is injected into the rat A H / P O (Sptawiflski, Wojtaszek, S w i e s - u n p u b l i s h e d results). This hypothesis would explain the long-term hyperthermia induced in rats and rabbits by A A as opposed to rather short-term PGF2~-induced hyperthermia and particularly to rapidly disappearing PGE2-induced fever in rats. Yet another explanation for the results presented is that exogenous AA does not follow the metabolic pathways of endogenous AA. However, studies done by Wolfe et al. (1976a) seem to exclude this possibility.

Endotoxin Fever and Arachidonic Acid Metabolism. The obtained results indicate that endotoxin in rats does not produce fever by increasing A A availability or by stimulation the A A cyclooxygenation. The former conclusion is supported by the fact that hydrocortisone, which prevents the release of membrane-bound A A (Gryglewski et al., 1975), does not affect endotoxin-induced fever in rats (Sptawifiski et al., 1977). Concerning the latter possibility, stimulation of cyclooxygenase by endotoxin would increase the level of PGF2~ as well as PGE2 since the rat hypothalamic tissue is capable of producing both PGs (see above). However, endotoxin fever in humans and rabbits is characterized by an increase in the P G E levels in cerebrospinal fluid while P G F levels remain unchanged (Philipp-Dormston and Siegert, 1974; PhilippDormston, 1976). Furthermore, PGG2 and P G H z which would be probably increased by stimulation

Pflfigers Arch. 374 (1978) of the A A c y c l o o x y g e n a t i o n - d o not induce a rise in rat body temperature as judged from the effect of their synthetic analogs (Hawkins and Lipton, 1977). Thus, the mechanism of endotoxin action based on the cyclooxygenase activation proposed by Ziel and K r u p p (1976) seems unlikely. Therefore, a m o n g the known metabolites of AA, only PGE2 fulfill the requirements tested for the role of mediator of endotoxin fever in rats. However, it is also possible that the increased level of PGE2 in the hypothalamus after endotoxin injection is not causaly related to the endotoxin activity to produce fever. The mechanism by which endotoxin raises the PGE2 level in the hypothalamus is not clear although the present study seems to indicate that is not mediated by the release of A A or the activation of cyclooxygenase, or both.

Acknowledgements. We are grateful to Professor R. Gryglewski for his help in this study, to Dr. J. Pike of Upjohn for providing us with samples of prostaglandins, and to Mr. W. Chwals for his help in preparing the English text. The expenses of this study were supported by NIH Special Foreign Currency Research Agreement No. 05-083-N. Some of the results were already presented at the joint Meeting of Polish and German Pharmacological Societies in Hannover, 1976. REFERENCES Clark, W. G., Cumby, H. R.: Antagonism by antipyretics of the hyperthermic effect of a prostaglandin precursor, sodium arachidonate, in the cat. J. Physiol. (Lond.) 257, 581--595 (1976) Cottee, F., Flower, R. J., Salmon, J. A., Vane, J, R. : Synthesis of 6-keto-PGFl~ by ram seminal vesicle microsomes. Prostaglandins 14, 413-424 (1977) Cranston, W. I., Duff, G. W., Hellon, R. F., MitchelI, D., Townsend, Y.: Evidence that brain prostaglandin synthesis is not essential in fever. J. Physiol. (Lond.) 259, 239-249 (1976) Crawshaw, L. I., Stitt, J. T. : Behavioural and autonomic induction of prostaglandin Ea fever in squirrel monkeys. J. Physiol. (Lond.) 244, 197-206 (1975) Feldberg, W., Saxena, P. N. : Prostaglandins, endotoxin and lipid A on body temperature in rats. J. Physiol. (Lond.) 249, 601 - 615 (1975) Flower, R. J., Blackwell, G. J. : The importance of phospholipaseA2 in prostaglandin biosynthesis. Biochem. Pharmacol. 25, 285-291 (1976) Flower, R. J., Vane, J. R. : Some pharmacologic and biochemical aspects of prostaglandin biosynthesis and its inhibition. In: Prostaglandin synthetase inhibitors (H. J. Robinson and,J. R. Vane, eds.). New York: Raven Press 1974 G6rka, Z., Sptawifiski,J., Katu2a, J., Suder, E.: Prostaglandins E2 and F2~ fever in rats. In: Symposium on prostaglandins (J. Knoll and K. Kelemen, eds.). Budapest: Akademiai Kiado 1976 Gornall, A. G,, Bardawill, C. J., David, M. M. : Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751-766 (1949) Gryglewski, R.: Prostaglandin and thromboxane biosynthesis inhibitors. N.aunyn-Schmiedeberg'sArch. Pharmacol. 297, $85$88 (1977)

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