Brain Research Bulktin, Vol. 33, pp. 403-409, 1994 0361-9230/94 $6.00 + .OO Printed in the USA. All rights reserved. Copyright 0 1993 Pergamon Press Ltd.

Actinomycin D Blocks Interleukin-lcu-induced Pial Arteriolar Dilation and Increased Prostanoid

Production in Newborn

MASM SHIBATA,’ ROBERT MIRRO,? ELLS M. ~MS~~

.

Pigs

AND CHARLES W. LEFFLER

Laboratory for Research in Neonatal Physiology, Departments of Physiology and Biophysics, Pediatrics, and Obstetrics and Gynecology, University of Tennessee, 894 Union Avenue, Memphis, TN 38163

Received 24 May 1993; Accepted 11 August 1993

SBIBATA, M., R. MIRRO, W. M. ARMSTEAD AND C. W. LEFFLER. ~~ti~y~~ D blocks interleaf-lo-i~ced pial arteriolar dilation and increasedprostanoidpmduction in newborn pigs. BRAIN RES BULL 33(4) 403-409,1994.-Effects of protein synthesis and cyclooxygenase inhibitors on interleukin-la (&la)- and histamine-induced pial arteriolar dilation and cerebrospinal fluid (CSF) prostauoid increases were examined in anesthetixed pigIets using closed cranial windows. Topical IL- la (10.8 yg) increased piaI arteriohtr diameter from 15 to 30 min after its infusion, and enhanced CSF prostanoids. Topical protein synthesis ~bitor, actinomycin D, at a concentration of lfl-* M attenuated and 1K6 M completely blocked both IL-la-induced vasodilation and CSF prostanoid increase. Inhibition of prostaghmdin H synthases with indomethacin blocked both vasodilation and CSF prostanoid increase by IL-la. Topical histamine (low6 M) also increased pial arteriolar diameter and CSF prostanoids but without the delay seen between IL-la infusion and responses. These histamine effects were not modified by coinfusion of actinomycin D but blocked by indomethacin. These resuhs suggest that, although IL-la and histamine do share the same mech- anism insofar as activation of prostaglandm synthesis is concerned, an additionat step appears to be involved for IL-la, likely involving de nova protein synthesis.

Actinomycin D Histamine Indomethacin Closed cranial window

THE most common cause of neonatal Gram-negative bacterial meningitis is Escherichia coli (13). The cell wall component of E. co& ~~~lysa~h~ide (LPS), is the most potent inducer of symptoms. When intracisternally injected into adult animals, bac- teria increase cerebral blood flow (CBF), cerebrospinal fluid prostanoids and neutrophil counts, and intracranial pressure, as well as permeability of blood-brain barrier (21,22,33,34). These alterations were believed to be caused by increased synthesis of brain cytokines induced by LPS since intracistemal or subarach- noid bacterial injection stimulates production of interleukin-1 (IL-l), IL-6, and tumor necrosis factor ar (TNFo) in cerebrospinal fluid (35), and since injection of these cytokines through the same route can mimic some of these alterations caused by bacteria (23,24,30). Intracistemal bacteria increase CBF without affecting systemic hemodynamics (21). Further, since topically applied bacteria onto the exposed cerebral cortex induce pial arteriolar dilation without altering systemic hem~yn~ics (4,17), it is likely that the observed CBF increase was caused by cerebral vasodilation through bacteria-induced enhanced cytokine syn- thesis.

Using newborn pigs, we have previously demonstrated that dilation of pial arterioles after topical IL-lo involves increased vasoactive preboil because indorne~a~~ blocked both the enhanced prostanoid synthesis and the dilation (32). However, little is known about cytokine signal transduction pathway(s) leading to the enhanced cerebrospinal fluid prostanoids and ce- rebral vasodilation.

The present study was designed to begin to address underly- ing mechanism of IL-la-induced pial arteriolar dilation and in- creased cerebrospinal fluid prostanoids in newborn pigs using closed cranial windows. The hypothesis was that while both IL- iru and histamine dilate pial arterioles pros~oid-de~nden~y, IL-lo but not histamine would require an additional step for its action involving local protein synthesis.

Surgery

METHODS

Newborn pigs (n = 39) of either sex weighing between 0.8- 2.4 kg (2-4 days old) were anesthetized with a mixture of ke-

t Deceased. ’ To whom requests for reprints should be addressed. ’ Present address: Department of ~~~~ioiogy and CriticaI Care Medicine, Children’s Hospital of Ph~adelphia, P~a~lphia, PA 191044399.

403

404 SHIBATA ET AL.

tamine hydr~hloride and acepromazine (33.0 + 3.3 mg&g, im, respectively, Ayerst Laboratory, Inc., New York, NY). Anesthe- sia was maintained with a-chloralose in an initial dose of 50 mg/ kg IV (Sigma Chemical Co., St. Louis, MO) followed by 7 mg/ kg every hour. Catheterization of both a femoral artery and vein were performed for, respectively, measuring arterial pH, Po2, PcoZ and blood pressure, and injecting anesthetic and fluids as required. After intubation, animals were ventilated mechanically, and mounted on stereotaxic head frame. After retracting the scalp, an opening 2 cm in diameter was made over the parieto- temporal cortex. The dura was cut and reflected to expose the cortical surface. Caution was taken to prevent damage or irrita- tion on the exposed surface. A cranial window made of circular stainless steel with a glass pane equipped with inlet and outlet ports was aseptically inserted into the opening and fixed to the skull with acrylic dental cement. The closed cranial window was then filled with warmed and appropriately gassed artificial cere- brospinal fluid (aCSP). The aCSF was maintained at 37°C and bubbled with a gas mixture of 6.3% Od5.8% COz. in Nz to pro- duce Po2, Pco2, and pH in the range of, respectively, 45-50 mmHg, 32-41 mmHg, and 7.39-7.45. The composition of aCSF was as follows (in mM): KC1 3.0; MgC& 1.5; CaClz 1.5; NaCl 132; urea 6.6; dextrose 3.7; NaHCO, 24.6.

Human Recombinant IL-f@

IL-ltr was a generous gift from Ho~ann-troche (Nutley, NJ). It is expressed in Escherich~ coti and consists of the car- boxyterminal 155 amino acids of the 271-amino acid human IL- la precursor. Lyophilized IL-la was reconstituted with sterile distilled water to a concentration of 10.8 &15 ~1 vehicle, and stored at 4°C. The vehicle contains 50 mM RI&PO4 and 0.1 M NaCl @H 6.5). Contamination of IL-la was minimized by ster- ilizing all materials used to handle this cytokine by autoclave.

Experimental Protocols

After surgery, the cranial window was repeatedly flushed with 5 ml aCSF during a 20 min period. Since the window contains maximally 500 ~1 CSF, this volume of aCSF would theoretically wash out the window 10 times. The dissection microscope and video camera were set above the cranial window, and images of exposed cortical surface were projected onto a video monitor via a dimensional analysis system (VPA 1000, FOR A Corp., Ltd., Natwick, MA).

The present study consisted of three experimental groups, I, II, and III, in which effects of (I) IL-la (vasodilator cytokine), and indomethacin (prostaglandin H synthase inhibitor), (II) IL- la, and actinomycin D (protein synthesis inhibitor), and (III) histamine [prostanoid-dependent vasodilator agent (18)], acti- nomycin D, and indomethacin on pial arteriolar diameter (range, 91- 117 pm) and CSF prostanoids were examined. The aims of these experiments were (I) to address the prostanoid-dependence of dilatory responses of cerebral vessels to IL-la, (II) to examine involvement of de nova protein synthesis in the IL-la response, and (III) to compare the IL-la response to prostanoid-dependent histamine-indu~d vasodilation. All substances except for indo- methacin were topically applied onto the exposed corticaf surface under the cranial window. The same experimental protocols were employed for every treatment in all groups in the following man- ner. Firstly, after flushing the space under the window with 5 ml aCSF, diameter measurements of a selected pial arteriole were taken at 5 min intervals for 10 min to establish the baseline value. No CSF sample was collected. The space under the window was flushed with 5 ml aCSF. Secondly, 5 ml aCSF containing no substance was infused under the window, and sequential mea-

surements of the pial arteriolar diameter were taken at S min intervals for 30 min for control treatment. Thirdly, after the di- ameter measurements had been completed, approximately 300 ~1 CSF were collected from under the window through the outlet port by injecting aCSF into the inlet port. Collected CSF samples were immediately frozen at -20°C for later radioimmunoassay to determine levels of prostanoids. The same procedures were repeated for the second and the third treatment periods in all groups with aCSF containing IL-la, IL-la vehicle, actinomycin D, or histamine with flushouts of the aCSF under the window between each treatment.

The group I was further divided into two subgroups, and pig- lets were tested as follows, group 1: control -+ IL-lo vehicle + IL-lo, and group 2: control -+ indomethacin -+ indomethacin + IL-la. In the first group, IL-la vehicle in a volume of 15 ~1 was added to 500 ~1 aCSF and infused under the window over a l- min period. IL-la at a dose of 10.8 &15 ~1 vehicle dissolved in 500 ,ul aCSF was infused under the window over a l-mm period. In the second group, a second control treatment with aCSF was performed 20 min after intravenous injection of in- domethacin trihydrate (5-7 m&g, Merck Sharp and Dohme, Rahway, NJ). Then, IL-la was tested as described in the first subgroup without additional indomethacin injection.

The group II was similarly divided into two subgroups, and newborn pigs were examined as follows, group 1: control -+ ac- tinomycin D 10-a M -+ actinomycin D 10-s M + IL-la, and group 2: control + actinomycin D 10e6 M -+ actinomy~in D W6 M + IL-la. Artificial CSF containing actinomycin D (Sigma Chemical Co.) at either lo-* or 10eh M was prepared immediately prior to its use. These solutions were prepared by adding actinomycin D, in a volume of 2.5 ~1 absolute ethyl al- cohol, to 5 ml aCSF. Hence, the concentration of the alcohol was 0.05%. Only one dose of actinomycin D was tested per animal. In each group, following either concentration of actinomycin D alone has been tested for 30 min, the window was flushed re- peatedly with 5 ml aCSF containing the same concentration of the substance. Then, 500 ~1 aCSF of either concentration of ac- tinomycin D containing 10.8 pg IL-la/l5 ~1 vehicle was infused under the window over a 1-min period.

The group III was further divided into three subgroups. Piglets were tested as follows, group 1: control + histamine 10e6 M, group 2: control --) actinomycin D 10e6 M + actinomycin D lo-” M + histamine lob6 M, and group 3: control -+ indometh- acin -+ indomethacin c histamine 1O-6 M. Testing histamine ef- fect twice in the same animal was avoided because of the reported tachyphylaxis to this substance in peripheral vessels (1). For the third treatment in the second group, these two substances were premixed prior to their infusion under the window. Procedures for indomethacin pretreatment were the same as those in the group I.

Levels of arterial blood gases, and pH were monitored (213 pH/Blood Gas Analyzer, Instrumentation Laboratory, Lexington, MA) once immediately before each control, once 20-30 min after initiation of treatment with each substance, and once im- mediately after completion of all treatment. Arterial blood pres- sure was recorded on-line by a pen-recorder (Recorder 24OOS, Gould Inc., Cleveland, OH) throughout the whole experimental period. Levels of blood pressure were read simultaneously as those of blood gases and pH. Specific attention was paid to con- firm that the observed changes in cerebral vasomotor tone were not related to those of blood gases, pH or systemic blood pressure.

Determination of CSF Prostanoid Levels

Levels of prostaglandin Ez (PG&) and 6-keto-PGF,,, the hydrolysis product of PG12, in sample CSF (unknown) from

IL-la INCREASES ARTERIOLAR DIAMETER AND PROSTANOIDS 405

TABLE 1

BLOOD GASES, pH, AND MEAN ARTERIAL BLOOD PRESSURE (MAP) OF PIGLETS BEFORE, DURING,

AND AFTER THE TREATMENTS

Pa02 PaC02 MAP

0”” Hg) (- W PH (mm Hg) n

Before 87 2 3 32 2 2 7.47 2 0.03 67 2 3 39 During-l 86 2 2 34 2 2 7.43 ? 0.04 67 2 5 39 During-2 86 2 4 3.5 t 3 7.43 2 0.05 66 2 4 32 After 84 2 3 35 2 3 7.41 t 0.02 65 ? 3 39

During-l: during the first treatment; During-& during the second treat- ment.

under the window were analyzed by radioimmunoassay against an aCSF background as described previously (32). Analyses were performed at three dilution’s to assess parallelism between the unknown dilution curve and the standard curve. The pros- tanoid antibodies used in this assay were produced in rabbits, and the bound fraction of prostanoids was separated from the free fraction using dextran-coated charcoal after 24 h incubation at 4°C. Our antibodies cross-react minimally (<l%) with the other known prostanoids and provide analysis of prostanoid

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FIG. 1. Changes of pial arteriolar diameter after topical applications of artificial cerebrospinal fluid (CONTROL), interleukin-la (ILla, 10.8 pg), its vehicle (VEHICLE) in the absence (A) and presence (B) of in- travenous indomethacin (INDO, 5-7 mg/kg). Indomethacin was injected 20 min prior to the measurement of the vessel diameter. Diameter changes were compared with those taken immediately prior to aplication of each substance at time zero, and expressed in percentage. Values are means 2 SEM ’ p < 0.05, compared to its baseline value, and b corre- sponding control value.

r-t=5

CNTL VHCL IL-1 a CNTL INDO INDO

la -I

FIG. 2. Levels of cerebrospinal fluid prostaglandin l!& (PG&) and 6-keto- PGF,, after topical application of artificial cerebrospinal fluid (CNTL), interleukin-la (IL-la, 10.8 pg), and its vehicle (VHCL) with (right half) and without (left half) intravenous indomethacin (INDO, 5-7 mg/kg). Each treatment corresponds to that shown in Fig. 1, and prostanoid levels were determined at the end of 30 min measurement of diameter changes. Values are means ? SEM. “p < 0.05, compared to its corresponding control value.

concentrations within the range of lOO-50,000 pg/ml CSF at the dilutions used. Target ligands are not displaced from the antibodies by arachidonate (20 &ml); 5hydroxyeicosatetrae- noic acid (HETE) or 15-HETE (1 pg/ml); leukotriene (LT) Bq, LTC,+ LTD4, or LTE4 (5 &ml) or lipoxin & or lipoxin B, (10 @ml).

Data Analysis

Diameter changes of pial arterioles recorded every 5 min for 30 min during the exposure of the vessels to various substances were compared with a corresponding value taken immediately prior to each treatment at time zero, and expressed in percentage. Those changes recorded every 5 min for 10 min before each treatment as the baseline value were also compared with a cor- responding value measured immediately prior to each treatment at time zero. Percentage values taken at each reading after treat- ments were compared with those of corresponding baseline as well as with those of corresponding control values. CSF samples collected from under the window were analyzed, and their pros- tanoid concentrations were expressed as picograms per milliliter CSF (pg/ml). Levels of CSF prostanoids were compared with a corresponding control value. All values are presented as means + SEM. Data were examined statistically using analysis of var- iance and Fisher PLSD for paired comparisons. The 95% level of confidence @ < 0.05) was accepted as statistically significant.

RESULTS

Significant changes in blood gasses, pH and mean arterial blood pressure were not observed either before, during or after topical administrations of &la, actinomycin D, histamine, or intravenous injection of indomethacin (Table 1).

Effects of Zndomethacin on Responses to IL-la

IL-la at a dose of 10.8 pg consistently increased pial arteri- olar diameter while control aCSF and IL-la vehicle had no effect

406 SHIBATA ET AL.

on the vasomotor tone (Fig. 1A). Significant diameter increases above the baseline levels were observed in all the animals from 15 to 30 min after IL-la infusion under the window (n = 5). The dose of IL-la used was selected because we have previously shown that 10.8 pg IL-la causes modest and constant increases in pial arteriolar diameter in piglets (32). CSF levels of prosta- glandin I!& (PGEQ and 6-keto-PGF1, increased significantly above the control levels by, respectively, 217 and 341% (n = 5) during H-lo-induced vasodilation (Fig. 2). Levels of CSF pros- tanoids after IL-la vehicle did not differ from those of controls. Indomethacin blocked IL-lo-educed va~ilation (INDG + IL- la in Fig. lB, n = 4), and lowered the resting prostanoid levels and prevented their increases by IL-la! (Fig. 2).

Efiects of Actinomycin D on Responses to IL-la

Actinomycin D attenuated IL-lo-induced pial arteriolar di- lation and CSF prostanoid increase in a dose dependent manner in animals pretreated with actinomycin D (Figs. 3 and 4). IL-lo in a dose of 10.8 pg following pretreatment with topical 1O-8 M actinomycin D (AM D + IL-la in Fig. 3A, n = 6) increased arteriolar diameter above the baseline levels from 15 min through 30 min postinfusion. A steady state of the vasodilation averaged from 20 to 30 mm postinfusion was 31% less than that observed with IL-lcr alone during the same period. IL-la following pre- treatment with IO-’ M actinomycin D (n = 6) increased PGE, far less than in piglets not pretreated with actinomycin D (AM D + IL-lo in Fig. 4). The apparent increase in 6keto-PGF,, after 10-a M actinomycin D was not significant. Pretreatment with the higher dose of actinomycin D (lOa M) completely

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FIG. 3. Changes of pial arteriolar diameter after topical application of artificial cerebrospinal fluid (CONTROL), actinomycin D (AM D), and interleukin-ln (IL-la, 10.8 pg) following topical pretreatment with ac- tinomycin D (AM D + &la) at lo-’ M (A) and 10e6 M (B). The dotted line in A is taken from Fig. 1A for comparison where ILla (10.8 pg) was tested alone. ’ p < 0.05, compared to its baseline value, and b cor- responding control value.

4000

n=6

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dM ACTINOMYCIN 0 I I

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IL-t * 121 * FIG. 4. Levels of cerebrospinal fluid prosta~an~ & (PC&) and 6&eto- PGF1, after topical applications of artikial cerebrospinal fluid for con- trol (ChTL), actinomycin D (AM D), and after interleukin-ln (IL-In, 10.8 pg) following topical pretreatment with actinomycin D (AM D t IL-la) at lo-* M and 10” M. Each treatment corresponds to that indi- cated in Fig. 3, and prostanoid levels were determined at the end of 30 min measurement of diameter changes. b p < 0.05, compared to its cor- responding control value.

blocked both IL-lo-induced vasodilation (AM D + IL-la in Fig. 3B, n = 7), and the enhanced synthesis of prostanoids by IL-la (Fig. 4, tz = 7). Actinomycin D alone at either lo-’ or 10m6 M produced no significant change on pial arteriolar diameter or CSF prostanoid levels as compared to those of the controls.

Eflects of Actinomycin D and Z~domet~ac~~ on Responses to Histamine

Only one dose of histamine (lo-’ M) was used in this exper- iment because we have previously shown that histamine at this concentration significantly increases pial arteroiolar diameter and levels of CSF prostanoids in piglets (18). Similarly, only lo-” M actinomicin D was used since this concentration proved to be effective to completely suppress IL-lo effect in the preceding experiment. Histamine immediately increased pial arteriolar di- ameter reaching a maximum of 22.9 -t 6.1% (HST in Fig. 5A, IE = 7) above the control levels within 5- 10 min after its infusion under the window. During the va~dilation, levels of CSF PG& and 6-keto-PGF,, were significantly increased by, respectively, 296 and 107% above the controls (Fig. 6). Actinomycin D alone affected neither pial arteriolar diameter nor CSF prostanoid levels (Figs. 5A and 6). Histamine following pretreatment with acti- nomycin D (AM D + HST in Fig. 5A, n = 6) increased arteriolar diameter 22.2 t 4.9% over its control level, as well as levels of PO& and &keto-PGFi, by, respectively, 355 and 161% (Fig. 6). Indomethacin alone did not affect pial arteriolar diameter (INDO in Fig. 5B) and markedly decreased CSF prostanoids (Fig_ 6). Indomethacin also blocked both histamine-induced vasodilation and CSF prostanoid increase (INDO + HST in Figs. 5B and 6, n = 4). There was a small but significant diameter increase (8.3% on average) in this treatment beginning at 20 min (Fig. 5B).

IL-lo INCREASES ARTERIOLAR DIAMETER AND PROSTANOIDS 407

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tr 25-1 ab r

B so 1

4

cc 25 E 1

a a a

. -s! . I . I . I . I . 1 . -I

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FIG. 5. Changes of pial arteriolar diameter after topical applications of artificial cerebrospinal fluid for controls (CONTROL), and histamine (HST, 10e6 M) with actinomycin D (Ah4 D, 10e6 M, in A), and intra- venous indomethacin (INW, 5-7 mg/kg, in B). Indomethacin was in- jected 20 min prior to the recording of diameter changes. Results shown in A consists of hvo groups, each with an independent control trial. How- ever, since data from these two controls were statistically similar, only one is shown in this figure. a p < 0.05, compared to its baseline value, and “corresponding control value.

DISCUSSION

The present study demonstrated that the delayed pial arteriolar dilation and the increased cerebral prostanoid by IGlo were completely blocked by inhibition of local protein synthesis with actinomycin D. In contrast, the immediate vasodilation and the increase in prostanoids by histamine were not affected by acti- nomycin D.

The results that topical IL-lo increased pial arteriolar diam- eter and CSF levels of prostanoids, and both responses were blocked by indomethacin, confirm our previous findings (32). They are also in good agreement with the report that IL-l-in- duced relaxation of the isolated mesenteric artery is prostanoid dependent, and inhibited by indomethacin (16). Increased CSF prostanoids following topical IL-la in the present study are con- sonant with the reported evidence that intracistemally injected cytokines enhanced CSF prostanoids in animal models of men- ingitis (24,30). The finding on actinomycin D is consistent with the reported observations that IL-l-induced adhesion of neutro- phi1 to peripheral vascular endothelium, release of arachidonic acid and its prostanoid metabolites from endothelium, fibroblasts, and macrophages, and induction of fever were all blocked by local protein synthesis inhibitors such as actinomycin D or cy- cloheximide (5,6,15,25,29,36). Previous studies demonstrated

that IL-1 locally induces protein synthesis (3), and these proteins are probably important for the cytokine to express its numerous biological properties (9). Accordingly, 4 to 6 distinct, yet over- lapping, sets of proteins with molecular weight ranging from 25 to 80 kDa, were detected in human fibroblasts after exposure to ILL Since other cytokines, such as tumor necrosis factor and interferon, also induce synthesis of a similar set of proteins (28) and analogous biological responses (3) similarities among these proteins may represent overlapping functions of these cytokines and indicate common biochemical pathways these cytokines share. Of particular interest in this regard is the evidence that local protein synthesis inhibitors also block IL-l-induced in- creased cGMP and nitrite formation, an oxidation product of ni- tric oxide, from vascular smooth muscle cells (2,12). These data, therefore, may suggest the possibility that local protein synthesis by IL-1 is indeed a critical mechanism which would take place at the very early stage of IL-1 signal transduction pathways. This may explain why inhibition of protein synthesis can block a va- riety of biological responses induced by IL-l. This assumption is also strengthened by the present result that while actinomycin D inhibited prostanoid synthesis by IL-lo, it did not inhibit the synthesis by histamine.

Large doses of intravenously injected IL-1 cause systemic hy- potension which is blocked by prostaglandin H synthase inhibi- tors (20). Vascular endothelial cells in in vitro preparations re- lease significant amount of prostanoids after exposure to IL-1 (8,27). It is believed that, unlike topically applied IL-l, circulat- ing IL-1 stimulates prostanoid synthesis from the endothelium and releases dilator prostanoids, such as PGE and PGI*, which relax vascular smooth muscle cells resulting in systemic hypo- tension. On the other hand, cerebral hemodynamic changes after systemic IL-1 are not well understood. It has been reported that intravenous injection of crude cytokines increased the content of blood in the brain (26). However, it is not known whether CBF increased following the intravenous cytokines.

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FIG. 6. Levels of cerebrospinal fluid prostaglandin Q (PC&) and 6-keto- PGF,, after topical applications of artificial cerebrosoinal fluid for con- trols (CNTL) &d hi&mine (HST, 10e6 M) with actiomycin D (Ah4 D, 10e6 M) or intravenous indomethacin (INDO, 5-7 mgkg). Each treat- ment corresponds to that shown in Fig. 5, and prostanoid levels were determined at the end of 30 min measurement of diameter changes. b p

< 0.05, compared to its corresponding control value.

408 SHIBATA ET AL.

Increased CSF prostanoids and pial arteriolar dilation ob- served after topical application of IL-la in the present study are most likely a localized response. This assumption can be sup- ported by the reported evidence that blood-born IL-l does not readily cross the blood-brain barrier because of its large molec- ular weight (17-21 kDa) (7,ll). Likewise, topically applied IL- la in the present study would be unlikely to cross the blood- brain barrier in the opposite direction to enter the blood stream. Topically applied IGla probably acts on, for example, abundant cerebral pial vessels directly. Results from in vitro studies, in- deed, support this presumption that vascular smooth muscle cells synthesize prostanoids when they are exposed to IL-1 (8,27). However, possible involvement of other elements under the cra- nial window, such as the endothelium, neurons, glia, and sensory nerves, in production of prostanoids should not be excluded. In fact, it has been shown that IL-1 stimulates prostanoid synthesis by most of these elements (8,10,27,31). Recently, a new form of prostaglandin H (PGH) synthase has been reported in fibroblasts (19,37). Expression of inducible PGH synthase (PGH synthase II) was selectively increased by LPS (14,15) and resulted in en- hanced PGE, synthesis (15). It is, therefore, likely that IL-la- induced increased CSF prostanoids in the present study result from induction of synthesis of PGH synthase II by this cytokine.

Although pial arteriolar dilation and enhanced prostanoid syn- thesis after IL-la and histamine appear to be analogous, and the responses by both substances were inhibited by indomethacin, the mechanism involved seems to be different. First, responses by IL-la were blocked by pretreatment with actinomycin D, but those by histamine were not. Second, the histamine effects on vasomotor tone begin almost instantaneously and reach a maxi-

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mum promptly while IL-la’s begin only after a prolonged pe- riod. The delay for IL-la to increase pial arteriolar diameter may have been caused by the time necessary for this polypeptide to induce local protein synthesis. It is, however, not clear in the present study how long exactly IL-la would need to synthesize proteins. Third, IL-la increases 6-keto-PGF,,, more than PGEz (Fig. 2, see also ref. 32) whereas histamine augments PGE, more than 6-keto-PGF,, (Fig. 6). This difference might suggest the possibility that target cells for IL-la and histamine to synthetize major prostanoids are different. Histamine has been found to act preferentially on the endothelial but not smooth muscle cells of isolated peripheral vessels (38). Topically applied IL-la in the present study, on the other hand, might have primarily acted on the smooth muscle cells rather than the endothelial cells because of a direct access for IL-la to the former. Alternatively, both IL- la and histamine might have stimulated the same target cells but increased PGE2 and 6-keto-PGF,,, differentially through different mechanisms.

In conclusion, the present results indicate that, although pial arteriolar dilation induced by IL-1~ and histamine were both prostanoid dependent, the mechanism underlying the responses to these two substances appear to be different since only IL-la

requires time and local protein synthesis to express its properties.

ACKNOWLEDGEMENTS

We acknowledge the technical assistance of M. Jackson, W. Gan- naway, J. Pirani, A. Fedinec, S. Tong, and A. M.-J. Shibata. This work was supported by the National Institutes of Health. We gratefully ac- knowledge the generous gift of the IL-la used in this study by Hoff- mann-La Roche.

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