Cyclooxygenase-2/microsomal prostaglandin E synthase-1 complex in the preoptic-anterior hypothalamus of the mouse: involvement through fever to intravenous lipopolysaccharide.

AIM: Prostaglandin E2 (PGE2) is now well established as a central effector of pyrogen fever. However, questions remain on the source, local vs. blood-borne, of the compound for the early phase of the typically biphasic fever (Phases 1 and 2) to i.v. pyrogens. To verify the role of centrally formed PGE2, we examined the cyclooxygenase (COX)/prostaglandin E synthase (PGES) complex through fever to i.v. lipopolysaccharide (LPS). METHODS: Experiments were carried out in the conscious mouse and LPS effect was ascertained on all steps of expression - gene, protein, catalytic activity - of individual enzymes. The analysis was limited to the preoptic-anterior hypothalamus (AH/POA). RESULTS: We found upregulation of the COX2 transcript together with an upward trend for the mPGES1 transcript during Phase 1. Coincidentally, there was a progressive increase in COX2 and mPGES1 protein expression through Phases 1 and 2. Catalytic activity for COX1 and COX2 combined was instead enhanced only in Phase 2, while mPGES1 activity remained steady at an intrinsically high level. Other COX and PGES enzymes were not modified through either Phase, and COX2/mPGES1 changes subsided with fever defervescence. CONCLUSION: The findings confirm a key function of COX2 and mPGES1 for the synthesis of pyrogenic PGE2 and, at the same time, document their early response to LPS. We conclude that locally formed PGE2 in AH/POA is qualified for a role in the initiation of fever.

Indomethacin promotes nitric oxide function in the ductus arteriosus in the mouse.

BACKGROUND AND PURPOSE: Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E(2) in concert with nitric oxide (NO) and carbon monoxide (CO). Accordingly, we have previously found that NO activity increases upon deletion of either COX. Here, we have examined whether COX inhibition by indomethacin mimics COX deletion in promoting NO. EXPERIMENTAL APPROACH: Experiments were performed in vitro and in vivo with wild-type (WT) and eNOS-/-, near-term mouse foetuses. Indomethacin was given p.o. to the mother as single (acute treatment) or repeated (daily for 3 days; chronic treatment) doses within a therapeutic range (2 mg kg(-1)). KEY RESULTS: Indomethacin promoted eNOS mRNA expression in the WT ductus. Coincidentally, the drug enhanced the contraction of the isolated ductus to the NOS inhibitor, N(G)-nitro-L-arginine methyl ester, and its effect augmented with the length of treatment. No such enhancement was seen with the eNOS-/- ductus. Chronic indomethacin also increased, albeit marginally, the contraction of the WT ductus to the CO synthesis inhibitor, zinc protoporphyrin. Whether given acutely or chronically, indomethacin induced a little narrowing of the ductus antenatally and had no effect on postnatal closure of the vessel. CONCLUSIONS AND IMPLICATIONS: We conclude that activation of NO and, to a much lesser degree, CO mechanisms is an integral part of the indomethacin effect on the ductus. This relaxing influence may oppose the contraction from PGE(2) suppression and could explain the failures of indomethacin therapy in premature infants with persistent duct.

Interactions between NO, CO and an endothelium-derived hyperpolarizing factor (EDHF) in maintaining patency of the ductus arteriosus in the mouse.

BACKGROUND AND PURPOSE: Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E(2), possibly along with nitric oxide (NO) and carbon monoxide (CO), and cyclooxygenase (COX) deletion upregulates NO. Here, we have examined enzyme source and action of NO for ductus patency and whether NO and CO are upregulated by deletion of, respectively, heme oxygenase 2 (HO-2) and COX1 or COX2. EXPERIMENTAL APPROACH: Experiments were performed in vitro and in vivo with wild-type and gene-deleted, near-term mouse fetuses. KEY RESULTS: N(G)-nitro-L-arginine methyl ester (L-NAME) contracted the isolated ductus and its effect was reduced by eNOS, but not iNOS, deletion. L-NAME contraction was not modified by HO-2 deletion. Zinc protoporphyrin (ZnPP) also contracted the ductus, an action unaffected by deletion of either COX isoform. Bradykinin (BK) relaxed indomethacin-contracted ductus similarly in wild-type and eNOS-/- or iNOS-/-. BK relaxation was suppressed by either L-NAME or ZnPP. However, it reappeared with combined L-NAME and ZnPP to subside again with K(+) increase or K(+) channel inhibition. In vivo, the ductus was patent in wild-type and NOS-deleted fetuses. Likewise, no genotype-related difference was noted in postnatal closure. CONCLUSIONS AND IMPLICATIONS: NO, formed mainly via eNOS, regulates ductal tone. NO and CO cooperatively mediate BK-induced relaxation in the absence of PGE(2). However, in the absence of PGE(2), NO and CO, BK induces a relaxant substance behaving as an endothelium-derived hyperpolarizing factor. Ductus patency is, therefore, sustained by a cohort of agents with PGE(2) and NO being preferentially coupled for reciprocal compensation.

Cardiopulmonary bypass in ewe's fetus: advances and setbacks in our learning curve.

Fetal cardiac surgery represents a surgical challenge and several centers are attempting to establish a suitable methodology in animals. We present our experience with extra-corporeal bypass procedures in preterm and term sheep fetuses. Twenty-two fetuses (103-139 days gestation, mean 115 days gestation) underwent a 1-hour period of right heart-to-pulmonary artery extracorporeal circulation followed by 1 hour of observation. Animals were divided into group 1 and group 2, according to gestational age (above and below 0.85). Three pumps were used: centrifugal without (group 1) reservoir, centrifugal with (group 2) reservoir, and roller with reservoir (group 2). Experiments were completed in 75% of fetuses in group 1 and in 37% of fetuses in group 2. Bleeding was the main cause of failure, especially for group 2. A slow deterioration of blood gas status was noted in group 1, while this trend could be partially reversed in group 2 with corrective measures. Complete heart bypass could not be achieved in either group, and residual fluctuations in arterial pressure were observed. During bypass, body temperature decreased more in group 2 than in group 1. We conclude that cardiac bypass is feasible over a short period in near-term fetuses. A successful outcome may also be obtained in younger fetuses, but better measures need to be implemented for the prevention of surgical bleeding.

Endothelin-induced constriction of the ductus venosus in fetal sheep: developmental aspects and possible interaction with vasodilatory prostaglandin.

1. The ductus venosus is actively regulated in the fetus, but questions remain on the presence of a functional sphincter at its inlet. Using fetal sheep (0.6-0.7 gestation onwards), we have examined the morphology of the vessel and have also determined whether endothelin-1 (ET-1) qualifies as a natural constrictor being modulated by prostaglandins (PGs). 2. Masson's staining and alpha-actin immunohistochemistry showed a muscular, sphincter-like formation at the ductus inlet and a muscle layer within the wall of the vessel proper. This muscle cell component increased with age. 3. ET-1 contracted dose-dependently isolated sphincter and extrasphincter preparations of the ductus from term fetus. This ET-1 effect also occurred in the premature, but its threshold was higher. 4. BQ123 (1 microm) caused a rightward shift in the ET-1 dose-response curve, while indomethacin at a threshold concentration (28 nm) tended to have an opposite effect. 5. Big ET-1 also contracted the ductus sphincter but differed from ET-1 for its lesser potency and inhibition by phosphoramidon (50 microm). 6. The ductus sphincter (term and preterm) and extrasphincter (term) released 6-keto-PGF(1alpha) (hence PGI(2)) and, to a lesser degree, PGE(2) at rest and their release increased dose-dependently upon ET-1 treatment. Both basal and stimulated release was curtailed by endothelium removal. 7. BQ123 and phosphoramidon reduced slightly the contraction of ductus sphincter to indomethacin (2.8 microm). 8. We conclude that the ductus contains a contractile mechanism in the sphincter and extrasphincter regions. ET-1 lends itself to a role in the generation of contractile tone and its action may be modulated by prostaglandins.

Cyclooxygenase-1 and cyclooxygenase-2 in the mouse ductus arteriosus: individual activity and functional coupling with nitric oxide synthase.

1. Prenatal patency of the ductus arteriosus is maintained by prostaglandin (PG) E(2), conceivably in concert with nitric oxide (NO). Local PGE(2) formation is sustained by cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2), a possible exception being the mouse in which COX1, or both COXs, are reportedly absent. Here, we have examined the occurrence of functional COX isoforms in the near-term mouse ductus and the possibility of COX deletion causing NO upregulation. 2. COX1 and COX2 were detected in smooth muscle cells by immunogold electronmicroscopy, both being located primarily in the perinuclear region. Cytosolic and microsomal PGE synthases (cPGES and mPGES) were also found, but they occurred diffusely across the cytosol. COX1 and, far more frequently, COX2 were colocalised with mPGES, while neither COX appeared to be colocalized with cPGES. 3. The isolated ductus from wild-type and COX1-/- mice contracted promptly to indomethacin (2.8 micro M). Conversely, the contraction of COX2-/- ductus to the same inhibitor started only after a delay and was slower. 4. N(G)-nitro-L-arginine methyl ester (L-NAME, 100 micro M) weakly contracted the isolated wild-type ductus. Its effect, however, increased three- to four-fold after deleting either COX, hence equalling that of indomethacin. 5. In vivo, the ductus was patent in all mice foetuses, whether wild-type or COX-deleted. Likewise, no genotype-related difference was noted in its postnatal closure. 6. We conclude that the mouse ductus has a complete system for PGE(2) synthesis comprising both COX1 and COX2. The two enzymes respond differently to indomethacin but, nevertheless, deletion of either one results in NO upregulation. PGE(2) and NO can function synergistically in keeping the ductus patent.

Function of cyclo-oxygenase-1 and cyclo-oxygenase-2 in the ductus arteriosus from foetal lamb: differential development and change by oxygen and endotoxin.

1. Prenatal patency of the ductus arteriosus is maintained mainly by prostaglandin(PG) E(2). Here we have examined the relative importance of cyclo-oxygenase-1 (COX1) and cyclo-oxygenase-2 (COX2) for PGE(2) formation in the foetal lamb ductus (0.65 gestation onwards). 2. Using fluorescence microscopy and immunogold staining, COX1 appeared more abundant than COX2 in endothelial and smooth muscle cells, and this difference was greater before-term. Inside muscle cells, COX1 and COX2 immunoreactivity was located primarily in the perinuclear region. Endotoxin, given to the lamb in utero (approximately 0.1 microg kg(-1)), caused COX2 upregulation, while an opposite effect with disappearance of the enzyme followed endotoxin treatment in vitro (100 ng ml(-1)). COX1 immunoreactivity remained virtually unchanged with either treatment; however, this isoform as well as any induced COX2 migrated towards the outer cytoplasm. 3. The COX2 inhibitor L-745,337 (1--10 microM) contracted the isolated ductus at term, the response being almost as high as that to indomethacin (dual COX1/COX2 inhibitor) over the same dose-range. Conversely, L-745,337 was relatively less effective in the premature. 4. Pretreatment of the premature in vivo with endotoxin enhanced the contraction of the ductus to L-745,337, while in vitro endotoxin had a variable effect. 5. The premature ductus exhibited a stronger contraction to L-745,337 following exposure to oxygen. On the other hand, the oxygen contraction, which is modest before-term, was enhanced by L-745,337. 6. We conclude that COX1 and COX2 develop unevenly in the ductus. While both enzymes contribute to PGE(2) formation at term, COX1 is the major isoform in the premature. COX2, however, may acquire greater importance before-term following physiological and pathophysiological stimuli.

Deletion of the endothelin-A-receptor suppresses oxygen-induced constriction but not postnatal closure of the ductus arteriosus.

Experiments were carried out in mutant 129/SvEv mice lacking the endothelin-A (ET(A))-receptor to determine whether endothelin-1 (ET-1), acting as a messenger for oxygen constriction, is responsible for closure of the ductus arteriosus at birth. The isolated ductus from ET(A) -/- fetuses, unlike that from ET(A) +/+ littermates, contracted marginally to oxygen and ET-1 but responded to a thromboxane analog. In vivo, reduction in ductus lumen was equally pronounced in tracheotomized ET(A) -/- and ET(A) +/+ newborns. Conversely, no such vessel narrowing was seen in hyperoxic ET(A), -/- fetuses, although it occurred in ET(A) +/+ littermates. Notwithstanding the uneven behaviour of the ductus in vitro and in vivo, no ET(A) genotype-related difference was noted in the morphology of the vessel on both light and electron microscopy. We conclude that ET-1 mediates the ductus constriction to oxygen. Without ET-1, however, the vessel still closes postnatally probably as a result of the withdrawal of the relaxing influence of prostaglandin E2 (PGE2).

The endothelin-A-receptor antagonist PD 180988 (CI-1034) selectively reverses the pulmonary vasoconstrictor response to hypoxia in the lamb.

Endothelin-1 (ET-1) is assigned a mediator role in the constrictor response of the pulmonary vasculature to hypoxia. Accordingly, a recently developed endothelin-A (ETA) antagonist, PD180988, was tested in the chronically instrumented newborn lamb to verify this possibility and, at the same time, to study a potential new treatment for pulmonary hypertension (PH). PD180988, given by infusion after a priming bolus, had an insignificant effect on the pulmonary circulation under normoxia, while it reversed the sustained pulmonary constriction caused by hypoxia. No appreciable change was noted under either experimental condition in the systemic circulation and cardiac contractility. We conclude that PD180988 is a selective inhibitor of hypoxic pulmonary vasoconstriction and lends itself to therapeutic use in infants.

Endothelin-1 release from the lamb ductus arteriosus: are carbon monoxide and nitric oxide regulatory agents?

We have proposed that endothelin-1 (ET-1), formed through the activation of a cytochrome P450 (CYP450)-based monooxygenase reaction, is important for generation of contractile tone in the ductus arteriosus and, consequently, for closure of the vessel at birth. The present investigation was undertaken to ascertain, using an isolated ductus preparation from near-term fetal lambs, whether carbon monoxide (CO) and nitric oxide (NO) qualify as regulators of the CYP450/ET-1 system. Preparations released ET-1 at rest and its amount showed no significant reduction following removal of the endothelium. Basal release was not changed by the NO synthesis inhibitor, N(G)-nitro-L-arginine methylester (L-NAME, 100 microM), nor by agents altering cyclic GMP content (i.e. increase; ONO-1505, 1 microM) and action (i.e. decrease; LY-83583, 10 microM). These findings extend previous work showing no effect of the CO synthesis inhibitor zinc protoporphyrin IX (ZnPP, 10 microM) under the same conditions (10). Conversely, both CO (65 microM) and the NO donor, sodium nitroprusside (SNP, 10 microM), curtailed ET-1 release. ET-1 release was increased by oxygen and reduced by pyrogens (endotoxin and IL-1, both at 100 ng mL(-1)). The endotoxin effect tended to be reversed by L-NAME and ZnPP, used singly or in combination. We conclude that ET-1 is formed naturally in the ductus and that its formation may change in response to physiological (oxygen) and pathophysiological (pyrogens) stimuli. Endogenous CO and NO, however, appear to have little or no role as ET-1 regulators.

Perinatal changes in choroidal 15-hydroxyprostaglandin dehydrogenase: implications for prostaglandin removal from brain.

We have previously shown in the sheep fetus at 0.7 and 0.9 gestation that the choroid plexus, unlike brain parenchyma, catabolizes prostaglandins (PGs). Peculiarly, in the choroid plexus, PGE(2) catabolism persists throughout the neonatal period to abate in the adult, while PGF(2alpha) catabolism abates shortly after birth. To explain this differential behavior and elucidate the function of catabolic enzymes, we examined the cellular location and activity of the rate-limiting enzyme for PGE(2) and PGF(2alpha) catabolism, 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Immunofluorescence histochemistry and immunogold electronmicroscopy revealed abundant 15-PGDH expression in the epithelial cytosol close to the brush-border membrane at 0.7 and 0.9 gestation. In contrast, at 5 and 15 days postnatal, 15-PGDH was found throughout the cytosol of stromal fibroblasts. No staining was observed at either location in pregnant adults. PGF(2alpha) catabolism was minimal in the total homogenate and 100000xg supernatant of the fetal choroid plexus at 0.7 and 0.9 gestation, while PGE(2) catabolism was evident at 0.7 gestation only. In contrast, both PGs were catabolized in minced specimens at either age. In conclusion, our study shows immunoreactive 15-PGDH in the choroid plexus from fetal and neonatal, but not pregnant adult, sheep. Results suggest that PGE(2) catabolism is not as critically dependent as that of PGF(2alpha) on tissue integrity and 15-PGDH location. Given the key role being assigned to the choroid plexus in PG removal from brain, we speculate that persistence of PGE(2) catabolism into the early postnatal period protects against central respiratory depression caused by the compound during this susceptible stage of development.

Contractile and relaxing mechanisms in pulmonary resistance arteries of the preterm fetal lamb.

Isolated pulmonary resistance arteries from term fetal lambs have nitric oxide (NO)- and prostaglandin-mediated relaxing mechanisms which are activated when PO(2) is raised from fetal to neonatal levels. The same vessels contract under hypoxia, and the contraction has been ascribed to endothelin-1 (ET-1). We have now studied these vasoeffector mechanisms before term (0.7 and 0.65 gestation) with the objective of determining whether their activity correlates with the development of susceptibility to oxygen changes. Experiments were carried out at neonatal PO(2), when expectedly relaxing mechanisms are maximally expressed, or under hypoxia. At either fetal age, the NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (100 microM), had no effect on basal tone, while indomethacin (2.8 microM) was a weak constrictor. Premature arteries did not contract when first exposed to hypoxia, but they responded marginally to a second exposure. The same arteries contracted strongly to a thromboxane A(2) analogue (ONO-11113, 0.1 microM) and ET-1 (10 nM), while their contraction to activating solution (5 mM Ca(2+) in K(+)-Krebs solution) was small and variable. At 0.7 gestation, bradykinin (0.1-100 nM), acetylcholine (0.01-10 microM), and sodium nitroprusside (0.1 nM to 10 microM) dose-dependently relaxed arteries precontracted with ONO-11113. Conversely, at 0.65 gestation the relaxation to bradykinin and acetylcholine was not dose-dependent and tended to be weaker. We conclude that preterm pulmonary arteries have viable effector mechanisms for contraction and relaxation. However, the capability for these mechanisms to be activated by PO(2) changes is markedly curtailed.

Differential uptake and catabolism of prostaglandin (PG)E(2) versus PGF(2alpha) in the sheep choroid plexus during development.

The early postnatal decrease in prostaglandin (PG)E(2) levels in cerebrospinal fluid (CSF) likely contributes to the establishment of continuous breathing. To elucidate mechanisms underlying this event, choroid plexuses from lateral (L-CP) and third/fourth (III/IV-CP) ventricles were incubated with [3H]-PGE(2) and label uptake (tissue-to-medium ratio for radioactivity, T/M) and catabolism (%radioactivity associated with metabolites, PGM) were measured. [3H]-PGF(2alpha) was a reference. Uptake of [3H]-PGE(2) was lower than [3H]-PGF(2alpha) in the term fetus (L-CP: 5.9+/-0.5 vs. 9.6+/-0. 9, n=11; III/IV-CP: 2.7+/-0.4 vs. 7.7+/-1.0, n=5) and 17 d lamb (L-CP: 5.3+/-0.8 vs. 11.0+/-1.2, n=7; III/IV-CP: 3.1+/-0.2 vs. 11. 6+/-2.8, n=3 and 4, respectively). This difference was not significant in the pregnant adult. Release of the two compounds was similar and did not change with age. [3H]-PGE(2) uptake was reduced by probenecid (1 mM) and excess PG (60 microM PGE(2) or PGF(2alpha)). Excess PG also reduced catabolism in the fetus, which was extensive for [3H]-PGE(2) and [3H]-PGF(2alpha)60%). In the lamb, catabolism remained high for [3H]-PGE(2) (L-CP: 64+/-4%, n=7; III/IV-CP: 41+/-4%, n=3), but not [3H]-PGF(2alpha) (L-CP: 26+/-4%, n=7; III/IV-CP: 4+/-1%, n=4). In the pregnant adult, catabolism was above background only for [3H]-PGE(2) in the L-CP (26+/-5%, n=11). Unlike the perinatal animal, this catabolism was reduced by probenecid. In conclusion, PGE(2) uptake and catabolism operate independently in the choroid plexus from perinatal sheep. Differences between PGE(2) and PGF(2alpha) are developmentally-regulated for both mechanisms. While neither process explains the postnatal decrease in CSF PGE(2), both may help keep CSF levels low during early postnatal development.

Endothelin A receptor is necessary for O(2) constriction but not closure of ductus arteriosus.

In vitro and in vivo techniques were developed with genetically modified mice to determine whether endothelin-1 (ET-1) functions as an O(2) mediator in closure of the ductus arteriosus (DA) at birth. Wild-type CD-1 and 129/SvEv mice with ET(A) receptor -/-, +/-, and +/+ genotypes were used. Isolated DA from term ET(A) +/+ fetuses contracted to O(2) (5-95%) and a thromboxane A(2) analog (ONO-11113, 0.1 microM). Instead, ET-1 elicited a dual response with weak relaxation (0.1 nM) preceding contraction (1-100 nM). Indomethacin (2.8 microM) was also a constrictor. ET(A) -/- DA, unlike ET(A) +/+ DA, contracted marginally to O(2) and ET-1 but responded to ONO-11113. O(2) contraction was also reduced in ET(A) +/- DA. In vivo, DA constricted equally in tracheotomized ET(A) -/- and ET(A) +/+ newborns. Conversely, no DA constriction was seen in hyperoxic ET(A) -/- fetuses in utero, although it occurred in ET(A) +/+ and +/- littermates. We conclude that ET-1 mediates the DA constrictor response to O(2). Without ET-1, however, the vessel still closes postnatally, conceivably caused by the withdrawal of relaxing influence(s).

Does endothelin-1 reduce superior mesenteric artery blood flow velocity in preterm neonates?

AIM: To compare plasma endothelin-1 (ET-1) concentrations in preterm neonates from pre-eclamptic and normal mothers; and to evaluate whether ET-1 has a role in altered arterial blood flow velocity. METHODS: Umbilical arterial blood and neonatal arterial blood were sampled on days 1 and 3 for gas analysis and measurement of plasma ET-1. Doppler ultrasonography of the middle cerebral, renal, and superior mesenteric arteries (SMA) was performed. RESULTS: Neonates in the pre-eclampsia (n = 18) and control (n = 18) groups had mean (SD) gestational ages of 31.1 (2.5) weeks and 30.4 (2.1) weeks; their birth-weights were 1432 (SD 676) g and 1692 (SD 500) g, respectively. In the pre-eclampsia group mean umbilical arterial PO2 was lower--1.88 (0.75) kPa compared with 3.27 (1.41) kPa (p < 0.01)--and mean plasma ET-1 concentration was higher in the umbilical artery--40.6 (SD 15.0) compared with 30.5 (SD 13.8) pg/ml (p = 0.04) and day 1 blood--54.9 (35.0) pg/ml compared with 33.6 (14.6) pg/ml (p = 0.03). Middle cerebral artery peak systolic velocity was higher and SMA time averaged, peak systolic, and mean peak velocities were lower in the pre-eclampsia group. SMA time averaged velocity was inversely related to plasma ET-1 concentration. CONCLUSION: The association between increased production of ET-1 and reduction in SMA time averaged velocity suggests a possible mechanism for hypoperfusion of the intestinal wall in neonates.

Formation of interleukin-6 in the brain of the febrile cat: relationship to interleukin-1.

Previous investigations have shown that interleukin-6 (IL-6), unlike other cytokines, is produced in larger amounts in the brain of the febrile animal regardless of the route, peripheral vs. central, of pyrogen administration. In addition, depending on the experimental condition IL-6 production may or may not require the prior induction of interleukin-1 (IL-1). The present study was carried out in the conscious cat to assess the importance of brain-derived IL-6 in the pathogenesis of fever and the interaction at that site between this cytokine and IL-1. IL-6 was detected in cerebrospinal fluid (CSF) collected at rest and its levels increased during the fever to intravenous (i.v.) endotoxin. The IL-6 elevation, but not the fever, was reversed by pretreatment with intracerebroventricular (i.c.v.) IL-1 receptor antagonist (hIL-1ra). Conversely, when pyrogens (endotoxin, IL-1) were given i.c.v., i.c.v. hIL-1ra reduced the fever without altering significantly the associated rise in CSF IL-6. We conclude that IL-6 is formed in brain in response to both i.v. and i.c.v. pyrogens; however, its formation, whether requiring the prior induction of IL-1 or not, does not appear to be critical for the development of the fever. Blood-borne IL-6, unlike brain-derived IL-6, may still play a role in fever as a trigger of signal-transducing mechanisms operating across the blood-brain barrier.

Effect of endothelium removal on prostaglandin and nitric oxide function in pulmonary resistance arteries in the lamb.

We have recently shown that isolated pulmonary resistance arteries of the fetal lamb have prostaglandin (PG) I2 based and nitric oxide (NO) based relaxing mechanisms, which are activated by oxygen (at neonatal levels) and bradykinin. The present study was carried out to ascertain whether these mechanisms remain operational after removal of the endothelium. Endothelium-denuded vessels pre-equilibrated at a neonatal Po2 were not affected by indomethacin (2.8 microM), while they contracted weakly to NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). However, the latter response did not reach significance and resembled that of intact vessels at fetal Po2. Bradykinin (0.1-100 nM) dose dependently (from 1-3 nM upwards) relaxed endothelium-denuded arteries that had been precontracted with a thromboxane (TX) A2 analog (ONO-11113, 0.1 microM) or excess potassium (5 mM Ca2+ in K(+)-Krebs) at a neonatal Po2. The response was the same under the two conditions, but it was smaller than that of intact arteries. Bradykinin relaxation of ONO-11113-contracted arteries was completely or nearly completely inhibited by indomethacin and L-NAME. We conclude that endothelium-denuded, pulmonary resistance arteries maintain PG (conceivably PGI2) mediated and NO-mediated relaxing mechanisms. These extra-endothelial mechanisms are activated by bradykinin but not by oxygen.

Effect of ciliary neurotrophic factor on body temperature and cerebrospinal fluid prostanoids in the cat.

It has been proposed that ciliary neurotrophic factor (CNTF) belongs to the group of cytokines causing fever in response to infectious and inflammatory noxae. The present investigation was undertaken in the conscious cat to verify whether CNTF (human type, hCNTF) is pyrogenic when given either intravenously (i.v.) or intracerebroventricularly (i.c.v.) and correlate at the same time body temperature with cerebrospinal fluid (CSF) levels of prostaglandin (PG) E2 (i.e., the putative fever mediator in brain) and thromboxane (TX) B2 (the stable TXA2 byproduct) in untreated vs. treated animals. hCNTF (10 microg/kg i.v.; 1 microg i.c.v.) caused fever by both routes and the increase in body temperature was associated with an upward change in CSF PGE2. Conversely, CSF TXB2 showed no elevation. Similarly unaffected was CSF TXB2 by human interleukin 6 (hIL-6, 1 microg i.c.v.), a cytokine with known pyrogenic and PGE2-promoting actions sharing the signal-transducing mechanism with hCNTF. We conclude that CNTF lends itself to a role in the pathogenesis of fever. The modest PGE2 elevation relatively to other cytokines, specifically hIL-1, is ascribed to the fact that CNTF activates the inducible isoform of arachidonate cyclooxygenase, which is constitutively expressed in brain, without concomitantly promoting the formation of new enzyme.

Prostaglandin uptake and catabolism by the choroid plexus during development in sheep.

We have previously reported that prostaglandin(PG) E2 levels in sheep cerebrospinal fluid (CSF) are high prenatally and abate rapidly after birth. This event may contribute to the establishment of continuous breathing. To explain this change, we have examined PG disposal mechanisms in the perinatal and adult (pregnant and non-pregnant animal) sheep brain by measuring the capacity of the isolated choroid plexus to concentrate [3H]PGF2alpha and [3H]PGE2. At 0.9 gestation, [3H]PGF2alpha uptake (expressed as the tissue-to-medium ratio, T/M) attained a steady-state by 15 min and was maintained thereafter (T/M at 60 min, 5.6 +/- 0.6; n = 16). Likewise, [3H]PGE2 was taken up by the tissue, but the actual accumulation was smaller (T/M at 60 min, 2.6 +/- 0.2; n = 8). Thin-layer radiochromatographic analysis of the tissue following incubation with [3H]PGF2alpha showed that 55 +/- 4% (n = 10) of radioactivity migrated as the 15-keto-13,14-dihydro metabolite. [3H]PGF2alpha uptake decreased upon treatment with probenecid (1 mM) (T/M, 2.5 +/- 0.2; n = 10) or after adding unlabelled PGF2alpha to the medium (1-60 microM) (T/M at 60 microM, 1.8 +/- 0.1; n = 13). The yield of labelled metabolite was also lower when using excess PGF2alpha (14% of control at 60 microM; n = 13), while it was not affected by probenecid. Uptake of both PGs did not change through development, from 0.7 gestation to day 18 postnatal, and attained higher values in the pregnant adult. Conversely, PGF2alpha catabolism decreased postnatally and became negligible by adult age. We conclude that during the perinatal period PGs can be removed from CSF by two distinct processes in the choroid plexus, active transport and catabolism. Neither process, however, can account for the birth-related change in CSF PGE2.