Preoptic nitric oxide attenuates endotoxic fever in guinea pigs by inhibiting the POA release of norepinephrine

Lipopolysaccharide (LPS) administration induces hypothalamic nitric oxide (NO); NO is antipyretic in the preoptic area (POA), but its mechanism of action is uncertain. LPS also stimulates the release of preoptic norepinephrine (NE), which mediates fever onset. Because NE upregulates NO synthases and NO induces cyclooxygenase (COX)-2-dependent PGE(2), we investigated whether NO mediates the production of this central fever mediator. Conscious guinea pigs with intra-POA microdialysis probes received LPS intravenously (2 mug/kg) and, thereafter, an NO donor (SIN-1) or scavenger (carboxy-PTIO) intra-POA (20 mug/mul each, 2 mul/min, 6 h). Core temperature (T(c)) was monitored constantly; dialysate NE and PGE(2) were analyzed in 30-min collections. To verify the reported involvement of alpha(2)-adrenoceptors (AR) in PGE(2) production, clonidine (alpha(2)-AR agonist, 2 mug/mul) was microdialyzed with and without SIN-1 or carboxy-PTIO. To assess the possible involvement of oxidative NE and/or NO products in the demonstrated initially COX-2-independent POA PGE(2) increase, (+)-catechin (an antioxidant, 3 mug/mul) was microdialyzed, and POA PGE(2), and T(c) were determined. SIN-1 and carboxy-PTIO reduced and enhanced, respectively, the rises in NE, PGE(2), and T(c) produced by intravenous LPS. Similarly, they prevented and increased, respectively, the delayed elevations of PGE(2) and T(c) induced by intra-POA clonidine. (+)-Catechin prevented the LPS-induced elevation of PGE(2), but not of T(c). We conclude that the antipyretic activity of NO derives from its inhibitory modulation of the LPS-induced release of POA NE. These data also implicate free radicals in POA PGE(2) production and raise questions about its role as a central LPS fever mediator.     

Preoptic norepinephrine mediates the febrile response of guinea pigs to lipopolysaccharide

Norepinephrine (NE) microdialyzed in the preoptic area (POA) raises core temperature (T(c)) via 1) alpha(1)-adrenoceptors (AR), quickly and independently of POA PGE(2), and 2) alpha(2)-AR, after a delay and PGE(2) dependently. Since systemic lipopolysaccharide (LPS) activates the central noradrenergic system, we investigated whether preoptic NE mediates LPS fever. We injected LPS (2 microg/kg iv) in guinea pigs prepared with intra-POA microdialysis probes and determined POA cerebrospinal (CSF) NE levels. We similarly microdialyzed prazosin (alpha(1) blocker, 1 microg/microl), yohimbine (alpha(2) blocker, 1 microg/microl), SC-560 [cyclooxygenase (COX)-1 blocker, 5 microg/microl], acetaminophen (presumptive COX-1v blocker, 5 microg/microl), or MK-0663 (COX-2 blocker, 0.5 microg/microl) in other animals before intravenous LPS and measured CSF PGE(2). All of the agents were perfused at 2 microg/min for 6 h. T(c) was monitored constantly. POA NE peaked within 30 min after LPS and then returned to baseline over the next 90 min. T(c) increased within 12 min to a first peak at approximately 60 min and to a second at approximately 150 min and then declined over the following 2.5 h. POA PGE(2) followed a concurrent course. Prazosin pretreatment eliminated the first T(c) rise but not the second; PGE(2) rose normally. Yohimbine pretreatment did not affect the first T(c) rise, which continued unchanged for 6 h; the second rise, however, was absent, and PGE(2) levels did not increase. SC-560 and acetaminophen did not alter the LPS-induced PGE(2) and T(c) rises; MK-0663 prevented both the late PGE(2) and T(c) rises. These results confirm that POA NE is pivotal in the development of LPS fever.     

Cytokines, PGE2 and endotoxic fever: a re-assessment

The innate immune system serves as the first line of host defense against the deleterious effects of invading infectious pathogens. Fever is the hallmark among the defense mechanisms evoked by the entry into the body of such pathogens. The conventional view of the steps that lead to fever production is that they begin with the biosynthesis of pyrogenic cytokines by mononuclear phagocytes stimulated by the pathogens, their release into the circulation and transport to the thermoregulatory center in the preoptic area (POA) of the anterior hypothalamus, and their induction there of cyclooxygenase (COX)-2-dependent prostaglandin (PG)E(2), the putative final mediator of the febrile response. But data accumulated over the past 5 years have gradually challenged this classical concept, due mostly to the temporal incompatibility of the newer findings with this concatenation of events. Thus, the former studies generally overlooked that the production of cytokines and the transduction of their pyrogenic signals into fever-mediating PGE(2) proceed at relatively slow rates, significantly slower certainly than the onset latency of fever produced by the i.v. injection of bacterial endotoxic lipopolysaccharides (LPS). Here, we review the conflicts between the earlier and the more recent findings and summarize new data that reconcile many of the contradictions. A unified model based on these data explicating the generation and maintenance of the febrile response is presented. It postulates that the steps in the production of LPS fever occur in the following sequence: the immediate activation by LPS of the complement (C) cascade, the stimulation by the anaphylatoxic C component C5a of Kupffer cells, their consequent, virtually instantaneous release of PGE(2), its excitation of hepatic vagal afferents, their transmission of the induced signals to the POA via the ventral noradrenergic bundle, and the activation by the thus, locally released norepinephrine (NE) of neural alpha(1)- and glial alpha(2)-adrenoceptors. The activation of the first causes an immediate, PGE(2)-independent rise in core temperature (T(c)) [the early phase of fever; an antioxidant-sensitive PGE(2) rise, however, accompanies this first phase], and of the second a delayed, PGE(2)-dependent T(c) rise [the late phase of fever]. Meanwhile-generated pyrogenic cytokines and their consequent upregulation of blood-brain barrier cells COX-2 also contribute to the latter rise. The consecutive steps that initiate the febrile response to LPS would now appear, therefore, to occur in an order different than conceived originally.     

Possible sequence of pyrogenic afferent processing in the POA

(1) It is generally considered that fever is modulated in the preoptic-anterior hypothalamic area (POA) in response to signaling by pyrogenic cytokines elaborated in the periphery by mononuclear phagocytes and the consequent induction of prostaglandin (PG)E₂ in the POA. The mechanism of the centripetal transmission of this pyrogenic signal, however, is controversial. One hypothesis suggests that it is conveyed via the vagus to the nucleus tractus solitarius and from there to the POA via the ventral noradrenergic bundle, causing the intraPOA release of norepinephrine (NE) which then stimulates the production of PGE₂. (2) In this article, we review recent data from our laboratory showing that NE microdialyzed into the POA of conscious guinea pigs or injected intracerebroventricularly into conscious mice indeed evokes two distinct core temperature (T_c) rises, viz., one ₁-adrenoceptor (AR)-mediated, rapid in onset and PGE₂-independent, and the other ₂-AR-mediated, delayed and COX-2/PGE₂-dependent. (3) We further present new data suggesting that the febrile response of conscious guinea pigs to intraperitoneally injected lipopolysaccharide (LPS) is mediated by intraPOA NE in accord with the above sequence, i.e., via ₁-AR to initiate the first, PGE₂-independent elevation of T_c, and via ₂-AR to induce the delayed production of COX-2-dependent PGE₂ and the continued rise of T_c. (4) These results thus validate the presumptive involvement of NE in LPS fever induction in guinea pigs.     

LPS-activated complement, not LPS per se, triggers the early release of PGE2 by Kupffer cells

The intravenous injection of LPS rapidly evokes fever. We have hypothesized that its onset is mediated by prostaglandin (PG)E(2) quickly released by Kupffer cells (Kc). LPS, however, does not stimulate PGE(2) production by Kc as rapidly as it induces fever; but complement (C) activated by LPS could be the exciting agent. To test this hypothesis, we injected LPS (2 or 8 microg/kg) or cobra venom factor (CVF, an immediate activator of the C cascade that depletes its substrate, ultimately causing hypocomplementemia; 25 U/animal) into the portal vein of anesthetized guinea pigs and measured the appearance of PGE(2), TNF-alpha, IL-1beta, and IL-6 in the inferior vena cava (IVC) over the following 60 min. LPS (at both doses) and CVF induced similar rises in PGE(2) within the first 5 min after treatment; the rises in PGE(2) due to CVF returned to control in 15 min, whereas PGE(2) rises due to LPS increased further, then stabilized. LPS given 3 h after CVF to the same animals also elevated PGE(2), but after a 30- to 45-min delay. CVF per se did not alter basal PGE(2) and cytokine levels and their responses to LPS. These in vivo effects were substantiated by the in vitro responses of primary Kc from guinea pigs to C (0.116 U/ml) and LPS (200 ng/ml). These results indicate that LPS-activated C rather than LPS itself triggers the early release of PGE(2) by Kc.     

Kupffer cell-generated PGE2 triggers the febrile response of guinea pigs to intravenously injected LPS

Because the onset of fever induced by intravenously (i.v.) injected bacterial endotoxic lipopolysaccharides (LPS) precedes the appearance in the bloodstream of pyrogenic cytokines, the presumptive peripheral triggers of the febrile response, we have postulated previously that, in their stead, PGE2 could be the peripheral fever trigger because it appears in blood coincidentally with the initial body core temperature (Tc) rise. To test this hypothesis, we injected Salmonella enteritidis LPS (2 microg/kg body wt i.v.) into conscious guinea pigs and measured their plasma levels of LPS, PGE2, TNF-alpha, IL-1beta, and IL-6 before and 15, 30, 60, 90, and 120 min after LPS administration; Tc was monitored continuously. The animals were untreated or Kupffer cell (KC) depleted; the essential involvement of KCs in LPS fever was shown previously. LPS very promptly (<10 min) induced a rise of Tc that was temporally correlated with the elevation of plasma PGE2. KC depletion prevented the Tc and plasma PGE2 rises and slowed the clearance of LPS from the blood. TNF-alpha was not detectable in plasma until 30 min and in IL-1beta and IL-6 until 60 min after LPS injection. KC depletion did not alter the times of appearance or magnitudes of rises of these cytokines, except TNF-alpha, the maximal level of which was increased approximately twofold in the KC-depleted animals. In a follow-up experiment, PGE2 antiserum administered i.v. 10 min before LPS significantly attenuated the febrile response to LPS. Together, these results support the view that, in guinea pigs, PGE2 rather than pyrogenic cytokines is generated by KCs in immediate response to i.v. LPS and triggers the febrile response.     

Different response of ANP secretion to adrenoceptor stimulation in renal hypertensive rat atria

Sympathetic nervous system and atrial natriuretic peptide (ANP) system play fundamental roles in the regulation of cardiovascular functions. Overactivity of sympathetic nervous system can lead into cardiovascular diseases such as heart failure and hypertension. The present study aimed to define which adrenergic receptors (ARs) affect atrial contractility and ANP release and to determine their modification in renal hypertensive rat atria. An alpha(1)-AR agonist, cirazoline increased ANP release with positive inotropism. These alpha(1)-AR agonist-mediated responses were attenuated by the alpha(1A)-AR antagonist, but not by the alpha(1B)- or alpha(1D)-AR antagonist. An alpha(2)-AR agonist, guanabenz and clonidine increased ANP release with negative inotropism and decreased cAMP level. The order of potency for the increased ANP release was cirazoline>phenylephrine=guanabenz>clonidine. In contrast, a beta-AR agonist, isoproterenol decreased ANP release with positive inotropism and these responses were blocked by the beta(1)-AR antagonist but not by the beta(2)-AR antagonist. The increased cAMP level by isoproterenol was suppressed by pretreatment with both beta(1)- and beta(2)-AR antagonists. In renal hypertensive rat atria, the effects of isoproterenol on atrial contractility, ANP release, and cAMP level were attenuated whereas the effect of cirazoline on ANP release was unaltered. Atrial beta(1)-AR mRNA level but not alpha(1A)-AR mRNA level was decreased in renal hypertensive rats. These findings suggest that alpha(1A)- and beta(1)-AR oppositely regulate atrial ANP release and that atrial beta(1)-AR expression/function is impaired in renal hypertensive rats.     

Carrageenan-induced paw edema in rat elicits a predominant prostaglandin E2 (PGE2) response in the central nervous system associated with the induction of microsomal PGE2 synthase-1

Peripheral inflammation involves an increase in cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) synthesis in the central nervous system (CNS), which contributes to allodynia and hyperalgesia. In the present study we have determined the changes in prostanoid tissue levels and in expression of terminal prostanoid synthases in both the CNS and inflamed peripheral tissue during carrageenan-induced paw inflammation in the rat. Prostanoid levels were measured by liquid chromatography-mass spectrometry and enzyme expression at the RNA level by quantitative PCR analysis during both the early (1-6 h) and late (12 and 24 h) phases of the inflammatory response. In the paw, the early phase was associated with increases in PGE(2) and thromboxane (TX)B(2) levels and with a peak of COX-2 expression that preceded that of microsomal prostaglandin-E(2) synthase-1 (mPGES-1). COX-2 and mPGES-1 remained elevated during the late phase, and PGE(2) continued to further increase through 24 h. The cytosolic PGE(2) synthase (cPGES) showed a small transient increase during the early phase, whereas mPGES-2 expression was not affected by inflammation. In the cerebrospinal fluid, elevated levels of PGE(2), 6-keto-PGF(1alpha), PGD(2), and TXB(2) were detected during the early phase. PGE(2) levels also increased in the spinal cord and, to a lesser extent, in the brain and remained elevated in both the cerebrospinal fluid and the spinal cord during the late phase. The expression of mPGES-1 was strongly up-regulated in the brain and spinal cord during inflammation, whereas no change was detected for the expression of cPGES, mPGES-2, COX-1, and terminal PGD, TX, or PGI synthases. The results show that the carrageenan-induced edema in the paw elicits an early phase of COX-2 induction in the CNS leading to an increase synthesis in PGD(2), 6-keto-PGF(1alpha), and TXB(2) in addition to the major PGE(2) response. The data also indicate that the up-regulation of mPGES-1 contributes to COX-2-mediated PGE(2) production in the CNS during peripheral inflammation.     

Bile duct ligation induced acute inflammation up-regulates cyclooxygenase-2 content and PGE2 release in guinea pig gallbladder smooth muscle cell cultures

INTRODUCTION: This study examines hypotheses that BDL induces increased guinea pig gallbladder smooth muscle PGE2 release by up-regulation of COX-2. METHODS: BDL, Sham and Control Hartley guinea pig gallbladders were placed in cell culture, grown to confluence and underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacylin Synthase, actin, caldesmon, vinculin, meta-vinculin and tropomyosin and were assayed for basal release of 6-keto-PGF(1alpha), PGE2 and TxB2 by EIA. RESULTS: BDL did not alter content of smooth muscle cytoskeletal proteins. BDL for 48 h increased smooth muscle cell release of PGE2 and 6-keto-PGF(1alpha) by 3-fold or more when compared to the Control and Sham groups. Western Blot analysis showed increased content of COX-2 in the BDL group. CONCLUSIONS: BDL for 48 h markedly increased endogenous guinea pig smooth muscle cell PG release, which was due to increased COX-2 synthesis.     

Pre- and postjunctional alpha(2)-adrenergic receptors in fetal and adult ovine cerebral arteries

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered alpha(2)-adrenergic receptor (alpha(2)-AR) density and/or affinity. In fetal (approximately 140 days) and adult sheep, we measured alpha(2)-AR density and affinity with the antagonist [(3)H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the alpha(2)-AR antagonists yohimbine and idazoxan and contractile responses to the alpha(2)-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, alpha(2)-AR density was 201 +/- 18 and 52 +/- 6 fmol/mg protein, respectively (P < 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, 10(-7) M yohimbine significantly decreased the pD(2) for NE-induced tension in the presence of 3 x 10(-5) M cocaine, 10(-5) M deoxycorticosterone, and 10(-6) M tetrodotoxin. In fetal, but not adult, MCA, UK-14304 induced a significant decrease in pD(2) for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of 10(-6) M idazoxan to block alpha(2)-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, alpha(2)-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional alpha(2)-AR.     

Clonidine Inhibition of Norepinephrine Release from Normal and Morphine-Tolerant Guinea Pig Cortical Slices

Endogenous norepinephrine (NE) release in cerebral cortex slices taken from normal and morphine-tolerant guinea pigs was measured by HPLC. In normal slices, a linear relationship was found between electrically evoked NE release and the log of the frequency of stimulation in the range of 1-20 Hz. The efficiency of the alpha 2-mediated autofeedback was tested by adding the alpha 2-agonist clonidine and the alpha 2 agonist idazoxan. NE release was dose-dependently reduced by clonidine (1 nmol/L-1 mumol/L) and increased by idazoxan (10-100 nmol/L). The inhibition by clonidine was significantly greater at 1 Hz than at 3 Hz, whereas the absolute increase in NE release induced by idazoxan was greater at 3 Hz than at 1 Hz. Morphine at 1 mumol/L (a concentration per se ineffective) shifted to the left the clonidine concentrations able to inhibit NE release at 3 and 1 Hz (1-10 nmol/L), but at both frequencies, the opiate reduced the maximal inhibition induced by clonidine at 1 mumol/L. In slices taken from morphine-tolerant guinea pigs (in the presence of morphine at 1 mumol/L), clonidine (1 nmol/L-1 mumol/L) was ineffective at the stimulation rate of 3 Hz, but it was more active than in normal slices at 1 Hz. Such a response pattern suggests a reduced availability of alpha 2 receptors and an increase in their sensitivity to clonidine. However, chronic morphine treatment did not influence the physiological autoinhibition because the increase in NE release elicited by idazoxan (10-100 nmol/L) at 1 and 3 Hz was the same in normal and in "morphine-tolerant" slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

We have previously shown that the cyclooxygenase (COX)-2/PGE2 pathway plays a key role in VEGF production in gastric fibroblasts. Recent studies have identified three PGE synthase (PGES) isozymes: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1 and -2, but little is known regarding the expression and roles of these enzymes in gastric fibroblasts. Thus we examined IL-1beta-stimulated mPGES-1 and cPGES mRNA and protein expression in gastric fibroblasts by quantitative PCR and Western blot analysis, respectively, and studied both their relationship to COX-1 and -2 and their roles in PGE2 and VEGF production in vitro. IL-1beta stimulated increases in both COX-2 and mPGES-1 mRNA and protein expression levels. However, COX-2 mRNA and protein expression were more rapidly induced than mPGES-1 mRNA and protein expression. Furthermore, MK-886, a nonselective mPGES-1 inhibitor, failed to inhibit IL-1beta-induced PGE2 release at the 8-h time point, while totally inhibiting PGE2 at the later stage. However, MK-886 did inhibit IL-1beta-stimulated PGES activity in vitro by 86.8%. N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398), a selective COX-2 inhibitor, totally inhibited PGE2 production at both the 8-h and 24-h time points, suggesting that COX-2-dependent PGE2 generation does not depend on mPGES-1 activity at the early stage. In contrast, NS-398 did not inhibit VEGF production at 8 h, and only partially at 24 h, whereas MK-886 totally inhibited VEGF production at each time point. These results suggest that IL-1beta-induced mPGES-1 protein expression preferentially coupled with COX-2 protein at late stages of PGE2 production and that IL-1beta-stimulated VEGF production was totally dependent on membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily proteins, which includes mPGES-1, but was partially dependent on the COX-2/PGE2 pathway.     

Presynaptic alpha 2 -adrenergic stimulation leads to growth hormone release in the dog

In previous studies we have shown that the alpha 2 -adrenergic receptor agonist clonidine (CLON) releases growth hormone (GH) in conscious dogs, an effect abolished by the selective alpha 2-receptor antagonist yohimbine (YOH) and by reserpine, but not by the alpha 1-receptor antagonist prazosin (1). In the present work intravenous (iv) administration of CLON in conscious dogs evoked a dose-related rise in plasma GH at doses of 2-8 /micrograms/Kg, but not at 16 and 32 /micrograms/Kg. Acute pretreatment with the selective inhibitor of norepinephrine (NE) synthesis, DU-18288, or with a potent antagonist of presynaptic alpha 2-receptors, mianserin abolished the GH rise induced by CLON (4 /micrograms/Kg iv). In contrast, a 10-day-pretreatment with YOH greatly enhanced the GH-releasing effect of CLON (2 /micrograms/Kg iv). In all these data indicate that in the dog: 1) CLON induces GH release via activation of alpha 2-adrenergic receptors; 2) these receptors are likely located on presynaptic sites [experiments with reserpine (1), DU-18288, mianserin, dose-response curve with CLON 2-32/micrograms/kg iv]; 3) the adrenergic receptors involved in GH release exhibit supersensitivity upon (YOH-induced) chronic pharmacologic denervation. In view of the inhibitory action of presynaptic alpha 2-adrenergic receptors (autoreceptors) on NE function, it may be envisioned that in the dog noradrenergic activation is inhibitory and not stimulatory to GH release.     

ChTX induces oscillatory contraction in guinea pig trachea: role of cyclooxygenase-2 and PGE2

We examined the possible role of cyclooxygenase (COX) in charybdotoxin (ChTX)-induced oscillatory contraction in guinea pig trachea. Involvement of prostaglandin E(2) (PGE(2)) in ChTX-induced oscillatory contraction was also investigated. ChTX (100 nM) induced oscillatory contraction in guinea pig trachea. The mean oscillatory frequency induced by ChTX was 10.7 +/- 0.8 counts/h. Maximum and minimum tensions within ChTX-induced oscillatory contractions were 68.4 +/- 1.8 and 14.3 +/- 1.7% compared with K(+) (72.7 mM) contractions. ChTX-induced oscillatory contraction was completely inhibited by indomethacin, a nonselective COX inhibitor. Valeryl salicylate, a selective COX-1 inhibitor, did not significantly inhibit this contraction, whereas N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide, a selective COX-2 inhibitor, abolished this contraction. Exogenously applied arachidonic acid enhanced ChTX-induced oscillatory contraction. SC-51322, a selective PGE receptor subtype EP(1) antagonist, significantly inhibited ChTX-induced oscillatory contraction. Exogenously applied PGE(2) induced only a slight phasic contraction in guinea pig trachea, but PGE(2) induced strong oscillatory contraction after pretreatment with indomethacin and ChTX. Moreover, ChTX time-dependently stimulated PGE(2) generation. These results suggest that ChTX specifically activates COX-2 and stimulates PGE(2) production and that ChTX-induced oscillatory contraction in guinea pig trachea is mediated by activation of EP(1) receptor.     

Inhibited skeletal muscle healing in cyclooxygenase-2 gene-deficient mice: the role of PGE2 and PGF2alpha.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat skeletal muscle injury. However, studies have shown that NSAIDs may be detrimental to the healing process. Mediated by prostaglandin F(2alpha) (PGF(2alpha)) and prostaglandin E(2) (PGE(2)), the cycloxygenase-2 (COX-2) pathway plays an important role in muscle healing. We hypothesize that the COX-2 pathway is important for the fusion of muscle cells and the regeneration of injured muscle. For the in vitro experiments, we isolated myogenic precursor cells from wild-type (Wt) and COX-2 gene-deficient (COX-2(-/-)) mice and examined the effect of PGE(2) and PGF(2alpha) on cell fusion. For the in vivo experiments, we created laceration injury on the tibialis anterior (TA) muscles of Wt and COX-2(-/-) mice. Five and 14 days after injury, we examined the TA muscles histologically and functionally. We found that the secondary fusion between nascent myotubes and myogenic precursor cells isolated from COX-2(-/-) mice was severely compromised compared with that of Wt controls but was restored by the addition of PGF(2alpha) or, to a lesser extent, PGE(2) to the culture. Histological and functional assessments of the TA muscles in COX-2(-/-) mice revealed decreased regeneration relative to that observed in the Wt mice. The findings reported here demonstrate that the COX-2 pathway plays an important role in muscle healing and that prostaglandins are key mediators of the COX-2 pathway. It suggests that the decision to use NSAIDs to treat muscle injuries warrants critical evaluation because NSAIDs might impair muscle healing by inhibiting the fusion of myogenic precursor cells.     

Hormonal regulation of PGE2 and COX-2 production in rabbit uterine cervical fibroblasts.

Prostaglandins (PGs) cause uterine contraction to initiate labor at term. We investigated the effect of progesterone and 17beta-estradiol on the production of PGE2 in rabbit uterine cervical fibroblasts. When the cervical fibroblasts were treated with interleukin-1alpha (IL-1alpha), the level of PGE2 was augmented in a time- and dose-dependent manner. The IL-1alpha-augmented PGE2 level was almost completely suppressed by progesterone and 17beta-estradiol at the physiological concentration (0.01 microM), whereas a slight decrease in the basal level of PGE2 was observed in the cervical fibroblasts treated with both hormones at a pharmacological concentration (1 microM). In addition, the level of PGE2 augmented by IL-1alpha was due to the increase of cyclooxygenase (COX) activity, which was inhibited by progesterone and 17beta-estradiol as well as by indomethacin and a specific COX-2 inhibitor, NS-398, but not by the well-known COX-1 inhibitor, aspirin. Furthermore, progesterone and 17beta-estradiol suppressed the IL-1alpha-augmented COX-2 production but not the constitutive production of COX-1 in rabbit uterine cervical fibroblasts. These results suggest that progesterone and 17beta-estradiol prevent the initiation of labor by inhibiting PGE2 production after the suppression of COX-2 production during pregnancy in the rabbit.     

Expression of enzymes of cyclooxygenase pathway and secretion of prostaglandin E2 and F2alpha by porcine myometrium during luteolysis and early pregnancy

Past studies of uterine prostaglandin (PGs) and pig reproduction have focused on endometrial rather than myometrial PGs. This study documents the synthesis and secretion of myometrial prostaglandins (PGs) in pigs and the involvement of oxytocin (OT) in these processes. Cyclooxygenase-2 (COX-2) expression was similar in myometrial explants from cyclic and pregnant pigs (days 14-16) and OT (10(-7) M) in vitro significantly increased COX-2 protein regardless of reproductive state. Basal expression of prostaglandin E2 synthase (PGES) was higher during pregnancy than during luteolysis. Conversely, prostaglandin F synthase (PGFS) was highest during luteolysis and lower in myometrium from gravid animals. OT had no influence on the expression of PGES and PGFS. In another tissue culture experiment, myometrial slices produced more PGE2 than PGF2alpha regardless of reproductive state of the female. OT stimulated PGE2 production in myometrium harvested during luteolysis and increased PGF2alpha production in all tissues examined. Progesterone (P4; 10(-5) M) blocked stimulatory effect of OT on myometrial PG release. Myometrial OTr mRNA was higher (P=0.03) during luteolysis than during pregnancy. In conclusion: (1) oxytocin increases myometrial COX-2 expression, but does not influence the expression of terminal enzymes of PGs synthesis (PGES and PGFS); (2) porcine myometrium preferentially produces PGs during early pregnancy and secretes more PGE2 than PGF2alpha; (3) myometrial OT and OTr support secretion of PGs from myometrium during luteolysis.     

The effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on chorioamnion secretion of prostaglandins (PG)F 2 alpha and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.