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.

Role of cyclooxygenases 1 and 2 in the modulation of neuromuscular functions in the distal colon of humans and mice.

BACKGROUND: Cyclooxygenase isoforms (COX-1, COX-2) may exert differential regulatory actions on enteric motor functions under normal or pathological conditions. AIMS: To examine the occurrence and functions of COX-1 and COX-2 in the neuromuscular compartment of normal distal colon using human and murine tissue. METHODS: Gene expression (human, mouse), protein expression (human), gene deletion (mouse), and the effects of dual and isoform specific COX inhibitors on in vitro motility (human, mouse) were investigated. RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) showed mRNA expression of COX-1 and COX-2 in human and wild-type mouse colonic muscle whereas only COX-2 or COX-1 was detected in COX-1 or COX-2 knockout animals. Immunohistochemistry localised both isoforms in neurones of myenteric ganglia, COX-1 in circular layer myocytes, and COX-2 in longitudinal muscle. Indomethacin (COX-1/COX-2 inhibitor), SC-560 (COX-1 inhibitor), or DFU (COX-2 inhibitor) enhanced atropine sensitive electrically induced contractions of human longitudinal muscle. The most prominent actions were recorded with indomethacin or SC-560 plus DFU. These results were confirmed under pharmacological blockade of non-cholinergic nerves. Atropine sensitive contractions evoked by carbachol in the presence of tetrodotoxin were enhanced by indomethacin or DFU but not by SC-560. In wild-type mice, contractile responses to electrical stimulation were enhanced by indomethacin, SC-560, or DFU. SC-560 potentiated electrically induced contractions in COX-2, but not COX-1, knockout mice. In contrast, DFU enhanced the contractions elicited by electrical stimuli in COX-1, but not in COX-2, knockout mice. CONCLUSIONS: These results indicate that COX-1 and COX-2 are expressed in the neuromuscular compartment of normal human colon where they modulate cholinergic excitatory control of colonic motility at prejunctional and postjunctional sites, respectively.

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.

Effect of the oxidative stress induced by adriamycin on rat hepatocyte bioenergetics during ageing.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on mitochondrial functions. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor's age, whilst the hydrogen peroxide is significantly higher in aged than in adult rat hepatocytes. Kinetic studies in isolated mitochondria show that the mitochondrial respiratory chain activity (NADH --> O2) of 50 microM adriamycin-treated hepatocytes is lowered both in adult and aged rats. The same adriamycin concentration induces a slight decrease of the maximal rate of ATP hydrolysis in both young and aged rats, without affecting the Km for the substrate. However, at drug concentrations lower than 50 microM, both ATPase and NADH oxidation activities decrease significantly in aged rats only. The results suggest that free radicals increase during ageing in rat hepatocytes but are unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that some effects of the drug may be due to other reasons besides oxidative stress.

Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a.

We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of succinate-sustained ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the succinate-sustained ATP synthesis rate of the particles was found, but both the ATP hydrolysis rate and ATP-driven proton translocation (when the protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human F(1)F(0)-ATPase that causes a severe impairment of ATP synthesis. This is possibly due to a defect in either the vectorial proton transport from the cytosol to the mitochondrial matrix or the coupling of proton flow through F(0) to ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired ATP synthesis. On the other hand, ATP hydrolysis that involves proton flow from the matrix to the cytosol is essentially unaffected.

The effect of aging and an oxidative stress on peroxide levels and the mitochondrial membrane potential in isolated rat hepatocytes.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on the mitochondrial membrane potential. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor age. The hydrogen peroxide is significantly increased in both aged and adult rat hepatocytes, however, due to the high basal level in the aged cells, it is higher in aged rat cells not subjected to oxidative stress than that elicited by 50 microM adriamycin in young rat hepatocytes. The results suggest that a hydrogen peroxide increase in hepatocytes of aged rats is unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that the effects of the drug may be due to the concomitant high level of both superoxide and hydrogen peroxide.

Capsaicin-responsive NADH oxidase activities from urine of cancer patients.

NADH oxidases of low specific activities from urine of cancer patients were found to be inhibited or stimulated by the vanilloid capsaicin (8-methyl-N-vanillyl-6-noneamide). Similar activities, inhibited or stimulated by capsaicin, were reported previously for sera of cancer patients but not for sera of normal volunteers or for patients with disorders other than cancer. Like those from sera, the activities from urine were resistant to heat and to digestion with proteinase K. Two different fractions with capsaicin-responsive NADH oxidase activities were obtained by FPLC. One fraction in which the 33-kDa band was the major component exhibited NADH oxidase activity stimulated by capsaicin. Another fraction in which 66-kDa and 45-kDa bands were major components exhibited NADH oxidase activities inhibited by capsaicin. A monoclonal antibody generated to a ca 34-kDa form of the NADH oxidase from sera reacted with a urine protein of a ca 33-kDa band in the capsaicin-stimulated fraction. The 33-kDa protein was of low abundance and was estimated to be present in amounts between 5 and 100 microgram/L, depending on the particular patient.

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing.

ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slightly decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF1. It is concluded that no activity changes to be directly ascribed to the catalytic sector F1 of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F0 occur as a consequence of mtDNA mutations.

Conformational changes of the mitochondrial F1-ATPase epsilon-subunit induced by nucleotide binding as observed by phosphorescence spectroscopy.

Changes in conformation of the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg2+ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the epsilon-subunit are evident at high ligand concentration added to both the nucleotide-depleted F1 (Nd-F1) and the F1 preparation containing the three tightly bound nucleotides (F1(2,1)). Since the effects observed are similar in both the F1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the epsilon-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F1. The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F1(3,0)). Phosphorescence lifetime measurements on this F1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the epsilon-subunit. This implies that the binding to the vacant noncatalytic site of F1(2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F1 and F1(2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F1(2,1) in the absence of Mg2+ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the epsilon-subunit is discussed.

A study of the mitochondrial F1-ATPase tryptophan phosphorescence at 273 K.

The bovine heart mitochondrial F1-ATPase complex exhibits an intrinsic tryptophan phosphorescence that can be used to monitor structural changes of the epsilon-subunit. The phosphorescence decay rate of F1 containing the tightly bound nucleotides increases upon addition of adenine nucleoside triphosphate in the presence of magnesium. The average phosphorescence lifetime of this enzyme preparation decreases from 10.2 to 7.8 ms upon Mg-ATP addition. Since increasing phosphorescence decay rate is related to increasing flexibility of proteins, Mg-ATP added to the F1-ATPase complex can enhance the flexibility of the protein structure surrounding the chromophore. Experiments carried out on F1 prepared with the three noncatalytic sites filled and the three catalytic sites vacant show a significant increase of the phosphorescence lifetime from 6.4 ms to 7.6 ms upon Mg-ATP addition. These results suggest that the mitochondrial F1-ATPase epsilon-subunit conformation senses differently the nucleoside triphosphate binding to catalytic or noncatalytic sites.

Interactions and effects of 2-hydroxy-5-nitrobenzyl bromide on the bovine heart mitochondrial F1-ATPase.

1. The F1-ATPase from bovine heart mitochondria was shown to chemically react and to absorb 2-hydroxy-5-nitrobenzyl bromide (HNB) with changes in catalytic properties. 2. The treatment of the enzyme with HNB at concentrations below 0.5 mM resulted in an increase of Vm and in an unchanged Km. Above 0.5 mM HNB elicited a concentration-dependent inhibition of F1. 3. HNB was found tightly bound to the enzyme epsilon-subunit whose tryptophan residue resulted modified. 4. The F1 activation appears the consequence of the covalent binding of the reagent to the enzyme, whilst inhibition results from non-covalent, reversible binding. 5. The possibility that the epsilon-subunit of mitochondrial F1-ATPase may influence the functional or regulating domain of the enzyme is discussed.