No abstract

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.

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.

Ductus arteriosus (DA) closure is initiated by oxygen rise postnatally and progresses in two, functional-to-permanent, stages. Here, using GeneChip Arrays in rats (normoxic and hyperoxic fetus, normoxic newborn), we examined whether oxygen alone duplicates the birth process in affecting DA genes. In addition, by comparing DA with aorta (Ao), we identified features in postnatal gene profile marking transitional adjustments in a closing (DA) vs. a persistent (Ao) vessel. We found changes in neonatal DA denoting enhanced formation and action of the constrictor endothelin-1 (ET-1). Likewise, ANG II type 1 receptor was upregulated, and the compound was a constrictor. Conversely, relaxant PGE2 became less effective. Among agents for functional closure, only ET-1 was affected similarly by oxygen and birth. Coincidentally, neonatal DA showed enhanced contractile drive with upregulation of Rho-Rho kinase and calcium signaling along with downregulation of contractile proteins. The latter effect was shared by oxygen. Changes denoting active remodeling were also seen in neonatal but not hyperoxic fetal DA. Ao, unlike DA, exhibited postnatal variations in noradrenergic, purinergic, and PGI2 systems with opposing effects on vasomotion. Contraction and remodeling processes were also less affected by birth, whereas lipid and glucose metabolism were upregulated. We conclude that several agents, including ANG II as novel effector, promote functional closure of DA, but only ET-1 is causally coupled with oxygen. Oxygen has no role in processes for permanent closure. Functional closure is associated with downregulation of contractile apparatus, and this may render neonatal DA less amenable to tone manipulation. Conceivably, activation of metabolism in neonatal Ao is a distinguishing feature for transitional adaptations in the permanent vasculature.

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 nearterm 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 COX1À/À mice contracted promptly to indomethacin (2.8 mm). Conversely, the contraction of COX2À/À ductus to the same inhibitor started only after a delay and was slower. 4 N G -nitro-l-arginine methyl ester (l-NAME, 100 mm) 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.

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 eNOSÀ/À or iNOSÀ/À. BK relaxation was suppressed by either L-NAME or ZnPP. However, it reappeared with combined L-NAME and ZnPP to subside again with K þ increase or K þ 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 BKinduced 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.

This study indicates a close relationship between tissue heteroplasmy, expression of the biochemical defect in platelets, and clinical involvement. The biochemical defect was greater than previously reported, and we found no evidence of a biochemical threshold. The uniform distribution of high mutant loads among our patients' tissues suggests a differential tissue-specific reliance on mitochondrial ATP synthesis.

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 eNOSÀ/À, 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 À1 ). 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 eNOSÀ/À 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.