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.