Genotoxicity of tetrahydrofolic acid to hematopoietic stem and progenitor cells.

Metabolically reactive formaldehyde is a genotoxin and a carcinogen. Mice lacking the main formaldehyde-detoxifying gene Adh5 combined with the loss of the Fanconi anemia (FA) DNA repair pathway rapidly succumbed to bone marrow failure (BMF) primarily due to the extensive ablation of the hematopoietic stem cell (HSC) pool. However, the mechanism by which formaldehyde mediates these toxic effects is still unknown. We uncover a detrimental role of tetrahydrofolic acid (THF) in cells lacking Adh5 or the FA repair pathway. We show that Adh5- or FA-deficient cells are hypersensitive to formaldehyde and to THF, presenting DNA damage and genome instability. THF cytotoxicity involved imbalance of the nucleotide pool by deregulation of the thymidylate synthase (TYMS) enzyme, which stalled replication forks. In mice, THF exposure had widespread effects on hematopoiesis, affecting the frequency and the viability of myeloid- and lymphoid-committed precursor cells. Moreover, the hematopoietic stem and progenitor cells (HSPC) showed genomic instability, reduced colony-forming capacity and increased frequency of cycling and apoptotic HSCs upon THF exposure. Overall, our data reveal that the physiological pool of THF and formaldehyde challenge the stability of the genome of HSPCs that might lead to blood disorders.

Sdhd ablation promotes thyroid tumorigenesis by inducing a stem-like phenotype.

Mutations in genes encoding enzymes in the tricarboxylic acid cycle (TCA, also known as the Krebs cycle) have been implicated as causative genetic lesions in a number of human cancers, including renal cell cancers, glioblastomas and pheochromocytomas. In recent studies, missense mutations in the succinate dehydrogenase (SDH) complex have also been proposed to cause differentiated thyroid cancer. In order to gain mechanistic insight into this process, we generated mice lacking the SDH subunit D (Sdhd) in the thyroid. We report that these mice develop enlarged thyroid glands with follicle hypercellularity and increased proliferation. In vitro, human thyroid cell lines with knockdown of SDHD exhibit an enhanced migratory capability, despite no change in proliferative capacity. Interestingly, these cells acquire stem-like features which are also observed in the mouse tumors. The stem-like characteristics are reversed by α-ketoglutarate, suggesting that SDH-associated tumorigenesis results from dedifferentiation driven by an imbalance in cellular metabolites of the TCA cycle. The results of this study reveal a metabolic vulnerability for potential future treatment of SDH-associated neoplasia.

Role of VHL, HIF1A and SDH on the expression of miR-210: Implications for tumoral pseudo-hypoxic fate.

The hypoxia-inducible factor 1α (HIF-1α) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1α knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHL-mutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1α in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1α-positive and HIF-1α-negative, tumor cell population. Thus, activation of HIF-1α is likely an early event in VHL-defective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1α/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.

Sensitivity of hematopoietic stem cells to mitochondrial dysfunction by SdhD gene deletion.

It is established that hematopoietic stem cells (HSC) in the hypoxic bone marrow have adapted their metabolism to oxygen-limiting conditions. This adaptation includes suppression of mitochondrial activity, induction of anerobic glycolysis, and activation of hypoxia-inducible transcription factor 1α (Hif1α)-dependent gene expression. During progression of hematopoiesis, a metabolic switch towards mitochondrial oxidative phosphorylation is observed, making this organelle essential for determining cell fate choice in bone marrow. However, given that HSC metabolism is essentially oxygen-independent, it is still unclear whether functional mitochondria are absolutely required for their survival. To assess the actual dependency of these undifferentiated cells on mitochondrial function, we have performed an analysis of the hematopoiesis in a mouse mutant, named SDHD-ESR, with inducible deletion of the mitochondrial protein-encoding SdhD gene. This gene encodes one of the subunits of the mitochondrial complex II (MCII). In this study, we demonstrate that, in contrast to what has been previously established, survival of HSC, and also myeloid and B-lymphoid progenitors, depends on proper mitochondrial activity. In addition, gene expression analysis of these hematopoietic lineages in SDHD-ESR mutants calls into question the proposed activation of Hif1α in response to MCII dysfunction.

Resistance of glia-like central and peripheral neural stem cells to genetically induced mitochondrial dysfunction--differential effects on neurogenesis.

Mitochondria play a central role in stem cell homeostasis. Reversible switching between aerobic and anaerobic metabolism is critical for stem cell quiescence, multipotency, and differentiation, as well as for cell reprogramming. However, the effect of mitochondrial dysfunction on neural stem cell (NSC) function is unstudied. We have generated an animal model with homozygous deletion of the succinate dehydrogenase subunit D gene restricted to cells of glial fibrillary acidic protein lineage (hGFAP-SDHD mouse). Genetic mitochondrial damage did not alter the generation, maintenance, or multipotency of glia-like central NSCs. However, differentiation to neurons and oligodendrocytes (but not to astrocytes) was impaired and, hence, hGFAP-SDHD mice showed extensive brain atrophy. Peripheral neuronal populations were normal in hGFAP-SDHD mice, thus highlighting their non-glial (non hGFAP(+)) lineage. An exception to this was the carotid body, an arterial chemoreceptor organ atrophied in hGFAP-SDHD mice. The carotid body contains glia-like adult stem cells, which, as for brain NSCs, are resistant to genetic mitochondrial damage.

Myelomatous plasma cells display an aberrant gene expression pattern similar to that observed in normal memory B cells.

Memory B cells (MBCs) remain in a quiescent state for years, expressing pro-survival and anti-apoptotic factors while repressing cell proliferation and activation genes. During their differentiation into plasma cells (PCs), their expression pattern is reversed, with a higher expression of genes related to cell proliferation and activation, and a lower expression of pro-survival genes. To determine whether myelomatous PCs (mPCs) share characteristics with normal PCs and MBCs and to identify genes involved in the pathophysiology of multiple myeloma (MM), we compared gene expression patterns in these three cell sub-types. We observed that mPCs had features intermediate between those of MBCs and normal PCs, and identified 3455 genes differentially expressed in mPCs relative to normal PCs but with a similar expression pattern to that in MBCs. Most of these genes are involved in cell death and survival, cell growth and proliferation and protein synthesis. According to our findings, mPCs have a gene expression pattern closer to a MBC than a PC with a high expression of genes involved in cell survival. These genes should be physiologically inactivated in the transit from MBC to PC, but remain overexpressed in mPCs and thus may play a role in the pathophysiology of the disease.

Overexpression of GYS1, MIF, and MYC is associated with adverse outcome and poor response to azacitidine in myelodysplastic syndromes and acute myeloid leukemia.

BACKGROUND: The prognosis of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) is very heterogeneous. PATIENTS AND METHODS: We analyzed the prognostic value of several genes in a cohort of 85 MDS and AML patients. RESULTS: Overexpression of glycogen synthase 1 and macrophage migration inhibitory factor genes had an adverse outcome in multivariate analysis (P = .003 and P < .001, respectively). Furthermore, the higher expression of myelocytomatosis oncogene was associated with a lower response to azacitidine (P = .03). CONCLUSION: In the current study we identified a specific gene expression profile as prognostic factors for response to azacitidine and survival in MDS and AML.

The CD27+ memory B cells display changes in the gene expression pattern in elderly individuals.

Memory B cells (MBCs) have a very long life-span as compared to naïve B cells (NBCs), remaining viable for years. It could predispose them to suffer misbalances in the gene expression pattern at the long term, which might be involved in the development of age-related B-cell disorders. In order to identify genes whose expression might change during life, we analyzed the gene expression patterns of CD27- NBCs versus CD27+ MBCs in young and old subjects. Using microarray assays we observed that the expression pattern of CD27- NBCs versus CD27+ MBCs is significantly different. Furthermore, in order to evaluate the age effect, we compared the gene expression pattern of young versus aged subjects in both cell populations. Interestingly, we did not find significant differences in the CD27- NBC population between young and aged individuals, whereas we found 925 genes differentially expressed in CD27+ MBCs. Among these genes, 193 were also differentially expressed in CD27+ MBCs as compared to CD27- NBCs, most of them involved in cell survival, cell growth and proliferation, cellular development and gene expression. We conclude that gene expression profiles of CD27- NBCs and CD27+ MBCs are different. Moreover, whereas the gene expression pattern of CD27+ MBCs varies with age, the same does not happen in CD27- NBCs. This suggests that MBCs undergo time-dependent changes which could underlie a higher susceptibility to dysfunction with age. This article is protected by copyright. All rights reserved.

Genetically modeled mice with mutations in mitochondrial metabolic enzymes for the study of cancer.

Mitochondrial dysfunction has long been implicated in progression of cancer. As a paradigm, the "Warburg effect," which by means of a switch toward anaerobic metabolism enables cancer cells to proliferate in oxygen limiting conditions, is well established. Besides this metabolic transformation of tumors, it has been discovered that mutations in genes encoding mitochondrial proteins are the etiological factors in different types of cancer. This confers to mitochondrial dysfunction a causative role, rather than resultant, in tumor genesis beyond its role in tumor progression and development. Mitochondrial proteins encoded by tumor-suppressor genes are part of the succinate-dehydrogenase, the fumarate-hydratase, and the mitochondrial isocitrate-dehydrogenase enzymes, all of them participating in the Krebs cycle. The spectrum of tumors associated with mutations in these genes is becoming larger and varies between each enzyme. Several mechanisms of tumorigenesis have been proposed for the different enzymatic defects, most of them based on studies using cellular and animal models. Regarding the molecular pathways implicated in the oncogenic transformation, one of the first accepted theories was based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as "pseudo-hypoxic drive." This mechanism has been linked to the three types of mutations, thus suggesting a central role in cancer. However, other alternative molecular processes, such as oxidative stress or altered chromatin remodeling, have been also proposed to play an onco-pathogenic role. In the recent years, the role of oncometabolites, a new concept emerged from biochemical studies upon these tumors, has acquired relevance as responsible for tumor formation. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. In this review, we summarize the results obtained from mouse strains genetically modified in the three different enzymes.

An O2-sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia.

Neural stem cells (NSCs) exist in germinal centers of the adult brain and in the carotid body (CB), an oxygen-sensing organ that grows under chronic hypoxemia. How stem cell lineage differentiation into mature glomus cells is coupled with changes in physiological demand is poorly understood. Here, we show that hypoxia does not affect CB NSC proliferation directly. Rather, mature glomus cells expressing endothelin-1, the O2-sensing elements in the CB that secrete neurotransmitters in response to hypoxia, establish abundant synaptic-like contacts with stem cells, which express endothelin receptors, and instruct their growth. Inhibition of glomus cell transmitter release or their selective destruction markedly diminishes CB cell growth during hypoxia, showing that CB NSCs are under the direct "synaptic" control of the mature O2-sensitive cells. Thus, glomus cells not only acutely activate the respiratory center but also induce NSC-dependent CB hypertrophy necessary for acclimatization to chronic hypoxemia.

A conditional mouse mutant in the tumor suppressor SdhD gene unveils a link between p21(WAF1/Cip1) induction and mitochondrial dysfunction.

Mutations in mitochondrial complex II (MCII; succinate dehydrogenase, Sdh) genes cause familiar pheochromocytoma/paraganglioma tumors. Several mechanisms have been proposed to account for Sdh-mutation-induced tumorigenesis, the most accepted of which is based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as "pseudo-hypoxic drive". Other molecular processes, such as oxidative stress, apoptosis, or chromatin remodeling have been also proposed to play a causative role. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. Moreover, the biological factors that determine the tissue-specificity of these tumors have not been identified. In this work, we made use of the inducible SDHD-ESR mouse, a conditional mutant in the SdhD gene, which encodes the small subunit of MCII, and that acts as a tumor suppressor gene in humans. The analysis of the Hif1α pathway in SDHD-ESR tissues and in two newly derived cell lines after complete SdhD loss -a requirement for hereditary paraganglioma type-1 tumor formation in humans- partially recapitulated the "pseudo-hypoxic" response and rendered inconsistent results. Therefore, we performed microarray analysis of adrenal medulla and kidney in order to identify other early gene expression changes elicited by SdhD deletion. Our results revealed that each mutant tissue displayed different variations in their gene expression profiles affecting to different biological processes. However, we found that the Cdkn1a gene was up-regulated in both tissues. This gene encodes the cyclin-dependent kinase inhibitor p21(WAF1/Cip1), a factor implicated in cell cycle, senescence, and cancer. The two SDHD-ESR cell lines also showed accumulation of this protein. This new and unprecedented evidence for a link between SdhD dysfunction and p21(WAF1/Cip1) will open new avenues for the study of the mechanisms that cause tumors in Sdh mutants. Finally, we discuss the actual role of Hif1α in tumorigenesis.

Gene and miRNA expression profiles of hematopoietic progenitor cells vary depending on their origin.

Hematopoietic progenitor cells (HPCs) from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (G-PB), bone marrow (BM), or umbilical cord blood (CB) have differing biological properties and differing kinetics of engraftment post-transplantation, which might be explained, at least in part, by differing gene and miRNA expression patterns. To assess the differences in gene and miRNA expression, we analyzed whole genome expression profiles as well as the expression of 384 miRNAs in CD34(+) cells isolated from 18 healthy individuals (6 individuals per subtype of HPC source). We identified 43 genes and 36 miRNAs differentially expressed in the various CD34(+) cell sources. We observed that CD34(+) cells from CB and BM showed similar gene and miRNA expression profiles, whereas CD34(+) cells from G-PB had a very different expression pattern. Remarkably, 20 of the differentially expressed genes are targets of the differentially expressed miRNAs. Of note, the majority of genes differentially expressed in CD34(+) cells from G-PB are involved in cell cycle regulation, promoting the process of proliferation, survival, hematopoiesis, and cell signaling, and are targets of overexpressed and underexpressed miRNAs in CD34(+) cells from the same source. These data suggest significant differences in gene and miRNA expression among the various HPC sources used in transplantation. We hypothesize that the differentially expressed genes and miRNAs involved in cell cycle and proliferation might explain the differing kinetics of engraftment observed after transplantation of hematopoietic stem cells obtained from these different sources.

Granulocyte colony-stimulating factor produces long-term changes in gene and microRNA expression profiles in CD34+ cells from healthy donors.

Granulocyte colony-stimulating factor is the most commonly used cytokine for the mobilization of hematopoietic progenitor cells from healthy donors for allogeneic stem cell transplantation. Although the administration of this cytokine is considered safe, knowledge about its long-term effects, especially in hematopoietic progenitor cells, is limited. On this background, the aim of our study was to analyze whether or not granulocyte colony-stimulating factor induces changes in gene and microRNA expression profiles in hematopoietic progenitor cells from healthy donors, and to determine whether or not these changes persist in the long-term. For this purpose, we analyzed the whole genome expression profile and the expression of 384 microRNA in CD34(+) cells isolated from peripheral blood of six healthy donors, before mobilization and at 5, 30 and 365 days after mobilization with granulocyte colony-stimulating factor. Six microRNA were differentially expressed at all time points analyzed after mobilization treatment as compared to the expression in samples obtained before exposure to the drug. In addition, 2424 genes were also differentially expressed for at least 1 year after mobilization. Of interest, 109 of these genes are targets of the differentially expressed microRNA also identified in this study. These data strongly suggest that granulocyte colony-stimulating factor modifies gene and microRNA expression profiles in hematopoietic progenitor cells from healthy donors. Remarkably, some changes are present from early time-points and persist for at least 1 year after exposure to the drug. This effect on hematopoietic progenitor cells has not been previously reported.

Differential impairment of catecholaminergic cell maturation and survival by genetic mitochondrial complex II dysfunction.

The SDHD gene (subunit D of succinate dehydrogenase) has been shown to be involved in the generation of paragangliomas and pheochromocytomas. Loss of heterozygosity of the normal allele is necessary for tumor transformation of the affected cells. As complete SdhD deletion is lethal, we have generated mouse models carrying a "floxed" SdhD allele and either an inducible (SDHD-ESR strain) or a catecholaminergic tissue-specific (TH-SDHD strain) CRE recombinase. Ablation of both SdhD alleles in adult SDHD-ESR mice did not result in generation of paragangliomas or pheochromocytomas. In contrast, carotid bodies from these animals showed smaller volume than controls. In accord with these observations, the TH-SDHD mice had decreased cell numbers in the adrenal medulla, carotid body, and superior cervical ganglion. They also manifested inhibited postnatal maturation of mesencephalic dopaminergic neurons and progressive cell loss during the first year of life. These alterations were particularly intense in the substantia nigra, the most affected neuronal population in Parkinson's disease. Unexpectedly, TH(+) neurons in the locus coeruleus and group A13, also lacking the SdhD gene, were unaltered. These data indicate that complete loss of SdhD is not sufficient to induce tumorigenesis in mice. They suggest that substantia nigra neurons are more susceptible to mitochondrial damage than other catecholaminergic cells, particularly during a critical postnatal maturation period.

Mitochondrial complex II is essential for hypoxia-induced pulmonary vasoconstriction of intra- but not of pre-acinar arteries.

AIMS: Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II (SDHD(+/-)). METHODS AND RESULTS: Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD(+/-) mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20-50 µm) intra-acinar and larger (51-100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD(+/-) mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD(+/-) than in wild-type mice. CONCLUSION: SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.

Mitochondrial complex II participates in normoxic and hypoxic regulation of α-keto acids in the murine heart.

α-Keto acids (α-KAs) are not just metabolic intermediates but are also powerful modulators of different cellular pathways. Here, we tested the hypothesis that α-KA concentrations are regulated by complex II (succinate dehydrogenase=SDH), which represents an intersection between the mitochondrial respiratory chain for which an important function in cardiopulmonary oxygen sensing has been demonstrated, and the Krebs cycle, a central element of α-KA metabolism. SDH subunit D heterozygous (SDHD(+/-)) and wild-type (WT) mice were housed at normoxia or hypoxia (10% O(2)) for 4 days or 3 weeks, and right ventricular pressure, right ventricle/(left ventricle+septum) ratio, cardiomyocyte ultrastructure, pulmonary vascular remodelling, ventricular complex II subunit expression, SDH activity and α-KA concentrations were analysed. In both strains, hypoxia induced increases in right ventricular pressure and enhanced muscularization of distal pulmonary arteries. Right ventricular hypertrophy was less severe in SDHD(+/-) mice although the cardiomyocyte ultrastructure and mitochondrial morphometric parameters were unchanged. Protein amounts of SDHA, SDHB and SDHC, and SDH activity were distinctly reduced in SDHD(+/-) mice. In normoxic SDHD(+/-) mice, α-ketoisocaproate concentration was lowered to 50% as compared to WT animals. Right/left ventricular concentration differences and the hypoxia-induced decline in individual α-KAs were less pronounced in SDHD(+/-) animals indicating that mitochondrial complex II participates in the adjustment of cardiac α-KA concentrations both under normoxic and hypoxic conditions. These characteristics are not related to the hemodynamic consequences of hypoxia-induced pulmonary vascular remodelling, since its extent and right ventricular pressure were not affected in SDHD(+/-) mice albeit right ventricular hypertrophy was attenuated.

Oxygen sensing in the carotid body.

The carotid body (CB) is a neural crest-derived organ whose function is to elicit hyperventilation in response to hypoxemia. The CB contains clusters of neuron-like glomus cells enveloped by glia-like sustentacular cells. CB responsiveness to acute hypoxia relies on the inhibition of O2-sensitive K+ channels in glomus cells, which leads to depolarization, Ca2+ entry and release of transmitters that activate afferent nerve fibers. The molecular mechanisms underlying K+ channel modulation by O2 tension are unknown. Putative hypoxia-sensing mechanisms can be studied in detail using genetically modified mice in conjunction with a thin carotid body slice preparation. We discuss here the role in CB oxygen sensing of the hypoxia-inducible factor 1alpha, the mitochondrial complex II subunit D, and heme oxygenase 2. In chronic hypoxia the CB grows with increase in glomus cell number. We identified CB stem cells of glial lineage, which can differentiate into functionally normal glomus cells.

Absolute requirement of GDNF for adult catecholaminergic neuron survival.

GDNF is a potent neurotrophic factor that protects catecholaminergic neurons from toxic damage and induces fiber outgrowth. However, the actual role of endogenous GDNF in the normal adult brain is unknown, even though GDNF-based therapies are considered promising for neurodegenerative disorders. We have generated a conditional GDNF-null mouse to suppress GDNF expression in adulthood, hence avoiding the developmental compensatory modifications masking its true physiologic action. After Gdnf ablation, mice showed a progressive hypokinesia and a selective decrease of brain tyrosine hydroxylase (Th) mRNA, accompanied by pronounced catecholaminergic cell death, affecting most notably the locus coeruleus, which practically disappears; the substantia nigra; and the ventral tegmental area. These data unequivocally demonstrate that GDNF is indispensable for adult catecholaminergic neuron survival and also show that, under physiologic conditions, downregulation of a single trophic factor can produce massive neuronal death.

Mechanisms of acute oxygen sensing by the carotid body: lessons from genetically modified animals.

We have studied carotid body (CB) glomus cell sensitivity to changes in O(2) tension in three different genetically engineered animals models using thin CB slices and monitoring the secretory response to hypoxia by amperometry. Glomus cells from partially HIF-1alpha deficient mice exhibited a normal sensitivity to hypoxia. Animals with complete deletion of the small membrane anchoring subunit of succinate dehydrogenase (SDHD) died during embryonic life but heterozygous SDHD +/- mice showed a normal CB response to low O(2) tension. SDHD +/- mice had, however, a clear CB phenotype characterized by a decrease of K(+) current amplitude, an increase of basal catecholamine release from glomus cells, and a slight organ growth. The lack of hemeoxygenase-2 (HO-2), a ubiquitous powerful antioxidant enzyme, produces a notable CB phenotype, characterized by hypertrophy and alteration in the level of CB expression of some stress-dependent genes (including down-regulation of the maxi-K(+) channel alpha-subunit). Nevertheless, in HO-2 deficient mice the exquisite intrinsic O(2) responsiveness of CB glomus cells remains unaltered. Therefore, HO-2 is not absolutely necessary for acute CB O(2) sensing. Although the nature of the CB acute O(2) sensor(s) is yet unknown, studies similar to those summarized here serve to test the existing hypothesis and help to distinguish between those that need to be explored further and those that definitively lack experimental support.

Oxygen-sensing by ion channels and mitochondrial function in carotid body glomus cells.

Carotid body glomus cells release transmitters in response to hypoxia due to the increase of excitability resulting from inhibition of O2-regulated K+ channels. The mechanisms involved in the detection of changes of O2 tension are unknown. Inhibition of the mitochondrial electron transport chain (ETC) at proximal and distal complexes induces external Ca(2+)-dependent catecholamine secretion. At saturating concentration of the ETC inhibitors, the cellular response to hypoxia is maintained. However, rotenone, a complex I blocker, selectively occludes the responsiveness to hypoxia of glomus cells in a dose-dependent manner. The effect of rotenone is not mimicked by complex I inhibitors acting on different sites. We have also generated a knock-out mouse lacking SDHD, the small membrane-anchoring protein of the succinate dehydrogenase (complex II) of the mitochondrial electron transport chain. Homozygous Sdhd(-/-) animals die at early embryonic stages. Heterozygous Sdhd(+/-) mice show a general, non-compensated, deficiency of complex II activity, and abnormal enhancement of resting carotid body secretion rate due to decrease of K+ conductance and persistent Ca2+ influx into glomus cells. However, responsiveness to hypoxia of carotid bodies from Sdhd(+/-) mice remains intact. These data strongly suggest that sensitivity to hypoxia of carotid body glomus cells is not linked in a simple way to mitochondrial electron flow. Nevertheless, it is possible that a rotenone-sensitive molecule critically participates in acute carotid body oxygen sensing.