Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.

KEY POINTS: Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. ABSTRACT: Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin-deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells.

T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal.

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na(+) currents as well as transient Ca(2+) currents abolished by the external application of Ni(2+). Biophysical and pharmacological data indicated that the Ca(2+) current is predominantly mediated by T-type (Ca(v)3.2) channels. The number of cells expressing T-type channels and Ca(v)3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca(2+) currents with Ni(2+) induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Ca(v)3.2) Ca(2+) channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Ca(v)3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca(2+) entry mediated by Ca(v)3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.

Developmental change of T-type Ca2+ channel expression and its role in rat chromaffin cell responsiveness to acute hypoxia.

Neonatal chromaffin cells of the adrenal medulla (AM) are intrinsic chemoreceptors that secrete catecholamines in response to hypoxia, thus contributing to fetal adaptation to extrauterine life. In most mammals studied, oxygen sensitivity of AM cells disappears a few days after birth, possibly due to innervation of the adrenal gland by the cholinergic fibres of the splanchnic nerve (approximately postnatal day 7 in the rat). The mechanisms underlying these homeostatic changes in chromaffin cells are unknown. Low voltage-activated, T-type, Ca(2+) channels regulate cell excitability and their expression is up-regulated by hypoxia. Hence, we hypothesized that these channels contribute to the developmental changes in the chemoreceptive properties of AM chromaffin cells. Using electrophysiological, immunocytochemical and molecular biology methodologies we show here that neonatal AM chromaffin cells express T-type Ca(2+) channels (of alpha1H or Ca(v)3.2 sub-type) and that the function of these channels is necessary for catecholamine release in response to acute hypoxia. T-type Ca(2+) channel expression, as well as chromaffin cell responsiveness to hypoxia, decrease with postnatal maturation. Adult chromaffin cell sensitivity to hypoxia reappears after AM denervation in parallel with the recruitment of T-type Ca(2+) channels. These observations indicate that T-type Ca(2+) channels are essential for the acute response of chromaffin cells to hypoxia and help explain the disappearance of O(2) sensitivity in adult AM chromaffin cells. Our results may also be relevant for understanding the pathogenesis of disorders associated with chronic hypoxia or maternal nicotine consumption.

Decreased expression of maxi-K+ channel beta1-subunit and altered vasoregulation in hypoxia.

BACKGROUND: Hypertension, a major cause of cardiovascular morbidity and mortality, can result from chronic hypoxia; however, the pathogenesis of this disorder is unknown. We hypothesized that downregulation of the maxi-K+ channel beta1-subunit by hypoxia decreases the ability of these channels to hyperpolarize arterial smooth muscle cells, thus favoring vasoconstriction and hypertension. METHODS AND RESULTS: Lowering O2 tension produced a decrease of maxi-K+ beta1-subunit mRNA levels in rat (aortic and basilar) and human (mammary) arterial myocytes. This was paralleled by a reduction of the beta1-subunit protein level as determined by immunocytochemistry and flow cytometry. Exposure to hypoxia also produced a decrease of open probability, mean open time, and sensitivity to the xenoestrogen tamoxifen of single maxi-K+ channels recorded from patch-clamped dispersed myocytes. The number of channels per patch and the single-channel conductance were not altered. The vasorelaxing force of maxi-K+ channels was diminished in rat and human arterial rings exposed to low oxygen tension. CONCLUSIONS: These results indicate that a decrease of the maxi-K+ channel beta1-subunit expression in arterial myocytes is a key factor in the vasomotor alterations induced by hypoxia.

Regulation of oxygen sensing by ion channels.

O(2) sensing is of critical importance for cell survival and adaptation of living organisms to changing environments or physiological conditions. O(2)-sensitive ion channels are major effectors of the cellular responses to hypoxia. These channels are preferentially found in excitable neurosecretory cells (glomus cells of the carotid body, cells in the neuroepithelial bodies of the lung, and neonatal adrenal chromaffin cells), which mediate fast cardiorespiratory adjustments to hypoxia. O(2)-sensitive channels are also expressed in the pulmonary and systemic arterial smooth muscle cells where they participate in the vasomotor responses to low O(2) tension (particularly in hypoxic pulmonary vasoconstriction). The mechanisms underlying O(2) sensing and how the O(2) sensors interact with the ion channels remain unknown. Recent advances in the field give different support to the various current hypotheses. Besides the participation of ion channels in acute O(2) sensing, they also contribute to the gene program developed under chronic hypoxia. Gene expression of T-type calcium channels is upregulated by hypoxia through the same hypoxia-inducible factor-dependent signaling pathway utilized by the classical O(2)-regulated genes. Alteration of acute or chronic O(2) sensing by ion channels could participate in the pathophysiology of human diseases, such as sudden infant death syndrome or primary pulmonary hypertension.

Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections.

X-gal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. However, this procedure does not permit the direct confocal acquisition of images, thus preventing the identification of labelled cells on the depth (Z) axis of tissue sections and leading sometimes to erroneous conclusions in co-localization and gene expression studies. Here we report a technique, based on X-gal fluorescence emission and mathematically-based optical correction, to obtain high quality fluorescence confocal images. This method, combined with immunofluorescence, makes it possible to unequivocally identify X-gal-labelled cells in tissue sections, emerging as a valuable tool in gene expression and cell tracing analysis.

Carotid body chemosensory responses in mice deficient of TASK channels.

Background K(+) channels of the TASK family are believed to participate in sensory transduction by chemoreceptor (glomus) cells of the carotid body (CB). However, studies on the systemic CB-mediated ventilatory response to hypoxia and hypercapnia in TASK1- and/or TASK3-deficient mice have yielded conflicting results. We have characterized the glomus cell phenotype of TASK-null mice and studied the responses of individual cells to hypoxia and other chemical stimuli. CB morphology and glomus cell size were normal in wild-type as well as in TASK1(-/-) or double TASK1/3(-/-) mice. Patch-clamped TASK1/3-null glomus cells had significantly higher membrane resistance and less hyperpolarized resting potential than their wild-type counterpart. These electrical parameters were practically normal in TASK1(-/-) cells. Sensitivity of background currents to changes of extracellular pH was drastically diminished in TASK1/3-null cells. In contrast with these observations, responsiveness to hypoxia or hypercapnia of either TASK1(-/-) or double TASK1/3(-/-) cells, as estimated by the amperometric measurement of catecholamine release, was apparently normal. TASK1/3 knockout cells showed an enhanced secretory rate in basal (normoxic) conditions compatible with their increased excitability. Responsiveness to hypoxia of TASK1/3-null cells was maintained after pharmacological blockade of maxi-K(+) channels. These data in the TASK-null mouse model indicate that TASK3 channels contribute to the background K(+) current in glomus cells and to their sensitivity to external pH. They also suggest that, although TASK1 channels might be dispensable for O(2)/CO(2) sensing in mouse CB cells, TASK3 channels (or TASK1/3 heteromers) could mediate hypoxic depolarization of normal glomus cells. The ability of TASK1/3(-/-) glomus cells to maintain a powerful response to hypoxia even after blockade of maxi-K(+) channels, suggests the existence of multiple sensor and/or effector mechanisms, which could confer upon the cells a high adaptability to maintain their chemosensory function.

Induction of T-type calcium channel gene expression by chronic hypoxia.

Cellular responses to hypoxia can be acute or chronic. Acute responses mainly depend on oxygen-sensitive ion channels, whereas chronic responses rely on the hypoxia-inducible transcription factors (HIFs), which up-regulate the expression of enzymes, transporters, and growth factors. It is unknown whether the expression of genes coding for ion channels is also influenced by hypoxia. We report here that the alpha1H gene of T-type voltage-gated calcium channels is highly induced by lowering oxygen tension in PC12 cells. Accumulation of alpha1H mRNA in response to hypoxia is time- and dose-dependent and paralleled by an increase in the density of T-type calcium channel current recorded in patch clamped cells. HIF appears to be involved in the response to hypoxia, since cobalt chloride, desferrioxamine, and dimethyloxalylglycine, compounds that mimic HIF-regulated gene expression, replicate the hypoxic effect. Moreover, functional inhibition of HIF-2alpha protein accumulation using antisense HIF-2alpha oligonucleotides reverses the effect of hypoxia on T-type Ca2+ channel expression. Importantly, regulation by oxygen tension is specific for T-type calcium channels, since it is not observed with the L-, N-, and P/Q-channel types. These findings show for the first time that hypoxia induces an ion channel gene via a HIF-dependent mechanism and define a new role for the T-type calcium channels as regulators of cellular excitability and calcium influx under chronic hypoxia.