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 O2/CO2 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.

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 (∼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 α1H 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.

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 upregulate 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 ␣ 1H gene of T-type voltage-gated calcium channels is highly induced by lowering oxygen tension in PC12 cells. Accumulation of ␣ 1H mRNA in response to hypoxia is time-and dose-dependent and paralleled by an increase in the density of Ttype 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-2␣ protein accumulation using antisense HIF-2␣ oligonucleotides reverses the effect of hypoxia on T-type Ca 2؉ 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.Maintaining optimal oxygen homeostasis is of paramount importance for cells. Reductions of oxygen supply trigger cell adaptive responses that minimize the deleterious effects of hypoxia. Cellular responses to hypoxia can be acute, occurring over a time scale of seconds or minutes, or chronic, with time courses of hours to days (1-5). The major effectors of the acute cellular responses to hypoxia are oxygen-sensitive ion channels. These channels mediate the fast adaptive changes in cell excitability, contractility, and secretory activity that occur in response to low ambient oxygen tension (PO 2 ) (1, 5). On the other hand, chronic cellular responses to hypoxia, studied in great detail in the past few years, are mediated by ubiquitously expressed hypoxia-inducible transcription factors (HIF-1␣ 1 and isoforms). Stabilization and transcriptional activity of HIF depend on oxygen-regulated hydroxylases (6 -8). Hypoxia-inducible factors regulate the expression of a wide repertoire of oxygen-sensitive genes with roles in diverse cellular functions such as angiogenesis, red blood cell production, glucose and energy metabolism, apoptosis, and cell proliferation (1-5).Despite progress in the understanding of the role of ion channels and gene expression in the cellular responses to hypoxia, long term regulation of ion channel expression by maintained low PO 2 is poorly known. There are previous reports showing that prolonged hypoxia down-regulates various voltage-gated K ϩ (Kv) channel genes in pulmonary artery smooth muscle cells (9), and the opposite has been observed with the ...

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.

Key points• The carotid body (CB) is a key chemoreceptor organ that mediates the hyperventilatory response to hypoxia, and contributes to the process of acclimatisation to chronic hypoxaemia.• Knowledge of CB physiology at the cellular and molecular levels has advanced considerably in recent times thanks to studies on lower mammals; however, information on humans is practically absent. Here we describe the properties of human CB cells in slice preparations or after enzymatic dispersion.• Besides glomus (type I) and glia-like, sustentacular (type II) cells, adult human CBs contain nestin-positive neural progenitor cells. The human CB also expresses high levels of glial cell line-derived neurotrophic factor. These properties are maintained at an advanced age.• Human glomus cells contain a relatively high density of voltage-dependent Na + , Ca 2+ and K + channels. Membrane depolarisation with high extracellular K + induces an increase of cytosolic [Ca 2+ ] and quantal catecholamine release.• Human glomus cells are responsive to hypoxia and hypoglycaemia, both of which induce an increase in cytosolic [Ca 2+ ] and transmitter release. Chemosensory responses of glomus cells are also preserved at an advanced age.• These findings on the cellular and molecular physiology of the CB provide novel perspectives for the systematic study of pathologies involving this organ in humans.Abstract The carotid body (CB) is the major peripheral arterial chemoreceptor in mammals that mediates the acute hyperventilatory response to hypoxia. The CB grows in response to sustained hypoxia and also participates in acclimatisation to chronic hypoxaemia. Knowledge of CB physiology at the cellular level has increased considerably in recent times thanks to studies performed on lower mammals, and rodents in particular. However, the functional characteristics of human CB cells remain practically unknown. Herein, we use tissue slices or enzymatically dispersed cells to determine the characteristics of human CB cells. The adult human CB parenchyma contains clusters of chemosensitive glomus (type I) and sustentacular (type II) cells as well as nestin-positive progenitor cells. This organ also expresses high levels of the dopaminotrophic glial cell line-derived neurotrophic factor (GDNF). We found that GDNF production and the number of progenitor and glomus cells were preserved in the CBs of human subjects of advanced age. qualitatively similar to those reported in lower mammals. These cells responded to hypoxia with an external Ca 2+ -dependent increase of cytosolic Ca 2+ and quantal catecholamine secretion, as reported for other mammalian species. Interestingly, human glomus cells are also responsive to hypoglycaemia and together these two stimuli can potentiate each other's effects. The chemosensory responses of glomus cells are also preserved at an advanced age. These new data on the cellular and molecular physiology of the CB pave the way for future pathophysiological studies involving this organ in humans.

Rodríguez. Biophysical and pharmacological data indicated that the Ca 2ϩ current is predominantly mediated by T-type (Ca v3.2) channels. The number of cells expressing T-type channels and Cav3.2 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 2ϩ currents with Ni 2ϩ 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 v3.2) Ca 2ϩ channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Cav3.2 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 2ϩ entry mediated by Ca v3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.voltage-dependent inward currents; proliferation; Cav3.2 channel expression

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 ϩ channel ␤ 1 -subunit by hypoxia decreases the ability of these channels to hyperpolarize arterial smooth muscle cells, thus favoring vasoconstriction and hypertension. Methods and Results-Lowering O 2 tension produced a decrease of maxi-K ϩ ␤ 1 -subunit mRNA levels in rat (aortic and basilar) and human (mammary) arterial myocytes. This was paralleled by a reduction of the ␤ 1 -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 ϩ 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 ϩ channels was diminished in rat and human arterial rings exposed to low oxygen tension. Conclusions-These

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.