Carotid body thin slices: responses of glomus cells to hypoxia and K(+)-channel blockers

We describe the rat carotid body thin slice preparation, which allows to perform patch-clamp recording of membrane ionic currents and to monitor catecholamine secretion by amperometry in single glomus cells under direct visual control. We observed several electrophysiologically distinct cell classes within the same glomerulus. A voltage- and Ca(2+)-dependent component of the whole cell K(+) current was reversibly inhibited by low P(O(2)) (20 mmHg). Exposure of the cells to hypoxia elicited the appearance of spike-like exocytotic events. This response to hypoxia was reversible and required extracellular Ca(2+) influx. Addition of tetraethylammonium (TEA, 2-5 mM) to the extracellular solution induced in most (>95%) cells tested a secretory response similar to that elicited by low P(O(2)). Cells non-responsive to hypoxia but activated by exposure to high external K(+) were also stimulated by TEA. A secretory response similar to that of hypoxia or TEA was also observed after treatment of the cells with iberiotoxin to block selectively maxi-K(+) channels. Our data further support the view that membrane ion channels are critically involved in sensory transduction in the carotid body. We demonstrate that in intact glomus cells inhibition of voltage-dependent K(+) channels can contribute to initiate the secretory response to low P(O(2)).     

Nitric oxide enhances Ca2+-dependent K+ channel activity in rat carotid body cells

The nitric oxide (NO) donor S-nitroso-acetylpenicillamine (SNAP) enhanced Ca(2+)-dependent K(+) channel activity in rat carotid body chemoreceptor cells. Ca(2+)-dependent K(+) channel activity was enhanced by SNAP in 38% (whole-cell configuration) and 67% (cell-attached mode) of the cells tested and was not affected by intracellular Ca(2+) chelation with BAPTA-AM. Enhancement of Ca(2+)-dependent K(+) channel activity by SNAP was blocked by the cGMP-dependent protein kinase G inhibitor 8-[(4-chlorophenyl)thio]-guanosine 3',5'-cyclic monophosphothioate Rp diastereomer (Rp-8-pCPT-cGMPS). NO thus enhances Ca(2+)-dependent K(+) channel activity through cGMP-dependent protein kinase G. The NO-mediated increase in Ca(2+)-dependent K(+) channel activity is likely to alter the function of carotid body chemoreceptor cells and could explain the decreased chemosensitivity of the carotid body in response to NO released from efferent nerves or vascular endothelial cells.     

Roles of ion channels in carotid body chemotransmission of acute hypoxia.

In this review, we have highlighted the roles of ion channels in carotid body chemotransmission of acute hypoxia. With the application of new technologies, significant breakthroughs have been made in the last decade. The discovery of oxygen-sensitive K(+) channels in rabbit glomus cells has generated the membrane model of hypoxic chemotransmission: the inhibition of oxygen-sensitive K(+) channels by hypoxia initiates the depolarization of glomus cells and increases the firing frequency of glomus cells. The depolarization of glomus cells activates voltage-gated Ca(2+) channels, elevating intracellular Ca(2+) which triggers the release of neurotransmitters. The correlation of these events in rabbit glomus cells has been shown. However, a large corpus of data indicates that various mechanisms may be involved in different species. In rats, Ca(2+)-activated K(+) channels are inhibited by hypoxia. The role of this inhibition on rat glomus cell function is controversial, and the contribution of leak-type K(+) channels to rat glomus cell depolarization has recently been proposed. On the other hand, in cats, nicotinic ACh receptors (ligand-gated cation channels) may play a key role in initiating the depolarization of glomus cells and increasing the cytosolic Ca(2+) of glomus cells in response to hypoxia. Hypoxic inhibition of oxygen-sensitive K(+) channels would participate to further depolarize cat glomus cells. Additionally, the activity of Cl(-) channels and the modulation of ion channels by neurotransmitters may influence the excitability of glomus cells. For generating action potentials in chemoreceptor afferent nerves, nicotinic ACh receptors appear to be involved in cats and rats.     

Closure of multiple types of K+ channels is necessary to induce changes in renal vascular resistance in vivo in rats

Inhibition of K(+) channels might mediate renal vasoconstriction. As inhibition of a single type of K(+) channel caused minor or no renal vasoconstriction in vivo in rats, we hypothesized that several classes of K(+) channels must be blocked to elicit renal vasoconstriction. We measured renal blood flow (RBF) in vivo in anesthetized Sprague-Dawley rats. Test agents were infused directly into the renal artery to avoid systemic effects. Inhibition of BK(Ca) and K(ir) channels (with TEA and Ba(2+), respectively) caused small and transient reductions in RBF (to 93?±?2% and 95?±?1% of baseline, respectively). K(ATP), SK(Ca) or K(v) channel blockade (with glibenclamide, apamin and 4-aminopyridine, respectively) was without effect. However, a cocktail of all blockers caused a massive reduction of RBF (to 15?±?10% of baseline). Nifedipine and mibefradil abolished and reduced, respectively, this RBF reduction. The effect of the cocktail of K(+) channel blockers was confirmed in mice using the isolated blood-perfused juxtamedullary nephron preparation. A cocktail of K(+) channel openers (K(+), NS309, NS1619 and pinacidil) had only a minor effect on baseline RBF in vivo in rats, but reduced the vasoconstriction induced by bolus injections of norepinephrine or angiotensin II (by 33?±?5% and 60?±?5%, respectively). Our results indicate that closure of numerous types of K(+) channels could participate in the mediation of agonist-induced renal vasoconstriction. Our results also suggest that renal vasoconstriction elicited by K(+) channel blockade is mediated by nifedipine-sensitive Ca(2+) channels and partly by mibefradil-sensitive Ca(2+) channels.     

Pronounced depression by propofol on carotid body response to CO2 and K+-induced carotid body activation

Propofol is a commonly used anesthetic agent, and it attenuates hypoxic ventilatory response in humans. Propofol reduce in vivo and in vitro carotid body responses to hypoxia as well as to nicotine in experimental animals. In the present study we examined the effects of propofol on carotid body responses to hypercapnia and K(+)-induced carotid body activation and compared these effects with hypoxia in an in vitro rabbit carotid body preparation. Hypoxia, hypercapnia and potassium increased the carotid sinus nerve activity and propofol attenuated the chemoreceptor responses to all three stimuli. However, the magnitude of propofol-induced attenuation was greater for hypercapnic and K(+)-induced carotid body activation compared to the hypoxic response. These observations suggest that propofol-induced attenuation of the hypoxic response is partly secondary to depression of chemoreceptor response to hypercapnia inhibiting the synergistic interactions between O(2) and CO(2) and may involve CO(2)/H(+) sensitive K(+) channels.     

Hypoxic suppression of K+ currents in type I carotid body cells: selective effect on the Ca2(+)-activated K+ current.

Whole-cell K+ currents were recorded in isolated type I carotid body cells using the patch-clamp technique. Hypoxia (pO2 25 torr) reversibly suppressed K+ currents in a voltage-dependent manner: maximal effects were seen at low, positive test potentials, where the Ca2(+)-activated component of K+ currents was greatest. Enhancing this component with 5 microM BAY K 8644 exaggerated the effects of hypoxia, and when the component was inhibited (100 microM Cd2+ or 5 microM nifedipine) hypoxic effects were abolished. As hypoxia does not affect Ca2+ currents directly, these data indicate the suppressive effect of hypoxia is selective for the Ca2(+)-activated component of K+ currents in type I cells.     

Oxygen sensitive Kv channels in the carotid body

Hypoxic inhibition of K(+) channels has been documented in many native chemoreceptor cells, and is crucial to initiate reflexes directed to improve tissue O(2) supply. In the carotid body (CB) chemoreceptors, there is a general consensus regarding the facts that a decrease in P(O2) leads to membrane depolarization, increase of Ca(2+) entry trough voltage-dependent Ca(2+) channels and Ca(2+)-dependent release of neurotransmitters. Central to this pathway is the modulation by hypoxia of K(+) channels that triggers depolarization. However, the details of this process are still controversial, and even the molecular nature of these oxygen-sensitive K(+) (K(O2)) channels in the CB is hotly debated. Clearly there are inter-species differences, and even in the same preparation more that one K(O2) may be present. Here we recapitulate our present knowledge of the role of voltage dependent K(+) channels as K(O2) in the CB from different species, and their functional contribution to cell excitability in response to acute and chronic exposure to hypoxia.     

Pinacidil prevents membrane depolarisation and intracellular Ca2+ loading in single cardiomyocytes exposed to severe metabolic stress

Recently, it has been proposed that, besides sarcolemmal K(ATP) channels, the activation of mitochondrial K(ATP) channels may also contribute to the cardioprotective action of potassium channel openers. In this respect, use of drugs that target both mitochondrial and sarcolemmal K(ATP) channels, such as pinacidil, may be a promising therapeutic strategy against metabolic injury of the heart. Therefore, the main objective of the present study was to determine whether pinacidil could maintain the value of resting membrane potential and intracellular Ca2+ homeostasis in cardiac cells exposed to metabolic stress. Experiments were performed on isolated ventricular cardiomyocytes. The membrane potential was monitored during experiments using whole cell patch clamp electrophysiology and the intracellular Ca2+ concentration was measured by a digital epifluorescence imaging. Chemical hypoxia-reoxygenation was induced by application and removal of the mitochondrial poison 2,4 dinitrophenol (DNP). Under hypoxia-reoxygenation, membrane depolarisation and intracellular Ca2+ loading was induced by Ca2+ influx during hypoxia and release of Ca2+ from intracellular stores during reoxygenation. The K(ATP) channel activator, pinacidil, prevented intracellular Ca2+ loading and membrane depolarisation, irrespective of whether the channel opener was applied throughout the duration of hypoxia-reoxygenation or transiently during the hypoxic or reoxygenation stage. Thus, the present study provides evidence that pinacidil, a non-selective K(ATP) channel opener, can handle membrane potential and intracellular Ca2+ homeostasis in cardiomyocytes under hypoxia-reoxygenation irrespective of the stage of the metabolic insult. This provides further evidence, at the single cell level, that targeting K(ATP) channels may be a valuable approach to protect the myocardium against metabolic challenge.     

ATP causes glomus cell [Ca2+]c increase without corresponding increases in CSN activity

The hypothesis that an increase in intracellular calcium [Ca(2+)](c) in carotid body (CB) glomus cells will cause enhanced afferent carotid sinus nerve (CSN) activities was tested in the rat CB in-vitro with the use of extracellular ATP. ATP caused a dose dependent [Ca(2+)](c) increase in identified glomus cells. A major part of total [Ca(2+)](c) increase (2/3) was due to the [Ca(2+)] influx. The rest of [Ca(2+)](c) increase (1/3) was due to the release of [Ca(2+)] from the endoplasmic reticulum (ER) [Ca(2+)] stores, and it was inhibited by the pretreatment of cells with cyclopiazonic acid (CPA), an intracellular Ca(2+)-ATPase blocker. Suramin, a purinergic P(2) receptor membrane blocker, blocked [Ca(2+)] influx due to ATP in the presence of extracellular [Ca(2+)]. Perfusion with 5 and 10 microM ATP stimulated CSN activities in both normoxia (Nx) and hypoxia (Hx). Above that level, 100 microM ATP induced slight initial stimulation in CSN activities which were subsided subsequently in Nx and partly diminished in Hx, while 500 microM ATP completely inhibited CSN activities in Nx and Hx after a slight initial stimulation. Electrophysiological measurements of the glomus cell membrane potential in the presence of ATP (100 microM) during Nx indicated cellular enhanced outward K(+) current and hyperpolarization, suggesting potential mechanism for the inhibition of CSN activities. Thus, ATP dependent linear increases in [Ca(2+)](c) did not give rise to a corresponding increase in CSN activities, contravening the normally expected increase in CSN activities following [Ca(2+)](c) rise.     

Modulation of action potential firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons

The present study investigated the effects of iberiotoxin (IbTx), a peptide toxin blocker of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and NS1619, a BK(Ca) channel opener, on action potential firing of small and medium size afferent neurons from L6 and S1 dorsal root ganglia of adult rats. Application of IbTx (100 nM) reduced whole-cell outward currents in 67% of small and medium size neurons. Analysis of action potential profile revealed that IbTx significantly prolonged the duration of action potential and increased firing frequency of afferent neurons. IbTx did not significantly alter the resting membrane potential, threshold for action potential activation and action potential amplitude. The benzimidazolone NS1619 (10 microM) increased opening activity of a Ca(2+)-dependent channel as assessed by single channel measurements. In contrast to IbTx, NS1619 reversibly suppressed action potential firing, attributable to increases in threshold for evoking action potential, reduction in action potential amplitude and increases in amplitude of afterhyperpolarization. The effect of NS1619 on neuronal firing was sensitive to IbTx, indicating the attenuation of neuronal firing by NS1619 was mediated by opening BK(Ca) channels. NS1619 also reduced neuronal hyperexcitability evoked by 4-aminopyridine (4-AP), a transient-inactivated K(+) channel (A-current) blocker, in an IbTx-sensitive manner.These results indicate that IbTx-sensitive BK(Ca) channels exist in both small and medium diameter dorsal root ganglion (DRG) neurons and play important roles in the repolarization of action potential and firing frequency. NS1619 modulates action potential firing and suppresses 4-AP-evoked hyperexcitability in DRG neurons, in part, by opening BK(Ca) channels. These results suggest that opening BK(Ca) channels might be sufficient to suppress hyperexcitability of afferent neurons as those evoked by stimulants or by disease states.     

Purines, the carotid body and respiration

The carotid body is essential to detecting levels of oxygen in the blood and initiating the compensatory response. Increasing evidence suggests that the purines ATP and adenosine make a key contribution to this signaling by the carotid body. The glomus cells release ATP in response to hypoxia. This released ATP can stimulate P2X receptors on the carotid body to elevate intracellular Ca(2+) and to produce an excitatory response. This released ATP can be dephosphorylated to adenosine by a series of extracellular enzymes, which in turn can stimulate A(1), A(2A) and A(2B) adenosine receptors. Levels of extracellular adenosine can also be altered by membrane transporters. Endogenous adenosine stimulates these receptors to increase the ventilation rate and may modulate the catecholamine release from the carotid sinus nerve. Prolonged hypoxic challenge can alter the expression of purinergic receptors, suggesting a role in the adaptation. This review discusses evidence for a key role of ATP and adenosine in the hypoxic response of the carotid body, and emphasizes areas of new contributions likely to be important in the future.     

Mitochondrial ATP-sensitive K+ channels regulate NMDAR activity in the cortex of the anoxic western painted turtle

Hypoxic mammalian neurons undergo excitotoxic cell death, whereas painted turtle neurons survive prolonged anoxia without apparent injury. Anoxic survival is possibly mediated by a decrease in N-methyl-d-aspartate receptor (NMDAR) activity and maintenance of cellular calcium concentrations ([Ca(2+)](c)) within a narrow range during anoxia. In mammalian ischaemic models, activation of mitochondrial ATP-sensitive K(+) (mK(ATP)) channels partially uncouples mitochondria resulting in a moderate increase in [Ca(2+)](c) and neuroprotection. The aim of this study was to determine the role of mK(ATP) channels in anoxic turtle NMDAR regulation and if mitochondrial uncoupling and [Ca(2+)](c) changes underlie this regulation. In isolated mitochondria, the K(ATP) channel activators diazoxide and levcromakalim increased mitochondrial respiration and decreased ATP production rates, indicating mitochondria were 'mildly' uncoupled by 10-20%. These changes were blocked by the mK(ATP) antagonist 5-hydroxydecanoic acid (5HD). During anoxia, [Ca(2+)](c) increased 9.3 +/- 0.3% and NMDAR currents decreased 48.9 +/- 4.1%. These changes were abolished by K(ATP) channel blockade with 5HD or glibenclamide, Ca(2+)(c) chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by activation of the mitochondrial Ca(2+) uniporter with spermine. Similar to anoxia, diazoxide or levcromakalim increased [Ca(2+)](c) 8.9 +/- 0.7% and 3.8 +/- 0.3%, while decreasing normoxic whole-cell NMDAR currents by 41.1 +/- 6.7% and 55.4 +/- 10.2%, respectively. These changes were also blocked by 5HD or glibenclamide, BAPTA, or spermine. Blockade of mitochondrial Ca(2+)-uptake decreased normoxic NMDAR currents 47.0 +/- 3.1% and this change was blocked by BAPTA but not by 5HD. Taken together, these data suggest mK(ATP) channel activation in the anoxic turtle cortex uncouples mitochondria and reduces mitochondrial Ca(2+) uptake via the uniporter, subsequently increasing [Ca(2+)](c) and decreasing NMDAR activity.     

Ca(2+)-dependent inhibition of inwardly rectifying K(+) channel in opossum kidney cells

The effect of intracellular Ca(2+) on the activity of the inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS was examined by using the patch-clamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K(+) channel rapidly declined with an application of ionomycin (1 microM) in the presence of 10(-6) M Ca(2+) in cell-attached patches. The application of 10 microM phorbor-12-myristate-acetate (PMA) with 10(-6) M Ca(2+) reduced the K(+) channel activity. Although the channel activity was not influenced by an increase of bath Ca(2+) from 10(-7.5) to 10(-6) M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10(-6) M Ca(2+) in inside-out patches. The inhibitory effect of Ca(2+) with ionomycin on the channel activity was diminished by the pretreatment with a specific PKC inhibitor, GF 109203X (5 microM), in cell-attached patches. By contrast, the application of Ca(2+)/calmodulin kinase II (CaMK II, 300 pM) dramatically increased this channel activity in inside-out patches. In cell-attached patches, the addition of both GF 109203X and cyclospolin A (5 microM), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K(+) channel activity with ionomycin and 10(-6) M Ca(2+). The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 microM) and Ni(2+) (10 microM) to stimulate calcineurin inhibited the channel activity in inside-out patches. Furthermore, the inhibitory effect of PKC or calcineurin on this channel activity was abolished by a removal of Ca(2+) from bath solution. These results suggest that Ca(2+)-dependent inhibitory effect on the inwardly rectifying K(+) channel in OKP cells was mainly mediated by Ca(2+)-PKC-mediated phosphorylation, and that the Ca(2+)-calmodulin-dependent phosphorylation process may be counterbalanced by the Ca(2+)-calmodulin-dependent dephosphorylation process.     

Postnatal development of carotid body glomus cell O2 sensitivity

In mammals, the main sensors of arterial oxygen level are the carotid chemoreceptors, which exhibit low sensitivity to hypoxia at birth and become more sensitive over the first few days or weeks of life. This postnatal increase in hypoxia sensitivity of the arterial chemoreceptors, termed "resetting", remains poorly understood. In the carotid body, hypoxia is transduced by glomus cells, which are secretory sensory neurons that respond to hypoxia at higher P(O2) levels than non-chemoreceptor cell types. Maturation or resetting of carotid body O2 sensitivity potentially involves numerous aspects of the O2 transduction cascade at the glomus cell level, including glomus cell neurotransmitter secretion, neuromodulator function, neurotransmitter receptor expression, glomus cell depolarization in response to hypoxia, [Ca2+]i responses to hypoxia, K+ and Ca2+ channel O2 sensitivity and K+ channel expression. However, although progress has been made in the understanding of carotid body development, the precise mechanisms underlying postnatal maturation of these numerous aspects of chemotransduction remain obscure.     

Inhibition effect of arachidonic acid on hypoxia-induced [Ca(2+)](i) elevation in PC12 cells and human pulmonary artery smooth muscle cells

[Ca(2+)](i) elevation is a key event when O(2) sensitive cells, e.g. PC12 cells and pulmonary artery smooth muscle cells, face hypoxia. Ca(2+) entry pathways in mediating hypoxia-induced [Ca(2+)](i) elevation include: voltage-gated Ca(2+) channels (VGCCs), transient receptor potential (TRP) channel and Na(+)-Ca(2+) ex-changer (NCX). In the pulmonary artery, accumulated evidence strongly suggests that prostaglandins (PGs) are involved in pulmonary inflammation and cause vasoconstriction during hypoxia. In this study, we investigated the effect of arachidonic acid (AA), the upstream substrate for PGs, on hypoxia response in O(2) sensitive cells. Exogenous application of AA significantly inhibited hypoxia-induced [Ca(2+)](i) elevation. This effect was due to AA itself rather than its degenerative products. The pharmacological modulation of endogenous AA showed that the prevention of AA generation by blockage of cPLA2, diacylglycerol (DAG) lipase and fatty acid hydrolysis (FAAH), augments hypoxia-induced [Ca(2+)](i) elevation, whereas prevention of AA degeneration attenuates hypoxia-induced [Ca(2+)](i) elevation. Over-expression of COX2 enhances hypoxia-induced [Ca(2+)](i) elevation and this enhancement is reversed by exogenous AA. Our results suggest that AA inhibits hypoxia response. The dynamic alterations in cellular lipids might determine cell response to hypoxia.     

Changes in oxygen sensitivity of TASK in carotid body glomus cells during early postnatal development

A post-natal increase in carotid body (CB) hypoxia responsiveness occurs at the level of carotid sinus nerve activity, intracellular calcium, cell membrane depolarization and hypoxic inhibition of O(2)-sensitive background K(+) conductance. TASK-1, TASK-1/3 and TASK-3 are functionally expressed in CB glomus cells, with TASK-1/3 providing the major part of the O(2)-sensitive TASK-like background K(+) conductance. Here we report the effects of graded hypoxia on TASK-like channel activity in CB glomus cells from rats aged 0 to 1, 6 to 7 and 16 to 18 days; the time frame of postnatal CB functional maturation. TASK was active in nearly all cell-attached patches and TASK activity during normoxia did not differ across ages. Hypoxia produced a progressive decrease in channel opening frequency with graded decreases in O(2) level and also produced glomus cell depolarization, as assessed by the shift in reversal potential of TASK single channel current. Hypoxic inhibition of TASK activity was least at P0-P1 and increased with age mainly between 6-7 and 16-18 days. The O(2)-sensitive TASK activity was significantly greater in glomus cells from P16 to P18 when compared to cells from P0 to P1 day old rats. These results support the hypothesis that postnatal carotid body functional maturation is due, at least in part, to changes in the sensitivity of TASK to the hypoxic signals generated in glomus cells.     

The role of NADPH oxidase in carotid body arterial chemoreceptors

O(2)-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca(2+) entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O(2)-sensitivity of these cells currently focus on the coupling between local P(O2) and the open-closed state of K(+)-channels. Amongst multiple competing hypotheses is the notion that K(+)-channel activity is mediated by a phagocytic-like multisubunit enzyme, NADPH oxidase, which produces reactive oxygen species (ROS) in proportion to the prevailing P(O2). In O(2)-sensitive cells of lung neuroepithelial bodies (NEB), multiple studies confirm that ROS levels decrease in hypoxia, and that E(M) and K(+)-channel activity are indeed controlled by ROS produced by NADPH oxidase. However, recent studies in our laboratories suggest that ROS generated by a non-phagocyte isoform of the oxidase are important contributors to chemotransduction, but that their role in type I cells differs fundamentally from the mechanism utilized by NEB chemoreceptors. Data indicate that in response to hypoxia, NADPH oxidase activity is increased in type I cells, and further, that increased ROS levels generated in response to low-O(2) facilitate cell repolarization via specific subsets of K(+)-channels.     

BK channel openers inhibit migration of human glioma cells

Large-conductance Ca(2+)-activated K(+) channels (BK channels) are highly expressed in human glioma cells. However, less is known about their biological function in these cells. We used the patch-clamp technique to investigate activation properties of BK channels and time-lapse microscopy to evaluate the role of BK channel activation in migration of 1321N1 human glioma cells. In whole cells, internal perfusion with a solution containing 500 nM free Ca(2+) and external application of the BK channel opener phloretin (100 micro M) shifted the activation threshold of BK channel currents toward more negative voltages of about -30 mV, which is close to the resting potential of the cells. The concentration of intracellular Ca(2+) in fura-2-loaded 1321N1 cells was measured to be 235+/-19 nM and was increased to 472+/-25 nM after treatment with phloretin. Phloretin and another BK channel opener NS1619 (100 micro M) reduced the migration velocity by about 50%. A similar reduction was observed following muscarinic stimulation of glioma cells with acetylcholine (100 micro M). The effects of phloretin, NS1619 and acetylcholine on cell migration were completely abolished by co-application of the specific BK channel blockers paxilline (5 micro M) and iberiotoxin (100 nM). The phloretin-induced increase in intracellular Ca(2+) was unaffected by the removal of extracellular Ca(2+) and co-application of paxilline. These findings indicate that glioma cell migration was inhibited through BK channel activation, independent of intracellular Ca(2+).     

Inner mitochondrial maxi-K⁺ channels in neonatal renal tubular cells: novel therapeutic targets to control apoptosis

In developing kidneys, the total cell population is partly regulated by apoptosis. Despite our understanding of the molecular involvement in the regulatory pathway of apoptosis, we know little about the physiological involvement. Cardiomyocytes express large conductance voltage- and Ca(2+)-activated K(+) (maxi-K(+)) channels in their inner mitochondrial membranes. Triggering the mitochondrial K(+) influx necessary to inhibit apoptosis, the channels play cytoprotective roles during ischemic injury. Since proximal tubular cells in neonatal kidneys are physiologically under hypoxic stress, and since the channel activity is stimulated by hypoxia, those cells would share the same regulatory mechanism of apoptosis with ischemic cardiomyocytes. Therefore, we hypothesize here that the proximal tubular cells in neonatal kidneys would also express the maxi-K(+) channels in their inner mitochondrial membranes, and that the channels would play regulatory roles in apoptosis. Our hypothesis is unique because it sheds light for the first time on a physiological mechanism that involves the mitochondrial membranes in developing kidneys. It is also important because the idea could have novel therapeutic implications for kidney diseases that are associated with apoptosis.