PubMed Health⌕ Search

Biomedical subjects

C Peers

Publications and source records attributed to C Peers.

At least 19 recordsLinked to original sources
PubMed2006

Selective inhibition of L-type Ca2+ channels in A7r5 cells by physiological levels of testosterone.

Testosterone has marked beneficial cardiovascular effects, many of which have been attributed to a vasodilatory action. However, the molecular target of testosterone underlying this effect is subject to debate. In this study, we have used microfluorimetry as a noninvasive means of examining whether testosterone could exert dilatory effects via inhibition of voltage-gated Ca2+ entry in the model vascular smooth muscle cell line, A7r5. Rises of [Ca2+]i evoked by 50 mm K+ -containing solution were suppressed in a concentration-dependent manner by testosterone (IC50, 3.1 nm) and by the nonaromatizable analog, 5beta-dihydrotestosterone (IC50, 6.9 nm). The effects of testosterone were apparent in the presence of pimozide (to block T-type Ca2+ channels) but not nifedipine (to block L-type Ca2+ channels). Testosterone did not alter Ca2+ mobilization from intracellular stores by the prostaglandin analog U46619 or capacitative Ca2+ entry in cells pretreated with thapsigargin. Our results indicate that testosterone, at physiological concentrations, can selectively suppress Ca2+ entry into A7r5 cells via L-type Ca2+ channels. We suggest this effect is a likely mechanism underlying its vasodilatory actions and beneficial cardiovascular effects.

Animals↗
PubMed2006

Amyloid peptides mediate hypoxic increase of L-type Ca2+ channels in central neurones.

Prolonged hypoxia, encountered in individuals suffering from various cardiorespiratory diseases, enhances the likelihood of subsequently developing Alzheimer's disease (AD). However, the underlying mechanisms are unknown, as are the mechanisms of neurodegeneration of amyloid beta peptides (AbetaPs), although the latter involves disruption of Ca2+ homeostasis. Here, immunohistochemistry demonstrated that hypoxia increased production of AbetaPs, an effect which was prevented by inhibition of either beta or gamma secretase, the enzymes required for liberation of AbetaP from its precursor protein. Whole-cell patch clamp recordings showed that hypoxia selectively increased functional expression of L-type Ca2+ channels. This was prevented by inhibition of either beta or gamma secretase, indicating that hypoxic channel up-regulation is dependent upon AbetaP formation. Our results indicate for the first time that hypoxia promotes AbetaP formation in central neurons, and show that this leads to abnormally high selective expression of L-type Ca2+ channels whose blockade has previously been shown to be neuroprotective in AD models. These findings provide a cellular basis for understanding the increased incidence of AD following prolonged hypoxia.

Amyloid beta-Peptides↗
PubMed2006

Hypoxic regulation of Ca2+ signalling in astrocytes and endothelial cells.

Acute hypoxia is well known to modulate plasmalemmal ion channels in specific tissue types, thereby modulating [Ca2+]i. Alternative mechanisms by which acute hypoxia could modulate [Ca2+]i are less well explored, particularly in non-excitable cells. Here, we describe experiments employing microfluorimetric recordings from Fura-2-loaded rat cortical astrocytes and human saphenous vein endothelial cells designed to explore any effects of hypoxia (pO2 20-30 mmHg) on [Ca2+]i. In both cell types, hypoxia evoked small rises of [Ca2+]i in the majority of cells during perfusion with a Ca(2+)-free solution, indicating hypoxia can release Ca2+ from an intracellular pool. Capacitative Ca2+ entry was observed when Ca2+ was subsequently restored to the extracellular solution. These effects were abolished by pre-treatment of cells with thapsigargin or prior application of inositol 1,4,5-trisphosphate (IP3)-generating agonists. Antioxidants fully prevented this effect of hypoxia in both cell types. Mitochondrial uncoupling significantly enhanced the effects of hypoxia in astrocytes, yet markedly suppressed the effects of hypoxia in endothelial cells. Our findings indicate that hypoxia can modulate [Ca2+]i in non-excitable cells; most importantly, it can evoke Ca2+ release from intracellular stores via a mechanism which involves reactive oxygen species. The involvement of mitochondria in this effect appears to be tissue specific.

Animals↗
PubMed2005

Structural requirements for O2 sensing by the human tandem-P domain channel, hTREK1.

TREK1 is a member of the tandem-P domain K+ channel family which is expressed almost exclusively in the nervous system. It is modulated by a number of important factors including arachidonic acid and cell swelling. Since both factors are associated with brain ischemia, it has been suggested that activation of TREK1 may confer neuroprotection. However, it has been reported that the stably expressed human homologue of TREK1 is inhibited by hypoxia, calling into question its neuroprotective role in ischemia. Here, using transient transfection of HEK 293 cells with several hTREK1 mutations and whole-cell patch-clamp, we show that: hypoxic inhibition: (a) requires the C-terminal domain of the channel; (b) does not involve redox modulation of the C-terminal domain cysteine residues C365 and C399; and (c) is critically dependent on the glutamate residue at position 306. These data suggest strongly that neuroprotection is unlikely to be provided by this channel in low O2 environments and continue to cast a shadow of doubt over the precise role that TREK may have during hypoxic episodes.

Cell Line↗
PubMed2005

Hypoxic regulation of Ca2+ signaling in cultured rat astrocytes.

Acute hypoxia modulates various cell processes, such as cell excitability, through the regulation of ion channel activity. Given the central role of Ca2+ signaling in the physiological functioning of astrocytes, we have investigated how acute hypoxia regulates such signaling, and compared results with those evoked by bradykinin (BK), an agonist whose ability to liberate Ca2+ from intracellular stores is well documented. In Ca2+-free perfusate, BK evoked rises of [Ca2+]i in all cells examined. Hypoxia produced smaller rises of [Ca2+]i in most cells, but always suppressed subsequent rises of [Ca2+]i induced by BK. Thapsigargin pre-treatment of cells prevented any rise of [Ca2+]i evoked by either BK or hypoxia. Restoration of Ca2+ to the perfusate following a period of acute hypoxia always evoked capacitative Ca2+ entry. During mitochondrial inhibition (due to exposure to carbonyl cyanide p-trifluromethoxyphenyl hydrazone (FCCP) and oligomycin), rises in [Ca2+]i (observed in Ca2+-free perfusate) evoked by hypoxia or by BK, were significantly enhanced, and hypoxia always evoked responses. Our data indicate that hypoxia triggers Ca2+ release from endoplasmic reticulum stores, efficiently buffered by mitochondria. Such liberation of Ca2+ is sufficient to trigger capacitative Ca2+ entry. These findings indicate that the local O2 level is a key determinant of astrocyte Ca2+ signaling, likely modulating Ca2+-dependent astrocyte functions in the central nervous system.

Animals↗
PubMed2004

siRNA knock-down of gamma-glutamyl transpeptidase does not affect hypoxic K+ channel inhibition.

Large conductance, Ca(2+)-sensitive potassium (BK) channels are critical components of the O(2) signalling cascade in a number of cells, including the carotid body and central neurones. Although the nature of the BK channel O(2) sensor is still unknown, evidence suggests redox modulators might form part of the O(2) sensing channel complex. By metabolising glutathione, gamma-glutamyl transpeptidase (gammaGT) could act as such an O(2) sensor. Western blotting and immunocytochemistry revealed high gammaGT expression in HEK293 cells expressing the alpha- and beta-subunits of human recombinant BK and gammaGT co-immunoprecipitated with BKalpha. Acivicin blockade of gammaGT reversibly inhibited BK channels, suggesting that this BKalpha protein partner contributes to tonic channel activity. However, knock-out of gammaGT using siRNA had no effect on hypoxic BK channel inhibition. Together, these data indicate that gammaGT is a BKalpha protein partner, that its activity regulates BK channels but that it is not the BK O(2) sensor.

Cell Hypoxia↗
PubMed2003

Acute hypoxia occludes hTREK-1 modulation: re-evaluation of the potential role of tandem P domain K+ channels in central neuroprotection.

The human tandem P domain K+ channel hTREK-1 (KCNK2) is distributed widely through the CNS. Here, whole-cell patch clamp recordings were employed to investigate the effects of hypoxia on hTREK-1 channels stably expressed in human embryonic kidney cells. Acute hypoxia caused a rapid and reversible inhibition of whole-cell K+ current amplitudes; this was PO2 dependent with a maximal inhibition achieved at 60 mmHg and below. In accordance with previous studies, hTREK-1 current amplitudes were enhanced by arachidonic acid. This effect was concentration dependent, with maximal enhancement observed at a concentration of 10 microM. Membrane deformation by the crenator trinitrophenol (to mimic cell swelling) or the cup former chlorpromazine (to mimic cell shrinkage) caused robust activation and inhibition of currents, respectively. However, current augmentation by either arachidonic acid or trinitrophenol was completely prevented during hypoxia; conversely, hypoxia blunted the inhibitory action of chlorpromazine. The abilities of arachidonic acid to augment currents and of hypoxia to completely abrogate this effect were also observed in cell-attached patches. Our data indicate that hypoxia interacts with hTREK-1, and occludes its modulation by arachidonic acid and membrane deformation. These findings also suggest that the potential neuroprotective role of TREK channels, which has recently been proposed, requires reconsideration since hTREK-1 activation is unlikely when ambient PO2 is below 60 mmHg - a situation which normally pertains in the CNS even during systemic normoxia.

Arachidonic Acid↗
PubMed2003

Chronic hypoxia potentiates capacitative Ca2+ entry in type-I cortical astrocytes.

Prolonged hypoxia exerts profound effects on cell function, and has been associated with increased production of amyloid beta peptides (A beta Ps) of Alzheimer's disease. Here, we have investigated the effects of chronic hypoxia (2.5% O2, 24 h) on capacitative Ca2+ entry (CCE) in primary cultures of rat type-I cortical astrocytes, and compared results with those obtained in astrocytes exposed to A beta Ps. Chronic hypoxia caused a marked enhancement of CCE that was observed after intracellular Ca2+ stores were depleted by bradykinin application or by exposure to thapsigargin (1 microM). Exposure of cells for 24 h to 1 microM A beta P(1-40) did not alter CCE. Enhancement of CCE was not attributable to cell hyperpolarization, as chronically hypoxic cells were significantly depolarized as compared with controls. Mitochondrial inhibition [by FCCP (10 microM) and oligomycin (2.5 microg/mL)] suppressed CCE in all three cell groups, but more importantly there were no significant differences in the magnitude of CCE in the three astrocyte groups under these conditions. Similarly, the antioxidants melatonin and Trolox abolished the enhancement of CCE in hypoxic cells. Our results indicate that chronic hypoxia augments CCE in cortical type-I astrocytes, a finding which is not mimicked by A beta P(1-40) and appears to be dependent on altered mitochondrial function.

Amyloid beta-Peptides↗
PubMed2002

Hypoxia inhibits human recombinant large conductance, Ca(2+)-activated K(+) (maxi-K) channels by a mechanism which is membrane delimited and Ca(2+) sensitive.

Large conductance, Ca(2+)-activated K(+) (maxi-K ) channel activity was recorded in excised, inside-out patches from HEK 293 cells stably co-expressing the alpha- and beta-subunits of human brain maxi-K channels. At +50 mV, and in the presence of 300 nM Ca2+i, single channel activity was acutely and reversibly suppressed upon reducing P(O(2)) from 150 to > 40 mmHg by over 30 %. The hypoxia-evoked reduction in current was due predominantly to suppression in NP(o), although a minor component was attributable to reduced unitary conductance of 8-12 %. Hypoxia caused an approximate doubling of the time constant for activation but was without effect on deactivation. At lower levels of Ca2+i(30 and 100 nM), hypoxic inhibition did not reach significance. In contrast, 300 nM and 1 microM Ca2+i both sustained significant hypoxic suppression of activity over the entire activating voltage range. At these two Ca2+i levels, hypoxia evoked a positive shift in the activating voltage (by approximately 10 mV at 300 nM and approximately 25 mV at 1 microM). At saturating [Ca(2+)](i) (100 microM), hypoxic inhibition was absent. Distinguishing between hypoxia-evoked changes in voltage- and/or Ca2+i-sensitivity was achieved by evoking maximal channel activity using high depolarising potentials (up to +200 mV) in the presence of 300 nM or 100 microM Ca2+i or in its virtual absence (> 1 nM). Under these experimental conditions, hypoxia caused significant channel inhibition only in the presence of 300 nM Ca2+i. Thus, since regulation was observed in excised patches, maxi-K channel inhibition by hypoxia does not require soluble intracellular components and, mechanistically, is voltage independent and Ca2+i sensitive.

Calcium↗
PubMed2002

Chronic hypoxia remodels voltage-gated Ca2+ entry in a human airway chemoreceptor cell line.

Arterial and airway chemoreceptors respond to acute hypoxia by depolarizing, thereby activating voltage-gated Ca2+ channels and so permitting Ca2+ entry to trigger transmitter release. Following periods of prolonged hypoxia, these cells undergo a form of remodelling which involves altered expression of ion channels. Here, we use microspectrofluorimetric recordings of voltage-gated Ca2+ entry (activated by exposure of cells to 50 mM K+) to show that chronic hypoxia suppresses such Ca2+ entry in model airway chemoreceptor (H146) cells. Furthermore, Ca2+ entry via L-type channels is suppressed, whilst entry via N-type channels is greatly enhanced. The suppressed response, together with dramatic remodelling of routes available for voltage-gated Ca2+ entry, is likely to alter significantly the acute O2 sensing properties of these cells.

Calcium↗
PubMed2001

Electrochemistry of chalcogen compounds: prediction of antioxidant activity.

Synthesis and characterisation of organochalcogens has demonstrated a high correlation between their electrochemical oxidation potential on the glassy carbon electrode, their activity in bioassays and an unprecedented antioxidant activity in neuronal cell culture (EC50 approximately 20 nM) making electrochemical methodology a valuable tool in drug design for Alzheimer's and related diseases.

Animals↗
PubMed2001

Recombinant hTASK1 is an O(2)-sensitive K(+) channel.

Hypoxic inhibition of background K(+) channels is crucial to O(2) sensing by chemoreceptor tissues, but direct demonstration of O(2) sensitivity by any member of this K(+) channel family is lacking. HEK293 cells were transfected with a pcDNA3.1-hTASK1 construct; expression of hTASK1 was verified using RT-PCR and immunocytochemistry. Whole-cell K(+) currents of cells stably expressing hTASK-1 were, as anticipated, extremely sensitive to extracellular pH, within the physiological range (IC(50) approximately 7.0). All cells expressing this signature pH sensitivity were acutely modulated by pO(2); reduction of pO(2) from 150 to <40 mmHg (at pH 7.4) caused rapid and reversible suppression of pH-sensitive K(+) currents. Furthermore, these two regulatory signals clearly acted at the same channel, since the magnitude of the O(2)-sensitive current was dependent on the extracellular pH. These data represent the first direct verification that hTASK1 is O(2)-sensitive and reinforce the idea that this K(+) channel is key to O(2) sensing in chemoreceptors.

Cell Hypoxia↗
PubMed2001

NADPH oxidase does not account fully for O2-sensing in model airway chemoreceptor cells.

A key feature of O2 sensing by chemoreceptor tissues is the hypoxic inhibition of K+ channels. However, mechanisms coupling a fall of pO2 to channel closure differ between tissues: O2 regulation of K+ channels in chemoreceptive neuroepithelial bodies and their immortal counterparts, H146 cells, involves altered reactive oxygen species generation by NADPH oxidase. In contrast, this enzyme complex is not involved in O2 sensing by the carotid body and pulmonary vasculature. Here, we provide pharmacological evidence to support a role for NADPH oxidase in hypoxic inhibition of K+ currents in H146 cells. Two structurally unrelated NADPH oxidase inhibitors, diphenylene iodonium and phenylarsine oxide, suppressed hypoxic inhibition of K+ currents recorded using the patch-clamp technique. Most importantly, however, neither inhibitor fully blocked this response. Our findings provide the first evidence that multiple mechanisms may coexist within a specific cell type to account for hypoxic suppression of K+ channel activity.

Arsenicals↗
PubMed2001

A standing Na+ conductance in rat carotid body type I cells.

Substitution of extracellular Na+ with N-methyl D-glucamine caused marked hyperpolarisation in rat isolated carotid body type I cells, suggesting the presence of a standing Na+ conductance. Choline substitution produced smaller hyperpolarisations, whilst Li+ was virtually without effect. This Na+ conductance was not blocked by amiloride, tetrodotoxin, Zn2+ or Gd3+ and did not arise from electrogenic Na-glucose co-transport, since substitution of glucose with sucrose could not mimic the effects of Na+ substitution. Hypoxia and acidosis did not modify the tonic Na+ influx. Our results suggest that Na+ influx provides a constant depolarising influence on type I cells which acts to shift membrane potential beyond that required for initiation of neurosecretion, an essential step in carotid body chemotransduction.

Acid-Base Equilibrium↗
PubMed2001

Combined antisense and pharmacological approaches implicate hTASK as an airway O(2) sensing K(+) channel.

Neuroepithelial bodies act as airway oxygen sensors. The lung carcinoma line H146 is an established model for neuroepithelial body cells. Although O(2) sensing in both cells is via NADPH oxidase H(2)O(2)/free radical production and acute hypoxia promotes K(+) channel closure and cell depolarization, the identity of the K(+) channel is still controversial. However, recent data point toward the involvement of a member of the tandem P domain family of K(+) channels. Reverse transcription-polymerase chain reaction screening indicates that all known channels other than hTWIK1 and hTRAAK are expressed in H146 cells. Our detailed pharmacological characterization of the O(2)-sensitive K(+) current described herein is compatible with the involvement of hTASK1 or hTASK3 (pH dependence, tetraethylammonium and dithiothreitol insensitivity, blockade by arachidonic acid, and halothane activation). Furthermore, we have used antisense oligodeoxynucleotides directed against hTASK1 and hTASK3 to suppress almost completely the hTASK1 protein and show that these cells no longer respond to acute hypoxia; this behavior was not mirrored in liposome-only or missense-treated cells. Finally, we have used Zn(2+) treatment as a maneuver able to discriminate between these two homologues of hTASK and show that the most likely candidate channel for O(2) sensing in these cells is hTASK3.

Humans↗
PubMed2001

Hypoxic enhancement of evoked noradrenaline release from the human neuroblastoma SH-SY5Y.

The effects of chronic hypoxia (2.5% O(2), 24 h) on [3H]noradrenaline ([3H]NA) release evoked from human neuroblastoma SH-SY5Y cells by depolarisation and by activation of muscarinic receptors was investigated. Depolarization of cells with 100 mM K(+) evoked [3H]NA release, and chronic hypoxia enhanced this release significantly. In fluorimetric studies, the K(+)-evoked rises of [Ca(2+)](i) observed in response to 100 mM K(+) were also significantly enhanced. Muscarine-evoked [3H]NA release was also dramatically enhanced by chronic hypoxia. However, muscarine-induced release of Ca(2+) from intracellular stores and subsequent capacitative Ca(2+) entry was unaffected. The protein kinase C inhibitors GF 109 203X and RO-31-8220 did not prevent the enhancement of muscarine-evoked release caused by chronic hypoxia. These findings indicate that chronic hypoxia increases release of [3H]NA from human neuroblastoma SH-SY5Y cells. Enhancement of K(+)-evoked release was attributable to an enhancement of depolarisation-mediated Ca(2+) influx. In contrast, the larger enhancement of muscarine-evoked [3H]NA release was not due to greater release of Ca(2+) from internal stores, nor due to enhanced Ca(2+) influx. Furthermore, it was not attributable to activation of protein kinase C. These findings suggest that enhancement of sympathetic output, known to occur following prolonged hypoxia, may be mediated in part by enhancement of exocytosis.

Calcium↗
PubMed2001

Differential coupling of voltage-gated Ca(2+) channels to catecholamine secretion from separate PC12 cell batches.

Amperometric recordings were employed to investigate the coupling of Ca(2+) channels to catecholamine secretion in two batches of pheochromocytoma (PC12) cells. In 'new' (freshly obtained) cells (PC12n cells), secretion was dependent on Ca(2+) influx through L-type and N-type Ca(2+) channels. By contrast, in 'aged' cells (maintained in liquid nitrogen for 6-8 years; PC12a cells), secretion was mostly dependent on Ca(2+) influx through N-type channels. Patch clamp recordings revealed that L-type channels accounted for only ca. 26% of total whole-cell current in PC12a cells (determined by blockade caused by 2 microM nifedipine). In contrast, nifedipine suppressed currents by ca. 59% in PC12n cells. Thus important differences in fundamental physiological properties can be observed in PC12 cell batches even when obtained from the same source and maintained under identical conditions.

Animals↗