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M R Duchen

Publications and source records attributed to M R Duchen.

At least 37 records · Page 2Linked to original sources
PubMed1996

The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.

1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .-1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase. 4. The rise in [Mg2+]i was correlated with cell-attached recordings of ATP-sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN-_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0-ATPase at the expense of ATP reserves. 6. In response to CN-_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re-establishment of millimolar concentrations of ATP.

Adenosine Triphosphate↗
PubMed1996

Energy metabolism of adult astrocytes in vitro.

In this study we established cultures of astrocytes from the forebrain of the adult rat. The homeostatic regulatory mechanisms of the aerobic and anaerobic pathways of energy metabolism in these cells showed that adult astrocytes express many of the regulatory properties previously demonstrated in neonatal astrocytes. Changes in mitochondrial respiration and ATP production were readily evident upon incubation with the relevant substrates. Inhibition of mitochondrial respiration led to a compensatory increase in anaerobic glycolysis as evidenced by an increased release of lactate. We assessed the role of cytosolic calcium in the regulation of the mitochondrial energy metabolism. Increases in cytosolic calcium concentration in response to ATP or stimulation of mechanical receptors were followed by depolarizations of the mitochondrial membrane potential, whose magnitude reflected the amplitude of the cytosolic calcium response. The changes in mitochondrial membrane potential were largely dependent on the presence of external calcium. These results provide the first evidence of a signalling mechanism in astrocytes by which changes in cytosolic calcium mediate changes in respiration, possibly through mitochondrial calcium uptake and subsequent activation of several mitochondrial dehydrogenases. This signalling pathway would thus ensure that energy demands due to changes in cytosolic calcium concentrations are met by increases in energy production through increases in mitochondrial oxidative phosphorylation.

Animals↗
PubMed1996

Mechanisms of intracellular calcium regulation in adult astrocytes.

Microfluorimetric techniques were used to measure changes in intracellular calcium in astrocytes cultured from the forebrain of the adult rat. Application of ATP consistently raised intracellular calcium. The response persisted in the absence of extracellular calcium, but then quickly declined upon repeated agonist application. Thapsigargin abolished responses to nucleotides following depletion of the endoplasmic reticular calcium stores. Calcium release was inhibited by caffeine, but was dramatically increased through inositol phosphate receptor sensitization by the sulphydryl reagent thimerosal. Responses to repeated nucleotide applications resulted in a gradual decline of peak calcium concentrations, suggesting a (post)receptor-mediated desensitization or gradual depletion of the internal calcium stores. Subsequent application of ionomycin suggested intracellular calcium depletion as the relevant mechanism. Depletion of the internal calcium stores with ATP, ionomycin or thapsigargin failed to reveal a calcium influx pathway. These results suggest that the capacitative mechanism of calcium entry does not operate in response to nucleotide receptor activation in these cells, and that the immediate refilling of the internal calcium stores is primarily determined by re-uptake of cytosolic calcium into the endoplasmic reticulum. A complete refilling of this calcium store by extracellular calcium may be a much slower process. Control of these signal transduction pathways is crucial to the maintenance of the calcium/energy homeostasis of the adult astrocyte in the central nervous system.

Adenosine Triphosphate↗
PubMed1995

Electrophysiological and metabolic effects of a convulsant barbiturate on dissociated mouse primary sensory neurons.

1. The convulsant barbiturate 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB) depolarizes dorsal root ganglion (DRG) neurons. We have applied microfluorimetric and whole-cell patch clamp techniques to investigate the mechanisms underlying this response in freshly dissociated mouse DRG cells. 2. Application of CHEB (2-200 microM) raised cytosolic calcium concentration ([Ca2+]i) rapidly and reversibly in 55% of eighty-three neurons tested. This population did not correlate with other classifications of sensory neurons based on either cell size or the expression of membrane currents. 3. The response was dependent on external calcium and was reduced by 81 +/- 22% by Ruthenium Red. A rise in [Ca2+]i was still seen with the membrane potential clamped at -70 mV, excluding membrane depolarization and activation of voltage-dependent Ca2+ channels as the principal mechanism for the response. 4. The rise in [Ca2+]i was associated with an increase in membrane conductance and a current, ICHEB, which was inward at -70 mV. Both the rise in [Ca2+]i and the current showed 'run-down' under whole-cell recording conditions. When K+ conductances were blocked, the reversal potential of ICHEB was close to 0 mV. This was independent of the Cl- reversal potential, suggesting that ICHEB is carried as a non-specific cation current. 5. In contrast to the change in [Ca2+]i, ICHEB was not dependent on external Ca2+ and the current was still seen when [Ca2+]i as strongly buffered by the pipette filling solution. These data suggest that CHEB opens a non-selective cation channel permeant to Ca2+, raising [Ca2+]i and further depolarizing the cell membrane potential. The exact nature of this conductance remains unknown. These actions could readily account for the convulsant actions of the drug, depolarizing neurons and increasing transmitter release. 6. It was also noted that CHEB increases autofluorescence derived from mitochondrial NAD(P)H. Further examination of this phenomenon using the dye rhodamine 123 to follow changes in mitochondrial potential (psi m) suggested that CHEB is a potent inhibitor of mitochondrial respiration, probably acting at complex I. These effects appeared to be quite distinct from the action of CHEB at the level of the plasma membrane.

Animals↗
PubMed1995

A fluorimetric and amperometric study of calcium and secretion in isolated mouse pancreatic beta-cells.

We have examined the temporal relationship between intracellular Ca2+ concentration ([Ca2+]i) and secretion in single intact pancreatic beta-cells. Secretion was detected as the release of 5-hydroxytryptamine from pre-loaded beta-cells, using amperometry, and changes in [Ca2+]i were monitored by microfluorimetry. Stimulation of beta-cells by elevation of the extracellular K+ concentration ([K+]o), acetylcholine or glucose increased [Ca2+]i and, after a delay of 2-7 s, evoked amperometric currents. In the presence of glucose, we observed oscillations in [Ca2+]i which were associated with oscillations in the amplitude and frequency of amperometric currents: however, the temporal correlation was not exact, suggesting that there is a significant latency between the increase in average [Ca2+]i and exocytosis. Both the amplitude and frequency of the amperometric currents elicited by 50 mM KCl declined with successive stimulation, but were restored by agents which elevate intracellular adenosine 3':5':cyclic monophosphate (cAMP). This suggests that beta-cells may possess a readily releasable pool of granules which is replenished by cAMP. The variable amplitude of the amperometric currents is discussed in terms of a model in which several secretory granules fuse simultaneously with the plasma membrane.

Acetylcholine↗
PubMed1994

Differential expression of membrane currents in dissociated mouse primary sensory neurons.

The whole cell configuration of the patch clamp technique has been applied to identify the membrane currents expressed by populations of dissociated mouse primary sensory neurons. Three discrete populations of cells were distinguished on the basis of cell size and the array of currents expressed. Group 1 cells (capacitance 10-30 pF) expressed a Na+ current resistant to tetrodotoxin (1 microM) and a prominent, low threshold, inactivating, K+ current sensitive to 4-aminopyridine (IA). A population (53%) of these small cells responded to capsaicin (10 microM) with an inward current, suggesting a functional correlate with nociceptive "C"-cells. The cells of Group 2 (capacitance 55-85 pF) were characterized by the expression of a Na+ current sensitive to tetrodotoxin and a prominent inward current activated by hyperpolarization (IH). They also showed a variant of the A-type K+ current, which was a low threshold, but sustained K+ current, sensitive to dendrotoxin (30 nM). Group 3 cells, of intermediate size (capacitance 30-55 pF) were similar to Group 2 cells, in that they expressed a tetrodotoxin-sensitive Na+ current and (through reduced in amplitude), IH. The most notable feature of Group 3 cells was the expression of a transient, low threshold Ca2+ current. The differential expression of these conductances was reflected in the behaviour of cells under current clamp control. Each group of cells could thus be distinguished by the selective expression of specific ionic conductances which correlated clearly with cell size, suggesting a correlation with well recognised functional differentiation of sensory neurons. The selective expression of specific subsets of membrane channels may provide valuable markers in studying the developmental regulation of phenotype in this population of cells.

Action Potentials↗
PubMed1993

Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells.

Microfluorimetric and patch-clamp techniques have been combined to determine the relationship between changes in mitochondrial metabolism, the activity of KATP channels and changes in intracellular free calcium concentration ([Ca2+]i) in isolated pancreatic beta-cells in response to glucose, ketoisocaproic acid (KIC) and the electron donor couple tetramethyl p-phenylenediamine (TMPD) and ascorbate. Exposure of cells to 20 mM glucose raised NAD(P)H autofluorescence after a delay of 28 +/- 1 s (mean +/- S.E.M., n = 30). The mitochondrial inner membrane potential, delta psi m (monitored using rhodamine 123 fluorescence), hyperpolarized with a latency of 49 +/- 6 s (n = 17), and the [Ca2+]i rose after 129 +/- 13 s (n = 5). The amplitudes of the metabolic changes were graded appropriately with glucose concentration over the range 2.5-20 mM. All variables responded to KIC with shorter latencies: NAD(P)H autofluorescence rose after a delay of 20 +/- 3 s (n = 5) and rhodamine 123 changed after 21 +/- 3 s (n = 6). The electron donor couple, TMPD with ascorbate, rapidly hyperpolarized delta psi m and raised [Ca2+]i. When [Ca2+]i was raised by sustained exposure to 20 mM glucose, TMPD had no further effect. TMPD also decreased whole-cell KATP currents and depolarized the cell membrane, measured with the perforated patch configuration. These data are consistent with a central role for mitochondrial oxidative phosphorylation in coupling changes in glucose concentration with the secretion of insulin.

Animals↗
PubMed1993

On the involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion injury.

Mammalian cardiomyocytes may withstand prolonged periods of ischaemia, only to die on reperfusion. We review data that implicate mitochondrial dysfunction as a basis for reperfusion induced cell injury, and present some new evidence that suggests that such a mechanism operates in intact cardiomyocytes. The mitochondrial dysfunction is the consequence of the opening of high conductance pores in the inner mitochondrial membrane, which uncouple mitochondrial oxidative phosphorylation, promoting ATP hydrolysis. The conditions required to open the pores correlate closely to conditions that prevail upon reperfusion of the ischaemic heart: a high [Ca2+]i and Pi, low [ATP], and oxidative stress. Pore opening is suppressed by physiological concentrations of ATP. Pore opening may be prevented by cyclosporin A. Studies in isolated myocytes show that mitochondria become uncoupled after reoxygenation, and that this is associated with the hypercontracture that signals cell death. Cyclosporin A reduces the proportion of hypercontracted myocytes in populations of cells rendered anoxic.

Adenosine Triphosphate↗
PubMed1992

Ca(2+)-dependent changes in the mitochondrial energetics in single dissociated mouse sensory neurons.

Depolarization of neurons promotes Ca2+ influx through voltage-activated channels, raising the intracellular Ca2+ concentration ([Ca2+]i). The consequences of such changes in [Ca2+]i for mitochondrial function were assessed in single, freshly dissociated mammalian neurons. Microfluorimetric techniques were used to measure [Ca2+]i, mitochondrial membrane potential [delta psi m, Rhodamine 123 (Rh 123) fluorescence], NAD(P)H/NAD(P)+ autofluorescence and flavoprotein autofluorescence combined with whole-cell voltage-clamp techniques. Brief (100-500 ms) depolarization of the cell membrane by high K+ or by voltage commands raised [Ca2+]i and depolarized delta psi m. The change in delta psi m was dependent on extracellular Ca2+. Under voltage-clamp control of the cell membrane, the voltage-dependence of the change in Rh 123 fluorescence reflected that of the Ca2+ current. The response was reduced by Ca2+ buffers introduced into the cell. The behaviour of this signal is thus consistent with a mitochondrial response to raised [Ca2+]i and does not reflect the change in cell membrane potential per se. Similar stimuli caused a rapid decrease of NAD(P)H autofluorescence, followed by an increase which could last several minutes. Flavoprotein fluorescence increased transiently, followed by a decrease lasting for several minutes. These signals indicate an initial oxidation of NAD(P)H and FADH, followed by a prolonged increase in the reduced state of both coenzymes. All these changes were dependent on extracellular [Ca2+]. Raising [Ca2+]i again during the period of NAD+ reduction caused an oxidizing response. Ruthenium Red applied to the cells (i) reduced both the Ca2+ current and the depolarization-induced [Ca2+]i transient and (ii) directly quenched Rh 123 fluorescence. When introduced into the cells with patch pipettes, it prevented the changes in autofluorescence without interfering with the Ca2+ conductance. Oligomycin blocked neither the response of delta psi m nor of NADH autofluorescence, suggesting that the signals do not reflect a response to falling ATP/ADP.Pi ratios as a consequence of the high [Ca2+]i. The changes in NADH autofluorescence were sustained in the presence of iodoacetic acid with pyruvate as substrate. Thus brief physiological elevations of [Ca2+]i depolarize delta psi m, probably through Ca2+ cycling across the mitochondrial inner membrane. The changes in autofluorescence are consistent with (i) increased respiration which could result from the depolarization of delta psi m, followed rapidly by (ii) increased activity of the Ca(2+)-dependent intramitochondrial enzymes. Changes in [Ca2+]i within a physiological range may thus promote significant and long-lasting changes in mitochondrial energy production.

Animals↗
PubMed1992

Mitochondrial function in type I cells isolated from rabbit arterial chemoreceptors.

1. In this, and the accompanying paper (Duchen & Biscoe, 1992), we test the hypothesis that the oxygen sensitivity of mitochondrial electron transport forms a basis for transduction in the carotid body, the primary peripheral arterial oxygen sensor. We here describe for isolated type I cells the changes in autofluorescence of mitochondrial NAD(P)H that accompany changes in PO2. 2. NAD(P)H autofluorescence (excitation, 340-360 nm; emission peak, 450 nm) increased with anoxia, reflecting a rise in the NAD(P)H/NAD(P) ratio. Graded increases in autofluorescence were seen in response to graded decreases in PO2, suggesting that mitochondrial function is progressively altered below a PO2 of about 60 mmHg. 3. A mitochondrial origin for the NAD(P)H autofluorescence was suggested by the mutual exclusion of the responses to anoxia and cyanide. 4. Oxidized flavoproteins fluoresce when excited at 450 nm with an emission peak at 550 nm. The small signals obtained under these conditions increased with uncoupler and showed a graded decrease with falling PO2 reflecting a rise in the FADH/FAD ratio. 5. Hypoxia raises [Ca2+]i. The hypoxia-induced changes in mitochondrial function were not secondary to this rise. A brief K(+)-induced depolarization leads to a transient increase in [Ca2+]i. At the same time there is a rapid decrease in NAD(P)H autofluorescence followed by an increase that far outlasts the rise in [Ca2+]i. This delayed increase in autofluorescence was smaller than was the increase with anoxia, even though K(+)-induced depolarization raised [Ca2+]i more than does anoxia. In Ca(2+)-free solutions the depolarization-induced changes were abolished, while those associated with hypoxia were maintained. 6. The changes of autofluorescence with K(+)-induced depolarization appear to reflect (i) oxidation of NAD(P)H by stimulation of respiration following mitochondrial Ca2+ uptake and (ii) reduction of NAD(P) by the Ca(2+)-dependent activation of mitochondrial dehydrogenases. This activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in PO2. 7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the PO2 fell below about 5 mmHg. 8. Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (delta FFCCP). Blockade of electron transport by anoxia or CN- decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (delta FCN). The fractional change in autofluorescence with FCCP, delta FFCCP/delta FFCCP+FCN), is thus a measure of resting O2 consumption.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body.

1. In the accompanying paper (Duchen & Biscoe, 1992) we have described graded changes in autofluorescence derived from mitochondrial NAD(P)H in type I cells of the carotid body in response to changes of PO2 over a physiologically significant range. These observations suggest that mitochondrial function in these cells is unusually sensitive to oxygen and could play a role in oxygen sensing. We have now explored further the relationships between hypoxia, mitochondrial membrane potential (delta psi m) and [Ca2+]i. 2. The fluorescence of Rhodamine 123 (Rh 123) accumulated within mitochondria is quenched by delta psi m. Mitochondrial depolarization thus increases the fluorescence signal. Blockade of electron transport (CN-, anoxia, rotenone) and uncoupling agents (e.g. carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; FCCP) increased fluorescence by up to 80-120%, while fluorescence was reduced by blockade of the F0 proton channel of the mitochondrial ATP synthase complex (oligomycin). 3. delta psi m depolarized rapidly with anoxia, and was usually completely dissipated within 1-2 min. The depolarization of delta psi m with anoxia (or CN-) and repolarization on reoxygenation both followed a time course well characterized as the sum of two exponential processes. Oligomycin (0.2-2 micrograms/ml) hyperpolarized delta psi m and abolished the slower components of both the depolarization with anoxia and of the subsequent repolarization. These data (i) illustrate the role of the F1-F0 ATP synthetase in slowing the rate of dissipation of delta psi m on cessation of electron transport, (ii) confirm blockade of the ATP synthetase by oligomycin at these concentrations, and (iii) indicate significant accumulation of intramitochondrial ADP during 1-2 min of anoxia. 4. Depolarization of delta psi m was graded with graded changes in PO2 below about 60 mmHg. The stimulus-response curves thus constructed strongly resemble those for [Ca2+]i and NAD(P)H with PO2. The change in delta psi m closely followed changes in PO2 with time. 5. The rate of rise of [Ca2+]i in response to anoxia is strongly temperature sensitive. The rate of depolarization of delta psi m with anoxia similarly increased at least two- to fivefold on warming from 22 to 36 degrees C. The change with FCCP was not significantly altered by temperature. 6. These data show that the mitochondrial membrane potential changes over a physiological range of PO2 values in type I cells. This contrasts with the behaviour in dissociated chromaffin cells and sensory neurons, in which no change was measurable until the PO2 fell close to zero.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Monitoring exocytosis from single mast cells by fast voltammetry.

We have used fast differential ramp voltammetry with carbon-fibre electrodes to monitor exocytotic secretion in single rat mast cells. The oxidation peak and other aspects of the electrochemical profile of the substance released were similar to those of 5-hydroxytryptamine (5-HT) and the signals were increased by preloading the secretory granules with exogenous 5-HT. Metabolic blockade inhibited both visible degranulation and the electrochemical signal. For comparison, quinacrine, which is fluorescent and accumulates in secretory vesicles, was used as an alternative means of detecting secretion in single cells. The amplitude of the electrochemical signals observed during degranulation correlated well with the loss of quinacrine fluorescence. Both methods were used to record successive rounds of secretion in single mast cells in response to repeated applications of compound 48/80.

Animals↗
PubMed1990

Effects of metabolic inhibition on the membrane properties of isolated mouse primary sensory neurones.

1. The patch-clamp technique has been used to investigate the mechanisms that couple membrane excitability to metabolism in neurones isolated from mouse dorsal root ganglia. 2. Blockade of electron transport by cyanide (CN-), reduction of the mitochondrial membrane potential with carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), removal of glucose or inhibition of glycolysis with idoacetic acid (IAA), all increased a K+ conductance (gK), which could be sufficient to shunt action potentials. 3. The K+ conductance was reduced by incubation of cells in Ca2(+)-free solutions or by increasing the Ca2+ buffering power of pipette-filling solutions. The Ca2+ ionophore, ionomycin, also increased a K+ conductance, and current fluctuation analysis showed that the channels carrying the current induced by both ionomycin and by CN- had a similar mean conductance of circa 9 pS. Thus, increased gK was a Ca2(+)-dependent K+ conductance, gK(Ca), reflecting a rise in resting [Ca2+]i. 4. The conductance was not affected by inclusion of ATP or an ATP-regenerating system in the pipette, suggesting that the underlying rise in [Ca2+] is not due directly to loss of ATP, and confirming that the increased gK is not carried through ATP-dependent K+ channels. 5. Voltage-gated K+ currents evoked by membrane depolarization were increased by CN- or glucose removal. The current-voltage relation of the increased gK mirrored the voltage dependence of Ca2+ entry, and thus reflects impaired cellular handling of the Ca2+ load imposed by depolarization. 6. The rise in [Ca2+]i and altered Ca2+ buffering capacity induced by metabolic blockade affected several other conductances: (i) a Ca2(+)-dependent chloride current was increased. (ii) Both the low-threshold transient and high-threshold sustained voltage-gated Ca2+ currents were attenuated and their thresholds were shifted in the hyperpolarizing direction. (iii) The inward current activated by hyperpolarization. IH, seen in large cells, was attenuated by either metabolic blockade or ionomycin. 7. The responses of these neurones to impaired metabolism thus depend largely on the effects of raised [Ca2+]i on the populations of channels expressed by the cells. These changes in membrane properties could account for some of the changes in neuronal behaviour seen during the clinical states of hypoxia or hypoglycaemia, underlying changes in central nervous system function.

Adenosine Triphosphate↗
PubMed1990

Effects of metabolic blockade on the regulation of intracellular calcium in dissociated mouse sensory neurones.

1. Impaired intracellular Ca2+ concentration ([Ca2+]i) regulation may underlie alterations in neuronal function during hypoxia or hypoglycaemia and may initiate cell damage. We have used the Ca2(+)-sensitive fluorophore, Fura-2, to study the regulation of [Ca2+]i in neurones isolated from mouse dorsal root ganglia. Mean resting [Ca2+]i was 163 +/- 11 nM (mean +/- S.E.M., n = 38). 2. Depolarization by exposure to 20 or 30 mM-K+ caused a rapid Co2(+)- and Cd2(+)-sensitive rise in [Ca2+]i, which subsequently declined with a time course usually fitted by the sum of two exponential functions. 3. Interference with mitochondrial function (by CN- or FCPP) or with glycolysis (by glucose removal) all raised [Ca2+]i by up to 220%. Addition of FCCP in the presence of CN- further increased [Ca2+]i. The response to CN- was still seen in the absence of extracellular Ca2+, although it attenuated rapidly, indicating release from an intracellular store. 4. Either CN- or glucose removal increased the rise in [Ca2+]i induced by K+ 2- to 3-fold and slowed recovery, suggesting interference with sequestration or extrusion of [Ca2+]i. 5. Resting [Ca2+]i rose when external Na+ was replaced by Li+ or N-methyl-D-glucamine, demonstrating the presence of a Na(+)-Ca2+ exchange process. However, Na+ replacement had only a slight effect on the handling of a Ca2+ load. 6. We conclude that (i) Ca2+ is released into the cytoplasm from intracellular organelles when energy supplies are reduced: (ii) that the extrusion or sequestration of Ca2+ entering the cell during electrical activity is rapidly impaired by interference with mitochondrial metabolism: and (iii) Na(+)-Ca2+ exchange makes only a small contribution to intracellular Ca2+ homeostasis. 7. [Ca2+]i would thus be expected to rise in vivo during hypoxia or hypoglycaemia and may initiate alterations in neuronal function. However, if a rise in Ca2+ is an important cause of cell damage in cerebral hypoxaemia, the combination of excitation and hypoxia will lead to the largest increases in [Ca2+]i, while hypoxia alone appears to cause only a small increase in [Ca2+]i in quiescent cells.

Animals↗
PubMed1990

Responses of type I cells dissociated from the rabbit carotid body to hypoxia.

1. The carotid body chemoreceptors are stimulated in situ by hypoxia. We have studied type I cells freshly dissociated from the carotid body of the rabbit. We have used microfluorimetric and patch clamp techniques to examine the responses to hypoxia, to anoxia, and to metabolic inhibition. 2. NADH autofluorescence measured at both 400 and 500 nm increased rapidly and reversibly in response to anoxia or to cyanide (CN-), reflecting a change in mitochondrial metabolism. 3. Indo-1 was used to measure changes in intracellular calcium, [Ca2+]i. Anoxia reversibly increased [Ca2+]i from approximately 50-100 to approximately 200-450 nM in all cells tested. The response showed a striking temperature sensitivity. Responses to hypoxic stimuli were barely detectable at 17-20 degrees C, and were dramatically increased on warming to 36 degrees C. In contrast, responses to K(+)-induced depolarization were only slightly increased in rate of onset and recovery by warming. 4. The rise in [Ca2+]i originated largely from an intracellular store which was slowly depleted by exposure to nominally Ca2(+)-free solutions. Responses were unaffected by blockade of Ca2+ channels with organic (D600, verapamil) or inorganic (Co2+) blockers, by blockade of Na+ channels with tetrodotoxin (TTX), or by increasing action potential duration with tetraethylammonium (TEA). Responses to anoxia were increased by the increased [Ca2+]i loading that follows prior exposure to Ca2(+)-free solutions. 5. Responses to anoxia, to blockade of electron transport by CN-, and to the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), were equivalent in amplitude. The response to anoxia was occluded by concurrent application of FCCP, suggesting that the Ca2+ originates from the same pool in each case. 6. At 35-36 degrees C, responses to graded levels of PO2 were also graded. Thresholds varied between cells, but were typically 30-50 mmHg. Stimulus-responses curves were essentially hyperbolic, increasing dramatically as the PO2 approached 0 mmHg. 7. The sensitivity of cells to hypoxic solutions was increased by acidification of the superfusate over the pH range from 7.3 to 6.85. 8. Cell-attached patch clamp recordings showed depression of spontaneous action potentials associated with a rise in [Ca2+]i during exposure to anoxic solutions. Whole-cell recordings showed that anoxia increased a voltage-gated gK as described previously for CN-, while producing no change in resting conductance. 9. These data suggest that the rise in [Ca2+]i originates largely from Ca2+ efflux from a mitochondrial pool.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1990

Cellular basis of transduction in carotid chemoreceptors.

Understanding transduction mechanisms is central to much of sensory physiology. The carotid chemoreceptors monitor the PO2 of arterial blood en route to the brain and are powerfully excited when the arterial PO2 falls to less than 60 mmHg. The type I cell is generally believed to be the transducer. These cells release catecholamines in response to agents that excite the receptor (hypoxia, cyanide, K(+)-induced depolarization, etc.). Adherent to the cells are the saucer-shaped nerve endings of the axons of the sinus nerve. We and others have used patch-clamp techniques to study the electrophysiological properties of the type I cells. We have also investigated type I cell chemistry with microfluorometric techniques, to measure intracellular Ca2+ concentration ([Ca2+]i), mitochondrial NADH, and mitochondrial membrane potential (delta psi m). During hypoxia there are graded increases in NADH, [Ca2+]i (which presumably will promote transmitter release), and graded depolarization of delta psi m. These results suggest that the Ca2+ is largely derived from an intracellular store, probably from mitochondria, and that release is entrained to delta psi m. Comparative studies with other cells indicate an increased sensitivity of the mitochondria of type I cells to changes in PO2. The data suggest that the electrophysiological responses of type I cells to hypoxia are not central to the response, although the excitability of the cells may provide a mechanism for the modulation of the response by varying voltage-gated Ca2+ influx.

Animals↗
PubMed1989

Electrophysiological responses of dissociated type I cells of the rabbit carotid body to cyanide.

1. The carotid body is the major peripheral sensor of arterial PO2 in the mammal and is excited by cyanide (CN-). Type I cells, the presumed sites for transduction, were freshly dissociated from the carotid body of the adult rabbit and studied with the whole-cell patch clamp technique. 2. Type I cells were hyperpolarized by CN-, the action potential was shortened, and there was an increased after-hyperpolarization. 3. Under voltage clamp control, CN- increased a voltage-dependent outward current, which showed pronounced outward rectification. Tail currents increased by CN- reversed close to the predicted EK, the reversal potential of the CN--induced current depended on extracellular [K+], and the current was blocked by intracellular TEA+ and Cs+. 4. The i-V relation of the CN--induced conductance strongly mirrored that of voltage-gated Ca2+ entry, and the response was abolished by removal of extracellular Ca2+. We conclude that the increased gK is Ca2+ -dependent (gK(Ca]. 5. The Ca2+ current was attenuated by CN-, and showed an increased rate of inactivation. Thus, the increased gK(Ca) must result from an alteration in Ca2+ homeostasis independent of the Ca2+ current, and not an increased Ca2+ entry through voltage-activated channels. 6. Carbachol also hyperpolarized cells and increased a K+ conductance. 7. At depolarized holding potentials a steady-state outward current was increased by CN-. The current reversed close to EK, and was associated with increased current fluctuations. Noise analysis showed that a channel conductance of 3 pS carries the current. 8. The response to CN- was not impaired by the inclusion of 5 mM-MgATP in the patch pipette. 9. If signals to the CNS are initiated by the calcium-dependent release of transmitters from type I cells, transduction would appear to be the direct consequence of the energy dependence of Ca2+ homeostasis.

Animals↗