Biomedical subjects

A Mathie

Publications and source records attributed to A Mathie.

At least 19 recordsLinked to original sources

PubMed2002

Inhibition of the potassium current IK(SO), in cerebellar granule cells, by the inhibitors of MEK1 activation, PD 98059 and U 0126.

IK(SO) is a standing-outward potassium current found in cerebellar granule neurons which is inhibited by the activation of muscarinic M(3) receptors. However the pathway between muscarinic receptor activation and current inhibition is unknown. Using two structurally distinct inhibitors of the activation of MEK1 (mitogen activated protein (MAP) kinase kinase 1), PD 98059 and U 0126, we have shown that the MAP kinase signalling cascade does not appear to underlie muscarinic inhibition of IK(SO), recorded using whole-cell patch-clamp methods. Nevertheless, both PD 98059 and U 0126 caused an inhibition of IK(SO) when applied acutely with 30 microM of each compound producing around 50% inhibition of the current. In addition, U 0125, which is structurally related to U 0126 but has a much lower potency for inhibiting MEK1 activation, was also able to inhibit IK(SO) to a similar degree. Neither the inhibition by PD 98059 nor that by U 0126 was found to be voltage dependent. This was true whether the IK(SO) current was outward or inward. Block of IK(SO) by these two compounds may compromise interpretation of studies in intact neuronal preparations when they are used as MEK1 inhibitors.

Animals↗

PubMed2000

The role of Ca2+ stores in the muscarinic inhibition of the K+ current IK(SO) in neonatal rat cerebellar granule cells.

Cerebellar granule neurons (CGNs) possess a standing outward potassium current (IK(SO)) which shares many similarities with current through the two-pore domain potassium channel TASK-1 and which is inhibited following activation of muscarinic acetylcholine receptors. The action of muscarine on IK(SO) was unaffected by the M2 receptor antagonist methoctramine (100 nM) but was blocked by the M3 antagonist zamifenacin, which, at a concentration of 100 nM, shifted the muscarine concentration-response curve to the right by around 50-fold. Surprisingly, M3 receptor activation rarely produced a detectable increase in [Ca2+]i unless preceded by depolarization of the cells with 25 mM K+. Experiments with thapsigargin and ionomycin suggested that the endoplasmic reticulum Ca2+ stores in CGNs were depleted at rest. In contrast, cerebellar glial cells in the same fields of cells possessed substantial endoplasmic reticulum Ca2+ stores at rest. Pretreatment of the cells with BAPTA AM, thapsigargin or the phospholipase C (PLC) inhibitor U-73122 all blocked the muscarine-induced Ca2+ signal but had little or no effect on muscarinic inhibition of IK(SO). Raising [Ca2+]i directly with ionomycin caused a small but significant inhibition of IK(SO). It is concluded that muscarine acts on M3 muscarinic acetylcholine receptors both to inhibit IK(SO) and to mobilize Ca2+ from intracellular stores in CGNs. While the mobilization of Ca2+ occurs through activation of PLC, this does not seem to be the primary mechanism underlying muscarinic inhibition of IK(SO).

Animals↗

PubMed2000

Purinergic and muscarinic receptor activation activates a common calcium entry pathway in rat neocortical neurons and glial cells.

The nature of metabotropic purinergic and muscarinic receptor-mediated increases in intracellular calcium in primary rat neocortical neurons and glial cells has been investigated using fluorescence imaging techniques. Bath-application of ATP and muscarine (10 microM) elicited a characteristic increase in intracellular calcium in both neurons and glial cells. The profile of this response consisted of an initial transient increase followed by a sustained elevation (the plateau phase) which was dependent on extracellular calcium. Examination of the pharmacological basis of the purinergic receptor-mediated calcium response using 10 microM 2-methyl-thio ATP (MeS-ATP) and UTP revealed that P(2Y) receptor activation underlies this response. The calcium influx pathway responsible for the sustained calcium response was inhibited by metal ions. In both cell types La(3+) and Zn(2+) (100 microM) effectively inhibited the plateau phase of the response, whilst 100 microM Ni(2+) had little or no effect. In conclusion, P(2Y) purinergic and muscarinic receptor activation evoke a sustained increase in intracellular calcium in neocortical neurons and glial cells. This response has similar characteristics to that we have previously described following mGlu(5) activation. We propose that in these cell types stimulation of metabotropic receptors coupled to phosphoinositide turnover activates a common calcium entry pathway that is distinct from voltage-gated calcium channels and resembles store-operated calcium entry.

Adenosine Triphosphate↗

PubMed2000

A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons.

Cerebellar granule neurons (CGNs) are one of the most populous cells in the mammalian brain. They express an outwardly rectifying potassium current, termed a "standing-outward" K(+) current, or IK(SO), which does not inactivate. It is active at the resting potential of CGNs, and blocking IK(SO) leads to cell depolarization. IK(SO) is blocked by Ba(2+) ions and is regulated by activation of muscarinic M(3) receptors, but it is insensitive to the classical broad-spectrum potassium channel blocking drugs 4-aminopyridine and tetraethylammonium ions. The molecular nature of this important current has yet to be established, but in this study, we provide strong evidence to suggest that IK(SO) is the functional correlate of the recently identified two-pore domain potassium channel TASK-1. We show that IK(SO) has no threshold for activation by voltage and that it is blocked by small extracellular acidifications. Both of these are properties that are diagnostic of TASK-1 channels. In addition, we show that TASK-1 currents expressed in Xenopus oocytes are inhibited after activation of endogenous M(3) muscarinic receptors. Finally, we demonstrate that mRNA for TASK-1 is found in CGNs and that TASK-1 protein is expressed in CGN membranes. This description of a functional two-pore domain potassium channel in the mammalian central nervous system indicates its physiological importance in controlling cell excitability and how agents that modify its activity, such as agonists at G protein-coupled receptors and hydrogen ions, can profoundly alter both the neuron's resting potential and its excitability.

Animals↗

PubMed2000

Inhibition of delayed rectifier K+ conductance in cultured rat cerebellar granule neurons by activation of calcium-permeable AMPA receptors.

Activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors in cerebellar granule cells during perforated-patch whole-cell recordings activated an inward current at negative voltages which was followed, after a delay, by the inhibition of an outward potassium current at voltages positive to -20 mV. The activated inward current was inwardly rectifying suggesting that the AMPA receptors were Ca2+-permeable. This was confirmed by direct measurements of intracellular calcium where Ca2+ rises were seen following AMPA receptor activation in Na+-free external solution. Ca2+ rises were equally large in the presence of 100 microM Cd2+ to block voltage-gated Ca2+ channels. Specific voltage-protocols, allowing selective activation of the delayed rectifier potassium current (KV) and the transient A current (KA), showed that kainate inhibited KV, but not to any great extent KA. The inhibition of KV was blocked by the AMPA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and was no longer observed when the KV current was abolished with high concentrations of Ba2+. The responses to kainate were not altered by pre-treating the cells with pertussis toxin, suggesting that the AMPA receptor stimulation of the G-protein Gi cannot account for the effects observed. Replacing extracellular Na+ with choline did not alter the inhibition of KV by kainate, however, removing extracellular Ca2+ reduced the kainate response. The inhibition of KV by kainate was unaffected by the presence of 100 microM Cd2+. The guanylyl cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), did not alter kainate inhibition of KV. It is concluded that ion influx (particularly Ca2+ ions) through AMPA receptor channels following receptor activation leads to an inhibition of KV currents in cerebellar granule neurons.

Animals↗

PubMed1999

Inhibition of neuronal KV potassium currents by the antidepressant drug, fluoxetine.

1. The effect of the antidepressant drug, fluoxetine on neuronal delayed rectifier (KV) potassium (K) currents was investigated using perforated-patch whole-cell electrophysiological recording methods. 2. Fluoxetine was an effective inhibitor of KV currents in cerebellar granule neurons (CGNs) and also inhibited recombinant KV1.1 channels expressed in Chinese hamster ovary (CHO) cells. 3. Fluoxetine had an IC50 of 11 microM in CGNs but was slightly less potent on KV1.1 channels (IC50=55 microM). Interestingly, fluoxetine was a much more potent inhibitor of KV1.1 expressed in mammalian cells than has been found previously for the same homomeric channel expressed in Xenopus oocytes. 4. At concentrations that produced around 50% block, the shape of the KV currents in the presence of fluoxetine was simply scaled down when compared to control currents. 5. The effect of fluoxetine on KV currents in CGNs was neither voltage-dependent nor dependent on the channels being in their open state. Both of these observations suggest that fluoxetine does not act as a simple open channel blocking agent. 6. It is concluded that block of KV currents in mammalian neurons can occur at therapeutic levels of fluoxetine. This could lead to an increase in neuronal excitability and this effect may contribute to the therapeutic antidepressant action of fluoxetine.

Animals↗

PubMed1998

Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons.

1. Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization-activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N-methyl-D-glucamine) or by addition of 10 mM caesium or barium ions. 2. The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl- > Br- > I- > cyclamate. All substituted permeant anions also blocked the current. 3. The current was blocked by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), 9AC (anthracene-9-carboxylic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) but was unaffected by SITS (4-acetamido-4'-isothiocyanatostilbene- 2,2'-disulphonic acid) and niflumic acid. The effective blockers were voltage dependent; DIDS and NPPB were more effective at depolarized potentials while 9AC was more effective at hyperpolarized potentials. 4. The current was enhanced by extracellular acidification and reduced by extracellular alkalinization. Reducing external osmolarity was without effect in conventional whole-cell recording but enhanced current amplitude in those perforated-patch recordings where little current was evident in control external solution. 5. The current in SCG neurons was blocked by external cadmium and zinc. ClC-2 chloride currents expressed in Xenopus oocytes were also sensitive to block by these divalent ions and by DIDS but the sensitivity of ClC-2 to block by cadmium ions was lower than that of the current in SCG neurons. 6. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed the presence of mRNA for ClC-2 in SCG neurons but not in rat cerebellar granule cells which do not possess a hyperpolarization-activated Cl- current. 7. The data suggest that ClC-2 may be functionally expressed in rat SCG neurons. This current may play a role in regulating the internal chloride concentration in these neurons and hence their response to activation of GABAA receptors.

Animals↗

PubMed1998

Voltage-activated potassium channels in mammalian neurons and their block by novel pharmacological agents.

1. Electrophysiological studies have shown that a number of different types of potassium (K) channel currents exist in mammalian neurons. Among them are the voltage-gated K channel-currents which have been classified as fast-inactivating A-type currents (KA) and slowly inactivating delayed-rectifier type currents (KDR). 2. Two major molecular superfamilies of K channel have been identified; the KIR superfamily and the Shaker-related superfamily with a number of different pore-forming alpha-subunits in each superfamily. 3. Within the Shaker-related superfamily are the KV family, comprising of at least 18 different alpha-subunits that almost certainly underlie classically defined KA and KDR currents. However, the relationship between each of these cloned alpha-subunits and native voltage-gated K currents remains, for the most part, to be established. 4. Classical pharmacological blockers of voltage-gated K channels such as tetraethylammonium ions (TEA), 4-aminopyridine (4-AP), and certain toxins lack selectivity between different native channel currents and between different cloned K channel currents. 5. A number of other agents block neuronal voltage-gated K channels. All of these compounds are used primarily for other actions they possess. They include organic calcium (Ca) channel blockers, divalent and trivalent metal ions and certain calcium signalling agents such as caffeine. 6. A number of clinically active tricyclic compounds such as imipramine, amitriptyline, and chlorpromazine are also potent inhibitors of neuronal voltage-gated K channels. These compounds are weak bases and it appears that their uncharged form is required for activity. These compounds may provide a useful starting point for the rational design of novel selective K channel blocking agents.

4-Aminopyridine↗

PubMed1998

Activation of group I metabotropic glutamate receptors elicits pH changes in cultured rat cortical glia and neurons.

Activation of metabotropic glutamate receptors is known to elicit a rise in intracellular Ca2+ and the present study was undertaken to see whether they also modulate the intracellular pH (pHi) of neurons and glia. Measurements of the pHi of neurons and astrocytes were made with the ratiometric fluorescent dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. In the absence of bicarbonate, stimulation with the specific metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid caused a fall in pHi in both astrocytes and neurons. In the presence of bicarbonate, stimulation with 25 microM 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid elicited a rise in pHi in the astrocytes, while the neurons responded with a small acidification. The astrocytic alkalinization could also be elicited by the specific group I metabotropic glutamate receptor agonist (S)-3-hydroxyphenylglycine but not by the group II agonist (2S,1'S,2'S)-(2-carboxycyclopropyl)glycine or by the group III agonist L(+)-2-amino-4-phosphonobutyric acid. The alkalinization of glial cells could be reduced by preloading the cells with BAPTA, but not by removal of extracellular Ca2+. Depolarization of the astrocytes with potassium elicited a small alkalinization, but stimulation with 100 microM 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid in high potassium medium elicited a further alkalinization. It is concluded that activation of group I metabotropic glutamate receptors leads to an alkalinization of astrocytes by a process that involves an elevation of intracellular Ca2+. The pHi changes that follow activation of the metabotropic glutamate receptors may play a role in initiation of glial proliferation following cerebral injury.

Animals↗

PubMed1998

Inhibition by inorganic ions of a sustained calcium signal evoked by activation of mGlu5 receptors in rat cortical neurons and glia.

The effect of mGlu receptor agonists on intracellular calcium (Ca2+) in rat cortical neurons and glial cells was studied. The responses evoked consisted of two phases; an initial transient response followed by a sustained plateau. In both cell types the order of potency of group I mGlu receptor agonists was DHPG > 1S,3R ACPD > 3-HPG. The selective mGlu5 agonist CHPG elicited responses in both cell types as did S4C3-HPG which is thought to be an mGlu5 agonist at high concentrations. S4-CPG had no effect on intracellular Ca2+ levels nor did it inhibit the action of IS,3R ACPD. These results suggest that the responses in both cell types are mediated by mGlu5 receptors. In the absence of extracellular Ca2+ ions, 1S,3R ACPD (100 microM) induced only a transient Ca2+ response which decayed to baseline with a time constant of approximately 20 s in both cell types. Subsequent readdition of Ca2+ (2 mM) to the external solution in the continued presence of 1S,3R ACPD induced a sustained Ca2+ plateau. The sustained Ca2+ plateau could be blocked by a number of inorganic cations, with an order of potency of Zn2+ > or = La3+ > Cd2+ > or = Co2+ > Ni2+ > Mg2+. Similar concentrations of Zn2+ had little effect on Ca2+-influx evoked by 25 mM K+. It is concluded that the Ca2+-entry pathway activated by mGlu5 receptors resembles store-operated Ca2+-entry pathways that have been described in other cell types.

Animals↗

PubMed1997

Is EAG the answer to the M-current?

The resemblance between the Drosophila EAG current and the mammalian M-current is very strong, but final confirmation for a member of the extended EAG family encoding the M-current remains to be determined.

Animals↗

PubMed1997

Team working.

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Education, Medical↗

PubMed1996

A non-inactivating K+ current sensitive to muscarinic receptor activation in rat cultured cerebellar granule neurons.

1. Whole-cell recordings were made from cultured cerebellar granule neurons using perforated patch clamp techniques. The primary cultures were prepared using 6- to 9-day-old Sprague-Dawley rats. 2. Neurons in culture for less than 48 h possessed resting membrane potentials of -29 mV. However, neurons in culture for 7 days had much more hyperpolarized resting membrane potentials (-89 mV). Over the same period, these neurons developed an additional component of outward current. 3. This non-inactivating current was activated by depolarization, exhibited outward rectification and reversed close to the potassium equilibrium potential. The kinetics of activation and deactivation were very rapid. 4. Muscarine ((+)-muscarine chloride) reversibly inhibited the current with an EC50 of 0.17 microM. The inhibition by muscarine was unaffected by pre-incubation for 17-20 h with 120 micrograms ml-1 pertussis toxin. 5. The current and its inhibition by muscarine were unaffected by 100 microM Cd2+. In Ca(2+)-free conditions, the current was significantly larger than in 0.5 mM Ca2+, but inhibition by 10 microM muscarine was significantly reduced. 6. The standing outward current was not obviously affected by 50 microM 5-HT, 50 microM noradrenaline, 50 microM 2-chloroadenosine or 5 mM tetraethylammonium. It was reduced by 10 microM La3+, 10 microM Zn2+ and 1 mM Ba2+. 7. Muscarinic agonists increased the input resistance of neurons and shifted the zero current level in the depolarized direction when voltage clamped. This enhanced excitability was evident under current clamp, where 10 microM muscarine depolarized granule neurons such that action potentials became evident.

Animals↗

PubMed1996

Effects on K+ currents in rat cerebellar granule neurones of a membrane-permeable analogue of the calcium chelator BAPTA.

1. Whole cell recordings of voltage-activated K+ currents were made with the amphotericin B perforated patch technique from cerebellar granule (CG) neurones of 6-8 days rats that had been in culture for 1 to 16 days. By use of appropriate voltage protocols, the effects of the membrane-permeant form of BAPTA, 1,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), on the transient A current (IKA), the delayed rectifier current (IKV) and a standing outward current (IKSO) were investigated. 2. Bath application of 25 microM BAPTA-AM inhibited both IKV and IKSO in cultured neurones, but did not seem to affect IKA. Neither 25 microM BAPTA (free acid) nor 25 microM ethylenediaminetetraacetic acid acetoxymethyl ester (EDTA-AM) had any significant effect on the magnitude of IKSO. Similarly in short-term (1-2 days) cultured CG neurones IKV, but not IKA, was inhibited by 25 microM BAPTA-AM. 3. BAPTA-AM (2.5 microM) reduced IKV in short-term culture CG neurones, with further inhibition being seen when the perfusate was changed to one containing 25 microM BAPTA-AM. 4. Tetraethylammonium ions (TEA) (10 mM) reversibly inhibited IKV in these cells with a similar rate of block of IKV to that induced by 25 microM BAPTA-AM. 5. The degree of inhibition of IKV by 25 microM BAPTA-AM was both time- and voltage-dependent, in contrast to the inhibition of this current by TEA. 6. These data indicate that BAPTA-AM reduces K+ currents in cerebellar granule neurones and that this inhibition cannot be explained in terms of intracellular Ca2+ chelation, but is a direct effect on the underlying channels.

Animals↗

PubMed1995

Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons.

Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.

Alkaloids↗

PubMed1995

Potent block of potassium currents in rat isolated sympathetic neurones by the uncharged form of amitriptyline and related tricyclic compounds.

1. The block of K+ currents by amitriptyline and the related tricyclic compounds cyproheptadine and dizocilpine was studied in dissociated rat sympathetic neurones by whole-cell voltage-clamp recording. 2. Cyproheptadine (30 microM) inhibited the delayed-rectifier current (Kv) by 92% and the transient current (KA) by 43%. For inhibition of Kv, cyproheptaidine had a KD of 2.2 microM. Dizocilpine (30 microM) inhibited Kv by 26% and KA by 22%. The stereoisomers of dizocilpine were equally potent at blocking Kv and KA. 3. Amitriptyline, a weak base, was significantly more effective in blocking Kv at pH 9.4 (KD = 0.46 microM) where the ratio of charged to uncharged drug was 50:50 compared with pH 7.4 (KD = 11.9 microM) where the ratio was 99:1. 4. N-methylamitriptyline (10 microM), the permanently charged analogue of amitriptyline, inhibited Kv by only 2% whereas in the same cells amitriptyline (10 microM) inhibited Kv by 36%. 5. Neither amitriptyline nor N-methylamitriptyline had a detectable effect on Kv when added to the intracellular solution. 6. It is concluded that the uncharged form of amitriptyline is approximately one hundred times more potent in blocking Kv than the charged form. However, this does not seem to be due to uncharged amitriptyline having better access to an intracellular binding site.

Amitriptyline↗

PubMed1995

Quantification of pneumothorax size on chest radiographs using interpleural distances: regression analysis based on volume measurements from helical CT.

OBJECTIVE: The aim of this study was to define the relationship between interpleural distance measurements on an erect posteroanterior chest radiograph and pneumothorax size as measured by helical CT in a series of patients. SUBJECTS AND METHODS: Twenty pneumothoraces from 19 patients (10 males, nine females) were analyzed. Most pneumothoraces were on the right side (n = 16). The etiology was spontaneous in seven patients and iatrogenic in thirteen. All patients underwent an erect inspiratory posteroanterior radiograph and a helical CT scan of the thorax on the same visit to the radiology department. The interpleural distance was measured at three locations and the figures added together. Following helical CT of the thorax, the percentage pneumothorax size was calculated by drawing regions of interest around the relevant hemithorax and lung on 10-mm reconstructed slices. A scattergram of the sum of interpleural distances in centimeters versus percentage pneumothorax size was plotted. RESULTS: Analysis of results showed that percentage pneumothorax size could be calculated by the formula Y = 4.2 + [4.7 x (A + B + C)], r = .98, p < .0001. CONCLUSION: This study identified a formula for accurately calculating percentage pneumothorax size as determined by helical CT from an erect posteroanterior radiograph. Using this formula with the clinical status of the patient should more easily identify patients requiring active intervention.

Adolescent↗

PubMed1994

Modulation of the gating of the transient outward potassium current of rat isolated cerebellar granule neurons by lanthanum.

The effects of the trivalent cation, lanthanum (La3+) on voltage-dependent K+ conductances were studied in rat isolated cerebellar granule neurons under whole-cell voltage-clamp conditions. La3+ at low micromolar concentrations caused a pronounced enhancement in the outward current evoked by depolarising steps from -50 mV, with the apparent recruitment of an inactivating component. The steady-state inactivation curve for the transient outward current, evoked by depolarising steps from -140 mV, was shifted by approximately 40 mV in the depolarising direction by 10 microM La3+, with a slight increase in the slope factor. The kinetics of activation and inactivation were slowed in the presence of La3+. A shift of 10 mV in the depolarising direction was seen for the activation curve of the delayed rectifier current in the presence of 10 microM La3+. These results indicate that La3+ has a potent effect on the gating characteristics of voltage-activated K+ currents. This effect cannot be explained by surface charge considerations.

Animals↗