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C Peers

Publications and source records attributed to C Peers.

At least 55 records · Page 3Linked to original sources
PubMed1999

Potentiation of quantal catecholamine secretion by glibenclamide: evidence for a novel role of sulphonylurea receptors in regulating the Ca(2+) sensitivity of exocytosis.

Electrochemical detection of quantal catecholamine release from PC-12 cells revealed that glibenclamide, an inhibitor of ATP-sensitive K(+) channels, potentiated Ca(2+)-dependent exocytosis evoked by raised extracellular [K(+)] and by exposure of cells to caffeine. Glibenclamide was without effect on voltage-gated Ca(2+) currents, membrane potential, or rises of [Ca(2+)](i) evoked by either raised extracellular [K(+)] or caffeine. The dependence of K(+)-evoked secretion on extracellular Ca(2+) was shifted leftward in the presence of glibenclamide, with a small increase in the plateau level of release, suggesting that glibenclamide primarily increased the Ca(2+) sensitivity of the exocytotic apparatus. Enhancement of secretion by glibenclamide was reversed by pinacidil and cromakalim, indicating that the effects of glibenclamide were mediated via an action on a sulfonylurea receptor. These results demonstrate that sulfonylurea receptors can modulate Ca(2+)-dependent exocytosis via a mechanism downstream of Ca(2+) influx or mobilization.

ATP-Binding Cassette Transporters↗
PubMed1999

Modulation of recombinant human cardiac L-type Ca2+ channel alpha1C subunits by redox agents and hypoxia.

1. Whole-cell patch clamp recordings were used to investigate the modulation by reducing and oxidizing agents of recombinant human cardiac L-type Ca2+ channel alpha1C subunits stably expressed in human embryonic kidney (HEK 293) cells. 2. The oxidizing agents thimerosal (10 microM) and p-chloromercuribenzene sulphonic acid (PCMBS; 2 microM to 2 mM) caused irreversible inhibition of Ca2+ channel currents. The reducing agent 1,4-dithiothreitol (DTT; 2 mM) was without effect on Ca2+ channel currents, but reversed the inhibitory actions of thimerosal and PCMBS. 3. Ca2+ channel currents were also inhibited by pretreatment with the methanethiosulphonate compound (2-aminoethyl)methanethiosulphonate (MTSEA, 2.5 mM), but were unaffected by identical pretreatment with (2-sulphonatoethyl)methanethiosulphonate (MTSES, 10 mM). The effects of MTSEA could be fully reversed by DTT (2 mM). The degree of current inhibition caused by 200 microM PCMBS was not significantly affected by pretreatment with MTSEA, and following PCMBS treatment, MTSEA caused a similar degree of inhibition to that observed in cells that were not previously treated with PCMBS. These findings suggested that distinct thiol groups were modulated by these two agents. 4. Hypoxic inhibition of Ca2+ channel currents was unaffected by pretreatment of cells with MTSEA but was fully prevented by treatment with PCMBS. Our results indicate that distinct cysteine residues on the alpha1C subunit can undergo redox modulation and in so doing alter channel function. Some, but not all, of these residues appear to be associated with the mechanism underlying inhibition of this channel by hypoxia.

Calcium Channel Blockers↗
PubMed1999

Chronic hypoxia enhances the secretory response of rat phaeochromocytoma cells to acute hypoxia.

1. Amperometric recordings were made from individual phaeochromocytoma (PC12) cells using carbon fibre microelectrodes to investigate the effects of chronic hypoxia (10% O2) on the secretory responses evoked by acute hypoxia. 2. Exposure to chronic hypoxia for 21-26 h increased the frequency of exocytotic events evoked in response to acute hypoxia (PO2 ca 10-60 mmHg). 3. Chronic hypoxia increased the value of Q1/3, determined by the integration of amperometric events, indicating an increase in quantal size: this reflects either an increase in vesicular dimensions or vesicular catecholamine concentration. 4. Exocytotic frequency evoked by bath application of tetraethylammonium (1-10 mM) was significantly enhanced following chronic hypoxia. 5. In both control and chronically hypoxic PC12 cells, exocytosis in response to acute hypoxia was completely abolished in Ca2+-free solutions. Cd2+ (200 microM) completely inhibited exocytosis from control cells, but left a significant residual release in chronically hypoxic PC12 cells. 6. The Cd2+-resistant release evoked by acute hypoxia in chronically hypoxic PC12 cells was inhibited by inorganic ions (0.01-10 mM) in a potency order of La3+ > Gd3+ > Zn2+. Ni2+ (10 mM) was without effect. 7. Our results suggest that chronic hypoxia enhances the secretory response of PC12 cells in part by increasing the depolarization mediated by an oxygen-sensitive K+ channel. In addition, acute hypoxia activates a Cd2+-resistant Ca2+ influx pathway in chronically hypoxic PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1999

Store-operated Ca2+ influx and voltage-gated Ca2+ channels coupled to exocytosis in pheochromocytoma (PC12) cells.

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.

Animals↗
PubMed1999

Potential identification of the O2-sensitive K+ current in a human neuroepithelial body-derived cell line.

Whole cell recording of H-146 cells revealed that the outward K+ current was completely inhibited by quinidine (IC50 approximately 17 microM). In contrast, maximal concentrations of 4-aminopyridine (4-AP; >/=10 mM) reversibly blocked only approximately 60% (IC50 approximately 1.52 mM). Ten millimolar 4-AP had no effect on the inhibition by hypoxia, which reduced current density from approximately 27 to approximately 13 pA/pF, whereas 1 mM quinidine abolished the hypoxic effect. In current clamp, 10 mM 4-AP depolarized the cell by approximately 18 mV and hypoxia caused further reversible depolarization of approximately 4 mV. One millimolar quinidine collapsed the membrane potential and abrogated any further hypoxic depolarization. RT-PCR revealed expression of the acid-sensitive, twin P domain K+ channel TASK but not of TWIK, TREK, or the known hypoxia-sensitive Kv2.1, which was confirmed by sequencing and further PCR with primers to the coding region of TASK. However, a reduction in extracellular pH had no effect on K+ current. Thus, although the current more closely resembles TWIK than TASK pharmacologically, structurally the reverse appears to be true. This suggests that a novel acid-insensitive channel related to TASK may be responsible for the hypoxia-sensitive K+ current of these cells.

Base Sequence↗
PubMed1998

Ionic currents in carotid body type I cells isolated from normoxic and chronically hypoxic adult rats.

Whole-cell recordings were used to investigate the effects of a 3-week period of hypoxia (10% O2) on the properties of K+ and Ca2+ currents in type I cells isolated from adult rat carotid bodies. Chronic hypoxia significantly increased whole-cell membrane capacitance. K+ current amplitudes were not affected by this period of hypoxia, but K+ current density was significantly reduced in cells from chronically hypoxic rats as compared with normoxically maintained, age-matched controls. K+ current density was separated into Ca2+-dependent and Ca2+-independent components by bath application of 200 microM Cd2+, which blocked Ca2+ currents and therefore, indirectly, Ca2+-dependent K+ currents. Ca2+-dependent K+ current density was not significantly different in control and chronically hypoxic type I cells. Cd2+-resistant (Ca2+-insensitive) K+ current densities were significantly reduced in type I cells from chronically hypoxic rats. Acute hypoxia (Po2 15-22 mmHg) caused reversible, selective inhibition of Ca2+-dependent K+ currents in both groups of cells and Ca2+-insensitive K+ currents were unaffected by acute hypoxia. Ca2+ channel current density was not significantly affected by chronic hypoxia, nor was the degree of Ca2+ channel current inhibition caused by nifedipine (5 microM). Acute hypoxia did not affect Ca2+ channel currents in either group. Our results indicate that adult rat type I cells undergo a selective suppression of Ca2+-insensitive, voltage-gated K+ currents in response to chronic hypoxia in vivo. These findings are discussed in relation to the known adaptations of the intact carotid body to chronic hypoxia.

Adaptation, Physiological↗
PubMed1998

Inhibition of Ca2+-dependent K+ channels in rat carotid body type I cells by protein kinase C.

1. Whole-cell patch clamp recordings were used to investigate the effects of protein kinase C (PKC) activation on K+ and Ca2+ currents in type I cells isolated from the rat carotid body. 2. Pretreatment of cells for 10 min at 37 C with 4alpha-phorbol 12,13-didecanoate (4alpha-PDD, 200 nM), a phorbol ester which does not activate PKC, did not affect K+ current density as compared with cells pretreated with vehicle alone. By contrast, identical pretreatment with 200 nM 12-O-teradecanoylphorbol-13-acetate (TPA, a PKC activator) dramatically reduced K+ current density in type I cells. This effect was prevented by co-incubation of cells with the PKC inhibitor bisindolylmaleimide (BIM, 3 microM). 3. The sensitivity of K+ currents to inhibition by 200 microM Cd2+ (indicative of the presence of Ca2+-dependent K+ channels) was markedly reduced in TPA-treated cells as compared with sham-treated cells, cells treated with 4alpha-PDD, and cells treated with both TPA and BIM. Cd2+-resistant K+ current densities were of similar magnitude in all four groups of cells, as were the input resistances determined over the voltage range -100 mV to -50 mV. 4. Ca2+ channel current density was not significantly different in type I cells pretreated with 200 nM 4alpha-PDD as compared with cells treated with the same concentration of TPA. 5. The degree of inhibition of K+ currents caused by hypoxia (Po2 15-20 mmHg) was unaltered by pretreatment of cells with 3 microM BIM. 6. The resting membrane potential of cells pretreated with TPA was depolarized as compared with controls, and the Ca2+-dependent K+ channel inhibitor iberiotoxin (20 nM) failed to depolarize these cells further. 7. Our results suggest that activation of PKC causes a marked, selective inhibition of Ca2+-dependent K+ currents in type I carotid body cells, but that PKC activation is unlikely to account for inhibition of these channels by acute hypoxia.

Animals↗
PubMed1998

Hypoxia evokes catecholamine secretion from rat pheochromocytoma PC-12 cells.

We have monitored exocytosis of catecholamines from individual PC-12 cells by amperometry using carbon fiber microelectrodes in order to investigate possible secretory responses to acute hypoxia. In normoxia, no secretion was detected from cells perfused with a solution containing 5 mM K+. However, when [K+] was raised (10-100 mM), exocytotic events were observed. Hypoxia (PO2 11 mmHg) stimulated secretion from PC-12 cells, and in hypoxic conditions exocytosis was greater at each [K+] studied as compared with normoxia. Hypoxia-evoked secretion was abolished in Ca2+ free solutions containing 1 mM EGTA and by the non-specific Ca2+ channel blocker, Cd2+ (200 microM). Secretion was also largely inhibited by omega-conotoxin GVIA (1 microM). Exocytosis was also observed in normoxia when cells were exposed to tetraethylammonium (1-10 mM), but not 4-aminopyridine (3 mM). Our findings indicate that hypoxia evokes exocytosis via depolarization arising from inhibition of a TEA-sensitive K+ conductance, leading to Ca2+ influx primarily via N-type Ca2+ channels.

Animals↗
PubMed1998

Arachidonic acid inhibits both K+ and Ca2+ currents in isolated type I cells of the rat carotid body.

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K+ and Ca2+ channels in isolated rat type I carotid body cells. AA (2-20 microM) produced a concentration-dependent inhibition of both K+ currents and Ca2+ channel currents. The effects of AA on K+ currents were unaffected by indomethacin (5 microM), phenidone (5 microM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.

Animals↗
PubMed1998

Inhibition of recombinant human cardiac L-type Ca2+ channel alpha1C subunits by 3-isobutyl-1-methylxanthine.

Inhibition of ion channels by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and related compounds has been demonstrated in various cell types, including the neuromuscular junction, GH3 cells and vascular smooth muscle cells. These effects may be unrelated to the actions of these compounds on cellular metabolism, intracellular Ca2+ stores and phosphodiesterase inhibition. In this study, the inhibition of recombinant human cardiac L-type Ca2+ channel alpha1C subunits by IBMX was examined using the whole-cell configuration of the patch clamp technique. Inhibition was repeatable, voltage-independent and associated with increased apparent channel inactivation. The actions of IBMX were unaffected in the presence of inhibitors of protein kinases A and G. The non-xanthine phosphodiesterase inhibitor rolipram had a small inhibitory effect on currents, but this was also unaffected by a protein kinase A inhibitor. These effects of IBMX could not be attributed to release of Ca2+ from intracellular stores. Our findings indicate that methylxanthines can inhibit the cardiac L-type Ca2+ channel alpha1C subunit in the absence of auxiliary subunits by an undetermined, possibly direct mechanism.

1-Methyl-3-isobutylxanthine↗
PubMed1998

The regulation of neurotransmitter secretion by protein kinase C.

The effect of protein kinase C (PKC) on the release of neurotransmitters from a number preparations, including sympathetic nerve endings, brain slices, synaptosomes, and neuronally derived cell lines, is considered. A comparison is drawn between effects of activation of PKC on neurotransmitter release from small synaptic vesicles and large dense-cored vesicles. The enhancement of neurotransmitter release is discussed in relation to the effect of PKC on: 1. Rearrangement of the F-actin-based cytoskeleton, including the possible role of MARCKS in this process, to allow access of large dense-cored vesicles to release sites on the plasma membrane. 2. Phosphorylation of key components in the SNAP/SNARE complex associated with the docking and fusion of vesicles at site of secretion. 3. Ion channel activity, particularly Ca2+ channels.

Animals↗
PubMed1998

Hypoxia enhances [3H]noradrenaline release evoked by nicotinic receptor activation from the human neuroblastoma SH-SY5Y.

We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K+]o (100 mM) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [3H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K+-evoked release was unaffected by hypoxia (PO2 approximately 30-38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca2+o with 1 mM EGTA abolished NA release. K+-evoked release was also dramatically reduced in the presence of 200 microM Cd2+ to block voltage-gated Ca2+ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd2+-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca2+ influx through the nAChR pore, or by activating an unidentified Cd2+-resistant Ca2+-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.

Calcium↗
PubMed1998

O2-sensitive K+ channels in neuroepithelial body-derived small cell carcinoma cells of the human lung.

Neuroepithelial bodies act as airway O2 sensors, but studies of their activity at the cellular level have been severely limited because they are present at such a low density in lung tissue. Small cell lung carcinoma (SCLC) cells are believed to be derived from neuroepithelial body cells and may represent a model system for investigating the mechanisms of airway chemoreception. Here we have used the whole cell patch-clamp technique to investigate the effects of acute hypoxia on voltage-gated ionic currents and membrane potential in H-146 SCLC cells. Step depolarizations evoked transient inward currents due to activation of Na+ and Ca2+ channels, followed by outward K+ currents. K+ currents were partially inhibited by 200 microM Cd2+ (indicative of the presence of a Ca2+-dependent component of the K+ current) and were inhibited by tetraethylammonium (TEA) in a concentration-dependent manner, although even at 100 mM TEA, a residual K+ current could be detected. Hypoxia (PO2 15-20 mmHg) caused a reversible inhibition of outward K+ currents without affecting inward currents. Inhibition by hypoxia was also observed in the presence of Cd2+. Hypoxia and TEA caused membrane depolarization in H-146 cells, and their effects appeared additive. These findings indicate that H-146 cells possess O2-sensitive, Ca2+-independent K+ channels that can influence cell membrane potential. SCLC cells may, therefore, represent a good model for investigating the mechanisms underlying O2 sensing by airway chemoreceptor cells.

Cadmium↗
PubMed1997

Swelling- and cAMP-activated Cl- currents in isolated rat carotid body type I cells.

1. In the whole-cell configuration of the patch clamp technique, isolated rat carotid body type I cells exhibited reversible activation of Cl- currents during cell swelling effected by hypotonic extracellular solutions. 2. Hypotonic solutions evoked outwardly rectifying, non-inactivating currents which showed time-independent activation. The reversal potential (E(rev)) for the hypotonically evoked current was 1.6 +/- 0.6 mV (n = 26). Reduction of extracellular Cl- from 133 to 65.5 mM caused a shift in E(rev) of +14.7 +/- 0.4 mV (n = 5). 3. The swelling-activated Cl- current could not activate when ATP was omitted from the patch pipette or when substituted for the non-hydrolysable ATP analogues 5'-adenylylimidodiphosphate, AMP-PNP (2 mM) or beta, gamma-methylene-adenosine 5'-triphosphate. AMP-PCP (2 mM). The current also failed to activate in the absence of free intracellular Ca2+. 4. The swelling-activated Cl- current was sensitive to blockade by the Cl- channel blockers niflumic acid (300 microM) and 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS; 200 microM), although the blockade by DIDS was voltage dependent. 5. A similar, non-inactivating, outwardly rectifying Cl- current was evoked by the inclusion of cAMP (200 microM) in the patch pipette. This current could be inhibited by niflumic acid (300 microM), DIDS (200 microM) and hypertonic solutions, and was virtually abolished in the absence of intracellular ATP. 6. In conclusion, carotid body type I cells possess Cl- currents activated by cell swelling and rises in intracellular cAMP concentration. These currents may be involved in cell volume regulation, blood volume and osmolarity regulation and the response of the type I cell to chemostimuli.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Developmental changes in isolated rat type I carotid body cell K+ currents and their modulation by hypoxia.

1. Whole-cell patch-clamp recordings were used to investigate possible age-related changes in K+ currents of type 1 carotid body cells isolated from the rat. K+ current density increased with age, as measured in cells isolated from 4-day-old, 10-day-old and adult rats (> or = 5 weeks old). 2. The proportion of current reversibly inhibited by high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions, indicative of the proportion of current attributable to activation of Ca(2+) -sensitive K+ (KCa) channels, was significantly smaller in cells of 4-day-old rats compared with 10-day-old rats, despite inward Ca2+ current densities being similar in these two age groups. Inhibition of K+ currents by high [Mg2+], low [Ca2+] solutions was similar in 10-day-old and adult type 1 cells. 3. Hypoxia (PO2, 16-23 mmHg) caused reversible reductions in type I cells from rats of all age groups. However, reductions seen in cells of 4-day-old rats were significantly smaller than those seen in cells of 10-day-olds and adults. The degree of hypoxic inhibition in these latter two groups was not significantly different. 4. In the presence of high [Mg2+], low [Ca2+] solutions, hypoxia (PO2, 16-23 mmHg) was without significant effect on residual K+ currents in cells from all age groups. 5. These observations indicate that K+ current density increases with postnatal age in the rat. Between days 4 and 10, there appears to be a predominant enhancement of KCa channels, and over the same age range hypoxic sensitivity of K+ currents increases. Our findings demonstrate that this latter observation arises because hypoxia selectively inhibits KCa channels in cells at all ages studied. These results suggest an important role for KCa channels in postnatal maturation of hypoxic chemoreception in the rat carotid body.

Aging↗
PubMed1997

Hypoxia inhibits the recombinant alpha 1C subunit of the human cardiac L-type Ca2+ channel.

1. Whole-cell patch clamp recordings were used to investigate the effects of hypoxia on recombinant human L-type Ca2+ channel alpha 1C subunits stably expressed in human embryonic kidney (HEK 293) cells. 2. Ca2+ channel currents were reversibly inhibited by hypoxia (PO2 < 90 mmHg). The degree of inhibition depended on the charge carrier used, Ca2+ currents being more O2 sensitive than Ba2+ currents. 3. Hypoxic inhibition of Ca2+ channel currents was more pronounced at lower activating membrane potentials (< or = +30 mV), and was associated with a slowing of activation kinetics. Current inactivation and deactivation were unaffected by hypoxia. 4. Since hypoxia similarly regulates native L-type Ca2+ channels in vascular smooth muscle cells, our results suggest that hypoxic regulation of L-type Ca2+ channels arises from modification of structural features of the alpha 1 subunit common to cardiac and smooth muscle L-type channels.

Animals↗
PubMed1997

Multiple effects of nordihydroguaiaretic acid on ionic currents in rat isolated type I carotid body cells.

1. The effects of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the ionic currents of rat carotid body type I cells were investigated by use of whole-cell and outside-out patch clamp techniques. 2. NDGA (5-50 microM) produced a concentration-dependent inhibition of whole-cell K+ currents at all activating test potentials (holding potential -70 mV). The time-course of the inhibition was also concentration-dependent and the effects of NDGA were only reversible following brief periods of exposure (<2 min). Another lipoxygenase inhibitor, phenidone (5 microM), was without effect on whole-cell K+ currents in carotid body type I cells. 3. NDGA (5-50 microM) also inhibited whole-cell Ca2+ channel currents (recorded with Ba2+ as charge carrier) in a concentration-dependent manner. 4. Isolation of voltage-gated K+ channels by use of high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions revealed a direct inhibition of the voltage-sensitive component of the whole-cell K+ current by NDGA (50 microM). 5. In excised, outside-out patches NDGA (20-50 microM) increased large conductance, Ca2+ activated K+ channel activity approximately 10 fold, an effect which could be reversed by either tetraethylammonium (10 mM) or charybdotoxin (30 nM). 6. It is concluded that NDGA activates maxi-K+ channels in carotid body type I cells and over the same concentration range inhibits voltage-sensitive K+ and Ca2+ channels. The inhibition of whole cell K+ currents seen is most likely due to a combination of direct inhibition of the voltage-sensitive K+ current and indirect inhibition of maxi-K+ channel activity through blockade of Ca2+ channels.

Animals↗