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C Peers

Publications and source records attributed to C Peers.

At least 37 records · Page 2Linked to original sources
PubMed2001

Acute oxygen sensing: diverse but convergent mechanisms in airway and arterial chemoreceptors.

Airway neuroepithelial bodies sense changes in inspired O2, whereas arterial O2 levels are monitored primarily by the carotid body. Both respond to hypoxia by initiating corrective cardiorespiratory reflexes, thereby optimising gas exchange in the face of a potentially deleterious O2 supply. One unifying theme underpinning chemotransduction in these tissues is K+ channel inhibition. However, the transduction components, from O2 sensor to K+ channel, display considerable tissue specificity yet result in analogous end points. Here we highlight how emerging data are contributing to a more complete understanding of O2 chemosensing at the molecular level.

Animals↗
PubMed2001

Inhibition of depolarisation-evoked [(3)H]noradrenaline release from SH-SYFY human neuroblastoma cells by muscarinic (M1) receptors is not mediated by changes in [Ca(2+)].

The aim of this study was to obtain further understanding of the mechanism by which activation of muscarinic M(1) receptors inhibits K(+)-evoked noradrenaline (NA) release in the human neuroblastoma SH-SY5Y. Previous studies have found that muscarinic M(1) and M(3) receptors couple to the activation of phospholipase C in SH-SY5Y cells leading to an increase in (a) intracellular calcium ([Ca(2+)](i)) and (b) activation of protein kinase C (PKC). This study used specific inhibitors of PKC and conditions which deplete Ca(2+)(i) stores to examine the role of protein kinase C and changes in [Ca(2+)](i) in mediating the inhibition of K(+)-evoked NA release by muscarine. Our data show that pretreatment of SH-SY5Y cell layers with bisindolylmaleimide I (BIM-I) (i) failed to reverse inhibition of K(+)-evoked NA release by muscarine but (ii) did overcome the attenuation of muscarine inhibition following pretreatment with TPA. Furthermore pretreating cell layers with Ca(2+)-free Hepes buffered saline in the presence of thapsigargin, conditions which prevented muscarine induced increases in [Ca(2+)](i), failed to prevent inhibition of K(+)-evoked NA release by muscarine. The effect of muscarine on K(+)-evoked uptake of Ca(2+)(e) was examined in SH-SY5Y cells loaded with Fura-2. Muscarine inhibited Ca(2+)(e)-uptake by decreasing the rate at which Ca(2+) entered SH-SY5Y cells via voltage sensitive Ca(2+)-channels. Thus this study shows that muscarine inhibits depolarisation-evoked NA release by a mechanism which is not dependent on activation of PKC or release of Ca(2+) from internal stores.

Calcium↗
PubMed2001

Amyloid beta peptides mediate hypoxic augmentation of Ca(2+) channels.

Clinical studies indicate that neurodegeneration caused by Alzheimer's amyloid beta peptide (AbetaP) formation can be triggered or induced by prolonged (chronic) hypoxia. Here, we demonstrate that 24-h culture of PC12 cells in 10% O(2) leads to induction of a Cd(2+)-resistant Ca(2+) influx pathway and selective potentiation of L-type Ca(2+) current. Both effects were suppressed or prevented by a monoclonal antibody raised against the N'-terminus of AbetaP, and were fully mimicked by AbetaP(1-40 and) AbetaP(1-42), but not by AbetaP(40-1). Potentiation of L-type currents was also induced by exposure to AbetaP(25-35). Our results indicate that hypoxia induces enhancement of Ca(2+) channels, which is mediated by increased AbetaP formation.

Amyloid beta-Peptides↗
PubMed2001

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.

Alzheimer Disease↗
PubMed2001

Prion protein fragment 106-126 potentiates catecholamine secretion from PC-12 cells.

The toxic actions of scrapie prion protein (PrP(sc)) are poorly understood. We investigated the ability of the toxic PrP(sc) fragment 106-126 to interfere with evoked catecholamine secretion from PC-12 cells. Prion protein fragment 106-126 (PrP106-126) caused a time- and concentration-dependent augmentation of exocytosis due to the emergence of a Ca(2+) influx pathway resistant to Cd(2+) but sensitive to other inorganic cations. In control cells, secretion was dependent on Ca(2+) influx through L- and N-type Ca(2+) channels, but after exposure to PrP106-126, secretion was unaffected by N-type channel blockade. Instead, selective L-type channel blockade was as effective as Cd(2+) in suppressing secretion. Patch-clamp recordings revealed no change in total Ca(2+) current density in PrP106-126-treated cells or in the contribution to total current of L-type channels, but a small Cd(2+)-resistant current was found only in PrP106-126-treated cells. Thus PrP106-126 augments secretion by inducing a Cd(2+)-resistant Ca(2+) influx pathway and alters coupling of native Ca(2+) channels to exocytosis. These effects are likely contributory factors in the toxic cellular actions of PrP(sc).

Animals↗
PubMed2000

Splice variants reveal the region involved in oxygen sensing by recombinant human L-type Ca(2+) channels.

Regulation of vascular smooth muscle Ca(2+) channels by oxygen tension contributes importantly to hypoxic vasodilatation. We previously described the inhibitory effects of hypoxia on the recombinant human cardiac L-type Ca(2+) channel alpha(1C) subunit (hHT isoform) expressed in HEK 293 cells. We now demonstrate that hypoxia inhibits only one of the three naturally occurring splice variants of this channel that differ only in the C-terminal domain, permitting identification of a 71-amino acid insert in the C-terminal region of the channel that confers oxygen sensitivity. Selective restriction of the spliced insert allowed determination of a 39-amino acid region essential for oxygen sensing. This represents the first identification of the structural region of an ion channel required for sensing changes in oxygen tension.

Alternative Splicing↗
PubMed2000

Protein kinase C evokes quantal catecholamine release from PC12 cells via activation of L-type Ca2+ channels.

Application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to PC12 cells under resting conditions evoked quantal catecholamine secretion, as detected amperometrically. This effect was not mimicked by 4alpha-phorbol-12,13-didecanoate, another phorbol ester, which is inactive with respect to protein kinase C activation, and was prevented by the protein kinase C inhibitor bisindolylmaleimide. TPA also caused a rise of [Ca(2+)](i) in Fura-2-loaded PC12 cells, and again this was not mimicked by 4alpha-phorbol-12,13-didecanoate and could be blocked by bisindolylmaleimide. TPA-evoked secretion was entirely dependent on extracellular Ca(2+) and was fully abolished by nifedipine, as were TPA-induced rises of [Ca(2+)](i). Resting membrane potential, monitored using perforated patch recordings, was unaffected by TPA. However, a small (6-8 mV) hyperpolarizing shift in the voltage dependence of Ca(2+) channel currents (determined using whole-cell patch clamp recordings) was induced by TPA, and this could be fully prevented by nifedipine. In contrast to results with depolarizing stimuli, which evoke exocytosis because of Ca(2+) influx through N-type channels in these cells, the present results indicate that protein kinase C activation leads directly to quantal catecholamine secretion in the absence of depolarizing stimuli via a selective shift in the activation of L-type Ca(2+) channels.

Animals↗
PubMed2000

Mitochondrial inhibitors evoke catecholamine release from pheochromocytoma cells.

Quantal catecholamine secretion evoked from individual pheochromocytoma (PC12) cells by exposure to mitochondrial inhibitors and uncouplers was monitored in real time using amperometry. Cyanide (0.05-5 mM) caused a concentration-dependent increase in the frequency of amperometric events. This secretory response was abolished by removal of extracellular Ca(2+) and by the application of Cd(2+) (200 microM), a nonselective blocker of voltage-gated Ca(2+) channels. Secretion was also inhibited by ca. 75% following pretreatment of cells with omega-conotoxin GVIA to inhibit N-type Ca(2+) channels selectively. Secretion was also detected when cells were exposed to rotenone (10 microM), dinitrophenol (250 microM) and p-trifluoromethoxyphenyl hydrazone (1 microM) and, as for cyanide, these secretory responses were abolished by removal of extracellular Ca(2+) or application of 200 microM Cd(2+). These results indicate that, like hypoxia, mitochondrial inhibitors and uncouplers evoke catecholamine secretion from PC12 cells which is wholly dependent on Ca(2+) influx through voltage-gated Ca(2+) channels.

2,4-Dinitrophenol↗
PubMed2000

O(2) sensing by airway chemoreceptor-derived cells. Protein kinase c activation reveals functional evidence for involvement of NADPH oxidase.

Accumulating evidence suggests that neuroepithelial bodies are airway O(2) sensors. Recently, we have established the H-146 small cell lung carcinoma line as a suitable model to study the biochemical basis of neuroepithelial body cell chemotransduction. Here we explore the possibility that hypoxic modulation of K(+) channels is intimately linked to activity of NADPH oxidase. Graded hypoxia caused graded inhibition of whole cell K(+) currents, which correlated well with membrane depolarization. Pretreatment with the phorbol ester, 12-O-tetradecanoyl (TPA), inhibited K(+) currents at all potentials. Although 4alpha-phorbol 12,13-didecanoate and TPA in the presence of bisindolylmaleimide were also able to depress K(+) currents, only TPA could significantly ameliorate hypoxic depression of these currents. Thus, protein kinase C (PKC) activation modulates the sensitivity of these cells to changes in pO(2). Furthermore, because the addition of H(2)O(2), a downstream product of NADPH oxidase, could only activate K(+) currents during hypoxia (when endogenous H(2)O(2) production is suppressed), it appears likely that PKC modulates the affinity of NADPH oxidase for O(2) potentially via phosphorylation of the p47(phox) subunit, which is present in these cells. These data show that PKC is an important regulator of the O(2)-transduction pathway and suggests that NADPH oxidase represents a significant component of the airway O(2) sensor.

4-Aminopyridine↗
PubMed2000

Effects of hypoxia and dithionite on catecholamine release from isolated type I cells of the rat carotid body.

1. Amperometric recordings were conducted to investigate the ability of hypoxia and anoxia to evoke quantal catecholamine secretion from isolated type I cells of the rat carotid body. 2. Hypoxia (PO2 8-14 mmHg) consistently failed to evoke catecholamine secretion from type I cells, when cells were perfused either at room temperature (21-24 C) or at 35-37 C, and regardless of whether Hepes- or HCO3-/CO2-buffered solutions were used. 3. Elevating extracellular [K+] caused concentration-dependent secretion from individual type I cells, with a threshold concentration of approximately 25 mM. In the presence of this level of extracellular K+, hypoxia (PO2 8-14 mmHg) caused a marked enhancement of secretion which was fully blocked by 200 microM Cd2+, a non-specific blocker of voltage-gated Ca2+ channels. 4. Anoxia (N2-equilibrated solution containing 0.5 mM dithionite) evoked exocytosis from type I cells when extracellular [K+] was 5 mM. This secretion was completely inhibited by removal of extracellular Ca2+, but was not significantly affected by Cd2+ (200 microM), Ni2+ (2 mM), Zn2+ (1 mM) or nifedipine (2 microM). Secretion was also observed when 0.5 mM dithionite was added to air-equilibrated solutions. 5. Anoxia also evoked secretion from chemoreceptive phaeochromocytoma (PC12) cells, which was wholly Ca2+ dependent, but unaffected by Cd2+ (200 microM). 6. Our results suggest that hypoxia can evoke catecholamine secretion from isolated type I cells, but only in the presence of elevated extracellular [K+]. This may be due to the cells being relatively hyperpolarized following dissociation. In addition, we have shown that dithionite evokes catecholamine release regardless of PO2 levels, and this release is due mainly to an artefactual Ca2+ influx pathway activated in the presence of dithionite.

Animals↗
PubMed2000

Modulation of recombinant T-type Ca2+ channels by hypoxia and glutathione.

T-type Ca2+ channels are expressed in a wide variety of central and peripheral neurons and play an important role in neuronal firing and rhythmicity. Here we examined the effects of hypoxia on the recently cloned T-type Ca2+ channel alpha1G, alpha1H and alpha1I subunits, stably expressed in HEK 293 cells. In cells expressing the human alpha1H or the rat alpha1I subunit, Ca2+ channel currents were inhibited reversibly by hypoxia (PO2<110 mm Hg). The degree of inhibition was more marked in cells expressing the a1H subunit. This hypoxic inhibition was not voltage dependent. In cells expressing the rat alpha1G subunit, hypoxia caused no detectable reduction in Ca2+ channel activity. Regardless of the channel type examined, hypoxia was without effect on the kinetic properties of the Ca2+ current (activation, inactivation and deactivation) or on steady-state inactivation. Ca2+ current through the alpha1H subunit was enhanced by the reducing agent reduced glutathione (GSH; 2 mM) and inhibited by oxidised glutathione (GSSG; 2 mM). In contrast, Ca2+ current through the alpha1G subunit was unaffected by GSH. In alpha1H cells, neither GSH nor GSSG had any effect on the ability of hypoxia to reduce Ca2+ current amplitudes. Thus, different members of the T-type Ca2+ channel family are differently regulated by hypoxia and redox agents. Hypoxic regulation of the alpha1H subunit appears to be independent of changes in levels of the intracellular redox couple GSSG:GSH.

Animals↗
PubMed2000

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

1. Clotrimazole (CLT) is an antimycotic agent with a potential role in the treatment of cancer. Whole-cell patch clamp recordings and Fura-2 AM fluorescence measurements were used to investigate the inhibition by CLT of recombinant human cardiac L-type Ca2+ channel alpha 1C subunits, stably expressed in human embryonic kidney (HEK 293) cells. 2. CLT (100 nmol l-1 to 25 mumol l-1) reduced Ca2+ channel currents in a concentration-dependent manner. Inhibition was neither use- or voltage-dependent. The effects of CLT were rapid and maximal effects were attained within 3 min. Application of CLT also caused an acceleration of apparent Ca2+ channel current inactivation. 3. Basal current density and the degree of inhibition due to CLT were not significantly altered by pretreating cells with 3 mmol l-1 1-aminobenzotriazole for 1 h, or by dialysing cells for 10 min with 2 mmol l-1 alpha-napthoflavone via the patch pipette, suggesting that the inhibitory action of CLT was not due to inhibition of cytochrome P-450. 4. CLT (10 mumol l-1) did not influence [Ca2+]i, as determined by Fura-2 AM fluorescence measurements. 5. Dialysing cells for 10 min with the non-specific serine/threonine kinase inhibitor H-7 (10 mumol l-1) was without effect on basal current density or on the inhibitory response to 10 mumol l-1 CLT, indicating that CLT is not acting via an indirect effect on these kinases. 6. These data suggest that CLT exerts a direct blocking effect on the alpha 1C subunit at therapeutic concentrations. This effect may explain the abbreviation of the action potential duration by CLT observed in cardiac myocytes.

Antifungal Agents↗
PubMed2000

Three distinct Ca(2+) influx pathways couple acetylcholine receptor activation to catecholamine secretion from PC12 cells.

Amperometry and microfluorimetry were employed to investigate the Ca(2+)-dependence of catecholamine release induced from PC12 cells by cholinergic agonists. Nicotine-evoked exocytosis was entirely dependent on extracellular Ca(2+) but was only partly blocked by Cd(2+), a nonselective blocker of voltage-gated Ca(2+) channels. Secretion and rises of [Ca(2+)](i) observed in response to nicotine could be almost completely blocked by methyllycaconitine and alpha-bungarotoxin, indicating that such release was mediated by receptors composed of alpha7 nicotinic acetylcholine receptor subunits. Secretion and [Ca(2+)](i) rises could also be fully blocked by co-application of Cd(2+) and Zn(2+). Release evoked by muscarine was also fully dependent on extracellular Ca(2+). Muscarinic receptor activation stimulated release of Ca(2+) from a caffeine-sensitive intracellular store, and release from this store induced capacitative Ca(2+) entry that could be blocked by La(3+) and Zn(2+). This Ca(2+) entry pathway mediated all secretion evoked by muscarine. Thus, activation of acetylcholine receptors stimulated rises of [Ca(2+)](i) and exocytosis via Ca(2+) influx through voltage-gated Ca(2+) channels, alpha7 subunit-containing nicotinic acetylcholine receptors, and channels underlying capacitative Ca(2+) entry.

Animals↗
PubMed1999

Hypoxic enhancement of quantal catecholamine secretion. Evidence for the involvement of amyloid beta-peptides.

Prolonged exposure to hypoxia (10% O(2)) enhanced quantal catecholamine release evoked from O(2)-sensing pheochromocytoma (PC12) cells, as monitored using single-cell amperometric recordings. The enhancement of exocytosis was apparent after 12 h of hypoxia and was maximal at 24 h. Elevated levels of secretion were due to the emergence of a Ca(2+) influx pathway that persisted during complete blockade of known voltage-gated Ca(2+) channels. Secretion triggered by this Ca(2+) influx was severely reduced by known inhibitors of Alzheimer's amyloid beta-peptides (AbetaPs), including an N terminus-directed monoclonal antibody. The enhancing effect on secretion of chronic hypoxia was mimicked closely by direct application of AbetaP to cells under normoxic conditions, although the effects of AbetaP were more rapid at onset, being maximal after only 6 h. The present results suggest that prolonged hypoxia can induce formation of Ca(2+)-permeable AbetaP channels and that such induction can lead directly to excessive neurosecretion. This is a potential contributory factor to AbetaP pathophysiology following cerebral ischemia.

Alzheimer Disease↗
PubMed1999

Acid-evoked quantal catecholamine secretion from rat phaeochromocytoma cells and its interaction with hypoxia-evoked secretion.

1. Amperometric recordings using polarized carbon fibre microelectrodes were used to detect exocytosis of catecholamines from rat phaeochromocytoma (PC12) cells in response to a reduction in pHo. 2. Exocytosis was detected at pHo levels of between 7.2 and 6.8. This was probably due to intracellular acidification, since acid-evoked secretion was enhanced by the Na+-H+ exchange blocker ethylisopropylamiloride (30 microM), and was mimicked by sodium propionate (10 mM), which causes selective intracellular acidosis. 3. Acid-evoked exocytosis was abolished by removal of Ca2+o or application of 200 microM Cd2+. It was unaffected by nifedipine, but significantly reduced by either omega-conotoxin GVIA (1 microM) or omega-agatoxin GIVA (200 nM). The two toxins applied together almost completely abolished (> 97 %) acid-evoked secretion. 4. Hypoxia-evoked catecholamine release was potentiated under acidic conditions and suppressed under alkaline conditions in a manner which indicated a greater than additive interaction of these two stimuli. 5. Our results indicate that, like carotid body arterial chemoreceptors, PC12 cells represent model chemoreceptor cells for both hypoxia and acidity and that the release of catecholamines in response to these physiological stimuli is dependent on Ca2+ influx through voltage-gated N- and P/Q-type Ca2+ channels.

Animals↗