Search PubMed⌕ Search

Biomedical subjects

C Peers

Publications and source records attributed to C Peers.

At least 109 records · Page 6Linked to original sources
PubMed1993

Nicotinic acetylcholine receptors in isolated type I cells of the neonatal rat carotid body.

Electrophysiological responses of enzymatically isolated type I cells from the neonatal rat carotid body to cholinergic agonists were examined using the whole-cell patch-clamp technique. Inward currents were evoked in cells clamped at -70 mV in response to bath-applied carbachol and two selective nicotinic agonists, nicotine and dimethylphenylpiperazinium. Muscarine failed to produce any change in membrane current. Responses to nicotine were concentration-dependent and also voltage-dependent, showing strong rectification positive to -40 mV. Currents evoked by nicotine were reduced or abolished in the presence of mecamylamine and also by high concentrations of atropine (10 or 100 microM). Under "current-clamp", nicotine was shown to depolarize type I cells, an effect which was only slowly reversible, but which could be rapidly attenuated by introduction of mecamylamine to the perfusate. In voltage-clamped cells, nicotine could evoke inward currents when extracellular Na+ was replaced by Ca2+. Our results demonstrate the presence of functional nicotinic acetylcholine receptors on type I cells of the neonatal rat carotid body. Activation of these receptors could lead to excitation of the intact carotid body by either of two possible mechanisms: depolarization of type I cells sufficient to open voltage-gated Ca2+ channels, or Ca2+ influx through the receptor pore itself. Either (or both) mechanisms could trigger catecholamine release from type I cells, which is a fundamental step in chemotransmission.

Animals↗
PubMed1993

Nicotinic receptor-mediated release of noradrenaline in the human neuroblastoma SH-SY5Y.

Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12-O-tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 microM mecamylamine but not by 1 microM atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 microM Bay K 8644 (an L-type calcium agonist) and inhibited by 1 microM nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 microM desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Nicotinic acetylcholine receptors in human neuroblastoma (SH-SY5Y) cells.

Whole-cell patch-clamp recordings were used to investigate nicotinic acetylcholine receptors (nAChRs) in the human neuroblastoma cell line, SH-SY5Y. Acetylcholine, nicotine and the neuronal nAChR agonist dimethylphenylpiperazinium iodide (DMPP), but not muscarine, all evoked inward currents in the cells (voltage-clamped at -60 mV). DMPP's actions were concentration- and voltage-dependent, and were antagonised by the neuronal nAChR antagonist mecamylamine (1-3 microM). Atropine was ineffective at 0.1 microM, but at 1 microM caused significant reductions in current amplitudes. Pre-incubation of cells with 2 microM alpha-cobratoxin had no effect on the actions of DMPP, and inward currents could also be induced when extracellular NaCl was replaced with CaCl2. DMPP also reversibly depolarized SH-SY5Y cells. These findings clearly identify nAChRs in SH-SY5Y cells, and provide two possible mechanisms by which receptor activation may lead to noradrenaline release, namely by triggering Ca2+ influx through the nAChR itself or by opening voltage-gated Ca2+ channels.

Acetylcholine↗
PubMed1992

Modulation of ionic currents in isolated type I cells of the neonatal rat carotid body by p-chloromercuribenzenesulfonic acid.

The sulphydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), irreversibly excites the carotid body in vivo. We tested the effects of PCMBS on ionic currents in isolated type I cells, using the whole-cell patch-clamp technique. PCMBS selectively and irreversibly inhibited the Ca(2+)-activated K+ current (IKCa) in a dose-dependent manner (0.01-1 mM). The same concentrations of PCMBS did not affect the Ca(2+)-independent K+ current (IKv), but caused a transient enhancement of the Ca2+ current (ICa). The inhibition of IKCa by PCMBS is similar to the previously reported effects of hypoxia, and suggests a central role for the channels underlying this current in chemotransduction.

4-Chloromercuribenzenesulfonate↗
PubMed1992

Glibenclamide inhibits a voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y.

The whole-cell patch-clamp technique was used to record outward K+ currents in the human neuroblastoma cell line SH-SY5Y. These K+ currents were inhibited by tetraethylammonium (20 mM) and by 4-aminopyridine (2 mM). The Ca2+ channel blocker Cd2+ (0.2 mM) also inhibited K+ currents, indicating that a component was Ca(2+)-activated. Glibenclamide, a presumed selective inhibitor of ATP-sensitive K+ channels, caused reversible inhibitions of the K+ currents and appeared to accelerate their inactivation. These effects were not significantly affected by intracellular ATP (ATPi), and were observed in the presence of 0.2 mM Cd2+. It is concluded that glibenclamide inhibits a voltage-gated, Ca(2+)- and ATPi-independent K+ current in SH-SY5Y cells.

4-Aminopyridine↗
PubMed1992

Blockade of delayed rectifier K+ currents in neuroblastoma x glioma hybrid (NG 108-15) cells by clofilium, a class III antidysrhythmic agent.

1. The whole-cell patch-clamp technique was used to examine the effects of the class III antidysrhythmic agent, clofilium, on voltage-activated delayed rectifier K+ currents (IKv) in undifferentiated mouse neuroblastoma x rat glioma hybrid (NG 108-15) cells. Ca(2+)-activated K+ currents also seen in these cells were abolished by bath application of 4 mM Co2+. 2. Bath application of clofilium (0.3 to 70 microM) caused dose-dependent, irreversible inhibition of IKv in these cells. Under control conditions, activated currents were sustained during 200 ms depolarizing steps, but in the presence of clofilium, or after its wash-out, currents were reduced in amplitude and showed a time-dependent decay. 3. Clofilium blockade of IKv was voltage-dependent; the degree of current inhibition increased with increasing depolarizations. The transient nature of IKv seen in the presence of clofilium was also more apparent at higher test potentials. 4. The effects of clofilium were use-dependent: when cells were left unstimulated during drug application, and then depolarizations were resumed, several pulses were required for clofilium blockade to reach a steady level. Similar results were obtained post-clofilium, when cells were unstimulated during application and then removal of clofilium, suggesting that although the blocking action of the drug was use-dependent, it bound to the closed, delayed rectifier K+ channel. 5. High concentrations (100 or 300 microM) of sotalol, another class III antidysrhythmic agent, were without discernible effects on IKv in NG 108-15 cells. 6. The effects of clofilium on a neuronal IKv described here, and its possible mechanism of action, are compared with previously reported effects of clofilium on the cardiac IKv.

Animals↗
PubMed1991

Effects of doxapram on ionic currents recorded in isolated type I cells of the neonatal rat carotid body.

Whole-cell patch-clamp recordings were used to investigate the effects of the respiratory stimulant doxapram on K+ and Ca2+ currents in isolated type I cells of the neonatal rat carotid body. Doxapram (1-100 microM) caused rapid, reversible and dose-dependent inhibitions of K+ currents recorded in type I cells (IC50 approximately 13 microM). Inhibition was voltage-dependent, in that the effects of doxapram were maximal at test potentials where a shoulder in the current-voltage relationship was maximal. These K+ currents were composed of both Ca(2+)-activated and Ca(2+)-independent components. Using high [Mg2+], low [Ca2+] solutions to inhibit Ca(2+)-activated K+ currents, doxapram was also seen to directly inhibit Ca(2+)-independent K+ currents. This effect was voltage-independent and was less potent (IC50 approximately 20 microM) than under control conditions, suggesting that doxapram was a more potent inhibitor of the Ca(2+)-activated K+ currents recorded under control conditions. Doxapram (10 microM) was without effect on L-type Ca2+ channel currents recorded under conditions where K+ channel activity was minimized and was also without significant effect on K+ currents recorded in the neuronal cell line NG-108 15, suggesting a selective effect on carotid body type I cells. The effects of doxapram on type I cells show similarities to those of the physiological stimuli of the carotid body, suggesting that doxapram may share a similar mechanism of action in stimulating the intact organ.

Animals↗
PubMed1991

Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.

1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced intracellular acidosis. Furthermore, the pre-treatment with 200 microM-DIDS of a pre-acidified cell in Na(+)-free Tyrode solution completely inhibited pHi recovery when Nao+ was reintroduced together with concomitant addition of 150 microM-EIPA. We conclude, that in the presence of CO2-HCO3-, a Na(+)- and HCO3-dependent (DIDS inhibitable) mechanism aids acid extrusion.(ABSTRACT TRUNCATED AT 400 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Inhibition of Ca(2+)-activated K+ currents by intracellular acidosis in isolated type I cells of the neonatal rat carotid body.

1. K+ and Ca2+ currents were recorded from enzymatically isolated type I cells of the neonatal rat carotid body, using the whole-cell configuration of the patch-clamp technique. The effects of intracellular acidosis, caused by bath application of anions of weak acids (propionate and acetate), were tested on these currents. 2. Bath application of propionate or acetate (10 or 20 mM) caused reversible reductions in K+ current amplitudes. These effects were maximal at low, positive test potentials where a shoulder in the current-voltage relationship occurs due to the activation of Ca(2+)-activated K+ currents. 3. Time-course studies showed propionate to cause a rapid initial reduction of K+ currents which recovered partially during its continued application. Removal of propionate produced small, transient overshoots of K+ current amplitudes. In the absence of propionate or acetate, bath application of the Na(+)-H+ exchange inhibitor amiloride caused slowly developing inhibition of K+ current amplitudes. 4. Changing extracellular pH from 7.4 to 8.0 increased K+ current amplitudes, but at this pHo propionate caused smaller reductions in K+ currents than at a pHo of 7.4. 5. In the presence of 0.1 mM-Cd2+, or in high-Mg2+ (6 mM), low-Ca2+ (0.1 mM) solutions, the residual, Ca(2+)-independent K+ currents were unaffected by 20 mM-propionate or acetate. 6. Ca2+ channel currents were also recorded, using 10 mM-Ba2+ as the charge carrier. These sustained currents were completely abolished by 0.1 mM-Cd2+ and were enlarged in the presence of 5 microM-Bay K 8644, suggesting that the currents passed through L-type Ca2+ channels. 7. Ca2+ channel currents were not significantly affected by intracellular acidosis caused by bath application of 10 mM-propionate or acetate. They were also unaffected by a reduction of the extracellular pH from 7.4 to 7.0. 8. It is concluded that intracellular acidosis selectively inhibits Ca(2+)-activated K+ currents in type I carotid body cells. The possible significance of this effect on chemotransduction in the intact carotid body is discussed.

Animals↗
PubMed1991

Effects of extracellular pH, PCO2 and HCO3- on intracellular pH in isolated type-I cells of the neonatal rat carotid body.

1. The effects of changing PCO2 extracellular pH (pHo) and HCO3- on intracellular pH (pHi) were studied in isolated neonatal rat type-I carotid body cells using the pH-sensitive fluoroprobe, carboxy-SNARF-1. 2. Simulated respiratory acidosis and alkalosis (i.e. changes in PCO2 at constant HCO3-) led to rapid (half-time t0.5 = 3 s) monotonic changes in pHi. The relationship between pHi and pHo under these conditions was linear, steep (0.63 pHi/pHo) and remarkably similar to the response predicted from a passive cell model (i.e. a cell lacking pHi regulation). 3. In order to model the above pHi changes (point 2), it was necessary to determine beta i (intrinsic intracellular buffering power). By using small incremental acid loads in the cell (progressive [NH4+]o removal), beta i was determined as a function of pHi to be: beta i = 127.6-16.04 pHi. 4. Changes in PCO2 at constant pHo (i.e. simultaneously changing HCO3-) caused rapid transient changes in pHi but did not significantly affect steady-state pHi over the range 1-10% CO2. 5. When PCO2 was held constant (5%), changing HCO3- and thus pHo (i.e. a simulated metabolic acidosis/alkalosis) led to much slower changes in pHi (t0.5 approximately 1 min). Steady-state pHi showed an almost identical dependence on pHo (slope 0.68) to that found for simulated respiratory acidosis/alkalosis. Therefore, over the range of pHo, PCO2 and [HCO3-]o tested, steady-state pHi appeared to be a unique function of pHo and independent of PCO2 and [HCO3-]o. 6. The effects on pHi of respiratory acidosis, metabolic acidosis and increases of PCO2 at constant pHo (present work) were compared with previously published work on the ability of similar manoeuvres to increase the carotid sinus nerve (CSN) discharge rate. The two sets of data showed several striking similarities: (i) in both cases, the response to a respiratory acidosis was rapid in onset, maintained and reversible; (ii) in both cases, the speed of response to a metabolic acidosis was significantly slower than in (i) but, again, it was maintained and reversible; (iii) in both cases, increases in PCO2 at constant pHo elicited a rapid response but one which was only transient with no change in the steady-state value. 7. The close correlation between the effects of changing pHo, PCO2 and [HCO3-]o on pHi and on CSN discharge suggests that a change in type-I cell pHi is the first step in the chemoreception of blood pH by the carotid body.(ABSTRACT TRUNCATED AT 400 WORDS)

Acidosis↗
PubMed1990

Hypoxic suppression of K+ currents in type I carotid body cells: selective effect on the Ca2(+)-activated K+ current.

Whole-cell K+ currents were recorded in isolated type I carotid body cells using the patch-clamp technique. Hypoxia (pO2 25 torr) reversibly suppressed K+ currents in a voltage-dependent manner: maximal effects were seen at low, positive test potentials, where the Ca2(+)-activated component of K+ currents was greatest. Enhancing this component with 5 microM BAY K 8644 exaggerated the effects of hypoxia, and when the component was inhibited (100 microM Cd2+ or 5 microM nifedipine) hypoxic effects were abolished. As hypoxia does not affect Ca2+ currents directly, these data indicate the suppressive effect of hypoxia is selective for the Ca2(+)-activated component of K+ currents in type I cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Effects of D600 on hypoxic suppression of K+ currents in isolated type I carotid body cells of the neonatal rat.

K+ currents recorded from isolated type I carotid body cells were reversibly suppressed by hypoxia (pO2 25 Torr). Inhibition of Ca2+ influx using high Mg2+, low Ca2+ solutions abolished this effect, indicating hypoxia selectively inhibits the Ca2(+)-activated component of K+ currents. The Ca2+ channel blockers D600 and verapamil reversed the suppressive effects of hypoxia. These drugs also partially inhibited Ca2(+)-independent K+ currents, but this effect was relieved by hypoxia, causing apparent reversal of hypoxic suppression of the K+ currents seen under control conditions.

Animals↗
PubMed1990

Potassium currents recorded in type I carotid body cells from the neonatal rat and their modulation by chemoexcitatory agents.

Whole cell patch clamp recordings were made from type I cells of the neonatal rat carotid body, isolated and maintained in primary culture for up to 48 h. Depolarizing voltage steps applied from a holding potential of -70 mV evoked outward currents positive to approximately -30 mV. Currents were strongly blocked by extracellular tetraethylammonium (25 mM), and were therefore attributed to activation of voltage-dependent K+ channels. Currents were also suppressed by 4-aminopyridine, removal of extracellular Ca2+, and replacement of extracellular Ca2+ with Ba2+. These results suggest there are Ca2(+)-dependent and Ca2(+)-independent components of the K+ currents. No evidence was found to suggest that ATP-sensitive K+ channels were present. The effects of 3 chemoexcitatory agents (NaCN, almitrine and reduced extracellular pH) on K+ currents in isolated type I cells were investigated. All three agents suppressed K+ currents to similar degrees. The effects of lowered pH and NaCN were reversible, and NaCN-induced reductions occurred regardless of the presence of intracellular ATP. The effect of almitrine was irreversible for up to 30 min of recording. It is concluded that the reduction of K+ currents by chemoexcitants may play a role in the mechanism of chemotransduction in the carotid body.

Adenosine Triphosphate↗
PubMed1990

Whole-cell K+ currents in type II pneumocytes freshly isolated from rat lung: pharmacological evidence for two subpopulations of cells.

The patch clamp technique was used to record whole cell K+ currents in type II pneumocytes freshly isolated from adult rats. Depolarizing voltage steps evoked outward K+ currents which were distinguished into low and high threshold types, only one type being apparent in any one cell. Low-threshold (LT) currents were activated at test potentials of -40 mV to -20 mV and were reduced in amplitude by 20 mM tetraethylammonium (TEA). High-threshold (HT) currents were activated only at test potentials positive to -20 mV and current noise was always greater than for LT currents. HT currents were also significantly more sensitive than were LT currents to block by TEA. Quinine (1 mM) blocked LT currents reversibly at all activating test potentials. HT currents were also reversibly blocked by 1 mM quinine, but in a voltage-dependent manner, the degree of block increasing with increasing test potential. 4-Aminopyridine (2 mM) further distinguished the two current types: it was virtually without effect on HT currents but caused large reductions in LT current amplitudes, apparently by acting on the open channels underlying this current. These data clearly distinguish type II pneumocytes into two subpopulations and suggest that they may play separate roles in the functioning of the intact alveolar epithelium.

4-Aminopyridine↗
PubMed1990

Ligustrazine selectively blocks ATP-sensitive K+ channels in mouse pancreatic beta cells.

Ligustrazine potently inhibits hypoxic pulmonary vasoconstriction. This effect can be mimicked by the sulphonylurea tolbutamide, a blocker of ATP-sensitive K+ channels. Here we demonstrate that ligustrazine is a specific blocker of ATP-sensitive K+ channels in mouse pancreatic beta cells. This suggests that ligustrazine may act in the pulmonary circulation by blocking ATP-sensitive K+ channels.

Adenosine Triphosphate↗
PubMed1990

Selective action of myasthenic syndrome antibodies on calcium channels in a rodent neuroblastoma x glioma cell line.

1. The effect of Lambert-Eaton myasthenic syndrome (LEMS) immunoglobulin G (IgG) on Ca2+ channels in undifferentiated mouse neuroblastoma x rat glioma hybrid cells (NG 108 15) was studied using the whole-cell patch clamp technique. 2. Sustained inward Ca2+ channel currents were evoked by depolarizing pulses from holding potentials of -80 and -40 mV, and were blocked by 5 microM-nitrendipine (L-type currents). Transient inward Ca2+ channel currents were activated from a holding potential of -80 mV by small depolarizing steps (T-type currents). Noradrenaline (10 microM) was without effect on transient currents. 3. LEMS IgG selectively reduced sustained (L-type) Ca2+ channel current amplitudes evoked from either holding potential used. In the presence of nitrendipine (5 microM), there was no significant effect of LEMS IgG on the remaining transient (T-type) Ca2+ channel current amplitudes. 4. Studies of the potential for maximal inward current indicated that voltage sensitivities of both L- and T-type Ca2+ channel current amplitudes were unaffected by LEMS IgG, whether recorded in the presence or absence of nitrendipine. LEMS IgG had no significant effect on the time-to-peak or decay of Ca2+ channel currents. 5. It is concluded that LEMS IgG acts selectively to cause functional loss of L-type, but not T-type, Ca2+ channels in NG 108 15 cells. Any effect of LEMS IgG on N-type channels (not present in these undifferentiated cells) was not studied here. LEMS IgG also acts at motor nerve terminal Ca2+ channels leading to muscle weakness. Thus antigenic similarities must exist between L-type channels in NG 108 15 cells and Ca2+ channels at motor nerve terminals.

Action Potentials↗
PubMed1990

Effect of lowered extracellular pH on Ca2(+)-dependent K+ currents in type I cells from the neonatal rat carotid body.

1. The whole-cell configuration of the patch-clamp technique was used to record K+ currents from type I cells enzymatically dispersed from the neonatal rat carotid body. The current-voltage (I-V) relationship for the K+ currents showed a prominent, outward shoulder at test potentials of between +10 and +30 mV. 2. The shoulder of the I-V curve could be enhanced by raising extracellular Ca2+ concentration or by bath application of 5 microM-Bay K 8644. It could also be suppressed by bath application of 100 microM-Cd2+ or 5 microM-methoxyverapamil (D600), indicating that a large component of the K+ current in these cells was activated by an influx of Ca2+ through its own channels during cell depolarization. 3. Potassium currents were also reversibly suppressed by 8 nM-charybdotoxin but unaffected by 100 nM-apamin, suggesting that the Ca2(+)-dependent K+ current was carried through large or intermediate conductance Ca2(+)-activated K+ channels. 4. Lowering the pH of the bathing medium from 7.40 to 7.00 reversibly reduced the K+ current amplitudes, and suppressed the shoulder normally seen in the I-V relationship. This effect was enhanced in the presence of 5 microM-Bay K 8644 and abolished in the presence of 5 microM-D600. 5. It is concluded that the Ca2(+)-dependent K+ channels of type I carotid body cells are selectively suppressed by extracellular acidity. Possible mechanisms underlying this effect, and its role in excitation of the carotid body are discussed.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗