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C Peers

Publications and source records attributed to C Peers.

At least 73 records · Page 4Linked to original sources
PubMed1997

Electrochemical detection of K(+)-evoked quantal secretory events from isolated rat type I carotid body cells.

Using amperometric techniques, electrochemical events associated with vesicular transmitter release were recorded from isolated rat type I carotid body cells when exposed to a solution containing 50 mM K+. Events were enhanced in amplitude by preloading cells with the catecholamine precursor, L-beta-3,4-dihydroxyphenylalanine (L-DOPA). K(+)-evoked secretion was abolished by the non-selective Ca2+ channel blocker Cd2+ (100 microM) and markedly reduced by the L-type Ca2+ channel blocker nifedipine (5 microM). Our results indicate that secretion from isolated rat type I cells can be monitored electrochemically and we demonstrate a major role for L-type Ca2+ channels in mediating K(+)-evoked secretion.

Animals↗
PubMed1997

Regulation of [Ca++]i in human neuroblastoma (SH-SY5Y) cells expressing recombinant rat angiotensin1A receptors by angiotensin II and carbachol.

The ability of angiotensin II (AII) to regulate [Ca++]i in human neuroblastoma (SH-SY5Y) cells stably expressing recombinant rat AT1A receptors was investigated using microfluorimetric methods, and compared to responses obtained by stimulation of native muscarinic receptors. Applications of AII or carbachol produced biphasic rises of [Ca++]i, but in Ca++-free solutions (containing 1 mM ethylene glycol-bis (beta-aminoethyl ether)N,N,N,'N'-tetraacetic acid), both agonists produced only transient monophasic rises of [Ca++]i, and second applications were without effect. Application of Ca++(o) (2.5 mM) to cells after exposure to either agonist produced a Ni2+-sensitive rise of [Ca++]i in the absence of agonist ("capacitative Ca++ influx"). After removal of Ca++(o), both AII and carbachol elicited a second rise of [Ca++]i. Thapsigargin (1 microM) prevented these second rises of [Ca++]i. During capacitative Ca++ influx, application of AII failed to produce a further rise of [Ca++]i. In contrast, carbachol produced a further rise of [Ca++]i, attributable to activation of both nicotinic and muscarinic receptors, because it was reduced (but not abolished) by mecamylamine (1 microM) and was observed when muscarine was used as the agonist. Thus, activation of recombinant AT1A and muscarinic receptors in SH-SY5Y cells leads to mobilization of Ca++ from a common intracellular pool, and stimulates capacitative Ca++ influx. Muscarinic (but not AII) receptor occupancy is capable of stimulating an additional Ca++ influx pathway.

Angiotensin II↗
PubMed1996

Neuropeptide Y elevates intracellular Ca2+ and evokes noradrenaline release in SH-SY5Y cells.

Exposure of human neuroblastoma SH-SY5Y cells to 300 nM neuropeptide Y (NPY) or 1 microM muscarine separately failed to evoke release of [3H]noradrenaline ([3H]NA). Both agonists, however, induced a modest rise in [Ca2+]i. When NPY and muscarine were applied simultaneously, the rise in [Ca2+]i was greater than the sum of the rises of either agonist applied alone, and also evoked [3H]NA release, NPY evoked a rise in [Ca2+]i when applied during prolonged exposure to muscarine, although the peak level of [Ca2+]i was significantly lower (p < 0.05) than that reached following simultaneous application, and [3H]NA release was not stimulated. Simultaneous exposure of SH-SY5Y cells to muscarine and NPY thus induces a greater than additive rise in [Ca2+]i exceeding a critical level required to evoke [3H]NA release.

Calcium↗
PubMed1996

Ca2+ channel currents in type I carotid body cells of normoxic and chronically hypoxic neonatal rats.

Whole-cell patch-clamp recordings were used to study voltage-gated Ca2+ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O2). Currents activated at potentials of -40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca2+ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 microM) always partially inhibited currents and Bay K 8644 (2-5 microM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker omega-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca2+ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca2+ channels in type I cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Modified, cyclic dodecapeptide analog of neuropeptide Y is the smallest full agonist at the human Y2 receptor.

In order to stabilize the C-terminal dodecapeptide of neuropeptide Y (NPY) we replaced Leu28 and Thr32 by Lys and Glu, respectively, and subsequently linked these residues by lactamization. This peptide analog of NPY shows a more than 100-fold increase in affinity compared to the C-terminal linear dodecapeptide in receptor binding studies performed at human neuroblastoma cells SMS-KAN, which exclusively express the Y2 receptor subtype. Signal transduction was investigated by measuring Ca2+ current inhibition in human SH-SY5Y cells and cyclic [Lys28-Glu32] NPY Ac-25-36 and NPY were shown to be equipotent in this assay. Thus, this molecule is the smallest Y2 receptor selective full agonist of NPY. Using 2D-NMR experiments and molecular modelling techniques, the structures of the linear and cyclic peptides have been investigated and significant differences have been found, which may explain the improvement in biological activity. Thus, a model of the bioactive conformation of NPY at the human Y2 receptor is suggested.

Amino Acid Sequence↗
PubMed1996

Opioid receptor independent inhibition of Ca2+ and K+ currents in NG108-15 cells by the kappa opioid receptor agonist U50488H.

The whole-cell patch clamp technique was used to investigate the actions of the opioid agonist U50488H on Ca2+ and K+ currents in differentiated NG108-15 cells. U50488H (5-50 microM) caused a concentration-dependent, reversible inhibition of high voltage-activated Ca2+ currents which persisted in the presence of nifedipine (2 microM), indicating a blockade of N-type Ca2+ channels. The actions of U50488H were also observed in the presence of 30 microM naloxone, which fully abolished current inhibition caused by a selective delta opioid receptor agonist. U50488H also inhibited Ca(2+)-insensitive, voltage-gated K+ currents in NG108-15 cells in the presence of naloxone. Our results indicate that U50488H can inhibit neuronal ionic channels via a mechanism which does not involve activation of kappa opioid receptors.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1996

Inhibition of Ca(2+)-sensitive K+ currents in NG 108-15 cells by substance P and related tachykinins.

1. The whole-cell patch-clamp technique was used to investigate the actions of substance P and other agonists at neurokinin (NK) receptors on voltage-gated K+ and Ca+ channel currents in undifferentiated mouse neuroblastoma x rat glioma NG 108-15 cells. 2. Both substance P (0.3-30 microM) and the NK1 receptor selective agonist GR73632 (10 nM-10 microM) caused concentration-dependent inhibition of K+ currents. GR64349 and senktide (agonists at NK2 and NK3 receptors respectively) also inhibited K+ currents, but only at concentrations which were several orders of magnitude greater than GR73632, suggesting that current inhibition was mediated via NK1 receptors. 3. Substance P and GR73632 were without effect on residual K+ currents recorded in the presence of extracellular Co2+ (4 mM) to abolish the Ca(2+)-sensitive component (IKca) of the K+ current. Ca2+ channel currents, recorded with either Ba2+ or Ca2+ as charge carrier, were unaffected by NK1, NK2 and NK3 receptor ligands. 4. Iontophoretic application of GR73632 produced a current-dependent reduction of K+ currents. In the presence of the non-peptide NK1 antagonists, CP-99,994 and RP67580, and the peptide antagonist, GR82334, the current-response relationship was reversibly shifted to the right. This indicates that the response is mediated by NK1 receptors. 5. Our results indicate that activation of NK1 receptors leads to the selective inhibition of IKca in undifferentiated NG 108-15 cells.

Animals↗
PubMed1996

Effects of cytochrome P-450 inhibitors on ionic currents in isolated rat type I carotid body cells.

Hypoxic chemoreception in the carotid body involves selective inhibition of K+ channels in type I cells. We have investigated whether cytochrome P-450 may act as an O2 sensor coupling hypoxia to K+ channel inhibition, by investigating the actions of P-450 inhibitors to modulate channel activity (recorded using patch-clamp techniques) in type I cells isolated from 8-to 12-day-old rat pups. The imidazole antimycotic P-450 inhibitors miconazole and clotrimazole (1-10 microM) inhibited the Ca(2+)-activated (KCa) and voltage-gated K+ (Kv) currents in isolated type I cells. Single-channel recordings indicated that the KCa channels could be inhibited directly by miconazole. Miconazole also irreversibly inhibited Ca2+ channel currents. By contrast, acute application of the suicide substrate P-450 inhibitor, 1-aminobenzotriazole (1-ABT; 3 mM) was without effect on K+ or Ca2+ currents. Hypoxia (16-23 mmHg) reversibly inhibited K+ currents and prevented the inhibitory actions of miconazole. Furthermore, the inhibitory actions of miconazole could be partially reversed by hypoxia. Pretreatment of cells for 60 min with 3 mM 1-ABT substantially reduced the inhibitory actions of hypoxia on K+ currents. Our results indicate that imidazole antimycotic P-450 inhibitors can directly and nonselectively inhibit ionic channels in type I cells but, more importantly, provide evidence to suggest that hypoxic inhibition of K+ currents in type I cells is mediated in part at least by cytochrome P-450.

Animals↗
PubMed1995

The effect of the angiotensin II (AT1A) receptor stably transfected into human neuroblastoma SH-SY5Y cells on noradrenaline release and changes in intracellular calcium.

A stable cell line expressing the angiotensin II (AII) receptor has been obtained by transfecting the human neuroblastoma SH-SY5Y with the plasmid pCEP4 containing the entire coding region of the rat angiotensin AII receptor AT1A. Angiotensin II (AII; 1-100 nM) evokes the release of [3H]noradrenaline ([3H]NA) in this cell line. Pretreatment with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances the AII-evoked release of [3H]NA approximately two-fold. Removal of extracellular Ca2+ ([Ca2+]o) decreases 100 nM AII-evoked release of [3H]NA by over 50% both in the presence and absence of TPA. AII increases intracellular Ca2+ ([Ca2+]i) in this cell line which is consistent with the AT1A receptor being coupled to phospholipase C. Pretreatment with 100 nM TPA for 8 min attenuated the effect of AII on [Ca2+]i. The effects of AT1A receptor stimulation are therefore regulated differently in this cell line by activation of protein kinase C (PKC). Thus a useful cell line has been obtained from the human neuroblastoma SH-SY5Y in which to study at the molecular level the mechanism(s) by which AII regulates NA release.

Angiotensin II↗
PubMed1995

Low potency inhibition of Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by [Ala31]NPY, an L-alanine substituted analogue of neuropeptide Y.

Whole-cell Ca2+ channel currents were recorded in human neuroblastoma (SH-SY5Y) cells, using the perforated-patch technique with 10 mM Ba2+ as charge carrier. Neuropeptide Y (NPY; 10 nM to 1 microM) caused concentration-dependent inhibition of Ca2+ channel currents which were associated with a slowing in current activation kinetics. [Ala31]NPY, a residue 31 L-alanine substituted analogue of NPY, also inhibited Ca2+ channel currents and caused slowing of activation kinetics, but with approximately 6-fold lower potency. In the presence of 100 nM [Ala31]NPY (which itself had little or no effect on currents), the actions of NPY were similar in magnitude to its effects in the absence of the analogue. Our results suggest that substitution of isoleucine for alanine at residue 31 results in a NPY analogue which is a full agonist but with lower affinity for Y2 receptors.

Barium↗
PubMed1995

The effect of barium on [3H]noradrenalin release from the human neuroblastoma SH-SY5Y.

Replacement of Ca2+ with Ba2+ in HEPES-buffered saline stimulated [3H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba(2+)-evoked release was inhibited by 85% by 3 microM tetrodotoxin and 95% by 5 microM nifedipine. Ba2+ also increased the potency of K(+)-evoked release of [3H]noradrenalin, as maximal release was observed with 60 mM K+ compared with the 100 mM K+ necessary to achieve maximal release in the presence of Ca2+. In contrast, replacing Ca2+ with Ba2+ had little effect on carbachol- and bradykinin-evoked release of [3H]noradrenalin. No evidence was obtained from studies on changes in [Ca2+]i (in response to 100 microM carbachol) using fura-2 that Ba2+ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba2+ depolarizes SH-SY5Y cells as well as enhancing inward Ca2+ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba2+ blocks K+ channels, leading to depolarization followed by opening of voltage-sensitive Na+ channels. This in turn opens voltage-sensitive L-type Ca2+ channels, which are coupled to the release of [3H]noradrenalin in SH-SY5Y cells.

Barium↗
PubMed1995

Ca(2+)-activated K+ channels in isolated type I cells of the neonatal rat carotid body.

1. Ca(2+)-activated K+ (K+Ca) channels in neonatal rat type I carotid body cells were studied using single channel patch clamp techniques. In outside-out patches, using symmetrical 120 mM [K+] solutions, channels were observed with a slope conductance of 190 pS and a reversal potential of 0 mV. Reducing [K+]o to 5 mM shifted the reversal potential as expected for a K(+)-selective channel. 2. With 100 nM Ca2+ bathing the cytosolic aspect of patches, channel activity (number of active channels in a patch x open probability, NPo) increased with depolarization. NPo also increased with increasing 'cytosolic' [Ca2+] at a fixed membrane potential (0 mV). Using outside-out patches, bath application of 20 or 100 nM charybdotoxin reduced NPo by > 85%. These data indicate the presence of K+Ca channels in type I cells. 3. At 0 mV, using solutions of identical composition (1 microM Ca2+ bathing the cytosolic aspect of the channels), NPo was higher in outside-out patches than in inside-out patches. NPo was greatest in recordings using the perforated-vesicle technique. 4. Hypoxia and anoxia were without effect on K+Ca channels in outside-out patches, but caused significant, reversible reductions of NPo in channels recorded in perforated vesicles. 5. The whole-cell perforated-patch technique was used to record membrane potential at 35-37 degrees C. Hypoxia, anoxia and charybdotoxin all depolarized type I cells. 6. Our results suggest an important role for K+Ca channels in type I carotid body cells, and their activity in relation to a model for chemotransduction is discussed.

Animals↗
PubMed1995

Doxapram stimulates dopamine release from the intact rat carotid body in vitro.

Hypoxic chemotransduction by the carotid body is believed to involve inhibition of K+ channels in type I cells, leading to depolarization and the opening of Ca2+ channels which triggers catecholamine release. We have investigated whether the clinically used ventilatory stimulant doxapram (which, like hypoxia, blocks K+ channels in isolated type I cells) also stimulates catecholamine release from the intact carotid body in vitro, by pre-incubating tissues with [3H]tyrosine. 3H overflow was evoked by raised extracellular [K+] (60 mM) and by cyanide (2 mM). Doxapram (15-150 microM) also evoked 3H overflow in a concentration dependent manner, and doxapram-evoked release was inhibited by the Ca2+ channel blocker nifedipine (5 microM). Analysis of released tritiated compounds suggested that doxapram preferentially stimulated the release of dopamine. Our results indicate that the mechanism of action of doxapram shares similarities with that of hypoxia in the carotid body.

Animals↗
PubMed1995

O2-sensitive K+ currents in carotid body chemoreceptor cells from normoxic and chronically hypoxic rats and their roles in hypoxic chemotransduction.

Carotid body-mediated ventilatory increases in response to acute hypoxia are attenuated in animals reared in an hypoxic environment. Normally, O2-sensitive K+ channels in neurosecretory type I carotid body cells are intimately involved in excitation of the intact organ by hypoxia. We have therefore studied K+ channels and their sensitivity to acute hypoxia (PO2 12-20 mmHg) in type I cells isolated from neonatal rats born and reared in normoxic and hypoxic environments. When compared with cells from normoxic rats, K+ current density in cells from hypoxic rats was significantly reduced, whereas Ca2+ current density was unaffected. Charybdotoxin (20 nM) inhibited K+ currents in cells from normoxic rats by approximately 25% but was without significant effect in cells from hypoxic rats. However, hypoxia caused similar, reversible inhibitions of K+ currents in cells from the two groups. Resting membrane potentials (measured at 37 degrees C using the perforated-patch technique) were similar in normoxic and hypoxic rats. However, although acute hypoxia depolarized type I cells of normoxic rats, it was without effect on membrane potential in type I cells from hypoxic animals. Charybdotoxin (20 nM) also depolarized cells from normoxic rats. Our results suggest that type I cells from chronically hypoxic rats, like normoxic rats, possess O2-sensing mechanisms. However, they lack charybdotoxin-sensitive K+ channels that contribute to resting membrane potential in normoxically reared rats, and this appears to prevent them from depolarizing (and hence triggering Ca2+ influx and neurosecretion) during acute hypoxia.

Animals↗
PubMed1995

Transduction of chemostimuli by the type I carotid body cell.

The postulated mechanisms for hypoxic and acidic chemotransduction by type I cells that we have described here are summarized in the diagrams of Fig. 4. Most if not all of these require more complete evaluation and, as we have described, there are obvious points of contention that need to be resolved. Nevertheless, it is apparent that studies of isolated type I cell preparations carried out over the last six years have provided significant advancements in our understanding of chemotransduction in the type I cell. Only when the functioning of these cells has been fully described can we hope to understand the mechanisms underlying the responses of the intact organ to chemostimuli.

Animals↗