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The effect of the neuropeptide substance P on desensitization of ATP receptors of PC12 cells
No full text1 Patch clamp recording (whole cell configuration) was employed to investigate the modulatory action of substance P on inward currents elicited by adenosine 5'-triphosphate (ATP, focally applied via a pressure pipette) from phaechromocytoma (PC12) cells usually held at -70 mV.
2 Bath-applied substance P (0.2-20 mu M) had no effect on baseline membrane current but reversibly reduced ATP peak currents in a concentration-dependent fashion. The depressant effect was not associated with a change in the ATP current reversal potential.
3 Equiamplitude peak responses induced by 50 mu M or 5 mM ATP were differentially affected by substance P which preferentially reduced currents evoked by 5 mM ATP. In the presence of substance P a conditioning pulse of ATP evoked a stronger depression to subsequent test pulses of the same agonist.
4 Combined patch clamping and confocal laser imaging of intracellular Ca2+ ([Ca2+](i)) of single PC12 cells showed that substance P (applied by a pressure pipette) pet se had no effect on [Ca2+](i) or current baseline, although it reduced the inward current and associated [Ca2+](i) rise elicited by ATP.
5 These results are interpreted as due to facilitation by substance P of desensitization of ATP-gated P-2X2 receptors of PC12 cells. It is proposed that the novel modulation by this peptide of ATP responses may serve as a model for further studies aimed at elucidating the action of substance P on purinergic neurotransmission
Role of intracellular calcium in fast and slow desensitization of P2-receptors in PC12 cells
No full text1 Combined whole-cell patch clamp recording and confocal laser scanning microscopy of [Ca2+](i) transients were performed on single PC12 cells to study any correlation between membrane currents induced by ATP and elevation in [Ca2+](i). ATP was applied by pressure from micropipettes near the recorded PC12 cells continuously superfused at a fast rate,
2 Brief (20 ms) pulses of ATP elicited monophasic inward currents and [Ca2+](i) increases. Long applications (2 s) of ATP (5 mM) evoked peak currents which rapidly faded during the pulse and were followed by a large rebound current, interpreted as due to rapid desensitization and recovery of P-2-receptors. The associated [Ca2+](i) increase grew monotonically to a peak reached only after the occurrence of the current rebound, indicating that it is unlikely this cation has a role in fast desensitization.
3 Both membrane currents and [Ca2+](i) transients were dependent on holding membrane potential, suggesting that Ca2+ influx is the predominant cause of [Ca2+](i) elevation. Tills view was supported by experiments carried out in Ca2+-free solution.
4 Brief pulses of ATP applied after a desensitizing pulse (2 s) of the same elicited smaller inward currents and [Ca2+](i) rises indicating a role for [Ca2+](i) in controlling slow desensitization of P-2-receptors.
5 This notion was confirmed in experiments with various [Ca2+](i) chelators which differentially affected slow desensitization in relation to their buffering capacity, while sparing fast receptor desensitization,
6 These results suggest a role for [Ca2+](i) in slow rather than fast desensitization of P-2-receptors, thus proposing this divalent cation as an intracellular factor able to provide an efficient and reversible control over receptor activity induced by ATP
Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells
No full textAlpha-Agatoxin prevents inhibition of calcium transients by IgGs from ALS patients in cultured rat hippocampal neurons
No full textFading and rebound of currents induced by ATP on PC12 cells
No full text1 Patch clamp recording (whole cell configuration) was used to study the action of ATP on rat phaeochromocytoma (PC12) cells usually held at -70 mV and rapidly superfused with buffered saline. ATP (0.5, 1 or 5 mM), applied from micropipettes by pressure application with brief (less than or equal to 50 ms) pulses, induced inward currents with rapid onset and decay. ADP and alpha,beta-methylene ATP were ineffective.
2 ATP (5 mM) applied with pulses >200 ms long elicited a complex current response characterized by a rapid peak which faded and was followed by a strong current rebound (lasting several s) as soon as the application was terminated. This type of response was readily replicated as long as ATP applications were spaced at 2-3 min intervals. The amplitude of peak and rebound currents was dependent on the length of pressure pulse and was similarly depressed by bath application of a threshold dose (25 mu M) of ATP. Rapid fading and rebound of ATP-induced membrane currents were also observed when the Y-tube method was used for applying this agonist.
3 The reversal potential for peak and rebound currents was the same while the time constant values for peak fading and rebound onset were insensitive to changes in membrane potential between -70 and -40 mV. When ATP was applied to a cell clamped at depolarized potential, no current was observed but rapid return of the membrane potential to -70 mV immediately at the end of ATP application was associated with a large rebound current.
4 Brief (20 ms) application of ATP during the onset of the rebound current strongly and transiently suppressed it. The same application performed during the gradual decay of the rebound wave elicited a transient inward current which was much smaller and shorter than the one observed when the cell was in its resting state. Application of 2 s ATP pulses at 20 s intervals equally reduced the initial peak and rebound currents which recovered at the same rate.
5 The present data are interpreted according to a scheme which suggests two types of ATP receptor desensitization. The first one (D1) would be characterized by fast kinetics and low agonist affinity; rapid recovery from D1 would then be manifested as current rebound presumably due to receptor reactivation. The second desensitized state (D2) has slow kinetics and high affinity for the agonist: it is therefore typically seen with sustained application of a low dose of ATP. It is proposed that desensitization and its recovery can influence the time course of membrane responses mediated by purinoceptors
Changes in intracellular calcium induced by NMDA in cultured rat hippocampal neurons require exogenous glycine
No full textConfocal laser scanning microscopy was used to study changes in intracellular free calcium concentration ([Ca2+](i)) at the level of the soma of cultured hippocampal neurones following pressure application of glutamate or N-methyl-D-aspartate (NMDA). [Ca2+](i) was imaged in the presence of tetrodotoxin after loading cells with the fluorescent dye indicator fluo-3/AM. Responses to glutamate were potently antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 mu M). They were also strongly and reversibly depressed by 3-((+/-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP; 20 mu M), leaving a small CNQX-sensitive component. Responses to NMDA were also blocked by CNQX. In the presence of saturating concentrations of glycine (100 mu M), the depression of glutamate or NMDA responses by CNQX was greatly reduced. Exogenously applied glycine also potentiated the NMDA response. These data indicate that the glycine binding site of the NMDA receptor channel is not saturated in cultured hippocampal neurones and thus is susceptible to the action of agonists or antagonists
Reactive oxygen species mediate the potentiating effects of ATP on GABAergic synaptic transmission in the immature hippocampus
No full textRecovery from desensitization of neuronal nicotinic acetylcholine receptors of rat chromaffin cells is modulated by intracellular calcium through distinct second messengers
No full textThe mechanisms through which changes in intracellular Ca2+ concentration ([Ca2+]i) might influence desensitization of neuronal nicotinic receptors (nAChRs) of rat chromaffin cells were investigated by simultaneous patch-clamp recording of membrane currents and confocal microscopy imaging of [Ca2+]i induced by nicotine. Increases in [Ca2+]i that were induced by membrane depolarization or occurred spontaneously did not influence inward currents elicited by focally applied test pulses (10 msec) of nicotine, indicating that raised [Ca2+]i per se did not trigger desensitization of nAChRs. Desensitization of nAChRs, evoked by 2 sec focal application of nicotine, which largely raised [Ca2+]i, was not affected by intracellular application of agents that activate or depress protein kinase C (PKC) or A (PKA) or inhibit phosphatase 1, 2 A and B. Conversely, recovery from desensitization was facilitated by the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the phosphatase 2 B inhibiting complex of cyclosporin A-cyclophilin A, whereas it was impaired by the broad spectrum kinase inhibitor staurosporine. The effects of PMA or staurosporine were prevented by the intracellularly applied Ca2+ chelator BAPTA. The adenylate cyclase activator forskolin accelerated recovery, whereas the selective PKA antagonist Rp-cAMPS had an opposite effect. The action of staurosporine and Rp-cAMPS on recovery from desensitization was additive. It is proposed that when nAChRs are desensitized, they become susceptible to modulation by [Ca2+]i via intracellular second messengers such as serine/threonine kinases and calcineurin. Thus, the phosphorylation state of neuronal nAChRs appears to regulate their rate of recovery from desensitization
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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