bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic analysis of the phosphorylation‐dependent interactions of synapsin I with rat brain synaptic vesicles
Authors: Stefani Giovanni , Onofri Franco , Valtorta Flavia , Vaccaro Paola , Greengard Paul , Benfenati Fabio 
Published: September 2004

Synapsin I, a major synaptic vesicle (SV)‐associated phosphoprotein, is involved in the regulation of neurotransmitter release and synapse formation. By binding to both phospholipid and protein components of SV with high affinity and in a phosphorylation‐dependent fashion, synapsin I is believed to cluster SV and to attach them to the actin‐based cytoskeleton of the nerve terminal.
In the present study we have investigated the kinetic aspects of synapsin I–SV interactions and the mechanisms of their modulation by ionic strength and site‐specific phosphorylation, using fluorescence resonance energy transfer between suitable fluorophores linked to synapsin I and to the membrane bilayer.
The binding of synapsin I to the phospholipid and protein components of SV has fast kinetics: mean time constants ranged between 1 and 4 s for association and 9 and 11s for ionic strength‐induced dissociation at 20°C. The interaction with the phospholipid component consists predominantly of a hydrophobic binding with the core of the membrane which may account for the membrane stabilizing effect of synapsin I.
Phosphorylation of synapsin I by either SV‐associated or purified exogenous Ca2+/calmodulin‐dependent protein kinase II (CaMPKII) inhibited the association rate and the binding to SV at steady state by acting on the ionic strength‐sensitive component of the binding. When dephosphorylated synapsin I was previously bound to SV, exposure of SV to Ca2+/calmodulin in the presence of ATP triggered a prompt dissociation of synapsin I with a time constant similar to that of ionic strength‐induced dissociation.
In conclusion, the reversible interactions between synapsin I and SV are highly regulated by site‐specific phosphorylation and have kinetics of the same order of magnitude as the kinetics of SV recycling determined in mammalian neurons under comparable temperature conditions. These findings are consistent with the hypothesis that synapsin I associates with, and dissociates from, SV during the exo–endocytotic cycle. The on‐vesicle phosphorylation of synapsin I by the SV‐associated CaMPKII, and the subsequent dissociation of the protein from the vesicle membrane, though not involved in mediating exocytosis of primed vesicles evoked by a single stimulus, may represent a prompt and efficient mechanism for the modulation of neurotransmitter release and presynaptic plasticity.
ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes–possible sensory mechanism?
Authors: Ferguson D. R. , Kennedy Ian , Burton T. J. 
Published: September 2004

The responses of rabbit urinary bladder to hydrostatic pressure changes and to electrical stimulation have been investigated using both the Ussing chamber and a superfusion apparatus. These experiments enabled us to monitor changes in both ionic transport across the tissue and cellular ATP release from it.
The urinary bladder of the rabbit maintains an electrical potential difference across its wall as a result largely of active sodium transport from the urinary (mucosal) to the serosal surface.
Small hydrostatic pressure differences produced by removal of bathing fluid from one side of the tissue caused reproducible changes in both potential difference and short‐circuit current. The magnitude of these changes increases as the volume of fluid removed increases.
Amiloride on the mucosal (urinary), but not the serosal, surface of the membrane reduces the transepithelial potential difference and short‐circuit current with an IC50 of 300 nm. Amiloride reduces the size of, but does not abolish, transepithelial potential changes caused by alterations in hydrostatic pressure.
Field electrical stimulation of strips of bladder tissue produces a reproducible release of ATP. Such release was demonstrated to occur largely from urothelial cells and is apparently non‐vesicular as it increases in the absence of calcium and is not abolished by tetrodotoxin.
It is proposed that ATP is released from the urothelium as a sensory mediator for the degree of distension of the rabbit urinary bladder and other sensory modalities.
Effect of the immunosupressant FK506 on excitation—contraction coupling and outward K+ currents in rat ventricular myocytes
Authors: DuBell William H. , Wright Pamela A. , Lederer W. J. , Rogers Terry B. 
Published: September 2004

We examined the effects of the immunosupressant drug FK506 on excitation–contraction coupling in isolated rat ventricular myocytes. [Ca2+]i transients were recorded using the intracellular Ca2+ indicators fluo‐3 and indo‐1 while action potentials (APs) or membrane currents were recorded using patch‐type microelectrodes in the whole cell mode.
FK506 (25 (μm) rapidly and reversibly increased the magnitude of the [Ca2+]i transient in intact cells without changing resting [Ca2+]i or the kinetics of the [Ca2+]i transient, a finding consistent with previous reports that investigated the actions of FK506 on the sarcoplasmic reticulum Ca2+ release channel.
The 36% increase in the [Ca2+]i transient produced by FK506 was accompanied by a 293% increase in AP duration (by 293%). Importantly, the addition of FK506 had no effect on the [Ca2+]i transient when the depolarizing duration was controlled in voltage clamp experiments. The increased AP duration could be explained by a marked inward shift in the net membrane current that was observed in these experiments.
The net inward current change was not directly responsible for a change in Ca2+ influx, since no change in L‐type Ca2+ current (ICa) was observed. Instead, FK506 inhibited both the transient outward K+ current (Ito) and the delayed rectifier K+ current (IK).
We conclude that FK506 increases the [Ca2+]i transient during normal contractions by an indirect action: it prolongs the action potential. This action does not appear to depend on the established action of FK506 on the ryanodine receptor. Instead, the inhibition of outward K+ currents prolongs the AP which secondarily increases Ca2+ influx and/or decreases Ca2+ efflux.
Effects of sympathetic nerve stimulation on membrane potential, [Ca2+]i and force in the arrested sinus venosus of the toad, Bufo marinus
Authors: Cousins Helen M. , Bramich Narelle J. 
Published: September 2004

The effects of sympathetic nerve stimulation on membrane potential and on the intracellular concentration of calcium ions, [Ca2+]i, were recorded concurrently from the sinus venosus of the toad, Bufo marinus, in preparations where beating had been abolished by adding an organic calcium antagonist to the physiological saline. In a separate set of experiments the effects of sympathetic nerve stimulation on force production were examined.
Stimulation of the sympathetic nerves caused a membrane depolarization and a simultaneous increase in [Ca2+]i. Both responses were reduced by dihydroergotamine (20 μm).
The membrane depolarization and increase in [Ca2+]i evoked by sympathetic nerve stimulation were abolished by ryanodine (10 μm), or caffeine (3 mm). The effects of caffeine, but not those of ryanodine, were fully reversible.
Although the Ca2+‐ATPase inhibitor thapsigargin (30 μm) itself had little effect on the responses to sympathetic nerve stimulation, in its presence caffeine (3 him) irreversibly abolished the responses.
In the presence of nifedipine (10 μm), sympathetic nerve stimulation caused contractions of the sinus venosus. These responses were abolished by either ryanodine (10 μm) or caffeine (3 mm).
The results suggest that neuronally released transmitter activates a complex biochemical pathway which triggers the release of Ca2+ from internal stores.
Control of rat GluR6 glutamate receptor open probability by protein kinase A and calcineurin
Authors: Traynelis Stephen F. , Wahl Philip 
Published: September 2004

We have used non‐stationary variance analysis to examine the single channel conductance and the probability of channel opening at the peak of the homomeric GluR6 response (Po,peak) to 100–200 ms application (10–90% exchange time, 0–3 ms) of glutamate onto excised membrane patches from transiently transfected human embryonic kidney cells (HEK 293).
Our determinations of both Po,peak and single channel conductance of simulated current responses are insensitive to system filtering, response rise time, desensitization rate and measured variation in our drug perfusion speed. Isolation of stochastic current fluctuations using the local mean response waveform minimizes problems associated with modest run‐down of response amplitude during the experiment.
The slope conductance calculated from the weighted mean unitary currents for the channels activated in response to glutamate application is 16 pS. Chord conductance between −40 and −80 mV is independent of agonist concentration. Conversion of the codon for glutamine621 to arginine (Q621R) by RNA editing reduces conductance by more than 35‐fold to less than 0.4 pS without changing response time course, desensitization, or Po,peak.
P
o,peak is high at saturating glutamate concentrations (0.65 ± 0.23; mean ±s.d.) and varies with agonist concentration. The half‐maximally effective glutamate concentration (EC50) determined for Po,peak (0.2 mm; Hill slope = 0.6) is similar to that determined for the macroscopic peak current amplitude (0.5 mm; Hill slope = 1.0) in response to rapid agonist application.
Inclusion of the purified catalytic subunit of cAMP‐dependent protein kinase A (PKA) in the patch pipette increases Po,peak to 0.85 ± 0.12 and co‐transfection of cells with a cDNA encoding the catalytic subunit of PKA (Cα‐PKA) increases Po,peak to 0.94 ± 0.09.
Inclusion of purified calcineurin plus its coactivators 200 nm Ca2+ and calmodulin in the patch pipette decreases Po,peak to 0.48 ± 0.10. The calcineurin‐stimulated decrease of Po,peak in cells co‐transfected with Cα‐PKA is blocked by 800 nm deltamethrin, a calcineurin inhibitor. Calmodulin, 200 nm Ca2+ and deltamethrin have no effect on Po,peak in the absence of calcineurin. As predicted from its effects on Po,peak, inclusion of calcineurin in the patch pipette accelerates the run‐down of whole cell GluR6 responses in cells co‐transfected with Cα‐PKA.
The effects of both calcineurin and PKA on Po,peak for GluR6 receptors in excised patches occur without any detectable changes to response time course, desensitization, or chord conductance.
We conclude that the binding of glutamate to homomeric GluR6 receptors is associated with a high probability of channel opening, which is under the control of two signalling systems that are known to be co‐localized at the neuronal membrane: PKA (Po,peak near 1.0) and calcineurin (Po,peak near 0.5).
Stimulation of anion secretion by β‐adrenoceptors in the mouse endometrial epithelium
Authors: Chan H. C. , Fong S. K. , So S. C. , Chung Y. W. , Wong P. Y. D. 
Published: September 2004

Regulation of anion secretion by adrenoceptors in primary culture of mouse endometrial epithelium was investigated using the short circuit current (ISC) technique.
Adrenaline stimulated a sustained increase in the ISC in a concentration‐dependent manner. The adrenaline‐induced ISC could be inhibited by pretreatment with diphenylamine 2,2'‐dicarboxylic acid (DPC) or replacement of external Cl− and HCO3−, but not by amiloride or replacement of Na+ in apical solution.
The concentration‐dependent responses of the adrenaline‐induced ISC to the CF channel blockers glibenclamide and DPC were examined and exhibited IC50 values of 380 and 960 μm, respectively.
The effect of various adrenoceptor agonists on the ISC was examined. The order of potency appeared to be isoprenaline > adrenaline > noradrenaline, while no response was elicited by the α‐adrenoceptor agonist methoxamine, indicating a predominant involvement of β‐adrenoceptors.
The β‐adrenoceptor antagonist propranolol was found to be much more effective than the α‐adrenoceptor antagonist phentolamine in inhibiting the ISC responses induced by all adrenoceptor agonists examined.
The effect of adrenaline on the ISC was mimicked by an adenylate cyclase activator, forskolin, but suppressed by the adenylate cyclase inhibitor MDL 12,330A, indicating the involvement of cAMP.
Our results demonstrate that anion secretion by the mouse endometrial epithelium is regulated by β‐adrenoceptors and involves a cAMP‐dependent mechanism.
Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition
Authors: Brain K. L. , Bennett M. R. 
Published: September 2004

The sympathetic nerve terminals of the mouse vas deferens were loaded with the calcium indicator Oregon Green 488 BAPTA‐1 by orthograde transport along the postganglionic nerves. Changes in the calcium concentration in the varicosity (Δ[Ca2+]v) were determined following single impulses, and short (5‐impulse) and long (200‐impulse) trains at 5 Hz.
All varicosities showed a significant Δ[Ca2+]v in response to every single impulse. The elevated Δ[Ca2+]v declined in two phases with similar kinetics for all varicosities: a fast phase (time constant, 0.42 ± 0.05 s) and a moderate phase (3.6 ± 0.4 s).
Line scanning confocal microscopy revealed that the Δ[Ca2+] of a single terminal following single impulses was smaller for the intervaricose regions than for the varicosities.
Blockade of the voltage‐sensitive calcium channels with Cd2+ (in calcium‐free solution) completely blocked the Δ[Ca2+]v on stimulation. The addition of either nifedipine (10 μm), ω‐conotoxin GVIA (100 nM) or ω‐agatoxin TK (100 nm) showed that 47 ± 6% of the evoked response was mediated by N‐type calcium channels.
Ryanodine (10 μm) did not significantly change the amplitude of Δ[Ca2+]v in response to short trains.
Spontaneous increases in Δ[Ca2+]v were observed in individual varicosities, with coupling in the increase of Δ[Ca2+]v between varicosities.
The presynaptic α2‐receptor antagonist yohimbine (10 μm) increased the amplitude of Δ[Ca2+]v in response to five impulses (5 Hz) by 54 ± 14%, while the α2‐receptor agonist clonidine (1 μm) decreased the Δ[Ca2+]v by 55 ± 4%.
These results are discussed in terms of the hypotheses that the increased probability for secretion at sympathetic nerve terminals which accompanies facilitation and augmentation is due to the residual Δ[Ca2+]v remaining after the calcium influx following impulses and that noradrenaline acts presynaptically to decrease the probability of secretion by modifying calcium influx.
Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells
Authors: Kamouchi M. , Van Den Bremt K. , Eggermont J. , Droogmans G. , Nilius B. 
Published: September 2004

We have used the patch‐clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole‐cell mode, IK(IR) was defined as the Ba2+‐sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K+‐selective channel with a conductance of 31 ± 3 pS.
Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1.
Intracellular ATP is required to prevent run‐down of IRK in whole‐cell mode. Single channel activity disappeared in inside‐out patches exposed to ATP‐free solution and in cell‐attached patches on cells exposed to metabolic inhibition (KCN, 2‐deoxyglucose).
The non‐hydrolysable ATP analogues, ATPγS and adenylyl imidodiphosphate (AMP‐PNP), did not prevent run‐down. Run‐down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A).
GTPγS and A1F4− inhibited IRK, also in the presence of ATP. GTPγS antagonized the GTPγS effect. Pretreatment of the cells with PTX did not affect the GTPγS‐induced inhibition. Okadaic acid, however, slowed this inhibition.
Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run‐down.
We conclude that run‐down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX‐insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein‐dependent activation of a phosphatase.
Identification of Regions That Regulate the Expression and Activity of G Protein‐Gated Inward Rectifier K+ Channels in Xenopus Oocytes
Authors: Stevens Edward B. , Woodward Robert , Ho Ivan H. M. , Murrell‐Lagnado Ruth 
Published: September 2004

The involvement of the cytoplasmic and core regions of K+ channel Kir3.1 and Kir3.2 subunits in determining the cell surface expression and G protein‐gated activity of homomeric and heteromeric channel complexes was investigated by heterologous expression of chimeric and wild‐type subunits together with the m2 muscarinic receptor in Xenopus oocytes.
Co‐expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression of these subunits. Immunofluorescence labelling indicated that Kir3.2 homomeric channels and Kir3.1–Kir3.2 heteromeric channels were expressed at high levels at the cell surface whereas Kir3.1 homomeric complexes were not expressed at the cell surface. Chimeric subunits composed of Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expression of channels at the plasma membrane.
Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chimeric subunit (121) which displayed dramatically increased acetylcholine‐induced channel activity compared with the wild‐type Kir3.2 homomeric channel. Cell‐attached, single‐channel recordings revealed that chimera 121 channel openings were longer than Kir3.2 openings.
Individually substituting the N‐ and C‐terminal tails of Kir3.1 for those of Kir3.2 showed that the C‐terminal tail of Kir3.1 enhanced the activity of heteromeric channels independently of the N‐terminal or core regions of this subunit.
The chimeric channel, 121, displayed a higher ratio of ACh‐induced to basal activity than the Kir3.1–Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be activated by the basal turnover of G proteins, implying that they have a lower affinity for Gβγ. Our results suggest that substituting the Kir3.1 C‐terminal tail for the Kir3.2 tail promotes the opening conformational change of the Gβγ‐bound channel.
The core and C‐terminal regions of Kir3.1 independently conferred time dependence on voltage‐dependent activation. The time constant (τ) was between 5 and 10 ms and varied little over the voltage range −60 to −120 mV.
In vivo downregulation of M2 receptors revealed by measurement of muscarinic K+ current in cultured guinea‐pig atrial myocytes
Authors: Bünemann Moritz , Brandts Bodo , Pott Lutz 
Published: September 2004

Muscarinic K+ current (IK(ACh)) elicited by acetylcholine (ACh) was measured in guinea‐pig atrial myocytes, which were either freshly isolated or cultured for up to 8 days.
The half‐time of activation of inward IK(ACh) by a saturating concentration (10 μm) of ACh decreased from ∼400 ms (in freshly isolated cells) to 250 ms after 6 days in culture. This was paralleled by an increase in the fast desensitizing component of IK(ACh). The density of steady‐state currents was not changed. Downregulation of M2 receptors by long‐term treatment of isolated myocytes with carbachol in vitro had opposite effects.
The EC50 of ACh for the activation of steady‐state −K(ACh) was reduced from 5 × 107 M (day 0) to 8 × 108 m (day 6). The shift in EC50 occurred with a half‐time of about 2 days, similar to the recovery from downregulation induced by treating atrial myocytes with carbachol in vitro.
The increase in sensitivity to ACh can be accounted for by an ∼6‐fold increase in the density of M2 receptors.
It is concluded that sensitization in culture reflects recovery from downregulation of M2 receptors due to the tonic vagal input to the heart in the intact animal.
Voltage‐induced slow activation and deactivation of mechanosensitive channels inXenopusoocytes
Authors: Silberberg Shai D. , Magleby Karl L. 
Published: September 2004

The relationship between stretch and voltage activation of mechanosensitive (MS) channels from Xenopus oocytes was studied in excised patches of membrane using the patch clamp technique.As is characteristic of MS channels in oocytes, stretching the membrane by applying negative pressure to the patch pipette at −50 mV activated the MS channels rapidly. The channels then deactivated rapidly when the stretch was removed. The stretch‐activated MS channels entered a main conductance level (45 pS) and one or more subconductance levels in the range of about 75–90% of the main conductance level.In the absence of stretch, a depolarizing step from −50 to +50 mV activated apparent MS channels after long delays of typically 1–20 s (range, 100 ms to 6 min). Upon repolarization, the channels deactivated slowly with a single exponential (mean time constant of 4 s) or double exponential (mean time constants of 0.8 and 3s) time course.Delayed activation with depolarization and slow deactivation upon repolarization were also observed for apparent MS channels in on‐cell patches.The voltage‐activated channels were cation selective and had the same selectivity and conductance levels as the stretch‐activated MS channels. Applying stretch during voltage‐induced channel activity did not activate any additional channels, and the same maximal number of channels were typically activated by either stretch or by voltage. These observations suggest that voltage activates the same MS channels that are activated by stretch.The opening of MS channels following steps to +50 mV occurred in an apparently co‐operative manner in 70% of the excised patches containing multiple MS channels.In the absence of stretch, the opening frequency and open probability of MS channels increased with depolarization in the examined voltage range of −60 to −20 mV.Applying a brief stretch during the delay to activation at +50 mV activated the MS channels rapidly, which then remained active when the stretch was removed. In contrast, applying a brief stretch during the slow deactivation induced by stepping from +50 to −50 mV abruptly terminated the voltage‐induced channel activity upon release of the stretch and inhibited subsequent depolarization‐induced activity.Depolarizing steps from −50 to +50 mV inhibited any spontaneous channel activity that was present before the depolarizing step. If the potential was stepped back to −50 mV before the channels activated at +50 mV, a delayed activation could occur at −50 mV, followed by normal deactivation, indicating that the depolarizing step initiated activation processes that were initially masked by inhibition.These observations suggest that voltage and stretch can induce different functional gating configurations of MS channels with associated structures, and that these different gating configurations can interconvert.