bluuebunny/crossref_metadata_2025_split huggingface dataset:
Frequency‐Dependent Actions of Benzodiazepines on GABAA Receptors in Cultured Murine Cerebellar Granule Cells
Authors: Mellor J. R. , Randall A. D. 
Published: September 2004

Miniature IPSCs recorded from cultured murine cerebellar granule cells increased in half‐width and amplitude following application of the benzodiazepine (BDZ) Flunitrazepam (Flu, 1 μm). The increase in the half‐width was much greater than that in the amplitude.
Five‐millisecond applications of 1 mm GABA to nucleated outside‐out patches elicited rapidly rising biexponentially decaying responses that resembled IPSCs. Flu had no effect on the amplitude of such responses, but consistently slowed their deactivation by ∼50%. This effect was reversed by Flu washout or application of the BDZ antagonist Ro 15‐1788. The partial inverse agonist Ro15‐4513 speeded deactivation and depressed peak current amplitude by 23 ± 12%.
The EC50 for GABA was between 45 and 50 μm. At submaximally effective agonist concentrations, Flu increased response amplitude and slowed response deactivation. Both effects were present in all cells taken from young cultures (4–7 days in vitro) but the latter was absent in 55% of the neurones obtained from older cultures (14–27 days in vitro).
With 120 ms applications of 20 μm GABA, responses activated monoexponentially (time constant, 39.8 ± 2.8 ms) and deactivated biexponentially (time constants, 40.4 ± 2.1 and 251 ± 15 ms). Application of Flu slowed both activation and deactivation. The latter effect arose from an increased contribution of the slower component of decay.
Desensitization of responses to 1 mm GABA was biexponential, with time constants of 47 ± 11 and 479 ± 49 ms. Flu speeded desensitization by decreasing both fast and slow time constants. GABAA receptor desensitization consistently slowed subsequent deactivation. No significant relationship between the level of desensitization and the amount of slowing of deactivation produced by Flu was found.
Responses to paired 5 ms applications of 1 mm GABA indicated that the slowing of deactivation and the speeding of desensitization produced by Flu combine to generate a marked frequency dependence in the actions of this BDZ. Thus when compared with control responses, GABA‐induced charge transfer was only enhanced by Flu during the first of two successive agonist applications.
Action potential propagation into the presynaptic dendrites of rat mitral cells
Authors: Bischofberger Josef , Jonas Peter 
Published: September 2004

Dendritic patch‐clamp recordings were obtained from mitral cells in rat olfactory bulb slices, up to 350 μm from the soma. Simultaneous dendritic and somatic whole‐cell recordings indicated that action potentials (APs) evoked by somatic or dendritic current injection were initiated near the soma. Both the large amplitude (100.7 ± 1.1 mV) and the short duration (1.38 ± 0.07 ms) of the AP were maintained as the AP propagated back into the primary mitral cell dendrites.
Outside‐out patches isolated from mitral cell dendrites contained voltage‐gated Na+ channels (peak conductance density, 90 pS μm−2 at −10 mV). When an AP was used as a somatic voltage‐clamp command in the presence of 1 μm tetrodotoxin (TTX), the amplitude of the dendritic potential was attenuated to 48 ± 14 mV. This shows that dendritic Na+ channels support the active back‐propagation of APs.
Dendritic patches contained voltage‐gated K+ channels with high density (conductance density, 513 pS μm−2 at 30 mV). Dendritic K+ currents were reduced to 35% by 1 mm external tetraethylammonium chloride (TEACl). When an AP was used as a somatic voltage‐clamp command in the presence of TEACl, the dendritic potential was markedly prolonged. This indicates that dendritic K+ channels mediate the fast repolarization of dendritic APs.
We conclude that voltage‐gated Na+ and K+ channels support dendritic APs with large amplitudes and short durations that may trigger fast transmitter release at dendrodendritic synapses in the olfactory bulb.
Amine precursor uptake and decarboxylation: significance for processing of the rat gastrin precursor
Authors: Voronina Svetlana , Henry Julie , Vaillant Camille , Dockray Graham J. , Varro Andrea 
Published: September 2004

Conversion of prohormone precursors to smaller active products occurs in secretory granules, which also have the capacity to concentrate biogenic amines. We have examined how processing of the gastrin precursor, progastrin, in rat antral mucosa is influenced by modulation of the biogenic amine content of secretory granules.
Newly synthesized progastrin‐derived peptides in rat antral mucosa were labelled in vitro with 35SO42− using a pulse–chase protocol and detected after immunoprecipitation by HPLC with on‐line liquid scintillation counting. Secretory granule morphology was examined by electron microscopy. The effects of experimentally manipulating secretory granule pH and amine content were examined.
The dopamine precursor l‐β‐3,4‐dihydroxyphenylalanine (l‐DOPA) inhibited cleavage of 35S‐labelled thirty‐four amino acid amidated gastrin, i.e. [35S]G34, and of [35S]G34 with COOH‐terminal glycine, i.e. [35S]G34–Gly, at a pair of lysine residues, but did not influence cleavage of progastrin at pairs of arginine residues. The effect of l‐DOPA was reversed by reserpine, which inhibits the amine–proton exchangers VMAT1 and VMAT2, and by carbidopa, which inhibits aromatic L‐amino acid decarboxylase.
Treatments that raise intragranular pH, e.g. the weak base chloroquine, the ionophore monensin and the vacuolar proton pump inhibitor bafilomycin A1, had similar effects to l‐DOPA.
Electron microscopical studies showed that the electron‐dense aggregrates in gastrin cell secretory granules were lost after inhibition of the vacuolar proton pump. Treatment with l‐DOPA produced reserpine‐sensitive dissipation of the electron‐dense aggregates, compatible with the idea that increased amine delivery raised intragranular pH.
The data suggest that the processes of amine precursor uptake, decarboxylation and sequestration in secretory granules are associated with selective modulation of progastrin cleavage, possibly by raising intragranular pH and thereby inhibiting pH‐sensitive prohormone convertases.
Calcium release from intracellular stores evoked by extracellular ATP in a Xenopus renal epithelial cell line
Authors: Mori Masahiro , Hosomi Hidenobu , Nishizaki Tomoyuki , Kawahara Katsumasa , Okada Yasuhiro 
Published: September 2004

The signal transduction mechanism mediating extracellular adenosine 5′‐triphosphate (ATP)‐induced calcium release in a renal epithelial cell line (A6) was investigated using the whole‐cell voltage‐clamp technique and fura‐2 fluorescence measurement.
ATP (10 μM) activated calcium‐dependent non‐selective cation channels in cells held under voltage clamp.
Guanosine 5′‐O‐(2‐thiodiphosphate) (GDPβS; 0.1–1.0 mM) in the pipette inhibited the ATP‐activated calcium‐dependent currents. With guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS; 0.1–1.0 mM) in the pipette, the currents were spontaneously elicited without application of ATP. Pretreatment with pertussis toxin (PTX) affected neither the ATP‐activated currents nor the increase in intracellular free calcium concentration ([Ca 2+])i evoked by ATP.
Intracellular application of neomycin or heparin inhibited the ATP‐activated currents. Inositol 1,4,5‐trisphosphate (IP3; 0.1–100μM) in the internal solution produced currents similar to those due to ATP activation.
These results suggest that a PTX ‐insensitive guanosine 5′‐triphosphate (GTP)‐binding regulatory protein (G protein) is involved in extracellular ATP‐induced phosphoinositide turnover and subsequent calcium release from IP3 ‐sensitive stores, which subsequently activates the calcium ‐dependent channels in A6 cells.
Mechanism underlying corticotropin‐releasing hormone (CRH) triggered cytosolic Ca2+ rise in identified rat corticotrophs
Authors: Lee Andy K. , Tse Amy 
Published: September 2004

The patch‐clamp technique was used in conjunction with the fluorescent Ca2+ indicator indo‐1 to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i) and membrane potential in single rat corticotrophs identified with the reverse haemolytic plaque assay.
Application of the adrenocorticotropin (ACTH) secretagogue, corticotropin‐releasing hormone (CRH), triggered a sustained [Ca2+]i elevation and membrane depolarization.
The CRH action was mediated via the cAMP‐dependent protein kinase cascade. Both the CRH‐induced depolarization and [Ca2+]i elevation could be mimicked by extracellular application of the adenylate cyclase activator forskolin or the membrane‐permeable cAMP analogue, 8‐(4‐chlorophenylthio)‐adenosine‐3’,5’‐cyclic monophosphate (8‐CPT‐cAMP). Intracellular adenosine cyclic 3’,5’‐(Rp)‐phosphothioate (Rp‐cAMPS), a protein kinase A inhibitor, abolished the CRH effects.
Voltage‐clamp studies suggest that the CRH‐triggered depolarization was due to the reduction of background K+ conductances. The CRH‐sensitive current was Ca2+ independent and was insensitive to the K+ channel blockers tetraethylammonium (TEA) or 4‐amino‐pyridine (4‐AP), but could be partially inhibited by Ba2+.
The CRH‐triggered steady‐state depolarization stimulated extracellular Ca2+ entry via voltage‐gated Ca2+ channels and raised [Ca2+]i. CRH failed to stimulate [Ca2+]i rise in cells that were voltage clamped at their resting potential. Removal of extracellular Ca2+ or inhibition of Ca2+ channels by Ni2+ abolished the [Ca2+]i rise.
Voltage‐clamp studies of voltage‐gated Ca2+ channels using Ba2+ as charge carrier show that ∼90% of the channels were available for activation at the resting potential. CRH did not enhance the voltage‐gated Ca2+ channels.
Intramembrane charge movement and sarcoplasmic calcium release in enzymatically isolated mammalian skeletal muscle fibres
Authors: Szentesi Peter , Jacquemond Vincent , Kovács László , Csernoch László 
Published: September 2004

Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea‐pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation–contraction coupling were studied under voltage clamp conditions.
The voltage dependence of intramembrane charge movement followed a two‐state Boltzmann distribution with maximal available charge of 26.1 ± 1.5 and 26.1 ± 1.3 nC μF−1, mid‐point voltage of –35.1 ± 5.0 and –42.2 ± 1.2 mV and steepness of 16.7 ± 2.2 and 17.0 ± 1.9 mV (means ±s.e.m., n= 7 and 4) in rats and guinea‐pigs, respectively.
Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium‐sensitive dyes antipyrylazo III, fura‐2 and mag‐fura‐5. Resting [Ca2+]i was similar in rats and guinea‐pigs with 125 ± 18 and 115 ± 8 nm (n= 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 ± 1.0 μm; n= 7), than in guinea‐pigs (2.8 ± 0.3; n= 4).
The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 ± 0.1 and 0.9 ± 0.1 mm in rats and guinea‐pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 ± 0.08 and 0.81 ± 0.06% ms−1 in rats and 2.43 ± 0.25 and 0.88 ± 0.01 % ms−1 in guinea‐pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians.
These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.
Functional Distribution of Three Types of Na+ Channel on Soma and Processes of Dorsal Horn Neurones of Rat Spinal Cord
Authors: Safronov Boris V. , Wolff Matthias , Vogel Werner 
Published: September 2004

Voltage‐gated Na+ channels and their distribution were studied by the patch‐clamp technique in intact dorsal horn neurones in slices of newborn rat spinal cord and in neurones isolated from the slice by slow withdrawal of the recording pipette. This new method of neurone isolation was further used to study the roles of soma and axon in generation of action potentials.
Tetrodotoxin (TTX)‐sensitive Na+ currents in intact neurones consisted of three components. A fast component with an inactivation time constant (τf) of 0.6–2.0 ms formed the major part (80–90%) of the total Na+ current. The remaining parts consisted of a slowly inactivating component (τs of 5–20 ms) and a steady‐state component.
Single fast and slow inactivating Na+ channels with conductances of 11.6 and 15.5 pS, respectively, were identified in the soma of intact neurones in the slice. Steady‐state Na+ channels were not found in the soma, suggesting an axonal or dendritic localization of these channels.
In the whole‐cell recording mode, the entire soma of a dorsal horn neurone could be isolated from the slice by slow withdrawal of the recording pipette, leaving all or nearly all of its processes in the slice. The isolated structure was classified as: (1) ‘soma’ if it lost all of its processes, (2) ‘soma+axon’ complex if it preserved one process and at least 85% of its original peak Na+ current or (3) ‘soma+dendrite’ complex if it preserved one process but the remaining Na+ current did not exceed those observed in the isolated ‘somata’.
The spatial distribution of Na+ channels in the neurone was studied by comparing Na+ currents recorded before and after isolation. The isolated ‘soma’ contained 13.8 ± 1.3% of inactivating Na+ current but no steady‐state Na+ current. ‘Soma+axon’ complexes contained 93.6 ± 1.4% of inactivating and 46% of steady‐state Na+ current.
In current‐clamp experiments, the intact neurones and isolated ‘soma+axon’ complexes responded with ‘all‐or‐nothing’ action potentials to current injections. In contrast, isolated ‘somata’ showed only passive or local responses and were unable to generate action potentials.
It is concluded that dorsal horn neurones of the spinal cord possess three types of TTX‐sensitive voltage‐gated Na+ channels. The method of entire soma isolation described here shows that the majority of inactivating Na+ channels are localized in the axon hillock and only a small proportion (ca 1/7) are distributed in the soma. Steady‐state Na+ channels are most probably expressed in the axonal and dendritic membranes. The soma itself is not able to generate action potentials. The axon or its initial segment plays a crucial role in the generation of action potentials.
The electrogenic effects of Na+–K+‐ATPase in rat auditory thalamus
Authors: Senatorov V. V. , Mooney D. , Hu B. 
Published: September 2004

The electrogenic effects of the Na+–K+‐ATPase in thalamic neurones were investigated by means of intracellular and whole‐cell patch‐clamp recording techniques in rat medial geniculate body (MGB) maintained in vitro.
In twenty‐six out of thirty‐one neurones recorded intracellularly, application of the Na+–K+ pump inhibitor strophanthidin induced two different types of membrane depolarization: a small, reversible depolarization with a peak amplitude of 4 ± 2.6 mV or a prolonged depolarization of large amplitude (48.6 ± 9.0 mV) with or without a decrease in apparent membrane resistance. Blockade of glutamate receptors with kynurenic acid or 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione and (±)‐2‐amino‐5‐phosphonopentanoic acid did not prevent either type of pump response, but the large depolarization was not seen when the medium contained the sodium channel blocker TTX.
Whole‐cell patch‐clamp recording showed that the small membrane depolarization is mediated by an inward membrane current (39.00 ± 5.70 pA) that exhibited a weak voltage dependence. An inward current of similar amplitude was also induced in MGB cells when the pipette solution contained nominally zero Na+ or when K+ was temporarily omitted from the extracellular medium. The large membrane depolarization or the corresponding membrane current was not observed in whole‐cell conditions.
Transient inhibition of the electrogenic Na+–K+‐ATPase consistently led to a change in the mode of synaptic transmission in MGB cells, during which the synaptically evoked burst response was either blocked or converted into a single spike discharge.
Taken together, these data suggest that blockade of the electrogenic pump produces a dual membrane effect in mammalian thalamic neurones: a small electrogenic membrane depolarization and a large depolarization response that can be prevented by artificially maintaining the transmembrane ionic gradients. The electrogenic activity of the Na+–K+‐ATPase may play an important role in setting the mode of synaptic transmission in sensory thalamus.
Contribution of Selectins to Leucocyte Sequestration in Pulmonary Microvessels by Intravital Microscopy in Rabbits
Authors: Kuebler W. M. , Kuhnle G. E. H. , Groh J. , Goetz A. E. 
Published: September 2004

Sequestration of leucocytes in the lung is the net result of leucocyte rolling and sticking in pulmonary arterioles and venules and their retention in alveolar capillaries.
In order to investigate whether adhesion molecules of the selectin family contribute to these phenomena the effects of fucoidin (an inhibitor of L‐ and P‐selectin) on micro‐haemodynamics and leucocyte kinetics were studied in pulmonary arterioles, capillaries and venules by means of intravital fluorescence microscopy in a rabbit model.
Fucoidin reduced leucocyte rolling in pulmonary arterioles and venules by 75 and 83%, respectively, without affecting leucocyte sticking. In alveolar capillaries, fucoidin reduced leucocyte retention and accelerated leucocyte passage, thus reducing the alveolar transit time of leucocytes by 62%.
It is concluded that rolling of leucocytes in pulmonary microvessels is mediated by selectins, whereas sticking relies on selectin‐independent mechanisms.
Leucocyte retention in alveolar capillaries is not due solely to mechanical hindrance of leucocyte passage through narrow vessel segments, as previously hypothesized, but also depends on interaction of leucocytes with the capillary endothelium.
Emptying of Intracellular Ca2+ Stores Stimulates Ca2+ Entry in Mouse Pancreatic β‐Cells by Both Direct and Indirect Mechanisms
Authors: Miura Yoshikazu , Henquin Jean‐Claude , Gilon Patrick 
Published: September 2004

In non‐excitable cells, the depletion of intracellular Ca2+ stores triggers Ca2+ influx by a process called capacitative Ca2+ entry. In the present study, we have investigated how the emptying of these stores by thapsigargin (1 μM) influences Ca2+ influx in electrically excitable pancreatic β‐cells. The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in clusters of mouse β‐cells or in whole islets loaded with fura‐2.
The membrane was first held hyperpolarized by diazoxide, an opener of ATP‐sensitive K+(KATP) channels, in the presence of 4.8 mM K+. Alternating between Ca2+‐free medium and medium containing 2.5 mM Ca2+ caused a minor rise in [Ca2+]i (∼14 nM) in clusters of β‐cells. A larger rise (∼65 nM), resistant to the blockade of voltage‐dependent Ca2+ channels by D600, occurred when extracellular Ca2+ was readmitted after emptying intracellular Ca2+ stores with thapsigargin or acetylcholine. Thus there exists a small capacitative Ca2+ entry in β‐cells.
When the membrane potential was clamped at depolarized levels with 10, 20 or 45 mM K+ in the presence of diazoxide, [Ca2+]i increased to different plateau levels ranging between 100 and 900 nM. Thapsigargin consistently caused a further transient rise in [Ca2+]i, but had little (at 10 mM K+) or no effect on the plateau level. This confirms that the capacitative Ca2+ entry is small.
In clusters of cells whose membrane potential was not clamped with diazoxide, 15 mM glucose (in 4.8 mM K+) induced [Ca2+]i oscillations by promoting Ca2+ influx through voltage‐dependent Ca2+ channels. The application of thapsigargin accelerated these oscillations and increased their amplitude, sometimes causing a sustained elevation of [Ca2+]i. Similar results were obtained from whole islets perifused with a medium containing ≥ 6 mM glucose. The effect of thapsigargin was always much larger than expected from the capacitative Ca2+ entry, probably because of a potentiation of Ca2+ influx through voltage‐dependent Ca2+ channels.
This potentiating effect of thapsigargin did not result from an acceleration of cell metabolism since the drug did not affect glucose‐induced changes in NAD(P)H fluorescence. It is also unlikely to involve the inhibition of KATP channels because thapsigargin steadily elevated [Ca2+]i in cells in which [Ca2+]i oscillations persisted in the presence of a maximally effective concentration of tolbutamide.
In conclusion, the emptying of intracellular Ca2+ stores in β‐cells induces a small capacitative Ca2+ entry and activates a depolarizing current which potentiates glucose‐induced Ca2+ influx through voltage‐dependent Ca2+ channels.
Hyperpolarization of isolated capillaries from guinea‐pig heart induced by K+ channel openers and glucose deprivation
Authors: Langheinrich Ulrike , Daut Jürgen 
Published: September 2004

The present study was designed to test if microvascular coronary endothelial cells express ATP–sensitive K+ channels (KATP channels). We performed microfluorometric measurements of the membrane potential of freshly isolated guinea‐pig coronary capillaries equilibrated with the voltage–sensitive dye bis‐oxonol (bis‐[1,3–dibutylbarbituric acid] trimethineoxonol, [DiBAC4(3)]).
The resting membrane potential of capillaries in physiological salt solution was −46±4.2 mV (n= 8) at room temperature (22 °C) as determined after calibration of the fluorescence using the Na+–K+ ionophore gramicidin in the presence of different K+ concentrations. Spontaneous membrane potential fluctuations of 10–20 mV amplitude were often observed.
A reversible, sustained hyperpolarization to a new membrane potential close to the K+ equilibrium potential (EK) could be induced by application of the K+ channel openers HOE 234 (100 nm to 1 μM), diazoxide (10 pm to 100 nM) or pinacidil (100 nM). Subsequent addition of glibenclamide (200 nm to 2 μM) reversed this hyperpolarization.
A glibenclamide–sensitive hyperpolarization of coronary capillaries to values near EK was also observed upon omission of D‐glucose (10 mM) from the superfusing solution or by substituting L‐glucose for D‐glucose. Maximum hyperpolarization was reached in less than 10 min.
Our results suggest that microvascular coronary endothelial cells express KATP channels which may be activated during hypoglycaemia.