bluuebunny/crossref_metadata_2025_split huggingface dataset:
Repetitive hypoxia rapidly depresses arousal from active sleep in newborn lambs
Authors: Johnston Renea V. , Grant Daniel A. , Wilkinson Malcolm H. , Walker Adrian M. 
Published: September 2004

Arousal from sleep is an important protective mechanism that is depressed by repeated episodes of hypoxia. We aimed to determine how rapidly arousal depression occurs during repeated hypoxia and to determine if the depression is sleep state specific.
Three successive 12 h overnight sleep recordings were performed in six newborn lambs instrumented to record sleep state, blood pressure, heart rate and blood gases. The first (control) and third (recovery) nights were baseline studies (inspired oxygen fraction, FI,O2 = 0.21) to determine the spontaneous arousal probability. During the second (test) study night, lambs were exposed to a 60 s episode of isocapnic hypoxia (FI,O2 = 0.10; inspired carbon dioxide fraction, FI,CO2 = 0.03) during every epoch of sleep.
During quiet sleep (QS), the probability of arousing to hypoxia (56 %) remained significantly higher than the probability of arousing spontaneously (18 %) throughout the repeated hypoxic exposures (χ2= 81.5, P < 0.001). By contrast, during active sleep (AS) arousal rapidly became depressed with repetition of the hypoxic stimulus; the probability of arousal in hypoxia (52 %) was significantly higher than the probability of spontaneous arousal (12 %) during the first ten hypoxic exposures (χ2= 18.2, P < 0.001), but there was no difference thereafter.
We conclude that, when repeated, moderate hypoxia very rapidly becomes ineffective as an arousing stimulus in AS, but not in QS. These results suggest that the arousal mechanism is particularly vulnerable to failure during AS.
Ion permeation and conduction in a human recombinant 5‐HT3 receptor subunit (h5‐HT3A)
Authors: Brown A. M. , Hope A. G. , Lambert J. J. , Peters J. A. 
Published: September 2004

A human recombinant homo‐oligomeric 5‐HT3 receptor (h5‐HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole‐cell recording configuration of the patch clamp technique.
5‐HT evoked transient inward currents (EC50= 3.4 μM; Hill coefficient = 1.8) that were blocked by the 5‐HT3 receptor antagonist ondansetron (IC50= 103 pM) and by the non‐selective agents metoclopramide (IC50= 69 nM), cocaine (IC50= 459 nM) and (+)‐tubocurarine (IC50= 2.8 μM).
5‐HT‐induced currents rectified inwardly and reversed in sign (E5‐HT) at a potential of −2.2 mV. N‐Methyl‐D‐glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively.
Permeability towards divalent cations was assessed from measurements of E5‐HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively.
Single channel chord conductance (γ) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at −40 mV to 742 fS at −100 mV.
Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole‐cell current evoked by 5‐HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage‐independent augmentation of γ contributes to this phenomenon.
The data indicate that homo‐oligomeric receptors composed of h5‐HT3A subunits form inwardly rectifying cation‐selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.
Role of the NO‐cGMP pathway in the muscarinic regulation of the L‐type Ca2+ current in human atrial myocytes
Authors: Vandecasteele Grégoire , Eschenhagen Thomas , Fischmeister Rodolphe 
Published: September 2004

The whole‐cell patch‐clamp technique was used to examine the participation of nitric oxide synthase (NOS) and soluble guanylyl cyclase in the muscarinic regulation of the L‐type Ca2+ current (ICa) in freshly isolated human atrial myocytes.
Acetylcholine (ACh, 1 μM) decreased basal ICa by 39.1 ± 5.5 % (n= 8) under control conditions, and by 38.0 ± 6.1 % (n= 6) in the presence of 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ, 10 μM), a potent guanylyl cyclase inhibitor, and NG‐monomethyl‐L‐arginine (L‐NMMA, 1 mM), a competitive NOS inhibitor. L‐NMMA alone had no effect on ICa, whilst ODQ increased ICa in 50 % of the cells.
The accentuated antagonism of ACh on ICa, i.e. its ability to antagonize the stimulatory effect of β‐adrenergic agonists and, by extension, of other cAMP‐elevating agents, was examined after the current was stimulated by either the β‐adrenergic agonist isoprenaline (Iso) or serotonin (5‐HT). ACh (100 nM or 1 μM) completely blocked the stimulatory effects of 10 nM Iso or 10 nM 5‐HT on ICa.
Extracellular application of Methylene Blue (MBlue, 10 μM), a guanylyl cyclase inhibitor, antagonized the inhibitory effect of 1 μM ACh on Iso‐ or 5‐HT‐stimulated ICa. However, this effect was overcome by a 100‐fold higher ACh concentration and was not mimicked by an intracellular application of MBlue.
Inhibition of NOS and soluble guanylyl cyclase activities by addition of ODQ (10 μM) and L‐NMMA (1 mM) to both extracellular and intracellular solutions, or by a 2 h pre‐incubation of the cells with these inhibitors, modified neither the Iso (10 nM) response nor the inhibitory effect of ACh (100 nM or 1 μM) on Iso‐stimulated ICa.
Extracellular application of the NO donor SNAP (S‐nitroso‐N‐acetyl‐D,L‐penicillamine) at 100 nM produced a stimulatory effect on ICa in control conditions. This stimulatory effect was abolished by intracellular MBlue (20 μM) or by intracellular and extracellular application of ODQ (10 μM) in combination with L‐NMMA (1 mM).
We conclude that the NO‐cGMP pathway does not contribute significantly to the muscarinic regulation of ICa in human atrial myocytes.
Two mutations linked to nocturnal frontal lobe epilepsy cause use‐dependent potentiation of the nicotinic ACh response
Authors: Figl Antonio , Viseshakul Nareerat , Shafaee Navid , Forsayeth John , Cohen Bruce N. 
Published: September 2004

We constructed rat homologues (S252F and +L264) of two human α4 nicotinic mutations ‐ α4(S248F) and α4(777ins3) ‐ that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co‐expressed them with wild‐type rat β2 subunits in Xenopus oocytes.
The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use‐dependent potentiation of the response during a train of brief 100 nm ACh pulses. Second, they delayed the rise times of the 5–15 nm (+L264) and 30 nm (S252F) ACh responses. Third, they reduced extracellular Ca2+‐induced increases in the 30 μm ACh response.
Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage‐jump relaxations, enhanced steady‐state desensitization and reduced the single‐channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single‐channel conductance. Neither mutation affected the number of surface receptors measured by antibody binding but the S252F mutation reduced the maximum ACh response.
The ACh concentration dependence of use‐dependent potentiation and the delay in the rising phase of the mutant ACh response suggest that these effects are caused by a slow unblocking of the closed mutant receptors. Use‐dependent potentiation of the mutant response during a series of high‐frequency cholinergic inputs to the presynaptic terminal could trigger ADNFLE seizures by suddenly increasing nicotinic‐mediated transmitter release.
Non‐genomic inhibition of human P2X7 purinoceptor by 17β‐oestradiol
Authors: Cario‐Toumaniantz Chrystelle , Loirand Gervaise , Ferrier Laurent , Pacaud Pierre 
Published: September 2004

Effects of oestrogen on the current evoked by ATP and benzoylbenzoyl ATP (BzATP) in CV‐1 monkey kidney cells transformed by SV 40 (COS cells) expressing the human P2X7 (hP2X7) purinoceptor were studied using standard patch‐clamp techniques.
17β‐Oestradiol rapidly and reversibly inhibited the whole‐cell hP2X7 receptor cation current. This inhibitory action resulted in a rightward shift of the dose‐response curve to ATP and BzATP in the presence of physiological as well as low divalent cation concentrations.
The inhibitory effect of 17β‐oestradiol on the BzATP‐ or ATP‐induced cation current was concentration dependent. The half‐maximal inhibition was obtained with 3 μM 17β‐oestradiol. Progesterone and 17α‐oestradiol had almost no effect on the hP2X7 receptor cation current.
The inhibition of the hP2X7 receptor cation current by 17β‐oestradiol did not depend on the membrane potential. 17β‐Oestradiol added to the extracellular side of outside‐out patches inhibited BzATP‐activated single‐channel currents.
Activation of the hP2X7 receptor in both COS and U937 (human macrophage) cells did not induce the formation of large non‐specific pores.
Since COS cells do not express endogenous nuclear oestrogen receptor, this study shows that, at pharmacological concentrations, 17β‐oestradiol inhibited the hP2X7 receptor cation channel in a non‐genomic manner.
Regulation of Ca2+‐dependent Cl− conductance in a human colonic epithelial cell line (T84): cross‐talk between Ins(3,4,5,6)P4 and protein phosphatases
Authors: Xie Weiwen , Solomons Kevin R. H. , Freeman Sally , Kaetzel Marcia A. , Bruzik Karol S. , Nelson Deborah J. , Shears Stephen B. 
Published: September 2004

We have studied the regulation of whole‐cell chloride current in T84 colonic epithelial cells by inositol 3,4,5,6‐tetrakisphosphate (Ins(3,4,5,6)P4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1,2‐bisdeoxy‐1,2‐bisfluoro‐Ins(3,4,5,6)P4 (i.e. F2‐Ins(3,4,5,6)P4).
Calmodulin‐dependent protein kinase II (CaMKII) increased chloride current 20‐fold. This current (ICl,CaMK) continued for 7 ± 1.2 min before its deactivation, or running down, by approximately 60 %. This run‐down was prevented by okadaic acid, whereupon ICl,CaMK remained near its maximum value for ≥ 14.3 ± 0.6 min.
F2‐Ins(3,4,5,6)P4 inhibited ICl,CaMK (IC50= 100 μM) stereo‐specifically, since its enantiomer, F2‐Ins(1,4,5,6)P4 had no effect at <= 500 μM. Dose‐response data (Hill coefficient = 1.3) showed that F2‐Ins(3,4,5,6)P4 imitated only the non‐co‐operative phase of inhibition by Ins(3,4,5,6)P4, and not the co‐operative phase.
Ins(3,4,5,6)P4 was prevented from blocking ICl,CaMK by okadaic acid (IC50= 1.5 nM) and microcystin (IC50= 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P4 to function. The IC50 values indicate that more than one species of phosphatase was required. One of these may be PP1, since F2‐Ins(3,4,5,6)P4‐dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.
Intracellular nucleotide‐mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil
Authors: Satoh Eisaku , Yamada Mitsuhiko , Kondo Chikako , Repunte Vez P. , Horio Yoshiyuki , Iijima Toshihiko , Kurachi Yoshihisa 
Published: September 2004

We have examined the properties of intracellular nucleotide‐mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell‐attached form, both types of K+ channel were activated by pinacidil.
In inside‐out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi‐mediated channel inhibition in a concentration‐dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di‐ or tri‐, but not mono‐, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil.
In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell‐shaped concentration‐dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil.
From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide‐independent fast channel gate and a nucleotide‐dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate.
We conclude that Kir6.0 subunits play a crucial role in the nucleotide‐mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.
Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Ranapipiens
Authors: Lutz Gordon J. , Cuizon Denise B. , Ryan Allen F. , Lieber Richard L. 
Published: September 2004

Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS‐PAGE and only one adult hindlimb MHC isoform has been cloned.
In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Ranapipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS‐PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co‐migrated with type 1 MHC. The combined data from immunohistochemistry and SDS‐PAGE suggests that Rana fibre types are composed of four different MHCs.
Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno‐identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types.
Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level.
These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system.
Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation.
Termination of Ca2+ release during Ca2+ sparks in rat ventricular myocytes
Authors: Lukyanenko Valeriy , Wiesner Theodore F. , Györke Sandor 
Published: September 2004

Confocal Ca2+ imaging was used to measure spontaneous release events (Ca2+ sparks) in fluo‐3‐loaded isolated rat ventricular myocytes.
The microscopic Ca2+ release flux underlying Ca2+ sparks was derived by adapting the methods used previously to describe macroscopic Ca2+ release from cell‐averaged Ca2+ transients.
The magnitude of the local release fluxes varied from 2 to 5 μM ms−1, depending on SR Ca2+ loading conditions. Following spontaneous activation, the release flux rapidly decayed (τ= 6–12 ms). The rate of termination of release flux was found to be directly related to the magnitude of the flux (r2= 0.88).
The rate of termination of local release flux was slowed in the presence of FK506, a compound that is known to reduce inactivation of SR Ca2+ channels in vitro.
These results suggest that termination of release flux during sparks is not due to a spontaneous stochastic decay process or local depletion of Ca2+ from the SR, but rather involves an active extinguishing mechanism such as Ca2+‐dependent inactivation or adaptation.
Functional interactions between nicotinic and P2X channels in short‐term cultures of guinea‐pig submucosal neurons
Authors: Barajas‐López Carlos , Espinosa‐Luna Rosa , Zhu Yaohui 
Published: September 2004

Functional interactions between nicotinic and P2X receptors in submucosal neurons were investigated. Whole‐cell currents induced by ACh (IACh) and ATP (IATP) were blocked by hexamethonium and PPADS), respectively. Currents induced by simultaneous application of the two transmitters (IACh+ATP) were only as large as the current induced by the most effective of these substances. This current occlusion indicates that activation of nicotinic and P2X channels is not independent.
Kinetic parameters of IACh+ATP indicate that they are carried through channels activated by either substance. In agreement with this interpretation, both IACh and IATP amplitudes were decreased when ATP and ACh were applied simultaneously, whereas no cross‐desensitization was observed when nicotinic and P2X receptors were desensitized individually.
Current occlusion was observed at membrane potentials of −60 and +10 mV, when IACh and IATP were inward. However, when these currents were outward (at +40 mV), current occlusion was not observed. Current occlusion was still observed at +40 mV in experiments in which the reversal potential of these currents had been adjusted to more positive values.
Current occlusion occurred as soon as currents were detected (< 5 ms), was still present in the absence of Ca2+, Na+ or Mg2+, and after adding staurosporine, genistein, K‐252a, or N‐ethylmaleimide to the pipette solution. Similar observations were noted after substituting α,β‐methylene ATP for ATP, or GTP for GTP‐γ‐S in the pipette and in experiments carried out at 36, 23 and 9 °C.
We propose that nicotinic and P2X channels are in functional clusters of at least two, and that the influx of ions through one activates (through allosteric interactions) a mechanism that inhibits the other channel.
Neutralization of aspartate residues in the murine inwardly rectifying K+ channel IRK1 affects the substate behaviour in Mg2+ block
Authors: Oishi Keiko , Omori Koichiro , Ohyama Hideo , Shingu Koh , Matsuda Hiroko 
Published: September 2004

To investigate the molecular basis of the sublevels induced in the outward current during block by intracellular Mg2+, single‐channel currents through inwardly rectifying K+ (IRK1) channels were studied.
cDNA encoding a functional murine IRK1 channel was transfected into COS‐1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48‐72 h.
Intracellular Mg2+ at micromolar concentrations induced sublevels in the outward current at one‐third and two‐thirds of the unitary amplitude seen in wild‐type channels. Replacing Asp 172 with Asn (D172N) and Gln (D172Q) abolished these sublevels, i.e. the channel showed only the fully open and fully blocked states.
Both mutations reduced the Mg2+ sensitivity of the channel at 2 μM Mg2+. However, the Mg2+ sensitivity did not differ significantly at higher concentrations (10 μM) and voltages (+70 mV).
Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behaviour.
Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two‐thirds amplitude, while those from tetramers with three D172N mutant subunits showed no sublevels.
These findings suggest that differences in Mg2+ binding patterns lead to different conductive states in a single‐barrelled channel.
Direct measurement of SR release flux by tracking ‘Ca2+ spikes’ in rat cardiac myocytes
Authors: Song Long‐Sheng , Sham James S. K. , Stern Michael D. , Lakatta Edward G. , Cheng Heping 
Published: September 2004

Ca2+ release flux across the sarcoplasmic reticulum (SR) during cardiac excitation‐contraction coupling was investigated using a novel fluorescence method. Under whole‐cell voltage‐clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 mm, 150 nm free Ca2+), to minimize the residence time of released Ca2+ in the cytoplasm, and a low‐affinity, fast Ca2+ indicator, Oregon Green 488 BAPTA‐5N (OG‐5N; 1.0 mm, Kd≈ 31 μm), to optimize the detection of localized high [Ca2+] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca2+] at high spatial and temporal resolution.
Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca2+] change is the sum of two terms: one major term proportional to the SR release flux/Ca2+ influx, and the other reflecting the running integral of the released Ca2+.
Indeed, the OG‐5N transients in EGTA‐containing cells consisted of a prominent spike followed by a small pedestal. The OG‐5N spike closely resembled the first derivative (d[Ca2+]/dt) of the conventional Ca2+ transient (with no EGTA), and mimicked the model‐derived SR Ca2+ release function reported previously. In SR Ca2+‐depleted cells, the OG‐5N transient also closely followed the waveform of L‐type Ca2+ current (ICa). Using ICa as a known source of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrapolation of the ICa‐elicited OG‐5N signal.
The OG‐5N image signal was localized to discrete release sites at the Z‐line level of sarcomeres, indicating that the local OG‐5N spike arises from ‘Ca2+ spikes’ at transverse (T) tubule‐SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules).
Both peak SR release flux and total amount of released Ca2+ exhibited a bell‐shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca2+ release was most synchronized and produced maximal peak release flux (4.2 mm s−1) at 0 mV; in contrast, maximal total release occurred at −20 mV (71 versus 61 μm at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca2+] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca2+ released, but also the synchronization among release sites affects the whole‐cell Ca2+ transient and the Ca2+‐myofilament interaction.
A neuronal C5a receptor and an associated apoptotic signal transduction pathway
Authors: Farkas I. , Baranyi L. , Takahashi M. , Fukuda A. , Liposits Zs. , Yamamoto T. , Okada H. 
Published: September 2004

We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase‐polymerase chain reaction (RT‐PCR) in TGW human neuroblastoma cells.
Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS).
To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U‐937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226‐MAP) induced rapid elevation of nuclear c‐fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells.
Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1–2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx.
We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin‐sensitive G‐proteins.
Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration.