bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of cell function by methionine oxidation and reduction
Authors: Hoshi Toshinori , Heinemann Stefan H.
Published: August 2004

Reactive oxygen species (ROS) are generated during normal cellular activity and may exist in excess in some pathophysiological conditions, such as inflammation or reperfusion injury. These molecules oxidize a variety of cellular constituents, but sulfur‐containing amino acid residues are especially susceptible. While reversible cysteine oxidation and reduction is part of well‐established signalling systems, the oxidation and the enzymatically catalysed reduction of methionine is just emerging as a novel molecular mechanism for cellular regulation. Here we discuss how the oxidation of methionine to methionine sulfoxide in signalling proteins such as ion channels affects the function of these target proteins. Methionine sulfoxide reductase, which reduces methionine sulfoxide to methionine in a thioredoxin‐dependent manner, is therefore not only an enzyme important for the repair of age‐ or degenerative disease‐related protein modifications. It is also a potential missing link in the post‐translational modification cycle involved in the specific oxidation and reduction of methionine residues in cellular signalling proteins, which may give rise to activity‐dependent plastic changes in cellular excitability.
State‐dependent modulation of sensory feedback
Authors: Hultborn H.
Published: August 2004

By tradition ‐ and for historical reasons ‐ reflex pathways and interneurones have been named by their dominating sensory input. Later studies have demonstrated that each individual interneurone, as a rule, receives a broad convergence from a large variety of sensory modalities, as well as inputs from one or more descending tracts. It is thus possible that the traditional nomenclature inadvertently has served as a ‘straightjacket’ for conceptual development in this field. Indeed, there is now much evidence in favour of the view that the many classes of spinal interneurones may be seen as ‘functional units’ representing different levels of muscle synergies, parts of movements, or even more integrated motor behaviour. Such ‘functional units’ may be used by (different) descending pathways to mediate the motor commands from the brain and integrate the appropriate (multimodal) sensory feedback into the central command. A given sensory stimulus would then be able to affect the motor output through a number of parallel, or alternative, segmental pathways belonging to different ‘functional units’. If this were correct it would indeed be predicted, rather than coming as a surprise, that a given sensory stimulus can result in different outputs ‐ even with a different sign ‐ depending on the preceding selection of active ‘functional units’, i.e. the type of motor activity initiated by the brain.
Ion channels of importance for the locomotor pattern generation in the lamprey brainstem‐spinal cord
Authors: Grillner Sten , Wallén Peter , Hill Russell , Cangiano Lorenzo , Manira Abdeljabbar El
Published: August 2004

The intrinsic function of the spinal network that generates locomotion can be studied in the isolated brainstem‐spinal cord of the lamprey, a lower vertebrate. The motor pattern underlying locomotion can be elicited in the isolated spinal cord. The network consists of excitatory glutamatergic and inhibitory glycinergic interneurones with known connectivity. The current review addresses the different subtypes of ion channels that are present in the cell types that constitute the network. In particular the roles of the different subtypes of Ca2+ channels and potassium channels that regulate integrated neuronal functions, like frequency regulation, spike frequency adaptation and properties that are important for generating features of the motor pattern (e.g. burst termination), are reviewed. By knowing the role of an ion channel at the cellular level, we also, based on previous knowledge of network connectivity, can understand which effect a given ion channel may exert at the different levels from molecule and cell to network and behaviour.
Spinal interneuronal systems: identification, multifunctional character and reconfigurations in mammals
Authors: Jankowska E.
Published: August 2004

This review focuses on the flexibility of operation of spinal interneuronal networks and their multifunctional character in mammals. It concerns, in particular, two ways in which spinal interneuronal networks may be functionally reorganised, namely by modulating the synaptic actions of primary afferents by monoamines and by GABAergic presynaptic inhibition. The evidence will be reviewed for topographical and target‐related differences in modulatory effects in various interneuronal networks and these will be related to differences in the intrinsic properties of different functional types of interneurones in these networks and to the role played by them.
Calcium currents and transients in co‐cultured contracting normal and Duchenne muscular dystrophy human myotubes
Authors: Imbert Nathalie , Vandebrouck Clarisse , Duport Gérard , Raymond Guy , Hassoni Abdul A. , Constantin Bruno , Cullen Michael J. , Cognard Christian
Published: August 2004

The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co‐cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T‐ and L‐type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co‐cultures was carried out.

The resting membrane potential was slightly less negative in DMD (‐61.4 ± 1.1 mV) than in normal myotubes (‐65.5 ± 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo‐1.

The time integral under the spontaneous Ca2+ transients was significantly greater in DMD myotubes (97 ± 8 nm s) than in normal myotubes (67 ± 13 nm s).

The L‐ and T‐type current densities estimated from patch‐clamp recordings were smaller in DMD cells (2.0 ± 0.5 and 0.90 ± 0.19 pA pF−1, respectively) than in normal cells (3.9 ± 0.7 and 1.39 ± 0.30 pA pF−1, respectively).

The voltage‐dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half‐maximal (Vh,0.5) of the T‐ and L‐type currents towards less negative potentials, from ‐72.1 ± 0.7 and ‐53.7 ± 1.5 mV in normal cells to ‐61.9 ± 1.4 and ‐29.2 ± 1.4 mV in DMD cells, respectively.

Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 ± 45 pF) than of the normal myotubes (299 ± 34 pF) of the same apparent size.

Taken together, these results show that differences in T‐ and L‐type calcium currents between normal and DMD myotubes cannot simply explain all observed alterations in calcium homeostasis in DMD myotubes, thus suggesting that other transmembrane calcium transport mechanisms must also be altered in DMD myotubes compared with normal myotubes.
Is myosin phosphatase regulated in vivo by inhibitor‐1? Evidence from inhibitor‐1 knockout mice
Authors: Carr Andrew N. , Sutliff Roy L. , Weber Craig S. , Allen Patrick B. , Greengard Paul , de Lanerolle Primal , Kranias Evangelia G. , Paul Richard J.
Published: August 2004

The Ca2+ sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor‐1 (I‐1) is the classic type‐1 phosphatase inhibitor, but its presence and role in cAMP‐dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I‐1 in vivo, we investigated smooth muscle function in a mouse model lacking the I‐1 protein (I‐1(‐/‐) mice).

Significant amounts of I‐1 protein were detected in the wild‐type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by 32P‐labelled aortic extracts from WT mice.

Despite the significant presence of I‐1 in WT aorta, phenylephrine and KCl concentration‐ isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I‐1(‐/‐) aorta. cGMP‐dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC20), measured in aortic extracts, were nearly identical between WT and I‐1(‐/‐) mice.

In the portal vein, I‐1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC50 of isoproterenol relaxation (EC50= 10.4 ± 1.4 nm) compared to the WT value (EC50= 3.5 ± 0.2 nm). Contraction in response to acetylcholine as well as Ca2+ sensitivity were similar between WT and I‐1(‐/‐) aorta.

Despite the prevalence of I‐1 and its activation by PKA in the aorta, I‐1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I‐1 may play a role as a fine‐tuning mechanism involved in regulating portal vein responsiveness to β‐adrenergic agonists.
Regulation of a G protein‐gated inwardly rectifying K+ channel by a Ca2+‐independent protein kinase C
Authors: Leaney Joanne L. , Dekker Lodewijk V. , Tinker Andrew
Published: August 2004

Members of the Kir3.0 family of inwardly rectifying K+ channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by Gβγ subunits released in response to Gi/o‐coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the Gq/11‐coupled M1 and M3 muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal‐type channels stably expressed in the human embryonic kidney cell line HEK293.

We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M1 and M3 receptors. This contrasts with the simple stimulation seen through activation of M2 and M4 receptors.

Channel stimulation via M1 but not M3 receptors was sensitive to pertussis toxin whereas channel inhibition through both M1 and M3 receptors was insensitive. In contrast over‐expression of the C‐terminus of phospholipase Cβ1 or a Gq/11‐specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase.

The inhibitory effects of M1 and M3 receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4αphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M3 receptor.

The receptor‐mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0‐669 nm).

The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCδ and PKCε.

We also demonstrate that activation of such a pathway via both receptor‐mediated and receptor‐independent means profoundly attenuated subsequent channel stimulation by Gi/o‐coupled receptors.

Our data support a role for a Ca2+‐independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M1 and M3 muscarinic receptor stimulation.
Differential regulation of Ca2+‐activated Cl− currents in rabbit arterial and portal vein smooth muscle cells by Ca2+‐calmodulin‐dependent kinase
Authors: Greenwood Iain A. , Ledoux Jonathan , Leblanc Normand
Published: August 2004

Ca2+‐activated chloride currents (ICl(Ca)) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca2+‐calmodulin‐dependent kinase II (CaMKII) were compared between the three cell types.

In PA and CA myocytes dialysed and superfused with K+‐free media, pipette solutions containing fixed levels of free Ca2+ in the range of 250 nm to 1 μm evoked well sustained, outwardly rectifying ICl(Ca) currents in about 90 % of cells. The CaMKII inhibitor KN‐93 (5 μm) increased the amplitude of ICl(Ca) in PA and CA myocytes. However, the threshold intracellular Ca2+ concentration for detecting this effect was different in the two arterial cell types. KN‐93 also enhanced the rate of activation of the time‐dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady‐state activation curve.

In PA myocytes, the effects of KN‐93 were not mirrored by its inactive analogue KN‐92 but were reproduced by the inclusion of autocamtide‐2‐related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nm) significantly reduced ICl(Ca) evoked by 500 nm Ca2+.

In PV myocytes, ICl(Ca) was evoked by pipette solutions containing up to 1 μm free Ca2+ in less than 40 % of cells. Application of KN‐93 to cells where ICl(Ca) was sustained produced a small inhibition (≈25 %) of the current in 70 % of the cells.

The present study shows that regulation of Ca2+‐dependent Cl− channels by CaMKII differs between arterial and portal vein myocytes.
Spinal circuitry of sensorimotor control of locomotion
Authors: McCrea David A.
Published: August 2004

During locomotion many segmental hindlimb reflex pathways serve not only to regulate the excitability of local groups of motoneurones, but also to control the basic operation of the central pattern‐generating circuitry responsible for locomotion. This is accomplished through a reorganization of reflexes that includes the suppression of reflex pathways operating at rest and the recruitment during locomotion of previously unrecognized types of spinal interneurones. In addition presynaptic inhibition of transmission from segmental afferents serves to regulate the gain of segmental reflexes and may contribute to the selection of particular reflex pathways during locomotion. The fictive locomotion preparation in adult decerebrate cats has proved to be an important tool in understanding reflex pathway reorganization. Further identification of the spinal interneurones involved in locomotor‐dependent reflexes will contribute to our understanding not only of reflex pathway organization but also of the organization of the mammalian central pattern generator.
Ionic selectivity of native ATP‐activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia
Authors: Liu Dong‐Mei , Adams David J.
Published: August 2004

The relative permeability of the native P2X receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements of ATP‐evoked currents in parasympathetic neurones dissociated from rat submandibular ganglia using the dialysed whole‐cell patch clamp technique.

The P2X receptor‐channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Na+ > Li+ > Cs+ > Rb+ > K+, and permeability ratios relative to Cs+ (PX/PCs) ranging from 1.11 to 0.86.

The selectivity for the divalent alkaline earth cations was also weak with the sequence Ca2+ > Sr2+ > Ba2+ > Mn2+ > Mg2+. ATP‐evoked currents were strongly inhibited when the extracellular divalent cation concentration was increased.

The calculated permeability ratios of different ammonium cations are higher than those of the alkali metal cations. The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations have a higher permeability than that predicted by molecular size.

Acidification to pH 6.2 increased the ATP‐induced current amplitude twofold, whereas alkalization to 8.2 and 9.2 markedly reduced current amplitude. Cell dialysis with either anti‐P2X2 and/or anti‐P2X4 but not anti‐P2X1 antibodies attenuated the ATP‐evoked current amplitude. Taken together, these data are consistent with homomeric and/or heteromeric P2X2 and P2X4 receptor subtypes expressed in rat submandibular neurones.

The permeability ratios for the series of monovalent organic cations, with the exception of unsaturated cations, were approximately related to the ionic size. The relative permeabilities of the monovalent inoganic and organic cations tested are similar to those reported previously for cloned rat P2X2 receptors expressed in mammalian cells.
Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin
Authors: Hamilton Sara G. , McMahon Stephen B. , Lewin Gary R.
Published: August 2004

ATP can elicit pain in humans and, together with other P2X channel agonists, can produce nocifensive responses in rodents. We used the rat in vitro skin‐nerve preparation to quantify primary afferent responses to ATP and its stable analogue α,β‐methylene ATP in normal and carrageenan‐inflamed skin.

Both ATP and α,β‐methylene ATP were found to specifically activate the peripheral terminals of Aδ and C‐fibre nociceptors in the skin. Thirty‐nine per cent of the nociceptors tested responded to the maximal dose of α,β‐methylene ATP (5 mm). In contrast, non‐nociceptive, low‐threshold mechano‐sensitive fibres were never activated by the same agonist concentrations.

Amongst the nociceptor population, C‐mechanoheat fibres (C‐MH or polymodal nociceptors) were markedly more responsive to P2X agonists than mechanonociceptors (C‐M nociceptors) with Aδ‐ or C‐fibre axons. Both C‐mechanoheat and C‐mechanonociceptors were activated by α,β‐methylene ATP doses as low as 50 μm.

In skin inflamed with carrageenan 3‐4 h before recording both the number of responsive C‐fibre nociceptors and their response magnitude increased. The increased neural response under inflammatory conditions was largely observed in C‐mechanoheat or polymodal nociceptors. After low doses of P2X agonists C‐MH fibres but not C‐M fibres developed elevated ongoing activity and this effect was only seen after carrageenan inflammation. The time course of α,β‐methylene ATP‐evoked discharges in nociceptors was found to correlate well with the time course of behavioural nocifensive responses in rats to the same agonist described in a previous study (Hamilton et al. 1999).

We conclude that the rapid increase in the number of α,β‐methylene ATP responsive nociceptors and the increased magnitude of the neural response following carrageenan inflammation explains why very low concentrations of such agonists can cause pain in inflammatory states.
Minimal force‐frequency modulation of inotropy and relaxation of in situ murine heart
Authors: Georgakopoulos Dimitrios , Kass David A.
Published: August 2004

The normal influence of heart rate (HR) on cardiac contraction and relaxation in the mouse remains uncertain despite its importance in interpreting many genetically engineered models. Prior in vivo data have repeatedly shown positive effects only at subphysiological heart rates, yet depressed basal conditions and use of load‐dependent parameters probably have an impact on these results.

Open‐chest mice of various strains (n = 16, etomidate/urethane anaesthesia) were instrumented with a miniaturized pressure‐volume catheter employing absolute left ventricular (LV) volume calibration. HR was slowed (< 400 beats min−1) using ULFS‐49, and atrial or ventricular pacing was achieved via an intra‐oesophageal catheter. Pressure‐volume data yielded cardiac‐specific contractile indexes minimally altered by vascular load.

At a resting HR of 600 beats min−1, peak pressure‐rise rate (dP/dtmax) was 16 871 ± 2941 mmHg s−1 (mean ±s.d.) and the relaxation time constant was 3.9 ± 0.8 ms, similar to values in conscious animals. Within the broad physiological range (500‐850 beats min−1), load‐insensitive contractile indexes and relaxation rate varied minimally, whereas dP/dtmax peaked at 600 ± 25 beats min−1 and decreased at higher rates due to preload sensitivity. Contraction and relaxation were enhanced modestly (13‐15 %) at HRs of between 400 and 500 beats min−1.

The minimal force‐frequency dependence was explained by rapid calcium cycling kinetics, with a mechanical restitution time constant of 9 ± 2.7 ms, and by dominant sarcoplasmic reticular buffering (recirculation fraction of 93 ± 1 %).

The mouse normally has a very limited force‐frequency reserve at physiological HRs, unlike larger mammals and man. This is important to consider when studying disease evolution and survival of genetic models that alter calcium homeostasis and SR function.
Sacral spinal interneurones and the control of urinary bladder and urethral striated sphincter muscle function
Authors: Shefchyk Susan J.
Published: August 2004

Normally, during bladder filling (continence) and expulsion (micturition) there is a reciprocity between the pattern of activity in the urinary bladder sacral parasympathetic efferents and the somatic motoneurones innervating the striated external urethral sphincter muscle. The co‐ordination of this pattern of reciprocal activity appears to be determined by excitatory and inhibitory actions of a variety of segmental afferents and descending systems with sacral spinal actions. These actions may in part be mediated through lower lumbar and sacral excitatory and inhibitory spinal interneurones. Over the past 30 years, both neuroanatomical and electrophysiological approaches have been used to reveal an ever‐increasing richness in the neuronal network in the lower spinal cord related to the bladder and striated external urethral sphincter muscle. The purpose of this review is to present an overview of the identified excitatory and inhibitory spinal interneurones hypothesized to be involved in the central networks controlling the sacral bladder parasympathetic preganglionic neurones and striated urethral sphincter motoneurones during continence and micturition.
Compartmental neurodegeneration and synaptic plasticity in the Wlds mutant mouse
Authors: Gillingwater Thomas H. , Ribchester Richard R.
Published: August 2004

This review focuses on recent developments in our understanding of neurodegeneration at the mammalian neuromuscular junction. We provide evidence to support a hypothesis of compartmental neurodegeneration, whereby synaptic degeneration occurs by a separate, distinct mechanism from cell body and axonal degeneration. Studies of the spontaneous mutant Wlds mouse, in which Wallerian degeneration is characteristically slow, provide key evidence in support of this hypothesis. Some features of synaptic degeneration in the absence of Wallerian degeneration resemble synapse elimination in neonatal muscle. This and other forms of synaptic plasticity may be accessible to further investigations, exploiting advantages afforded by the Wlds mutant, or transgenic mice that express the Wlds gene.
Learning from the spinal cord
Authors: Loeb Gerald E.
Published: August 2004

The graceful control of multiarticulated limbs equipped with slow, non‐linear actuators (muscles) is a difficult problem for which robotic engineering affords no general solution. The vertebrate spinal cord provides an existence proof that such control is, indeed, possible. The biological solution is complex and incompletely known, despite a century of meticulous neurophysiological research, celebrated in part by this symposium. This is frustrating for those who would reanimate paralysed limbs either through promoting regeneration of the injured spinal cord or by functional electrical stimulation. The importance of and general role played by the spinal cord might be more easily recognized by analogy to marionette puppets, another system in which a brain (the puppeteer's) must cope with a large number of partially redundant actuators (strings) moving a mechanical linkage with complex intrinsic properties.
Two open states and rate‐limiting gating steps revealed by intracellular Na+ block of human KCNQ1 and KCNQ1/KCNE1 K+ channels
Authors: Pusch Michael , Ferrera Loretta , Friedrich Thomas
Published: August 2004

KCNQ1, the first member of a new K+ channel family, associates with the small KCNE1 subunit to form the slow cardiac delayed rectifier current, IKs. Mutations in both genes encoding these channels lead to cardiac arrhythmia. We studied the block by intracellular Na+ of human homomeric KCNQ1 (homomers) and heteromeric KCNQ1/KCNE1 (heteromers) expressed in CHO cells (Chinese hamster ovary cell line) using whole‐cell patch recording.

In the nominal absence of extracellular K+ and with 65 mm intracellular K+, the replacement of 65 mm intracellular N‐methyl‐d‐glucamine (NMDG+) by 65 mm Na+ induced a decay of outward (K+) currents through homomers after maximal activation reminiscent of an inactivation process. The decay had a time constant in the hundreds of milliseconds range.

The inactivation process of homomers was, however, not directly dependent on [Na+]i, as evidenced by unaltered biphasic deactivation at negative voltages.

An instantaneous voltage‐dependent Na+ block of homomers was revealed using tail current protocols with activating prepulses that saturated the gating processes of the channel. The instantaneous block was partially relieved at very large positive voltages (≥ 60 mV) and in 20 mm extracellular K+. The instantaneous block of homomers was much less pronounced if the tail currents were measured after short activating prepulses, demonstrating the presence of (at least) two open states: a first, relatively [Na+]i‐insensitive and a subsequent [Na+]i‐sensitive open state; the current decay reflects the transition between the two open states.

Heteromers exhibited a very similar instantaneous block by Na+i independently of the prepulse duration. Heteromers did not show a Na+i‐induced current decay.

Our results demonstrate the presence of two open states of KCNQ1 channels with different [Na+]i sensitivities. The rate‐limiting step of homomeric KCNQ1 gating at positive voltages is the transition between these two open states. The rate‐limiting step of the gating of KCNQ1/KCNE1 channels appears to be the entry into the first open state.
Depletion of Ca2+ in the sarcoplasmic reticulum stimulates Ca2+ entry into mouse skeletal muscle fibres
Authors: Kurebayashi Nagomi , Ogawa Yasuo
Published: August 2004

To examine whether a capacitative Ca2+ entry pathway is present in skeletal muscle, thin muscle fibre bundles were isolated from extensor digitorum longus (EDL) muscle of adult mice, and isometric tension and fura‐2 signals were simultaneously measured.

The sarcoplasmic reticulum (SR) in the muscle fibres was successfully depleted of Ca2+ by repetitive treatments with high‐K+ solutions, initially in the absence and then in the presence of a sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor.

Depletion of the SR of Ca2+ enabled us for the first time to show convincingly that the vast majority of the voltage‐sensitive Ca2+ store overlaps the caffeine‐sensitive Ca2+ store in intact fibres from mouse EDL muscle. This conclusion was based on the observation that both high‐K+ solution and caffeine failed to cause a contracture in the depleted muscle fibres.

The existence of a Ca2+ influx pathway active enough to refill the depleted SR within several minutes was shown in skeletal muscle fibres. Ca2+ entry was sensitive to Ni2+, but resistant to nifedipine and was suppressed by plasma membrane depolarisation.

Evidence for store‐operated Ca2+ entry was provided by measurements of Mn2+ entry. Significant acceleration of Mn2+ entry was observed only when the SR was severely depleted of Ca2+. The Mn2+ influx, which was blocked by Ni2+ but not by nifedipine, was inwardly rectifying, as is the case with the Ca2+ entry. These results indicate that the store‐operated Ca2+ entry is similar to the Ca2+ release‐activated Ca2+ channel (CRAC) current described in other preparations.
Atrial natriuretic factor increases splenic microvascular pressure and fluid extravasation in the rat
Authors: Sultanian Richard , Deng Yiming , Kaufman Susan
Published: August 2004

The spleen is an important site of atrial natriuretic factor (ANF)‐induced fluid extravasation into the systemic lymphatic system. The mechanism underlying this process was studied in a blood‐perfused (1 ml min−1) rat spleen using the double occlusion technique. To ensure that our observations were spleen specific, a similar protocol was repeated in the hindquarters.

Rat ANF(1‐28), infused into the splenic artery of anaesthetized male rats, caused a dose‐dependent (0.3‐59 pmol min−1) increase in microvascular pressure from 11.3 ± 0.7 to 14.9 ± 0.5 mmHg and in post‐capillary resistance from 7.2 ± 0.6 to 10.1 ± 1.1 mmHg ml−1. ANF elicited no change in splenic pre‐capillary resistance or in hindquarter haemodynamics.

Intrasplenic ANF (6.5 pmol min−1) caused a sustained increase in intrasplenic fluid efflux from 0.1 ± 0.1 to 0.3 ± 0.1 ml min−1, and in capillary filtration coefficient (Kf) from 1.2 ± 0.5 to 2.4 ± 0.6 ml mmHg−1 min−1 (100 g tissue)−1.

Mechanical elevation of splenic intravascular pressure (from 11.3 ± 0.7 to 22.4 ± 0.2 mmHg) significantly increased intrasplenic fluid extravasation (from 0.4 ± 0.3 to 1.4 ± 0.3 ml min−1).

The natriuretic peptide receptor‐C (NPRC)‐specific agonist C‐ANF(4‐23) (12.5 and 125 pmol min−1) did not alter splenic intravascular pressure or pre‐/post‐capillary resistance.

The ANF antagonist A71915 (8.3 and 83 pmol min−1), which blocks ANF‐stimulated cGMP production via natriuretic peptide receptor‐A (NPRA), inhibited the ANF‐induced changes in splenic microvascular pressure and post‐capillary resistance.

It is concluded that ANF enhances the extravasation of isoncotic fluid from the splenic vasculature both by raising intrasplenic microvascular pressure (increased post‐capillary resistance) and by increasing filtration area. The constrictive activity of ANF on the splenic vasculature is mediated through NPRA.
Role of myoplasmic phosphate in contractile function of skeletal muscle: studies on creatine kinase‐deficient mice
Authors: Dahlstedt Anders J. , Katz Abram , Westerblad Håkan
Published: August 2004

Increased myoplasmic inorganic phosphate (Pi) has been suggested to have an important role in skeletal muscle fatigue, especially in the early phase. In the present study we used intact fast‐twitch muscle cells from mice completely deficient in creatine kinase (CK‐/‐) to test this suggestion. These CK‐/‐ muscle cells provide a good model since they display a higher Pi concentration in the unfatigued state and fatigue without significant increase of Pi.

Tetanic contractions (350 ms duration) were produced in intact single muscle fibres. The free myoplasmic [Ca2+] ([Ca2+]i) was measured with the fluorescent indicator indo‐1. The force‐[Ca2+]i relationship was constructed from tetani at different frequencies.

Compared with wild‐type fibres, CK‐/‐ fibres displayed lower force in 100 Hz tetani and at saturating [Ca2+]i (i.e. 100 Hz stimulation during caffeine exposure), higher tetanic [Ca2+]i during the first 100 ms of tetanic stimulation, reduced myofibrillar Ca2+ sensitivity when measurements were performed 100–200 ms into tetani, and slowed force relaxation that was due to altered cross‐bridge kinetics rather than delayed Ca2+ removal from the myoplasm.

In wild‐type fibres, a series of 10 tetani resulted in reduced tetanic force, slowed force relaxation, and increased amplitude of [Ca2+]i tails after tetani. None of these changes were observed in CK‐/‐ fibres.

Complementary experiments on isolated fast‐twitch extensor digitorum longus muscles showed a reduction of tetanic force and relaxation speed in CK‐/‐ muscles similar to those observed in single fibres.

In conclusion, increased Pi concentration can explain changes observed in the early phase of skeletal muscle fatigue. Increased Pi appears to be involved in both fatigue‐induced changes of cross‐bridge function and SR Ca2+ handling.
Endocytosis in identified rat corticotrophs
Authors: Lee Andy K. , Tse Amy
Published: August 2004

We used the patch‐clamp technique, in conjunction with membrane capacitance measurement, fluorescence measurement of intracellular calcium concentration ([Ca2+]i), and flash photolysis of caged Ca2+ to study exo‐ and endocytosis in identified rat corticotrophs.

Exocytosis stimulated by depolarization pulses was typically followed by a ‘slow’ endocytosis that retrieved the membrane with a time constant of ∼6 s. The efficiency (the endocytosis/exocytosis amplitude ratio) of ‘slow’ endocytosis was ∼1.2 at [Ca2+]i < 3 μm and increased to ∼1.6 at [Ca2+]i > 3 μm.

Whole‐cell dialysis through a patch pipette did not affect the kinetics and the efficiency of ‘slow’ endocytosis, but the amplitude of exocytosis was reduced.

‘Slow’ endocytosis did not require sustained [Ca2+]i elevation and its kinetics was only weakly [Ca2+]i dependent. Our results suggest that ‘slow’ endocytosis involves a Ca2+ sensor with a high Ca2+ affinity (∼500 nm).

At high [Ca2+]i (> 10 μm), the ‘slow’ endocytosis was frequently preceded by a ‘fast’ endocytosis that comprised multiple steps of rapid decrease in membrane capacitance.

Neither calmodulin nor calcineurin appeared to be the Ca2+ sensor for endocytosis because the two forms of endocytosis were not affected by the calmodulin inhibitor calmidazolium (500 μm) or the calcineurin inhibitors cyclosporin A (1 μm) and calcineurin autoinhibitory peptide (1 mg ml−1). Ba2+, a poor activator of calmodulin, could support both forms of endocytosis but slowed the kinetics of ‘slow’ endocytosis ∼2‐fold.

Non‐hydrolysable analogues of GTP (GDP‐β‐S) and ATP (ATP‐γ‐S) also failed to inhibit either form of endocytosis, indicating that neither GTP nor ATP was essential for endocytosis.

We suggest that the high Ca2+ affinity of ‘slow’ endocytosis may be important for maintaining continuous cycles of exocytosis‐endocytosis during sustained adrenocorticotropin secretion in corticotrophs.
Dendritic mechanisms underlying the coupling of the dendritic with the axonal action potential initiation zone of adult rat layer 5 pyramidal neurons
Authors: Larkum M. E. , Zhu J. J. , Sakmann B.
Published: August 2004

Double, triple and quadruple whole‐cell voltage recordings were made simultaneously from different parts of the apical dendritic arbor and the soma of adult layer 5 (L5) pyramidal neurons. We investigated the membrane mechanisms that support the conduction of dendritic action potentials (APs) between the dendritic and axonal AP initiation zones and their influence on the subsequent AP pattern.

The duration of the current injection to the distal dendritic initiation zone controlled the degree of coupling with the axonal initiation zone and the AP pattern.

Two components of the distally evoked regenerative potential were pharmacologically distinguished: a rapidly rising peak potential that was TTX sensitive and a slowly rising plateau‐like potential that was Cd2+ and Ni2+ sensitive and present only with longer‐duration current injection.

The amplitude of the faster forward‐propagating Na+‐dependent component and the amplitude of the back‐propagating AP fell into two classes (more distinctly in the forward‐propagating case). Current injection into the dendrite altered propagation in both directions.

Somatic current injections that elicited single Na+ APs evoked bursts of Na+ APs when current was injected simultaneously into the proximal apical dendrite. The mechanism did not depend on dendritic Na+–Ca2+ APs.

A three‐compartment model of a L5 pyramidal neuron is proposed. It comprises the distal dendritic and axonal AP initiation zones and the proximal apical dendrite. Each compartment contributes to the initiation and to the pattern of AP discharge in a distinct manner. Input to the three main dendritic arbors (tuft dendrites, apical oblique dendrites and basal dendrites) has a dominant influence on only one of these compartments. Thus, the AP pattern of L5 pyramids reflects the laminar distribution of synaptic activity in a cortical column.
Dopamine D2‐like receptors selectively block N‐type Ca2+ channels to reduce GABA release onto rat striatal cholinergic interneurones
Authors: Momiyama Toshihiko , Koga Eiko
Published: August 2004

The modulatory roles of dopamine (DA) in inhibitory transmission onto striatal large cholinergic interneurones were investigated in rat brain slices using patch‐clamp recording.

Pharmacologically isolated GABAA receptor‐mediated IPSCs were recorded by focal stimulation within the striatum. Bath application of DA reversibly suppressed the amplitude of evoked IPSCs in a concentration‐dependent manner (IC50, 10.0 μm).

A D2‐like receptor agonist, quinpirole (3–30 μm), also suppressed the IPSCs, whereas a D1‐like receptor agonist, SKF 81297, did not affect IPSCs. Sulpiride, a D2‐like receptor antagonist, blocked the DA‐induced suppression of IPSCs (apparent dissociation constant (KB), 0.36 μm), while a D1‐like receptor antagonist, SCH 23390 (10 μm), had no effect.

DA (30 μm) reduced the frequency of spontaneous miniature IPSCs (mIPSCs) without changing their amplitude distribution, suggesting that GABA release was inhibited, whereas the sensitivity of postsynaptic GABAA receptors was not affected. The effect of DA on the frequency of mIPSCs was diminished when extracellular Ca2+ was replaced by Mg2+ (5 mm), indicating that DA affected the Ca2+ entry into the presynaptic terminal.

An N‐type Ca2+ channel selective blocker, ω‐conotoxin GVIA (ω‐CgTX, 3 μm), suppressed IPSCs by 65.4%, whereas a P/Q‐type Ca2+ channel selective blocker, ω‐agatoxin IVA (ω‐Aga‐IVA, 200 nm), suppressed IPSCs by 78.4%. Simultaneous application of both blockers suppressed IPSCs by 95.9%. Assuming a 3rd power relationship between Ca2+ concentration and transmitter release, the contribution of N‐, P/Q‐ and other types of Ca2+ channels to presynaptic Ca2+ entry is estimated to be, respectively, 29.8, 40.0 and 34.5% at this synapse. After the application of ω‐CgTX, DA (30 μm) no longer affected IPSCs. In contrast, ω‐Aga‐IVA did not alter the level of suppression by DA, suggesting that the action of DA was selective for N‐type Ca2+ channels.

A G protein alkylating agent, N‐ethylmaleimide (NEM), significantly reduced the DA‐induced suppression of IPSCs.

These results suggest that presynaptic D2‐like receptors are present on the terminals of GABAergic afferents to striatal cholinergic interneurones, and down‐regulate GABA release by selectively blocking N‐type Ca2+ channels through NEM‐sensitive G proteins.
Sequence dependence of post‐tetanic potentiation after sequential heterosynaptic stimulation in the rat auditory cortex
Authors: Seki Kenjiro , Kudoh Masaharu , Shibuki Katsuei
Published: August 2004

To investigate the mechanisms for the coding stimulus sequence in the auditory cortex (AC), post‐tetanic potentiation (PTP) was recorded after sequentially combined heterosynaptic stimulation was applied in rat AC slices.

Brief tetanic stimulation (TS) was applied at two sites on AC slices at intervals of 0.5–10 s. PTP of field potentials was induced by the earlier TS, rather than the later TS. PTP was followed by sequence‐dependent long‐term potentiation (LTP).

Using Ca2+ imaging in the slices loaded with rhod‐2, a Ca2+ indicator, a sequence‐dependent distribution of PTP was found in AC slices.

The sequence‐dependent PTP in excitatory postsynaptic potentials (EPSPs) was observed in supragranular pyramidal neurons.

The sequence dependence of PTP was not significantly affected by 1 μm bicuculline, an antagonist of GABAA receptors, or 100 μm 2‐hydroxysaclofen, an antagonist of GABAB receptors.

Depolarization and firing recorded in pyramidal neurons during the later TS were less vigorous than when the slices were incubated in the control medium. However, this suppression of the responses during the later TS was not observed in the presence of 50 μm atropine, an antagonist of muscarinic receptors.

PTP was induced by the earlier and later TS in the presence of 50 μm atropine, so that the sequence dependence of PTP was abolished. Pirenzepine (50 μm), an antagonist of muscarinic M1 receptors, but not methoctramine (30 μm), an antagonist of M2 receptors, eliminated the sequence dependence of PTP.

These findings suggest that the sequence dependence of PTP in AC might have a role in the temporal processing of auditory information on the scale of seconds.
Spatial properties of koniocellular cells in the lateral geniculate nucleus of the marmoset Callithrix jacchus
Authors: White Andrew J. R. , Solomon Samuel G. , Martin Paul R.
Published: August 2004

The receptive field dimensions, contrast sensitivity and linearity of spatial summation of koniocellular (KC), parvocellular (PC) and magnocellular (MC) cells in the lateral geniculate nucleus (LGN) of 11 adult marmosets were measured using achromatic sinusoidal gratings.

The receptive field centre diameter of cells in each (PC, KC and MC) class increases with distance from the fovea. There is substantial overlap in centre size between the three cell classes at any eccentricity, but the PC cells have, on average, the smallest centres and the KC cells have the largest. Some PC and KC cells did not respond at all to the grating stimulus.

The contrast sensitivity of the receptive field centre mechanism in KC cells decreases in proportion to the centre area. A similar trend was seen for the surround mechanism. These characteristics are common to PC and MC cells, suggesting that they originate at an early stage of visual processing in the retina.

The KC cells showed, in general, lower peak evoked discharge rates than PC or MC cells. The spontaneous discharge rate of KC cells was lower than that of PC cells and similar to that of MC cells.

The majority of cells in all divisions of the LGN show linear spatial summation. A few cells did show non‐linear spatial summation; these cells were predominantly located in the MC and ventral KC layers.

The ventral KC layers below and between the MC layers contain cells with larger and more transiently responding receptive fields than cells in the more dorsal KC layers.

We conclude that many of the contrast‐dependent spatial properties of cells in the marmoset LGN are common to PC, MC and KC cells. The main difference between KC cells and the other two classes is that there is more variability in their response properties, and they are less responsive to high spatial frequencies.
Maternal undernutrition increases arterial blood pressure in the sheep fetus during late gestation
Authors: Edwards L. J. , McMillen I. C.
Published: August 2004

We have investigated the effect of a 50% reduction in maternal nutrient intake during the last 30 days of pregnancy on arterial blood pressure and on arterial blood pressure responses to angiotensin II (AII) and the angiotensin converting enzyme (ACE) inhibitor captopril in the sheep fetus at 115–125 and at 135–145 days gestation (term = 147 ± 3 days gestation).

Fetal plasma glucose concentrations were lower in the undernourished (UN) group compared to the control animals. There was no difference, however, in fetal plasma cortisol or adrenocorticotrophic hormone (ACTH) concentrations between the UN and control groups between 115 and 145 days gestation.

During the first 10 days of undernutrition, maternal plasma concentrations of cortisol were increased in the UN group compared to controls. At 115–125 days gestation, fetal arterial blood pressure was also higher in the UN group compared with controls and there was an inverse relationship (r=−0.62, P < 0.05) between mean arterial pressure and the fetal plasma concentrations of ACTH in the UN group. Fetal blood pressure responses to increasing doses of angiotensin II were also higher (P < 0.05) in UN compared to control animals at 115–125 days gestation.

Between 135 and 145 days gestation, fetal arterial blood pressure was increased in UN fetal sheep and mean arterial blood pressure was correlated with fetal plasma concentrations of cortisol.

Increased arterial blood pressure and responsiveness to AII measured in the fetuses of nutrient‐restricted ewes may be related in part to fetal exposure to the actions of cortisol derived from transplacental transfer during the first 10 days after the start of the restricted feeding regime.
Eccentric exercise‐induced morphological changes in the membrane systems involved in excitation—contraction coupling in rat skeletal muscle
Authors: Takekura Hiroaki , Fujinami Nahoko , Nishizawa Tomie , Ogasawara Hitomi , Kasuga Norikatsu
Published: August 2004

Physiological evidence suggests that excitation‐contraction (E—C) coupling failure results from eccentric contraction‐induced muscle injury because of structural and morphological damage to membrane systems directly associated with the E—C coupling processes within skeletal muscle fibres. In this study using rats, we observed the ultrastructural features of the membrane systems of fast‐twitch (FT) and slow‐twitch (ST) muscle fibres involved in E—C coupling following level and downhill running exercise. Our aim was to find out whether mechanically mediated events following eccentric exercise caused disorder in the membrane systems involved in E—C coupling, and how soon after exercise such disorder occurred. We also compared the morphological changes of the membrane systems between ST and FT muscle fibres within the same muscles.

Single muscle fibres were dissected from triceps brachii muscles of male Fischer 344 rats after level or downhill (16 deg decline) motor‐driven treadmill running (18 m min−1, 5 min running with 2 min rest interval, 18 bouts). All single muscle fibres were histochemically classified into ST or FT fibres. The membrane systems were visualized using Ca2+–K3Fe(CN)6–OsO4 techniques, and observed by high voltage electron microscopy (120–200 kV).

There were four obvious ultrastructural changes in the arrangement of the transverse (t)‐tubules and the disposition of triads after the downhill running exercise: (1) an increase in the number of longitudinal segments of the t‐tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolar clusters, and (4) the appearance of pentads and heptads (close apposition of two or three t‐tubule elements with three or four elements of terminal cisternae of the sarcoplasmic reticulum). The caveolar clusters appeared almost exclusively in the ST fibres immediately after downhill running exercise and again 16 h later. The pentads and heptads appeared almost exclusively in the FT fibres, and their numbers increased dramatically 2–3 days after the downhill running exercise.

The eccentric exercise led to the formation of abnormal membrane systems involved in E–C coupling processes. These systems have unique morphological features, which differ between ST and FT fibres, even within the same skeletal muscle, and the damage appears to be concentrated in the FT fibres. These observations also support the idea that eccentric exercise‐ induced E–C coupling failure is due to physical and chemical disruption of the membrane systems involved in the E–C coupling process in skeletal muscle.