bluuebunny/crossref_metadata_2025 huggingface dataset:
Energetics of lengthening in mouse and toad skeletal muscles
Authors: Constable J. K. , Barclay C. J. , Gibbs C. L.
Published: September 2004

The energetics of lengthening were studied in amphibian and mammalian skeletal muscle. The aims were to determine whether energy absorption during stretch is a general property of skeletal muscle and to investigate the influence of lengthening velocity on energy absorption.

Experiments were performed in vitro (21 °C) using bundles of muscle fibres from fast‐twitch extensor digitorum longus and slow‐twitch soleus muscles of the mouse and tibialis anterior muscles of a toad, Bufo marìnus. Initial heat production and mechanical work done on muscles were measured during isovelocity lengthening. Enthalpy output during lengthening was calculated as the difference between the amount of heat produced and the work done.

For all three muscle types, more energy was put into muscles as work than was produced as heat. Thus, part of the energy put into muscles to stretch them must have been absorbed.

For all three muscle types, the amount of energy absorbed was constant at velocities exceeding ∼0.5Vmax (Vmax is the maximum shortening velocity), but was significantly lower at slow velocities of lengthening. The same amount of energy was absorbed by all three muscles when lengthened at ≥ 0.5 Vmax.

It was concluded that absorption of energy during lengthening occurs in mammalian as well as amphibian muscle and that lengthening velocity has only a small effect on the amount of energy absorbed.
Energetic cost of activation processes during contraction of swine arterial smooth muscle
Authors: Wingard Christopher J. , Paul Richard J. , Murphy Richard A.
Published: September 2004

The objective of this study was to partition the increase in ATP consumption during contraction of swine carotid arterial smooth muscle estimated from suprabasal oxygen consumption (suprabasal JO2) and lactate release (Jlactate) into a component associated with cross‐bridge cycling (Jx) and one reflecting activation (JA).

Two experimental approaches – varying length under constant activation, and varying activation at a long length (1.8 times the optimal length for force development (L0)) where force generation is minimal – revealed a linear dependence of J0 and activation energy (JA) on cross‐bridge phosphorylation. Protocols inducing a large increase in myosin regulatory light chain (MRLC) phosphorylation at 1.8L0 resulted in significant elevations of JO2 and marked reductions in the economy of force maintenance. Our evidence suggests that this is primarily due to the increased cost of cross‐bridge phosphorylation.

The extrapolated estimate of JA during maximal K+‐induced depolarization made by varying length was 16%, while at 1.8LO it was 33% of the suprabasal JO2 at L0. Calculated activation energies ranged from 17 to 45% of the suprabasal JO2 at L0 and from 72 to 87% of the suprabasal JO2 at 1.8L0 under stimulation conditions that varied steady‐state MRLC phosphorylation from 15 to 50%.

The results suggest that the kinetics of cross‐bridge phosphorylation‐dephosphorylation can rival those of cross‐bridge cycling during isometric contractions in swine arterial smooth muscle.
The Effects of Breathing He–O2 Mixtures on Maximal Oxygen Consumption in Normoxic and Hypoxic Men
Authors: Esposito Fabio , Ferretti Guido
Published: September 2004

The hypothesis that the ventilatory resistance to O2 flow (RV) does limit maximal O2 consumption (V̇O2 max) in hypoxia, but not in normoxia, at least in non‐athletic subjects, was tested. RV was reduced by using He–O2 mixtures.

V̇O2, max was measured during graded cyclo‐ergometric exercise in eight men (aged 30 ± 3 years) who breathed N2–O2 and He–O2 mixtures in normoxia (inspired oxygen fraction (FI,O2= 0.21) and hypoxia (FI,O2= 0.11). O2 consumption, expired and alveolar ventilations (V̇E and V̇A, respectively), blood lactate and haemoglobin concentrations, heart rate and arterial oxygen saturation (Sa,O2) were determined at the steady state of each work load. Arterial O2 and CO2 partial pressures (Pa,O2) and Pa,CO2, respectively) were measured at rest and at the end of the highest work load.

Maximal V̇E and V̇A were significantly increased by He—O2 breathing in normoxia (+27 and +18%, respectively), without significant changes in Pa, O2, Sa, O2V̇O2, max In hypoxia, V̇E and V̇A increased (+31 and +24%, respectively), together with Pa,O2 (+17%), Sa, O2 (+6%) and V̇O2, max (+14%).

The results support the hypothesis that the role of RV in limiting V̇O2,max is negligible in normoxia. In hypoxia, the finding that higher V̇E and V̇A values during He–O2 breathing led to higher V̇O2,max values suggests a greater role of RV as a limiting factor. It is unclear whether the finding that the V̇O2 max values were the same during He–O2 and N2–O2 breathing in normoxia is due to a non‐linear response of the O2 transfer system, as previously proposed.
Perception and gut reflexes induced by stimulation of gastrointestinal thermoreceptors in humans
Authors: Villanova Nicola , Azpiroz Fernando , Malagelada Juan‐R.
Published: September 2004

Experimental studies in animals suggest the existence of thermoreceptors in the gastrointestinal tract. Our aim was to investigate the distribution and specificity of upper gut thermoreceptors in humans.

In healthy subjects, thermal stimulation of the stomach (n= 8) and the small intestine (n= 6) was produced by means of a thermostat, which recirculates water at adjusted temperatures through an ultrathin intraluminal bag. Progressively warm (42, 47 and 52 °C) and cold (32, 22 and 12 °C) stimuli of 3 min duration were alternately applied at 13 min intervals. Perception was scored on a scale of 0–6 and gastric tone responses were measured with a barostat.

Thermal stimuli induced specific responses: cold stimuli induced abdominal cold sensation and a reflex contraction of the stomach, whereas warm stimuli induced warm sensation and a reflex gastric relaxation.

Thermal stimuli induced similar stimulus‐related perception in the stomach and small intestine (temperatures between 12 and 49.5 ± 0.5 °C were tolerated).

The reflex responses were site specific. Warm and cold stimulation of the stomach induced gastric reflexes (76 ± 26 ml isobaric expansion at 47 °C, and 68 ± 10 ml contraction at 12°C; P > 0.05 for both). However, only warm, not cold, stimulation of the intestine induced enterogastric reflexes.

These results indicate that in humans, warm and cold receptors are distributed along the gastrointestinal tract and project afferent input both into perception and reflex circuits with specific topographic organization.
Mechanism of action of endothelin in rat cardiac muscle: cross‐bridge kinetics and myosin light chain phosphorylation
Authors: Rossmanith Gunther H. , Hoh Joseph F. Y. , Turnbull Lynne , Ludowyke Russell I.
Published: September 2004

The molecular mechanism of inotropic action of endothelin was investigated in rat ventricular muscle by studying its effects on characteristics of isometric twitch, barium‐induced steady contracture and the level of incorporation of 32Pi into myosin light chain 2.

Exposure of rat papillary muscle to endothelin caused an increase in isometric twitch force but did not alter the twitch‐time parameters.

Endothelin did not significantly change the maximum contracture tension but did cause an increase in contracture tension at submaximal levels of activation, without changes in the tension‐to‐stiffness ratio and kinetics of attached cross‐bridges. Kinetics of attached cross‐bridges were deduced during steady contracture from complex‐stiffness values, and in particular from the frequency at which muscle stiffness assumes a minimum value, fmin. Endothelin did not alter fmin.

Endothelin caused an increase in the level of incorporation of 32Pi into myosin light chain 2 without a concurrent change in the level of incorporation of 32Pi into troponin I.

We conclude that the inotropic action of endothelin is not due to an increase in the kinetics of attached cross‐bridges, nor due to a change in the force per unit cross‐bridge, but may result from an increased divalent cation sensitivity caused by elevated myosin light chain 2 phosphorylation, resembling post‐tetanic potentiation in fast skeletal muscle fibres.
Discharge of human muscle spindle afferents innervating ankle dorsiflexors during target isometric contractions
Authors: Wilson Linda R , Gandevia Simon C , Burke David
Published: September 2004

There are discrepancies in the literature about the reproducibility of forces at which human muscle spindle afferents accelerate their discharge during isometric voluntary contractions. The aim of this study was to determine for single muscle spindle afferents both the reproducibility of the ‘acceleration threshold’ and the factors contributing to variability of ‘acceleration threshold’.

Microneurographic recordings were made from muscle spindle afferents innervating tibialis anterior while subjects performed isometric ankle dorsiflexions. Subjects matched the force of their contractions with a visually displayed ‘ramp‐and‐hol? template. Template parameters were determined by the force of maximal isometric ankle dorsiflexion (MVC), and expressed as per cent MVC. The required ‘ramp’ rate and ‘hold’ force was adjusted between trials (range, 0.5‐5% MVC s− and 0.5‐20% MVC, respectively). The duration of the hold phase was 4 s and, following each contraction, stretch was applied transversely to the tendon to minimize the influence of any ‘after‐effects’ on spindle afferent responses in subsequent contractions.

For each contraction, the force at which the rate of muscle spindle discharge increased was defined as the ‘acceleration threshold’. Of twenty‐six muscle spindle afferents innervating tibialis anterior, all but two increased their discharge in the test contractions. In 90% of contractions, acceleration thresholds were less than 3.2% MVC (range, 0.01–11.9% MVC).

Individual muscle spindle afferents increased their discharge at similar but not identical forces in repeated contractions. There was a positive correlation between the rate of contraction and the acceleration threshold (P < 0.001), but the strength of the target contraction had no effect on the threshold, and there was no trend for thresholds to change over time.

The results suggest, first, that most muscle spindle endings in the human pretibial muscles receive a significant increase in fusimotor drive during relatively weak isometric efforts and secondly, that when fusimotor after‐effects are controlled, much of the residual variability in ‘acceleration threshold’ for any one spindle in repeated contractions is due to extrafusal factors, particularly variability in contraction rate.
Lymph Flow Pattern in the Intact Thoracic Duct in Sheep
Authors: Onizuka Masataka , FlatebØ Torun , Nicolaysen Gunnar
Published: September 2004

To study the lymph flow dynamics in the intact thoracic duct, we applied an ultrasound transit‐time flow probe in seven anaesthetized and four unanaesthetized adult sheep (.60 kg). In unanaesthetized non‐fasting animals we found that lymph flow in the thoracic duct was always regular pulsatile (pulsation frequency, 5.2 ± 0.8 min−1) with no relation to heart or respiratory activity. At baseline the peak level of the thoracic duct pulse flow was 11.6–20.7 ml min−1 with a nadir of 0–3.6 ml min−1. Mean lymph flow was 5.4 ± 3.1 ml min−1. The flow pattern of lymph in the thoracic duct was essentially the same in the anaesthetized animals.

In both the anaesthetized and unanaesthetized animals, the lymph flow response to a stepwise increase in the outflow venous pressure showed interindividual variation. Some were sensitive to any increase in outflow venous pressure, but others were resistant in that lymph flow did not decrease until outflow venous pressure was increased to higher levels. This resistance was also observed in the high lymph flow condition produced by fluid infusion in the anaesthetized animal and mechanical constriction of the caudal vena cava in the unanaesthetized animals. Pulsation frequency of the thoracic duct flow initially increased and then decreased with a stepwise increase in the outflow venous pressure. This initial increase might be a compensatory response to maintain lymph flow against elevated outflow venous pressure.

To test the effect of long‐term outflow venous pressure elevation in unanaesthetized sheep, outflow venous pressure was increased by inflation of a cuff around the cranial vena cava for 1, 5 or 25 h. The cuff was inflated to a level where lymph flow was reduced. Lymph flow remained low or decreased further during the entire cuff‐inflation period. We calculated the lymph debt caused by the outflow venous pressure elevation and the amount ‘repaid’ when venous pressure returned to normal. Lymph debt for 25 h was 6400 ml but only 200 ml was repaid. Since we observed no visible oedema formation in the lower body of the sheep, the non‐colloidal components of the lymph must have been reabsorbed into the bloodstream, most likely in the lymph nodes.
Lymph flow dynamics in exercising human skeletal muscle as detected by scintography
Authors: Havas Eino , Parviainen Tapani , Vuorela Juha , Toivanen Jarmo , Nikula Tuomo , Vihko Veikko
Published: September 2004

The effects of dynamic and isometric muscle contractions on the lymph flow dynamics in human skeletal muscle were studied with a scintographic method.

Radioactively labelled human serum albumin (99mTc‐HSA) was injected bilaterally into the vastus lateralis muscles of eight men (n= 16), four of whom had had an endurance training background. The subjects performed 100 submaximal contractions in 10 min as (i) dynamic knee extensions (CONS), (ii) isometric contractions with the knees at full extension (IMExt), or (iii) isometric contractions with knees fixed at 90 deg angle flexion (IMFlex). The exercises were separated by 65 min periods in supine rest. The level of radioactivity at the injection site was monitored by a gamma‐camera, and the clearance rate of radioactivity (CR) was calculated as the fractional decrease during the periods of interest (CR unit =% min−).

The clearance rate was low during the rest periods (0.04 ± 0.05% min−1), though higher in the trained than in the sedentary subjects (0.06 ± 0.05 vs. 0.03 ± 0.03% min−1; P= 0.008). Exercise increased the clearance rate three‐ to sixfold, to 0.16 ± 0.16% min−1 during CONS, 0.20 ± 0.15% min− during IMExt and 0.09 ± 0.11 % min−1 during IMFlex. There were no differences between the subject subgroups.

The higher clearance rate during IMExt than during IMFlex (P= 0.02) demonstrates the importance of muscle deformations on lymph propulsion and experimentally confirms the current concepts of lymph formation and propulsion in voluntarily active skeletal muscle. It is suggested that lymph propulsion by working muscle is most efficient when the muscle is able to shorten close to its minimum length.
Extracellular divalent cations block a cation non‐selective conductance unrelated to calcium channels in rat cardiac muscle
Authors: Mubagwa Kanigula , Stengl Milan , Flameng Willem
Published: September 2004

The effect of removing extracellular divalent cations on resting potential (Vrest) and background conductance of rat cardiac muscle was studied. Vrest was measured with 3 M KCl‐filled microelectrodes in papillary muscles, or with a patch electrode in ventricular myocytes. Whole‐cell membrane currents were measured in myocytes using step or ramp voltage commands.

In both muscles and single cells, decrease or removal of Ca2+o and Mg2+o caused a nifedipine‐resistant depolarization, which was reversed upon readmission of Ca2+o or Mg2+o (half‐maximal effect at 0.8 mM Ca2+o or 3 mM Mg2+o in muscles).

In single myocytes, removal of Ca2+o and Mg2+o had no effect on the seal resistance in non‐ruptured cell‐attached recordings, but reversibly induced a current with a reversal potential (Vrev) of −8 ± 3.4 mV (with internal Cs+; mean. s.e.m., n= 23) during whole‐cell recordings. The current was insensitive to nifedipine (3–100 μM) or amiloride (1 mM). Vrev was insensitive to changes in the equilibrium potential for chloride ions (Ecl.

The current induced in the absence of extracellular divalent cations was blocked in a concentration‐dependent manner by Ca2+o. (At −80 mV, the affinity constant Kca was 60 μM with a Hill coefficient of 0.9.) KCa was voltage dependent at positive but not negative potentials. Mg2+o, Ni2+o, Sr2+o, Ba2+o, Cd2+o and Gd3+o also blocked the current.

In 0 mM Na+ (145 mM NMDG+), the inward component of the divalent cation‐sensitive current was decreased and Vrev shifted to more negative potentials.

These results suggest that a novel conductance pathway, permeable to monovalent cations but not to OF and blocked by divalent cations, exists in ventricular myocytes.
Effects of Extracellular pH on Receptor‐Mediated Ca2+ Influx in A7r5 Rat Smooth Muscle Cells: Involvement of Two Different Types of Channel
Authors: Iwasawa Kuniaki , Nakajima Toshiaki , Hazama Hisanori , Goto Atsuro , Shin Wee Soo , Toyo‐oka Teruhiko , Omata Masao
Published: September 2004

The effects of extracellular pH (pHo) on receptor (vasopressin or endothelin‐l)‐mediated Ca2+ entry and Ca2+‐permeable channels were investigated in aortic smooth muscle cells (A7r5) from rat embryonic thoracic aorta. Intracellular Ca2+ ([Ca2+]i) was measured using fura‐2 AM and whole‐cell voltage clamp techniques were employed.

Vasopressin and endothelin‐1 (100 nM) in the presence of nicardipine (10 μM) evoked a sustained rise in [Ca2+]i due to calcium entry. Extracellular acidosis decreased receptor (vasopressin or endothelin‐l)‐mediated Ca2+ entry, while extracellular alkalosis potentiated it.

Depletion of intracellular Ca2+ stores with thapsigargin (1 μM) also evoked Ca2+ entry activated by emptying of intracellular Ca2+ stores (capacitative Ca2+ entry). Extracellular acidosis decreased this capacitative Ca2+ entry, while extracellular alkalosis potentiated it.

Under voltage‐clamp conditions with Cs+ internal solution, vasopressin and endothelin‐1 activated non‐selective cation currents (Icat). Ba2+ or Ca2+ were also charge carriers of Icat. Reducing the pHo inhibited Icat, while increasing pHo potentiated it in a reversible manner.

Intracellular pH (pHi) changes did not cause the same marked effects as pHo changes, and a high concentration of Hepes (50 mM) in the patch pipette did not inhibit the effects of pHo on Icat.

Similar results were obtained when Icat was activated by GTPγS (1 mM) applied through the patch pipette, even in the absence of agonists, probably because of direct activation of GTP‐binding proteins coupled to the receptors.

In cells treated with thapsigargin, addition of Ca2+ to the bath solution induced Ca2+‐dependent K+ currents activated by capacitative Ca2+ entry. However, no measurable ionic currents activated by capacitative Ca2+ entry (ICRAC) were observed under conditions with Cs+ internal solution and EGTA (5 mM), although vasopressin still activated Icat.

These results suggest that the contractile agonists vasopressin and endothelin‐1 evoke Ca2+ entry through two different types of Ca2+‐permeable channel (Icat and ICRAC) and pHo affects these channels, which may modulate receptor‐mediated Ca2+ influx in A7r5 cells. Thus, pH‐induced changes of these channels may play a pathophysiological role in the control of receptor‐mediated contractions.
The effect of altitude hypoxia on glucose homeostasis in men
Authors: Larsen Jens Jørn , Hansen Jesper Melchior , Olsen Niels Vidiendal , Galbo Henrik , Dela Flemming
Published: September 2004

Exposure to altitude hypoxia elicits changes in glucose homeostasis with increases in glucose and insulin concentrations within the first few days at altitude. Both increased and unchanged hepatic glucose production (HGP) have previously been reported in response to acute altitude hypoxia. Insulin action on glucose uptake has never been investigated during altitude hypoxia.

In eight healthy, sea level resident men (27 ± 1 years (mean ± S.E.M.); weight, 72 ± 2 kg; height, 182 ± 2 cm) hyperinsulinaemic (50 mU min−1 m−2), euglycaemic clamps were carried out at sea level, and subsequently on days 2 and 7 after a rapid passive ascent to an altitude of 4559 m.

Acute mountain sickness scores increased in the first days of altitude exposure, with a peak on day 2. Basal HGP did not change with the transition from sea level (2.2 ± 0.2 mg min− kg−1) to altitude (2.0 ± 0.1 and 2.1 ± 0.2 mg min−1 kg−1, days 2 and 7, respectively). Insulin‐stimulated glucose uptake rate was halved on day two compared with sea level (4.5 ± 0.6 and 9.8 ± 1.1 mg min−1 kg−1, respectively; P < 0.05), and was partly restored on day 7 (7.4 ± 1.4 mg min−1 kg−1; P < 0.05vs. day two and sea level). Concentrations of glucagon and growth hormone remained unchanged, whereas glucose, C‐peptide and cortisol increased on day 2. Noradrenaline concentrations increased during the stay at altitude, while adrenaline concentrations remained unchanged. In response to insulin infusion, catecholamines increased on day 2 (noradrenaline and adrenaline) and day 7 (adrenaline), but not at sea level.

In conclusion, insulin action decreases markedly in response to two days of altitude hypoxia, but improves with more prolonged exposure. HGP is always unchanged. The changes in insulin action may in part be explained by the changes in counter‐regulatory hormones.
Voltage‐Dependent Interaction Between the Muscarinic ACh Receptor and Proteins of the Exocytic Machinery
Authors: Linial Michal , Ilouz Nili , Parnas Hanna
Published: September 2004

Release of neurotransmitter into the synaptic cleft is the last step in the chain of molecular events following the arrival of an action potential at the nerve terminal. The neurotransmitter exerts negative feedback on its own release. This inhibition would be most effective if exerted on the first step in this chain of events, i.e. a step that is mediated by membrane depolarization. Indeed, in numerous studies feedback inhibition was found to be voltage dependent.

The purpose of this study is to investigate whether the mechanism underlying feedback inhibition of transmitter release resides in interaction between the presynaptic autoreceptors and the exocytic apparatus, specifically the soluble NSF‐attachment protein receptor (SNARE) complex.

Using rat synaptosomes we show that the muscarinic ACh autoreceptor (mAChR) is an integral component of the exocytic machinery. It interacts with syntaxin, synaptosomal‐associated protein of 25 kDa (SNAP‐25), vesicle‐associated membrane protein (VAMP) and synaptotagmin as shown using both cross‐linking and immunoprecipitation.

The interaction between mAChRs and both syntaxin and SNAP‐25 is modulated by depolarization levels; binding is maximal at resting potential and disassembly occurs at higher depolarization.

This voltage‐dependent interaction of mAChRs with the secretory core complex appears suitable for controlling the rapid, synchronous neurotransmitter release at nerve terminals.
Mental rehearsal of motor tasks recruits α‐motoneurones but fails to recruit human fusimotor neurones selectively
Authors: Gandevia Simon C. , Wilson Linda R. , Inglis J. Timothy , Burke David
Published: September 2004

As mental rehearsal of movements activates multiple cortical areas associated with movement, we assessed whether this increases fusimotor drive and whether enhanced muscle spindle activity could contribute to the improvement in skill that accompanies mental rehearsal.

Microneurographic recordings were made from six muscle spindle afferents innervating extensor muscles in the forearm or tibialis anterior, which were selected because their discharge increased during very weak contractions. Activity was monitored while subjects imagined performing a range of activities including simple and complex movements involving the relevant muscles.

No activation of muscle spindle afferents occurred during imagined motor tasks without EMG. When the relevant muscles contracted during mental rehearsal, spindle discharge increased, much as in weak contractions.

Mental rehearsal increased background EMG in the involved muscles and also increased H reflex amplitude independently of EMG changes.

Although there was no evidence for selective fusimotor activation during imagined movement, skeletomotor activity and reflex excitability increased. Similar changes occur with preparation for movement following a cue. It is likely that mental rehearsal usually involves unintentional performance of the planned motor task.
Chloride channel blockers inhibit myogenic tone in rat cerebral arteries
Authors: Nelson Mark T. , Conway Mathew A. , Knot Harm J. , Brayden Joseph E.
Published: September 2004

We have investigated the role of chloride channels in pressure‐induced depolarization and contraction of cerebral artery smooth muscle cells.

Two chloride channel blockers, indanyloxyacetic acid (IAA‐94) and 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulphonic acid (DIDS), caused hyperpolarizations (10–15 mV) and dilatations (up to 90 %) of pressurized (80 mmHg), rat posterior cerebral arteries. Niflumic acid, a blocker of calcium‐activated chloride channels, did not affect arterial tone.

Dilatations to IAA‐94 and DIDS were unaffected by potassium channel blockers, but were prevented by elevated potassium. IAA‐94 and DIDS had no effect on membrane potential or diameter of arteries at low intravascular pressure, where myogenic tone is absent. Reduction of extracellular chloride (60 mm Cl−) increased the pressure‐induced contractions. Removal of extracellular sodium did not affect the pressure‐induced responses.

Our results suggest that intravascular pressure activates DIDS‐ and IAA‐94‐sensitive chloride channels to depolarize arterial smooth muscle, thereby contributing to the myogenic constriction.
Sodium Channel Distribution Within the Rabbit Atrioventricular Node as Analysed by Confocal Microscopy
Authors: Petrecca Kevin , Amellal Farid , Laird Dale W. , Cohen Sidney A. , Shrier Alvin
Published: September 2004

Paired 20 μm thick sections of fresh frozen tissue taken from the frontal plane of the rabbit atrioventricular (AV) nodal region were processed for histology and immunohistochemistry. Confocal microscopy was used to image the distribution of sodium channels using IgG (R12) developed against a highly conserved sequence in the interdomain 3–4 region of cloned sodium channels.

In ventricular and atrial cells, sodium channel immunofluorescence was localized to lateral membranes and T‐tubules. In the open AV node, levels of sodium channel immunofluorescence in the transitional cell zone and in the lower nodal cell tract were comparable to that found in the atrial and ventricular myocardium.

In the enclosed AV node a gradation of sodium channel immunofluorescence is present such that peripherally located circumferential transitional cells display high levels of immunofluorescence, comparable to that of atrial and ventricular myocardium, while centrally located midnodal cells display decreased levels of or no immunofluorescence.

In order to correlate the distribution of sodium channels with the distribution of gap junctions, we used IgG directed against the carboxyl terminus of connexin43 (CT‐360). Ventricular cell immunofluorescence was localized primarily to the intercalated disk region, while in the AV node, the pattern of distribution was found to be similar to that of sodium channels.

The reduced levels of and/or absence of immunofluorescence in the midnodal cell region indicates a paucity of sodium channel and connexin43 protein expression in this region of the AV node that would favour slow impulse conduction.
Corticotropin releasing hormone inhibits an inwardly rectifying potassium current in rat corticotropes
Authors: Kuryshev Yuri A. , Haak Lee , Childs Gwen V. , Ritchie Aileen K.
Published: September 2004

The perforated‐patch‐clamp technique was used to identify an inwardly rectifying K+ current (IK(IR)) in cultured rat anterior pituitary cells highly enriched in corticotropes. IK(IR) was rapidly activating and highly selective for K+. The K+ conductance was approximately proportional to the square root of the extracellular K+ concentration.

I
K(IR) was blocked in a voltage‐dependent manner by external Ba2+ and Cs+, slightly attenuated by 5 mM 4‐aminopyridine (15% inhibition) and insensitive to 10 mM tetra‐ethylammonium, 2 mM Ca2+, 1 mM Cd2+ and 50 μM La3+.

In physiological saline, 100 μM Ba2+, which inhibits 86% of IK(IR) at the cell resting potential, depolarized cells by 6.1 ± 0.7 mV from a mean resting potential of −59.6 ± 0.8 mV.

Corticotropin releasing hormone (CRH), which activates adenylyl cyclase and stimulates adrenocorticotropic hormone (ACTH) secretion from corticotropes, inhibited IK(IR) by 25% and depolarized the cells by 10.2 ± 1.0 mV. Dibutyryl cAMP ((Bu)2cAMP) mimicked these effects.

The membrane depolarization evoked by Ba2+ or CRH increased the cell firing frequency. Comparison of cells exhibiting a membrane potential of approximately −50 mV revealed that spike frequency in the presence of CRH (109 ± 7 spikes (5 min)−1) was greater than in control (60 ± 5 spikes (5 min)−1) or Ba2+‐treated (77 ± 15 spikes (5 min)−1) corticotropes.

The data suggest that IK(IR) contributes to maintenance of the resting membrane potential of rat corticotropes. Inhibition of IK(IR) plays a role in, but does not account for all of, the membrane depolarization and enhancement of firing frequency evoked by CRH.
The Prostacyclin Analogue Carbacyclin Inhibits Ca2+‐Activated K+ Current in Aortic Baroreceptor Neurones of Rats
Authors: Li Zhi , Lee Hon‐Chi , Bielefeldt Klaus , Chapleau Mark W. , Abboud Francois M.
Published: September 2004

Previous studies indicate that prostacyclin (PGI2) increases the activity of baroreceptor afferent fibres. The purpose of this study was to test the hypothesis that PGI2 inhibits Ca2+‐activated K+ current (IK(Ca)) in isolated baroreceptor neurones in culture.

Rat aortic baroreceptor neurones in the nodose ganglia were labelled in vivo by applying a fluorescent dye (DiI) to the aortic arch 1–2 weeks before dissociation of the neurones. Outward K+ currents in baroreceptor neurones evoked by depolarizing voltage steps from a holding potential of −40 mV were recorded using the whole‐cell patch‐clamp technique.

Exposure of baroreceptor neurones to the stable PGI2 analogue carbacyclin significantly inhibited the steady‐state K+ current in a dose‐dependent and reversible manner. The inhibition of K+ current was not caused indirectly by changes in cytosolic Ca2+ concentration. The Ca2+‐activated K+ channel blocker charybdotoxin (ChTX, 10−7m) also inhibited the K+ current. In the presence of ChTX or in the absence of Ca2+, carbacyclin failed to inhibit the residual K+ current. Furthermore, in the presence of high concentrations of carbacyclin, ChTX did not cause further reduction of K+ current.

Carbacyclin‐induced inhibition of IK(Ca) was mimicked by 8‐bromo‐cAMP and by activation of G‐protein with GTPγS. The inhibitory effect of carbacyclin on IK(Ca) was abolished by GDPβS, which blocks G‐protein activation, and by a selective inhibitor of cAMP‐dependent protein kinase, PKI5–24.

The results demonstrate that carbacyclin inhibits ChTX‐sensitive IK(Ca) in isolated aortic baroreceptor neurones by a G‐protein‐coupled activation of cAMP‐dependent protein kinase. This mechanism may contribute to the PGI2‐induced increase in baroreceptor activity demonstrated previously.
‘Quantal’ calcium release operated by membrane voltage in frog skeletal muscle
Authors: Pizarro Gonzalo , Shirokova Natalia , Tsugorka Alexander , Ríos Eduardo
Published: September 2004

Ca2+ transients and Ca2+ release flux were determined optically in cut skeletal muscle fibres under voltage clamp. ‘Decay’ of release during a depolarizing pulse was defined as the difference between the peak value of release and the much lower steady level reached after about 100 ms of depolarization. Using a double‐pulse protocol, the inactivating effect of release was measured by ‘suppression’, the difference between the peak values of release in the test pulse, in the absence and presence of a conditioning pulse that closely preceded the test pulse.

The relationship between decay and suppression was found to follow two simple arithmetic rules. Whenever the conditioning depolarization was less than or equal to the test depolarization, decay in the conditioning release was approximately equal to suppression of the test release. Whenever the conditioning depolarization was greater than that of the test, suppression was complete, i.e. test release was reduced to a function that increased monotonically to a steady level. The steady level was the same with or without conditioning.

These arithmetic rules suggest that inactivation of Ca2+ release channels is strictly and fatally linked to their activation. More than a strict linkage, however, is required to explain the arithmetic properties.

The arithmetic rules of inactivation result in three other properties that are inexplicable with classical models of channel gating: constant suppression, incremental inactivation and increment detection. These properties were first demonstrated for inositol trisphosphate (IP3)‐sensitive channels and used to define IP3‐induced release as quantal. In this sense, it can now be stated that skeletal muscle Ca2+ release is activated by membrane voltage in a quantal manner.

For both classes of intracellular Ca2+ channels, one explanation of the observations is the existence of subsets of channels with different sensitivities (to voltage or agonist dose). In an alternative explanation, channels are identical, but have a complex repertoire of voltage‐ or dose‐dependent responses.
Selective inhibition of a slow‐inactivating voltage‐dependent K+ channel in rat PC 12 cells by hypoxia
Authors: Conforti Laura , Millhorn David E.
Published: September 2004

Electrophysiological (single‐channel patch clamp) and molecular biological experiments (reverse transcriptase‐polymerase chain reaction) were performed to attempt to identify the O2‐sensitive K+ channel in rat phaeochromocytoma (PC12) cells.

Four types of K+ channels were recorded in PC12 cells: a small‐conductance K+ channel (14 pS), a calcium‐activated K+ channel (KCa; 102 pS) and two K+ channels with similar conductance (20 pS). These last two channels differed in their time‐dependent inactivation: one was a slow‐inactivating channel, while the other belonged to the family of fast transient K+ channels.

The slow‐inactivating 20 pS K+ channel was inhibited by hypoxia. Exposure to hypoxia produced a 50% reduction in channel activity (number of active channels in the patch × open probability). Hypoxia had no effect on the 20 pS transient K+ channels, whereas reduced O2 stimulated the KCa channels.

The genes encoding the α‐subunits of slow‐inactivating K+ channels for two members of the Shaker subfamily of K+ channels (Kvl.2 and Kvl.3) together with the Kv2.1, Kv3.1 and Kv3.2 channel genes were identified in PC12 cells.

The expression of the Shaker Kv1.2, but none of the other K+ channel genes, increased in cells exposed to prolonged hypoxia (18 h). The same cells were more resuponsive to a subsequent exposure to hypoxia (35% inhibition of K+ current measured in whole‐cell voltage clamp) compared with the cells maintained in normoxia (19% inhibition).

These results indicate that the O2‐sensitive K+ channel in PC12 cells is a 20 pS slow‐inactivating K+ channel that is upregulated by hypoxia. This channel appears to belong to the Shaker subfamily of voltage‐gated K+ channels.
The ion channel properties of a rat recombinant neuronal nicotinic receptor are dependent on the host cell type
Authors: Lewis Trevor M. , Harkness Patricia C. , Sivilotti Lucia G. , Colquhoun David , Millar Neil S.
Published: September 2004

A stable mammalian cell line (L‐α3β4) has been established which expresses the cloned rat neuronal nicotinic acetylcholine receptor (nAChR) subunits α3 and β4, which are the most abundant in autonomic ganglia. Ion channel properties of nAChRs expressed in L‐α3β4 cells were investigated by single‐channel and whole‐cell recording techniques, and compared with both rat α3β4 nAChRs expressed in Xenopus oocytes, and endogenous nicotinic receptors in rat superior cervical ganglion (SCO) neurones, using identical solutions for all cell types.

Acetylcholine (ACh) caused activation of single ion channel currents with a range of amplitudes. Some channels had high conductances (30–40 pS), and relatively brief lifetimes; these resembled the predominant native channel from SCG. Other channels had low conductances (20–26 pS) and long bursts of openings which were quite unlike native channels, but which were similar to channels formed by α3β4 in oocytes. Both types often occurred in the same patch.

Cytisine was about 3 times more potent than ACh (low‐concentration potency ratio) in L‐α3β4 cells, which is not dissimilar to the 5‐fold potency ratio found in both SCG and oocytes, whereas 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) was less potent than ACh in some cells (as in the oocyte), but more potent in others (as in SCG).

While the channels expressed in L‐α3β4 cells do not mimic exactly those expressed in rat SCG, they differ considerably from the same subunit combination expressed in oocytes. Larger conductance, SCG‐like channels were detected frequently in L‐α3β4, but were rarely, if ever, seen in oocytes injected with α3 and β4 mRNA. Our results indicate that ion channel properties such as single‐channel conductance can be influenced by the choice of heterologous expression system.
The Role of Na+−Ca2+ Exchange Current in Electrical Restitution in Ferret Ventricular Cells
Authors: Janvier N. C. , McMorn S. O. , Harrison S. M. , Taggart P. , Boyett M. R.
Published: September 2004

The mechanisms underlying electrical restitution (recovery of action potential duration after a preceding beat) were investigated in ferret ventricular cells. The time to 80% recovery (t80) of action potential duration was ∼204 ms.

At a holding potential of −80 mV, the Ca2+ current (ICa) reactivated and the delayed rectifier K+ current (IK) deactivated very rapidly (t80: ∼32 and ∼93 ms, respectively). The kinetics of both currents are too fast to account for electrical restitution alone.

The putative inward Na+−Ca2+ exchange current (INa‐Ca) produced by the Na+−Ca2+ exchanger in response to the intracellular Ca2+ transient reprimed (t80: 189 ms) with the same time course as mechanical restitution (recovery of contraction) and with a similar time course to electrical restitution.

Substantial reduction of inward INa‐Ca, by buffering intracellular Ca2+ with the acetyl methyl ester form of BAPTA, shortened the action potential and greatly altered the electrical restitution curve. Subsequent addition of nifedipine (to block ICa) or 4–aminopyridine (4–AP) (to block the transient outward current, ITO) further altered the electrical restitution curve.

Any time‐dependent current that contributes to the action potential is likely to affect the time course of electrical restitution. Although ICa:, IK and ITO were previously thought to be the only currents involved in electrical restitution, we conclude that inward INa‐Ca also plays an important role.
Extracellular Nucleotides Activate non‐Selective Cation and Ca2+‐Dependent K+ Channels in Rat Osteoclasts
Authors: Weidema A. Frederik , Barbera Joseph , Dixon S. Jeffrey , Sims Stephen M.
Published: September 2004

Extracellular ATP elevates cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, but its effects on ion channels have not been reported previously. Membrane currents and [Ca2+]i were recorded in isolated rat osteoclasts using patch clamp and fluorescence techniques.

At negative membrane potentials, ATP (1–100 μm) activated an inward current that peaked rapidly and then declined. A later current was outward at potentials positive to the equilibrium potential for K+ (EK) and showed oscillations.

The initial inward current, studied in isolation using Cs+ in the electrode solution, showed rapid activation, inward rectification and reversal at +3 ± 4 mV. Reduction of [Na+]o to 10 mM shifted the reversal potential to –21 ± 3 mV, indicating that ATP activates a non‐selective cation current, consistent with involvement of P2X receptors.

The later current activated by ATP, studied with K+ in the electrode solution, exhibited a linear I–V relationship, and reversed at –71 ± 4 mV. The reversal potential shifted 51 mV per 10‐fold change of [K+]o, indicating that ATP activates a K+ current (IK).

In fura‐2‐loaded cells, ATP caused elevation of [Ca2+]i that persisted in Ca2+‐free solution, indicating that ATP induced release of Ca2+ from intracellular stores, consistent with involvement of P2Y receptors. Simultaneous patch clamp and fluorescence recordings revealed that IK was associated with the elevation of [Ca2+]i. Using a Ca2+ ionophore (4Br‐A23l87) to elevate [Ca2+]iIK activated when [Ca2+]i exceeded ∼400 nm, with half‐maximal activation at 580 ± 50 nM.

In cell‐attached patches, ATP activated a channel with a conductance of 48 ± 6 pS, that reversed direction near EK Channel open probability increased with elevation of [Ca2+]i, indicating the Ca2+ dependence of this channel.

These results demonstrate that rat osteoclasts express two types of purinoceptors. P2X receptors give rise to non‐selective cation current. P2Y receptors mediate Ca2+ release from stores, causing activation of a Ca2+‐dependent K+ channel.
Functional and Morphological Features of Skeletal Muscle from Mutant Mice Lacking Both Type 1 and Type 3 Ryanodine Receptors
Authors: Ikemoto Takaaki , Komazaki Shinji , Takeshima Hiroshi , Nishi Miyuki , Noda Tetsuo , Iino Masamitsu , Endo Makoto
Published: September 2004

We generated mice with targeted disruptions in the genes for both ryanodine receptor type 1 (RyR‐1) and type 3 (RyR‐3) to study the functional roles of RyR subtypes in skeletal muscle.

In permeabilized myocytes lacking both the RyRs, the Ca2+‐induced Ca2+ release (CICR) mechanism was completely lost, and caffeine failed to induce Ca2+ release.

Replacement of potassium methanesulphonate in an experimental intracellular solution with choline chloride resulted in Ca2+ release in the wild‐type muscle but not in the mutant muscle lacking RyR‐1.

The double‐mutant mice exhibited more severe muscular degeneration than RyR‐1‐deficient mice with formation of large vacuoles and swollen mitochondria while structural coupling between T‐tubules and the sarcoplasmic reticulum was retained.

These results demonstrate that CICR is mediated solely by RyR‐1 and RyR‐3 in skeletal muscle cells, and suggest that RyR‐1 is involved in Cl−‐induced Ca2+ release. The results also suggest the presence of molecular components other than RyRs responsible for the triad formation. RyR‐3 may have a role in the normal morphogenesis of skeletal muscle cells, although functionally it can be replaced by RyR‐1.
Voltage‐dependent binding and calcium channel current inhibition by an anti‐α1D subunit antibody in rat dorsal root ganglion neurones and guinea‐pig myocytes
Authors: Wyatt C. N. , Campbell V. , Brodbeck J. , Brice N. L. , Page K. M. , Berrow N. S. , Brickley K. , Terracciano C. M. N. , Naqvi R. U. , MacLeod K. T. , Dolphin A. C.
Published: September 2004

The presence of calcium channel α1D subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea‐pig cardiac myocytes was demonstrated using the reverse transcriptase‐polymerase chain reaction.

An antipeptide antibody targeted at a region of the voltage‐dependent calcium channel α1Dsubunit C‐terminal to the pore‐forming SS1–SS2 loop in domain IV (amino acids 1417–1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+.

Incubation of cells under depolarizing conditions for 2–4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.05) for cardiac myocytes when compared with controls.

S‐(–)‐Bay K 8644 (1 μM) enhanced calcium channel currents in DRGs by 75 ± 19% (n= 5) in neurones incubated under depolarizing conditions with antibody that had been pre‐adsorbed with its immunizing peptide (100 μg ml−1). This was significantly (P < 0.05) larger than the enhancement by S‐(–)‐Bay K 8644 that was seen with cells incubated under identical conditions but with antibody alone, which was 15 ± 4% (n= 5).

These results demonstrate the presence of calcium channel α1D subunits in rat DRG neurones and guinea‐pig cardiac myocytes. They also show that amino acids 1417–1434 of the α1D subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calcium channel current and reduces the ability of S‐(–)‐Bay K 8644 to enhance this current, indicating that it is an L‐type current that is attenuated.
A Novel Role for HERG K+ Channels: Spike‐Frequency Adaptation
Authors: Chiesa Nicoletta , Rosati Barbara , Arcangeli Annarosa , Olivotto Massimo , Wanke Enzo
Published: September 2004

The regular firing of a Hodgkin‐Huxley neurone endowed with fast Na+ and delayed K+ channels can be converted into adapting firing by appending HERG (human eag‐related gene) channels.

The computer model predictions were verified by studying the firing properties of F‐11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long‐term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike‐frequency adaptation of their firing when current clamped with long depolarizations.

In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike‐frequency adaptation, and the conversion of adapting firing to regular firing.

It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike‐frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents, IAHP, IC and IM. In addition to the known cardiac arrhythmia syndrome (LQT2), genetic mutations or an altered HERG expression could lead to continuous hyperexcitable states sustained by the inability of nerve or endocrine cells to accommodate to repetitive stimuli. This might help in clarifying the pathogenesis of still undefined idiopathic familial epilepsies.
A Charybdotoxin‐Insensitive Conductance in Human T Lymphocytes: T Cell Membrane Potential is Set by Distinct K+ Channels
Authors: Verheugen Jos A. H. , Korn Henri
Published: September 2004

Changes in the membrane potential (Vm) of human T lymphocytes upon K+ channel block were inferred from alterations in K+ current reversal potential in cell‐attached patches. It was found that a high concentration of charybdotoxin (100 nm, CTX), which blocks both voltage‐gated (K(V)) and Ca2+‐activated (K(Ca)) potassium channels in these cells, depolarizes Vm of lymphocytes only partially. Subsequent whole‐cell measurements of the same cells showed that 39 ± 25% of the voltage‐gated current remains in the presence of CTX.

The CTX‐resistant current reverses at potentials between −80 and −90 mV, indicating that it is K+ selective. The current is activated at more depolarized potentials compared with the unblocked −IK(V) current with a threshold between −40 and −20 mV and a half‐maximal activation at +50 mV. Inactivation during prolonged depolarization is slow. Steady‐state inactivation is half‐maximal at ‐45 mV and complete at potentials > − 20 mV. The CTX‐resistant IK(V) is completely blocked by nifedipine and is not sensitive to dendrotoxin.

The effect of nifedipine on the Vm of lymphocytes varies between cells depending on the contribution of the nifedipine‐sensitive current to whole‐cell IK(V) Combined application of CTX and nifedipine completely depolarizes Vm.

The extent to which T cell receptor‐evoked Ca2+ signals of resting cells are inhibited by K+ channel blockers correlates with the magnitude of the depolarization induced by the drugs. Complete suppression of the response is achieved only by combined block of the CTX‐sensitive and ‐insensitive IK(V). The enhanced Ca2+ response of activated cells, which express increased numbers of K(Ca) channels, is in addition subject to modulation by blockers which prevent the hyperpolarization during the Ca2+ rise mediated by these channels.