bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of an inward rectifier K+ current and of hyperpolarization in human myoblast fusion
Authors: Liu J.‐H. , Bijlenga P. , Fischer‐Lougheed J. , Occhiodoro T. , Kaelin A. , Bader C. R. , Bernheim L.
Published: September 2004

The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion‐competent myoblasts (FCMBs), and freshly formed myotubes.

Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non‐inactivating delayed rectifier, and IK(IR), an inward rectifier.

I
K(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28 % of the FCMBs and in all myotubes.

I
K(IR) is reversibly blocked by Ba2+ or Cs+.

Cells expressing IK(IR) have resting membrane potentials of −65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (‐32 mV).

Cs+ and Ba2+ ions reduce myoblast fusion.

It is hypothesized that the IK(IR)‐mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T‐type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion.
Out‐of‐equilibrium pH transients in the guinea‐pig ventricular myocyte
Authors: Leem Chae‐Hun , Vaughan‐Jones Richard D.
Published: September 2004

Following an intracellular alkali load (imposed by acetate prepulsing in CO2/HCO3− buffer), intracellular pH (pHi) of the guinea‐pig ventricular myocyte (recorded from intracellular SNARF fluorescence) recovers to control levels. Recovery has two phases. An initial rapid phase (lasting up to 2 min) is followed by a later slow phase (several minutes). Inhibition of sarcolemmal acid‐loading carriers (by removal of extracellular Cl−) inhibits the later, slow phase but the initial rapid recovery phase persists. It also persists in the absence of extracellular Na+ and in the presence of the HCO3− transport inhibitor DIDS (4,4‐di‐isothiocyanatostilbene‐2,2‐disulphonic acid).

The rapid recovery phase is not evident if the alkali load has been induced by reducing PCO2 (from 10 to 5 %), and it is inhibited in the absence of CO2/HCO3− buffer (i.e. Hepes buffer). It is also slowed by the carbonic anhydrase (CA) inhibitor acetazolamide (ATZ). We conclude that it is caused by buffering of the alkali load through the hydration of intracellular CO2 (CO2‐dependent buffering).

The time course of rapid recovery is consistent with an intracellular CO2 hydration rate constant (k1) of 0.36 s−1 in the presence of CA activity, and 0.14 s−1 in the absence of CA activity. This latter k1 value matches the literature value for uncatalysed CO2 hydration in free solution. Natural CO2 hydration is accelerated 2.6‐fold in the ventricular myocyte by endogenous CA.

The rapid recovery phase represents a period when the intracellular CO2/HCO3− buffer is out of equilibrium (OOE). Modelling of the recovery phase using our k1 value, indicates that OOE conditions will normally extend for at least 2 min following a step rise in pHi (at constant PCO2). If CA is inactive, this period can be as long as 5 min. During normal pHi regulation, the recovery rate during these periods cannot be used as a measure of sarcolemmal acid loading since it is a mixture of slow CO2‐dependent buffering and transmembrane acid loading. The implication of this finding for quantification of pHi regulation during alkalosis is discussed.
Origin of slow and fast oscillations of Ca2+ in mouse pancreatic islets
Authors: Liu Yi‐Jia , Tengholm Anders , Grapengiesser Eva , Hellman Bo , Gylfe Erik
Published: September 2004

Pancreatic islets exposed to 11 mM glucose exhibited complex variations of cytoplasmic Ca2+ concentration ([Ca2+]i) with slow (0.3‐0.9 min−1) or fast (2‐7 min−1) oscillations or with a mixed pattern.

Using digital imaging and confocal microscopy we demonstrated that the mixed pattern with slow and superimposed fast oscillations was due to separate cell populations with the respective responses.

In islets with mixed [Ca2+]i oscillations, exposure to the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (DTBHQ) resulted in a selective disappearance of the fast pattern and amplification of the slow pattern.

In addition, the protein kinase A inhibitor RP‐cyclic adenosine 3′,5′‐monophosphorothioate sodium salt transformed the mixed [Ca2+]i oscillations into slow oscillations with larger amplitude.

Islets exhibiting only slow oscillations reacted to low concentrations of glucagon with induction of the fast or the mixed pattern. In this case the fast oscillations were also counteracted by DTBHQ.

The spontaneously occurring fast oscillations seemed to require the presence of cAMP‐elevating glucagon, since they were more common in large islets and suppressed during culture.

Image analysis revealed [Ca2+]i spikes occurring irregularly in time and space within an islet. These spikes were preferentially observed together with fast [Ca2+]i oscillations, and they became more common after exposure to glucagon.

Both the slow and fast oscillations of [Ca2+]i in pancreatic islets rely on periodic entry of Ca2+. However, the fast oscillations also depend in some way on paracrine factors promoting mobilization of Ca2+ from intracellular stores. It is proposed that such a mobilization in different cells within a tightly coupled islet syncytium generates spikes which co‐ordinate the regular bursts of action potentials underlying the fast oscillations.
Glycine and GABAA receptor‐mediated synaptic transmission in rat substantia gelatinosa: inhibition by μ‐opioid and GABAB agonists
Authors: Grudt Timothy J. , Henderson Graeme
Published: September 2004

Bicuculline‐sensitive and strychnine‐sensitive inhibitory postsynaptic currents (IPSCs) could be evoked in neurones of the rat substantia gelatinosa of the spinal trigeminal nucleus pars caudalis.

Spontaneous tetrodotoxin (TTX)‐insensitive‐mediated miniature IPSCs (mIPSCs) blocked by strychnine or bicuculline were also present in many neurones. The decay of the glycine receptor‐mediated mIPSCs was fitted by a single exponential, whereas the decay of the GABAA receptor‐mediated mIPSCs could in some instances be fitted by a single exponential, but in other instances required two exponentials.

An increase in baseline current noise developed during the course of the recording. This noise was abolished by strychnine (1 μm) but was insensitive to bicuculline (10 μm), TTX (0.5 μm), [D‐Ala2, N‐Me‐Phe4, Gly5‐ol]‐enkephalin (DAMGO, 1 μm) or baclofen (30 μm). The single‐channel conductance underlying the noise was estimated to be 21 pS.

The μ‐opioid agonist DAMGO (1‐10 μm) reduced the amplitude of the evoked glycine receptor‐mediated IPSC and the evoked GABAA receptor‐mediated IPSC. The μ‐opioid antagonist D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2 (CTAP, 1 μm) reversed the DAMGO inhibition.

The GABAB agonist baclofen (30 μm) reduced the amplitude of the evoked glycine receptor‐ mediated IPSC and the GABAA receptor‐mediated IPSC. The inhibition was reversed by the selective GABAB antagonist 3‐N[1‐(S)‐(3,4‐dichlorophenyl) ethyl] amino‐2‐(S)‐hydroxypropyl‐P‐benzyl‐phosphinic acid (CGP 55845A, 1 μm).

Both DAMGO and baclofen reduced the frequency of glycine and GABAA receptor‐mediated mIPSCs without affecting average amplitude, and increased the percentage of failures of the evoked glycine and GABAA receptor‐mediated IPSCs, suggesting a presynaptic site of action.
Field sensitivity action spectra of cone photoreceptors in the turtle retina
Authors: Perlman Ido , Itzhaki Aviran , Asi Husam , Alpern Mathew
Published: September 2004

The Stiles two‐colour increment threshold technique was applied to turtle cone photoreceptors in order to derive their field sensitivity action spectra.

Photoresponses of cone photoreceptors were recorded intracellularly. Flash sensitivities were calculated from small amplitude (< 1 mV) responses. The desensitizing effects of backgrounds of different wavelengths were measured and the background irradiance needed to desensitize the cone by a factor of 10 (1 log unit) was defined as threshold. The reciprocals of these thresholds were used to construct the field sensitivity action spectrum.

The field sensitivity action spectra of long‐wavelength‐sensitive (L) and medium‐wavelength‐sensitive (M) cones depended upon the wavelength of the test flash used to measure them. This excludes the possibility that turtle cones can function as single‐colour mechanisms in the Stiles sense.

In fourteen L‐cones, the average wavelength of peak sensitivity of the field sensitivity action spectrum was 613.7 ± 7.7 nm for the 500 nm test and 635.6 ± 9.6 nm for the 700 nm test. For six M‐cones, these values were 558.5 ± 6.8 and 628.8 ± 10.6 nm for the 500 and 700 nm tests, respectively.

Two physiological mechanisms are suggested as contributing to the dependency of the field sensitivity action spectrum upon test wavelength. One is based upon the transmissivity properties of the coloured oil droplets, while the other hypothesizes excitatory interactions between cones of different spectral type.

Computer simulations of the field sensitivity action spectra indicate that both mechanisms are needed in order to account for the dependency of the field sensitivity action spectrum upon the wavelength of the test flash.
Integrin and cytoskeletal involvement in signalling cell volume changes to glutamine transport in rat skeletal muscle
Authors: Low Sylvia Y. , Taylor Peter M.
Published: September 2004

Muscle glutamine transport is modulated in response to changes in cell volume by a mechanism dependent on active phosphatidylinositol 3‐kinase. We investigated the possibility that this mechanism requires interactions between the extracellular matrix (ECM), integrins and the cytoskeleton as components of a mechanochemical transduction system.

Using skeletal muscle cells, we studied effects of (a) inactivating integrin‐substratum interactions by using integrin‐binding peptide GRGDTP with inactive peptide GRGESP as control, and (b) disrupting the cytoskeleton using colchicine or cytochalasin D, on glutamine transport after brief exposure to hypo‐osmotic, isosmotic or hyperosmotic medium (170, 300 and 430 mosmol kg−1, respectively).

Neither GRGDTP nor GRGESP significantly affected basal glutamine uptake (0.05 mm; 338 ± 58 pmol min−1 (mg protein)−1) but GRGDTP specifically prevented the increase (71 %) and decrease (39 %) in glutamine uptake in response to hypo‐ and hyperosmotic exposure, respectively.

Colchicine and cytochalasin D prevented the increase and decrease in glutamine uptake in response to changes in external osmolality. They also increased basal glutamine uptake by 59 ± 19 and 85 ± 16 %, respectively, in a wortmannin‐sensitive manner.

These results indicate involvement of ECM‐integrin‐mediated cell adhesion and the cytoskeleton in mechanochemical transduction of cell volume changes to chemical signals modulating glutamine transport in skeletal muscle. Phosphatidylinositol 3‐kinase may function to maintain the mechanotransducer in an active state.
Barium triggers rapid endocytosis in calf adrenal chromaffin cells
Authors: Nucifora Paolo G. P. , Fox Aaron P.
Published: September 2004

Changes in cell capacitance were monitored in whole‐cell patch‐clamp recordings from calf adrenal chromaffin cells using a software‐based phase‐tracking technique. Rapid endocytosis and exocytosis were observed in extracellular solutions containing either Ca2+ or Ba2+.

There was no significant difference in the magnitude or the time course of rapid endocytosis of cells stimulated in Ca2+ as compared to Ba2+. When cells were pretreated with caffeine and thapsigargin in order to deplete intracellular Ca2+ stores, rapid endocytosis in Ba2+ was not affected. This indicates that Ba2+ itself is capable of supporting rapid endocytosis.

The application of the calmodulin inhibitor calmidazolium via the intracellular pipette solution did not inhibit rapid endocytosis. Although our findings are inconsistent with an immediate requirement for calmodulin in rapid endocytosis, they do not rule out an involvement on a longer time scale.

While rapid endocytosis was not affected by the substitution of Ca2+ with Ba2+, the maximum rate of exocytosis was higher in cells stimulated in Ca2+ than in Ba2+. Since Ba2+ currents were much larger than Ca2+ currents during depolarizations to +10 mV (the test potential used in these experiments), Ba2+ appears to be less efficient at promoting exocytosis than Ca2+.
ATP is a potent stimulator of the activation and formation of rodent osteoclasts
Authors: Morrison Matthew S. , Turin Luca , King Brian F. , Burnstock Geoffrey , Arnett Timothy R.
Published: September 2004

There is increasing evidence that extracellular ATP acts directly on bone cells via P2 receptors. In normal rat osteoclasts, ATP activates both non‐selective cation channels and Ca2+‐dependent K+ channels. In this study we investigated the action of ATP on the formation of osteoclasts and on the ultimate function of these cells, namely resorption pit formation.

We found that ATP stimulated resorption pit formation up to 5.6‐fold when osteoclast‐containing bone cell populations from neonatal rats were cultured for 26 h on ivory discs, with a maximum effect occurring at relatively low concentrations (0.2‐2 μM). The stimulatory effect of ATP was amplified greatly when osteoclasts were activated by culture in acidified media (pH 6.9‐7.0). Pit formation by acid‐activated osteoclasts in the absence of ATP was inhibited by apyrase, an ecto‐ATPase and by suramin, an antagonist of P2 receptors.

Over the same concentration range at which rat osteoclast activation occurred (0.2‐2 μM), ATP also enhanced osteoclast formation in 10 day mouse marrow cultures, by up to 3.3‐fold, with corresponding increases in resorption pit formation. Higher concentrations of ATP (20–200 μM) reduced or blocked osteoclast formation. Adenosine, a P1 receptor agonist, was without effect on either osteoclast activation or formation.

These results suggest that low levels of extracellular ATP may play a fundamental role in modulating both the resorptive function and formation of mammalian osteoclasts.
Release of acetylcholine from embryonic myocytes in Xenopus cell cultures
Authors: Fu Wen‐Mei , Liou Houng‐Chi , Chen Yu‐Hwa , Wang Seu‐Mei
Published: September 2004

Acetylcholine (ACh) is important as the transmitter responsible for neuromuscular transmission. Here we report the non‐quantal release of ACh from embryonic myocytes.

Co‐cultures of spinal neurons and myotomal muscle cells were prepared from 1‐day‐old Xenopus embryos. Single channel currents were recorded in the non‐innervated myocytes. When the patch pipette was filled with Ringer solution alone, spontaneous single channel currents occurred, which were inhibited by d‐tubocurarine (d‐Tc).

The channel conductance appearing in Ringer solution (37.3 pS) was similar to that of an embryonic‐type ACh channel (36.9 pS), indicating that ACh is probably released from myocytes in normal Ringer solution.

When the patch pipette was filled with anticholinesterase alone to prevent hydrolysis of ACh released from myocytes, both physostigmine and neostigmine in a concentration‐dependent manner increased channel open probability; it was reduced by d‐Tc or α‐bungarotoxin.

Vesamicol and quinacrine, vesicular transporter inhibitors, reduced the channel open probability caused by ACh released from myocytes in the presence of neostigmine or physostigmine.

Intracellular alkalinization with NH4Cl inhibited the ACh release from myocytes, whereas, extracellular alkalinization, brought about by replacing normal Ringer solution, with pH 8.6 Ringer solution enhanced ACh release.

The immunocytochemistry of choline acetyltransferase (ChAT) showed that ChAT exists in both myocytes and neuronal cells but not in fibroblasts.

These results suggest that embryonic myocytes are capable of synthesizing and releasing ACh in a non‐quantal manner. Extracellular alkalinization enhanced and intracellular alkalinization inhibited ACh release from myocytes.
Blockade by 18β‐glycyrrhetinic acid of intercellular electrical coupling in guinea‐pig arterioles
Authors: Yamamoto Yoshimichi , Fukuta Hiroyasu , Nakahira Yoko , Suzuki Hikaru
Published: September 2004

Intercellular electrical communication between smooth muscle and endothelial cells was examined in guinea‐pig mesenteric arterioles using the whole‐cell patch‐clamp method. The time course of the current required to impose a 10 mV voltage clamp step was used to determine the extent of electrical coupling between them. Currents recorded from both smooth muscle and endothelial cells relaxed in a multi‐exponential manner, indicating the existence of electrical coupling between cells.

18β‐Glycyrrhetinic acid, a gap junction blocker, quickly blocked electrical communication at 40 μM, while neither heptanol nor octanol did so at concentrations of up to 1 mM.

In the current clamp mode, repetitive spikes, induced by 10 mM Ba2+ solutions, could be recorded from both kinds of cells. After blocking gap junctions, spikes could only be recorded from the smooth muscle cell layer, indicating that they had been conducted through myoendothelial junctions.

In endothelial cells, acetylcholine (ACh, 3 μM) induced hyperpolarizing responses, which had two phases (an initial fast and a second slower phase) in the current clamp condition. This ACh response persisted in the presence of 18β‐glycyrrhetinic acid, although this compound seemed to make the membrane slightly leaky.

After blocking gap junctions, the membrane potential of a single cell in a multicellular preparation could be well clamped. Thus, 18β‐glycyrrhetinic acid may be useful in studying the function of both arteriolar smooth muscle and endothelial cells while they remain located within a multicellular preparation.
Short‐term facilitation evoked during brief afferent tetani is not altered by long‐term potentiation in the guinea‐pig hippocampal CA1 region
Authors: Pananceau M. , Chen H.‐X. , Gustafsson B.
Published: September 2004

The aim was to examine whether long‐term potentiation (LTP) had effects on short‐term synaptic plasticity outside those predicted from its effect on single volley‐induced responses. Field recordings from the CA1 region of guinea‐pig hippocampal slices were used, and short‐ term plasticity was evoked by five‐impulse trains of 20 and 50 Hz.

The five‐impulse trains were evoked in the presence of D(‐)‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; 20‐50 μM), picrotoxin (100 μM), and 2‐OH‐saclofen (200 μM), and care was taken to avoid initiation of postsynaptic spike activation. Field responses were thus considered to reflect non‐NMDA receptor‐mediated activity only, and demonstrated a net facilitation during the trains.

The facilitation was found, on average, to be unaffected by LTP, evoked by strong afferent tetanization. This was true also when release probability had been altered either by the adenosine agonist N‐cyclohexyladenosine (CHA; 100 nM) or the antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 200 nM). When examined for individual experiments, cases with increases, or decreases, of facilitation following LTP were observed. These deviations showed no relation to initial release probability or to LTP magnitude, and they were also observed in control inputs not subjected to LTP.

Impairment of non‐NMDA receptor desensitization by cyclothiazide (30 μM) increased facilitation observed during a 50 Hz, but not a 20 Hz, train. LTP had no effect on facilitation, in the presence of this drug, either during 20 or 50 Hz trains.

The results suggest that the effect of LTP in the hippocampal CA1 region on non‐NMDA receptor‐mediated synaptic responses to a brief afferent tetanus does not differ from that on a low‐frequency, single volley‐induced response. They do not support the notion that LTP is based on changes in release probability of previously active synapses. If LTP is based on recruitment of previously, pre‐ or postsynaptically, silent synapses, these synapses must have, on average, release characteristics similar to the previously active ones.
Visual responses of neurones in the second visual area of flying foxes (Pteropus poliocephalus) after lesions of striate cortex
Authors: Funk Agnes P. , Rosa Marcello G. P.
Published: September 2004

The first (V1) and second (V2) cortical visual areas exist in all mammals. However, the functional relationship between these areas varies between species. While in monkeys the responses of V2 cells depend on inputs from V1, in all non‐primates studied so far V2 cells largely retain responsiveness to photic stimuli after destruction of V1.

We studied the visual responsiveness of neurones in V2 of flying foxes after total or partial lesions of the primary visual cortex (V1). The main finding was that visual responses can be evoked in the region of V2 corresponding, in visuotopic co‐ordinates, to the lesioned portion of V1 (‘lesion projection zone’; LPZ).

The visuotopic organization of V2 was not altered by V1 lesions.

The proportion of neurones with strong visual responses was significantly lower within the LPZs (31.5 %) than outside these zones, or in non‐lesioned control hemispheres (> 70 %). LPZ cells showed weak direction and orientation bias, and responded consistently only at low spatial and temporal frequencies.

The data demonstrate that the functional relationship between V1 and V2 of flying foxes resembles that observed in non‐primate mammals. This observation contrasts with the ‘primate‐like’ characteristics of the flying fox visual system reported by previous studies.
New observations on coupling between group II muscle afferents and feline γ‐motoneurones
Authors: Gladden M. H. , Jankowska E. , Czarkowska‐Bauch J.
Published: September 2004

Extra‐ or intracellular recordings were made from seventy‐six γ‐motoneurones of hindlimb muscles in chloralose anaesthetized cats to re‐assess the coupling between secondary muscle spindle afferents (group II muscle afferents) and these neurones. The latencies of a number of responses evoked by group II muscle afferents in γ‐motoneurones were shorter than minimal latencies of responses induced disynaptically in other spinal neurones. These latencies are therefore compatible with monosynaptic coupling between muscle spindle secondaries and γ‐motoneurones.

Responses fulfilling criteria for monosynaptically evoked responses were seen in about one third of γ‐motoneurones with input from the group II muscle afferents tested (in 6 of 18 motoneurones recorded intracellularly and in 26 of 74 motoneurones recorded extracellularly). They were usually evoked from only one of the stimulated nerves, stimulation of group II afferents of other nerves being followed by responses at longer latencies.

Most γ‐motoneurones were excited by group II afferents from several muscles, both flexors and extensors. However, a comparison of group II input to γ‐motoneurones innervating medial gastrocnemius and four other hindlimb muscles revealed differences in both incidence and sources.

This study extends results of previous studies by providing evidence that some synaptic actions of group II afferents, including afferents from the same muscle, are evoked monosynaptically, and may assist in sustaining the activation of γ‐motoneurones by positive feedback.
Role of adenosine and its receptors in the vasodilatation induced in the cerebral cortex of the rat by systemic hypoxia
Authors: Coney A. M. , Marshall Janice M.
Published: September 2004

In anaesthetized rats, we have examined the role of adenosine in vasodilatation evoked in the cerebral cortex by systemic hypoxia (breathing 8 % O2). Red cell flux was recorded from the surface of the exposed parietal cortex (CoRCF) by a laser Doppler probe, cortical vascular conductance (CoVC) being computed as CoRCF divided by mean arterial blood pressure. All agonists and antagonists were applied topically to the cortex.

Systemic hypoxia or adenosine application for 5 or 10 min, respectively, induced an increase in CoRCF and CoVC. These responses were substantially reduced by 8‐phenyltheophylline (8‐PT), an adenosine receptor antagonist which is non‐selective between the adenosine A1 and A2A receptor subtypes. By contrast, the adenosine receptor antagonist 8‐sulphophenyltheophylline (8‐SPT) which is similarly non‐selective, but unlike 8‐PT, does not cross the blood‐brain barrier, reduced the increases in CoRCF and CoVC induced by adenosine, but had no effect on those induced by hypoxia.

The A2A receptor agonist CGS21680 produced a substantial increase in CoRCF and CoVC, but the A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine had minimal effects.

The A2A receptor antagonist ZM241385 reduced the increase in CoRCF and CoVC induced by adenosine and reduced the increase in CoRCF induced by hypoxia.

We propose that exogenous adenosine that is topically applied to the cerebral cortex produces vasodilatation by acting on A2A receptors on the vascular smooth muscle. However, during systemic hypoxia, we propose that adenosine is released from endothelial cells and acts on endothelial A2A receptors to produce the major part of the hypoxia‐induced dilatation in the cerebral cortex.
Fast voltage gating of Ca2+ release in frog skeletal muscle revealed by supercharging pulses
Authors: Kim Albert M. , Vergara Julio L.
Published: September 2004

In single frog skeletal muscle fibres, we utilized supercharging voltage clamp command pulses to boost the rate of depolarization in the transverse tubular system (T‐system) such that 95 % of steady‐state potential is achieved in < 2 ms (as indicated by fluorescent potentiometric dye signals detected from a global illumination region). Signals detected near the edge of muscle fibres indicate that peripheral regions of the T‐system are not significantly overcompensated under these conditions.

We explored the impact of accelerating T‐system depolarization on voltage‐dependent events of excitation‐contraction (E‐C) coupling by measuring charge movement currents (CMCs) and Ca2+ fluorescence transients in response to both supercharging and conventional step pulses.

When compared with CMCs elicited by step pulses, supercharging CMCs are larger, and their kinetics more closely resemble those of gating current records reported for ionic channels. Furthermore, they decay bi‐exponentially (τfast range, 1.3‐1.8 ms; τslow range, 7.3‐11.9 ms), whereas step CMCs fall with a single exponential time course (τ range, 12.5‐26.7 ms).

Similarly, supercharging produces a distinct acceleration in Ca2+ release transients, which show little evidence of the voltage‐dependent onset latencies previously encountered using step pulses.

The use of this novel methodology in skeletal muscle unveils a previously undetected component of charge movement, the rapid, voltage‐dependent recruitment of which may provide the basis for understanding the fast gating of physiological E‐C coupling.
Distribution of photon absorption rates across the rat retina
Authors: Williams T. P. , Webbers J. P. P. , Giordano L. , Henderson R. P.
Published: September 2004

An investigation into the distribution of light intensity across the rat retina was carried out on excised, intact rat eyes exposed to Ganzfeld illumination from a helium‐neon laser (543 nm).

Some of the light entering the eyes exits through the sclera where its intensity can be monitored with an optical ‘pick‐up’ that samples the intensity coming from a small region of external sclera and underlying retina. The spatial resolution of the pick‐up is such that it samples light that has passed through ca 2 % of the rods in the rat eye.

Some of the laser light is absorbed by the rod pigment, rhodopsin, which gradually bleaches. Bleaching in the retina, in turn, causes an exponential increase in intensity emanating from the sclera. By monitoring this intensity increase, we are able to measure two important parameters in a single bleaching run: the local rhodopsin concentration and the local intensity falling on the rods.

With an ocular transmission photometer, we have measured both the local intensity and the local rhodopsin concentration across wide regions of rat retina. Both pigmented and albino rats were studied.

The distributions of rhodopsin and intensity were both nearly uniform; consequently, the product, (rhodopsin concentration) × (intensity), was similarly nearly equal across the retina. This means that the initial rate of photon absorption is about the same at all retinal locations.

Interpreted in terms of photostasis (the regulation of daily photon catch), this means that the rate of photon absorption is about the same in each rod, viz. 14 400 photons absorbed per rod per second. Since this rate of absorption is sufficient to saturate the rod, one possible purpose of photostasis is to maintain the rod system in a saturated state during daylight hours.
Force‐velocity and power‐load curves in rat skinned cardiac myocytes
Authors: McDonald Kerry S. , Wolff Matthew R. , Moss Richard L.
Published: September 2004

This study utilized a skinned myocyte preparation with low end compliance to examine force‐velocity and power‐load curves at 12 °C in myocytes from rat hearts.

In maximally activated myocyte preparations, shortening velocities appeared to remain constant during load clamps in which shortening took place over a sarcomere length range of ≈2.30‐2.00 μm. These results suggest that previously reported curvilinear length traces during load clamps of multicellular preparations were due in part to extracellular viscoelastic structures that give rise to restoring forces during myocardial shortening.

During submaximal Ca2+ activations, the velocity of shortening at low loads slowed and the time course of shortening became curvilinear, i.e. velocity progressively slowed as shortening continued. This result implies that cross‐bridge cycling kinetics are slower at low levels of activation and that an internal load arises during shortening of submaximally activated myocytes, perhaps due to slowly detaching cross‐bridges.

Reduced levels of activator Ca2+ also reduced maximal power output and increased the relative load at which power output was optimal. For a given absolute load, the shift has the effect of maintaining power output near the optimum level despite reductions in cross‐bridge number and force generating capability at lower levels of Ca2+.
Chemoafferent degeneration and carotid body hypoplasia following chronic hyperoxia in newborn rats
Authors: Erickson Jeffery T. , Mayer Catherine , Jawa Andrew , Ling Liming , Olson E. Burt , Vidruk Edward H. , Mitchell Gordon S. , Katz David M.
Published: September 2004

To define the role of environmental oxygen in regulating postnatal maturation of the carotid body afferent pathway, light and electron microscopic methods were used to compare chemoafferent neurone survival and carotid body development in newborn rats reared from birth in normoxia (21 % O2) or chronic hyperoxia (60 % O2).

Four weeks of chronic hyperoxia resulted in a significant 41 % decrease in the number of unmyelinated axons in the carotid sinus nerve, compared with age‐matched normoxic controls. In contrast, the number of myelinated axons was unaffected by hyperoxic exposure.

Chemoafferent neurones, located in the glossopharyngeal petrosal ganglion, already exhibited degenerative changes following 1 week of hyperoxia from birth, indicating that even a relatively short hyperoxic exposure was sufficient to derange normal chemoafferent development. In contrast, no such changes were observed in the vagal nodose ganglion, demonstrating that the effect of high oxygen levels was specific to sensory neurones in the carotid body afferent pathway. Moreover, petrosal ganglion neurones were sensitive to hyperoxic exposure only during the early postnatal period.

Chemoafferent degeneration in chronically hyperoxic animals was accompanied by marked hypoplasia of the carotid body. In view of previous findings from our laboratory that chemoafferent neurones require trophic support from the carotid body for survival after birth, we propose that chemoafferent degeneration following chronic hyperoxia is due specifically to the loss of target tissue in the carotid body.
Influence of the hypothalamic paraventricular nucleus on cardiovascular neurones in the rostral ventrolateral medulla of the rat
Authors: Yang Z. , Coote J. H.
Published: September 2004

The question of whether neurones in the paraventricular nucleus (PVN) of the hypothalamus have an excitatory influence on reticulo‐spinal vasomotor neurones of the rostral ventrolateral medulla (RVL) has been addressed in this study using anaesthetized rats.

Extracellular microelectrode recordings were made from sixty vasomotor neurones in the RVL, identified by their cardiac cycle‐related probability of discharge, by the decrease in activity in response to an increase in arterial blood pressure produced by intravenous phenylephrine and by the increase in activity in response to a decrease in blood pressure produced by intravenous nitroprusside.

More than 70 % of these RVL vasomotor neurones were identified as spinally projecting by antidromically activating their axons via a stimulating electrode in the lateral funiculus of the T2 or T10 segment of spinal cord.

Activation of neurones at different sites in the PVN with a microinjection of d,l‐homocysteic acid (DLH) elicited either pressor or depressor responses.

At PVN pressor sites fifteen RVL vasomotor neurones were shown to be activated prior to the blood pressure change. A further twenty RVL vasomotor neurones were observed to decrease activity following the blood pressure rise. At PVN depressor sites twelve RVL neurones were inhibited prior to the blood pressure change whereas another thirteen identified RVL neurones increased their discharge following the fall in blood pressure.

In three rats single shock electrical stimulation at a PVN pressor site, first identified with DLH, elicited a single or double action potential in thirteen RVL neurones with a latency of 27 ± 1 ms.

It is concluded that PVN neurones may elicit increases in blood pressure via excitatory connections with RVL‐spinal vasomotor neurones, and that other PVN neurones may elicit decreases in blood pressure via inhibitory connections with these RVL neurones.
Modulation of responses of feline γ‐motoneurones by noradrenaline, tizanidine and clonidine
Authors: Jankowska E. , Gladden M. H. , Czarkowska‐Bauch J.
Published: September 2004

Effects of noradrenaline (NA) and the α2 agonists tizanidine and clonidine were tested on extracellularly recorded responses of γ‐motoneurones in deeply anaesthetized cats. Two types of responses were used; firstly, short latency phasic responses evoked by electrical stimulation of group II afferents in a muscle nerve and, secondly, tonic background discharges.

Responses evoked by group II muscle afferents were depressed when NA and tizanidine were applied ionophoretically close to a γ‐motoneurone and when clonidine was applied systemically. The number of spike potentials evoked by stimulation of these afferents decreased and their latencies increased. Responses evoked by flexor or extensor afferents in γ‐motoneurones innervating flexors or extensors were similarly depressed.

Tonic discharges were inconsistently and/or insignificantly affected by locally applied NA and tizanidine but were depressed by systemically applied clonidine.

Control tests indicate specific effects of NA and tizanidine application since similarly ionophoresed H+ ions did not change responses of γ‐motoneurones to stimulation of group II afferents, or only weakly enhanced their background discharges. Furthermore, serotonin ejected from a solution with a similar pH facilitated rather than depressed responses of γ‐motoneurones.

The results indicate that some antispastic effects of clonidine and tizanidine may be due to the depression of group II‐evoked responses of γ‐motoneurones, resulting in weaker responses of muscle spindles to muscle stretches.
Multiple pathways for L‐methionine transport in brush‐border membrane vesicles from chicken jejunum
Authors: Soriano‐García Juan F. , Torras‐Llort Mònica , Ferrer Ruth , Moretó Miquel
Published: September 2004

The intestinal transport of L‐methionine has been investigated in brush‐border membrane vesicles isolated from the jejunum of 6‐week‐old chickens. L‐Methionine influx is mediated by passive diffusion and by Na+‐dependent and Na+‐independent carrier‐mediated mechanisms.

In the absence of Na+, cis‐inhibition experiments with neutral and cationic amino acids indicate that two transport components are involved in L‐methionine influx: one sensitive to L‐lysine and the other sensitive to 2‐aminobicyclo[2.2.1]heptane‐2‐carboxylic acid (BCH). The L‐lysine‐sensitive flux is strongly inhibited by L‐phenylalanine and can be broken down into two pathways, one sensitive to N‐ethylmaleimide (NEM) and the other to L‐glutamine and L‐cystine.

The kinetics of L‐methionine influx in Na+‐free conditions is described by a model involving three transport systems, here called a,b and c: systems a and b are able to interact with cationic amino acids but differ in their kinetic characteristics (system a: Km= 2.2 ± 0.3 μM and Vmax= 0.13 ± 0.005 pmol (mg protein)−1 (2 s)−1; system b: Km= 3.0 ± 0.3 mM and Vmax= 465 ± 4.3 pmol (mg protein)−1 (2 s)−1); system c is specific for neutral amino acids, has a Km of 1.29 ± 0.08 mM and a Vmax of 229 ± 5.0 pmol (mg protein)−1 (2 s)−1 and is sensitive to BCH inhibition.

The Na+‐dependent component can be inhibited by BCH and L‐phenylalanine but cannot interact either with cationic amino acids or with α‐(methylamino)isobutyrate (MeAIB).

The kinetic analysis of L‐methionine influx under a Na+ gradient confirms the activity of the above described transport systems a and b. System a is not affected by the presence of Na+ while system b shows a 3‐fold decrease in the Michaelis constant and a 1.4‐fold increase in Vmax. In the presence of Na+, the BCH‐sensitive component can be subdivided into two pathways: one corresponds to system c and the other is Na+ dependent and has a Km of 0.64 ± 0.013 mM and a Vmax of 391 ± 2.3 pmol (mg protein)−1 (2 s)−1.

It is concluded that L‐methionine is transported in the chicken jejunum by four transport systems, one with functional characteristics similar to those of system bo, + (system a); a second (system b) similar to system y+, which we suggest naming y+m to account for its high Vmax for L‐methionine transport in the absence of Na+; a third (system c) which is Na+ independent and has similar properties to system L; and a fourth showing Na+ dependence and tentatively identified with system B.
Na+‐K+ pump cycle during β‐adrenergic stimulation of adult rat cardiac myocytes
Authors: Dobretsov Maxim , Hastings Stephanie L. , Stimers Joseph R.
Published: September 2004

The mechanisms underlying the increase in Na+‐K+ pump current (Ip) caused by adrenergic stimulation were investigated in cultured adult rat cardiac myocytes using the whole‐cell patch‐clamp technique at 31‐33 °C.

In myocytes perfused internally with 50 mm Na+ (0 Ki+, 20 nM Ca2+, caesium aspartate solution) and externally with 5.4 mm K+o, noradrenaline (NA) and isoprenaline (Iso) (1‐50 μm) stimulated Ip by 40‐45 %.

Na+‐dependent transient Ip measurements with 0 mm K+i and 0 mm K+o revealed no change in the total charge transferred by the Na+‐K+ pump during the conformational change, suggesting that the pump site density was not changed by adrenergic stimulation (2630 ± 370 pumps μm−2 in control and 2540 ± 190 pumps μm−2 in the presence of 10 μm NA).

With saturating Na+i or K+o (150 and 15‐20 mm, respectively), Ip was still stimulated by NA and Iso. Thus, there was no indication that adrenergic activation of the Na+‐K+ pump was mediated by accumulation of Na+i and K+o or changes in the Na+‐K+ pump affinity for Na+i and K+o.

Both Ip and its increase under adrenergic stimulation were found to depend on [K+]i. While steady‐state Ip decreased from 2.2 ± 0.1 to 1.2 ± 0.1 pA pF−1 (P < 0.05), the stimulation of Ip by 10 μm Iso increased from 0.38 ± 0.04 to 0.67 ± 0.06 pA pF−1 (P < 0.05) with an increase in [K+]i from 0 to 100 mm.

Under conditions that cause the Ip‐Vm (membrane potential) relationship to express a positive slope ([Na+]o, 150 mm; [K+]o, 5.4 mm) or a negative slope ([Na+]o, 0; [K+]o, 0.3 mm) Iso stimulated Ip with no change in the shape of Ip‐Vm curves. Thus, adrenergic stimulation of the Na+‐K+ pump was not due to an alteration of voltage‐dependent steps of the pump cycle.

Simulation of these data with a six‐step model of the Na+‐K+ pump cycle suggested that in rat ventricular myocytes a signal from adrenergic receptors increased the Na+‐K+ pump rate by modulating the rate of K+ de‐occlusion and release by the pump.
Reappearance of activity in the vestibular neurones of labyrinthectomized guinea‐pigs is not delayed by cycloheximide
Authors: Ris Laurence , Wattiez Ruddy , de Waele Catherine , Vidal Pierre‐Paul , Godaux Emile
Published: September 2004

In mammals, unilateral labyrinthectomy induces an immediate depression of the resting discharges in the neurones of the ipsilateral vestibular nuclei. Later on, a spontaneous restoration of this activity occurs. The aim of the present study was to test the possibility that protein synthesis could be involved in the start of this process in the guinea‐pig.

Cycloheximide (CHX), a protein synthesis inhibitor, was injected intramuscularly 1 h before (30 mg kg−1) and 5 h after (15 mg kg−1) labyrinthectomy.

In a first group of animals, CHX was found to induce an inhibition of protein synthesis at levels ranging from 71 to 93 % for 9 h after labyrinthectomy.

In a second group of alert animals, we studied single unit activity of second‐order vestibular neurones. It was found that, in the 12–16 h post‐labyrinthectomy period, at a time when restoration began in guinea‐pigs not treated with CHX, the discharges in the labyrinthectomized group treated with CHX were not different from those observed in a previous study in labyrinthectomized animals not treated with CHX.

We conclude that protein synthesis is not required for the start of restoration of activity in the vestibular neurones deprived of their ipsilateral labyrinthine input.
Age and gender differences in excitation‐contraction coupling of the rat ventricle
Authors: Leblanc Normand , Chartier Denis , Gosselin Hugues , Rouleau Jean‐Lucien
Published: September 2004

The objective of this study was to determine potential post‐pubertal gender‐specific differences in the contractility of papillary muscles, the electrophysiological properties and Ca2+ transients of freshly dissociated ventricular myocytes from the rat heart.

The contractions of rat papillary muscles from 2‐ to 14‐month‐old male and female rats were studied under isometric and isotonic conditions (29 °C). While the hearts of young (2–4 months) male and female rats displayed a similar contractile profile, papillary muscles of female rats aged 6 months and older exhibited smaller isometric and isotonic contractions, smaller maximal rates of tension and shortening development and decline (±DT/dt and ±DL/dt) velocities during both the onset and relaxation phases, and shorter contractions than age‐matched males.

To explore the possible cellular basis accounting for these differences, action potentials and macroscopic currents were recorded from freshly dissociated myocytes using the whole‐cell patch clamp technique (35 °C). Action potentials from male and female myocytes of 3‐ and 9‐month‐old rats did not vary as a function of age or gender. Consistent with these results, the magnitude (expressed in pA pF−1), voltage‐dependence and kinetics of the inward rectifier (IK1), transient outward (Ito) and sustained (IK) K+ currents displayed little, if any dependence on age or gender.

L‐type Ca2+ current (ICa(L)) measured in caesium‐loaded myocytes (35 °C) from male and female rats of 3, 6 and 9 months of age exhibited similar characteristics. In contrast, while Ca2+ transients measured with indo‐1 were similar between 3‐month‐old male and female rat myocytes, Ca2+ transients of 10‐month‐old female myocytes were significantly reduced and showed a diminished rate of relaxation in comparison with those recorded in male rats of similar age.

These results suggest that important gender‐related changes in excitation‐contraction coupling occur following puberty, probably due to differences in Ca2+ handling capabilities at the level of the sarcoplasmic reticulum.
Extracellular ATP raises cytosolic calcium and activates basolateral chloride conductance in Necturus proximal tubule
Authors: Bouyer Patrice , Paulais Marc , Cougnon Marc , Hulin Philippe , Anagnostopoulos Takis , Planelles Gabrielle
Published: September 2004

Extracellular nucleotides modulate ionic transport mechanisms in various epithelia. In the present study, we investigated the effects of extracellular ATP on the intracellular free Ca2+ concentration ([Ca2+]i) and electrophysiological properties of Necturus maculosus proximal convoluted tubule (PCT).

ATP raised [Ca2+]i in microdissected fura‐2‐loaded PCTs (half‐maximal effect, ≈15 μmol l−1 ATP). The initial ATP‐induced changes in [Ca2+]i were not blunted by the removal of external Ca2+ nor by the presence of Ca2+ channel blockers, but were abolished by thapsigargin and suramin. The sequence for the potency of various agonists on [Ca2+]i was 2‐methylthioATP (2MeSATP) = ADP = ATP ≫ UTP, 2′,3′‐O‐(4‐benzoilbenzoil) ATP (BzATP), α,β‐methylene ATP (AMPCPP), adenosine.

In vivo electrophysiological measurements showed that 100 μmol l−1 peritubular ATP added to a Ringer solution reduced the basolateral cell membrane potential (Vm) and increased the cell membrane input conductance. In a low Cl− solution, this ATP‐induced depolarization was enhanced. These effects were inhibited by 1 mmol l−1SITS, consistent with the activation of a basolateral Cl− conductance.

The ATP‐induced change in Vm was reproduced by ADP but not by UTP or adenosine, and was prevented by suramin.

The ATP‐induced membrane depolarization was not influenced by thapsigargin, BAPTA AM, or staurosporin and was not reproduced by manoeuvres increasing [Ca2+]i or intracellular cAMP content.

We conclude that, in Necturus PCT, a P2y receptor mobilizes Ca2+ mainly from intracellular pools and increases a basolateral Cl− conductance, GCl. The activation of GCl occurs by a mechanism which is not related either to an increase in [Ca2+]i or cAMP content, or to PKC activation.