bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of mEPSCs recorded in layer II neurones of rat barrel cortex
Authors: Simkus Christopher R. L. , Stricker Christian
Published: November 2002

Voltage‐clamp recordings from layer II neurones in somatosensory cortex of rats aged between 12 and 17 days showed a high frequency of spontaneous postsynaptic currents (sPSCs), which on average was 33 ± 13 Hz (s.d.). sPSCs were mediated largely by glutamatergic AMPA receptors. Their rates and amplitudes were independent of blocking sodium channels with 1 μm tetrodotoxin (TTX). Most of them, therefore, represent genuine miniature excitatory postsynaptic currents (mEPSCs). The rise time of the fastest (10 %) mEPSCs was 288 ± 86 μs (10‐90 %) and the half‐width was 1073 ± 532 μs. The amplitude was −5.9 ± 1.1 pA with a coefficient of variation (CV) of 0.44 ± 0.14. The rate of mEPSCs was very temperature sensitive with a Q10 (33‐37 °C) of 8.9 ± 0.9. Due to this temperature sensitivity, we estimated that the microscope lamp contributed an increase in temperature of about 4 °C to the tissue in the focal volume of the condenser. Cell‐type differences in the rate of mEPSCs were found between pyramidal/multipolar and bipolar cells. The latter had a frequency of about a third of that seen in the other cell groups. Recordings in layer II are ideally suited to investigate mechanisms of spontaneous transmitter release.
The contribution of intracellular calcium stores to mEPSCs recorded in layer II neurones of rat barrel cortex
Authors: Simkus Christopher R. L. , Stricker Christian
Published: November 2002

Loading slices of rat barrel cortex with 50 μm BAPTA‐AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca2+ stores with 20 μm cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca2+ stores, results in a similar reduction of mEPSCs to that observed with BAPTA‐AM, indicating that the source for intracellular Ca2+ is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 μm ryanodine or 10 mm caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca2+ release from ryanodine stores. Blocking IP3 receptors with 14 μm 2‐aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP3 stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 μm (RS)‐3,5‐dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP3 receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca2+‐induced Ca2+ release (CICR) from ryanodine stores after the spontaneous opening of IP3 stores.
A cold‐ and menthol‐activated current in rat dorsal root ganglion neurones: properties and role in cold transduction
Authors: Reid Gordon , Babes Alexandru , Pluteanu Florentina
Published: November 2002

Skin temperature is sensed by peripheral thermoreceptors. Using the neuronal soma in primary culture as a model of the receptor terminal, we have investigated the mechanisms of cold transduction in thermoreceptive neurones from rat dorsal root ganglia. Cold‐sensitive neurones were pre‐selected by screening for an increase in [Ca2+]ion cooling; 49 % of them were also excited by 0.5 μmcapsaicin. Action potentials and voltage‐gated currents of cold‐sensitive neurones were clearly distinct from those of cold‐insensitive neurones. All cold‐sensitive neurones expressed an inward current activated by cold and sensitised by (‐)‐menthol, which was absent from cold‐insensitive neurones. This current was carried mainly by Na+ions and caused a depolarisation on cooling accompanied by action potentials, inducing voltage‐gated Ca2+entry; a minor fraction of Ca2+entry was voltage‐independent. Application of (‐)‐menthol shifted the threshold temperatures of the cold‐induced depolarisation and the inward current to the same extent, indicating that the cold‐ and menthol‐activated current normally sets the threshold temperature for depolarisation during cooling. The action of menthol was stereospecific, with the (+)‐isomer being a less effective agonist than the (‐)‐isomer. Extracellular Ca2+modulated the cold‐ and menthol‐activated current in a similar way to its action on intact cold receptors: lowered [Ca2+]osensitised the current, while raised [Ca2+]oantagonised the menthol‐induced sensitisation. During long cooling pulses the current showed adaptation, which depended on extracellular Ca2+and was mediated by a rise in [Ca2+]i. This adaptation consisted of a shift in the temperature sensitivity of the channel. In capsaicin‐sensitive neurones, capsaicin application caused a profound depression of the cold‐activated current. Inclusion of nerve growth factor in the culture medium shifted the threshold of the cold‐activated current towards warmer temperatures. The current was blocked by 50 μmcapsazepine and 100 μmSKF 96365. We conclude that the cold‐ and menthol‐activated current is the major mechanism responsible for cold‐induced depolarisation in DRG neurones, and largely accounts for the known transduction properties of intact cold receptors.
Effect of caffeine on self‐sustained firing in human motor units
Authors: Walton Christine , Kalmar Jayne M. , Cafarelli E.
Published: November 2002

This study examined the effect of caffeine on self‐sustained firing (SSF) of human motor units. At physiological doses, caffeine acts as a competitive antagonist to the inhibitory effects of adenosine. This antagonism has many possible effects on the central nervous system. One of these effects is to increase the release of the excitatory neurotransmitters serotonin and noradrenaline. In addition, caffeine increases serotonin concentration in brainstem regions that have excitatory projections to spinal motor neurons. Since plateau potentials, which are responsible for SSF, are facilitated by these neurotransmitters, we hypothesized that caffeine would increase the frequency at which SSF occurs. A double‐blind, repeated‐measures design using either drug (6 mg kg−1 caffeine) or placebo (flour) was carried out on seven male subjects who reported ingesting less than 200 mg week−1 caffeine. We investigated the occurrence of SSF in tibialis anterior motor units (214 trials) and found a significant (P < 0.05) increase in the occurrence of SSF in the caffeine trial (87.0 ± 5.8 %) compared to the placebo (64.6 ± 9.7 %). These data further verify the presence of SSF in the tibialis anterior motor units of young men and provide indirect evidence of the facilitation of plateau potentials by monoamines in the human neuromuscular system.
Stoichiometry of NA+−CA2+ exchange is 3:1 in guinea‐pig ventricular myocytes
Authors: Hinata Masamitsu , Yamamura Hisao , Li Libing , Watanabe Yasuhide , Watano Tomokazu , Imaizumi Yuji , Kimura Junko
Published: November 2002

In single guinea‐pig ventricular myocytes, we examined the stoichiometry of Na+‐Ca2+ exchange (NCX) by measuring the reversal potential (ENCX) of NCX current (INCX) and intracellular Ca2+ concentration ([Ca2+]i) with the whole‐cell voltage‐clamp technique and confocal microscopy, respectively. With given ionic concentrations in the external and pipette solutions, the predicted ENCX were −73 and −11 mV at 3:1 and 4:1 stoichiometries, respectively. ENCX measured were −69 ± 2 mV (n= 11), −47 ± 1 mV (n= 14) and −15 ± 1 mV (n= 15) at holding potentials (HP) of −73, −42 and −11 mV, respectively. Thus, ENCX almost coincided with HP, indicating that [Ca2+]i and/or [Na+]i changed due to INCX flow. Shifts of ENCX (ΔENCX) were measured by changing [Ca2+]o or [Na+]o. The measured values of ΔENCX were almost always smaller than those expected theoretically at a stoichiometry of either 3:1 or 4:1. Using indo‐1 fluorescence, [Ca2+]i measured under the whole‐cell voltage‐clamp supported a 3:1 but not 4:1 stoichiometry. To prevent Ca2+ accumulation, we inhibited INCX with Ni2+ and re‐examined ENCX during washing out Ni2+. With HP at predicted ENCX at a 3:1 stoichiometry, ENCX developed was close to predicted ENCX and did not change with time. However, with HP at predicted ENCX for a 4:1 stoichiometry, ENCX developed initially near a predicted ENCX for a 3:1 stoichiometry and shifted toward ENCX for a 4:1 stoichiometry with time. We conclude that the stoichiometry of cardiac NCX is 3:1.
Excitability of human muscle afferents studied using threshold tracking of the H reflex
Authors: Lin Cindy S.‐Y. , Chan Jane H. L. , Pierrot‐Deseilligny Emmanuel , Burke David
Published: November 2002

In human peripheral nerves, physiological evidence has been presented for a number of biophysical differences between cutaneous afferents and α motor axons. The differences in strength‐duration properties for cutaneous afferents and motor axons in the median nerve have been attributed to greater expression of a persistent Na+ conductance (INa,P) on cutaneous afferents. However, it is unclear whether the biophysical properties of human group Ia afferents differ from those of cutaneous afferents. The present studies were undertaken to determine whether the properties of human group Ia afferents can be studied indirectly using ‘threshold tracking’ to measure the excitability changes in the H reflex, and to determine whether the excitability of group Ia afferents differs from that of cutaneous afferents. The strength‐duration properties of the soleus H reflex and soleus motor axons were measured at rest and during sustained voluntary contractions. Similar experiments were performed on the median nerve at the wrist to study the strength‐duration properties of cutaneous afferents, α motor axons and H reflex of the thenar muscles. In addition, the technique of ‘latent addition’ was used to determine whether there was a difference in a low‐threshold conductance on soleus Ia afferent and motor axons. The present findings indicate that the strength‐duration time constant (τSD) for the H reflex is longer than that for α motor axons, but similar to that for cutaneous afferents. There were no differences in τSD for the soleus H reflex at rest and during contractions, suggesting that τSD for the H reflex is largely unaffected by changes in synaptic or motoneurone properties. Finally, the difference in latent addition suggests that the longer τSD of the soleus H reflex may indeed be due to greater activity of a persistent Na+ conductance on Ia afferents than on soleus α motor axons.
Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6)
Authors: Jans Danny , Srinivas S. P. , Waelkens Etienne , Segal Andrei , Larivière Els , Simaels Jeannine , Van Driessche Willy
Published: November 2002

In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin‐luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing‐type chamber. We developed a LL pulse protocol to determine the rate of ATP release (RATP) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure RATP as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 μl of A6 cells released ATP at a rate of 66 ± 8 fmol min−1. A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H2O)−1 elevated RATP rapidly to a peak value of 1.89 ± 0.11 pmol min−1 (RATPpeak) followed by a plateau phase reaching 0.51 ± 0.07 pmol min−1 (RATPplat). Both RATPpeak and RATPplat values increased with the degree of dilution. The magnitude of RATPplat remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 ± 0.08 pmol min−1 was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H2O)−1. This RATP value, induced in the absence of cell swelling, is comparable to RATPplat. Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose‐response curves for peak [Ca2+]i increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with RATPpeak. The link between the ATP release and the fast [Ca2+]i transient was also demonstrated by the depression of both phenomena by Cl− removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin‐sensitive rise of [Ca2+]i during the hyposmotic shock.
Motor unit synchronisation is enhanced during slow lengthening contractions of a hand muscle
Authors: Semmler John G. , Kornatz Kurt W. , Dinenno Devin V. , Zhou Shi , Enoka Roger M.
Published: November 2002

This study examined the strength of motor unit synchronisation based on time‐ and frequency‐domain measures during postural, shortening and lengthening contractions of a hand muscle in young adults. Single motor unit activity was recorded with intramuscular electrodes in the left first dorsal interosseus muscle as the subject held the index finger at a constant position while supporting a light load for 2‐5 min. The subject then performed slow (1.7 deg s−1) shortening and lengthening contractions to lift and lower the load. The movement required subjects to perform 10‐25 constant‐velocity contractions with the index finger over a 10 deg range of motion by using 6 s shortening and lengthening contractions. Individual discharge times were obtained from 23 pairs of motor units in 14 subjects to assess the strength of motor unit synchronisation and coherence during the three tasks. The strength of motor unit synchronisation was approximately 50 % greater during the lengthening contractions compared with the postural and shortening contractions, and the width of the central synchronous peak in the cross‐correlation histogram was ≈4 ms narrower during shortening contractions. These findings reveal that there is an increase in common input to motoneurones during lengthening contractions and a greater relative contribution of direct common inputs to motoneurones during shortening contractions compared with postural tasks. Furthermore, the amount of motor unit coherence in the low‐frequency band (2‐12 Hz) was reduced during shortening contractions compared with postural and lengthening contractions. These data indicate that the timing of inputs received by the motoneurones innervating the first dorsal interosseus of young adults differs during postural, shortening and lengthening contractions against a light load.
Functional regulation of xanthine oxidoreductase expression and localization in the mouse mammary gland: evidence of a role in lipid secretion
Authors: McManaman J. L. , Palmer C. A. , Wright R. M. , Neville M. C.
Published: November 2002

Xanthine oxidoreductase (XOR), a key enzyme of purine metabolism, has been implicated in the secretion of the milk fat droplet in lactating mammary epithelial cells, possibly through structural interactions with other milk fat globule proteins including butyrophilin (Btn) and adipophilin (ADPH). To help determine the mechanism by which XOR is regulated, we examined the expression and localization of XOR in the non‐secretory states of late pregnancy and induced involution compared with the state of active secretion. XOR mRNA levels started to increase at mid‐pregnancy, turned sharply upwards at the onset of lactation and decreased rapidly with forced involution, indicating transcriptional control of the enzyme level by differentiation and secretory function. During pregnancy and involution the enzyme was diffusely distributed in the cytoplasm, but moved rapidly to the apical membrane of the cells when secretion was activated, where it colocalized with both Btn and ADPH, similar to the situation in the milk fat globule itself. Size‐exclusion chromatography of solubilized milk fat globule membrane proteins showed that XOR formed a sulphydryl‐bond‐dependent complex with Btn and ADPH in the milk fat globule membrane. XOR returned to a diffuse cytoplasmic localization shortly after induced involution, while Btn remained localized to the apical membrane, suggesting that localization of XOR is not dependent on the presence of Btn in the apical membrane. Our findings indicate that the expression and membrane association of XOR in the mammary gland are tightly regulated by secretory activity, and suggest that the apical membrane association of XOR regulates the coupling of lipid droplets to the apical plasma membrane during milk lipid secretion.
The β1a subunit regulates the functional properties of adult frog and mouse L‐type Ca2+ channels of skeletal muscle
Authors: García Rubén , Carrillo Elba , Rebolledo Santiago , García María C. , Sánchez Jorge A.
Published: November 2002

The β1a subunit, one of the auxiliary subunits of CaV1.1 channels, was expressed in COS‐1 cells, purified by electroelution and electrodialysis techniques and identified by Western blot using monoclonal antibodies. The purified β1a subunit strongly interacted in vitro with the alpha interaction domain (AID) of CaV1.1 channels. The actions of the purified β1a subunit on CaV1.1 channel currents were assessed in whole cell voltage clamp experiments performed in vesicles derived from frog and mouse adult skeletal muscle plasma membranes. L‐type inward currents were recorded in solutions containing Ba2+ (IBa). Values of peak IBa were doubled by the β1a subunit in frog and mouse muscle vesicles and the amplitude of the slow component of tail currents was greatly increased. The actions of the β1a subunit on CaV1.1 channel currents reached a steady state within 20 min. The β1a subunit had no effect on the time courses of activation or inactivation of IBa or shifted the current‐voltage relation. Non‐linear capacitive currents were recorded in solutions that contained mostly impermeant ions. Charge movement depended on voltage with average Boltzmann parameters: Qmax+ 28.0 ± 6.6 nC μF−1, V+−58.0 ± 2.0 mV and k+ 15.3± 1.1 mV (n= 24). In the presence of the β1a subunit, these parameters remained unchanged: Qmax+ 29.8 ± 3.5 nC μF−1, V+−54.5 ± 2.2 mV and k+ 16.4± 1.3 mV (n= 21). Overall, the work describes a novel preparation to explore in situ the role of the β1a subunit on the function of adult CaV1.1 channels.
Differential activation of ion channels by inositol 1,4,5‐trisphosphate (IP3)‐ and ryanodine‐sensitive calcium stores in rat basilar artery vasomotion
Authors: Haddock R. E. , Hill C. E.
Published: November 2002

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. Using electrophysiology to study changes in membrane potential, the ratiometric calcium indicator Fura‐2 AM to study changes in [Ca2+]i in both the arterial wall and in individual smooth muscle cells (SMCs), and video microscopy to study changes in vessel diameter, we have investigated the cellular mechanisms underlying vasomotion in the juvenile rat basilar artery. During vasomotion, rhythmical oscillations in both membrane potential and [Ca2+]i were found to precede rhythmical contractions. Nifedipine depolarized SMCs and abolished rhythmical contractions and depolarizations. [Ca2+]i oscillations in the arterial wall became reduced and irregular, while [Ca2+]i oscillations in adjacent SMCs were no longer synchronized. BAPTA‐AM, thapsigargin and U73122 hyperpolarized SMCs, relaxed the vessel, decreased basal calcium levels and abolished vasomotion. Chloride substitution abolished rhythmical activity, depolarized SMCs, increased basal calcium levels and constricted the vessel, while niflumic acid and DIDS abolished vasomotion. Ryanodine, charybdotoxin and TRAM‐34, but not iberiotoxin, 4‐aminopyridine or apamin, each depolarized SMCs and increased the frequency of rhythmical depolarizations and [Ca2+]i oscillations. We conclude that vasomotion in the basilar artery depends on the release of intracellular calcium from IP3 (inositol 1,4,5,‐trisphosphate)‐sensitive stores which activates calcium‐dependent chloride channels to depolarize SMCs. Depolarization in turn activates voltage‐dependent calcium channels, synchronizing contractions of adjacent cells through influx of extracellular calcium. Subsequent calcium‐induced calcium release from ryanodine‐sensitive stores activates an intermediate conductance potassium channel, hyperpolarizing the SMCs and providing a negative feedback pathway for regeneration of the contractile cycle.
Dissociation between muscle tricarboxylic acid cycle pool size and aerobic energy provision during prolonged exercise in humans
Authors: Gibala Martin J. , González‐Alonso José , Saltin Bengt
Published: November 2002

It has been suggested that a decrease in the total concentration of tricarboxylic acid cycle intermediates (TCAIs) ‐ secondary to a reduction in glycogen availability ‐ compromises oxidative energy provision in skeletal muscle during prolonged exercise. However, no study has directly tested this hypothesis. We therefore studied six men (28 ± 2 years) during 90 min of leg kicking exercise at an intensity equivalent to 70 % of maximum. Biopsies (vastus lateralis) were obtained at rest and after 5, 10, 15, 30, 60 and 90 min of exercise, and thigh oxygen uptake (VO2,thigh) was calculated according to the Fick principle. The sum of six measured TCAIs (≈95 % of total pool size) was 1.30 ± 0.15 mmol (kg dry wt)−1 at rest and increased (P≤ 0.05) rapidly during exercise to a peak value of 3.15 ± 0.23 mmol (kg dry wt)−1 after 10 min. Thereafter, the [TCAI] declined to 2.14 ± 0.23, 1.73 ± 0.32 and 1.62 ± 0.10 mmol (kg dry wt)−1 after 30, 60 and 90 min, respectively (P≤ 0.05vs.10 min). Despite the 50 % decrease in [TCAI], aerobic energy provision was not compromised, as evidenced by stable VO2,thigh values throughout the entire exercise bout and little change in muscle [phosphocreatine] after 10 min. The largest decrease in [TCAI] (Δ+ 1.00 ± 0.24 mmol (kg dry wt)−1) occurred from 10 to 30 min of exercise despite the fact that muscle [glycogen] remained relatively high at this point in exercise (≈274 ± 24 mmol (kg dry wt)−1 after 30 min; ≈65 % of rest value). Conversely, there was little change in [TCAI] during the final 30 min of exercise (Δ+ 0.11 ± 0.29 mmol (kg dry wt)−1) despite a decrease in [glycogen] to ≈72 ± 3 mmol (kg dry wt)−1 after 90 min (≈13 % of rest value). We conclude that there is a progressive decrease in muscle [TCAI] during prolonged exercise in humans; however this decrease does not compromise aerobic energy provision and is not attributable to the depletion of muscle [glycogen].
Long‐lasting modulation of synaptic input to Purkinje neurons by Bergmann glia stimulation in rat brain slices
Authors: Brockhaus Johannes , Deitmer Joachim W.
Published: November 2002

Information processing in the nervous system is achieved primarily at chemical synapses between neurons. Recent evidence suggests that glia‐neuron interactions contribute in multiple ways to the synaptic process. In the present study we used the frequency of spontaneous postsynaptic currents (sPSC) in Purkinje neurons in acute cerebellar brain slices from juvenile rats (13‐19 days old) as a measure of synaptic activity. Following 50 depolarizing pulses to an adjacent Bergmann glial cell (50 mV; duration 0.5 s; 1 Hz) the sPSC frequency of the Purkinje neuron was reduced to 65 ± 7 % of control values within 10 min after glial stimulation and remained depressed for at least 40 min. Depolarizing pulses to 0 mV had a comparable effect (70 ± 5 % of control). The frequency of miniature PSCs, as recorded in 300 nm TTX, was not modulated after glial stimulation. Blockade of ionotropic glutamate receptors (iGluRs) with kynurenic acid (1 mm) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 5 μm) suppressed the reduction of neuronal activity induced by glial depolarization, whereas the glial modulation of synaptic activity was not inhibited by a block of N‐methyl‐d‐aspartate iGluRs, metabotropic glutamate receptors, cannabinoid receptors or GABAB receptors. Fluorometric measurements of the intraglial Ca2+ concentration revealed no glial Ca2+ transients during the depolarization series, and glial cell stimulation reduced the neuronal sPSC frequency even after loading the glial cell with 20 mm of the Ca2+ chelator BAPTA. Our results indicate a glia‐induced long‐lasting depression of neuronal communication mediated by iGluRs.
β‐adrenergic and muscarinic agonists modulate inactivation of l‐type ca2+ Channel Currents in Guinea‐Pig Ventricular Myocytes
Authors: Findlay Ian
Published: November 2002

The objective of this study was to examine the effects of isoproterenol (isoprenaline) and carbachol upon voltage‐dependent inactivation of L‐type Ca2+ current (ICa,L). ICa,L was recorded in guinea‐pig isolated ventricular myocytes in the presence and absence of extracellular Ca2+ to separate total inactivation and voltage‐dependent inactivation. In the presence of Ca2+, isoproterenol and carbachol had ‘competitive’ effects upon the relationships between membrane voltage and ICa,L amplitude and inactivation. Neither agonist had a marked effect upon the decay of inward ICa,L carried by Ca2+. In the absence of Ca2+, isoproterenol severely reduced and slowed ICa,L inactivation; this effect was reversed by carbachol. Under control conditions decay was dominated by fast inactivation. Isoproterenol reduced fast‐inactivating and increased time‐independent currents in a dose‐dependent manner. These effects were counteracted by carbachol. There was a reciprocal relationship between the amplitude of fast‐inactivating and time‐independent currents with agonist stimulation. It is concluded that agonist modulation of rapid voltage‐dependent inactivation of L‐type Ca2+ channels involves an ‘on‐off’ switch.
A rhythmic motor pattern activated by circumferential stretch in guinea‐pig distal colon
Authors: Spencer Nick J. , Hennig Grant W. , Smith Terence K.
Published: November 2002

Simultaneous intracellular recordings were made from pairs of circular muscle (CM) cells, at the oral and anal ends of a segment of guinea‐pig distal colon, to investigate the neuronal mechanisms underlying faecal pellet propulsion. When a minimum degree of circumferential stretch was applied to sheet preparations of colon, recordings from CM cells revealed either no ongoing junction potentials, or alternatively, small potentials usually < 5 mV in amplitude. Maintained circumferential stretch applied to these preparations evoked an ongoing discharge of excitatory junction potentials (EJPs) at the oral recording site (range: 1‐25 mV), which lasted for up to 6 h. The onset of each large oral EJP was time‐locked with the onset of an inhibitory junction potential (IJP) at an anal recording electrode, located 2 cm from the oral recording. Similar results were obtained in isolated intact tube preparations of colon, when recordings were made immediately oral and anal of an artificial faecal pellet. The amplitudes of many large (> 5 mV) oral EJPs were linearly related to the amplitudes of anal IJPs occurring 20 mm apart. In the absence of an L‐type Ca2+ channel blocker, action potentials occurred on each large oral EJP. Synchronized discharges of stretch‐activated EJPs and IJPs were preserved following pretreatment with capsaicin (10 μm), were unaffected by nifedipine (1 μm) and did not require the mucosa or submucous plexus. EJPs and IJPs were abolished by hexamethonium (300 μm) or tetrodotoxin (1 μm), but persisted in the presence of pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS; 10 μm) or an NK3 tachykinin receptor antagonist (Neurokinin A 4‐10; 100 nm to 5 μm). In summary, maintained circumferential stretch of the distal colon activates a population of intrinsic mechanosensory neurons that generate repetitive firing of ascending excitatory and descending inhibitory pathways to CM. These mechanosensory neurons, which may be interneurons, are stretch sensitive, rather than muscle tension sensitive, since they are resistant to muscular paralysis. We suggest the synchrony in onset of oral EJPs and anal IJPs over large regions of colon is due to synchronous synaptic activation of ascending and descending interneurons.
Effect of blood haemoglobin concentration on V̇o2,max and cardiovascular function in lowlanders acclimatised to 5260 m
Authors: Calbet J. A. L. , Rådegran G. , Boushel R. , Søndergaard H. , Saltin B. , Wagner P. D.
Published: November 2002

The principal aim of this investigation was to determine the influence of blood haemoglobin concentration ([Hb]) on maximal exercise capacity and maximal O2 consumption (V̇O2,max) in healthy subjects acclimatised to high altitude. Secondarily, we examined the effects of [Hb] on the regulation of cardiac output (CO), blood pressure and muscular blood flow (LBF) during exercise. Eight Danish lowlanders (three females and five males; 24 ± 0.6 years, mean ±s.e.m.) performed submaximal and maximal exercise on a cycle ergometer after 9 weeks at an altitude of 5260 m (Mt Chacaltaya, Bolivia). This was done first with the high [Hb] resulting from acclimatisation and again 2‐4 days later, 1 h after isovolaemic haemodilution with Dextran 70 to near sea level [Hb]. After measurements at maximal exercise while breathing air at each [Hb], subjects were switched to hyperoxia (55 % O2 in N2) and the measurements were repeated, increasing the work rate as tolerated. Hyperoxia increased maximal power output and leg V̇O2,max, showing that breathing ambient air at 5260 m, V̇O2,max is limited by the availability of O2 rather than by muscular oxidative capacity. Altitude increased [Hb] by 36 % from 136 ± 5 to 185 ± 5 g l−1 (P < 0.001), while haemodilution (replacing 1 l of blood with 1 l of 6 % Dextran) lowered [Hb] by 24 % to 142 ± 6 g l−1 (P < 0.001). Haemodilution had no effect on maximal pulmonary or leg V̇O2,max, or power output. Despite higher LBF, leg O2 delivery was reduced and maximal V̇O2 was thus maintained by higher O2 extraction. While CO increased linearly with work rate irrespective of [Hb] or inspired oxygen fraction (FI,O2), both LBF and leg vascular conductance were systematically higher when [Hb] was low. Close and significant relationships were seen between LBF (and CO) and both plasma noradrenaline and K+ concentrations, independently of [Hb] and FI,O2. In summary, under conditions where O2 supply limits maximal exercise, the increase in [Hb] with altitude acclimatisation does not improve maximal exercise capacity or V̇O2,max, and does not alter peak CO. However, LBF and vascular conductance are higher at altitude when [Hb] is lowered to sea level values, with both relating closely to catecholamine and potassium concentrations. This suggests that the lack of effect of [Hb] on V̇O2,max may involve reciprocal changes in LBF via local metabolic control of the muscle vasculature.
Voltage‐dependent priming of rat vanilloid receptor: effects of agonist and protein kinase C activation
Authors: Ahern Gerard P. , Premkumar Louis S.
Published: November 2002

The responses of vanilloid receptor (VR) channels to changing membrane potential were studied in Xenopus oocytes and rat dorsal root ganglion (DRG) neurons. In oocytes, capsaicin‐evoked VR currents increased instantaneously upon a step depolarization and thereafter rose biexponentially with time constants of ≈20 and 1000 ms. Similarly, upon repolarization the current abruptly decreased, followed by a biexponential decay with time constants of ≈4 and 200 ms. Qualitatively similar effects were observed in single channel recordings of native VR channels from DRG neurons and with endogenous VR activators, including heat (43 °C), H+, anandamide and protein kinase C (PKC). The magnitude of the time‐dependent current rise increased with membrane depolarization. This effect was accompanied by an increase in the relative proportion of the fast kinetic component, A1. In contrast, the time constants of the activation and deactivation processes were not strongly voltage dependent. Increasing the agonist concentration both reduced the magnitude of the current rise and increased its overall rate, without significantly altering the deactivation rate. In contrast, PKC both speeded the current rise and slowed its decay. These results suggest that voltage interacts with agonists in a synergistic manner to augment VR current and this mechanism will be enhanced under conditions of inflammation when VRs are likely to be phosphorylated.
Voltage‐dependent inactivation of l‐type ca2+ Currents in Guinea‐Pig Ventricular Myocytes
Authors: Findlay Ian
Published: November 2002

The objective of this study was to describe the kinetics of voltage‐dependent inactivation of native cardiac L‐type Ca2+ currents. Whole‐cell currents were recorded from guinea‐pig isolated ventricular myocytes. Voltage‐dependent inactivation was separated from Ca2+‐dependent inactivation by replacing extracellular Ca2+ with Mg2+ and recording outward currents through Ca2+ channels. Voltage‐dependent inactivation accelerated from slow monophasic decay at −30 mV to maximal rapid biphasic decay at +20 mV. Maximal voltage‐dependent inactivation occurred with τf≈30 ms and τs≈300 ms, the fast component of decay accounted for 70 % of the current amplitude. In basal conditions Ca2+ current availability was sigmoid. Isoproterenol (isoprenaline) evoked a large increase in a time‐independent component of the Ca2+ current which also increased with depolarisation. This was responsible for the apparent recovery of Ca2+ channel current availability at positive membrane potentials and thus a U‐shaped availability‐voltage (A‐V) relationship. It is concluded that β‐adrenergic stimulation altered the reaction of native cardiac L‐type Ca2+ channels to membrane voltage. In basal conditions, voltage accelerated inactivation. In isoproterenol, voltage could also reduce inactivation.
Inadequate heat release from the human brain during prolonged exercise with hyperthermia
Authors: Nybo Lars , Secher Niels H. , Nielsen Bodil
Published: November 2002

Brain temperature appears to be an important factor affecting motor activity, but it is not known to what extent brain temperature increases during prolonged exercise in humans. Cerebral heat exchange was therefore evaluated in seven males during exercise with and without hyperthermia. Middle cerebral artery mean blood velocity (MCA Vmean) was continuously monitored while global cerebral blood flow (CBF) and cerebral energy turnover were determined at the end of the two exercise trials in three subjects. The arterial to venous temperature difference across the brain (v‐aDtemp) was determined via thermocouples placed in the internal jugular vein and in the aorta. The jugular venous blood temperature was always higher than that of the arterial blood, demonstrating that heat was released via the CBF during the normothermic as well as the hyperthermic exercise condition. However, heat removal via the jugular venous blood was 30 ± 6 % lower during hyperthermia compared to the control trial. The reduced heat removal from the brain was mainly a result of a 20 ± 6 % lower CBF (22 ± 9 % reduction in MCA Vmean), because the v‐aDtemp was not significantly different in the hyperthermic (0.20 ± 0.05 °C) compared to the control trial (0.22 ± 0.05 °C). During hyperthermia, the impaired heat removal via the blood was combined with a 7 ± 2 % higher heat production in the brain and heat was consequently stored in the brain at a rate of 0.20 ± 0.06 J g−1 min−1. The present results indicate that the average brain temperature is at least 0.2 °C higher than that of the body core during exercise with or without hyperthermia.
Growth hormone‐releasing peptide‐2 reduces inward rectifying K+ currents via a PKA‐cAMP‐mediated signalling pathway in ovine somatotropes
Authors: Xu Ruwei , Zhao Yufeng , Chen Chen
Published: November 2002

Inward‐rectifying potassium (Kir) channels are essential for maintaining the resting membrane potential near the K+ equilibrium and they are responsible for hyperpolarisation‐induced K+ influx. We characterised the Kir current in primary cultured ovine somatotropes and examined the effect of growth hormone‐releasing peptide‐2 (GHRP‐2) on this current and its related intracellular signalling pathways. The Kir current was, in most cases, isolated using nystatin‐perforated patch‐clamp techniques. In bath solution containing 5 mm K+, the Kir current was composed of both transient (fast activated) and delayed (slowly activated) components. An increase in the external K+ concentration from 5 to 25 mm induced an augmentation of ≈4‐fold in the delayed part of the Kir current and both BaCl2 and CsCl dose‐dependently inhibited this current, confirming the presence of the Kir current in ovine somatotropes. Moreover, this specific effect of high K+ on the Kir current was only observed in the cells that showed positive staining with anti‐growth hormone (GH) antibodies, or in GC cells that belong to a rat somatotrope cell line. Application of GHRP‐2 (100 nm) reversibly and significantly reduced the Kir current in bath solutions with 5 or 25 mm K+ in ovine somatotropes. In addition, we found that the reduction in the Kir current mediated by GHRP‐2 was totally abolished by the pretreatments with H89 (1 μm) or Rp‐cAMP (100 μm) or by intracellular dialysis of a specific protein kinase A (PKA) inhibitory peptide PKI (10 μm). The specific PKC blocker chelerythrine (1 μm) or inhibitory peptide PKC19‐36 (10 μm) did not show any effects on the GHRP‐2‐induced decrease in the Kir current. These results suggest that the inhibition of Kir current through PKA‐cAMP pathways may play an integral role in GHRP‐2‐induced depolarisation and GH release in ovine somatotropes.
Hyperpolarization and lysophosphatidylcholine induce inward currents and ethidium fluorescence in rabbit ventricular myocytes
Authors: Song Y.‐M. , Ochi R.
Published: November 2002

Strong electric pulses produce reversible or irreversible membrane breakdown (electroporation). We analysed the permeation properties of minute pores caused by hyperpolarization or lysophosphatidylcholine (LPC) by comparing the amount of charge carried by irregular inward currents (Ihi) with changes in ethidium bromide (EB) fluorescence in isolated rabbit ventricular myocytes. Forty‐second negative pulses from a holding potential of −20 mV induced Ihi whose conductance increased with hyperpolarization; the mean conductance (Ghi) was 63.6 ± 9.9 pS pF−1 (mean ±s.e.m., n= 9) at −160 mV. EB fluorescence increased during voltage pulses in parallel with the time integral of Ihi (Qhi), with the magnitude of the increases in nuclear EB fluorescence being 5.3 times greater than in the cytoplasm at −160 mV. Similar hyperpolarization‐induced parallel increases in Ihi and EB fluorescence were also obtained in Na+‐free, N‐methyl‐d‐glucamine (NMDG) solution. LPC (10 μm) induced large (101.2 ± 21.2 pS pF−1, n= 16), rapid (rise times, 1‐10 ms) Ihi with slow relaxation rates at −80 mV that reflected increases in Ghi to 94.3 ± 24.8 pS pF−1 (n= 8) at 6 min. Plots of EB fluorescence vs. Qhi were well fitted by a common Hill's equation with a Hill coefficient of 0.97. Taken together, our findings indicate that hyperpolarization and LPC produced pores having the same filter properties for the permeation of small ions, including ethidium+, and that Ihi (carried in part by Ca2+) generated by membrane breakdown are capable of supplying sufficient ions to evoke abnormal excitation and contraction in cardiac myocytes.
Single‐cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions
Authors: Florian P. , Schöneberg T. , Schulzke J. D. , Fromm M. , Gitter A. H.
Published: November 2002

Restitution of single‐cell defects, a frequent event in epithelia with high turnover, is poorly understood. Morphological and functional changes were recorded, using intravital time‐lapse video microscopy, confocal fluorescence microscopy, and conductance scanning techniques. After artificial single‐cell loss from an HT‐29/B6 colonic cell monolayer, the basal ends of adjacent cells extended. Concurrently, the local conductive leak associated with the defect sealed with an exponential time course (from 0.48 ± 0.05 μS 2 min post lesion to 0.17 ± 0.02 μS 8 min post lesion, n= 17). Between 3 and 10 min post lesion, a band of actin arose around the gap, which colocalized with a ring of ZO‐1 and occludin. Hence, tight junction proteins bound to the actin band facing the gap, and competent tight junctions assembled in the adjoining cell membranes. Closure and sealing were inhibited when actin polymerization was blocked by cytochalasin D, delayed following decrease of myosin‐ATPase activity by butanedione monoxime, and blocked after myosin light chain kinase inhibition by ML‐7. The Rho‐associated protein kinase inhibitor Y‐27632 did not affect restitution. After loosening of intercellular contacts in low Ca2+ Ringer solution, the time course of restitution was not significantly altered. Albeit epithelial conductivity was 12‐fold higher in low Ca2+ Ringer solution than in controls, under both conditions the repaired epithelium assumed the same conductivity as distant intact epithelium. In conclusion, epithelial restitution of single‐cell defects comprises rapid closure by an actinomyosin ‘purse‐string’ mechanism and simultaneous formation of a functional barrier from tight junction proteins also associated with the purse string.
Glucose‐dependent regulation of rhythmic action potential firing in pancreatic β‐cells by kATP‐channel modulation
Authors: Kanno T. , Rorsman P. , Göpel S. O.
Published: November 2002

The regulation of a K+ current activating during oscillatory electrical activity (IK,slow) in an insulin‐releasing β‐cell was studied by applying the perforated patch whole‐cell technique to intact mouse pancreatic islets. The resting whole‐cell conductance in the presence of 10 mm glucose amounted to 1.3 nS, which rose by 50 % during a series of 26 simulated action potentials. Application of the KATP‐channel blocker tolbutamide produced uninterrupted action potential firing and reduced IK,slow by ≈50 %. Increasing glucose from 15 to 30 mm, which likewise converted oscillatory electrical activity into continuous action potential firing, reduced IK,slow by ≈30 % whilst not affecting the resting conductance. Action potential firing may culminate in opening of KATP channels by activation of ATP‐dependent Ca2+ pumping as suggested by the observation that the sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor thapsigargin (4 μm) inhibited IK,slow by 25 % and abolished bursting electrical activity. We conclude that oscillatory glucose‐induced electrical activity in the β‐cell involves the opening of KATP‐channel activity and that these channels, in addition to constituting the glucose‐regulated K+ conductance, also play a role in the graded response to supra‐threshold glucose concentrations.
The role of the sarcoplasmic reticulum in neonatal uterine smooth muscle: enhanced role compared to adult rat
Authors: Noble Karen , Wray Susan
Published: November 2002

Little is known about contractile activity, response to agonists or excitation‐contraction coupling in neonatal smooth muscle. We have therefore investigated 10‐day rat uterus to better understand these processes, and compared it to adult uterus to elucidate how control of contractility develops. Spontaneous contractions are present in the 10‐day neonatal uterus, although they are not as large or as regular as those present in adult tissues. External Ca2+ entry via L‐type Ca2+ channels is the sole source of Ca2+ and is essential for the spontaneous activity. The neonatal uterus was responsive to carbachol or prostaglandin F2α application; it showed a marked stimulation and a clear dissociation between the force and Ca2+ changes. Such sensitization was not apparent in adult rat myometrium. The sarcoplasmic reticulum (SR) had more releasable Ca2+ and contributed more to the response to agonists in neonatal compared to adult tissues. Thus, Ca2+ entry as opposed to SR Ca2+ release contributed much less to the uterine response to agonists in the neonatal, compared to adult tissues. Inhibition of the SR by cyclopiazonic acid also caused a more vigorous increase in Ca2+ and contractile activity, particularly frequency, in the neonatal compared to the adult uterus. Taken together these data suggest that: (1) spontaneous activity is already present by day 10, (2) receptor‐coupling and excitation‐contraction signalling pathways are functional, (3) the SR and Ca2+ sensitization mechanisms play a more prominent role in the neonate, and (4) there is a shift to a greater reliance on Ca2+ entry and excitability with development of the myometrium.
cAMP‐independent responses of olfactory neurons in Xenopus laevis tadpoles and their projection onto olfactory bulb neurons
Authors: Manzini Ivan , Rössler Wolfgang , Schild Detlev
Published: November 2002

We report on responses of olfactory receptor neurons (ORNs) upon application of amino acids and forskolin using a novel slice preparation of the olfactory epithelium of Xenopus laevis tadpoles. Responses were measured using the patch‐clamp technique. Both amino acids and forskolin proved to be potent stimuli. Interestingly, a number of ORNs that responded to amino acids did not respond to forskolin. This suggests that some amino acids activate transduction pathways other than the well‐known cAMP‐mediated one. The differential processing of cAMP‐mediated stimuli on the one hand and amino acid stimuli on the other was further elucidated by calcium‐imaging of olfactory bulb neurons using a novel nose‐olfactory bulb preparation of Xenopus laevis tadpoles. The projection pattern of amino acid‐sensitive ORNs to olfactory bulb neurons differed markedly from the projection pattern of forskolin‐sensitive ORNs. Olfactory bulb neurons activated by amino acids were located laterally compared to those activated by forskolin, and only a small proportion responded to both stimuli. The ensemble of neurons activated by forskolin was also activated by the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX) and the membrane‐permeant cAMP analogue 8‐(4‐chlorophenylthio)adenosine 3′,5′‐cyclic monophosphate (pCPT‐cAMP). We therefore conclude that sensory transduction of a number of amino acids is cAMP independent, and amino acid‐ and cAMP‐mediated responses are processed differentially at the level of the olfactory bulb.