bluuebunny/crossref_metadata_2025 huggingface dataset:
Function of sural nerve reflexes during human walking
Authors: Zehr E. P. , Stein R. B. , Komiyama T.
Published: September 2004

The functions of ipsilateral cutaneous reflexes were studied with short trains of stimuli presented pseudorandomly to the sural nerve during human walking. Electromyograms (EMG) of lower (tibialis anterior (TA), soleus, lateral (LG) and medial (MG) gastrocnemius) and upper leg (vastus lateralis and biceps femoris) muscles were recorded, together with ankle, knee and hip joint angles. Net reflex EMG responses were quantified in each of the sixteen parts of the step cycle. The kinematic measurements included ankle eversion‐ inversion, and ankle, knee and hip flexion‐extension.

The function of the sural reflexes depended upon the part of the step cycle in which the nerve was stimulated and the intensity of stimulation. During stance, reflexes in MG and TA muscles in response to a medium intensity of stimulation (1.9 × radiating threshold, × RT) were closely associated with ankle eversion and dorsiflexion responses, respectively. These responses could assist in accommodation to uneven terrain that applies pressure to the lateral side of the foot (sural innervation area). Non‐noxious, high intensity (2.3 × RT) stimulation resulted in strong suppression of LG and MG during stance which was correlated to a small reduction in ankle plantarflexion. At this higher intensity the response would function to prevent the foot from moving more forcefully onto a potentially harmful obstacle.

During swing, ankle dorsiflexion increased and was significantly correlated to the net TA EMG response after both medium and high intensity stimulation. Knee flexion was increased throughout swing at both intensities of stimulation. These responses may serve in an avoidance response in which the swing limb is brought past an obstacle without destabilizing contact.

The net EMG and kinematic responses suggest that cutaneous reflexes stabilize human gait against external perturbations produced by an uneven surface in stance or obstacles encountered during swing.
Epiglottic movements during breathing in humans
Authors: Amis T. C. , O'Neill N. , Somma E. Di , Wheatley J. R.
Published: September 2004

Using X‐ray fluoroscopy we measured antero‐posterior (A–P) and cranio‐caudal (C–C) displacements of the epiglottic tip (ET), corniculate cartilage and hyoid bone in seven seated, normal human subjects (age 34 ± 3 years; mean ±s.e.m.; 4 males, 3 females) breathing via a nasal mask or mouthpiece with (RL) and without (UB) a fixed resistive load.

During UB, via either mouth or nose, there were no significant A‐P ET movements. During RL via the nose the ET at peak expiratory flow was 2.6 ± 1.3 mm cranial to its position at peak inspiratory flow (P < 0.05, ANOVA). C–C movements of the ET correlated strongly with C‐C movements of the corniculate cartilage and hyoid bone.

The ET, corniculate cartilage and hyoid bone (at zero airflow) were situated more caudally during oral UB than for any other condition.

When present, epiglottic movements during breathing do not appear to be independent of those of the larynx and hyoid. Furthermore, epiglottic position may be related to the level of upper airway resistance.
Movement detection at the human big toe
Authors: Refshauge Kathryn M. , Taylor Janet L. , McCloskey D. I. , Gianoutsos M. , Mathews P. , Fitzpatrick Richard C.
Published: September 2004

To be detected, movements of the interphalangeal joint of the big toe must be greater than at other joints. This poor acuity may arise because the anatomy of the foot and ankle results in poor coupling between the toe and the muscles that operate it. To vary this coupling, the effect of ankle position on proprioceptive acuity at the toe was measured.

We measured proprioceptive acuity at the toe with the ankle in different positions and found that ankle plantarflexion did improve acuity. This implies that, with the ankle at mid‐range or dorsiflexed, toe movement is inadequately transferred to muscle fascicles.

To determine actual changes in fascicle length of the toe extensor, movements of extensor hallucis longus near the toe and at the muscle‐tendon junction were measured during surgical exposure in one subject. Ankle position greatly affected movement transfer from toe to muscle‐tendon junction: no tendon movement was transferred with the ankle dorsiflexed, but all movement was transferred with the ankle plantarflexed.

When the relationship between joint rotation and muscle fascicle length measured in vivo was used to express the smallest detectable movements of the toe as proportional changes in muscle fascicle length, these detectable changes were similar to those at all other limb joints. This suggests that change in muscle fascicle length is of major interest to the nervous system.
Control of motility patterns in the human colonic circular muscle layer by pacemaker activity
Authors: Rae M. G. , Fleming N. , McGregor D. B. , Sanders K. M. , Keef K. D.
Published: September 2004

This study characterized the electrical and mechanical activities of human colonic muscle strips obtained from either the ascending, descending or sigmoid colon of patient volunteers during elective colon resections.

Rhythmic contractile activity was observed in colonic circular muscle strips in the absence of external stimuli. This activity persisted in the presence of atropine, phentolamine, propranolol, tetrodotoxin and Nω‐nitro‐L‐arginine but was abolished by nifedipine.

The activity of whole circular muscle (WCM) was compared with that of the myenteric half (MCM), the submucosal half (SCM) and the interior (ICM) of the circular muscle layer. WCM exhibited a prominent 2–4 contractions min−1 contractile pattern which was also present in strips of SCM. In contrast, MCM and ICM exhibited slow (0.3–0.6 contractions min−1), long duration contractions with superimposed higher frequency contractions (17–18 contractions min−1).

Resting membrane potential (Vm), recorded at various positions through the thickness of WCM strips did not differ and averaged −50 mV.

Slow waves were observed in 83 % of muscles. They averaged 12 mV in amplitude, 9.4 s in duration and had a frequency of 2–4 contractions min−1. Slow waves were greatest in amplitude near the submucosal edge and decreased with distance away from this edge. Each slow wave was associated with a transient contraction.

Near the myenteric edge, rapid fluctuations of Vm with a mean frequency of 18 contractions min−1 were recorded in 67 % of muscles. Spiking activity was common and was superimposed upon slow waves and rapid Vm fluctuations.

In summary, slow waves were identified in the human colonic circular muscle layer which arise at or near the submucosal edge. These electrical events give rise to a 2–4 contractions min−1 contractile rhythm which is characteristic of the intact muscle layer. Thus, the nature and spatial organization of pacemaker activity in the human colon bears significant resemblance to other animal models, such as the dog and pig.
Hypoxia and smooth muscle function: key regulatory events during metabolic stress
Authors: Taggart Michael J. , Wray Susan
Published: September 2004

Hypoxia rapidly reduces force in many smooth muscles and we review recent data that shed light on the mechanisms involved. As many regulated cellular processes are integrated to co‐ordinate smooth muscle contractility, the processes responsible for decreased force output with altered metabolism are also likely to be many, acting in concert, rather than the actions of one altered parameter. Nevertheless the aim of this study is to elucidate the hierarchical series of events that contribute to reduced smooth muscle force production during altered metabolism. We conclude that in many phasic smooth muscles the decrease in force can be attributed to impaired electro‐mechanical coupling whereby the Ca2+ transient is reduced. A direct effect of hypoxia on the Ca2+ channel may be of key importance. In tonic vascular smooth muscles KATP channels may also play a role in the integrated functional responses to hypoxia. There are also many examples of force being reduced, in tonically activated preparations, without a fall in steady‐state Ca2+; indeed it usually increases. We examine the roles of altered [ATP], pH, myosin phosphorylation, inorganic phosphate and proteolytic activity on the [Ca2+]‐force relationship during hypoxia. We find no defining force‐inhibitory role for any one factor acting alone, and suggest that force most probably falls as a result of the combination of myriad factors.
Vasopressin, angiotensin II and renal responses during water immersion in hydrated humans
Authors: Hammerum Mette Schou , Bie Peter , Pump Bettina , Johansen Lars Bo , Christensen Niels Juel , Norsk Peter
Published: September 2004

The hypothesis was tested that in hydrated humans the release of arginine vasopressin and angiotensin II is suppressed by water immersion (WI) and that this is a mechanism of the immersion‐induced diuresis and natriuresis. Seven male subjects on controlled sodium (65‐75 mmol per 24 h for 4 days) and water intake were studied.

Plasma vasopressin was promptly suppressed by WI, declining from 0·76 ± 0·13 to 0·23 ± 0·08 pg ml−1 (P < 0·05), with a concomitant increase in renal water output (CH2O) from ‐0·4 ± 0·2 to 4·4 ± 0·7 ml min−1 (P < 0·05). Subsequently, CH2O returned to the level of control, whereas plasma vasopressin remained suppressed. Plasma osmolality gradually increased from 285 ± 1 to 289 ± 1 mosmol kg−1 (P < 0·05). WI caused a 9‐fold increase in renal sodium excretion. Plasma angiotensin II decreased from 27·1 ± 5·3 to 4·3 ± 0·7 pg ml−1 (P < 0·05), and the intraindividual correlation coefficients between sodium excretion rates and angiotensin II concentrations varied between 0·73 and 0·96 (P < 0·002).

The data demonstrate that plasma vasopressin and angiotensin II concentrations decrease during WI in hydrated humans, concomitantly with initial increases in CH2O and sodium excretion. Therefore, vasopressin could constitute a mediator of CH2O and angiotensin II of the natriuresis of WI. The subsequent return of CH2O to the level of control is, however, also caused by other factors.
Role of receptor kinase in short‐term desensitization of cardiac muscarinic K+ channels expressed in Chinese hamster ovary cells
Authors: Shui Z. , Khan I. A. , Tsuga H. , Haga T. , Boyett M. R.
Published: September 2004

The cardiac muscarinic receptor‐K+ channel system was reconstructed in Chinese hamster ovary (CHO) cells by transfecting the cells with the various components of the system. The activity of the muscarinic K+ channel was measured with the cell‐attached configuration of the patch clamp technique.

In CHO cells transfected with the channel (Kir3.1/Kir3.4), receptor (hm2) and receptor kinase (GRK2), on exposure to agonist, there was a decline in channel activity as a result of desensitization, similar to that in atrial cells.

Whereas the desensitization was almost abolished by not transfecting with the receptor kinase or by transfecting with a mutant receptor lacking phosphorylation sites, it was only reduced (by ≈39 %) by transfecting with a mutant receptor kinase with little kinase activity.

These results suggest that the receptor kinase is responsible for desensitization of the muscarinic K+ channel and that this involves phosphorylation‐dependent and ‐independent mechanisms.
Pore mutations alter closing and opening kinetics in Shaker K+ channels
Authors: Molina A. , Ortega‐Sáenz P. , López‐Barneo J.
Published: September 2004

We have studied the effects of mutations of amino acids in the pore (positions 447 and 449) and the elevation of extracellular [K+] on the closing and opening kinetics of Shaker B K+ channels transiently expressed in Chinese hamster ovary (CHO) cells.

Mutant D447E had closing and C‐type inactivation kinetics which were faster than the wild‐type channel. These processes were slowed by increasing extracellular [K+] and in these conditions the channels exhibited linear instantaneous current‐voltage relationships. Thus, the mutation seems to produce uniform decrease of occupancy by K+ in sites along the channel pore where the cation competes with closing and C‐type inactivation.

In other mutants also showing K+‐dependent fast C‐type inactivation, closing was found to be slower than in the wild‐type channel and insensitive to variations in external [K+]. These characteristics were particularly apparent in mutant T449K which even in high [K+] has a non‐linear instantaneous current‐voltage relationship with marked saturation of the inward current recorded at negative membrane potentials. Hence, in this channel type occupation by K+ of the pore appears to be non‐uniform with low occupancy of sites near the outer entrance and saturation of the sites accessible from the internal solution.

The results show that channel closing is influenced by changes in the pore structure leading to alterations in the occupation of the channels by permeant cations. The differential effects of pore mutations and high external [K+] on closing and C‐type inactivation indicate that the respective gates are associated with separate domains of the molecule.

Point mutations in the pore sequence can also lead to modifications in channel opening. In general, channels with fast C‐type inactivation also show a fast rising phase of activation. However, these effects appear not to be due to primary modifications of the activation process but to arise from the coupling of activation and C‐type inactivation.

These data, demonstrating that the pore structure influences most of the gating parameters of K+ channels, give further insight into the mechanisms underlying the modulation of K+ channel function by changes in the ionic composition in the extracellular milieu.
Learning to cope with repeated stress
Authors: Leng Gareth , Russell John A.
Published: September 2004

stress (stres). 1. Any potentially damaging strain, force, or agent, which stimulates a physiological defence reaction and is capable under certain circumstances of producing pathological lesions. Adaptation to the various kinds of stress to which man is exposed is a condition of his survival …. (British Medical Dictionary, 1961)
The sulphonylurea receptor SUR1 regulates ATP‐sensitive mouse Kir6.2 K+ channels linked to the green fluorescent protein in human embryonic kidney cells (HEK 293)
Authors: John Scott A. , Monck Jonathan R. , Weiss James N. , Ribalet Bernard
Published: September 2004

Using a chimeric protein comprising the green fluorescent protein (GFP) linked to the C‐terminus of the K+ channel protein mouse Kir6.2 (Kir6.2‐C‐GFP), the interactions between the sulphonylurea receptor SUR1 and Kir6.2 were investigated in transfected human embryonic kidney cells (HEK 293) by combined imaging and patch clamp techniques.

HEK 293 cells transfected with mouse Kir6.2‐C‐GFP and wild‐type Kir6.2 exhibited functional K+ channels independently of SUR1. These channels were inhibited by ATP (IC50= 150 μM), but were not responsive to stimulation by ADP or inhibition by sulphonylureas. Typically, 15 ± 7 active channels were found in an excised patch.

The distribution of Kir6.2‐C‐GFP protein was investigated by imaging of GFP fluorescence. There was a lamellar pattern of fluorescence labelling inside the cytoplasm (presumably associated with the endoplasmic reticulum and the Golgi apparatus) and intense punctate labelling near the cell membrane, but little fluorescence was associated with the plasma membrane.

In contrast, cells co‐transfected with Kir6.2‐C‐GFP and SUR1 exhibited intense uniform plasma membrane labelling, and the lamellar and punctate labelling seen without SUR1 was no longer prominent.

In cells co‐transfected with Kir6.2‐C‐GFP and SUR1, strong membrane labelling was associated with very high channel activity, with 484 ± 311 active channels per excised patch. These K+ channels were sensitive to inhibition by ATP (IC50= 17 μM), stimulated by ADP and inhibited by sulphonylureas.

We conclude that co‐expression of SUR1 and Kir6.2 generates channels with the properties of native KATP channels. In addition, SUR1 promotes uniform insertion of Kir6.2‐C‐GFP into the plasma membrane and a 35‐fold increase in channel activity, suggesting that SUR1 facilitates protein trafficking of Kir6.2 into the plasma membrane.
KATP channel formation by the sulphonylurea receptors SUR1 with Kir6.2 subunits in rat dorsal vagal neurons in situ
Authors: Karschin Andreas , Brockhaus Johannes , Ballanyi Klaus
Published: September 2004

Functional and molecular properties of ATP‐sensitive K+ (KATP) channels were studied in dorsal vagal neurons (DVNs) of rat brainstem slices using patch‐clamp and single‐cell antisense RNA amplification‐polymerase chain reaction (PCR) techniques.

In the cell‐attached configuration, 1 mM cyanide resulted in block of spontaneous firing and concomitant opening of single channels with a mean single open time of 2‐3 ms and a burst duration of up to several hundred milliseconds. Inhibition of such single‐channel activity with 200 μM tolbutamide led to the reappearance of spontaneous discharge.

Whole‐cell recordings during anoxia revealed a hyperpolarization of the DVNs. Harvesting of cytoplasm, antisense RNA amplification and subsequent PCR showed coexpression for single DVNs of mRNA for the sulphonylurea receptor SUR1 isoform and for the inwardly rectifying K+ channel subunit Kir6.2, but not for the SUR2 or Kir6.1 isoforms of these channel/receptor subclasses.

Upon anoxia, a stable depolarization by less than 10 mV was observed in non‐excitable cells in the dorsal vagal nucleus. These cells, which expressed glial fibrillary acidic protein (GFAP), showed a high level of mRNA for Kir6.2, a weak signal for SUR1, whereas SUR2 or Kir6.1 were not detected.

The results suggest that functional KATP channels in DVNs are constituted by the formation of Kir6.2 subunits with SUR1 receptors.
The effect of overexpression of auxiliary Ca2+ channel subunits on native Ca2+ channel currents in undifferentiated mammalian NG108–15 cells
Authors: Wyatt Christopher N. , Page Karen M. , Berrow Nicholas S. , Brice Nicola L. , Dolphin Annette C.
Published: September 2004

High voltage activated (HVA) Ca2+ channels are composed of a pore‐forming α1 subunit and the accessory β and α2‐δ subunits. However, the subunit composition of low voltage activated (LVA), or T‐type, Ca2+ channels has yet to be elucidated. We have examined whether native calcium channels in NG108–15 mouse neuroblastoma × rat glioma hybrid cells, which express predominantly LVA currents when undifferentiated, are modulated by overexpression of accessory calcium channel subunits.

Endogenous α1A, B, C, D and E, and low levels of β and α2‐δ subunit protein were demonstrated in undifferentiated NG108–15 cells.

The α2‐δ, β2a or β1b accessory subunits were overexpressed by transfection of the cDNAs into these cells, and the effect examined on the endogenous Ca2+ channel currents. Heterologous expression, particularly of α2‐δ but also of β2a subunits clearly affected the profile of these currents. Both subunits induced a sustained component in the currents evoked by depolarizing voltages above −30 mV, and α2‐δ additionally caused a depolarization in the voltage dependence of current activation, suggesting that it also affected the native T‐type currents. In contrast, β1b overexpression had no effect on the endogenous Ca2+ currents, despite immunocytochemical evidence for its expression in the transfected cells.

These results suggest that in NG108–15 cells, overexpression of the Ca2+ channel accessory subunits α2‐δ and β2a induce a sustained component of HVA current, and α2‐δ also influences the voltage dependence of activation of the LVA current. It is possible that native T‐type α1 subunits are not associated with β subunits.
Chronic hypoxia reduces adenosine A2A receptor‐mediated inhibition of calcium current in rat PC12 cells via downregulation of protein kinase A
Authors: Kobayashi Shuichi , Beitner‐Johnson Dana , Conforti Laura , Millhorn David E.
Published: September 2004

Adenosine has been shown to decrease Ca2+ current (ICa) and attenuate the hypoxia‐induced enhancement of intracellular free Ca2+ ([Ca2+]i) in oxygen‐sensitive rat phaeochromocytoma (PC12) cells. These effects are mediated via the adenosine A2A receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia‐induced change in [Ca2+]i during chronic hypoxia.

Whole cell patch‐clamp studies revealed that the effect of adenosine on ICa was significantly reduced when PC12 cells were exposed to hypoxia (10 % O2) for 24 and 48 h.

Ca2+ imaging studies using fura‐2 revealed that the anoxia‐induced increase in [Ca2+]i was significantly enhanced when PC12 cells were exposed to 10 % O2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia‐induced elevation of [Ca2+]i was significantly blunted in PC12 cells exposed to hypoxia for 48 h.

Northern blot analysis revealed that mRNA for the A2A receptor, which is the only adenosine receptor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [3H]CGS21680, a selective A2A receptor ligand, showed that the number of adenosine A2A receptor binding sites was similarly increased during exposure to 10 % O2 for 48 h.

PKA enzyme activity was significantly inhibited when PC12 cells were exposed to 10 % O2 for 24 and 48 h. However, we found that hypoxia failed to induce change in adenosine‐ and forskolin‐stimulated adenylate cyclase enzyme activity. Chronic hypoxia also did not alter the immunoreactivity level of the G protein Gsα, an effector of the A2 signalling pathway.

Whole cell patch‐clamp analysis showed that the effect of 8‐bromo‐cAMP, an activator of PKA, on ICa was significantly attenuated during 48 h exposure to 10 % O2.

We conclude therefore that the reduced effect of adenosine on ICa and [Ca2+]i in PC12 cells exposed to chronic hypoxia is due to hypoxia‐induced downregulation of PKA. This mechanism may serve to reduce the negative feedback on ICa and [Ca2+]i by adenosine and therefore maintain enhanced membrane excitability of PC12 cells during long‐term hypoxia.
Synaptic interactions between smooth and spiny neurones in layer 4 of cat visual cortex in vitro
Authors: Tarczy‐Hornoch K. , Martin K. A. C. , Jack J. J. B. , Stratford K. J.
Published: September 2004

Dual intracellular recording was used to examine the interactions between neighbouring spiny (excitatory) and smooth (inhibitory) neurones in layer 4 of cat visual cortex in vitro. Synaptic connections were found in seventeen excitatory‐inhibitory neurone pairs, along with one inhibitory‐inhibitory connection.

Fast excitatory inputs onto smooth neurones (basket cells) from spiny cells (spiny stellate or pyramidal cells) (n= 6) produce large excitatory postsynaptic potentials (EPSPs) of up to 4 mV mean amplitude, whereas basket cells evoke slower inhibitory postsynaptic potentials (IPSPs) in their postsynaptic targets (n= 17), of smaller amplitude (up to 1.6 mV at membrane potentials of ‐60 mV).

Both types of PSP appear to be multiquantal, and both may exhibit depression of up to 60 % during short trains of presynaptic spikes. This depression can involve presynaptic and/or postsynaptic factors.

One‐third (n= 5) of the spiny cell‐smooth cell pairs tested were reciprocally connected, and in the one pair for which the suprathreshold interactions were comprehensively investigated, the pattern of basket cell firing was strongly influenced by the activity in the connected excitatory neurone. The basket cell was only effective in inhibiting spiny cell firing when the excitatory neurone was weakly driven.
Involvement of tyrosine kinases, Ca2+ and PKC in activation of mitogen‐activated protein (MAP) kinase in human polymorphonuclear neutrophils
Authors: Zhang Hong , Garlichs Christoph D. , Mügge Andreas , Daniel Werner G.
Published: September 2004

Activation of mitogen‐activated protein (MAP) kinase is an early response to a wide variety of stimuli and plays an important role in the regulation of cellular functions. In the present study we investigated the activation of MAP kinase in human polymorphonuclear neutrophils (PMNs).

Activity of MAP kinase and protein kinase C (PKC) was measured radiometrically from the rate of phosphorylation of specific peptide substrates. Protein phosphorylation was measured by immunoprecipitation and Western blot analysis.

N‐Formyl‐Met‐Leu‐Phe (fMLP), phorbol 12‐myristate, 13‐acetate (PMA) and the Ca2+‐ATPase inhibitors thapsigargin (Tg) and cyclopiazonic acid (CPA) increased MAP kinase activity significantly. The tyrosine kinase inhibitors erbstatin and herbimycin A partially inhibited the effects of fMLP and PMA, and completely abolished the effects of both Tg and CPA. The specific PKC inhibitor calphostin C suppressed activation of MAP kinase produced by fMLP and PMA, but had no effect on that produced by Tg and CPA. Tg and CPA were without effect on PKC activity.

Immunoprecipitation and Western blot analysis indicated that the 42 and 44 kDa tyrosine‐phosphorylated proteins found after stimulation of PMNs were both members of the MAP kinase family. Pretreatment of PMNs with staurosporine, EGTA or erbstatin significantly reduced the tyrosine phosphorylation of MAP kinase(s).

These results suggest that in human PMNs, MAP kinase can be stimulated in both a PKC‐dependent and a PKC‐independent manner. The Ca2+ signal leads to activation of tyrosine kinases, which contribute to the activation of MAP kinase. However, a PMA‐sensitive Ca2+‐independent pathway also exists. Mobilization of Ca2+ and activation of PKC synergistically induce maximal MAP kinase activation and tyrosine phosphorylation.
Facilitation of currents through rat Ca2+‐permeable AMPA receptor channels by activity‐dependent relief from polyamine block
Authors: Rozov Andrei , Zilberter Yuri , Wollmuth Lonnie P. , Burnashev Nail
Published: September 2004

In outside‐out patches excised from human embryonic kidney (HEK) 293 cells expressing Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate receptor (AMPAR) channels, currents activated by 1 ms glutamate pulses at negative membrane potentials facilitated during and following a repetitive (2 to 100 Hz) agonist application. The degree of facilitation depended on subunit type, membrane potential and stimulation frequency being antagonized by a slow recovery from desensitization.

Activity‐dependent current facilitation occurred in Ca2+‐permeable but not in Ca2+‐impermeable AMPAR channels. Current facilitation, however, does not depend on Ca2+ flux. Rather it reflects a relief from the block of Ca2+‐permeable AMPARs by intracellular polyamines since facilitation occurred only in the presence of polyamines and since facilitated currents had a nearly linear current‐voltage relation (I‐V).

Relief from polyamine block was use dependent and occurred mainly in open channels. The relief mechanism was determined primarily by membrane potential rather than by current flow.

In closed channels the degree of polyamine block was independent of membrane potential. The voltage dependence of the rate of relief from the block in open channels rather than the voltage dependence of the block underlies the inwardly rectifying shape of the I‐V at negative potentials.

Currents through native Ca2+‐permeable AMPAR channels in outside‐out or nucleated patches from either hippocampal basket cells or a subtype of neocortical layer II nonpyramidal cells also showed facilitation.

It is concluded that a use‐dependent relief from polyamine block during consecutive AMPAR channel openings underlies current facilitation. This polyamine‐AMPAR interaction may represent a new activity‐dependent postsynaptic mechanism for control of synaptic signalling.
Ca2+ influx through voltage‐gated Ca2+ channels regulates 5‐HT3 receptor channel desensitization in rat glioma × mouse neuroblastoma hybrid NG108–15 cells
Authors: Jones Susan , Yakel Jerrel L.
Published: September 2004

The kinetics of desensitization of the 5‐HT3 receptor (5‐HT3R)‐gated ion channel were investigated using whole‐cell and perforated‐patch recording techniques in NG108–15 cells.

Rapid application of 5‐HT (50 μM) elicited a 5‐HT3R‐mediated inward current response that desensitized completely in the continued presence of agonist. In the whole‐cell recording configuration (holding potential of −70 mV) while buffering internal calcium (Ca2+i) with 5 mM EGTA (0.5 mM added Ca2+; with an estimated free [Ca2+] of 30 nM), the rate of desensitization was initially rapid (with a half‐time of ≈230 ms), but dramatically slowed with time by 1120 ± 160 %.

This slowing in the rate of desensitization was reduced by stronger Ca2+ buffering (20 mM BAPTA, without added Ca2+), or by the bath application of cadmium (100 μM) to block voltage‐gated Ca2+ channels. The rate of desensitization was also dependent on membrane potential.

In perforated‐patch recordings, the rate of desensitization remained constant. However, a slowing in the desensitization rate could be induced by depolarizing cells immediately prior to the application of 5‐HT.

The depolarization‐induced slowing was blocked by incubating cells with BAPTA‐AM (a membrane‐permeant analogue of BAPTA) or by the bath application of cadmium.

These data suggest that Ca2+ influx through a cadmium‐sensitive voltage‐gated Ca2+ channel increases the cytoplasmic Ca2+ concentration ([Ca2+]i) and induces a dramatic slowing in the kinetics of desensitization of the 5‐HT3R channel. These data provide evidence for cross‐talk between voltage‐gated Ca2+ channels and 5‐HT3Rs in NG108–15 cells.
Contrasting Ca2+ channel subtypes at cell bodies and synaptic terminals of rat anterioventral cochlear bushy neurones
Authors: Doughty Joanne M. , Barnes‐Davies Margaret , Rusznák Zoltan , Harasztosi Csaba , Forsythe Ian D.
Published: September 2004

Whole‐cell patch clamp recordings were made from bushy cells of the anterioventral cochlear nucleus (aVCN) and their synaptic terminals (calyx of Held) in the medial nucleus of the trapezoid body (MNTB).

Both high voltage‐activated (HVA) and low voltage‐activated (LVA) calcium currents were present in acutely dissociated aVCN neurones and in identified bushy neurones from a cochlear nucleus slice.

The transient LVA calcium current activated rapidly on depolarization (half‐activation, −59 mV) and inactivated during maintained depolarization (half‐inactivation, −89 mV). This T‐type current was observed in somatic recordings but was absent from presynaptic terminals.

On the basis of their pharmacological sensitivity, P/Q‐type Ca2+ channels accounted for only 6 % of the somatic HVA, while L‐, N‐ and R‐type Ca2+ channels each accounted for around one‐third of the somatic calcium current.

The divalent permeabilities of these native calcium channels were compared. The Ba2+/Ca2+ conductance ratios of the somatic HVA and LVA channels were 1.4 and 0.7, respectively. The conductance ratio of the presynaptic HVA current was 0.9, significantly lower that that of the somatic HVA current.

We conclude that LVA currents are expressed in the bushy cell body, but are not localized to the excitatory synaptic terminal. All of the HVA current subtypes are expressed in bushy cells, but there is a strong polarity to their localization; P‐type contribute little to somatic currents but predominate at the synaptic terminal; L‐, N‐ and R‐types dominate at the soma, but contribute negligibly to calcium currents in the terminal.
Near‐visible ultraviolet light induces a novel ubiquitous calcium‐permeable cation current in mammalian cell lines
Authors: Mendez Francisco , Penner Reinhold
Published: September 2004

We studied the immediate and short‐term effects of UV light in the near‐visible range at the cellular and membrane level using the whole‐cell patch‐clamp technique in combination with digital fluorescence imaging.

Illumination with monochromatic UVA light (340‐380 nm) induced a sustained non‐saturable increase in membrane conductance dependent on wavelength and light intensity in several different mammalian cell types including RBL, mast, HEK, PC12 and 3T3 cells.

The current was non‐selective for cations and permeable to Ca2+, but was inhibited by trivalent cations and was not due to the activation of an endogenous ion channel. We termed this novel current ILiNC for light‐induced non‐selective cation current.

A similar current was evoked by chemical peroxidants such as hydrogen peroxide and tert‐butylhydroperoxide, but not by cytosolic oxidized glutathione.

The free‐radical scavengers tocopherol (vitamin E) and ascorbic acid (vitamin C) significantly reduced the UV light effect.

The generation of the current was membrane delimited since it could be induced by the same UVA treatment in cell‐free membrane patches showing a similar wavelength dependence.

These results suggest that ILiNC is activated by UVA light‐induced generation of free radicals acting through lipid or protein peroxidation, and may represent a ubiquitous mechanism by which Na+ and Ca2+ can enter cells after phototoxic or free radical‐induced membrane damage.
Calcineurin involvement in the regulation of high‐threshold Ca2+ channels in NG108–15 (rodent neuroblastoma × glioma hybrid) cells
Authors: Lukyanetz E. A. , Piper T. P. , Sihra T. S.
Published: September 2004

We examined the relationship between calcineurin (protein phosphatase 2B (PP2B)) and voltage‐operated Ca2+ channels (VOCCs) in NG108–15 cells. PP2B expression in NG108–15 cells was altered by transfection with plasmid constructs containing a full length cDNA of human PP2Bβ3 in sense (CN‐15) and antisense (CN‐21) orientation.

Confocal immunocytochemical localization showed that in wild‐type cells, PP2B immunoreactivity is uniformly distributed in undifferentiated cells and located at the inner surface of soma membrane and neurites in differentiated cells.

To test the Ca2+ dependence of the VOCC, we used high‐frequency stimulation (HFS). The L‐ and N‐type VOCCs decreased by 37 and 52 %, respectively, whereas the T‐type current was only marginally sensitive to this procedure. FK‐506 (2 μM), a specific blocker of PP2B, reduced the inhibition of L‐ and N‐type VOCCs induced by HFS by 30 and 33 %, respectively.

In CN‐15‐transfected cells overexpressing PP2B, total high‐voltage‐activated (HVA) VOCCs were suppressed by about 60 % at a test potential of +20 mV. Intracellular addition of EGTA or FK‐506 into CN‐15‐transfected cells induced an up to 5‐fold increase of HVA VOCCs.

These findings indicate that PP2B activity does not influence the expression of HVA Ca2+ channels, but modulates their function by Ca2+‐dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylated state under control conditions, are downregulated by PP2B upon stimulation, with the major effect being on N‐type VOCCs.
Inhibition of N‐ and P/Q‐type calcium channels by postsynaptic GABAB receptor activation in rat supraoptic neurones
Authors: Harayama Nobuya , Shibuya Izumi , Tanaka Keiko , Kabashima Narutoshi , Ueta Yoichi , Yamashita Hiroshi
Published: September 2004

Voltage‐dependent Ca2+ currents of dissociated rat supraoptic nucleus (SON) neurones were measured using the whole‐cell configuration of the patch‐clamp technique to examine direct postsynaptic effects of GABAB receptor activation on SON magnocellular neurones.

The selective GABAB agonist baclofen reversibly inhibited voltage‐dependent Ca2+ currents elicited by voltage steps from a holding potential of −80 mV to depolarized potentials in a dose‐dependent manner. The ED50 of baclofen for inhibiting Ca2+ currents was 1.4 × 10−6 M. Baclofen did not inhibit low threshold Ca2+ currents elicited by voltage steps from −120 to −40 mV.

Inhibition of high threshold Ca2+ currents by baclofen was rapidly and completely reversed by the selective GABAB antagonists, CGP 35348 and CGP 55845A, when the antagonists were added at the molar ratio vs. baclofen of 10 : 1 and 0.01 : 1, respectively. It was also reversed by a prepulse to +150 mV lasting for 100 ms.

The inhibition of Ca2+ currents was abolished when the cells were pretreated with pertussis toxin for longer than 20 h or with N‐ethylmaleimide for 2 min. It was also abolished when GDPβS was included in the patch pipette. When GTPγS was included in the patch pipette, baclofen produced irreversible inhibition of Ca2+ currents and this inhibition was again reversed by the prepulse procedure.

The inhibition of N‐, P/Q‐, L‐ and R‐type Ca2+ channels by baclofen (10−5 M) was 24.1, 10.5, 3.1 and 3.6 %, respectively, of the total Ca2+ currents. Only the inhibition of N‐ and P/Q‐types was significant.

These results suggest that GABAB receptors exist in the postsynaptic sites of the SON magnocellular neurones and mediate selective inhibitory actions on voltage‐dependent Ca2+ channels of N‐ and P/Q‐types via pertussis toxin‐sensitive G proteins, and that such inhibitory mechanisms may play a role in the regulation of SON neurones by the GABA neurones.
α‐Adrenergic stimulation of cytosolic Ca2+ oscillations and exocytosis in identified rat corticotrophs
Authors: Tse Amy , Tse Frederick W.
Published: September 2004

The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo‐1, or indo‐1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i), ionic current and changes in membrane capacitance in single rat corticotrophs identified with the reverse haemolytic plaque assay.

Application of the adrenocorticotropin (ACTH) secretagogue noradrenaline (NA; norepinephrine), triggered [Ca2+]i oscillation in corticotrophs via α‐adrenergic receptors and the guanosine trisphosphate (GTP) binding protein‐coupled phosphoinositide pathway.

Simultaneous measurement of [Ca2+]i and capacitance shows that exocytosis was triggered during the first cycle of NA‐induced [Ca2+]i oscillation and the mean increase in cell membrane surface area was 1.4 ± 0.3 % (n= 6).

When Ca2+ was directly released from the inositol 1,4,5 trisphosphate (IP3)‐sensitive store via flash photolysis of caged IP3, the mean increase in cell surface area was 1.5 ± 0.5 % (n= 6). Thus, NA‐stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release.

Large and rapid elevation of [Ca2+]i (>15 μm) via flash photolysis of caged Ca2+ triggered two phases of exocytosis: a rapid exocytic burst that was complete in ∼100 ms and a slow burst that continued for many seconds.

The rapid exocytic burst reflected the exhaustion of a pool of readily releasable granules and, on average, increased the cell surface by 2.8 ± 0.1 % (n= 14).

We suggest that the relatively weak exocytic response in corticotrophs during intracellular Ca2+ release may be partially attributed to a smaller pool of readily releasable granules.
Multiple modulatory effects of dopamine on calcium channel kinetics in adult rat sensory neurons
Authors: Formenti Alessandro , Martina Marzia , Plebani Alessio , Mancia Mauro
Published: September 2004

The aim of this research was to study the modulatory effects induced on high‐voltage‐activated (HVA) calcium channels and pharmacologically isolated subtypes through dopamine receptor activation.

The experiments were carried out on acutely isolated adult rat sensory neurons, recorded by means of the whole‐cell patch‐clamp technique.

At saturating concentrations dopamine was effective in inducing: (a) a voltage‐dependent prolongation of activation kinetics, (b) a voltage‐independent scaling down of the currents without any changes in activation and inactivation kinetics, and (c) an acceleration of inactivation kinetics, not affected by a positive conditioning prepulse.

These three inhibitory effects were observed on N‐ and P/Q‐type currents, whereas only a voltage‐independent scaling up and/or scaling down was observed on L‐type current.

The inhibitory effects were sometimes observed in isolation in different neurons, but more frequently they were variously combined in the same cell. A correlation analysis of these effects shows no relationship between them, corroborating the conclusion that they are mechanistically distinct.

The existence of an inactivating effect accounts for the occurrence of a voltage‐dependent inhibitory effect in some cells without an apparent slowing down of activation kinetics, since the increased inactivation may mask the slow component of the activation.

The multiple modulatory effects on calcium channels, even on pharmacologically separated N‐, L‐ and P/Q‐currents, suggest that pharmacological and functional classifications do not necessarily match completely.

The multiple modulatory effects on HVA calcium currents may play a prominent role both in controlling the integrative properties of neurons and in regulating output at a presynaptic level.
The effect of extracellularly applied divalent cations on cytosolic Ca2+ in murine Leydig cells: evidence for a Ca2+‐sensing receptor
Authors: Adebanjo Olugbenga A. , Igietseme Joseph , Huang Christopher L.‐H. , Zaidi Mone
Published: September 2004

The effect of extracellularly applied divalent cations upon cytosolic Ca2+ levels ([Ca2+]) was investigated in fura‐2‐loaded mouse Leydig (TM3) cells.

The extracellular application of Ca2+ (2.5–15 mM) or Ni2+ (0.5–5 mM) elicited concentration‐dependent elevations in cytosolic [Ca2+] that were followed by decays to baseline levels. Extracellular Mg2+ (0.8–15 mM) failed to influence cytosolic [Ca2+].

Conditioning applications of Ca2+ (2.5–10 mM), Mg2+ (2.5–15 mM) or Ni2+ (0.5–5 mM) all attenuated the cytosolic Ca2+ response to a subsequent test application of 5 mM [Ni2+].

The amplitude of Ni2+‐induced cytosolic Ca2+ signals remained constant in low‐Ca2+ solutions. Such findings suggest a participation of Ca2+ release from intracellular stores. In parallel, depletion of Ca2+ stores by either ionomycin (5 μM, in low‐Ca2+ solutions) or thapsigargin (4 μM) abolished or attenuated Ni2+‐induced Ca2+ transients.

Ionomycin (5 μM) elevated cytosolic [Ca2+] in Ca2+‐free solutions even after prior Ni2+ application, indicating the presence of Ni2+‐insensitive stores.

Caffeine (250 and 500 μM) elevated cytosolic [Ca2+] and attenuated Ni2+‐induced Ca2+ release. Furthermore, TM3 cells stained intensely with a specific anti‐ryanodine receptor antiserum, Ab34. These findings suggest that Ca2+ release is regulated by ryanodine receptors.

Both membrane depolarization and hyperpolarization, brought about by changes in extracellular [K+] ([K+]e) in the presence of valinomycin (5 μM), altered the waveform of the Ni2+‐induced cytosolic Ca2+ signal. Hyperpolarization, in addition, diminished the response magnitude. Such voltage‐induced response modulation localizes the regulatory events to the Leydig cell plasma membrane.

We propose the existence of a cell surface divalent cation (Ca2+) receptor in Leydig cells, the activation of which triggers Ca2+ fluxes through ryanodine receptors.
The effect of polyamines on KATP channels in guinea‐pig ventricular myocytes
Authors: Niu X. W. , Meech R. W.
Published: September 2004

The effect of natural polyamines on KATP channels was studied using inside‐out patches from guinea‐pig ventricular myocytes.

At a holding potential of +40 mV, spermine at the intracellular membrane surface reduced the KATP channel open probability (Popen) in a dose‐dependent manner. Half‐maximal inhibition occurred at 29 μM with a Hill coefficient of 1.2.

The effect of spermine on Popen was not greatly influenced by the membrane potential but there appeared to be a small reduction in unitary current amplitude during strong depolarizations.

Analysis of KATP single channel kinetics showed that spermine inhibited the channel by decreasing the mean open time and introducing transitions to a long closed state.

Spermidine (0.1 mM) was found to have a similar effect to spermine. Putrescine (10 mM) was found to block more effectively at positive membrane potentials. Up to 20 mM arginine had no significant effect on KATP channels.

Our results indicate that natural polyamines influence native KATP channel gating in cardiac myocytes.
Presynaptic calcium channels mediating synaptic transmission in submucosal neurones of the guinea‐pig caecum
Authors: Cunningham Shauna M. C. , Mihara Satoshi , Higashi Hideho
Published: September 2004

Intracellular recording techniques were used to examine the voltage‐activated calcium channels mediating neurotransmitter release from nerve terminals of extrinsic, sympathetic origin and intrinsic (enteric) origin innervating submucosal neurones of the guinea‐pig caecum.

The noradrenergic slow inhibitory postsynaptic potential (IPSP) was abolished by superfusion of ω‐conotoxin (ω‐CTX) GVIA (3‐300 nM), with an apparent IC50 of 8.6 nM. Superfusion of ω‐CTX MVIIC (500 nM) also suppressed the amplitude of slow IPSPs, but both ω‐agatoxin IVA (100 nM) and nicardipine (1‐10 μM) were ineffective. The hyperpolarization induced by exogenous noradrenaline was not affected by ω‐CTX GVIA (100 nM).

In contrast to the slow IPSP, the amplitude of the cholinergic fast excitatory postsynaptic potential (EPSP) was partially inhibited, but not abolished, by ω‐CTX GVIA (0.1‐1 μM). Furthermore, ω‐agatoxin IVA (0.1‐1 μM) or ω‐CTX MVIIC (0.1‐1 μM) also affected the fast EPSP, but nicardipine (1–10 μM) was ineffective. In combination, ω‐CTX GVIA (100 nM) and ω‐agatoxin IVA (100 nM) inhibited the fast EPSP by 74 ± 6 %; the residual fast EPSP was not affected by ω‐CTX MVIIC (100 nM). The fast EPSP was completely abolished by low Ca2+, high Mg2+ Krebs solution or Krebs solution containing Co2+ (2 mM) and Cd2+ (400 μM). The depolarization induced by exogenous acetylcholine was not affected by either ω‐CTX GVIA (100 nM), ω‐agatoxin IVA (100 nM) or ω‐CTX MVIIC (100 nM).

Taken together, these results suggest that, in the submucosal plexus of the guinea‐pig caecum, release of noradrenaline from extrinsic nerve terminals is regulated by N‐type calcium channels, whereas release of acetylcholine from intrinsic nerve terminals involves several types of calcium channel.
Gap junctional communication and pharmacological heterogeneity in astrocytes cultured from the rat striatum
Authors: Venance Laurent , Prémont Joël , Glowinski Jacques , Giaume Christian
Published: September 2004

Indo‐1 and fluo‐3 imaging techniques were used to investigate the role of gap junctions in the changes in cytosolic calcium concentrations ([Ca2+]i) induced by several receptor agonists. Subpopulations of confluent cultured astrocytes from the rat striatum were superfused with submaximal concentrations of endothelin‐1 (Et1) and the α1‐adrenergic and muscarinic receptor agonists, methoxamine and carbachol, respectively.

Combined binding and autoradiographic studies indicated that all striatal astrocytes possess binding sites for Et1. In contrast, α1‐adrenergic and muscarinic binding sites were found to be heterogeneously distributed. In agreement with these findings, Et1 induced fast calcium responses in all cells while only subsets of striatal astrocytes responded to the application of either methoxamine or carbachol.

Halothane, heptanol and octanol, which are commonly used as gap junction inhibitors, drastically reduced the amplitude of Et1‐induced calcium responses. In contrast, 18‐α‐glycyrrhetinic acid (αGA) used at a concentration known to block gap junction permeability in astrocytes had no significant effect on the amplitude of these calcium responses.

As demonstrated by quantitative and topological analysis, Et1 application similarly increased [Ca2+]i levels in all astrocytes in both the absence and presence of αGA.

In control conditions, subpopulations of cells responding to methoxamine or carbachol exhibited two main types of calcium responses which differed in their shape and kinetic characteristics. In the presence of αGA the number of cells responding to these receptor agonists was significantly reduced. Indeed, responses characterized by their long latency, slow rise time and weak amplitude disappeared in the presence of αGA while responses with short latency and fast rise time were preserved.

These results indicate that permeable gap junction channels tend to attenuate the pharmacological and functional heterogeneity of populations of astrocytes, while their inhibition restricts calcium responses in astrocytes expressing high densities of transmitter receptors coupled to phospholipase C.