bluuebunny/crossref_metadata_2025 huggingface dataset:
Dependence of release of [3H]noradrenaline from rabbit pulmonary artery on internal sodium.
Authors: Török T L , Tóth P T , Tóthfalusi L , Azzidani A M , Magyar K
Published: December 2014

1. [3H]Noradrenaline ([3H]NA) release from the isolated main pulmonary artery of the rabbit has been measured in the presence of uptake blockers (cocaine, 3 x 10(‐5) M, and corticosterone, 5 x 10(‐5) M) and after blocking the monoamine oxidase enzyme by pargyline (1.2 x 10(‐4) M). 2. In normal Krebs solution Mn2+ (2 mM) significantly inhibited both [3H]NA release (approximately 80%; P < 0.001) and the contraction following 2 Hz field stimulation. 3. In Ca(2+)‐free, EGTA (1 mM)‐containing solution, the Na+ pump was inhibited by removal of K+ from the external medium. In Na+ pump‐inhibited arteries, 2 mM Mn2+ (free Mn2+, 1 mM) increased the spontaneous release of [3H]NA according to the time of Na+ loading. TTX (10(‐7) M) did not inhibit significantly the Mn(2+)‐induced [3H]NA release from Na(+)‐loaded preparations (percentage inhibition, approximately 24; P > 0.30). 4. Without Na+ loading (Ca2+ free, EGTA alone), Mn2+ failed to promote 3H release from arteries. 5. With constant Na+ loading (120 min ‘K(+)‐free’ perfusion in Ca(2+)‐free, 1 mM EGTA‐containing solution), the release of 3H was also directly dependent on free Mn2+ concentration (0.2, 0.6 and 1 mM). 6. The Mn2+ (2 mM; free Mn2+, 1 mM)‐induced 3H release from Na(+)‐loaded nerves (120 min ‘K(+)‐free’, perfusion) was further enhanced, when external Na+ was simultaneously reduced from 139.2 to 26.2 mM (choline+ or sucrose substitution). 7. Diphenylhydantoin (DPH, 10(‐4) M) significantly reduced the Mn(2+)‐evoked 3H release (approximately 44%; P < 0.02) when it was present during ‘K(+)‐free’, perfusion. 8. Mn2+ was ineffective in releasing 3H if the Na+ pump was previously reactivated by readmission of K+ to Na(+)‐loaded arteries. 9. It is concluded that in Ca(2+)‐free solution Mn2+ releases neurotransmitter in a manner which depends on the degree of loading with internal Na+. The results suggest this depends at least partly on a block of Ca2+ efflux.
The effect of caged calcium release on the adaptation of the transduction current in chick hair cells.
Authors: Kimitsuki T , Ohmori H
Published: December 2014

1. Intracellular Ca2+ concentration ([Ca2+]i) was raised by photolysis of a caged calcium compound, nitr‐5, and its effects on the mechano‐electrical transduction (MET) current were studied by a whole‐cell patch electrode voltage clamp technique in dissociated hair cells of a chick. Nitr‐5 was loaded into the hair cell by incubation with the membrane‐permeable form of the compound (nitr‐5 AM). 2. Photolysis of nitr‐5 by ultraviolet (UV) light irradiation induced outward currents at ‐50 mV when recorded with a KCl‐based intracellular medium without Ca2+ chelating compounds. The average amplitude of the photo‐activated outward current was 115 +/‐ 82 pA (mean +/‐ S.D., n = 5). 3. The MET current generated at ‐50 mV showed a decay after step displacement of the hair bundle. This adaptation was accelerated after UV exposure of the cell. The adaptation was further accelerated by hyperpolarization of the membrane and was eliminated in 20‐100 microM Ca2+ extracellular media. 4. The displacement‐response relationship was shifted towards the positive direction after the UV irradiation. 5. The recovery of the transducer current after step displacement of the hair bundle was accelerated after UV irradiation, for both the inward‐going MET current recorded at ‐50 mV and the outward‐going MET current at +54 mV. However, the adaptation was not observed at positive membrane potentials even after the photolysis of nitr‐5. 6. The extent of MET current decay was reduced or disappeared in 20‐100 microM Ca2+ extracellular media and the offset time course was prolonged at the membrane potential of ‐50 mV. The current decay was not observed even after the photo‐release of intracellular Ca2+ in 50‐100 microM Ca2+ extracellular media. 7. These results (paragraphs 3‐6) suggest that the MET current adaptation is accelerated by the increase of [Ca2+]i, and that Ca2+ ions entering through MET channels are essential in the development of adaptation. 8. The adaptation of the MET current was reversibly reduced in a dihydrostreptomycin (DHSM, 20‐50 microM) medium. The time course of the adaptation changes lagged the changes in the MET current amplitude. 9. The adaptation developed or disappeared with a delay of 10‐20 s after the introduction of either the normal‐Ca2+ (2.5 mM) or the low‐Ca2+ (50‐100 microM) extracellular medium, respectively. These delays in the development and the subsidence of adaptation suggest a presence of a Ca2+ buffer site intracellularly between the adaptative site and the MET channel.
Caffeine‐induced release and reuptake of Ca2+ by Ca2+ stores in myocytes from guinea‐pig urinary bladder.
Authors: Ganitkevich VYa  , Isenberg G
Published: December 2014

1. Voltage‐clamped isolated smooth muscle cells from guinea‐pig urinary bladder were studied with 3.6 mM extracellular Ca2+ at 36 degrees C. The fluorescence of the Ca(2+)‐sensitive dye Indo‐1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transient). Fast application of caffeine (10 mM) to the cell was used to release the intracellular Ca2+ from a ‘caffeine‐sensitive Ca2+ store’. 2. At the holding potential ‐60 mV, a short (1 s) caffeine application increased [Ca2+]i within less than 1 s from the resting 118 +/‐ 22 nM to 1490 +/‐ 332 nM. Following the caffeine wash‐out, [Ca2+]i fell from this peak to a subresting level of 47 +/‐ 12 nM, i.e. an ‘undershoot’ of [Ca2+]i occurred. Subsequent caffeine‐induced [Ca2+]i transients had attenuated peaks suggesting that the caffeine‐sensitive Ca2+ store had lost a part of the releasable Ca2+. 3. In the continuous presence of caffeine, [Ca2+]i decayed from its peak to control resting [Ca2+]i values. The wash‐out of caffeine following prolonged (10‐30 s) treatment also resulted in [Ca2+]i undershoot. Subsequent caffeine‐induced [Ca2+]i transients were largely abolished as if the caffeine‐sensitive Ca2+ store had lost a large part of releasable Ca2+. During the undershoot, hyperpolarization to ‐100 mV did not affect [Ca2+]i. In most cells studied, recovery of [Ca2+]i from the undershoot to the resting level required depolarizations inducing Ca2+ influx through L‐type Ca2+ channels. 4. Block of plasmalemmal Ca(2+)‐ATPase (PMCa) with extracellular La3+ (3 mM) did not modify the decay of the [Ca2+]i transients induced by depolarization or by a 1 s caffeine application suggesting that decay rate of both is not limited by PMCa rate. La3+ abolished the undershoot of [Ca2+]i. In the continuous presence of caffeine, La3+ largely prevented the decay of [Ca2+]i. 5. When the depolarizing steps from ‐60 to 0 mV (160 ms duration) were applied during the period of [Ca2+]i undershoot, the half‐time of decay of the corresponding [Ca2+]i transients was up to three times faster than in control. Repetitive depolarizations restored the rate of decay and [Ca2+]i recovered to the resting value. Both processes recovered along a similar time course. 6. Application of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX; 0.1 mM) or of 8‐Br‐cAMP (0.1 mM) did not mimic the above caffeine effects suggesting that stimulation of sarcoplasmic reticulum (SR) Ca(2+)‐ATPase (SERCa) by cAMP‐dependent phosphorylation is not the underlying mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
Ca2+ levels in myotubes grown from the skeletal muscle of dystrophic (mdx) and normal mice.
Authors: Bakker A J , Head S I , Williams D A , Stephenson D G
Published: December 2014

1. Myotubes were grown in culture from normal (C57BL/ScSn) and mdx mice and the cytosolic [Ca2+] was monitored through development (5‐21 days in culture) using fura‐2 loaded via ionophoresis. Simultaneous measurements of the membrane potential and cytosolic [Ca2+] were made in normal and mdx myotubes before, during and after stimulation by action potentials elicited following anode break excitation. All experiments were undertaken at 22 degrees C. All data are expressed as means +/‐ S.E.M. 2. A new method was developed which enabled accurate determination of the fluorescence characteristics of fura‐2 in murine skeletal muscle fibres. In the under in vitro conditions by 14.60 +/‐ 0.05, 9.40 +/‐ 0.15 and 6.90 +/‐ 0.43% respectively. 3. The resting cytosolic [Ca2+] in the mdx myotubes was consistently higher than in the normal myotubes throughout the developmental period measured. Overall, the resting cytosolic [Ca2+] in mdx myotubes (134 +/‐ 9 nM, n = 22) was twofold higher than in normal myotubes (66 +/‐ 6 nM, n = 26). After stimulation (one to three action potentials) the cytosolic [Ca2+] of both mdx and normal myotubes remained elevated. The mdx myotubes (236 +/‐ 55 nM, n = 5) again had approximately double the cytosolic [Ca2+] of normal myotubes (109 +/‐ 19 nM, n = 9). 4. The time course and amplitude of the Ca2+ responses measured in the mdx and normal myotubes after action potential stimulation were variable. Two categories of Ca2+ response were observed in mdx and normal myotubes, the first consisted of a small, slow rise in [Ca2+] that remained elevated and the second consisted of a rapid (time to peak 7.4 +/‐ 1.5 ms) (n = 8) rise in [Ca2+] with amplitudes in the range 61‐773 nM and a [Ca2+] decay rate constant of 4.35 +/‐ 1.57 s‐1 (n = 8) (range 0.96‐15 s‐1). 5. In conclusion, the elevated cytosolic [Ca2+] reported here through development of cultured mdx myotubes suggests that this genetic disorder results in a defect which compromises the ability of the myotubes to strictly regulate cytosolic [Ca2+]. The results are consistent with the presence of functionally abnormal Ca2+ channels recently reported in mdx myotubes.
Ca2+ and Ca(2+)‐activated Cl‐ currents in rabbit oesophageal smooth muscle.
Authors: Akbarali H I , Giles W R
Published: December 2014

1. An inward current carried by Ca2+ was recorded from single smooth muscle cells of rabbit oesophageal muscularis mucosae using a whole‐cell gigaseal technique with physiological (2 mM) external calcium concentration ([Ca2+]o) in the presence of intracellular Cs+ ([Cs+]i 130 mM). Only one type of Ca2+ current could be identified. The threshold for its activation was approximately ‐30 mV and maximum inward current (approximately 300 pA) was recorded at 0 mV. 2. This inward current was blocked by Co2+ (4 mM), Cd2+ (0.5 mM) and nifedipine (1 microM) and was enhanced by Bay K 8644 (5 microM). We therefore classify it as a L‐type Ca2+ current and denote it ICa. 3. Steady‐state inactivation data were well‐fitted by a Boltzmann distribution, indicating that inactivation of the Ca2+ current is strongly modulated by membrane potential. However, the inactivation of ICa slowed significantly and became less complete when BaCl2 replaced CaCl2 in the Tyrode solution suggesting that the inactivation of ICa may also be dependent on [Ca2+]i. The steady‐state activation and inactivation curves for ICa overlap between ‐40 and 0 mV indicating that there may be a Ca2+ window current in this range of potentials. 4. When EGTA was omitted from the pipette‐filling solution, depolarizations positive to ‐10 mV resulted in a transient as opposed to a maintained inward Ca2+ current which was followed by a relatively large outward current. Under these conditions, slowly decaying inward tail currents were also recorded upon repolarization to the holding potential, ‐60 mV. However, when EGTA was omitted from the pipette, marked ‘run‐down’ of the Ca2+ current occurred within 10 min after starting the whole‐cell recording. 5. This run‐down of ICa was reduced significantly when the nystatin perforated patch technique was used. Under these conditions stable ICa records could be obtained for over 1 h. Outward currents and slow decaying inward tail currents similar to those recorded with no EGTA in the pipette were also obtained consistently using the nystatin recording technique. 6. In nystatin perforated patch recordings, CoCl2 (2 mM) completely abolished the Ca2+ current, the outward currents and the slow inward tails. These findings suggest that the outward currents and slow inward tails are activated by a transmembrane influx of Ca2+. 7. Ion replacement and pharmacological tests provided evidence that both the outward currents and the slow inward tails are due to Ca(2+)‐activated Cl‐ current (ICl(Ca)).(ABSTRACT TRUNCATED AT 400 WORDS)
Activation of muscarinic K+ current in guinea‐pig atrial myocytes by a serum factor.
Authors: Banach K , Hüser J , Lipp P , Wellner M C , Pott L
Published: December 2014

1. Atrial myocytes obtained by enzymatic perfusion of hearts from adult guinea‐pigs and cultured for 0‐14 days were studied using the whole‐cell voltage‐clamp technique. 2. Superfusion of the myocytes with diluted sera (1:100 to 1:10,000) from different species (human, horse, guinea‐pig) evoked an inward rectifying K+ current. The voltage‐dependent properties of this current were identical to those of the K+ current activated by acetylcholine (IK(ACh)). Current density in the presence of horse serum (1:100) approximately corresponded to the non‐desensitizing fraction of IK(ACh) during superfusion with 1‐2 x 10(‐6) M ACh. 3. During a maximal serum‐evoked current, application of ACh (10(‐6) M) failed to evoke additional K+ current. After switching superfusion from serum‐containing to serum‐free solution, the K+ current decayed 1‐2 orders of magnitude slower than ACh‐activated IK(ACh). During the decay of the serum‐evoked current, a proportional increase in responsiveness to ACh was recorded. During submaximal activation of K+ current by serum, a saturating concentration of ACh resulted in a total current that was identical to the current evoked by ACh alone minus the desensitizing component. Thus, activation of K+ current by serum caused desensitization of IK(ACh). From these results it is concluded that sera contain a factor that activates the same population of K+ channels as ACh. 4. Irreversible activation of IK(ACh) by ACh in myocytes dialysed with the GTP‐analogue GTP‐gamma‐S abolished sensitivity to serum and vice versa. 5. The effect of serum was not modified by atropine (10(‐6) M) which completely blocked the response to 2 x 10(‐6) M ACh. Furthermore, theophylline (1 mM), which completely inhibited IK(ACh) activation by adenosine (100 microM), failed to inhibit the effect of serum. Thus, neither muscarinic nor purinergic (A1) receptors are involved. 6. The peptide somatostatin (10(‐6) M) and the alpha 1‐agonist phenylephrine (1 microM) which previously have been shown to cause activation of IK(ACh) channels, in the present study failed to evoke any measurable current, which excludes the involvement of the corresponding receptors. 7. Pre‐incubation of the cells with pertussis toxin completely abolished IK(ACh) evoked by ACh, adenosine and serum, suggesting that the activating factor, like the classical agonists, causes opening of IK(ACh) channels via a G protein (Gi, GK). 8. The potency of serum to activate IK(ACh) was not reduced by dialysis, suggesting the molecular mass of the unknown factor to be > or = 5 kDa. No activating potency was found in the dialysing solutions.(ABSTRACT TRUNCATED AT 400 WORDS)
The locomotor discharge characteristics of ankle flexor gamma‐motoneurones in the decerebrate cat.
Authors: Murphy P R , Hammond G R
Published: December 2014

1. The discharge patterns of ankle flexor, tibialis anterior (TA), gamma‐motoneurones were recorded during locomotion in the decerebrate cat. 2. At rest gamma‐efferents had no background discharge. During locomotion two patterns of gamma activity could be distinguished. Most units (16) were phasically recruited with homonymous electroneurogram (ENG) activity, while the remainder (5) were tonically active throughout the step cycle. 3. The modulation of phasic units was greater (P < 0.01) than tonic neurones. Phasic units had lower (P < 0.02) mean, but higher (P < 0.01) peak, rates during the step cycle. 4. The discharge rate of both types of efferent increased around the onset of ENG activity and peaked during ENG activity, or shortly after its cessation. The conduction velocities of phasic and tonic units overlapped widely. 5. It is proposed, on the basis of muscle spindle afferent recordings during locomotion, that TA phasic and tonic units correspond to static and dynamic gamma‐motoneurones, respectively. This correspondence is functionally advantageous for the role of ankle flexor muscles during locomotion. Thus phasic static gamma discharge during flexion would aid muscle contraction via increased Ia afferent activity, while tonic dynamic gamma firing would enhance Ia afferent stretch sensitivity throughout the step cycle. Such enhancement during flexion would oppose unexpected muscle lengthening while, during extension, it would contribute to reciprocal inhibition of ankle extensor muscles. 6. The results are discussed in relation to strategies of gamma usage during rhythmic movements. It is postulated that, for such behaviour, muscle contraction is accompanied by coactivity in static and dynamic gamma‐motoneurones. A functional rationale is suggested for this strategy.
Sodium‐dependent regulation of epithelial sodium channel densities in frog skin; a role for the cytoskeleton.
Authors: Els W J , Chou K Y
Published: December 2014

1. A weak electroneutral sodium channel blocker 6‐chloro‐3,5‐diamino‐pyrazine‐2‐carboxamide was used to perform noise analysis on isolated epithelium from Rana fuscigula to determine the cellular mechanism underlying autoregulation of Na+ channel densities in response to a reduction in the mucosal Na+ concentration. 2. The inherent transport rates of these tissues were generally lower than in other frog skins. The macroscopic sodium current, INa, averaged 10.71 microA/cm2 and was mainly determined by the number of open channels (N(o)) which averaged 21.6 million/cm2. The calculated mean channel open probability (beta') was 0.38, and corresponded very closely to values previously determined by patch clamp. 3. Reducing the mucosal Na+ from 110 to 10 mM caused large increases in the open channel density, which stabilized the Na+ transport rate. N(o) increased from a mean value of 26.6 to 64.3 million/cm2 within 2 min. 4. Autoregulatory changes were induced primarily by increasing beta' by about 60% and to a lesser extent by an increase in NT, the total number of open and closed channels. 5. We also examined the role of the cytoskeleton in the regulation of Na+ channel densities. Colchicine treatment, which disrupted microtubules, had no apparent effect on the ability of the tissues to autoregulate their Na+ channel densities. 6. The integrity of the microfilaments were essential for autoregulatory changes in N(o). After we had disrupted the microfilaments with cytochalasin B, we observed a marked reduction in the ability of the tissues to increase N(o). 7. The mean N(o) did not increase in response to a drop in mucosal Na+ despite the fact that beta' increased by 69%. We, therefore, assumed that cytochalasin B did not affect Na+ channels already present in the membrane but interfered with recruitment of new channels. Significantly, we did not observe any increase in NT. 8. In kidney and other tight epithelia, microfilaments are responsible for regulating the delivery of newly synthesized membrane proteins. We believe that our results with cytochalasin‐treated tissues support the theory that autoregulatory changes in N(o) are also regulated by the recruitment of channels from a cytoplasmic pool.
Evidence for a K(+)‐H+ exchange in trout red blood cells.
Authors: Fievet B , Guizouarn H , Pellissier B , Garcia‐Romeu F , Motais R
Published: December 2014

1. Exposure of trout red blood cells to beta‐adrenergic agonist isoprenaline activates a cAMP‐dependent Na(+)‐H+ antiport, the movements of protons being compensated by a Cl(‐)‐OH‐ (or HCO3‐) exchange mediated by band 3 protein. The absorption of water osmotically linked to sodium and chloride induces cell swelling. 2. In the presence of acetazolamide, anionic exchange is inhibited and activation of cationic exchange resulted in the first 2 min in a strong external acidification and a large internal alkalinization leading to a reversal of the transmembrane pH gradient. Then, for at least 1 h and despite the inhibition of Cl‐ entry, a net Na+ uptake occurred which was balanced by an equivalent K+ loss, with the result that cell volume and pH gradient remained unchanged. 3. In such conditions, the inactivation of the Na(+)‐H+ exchanger by a beta‐antagonist, propranolol, blocked Na+ entry while K+ continued to be lost. This volume‐independent K+ efflux, which is thus independent of the Na(+)‐H+ exchanger, was not accompanied by a Cl‐ efflux but was associated with large internal and external pH changes consistent with K(+)‐H+ exchange. 4. The K+ loss and the related pH changes are inhibited by compounds which are known to inhibit the K(+)‐anion co‐transporter in trout red cells, i.e. 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid (Dids) and niflumic acid.
Volume‐activated Cl(‐)‐independent and Cl(‐)‐dependent K+ pathways in trout red blood cells.
Authors: Guizouarn H , Harvey B J , Borgese F , Gabillat N , Garcia‐Romeu F , Motais R
Published: December 2014

1. Swelling of trout erythrocytes can be induced either by addition of catecholamine to the cell suspension, thus promoting NaCl uptake via beta‐adrenergic‐stimulated Na(+)‐H+ exchange (isotonic swelling) or by suspending red blood cells in a hypotonic medium (hypotonic swelling). In both cases cells tend to regulate their volume by losing K+, but the characteristics of the volume‐activated K+ pathways are different: after hormonally induced swelling the K+ loss is strictly Cl‐ dependent; after hypotonic swelling the K+ loss is essentially Cl‐ independent. 2. In order to determine the nature of these volume regulatory pathways (i.e. whether the net K+ loss was conductive or was by electroneutral K(+)‐H+ exchange or KCl co‐transport), studies were performed to analyse ion fluxes and associated electrical phenomena. The cell membrane potential and intracellular ionic activities of volume‐regulating and volume‐static cells were measured by impalement with conventional microelectrodes and double‐barrelled ion‐sensitive microelectrodes. 3. The information gained from the electrical and ion flux studies leads to the conclusion that both Cl(‐)‐independent and Cl(‐)‐dependent K+ loss proceed via electrically silent pathways. 4. Experiments were designed to distinguish between electroneutral K(+)‐H+ exchange or KCl co‐transport. These were based upon the inhibition of Cl(‐)‐OH‐ exchange to evaluate the degree of coupling between K+ and Cl‐ (KCl stoichiometry, pH change). The experimental observations are consistent with the fact that both Cl(‐)‐independent and Cl(‐)‐dependent K+ loss are mediated by coupled K(+)‐anion co‐transport and not by K(+)‐H+ exchange. 5. On the basis of previous data, we suggest that only one type of K(+)‐anion co‐transport exists in the cell membrane, for which the selectivity for anions varies according to the change in cellular ionic strength induced by swelling.
Neither changes in phosphorus metabolite levels nor myosin isoforms can explain the weakness in aged mouse muscle.
Authors: Phillips S K , Wiseman R W , Woledge R C , Kushmerick M J
Published: December 2014

1. The contractile force, phosphorus metabolite levels, intracellular pH and myosin isoforms were compared in isolated soleus and extensor digitorum longus (EDL) muscles from young (6 month old) and aged (28 month old) mice, at 23 degrees C. 2. The isometric force per unit cross‐sectional area was significantly lower by 21 +/‐ 5% in soleus muscles from aged mice compared to those from young mice (mean +/‐ S.E.M., n = 11 and 9 respectively). 3. The EDL muscle contained twice as much total creatine and phosphocreatine as the soleus, 1.7 times as much ATP, and 0.4 times the inorganic phosphate (Pi) per unit weight. The intracellular pH and free ADP levels were not significantly different between these muscle types. 4. There was no significant difference in resting metabolite levels between young and old EDL or soleus despite the difference in mechanical strength. 5. Examination of the expression of myosin isoforms by non‐denaturing gel electrophoresis has shown that the percentage of each isoform does not change with respect to age; thus, if there is an atrophic process occurring, it is not fibre type specific. 6. We have determined that neither the Pi levels nor the intracellular pH can explain the differences seen in muscle strength with age. There is also no correlation between muscle weakness and any of the other metabolites responsible for energy transduction (phosphocreatine, ATP or ADP).
Neural induction suppresses early expression of the inward‐rectifier K+ channel in the ascidian blastomere.
Authors: Okamura Y , Takahashi K
Published: December 2014

1. Early expression of ion channels following neural induction was examined in isolated, cleavage‐arrested blastomeres from the ascidian embryo using a two‐electrode voltage clamp. Currents were recorded from the isolated, cleavage‐arrested blastomere, a4‐2, after treatment with serine protease, subtilisin, which induces neural differentiation as consistently as cell contact. 2. The inward‐rectifier K+ current increased at the late gastrula stage shortly after the sensitive period for neural induction both in the induced (protease‐treated) and uninduced cells. Ca2+ channels, characteristic of epidermal‐type differentiation, and delayed‐rectifier K+ channels and differentiated‐type Na+ channels, characteristic of neural‐type differentiation appeared much later than the inward‐rectifier K+ channels, at a time corresponding to the tail bud stage of the intact embryo. 3. When cells were treated with subtilisin during the critical period for neural induction, the increase in the inward‐rectifier K+ current from the late gastrula stage to the neurula stage was about three times smaller (3.67 +/‐ 1.74 nA, mean +/‐ S.D., n = 14) than in untreated cells (11.25 +/‐ 3.10 nA, n = 26). The same changes in the inward‐rectifier K+ channel were also observed in a4 2 blastomeres which were induced by cell contact with an A4‐1 blastomere. However, when cells were treated with subtilisin after the critical period for neural induction, the amplitude of the inward‐rectifier K+ current was the same as in untreated cells. Thus the expressed level of the inward‐rectifier K+ channel was linked to the determination of neural or epidermal cell types. 4. There was no significant difference in the input capacitance of induced and uninduced cells, indicating that the difference in the amplitude of the inward‐rectifier K+ currents derived from a difference in the channel density rather than a difference in cell surface area. 5. The expression of the inward‐rectifier K+ channel at the late gastrula stage was sensitive to alpha‐amanitin, a highly specific transcription inhibitor. In both induced and uninduced cells, injection of alpha‐amanitin at the 32‐cell stage reduced the current density of the inward‐rectifier K+ channel to about 2 nA/nF, corresponding to 13% of that recorded from uninjected cells. By contrast, the expression of the fast‐inactivating‐type Na+ current, which transiently increased along with the inward‐rectifier K+ channel, was resistant to alpha‐amanitin injection. 6. The dose of alpha‐amanitin injected was controlled by monitoring co‐injected fluorescent dye, fura‐2.(ABSTRACT TRUNCATED AT 400 WORDS)
Time course of spontaneous calcium‐activated chloride currents in smooth muscle cells from the rabbit portal vein.
Authors: Hogg R C , Wang Q , Large W A
Published: December 2014

1. The time course of spontaneous calcium‐activated chloride currents was studied with the perforated patch technique in freshly dispersed smooth muscle cells from the rabbit portal vein. 2. In potassium‐containing solutions the spontaneous transient outward current (STOC, a calcium‐activated potassium current) was more commonly recorded than spontaneous transient inward currents (STICs, a calcium‐activated chloride current). In addition the duration of STOCs was much briefer (about 100 ms) than the duration of STICs (about 400 ms). 3. The decay of STICs could be described by a single exponential but the STOC decay appeared to be more complex. The decay time constant of STICs was not determined significantly by amplitude. 4. The time constant of decay of STICs (tau) was 86 ms at ‐50 mV and was increased by depolarization. Between ‐90 and +50 mV the relationship between tau and membrane potential was exponential and tau changed e‐fold for a change of membrane potential of 120 mV. 5. The I‐V relationship of STIC amplitude was linear between ‐10 and +50 mV but at more negative potentials the chord conductance was reduced and the I‐V relationship exhibited negative slope conductance between ‐50 and ‐90 mV. 6. There was good agreement between the STIC tau values and the exponential relaxations to voltage steps evoked during caffeine‐induced calcium‐activated chloride currents. 7. In the presence of the chloride channel blocking agent anthracene‐9‐carboxylic acid the STIC amplitude was reduced and tau was increased. This effect was voltage dependent with a much greater effect at positive potentials. 8. The evidence suggests that the decay of STICs represents closure of chloride channels and tau approximates to the channel mean open time.
Galanin antagonist effects on cardiac vagal inhibitory actions of sympathetic stimulation in anaesthetized cats and dogs.
Authors: Ulman L G , Moriarty M , Potter E K , McCloskey D I
Published: December 2014

1. Galantide, a putative galanin antagonist composed of twenty amino acids, caused a significant reduction in the vagal attenuating action of galanin injection (20 micrograms/kg; 6.2 nmol/kg) in anaesthetized cats at both ten times (137 micrograms/kg; 62 nmol/kg) and twenty‐five times (343 micrograms/kg: 156 nmol/kg) the molar dose of galanin. Galantide did not block the depressor action of galanin in these animals. 2. Galantide, at both doses, also significantly reduced the vagal attenuating action of a 5 min period of cardiac sympathetic stimulation at 16 Hz in anaesthetized cats. 3. In anaesthetized dogs, galantide, at the same dose used in cats (137 micrograms/kg; 62 nmol/kg) had no significant effect on the vagal attenuation evoked by cardiac sympathetic stimulation or injection of neuropeptide Y (35 micrograms/kg; 8.2 nmol/kg). 4. This study therefore demonstrates antagonist properties of galantide on the vagal inhibitory action of galanin. It supports the hypothesis that the vagal inhibitory factor released by cardiac sympathetic nerve stimulation in the cat, but not the dog, is galanin, although it does not exclude the possibility of other factors playing a more minor role. Because galantide was not shown to block the depressor action of galanin, this study also suggests that there may be more than one galanin receptor subtype.
Effects of metabolic inhibition and changes of intracellular pH on potassium permeability and contraction of rat uterus.
Authors: Heaton R C , Wray S , Eisner D A
Published: December 2014

1. We have investigated the role of changes of potassium efflux in the inhibition of uterine force produced by cyanide. K+ efflux (86Rb) was measured from pregnant and non‐pregnant rat myometrial strips during metabolic inhibition with cyanide and following manoeuvres to displace intracellular pH (pHi). 2. Cyanide greatly reduced or abolished spontaneous contractions. If the membrane was depolarized directly at this stage (by elevating external K+) then contraction redeveloped. This suggests that the initial depression of force is due to a failure of membrane excitation. 3. Cyanide reversibly increased 86Rb efflux (30‐35%) in both pregnant and nonpregnant uteri and contraction was reduced. The increase in 86Rb efflux with cyanide was not secondary to changes of membrane potential as it also occurred in both high‐K+ and Ca(2+)‐free solutions. 4. Glibenclamide (20 microM), an antagonist of K+ATP channels, reduced the cyanide‐evoked increase of 86Rb efflux by about 50%. The glibenclamide‐insensitive component of efflux persisted in a Ca(2+)‐free solution. Despite its action on 86Rb efflux, glibenclamide did not restore contraction. 5. Intracellular pH falls during metabolic inhibition. We therefore investigated whether reducing pHi (in the absence of cyanide) had an effect on 86Rb efflux. Application of the weak acid butyrate (60 mM, at constant external pH, 7.4) had no significant effect on 86Rb efflux. Thus it is unlikely that the acidification in hypoxia contributes to the increased K+ efflux. 6. Intracellular alkalinization produced by the weak base trimethylamine (60 mM) increased the frequency of uterine contraction and the 86Rb efflux. However, there was no effect on the 86Rb efflux in a Ca(2+)‐free solution. The increased efflux is therefore presumably a consequence of the increased frequency. 7. It is concluded that metabolic inhibition produced by cyanide, produces an increase in K+ efflux from the myometrium. Part of this efflux is glibenclamide sensitive. This increased K+ efflux will lead to hyperpolarization of the myometrial membrane and thus decrease excitation. Thus reduced surface membrane excitability will contribute to the fall of force in hypoxia; specifically it may cause the initial loss of spontaneous contractions in the uterus.
Calcium entry through nicotinic receptor channels and calcium channels in cultured rat superior cervical ganglion cells.
Authors: Trouslard J , Marsh S J , Brown D A
Published: December 2014

1. Patch‐clamp techniques in conjunction with indo‐1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage‐clamp conditions, pressure application of the nicotinic agonist DMPP (1,1‐dimethyl‐4‐phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/‐ 26 nM at a clamp potential of ‐60 mV. This was accompanied by an inward current of ‐4.53 +/‐ 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP‐induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM‐10 microM) but were unaffected by alpha‐bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from ‐80 to ‐40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine‐sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage‐dependent calcium current. On average at ‐60 mV, the M/q ratio of this highly calcium‐selective permeability was 1961 mM nC‐1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant‐field theory, ion‐substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.
Charge inactivation in the membrane of intact frog striated muscle fibers.
Authors: Huang C L
Published: December 2014

1. Charge movements were compared in normally polarized and depolarized intact frog muscle fibres under voltage clamp. 2. The membrane capacitance was linear through positive control steps made consistently from a holding voltage of ‐10 mV, in agreement with earlier reports from cut fibres. 3. A shift in holding voltage from ‐90 to ‐10 mV reduced both the absolute amount and the voltage dependence of charge movement elicited by voltage steps imposed from a fixed conditioning voltage of ‐180 mV. The charge transferred by steps from ‐180 to ‐20 mV was 43.8 +/‐ 1.14 nC/microF in fully polarized fibres and 21.7 +/‐ 1.49 nC/microF in the same depolarized fibres (means +/‐ S.E. of the mean; four fibres). 4. Charge movement in response to steps from ‐90 to ‐20 mV increased from 10.4 +/‐ 1.60 nC/microF to 28.4 +/‐ 2.42 nC/microF (five fibres) within 30s of changing the holding voltage from ‐10 to ‐90 mV. 5. The same fibres also showed significant charge movement between voltages of ‐180 and ‐90 mV. However, shifts in holding voltage did not significantly alter the maximum value of this charge, around 10‐11 nC/microF. 6. Membrane capacitance as measured by small steps to a voltage of ‐90 mV remained constant despite holding potential changes, or lidocaine (10 mM) treatment. 7. The same results were obtained whether the above procedures were applied to fibres exposed to normal extracellular [Ca2+], or in Ca(2+)‐free media. In both cases tubular cable corrections did not affect the results. 8. These findings suggest independent charge I and charge II systems in which inactivation of charge I is not associated with its interconversion into charge II.
Thiamine outflow from the enterocyte: a study using basolateral membrane vesicles from rat small intestine.
Authors: Laforenza U , Gastaldi G , Rindi G
Published: December 2014

1. Rat small intestinal basolateral membrane vesicles (BLMVs) were prepared and found to be 31% non‐vesiculated and 69% vesiculated, 4.9% right side out and 63.8% inside out. 2. Thiamine uptake by BLMVs followed a hyperbolic time course reaching equilibrium after 60‐90 min incubation. Uptake was not affected by the transmembrane potential or by the presence or absence of Na+ or K+ in the incubation medium. 3. At concentrations below 1.25 microM, [3H]thiamine was taken up mainly by a saturable mechanism with an apparent Michaelis‐Menten constant (Km) = 1.32 microM and maximal flux (Jmax) = 1.93 pmol (mg protein)‐1 (4 s)‐1. At higher concentrations, a non‐saturable mechanism prevailed. 4. Only 29% of [3H]thiamine taken up by the vesicles was membrane bound, the remaining being translocated into the vesicular space. No thiamine phosphoesters could be detected inside the vesicles. 5. In the absence of ATP, the Na(+)‐K(+)‐ATPase inhibitors ouabain, frusemide and vanadate reduced thiamine uptake by 35, 30 and 15% respectively. 6. In experiments conducted with K+ inside the vesicles and Na+, Mg2+ and ATP outside, the time course of thiamine uptake by BLMVs displayed an overshoot (80‐90% increment) at 30 s incubation as compared to controls. When ATP was replaced with phosphocreatine, or when NaCl was replaced with isosmotic amounts of KCl, the overshoot disappeared. 7. The thiamine analogues pyrithiamine, amprolium and 4'‐oxythiamine decreased the ATPase‐dependent transport of [3H]thiamine by 100, 86 and 31% respectively. 8. These results provide evidence that the transport of thiamine by BLMVs is coupled directly to the hydrolysis of ATP (primary active transport).
Membrane potential, resting calcium and calcium transients in isolated muscle fibres from normal and dystrophic mice.
Authors: Head S I
Published: December 2014

1. Single skeletal muscle fibres were enzymatically isolated from the flexor digitorum brevis muscles (FDB) of dystrophic mdx and control C57BL/10 mice aged 3‐9 weeks. In this age range the majority (> 95%) of the mdx fibres were morphologically normal. 2. There was no significant difference between the resting membrane potential (RMP) of mdx and control mice, ‐71.2 +/‐ 1.21 (n = 26) and ‐70.6 +/‐ 1.15 mV (n = 42), respectively. 3. At RMP more negative than ‐60 mV the resting calcium (recorded with fura‐2, free acid ionophoresed into cell) in the dystrophic mdx cells was not significantly different from the normal animals, 45.7 +/‐ 4.1 (n = 10) and 46.2 +/‐ 3.9 nM (n = 9), respectively. 4. The resting cytosolic calcium concentration was measured simultaneously with the RMP. At RMP between ‐60 to ‐17 mV there was an increase in the resting calcium concentration in both mdx and control ranging from 79.3 to 252 nM. This increase was most probably due to the activation of the slow calcium current. 5. Fura‐2 calcium transients were produced via single action potential stimulation using an intracellular microelectrode both to stimulate the cell and record potential changes. There was no significant difference between the rise time (Tp) or half‐decay time (T1/2) at 22 degrees C of the calcium transient in response to a single action potential in mdx compared to normal animals, 5.9 +/‐ 0.34 (n = 8) and 5.4 +/‐ 0.36 ms (n = 7); 39.5 +/‐ 2.9 (n = 8) and 40.75 +/‐ 3.7 ms (n = 7), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Synapse formation and elimination during growth of the pectoral muscle in Xenopus laevis.
Authors: Dunia R , Herrera A A
Published: December 2014

1. Synapse formation and synapse elimination were studied in the pectoral muscle of Xenopus laevis. 2. Histology showed that fibres were not added during postmetamorphic growth. Most fibres were innervated at two widely separated junctions and this number did not change as frogs grew. 3. Intracellular recording revealed that fibres with two junctions could be mononeuronally innervated, or innervated in one of three different polyneuronal patterns. A growth‐related shift in innervation pattern was observed, with the polyneuronal patterns replaced by mononeuronal innervation. 4. Endplate potentials (EPPs) evoked by low‐frequency nerve stimulation were simultaneously measured at both junctions on individual fibres. For each fibre, the ratio of EPP amplitudes (smaller/larger) was calculated. When the two junctions were innervated by different motoneurones (A‐B), the median EPP ratio was smaller than when the two junctions were innervated by the same motoneurone (A‐A), although the difference was not significant. 5. The difference in the ratio of EPP amplitudes became significant, however, if junctions were conditioned by a train of fifty stimuli at 10 Hz. Immediately after such a train, EPP ratios for A‐B fibres were significantly smaller than ratios for A‐A fibres. This difference was due to greater synaptic depression at one of the junctions on A‐B fibres. 6. We concluded that enhanced depression of the EPP upon repetitive stimulation is a physiological correlate of the competition that underlies synapse elimination.
The effects of low calcium on the voltage‐dependent conductances involved in tuning of turtle hair cells.
Authors: Art J J , Fettiplace R , Wu Y C
Published: December 2014

1. The voltage‐dependent conductances of turtle cochlear hair cells of known resonant frequency were characterized by tight‐seal, whole‐cell recording during superfusion with solutions containing normal (2.8 mM) and reduced (0.1‐10 microM) Ca2+. 2. In 1 microM Ca2+, the current flowing through the voltage‐dependent Ca2+ channels was increased roughly fivefold and had a reversal potential near 0 mV. This observation may be explained by the Ca2+ channels becoming non‐selectively permeable to monovalent cations in low‐Ca2+ solutions. Lowering the Ca2+ further to 0.1 microM produced little increase in the current. 3. The size of the non‐selective current increased systematically with the resonant frequency of the hair cell over the range from 10 to 320 Hz. This suggests that hair cells tuned to higher frequencies contain more voltage‐dependent Ca2+ channels. 4. There was a good correlation between the amplitudes of the non‐selective current and the K+ current which underlies electrical tuning of these hair cells. The amplitude of the K+ current also increased systematically with resonant frequency. 5. In cells with resonant frequencies between 120 and 320 Hz, the K+ current was completely abolished in 1 microM Ca2+, consistent with prior evidence that this current flows through Ca2+ activated K+ channels. In a majority of cells tuned between 50 and 120 Hz, the K+ current was incompletely blocked in 1 microM Ca2+, but was eliminated in 0.1 microM Ca2+. In all hair cells the K+ current was abolished by 25 mM tetraethylammonium chloride. 6. In cells tuned to 10‐20 Hz, the K+ current was not substantially diminished even in 0.1 microM Ca2+, which argues that it may not be Ca2+ activated. 7. In cells tuned to frequencies above 100 Hz, the K+ current could still be evoked by depolarization during superfusion with 10 microM Ca2+. However, its half‐activation voltage was shifted to more depolarized levels and its maximum amplitude was systematically reduced with increasing resonant frequency. 8. These observations are consistent with the notion that in cells tuned to more than 50 Hz, there is a fixed ratio of the number of voltage‐dependent Ca2+ channels to Ca(2+)‐activated K+ channels, the numbers of each increasing in proportion to resonant frequency. The results also provide indirect evidence that the Ca(2+)‐activated K+ channels in cells tuned to higher frequencies may be less sensitive to intracellular Ca2+.
Mechanism of mu‐opioid receptor‐mediated presynaptic inhibition in the rat hippocampus in vitro.
Authors: Capogna M , Gähwiler B H , Thompson S M
Published: December 2014

1. The electrophysiological action of the mu‐opioid receptor‐preferring agonist D‐Ala2, MePhe4, Met(O)5‐ol‐enkephalin (FK 33‐824) on synaptic transmission has been studied in area CA3 of organotypic rat hippocampal slice cultures. 2. FK 33‐824 (1 microM) had no effect on the amplitude of pharmacologically isolated N‐methyl‐D‐aspartate (NMDA) or non‐NMDA receptor‐mediated EPSPs. 3. FK 33‐824 (10 nM to 10 microM) reduced the amplitude of monosynaptic inhibitory postsynaptic potentials (IPSPs) that were elicited in pyramidal cells with local stimulation after pharmacological blockade of excitatory amino acid receptors. This effect was reversible, dose‐dependent, and sensitive to naloxone and the mu‐receptor antagonist Cys2,Tyr3,Orn5,Pen7‐amide (CTOP). FK 33‐824 at 1 microM caused a mean reduction in the amplitude of the monosynaptic IPSP of 70%. 4. Neither delta‐ nor kappa‐receptor‐preferring agonists had any effect on excitatory or inhibitory synaptic potentials. 5. The disinhibitory action of FK 33‐824 was blocked by incubating the cultures with pertussis toxin (500 ng/ml for 48 h) or by stimulation of protein kinase C with phorbol 12,13‐dibutyrate (PDBu, 0.5 microM). 6. The depression of monosynaptic IPSPs by FK 33‐824 was unaffected by extracellular application of the K+ channel blockers Ba2+ or Cs+ (1 mM each). 7. FK 33‐824 produced a decrease in the frequency of miniature, action potential‐independent, spontaneous inhibitory synaptic currents (mIPSCs) recorded with whole‐cell voltage‐clamp techniques, but did not change their mean amplitude. Application of the Ca2+ channel blocker Cd2+ (100 microM) or of nominally Ca(2+)‐free solutions did not alter either the frequency and amplitude of mIPSCs or the reduction of mIPSC frequency induced by FK 33‐824. 8. The effect of FK 33‐824 on spontaneous mIPSCs was prevented by naloxone, and by incubation of cultures with pertussis toxin. 9. These results indicate that mu‐opioid receptors decrease GABA release presynaptically by a G protein‐mediated inhibition of the vesicular GABA release process, and not by changes in axon terminal K+ or Ca2+ conductances that are sensitive to extracellular Ba2+, Cs+ or Cd2+.