bluuebunny/crossref_metadata_2025 huggingface dataset:
Acceleration of puberty onset in female mice by male urinary proteins.
Authors: Mucignat-Caretta C , Caretta A , Cavaggioni A
Published: December 2014

1. Puberty onset in female mice is accelerated by exposure to conspecific adult male urine, which acts through the vomeronasal organ and the accessory olfactory system. A distinctive component of adult male mouse urine is the major urinary protein complex (MUP), which is a lipocalin; it has a hydrophobic pocket that binds small endogenous volatile molecules. The MUP gene family also codes for a hexapeptide, which has four residues in common with the N‐terminal region of MUP. 2. MUP, the volatiles bound to MUP and the MUP‐related hexapeptide have been tested for the induction of puberty acceleration by measuring the increase in uterus weight related to the first pro‐oestrus phase. MUP, together with its bound volatiles, induces puberty acceleration. Its activity is retained even when the volatiles have been removed either by organic extraction or competition displacement with a high‐affinity ligand. 3. MUP‐related hexapeptide also induces puberty acceleration in female mice. In contrast, the odorants bound to MUP do not exert this effect. 4. It is proposed that the vomeronasal organ contains receptors that recognize the short N‐terminal consensus sequence, N‐Glu‐Glu‐Ala‐X‐Ser (where X is a polar residue), common to both MUP and the hexapeptide.
Ankle extensor group I afferents excite extensors throughout the hindlimb during fictive locomotion in the cat.
Authors: Guertin P , Angel M J , Perreault M C , McCrea D A
Published: December 2014

1. The effects of stimulating hindlimb extensor nerves (100‐200 ms trains, 100 Hz, < or = 2 times threshold) during the flexor and extensor phases of the locomotor step cycle were analysed in the decerebrate, paralysed cat during fictive locomotion evoked by stimulation of the mesencephalic locomotor region. 2. Stimulation during extension of either the medial gastrocnemius (MG), lateral gastrocnemius‐soleus (LGS) or plantaris (Pl) nerves was equally effective in increasing the duration and amplitude of electroneurogram (ENG) activity recorded in ipsilateral ankle, knee and hip extensor nerves. Enhancement of extensor ENG activity could be evoked with near threshold stimulation intensity and appeared within 10‐40 ms of the onset of ankle extensor nerve stimulation. Stimulation of anterior biceps during extension occasionally evoked a modest increase in the duration of activity of hip, knee and ankle extensors. Stimulation of quadriceps during extension enhanced the activity of proximal extensors and soleus, but inhibited other ankle extensors. 3. Selective activation of ankle extensor Ia spindle afferents by muscle stretch also enhanced ipsilateral extension. It is argued that both muscle spindle and tendon organ afferents can contribute to the increase in extensor nerve activity evoked by group I stimulation intensity during fictive locomotion. 4. During flexion, stimulation of either the MG, Pl or LGS nerves at group I strength terminated on‐going activity in ipsilateral flexors and initiated a burst of activity in ipsilateral hip, knee and ankle extensors, i.e. reset the step cycle to extension. 5. Low strength stimulation of the mixed muscle and cutaneous nerve innervating the plantar aspect of the foot produced extension enhancement and resetting similar to that evoked by group I muscle afferent stimulation. Stimulation of the cutaneous nerve supplying the dorsal aspect of the foot during extension enhanced extensor activity, and during flexion, enhanced the activity of flexors. 6. The effects reported here during fictive locomotion may also occur during overground locomotion with natural activation of group I muscle spindle and tendon organ afferents. Extensor spindle and tendon organ afferents may thus serve as an excitatory reflex system helping to shape the amplitude, duration and timing of ipsilateral extensor activity. Increased or unexpected activation of group I ankle extensor afferents or plantar foot afferents during locomotion could also compensate for increased loading of the limb.
Non‐selective cation current of guinea‐pig endocardial endothelial cells.
Authors: Manabe K , Takano M , Noma A
Published: December 2014

1. Endocardial endothelial (EE) cells, isolated by the enzymatic treatment of guinea‐pig heart, were used for whole‐cell voltage clamp experiments. 2. The inward rectifier K+ current was observed in about half of the experiments. The contribution of Ca(2+)‐dependent K+ current to the resting membrane conductance was also suggested. 3. After the K+ conductances were suppressed, removal of external Na+ revealed an inward cation current (1.2 pA pF‐1, at ‐45 mV), whose slope conductance was a saturable function of external Na+ concentration. When Na+ was totally replaced by various monovalent cations, the order of the membrane conductances was K+ > Rb+ > Cs+ > Na+ > Li+. 4. This basal non‐selective cation current was blocked by either Gd3+ or La3+, and showed slight outward rectification. 5. Addition of 20 mM Ca2+ or Ba2+, but not Mg2+ or Mn2+, to the Na(+)‐free solution, induced an inward current, indicating that this current possesses a significant Ca2+ permeability. 6. In approximately 15% of the experiments, ATP and histamine induced another type of non‐selective cation current, which showed different ion selectivity (Na+ > K+, Cs+) and rectification (inward). 7. The basal non‐selective cation current is responsible for both the low resting potential and the leak Ca2+ influx of EE cells.
Inhibition of calcium currents and exocytosis by Lambert‐Eaton syndrome antibodies in human lung cancer cells.
Authors: Viglione M P , O'Shaughnessy T J , Kim Y I
Published: December 2014

1. Human small‐cell lung cancer (SCLC) cells are believed to express the antigens responsible for the production of pathological antibodies in the Lambert‐Eaton syndrome (LES), a Ca2+ channel disorder in which quantal transmitter release from the motor nerve terminal is impaired. Whole‐cell patch‐clamp techniques were used to study the voltage‐dependent Ca2+ channels expressed by H146 SCLC cells and the effects of LES antibodies on these channels. The types of Ca2+ channels were determined using biophysical properties and pharmacological sensitivity to several antagonists. 2. Whole‐cell Ca2+ currents (ICa) in SCLC cells are sensitive to the dihydropyridine (DHP) nicardipine, omega‐conotoxin GVIA (omega‐CgTX GVIA) and omega‐agatoxin IVA (omega‐AgTX IVA). Nicardipine at 100 nM and 10 microM reduced ICa by 35 and 45% (n = 38 cells), respectively, while omega‐CgTX GVIA (1 microM) inhibited ICa by 32% (n = 31). Application of omega‐AgTX IVA at 50 and 100 nM to the cancer cells decreased ICa by 41 and 42%, respectively (n = 22). 3. Measurement of cell membrane capacitance (Cm) revealed that Ca(2+)‐dependent exocytosis underlies the secretory activity of SCLC cells. Exocytosis, when induced by step depolarizing pulses and measured by increases in Cm, was markedly inhibited by nicardipine (10 microM) and omega‐AgTX IVA (100 nM). In contrast, omega‐CgTX GVIA (1 microM) was not as effective in altering increases in Cm. 4. From negative (‐80 mV) and depolarized (‐40 mV) holding potentials, both peak and plateau ICa were inhibited by the presence of LES antibodies (1 mg ml‐1 IgG). LES serum also reduced depolarization‐induced increases in Cm by 48% (n = 15). 5. To determine whether the LES antibodies are downregulating a specific type(s) of Ca2+ channel, nicardipine (10 microM), omega‐CgTX GVIA (1 microM) or omega‐AgTX IVA (100 nM) was applied to tumour cells that had been previously exposed to LES serum for 24 h. The most pronounced change was that omega‐AgTX IVA was 38‐84% less effective at reducing ICa after the IgG treatment. The effectiveness of nicardipine was diminished by 18% after incubation with the LES antibodies, whereas the omega‐CgTX GVIA was seen to be more effective. These results suggest that LES IgG downregulates P‐type Ca2+ channels and, possibly, to a lesser extent L‐type channels. 6. In view of recent evidence that P‐type Ca2+ channels mediate cholinergic transmitter release at the mammalian neuromuscular junction (NMJ), the expression of P‐type Ca2+ channels in the SCLC cells and the reactivity of LES IgG with these channels support the hypothesis that P‐type Ca2+ channels in these cancer cells may trigger the autoantibody production in this disorder. The antibodies so produced are implicated in the functional impairment of the Ca2+ channels characteristic of LES.
Species differences in the activity of the Na(+)‐Ca2+ exchanger in mammalian cardiac myocytes.
Authors: Sham J S , Hatem S N , Morad M
Published: December 2014

1. Species differences in the activity of the exchanger were evaluated in isolated myocytes from rat, guinea‐pig, hamster ventricles and human atria. Fluorescence measurements using fura‐2 were carried out in conjunction with the whole‐cell patch‐clamp technique for simultaneous recording of membrane currents and intracellular Ca2+ concentration. 2. Ca2+ release from sarcoplasmic reticulum (SR) induced either by rapid application of caffeine or by Ca2+ current elicited inward Na(+)‐Ca2+ exchange currents (INa‐Ca). The magnitude of INa‐Ca was largest in hamster, smallest in rat, with guinea‐pig and human myocytes having intermediate values. The ratio of caffeine‐induced exchanger current densities, normalized with respect to the peak Ca2+ release, was 4:2:1.5:1 for hamster > guinea‐pig > or = human > or = rat myocytes. 3. The rates of Ca2+ removal in the presence of caffeine, which reflect primarily the Ca2+ extruding activity of the Na(+)‐Ca2+ exchanger, followed the same order of hamster > guinea‐pig > or = human > or = rat. 4. The kinetics of INa‐Ca vs. Ca2+ transients were different among species. In rat myocytes, the kinetics of the INa‐Ca and the Ca2+ transients were similar, with INa‐Ca linearly proportional to intracellular Ca2+ concentration ([Ca2+]i). In hamster myocytes, the time course of INa‐Ca tracked only the declining phase of the Ca2+ transient with INa‐Ca having faster kinetics during the Ca2+ release. These findings suggest that the Ca2+ concentrations in the vicinity of the exchanger were significantly higher than those of the cytosol during Ca2+ release in hamster myocytes. 5. We concluded that there are significant species differences in the exchanger activity of cardiac myocytes, arising from differences in exchanger densities, their modulation and/or their spatial distribution with respect to the ryanodine receptors of cardiac myocytes.
Increased resting discharge of human spindle afferents following voluntary contractions.
Authors: Wilson L R , Gandevia S C , Burke D
Published: December 2014

1. The aim of this study was to assess the incidence of lasting alterations in discharge rate of muscle spindle afferents innervating human ankle and toe dorsiflexor muscles following isometric contractions. 2. The subjects performed controlled isometric ankle dorsiflexions maintained for approximately 5 s. During the contraction the discharge of all but one spindle afferent increased above the precontraction level. After complete relaxation, there was prolonged enhancement of the discharge rate of nineteen of fifty‐five muscle spindle afferents and none of three Golgi tendon organ afferents. Ten of the nineteen spindle afferents had been silent prior to the contraction. For the population of fifty‐five spindle afferents, the mean ‘postcontraction’ discharge rate was 65% higher than the mean precontraction discharge rate, with the mean rate increasing from 2.3 to 3.9 Hz (P < 0.001). The mean duration of the enhanced postcontraction discharge was 52 s (range, 8‐240 s). 3. Stretch applied to the tendon of the receptor‐bearing muscle in twelve of fourteen spindle afferents with an enhanced postcontraction discharge rate eliminated or reduced the enhanced discharge rate. 4. The high incidence of an enhanced spindle discharge after voluntary contraction (35% of spindle afferents) suggests that muscle ‘history’ should be taken into account when interpreting changes in spindle discharge rates. The enhanced discharge rates following contraction probably reflect a long‐lasting effect of the contraction‐associated increase in fusimotor drive on intrafusal stiffness, rather than the persistence of fusimotor drive following relaxation.
C‐type inactivation controls recovery in a fast inactivating cardiac K+ channel (Kv1.4) expressed in Xenopus oocytes.
Authors: Rasmusson R L , Morales M J , Castellino R C , Zhang Y , Campbell D L , Strauss H C
Published: December 2014

1. A fast inactivating transient K+ current (FK1) cloned from ferret ventricle and expressed in Xenopus oocytes was studied using the two‐electrode voltage clamp technique. Removal of the NH2‐terminal domain of FK1 (FK1 delta 2‐146) removed fast inactivation consistent with previous findings in Kv1.4 channels. The NH2‐terminal deletion mutation revealed a slow inactivation process, which matches the criteria for C‐type inactivation described for Shaker B channels. 2. Inactivation of FK1 delta 2‐146 at depolarized potentials was well described by a single exponential process with a voltage‐insensitive time constant. In the range ‐90 to +20 mV, steady‐state C‐type inactivation was well described by a Boltzmann relationship that compares closely with inactivation measured in the presence of the NH2‐terminus. These results suggest that C‐type inactivation is coupled to activation. 3. The coupling of C‐type inactivation to activation was assessed by mutation of the fourth positively charged residue (arginine 454) in the S4 voltage sensor to glutamine (R454Q). This mutation produced a hyperpolarizing shift in the inactivation relationship of both FK1 and FK1 delta 2‐146 without altering the rate of inactivation of either clone. 4. The rates of recovery from inactivation are nearly identical in FK1 and FK1 delta 2‐146. 5. To assess the mechanisms underlying recovery from inactivation the effects of elevated [K+]o and selective mutations in the extracellular pore and the S4 voltage sensor were compared in FK1 and FK1 delta 2‐146. The similarity in recovery rates in response to these perturbations suggests that recovery from C‐type inactivation governs the overall rate of recovery of inactivated channels for both FK1 and FK1 delta 2‐146. 6. Analysis of the rate of recovery of FK1 channels for inactivating pulses of different durations (70‐2000 ms) indicates that recovery rate is insensitive to the duration of the inactivating pulse.
Changes in IP3 and cytosolic Ca2+ in response to sugars and non‐sugar sweeteners in transduction of sweet taste in the rat.
Authors: Bernhardt S J , Naim M , Zehavi U , Lindemann B
Published: December 2014

1. The transduction pathways of sweet‐sensitive cells in rat circumvallate (CV) taste buds were investigated with assays for inositol 1,4,5‐trisphosphate (IP3) and with Ca2+ imaging. Stimulation with the non‐sugar sweeteners SC‐45647 and saccharin rapidly increased the cellular content of IP3 by 400 pmol (mg protein)‐1, while sucrose had a much smaller effect on IP3. As shown previously, sucrose, but not saccharin, increased the content of cyclic adenosine monophosphate (cAMP) of this preparation. 2. Stimulation of isolated CV taste buds with SC‐45647 increased the cytosolic Ca2+ concentration ([Ca2+]i) by 56.7 +/‐ 3.2 nM (n = 181). Due to the non‐confocality of the measuring system, these concentrations are underestimates. The increase in [Ca2+]i did not require the presence of extracellular Ca2+, suggesting that the Ca2+ release was from intracellular stores. 3. Individual cells responding to the non‐sugar sweeteners with Ca2+ release also responded to sucrose and to forskolin with an increase in [Ca2+]i. Such cells did not respond to the bitter tastant denatonium chloride. 4. Responses to sucrose were abolished by lowering the Ca2+ concentration of the stimulus solution, indicating Ca2+ uptake from the extracellular medium. 5. The responses of sweet‐sensitive cells to forskolin were also abolished when Ca2+ ions were omitted from the stimulus solution. They were partially inhibited by the presence of Co2+, Ni2+, D600 (methoxyverapamil) and amiloride, indicating multiple pathways of Ca2+ uptake activated by cAMP. 6. In conclusion, a sweet‐sensitive cell of the rat responds to sucrose with an increase in cAMP and Ca2+ uptake, but to non‐sugar sweeteners with an increase in IP3 and Ca2+ release. The increase in [Ca2+]i, common to both pathways, is presumably required for synaptic exocytosis and for signal termination.
Supraspinal factors in human muscle fatigue: evidence for suboptimal output from the motor cortex.
Authors: Gandevia S C , Allen G M , Butler J E , Taylor J L
Published: December 2014

1. Voluntary activation of elbow flexor muscles can be optimal during brief maximal voluntary contractions (MVCs), although central fatigue, a progressive decline in the ability to drive the muscle maximally, develops during sustained or repeated efforts. We stimulated the motor cortex and motor point in human subjects to investigate motor output during fatigue. 2. The increment in force (relative to the voluntary force) produced by stimulation of the motor point of biceps brachii increased during sustained isometric MVCs of the elbow flexors. Motoneuronal output became suboptimal during the contraction, i.e. central fatigue developed and accounted for a small but significant loss of maximal voluntary force. During 3 min MVCs, voluntary activation of biceps fell to an average of 90.7% from an average of > 99%. 3. The increment in force (relative to the voluntary force) produced by magnetic cortical stimulation was initially small (1.0%) but also increased during sustained MVCs to 9.8% (with a 2 min MVC). Thus, cortical output was not optimal at the time of stimulation nor were sites distal to the motor cortex already acting maximally. 4. A sphygmomanometer cuff around the upper arm blocked blood supply to brachioradialis near the end of a sustained MVC and throughout subsequent brief MVCs. Neither maximal voluntary force nor voluntary activation recovered during ischaemia after the sustained MVC. However, fatigue‐induced changes in EMG responses to magnetic cortical stimulation recovered rapidly despite maintained ischaemia. 5. In conclusion, during sustained MVCs, voluntary activation becomes less than optimal so that force can be increased by stimulation of the motor cortex or the motor nerve. Complex changes in excitability of the motor cortex also occur with fatigue, but can be dissociated from the impairment of voluntary activation. We argue that inadequate neural drive effectively ‘upstream’ of the motor cortex must be one site involved in the genesis of central fatigue.
Calcium dependence of calcium extrusion and calcium uptake in mouse pancreatic acinar cells.
Authors: Camello P , Gardner J , Petersen O H , Tepikin A V
Published: December 2014

1. The droplet technique was used to investigate the calcium dependence of calcium extrusion from pancreatic acinar cells with preserved intracellular environments. The calcium dependence of calcium extrusion indicated a strong co‐operativity (Hill coefficient, 3). The half‐maximal rate of calcium extrusion occurred at an intracellular free calcium concentration ([Ca2+]i) of approximately 200 nM. At [Ca2+]i levels higher than 400 nM the calcium extrusion mechanism was almost completely saturated. 2. The rate of [Ca2+]i recovery was measured with the same cells under conditions where both calcium extrusion and calcium reuptake occurred simultaneously and under conditions when calcium reuptake was prevented and recovery depended entirely upon calcium extrusion. The rate of [Ca2+]i recovery due to calcium reuptake displayed a very sharp dependence on [Ca2+]i. The rate of [Ca2+]i recovery due to reuptake increased approximately 10 times (from 4.3 to 44.1 nM s‐1) for an increase of [Ca2+]i of only 100 nM (from 120 to 220 nM). 3. With a decrease of [Ca2+]i the ratio of rate of calcium extrusion to rate of calcium uptake into internal stores increased, indicating that extrusion plays a more important role at low [Ca2+]i levels. Data for [Ca2+]i recovery rates due to extrusion and due to reuptake allowed us to evaluate the absolute rate of calcium translocation into the internal stores during the recovery process. When [Ca2+]i = 350 nM the total (i.e. bound and free) calcium concentration in the cytosol decreased by approximately 100 microM s‐1 due to calcium uptake into internal stores. The rate of uptake was approximately 20 times slower when [Ca2+]i = 120 nM.
Arginine metabolism in cat kidney.
Authors: Levillain O , Parvy P , Hus-Citharel A
Published: December 2014

1. Arginine is essential for growth in the kitten and, because of the resulting hyperammonaemia, in the adult cat an arginine‐free diet is life threatening. 2. The kidney is the main site of arginine synthesis. 3. This study was performed to determine whether the cat kidney synthesizes arginine and to establish which factors, such as low citrullinaemia, defects of argininosuccinate synthase and lyase activities or high renal arginase activity, might limit renal arginine production. 4. Identified nephron segments were isolated by microdissection from collagenase‐treated cat kidney. 5. Arginine metabolism was studied by incubating the nephron segments with either physiological concentrations of L‐[ureido‐14C]citrulline (anabolism) or L‐[guanido‐14C]‐arginine (catabolism). Arginine and urea were measured by a micro‐enzymatic method. Amino acids were measured by HPLC. 6. In cat blood, the citrulline, but not the arginine, concentration was very low by comparison with other species. 7. Arginine synthesis occurred almost entirely in the proximal tubule, the highest rate occurring in the proximal convoluted tubule and the lowest in the medullary straight proximal tubule. 8. Arginase activity was restricted to the proximal tubule. Urea production increased from the convoluted towards the medullary straight tubule. 9. The limited capacity of the cat kidney to produce arginine in vivo may result from the low blood concentration of citrulline and from the high arginase activity in the various proximal cells with the ability to synthesize arginine.
Ionic basis of spike after‐depolarization and burst generation in adult rat hippocampal CA1 pyramidal cells.
Authors: Azouz R , Jensen M S , Yaari Y
Published: December 2014

1. Intracellular recordings in adult rat hippocampal slices were used to identify the ionic conductances underlying active spike after‐depolarization (ADP) and intrinsic burst firing in the somata of CA1 pyramidal cells (PCs). To test the ‘Ca2+ hypothesis’, Ca2+ currents were suppressed by replacing the Ca2+ in the saline with either Mn2+ or Mg2+. Alternatively, the inorganic Ca2+ channel blockers Cd2+ (0.5 mM) or Ni2+ (2 mM) were added to the saline. To test the ‘Na+ hypothesis’, Na+ currents were blocked with tetrodotoxin (TTX; 0.5 microM). 2. The suppression of Ca2+ currents blocked the fast after‐hyperpolarization (AHP) generated by the fast Ca(2+)‐gated K+ current Ic, while enhancing the amplitude and duration of active spike ADPS. 3. Evoked and spontaneous burst firing was preserved undiminished following Ca2+ current suppression, while the propensity to fire bursts increased in many cases. The postburst medium AHP (generated primarily by the muscarine‐sensitive voltage‐gated K+ current, IM) was not affected by this treatment, which blocked the slow AHP (generated by the slow Ca(2+)‐gated K+ current, IAHP). 4. TTX strongly suppressed active ADPs and intrinsic bursts before substantially reducing the threshold, rate of rise and amplitude of solitary spikes. 5. In Ca(2+)‐free saline, caesium‐filled PCs generated large, plateau ADPs following an initial burst of fast spikes. Application of TTX suppressed these ADPs before solitary fast spikes appeared to be reduced. 6. Injection of brief, just subthreshold depolarizing current pulses into bursters evoked slow depolarizing potentials lasting up to 50 ms. These persisted after suppression of Ca2+ currents and were entirely blocked by TTX. 7. We conclude that active spike ADPs and intrinsic bursts in the somata of adult CA1 PCs are generated by a low voltage‐gated, persistent Na+ current. Burst termination is mediated by voltage‐gated K+ currents activated during the burst (most likely IM), rather than by the Ca(2+)‐gated K+ currents Ic and IAHP. The latter currents downregulate the innate tendency of CA1 PCs to burst (Ic) and limit the rate of spontaneous burst firing (IAHP).
Relationship of firing intervals of human motor units to the trajectory of post‐spike after‐hyperpolarization and synaptic noise.
Authors: Matthews P B
Published: December 2014

1. Interspike interval distributions from human motor units of a variety of muscles were analysed to assess the role of synaptic noise in excitation. The time course of the underlying post‐spike after‐hyperpolarization (AHP) was deduced by applying a specially developed transform to the interval data. Different firing rates were studied both by varying the firing voluntarily, and by selecting subpopulations of spikes for a given firing rate from long recordings with slight variations in frequency. 2. At low firing rates the interval histograms had an exponential tail. Thus at long intervals, the motoneurone was randomly excited by noise and its post‐spike AHP was complete. This contrasts with the firing produced by intracellular current injection in the cat, when the membrane potential increases linearly until threshold is reached. The interval histogram was therefore analysed with the aid of a model of synaptic excitation to deduce the mean ‘trajectory’ of membrane voltage in the last part of the interspike interval. 3. The computer model, described in the Appendix, was used to determine the effect of the mean level of membrane potential on the probability of a spike being excited, per unit time, during an on‐going interspike interval. All variables were treated as voltages, with synaptic noise simulated by time‐smoothed Gaussian noise. This enabled an interval distribution to be transformed into a segment of the underlying trajectory of the membrane potential; the potential was expressed in terms of the noise amplitude and the spike threshold. 4. At low firing rates, the equilibrium value of the membrane voltage trajectory lay well below threshold; the deviation typically corresponded to the standard deviation of the noise or more. The noise standard deviation was estimated to be about 2 mV. 5. With increasing mean firing rate, the near‐threshold portion of the trajectory obtainable from the histogram occurred earlier, was steeper and rose to a higher level. Trajectories for different firing rates fell on the same curve after shifting them vertically by varying amounts. The curve was taken to represent the AHP of the motoneurone and was closely exponential. The shift of the trajectory gave its mean synaptic drive. The duration of the AHP varied between units and was longer than average for units from soleus muscle. 6. Further modelling showed that summation of noise with the AHP can explain the well‐known changes in discharge variability that occur as firing rate increases. 7. It is concluded that synaptic noise plays a major role in the excitation of tonically firing human motoneurones and that the noiseless motoneurone with a linear trajectory provides an inadequate model for the conscious human. This is of interest in relation to various standard measures of human motor unit activity such as short‐term synchronization.
Role of calbindin‐D9k in buffering cytosolic free Ca2+ ions in pig duodenal enterocytes.
Authors: Schröder B , Schlumbohm C , Kaune R , Breves G
Published: December 2014

1. The aim of the present study was to test whether the vitamin D‐dependent Ca(2+)‐binding protein calbindin‐D9k could function as an important cytosolic Ca2+ buffer in duodenal enterocytes while facilitating transepithelial active transport of Ca2+ ions. For the investigations we used dual‐wavelength, fluorescence ratio imaging, with fura‐2 as the Ca(2+)‐sensitive dye, to measure changes in cytosolic concentrations of free Ca2+ ions ([Ca2+]i) in isolated pig duodenal enterocytes affected by different cytosolic calbindin‐D9k concentrations. 2. Epithelial cells were obtained from weaned piglets with normal calbindin‐D9k concentrations (con‐piglets), from piglets with low calbindin‐D9k levels due to inherited calcitriol deficiency caused by defective renal 25‐hydroxycholecalciferol D3‐1 alpha‐hydroxylase activity (def‐piglets), and from piglets with reconstituted calbindin‐D9k concentrations, i.e. def‐animals treated with high doses of vitamin D3 which elevated plasma calcitriol levels by extrarenal production (def‐D3‐piglets). Basal levels of [Ca2+]i ranged between 170 and 205 nM and did not differ significantly between the groups. 3. After addition of 5 mM theophylline, the [Ca2+]i in enterocytes from con‐piglets doubled during the 10 min incubation. This effect, however, was three times higher in enterocytes from def‐piglets compared with those from con‐piglets. Similar results were obtained after 4 min incubation of enterocytes from con‐ and def‐piglets in the presence of 1 microM ionomycin. In preparations from def‐D3‐piglets, ionomycin‐induced increases in [Ca2+]i were significantly lower compared with enterocytes from def‐piglets and were not different from the control values. 4. From the results, substantial support is given for the hypothesis that one of the major functions of mucosal calbindin‐D9k is the effective buffering of Ca2+ ions.
Ischaemia‐sensitive sympathetic afferents innervating the gastrointestinal tract function as nociceptors in cats.
Authors: Pan H L , Longhurst J C
Published: December 2014

1. Activation of sympathetic visceral afferents during mesenteric ischaemia induces visceral pain and reflexly excites the cardiovascular system. The present study investigated the differential responses of ischaemically sensitive and insensitive sympathetic C fibre afferents to graded distension of the gastrointestinal tract. 2. Single‐unit activity of C fibre afferents innervating the stomach, duodenum and jejunum was recorded from the right thoracic sympathetic chain of anaesthetized cats. Ischaemically sensitive and insensitive C fibre afferents were identified according to their response to 5‐20 min of ischaemia. The functional characteristics of the stimulus‐response relationships of afferents were determined by distension of a balloon placed in the corresponding segment of gastrointestinal tract. 3. The results show that ischaemically insensitive C fibre afferents had a lower threshold in response to distension (13 +/‐ 5 mmHg, n = 10). The discharge frequency of these afferents was saturated within a low pressure range of distension. However, ischaemically sensitive C fibre afferents had a high threshold (86 +/‐ 12 mmHg, n = 10) and a larger peak response to mechanical distension in the noxious range (60‐180 mmHg). There were no differences between ischaemically sensitive and insensitive C fibre afferents with regard to their testing activity or responses to bradykinin (10 micrograms I.A.). 4. This study demonstrates that the gastrointestinal system is innervated by low and high threshold sympathetic C fibre afferents, the latter having the distinct ability to encode nociceptive information such as excessive distension and ischaemia.
Total and sarcoplasmic reticulum calcium contents of skinned fibres from rat skeletal muscle.
Authors: Fryer M W , Stephenson D G
Published: December 2014

1. The Ca2+ content of single mammalian skeletal muscle fibres was determined using a novel technique. Mechanically skinned fibres were equilibrated with varying amounts of the Ca2+ buffer BAPTA and were then lysed in a detergent‐paraffin oil emulsion. The subsequent myofilament force response was used to estimate the additional amount of Ca2+ bound to BAPTA following lysis of intracellular membranes. 2. The total endogenous Ca2+ content (corrected for endogenous Ca2+ buffering) of fast‐twitch (FT) and slow‐twitch (ST) fibres at a myoplasmic pCa (‐log[Ca2+]) of 7.15 was 1.32 +/‐ 0.02 and 1.35 +/‐ 0.08 mM per fibre volume, respectively. The sarcoplasmic reticulum (SR) component of these estimates was calculated as 1.01 and 1.14 mM, respectively, which normalized to SR volume corresponds to resting SR Ca2+ contents of 11 and 21 mM, respectively. 3. Equilibration of ‘resting’ fibres with low myoplasmic [Ca2+] (pCa 7.67‐9.00) elicited a time‐dependent decrease in Ca2+ content in both fibre types. Equilibration of resting fibres with higher myoplasmic [Ca2+] (pCa 5.96‐6.32) had no effect on the Ca2+ content of ST fibres but increased the Ca2+ content of FT fibres. The maximum steady‐state total Ca2+ content (3.85 mM) was achieved in FT fibres after 3 min equilibration at pCa 5.96. Equilibration at higher myoplasmic [Ca2+] was less effective, probably due to Ca2(+)‐induced Ca2+ release from the SR. 4. Exposure of fibres to either caffeine (30 mM, pCa approximately 8, 2 min) or low myoplasmic [Mg2+] (0.05 mM, pCa approximately 9, 1 min) released approximately 85% of the resting SR Ca2+ content. The ability of caffeine to release SR Ca2+ was dependent on the myoplasmic Ca2+ buffering conditions. 5. The results demonstrate that the SR of ST fibres is saturated with Ca2+ at resting myoplasmic [Ca2+] while the SR of FT fibres is only about one‐third saturated with Ca2+ under equivalent conditions. These differences suggest that the rate of SR Ca2+ uptake in FT fibres is predominantly controlled by myoplasmic [Ca2+] while that of ST fibres is more likely to be limited by the [Ca2+] within the SR lumen.
Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells.
Authors: Chizhmakov I V , Geraghty F M , Ogden D C , Hayhurst A , Antoniou M , Hay A J
Published: December 2014

1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole‐cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti‐influenza drug rimantadine. 2. The ion selectivity of the rimantadine‐sensitive current through M2 was determined. Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl‐ concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10(‐7). 3. The M2 conductance increased as external pH decreased below 8.5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 microM and 1 microM, respectively, under physiological conditions. 4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans‐Golgi pH during virus infection.
Modulation of excitatory synaptic transmission by low concentrations of glutamate in cultured rat hippocampal neurons.
Authors: Zorumski C F , Mennerick S , Que J
Published: December 2014

1. The effects of low micromolar concentrations of glutamate on fast excitatory synaptic responses were studied in microcultures of postnatal rat hippocampal neurons using whole‐cell patch clamp recordings. 2. Glutamate depressed the alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor component of excitatory autaptic currents (EACs) with an EC50 of 3.8 microM. 3. Both pre‐ and postsynaptic effects contributed to the depression of AMPA receptor‐mediated EACs. Cyclothiazide and wheatgerm agglutinin, agents which inhibit AMPA receptor desensitization, partially reversed the depression produced by glutamate, as did pertussis toxin, an agent that blocks presynaptic inhibition mediated by metabotropic glutamate receptors. 4. In neurons in which both the AMPA and N‐methyl‐D‐aspartate (NMDA) receptor components of EACs were examined, low concentrations of glutamate depressed the NMDA component of EACs to a greater extent. The EC50 for inhibiting the NMDA component was 1.3 microM. 5. Calcium‐dependent desensitization of postsynaptic NMDA receptors contributed to the depression of NMDA receptor‐mediated synaptic responses. Both depolarization of postsynaptic neurons to +70 mV to decrease Ca2+ influx via NMDA channels and inclusion of high concentrations of a calcium chelator in recording pipettes decreased the depression of NMDA receptor‐mediated EACs. 6. Threo‐3‐hydroxy‐aspartate (THA), an inhibitor of glutamate transport, depressed EACs by about 10% and increased the degree of depression produced by 2.5 microM glutamate, suggesting that glutamate transport in microcultures helps to control ambient glutamate levels. 7. Because the normal extracellular concentration of glutamate is about 1 microM, these results suggest that the ambient glutamate level is an important determinant of synaptic efficacy. Relatively small changes in extracellular glutamate can alter fast excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms.
The properties and distribution of inward rectifier potassium currents in pig coronary arterial smooth muscle.
Authors: Quayle J M , Dart C , Standen N B
Published: December 2014

1. Whole‐cell potassium currents were studied in single smooth muscle cells enzymatically isolated from pig coronary arteries. 2. In cells isolated from small diameter branches of the left anterior descending coronary artery (LAD), an inward rectifier potassium current (IK(IR)) was identified, which was inhibited by extracellular barium ions, suggesting the presence of inward rectifier potassium (KIR) channels. 3. The conductance for IK(IR) measured in 6, 12, 60 and 140 mM extracellular potassium was a function of membrane potential and the extracellular potassium concentration. 4. On hyperpolarization, IK(IR) activated along an exponential time course with a time constant that was voltage dependent. 5. Inward rectifier current was compared in cells isolated from coronary vessels taken from different points along the vascular tree. Current density was greater in cells isolated from small diameter coronary arteries; at ‐140 mV it was ‐20.5 +/‐ 4.4 pA pF‐1 (n = 23) in 4th order branches of the LAD, but ‐0.8 +/‐ 0.2 pA pF‐1 (n = 11) in the LAD itself. 6. In contrast to IK(IR), there was little effect of arterial diameter on the density of voltage‐dependent potassium current; densities at +30 mV were 12.8 +/‐ 1.3 pA pF‐1 (n = 19) in 4th order branches and 17.4 +/‐ 3.1 pA pF‐1 (n = 11) in the LAD. 7. We conclude that KIR channels are present in pig coronary arteries, and that they are expressed at a higher density in small diameter arteries. The presence of an enhanced IK(IR) may have functional consequences for the regulation of cell membrane potential and tone in small coronary arteries.
Impaired sympathetic vascular regulation in humans after acute dynamic exercise.
Authors: Halliwill J R , Taylor J A , Eckberg D L
Published: December 2014

1. The reduction in vascular resistance which accompanies acute dynamic exercise does not subside immediately during recovery, resulting in a post‐exercise hypotension. This sustained vasodilatation suggests that sympathetic vascular regulation is altered after exercise. 2. Therefore, we assessed the baroreflex control of sympathetic outflow in response to arterial pressure changes, and transduction of sympathetic activity into vascular resistance during a sympatho‐excitatory stimulus (isometric handgrip exercise) after either exercise (60 min cycling at 60% peak aerobic power (VO2,peak)) or sham treatment (60 min seated rest) in nine healthy subjects. 3. Both muscle sympathetic nerve activity and calf vascular resistance were reduced after exercise (‐29.7 +/‐ 8.8 and ‐25.3 +/‐ 9.1%, both P < 0.05). The baroreflex relation between diastolic pressure and sympathetic outflow was shifted downward after exercise (post‐exercise intercept, 218 +/‐ 38 total integrated activity (heartbeat)‐1; post‐sham intercept, 318 +/‐ 51 total integrated activity (heartbeat)‐1, P < 0.05), indicating less sympathetic outflow across all diastolic pressures. Further, the relation between sympathetic activity and vascular resistance was attenuated after exercise (post‐exercise slope, 0.0031 +/‐ 0.0007 units (total integrated activity)‐1 min; post‐sham slope, 0.0100 +/‐ 0.0033 units (total integrated activity)‐1 min, P < 0.05), indicating less vasoconstriction with any increase in sympathetic activity. 4. Thus, both baroreflex control of sympathetic outflow and the transduction of sympathetic activity into vascular resistance are altered after dynamic exercise. We conclude that the vasodilation which underlies post‐exercise hypotension results from both neural and vascular phenomena.
Possible role of diadenosine polyphosphates as modulators of cardiac sensory‐motor neurotransmission in guinea‐pigs.
Authors: Rubino A , Burnstock G
Published: December 2014

1. Isolated guinea‐pig atria were used to study the neuromodulatory effect of diadenosine polyphosphates (APnA) on cardiac capsaicin‐sensitive sensory‐motor neurotransmission. 2. In the presence of atropine, guanethidine and propranolol, electrical field stimulation (EFS) of the atrial preparations evoked a positive inotropic response which is known to be mediated by release of calcitonin gene‐related peptide (CGRP) from sensory‐motor nerves. P1,P2‐diadenosine pyrophosphate (AP2A), P1,P3‐diadenosine triphosphate (AP3A), P1,P4‐diadenosine tetraphosphate (AP4A), P1,P5‐diadenosine pentaphosphate (AP5A) and P1,P6‐diadenosine hexaphosphate (AP6A) inhibited in a concentration‐dependent way (0.1‐30 microM) cardiac responses to EFS. The inhibitory effect of APnA was mimicked by adenosine. 3. All the APnA tested had a direct negative inotropic effect, by reducing in a concentration‐dependent manner the basal contractile tension. The inotropism of APnA was comparable to that of adenosine. 4. Both inhibition of cardiac responses to EFS and negative inotropism of AP2A, AP3A and AP4A were sensitive to the antagonism by the A1 adenosine receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 0.1‐1 nM). The extent of antagonism of DPCPX for the APnA tested was comparable to that for adenosine. 5. Despite the direct negative inotropism, AP4A tested at the highest concentration used did not affect the cardiac responses to the neurotransmitter CGRP, applied exogenously. 6. These results have demonstrated that in isolated guinea‐pig atria APnA inhibited sensory‐motor neurotransmission, without affecting cardiac responses to exogenous CGRP. The effect of APnA was sensitive to antagonism by DPCPX, which suggests it operates via the activation of prejunctional A1 adenosine receptors. A postjunctional negative inotropism was also shown, mediated by myocardial A1 adenosine receptors.
Neuropeptide Y‐enhanced diuresis and natriuresis in anaesthetized rats is independent of renal blood flow reduction.
Authors: Bischoff A , Erdbrügger W , Smits J , Michel M C
Published: December 2014

1. Neuropeptide Y (NPY) has been reported to enhance diuresis and natriuresis in anaesthetized rats although it is a potent renal vasoconstrictor in vitro in vivo in several species. Therefore, we have investigated anaesthetized rats to see whether reduction in renal blood flow (RBF) and enhancement of diuresis and natriuresis can occur concomitantly, and how diuresis and natriuresis might be enhanced despite reduced RBF. 2. Systemic or intrarenal NPY infusion (0.03‐3 micrograms kg‐1 min‐1) had only a small effect on mean arterial pressure (maximal increase 15‐20 mmHg) and heart rate (maximal decrease 30 beats min‐1) but dose‐dependently reduced RBF (maximal peak reduction 3 ml min‐1) Endogenous creatinine clearance was not significantly altered. 3. In anaesthetized rats systemic infusion of 1 or 3 micrograms kg‐1 min‐1 NPY enhanced urine formation and sodium and calcium excretion by a maximum of 110, 110 and 45%, respectively, but did not alter potassium excretion. Enhancement of diuresis was also detectable in conscious rats. 4. The diuretic and natriuretic effects of systemically infused NPY were at least partly maintained in rats with decapsulated kidneys and in rats where NPY‐induced increase of renal perfusion pressure was excluded mechanically by an adjustable clamp placed on the abdominal aorta. 5. Intrarenal infusion of 0.3 or 1 microgram kg‐1 min‐1 NPY reduced RBF to a greater extent than systemic infusion (maximal peak reduction 4 ml min‐1) but caused a smaller enhancement or even a reduction of urine formation and sodium excretion. 6. We conclude that systemic infusion of NPY reduces RBF by a direct effect on the renal vasculature. Systemic NPY infusion enhances urine formation and sodium and calcium excretion. This occurs independently (at least in part) of pressure natriuresis by formation and/or release of an extrarenal factor which might act on distal tubules and/or collecting ducts.
Presynaptic calcium currents at voltage‐clamped excitor and inhibitor nerve terminals of crayfish.
Authors: Wright S N , Brodwick M S , Bittner G D
Published: December 2014

1. A two‐electrode voltage clamp was used to record calcium currents from the excitatory and inhibitory nerve terminals that innervate the crayfish (Procambarus spp.) opener muscle. Other voltage‐dependent currents were blocked with tetrodotoxin, 3,4‐diaminopyridine, 4‐aminopyridine and tetraethylammonium. 2. The presynaptic calcium current at both excitatory and inhibitory synapses was blocked by cadmium and omega‐agatoxin IVA but was not affected by omega‐conotoxin GVIA, omega‐conotoxin MVIIC or nifedipine, suggesting that the calcium currents flow through P‐type calcium channels. 3. Current‐voltage (I‐V) relations at both excitatory and inhibitory synapses are similar, with current activation near ‐40 mV, peak current near ‐10 mV and current reversal at membrane potentials greater than +25 mV. I‐V relations were scaled along the current axis by partial calcium current blockade with cobalt, suggesting that series resistance and space‐clamp errors were small. 4. A subset of terminals on one muscle fibre was locally superfused with a physiological saline containing barium; the rest of the preparation was superfused with a physiological saline containing calcium channel antagonists. Under such conditions the characteristics of the I‐V relation were very similar to the I‐V relations recorded when the entire preparation was bathed in physiological levels of calcium, suggesting that the space clamp was adequate. 5. Calcium channel activation, as determined from tail current analyses, was similar when the entire preparation was bathed in physiological levels of calcium or if terminals on one muscle fibre were locally superfused with barium. 6. During a 30 ms depolarization, calcium currents inactivated to a greater extent in inhibitory than in excitatory terminals. The inactivation was of small magnitude (< 20%) and was eliminated by intracellular injection of the calcium chelator BAPTA, suggesting that the inactivation was calcium dependent. 7. These data show that biophysical and pharmacological properties of calcium currents at crayfish neuromuscular junctions resemble those found at stellate synapses in squid.