bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuropeptide gene expression and capsaicin‐sensitive primary afferents: maintenance and spread of adjuvant arthritis in the rat.
Authors: Donaldson L F , McQueen D S , Seckl J R
Published: December 2014

1. Many experimental and clinical arthritides are characterized by their bilateral nature. There is strong evidence to suggest that this bilateral spread may be mediated by a neuronal mechanism. We have previously shown early and sustained induction of mRNAs encoding preprotachykinin (PPT) and calcitonin gene‐related peptide (CGRP) in dorsal root ganglion (DRG) neurons innervating an inflamed, arthritic joint. We have now investigated the involvement of capsaicin‐sensitive primary afferents and the expression of neuropeptide mRNAs in the maintenance and bilateral spread of mild adjuvant‐induced arthritis in the rat. 2. Capsaicin was applied perineurally to either the left (Cap‐L) or right (Cap‐R) sciatic nerve of halothane‐anaesthetized male Han Wistar rats. Two weeks after capsaicin lesioning, arthritis was induced by injection of Freund's complete adjuvant (FCA) around the left ankle at a dose that caused inflammation of the left ankle joint, and a delayed (14 days) contralateral (right) ankle arthritis. Arthritis was monitored for 15 days after injection, when animals were killed and the lumbar DRG dissected. PPT, CGRP, somatostatin (SS), and vasoactive intestinal polypeptide (VIP) mRNA expression was determined in L5 DRG using in situ hybridization. 3. Spread of inflammation/arthritis to the right limb was associated with bilateral rises in PPT and CGRP mRNA expression in L5 DRG. SS mRNA expression in right DRG was unaffected by spread of inflammation. FCA‐L+Cap‐L reduced left joint swelling and prevented spread of arthritis to the right joint when assessed by joint swelling. This inhibition of spread of arthritis was associated with significant reductions in all left L5 DRG neuropeptide mRNAs compared with controls, and the rise in right L5 DRG PPT mRNA expression seen in FCA‐L‐alone animals was blocked. FCA‐L+Cap‐R also reduced left joint swelling and prevented the spread of inflammation to the right ankle. This lesion prevented the rise in PPT and CGRP mRNA expression seen in right DRG with FCA‐L alone. 4. These findings suggest a role for capsaicin‐sensitive primary afferents and the primary afferent neuropeptides encoded by PPT and CGRP mRNA in the maintenance and spread of arthritis.
Estimate of net calcium fluxes and sarcoplasmic reticulum calcium content during systole in rat ventricular myocytes.
Authors: Negretti N , Varro A , Eisner D A
Published: December 2014

1. The experiments were performed on voltage‐clamped cells in which intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo‐1 (acetoxymethyl ester (AM) loading). When cells were stimulated with a short (100 ms) depolarizing pulse, following a rest, the magnitude of the first systolic calcium transient was greater than that in the steady state (rest potentiation) and decayed to its steady level over a few stimuli. If a longer pulse (800 ms) was used then the systolic calcium transient was either unaffected or increased in magnitude following a rest. During constant stimulation, if the length of the pulse is decreased, then the magnitude of the calcium transient decreased reversibly over several beats. 2. The calcium entry into the cell was measured from the integral of the inward calcium current and the efflux from the Na(+)‐Ca2+ exchange current on repolarization. During the negative staircase the calcium current was approximately constant whilst the Na(+)‐Ca2+ exchange current decayed in parallel with the systolic calcium transient. A net loss of calcium from the cell can be calculated from the extra Na(+)‐Ca2+ exchange current following the initial pulses. 3. The application of caffeine produces a transient increase of both [Ca2+]i and an inward Na(+)‐Ca2+ exchange current. The integral of this current can be used to estimate the caffeine‐releasable calcium content of the sarcoplasmic reticulum (SR), which decreases following stimulation with short compared to long pulses. This difference in SR calcium content is quantitatively similar to that estimated from the sarcolemmal currents. 4. At a given membrane potential, the relationship between [Ca2+]i and current during the caffeine exposure can be used to estimate the Na(+)‐Ca2+ exchange flux from the measured [Ca2+]i and thence the Na(+)‐Ca2+ exchange flux during depolarization. 5. For a long depolarizing pulse the extrusion of calcium from the cell on Na(+)‐Ca2+ exchange is comparable to the entry on the calcium current. In contrast, for short pulses the extrusion of calcium on the Na(+)‐Ca2+ exchange immediately after the pulse is greater than the entry during the pulse on the calcium current. 6. These results show that rest potentiation can be correlated with changes in the amount of calcium stored in the SR and this, in turn, can be accounted for by sarcolemmal fluxes.
Modification by protons of frog skeletal muscle KATP channels: effects on ion conduction and nucleotide inhibition.
Authors: Vivaudou M , Forestier C
Published: December 2014

1. The molecular mechanisms underlying pH regulation of skeletal muscle ATP‐sensitive K+ (KATP) channels were studied using the patch clamp technique in the inside‐out configuration. Two effects of intracellular protons were studied in detail: the decrease in magnitude of single‐channel currents and the increase in open probability (Po) of nucleotide‐inhibited channels. 2. The pH dependence of inward unit currents under different ionic conditions was in poor agreement with either a direct block of the pore by protons or an indirect proton‐induced conformational change, but was compatible with the protonation of surface charges located near the cytoplasmic entrance of the pore. This latter electrostatic mechanism was modelled using Gouy‐Chapman‐Stern theory, which predicted the data accurately with a surface charge density of about 0.1 negative elementary charges per square nanometre and a pK (pH value for 50% effect) value for protonation of these charges of 6.25. The same mechanism, i.e. neutralization of negative surface charges by cation binding, could also account for the previously reported reduction of inward unit currents by Mg2+. 3. Intracellular alkalization did not affect Po of the KATP channels. Acidification increased Po. In the presence of 0.1 mM ATP (no Mg2+), the channel activation vs. pH relationship could be fitted with a sigmoid curve with a Hill coefficient slightly above 2 and a pK value of 6. This latter value was dependent on the ATP concentration, decreasing from 6.3 in 30 microM ATP to 5.3 in 1 microM ATP. 4. Conversely, the channel inhibition vs. ATP concentration curve was shifted to the right when the pH was lowered. At pH 7.1, the ATP concentration causing half‐maximal inhibition was about 10 microM. At pH 5.4, it was about 400 microM. The Hill coefficient values remained slightly below 2. Similar effects were observed when ADP was used as the inhibitory nucleotide. 5. These results confirm that a reciprocal competitive link exists between proton and nucleotide binding sites. Quantitatively, they are in full agreement with a steady‐state model of a KATP channel possessing four identical protonation sites (microscopic pK, 6) allosterically connected to the channel open state and two identical nucleotide sites (microscopic ATP dissociation constant, approximately 30 microM) connected to the closed state.
Enhancing effect of calmodulin on Ca(2+)‐induced Ca2+ release in the sarcoplasmic reticulum of rabbit skeletal muscle fibres.
Authors: Ikemoto T , Iino M , Endo M
Published: December 2014

1. We analysed the effect of calmodulin on Ca(2+)‐induced Ca2+ release (CICR) in the sarcoplasmic reticulum (SR) using chemically skinned fibres of rabbit psoas muscle. Ca2+ release was measured using fura‐2 microfluorometry. 2. In saponin‐skinned fibres, calmodulin potentiated Ca2+ release at low Ca2+ concentrations (< 3 microM), while it showed an inhibitory effect at high Ca2+ concentrations (3‐30 microM). 3. Co‐application of ryanodine and calmodulin at 0.3 microM Ca2+, but not ryanodine alone, induced a decline in the Ca2+ uptake capacity of the SR, an effect expected from the open‐lock of active CICR channels by ryanodine. Thus, potentiation of Ca2+ release by calmodulin at low Ca2+ concentrations can be regarded as a result of the activation of the ryanodine receptor. 4. Greater concentrations of calmodulin were required for potentiation of CICR at low Ca2+ concentrations (1 microM) than for inhibition at high Ca2+ concentrations (10 microM). 5. In beta‐escin‐permeabilized fibres in which intrinsic calmodulin was retained, the rates of CICR were similar to those measured in the presence of 1 microM calmodulin in saponin‐permeabilized fibres. 6. These results suggest that calmodulin plays an important role in the regulation of CICR channels in intact skeletal muscle fibres.
Rhythmical bursts induced by NMDA in guinea‐pig cholinergic nucleus basalis neurones in vitro.
Authors: Khateb A , Fort P , Serafin M , Jones B E , Mühlethaler M
Published: December 2014

1. Intracellular recordings were performed in neurones within the basal forebrain of guinea‐pig brain slices. Following injection of biocytin (or biotinamide), a subset of recorded neurones which displayed distinct intrinsic membrane properties were confirmed as being cholinergic by immunohistochemical staining for choline acetyltransferase (ChAT). They were all located within the nucleus basalis magnocellularis. The response of the cholinergic cells to NMDA and to the agonists of the other glutamate receptors was tested by bath application of NMDA, t‐ACPD, AMPA and kainate. 2. When depolarized from a hyperpolarized level, cholinergic basalis neurones display the intrinsic ability to discharge in rhythmic bursts that are generated by low‐threshold Ca2+ spikes. In control solution, these rhythmic bursts were not sustained for more than 5‐6 cycles. However, in the presence of NMDA when the membrane was held at a hyperpolarized level, low‐threshold bursting activity was sustained for prolonged periods of time. This activity could be reversibly eliminated by D(‐)‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5), showing that it depended upon specific activation of NMDA receptors. 3. NMDA‐induced, voltage‐dependent, rhythmic depolarizations persisted in the presence of tetrodotoxin (TTX), indicating that they did not depend upon a TTX‐sensitive Na+ current and were generated postsynaptically. The rhythmic depolarizations were, however, eliminated by the partial replacement of Na+ with choline, demonstrating that they did depend upon Na+, the major carrier of the NMDA current. 4. In the presence of TTX, the NMDA‐induced rhythmic depolarizations were also eliminated by removal of Ca2+ from or addition of Ni2+ to the bath, indicating that they also depended upon Ca2+, which is carried by both the NMDA current and the low‐threshold Ca2+ current. The duration of the rhythmic depolarizations was increased in the presence of apamin, suggesting that the repolarization of the cells depended in part upon a Ca(2+)‐activated K+ (SK) conductance, but that other mechanisms were additionally involved in the repolarization phase of the bursting. 5. In both the absence and presence of TTX, the NMDA‐induced rhythmic activity persisted when Mg2+ was removed from the medium, indicating that the sustained rhythmic depolarizations did not hinge upon the Mg2+ block of the NMDA channels during hyperpolarization. The voltage dependence of the NMDA‐induced rhythmic depolarizations in the absence of Mg2+ appeared to be determined by the properties of the low‐threshold Ca2+ spike in the cholinergic basalis neurones.(ABSTRACT TRUNCATED AT 400 WORDS)
Hypothalamic modulation of laryngeal reflexes in the anaesthetized cat: role of the nucleus tractus solitarii.
Authors: Dawid‐Milner M S , Silva-Carvalho L , Goldsmith G E , Spyer K M
Published: December 2014

1. This investigation was initiated because activation of laryngeal afferents, either by electrical stimulation of the superior laryngeal nerve (SLN) or by natural stimulation of receptors in the laryngeal mucosa, results in a cardiorespiratory response comprising bradycardia, hypotension and apnoea (phrenic nerve activity was suppressed). This pattern of response is qualitatively equivalent to the response that is evoked on activation of the arterial baroreceptors. 2. Preliminary studies indicated that the effects of activating the SLN were suppressed during stimulation in the hypothalamic defence area (HDA) at points that also blocked the effects of baroreceptor stimulation. 3. Recordings were taken from seventy‐two neurones localized within the ipsilateral nucleus tractus solitarii (NTS) whose activity was modified by SLN stimulation. Sixty neurones responded with an EPSP on SLN stimulation; nine of these had an inspiratory firing pattern. Five neurones were seen to receive an IPSP on SLN stimulation. 4. Five respiratory SLN‐activated neurones were unresponsive to stimulation of the other nerve inputs, whilst four received convergent EPSP inputs on sinus nerve (SN) stimulation. One cell of these four also received inputs from the aortic and the vagus nerves. Sixty‐one non‐respiratory SLN‐activated neurones also received convergent inputs from the sinus nerve. Of these, fifty displayed an EPSP, four an IPSP and seven an EPSP‐IPSP. Fifteen neurones also received inputs from the aortic nerve and seventeen from the vagus. 5. From the population of neurones affected by SLN stimulation, twenty‐four of seventy were also influenced by HDA stimulation (3 were respiratory cells). Sixteen of these responses consisted of an EPSP (2 respiratory cells), five of an IPSP (1 respiratory cell) and three of an EPSP‐IPSP. 6. In neurones receiving an IPSP on HDA stimulation, the SLN‐evoked excitatory response was reduced throughout the period of HDA‐evoked inhibition. These neurones were all shown to receive excitatory inputs from the arterial baroreceptors and laryngeal mechanoreceptors. 7. Additionally, in the thirty‐seven neurones that were excited by SLN stimulation but received no direct synaptic input on HDA stimulation, a conditioning stimulus to the HDA evoked a block of SLN‐evoked responses without an accompanying change in membrane potential. Several of these neurones were also affected by both baroreceptor and laryngeal mechanoreceptor stimulation. 8. These observations are discussed in the context of the role of the NTS in cardiorespiratory control. The potential importance of these interactions in respiratory distress are highlighted and the implications for the organization of central pathways for the control of autonomic and respiratory function are discussed.
Hypothalamic modulation of the arterial chemoreceptor reflex in the anaesthetized cat: role of the nucleus tractus solitarii.
Authors: Silva-Carvalho L , Dawid‐Milner M S , Goldsmith G E , Spyer K M
Published: December 2014

1. There is evidence in the literature of a mutual facilitatory interaction between the arterial chemoreceptor reflex and the alerting stage of the defence reaction, particularly in relation to the patterning of cardiorespiratory activity. The present study has been designed to test the hypothesis that a portion of this interaction involves synaptic interactions within the nucleus tractus solitarii (NTS). 2. The study has involved an analysis of the effective interactions between the stimulation of the arterial chemoreceptors and the hypothalamic defence area (HDA) on the activity of NTS neurones recorded in anaesthetized, paralysed and artificially ventilated cats. 3. A group of eighteen NTS neurones was classified as chemosensitive, on the basis of displaying EPSPs on sinus nerve stimulation (SN) and their failure to show an excitatory response to baroreceptor stimulation. Thirteen of these neurones displayed pronounced excitatory responses to chemoreceptor stimulation. In sixteen of these neurones HDA stimulation elicited an EPSP; in four of these sixteen neurones this early EPSP was followed by an IPSP. In the remaining two (of 18) neurones HDA stimulation provoked no obvious synaptic response but facilitated the efficacy of both chemoreceptor inputs and SN stimulation. 4. Neurones shown to receive convergent inputs from the arterial chemoreceptors (and SN stimulation) and HDA, often displayed excitatory responses to stimulation of other peripheral inputs. Vagally evoked EPSPs were observed in nine neurones, SLN‐evoked responses in seven neurones and aortic nerve‐evoked EPSPs in three neurones. 5. The organization of these synaptic interactions is discussed and these data are used to explain the pattern of interaction between chemoreceptor, baroreceptor and HDA inputs within the NTS. Conclusions are drawn regarding the functional role of different classes of NTS neurone, based on the findings in this and the accompanying two papers.
Cardiac cells control transmitter release and calcium homeostasis in sympathetic neurons cultured from embryonic chick.
Authors: Wakade A R , Przywara D A , Bhave S V , Mashalkar V , Wakade T D
Published: December 2014

1. The contribution of target cells in controlling the functional properties of sympathetic neurons was investigated using pure neuronal cultures and co‐cultures of neurons with their physiological target cells. 2. Chick embryo sympathetic neurons cultured alone exhibited maximal elevation of cytosolic free Ca2+ ([Ca2+]i) and release of tritiated noradrenaline ([3H]NA) when given ten stimulating pulses at 1 Hz but not at 10 Hz, yielding a negative frequency‐release response. Stimulation‐evoked release was only slightly enhanced by the K+ channel blocker tetraethylammonium (TEA, 10 mM). 3. When sympathetic neurons were co‐cultured with cardiac cells of the chick embryo, electrically stimulated transmitter release and neuronal [Ca2+]i were reduced by 3‐ to 5‐fold. Co‐cultured neurons had a positive stimulation frequency‐‐[3H]NA release response and 5‐ to 7‐fold facilitation of release by TEA. 4. Voltage‐clamped Ca2+ current density was decreased from 0.61 +/‐ 0.13 pA micron‐2 in neurons alone to 0.19 +/‐ 0.03 pA micron‐2 in co‐cultured neurons. 5. Neonatal rat superior cervical ganglion (SCG) neurons were also relatively insensitive to TEA when cultured alone, but [3H]NA release was greatly facilitated by TEA when tested in SCG neurons co‐cultured with rat neonatal cardiac myocytes. 6. The cardiac cell‐induced changes in Ca2+ handling and release properties were produced within 24 h by sympathetic neuroeffector cells, but not by skeletal muscle cells or sensory neurons, and did not occur spontaneously in neurons grown alone for up to 6 days. 7. The frequency and TEA responses of neurons grown with cardiac cells are characteristic of responses seen in sympathetic neuroeffector organs. We conclude that physiological targets play a crucial role in development of normal transmitter‐release properties by controlling Ca2+ homeostasis in sympathetic neurons.
Thermogenesis associated with spontaneous activity: an important component of thermoregulatory needs in rats.
Authors: Girardier L , Clark M G , Seydoux J
Published: December 2014

1. Quantification of the energy cost of spontaneous activity in freely moving lean and obese Zucker rats was performed at 28 degrees C and during acute cold exposure (from 28 to 5 degrees C). 2. An open‐circuit metabolic chamber was supplemented with an ultrasensitive ergometric platform equipped with six undirectional accelerometers and with an opto‐electronic device for location of the rat's centre of mass. 3. Resting and mean metabolic rates during control and cold‐exposure periods were similar in both groups of rats. The ‘extra thermogenesis’ (ET), i.e. the difference between mean and resting metabolic rate, amounted to 11.7 +/‐ 1.1 and 8.6 +/‐ 0.7% of resting metabolic rate at 28 degrees C, and 39.7 +/‐ 2.9 and 34.1 +/‐ 2.9% of resting metabolic rate during cold exposure for lean and obese rats, respectively. 4. During the control period obese rats moved 3.71 +/‐ 0.61 m h‐1 and lean rats 8.69 +/‐ 0.57 m h‐1, but during cold exposure the distance moved by obese rats increased 3.58 +/‐ 0.33‐fold whereas that moved by lean rats only increased 1.40 +/‐ 0.06‐fold. The external work performed during spontaneous activity seldom reached 1.0% of the increase in metabolic rate. 5. In obese rats, weight was a good predictor of the distance covered, and cold exposure induced the same percentage increase in both distance and ET. Activity‐associated thermogenesis of obese rats was the predominant thermogenic source that substituted for their atrophied brown fat thermogenesis whereas in lean rats with active brown fat these correlations were not found.
The metabolic costs of different types of contractile activity of the human adductor pollicis muscle.
Authors: Newham D J , Jones D A , Turner D L , McIntyre D
Published: December 2014

1. The metabolic costs and physiological consequences of shortening contractions of a human muscle working in situ have been compared with those of the muscle maintaining a continuous isometric contraction and when performing repeated brief isometric contractions. 2. After a total of 10 s stimulation, the shortening and intermittent brief isometric protocols had very similar effects, causing a 30% loss of force and a threefold increase in the half‐time of relaxation. This was in contrast to the continuous isometric contraction protocol where there was less than 10% loss of force or slowing of relaxation. 3. The ATP cost over the first 5 s of the continuous isometric protocol was 27 mmol (l intracellular water)‐1 while for the shortening and repeated brief isometric protocols the costs were 48 and 46 mmol (l intracellular water)‐1, respectively. 4. The results show that shortening and repeated brief isometric contractions are considerably more energetically demanding, and hence more fatiguing, than sustained isometric contractions.
Pre‐ and postsynaptic whole‐cell recordings in the medial nucleus of the trapezoid body of the rat.
Authors: Borst J G , Helmchen F , Sakmann B
Published: December 2014

1. Simultaneous whole‐cell recordings in a rat brain slice preparation are described from presynaptic terminals (calyces of Held) and postsynaptic somata which form an axosomatic synapse in the medial nucleus of the trapezoid body (MNTB). 2. Presynaptic action potentials evoked suprathreshold excitatory postsynaptic potentials (EPSPs). The minimum synaptic delay was around 0.4 ms at 36 degrees C and 0.9 ms at 23‐24 degrees C. The amplitude of the L‐alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor‐mediated component of the excitatory postsynaptic currents (EPSCs) was 2‐13 nA (at ‐80 mV). 3. Current‐voltage relations showed that presynaptic Ca2+ channels were of the high voltage‐activated type. 4. A single action potential evoked a presynaptic fluorescence transient that decayed with a time constant of 0.3‐0.7 s, depending on the concentration (60‐200 microM) of the Ca2+ indicator Calcium Green‐5N (CG‐5N). The peak amplitude of the [Ca2+]i transient was severalfold larger in the terminal than in the preterminal axon. 5. EPSC peak amplitudes were stable for more than 30 min after establishing the whole‐cell configuration in the presynaptic terminal when the pipette contained 50 microM BAPTA. In contrast, with 1 mM BAPTA, peak amplitudes of EPSCs were reduced to one‐third. 6. Trains of presynaptic action potentials evoked EPSCs with progressively smaller amplitudes. Little change was observed in the depression when the terminals were dialysed with 50 microM BAPTA, whereas depression was reduced with 1 mM BAPTA. 7. In low (1 mM) [Ca2+]o, facilitation instead of depression of EPSCs was observed. 8. The effects of presynaptic BAPTA suggest that the endogenous mobile Ca2+ buffer capacity of giant presynaptic terminals in the MNTB is lower than in other terminals of fast transmitting synapses.
Control of the head in response to tilt of the body in normal and labyrinthine‐defective human subjects.
Authors: Kanaya T , Gresty M A , Bronstein A M , Buckwell D , Day B
Published: December 2014

1. Head movement responses to discrete, unpredictable tilts of the trunk from earth upright were studied in normal and labyrinthine‐defective (LD) subjects. Tilts of the seated, restrained trunk, were delivered in pitch and roll about head‐centred axes and approximated raised cosine displacements with peak amplitudes of 20‐30 deg and durations of 1.5‐2 s. Subjects performed mental arithmetic with eyes closed or read earth‐fixed text. 2. At the onset of tilt the head momentarily lagged behind the trunk because of inertia. Subsequently, head control varied widely with three broad types: (i) head relatively fixed to the trunk (in normal subjects and some patients); (ii) head unstable, falling in the direction of gimbal tilt (typical of acute patients for pitch motion); (iii) compensatory head movement in the opposite direction to gimbal tilt (observed consistently in normal subjects and in well‐adapted patients). 3. EMG was well developed in subjects with compensatory head movement and consisted of an initial burst of activity at minimum latencies of 25‐50 ms (means 72‐108 ms), followed by a prolonged peak; both occurring in the ‘side up’ neck muscles, appropriate for righting the head. These muscles are shortened during the initial head lag so the responses cannot be stretch reflexes. In normal subjects their origin is predominantly labyrinthine but in patients they may be an ‘unloading response’ of the neck. 4. Head stability in space was superior with the visual task for all subjects but vision only partially compensated for labyrinthine signals in unstable patients. 5. Modelling the responses to tilt suggests that, in LD subjects, the short‐latency burst could be driven by signals from the neck of the relative acceleration between head and trunk tilt. The longer latency EMG could be driven by a signal of head tilt in space. Normally, this signal is probably otolithic. In patients it could be synthesized from summing proprioceptive signals of position of head on trunk with trunk tilt.
Effects of diaphragm shortening on the mean action potential conduction velocity in canines.
Authors: Sinderby C , Lindström L , Comtois N , Grassino A E
Published: December 2014

1. The present study was designed to test if the mean muscle fibre action potential conduction velocity (VAPC) in the costal diaphragm changes with muscle length, in spontaneously breathing mongrel dogs. 2. VAPC was determined by the electromyogram (EMG) power spectrum ‘dip’ method, which is based on the bipolar electrode transfer function. A bipolar EMG electrode with a 20 mm fixed interelectrode distance was sutured to the costal diaphragm in the fibre direction, and in a region with a low density of motor endplates. Diaphragm length was measured with piezoelectric crystals positioned next to the EMG electrode. Seven dogs were vagotomized and spinally anaesthetized in order to increase diaphragmatic shortening, reduce velocity of shortening and abolish possible cross‐talk signals from adjacent muscles. 3. Our results showed that VAPC in the canine costal diaphragm was 3.4 m s‐1 and was not significantly related to diaphragmatic shortening.
Contribution of shrinkage of extracellular space to extracellular K+ accumulation in myocardial ischaemia of the rabbit.
Authors: Yan G X , Chen J , Yamada K A , Kléber A G , Corr P B
Published: December 2014

1. The contribution of the concentrating effect due to shrinkage of the extracellular space (ECS) to cellular K+ efflux on extracellular potassium ([K+]o) accumulation in response to ischaemia was investigated in an isolated, blood‐perfused rabbit papillary muscle preparation with a confined extracellular space. 2. The ECS was quantified using either of two extracellular markers, choline or tetramethyl ammonium (TMA), each with specific ion‐selective electrodes, as well as by measurement of extracellular resistance (ro). [K+]o and [Na+]o were also measured simultaneously using K(+)‐ and Na(+)‐selective electrodes. 3. During ischaemia, [K+]o increased 3‐fold from 4.2 +/‐ 0.1 to 12.6 +/‐ 1.0 mM at 10 min (n = 10) analogous to changes in the ischaemic heart in vivo. The ECS decreased to 83.9 +/‐ 3.2% of control measured using 1 mM choline extracellularly (n = 9, P < 0.01) or to 85.7 +/‐ 0.7% of control using 1 mM TMA (n = 6, P < 0.01). Nearly identical decreases in ro (84.1 +/‐ 2.4%, n = 15, P < 0.01) occurred simultaneously. 4. The small decrease in the ECS contributed only 0.8‐0.9 mM to the total increase in [K+]o of 8.4 mM and had a minor effect on transmembrane K+ flux. No significant differences between the relative changes in [choline] and [Na+]o were observed. This excluded a major transmembrane Na+ movement during early ischaemia. 5. Bumetanide (10 mM), an inhibitor of K(+)‐Cl‐ cotransport, a process which is involved in cell volume regulation consequent to osmotic cell swelling, significantly attenuated the increase in [K+]o after 6 min of ischaemia (8.3 +/‐ 0.6 mM, n = 5 vs. 10.3 +/‐ 0.4 mM in the control group, n = 6, P < 0.05), whereas N‐ethylmaleimide (1 mM), a stimulator of this cotransporter, augmented [K+]o accumulation (12.0 +/‐ 0.6 mM at 6 min, P < 0.05). 6. We conclude that during early myocardial ischaemia, a major component of [K+]o accumulation is not caused by diminution of ECS per se, but rather by increased net K+ efflux due in part to K(+)‐Cl cotransport secondary to myocyte volume regulation.
Transferable circulating factors and epithelial sodium transport after unilateral nephrectomy in the rat.
Authors: Pollock C A , Nobes M S , Gyory A Z , Heng P T , Field M J
Published: December 2014

1. In order to investigate the role of circulating serum factors in the altered renal haemodynamics and enhanced renal tubular transport observed in renal growth, micropuncture experiments were performed on normal animals infused with 20% plasma derived from animals in whom unilateral nephrectomy had been performed 3 days previously. 2. When animals infused with plasma from uninephrectomized animals (NxP) were compared with those infused with control plasma, the former had a higher tubular fluid flow rate measured at both the late proximal (LP; 26.7 +/‐ 1.6 vs. 18.4 +/‐ 1.4 nl min‐1; P < 0.001) and early distal (ED; 14.9 +/‐ 1.0 vs. 7.8 +/‐ 1.0 nl min‐1; P < 0.0001) sites, which was reflected in the final urine flow rate (16.1 +/‐ 3.4 vs. 5.1 +/‐ 0.8 microliter min‐1; P < 0.005). 3. The single nephron glomerular filtration rate (SNGFR) was higher in animals infused with NxP as determined from samples taken at the LP (45.8 +/‐ 2.8 vs. 35.7 +/‐ 2.3 nl min‐1; P < 0.01) and at the ED (34.5 +/‐ 2.5 vs. 28.1 +/‐ 1.8 nl min‐1; P = 0.05) sites. However, this increase was not reflected in the whole kidney GFR (1.04 +/‐ 0.06 vs. 0.89 +/‐ 0.06; P = 0.07), suggestive of a preferential increase in filtration in the outer cortical nephrons. 4. Tubular Na+ transport was higher in the animals infused with NxP as evidenced by a decrease in the fractional delivery of Na+ at the ED site (4.5 +/‐ 0.4 vs. 6.5 +/‐ 0.6% of the filtered load; P < 0.05). However, in the final urine there was a significant increase in the urinary sodium excretion in animals infused with NxP (0.67 +/‐ 0.14 vs. 0.29 +/‐ 0.09%; P < 0.05) indicating that natriuresis and probably diuresis was a result of inhibition of Na+ and water transport in the late distal tubule and collecting duct. 5. In conclusion, these experiments demonstrate that circulating factors induced by a reduction in renal mass significantly alter glomerular filtration and tubular Na+ transport.
Selective inhibition of high voltage‐activated L‐type and Q‐type Ca2+ currents by serotonin in rat melanotrophs.
Authors: Ciranna L , Feltz P , Schlichter R
Published: December 2014

1. Whole‐cell Ca2+ currents (ICa) from cultured rat melanotrophs were identified by their sensitivity to Ca2+ channel blockers, and their modulation by serotonin (5‐HT) was studied. All cells displayed high voltage‐activated (HVA; > ‐30 mV) Ca2+ currents. A low voltage‐activated (LVA; > ‐60 mV) Ca2+ current was detected in 92% of the cells. 2. The whole‐cell ICa was insensitive to omega‐conotoxin GVIA (0.5‐1 microM) indicating the absence of N‐type Ca2+ channels. 3. At a holding potential (Vh) of ‐70 mV, the L‐type channel blocker nifedipine reduced ICa in a dose‐dependent manner with a half‐maximal effective concentration (IC50) of 28 nM. The L‐type current represented 39% of the total ICa. 4. omega‐Agatoxin IVA (omega‐Aga IVA) produced a biphasic dose‐dependent inhibition of ICa, with IC50 values of 0.4 and 91 nM, indicating the presence of P‐type and Q‐type Ca2+ channels, which accounted respectively for 16 and 45% of the total ICa. The P‐type current was also blocked by synthetic funnel‐web spider toxin (sFTX 3.3; 1‐10 microM) and was present only in a subpopulation (60‐70%) of cells. 5. All cells possessed a Ca2+ current which was resistant to nifedipine (10 microM) and omega‐Aga IVA (50 nM). This current was not affected by Ni2+ (40 microM) but was abolished by a low concentration of Cd2+ (10 microM) and by omega‐conotoxin MVIIC (1 microM) indicating that it was a Q‐type Ca2+ current. 6. 5‐HT (10 microM) inhibited the whole‐cell ICa in 70% of the cells tested (n = 120) by activating 5‐HT1A and 5‐HT2C receptors. 5‐HT produced either a kinetic slowing of the activation phase (37% of the cells) or a scaling down (14% of the cells) of ICa. In the majority of cells (49%) both types of inhibition were found to coexist. 7. The effects of 5‐HT were voltage dependent, rendered irreversible when GTP‐gamma‐S (30 microM) was present in the pipette solution and abolished by pretreatment of the cells with pertussis toxin (PTX; 150 ng ml‐1, 18 h). 8. Low concentrations of omega‐Aga IVA (20 nM), which blocked mainly P‐type channels, did not reduce the effect of 5‐HT on ICa. The scaling down effect of 5‐HT on ICa was eliminated in the presence of nifedipine (10 microM) and the kinetic slowing effect of 5‐HT persisted after blockade of L‐ and P‐type channels but was abolished by omega‐conotoxin MVIIC (1 microM). 9. We conclude that rat melanotrophs possess functional L‐, P‐ and Q‐type Ca2+ channels and that 5‐HT inhibits selectively L‐type and Q‐type Ca2+ currents with different modalities. These effects are voltage dependent and mediated by a PTX‐sensitive G‐protein.
Influence of actinomycin D, a RNA synthesis inhibitor, on long‐term potentiation in rat hippocampal neurons in vivo and in vitro.
Authors: Frey U , Frey S , Schollmeier F , Krug M
Published: December 2014

1. Hippocampal long‐term potentiation (LTP) may serve as an elementary process underlying certain forms of learning and memory in vertebrates. As is the case with behavioural memory, hippocampal LTP in the rat CA1 region and in the dentate gyrus occurs in stages, which can be separated by an inhibitor of RNA synthesis. 2. Experiments have been performed in two brain regions, in the hippocampal CA1 region in vitro and in the dentate gyrus in vivo. 3. Maintenance of hippocampal LTP in the CA1 region in vitro was influenced by the RNA synthesis inhibitor actinomycin D from 4 h onwards. 4. The effect of actinomycin D on the time course of the population spike potentiation was more pronounced than the effect on the time course of the EPSP component, suggesting different mechanisms for the two forms of potentiation. 5. Intrahippocampal and intracerebroventricular injection of actinomycin D into rats prevented a late stage of LTP in the dentate gyrus in vivo measured as the population spike amplitude. 6. Since actinomycin D was only effective in influencing the maintenance of LTP when applied before tetanization, the requirement for transcription during LTP may have a critical time window. 7. Actinomycin D influenced the maintenance of LTP specifically, since the drug did not alter any potentials in control experiments after its removal or when it was administered shortly after tetanization. A second, structurally different RNA synthesis inhibitor, 5,6‐dichloro‐1‐beta‐D‐ribofuranosyl benzimidazole, mimicked the effect of actinomycin D in vitro.
Inhibition of a small‐conductance cAMP‐dependent Cl‐ channel in the mouse thick ascending limb at low internal pH.
Authors: Guinamard R , Paulais M , Teulon J
Published: December 2014

1. A small‐conductance Cl‐ channel that is stimulated by ATP and protein kinase A has been identified in the basolateral membranes of cortical thick ascending limbs (CTALs) of the mouse nephron. The present study uses the cell‐attached and inside‐out variants of the patch‐clamp technique to investigate the pH sensitivity of this channel. 2. The open‐state probability (Po) was dependent upon the internal pH in inside‐out patches. Expressed as a percentage of the Po value at pH 7.2, Po increased to about 180% at pH 7.6, and decreased to 25% at pH 6.8. Po was close to zero at pH 6.4. The internal pH had no effect on the channel unit conductance. 3. The effect of pH on the CTAL Cl‐ channel was assessed in intact cells using NH4Cl to acidify the intracellular compartment. Experiments with the pH‐sensitive fluorescent dye 2',7'‐(carboxyethyl)‐5'(6')‐carboxy fluorescein penta‐acetoxymethyl ester (BCECF) indicated that 1 mmol l‐1 NH4Cl acidified the cytoplasm by 0.15 pH units and 5 mmol l‐1 NH4Cl by 0.34 pH units. These concentrations of NH4Cl reduced the activity of the CTAL Cl‐ channel by 24 and 82% in cell‐attached patches, showing that moderate changes in internal pH substantially altered the activity of this channel. NH4+ had no direct effect on channel activity. 4. Inhibition at low pH is a newly discovered property of small‐conductance Cl‐ channels in epithelia, which might help discriminate between types of Cl‐ channel.
Differences between outward currents of human atrial and subepicardial ventricular myocytes.
Authors: Amos G J , Wettwer E , Metzger F , Li Q , Himmel H M , Ravens U
Published: December 2014

1. Outward currents were studied in myocytes isolated from human atrial and subepicardial ventricular myocardium using the whole‐cell voltage clamp technique at 22 degrees C. The Na+ current was inactivated with prepulses to ‐40 mV and the Ca2+ current was eliminated by both reducing extracellular [Ca2+] to 0.5 mM and addition of 100 microM CdCl2 to the bath solution. 2. In human myocytes, three different outward currents were observed. A slowly inactivating sustained outward current, I(so), was found in atrial but not ventricular myocytes. A rapidly inactivating outward current, I(to), of similar current density was observed in cells from the two tissues. An additional uncharacterized non‐inactivating background current of similar size was observed in atrial and in ventricular myocytes. 3. I(to) and I(so) could be differentiated in atrial myocytes by their different kinetics and potential dependence of inactivation, and their different sensitivities to block by 4‐amino‐pyridine, suggesting that two individual channel types were involved. 4. In atrial cells, inactivation of I(to) was more rapid and steady‐state inactivation occurred at more negative membrane potentials than in ventricular cells. Furthermore, the recovery of I(to) from inactivation was slower and without overshoot in atrial myocytes. In addition, 4‐aminopyridine‐induced block of I(to) was more efficient in atrial than in ventricular cells. These observations suggest that the channels responsible for atrial and ventricular I(to) were not identical. 5. We conclude that the differences in outward currents substantially contribute to the particular shapes of human atrial and ventricular action potentials. The existence of I(so) in atrial cells only provides a clinically interesting target for anti‐arrhythmic drug action, since blockers of I(so) would selectively prolong the atrial refractory period, leaving ventricular refractoriness unaltered.
Presynaptic muscarinic inhibition in bullfrog sympathetic ganglia.
Authors: Shen W X , Horn J P
Published: December 2014

1. Muscarinic modulation of nicotinic transmission was studied in bullfrog sympathetic ganglia by recording synaptic currents from B and C neurones. 2. Bath‐applied muscarine reduced the amplitude of EPSCs recorded at < 0.2 Hz from B neurones by up to 57%. The action was reversible, showed no apparent desensitization, and had an EC50 of 102 nM. Muscarine had no effect on EPSCs in C neurones. 3. Currents evoked by ionophoretic application of ACh to B neurones were unchanged by muscarine. Muscarine increased the coefficient of variation (c.v.) of EPSC amplitude. The effect upon the ratio of c.v.2control to c.v.2muscarine was proportional to the change in mean EPSC amplitude. 4. Activation of muscarinic receptors by ACh from nerve terminals was observed by comparing trains of EPSCs in normal Ringer solution and atropine. Inhibition of EPSC amplitude by 15‐40% was seen as frequency was increased from 1 to 5 Hz. The minimal latency for onset of inhibition was approximately 2 s. Stimulation at 20 Hz did not produce inhibition. 5. The results indicate that presynaptic muscarinic receptors are selectively expressed by a functional subclass of preganglionic sympathetic nerve terminals. Physiological activation of the receptors occurs during repetitive activity. The extent of autoreceptor‐mediated inhibition varies as a biphasic function of stimulus frequency.
Tonic sympathetic chemoreflex after blockade of respiratory rhythmogenesis in the rat.
Authors: Koshiya N , Guyenet P G
Published: December 2014

1. We sought to determine whether the increase in sympathetic nerve discharge (SND) caused by carotid chemoreceptor stimulation requires the integrity of ventrolateral medullary structures involved in generating respiratory rhythm and pattern. Experiments were done in urethane‐anaesthetized, vagotomized, aortic deafferented, ventilated rats except when indicated (see paragraph 3). 2. Brief hypoxia (N2 for 5‐12 s) or I.V. NaCN (50‐100 micrograms kg‐1) activated SND in bursts synchronized with the phrenic nerve discharge (PND). No effect was produced in chemo‐deafferented rats. 3. In unanaesthetized vagotomized decerebrated rats, ligation of the internal carotid arteries preserved peripheral chemoreceptor function but abolished baroreflexes. In this preparation, stimulation of peripheral chemoreceptors (N2 for 2‐6 s) also activated SND in bursts synchronized with PND. 4. Bilateral microinjection of the GABAA receptor agonist muscimol into the caudal ventrolateral medulla (CVLM) instantly blocked the sympathetic baroreflex, eliminated PND at rest and during chemoreceptor stimulation but did not change the mean increase in SND produced by chemoreceptor stimulation. Sympathoactivation in response to chemoreceptor stimulation became tonic after 1‐13 min and was still totally dependent on the integrity of the carotid sinus nerves. 5. Muscimol injection instantly eliminated the respiratory outflow of the Xth and XIIth cranial nerves, both at rest and during chemoreceptor stimulation. 6. Muscimol eliminated the on‐off respiratory pattern of neurons in the rostral ventrolateral medulla (RVLM). During chemoreceptor stimulation, these cells became activated or inhibited tonically. 7. Muscimol injection raised the resting discharge rate of vasomotor presympathetic cells in RVLM, blocked their baroreceptor inputs but did not change the magnitude of their excitation by chemoreceptor stimulation. Muscimol injection eliminated their respiratory modulation. 8. In conclusion, the sympathetic response to chemoreceptor stimulation may be due to convergence and integration in RVLM of two processes: respiration‐independent excitatory input to RVLM neurons and respiratory patterning of their activities via inputs from the pre‐Bötzinger complex.
Calcium‐activated potassium channels in the endothelium of intact rat aorta.
Authors: Marchenko S M , Sage S O
Published: December 2014

1. Single K+ channel currents and membrane potential were recorded in the endothelium of excised intact rat aorta. 2. Two types of K+ channel were found in excised patches, KCh and KAp. With Na+ and K+ as the main external and internal cations, outward conductances were 6.7 pS (KCh) and 2.8 pS (KAp). In symmetric 150 mM K+, the inward conductances were 18 and 9.1 pS. 3. Activation by Ca2+ was concentration dependent. KCh channels were activated by [Ca2+] > 0.1 microM and KAp by [Ca2+] > 0.5 microM. 4. Apamin at concentrations > 1 nM inhibited KAp Channels. Block was complete at 10 nM. KAp channels were insensitive to charybdotoxin. KCh channels were inhibited by charybdotoxin at concentrations > 50 nM, but were insensitive to apamin. 5. d‐Tubocurarine (dTC) evoked flickering activity of KAp channels at concentrations > 5 microM and complete block at 100 microM. At these doses, dTC did not affect KCh channels, but at concentrations > 1 mM it decreased the single channel amplitude. 6. Hyperpolarization evoked by acetylcholine was unaffected by apamin or dTC at low concentrations ( < or = 100 microM), but inhibited by high concentrations of charybdotoxin ( > 50 nM) or dTC ( > 1 mM). 7. These data suggest that KCh channels are novel Ca(2+)‐activated K+ channels responsible for the ACh‐evoked hyperpolarization in the endothelium of rat aorta.
Subtype‐specific regulation of recombinant NMDA receptor‐channels by protein tyrosine kinases of the src family.
Authors: Köhr G , Seeburg P H
Published: December 2014

1. Tyrosine kinases regulate NMDA receptor‐channel activity in cultured neurons, and NMDA receptor subunits are tyrosine phosphorylated in the brain. 2. Heteromeric NMDA receptor‐channels were transiently expressed in human embryonic kidney (HEK) 293 cells and glutamate (100 microM)‐activated whole‐cell currents (500 ms) were studied when tyrosine kinases of the src gene family were included in the pipette solution. 3. Glutamate‐activated currents (evoked every 20 s for up to 20 min) were increased by src and fyn kinases without affecting the desensitization and deactivation kinetics in NR1‐NR2A but the kinases had no effects in NR1‐NR2B, NR1‐NR2C and NR1‐NR2D receptor‐channels, suggesting that a phosphorylation site in NR2A is targeted. 4. In a mutant channel consisting of NR1 and a C‐terminal deletion mutant of NR2A (NR2A delta C), src and fyn kinases lost their potentiating effects indicating that the phosphorylation of tyrosine(s) in the C‐terminal domain of NR2A affects the current flux through native NMDA receptor‐channels.