bluuebunny/crossref_metadata_2025 huggingface dataset:
Volatile anaesthetic enhancement of paired‐pulse depression investigated in the rat hippocampus in vitro.
Authors: Pearce R A
Published: December 2014

1. A prominent in vivo effect of general anaesthetics, including volatile anaesthetics such as halothane, is the prolonging of paired‐pulse depression of the hippocampal CA1 population spike. The mechanisms by which volatile anaesthetics produce this effect were investigated in the hippocampal brain slice preparation by testing the effect of halothane on several long‐lasting inhibitory processes, including the calcium‐activated potassium current that underlies the slow after‐hyperpolarization (IAHP), the GABAB‐mediated potassium current that underlies the late IPSP, and the fast and slow components of the early GABAA‐mediated IPSP. 2. Halothane produced a dose‐dependent block of IAHP at concentrations between 0.5 and 1.5%. This block was manifested as a reduction in spike frequency adaptation, a reduction in the amplitude of the slow after‐hyperpolarization following a train of action potentials, and a reduction in the amplitude of the voltage‐clamped current following a calcium spike elicited in the presence of tetraethylammonium and tetrodotoxin. The effect did not appear to be caused by blockade of voltage‐sensitive calcium channels, since halothane markedly reduced IAHP at a concentration (1.5%) that had little effect on the depolarization‐evoked calcium spike. 3. Halothane reduced the amplitude of the late GABAB‐mediated IPSP by approximately 50% at concentrations between 1 and 2%. Similar results were obtained for polysynaptic and monosynaptic responses, and with current‐clamp and voltage‐clamp recordings. However, halothane, at concentrations up to 3%, had no effect on the presynaptic GABAB response, as indicated by no reduction in paired‐pulse depression of the monosynaptic GABAA response. 4. Halothane (2%) and enflurane (4%) prolonged the decay phase of the slow component of the monosynaptic GABAA‐mediated IPSC approximately twofold, but did not alter the amplitude of the response. Halothane also prolonged the decay phase of the fast component of the GABAA‐mediated IPSC, with no effect on the amplitude. However, enflurane markedly reduced the amplitude of the fast component of the GABAA IPSC, so that only a small slow current remained in response to a selective stimulus. 5. It is concluded that the effects of halothane on IAHP and on GABAB responses cannot account for its effects on paired‐pulse depression, but that volatile anaesthetics enhance paired‐pulse depression by prolonging the decay of the slow dendritic GABAA response. Furthermore, it is speculated that the proconvulsant property of enflurane is related to its depression of the fast somatic component of GABAA inhibition.
Effect of arachidonic acid on the L‐type calcium current in frog cardiac myocytes.
Authors: Petit-Jacques J , Hartzell H C
Published: December 2014

1. External application of the unsaturated fatty acid arachidonic acid (AA) to frog ventricular cells caused a large inhibition (approximately 85%) of the L‐type calcium current (ICa,L) previously stimulated by the beta‐adrenergic agonist isoprenaline (Iso). The concentration producing half‐maximal inhibition (K1/2) was 1.52 microM. The inhibitory effect did not affect the peak current‐voltage relationship but produced a negative shift in the inactivation curve. 2. The inhibitory effect of AA also occurred in cells internally perfused with cAMP and non‐hydrolysable analogues of cAMP. These data suggest that AA is acting by a mechanism located beyond adenylyl cyclase and does not involve changes in intracellular cAMP levels. 3. AA also inhibited the calcium current stimulated by internal perfusion with the catalytic subunit of protein kinase A (PKA), suggesting that AA acts downstream of channel phosphorylation. 4. The inhibitory effect of AA on the isoprenaline‐ or cAMP‐stimulated ICa,L is largely reduced in cells internally perfused with the thiophosphate donor analogue of ATP, ATP gamma S, or protein phosphatase 1 and 2A inhibitors like microcystin (MC) or okadaic acid (OA). External application of the phosphatase inhibitor calyculin (Caly) also reduced the AA effect. These data suggested that the AA effect on ICa,L involves activation of protein phosphatase activity. 5. The effect of AA on ICa,L was not affected by staurosporine, an inhibitor of protein kinases. It was also unaffected in cells internally perfused with GTP gamma S. These results suggest that neither a PKC‐ nor a G‐protein‐mediated mechanism are likely to be involved in the effect of AA on ICa,L. 6. A saturated fatty acid, myristic acid (MA), had no inhibitory effect on the isoprenaline‐stimulated Ca2+ current, whereas, in the same cells arachidonic acid produced approximately 85% inhibition of ICa,L. 7. The inhibitory effect of AA was not affected by exposing the cells to indomethacin (Indo), an inhibitor of the metabolism of AA by cyclo‐oxygenase, nor nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway. However, the non‐metabolizable analogue of AA, 5,8,11,14‐eicosatetraynoic acid (ETYA), was without effect on the isoprenaline‐stimulated ICa,L. 8. These results suggest that AA inhibits ICa,L via a mechanism which involves, in part, stimulation of protein phosphatase activity. This process could provide a new mechanism in the modulation of calcium channel activity.
Augmented force output in skeletal muscle fibres of Xenopus following a preceding bout of activity.
Authors: Bruton J D , Westerblad H , Katz A , Lännergren J
Published: December 2014

1. The effect of a brief period of activity on subsequent isometric tetanic force production was investigated in single muscle fibres of Xenopus laevis. 2. Following a train of ten tetani separated by 4 s intervals, tetanic force was significantly augmented by about 10%. The tetanic force augmentation persisted for at least 15 min and then slowly subsided. A similar potentiation was seen following trains of five and twenty tetani. 3. During the period of tetanic force potentiation, tetanic calcium was reduced by more than 30%, and intracellular pH was reduced from 7.15 +/‐ 0.07 to 7.03 +/‐ 0.11 (n = 4). 4. Fibre swelling was greatest at 1 min and then subsided over 15‐20 min and possibly accounted for a small part of the observed force potentiation. 5. A reduction in the inorganic phosphate (P1) concentration of more than 40% was found in fibres frozen in liquid nitrogen at the peak of force potentiation compared with resting fibres. 6. It is concluded that the augmentation of tetanic force found after a brief preceding bout of activity is due to a reduction in inorganic phosphate. This mechanism may underlie the improved performance observed in athletes after warm‐up.
Active potassium transport across guinea‐pig distal colon: action of secretagogues.
Authors: Rechkemmer G , Frizzell R A , Halm D R
Published: December 2014

1. Adrenaline (5 microM) stimulated a K+ secretory current by 2.2 mu equiv h‐1 cm‐2 in isolated guinea‐pig distal colonic epithelium. This secretory activity was inhibited entirely by addition of the loop diuretic bumetanide to the serosal solution. On‐going K+ uptake via the absorptive pathway was unaltered by these changes. 2. Prostaglandin E2 (PGE2, 2 microM) stimulated electrogenic K+ secretion and Cl‐ secretion by 3.0 and 3.6 mu equiv h‐1 cm‐2, respectively. Serosal addition of bumetanide completely inhibited this K+ secretion but blocked only approximately 70% of Cl‐ secretion. The bumetanide‐insensitive Cl‐ secretory current was dependent on the presence of Cl‐ and HCO3‐ in the bathing solutions. 3. Stimulation of electrogenic K+ secretion by PGE2 occurred with a half‐maximal concentration of 4 nM, an affinity approximately 300 times higher than that for stimulation of Cl‐ secretion by PGE2. 4. Forskolin (10 microM) stimulated Cl‐ secretion by 4.9 mu equiv h‐1 cm‐2. The apparent K+ secretory rate was increased by only 1.5 mu equiv h‐1 cm‐2. A bumetanide‐insensitive short‐circuit current (ISC) was apparent and of the same size as that stimulated by PGE2. 5. Addition of the Ca2+ ionophore A23187 (10 microM), in the presence of indomethacin (1 microM) to reduce prostaglandin production, inhibited the K+ absorptive pathway by 40% and concurrently stimulated a small rate of electrogenic K+ secretion. 6. Active K+ absorption was inhibited by the addition of ouabain, omeprazole or SCH28080 to the mucosal solution. Both omeprazole and SCH28080 also stimulated a small negative ISC, consistent with electrogenic K+ secretion. 7. Association of K+ absorption, K+ secretion and Cl‐ secretion is indicated by similarities in transport mechanism and by secretagogue regulation. In particular, maximal rates of K+ secretory current require uptake via apical membrane K+ pumps. Such interrelations support a common cellular locus for these ion transport pathways.
Evidence of facilitation of soleus‐coupled Renshaw cells during voluntary co‐contraction of antagonistic ankle muscles in man.
Authors: Nielsen J , Pierrot‐Deseilligny E
Published: December 2014

1. The amount of recurrent inhibition onto soleus motoneurones was compared during plantar flexion and co‐contraction of antagonistic ankle plantar and dorsiflexors at matched levels of background activity in the soleus muscle. 2. During weak plantar flexion and co‐contraction (less than 10% of maximal voluntary plantar flexion effort) a test reflex discharge (H' reflex), which was conditioned by a previous reflex discharge, was found to be significantly more depressed in relation to rest than an unconditioned reference H reflex. During strong plantar flexion (more than 50% of maximal voluntary plantar flexion effort) the H' reflex either increased more or to the same extent as the reference H reflex in relation to rest. In contrast to this, the H' reflex was strongly depressed during co‐contraction, whereas the reference H reflex was not significantly different from its resting value. 3. At the end of the ramp phase of a phasic contraction, large variations of the H' reflex were observed during plantar flexion (large increase in relation to rest) and during co‐contraction (marked decrease), whereas the reference H reflex was facilitated in the two situations. 4. These observations provide evidence that soleus‐coupled Renshaw cells are differently regulated during co‐contraction and plantar flexion. It is suggested that the Renshaw cells are inhibited during strong plantar flexion but not during strong co‐contraction. The functional significance of the findings is discussed.
Ca(2+)‐dependent non‐selective cation and potassium channels activated by bradykinin in pig coronary artery endothelial cells.
Authors: Baron A , Frieden M , Chabaud F , Bény J L
Published: December 2014

1. Using the cell‐attached and inside‐out modes of the patch‐clamp technique, we studied the Ca(2+)‐dependent ionic channels activated by bradykinin in cultured pig coronary artery endothelial cells to further understand electrophysiological events underlying cellular activation. 2. In the cell‐attached mode, bradykinin (94 nM) activated two types of Ca(2+)‐dependent channels: a high conductance K+ channel (285 pS in high symmetrical K+), whose open state probability was increased by depolarization, and a lower conductance inwardly rectifying non‐selective cation channel (44 pS in high symmetrical K+). 3. The 285 pS K+ channel was half‐maximally activated by cytosolic Ca2+ levels of 1.6 and 4.5 microM at +10 and ‐30 mV, respectively. Such local concentrations should be reached in the presence of bradykinin, which induces a mean maximal cytosolic Ca2+ rise of 1.3 microM. 4. The 285 pS K+ channel was inhibited by d‐tubocurarine, which acted by reducing the mean open time duration (flickering pattern), finally reducing the channel conductance. 5. Divalent cations such as Ca2+ could flow through the 44 pS non‐selective cation channel, with nearly the same permeability (P) as monovalent cations (PK: PNa: PCa = 1:1:0.7). 6. The cation channel appeared to be more sensitive to Ca2+ than the K+ channel, with a half‐maximal open probability induced by 0.7 microM Ca2+ on the intracellular side of the membrane. 7. In contrast to the K+ channel, the cation channel mean open time was clearly increased by bradykinin. This effect was delayed compared with the increase in the channel open state probability and was rapidly lost in the inside‐out configuration. Caffeine also activated the cation channel but more transiently than bradykinin and without any effect on the open duration. 8. In the absence of extracellular Ca2+, the bradykinin‐induced increase in cytosolic free Ca2+ was shortened temporally by 52% and reduced in amplitude by 88%, whereas the bradykinin‐induced hyperpolarization was not significantly reduced in amplitude but was shortened by 70%, thus illustrating the major role of Ca2+ influx in endothelial cell activation by bradykinin. 9. We conclude that bradykinin activates two types of Ca(2+)‐dependent channels in coronary endothelial cells: a high conductance K+ channel regulated by membrane potential, and an inwardly rectifying cation channel allowing Ca2+ entry, the cation channel being about 6 times more sensitive to Ca2+ than the K+ channel. The increase in cation channel open state probability involves an increase in open number, like the K+ channel, but also involves a rise in channel open duration. Ca2+ entry via cation channels could contribute to increase the cytoplasmic Ca2+ level, activate Ca(2+)‐dependent K+ channels, thus triggering membrane hyperpolarization when the endothelial cell is stimulated by a vasoactive agonist such as bradykinin.
Comparison of sarcolemmal calcium channel current in rabbit and rat ventricular myocytes.
Authors: Yuan W , Ginsburg K S , Bers D M
Published: December 2014

1. Fundamental properties of Ca2+ channel currents in rat and rabbit ventricular myocytes were measured using whole cell voltage clamp. 2. In rat, as compared with rabbit myocytes, Ca2+ channel current (ICa) was half‐activated at about 10 mV more negative potential, decayed slower, was half‐inactivated (in steady state) at about 5 mV more positive potential, and recovered faster from inactivation. 3. These features result in a larger steady‐state window current in rat, and also suggest that under comparable voltage clamp conditions, including action potential (AP) clamp, more Ca2+ influx would be expected in rat myocytes. 4. Ca2+ channel current carried by Na+ and Cs+ in the absence of divalent ions (Ins) also activated at more negative potential and decayed more slowly in rat. 5. The reversal potential for Ins was 6 mV more positive in rabbit, consistent with a larger permeability ratio (PNa/PCs) in rabbit than in rat. ICa also reversed at slightly more positive potentials in rabbit (such that PCa/PCs might also be higher). 6. Ca2+ influx was calculated by integration of ICa evoked by voltage clamp pulses (either square pulses or pulses based on recorded rabbit or rat APs). For a given clamp waveform, the Ca2+ influx was up to 25% greater in rat, as predicted from the fundamental properties of ICa and Ins. 7. However, the longer duration of the AP in rabbit myocytes compensated for the difference in influx, such that the integrated Ca2+ influx via ICa in response to the species‐appropriate waveform was about twice as large as that seen in rat.
Ionic mechanisms of Ca(2+)‐dependent electrolyte transport across equine sweat gland epithelium.
Authors: Ko W H , Chan H C , Chew S B , Wong P Y
Published: December 2014

1. The ionic mechanism involved in Ca(2+)‐stimulated electrolyte transport in cultured equine sweat gland epithelial cells was studied using the short‐circuit current (ISC) technique. 2. Microscopy revealed that the cultured cells grown on Millipore filters formed polarized monolayers with tight junctions. Monolayers exhibited a mean transepithelial resistance of 333.9 +/‐ 40.4 omega cm2. 3. Ca(2+)‐mobilizing agents, A23187 (1 microM) or thapsigargin (0.01‐1 microM), stimulated ISC while forskolin exerted little effect on the ISC. 4. Replacement of external Cl‐ by gluconate significantly reduced the ISC by 63% when stimulated by 0.1 microM thapsigargin. Residual ISC could be abolished (> 99%) by elimination of HCO3‐ from the bathing solution. 5. Basolateral addition of bumetanide (0.1 mM), ouabain (0.01 mM) and acetazolamide (45 microM) and apical addition of methyl isobutyl amiloride (MIA, 1‐100 microM) all had inhibitory effects on the thapsigargin‐stimulated ISC to various extents. 6. Substantial current inhibition could be obtained using 4, 4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS) and diphenylamine‐2‐carboxylate (DPC) in a concentration‐dependent manner. 7. The K+ channel blocker barium (5 mM) was effective on both sides of the epithelium with a much larger effect on the basolateral side. 8. The inhibitory effects of acetazolamide, amiloride, MIA, DIDS and DPC on the thapsigargin‐stimulated ISC were also observed when a Cl(‐)‐free solution was used. 9. The results provide evidence for Ca(2+)‐stimulated HCO3‐ as well as Cl‐ secretion by equine sweat gland epithelium.
Pulsatile motor output in human finger movements is not dependent on the stretch reflex.
Authors: Wessberg J , Vallbo A B
Published: December 2014

1. Stretch perturbations were delivered during slow voluntary finger movements with the aim of exploring the role of the stretch reflex in generating the 8‐10 Hz discontinuities that characterize these movements. Afferent activity from muscle spindle primary endings in the finger extensor muscles was recorded from the radial nerve, along with the EMG activity of these muscles, and kinematics of the relevant metacarpo‐phalangeal joint. 2. Perturbations elicited a distinct response from the muscle spindles appearing at the recording electrode after 13 ms, and weak reflex responses from the muscle with peak values at 53 and 63 ms during flexion and extension, respectively. 3. The time relations between kinematics, spindle firing and modulations of EMG activity elicited by the perturbations were compared with those of the self‐generated discontinuities. These analyses indicate that stretch reflex mechanisms cannot account for the modulations of EMG activity that give rise to successive 8‐10 Hz discontinuities. 4. A comparison of the reflex responses to perturbations with the EMG modulations during self‐generated movements indicates that the reflex was too weak to account for the pulsatile motor output during voluntary movements. 5. By inference it was concluded that the 8‐10 Hz discontinuities during self‐generated movements are probably generated by mechanisms within the central nervous system.
Noradrenaline transport and transporter mRNA of rat chromaffin cells are controlled by dexamethasone and nerve growth factor.
Authors: Wakade A R , Wakade T D , Poosch M , Bannon M J
Published: December 2014

1. The biochemical basis for differences in noradrenaline (NA) transporter function between chromaffin cells in the adrenal medulla and those maintained in primary culture was investigated. 2. Intact adrenal medullae of neonatal rats accumulated small amounts of [3H]NA. In contrast, dissociated chromaffin cells placed in culture for 2‐6 days accumulated 100‐1000 times more [3H]NA. 3. Nerve growth factor (NGF) stimulated, whereas glucocorticoids dose dependently and reversibly inhibited, [3H]NA transport in chromaffin cells maintained in culture up to 6 days. During this period, no change in the morphological or biochemical characteristics of either NGF‐treated or ‐untreated chromaffin cells was evident. 4. A rat NA transporter cDNA clone was isolated for use in the quantification of NA transporter mRNA. Intact adrenal medullae contained 40% less NA transporter mRNA than an equivalent number of chromaffin cells in culture. Furthermore, dexamethasone produced nearly 90% loss and NGF elicited approximately 60% increase in NA transporter mRNA levels in cultured cells. 5. In cultured cells, and possibly in vivo, glucocorticoids inhibit NA transporter function of chromaffin cells at least in part through a decrease in NA transporter mRNA.
Protein kinase C pathways modulate respiratory pattern generation in the cat.
Authors: Haji A , Pierrefiche O , Lalley P M , Richter D W
Published: December 2014

1. The significance of protein kinase C (PKC) in respiratory pattern generation was investigated in forty‐three expiratory neurones of anaesthetized cats. 2. Intracellular injection of R‐2,6‐diamino‐N‐([1‐(oxotridecyl)‐2‐piperidinyl]‐methyl)‐hexana mide dihydrochloride reversibly hyperpolarized twenty‐six neurones. Respiratory drive potentials decreased to 92% of control, and action potential discharges were reduced. Neuronal input resistance (Rin) decreased during inspiration and increased during expiration. 3. Voltage clamp revealed that blockade of PKC induced an increase of inhibitory drive currents and a decrease of excitatory drive currents in sixteen neurones. The amplitude of respiratory drive currents was decreased to 91% of control. The slope of synaptic inward currents during postinspiration was reduced. 4. After blockade of K+ conductances by TEA, additional blockade of PKC caused a hyperpolarization during postinspiration and expiration, but depolarization during inspiration in fourteen neurones. The respiratory drive currents were reduced to 61% of control. Respiratory drive potentials decreased to 72% of control, leading to reduced spontaneous discharge. Rin was increased throughout the respiratory cycle. 5. Stimulus‐evoked postsynaptic currents and potentials decreased after blockade of PKC with and without TEA. 6. The results indicate that PKC is endogenously active in expiratory neurones, modulating their excitability in three different ways: (a) it downregulates persistent K+ currents, (b) it upregulates Cl(‐)‐mediated inhibitory postsynaptic currents (IPSCs), and (c) it upregulates excitatory postsynaptic currents (EPSCs).
Monitoring calcium in turtle hair cells with a calcium‐activated potassium channel.
Authors: Tucker T R , Fettiplace R
Published: December 2014

1. An apamin‐sensitive Ca(2+)‐activated K+ channel was characterized in turtle hair cells and utilized to monitor submembranous intracellular Ca2+ and to evaluate the concentration of the mobile endogenous calcium buffer. 2. Isolated hair cells were voltage clamped with whole‐cell patch electrodes filled with a Cs(+)‐based intracellular solution to block the large‐conductance Ca(2+)‐activated K+ (BK) channel. Ca2+ currents evoked by depolarization were followed by inward tail currents lasting several hundred milliseconds. Both the Ca2+ current and slow tail current were abolished by nifedipine. 3. The tail current was carried by K+ and Cs+ (relative permeabilities PCa/PK = 0.22), and was fully blocked by 0.1 microM apamin and half blocked by 5 mM external TEA. These properties suggest the tail current flows through a Ca(2+)‐activated K+ channel distinct from the BK channels. 4. Intracellular Ca2+ was imaged with a confocal microscope in hair cells filled with the indicator Calcium Green‐5N introduced via the patch pipette. Increases in Ca2+ evoked by depolarization were localized to hotspots on the basolateral surface of the cell. The time course of the tail current closely matched the fast component of the fluorescenece monitored at a hotspot. 5. Ca(2+)‐ATPase pump inhibitors thapsigargin, 2,4‐di‐(t‐butyl)hydroquinone (BHQ) and vanadate, which are known to influence calcium regulation in turtle hair cells, prolonged the time course of the tail current, supporting the idea that the channel monitors cytoplasmic Ca2+. 6. The mobile endogenous buffer was estimated by combining perforated‐patch and whole‐cell recordings on a single cell. After recording tail currents with an amphotericin‐perforated patch, the patch was ruptured to obtain the whole‐cell mode, thus allowing washout of soluble cytoplasmic proteins and exchange with pipette buffers. By varying the concentration of Ca2+ buffer in the pipette, the mobile endogenous buffer was found to be equivalent to about 1 mM BAPTA.
Rostrocaudal gradient of mechanical advantage in the parasternal intercostal muscles of the dog.
Authors: De Troyer A , Legrand A , Wilson T A
Published: December 2014

1. Previous theoretical studies have led to the predictions that, in the dog, the parasternal intercostal muscles in the rostral interspaces shorten more during passive inflation than those in the caudal interspaces and have, therefore, a greater inspiratory mechanical advantage. The present studies were undertaken to test these predictions. 2. The effects of passive inflation on the length of the parasternal intercostals interspaces 1 to 7 were evaluated with markers implanted in the costal cartilages. Although the muscles in all interspaces shortened with passive inflation, the fractional shortening increased from the first to the second and third interspaces and then decreased continuously to the seventh interspace. 3. To understand this peculiar distribution, a geometric model of the parasternal area was then developed and a relation was obtained between muscle shortening and the angles that describe the orientation of the muscle and costal cartilage relative to the sternum. Measurement of these angles indicated that the rostrocaudal gradient of parasternal shortening resulted from the different orientations of the costal cartilages and their different rotations during passive inflation. 4. The changes in airway pressure generated by the parasternal intercostals in interspaces 3, 5 and 7 were finally measured during selective, maximal stimulation. The fall in pressure was invariably greatest during contraction of the third interspace and smallest during contraction of the seventh. 5. These observations indicate that, in the dog, the rostrocaudal gradient in rib rotation induces a rostrocaudal gradient of mechanical advantage in the parasternal intercostals, which has its climax in the second and third interspaces. These observations also support the concept that the respiratory effect of a given respiratory muscle can be computed from its behaviour during passive inflation.
Calcium‐induced inactivation of NMDA receptor‐channels evolves independently of run‐down in cultured rat brain neurones.
Authors: Medina I , Filippova N , Bakhramov A , Bregestovski P
Published: December 2014

1. Calcium‐induced transient inactivation of NMDA receptor (NMDAR) channels was studied in cultured rat hippocampal and cerebellar granule neurones using patch‐clamp techniques and confocal scanning microscopy. 2. During whole‐cell recordings, in the presence of 2 mM external Ca2+, conditioning (2‐20 s) pulses of NMDA (20‐100 microM) caused a transient decrease in NMDA responses. Recovery developed in two phases with time constants of 0.6 and 40 s. The slow phase of the recovery could be prevented either by strong intracellular Ca2+ ([Ca2+]i) buffering with 30 mM BAPTA or by using Ca(2+)‐free extracellular solution. 3. Simultaneous measurement of currents and Ca(2+)‐dependent fluorescence revealed a close correlation between the time constants of [Ca2+]i decay and the slow component of NMDA‐activated test current recovery. 4. During prolonged recordings, the transient inactivation was not related to irreversible NMDA‐activated current run‐down. After 25 min of recording with ATP‐free intracellular solution, NMDA‐activated currents in hippocampal neurones irreversibly decreased by 49 +/‐ 5% while inactivation decreased by 8% (n = 9). Calyculin A and FK‐506 (phosphatase inhibitors) significantly delayed run‐down but did not modulate the transient inactivation. 5. In cerebellar granule cells that did not show run‐down (4 mM MgATP in the pipette) the percentage of transient inactivation strongly decreased during 25 min of recording (from 28 +/‐ 6 to 7 +/‐ 5%, n = 15). 6. In cell‐attached recordings (5 microM NMDA in the pipette), elevation of [Ca2+]i (application of 100 microM NMDA to the soma) caused a reversible reduction of single NMDAR channel open probability (NPo) due to a decrease in the frequency of channel opening. 7. In inside‐out patches, application of Ca2+ to the cytoplasmic side of the membrane caused a rapid and reversible decrease in NPo (13 out of 29 patches). In the absence of run‐down, the ability of Ca2+ to transiently inhibit NMDAR channel activity disappeared after 3‐5 min of recording. 8. These results indicate that Ca(2+)‐induced transient inactivation of NMDAR currents develops independently from the run‐down and suggest that a diffusible Ca2+ ‐dependent factor mediates NMDAR channel inactivation.
Release properties of isolated neuromuscular boutons of the garter snake.
Authors: Wilkinson R S , Son Y J , Lunin S D
Published: December 2014

1. Motor nerve terminals innervating fibres in the transversus abdominis muscle of the garter snake comprise discrete boutons. Using a combination of enzymatic digestion and mechanical manipulation, individual boutons were removed from living terminals for study in isolation. 2. Boutons freed from terminals were usually allowed to remain in their original location on the endplate (‘attached’ one‐bouton synapse). Alternatively, they were removed from the endplate, and then placed on the same or another vacant endplate site to form a ‘reconstructed’ one‐bouton synapse. When removed from the endplate, boutons were 2‐4 microns in diameter and nearly spherical in shape, in contrast to the variety of complex shapes seen among boutons still in contact with muscle fibre endplates. 3. Transmitter release was assessed by intracellular recording from the postsynaptic fibre. Boutons produced spontaneous miniature endplate potentials (MEPPs) of nearly normal amplitude; extracellular stimulation elicited endplate potentials (EPPs) which resembled MEPPs. Typical EPP amplitudes fluctuated between zero and five quanta per stimulus. For low‐frequency stimulation under normal physiological conditions, mean quantal content, m, averaged 1.4; the binomial number of release sites, n, averaged 2.4; and the binomial probability of release, p, averaged 0.57. Statistics of the quantal fluctuations recorded from single boutons agreed only approximately with predictions of simple binomial theory, the discrepancy being that the apparent number of quanta released exceeded n in 5% of the events. 4. In separate experiments, activity‐dependent probes were used to locate rare naturally occurring nerve terminals comprising a single bouton. Activation of these small synapses evoked quantal responses similar to those of attached and reconstructed one‐bouton synapses described above.
Differential effects of protein kinase C activation on 5‐HT1A receptor coupling to Ca2+ and K+ currents in rat serotonergic neurones.
Authors: Chen Y , Penington N J
Published: December 2014

1. Activation of the enzyme protein kinase C (PKC) partially uncouples receptors from the inhibition of Ca2+ current. We have studied the effect of PKC activation on 5‐HT1A receptor coupling of Ca2+ currents and 5‐HT‐induced K+ current (IK,5‐HT) in acutely isolated adult rat dorsal raphe neurones. 2. The phorbol ester 4 beta‐phorbol 12‐myristate, 13‐acetate (PMA; 1 microM) did not significantly alter the peak Ca2+ current. A maximal dose of 5‐HT inhibited Ca2+ current on average by 52%; after application of PMA, the inhibition was only 30% and the effect was irreversible for the duration of the experiment. 3. The inactive phorbol ester 4 alpha‐phorbol (1 microM) did not reduce the effectiveness of 5‐HT. When the kinase inhibitor staurosporine (ST; 200 nM) was added, PMA reduced the effect of 5‐HT by only 13.9%. ST partially prevented or reversed the effect of PMA, depending on the order of addition. 4. The voltage‐dependent rate or re‐inhibition by 5‐HT was reduced by PMA, suggesting that fewer activated G‐protein subunits are available to interact with Ca2+ channel after the action of PMA. 5. In contrast, PMA (1 microM) did not have a significant effect on IK,5‐HT. 6. PKC activation has an inhibitory effect on one branch of the 5‐HT1A receptor transduction fork, namely inhibition of Ca2+ influx, but not on the activation of IK,5‐HT.
Salbutamol changes the molecular and mechanical properties of canine skeletal muscle.
Authors: Zhang K M , Hu P , Wang S W , Feher J J , Wright L D , Wechsler A S , Spratt J A , Briggs F N
Published: December 2014

1. Salbutamol, a beta 2‐agonist, increased the weight of the canine latissimus dorsi muscle. It also increased fusion frequency, and decreased time‐to‐peak tension, half‐relaxation time, and total contraction time. These changes in twitch times and fusion frequency were associated with changes in the levels of proteins expressed in slow‐ and fast‐twitch fibres. Salbutamol decreased the levels of the slow‐twitch cardiac isoform of sarco‐/endoplasmic reticulum Ca(2+)‐ATPase (SERCA2a) and phospholamban proteins, and increased the level of the fast‐twitch isoform of sarco‐/endoplasmic reticulum Ca(2+)‐ATPase (SERCA1a). 2. Changes in the levels of SERCA proteins, particularly SERCA1a, could account for most of the increases in calcium uptake rate observed in homogenates of muscles from the salbutamol‐treated animals and could partially account for the changes in half‐relaxation rates and other twitch times. 3. Changes in the levels of SERCA1a, SERCA2a and phospholamban protein did not always follow changes in the levels of their corresponding mRNAs. Divergence depended upon the SERCA isoform and muscle. The muscles studied were latissimus dorsi and vastus intermedius. 4. Salbutamol did not change the level of myosin heavy chain (HC)‐I isoforms in either muscle, suggesting that it did not increase the proportion of slow‐twitch fibres in these muscles. It did increase the level of HC‐IIx and decrease the level of HC‐IIa isoforms in the latissimus dorsi. Salbutamol did not produce these effects in the vastus intermedius. It is of particular interest that salbutamol changed the relative levels of SERCA proteins in the latissimus dorsi muscle without producing significant change in the level of HC‐I isoform.
The viscous, viscoelastic and elastic characteristics of resting fast and slow mammalian (rat) muscle fibres.
Authors: Mutungi G , Ranatunga K W
Published: December 2014

1. The tension and sarcomere length responses induced by ramp stretches (amplitude 1‐3% of initial fibre length (Lzero) and speeds of 0.01‐12 Lzero s‐1) were examined, at 10 degrees C and sarcomere lengths of approximately 2.7 microns, in resting intact muscle fibre bundles isolated from the soleus (a slow muscle) and extensor digitorum longus (a fast muscle) of the rat. 2. In both fibre types, the tension response to moderately fast ramp stretches consists of a viscous, a viscoelastic and an elastic component. At low stretch velocities, where the viscous component is very small, the tension response consists of only the viscoelastic and elastic components. 3. The viscosity coefficient (mean +/‐ S.E.M., 2 +/‐ 0.01 kN s m‐2, n = 12) and the relaxation time of the viscoelasticity (44 +/‐ 2 ms, n = 12) of the slow muscle fibres were significantly larger than those of the fast muscle fibres (0.8 +/‐ 0.1 kN s m‐2 and 11 +/‐ 1 ms, respectively, n = 20). 4. The relaxation time, in either fibre type, is too long for the viscoelasticity to be due to rapidly cycling, weakly attached cross‐bridges. Moreover, the tension components increased with sarcomere length and were insensitive to 5‐10 mM 2,3‐butanedione 2‐monoxime (BDM), which inhibited active contractions. 5. The possibility that the fast‐slow fibre differences may reflect differences in myoplasmic viscosity and connectin (titin) isoforms (in their gap filaments) is discussed.
Effects of long‐term conduction block on membrane properties of reinnervated and normally innervated rat skeletal muscle.
Authors: Pasino E , Buffelli M , Arancio O , Busetto G , Salviati A , Cangiano A
Published: December 2014

1. Do motoneurons regulate muscle extrajunctional membrane properties through chemical (trophic) factors in addition to evoked activity? We addressed this question by comparing the effects of denervation and nerve conduction block by tetrodotoxin (TTX) on extrajunctional acetylcholine (ACh) sensitivity and action potential resistance to TTX in adult rats. 2. We applied TTX to sciatic or tibial nerves for up to 5 weeks using an improved blocking technique which completely suppresses conduction but avoids nerve damage. 3. Reinnervation by TTX‐blocked axons had no effect on the high ACh sensitivity and TTX resistance induced by nerve crush. 4. Long‐lasting block of intact nerves (up to 38 days) induced extrajunctional changes as pronounced as after denervation. At shorter times (3 days), however, denervation induced much larger changes than TTX block; such a difference is thus only transiently present in muscle. 5. The effects of long‐lasting block were dose dependent. Dose levels (6.6 micrograms day‐1) corresponding to those used in the literature to block the rat sciatic nerve induced muscle effects much smaller than those induced by denervation, confirming published data. Our novel finding is that equal effects are obtained using doses substantially higher (up to 10.5 micrograms day‐1). For the soleus it was necessary in addition to apply the TTX directly to the smaller tibial nerve. 6. The TTX‐blocked nerves were normal in their histological appearance and capacity to transport anterogradely 3H‐labelled proteins, to release ACh in quantal and non‐quantal form or cluster ACh receptors and induce functional ectopic junctions on denervated soleus muscles. 7. We conclude that muscle evoked activity is the physiological regulator of extrajunctional membrane properties. Chemical factors from the nerve do not appear to participate in this regulation. The stronger response to denervation at short times only is best accounted for by factors produced by degenerating nerves.
Calcium channels involved in synaptic transmission at the mature and regenerating mouse neuromuscular junction.
Authors: Katz E , Ferro P A , Weisz G , Uchitel O D
Published: December 2014

1. The involvement of the different types of voltage‐dependent calcium channels (VDCCs) in synaptic transmission at the mature and newly formed mammalian neuromuscular junction was studied by evaluating the effects of L‐, P/Q‐ and N‐type VDCC antagonists on transmitter release in normal and reinnervating levator auris preparations of adult mice. 2. Nerve‐evoked transmitter release was blocked by omega‐agatoxin IVA (omega‐AgaIVA), a P/Q‐type VDCC blocker, both in normal and reinnervating endplates (100 nM omega‐AgaIVA caused > 90% inhibition). The N‐type VDCC antagonist omega‐conotoxin GVIA (omega‐CgTX; 1 and 5 microM), as occurs in normal preparations, did not significantly affect this type of release during reinnervation. Nitrendipine (1‐10 microM), an L‐type VDCC blocker, strongly antagonized release in reinnervating muscles (approximately 40‐69% blockade) and lacked any effect in normal preparations. 3. In reinnervating muscles, spontaneous release was not dependent on Ca2+ entry through either P‐ or L‐type VDCCs. Neither 100 nM omega‐AgaIVA nor 10 microM nitrendipine affected the miniature endplate potential (MEPP) frequency or amplitude. 4. At the newly formed endplates, K(+)‐evoked release was dependent on Ca2+ entry through VDCCs of the P‐type family (100 nM omega‐AgaIVA reduced approximately 70% of the K(+)‐evoked MEPP frequency). L‐type VDCCs were found not to participate in this type of release (10 microM nitrendipine lacked any effect). 5. In reinnervating muscles, the L‐type VDCC blocker, nitrendipine (10 microM), provoked a significant increase (approximately 25%) in the latency of the evoked endplate potential (EPP). This drug also caused an increase (approximately 0.3 ms) in the latency of the presynaptic currents. The P/Q‐ and Ny‐type VDCC blockers did not affect the latency of the EPP. 6. These results show that at newly formed mouse neuromuscular junctions, as occurs in mature preparations, VDCCs of the P‐type family play a prominent role in evoked transmitter release whereas N‐type channels are not involved in this process. In addition, signal conduction and transmitter release become highly sensitive to nitrendipine during reinnervation. This suggests that L‐type VDCCs may play a role in synaptic transmission at the immature mammalian neuromuscular junction.
Channel modulation by tyrosine phosphorylation in an identified leech neuron.
Authors: Aniksztejn L , Catarsi S , Drapeau P
Published: December 2014

1. We have examined the effects of tyrosine phosphorylation on a spontaneously active cation channel that also participates in the modulation of pressure‐sensitive (P) neurons in the leech. Cation channel activity in cell‐attached or isolated, inside‐out membrane patches from P cells in culture was monitored before and after treatments that altered the level of tyrosine phosphorylation. 2. In cell‐attached recordings from intact P cells, bath application of genistein, an inhibitor of tyrosine kinases, resulted in a 6.6 +/‐ 2.6‐fold increase in channel activity with no change in the mean open time or amplitude. Daidzein, an inactive form of genistein, was without effect. Addition of pervanadate, a membrane‐permeant inhibitor of tyrosine phosphatases, had no effect on its own and blocked the effect of subsequent addition of genistein. 3. In inside‐out P cell membrane patch recordings, exposure to a catalytically active fragment of a tyrosine phosphatase resulted in a 10.3 +/‐ 3.6‐fold increase in channel activity with no change in the mean open time or amplitude. Orthovanadate had no effect on channel activity and, when added with the phosphatase, prevented the increase in activity. 4. Our results demonstrate that the basal activity of cation channels is increased by tyrosine dephosphorylation, suggesting a constitutive modulation of channel activity under resting conditions.
Temperature sensitivity of neurones in slices of the rat spinal cord.
Authors: Pehl U , Schmid H A , Simon E
Published: December 2014

1. The inherent temperature sensitivity of 343 spontaneously active neurones recorded from rat spinal cord (SC) slices was investigated electrophysiologically. Recordings were made from 321 neurons from transverse and 22 neurons from longitudinal slices and their thermosensitivity was determined by relating changes in firing rate to changes in slice temperature. 2. Of the neurones from transverse slices, 53% were warm sensitive, 2% were cold sensitive and 45% were temperature insensitive. In longitudinal slices, 68% were warm sensitive and the remaining neurones were temperature insensitive. 3. When classified according to their recording sites in transverse slices, warm‐sensitive neurones in laminae I and II had the same mean temperature coefficient compared with those recorded from lamina X, despite the fact that the latter had a significantly higher spontaneous activity. 4. The intrinsic temperature sensitivity of the majority of warm‐sensitive neurones was confirmed by blocking their synaptic input. 5. A transient overshoot in activity, i.e. a dynamic response characteristic following rapid temperature stimuli (0.4 degree C s‐1) was observed in 73% of the warm‐sensitive and 59% of the temperature‐insensitive neurones in laminae I and II in response to rapid warming, but only rarely (< 10%) in lamina X. 6. Temperature‐sensitive SC neurones share response characteristics with temperature‐sensitive neurones in the preoptic and anterior hypothalamic (PO/AH) area and with peripheral temperature receptors. Functionally, these neurones may represent the cellular basis for the temperature sensory function of the spinal cord that has been well characterized in vivo in homeothermic species.
Evaluation of purine nucleotide loss, lipid peroxidation and ultrastructural alterations in post‐hypoxic hepatocytes.
Authors: Grune T , Müller K , Zöllner S , Haseloff R , Blasig I E , David H , Siems W
Published: December 2014

1. Hypoxic alterations in isolated rat hepatocytes were demonstrated by a 90% ATP loss during 60 min of ischaemia and temporary increases of nucleotide degradation products. 2. The oxidative stress during reoxygenation was demonstrated in these cells by a decrease in reduced glutathione (GSH) concentration (30%) and a threefold increase in lipid peroxidation products such as 4‐hydroxynonenal and thiobarbituric acid‐reactive substances (TBA‐RSs). The tremendous GSH loss could not be balanced by the slight oxidized glutathione (GSSG) increase during reoxygenation. 3. For the first time the involvement of free radicals was directly demonstrated using electron spin resonance (ESR) spectroscopy in reoxygenated liver cells. Using the spin trap 5,5‐dimethylpyrroline‐1‐oxide (DMPO), a carbon‐centred radical and the adduct of the hydroxyl radical could be detected during early reoxygenation. 4. Morphological alteration of cells was observed, beginning during hypoxia and increasing during post‐hypoxic reoxygenation. Electron microscopic findings of hypoxic and post‐hypoxic cell damage included pyknosis of nuclei, spherical transformation of mitochondria and increased number of vesicles.
Evidence for nitric oxide‐mediated sympathetic forearm vasodiolatation in humans.
Authors: Dietz N M , Engelke K A , Samuel T T , Fix R T , Joyner M J
Published: December 2014

1. Our aim was to determine if sympathetic vasodilatation occurs in the human forearm, and if the vasodilating substance nitric oxide contributes to this dilatation. We also sought to determine if the nitric oxide might be released as a result of cholinergic stimulation of the vascular endothelium. 2. Blood flow was measured in the resting non‐dominant forearm with venous occlusion plethysmography. To increase sympathetic traffic to the resting forearm, rhythmic handgrip exercise to fatigue followed by post‐exercise ischaemia was performed by the dominant forearm. A brachial artery catheter in the non‐dominant arm was used to selectively infuse drugs. 3. During control conditions, there was mild vasodilatation in the resting forearm during exercise followed by constriction during post‐exercise ischaemia. When exercise was performed after brachial artery administration of bretylium (to block noradrenaline release) and phentolamine (an alpha‐adrenergic antagonist), profound vasodilatation was seen in the resting forearm during both exercise and post‐exercise ischaemia. 4. When the nitric oxide synthase blocker NG‐monomethyl‐L‐arginine (L‐NMMA) was administered in the presence of bretylium and phentolamine prior to another bout of handgripping, little or no vasodilatation was seen either during exercise or post‐exercise ischaemia. Atropine also blunted the vasodilator responses to exercise and post‐exercise ischaemia after bretylium and phentolamine. 5. These results support the existence of active sympathetic vasodilatation in the human forearm and the involvement of nitric oxide in this phenomenon. They also suggest nitric oxide might be released as a result of cholinergic stimulation of the vascular endothelium.