bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophysiological characteristics of immunochemically identified rat oxytocin and vasopressin neurones in vitro.
Authors: Armstrong W E , Smith B N , Tian M
Published: December 2014

1. Intracellular recordings were made from supraoptic neurones in vitro from hypothalamic explants prepared from adult male rats. Neurones were injected with biotinylated markers, and of thirty‐nine labelled neurones, nineteen were identified immunocytochemically as containing oxytocin‐neurophysin and twenty as containing vasopressin‐neurophysin. 2. Vasopressin and oxytocin neurones did not differ in their resting membrane potential, input resistance, membrane time constant, action potential height from threshold, action potential width at half‐amplitude, and spike hyperpolarizing after‐potential amplitude. Both cell types exhibited spike broadening during brief, evoked spike trains (6‐8 spikes), but the degree of broadening was slightly greater for vasopressin neurones. When hyperpolarized below ‐75 mV, all but one neurone exhibited a transient outward rectification to depolarizing pulses, which delayed the occurrence of the first spike. 3. Both cell types exhibited a long after‐hyperpolarizing potential (AHP) following brief spike trains evoked either with a square wave pulse or using 5 ms pulses in a train. There were no significant differences between cell types in the size of the AHP evoked with nine spikes, or in the time constant of its decay. The maximal AHP evoked by a 180 ms pulse was elicited by an average of twelve to thirteen spikes, and neither the size of this maximal AHP nor its time constant of decay were different for the two cell types. 4. In most oxytocin and vasopressin neurones the AHP, and concomitantly spike frequency adaptation, were markedly reduced by the bee venom apamin and by d‐tubocurarine, known blockers of a Ca(2+)‐mediated K+ conductance. However, a minority of neurones, of both cell types, were relatively resistant to both agents. 5. In untreated neurones, 55% of vasopressin neurones and 32% of oxytocin neurones exhibited a depolarizing after‐potential (DAP) after individual spikes or, more commonly, after brief trains of spikes evoked with current pulses. For each neurone with a DAP, bursts of spikes could be evoked if the membrane potential was sufficiently depolarized such that the DAP reached spike threshold. In four out of five vasopressin neurones a DAP became evident only after pharmacological blockade of the AHP, whereas in six oxytocin neurones tested no such masking was found. 6. The firing patterns of neurones were examined at rest and after varying the membrane potential with continuous current injection. No identifying pattern was strictly associated with either cell type, and a substantial number of neurones were silent at rest.(ABSTRACT TRUNCATED AT 400 WORDS)
Relaxation mechanisms induced by stimulation of nerves and by nitric oxide in sheep urethral muscle.
Authors: García‐Pascual A , Triguero D
Published: December 2014

Isolated transverse and longitudinally oriented preparations of sheep urethra precontracted with noradrenaline responded to electrical field stimulation (EFS) with stimulus‐dependent non‐adrenergic, non‐cholinergic (NANC) relaxations. Exogenous nitric oxide (NO) (acidified NaNO2), S‐nitroso‐L‐cysteine (NC), sodium nitroprusside (SNP), 8‐Br‐cGMP, dibutyryl‐cAMP, forskolin and isoprenaline each relaxed precontracted transverse urethral preparations in a concentration‐dependent manner in order of protency: NC > forskolin > isoprenaline = SNP > NO > 8‐Br‐cGMP = dibutyryl‐cAMP. Longitudinally oriented preparations responded to NO and NC with concentration‐dependent relaxation, no different from that observed in transverse strips. Methylene blue (MB) and oxyhaemoglobin (HbO2) each shifted the concentration‐response curve for NO to the right without affecting EFS‐induced relaxation. Similarly, concentration‐dependent responses to NC were not affected by MB. The inhibition of relaxation to NO by MB was prevented by superoxide dismutase, suggesting the inhibition was caused by extracellular generation of superoxide anions. EFS‐induced relaxation was accompanied by elevation of cGMP. However, for the same level of relaxation, exogenous NO and NC induced 15‐ and 23‐times higher increases in cGMP values, respectively, than EFS. cAMP levels were not affected by EFS‐ or NO‐induced relaxation, although a large increase accompanied relaxation induced by forskolin. Forskolin also increased cGMP content. Pretreatment with MB reduced basal levels of cGMP and inhibited both relaxation and rise in cGMP levels induced by NO. SNP‐elicited relaxant responses, in the presence of MB, were accompanied by an accumulation of cGMP; cAMP levels were unaffected. MB reduced cGMP levels induced by NC, while the relaxant response was unchanged. In urethral preparations prelabelled with [3H]myoinositol, exposure to NA caused an accumulation of [3H]inositol phosphates, which was unaffected by pretreatment with 8‐Br‐cGMP or dibutyryl‐cAMP. EFS failed to induce a relaxant response in excess [K+]o‐contracted preparations, while relaxation with exogenous NO was unaffected. Ouabain abolished EFS‐induced relaxation and reduced responses to NO. Neither TEA nor glibenclamide affected relaxation to either EFS or NO. Relaxation elicited by SNP was not accompanied by any change in cGMP or cAMP levels, and was unaffected by MB, HbO2, K+ channel blockers (TEA and glibenclamide), ouabain or high [K+]o solution. This suggested that relaxation was caused by a mechanism independent of NO generation. A dense network of NADPH diaphorase‐positive fibres associated with both the circular and longitudinal smooth muscle layers of sheep urethra was found.(ABSTRACT TRUNCATED AT 400 WORDS)
Gating and conductance properties of a human delayed rectifier K+ channel expressed in frog oocytes.
Authors: Benndorf K , Koopmann R , Lorra C , Pongs O
Published: December 2014

1. The human delayed rectifier K+ channel h‐DRK1, a homologue to the DRK1 channel in the rat, was expressed in Xenopus oocytes. Single‐channel currents were measured in micropatches; macroscopic currents were measured either in macropatches, giant patches, or whole oocytes. 2. Macroscopic currents activated at ‐20 mV and more positive. The instantaneous current‐voltage relationship rectified outwardly to a higher degree than predicted by the Goldman‐Hodgkin‐Katz equation. 3. With the giant patch technique, ionic and putative on‐ and off‐gating currents were recorded simultaneously. The large ratio of the moved gating charges to the amplitude of the ionic current indicated that less than 1% of the gating channels actually opened. 4. The single‐channel conductance between 0 and +80 mV was calculated to be 9.4 pS. The channels opened with sublevels which appeared either independently of the fully open level as separate openings, in conjunction with the opening to and closing from the fully open level, or by starting from and ending at the fully open level. 5. The channels opened with two voltage‐independent open time constants in the range 1‐10 ms (filter 1 kHz). The burst open probability was fitted monoexponentially with time constants in the range of tens of milliseconds. 6. Assuming a sequential Markovian model with four independent voltage‐controlled transitions, fit of the steady‐state open probability of macroscopic currents showed two components of activation differing in their half‐maximal value. 7. The fit of time courses of cumulative first latency and ensemble‐averaged currents in single‐channel patches suggested that even a single channel may operate with the two different components of activation. 8. It is concluded that h‐DRK1 channels considerably rectify in an outward direction and that an apparently flat voltage dependence of activation may be explained by the overlap of two different components.
Suppression of voluntary motor activity revealed using transcranial magnetic stimulation of the motor cortex in man.
Authors: Davey N J , Romaiguère P , Maskill D W , Ellaway P H
Published: December 2014

1. Suppression of voluntary muscle activity of hand and arm muscles in response to transcranial magnetic stimulation (TMS) of the motor cortex has been investigated in man. 2. Suppression could be elicited by low levels of TMS without any prior excitatory response. The latency of the suppression was 3‐8 ms longer than the excitation observed at a higher stimulus intensity. The duration of the suppression ranged from 8 to 26 ms. 3. A circular stimulating coil was used to determine threshold intensity for excitation and suppression of contraction of thenar muscles in response to TMS at different locations over the motor cortex. The locations for lowest threshold excitation coincided with those for lowest threshold suppression. Suppression was elicited at a lower threshold than excitation at all locations. 4. A figure‐of‐eight stimulating coil was positioned over the left motor cortex at the lowest threshold point for excitation of the right thenar muscles. The orientation for the lowest threshold excitatory and inhibitory responses was the same for all subjects. That orientation induced a stimulating current travelling in an antero‐medial direction. Suppression was invariably elicited at lower thresholds than excitation. 5. When antagonistic muscles (second and third dorsal interosseus) were co‐contracted, TMS evoked coincident suppression of voluntary EMG in the two muscles without prior excitation of either muscle. This suggests that the suppression is not mediated via corticospinal activation of spinal interneurones. 6. Test responses to electrical stimulation of the cervical spinal cord were evoked in both relaxed and activated thenar muscles. In the relaxed muscle, prior TMS at an intensity that would suppress voluntary activity failed to influence the test responses, suggesting absence of inhibition at a spinal level. However, in the activated muscle, prior TMS could reduce the test response. This may be explained by disfacilitation of motoneurones due to inhibition of corticospinal output. 7. We propose that suppression of voluntary muscle activity by TMS is due in large part to activation of a mechanism within the motor cortex that reduces the corticospinal output to the muscle. It is concluded that TMS evokes excitation and inhibition via neuronal structures lying close to one another and having similar orientations.
Modulation of GABA‐mediated synaptic transmission by endogenous zinc in the immature rat hippocampus in vitro.
Authors: Xie X , Hider R C , Smart T G
Published: December 2014

1. Intracellular recordings from postnatal 2‐ to 12‐day‐old (P2‐12) rat hippocampal CA3 pyramidal neurones exhibited spontaneous synaptic potentials mediated by GABAA receptors. These potentials can be separated on the basis of amplitude into two classes which are referred to as small and large. 2. The large depolarizing potentials were reversibly inhibited by the Zn2+ chelator 1,2‐diethyl‐3‐hydroxypyridin‐4‐one (CP94). The small inhibitory postsynaptic potentials. (IPSPs) were apparently unaffected. 3. Stimulation of the mossy fibre pathway evoked composite excitatory postsynaptic potentials (EPSPs) and IPSPs. Threshold stimulus‐evoked synaptic potentials were mediated by GABAA receptors and were reversibly blocked by CP94. The responses evoked by suprathreshold stimulation and persisting in the presence of bicuculline or CP94 were partially inhibited by 2‐amino‐5‐phosphonopropionic acid (AP5) and were completely blocked with 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). 4. L‐Histidine, which preferentially forms complexes with Cu2+ > Zn2+ > Fe2+ > Mn2+, inhibited both naturally occurring spontaneous and evoked GABAA‐mediated large synaptic potentials without affecting the neuronal resting membrane properties. Exogenously applied Zn2+ induced large spontaneous synaptic potentials and prolonged the duration of the evoked potentials. These effects were reversibly blocked by histidine. 5. The metal chelating agent diethyldithiocarbamate had little effect on the large amplitude synaptic potentials. 6. The transition metal divalent cations Fe2+ and Mn2+ did not initiate large synaptic potentials in CA3 neurones; however, Cu2+ depolarized the membrane and enhanced both excitatory and inhibitory synaptic transmission, resulting in a transient increase in the frequency of the large amplitude events. In comparison, zinc increased the frequency of the large potentials and also induced such events in neurons (P4‐21) where innate potentials were absent. The postsynaptic response to ionophoretically applied GABA was either unaffected or slightly enhanced by Zn2+. 7. Under conditions favouring the activation of non‐NMDA receptors, excitatory synaptic transmission was unaffected by CP94 but was depressed by Zn2+. Responses to ionophoretically applied glutamate were not inhibited by Zn2+, indicating that Zn2+ affects excitatory synaptic transmission via a presynaptic mechanism. 8. We conclude that the naturally occurring large synaptic potentials in young CA3 neurones are apparently induced by endogenous Zn2+ which can promote or synchronize the release of GABA in the immature hippocampus.
Synaptic transmission from splanchnic nerves to the adrenal medulla of guinea‐pigs.
Authors: Holman M E , Coleman H A , Tonta M A , Parkington H C
Published: December 2014

1. Membrane potentials were recorded with conventional intracellular microelectrodes from chromaffin cells in isolated, bisected adrenal glands from guinea‐pigs. 2. All cells were electrically excitable and responded to depolarizing current with all‐or‐nothing action potentials that were blocked by tetrodotoxin. 3. Input resistance was 180 +/‐ 14 M omega and this was lower than that reported for isolated chromaffin cells using patch electrodes. 4. All cells responded to transmural stimulation with action potentials that arose from excitatory synaptic potentials in response to the excitation of one or more preganglionic fibres, many having strong synaptic action. Other fibres had weaker synaptic action but in all cases, maximal transmural stimulation caused depolarization well above threshold for action potential initiation. 5. Spontaneous excitatory synaptic potentials were observed whose frequency was greatly increased by repetitive stimulation at 10 or 30 Hz. 6. No evidence was found for the desensitization of nicotinic receptors in response to acetylcholine released from presynaptic nerve terminals. 7. These experiments show that there are many similarities between the responses to splanchnic nerve stimulation of guinea‐pig chromaffin cells in situ and sympathetic ganglion cells from the same species.
The upregulation of acetylcholine release at endplates of alpha‐bungarotoxin‐treated rats: its dependency on calcium.
Authors: Plomp J J , van Kempen G T , Molenaar P C
Published: December 2014

1. The presynaptic component of an adaptive feedback mechanism leading to increased acetylcholine (ACh) release was studied in endplates of diaphragms from rats treated chronically with alpha‐bungarotoxin (alpha BTX). 2. Quantal contents were calculated ‘directly’ from the amplitude of miniature endplate potentials (MEPPs) and endplate potentials (EPPs) which were recorded after mu‐conotoxin treatment to prevent muscle action potentials. 3. In vitro application of the Ca2+ channel blockers nifedipine (10 microM) or omega‐conotoxin (40 nM) had no significant effect on the increased quantal content of endplates from alpha BTX‐treated rats. 4. At control endplates, in vitro block of presynaptic K+ channels by 5 microM 3,4‐diaminopyridine did increase the quantal content to a level which was similar to that found in endplates of alpha BTX‐treated rats but also induced a broadening of EPPs, which was not found at endplates after alpha BTX treatment. 5. The difference between quantal contents of alpha BTX‐treated and control rats was highly dependent on the [Ca2+]o/[Mg2+]o ratio when [Mg2+]o was fixed at 1 mM. At low [Ca2+]o, the quantal content of endplates from alpha BTX‐treated rats was lower than that of controls while at [Ca2+]o in the normal and high range this was reversed. However, changing the [Ca2+]o/[Mg2+]o ratio by means of [Mg2+]o, at a fixed [Ca2+]o of 2 mM, did not influence the relative increase of quantal contents at endplates from alpha BTX‐treated rats. Double logarithmic plots of the ‘toxin‐induced’ myasthenia gravis (TIMG) and control quantal content versus [Ca2+]o had an approximately linear part between 0.2 and 1.5 mM [Ca2+]o. The slopes of the TIMG and control lines were 1.81 and 0.96, indicating that the ACh release in TIMG muscles was more sensitive to changes of [Ca2+]o than controls. 6. At normal [Ca2+]o and [Mg2+]o, the depression of EPP amplitude during stimulation of the phrenic nerve at 30‐50 Hz was somewhat larger at endplates from alpha BTX‐treated rats than at control endplates. At low [Ca2+]o, the potentiation of EPP amplitudes during a stimulus train was much larger at endplates from alpha BTX‐treated rats than from controls. 7. The results do not support the idea that the increased release of ACh is caused via regulatory effects on the presynaptic Ca2+ or K+ channels. Instead, the anomalous dependency of ACh release on Ca2+ in muscles of alpha BTX‐treated rats suggests that a cytoplasmic, Ca(2+)‐dependent, component is involved in the adaptive change of transmitter release.
Variation in myoplasmic Ca2+ concentration during contraction and relaxation studied by the indicator fluo‐3 in frog muscle fibres.
Authors: Caputo C , Edman K A , Lou F , Sun Y B
Published: December 2014

1. The fluorescent dye fluo‐3, in its permeant acetoxymethyl form, was used to monitor calcium transients during twitch and tetanus of single fibres isolated from the anterior tibialis muscle of Rana temporaria (2‐5 degrees C). 2. Fluo‐3 was loaded into the muscle fibre by diffusion. Under the experimental conditions used, approximately 45% of maximal fluorescence was reached during a 1 s fused isometric tetanus. Fluo‐3 had no detectable effect on the mechanical response of the fibre. 3. The free calcium concentration in the myoplasm, [Ca2+]i, and its variation with time, was calculated from the fluorescence signal by accounting for the on‐ and off‐rate constants for the binding of calcium to the dye. The time course of the calcium transient during twitch and tetanus determined in this way agreed well with previous measurements based on fast‐reacting calcium‐sensitive dyes. 4. [Ca2+]i declined steeply during the initial phase of force relaxation in both twitch and tetanus, but exhibited a secondary rise that closely coincided with the pseudoexponential fall of tension after the shoulder in the tetanus myogram. The rate of decay of [Ca2+]i during relaxation and the rate of decline of force both became progressively reduced by repetitive stimulation. 5. Stretch and shortening ramps performed during the plateau of an isometric tetanus had no detectable effect upon the calcium transient during the movement. By contrast, shortening and stretch imposed during the linear phase of relaxation both led to an increase of [Ca2+]i and to a steepening of the relaxation phase. 6. The results strongly suggest that the non‐uniform length changes that are known to occur along a muscle fibre during relaxation enhance the release of calcium from the contractile system. The calcium mobilized in this way probably accounts for the transitory increase of [Ca2+]i that is observed during the latter part of force relaxation.
Fast presynaptic GABAA receptor‐mediated Cl‐ conductance in cultured rat hippocampal neurones.
Authors: Vautrin J , Schaffner A E , Barker J L
Published: December 2014

1. Hippocampal neurones cultured from the 18‐day‐old embryonic rat for 3 days to 3 weeks were recorded with Cl(‐)‐filled patch pipettes. Spontaneous synaptic currents, which reversed at the equilibrium potential for Cl‐ ions (ECl) and were blocked by the GABAA (gamma‐aminobutyric acid) receptor antagonists bicuculline or picrotoxin, were recorded in every culture. At 25 degrees C and ‐80 mV they decayed with a time constant > or = 20 ms that invariably increased at positive potentials. After 2 weeks, 50‐75% of all neurones were GABA immunoreactive. 2. In pairs‐recordings, coincident synaptic currents in both cells were either spontaneous or evoked by stimulation of one cell. In the presence of tetrodotoxin and using pipettes containing lidocaine (lignocaine) N‐ethyl bromide, coincident spontaneous Cl‐ transients still occurred in both neurones far more frequently than expected by chance. 3. Holding the potential of one neurone at a positive value reversed the synaptic transients in that cell and, in half of the cells, increased the frequency of coincident events in both cells. 4. In neurones where depolarization increased the frequency of coinciding events and all regenerative current apparent at the soma was abolished, short depolarizing pulses occasionally evoked all‐or‐none, pre‐ and postsynaptic currents with matching transmission failures and identical delays in transmission. 5. The results suggest that the same pulse of GABA simultaneously activates GABAA receptor‐coupled Cl‐ channels on both sides of the same synaptic cleft, producing immediate auto‐transmission in the absence of collaterals or interneurones.
5‐Hydoxytryptamine evokes depolarizations and membrane potential oscillations in rat sympathetic preganglionic neurones.
Authors: Pickering A E , Spanswick D , Logan S D
Published: December 2014

1. Whole‐cell recordings were made from seventy‐seven identified rat sympathetic preganglionic neurones (SPN) in spinal cord slices. Perfusion of 5‐HT (0.5‐30 microM) strongly depolarized 90% of neurones. The response was slow in onset, could last over 10 min and was associated with an increase in input resistance. 5‐HT could also evoke rhythmical membrane potential oscillations in a population of previously quiescent neurones. 2. The 5‐HT response persisted in TTX and also in low‐Ca(2+)‐high‐Mg2+ artificial cerebrospinal fluid (ACSF), suggesting that the receptors are on SPN. The 5‐HT uptake inhibitor 6‐nitroquipazine potentiated the 5‐HT‐induced depolarization. 3. The 5‐HT‐induced depolarization was reduced and then abolished by membrane hyperpolarization to potentials of about ‐100 mV, but was not reversed in sign by further hyperpolarization. In voltage clamp, 5‐HT evoked inward currents associated with the reduction of an outwardly rectifying potassium conductance. 4. The 5‐HT2 receptor agonist alpha‐methyl‐5‐HT mimicked the 5‐HT response on all neurones, as did the 5‐HT1 receptor agonist 5‐carboxamidotryptamine (5‐CT) on 71% of SPN. The responses to 5‐HT, alpha‐methyl‐5‐HT and 5‐CT were inhibited by the 5‐HT2 antagonists ketanserin and ritanserin. 5. Pressure ejection of 5‐HT over the central canal region could evoke a biphasic inhibitory‐excitatory response. This response persisted in TTX, suggesting that an inhibitory 5‐HT receptor may be located on the medial dendrites. 6. SPN are powerfully depolarized by 5‐HT acting at 5‐HT2 receptors, via the closure of an outwardly rectifying potassium conductance. The long duration of the response and the ability of 5‐HT to induce rhythmical oscillations suggest that 5‐HT may have an important role in regulating SPN excitability.
Nitric oxide contributes to the rise in forearm blood flow during mental stress in humans.
Authors: Dietz N M , Rivera J M , Eggener S E , Fix R T , Warner D O , Joyner M J
Published: December 2014

1. Our aim was to determine whether the vasodilating substance nitric oxide (NO) contributes to the rise in forearm blood flow observed during mental stress in humans. We also determined whether the NO might be released as a result of cholinergic stimulation of the vascular endothelium. 2. Blood flow was measured in both forearms using plethysmography during several 3‐5 min bouts of a colour word test. In one forearm the nitric oxide synthase blocker NG‐monomethyl‐L‐arginine (L‐NMMA) and other drugs were infused via a brachial artery catheter. The contralateral forearm served as a control. 3. When L‐NMMA was given prior to mental stress it blunted the rise in blood flow in the treated forearm almost completely. The normal blood flow response returned during a second bout of stress conducted after a wash‐out period. During a third bout of mental stress, administration of more L‐NMMA again blunted the blood flow responses to mental stress. 4. When atropine was given prior to mental stress, the increases in blood flow were reduced in the treated forearm. Subsequent administration of both atropine and L‐NMMA caused a somewhat greater reduction in the blood flow responses than those observed with atropine alone. 5. These data demonstrate that NO plays a role in forearm vasodilatation during mental stress in humans. It is likely that most of the NO is released by cholinergic stimulation of the vascular endothelium.
Enhancement by prostaglandin E2 of bradykinin activation of embryonic rat sensory neurones.
Authors: Nicol G D , Cui M
Published: December 2014

1. The capacity of prostaglandin E2 (PGE2) to enhance the excitatory response elicited by bradykinin in embryonic rat sensory neurones grown in culture was investigated using the whole‐cell patch‐clamp recording technique. 2. The focal application of bradykinin (BK) produced a small concentration‐dependent depolarization that was associated with an inward current and was described by a ligand‐binding isotherm having an EC50 of 230 nM. Typically the depolarization was accompanied by action potentials (APs). 3. After pretreatment with 1 microM PGE2 for 10 min, the number of APs elicited by 100 nM BK was increased by about 3‐fold. However, PGE2 had no effect on the amplitude of either the BK‐elicited depolarization or inward current. The addition of 1 or 10 microM PGE2 had no effect on the resting membrane potential. 4. In all neurones exhibiting PGE2‐enhanced excitability, there was a decrease in the amount of injected current necessary to elicit an AP. 5. The enhanced excitability was not due to repeated exposure to BK since neither the amplitude of the BK‐evoked depolarization nor the number of APs was altered by the application of BK at 2 min intervals over a period of 30 min. 6. These results are consistent with the notion that PGE2 acts directly on sensory neurones to enhance the response to chemical excitatory agents, like BK, by lowering the AP firing threshold. The PGE2‐mediated sensitization does not result from an alteration of the resting potential or modulation of the neuronal response to the chemical agonist.
Modulation of GABAC receptors in rat retinal bipolar cells by protein kinase C.
Authors: Feigenspan A , Bormann J
Published: December 2014

1. The intracellular phosphorylation of bicuculline‐ and baclofen‐insensitive GABAC receptors was investigated in rat retinal bipolar cells. The cells were recorded in organotypic slice cultures by using the whole‐cell configuration of the patch‐clamp technique. 2. Peak GABA responses recorded in the presence of bicuculline decreased with repetitive GABA applications. Intracellular application of the phorbol ester, phorbol 12‐myristate, 13‐acetate (PMA) increased this run‐down, whilst it was prevented by both tamoxifen and phosphatase. 3. Perfusing the cells extracellularly with L‐AP4, trans‐(+/‐)‐1‐amino‐1,3‐cyclopentane dicarboxylate (ACPD) or alpha‐methyl serotonin accelerated the run‐down of GABAC responses. 4. Modulation of GABAC responses could be induced by intracellular application of GTP gamma S, indicating involvement of G‐proteins in the transduction cascade. 5. These results suggest that retinal GABAC receptors in bipolar cells are modulated by protein kinase C. Receptors which stimulate phospholipase C, presumably via Gi or Go, such as some of the metabotropic glutamate receptors or the 5‐HT2 receptor, appear to be linked to this regulatory pathway.
Characterization of endothelium‐derived hyperpolarizing factor as a cytochrome P450‐derived arachidonic acid metabolite in mammals.
Authors: Hecker M , Bara A T , Bauersachs J , Busse R
Published: December 2014

1. In addition to nitric oxide (NO) and prostacyclin (PGI2) an as yet unidentified endothelium‐derived hyperpolarizing factor (EDHF) contributes to the dilator effect of bradykinin in different vascular beds. We have investigated the nature and mechanism of action of this factor in freshly isolated bovine and porcine coronary artery segments which were preconstricted with the thromboxane mimetic U46619 (9,11‐dideoxy‐11 alpha, 9 alpha‐epoxymethano‐prostaglandin F2 alpha, 10‐30 nM). 2. The concentration‐response curve of bradykinin was significantly shifted to the right after inhibition of NO synthesis with NG‐nitro‐L‐arginine (L‐NNA, 30 microM), whereas cyclo‐oxygenase blockade with diclofenac (1 microM) had no effect. Preconstriction of the segments with potassium chloride (40‐60 mM) completely abrogated the NO/PGI2‐independent dilator response to bradykinin. In sandwich bioassay experiments, both the luminal and abluminal release of NO, but not that of EDHF, was readily detectable. 3. Inhibitors of Ca(2+)‐activated K+ channels (K+Ca), such as apamin (1 microM) and tetrabutylammonium (TBA, 3 mM), strongly attenuated the EDHF‐mediated bradykinin‐induced relaxation, while glibenclamide (3 microM), an inhibitor of K+ATP channels, had no effect. 4. These relaxations were also significantly inhibited by the phospholipase A2 inhibitor, quinacrine (30 microM), and the cytochrome P450 inhibitors, SKF525a (30‐100 microM) and clotrimazole (100 microM). Moreover, incubation of endothelium‐denuded coronary artery rings with a cytochrome P450‐derived arachidonic acid metabolite, 11,12‐epoxyeicosatetraenoic acid, elicited a concentration‐dependent (1‐10 microM) dilatation which was abolished both in the presence of TBA (3 mM) and following preconstriction of the segments with potassium chloride instead of U46619.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of the hyperpolarization‐activated current, I(f), in ventricular myocytes isolated from hypertensive rats.
Authors: Cerbai E , Barbieri M , Mugelli A
Published: December 2014

1. Left ventricular myocytes isolated from the heart of young (2‐month‐old) and old (18‐ to 20‐month‐old) spontaneously hypertensive rats (SHRs) were studied in the whole‐cell configuration. Since multicellular preparations from old SHRs show a diastolic depolarization phase, we performed experiments to test whether it was associated with the presence of a hyperpolarization‐activated If‐like current. 2. In control Tyrode solution, a time‐dependent increasing inward current activated by hyperpolarization was recorded in myocytes from old SHRs showing a diastolic depolarization phase. A barium‐insensitive, caesium‐sensitive, time‐dependent inward current was recorded in a minority (4 of 33) of cells from young SHRs (membrane capacitance, 160 +/‐ 7 pF) but in 93% (25 of 27, P < 0.01) of myocytes from old SHRs (membrane capacitance, 355 +/‐ 19 pF, P < 0.01). 3. The current was fully activated at ‐120 mV and voltage of half‐maximal activation was ‐88.1 +/‐ 1.5 mV; it was blocked by extracellular CsCl (4 mM) in a voltage‐dependent manner. Reducing [K+]o from 25 to 5.4 mM caused a shift of the reversal potential from ‐17.3 +/‐ 3.8 to ‐25.7 +/‐ 2.7 mV and a 60% decrease of current conductance. 4. These findings suggest that an If‐like current is present in rat ventricular myocytes from old SHRs, where it might favour the occurrence of spontaneous action potentials.
Myofibrillar ATPase activity during isometric contraction and isomyosin composition in rat single skinned muscle fibres.
Authors: Bottinelli R , Canepari M , Reggiani C , Stienen G J
Published: December 2014

1. Myofibrillar ATPase activity, isometric tension (Po) and unloaded shortening velocity (Vo) were determined in single skinned fibres isolated from rat hindlimb muscles during maximal calcium activation at 12 degrees C. In each fibre, myosin heavy chain (MHC) isoforms were identified using electrophoresis and immunocytochemistry. ATPase activity was determined spectrophotometrically from NADH oxidation in a coupled enzyme assay. 2. On the basis of their MHC isoform composition, the fibres (n = 102) were divided into five groups containing the slow isoform, I MHC, or one of the fast isoforms, IIB MHC, IIA MHC, IIX MHC, or a mixture of the latter three. ATPase activity was significantly higher in IIB than in 2X and IIA fibres (0.230 +/‐ 0.010, 0.178 +/‐ 0.023 and 0.168 +/‐ 0.026 nmol mm‐3 s‐1, respectively). Mixed fibres had intermediate values. ATPase activity in slow fibres was considerably less (0.045 +/‐ 0.006 nmol mm‐3 s‐1). 3. The ratio between ATPase activity and Po, i.e. tension cost, was found to be 2.90 +/‐ 0.09, 2.56 +/‐ 0.14, 1.89 +/‐ 0.22, 1.52 +/‐ 0.13 and 0.66 +/‐ 0.004 pmol ATP nM‐1 mm‐1 s‐1 in IIB, mixed, IIX, IIA and slow fibres, respectively. All the differences were statistically significant except that between IIA and IIX fibres. 4. Within each group of fibres with the same MHC composition, ATPase activity was found to correlate with Po, but not Vo. However, ATPase activity was found to correlate with Vo when all the fibre types were pooled together. 5. In thirty‐seven fast fibres the MLC ratio, i.e. the proportion of the fast alkali light chain isoform, MLC3f, to the amount of the regulatory light chain, MLC2f, was determined. IIB fibres had the highest proportion of MLC3f and IIA fibres, the lowest. 6. A multiple regression analysis, used to distinguish between the effects of MHC and MLC composition, showed that ATPase activity was insensitive to the MLC ratio, whereas it had a significant impact on Vo. 7. The results obtained in this study indicate that in rat skeletal muscle fibres: (a) ATPase activity during isometric contractions and tension cost are strongly dependent on MHC isoform composition, and (b) there is no evidence that the alkali MLC ratio is a determinant of ATPase activity.
After‐effects on stiffness and stretch reflexes of human finger flexor muscles attributed to muscle thixotropy.
Authors: Hagbarth K E , Nordin M , Bongiovanni L G
Published: December 2014

1. While the subject maintained a weak contraction in his finger flexor muscles, holding the metacarpophalangeal joints in 45 deg flexion, test torque pulses were applied which caused rapid finger extension movements and electromyographic (EMG) stretch reflex responses. Before each test pulse the fingers were passively flexed or extended (‘post‐short’ and ‘post‐long’ trials) for about 10 s. The EMG and joint deflection responses in the two types of trial were compared after averaging. 2. In the ‘post‐long’ trials, the EMG reflex response showed a comparative increase in latency, with a reduction of the short‐latency (M1) component and an enhancement of the medium‐latency (M2) component. 3. The angular deflections were larger, and the turning points of the deflections, which indicated the start of the mechanical reflex responses, occurred later in the ‘post‐long’ trials. These differences were not seen when the torque pulse was immediately preceded by a strong, brief isometric finger flexor contraction in the test position. 4. Immediately following the return to the test position the background finger flexor EMG activity was larger in the ‘post‐long’ trials, a difference which gradually subsided over 15‐20 s. A strong, brief contraction in the test position also eliminated this inter‐trial difference. 5. The results are interpreted as manifestations of thixotropic after‐effects in intra‐ and extrafusal muscle fibres. It is proposed that the M1 component of the stretch reflex is largely a response to the ‘initial burst’ of impulses in primary spindle afferents.(ABSTRACT TRUNCATED AT 250 WORDS)
Skeletal muscle DHP receptor mutations alter calcium currents in human hypokalaemic periodic paralysis myotubes.
Authors: Sipos I , Jurkat-Rott K , Harasztosi C , Fontaine B , Kovacs L , Melzer W , Lehmann‐Horn F
Published: December 2014

1. Mutations in the gene encoding the alpha 1‐subunit of the skeletal muscle dihydropyridine (DHP) receptor are responsible for familial hypokalaemic periodic paralysis (HypoPP), an autosomal dominant muscle disease. We investigated myotubes cultured from muscle of patients with arginine‐to‐histidine substitutions in putative voltage sensors, IIS4 (R528H) and IVS4 (R1239H), of the DHP receptor alpha 1‐subunit. 2. Analysis of the messenger ribonucleic acid (mRNA) in the myotubes from such patients indicated transcription from both the normal and mutant genes. 3. In control myotubes, the existence of the slow L‐type current and of two rapidly activating and inactivating calcium current components (T‐type with a maximum at about ‐20 mV and ‘third type’ with a maximum at +10 to +20 mV) was confirmed. In the myotubes from patients with either mutation, the third‐type current component was seen more frequently and, on average, with larger amplitude. 4. In myotubes with the IVS4 mutation (R1239H) the maximum L‐type current density was smaller than control (‐0.53 +/‐ 0.31 vs. ‐1.41 +/‐ 0.71 pA pF‐1). The voltage dependence of activation was normal, and hyperpolarizing prepulses to ‐120 mV for 20 s did not increase the reduced current amplitude during test pulses. 5. In myotubes with the IIS4 mutation (R528H) the L‐type current‐voltage relation, determined at a holding potential of ‐90 mV, was normal. However, the voltage dependence of inactivation was shifted by about 40 mV to more negative potentials (voltage at half‐maximum inactivation, V1/2 = ‐41.5 +/‐ 8.2 vs. ‐4.9 +/‐ 4.3 mV in normal controls).(ABSTRACT TRUNCATED AT 250 WORDS)
Ca(2+)‐activated K+ channels in isolated type I cells of the neonatal rat carotid body.
Authors: Wyatt C N , Peers C
Published: December 2014

1. Ca(2+)‐activated K+ (K+Ca) channels in neonatal rat type I carotid body cells were studied using single channel patch clamp techniques. In outside‐out patches, using symmetrical 120 mM [K+] solutions, channels were observed with a slope conductance of 190 pS and a reversal potential of 0 mV. Reducing [K+]o to 5 mM shifted the reversal potential as expected for a K(+)‐selective channel. 2. With 100 nM Ca2+ bathing the cytosolic aspect of patches, channel activity (number of active channels in a patch x open probability, NPo) increased with depolarization. NPo also increased with increasing ‘cytosolic’ [Ca2+] at a fixed membrane potential (0 mV). Using outside‐out patches, bath application of 20 or 100 nM charybdotoxin reduced NPo by > 85%. These data indicate the presence of K+Ca channels in type I cells. 3. At 0 mV, using solutions of identical composition (1 microM Ca2+ bathing the cytosolic aspect of the channels), NPo was higher in outside‐out patches than in inside‐out patches. NPo was greatest in recordings using the perforated‐vesicle technique. 4. Hypoxia and anoxia were without effect on K+Ca channels in outside‐out patches, but caused significant, reversible reductions of NPo in channels recorded in perforated vesicles. 5. The whole‐cell perforated‐patch technique was used to record membrane potential at 35‐37 degrees C. Hypoxia, anoxia and charybdotoxin all depolarized type I cells. 6. Our results suggest an important role for K+Ca channels in type I carotid body cells, and their activity in relation to a model for chemotransduction is discussed.
Flow‐dependent regulation of arteriolar diameter in rat skeletal muscle in situ: role of endothelium‐derived relaxing factor and prostanoids.
Authors: Friebel M , Klotz K F , Ley K , Gaehtgens P , Pries A R
Published: December 2014

1. Arteriolar diameter in the resting rat spinotrapezius muscle was studied by intravital video microscopy before and after blockade of the L‐arginine‐EDRF (NG‐nitro‐L‐arginine, L‐NNA) or the cyclo‐oxygenase‐prostacyclin (indomethacin) pathway. Blockade of either pathway leads to a decrease of arteriolar diameter of 25‐40%, while the combined blockade of both results in vasoconstriction of 50‐60%. 2. Alteration of blood flow velocity elicited by partial micropipette occlusion induces corresponding changes of vessel diameter. The flow‐dependent diameter response is reduced by about 80% by L‐NNA. By contrast, blockade of prostanoid production shows no significant influence on vessel response to blood flow alteration in the range tested. 3. Transient overshooting vasodilatation is seen for about 1 min following the sudden restoration of flow velocity subsequent to occlusion. In contrast to the initial phase of this response, the late phase is blocked by L‐NNA. 4. The findings suggest that basal release of endothelium‐derived relaxing factor (EDRF) and prostanoids leads to additive and independent dilator effects, and that flow‐dependent diameter changes are primarily mediated by EDRF. 5. If present data are compared with literature reports, it appears that arterial flow sensitivity is most pronounced in the smallest vessels. In such vessels, flow‐dependent dilatation will amplify even small changes of volume flow by more than four times.
Substance P effects on blood flow, fluid transport and vasoactive intestinal polypeptide release in the feline small intestine.
Authors: Brunsson I , Fahrenkrug J , Jodal M , Sjöqvist A , Lundgren O
Published: December 2014

1. Substance P (SP) infusions were given close I.A. to the feline small intestine in vivo in a dose that produced plasma concentrations of 1‐5 microM. This infusion regularly evoked a net fluid secretion measured with a gravimetric technique. Concomitantly, the release into blood of vasoactive intestinal polypeptide (VIP), a putative neurotransmitter of the enteric nervous system, increased. 2. The SP‐induced fluid secretion was blocked by tetrodotoxin (7 micrograms close I.A.), a blocker of fast sodium channels in excitable tissues, and hexamethonium (10 mg (kg body wt)‐1, I.V.), a nicotinic receptor antagonist, suggesting that the SP effect was mediated by the enteric nervous system. In line with this it was shown that the SP‐evoked release of VIP was also significantly diminished by hexamethonium. 3. Close I.A. infusions of methionine enkephalin (Met‐enkephalin; 7‐23 nmol min‐1) or electrical stimulation of the sympathetic nerve fibres (6 Hz) to the intestine markedly diminished net fluid secretion and the release of VIP caused by SP given close I.A. 4. The cyclo‐oxygenase inhibitor diclofenac (5 mg (kg body wt)‐1, I.V.) or the histamine‐1 receptor antagonist pyrilamine (10 mg (kg body wt)‐1, I.V.) did not influence the fluid secretion caused by SP, indicating that the effects of SP were not due to the actions of prostaglandins or histamine. 5. It is proposed that SP activates a nervous reflex arch that we have shown to be activated by various luminal stimuli, including cholera toxin.(ABSTRACT TRUNCATED AT 250 WORDS)
Functional availability of sodium channels modulated by cytosolic free Ca2+ in cultured mammalian neurons (N1E‐115).
Authors: Bulatko A K , Greeff N G
Published: December 2014

1. Whole‐cell sodium currents (INa) were measured in mouse neuroblastoma cells (N1E‐115) at different [Ca2+]i values using appropriate Ca‐EGTA buffers in the pipettes. 2. INa was found to be larger at pCa 7 than at pCa 8 or 9 with a ratio of 1:0.65 or 0.55, respectively. The steady‐state inactivation (h infinity curve) was independent of [Ca2+]i, thus excluding surface charge effects as a cause of the Ca2+ effect. 3. Recovery of INa from slow inactivation after changing from resting (‐30 to ‐40 mV) to holding potential (‐70 mV) occurred in a similar way at all pCa values. The Ca2+ effect appears to be independent of slow inactivation and to occur within the first 2 min of pipette buffer‐cytoplasm equilibration. 4. The cell membrane capacitance (Cm) was independent of [Ca2+]i, thus excluding exo‐ or endocytosis of sodium channel‐containing membrane as a cause of the Ca2+ effect. 5. Non‐stationary fluctuation analysis was used to determine simultaneously the single channel current (iNa) and the size of INa. At pCa values of 7 and 9, iNa was identical, i.e. 0.59 and 0.58 pA, while INa/Cm differed, i.e. 41.1 and 22.2 pA pF‐1, respectively. The peak open probability at 0 mV was about 0.5 for both pCa values indicating that [Ca2+]i controls the fraction of channels available for activation. 6. Since [Ca2+]i in other neurons varies between 30 and 100 nM in the resting and active state, respectively, the present data suggest a modulatory role for [Ca2+]i in neuronal excitability.
Role of membrane potential in endothelium‐dependent relaxation of guinea‐pig coronary arterial smooth muscle.
Authors: Parkington H C , Tonta M A , Coleman H A , Tare M
Published: December 2014

1. Membrane potential and tension were measured simultaneously in ring segments of main coronary artery of guinea‐pigs. The synthetic thromboxane A2 analogue U46619 depolarized the tissues from ‐58 +/‐ 2 to ‐40 +/‐ 1 mV and increased tension by 12 +/‐ 1 mN mm‐1. Nitric oxide (NO) and Iloprost, the stable analogue of prostacyclin, evoked hyperpolarization and relaxation. 2. The concentration of NO required to evoke half‐maximal hyperpolarization (EC50 of 2 x 10(‐5) M) was 40‐fold higher than that which was required to induce relaxation (EC50 of 5 x 10(‐7) M). The EC50 for Iloprost‐induced hyperpolarization (3 x 10(‐8) M) was similar to that for relaxation (4 x 10(‐8) M). 3. Glibenclamide (10(‐6) M) abolished the hyperpolarization in response to both NO and Iloprost but was without effect on the amplitudes of the relaxations over the complete concentration‐response curves. 4. Acetylcholine evoked concentration‐dependent hyperpolarization and relaxation in the presence of N omega‐nitro‐L‐arginine methyl ester (NAME; 10(‐5) M) and indomethacin (10(‐6) M), and these responses were attributed to endothelium‐derived hyperpolarizing factor (EDHF). The hyperpolarization produced by EDHF always preceded relaxation, and relaxation never occurred at concentrations of acetylcholine that were insufficient to evoke hyperpolarization. 5. The concentration‐hyperpolarization and concentration‐relaxation curves in response to acetylcholine were not affected by glibenclamide or barium (1‐3 mM) but were shifted to the right 4‐ and 5‐fold, respectively, by 1 mM tetraethylammonium. The hyperpolarization and relaxation evoked by acetylcholine were also reduced in a parallel manner when the potassium concentration in the superfusate was increased. 6. Hyperpolarizing current steps, applied to spiral strips of coronary artery denuded of endothelium and depolarized and constricted with U46619, caused relaxation. The relationship between hyperpolarization and relaxation evoked electronically was similar to that which was due to EDHF in intact tissues stimulated with acetylcholine. 7. It is concluded that the ability of NO or Iloprost to relax guinea‐pig coronary artery does not depend upon hyperpolarization of the smooth muscle. In contrast, hyperpolarization is likely to play a major, if not the only, role in the relaxation in response to EDHF in this tissue.