bluuebunny/crossref_metadata_2025 huggingface dataset:
The importance of sodium for anoxic transmission damage in rat hippocampal slices: mechanisms of protection by lidocaine.
Authors: Fried E , Amorim P , Chambers G , Cottrell J E , Kass I S
Published: December 2014

1. The effect of sodium influx on anoxic damage was investigated in rat hippocampal slices. Previous experiments demonstrated that a concentration of tetrodotoxin which blocks neuronal transmission protects against anoxic damage. In this study we examined low concentrations of lidocaine (lignocaine; which do not block neuronal transmission), for their effect on recovery of the evoked population spike recorded from the CA1 pyramidal cell layer. 2. Recovery of the population spike, measured 60 min after a 5 min anoxic period, was 4 +/‐ 2% of its preanoxic, predrug level. Lidocaine concentrations of 10, 50, and 100 microM significantly improved recovery to 56 +/‐ 12, 80 +/‐ 7 and 70 +/‐ 14%, respectively. 3. Lidocaine (10 microM) did not alter the size of the evoked response before anoxia and had no significant effect on potassium levels or calcium influx during anoxia. It did, however, reduce cellular sodium levels (146 +/‐ 7 vs. 202 +/‐ 12 nmol mg‐1) and preserve ATP levels (2.17 +/‐ 0.07 vs. 1.78 +/‐ 0.07 nmol mg‐1) during anoxia. All values were measured at the end of 5 min of anoxia except those for Ca2+ influx which were measured during 10 min of anoxia. 4. High concentrations of lidocaine (100 microM) did not improve recovery significantly over that observed with 10 microM. They also had no significantly greater effects on sodium levels than 10 microM lidocaine (137 +/‐ 12 vs. 146 +/‐ 7 nmol mg‐1); however, 100 microM lidocaine significantly improved potassium (202 +/‐ 18 vs. 145 +/‐ 6 nmol mg‐1) and ATP (2.57 +/‐ 0.06 vs. 2.17 +/‐ 0.07 nmol mg‐1) levels, while reducing calcium influx (7.76 +/‐ 0.12 vs. 9.24 +/‐ 0.39 nmol mg‐1 (10 min)‐1) when compared with 10 microM lidocaine. 5. We conclude that sodium influx and ATP depletion are of major importance in anoxic damage since 10 microM lidocaine reduced these changes during anoxia and improved recovery of the population spike. In addition, our results indicate that the properties of the sodium channel are altered during anoxia, since sodium influx is blocked by a concentration of lidocaine that does not affect the population spike in the preanoxic period.
Tension‐dependent changes of the intracellular Ca2+ transients in ferret ventricular muscles.
Authors: Kurihara S , Komukai K
Published: December 2014

1. We measured the change in intracellular Ca2+ transients, using aequorin, in response to muscle length change during twitch contraction in ferret ventricular muscles. 2. Intracellular Ca2+ concentration ([Ca2+]i) was transiently increased when the muscle length was quickly shortened to 92% of maximum length (Lmax) at various times after stimulation (this increase in [Ca2+] is termed extra‐Ca2+). The magnitude of extra‐Ca2+, measured at different extracellular Ca2+ concentrations ([Ca2+]o), showed a dependence upon the magnitude of tension reduction and upon [Ca2+]i immediately before the length change. 3. In the presence of caffeine (5 mM), the difference between the Ca2+ transient at Lmax and at shorter lengths showed a time course similar to the difference between the developed tension at both lengths. A quick release in the caffeine‐treated preparation produced the extra‐Ca2+ with a slower time course compared with that observed in the absence of caffeine. Stretching the muscle from 96% Lmax to Lmax produced more active tension and decreased [Ca2+]i. 4. These results indicate that the affinity of troponin‐C, a major Ca2+ binding protein, which controls contraction, is influenced by developed tension i.e. cross‐bridge attachment and detachment.
Activation of a novel organic solute transporter in mammalian red blood cells.
Authors: Culliford S J , Bernhardt I , Ellory J C
Published: December 2014

1. Suspending human red blood cells in isotonic sucrose (low ionic strength, LIS) medium induces a significant increase in membrane transport of glutamine, glutamate, lactate, histidine, taurine, glycine, serine, choline and carnitine but not sorbitol or sucrose. 2. Progressive lowering of ionic strength by sucrose or NaCl replacement gave a similar activation profile for taurine influx as found earlier for residual K+(86Rb+) flux. 3. The induced taurine transport could be measured as enhanced influx and efflux. Influx was linear with external concentration up to 10 mM, largely insensitive to alteration in cell volume, and did not vary with red blood cell age. 4. Unlike previous results for residual K+ transport, altering transmembrane potential with gluconate or glucuronate media led to an increase in taurine influx similar to that observed in LIS media. Varying medium pH confirmed the effect was not due to alteration in pH. 5. The LIS‐induced flux was sensitive to a variety of ‘classical’ anion transport inhibitors in the order of potency DNDS, DIDS, NPPB, DIOA, niflumic acid, furosemide (frusemide), glibenclamide, nitrendipine and bumetanide. 6. The taurine flux showed a temperature dependence similar to that of the LIS‐induced residual K+ flux. High hydrostatic pressure (40 MPa), however, inhibited taurine flux but stimulated residual K+ influx in LIS media. 7. A significant enhanced taurine flux could be demonstrated in red blood cells of other species, including horse, cattle, pig and high and low potassium type sheep. 8. It is concluded that lowering ionic strength activates a transport pathway for organic molecules sharing some similarities with background Cl‐ channels and LIS‐induced residual K+ fluxes. In the latter context, however, there are certain significant differences (effect of transmembrane potential; volume; pressure sensitivity; species distribution) which may be important, and the unequivocal identity of the two transport processes remains to be confirmed.
Role of the parabrachial nucleus in ventilatory responses of awake rats.
Authors: Mizusawa A , Ogawa H , Kikuchi Y , Hida W , Shirato K
Published: December 2014

1. The parabrachial nucleus (PBN) is thought to play an important role in cardiorespiratory control. However, the circumstances under which it affects ventilation are still not known. The purpose of the present study was to investigate how the PBN modulates the ventilatory responses to hypercapnia, hypoxia or a resistive load in awake rats with chemical lesions of the PBN. 2. In three groups of rats (with lateral PBN lesion, with Kölliker‐Fuse nucleus lesion and control), ventilation was measured under various conditions. 3. There was no difference in the breathing of normal room air in any of the groups. However, the lesioned groups showed a reduced ventilatory response to hyperoxic hypercapnia (inspired CO2 fractions (FI,CO2) of 3, 5, 8 and 10%) and to graded hypoxia (inspired O2 fractions (FI,O2) of 16, 12, 10 and 8%) compared with the control group. The control group showed a biphasic response to sustained hypoxia (FI,O2 at 10% for 30 min), known as ‘hypoxic depression’, while the lesioned groups showed moderate ventilatory exaggeration throughout hypoxia. In response to a resistive load, the lateral PBN lesion group showed no change in ventilatory compensation. 4. The PBN appeared to have a considerable influence on ventilation stimulated in various ways during wakefulness.
Opioid inhibition of rat periaqueductal grey neurones with identified projections to rostral ventromedial medulla in vitro.
Authors: Osborne P B , Vaughan C W , Wilson H I , Christie M J
Published: December 2014

1. Rat caudal periaqueductal grey (PAG) output neurones containing rhodamine microspheres, retrogradely transported from an injection site in the rostral ventromedial medulla (RVM), were visualized in brain slices and recorded from using whole‐cell patch clamp techniques. 2. The specific GABAB receptor agonist baclofen (10 microM) produced an outward current or hyperpolarization in fifty out of fifty‐six caudal PAG output neurones. In 44% of these baclofen‐sensitive neurones, the opioid agonist methionine enkephalin (30 microM) also produced an outward current or hyperpolarization. The opioid current reversed polarity at ‐104 mV and could also be produced by DAMGO, an agonist selective for the mu‐subtype of opioid receptor. 3. Opioid‐responding output neurones were not distributed uniformly in the caudal PAG. In horizontal slices containing lateral PAG, 56% of output neurones were inhibited by opioids, as compared with only 14% of the output neurones in slices containing ventrolateral PAG. 4. These observations are consistent with opioid disinhibition of ventrolateral PAG neurones projecting to the RVM as the predominant mechanism underlying opioid‐induced analgesia in the PAG. The role of opioid receptors found on a major proportion of the output neurones in the lateral PAG remains to be established, but is assumed not be related to modulation of nociceptive function.
Splenic blood flow and fluid efflux from the intravascular space in the rat.
Authors: Chen A , Kaufman S
Published: December 2014

1. Previous evidence has suggested that fluid is extracted from the blood during its passage through the splenic circulation. In order to further investigate this phenomenon, flow probes were placed around the splenic artery and vein of rats. Splenic blood flow and mean arterial blood pressure were measured immediately after surgery, while the rats were still anaesthetized. Five days later, the measurements were repeated first in conscious rats, and then after administering sodium pentobarbitone. Final measurements were made in the conscious animals at day 10 post surgery. 2. Splenic arterial blood flow was lowest at the time of surgery. It had doubled by day 5, and remained stable thereafter at about 8 ml min‐1. Splenic venous blood flow also increased after surgery, but less so than the arterial flow, so that the arteriovenous difference had increased to 2.0 +/‐ 0.4 ml min‐1 by day 5. 3. In response to sodium pentobarbitone (20 mg I.V.), there was a transient fall in splenic arterial blood flow from 6.7 +/‐ 0.9 to 4.9 +/‐ 0.7 ml min‐1. There was no significant change in the arteriovenous difference of blood flow. 4. In conclusion, we have shown that splenic blood flow is considerably higher than previously reported. We have also revealed a significant new path for fluid efflux from the intravascular space; at least 25% of fluid volume flowing into the splen is removed from the circulating blood. 5. Although anaesthesia modulates splenic blood flow, the changes are not as pronounced as those observed in the acute surgically prepared animal.
Local inhibition of nitric oxide generation in man reduces blood flow in finger pulp but not in hand dorsum skin.
Authors: Noon J P , Haynes W G , Webb D J , Shore A C
Published: December 2014

1. Nitric oxide generation is important in the regulation of resistance vessel tone. Until now, however, there has been no evidence of such a role for basal generation of nitric oxide in the skin microcirculation of humans. 2. To investigate this, L‐NG‐monomethylarginine (L‐NMMA), a competitive inhibitor of nitric oxide synthase, was administered at 1, 2 and 4 mumol min‐1 (each for 10 min), via the brachial artery, in six healthy male subjects. 3. At each dose, using laser Doppler fluximetry, red blood cell flux was measured as an index of blood flow in the pulp of the thumb, an area rich in arteriovenous anastomoses, and on the dorsal surface of the hand, where arteriovenous anastomoses are rare. Finger nailfold capillary blood velocity was monitored at each dose using videomicroscopy. Forearm blood flow was measured by venous occlusion plethysmography, before, and 8 min after, completing infusion of L‐NMMA. All data were obtained from both the infused and control arms. 4. L‐NMMA reduced blood flow in the infused forearm by 37% (P = 0.005). In contrast, dorsum red cell flux, capillary blood velocity, and skin temperature were unchanged. There was, however, a significant reduction in thumb red cell flux (ANOVA, P = 0.0001), reaching a maximum reduction of 33% with 4 mumol min‐1 L‐NMMA. There were no effects apparent in the opposite arm. 5. These results suggest that endogenous nitric oxide production may be more important in regulating microvascular skin blood flow in regions rich in arteriovenous anastomoses than in areas containing mainly nutritive vessels.
Lack of importance of respiratory muscle load in ventilatory regulation during heavy exercise in humans.
Authors: Krishnan B , Zintel T , McParland C , Gallagher C G
Published: December 2014

1. Seven active subjects (24 +/‐ 1 years; maximal oxygen uptake (VO2,max), 3.77 +/‐ 0.2 l min‐1; mean +/‐ S.E.M.) performed constant work rate heavy exercise (CWHE, approximately 80% of maximal incremental work rate) to exhaustion on 2 days, one with (unload) and one without (control) respiratory muscle unloading. 2. With unloading, a special device applied flow‐proportional mouth pressure assist (positive with inspiratory (I), negative with expiratory (E) flows) throughout each breath. No pressure assist occurred during control CWHE. To confirm unloading, respiratory muscle pressures (Pmus) were derived (n = 5) from measured pleural pressure and chest wall elastic and resistive pressures. 3. Other than minor differences in early exercise, the temporal course of minute ventilation (VE) was similar in both tests as exercise progressed. The fall in estimated mean alveolar CO2 (PA,CO2) throughout CWHE was identical in both tests. There were no significant differences (ANOVA) in VE, tidal volume, frequency, oxygen consumption rate (VO2), heart rate or PA,CO2, between unload and control CWHE, at matched times (at 50% of control duration and at the end of exercise). Unloading reduced Pmus significantly throughout CWHE; at 50% control duration, peak Pmus,I and Pmus,E fell by 24 and 41%, respectively, with unloading, as did mean Pmus,I and Pmus,E (21 and 44%). 4. The lack of any significant changes in VE, PA,CO2 or breathing pattern, despite a marked reduction in respiratory muscle load throughout CWHE, indicates that the load on the respiratory muscles has only a minor role in the regulation of ventilation during heavy exercise. 5. The absence of improvement in CWHE duration (control, 11.4 +/‐ 1.2 min; unload, 12.6 +/‐ 2.1 min, n.s.) with unloading implies that respiratory muscle function does not limit endurance exercise performance during cycling in healthy humans.
Effects of cytochalasin treatment on short‐term synaptic plasticity at developing neuromuscular junctions in frogs.
Authors: Wang X H , Zheng J Q , Poo M M
Published: December 2014

1. The role of actin microfilaments in synaptic transmission was tested by monitoring spontaneous and evoked transmitter release from developing neuromuscular synapses in Xenopus nerve‐muscle cultures, using whole‐cell recording of synaptic currents in the absence and presence of microfilament‐disrupting agents cytochalasins B and D. 2. Treatment with cytochalasins resulted in disruption of microfilament networks in the growth cone and the presynaptic nerve terminal of spinal neurons in Xenopus nerve‐muscle cultures, as revealed by rhodamine‐phalloidin staining. 3. The same cytochalasin treatment did not significantly affect the spontaneous or evoked synaptic currents during low‐frequency stimulation at 0.05 Hz in these Xenopus cultures. Synaptic depression induced by high‐frequency (5 Hz) stimulation, however, was reduced by this treatment. Paired‐pulse facilitation for short interpulse intervals was also increased by the treatment. 4. These results indicate that disruption of microfilaments alters short‐term changes in transmitter release induced by repetitive activity, without affecting normal synaptic transmission at low frequency. 5. Our results support the notion that actin microfilaments impose a barrier for mobilization of synaptic vesicles from the reserve pool, but do not affect the exocytosis of immediately available synaptic vesicles at the active zone.
Role of de novo protein synthesis and calmodulin in rapid activation of Na(+)‐H+ exchange of aldosterone in frog diluting segment.
Authors: Cooper G J , Hunter M
Published: December 2014

1. In the amphibian early distal tubule aldosterone activates the Na(+)‐H+ exchangers, resulting in an increase in intracellular pH (pHi). Since this activation is rapid (within 30 min), it may be mediated by either a genomic or non‐genomic pathway. 2. pHi was measured in single microperfused early distal tubule segments using the fluorescent probe 2',7'‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF). 3. A 30 min incubation in aldosterone increased both resting pHi and the setpoint of the Na(+‐H+ exchanger. These changes were prevented by the mineralocorticoid receptor antagonist, spironolactone. 4. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, were without effect on resting pHi, but inhibited activation of the Na(+)‐H+ exchanger by aldosterone. 5. The effect of aldosterone upon pHi and setpoint was also prevented by the calcium‐calmodulin antagonist, W‐7. 6. These results indicate that, although the response to aldosterone is rapid, aldosterone binds to a specific mineralocorticoid receptor which then triggers gene activation followed by de novo protein synthesis. Furthermore, since calmodulin is a known activator of the Na(+)‐H+ exchanger, and the response is inhibited by W‐7, it is suggested that this protein may be calmodulin.
Phospholipase A2 and protein kinase C contribute to myofilament sensitization to 5‐HT in the rabbit mesenteric artery.
Authors: Parsons S J , Sumner M J , Garland C J
Published: December 2014

1. Calcium (Ca2+, 0.1‐100 microM) stimulated concentration‐dependent contractions in small strips from the rabbit mesenteric artery in which the smooth muscle cells had been permeabilized with Staphylococcus aureus alpha‐toxin. 2. 5‐Hydroxytryptamine (5‐HT) and phenylephrine, each in the presence of 10 microM guanosine 5'‐triphosphate (GTP), concentration‐dependently stimulated additional contractions in strips sub‐maximally contracted by the presence of a buffered concentration of calcium (0.3 microM). All the additional contraction was abolished with the selective inhibitor of protein kinase C, Ro 31‐8220 (10 microM). 3. Quinacrine (10‐50 microM), an inhibitor of phospholipase A2, selectively inhibited the sensitization to 5‐HT, but did not alter the sensitization to either phenylephrine or GTP. 4. Myofilament sensitization to calcium was mimicked by exogenous arachidonic acid (300 microM, in the presence of indomethacin, miconazole and BW755c) and the stable analogue of arachidonic acid, 5,8,11,14‐eicosatetrayonic acid (ETYA, 100 microM), and in both cases did not require the additional presence of GTP. Ro 31‐8220, but not quinacrine, reduced the sensitization to arachidonic acid by around 30%. 5. These results indicate that G protein‐linked myofilament sensitization to calcium in the mesenteric artery that follows the activation of 5‐HT receptors, but not alpha 1‐receptors, involves phospholipase A2. The sensitization stimulated by each of these different receptors, and a component of the response to arachidonic acid, also appears to involve the activation of protein kinase C.
Minimal role of nitric oxide in basal coronary flow regulation and cardiac energetics of blood‐perfused isolated canine heart.
Authors: Saeki A , Recchia F A , Senzaki H , Kass D A
Published: December 2014

1. The role of nitric oxide (NO) in the regulation of basal coronary perfusion and ventricular chamber energetics was studied in isovolumetrically contracting isolated blood‐perfused canine hearts. Hearts were cross‐perfused by a donor animal prior to isolation, and chamber volume controlled by a servo‐pump. Coronary sinus flow and arterial‐coronary sinus oxygen difference were measured to determine energetic efficiency. 2. NO synthase (NOS) was competitively inhibited by NG‐monomethyl‐L‐arginine (L‐NMMA; 0.5 mg kg‐1, intracoronary), resulting in a reduction of acetylcholine (50 micrograms min‐1)‐induced flow augmentation from 143 to 62% (P < 0.001). 3. NOS inhibition had no significant effect on basal coronary flow. Coronary pressure‐flow relationships were determined at a constant cardiac workload by varying mean perfusion pressure between 20 and 150 mmHg. Neither the shape of the relationship, nor the low‐pressure value at which flow regulation was substantially diminished were altered by NOS inhibition. 4. Myocardial efficiency was assessed by the relationship between myocardial oxygen consumption and total pressure‐volume area (PVA), with cavity volume altered to generate varying PVAs. This relative load‐independent measure of energetic efficiency was minimally altered by NOS inhibition. 5. These results contrast with isolated crystalloid‐perfused heart experiments and suggest that in hearts with highly controlled ventricular loading and whole‐blood perfusion, effects of basal NO production on coronary perfusion and left ventricular energetics are minimal.
Sodium‐magnesium antiport in Retzius neurones of the leech Hirudo medicinalis.
Authors: Günzel D , Schlue W R
Published: December 2014

1. Intracellular free magnesium ([Mg2+]i) and sodium ([Na+]i) concentrations were measured in Retzius neurones of the leech Hirudo medicinalis using ion‐sensitive microelectrodes. 2. The mean steady‐state values for [Mg2+]i and [Na+]i were 0.46 mM (pMg, 3.34 +/‐ 0.23; range, 0.1‐1.2 mM; n = 32) and 8.95 mM (pNa, 2.05 +/‐ 0.15; range, 5.1‐15.5 mM, n = 21), respectively, at a mean membrane potential (Em) of ‐35.6 +/‐ 6.1 mV (n = 32). Thus, [Mg2+]i is far below the value calculated for a passive distribution (16.9 mM) but close to the equilibrium value calculated for a hypothetical 1 Na(+)‐1 Mg2+ antiport (0.41 mM). 3. Simultaneous measurements of [Mg2+]i, [Na+]i and Em in Retzius neurones showed that an increase in the extracellular Mg2+ concentration ([Mg2+]o) resulted in an increase in [Mg2+]i, a parallel decrease in [Na+]i and a membrane depolarization, while a decrease in [Mg2+]o had opposite effects. These results are compatible with calculations based on a 1 Na(+)‐1 Mg2+ antiport. 4. Na+ efflux at high [Mg2+]o still occurred when the Na(+)‐K+ pump was inhibited by the application of ouabain or in K(+)‐free solutions. This efflux was blocked by amiloride. 5. In the absence of extracellular Na+ ([Na+]o), no Mg2+ influx occurred. Mg2+ influx at high [Mg2+]o was even lower than in the presence of [Na+]o. Mg2+ efflux was blocked in the absence of [Na+]o. 6. The rate of Mg2+ extrusion was reduced by lowering [Na+]o, even if the Na+ gradient across the membrane remained almost unchanged. 7. Mg2+ efflux was blocked by amiloride (half‐maximal effect at 0.25 mM amiloride; Hill coefficient, 1.3) but not by 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA). 8. No changes in intracellular Ca2+ and pH (pHi) could be detected when [Mg2+]o was varied between 1 and 30 mM. 9. Changing pHi by up to 0.4 pH units had no effect on [Mg2+]i. 10. The results suggest the presence of an electrogenic 1 Na(+)‐1 Mg2+ antiport in leech Retzius neurones. This antiport can be reversed and is inhibited by low extracellular and/or intracellular Na+ and by amiloride.
Changes in [Ca2+]i, [Na+]i and Ca2+ current in isolated rat ventricular myocytes following an increase in cell length.
Authors: Hongo K , White E , Le Guennec J Y , Orchard C H
Published: December 2014

Isolated rat ventricular myocytes were stretched using carbon fibres to investigate the mechanisms underlying the increase in contraction following stretch. 2. [Ca2+]i and [Na+]i were monitored using the fluorescent indicators fura‐2 and sodium‐binding benzofuran isophthalate, respectively. The L‐type Ca2+ current was recorded simultaneously with contraction using the perforated patch‐clamp technique. 3. Mechanical stretch caused an immediate increase in contraction, followed by a slow increase. Contraction was prolonged immediately after the stretch, but did not change during the slow phase. 4. The Ca2+ transient did not change immediately after the stretch. The slow increase in contraction was accompanied by an increase in the amplitude of the Ca2+ transient. However, diastolic [Ca2+]i did not change significantly following stretch. 5. [Na+]i did not change significantly either immediately, or during the slow increase in contraction, after the stretch. 6. The L‐type Ca2+ current was not significantly altered either by mechanical loading of the cell with carbon fibres or by stretching the cell. 7. These results suggest that: (1) the rapid increase in contraction following a stretch is due to an increase in myofilament Ca2+ sensitivity rather than to changes in the L‐type Ca2+ current or [Na+]i; and (2) a slow increase in the Ca2+ transient underlies the slow increase in contraction in isolated myocytes, but is not caused by either an increase in diastolic [Ca2+]i or a change in [Na+]i (and hence Ca2+ influx via Na(+)‐Ca2+ exchange) or a change in myofilament Ca2+ sensitivity.
Effect of isosorbiddinitrate on exogenously expressed slowly activating K+ channels and endogenous K+ channels in Xenopus oocytes.
Authors: Busch A E , Kopp H G , Waldegger S , Samarzija I , Süssbrich H , Raber G , Kunzelmann K , Ruppersberg J P , Lang F
Published: December 2014

1. The effects of isosorbiddinitrate (ISDN) were tested on membrane currents and resting potential in Xenopus laevis oocytes which were either uninjected or injected with cRNA encoding for K+ channels from three distinct families (slowly activating IsK channels, delayed‐rectifying Kv1.1 or inwardly rectifying IRK1 K+ channels). 2. In uninjected oocytes ISDN (1 mM) resulted in a decrease of the holding current at potentials more positive than ‐100 mV and in an increase at potentials below ‐100 mV. Increasing extracellular K+ to 100 mM shifted the reversal potential for ISDN‐mediated effects to approximately ‐12 mV, suggesting an inhibition of a K+ conductance by ISDN. 3. In current clamp studies ISDN (1 mM) and Ba2+ (3 mM) depolarized cell membrane. ISDN and Ba2+ had no additive effects on membrane potential when applied simultaneously. In voltage clamp studies, corresponding results were observed for the effects of ISDN and Ba2+ on the holding current with an apparent K(m) of 0.21 and 0.08 mM, respectively. 4. In contrast to ISDN, the nitric oxide (NO) donors isosorbidmononitrate (ISMN) and S‐nitrosocysteine (SNOC) had no effects on the holding currents in Xenopus oocytes. Moreover, the guanylate inhibitor LY 83583 did not affect ISDN‐mediated holding current alterations, suggesting that ISDN acts independently of the second messenger NO. 5. ISDN inhibited exogenously expressed IsK channels with an apparent K(m) of 0.15 mM, but at 1 mM only weakly inhibited Kv1.1 and IRK1 channels. 6. It is concluded that ISDN inhibits an endogenous K+ conductance in Xenopus oocytes with a similar potency to that shown by expressed IsK channels. These effects are independent of the second messenger NO.
Fractional Ca2+ currents through somatic and dendritic glutamate receptor channels of rat hippocampal CA1 pyramidal neurones.
Authors: Garaschuk O , Schneggenburger R , Schirra C , Tempia F , Konnerth A
Published: December 2014

1. The Ca2+ permeability of non‐NMDA and NMDA receptor channels was studied using a fluorometric flux measurement approach in somata and dendrites of CA1 pyramidal neurones in rat hippocampal slices. For this purpose, the Ca2+ fraction of the total cation current (named ‘fractional Ca2+ current') was measured directly from the change in the Ca(2+)‐sensitive fura‐2 fluorescence at 380 nm excitation wavelength. 2. The fractional Ca2+ current through the somatic NMDA receptor channels was 10.69 +/‐ 2.13% (mean +/‐ S.D.) and that through dendritic receptor channels was 10.70 +/‐ 1.96%. The fractional Ca2+ current was not dependent on the extracellular Mg2+ concentration and its voltage dependence was in agreement with the Goldman‐Hodgkin‐Katz current equation. 3. AMPA (alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate) or kainate applications produced small but significant Ca2+ entry. Fractional Ca2+ currents of 0.58 +/‐ 0.34% were measured for somatic AMPA applications, 0.68 +/‐ 0.20% for somatic kainate applications, 0.66 +/‐ 0.25% for dendritic AMPA applications and 0.61 +/‐ 0.16% for dendritic kainate applications. 4. The expression pattern of glutamate receptor subunits encoding messenger ribonucleic acids (mRNAs) was analysed with the single‐cell reverse transcriptase‐polymerase chain reaction (RT‐PCR) approach applied to CA1 pyramidal neurones. The AMPA receptor subunits GluR‐A, GluR‐B and GluR‐C, and the NMDA receptor subunits NR2A and NR2B were found to be abundantly expressed in all CA1 pyramidal neurones tested. 5. This study establishes the fractional Ca2+ current through somatic and dendritic NMDA and non‐NMDA receptor channels in CA1 pyramidal neurones. The dendritic, presumably synaptic, NMDA receptor channels are highly Ca2+ permeable and have a fractional Ca2+ current closely resembling that of somatic extrasynaptic NMDA receptor channels. Both somatic and dendritic non‐NMDA receptor channels are of the ‘low Ca2+ permeable’ type and have a fractional Ca2+ current that is about twenty times smaller than that of NMDA receptor channels.
The effects of hypertonic saline solution on coronary blood flow in anaesthetized pigs.
Authors: Vacca G , Papillo B , Battaglia A , Grossini E , Mary D A , Pelosi G
Published: December 2014

1. The effects of intracoronary bolus infusion of hypertonic saline solution on left circumflex coronary blood flow were examined in sixteen anaesthetized and artificially ventilated pigs whilst preventing changes in heart rate and arterial blood pressure. 2. In fourteen pigs, bolus infusion of 7.5% hypertonic saline solution (2 ml within 30 s) caused a steady‐state increase in coronary blood flow without significantly affecting right atrial or left ventricular pressure and its rate of rise (dP/dtmax). Infusing normal saline solution (0.9%) at the same rate and volume in seven pigs did not have this effect. 3. In five pigs, the magnitude and the duration of the response of increase in coronary blood flow were increased in a graded manner by graded increases in the concentration of the hypertonic saline solution between 2.5, 5 and 7.5%. 4. In nine pigs, the response of increase in coronary blood flow to the bolus infusion of hypertonic saline solution was not affected by the blocking agents atropine, propranolol and phentolamine, but it was completely abolished in the same nine pigs by the subsequent intracoronary administration of N omega‐nitro‐L‐arginine methyl ester (L‐NAME) which blocks the synthesis of endothelium‐derived relaxing factor (EDRF) and in seven pigs by solely giving L‐NAME. 5. These results showed that the intracoronary bolus infusion of hypertonic saline solution in anaesthetized pigs caused a coronary vasodilatation which involved mechanisms dependent on the release of EDRF.
Modulation of the hyperpolarization‐activated current (Ih) by cyclic nucleotides in guinea‐pig primary afferent neurons.
Authors: Ingram S L , Williams J T
Published: December 2014

1. Whole‐cell patch‐clamp recordings were made from dissociated guinea‐pig nodose and trigeminal ganglion neurons in culture to study second messenger mechanisms of the hyperpolarization‐activated current (Ih) modulation. 2. Prostaglandin E2 (PGE2) and forskolin modulate Ih in primary afferents by shifting the activation curve in the depolarizing direction and increasing the maximum amplitude. 3. The cAMP analogues, RP‐cAMP‐S (an inhibitor of protein kinase A (PKA)) and SP‐cAMP‐S (an activator of PKA), both shifted the activation curve of Ih to more depolarized potentials and occluded the effects of forskolin. These results suggest that Ih is modulated by a direct action of the cAMP analogues. 4. Superfusion of other cyclic nucleotide analogues (8‐Br‐cAMP, 8‐(4‐chlorophenylthio)‐cAMP and 8‐Br‐cGMP) mimicked the actions of forskolin and PGE2, but dibutyryl cGMP, 5'‐AMP and adenosine had no effect on Ih. 8‐Br‐cAMP and 8‐Br‐cGMP had similar concentration response profiles, suggesting that Ih has little nucleotide selectivity. 5. The inhibitor peptide (PKI), the catalytic subunit of PKA (C subunit) and phosphatase inhibitors (microcystin and okadaic acid) had no effect on forskolin modulation of Ih. 6. These results indicate that Ih is regulated by cyclic nucleotides in sensory neurons. Positive regulation of Ih by prostaglandins produced during inflammation may lead to depolarization and facilitation of repetitive activity, and thus contribute to sensitization to painful stimuli.
High‐threshold Ca2+ currents in rat hippocampal interneurones and their selective inhibition by activation of GABA(B) receptors.
Authors: Lambert N A , Wilson W A
Published: December 2014

1. Whole‐cell calcium currents were recorded from visually identified inhibitory interneurones located in stratum radiatum (near the border with stratum lacunosum‐moleculare) of area. CA1 in rat hippocampal slices. Current‐voltage (I‐V) relationships in relatively well‐clamped neurones showed that inward current activated between ‐50 and ‐40 mV (holding potential, ‐80 mV) and was maximal near ‐10 mV. Currents showed little inactivation over the course of 85 ms steps, and were completely blocked by removal of Ca2+ or addition of Cd2+. Prominent low‐threshold currents were not observed under these conditions. 2. The calcium channels contributing to whole‐cell currents in interneurones were examined using selective channel antagonists. The selective N‐type calcium channel blocker omega‐conotoxin GVIA (omega‐CgTX‐GVIA; 10 microM) irreversibly blocked 23.2 +/‐ 2.8% of whole‐cell currents. The P/Q‐type antagonist omega‐agatoxin IVA (omega‐Aga‐IVA; 1‐5 microM) blocked 10.4 +/‐ 3.3% of whole‐cell currents. Block by omega‐Aga‐IVA was highly variable, ranging from 0 to 30%. The less selective conotoxin, omega‐conotoxin MVIIC (omega‐CTX‐MVIIC; 5 microM) blocked 31.0 +/‐ 2.7% of whole‐cell currents. The selective L‐type channel antagonist nifedipine (20 microM) blocked 27.5 +/‐ 3.5% of whole‐cell currents. 3. Whole‐cell calcium currents were reversibly inhibited by the selective GABA(B) receptor agonists (+/‐)‐baclofen or CGP 27492 (1‐3 microM; 18.9 +/‐ 1.4%). This inhibition was reversed or prevented by the selective GABAB receptor antagonist CGP 55845A (1 microM). Inhibition of inward current activated by voltage ramps was voltage dependent, being greatest near ‐10 mV, and less pronounced at more positive or negative potentials. Inhibition of calcium currents by GABAB receptor agonists was accompanied by an apparent change in the kinetics of whole‐cell currents consistent with a slowing of the rate of activation. CGP 27492 depressed calcium currents by 16.1 +/‐ 1.9% before application of omega‐CgTX‐GVIA, and by 3.9 +/‐ 2.0% after application of omega‐CgTX‐GVIA in the same cells (P < 0.005), consistent with preferential block of N‐type calcium channels. 4. Neither adenosine (200 microM) nor the selective mu‐opioid receptor agonist Tyr‐D‐Ala‐Gly‐MePhe‐Gly‐ol (DAMGO; 2 microM) inhibited calcium currents. Similarly, CGP 27492, but not adenosine or DAMGO, induced an outward current (at ‐ 70 mV) consistent with activation of inwardly rectifying potassium channels. 5. These results indicate that hippocampal inhibitory neurones located in stratum radiatum possess multiple calcium channel subtypes, including N‐type, L‐type, and at least two other types of high‐threshold channels. Activation of GABAB receptors (but not adenosine or mu‐opioid receptors) preferentially inhibits N‐type channels in these neurones. Similar inhibition occurring in the terminals of interneurones could contribute to depression of inhibitory synaptic transmission by activation of GABAB autoreceptors.
Endogenous and exogenous dopamine depress EPSCs in rat nucleus accumbens in vitro via D1 receptors activation.
Authors: Harvey J , Lacey M G
Published: December 2014

1. Whole‐cell patch clamp recordings were made from nucleus accumbens neurones in slices of rat ventral forebrain. In the presence of picrotoxin (50 microM), the amplitude of 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione‐sensitive glutamate EPSCs, recorded at holding potentials between ‐80 and ‐90 mV, was reversibly reduced by 56 +/‐ 11% (n = 6) by dopamine (30 microM). The selective dopamine D1 receptor agonists SKF 38393 (10 microM) and SKF 81297A (10 microM), but not the selective D2 receptor agonist quinpirole (10 microM), also reversibly depressed the EPSC by 36‐48%. The depression of the EPSC by dopamine was completely blocked by the D1 receptor antagonist SCH 23390 (1 microM), whereas the D2 antagonist (‐)‐sulpiride (1 microM) was without effect. 2. EPSCs were reversibly depressed by the dopamine mimetic psychostimulants cocaine (1‐20 microM) and amphetamine (10‐30 microM) by 40 +/‐ 16 and 62 +/‐ 9%, respectively, but only in about half of the cells tested (11/23 and 6/13, respectively). Their actions were completely reversed by SCH 23390 (1 microM), indicating that endogenous dopamine can also depress the EPSC via D1 receptors. 3. No discernable effects of dopamine, SKF 81297A, SKF 38393, quinpirole, cocaine or amphetamine were observed on membrane conductance or holding current (at holding potentials of ‐80 to ‐90 mV), suggesting that the depression of the EPSC was solely due to an action on presynaptic D1 receptors. 4. In contrast, agents that elevate intracellular levels of adenosine‐3':5'‐cyclic monophosphate (cAMP) (forskolin (1‐10 microM), 3‐isobutyl‐1‐methylxanthine (0.1‐1 mM), rolipram (10 microM), and dibutyryl cAMP (0.5‐1 mM)) caused a reversible increase in the EPSC amplitude (by 21‐150%). Furthermore, in the presence of forskolin (10 microM), the ability of dopamine to depress synaptic transmission was unaffected. 5. Together these data suggest that both exogenous dopamine and dopamine released from intrinsic nerve terminals attenuate glutamate receptor‐mediated synaptic transmission in the nucleus accumbens by presynaptic D1 receptor activation. The transduction mechanism underlying this effect does not appear to involve the formation of cAMP.
Electrical properties of smooth muscle in the guinea‐pig urinary bladder.
Authors: Bramich N J , Brading A F
Published: December 2014

1. The effects of transmural nerve stimulation were examined on preparations of detrusor smooth muscle from guinea‐pig urinary bladder using intracellular recording techniques. Most recordings were made from preparations in which spontaneous and evoked action potentials had been inhibited by nifedipine (10 microM), a dihydropyridine that blocks L‐type Ca2+ channels. 2. Supramaximal stimuli evoked excitatory junction potentials (EJPs) which could be divided into three basic types. Type 1 EJPs had short latencies (< 30 ms) and fast rise times (< 60 ms). Type 2 EJPs consisted of two components: a small depolarization that was followed by a second depolarization with a faster rise time. In a third type of cell, at high strengths of stimulation, EJPs resembled type 1 EJPs but at lower strengths of stimulation were similar in time course to type 2 EJPs. 3. All EJPs were abolished by tetrodotoxin (1 microM) and reduced by omega‐conotoxin (0.1 microM), but were unaffected by hexamethonium (0.1 mM), suggesting that they result from the release of transmitter from post‐ganglionic nerve fibres. All responses persisted in the presence of atropine (1 microM) but were abolished following the desensitization of P2‐purinoceptors with alpha, beta‐methylene ATP (m‐ATP; 10 microM). 4. Spontaneous excitatory junction potentials (SEJPs) were also recorded from most cells. SEJPs were similar in appearance to fast single‐component EJPs; however, in general they had a briefer time course. SEJPs persisted in the presence of tetrodotoxin (1 microM). 5. The electrical properties of urinary bladder smooth muscle were also examined. Voltage changes induced by point current injection into cells had fast rates of rise and decay (time constant, 5‐20 ms). The input resistance of cells ranged between 12 and 108 M omega. When recordings were taken from cells near the point of current injection, resultant electrotonic potentials could be detected in only a small proportions of cells. 6. The results are discussed in relation to the idea that transmural nerve stimulation in the guinea‐pig urinary bladder causes the activation of at least two different membrane conductances. Cells appear to be electrically coupled with one another. However, it is likely that coupling exists within discrete bundles of the smooth muscle.
Cellular mechanisms for neuronal thermosensitivity in the rat hypothalamus.
Authors: Griffin J D , Kaple M L , Chow A R , Boulant J A
Published: December 2014

1. To study the basic mechanisms of neuronal thermosensitivity, rat hypothalamic tissue slices were used to record and compare intracellular activity of temperature‐sensitive and ‐insensitive neurones. This study tested the hypothesis that different neuronal types have thermally dependent differences in the transient potentials that determine the interspike interval. 2. Most spontaneously firing neurones displayed depolarizing prepotentials that preceded each action potential. In warm‐sensitive neurones, warming increased the rate of rise of the depolarizing prepotential which, in turn, decreased the interspike interval and increased the firing rate. In contrast, temperature had little or no effect on the rate of rise in prepotentials of temperature‐insensitive neurones. 3. Prepotential depolarization can be due to increasing depolarizing conductances or decreasing hyperpolarizing conductances. These are differences in the ionic conductances responsible for prepotentials in temperature‐sensitive and ‐insensitive neurones. In warm‐sensitive neurones, the net ionic conductance decreased as the prepotential depolarized towards threshold, suggesting that the prepotential is primarily determined by a decrease in outward potassium conductances. In contrast, in low‐slope temperature‐insensitive neurones, the net conductance remained constant during the interspike interval, suggesting a more balanced combination of both depolarizing and hyperpolarizing conductances. 4. Transient outward potassium currents, including A‐currents, are important determinants of neuronal firing rates. These currents were identified in all warm‐sensitive neurones tested, as well as in many temperature‐insensitive and silent neurones. Since warming increased the rates of inactivation of these currents, transient K+ currents may contribute to the temperature‐dependent prepotentials of some hypothalamic neurones.
Calcium handling and purinoceptor subtypes involved in ATP‐induced contraction in rat small mesenteric arteries.
Authors: Lagaud G J , Stoclet J C , Andriantsitohaina R
Published: December 2014

1. The relationship between the stimulation of ATP receptors, the increase in intracellular free calcium concentration ([Ca2+]i; measured using the fluorescent indicator fura‐2), contraction and the subtypes of purinoceptors involved were investigated in the small mesenteric artery of the rat. 2. In normal physiological solution, ATP (0.001‐3 mM) caused concentration‐dependent increases in both [Ca2+]i and contraction. Both responses produced by ATP (1 mM) were inhibited by 50% in the presence of nitrendipine (1 microM) and were abolished in the presence of nitrendipine plus SK&F 96365 (30 microM). 3. In Ca(2+)‐free medium, ATP (3 mM) elicited a transient increase in both [Ca2+]i and tension which were abolished by caffeine and decreased by 65% by thapsigargin (1 microM). Moreover, ATP (1 and 3 mM) produced increases in the [3H]D‐myo‐inositol 1,4,5‐trisphosphate ([3H]IP3) content of vessels in a concentration‐dependent manner. 4. Treatment of the vessels with Bordetella pertussis toxin (PTX) inhibited contractions to ATP linked to the influx of calcium through nitrendipine‐sensitive mechanisms, but not those linked to the release of Ca2+ from intracellular stores nor the capacity of ATP in increasing IP3 content of the vessels. 5. The order of potency of ATP and its analogues in eliciting contraction was alpha, beta‐methylene‐ATP (alpha, beta‐MeATP) > 2‐methylthio‐ATP (2‐MeSATP) > ATP = ADP. The response to ATP was inhibited by suramin. Reactive Blue 2 (up to 100 microM) did not affect the contractile response to ATP. Pyridoxal‐phosphate‐6‐azophenyl‐2',4'‐disulphonic acid 4‐sodium (PPADS) and alpha, beta‐MeATP abolished the response to low concentrations of ATP and reduced contractions elicited by high concentrations of ATP. 6. After blockade of P2X‐purinoceptors with PPADS, the order of potency of ATP and its analogues was 2‐MeSATP > ATP = ADP. UTP produced concentration‐dependent contractions which were not affected by suramin, Reactive Blue 2, PPADS or alpha, beta‐MeATP, suggesting the presence of P2U‐purinoceptors. 7. The results suggest that low concentrations of ATP activate P2X‐purinoceptors and produce an influx of calcium through both voltage‐dependent calcium channels sensitive to nitrendipine and through receptor‐operated calcium channels sensitive to SK&F 96365. High concentrations of ATP activate P2Y‐purinoceptors which promote firstly a nitrendipine‐sensitive calcium influx via a PTX‐sensitive G protein and secondly a release of Ca2+ from an internal source via the production of IP3.
Slowing of relaxation and [Ca2+]i during prolonged tetanic stimulation of single fibres from Xenopus skeletal muscle.
Authors: Westerblad H , Allen D G
Published: December 2014

1. Parvalbumin (PA) has been proposed to take up Ca2+ and enhance skeletal muscle relaxation in brief contractions; as the duration of the contraction is increased, PA will become saturated with Ca2+ and no longer contribute to relaxation which therefore will be slowed. The rate of Ca2+ loading of PA is determined by the Mg2+ off rate (about 4 s‐1 at 22 degrees C). In the present study we produced prolonged tetani in intact, single fibres of Xenopus frogs while measuring force and the free myoplasmic [Ca2+] ([Ca2+]i) with indo‐1. 2. Mean rate constants of slowing of force relaxation with increasing tetanus duration ranged between 3.2 and 4.8 s‐1, thus, similar to the Mg2+ off rate of PA. 3. The amplitude of the tail of [Ca2+]i after tetani increased with tetanus duration. This increase developed with a rate constant similar to the Mg2+ off rate of PA 4. Steady‐state force‐[Ca2+]i curves were produced from tetani of various frequencies and tetani produced when force was depressed after fatiguing stimulation. These curves were used to convert [Ca2+]i records into Ca(2+)‐derived force. Relaxation of Ca(2+)‐derived force was slowed following a time course similar to that of real force. The lag between Ca(2+)‐derived and real force during relaxation was not affected by tetanus duration. 5. Tails of elevated [Ca2+]i after tetani were used to analyse the function of the SR Ca2+ pumps. This analysis showed a marked decline in the rate of Ca2+ uptake with prolonged tetani. 6. In conclusion, in Xenopus fibres the slowing of relaxation with increasing tetanus duration can be explained by altered Ca2+ handling due to PA Ca2+ loading and impaired SR Ca2+ uptake. This contrasts to our previous results in mouse fibres and the difference can be explained by a markedly lower rate of SR Ca2+ uptake resulting in higher tetanic [Ca2+]i in Xenopus fibres.