bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of route of breathing on the ventilatory and arousal responses to hypercapnia in awake and sleeping dogs.
Authors: Issa F G , Bitner S
Published: December 2014

1. The influence of the upper airway on the ventilatory and arousal responses to hypercapnia in wakefulness and sleep was investigated using a chronic animal model. 2. Experiments were performed in five unrestrained dogs trained to sleep naturally in the laboratory. The animal rebreathed through a chronic tracheostoma (thus excluding the upper airway from the breathing circuit), or through the snout (intact upper airway). Resistance to breathing and volume of dead space during quiet tracheal breathing were matched to those in quiet nasal breathing during wakefulness and sleep. CO2 rebreathing tests were performed during wakefulness, rapid eye movement (REM) and non‐REM (NREM) sleep, during nasal and tracheal breathing. 3. The ventilatory response to hypercapnia was significantly lower in nasal breathing compared with tracheal breathing, in all behavioural states. This was due to a smaller tidal volume and lower breathing frequency. 4. The ventilatory response to CO2 was lowest during REM sleep, irrespective of route used for breathing. 5. Alveolar partial pressure of CO2 (PA,CO2) level at arousal was identical in NREM nasal and tracheal rebreathing tests. Differences in PA,CO2 levels at arousal between NREM and REM sleep were not significant in nasal tests and only marginally different during tracheal breathing. 6. We conclude that nasal breathing influences the hypercapnic ventilatory response in wakefulness and sleep, and that the presence of CO2 in the upper airway does not affect arousal in NREM and REM sleep.
Effects of pH and inorganic phosphate on force production in alpha‐toxin‐permeabilized isolated rat uterine smooth muscle.
Authors: Crichton C A , Taggart M J , Wray S , Smith G L
Published: December 2014

1. Strips of longitudinal smooth muscle isolated from rat uterus were permeabilized using crude alpha‐toxin from the bacterium Staphylococcus aureus. This treatment rendered the surface membrane permeable to small molecular weight substances. Simultaneous measurements of tension and calcium concentration ([Ca2+]) (using indo‐1 fluorescence) were used to investigate the effects of pH and inorganic phosphate concentration ([Pi]) on Ca(2+)‐activated force generated by the contractile proteins. 2. Raising the [Pi] from 1 to 11 mM at a pH of 7.2 depressed both maximal and submaximal Ca(2+)‐activated force. This effect of Pi was concentration dependent having the majority of its effect by 6 mM. 3. Further experiments at a submaximal [Ca2+] showed that Ca(2+)‐activated force was enhanced by raising [Pi] from 6 to 11 mM suggesting that Pi increased the Ca2+ sensitivity of tension production. Based on these results, calculations indicate that the apparent affinity constant of Ca2+ for the contractile proteins increased from 4 x 10(6) M‐1 to 6 x 10(6) M‐1 on raising [Pi] from 1 to 11 mM. 4. Lowering pH from 7.2 to 6.7 at a [Pi] of 1 mM potentiated Ca(2+)‐activated force with a small depression in the apparent Ca2+ sensitivity of tension production. This effect of pH on maximum (100 microM Ca2+) and submaximum (0.3 microM Ca2+) Ca(2+)‐activated force was observed over a range of acidic pHs (7.0‐6.7). 5. Increasing pH from 7.2 to 7.7 at a [Pi] of 1 mM depressed Ca(2+)‐activated force with no effect on Ca2+ sensitivity of tension production. 6. Spontaneous contractions in intact rat myometrium are abolished under hypoxic conditions. Under these same conditions intracellular [Pi] rises and pH falls. The results of this study suggest that taken individually neither the effect of a rise in [Pi] nor a fall in pH on Ca(2+)‐activated force generated by the contractile proteins can account for the effect of hypoxia on spontaneous contractions.
Differences between contractions in vitro of slow and fast rat skeletal muscle persist after random reinnervation.
Authors: Lewis D M , Chamberlain S
Published: December 2014

1. The medial popliteal nerve was sectioned in adult rats, and reinnervation was permitted after nerve reunion. Reinnervation was observed in soleus (slow twitch) and plantaris (fast twitch) muscles for periods of 4‐21 months after the initial operation. 2. In the reinnervated muscles, isometric twitch time to peak (contraction time) and time to half‐relaxation were longer in soleus than plantaris in every muscle examined. 3. Some of the muscles were stained for actomyosin ATPase activity. The proportion of type I fibres (presumed slow) was at least twice as high in soleus compared with plantaris muscles. The proportion in the latter was similar to that predicted from random reinnervation. 4. The effects of differing proportions of fast and slow fibres on the twitch contraction and half‐relaxation times were modelled, and the experimental twitch data were found to be compatible with the proportions of fibre types in individual muscles. 5. It is speculated that the mechanism accounting for the higher proportion of slow fibres in soleus muscle probably does not involve selective reinnervation. It is suggested that fast motoneurones may be transformed to slow by a trophic chemical released by slow muscle fibres; such a process would be more extensive in predominantly slow twitch soleus than a fast muscle, and the resulting decrease in the proportion of fast motoneurones would account for the less extensive conversion in soleus.
Cytosolic calcium homeostasis in bovine parathyroid cells and its modulation by protein kinase C.
Authors: Racke F K , Nemeth E F
Published: December 2014

1. The effects of protein kinase C (PKC) activators and inhibitors on the mechanisms regulating cytosolic Ca2+ homeostasis in dissociated bovine parathyroid cells loaded with fura‐2 were examined. 2. Stepwise increases in the concentration of extracellular Ca2+ (from 0.5 to 2 or 3 mM) elicited transient followed by sustained increases in the concentration of intracellular free Ca2+ ([Ca2+]i). Cytosolic Ca2+ transients reflected the mobilization of intracellular Ca2+ and influx of extracellular Ca2+ whereas sustained increases in [Ca2+]i resulted from the influx of extracellular Ca2+. Brief (1‐2 min) pretreatment with phorbol myristate acetate (PMA) shifted the concentration‐response curve for extracellular Ca(2+)‐induced cytosolic Ca2+ transients to the right without affecting the maximal response. Cytosolic Ca2+ transients elicited by extracellular Mg2+ were similarly affected by PMA. 3. These effects of PMA were mimicked by various other activators of PKC with the rank order of potency PMA > phorbol dibutyrate > bryostatin , > (‐)indolactam V > mezerein. Isomers or analogues of these compounds that do not alter PKC activity (4 alpha‐phorbols and (+)indolactam V) did not alter [Ca2+]i. 4. PKC activators depressed evoked increases in [Ca2+]i when influx of extracellular Ca2+ was blocked with Gd3+. Cytosolic Ca2+ transients elicited by extracellular Mg2+ in the absence of extracellular Ca2+ were similarly inhibited by PKC activators. Activation of PKC thus inhibits the mobilization of intracellular Ca2+ elicited by extracellular divalent cations. 5. Increases in the concentration of extracellular Ca2+ caused corresponding increases in the formation of [3H]inositol 1,4,5‐trisphosphate ([3H]InsP3). Pretreatment with PMA shifted the concentration‐response curve for extracellular Ca(2+)‐induced [3H]InsP3 formation to the right without affecting the maximal response. 6. PKC activators also caused some depression of steady‐state increases in [Ca2+]i elicited by extracellular Ca2+. In contrast, PMA did not affect increases in [Ca2+]i elicited by ionomycin or thapsigargin. 7. Ba2+ was used to monitor divalent cation influx. PMA decreased the rate of rise of the fluorescent signal elicited by extracellular Ba2+. 8. All these effects of PKC activators on [Ca2+]i were blocked or reversed by staurosporine at concentrations (30‐100 nM) that inhibited PKC activity in parathyroid cells. Staurosporine alone potentiated cytosolic Ca2+ responses evoked by submaximal concentrations of extracellular divalent cations. 9. PKC thus depresses both the mobilization of intracellular Ca2+ and the influx of extracellular Ca2+ in parathyroid cells. The effects on [Ca2+]i provide evidence for a Ca2+ receptor on the surface of parathyroid cells that uses transmembrane signalling mechanisms common to some other Ca(2+)‐mobilizing receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Beta‐adrenergic enhancement of inhibitory synaptic activity in rat cerebellar stellate and Purkinje cells.
Authors: Llano I , Gerschenfeld H M
Published: December 2014

1. Using the tight‐seal whole‐cell recording technique, we studied the effects of noradrenaline (NA) on the spontaneous inhibitory synaptic currents (IPSCs) of stellate and Purkinje cells in rat cerebellar slices. 2. In both types of cells, NA (10 microM) induced a marked increase in the frequency of the IPSCs. This effect was observed both in the absence and in the presence of TTX in the saline bathing the cerebellar slices. 3. The NA‐induced increase in frequency of IPSCs and miniature IPSCs (mIPSCs) in the two cell types was mimicked by bath applications of isoprenaline (10 microM) and of the adenylyl cyclase activator forskolin (20 microM). Neither phenylephrine nor clonidine changed the frequency of IPSCs in stellate or Purkinje cells. 4. In stellate cells, the beta‐agonists and forskolin had variable effects on the amplitudes of both IPSCs and mIPSCs. None of these compounds altered the amplitude of mIPSCs in Purkinje cells. 5. The responses to local applications of GABA to Purkinje cells were unchanged by bath applications of beta‐adrenergic agonists or forskolin. A decrease in the response to GABA after treatment with these agents was observed in half the stellate cells examined. 6. We conclude that the major effect of NA on stellate and Purkinje cells is an increase in the frequency of occurrence of spontaneous inhibitory synaptic currents. This action is exerted through the activation of beta‐adrenergic receptors and is probably mediated by an intracellular mechanism involving cAMP. The beta‐adrenergic modulation of IPSC frequency takes place at the presynaptic level and may involve a change in the process of transmitter release.
Characterization of an outward potassium current in canine jejunal circular smooth muscle and its activation by fenamates.
Authors: Farrugia G , Rae J L , Szurszewski J H
Published: December 2014

1. A perforated patch clamp technique was used to study an outward potassium current in freshly dissociated circular smooth muscle cells of the canine jejunum. 2. A voltage‐dependent outward current was identified which was highly potassium selective, weakly holding voltage sensitive, increased its open probability at ‐65 mV, and reached unit open probability at +5 mV. 3. Quinidine (0.1‐1 mM) and tetraethylammonium ion (TEA) (10‐50 mM), blocked the potassium current in a dose‐dependent manner. Blockade of the outward potassium current was accompanied by membrane depolarization which reversed on removal of the blocker from the bathing solution. 4. Mefenamic and flufenamic acids, non‐steroidal anti‐inflammatory agents in the fenamate group, were potent activators of the current. Activation was accompanied by hyperpolarization of the membrane with a mean shift in the membrane voltage of 22 mV. 5. It was concluded that the outward potassium current is a major regulator of the resting membrane voltage in isolated circular smooth muscle cells of the canine jejunum. Fenamates activated this current with potentially profound effects on cellular excitability.
Evidence that capsaicin hyperaemia of rat sciatic vasa nervorum is local, opiate‐sensitive and involves mast cells.
Authors: Zochodne D W , Ho L T
Published: December 2014

1. In previous work, we identified a prolonged and intense hyperaemic response of rat sciatic endoneurial vasa nervorum produced by epineurial application of capsaicin. We postulated that this response, which was blocked by substance P (SP) or calcitonin gene‐related peptide (CGRP) antagonists, was a result of local release of neuropeptides on the ‘feeding’ epineurial vascular plexus. 2. In the present study, we evaluated factors that might influence capsaicin‐induced hyperaemia of the rat sciatic endoneurium as measured by hydrogen clearance: central afferent connections, the epineurial vascular plexus, the release of histamine and administration of opiates. 3. Interruption of central afferent connections by proximal nerve section or removal of the epineurial vascular plexus did not influence baseline endoneurial perfusion. Plexus removal, but not proximal section, prevented capsaicin hyperaemia. 4. The epineurial vascular plexus was desensitized to the effect of capsaicin by prior application of capsaicin. Capsaicin hyperaemia was also prevented by: topical treatment with Spantide II ((D‐NicLys1,3‐Pal3,D‐Cl2Phe5,Asn6,D‐Trp7,9,Nl e11) substance P) an SP antagonist, systemic pretreatment with a combination of H1 and H2 histamine receptor antagonists, systemic pretreatment with cromolyn sodium or systemic pretreatment with morphine. None of these pretreatments influenced baseline perfusion. When systemic morphine was given together with systemic naloxone, an opiate antagonist, capsaicin‐induced hyperaemia was restored. 5. These findings indicate that the capsaicin hyperaemia of vasa nervorum is locally mediated, is independent of central afferent connections and is sensitive to a variety of interventions. It requires an intact epineurial plexus that ‘feeds’ endoneurial microvessels and the release of histamine by mast cells. Its inhibition by morphine suggests that there are local opiate receptors on epineurial perivascular peptidergic fibres.
Selection of transmitter responses at sites of neurite contact during synapse formation between identified leech neurons.
Authors: Ching S , Catarsi S , Drapeau P
Published: December 2014

1. Pressure sensitive (P) neurons of the leech Hirudo medicinalis show both an inhibitory, Cl(‐)‐dependent response and a depolarizing, cationic response to pipette application of serotonin (5‐HT). Serotonergic Retzius (R) neurons in culture reform inhibitory, Cl(‐)‐dependent synapses with P neurons but fail to elicit the extrasynaptic, depolarizing response to 5‐HT. We have examined the localization of the selection of 5‐HT responses by testing the sensitivity of P cell growth cones and neurites to 5‐HT application. 2. As measured by intracellular recording at the P cell soma, synaptic release of 5‐HT from R cell processes activated only the Cl(‐)‐dependent response in P cell neurites. Focal application of 5‐HT from a micropipette depolarized uncontacted P cell growth cones and neurites. In contrast, processes from the same P cells that were contacted by R cells were rarely depolarized by 5‐HT application unless the application pipette was moved along the neurites away from the sites of contact. 3. The channels underlying the depolarizing response to 5‐HT were identified in patch clamp recordings from P cell growth cones. These cation channels showed rare, brief openings in the absence of 5‐HT. Application of 5‐HT in the bath (outside the patch pipette) increased channel activity in uncontacted P cell growth cones but not in growth cones of the same P cells contacted by R cells. 4. We conclude that the selection of transmitter responses during synapse formation was localized to discrete sites of contact between the synaptic partners.
GABAB receptors modulate glycinergic inhibition and spike threshold in Xenopus embryo spinal neurones.
Authors: Wall M J , Dale N
Published: December 2014

1. The actions of GABAB receptors in the generation of the neuronal pattern underlying swimming in the Xenopus embryo have been investigated using the agonist baclofen. 2. Baclofen (10‐100 microM) greatly reduced the length of swimming episodes and ventral root spike amplitude in a reversible manner. These effects were blocked by CGP 35348 (200‐300 microm) and hydroxysaclofen (200‐300 microM). 3. Baclofen (10‐100 microM) reduced the amplitude of glycinergic IPSPs in motoneurones during fictive swimming. 4. Strychnine‐sensitive spontaneous miniature inhibitory postsynaptic potentials (mIPSPs) were recorded from motoneurones. While baclofen (10‐100 microM) had no effect on the amplitude of the mIPSPs it greatly decreased their frequency of occurrence. 5. GABAB receptors may therefore be present on the terminals of commissural interneurones, the only glycinergic neurones in the Xenopus embryo's nervous system, and act to reduce neurotransmitter release. 6. Baclofen reduced the reliability of action potential firing in motoneurones during fictive swimming without an apparent effect on excitation. 7. Baclofen increased the threshold to action potential firing in response to the injection of depolarizing current in motoneurones. 8. The current‐voltage relationships of motoneurones were investigated. Baclofen (10‐100 microM) did not change the resting membrane potential, slope conductance or the membrane rectification.
Unitary properties of potassium channels activated by 5‐HT in acutely isolated rat dorsal raphe neurones.
Authors: Penington N J , Kelly J S , Fox A P
Published: December 2014

1. Single inwardly rectifying K+ channel currents were recorded from acutely isolated adult serotonergic dorsal raphe (DR) neurones using the cell‐attached and outside‐out patch clamp configuration. 2. Four equally spaced conductance levels were observed in both outside‐out and cell‐attached patch recordings with conductance levels averaging 11, 21, 30 and 40 pS. Larger conductance openings (50‐120 pS) were seen less frequently. 3. When using 136 [K+]0 the single channel I‐V relation was linear in the range 0 mV to ‐100 mV in all cases. 4. Transitions between the various conductance levels were observed, as were apparent direct opening and closing to each individual conductance level. Furthermore openings of 11, 21 and 30 pS were observed in almost all the patches. These results suggest that the different‐sized events result from substrates of a single channel rather than several different channels with different conductances. 5. Unitary K+ channel current probability of opening, recorded in cell‐attached patch, was unchanged after 5‐hydroxytryptamine (5‐HT) was added to the bath outside the patch pipette which suggests that no easily diffusible second messenger was involved. 6. The single K+ channel activity, however, was increased on average by 670% following the addition of 5‐HT to the bath when recording channel activity in the outside‐out configuration. Usually all K+ channel subconductance levels increased in activity but the largest increases occurred in the events with 30 and 40 pS conductance. 7. These results suggest that 5‐HT enhances the probability of opening of the resting K+ channel activity, which can open to several levels of conductance, and that no new channel or freely diffusible second messenger is involved in the response.
Calcium currents elicited by voltage steps and steady voltages in myocytes isolated from the rat basilar artery.
Authors: Langton P D , Standen N B
Published: December 2014

1. Whole‐cell patch clamp methods were used to record Ca2+ channel currents from isolated rat basilar arterial myocytes either in response to voltage steps or at steady holding potentials. Inward currents were rapidly and reversibly blocked by 2 mM Co2+, and this solution was routinely used to subtract leakage currents. 2. Peak currents measured in response to voltage steps were ‐85 +/‐ 23 pA (n = 7) in physiological Ca2+ (1.8 mM) and ‐256 +/‐ 39 pA (n = 19) in 10 mM Ba2+. The time course of activation and inactivation were unaffected by changing the holding potential from ‐88 to ‐48 mV, and thus we found no evidence for a component of current flowing through transient Ca2+ channels. 3. Activation curves were constructed by dividing the macroscopic current by the single Ca2+ channel current. The product of the number of functional channels and the open‐state probability (NPo) measured in this way rose to around 2000. Its dependence on voltage was fitted by a Boltzmann function with half‐activation at ‐8.6 mV in physiological Ca2+ concentrations and a steepness factor in the range 6‐8 mV. 4. In physiological solution, block by external Mg2+ reduced peak current through Ca2+ channels by 17%. 5. Steady‐state currents were measured by holding the cell at a fixed voltage and rapidly applying Co2+ to block the current through Ca2+ channels. Steady‐state currents could be detected at voltages as negative as ‐58 mV, and persisted for more than 15 min, suggesting that Ca2+ channels in this artery may provide a steady‐state Ca2+ influx that contributes to resting contractile tone.
Testing the common neural integrator hypothesis at the level of the individual abducens motoneurones in the alert cat.
Authors: Godaux E , Cheron G
Published: December 2014

1. As far as horizontal eye movements are concerned, the well‐known hypothesis of a common neural integrator states that the eye‐position signal is generated by a common network, regardless of the type of versional movement. The aim of this study was to evaluate the validity of this hypothesis by analysing the behaviour of the abducens motoneurones, the system into which the horizontal neural integrator(s) project(s). If there were a common neural integrator, the different motoneurones would receive the eye position signal through the same pathway and the sensitivity to eye position would be the same regardless of the type of versional movement. If there were multiple integrators, the sensitivity to eye position in one type of versional movement might be different from the sensitivity to eye position in another type of versional movement, at least for occasional motoneurones. 2. The discharge of thirty‐one antidromically identified abducens motoneurones was recorded in the alert cat during spontaneous eye movements made in the light and in response to sinusoidal rotations of the head in complete darkness. 3. All of the abducens motoneurones exhibited a burst of action potentials for lateral saccades. During fixation between saccades, they maintained a steady firing rate that increased as the cat fixated increasingly lateral eye positions. 4. For each abducens motoneurone, the sensitivity to eye position (Kf) was determined from measurements carried out during intersaccadic fixations. Kf was calculated from the slope of the firing rate‐eye position linear regression line. 5. The discharge rate of the identified motoneurones was observed during four sinusoidal vestibular stimulations (+/‐ 10 deg, 0.10 Hz; +/‐ 20 deg, 0.10 Hz; +/‐ 30 deg, 0.10 Hz; +/‐ 40 deg, 0.10 Hz). The motoneurones exhibited a burst of activity during fast phases in the lateral direction and paused during fast phases in the opposite direction. During slow phases, motoneurones modulated their activity as a function of the vestibularly induced eye movements except for slow phases that occurred in position ranges below their recruitment threshold. In these cases their activity was cut off. 6. A new method was developed to measure the sensitivity to eye position of neurones during vestibular slow phases. The difficulty came from the fact that, during slow phases, eye velocity and eye position changed simultaneously and that each of those two variables could influence neuronal activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Membrane potential modulates inositol 1,4,5‐trisphosphate‐mediated Ca2+ transients in guinea‐pig coronary myocytes.
Authors: Ganitkevich VYa  , Isenberg G
Published: December 2014

1. Vascular smooth muscle cells were isolated from the coronary artery of the guinea‐pig. At 2.5 mM [Ca2+]o and 36 degrees C, whole cell membrane currents were recorded under voltage‐clamp and the concentration of ionized calcium in the cytoplasm ([Ca2+]i) was monitored by indo‐1 fluorescence. 2. At ‐60 mV, [Ca2+]i was 143 +/‐ 36 mM (mean +/‐ S.D.) and was insensitive to clamp steps to +100 mV. During 1 min application of acetylcholine (ACh, 10 microM) [Ca2+]i increased within approximately 2 s to 1480 +/‐ 250 nM. During the subsequent slow decay, [Ca2+]i was transiently increased by depolarizing clamp steps and decreased during hyperpolarizing steps. [Ca2+]i transients in response to caffeine (10 mM) could not be modulated by voltage steps. The results suggest that modulation of [Ca2+]i by membrane potential involves inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3)‐induced Ca2+ release (IICR). 3. Modulation of IICR by membrane potential did not depend on sarcolemmal Ca2+ fluxes; it persisted after block of sarcolemmal Ca2+ fluxes with 3 mM lanthanum or after a change to nominally Ca(2+)‐free bathing solutions. 4. Modulation of [Ca2+]i by membrane potential was recorded during cell dialysis of 50 microM GTP‐gamma‐S in the absence of ACh. Cell dialysis of exogenous Ins(1,4,5)P3 (50 or 100 microM) did not mimic the effects. The sensitivity of [Ca2+]i to depolarizing clamp steps was also induced by cell dialysis of lithium ions which, presumably, inhibited the breakdown of Ins(1,4,5)P3. The results are compatible with the idea that the membrane potential modulates the liberation of Ins(1,4,5)P3. 5. Modulation of IICR by membrane potential is discussed as a new mechanism that contributes to the regulation of activator calcium and to the modulation of contraction in vascular smooth muscle cells.
Thyroliberin‐induced changes in the fluorescence of a membrane probe in individual bovine anterior pituitary cells.
Authors: Shorte S L , Stafford S J , Bamford M , Collett V J , Schofield J G
Published: December 2014

1. We have investigated the use of TMA‐DPH (1‐[4‐(trimethylammonio) phenyl]‐6‐phenylhexa‐1,3,5‐triene) as an indicator of exocytosis in individual bovine anterior pituitary cells using microfluorimetric imaging. 2. TMA‐DPH was photolabile in artificial and cell membranes. In cells incubated in TMA‐DPH the distribution of fluorescence depended both on the incubation time and the illumination schedule. If the dye was added while the cells were subjected to repeated cycles of 0.36 s light intermittent with 1‐15 s dark, the fluorescence of the peripheral annulus and the central region of individual cells rose in parallel and reached a steady state within 200 s; the annulus was always brighter than the central region. However, using long intervening dark periods (200 s), the central region continued to incorporate dye after the annulus had reached a plateau. 3. When the cells were loaded with TMA‐DPH using intermittent light with short dark periods, the dye washed out of the central region and the annulus in parallel when external dye was removed. However, if the cells had been loaded using long dark periods, the dye was washed out of the central region more slowly than from the annulus. 4. When cells were incubated in TMA‐DPH in the dark for 1 min and then exposed to constant illumination in the presence of external dye, the fluorescence of the central region and the annulus both decayed in parallel to a new steady state. If the cells were incubated in TMA‐DPH in the dark for 240 min the fluorescence from each region fell to a steady state but the falls were larger and were not in parallel. 5. We suggest that TMA‐DPH fluorescence was derived from plasma membrane‐associated and internalized dye and that the amount of fluorescence from the latter varied because TMA‐DPH was photobleached. Thus, when illumination was interrupted by short dark intervals, annular fluorescence was high compared to central fluorescence because bleached dye in the plasma membrane was rapidly replaced by unbleached dye from the medium. However, long dark intervals permitted the dye to be internalized before it was bleached and fluorescence was therefore also present in central regions. 6. The total cell fluorescence, observed using 15 s dark intervals, was increased 5‐40% (in single cells) in a dose‐dependent fashion by addition of TRH (tripeptide thyrotrophin‐releasing hormone; 1‐200 nM).(ABSTRACT TRUNCATED AT 400 WORDS)
The influence of thermoregulatory mechanisms on post‐exercise hypotension in humans.
Authors: Franklin P J , Green D J , Cable N T
Published: December 2014

1. To examine the influence of the rate of heat loss on the magnitude of post‐exercise hypotension, subjects were exposed to three different environmental conditions during recovery from bicycle exercise. 2. When subjects recovered in warm conditions both core temperature (measured in the external auditory meatus) and mean skin temperature were significantly elevated 60 min after the cessation of exercise. This attenuation of heat loss was associated with a significant reduction in post‐exercise mean arterial pressure. 3. In contrast, when subjects recovered in neutral or cool conditions both core temperature and mean arterial pressure had returned to baseline levels 60 min after exercise. 4. These results indicate that persistence of peripheral vasodilatation during recovery from exercise, and therefore the magnitude of post‐exercise hypotension, is dependent upon thermoregulatory‐induced changes in skin blood flow.
Modification of the transient outward current of rat atrial myocytes by metabolic inhibition and oxidant stress.
Authors: Pike G K , Bretag A H , Roberts M L
Published: December 2014

1. A putative function of the transient outward current (ITO) in cardiac myocytes is to modulate the shape of the action potential and, consequently, cardiac contractility. In addition, it has been suggested that this current may help protect against arrhythmias during periods of cardiac ischaemia. In our investigation of the possible anti‐arrhythmic action of ITO, we have examined its response to metabolic inhibition and oxidant stress. 2. Whole‐cell recordings were obtained from rat atrial myocytes using standard patch‐clamp techniques. Inhibition of metabolism, using 10 mM 2‐deoxy‐D‐glucose (2‐DG) to block glycolysis with or without the addition of 2 mM cyanide to block oxidative phosphorylation, led to inhibition of ITO at a holding potential of ‐70 mV. Shifting the holding potential to ‐80 mV restored ITO, suggesting that metabolic inhibition had shifted the inactivation curve of ITO in a negative direction. 3. Quasi steady‐state inactivation curves revealed a shift in ITO inactivation induced by complete metabolic inhibition with 2‐DG and cyanide. Myocytes typically contracted shortly after the shift was observed. In the presence of Ruthenium Red, contraction was delayed and myocytes could undergo several exposures to the metabolic inhibitors, each time displaying a shift in ITO inactivation. The shifts ranged between ‐7 and ‐20 mV. 4. Recovery from inactivation was determined using a two‐pulse protocol. The time constant of recovery at a holding potential of ‐80 mV reversibly shifted from 48 +/‐ 8 to 129 +/‐ 21 ms during metabolic inhibition (n = 4). 5. The activation of ITO from a holding potential of ‐100 mV shifted in a negative direction during metabolic inhibition, from a half‐activation voltage of 0.3 +/‐ 3.0 to ‐14.7 +/‐ 2.5 mV (n = 5). Such a ‐15 mV shift increases the amplitude of ITO by approximately 30% at 0 mV. 6. A shift in ITO inactivation similar to that produced by metabolic inhibition could be shown when myocytes were subjected to oxidant stress induced by either 1 mM t‐butyl hydroperoxide (TBHP) or the photoactivation of 100 nM Rose Bengal. Furthermore, an increase in pipette concentration of free Ca2+ from 20 to 200 nM also shifted ITO inactivation in a negative direction. 7. These results raise the possibility that the rise in intracellular [Ca2+] occurring during both metabolic inhibition and oxidant stress modifies activation and inactivation of ITO.(ABSTRACT TRUNCATED AT 400 WORDS)
A comparison of effect of temperature on phosphorus metabolites, pH and Mg2+ in human and ground squirrel red cells.
Authors: Marjanovic M , Gregory C , Ghosh P , Willis J S , Dawson M J
Published: December 2014

1. 31P NMR spectra were obtained at temperatures ranging from 2 to 30 degrees C from freshly drawn human (cold‐sensitive) and ground squirrel (cold‐tolerant) red cells. The concentration of ATP was also determined by luciferin‐luciferase assay over the same temperature range. 2. The concentration of ATP as determined by NMR or by the luciferin‐luciferase assay did not change with temperature in either species. The absolute concentration of ATP in human cells determined by NMR was not significantly different from the total ATP determined enzymatically. 3. The concentration of 2,3‐diphosphoglycerate was higher and that of pyridine nucleotides lower in human than in ground squirrel red cells. This species difference was independent of temperature. 4. Intracellular pH, as determined from the positions of the NMR peaks of 2‐ and 3‐phosphates of diphosphoglycerate, became more alkaline as the temperature was lowered. 5. Free intracellular magnesium, determined from the difference in the positions of the peaks for alpha‐ and beta‐phosphorus of ATP, increased in the ground squirrel red cells and decreased in the human red cells with cooling from 30 to 2 degrees C. Total magnesium, as determined by atomic emission spectroscopy, did not change with temperature in red cells of either species. 6. The intensities of all phosphorus metabolite signals from the ground squirrel cells increased with decreasing temperature, while those from the human cells were unaffected. Since chemical shift anisotropy in the presence of magnesium is a powerful spin‐lattice relaxation mechanism for phosphates, this is additional evidence for the temperature dependence of free magnesium concentration in the ground squirrel cells. 7. We conclude that there is no difference in phosphorus metabolites or intracellular pH which could account for the differential cold sensitivity in human and ground squirrel red cells. We suggest that, in the cold‐tolerant red cells from the ground squirrel, magnesium is released from binding sites as the temperature is lowered. The change in free intracellular Mg2+ may account at least in part for the unusually low temperature sensitivity of the Na(+)‐K+ pump in the red cells of this species.
Transport into retina measured by short vascular perfusion in the rat.
Authors: Gratton J A , Lightman S L , Bradbury M W
Published: December 2014

1. The short duration cerebrovascular perfusion method for measuring permeability of the blood‐brain barrier has been adapted to measuring transport into the retina. 2. The method has been characterized on the one hand by comparing uptakes of radiotracers during HCO3(‐)‐buffered saline perfusion with those occurring after intravenous bolus injection of radioisotopes, and on the other by comparing uptake into retina with the uptake into frontal cerebral cortex. The mean permeability‐surface area (PS) products (ml s‐1 g‐1) for [14C]urea and [14C]thiourea in the perfused retina were 1.2 +/‐ 0.26 x 10(‐3) and 2.1 +/‐ 0.01 x 10(‐3) respectively. The intravenous injection method gave comparable values for [14C]urea and [14C]thiourea of 1.6 +/‐ 0.28 x 10(‐3) and 3.24 +/‐ 0.55 x 10(‐3). The rates of uptake of the hydrophilic solutes were 2‐ to 7‐fold greater than in brain. 3. Retinal and choroidal capillary perfusion fluid flow rates were measured using a diffusible flow marker ([14C]diazepam) and a particulate indicator (15 microns cerium141‐labelled microspheres). Results using both flow markers confirmed that both capillary networks supplying the retina were being adequately perfused.
Electrophysiological effects of tachykinins and capsaicin on guinea‐pig bronchial parasympathetic ganglion neurones.
Authors: Myers A C , Undem B J
Published: December 2014

1. We evaluated the effects of neurokinins, tachykinin analogues, or capsaicin on passive membrane properties of guinea‐pig bronchial parasympathetic neurones using intracellular recording techniques. 2. Substance P (SP) and the tachykinin analogue, acetyl‐[Arg6,Sar9,Met(O2)11]‐SP(6‐11) (ASMSP), at concentrations selective for the neurokinin (NK)‐1 receptor subtype, depolarized the resting potential (3 and 5 mV, respectively) with no change in input resistance. Neurokinin A and beta Ala8NKA(4‐10), at concentrations selective for the NK‐2 receptor subtype (0.1 microM), were without effect. 3. Neurokinin B (NKB) and [Asp5,6,methyl‐Phe8]SP(5‐11) (senktide analogue), at concentrations selective for NK‐3 receptor subtype, elicited maximum depolarizations of 16 +/‐ 2 mV for both agonists. The peak of the depolarization was associated with an decrease in membrane resistance (35 +/‐ 4 and 50 +/‐ 7%, respectively). 4. Capsaicin (1 microM) elicited a 3‐24 mV depolarization of the resting potential of thirteen of eighteen bronchial ganglion neurones and decreased the input resistance of seven of thirteen of these neurones. The effects of capsaicin were reduced by desensitization with senktide analogue at a concentration selective for the NK‐3 receptor subtype, whereas a non‐peptide NK‐1 receptor antagonist had no effect. 5. Using voltage clamp analysis, capsaicin and senktide analogue evoked an inward current and an increase in membrane conductance at the resting membrane potential. The reversal potential for senktide analogue was estimated to be + 4 mV. 6. These data support the hypothesis that neurokinin‐containing nerve terminals are localized within guinea‐pig bronchial parasympathetic ganglia and, when released, the predominant effect of the neurokinins is by activation of NK‐3 receptors.
Role of 5‐HT3 receptors in peristaltic reflex elicited by stroking the mucosa in the canine jejunum.
Authors: Neya T , Mizutani M , Yamasato T
Published: December 2014

1. The role played by the 5‐HT3 receptor, a serotonin subtype receptor, in peristaltic reflexes was studied in dogs first given ketamine, then anaesthetized with urethane (1.0 g kg‐1, I.V.) and alpha‐chloralose (100 mg kg‐1, I.V.). The jejunal loop was partitioned into two segments with respect to blood supply. Drugs were infused intra‐arterially into each segment. 2. Stroking of the mucosa of the aboral and oral segments elicited an ascending contraction and a descending relaxation, respectively. 3. The ascending contraction was concentration‐dependently inhibited by treatment of the aboral segment with the 5‐HT3 receptor antagonists ICS 205‐930 and ondansetron (1.4 pmol min‐1 to 14 nmol min‐1 for both). The maximal inhibition was 49.5 and 69.3%, respectively. The response was not affected by treatment of the oral segment with these drugs. The descending relaxation was inhibited by 51.4 and 60.8%, respectively, by treatment of the oral segment with ICS 205‐930 and ondansetron (1.4 nmol min‐1 for both). 4. The ascending contraction was markedly inhibited by treatment of either segment with hexamethonium (140 nmol min‐1). The response was abolished by treating both segments with hexamethonium and by treating the oral segment with atropine (14 nmol min‐1). 5. These results suggest firstly that, in the canine jejunum, enteric neurons with 5‐HT3 receptors play a role as sensory neurons or interneurons in the ascending excitatory and the descending inhibitory pathways of the peristaltic reflex elicited by stroking the mucosa, and secondly, that the ascending limb is composed of cholinergic interneurons and motoneurons.
Turnover rate of the cardiac Na(+)‐Ca2+ exchanger in guinea‐pig ventricular myocytes.
Authors: Powell T , Noma A , Shioya T , Kozlowski R Z
Published: December 2014

1. Single guinea‐pig ventricular myocytes were voltage clamped using the whole‐cell configuration of the patch‐clamp technique and membrane current generated by the Na(+)‐Ca2+ exchange mechanism recorded. 2. Rapid increases in cytosolic free calcium ([Ca2+]i) evoked by flash photolysis of either nitr‐5 or DM‐nitrophen resulted in current relaxations, arising from a redistribution of exchanger carrier conformations induced by the changes in [Ca2+]i. 3. Relaxation time constants were temperature dependent with a temperature coefficient over a 10 degrees C range (Q10) of approximately 3 and also voltage dependent, decreasing on hyperpolarization for membrane potentials in the range +40 to ‐80 mV. 4. The experimental results are consistent with consecutive exchange models having electrogenic Na+ translocation steps, together with a site density and turnover rate similar to that for the Na(+)‐K+ pump.
Mild hypoxaemia does not alter red blood cell production in fetal sheep.
Authors: Anderson D F , Binder N D , Clemons G K
Published: December 2014

1. Fetal sheep at 120 days gestation were fitted with upper and lower body arterial and venous catheters in addition to a flow sensor and occluder placed around the aorta below the renal arteries. 2. After 7 days of recovery, the occluder was partially inflated to reduce aortic blood flow to 70% of control. Blood flow reduction was maintained at this level for the remainder of the experiment. 3. Blood samples were taken after 60 min of blood flow reduction and again after 3 or more days of blood flow reduction. 4. There was no change in upper body arterial or venous blood pressure. Lower body arterial blood pressure decreased, as expected. Arterial PO2 decreased while packed cell volume and haemoglobin concentration increased. There was no change in plasma erythropoietin concentrations or plasma renin activity. 5. While both red cell mass and haemoglobin mass increased during the period of the study, the rate of increase was no different from the rate of blood volume increase.
G protein‐mediated inhibition of phosphoinositide metabolism evoked by metabotropic glutamate receptors in frog oocytes.
Authors: Nakamura K , Nukada T , Haga T , Sugiyama H
Published: December 2014

1. Metabotropic glutamate receptor subtype 1 (mGluR1), when expressed in Xenopus oocytes, activates phosphoinositide‐specific phospholipase C (PLC) in a G protein‐dependent manner. This reaction results in the activation of chloride channels in the oocytes, and can be monitored electrophysiologically. We expressed different G protein alpha‐subunits together with mGluR1 in oocytes, and examined the effects of these G protein subunits on the PLC‐mediated reaction. 2. The expression of the alpha‐subunit of GL2, a bovine version of G11, which is a member of the Gq subgroup, potentiated the mGluR1‐evoked reaction, whereas the alpha‐subunit of GL1, a bovine G14, which is also a member of the Gq subgroup, strongly suppressed it. The expression of Gs alpha also suppressed this reaction. 3. We then expressed G beta 1 gamma 2‐subunits in addition to the G alpha‐subunits, and examined the mGluR1‐evoked reactions. Both the potentiation and suppression by GL2 alpha and GL1 alpha, respectively, were more pronounced in the presence of the G beta 1 gamma 2‐subunits. In contrast, the suppression by Gs alpha was completely reversed by G beta 1 gamma 2. 4. The direct activation of G proteins by the intracellular injection of either fluoride ions or guanosine‐5'‐O‐(3‐thiotriphosphate) (GTP gamma S) causes similar PLC‐mediated reactions. The expression of GL2 alpha, GL1 alpha or Gs alpha caused potentiation, suppression and no change, respectively, on the fluoride‐ (or GTP gamma S‐) evoked reactions.(ABSTRACT TRUNCATED AT 250 WORDS)
Sodium‐bicarbonate cotransport current in identified leech glial cells.
Authors: Munsch T , Deitmer J W
Published: December 2014

1. The membrane current associated with the cotransport of Na+ and HCO3‐ was investigated in neuropil glial cells in isolated ganglia of the leech Hirudo medicinalis L. using the two‐electrode voltage‐clamp technique. 2. The addition of 5% CO2‐24 mM HCO3‐ evoked an outward current, which slowly decayed, and which was dependent upon the presence of external Na+. Removal of CO2‐HCO3‐ elicited a transient inward current. Re‐addition of Na+ to Na(+)‐free saline in the presence of CO2‐HCO3‐ also produced an outward current. Under these conditions an intracellular alkalinization and a rise in intracellular [Na+] were recorded using triple‐barrelled, ion‐sensitive microelectrodes. Addition or removal of HCO3‐, in the absence of external Na+, caused little or no change in membrane voltage, membrane current and intracellular pH, indicating that the glial membrane has a very low HCO3‐ conductance. 3. Voltage steps revealed nearly linear current‐voltage relationships both in the absence and presence of CO2‐HCO3‐, with an intersection at the assumed reversal potential of the HCO(3‐)‐dependent current. These results suggest a cotransport stoichiometry of 2HCO3‐: 1 Na+. The HCO(3‐)‐dependent current could be inhibited by diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS). 4. Simultaneous recording of current and intracellular pH showed a correlation of the maximal acid‐base flux with the transient HCO(3‐)‐dependent current during voltage steps in the presence of CO2‐HCO3‐. The maximum rate of acid‐base flux and the HCO(3‐)‐dependent peak current showed a similar dependence on membrane voltage. Lowering the external pH from 7.4 to 7.0 produced an inward current, which increased twofold in the presence of CO2‐HCO3‐. This current was largely inhibited by DIDS, indicating outward‐going electrogenic Na(+)‐HCO3‐ cotransport during external acidification. 5. When external Na+ was replaced by Li+, a similar outward current and intracellular alkalinization were observed in the presence of CO2‐HCO3‐. The Li(+)‐induced intracellular alkalinization was not inhibited by amiloride, a blocker of Na+(Li+)‐H+ exchange, but was sensitive to DIDS. These results suggest that Li+ could, at least partly, substitute for Na+ at the cotransporter site. 6. Our results indicate that the Na(+)‐HCO3‐ cotransport produces a current across the glial cell membrane in both directions with a reversal potential near the membrane resting potential, rendering pHi a function of the glial membrane potential.
Microscopic heterogeneity in unitary N‐type calcium currents in rat sympathetic neurons.
Authors: Rittenhouse A R , Hess P
Published: December 2014

1. Single N‐type calcium (Ca2+) channels in rat superior cervical ganglion neurons display complex patterns of activity in both inactivating and non‐inactivating gating modes. Unitary currents were elicited by holding the patch at ‐90 mV and stepping to +30 mV for 740 ms. Barium (110 mM) was used as the charge carrier. The dihydropyridine agonist (+)‐202‐791 was included in the bath to ensure that single channel recordings showed no L‐type Ca2+ channel mode 2 activity. Using this protocol, we characterized three additional patterns of N‐type Ca2+ channel activity named: (1) LLP for large unitary current amplitude (i = ‐0.92 pA) and low open probability (Po = 0.26); (2) SLP for small unitary current amplitude (i = ‐0.77 pA) and low open probability (Po = 0.25); and (3) SHP for its small unitary current (i = ‐0.77 pA) and higher open probability (Po = 0.39). 2. Transitions among these patterns of activity occur more slowly than transitions between closed and open states, resulting in significant clustering of like sweeps. Thus, the complicated gating of single N‐type Ca2+ channels can be dissected into multiple, independent modes, each with the same reproducible pattern of activity. 3. This heterogeneous activity is not unique to sympathetic neurons, for inactivating (4), non‐inactivating (4), SLP (4) and SHP (3 patches) gating modes were also observed in cell‐attached patch recordings (n = 4) of single N‐type Ca2+ channels in differentiated phaeochromocytoma (PC12) cells. 4. The 1568 sweeps from four single N‐type Ca2+ channel recordings that used the same voltage protocol were categorized by mode to determine the frequency of occurrence of each. Of the 54% of sweeps that showed activity, 42% were inactivating and 58% were non‐inactivating. The contribution by each mode to the sustained current was estimated using the equation: I = NPoi, where N is the frequency of occurrence of each mode and Po and i are the mean values of open probability and unitary current amplitude respectively. The LLP mode contributed 18%, the SLP mode 16%, and the SHP mode 66% of the sustained whole cell N‐type Ba2+ current. 5. The variability in the incidence among these modes in other cell types may resolve some of the controversy surrounding the characterization of N‐ and L‐type whole cell Ca2+ current components in peripheral neurons. In addition, the number of different modes provides a source of plasticity that may be a target of modulation by neurotransmitters and cellular signals.