bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium homeostasis in identified rat gonadotrophs.
Authors: Tse A , Tse F W , Hille B
Published: December 2014

1. Whole‐cell voltage clamp was used in conjunction with the fluorescent Ca2+ indicator indo‐1 to measure extracellular Ca2+ entry and intracellular Ca2+ concentrations ([Ca2+]i) in rat gonadotrophs identified with the reverse haemolytic plaque assay. 2. Depolarizations to potentials more positive than ‐40 mV elicited inward Ca2+ current (ICa) and transient elevations of [Ca2+]i. 3. The relationship between [Ca2+]i elevations and Ca2+ entry with different Ca2+ buffer concentrations in the pipette showed that endogenous Ca2+ buffers normally bind approximately 99% of the Ca2+ entering the cell. 4. With [Ca2+]i elevations less than 500 nM, decay of [Ca2+]i could be approximated by an exponential whose time constant increased with the concentration of exogenous Ca2+ buffers. 5. Inhibitors of intracellular Ca(2+)‐ATPases, thapsigargin, cyclopiazonic acid (CPA) and 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone (BHQ), caused [Ca2+]i to rise. Application of BHQ during [Ca2+]i oscillations induced by gonadotrophin‐releasing hormone (GnRH) terminated the oscillation in a slowly decaying elevation. BHQ slowed the decay of depolarization‐induced [Ca2+]i elevations about 3‐fold. 6. Taking into account the Ca2+ buffering properties of the cytoplasm permitted estimation of the fluxes and rate constants for Ca2+ movements in gonadotrophs. The intracellular store is a major determinant of Ca2+ homeostasis in gonadotrophs.
Chloride currents of single mitochondria‐rich cells of toad skin epithelium.
Authors: Larsen E H , Harvey B J
Published: December 2014

1. Mitochondria‐rich (MR) cells in free suspension were obtained by collagenase and trypsin treatments of toad skin epithelium and studied by whole‐cell voltage clamp and membrane current fluctuation analysis. 2. Cells studied with a 100 microM amiloride and 5 mM Ba2+ Ringer solution on the outside and 10 mM Cs+ in the pipette generated large membrane currents with reversal potentials varying in a Nernstian way with pipette [Cl‐]. 3. The membrane chloride currents were activated in excess of Goldman‐Hodgkin‐Katz rectification by cell depolarization and clamping to positive cell potentials (VC). The resulting Cl‐ permeability was presented as an S‐shaped function of membrane potential with half‐maximal activation in the range 0 mV < VC < 50 mV. 4. The power density spectrum of Cl‐ current fluctuations could be fitted with a single Lorentzian component with a corner frequency, fc, of 34.9 +/‐ 2.6 Hz, and a low frequency asymptote, S(o), or 14.6 +/‐ 1.3 pA2 s per cell (mean +/‐ S.E.M.; VC = 25 mV, ECl = 0 mV, n = 6). 5. The lower‐limit single channel conductance, gamma Cl(1 ‐ Po), was 128 +/‐ 9 pS (VC = 25 mV, n = 6), where Po is the open channel probability. With Po = 0.5, this result indicates that Cl‐ channels of large unitary conductance (200‐300 pS) are present in the mitochondria‐rich cell membrane.
In vivo release of glutamate in nucleus tractus solitarii of the rat during hypoxia.
Authors: Mizusawa A , Ogawa H , Kikuchi Y , Hida W , Kurosawa H , Okabe S , Takishima T , Shirato K
Published: December 2014

1. An attempt has been made to test the hypothesis that, in the caudal part of nucleus tractus solitarii (NTS) where carotid sinus nerve (CSN) afferents project, L‐glutamate (Glut) modulates the hypoxic ventilatory response. 2. Unanaesthetized, peripherally chemodenervated (carotid body denervated; CBD) and sham‐operated, freely moving rats were used. During peripheral chemoreceptor stimulation by hypoxia (10% O2 for 30 min) or doxapram (Dox) infusion (2 mg kg‐1 (30 min)‐1), ventilation was recorded and successively, under the same conditions, the extracellular Glut concentration ([Glut]o) in the caudal NTS was measured by in vivo microdialysis. [Glut]o was also measured during hyperoxic hypercapnia (10% CO2‐30% O2 for 30 min). 3. Furthermore, the effects on ventilation of exogenous Glut, the NMDA (N‐methyl‐D‐aspartate) receptor antagonist MK‐801 or the ionotropic receptor antagonist kynurenate microinjected into the caudal NTS were investigated in sham‐operated rats. 4. In sham‐operated rats, both ventilation and [Glut]o in NTS were increased during peripheral chemoreceptor stimulation. On the other hand, no increases in either ventilation or Glut release were observed in CBD rats. In spite of ventilatory augmentation during hypercapnia, no response of [Glut]o to hypercapnia was observed in either group. 5. Local Glut application into NTS increased ventilation. Pretreatment with MK‐801 or kynurenate reduced the hypoxic ventilatory response. This reduction in ventilation was mainly due to the decrease in tidal volume. 6. These results suggest that hypoxia induced the release of Glut in NTS and that this effect was mediated by arterial chemosensory input.
Mode of regulation by G protein of the ATP‐sensitive K+ channel in guinea‐pig ventricular cell membrane.
Authors: Ito H , Vereecke J , Carmeliet E
Published: December 2014

1. The effect of G protein activation on the ATP‐sensitive K+ (K+ATP) channel was examined in inside‐out patches from guinea‐pig ventricular myocytes. At low (0.3 mM) intracellular ATP concentration ([ATP]i) in the bathing solution, in the absence of agonists in the pipette, guanosine 5'‐O‐(3‐thiotriphosphate) (GTP gamma S) or AlF4‐ applied to the intracellular side of the patch membrane gradually activated the K+ATP channel. The activation by GTP gamma S was irreversible, although high [ATP]i could completely close the channel. 2. In ATP‐free media GTP gamma S did not increase further the activity of the fully active channel, and was unable to reactivate the channel in the non‐operative state after rundown. [ATP]i‐channel activity curves constructed before and after GTP gamma S application demonstrated that GTP gamma S shifts the half‐inhibitory [ATP]i from 19.5 to 110 microM without changing the Hill coefficient. 3. When acetylcholine or adenosine was included in the pipette, intracellular GTP reversibly activated the K+ATP channel which was partially inhibited by [ATP]i. 4. These results indicate that G protein may stimulate myocardial K+ATP channels in the operative state by reducing the potency of ATP inhibition. The possible coupling of the G protein with muscarinic as well as A1 adenosine receptors is suggested.
Sulphorhodamine‐labelled cells in the neonatal rat spinal cord following chemically induced locomotor activity in vitro.
Authors: Kjaerulff O , Barajon I , Kiehn O
Published: December 2014

1. Sulphorhodamine 101, a fluorescent dye and newly identified activity marker, was used to localize potential spinal locomotor networks in the neonatal rat spinal cord. 2. Preparations of the spinal cord with one entire hindlimb attached or the spinal cord in isolation were kept in vitro. Spinal locomotor activity was maintained chemically with NMDA (5‐7.5 microM), in combination with 5‐HT (7.5‐20 microM), for 4‐4.5 h in the presence of 0.0001‐0.0005% sulphorhodamine 101. Matched non‐locomoting controls were exposed to the dye in the absence of transmitters for a comparable time. Transverse sections of the lumbar spinal cord (L1‐L6) were screened for rhodamine emission using an epifluorescence microscope. 3. In hindlimb‐attached locomoting preparations with intact dorsal roots, labelled cells were found on the leg side in the dorsal horn (mainly laminae II‐IV), in the intermediate grey (lamina VI‐VII) and around the central canal (lamina X). Dorsal rhizotomy was performed on the leg side, to prevent synaptic activity due to afferent inflow. This largely reduced the number of labelled cells in the dorsal horn and in the lateral part of the intermediate grey matter. A further reduction of labelling in these areas was seen after complete isolation of the cord or when compared to the legless side, with the majority of labelled cells persisting in a bilateral cluster close to the central canal and in the medial intermediate grey. Few labelled cells were observed in non‐locomoting preparations. The intensity of motoneuronal labelling was variable.(ABSTRACT TRUNCATED AT 250 WORDS)
Load dependence of changes in forearm and peripheral vascular resistance after acute leg exercise in man.
Authors: Piepoli M , Isea J E , Pannarale G , Adamopoulos S , Sleight P , Coats A J
Published: December 2014

1. It is known that acute exercise is often followed by a reduction in arterial blood pressure. Little is known about the time course of the recovery of the blood pressure or the influence of the intensity of the exercise on this response. Controversy exists, in particular, concerning the changes in peripheral resistance that occur during this period. 2. Eight normal volunteers performed, in random order on separate days, voluntary upright bicycle exercise of three different intensities (maximal, moderate and minimal load) and, on another day, a control period of sitting on a bicycle. They were monitored for 60 min after each test. 3. Diastolic pressure fell after maximal exercise at 5 min (‐15.45 mmHg) and 60 min (‐9.45 mmHg), compared with the control day. Systolic and mean pressure also fell (non‐significantly) after 45 min; heart rate was significantly elevated for the whole hour of recovery (at 60 min, +7.23 beats min‐1). No changes in post‐exercise blood pressure and heart rate were observed on the days of moderate and minimal exercises. 4. An increase in cardiac index was observed after maximal exercise compared with control (at 60 min, 2.6 +/‐ 0.3 vs. 1.9 +/‐ 0.2 l min‐1 m‐2). This was entirely accounted for by the persistent increase in heart rate, with no significant alteration in stroke volume after exercise on any day.(ABSTRACT TRUNCATED AT 250 WORDS)
Flash photolysis studies of the localization of calcium release sites in rat parotid isolated acinar cells.
Authors: Hassoni A A , Gray P T
Published: December 2014

1. The temporal relationship between cytosolic free Ca2+ concentration ([Ca2+]i) and activation of membrane current responses in single rat parotid acinar cells has been examined. Activation of muscarinic receptors by carbachol (CCh) at ‐40 mV (midway between EK and ECl under our experimental conditions) frequently evoked biphasic current responses, application of 2 microM CCh leading to rapid activation of an inward current followed by a slower outward current. 2. Photochemical release of inositol 1,4,5‐trisphosphate (InsP3), from ‘caged’ InsP3, by a brief near‐UV flash, evoked similar biphasic current responses at ‐40 mV. In contrast, elevation of [Ca2+]i by photolysis of the caged calcium compound nitr‐5 at ‐40 mV activated only monophasic current responses. 3. These results can be explained by a model in which the InsP3‐sensitive Ca2+ release sites are localized at the luminal pole of the cell, combined with a relative preponderance of Ca(2+)‐activated Cl‐ channels at that pole, and a relative preponderance of Ca(2+)‐activated K+ channels at the basal end.
Effects of pH and inorganic phosphate on rigor tension in chemically skinned rat ventricular trabeculae.
Authors: Smith G L , Steele D S
Published: December 2014

1. Ventricular trabeculae from rat heart were chemically skinned with Triton X‐100, which disrupts all cellular membranes including the sarcoplasmic reticulum. In the effective absence of Ca2+ (10(‐9) M), trabeculae developed a maintained rigor contracture when ATP was withdrawn from the bathing solution. 2. The final level of tension obtained following withdrawal of ATP was dependent upon the pH of the bathing solution during development of rigor. Rigor tension at pH 5.5 was 10.1 +/‐ 0.9% (n = 8, mean +/‐ S.E.M.) of that at pH 7.0. Bathing the preparation in alkaline solution increased rigor force. At pH 8.0, rigor force increased to 218 +/‐ 6.7% (n = 4) of control responses developed at pH 7.0. The rate of development of rigor tension increased as the pH of the bathing solution was increased. Once established, rigor tension was unaffected by subsequent changes in pH. These effects of pH were fully reversible within the range 5.5‐8.0. 3. The final level of rigor tension was slightly reduced when inorganic phosphate (P(i)) was included in the bathing solution prior to withdrawal of ATP. P(i) concentrations of 10, 20 and 30 mM reduced rigor tension to 87 +/‐ 2, 83 +/‐ 3 and 82 +/‐ 4% respectively. There was no significant effect of P(i) on the rate of development of rigor. The effect of P(i) at pH 6.0 was not significantly different from that observed at the control pH of 7.0. 4. These results suggest that the fall of intracellular pH and, to a lesser extent, the rise in [P(i)] that occurs during ischaemia will partially inhibit the development of a rigor contracture.
Mechanism for reactivation of the ATP‐sensitive K+ channel by MgATP complexes in guinea‐pig ventricular myocytes.
Authors: Furukawa T , Virág L , Furukawa N , Sawanobori T , Hiraoka M
Published: December 2014

1. A mechanism underlying reactivation of the adenosine 5'‐triphosphate‐sensitive K+ (K+ATP) channels by MgATP complexes after run‐down was examined in guinea‐pig ventricular myocytes using the patch‐clamp technique with inside‐out patch configuration. 2. After run‐down was induced by exposure of the intracellular side of the membrane patch to Ca2+ (1 mM), channel activity was reactivated by exposure and subsequent wash‐out of MgATP (2 mM). Addition of inhibitors of various serine/threonine protein kinases to the MgATP solution did not suppress reactivation of the run‐down channels. 3. Non‐ or poorly hydrolysable ATP analogues were unable to reactivate run‐down channels. 4. The degree of channel recovery was dependent upon the duration of MgATP exposure. The apparent half‐activation value (K1/2) of MgATP for reactivation was decreased with increasing exposure time. 5. Various products of ATP hydrolysis were unable to reactivate run‐down channels except a relatively low concentration (100 microM) of ADP exposure. 6. Other nucleotide triphosphates, in the presence of Mg2+, were unable to reactivate rundown channels. 7. Fluorescein 5‐isothiocyanate (50 microM), which interacts with lysine residues of the nucleotide‐binding site on various ATPases, inhibited K+ATP channel activity. After wash‐out, channel activity recovered only slightly. 8. These data suggest that the hydrolysis of ATP is important for reactivation of run‐down K+ATP channels but that protein phosphorylation by serine/threonine protein kinases may not be involved. Since no products of ATP hydrolysis could reproduce MgATP‐induced channel reactivation and since the degree of channel recovery was dependent upon the duration of MgATP application, the hydrolysis energy appears to be utilized for channel reactivation.
Na(+)‐H+ exchange in frog early distal tubule: effect of aldosterone on the set‐point.
Authors: Cooper G J , Hunter M
Published: December 2014

1. Intracellular pH (pHi) regulation was investigated in frog early distal tubule. Single tubules were dissected and perfused, such that the compositions of apical and basolateral solutions could be varied independently. pHi was measured using the fluorescent probe 2',7'‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF). 2. Brief exposure to NH4+ on the basolateral aspect of the tubules elicited an intracellular acidification, followed by an active recovery. The recovery was inhibited by amiloride and its analogue 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA) when added to the basolateral, but not the apical, solution. Omission of Na+ from the basolateral solution alone completely inhibited pHi recovery. Thus the Na(+)‐H+ exchangers appear to be located on the basolateral membrane. 3. Neither amiloride nor EIPA had any effect on pHi under control conditions, suggesting that the activity of the Na(+)‐H+ exchangers at the resting pHi is low. However, removal of basolateral Na+ caused an acidification that was blocked by amiloride, indicating that the Na(+)‐H+ exchangers can be activated from the resting state. 4. Intrinsic buffering power (beta i) was determined by stepwise removal of ammonium from the cells in Na(+)‐free conditions, to prevent pH regulation, and in the presence of Ba2+ and furosemide (frusemide), to inhibit ammonium transport. beta i was a function of pHi, increasing as pHi decreased. 5. Proton efflux was calculated during the recovery from an acid load in tubules from normal and K(+)‐loaded frogs and in tubules which had been incubated for 30 min with aldosterone. Potassium loading produces a chronic increase in plasma aldosterone. Both acute and chronic aldosterone treatment caused an intracellular alkalinization. This was due to an alkaline shift in the set‐point of the basolateral Na(+)‐H+ exchanger, with no change in the density and/or turnover rate.
Primary afferent depolarization of cat pudendal afferents during micturition and segmental afferent stimulation.
Authors: Angel M J , Fyda D , McCrea D A , Shefchyk S J
Published: December 2014

1. This investigation examined primary afferent depolarization (PAD) of perineal afferents during micturition and evoked by electrical stimulation of perineal, hindlimb cutaneous and muscle‐nerves. PAD was inferred from changes in excitability of spinal terminals of single afferents in decerebrate and chloralose‐anaesthetized paralysed male cats. Observations were made on perineal afferent fibres travelling in the sensory branch of the pudendal (SPud) and superficial perineal (SPeri) nerves. 2. Micturition was evoked by distension of the bladder and excitability changes were measured in twenty‐seven SPud afferents. In ten afferents, there was evidence of PAD during micturition. The time course of PAD was similar to the period of decreased activity in sphincter muscle efferents during micturition. In four afferents, there was decreased excitability during voiding that was interpreted as removal of tonic PAD. In the remaining thirteen afferents there were no detectable changes in excitability. Bladder distension in the absence of micturition failed to change the excitability of any SPud afferents tested. 3. Almost all SPud afferents were subject to PAD upon stimulation of cutaneous nerves. Superficial perineal, long saphenous, caudal cutaneous sural and the predominantly cutaneous posterior tibial nerves were particularly effective in evoking PAD. While group I strength stimulation of hindlimb muscle‐nerves produced PAD of some SPud fibres, group II stimulation often increased the magnitude or incidence of PAD. The patterns and magnitude of PAD observed in SPeri afferents were similar to those observed in SPud afferents. 4. Since some SPud afferents were subject to PAD during micturition, PAD is probably one mechanism responsible for suppression of sphincter reflexes during micturition. Additional roles of PAD of perineal afferents evoked by activation of hindlimb cutaneous and muscle afferents are discussed.
Effect of pulmonary lymphatic obstruction on respiratory rate and airway rapidly adapting receptor activity in rabbits.
Authors: Ravi K , Bonham A C , Kappagoda C T
Published: December 2014

1. The effects on respiratory rate of obstruction of pulmonary lymph flow, reduction of plasma protein concentration and a combination of the two procedures were examined in anaesthetized rabbits. The former was achieved by raising the pressure in a pouch created from the right external jugular vein and the latter by batch plasmapheresis. 2. In spontaneously breathing rabbits, neither pulmonary lymphatic obstruction (n = 6) nor plasmapheresis (n = 5) produced a significant change in respiratory rate. However, their combination (n = 8) produced a significant increase in respiratory rate (P < 0.05). 3. Cooling of the cervical vagi to 8‐9 degrees C (n = 4) and vagotomy (n = 7) abolished this response. 4. There was a significant increase in the activity of the airway rapidly adapting receptors (RARs; n = 9) during pulmonary lymphatic obstruction, plasmapheresis and their combination (P < 0.05). 5. It is concluded that in the rabbit, obstruction of lymphatic drainage from the lung after plasmapheresis causes a reflex increase in respiratory rate. The afferent pathway for this reflex response lies in the vagus nerve and the RARs are likely to be the receptors involved in this response.
Electrophysiological studies of the interaction between ventricular myocardium and coronary artery in monkeys.
Authors: Mekata F
Published: December 2014

1. The electrical influence of the coronary arteries on ventricular muscle was investigated using strips of ventricle that included a section of coronary artery (cardiac preparation) and isolated coronary arteries dissected from the ventricle (arterial preparation). 2. In cardiac preparations, a hyperpolarizing response was recorded from the epicardial surface of the ventricular muscle when acetylcholine (ACh) was added to the organ bath, on condition that the internal diameter of the coronary artery was between 0.15 and 0.6 mm, that the vessel ran at a depth of 0.2 mm or less below the surface of the preparation, and that the recording microelectrode was immediately adjacent to the artery. 3. ACh‐induced hyperpolarization was not detected in cardiac preparations which had no detectable arteries, or at sites distant from visible arteries. 4. In arterial preparations, a similar hyperpolarizing response was evoked by ACh in all vessels with an i.d. of 0.15‐1.2 mm. 5. In a preparation combining ventricular muscle and a strip of coronary artery (with the vascular endothelium in direct contact with the epicardial surface of the ventricular myocardium), hyperpolarization was also observed from the ventricular muscle after application of ACh. 6. The hyperpolarizing response of the ventricular myocardium in the cardiac preparation and in combined preparations of ventricular muscle and coronary artery was weakened or abolished by removal of the arterial endothelium. 7. These results indicate that some substance released from the coronary arterial endothelium after stimulation by ACh induces hyperpolarization of the ventricular myocardium.
Cardiac contraction and intramyocardial venous pressure generation in the anaesthetized dog.
Authors: Vergroesen I , Han Y , Goto M , Spaan J A
Published: December 2014

1. Two hypotheses relating to the influence of contraction of the heart on coronary venous pressure (Pv) were tested. The first assumes a direct transmission of left ventricular pressure (PLV). According to the alternative hypothesis the Pv is caused by cyclical changes in the elastance of the surrounding tissue. 2. A small epicardial vein was cannulated retrogradely in eight open‐chest dogs deeply anaesthetized with fentanyl. The duration of diastoles was varied after induction of a heart block with formaldehyde. Coronary arterial inflow and perfusion pressure were controlled by a perfusion system connected to the left main coronary artery by a Gregg cannula. Stopped‐flow Pv was studied with intrinsic coronary tone (IT) and after maximal dilatation with adenosine. 3. The Pv pulse in the first contraction after a long diastole was not significantly correlated to the PLV pulse, with a slope of 0.5, in any dog, either with IT or during adenosine treatment. Comparing the first contraction after the long diastole with the last beat before, systolic Pv pulse decreased significantly in seven out of eight dogs, but systolic PLV pulse increased in five dogs and was unaltered in three dogs in both conditions. In contrast, end‐diastolic Pv was significantly correlated to the systolic Pv in each individual animal under either condition. 4. The results indicate that pressure generation in the small coronary veins can be explained on the basis of the time‐varying elastance hypothesis and that a direct transmission of PLV to Pv is absent.
The quantal size at retinogeniculate synapses determined from spontaneous and evoked EPSCs in guinea‐pig thalamic slices.
Authors: Paulsen O , Heggelund P
Published: December 2014

1. To determine the quantal size at retinogeniculate synapses, spontaneous and evoked excitatory postsynaptic currents (EPSCs) were recorded in twelve neurones of the dorsal lateral geniculate nucleus in guinea‐pig thalamic slices using the whole‐cell patch‐clamp technique. We limited our study to the fast non‐N‐methyl‐D‐aspartate (NMDA) component of the EPSCs by adding the NMDA receptor antagonist DL‐2‐amino‐5‐phosphonovaleric acid to the perfusion medium. 2. Spontaneous EPSCs occurred at a frequency between 0.5 and 6.6 Hz (mean 2.5 Hz). The modal value of the peak conductance change of spontaneous excitatory events varied between cells from 102 to 179 pS. 3. EPSCs were evoked by electrical stimulation in the optic tract. The peak conductance change of EPSCs evoked by stimulation of a putative single input fibre ranged from 0.6 to 3.4 nS (mean 1.7 nS). 4. To resolve the quantal components of evoked EPSCs the external Ca2+ concentration was reduced and the external Mg2+ concentration increased for four cells. In this condition failures occurred and the amplitude histograms were multimodal with approximately equidistant peaks. 5. These multimodal histograms could be fitted by a sum of Gaussian functions with mean values corresponding to integer multiples of the modal value of the spontaneous EPSCs for the same cell. Thus, the quantal size of evoked EPSCs was the same as the modal value of spontaneous EPSCs. The mean of the apparent quantal conductance change was 138 pS. The estimated number of quanta released by stimulating a putative single input fibre in the control condition ranged from 4 to 27 (mean 13).(ABSTRACT TRUNCATED AT 250 WORDS)
The separation of exocytosis from endocytosis in rat melanotroph membrane capacitance records.
Authors: Zupancic G , Kocmur L , Veranic P , Grilc S , Kordas M , Zorec R
Published: December 2014

1. Using the patch‐clamp technique, we have monitored the secretory activity of single rat melanotrophs. Changes in membrane capacitance (Cm) were measured to detect small discrete femtofarad steps. These are believed to be due to interactions between single secretory organelles (granules) and plasmalemma. 2. A new approach was introduced to measure the amplitude of discrete steps in Cm. Records of Cm were converted into time derivatives, where discrete steps appeared as transients. A transient due to a 2 fF discrete step in Cm was easily distinguished from random noise, since the probability of such a transient being due to random noise was less than 0.01. To distinguish apparent steps from noise the computer‐based analysis employed a threshold of 3 times the standard deviation of the noise time derivative (dCm/dt). A phase diagram was created by plotting dCm/dt versus Cm, from which the magnitude and direction of transients were determined. Transients due to 2 fF steps (equivalent to a signal‐to‐noise ratio of 1) were detected with a reliability of 100%, whereas steps of 1 fF were detected with a reliability of more than 60%. The amplitude of false steps detected by the program was less than 1 fF, and the frequency of false detections of 0.075 S‐1 was equal for exocytotic and endocytotic events. 3. Electron microscopy was used to measure secretory organelle size and an immunogold technique was used to label the electron micrographs with an anti‐adrenocorticotrophin (ACTH) antibody. Secretory organelles in cultured and non‐cultured cells were of similar diameter. All sizes of secretory granules appear to contain ACTH, since secretory organelles of similar diameter stained positively with the anti‐ACTH antibodies. 4. Small discrete steps in Cm, recorded with the whole‐cell configuration and loosely buffered cytosolic calcium, were similar to the estimated Cm of secretory organelles from morphological data. Thus, measured discrete steps in Cm reflect interactions between single organelle size and plasma membrane. Exocytotic and endocytotic steps were found to be of similar size. 5. To separate exocytosis from endocytosis in Cm records, we assumed that the rates of exocytosis and endocytosis were related to the respective frequencies of discrete steps in Cm. A relationship between the frequency of exocytotic, but not endocytotic events, and the rate of change in Cm was observed. Thus, under our experimental conditions, an increase in Cm could be explained by an increased rate of exocytosis in rat melanotrophs.
Vagal influence on the motility of the feline jejunum.
Authors: Gustafsson B I , Delbro D S
Published: December 2014

1. The effects of electrical stimulation of the peripheral end of the cervical vagal nerve on jejunal motility were investigated in anaesthetized cats, pretreated with guanethidine, with sectioned splanchnic nerves and ligated adrenal vessels. Motility was monitored as volume changes of an intraluminal balloon. 2. Vagal stimulation elicited frequency‐dependent hypermotility with a short latency. Relaxatory events were also observed, which could indicate the presence of a non‐adrenergic inhibitory pathway. 3. After atropine treatment, contractions and relaxations could still be elicited. The former were compared to cholinergic contractions and showed a lower maximal amplitude and a longer latency to onset. Moreover, they were antagonized by 80‐100% by the opioid receptor antagonist, naloxone. 4. Vagal stimulation after hemicholinium, given in order to deplete the preganglionic acetylcholine content, elicited naloxone‐sensitive contractions. This suggests that a subpopulation of the vagal preganglionic fibres is non‐cholinergic. 5. Isolation of the balloon‐containing segment did not qualitatively alter the responses, indicating that the vagal fibres reach the small intestine via the paravascular mesenteric nerves. 6. It is concluded that cholinergic and non‐adrenergic, non‐cholinergic (NANC) contractions, as well as relaxations, could be elicited by efferent vagal stimulation. The NANC contractions seem to result from the activation of opioid receptors causing disinhibition of a tonic neurogenic restraint on the gut muscle.
Contribution of chloride conductance increase to slow EPSC and tachykinin current in guinea‐pig myenteric neurones.
Authors: Bertrand P P , Galligan J J
Published: December 2014

1. Single electrode voltage clamp recordings were obtained from myenteric neurones of guinea‐pig ileum in vitro. Slow excitatory postsynaptic currents (sEPSCs) were elicited by focal stimulation of interganglionic nerve strands in twenty‐four of thirty neurones more than 30 min after impalement. In seventeen of twenty‐four neurones, sEPSCs were associated with a conductance decrease and reversed polarity at ‐96 +/‐ 3 mV (near the reversal potential for potassium, EK); this response was due to inhibition of resting potassium conductance, gK. In seven of twenty‐four neurones, there was either no net conductance change or a biphasic conductance change during the sEPSC; a reversal potential for peak currents could not be determined. 2. Application of senktide (3 microM), a neurokinin‐3 receptor agonist, caused an inward current in forty‐one of fifty‐three neurones more than 30 min after impalement. In twenty of forty‐one neurones, senktide‐induced currents were due to inhibition of resting gK. In eleven of forty‐one neurones there was either no net conductance change or a biphasic conductance change; a reversal potential for peak currents could not be determined. In ten out of forty‐one neurones, senktide‐induced currents were associated with a conductance increase (ginc); the estimated reversal potential was ‐17 +/‐ 3 mV. 3. Application of forskolin (1 microM) caused an inward current that occluded the decrease in gK caused by senktide and the sEPSC. In neurones in which sESPCs and senktide responses were associated with an unclear or biphasic conductance change, forskolin did not reduce the peak current and residual currents were usually associated with a ginc. 4. In neurones in which senktide‐induced currents were associated with a ginc, reducing extracellular Cl‐ to 13 mM reduced senktide‐induced currents by 79%. Reducing extracellular Na+, or adding tetraethylammonium (TEA, 50 mM), cobalt (2 mM) or picrotoxin (30 microM) did not change senktide‐induced currents. The chloride transport/channel blockers niflumic acid and mefenamic acid (both at 100 microM) blocked senktide‐induced currents. It was concluded that senktide increases chloride conductance (gCl). 5. Chord conductance measurements made between ‐70 and ‐90 mV during sEPSCs were used to determine the contribution of an increase in gCl to sEPSCs. These measurements indicated that the peak sEPSC is composed of a 90% decrease in gK and a 10% increase in gCl. Similar data were obtained from measurements made during senktide responses.(ABSTRACT TRUNCATED AT 400 WORDS)
The effect of cardiac glycosides on the Na+ pump current‐voltage relationship of isolated rat and guinea‐pig heart cells.
Authors: Hermans A N , Glitsch H G , Verdonck F
Published: December 2014

1. Whole‐cell recording from isolated rat and guinea‐pig ventricular myocytes revealed a change of the cardiac Na+ pump current (Ip)‐voltage (V) relationship by cardiac glycosides, specific inhibitors of the Na(+)‐K+ pump. 2. Dihydro‐ouabain (DHO) diminished Ip in rat ventricular cells at 0 mV in a concentration‐dependent manner. 3. The concentration‐response curve of Ip inhibition caused by DHO was shifted to higher [DHO] at higher extracellular K+ concentrations ([K+]o) or at more negative membrane potentials. 4. In rat myocytes, DHO immediately flattened the normalized cardiac Ip‐V curve and evoked or enhanced a region of negative slope. 5. Ouabain, at concentrations which caused a comparable inhibition of Ip, exerted DHO‐like effects on the Ip‐V relationship of rat ventricular myocytes. However, the effects developed more slowly. 6. A slowly developing alteration of the Ip‐V curve was also observed upon application of DHO to guinea‐pig ventricular cells. The range of [DHO] used was about 100‐fold lower than that applied to rat ventricular cells, but was equally effective for Ip inhibition. 7. Increasing the K+ concentration of DHO‐containing media affected the existing equilibrium of DHO binding to the cardiac Na(+)‐K+ pump. A new equilibrium was reached within about 3 s in rat ventricular myocytes, but only within about 50 s in guinea‐pig ventricular cells under the experimental conditions chosen. 8. It is concluded that the changes of the cardiac Ip‐V curve induced by cardiac glycosides are mediated by voltage‐dependent variations of the local [K+]o at the K+ binding sites of the Na(+)‐K+ pump in an ‘access channel’. The variations were estimated by means of the Boltzmann equation. The estimations agreed with those derived from the measured DHO binding to the Na(+)‐K+ pump at various [K+]o. A new equilibrium of glycoside binding to the pump is established at the altered [K+]o. The time necessary to reach the new binding equilibrium varies with the cardioactive steroid, its concentration and the glycoside sensitivity of the cardiac cells.
Facilitation of an NMDA receptor‐mediated EPSP by paired‐pulse stimulation in rat neocortex via depression of GABAergic IPSPs.
Authors: Metherate R , Ashe J H
Published: December 2014

1. Tight seal, whole‐cell recordings from auditory cortex in vivo and in vitro were obtained to investigate modification of N‐methyl‐D‐aspartate (NMDA) receptor‐mediated synaptic activity by paired‐pulse afferent stimulation. 2. In recordings from urethane‐anaesthetized rats (at 37 degrees C), or from cortical slices maintained in vitro (32 degrees C), afferent stimulation elicited a monosynaptic early EPSP and polysynaptic early and late IPSPs. In addition, a late EPSP could be elicited when the stimulus was preceded by an identical priming stimulus (interval approximately 200 ms). The late EPSP was attenuated by the NMDA receptor antagonist DL‐2‐amino‐5‐phosphonovalerate (APV, 50 microM). 3. Bath application of the gamma‐aminobutyric acid‐B (GABAB) receptor antagonist 3‐amino‐2‐(4‐chlorophenyl)‐2‐hydroxy‐propylsulphonic acid (2‐OH‐saclofen; 50 microM) attenuated the late IPSP and clearly revealed a late EPSP. However, 2‐OH‐saclofen had lesser effects on the second late EPSP elicited during paired‐pulse stimulation. Membrane depolarization in 2‐OH‐saclofen increased the magnitude of the early IPSP, which suppressed the late EPSP once again. Since pharmacological blockade of EPSPs revealed paired‐pulse depression of monosynaptically elicited early and late IPSPs, these data indicate that (1) both early and late IPSPs were capable of suppressing the late EPSP, and (2) these effects were reduced during paired‐pulse stimulation. 4. Pharmacological isolation of the late EPSP allowed testing of the direct effect of paired‐pulse stimulation. Application of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 20 microM), picrotoxin (10 microM) and 2‐OH‐saclofen (50 microM) isolated the late EPSP (onset, 3 ms; peak latency, 28 ms; peak amplitude, 7 mV; duration, 240 ms), which grew in magnitude with membrane depolarization and was largely (> 90%) blocked by APV. Paired‐pulse stimulation depressed the isolated late EPSP by 30%. 5. Thus, apparent paired‐pulse facilitation of the late EPSP is attributable to release from GABAergic inhibition, and not to direct facilitation. Facilitation of the late EPSP is a functional consequence of IPSP depression. The results indicate the importance of inhibition in regulating synaptic activity mediated by NMDA receptors.
Tetrodotoxin‐resistant persistent Na+ current underlying pacemaker potentials of fish gonadotrophin‐releasing hormone neurones.
Authors: Oka Y
Published: December 2014

1. Gonadotrophin‐releasing hormone (GnRH)‐immunoreactive terminal nerve (TN) cells show endogenous regular beating discharges, which may be related to their putative neuromodulator functions. The ionic mechanism underlying the pacemaker potential was studied using intracellular and patch‐pipette current clamp recordings from a whole brain in vitro preparation of a small fish brain. 2. The pacemaker potentials were resistant to 1.5‐3 microM tetrodotoxin (TTX) and were not affected by Ca2+ channel blockers (amiloride, Ni2+, Co2+, Cd2+) or in Ca(2+)‐free solution. In contrast, the pacemaker potentials were readily blocked by substituting tetramethylammonium or choline for Na+ in the perfusing solution, and the resting membrane potential became more hyperpolarized than the control level. 3. The present results suggest that the TTX‐resistant persistent Na+ current, INa(slow), supplies the persistent depolarizing drive and plays an important role in the generation of pacemaker potentials in TN GnRH cells.
Primary afferent‐evoked glycine‐ and GABA‐mediated IPSPs in substantia gelatinosa neurones in the rat spinal cord in vitro.
Authors: Yoshimura M , Nishi S
Published: December 2014

1. The possible roles of glycine and gamma‐aminobutyric acid (GABA) as inhibitory transmitters in the spinal dorsal horn were studied by intracellular recordings from substantia gelatinosa (SG) neurones in transverse slices of the adult rat spinal cord which retained an attached dorsal root. 2. Stimulation of primary afferent A delta fibres evoked an initial excitatory postsynaptic potential (fast EPSP) followed by a short and/or long inhibitory postsynaptic potential (short and long IPSP). The short IPSP, observed in twenty‐nine SG neurones (37%) which received inhibitory inputs, had a mean latency of 3.6 ms and a half‐decay time of 11 ms, while the long IPSP had a mean latency of 3.7 ms and a half‐decay time of 42 ms and was observed in thirty‐seven SG neurones (47%). The remaining twelve neurones (16%) exhibited both short and long IPSPs. Both IPSPs reversed polarity at a membrane potential of ‐70 +/‐ 4 mV. The short IPSP was reversibly blocked by the glycine receptor antagonist strychnine (0.5‐2 microM), while the long IPSP was reversibly blocked by the GABAA receptor antagonist bicuculline (10‐20 microM). 3. In the majority of SG neurones, the short and long IPSPs appeared to be disynaptic and were blocked by the non‐N‐methyl‐D‐aspartic acid (non‐NMDA) receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 5‐10 microM). Both IPSPs were less sensitive (depressed by less than 30%) to the NMDA receptor antagonist DL‐2‐amino‐5‐phosphonovaleric acid (APV; 50‐100 microM). 4. In ten SG neurones (13%), bath‐applied glutamate (0.5‐2 mM) increased the amplitude and frequency of IPSPs, which had a similar time course to that of the short IPSP evoked by afferent A delta fibres. The glutamate‐induced short IPSPs were blocked by tetrodotoxin (0.5 microM) or strychnine (0.5‐1 microM). In twelve neurones (16%), glutamate hyperpolarized the membrane or increased the amplitude and frequency of IPSPs that had a similar time course to that of the A delta fibre‐evoked long IPSPs. The glutamate‐induced membrane hyperpolarization and long IPSPs decreased in amplitude with membrane hyperpolarization and reversed polarity at ‐70 +/‐ 6 mV. These hyperpolarizing responses were blocked by tetrodotoxin (0.5 microM) or bicuculline (10 microM). 5. These observations suggest that primary afferent A delta fibres activate glycinergic and/or GABAergic interneurones primarily through the non‐NMDA receptor subclass and result in inhibition of nearby SG neurones in the dorsal horn of the spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)
Antisense depletion of beta‐subunits modulates the biophysical and pharmacological properties of neuronal calcium channels.
Authors: Berrow N S , Campbell V , Fitzgerald E M , Brickley K , Dolphin A C
Published: December 2014

1. The role of the voltage‐dependent calcium channel (VDCC) beta‐subunit has been examined in cultured rat dorsal root ganglion neurones (DRGs). An antipeptide antibody was raised and this recognized proteins corresponding to beta‐subunits in a number of preparations. Immunoreactivity for the VDCC beta‐subunit in DRGs was concentrated on the internal side of the plasma membrane but was also present in the cytoplasm. 2. A twenty‐six‐mer antisense oligonucleotide with homology to all published VDCC beta‐subunit sequences was microinjected into individual cells, and maximal depletion of VDCC beta‐subunit immunoreactivity was observed after 108 h suggesting a half‐life for the turnover of the beta‐subunit greater than 50 h. No depletion was obtained with nonsense oligonucleotide. 3. The effect of depletion of VDCC beta‐subunit immunoreactivity on calcium channel currents in these cells was a reduction in amplitude of the maximum current of about 47%, and a shift in the voltage dependence of current activation of about +7 mV. These effects are the converse of those observed following co‐expression of cloned beta‐ with alpha 1‐subunits in oocytes and other expression systems. 4. The ability of the 1,4‐dihydropyridine (DHP) agonist Bay K 8644 to enhance calcium channel currents was greatly reduced following depletion of beta‐subunit immunoreactivity. This result is in agreement with the finding in several systems that co‐expression of the beta‐subunit with alpha 1‐subunits results in an increased number of DHP binding sites. 5. These results show that calcium channel beta‐subunits form part of native neuronal calcium channels and modify their biophysical and pharmacological properties.
Structure/function studies of mammalian Na‐H exchangers‐‐an update.
Authors: Yun C H , Tse C M , Nath S , Levine S L , Donowitz M
Published: December 2014

Four mammalian Na+/H+ exchangers have recently been cloned. Despite the structural similarity, these Na+/H+ exchanger isoforms differ in kinetic characteristics and their response to external stimuli. The present review deals with the recent developments in their functional characterization and their short‐term regulation.
Vesicular targeting and the control of ion secretion in epithelial cells: implications for cystic fibrosis.
Authors: Cunningham S A , Frizzell R A , Morris A P
Published: December 2014

Non‐polarized HT‐29 colonic epithelial cells fail to respond to cyclic AMP‐generating agonists with increases in plasma membrane anion conduction. Radio‐isotopic efflux and patch‐clamp experiments revealed that both undifferentiated and differentiated HT‐29 colonocytes possess volume‐ and Ca(2+)‐activated Cl‐ channels. However, only within the apical plasma membranes of the latter were cystic fibrosis transmembrane conductance regulator (CFTR) Cl‐ channels found. CFTR was expressed equally well in both non‐polarized and polarized colonocytes. Lack of CFTR‐dependent anion conduction was shown to be the result of CFTR retention within a peripheral intracellular compartment. We demonstrate that upon polarization, CFTR moves to the apical plasma membrane via a Brefeldin A (BFA)‐sensitive intracellular trafficking pathway.
The slow Ca(2+)‐activated K+ current, IAHP, in the rat sympathetic neurone.
Authors: Sacchi O , Rossi M L , Canella R
Published: December 2014

1. Adult and intact sympathetic neurones of the rat superior cervical ganglion maintained in vitro at 37 degrees C were analysed using the two‐electrode voltage‐clamp technique in order to investigate the slow component of the Ca(2+)‐dependent K+ current, IAHP. 2. The relationship between the after‐hyperpolarization (AHP) conductance, gAHP, and estimated Ca2+ influx resulting from short‐duration calcium currents evoked at various voltages proved to be linear over a wide range of injected Ca2+ charge. An inflow of about 1.7 x 10(7) Ca2+ ions was required before significant activation of gAHP occurred. After priming, the gAHP sensitivity was about 0.3 nS pC‐1 of Ca2+ inward charge. 3. IAHP was repeatedly measured at different membrane potentials; its amplitude decreased linearly with membrane hyperpolarization and was mostly abolished close to the K+ reversal potential, EK (‐93 mV). The monoexponential decay rate of IAHP was a linear function of total Ca2+ entry and was not significantly altered by membrane potential in the ‐40 to ‐80 mV range. 4. Voltage‐clamp tracings of IAHP could be modelled as a difference between two exponentials with tau on approximately 5 ms and tau off = 50‐250 ms. 5. Sympathetic neurones discharged only once at the onset of a long‐lasting depolarizing step. If IAHP was selectively blocked by apamin or D‐tubocurarine treatments, accommodation was abolished and an unusual repetitive firing appeared. 6. Summation of IAHP was demonstrated under voltage‐clamp conditions when the depolarizing steps were repeated sufficiently close to one another. Under current‐clamp conditions the threshold depolarizing charge for action potential discharge significantly increased with progressive pulse numbers in the train, suggesting that an opposing conductance was accumulating with repetitive firing. This frequency‐dependent spike firing ability was eliminated by pharmacological inhibition of the slow IAHP. 7. The IAHP was significantly activated by a single action potential; it was turned on cumulatively by Ca2+ load during successive action potential discharge and acted to further limit cell excitability.