bluuebunny/crossref_metadata_2025 huggingface dataset:
Somatostatin activates an inwardly rectifying K+ conductance in freshly dispersed rat somatotrophs.
Authors: Sims S M , Lussier B T , Kraicer J
Published: December 2014

1. Somatotrophs from enzymatically dispersed anterior pituitary glands of rats, enriched to greater than 94% purity by density gradient centrifugation, were studied within 16 h of isolation using patch clamp recording methods in the conventional whole‐cell and the perforated‐patch configurations. 2. Rhythmic oscillations of membrane potential gave rise to action potentials in thirty‐six of fifty‐two cells studied with the perforated‐patch technique. Membrane potential oscillated between approximately ‐70 mV and approximately ‐25 mV with an average frequency (mean +/‐ S.D.) of 0.9 +/‐ 0.9 s‐1. 3. The current‐voltage (I‐V) relationship of cells was linear at negative potentials with outward rectification at potentials positive to ‐40 mV. Evidence that the outward current was due to K+ channels came from the deactivation tail currents, which reversed direction close to the K+ equilibrium potential (EK). The reversal potential shifted 60 mV per tenfold change of external K+ concentration ([K+]o), as expected for K+ current. 4. Suppression of outward current by tetraethylammonium (TEA) provided additional evidence for K+ current. Cd2+ reduced outward current, suggesting the presence of Ca(2+)‐activated K+ conductance. 5. Depolarizing commands elicited transient inward Na+ current and a sustained Ca2+ current (ICa). ICa was recorded in isolation with Cs+ and TEA in the recording pipette and 10 mM‐Ba2+ as the charge carrier. Activation of ICa began at approximately ‐40 mV, with peak inward current at 0 to +10 mV. The half‐inactivation potential was approximately ‐35 mV. In addition, ICa was blocked by nifedipine. These characteristics indicate the presence of L‐type Ca2+ channels in somatotrophs. 6. Somatostatin caused hyperpolarization and suppressed the spontaneous bursts of action potentials. Under voltage clamp, somatostatin activated an inwardly rectifying current that reversed direction near EK. When EK was altered by elevation of [K+]o, the reversal potential of the somatostatin‐induced current shifted 55 mV per tenfold change of [K+]o, as predicted for a K+ current by the Nernst relation. The somatostatin‐induced conductance (gK) was greater at more negative potentials, and the activation range shifted positive with elevation of [K+]o. 7. We conclude that freshly isolated rat somatotrophs possess Na+, Ca2+ and K+ currents. A large proportion of the cells exhibit spontaneous bursts of action potentials. Somatostatin activates an inwardly rectifying K+ conductance, causing hyperpolarization and cessation of spontaneous action potential activity, actions that would contribute to suppression of growth hormone release.
Decline of myoplasmic Ca2+, recovery of calcium release and sarcoplasmic Ca2+ pump properties in frog skeletal muscle.
Authors: Klein M G , Kovacs L , Simon B J , Schneider M F
Published: December 2014

1. The two calcium indicators Antipyrylazo III (AP III) and Fura‐2 were used simultaneously to monitor free myoplasmic [Ca2+] in voltage‐clamped cut segments of frog skeletal muscle fibres (8‐10 degrees C). Antipyrylazo III was used for the relatively large [Ca2+] transients during 100‐200 ms depolarizing pulses to ‐20 to 0 mV and for the rapid decline of [Ca2+] during the 200 ms after the pulses. Fura‐2 was used to follow the slow decline of the small remaining elevation of [Ca2+] during the following 16 s (slow recovery period) and to monitor resting [Ca2+]. 2. From 1 to 16 s of the slow recovery period [Ca2+] declined with two exponential components, having time constants of 1.9 +/‐ 0.3 and 13.5 +/‐ 1.5 s (these and all other values are means +/‐ S.E.M. of eleven runs from seven fibres). At 1.2 s after the end of the pulses the amplitudes of the fast and slow exponential components of decline of [Ca2+] were 34 +/‐ 7 and 31 +/‐ 4 nM, respectively. The resting [Ca2+] in these runs was 40 +/‐ 4 nM. 3. The time course of calcium bound to parvalbumin [( Ca‐Parv]) was calculated from the [Ca2+] records using literature values for the parvalbumin kinetic constants. From 1 to 16 s of the slow recovery period the total calcium [Ca]T outside the sarcoplasmic reticulum (SR) was assumed to equal [Ca‐Parv] + [Ca‐Fura]. During this period [Ca]T declined with two exponential components having time constants of 1.7 +/‐ 0.2 and 14.2 +/‐ 1.4 s, the same as those for [Ca2+]. Assuming the total concentration of parvalbumin cation binding sites to be 1000 microM, the fast and slow components of [Ca]T had amplitudes of 117 +/‐ 21 and 147 +/‐ 16 microM, respectively, at 1.2 s after the pulses. 4. The rate of decline of [Ca]T, ‐d[Ca]T/dt, was used as a measure of the net rate of removal of calcium from the myoplasm by the SR. From 3 to 16 s of the slow recovery period and in the resting fibre ‐d[Ca]T/dt varied with [Ca2+] according to A[Ca2+]n‐L. The term A[Ca2+]n represents the pump rate and L represents a constant rate of calcium leak from the SR. 5. For 40 nM less than or equal to [Ca2+] less than or equal to 80 nM, the power n for the [Ca2+] dependence of pump rate was 3.9 +/‐ 0.6.(ABSTRACT TRUNCATED AT 400 WORDS)
Generation of slow‐wave‐type action potentials in canine colon smooth muscle involves a non‐L‐type Ca2+ conductance.
Authors: Huizinga J D , Farraway L , Den Hertog A
Published: December 2014

1. The hypothesis was addressed that a non‐L‐type calcium conductance is involved in the generation of the initial part of the slow‐wave‐type action potential in the canine colon. 2. In the absence of a sodium and chloride gradient (NaCl replaced by glucamine), and in the presence of nitrendipine (in ‘glucamine‐nitrendipine’ Krebs solution), a major portion of the upstroke potential of the slow wave persists at unchanged frequency. 3. In ‘glucamine‐nitrendipine’ Krebs solution, the rate of rise and amplitude of the upstroke potential is reduced by removal of extracellular calcium in a concentration‐dependent manner. 4. The rate of rise and the amplitude of the upstroke potential is in a concentration‐dependent manner reduced by Ni2+ greater than Cd2+ greater than Co2+ greater than Mg2+. 5. In ‘glucamine‐nitrendipine’ Krebs solution, Ba2+ cannot replace Ca2+ in the generation of the upstroke potential. 6. Positive evidence was obtained for the hypothesis that a non‐L‐type calcium conductance is involved in the initiation of the slow‐wave‐type action potential in colonic smooth muscle.
Multiple actions of 2,3‐butanedione monoxime on contractile activation in frog twitch fibres.
Authors: Hui C S , Maylie J
Published: December 2014

1. The effects of 2,3‐butanedione monoxime (BDM) on various steps in the excitation‐contraction coupling sequence, including action potential, charge movement and twitch tension, were studied in twitch fibres of Rana temporaria. 2. The resting potential of intact fibres in whole muscle bathed in 20 mM‐BDM was the same as control. The resting potential also remained stable after more than 100 min in 20 mM‐BDM. 3. The action potential was measured in intact fibres of fibre bundles with an intracellular microelectrode. Applications of 5 and 7.5 mM‐BDM had no effect on its amplitude, whereas 10 and 20 mM suppressed its amplitude by about 4 and 10%, respectively. Increasing concentrations of BDM prolonged the half‐width and elevated the after‐potential of the action potential progressively. The action potential was also measured in cut fibres mounted in a double Vaseline‐gap chamber. Results were similar to those in intact fibres. 4. Charge movement was measured in intact fibres of halved muscles with the three‐microelectrode voltage‐clamp technique. The steady‐state Q‐V plot of the total charge measured in isotonic tetraethylammonium (TEA) Ringer solution with 20 mM‐BDM appeared to be shifted about 10 mV in the depolarizing direction and to be slightly more shallow when compared with the control Q‐V plot measured in hypertonic TEA Ringer solution with 350 mM‐sucrose. After allowing for the voltage shift, 20 mM‐BDM did not appear to affect the kinetics of both components of charge movement, but suppressed the maximum amount of total charge by about one‐quarter. 5. Charge movement was also measured in cut fibres with the double Vaseline‐gap voltage‐clamp technique. In the presence of 20 mM‐BDM, charge movement traces resembled those from intact fibres. Twenty millimolar BDM suppressed the maximum amount of total charge by about one‐quarter, as in intact fibres. The steady‐state Q‐V plots from cut fibres were separated into Q beta (early current) and Q gamma (late hump current) components by least‐squares fitting with a sum of two Boltzmann distribution functions. On average, 20 mM‐BDM suppressed Q beta and Q gamma in roughly equal proportion, but did not affect the individual voltage distributions of Q beta and Q gamma. 6. Twitch tension was measured in single intact fibres stimulated extracellularly. BDM effectively reduced the peak amplitude, the time‐to‐peak and the half‐width of twitch tension. The interaction of BDM with receptors appeared to follow more or less a simple 1:1 binding in fibres stretched to sarcomere lengths of about 3.6 microns.(ABSTRACT TRUNCATED AT 400 WORDS)
The flow of human blood through capillary tubes.
Authors: Sirs J A
Published: December 2014

1. The current interpretation of in vivo blood flow is mainly based on the Hagen‐Poiseuille equation, although blood is not a Newtonian fluid. In this paper, experimental pressure‐flow curves of blood are explained on the basis that the viscosity of the blood is the sum of two components, a Newtonian viscosity term, N, and an anomalous viscosity term equal to A/(B + D), where A and B are constants, and D the shear rate. 2. To a first approximation, blood flow in capillary tubes, comparable to that in vivo, can be deduced if the applied pressure in Poiseuille's equation is reduced by an effective back‐pressure, p, equal to 8Al/3R, where l is the length of the capillary tube, and R its radius. 3. The theory explains the progressive change, from a parabolic velocity profile in large vessels, to a flattened profile in small vessels, as observed in vivo. 4. Experimental evidence is given that p is proportional to the length, and increases with decrease of R. The effect of the anomalous viscosity coefficient A was studied by varying the haematocrit, fibrinogen level, erythrocyte flexibility and temperature. 5. As the tube bore is decreased, the Fahraeus‐Lindqvist effect decreases N, but this is offset by an increase of the anomalous component, A. This results, at lower pressures, in an increase of the effective blood viscosity in small vessels and of the peripheral resistance, and, at higher pressures, in a decrease of the effective blood viscosity. 5. Blood flow is proportional to the radius to the power n, where n is a variable that increases with increase of A and decrease of the applied pressure.
Possible involvement of prostaglandins in psychological stress‐induced responses in rats.
Authors: Morimoto A , Watanabe T , Morimoto K , Nakamori T , Murakami N
Published: December 2014

1. We investigated the effect of pre‐treatment with intraperitoneal (I.P.) injection of indomethacin, an inhibitor of prostaglandin synthesis, on psychological stress‐induced responses including cardiovascular, thermoregulatory and hormonal responses in free‐moving rats. 2. Psychological stress was induced by cage‐switch stress. After the rats were placed in the novel environment, blood pressure, heart rate and body temperature significantly increased. Plasma levels of adrenocorticotrophic hormone (ACTH) and prostaglandin E2 were significantly higher 30 min after exposure to stress, in comparison to normal levels. 3. Pre‐treatment with I.P. indomethacin significantly suppressed the increases in body temperature induced by cage‐switch stress, but had no effect on increases in blood pressure and heart rate induced by this stress. Indomethacin also significantly suppressed the increases in the plasma levels of ACTH and prostaglandin E2 induced by cage‐switch stress. 4. The present results suggest that prostaglandins are involved in the development of hyperthermia and the ACTH response induced by psychological stress.
Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study.
Authors: Errchidi S , Monteau R , Hilaire G
Published: December 2014

1. Superfused brain stem‐spinal cord preparations of newborn rats, which continue to show a rhythmic respiratory activity in vitro, were used to analyse the mechanisms whereby the A5 noradrenergic area modulates the activity of the medullary respiratory rhythm generator in the newborn. 2. In preparations including the pons (ponto‐medullary preparations), noradrenaline (NA, 25‐100 microM) added to the bathing medium either increased (n = 29/50) or decreased (n = 21/50) the respiratory frequency and elicited a tonic discharge in the cervical ventral roots in 50% of the experiments. Double‐bath experiments showed that the increases in respiratory frequency were due to NA acting on the pons, whereas the decreases in respiratory frequency were due to NA acting on the medulla. The NA‐induced increases in respiratory frequency were attributed to inhibition of A5 neurons by NA and therefore to withdrawal of A5 inhibition on the medullary rhythm respiratory generator. The NA‐induced decreases in respiratory frequency seemed to mimic the effects of endogenous NA on the A5 medullary targets. 3. Noradrenaline‐induced tonic activity (i) could be induced after elimination of the pons but not on isolated spinal cord, (ii) could be elicited by alpha 1‐ but not alpha 2‐agonists, (iii) could be blocked by alpha 1‐ but not alpha 2‐antagonists. The tonic activity therefore originated from activation of alpha 1 receptors located in the medulla but its importance in respiratory function is doubtful. 4. In medullary preparations (elimination of the pons by transection), the effects of NA agonists and antagonists on respiratory frequency were analysed. Significant decreases in respiratory frequency were induced by NA, adrenaline, phenylephrine and alpha‐methyl‐NA, but not by the agonists classified as alpha 2 (clonidine and guanfacine), alpha 1 (6‐fluoro‐NA) and beta (isoprenaline). Since yohimbine, idazoxan and piperoxane (alpha 2 antagonists) blocked the NA‐induced decreases in respiratory frequency whereas prazosin (alpha 1‐antagonist) did not, it is postulated that alpha 2‐receptors may be involved in modulating respiratory frequency. 5. Stimulation, lesion and NA microejection experiments showed the complexity of the mechanisms mediating NA‐induced changes in respiratory activity but suggested that the main site of NA action is located in the rostral ventrolateral medulla, where electrical stimulations triggered inspiration prematurely, lesions suppressed the NA‐induced decrease in respiratory frequency, and localized application of NA led to an immediate decrease in the respiratory frequency.(ABSTRACT TRUNCATED AT 400 WORDS)
Halide transport in Xenopus oocytes.
Authors: Katayama Y , Widdicombe J H
Published: December 2014

1. Radioisotopes and intracellular microelectrodes were used to characterize the permeability of Xenopus oocytes to chloride and other halides. 2. Uptake of 36Cl had a half‐time for equilibration of approximately 3 h, with an initial rate of Cl‐ entry corresponding to a permeability coefficient of 3.9 x 10(‐7) cm/s, and an equilibrium uptake of 36Cl of 33 mM. 3. Replacement of bathing Na+ by K+ depolarized the oocytes from ‐46 to ‐7 mV and stimulated influx approximately 3‐fold. 4. Influx was linearly dependent on bathing [Cl‐] and was temperature dependent with an activation energy of 46 kJ/mol. Influx of 125I of 36Cl was not affected by the presence of equal concentrations of other halides or thiocyanate. These results are consistent with a channel‐mediated entry mechanism. 5. Diphenylamine‐2‐carboxylate (DPAC) and 9‐anthracene carboxylate (9‐AC), blockers of Cl‐ channels in other cells, inhibited Cl‐ entry with dissociation constants (Kds) of approximately 5 x 10(‐4) and approximately 10(‐3) M, respectively. Inhibitors of Cl(‐)‐HCO3‐ exchange or Na(+)‐K(+)‐2Cl‐ co‐transport did not affect Cl‐ influx. 6. Attempts to lower or raise intracellular Ca2+ with BAPTA or A23187, respectively, were also without effect on Cl‐ influx. 7. The halide selectivity sequence determined with isotopes was I‐ (3.2) greater than Br‐ (1.3) greater than Cl‐ (1.0). However, DPAC inhibited almost all of the 36Cl influx but only a small fraction of 125I influx. 8. Replacement of bathing Cl‐ by I‐ or Br‐resulted in hyperpolarizations, from which the same selectivity sequence was determined. 9. Replacement of bathing Cl‐ by gluconate caused a marked depolarization, which was inhibited by DPAC and, less potently, by 9‐AC.
Accounting for the Ca(2+)‐dependent kinetics of single large‐conductance Ca(2+)‐activated K+ channels in rat skeletal muscle.
Authors: McManus O B , Magleby K L
Published: December 2014

1. The Ca(2+)‐dependent kinetics of large‐conductance Ca(2+)‐activated K+ channels from cultured rat skeletal muscle were studied with the patch clamp technique. Data were collected in the absence of Na+ and Mg2+, which can alter the kinetics. About 2 x 10(5) open and shut intervals were analysed from each of five different excised membrane patches containing a single active channel. Analysis was restricted to activity in the normal mode, which includes 96% of the intervals. 2. The open probability (Popen) and dwell‐time distributions of open and shut intervals were obtained at three to four different [Ca2+]i for each of the channels. Popen data were also obtained from some multichannel patches. 3. Increasing [Ca2+]i increased Popen. At a pH of 7.0 the Hill coefficient was 3.7 +/‐ 0.8 (range of 3.0‐5.0) and a Popen of 0.5 occurred at 14 +/‐ 7 microM [Ca2+]i (K0.5) for data obtained at +30 mV (n = 6). At a pH of 7.2 the Hill coefficient was 3.0 +/‐ 0.5 (range of 2.2‐3.7) and K0.5 was 9 +/‐ 6 microM‐Ca2+ (n = 7). The large standard deviations for K0.5 reflect the observation that fourfold differences in K0.5 could be observed for different channels studied under the same experimental conditions. 4. Hill coefficients that can be greater than 3 suggest that the channel may bind four or more Ca2+ to become fully activated. The binding of four Ca2+ before opening would require a minimum of five shut states. This estimate of the minimum number of shut states is in general agreement with that obtained from the number of exponential components in the dwell‐time distributions of shut intervals. Thus, two different methods give similar estimates of the minimum number of shut states. If the channel can open with different numbers of bound Ca2+, then this could give rise to the three to four open states suggested by the three to four exponential components in the open dwell‐time distributions. 5. Kinetic schemes consistent with the Ca(2+)‐dependent kinetics were developed by simultaneously fitting open and shut dwell‐time distributions obtained at three to four different [Ca2+]i, using maximum likelihood techniques and corrections for missed events. Such simultaneous fitting can provide an increased ability to define models and rate constants.(ABSTRACT TRUNCATED AT 400 WORDS)
Water permeability of ventricular cell membrane in choroid plexus epithelium from Necturus maculosus.
Authors: Zeuthen T
Published: December 2014

1. The osmotic water permeability Lp and the relations between the flows of H2O, K+ and Cl‐ were studied in the ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus. 2. The flows were induced by abrupt changes in external osmolarity of the ventricular solution. Solution changes were convective and no effects of unstirred layers could be detected on measured parameters. 3. The initial rate of change in intracellular concentrations of K+ and Cl‐ was monitored by double‐barrelled ion‐selective microelectrodes. 4. The initial rate of flux of H2O could be monitored as the changes in the concentration of intracellular choline ions (Ch+i). When 0.5 mmol l‐1 of choline chloride was added to the external solutions, Ch+i attained values of 1‐5 mmol l‐1. The dilution or concentration of Ch+i could be recorded by K+ electrodes since the sensitivity of these to Ch+ is more than 50 times greater than to K+. 5. The Lp of the ventricular membrane of the epithelium was 1.4‐2.1 x 10(‐4) cm s‐1 (osmol l‐1)‐1 and independent of the direction of the induced water flux. Lp was unchanged in tissues adapted to osmolarities of half the physiological value. 6. The efflux of H2O induced by mannitol was associated with an instantaneous efflux of K+ which was inhibited by furosemide. The fluxes had a ratio of 40 mmol l‐1. The influx of H2O induced by the removal of NaCl from the ventricular solution was associated with an instantaneous influx of K+. The H2O influx had a ratio to the flux of K+ of 70 mmol l‐1. 7. The efflux of H2O induced by mannitol was associated with an efflux of Cl‐ which was inhibited by furosemide. The ratio of the two fluxes was in the range 15‐44 mmol l‐1. 8. The conclusion is that the Ch+ method gives a reliable measure of the movement of H2O across the ventricular membrane. The magnitude of the Lp and its relevance to transepithelial transport are discussed. The osmotically induced H2O movement is accompanied by furosemide‐sensitive fluxes of K+ and Cl‐ of the same magnitude. This suggests that co‐transport between H2O and KCl can take place in the membrane.
Differential effect of neuropeptide‐Y on membrane potential of cells in renal arterioles of the hydronephrotic mouse.
Authors: Nobiling R , Gabel M , Persson P B , Dietrich M S , Bührle C P
Published: December 2014

1. The effects of neuropeptide‐Y (NPY) on the membrane potential of vascular smooth muscle cells were studied in renal arterioles of hydronephrotic mouse kidneys. 2. Kidney vessels are only weakly coupled with length constants of less than 10 microns and are most probably ‘multiunit’ vessels. 3. The vasoconstrictor peptide NPY reversibly depolarizes only smooth muscle cells in arterioles at distances greater than 200 microns from the glomerulus, whereas no changes of the membrane potential can be evoked close to the glomerulus (distance less than 50 microns). 4. The depolarizations, when present, are dose dependent. 5. Regardless of distance from the glomerulus cells respond uniformly to application of the vasoconstrictor angiotensin II.
Guanosine diphosphate activates an adenosine 5'‐triphosphate‐sensitive K+ channel in the rabbit portal vein.
Authors: Kajioka S , Kitamura K , Kuriyama H
Published: December 2014

1. Properties of the pinacidil‐sensitive K+ channel in the smooth muscle of the rabbit portal vein were investigated using cell‐attached and inside‐ and outside‐out patch clamp techniques. 2. In the cell‐attached patch configuration, a K+ channel with a unitary conductance of 150 pS could be recorded when physiological salt solution (PSS) was in the pipette and high‐K+ solution was in the bath. Tetraethylammonium (TEA; less than 1 mM) and charybdotoxin (CTX; greater than 50 nM) inhibited the 150 pS K+ channel from the outside of the membrane. This channel was activated by an increase in the concentrations of intracellular Ca2+ but not by pinacidil (less than or equal to 500 microM). 3. In the cell‐attached patch configuration, bath application of pinacidil (greater than 3 microM) activated a K+ channel (ATP‐sensitive K+ channel) with a unitary conductance of 15 pS and the enhancing action of pinacidil was blocked by glibenclamide. However, in the cell‐free patch configuration, pinacidil (100 microM) failed to open the 15 pS K+ channel. With pinacidil in the pipette, the 15 pS K+ channel was completely inactivated within 5 s of the excision of the membrane. Opening of the 15 pS K+ channel also disappeared after saponin treatment (50 micrograms/ml). 4. In the cell‐free patch configuration, application of guanosine 5'‐diphosphate (GDP; greater than 100 microM) re‐activated the inactivated 15 pS K+ channel only when pinacidil was present either in the pipette or bath. GDP increased the mean open time and open probability of the 15 pS K+ channel in a concentration‐dependent manner. Simultaneous application of MgCl2 (less than or equal to 1 mM) with GDP did not modify the GDP‐induced activation. Neither GDP nor GTP (1 mM) had any effect on the 150 pS K+ channel. 5. Guanosine 5'‐triphosphate (GTP; 1 mM) activated the 15 pS K+ channel to a lesser extent that did GDP. Other guanine nucleotides (guanosine 5'‐monophosphate, GMP, 1 mM; guanosine 5'‐O‐(3‐thiotriphosphate), GTP gamma S, 100 microM; and guanosine 5'‐O‐(2‐thiodiphosphate), GDP beta S, 1 mM) failed to activate the 15 pS K+ channel. However, GDP beta S, but not GMP or GTP gamma S, inhibited this channel when it was activated by 1 mM‐GDP. 6. In the presence of pinacidil, adenosine 5'‐triphosphate (ATP; greater than or equal to 10 microM) inhibited the ATP‐sensitive K+ channel when it was activated by 1 mM‐GDP.(ABSTRACT TRUNCATED AT 400 WORDS)
A paradox concerning ion permeation of the delayed rectifier potassium ion channel in squid giant axons.
Authors: Clay J R
Published: December 2014

1. The fully activated current‐voltage relation (I‐V) of the delayed rectifier potassium ion channel in squid giant axons has a non‐linear dependence upon the driving force, V‐EK, as I have previously demonstrated, where V is membrane potential and EK is the equilibrium potential for potassium ions. 2. The non‐linearity of the I‐V relation and its dependence upon external potassium ion concentration are both well described, phenomenologically, by the Goldman‐Hodgkin‐Katz (GHK) flux equation, as I have also previously demonstrated. As illustrated below, this result can be modelled using the Eyring rate theory of single‐file diffusion of ions through a channel in the low‐occupancy limit of the theory. 3. The GHK equation analysis and the low‐occupancy limit of the Eyring rate theory are both consistent with the independence principle for movement of ions through the channel, which is at odds with tracer flux ratio results from the delayed rectifier, published elsewhere. Those results suggest that the channel is multiply occupied by two, or perhaps three, ions. 4. The resolution of this paradox is provided by a triple‐binding site, multiple‐occupancy model in which only one vacancy, at most, is allowed in the channel. This model predicts current‐voltage relations which are consistent with the data (and with the phenomenological prediction of the GHK flux equation). The model is also consistent, approximately, with the tracer flux ratio results.
Voltage‐gated calcium currents in cultured embryonic Xenopus spinal neurones.
Authors: Barish M E
Published: December 2014

1. Voltage‐gated Ca2+ currents were studied in cultured embryonic Xenopus spinal neurones using whole‐cell gigaohm seal techniques. Cultures of neural plate cells were established from stage 15‐17 embryos (see Methods), and were studied for up to 80 h in vitro. During this period neural precursor cells morphologically differentiate and commence expression of multiple types of voltage‐ and ligand‐gated ion channels. 2. Embryonic Xenopus neurones studied during the first 20‐40 h in culture display Ca2+ currents that correspond to the low‐voltage‐activated (T‐type) and high‐voltage‐activated forms described in other neurones and excitable cells. These Ca2+ current types could be separated based on voltage dependencies and pharmacological sensitivities. 3. T‐type Ca2+ current was activated at voltages positive to ‐50 mV, and was selectively blocked by 200 microM‐Ni2+. Curves describing the voltage dependencies of activation and steady‐state inactivation overlapped in a region centred on ‐40 mV. A small sustained Ca2+ current could be recorded within this voltage region. 4. High‐voltage‐activated (HVA) Ca2+ currents were observed at voltages positive to ‐10 mV, and could be separated into relaxing and sustained components (denoted as HVA‐relaxing and HVA‐sustained). HVA‐relaxing current was selectively reduced by Met‐enkephalin (17.5 microM). Both components of HVA current were sensitive to verapamil (100 microM), were almost completely blocked by omega‐conotoxin (3 microM) and were insensitive to nifedipine (20 microM). 5. The data indicate that T‐type Ca2+ current is present in the membrane during the initial period of channel and receptor expression, process outgrowth, and synaptogenesis, and is the dominant influence on voltage‐gated Ca2+ influx during subthreshold voltage excursions. Further, at more positive voltages, T‐type Ca2+ current contributes to inward Ca2+ current during the first 5‐10 ms after depolarizing voltage steps, and thus to inward Ca2+ current during the rising phase of the long‐lasting Ca(2+)‐dependent embryonic action potential. HVA Ca2+ currents (particularly the relaxing component) influence the plateau phase.
Effect of nitric oxide on circular muscle of the canine small intestine.
Authors: Stark M E , Bauer A J , Szurszewski J H
Published: December 2014

1. Experiments were designed to determine in circular muscle of the canine jejunum whether exogenous nitric oxide (NO) mimics the non‐adrenergic, non‐cholinergic inhibitory junction potential (NANC IJP), and whether changes in the availability of endogenous NO affects IJP amplitude. 2. Mechanical and intracellular electrical activity were recorded simultaneously from circular muscle of the canine jejunum. Electrical field stimulation evoked NANC IJPs and inhibited spontaneous contractions. 3. Infusions of NO solutions evoked immediate dose‐dependent and transient hyperpolarizations and transiently inhibited spontaneous contractions. NO‐evoked hyperpolarizations were unaffected by atropine, propranolol, phentolamine and tetrodotoxin. 4. The maximum IJP amplitude and the maximum amplitude of NO‐evoked hyperpolarization were similar. 5. NG‐Mono‐methyl‐L‐arginine (L‐NMMA), which inhibits synthesis of NO from L‐arginine, reduced IJP amplitudes but did not reduce the response to exogenous NO. L‐Arginine, but not D‐arginine, reversed the effect of L‐NMMA on IJP amplitude. 6. Oxyhaemoglobin, which binds and inactivates NO, reduced IJP amplitude and abolished the response to exogenous NO. 7. Exogenous NO mimicked the effects of NANC inhibitory input. Reducing the availability of endogenous NO reduced NANC inhibitory input. 8. It was concluded that NO mediates NANC neural inhibition and may act as a NANC inhibitory neurotransmitter in the canine jejunum.
Effects of amino acids on zinc transport in rat erythrocytes.
Authors: Aiken S P , Horn N M , Saunders N R
Published: December 2014

1. A significant proportion of plasma zinc exists complexed with amino acids. The effect of amino acids on the accumulation of radioactive zinc by rat erythrocytes was studied in vitro, to investigate the hypothesis that zinc might be transported into cells as an amino acid‐zinc complex. 2. L‐Histidine (500 microM‐10 mM) stimulated 65Zn uptake; 50 mM‐L‐histidine gave a slight inhibition of uptake. D‐Histidine (500 microM‐10 mM) inhibited uptake in a dose‐dependent manner. A non‐zinc‐binding amino acid, L‐alanine, did not affect 65Zn uptake. 3. The effect of L‐histidine was sodium dependent and temperature dependent, but was DIDS insensitive. These properties suggest that zinc is being transported as a zinc‐histidine complex, utilizing an amino acid carrier system. Uptake of zinc in the presence of L‐histidine differed from the previously described ionic mechanism, and may represent a physiological route of uptake. 4. L‐Histidine stimulated efflux of 65Zn from pre‐loaded cells. 5. The relevance of transport of a zinc‐histidine complex is discussed with reference to histidinaemia, and as a significant zinc transport system in the presence of the very low ionic zinc concentrations found in plasma.
Synaptic behaviour in the myenteric plexus of the guinea‐pig gastric antrum.
Authors: Tack J F , Wood J D
Published: December 2014

1. Intracellular recording methods were used to study the synaptic behaviour of neurones in the myenteric plexus of the guinea‐pig gastric antrum. Synaptic potentials occurred spontaneously or were evoked by focal electrical stimulation of interganglionic fibre tracts. Synaptic events consisted of fast and slow excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs). 2. Fast EPSPs with durations less than 20 ms were evoked in every antral neurone in a population sample of 370 cells. Most of the ganglion cells received multiple inputs from axons entering the individual ganglia in several different interganglionic fibre tracts. Many of the neurones also received input from multiple axons projecting in individual fibre tracts. The fast EPSPs behaved like nicotinic cholinergic EPSPs. They were evoked at stimulus frequencies up to 60 Hz without evidence of the run‐down characteristic of fast EPSPs in the intestine. 3. Slow EPSPs were evoked by repetitive stimulation of the interganglionic connectives. They consisted of a slowly activating depolarization which persisted for several seconds after termination of the stimulus. The depolarizing responses were associated with an increase in the input resistance, with enhanced excitability and with suppression of hyperpolarizing after‐potentials in AH/type 2 neurones. They were observed in 14.4% of the neurones of which 89% were AH/type 2 neurones. AH/type 2 neurones, unlike other myenteric neurones, were identified by action potentials with long lasting after‐hyperpolarization. 4. IPSPs were hyperpolarizing potentials evoked by repetitive stimulation of interganglionic fibre tracts. The hyperpolarizing responses were associated with decreased input resistance. They occurred in 1.4% of the antral neurones. 5. Application of acetylcholine (ACh) by micro‐ejection mimicked the fast EPSPs in all neurones. This fast nicotinic response to ACh was followed by a slowly activating, long‐lasting muscarinic depolarization in 32% of the neurones. The slow muscarinic response was associated with increased input resistance, suppression of hyperpolarizing after‐potentials and enhanced excitability. 6. Fast EPSPs were not suppressed by accumulation of ACh at presynaptic transmitter release sites. Unlike the intestine, presynaptic muscarinic autoreceptors appeared to be absent from the microcircuits in the antrum. 7. Synaptic behaviour in the local circuits of the gastric antrum differed from the gastric corpus. This may be a reflection of specialization of the circuits for organization of the distinctive patterns of digestive behaviour found in this region of the stomach.
Potassium channels opened by noradrenaline and other transmitters in excised membrane patches of guinea‐pig submucosal neurones.
Authors: Shen K Z , North R A , Surprenant A
Published: December 2014

1. Unitary potassium currents were recorded in outside‐out patches of membrane from guinea‐pig submucosal neurones. The actions of alpha 2‐adrenoceptor agonists, somatostatin and [Met5]enkephalin were studied. 2. Three main groups of background potassium channels were active. At ‐70 mV with 160 mM‐potassium on both sides of the membrane, they had conductances of 30‐65 (small), 120‐160 (intermediate) and 220‐260 pS (large). 3. The open channel current‐voltage relation showed only constant‐field rectification. Extracellular barium (2 mM) and caesium (2 mM) decreased inward but not outward currents. Tetraethylammonium (10 mM) had no effect. 4. Noradrenaline, somatostatin and [Met5]enkephalin each increased the open probability of all three classes of channel when two or more unitary amplitude channels were active in the membrane patch. Agonists were ineffective when no channel, or a single channel, was discernible in the patch. Agonists did not cause the appearance of unitary currents distinct from those seen prior to their application. 5. The effect of the agonists required intracellular guanosine 5'‐triphosphate. 6. The results show that the hyperpolarization of submucosal plexus neurones by noradrenaline, somatostatin and [Met5]enkephalin results from the increased opening of at least three types of background potassium channel, and that the coupling from the receptors to the channels is maintained in excised membrane patches.
The nature of fuel provision for the Na+,K(+)‐ATPase in porcine vascular smooth muscle.
Authors: Campbell J D , Paul R J
Published: December 2014

1. The specific contributions of aerobic glycolysis and oxidative metabolism to Na+ pump activity were quantitated in porcine carotid arteries under aerobic conditions. 2. Active reaccumulation of potassium by potassium‐depleted tissues could be supported by oxidative metabolism alone, anaerobic metabolism in the presence of glucose, or a combination of oxidative metabolism and aerobic glycolysis, but not under anaerobic conditions in the absence of glucose. 3. Increasing levels of potassium added to potassium‐depleted arteries under aerobic conditions resulted first in stimulation of aerobic lactate release which saturated at 0.028‐0.036 mumol min‐1 g‐1, which was then followed by a stimulation of oxidative metabolism. This behaviour is opposite to the classic Pasteur effect. 4. The dependence of potassium uptake and lactate release on the concentration of potassium added to potassium‐depleted arteries ('potassium re‐entry concentration') under aerobic conditions were qualitatively similar. The K0.5 (concentration at which the velocity is half‐maximally activated) and Vmax (the maximum velocity) for lactate release were 1.2 +/‐ 0.3 mM and 0.037 mumol min‐1 g‐1, respectively; those for K+ uptake were 4.3 +/‐ 0.4 mM and 0.399 mumol min‐1 g‐1. 5. The stoichiometric ratio between potassium uptake and ATP as calculated from lactate release approximated theoretical values of 2:1 (assuming 1 ATP per lactate) when potassium re‐entry concentrations were less than 2 mM; higher concentrations of potassium produced ratios up to 9:1. 6. Physiological pump rates, as determined by potassium efflux studies, corresponded to potassium re‐entry concentrations of less than or equal to 2 mM, the same potassium re‐entry concentrations where the stoichiometry between potassium transport and aerobic glycolysis approximated the theoretical ratio of 2:1. Increases in oxidative metabolism were not detected in this range, but were detected at potassium re‐entry concentrations of greater than or equal to 4 mM. 7. It was concluded that at physiological Na+ pump rates, aerobic glycolytic metabolism supported the N+,K(+)‐ATPase; at higher pump rates, oxidative metabolism was required for pump support as well.
Properties of a cell volume‐sensitive potassium conductance in isolated guinea‐pig and rat hepatocytes.
Authors: Sandford C A , Sweiry J H , Jenkinson D H
Published: December 2014

1. Whole‐cell voltage clamp and intracellular recording techniques were used to study the increase in K+ conductance that accompanies swelling in isolated guinea‐pig and rat hepatocytes in short‐term culture at 37 degrees C. 2. Swelling was induced (i) by the application of pressure (15 cmH2O) to the shank of the patch pipette, (ii) by exposing the cells to hypotonic solutions and (iii) as a consequence of leakage of electrolyte from an intracellular microelectrode. 3. Applying pressure to the patch pipette caused a large outward current (approximately 600 pA) to develop in guinea‐pig hepatocytes voltage clamped to 0 mV. This current reversed direction at ‐86 mV, close to the reversal potential for K+, EK (‐93 mV), and is attributable to the activation of a K+ conductance. 4. Spectral analysis of current noise during this response suggested a single‐channel conductance of 7 pS, though this may well be an underestimate. The power spectrum could be fitted by the sum of two Lorentzian components, with half‐power frequencies of 7 and 152 Hz. Seventy per cent of the variance was associated with the lower frequency component. 5. The steady‐state current‐voltage relationship for guinea‐pig hepatocytes, as determined by whole‐cell recording, was linear over the range ‐70 to +40 mV both before and during the increase in K+ conductance induced by swelling. 6. Confirming earlier work, intracellular recording using microelectrodes filled with 1 M‐potassium citrate sometimes resulted in a slow hyperpolarization and a large rise in input conductance. These changes are also attributable to an increase in K+ conductance as the cell swelled because of leakage from the electrode. 7. Application of hypotonic external solutions during intracellular recording caused hyperpolarization and an increase in conductance. Conversely, hypertonic solution evoked depolarization and a fall in conductance in partly swollen cells. 8. The volume‐activated K+ conductance was reversibly blocked by cetiedil, which caused half‐maximal inhibition at 2.3 microM. Bepridil, quinine and barium were also effective, with IC50s (concentrations giving 50% maximal inhibition) of 2.7, 12 and 67 microM respectively. 9. Much greater concentrations of cetiedil and bepridil (IC50 approximately 1 mM and 77 microM respectively) were required to inhibit the loss of K+ which follows the application of angiotensin II (100 nM) to guinea‐pig hepatocytes, and which occurs via Ca(2+)‐activated K+ channels. Our evidence suggests that the activation of K+ channels by cell swelling is Ca2+ independent.(ABSTRACT TRUNCATED AT 400 WORDS)
Cyclic AMP‐mediated regulation of excitation‐contraction coupling in canine gastric smooth muscle.
Authors: Ozaki H , Blondfield D P , Hori M , Sanders K M , Publicover N G
Published: December 2014

1. Agonists known to increase cyclic AMP levels in gastrointestinal smooth muscles were studied in isolated circular muscles of the canine antrum to investigate the mechanisms of the inhibitory effects of these agents. 2. Muscles were electrically active, generating typical slow wave activity. Cytosolic Ca2+ ([Ca2+]cyt; measured by Indo‐1 fluorescence) and tension increased in response to slow waves. 3. Stimulation by isoprenaline (via beta 2‐receptors) or forskolin, in the presence or absence of acetylcholine, inhibited the plateau phase and reduced phasic [Ca2+]cyt and contractile responses. 4. Vasoactive intestinal peptide (VIP) and calcitonin gene‐related peptide (CGRP), had similar effects to isoprenaline and forskolin. 5. Increases in the plateau phase of slow waves and the associated increases in [Ca2+]cyt and tension caused by direct activation of voltage‐dependent Ca2+ channels by Bay K 8644 (0.1 microM) were also reduced by forskolin. 6. Isoprenaline and forskolin induced negative chronotropic effects, but VIP increased frequency. 7. At a given level of [Ca2+]cyt, contractions were greater under control conditions than in the presence of isoprenaline, VIP and CGRP, suggesting that part of the inhibition produced by these agents may be due to decreased Ca2+ sensitivity of the contractile apparatus. 8. Experiments performed on alpha‐toxin‐permeabilized muscles confirmed that cyclic AMP‐dependent effects involve reduced Ca2+ sensitivity of the contractile apparatus. Addition of cyclic AMP (3‐300 microM) caused a reduction in Ca(2+)‐induced contraction at a constant level of Ca2+ (pCa 5.5). 9. These results suggest that increased cyclic AMP and probably subsequent activation of protein kinase A: (i) decrease [Ca2+]cyt and contraction by an inhibition of Ca2+ influx during slow waves, and (ii) decrease the sensitivity of the contractile apparatus to [Ca2+]cyt. The membrane effects might occur directly by inhibition of Ca2+ channels or indirectly by increasing the open probability of K+ channels which would tend to cause premature repolarization of slow waves.
Kinetic analysis of the GABAB‐mediated inhibition of the high‐threshold Ca2+ current in cultured rat sensory neurones.
Authors: Tatebayashi H , Ogata N
Published: December 2014

1. The action of baclofen on the voltage‐gated Ca2+ current (ICa) was studied, using cultured neurones of the newborn rat dorsal root ganglia (DRG). Two major categories of ICa were identified: a small transient current activated positive to ‐60 mV (low voltage‐activated ICa) and a large and slowly inactivating current activated positive to ‐30 mV (high voltage‐activated ICa). 2. Baclofen reversibly blocked the high voltage‐activated ICa and slowed the activation phase of the current in a concentration‐dependent manner (0.5‐50 microM). The half‐maximal effective concentration was about 1.5 microM as measured by a peak of ICa. On the contrary, a high concentration of baclofen (100 microM) had no detectable effect on the low voltage‐activated ICa. 3. The baclofen‐sensitive component of the high voltage‐activated ICa was largely inactivated by a depolarized holding potential (Vh) of ‐40 mV, whereas the baclofen‐resistant component was not affected by a change in Vh ranging from ‐110 to ‐30 mV. 4. The high voltage‐activated ICa had two components of current decay: an inactivating component and a quasi‐sustained component, with time constants about 420 and 1220 ms, respectively. The time constant of decay for the inactivating component was not affected by replacement of external Ca2+ with Ba2+, whereas that for the quasi‐sustained component was markedly prolonged, suggesting that the decay of this component may be due to Ca(2+)‐induced block rather than voltage‐dependent inactivation. A high concentration of baclofen (50 microM) selectively blocked the inactivating component. 5. The decay phase of the baclofen‐sensitive component of the high voltage‐activated ICa was best fitted by a sum of two exponentials, with 29.2 and 481 ms for the fast and slow components, respectively. The time constants of the two components were not affected by an increase in the concentration of baclofen, whereas the amplitudes changed concentration‐dependently. 6. The slowed activation of the high voltage‐activated ICa by baclofen was partially reversed by a large depolarizing pre‐pulse. However, such an acceleration of the current was similarly observed in the control solution. Furthermore, the actual current size increased by the pre‐pulse was similar in both the control and baclofen‐containing solutions. 7. These results suggest that baclofen selectively blocks the inactivating component of the high voltage‐activated ICa which forms a rapid rising phase of this current, thus slowing the activation phase of the total high voltage‐activated ICa.
Voltage‐ and time‐dependent inhibition of neuronal calcium channels by a GTP‐binding protein in a mammalian cell line.
Authors: Kasai H
Published: December 2014

1. Inhibitory modualtion of the three Ca2+ channel current components by neurotransmitters was studied using the whole‐cell patch‐clamp method in a mammalian cell line, NG108‐15. 2. In cells differentiated with dibutyryl cyclic AMP, both the low‐voltage‐activated current (ILVA) and omega CgTX‐sensitive high‐voltage‐activated current (I omega CgTX) could be inhibited by [D‐phen2, D‐phen5]enkephalin, acetylcholine and noradrenaline. In contrast, differentiation with prostaglandin E1 and theophylline eliminated the agonist‐induced inhibition of ILVA, but enhanced that of I omega CgTX. The DHP‐sensitive high‐voltage‐activated current was unaffected by the transmitters in most of the cells. 3. The inhibition was prevented by pre‐treatment of cells with pertussis toxin, suggesting involvement of a G‐protein. Long treatment of the cells with phorbol ester did not prevent the inhibition. 4. The inhibition was always partial: the maximal inhibition was 40% for ILVA and 70% for I omega CgTX. 5. The inhibition of ILVA and I omega CgTX was relieved during depolarization. Half‐maximal relief of inhibition of I omega CgTX was attained at 0 mV, irrespective of agonist concentration. 6. The kinetics of removal and re‐establishment of inhibition were voltage dependent. Both processes were single exponentials and had identical time constants at a given membrane potential. Time constants were 124 ms at ‐40 mV, 160 ms at 0 mV and 8 ms at 60 mV, at any agonist concentration. 7. Time courses of tail currents were unaltered by the inhibition. 8. The inhibition of the omega CgTX‐sensitive Ca2+ channel can be described as a shift in gating modes; with an additional voltage‐dependent gating state activated by the agonists. The voltage‐dependent properties of this modulation allow inhibition of Ca2+ channel to be overcome by high‐frequency trains of action potentials.