bluuebunny/crossref_metadata_2025 huggingface dataset:
Single voltage‐gated K+ channels and their functions in small dorsal root ganglion neurones of rat.
Authors: Safronov B V , Bischoff U , Vogel W
Published: December 2014

1. Single voltage‐activated K+ channels were investigated by means of the patch‐clamp technique in small dorsal root ganglion (DRG) neurones in 150 microns thin slices of new‐born rat DRG. It was found that K+ conductance in small DRG neurones is formed by one type of fast inactivating A‐channel and four types of delayed rectifier K+ channels, which could be separated on the basis of their single‐channel conductance, kinetics and sensitivity to external tetraethylammonium (TEA). 2. Potassium A‐channels were observed at relatively moderate density. They were weakly sensitive to TEA and activated between ‐70 and +20 mV. The conductance of A‐channels was about 40 pS for inward currents in symmetrical high‐K+ solutions with external 5 mM TEA added to suppress other types of K+ channels. The time constant of channel inactivation (tau in) was 18.8 ms at ‐70 mV and 6 ms at potentials positive to ‐20 mV. 3. A fast delayed rectifier (DRF) channel with a conductance of 55 pS in symmetrical high‐K+ solutions was the most frequent type of K+ channel. The channel activated in a broad potential range between ‐50 and +60 mV and demonstrated a fast deactivation within 1‐3 ms after potential return to ‐80 mV in high‐Ko+ solution. The tau in value was 90‐150 ms at positive membrane potentials. The single‐channel current amplitudes were blocked to 55% by 1 mM TEA. 4. Three further types of delayed rectifier K+ channels were called DR1‐, DR2‐ and DR3‐ channels. Their single‐channel conductances for inward currents in symmetrical high‐K+ solutions were distributed between 30 and 44 pS. The channels activated in almost the same voltage range between ‐60 and ‐10 mV. Deactivation of the channels at ‐80 mV lasted tens of milliseconds. The channels were separated on the basis of their sensitivities to TEA. DR1‐channel currents were reduced to 50% in the presence of 1 mM TEA, DR2‐channel currents were reduced to about 50% by 5 mM TEA, whereas the amplitudes of currents through DR3‐channels were almost unaffected by 5 mM TEA. 5. Addition of external 1 and 5 mM TEA to whole cells under current‐clamp condition depolarized the cell membrane, lowered the threshold for action potential firing, prolonged action potential duration and reduced the amplitude of after‐hyperpolarization. 6. It is concluded that potassium A‐, DRF‐, DR1‐, DR2‐ and DR3‐channels play multiple roles in the excitability of DRG neurones. Possible influences of these channels on the shape of the action potential, its firing threshold and the resting membrane potential of small DRG neurones are discussed.
Neuronal mediators of inhibitory junction potentials and relaxation in the guinea‐pig internal anal sphincter.
Authors: Rae M G , Muir T C
Published: December 2014

1. Inhibitory junction potentials (IJPs) and relaxations evoked in response to field stimulation (supramaximal voltage, 0.1 ms, single stimulus and 5 stimuli at 5‐40 Hz) of non‐adrenergic non‐cholinergic (NANC) nerves with atropine and phentolamine (each 1 microM) were measured in the guinea‐pig internal anal sphincter (gpIAS). The mean resting membrane potential was ‐44.2 +/‐ 0.2 mV (n = 1119 cells from 260 preparations). 2. NANC nerve stimulation evoked frequency‐dependent IJPs (19.7 +/‐ 1.1 mV, n = 165, 33 tissues to a single stimulus) and relaxations. IJPs consisted of two tetrodotoxin (1 microM)‐sensitive components: one was abolished by apamin (0.3 microM) and the P2‐purinoceptor antagonist suramin (100 microM); the other, smaller in amplitude, was sensitive to inhibitors of nitric oxide synthase (NOS, e.g. L‐NAME, 100 microM) and the nitric oxide (NO) scavenger oxyhaemoglobin (HbO, 10 microM). 3. ATP (1 mM), vasoactive intestinal polypeptide (VIP, 0.01‐0.25 microM) and pituitary adenylate cyclase‐activating peptide (PACAP(1‐27), 0.84 microM) each hyperpolarized and relaxed the gpIAS; only ATP responses resembled the evoked IJPs in time course. 4. The guanylyl cyclase inhibitor LY83583 (10 microM) abolished apamin‐insensitive IJPs and relaxations. The cGMP phosphodiesterase inhibitor M&B 22948 (30 microM) and 8‐Br‐cGMP (100 microM) each hyperpolarized the gpIAS. 5. Two components comprise the IJP and relaxation evoked in response to NANC nerve stimulation in the gpIAS. One, sensitive to apamin, resembles the response to ATP and is modulated by purinoceptor antagonists; the other, apamin and suramin insensitive, is inhibited by NO antagonists.
Endothelium‐dependent modulation of pacemaking in lymphatic vessels of the guinea‐pig mesentery.
Authors: von der Weid P Y , Crowe M J , Van Helden D F
Published: December 2014

1. Endothelial control of the rate of constrictions and the underlying pacemaker potentials has been studied in vitro in guinea‐pig mesenteric lymphatic vessels. 2. ACh stimulated 60% of intraluminally perfused vessels to slow or abolish lymphatic constrictions. This action was inhibited by atropine and was likely to be due to the release of endothelium‐derived nitric oxide (EDNO) as the effect was absent after endothelial lysis, mimicked by sodium nitroprusside (SNP), blocked by N omega‐nitro L‐arginine (NOLA) and partially inhibited by Methylene Blue (MB). 3. The remaining 40% of perfused vessels did not mechanically respond to ACh or SNP. In four of seven such vessels this appeared to be due to excessive perfusion‐associated release of EDNO, as incubation with NOLA restored the response to SNP. 4. Application of NOLA or MB in perfused vessels significantly increased constriction frequency, further indicating perfusion‐associated release of EDNO. 5. ACh induced a marked increase in endothelial [Ca2+]i of both mechanically responding and non‐responding vessels. This ACh‐induced increase could be repetitively induced when Ca2+ was present in the perfusate, but rapidly ran down when a Ca(2+)‐free EGTA perfusate was used. 6. Intracellular recordings from the smooth muscle of non‐perfused vessel segments demonstrated an ACh‐induced hyperpolarization and decrease in membrane resistance, changes which were prevented by atropine, NOLA, MB and endothelial lysis and mimicked by SNP. 7. ACh directly reduced the size of the underlying pacemaker potentials termed spontaneous transient depolarizations (STDs). 8. NOLA and MB enhanced STDs in non‐perfused vessel segments indicating an endogenous release of EDNO. 9. It is concluded that the lymphatic endothelium produces and releases EDNO endogenously, during perfusion or after stimulation with ACh, to decrease the efficacy of STDs to generate action potentials and resultant constrictions.
Endocytosis of secretory granules in mouse pancreatic beta‐cells evoked by transient elevation of cytosolic calcium.
Authors: Eliasson L , Proks P , Ammälä C , Ashcroft F M , Bokvist K , Renström E , Rorsman P , Smith P A
Published: December 2014

1. To investigate the mechanisms regulating the reuptake of secretory granule membranes following regulated exocytosis, we have monitored changes in cell capacitance in single pancreatic beta‐cells. 2. Membrane retrieval (endocytosis) occurred both in a continuous manner and in abrupt steps, corresponding to the simultaneous retrieval of 50‐100 granules. The large endocytotic steps were associated with a conductance change of about 1 nS which we attribute to the formation of a fission pore with a pore radius of approximately 1 nm. 3. In some cells, we observed large amplitude capacitance fluctuations, suggesting that aggregates of granules are connected to the plasma membrane by a single pore and are subsequently retrieved as a single unit. 4. Endocytosis was evoked by elevation of cytosolic [Ca2+]i, but once initiated, a sustained increase in [Ca2+]i was not required for endocytosis to continue. 5. The [Ca2+]i dependence of exo‐ and endocytosis was studied by photorelease of Ca2+ from the ‘caged’ precursor Ca(2+)‐nitrophenyl‐EGTA (Ca(2+)‐NP‐EGTA). Both exo‐ and endocytosis were initiated at between 0.5 and 2 microM Cai(2+). The rate of endocytosis saturated above 2 microM Cai(2+), whereas exocytosis continued to increase up to 4 microM Cai(2+). The maximum rate of endocytosis was < 25% of that of exocytosis. 6. Unlike exocytosis, endocytosis proceeded equally well in the presence or absence of Mg‐ATP. 7. Our data indicate that in the pancreatic beta‐cell, exocytosis and endocytosis are regulated by different mechanisms.
Electrophysiological properties of morphologically distinct cells isolated from the rabbit atrioventricular node.
Authors: Munk A A , Adjemian R A , Zhao J , Ogbaghebriel A , Shrier A
Published: December 2014

1. Experiments were conducted using the whole‐cell patch clamp technique to determine the electrophysiological properties and ionic currents of ovoid and rod‐shaped single isolated calcium‐tolerant rabbit atrioventricular (AV) nodal cells. 2. Action potential morphologies observed in these cells were similar to those obtained previously from intracellular recordings of intact atrioventricular nodal preparations: ovoid cells had N‐ or NH‐like action potential configurations (see below), whereas rod‐shaped cells had AN‐like configurations. 3. Action potential restitution in AV nodal cells was characterized by a progressive increase in overshoot potential, maximal upstroke velocity (Vmax) and action potential duration, as well as a decrease in latency from stimulus to Vmax. In rod‐shaped cells, premature stimuli could induce regenerative membrane responses before full action potential repolarization, whereas ovoid cells showed only post‐repolarization refractoriness. In ovoid cells stimulated at the low stimulus intensities there was no shortening of the action potential duration and the most premature action potentials were often prolonged. 4. The quasi‐steady‐state current‐voltage relationship of ovoid cells was significantly steeper, at both depolarized and hyperpolarized potentials, than that of either the rod‐shaped AV nodal cells or atrial cells. The rod‐shaped AV nodal cells and the atrial cells had similar current‐voltage (I‐V) relationships in the positive potential range, but the I‐V curves crossed over at potentials of about‐90 mV. 5. A hyperpolarization‐activated inward current (I(f)) was apparent in the range between ‐60 and ‐90 mV in 95% of the ovoid cells (n = 75), whereas in 88% of rod‐shaped cells (n = 16) I(f) was activated at more negative potentials. The magnitude of I(f) in ovoid cells, measured at ‐100 mV, was approximately 25 times that in rod‐shaped cells. 6. A rapid inward current (INa) greater than 1 nA was found in all rod‐shaped cells (n = 16) but in only 30% of ovoid cells (n = 75). A transient outward current (I(to)) was found in 93% of rod‐shaped cells (n = 14) and in 42% of ovoid cells (n = 54). The combination of I(to) and INa was found in 93% of rod‐shaped cells but in only 24% of ovoid cells. 7. These results suggest that there are at least two populations of isolated AV nodal cells with distinct action potentials and ionic current profiles that may contribute to the complex electrophysiological properties observed in the intact AV node.
Spatiotemporal analysis of calcium dynamics in the nucleus of hamster oocytes.
Authors: Shirakawa H , Miyazaki S
Published: December 2014

1. Subcellular Ca2+ dynamics inside and around the nucleus of immature hamster oocytes were analysed with confocal Ca2+ imaging. 2. The ratio value between emission intensity of two injected fluorescent Ca2+ indicators, Calcium Green and Fura Red, was almost uniform over the entire oocyte, suggesting that nucleoplasmic Ca2+ concentration ([Ca2+]n) is comparable to cytoplasmic Ca2+ concentration ([Ca2+]c) at the resting state. 3. When Ca2+ was iontophoretically injected into the nucleoplasm or the perinuclear cytoplasm, it diffused across the nuclear envelope (NE), and perinuclear [Ca2+]c and [Ca2+]n reached the same level within 2 s, although the NE worked as a weak but detectable barrier for Ca2+ diffusion. 4. Inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ release from the NE through the inner membrane was not detected, even when a large amount of IP3 was delivered in close proximity to the inner nuclear membrane. 5. When an oocyte was uniformly stimulated by photolysis of caged IP3, a Ca2+ rise was initiated in the perinuclear cytoplasm. The [Ca2+]n rise was always delayed with respect to, but rapidly equilibrated with, the [Ca2+]c rise. 6. Clusters of the endoplasmic reticulum were located in the perinuclear cytoplasm and served as the trigger zone of IP3‐induced Ca2+ release. 7. The results indicate that the [Ca2+]n rise occurs as the consequence of the influx of Ca2+ which was released in the perinuclear cytoplasm, not Ca2+ release from NE to the nucleoplasm.
Two components of exocytosis and endocytosis in phaeochromocytoma cells studied using caged Ca2+ compounds.
Authors: Kasai H , Takagi H , Ninomiya Y , Kishimoto T , Ito K , Yoshida A , Yoshioka T , Miyashita Y
Published: December 2014

1. Changes in membrane capacitance evoked by the rapid photolysis of a caged Ca2+ compound, DM‐nitrophen or nitrophenyl‐EGTA, were investigated in undifferentiated PC12 cells. They were interpreted as representing exocytosis and endocytosis. 2. The Ca2+ jumps evoked two components of exocytosis. Slow exocytosis was selectively evoked with small increases in intracellular Ca2+ concentration between 5 and 10 microM, while fast exocytosis preceded the slow one at [Ca2+]i greater than 10 microM. 3. The release rates of the two components of exocytosis depended steeply on [Ca2+]i. A half‐maximal release rate was achieved at 8 and 24 microM for the slow and fast exocytoses, respectively. 4. Prior Ca2+ rises did not augment the fast exocytosis. 5. The fast exocytosis was often followed by a rapid decrease in membrane capacitance, representing endocytosis, after a delay of 0.5‐2 s. The speed and delay in the fast endocytosis were Ca2+ dependent. Amounts of the fast endocytosis tended to balance with those of the fast exocytosis evoked by the same Ca2+ jumps. 6. The slow exocytosis was followed by a sluggish endocytosis that was associated with large capacitance steps indicative of secretory processes involving large dense‐core vesicles. The onset of the slow endocytosis exhibited a complex Ca2+ dependence. The amounts of the slow endocytosis appeared to parallel those of the slow exocytosis. Prior induction of the slow exocytosis gave rise to selective excess retrieval of membrane during the slow endocytosis. 7. These data indicate the existence of two distinct populations of secretory vesicles in PC12 cells. They seem to couple selectively with specific endocytotic mechanisms. Our data suggest that the two vesicles belong to two distinct secretory pathways.
Halothane and isoflurane preferentially depress a slowly inactivating component of Ca2+ channel current in guinea‐pig myocytes.
Authors: Pancrazio J J
Published: December 2014

1. The effects of the inhalational anaesthetics halothane and isoflurane on the high‐voltage‐activated Ca2+ channels were determined in isolated guinea‐pig ventricular myocytes using the patch‐clamp technique. 2. Recording solutions were equilibrated with inhalational anaesthetic vapour delivered from a calibrated vaporizer set at clinically relevant ranges of partial pressure. Anaesthetic concentrations in solution were determined using gas chromatography. 3. Halothane (0.9 mM in solution) and isoflurane (0.8 mM in solution) decreased peak whole‐cell CA2+ current (ICa) by approximately 40 and approximately 20%, respectively, while increasing the apparent rate of inactivation. 4. The sum of fast and slow exponential decay functions was required to fit the inactivation phase of ICa. The anaesthetics preferentially affected the slow component of inactivation while also increasing the rate of slow inactivation. The physiological significance of these effects was addressed by examining ICa evoked by a ventricular action potential waveform. 5. Measurement of the current carried by Ba2+ through Ca2+ channels (IBa) permitted the isolation of the slow component of inactivation. Halothane and isoflurane diminished peak IBa at 0 mV by approximately 45 and approximately 20% respectively, with similar changes in rate and magnitude of the slowly inactivating component as with ICa. 6. Cell‐attached patch‐clamp measurements of Ca2+ channel activity revealed that halothane did not alter single‐channel conductance. Instead, the anaesthetic reduced channel open probability to the same extent as observed during the whole‐cell recording, an effect partially due to an increase in null sweeps. In patches with a single channel present, the open‐time distribution, fitted by a single exponential, showed a decrease in mean open time. The closed‐time distribution, fitted by the sum of slow and fast exponential components, revealed an anaesthetic‐induced increase in the duration of the slow component with no effect on the fast component. Results are presented in terms of a channel‐gating model, and model predictions are examined with a computer simulation.
Silent calcium channels generate excessive tail currents and facilitation of calcium currents in rat skeletal myoballs.
Authors: Fleig A , Penner R
Published: December 2014

1. Whole‐cell patch‐clamp recording were employed to study facilitation of Ca2+ currents and excessive Ca2+ tail currents evoked by strong and long‐lasting conditioning depolarizations in skeletal myoballs cultured from newborn rats. 2. Paired‐pulse facilitation and excessive tail currents showed the same voltage dependence, becoming prominent at conditioning potentials above +30 mV. 3. Recruitment of excessive tail currents and facilitation occurred with the same time dependence (time constant (tau), approximately 200 ms to approximately 1 s), accelerating with the depolarization strength of conditioning pulses. 4. Reversal of Ca2+ current facilitation during the repolarization period between conditioning and test pulses was time‐ and voltage dependent. The time window of recruitment of facilitated Ca2+ currents narrowed considerably at more negative repolarization potentials (tau: approximately 10 ms at ‐100 mV, but approximately 1.5 at 0 mV). 5. Neither omission of internal ATP nor perfusion of the cells with the peptide inhibitor of protein kinase A (PKI) had significant effects on Ca2+ current facilitation, although internal perfusion with ATP gamma S slowly suppressed the facilitation currents by about 30%. External application of either ryanodine or caffeine under control conditions selectively and significantly suppressed the facilitated Ca2+ currents by about 30‐40%. 6. We propose that facilitation of Ca2+ currents and excessive tail currents are consequences of a common mechanism linked to ryanodine receptors.
ATP‐sensitive K+ channels are functional in expiratory neurones of normoxic cats.
Authors: Pierrefiche O , Bischoff A M , Richter D W
Published: December 2014

1. We analysed spontaneously active expiratory neurones (n = 48) of anaesthetized cats for the presence of ATP‐sensitive K+ (KATP) channels. 2. Intracellular injection of ATP reversibly depolarized neurones during all phases of the respiratory cycle. During expiration, membrane potential depolarized by an average of 1.5 +/‐ 0.1 mV leading to a 25% increase of discharge frequency. During inspiration, ATP induced a 1.8 +/‐ 0.2 mV depolarization, which was accompanied by a maximum of 20% increase of input resistance (Rn). 3. Extracellular application of diazoxide, an agonist of KATP channels, resulted in reversible membrane hyperpolarization in 68% of neurones (n = 19). This hyperpolarization (2.5 mV during expiration and 3.1 mV during inspiration) was accompanied by a 22% decrease in Rn. 4. Extracellular application of tolbutamide and glibenclamide, two antagonists of KATP channels, evoked reversible depolarizations in 76% of neurones (n = 21). The depolarization was relatively constant throughout the respiratory cycle (1.4 mV during expiration and 2.3 mV during inspiration). Rn increased by 22%. 5. The same sulphonylureas also changed the steepness of membrane depolarization when neurones escaped spontaneous synaptic inhibition during postinspiration. Extracellularly applied tolbutamide and glibenclamide increased the steepness of depolarization by 21%, while diazoxide reduced it by 20%. 6. Antagonism of drugs was verified by simultaneous extra‐ and intracellular application of diazoxide and glibenclamide, respectively. 7. During voltage clamp at holding potential at ‐60 to ‐67 mV, intracellular or extracellular application of tolbutamide and glibenclamide blocked a persistent outward current. 8. We conclude that KATP channels are functional in expiratory neurones of adult cats and contribute to the control of excitability even during normoxia.
Rapid exocytosis and endocytosis in nerve terminals of the rat posterior pituitary.
Authors: Hsu S F , Jackson M B
Published: December 2014

1. Ca(2+)‐induced exocytosis and endocytosis were studied by measuring the membrane capacitance of voltage‐clamped peptidergic nerve terminals in slices prepared from the rat posterior pituitary. 2. Depolarizing pulses produced rapid increases in capacitance. These increases varied in parallel with Ca2+ current as voltage was varied. Elimination of Ca2+ current blocked depolarization‐induced capacitance changes. 3. Depolarization‐induced capacitance changes increased with pulse duration. Capacitance changes also increased with integrated Ca2+ influx, but saturated at high levels of Ca2+ entry. This saturation allowed us to estimate a pool size of 190 vesicles, assuming each vesicle has a capacitance of 1 fF. Vesicles from this pool fused with a time constant of 0.43 s. The capacitance change increased with the first power of integrated Ca2+ influx. 4. Experiments with briefer pulses revealed a rapid component of exocytosis comprising a pool of forty vesicles that fuse with a time constant of 14 ms. This rapid process may reflect a final Ca(2+)‐regulated triggering step, which is distinct from the slower kinetic step revealed by longer duration pulses. The slower step may reflect a priming of vesicles prior to exocytosis. 5. Depolarization‐induced capacitance increases in most cases were followed by a rapid decay in capacitance, reflecting membrane reuptake tightly coupled to exocytosis. A variable amount of rapid endocytosis followed depolarization‐induced capacitance increases. The time constant for rapid endocytosis to baseline was 0.44 s. Excess endocytosis was occasionally observed, with capacitance decaying below the pre‐stimulus baseline with a time constant of 2.1 s. 6. Rapid endocytosis was slower after pulses that produced greater increases in intracellular Ca2+, consistent with the hypothesis that intracellular Ca2+ inhibits rapid endocytosis. 7. Exocytosis follows depolarization with no detectable delay, indicating that Ca2+ triggers neuropeptide secretion from nerve terminals with kinetics comparable to that observed in other rapidly secreting systems.
On muscarinic control of neurogenic mucus secretion in ferret trachea.
Authors: Ramnarine S I , Haddad E B , Khawaja A M , Mak J C , Rogers D F
Published: December 2014

1. Muscarinic receptor subtypes mediating neurogenic mucus secretion in ferret trachea were characterized in vitro and in vivo using 35SO4 as a label for secreted mucus, and the muscarinic receptor antagonists telenzepine for the M1 receptor subtype, methoctramine for the M2 subtype and 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP) for the M3 receptor. We also performed receptor binding and mapping studies. 2. Each muscarinic antagonist displaced [N‐methyl‐3H]scopolamine binding with high‐affinity binding constant (KH) values of 1.9, 2.7 and 5.0 nM for telenzepine, methoctramine and 4‐DAMP, respectively. Muscarinic M1 and M3 receptors localized to submucosal glands, whereas M2 receptors did not. 3. In vitro, electrical stimulation (50 V, 10 Hz, 0.5 ms for 5 min) increased 35SO4 output by 160%. Telenzepine did not inhibit the neurogenic secretory response at concentrations two‐or twentyfold its KH value, nor did it inhibit secretion induced by acetylcholine (ACh). 4‐DAMP inhibited neurogenic secretion by 80 and 95%, respectively, at concentrations two‐and twentyfold its KH value, and also inhibited ACh‐induced secretion. Methoctramine potentiated neurogenic secretion induced at 2.5 Hz (50 V, 0.5 ms for 5 min) in a dose‐related (5.4‐100 nM) manner with increases of 33‐451% above electrically stimulated values. Methoctramine did not potentiate secretion induced at 10 Hz and did not have any effect on ACh‐induced secretion. 4. In vivo, vagal stimulation (10 V, 10 Hz, 2 ms for 8 min) increased output of 35SO4 by approximately 120%. Telenzepine had no significant effect on neurogenic secretion. Methoctramine approximately doubled the stimulated response, whereas 4‐DAMP abolished the stimulated secretory response. 5. We conclude that in ferret trachea, cholinergic nerve stimulation increases mucus secretion via muscarinic M3 receptors on the submucosal glands. The magnitude of the secretory response is regulated by neuronal M2 muscarinic receptors. The muscarinic M1 receptors localized to the submucosal glands do not appear to be involved with mucus secretion.
The mechanism of calcium channel facilitation in bovine chromaffin cells.
Authors: Albillos A , Gandía L , Michelena P , Gilabert J A , del Valle M , Carbone E , García A G
Published: December 2014

1. This study was planned to clarify the mechanism of Ca2+ channel facilitation by depolarizing prepulses given to voltage‐clamped bovine chromaffin cells. The hypothesis for an autocrine modulation of such channels was tested by studying the effects of a soluble vesicle lysate (SVL) on whole‐cell Ba2+ currents (IBa). 2. SVL was prepared from a bovine adrenal medullary homogenate. The ATP content in this concentrated SVL amounted to 3.18 +/‐ 0.12 mM (n = 4). The concentration of noradrenaline and adrenaline present in the SVL was 11.2 +/‐ 0.97 and 15.2 +/‐ 2 mM, respectively (n = 5). A 1:1000 dilution of SVL in the external solution halved the magnitude of IBa and produced a 7‐fold slowing of its activation kinetics. The blocking effects of SVL were concentration dependent and quickly reversed upon washout. 3. Inhibition and slowing of the kinetics of IBa by SVL could be partially reversed by strong depolarizing prepulses (+90 mV, 45 ms). This reversal of inhibition, called Ca2+ channel facilitation, persisted in the presence of 3 microM nifedipine. 4. Intracellular dialysis of GDP‐beta‐S (0.5 mM) or pretreatment of the cells with pertussis toxin (100 ng ml‐1 for 18‐24 h) prevented the reduction in peak current caused by a 1:100 dilution of SVL; no prepulse facilitation could be observed under these conditions. 5. The receptor blockers naloxone (10 microM) or suramin (100 microM) and PPADS (100 microM) largely antagonized the effects of SVL. Treatment of SVL with alkaline phosphatase or dialysis against a saline buffer to remove low molecular mass materials (< 10 kDa) considerably reduced the activity of SVL. 6. Stopping the flow of the external solution (10 mM Ba2+) gradually reduced the size, and slowed down the activation phase, of the current. Prepulse facilitation of IBa was absent or weak in a superfused cell, but was massive upon flow‐stop conditions in the presence or absence of 3 microM nifedipine. 7. Our experiments suggest that facilitation by prepulses of whole‐cell current through Ca2+ channels is due to the suppression of an autoinhibitory autocrine loop present in bovine chromaffin cells. By acting at least on purinergic and opiate receptors, the exocytotic release of ATP and opiates will cause a tonic inhibition of the current through a G‐protein‐mediated mechanism. Such a mechanism will be removed by strong depolarizing prepulses, and will involve preferentially non‐L‐type channels. In the light of these and other recent results, previously held views on the selective recruitment by prepulses of dihydropyridine‐sensitive Ca2+ channels are not tenable.
Mechanisms of activation of muscle branched‐chain alpha‐keto acid dehydrogenase during exercise in man.
Authors: van Hall G , MacLean D A , Saltin B , Wagenmakers A J
Published: December 2014

1. Exercise leads to activation (dephosphorylation) of the branched‐chain alpha‐keto acid dehydrogenase (BCKADH). Here we investigate the effect of low pre‐exercise muscle glycogen content and of branched‐chain amino acid (BCAA) ingestion on the activity of BCKADH at rest and after 90 min of one‐leg knee‐extensor exercise at 65% maximal one‐leg power output in five subjects. 2. Pre‐exercise BCAA ingestion (308 mg BCAAs (kg body wt)‐1) caused an increased muscle BCAA uptake, a higher intramuscular BCAA concentration and activation of BCKADH both at rest (9 +/‐ 1 versus 25 +/‐ 5% for the control and BCAA test, respectively) and after exercise (27 +/‐ 4 versus 54 +/‐ 7%). 3. At rest the percentage active BCKADH was not different, 6 +/‐ 2% versus 5 +/‐ 1%, in the normal and low glycogen content leg (392 +/‐ 21 and 147 +/‐ 34 mumol glycosyl units (g dry muscle)‐1, respectively). The post‐exercise BCKADH activity was higher in the low (46 +/‐ 2%) than in the normal glycogen content leg (26 +/‐ 2%). 4. It is concluded that: (1) the mechanism of activation by BCAA ingestion probably involves an increase of the muscle BCAA concentration; (2) BCKADH activation caused by exercise and BCAA ingestion are additive; (3) low pre‐exercise muscle glycogen content augments the exercise‐induced BCKADH activation without an increase in muscle BCAA concentration; and (4) the mechanism of BCKADH activation via BCAA ingestion and low muscle glycogen content are different.
Phase‐shifting human circadian rhythms: influence of sleep timing, social contact and light exposure.
Authors: Duffy J F , Kronauer R E , Czeisler C A
Published: December 2014

1. Both the timing of behavioural events (activity, sleep and social interactions) and the environmental light‐dark cycle have been reported to contribute to entrainment of human circadian rhythms to the 24 h day. Yet, the relative contribution of those putative behavioural synchronizers to that of light exposure remains unclear. 2. To investigate this, we inverted the schedule of rest, sedentary activity and social contact of thirty‐two young men either with or without exposure to bright light. 3. On this inverted schedule, the endogenous component of the core temperature rhythm of subjects who were exposed to bright light showed a significant phase shift, demonstrating that they were adapting to the new schedule. In contrast, the core temperature rhythm of subjects who were not exposed to bright light moved on average 0.2 h later per day and after 10 days had not significantly adapted to the new schedule. 4. The direction of phase shift in the groups exposed to bright light was dependent on the time of bright light exposure, while control subjects drifted to a later hour regardless of the timing of their schedule of sleep timing, social contact and meals. 5. These results support the concept that the light‐dark cycle is the most important synchronizer of the human circadian system. They suggest that inversion of the sleep‐wake, rest‐activity and social contact cycles provides relatively minimal drive for resetting the human circadian pacemaker. 6. These data indicate that interventions designed to phase shift human circadian rhythms for adjustment to time zone changes or altered work schedules should focus on properly timed light exposure.
Ca(2+)‐dependent inactivation of NMDA receptors: fast kinetics and high Ca2+ sensitivity in rat dorsal horn neurons.
Authors: Kyrozis A , Albuquerque C , Gu J , MacDermott A B
Published: December 2014

1. Ca(2+)‐dependent inactivation (CDI) of NMDA receptors was studied using both cultured embryonic rat dorsal horn neurons and acutely dissociated postnatal rat dorsal horn neurons. The perforated patch recording method was employed in order to preserve intracellular Ca2+ buffers and other cellular constituents. In this way, the kinetics of intracellular Ca2+ concentration ([Ca2+]i) transients and other second messenger signalling systems were maintained in a relatively normal condition. 2. Continuous application of 30 microM NMDA to cultured dorsal horn neurons voltage clamped at ‐70 mV evoked currents that inactivated to about 40% of the peak value with time constants between 200 and 600 ms. CDI with similar kinetics was also observed in acutely dissociated postnatal rat dorsal horn neurons. 3. When NMDA was applied in a low (20 microM) Ca2+ bath or when dorsal horn neurons were held at +70 mV, inactivation was either very weak or absent. The peaks of NMDA currents were significantly suppressed when preceded by voltage steps to 0 mV or by evoked action potentials. The suppression was dependent on the presence of Ca2+ in the extracellular solution. Voltage steps to +100 mV were ineffective in suppressing NMDA responses. Therefore, the observed inactivation was caused by an increase in [Ca2+]i following Ca2+ entry through NMDA channels or through voltage gated Ca2+ channels. 4. Caffeine application reduced currents evoked by subsequent NMDA applications. This reduction was not dependent on the presence of extracellular Ca2+ but was abolished after incubation of the cells with ryanodine, suggesting that Ca2+ release from intracellular stores also induced CDI. 5. Simultaneous measurements of somal [Ca2+]i and of currents evoked by somal NMDA applications showed that the magnitude of CDI was correlated with [Ca2+]i levels and that [Ca2+]i elevations of 100‐300 nM were usually sufficient to inactivate NMDA currents by more than 30%. 6. Dose‐response curves of non inactivated and inactivated NMDA responses showed that the apparent receptor affinity for NMDA is not different under the two conditions. CDI is caused instead by non‐competitive inhibition of NMDA receptors. CDI was not overcome by increasing glycine concentration, suggesting that it is not mediated by glycine dissociation from the receptor. 7. These results show that, with an intact intracellular environment, CDI in dorsal horn neurons constitutes a potent, inhibitory control of NMDA currents with a faster onset than previously demonstrated. CDI is induced by a variety of [Ca2+]i‐elevating stimuli of physiological relevance including Ca2+ entry through ligand‐ and voltage‐gated channels and Ca2+ release from intracellular stores. Our demonstration that CDI is strongly expressed in neurons maturing in vivo supports the hypothesis that CDI may regulate, in part, the postsynaptic integration of excitatory input in the mature or maturing nervous system.
Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes.
Authors: Clément-Chomienne O , Walsh M P , Cole W C
Published: December 2014

1. The effect of angiotension II (Ang) on delayed rectifier K+ current (IK(V)) was studied in isolated rabbit portal vein smooth muscle cells using standard whole‐cell voltage clamp technique. The effect of 100 nM Ang on macroscopic, whole‐cell IK(V) was assessed in myocytes dialysed with 10 mM BAPTA, 5 mM ATP and 1 mM GTP either at room temperature or at 30 degrees C. 2. Application of Ang caused a decline in IK(V) which was reversed upon washout of the drug. Tail current recorded after 250 ms pulses to +30 mV and repolarization to ‐40 mV was reduced from 3.9 +/‐ 0.7 to 2.5 +/‐ 0.5 pA pF‐1 at 20 degrees C (n = 6) and from 4.5 +/‐ 0.5 to 3.13 +/‐ 0.4 pA pF‐1 at 30 degrees C(n = 17). 3. Ang had no effect on outward current in the presence of an AT1 selective antagonist, losartan (1 microM), which alone had no direct effect on the amplitude of IK(V). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 microM intracellular BAPTA did not affect the suppression of IK(V) by Ang. 4. Ang induced a decrease in time constant for the rapid phase of inactivation of the macroscopic current (tau 1 reduced from 377 +/‐ 32 to 245 +/‐ 11 ms; tau 2 unchanged, n = 17). Neither the voltage dependence of activation nor inactivation were affected by Ang. 5. The inhibition of IK(V) by Ang was abolished by intracellular dialysis with the selective PKC inhibitors, calphostin C (1 microM) and chelerythrine (50 microM). These data provide strong evidence that the decline in IK(V) due to Ang treatment is due to PKC activation. 6. The pattern of expression of PKC isoforms was examined in rabbit portal vein using isoenzyme‐specific antibodies: alpha, epsilon and zeta isoenzymes were detected, but beta, gamma, delta and eta isoenzymes were not. 7. The lack of requirement for Ca2+, as well as the sensitivity of the Ang response to chelerythrine, suggest the involvement of the Ca(2+)‐independent PKC isoenzyme epsilon in the signal transduction pathway responsible for IK(V) inhibition by Ang.
Block of large conductance Ca(2+)‐activated K+ channels in rabbit vascular myocytes by internal Mg2+ and Na+.
Authors: Morales E , Cole W C , Remillard C V , Leblane N
Published: December 2014

1. We studied the biophysical properties of single large conductance (> 200 pS in symmetrical K+ pipette and bath solutions) Ca(2+)‐activated K+ (BKca) channels of rabbit portal vein and coronary arterial smooth muscle cells using the cell‐attached and inside‐out variants of the patch‐clamp technique (at 22 degrees C). 2. The unitary conductance of BKca channels recorded in cell‐attached patches with K+ concentrations in the range 5.4‐140 mM was significantly lower than that predicted on the basis of the conductance measured in inside‐out patches with symmetrical K+ pipette and bath solutions (140 mM) and the constant field equation. In cell‐attached patches from cells bathed in depolarizing medium (140 mM) with 5.4 mM K+ in the pipette solution, BKca channels were difficult to detect on the physiological range of membrane potentials (approximately ‐50 mV). Unitary currents were smaller at all voltages in the range ‐50 to 0 mV and the i‐V relationship exhibited strong inward rectification at potentials > 0 mV. These channels were unequivocally identified as BKca channels due to their sensitivity to caffeine (10 mM) and iberiotoxin (20 nM), and their non‐stationary kinetic properties. 3. Exposure of the cytoplasmic side of excised patches to [Mg2+] in the range 0‐15 mM produced two effects on BKca channel activity: the slope conductance and open probability were reduced and enhanced, respectively, in a concentration‐dependent manner by this cation. The Mg(2+)‐induced reduction in conductance exhibited weak voltage dependence. 4. Application of 20 mM Na+ to the internal face of BKca channels recorded in the inside‐out configuration produced a flickery block at potentials > or = +20 mV resulting in reduced unitary current amplitudes and strong inward rectification of the i‐V relationship. Exposure of inside‐out patches to a combination of 20 mM Na+ and 2 mM Mg2+ further reduced unitary current amplitude to a level similar to the algebraic sum of the effect of each cation in isolation. 5. We conclude that Ca(2+)‐dependent K+ channels of vascular smooth muscle cells display a lower unitary conductance when recorded under physiological conditions than that previously estimated on the basis of their behaviour in excised membrane patches. Our data indicate that the decreased permeation through BKca channels may be partly attributed to block by intracellular Mg2+ and Na+, which appear to interact with distinct binding sites along the inner side of the pore.
Ryanodine produces a low frequency stimulation‐induced NMDA receptor‐independent long‐term potentiation in the rat dentate gyrus in vitro.
Authors: Wang Y , Wu J , Rowan M J , Anwyl R
Published: December 2014

1. The induction of long‐term potentiation (LTP) was investigated in the rat dentate gyrus in the presence of ryanodine, an agent which is known to selectively bind to the ryanodine receptor (RyR) Ca2+ channels which regulate Ca2+ release from intracellular Ca2+ stores. 2. In control media, high frequency stimulation (HFS) induced LTP, and prolonged low frequency stimulation (LFS) induced long‐term depression (LTD), of field excitatory postsynaptic potentials (EPSPs) and patch clamped excitatory postsynaptic currents (EPSCs). 3. In the presence of ryanodine, at a threshold concentration of about 1 microM, HFS‐induced LTP was inhibited, whereas LFS (5 Hz, 900 pulses) now induced LTP. 4. The N‐methyl‐D‐aspartate receptor (NMDAR) antagonist D‐2‐amino‐phosphonopentanoate (D‐AP5), at both 50 and 200 microM, did not prevent the induction of LTP by 5 Hz LFS in the presence of ryanodine. This demonstrates the NMDAR independence of LTP induction in the presence of ryanodine. Furthermore, D AP5 reversed the block of HFS‐induced LTP by ryanodine. 5. The induction of LTP by 5 Hz LFS in the presence of ryanodine was blocked by lowering extracellular Ca2+, or by rapidly buffering intracellular Ca2+ to very low levels with BAPTA. 6. The induction of LTP by 5 Hz LFS was inhibited by the L‐type voltage‐gated Ca2+ channel blocker nifedipine, and also by Ni2+ a commonly used T type voltage‐gated Ca2+ channel blocker. 7. The 5 Hz LFS‐induced LTP in the presence of ryanodine was inhibited by the metabotropic glutamate receptor (mGluR) antagonist (+)‐alpha‐methyl 4‐carboxyphenylglycine (MCPG). 8. The 5 Hz LFS‐induced LTP in the presence of ryanodine was blocked by Ruthenium Red, an agent known to block RyR channel opening, and also by thapsigargin, an agent known to block‐ATP‐dependent Ca2+ uptake into endoplasmic reticulum. 9. The results of the present studies emphasize the importance of intracellular Ca2+ stores in the induction of LTP.
Regional differences in the response of the isolated sino‐atrial node of the rabbit to vagal stimulation.
Authors: Kodama I , Boyett M R , Suzuki R , Honjo H , Toyama J
Published: December 2014

1. The effects of brief postganglionic vagal nerve stimulation on electrical activity in different regions of the rabbit sino‐atrial node and surrounding atrial muscle were recorded. 2. At the centre of the node (the leading pacemaker site), the brief stimulation resulted in a large hyperpolarization followed by a depolarization and a shortening of the action potential. All effects were short lasting (time to 90% recovery of membrane potential, 0.8 s). 3. At other sites within the node and in the surrounding atrial muscle, although there was still a substantial action potential shortening, the hyperpolarization was smaller and the depolarization was small or absent. All effects were longer lasting (time to 90% recovery in atrial muscle, 11.4s). 4. The depolarization in the centre of the node was abolished by block of the hyperpolarization‐activated current (i(f)) by Cs+ or zatebradine (UL‐FS 49). It could, therefore, result from the activation of i(f) during the preceding hyperpolarization. 5. Block of acetylcholinesterase by eserine greatly slowed recovery from vagal stimulation at all sites, demonstrating that recovery is dependent on acetylcholinesterase. The longer lasting effects of vagal stimulation in atrial muscle, therefore, result from lower acetylcholinesterase activity. 6. Vagal stimulation resulted in a short lasting initial slowing of spontaneous action potentials followed by a long‐lasting secondary slowing. Whereas the initial slowing coincided with the effects of vagal stimulation on the centre of the node, the secondary slowing coincided with the slower effects of vagal stimulation on the surrounding atrial muscle. The secondary slowing was reduced by 68 +/‐ 11% (n = 5) by cutting the atrial muscle away from the node. 7. It is concluded that the short‐lasting initial slowing of spontaneous action potentials is the direct effect of vagal stimulation on the centre of the sino atrial node, whereas the secondary slowing is the result of the longer lasting effects of vagal stimulation on the surrounding atrial muscle and the electrotonic suppression of the node by the muscle.
Effects of arterial perfusion pressure on force production in working human hand muscles.
Authors: Fitzpatrick R , Taylor J L , McCloskey D I
Published: December 2014

1. The effects of hydrostatic changes in perfusion pressure on performance of working fatigue‐resistant muscle fibres in the hand were studied in six normal subjects. 2. Supramaximal stimuli were delivered in trains of 200 ms duration, at 1 train S‐1, to the ulnar nerve proximal to the wrist to produce isometric contractions of adductor pollicis. The force of contraction was measured and, after a level contraction force was achieved, the arm was passively raised or lowered. 3. Lifting the hand 45 cm above the heart produced a decline in force output from the muscle within several seconds which, after 4 min, fell by 22% below the steady‐state level. Lowering the hand 45 cm below heart level produced an 8% increase in force output. Greater changes in force output occur at higher workloads. 4. It is concluded that in human subjects, muscle performance is sensitive to changes in perfusion pressure that occur across the physiological range. At moderate work levels, force output of the working muscles in the hand can vary by up to 30% over the physiological range of blood pressure. This dependence of muscular force on blood perfusion is of potential importance to motor control during normal activities.
M1 muscarinic receptor‐induced facilitation of ACh and noradrenaline release in the rat bladder is mediated by protein kinase C.
Authors: Somogyi G T , Tanowitz M , Zernova G , de Groat W C
Published: December 2014

1. [3H]Noradrenaline (NA) AND [14C]acetylcholine (ACh) released by electrical field stimulation were measured simultaneously in strips from the body of rat urinary bladder. 2. [3H]NA and [14C]ACh release was greater during continuous stimulation (CS; 10 Hz, 100 shocks) or in the presence of eserine than during intermittent train stimulation (IS; 10 Hz, 10 shocks every 5 s, 10 times). Atropine (1 microM) or pirenzepine (0.05‐0.1 microM) blocked the CS‐ or eserine‐facilitated release. 3. The protein kinase C (PKC) activator phorbol dibutyrate (PDB; 0.05 and 0.5 microM) increased the release of both [3H]NA and [14C]ACh in a concentration‐dependent manner. Atropine blocked the PDB‐induced facilitation of ACh release but not the facilitation of NA release. 4. The protein kinase A (PKA) activator 8‐Br‐cAMP did not affect ACh release but enhanced NA release. 5. The PKC inhibitor H‐7 (50‐100 microM) inhibited the CS‐ or eserine‐facilitated release of both ACh and NA, but did not affect the non‐facilitated release evoked by IS. H‐7 also inhibited 0.5 microM PDB‐induced facilitation of ACh release but not NA release. 6. Down‐regulating PKC by pretreatment for 30 min with 5 microM PDB decreased the facilitated release of ACh and the eserine‐induced facilitation of NA release. 7. Electrically evoked contractions of the bladder strips exhibited a biphasic response to PDB (2.5 microM), which consisted of an initial enhancement of the peak amplitude and area followed after 20 min by an inhibition of contractions. H‐7 inhibited the electrically evoked contractions in a dose‐dependent fashion. 8. It is concluded that a phospholipase C‐PKC signal transduction pathway is essential for muscarinic receptor‐induced facilitation of ACh and NA release but is not involved in the non‐facilitated release of transmitters in the rat urinary bladder.