bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of forskolin with the effect of atropine on [3H]acetylcholine secretion in guinea‐pig ileum myenteric plexus.
Authors: Alberts P , Ogren V R
Published: December 2014

1. Secretion of [3H]acetylcholine was studied in the guinea‐pig ileum longitudinal muscle‐myenteric plexus preparation. The transmitter stores of the cholinergic nerves were labelled by pre‐incubation with [3H]choline. The preparation was mounted in an organ bath and superfused with Tyrode solution containing hemicholinium‐3 and eserine. [3H]Acetylcholine secretion was evoked by electrical stimulation (0.5 Hz, 150 shocks). 2. 8‐Bromo cyclic AMP, the adenylate cyclase activator forskolin, and the cyclic nucleotide phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine enhanced the [3H]acetylcholine secretion in a concentration‐dependent manner. The values of 'maximal enhancement' calculated were similar, viz. 200‐300% of control. 3. 8‐Bromo cyclic GMP reduced the [3H]acetylcholine secretion. 4. The 'maximal enhancement' of 3‐isobutyl‐1‐methylxanthine was not altered by the presence of forskolin (25 mumol/l) suggesting a common mechanism of action, i.e. elevation of endogenous cyclic AMP levels. 5. The muscarinic acetylcholine receptor antagonist atropine enhanced the [3H]acetylcholine secretion with a 'maximal enhancement' of 506% of control. Presence of neither forskolin (25 mumol/l) nor 3‐isobutyl‐1‐methylxanthine (5 mmol/l) altered the 'maximal enhancement' for atropine. 6. In contrast, atropine (1 mumol/l) and 4‐aminopyridine (0.5 mmol/l) additively enhanced the [3H]acetylcholine secretion. 7. The results suggest that neuronal cyclic AMP may be involved in muscarinic acetylcholine receptor‐mediated control of [3H]acetylcholine secretion in guinea‐pig ileum myenteric plexus.
A chloride conductance activated by adenosine 3',5'‐cyclic monophosphate in the apical membrane of Necturus enterocytes.
Authors: Giraldez F , Sepúlveda F V , Sheppard D N
Published: December 2014

1. Intracellular potentials, Cl‐ activity and membrane resistances were measured in Necturus small intestinal epithelium during Cl‐ replacement experiments using conventional or Cl‐ ‐selective double‐barrelled microelectrodes. A Cl‐ conductance, located in the apical membrane and activated by cyclic nucleotides is demonstrated by ion‐substitution experiments. 2. The mean mucosal membrane potential (Em) was ‐35.5 mV. Removal of Cl‐ from the mucosal medium by replacement with gluconate, evoked a sudden depolarization of Em and an immediate increase in the fractional resistance of the mucosal membrane (f(Rm)). The size of the change in Em varied between 3 and 65 mV, corresponding to Cl‐ to K+ permeability ratios between 0.2 and 20. It was inversely related to the initial f(Rm), which ranged from 0.04 to 0.50. 3. Prolonged incubation in low‐Cl‐ solutions led to a reversal of the initial depolarization and to a sustained hyperpolarization accompanied by a marked increase in f(Rm). The new value of Em was close to the K+ equilibrium potential, consistent with a depletion of cellular Cl‐ and the preponderance of a K+ membrane permeability in the absence of Cl‐. This emphasizes the role of Cl‐ in establishing Em. 4. Removal of mucosal Cl‐ produced a fast decrease in intracellular Cl‐, as measured with Cl‐ ‐selective microelectrodes. The efflux was consistent with electrodiffusion across the mucosal membrane. Changes in Em paralleled changes in intracellular Cl‐ activity, indicating the presence of a large Cl‐ conductance. 5. Dibutyryl cyclic AMP or forskolin produced a slow depolarization, a decrease in f(Rm) and an increased change in intracellular potential in low mucosal Cl‐ which on average corresponds to an approximately 15‐fold increase in the relative Cl‐ permeability. These results are consistent with an activation of apical Cl‐ conductance. 6. The selectivity of Cl‐ channels of Necturus enterocytes to different anions was obtained from potential measurements. The sequence of permeabilities was SCN‐ greater than I‐ greater than or equal to Br‐ greater than NO3‐ greater than Cl‐ much greater than HCO3‐ greater than gluconate. This is consistent with a model involving a weak interaction of the anions with the selectivity filter. 7. The selectivity of the anion conductance was maintained after activation with cyclic nucleotides, suggesting a single channel for the permeation of the different anions tested, rather than parallel channels. 8. Derivatives of 9‐anthracene which are potent inhibitors of Cl‐ channels in other systems failed to block the apical Cl‐ conductance of Necturus enterocytes. Chloride conductance was also insensitive to furosemide.(ABSTRACT TRUNCATED AT 400 WORDS)
Cations that alter surface potentials of lipid bilayers increase the calcium requirement for exocytosis in sea urchin eggs.
Authors: McLaughlin S , Whitaker M
Published: December 2014

1. We examined the effects of cations that bind to phospholipid bilayers on the calcium requirement for exocytosis in vitro. 2. Tetracaine and trifluoperazine, two monovalent cations that bind hydrophobically to lipid bilayers, decreased the zeta‐potential of lipid vesicles with a phospholipid composition similar to sea urchin plasma membranes; the decrease in the magnitude of the negative zeta‐potential was consistent with the Gouy‐Chapman‐Stern theory. 3. Trifluoperazine and tetracaine also increased the concentration of calcium required to produce half‐maximal exocytosis (Ca50) in isolated fragments of plasma membranes from sea urchin eggs. 4. The effects of trifluoperazine and tetracaine on the Ca50 of egg fragments can be explained quantitatively from the zeta‐potential data obtained with phospholipid vesicles if we assume the calcium binding sites responsible for triggering exocytosis lie within a Debye length (less than 1 nm) of the surface of the plasma membranes, such that the concentration of calcium at the sites is affected by the surface potential of the membrane. 5. The divalent cation magnesium, which binds specifically to the phosphate group of lipids, affected Ca50 in a manner that can also be explained quantitatively from its effects on the zeta‐potential of phospholipid vesicles. 6. The polyvalent cation neomycin, which adsorbs to the lipid phosphatidylinositol 4,5‐bisphosphate with high affinity, also binds co‐operatively to phosphatidylinositol; its effect on Ca50 can be explained quantitatively from its effects on the zeta‐potential of vesicles containing the latter lipid. 7. A decrease in the ionic strength increases the magnitude of the surface potential of membranes; the effect of ionic strength on Ca50 can be explained qualitatively from its effect on the zeta‐potential of phospholipid vesicles. 8. All our results are consistent with the hypothesis that calcium binding sites lie within a Debye length of the plasma membrane; they also indicate that drugs with specific, high‐affinity sites may affect exocytosis by a non‐specific mechanism.
Kinetic differences between embryonic‐ and adult‐type acetylcholine receptors in rat myotubes.
Authors: Jaramillo F , Schuetze S M
Published: December 2014

1. The burst structures of embryonic‐type (low‐gamma) and adult‐type (high‐gamma) nicotinic acetylcholine (ACh) receptors (AChRs) in rat myotubes were investigated with the patch clamp technique. The channels were activated with the agonists ACh and suberyldicholine (SubCh). 2. With either agonist, the distribution of burst durations showed two exponential components for both channel types: a 'long' component that corresponds to bursts of one or more openings and a 'brief' component that includes short, isolated openings. 3. For low‐gamma channels, the percentage of all openings associated with the brief component decreased from approximately 40% at 10‐100 nM‐ACh to less than 10% at 10‐100 microM‐ACh. 4. Both high‐gamma and low‐gamma long bursts were interrupted by brief (30‐90 microseconds) closures and longer (approximately 1 ms) transitions to a partially open (subconductance) state. The duration of brief closures and partial openings was relatively independent of the agonist, but their frequency within low‐gamma bursts was 3‐fold higher with SubCh than with ACh. 5. Brief closures are interpreted as transitions to a closed, doubly liganded state from which the channel can reopen. This model predicts that the channel opening rate is greater than 10,000 s‐1 for both channel types at room temperature. 6. Estimates of the channel opening rate inferred from the rising phase of miniature end‐plate currents recorded from rat soleus fibres are consistent with this interpretation. 7. Both high‐gamma and low‐gamma channels apparently operate via similar gating mechanisms. Differences in their gating behaviour can be explained in terms of faster kinetic rate constants for high‐gamma channels.
A modulatory action of divalent cations on transient outward current in cultured rat sensory neurones.
Authors: Mayer M L , Sugiyama K
Published: December 2014

1. The effects of some divalent cations on the A‐current (IA) in cultured rat dorsal root ganglion cells (DRGs) were studied using whole‐cell patch recording. 2. IA was not affected by omission of calcium from the external medium; however it was significantly depressed by manganese (10 mM) applied by pressure ejection. This depressant effect of manganese resulted from a depolarizing shift of the activation curve by 17 mV, associated with only a slight reduction of the maximum conductance. At 10 mM manganese also caused a depolarizing shift of the steady‐state inactivation curve by 34 mV. Divalent cations other than manganese also gave positive shifts of the steady‐state activation and inactivation curves for IA but were of different potency; the sequence was: Cd2+ greater than Mn2+ = Co2+ greater than Mg2+. 3. A dose‐response curve for the depolarizing shift of the activation and inactivation curves of IA, as a function of manganese concentration, could be fitted by a single binding site model with an apparent dissociation constant of approximately 17 mM. The depolarizing shift of the inactivation curve was on average twice as large as that of the activation curve. 4. In contrast to its effect on IA, manganese (10 mM) did not cause any appreciable change in the voltage dependence of the activation curve for the delayed rectifier K+ current. 5. A low concentration of manganese (1 mM) increased the amplitude of IA recorded at pre‐pulse potentials ranging from ‐50 to ‐70 mV. This augmentation of IA resulted from a positive shift of the inactivation curve by 6 mV without an appreciable shift of the activation curve; as a result a population of A‐channels is released from inactivation over pre‐pulse potentials from ‐50 to ‐70 mV. 6. These results show that divalent cations can evoke a depolarizing shift of both the activation and inactivation gates controlling IA; this causes either depression or augmentation of IA, depending on the species and concentration of the divalent cation, and also on the pre‐ pulse potential used to de‐inactivate IA. This modulatory effect of divalent cations on the gating of IA appears to reflect binding to a specific, saturable site, either the A‐channel protein itself, or phospholipids electrically close to the gating apparatus.
The interactions between potassium and sodium currents in generating action potentials in the rat sympathetic neurone.
Authors: Belluzzi O , Sacchi O
Published: December 2014

1. Membrane conductance parameters for the rat sympathetic neurone in vitro at 37 degrees C have been determined by two‐electrode voltage‐clamp analysis. The activation kinetics of two ionic currents, IA and IK(V), has been considered. Data for both currents are expressed in terms of Hodgkin‐Huxley equations. 2. The isolated IA developed following third‐order kinetics. The activation time constant, tau a, was estimated from the current time‐to‐peak and, for V less than or equal to ‐40 mV, from the IA tail current analysis upon membrane repolarization to various potentials. The maximum tau a occurred at ‐55 mV and varied from 0.26 to 0.82 ms in the range of potentials between ‐100 and +10 mV. The steady‐state value of the variable a, corrected for inactivation, was evaluated in the voltage range from ‐60 to 0 mV; 14.4 mV are required to change a infinity e‐fold. Steady‐state gA was voltage dependent, increasing with depolarization to a maximum of 1.40 microS at +10 mV. 3. IK(V) was similarly analysed in isolation. The current proved to develop as a first‐order process. tau n was determined by fitting a single exponential to the IK(V) rising phase and to the tail currents at the end of short depolarizing pulses. The bell‐shaped voltage dependence of tau n exhibited a maximum (25.5 ms) at ‐30 mV, becoming minimal (1.8 ms) at ‐80 and +20 mV. The n infinity curve was obtained (n infinity = 0.5 at ‐6.54 mV; k = 8.91 mV). The mean maximum conductance, gK(V), was 0.33 microS per neurone at +10 mV. 4. Single spikes have been elicited by brief current pulses at membrane potentials from ‐40 to ‐100 mV under two‐electrode current‐clamp conditions in normal saline and in the presence of blockers of the ICa‐IK(Ca) (Cd2+) and/or IK(V) (TEA, tetraethylammonium) systems. Spike repolarization was affected by the suppression of either current in the depolarized neurone, but was insensitive to both treatments when the spike arose from holding levels negative to ‐75 to ‐80 mV, indicating that at these membrane potentials the IA current mainly, if not exclusively, contributes to the action potential falling phase. 5. The basic features of the sympathetic neurone action potential were reconstructed by simulations based on present and previous voltage‐clamp characterization of the IA, IK(V) and INa conductances.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of a phorbol ester on acetylcholine‐induced Ca2+ mobilization and contraction in the porcine coronary artery.
Authors: Itoh T , Kubota Y , Kuriyama H
Published: December 2014

1. The effects of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), an activator of protein kinase C, have been investigated on intact and chemically skinned muscle strips of the porcine coronary artery. 2. In the presence or absence of extracellular Ca2+, TPA (0.1‐1 nM) slightly enhanced the amplitude of ACh (10 microM)‐induced contractions but at 100 nM, inhibited the contractions by approximately 50%. 3. ACh (10 microM) reduced the amount of [32P]phosphatidylinositol 4,5‐bisphosphate (PIP2) and increased the amount of [32P]phosphatidic acid (PA) in the presence or absence of Ca2+. TPA (over 1 nM) dose‐dependently inhibited the hydrolysis of PIP2 induced by ACh. 4. ACh (over 0.1 microM) dose‐dependently increased the intensity of fura‐2 fluorescence in dispersed single‐cell suspensions. TPA (over 1 nM) dose‐dependently inhibited the increase of the Ca2+ transient evoked by ACh, but it did not modify the ionomycin‐induced Ca2+ transient or the resting fluorescence. These inhibitory effects of TPA occurred over a similar dose range to that which inhibited ACh‐induced PIP2 break‐down. 5. When the relationship between ACh‐induced contraction amplitude and Ca2+ transient was observed in the presence or absence of 10 nM‐TPA, TPA greatly reduced the Ca2+ transient but did not modify the amplitude of contraction. 6. In saponin‐treated skinned muscle strips, TPA (10 nM) or 1,2‐diolein (50 micrograms/ml) with phosphatidylserine (PS; 50 micrograms/ml) increased the amplitude of contraction evoked by various concentrations of Ca2+ (0.1‐1.0 microM) without any change in the maximum amplitude of the Ca2+‐induced contraction. 7. TPA (10 nM) with PS (50 micrograms/ml) increased the amplitude of contraction evoked by 10 microM‐inositol 1,4,5‐trisphosphate in chemically skinned muscle strips. 8. It is concluded that TPA inhibits the ACh‐induced [Ca2+]i increase by inhibiting the hydrolysis of PIP2, but that it enhances the Ca2+ sensitivity of the contractile proteins. These results indicate that ACh‐induced contractions are controlled by negative feed‐back regulation of PIP2 hydrolysis together with a positive feed‐back regulation of the Ca2+ sensitivity of the contractile proteins. This may depend on the on‐going level of protein kinase C activation.
The effects of magnesium on potassium transport in ferret red cells.
Authors: Flatman P W
Published: December 2014

1. The magnesium dependence of net and isotopic (using 86Rb as tracer) potassium transport was measured in fed ferret red cells. Bumetanide (0.1 mM) was used to dissect total flux into two components: bumetanide sensitive and bumetanide resistant. 2. Increasing the external magnesium concentration from zero (added) to 2 mM stimulated bumetanide‐sensitive uptake by 16% but inhibited the bumetanide‐resistant component by about 20%. 3. Ionophore A23187 was used to control internal magnesium concentration. A23187 was usually present in the cells during measurement of isotopic fluxes but was washed away before measurement of net fluxes. The magnesium‐buffering characteristics of fed ferret red cells were assessed during these experiments. The cytoplasm acts as a high‐capacity, low‐affinity magnesium buffer over most of the range. Some high‐affinity binding was seen in the presence of A23187 and 2 mM‐EDTA. 4. A23187 itself slightly inhibits bumetanide‐sensitive potassium transport. 5. Bumetanide‐sensitive potassium transport is strongly dependent on the concentration of internal ionized magnesium. Transport is 35% maximal at 10(‐7) M and increases up to the maximal rate at 1.3 mM. Further increase in ionized magnesium concentration to 3.5 mM has no additional effect. The curve relating activity to magnesium concentration is steepest at the physiological magnesium concentration. The effects of changing magnesium concentration are fully reversible. 6. Reduction of internal ionized magnesium concentration to 10(‐7) M with A23187 and EDTA approximately doubles bumetanide‐resistant potassium transport. 7. Bumetanide‐sensitive fluxes occur via the sodium‐potassium‐chloride co‐transport system under the conditions used. Results described in this paper thus suggest that internal magnesium may be an important physiological controller of sodium‐potassium‐chloride co‐transport activity.
The adrenal contribution to the neuroendocrine responses to splanchnic nerve stimulation in conscious calves.
Authors: Bloom S R , Edwards A V , Jones C T
Published: December 2014

1. The extent to which the adrenal gland contributes to neuroendocrine responses to electrical stimulation of the peripheral end of the splanchnic nerve has been investigated in conscious calves in which the right nerve was stimulated either at 4 Hz continuously for 10 min or at 40 Hz in 1 s bursts at 10 s intervals for the same period. 2. It was confirmed that the release of neuropeptide Y (NPY) and of gastrin‐releasing peptide (GRP) is potentiated by stimulation in bursts at a relatively high frequency and shown that the adrenal gland made a negligible contribution to these responses. 3. There was no detectable change in the concentration of vasoactive intestinal peptide (VIP) in the arterial plasma but the existence of a very small but highly significant rise in the output of VIP from the adrenal provided evidence that it was released within the gland in response to splanchnic nerve stimulation. 4. The concentration of calcitonin gene‐related peptide (CGRP) in the arterial and adrenal venous effluent plasma was consistently below the level of detection of the assay. 5. Splanchnic nerve stimulation resulted in an abrupt rise in the output of both free and total met5‐enkephalin‐like immunoreactivity from the adrenal gland which was substantially potentiated by stimulating in bursts. This pattern of stimulation also increased the proportion released in a high‐molecular‐weight form. 6. Stimulation in bursts significantly enhanced the output of both adrenaline and noradrenaline from the adrenal and resulted in the release of proportionately more noradrenaline. Small amounts of dopamine and DOPAC were also released during splanchnic nerve stimulation and the output of dopamine was significantly increased by stimulating in bursts. 7. Both patterns of stimulation elicited an abrupt rise in mean plasma adrenocorticotrophic hormone (ACTH) concentration, which was associated with an increase in mean adrenal cortisol output and the former effect was significantly enhanced by stimulating in bursts. 8. It is concluded that certain responses to splanchnic nerve stimulation are significantly potentiated by an intermittent high‐frequency pattern of stimulation, including all those that are attributable to adrenal medullary activity, whereas others are apparently unaffected by changes in stimulus pattern.
Adrenoceptor‐mediated changes of excitation and contraction in ventricular heart muscle from guinea‐pigs and rabbits.
Authors: Hescheler J , Nawrath H , Tang M , Trautwein W
Published: December 2014

1. The influence of alpha‐adrenoceptor stimulation on mechanical and electrophysiological parameters was investigated in ventricular preparations from guinea‐pigs and rabbits. Action potential and force of contraction were measured in papillary muscles and ionic currents were measured in isolated myocytes. 2. The effects of alpha‐adrenoceptor stimulation were compared with those of beta‐adrenoceptor stimulation. 3. In the guinea‐pig the stimulation of alpha‐adrenoceptors caused a small increase in the force of contraction (less than 10% of the response to beta‐adrenoceptor stimulation) which was not accompanied by any increase of the slow calcium inward current. beta‐Adrenoceptor stimulation produced large increases in both force of contraction and slow inward calcium current. The noradrenaline‐induced increase in the slow inward calcium current was not affected by phentolamine. 4. In the rabbit, alpha‐adrenoceptor stimulation produced large increases in the force of contraction (about two thirds of those seen in response to beta‐adrenoceptor stimulation). Whereas beta‐adrenoceptor stimulation also produced large increases in both maximal upstroke velocity of slow‐response action potentials and slow inward calcium current, there was almost no change of both parameters in response to alpha‐adrenoceptor stimulation. 5. We conclude that, first, the contribution of alpha‐adrenoceptors to adrenoceptor‐mediated changes of force of contraction is minimal in the guinea‐pig ventricle, and second, the pronounced changes of force of contraction in the rabbit ventricle in response to alpha‐adrenoceptor stimulation are unrelated to changes in the slow inward calcium current.
Role of calcium‐activated potassium channels in transmitter release at the squid giant synapse.
Authors: Augustine G J , Charlton M P , Horn R
Published: December 2014

1. Several compounds known to block Ca2+‐activated K+ channels were microinjected into squid 'giant' presynaptic terminals to test the hypothesis that these channels mediate Ca2+‐dependent neurotransmitter release. 2. Injection of tetrapentylammonium, nonyl‐triethylammonium and decamethonium all reversibly blocked transmission evoked by presynaptic action potentials. 3. All three of these compounds blocked presynaptic Ca2+ channels. The actions of tetrapentylammonium on presynaptic Ca2+ influx were examined in detail and found to be quantitatively sufficient to account for the ability of this compound to inhibit transmitter release. 4. Injection of Ba2+, another agent known to block Ca2+‐activated K+ channels, also reversibly blocked evoked transmitter release. Ba2+ simultaneously enhanced basal (asynchronous) transmitter release and thus may be decreasing evoked release by depleting transmitter quanta available for release. 5. None of these results provide any support for the hypothesis that Ca2+‐activated K+ channels mediate Ca2+‐dependent release of transmitter at the squid synapse. However, our results have identified a new class of compounds that block Ca2+ channels from their cytoplasmic surface.
The organization and development of compartmentalized innervation in rat extensor digitorum longus muscle.
Authors: Balice-Gordon R J , Thompson W J
Published: December 2014

1. We have examined the innervation of the rat extensor digitorum longus (EDL) muscle by the two extramuscular branches formed from the bifurcation of its muscle nerve. Observations of muscle contractions, recordings of end‐plate potentials, and glycogen depletion of young adult muscles show that each branch innervates a separate region or 'compartment' in the muscle. The branch entering the muscle nearer the knee (the K branch) innervates fibres in the anteromedial half of the muscle whereas the branch entering closer to the foot (the F branch) innervates fibres located posterolaterally. Individual EDL motoneurones project either into the K or the F branch and therefore innervate fibres located in one compartment. The boundary between the compartments is usually sharply delineated. No obvious anatomical feature exists within the muscle which would explain the division of the muscle into two distinct regions. 2. The presence of a segmentotopic projection from the spinal cord to the muscle was investigated to evaluate its possible contribution to the compartmental pattern. The most posterior neurones of the EDL motor pool were found to project more frequently to the posterolateral F compartment; similarly, the most anterior neurones most frequently project to the anteromedial K compartment. However, each compartment is innervated by both anteriorly and posteriorly located motoneurones. The segmentotopic projection is too weak to explain the presence of neuromuscular compartments. 3. The post‐natal period of synapse elimination appears to play at best a minor role in setting up the compartmentalized innervation. Glycogen depletion and intracellular recording in 1‐2‐day‐old muscles show that each nerve branch innervates fibres in the same region of the muscle as in the adult. Most of the fibres in each compartment are polyneuronally innervated by axons in their own particular nerve branch, although fibres located near the boundary between the two compartments are innervated by axons from both nerve branches. This convergent innervation from the two branches disappears in concert with the elimination of polyneuronal innervation throughout the muscle. A random elimination of these convergent inputs appears adequate to explain the final compartmental pattern. 4. Our findings suggest that the compartmental pattern is primarily the consequence of te segregation of EDL motoneurones into two nerve branches which are directed into separate regions of the muscle.
Electrophysiological and mechanical effects of substance P and acetylcholine on rabbit aorta.
Authors: Bény J L , Brunet P C
Published: December 2014

1. The mechanical and electrical properties of smooth muscle cells of the rabbit aorta were recorded simultaneously using respectively a force transducer and a 3 M‐KCl‐filled glass microelectrode. 2. Acetylcholine had two effects depending on concentration. At low concentration, it caused a persistent endothelium‐dependent relaxation and hyperpolarization. At higher concentrations the acetylcholine endothelium‐dependent relaxation summed with an endothelium‐independent contraction. 3. Substance P caused a transient endothelium‐dependent relaxation and hyperpolarization. 4. Acetylcholine and substance P depolarized and contracted de‐endothelialized smooth muscle. When the de‐endothelialized strip was pre‐contracted by noradrenaline, acetylcholine depolarized the muscle but substance P did not. 5. In a 'cascade' experiment, the perfusate from an upstream intact aorta passed over a downstream de‐endothelialized strip. Acetylcholine and substance P relaxed the downstream strip showing that they released an endothelial humoral factor which relaxes smooth muscle. 6. The results suggest a constant release of a factor from the endothelial cells which hyperpolarizes the smooth muscle cells in the media. Activation of acetylcholine and substance P receptors on the endothelium accelerates the release of this factor and causes vasodilatation.
Voltage‐gated sodium and calcium currents in rat osteoblasts.
Authors: Chesnoy-Marchais D , Fritsch J
Published: December 2014

1. The whole‐cell voltage‐clamp mode of the patch‐clamp technique was used to investigate the presence of voltage‐gated inward currents in osteoblasts from newborn rat calvaria. 2. In K+‐free solutions, three kinds of inward currents could be activated by depolarization: a voltage‐gated Na+ current and two different types of Ca2+ currents. 3. The Na+ current was activated by depolarization above ‐40 mV in all the cells. It was reduced by half by 10 nM‐TTX (tetrodotoxin). 4. In an isotonic Ba2+ external solution containing TTX, and with a Cs‐EGTA internal solution buffered at pCa 8, depolarizing jumps induced both a transient Ba2+ current and a sustained Ba2+ current. The relative proportions of these two currents varied greatly among cells. 5. The transient and sustained Ba2+ currents differ with respect to their time course and their voltage dependence. 6. The depolarization‐activated inward currents were also observed under more physiological conditions, in the presence of only 2 mM‐external Ca2+ and with a K+ internal solution buffered at pCa 7. 7. A few records obtained in current clamp showed that it is possible to induce action potentials in osteoblasts.
Effect of repetitive activity upon intracellular pH, sodium and contraction in sheep cardiac Purkinje fibres.
Authors: Bountra C , Kaila K , Vaughan-Jones R D
Published: December 2014

1. The influence of repetitive activity upon intracellular pH (pHi), intracellular Na+ activity (aNA(i)) and contraction was examined in isolated sheep cardiac Purkinje fibres. Ion‐selective microelectrodes were used to measure intracellular Na+ and H+ ion activity. Twitch tension was elicited by field stimulation or by depolarizing pulses applied using a two‐microelectrode voltage clamp. Experiments were performed in HEPES‐buffered solution equilibrated either with air or 100% O2. 2. An increase in action potential frequency from a basal rate of 0.1 to 1‐4 Hz induced a reversible fall in pHi and a reversible rise in aNa(i). These effects reached a steady state 3‐10 min following an increase in stimulation frequency, and showed a linear dependence on frequency with a mean slope of 0.023 pH units Hz‐1 and 0.57 mmol l‐1 Hz‐1, respectively. The rise in total intracellular acid and aNa(i) associated with a single action potential was estimated as 5.3 mu equiv l‐1 of acid and 3.5 mu equiv l‐1 of Na+. 3. At action potential frequencies greater than 1 Hz, the rate‐dependent rise in aNa(i) was usually accompanied by a positive force staircase. 4. The fall in pHi following a rate increase also occurred when fibres were bathed in Tyrode solution equilibrated with 23 mM‐HCO3‐ plus nominally 5% CO2/95% O2. In these cases, however, the fall in pHi was halved in magnitude. 5. In fibres exposed to strophanthidin (0.5 microM), the rate‐dependent fall in pHi was doubled in magnitude and its time course was more variable than under drug‐free conditions. The rate‐dependent rise in aiNa was also usually larger in strophanthidin. 6. In order to examine the influence of the rate‐dependent acidosis on developed tension, the acidosis was reversed experimentally by adding 2 mmol l‐1 NH4Cl to the bathing solution. This produced a rise in pHi accompanied by a large increase in twitch tension. Such an effect of pHi upon tension was quantitatively similar to that observed in previous work on Purkinje fibres (Vaughan‐Jones, Eisner & Lederer, 1987). 7. It is concluded that the rate dependence of pHi will influence both the magnitude and the time course of an inotropic response to a change in heart rate.
Endothelium‐derived relaxing factor and nitroprusside compared in noradrenaline‐ and K+‐contracted rabbit and rat aortae.
Authors: Collins P , Henderson A H , Lang D , Lewis M J
Published: December 2014

1. The effects of endothelium‐derived relaxing factor (EDRF) (as stimulated by acetylcholine in the presence of endothelium), sodium nitroprusside and 8‐bromocyclic GMP on mechanical relaxation, calcium (45Ca) influx and cyclic GMP levels were studied in isolated rabbit aortic preparations pre‐contracted either by noradrenaline or by high (120 mM) extracellular potassium. 2. The results confirmed a relatively greater effect of these three interventions on mechanical relaxation and on reducing calcium influx in noradrenaline‐contracted than in potassium‐contracted preparations. 3. The increase in cyclic GMP levels induced by sodium nitroprusside, contrary to previous reports, was no greater in noradrenaline‐stimulated preparations than in potassium‐stimulated preparations, a finding confirmed in rat aortic preparations, and relaxation was not associated with a significant reduction of calcium influx in the potassium‐stimulated preparations. 4. Cyclic GMP‐mediated relaxation of potassium contraction thus appears to be due to actions of cyclic GMP other than on calcium influx. 5. These findings suggest that cyclic GMP reduces calcium influx more through receptor‐operated channels than through voltage‐operated channels. 6. The endothelium‐dependent acetylcholine‐induced elevation of cyclic GMP was reduced both by noradrenaline and by high extracellular potassium, possibly by altering release or activity of EDRF. 7. The sensitivity of the soluble guanylate cyclase system to stimulation by EDRF and nitrovasodilators appears to be greater in rat than rabbit aortic preparations.
Corticocortical synaptic influences on morphologically identified pyramidal neurones in the motor cortex of the monkey.
Authors: Ghosh S , Porter R
Published: December 2014

1. Corticocortical synaptic influences on pyramidal neurones in the precentral motor cortex of monkeys were examined using intracellular recordings. Corticocortical afferents from the postarcuate premotor area and the somatic sensory cortical areas were activated by bifocal stimulation of the cortical surface. Neurones that were found to respond orthodromically to such stimuli were labelled by intracellular ionophoresis of horseradish peroxidase. 2. Almost all neurones that were penetrated satisfactorily and labelled successfully were found to be pyramidal neurones located in lamina III or lamina V. Some labelled neurones in lamina V were also characterized as pyramidal tract neurones (PTNs) by antidromic activation from the cerebral peduncles or medullary pyramids. 3. Pyramidal neurones located in lamina III and lamina V (including PTNs) were excited at short latency by stimulation of the premotor cortex (1.1‐4.0 ms) and somatosensory cortex (1.1‐6.5 ms). There were no statistical differences in the distributions of latencies of corticocortical EPSPs between those evoked in lamina III neurones and those recorded in lamina V neurones, or between corticocortical EPSPs evoked from the premotor cortex in comparison with those from the somatosensory cortex. Excitatory responses to stimulation of the premotor area were usually more difficult to evoke and smaller in amplitude than those produced by stimulation of the somatosensory areas. 4. Corticocortical EPSPs were often followed by IPSPs. The amplitudes of the EPSPs and IPSPs could be increased by increasing the stimulus intensity. In a few neurones IPSPs that were not preceded by EPSPs were recorded.
Characteristics of spontaneous miniature and subminiature end‐plate currents at the mouse neuromuscular junction.
Authors: Erxleben C , Kriebel M E
Published: December 2014

1. Neuromuscular junctions of the mouse diaphragm were voltage clamped with a two‐electrode voltage clamp in order to evaluate time characteristics of miniature end‐plate currents (MEPCs). 2. The MEPCs fell into two amplitude classes: a larger class with an overall bell‐shaped distribution (bell MEPCs) and a smaller class which forms a right‐hand skew distribution (skew MEPCs). The mean MEPC amplitudes varied greatly because of the large range in the ratio of skew to bell MEPCs. This variation was greatest in neonate mice. 3. Rise time and time‐to‐peak were the same for MEPCs of the skew and bell classes. The MEPCs of both classes in neonate and adult mice had the same ratio of area (charge) over amplitude and the same time constant of decay. The absolute values changed with maturation (at 30 degrees C the ratio of area/amplitude was 4.5 +/‐ 0.8 ms in the newborn and 1.2 +/‐ 0.05 ms in the adult; the time constant of decay was 5.6 +/‐ 1.2 ms in the newborn and 0.8 +/‐ 0.05 ms in the adult). 4. Atypical MEPCs were found at all junctions. These had slow rising and falling phases, notches on the rising phases or a step the size of the sub‐MEPC class. The number of atypical MEPCs increased during the experiment. 5. The data suggest that both skew and bell MEPC classes are released from the same presynaptic region and are generated by the same postsynaptic mechanism.
Sodium channel distribution in normal and denervated rodent and snake skeletal muscle.
Authors: Caldwell J H , Milton R L
Published: December 2014

1. Sodium channel current density was measured using the loose‐patch voltage clamp technique. Innervated rat, mouse and snake muscle had the highest density of Na+ channels in the end‐plate region. These high Na+ channel densities were maintained in denervated muscle. 2. Perijunctional membrane had a Na+ current density 5‐ to 10‐fold greater than the density several hundred micrometres from the end‐plate. In all muscles this concentration of channels near the end‐plate persisted following denervation. 3. At the tendon Na+ current density fell to low values (approximately 1 mA/cm2). The decrease in density began about 300‐500 microns from the tendon. This pattern was found in all snake twitch fibres and fast‐twitch (EDL) rat and mouse muscle fibres. This reduction in channel density near the tendon was not affected by denervation. 4. Sodium channels in all regions of innervated rat and snake muscle fibres were highly sensitive to tetrodotoxin (TTX). Sodium channels in snake muscle remained sensitive to TTX after denervation. Sodium channels that are relatively resistant to TTX appeared in rat muscle after denervation. TTX‐resistant channels were even more concentrated near the end‐plate than were TTX‐sensitive channels in innervated muscle. At the tendon TTX‐resistant Na+ channel density decreased. 5. We conclude that although the nerve presumably directs the localization of Na+ channels during development, the ability to maintain this distribution and to control the distribution of newly appearing channels persists long after the nerve has been removed.
A study of the electrical characteristics of sodium currents in single ventricular cells of the frog.
Authors: Seyama I , Yamaoka K
Published: December 2014

1. The generation of action potentials elicited from enzymatically dispersed ventricular cells from the frog, Rana catesbeiana, has been shown to be due to the influx of both Na+ and Ca2+. The maximum rate of rise, the amplitude and the duration at 50% repolarization of the action potential were estimated to be 26.4 +/‐ 5.1 V/s (n = 8), 110 +/‐ 2.7 mV (n = 8) and 601 +/‐ 180 ms (n = 8) at 15 degrees C, respectively. 2. Inward Na+ current (INa) was studied in these ventricular cells by the whole‐cell patch clamp technique in a medium where Ca2+ current was eliminated by substituting extracellular Mg2+ for Ca2+ and K+ current was suppressed by applying Cs+ intracellularly. All the voltage clamp experiments were carried out at 4 degrees C. 3. INa elicited by single depolarizing steps from a holding potential (VH) of ‐80 mV had a threshold of ‐50 mV and was maximal at ‐20 mV. Peak currents in normal Ringer solution containing 113.5 mM‐Na+ were of the order of 0.01‐0.02 mA/cm2. Maximum Na+ conductance (gNa) was calculated to be 5.9 mS/cm2. 4. Under normal conditions the reversal potential for INa was determined to be 50 mV, which is close to the value predicted from the Nernst equation. The reversal potential changed by 59 mV per tenfold change in the activity of extracellular Na+ (aNa). 5. The instantaneous relation between INa tail currents and membrane potential is linear, crossing the abscissa at the reversal potential for INa. 6. Reconstructions of INa were made in terms of the parameters of the Hodgkin‐Huxley model for the squid axon, using constants obtained from the frog ventricular cells. 7. The falling phase of INa and the development of inactivation measured by the double‐pulse method could be well fitted by a single‐exponential function. 8. The time course for recovery of INa from inactivation exhibited a single time constant.
An ATP‐sensitive conductance in single smooth muscle cells from the rat vas deferens.
Authors: Friel D D
Published: December 2014

1. The whole‐cell voltage‐clamp technique was used to study the effects of extracellular ATP on smooth muscle cells isolated from the rat vas deferens. 2. ATP (1‐200 microM) elicited an inward‐rectifying current that was rapid in onset (less than or equal to 100 ms), reached a peak value that depended on [ATP], and desensitized in the continued presence of ATP (half‐time approximately 2 s). 3. Cells recovered from desensitization when incubated in the absence of ATP (resensitization half‐time approximately 2 min). 4. A comparison was made of the ability of ATP and several of its structural analogues to stimulate inward current at a negative holding potential. ATP was by far the most effective compound among the series ATP, ADP, AMP, adenosine, GTP, UTP and ITP. ADP elicited a current that was 20‐25% as large as that produced by ATP, while the other compounds were ineffective at a concentration which produced a maximal ATP response. 5. AMP‐CPP (alpha, beta‐methylene ATP), AMP‐PCP (beta, gamma‐methylene ATP), and AMP‐PNP (beta, gamma‐imido ATP), which are relatively resistant to hydrolysis, were similarly compared to ATP. While none of these analogues elicited a current resembling the ATP‐induced current, AMP‐CPP and AMP‐PNP each produced a small, relatively sustained inward current; AMP‐PCP had little or no effect. 6. The ATP‐sensitive conductance is cation selective, but does not appear to discriminate strongly between Na+, K+ and Mg2+. 7. Analysis of the fluctuations which accompany the ATP‐induced current suggests that ATP controls a population of channels with a unitary current greater than 0.5 pA at ‐130 mV. 8. The ATP‐evoked current discussed in this report may be responsible for the depolarizing effect of ATP previously described in multicellular preparations of the vas deferens.
Ionic regulation of intracellular pH in rat calvarial osteoblasts.
Authors: Redhead C R
Published: December 2014

1. Intracellular pH of cultured rat calvarial osteoblasts was monitored continuously using the pH‐sensitive fluorescent probe bis‐carboxyethyl carboxyfluorescein. 2. Recovery from an intracellular acid load brought about by exposure to ammonium chloride was dependent on external sodium and blocked by the sodium‐hydrogen exchange inhibitor amiloride (1 mM), indicating the presence of a plasma membrane sodium‐hydrogen exchanger. 3. A SITS‐ (4‐acetamido‐4'‐isothiocyanostilbene‐2,2'‐disulphonic acid) sensitive alkalinization occurred on the isosmotic replacement of external chloride by gluconate, suggesting the presence of chloride‐bicarbonate exchange. 4. The dependence of the rate of sodium‐hydrogen exchange on external sodium followed first‐order kinetics, but the rate of exchange appeared to be sensitive to intracellular pH. 5. The rate of alkalinization brought about by the isosmotic replacement of chloride was sensitive to external bicarbonate concentration, but independent of external sodium. 6. Sodium‐hydrogen exchange appeared to be inhibited and chloride‐bicarbonate exchange stimulated by 1‐34 parathyroid hormone.
Cardiovascular responses to stimulation of pulmonary C fibres in the cat: their modulation by changes in respiration.
Authors: Daly M D , Kirkman E
Published: December 2014

1. In cats anaesthetized with a mixture of chloralose and urethane, stimulation of pulmonary C fibres by right atrial injections of phenylbiguanide caused, after a latency of about 3 s, a reduction in pulmonary ventilation or apnoea, bradycardia and systemic hypotension, confirming previous work. 2. A decrease in femoral artery perfusion pressure also occurred under conditions in which the hindlimb was vascularly isolated, the blood flow was maintained constant and the inferior vena caval pressure did not change. This indicates a reduction in vascular resistance due to vasodilatation. The response was unaffected by atropine and propranolol, but was reduced or abolished by guanethidine, hexamethonium and denervation of the limb, indicating that it is due to a reduction in activity in sympathetic vasoconstrictor fibres. 3. Similar cardiovascular responses were observed when the arterial blood pressure was maintained constant, and also in artificially ventilated animals. 4. Evidence is presented that the receptors responsible for the respiratory and cardiovascular responses to right atrial injections of phenylbiguanide lie in the pulmonary vascular bed. 5. When the pulmonary C fibres were excited during a period of apnoea which was induced reflexly by electrical stimulation of the central end of a superior laryngeal nerve, there were no consistent differences in the size of the cardiac or vascular responses compared to the control responses in the absence of the laryngeal input. This result occurred irrespective of the size of the control ventilatory response to phenylbiguanide. 6. By contrast in the same experiments, the cardio‐inhibitory and vasoconstrictor responses to excitation of the carotid body chemoreceptors were invariably potentiated by electrical stimulation of a superior laryngeal nerve, as found previously. 7. The possible central mechanisms responsible for the differential modulation of pulmonary C fibre and carotid chemoreceptor reflexes by respiration are discussed.
Voltage‐dependent decrease in the availability of single calcium channels by nitrendipine in guinea‐pig ventricular cells.
Authors: Kawashima Y , Ochi R
Published: December 2014

1. The mechanism of Ca2+ channel block by nitrendipine was studied by recording single‐channel activity from cell‐attached patches on guinea‐pig ventricular cells using patch pipettes containing 50 mM‐Ba2+. Test depolarization pulses to around 10 mV with a duration of 100 ms were applied repetitively at 2 Hz. 2. The percentage of non‐blank sweeps was maximal (about 40%) at a holding potential between ‐65 and ‐130 mV and decreased sigmoidally with its depolarization. Nitrendipine shifted the availability‐voltage relationship in a hyperpolarizing direction. 3. From the number of consecutive non‐blank sweeps and that of blank sweeps, the duration of the available state and that of the unavailable state were estimated. 4. The histogram of the duration of the available state showed a single‐exponential distribution. Its mean duration was about 1.5 s and was shortened by nitrendipine. Correspondingly, the decay of the mean current during the depolarization step was accelerated by nitrendipine. 5. In the presence of 100 nM‐nitrendipine the histogram of the duration of the unavailable state at large negative holding potentials was simulated as the sum of two exponential components, one with a time constant similar to that in the control and the other with a time constant of 6‐7 s. 6. The histogram of the duration of the unavailable state at depolarized holding potentials was simulated by a double‐exponential curve also in the control. The duration of the slow component was prolonged by nitrendipine. 7. The prolongation of the unavailable states initiated by drug binding during depolarization steps and maintained during depolarized holding potentials is the mechanism of the blockade. The rate constants of the state transitions between an available state and two unavailable states were estimated.