bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties and ionic basis of the action potentials in the periaqueductal grey neurones of the guinea‐pig.
Authors: Sánchez D , Ribas J
Published: December 2014

1. Action potentials of neurones of the ventral part of the guinea‐pig periaqueductal grey (PAG) were studied by intracellular recording in a mesencephalic slice preparation maintained in vitro. 2. Fast spikes spontaneously fired last 2.8 +/‐ 0.6 ms (mean +/‐ S.D.) and have an amplitude of 72.3 +/‐ 5.3 mV (n = 28). The neurones could be antidromically activated from the neighbouring white matter and these spikes show an initial segment component that triggers the soma‐dendritic spike. These two components were dissociated by hyperpolarization. Action potentials are Na+ dependent and a Ca2+ conductance is responsible for the hump on the falling phase. Hyperpolarization makes the hump disappear and a faster rate of rise and fall are seen. Accommodation of the firing threshold is observed in response to depolarizing ramps, which is eliminated with hyperpolarization. 3. High‐threshold Ca2+ spikes are evoked in either Na(+)‐free solution or in the presence of tetrodotoxin (TTX). These presumed dendritic action potentials display a fast repolarization and a large after‐hyperpolarization (AHP) that prevent repetitive firing. This AHP is mainly generated by Ca(2+)‐dependent K+ conductances. 4. The repolarization of fast action potentials depends on the activation of K+ conductances as well as a Na+ inactivation process. A fast‐activated tetraethyl‐ammonium (TEA)‐sensitive K+ conductance, that could be Ca2+ dependent, and a K+ conductance blocked by apamin seem to be involved in the repolarization. 5. Each fast action potential is followed by a pronounced AHP with two components, an initial fast and a slow decaying phase. Membrane hyperpolarization around ‐60 mV eliminated the first component and the AHP acquired a plateau‐like shape. At ‐90 mV the AHP was nullified. The slow phase was Ca2+ dependent and an apamin‐sensitive K+ conductance is involved in its generation. This conductance may be active during the early part of the AHP, but a fast‐activated TEA‐sensitive K+ conductance and other voltage‐dependent K+ conductances might also be present. A Ca2+ conductance is hypothesized to account for the fast depolarizing change after the AHP peak. 6. A delayed return to the baseline is observed after hyperpolarizing pulses. It is generated by the activation of a transient voltage‐dependent K+ conductance that is inactive at resting membrane potential (RMP, around ‐50 mV). This transient hyperpolarization is abolished by Ca2+ channel blockers and insensitive to high external concentrations of 4‐aminopyridine, TEA and Cs+.(ABSTRACT TRUNCATED AT 400 WORDS)
Substitution of cardiac troponin C into rabbit muscle does not alter the length dependence of Ca2+ sensitivity of tension.
Authors: Moss R L , Nwoye L O , Greaser M L
Published: December 2014

1. The isometric length‐tension relationship for cardiac muscle is generally steeper than for skeletal muscle in the physiological range of sarcomere lengths. Recent studies suggest that cardiac troponin C (cTnC) may have intrinsic properties that confer greater length‐dependent changes in Ca2+ sensitivity of tension than for skeletal troponin C (sTnC). We tested this hypothesis by characterizing tension‐pCa (pCa is ‐log[Ca2+]) relationships in rabbit skinned psoas muscle fibres at mean sarcomere lengths of 2.32 and 1.87 microns both before and after partial replacement of endogenous sTnC with cTnC. 2. In untreated control fibres, the mid‐point (pCa50) of the tension‐pCa relationship shifted to lower pCa by 0.15 +/‐ 0.02 pCa units, i.e. became less sensitive to Ca2+, when sarcomere length was reduced, and the relationship became steeper. 3. Partial extraction of endogenous sTnC and reconstitution with cTnC resulted in no change in the length‐dependent shift of pCa50 when reconstitution with cTnC was more than 95% complete; however, when reconstitution was less than 95% complete, there were significant increases in the length‐dependent shift in pCa50. 4. An increase in the length‐dependent shift of pCa50 was also observed in fibres from which sTnC was partially extracted, but no cTnC was subsequently re‐added. 5. We conclude that differences in type of TnC alone are not sufficient to explain differences between skeletal and cardiac muscles in the length dependence of Ca2+ sensitivity of tension.
Magnesium and ATP dependence of K‐Cl co‐transport in low K+ sheep red blood cells.
Authors: Delpire E , Lauf P K
Published: December 2014

1. In low K+ (LK) sheep red blood cells, depletion of adenosine triphosphate (ATP) by glycolysis inhibition induced specific effects on ouabain‐resistant Cl(‐)‐dependent K+ transport (K‐Cl co‐transport), depending on the osmolarity: stimulation in isosmotic while inhibition in hyposmotic solutions. However, these effects depended upon the presence of internal Mg2+. 2. In LK sheep red blood cells, ATP constituted nearly 90% of the Mg2+ buffering capacity. As no significant reduction of total Mg2+ was observed after ATP depletion, the overall internal Mg2+ in ATP‐depleted cells exists in the free form. 3. The dependence of K+ efflux on internal Mg2+ was also directly related to the presence of ATP. In control cells, Mg2+ constituted an endogenous inhibitor, inducing a 70% inhibition of K‐Cl fluxes but only 30% in ATP‐depleted cells. The Cl(‐)‐insensitive component of K+ efflux was unaffected by the divalent cation. 4. After Mg2+ removal, the rate of K+ efflux was significantly increased at all osmolarities, between 240 mosM (swollen cells) and 440 mosM (shrunken cells). Hence, Mg(2+)‐depleted LK sheep red cells lose volume sensitivity of K‐Cl co‐transport. 5. Internal K+ or Cl‐ were not required for the Mg2+ inhibition, and Mg2+ did not interfere with the internal binding sites for Cl‐ or K+. Hence, the sites for Mg2+ or MgATP, and for K+ and Cl‐ are independent of each other.
The role of troponin C in the length dependence of Ca(2+)‐sensitive force of mammalian skeletal and cardiac muscles.
Authors: Gulati J , Sonnenblick E , Babu A
Published: December 2014

1. Skinned fibre preparations of right ventricular trabeculae, psoas and soleus muscles from hamster and rabbit were activated by Ca2+ and the length dependencies of their pCa (‐log [Ca2+])‐force relationships were compared. 2. Ca2+ sensitivity of the myocardium was higher at 2.2‐2.4 microns than that at 1.7‐1.9 microns. The length dependence was at least twofold greater in cardiac muscle than in fast skeletal fibres at identical temperatures and salt concentrations. Slow‐twitch fibres gave a response similar to that in the myocardium. 3. The effect of the troponin C (TnC) phenotype on the length dependence of Ca2+ sensitivity was measured on both fast skeletal fibres and cardiac muscle with TnC exchange in situ. The length‐induced increase in Ca2+ sensitivity was found to be greater in the presence of cardiac TnC than with fast skeletal TnC. Thus the results indicate that a certain domain of TnC is specialized in this length function, and that this domain is different in the two phenotypes. 4. The possibility that the enhanced length dependence of Ca2+ sensitivity after cardiac TnC reconstitution was attributable to reduced TnC binding was excluded when the length dependence of partially extracted fast fibres was reduced to one‐half the normal value after a 50% deletion of the native TnC. 5. Two recombinant forms of cardiac TnC (kindly provided by Dr John Putkey, Houston, TX, USA) were used next, to investigate the roles of two specific domains in TnC in the control of length dependence of Ca2+ sensitivity and in the contraction‐relaxation switching of cardiac muscle: 6. Using mutant CBM1 [corrected], in which site 1 was modified such as to bind the 4th Ca2+ ion, as in skeletal TnC, the length‐induced Ca2+ sensitivity in cardiac muscle was suppressed. The effect was intermediate between cardiac and skeletal TnCs under the same conditions. The pSr (‐log [Sr2+])‐force relationship of cardiac muscle was also measured. In the presence of the mutant, skinned trabeculae manifest pSr‐activation curves identical to those of fast fibres. This indicates that the metal ion binding properties of site 1 in TnC modulate the regulatory action of site 2. 7. Using mutant CBM2A, in which site 2 was inactivated, the activation of cardiac muscle by both Ca2+ and Sr2+ ions was completely blocked. This is the expected result, since both regulatory sites were now inactive, regulatory site 1 being normally inactive in cardiac muscle.(ABSTRACT TRUNCATED AT 400 WORDS)
Kinetics of adenosine triphosphate hydrolysis by shortening myofibrils from rabbit psoas muscle.
Authors: Ohno T , Kodama T
Published: December 2014

1. Using a solenoid‐operated mixing device, time‐resolved measurements were made of shortening and accompanying ATP hydrolysis at 20 degrees C by myofibrils prepared from rabbit psoas muscle. 2. The extent of ATP hydrolysis was determined by an improved Malachite Green method for determination of inorganic phosphate (Pi) in the presence of a large excess of ATP. For the measurement of the change in sarcomere length by phase contrast microscopy, shortening was terminated without delay and artifact by a mixture of 0.2 M‐acetate (pH 4.6) and 1.25% (v/v) glutaraldehyde. 3. The shortening velocity per half‐sarcomere was 10 microns s‐1 in 25 mM‐KCl for sarcomere lengths above 1.4 microns, and at least 12 microns s‐1 in 150 mM‐KCl for sarcomere lengths above 1.7 microns. During this rapid shortening, there was no significant ATP turnover by cross‐bridges (upper 95% confidence limit: 0.14 mol (mol of myosin head)‐1 in 25 mM‐KCl; 0.12 mol mol‐1 in KCl solutions greater than or equal to 100 mM). 4. When the sarcomeres shortened below 1.7 microns in KCl concentrations greater than 100 mM or below 1.4 microns in 25 mM‐KCl, there was a transient acceleration of ATP hydrolysis (delayed ATP hydrolysis), which was then followed by a steady slow hydrolysis. 5. The magnitudes (+/‐ estimated standard deviation) of delayed ATP hydrolysis by myofibrils of initial sarcomere length 2.4 microns were 0.42 +/‐ 0.19, 0.31 +/‐ 0.10 and 0.17 +/‐ 0.09 mol (mol myosin head)‐1 in 25 mM, 100 mM and 150 mM‐KCl, respectively. For myofibrils of sarcomere length 2.0 microns, however, it decreased to 0.24 +/‐ 0.10 mol mol‐1 in 25 mM‐KCl or to an insignificant level in 150 mM‐KCl. 6. These results indicate that most of the ATP hydrolysis products remain bound to cross‐bridges during rapid shortening, and that when the force opposing shortening increases, a proportion of cross‐bridges rapidly dissociate the products and enter the next ATP cycle, which diminishes with the decrease in shortening distance as well as the increase in ionic strength. Such behaviour of the cross‐bridge is probably a manifestation of its energetic and kinetic properties in the state with bound ADP and Pi interacting with actin filaments at zero load and at a transition from zero to non‐zero loads.
Analysis of the horizontal cell contribution to the receptive field surround of ganglion cells in the rabbit retina.
Authors: Mangel S C
Published: December 2014

1. The influence of horizontal cells on ganglion cells, the output neuron of the retina, was examined in an in vitro rabbit eyecup preparation. The extracellular spike activity of ganglion cells was monitored while pulsatile DC or sinusoidally modulated current was injected intracellularly into nearby horizontal cells. Interactions between the effects of light stimulation and horizontal cell current injections on ganglion cell responses were also examined. 2. Horizontal cells were found to contribute to the receptive field surround of ganglion cells. In particular, horizontal cells contributed to surround excitability and to surround antagonism of the centre light response. 3. Brisk, sluggish and direction‐selective ganglion cells were all affected by current injections into horizontal cells. However, brisk ganglion cells responded to lower amplitude currents than did sluggish or direction‐selective cells. 4. Horizontal cells with receptive fields that overlap those of ganglion cells were able to affect ganglion cell discharge. Moreover, the closer a horizontal cell was to the receptive field centre of a ganglion cell, the more effective were current injections in modulating ganglion cell discharge rate. The length constant of the horizontal cell contribution to the ganglion cell receptive field was approximately 200 microns. These results indicate that horizontal cells which are located within or outside of a ganglion cell's receptive field centre can influence that ganglion cell's activity. 5. The influence of horizontal cells on ganglion cell discharges was relatively weak at low temporal frequencies of sinusoidally modulated current. 6. Application of 2‐amino‐4‐phosphonobutyrate (APB), a glutamate analogue, blocked the modulation of spike activity of on‐centre ganglion cells that was induced by sinusoidally modulated current injected into nearby horizontal cells. The spike activity of off‐centre ganglion cells was not blocked. 7. These findings suggest that horizontal cells contribute to the surround of ganglion cells and bipolar cells primarily through a feedback pathway onto cone photoreceptor cells.
Independent digit control: failure to partition perceived heaviness of weights lifted by digits of the human hand.
Authors: Kilbreath S L , Gandevia S C
Published: December 2014

1. A weight‐matching task was used to investigate the ability to estimate heaviness when weight lifting was isolated to the extrinsic flexor muscles (or portions thereof) that act on the digits of the hand. 2. Subjects matched a reference weight (200 g) lifted by a digit on the right with a variable weight lifted by the left thumb; a concurrent weight was simultaneously lifted by a digit on the right (reference) side. The reference and concurrent weights were lifted either by the same muscle (digital portions of flexor digitorum profundus), or anatomically separate but functionally related muscles (flexor digitorum profundus and flexor pollicis longus). Anaesthesia of the radial nerve and/or posturing of the hand was used to eliminate any small forces generated by co‐contraction of the extensor muscles of the digits. 3. When the concurrent weight was equal to or greater than the reference weight, the perceived heaviness of the reference weight increased significantly from control trials (in which no concurrent weight was lifted). Although perceived heaviness of the reference weight increased progressively as the concurrent weight increased, reproducibility (expressed as the coefficient of variation) did not deteriorate when a weight was lifted concurrently. These findings were qualitatively similar when the reference and concurrent weights were lifted by two digital portions of flexor digitorum profundus or when the weights were lifted by flexion of the thumb and index finger. Also, anaesthesia of the digits which lifted the reference and concurrent weights did not alter the changes in perceived heaviness. 4. Perceived heaviness of the reference weight lifted by flexor digitorum profundus did not change when subjects lifted the concurrent weight with a remote muscle group (ankle dorsiflexors). 5. This study shows that signals of heaviness are systematically overestimated whenever more than one portion of the extrinsic muscles which flex the different digits are simultaneously active. Given that estimates of heaviness are biased by the central motor command, one explanation is that when the total motor drive increases, the central nervous system is unable to partition precisely the destination of motor commands to functionally related ‘muscles’.
The effect of permeant ions on single calcium channel activation in mouse neuroblastoma cells: ion‐channel interaction.
Authors: Shuba Y M , Teslenko V I , Savchenko A N , Pogorelaya N H
Published: December 2014

1. Single low‐threshold inactivating (LTI or T‐type) Ca2+ channels of undifferentiated neuroblastoma cells (clone N1E‐115) were investigated using the patch‐clamp technique. 2. Single‐channel conductance, gi, for Ca2+, Sr2+ or Ba2+ as a permeant cation was similar (7.2 pS). Mean channel open time, tau op, was also practically independent of the divalent ion species; it decreased from 0.7 to 0.3 ms between ‐40 and 0 mV. 3. Modification of the calcium channel selectivity by lowering the external Ca2+ concentration to 10(‐8) M produced an increase in gi for Na+ and Li+ ions and a shift of potential‐dependent characteristics in the hyperpolarizing direction. Voltage sensitivity and absolute values of tau op were also changed. These changes were dependent on both permeant monovalent ion type and concentration. 4. At high [Na+]o, tau op was almost potential independent (congruent to 0.3 ms). Decrease in [Na+]o and substitution of Li+ for Na+ increased tau op and the steepness of its potential dependency. 5. The divalent and monovalent cations that were tested had much smaller effect on the mean intraburst shut time, tau cl(f), which was nearly independent of membrane potential (congruent to 0.6 ms). By contrast, mean burst duration was strongly potential dependent and noticeably affected by permeant ion type. 6. All kinetic changes were analysed in terms of a four‐state sequential model for channel activation. According to this model the channel enters the open state through three closed states. Transitions between closed states can be formally related to the transmembrane movement of two charged gating particles (m2 process). The interaction between ion flux and a sterical region of the Ca2+ channel selectivity filter may, depending on ion transfer rate and ionic radius, lead to a local increase of the dielectric constant, resulting in redistribution of the electric field and changes in potential dependency of tau op.
Effect of bradykinin, ATP and adrenaline on cell membrane resistances of Madin‐Darby canine kidney cells.
Authors: Ritter M , Lang F
Published: December 2014

1. Previous studies have shown that bradykinin, ATP and adrenaline hyperpolarize the cell membrane of Madin‐Darby canine kidney (MDCK) cells by activation of calcium‐sensitive K+ channels. The present study has been performed to determine the effect of these hormones on the resistance of the cell membrane and the cellular coupling. To this end, cellular cable analysis has been performed. 2. As a result, all three hormones lead to the expected, marked decrease of cell membrane resistance. 3. However, the bradykinin‐induced reduction of cell membrane resistance was sustained, contrasting with only transient hyperpolarization induced by bradykinin and only transient activation of the K+ channels. Thus, the cable analysis reveals the sustained activation of an additional conductance. 4. ATP, but not the other two hormones, leads to a delayed increase of the intercellular coupling resistances. 5. Prolonged exposure of the cells to adrenaline leads to oscillations of the cell membrane potential, apparently by oscillatory activation of the K+ channels.
Cadmium uptake through the anion exchanger in human red blood cells.
Authors: Lou M , Garay R , Alda J O
Published: December 2014

1. The initial rate of Cd2+ uptake in human red cells was measured by atomic absorption spectrophotometry. 2. About 96% of Cd2+ uptake was inhibited by DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid) with IC50 (concentration giving 50% of maximal inhibition) of 0.3 microM and by furosemide with IC50 of 500 microM and was resistant to ouabain and amiloride. This indicates the implication of the [Cl(‐)‐HCO3‐] anion exchanger in Cd2+ uptake. 3. DIDS‐sensitive Cd2+ uptake required the presence of external HCO3‐. HCO3‐ ions had a biphasic effect on Cd2+ uptake. Low bicarbonate concentrations were stimulatory, suggesting formation of translocating bicarbonate‐cadmium complexes. Higher bicarbonate concentrations were inhibitory, suggesting further bicarbonate complexation with formation of non‐translocating species. Depending on the presence or absence of external Cl‐, a maximal Cd2+ uptake of 1.7 or 0.37 mmol (l cells)‐1 h‐1 was observed at bicarbonate concentrations of 15.6 or 11 mM respectively. 4. In the presence of bicarbonate, external Cl‐ ions strongly stimulated Cd2+ uptake, with linear increase between 70 and 125 mM. This suggests that one translocating species may have chloride as ligand. 5. DIDS‐sensitive Cd2+ uptake was modestly inhibited by physiological concentrations of external phosphate and was resistant to external K+, Mg2+ and Ca2+. 6. In conclusion, the anion exchanger is the major transport mechanism for red cell cadmium uptake. Translocating species appear to be monovalent anion complexes of cadmium with HCO3‐ such as [Cd(OH)(HCO3)2]‐ and [Cd(OH)(HCO3)Cl]‐.
Characterization of a Ca(2+)‐dependent anion channel from sheep tracheal epithelium incorporated into planar bilayers.
Authors: Alton E W , Manning S D , Schlatter P J , Geddes D M , Williams A J
Published: December 2014

1. Anion‐selective channels from the apical membrane of respiratory epithelia are involved in the secretion of chloride into the airway lumen. In cystic fibrosis (CF) there is an abnormality of phosphorylation‐regulated chloride transport in this tissue, whilst a calcium‐dependent pathway appears to function normally. 2. Using incorporation of apical membrane vesicles into planar phospholipid bilayers, we have characterized the most commonly seen anion‐selective channel from sheep tracheal epithelium. 3. In symmetrical 200 mM‐NaCl solutions the channel showed rectification, with a chord conductance at negative voltages of 107 pS and at positive voltages of 67 pS. The channel characteristically demonstrated subconductance states at 1/3 and 3/4 of the fully open level. Selectivity for chloride over sodium was approximately 6:1. 4. The channel required a minimum of approximately 100 microM‐calcium on the presumed cytoplasmic surface (cis) for opening events to be observed. Open probability (Po) of the fully open state was markedly voltage dependent, but little effect of voltage was seen on the 1/3 subconductance state. 5. The relative permeabilities of monovalent anions monitored under bi‐ionic conditions gave the following sequence: NO3‐ greater than I‐ greater than Cl‐ = Br‐ much much greater than F‐. The order of conductances in symmetrical solutions was Cl‐ = NO3‐ greater than Br‐ greater than I‐ much much greater than F‐. 6. The chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) produced a dose‐related reduction in Po with a flickering block at 10‐50 microM and complete block at higher concentrations. 7. ATP produced a dose‐related reduction in Po with effects at 1 microM and complete closing at 1 mM. These effects were only seen with addition to the cis chamber. 8. The catalytic subunit of protein kinase A, either when incubated with vesicles prior to incorporation into bilayers, or when added directly to either chamber, produced no effect. 9. Channels with very similar properties were seen from transfected human tracheo‐bronchial cells. 10. Recent whole‐cell patch‐clamp studies have suggested a distinct calcium‐activated chloride current in secretory epithelia. The described channel has properties in common with this current and may be a candidate for its single‐channel basis.
Properties of the late transient outward current in isolated intestinal smooth muscle cells of the guinea‐pig.
Authors: Zholos A V , Baidan L V , Shuba M F
Published: December 2014

1. Whole‐cell membrane currents in voltage‐clamped single isolated cells of longitudinal smooth muscle of guinea‐pig ileum were studied at room temperature using patch pipettes filled with either high‐K+ solution or high‐Cs+ solution, to suppress K+ outward current, and containing 0.3 mM‐EGTA. 2. In the presence of high‐K+ solution in the pipette, membrane depolarization from the holding potential of ‐50 mV evoked an initial inward calcium current (ICa) followed by a large initial transient outward current and a sustained outward current with spontaneous oscillations superimposed. Prolonged depolarization above ‐20 mV produced a late transient outward current which reached a maximum (up to several nanoamps at +10 mV) within approximately 1 s and lasted several seconds. 3. The late outward current (ILTO) was voltage dependent and reversed at the EK (potassium equilibrium potential) in cells exposed to high‐K+ external solution. It was blocked by TEA+ (tetraethylammonium) or Ba2+ applied externally (calculated Kd (dissociation constant) values were 0.67 and 4.43 mM, respectively) or by high‐Cs+ solution perfusing the cell. The removal of extracellular Ca2+, application of Ca2+ channel blockers (3 mM‐Co2+, 0.2 mM‐Cd2+ or 1 microM‐nifedipine) or perfusion of 5 mM‐EGTA inside the cell also abolished the current. Thus, the current seems to be a Ca(2+)‐activated K+ current. 4. There is a great discrepancy between the time course of the ICa and that of the late ILTO, which suggests that Ca2+ release from intracellular storage sites may contribute to the generation of the ILTO. 5. Bath application of caffeine (10 mM) during the development of ILTO enhanced the current. However, in the presence of caffeine ILTO was inhibited. Moderate inhibition of ICa by caffeine was also observed. 6. Ryanodine (5 microM) applied to the bathing solution completely inhibited ILTO within 3.5 min; however, it had no or little effect on the ICa. 7. Ruthenium Red (10 microM) completely blocked the ILTO and slightly and more slowly inhibited the ICa. 8. Increasing Mg2+ concentration in the pipette solution from 1 to 6 mM abolished the ILTO. 9. It was concluded that the ILTO was activated mainly by Ca2+ released from the intracellular storage sites following Ca2+ entry, presumably by a Ca(2+)‐induced Ca2+ release mechanism.
Excitation‐contraction coupling in skeletal muscle fibres of rat and toad in the presence of GTP gamma S.
Authors: Lamb G D , Stephenson D G
Published: December 2014

1. Rapid force responses were elicited in single mechanically skinned fibres from extensor digitorum longus (EDL) muscle of the rat when the fibres were depolarized by substituting K+ in the bathing solution with Na+. The properties of these depolarization‐induced responses, the responses to lowered [Mg2+], and the characteristics of the slow prolonged response ('second component’) produced in ‘loaded’ fibres by choline chloride (ChCl) substitution, were virtually identical to those observed previously in skinned fibres from toad muscle. 2. At physiological levels of [Mg2+] (1 mM) and Ca2+ loading, application of 50 microM‐ to 1 mM‐GTP gamma S (guanosine‐5'‐O‐(3‐thiotriphosphate), a non‐hydrolysable analogue of GTP) did not produce a response in any mammalian or amphibian fibre, even though the depolarization‐induced coupling was totally functional. Furthermore, the presence of GTP gamma S had no apparent effect on the size, the threshold or the maximum number of responses which could be elicited by depolarization. 3. GTP gamma S did not elicit any response when excitation‐contraction coupling was abolished by prolonged depolarization or by chemically skinning the fibre with saponin or by 24 h exposure to low [Ca2+] (5 mM‐EGTA). 4. GDP beta S (guanosine‐5'‐O‐(2‐thiodiphosphate), 250 microM or 1 mM) neither evoked a response nor affected the responses to depolarization or caffeine. 5. When the [Mg2+] was lowered to 0.2 mM and the fibres were heavily loaded with Ca2+, addition of GTP gamma S (250 microM or 1 mM) induced a small response in about 50% of fibres, but depolarization‐induced responses were not affected in any fibres. 6. Asymmetric charge movement recorded in EDL fibres with the vaseline‐gap voltage clamp was not affected by the application of 1 mM‐GTP gamma S to the cut ends of the fibres for up to 1 h. 7. These data imply that GTP‐binding proteins (G‐proteins) are not involved in coupling the voltage sensors to Ca2+ release in skeletal muscle. Furthermore, there was no evidence that G‐proteins play any role in modulating the voltage sensors, though this possibility could not be totally excluded.
Intracellular electrophysiological study of suprachiasmatic nucleus neurons in rodents: excitatory synaptic mechanisms.
Authors: Kim Y I , Dudek F E
Published: December 2014

1. To study the synaptic mechanisms of excitatory transmission in the suprachiasmatic nucleus (SCN), we assessed the effects of excitatory amino acid receptor antagonists on excitatory postsynaptic potentials (EPSPs) recorded from SCN neurons in horizontal and parasagittal hypothalamic slice preparations from rats and guinea‐pigs. The EPSPs were evoked by electrical stimulation of either optic nerve or a site near the SCN. 2. When evoked at membrane potentials between ‐60 and ‐100 mV, the EPSPs from optic nerve stimulation were conventional in shape; they rose to the peak quickly (6.2 +/‐ 0.5 ms, mean +/‐ S.E.M.; n = 45) and decayed gradually over 50‐250 ms. When evoked at membrane potentials between ‐20 and ‐55 mV after blockade of outward K+ currents and fast Na+ spikes by intracellular injection of Cs+ and QX‐314 (n = 5 neurons), a slow depolarizing potential emerged near the fast peak of the EPSP. This slow potential, unlike the fast peak, was not linearly related to membrane potential. 3. An antagonist for kainate‐ and quisqualate‐type excitatory amino acid receptors, 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX 1‐10 microM), depressed in a concentration‐dependent and reversible manner the EPSPs evoked by optic nerve stimulation at membrane potentials between ‐60 and ‐100 mV (n = 9). The effects of DNQX were not associated with any significant changes in the baseline input resistance or membrane potential of the postsynaptic neurons. The selective N‐methyl‐D‐aspartate (NMDA) receptor antagonist, DL‐2‐amino‐5‐phosphonopentanoic acid (AP5, 50‐100 microM), did not affect significantly and consistently the EPSPs evoked at these membrane potentials (n = 7). On the other hand, AP5 (50 microM) blocked or depressed the slow depolarizing component of the EPSPs evoked at membrane potentials between ‐20 and ‐55 mV (n = 4). No significant changes in baseline input resistance or membrane potential accompanied the effects of AP5. 4. Stimulation of a site lateral or dorsocaudal to the SCN evoked EPSPs distinct from those evoked by optic nerve stimulation. Again, DNQX (0.3‐10 microM) depressed the EPSPs evoked at membrane potentials between ‐60 and ‐100 mV (n = 4) whereas AP5 (50 microM) had no effect (n = 5). When evoked at less negative membrane potentials (i.e. ‐20 to ‐55 mV) after intracellular injection of Cs+ and QX‐314, the EPSPs had a slow depolarizing potential, similar to the EPSPs from optic nerve stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Calcium oscillations in guinea‐pig pancreatic acinar cells exposed to carbachol, cholecystokinin and substance P.
Authors: Sjödin L , Dahlén H G , Gylfe E
Published: December 2014

1. Cytoplasmic Ca2+ ([Ca2+]i) responses were studied in guinea‐pig pancreatic acinar cells during stimulation with cholecystokinin octapeptide (CCK‐8), substance P (SP) and carbachol. 2. Individual cells exhibited [Ca2+]i responses to all three agonists. 3. In the absence of external Ca2+, all the agonists initiated [Ca2+]i peaks which, particularly at high agonist concentrations, rapidly declined. 4. SP induced repetitive monophasic [Ca2+]i transients which started from basal [Ca2+]i even after elevation of the external Ca2+ concentration. 5. CCK‐8 triggered similar oscillations, which particularly at high agonist concentration or after elevating external Ca2+ became superimposed upon a sustained elevation of [Ca2+]i. 6. Carbachol‐induced oscillations were more complex with [Ca2+]i transients superimposed on slower waves. 7. At high carbachol concentrations or elevation of external Ca2+ the slow waves fused into a sustained increase of [Ca2+]i. 8. The protein kinase C (PKC) activator 12‐O‐tetradecanoylphorbol‐13‐acetate attenuated the agonist‐induced [Ca2+]i responses, and this effect was reversed by the PKC activator staurosporine. 9. The results indicate that oscillations of [Ca2+]i induced by SP, CCK‐8 and carbachol involve intracellular mobilization of Ca2+. 10. CCK‐8 and carbachol also cause a rise of [Ca2+]i by a mechanism more directly dependent on the presence of extracellular Ca2+. 11. In the case of carbachol the latter component is subject to oscillatory control. 12. The transition from oscillatory [Ca2+]i to sustained increase may be associated with inhibition of amylase release.
Modulation of potassium channels in intact human T lymphocytes.
Authors: Pahapill P A , Schlichter L C
Published: December 2014

1. A voltage‐dependent K+ channel called the ‘n’ type (for ‘normal’) is the most prevalent ion channel found in whole‐cell recordings from T lymphocytes. In whole‐cell patch‐clamp recordings activity of the n‐type channel is affected by mitogenic agents, pH, Ca2+ and temperature but not by cyclic nucleotides. Because channel properties and regulation can depend on cytoplasmic components we sought to reassess the properties of K+ channels in intact, normal human T lymphocytes using cell‐attached, patch‐clamp recordings. In the present study, we show that the predominant K+ channel in resting, intact cells is the n type and is affected by voltage, temperature and Ca2+ in ways similar to the disrupted cell. Moreover, K+ channels are activated by agents that raise cyclic AMP in intact cells. 2. In cell‐attached recordings, we found voltage‐activated K+ channels in about 60% of patches at room temperature. The channel was K+ selective as judged from the reversal potential under different Ka(+)‐K+ gradients and at different resting membrane potentials. Some patches were subsequently excised and the selectivity further confirmed. The current‐voltage relation was inwardly rectifying under symmetrical K+ concentrations and had a slope conductance of 9.4 pS at 50 mV depolarized and 23.8 pS at 50 mV hyperpolarized from the resting potential. From the reversal potentials under various conditions the cell resting potential was ‐51 +/‐ 1 mV in normal NaCl saline and about 0 mV when the bath contained 150 mM‐KCl saline. Two other types of K+ channel were seen in resting, intact cells, but were much less common (less than 5% and 11% of patches). A large‐conductance K+ channel was seen in less than 1% of inside‐out patches. 3. The predominant K+ channel in intact, resting T lymphocytes was confirmed as the n type underlying the whole‐cell K+ current evoked by voltage steps. In cell‐attached patches there was a low, steady‐state level of activity at the resting potential but activity was greatly increased by depolarizing voltage jumps. Steady‐state inactivation could be removed by a hyperpolarizing pre‐pulse. Ensemble currents constructed by summing channel openings during repeated voltage jumps showed sigmoid kinetics of current activation and a monoexponential decay phase. These kinetics were well fitted by a Hodgkin‐Huxley‐type n4j kinetic model with time constants very similar to the whole‐cell current of disrupted cells. Moreover, the kinetics depended on the external K+ concentration as previous research has shown.(ABSTRACT TRUNCATED AT 400 WORDS)
Involvement of sarcoplasmic reticulum ‘Ca2+ release channels’ in excitation‐contraction coupling in vertebrate skeletal muscle.
Authors: Brunder D G , Györke S , Dettbarn C , Palade P
Published: December 2014

1. Pharmacological blockers of calcium‐induced calcium release from isolated skeletal sarcoplasmic reticulum (SR) vesicles have been introduced into frog skeletal muscle fibres to determine their effects on excitation‐contraction coupling. 2. Among the blockers tested, Ruthenium Red, neomycin, gentamicin and 9‐aminoacridine inhibited the SR Ca2+ release associated with excitation‐contraction (E‐C) coupling as much as they inhibited caffeine potentiation of that release. Protamine, certain of its derivatives, and spermine were ineffective in both in situ tests. 3. Alternative sites of polyamine action on the contractile proteins, SR Ca2+ uptake or charge movements were ruled out. 4. All polyamines tested required considerably higher concentrations to inhibit excitation‐contraction coupling than to block Ca2+ release from isolated SR vesicles. 5. The quantitative pharmacological difference in sensitivity between isolated and intact systems serves as a reminder that results on isolated systems cannot generally be used to predict results of the same substances on more physiological systems. 6. Since caffeine is known to open the SR ‘Ca2+ release channels’ (the ryanodine receptors that mediate Ca(2+)‐induced Ca2+ release), the equal effectiveness of these blockers at inhibiting excitation‐contraction (E‐C) coupling and its potentiation by caffeine suggests that the SR ‘Ca2+ release channels’ are indeed involved in excitation‐concentration coupling in skeletal muscle, although the results do not indicate how the channel is gated open during E‐C coupling.
The ATP‐induced inward current in mouse lacrimal acinar cells is potentiated by isoprenaline and GTP.
Authors: Sasaki T , Gallacher D V
Published: December 2014

1. ATP activates calcium (Ca2+) influx in mouse lacrimal acinar cells in the absence of phosphoinositide hydrolysis. Extracellular ATP (1 mM) activates receptor‐operated cation channels, promoting entry of Na+ and Ca2+ (inward current). This Ca2+ influx in turn activates K+ channels resulting in a delayed, outward, current component. The present study uses patch‐clamp current recording techniques to investigate the role of beta‐adrenoceptor mechanisms, intracellular cyclic AMP and GTP in the regulation of the ATP‐induced inward currents. 2. The beta‐adrenoceptor agonist, isoprenaline (1 microM), does not increase the resting membrane currents but markedly enhances the ATP‐induced inward and outward currents. This effect of isoprenaline is blocked by the beta‐adrenoceptor antagonist propranolol. 3. Internal application of cyclic AMP mimics the potentiating effect of isoprenaline. 100 microM‐cyclic AMP increases the ATP‐induced inward and outward currents to about 200% as compared to control responses. 4. Pre‐treatment of the cells with the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX; 1 mM), also results in a marked potentiation of the ATP‐induced inward currents to 170% as compared to control responses. 5. The ATP‐induced inward current responses are not blocked by either the removal of extracellular Ca2+ or by chelation of intracellular Ca2+ (by inclusion of 10 mM‐EGTA in the recording pipette). Both protocols did however block the potentiating effect of internal cyclic AMP on the ATP‐induced inward current responses. 6. Intracellular ATP (10 mM) reduces the amplitude of the inward currents evoked by external ATP application by about 60% and the currents were no longer potentiated by internal cyclic AMP. 7. Intracellular GTP or GTP‐gamma‐S (100 microM in the pipette solution) potentiates the current responses to ATP, increasing both the amplitude and duration of the inward currents. 8. In excised inside‐out patches, with ATP in the recording pipette (i.e. external ATP), the catalytic subunit of the cyclic AMP‐dependent protein kinase activated the cation channels. The effect of the catalytic subunit was readily reversible and abolished by an inhibitor of the protein kinase. 9. External ATP activates Ca2+ influx in lacrimal acinar cells by a mechanism that is distinct from that activated by phosphoinositide‐coupled receptors. The effect is mediated by direct activation of cation channels in the cell surface membrane which allow for significant entry of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Substance P inhibits activation of calcium‐dependent potassium conductances in guinea‐pig myenteric neurones.
Authors: Morita K , Katayama Y
Published: December 2014

1. Intracellular recordings were made from myenteric AH neurones of the guinea‐pig ileum in vitro. Some experiments were done with a single‐electrode voltage clamp to measure membrane currents. 2. Substance P (SP) applied by superfusion (10 nM‐300 nM), pressure ejection (100 nM‐10 microM, 760 mmHg, for 10‐20 ms) or ionophoresis (1 mM, 100 nA, for 0.2 s) caused a membrane depolarization and an inward current, associated with a decrease in potassium conductance. 3. The SP‐induced depolarization was abolished within 15 min by superfusion with calcium‐free/high‐magnesium (10 mM) solution or solutions containing cobalt, manganese or nickel at 1‐3 mM. The response persisted even after 40‐60 min of superfusion with calcium‐free/normal‐magnesium (1.2 mM) solution. In all these solutions, synaptic potentials were abolished within 5 min. 4. SP inhibited a slowly developing outward current and an outward tail current during and after a long depolarizing command pulse (2‐10 s), and an outward after‐current following single or multiple brief depolarizing command pulses (10‐50 ms). These outward currents were suppressed in calcium‐free/high‐magnesium solution. 5. SP depressed both a calcium‐dependent slow after‐hyperpolarization following the action potential and an outward after‐current preceded by a brief depolarizing command. Both the SP‐induced depolarization and the SP‐induced inward current were augmented when the peptide was pressure‐ejected during the recovery phase of the slow after‐hyperpolarization and during that of the slow outward after‐current, but both of them were inhibited or almost abolished when SP was applied immediately after spike initiation or a brief depolarizing command. 6. The SP‐induced response was depressed by barium (1‐2 mM). The SP response was not inhibited by tetraethylammonium at low concentrations (5‐10 mM), but was depressed at high concentration (20 mM). 7. Superfusion (1‐10 nM) or pressure application of a calcium ionophore, A23187, inhibited or even reversed the SP depolarization and the SP‐induced inward current. 8. These results indicate that SP inhibits activation of a calcium‐dependent potassium conductance which contributes to both the slow after‐hyperpolarization and the resting membrane potential. SP may affect the process by which calcium activates this potassium conductance.
5‐HT3 receptor channels in dissociated rat superior cervical ganglion neurons.
Authors: Yang J , Mathie A , Hille B
Published: December 2014

1. Whole‐cell and single‐channel voltage‐clamp techniques were used to record the 5‐HT3 receptor‐mediated currents in neurons freshly dissociated from rat superior cervical ganglia. 2. Whole‐cell currents elicited by brief pressure ejection of 5‐HT (10 microM) reversed at ‐4.5 mV when extracellular and intracellular solutions mainly contained NaCl and CsCl. The peak current‐voltage relation showed modest inward rectification that was fully developed within less than 2 ms of the applied voltage step. 3. With prolonged application of 5‐HT (10 microM) using a fast perfusion system, the response desensitized in two phases with fast and slow time constants of 0.57 and 6.0 s at ‐74 mV. The time constants showed little voltage dependence; however, the relative amplitude of the two components was significantly dependent on voltage. The time course of desensitization was not affected by agents that increase the levels of intracellular cyclic AMP. 4. The relative permeability of the channel was determined from reversal potential changes. The channel passed small cations non‐selectively, with permeability ratios (PX/PNa) of 0.93 and 1.24 for Cs+ and K+. The organic cations Tris and glucosamine were measurably permeant with permeability ratios of 0.19 and 0.06. Ca2+ was fairly permeant with a relative permeability of 0.55 in 20 mM solution and of 0.16 when the concentration of CaCl2 was increased to 115 mM. No permeability was detected for Cl‐. 5. Fluctuation analysis of the whole‐cell current revealed an apparent single‐channel current of approximately 0.18 pA at ‐74 mV. 6. 5‐HT‐activated single‐channel currents were recorded in excised outside‐out patches. When 5‐HT (10 microM) was delivered by pressure ejection, channel openings appeared rapidly with a delay of 28 ms. The unitary current was about approximately 0.80 pA at ‐74 mV. The channel activity induced by bath perfusion of 5‐HT (0.8 microM) was significantly reduced by 100 nM of the 5‐HT3 receptor‐specific antagonists 3‐tropanyl‐3,5‐dichlorobenzoate (MDL 72222) or 3‐tropanyl‐indole‐3‐carboxylate (ICS 205‐930). 7. The single‐channel current‐voltage relation was non‐linear, with moderate inward rectification similar to that of the whole‐cell current. The chord conductance of the channel decreased with membrane depolarization from 14.6 pS at ‐104 mV to only 9.9 pS at ‐54 mV. Open‐time distributions consisted of two components with mean time constants of 0.45 and 2.8 ms at ‐104 mV. Burst‐length distributions were also made up of two components with time constants of 0.45 and 4.6 ms.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulation of single quantal efficacy at the snake neuromuscular junction.
Authors: Wilkinson R S , Lunin S D , Stevermer J J
Published: December 2014

1. Postsynaptic responses to spontaneous quantal transmitter release have been compared among neuromuscular junctions in a thin snake muscle. For each junction the type, diameter, and input conductance, G(in) of the postsynaptic muscle fibre were determined. Particularly among fibres of a given type, G(in) was directly correlated with fibre diameter. 2. Miniature endplate potentials (MEPPs) were recorded intracellularly near endplates visualized with Nomarski optics. Mean MEPP amplitude decreased with increasing G(in) among fibres in one muscle. However, the dependence of mean amplitude upon G(in) was not ohmic, as would be expected if the underlying single quantal currents (miniature endplate currents, MEPCs) were of similar amplitude at all junctions. Instead, the relation between MEPPs and G(in) suggested that mean MEPC amplitudes, calculated as mean MEPP amplitude x G(in), increased with increasing G(in). 3. MEPCs were recorded directly using the two‐microelectrode voltage clamp technique. Mean MEPC amplitudes depended systematically on G(in), again such that MEPCs were on average larger in fibres with higher G(in). 4. MEPCs were recorded extracellularly from small regions of endplates (underlying a few nerve terminal boutons). Amplitudes of MEPCs depended on G(in) or fibre diameter in the same manner as amplitudes of MEPCs recorded by intracellular voltage clamp. 5. When the anticholinesterase agent neostigmine was added to the bath, amplitude and duration of MEPPs, MEPCs, and extracellular MEPCs increased. However, the systematic dependence of mean MEPC amplitude on G(in) or fibre diameter remained. 6. Evoked subthreshold endplate potentials (EPPs) were recorded under conditions of low extracellular Ca2+. Endplate currents (EPP amplitudes x G(in)) were systematically larger in fibres with larger G(in), indicating regulation of evoked synaptic current in the muscle. The regulation was found to be due to a combination of increased quantal content and larger single quantal currents in larger (higher G(in)) fibres. 7. Synaptic size, assessed either by area of cholinesterase staining or number of terminal boutons, increased with increasing fibre diameter. Assuming that quantal content is proportional to synaptic size, this relation was sufficient to account for the observed increase in quantal content with increasing G(in) among fibres in the muscle, but was not alone sufficient to account for the observed regulation of evoked current. 8. It is concluded that the efficacy of individual transmitter quanta released at the snake neuromuscular junction is regulated such that large muscle fibres receive larger single quantal currents. Regulation of single quantal current contributes substantially to overall regulation of synaptic strength in the muscle.