bluuebunny/crossref_metadata_2025 huggingface dataset:
Ionic currents responsible for the generation of pace‐maker current in the rabbit sino‐atrial node.
Authors: Maylie J , Morad M
Published: December 2014

The ionic nature of the pace‐maker current (delta Ip, If, Ih) was investigated in rabbit sino‐atrial node using a single sucrose‐gap voltage‐clamp technique. The pace‐maker current was activated by hyperpolarizing clamp steps negative to ‐50 mV and the pace‐maker potential was activated by an action potential or a depolarizing clamp step. Neither pace‐maker current nor pace‐maker potential were altered by addition of tetrodotoxin, but a tetrodotoxin‐sensitive channel could be activated in sino‐atrial nodal strips following hyperpolarizing clamp steps. Ca2+‐channel blockers did not affect the voltage dependence of delta Ip or the maximum diastolic potential (m.d.p.) significantly. Removal of Ca2+ did not affect the pace‐maker current at clamp potentials near the pace‐maker potential range (‐60 to ‐80 mV), but it did reduce the potential dependence of the m.d.p. Removal of Na+ suppressed completely the pace‐maker current and hyperpolarized the membrane. Removal of Na+ also increased membrane conductance, most likely through an increase in resting K+ permeability. Low concentration of Cs+ (less than 5 mM), but not Ba2+ or tetraethylammonium, markedly suppressed activation delta Ip and reduced the rate of pacing slightly. Cs+ also decreased the membrane conductance and hyperpolarized the membrane. In 50% of experiments designed to determine contribution of IK to pace‐maker current, a double‐pulse procedure revealed a time‐dependent component of delta Ip which reversed near the K+ equilibrium potential, EK. Release of depolarizing or hyperpolarizing test clamps was followed by pace‐maker potentials, the magnitudes of which were dependent on the test‐clamp potential. The m.d.p. approached values near EK following depolarizing clamps and near ‐45 mV following hyperpolarizing clamps. The results suggest that delta Ip is carried primarily by Na+ and is blocked by Cs+. It is likely, however, that Ca2+ alters the rate of pacing not only through its contribution to the Isi system, but also through activation of a K+ conductance.
Asymmetric calcium influx and efflux at maternal and fetal sides of the guinea‐pig placenta: kinetics and specificity.
Authors: Sweiry J H , Yudilevich D L
Published: December 2014

Unidirectional influx of calcium across maternal and fetal sides of the syncytiotrophoblast was investigated in the guinea‐pig placenta by using a rapid (less than 30 s) paired‐tracer dilution technique. Experiments were performed in an in situ placenta artificially perfused through the umbilical vessels or in an isolated placenta in which both the maternal and fetal circulations were perfused. At equimolar Ca2+ concentrations, unidirectional calcium influx was always significantly lower on the maternal side than on the fetal side. Saturation kinetics were observed: on the fetal side the estimated Km was 1.8 +/‐ 0.7 mM and Vmax was 1.66 +/‐ 0.28 mumol/min X g (mean +/‐ S.E. of mean) and on the maternal side Km ranged from 0.18 to 1.15 mM and Vmax ranged from 0.12 to 0.59 mumol/min X g. When the inhibition of calcium influx was investigated on the fetal side of the trophoblast by using competing cations, the following sequence was observed: Ba2+ greater than Ca2+ congruent to Ni2+ greater than Sr2+ greater than Mg2+ congruent to Li+. Efflux of 45Ca2+ from the trophoblast into the ipsilateral circulation (backflux) was rapid (20‐100% in 6 min) and asymmetric since the fetal:maternal ratio was 1.35 +/‐ 0.11 (mean +/‐ S.E. of mean) in the presence of 0.1 mM‐Ca2+. In the dually perfused placenta, transplacental transfer (6 min) of 45Ca2+ varied over a wide range (0‐80%); however, it was similar to that of the extracellular reference tracer, 22Na+, in either maternal‐to‐fetal or fetal‐to‐maternal directions. It is suggested that this is a consequence of the 'leakiness' of the dually perfused placenta since the transplacental transfer of 22Na+ and D‐[3H]mannitol (or L‐[14C]glucose) measured simultaneously were also variable but similar. Transplacental transfer of 45Ca2+ could not be used to characterize specific calcium‐transport mechanisms, whereas highly sensitive trophoblast uptake measurements were provided by the single‐circulation, paired‐tracer technique. Our findings suggest the presence of a specific carrier‐mediated transport system for calcium on both maternal and fetal surfaces of the trophoblast. The asymmetries in unidirectional influx into the trophoblast and rapid backflux indicate a mechanism by which the net transfer of calcium from the maternal to the fetal circulation is maintained in favour of the fetus.
Lesions of the locus coeruleus abolish baroreceptor‐induced depression of supraoptic neurones in the rat.
Authors: Banks D , Harris M C
Published: December 2014

Urethane‐anaesthetized rats were used to investigate the influence of lesions within the locus coeruleus on the inhibition of phasically discharging supraoptic neurones that normally follows the activation of arterial baroreceptors. Carotid sinus baroreceptors were stimulated by the inflation of a blind sac of the carotid bifurcation. A general activation of arterial baroreceptors was evoked by increasing arterial blood pressure following the intravenous injection of the pure alpha‐adrenoreceptor agonist phenylephrine. The locus coeruleus of one side only was destroyed either by thermal (radio‐frequency) lesions, or by the injection of 6‐hydroxydopamine (1 microliter, 0.5 mg/ml). The extent of each lesion was assessed histologically in stained tissue and with fluorescence histochemistry. Lesions in locus coeruleus abolished all baroreceptor input to supraoptic neurones on the side ipsilateral to the lesion. The lesions had no effect on the cardiovascular responses to the stimulus, and did not abolish the excitation of supraoptic neurones after ipsilateral carotid body chemoreceptor activation. 6‐Hydroxydopamine lesions (1 microliter, mg/ml) in the rostral part of the ventrolateral A1 catecholamine neurones were less consistent in their abolition of baroreceptor input to the supraoptic nucleus. When the input from ipsilateral carotid sinus baroreceptors was abolished, there was an equivalent effect on the influence of the carotid body chemoreceptors. Input from other arterial baroreceptors, activated by phenylephrine injection, was not affected. From these results, it is proposed that the baroreceptor‐induced depression of‐phasically discharging supraoptic neurones is mediated via a direct noradrenergic input from the locus coeruleus.
Biochemistry of rat single muscle fibres in newly assembled motor units following nerve crush.
Authors: Nemeth P M , Turk W R
Published: December 2014

A partial crush was applied surgically to the common peroneal nerves of rats, producing motor deficits lasting 4 weeks; the tibialis anterior muscles supplied by the crushed nerves were removed 5 weeks after recovery along with the contralateral control muscles. Myosin ATPase staining following pre‐incubation at pH 4.5 was used to determine fibre types and to demonstrate areas of fibre‐type grouping in the reinnervated areas of the muscles. Enzyme activities of lactate dehydrogenase (LDH), adenylokinase (AK) and malate dehydrogenase (MDH) were measured using micro‐analytical techniques on the individual fibres within the histochemically identical groups and on fibres of the same types selected from areas of the test muscle or the contralateral control which appeared normal. The results show that the degree of enzymatic variation among single fibres reinnervated by a common axon is very small when compared to the general fibre population and, moreover, to fibres of the same histochemical type. Enzyme variability within the newly formed motor units was only slightly greater than the variability reported for normal motor units (Nemeth, Pette & Vrbová, 1981). The results indicate that skeletal muscle fibres originally having great differences in levels of enzyme activity, as demonstrated in the general fibre population, acquire considerable enzymatic similarity following reinnervation by a common motor neurone.
Induction and disappearance of excitability in the oocyte of Xenopus laevis: a voltage‐clamp study.
Authors: Baud C , Kado R T
Published: December 2014

Electrically excitable, sodium‐selective channels are induced in the membrane of the oocytes of Xenopus laevis when it is submitted to prolonged positive potentials (Kado, Marcher & Ozon, 1979; Baud, Kado & Marcher, 1982). Under a long positive voltage‐clamp step, the membrane current, initially outward, becomes inward with a sigmoidal time course. The mean time to half‐maximal inward current (t 1/2) is about 18 s at 16 degrees C when stepping the membrane potential to +55 mV. The rate of channel induction was very temperature dependent (Q10 about 5). In an Arrhenius plot, the t 1/2 for induction at temperatures between 5 and 22 degrees C showed a single slope. The rate of induction was dependent on the membrane potential, increasing exponentially with positive membrane potential (e‐fold for a 20 mV change). When the membrane was maintained at resting potential after induction, the ability to produce inward currents with short depolarizing steps slowly disappeared with a t 1/2 of 4 min at 16 degrees C. The temperature dependence for disappearance was larger than that found for induction (Q10 about 7). The rate of disappearance was not dependent on holding the membrane potential in the range ‐30 to ‐100 mV. Induction proceeded in calcium‐free medium. Cycloheximide, a potent protein synthesis inhibitor had no effect (100 micrograms/ml) on the induction rate. Isobutylmethylxanthine (IBMX) or theophyllin (phosphodiesterase inhibitors) applied externally (10(‐4) M) did not affect the induction or disappearance rates. From the present results, mechanisms such as protein synthesis or a second messenger (such as calcium or cyclic AMP) do not appear to be involved. During the depolarization necessary to obtain induction, another conductance was also activated. It was more slowly established, appeared to be non‐saturable and had a reversal potential between zero and ‐10 mV. It was found to be very much reduced at temperatures below about 16 degrees C.
Muscle size and motor unit survival in mice.
Authors: Habgood M D , Hopkins W G , Slack J R
Published: December 2014

The soleus muscles in neonatal and adult mice were surgically reduced in size on one side of the animal. The experimental and control muscles were excised 6‐48 weeks later and the number of motor units in each muscle was estimated by stimulating the muscle nerve and counting step increments in the electromyogram recorded in vitro. Multiple innervation in individual muscle fibres was then assessed by intracellular recording and by visualization of end‐plates in the light microscope with cholinesterase stain. Muscle fibres were counted in cross‐sections of each muscle in the light microscope. Surgical reductions in the size of the muscle during the first 3 weeks of life produced correlated reductions in the number of motor units in the muscle. This could not be attributed to masking of motor units by multiple innervation, which was always less than 10% in these muscles. The loss of motor units was greatest following reduction in muscle size in newborn mice, whereas in 6‐week‐old mice there was no significant loss of motor units following the operation. Thus, survival of neonatal motor units shows an age‐related dependence on the number of muscle fibres available for innervation. In control muscles there was a highly significant correlation between motor unit and muscle fibre numbers, which is consistent with the hypothesis that motor neurone survival during the embryonic period of cell death is dependent upon the number of muscle fibres available for innervation.
Multiple actions of a molluscan cardioexcitatory neuropeptide and related peptides on identified Helix neurones.
Authors: Cottrell G A , Davies N W , Green K A
Published: December 2014

The effects of the molluscan neuropeptide Phe‐Met‐Arg‐Phe‐NH2 (FMRF amide) and related peptides (Price & Greenberg, 1977) were tested on Helix aspersa neurones. Ionophoretic application of FMRFamide depolarized and excited some neurones, but hyperpolarized and inhibited others. In some neurones the sign of the response was dependent on the membrane potential. Two responses resulted from an increase in membrane conductance, a depolarizing response mediated mainly by an increase in Na+ ion permeability, and a hyperpolarizing response mediated by an increase in K+ ion permeability. In the C1 neurone a voltage‐dependent response was observed, which only occurred when the neurone was depolarized from its resting level. This response was recorded as an inward current during voltage clamp and resulted from a decrease in K current(s), possibly Ca‐activated K current. More than one response may occur in a single neurone. In the C1 neurone, the K‐mediated hyperpolarization occurred as well as the voltage‐dependent response, while the depolarization seen in the F2 neurone was a combination of an increase in Na conductance and an increase in K conductance.
Sodium‐selective micro‐electrode study of apical permeability in frog skin: effects of sodium, amiloride and ouabain.
Authors: Harvey B J , Kernan R P
Published: December 2014

The intracellular sodium ion activity (aiNa), apical membrane potential (psi ac) and apical sodium electrochemical driving force (delta mu Na) in Rana temporaria skin were measured using double‐barrelled sodium‐sensitive micro‐electrodes, in the presence of various apical sodium activities (aoNa), amiloride, ouabain, and during voltage clamp of psi ac. The permeability and specific conductance of the apical cell membrane to sodium entry (PaNa and GaNa respectively) were calculated from the Goldman‐Hodgkin‐Katz equation and the Nernst‐Planck (electrodiffusion) permeability equations respectively. The roles of aoNa and aiNa in the control of apical sodium entry were studied. PaNa increased linearly with log decrease in aoNa between 79 and 0.01 mM. Under short‐circuit conditions, aiNa remained constant over the aoNa range of 10‐79 mM, but decreased when aoNa was lower than 10 mM, due to a fall in delta mu Na and GaNa. Amiloride decreased PaNa, GaNa and aiNa, a result analogous to that observed in spontaneous low‐transporting skins. Ouabain inhibited sodium transport and increased aiNa before any changes in PaNa occurred. The latter decreased only when aiNa rose above 15 mM. Increasing delta mu Na by hyperpolarizing voltage clamp of the apical cell membrane elicited a saturable increase in aiNa. The opposite effect was elicited by depolarizing psi ac. Electrodiffusion appears to be the sole mode of apical sodium entry.
Relations among passive electrical properties of lumbar alpha‐motoneurones of the cat.
Authors: Gustafsson B , Pinter M J
Published: December 2014

The relations among passive membrane properties have been examined in cat motoneurones utilizing exclusively electrophysiological techniques. A significant relation was found to exist between the input resistance and the membrane time constant. The estimated electrotonic length showed no evident tendency to vary with input resistance but did show a tendency to decrease with increasing time constant. Detailed analysis of this trend suggests, however, that a variation in dendritic geometry is likely to exist among cat motoneurones, such that the dendritic trees of motoneurones projecting to fast‐twitch muscle units are relatively more expansive than those of motoneurones projecting to slow‐twitch units. Utilizing an expression derived from the Rall neurone model, the total capacitance of the equivalent cylinder corresponding to a motoneurone has been estimated. With the assumption of a constant and uniform specific capacitance of 1 mu F/cm2, the resulting values have been used as estimates of cell surface area. These estimates agree well with morphologically obtained measurements from cat motoneurones reported by others. Both membrane time constant (and thus likely specific membrane resistivity) and electrotonic length showed little tendency to vary with surface area. However, after‐hyperpolarization (a.h.p.) duration showed some tendency to vary such that cells with brief a.h.p. duration were, on average, larger than those with longer a.h.p. durations. Apart from motoneurones with the lowest values, axonal conduction velocity was only weakly related to variations in estimated surface area. Input resistance and membrane time constant were found to vary systematically with the a.h.p. duration. Analysis suggested that the major part of the increase in input resistance with a.h.p. duration was related to an increase in membrane resistivity and a variation in dendritic geometry rather than to differences in surface area among the motoneurones. The possible effects of imperfect electrode seals have been considered. According to an analysis of a passive membrane model, soma leaks caused by impalement injury will result in underestimates of input resistance and time constant and over‐estimates of electrotonic length and total capacitance. Assuming a non‐injured resting potential of ‐80 mV, a comparison of membrane potentials predicted by various relative leaks (leak conductance/input conductance) with those actually observed suggests that the magnitude of these errors in the present material will not unduly affect the presented results.+4
Acetylcholine receptor channel properties during development of Xenopus muscle cells in culture.
Authors: Brehm P , Kidokoro Y , Moody-Corbett F
Published: December 2014

Developmental changes in acetylcholine (ACh) receptor channel function on aneural cultures of embryonic myotomal muscle cells were examined using the patch‐clamp technique. At all stages of differentiation two different unitary‐event amplitudes were observed, corresponding to high‐gamma (single‐channel conductance) (64 pS) and low‐gamma (46 pS) channel types. No change in conductance occurred for either channel type during the 6‐day in vitro period examined. At resting membrane potential (‐85 mV) the low‐gamma channel exhibited a mean open time of approximately 2 ms which, on the average, was 2‐3‐fold longer than that measured for the high‐gamma channel. Neither the estimated mean channel open time nor the voltage dependence of the open state measured for either channel type changed during development. In recordings with low ACh concentration (0.1‐0.25 microM) both high‐gamma and low‐gamma channel types exhibited non‐stationary opening probabilities over the recording period. Usually the opening rate of both channel types decreased with time following seal formation, however, the 'drop‐out' rate was faster for the low‐gamma channel. A developmental increase in the proportion of high‐gamma events occurred between day 1 (16%) and day 5 (56%) in culture, paralleling the time‐dependent changes in the channel kinetics based on ACh‐activated membrane noise. We conclude that the development of non‐junctional muscle membrane is associated with increased expression of high‐gamma channels and that this process is primarily responsible for the previously reported developmental alterations in macroscopic ACh receptor channel currents.
A transient outward current related to calcium release and development of tension in elephant seal atrial fibres.
Authors: Maylie J , Morad M
Published: December 2014

Membrane currents and development of tension in atrial trabeculae from elephant seal hearts were studied using a single sucrose‐gap voltage‐clamp technique. A transient outward current (Ito) was observed with kinetics, voltage and beat dependence, similar to those of tension. Ito had a bell‐shaped voltage dependence similar to that of tension and the slow inward current (Isi). Ito, unlike Isi, showed beat dependence quite similar to developed tension. Increases in [Ca]o, frequency of stimulation, and addition of adrenaline enhanced Ito and developed tension. Ito was suppressed by addition of Mn2+, tetracaine, or by depolarizing pre‐pulses (to ‐40 mV for 250 ms). Caffeine at low concentrations (1 mM) blocked beat dependence of Ito. At higher concentrations (greater than 5 mM) caffeine suppressed the activation of Ito, phasic tension, and the second component of the birefringence signal (related to Ca2+‐releasing activity of the sarcoplasmic reticulum (s.r.]. Similar to Isi phasic tension and Ito, the voltage dependence of the second component of the birefringence signal was bell‐shaped. Our studies suggest that activation of Ito is related to triggered release of Ca2+ from the s.r. which generates the phasic tension. An excitation‐contraction coupling scheme is presented which incorporates these findings and suggests that Ito may be responsible for shorter action potentials found in atrial fibres.
Three types of calcium‐dependent channel in rat lacrimal glands.
Authors: Marty A , Tan Y P , Trautmann A
Published: December 2014

Isolated cells from rat lacrimal glands were studied with patch‐clamp techniques. Whole‐cell and cell‐attached recordings were obtained while the cells were stimulated by application of carbamylcholine or of the Ca ionophore, A23187. The results were compared with recordings of Ca‐dependent channels obtained in isolated patches. Whole‐cell recordings revealed two types of carbamylcholine‐induced current. At low levels of stimulation, a specific class of Ca‐dependent K channels was selectively activated ('BK channels'). With more intense stimulation an inward current, Ii, was obtained at the cell resting potential. Ii rose rather abruptly after a long delay. In several cells, Ii currents presented spontaneous oscillations. Both K and Ii current responses to carbamylcholine were due to activation of muscarinic receptors. Both responses were elicited by a rise of the intracellular Ca concentration. The immediate source of Ca was intracellular. Replacement of intracellular K with either Na or Cs blocked BK channels entirely, thus allowing the study of Ii currents free from K currents. Ii responses to carbamylcholine were, however, less frequently obtained in Na‐ or Cs‐dialysed cells than in K‐dialysed cells. In symmetrical NaCl solutions, Ii inverted at 0 mV. When replacing part of the intracellular or extracellular Cl with glutamate the reversal potential, Ei, was found to vary in the same direction as the equilibrium potential for Cl ions, ECl. In some experiments, Ei was close to ECl but in others Ei deviated strongly from ECl. These experiments suggested that Ii was mainly due to a Cl‐selective conductance, and that another conductance type was contributing to Ii in variable proportions. It was found that, in K‐free solutions, Ii had a reversal potential very close to ECl. Noise analysis showed that the Cl channels involved in Ii current had a unit conductance of about 1‐2 pS in symmetrical NaCl solutions. At ‐60 mV, the mean channel open time derived from noise power spectra was about 200 ms. The activation of the Ca‐dependent Cl channels was increased by depolarization. Voltage jumps elicited slow exponential relaxations. At ‐60 mV, the time constants of the relaxations were in the range 100‐250 ms. Cell‐attached recordings suggested that internal Ca activated three types of channel, depending on the Ca concentration: BK channels, 2‐4 pS channels and 25 pS channels. Inside‐out and outside‐out patch conditions allowed a rough estimate to be made of the Ca concentration needed to activate each class of channel.(ABSTRACT TRUNCATED AT 400 WORDS)
The role of gamma‐aminobutyric acid mediated inhibition in the response properties of cat lateral geniculate nucleus neurones.
Authors: Berardi N , Morrone M C
Published: December 2014

We studied the effect of local ionophoretic application of bicuculline on the response of cat lateral geniculate nucleus (laminae A) cells to stimulation by sinusoidal gratings and spots of light. Application of bicuculline produced an increase both of spontaneous and visually driven discharge of both X and Y cells. On stimulation by drifting sinusoidal gratings, the average discharge of both X and Y cells remained constant with increasing contrast under normal conditions. Application of bicuculline caused the average discharge to increase with contrast, indicating that the constancy of the average discharge was maintained by gamma‐aminobutyric acid mediated inhibition. Under normal conditions, the amplitude of response modulation of both X and Y cells to sinusoidal grating stimulation increased monotonically with stimulus contrast. During bicuculline application, the slope of the contrast‐response curve for X cells but not for Y cells increased, indicating that the inhibition which dampened the modulation of X cells (but not Y cells) was contrast dependent. Application of acetylcholine also increased the average discharge and the amplitude of modulation of the cell responses, but this increase did not depend on stimulus contrast. Under normal conditions, X but not Y cells showed an attenuation of response and an increase in contrast threshold to low spatial frequencies. This attenuation vanished during bicuculline application. The shape of Y‐cell response curves was unaffected by bicuculline. Bicuculline had the same effect on the non‐linear component of Y‐cell response as on the linear component. Although bicuculline had a different effect on the response of X and Y cells to stimulation by gratings, it reduced the antagonistic surround of both X and Y cells to a similar extent (revealed by plotting the cell receptive fields with flashed spots of light).
Isolation of calcium current and its sensitivity to monovalent cations in dialysed ventricular cells of guinea‐pig.
Authors: Matsuda H , Noma A
Published: December 2014

The ion selectivity of the Ca2+ channels in single ventricular cells of guinea‐pig was studied using a 'giga‐ohm seal' patch electrode for voltage clamp and internal dialysis. To isolate the Ca2+ channel current, currents through the Na+ channel and K+ channels were minimized by replacing external Na+ with Tris+ and removing K+ from both sides of the membrane. With 5 mM‐ATP and 5 mM‐EGTA in the pipette solution, the Ca2+ current was well maintained for more than 30 min in K+‐ and/or Na+‐free external solution. Substitution of Cs+ for intracellular K+ eliminated the region of negative slope conductance in the steady‐state current‐voltage curve and shifted the zero‐current potential or resting potential from ‐80 to ‐31 mV. After Cs+ substitution, a large inward current still flowed via inwardly rectifying K+ channels, but was abolished by removing external K+, which resulted in reduction of the resting membrane slope conductance to 1% of the control value. A decaying outward current attributable to the inwardly rectifying K+ channel was observed on depolarization in 5.4 mM‐external K+ solution with Cs+‐rich internal solution after blocking Ca2+ current. The induction of that current caused an apparent decrease of Ca2+ channel current when the K+‐rich internal solution was switched to the Cs+‐rich one at an external K+ concentration of 5.4 mM. When inwardly rectifying K+ current was suppressed by exposure to K+‐free external solution, replacement of intracellular K+ with Cs+ caused no significant change in the Ca2+ current. With Cs+‐rich solution in the electrode, the decaying outward current was responsible for an apparent depression of the Ca2+ current observed when extracellular K+ was increased. When the K+ current was abolished by 0.2 mM‐extracellular Ba2+, changes in external K+ concentration did not affect the Ca2+ current, excluding the possibility of a direct inhibitory action of K+ on the Ca2+ channel. A time‐ and voltage‐dependent outward current attributed to Cs+ was observed at potentials above +30 mV in Na+‐, K+‐free external solution with Cs+‐rich internal solution. This current persisted in the presence of 20 mM‐intracellular TEA Cl and 5 mM‐extracellular 4‐aminopyridine. Inorganic Ca2+ channel blockers, such as Co2+ or Cd2+, not only suppressed the inward Ca2+ current but also caused some reduction in outward current. Thus the blocker‐sensitive peak current reversed at around +75 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
The photocurrent, noise and spectral sensitivity of rods of the monkey Macaca fascicularis.
Authors: Baylor D A , Nunn B J , Schnapf J L
Published: December 2014

Visual transduction in rods of the cynomolgus monkey, Macaca fascicularis, was studied by recording membrane current from single outer segments projecting from small pieces of retina. Light flashes evoked transient outward‐going photocurrents with saturating amplitudes of up to 34 pA. A flash causing twenty to fifty photoisomerizations gave a response of half the saturating amplitude. The response‐stimulus relation was of the form 1‐e‐x where x is flash strength. The response to a dim flash usually had a time to peak of 150‐250 ms and resembled the impulse response of a series of six low‐pass filters. From the average spectral sensitivity of ten rods the rhodopsin was estimated to have a peak absorption near 491 nm. The spectral sensitivity of the rods was in good agreement with the average human scotopic visibility curve determined by Crawford (1949), when the human curve was corrected for lens absorption and self‐screening of rhodopsin. Fluctuations in the photocurrent evoked by dim lights were consistent with a quantal event about 0.7 pA in peak amplitude. A steady light causing about 100 photoisomerizations s‐1 reduced the flash sensitivity to half the dark‐adapted value. At higher background levels the rod rapidly saturated. These results support the idea that dim background light desensitizes human scotopic vision by a mechanism central to the rod outer segments while scotopic saturation may occur within the outer segments. Recovery of the photocurrent after bright flashes was marked by quantized step‐like events. The events had the properties expected if bleached rhodopsin in the disks occasionally caused an abrupt blockage of the dark current over about one‐twentieth of the length of the outer segment. It is suggested that superposition of these events after bleaching may contribute to the threshold elevation measured psychophysically. The current in darkness showed random fluctuations which disappeared in bright light. The continuous component of the noise had a variance of about 0.03 pA2 and a power spectrum that fell to half near 3 Hz. A second component, consisting of discrete events resembling single‐photon responses, was estimated to occur at a rate of 0.006 s‐1. It is suggested that the continuous component of the noise may be removed from scotopic vision by a thresholding operation near the rod output.
Excitation‐contraction coupling and charge movement in denervated rat extensor digitorum longus and soleus muscles.
Authors: Dulhunty A F , Gage P W
Published: December 2014

K contractures and asymmetrical charge movement were recorded in extensor digitorum longus (e.d.l.) and soleus muscles that had been denervated for 2‐68 days. The relationship between maximum tension during a K contracture and membrane potential shifted to more negative potentials in denervated e.d.l. muscles (by ‐25 mV on average) and to a lesser extent in soleus (by ‐8 mV on average), and became steeper, more so in e.d.l. than soleus. Apart from an early negative shift of ‐11 mV in the voltage dependence of tension in e.d.l. muscles during the first week, the other changes in K contractures following denervation occurred progressively during the first 3 weeks and then stabilized. There was a clear difference in charge movement in denervated e.d.l. fibres but little change in denervated soleus fibres, so that the characteristics of charge movement in e.d.l. and soleus became very similar. The maximum amount of charge movement fell from an average normal value of 23 nC/microF to 6 nC/microF in e.d.l. within the first 2 weeks. The voltage sensitivity shifted to more negative potentials (by about ‐12 mV on average) within the first week. There was no significant change in the slope of the relationship between charge and membrane potential. The effects of denervation on charge movement could only partly explain the changes in K contractures. The only obvious parallels were the early negative shift in the voltage dependence of charge movement and tension in denervated e.d.l. fibres. The other changes in K contractures in denervated fibres could be due to a change in the relationship between charge movement and Ca concentration in the myoplasm or an increase in the Ca affinity of the myofilaments. Although charge movement fell to about a quarter of normal in denervated e.d.l. fibres, membrane capacity increased approximately 3‐fold. A similar increase in capacity in soleus fibres was not associated with a change in charge movement. Fewer indentations were seen in denervated than in normal e.d.l. fibres. The decrease paralleled the fall in charge movement.
Identification of delayed potassium and calcium currents in the rat sympathetic neurone under voltage clamp.
Authors: Belluzzi O , Sacchi O , Wanke E
Published: December 2014

Post‐ganglionic neurones of the isolated rat superior cervical ganglion were studied at 37 degrees C under two‐electrode voltage‐clamp conditions. Membrane depolarization beyond ‐40 mV from holding levels between ‐50 and ‐100 mV produced a delayed outward current which exhibited no inactivation within this voltage range. The current is carried primarily by K+ ions and its instantaneous I‐V relation is linear. The total outward current could be separated into two distinct components on the basis of ion‐substitution experiments. A voltage‐dependent component of the delayed current, termed IK(V), is activated by membrane depolarization beyond ‐40 mV when Ca2+ fluxes are selectively blocked by Cd2+ or in Ca2+‐free solution. IK(V) develops following first‐order kinetics and rises to a peak with a voltage‐dependent delay (239 ms at ‐30 mV and 23 ms at +10 mV). GK(V) attains a saturating value of the order of 17 mS/cm2 at about +20 mV and can be described in terms of a simple Boltzmann distribution for a single gating particle with a valency equal to +2.5. A second component of the delayed outward current, termed IK(Ca), depends on Ca2+ entry for its activation and was isolated as difference current before and after block of Ca2+ movements across the membrane. IK(Ca) is larger and faster than IK(V): it is strictly related to Ca2+ influx and also depends on membrane potential depolarization. A distinct Ca2+ current, ICa, was recorded from the neurone exposed to Na+‐free or tetrodotoxin solution. ICa was activated by membrane depolarization beyond ‐30 mV and reached a maximum value near 0 mV. Its activation agrees with fourth‐order kinetics and becomes faster with increasing depolarization. The Ca2+ current developed with a voltage‐dependent time to peak of 2.9‐1.8 ms and thereafter completely inactivated. The relationship between ICa and IK(Ca) is discussed. The Ca2+‐k+ repolarizing system is expected to be mainly associated with action potentials arising from a depolarized neurone, whereas the IA current (Belluzzi, Sacchi & Wanke, 1985) dominates the repolarization mechanism at the normal membrane potential. The effect of muscarine was examined. Muscarine (10‐50 microM) produced a fall in conductance with a voltage dependence similar to that exhibited by GK(Ca) and was ineffective when removing extracellular Ca2+ or adding Cd2+. A partial suppression of ICa by muscarine is demonstrated. It is suggested that the decrease of the outward current magnitude in the presence of muscarine may be accounted for qualitatively by the reduction in ICa.
Elemental distribution in Rana pipiens retinal rods: quantitative electron probe analysis.
Authors: Somlyo A P , Walz B
Published: December 2014

The composition of dark‐adapted and illuminated retinal rod outer and inner segments and mitochondria was determined with electron probe X‐ray micro‐analysis of cryosections. The concentration of Ca in the outer segment was 0.4 mmol/kg dry wt. (0.1 Ca/rhodopsin) and did not measurably change upon illumination with saturating light for 5 min. The non‐mitochondrial regions of the inner segment contained the highest concentrations (up to 13 mmol/kg dry wt.) of Ca in rods; these regions probably represent the endoplasmic reticulum. The equilibrium potentials estimated from the measured elemental concentrations and the known water content of dark‐adapted outer segments were (mV): ENa = +17, EK = ‐83, ECl = ‐27. The respective values in the inner segment were: ENa = +20, EK = ‐89, ECl = ‐26. The above values were obtained in frog rods bathed in 0.18 mM‐Ca Ringer solution. In the outer segment of toad rods bathed in 1.8 mM‐Ca Ringer, ENa = +33 mV. The Mg content of the rods was high. The (computed) concentration in the dark‐adapted retinae was 11 mM in the outer segment and 24 mM in the inner segment. Illumination caused a reduction in Mg to 9 mM (outer segment) and 16 mM (inner segment). Illumination caused a highly significant reduction in Na and Cl concentrations, and an increase in K concentration in both outer and inner segments. Exposure to Na‐free (choline Ringer) solution resulted in reduction in Na to just‐detectable levels (3 +/‐ 1 mmol/kg dry wt.) in the outer segment and to 5 +/‐ 1 mM in the inner segment. This was associated with a significant loss of Cl and decrease in ECl to ‐50 mV. The low Na content of the outer segment in the Na‐depleted rods is not compatible with an extracellular concentration (105 mM) of inexchangeable Na in the intradiskal space. Mitochondrial Na and Mg paralleled the changes in the cytoplasmic concentrations: both mitochondrial Na and Mg were significantly decreased in illuminated, compared to dark‐adapted rods. There was no detectable Ca (0 +/‐ 0.2 mmol/kg dry wt.) in mitochondria of dark‐adapted rods containing high concentrations of Na; mitochondrial Ca was slightly higher (0.5 +/‐ 0.2 mmol/kg dry wt.) in the mitochondria that contained low Na following illumination.
Respiratory responses to medullary hydrogen ion changes in cats: different effects of respiratory and metabolic acidoses.
Authors: Eldridge F L , Kiley J P , Millhorn D E
Published: December 2014

The steady‐state responses of respiration, measured as integrated phrenic nerve activity, to hypercapnic acidosis of the medullary extracellular fluid (e.c.f.) and to metabolically generated acidosis were compared in paralysed, vagotomized and glomectomized cats. E.c.f. hydrogen ion concentration [( H+]) was measured directly by means of a small (2 mm diameter) pH electrode placed on the ventral medulla. The results in ten cats show that changes of medullary e.c.f. [H+] were linearly related to changes of end‐tidal PCO2 both before (r = 0.999) and after (r = 0.996) development of metabolic acidosis. There was a curvilinear relation between hypercapnic e.c.f. [H+] changes and the respiratory response that reflects progressive saturation of a central neural pathway between the chemoreceptors and the respiratory controller. This relation was similar in form both before and after development of metabolic acidosis. When acidosis of metabolic origin was present, apnea occurred with only small decreases of CO2 despite a high [H+]. The respiratory responses to the same e.c.f. [H+] change were only about one‐half as large when they were generated metabolically as when they were generated by raising PCO2. Both exogenously induced metabolic acidosis (HCl infusion) and endogenous acidosis yielded similar results. We conclude that the e.c.f. [H+] does not represent the unique stimulus to the central chemoreceptors. We discuss several alternate mechanisms for the action of CO2 and [H+] on central chemoreceptors but none can be considered definitive at the present time.
Two components of muscarine‐sensitive membrane current in rat sympathetic neurones.
Authors: Brown D A , Selyanko A A
Published: December 2014

Membrane currents induced by muscarine (Imus) were recorded in voltage‐clamped neurones in isolated rat superior cervical ganglia. Two components of Imus were regularly recorded: an inward current resulting from inhibition of the outward K+ current, IM; and an outward current attributable to the reduction of a steady inward current. The presence of these two components caused a 'cross‐over' in the current‐voltage curves at ‐50 +/‐ 3 mV in neurones impaled with KCl‐filled micro‐electrodes or at ‐63 +/‐ 4 mV in neurones impaled with K‐acetate‐filled electrodes. Both components of Imus were prevented by atropine. Both persisted in Krebs solution containing tetrodotoxin (1 microM), Cd2+ (200 microM) or 0 Ca2+. When IM was inhibited by external Ba2+ or internal Cs+ only the outward component of Imus could be detected. This component reversed at +3 +/‐ 2 mV in cells impaled with CsCl‐filled electrodes or at ‐20 +/‐ 3 mV in cells impaled with Cs‐acetate‐filled electrodes. The reversal potentials agreed with those for the currents induced by gamma‐aminobutyric acid (+4 +/‐ 2 mV and ‐16 +/‐ 3 mV with CsCl and Cs acetate electrodes respectively). Replacement of external NaCl with Na acetate (so reducing external Cl‐ concentration ( [Cl‐]o) from 155 to 22 mM) shifted the reversal potential for Imus by +25 and +14.5 mV in two cells impaled with CsCl‐filled electrodes. A tenfold reduction of external [Na+] (by glucosamine replacement) did not significantly alter the reversal potential for Imus in KCl or CsCl‐impaled cells. Under conditions where IM is already inhibited, the residual outward component of Imus can lead to hyperpolarization and inhibition of neuronal activity in unclamped cells. We conclude that both inward and outward components of Imus result from direct activation of muscarinic receptors on the ganglion cells. The inward component results from IM inhibition. We suggest that the outward component results from inhibition of another, voltage‐independent current IX which largely comprises a Cl‐ current. The inward component induces membrane depolarization and an increased excitability; the outward component can lead to hyperpolarization and reduced excitability.
Effects of apamin, quinine and neuromuscular blockers on calcium‐activated potassium channels in guinea‐pig hepatocytes.
Authors: Cook N S , Haylett D G
Published: December 2014

The bee venom peptide, apamin, has been radiolabelled with 125I, the monoiodinated derivative purified, and its binding to intact guinea‐pig liver cells studied. At 37 degrees C 125I‐monoiodoapamin associated with, and dissociated from, guinea‐pig hepatocytes remarkably rapidly. The association and dissociation rate constants were 1.4 X 10(8) M‐1 s‐1 and 0.035 s‐1 respectively. Equilibrium binding studies demonstrated a saturable binding component compatible with 1:1 binding to a single class of site and having an equilibrium dissociation constant (KL) of 390 pM. The maximal binding capacity was 1.1 fmol mg‐1 dry wt. of tissue. Unlabelled apamin displaced bound 125I‐monoiodoapamin with a KI of 380 pM, which is consistent with the concentration of apamin required to inhibit Ca2+‐activated K+ permeability (PK(Ca) ) in these cells. Inhibitable binding of 125I‐monoiodoapamin to rat hepatocytes was much less than to guinea‐pig hepatocytes and could not be reliably quantified. Neither was there any discernible inhibitable binding to human erythrocytes. This is in keeping with the reported lack of apamin‐sensitive Ca2+‐activated K+ channels in these cell types. Various agents were tested for their ability to inhibit monoiodoapamin binding to, and Ca2+‐mediated K+ efflux from, guinea‐pig hepatocytes. All compounds tested which inhibited binding also blocked K+ efflux at similar concentrations. TEA and quinine affected hepatocytes only at high concentration (KI = 5.8 and 0.51 mM respectively). 9‐aminoacridine, quinacrine and chloroquine were slightly more effective (KI = 70‐180 microM). By far the most active compounds (apart from apamin) were the neuromuscular blocking agents; tubocurarine, pancuronium and atracurium (KI = 7.5, 6.8 and 4.5 microM respectively). Gallamine was slightly less effective (KI = 14 microM) and decamethonium and hexamethonium much less so (KI = 620 and 760 microM respectively). 3,4‐diaminopyridine, alpha‐bungarotoxin and tetrodotoxin were among several compounds which showed little or no affinity for apamin binding sites or inhibition of K+ efflux in guinea‐pig hepatocytes. The saturable binding of 125I‐monoiodoapamin to guinea‐pig hepatocytes corresponds to about 1700 sites per cell. Assuming, tentatively, that binding sites correspond to channels the rate of K+ loss observed following agonist action can readily be explained if these channels have unitary conductances in the range reported for PK(Ca) in other tissues.
Cation activation of the pig kidney sodium pump: transmembrane allosteric effects of sodium.
Authors: Karlish S J , Stein W D
Published: December 2014

We have studied activation by Na or Rb ions of different transport modes of the Na‐K pump, using phospholipid vesicles reconstituted with pig kidney Na‐K‐ATPase. The shape of the activation curves, sigmoid or quasi‐hyperbolic, depends on the nature of the cation at the opposite surface and not on the specific mode of transport. ATP‐dependent Na uptake into K‐containing vesicles (Na‐K exchange) is activated by cytoplasmic Na along a highly sigmoid curve in the absence of extracellular Na (Hill number, nH = 1.9). Activation displays progressively less‐sigmoid curves as extracellular Na is raised to 150 mM (nH = 1.2). The maximal rate of the Na‐K exchange is not affected. Na is not transported from the extracellular face by the pump in the presence of excess extracellular K, and the transmembrane effects of the extracellular Na are therefore 'allosteric' in nature. ATP‐dependent Na‐Na exchange (Lee & Blostein, 1980) and classical ATP‐plus‐ADP‐dependent Na‐Na exchange are activated by cytoplasmic Na along hyperbolic curves. ATP‐dependent Na uptake into Tris‐containing vesicles is activated by cytoplasmic Na along a somewhat sigmoidal curve. (ATP + Pi)‐dependent Rb‐Rb exchange is activated by cytoplasmic and extracellular Rb along strictly hyperbolic curves. The same applies for Rb‐Rb exchange in the presence or absence of ATP or Pi alone. The presence of a high concentration of extracellular Na together with extracellular Rb induces a sigmoidal activation by cytoplasmic Rb of (ATP + Pi)‐dependent Rb‐Rb exchange (nH = 1.45) but does not affect the maximal rate of exchange. Slow passive Rb fluxes through the pump observed in the absence of other pump ligands (see Karlish & Stein, 1982 alpha) are activated by cytoplasmic Rb along a strictly hyperbolic curve with extracellular Rb, nH = 1.0 (Rb‐Rb exchange), along a strongly sigmoid curve with extracellular Na, nH = 1.5 (Rb‐Na exchange), and along less‐sigmoid curves with extracellular Tris, nH = 1.24 (net Rb flux) or extracellular Li, nH = 1.2 (Rb‐Li exchange). Activation of the passive Rb fluxes by extracellular Rb is hyperbolic in the presence of cytoplasmic Rb, Li or Tris but is sigmoid in the presence of cytoplasmic Na (nH = 1.36). Inhibition by cytoplasmic Na of passive Rb fluxes from the cytoplasmic to the extracellular face of the pump depends on the nature of the cation at the extracellular surface.(ABSTRACT TRUNCATED AT 400 WORDS)