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J J Singer

Publications and source records attributed to J J Singer.

At least 37 records · Page 2Linked to original sources
PubMed1986

An automated technique for analysis of current transitions in multilevel single-channel recordings.

Detailed kinetic studies of ion channel gating are best carried out using the patch-clamp technique which permits the measurement of the ionic current through individual channels. Typical patch-clamp recordings show the current signal, in the form of a sequence of rectangular pulses (analogous to a random telegraph signal), riding on slow baseline drift, partially obscured by high-frequency noise and distorted by filtering. In order to analyze such recordings, we have developed a set of interactive Pascal programs based on a feature-detection algorithm capable of identifying current transitions in multiple-channel recordings in the presence of substantial levels of noise and drift. Software operation is largely automated but includes provisions for examination and correction of the output. The software was optimized and systematically evaluated using simulated data with variable amounts of noise and drift. Results indicate that satisfactory performance is obtained for signal-to-noise ratio as low as four even with uncommonly large baseline drift. Steady-state processing speeds varied from 1,000 to 4,000 samples per second depending on data complexity.

Automation↗
PubMed1986

Multiple neuropeptides exert a direct effect on the same isolated single smooth muscle cell.

The contractile effect of various neuropeptides was examined by pressure ejecting these agents from a pipette onto single smooth muscle cells freshly dissociated from the stomach of Bufo marinus. Substance P, cholecystokinin-octapeptide, and bombesin caused contraction, whereas vasoactive intestinal peptide, secretin, and dopamine inhibited acetylcholine-induced contractions. Acetylcholine and the three peptides which produced contraction were found in some instances to act on the same cell, suggesting that receptors for these agents exist on one and the same cell.

Acetylcholine↗
PubMed1986

Substance P and acetylcholine both suppress the same K+ current in dissociated smooth muscle cells.

The effect of substance P on freshly dissociated gastric smooth muscle cells was examined electrophysiologically. Substance P caused depolarization, associated with a membrane conductance decrease, which led to the generation of action potentials and contraction. When the membrane potential was held constant under voltage clamp, substance P induced a net inward current, also associated with a conductance decrease. The net inward current resulted from suppression of an outward K+ current, one which resembled the acetylcholine-sensitive M-current in these cells. When substance P maximally suppressed this outward K+ current, acetylcholine (ACh) had no additional effect. Conversely, when ACh fully suppressed the M-current, substance P was without additional effect. These results indicate that substance P suppresses the same outward K+ current affected by ACh. Suppression of M-current by substance P was observed in approximately half (44 of 85) of the cells studied in these experiments. In those cells that did not respond to substance P, ACh was nevertheless capable of suppressing the M-current. Thus both substance P and cholinergic agonists appear to exert their excitatory effects on smooth muscle cells by inhibiting a common K+ current.

Acetylcholine↗
PubMed1986

Large-conductance Ca2+-activated K+ channels in freshly dissociated smooth muscle cells.

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated. TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.

Animals↗
PubMed1985

Possible forms for dwell-time histograms from single-channel current records.

Certain macromolecules embedded in the cell membranes of a variety of cells behave as gated ion-selective pores or channels. The length of time that a channel remains open or closed is not deterministic in nature and must be described in terms of relative probabilities. If channels act independently of each other and appropriate experimental conditions can be maintained, the behavior of a channel can be described by a homogeneous Markov process. Using this representation, the relative probability of observing openings (or closings) of various durations can be described by a sum of discrete components which are related to the underlying model of the kinetic behavior of the channel. Generally, these discrete components are taken to be simple decaying exponentials; however, exponentially decaying oscillatory components (as well as certain others which are discussed) are consistent with the Markov process representation. The presence of components other than simple decaying exponentials is shown to imply the violation of detailed balance in the steady-state (which requires energy), and thus, the presence of cyclic pathways in models which accurately represent the kinetic behavior of the channel. Oscillatory components, if present, will in general decay at a faster rate than the slowest decaying component, which, except under a very restricted set of conditions, will be a simple exponential.

Cell Membrane Permeability↗
PubMed1985

Cholinergic agonists suppress a potassium current in freshly dissociated smooth muscle cells of the toad.

Single micro-electrode voltage-clamp and current-clamp techniques were used to study cholinergic responses in single freshly isolated gastric smooth muscle cells from the toad Bufo marinus. Acetylcholine (ACh) or muscarine caused membrane depolarization, which sometimes gave rise to action potentials and contractions. The agonist-induced depolarization is due to the suppression of a voltage-dependent K+ conductance, a conclusion based on the following observations. Depolarization was accompanied by an apparent membrane conductance decrease, seen as the increased size of voltage deflexions in response to constant current pulses. The conductance decrease was confirmed under voltage clamp, where current deflexions in response to constant voltage jumps were smaller in the presence of cholinergic agonists. Muscarine induced net inward currents at potentials positive to the K+ equilibrium potential (EK), and net outward currents at potentials negative to EK. In experiments where external K+ concentration ([K+]o) ranged from 20 to 90 mM the reversal potentials shifted 58 mV positive per tenfold elevation of [K+]o, as expected for a K+ current. The steady-state current-voltage relationship revealed that the K+ current inhibited by muscarine was larger at more positive potentials than expected from driving force considerations alone. Therefore, the underlying conductance suppressed by cholinergic agonists was voltage dependent, with almost complete deactivation at potentials more negative than approximately -70 mV and exhibiting a sigmoidal activation curve upon depolarization. The deactivation of this voltage-dependent K+ conductance caused slow current relaxations to occur in response to hyperpolarizing voltage commands from depolarized holding potentials. In experiments where [K+]o ranged from 3 to 30 mM, these current relaxations reversed direction at potentials near EK and the reversal potential shifted 52 mV positive per tenfold elevation of [K+]o, indicating that K ions carry most of the charge. The current relaxations that occurred in response to hyperpolarizing voltage commands were suppressed by ACh, muscarine and oxotremorine. The effects of muscarine persisted in nominally Ca2+-free solutions containing Mn2+. Ba2+ mimicked the effects of muscarinic agonists. Thus, isolated smooth muscle cells exhibit a K+ current resembling the M-current of sympathetic and other neurones, which is reversibly suppressed by cholinergic agonists. The existence of a cholinergic K+ conductance decrease is of interest because it has not previously been demonstrated in smooth muscle.

Action Potentials↗
PubMed1984

Landmark article Oct 28, 1933. Successful removal of an entire lung for carcinoma of the bronchus. By Evarts A. Graham and J. J. Singer.

The left lung and many of the tracheobronchial mediastinal glands were removed in a one stage operation because of a carcinoma that originated in the bronchus of the upper lobe but which was so close to the bronchus of the lower lobe that, in order to remove it completely, it was necessary to remove the entire lung. This is apparently the first case in which an entire lung has been removed successfully at one stage. 600 South Kingshighway.

Carcinoma, Bronchogenic↗
PubMed1981

Voltage clamp of single freshly dissociated smooth muscle cells: current-voltage relationships for three currents.

Voltage-clamp experiments on single freshly dissociated (i.e. uncultured) vertebrate smooth muscle cells were carried out under conditions where the initial inward current, as well as various phases of outward current, could be studied. Current-voltage relationships were obtained for the initial current, the peak outward current, and a later, steady-state current, over a potential range of approximately -130 mV to +50 mV. Evidence is presented that the initial current is carried by Ca+++ ions and is responsible for the rising phase of the action potential and that the peak in the outward current is due to Ca++ activation of K+ conductance.

Action Potentials↗
PubMed1980

Passive properties of the membrane of single freshly isolated smooth muscle cells.

Single, smooth muscle cells were isolated from the stomach muscularis of the toad Bufo marinus and studied on the same day as isolation using standard electrophysiological techniques and direct microscopic observation at high magnification. Following penetration a period of hyperpolarization occurred that appeared to be caused by an increase in K+ conductance activated by Ca2+ entering the cell upon penetration. Ion substitution studies showed that the stable steady-state resting potential was dependent on both [Na+]0 and [K+]0. At [Ca2+]0 = 1.8 mM, active responses could be elicited which, at the higher [Ca2+]0 (< 8mM) generally employed, became action potentials with overshoots. Calculations employing the equations for a short cable and the observed change of membrane potential as a single exponential in response to a small hyperpolarizing current step both indicated that the length constant (lambda) was sufficiently greater than the cell length so that the cell behaved as an isopotential surface during subthreshold perturbations. From photomicrographic measurements of each cell studied and the input resistance, values of specific membrane resistance (Rm) were obtained that ranged as high as 152 k omega x cm2 depending on the ionic environment, most notably on [Ca2+]0. The membrane capacity (Cm) referred to the surface area measured with light microscopy was 1.3 +/- 0.3 microF/cm2 (mean +/- SD). When the best estimate of caveolar membrane area was included, Cm referred to total membrane area (caveolar plus noncaveolar) was approximately 0.8 microF/cm2.

Animals↗
PubMed1980

Calcium action potentials in single freshly isolated smooth muscle cells.

The ionic basis of the action potential was investigated using intracellular microelectrodes in single smooth muscle cells freshly isolated from the stomach of the toad Bufo marinus. When [Ca2+]0 was elevated (> 8mM), action potentials were readily elicited, which had similar characteristics to those found in many tissue preparations of visceral smooth muscle. There was a decrease in membrane resistance at the peak of the action potential and during the undershoot. The following evidence indicated that the inward current is carried by Ca2+: 1) Raising [Ca2+]0 from 15 to 49.6 mM in the presence of 18.2 mM tetraethylammonium chloride (TEA) increased the maximum rate of rise and the overshoot amplitude, the latter by 15 mV, i.e., 29.5 mV/10-fold change in [Ca2+]0. Changing [Na2+]0 from 11.8 to 81.8 mM had no significant effect on the maximum rate of rise or the overshoot. 2) The action potentials were blocked by 8 mM Mn2+ ([Ca2+]0 = 14.6 mM) but not by 14.3 microM tetrodotoxin (TTX) ([Na2+]0 = 100 mM). 3) Action potentials could be elicited when [Ba2+]0 or [Sr2+]0 were present in high concentrations ([Ca2+]0 less than or equal to 31 microM,[Na2+]0 = 11.8 mM). Both the maximum rate of rise and overshoot amplitude of the action potential increased as the membrane potential became more negative, suggesting increased activation of the inward current. Both TEA and Ba2+ prolonged the action potential, suggesting that a K+ current is responsible for repolarization. Action potentials could also be elicited on anode break at elevated [K+]0 (91 mM).

Action Potentials↗
PubMed1980

Rectifying properties of the membrane of single freshly isolated smooth muscle cells.

Single smooth muscle cells freshly isolated from the stomach muscularis of the toad Bufo marinus were studied under direct microscopic observation using intracellular microelectrodes. The deviation of the membrane potential from rest was recorded when steps of current were injected into the cell. Outward-going rectification was consistently observed both in the presence of 1.8 mM and higher external concentrations of Ca2+. There was no indication of inward-going rectification even under conditions favoring its demonstration, i.e., when the external concentration of K+ was high (108 mM) and Cl-, low (39.6 mM). In the presence of tetraethylammonium chloride (TEA), there was a marked decrease in the rectification normally observed with depolarizing currents, suggesting that a K+ conductance contributes to the outward-going rectification. This K+ conductance increased by almost two orders of magnitude over the range from -20 to 0 mV, and displayed an e-fold increase with a depolarization as small as 4-7 mV. In response to hyperpolarizing currents, the membrane potential did not always reach a plateau but at times continued to become more negative. The feasibility of the depletion of ions from the caveolae as an explanation for this observation is discussed.

Animals↗
PubMed1980

Penetration-induced hyperpolarization as evidence for Ca2+ activation of K+ conductance in isolated smooth muscle cells.

Single smooth muscle cells, freshly isolated by enzymatic digestion of the stomach muscularis of the toad Bufo marinus were studied under direct microscopic observation using standard electrophysiological techniques. Following penetration with a microelectrode, a hyperpolarization lasting many seconds occurred before the membrane depolarized to a steady-state level. The following lines of evidence indicate that the penetration-induced hyperpolarization results from an increase in K+ conductance caused by Ca2+ that enters the cell at the time of penetration: 1) The cell contracted at the time of penetration indicating that [Ca2+]i was elevated even though no action potential had occurred; the cell subsequently relaxed. 2) The input resistance was much lower during the hyperpolarization than during the steady-state resting potential. In the steady state all cells displayed outward-going rectification. 3) At constant [Ca2+]0, the amplitude of the hyperpolarization varied with log[K+]0 (1.3-56 mM) to a much greater degree than did the steady-state potential. Tetraethylammonium chloride (TEA) (18.2 mM) reduced the hyperpolarization. 4) At constant [K+]0, the amplitude of the hyperpolarization increased as the [Ca2+]0 was raised (1.8-52.1 mM). 5) With [Ca2+]0 low (less than or equal to 0.16 mM), the hyperpolarization was almost completely abolished in the presence of a high concentration of Ba2+ (80 mM) or Mn2+ (79.2 mM); this was not the case with Sr2+.

Animals↗
PubMed1977

Detection of contraction of isolated smooth muscle cells in suspension.

A Coulter counter has been utilized to assess the contractile state of suspensions of isolated smooth muscle cells prepared by enzymatic digestion of stomach muscularis of Bufo marinus. Pulse-duration and pulse-hieght histograms were constructed from the pulses that occur as cells pass through the Coulter counter orifice. Contraction of cells in suspension causes shifts in these pulse histograms to a greater percent of shorter duration pulses and of larger pulse heights. These shifts are consistent with teh assumption made in out studies that the duration and amplitude of the pulses generated are related to the length and cross-sectional area (or square of the diameter), respectively, of the cell. Shifts in the pulse-height histogram were found to be more reliable. The results of a calibration experiment in which shifts in the histograms of pulse heights were linerrly related to shifts in the histograms of all lengths measured from photomicrographs suggested that the shifts in the pulse-height histogram could be used as a continuous index of changes in the contractile state of a population of cells in suspension.

Animals↗
PubMed1977

Characteristics of response of isolated smooth muscle cells to cholinergic drugs.

The contractile responses of suspensions of isolated smooth muscle cells from the stomach muscularis of Bufo marinus were assessed with a Coulter counter. Contractile responses of strips from the same tissue were recorded isotonically. Suspensions of isolated smooth muscle cells exhibit a dose-dependent graded response to cholinergic agonists. The intact tissue has a lower sensitivity than the isolated cells to acetycholine but a higher sensitivity to carbachol. The higher sensitivity of isolated cells to acetycholine probably reflected decreased efficiency of cholinesterases upon removal of diffusion barriers. Because the isolated smooth muscle cells have affinity constants for atropine (K1 equals 0.07 +/- 0.02NM) and carbachol (9.5 +/- 3.7 muM) similar to that in this and other intact tissues, the affinity of the cholinergic receptor appears unaffected by cell isolation. The rate constant for dissociation (k2) of atropine was estimated from the slowing of response to carbachol by atropine; k2 in the isolated cells is 100 times larger than seen in intact tissues. Further insight into the interaction of cholinergic substances with their smooth muscle receptors might by obtained using this system. The isolated cells contain intact receptors, and diffusion limitations inherent to intact tissue have been removed.

Acetylcholine↗