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Biomedical subjects

J J Singer

Publications and source records attributed to J J Singer.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1969

Evidence for a role of cyclic AMP in neuromuscular transmission.

Experiments were undertaken to determine if the effects of epinephrine in promoting neuromuscular transmission were mediated by adenosine 3':5'-cyclic phosphate (cyclic AMP). Dibutyryl cyclic AMP and the methyl xanthines, theophylline and caffeine (which inhibit cyclic AMP hydrolysis), were found to increase the amplitude of the end plate potential in the isolated rat diaphragm. Like epinephrine (which is known to promote cyclic AMP synthesis), these agents appear to facilitate the release of acetylcholine from the motor neuron. This interpretation is supported by the observation that theophylline and dibutyryl cyclic AMP markedly increased the frequency but not the amplitude of the spontaneous miniature end plate potentials. In addition, these drugs increased the number of transmitter packets released in response to nerve stimulation. These results are consistent with the view that cyclic AMP plays a role in the release of acetylcholine and in the "defatiguing effect" of epinephrine.

Acetylcholine↗