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F S Fay

Publications and source records attributed to F S Fay.

At least 91 records · Page 5Linked to original sources
PubMed1986

Calcium transients and resting levels in isolated smooth muscle cells as monitored with quin 2.

The Ca2+-sensitive dye quin 2 was used to monitor Ca2+ levels and to follow Ca2+ transients in suspensions of isolated toad (Bufo marinus) smooth muscle cells, whose contractile activity was monitored with a Coulter counter. Single cells were not utilized to follow Ca2+ changes involved with contraction because of problems of rapid dye bleaching and due to photosensitization of smooth muscle cells loaded with quin 2. High levels of quin 2 loading (2-5 mM) severely prolonged (time course increase greater than 3 times), or completely inhibited, contractile responses to carbachol or potassium depolarization. Lower levels (less than 1 mM) produced adequate fluorescent signals, even at the single cell level, and allowed contractile responses of normal magnitude, although with somewhat prolonged (2-3 times) time course. Resting Ca2+ concentrations determined using quin 2 at these lower levels were 129 +/- 3 nM, a value that closely coincided with that measured in the same cell type using Ca-sensitive microelectrodes, or an alternate, more highly fluorescent dye, Fura-2. Resting Ca2+ was highly dependent on the extracellular Ca2+ concentration that appeared to effect intracellular Ca2+ (Cai2+) both by altering the driving force on Ca2+ cross the membrane, as well as the Ca2+ permeability of the cell itself. A small but significant relaxation was observed in response to the lowering of cytoplasmic Ca2+ below resting levels. After carbachol or K+ addition, fluorescent transients peaked significantly before the onset of contraction, which was also transient. Isoproterenol, a known relaxant of these cells, caused a small decrease in Cai2+ (approximately 40 nM) below rest, when applied in maximal dosage (10(-4) M). Isoproterenol also consistently diminished the Ca2+ transient induced by excitatory stimuli such as carbachol or K+. These results indicate that changes in contractility may be directly linked to changes in free cytoplasmic Ca2+ in smooth muscle cells.

Aminoquinolines↗
PubMed1985

Computerized analysis of TV images for ultrasensitive monitoring of the reaction of fluorochrome with protein.

Preparation of protein-specific fluorescent probes with the desired degree of fluorochrome can be greatly facilitated by a technique that combines thin-layer chromatography (TLC) with quantitative image analysis (QIA). Using TLC/QIA, the investigator can determine the fluorochrome/protein ratio on-line with only a few micrograms of protein as fluorochrome is conjugated to protein. In addition, this technique allows rapid quantitation of dye noncovalently adsorbed to fluorochrome-labeled protein.

Chemical Phenomena↗
PubMed1984

Tension transients in single isolated smooth muscle cells.

Tension transients have been recorded for the first time in a single smooth muscle cell. The transient contains a linear elastic response and a biphasic recovery which appear to originate from the cross-bridges. A comparison of transients in smooth and fast skeletal muscle fibers suggests that the cross-bridge in smooth muscle is more compliant than in striated muscle and that transitions between several cross-bridge states occur more slowly.

Animals↗
PubMed1984

Beta-adrenergic stimulation of 42K influx in isolated smooth muscle cells.

The present studies were designed to examine the effect of the beta-adrenergic agent isoproterenol (ISO) on 42K influx into isolated smooth muscle cells. Unidirectional 42K influx studies were carried out as described previously using suspensions of smooth muscle cells derived from the toad stomach. Results from these studies indicated that ISO (10(-4) M) produces an approximate threefold increase in 42K influx within 1 min after drug addition. This response is transient such that the flux rate returns to near basal levels within 5 min after drug addition. The stimulatory effect of ISO on 42K influx is blocked by pretreatment with ouabain (1-7.5 X 10(-4) M). ISO thus appears to increase 42K influx by stimulating the Na+-K+ pump in the smooth muscle cells. Other findings indicate that the stimulatory effect of ISO on Na+-K+ pumping is dose dependent, blocked by the beta-antagonist pindolol, and mimicked by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP). These findings support our hypothesis that ISO elicits a beta-adrenergic, cAMP-dependent activation of the Na+-K+ pump in isolated smooth muscle cells.

Animals↗
PubMed1984

Transmembrane 45Ca fluxes in isolated smooth muscle cells: basal Ca2+ fluxes.

Methods are described for monitoring unidirectional fluxes of 45Ca in smooth muscle cells in suspension. Compartmental analysis of 45Ca influx data indicate that 45Ca exchange consists of two kinetically distinguishable components in the toad stomach muscle cells: a relatively small (approximately 100-pmol X cm-2) component with a rapid rate of exchange (t1/2 approximately 1.4 min) and a larger (approximately 930-pmol X cm-2) component, which exchanges more slowly (t1/2 congruent to 87 min). The rate of exchange of the latter but not the former Ca2+ pool is increased on exposure of the cells to elevated K+ levels; thus the "slow" component of uptake appears to reflect transmembrane Ca2+ flux, whereas the "rapid" component may reflect exchange of surface-bound label. Consistent with this interpretation is the finding that under conditions in which surface 45Ca is rapidly and completely displaced, 45Ca efflux occurs as a simple monoexponential process with a slow rate of exchange (k = 7.80 X 10(-5) s-1). The apparent rate of transmembrane Ca2+ flux in smooth muscle cells at rest is approximately 0.1 pmol X cm-2 X s-1. Cellular processes that could give rise to an apparent transmembrane flux rate of this magnitude are discussed, and a model is presented which appears to describe cellular 45Ca exchange in the isolated smooth muscle cells.

Animals↗
PubMed1984

Beta-adrenergic effects on transmembrane 45Ca fluxes in isolated smooth muscle cells.

The effect of the beta-adrenergic agent isoproterenol (ISO) on transmembrane fluxes of 45Ca was assessed in smooth muscle cells in suspension. Exposure of the cells to this smooth muscle relaxant provoked a large (13-fold) transient increase in 45Ca efflux from the isolated cells. This effect was blocked by the beta-adrenergic antagonist pindolol and mimicked by exposure of the cells to dibutyryl adenosine 3',5'-cyclic monophosphate. ISO had no significant effect on 45Ca binding or on 45Ca influx in those cells, thus the ISO-induced increase in 45Ca efflux appears to reflect increased Ca2+ extrusion from the smooth muscle cells. Because the ISO-induced increase in 45Ca efflux was abolished by pretreatment of the cells with the Na+-K+ pump inhibitor ouabain and by lowering extracellular Na+ levels, we propose that the increased Ca2+ extrusion in response to ISO is coupled to increased activity of the plasmalemmal Na+-K+ pump via increased Na+-Ca2+ exchange.

Animals↗
PubMed1983

Tension transients in single isolated smooth muscle cells.

Tension transients were recorded in a single smooth muscle cell. The transient contains a linear elastic response and a biphasic recovery that appear to originate from the cross-bridges. A comparison of transients in smooth and fast skeletal muscle fibers suggests that the cross-bridge in smooth muscle is more compliant than the cross-bridge in striated muscle and that transitions between several cross-bridge states occur more slowly in smooth muscle than in striated muscle.

Animals↗
PubMed1983

Distribution of alpha-actinin in single isolated smooth muscle cells.

In order to probe the organization of the contractile machinery in smooth muscle, we have studied the distribution of alpha-actinin, a protein present in high concentration in dense bodies, structures apparently analogous to the Z-disks of striated muscle. Localization of alpha-actinin in single isolated smooth muscle cells of the stomach muscularis of Bufo marinus was determined by analysis of the pattern of anti-alpha-actinin staining in single fluorescence photomicrographs, stereo pair micrographs, and computerized three-dimensional reconstructions from multiple image planes. The distribution of anti-alpha-actinin and antitubulin staining was compared in contracted and relaxed cells. The studies revealed that alpha-actinin is present in high concentrations in fusiform elements (mean axial ratio = 4.82) throughout the cytoplasm and in larger, more irregularly shaped plaques along the cell margins. Many of the fusiform-stained elements are organized into stringlike arrays characterized by a regular repeating pattern (mean center-to-center interspace = 2.2 +/- 0.1 micron). These linear arrays appear to terminate at the anti-alpha-actinin stained larger plaques along the cell margin; several of these strings often run in parallel with their elements in lateral register. While this general pattern of organization is maintained in cells during contraction, the distance between successive stained elements in stringlike arrays is decreased. We suggest that the decrease in the distance between elements in these strings results from shortening of materials that constitute these linear arrays. We do not believe that the shortening within these arrays reflects compression by forces generated elsewhere within the cell, as the reorganization of noncontractile microtubules is qualitatively different from the changes in the pattern of anti-alpha-actinin staining.

Actinin↗
PubMed1983

Cross-bridge elasticity in single smooth muscle cells.

In smooth muscle, a cross-bridge mechanism is believed to be responsible for active force generation and fiber shortening. In the present studies, the viscoelastic and kinetic properties of the cross-bridge were probed by eliciting tension transients in response to small, rapid, step length changes (delta L = 0.3-1.0% Lcell in 2 ms). Tension transients were obtained in a single smooth muscle cell isolated from the toad (Bufo marinus) stomach muscularis, which was tied between a force transducer and a displacement device. To record the transients, which were of extremely small magnitude (0.1 microN), a high-frequency (400 Hz), ultrasensitive force transducer (18 mV/microN) was designed and built. The transients obtained during maximal force generation (Fmax = 2.26 microN) were characterized by a linear elastic response (Emax = 1.26 X 10(4) mN/mm2) coincident with the length step, which was followed by a biphasic tension recovery made up of two exponentials (tau fast = 5-20 ms, tau slow = 50-300 ms). During the development of force upon activation, transients were elicited. The relationship between stiffness and force was linear, which suggests that the transients originate within the cross-bridge and reflect the cross-bridge's viscoelastic and kinetic properties. The observed fiber elasticity suggests that the smooth muscle cross-bridge is considerably more compliant than in fast striated muscle. A thermodynamic model is presented that allows for an analysis of the factors contributing to the increased compliance of the smooth muscle cross-bridge.

Animals↗
PubMed1980

Control of ion distribution in isolated smooth muscle cells. I. Potassium.

We describe a technique for examining unidirectional ion movements in suspensions of enzymatically disaggregated smooth muscle cells derived from stomach muscle of the toad. This technique has been used to analyze the movement of 42K across these cells. This analysis was greatly simplified by the finding that the cells were in a steady state with respect to K+ distribution after isolation. The potassium contents of the isolated cells were identical to those of intact smooth muscle (131 mM/liter intracellular fluid) and stable for over 4 h; moreover, the unidirectional influx and efflux rates were equal. An additional simplification was provided by the finding that virtually all the K+ exchanges in a manner predicted for a simple two-compartment system consisting of an extracellular and an intracellular space. Transmembrane K+ flux in these cells averaged 1.2 pmol.cm-2.s-1 at room temperature. A large portion (approximately 80%) of 42K influx appeared to be mediated by a saturable transport system with an apparent Km of 0.6 mM and an apparent Vmax of 1.3 pmol.cm-2.s-1. The calculated resting membrane permeability to K+ in these isolated smooth muscle cells, assuming a membrane potential of -50 mV, was 2.9 X 10(-8) cm/s. The calculated gK+ was 2.7 mumho/cm2 constituting only a small fraction of the total membrane conductance as measured electrophysiologically. The latter finding suggests that the resting membrane potential in the isolated cells must be determined by ions in addition to K+. We propose that these methods for studying ion movements in smooth muscle should aid in unraveling the mechanisms responsible for controlling the distribution of ions both at rest, as in the present study, as well as in response to neurotransmitters.

Animals↗
PubMed1979

Mechanism of beta-adrenergic relaxation of smooth muscle.

The mechanism of beta-adrenergic relaxation was investigated in isolated smooth muscle cells. Beta-adrenergic agents stimulate cyclic AMP-dependent phosphorylation, enhance Na+/K+ transport and induce relaxation. The stimulation of Na+/K+ transport is obligatory for relaxation, and we suggest that this stimulation induces relaxation through enhanced Na+/Ca2+ exchange.

Animals↗