Biomedical subjects

E F Etter

Publications and source records attributed to E F Etter.

9 recordsLinked to original sources

PubMed2001

Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation.

We investigated whether myosin light chain phosphatase activity changes during nitric oxide-induced relaxation of contracted intact carotid media and how changes in phosphatase activity mediate this relaxation. We also investigated one mechanism for regulating this phosphatase. Myosin phosphatase activity, myosin light chain phosphorylation, guanosine 3',5'-cyclic monophosphate (cGMP) concentration, and phosphorylation of the inhibitory protein CPI-17 were all assayed in homogenates of one carotid media ring at each time point during nitric oxide-induced relaxation. The application of sodium nitroprusside to histamine-contracted media caused rapid declines in light chain phosphorylation and force. These were temporally correlated with a rapid elevation of cGMP and a large transient increase in myosin phosphatase activity. During the early response to nitroprusside, when force declined, increases in myosin phosphatase activity, concurrent with cGMP-mediated decreases in calcium and myosin light chain kinase activity, could accelerate light chain dephosphorylation. CPI-17 was dephosphorylated upon application of nitroprusside at the same time that myosin phosphatase activity increased, suggesting that the removal of inhibition by phospho-CPI-17 contributed to the increase in myosin phosphatase activity. After 20 min of nitroprusside, myosin phosphatase activity had declined to basal levels, however low force was sustained. Additional light chain phosphorylation-independent mechanisms may be involved in sustaining the relaxation.

Animals↗

PubMed2001

Relationship of Ca2+ sparks to STOCs studied with 2D and 3D imaging in feline oesophageal smooth muscle cells.

We recorded Ca2+ sparks and spontaneous transient outward currents (STOCs) simultaneously in smooth muscle cells using whole-cell patch recording and a unique, high-speed widefield digital imaging system to monitor fluo-3 fluorescence in both two and three dimensions (2D and 3D). In 2D imaging, the correlation between the amplitude of a spark and its corresponding STOC was a weak one, and 27 % of the sparks failed to cause STOCs. The STOCless sparks were not significantly different in amplitude from those that caused STOCs. Three-dimensional imaging disclosed that STOCless sparks were located close to the cell surface, and on average their apparent distance from the cell surface was not significantly different from the sparks that cause STOCs. Statistical evaluation of spark clusters disclosed that there were regions of the cell where the probability of spark occurrence was high and others where it was quite low.

Aniline Compounds↗

PubMed2000

A dilution immunoassay to measure myosin regulatory light chain phosphorylation.

We examined the quantitation of myosin regulatory light chain phosphorylation (MRLCP) by Western blot and found both offset and saturation errors. The desirable characteristics of an MRLCP assay are that the dynamic range be 60- to 100-fold and that the detection threshold be known and preferably very small relative to total MRLC concentration. No technique examined provided all these characteristics. However, accurate measurements can be obtained by including serial dilutions of the sample to provide a fractional calibration scale in terms of the dephosphorylated light chain and by using interpolation of the phosphorylated band signal intensity to provide values for the relative phosphorylation ratio. We found that this method offers several advantages over methods that rely on signal ratios from single samples: The dilution ratio method is less subject to errors from differences in protein load, it offers estimates of the error in the individual measurement, and has some redundancy that increases the likelihood of obtaining a valid measurement despite gel or membrane artifacts.

Animals↗

PubMed1996

Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

Animals↗

PubMed1996

Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.

Animals↗

PubMed1994

Detection of changes in near-membrane Ca2+ concentration using a novel membrane-associated Ca2+ indicator.

A Ca2+ indicator has been synthesized and characterized which can be used to monitor rapid changes in the free Ca2+ concentration ([Ca2+]) immediately adjacent to cell membranes. This indicator, referred to as C18-Fura-2, consists of a Fura-2 molecule conjugated to a lipophilic alkyl chain which will insert into cell membranes. When associated with cell membranes in low concentrations, C18-Fura-2 exhibits an excitation spectrum with a large Stokes shift and a single isobestic point, thus [Ca2+] can be calculated ratiometrically. The apparent Ca2+ dissociation constant of cell-associated C18-Fura-2 is around 150 nM. C18-Fura-2 orients in the cell membrane so that the fluorophore is facing the side to which it was applied. C18-Fura-2 was used to record rapid changes in intracellular [Ca2+] which occurred in response to membrane depolarization in isolated smooth muscle cells. The initial rise of the [Ca2+] transient reported by C18-Fura-2 was four to six times faster than the rise of the [Ca2+] transient reported by cytosolic Fura-2. This result suggests that C18-Fura-2 was located at the plasma membrane near sites of Ca2+ influx and indicates that membrane-associated Ca2+ indicators can be used to detect rapid, localized changes in [Ca2+] which are obscured in signals recorded using water-soluble, bulk cytosolic fluorescent Ca2+ indicators.

Animals↗

PubMed1993

Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle.

The Na+/Ca2+ exchanger, driven by a transmembrane Na+ gradient, plays a key role in regulating Ca2+ concentration in many cells. Although the exchanger influences Ca2+ concentration, its activity in smooth muscle appears to be closely coupled to Ca2+ availability from intracellular stores. This linkage might result if the exchanger were positioned close to Ca2+ storage sites within the sarcoplasmic reticulum. To test this hypothesis we have developed methods to assess the relative three-dimensional distribution of proteins involved in Na+/K+ pumping, Na+/Ca2+ exchange, Ca2+ storage within the sarcoplasmic reticulum, and attachment of contractile filaments to the membrane in smooth muscle. Here we report that the Na+/Ca2+ exchanger is largely co-distributed with the Na+/K+ pump on unique regions of the plasma membrane in register with, and close to, calsequestrin-containing regions of the sarcoplasmic reticulum in sites distinct from the sites where contractile filaments attach to the membrane. This molecular organization suggests that the plasma membrane is divided into at least two functional domains, and appear to provide a mechanism for the strong linkage seen in smooth muscle between Na+/K+ pumping and Na+/Ca2+ exchange, and between Na+/Ca2+ exchange and Ca2+ release from the sarcoplasmic reticulum.

Animals↗

PubMed1990

The effect of phenylglyoxal on contraction and intramembrane charge movement in frog skeletal muscle.

1. The effects of the arginine-specific protein-modifying reagent, phenylglyoxal, on contraction and intramembrane charge movement were studied in cut single fibres from frog skeletal muscle, using the double-Vaseline-gap voltage clamp technique. 2. The strength-duration curve for pulses which produced microscopically just-detectable contractions was shifted to more positive potentials and longer durations following treatment of fibres with phenylglyoxal. Caffeine-induced contractures were not blocked. 3. The amount of charge moved by large depolarizing pulses from -100 mV holding potential (charge 1) declined during the phenylglyoxal treatment with a single-exponential time course (tau = 7 min). Linear capacitance did not change significantly over the entire experiment. Inhibition of charge movement was predominantly irreversible. 4. Slow bumps (Q gamma) observed in charge movement current transients recorded before phenylglyoxal treatment, using either large test pulses or small steps superimposed on test pulses, were absent from currents recorded after treatment. The current removed by phenylglyoxal contained the bump (Q gamma) and a small fast transient (Q beta). 5. The amount of charge moved by large depolarizing pulses from -100 mV was reduced 20-50% following phenylglyoxal treatment. Charge moved by pulses to potentials more negative than -40 mV was relatively unaffected. The magnitude and voltage range of this inhibitory effect were the same whether the reagent was applied at -100 mV or at 0 mV holding potential. 6. A phenylglyoxal-sensitive component of charge was isolated which had a much steeper voltage dependence than the total charge movement or the charge remaining after treatment. 7. Charge recorded during hyperpolarizing pulses from 0 mV holding potential (charge 2) was reduced very little (less than 5%) at any potential by phenylglyoxal treatments at either 0 or -100 mV. 8. The phenylglyoxal reaction with charge 2 was kinetically different from the reaction with charge 1. 9. The effects of phenylglyoxal on charge 1 and charge 2 both measured in the same fibre were compared. Whether phenylglyoxal was applied at -100 mV, or at 0 mV the results were the same: charge 1 was inhibited much more (25-60%) than charge 2(2-12%). 10. The results presented here indicate that arginyl residues have a functional role in the voltage-sensing mechanism of excitation-contraction coupling and support the hypothesis that the dihydropyridine receptor is the voltage sensor molecule.

Action Potentials↗

PubMed1983

Modulated anticonvulsant block of sodium channels in nerve and muscle.

We have looked at several anticonvulsant drugs regarding their potency for basal block of sodium channels in both skeletal muscle and myelinated nerve preparations under voltage clamp conditions. There is an inverse relationship observed between the half-blocking concentration of each drug and its lipid distribution coefficient. Furthermore the anticonvulsants which are cyclic amides show a systematically weaker potency for blocking channels than do local anesthetics (linear amides) of comparable solubility. Additional kinetic characterizations of drug block are described that may contribute to accumulation of frequency-dependent block and to the appearance of 'inactivation shift' phenomena. Finally, evidence is provided showing that phenytoin and carbamazepine (as well as phenobarbital) selectively block the inactive form of closed sodium channels, thus giving these anticonvulsants a capability for selective action in depolarized tissue.

Anesthetics, Local↗