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Biomedical subjects

E E Daniel

Publications and source records attributed to E E Daniel.

At least 361 records · Page 20Linked to original sources
PubMed1981

Effect of digitonin on rat myometrium subcellular membrane fractions.

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.

5'-Nucleotidase↗
PubMed1981

Thallous ion activation of p-nitrophenylphosphatase of rat myometrium plasma membrane.

Addition of thallous ion (Tl+) inhibited the spontaneous mechanical activity of rat myometrium in K-free Krebs solutions with an I50 value of 30 microM. The corresponding value for I50 for similar inhibition by addition of K+ was 150 microM. Tl+ as well as K+-activated p-nitrophenylphosphatase (PNPPase) of isolated rat myometrium plasma membrane vesicles. Half maximal activation was caused by 0.47 mM Tl+ or 1.6 mM K+. Maximal enzyme activities obtained using Tl+ and K+ were comparable. The Km values for the substance p-nitrophenylphosphatase using Tl+ (1.24 mM) and K+ (1.46 mM) were also similar. Activation by either ion was inhibited by ouabain, Na+, inorganic phosphate, and vanadate (V +5). The results suggest that Tl+ can substitute for K+ for activation of the Na-K pump of rat myometrium plasma membrane.

4-Nitrophenylphosphatase↗
PubMed1981

Isolation and characterization of subcellular membranes from canine stomach smooth muscle.

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.

Adenosine Triphosphate↗
PubMed1981

Na--Ca exchange in rat myometrium membrane vesicles highly enriched in plasma membranes.

When isolated rat myometrium vesicles highly enriched in plasma membranes were preincubated with 100 mM NaCl and then diluted 21-fold in Na-free media, an ATP-independent Ca uptake value of 4.10 +/- 0.23 mumol/g protein occurred, compared to a value of 2.87 +/- 0.16 for a similar uptake by vesicles preincubated in Na-free media. Brief (less than 10 s) exposure of the membrane vesicles to 5 mM ethyleneglycol-bis(beta-aminoethyl)-N,N'-tetraacetic acid (EGTA) after the Ca uptake showed that the NaCl preincubated vesicles retained more Ca than the sucrose or KCl preincubated vesicles. A NaCl concentration in the membrane fractions identical to its concentration in the Ca uptake medium did not enhance the Ca uptake by the vesicles did not show an increased Ca uptake. NaCl added to plasma membrane vesicles actively loaded with Ca caused retention of less Ca than the control. NaCl added to actively loaded vesicles along with EGTA also enhanced calcium efflux compared to EGTA alone. Sucrose, K+, Rb+, or Cs+ could not replace Na+ for the Na+-dependent Ca uptake or release, while Li+ was a poor substitute in both the instances. Na+-dependent Ca-uptake distribution in the various fractions correlated very well with their 5'-nucleotidase activity but not with their NADPH- or succinate-dependent cytochrome c reductase activities. The results have been discussed using a Na--Ca exchange model as well as by a model in which Na+ competes for calcium binding to the membranes.

Adenosine Triphosphate↗
PubMed1981

Isolation of plasma membranes from rat mesenteric veins: a comparison of their physical and biochemical properties with arterial membranes.

Plasma membranes were isolated from smooth muscles of rat mesenteric veins. The plasma membrane fraction is relatively pure according to its morphological and enzymatic characteristics. The membrane distribution, enzymatic activities, as well as calcium accumulation by the plasma membrane fraction from venous smooth muscle were compared to those from arterial smooth muscle. The isolated venous smooth muscle plasma membranes formed primarily closed vesicles which were capable of accumulating Ca2+ in the presence of ATP suggesting active transport of Ca2+ across the membrane vesicles. Evidence obtained from several approaches by studying the effect of A23187, phosphate ions and hypotonic shock on the Ca2+ accumulation in the presence of ATP revealed that there is an active transport of Ca2+ across isolated vascular smooth muscle membrane vesicles in addition to binding of Ca2+. However, venous smooth muscle plasma membrane fraction appears to be different from arterial smooth muscle plasma membrane fraction in its low activity of alkaline phosphatase, greater Ca2+ binding and lower Ca2+ transport. These and previous studies show that the plasma membrane of vascular muscles may play an important role in the steady state regulation of cellular calcium concentration during excitation-contraction coupling, especially in small arteries and veins.

Adenosine Triphosphate↗
PubMed1981

Colonic motor and myoelectrical activity: a comparative study of normal subjects, psychoneurotic patients, and patients with irritable bowel syndrome.

Colonic electrical control activity and colonic motor activity in patients with IBS were compared to that found in patients who were equally psychologically disturbed but without bowel symptoms and to the colonic activity of normal subjects. A psychologic assessment was followed by measurement of activity in the rectosigmoid colon during baseline, a neutral interview, a stressful interview, and after a meal or neostigmine. Myoelectrical activity was recorded from two intraluminal bipolar suction electrodes; motor activity was recorded from two intraluminal strain gauges at the same sites. The number and duration of contractions per minute were determined by inspection of the motor activity record; the frequency content of both the motor and myoelectrical records was determined using the Fast Fourier Transform method. Patient selection was successful in producing two groups psychologically similar to each other and more disturbed than the normals. The IBS group had a greater number and duration of contractions than the normals at one recording site during baseline; there were no differences in electrical control (slow wave) activity between these same two groups. The IBS group did not differ significantly from the neurotic group on measures of either electrical control activity or motor activity. Possible reasons for the discrepancies between this and some previous studies are discussed.

Adolescent↗
PubMed1981

Gastroduodenal motility in a case of dystrophia myotonica.

A 53-year-old man with dystrophia myotonica developed symptoms of a chronic intestinal motility disorder. Incoordinate small bowel contractions as well as dilated segments were observed on x-ray examination. Gastroduodenal manometry in the fasted state showed contractions of reduced amplitude. A migrating motor complex occurred in the basal state. Pentagastrin and edrophonium injections increased motility; a liquid meal was followed by a normal motor response. The maximum gastric contraction rate (after pentagastrin injection) was 3.8/min. The maximum duodenal contraction rate observed at each duodenal recording site varied between 11.0 and 18.5/min. The lowest rate at each recording site was 11.1, 11.5, 11.0, and 11.2/min, while the fastest rate was 17.6, 16.6, 18.5, and 17.6/min. These maximum rates occurred independently of the rates at adjacent sites and of the drug infusions. The gastroduodenal motor abnormality in this patient thus predominantly affects smooth muscle, with the intrinsic neurons able to release acetylcholine and the muscle capable of responding to it. The results may indicate that electrical control activity in the duodenum intermittently oscillates more rapidly than normal, and when this occurs, phase-locking is absent. We conclude that in dystrophia myotonica there may be a defect in the cell membrane, which can cause more frequent electrical oscillations of the cell; alternatively, there may be a defect in cell-to-cell coupling.

Duodenum↗
PubMed1980

Imidazole inhibits a temperature-dependent component of mammalial skeletal muscle action potential.

Temperature-dependent transition of conductance in ionic channels has been described in several membrane systems. We report here an abrupt change in the maximal rate of rise of the action potentials of the rat extensor digitorum longus (EDL) at 32 degrees C, indicating alteration in the functional characteristics of the sodium channels. Tetrodotoxin (TTX) and imidazole were used to investigate further the change in properties of the sodium channels with respect to temperature. TTX is known for its specificity in blocking sodium channels. It has been proposed that the guanidinium group of the TTX molecule is essential for its activity. Other guanidinium compounds have also been shown to be effective in blocking the sodium channels. In this study, imidazole, a compound structurally similar to guanidine, was also tested. We find that TTX and imidazole affect the maximal rate of rise of the action potential in a temperature-dependent manner.

Action Potentials↗
PubMed1980

Dimensional changes of cultured smooth muscle cells due to preparatory processes for transmission electron microscopy.

The change in volume of cultured smooth muscle cells prepared with four different fixation procedures for transmission electron microscopy was studied. Although the cells showed swelling after being embedded in Spurr's embedding medium, the degree of swelling depended on the particular method of fixation procedure used. When the volume of the cells measured using transmission electron microscopy was compared with that of the fresh cell volume, cells prepared by two of the methods showed swelling, and cellular shrinkage was noted in the other two methods. One method which caused the least amount of volume change is recommended for quantitative electron microscopic study of vertebrate smooth muscle cell systems.

Animals↗
PubMed1980

Comparative study of the smooth muscle layers of the rabbit duodenum.

1. Intracellular electrodes were used to compare the electrical activity of smooth muscle cells from the longitudinal and circular layers of the rabbit duodenum and their responses to stimulation of the intramural nerves. 2. The longitudinal muscle cells had an average membrane potential of 52 mV when measured between slow waves. Spontaneous action potentials were superimposed on every slow wave. 3. The circular muscle cells had a higher membrane potential of 64 mV although the amplitude of the slow waves was similar to that of the longitudinal muscle cells. Spontaneous action potentials were rarely observed in the circular muscle cells. 4. Lowering the temperature from 36 to 30 degrees C caused a reduction in the membrane potential of the longitudinal muscle cells but not in the circular muscle cells. However, the amplitude of the slow waves of the two layers was reduced to a similar extent. 5. Electrical stimulation produced advances of the slow wave cycles if the stimuli were applied between slow waves. The responses of the cells from the two layers were identical. 6. Under normal conditions, electrotonic coupling was observed only in cells of the muscle layer whose long axis was aligned along the direction of the applied current. 7. In the longitudinal muscle, cholinergic responses blocked by atropine were observed. Inhibitory potentials were the predominant response in the circular muscle. 8. Excitatory responses were recorded in 9% of the circular muscle cells. "Off' excitation following termination of a train of repetitive stimulation pulses was also observed. 9. The differences in membrane potentials, spontaneous spiking activities, neural responses, and the failure to demonstrate good electrotonic coupling between the muscle layers suggest that there was poor electrotonic interaction between the muscle layers. The amplitude of the slow waves of the two layers was nevertheless similar. Thus the validity of the hypothesis that slow waves were transmitted passively from the longitudinal layer into the circular layer through electrotonic coupling must be questioned.

Animals↗
PubMed1980

Ultrastructural study of guinea pig tracheal smooth muscle and its innervation.

The ultrastructure of guinea pig tracheal smooth muscle and its innervation was studied by transmission electron microscopy. Smooth muscle cells were arranged into bundles separated by collagen and connective tissue cells. Gap junctions were found only rarely between adjacent smooth muscle cells. Bundles of axons (predominantly unmyelinated) were seen on the periphery of the muscle bundles. Some penetrated deep into the bundles and a few made close neuromuscular contacts (less than 20 nm). Such a spatial arrangement of nerves and smooth muscle suggests a potential for nervous control and limited myogenic activity. Axon profiles containing small agranular vesicles characteristic of cholinergic nerves and small granular vesicles characteristic of adrenergic nerves were seen in close association with airway smooth muscle and with one another. Some large granular vesicles were associated with varicosities of both nerve types. Quantitative studies demonstrated a preponderance of cholinergic over adrenergic nerve profiles in both upper and lower segments. Varicosities, characteristic of adrenergic nerves, were observed more frequently in upper compared with lower segments; however, observed differences were not significant. The morphological findings are consistent with the control functions exerted by cholinergic and adrenergic nerves but provide no basis for control by nonadrenergic inhibitory nerves.

Adrenergic Fibers↗
PubMed1980

Smooth muscle membrane vesicle orientation: a study on intactness and sidedness of rat myometrium plasma membrane vesicles.

Plasma membrane vesicles of rat myometrium were prepared in media containing 240 mM sucrose. The vesicles were exposed to isotonic, hypertonic, and hypotonic sucrose concentrations, fixed, sectioned, and studied using the electron microscope. The vesicles fixed in isotonic media were circular in appearance. Vesicles fixed in hypertonic media were distorted and showed a reduced volume to surface ratio consistent with the hypothesis that greater than 80% of the vesicles were osmotically active to sucrose. Cationized ferritin binding studies and Ca binding and release studies were also consistent with this finding. Exposure to hypotonic media also yielded membranes with distorted profiles indicating that they had been ruptured. [3H]Sucrose trapping experiments revealed that the vesicles had an internal volume of 1.20-1.44 mL/g protein. Hypotonic shock treatment reduced this intravesicular volume to 0.20-0.28 mL/g protein. The hypotonic shock treatment also led to enhanced galactose oxidase catalyzed Na3B3H4 labelling of the membranes and to increased K+-activated ouabain-sensitive p-nitrophenyl phosphatase activity. The enhancement was the same (55 +/- 10%) in the various membrane preparations for both the parameters. The data are interpreted to conclude that the rat myometrium plasma membrane vesicles consisted of 20% broken vesicles and equal proportions of intact vesicles of inside-out and rightside-out orientations.

4-Nitrophenylphosphatase↗
PubMed1980

Effect of vanadate on rat myometrium plasma membrane enzyme activities.

Vanadate inhibited K+-activated and K+-activated ouabain-sensitive p-nitrophenyl phosphatases of rat myometrium at nanomolar concentrations. The vanadate concentrations required for 50% inhibition were 220 +/- 30 nM for the K+-activated component of the enzyme and 200 +/- 30 nM for the K+-activated ouabain-sensitive component. Micromolar concentrations of vanadate inhibited acid and alkaline p-nitrophenyl phosphatases. ATP-dependent Ca uptake by the plasma membrane vesicles was not inhibited by 10nM - 1 mM vanadate. Mg2+-ATPase was also not affected. Thus K+-activated and K+-activated ouabain-sensitive p-nitrophenyl phosphatase activities of the plasma membrane were most sensitive to inhibition by vanadate. Preliminary experiments demonstrated that similar to ouabain, vanadate inhibited potassium-induced abolition of spontaneous contractile activity of isolated rat myometrium in K-free Krebs. This effect of vanadate is consistent with vanadate inhibition of K+-activated ouabain-sensitive p-nitrophenyl phosphatase.

Animals↗
PubMed1980

Structural and functional study of control of canine tracheal smooth muscle.

The structural bases for myogenic and neurogenic control of canine tracheal smooth muscle were studied. At optimum lengths, strips of muscle showed insignificant neurogenic or myogenic tone. Atropine and/or tetrodotoxin blocked the contractile responses elicited on electrical field stimulation of intrinsic nerves. After raising the tone with tetraethylammonium ion and in the presence of atropine, field stimulation of nerves caused a relaxation, a major component of which was blocked by propranolol and/or tetrodotoxin, suggesting an effect mediated through interaction of mediator released from sympathetic nerves with beta-adrenergic receptors. Electron microscopic studies revealed gap junctions between extensions of smooth-muscle cells and a sparse innervation. The axonal varicosities, corresponding to cholinergic (predominantly) and adrenergic (occasionally) nerves, were seen predominantly in the clefts between cell bundles. The physiological responses were compared with the morphological features. Although this muscle exhibits multiunit behavior in vitro, implying that nerves initiate the coordinate activity, its ultrastructural features suggest a potential for single-unit behavior.

Animals↗
PubMed1980

Characteristics of calcium transport and binding by rat myometrium plasma membrane subfractions.

A gradient has been designed to yield two subfractions of plasma membrane vesicles from rat myometrium, a low buoyant density (8-24% sucrose) fraction N1 richer in 5'-nucleotidase and a higher buoyant density (24-30% sucrose) fraction N2, instead of a previously described fraction F1. Both N1 and N2 had very low activities of NADPH-cytochrome c reductase and succinate-cytochrome c reductase. Electron micrographs of thin sections of N1 showed clear vesicles, whereas N2 consisted of vesicles with electron-dense bodies attached to them. These plasma membrane vesicles can actively take up Ca. The active uptake of Ca was potentiated by oxalate and phosphate and abolished by the Ca ionophore A23187. Dilution of actively loaded vesicles in isotonic media containing EGTA led to loss of a small proportion of the stored Ca instantaneously and the remainder more slowly in a biphasic manner. Dilution in hypotonic media with EGTA led to a release of a much larger proportion of the accumulated Ca. A23187 at high concentrations (10 microM) caused a release of all the sequestered Ca whether the active Ca uptake had been carried out in the presence or in the absence of oxalate. A23187, 0.5 microM, released all the sequestered Ca from the vesicles that were actively loaded in the absence of oxalate, but only 37% when the vesicles were actively loaded with Ca in the presence of oxalate. Comparison of the composite plasma membrane fraction F1 (8-30% sucrose) and the subfractions N1 and N2 showed that they had different capacities for Ca uptake in the presence and absence of ATP. An attempt has been made to analyze the active Ca-uptake data in terms of various Ca pools.

Adenosine Triphosphatases↗