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Biomedical subjects

A Scarpa

Publications and source records attributed to A Scarpa.

At least 199 records · Page 11Linked to original sources
PubMed1985

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum.

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).

Adenosine Triphosphate↗
PubMed1985

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction.

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.

Animals↗
PubMed1985

Amine transport into chromaffin ghosts. Kinetic measurements of net uptake of biologically and pharmacologically relevant amines using an on-line amperometric technique.

The kinetic parameters for net transport of dopamine, epinephrine, norepinephrine, 5-hydroxytryptamine, S alpha-methyldopamine, R alpha-methyldopamine, and 1R,2S alpha-methylnorepinephrine into highly purified bovine chromaffin ghosts were determined using an on-line amperometric technique. Chromaffin ghosts devoid of endogenous amines were formed from lysis of chromaffin granules under hypotonic conditions, extensive washing of the scattered membranes, followed by resuspension in iso-osmotic media and overnight dialysis. When chromaffin ghosts formed so as to generate and maintain a large delta pH were suspended in 185 mM KCl, 10 mM Hepes at pH 7.0, 37 degrees C, the addition of MgATP resulted in rapid acidification of the intravesicular space, which was maintained at pH 6.0 (+/- 0.1) for over 30 min. Kinetic net amine transport was subsequently measured with a glassy carbon electrode. The initial rates of uptake were found to follow Michaelis-Menten kinetics. Computer based statistical analysis of the data using distribution-free procedures yielded Km (and V) values as follows: in microM (nmol X mg protein-1 X min-1) dopamine, 16.2 (14.0); R-norepinephrine, 32.5 (12.9); R-epinephrine, 35.1 (15.2); 5-hydroxytryptamine, 4.7 (5.1); S alpha-methyldopamine, 17.7 (11.2); R alpha-methyldopamine, 44.2 (9.9); 1R,2S alpha-methylnorepinephrine, 76.5 (12.5). The physiologic and pharmacologic implications of these kinetic parameters are discussed.

Adrenal Medulla↗
PubMed1985

Transmembrane calcium movement in 20,25-diazacholesterol myotonia.

An abnormality in myoplasmic Ca2+ regulation has frequently been proposed in 20,25-diazacholesterol (20,25-D) myotonia. We report here the results of several studies of transmembrane Ca2+ movement in this animal model. (i) Physiologic Ca2+ release by intact sarcoplasmic reticulum (SR) was examined in chemically skinned single muscle fibers preloaded in EGTA-buffered Ca2+ solutions (pCa2+7.0 to 6.4). Isometric tension development and Ca2+ release thresholds in response to Cl- or caffeine showed no differences between control and 20,25-D fibers at any pCa2+. (ii) The kinetics of energy-dependent Ca2+ accumulation in purified SR vesicles were followed spectrophotometrically using Ca2+-sensitive dyes. The apparent rate for ATP-dependent Ca2+ uptake and Ca2+ sequestering capacity were unchanged in SR from 20,25-D animals vs. controls. (iii) Surface membrane Ca2+ATPase activity was measured in red blood cell ghosts and sarcolemma. Enzyme Vmax was decreased by 25 to 50% in both membranes in the 20,25-D-treated animals with a compensatory increase in the number of Ca2+ATPase molecules. In general, the SR handling of Ca2+ appears normal in 20,25-D myotonia, although the activity of Ca2+ATPase in membranes with high sterol content may be altered in response to changes in the lipid environment in this model.

Adenosine Triphosphate↗
PubMed1985

Internalization of metallochromic Ca2+ indicators in mammalian cells.

Two new techniques for internalizing metallochromic indicators into the cytosol of mammalian cells are described. One method consists of hypertonically treating the cells in the presence of the indicator, followed by a hypoosmotic treatment. The second method consists of incubating the cells at high density in a concentrated indicator solution in physiological saline. Using either method, arsenazo III or antipyrylazo III was internalized into Ehrlich Ascites tumor (EAT) cells at concentrations yielding measurable differential absorbance changes which correspond to changes in the intracellular Ca2+ concentration. In the case of antipyrylazo III, the amount of indicator internalized ranged between 140 and 350 microM, and was dependent on the metabolic state of the cell during loading. Control and loaded cells possessed virtually identical ATP/ADP ratios, as measured by high performance liquid chromatography (HPLC) in cell extracts. Antipyrylazo III was also internalized by rat hepatocytes without detectable cell damage. Treatment of metabolically active EAT cells with the calcium ionophore A23187 results in only a slight increase in the intracellular free Ca2+ concentration, [Ca2+]i, whereas treatment with the calcium ionophore ionomycin induces a substantial but transient increase in the [Ca2+]i. In contrast, metabolically inhibited EAT cells show a large rise in the [Ca2+]i upon addition of A23187. Thus, these techniques offer another way of measuring intracellular free Ca2+ changes in mammalian cells and may prove useful, especially where concentrations of free cytosolic Ca2+ larger than 1 microM are expected.

Adenine Nucleotides↗
PubMed1985

Time-resolved x-ray diffraction studies of the sarcoplasmic reticulum membrane during active transport.

X-ray and neutron diffraction studies of oriented multilayers of a highly purified fraction of isolated sarcoplasmic reticulum (SR) have previously provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10-A resolution. These studies used biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via phospholipid transfer proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have detected significant changes in the membrane profile structure associated with phosphorylation of the Ca2+-ATPase within a single turnover of the Ca2+-transport cycle. These studies used the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100-500-ms data collection times, and double-beam spectrophotometry to monitor the Ca2+-transport process directly in the oriented SR membrane multilayer.

Animals↗
PubMed1985

Inositol trisphosphate-induced calcium release and contraction in vascular smooth muscle.

Inositol 1,4,5-trisphosphate (InsP3) caused Ca release and tension development in rabbit main pulmonary artery smooth muscle permeabilized with saponin or digitonin. Both of these responses to single additions of InsP3 (0.5-30 microM) were repeatable and occurred in the presence of 0.0-1.9 mM free Mg2+. Sustained contractions were induced by InsP3. The amount of Ca released by InsP3, measured with a Ca2+-selective electrode, was also estimated to be sufficient to stimulate contraction in intact smooth muscle. Ca release was not influenced by inhibitors of mitochondrial oxidative phosphorylation. The uptake of Ca2+ from the medium into the InsP3-sensitive pool was ATP-dependent. The present results support the hypothesis that, in smooth muscle, InsP3 is the messenger, or one of the messengers, involved in transmitter-induced (pharmacomechanical) Ca release from the sarcoplasmic reticulum, which is the intracellular Ca store identified previously as the source of Ca released by norepinephrine in main pulmonary artery.

Animals↗
PubMed1984

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells.

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.

5'-Nucleotidase↗
PubMed1984

Hyperplastic (metaplastic) polyps of the colon. A histologic and histochemical study.

One hundred seventy-three hyperplastic polyps removed from the colon of 146 patients were carefully examined by light microscopy in order to evaluate the histologic variants. Seventy polyps were also studied histochemically in order to assess the mucin distribution. Seven polyps measured greater than 1 cm. The frequency of hyperplastic foci has been assessed in a review of 1000 colonic adenomas, 50 juvenile, 30 inflammatory, and six Peutz-Jeghers polyps. The vast majority of the hyperplastic polyps showed features similar to those of inflammatory and ischemic bowel mucosa, leading to the suggestion of an inflammatory-ischemic origin of the polyps. The frequent finding of dysplastic features within hyperplastic polyps greater than 1 cm suggests that at least the large polyps are dysplasia-prone.

Adenoma↗
PubMed1984

Difference in glucose dependency of insulin and somatostatin release.

The splenic Brockmann body of channel catfish (Ictalurus punctatus) was isolated and perfused in vitro. It has been shown previously that the hormone release from the catfish pancreas in response to various stimuli resembled that of a mammalian pancreas. The release of insulin and somatostatin was measured at various concentrations of glucose in the perfusate, and the corresponding dose-response curves were derived. As in a variety of mammalian pancreases, insulin release was stimulated half-maximally at ca. 9 mM glucose. In contrast, half-maximal somatostatin release occurred already at ca. 5 mM glucose. Although a comparable finding with mammalian D cells has not yet been reported, histological and physiological resemblances of the mammalian and the fish pancreas suggest that mammalian D cells may also have a glucose sensitivity in the 5 mM range.

Animals↗
PubMed1984

Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon.

We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O-thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O-thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5' flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta-mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

Base Sequence↗
PubMed1983

Mode of action of diethyl ether on ATP-dependent Ca2+ transport by sarcoplasmic reticulum vesicles.

The mode of action of diethyl ether on the sarcoplasmic reticulum Ca2+ pump and ATPase activity was investigated in sarcoplasmic reticulum vesicles (SR). The addition of ether (5%, v/v) at 23 degrees nearly doubled the rates of ATP-dependent Ca2+ uptake and ATP hydrolysis by SR for a wide range of ATP concentrations (20 microM to 10 mM). By contrast, the Ca2+-independent ATPase activity of SR decreased with increasing ether concentrations and practically ceased at higher ether solutions (5-7%, v/v). Ether not only enhanced the forward transport of Ca2+ into vesicles but also the reversal of the Ca2+ pump, and higher rates of Ca2+ efflux coupled to ATP synthesis were observed from Ca2+-loaded vesicles. Electron micrographs of SR pellets showed that the average radius of the vesicles increased by about 20% upon exposure to ether. In the range of 5-40 degree the rate of Ca2+ transport increased with temperature; at about 4 degrees active Ca2+ transport by SR normally ceased, but with the addition of ether (5%, v/v) significant Ca2+ transport (1-2 nmoles Ca2+ per mg per sec) occurred at 0 degree. The further stimulation of SR Ca2+ transport induced by ether was particularly effective at low temperatures; ether increased the rate of Ca2+ uptake by a factor of 10 at 5 degrees but only by a factor of 1.5 at 40 degrees. The effect of ether on the (Ca2+-Mg2+)ATPase of SR could be reversed by resuspending the vesicles in ether-free media. The centrifugation and resuspension of SR in ether-free solutions did not make the vesicles leaky nor did it lyse them irreversibly unless they were also mechanically disrupted. The results indicate that, in ether, there was an increase of intravesicular volume and an increase in membrane fluidity of SR, which could account for the dramatic increase in Ca2+ capacity and rate of Ca2+ transport of ether-treated SR.

Adenosine Triphosphatases↗