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Biomedical subjects

A Scarpa

Publications and source records attributed to A Scarpa.

At least 217 records · Page 12Linked to original sources
PubMed1983

Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate.

The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.

Adenosine Diphosphate↗
PubMed1983

Similarities of Li+ and low Ca2+ in the modulation of secretion by parathyroid cells in vitro.

Parathyroid hormone (PTH) release from parathyroid cells in vitro was found to be stimulated by the presence of extracellular lithium ions through a specific secretory response which was comparable to that elicited by low extracellular Ca2+. Li+ (5-40 mM) caused a dramatic increase in the basal PTH secretory rate (up to 2.5-fold), as well as a further enhancement of the low Ca2+-stimulated PTH secretory rate (up to 0.5-fold). Cellular mediators commonly implicated in hormone release from endocrine cells, such as Ca2+ influx, cAMP, and changes in the activity of (Na+, K+) ATPase, were not involved in Li+-stimulated PTH release. The PTH secretory response induced by Li+ was fully maintained in the virtual absence of external Ca2+. Parathyroid cellular cAMP levels were unchanged in buffers varied with respect to both Li+ and Ca2+ and remained constant throughout the time course of Li+ uptake. Ouabain, in concentrations sufficient to block Na+, K+ activity, was also without effect on PTH release elicited by Li+. To identify the site and possible effectors of this stimulatory effect, experimental protocols were devised to modulate the level of intracellular Li+ at constant external Li+ concentrations. These results showed that the enhancement of PTH secretion by Li+ was independent of a 3-fold variation in intracellular Li+, but was directly related to the external Li+ concentration. This and previous evidence suggest that the regulation of PTH secretion by both lithium and calcium ions does not require their transmembrane influx but occurs instead in the parathyroid plasma membrane.

Animals↗
PubMed1983

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction.

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47-72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.

Animals↗
PubMed1983

Parathyroid hormone secretion in the absence of extracellular free Ca2+ and transmembrane Ca2+ influx.

The involvement of extracellular Ca2+ and Ca2+ influx across the plasma membrane in parathyroid hormone (PTH) secretion was investigated in vitro using a new preparation of bovine parathyroid cells. Incubation of these cells in the presence of 25 microM or 2.5 microM free ambient Ca2+ induced a maximal rate of PTH secretion. Low free Ca2+ secretion is not associated with changes in membrane permeability, requires metabolic energy, and is reversible. The Ca2+ channel blocker D600 had no effect on either 45Ca-influx or PTH secretion in these cells. These results, showing that extracellular Ca2+ and Ca2+ influx across the plasma membrane are not required for PTH secretion by parathyroid cells, emphasize the differences in the cellular mechanisms underlying the secretion of PTH vs that of other secretory cells.

Animals↗
PubMed1983

Intracellular Mg2+ homeostasis of Ehrlich ascites tumor cells.

Treatment of Ehrlich ascites tumor cells with the cation ionophore A23187 results in cellular accumulation of calcium from the extracellular medium only when cellular metabolism is inhibited by uncouplers and iodoacetate. In the metabolically active cells A23187 induces a substantial loss of intracellular magnesium at extracellular Mg2+ concentrations below 1 mM. By contrast, in the inhibited cells A23187 induces a conspicuous loss of magnesium at concentrations of magnesium in the medium ranging from 0.2 to 5 mM. This cellular magnesium loss is accompanied by an increase in intracellular calcium. At 10 mM external Mg2+, A23187 provokes an intracellular magnesium increase in both metabolically active and inhibited cells with a corresponding H+ ejection. Intracellular free Mg2+ in both control and metabolically inhibited cells was estimated through a null-point titration by monitoring changes in extracellular Mg2+ concentration due to the addition of A23187 to cell suspensions with the metallochromic indicator Antipyrylazo III. Free intracellular Mg2+ was 0.4 and 1.2 mM in metabolically active cells and in cells treated with metabolic inhibitors, respectively. Under these conditions the cellular ATP was 30.9 and 2.3 nmol/mg dry wt, respectively. The results lead to the conclusion that in intact Ehrlich ascites tumor cells the level of ATP mainly regulates the intracellular free Mg2+ concentration and, in turn, intracellular free Mg2+ determines the entry of calcium into the cell after A23187 addition.

Adenine Nucleotides↗
PubMed1983

Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions. The slow phase, generally believed to be associated with the translocation of Ca+2, across the membrane profile, appears to be the same process in SR dispersions and multilayers through their virtually identical rate constants and their identical activation energies of 22 +/-1 kcal mol -1. The stoichiometry of ~2 mol Ca2+/mol ATP hydrolyzed was measured in dispersions for the slow phase of Ca2+ uptake. Photolysis of caged ATP with the lamp and the laser provides comparable results for the Ca2+ uptake kinetics in SR dispersions and multilayers. Laser flash photolysis, however, has the advantages of optimal time resolution and effective synchronization of the ensemble of Ca2+-ATPase molecules in the ATP initiated Ca2+ transport process.

Adenosine Triphosphate↗
PubMed1983

Mucin secretion and morphological changes of the mucosa in non-neoplastic diseases of the colon.

Changes in mucin secretion and increase in height of the colonic mucosa adjacent to colorectal carcinoma (transitional mucosa) have been considered pre-malignant. In this study similar changes (both morphological and histochemical) have been found in some cases of ulcerative colitis and ischaemic colitis, as well as in juvenile, inflammatory and hyperplastic (metaplastic) polyps. 'Transitional' patterns of mucin secretion also occur in some other cases of ulcerative colitis, colostomies and Crohn's disease of the colon in which the mucosa has a normal height, suggesting the changes in mucin secretion are independent of mucosal morphology. In all these pathological conditions, hyperplastic (metaplastic) mucosa also coexisted. These findings seem to suggest that: (1) 'transitional' changes more likely represent a secondary regenerative phenomenon rather than a premalignant one; (2) the pattern of mucin secretion is not selective enough to serve as a premalignant marker; therefore is not a valid prognostic indicator in colonic biopsies; (3) hyperplastic (metaplastic) changes might derive from 'transitional' mucosa as a result of a more mature phase of this exaggerated regenerative phenomenon. However, in some patients longstanding 'transitional' mucosa may lead to dysplasia under the influence of environmental and genetic factors.

Biopsy↗
PubMed1983

Juvenile and inflammatory polyps of the colon--a histological and histochemical study.

A light microscopy and histochemical study of 24 juvenile and 27 inflammatory polyps showed that both may derive from inflammatory processes. Granulation tissue, secondary to spontaneous local inflammation or due to surgical procedures may subsequently be covered by regenerating epithelium which lines haemorrhagic cavities and mucus lakes to form irregular, elongated and cystic glands, which are characteristic of juvenile polyps. Both juvenile and inflammatory polyps showed cystic, metaplastic and 'transitional-type' glands. The mucin distribution was identical in both types of polyps. All these findings suggest a common origin of the polyps. The presence of 'transitional-type' glands seems to confirm these as a secondary regenerative phenomenon rather than pre-neoplastic, although dysplastic changes in juvenile polyps have been described. It is suggested that both the juvenile and inflammatory polyps may undergo dysplasia only in genetically predisposed subjects. However, this event seems to be very rare.

Adolescent↗
PubMed1982

H+ ATPase of chromaffin granules. Kinetics, regulation, and stoichiometry.

The chromaffin granule ATPase mediates an inwardly directed transport of H+ against concentration gradients, thereby forming and maintaining an electrochemical transmembrane H+ gradient. The kinetics of this ATPase, its activity modulation by changes in electrochemical H+ gradients, and the stoichiometry between H+ transport and ATP hydrolysis were studied in intact bovine chromaffin granules, resealed chromaffin granule ghosts, and highly purified fragmented chromaffin granule membranes. In fragmented membranes the H+ ATPase has a KM for ATP of 69 microM, a maximum of activity at pH 7.3, and a Vmax of 111 nmol/min/mg of protein at 20 degrees C. Trimethyl tin inhibits the ATPase at much lower concentrations than dicyclohexylcarbodiimide, whereas oligomycin, reserpine, and other inhibitors were without effect. In intact chromaffin granules, the ATPase activity was stimulated up to 300% by collapsing the H+ transmembrane gradients. H+/ATP stoichiometry was measured in resealed chromaffin ghosts devoid of ATP and catecholamines under conditions where no net pH changes occur upon ATP hydrolysis. After addition of ATP, the rates of H+ accumulation in the ghosts and ATP hydrolysis were both linear for about 60-100 s, and the ratio of H+ to ATP was 1.71. These data indicate that the H+ ATPase of chromaffin granules has both kinetic similarities and dissimilarities with other known H+ ATPases. The regulation by changes in H+ gradients and the fixed H+/ATP ratio of this ATPase is further evidence of its primary role in establishing electrogenic H+ translocation and H+ gradients in chromaffin granules.

Adenosine Triphosphatases↗
PubMed1982

Regulation of parathyroid hormone secretion in vitro by divalent cations and cellular metabolism.

Parathyroid hormone (PTH) secretion was investigated in intact cells isolated in vitro. Parathyroid cells from bovine parathyroid glands were obtained through tissue dispersion and cell purification through isotonic Percoll gradients, a newly developed protocol enhancing cell homogeneity and viability. Isolated cells maintained both metabolic viability and plasma membrane intactness for over 3 h at 37 degrees C, as shown by the large ATP/ADP ratios and the high intracellular K+ content (ouabain-sensitive) measured. The rate of PTH secretion was inversely related to the Ca2+ concentrations in the medium; secretion was 54 and 18 ng PTH/mg of protein/min when free Ca2+ in the buffer was 0.8 and 2 mM, respectively. At either Ca2+ concentration, PTH secretion was strongly dependent on cell metabolism; it was inhibited by 80-85% within 10 min when cells were suspended in glucose-free buffer containing either cyanide or oxidative phosphorylation uncoupler. Under these conditions, both cellular ATP production and calcium-dependent PTH release could be partially restored by addition of 5 mM glucose. La3+, Mn2+, Sr2+, Ba2+, and Mg2+ were each tested in a range of 0.5-2.5 mM for their effects in suppressing low calcium-stimulated secretion. La3+ and Mn2+ were about twice as effective as Ca2+ on a molar basis, Sr2+ was similar to Ca2+, Mg2+ was about half as effective, and Ba2+ had almost no effect. These results suggest that the Ca2+-dependent stimulus-secretion coupling of these cells is largely different from that established in other secretory cells and provide an in vitro system to further investigate the regulation of PTH secretion.

Adenosine Diphosphate↗
PubMed1982

Catecholamine transport and energy-linked function of chromafffin granules isolated from a human pheochromocytoma.

The structure and function of chromaffin granules of human pheochromocytoma was extensively investigated in a highly purified granule fraction obtained from a single specimen of human pheochromocytoma tissue. Pheochromocytoma chromaffin granules were analyzed for catecholamine, ATP, enkephalin, phospholipid, cytochrome and ion content. Using a variety of techniques it was found that the membrane of these granules is highly impermeable to Na+, K+, and H+, and that the intragranular pH was maintained at 5.1 irrespective of suspending media. The presence of MgATP induces a transmembrane potential (delta psi) across the membrane of these granules which is positive inside and which corresponds to 90 mV. Both delta pH and delta psi are coupled to biogenic amine accumulation into the granules in a process which is reserpine sensitive. These properties are compared with those of chromaffin granules isolated from normal human tissue or from other animal species and are discussed in terms of possible explanation at a biochemical or subcellular level of the clinical manifestation of the pheochromocytoma.

Adenosine Triphosphate↗
PubMed1982

Mechanisms of accumulation of tyramine, metaraminol, and isoproterenol in isolated chromaffin granules and ghosts.

The effects of the transmembrane pH gradient (delta pH) and the transmembrane potential gradient (delta psi) on the uptake of several sympathomimetic amines were investigated, using bovine adrenal chromaffin granules isolated in isotonic sucrose. As previously described [R. Johnson and A. Scarpa, J. Biol. Chem. 254 3750 (1979)], freshly isolated chromaffin granules maintain an intragranular pH of 5.5 as measured by [14C] methylamine distribution and, in the presence of ATP, generate a delta psi of 80 mV, positive inside, as measured by [14C] methylamine distribution. When tyramine, metaraminol, and isoproterenol (1-50 mM) were added to well-buffered suspensions of granules at pH 7.0, a dose-related alkalinization of the granule interior was observed. Study of the time-resolved influx of the same amines labeled radiochemically (5-21 microM) revealed that all the amines were accumulated against an apparent concentration gradient. However, while accumulation of [14C] serotonin and [3H] isoproterenol was totally inhibited by reserpine, [14C] tryramine accumulation was inhibited by only 60% and [14C[ metaraminol uptake was unaffected. The ATP-dependent generation of a delta psi produced a stimulation of amine uptake in the order: serotonin greater than isoproterenol greater than tyramine; metaraminol accumulation was not enhanced by ATP addition. The relationship between the electrochemical proton gradient (delta micro H+) and the electrochemical gradient for each of the sympathomimetic amines (delta micro A) was investigated utilizing chromaffin ghosts devoid of endogenous matrix gradients or components. All amines were accumulated in the presence of delta pH alone. In the presence of delta psi alone, [14C] serotonin, (14C] tyramine, and [3H] isoproterenol were accumulated, but no [3H] metaraminol uptake was demonstrable. The results indicate that serotonin and isoproterenol accumulated in isolated chromaffin granules and ghosts via a reserpine-sensitive mechanism, driven by the magnitude of the electrochemical proton gradient. Conversely, metaraminol permeated the membrane of the chromaffin granule through the apolar lipid phase and distributed according to the delta pH alone. Tyramine uptake proceeded by both mechanisms. The implications of the mechanism of accumulation of these potent physiologic and pharmacologic agents for their in vivo action are discussed.

Adenosine Triphosphate↗
PubMed1982

Sarcoplasmic Ca2+ transients during the contractile cycle of single barnacle muscle fibres: measurements with arsenazo III-injected fibres.

Single muscle fibres from Balanus nubilus were injected with the metallochromic Ca2+ indicator arsenazo III; multi-wavelength microspectrophotometry was used to monitor changes in the absorption spectrum of such fibres during isometric tension transients elicited by electrical stimulation. Differential absorbance changes, recorded in the 660-720 nm region of the spectrum, were characterized by rapid, nearly linear increases during constant current depolarizing pulses (25-200 ms); upon termination of electrical stimuli the increased differential absorbance described an exponential (k = 0.6-2.0 s-1) decay toward baseline levels. The maximal increases in these absorbance signals preceded the peaks of the tension transients by 300-400 ms at 15-17 degrees C. Calibration of these optical transients indicated that the initial velocity of the rising phase corresponded to a 0.03 microM ms-1 increase in sarcoplasmic free Ca2+. Appreciable tension was not generated until the apparent peak magnitude of the free Ca2+ transient exceeded 2 microM; a further three-fold increase (from 2 to 6 micrometers) in the maximal value of the free Ca2+ transient was accompanied by a 20-fold increase in the magnitude of the tension transient.

Animals↗