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P Volpe

Publications and source records attributed to P Volpe.

At least 91 records · Page 5Linked to original sources
PubMed1992

Postnatal expression of the inositol 1,4,5-trisphosphate receptor in canine cerebellum.

1. Inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, has been shown to be the link between activation of several plasma membrane receptors and Ca2+ release from intracellular, membrane-bound compartments. In this study, the postnatal expression of the canine cerebellum IP3 receptor was investigated by biochemical, ligand binding and immunocytochemical methods. 2. Specific receptor sites for IP3 and the extent of IP3-induced Ca2+ release were quantitated in microsomal fractions isolated from cerebella of developing (0-28 day-old) and adult dogs. The IP3 receptor was detected in newborn animals and adult levels were attained within 3-4 weeks. 3. The time-course of IP3 receptor ontogeny paralleled both growth of Purkinje neurons, as indicated by immunofluorescence of cerebellum cortex cryosections with anti-IP3 receptor antibodies, and synaptogenesis, as judged by Western blotting of the microsomal fractions with anti-synaptophysin antibodies.

Animals↗
PubMed1992

Ca2+ stores in Purkinje neurons: endoplasmic reticulum subcompartments demonstrated by the heterogeneous distribution of the InsP3 receptor, Ca(2+)-ATPase, and calsequestrin.

The nature of second messenger-responsive intracellular Ca2+ stores in neurons remains open for discussion. Here, we demonstrate the existence in Purkinje cells (PCs) of endoplastic reticulum (ER) subcompartments characterized by an uneven distribution of three proteins involved in Ca2+ storage and release: the inositol 1,4,5-trisphosphate (InsP3) receptor, Ca(2+)-ATPase, and calsequestrin. Ca(2+)-ATPase and the InsP3 receptor have a widespread, although not identical, distribution throughout the ER. Calsequestrin is localized throughout the smooth ER and is particularly concentrated in pleiomorphic vesicles with a moderately electron-dense core, which appear to represent a subcompartment of the smooth ER. In double-labeling experiments many of these vesicles were unlabeled by InsP3 receptor antibodies. These results suggest a key role of the ER as an intracellular Ca2+ store and demonstrate a possible structural basis for distinct intracellular Ca2+ pools regulated by different second messengers.

Animals↗
PubMed1991

Purification and characterization of calsequestrin from chicken cerebellum.

Chicken cerebellum microsomal fractions contain a protein tentatively identified as calsequestrin (CS) (Volpe et al., Neuron 5, 713-721, 1990). Here we report, for the first time, the purification of cerebellum CS from whole tissue homogenate by DEAE-Cellulose chromatography and Ca(2+)-dependent elution from phenyl-Sepharose. The purified cerebellum CS displays the shift and increase in intrinsic fluorescence characteristic of skeletal muscle CS, and is shown to be a high-capacity, low-affinity Ca2+ binding protein (Kd = 1 mM).

Animals↗
PubMed1991

Quantitation of ryanodine receptor of rabbit skeletal muscle, heart and brain.

The total number of high-affinity ryanodine receptor (RyR) binding sites present in skeletal and cardiac muscle and in brain tissue of the rabbit was determined by [3H]ryanodine binding to subfractions obtained by differential centrifugation of homogenates prepared in a low-ionic strength medium, containing 0.5% Chaps. In all three tissues at least 80% of [3H]ryanodine binding was recovered in the total membrane (TM) fraction obtained by centrifuging between 650 g for 10 min and 120,000 x g for 90 min. Skeletal muscle displayed higher contents of high-affinity RyR sites (about 49 pmol/g wet wt) than heart and brain (about 12 pmol and 3.5 pmol/g wet wt, respectively). The affinity for ryanodine, as well as the affinity for Ca2+, in the absence or presence of Ca2(+)-releasing drugs (caffeine and doxorubicin) of TM from skeletal muscle, were found to be identical to those of purified terminal cisternae. As low as 1 g of tissue was sufficient to perform several experiments.

Animals↗
PubMed1991

Frog brain expresses a 60 KDa Ca2+ binding protein similar to mammalian calreticulin.

The present report was undertaken in an effort to characterize the nature of Ca2+ binding protein(s) in the central nervous system of less evolved vertebrates. In particular we investigated whether the brain microsomal fraction of Rana esculenta expresses calsequestrin, calreticulin and/or other related Ca2+ binding protein(s). We found that a 60 KDa protein having an NH2-terminal amino acid sequence similar to mammalian calreticulin is the major microsomal Ca2(+)-binding protein.

Amino Acid Sequence↗
PubMed1991

The bulk of Ca2+ released to the myoplasm is free in the sarcoplasmic reticulum and does not unbind from calsequestrin.

Calsequestrin (CS) is the major Ca2+ binding protein contained in the lumen of sarcoplasmic reticulum (SR). Ca2+ binding properties and tissue concentration of CS of frog skeletal muscle were measured. At equilibrium, maximal Ca2+ binding capacity of purified CS was about 1.2 mumol Ca2+/mg protein. Apparent Kds for Ca2+ were around 50 microM in the absence of salts, around 0.9 mM in the presence of 100 mM KCl, and around 1.1 mM under 'physiological' conditions. Quantitation of CS in homogenates was accomplished by three methods (Stains-all staining, immunoblotting and 45Ca ligand overlay). Frog muscle contained about 0.5 mg of CS/g wet weight, that is 6.1 mM CS inside the SR. At rest the in situ free [Ca2+] of SR was calculated to be 3.6 mM, and, thus, CS is largely saturated with Ca2+. Moreover, computer simulations of Ca2+ release indicated that about 75% of Ca2+ released during a twitch is free in the SR and does not unbind from CS.

Animals↗
PubMed1991

Antibodies as probes for ligand gating of single sarcoplasmic reticulum Ca2(+)-release channels.

A large (565 kDa) junctional sarcoplasmic reticulum (SR) protein, the ryanodine receptor (RYR), may play both a structural and a functional role in the mechanism of skeletal muscle excitation-contraction coupling. Recently, the primary amino acid sequence of the RYR has been elucidated. In this paper, we introduce an immunological approach to examine the functional (electrophysiological) properties of the RYR when it is incorporated into planar lipid bilayers. The effects of two polyclonal antibodies against the SR junctional face membrane (JFM) and the RYR (anti-JFM and anti-RYR) were tested on the single-channel gating properties of the RYR SR Ca2(+)-release channel. Junctional SR vesicles were fused into planar lipid bilayers in solutions containing caesium salts. Solutions were designed to minimize the background conductances of the SR K+ and Cl- channels. Three actions of the anti-JFM antibody were distinguished on the basis of single-channel gating and conductance. The anti-RYR antibody had a single action, a simultaneous decrease in single-channel open probability (Po) and conductance. Both antibodies appear to alter single-channel gating by disrupting the Ca2(+)-activation mechanism of the channel. Anti-RYR-antibody-induced gating abnormalities were reversed by ATP, although the ATP-re-activated channels had altered gating kinetics. Two antigenic regions, recognizing the anti-RYR antibody, in the C-terminal end of the RYR primary amino acid sequence contain or are closely associated with putative ligand (Ca2+ and ATP)-binding sites identified previously. Our results demonstrate (1) that the antibodies induced abnormal gating (decreased open probability and stabilization of subconducting states) of SR release channels, and (2) that abnormal gating is not associated with physical obstruction or alteration of the conduction pathway. Thus antibodies directed at specific regions of the RYR (e.g. putative ligand-binding sites) can be used as effective probes with which to study the structural and functional properties of the SR Ca2(+)-release channel gating at the single-channel level.

Adenosine Triphosphate↗
PubMed1991

Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons.

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.

Animals↗
PubMed1991

Caffeine- and ryanodine-sensitive Ca2+ stores of canine cerebrum and cerebellum neurons.

[3H]ryanodine binding to and Ca2+ release from microsomal fractions derived from canine cerebrum (CBR) and cerebellum (CBL) were investigated. High-affinity ryanodine binding sites were detected in both cerebrum and cerebellum microsomes [CBR: maximal binding capacity (Bmax) = 446 fmol/mg protein, dissociation constant (Kd) = 9 nM, Hill coefficient (n) = 0.95; CBL: Bmax = 650, Kd = 12, n = 1.8]. Ryanodine binding in both fractions was increased by millimolar concentrations of ATP [or its nonhydrolyzable analogue beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP)] and micromolar concentrations of Ca2+ but was decreased by micromolar concentrations of ruthenium red, similar to that found in sarcoplasmic reticulum (SR) of striated muscle. The addition of caffeine or the sudden elevation of extravesicular Ca2+ induced a rapid La(3+)-sensitive Ca2+ release from both CBR and CBL microsomal fractions with rate constants of approximately 100 s-1, as determined by stopped-flow photometry of the Ca2+ indicator arsenazo III. The release of Ca2+ was activated by either millimolar ATP or AMP-PCP, blocked by micromolar concentrations of La3+, and significantly inhibited by 50 microM ryanodine. Mg2+ and ruthenium red in millimolar and micromolar concentrations, respectively, caused only a slight inhibition of Ca2+ release. These results indicate that rapid Ca2+ release occurs from caffeine-, Ca2+- and ryanodine-sensitive Ca2+ stores in both CBR and CBL microsomal fractions.

Animals↗
PubMed1991

Evidence for intraluminal Ca++ regulatory site defect in sarcoplasmic reticulum from malignant hyperthermia pig muscle.

Malignant hyperthermia (MH) is a pharmacogenetic disease of humans and various animal species that predisposes to a life-threatening, anesthetic agent-induced syndrome. MH is thought to be a consequence of abnormal, sustained increases in myoplasmic Ca++ and sarcoplasmic reticulum (SR) membranes from MH muscle have been shown to have a Ca++ release channel defect. In the present study we have tested a hypothesis that the abnormal Ca++ release mechanism in MH can be expressed when Ca++ is loaded in the presence of pyrophosphate. SR membrane vesicles isolated from normal and MH pig muscle were loaded with Ca++ in the presence and absence of pyrophosphate until Ca(++)-induced Ca++ release occurred. Under both circumstances the threshold amount of Ca++ loaded until Ca++ release occurred was lower in the SR from MH pig skeletal muscle. This difference in amount of Ca++ preload is not explained by results obtained comparing rates of Ca++ uptake, number of ryanodine binding sites or the amounts of calsequestrin among SR vesicles from MH and normal muscle. We conclude from this study that use of pyrophosphate for Ca++ loading does not ablate the abnormal Ca++ release in SR from MH muscle, suggesting the study can be done on small amounts of SR from biopsied human muscle. The data also suggest that abnormality in an intraluminal, low affinity Ca++ binding site regulating Ca++ release occurs in the SR membrane of MH pig muscle.

Animals↗
PubMed1990

Muscle-type MM creatine kinase is specifically bound to sarcoplasmic reticulum and can support Ca2+ uptake and regulate local ATP/ADP ratios.

Highly purified fractions of sarcoplasmic reticulum (SR) were prepared from chicken pectoralis muscles (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) and analyzed for the presence of creatine kinase (CK). Vesicles derived from longitudinal SR contained 0.703 +/- 0.428 IU of CK/mg of (SR) protein. Immunogold localization of muscle-type MM-CK on ultrathin cryosections of muscle, after removal of soluble CK, revealed relatively strong in situ labeling of M-CK remaining bound to the M band as well as to the SR membranes. In addition, purified SR vesicles were also labeled by anti-M-CK antibodies, and the peripheral labeling was similar to that observed with anti-Ca2(+)-ATPase antibodies. Only some particulate CK enzyme was released from isolated SR membranes by EDTA/low salt buffer, and CK was resistant to extraction by 0.6 M KCl. Thus, some of the MM-CK present in muscle displays strong associative behavior to the SR membranes. The SR-bound CK was sufficient to support, in the presence of phosphocreatine plus ADP, a significant portion of the maximal in vitro Ca2+ uptake rate. The ATP regeneration potential of SR-bound CK was similar to the rate of Ca2(+)-stimulated ATP hydrolysis of isolated SR vesicles. Thus, CK bound to SR may be physiologically relevant in vivo for regeneration of ATP used by the Ca2(+)-ATPase, as well as for regulation of local ATP/ADP ratios in the proximity of the Ca2+ pump and of other ATP-requiring reactions in the excitation-contraction coupling pathway.

Adenosine Diphosphate↗
PubMed1990

Coexpression of two isoforms of calsequestrin in rabbit slow-twitch muscle.

The cardiac and fast-twitch skeletal muscle forms of the Ca2(+)-binding protein calsequestrin (CS) are the products of two different genes, both of which are transcribed in slow-twitch skeletal muscle, though at much different rates (Scott et al., 1988., Fliegel et al., 1989). We have investigated this problem more closely at the protein level, on isolated terminal cisternae (TC) of the sarcoplasmic reticulum (SR) of rabbit slow-twitch muscle, and following purification of two distinct forms of CS from whole tissue by DEAE-Cellulose chromatography and CA2(+)-dependent elution from phenyl-Sepharose. Two electrophoretically (apparent molecular mass of 64 kDa and 54 kDa, respectively), and antigenically distinct forms of CS, here shown to be related to the fast-twitch skeletal muscle and to cardiac-type isoform of CS, respectively, colocalize to junctional TC of slow-twitch muscle. The cardiac-type isoform that is expressed in slow-twitch muscle accounts for about 25% of total CS present in isolated TC, it binds Ca2+ as effectively as the major CS form, using a 45Ca-overlay technique, and it shares extensive similarities with dog cardiac CS, not only in size and antigenically, but also in pl, as well as in the DEAE-elution characteristics. No difference in behaviour with phenyl-Sepharose resin were observed between the two CS isoforms from slow-twitch muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1990

Gastrointestinal tuberculosis. Report of four cases.

Gastrointestinal tuberculosis is a rare disease in the United States. Correct identification is often delayed because it is not considered early on in the differential diagnosis. Four patients with gastrointestinal tuberculosis and the symptoms, diagnosis, complications, and treatment of the disease are discussed. Gastrointestinal tuberculosis should be considered in Asian immigrant patients who present with symptoms and signs of inflammatory bowel disease.

Adult↗
PubMed1990

Calsequestrin, a component of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of chicken cerebellum.

The presence and distribution of calsequestrin (CS), Ca2+ pump, and inositol 1,4,5-trisphosphate (IP3) receptor were investigated biochemically and immunologically in microsomal (P3) fractions isolated from chicken cerebrum and cerebellum. Two different batches of polyclonal antibodies specific for chicken skeletal muscle CS identified a Ca2+ binding, CS-like protein that was extremely enriched in cerebellum P3 fractions and absent from all cerebrum fractions. The cerebellum CS-like protein was deemed authentic CS because the N-terminal amino acid domain and peptide mapping were identical to those of skeletal muscle CS in the same species. CS was detected in striated muscles and cerebellum only. Cerebellum P3 fractions were also found to be considerably enriched in Ca2+ pump and IP3 receptor compared with the homologous cerebrum fractions, as judged by measurements of Ca2+ uptake, Ca2(+)-ATPase activity, IP3-induced Ca2+ release, and [3H]IP3 binding, respectively. Cerebellum microsomal fractions therefore appear to contain membrane fragments endowed with Ca2+ pump, IP3 receptor, and CS, i.e., three key components of a Ca2+ storage organelle.

Animals↗
PubMed1990

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. I. Effect of Mg2+.

Canine cerebellar membranes were fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). The effect of Mg2+ on inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release and [3H]IP3 binding was assessed. Mg2+ inhibited IP3-induced Ca2+ release in a concentration-dependent manner. Mg2+ influenced both the extent of IP3-induced Ca2+ release and the apparent affinity for IP3. A 10-fold change of free Mg2+ (from approximately 30 to approximately 300 microM) reduced the extent of Ca2+ release by two- to threefold and shifted the apparent Michaelis constant from approximately 0.5 to approximately 0.9 microM IP3. Thus Mg2+ seemed to be noncompetitive inhibitor of IP3-induced Ca2+ release. Mg2+ also inhibited Ca2+ release elicited by glycerophosphoinositol 4,5-bisphosphate, a poorly metabolized analogue of IP3. Mg2+ and heparin sodium were shown to be additive inhibitors of IP3-induced Ca2+ release. Mg2+ inhibited [3H]IP3 binding under experimental conditions designed to minimize IP3 hydrolysis. Scatchard plots indicated that 0.5 mM free Mg2+ reduced maximum binding from 10.9 to 3.5 pmol IP3 bound/mg protein and increased the dissociation constant from 136 to 227 nM. The modulation of [3H]IP3 binding and IP3-induced Ca2+ release by Mg2+ could be physiologically relevant.

Adenosine Triphosphate↗
PubMed1990

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. II. Effect of cAMP-dependent protein kinase.

The effect of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) on Ca2+ loading, inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release, and [3H]IP3 binding of canine cerebellar membrane fractions was investigated. PKA in the presence of cAMP and the catalytic subunit of PKA did not change Ca2+ loading yet increased the extent of IP3-induced Ca2+ release by approximately 35%. Hill plot analysis indicated that the catalytic subunit of PKA increased the apparent Michaelis constant of IP3-induced Ca2+ release twofold, from 0.3 to 0.7 microM IP3. The protein kinase inhibitor reversed these changes. cAMP affected neither Ca2+ loading nor IP3-induced Ca2+ release. The catalytic subunit of PKA did not appreciably affect the maximum binding and dissociation constant of [3H]IP3 binding, as judged by Scatchard analysis. Thus the catalytic subunit of PKA influences the opening of Ca2+ channels by IP3 without interfering with the binding of IP3 to its receptor sites.

Animals↗
PubMed1990

Rapidly exchanging Ca2+ stores of non-muscle cells.

The rapid and transient redistribution of calcium from intracellular stores is a key event of cell activation. The nature and molecular composition of intracellular Ca2+ stores of non-muscle cells are the object of intense investigation. In this paper, we review: (a) the experimental evidence in favor of the existence of intracellular, membrane-bound compartments specialized for uptake, storage and release of calcium, (b) the main protein components of rapidly exchanging Ca2+ stores, i.e. Ca2+ pump, intralumenal Ca2+ binding proteins (calsequestrin, calreticulin, etc.) and Ca2+ channels sensitive to either inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine, and (c) the relationship between Ca2+ stores and the endoplasmic reticulum.

Animals↗