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PubMed2001

Endoplasmic reticulum stress prolongs GH-induced Janus kinase (JAK2)/signal transducer and activator of transcription (STAT5) signaling pathway.

The desensitization of the GH-induced Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) signaling pathway plays a crucial role in GH regulation of hepatic genes. Previous studies have demonstrated that the inactivation of the GH-induced JAK2/STAT5 pathway is regulated by protein translation and suppressors of cytokine signaling (SOCS). In this study we sought to explore the relationships between endoplasmic reticulum stress, GH-induced JAK2/STAT5 activity and SOCS expression. 1,2-bis(o-Aminophenoxy)ethane-N,N,N,N-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM), used to provoke endoplasmic reticulum stress, caused a drastic inhibition of protein translation that correlated with the phosphorylation of the eukaryotic translation initiation factor 2alpha. Both GH and BAPTA-AM caused a rapid induction of the transcription factor C/EBP homology protein (CHOP) and an additive effect was observed with combined treatment, which suggests a regulatory role of GH on endoplasmic reticulum stress. Endoplasmic reticulum stress did not interfere with the rapid GH activation of STAT5 DNA binding activity. However, BAPTA-AM prolonged the DNA binding activity of STAT5 without affecting STAT5 or JAK2 protein levels. GH-induced phosphorylation of JAK2 and STAT5 DNA binding activity were prolonged in the presence of BAPTA-AM, suggesting that endoplasmic reticulum stress prevents the inactivation of STAT5 DNA binding activity by modulating the rate of JAK2/STAT5 dephosphorylation. Like BAPTA-AM, the endoplasmic reticulum stressors dithiothreitol and A23187 also prolonged the GH-induced STAT5 DNA binding activity. We were not able to correlate BAPTA-AM effects to the GH-dependent expression of SOCS proteins or SOCS mRNA, suggesting that endoplasmic reticulum stress modulates the rate of JAK2/STAT5 dephosphorylation through mechanisms other than inhibition of SOCS expression. This study indicates that cellular stress may modulate transcription through the JAK/STAT pathway.

Animals↗

PubMedDate not supplied

Low levels of glucose-6-phosphate hydrolysis in the sarcoplasmic reticulum of skeletal muscle: involvement of glucose-6-phosphatase.

Glucose-6-phosphate hydrolysis was measured in a fraction obtained from rabbit fast-twitch skeletal muscle and corresponding to total sarcoplasmic reticulum, as well as in three subfractions containing longitudinal tubules, terminal cisternae or both structures. In all cases the levels of hydrolysis measured both in native and disrupted membranes were approximately 60-100 times lower than the microsomal glucose-6-phosphatase activity of the corresponding livers. In contrast to liver microsomes, most (up to 80%) of the glucose-6-phosphate hydrolysing activity in muscle sarcoplasmic reticulum membranes was not inactivated by pH 5.0 pre-incubation indicating that it was not catalysed by the specific glucose-6-phosphatase enzyme. Osmotically induced changes in light-scattering intensity of sarcoplasmic reticulum vesicles revealed that, in contrast to liver microsomes, sarcoplasmic reticulum vesicles were not selectively permeable to glucose-6-phosphate as mannose-6-phosphate was also permeable and in addition they were poorly permeable to glucose. Immunoblot experiments using antibodies raised against the glucose-6-phosphatase enzyme, and liver endoplasmic reticulum glucose and Pi translocases, failed to detect the presence of these protein components in sarcoplasmic reticulum membranes. Southern blot analysis of reverse transcriptase-polymerase chain reaction products from rat muscle revealed that glucose-6-phosphatase mRNA is present in muscle. Quantification of Northern blot analysis of liver and muscle mRNA indicated that muscle contains less than 2% of the amount of glucose-6-phosphate mRNA found in corresponding livers. We conclude that very low levels of specific glucose-6-phosphatase (e.g. as in liver; E.C. 3.1.3.9) are present in muscle sarcoplasmic reticulum and that the muscle and liver glucose-6-phosphatase systems have several different properties.

Animals↗

PubMed1976

[Ultrastructural changes in cells of the inner enamel epithelium, stratum intermedium and reticulum stellatum in the enamel organ of the upper incisor of the Wistar rat at different phases of amelogenesis].

An electronmicroscopical study of the enamel organ of the upper incisors germs of Wistar rats was performed to analyse the ultrastructural features of the cells of the inner epithelium, the intermediate layer and the stellate reticulum, during preimary, young, transitional and mineralized enamel phases of amelogenesis. So, it was observed that the mitochondria in the ameloblasts are ovoid before the beginning of the enamel matrix formation and in the primary and young enamel phases. However, in the transitional and mineralized phases, these organelles are long and tortuous and some are characterized by a compact structure. In the cells of intermediate layer and stellate reticulum, the mitochondria are ovoid until the beginning of the mineralized phase. At the ending of this phase, these organelles are very long and present irregular form; many of them show also a compact structure. The "zonula adhaerens" could be observed only in the ameloblasts of the primary and young enamel phase. The cytoplasm of ameloblasts, during primary and young enamel phases is characterized by an abundance of free ribosomes and a branular endoplasmic reticulum; but during transitional and mineralized enamel phases, the cytoplasm of these cells shows little granular endoplasmic reticulum and free ribosomes, but ehe agranular endoplasmic reticulum is present. The granular endoplasmic reticulum and free ribosomes are abundant in the cells of the intermediate layer and stellate reticulum at the ending of the young enamel phase, in the transitional enamel phase and in the beginning of the minieralized phase. During different phases of amelogenesis, in the three above referred layers of the enamel organ, were also studied the features of the Golgi apparatus the presence and topographic distribution of the pigment granules, as well as the lysosomes, desmosomes and the tonophibriles.

Ameloblasts↗

PubMedDate not supplied

[Effect of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. I. Comparative study of Ca2+ accumulation in mitochondria and sarcoplasmic reticulum].

In experiments, which were carried out with the use of a radioactive label (45Ca2+) on the suspension of rat uterus myocytes treated by digitonin solution (0.1 mg/ml), influence of Mg ions and spermine on Mg2+, ATP-dependent Ca2+ transport in mitochondria and sarcoplasmic reticulum was investigated. Ca2+ accumulation in mitochondria (1324 +/- 174 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). Oxalate-stimulated Ca2+ accumulation in sarcoplasmic reticulum (136 +/- 17 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to ruthenium red and was blocked by thapsigargin. It has been shown, that initial speed and level of energy-dependent Ca2+ accumulation in mitochondria considerably exceeded the values of these parameters for sarcoplasmic reticulum Ca2+-accumulation system. Ca2+ accumulation kinetic in mitochondria was characterized by a steady-state phase (for 5-10 min. of incubation) while accumulation kinetic of this cation in sarcoplasmic reticulum corresponded to zero order reaction. Increase of Mg2+ concentration up to 5 mM led to activation of Ca2+-accumulation systems in mitochondria and sarcoplasmic reticulum (values of activation constants K(Mg) for Mg2+ were 2.8 and 0.6 mM, accordingly). Concentration dependence of spermine action on Ca2+ accumulation in mitochondria was described by a dome-shaped curve with a maximum at 1 mM spermine. In case of sarcoplasmic reticulum Ca2+ pump only the inhibition phase was tested at spermine concentration above 1 mM. However values of inhibition constants for both transporting systems were practically identical--5.2 +/- 0.6 and 5.7 +/- 0.7 mM, accordingly. Hence, Mg ions carry out the important role in regulation of energy-dependent Ca2+ transporting systems both in uterus smooth muscle mitochondria and sarcoplasmic reticulum. Spermine acts first of all on mitochondrial calcium uniporter.

Adenosine Triphosphate↗

PubMed1991

Depletion of cellular calcium accelerates protein degradation in the endoplasmic reticulum.

In this study the effects of A23187 and thapsigargin on the degradation of T-cell antigen receptor-beta (TCR-beta) and CD3-delta in the endoplasmic reticulum have been studied. Preliminary experiments showed that these drugs had different effects on the secretory pathway. Depletion of cellular calcium pools by incubation of cells with A23187 in calcium-free medium blocked transport between the endoplasmic reticulum and the Golgi apparatus whereas thapsigargin caused a modest increase in transport. When added to cells transfected with TCR-beta or CD3-delta the drugs caused an immediate stimulation of proteolysis of presynthesized protein and at maximum effective concentrations caused a 3-fold increase in the rate of degradation. They did not affect the lag period of 1 h which precedes degradation of newly synthesized proteins. Chelation of cytosolic calcium also accelerated degradation, suggesting that depletion of calcium from the endoplasmic reticulum was the main stimulus of proteolysis and that increased degradation was not caused by a transient increase in cytosolic calcium levels. The selectivity of degradation in the endoplasmic reticulum was maintained. A23187 had no effect on the stability of CD3-gamma nor co-transfected epsilon-beta dimers. Calcium depletion increased the overall rate of degradation in the endoplasmic reticulum and increased the rate of proteolysis of an "anchor minus" beta chain. The results suggested that proteolysis within the endoplasmic reticulum may be regulated by the high concentrations of Ca2+ which are stored in the organelle. Ca2+ may be required for protein folding. Calcium depletion may have caused the beta and delta chains to adopt a conformation that was more susceptible to proteolysis. Alternatively, calcium depletion may have disrupted the lumenal content of the endoplasmic reticulum and increased the access of proteases to potential substrates.

Animals↗

PubMed1989

Ethanol inhibition of spontaneous calcium release from isolated sarcoplasmic reticulum.

Ethanol, in vitro, decreases muscle fiber twitch tension by a mechanism unrelated to the electrical events of the motor end-plate or muscle surface membranes, an effect which may be attributable to a primary ethanol effect on the calcium release process of the sarcoplasmic reticulum. In this study, ethanol was found to progressively decrease a form of calcium release from isolated sarcoplasmic reticulum vesicles seen after calcium loading in high-phosphate media (spontaneous calcium release). Elevated extravesicular free calcium concentrations are known to inhibit spontaneous calcium release, and the ability of sarcoplasmic reticulum to achieve and maintain submicromolar extravesicular free calcium concentrations in the presence of ethanol was therefore determined. Ethanol had no effect on the low extravesicular free calcium concentrations achieved by isolated sarcoplasmic reticulum, and had no effect on residual ATPase activity remaining after cessation of calcium pumping. This latter result suggests that efflux of vesicular calcium and persistent calcium reaccumulation do not occur in the presence of ethanol. These results suggest that ethanol-induced depression of spontaneous calcium release is not attributable to ethanol effects on sarcoplasmic reticulum membrane calcium leakage or on sarcoplasmic reticulum calcium pumping. Ethanol inhibition of spontaneous calcium release from isolated sarcopasmic reticulum may reflect an effect of ethanol on the calcium release process in intact muscle fibers responsible for ethanol-induced decreases in muscle fiber twitch tension.

Adenosine Triphosphatases↗

PubMed1985

Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct.

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.

Animals↗

PubMed1982

Modification of calcium fluxes by dimethyl sulfoxide and 2-butoxyethanol in sarcoplasmic reticulum vesicles: a possible mechanism for skeletal muscle relaxation induced by dimethyl sulfoxide.

In order to investigate the mechanism of skeletal muscle relaxation induced by dimethyl sulfoxide, 2-butoxyethanol and dimethyl sulfoxide were examined for their effects on 1) Ca2+ uptake into and efflux from sarcoplasmic reticulum vesicles prepared from rabbit fast skeletal muscle and crayfish tail muscle by the murexide method, 2) ATPase activities of rabbit reticulum vesicles, 3) the isolated phrenic nerve-diaphragm preparation of the rat and 4) crayfish opener muscle preparation. Ca2+ efflux rate from rabbit reticulum vesicles was markedly decreased with increasing concentrations (5-20% v/v) of dimethyl sulfoxide without affecting the maximum Ca2+ uptake by the reticulum. 2-Butoxyethanol showed quite contrary effects. Dimethyl sulfoxide strongly inhibited the activity of basal ATPase rather than of Ca2+-dependent ATPase. 2-Butoxyethanol did not significantly inhibit the activity of basal ATPase, but markedly increased Ca2+-dependent ATPase activity. Antagonisms between dimethyl sulfoxide and caffeine were demonstrated either in contractions of crayfish opener muscles or in the Ca2+ release from crayfish sarcoplasmic reticulum vesicles. These results indicate a possibility that dimethyl sulfoxide reversibly induces skeletal muscle relaxation mainly in the sarcoplasmic reticulum by means of decreasing the rate and the amount of Ca2+ release from the reticulum.

Adenosine Triphosphatases↗

PubMed1983

Regulation of cardiac sarcoplasmic reticulum calcium transport by calcium-calmodulin-dependent phosphorylation.

Cardiac sarcoplasmic reticulum contains an endogenous calcium-calmodulin-dependent protein kinase and a 22,000-Da substrate, phospholamban. This kinase is half-maximally activated (EC50) by 3.8 +/- 0.3 microM calcium and is absolutely dependent on exogenous calmodulin (EC50 = 49 nM). To determine the effect of this phosphorylation on calcium transport, sarcoplasmic reticulum vesicles (0.5 mg/ml) were preincubated under conditions for optimal phosphorylation (50 mM potassium phosphate, pH 7.0, 10 mM MgCl2, 0.5 mM EGTA, 0.478 mM CACl2, 0.1 microM calmodulin, 0.5 mM ATP). Control sarcoplasmic reticulum was preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged and resuspended in 0.3 M sucrose, 20 mM Tris-HCl, 100 mM KCl, pH 7.0, to remove calmodulin and subsequently assayed for calcium (45Ca) transport in the presence of 2.5 mM Tris-oxalate. Phosphorylation of sarcoplasmic reticulum vesicles by calcium-calmodulin-dependent protein kinase resulted in a significant increase (2- to 4-fold) in the rate of calcium transport at low calcium concentrations (less than 3 microM), while calcium transport was minimally affected at higher calcium. Hill coefficients (n) derived from Hill plots of transport data showed no difference between control and phosphorylated sarcoplasmic reticulum (n = 2.0), indicating that phosphorylation does not alter the cooperativity between calcium sites on the calcium pump. The EC50 for calcium activation of calcium transport by control vesicles was 0.86 +/- 0.1 microM calcium, and phosphorylation of phospholamban decreased this value to 0.61 +/- 0.07 microM calcium (n = 7, p less than 0.028), indicating an increase in the apparent affinity for calcium upon phosphorylation. These results were found to be specific for calcium-calmodulin-dependent phosphorylation of phospholamban. Control experiments on the effects of the reactants used in the phosphorylation assay and subsequent centrifugation of sarcoplasmic reticulum showed no alteration of the rate of calcium transport. Therefore, the calcium pump in cardiac sarcoplasmic reticulum appears to be regulated by an endogenous calcium-calmodulin-dependent protein kinase, and this may provide an important regulatory mechanism for the myocardium.

Adenosine Triphosphatases↗

PubMedDate not supplied

Alterations of membrane potential and Ca2+ flux of sarcoplasmic reticulum vesicles in ischemic myocardium.

The sequence of contraction-relaxation for myocardial cells is believed to be linked to Ca2+ flux across the sarcoplasmic reticulum. Alterations of sarcoplasmic reticulum function during ischemia may result in depressing the myocardial contraction-relaxation sequence. This study examines the relationship between the membrane potential and Ca2+ flux across sarcoplasmic reticulum vesicles isolated from non-ischemic and ischemic myocardium. Ischemic myocardium was produced by ligating the coronary artery of swine hearts for 15 and 30 minutes. Membrane potential was determined by use of the fluorescence-sensitive dye, 3,3'-diethylthiadicarbocyanine, and Ca2+ uptake was studied spectrophotometrically with the use of murexide. Results are as follows: (1) membrane potential and Ca2+ uptake by sarcoplasmic reticulum from ischemic myocardium progressively decreased with the length of ischemia; and (2) preincubation of sarcoplasmic reticulum from non-ischemic myocardium with deoxycholate (0.01 approximately 0.09 percent) resulted in progressively decreasing membrane potential and Ca2+ uptake. Apparently a correlation exists between membrane potential and the rate of Ca2+ uptake. These results suggest that membrane characteristics of sarcoplasmic reticulum are altered within as early as 15 minutes of the onset of ischemia. Alteration of membrane permeability in sarcoplasmic reticulum from ischemic myocardium may be responsible for the observed decrease in membrane potential and Ca2+ uptake.

Animals↗

PubMed2006

Decreased cardiac sarcoplasmic reticulum Ca2+ -ATPase activity contributes to cardiac dysfunction in streptozotocin-induced diabetic rats.

Diabetic cardiomyopathy is characterized by reduced cardiac contractility due to direct changes in myocardium function independent of vascular disease. This study is to investigate the alterations of cardiac sarcoplasmic reticulum Ca2+ -ATPase activity and cardiac function in streptozotocin-induced diabetic rats. Diabetes mellitus (DM) was induced in male Wistar rats by intraperitoneal injection of streptozotocin. The activity of myocardium sarcoplasmic reticulum Ca2+ -ATPase and the left ventricular hemodynamic parameters were measured in DM rats 4 weeks, 6 weeks and 8 weeks after streptozotocin was administered. Phospholamban mRNA expression was detected by reverse transcription-polymerase chain reaction, and the protein levels of phospholamban and sarcoplasmic reticulum Ca2+ -ATPase were determined by Western blot. Normal rats served as control group. It was found that in DM rats 4 weeks after streptozotocin injection, the cardiac function, myocardium sarcoplasmic reticulum Ca2+ -ATPase activity, phospholamban mRNA and phospholamban protein were not significantly changed compared with those in the control rats. At 6 and 8 weeks after the streptozotocin injection, DM rats showed a significant decrease in sarcoplasmic reticulum Ca2+ -ATPase activity and cardiac function, as indicated by an increase of LVEDP and a marked depression in LVSP and +/- dP/dtmax. At the same time points, increases in phospholamban mRNA and protein levels were observed in DM rats. Sarcoplasmic reticulum Ca2+ -ATPase protein level showed no significant alterations in all DM rats compared with that in control rats. Our work confirms that sarcoplasmic reticulum Ca2+ -ATPase activity is depressed in rats with streptozotocin-induced DM, which is accompanied by elevated phospholamban protein level thus contribute to the pathogenesis of cardiac dysfunction in diabetic rats.

Animals↗

PubMed1999

Apoptosis in the early involuting stellate reticulum of rat molar tooth germs.

When the enamel organ of the rat tooth germ is fully developed at the tip of the prospective cusp, amelogenesis begins, and at this site the overlaying stellate reticulum begins its involution. During the involution process, there is a gradual decrease in intercellular spaces, invasion by blood vessels, appearance of macrophage-like cells and reduction in the number of stellate reticulum cells. Since reduction or disappearance of cells during embryonic development in organs and tissues has been shown to occur by apoptosis, we decided to examine early involuting regions of the stellate reticulum in the hope of detecting apoptosis. For this purpose, upper first molars of Wistar newborn rats aged 1 and 3 days were fixed in formaldehyde for the TUNEL method and in glutaraldehyde-formaldehyde for light and electron microscopy. Paraffin sections revealed TUNEL-positive structures, i.e. brown-yellow-stained bodies, in the central portion of the stellate reticulum, and next to the outer enamel epithelium and stratum intermedium. Examination of ultrathin sections confirmed the TUNEL findings: some stellate reticulum cells showed nuclei containing crescent-like electron-opaque condensed masses of peripheral chromatin, typical of apoptosis. Also, apoptotic bodies of various sizes and appearances were frequently observed within stellate reticulum cells. We should like to suggest that apoptosis is associated with the reduction in the number of cells during regression of the reticulum.

Amelogenesis↗

PubMed1993

Ultrastructural localization of neuropeptide Y-immunoreactivity in the axonal reticulum elements, accumulating anterogradely in transected rat sciatic nerve.

The detection of dopamine-beta-hydroxylase and cytochrome B561 on the membranes of the axonal reticulum demonstrated that in sympathetic neurons the different compartments of the axonal reticulum participate in the formation of neurosecretory vesicles. In the present study we tried to reveal that the components of the vesicular content are also channeled along the axonal reticulum, by examining whether neuropeptide Y could be localized in elements of the axonal reticulum. Therefore 6 h transected rat sciatic nerve was embedded in glycolmethacrylate and an immunogold labeling was performed. Counterstaining with phosphotungstic acid at low pH selectively contrasted the accumulated axonal reticulum elements and associated granules. In the non-myelinated axons gold labeling was localized on granules and on tubular and granular profiles, demonstrating the presence of neuropeptide Y in the accumulated axonal reticulum elements. This indicates that neuropeptides are indeed transported via the axonal reticulum to the nerve ending and suggests that the accumulation of large dense-cored vesicles at a block is mainly due to local new formation rather than down transport.

Animals↗

PubMed1968

The effect of quinidine on calcium accumulation by isolated sarcoplasmic reticulum of skeletal and cardiac muscle.

Quinidine potentiates twitch tension and (at higher concentrations) causes contracture of skeletal muscle whereas the same drug reduces tension development of cardiac muscle. To gain insight into the possible differences in the excitation-contraction coupling mechanism of the two types of muscle the effect of quinidine on calcium accumulation by isolated sarcoplasmic reticulum from skeletal and cardiac muscle was investigated. In a medium containing ATP, Mg(++), oxalate, and (45)Ca, pharmacologically active concentrations of the drug inhibited calcium accumulation by both skeletal and cardiac sarcoplasmic reticulum. The inhibition of the rates of calcium, uptake by the skeletal muscle preparation ranged from 11% with 10(-4)M quinidine to 90% with 10(-3)M quinidine. With the cardiac muscle preparation the inhibition ranged from 16% with 3 x 10(-6)M quinidine to 100% with 10(-3)M quinidine. With both preparations the inhibition of calcium transport was accompanied by an inhibition of the Ca(++)-activated ATPase activity of the sarcoplasmic reticulum. The effect of quinidine on the skeletal sarcoplasmic reticulum supports the hypothesis that this compound produces twitch potentiation and contracture by interfering with intracellular calcium, sequestration. Its effect on cardiac sarcoplasmic reticulum. has been interpreted in terms of the hypothesis that cardiac contractility is a function of the amount of calcium released from the sarcoplasmic reticulum which is in turn dependent upon the absolute calcium content of the reticulum. Hence, following inhibition of calcium transport there would be less calcium available for coupling.

Adenosine Triphosphatases↗

PubMed1987

Cytoskeletal components of lymphoid organs. I. Synthesis of cytokeratins 8 and 18 and desmin in subpopulations of extrafollicular reticulum cells of human lymph nodes, tonsils, and spleen.

Using light and electron microscopic immunolocalization with antibodies to cytoskeletal proteins, we have characterized the nonlymphoid cells of various human lymphoid organs (lymph nodes, tonsils, spleen). In all these tissues, the lymphoid follicles contain a three-dimensional meshwork of "dendritic reticulum cells" which are characterized by the presence of desmosomal junctions, as demonstrated by positive punctate staining with antibodies to the desmosome-specific proteins desmoplakin I and desmoglein, and by intermediate-sized filaments (IFs) of the vimentin type only. In contrast, the extrafollicular regions are characterized by an extended meshwork of other types of reticulum cells, which also contain vimentin IFs but lack desmosomal proteins. In addition, a considerable, although variable proportion of these extrafollicular reticulum cells forms IFs containing cytokeratins 8 and 18 and/or desmin-containing IFs. The occurrence of cytokeratins 8 and 18 in lymph nodes has also been shown by gel electrophoresis and immunoblotting. Results of double-label immunolocalization indicate that some of the extrafollicular reticulum cells coexpress all three kinds of IF protein. A large proportion of these cells also synthesizes another marker of myogenic differentiation, i.e., the isoform of alpha-actin specific for smooth muscle. This proportion includes some cells that are negative for desmin. Comparison of the distribution of cells expressing cytokeratins and/or desmin with that of reticulum cells showing strong alkaline phosphatase activity (as a marker for the so-called "fiber-associated (fibroblastic) reticulum cells") suggests that the former represent a subset of the latter. The biological meaning of these different patterns of expression in reticulum cells and of the resulting cell-type heterogeneity as well as possible implications of these observations for tumor diagnosis, notably of lymph-node metastases and lymphomas, are discussed.

Adult↗

PubMed1993

Interactions of calreticulin with proteins of the endoplasmic and sarcoplasmic reticulum membranes.

The ability of [125I]calreticulin to bind to membrane fractions isolated from different muscle and non-muscle tissues was examined by a protein overlay technique. Specific [125I]calreticulin binding proteins were detected in rat liver smooth and rough endoplasmic reticulum and Golgi, in canine pancreatic microsomes, and in rabbit skeletal muscle sarcoplasmic reticulum. These proteins were confined only to membranes that contain calreticulin; they were not found in rat liver mitochondria or cytosol. [125I]Calreticulin binds to a 50-kDa protein and a number of lower M(r) (20,000-38,000) endoplasmic reticulum membrane proteins and to 30-kDa protein in skeletal muscle sarcoplasmic reticulum. Full-length calreticulin and the carboxyl-terminal region (C-domain) of the protein both competed with [125I]calreticulin for binding to the membrane proteins. Binding of [125I]calreticulin to pancreatic microsomes was also partially inhibited by the N-domain and to a lesser extent by the P-domain of the protein. We conclude that calreticulin interacts with the endoplasmic reticulum membrane proteins mainly through its carboxyl-terminal domain and that the endoplasmic and sarcoplasmic reticulum membranes may contain different calreticulin binding proteins.

Animals↗

PubMed2001

TcSCA complements yeast mutants defective in Ca2+ pumps and encodes a Ca2+-ATPase that localizes to the endoplasmic reticulum of Trypanosoma cruzi.

Intracellular Ca(2+) in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca(2+)-ATPase. Evidence for an endoplasmic reticulum-located Ca(2+) uptake has been more elusive and based on indirect results. Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPase from T. cruzi. The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa. Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca(2+) pumps (pmr1 pmc1 cnb 1) restored growth on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca(2+)-dependent and hydroxylamine-sensitive (32)P-labeled phosphoprotein of 110 kDa in the presence of [gamma-(32)P]ATP. Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation. Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin. An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein. The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb 1 on medium containing Mn(2+) suggests that TcSCA may also regulate Mn(2+) homeostasis by pumping Mn(2+) into the endoplasmic reticulum of T. cruzi.

Amino Acid Sequence↗

PubMed2002

Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons.

We have studied the Ca(2+) leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca(2+) in the endoplasmic reticulum ([Ca(2+)](ER)). Cytosolic Ca(2+) ([Ca(2+)](C)) was clamped to the resting level by a BAPTA-Ca(2+) mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca(2+)](ER), and the rate of Ca(2+) release generated by 10 pm cholecystokinin is at least 3x as fast as the basal Ca(2+) leak revealed by inhibition of the endoplasmic reticulum Ca(2+)-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca(2+)](ER), with an EC(50) of 0.98 +/- 0.06 microm. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP(3)) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH(2) cyclic ADP-ribose (20 microm) inhibits the action of cholecystokinin, but not of acetylcholine(.) The basal Ca(2+) leak from the endoplasmic reticulum is not blocked by antagonists of the IP(3) receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with puromycin (0.1-1 mm) to remove nascent polypeptides from ribosomes increases Ca(2+) leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca(2+) pumps and of the receptors for IP(3) or ryanodine.

Acetylcholine↗