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PubMed1984

Evidence for the presence of calsequestrin in two structurally different regions of myocardial sarcoplasmic reticulum.

Localization of calsequestrin in chicken ventricular muscle cells was determined by indirect immunofluorescence and immuno-Protein A-colloidal gold labeling of cryostat and ultracryotomy sections, respectively. Calsequestrin was localized in the lumen of peripheral junctional sarcoplasmic reticulum, as well as in the lumen of membrane-bound structures present in the central region of the I-band, while being absent from the lumen of the sarcoplasmic reticulum in the A-band region of the cardiac muscle cells. Since chicken ventricular muscle cells lack transverse tubules, the presence of calsequestrin in membrane bound structures in the central region of the I-band suggests that these cells contain nonjunctional regions of sarcoplasmic reticulum that are involved in Ca2+ storage and possibly Ca2+ release. It is likely that the calsequestrin containing structures present throughout the I-band region of the muscle cells correspond to specialized regions of the free sarcoplasmic reticulum in the I-band called corbular sarcoplasmic reticulum. It will be of interest to determine whether Ca2+ storage and possibly Ca2+ release from junctional and nonjunctional regions of the sarcoplasmic reticulum in chicken ventricular muscle cells are regulated by the same or different physiological signals.

Animals↗
PubMed1982

Calcium accumulation by the sarcoplasmic reticulum in two populations of chemically skinned human muscle fibers. Effects of calcium and cyclic AMP.

In previous efforts to characterize sarcoplasmic reticulum function in human muscles, it has not been possible to distinguish the relative contributions of fast-twitch and slow-twitch fibers. In this study, we have used light scattering and 45Ca to monitor Ca accumulation by the sarcoplasmic reticulum of isolated, chemically skinned human muscle fibers in the presence and absence of oxalate. Oxalate (5 mM) increased the capacity for Ca accumulation by a factor of 35 and made it possible to assess both rate of Ca uptake and relative sarcoplasmic reticulum volume in individual fibers. At a fixed ionized Ca concentration, the rate and maximal capacity (an index of sarcoplasmic reticulum volume) both varied over a wide range, but fibers fell into two distinct groups (fast and slow). Between the two groups, there was a 2- to 2.5-fold difference in oxalate-supported Ca uptake rates, but no difference in average sarcoplasmic reticulum volumes. Intrinsic differences in sarcoplasmic reticulum function (Vmax, K0.5, and n) were sought to account for the distinction between fast and slow groups. In both groups, rate of Ca accumulation increased sigmoidally as [Ca++] was increased from 0.1 to 1 microM. Apparent affinities for Ca++ (K0.5) were similar in the two groups, but slow fibers had a lower Vmax and larger n values. Slow fibers also differed from fast fibers in responding with enhanced Ca uptake upon addition of cyclic AMP (10(-6) M, alone or with protein kinase). Acceleration by cyclic AMP was adequate to account for adrenaline-induced increases in relaxation rates previously observed in human muscles containing mixtures in fast-twitch and slow-twitch fibers.

Biological Transport↗
PubMed1991

Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.

Adenosine Triphosphate↗
PubMed1998

Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles.

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 x g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Delta24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

Amino Acid Sequence↗
PubMed1980

Donnan potential in sarcoplasmic reticulum vesicles measured by using a fluorescent cyanine dye.

The Donnan potential in sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle was measured by using a fluorescent cyanine dye, diS-C3-(5) (3,3'-dipropylthiadicarbocyanine iodide). The Donnan potential is caused by the existence of negative fixed charges inside the vesicles. On average, sarcoplasmic reticulum vesicles have internal negative fixed charges of 420 nmol/mg protein at pH 6.8. The fixed charges are mainly due to low affinity Ca2+ binding proteins. The Donnan potential in sarcoplasmic reticulum vesicles was about -72 mV in 5 mM K-MES buffer (pH 6.8). When Ca2+ was accumulated into sarcoplasmic reticulum vesicles with ATP, the concentration of negative fixed charges decreased from 140 mM to 95 mM due to Ca2+ binding to the negative fixed charges, and sarcoplasmic reticulum vesicles were depolarized by about 10 mV. It was found that there are two types of sarcoplasmic reticulum vesicles, one type (60%) having many negative fixed charges inside the vesicles and the other (40%) having few fixed charges.

Animals↗
PubMed1990

Calcium accumulation by sarcoplasmic reticulum in whole muscle homogenate preparations of malignant hyperthermia diagnostic patients and pigs.

Calcium accumulation by the sarcoplasmic reticulum in whole muscle homogenate preparations of malignant hyperthermia-susceptible (MH+) and non-susceptible (MH-) humans and pigs was investigated using a calcium electrode at 35 degrees C. Sarcoplasmic reticulum of MH+ humans and pigs showed normal Ca2+ accumulation, with no difference being observed in the rate and the time taken to achieve maximal accumulation. However, the capacity for Ca2+ accumulation by the sarcoplasmic reticulum in MH+ humans and pigs is considerably less stable than normal after prolonged ageing of the whole muscle homogenate preparations in ice. In MH+ patients, the capacity for Ca2+ accumulation by the sarcoplasmic reticulum showed a decline of 62% at 22 h ageing and 70% at 48 h ageing, as compared with a reduction of only 23% in MH- patients. In MH+ pigs, the sarcoplasmic reticulum showed a 96% deterioration in the capacity for Ca2+ accumulation as compared with a loss of only 40% in MH- pigs at 7 h ageing in ice. In both humans and pigs, the decline in Ca2+ accumulation was prevented by incubating the whole muscle homogenate preparations for 2 h at 35 degrees C prior to ageing the preparations. The diminished Ca2+ accumulating capacity of the sarcoplasmic reticulum in whole muscle homogenate preparations of MH-susceptible individuals in our experimental protocol provides a potential diagnostic test for malignant hyperthermia susceptibility.

Animals↗
PubMed1979

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.

Adenosine Triphosphate↗
PubMed1984

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of fast and slow skeletal and cardiac muscles.

Crystalline arrays of Ca2+ transport ATPase develop in sarcoplasmic reticulum membranes after treatment with Na3VO4 in a calcium-free medium [ Dux , L. and Martonosi , A. (1983) J. Biol. Chem. 258, 2599-2603]. The proportion of vesicles containing Ca2+-ATPase crystals in microsome preparations isolated from rat muscle of different fiber types (semimembranosus, levator ani, extensor digitorum longus, diaphragm, soleus, and heart) correlates well with the Ca2+-ATPase content and Ca2+-modulated ATPase activity. This implies that the concentration of Ca2+-ATPase in sarcoplasmic reticulum membranes of fast and slow skeletal or cardiac muscles differs only slightly, and the low Ca2+ transport activity of 'sarcoplasmic reticulum' preparations isolated from slow-twitch skeletal and cardiac muscles is due to the presence of large amount of non-sarcoplasmic-reticulum membrane elements. This is in accord with the relatively small differences in the density of 8.5-nm intramembranous particles seen by freeze-etch electron microscopy in sarcoplasmic reticulum of red and white muscles. The dimensions of the Ca2+-ATPase crystal lattice are similar in sarcoplasmic reticulum membranes of different fiber types; therefore if structural differences exist between 'isoenzymes' of Ca2+-ATPase, these are not reflected in the crystal-lattice.

Animals↗
PubMed1997

Calcium-induced release of strontium ions from the sarcoplasmic reticulum of rat cardiac ventricular myocytes.

1. The effects of strontium ions, Sr2+, on Ca(2+)-dependent feedback mechanisms during excitation-contraction coupling were examined in voltage-clamped rat ventricular myocytes in which intracellular [Ca2+] and [Sr2+] were monitored with the fluorescent indicator, indo-1. 2. Voltage clamp depolarizations and caffeine applications during superfusion in Ca(2+)-free, Sr(2+)-containing solutions were employed to exchange intracellular Ca2+ with Sr2+. Myocytes were loaded with Sr2+ by applying voltage clamp depolarizations during superfusion in Na(+)-free, Sr(2+)-containing solutions. 3. Caffeine applications produced large fluorescence transients in Sr(2+)-loaded cells. Thus, Sr2+ could be sequestered and released from the sarcoplasmic reticulum. 4. Ca2+ influx, but not Sr2+ influx, via sarcolemmal Ca2+ channels evoked ryanodine-sensitive fluorescence transients in Sr(2+)-loaded cells. These results demonstrated that Ca2+ influx-induced Sr2+ release (CISR) from the sarcoplasmic reticulum occurred in these experiments, even though Sr2+ influx-induced Sr2+ release was not observed. 5. The amplitude of the Ca2+ influx-induced fluorescence transient was 17 +/- 1% of the caffeine-induced transient (n = 5 cells), an indication that fractional utilization of Sr2+ sequestered in the sarcoplasmic reticulum during CISR was low. 6. With increased Sr2+ loading, the amplitude of Ca2+ influx- and caffeine-induced fluorescence transients increased, but fractional utilization of sarcoplasmic reticulum divalent cation stores was independent of the degree of Sr2+ loading. These data suggest that Ca2+ influx directly activated the release of divalent cations from the sarcoplasmic reticulum, but mechanisms promoting positive feedback of Sr2+ release were minimal during CISR. 7. By comparison, in Ca(2+)-loaded myocytes, Ca2+ influx-induced Ca2+ release (CICR) utilized a greater fraction of caffeine-releasable stores than CISR. Fractional utilization of Ca2+ stores during CICR increased with the degree of Ca2+ loading. 8. Taken together, these results suggest that Ca(2+)-dependent feedback mechanisms play a major role in determining the extent of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling under a wide range of conditions.

Animals↗
PubMed2006

Greater susceptibility of the sarcoplasmic reticulum to H2O2 injuries in diaphragm muscle from mdx mice.

The aim of the present study was to investigate the direct effects of a reactive oxygen species, H(2)O(2), on the contractile function and sarcoplasmic reticulum properties of dystrophin-deficient diaphragm using chemically skinned fibers and sarcoplasmic reticulum vesicle preparations. The results obtained using Triton X-100-skinned fibers demonstrate that exposure to 1 mM H(2)O(2) had similar effects on the maximal Ca(2+)-activated tension and on the Ca(2+) sensitivity of the contractile apparatus of diaphragm fibers in Bl10 and mdx mice. The effects of H(2)O(2) were also assessed on sarcoplasmic reticulum function using saponin-skinned fibers and sarcoplasmic reticulum vesicle preparations. We found that H(2)O(2) induced changes in sarcoplasmic reticulum properties, particularly in the Ca(2+) pump function. The most important finding was that diaphragm muscle from mdx mice displayed increased sensitivity to the oxidant. Furthermore, in isolated superfused diaphragm muscle from mdx mice, the data demonstrate that the amount of superoxide anion produced under fatiguing conditions was increased. Our study shows that the sarcoplasmic reticulum, and the Ca(2+) pump in particular, in dystrophin-deficient muscles display increased susceptibility to H(2)O(2) injuries. This suggests that free radicals might, therefore, be involved in the pathophysiological pathway and dysregulation of Ca(2+) homeostasis of muscular dystrophy.

Animals↗
PubMed2003

Determinant for endoplasmic reticulum retention in the luminal domain of the human cytomegalovirus US3 glycoprotein.

US3 of human cytomegalovirus is an endoplasmic reticulum resident transmembrane glycoprotein that binds to major histocompatibility complex class I molecules and prevents their departure. The endoplasmic reticulum retention signal of the US3 protein is contained in the luminal domain of the protein. To define the endoplasmic reticulum retention sequence in more detail, we have generated a series of deletion and point mutants of the US3 protein. By analyzing the rate of intracellular transport and immunolocalization of the mutants, we have identified Ser58, Glu63, and Lys64 as crucial for retention, suggesting that the retention signal of the US3 protein has a complex spatial arrangement and does not comprise a contiguous sequence of amino acids. We also show that a modified US3 protein with a mutation in any of these amino acids maintains its ability to bind class I molecules; however, such mutated proteins are no longer retained in the endoplasmic reticulum and are not able to block the cell surface expression of class I molecules. These findings indicate that the properties that allow the US3 glycoprotein to be localized in the endoplasmic reticulum and bind major histocompatibility complex class I molecules are located in different parts of the molecule and that the ability of US3 to block antigen presentation is due solely to its ability to retain class I molecules in the endoplasmic reticulum.

Amino Acid Motifs↗
PubMed1987

Differentiation of sarcoplasmic reticulum during cardiac myogenesis.

The composition and function of fetal and mature sheep cardiac sarcoplasmic reticulum membranes were investigated. Phospholamban, a major phosphoprotein in the mature sarcoplasmic reticulum membranes, was present in early stages of cardiac myogenesis. This fetal form of phospholamban was phosphorylated by cAMP-dependent protein kinase but not in the presence of Ca2+ and calmodulin. Ca2+ uptake and Ca2+-dependent ATPase activity were low in fetal sarcoplasmic reticulum compared with the adult controls, although the apparent affinities for Ca2+ were similar. Sarcoplasmic reticulum vesicles isolated at all developmental stages had very low levels of plasma membrane (as determined by Na+-K+-ATPase and Na+-Ca2+ exchanger activities) and mitochondrial contamination. Sarcoplasmic reticulum Ca2+ uptake and Ca2+-dependent ATPase activities were not affected by micromolar concentrations of vanadate, and the accumulated Ca2+ could not be released by the addition of NaCl. The amount of both the 110- and 55-kDa protein bands, identified with specific antibodies as Ca2+-ATPase and calsequestrin, respectively, was low in early stages of cardiac myogenesis. Age-related differences in the Ca2+ transport properties of cardiac sarcoplasmic reticulum and in the amount of the Ca2+-ATPase and calsequestrin may explain alterations in the regulation of intracellular Ca2+ concentrations in the fetal heart. This may contribute to the developmental changes in myocardial function.

Animals↗
PubMed1988

Calcium transport properties of cardiac sarcoplasmic reticulum from cardiomyopathic Syrian hamsters (BIO 53.58 and 14.6): evidence for a quantitative defect in dilated myopathic hearts not evident in hypertrophic hearts.

Calcium uptake was measured in homogenates and microsomal preparations enriched in sarcoplasmic reticulum vesicles isolated from hearts of hypertrophic (BIO 14.6) and dilated (BIO 53.58) cardiomyopathic as well as control (F1B) Syrian hamsters at 3, 7, 9, and 11 months of age. Calcium uptake studies were done using the Millipore filtration technique under conditions known to restrict transport to the sarcoplasmic reticulum. Steady-state calcium uptake capacity was used as a measure of the relative amounts of sarcoplasmic reticulum in homogenates prepared from individual hearts. At 3 months of age, there were no differences in calcium uptake in homogenates from control or myopathic hearts. However, by 9 months, although calcium uptake of homogenates from control and hypertrophic hearts was the same, calcium uptake by homogenates from dilated hearts was significantly depressed both in initial rate and capacity. Similar trends were seen in the microsomal vesicle preparations, but the decrease in calcium uptake in the dilated hearts was significantly lower by 3 months of age. The catalytic activity of the sarcoplasmic reticulum transport enzyme was estimated from the ratio of velocity to capacity, which provides a measure of the fractional rate of filling of the sarcoplasmic reticulum with calcium. The velocity-to-capacity ratios were not different at any of the ages in both the homogenate and microsomal preparations. The results of this study demonstrate that a major defect in the dilated cardiomyopathy may be due to a decrease in the volume or number of sarcoplasmic reticulum calcium transport sites rather than a decrease in specific activity of the transport enzyme.

Algorithms↗
PubMed1998

Sulfhydryl redox state affects susceptibility to ischemia and sarcoplasmic reticulum Ca2+ release in rat heart. Implications for ischemic preconditioning.

We investigated the effect of sulfhydryl and disulfide reagents on ischemic preconditioning and on sarcoplasmic reticulum Ca2+ release. Isolated working rat hearts were subjected to ischemic preconditioning (three 3-minute periods of global ischemia) or to control aerobic perfusion, which was followed by 30 minutes of global ischemia and 120 minutes of retrograde reperfusion. Necrosis was evaluated on the basis of lactate dehydrogenase release and triphenyltetrazolium chloride staining. In parallel experiments, sarcoplasmic reticulum Ca2+ release and [3H]-ryanodine binding were determined before the sustained ischemia. Ischemic preconditioning was associated with protection versus ischemic injury, decreased Ca2+ release and reduced [3H]-ryanodine binding. The disulfide reducing agent dithiothreitol (1 mmol/L) removed the protection provided by ischemic preconditioning, if added to the perfusion buffer either before or after the preconditioning procedure. In preconditioned hearts, dithiothreitol increased sarcoplasmic reticulum Ca2+ release and ryanodine binding, whereas in control hearts it had no effect on either tissue injury or sarcoplasmic reticulum function. Perfusion of control hearts with the sulfhydryl blocking agents 4,4'-dithiodipyridine (25 micromol/L) and N-ethylmaleimide (16 micromol/L) increased the resistance to ischemia and reduced sarcoplasmic reticulum Ca2+ release and [3H]-ryanodine binding. These effects were not additive with those induced by preconditioning. Sulfhydryl and disulfide reagents produced similar effects on Ca2+ release and [3H]-ryanodine binding if added in vitro to preparations obtained from control and preconditioned hearts. We conclude that ischemic preconditioning is associated with the oxidation of sulfhydryl groups involved in the modulation of sarcoplasmic reticulum Ca2+ release.

Animals↗
PubMed1990

Phospholamban-mediated stimulation of Ca2+ uptake in sarcoplasmic reticulum from normal and failing hearts.

Studies in animal models have suggested that alterations affecting phospholamban-mediated stimulation of Ca2+ uptake by sarcoplasmic reticulum are involved in the pathophysiology of heart disease. A monoclonal antibody that binds to phospholamban and stimulates Ca2+ uptake was used to characterize phospholamban-mediated effects in human cardiac sarcoplasmic reticulum and to compare these effects in tissue from normal and failing hearts. Stimulation of Ca2+ uptake by anti-phospholamban monoclonal antibody simulated the effect of phosphorylation of phospholamban by cAMP-dependent protein kinase. Binding of anti-phospholamban antibody reduced the K0.5 of the Ca2(+)-transporting ATPase from 0.53 microM [( Ca2+]) to 0.29 microM [( Ca2+]), without affecting Vmax or nHill. At 0.2 microM Ca2+, stimulation was 1.93-fold in sarcoplasmic reticulum prepared from normal human left ventricular myocardium and 1.94-fold in sarcoplasmic reticulum prepared from the left ventricular myocardium of patients with heart failure resulting from idiopathic dilated cardiomyopathy. Stimulation of Ca2+ uptake in canine cardiac sarcoplasmic reticulum under identical conditions was 1.89-fold. Phospholamban-mediated stimulation of Ca2+ uptake in human cardiac sarcoplasmic reticulum is thus comparable in magnitude to that observed in other species and results from an increase in the apparent affinity of the Ca2(+)-transporting ATPase for Ca2+. The pathogenesis of heart failure in idiopathic dilated cardiomyopathy does not, however, appear to involve intrinsic alterations of this mechanism.

Adenosine Triphosphatases↗
PubMed1993

Doxorubicin cardiomyopathy is associated with a decrease in calcium release channel of the sarcoplasmic reticulum in a chronic rabbit model.

Doxorubicin is a highly effective cancer chemotherapeutic agent that produces a dose-dependent cardiomyopathy that limits its clinical usefulness. Clinical and animal studies of morphological changes during the early stages of doxorubicin-induced cardiomyopathy have suggested that the sarcoplasmic reticulum, the intracellular membrane system responsible for myoplasmic calcium regulation in adult mammalian heart, may be the early target of doxorubicin. To detect changes in the calcium pump protein or the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum during chronic doxorubicin treatment, rabbits were treated with intravenous doxorubicin (1 mg/kg) twice weekly for 12 to 18 doses. Pair-fed controls received intravenous normal saline. The severity of cardiomyopathy was scored by light and electron microscopy of left ventricular papillary muscles. Developed tension was measured in isolated atrial strips. In subcellular fractions from heart, [3H]ryanodine binding was decreased in doxorubicin-treated rabbits (0.33 +/- 0.03 pmol/mg) compared with control rabbits (0.66 +/- 0.02 pmol/mg; P < 0.0001). The magnitude of the decrease in [3H]ryanodine binding correlated with both the severity of the cardiomyopathy graded by pathology score (light and electron microscopy) and the decrease in developed tension in isolated atrial strips. Bmax for [3H]ryanodine binding and the amount of immunoreactive ryanodine receptor by Western blot analysis using sequence-specific antibody were both decreased, consistent with a decrease in the amount of calcium release channel of sarcoplasmic reticulum in doxorubicin-treated rabbits. In contrast, there was no decrease in the amount or the activity of the calcium pump protein of the sarcoplasmic reticulum in doxorubicin-treated rabbits. Doxorubicin treatment did not decrease [3H]ryanodine binding or the amount of immunoreactive calcium release channel of sarcoplasmic reticulum in skeletal muscle. Since the sarcoplasmic reticulum regulates muscle contraction by the cyclic uptake and release of a large internal calcium pool, altered function of the calcium release channel could lead to the abnormalities of contraction and relaxation observed in the doxorubicin cardiomyopathy.

Animals↗
PubMed2005

Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of proteins associated with the translocation channel.

Steroid-secreting cells are characterized by abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. Yet they have relatively little morphologically identifiable rough endoplasmic reticulum, presumably required for synthesis and maintenance of the smooth membranes. In this study, we demonstrate that adrenal smooth microsomal subfractions enriched in smooth endoplasmic reticulum membranes contain high levels of translocation apparatus and oligosaccharyltransferase complex proteins, previously thought confined to rough endoplasmic reticulum. We further demonstrate that these smooth microsomal subfractions are capable of effecting cotranslational translocation, signal peptide cleavage, and N-glycosylation of newly synthesized polypeptides. This shifts the paradigm for distinction between smooth and rough endoplasmic reticulum. Confocal microscopy revealed the proteins to be distributed throughout the abundant tubular endoplasmic reticulum in these cells, which is predominantly smooth surfaced. We hypothesize that the broadly distributed translocon and oligosaccharyltransferase proteins participate in local synthesis and/or quality control of membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally regulated manner.

Adrenal Cortex↗
PubMed2005

Endoplasmic reticulum stress increases glucose-6-phosphatase and glucose cycling in liver cells.

Impaired regulation of hepatic glucose production is a characteristic feature of the metabolic syndrome, a cluster of diseases that includes obesity, insulin resistance, type 2 diabetes, and cardiovascular disease. It has been proposed that sustained endoplasmic reticulum stress, which appears to occur in obesity and diabetes, modulates insulin action in the liver. In this study, we show that experimental induction of endoplasmic reticulum stress increases expression and activity of glucose-6-phosphatase and the capacity for glucose release and glucose cycling in primary rat hepatocytes and H4IIE liver cells. Increased expression of the catalytic subunit of glucose-6-phosphatase was largely a result of increased transcription. Deletion analysis of the glucose-6-phosphatase promoter identified an endoplasmic reticulum stress-responsive region located between -233 and -187 with respect to the transcriptional start site. Experimental induction of endoplasmic reticulum stress increased the activity of c-jun N-terminal kinase. Prevention of endoplasmic reticulum stress-mediated activation of c-jun N-terminal kinase reduced the expression of the catalytic subunit of glucose-6-phosphatase, glucose-6-phosphatase activity, glucose release, and glucose cycling. These data demonstrate that sustained endoplasmic reticulum stress in the hepatocyte provokes adaptations, mediated in part via activation of c-jun N-terminal kinase, that act to increase hepatocellular capacity for glucose release and glucose cycling.

Animals↗