Studies on phosphorylase b kinase from neutral tissues

Abstract— Phosphorylase b kinase (ATP: phosphorylase phosphotransferase; EC 2.7.1.38), the enzyme which converts phosphorylase b to phosphorylase a (α-1,4-glucan: orthophosphate glucosyltransferase; EC2.4.1.1) was examined in nerve tissue. Both phosphorylase and phosphorylase kinase were present in all nerve tissues tested, with central tissues about ten times as active as peripheral nerve. Exceptions were the superior cervical and stellate ganglia, tissues rich in synapses, which displayed activity similar to brain. Phosphorylase kinase in brain had properties similar to those of the enzyme in skeletal and cardiac muscle; it was activated in vitro by ATP and adenosine 3′,5′-monophosphate (cyclic AMP) and by Ca²⁺. Subconvulsive doses of insulin or of amphetamine administered to mice produced some activation of the enzyme. It is concluded that the mechanism for activation of phosphorylase in nerve tissue is similar to that in muscle.     

Adenosine 5'-O(3-thiotriphosphate) in the control of phosphorylase activity

Rabbit muscle phosphorylase b (EC 2.4.1.1) is converted to a thio-analog of phosphorylase a by phosphorylase kinase, Mg²⁺ and adenosine 5′-O(3-thiotriphosphate)(ATPγS). Conversion proceeds at one-fifth the rate obtained with ATP though the extent of reaction and final level of activation of the enzyme are the same. However, the thiophosphorylase a produced is resistant to phosphorylase phosphatase and, therefore, behaves as a competitive inhibitor with a K_I of 3 μM, similar to the K_M obtained with normal phosphorylase a. ATPγS can also be utilized by protein kinase in the activation of phosphorylase kinase at a rate similar to that obtained with ATP. It is hydrolyzed at 5 to 10 times the normal rate by the sarcoplasmic reticulum ATPase. When added to a muscle glycogen-particulate complex in the presence of Ca²⁺ and Mg²⁺, ATPγS triggers an activation of phosphorylase with simultaneous inhibition of phosphorylase phosphatase as previously observed with ATP.     

Control of platelet glycogenolysis activation of phosporylase kinase by calcium

An apparent enigma during platelet aggregation is that increased glycogenolysis occurs despite a fall in cyclic AMP levels. Activation by a classical cascade is therefore unlikely, and an alternative stimulus for phosphorylase a formation was sought. It was found that low levels of Ca²⁺ markedly activate phosphorylase b kinase from human platelets, with a K_a of 0.89 μM Ca²⁺, which is similar to that for the skeletal muscle enzyme. The kinase activity is unstable, and on enzyme ageing there is a 50% loss in activity with the K_a decreasing to 0.33 μM Ca²⁺.In unstimulated platelets, phosphorylase a was 13.3% of total measured activity, and glycogen synthetase I was 32.3%. Aggregation induced by ADP did not change the percentage of I synthetase, while increasing that for phosphorylase a. Dibutyryl cyclic AMP did, as expected, increase the percentage of both phosphorylated enzymes.These findings suggest that the natural activator of platelet glycogenolysis during aggregation is Ca²⁺, which directly stimulates phosphorylase b kinase without altering glycogen synthetase activity. The cyclic AMP-dependent protein kinase does not appear to be involved.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca²⁺-dependent ATPase and are also able to accumulate up to 10 μmol Ca²⁺ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a₃ and an increase in the content of [³²P]phosphoenzyme. The basic components in “calcium-oxalate preparation” from hearts are proteins with molecular weights of about 100 000 (Ca²⁺-dependent ATPase) and 55 000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca²⁺-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12–16 μmol P_i/min per mg protein. Enzyme activity of purified Ca²⁺-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca²⁺-dependent ATPases isolated from heart and skeletal muscles.     

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.     

Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca²⁺-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca²⁺-binding protein and a M₅₅ protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca²⁺-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of ³²P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca²⁺ uptake. The Ca²⁺-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein.The Ca²⁺-pump and Ca²⁺-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca²⁺-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles ³²P-labelled phosphoenzyme per mg protein. The Ca²⁺-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues).Ca²⁺ binding by sarcoplasmic reticulum vesicles and by the Ca²⁺-pump and Ca²⁺-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca²⁺-pump protein contained nonspecific high-affinity Ca²⁺ binding sites with a capacity of 90–100 and 55–70 nmoles Ca²⁺ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca²⁺ per mg protein. The binding constants for nonspecific and specific Ca²⁺ binding by both preparations were approx. 1 μM^?1. The Ca²⁺-binding protein nonspecifically bound 900–1000 nmoles Ca²⁺ per mg protein with a binding constant of about 0.25 μM^?1.     

Cyclic AMP stimulation of membrane phosphorylation and Ca2+-activated,Mg2+-dependent ATPase in cardiac sarcoplasmic reticulum

Ca²⁺-activated ATPase (EC 3.6.1.15) in canine cardiac sarcoplasmic reticulum was stimulated 50–80% by cyclic adenosine 3′ : 5′-monophosphate. The relationship of this stimulation to cyclic AMP-dependent membrane phosphorylation with phosphoester bands was studied. Cyclic AMP stimulation of ATPase activity was specific for Ca²⁺-activated ATPase and was half-maximal at about 0.1 μM which is similar to the concentration required for half-maximal stimulation of membrane phosphorylation by endogenous cyclic AMP-stimulated protein kinase (EC 2.7.1.37). Cyclic AMP stimulation of Ca²⁺-activated ATPase was calcium dependent and maximal at calculated Ca²⁺ concentrations of 2.0 μM. Cyclic AMP-dependent Ca²⁺-activated ATPase correlated well with the cyclic AMP-dependent membrane phosphorylation of which 80% was 20 000 molecular weight protein identified by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. In trypsin-treated microsomes, cyclic AMP did not stimulate Ca²⁺-activated ATPase or phosphorylation of the 20 000 molecular weight membrane protein. An endogenous calcium-stimulated protein kinase (probably phosphorylase b kinase) with an apparent K_m for ATP of 0.21–0.32 mM was present and appeared to be involved in the cyclic AMP-dependent phosphorylation of the 20 000 molecular weight protein which was calcium dependent. Cyclic guanosine 3′ : 5′-monophosphate did not inhibit any of the stimulatory effects of cyclic AMP. These data suggest that the cyclic AMP stimulation of Ca²⁺-activated ATPase in cardiac sarcoplasmic reticulum is mediated by the 20 000 molecular weight phosphoprotein product of a series of kinase reactions similar to those activating phosphorylase b.     

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

Summary We have previously shown that inositol-1,4,5-trisphosphate (IP₃) releases Ca²⁺ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983,Nature (London) 306:67–69). This observation suggests that IP₃ might provide the missing link between activation of the muscarinic receptor and Ca²⁺ release from intracellular stores during stimulation. In order to localize the intracellular IP₃-sensitive calcium pool, IP₃-induced Ca²⁺ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl₂. IP₃-induced Ca²⁺ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000×g, sucrose-density centrifugation, or MgCl₂ precipitation there was a close correlation of IP₃-induced Ca²⁺ release with the endoplasmic reticulum markers ribonucleic acid (r=0.96, 1.00, 0.91, respectively) and NADPH cytochromec reductase (r=0.63, 0.98, 090, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochromec oxidase (r=–0.64) and glutamate dehydrogenase (r=–0.75) and with the plasma membrane markers (Na⁺+K⁺)-ATPase (r=–0.81) and alkaline phosphatase (r=–0.77) in all fractions analyzed. IP₃-induced Ca²⁺ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca²⁺ from endoplasmic reticulum in pancreatic acinar cells.     

Properties of the complexes formed by 1-anilinonaphthalene-8-sulfonate with phosphorylase kinase and calmodulin

Phosphorylase kinase contains four approximately equivalent binding sites for 1-anilinonaphthalene-8-sulfonate (1,8-ANS). Measurements of the time decay of fluorescence anisotropy have failed to give any indication of internal degrees of rotational freedom involving a significant portion of the tertiary structure. In the presence of 1 mM Ca²⁺, calmodulin binds one molecule of 1,8-ANS. No binding occurs in the absence of Ca²⁺. The binding is strongly temperature-dependent, a decrease in binding occurring with increasing temperature. Determinations of the time decay of fluorescence anisotropy indicate the presence of internal rotations, which become more important with increasing temperature. Complex formation between phosphorylase kinase and calmodulin reduces the binding of 1,8-ANS.     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals

Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca²⁺ in the presence of ATP. The specific activity of ATP-dependent uptake is ~20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca²⁺ stimulation of ATP hydrolysis was not discernible above the high background of Ca²⁺-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca²⁺ concentration, suggesting that Ca²⁺ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca²⁺ at half maximal velocity of uptake (S_0.5) was 12.5 micromolar and the Hill coefficient (n_H) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca²⁺, although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca²⁺ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca²⁺ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca²⁺ into microsomal vesicles declined by ~70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S_0.5 or n_H, but V_max for ATP-dependent Ca²⁺ uptake decreased by ~40%. A similar decline in ATP-dependent uptake of Ca²⁺ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene.     

ATP-dependent CA2+ transport in endoplasmic reticulum isolated from roots ofLepidium sativum L.

Endoplasmic reticulum membranes were isolated from roots of garden cress (Lepidium sativum L. cv Krause) using differential and discontinuous sucrose gradient centrifugation. The endoplasmic reticulum fraction was 80% rough endoplasmic reticulum oriented with the cytoplasmic surface directed outward and contaminated with 12% unidentified smooth membranes and 8% mitochondria. Marker enzyme analysis showed that the activity for endoplasmic reticulum was enriched 2.4-fold over total membrane activity while no other organelle activity showed an enrichment. All evidence indicated that the fraction was composed of highly enriched endoplasmic reticulum membranes. Ca²⁺ uptake activity was measured using the filter technique described by Gross and Marmé (1978). The results of these experiments showed an ATP-dependent, oxalate-stimulated Ca²⁺ uptake into vesicles of the endoplasmic reticulum fraction. The majority of the transport activity was microsomal since specific inhibitors of mitochondrial Ca²⁺ transport (ruthenium red, LaCl₃ and oligomycin) inhibited the activity by only 25%. Sodium azide showed no inhibition. The transport was likely directly coupled to ATP hydrolysis since there was no inhibition with carbonylcyanidem-chlorophenylhydrazone. The transport activity was specific for ATP showing only 36% and 29% of the activity with inosine diphosphate and guanosine 5′-triphosphate, respectively. The results indicate a Ca²⁺ transport function located on the endoplasmic reciculum of garden cress roots.     

Biochemical changes in isolated hepatocytes exposed to tert-butyl hydroperoxide. implications for its cytotoxicity.

When isolated hepatocytes were exposed to tert-butyl hydroperoxide (tBOOH) they lost their cellular membrane integrity. Decreased levels of GSH, increased phosphorylase a activity (an indirect index of the amount of free cytosolic Ca²⁺), and increase in the formation of malondialdehyde (MDA)-like products (an index of lipid peroxidation) preceeded the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH), indicating that this later process was the consequence of the former intracellular events. While ATP levels were not modified during the incubation of cells with increasing concentrations of tBOOH, protein synthesis was decreased in a concentration-dependent manner. The glycogen content decreased at the same time as the increase in LDH leakage. The addition of promethazine (PMZ) an antioxidant molecule, prevented the lipid peroxidation, but did not protect cells against the oxidative effects of tBOOH, including loss of membrane integrity. Nevertheless, the addition of GSH to cell suspensions incubated with tBOOH, decreased the formation of MDA-like products, restored the protein synthesis rate, prevented partially the activation of phosphorylase a and preserved cell viability. On the basis of these results, we postulate that both GSH depletion and modification in phosphorylase a activity (Ca²⁺ levels) were the most relevant intracellular events to explain the cytotoxicity of tBOOH.Abbreviations tBOOH tert-butyl hydroperoxides - GSH reduced glutathione - LDH lactate dehydrogenase - MDA malondialdehyde - TBA thiobarbituric acid - PMZ promethazin - BSA bovine serum albumin     

酿酒酵母细胞中内质网应激与未折叠蛋白反应的研究进展

内质网应激激活的未折叠蛋白反应(Unfolded protein response,UPR)途径在酿酒酵母和哺乳动物细胞中是非常保守的。内质网(Endoplasmic reticulum,ER)是蛋白质合成、折叠和修饰的细胞器,也是贮存钙的主要场所之一。酵母细胞内质网钙平衡与UPR的作用是相互的;两个MAPK途径——HOG途径和CWI途径都是细胞应答内质网应激压力时生存所必需的;重金属镉离子能够激活UPR途径,它通过激活钙离子通道Cch1/Mid1进入细胞影响钙离子的功能。本文结合最新研究进展对酿酒酵母细胞中的两个MAPK途径、镉离子和钙离子稳态与内质网应激激活的UPR途径之间相互关系进行综述。     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: Studies with extracellular adenosine 5′-triphosphate

The incubation of isolated rat hepatocytes with extracellular adenosine 5′-trihosphate (ATP) resulted in an inhibition of Ca²⁺ efflux. The ATP-induced Ca²⁺ accumulation as determined by the increase in phosphorylase a activity and the Ca²⁺ -sensitive fluorescent indicator (2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis-[carboxymethyl] aminoquinoline-tetrakis-[acetoxymethyl]ester) (Quin 2-AM) was associated with both the hydrolysis of ATP and the phosphorylation of a 110 kDa protein. No significant alteration in the intracellular ATP level was observed. The appearance of surface blebs and cytotoxicity followed the rise in cytosolic Ca²⁺, suggesting that the increased free Ca²⁺ may be responsible for the loss of viability. When a calmodulin inhibitor, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] ethyl]-1H-imidazolium chloride (calmidazolium), was included in the medium prior to ATP addition, bleb formation was reduced and the loss of viability was completely prevented, indicating that a Ca²⁺ -calmodulin process may be involved in the initiation of cytotoxicity.     

Calcium Transport in the Green Alga Mesotaenium caldariorum: Preliminary Characterization and Subcellular Distribution

The subcellular localization and biochemical characterization of calcium transport were studied in the unicellular green alga Mesotaenium caldariorum. Membrane fractions prepared by osmotic lysis of Mesotaenium protoplasts exhibit high rates of ATP-dependent calcium uptake. Sucrose gradient centrifugation separates two pools of activity, which display specific activities for calcium transport as high as 15 nanomoles Ca²⁺ per minute per milligram of protein. Marker enzyme analysis shows that this dual distribution of calcium transport activity is similar to that of vanadate-insensitive ATPase and pyrophosphatase, activities considered to be associated with the tonoplast. Plasma membranes, endoplasmic reticulum vesicles, mitochondrial membranes, and thylakoids band at higher densities than either calcium transport fraction. Both pools of ATP-dependent calcium uptake contain two components which are not separable on sucrose gradients but can be distinguished on the basis of inhibitor sensitivity. One component is inhibited by nigericin or trimethyltin chloride (I₅₀ values of 3 nanomolar and 4 micromolar, respectively), while the other component is vanadate sensitive (I₅₀ of 25 micromolar). These results suggest that direct Ca²⁺ transport and Ca²⁺/H⁺ antiport activities are present in both sucrose gradient fractions.     

Characterisation of a human liver cystathionine beta synthase mRNA sequence corresponding to the c.[833T>C;844_845ins68] mutation in CBS gene

Endoplasmic reticulum (ER) homeostasis is crucial for β-cell function and survival. Direct as well as indirect evidence has pointed toward Ca²⁺ as an important determinant of interleukin-1β (IL-1β)-induced β-cell dysfunction and apoptosis. In the present study, we show that IL-1β-induced apoptosis and necrosis in primary rat β-cells and MIN6 cells largely depends on ER stress, ER Ca²⁺ release, and c-Jun N-terminal kinase (JNK) activation. β-cells also showed marked sensitivity to apoptosis induced by sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA) blockers, thapsigargin and cyclopiazonic acid (CPA). IL-1β induced ER Ca²⁺ release, which was paralleled by an IL-1β-dependent induction of JNK activation and the ER stress response, including activation of PRK (RNA-dependent protein kinase)-like ER kinase (PERK). Furthermore, reduced activation of JNK, utilizing JNK inhibitor SP600125, resulted in significant protection from IL-1β- or thapsigargin-induced apoptosis via ER stress. In conclusion, our results suggest that the IL-1β-induced depletion of ER Ca²⁺ and activation of the ER stress via JNK pathway are potential contributory mechanisms to β-cell death.     

Antioxidant metallothionein alleviates endoplasmic reticulum stress-induced myocardial apoptosis and contractile dysfunction

Abstract

Aims. Endoplasmic reticulum (ER) stress exerts myocardial oxidative stress, apoptosis, and contractile anomalies, although the precise interplay between ER stress and apoptosis remains elusive. This study was designed to examine the impact of the cysteine-rich free radical scavenger metallothionein on ER stress-induced myocardial contractile defect and underlying mechanisms. Methods and results. Wild-type friendly virus B and transgenic mice with cardiac-specific overexpression of metallothionein were challenged with the ER stress inducer tunicamycin (1 mg/kg, intraperitoneal, 48 h) prior to the assessment of myocardial function, oxidative stress, and apoptosis. Our results revealed that tunicamycin promoted cardiac remodeling (enlarged left ventricular end systolic/diastolic diameters with little changes in left ventricular wall thickness), suppressed fractional shortening and cardiomyocyte contractile function, elevated resting Ca²⁺, decreased stimulated Ca²⁺ release, prolonged intracellular Ca²⁺ clearance, and downregulated sarco(endo)plasmic reticulum Ca²⁺-ATPase levels, the effects of which were negated by metallothionein. Treatment with tunicamycin caused cardiomyocyte mitochondrial injury, as evidenced by decreased mitochondrial membrane potential (??m, assessed by JC-1 staining), the effect of which was negated by the antioxidant. Moreover, tunicamycin challenge dramatically facilitated myocardial apoptosis as manifested by increased Bax, caspase 9, and caspase 12 protein levels, as well as elevated caspase 3 activity. Interestingly, metallothionein transgene significantly alleviated tunicamycin-induced myocardial apoptosis. Conclusion. Taken together, our data favor a beneficial effect of metallothionein against ER stress-induced cardiac dysfunction possibly associated with attenuation of myocardial apoptosis.     

The phosphorylation of troponin B by phosphorylase b kinase in skeletal muscle of mice carrying the phosphorylase b kinase deficiency gene

In skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase $\text{b}$ to $\text{a}$ in the presence of Ca⁺⁺, Mg⁺⁺, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase $\text{b}$. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase $\text{b}$ converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA¹ (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca⁺⁺. Thus the phosphorylase $\text{b}$ kinase in skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.