Studies on the in Vitro Interaction of Electrical Stimulation and Ca++ Movement in Sarcoplasmic Reticulum

Sarcoplasmic reticulum fragments (S.R.F.) were isolated from skeletal and heart muscles. These fragments were found to take up Ca⁺⁺ very actively from media. When monophasic square waves were passed through the S.R.F. suspension, the Ca⁺⁺ uptake by S.R.F. was decreased. When the suspension was stimulated electrically after the Ca⁺⁺ was taken up by S.R.F., the initiation and the cessation of the stimulation were followed by the release and re-uptake of Ca⁺⁺ by S.R.F., respectively. The degree of inhibition of the Ca⁺⁺ uptake as well as of the Ca⁺⁺ release by electrical stimulation was dependent on the voltage and the frequency of stimulation. The presence of inorganic phosphate or oxalate modified the influence of electrical stimulation on the release and the uptake of Ca⁺⁺ by S.R.F. Attempts were made to observe the release of Ca⁺⁺ by electrical stimulation from unfractionated sarcoplasmic reticulum remaining in myofibers, and the interaction of the released Ca⁺⁺ with myofibrils in vitro. For this purpose, the glycerol-extracted fiber was selected as a muscle model, since it contains both sarcoplasmic reticulum and myofibrils. It was found that electrical stimulation of skeletal and heart glycerol-extracted fibers resulted in the contraction of fibers. It appeared that the contraction of glycerol fibers by electrical stimulation was caused by the Ca⁺⁺ release from sarcoplasmic reticulum by stimulation.     

The Effect of Calcium Ionophores on Fragmented Sarcoplasmic Reticulum

X-537 A and A 23187, two antibiotics which form liphophilic complexes with divalent cations, function as ionophores in vesicular fragments of sarcoplasmic reticulum (SR). Addition of either ionophore to SR preloaded with calcium in the presence of adenosine triphosphate (ATP), causes rapid release of calcium. Furthermore, net calcium accumulation by SR is prevented, when the ionophores are added to the reaction mixture before ATP. On the contrary, ATP-independent calcium binding to SR is not inhibited. This effect is specific for the two antibiotics and could not be reproduced, either by inactive derivatives, or by other known ionophores. Neither ionophore produces alterations of the electron microscopic appearance of SR membranes or inhibition of the calcium-dependent ATPase. In fact, the burst of ATP hydrolysis obtained on addition of calcium, is prolonged in the presence of the ionophores. Lanthanum inhibits ATP-independent calcium binding to SR, ATP-dependent calcium accumulation and calcium-dependent ATPase. However, addition of lanthanum to SR preloaded in the presence of ATP, does not cause calcium release. The reported experiments indicated that: (a) ATP-dependent calcium accumulation by SR results in primary formation of calcium ion gradients across the membrane. (b) Most of the accumulated calcium is not available for displacement by lanthanum on the outer surface of the membrane. (c) Calcium ionophores induce rapid equilibration of the gradients, by facilitating cation diffusion across the membrane.     

Neoplastic Transformation-Associated Stimulation of the In Vitro Resolution of Concatemer Junction Fragments from Minute Virus of Mice DNA

Minute virus of mice (MVM) shows an oncotropic behavior reflected by its ability to amplify its genome more efficiently in a number of transformed versus normal cells. In vivo and in vitro studies revealed that the major effect of cell transformation on MVM DNA replication occurs at the level of double-stranded replicative-form amplification. In particular, resolution of MVM DNA concatemers into monomers was found to be highly sensitive to neoplastic transformation.     

Ion Effects on Calcium Accumulation by Cardiac Sarcoplasmic Reticulum

The effects of monovalent cations on the active calcium-accumulating ability of cardiac sarcoplasmic reticulum were assessed. Grana prepared in an ion-free system accumulated calcium when ATP and Mg⁺⁺ were present. Sodium ion and to a lesser extent lithium but not K⁺ reduced the amount of calcium taken up. The reduction of calcium binding by Na⁺ is not due to inhibition of uptake but to a rapid release of the radiocalcium bound. The amount of calcium released by sodium does not appear to be enough to explain contraction on the basis of sodium influx into muscle, but may be significant in the regulation of tension.     

脂肪族类两亲物对肌浆网膜蛋白结构的影响

本文观察和比较了六种C_(18)脂肪族类两亲物(fatty amphiphile,FA),包括硬脂酸(stearic acid)、硬脂胺(stearyl amine)、硬脂醇(stearyl alcohol)、油酸(oleic acid)、油胺(oleylamine)和油醇(oleyl alcohol),对肌浆网(sarcoplasmic reticulum,SR)钙泵蛋白结构的影响。当FA∶SR(μmol∶mg)的比例为2.67∶1—21.33∶1时,除油醇(oleyl alcohol)外,其余五种FA引起天然兔骨骼肌肌浆网蛋白内源荧光强度降低。随FA∶SR比例升高,降低幅度加大。五种FA的最大降低幅度在10—32％之间。其中带电荷FA比不带电荷FA的作用强,但均未见峰位位移。当FA∶SR的比例为2.67∶1时,除硬脂醇(stearyl alcohol)外,其余五种FA使N-(3-芘)-马来酰胺(N-(3-pyrene)maleimide,N-(3-p)-M)修饰的SR蛋白巯基荧光强度分别上升9％,40％,150％,193％和5％,但也未见峰位位移。ATP可减弱胺类FA引起的SR蛋白内源荧光降低和巯基荧光升高的幅度。油酸、油胺和硬脂胺既抑制SRCa~(2+),Mg~(2+)-ATPase活力和SR钙蓄积,又使SR钙泵蛋白的巯基修饰荧光显著上升。提示C_(18)脂肪族类两亲物使SR功能受损与其引起钙泵蛋白构象的显著变化有关。     

The Mechanism of the Action of Caffeine on Sarcoplasmic Reticulum

Evidence is presented that caffeine does not act on the mitochondrial Ca uptake system and that its effect cannot be attributed to the accumulation of adenosine 3',5'-phosphate. Two distinct caffeine effects are described. At high ATP concentrations caffeine decreases the coupling between ATP hydrolysis and Ca inflow. It either inhibits inflow without any inhibition of the rate of ATP hydrolysis, or it stimulates the ATPase activity without stimulating Ca inflow. These high ATP concentrations (much higher than needed for the saturation of the transport ATPase) greatly reduce the control of the turnover rate of the transport system, by accumulated Ca. At low ATP concentrations when the transport system is under maximal control by accumulated Ca, caffeine inhibits the ATPase activity without affecting the rate of Ca inflow.     

大鼠心肌肌浆网的纯化及其性质研究

 用超声波破碎心肌细胞,差速离心法纯化大鼠心肌肌浆网(CSR)。SDS-聚丙烯酰胺凝胶电泳测得Ca~(2+)-ATPase分子量为98kD;电镜观察膜制备为完整的CSR微囊;标志酶哇巴因敏感型Na~(+),K~(+)-ATPase和叠氮化钠敏感型Mg~(2+)-ATPase活性表明膜制备中肌膜含量很低,但仍有线粒体污染。 用~(45)Ca~(2+)示踪微孔滤膜法研究Ca~(2+)跨膜转运,CSRCa~(2+)蓄集最大值为57nmol/mg蛋白。CSR Ca~(2+)-ATPase在4℃—21℃和21℃—49℃两区间反应活化能不同,前者大于后者。酶的最适pH为7.4。以ATP为底物,该酶有两个表观Km值:Km_1为3.7μmol/LKm_2为713μmol/L。     

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.     

Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum

Calumenin is a multiple EF-hand Ca²⁺-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265–269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca²⁺-cycling proteins. But it enhanced Ca²⁺ transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca²⁺ uptake showed increased Ca²⁺ sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 μm Ca²⁺, suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132–222 amino acids of calumenin and 853–892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca²⁺-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca²⁺-cycling of cardiomyocytes.     

Immunolocalization of Sarcoplasmic Reticulum Proteins in Mammalian Skeletal Muscle Fibers

SYNOPSIS. The sarcoplasmic reticulum is the intracellular membranesystem in skeletal muscle fibers which regulates the Ca^2$ concentrationof the myofibril and thereby the contraction relaxation cycle. In the past the proposed explanation for the differences inthe contractile properties of fast and slow skeletal fibershas been attributed mainly to quantitative rather than qualitativedifferences in the structure, function and molecular compositionof the sarcoplasmic reticulum of these two fiber types. Recentimmunocytochemical and biochemical studies have, however, clearlydemonstrated that the Ca^2$-ATPase of the sarcoplasmic reticulumin slow skeletal fibers is structurally and thus perhaps alsofunctionally related to that of the cardiac fibers, but distinctlydifferent from that of fast skeletal fibers. Furthermore similarstudies have shown that phospholamban, a cardiac sarcoplasmicreticulum protein believed to modulate the activity of the cardiacCa^2$-ATPase, is also present in slow but not fast skeletal fibers. The availability of antibodies specific to the fast and slowisoforms of the Ca^2$-ATPase, and to phospholamban will now enableus to apply immunocytochemical labeling techniques to examinethe effect of neuronal and other physiological signals on theregulation of the gene expression of sarcoplasmic reticulumproteins at the cellular level.     

Neuritin Attenuates Neuronal Apoptosis Mediated by Endoplasmic Reticulum Stress In Vitro

Neuritin is an extracellular glycophosphatidylinositol-linked protein that promotes neuronal survival, differentiation, function, and repair, but the exact mechanism of this neuroprotective effect remains unclear. Meanwhile, endoplasmic reticulum stress (ERS) induced apoptosis is attracting increased attention. In this work, we hypothesized that neuritin inhibited ERS to protect cortical neurons. To check this hypothesis, we exposed primary cultured cortical neurons to oxygen and glucose deprivation (OGD) for 45 min followed by reperfusion (R) to activate ERS. We then performed resuscitation for 6, 12, 24, and 48 h. ERS-related factors such as glucose-regulated protein 78 (GRP78), caspase-12 and CHOP were detected by Western blotting and quantitative real-time polymerase chain reaction assay. Apoptosis was assessed by Annexin V binding and propidium iodide staining. Ultrastructural changes of endoplasmic reticulum were observed under a transmission electron microscope. Results showed that GRP78 expression significantly increased at 12, 24, and 48 h and peaked at 24 h. Caspase-12 and CHOP expression significantly increased in a time-dependent manner at 12, 24, and 48 h. GRP78, caspase-12 and CHOP expression as well as apoptosis rate of primary cultured neurons and the ultrastructural changes of endoplasmic reticulum in the OGD/R?+?neuritin group significantly improved compared with the OGD/R group. In conclusion, the neuroprotection function of neuritin may be involved in ERS pathways.     

In Vitro Stimulation of Protein Kinase C by Melatonin

It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca²⁺ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca²⁺ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [³H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 M) and protein kinase C inhibitors (100 M) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [³H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca²⁺. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca²⁺ dependent proteins.     

Stimulation of Melatonin Synthesis in Ovine Pineals In Vitro

Static and superfused pineal slices (750 micron) have been used to study the control of melatonin synthesis by ovine pineals. Static incubates show a time-dependent accumulation of melatonin in the medium; this is significantly increased by stimulation with norepinephrine (NE) (10(-5) M), reaching 300% above control levels after 4 h. Perifused pineal slices show a rapid rise in melatonin release within 12-18 min in response to NE stimulation. This reaches a 3.5-4.5-fold increase in melatonin released within 30 min. Withdrawal of NE is associated with a rapid return to prestimulated levels within 12-18 min. These time-course characteristics compare favorably to those changes seen in vivo. The formation of [14C]melatonin from [14C]-tryptophan shows a linear increase with time. In the presence of NE (10(-5) M), the rate of synthesis is increased, albeit after an initial time lag of at least 30 min. The latter may reflect an N-acetyltransferase-independent mechanism of synthesis and release. In static incubations, propranolol (10(-5) M) inhibited NE-induced melatonin production by about 60%, but prazosin (10(-5) M) had no effect. As dibutyryl cyclic AMP (10(-3) M) stimulated melatonin production, it is concluded that beta-receptors are of primary importance to the control of melatonin production, as in the rat. The role of alpha 1-receptors is less clear, but the stimulatory action of phorbol 12-myristate 13-acetate on melatonin release implicates a receptor linked to phosphatidylinositol turnover.     

Isolation of two Proteolipids from Rabbit Skeletal Muscle Sarcoplasmic Reticulum

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteo-lipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na, K)-ATPase.     

Effects of Potentiators of Muscular Contraction on Binding of Cations by Sarcoplasmic Reticulum

Anionic (NO₃^-, Br^-, I^-, and SCN^-) and cationic (Zn⁺⁺ and Cd⁺⁺) potentiators of the twitch output of skeletal muscle depress the active binding of Ca by sarcoplasmic reticulum isolated from rabbit skeletal muscle. Zinc and Cd exchange for Ca and Mg at the binding sites of the reticular membranes, whereas the anions effectively induce a replacement by Mg of Ca bound actively in the presence of ATP. In the absence of ATP, the passive binding of both Ca and Mg is increased by the anions tested. Furthermore, the anions increase the total capacity of the membrane fragments for passive cation binding. The Ca-stimulated ATPase activity of the membranes is inhibited by Zn and Cd, but not by the anions. Shifts in cations bound to muscle membrane systems caused by agents that increase the force of contraction developed during the twitch are considered to be the primary event modifying excitation-contraction coupling, and thus leading to potentiation.     

pH-Modulation of Chloride Channels from the Sarcoplasmic Reticulum of Skeletal Muscle

The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca²⁺ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH _cis ) and luminal pH (pH _trans ) was investigated using the lipid bilayer-vesicle fusion technique. Low pH _cis 6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH _cis 7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas at low pH _cis 6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low pH _trans 4.07, but not pH _trans 6.28, modified the activity of SCl channels. The effects of low pH _cis are pronounced at 10⁻³ and 10⁻⁴ m [Ca²⁺] _cis but are not apparent at 10⁻⁵ m [Ca²⁺] _cis , where the subconductances of the channel are already prominent. Low pH _cis -induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pH _cis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels. Received: 20 May 1998/Revised: 24 September 1998     

Effects of TRIS and HEPES on Function of Rabbit Muscle Light Sarcoplasmic Reticulum

ATP hydrolysis activity and calcium transport activity were determined on light sarcoplasmic reticulum from rabbit skeletal muscle. The effects of two buffers, TRIS and HEPES, were compared. Titration of TRIS into sarcoplasmic reticulum preparations in HEPES provided evidence for TRIS inhibition of ATPase activity and TRIS stimulation of calcium transport activity.     

Electrokinetic Properties of the Sarcoplasmic Reticulum Membrane Obtained from Reconstitution Studies

Electrophoretic mobility data of SR vesicles reconstituted with uncharged and two mixtures of charged and uncharged lipids (Brethes, D., Dulon, D., Johannin, G., Arrio, B., Gulik-Krzywicki, T., Chevallier, J. 1986. Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles. Arch. Biochem. Biophys. 246:355–356) were analyzed in terms of four models of the membrane-water interface: (I) a smooth, negatively charged surface; (II) a negatively charged surface of lipid bilayer covered with an electrically neutral surface frictional layer; (III) an electrically neutral lipid bilayer covered with a neutral frictional layer containing a sheet of negative charge at some distance above the surface of the bilayer; (IV) an electrically neutral lipid bilayer covered with a homogeneously charged frictional layer. The electrophoretic mobility was predicted from the numerical integration of Poisson-Boltzmann and Navier-Stokes equations. Experimental results were consistent only with predictions based on Model-III with charged sheet about 4 nm above the bilayer and frictional layer about 10 nm thick. Assuming that the charge of the SR membrane is solely due to that on Ca⁺⁺-ATPase pumps, the dominant SR protein, the mobility data of SR and reconstituted SR vesicles are consistent with 12 electron charges/ATPase. This value compares well to the net charge of the cytoplasmic portion of ATPase estimated from the amino acid sequence (-11e). The position of the charged sheet suggests that the charge on the ATPase is concentrated in the middle of the cytoplasmic portion. The frictional layer of SR can be also assigned to the cytoplasmic portion of Ca⁺⁺-ATPase. The layer has been characterized with hydrodynamic shielding length of 1.1 nm. Its thickness is comparable to the height of the cytoplasmic portion of Ca⁺⁺-ATPase. Received: 15 June 1998/Revised: 8 October 1998     

Control of Locomotion In Vitro: II. Chemical Stimulation

Previous studies have described the presence of alternating activity induced in left and right ventral roots of the neonate rat in vitro brainstem-spinal cord preparation, following application of certain neuroactive substances to the bathing solution. The present findings show the presence of chemically induced, adult-like coordinated airstepping demonstrated by electromyographic recordings in the hindlimb-attached in vitro brainstem-spinal cord preparation. Analysis of muscular activity demonstrated alternation between antagonists of one limb and between agonists of different limbs, as well as a proximodistal delay in agonists active at different joints of the same limb. Neuroactive agents were applied independently to either the brainstem or spinal cord bath. The substances surveyed in the present studies included some of those used previously, as well as additional compounds: bicuculline and picrotoxin (γ-aminobutyric acid-ergic antagonists), N-methyl-D-aspartic acid (excitatory amino acid agonist), substance P, acetylcholine, carbachol (cholinergic agonist), and serotonin. Application of these substances to the brainstem bath produced rhythmic airstepping. Application of dopamine, aspartate, glutamate, and N-methyl-D-aspartic acid to the spinal cord bath also produced rhythmic airstepping, while application of acetylcholine produced tonic, long-lasting co-contractions. These findings reveal the presence of several neurochemical systems in the central nervous system that can be activated at birth to induce coordinated airstepping in the neonate rat in vitro brainstem-spinal cord preparation.