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PubMed2004

Calreticulin, Ca2+, and calcineurin - signaling from the endoplasmic reticulum.

Calcium (Ca2+) is a universal signalling molecule involved in many aspects of cellular function. The majority of intracellular Ca2+ is stored in the endoplasmic reticulum and once Ca2+ is released from the endoplasmic reticulum, specific plasma membrane Ca2+ channels are activated, resulting in increased intracellular Ca2+. In the lumen of the endoplasmic reticulum, Ca2+ is buffered by Ca2+ binding chaperones such as calreticulin. Calreticulin-deficiency is lethal in utero due to impaired cardiac development and in the absence of calreticulin, Ca2+ storage capacity within the endoplasmic reticulum and inositol 1,4,5-trisphosphate (InsP3) receptor mediated Ca2+ release from the endoplasmic reticulum are compromised. Over-expression of constitutively active calcineurin in the heart rescues calreticulin-deficient mice from embryonic lethality. This observation indicates that calreticulin is a key upstream regulator of calcineurin in Ca2+-signalling pathways and highlights the importance of the endoplasmic reticulum and endoplasmic reticulum-dependent Ca2+ homeostasis for cellular commitment and tissue development during organogenesis. Furthermore, Ca2+ handling by the endoplasmic reticulum has profound effects on cell sensitivity to apoptosis. Signalling between calreticulin in the lumen of the endoplasmic reticulum and calcineurin in the cytoplasm may play a role in the modulation of cell sensitivity to apoptosis and the regulation of Ca2+-dependent apoptotic pathways.

Animals↗
PubMed1990

Distinct immunopeptide maps of the sarcoplasmic reticulum Ca2+ release channel in malignant hyperthermia.

Sarcoplasmic reticulum isolated from malignant hyperthermia-susceptible (MHS) muscle exhibits abnormalities in the regulation of calcium release. To identify the molecular basis of this abnormality, the Ca2+ release channel from both normal and MHS sarcoplasmic reticulum was examined using proteolytic digestion followed by immunoblot staining with a polyclonal antibody against the rabbit Ca2+ release channel protein. Under appropriate conditions, trypsin digestion of isolated sarcoplasmic reticulum vesicles from the two types of pigs revealed a distinct difference in the immunostaining pattern of the Ca2+ release channel-derived peptides. An approximate 86-kDa peptide was the predominant fragment in normal sarcoplasmic reticulum while an approximate 99-kDa peptide fragment was the major peptide detected in MHS sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum vesicles isolated from four normal and four MHS pigs showed that the differences were highly reproducible. Trypsin digestion of sarcoplasmic reticulum isolated from heterozygous pigs, which contain one normal and one MHS allele, showed an antibody staining pattern that was intermediate between MHS and normal sarcoplasmic reticulum. These results can be explained by a primary amino acid sequence difference between the normal and MHS Ca2+ release channels and support the hypothesis that a mutation in the gene coding for the sarcoplasmic reticulum Ca2+ release channel is responsible for malignant hyperthermia.

Animals↗
PubMed1979

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.

Animals↗
PubMed1989

[Development of endoplasmic reticulum and its enzymic marker in cultured mouse spinal neurons].

In an attempt to elucidate the relationship between synapse formation and cell development, the morphology and cytochemistry of the endoplasmic reticulum and its enzymic marker, glucose-6-phosphatase (G-6-Pase), in cultured mouse spinal neurons were investigated ultrastructurally. It was found that in the early period of the development, neurons were characterized by scarceness of organelles; only a few of granular or agranular endoplasmic reticulum and mitochondria were seen. The endoplasmic reticulum and nuclear envelope were packed specifically with G-6-Pase resection product but the product was weak. After a period of culture, most of the neurons had well-developed endoplasmic reticulum, Golgi apparatus, mitochondria and microtubules, etc. The Golgi apparatus was relatively large, having some cisternae associated with vesicles. Either concave of convex face of the saccules was labeled by thiamine pyrophosphatase (TPPase) specifically. GERL, labeled by cytidine monophosphatase (CMPase), was also seen close to the inner or outer face of some Golgi apparatus. The endoplasmic reticulum at this stage was distributed throughout the cytoplasm, including that in dendrites; its enzyme marker (G-6-Pase) localized consistently within the lumen of all endoplasmic reticulum, nuclear space and subsurface cisternae, and frequently in the concave saccules of the Golgi apparatus. After a long-term culture, some neurons became "aged". The endoplasmic reticulum cisternae enlarged and G-6-Pase reaction reduced. Along with the neuronal development, especially maturation of the endoplasmic reticulum and its enzymic marker, synapse formation was begun at the neuropile area. The axo-dendritic synapses always occurred between the axonal terminals and dendrites where the endoplasmic reticulum had showed positive G-6-Pase reactions. Considering the fact, it suggests that the appearance and change of these specific enzymes may be related to the maturation of the neurons in vitro, and also related to the synapse formation between neurons.

Animals↗
PubMed1994

Ultrasonography of the reticulum in cows.

The reticulum and adjacent organs were examined ultrasonographically in 51 cows by use of a 3.5-MHz linear transducer applied to the ventral aspect of the thorax over the sixth and seventh intercostal spaces. Examination included assessment of the contour of the reticulum, of reticular contractions, and of the organs adjacent to the reticulum. The normal reticulum appeared as a half-moon-shaped structure with a smooth contour; it contracted at regular intervals and was situated immediately adjacent to the diaphragm and ventral portion of the abdominal wall when relaxed. Contents of the reticulum could not normally be imaged because of its partly gaseous composition. The ruminoreticular groove, craniodorsal blind sac of the rumen, and the ventral sac of the rumen were observed caudally. The distal aspect of the spleen and parts of the omasum, abomasum, and liver could be imaged. Reticular motility was characterized by a biphasic contraction pattern. Four biphasic reticular contractions usually were observed during a 4-minute period. During the first (incomplete) contraction, the reticulum contracted by a mean of 7.2 +/- 2.30 cm. There was then low-grade, incomplete relaxation of the reticulum, followed immediately by the second reticular contraction, during which the reticulum usually disappeared from the 17.5-cm-deep screen. The reticulum then reappeared in its normal position. The first reticular contraction lasted a mean of 2.6 +/- 0.33 seconds and the second contraction lasted 3.9 +/- 0.55 seconds. The mean interval between 2 biphasic contractions was 44.9 +/- 10.53 seconds. The speed of the first reticular contraction was 5.4 +/- 1.32 cm/s.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Reticular groove and reticulum are innervated by myenteric neurons with different neurochemical codes.

The reticulum and the reticular groove are functional distinct compartments within the ovine forestomach. While the reticulum takes part in various motor functions, such as mixing, retaining, and rejecting the forestomach ingesta, the reticular groove serves mainly as a bypass between the esophagus and the abomasum. To accomplish these different tasks, the compartments develop specific motility patterns that are controlled by intrinsic neural circuits. In this study the intrinsic innervation by myenteric neurons was analyzed by quadruple immunohistochemistry against cholineacetyl transferase (ChAT), nitric oxide synthase (NOS), substance P (SP), and vasoactive intestinal peptide (VIP). Four neurochemically different subpopulations of myenteric neurons were found in the reticulum and the floor of the reticular groove: ChAT/-, ChAT/SP, NOS/-, and NOS/VIP. The neuronal proportions were calculated relative to all myenteric neurons. Neurons of the reticulum were mostly immunoreactive for ChAT (89% +/- 3%), whereas neurons adjacent to the reticular groove predominantly expressed a nitrergic phenotype (62% +/- 4%). ChAT-positive neurons were also immunoreactive for SP (ChAT/SP: 64% +/- 3% reticulum; 25% +/- 1% reticular groove) or were purely cholinergic (ChAT/-: 25% +/- 4% reticulum; 13% +/- 3% reticular groove). NOS-positive neurons colocalized VIP (NOS/VIP: 10% +/- 3% reticulum; 46% +/- 1% reticular groove) or none of the other neurotransmitters (NOS/-: 1% +/- 1% reticulum; 17% +/- 3% reticular groove). Analysis of the soma sizes revealed that in both compartments the nitrergic neurons were significantly larger than the cholinergic neurons. It is suggested that the specific neurochemical code in combination with a specific morphology leads to a precise regulation of the specialized tasks of the reticulum and reticular groove by subpopulations of myenteric neurons.

Animals↗
PubMed1983

The axoplasmic reticulum within myelinated axons is not transported rapidly.

The axoplasmic reticulum in myelinated axons is an extensive system of branched smooth membranous tubules which is found throughout the length of large axons. To investigate its motility and possible role in fast axonal transport, a focal chilling method was used to arrest transport at two sites separated by a 3 mm wide warm region along the saphenous nerve of mice. The experiments ran for 3-4 h since axoplasmic material travelling faster than 25 mm/day would clear from the central warm region. The nerve was subsequently fixed and processed by a technique that enhances the electron density of the axoplasmic reticulum. Thin and thick sections from several regions along the nerve were then systematically studied using conventional and high voltage electron microscopy. In these studies we found that: 1. the axoplasmic reticulum does not accumulate against the proximal sides of the cold blocks; 2. although often closely associated, there is no evidence of continuity between the axoplasmic reticulum and the discrete membranous compartments that do accumulate proximal to the chilled regions; 3. the axoplasmic reticulum remains in the central 3 mm wide warm region; 4. the axoplasmic reticulum does not accumulate against the distal sides of the cold blocks; 5. retrogradely moving elements that do accumulate distal to the cold blocks do not fuse with the axoplasmic reticulum and are not contained in it; and 6. both retrograde and anterograde vector types are often closely associated with elements of axoplasmic reticulum. These results were supported by quantitative morphometric analysis. We conclude that the axoplasmic reticulum represents a discrete membrane system, separate from either anterogradely or retrogradely moving rapid transport vectors, and that this interconnected cisternal system itself is not rapidly transported.

Animals↗
PubMed1973

Effect of gibberellic Acid and actinomycin d on the formation and distribution of rough endoplasmic reticulum in barley aleurone cells.

Analysis of structural changes in barley aleurone cells during germination or following incubation of isolated layers in gibberellic acid with or without actinomycin D revealed extensive development of rough endoplasmic reticulum. Following the assembly of stacked rough endoplasmic reticulum, vesiculation occurred mainly in basal regions of the cell, resulting in a polar distribution of rough endoplasmic reticulum vesicles. It is postulated that these vesicles are involved in protein secretion, because smooth vesicles, derived from the rough endoplasmic reticulum, apparently become appressed to the plasma membrane. The increased alpha-amylase in the ambient medium and in cell homogenates correlated directly with formation and subsequent vesiculation of the rough endoplasmic reticulum. Furthermore, when cells were treated with actinomycin D and gibberellic acid, alpha-amylase synthesis was inhibited by 45% and secretion by 63%. These cells were characterized cytologically by large areas of disarrayed segments of fragmented rough endoplasmic reticulum, corresponding to a high intracellular level of alpha-amylase. In addition, small lipid bodies common to the segmented regions of rough endoplasmic reticulum were surrounded by fine fibrous material, short segments of rough endoplasmic reticulum, and free ribosomes, suggesting that actinomycin D had interfered with development and organization of rough endoplasmic reticulum.

Journal Article↗
PubMed1989

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences.

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.

Amino Acid Sequence↗
PubMed2000

Sarcoplasmic reticulum Ca content, sarcolemmal Ca influx and the genesis of arrhythmias in isolated guinea-pig cardiomyocytes.

Early afterdepolarizations are seen during the repolarization phases of the action potential and delayed afterdepolarizations appear later, usually following complete repolarization of the cell membrane potential. Both forms of afterdepolarization are linked to the occurrence of aftercontractions, seem to play a role in the generation of ventricular arrhythmias and are believed to be the result of abnormalities of intracellular Ca handling. Suggestions for the mechanisms responsible vary from both types of afterpotential being mediated by Ca release from the sarcoplasmic reticulum, to early afterdepolarization formation being due to reactivation of the L-type sarcolemmal Ca channels during the action potential. We tried to assess the functional importance of the sarcoplasmic reticulum or Ca influx in the development of afterpotentials and abnormal contractile activity in guinea-pig cardiac myocytes. Ca influx was increased using isoproterenol, Bay K8644 or increasing extracellular [Ca]. Sarcoplasmic reticulum Ca content was measured using rapid cooling contractures or caffeine-induced Na/Ca exchange current and the sarcoplasmic reticulum was inhibited using caffeine or thapsigargin. Aftercontractions associated with either early or delayed afterdepolarizations could be induced by increasing Ca influx. The increased Ca influx produced increases in sarcoplasmic reticulum Ca content and aftercontractions were associated with a larger SR Ca content. However, the sarcoplasmic reticulum was no more loaded with Ca when aftercontractions occurred than when aftercontractions did not occur. Preventing Ca sequestration by the sarcoplasmic reticulum inhibited the formation of aftercontractions. The results suggest that alterations to both Ca influx and sarcoplasmic reticulum Ca content are required to produce aftercontractions.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle.

Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP3) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP3 receptors is unknown. InsP3 receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth muscle. InsP3 receptor labelling was predominantly localized at the cell periphery, where most of the sarcoplasmic reticulum is localized in vas deferens (phasic muscle). Elements of central sarcoplasmic reticulum, where present, were also labelled. Distribution of calsequestrin in vas deferens was similar to that of the InsP3 receptor. In aorta (tonic muscle) the InsP3 receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with the anti-calsequestrin antibody in aorta. InsP3 and caffeine, but not cyclic ADP-ribose, released intracellular Ca2+ in permeabilized vas deferens and aorta. The ultrastructure of the sarcoplasmic reticulum, investigated in stereo views of semi-thick and thin sections of osmium ferricyanide stained tissue, is shown to have several distinctive features, such as fenestrated sheets (single or in stacks), as well as numerous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle cell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP3 receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe analysis results showing calcium release from both regions.

Animals↗
PubMed1995

Porin-type 1 proteins in sarcoplasmic reticulum and plasmalemma of striated muscle fibres.

The location of porin-type 1 proteins in mammalian striated muscle has been assessed using immunogold electron microscopy with an anti-porin 31HL monoclonal antibody as the primary antibody. Gold particles were found on the mitochondrial outer membrane, the sarcoplasmic reticulum and plasmalemma in longitudinal sections of rat and rabbit skeletal muscle and rabbit and sheep cardiac muscle. The relative densities of gold particles in the mitochondrial outer membrane, sarcoplasmic reticulum and plasmalemma were 7:3:1 in white sternomastoid muscle, for example. Skeletal and cardiac sarcoplasmic reticulum vesicles, which had been fractionated by discontinuous sucrose density centrifugation, were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. The anti-porin 31HL monoclonal antibody detected a band of relative molecular mass (M(r)) 31,000 in all muscle sarcoplasmic reticulum vesicle fractions and also in liver mitochondria. The intensity of immunostaining of the sarcoplasmic reticulum fractions was 2.5-10% that of mitochondrial outer membranes per microgram of membrane protein blotted. Contamination of the sarcoplasmic reticulum fractions by mitochondrial outer membrane was < 0.75% as determined from the specific activity of monoamine oxidase. Thus, only a small part of the porin detected in sarcoplasmic reticulum vesicles can be attributed to mitochondrial contamination. These results show that porin-type1 immunoreactivity is not restricted to mitochondria but found in the sarcoplasmic reticulum and plasmalemma of both mammalian skeletal and cardiac muscle.

Animals↗
PubMed1994

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1.

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC6 (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10-70 nm for MDCK cells and 20-90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.

Animals↗
PubMedDate not supplied

Activation and deactivation of sarcoplasmic reticulum calcium release channels: molecular dissection of mechanisms via novel semi-synthetic ryanoids.

The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium-regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C10-Oeq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel. Binding affinities of these C10-Oeq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (Kd values > 53.9 nM, Kd values 0.3-0.7 nM and Kd values 1.3-20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration that could be tested. Half-maximal concentrations for activation (EC50act values) ranged from 0.87-4.2 microM, compared with an EC50act of 1.3 microM for ryanodine. Using a low concentration (0.5 nM) of a high specific activity, radioiodinated derivative of ryanodine, C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine (1400 Ci/mmol) as the radioligand in displacement binding affinity assays, two distinct, sequential ryanodine binding isotherms were demonstrated within the normal 0-300 nM ryanodine sigmoidal displacement curve. A high affinity site had an IC50 of 0.5 nM (Kd = 0.26 +/- 0.02 nM). Above this concentration, an apparent plateau occurred between 3 and 6 nM ryanodine, and at higher concentrations a lower affinity site was revealed that demonstrated an IC50 of about 25 nM (Kd = 11.7 +/- 1.2 nM). Scatchard analysis from direct binding of C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine to junctional sarcoplasmic reticulum vesicles also suggests the presence of more than one class of binding sites within the nanomolar concentration range. The high affinity site demonstrated a Bmax of 3 pmol/mg protein. We were unable to saturate the lower affinity binding sites with this ligand. To evaluate the functional effects occurring among sarcoplasmic reticulum calcium release channel monomers as a consequence of ryanodine's binding, we utilized a photo-activatable derivative of ryanodine, C10-Oeq N-(4-azido salicyloyl) glycyl ryanodine that demonstrates channel modulating characteristics similar to ryanodine. Covalently labeling the sarcoplasmic reticulum calcium-release channels with this ligand, followed by measurements of rates of calcium efflux and SDS-PAGE of the labeled protein, revealed that deactivation of the sarcoplasmic reticulum calcium release channels of skeletal muscle by this ryanoid occurred at concentrations which apparently produce virtually irreversibly interactions between receptor monomers. This 'polymerization' was indicated by the progressive appearance of two higher molecular weight protein bands on SDS-PAGE, concomitant with progressive decreases in the ryanodine receptor monomer band that runs at an apparent molecular mass of 365 kDa. In summary, we have prepared and utilized novel C10-Oeq ester derivatives of ryanodine and dehydroryanodine in studies aimed at better understanding the molecular basis for the complex biphasic actions of ryanodine on the sarcoplasmic reticulum calcium release channels from rabbit skeletal muscle cells. The described studies presage correlations that may be useful in furthering our understa

Animals↗
PubMed1999

Na(+)-Ca2+ exchange induces low Na+ contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase.

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.

Animals↗
PubMed1981

Enzymatic activity of dystrophic chicken sarcoplasmic reticulum.

We have isolated sarcoplasmic reticulum from normal and dystrophic chicken muscle, using an improved isolation procedure. Dystrophic sarcoplasmic reticulum has a reduced level of calcium-sensitive ATPase activity, phosphoenzyme formation, and steady-state calcium transport. Anion-stimulated calcium transport by dystrophic sarcoplasmic reticulum is also reduced when measured under the proper conditions, and dystrophic sarcoplasmic reticulum shows no alteration in calcium efflux rate. Active calcium phosphate loading of the normal and dystrophic sarcoplasmic reticulum preparations indicates that a reduced percentage jof the dystrophic vesicles are capable of active calcium transport. The loaded dystrophic sarcoplasmic reticulum vesicles exhibit the same relative reductions in enzymatic activity as the starting sarcoplasmic reticulum preparations. However, the enzyme activities of normal and dystrophic sarcoplasmic reticulum are similar in the presence of detergent and exogenous phospholipid. On the basis of these results, we suggest that the lipid microenvironment of the dystrophic enzyme is altered.

Animals↗
PubMed1989

Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum.

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12 E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.

Animals↗
PubMed1985

The effect of hypothyroidism on sarcoplasmic reticulum in fast-twitch muscle of the rat.

The effects of hypothyroidism on the Ca2+-transport capabilities of fast-twitch muscle (m. gastrocnemius) of the rat were studied in whole-muscle homogenate and isolated sarcoplasmic reticulum. Hypothyroidism did not affect the percentage recovery and the vesicle composition of the sarcoplasmic reticulum fraction, the total lipid and phospholipid-to-protein ratios and the protein composition (both qualitative and quantitative). Also the Ca2+-loading capacity of purified sarcoplasmic reticulum, in the presence of oxalate, and the Ca2+ and pH dependence of both the uptake reaction and the coupled ATPase activity were unchanged. However, the homogenate Ca2+-loading capacity and the Ca2+-uptake activity were depressed, as was the yield of purified sarcoplasmic reticulum. The results indicate a 31% reduction of the entire sarcoplasmic reticulum membrane system per volume of muscle. Ca2+/ATP coupling ratios, determined in purified sarcoplasmic reticulum vesicles by measurement of initial rates of net Ca2+ uptake and Ca2+-Mg2+-dependent hydrolysis of ATP, were found to be 1.48 +/- 0.06 and 2.08 +/- 0.05 in the euthyroid and hypothyroid groups, respectively. Identical values were obtained with a recently described Ca2+-pulse method (Meltzer, S. and Berman, M.C. (1984) Anal. Biochem. 138, 458-464), i.e., 1.53 +/- 0.06 and 2.01 +/- 0.03 in the euthyroid and hypothyroid groups, respectively. Passive Ca2+ efflux from sarcoplasmic reticulum was the same in both groups (30 nmol/mg per min), as was the fraction of vesicles that did not show net uptake of Ca2+ (less than 10%), which makes it unlikely that these parameters provide an explanation for the differences in the coupling ratio. The energy of activation of the (Ca2+ + Mg2+)-ATPase was increased in hypothyroidism, which may point to changes in the phospholipid environment of the enzyme. Physiological concentrations of T3 and T4 had no effect on the (Ca2+ + Mg2+)-ATPase in vitro, but all observed changes in the hypothyroid state could be reversed within 14 days by administration of T3 to hypothyroid animals. Approximate calculations indicate that the observed changes in the sarcoplasmic reticulum as a result of thyroid-hormone depletion may contribute significantly to the decrease in relaxation rate and the decrease in energy consumption during contraction.

Animals↗