Diabetic alterations in cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban protein expression.

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular Ca2+ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR Ca2+-ATPase and phospholamban (the inhibitor of SR Ca2+-ATPase). The maximal Ca2+ uptake and the affinity of Ca2+-ATPase for Ca2+ were decreased, and exogenous phosphorylation level of phospholamban was higher in streptozotocin-induced diabetic rat SR. Levels of both mRNA and protein of phospholamban were significantly increased in the diabetic hearts, whereas those of SR Ca2+-ATPase were significantly decreased. Consequently, the relative phospholamban/Ca2+-ATPase ratio was 1.88 in the diabetic hearts, and these changes were correlated with changes in the rates of SR Ca2+ uptake. However, phosphatase pretreatment of phospholamban for dephosphorylation of the sites phosphorylated in vivo did not change the levels of subsequent phospholamban phosphorylation in either control or diabetic rat hearts. The above data indicated that the increased phospholamban phosphorylation was not due to autonomic dysfunction but possibly due to increased phospholamban expression. These findings suggest that reduction of the SR Ca2+-ATPase level would contribute to decreased rates of SR Ca2+ uptake and that this function is further impaired by the enhanced inhibition by phospholamban due to its increased expression in the diabetic heart.     

Interactions between Ca2+-ATPase and the pentameric form of phospholamban in two-dimensional co-crystals

Phospholamban (PLB) physically interacts with Ca(2+)-ATPase and regulates contractility of the heart. We have studied this interaction using electron microscopy of large two-dimensional co-crystals of Ca(2+)-ATPase and the I40A mutant of PLB. Crystallization conditions were derived from those previously used for thin, helical crystals, but the addition of a 10-fold higher concentration of magnesium had a dramatic effect on the crystal morphology and packing. Two types of crystals were observed, and were characterized both by standard crystallographic methods and by electron tomography. The two crystal types had the same underlying lattice, which comprised antiparallel dimer ribbons of Ca(2+)-ATPase molecules previously seen in thin, helical crystals, but packed into a novel lattice with p22(1)2(1) symmetry. One crystal type was single-layered, whereas the other was a flattened tube and therefore double-layered. Additional features were observed between the dimer ribbons, which were substantially farther apart than in previous helical crystals. We attributed these additional densities to PLB, and built a three-dimensional model to show potential interactions with Ca(2+)-ATPase. These densities are most consistent with the pentameric form of PLB, despite the use of the presumed monomeric I40A mutant. Furthermore, our results indicate that this pentameric form of PLB is capable of a direct interaction with Ca(2+)-ATPase.     

Ca2+/calmodulin-dependent phosphorylation of the Ca2+-ATPase, uncoupled from phospholamban, stimulates Ca2+-pumping in native cardiac sarcoplasmic reticulum

Recent studies have demonstrated phosphorylation of the cardiac and slow-twitch muscle isoform (SERCA2a) of the sarcoplasmic reticulum (SR) Ca2+-ATPase (at Ser38) by a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase). Analysis of the functional consequence of Ca2+-ATPase phosphorylation in the native SR membranes, however, is complicated by the concurrent phosphorylation of the SR proteins phospholamban (PLN) which stimulates Ca2+ sequestration by the Ca2+-ATPase, and the ryanodine receptor-Ca2+ release channel (RYR-CRC) which likely augments Ca2+ release from the SR. In the present study, we achieved selective phosphorylation of the Ca2+-ATPase by endogenous CaM kinase in isolated rabbit cardiac SR vesicles utilizing a PLN monoclonal antibody (PLN AB) which inhibits PLN phosphorylation, and the RYR-CRC blocking drug, ruthenium red, which inhibits phosphorylation of RYR-CRC. Analysis of the Ca2+ concentration-dependence of ATP-energized Ca2+ uptake by SR showed that endogenous CaM kinase mediated phosphorylation of the Ca2+-ATPase, in the absence of PLN and/or RYR-CRC phosphorylation, results in a significant increase (approximately 50-70%) in the Vmax of Ca2+ sequestration without any change in the k0.5 for Ca2+ activation of the Ca2+ transport rate. On the other hand, treatment of SR with PLN AB (which mimics the effect of PLN phosphorylation by uncoupling Ca2+-ATPase from PLN) resulted in approximately 2-fold decrease in k0.5 for Ca2+ without any change in Vmax of Ca2+ sequestration. These findings suggest that, besides PLN phosphorylation, direct phosphorylation of the Ca2+-ATPase by SR-associated CaM kinase serves to enhance the speed of cardiac muscle relaxation.     

Solid-state NMR reveals structural changes in phospholamban accompanying the functional regulation of Ca2+-ATPase

Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca2+-ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca2+-ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. Novel backbone amide proton-deuterium exchange CP-MAS NMR experiments on the two PLB analogues co-reconstituted with SERCA1 indicated that labeled residues Leu42 and Leu44 were situated well within the membrane interior, whereas Pro21 and Ala24 lie exposed outside the membrane. Internuclear distance measurements on PLB using rotational resonance NMR indicated that the sequences Pro21-Ala24 and Leu42-Leu44 adopt an alpha-helical structure in pure lipid bilayers, which is unchanged in the presence of Ca2+-ATPase. By contrast, rotational echo double resonance (REDOR) NMR experiments revealed that the sequence Ala24-Gln26 switches from an alpha-helix in pure lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca2+-ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins.     

Controlling the inhibition of the sarcoplasmic Ca2+-ATPase by tuning phospholamban structural dynamics

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.     

Heterologous expression of sarcoplasmic reticulum Ca2+-ATPase

In recent years, expression of rabbit sarcoplasmic reticulum (SR) Ca²⁺-ATPase in heterologous systems has been a widely used strategy to study altered enzymes generated by site-directed mutagenesis. Various eukaryotic expression systems have been tested, all of them yielding comparable amounts of recombinant protein. However, the relatively low yield of recombinant protein obtained so far suggests that novel purification techniques will be required to allow further characterization of this enzyme based on direct ligand-binding measurements.     

Ca2+-binding sites and Ca2+-ATPase activity in barley root tip cells

Summary Different cytochemical methods were employed to demonstrate the existence of Ca²⁺-binding sites (Ca²⁺-bs) at the membranes of barley root tip cells, involving addition of CaCl₂ (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca²⁺-dependent ATPase activity. Addition of 10 mM CaCl₂ to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl₂ electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl₂) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca²⁺-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion.     

Ca2+ -dependent interaction of S100A1 with the sarcoplasmic reticulum Ca2+ -ATPase2a and phospholamban in the human heart

The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.     

Differential expression of ectoMg2+-ATPase and ectoCa2+-ATPase activities in human hepatoma cells

A human hepatoma cell line (Li-7A) possesses ectoATPase activity which is activated by either Mg2+ or Ca2+. Both ectoMg2+-ATPase and ectoCa2+-ATPase hydrolyze other nucleoside triphosphates, are inactive with ADP and AMP, and are inhibited by both p-chloromercuriphenyl sulfonate (pCMPS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Different Km values for ATP and pH curves are obtained for ectoMg2+-ATPase and ectoCa2+-ATPase. The specific activities of the two ATPases remain relatively constant through several days of cell growth after an initial decrease. In contrast, the specific activities of the two ATPases, especially the ectoCa2+-ATPase, increases continuously in Li-7A cells cultured in the presence of EGF, cholera toxin, and hydrocortisone. The ATPases of the factor-treated cells are also indiscriminate with respect to nucleoside triphosphate substrates; however, the kinetic constants for substrates are altered when compared to that of the untreated cells. Most strikingly, the sensitivity to inhibitors is greatly reduced. It is concluded that the long-term effect of EGF, cholera toxin, and hydrocortisone on the Li-7A cells is the induction or activation of a new or minor component of the ectoATPases, which is preferentially activated by Ca2+ and insensitive to pCMPS.     

Solid-state NMR measurements of the kinetics of the interaction between phospholamban and Ca2+-ATPase in lipid bilayers

Phospholamban (PLB) is a small transmembrane protein that regulates calcium transport across the sarcoplasmic reticulum (SR) of cardiac cells via a reversible inhibitory interaction with Ca2+-ATPase. In this work solid-state NMR methods have been used to investigate the dynamics of the inhibitory association between PLB and Ca2+-ATPase. Skeletal muscle Ca2+-ATPase was incorporated into phosphatidylcholine membranes together with a ten-fold excess of a null-cysteine mutant of PLB labelled with 13C at Leu-44 in the transmembrane domain ([alpha-13C-L44]AAA-PLB). In these membranes the PLB variant was found to partially inhibit Ca2+-ATPase by reducing the affinity of the enzyme for calcium. Cross-polarization magic angle spinning (CP-MAS) 13C NMR spectra of the membranes exhibited a signature peak from [alpha-13C-L44]AAA-PLB at 56 ppm. Changes in the intensity of the peak were observed at different temperatures, which was diagnostic of direct interaction between [alpha-13C-L44]AAA-PLB and Ca2+-ATPase. Measurements of dipolar couplings between the 13C label and neighbouring protons were analysed to show that the mean residency time for the association of AAA-PLB with Ca2+-ATPase was on the order of 2.5 ms at temperatures between 0 degrees C and 30 degrees C. This new NMR approach will be useful for examining how the association of the two proteins is affected by physiological stimuli such as kinases and the elevation of calcium concentration.     

Comparison of (Ca2+-Mg2+)-ATPase and Ca2+-ATPase in rat cardiac sarcolemma

The calcium dependency of the Ca2+-pump ATPase of rat cardiac sarcolemma was investigated in the presence and absence of EGTA and EDTA in combination with two free Mg2+-ion concentrations. The results showed: that Mg2+-ions are not essential for the turnover of the Ca2+-pump ATPase; that the Ca2+-affinity is regulated by the concentration of the calcium-chelator complex present in the medium; that (Ca2+-Mg2+)-ATPase and Ca2+-ATPase are probably expressions of the same Ca2+-pump ATPase in the plasma membrane of the cell.     

Reduced sarcoplasmic reticulum Ca2+-ATPase activity and dephosphorylated phospholamban contribute to contractile dysfunction in human hibernating myocardium

Human hibernating myocardium (HHM) is characterized by reversible contractile dysfunction during chronic ischemia. A disturbed calcium-homeostasis is a decisive factor for reduced functional capacity in heart diseases. We therefore investigated calcium-handling proteins in HHM. In 12 patients suffering from multi-vessel coronary artery disease and contractile dysfunction with indication for bypass surgery, HHM was detected preoperatively by thallium scintigraphy, radionuclide ventriculography and dobutamine echocardiography. Transmural biopsies of these regions were taken and analyzed by immunohistochemistry and electron microscopy. Furthermore, SR-calcium ATPase (SERCA2a), phospholamban (PLN), the phosphorylated forms of PLN (PLN-Ser16, PLN-Thr17) as well as sodium-calcium exchanger (NCX) and ryanodine receptor (RyR2) were investigated by RT-PCR and Western-blotting. Additionally, SERCA2a activity was measured by an enzyme-coupled assay. In all patients complete functional recovery could be documented 3 months after revascularization by repeating all preoperative investigations. In HHM maximal SERCA2a activity was significantly reduced (HHM: 424.5± 33.9, control: 609.0± 48.5 nmol ATP mg protein⁻¹ min⁻¹, p≤ 0.05), whereas SERCA2a protein levels were unchanged. mRNA levels (HHM: 1.36± 0.08 vs. control: 0.78± 0.04, p≤ 0.05) and protein amount (HHM:1.67± 0.14 vs. control: 1.00± 0.04, p≤ 0.05) of PLN (A1) were increased resulting in an increased PLN:SERCA2a-ratio. PLN-Ser16 (HHM: 0.60± 0.08 vs. control: 1.00± 0.11, p≤ 0.05) and PLN-Thr17 (HHM: 0.63± 0.11 vs. control: 1.00± 0.06, p≤ 0.05) phosphorylation was significantly decreased. RyR2 and NCX showed no significant alteration. In HHM a decreased activity of SERCA2a due to an impaired phosphorylation of PLN contributes to contractile dysfunction. The increase in the relative ratio of PLN/SERCA2a leads to a decreased calcium affinity of SERCA2a.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.     

Ca2+-dependent protein kinase--a modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells

Cross-talk between cAMP and [Ca(2+)](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca(2+) release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340-1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca(2+) clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca(2+)](i) the rate of Ca(2+) clearance, after the removal of the stimulus, was potentiated substantially ( approximately 2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) with La(3+). PMCA activity, when isolated pharmacologically, was also potentiated ( approximately 2-fold) by forskolin. Ca(2+) uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca(2+)-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca(2+)-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca(2+)](i) was elevated by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca(2+) chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca(2+) clearance because of a complex modulation of PMCA activity that involves a Ca(2+)-dependent step. Tight regulation of both Ca(2+) release and Ca(2+) efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca(2+) signaling.     

Lipid-mediated folding/unfolding of phospholamban as a regulatory mechanism for the sarcoplasmic reticulum Ca2+-ATPase

The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca(2+) flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T' state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.     

The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.     

Ca2+-ATPase in mucous and oxyntico-peptic cells of the fowl proventriculus

Summary Calcium adenosine triphosphatase (Ca²⁺-ATPase) was localized by means of histo- and ultracytochemistry in the secretory cells of the proventriculus of the domestic fowl. The mucous cells exhibited plasmalemmal-associated enzyme activity on the external aspect of the basolateral cell membrane. Intracellularly, the luminal aspect of Golgi-membranes and of secretory vesicle membranes reacted positively for Ca²⁺-ATPase activity, as did the apical cytosol and the matrix of lysosomes. Oxyntico-peptic cells were characterized by apical and apico-lateral plasmalemmal activity and by an organelle-associated distributional pattern similar to that in the mucous cells. In addition, Ca²⁺-ATPase was associated either with the matrix of mitochondria or with tubuli of the rough-surfaced endoplasmic reticulum. The results are discussed with respect to messenger and effector functions of calcium in the process of proventricular mucus secretion. In addition, Ca²⁺-ATPase distributional patterns in the oxyntico-peptic cell are related to the unique structure and function of these cells.     

Occlusion of Ca2+ in soluble monomeric sarcoplasmic reticulum Ca2+-ATPase

Sarcoplasmic reticulum Ca2+-ATPase solubilized in monomeric form by nonionic detergent was reacted with CrATP in the presence of 45Ca2+. A Ca2+-occluded complex formed, which was stable during high performance liquid chromatography in the presence of excess non-radioactive Ca2+. The elution position corresponded to monomeric Ca2+-ATPase. It is concluded that a single Ca2+-ATPase polypeptide chain provides the full structural basis for Ca2+ occlusion.     

Slow/cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNAs are expressed in chronically stimulated rabbit fast-twitch muscle

Fast-twitch extensor digitorum longus muscles of the rabbit were subjected to chronic low-frequency stimulation during different time periods. Changes in the relative amounts of mRNAs encoding fast and slow/cardiac Ca2+-ATPase isoforms were assessed through the use of an RNase-protection assay. Stimulation-induced increases in slow cardiac Ca2+-ATPase and phospholamban mRNAs were quantified by mRNA hybridization. Prolonged stimulation resulted in an exchange of the fast with the slow/cardiac Ca2+-ATPase isoform mRNAs. The exchange was complete after 72 d of stimulation as compared with normal slow-twitch soleus muscle. The tissue content of phospholamban mRNA reached levels similar to that found in normal slow-twitch soleus muscle by the same time. The conversion of the sarcoplasmic reticulum coincided with the fast-to-slow troponin C isoform transition, previously investigated in the same muscles.