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At least 19 recordsLinked to original sources
PubMed2000

Maximal inhibition of SERCA2 Ca(2+) affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban.

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca(2+) affinity of the SR Ca(2+)-ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca(2+) uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca(2+) with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (>/=2.6:1) exhibited increases in beta-myosin heavy chain expression, associated with cardiac hypertrophy. These findings demonstrate that overexpression of a non-phosphorylatable form of phospholamban in transgenic mouse hearts resulted in saturation of the functional phospholamban:SERCA2 ratio at 2.6:1 and suggest that approximately 40% of the SR Ca(2+) pumps are functionally regulated by phospholamban in vivo.

Animals↗
PubMed1982

Ca2+/calmodulin-dependent phospholamban kinase from cardiac sarcoplasmic reticulum is distinct from phosphorylase kinase and forms a regulatory complex with phospholamban and the Ca2+-ATPase.

We recently reported that phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, is phosphorylated by both cAMP-dependent protein kinase and a membrane-bound, Ca2+/calmodulin-dependent phospholamban kinase. Phospholamban kinase and glycogen phosphorylase b kinase share the same substrate specificity. They differ however in that phospholamban kinase exhibits an absolute requirement for exogenous calmodulin. In line with the latter observation, phospholamban kinase is shown in this report to be inhibited by fluphenazine. Lower concentrations of the drug induced an activation of the kinase, presumably by hydrophobic interaction with either membrane phospholipids or integral proteins. Also, phospholamban kinase was found to be totally insensitive to antibodies elicited against phosphorylase kinase. Since antipsychotic drugs fail to inhibit the delta-subunit-dependent activity of phosphorylase kinase, the above findings confirm that the two kinases are distinct molecular entities. After detergent solubilization of the sarcoplasmic reticulum, the phospholamban-ATPase complex remains a substrate for phospholamban kinase activity, which retains the ability to catalyze the phosphorylation of exogenous phosphorylase b. However, the Ca2+ dependence is entirely lost upon solubilization and no kinase activity is retained on calmodulin-Sepharose in the presence of Ca2+ ions. Phospholamban and phosphorylase kinase activities copurify with the pump-phospholamban complex upon fractionation of the solubilized proteins by density gradient ultracentrifugation, suggesting a tight interaction between the ATPase, its activator, and the phospholamban kinase. A tentative schematic representation of this supramolecular assembly is based upon the results described in this and preceding papers.

Adenosine Triphosphatases↗
PubMed1992

Molecular mechanism of regulation of Ca2+ pump ATPase by phospholamban in cardiac sarcoplasmic reticulum. Effects of synthetic phospholamban peptides on Ca2+ pump ATPase.

The molecular mechanism of the regulation of Ca2+ pump ATPase by phospholamban in cardiac sarcoplasmic reticulum was examined using synthetic peptides of phospholamban and purified Ca2+ pump ATPase from cardiac sarcoplasmic reticulum. The phospholamban monomer of 52 amino acid residues contains two distinct domains, the cytoplasmic (amino acids 1-30) and the transmembrane (amino acids 31-52) domains. The peptide corresponding to the amino acids 1-31 of phospholamban (PLN 1-31) decreased the Vmax of the Ca(2+)-dependent ATPase activity in dose-dependent manner, while it had no effect on the affinity of the ATPase for Ca2+ (KCa). On the other hand, the peptide corresponding to the amino acids 28-47 of phospholamban (PLN 28-47) increased the KCa from 0.52 to 1.33 microM without significant change in the Vmax value when reconstituted into vesicles with the ATPase. Essentially the same results as PLN 28-47 were obtained with the peptide corresponding to the amino acids 8-47 of phospholamban (PLN 8-47). The inhibitory effects of PLN 1-31 and PLN 8-47 on the ATPase were reversed by cAMP-dependent phosphorylation of the peptides (Ser16). These results indicate that phospholamban suppresses Ca2+ pump ATPase at two different sites, the cytoplasmic domain for Vmax and the transmembrane domain for KCa, and that cAMP-dependent phosphorylation de-suppresses these inhibitory effects on the ATPase.

Amino Acid Sequence↗
PubMed1999

Structure of the 1-36 amino-terminal fragment of human phospholamban by nuclear magnetic resonance and modeling of the phospholamban pentamer.

The structure of a 36-amino-acid-long amino-terminal fragment of phospholamban (phospholamban[1-36]) in aqueous solution containing 30% trifluoroethanol was determined by nuclear magnetic resonance. The peptide, which comprises the cytoplasmic domain and six residues of the transmembrane domain of phospholamban, assumes a conformation characterized by two alpha-helices connected by a turn. The residues of the turn are Ile18, Glu19, Met20, and Pro21, which are adjacent to the two phosphorylation sites Ser16 and Thr17. The proline is in a trans conformation. The helix comprising amino acids 22-36 is well determined (the root mean square deviation for the backbone atoms, calculated for a family of 18 nuclear magnetic resonance structures is 0.57 A). Recently, two molecular models of the transmembrane domain of phospholamban were proposed in which a symmetric homopentamer is composed of a left-handed coiled coil of alpha-helices. The two models differ by the relative orientation of the helices. The model proposed by,Simmerman et al. (H.K. Simmerman, Y.M. Kobayashi, J.M. Autry, and L.R. Jones, 1996, J. Biol. Chem. 271:5941-5946), in which the coiled coil is stabilized by a leucine-isoleucine zipper, is similar to the transmembrane pentamer structure of the cartilage oligomeric membrane protein determined recently by x-ray (V. Malashkevich, R. Kammerer, V Efimov, T. Schulthess, and J. Engel, 1996, Science 274:761-765). In the model proposed by Adams et al. (P.D. Adams, I.T. Arkin, D.M. Engelman, and A.T. Brunger, 1995, Nature Struct. Biol. 2:154-162), the helices in the coiled coil have a different relative orientation, i.e., are rotated clockwise by approximately 50 degrees. It was possible to overlap and connect the structure of phospholamban[1-36] derived in the present study to the two transmembrane pentamer models proposed. In this way two models of the whole phospholamban in its pentameric form were generated. When our structure was connected to the leucine-isoleucine zipper model, the inner side of the cytoplasmic domain of the pentamer (where the helices face one another) was lined by polar residues (Gln23, Gln26, and Asn30), whereas the five Arg25 side chains were on the outer side. On the contrary, when our structure was connected to the other transmembrane model, in the inner side of the cytoplasmic domain of the pentamer, the five Arg25 residues formed a highly charged cluster.

Amino Acid Sequence↗
PubMed1997

Comparison of the effects of phospholamban and jasmone on the calcium pump of cardiac sarcoplasmic reticulum. Evidence for modulation by phospholamban of both Ca2+ affinity and Vmax (Ca) of calcium transport.

Regulation of the calcium pump of the cardiac sarcoplasmic reticulum by phosphorylation/dephosphorylation of phospholamban is central to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. In order to study the mechanism of this regulation, we first obtained purified ruthenium red-insensitive microsomes enriched in sarcoplasmic reticulum membranes. The kinetics of microsomal Ca2+ uptake after phospholamban phosphorylation or trypsin treatment, which cleaves the inhibitory cytoplasmic domain of phospholamban, were then compared with those in the presence of jasmone, whose effects on the kinetics of fast skeletal muscle Ca2+-ATPase are largely known. All three treatments increased Vmax (Ca) at 25 degrees C and millimolar ATP; phosphorylation and trypsin decreased the Km (Ca), while jasmone increased it. Trypsin and jasmone increased the rate of E2P decomposition 1.8- and 3. 0-fold, respectively. The effects of phospholamban phosphorylation and jasmone on the Ca2+-ATPase activity paralleled their effects on Ca2+ uptake. Our data demonstrate that phospholamban regulates E2P decomposition in addition to the known increase in the rate of a conformational change in the Ca2+-ATPase upon binding the first of two Ca2+. These steps in the catalytic cycle of the Ca2+-ATPase may contribute to or account for phospholamban's effects on both Vmax (Ca) and Km (Ca), whose relative magnitude may vary under different experimental and, presumably, physiological conditions.

Adenosine Triphosphate↗
PubMed1986

Stimulation of bovine cardiac sarcoplasmic reticulum Ca2+ pump and blocking of phospholamban phosphorylation and dephosphorylation by a phospholamban monoclonal antibody.

A monoclonal antibody, A1, was produced against sodium dodecyl sulfate-polyacrylamide gel electrophoresis purified canine phospholamban and isolated from mouse ascites by chromatography on a hydroxylapatite column. Western immunoblotting experiments showed that the antibody was specific for phospholamban and cross-reacted with the protein from a bovine source. Incubation of bovine cardiac sarcoplasmic reticulum (SR) vesicles with the antibody resulted in a marked increase in the ATP-dependent Ca2+ pump activity which was slightly higher than that brought about by cyclic AMP-dependent protein kinase. This observation provides direct proof for the involvement of phospholamban as a SR Ca2+ pump regulatory protein. In addition to stimulating the Ca2+ pump activity, antibody A1 was capable of blocking the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase and by an endogenous SR Ca2+/calmodulin-dependent protein kinase. It was also capable of blocking the dephosphorylation of phosphorylated phospholamban by an endogenous SR protein phosphatase. From these observations, it may be suggested that the antigenic site of A1 antibody is proximal to the phosphorylation sites of phospholamban.

Adenosine Triphosphatases↗
PubMed1998

Preservation of the in vivo phosphorylation status of phospholamban in the heart: evidence for a site-specific difference in the dephosphorylation of phospholamban.

The phosphorylation status of the cardiac sarcoplasmic reticular (SR) protein phospholamban determines the activity of the SR Ca(2+)-ATPase. In order to predict SR Ca2+ transport in vivo, it is vital that techniques used to measure the phosphorylation status of phospholamban adequately clamp the endogenous kinases and phosphatases which modify phosphorylation during sample preparation. A recent study (Boateng, S., Seymour, A-M., Dunn, M., Yacoub, M., and Boheler, K. (1997) Biochem. Biophys. Res. Comm. 239, 701-705) has suggested that phosphatase inhibitors must be present in quenching media to prevent almost total dephosphorylation of phospholamban. We addressed this issue by assessing the effect of both kinase and phosphatase inhibition on site-specific phosphorylation of phospholamban in ferret ventricular muscle and isolated rat ventricular myocytes quenched with Laemmli sample buffer. Under these clearly defined quenching conditions in isolated myocytes, we demonstrated that the phosphorylation status of phospholamban was low under basal conditions, and high following exposure to the beta-agonist isoprenaline. The only significant effect of inhibitor inclusion in the quench solution was in isolated myocyte preparations where phosphatase inhibition increased phosphorylation at Ser16 by about a third. The differential effect of phosphatase inclusion on phosphorylation at Ser16 and Thr17 may indicate that different enzymes are involved in dephosphorylation of the two sites.

Amino Acid Sequence↗
PubMed1986

Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure.

Purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles was subjected to proteolysis and peptide mapping to localize the different sites of phosphorylation on the protein and to gain further information on its subunit structure. Five different proteases (trypsin, papain, chymotrypsin, elastase, and Pronase) degraded the oligomeric 27-kDa phosphoprotein into a major 21-22-kDa protease-resistant fragment. No 32P was retained by this protease-resistant fragment, regardless of whether phospholamban had been phosphorylated by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, or protein kinase C. Phosphoamino acid analysis and thin-layer electrophoresis of liberated phosphopeptides revealed that 1 threonine and 2 serine residues were phosphorylated in phospholamban and that 1 of these serine residues and the threonine residue were in close proximity. Only serine was phosphorylated by cAMP-dependent protein kinase, whereas Ca2+-calmodulin-dependent protein kinase phosphorylated exclusively threonine. The results demonstrate that phospholamban has a large protease-resistant domain and a smaller protease-sensitive domain, the latter of which contains all of the sites of phosphorylation. The 21-22-kDa protease-resistant domain, although devoid of incorporated 32P, was completely dissociated into identical lower molecular weight subunits by boiling in sodium dodecyl sulfate, suggesting that this region of the molecule promotes the relatively strong interactions that hold the subunits together. The data presented lend further support for a model of phospholamban structure in which several identical low molecular weight subunits are noncovalently bound to one another, each containing one site of phosphorylation for cAMP-dependent protein kinase and another site of phosphorylation for Ca2+/calmodulin-dependent protein kinase.

Animals↗
PubMed2002

Time course and mechanisms of phosphorylation of phospholamban residues in ischemia-reperfused rat hearts. Dissociation of phospholamban phosphorylation pathways.

Sarcoplasmic reticulum (SR) dysfunction is one of the multiple alterations that occurs in ischemia-reperfused hearts. Because SR function is regulated by phosphorylation of phospholamban (PLB), a SR protein phosphorylated by cAMP-dependent protein kinase (PKA) at Ser(16)and Ca(2+)-calmodulin-dependent protein kinase (CaMKII) at Thr(17), the phosphorylation of these residues during ischemia and reperfusion was examined in Langendorff-perfused rat hearts. Ser(16)phosphorylation increased significantly after 20 min of ischemia from 2.5+/-0.6% to 99.8+/-25.5% of maximal isoproterenol-induced site-specific phosphorylation and decreased to control values immediately after reperfusion. Thr(17)phosphorylation transiently increased at 2-5 min of ischemia and at 1 min of reperfusion (R1, 166.2+/-28.2%). The ischemia-induced increase in Ser(16)phosphorylation was significantly diminished in hearts from catecholamine-depleted animals and/or after beta-blockade and abolished in the presence of the PKA-inhibitor, H-89. Thr(17)phosphorylation at the beginning of ischemia was blunted by nifedipine, whereas at R1 it was significantly diminished by perfusion with 0 m m Ca(2+)in the presence of EGTA and by the Na(+)/Ca(2+)exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit CaMKII, decreased Thr(17)phosphorylation at R1 and significantly prolonged half relaxation time. The results demonstrated a dissociation between the phosphorylation of PLB sites, being phosphorylation of Ser(16)dependent on the beta-adrenergic cascade during ischemia and phosphorylation of Thr(17)on Ca(2+)influx both, at the beginning of ischemia and reperfusion. Phosphorylation of Thr(17)at the onset of reflow may provide the cell a mechanism to cope with Ca(2+)overload, transiently favoring the recovery of relaxation during early reperfusion.

Animals↗
PubMed1986

Concerted phosphorylation of the 26-kilodalton phospholamban oligomer and of the low molecular weight phospholamban subunits.

Phospholamban (PLB) from cardiac sarcoplasmic reticulum (SR) was phosphorylated under various conditions by the adenosine cyclic 3',5'-phosphate (cAMP)-dependent and/or the calmodulin-dependent protein kinase. The small shifts in apparent molecular weight resulting from the incorporation of Pi groups in the PLB complexes were analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In parallel experiments, PLB was dissociated into its subunits and analyzed by using a newly developed isoelectric focusing system. The pI values of the PLB subunits phosphorylated by the cAMP- or calmodulin-dependent kinase were 6.2 and 6.4, respectively. Double phosphorylation of the same subunit resulted in an acidic shift of the pI to 5.2. The combined analysis of the behavior of the PLB complex and of its subunits has greatly simplified the interpretation of the complex phosphorylation pattern and has led to the following conclusions: The PLB complex is composed of five probably identical subunits, each of them containing a distinct phosphorylation site for the calmodulin- and the cAMP-dependent kinase. The population of PLB interacting with the endogenous calmodulin-dependent kinase cannot be phosphorylated by the cAMP-dependent kinase unless previously phosphorylated in the presence of calmodulin. It was also observed that after maximal phosphorylation of PLB in the presence of very large amounts of the cAMP-dependent protein kinase, the Ca2+ pumping rate of the cardiac SR ATPase is stimulated up to 5-fold, i.e., a level of a stimulation which exceeds considerably the values so far reported in the literature.

Adenosine Triphosphatases↗
PubMed1997

Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes.

Protein kinase A- (PKA-) catalyzed phosphorylation of phospholamban (PLN), the protein regulator of the cardiac Ca pump, mediates abbreviation of systole in response to beta-adrenergic agonists. Investigators previously, however, have been unsuccessful in demonstrating an effect of PLN phosphorylation or anti-PLN monoclonal antibody (mAb), which is considered to mimic phosphorylation's well-known effect on Km(Ca), on microsomal Ca uptake at the (high) Ca2+ concentrations found intracellularly at peak systole. We therefore compared the effects of the catalytic subunit of PKA and anti-PLN mAb on the kinetics of Ca uptake in sucrose gradient-purified cardiac microsomes. Both treatments produced a 33-44% increase in Vmax(Ca) at 25 and 37 degrees C, and an 11-31% decrease in Km(Ca) with comparable changes in Ca2+-ATPase activity. An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increased Vmax(Ca) of Ca uptake at 25 degrees C but not at 37 degrees C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from Pi at different temperatures. Our data document almost identical increases in Vmax(Ca) of microsomal Ca uptake with PLN phosphorylation or addition of anti-PLN mAb and hence provide insight into the kinetic mechanism of PLN's regulation of the cardiac sarcoplasmic reticulum Ca pump protein.

Animals↗
PubMed1996

Biochemical and biophysical comparison of native and chemically synthesized phospholamban and a monomeric phospholamban analog.

Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplasmic reticulum using immunoaffinity chromatography and prepared by solid phase peptide synthesis. The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentameric oligomeric states. They are similarly detected on Western blots, are phosphorylation substrates, have identical amino acid compositions that directly reflect their predicted values, yield the same internal amino acid sequences upon CNBr digestion, and have molecular mass values agreeing with the expected value (approximately 6123 Da). Native and synthetic PLB reduced the calcium sensitivity of Ca2+ATPase, which is reversed by anti-PLB antibody. A Cys-to-Ser PLB analog, where the cysteines (36, 41, and 46) were substituted by serines, is monomeric on SDS-polyacrylamide gel electrophoresis, can be phosphorylated, and is recognized by polyclonal antisera. PLB migrates with a sedimentation coefficient of 4.8 S in sedimentation velocity ultracentrifugation experiments, whereas Cys-to-Ser PLB does not sediment, consistent with a monomeric state. Circular dichroism spectral analysis of PLB indicates about 70% alpha-helical structure, whereas Cys-to-Ser PLB manifests only about 30%. Because the physiochemical properties of native and synthetic PLB appear identical, the more readily available synthetic protein should be suitable for more extensive structural studies.

Adenosine Triphosphatases↗
PubMed1999

Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure.

Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by beta-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt-PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25 degrees C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25 degrees C, has no inhibitory activity when assayed at 25 degrees C. However, at 37 degrees C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37 degrees C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase.

Adenosine Triphosphate↗
PubMed2000

Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP.

Previous co-immunoprecipitation studies (Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 32855-32862) revealed that physical interactions between phospholamban (PLN) and the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) were retained, even with PLN monoclonal antibody 1D11 bound to an epitope lying between PLN residues 7 and 17. Because the 1D11 antibody relieves inhibitory interaction between the two proteins, it was of interest to determine whether PLN phosphorylation or elevation of Ca(2+), which also relieves inhibitory interactions between PLN and SERCA, would disrupt physical interactions. Co-immunoprecipitation was measured in the presence of increasing concentrations of Ca(2+) or after phosphorylation of PLN by protein kinase A. Physical interactions were dissociated by elevated Ca(2+) but not by PLN phosphorylation. The addition of ATP enhanced interactions between PLN and SERCA. The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA. These data suggest that physical interactions between PLN and SERCA are stable when SERCA is in the Ca(2+)-free E(2) conformation but not when it is in the E(1) conformation and that phosphorylation of PLN does not dissociate physical interactions between PLN and SERCA.

Adenosine Triphosphate↗
PubMed2003

Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants.

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on beta-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 microM) or the beta-adrenergic blocker dl-propranolol (1 microM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.

Amino Acid Substitution↗
PubMed1999

Phospholamban is present in endothelial cells and modulates endothelium-dependent relaxation. Evidence from phospholamban gene-ablated mice.

Vascular endothelial cells regulate vascular smooth muscle tone through Ca2+-dependent production and release of vasoactive molecules. Phospholamban (PLB) is a 24- to 27-kDa phosphoprotein that modulates activity of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). Expression of PLB is reportedly limited to cardiac, slow-twitch skeletal and smooth muscle in which PLB is an important regulator of [Ca2+]i and contractility in these muscles. In the present study, we report the existence of PLB in the vascular endothelium, a nonmuscle tissue, and provide functional data on PLB regulation of vascular contractility through its actions in the endothelium. Endothelium-dependent relaxation to acetylcholine was attenuated in aorta of PLB-deficient (PLB-KO) mice compared with wild-type (WT) controls. This effect was not due to actions of nitric oxide on the smooth muscle, because sodium nitroprusside-mediated relaxation in either denuded or endothelium-intact aortas was unaffected by PLB ablation. Relative to denuded vessels, relaxation to forskolin was enhanced in WT endothelium-intact aortas. The endothelium-dependent component of this relaxation was attenuated in PLB-KO aortas. To investigate whether these changes were due to PLB, WT mouse aorta endothelial cells were isolated. Both reverse transcriptase-polymerase chain reaction and Western blot analyses revealed the presence of PLB in endothelial cells, which were shown to be >98% pure by diI-acetylated LDL uptake and nuclear counterstaining. These data indicate that PLB is present and modulates vascular function as a result of its actions in endothelial cells. The presence of PLB in endothelial cells opens new fields for investigation of Ca2+ regulatory pathways in nonmuscle cells and for modulation of endothelial-vascular interactions.

Animals↗