Control of sarcoplasmic/endoplasmic-reticulum Ca2+ pump expression in cardiac and smooth muscle.
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ABSTRACT:
SUBMITTER: Misquitta CM
PROVIDER: S-EPMC1220039 | biostudies-other | 1999 Feb
REPOSITORIES: biostudies-other
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Project description:The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.
| S-EPMC1149611 | biostudies-other
Project description:cDNA clones coding for the endoplasmic reticulum Ca2+-transport ATPase have been cloned from a pig smooth-muscle cDNA library. The transcripts can be divided into two classes which differ in their 3' ends due to alternative splicing of the primary gene transcript. The class 1 cDNA encodes a protein of 997 amino acids (Mr 110,000). The class 2 protein (1042 amino acids; Mr 115,000) is completely identical to the class 1 protein except that the four C-terminal amino acids of the class 1 protein are replaced in the class 2 protein with a tail of 49 amino acids. Comparison of these sequences with other Ca2+ pump sequences reveals that the class 1 isoform corresponds to the sarcoplasmic reticulum Ca2+ pump of slow-twitch skeletal/cardiac muscle, whereas the class 2 protein corresponds to a Ca2+ pump recently detected in non-muscle tissues.
| S-EPMC1138741 | biostudies-other
Project description:The melastatin transient receptor potential (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. Activation of the channel is Ca(2+)-dependent, but prolonged exposure to high (>1 microM) levels of intracellular Ca(2+) causes rapid (within approximately 2 min) desensitization of TRPM4 currents under conventional whole cell and inside-out patch-clamp conditions. The goal of the present study was to establish a novel method to record sustained TRPM4 currents in smooth muscle cells under near-physiological conditions. Using the amphotericin B-perforated patch-clamp technique, we recorded and characterized sustained (up to 30 min) transient inward cation currents (TICCs) in freshly isolated cerebral artery myocytes. In symmetrical cation solutions, TICCs reversed at 0 mV and had an apparent unitary conductance of 25 pS. Replacement of extracellular Na(+) with the nonpermeable cation N-methyl-d-glucamine abolished the current. TICC activity was attenuated by the TRPM4 blockers fluflenamic acid and 9-phenanthrol. Selective silencing of TRPM4 expression using small interfering RNA diminished TICC activity, suggesting that the molecular identity of the responsible ion channel is TRPM4. We used the perforated patch-clamp method to test the hypothesis that TRPM4 is activated by intracellular Ca(2+) signaling events. We found that TICC activity is independent of Ca(2+) influx and ryanodine receptor activity but is attenuated by sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibition and blockade of inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release from the sarcoplasmic reticulum. Our findings suggest that TRPM4 channels in cerebral artery myocytes are regulated by Ca(2+) release from inositol 1,4,5-trisphosphate receptor on the sarcoplasmic reticulum.
| S-EPMC2928641 | biostudies-literature
Project description:Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.
| S-EPMC1135545 | biostudies-other
Project description:Agonist-stimulated smooth muscle Ca2+ waves regulate blood vessel tone and vasomotion. Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were stimulated by a combination of inositol 1,4,5-trisphosphate- and Ca2+ -induced Ca2+ release from the endo/sarcoplasmic reticulum. Herein, we present the first report of endothelin-1 stimulated waves of Ca2+ depletion from the sarcoplasmic reticulum of vascular smooth muscle cells using a calsequestrin-targeted Ca2+ indicator. Our findings confirm that these waves are due to regenerative Ca2+ -induced Ca2+ release by the receptors for inositol 1,4,5-trisphosphate. Our main new finding is a transient elevation in SR luminal Ca2+ concentration ([Ca2+](SR)) both at the site of wave initiation, just before regenerative Ca2+ release commences, and at the advancing wave front, during propagation. This strongly suggests a role for [Ca2+](SR) in the activation of inositol 1,4,5-trisphosphate receptors during agonist-induced calcium waves. In addition, quantitative analysis of the gradual decrease in the velocity of the depletion wave, observed in the absence of external Ca2+, indicates continuity of the lumen of the sarcoplasmic reticulum network. Finally, our observation that the depletion wave was arrested by the nuclear envelope may have implications for selective Ca2+ signalling.
| S-EPMC3567057 | biostudies-literature
Project description:Nonmuscle cells have almost ubiquitously evolved a mechanism to detect and prevent Ca2+ store depletion-store operated calcium entry. No such mechanism has, as yet, been reported in cardiac myocytes. However, it is conceivable that such a mechanism may play an important role in cardiac Ca2+ homeostasis to ensure the availability of sufficient stored Ca2+ to maintain normal excitation contraction coupling. We present data that confirms the presence of a mechanism that is able to monitor the Ca2+ load of the SR and initiate a signaling process to accelerate Ca2+ uptake by the SR when store depletion is detected. Depletion of SR Ca2+ activates a protein kinase, the principal SR substrate of which is phospholamban. Phosphorylation of this SR protein promotes Ca2+ pump activity and therefore store refilling. Furthermore, a protein kinase activity associated with the SR that is inhibited by Ca2+ ions has been identified. We have measured lumenal [Ca2+] by using a fluorescent Ca2+ indicator and found that by initiating Ca2+ uptake and increasing Ca2+ load, we can inhibit the protein kinase activity associated with the SR. This confirms that a protein kinase, that is regulated by lumenal [Ca2+], has been identified and represents part of a previously unidentified signalling cascade. This local feedback mechanism would allow the myocyte to detect and prevent SR Ca2+ load depletion.
| S-EPMC19055 | biostudies-literature
Project description:?-Adrenergic receptor (?-AR) activation can provoke cardiac arrhythmias mediated by cAMP-dependent alterations of Ca(2+) signaling. However, cAMP can activate both protein kinase A and an exchange protein directly activated by cAMP (Epac), but their functional interaction is unclear. In heart, selective Epac activation can induce potentially arrhythmogenic sarcoplasmic reticulum (SR) Ca(2+) release that involves Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) effects on the ryanodine receptor (RyR).We tested whether physiological ?-AR activation causes Epac-mediated SR Ca(2+) leak and arrhythmias and whether it requires Epac1 versus Epac2, ?(1)-AR versus ?(2)-AR, and CaMKII?-dependent phosphorylation of RyR2-S2814. We used knockout (KO) mice for Epac1, Epac2, or both. All KOs exhibited unaltered basal cardiac function, Ca(2+) handling, and hypertrophy in response to pressure overload. However, SR Ca(2+) leak induced by the specific Epac activator 8-CPT in wild-type mice was abolished in Epac2-KO and double-KO mice but was unaltered in Epac1-KO mice. ?-AR-induced arrhythmias were also less inducible in Epac2-KO versus wild-type mice. ?-AR activation with protein kinase A inhibition mimicked 8-CPT effects on SR Ca(2+) leak and was prevented by blockade of ?(1)-AR but not ?(2)-AR. CaMKII inhibition (KN93) and genetic ablation of either CaMKII? or CaMKII phosphorylation on RyR2-S2814 prevented 8-CPT-induced SR Ca(2+) leak.?(1)-AR activates Epac2 to induce SR Ca(2+) leak via CaMKII?-dependent phosphorylation of RyR2-S2814. This pathway contributes to ?-AR-induced arrhythmias and reduced cardiac function.
| S-EPMC3690126 | biostudies-literature
Project description:RationaleModulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca(2+) homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown.ObjectivesThis study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken.MethodsLung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots.Measurements and main resultsSERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTR(inh)172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-alpha.ConclusionsReduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF.
| S-EPMC2675566 | biostudies-literature
Project description:Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca(2+). A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca(2+) pump and the ryanodine receptor 2, leading to increased Ca(2+) uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca(2+)-release channels and accelerates Ca(2+) reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca(2+) transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca(2+) cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure.
| S-EPMC2769513 | biostudies-literature
Project description:BackgroundMitochondria dynamically buffer cytosolic Ca(2+) in cardiac ventricular cells and this affects the Ca(2+) load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca(2+) releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+)-Ca(2+) exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).ObjectiveTo determine if mitochondrial Ca(2+) (Ca(2+) (m)), cytosolic Ca(2+) (Ca(2+) (c))-SR-Ca(2+) crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.ResultsInhibition of mitochondrial Ca(2+) influx into (Ru360) or Ca(2+) efflux from (CGP-37157) decreased [Ca(2+)](m) to 80 ± 8% control or increased [Ca(2+)](m) to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+) influx or efflux, the SR Ca(2+) load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+) signal were highly correlated with the change in the SR Ca(2+) load (r(2)?=?0.97). Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control) in response to changes in [Ca(2+)](m) were predicted by concurrent changes in LCR period (r(2) = 0.84).ConclusionA change in SANC Ca(2+) (m) flux translates into a change in the AP firing rate by effecting changes in Ca(2+) (c) and SR Ca(2+) loading, which affects the characteristics of spontaneous SR Ca(2+) release.
| S-EPMC3362629 | biostudies-literature