Expression of endoplasmic-reticulum Ca2(+)-pump isoforms and of phospholamban in pig smooth-muscle tissues.

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.     

Isoform switching of the sarco(endo)plasmic reticulum Ca2+ pump during differentiation of BC3H1 myoblasts

We have studied the expression of the gene 2 for the sarco(endo)plasmic reticulum Ca2+ pump (SERCA2) in BC3H1 cells. Myogenic differentiation not only activated the SERCA2 expression but it also induced an isoform switch. Undifferentiated myoblasts only expressed the SERCA2b isoform (non-muscle) whereas differentiated myocytes predominantly contained the SERCA2a isoform (cardiac/slow skeletal muscle). The isoform switch was documented by immunoblot analysis with isoform-specific antibodies. This observation was confirmed at the mRNA level by using antisense RNA probes specific for class 1 (SERCA2a) or class 2 (SERCA2b) messengers. The expression of the SERCA2a isoform after differentiation was accompanied by a decreased sensitivity of the Ca2+ uptake in permeabilized cells to the Ca2+ pump inhibitor thapsigargin.     

(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles

We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle.     

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.     

The sarcoplasmic reticulum Ca(2+)-ATPase, SERCA1a, contains endoplasmic reticulum targeting information.

The fast-twitch skeletal muscle Ca(2+)-ATPase isoenzyme, SERCA1a, is localized in chick skeletal myotubes to both the sarcoplasmic reticulum (SR) and to the nuclear envelope, an extension of the endoplasmic reticulum (ER). The ER labeling remained after cycloheximide treatment, indicating that it did not represent newly synthesized SERCA1a in transit to the SR. Expression of the cDNA encoding SERCA1a in cultured non-muscle cells led to the localization of the enzyme in the ER, as indicated by organelle morphology and the co-localization of SERCA1a with the endogenous ER luminal protein, BiP. Immunopurification analysis showed that SERCA1a was not bound to BiP, nor was any degradation apparent. Thus, the SR Ca(2+)-ATPase appears to contain ER targeting information.     

Characterization and expression of the rat heart sarcoplasmic reticulum Ca2+-ATPase mRNA

Sarcoplasmic reticulum Ca2+-ATPase cDNA clones have been isolated from an adult rat heart cDNA library and the nucleotide sequence of the Ca2+-ATPase mRNA determined. The sequence has an open reading frame of 997 codons. It is identical to a cDNA isolated from a rat stomach cDNA library and 90% isologous to the rabbit and human slow/cardiac cDNAs. Nuclease S1 mapping analysis indicates that this sequence corresponds to the main Ca2+-ATPase mRNA present in heart and in slow skeletal muscle and that it is expressed in various proportions in smooth and non-muscle tissues, together with another isoform which differs from the cardiac form in the sequence of its 3'-end.     

Direct measurement of Ca2+ uptake and release by the sarcoplasmic reticulum of saponin permeabilized isolated smooth muscle cells

To make direct measurements of Ca2+ uptake and release by the sarcoplasmic reticulum (SR) of isolated smooth muscle cells, a fluorometric method for monitoring Ca2+ uptake by striated muscle SR vesicles (Kargacin, M.E., C.R. Scheid, and T.W. Honeyman. 1988. American Journal of Physiology. 245:C694-C698) was modified. With the method, it was possible to make continuous measurements of SR function in saponin-skinned smooth muscle cells in suspension. Calcium uptake by the SR was inhibited by thapsigargin and sequestered Ca2+ could be released by Br-A23187 and thapsigargin. From the rate of Ca2+ uptake by the skinned cells and the density of cells in suspension, it was possible to calculate the Ca2+ uptake rate for the SR of a single cell. Our results indicate that the SR Ca2+ pump in smooth muscle cells can remove Ca2+ at a rate that is 45-75% of the rate at which Ca2+ is removed from the cytoplasm of intact cells during transient Ca2+ signals. From estimates of SR volume reported by others and our measurements of the amount of Ca2+ taken up by the skinned cells, we conclude that the SR of a single cell can store greater than 10 times the amount of Ca2+ needed to elicit a single transient contractile response.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+ pump by phospholamban in reconstituted phospholipid vesicles

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban, and phosphorylation may relieve this inhibition. Fast-twitch skeletal muscle SR does not contain phospholamban, and it is not known whether the Ca(2+)-ATPase isoform from this muscle may be also subject to regulation by phospholamban in a similar manner as the cardiac isoform. To determine this we reconstituted the skeletal isoform of the SR Ca(2+)-ATPase with phospholamban in phosphatidylcholine proteoliposomes. Inclusion of phospholamban was associated with significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0, and phosphorylation of phospholamban by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects on the Ca2+ pump. Similar effects of phospholamban were also observed using phosphatidylcholine:phosphatidylserine proteoliposomes, in which the Ca2+ pump was activated by the negatively charged phospholipids (24). Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by cross-linking experiments, using a synthetic peptide that corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban, although this regulator is not expressed in fast-twitch skeletal muscles.     

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.     

Structure, function and subcellular localization of a human platelet Ca2+-ATPase

Human platelets contain a Ca2+-ATPase in internal membranes that is essential for Ca2+ homeostasis. This Ca2+ pump has enzymatic properties quite similar to the sarcoplasmic reticulum (SR) Ca2+ pumps. Antibodies against the SR Ca2+ pump crossreact with the human platelet protein. However, the platelet Ca2+-ATPase is approximately 10 kD larger than the SR pumps and exhibits a larger mRNA coding for the protein in a megakaryocyte tumor cell line. In addition, the platelet Ca2+-pump may be localized in specialized internal membrane structures that function in Ca2+ uptake and release. These results suggest that the platelet Ca2+-ATPase may represent a new class of internal membrane Ca2+-pumps.     

Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca2+ pump of bovine pulmonary artery

This paper describes the stimulation by cyclic nucleotide dependent protein kinases on the Ca2+ uptake by isolated endoplasmic reticulum (ER) vesicles from the bovine main pulmonary artery. This ER fraction has previously been shown to be highly enriched in phospholamban, a protein kinase substrate that has been well characterized in cardiac sarcoplasmic reticulum (SR), where its phosphorylation is accompanied by an increased rate of Ca2+ uptake. As previously observed for the phosphorylation of phospholamban, the stimulation of the rate of Ca uptake was as high with cGMP dependent protein kinase as with cAMP dependent protein kinase. The effect of phosphorylation of the ER membranes from smooth muscle on the Ca2+ uptake was smaller than that seen in cardiac SR, and it was only observed if albumin was included during the isolation of the membranes. This relatively small effect is probably not due to a lower ratio of phospholamban to Ca2(+)-transport enzyme in the ER membranes as compared to cardiac SR. Several alternative explanations are discussed.     

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.     

Drug action of thapsigargin on the Ca2+ pump protein of sarcoplasmic reticulum.

Thapsigargin is found to be a potent inhibitor of the intracellular Ca2+ pump proteins from skeletal muscle sarcoplasmic reticulum (SR), cardiac SR, and brain microsomes. For skeletal muscle SR, the molar ratio of thapsigargin to Ca2+ pump protein for complete inhibition (MRc) of the Ca2+ loading rate, Ca(2+)-dependent ATPase activity, and formation of phosphorylated intermediate (EP) was approximately 1. When the Ca2+ pump protein of low affinity to Ca2+ (E2 state) was pretreated with thapsigargin, ATP and Ca2+ binding to the Ca2+ pump protein was completely inhibited. In the presence of Ca2+ (E1 state), Ca2+ pump protein was protected from inactivation by thapsigargin with respect to Ca2+ binding and EP formation. The MRc for brain microsomes, which mediate Ca2+ uptake into intracellular (inositol 1,4,5-trisphosphate-releasable) Ca2+ pools, is likewise stoichiometric. Approximately 30% of Ca2+ loading activity of brain microsomes was insensitive to thapsigargin, indicating the presence of other Ca2+ pumping system(s). The MRc for heart is 3.8, indicating that the Ca2+ pump of cardiac SR is less sensitive to thapsigargin. Phosphorylation of cardiac SR with protein kinase A increased the sensitivity to thapsigargin to MRc of 2.8. In summary, we find that: 1) thapsigargin is the most effective inhibitor of the Ca2+ pump protein of intracellular membranes (SR and endoplasmic reticulum); 2) its primary inhibitory action appears to inactivate the E2 form of the enzyme preferentially; 3) cardiac SR shows lesser sensitivity to thapsigargin than skeletal muscle SR and brain microsomes; protein kinase A treatment of cardiac SR enhances the sensitivity to the drug.     

Molecular cloning and sequencing of the plasma-membrane Ca2+ pump of pig smooth muscle.

cDNAs coding for the plasma-membrane Ca2+ pump have been isolated from a pig smooth-muscle cDNA library and sequenced. The open reading frame encodes a protein of 1220 amino acids, which corresponds to the one already described in a human teratoma cell line. We demonstrate here that this cDNA probably represents the only isoform of the plasma-membrane Ca2(+)-transport ATPase expressed in this smooth muscle. There is no evidence for the expression of any other plasma-membrane Ca2(+)-pump gene, or for the presence of other alternatively spliced isoforms. These results are in apparent contradiction to those obtained on protein levels which demonstrate the reaction of at least two different polypeptides with a panel of antibodies against the plasma-membrane ATPase. It is suggested that these two polypeptides could result from a post-translational modification of one single enzyme.     

Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.