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N Narayanan

Publications and source records attributed to N Narayanan.

At least 19 recordsLinked to original sources
PubMed2000

Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration.

Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.

Amino Acid Sequence↗
PubMed2000

Reversible inhibition of the calcium-pumping ATPase in native cardiac sarcoplasmic reticulum by a calmodulin-binding peptide. Evidence for calmodulin-dependent regulation of the V(max) of calcium transport.

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca(2+) cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca(2+) pump (Ca(2+)-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca(2+) uptake, Ca(2+)-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca(2+) uptake and Ca(2+)-ATPase activities of SR (50% inhibition at approximately 2-3 microM CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V(max) of Ca(2+) uptake, unaltered K(0.5) for Ca(2+) activation of Ca(2+) transport, and a 10-fold decrease in the apparent affinity of the Ca(2+)-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca(2+)-binding sites of the Ca(2+)-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca(2+)-ATPase, phospholamban, and ryanodine receptor-Ca(2+) release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca(2+) pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 microM). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca(2+) transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca(2+) pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca(2+) pump function, CaM BP (1-2.5 microM) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM.

Adenosine Triphosphate↗
PubMed2000

Antipyretic, antinociceptive and anti-inflammatory activity of Premna herbacea roots.

The alcoholic extract of the roots of Premna herbacea was investigated for its antipyretic, antinociceptive and anti-inflammatory potential in animal models. The extract, when administered orally to mice has been found to be safe up to a dose of 8.0 g/kg. A significant antipyretic effect has been observed in rabbits while mild antinociceptive effects were evidenced in mice when tested by chemical as well as thermal methods. The extract did not exhibit any anti-inflammatory activity in acute but significantly reduced the chronic inflammation.

Acetic Acid↗
PubMed2000

Thyroid hormone-induced overexpression of functional ryanodine receptors in the rabbit heart.

Modifications in the Ca(2+)-uptake and -release functions of the sarcoplasmic reticulum (SR) may be a major component of the mechanisms underlying thyroid state-dependent alterations in heart rate, myocardial contractility, and metabolism. We investigated the influence of hyperthyroid state on the expression and functional properties of the ryanodine receptor (RyR), a major protein in the junctional SR (JSR), which mediates Ca(2+) release to trigger muscle contraction. Experiments were performed using homogenates and JSR vesicles derived from ventricular myocardium of euthyroid and hyperthyroid rabbits. Hyperthyroidism, with attendant cardiac hypertrophy, was induced by the injection of L-thyroxine (200 microg/kg body wt) daily for 7 days. Western blotting analysis using cardiac RyR-specific antibody revealed a significant increase (>50%) in the relative amount of RyR in the hyperthyroid compared with euthyroid rabbits. Ca(2+)-dependent, high-affinity [(3)H]ryanodine binding was also significantly greater ( approximately 40%) in JSR from hyperthyroid rabbits. The Ca(2+ )sensitivity of [(3)H]ryanodine binding and the dissociation constant for [(3)H]ryanodine did not differ significantly between euthyroid and hyperthyroid hearts. Measurement of Ca(2+)-release rates from passively Ca(2+)-preloaded JSR vesicles and assessment of the effect of RyR-Ca(2+)-release channel (CRC) blockade on active Ca(2+)-uptake rates revealed significantly enhanced (>2-fold) CRC activity in the hyperthyroid, compared with euthyroid, JSR. These results demonstrate overexpression of functional RyR in thyroid hormone-induced cardiac hypertrophy. Relative abundance of RyR may be responsible, in part, for the changes in SR Ca(2+) release, cytosolic Ca(2+) transient, and cardiac systolic function associated with thyroid hormone-induced cardiac hypertrophy.

Animals↗
PubMed1999

Serine phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase in the intact beating rabbit heart.

Recent studies have demonstrated that Ca(2+)/calmodulin-dependent protein kinase phosphorylates the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR) in vitro. Also, evidence from in vitro studies suggested that this phosphorylation, occurring at Ser(38), results in stimulation of Ca(2+) transport. In the present study, we investigated whether serine phosphorylation of the SR Ca(2+)-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing (32)P(i) and contractions were monitored by recording systolic left ventricular pressure development. Following 45-50 min of (32)P perfusion, the hearts were freeze-clamped, SR isolated, and analyzed for protein phosphorylation. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosphorylation of several peptides including the Ca(2+)-ATPase and Ca(2+) release channel (ryanodine receptor). The identity of Ca(2+)-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipitation using a cardiac SR Ca(2+)-ATPase-specific monoclonal antibody. The Ca(2+)-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody indicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca(2+)-ATPase phosphorylation in situ yielded a value of 208 +/- 12 pmol (32)P/mg SR protein which corresponded to the phosphorylation of approximately 20% of the Ca(2+) pump units in the SR membrane. Since this phosphorylation occurred under basal conditions (i.e., in the absence of any inotropic intervention), a considerable steady-state pool of serine-phosphorylated Ca(2+)-ATPase likely exists in the normally beating heart. These findings demonstrate that serine phosphorylation of the Ca(2+)-ATPase is a physiological event which may be important in the regulation of SR function.

Animals↗
PubMed1999

Detection of biomolecules in the near-infrared spectral region via a fiber-optic immunosensor.

The design, development, and application of a fluorescent fiber-optic immunosensor (FFOI) procedure for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region is reported. The technique was developed through the combined use of fiber-optics, semiconductor laser excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI's sensing tip and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize antibody sandwich technique. Three individual immunoassays are reported. The first two assays utilize the FFOI and NN382, a commercial NIR dye, for the detection of human immunoglobulin G (IgG). In these assays, goat anti-human IgG antibody (GAHG) is immobilized on the sensitive terminal of the FFOI followed by the exposure of the antibody-coated terminal to human IgG. The probe is then introduced to GAHG labeled with NN382, generating a signal. The third assay utilizes the FFOI for the detection of trace amounts of Legionella pneumophila serogroup 1 (LPS1). In this assay, rabbit anti-LPS1 antibody is immobilized on the sensitive terminal of the FFOI followed by exposure to LPS1. The antigen-coated probe is then treated with monoclonal anti-LPS1 antibody followed by incubation with GAHG labeled with NN382. The assays are optimized to detect the corresponding antigen via the NIR-FFOI. Typical measurements are performed in 10-15 min. A 780-nm semiconductor laser provides the excitation of the immune complex and the resulting emission is detected by a 820-nm silicon photodiode detector. The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. Solutions of IgG and LPS1 with concentrations as low as 10(-11) M and 0.5 ng/ml, respectively, have been detected with a minimum interference.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

The antifungal antibiotic clotrimazole potently inhibits L-type calcium current in guinea-pig ventricular myocytes.

The antimycotic agent clotrimazole (CLT) is a promising potential therapeutic agent for a variety of diseases including cancer. Although it is known that CLT alters calcium homeostasis in many cell types, its cardiac effects are virtually unknown. We investigated the effects of CLT on L-type calcium current (ICa,L) and action potentials in guinea-pig ventricular myocytes. CLT (5, 25 and 50 microM) inhibited basal ICa,L by 16, 59 and 93%, respectively. The inhibitory effect of CLT was rapid and the peak effect was attained within 3 min. At a concentration of 25 microM, the inhibitory effect of CLT was partially reversible whereas the response to 50 microM CLT persisted following drug withdrawal. CLT abbreviated action potential duration at 50 and 90% of repolarization and suppressed the plateau significantly. These results indicate that CLT may have important cardiac effects at concentrations used to induce the antiproliferative action of the drug.

Action Potentials↗
PubMed1998

Clotrimazole, an antimycotic drug, inhibits the sarcoplasmic reticulum calcium pump and contractile function in heart muscle.

Clotrimazole (CLT), an antimycotic drug, has been shown to inhibit proliferation of normal and cancer cell lines and its systemic use as a new tool in the treatment of proliferative disorders is presently under scrutiny (Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) Nature Med. 1, 534-540). The action of CLT is thought to involve depletion of intracellular Ca2+ stores but the underlying mechanism has not been defined. The present study utilized membrane vesicles of rabbit cardiac sarcoplasmic reticulum (SR) to determine the mechanism by which CLT depletes intracellular Ca2+ stores. The results revealed a strong, concentration-dependent inhibitory action of CLT on the ATP-energized Ca2+ uptake activity of SR (50% inhibition with approximately 35 microM CLT). The inhibition was of rapid onset (manifested in <15 s), and was accompanied by a 7-fold decrease in the apparent affinity of the SR Ca2+-ATPase for Ca2+ and a minor decrement in the enzyme's apparent affinity toward ATP. Exposure of SR to CLT in the absence or presence of Ca2+ resulted in irreversible inhibition of Ca2+ uptake demonstrating that the Ca2+-bound and Ca2+-free conformations of the Ca2+-ATPase are CLT-sensitive. Introduction of CLT to the reaction medium subsequent to induction of enzyme turnover with Ca2+ and ATP resulted in instantaneous cessation of Ca2+ transport indicating that an intermediate enzyme species generated during turnover undergoes rapid inactivation by CLT. The inhibition of Ca2+ uptake by CLT was accompanied by inhibition of Ca2+-stimulated ATP hydrolysis and Ca2+-induced phosphoenzyme intermediate formation from ATP in the ATPase catalytic cycle. Phosphorylation of the Ca2+-deprived enzyme with Pi in the reverse direction of catalytic cycle and Ca2+ release from Ca2+-preloaded SR vesicles were unaffected by CLT. It is concluded that CLT depletes intracellular Ca2+ stores by inhibiting Ca2+ sequestration by the Ca2+-ATPase. The mechanism of ATPase inhibition involves a drug-induced alteration in the Ca2+-binding site(s) resulting in paralysis of the enzyme's catalytic and ion transport cycle. CLT (50 microM) caused marked depression of contractile function in isolated perfused, electrically paced rabbit heart preparations. The contractile function recovered gradually following withdrawal of CLT from the perfusate indicating the existence of mechanisms in the intact cell to inactivate, metabolize, or clear CLT from its target site.

Adenosine Triphosphate↗
PubMed1998

Defibrillation depresses heart sarcoplasmic reticulum calcium pump: a mechanism of postshock dysfunction.

Presently, the only therapy for ventricular fibrillation is delivery of high-voltage shocks. Despite "successful defibrillation," patients may have poor cardiac contractility, the mechanisms of which are unknown. Intracellular Ca2+ handling by the sarcoplasmic reticulum (SR) plays a major role in contractility. We tested the hypothesis that defibrillation shocks interfere with Ca2+ transport function of cardiac SR. Rats anesthetized with pentobarbital sodium had bilateral electrodes implanted subcutaneously for transthoracic shocks. A series of 10 shocks, 10 s apart, at 0-250 V was delivered from a trapezoidal defibrilator. The hearts were rapidly removed, SR-enriched membrane vesicles were isolated, and ATP-dependent Ca2+ uptake and Ca(2+)-stimulated ATP hydrolysis were determined. There was a marked, shock-related decline in Ca2+ uptake, whereas adenosinetriphosphatase activity remained unaltered. The polypeptide compositions were similar in control and shocked SR. In Langendorff hearts, shocks also decreased contractility and slowed relaxation. These data indicate that shocks with current densities similar to defibrillation depress Ca(2+)-pumping function of cardiac SR because of uncoupling of ATP hydrolysis and Ca2+ transport. Shock-induced impairment of Ca2+ pump function may underlie postshock myocardial dysfunction.

Animals↗
PubMed1998

Effects of aging on sarcoplasmic reticulum Ca2+-cycling proteins and their phosphorylation in rat myocardium.

Diminished Ca2+-sequestering activity of the sarcoplasmic reticulum (SR) is implicated in the age-associated slowing of cardiac muscle relaxation. In attempting to further define the underlying mechanisms, the present study investigated the impact of aging on the contents of major SR Ca2+-cycling proteins and SR protein phosphorylation by endogenous Ca2+/calmodulin-dependent protein kinase (CaM kinase). The studies were performed using homogenates and SR vesicles derived from the ventricular myocardium of adult (6-8 mo old) and aged (26-28 mo old) Fischer 344 rats. Western immunoblotting analysis showed no significant age-related difference in the relative amounts of ryanodine receptor-Ca2+-release channel (RyR-CRC), the Ca2+-storage protein calsequestrin, Ca2+-pumping ATPase (Ca2+-ATPase), and Ca2+-ATPase-regulatory protein phospholamban (PLB) in SR or homogenate. On the other hand, the relative amount of immunoreactive CaM kinase II (delta-isoform) was approximately 50% lower in the aged heart. CaM kinase-mediated phosphorylation of RyR-CRC, Ca2+-ATPase, and PLB was reduced significantly ( approximately 25-40%) in the aged compared with adult rat. ATP-dependent Ca2+-uptake activity of SR and the stimulatory effect of calmodulin on Ca2+ uptake were also reduced significantly with aging. Treatment of SR vesicles with anti-PLB antibody (PLBab) invoked relatively less stimulation of Ca2+ uptake in the aged (</=26%) compared with the adult (</=65%) rat. Ca2+-ATPase but not PLB underwent phosphorylation by CaM kinase in PLBab-treated SR with resultant stimulation of Ca2+ uptake. The rates of Ca2+ uptake by PLBab-treated SR were significantly lower (45-55%) in the aged compared with adult rat in the absence and presence of calmodulin. These findings imply that changes in the intrinsic functional properties of SR Ca2+-cycling proteins and/or their phosphorylation-dependent regulation contribute to impaired SR function in the aging heart.

Aging↗
PubMed1997

Ontogeny of sarcoplasmic reticulum protein phosphorylation by Ca2+--calmodulin-dependent protein kinase.

In the adult myocardium the Ca2+ uptake and release functions of the sarcoplasmic reticulum (SR) are known to be regulated by a membrane-associated Ca2+-calmodulin-dependent protein kinase (CaM kinase) which phosphorylates the Ca2+-pumping ATPase (Ca2+ pump), Ca2+ release channel (ryanodine receptor) and the Ca2+ pump-regulatory protein, phospholamban. The role of CaM kinase during development, however, has not been examined previously. The present study investigated the ontogenetic expression of SR-associated CaM kinase in the rabbit myocardium as well as development-related changes in CaM kinase-mediated phosphorylation of the SR proteins (Ca2+ pump, Ca2+ release channel and phospholamban) involved in transmembrane Ca2+ cycling. For these experiments, cardiac muscle homogenate and SR-enriched membrane fraction derived from fetal (21- and 28-days gestation), newborn (2 days postnatal) and adult New Zealand White rabbits were used. Western immunoblotting analysis detected the presence of phospholamban, Ca2+ pump and Ca2+ release channel in homogenate and SR at all ages tested. The amount of these proteins in the SR increased substantially during fetal and postnatal development. Phosphorylation studies revealed the presence of CaM kinase-dependent phosphorylation of the Ca2+ pump, Ca2+ release channel and phospholamban as early as 21-days gestation. This phosphorylation could be elicited with the addition of only Ca2+ and calmodulin indicating the presence of a SR-associated CaM kinase as early as 21-days gestation. This was confirmed using a delta-CaM kinase II-specific antibody. Phosphorylation per unit amount of each substrate was greater in the fetus and newborn compared to adult. Phosphorylation of phospholamban could be elicited by exogenous cAMP-dependent protein kinase (PKA) at all developmental stages studied. Activation of SR CaM kinase with Ca2+ and calmodulin, or induction of phospholamban phosphorylation by exogenous PKA, resulted in stimulation of the Ca2+ uptake activity of SR in fetal, newborn and adult heart. These results demonstrate early ontogenetic expression of the Ca2+ cycling proteins and CaM kinase in the SR and the concurrent development of phosphorylation-dependent regulation of SR Ca2+ cycling.

Animals↗
PubMed1997

Phosphorylation and regulation of the Ca(2+)-pumping ATPase in cardiac sarcoplasmic reticulum by calcium/calmodulin-dependent protein kinase.

In cardiac muscle, a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates the Ca(2+)-pumping ATPase in addition to its previously characterized substrates, phospholamban and Ca(2+)-release channel (ryanodine receptor). The phosphorylated amino acid in the Ca(2+)-ATPase has been identified as serine. Posphorylation of the Ca(2+)-ATPase is rapid and is reversible by a membrane-associated protein phosphatase, Ca(2+)-ATPase purified from cardiac SR underwent phosphorylation by exogenous CaM kinase, and the phosphorylated enzyme displayed twofold greater catalytic activity without alteration in its Ca(2+)-sensitivity. The phosphorylation of the Ca(2+)-ATPase was found to be isoform-specific in that the cardiac and slow-twitch skeletal muscle isoform (SERCA 2), but not the fast-twitch skeletal muscle isoform (SERCA 1), underwent phosphorylation by CaM kinase. Studies using SERCA 1 and SERCA 2 isoforms and their mutants expressed in a heterelogous cell system have resulted in i) confirmation of the isoform specificity of Ca(2+)-ATPase phosphorylation by CaM kinase, ii) identification of Ser38 as the site in SERCA 2 phosphorylated by CaM kinase, and iii) demonstration of phosphorylation-induced increase in Vmax of Ca2+ transport by the SERCA 2 enzyme. These observations suggest that in cardiac and slow-twitch skeletal muscle direct phosphorylation of the SR Ca(2+)-ATPase by the membrane-bound CaM kinase may serve to stimulate Ca2+ sequestration and therefore, the speed of muscle relaxation.

Animals↗
PubMed1996

Divergent effects of ruthenium red and ryanodine on Ca2+/calmodulin-dependent phosphorylation of the Ca2+ release channel (ryanodine receptor) in cardiac sarcoplasmic reticulum.

In cardiac muscle, a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the sarcoplasmic reticulum (SR) is known to phosphorylate the membrane proteins phospholamban, Ca(2+)-ATPase, and Ca(2+)-release channel (ryanodine receptor). Phosphorylation of phospholamban and Ca(2+)-ATPase is recognized to stimulate Ca2+ sequestration by the SR but the functional consequence of Ca2+ channel phosphorylation has not been clearly established. In this study, we investigated the effects of the SR Ca(2+)-release inhibitor, ruthenium red (RR), and the SR Ca(2+)-release activator, ryanodine (at submicromolar concentrations), on CaM kinase-mediated phosphorylation of the Ca(2+)-cycling proteins in rabbit cardiac SR. Incubation of SR with RR (5-30 microM) for 3 min at 37 degrees C resulted in marked (up to 85%) inhibition of Ca2+ channel phosphorylation (50% inhibition with 15 +/- 2 microM RR) by the endogenous membrane-associated CaM kinase. Phosphorylation of the Ca2+ channel by exogenously added multifunctional alpha CaM kinase II was also inhibited similarly by RR. Phosphorylation of the Ca(2+)-ATPase by endogenous and exogenous CaM kinase was inhibited only modestly (25-30%) by RR, and phospholamban phosphorylation was unaffected by RR. The magnitude of RR-induced inhibition of Ca2+ channel phosphorylation did not differ appreciably at saturating or subsaturating concentrations of Ca2+ or calmodulin, and in the absence or presence of protein phosphatase inhibitors. In contrast to the effects of RR, low concentrations of ryanodine (0.25-1 microM) caused significant stimulation (up to approximately 50%) of Ca2+ channel phosphorylation but had no effect on Ca(2+)-ATPase and phospholamban phosphorylation. These findings suggest that interaction of RR with the ryanodine receptor induces a "nonphosphorylatable state" of the Ca(2+)-release channel, likely through a conformational change involving occlusion of the CaM kinase phosphorylation site. On the other hand, ryanodine binding to the receptor may serve to maintain an open, "phosphorylatable state" of the channel.

Animals↗
PubMed1996

Sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle is regulated by a novel pathway.

Coronary artery smooth muscle expresses an alternative splice (SERCA2b) of the sarcoplasmic reticulum (SR) Ca2+ pump gene SERCA2, which is also expressed in cardiac muscle (SERCA2a), but how the activity of this transporter is regulated in the coronary artery is not known. SERCA2a in the cardiac muscle can be regulated via phospholamban or, as recently reported, by a direct phosphorylation of this protein by calmodulin kinase (Xu, A., C. Hawkins, and N. Narayanan. J.Biol. Chem. 268:8394-8397, 1993). Because both SERCA2a and SERCA2b contain this calmodulin kinase phosphorylation site, we examined the effect of endogenous calmodulin kinase phosphorylation of the SR Ca2+ pump in the coronary artery. SR-enriched membranes were isolated from coronary artery smooth muscle and washed in ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to remove bound calmodulin. When these membranes were incubated with MgATP2- in the presence of Ca2+/calmodulin, a 115-kDa protein was phosphorylated. This phosphorylated 115-kDa protein was identified as SERCA2b in Western blots and by immunoprecipitation using a SERCA2-selective antibody. Preincubating the membranes in MgATP2- in the presence of Ca2+/calmodulin stimulated the subsequent Ca2+ uptake in the presence of oxalate plus MgATP2- and azide. The stimulation of Ca2+ uptake was inhibited by including the SR Ca2+ pump inhibitors thapsigargin and cyclopiazonic acid in the Ca2+ uptake medium or by including the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or the calmodulin kinase II peptide fragment 290-309 in the phosphorylation solution. Thus an endogenous calmodulin-dependent kinase phosphorylated SERCA2b and activated it. Phospholamban could not be detected in these membranes in Western blots. Therefore, the regulation of the SR Ca2+ pump activity in coronary artery smooth muscle may involve a direct phosphorylation of the pump protein by an endogenous calmodulin-dependent kinase.

Alternative Splicing↗
PubMed1996

Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat.

The impact of aging on the Ca2+ pump function of skeletal muscle sarcoplasmic reticulum (SR) was investigated using SR-enriched membrane vesicles isolated from the slow-twitch soleus muscle (SM) and the relatively fast-twitch gastrocnemius muscle (GM) isolated from adult (6-8 mo old) and aged (26-28 mo old) Fischer 344 rats. In addition, isometric twitch characteristics of SM and GM were determined in situ in adult and aged rats under anesthesia. The rates of ATP-supported Ca2+ uptake by SM SR was markedly lower ( approximately 50%) in the aged compared with adult at varying Ca2+ (0.11-8.24 microM) concentrations. Kinetic analysis of the data revealed age-associated decrease in maximum activity reached (Vmax) and increase in the concentration of Ca2+ giving half of Vmax. In contrast, no significant age-related difference was observed in ATP-supported Ca2+ uptake activity of GM SR. The Ca(2+)-stimulated adenosinetriphosphatase (ATPase) activities and the amount of Ca(2+)-ATPase protein did not vary significantly with aging in SM or GM SR. Also, no significant age-related difference was observed in the content of the ryanodine receptor (Ca(2+)-release channel) or the Ca2+ binding protein, calsequestrin in SM and GM SR. In isometrically contracting SM, the time to peak force, half-relaxation time, and contraction duration were significantly prolonged in the aged compared with adult, whereas there was no age-related difference in maximum developed force. None of these isometric twitch parameters differed significantly with age in the GM. These results demonstrate that the effects of aging on skeletal muscle contractile properties and SR function are muscle specific. Furthermore, the data strongly suggest that impairment in SR Ca2+ pump function, apparently due to uncoupling of ATP hydrolysis from Ca2+ transport, contributes to the age-associated slowing of relaxation in the soleus muscle.

Aging↗