CaM kinase II activation and phospholamban phosphorylation by SNP in murine gastric antrum smooth muscles

Elevations in the intracellular Ca(2+) concentration activate the serine/threonine protein kinase Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). We tested the hypothesis that increased sarco(endo)plasmic reticulum Ca(2+)-ATPase activity by phospholamban (PLB) phosphorylation contributes to smooth muscle relaxation by elevating the sarcoplasmic reticulum (SR) Ca(2+) load and increasing the frequency of Ca(2+) release events from the SR. We have previously shown that caffeine or sodium nitroprusside (SNP) relaxes murine gastric fundus smooth muscles and increases PLB phosphorylation by CaM kinase II. These findings suggest that an increased SR Ca(2+) load increases the frequency of Ca(2+) transients from the SR and results in PLB phosphorylation by CaM kinase II, contributing to caffeine- or SNP-induced relaxation. The aim of the present study was to investigate the effects of SNP on CaM kinase II and PLB phosphorylation in gastric antrum smooth muscles. SNP or 8-bromo-cGMP decreased the basal tone and amplitudes of spontaneous phasic contractions and activated CaM kinase II. SNP-induced relaxation and CaM kinase II activation were blocked by [1,2,4]oxadizolo-[4,3alpha]quinoxaline-1-one (ODQ) and inhibited by cyclopiazonic acid (CPA) or KN-93. SNP also increased PLBSer(16) and PLBThr(17) phosphorylation. Both PLBSer(16) and Thr(17) phosphorylation were ODQ sensitive. However, only PLBThr(17) phosphorylation was inhibited by CPA or KN-93. These results suggest that CaM kinase II activation and PLB phosphorylation participate in the relaxant effect of SNP on murine gastric antrum smooth muscles through a nitric oxide/guanylyl cyclase/cGMP pathway.     

Roles of CaM kinase II and phospholamban in SNP-induced relaxation of murine gastric fundus smooth muscles

The mechanisms by which nitric oxide (NO) relaxes smooth muscles are unclear. The NO donor sodium nitroprusside (SNP) has been reported to increase the Ca²⁺ release frequency (Ca²⁺ sparks) through ryanodine receptors (RyRs) and activate spontaneous transient outward currents (STOCs), resulting in smooth muscle relaxation. Our findings that caffeine relaxes and hyperpolarizes murine gastric fundus smooth muscles and increases phospholamban (PLB) phosphorylation by Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) suggest that PLB phosphorylation by CaM kinase II participates in smooth muscle relaxation by increasing sarcoplasmic reticulum (SR) Ca²⁺ uptake and the frequencies of SR Ca²⁺ release events and STOCs. Thus, in the present study, we investigated the roles of CaM kinase II and PLB in SNP-induced relaxation of murine gastric fundus smooth muscles. SNP hyperpolarized and relaxed gastric fundus circular smooth muscles and activated CaM kinase II. SNP-induced CaM kinase II activation was prevented by KN-93. Ryanodine, tetracaine, 2-aminoethoxydiphenylborate, and cyclopiazonic acid inhibited SNP-induced fundus smooth muscle relaxation and CaM kinase II activation. The Ca²⁺-activated K⁺ channel blockers iberiotoxin and apamin inhibited SNP-induced hyperpolarization and relaxation. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-]quinoxalin-1-one inhibited SNP-induced relaxation and CaM kinase II activation. The membrane-permeable cGMP analog 8-bromo-cGMP relaxed gastric fundus smooth muscles and activated CaM kinase II. SNP increased phosphorylation of PLB at Ser¹⁶ and Thr¹⁷. Thr¹⁷ phosphorylation of PLB was inhibited by cyclopiazonic acid and KN-93. Ser¹⁶ and Thr¹⁷ phosphorylation of PLB was sensitive to 1H-[1,2,4]oxadiazolo-[4,3-]quinoxalin-1-one. These results demonstrate a novel pathway linking the NO-soluble guanylyl cyclase-cGMP pathway, SR Ca²⁺ release, PLB, and CaM kinase II to relaxation in gastric fundus smooth muscles. calcium signaling; nitric oxide; sodium nitroprusside; calmodulin     

Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the gamma and delta isoforms are expressed in myocytes. Relative gamma and delta message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles.     

Coordinate expression of Ca2+-ATPase slow-twitch isoform and of beta calmodulin-dependent protein kinase in phospholamban-deficient sarcoplasmic reticulum of rabbit masseter muscle

Modulation of sarcoplasmic reticulum (SR) Ca(2+) transport by endogenous calmodulin-dependent protein kinase II (CaM K II) involves covalent changes of regulatory protein phospholamban (PLB), as a common, but not the only mechanism, in limb slow-twitch muscles of certain mammalian species, such as the rabbit. Here, using immunofluorescent techniques in situ, and biochemical and immunological methods on the isolated SR, we have demonstrated that rabbit masseter, a muscle with a distinct embryological origin, lacks PLB. Accommodating embryological heterogeneity in the paradigm of neural-dependent expression of specific isogenes in skeletal muscle fibers, our results provide novel evidence for the differential expression in the SR of 72 kDa beta components of CaM K II, together with the expression of a slow-twitch sarcoendoplasmic reticulum Ca(2+)-ATPase isoform, both in limb muscle and in the masseter.     

CaM kinase II and phospholamban contribute to caffeine-induced relaxation of murine gastric fundus smooth muscle

Caffeine has been shown to increase the Ca²⁺ release frequency (Ca²⁺ sparks) from the sarcoplasmic reticulum (SR) through ryanodine-sensitive stores and relax gastric fundus smooth muscle. Increased Ca²⁺ store refilling increases the frequency of Ca²⁺ release events and store refilling is enhanced by CaM kinase II (CaMKII) phosphorylation of phospholamban (PLB). These findings suggest that transient, localized Ca²⁺ release events from the SR may activate CaMKII and contribute to relaxation by enhancing store refilling due to PLB Thr17 phosphorylation. To investigate this possibility, we examined the effects of caffeine on CaMKII, muscle tone, and PLB phosphorylation in murine gastric fundus smooth muscle. Caffeine (1 mM) hyperpolarized and relaxed murine gastric fundus smooth muscle and activated CaMKII. Ryanodine, tetracaine, or cyclopiazonic acid each prevented CaMKII activation and significantly inhibited caffeine-induced relaxation. The large-conductance Ca²⁺-activated K⁺ channel blocker iberiotoxin, but not apamin, partially inhibited caffeine-induced relaxation. Caffeine-induced CaMKII activation increased PLB Thr17, but not PLB Ser16 phosphorylation. 3-Isobutyl-1-methylxanthine increased PLB Ser16 phosphorylation, but not PLB Thr17 phosphorylation. The CaMKII inhibitor KN-93 inhibited caffeine-induced relaxation and PLB Thr17 phosphorylation. These results show that caffeine-induced CaMKII activation and PLB phosphorylation play a role in the relaxation of gastric fundus smooth muscles. Ca²⁺/CaM-dependent protein kinase II     

Variation of phospholamban in slow-twitch muscle sarcoplasmic reticulum between mammalian species and a link to the substrate specificity of endogenous Ca(2+)-calmodulin-dependent protein kinase

Systematic immunological and biochemical studies indicate that the level of expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase regulatory protein phospholamban (PLB) in mammalian slow-twitch fibers varies from zero, in the rat, to significant levels in the rabbit, and even higher in humans. The lack of PLB expression in the rat, at the mRNA level, is shown to be exclusive to slow-twitch skeletal muscle, and not to be shared by the heart, thus suggesting a tissue-specific, in addition to a species-specific regulation of PLB. A comparison of sucrose density-purified SR of rat and rabbit slow-twitch muscle, with regard to protein compositional and phosphorylation properties, demonstrates that the biodiversity is two-fold, i.e. (a) in PLB membrane density; and (b) in the ability of membrane-bound Ca(2+)-calmodulin (CaM)-dependent protein kinase II to phosphorylate both PLB and SERCA2a (slow-twitch isoform of Ca(2+)-ATPase). The basal phosphorylation state of PLB at Thr-17 in isolated SR vesicles from rabbit slow-twitch muscle, colocalization of CaM K II with PLB and SERCA2a at the same membrane domain, and the divergent subcellular distribution of PKA, taken together, seem to argue for a differential heterogeneity in the regulation of Ca(2+) transport between such muscle and heart muscle.     

Phosphorylation of phospholamban and troponin I in beta-adrenergic-induced acceleration of cardiac relaxation

Activation of cAMP-dependent protein kinase A (PKA) in ventricular myocytes by isoproterenol (Iso) causes phosphorylation of both phospholamban (PLB) and troponin I (TnI) and accelerates relaxation by up to twofold. Because PLB phosphorylation increases sarcoplasmic reticulum (SR) Ca pumping and TnI phosphorylation increases the rate of Ca dissociation from the myofilaments, both factors could contribute to the acceleration of relaxation seen with PKA activation. To compare quantitatively the role of TnI versus PLB phosphorylation, we measured relaxation rates before and after maximal Iso treatment for twitches of matched amplitudes in ventricular myocytes and muscle from wild-type (WT) mice and from mice in which the PLB gene was knocked out (PLB-KO). Because Iso increases contractions, even in the PLB-KO mouse, extracellular [Ca] or sarcomere length was adjusted to obtain matching twitch amplitudes (in the presence and absence of Iso). In PLB-KO myocytes and muscles (which were allowed to shorten), Iso did not alter the time constant (tau) of relaxation ( approximately 29 ms). However, with increasing isometric force development in the PLB-KO muscles, Iso progressively but modestly accelerated relaxation (by 17%). These results contrast with WT myocytes and muscles where Iso greatly reduced tau of cell relaxation and intracellular Ca concentration decline (by 30-50%), independent of mechanical load. The Iso treatment used produced comparable increases in phosphorylation of TnI and PLB in WT. We conclude that the effect of beta-adrenergic activation on relaxation is mediated entirely by PLB phosphorylation in the absence of external load. However, TnI phosphorylation could contribute up to 14-18% of this lusitropic effect in the WT mouse during maximal isometric contractions.     

Role of phospholamban in the modulation of arterial Ca(2+) sparks and Ca(2+)-activated K(+) channels by cAMP

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR)Ca²⁺-ATPase, and this inhibition is relieved bycAMP-dependent protein kinase (PKA)-mediated phosphorylation. The roleof PLB in regulating Ca²⁺ release throughryanodine-sensitive Ca²⁺ release channels, measured asCa²⁺ sparks, was examined using smooth muscle cells ofcerebral arteries from PLB-deficient ("knockout") mice(PLB-KO). Ca²⁺ sparks were monitored opticallyusing the fluorescent Ca²⁺ indicator fluo 3 or electricallyby measuring transient large-conductance Ca²⁺-activatedK⁺ (BK) channel currents activated by Ca²⁺sparks. Basal Ca²⁺ spark and transient BK current frequencywere elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, anactivator of adenylyl cyclase, increased the frequency ofCa²⁺ sparks and transient BK currents in cerebral arteriesfrom control mice. However, forskolin had little effect on thefrequency of Ca²⁺ sparks and transient BK currents fromPLB-KO cerebral arteries. Forskolin or PLB-KO increased SRCa²⁺ load, as measured by caffeine-induced Ca²⁺transients. This study provides the first evidence that PLB is criticalfor frequency modulation of Ca²⁺ sparks and associated BKcurrents by PKA in smooth muscle.

     

Dynamic regulation of sarcoplasmic reticulum Ca(2+) content and release by luminal Ca(2+)-sensitive leak in rat ventricular myocytes.

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.     

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.     

Phospholamban decreases the energetic efficiency of the sarcoplasmic reticulum Ca pump

We tested the hypothesis that increased Sarcoplasmic reticulum (SR) Ca content ([Ca](SRT)) in phospholamban knockout mice (PLB-KO) is because of increased SR Ca pump efficiency defined by the steady-state SR [Ca] gradient. The time course of thapsigargin-sensitive ATP-dependent (45)Ca influx into and efflux out of cardiac SR vesicles from PLB-KO and wild-type (WT) mice was measured at 100 nm free [Ca]. We found that PLB decreased the initial SR Ca uptake rate (0.13 versus 0.31 nmol/mg/s) and decreased steady-state (45)Ca content (0.9 versus 4.1 nmol/mg protein). Furthermore, at similar total SR [Ca], the pump-mediated Ca efflux rate was higher in WT (0.065 versus 0.037 nmol/mg/s). The pump-independent leak rate constant (k(leak)) was also measured at 100 nm free [Ca]. The results indicate that k(leak) was < 1% of pump-mediated backflux and was not different among nonpentameric mutant PLB (PLB-C41F), WT pentameric PLB (same expression level), and PLB-KO. Therefore differences in passive SR Ca leak cannot be the cause of the higher thapsigargin-sensitive Ca efflux from the WT membranes. We conclude that the decreased total SR [Ca] in WT mice is caused by decreased SR Ca influx rate, an increased Ca-pump backflux, and unaltered leak. Based upon both thermodynamic and kinetic analysis, we conclude that PLB decreases the energetic efficiency of the SR Ca pump.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

Differences in calmodulin and calmodulin-binding proteins in phasic and tonic smooth muscles

To determine whether densities ofcalmodulin (CaM) and CaM-binding proteins are related to phasic andtonic behavior of smooth muscles, we quantified these proteins in theopossum esophageal body (EB) and lower esophageal sphincter (LES),which represent phasic and tonic smooth muscles, respectively. Gelelectrophoresis, immunoprecipitation, Western blot, and hemagglutininepitope-tagged CaM (HA-CaM) overlay assay with quantitative scanningdensitometry and phosphorylation measurements were used. Total proteincontent in the two smooth muscles was similar (~30 mg protein/gfrozen tissue). Total tissue concentration of CaM was significantly(25%) higher in EB than in LES (P < 0.05).HA-CaM-binding proteins were qualitatively similar in LES and EBextracts. Myosin, myristoylated alanine-rich C kinase substrateprotein, Ca²⁺/CaM kinase II, and calponin contents werealso similar in the two muscles. However, content and total activity ofmyosin light chain kinase (MLCK) and content of caldesmon (CaD) werethree- to fourfold higher in EB than in LES. Increased CaM and MLCKcontent may allow for a wide range of contractile force varying fromcomplete relaxation in the basal state to a large-amplitude,high-velocity contraction in EB phasic muscle. Increased content ofCaD, which provides a braking mechanism on contraction, may furthercontribute to the phasic contractile behavior. In contrast, low CaM,MLCK, and CaD content may be responsible for a small range ofcontractile force seen in tonic muscle of LES.

     

PKC-alpha regulates cardiac contractility and propensity toward heart failure

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.     

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca2+) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg(-1) i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca2+-uptake activity in diabetic animals. The reduction in SR Ca2+-uptake was consistent with a significant decrease in the level of SR Ca2+-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca2+-uptake was also due to a reduction in the phosphorylation of PLB by the Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca2+-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart.     

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.     

alphaKAP is an anchoring protein for a novel CaM kinase II isoform in skeletal muscle.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is present in a membrane-bound form that phosphorylates synapsin I on neuronal synaptic vesicles and the ryanodine receptor at skeletal muscle sarcoplasmic reticulum (SR), but it is unclear how this soluble enzyme is targeted to membranes. We demonstrate that alphaKAP, a non-kinase protein encoded by a gene within the gene of alpha-CaM kinase II, can target the CaM kinase II holoenzyme to the SR membrane. Our results indicate that alphaKAP (i) is anchored to the membrane via its N-terminal hydrophobic domain, (ii) can co-assemble with catalytically competent CaM kinase II isoforms and target them to the membrane regardless of their state of activation, and (iii) is co-localized and associated with rat skeletal muscle CaM kinase II in vivo. alphaKAP is therefore the first demonstrated anchoring protein for CaM kinase II. CaM kinase II assembled with alphaKAP retains normal enzymatic activity and the ability to become Ca2+-independent following autophosphorylation. A new variant of beta-CaM kinase II, termed betaM-CaM kinase II, is one of the predominant CaM kinase II isoforms associated with alphaKAP in skeletal muscle SR.     

Role of phospholamban in cyclic GMP mediated signaling in cardiac myocytes.

We tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes were mediated through phospholamban (PLB) and activation of sarcoplasmic reticulum Ca(2+)-ATPase. Using ventricular myocytes from wild type (WT, n=10) and PLB knockout (PLB-KO, n=10) mouse hearts, functional changes were measured using a video edge detector at baseline and after 10(-6), 10(-5)M 8-bromo-cyclic GMP (cGMP), 10(-8), 10(-7)M C-type natriuretic peptide (CNP), or 10(-6), 10(-5)M S-nitroso-N-acetyl-penicillamine (SNAP, nitric oxide donor). Changes in cytosolic Ca(2+) concentration were assessed in fura 2-loaded WT and PLB-KO myocytes. Cyclic GMP dependent phosphorylation analysis was also performed in WT and PLB-KO myocytes. 8-bromo-cGMP 10(-5)M caused a significant decrease in %shortening (3.6+/-0.2% to 2.3+/-0.1%) in WT, but little change in PLB-KO myocytes (3.4+/-0.1% to 3.2+/-0.2%). Similarly, CNP and SNAP reduced %shortening of WT, but not PLB-KO myocyte. Changes in other contractile parameters such as maximum rate of shortening and relaxation were consistent with the changes in % shortening. Intracellular Ca(2+) transients changed similarly to cell contractility in WT and PLB-KO myocytes treated with cGMP and CNP; i.e. Ca(2+) transients decreased with cGMP or CNP in WT myocytes, but were unchanged in PLB-KO myocytes. cGMP dependent phosphorylation analysis showed that some proteins were phosphorylated by cGMP to a lesser extent in PLB-KO compared with WT myocytes, suggesting impaired cGMP-kinase function in PLB-KO cardiac myocytes. These results indicated that cGMP-induced reductions in cardiac myocyte function were at least partially mediated through the action of phospholamban.     

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.