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1.
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  相似文献   

2.
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.  相似文献   

3.
Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and this inhibition is relieved by Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca(2+) release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular Ca(2+) waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, but the effects were less pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular Ca(2+) wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that PLB is an important modulator of gastric antrum smooth muscle contractility by modulation of SR Ca(2+) release and CaM kinase II activity.  相似文献   

4.
Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser16 by PKA and/or at Thr17 by CaMKII increases the affinity of the SR Ca2+ pump for Ca2+. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr17 PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser16 and Thr17 residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr17 phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr17 phosphorylation was dependent on Ca2+ entry to the cardiac cell. This Ca2+ influx would mainly occur by the coupled activation of the Na+/H+ exchanger and the Na+/Ca2+ exchanger working in the reverse mode, since phosphorylation of Thr17 was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca2+ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr17 phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr17 of PLB upon reflow, may favor the full recovery of relaxation after ischemia. (Mol Cell Biochem 263: 131–136, 2004)  相似文献   

5.
In vascular smooth muscle (VSM) and manyother cells, G protein receptor-coupled activation of mitogen-activatedprotein kinases has been linked, in part, to increases in freeintracellular Ca2+. Previously, we demonstrated thationomycin-, angiotensin II-, and thrombin-induced activation ofextracellular signal-regulated kinase (ERK)1/2 in VSM cells wasattenuated by pretreatment with KN-93, a selective inhibitor of themultifunctional Ca2+/calmodulin-dependent protein kinase(CaM kinase II). In the present study, we show that theCa2+-dependent pathway leading to activation of ERK1/2 ispreceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation andis attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibitsCa2+-dependent PYK2 activation and EGF receptor tyrosinephosphorylation in response to ionomycin, ATP, and platelet-derivedgrowth factor but has no effect on phorbol 12,13-dibutyrate- orEGF-induced responses. The results implicate CaM kinase II as anintermediate in the Ca2+/calmodulin-dependent activation of PYK2.

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6.
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.

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7.
This study investigated the effects of L-thyroxine-induced hyperthyroidism on Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca2+ pump (Ca2+-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca2+-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30–50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 (150%) Ca2+ pump isoform, unaltered levels of SERCA2 Ca2+ pump isoform, and lower levels of PLN (50%) and -, -, and -CaM kinase II (40 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca2+ uptake and Ca2+-stimulated ATPase activities compared with that from euthyroid control. The Vmax of Ca2+ uptake (in nmol Ca2+·mg SR protein–1·min–1: euthyroid, 818 ± 73; hyperthyroid, 1,649 ± 90) but not the apparent affinity of the Ca2+-ATPase for Ca2+ (euthyroid, 0.97 ± 0.02 µM, hyperthyroid, 1.09 ± 0.04 µM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca2+ uptake by soleus muscle SR was 60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater (36%), and the time to peak force and relaxation time were significantly lower (30–40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca2+ pump. calmodulin kinase II; phospholamban ; calcium ion-adenosinetriphosphatase; sarcoplasmic reticulum  相似文献   

8.
Ca+/calmodulin-dependent protein kinase II(CaM kinase II) has been implicated in the regulation of smooth musclecontractility. The goals of this study were to determine: 1) towhat extent CaM kinase II is activated by contractile stimuli in intactarterial smooth muscle, and 2) the effect of a CaM kinase IIinhibitor (KN-93) on CaM kinase II activation, phosphorylation ofmyosin regulatory light chains (MLC20), and force. Bothhistamine (1 µM) and KCl depolarization activated CaM kinase II witha time course preceding maximal force development, and suprabasal CaM kinase II activation was sustained during tonic contractions. CaMkinase II activation was inhibited by KN-93 pretreatment(IC50 ~1 µM). KN-93 inhibited histamine-induced tonicforce maintenance, whereas early force development andMLC20 phosphorylation responses during the entire timecourse were unaffected. Both force development and maintenance inresponse to KCl were inhibited by KN-93. Rapid increases in KCl-inducedMLC20 phosphorylation were also inhibited by KN-93, whereassteady-state MLC20 phosphorylation responses wereunaffected. In contrast, phorbol 12,13-dibutyrate (PDBu) did notactivate CaM kinase II and PDBu-stimulated force development wasunaffected by KN-93. Thus KN-93 appears to target a step(s) essentialfor force maintenance in response to physiological stimuli, suggestinga role for CaM kinase II in regulating tonic contractile responses inarterial smooth muscle. Pharmacological activation of protein kinase Cbypasses the KN-93 sensitive step.

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9.
Although dilated cardiomyopathy (DCM) is known to result in cardiac contractile dysfunction, the underlying mechanisms are unclear. The sarcoplasmic reticulum (SR) is the main regulator of intracellular Ca2+ required for cardiac contraction and relaxation. We therefore hypothesized that abnormalities in both SR function and regulation will contribute to cardiac contractile dysfunction of the J2N-k cardiomyopathic hamster, an appropriate model of DCM. Echocardiographic assessment indicated contractile dysfunction, because the ejection fraction, fractional shortening, cardiac output, and heart rate were all significantly reduced in J2N-k hamsters compared with controls. Depressed cardiac function was associated with decreased cardiac SR Ca2+ uptake in the cardiomyopathic hamsters. Reduced SR Ca2+ uptake could be further linked to a decrease in the expression of the SR Ca2+-ATPase and cAMP-dependent protein kinase (PKA)-mediated phospholamban (PLB) phosphorylation at serine-16. Depressed PLB phosphorylation was paralleled with a reduction in the activity of SR-associated PKA, as well as an elevation in protein phosphatase activity in J2N-k hamster. The results of this study suggest that an alteration in SR function and its regulation contribute to cardiac contractile dysfunction in the J2N-k cardiomyopathic hamster. sarcoplasmic reticulum; cardiomyopathy; cAMP-dependent protein kinase; Ca2+/calmodulin-dependent protein kinase; sarco(endo)plasmic reticulum ATPase; phospholamban  相似文献   

10.
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.

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11.
Although the sarcoplasmic reticulum (SR) is known to regulatethe intracellular concentration ofCa2+ and the SR function has beenshown to become abnormal during ischemia-reperfusion in theheart, the mechanisms for this defect are not fully understood. Becausephosphorylation of SR proteins plays a crucial role in the regulationof SR function, we investigated the status of endogenousCa2+/calmodulin-dependent proteinkinase (CaMK) and exogenous cAMP-dependent protein kinase (PKA)phosphorylation of the SR proteins in control, ischemic (I), andischemia-reperfused (I/R) hearts treated or not treated withsuperoxide dismutase (SOD) plus catalase (CAT). SR and cytosolicfractions were isolated from control, I, and I/R hearts treated or nottreated with SOD plus CAT, and the SR protein phosphorylation by CaMKand PKA, the CaMK- and PKA-stimulated Ca2+ uptake, and the CaMK, PKA,and phosphatase activities were studied. The SR CaMK andCaMK-stimulated Ca2+ uptakeactivities, as well as CaMK phosphorylation ofCa2+ pump ATPase (SERCA2a) andphospholamban (PLB), were significantly decreased in both I and I/Rhearts. The PKA phosphorylation of PLB and PKA-stimulatedCa2+ uptake were reducedsignificantly in the I/R hearts only. Cytosolic CaMK and PKA activitieswere unaltered, whereas SR phosphatase activity in the I and I/R heartswas depressed. SOD plus CAT treatment prevented the observedalterations in SR CaMK and phosphatase activities, CaMK and PKAphosphorylations, and CaMK- and PKA-stimulated Ca2+ uptake. These resultsindicate that depressed CaMK phosphorylation and CaMK-stimulatedCa2+ uptake in I/R hearts may bedue to a depression in the SR CaMK activity. Furthermore, prevention ofthe I/R-induced alterations in SR protein phosphorylation by SOD plusCAT treatment is consistent with the role of oxidative stress duringischemia-reperfusion injury in the heart.

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12.
Localized Ca2+ transients resulting from inositoltrisphosphate (IP3)-dependent Ca2+ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa2+ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca2+([Ca2+]o) reduced localized Ca2+transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca2+ transients and STOCs showed increasedcoupling strength between Ca2+ transients and STOCs when[Ca2+]o was reduced. Gd3+ (10 µM) did not affect Ca2+ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca2+influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca2+ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca2+ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca2+ transients. Single channelstudies indicated that large-conductance Ca2+-activatedK+ (BK) channels were inhibited by aCa2+-dependent PKC. In summary 1)Ca2+ release from IP3 receptor-operated storesactivates Ca2+-activated K+ channels;2) Ca2+ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca2+ sensitivity of BK channels, reducing the couplingstrength between localized Ca2+ transients and BK channels.

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13.
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.  相似文献   

14.
The Ca2+-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca2+-uptake and Ca2+-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca2+-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca2+-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca2+-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca2+-ATPase. Regulation of the Ca2+-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca2+-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.  相似文献   

15.
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. (Mol Cell Biochem 261: 245–249, 2004)  相似文献   

16.
ATP is a candidate enteric inhibitory neurotransmitterin visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca2+-activatedK+ (SK) channels. Coupling between ATP stimulation and SKchannels may be mediated by localized Ca2+ release.Isolated myocytes of the murine colon produced spontaneous, localizedCa2+ release events. These events corresponded tospontaneous transient outward currents (STOCs) consisting ofcharybdotoxin (ChTX)-sensitive and -insensitive events.ChTX-insensitive STOCs were inhibited by apamin. LocalizedCa2+ transients were not blocked by ryanodine, but theseevents were reduced in magnitude and frequency by xestospongin C(Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus wehave termed the localized Ca2+ events in colonic myocytes"Ca2+ puffs." The P2Y receptor agonist2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency ofCa2+ puffs. 2-MeS-ATP also increased STOCs in associationwith the increase in Ca2+ puffs.Pyridoxal-phospate-6-azophenyl-2',4'-disculfonic acid tetrasodium, aP2 receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca2+ transients and the effects of 2-MeS-ATP onCa2+ puffs and STOCs were blocked by U-73122, an inhibitorof phospholipase C. Xe-C and ryanodine also blocked responses to2-MeS-ATP, suggesting that, in addition to release from IP3receptor-operated stores, ryanodine receptors may be recruited duringagonist stimulation to amplify release of Ca2+. These datasuggest that localized Ca2+ release modulatesCa2+-dependent ionic conductances in the plasma membrane.Localized Ca2+ release may contribute to the electricalresponses resulting from purinergic stimulation.

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17.
Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1–/–) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca2+ ([Ca2+]i) decline and relengthening. Contraction and [Ca2+]i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1–/– and wild-type (WT) mice. We also simultaneously measured Ca2+ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1–/–, and PLB–/– mice. NOS1–/– trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-L-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca2+ transient and cell shortening amplitudes and prolonged decline of [Ca2+]i). Alternatively, NOS1 inhibition with SMLT in PLB–/– myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca2+ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. β-Adrenergic stimulation restored the slowed [Ca2+]i decline in NOS1–/– myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca2+]i decline, and this effect is absent in PLB–/– myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite. NOS1; peroxynitrite; force-frequency response  相似文献   

18.
Calmodulin (CaM) activates the skeletal muscle ryanodine receptorCa2+ release channel (RyR1) in the presence of nanomolarCa2+ concentrations. However, the role of CaM activation inthe mechanisms that control Ca2+ release from thesarcoplasmic reticulum (SR) in skeletal muscle and in the heart remainsunclear. In media that contained 100 nM Ca2+, the rate of45Ca2+ release from porcine skeletal muscle SRvesicles was increased approximately threefold in the presence of CaM(1 µM). In contrast, cardiac SR vesicle45Ca2+ release was unaffected by CaM,suggesting that CaM activated the skeletal RyR1 but not the cardiacRyR2 channel isoform. The activation of RyR1 by CaM was associated withan approximately sixfold increase in the Ca2+ sensitivityof [3H]ryanodine binding to skeletal muscle SR, whereasthe Ca2+ sensitivity of cardiac SR[3H]ryanodine binding was similar in the absence andpresence of CaM. Cross-linking experiments identified both RyR1 andRyR2 as predominant CaM binding proteins in skeletal and cardiac SR,respectively, and [35S]CaM binding determinations furtherindicated comparable CaM binding to the two isoforms in the presence ofmicromolar Ca2+. In nanomolar Ca2+, however,the affinity and stoichiometry of RyR2 [35S]CaM bindingwas reduced compared with that of RyR1. Together, our results indicatethat CaM activates RyR1 by increasing the Ca2+ sensitivityof the channel, and further suggest differences in CaM's functionalinteractions with the RyR1 and RyR2 isoforms that may potentiallycontribute to differences in the Ca2+ dependence of channelactivation in skeletal and cardiac muscle.

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19.
The G protein-coupled receptor agonistsangiotensin II (ANG II) and lysophosphatidic acid (LPA) rapidly inducetyrosine phosphorylation of the cytosolic proline-rich tyrosine kinase2 (Pyk2) in IEC-18 intestinal epithelial cells. The combined Pyk2tyrosine phosphorylation induced by phorbol 12,13-dibutyrate, a directagonist of protein kinase C (PKC), and ionomycin, a Ca2+ionophore, was equal to that induced by ANG II. Inhibition of eitherPKC or Ca2+ signaling attenuated the effect of ANG II andLPA, although simultaneous inhibition of both pathways failed tocompletely abolish Pyk2 tyrosine phosphorylation. Cytochalasin D, whichdisrupts stress fibers, strongly inhibited the response of Pyk2 to ANGII or LPA. The distinct Rho-associated kinase (ROK) inhibitors HA-1077and Y-27632, as well as the Rho inhibitor Clostridiumbotulinum C3 exoenzyme, also significantly attenuated ANG II- andLPA-stimulated Pyk2 tyrosine phosphorylation. Simultaneous inhibitionof PKC, Ca2+, and either actin assembly or ROK completelyabolished the Pyk2 response. Together, these results show that ANG IIand LPA rapidly induce Pyk2 tyrosine phosphorylation in intestinalepithelial cells via separate Ca2+-, PKC-, and Rho-mediated pathways.

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20.
Phospholamban(PLB) ablation is associated with enhanced sarcoplasmic reticulum (SR)Ca2+ uptake and attenuation of thecardiac contractile responses to -adrenergic agonists. In thepresent study, we compared the effects of isoproterenol (Iso) on theCa2+ currents(ICa) ofventricular myocytes isolated from wild-type (WT) and PLB knockout(PLB-KO) mice. Current density and voltage dependence ofICa were similarbetween WT and PLB-KO cells. However, ICa recorded fromPLB-KO myocytes had significantly faster decay kinetics. Iso increasedICa amplitude inboth groups in a dose-dependent manner (50% effective concentration,57.1 nM). Iso did not alter the rate ofICa inactivationin WT cells but significantly prolonged the rate of inactivation inPLB-KO cells. When Ba2+ was usedas the charge carrier, Iso slowed the decay of the current in both WTand PLB-KO cells. Depletion of SRCa2+ by ryanodine also slowed therate of inactivation ofICa, and subsequent application of Iso further reduced the inactivation rate ofboth groups. These results suggest that enhancedCa2+ release from the SR offsetsthe slowing effects of -adrenergic receptor stimulation on the rateof inactivation ofICa.

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