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

2005 ◽  
Vol 288 (6) ◽  
pp. C1202-C1210 ◽  
Author(s):  
Minkyung Kim ◽  
Sang Yun Cho ◽  
In Soo Han ◽  
Sang Don Koh ◽  
Brian A. Perrino
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Channel Blocker ◽  
Muscle Tone ◽  
Smooth Muscles ◽  
Cyclopiazonic Acid ◽  
Gastric Fundus ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
Camkii Activation

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.

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

2006 ◽  
Vol 291 (2) ◽  
pp. C337-C347 ◽  
Author(s):  
Minkyung Kim ◽  
In Soo Han ◽  
Sang Don Koh ◽  
Brian A. Perrino
Keyword(s):  
Smooth Muscle ◽  
Soluble Guanylate Cyclase ◽  
Muscle Relaxation ◽  
Smooth Muscles ◽  
Cyclopiazonic Acid ◽  
Gastric Fundus ◽  
Smooth Muscle Relaxation ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
No Donor

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 1 H-[1,2,4]oxadiazolo-[4,3-α]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 1 H-[1,2,4]oxadiazolo-[4,3-α]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.

Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum

2008 ◽  
Vol 294 (2) ◽  
pp. C432-C441 ◽  
Author(s):  
Minkyung Kim ◽  
Grant W. Hennig ◽  
Terence K. Smith ◽  
Brian A. Perrino
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscles ◽  
Cyclopiazonic Acid ◽  
Gastric Fundus ◽  
Gastric Antrum ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
Smooth Muscle Contractility ◽  
Intracellular Ca ◽  
Dependent Protein

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), and this inhibition is relieved by Ca2+ calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ release and CaM kinase II activity.

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

2007 ◽  
Vol 292 (4) ◽  
pp. G1045-G1054 ◽  
Author(s):  
Minkyung Kim ◽  
Brian A. Perrino
Keyword(s):  
Protein Kinase ◽  
Muscle Relaxation ◽  
Smooth Muscles ◽  
Cyclopiazonic Acid ◽  
Gastric Fundus ◽  
Gastric Antrum ◽  
Smooth Muscle Relaxation ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
Intracellular Ca

Elevations in the intracellular Ca2+ concentration activate the serine/threonine protein kinase Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). We tested the hypothesis that increased sarco(endo)plasmic reticulum Ca2+-ATPase activity by phospholamban (PLB) phosphorylation contributes to smooth muscle relaxation by elevating the sarcoplasmic reticulum (SR) Ca2+ load and increasing the frequency of Ca2+ 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 Ca2+ load increases the frequency of Ca2+ 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-[4,3α]quinoxaline-1-one (ODQ) and inhibited by cyclopiazonic acid (CPA) or KN-93. SNP also increased PLBSer16 and PLBThr17 phosphorylation. Both PLBSer16 and Thr17 phosphorylation were ODQ sensitive. However, only PLBThr17 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.

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

2002 ◽  
Vol 283 (5) ◽  
pp. C1399-C1413 ◽  
Author(s):  
Jillinda M. Lorenz ◽  
Marilyn H. Riddervold ◽  
Elizabeth A. H. Beckett ◽  
Salah A. Baker ◽  
Brian A. Perrino
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscles ◽  
Proximal Colon ◽  
Gastric Fundus ◽  
Calcium Oscillations ◽  
Cam Kinase Ii ◽  
Activity Levels ◽  
Dependent Manner ◽  
Cam Kinase ◽  
Autonomous Activity

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 γ and δ isoforms are expressed in myocytes. Relative γ and δ 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.

Sarcoplasmic reticulum Ca2+ uptake is impaired in coronary smooth muscle distal to coronary occlusion

2001 ◽  
Vol 281 (1) ◽  
pp. H223-H231 ◽  
Author(s):  
Cristine L. Heaps ◽  
Michael Sturek ◽  
Elmer M. Price ◽  
M. Harold Laughlin ◽  
Janet L. Parker
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Exercise Training ◽  
Coronary Occlusion ◽  
Smooth Muscles ◽  
Cyclopiazonic Acid ◽  
Coronary Smooth Muscle ◽  
Protein Levels ◽  
Chronic Occlusion ◽  
Plasmic Reticulum

After chronic occlusion, collateral-dependent coronary arteries exhibit alterations in both vasomotor reactivity and associated myoplasmic free Ca2+ levels that are prevented by chronic exercise training. We tested the hypotheses that coronary occlusion diminishes Ca2+ uptake by the sarcoplasmic reticulum (SR) and that exercise training would prevent impaired SR Ca2+ uptake. Ameroid constrictors were surgically placed around the proximal left circumflex (LCx) artery of female swine 8 wk before initiating 16-wk sedentary (pen confined) or exercise-training (treadmill run) protocols. Twenty-four weeks after Ameroid placement, smooth muscles cells were enzymatically dissociated from both the LCx and nonoccluded left anterior descending (LAD) arteries of sedentary and exercise-trained pigs, and myoplasmic free Ca2+ was studied using fura 2 microfluorometry. After the SR Ca2+ store was partially depleted with caffeine (5 mM), KCl-induced membrane depolarization produced a significant decrease in the time to half-maximal ( t ½) myoplasmic free Ca2+ accumulation in LCx versus LAD cells of sedentary pigs. Furthermore, inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA; 10 μM cyclopiazonic acid) significantly reduced t ½ in cells isolated from the LAD but not from the LCx. Exercise training did not prevent the differences in t ½ myoplasmic free Ca2+ accumulation observed between LCx and LAD cells. Occlusion or exercise training did not alter SERCA protein levels. These results support our hypothesis of impaired SR Ca2+ uptake in coronary smooth muscle cells isolated distal to chronic occlusion. Impaired SR Ca2+ uptake was independent of SERCA protein levels and was not prevented by exercise training.

Signal transduction in smooth muscle as studied by the subcellular membrane approach

1992 ◽  
Vol 70 (4) ◽  
pp. 501-508 ◽  
Author(s):  
Chiu-Yin Kwan
Keyword(s):  
Smooth Muscle ◽  
Ion Channels ◽  
Sarcoplasmic Reticulum ◽  
Second Messengers ◽  
Smooth Muscles ◽  
Calcium Signalling ◽  
Cyclopiazonic Acid ◽  
Membrane Receptors ◽  
Subcellular Membrane ◽  
Neuronal Membranes

Many of the contractile regulatory events in smooth muscle reside in various cellular membrane components as functional membrane constituents that interact in a variably complex manner. The physiological handling of ionized calcium (Ca2+), which serves multiple roles as an extracellular signal, a second messenger, and an activator interacting directly with myofilaments to effectuate contractile responses, referred to as Ca2+ signalling processes, represents an integral part of a more complicated membrane transduction mechanism. The subcellular membrane approach toward the understanding of Ca2+ signalling as well as the transduction mechanisms involving membrane receptors, GTP binding proteins, ion channels, membrane-bound enzymes, and the production of intracellular second messengers has made a significant contribution in smooth muscle research for the past decade. This review summarizes the current state of knowledge about the multiplicity of interactions between Ca2+ and various membrane constituents in the surface membranes and sarcoplasmic reticulum, such as Ca2+ binding, Ca2+ ATPase pumps, Ca2+ channels, and Ca2+Na+ or related ion exchangers. A number of recent novel findings from this laboratory have also been discussed. First of all, the technical refinement of membrane separation and characterization, which permits better identification of neuronal membranes in highly innervated smooth muscle tissues, led to the distinction of prejunctional and postjunctional membrane receptors. Secondly, unlike the Ca2+-release channels labelled with [3H]inositol 1,4,5-trisphosphate, the other type of internal membrane Ca2+-release channels labelled by [3H]ryanodine has been identified only recently in smooth muscle. Thirdly, the successful use of thapsigargin and cyclopiazonic acid as selective inhibitors for the sarcoplasmic reticulum Ca2+ pump in smooth muscle provided novel pharmacological tools to probe the refilling mechanisms of internal Ca2+ stores. Finally, the intriguing relationship between, and regulation of, α-adrenoceptor subtypes in vascular smooth muscles underscores the complexity of their transducing mechanisms and associated utilization of Ca2+.Key words: smooth muscle, sarcoplasmic reticulum, membrane receptors, calcium signalling, ion channels.

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