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NEUROREGULATION AND MOTILITY

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

Department of Physiology and Cell Biology, Center of Biomedical Research Excellence, University of Nevada School of Medicine, Reno, Nevada

Submitted 10 May 2006 ; accepted in final form 19 December 2006

	ABSTRACT

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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-[4,3]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.

Ca²⁺/calmodulin-dependent protein kinase II; phospholamban; nitric oxide

The relaxant effects of nitric oxide (NO) and NO donors on smooth muscles are well established, but many details of the mechanism of NO-induced relaxation are still under investigation (54). One possible mechanism by which NO relaxes smooth muscle is by hyperpolarization of the smooth muscle cell plasma membrane. Porter et al. (43) reported that the NO donor sodium nitroprusside (SNP) increases Ca²⁺ spark frequency and activates spontaneous transient outward currents (STOCs) via a NO/soluble guanylyl cyclase (sGC)/cGMP pathway in cerebral and coronary artery myocytes. Furthermore, SNP-induced relaxation of the rat gastric fundus is ryanodine sensitive and involves small-conductance Ca²⁺-activated K⁺ (K_Ca) channels (15). Similarly, in guinea pig gastric antrum myocytes, SNP increases intermediate-conductance K_Ca current and enhances STOCs, which are sensitive to inositol (1,4,5)-trisphosphate receptor (IP₃R) inhibitors (58). These findings suggest that sarcoplasmic reticulum (SR) Ca²⁺ release plays a role in the nitrergic relaxation of smooth muscles.

In several smooth muscles, it has been shown that nitrergic relaxation involves lowering the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and elevating the SR Ca²⁺ concentration ([Ca²⁺]_SR) by the activation of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) (7, 24, 42, 45, 55). SERCA activity is regulated by the SR membrane protein phospholamban (PLB). Phosphorylation of PLB by PKA or PKG at Ser¹⁶ or Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) at Thr¹⁷ removes its inhibitory effects on SERCA, thereby enhancing Ca²⁺ uptake into the SR. The increased SERCA activity elevates the SR Ca²⁺ load and lowers cytosolic Ca²⁺ levels, resulting in relaxation (19, 24). The SR Ca²⁺ load is important in the regulation of the frequency of Ca²⁺-release events from the SR (5, 59). Higher levels of SR Ca²⁺ are correlated with increased Ca²⁺ spark frequencies, which lead to increased STOC frequencies, membrane potential hyperpolarization, and relaxation (3, 36, 49, 59). PLB transgenic animal studies (47, 57) have supported the relationship between SR Ca²⁺ load and Ca²⁺ spark frequency and suggest that PLB plays a key role in the regulation of SR Ca²⁺ load and local Ca²⁺-release events from the SR in smooth muscles. These findings suggest that CaM kinase II activation and PLB phosphorylation due to an increase in SR Ca²⁺-release events could be involved in NO- or NO donor-induced relaxation of smooth muscles.

We (27, 28) have previously shown that 1 mM caffeine or 10 µM SNP relaxes gastric fundus smooth muscle strips in vitro. These studies (27, 28) also showed that CaM kinase II activation by caffeine or SNP in murine gastric fundus smooth muscles is blocked by SR Ca²⁺ channel inhibitors and suggested that PLB phosphorylation by CaM kinase II is involved in the relaxation induced by caffeine or SNP. The purpose of the present study was to extend these studies to a portion of the stomach that exhibits spontaneous phasic contractions and to investigate the roles of CaM kinase II and PLB phosphorylation in SNP-induced relaxation of murine antrum smooth muscles.

	METHODS

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Tissue preparation for the CaM kinase II assay. Gastric antrum smooth muscles were obtained from adult CD-1 mice (6–8 wk old, Charles River Laboratories, Wilmington, MA). Animals were anesthetized by isofluorane inhalation and euthanized by decapitation. Mice were maintained and experiments carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by University of Nevada Institutional Animal Care and Use Committee. The gastric antrum tissues were pinned out in a Sylgard-lined dish containing oxygenated Krebs solution (120 mM NaCl, 6 mM KCl, 15 mM NaHCO₃, 12 mM glucose, 3 mM MgCl₂, 1.5 mM NaH₂PO₄, and 3.5 mM CaCl₂; pH 7.2). Mucosa and submucosa layers were removed using fine-tipped forceps. To determine the effects of SNP or 8-bromo-cGMP on CaM kinase II activity, tissues were equilibrated in Krebs buffer for 45 min at 37°C and then incubated at 37°C in the absence or presence of each compound for 15 min. To determine the effects of various blockers on SNP-induced CaM kinase II activity, tissues were equilibrated in Krebs buffer for 45 min at 37°C and then incubated at 37°C in the presence of each blocker for 20 min, followed by further incubations with SNP for 15 min in the presence of each blocker. After treatment, tissues were collected, frozen in liquid nitrogen, and stored at –80°C. For the activity assays, each frozen tissue was homogenized at 4°C with a glass tissue grinder in 0.3 ml lysis buffer (50 mM MOPS, 0.2% Nonidet P-40, 100 mM Na₄P₂O₇, 100 mM NaF, 250 mM NaCl, 3 mM EGTA, 1 mM DTT, 1 mM PMSF, and protease inhibitors) and centrifuged at 16,000 g for 15 min at 4°C, and the supernatant was aliquotted and stored at –80°C. Protein concentrations were determined using the Bradford assay with bovine -globulin as the standard.

CaM kinase II activity assays. CaM kinase II activity in the lysates was assayed in a total volume of 30 µl containing 50 mM HEPES (pH7.4), 10 mM magnesium acetate, 0.2 mM [-³²P]ATP (500–1,000 counts·min^–1·pmol^–1) (MP Biomedicals, Irvine, CA), and 20 µM autocamtide-2 (a specific CaM kinase II peptide substrate, KKALRRQETVDAL) (BioMol, Plymouth Meeting, PA) plus 600 nM CaM (EMD Biosciences, La Jolla, CA) and 0.8 mM CaCl₂ (for total activity) or 1.0 mM EGTA (for autonomous activity) as previously described (27, 28). Total (Ca²⁺/CaM stimulated) and autonomous (Ca²⁺/CaM independent) CaM kinase II activities from the cytosolic fraction of control and treated gastric antrum smooth muscles from at least three animals were assayed in triplicate from each tissue. Kinase activity was calculated and expressed as nanomoles of P_i incorporated per minute per milligram of lysate protein.

Mechanical responses of gastric antrum smooth muscles. Standard organ bath techniques were employed to measure the changes in force provided by antrum smooth muscle strips (6 x 3 mm). One end of each smooth muscle strip was attached to a fixed mount, and the opposite end of each muscle strip was attached to a Fort 10 isometric strain gauge (WPI, Sarasota, FL) in parallel with the circular smooth muscle layer. Muscle strips were immersed in organ baths containing Krebs buffer (3 ml) maintained at 37 ± 0.5°C. The pH was kept constant at 7.4 by bubbling the Krebs solution with 97% O₂-3% CO₂. A resting force of 6 mN was applied to set the muscles at optimum length. This was followed by an equilibration period of at least 1 h, during which time the bath was continuously perfused with oxygenated Krebs solution. Following the equilibration period, muscle strips were incubated in Krebs solution containing the compounds as indicated in the figures. Mechanical responses were recorded on a personal computer running Acqknowledge 3.2.6 (BIOPAC Systems, Santa Barbara, CA).

SDS-PAGE and Western blot analysis of PLB phospho-Thr¹⁷ and phospho-Ser¹⁶ from gastric antrum smooth muscle SR fractions. Gastric antrum smooth muscles obtained from adult CD-1 mice (as described above) were placed into the following groups: oxygenated Krebs solution treated (control), SNP treated, or SNP treated in the presence of each blocker. All antrum smooth muscle strips were equilibrated in oxygenated Krebs buffer for 45 min at 37°C. For SNP treatment, smooth muscle strips were then perfused with oxygenated Krebs buffer containing SNP for 15 min at 37°C. To determine the effects of the various blockers, smooth muscle strips were perfused with oxygenated Krebs buffer containing the appropriate blocker for 20 min, followed by further incubations with SNP for 15 min in the presence of the appropriate blocker. Tissues were collected, frozen in liquid nitrogen, and stored at –80°C. Gastric antrum smooth muscle SR fractions were obtained from CD-1 mice as previously described (21). Briefly, frozen tissues were homogenized at 4°C with a glass tissue grinder in 6 volumes by weight of ice-cold lysis buffer [10 mM Tris·HCl (pH 6.8), 100 mM NaF, 20 mM sodium pyrophosphate, and protease inhibitor tablet], the homogenate was centrifuged at 1,000 g for 10 min at 4°C, and the supernatant was decanted and placed on ice. The pellet was resuspended in 4 volumes by weight of ice-cold lysis buffer and spun at 1,000 g for 10 min at 4°C. The two supernatants were combined and spun at 8,000 g for 20 min at 4°C. The supernatant was brought to a total volume of 2 ml with lysis buffer. Solid KCl was added to a final concentration of 0.6 M, and samples were placed on ice for 25 min. Samples were then centrifuged at 40,000 g for 60 min at 4°C to pellet the SR fraction. Pellets were resuspended in 200 µl of SR buffer [10 mM Tris·HCl (pH 6.8), 100 mM KCl, 100 mM NaF, 20 mM sodium pyrophosphate, and protease inhibitor tablet] and stored at –80°C. Protein concentrations were determined with the Bradford assay using bovine -globulin as the standard. Smooth muscle SR proteins were separated by SDS-PAGE (15% high-salt gel) and transferred to nitrocellulose membranes by Western blotting. Blots were incubated with primary and secondary antibodies, washed, and processed for enhanced chemiluminescence image detection using ECL Advantage (Amersham Biosciences, Piscataway, NJ). PLB-PO₄-Thr¹⁷, PLB-PO₄-Ser¹⁶, and PLB antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1:5,000 dilution, and horseradish-peroxidase-conjugated secondary antibody (Chemicon, Temecula, CA) was used at 1:50,000 dilution. Protein bands were visualized with a charge-coupled device camera-based detection system (Epi Chem II, UVP Laboratory Products). Collected TIFF images were analyzed using Adobe Photoshop. The Western blot data shown in each figure are representative of four separate experiments. Densitometry of the immunostained phospho-PLB and PLB protein bands was carried out with Un-Scan-It software (Silk Scientific, Orem,UT) using lane analysis for background subtraction. Signal intensities of anti-phospho-PLBSer¹⁶ and anti-phospho-PLBThr¹⁷ immunoblots from 1 mM cGMP-treated antrum smooth muscle tissues were normalized to 1 (n = 5). The ratio of SNP-evoked PLB phosphorylation to cGMP-evoked PLB phosphorylation was determibed by densitometry of the signal intensities of the immunoblots.

Materials. The following drugs were used: TTX, iberiotoxin (IbTX), and apamin were obtained from Sigma (St. Louis, MO). Cyclopiazonic acid (CPA) was purchased from BioMol. KN-93, 8-bromo-cGMP, 1H-[1,2,4]oxadiazolo-[4,3-]quinoxalin-1-one (ODQ), and SNP were purchased from EMD Bioscience (La Jolla, CA). Mini-EDTA free protease inhibitor pills were obtained from Roche Applied Science (Indianapolis, IN). All other chemicals and materials were of reagent grade.

Statistical analysis. Data are expressed as means ± SD. Data sets were tested for significance using ANOVA to analyze multiple groups. Data were considered significantly different from control values when P < 0.05.

	RESULTS

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Effect of SNP on mechanical responses of murine antrum smooth muscles. As shown in Fig. 1A, 10 µM SNP reduced the average amplitude of contractions from 1.4 ± 0.6 to 0.16 ± 0.05 mN (n = 8, P < 0.001), representing an 89 ± 1% inhibition. SNP (10 µM) also decreased the basal tone by 0.6 ± 0.1 mN (n = 8, P < 0.001). The effects of SNP on the mechanical responses of the antrum smooth muscle strips were fully reversible upon washout. In addition, the effects of SNP on the mechanical activity of antrum smooth muscle strips were TTX (1 µM) insensitive (data not shown), indicating that the effects of SNP on the mechanical responses are myogenic in origin.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Effects of large- and small-conductance Ca2+-activated K+ channel blockers on sodium nitroprusside (SNP)-induced relaxation of gastric antrum smooth muscle strips. A and B: representative traces of the changes in mechanical responses of gastric antrum smooth muscle strips incubated with 10 µM SNP alone followed by incubation with 10 µM SNP and 100 nM iberiotoxin (IbTX; A) or with 10 µM SNP alone followed by incubation with 10 µM SNP and 300 nM apamin (B).

Effect of SNP on autonomous CaM kinase II activity in antrum smooth muscles. Since we (28) have previously reported that SNP-induced SR Ca²⁺ release activates CaM kinase II in gastric fundus smooth muscles, CaM kinase II activity was measured in lysates of untreated and SNP-treated antrum smooth muscles. ³²P incorporation into the autocamtide-2 peptide substrate was used to measure CaM kinase II activity (18). Total (Ca²⁺/CaM stimulated) CaM kinase II activities were not significantly affected by 10 µM SNP. Total CaM kinase II activities in control and SNP-treated antrum smooth muscle tissues were 8.7 ± 1.4 and 8.2 ± 0.7 nmol·min^–1·mg^–1, respectively (n = 6). However, as shown in Fig. 2A, 10 µM SNP increased autonomous CaM kinase II activity from 2.7 ± 0.2 to 3.6 ± 0.3 nmol·min^–1·mg^–1 (n = 6, P < 0.001). This represents an increase in autonomous CaM kinase II activity from 33% to 44% of the total CaM kinase II activity. KN-93 (10 µM), the inhibitor of Ca²⁺/CaM binding to CaM kinase II, prevented the SNP-induced increase in ³²P incorporation into the autocamtide-2 peptide substrate, supporting the conclusion that the kinase activity measured in response to SNP is due to CaM kinase II. As shown in Fig. 2B, by itself KN-93 decreased the muscle tone by 0.23 ± 0.06 mN but inhibited the SNP-induced reduction in basal tone by 84 ± 5% (n = 5, P < 0.001). In contrast, KN-93 did not affect the SNP-induced reduction in the amplitudes of phasic contractions but delayed the recovery of spontaneous phasic contractions (Fig. 2B).

View larger version (14K): [in this window] [in a new window]   Fig. 2. SNP activates Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in gastric antrum smooth muscle strips. A: autonomous (Ca2+/CaM independent) CaM kinase II activities were assayed as described in METHODS. Average values ± SD of autonomous CaM kinase II activities were obtained from gastric antrum smooth muscle strips incubated without (control) or with 10 µM SNP for 15 min, with 10 µM KN-93 for 20 min followed by 10 µM SNP with KN-93 for 15 min, or with 10 µM KN-93 alone for 20 min. **P < 0.001. B: representative traces of the changes in mechanical responses of gastric antrum smooth muscle strips incubated with 10 µM SNP alone or with 10 µM SNP in the presence of 10 µM KN-93.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Cyclopiazonic acid (CPA) inhibits SNP-induced relaxation and CaM kinase II activation in gastric antrum smooth muscle strips. A: representative trace of the change in the mechanical response of gastric antrum smooth muscle strips incubated with 10 µM SNP alone or with 10 µM CPA followed by the addition of 10 µM SNP. B: representative trace of the change in the mechanical response of gastric antrum smooth muscle strips incubated with 10 µM SNP alone or with 10 µM SNP followed by the addition of 10 µM CPA. C: average values ± SD of autonomous CaM kinase II activities obtained from gastric antrum smooth muscle strips incubated without (control) or with 10 µM SNP for 15 min, with 10 µM CPA for 20 min followed by 10 µM SNP with CPA for 15 min, or with 10 µM CPA alone for 20 min. **P < 0.001.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effects of 1H-[1,2,4]oxadiazolo-[4,3]quinoxaline-1-one (ODQ) or 8-bromo-cGMP (8-Br-cGMP) on SNP-induced relaxation and CaM kinase II activation in gastric antrum smooth muscle strips. A and B: representative traces of the changes in mechanical responses of gastric antrum smooth muscle strips incubated with 10 µM SNP alone followed by incubation with 10 µM SNP in the presence of 10 µM ODQ (A) or incubated with 10 µM SNP alone or with 1 mM 8-Br-cGMP alone (B). C: average values ± SD of autonomous CaM kinase II activities obtained from gastric antrum smooth muscle strips incubated without (control) or with 10 µM SNP for 15 min, with 10 µM ODQ for 20 min followed by 10 µM SNP with ODQ for 15 min, or with 10 µM ODQ alone for 20 min. **P < 0.001.

View larger version (26K): [in this window] [in a new window]   Fig. 5. SNP increases phospholamban (PLB) phosphorylation at Ser16 and Thr17. SDS-PAGE and immunoblot analysis using phospho-PLBSer16 (P-Ser16) and phospho-PLBThr17 (P-Thr17) antibodies were carried out as described in METHODS. A and B: representative immunoblots of P-Ser16 (A) or P-Thr17 (B) from the sarcoplasmic reticulum (SR)-enriched fraction of gastric antrum smooth muscle strips incubated without (control) or with 10 µM SNP for 15 min, with 10 µM KN-93 for 20 min followed by 10 µM SNP with KN-93 for 15 min, or with 10 µM ODQ for 20 min followed by 10 µM SNP with ODQ for 15 min. C and D: representative immunoblots of P-Ser16 (C) or P-Thr17 (D) from the SR-enriched fraction of gastric antrum smooth muscle strips incubated without (control) or with 1 mM 8-Br-cGMP for 15 min. Each lane contained 50 µg protein. Bottom blots in A–D show samples immunostained with the anti-PLB antibody as an additional control for equal protein loading.

	DISCUSSION

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The NO donor SNP (10 µM) almost completely abolished the spontaneous contractions of murine antrum smooth muscle strips. The amplitudes of spontaneous contractions were reduced by 90%. The involvement of SR Ca²⁺ levels in the relaxation of antrum smooth muscles by SNP is suggested by the results shown in Fig. 3 demonstrating that the SERCA inhibitor CPA partially inhibited SNP-induced relaxation. Lowering the level of SR Ca²⁺ by SERCA pump inhibition is correlated with decreased frequencies of Ca²⁺ sparks and a resistance to relaxation (3, 36, 49, 59). The amplitudes of the spontaneous contractions observed in the presence of CPA and SNP were fivefold higher than the amplitudes measured in the presence of SNP alone. The frequency of spontaneous contractions in the presence of CPA and SNP was 20% of the frequency observed in untreated antrum smooth muscle strips. Similar results were obtained when we incubated antrum smooth muscle strips with SNP and then added CPA. A recovery of spontaneous phasic contractions occurred when CPA was added to antrum smooth muscle strips in the presence of SNP. Studies (22, 23) in other muscle types have indicated that SERCA activity influences contractile behavior by modulating the rate of Ca²⁺ clearance from the cytosol and the rate of subsequent SR Ca²⁺ release. When cytosolic Ca²⁺ removal is inhibited by CPA, more Ca²⁺ is available for contraction (31). In addition, when SR Ca²⁺ refilling is inhibited by CPA, less Ca²⁺ is available for release (20). The findings shown in Fig. 3 suggest that SERCA pump activity is involved in the SNP- induced relaxation of antrum smooth muscle. However, the lack of complete inhibition of SNP-induced relaxation by CPA indicates that parallel cGMP-dependent pathways, such as increased myosin light chain phosphatase activity due to PKG inhibition of RhoA pathways, are likely contributing to the relaxation (35).

Localized Ca²⁺ transient release from intracellular stores can activate K_Ca channels in the plasma membrane where the SR and plasma membranes are in close apposition (12–20 nm) (56). Porter et al. (43) showed that SNP increased Ca²⁺ spark frequency twofold and STOC activities in isolated rat cerebral and coronary arterial smooth muscle cells. Thus, we treated antrum smooth muscle strips with IbTX or apamin to examine whether large- or small-conductance K_Ca channels were involved in mechanical responses to SNP. IbTX alone slightly increased the basal tone with no changes in the contractile amplitude and frequency of spontaneous phasic contractions, whereas apamin evoked larger increases in the basal tone and amplitudes of phasic contractions. These results suggest that small-conductance K_Ca channels may be more important than large-conductance K_Ca channels in setting the basal tone and amplitudes of phasic contractions of antral smooth muscle. These findings are different from our previous report (28), in which large-conductance K_Ca channels were more involved in setting the basal fundus tone. As shown in Fig. 1, neither K_Ca channel blocker was particularly effective at attenuating the inhibitory effects of SNP on the contractile activity of antrum smooth muscle strips, although small but measurable phasic contractions could be observed in strips treated with SNP in the presence of either K_Ca channel blocker. These results are consistent with findings from other smooth muscle tissues showing that removal of cytosolic Ca²⁺ by increased SERCA activity is more important than K_Ca channel activation in NO-induced relaxations (7).

CaM kinase II activation is dependent on the duration, amplitude, and frequency of Ca²⁺ oscillations (10, 14, 34). Although bulk increases in cytosolic Ca²⁺ can activate CaM kinase II, frequency-dependent activation by transient Ca²⁺ release resulting in localized high Ca²⁺ concentrations are likely to activate nearby CaM kinase II (1). Evidence from studies (1, 27, 28, 41) of tonic vascular and gastric fundus smooth muscles have suggested that Ca²⁺-release events from the SR (Ca²⁺ sparks or puffs) activate CaM kinase II. These findings led us to investigate CaM kinase II activation by SR Ca²⁺ release in gastric smooth muscles that undergo spontaneous phasic contractions. The results shown in Fig. 2 demonstrate that incubation of gastric antrum smooth muscle strips with 10 µM SNP activated CaM kinase II, as indicated by the increase in autonomous CaM kinase II activity. The CaM kinase II inhibitor KN-93 prevented CaM kinase II activation by SNP, indicating that the increase in kinase activity measured in lysates from SNP-treated antrum smooth muscle strips is due to CaM kinase II. The results shown in Fig. 3 demonstrate that CPA inhibited SNP-induced relaxation of antrum smooth muscle strips and suggest that SNP evokes SR Ca²⁺ release. Similarly, CPA prevented the activation of CaM kinase II by SNP. The lack of CaM kinase II activation by SNP in the presence of CPA is consistent with previous reports (5, 20, 47, 59) showing that decreasing the SR Ca²⁺ load by blockade of SERCA decreases the concentration of Ca²⁺ released from the SR.

Cardiac, skeletal, and smooth muscle SERCA activity is regulated by the integral SR membrane protein PLB (4, 44, 46, 50). Dephosphorylated PLB inhibits SERCA activity by 50% (53). PLB phosphorylation by PKA or PKG at Ser¹⁶, or CaM kinase II at Thr¹⁷, relieves its inhibitory effect on the SERCA pump, enhancing SERCA activity and contributing to relaxation by decreasing [Ca²⁺]_i and increasing [Ca²⁺]_SR (30). The Western blot analysis results shown in Fig. 5 demonstrate that the sGC inhibitor ODQ prevented the SNP-induced increase in PLBSer¹⁶ phosphorylation, suggesting that PLBSer¹⁶ phosphorylation is due to PKG activation by cGMP. KN-93 or CPA had no effect on PLBSer¹⁶ phosphorylation, indicating that the SNP-induced increase in PLBSer¹⁶ is not due to CaM kinase II and is not affected by the SR Ca²⁺ load. The results shown in Fig. 5 also demonstrate that PLBThr¹⁷ phosphorylation also increased in response to SNP and was inhibited by KN-93 or CPA. These findings are consistent with the results shown in Figs. 2 and 3, which demonstrated that KN-93 or CPA prevented CaM kinase II activation by SNP, and further strengthen the conclusion that CaM kinase II activation by SNP increased PLBThr¹⁷ phosphorylation. The SNP-induced increase in PLBThr¹⁷ phosphorylation was also prevented by ODQ, suggesting that PKG activation by the NO/sGC/cGMP pathway is a prerequisite for CaM kinase II activation. The levels of SNP-evoked PLB phosphorylation were comparable to the levels of PLB phosphorylation evoked by 1 mM cGMP. Signal intensities of anti-phospho-PLBSer¹⁶ and anti-phospho-PLBThr¹⁷ immunoblots from untreated antrum smooth muscle tissues increased from 31% to 92% and 85%, respectively, of the signal intensities of anti-phospho-PLBSer¹⁶ and anti-phospho-PLBThr¹⁷ immunoblots from cGMP-treated tissues. These findings suggest that the ODQ-sensitive PLB phosphorylation in antrum smooth muscles due to SNP is physiologically relevant. Furthermore, these findings are similar to those of a study (33) investigating -adrenergic-induced PLB phosphorylation in the heart, which indicated about a fourfold increase in PLBSer¹⁶ phosphorylation.

The physiological role of CaM kinase II activation in antrum smooth muscles is not fully known. The CaM kinase II inhibitor KN-93 inhibited the relaxation of gastric fundus smooth muscle strips (27, 28). The results shown in Fig. 2 demonstrate that KN-93 also inhibited the SNP-induced decrease in basal tone of gastric antrum smooth muscle strips. KN-93 had no effect on the SNP-induced inhibition of phasic contractions but lengthened the time for recovery of spontaneous phasic contractions. The findings shown in Fig. 3 demonstrate that basal tone increased and phasic contractions were partially restored with CPA, suggesting that cytosolic Ca²⁺ is important for spontaneous contractions. Although CaM kinase II activity is inhibited by KN-93, PKG activity is not, and phosphorylation of PLB by PKG may be more important during SNP treatment. In cardiac myocytes, Ca²⁺ transients modulate the phosphorylation of PLB by CaM kinase II and, thus, SERCA pump activity and SR Ca²⁺ load (17). The ability of CaM kinase II to phosphorylate PLBThr¹⁷ in response to different stimulation frequencies warrants further investigations into the modulation of smooth muscle contractility by differential activation by CaM kinase II (17). In neuronal and cardiac tissues, autonomous CaM kinase II (Ca²⁺ independent) activity persists after the initial Ca²⁺ stimulus has ceased (16, 32). Similarly, its Ca²⁺-independent activity in antrum smooth muscles may provide a mechanism for prolonging the effects of NO/cGMP on SERCA activity after the cGMP and Ca²⁺ signals have ceased. Additional studies are underway to determine the respective contributions of SERCA activity, PLB phosphorylation, and CaM kinase II in the response of antrum smooth muscles to nitrergic relaxation.

In summary, the results of the present study suggest that the relaxant effect of SNP on antrum smooth muscles involves PLB phosphorylation by PKG through a NO/sGC/cGMP pathway. These results also suggest that the activation of CaM kinase II by SNP involves increased Ca²⁺-release events from the SR through PLB phosphorylation by PKG and indicate that CaM kinase II may also play a role in the relaxant effect of SNP by phosphorylation of PLB at Thr¹⁷. Furthermore, these findings demonstrate a novel link between nitrergic and Ca²⁺/calmodulin-dependent signaling pathways in murine antrum smooth muscle tissues.

	GRANT

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This work was supported by National Center for Research Resources Grant RR-018751.

	ACKNOWLEDGMENTS

 
A preliminary version of this work was presented at the 20^th International Symposium on Neurogastroenterology and Motility, July 3–7, 2005, in Toulouse, France.

	FOOTNOTES

 

Address for reprint requests and other correspondence: B. A. Perrino, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Anderson Bldg., MS352, Reno, NV 89557 (e-mail: perrinob{at}unr.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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1. Abraham ST, Benscoter H, Schworer CM, Singer HA. In situ Ca²⁺ dependence for activation of Ca²⁺/calmodulin dependent protein kinase II in vascular smooth muscle cells. J Biol Chem 271: 2506–2513, 1996.[Abstract/Free Full Text]
2. Arnold WP, Mittal CK, Katsuki S, Murad F. Nitric oxide activates guanylate cyclase and increases guanosine 3':5'-cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci USA 74: 3203–3207, 1977.[Abstract/Free Full Text]
3. Bayguinov O, Hagen B, Bonev AD, Nelson MT, Sanders KM. Intracellular calcium events activated by ATP in murine colonic myocytes. Am J Physiol Cell Physiol 279: C126–C135, 2000.[Abstract/Free Full Text]
4. Briggs FN, Lee KF, Wechsler AW, Jones LR. Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca²⁺-ATPase. J Biol Chem 267 : 26056–26061, 1992.[Abstract/Free Full Text]
5. Cheranov SY, Jaggar JH. Sarcoplasmic reticulum calcium load regulates rat arterial smooth muscle calcium sparks and transient K_Ca currents. J Physiol 544: 71–84, 2002.[Abstract/Free Full Text]
6. Code CF, Marlett JA. Canine tachygastria. Mayo Clin Proc 49: 325–332, 1974.[Web of Science][Medline]
7. Cohen RA, Weisbrod RM, Gericke M, Yaghoubi M, Bierl C, Bolotina WM. Mechanism of nitric oxided-induced vasodilation. Refilling of intracellular by sarcoplasmic reticulum Ca²⁺ ATPase and inhibition of store-operated Ca²⁺ influx. Circ Res 84: 210–219, 1999.[Abstract/Free Full Text]
8. Cornwell TL, Lincoln TM. Regulation of intracellular Ca²⁺ levels in cultured vascular smooth muscle cells. Reduction of Ca²⁺ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase. J Biol Chem 264: 1146–1155, 1989.[Abstract/Free Full Text]
9. Cousins HM, Edwards FR, Hickey H, Hill CE, Hirst GD. Electrical coupling between the myenteric interstitial cells of Cajal and adjacent muscle layers in the guinea-pig gastric antrum. J Physiol 550: 829–844, 2003.[Abstract/Free Full Text]
10. DeKoninck P, Schulman H. Sensitivity of CaM kinase II to the frequency of Ca²⁺ oscillations. Science 279: 227–230, 1998.[Abstract/Free Full Text]
11. Dickens EJ, Hirst GD, Tomita T. Identification of rhythmically active cells in guinea-pig stomach. J Physiol 514: 515–531, 1999.[Abstract/Free Full Text]
12. El Sharkawy TY, Morgan KG, Szurszewski JH. Intracellular electrical activity of canine and human gastric smooth muscle. J Physiol 279: 291–307, 1978.[Abstract/Free Full Text]
13. Fritsch RM, Saur D, Kurjak M, Qesterle D, Schlossmann J, Geiselhoringer A, Hofmann F, Allescher HD. InsP₃R-associated cGMP kinase substrate (IRAG) is essential for nitric oxide-induced inhibition of calcium signaling in human colonic smooth muscle. J Biol Chem 279: 12551–12559, 2004.[Abstract/Free Full Text]
14. Gaertner TR, Kolodziej SJ, Wang D, Kobayashi R, Koomen JM, Stoops JK, Waxham MN. Comparative analyses of the three-dimensional structures and enzymatic properties of alpha, beta, gamma and delta isoforms of Ca²⁺-calmodulin-dependent protein kinase II. J Biol Chem 279: 12484–12494, 2004.[Abstract/Free Full Text]
15. Geeson J, Larsson K, Hourani SMO, Toms NJ. Sodium nitroprusside-induced rat fundus relaxation is ryanodine-sensitive and involves L-type Ca²⁺ channel and small conductance Ca²⁺-sensitive K⁺ channel components. Auton Autacoid Pharmacol 22: 297–301, 2002.[CrossRef][Medline]
16. Giese KP, Fedorov NB, Filipkowski RK, Silva AJ. Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science 279: 870–873, 1998.[Abstract/Free Full Text]
17. Hagemann D, Kuschel M, Kuramochi T, Zhu W, Cheng H, Xiao RP. Frequency-encoding Thr17 phospholamban phosphorylation is independent of Ser16 phosphorylation in cardiac myocytes. J Biol Chem 275: 22532–22536, 2000.[Abstract/Free Full Text]
18. Hanson PI, Kapiloff MS, Lou LL, Rosenfeld MG, Schulman H. Expression of a multifunctional Ca²⁺/calmodulin-dependent protein kinase and mutational analysis of its autoregulation. Neuron 3: 59–70, 1989.[CrossRef][Web of Science][Medline]
19. Inesi G, Sumbilla C, Kirtley ME. Relationships of molecular structure and function in Ca²⁺-transport ATPase. Physiol Rev 70: 749–760, 1990.[Free Full Text]
20. Janiak R, Wilson SM, Montague S, Hume JR. Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells. Am J Physiol Cell Physiol 280: C22–C33, 2001.[Abstract/Free Full Text]
21. Jones DL, Narayann N. Defibrillation depresses heart sarcoplasmic reticulum calcium pump: a mechanism of postshock dysfunction. Am J Physiol Heart Circ Physiol 274: H98–H105, 1998.[Abstract/Free Full Text]
22. Kadambi VL, Kranias EG. Phospholamban: a protein coming of age. Biochem Biophys Res Commun 239: 1–5, 1997.[CrossRef][Web of Science][Medline]
23. Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano KI, Harada KI, Miyamoto S, Nakazawa H, Won KJ, Sato K. Calcium movements, distribution, and functions in smooth muscle. Pharmacol Rev 49: 157–230, 1997.[Abstract/Free Full Text]
24. Karczewski P, Hendrischke T, Wolf WP, Morano I, Bartel S, Schrader J. Phosphorylation of phospholamban correlates with relaxation of coronary artery induced by nitric oxide, adenosine, and prostacyclin in the pig. J Cell Biochem 70: 49–59, 1998.[CrossRef][Web of Science][Medline]
25. Kelly KA, Code CF. Canine gastric pacemaker. Am J Physiol 220: 112–118, 1971.[Free Full Text]
26. Kim CH, Azpiroz F, Malagelada JR. Characteristics of spontaneous and drug-induced gastric dysrhythmias in a chronic canine model. Gastroenterology 90: 421–427, 1986.[Web of Science][Medline]
27. Kim M, Cho SY, Han IS, Koh SD, Perrino BA. CaM kinase II and phospholamban contribute to caffeine-induced relaxation of murine gastric fundus smooth muscle. Am J Physiol Cell Physiol 288: C1202–C1210, 2005.[Abstract/Free Full Text]
28. Kim M, Han IS, Koh SD, Perrino BA. Roles of CaM kinase II and phospholamban in SNP-induced relaxation of murine gastric fundus smooth muscles. Am J Physiol Cell Physiol 291: C337–C347, 2006.[Abstract/Free Full Text]
29. Kuiken SD, Vergeer M, Heisterkamp SH, Tytgat GN, Boeckxstaens GE. Role of nitric oxide in gastric motor and sensory functions in healthy subjects. Gut 51: 212–218, 2002.[Abstract/Free Full Text]
30. Kuschel M, Karczewski P, Hempel P, Schlegel WP, Krause EG, Bartel S. Ser16 prevails over Thr17 phospholamban phosphorylation in the -adrenergic regulation of cardiac relaxation. Am J Physiol Heart Circ Physiol 276: H1625–H1633, 1999.[Abstract/Free Full Text]
31. Laporte R, Hui A, Laher I. Pharmacological modulation of sarcoplasmic reticulum function in smooth muscle. Pharmacol Rev 56: 439–513, 2004.[Abstract/Free Full Text]
32. Maier LS, Bers DM. Calcium, calmodulin, and calcium-calmodulin kinase II: heartbeat to heartbeat and beyond. J Mol Cell Cardiol 34: 919–939, 2002.[CrossRef][Web of Science][Medline]
33. Mayer EJ, Huckle W, Johnson RG, McKenna E. Characterization and quantitation of phospholamban and its phosphorylation state using antibodies. Biochem Biophys Res Commun 267: 40–48, 2000.[CrossRef][Web of Science][Medline]
34. Meyer T, Hanson PI, Schulman H. Calmodulin trapping by calcium-calmodulin-dependent protein kinase. Science 256: 1199–1202, 1992.[Abstract/Free Full Text]
35. Murthy KS. Signaling for contraction and relaxation in smooth muscle of the gut. Annu Rev Physiol 68: 345–374, 2006.[CrossRef][Web of Science][Medline]
36. Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633–637, 1995.[Abstract/Free Full Text]
37. Nishimura J, van Breemen C. Possible involvement of actomyosin ADP complex in regulation of Ca²⁺ sensitivity in alpha-toxin permeabilized smooth muscle. Biochem Biophys Res Commun 165: 408–415, 1989.[CrossRef][Web of Science][Medline]
38. Ordog T, Baldo M, Danko R, Sanders KM. Plasticity of electrical pacemaking by interstitial cells of Cajal and gastric dysrhythmias in W/W mutant mice. Gastroenterology 123: 2028–2040, 2002.[CrossRef][Web of Science][Medline]
39. Ordog T, Ward SM, Sanders KM. Interstitial cells of Cajal generate electrical slow waves in the murine stomach. J Physiol 518: 257–269, 1999.[Abstract/Free Full Text]
40. Ozaki H, Stevens RJ, Blondfield DP, Publicover NG, Sanders KM. Simultaneous measurement of membrane potential, cytosolic Ca²⁺, and tension in intact smooth muscles. Am J Physiol Cell Physiol 260: C917–C925, 1991.[Abstract/Free Full Text]
41. Perez GJ, Bonev AD, Nelson MT. Micromolar Ca²⁺ from sparks activates Ca²⁺-sensitive K⁺ channels in rat cerebral artery smooth muscle. Am J Physiol Cell Physiol 281: C1769–C1775, 2001.[Abstract/Free Full Text]
42. Petkov GV, Boev K. The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca²⁺-ATPase in the smooth muscle tone of the cat gastric fundus. Pflügers Arch 431: 928–935, 1996.[Web of Science][Medline]
43. Porter VA, Bonev AD, Knot HJ, Heppner TJ, Stevenson AS, Kleppisch T, Lederer WJ, Nelson MT. Frequency modulation of Ca²⁺ sparks is involved in regulation of arterial diameter by cyclic nucleotides. Am J Physiol Cell Physiol 274: C1346–C1355, 1998.[Abstract/Free Full Text]
44. Raeymaekers L, Eggermont JA, Wuytack F, Casteels R. Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca²⁺ pump of bovine pulmonary artery. Cell Calcium 11: 261–268, 1990.[CrossRef][Web of Science][Medline]
45. Raymond GL, Wendt IR. Force and intracellular Ca²⁺ during cyclic nucleotide-mediated relaxation of rat anococcygeus muscle and the effects of cyclopiazonic acid. Br J Pharmacol 119: 1029–1037, 1996.[Web of Science][Medline]
46. Rodriguez P, Kranias EG. Phospholamban: a key determinant of cardiac function and dysfunction. Arch Mal Coeur Vaiss 98: 1239–1243, 2005.[Web of Science][Medline]
47. Santana LF, Kranias EG, Lederer WJ. Calcium sparks and excitation-contraction coupling in phospholamban-deficient mouse ventricular myocytes. J Physiol 503: 21–29, 1997.[Abstract/Free Full Text]
48. Selemidis S, Cocks TM. Nitrergic relaxation of the mouse gastric fundus is mediated by cyclic GMP-dependent and ryanodine-sensitive mechanisms. Br J Pharmacol 129: 1315–1322, 2000.[CrossRef][Web of Science][Medline]
49. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231–236, 1994.[CrossRef][Medline]
50. Sutliff RL, Hoying JB, Kadambi VJ, Kranias EG, Paul RJ. Phospholamban is present in endothelial cells and modulates endothelium-dependent relaxation. Evidence from phospholamban gene-ablated mice. Circ Res 84: 360–364, 1999.[Abstract/Free Full Text]
51. Tack J, Piessevaux H, Coulie B. Role of impaired gastric accommodation to a meal in functional dyspepsia. Gastroenterology 115: 1346–1352, 1998.[CrossRef][Web of Science][Medline]
52. Thornbury KD, Ward SM, Dalziel HH, Carl A, Westfall DP, Sanders KM. Nitric oxide and nitrosocysteine mimic nonadrenergic, noncholinergic hyperpolarization in canine proximal colon. Am J Physiol Gastrointest Liver Physiol 261: G553–G557, 1991.[Abstract/Free Full Text]
53. Verboomen H, Wuytack F, De Smedt H, Himpens B, Casteels R. Functional differences between SERCA2a and SERCA2b Ca²⁺ pumps and their modulation by phospholamban. Biochem J 286: 591–596, 1992.[Web of Science][Medline]
54. Wanstall JC, Homer KL, Doggrell SA. Evidence for, and importance of, cGMP-independent mechanisms with NO and NO donors on blood vessels and platelets. Curr Vasc Pharmacol 3: 41–53, 2005.[CrossRef][Medline]
55. Wayman CP, McFadzean I, Gibson A, Tucker JF. Inhibition by sodium nitroprusside of a calcium store depletion-activated non-selective cation current in smooth muscle cells of the mouse anococcygeus. Br J Pharmacol 118: 2001–2008, 1996.[Web of Science][Medline]
56. Wellman GC, Nelson MT. Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca²⁺-sensitive ion channels. Cell Calcium 34: 211–229, 2003.[CrossRef][Web of Science][Medline]
57. Wellman GC, Santana LF, Bonev AD, Nelson MT. Role of phospholamban in the modulation of arterial Ca²⁺ sparks and Ca²⁺-activated K⁺ channels by cAMP. Am J Physiol Cell Physiol 281: C1029–C1037, 2001.[Abstract/Free Full Text]
58. Yu YC, Guo HS, Li Y, Piao L, Li L, Li ZL, Xu WX. Role of calcium mobilization in sodium nitroprusside-induced increase of calcium-activated potassium currents in gastric antrum circular myocytes of guinea pig. Acta Pharmacol Sin 24: 819–825, 2003.[Web of Science][Medline]
59. ZhuGe R, Tuft RA, Fogarty KE, Bellve K, Fay FS, Walsh JV Jr. The influence of sarcoplasmic reticulum Ca²⁺ concentration on Ca²⁺ sparks and spontaneous transient outward currents in single smooth muscle cells. J Gen Physiol 113: 215–228, 1999.[Abstract/Free Full Text]

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