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

Minkyung Kim, Sang Yun Cho, In Soo Han, Sang Don Koh, and Brian A. Perrino

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

Submitted 24 June 2004 ; accepted in final form 13 January 2005


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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


LOCAL CALCIUM RELEASE EVENTS through ryanodine receptors and IP3 receptors in the sarcoplasmic reticulum (SR) membrane of smooth muscle cells are important regulators of membrane potential and thus smooth muscle excitability and contractility (2, 29). Gastric fundus smooth muscle relaxation is critical to the accommodation reflex, which allows the stomach to expand during food intake with no change in pressure (47). Insufficient gastric accommodation is a frequent pathophysiological mechanism underlying functional dyspepsia (45). Investigating the intrinsic excitation-contraction mechanisms of gastric fundus smooth muscles will enhance our understanding of stomach smooth muscle physiology. In gastric fundus smooth muscles, caffeine can increase the frequency of ryanodine-sensitive Ca2+ release and cause relaxation that is sensitive to the Ca2+-sensitive K+ channel (KCa) blockers tetraethylammonium (TEA) and iberiotoxin, suggesting that membrane hyperpolarization and spontaneous transient outward currents (STOCs) induced by large-conductance Ca2+-activated K+ channels contribute to the relaxation in response to caffeine (35, 41, 48). The frequency of spontaneous or caffeine-induced Ca2+ transients through ryanodine-sensitive Ca2+ release channels is directly related to the SR Ca2+ load (39, 56, 58). Increasing the luminal SR Ca2+ content increases ryanodine receptor open probability, resulting in increased Ca2+ spark frequency (5).

The level of luminal SR Ca2+ depends on the balance between Ca2+ exit through the Ca2+ release channels and Ca2+ entry through the SR Ca2+-ATPase (SERCA pump) (27). SERCA pump activation decreases intracellular [Ca2+] ([Ca2+]i) and elevates the SR Ca2+ load (14). SERCA pump activation can contribute to relaxation by lowering the [Ca2+]i and decreasing the amount of Ca2+ available for contraction (18). However, elevations in luminal SR Ca2+ content by SERCA pump activation also increase Ca2+ spark and STOC frequencies (5). Transgenic techniques have revealed the important role of SERCA pump regulation by phospholamban (PLB) in cardiac, skeletal, and smooth muscles. In hearts from PLB knockout mice, cytosolic Ca2+ clearance is enhanced and rates of contraction and relaxation are increased (40). In bladder smooth muscle, Ca2+ clearance is enhanced, the SR Ca2+ load is increased, and maximum contractile force is decreased in PLB knockout mice (30). In cerebral arteries of PLB knockout mice, Ca2+ spark frequency and transient large-conductance Ca2+-activated K+ channel current frequency are increased (56). In cardiac, skeletal, and several types of smooth muscle, SERCA pump activity and the luminal SR Ca2+ content is increased by PLB phosphorylation by cAMP-dependent protein kinase (PKA) or CaM kinase II (CaMKII) (8, 30, 56). These studies indicate that PLB phosphorylation by PKA or CaMKII is an important modulator of SERCA pump activity and contractile activity and suggest that CaMKII activation and PLB phosphorylation could be involved in the caffeine-induced relaxation of the gastric fundus by ryanodine-sensitive Ca2+ release.

CaMKII holoenzymes are multimers composed of 12 kinase subunits arranged as a stacked pair of rings around a central core complex (9, 13). Four genes encode the kinase subunit isoforms ({alpha}, {beta}, {gamma}, {delta}) and alternative splicing generates additional diversity (49). The nonneuronal {gamma}- and {delta}-subunit isoforms are expressed in smooth muscles, including gastrointestinal smooth muscles (23). CaMKII holoenzymes display complex activation properties that give rise to different levels of Ca2+/CaM-dependent kinase activity (43). Although each kinase subunit binds Ca2+/CaM and becomes activated independently, two or more adjacent Ca2+/CaM-bound subunits are required for Thr287 autophosphorylation to occur (13). Autophosphorylation at Thr287 results in the generation of Ca2+/CaM-independent (autonomous) kinase activity, which extends the physiological response to a transient increase in [Ca2+]i (43). The magnitude of autonomous activity depends on the duration, amplitude, and frequency of the transient increases of [Ca2+]i (7). Thus different frequencies of Ca2+ transients can give rise to different levels of autonomous CaMKII activity. We have shown that the CaMKII inhibitor KN-93 enhances ACh-induced gastric fundus contraction, suggesting that CaMKII activation during contraction inhibits the development of tone (21, 23). Because the [Ca2+] in the vicinity of localized Ca2+ release through ryanodine-sensitive Ca2+ channels is in the range that will activate CaMKII (33), these findings suggest that localized Ca2+ release events from internal Ca2+ stores of smooth muscle may activate CaMKII. Therefore, we investigated the effects of caffeine-sensitive Ca2+ release on the activation of CaMKII, PLB phosphorylation, and tone in gastric fundus smooth muscles.


    METHODS
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 ABSTRACT
 METHODS
 MATERIALS
 RESULTS
 DISCUSSION
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Tissue preparation for CaMKII assay. Gastric fundus smooth muscles were obtained from adult CD-1 mice (6 to 8 wk old) (Charles River Laboratories, Wilmington, MA). The animals were anesthetized by isofluorane inhalation and euthanized by decapitation. Mice were maintained and the 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 the University of Nevada at Reno Institutional Animal Care and Use Committee. The gastric fundus tissues were pinned out in a Sylgard-lined dish containing Krebs solution composed of (in mM) 120 NaCl, 6 KCl, 15 NaHCO3, 12 glucose, 3 MgCl2, 1.5 NaH2PO4, and 3.5 CaCl2, pH 7.2. The mucosa and submucosa layers were removed with the use of fine-tipped forceps. For determining the effects of caffeine or cyclopiazonic acid (CPA) on CaMKII activity, the 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. For determining the effects of various blockers on caffeine-induced CaMKII activity, the 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 caffeine for 15 min in the presence of each blocker. After treatment, the tissues were collected, frozen in liquid nitrogen, and stored at –80°C. When needed for assays, the frozen tissues were homogenized at 4°C with a glass tissue grinder in lysis buffer (50 mM MOPS, 0.2% Nonidet P-40, 100 mM Na4P2O7, 100 mM NaF, 250 mM NaCl, 3 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors). Homogenates were centrifuged at 16,000 g for 15 min at 4°C, and the supernatants were aliquotted and stored at –80°C. Protein concentrations were determined using the Bradford assay with bovine {gamma}-globulin as a standard.

CaMKII activity assays. CaMKII activity in the lysates was assayed in a total volume of 30 µl containing 50 mM HEPES (pH 7.4), 10 mM magnesium acetate, 0.2 mM [{gamma}-32P]ATP (500–1,000 cpm/pmol) (DuPont, Boston, MA), 20 µM autocamtide-2 (a specific CaMKII 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). Reactions were initiated by the addition of 3 µg of lysate proteins, allowed to proceed at 30°C for 2 min, and terminated by spotting 10 µl of the reaction onto P-81 paper. The papers were washed thoroughly in 75 mM phosphoric acid, rinsed in ethanol, and dried. The papers were added to vials of Ecoscint O (National Diagnostics, Atlanta, GA), and affixed radioactivity was quantified by scintillation counting. Kinase activity was calculated and expressed as picomoles of Pi incorporated per minute per microgram of lysate protein. Total (Ca2+/CaM stimulated) and autonomous (Ca2+/CaM independent) CaMKII activities from the cytosolic fraction of control and treated gastric fundus smooth muscles from at least three animals were assayed in triplicate from each tissue as described. Autonomous activity is expressed as a percentage of the total Ca2+/CaM-dependent activity.

Simultaneous measurement of cytosolic [Ca2+] and tension in fundus smooth muscles. Gastric fundus smooth muscles were obtained from adult CD-1 mice (as described above). Muscle strips (~3 mm x 10 mm) were cut parallel to the circular muscle layer. The muscle strips were placed in solutions of 10 µg/ml fura-2 AM (Molecular Probes, Eugene, OR) with 0.01% cremofor EL for 2.5 h at 37°C. After being loaded, the tissues were transferred to the recording chamber maintained at 37 ± 0.5°C with oxygenated Krebs-Ringer bicarbonate (KRB) solution. One end of the tissue was attached to a small isometric force transducer using thread. A resting force of 400 mg was applied to set the muscles at optimum length. After a 1-h equilibration period with KRB solution, the muscle strips were incubated in KRB solution containing the compounds. Cytosolic [Ca2+] and contractile tension were measured using a modified organ bath interfaced with a photometer (model CAF102, Jasco), as previously described (32). Signals from photometer and force responses were recorded on a personal computer running Acqknowlege 3.2.6 (model MP100, BIOPAC Systems, Santa Barbara, CA).

Mechanical responses of gastric fundus smooth muscles. A standard organ bath technique was employed to measure changes in force provided by fundus smooth muscle strips (~6 mm x 3 mm). One end of the tissue was attached to a fixed mount, and the opposite end to a Fort 10 isometric strain gauge (WPI, Sarasota, FL). The muscles were immersed in organ baths maintained at 37 ± 0.5°C with oxygenated KRB. A resting force of 400 mg was applied to set the muscles at optimum length. This was followed by an equilibration period of 1 h, during which time the bath was continuously perfused with oxygenated KRB. After the equilibration period, the muscle strips were incubated in Krebs containing the compounds, as indicated in the figure legends. Mechanical responses were recorded on a personal computer running Acqknowlege 3.2.6 (BIOPAC Systems). At the end of the experiments, the wet weight of each muscle strip was measured. Contractile force is indicated as mN/g of the gastric fundus smooth muscle tissues, as previously described (3, 31, 51, 52).

SDS-PAGE and Western blot analysis of PLB phospho-Thr17 and phospho-Ser16 from gastric fundus smooth muscle SR fraction. Gastric fundus smooth muscles obtained from adult CD-1 mice (as described above) were grouped into Krebs control or caffeine treated. For Krebs control, the tissues were equilibrated in Krebs buffer for 45 min at 37°C and for caffeine or 3-isobutyl-1-methylxanthine (IBMX) treatment, the tissues were equilibrated in Krebs buffer for 45 min at 37°C and then incubated at 37°C for 15 min. For KN-93 treatment, tissues were preequilibrated for 20 min. After treatment, the tissues were collected, frozen in liquid nitrogen, and stored at –80°C until used. Gastric fundus smooth muscle SR fractions were obtained from CD-1 mice, as described previously (17). Briefly, the frozen tissues were homogenized at 4°C with a glass tissue grinder in 6 volumes by weight 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 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 the samples placed on ice for 25 min. The samples were then centrifuged at 40,000 g for 60 min at 4°C to pellet the SR fraction. The pellets were resuspended in 200 µl of SR buffer composed of (in mM) 10 Tris·HCl, pH 6.8, 100 KCl, 100 NaF, 20 sodium pyrophosphate, and protease inhibitor tablet, and stored at –80°C. Protein concentrations were determined with the Bradford assay using bovine {gamma}-globulin as standard. Gastric fundus smooth muscle SR proteins were separated by SDS-PAGE (15% high-salt gel) and transferred to nitrocellulose by Western blot analysis. The blots were incubated with primary and secondary antibodies, washed, and processed for enhanced chemiluminescence image detection using ECL Advantage (Amersham Biosciences, Piscataway, NJ). The PLB-PO4-Thr17 and PLB-PO4-Ser16 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used at a 1:5,000 dilution ratio, and the horseradish peroxidase-conjugated secondary antibody (Chemicon, Temecula, CA) was used at a 1:50,000 dilution ratio. Protein bands were visualized with a charge-coupled device camera-based detection system (Epi Chem II, UVP Laboratory Products). The collected Tiff images were analyzed with the use of Adobe Photoshop. Densitometry of the immunostained PLB protein bands was carried out with Un-Scan-It software (Silk Scientific, Orem, UT) using lane analysis for background subtraction. The control signal values were normalized to a value of 1.

Intracellular microelectrode recordings. Gastric fundus smooth muscles obtained from adult CD-1 mice (as described above) were maintained at 37.5 ± 0.5°C by a flowing KRB solution containing (in mM) 120.4 NaCl, 5.9 KCl, 15.5 NaHCO3, 11.5 glucose, 1.2 MgCl2, 1.2 NaH2PO4, and 2.5 CaCl2. The solution was bubbled with 97% O2-3% CO2 and had a pH of 7.3–7.4. Smooth muscle cells were impaled with glass microelectrodes filled with 3 M KCl having resistances between 50 and 80 M{Omega}. Transmembrane potential was measured with a high input impedance amplifier (model S-7071, WPI). Tissues were equilibrated for ~1 h before recordings were begun.


    MATERIALS
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 ABSTRACT
 METHODS
 MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Caffeine, ryanodine, tetracaine, sodium nitroprusside, iberiotoxin, apamin, and IBMX were obtained from Sigma (St. Louis, MO). CPA was purchased from BioMol. KN-93 was purchased from EMD Biosciences, and 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 were tabulated as means ± SE. Data sets were tested for significance using Student's t-test. Data were considered significantly different from control values when P < 0.05.


    RESULTS
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 ABSTRACT
 METHODS
 MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of caffeine on fundus smooth muscle tone and CaMKII activity. Incubation of murine gastric fundus smooth muscles with 1 mM caffeine resulted in a 70.82 ± 9.84 mN/g decrease in basal tone (n = 20). A typical response is shown in Fig. 1A. This extent of relaxation was compared with the relaxation evoked by 10 µM sodium nitroprusside. The level of relaxation induced by 1 mM caffeine was 82 ± 6.2% of the sodium nitroprusside-induced relaxation, as shown in the representative isometric force recording in Fig. 1B (n = 15). After ryanodine pretreatment (10 µM), the caffeine-induced relaxation was inhibited by 32.9 ± 5.5% (n = 6). A typical response is shown in (Fig. 1C). Similarly, tetracaine (100 µM) also inhibited caffeine-induced relaxation by 38.2 ± 9.4% (n = 9). A representative mechanical recording is shown in Fig. 1D. These results suggest that 1 mM caffeine relaxes gastric fundus smooth muscle by increasing Ca2+ release through ryanodine and tetracaine-sensitive Ca2+ channels in the SR membrane. To investigate whether CaMKII is activated in caffeine-treated gastric fundus smooth muscles, we measured CaMKII activity in gastric fundus smooth muscle strips incubated without and with 1 mM caffeine. After 15 min of caffeine treatment to relax the gastric fundus smooth muscle tissues, CaMKII activity was increased, as indicated by the increase in CaMKII autonomous activity from 35.6 ± 2.99% to 51.2 ± 2.39% of total activity (Fig. 2) (n = 8; P < 0.05). To test the possibility that caffeine-induced CaMKII activation may be due to ryanodine-sensitive Ca2+ release from the SR, we examined the effects of ryanodine or tetracaine on caffeine-induced CaMKII activation. As shown in Fig. 2, in the presence of ryanodine (10 µM) or tetracaine (100 µM), 1 mM caffeine did not increase CaMKII autonomous activity. As a percentage of total CaMKII activity, autonomous activity levels in the presence of caffeine plus ryanodine (36.2 ± 2.9%) or caffeine plus tetracaine (31.3 ± 1.8%) were similar to autonomous activity levels from control smooth muscles (35.6 ± 2.9%) (n = 8). In addition, ryanodine or tetracaine alone had no effect on both total and autonomous CaMKII activities compared with control.



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Fig. 1. Caffeine relaxes gastric fundus smooth muscle in a ryanodine- or tetracaine-sensitive manner. Isometric force was measured, as described in METHODS. Typical changes in tension of murine gastric fundus smooth muscle strips incubated with 1 mM caffeine (A) or 10 µM sodium nitroprusside (SNP) (B). C: typical changes in tension of murine gastric fundus smooth muscle strips incubated with 10 µM ryanodine alone, followed by caffeine in the presence of ryanodine. D: typical changes in tension of murine gastric fundus smooth muscle strips incubated with 1 mM caffeine alone or caffeine in the presence of 100 µM tetracaine.

 


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Fig. 2. Caffeine activates CaM kinase II (CaMKII) in a ryanodine- or tetracaine-sensitive manner. Total and autonomous CaMKII activities were measured as described in METHODS. Averaged values of total (open bars) and autonomous (gray bars) CaMKII activities from gastric fundus smooth muscles that were untreated, or treated with caffeine alone (1 mM), ryanodine alone (10 µM), or tetracaine alone (100 µM) for 15 min; or after 20 min of ryanodine or tetracaine pretreatment, caffeine in the presence of ryanodine (10 µM) or tetracaine (100 µM) for 15 min. *P < 0.05 for the autonomous activity as a percentage of the total activity.

 
Effects of KCa channel blockers on caffeine-induced hyperpolarization and relaxation. Consistent with the findings in Fig. 1 showing caffeine-induced relaxation of murine gastric fundus smooth muscles, we found that 1 mM caffeine hyperpolarized the membrane potential of gastric fundus smooth muscle from –41.32 ± 2.67 to –54.8 ± 3.18 mV (Fig. 3A) (P < 0.05; n = 6). Ryanodine and tetracaine each blocked the caffeine-induced hyperpolarization. As shown in Fig. 3B, in the presence of ryanodine the membrane potential depolarized from –40.93 ± 1.29 to –33.2 ± 1.1 mV (P < 0.05) (n = 5), and hyperpolarization did not occur when caffeine was present. Similarly, tetracaine depolarized the membrane potential from –42.79 ± 2.84 to –28.98 ± 2.67 mV and after the addition of caffeine, the membrane potential remained relatively unchanged (–32.34 ± 1.17 mV) (Fig. 3C) (n = 4).



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Fig. 3. Effects of ryanodine, tetracaine, iberiotoxin, or apamin on caffeine-induced membrane potential hyperpolarization and relaxation. Membrane potential and isometric force were measured as described in METHODS. Typical changes in membrane potential of murine gastric fundus smooth muscle strips incubated with 1 mM caffeine (A), caffeine in the presence of 10 µM ryanodine (B), or caffeine in the presence of 100 µM tetracaine (C). Typical changes in tension of murine gastric fundus smooth muscle strips incubated with caffeine in the presence of 100 nM iberiotoxin (IbTx) (D) or 300 nM apamin (E). Typical changes in membrane potential of murine gastric fundus smooth muscle strips incubated with 100 nM iberiotoxin (F).

 
Experiments were carried out to determine the involvement of KCa channels in the caffeine-induced relaxation and membrane hyperpolarization of gastric fundus smooth muscles. Iberiotoxin, but not apamin, inhibited caffeine-induced relaxation, although both iberiotoxin and apamin each evoked increases in fundus smooth muscle tone. As indicated in Fig. 1, 1 mM caffeine decreased fundus tone by 70.82 ± 9.84 mN/g. In the presence of 100 nM iberiotoxin, the relaxation evoked by caffeine was inhibited by 60.7 ± 3.9% (Fig. 3D) (n = 6), as indicated by a 27.63 ± 1.72 mN/g decrease in fundus tone. In contrast, in the presence of apamin (300 nM) caffeine decreased the fundus tone by 62.17 ± 8.3 mN/g (Fig. 3E) (n = 6). Iberiotoxin depolarized the membrane potential from –41.55 ± 0.77 to –35.25 ± 0.35 mV, and inhibited the hyperpolarization induced by caffeine (Fig. 3F) (n = 2).

Effect of CPA on fundus smooth muscle tone and caffeine-induced relaxation and CaMKII activation. The results shown in Fig. 2 indicate that 1 mM caffeine activates CaMKII in a ryanodine-sensitive manner, suggesting that CaMKII activation involves Ca2+ influx into the cytosol through ryanodine receptors. It has been shown previously that Ca2+ release through ryanodine receptors depends on a Ca2+ supply maintained by SERCA pump-dependent refilling of SR Ca2+ stores (15, 39, 58). These findings suggest that SERCA pump inhibition would inhibit caffeine-induced relaxation and CaMKII activation. CPA by itself increased fundus smooth muscle tone by 224.52 ± 21.24 mN/g (n = 15). A typical response is shown in Fig. 4A. These increases in tension were accompanied by increased cytosolic Ca2+ levels, as measured by the change in the Ca2+ indicator fura-2 ratio (Fig. 4A) (n = 5). In addition, CPA significantly inhibited caffeine-induced relaxation of gastric fundus smooth muscle. In the presence of CPA, the level of relaxation was only 33.32 ± 8.8% (P < 0.05) of the relaxation from caffeine alone (n = 10), as shown by a representative mechanical recording in Fig. 4B. As shown in Fig. 4C, caffeine alone increased CaMKII autonomous activity from 34.7 ± 4.1% of total activity to 51.2 ± 2.39% (P < 0.05) of total activity, but in the presence of CPA and caffeine, CaMKII autonomous activity remained at 34.2 ± 3.2% of total activity (n = 5). Furthermore, similar to ryanodine, both total and autonomous CaMKII activity levels in the presence of CPA were similar to control levels (Fig. 4C) (n = 5), indicating that CPA does not activate CaMKII even though cytosolic Ca2+ levels increased.



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Fig. 4. Effects of cyclopiazonic acid (CPA) on murine gastric fundus smooth muscle tension, intracellular [Ca2+] ([Ca2+]i), and caffeine-induced relaxation and CaMKII activation. Isometric force, [Ca2+]i, and CaMKII activity were measured as described in METHODS. A: typical changes in tension and averaged values of the fura 2 ratios from murine gastric fundus smooth muscle strips incubated with 10 µM CPA. B: typical changes in tension of murine gastric fundus smooth muscle strips incubated with 1 mM caffeine in the presence of 10 µM CPA, or caffeine alone. C: averaged values of total (open bars) and autonomous (gray bars) CaMKII activities in lysates from murine gastric fundus smooth muscle strips incubated with 1 mM caffeine for 15 min; or after 20 min of CPA pretreatment, caffeine in the presence of CPA (10 µM) for 15 min. *P < 0.05 for the autonomous activity as a percentage of the total activity.

 
Effect of caffeine-induced CaMKII activation on PLB phosphorylation. The findings shown in Fig. 4 suggest that refilling of SR Ca2+ stores by the SERCA pump is necessary for caffeine-induced, ryanodine-sensitive CaMKII activation. Because phosphorylation of PLB Thr17 by CaMKII enhances SERCA pump activity (42, 44, 46), we investigated whether PLB Thr17 phosphorylation is increased by caffeine-induced CaMKII activation. PLB phosphorylation was monitored by Western blot analysis using an antibody specific for phospho-Thr17. As shown in the Western blots of Figs. 5A and 6B, compared with control, PLB Thr17 phosphorylation is increased by incubation of gastric fundus smooth muscles with 1 mM caffeine. Densitometry of the signals obtained with the anti-phospho-Thr17 antibody indicates a 1.71 ± 0.23-fold increase in signal intensity with caffeine treatment compared with untreated tissues. The CaMKII inhibitor KN-93 (10 µM) inhibited the caffeine-induced increase in PLB Thr17 phosphorylation (n = 8). In the presence of caffeine plus KN-93 (10 µM) a signal intensity value of 1.01 ± 0.26 relative to the normalized signal intensity values of 1 obtained from untreated tissues was measured (Fig. 5A). As shown in Fig. 5B, KN-93 (10 µM) inhibited caffeine-induced CaMKII activation (n = 5), suggesting that the increase in PLB phospho-Thr17 staining observed with caffeine treatment of gastric fundus smooth muscles is due to CaMKII activation.



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Fig. 5. Effects of caffeine and KN-93 on phospholamban (PLB) Thr17 phosphorylation and CaMKII activation. SDS-PAGE and immunoblotting using antibodies specific for phospho-Thr17 were carried out as described in METHODS. A: representative blot of PLB phospho-Thr17 in the SR-enriched fraction from control, caffeine-treated for 15 min; or after 20-min pretreatment with KN-93, caffeine in the presence of KN-93 (10 µM), gastric fundus smooth muscle tissues (P = PLB pentamer). B: averaged values of total (open bars) and autonomous (gray bars) CaMKII activities in lysates from murine gastric fundus smooth muscle strips incubated with 1 mM caffeine for 15 min; or after 20 min of 10 µM KN-93 pretreatment, caffeine in the presence of KN-93 for 15 min. *P < 0.05 for the autonomous activity as a percentage of the total activity.

 


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Fig. 6. Effects of caffeine and 3-isobutyl-1-methylxanthine (IBMX) on PLB Ser16 and Thr17 phosphorylation. SDS-PAGE and immunoblotting using antibodies specific for phospho-Ser16 or phospho-Thr17 were carried out, as described in METHODS. A: representative blot of PLB phospho-Ser16 in the SR-enriched fraction from control, caffeine-treated for 15 min, or IBMX-treated for 15 min, gastric fundus smooth muscle tissues. B: representative blot of PLB phospho-Thr17 in the SR-enriched fraction from caffeine-treated for 15 min, control, or IBMX-treated for 15 min, gastric fundus smooth muscle tissues (P = PLB pentamer).

 
Because caffeine is also a phosphodiesterase inhibitor, we determined the effect of 1 mM IBMX treatment on PLB Ser16, and PLB Ser Thr17 phosphorylation. IBMX increased PLB phospho-Ser16 staining (Fig. 6A), but did not increase PLB phospho-Thr17 staining (Fig. 6B). In contrast, caffeine did not increase PLB phospho-Ser16 staining (Fig. 6A), but did increase PLB phospho-Thr17 staining (Fig. 6B) (n = 5). We also measured CaMKII activity and isometric force from IBMX-treated gastric fundus smooth muscles. Although 1 mM IBMX decreased the tone by 98.45 ± 12.09 mN/g (n = 4) within 5 min, the CaMKII autonomous activity levels of 32.13 ± 2.8% of total CaMKII activity in the presence of 1 mM IBMX (n = 4) were similar to control levels.

To investigate the role of CaMKII in caffeine-induced relaxation, gastric fundus smooth muscle tone was measured from tissues incubated with caffeine in the absence or presence of KN-93 (10 µM). As shown by the representative mechanical recording in Fig. 7A, KN-93 inhibited caffeine-induced relaxation by 50.6 ± 9.2% (P < 0.05; n = 8). In addition, the hyperpolarization evoked by caffeine alone was prevented by KN-93. The resting membrane potential of –40.61 ± 2.48 mV was not significantly affected by KN-93 alone (–36.18 ± 2.35 mV), and after the addition of caffeine a membrane potential value of –39.99 ± 2.37 mV was measured (Fig. 7B) (n = 4). Together, these findings suggest that PLB Thr17 phosphorylation by CaMKII is involved in caffeine-induced relaxation of murine gastric fundus smooth muscles.



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Fig. 7. Effects of caffeine and KN-93 on gastric fundus smooth muscle relaxation and hyperpolarization. A: typical changes in tension of gastric fundus smooth muscle strips incubated with 1 mM caffeine or caffeine in the presence of 10 µM KN-93. B: typical changes in membrane potential of gastric fundus smooth muscle strips incubated with 10 µM KN-93, followed by 1 mM caffeine in the presence of 10 µM KN-93.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported that ACh activates CaMKII in murine gastric fundus smooth muscle, but the mechanism of CaMKII activation was not addressed (23). Because CaMKII activation varies with the frequencies of Ca2+ oscillations, different Ca2+ sources are likely to give rise to different activation states of CaMKII (13). Gastrointestinal smooth muscle cells utilize extra- and intracellular Ca2+ to regulate membrane potential, sensitivity, and contractility (4, 38, 55). Transient localized Ca2+ release events from the SR play an important role in modulating gastrointestinal smooth muscle contractility (2, 11). In stomach smooth muscles, NO and caffeine increase Ca2+ sparks and STOCs, in part, via ryanodine-sensitive mechanisms (41, 48). The [Ca2+] in the vicinity of ryanodine-sensitive Ca2+ sparks in vascular smooth muscle is in the range that will activate CaMKII (33). These findings led us to investigate whether CaMKII is activated by caffeine and contributes to gastric fundus smooth muscle relaxation.

We found that caffeine activates CaMKII in a tetracaine- and ryanodine-sensitive manner. Caffeine increases Ca2+ spark and STOC frequencies in vascular and gastric fundus smooth muscles, suggesting that the caffeine-induced CaMKII activation we observed in gastric fundus smooth muscles involved increased Ca2+ spark activity (20, 26, 41, 48). Ca2+ release from the SR partly depends on a Ca2+ supply in the SR lumen that is maintained by the SERCA pump (5, 39, 58). The finding that CPA prevented caffeine-induced CaMKII activation suggests that SR luminal Ca2+ levels were lowered and insufficient to support caffeine-induced Ca2+ spark activity and CaMKII activation (15, 50). These findings also suggest that CaMKII autonomous activity levels in unstimulated gastric fundus smooth muscles are sensitive to Ca2+ release from the SR. However, ryanodine, tetracaine, or CPA alone did not affect total and autonomous CaMKII activity levels. Spark frequencies of 0.5–1.5 Hz have been reported in isolated stomach smooth muscle cells (58), which may be too low to activate CaMKII. The mechanisms involved in maintaining the CaMKII autonomous activity levels in unstimulated gastric fundus smooth muscles are currently under investigation. Similar to previous reports, SERCA pump inhibition with CPA raised [Ca2+]i and induced contraction (25, 35, 36). Other studies (34, 55) have reported that nifedipine-sensitive Ca2+ influx is partly necessary for the contractions induced by CPA. However, CPA did not result in CaMKII activation, suggesting that the type of elevation of [Ca2+]i induced by CPA is unable to activate CaMKII.

SERCA pump activity also contributes to relaxation by clearing Ca2+ from the cytosol (5, 25, 34, 56). Similar to cyclic-nucleotide mediated SERCA pump activation by PLB phosphorylation at Ser16, elevation of [Ca2+]i can activate the SERCA pump via CaMKII phosphorylation of PLB at Thr17 (42, 44, 46). Caffeine increased PLB phospho-Thr17 staining, indicating that PLB is a substrate of CaMKII activated by caffeine. In addition, both caffeine-induced relaxation and PLB Thr17 phosphorylation were inhibited by treatment of fundus smooth muscles with KN-93. These findings suggest that KN-93 inhibition of CaMKII and PLB Thr17 phosphorylation mimics the effects of CPA on caffeine-induced relaxation by causing inhibition of SERCA pump by nonphosphorylated PLB. KN-93 was shown to decrease resting Ca2+ spark frequency by 88% and SR Ca2+ content by 37% in isolated ferret aorta smooth muscle cells (19). Together, these results suggest that the inhibition of caffeine-induced relaxation of gastric fundus smooth muscle by KN-93 involves inhibition of Ca2+ spark activity by decreased SERCA pump activity due to lowered PLB Thr17 phosphorylation by CaMKII. However, it has also been reported that CaMKII phosphorylates SERCA 2 and increases the Vmax of Ca2+ transport (28, 57). Furthermore, CaMKII phosphorylation of RyR2 increases its open probability (53). Thus a role for direct phosphorylation of SERCA2 and RyR2 by caffeine-activated CaMKII in the relaxation evoked by caffeine in gastric smooth muscle tissues cannot be ruled out. RyR1, RyR2, and RyR3 mRNAs are expressed in murine stomach, although their locations within specific stomach regions were not analyzed (10).

Caffeine-evoked relaxations were only partially inhibited by iberiotoxin, suggesting that enhanced Ca2+ clearance from the cytosol also contributes to the caffeine-induced relaxation. These findings are different from a previous report (48) showing that TEA had no effect on caffeine-induced relaxation of murine gastric corpus smooth muscle strips. The differences between the two studies are likely due to the differences in caffeine concentrations used, and the use of different stomach smooth muscles. In their study, 3 mM caffeine was used and produced a transient initial contraction, followed by relaxation. We used 1 mM caffeine, which did not produce an initial contraction. In addition, the TEA-sensitive STOCs evoked by caffeine were obtained from corpus smooth muscle cells held at –20 mV (48). However, the effects of TEA on caffeine-induced relaxation were examined in corpus smooth muscle strips, presumably characterized by an initial resting membrane potential of –51 mV (48). The results of the current-voltage relationships of the caffeine-induced STOCs from isolated gastric corpus smooth muscle cells at more negative potentials suggest that TEA-sensitive Ca2+-activated K+ channel activity would be lower in the gastric corpus smooth muscle strips (48). In these conditions, an enhanced rate of Ca2+ uptake by the SERCA pump may play a predominant role in the caffeine-induced relaxation. In contrast, we measured the effects of iberiotoxin on caffeine-evoked relaxations from gastric fundus smooth muscle strips exhibiting a resting membrane potential of –41.55 ± 0.77 mV. At –41.55 ± 0.77 mV, TEA- or iberiotoxin-sensitive Ca2+-activated K+ channel activity could play a greater role in caffeine evoked relaxation. It is likely both mechanisms are involved in the effects of caffeine on gastric fundus tone, but the relative importance of each is unknown. Nevertheless, the findings that iberiotoxin, but not apamin, partially inhibited caffeine-induced relaxation of gastric fundus smooth muscle strips suggest that large-conductance Ca2+-activated K+ channels play a role in the caffeine-induced membrane hyperpolarization and relaxation of gastric fundus smooth muscles cells.

Caffeine has many pharmacological effects, including adenosine receptor blockade, elevation of cAMP levels by inhibition of phosphodiesterases, and augmentation of Ca2+ release from intracellular stores (6). Similar to caffeine, IBMX relaxed murine gastric fundus smooth muscle strips. However, CaMKII activity and PLB Thr17 phosphorylation were unaffected by IBMX. In addition, the finding that PLB Ser16 phosphorylation was not increased by caffeine does not support a mechanism of elevated cAMP levels due to phosphodiesterase inhibition in caffeine-induced relaxation of gastric fundus smooth muscles (6, 41). Thus our findings showing that relaxation of gastric fundus smooth muscles by caffeine is ryanodine sensitive, and involves CaMKII activation and PLB Thr17 phosphorylation point to a role for increased Ca2+ clearance from the cytosol and Ca2+ release from the SR in the gastric fundus smooth muscle relaxation.

Smooth muscle excitability and contractility involves several integrated signaling systems (4, 22, 24). Transient localized Ca2+ release events (Ca2+ sparks or puffs) can lead to smooth muscle relaxation by activating STOCs through Ca2+-activated K+ channels (2, 11, 29, 55). Increased clearance of Ca2+ from the cytosol into the SR lumen relaxes smooth muscles and decreases the contractile force by decreasing the Ca2+ available for contraction and increasing Ca2+ spark and STOC frequencies (25). Ca2+ sparks, STOCs, and vasodilation are all augmented in cerebral arteries of PLB knockout mice, suggesting that enhanced refilling of intracellular Ca2+ stores promotes Ca2+ spark activity (56). In bladder smooth muscle, in which relaxation is also characterized by Ca2+ sparks and STOCS, PLB knockout mice show increased Ca2+ clearance from the cytoplasm and decreased detrussor muscle contraction (16, 30, 54). In cardiac myocytes, Ca2+ transients can modulate the phosphorylation of PLB by CaMKII, and thus SERCA pump activity and SR Ca2+ load (12). The ability of CaMKII to function as a frequency decoder of Ca2+ signals and phosphorylate PLB Thr17 in response to different stimulation frequencies warrants further investigations into the modulation of smooth muscle contractility by differential activation by CaMKII (12). The response of CaMKII to increases in cytosolic Ca2+ levels is influenced by the subunit composition of the holoenzymes (7, 9). The {delta}- and {gamma}-holoenzymes expressed in gastrointestinal smooth muscles display different kinetics of Ca2+/CaM binding and rates of autophosphorylation (9, 23). Although bulk increases in cytosolic Ca2+ activate CaMKII, frequency-dependent activation by transient Ca2+ release resulting in localized high Ca2+ concentrations are likely to activate nearby CaMKII (1). SR-associated CaMKII is a candidate for activation by Ca2+ release from the SR through ryanodine receptors (37), and we are investigating the characteristics of CaMKII association with the SR of gastric fundus smooth muscles. In summary, the results of this study show that caffeine activates CaMKII in a ryanodine-sensitive manner, and that CaMKII activation and PLB Thr17 phosphorylation are involved in the mechanisms by which caffeine relaxes gastric fundus smooth muscles. These findings provide further insights into the Ca2+-sensitive processes that modulate smooth muscle excitation-contraction.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Center for Research Resources Grant NCRR018751.


    ACKNOWLEDGMENTS
 
We thank Kent Sanders, Jim Kenyon, and Brian Hagen for many helpful discussions and help with Ca2+ measurements.


    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}physiology.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.


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