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Am J Physiol Heart Circ Physiol 278: H1856-H1865, 2000;
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Vol. 278, Issue 6, H1856-H1865, June 2000

Endoplasmic reticulum contribution to the relaxant effect of cGMP- and cAMP-elevating agents in feline aorta

Cecilia Mundiña-Weilenmann1, Leticia Vittone1, Gustavo Rinaldi2, Matilde Said1, Gladys Chiappe de Cingolani1, and Alicia Mattiazzi1

1 Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, and 2 Cátedra de Fisiología, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1900 La Plata, Argentina


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The contribution of endoplasmic reticulum (ER) and phosphorylation of phospholamban (PLB) to the relaxant effect of cGMP- and cAMP-elevating agents was studied in feline aorta. Sodium nitroprusside (NP, 100 µM) completely relaxed contracture induced by 10 µM norepinephrine. This NP-induced relaxation was partially prevented by tetraethylammonium, suggesting that a fraction of NP-induced relaxation was mediated by activation of K+ channels. In the absence and presence of tetraethylammonium, the relaxant effect of NP was associated with a significant increase in Ser16 phosphorylation of PLB immunodetected by phosphorylation site-specific antibodies. The relaxant effect of NP on aortic strips precontracted with 80 mM KCl was significantly reduced by 1 µM thapsigargin. This decrease, which represents the ER contribution to the relaxant effect of NP, reached 23 ± 9% at 100 µM NP and was closely associated with a dose-dependent increase in Ser16 phosphorylation (128 ± 49% over control at 100 µM NP). Effects of NP were associated with a significant increase in activity of protein kinase G and were mimicked by 8-bromo-cGMP. Forskolin produced a dose-dependent relaxant effect on KCl-induced contracture, which reached 64 ± 8% at 50 µM and was associated with an increase in phosphorylation of Ser16 residue of PLB (88 ± 18% over control). Thapsigargin reduced this relaxant effect by 38 ± 9%. 8-Bromo-cAMP mimicked effects of forskolin. The ER-mediated relaxant effect and the increase in Ser16 phosphorylation produced by forskolin were partially blocked by the protein kinase A inhibitor H-89 (5 µM). The results indicate that ER partially contributes to the relaxant effect of NP and forskolin in feline aorta. This effect may be mediated by the associated increase in Ser16 phosphorylation of PLB.

vascular smooth muscle; relaxation; phospholamban


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PHOSPHOLAMBAN (PLB) is a phosphorylatable protein component of cardiac sarcoplasmic reticulum (SR) that reversibly inhibits the activity of the SR Ca2+-ATPase (SERCA2) and SR Ca2+ transport. Phosphorylation of PLB by cAMP-dependent protein kinase (PKA) at the Ser16 residue or Ca2+/calmodulin-dependent protein kinase at the Thr17 residue relieves this inhibition, thus increasing ATPase activity and the rate of Ca2+ uptake by the SR (13, 34, 36). In smooth muscle membrane vesicles, it has also been shown that phosphorylation of PLB by PKA or cGMP-dependent protein kinase (PKG) produced an increase in the activity of the Ca2+ pump of the sarco(endo)plasmic reticulum and an increase in Ca2+ transport (29, 42). Although these in vitro studies indicated that the behavior of PLB is similar in both types of muscles, the physiological role of PLB phosphorylation in smooth muscle is less well defined than in cardiac muscle (22, 23, 25, 40). Karczewski et al. (15, 16) demonstrated in rat aorta and pig coronary smooth muscle that an increase in PLB phosphorylation was associated with an increase in the relaxation produced by nitric oxide (NO). These results contrast with earlier experiments by Huggins et al. (12), in which PLB was not phosphorylated in sheep pulmonary artery, and with more recent findings obtained in rat tail artery, in which PLB seems not to have any significant role in the relaxant effect of PKG- and PKA-activating agents (4, 32, 38). The physiological meaning of PLB in smooth muscle has also been explored in PLB knockout mice. The absence of PLB in these animals should lead to a faster Ca2+ uptake by the endoplasmic reticulum (ER), which would produce in turn a faster relaxation and eventually an increase in contractility. The results of these experiments did indicate that PLB modulates smooth muscle contractility but unexpectedly failed to demonstrate a significant role of this protein in the regulation of smooth muscle relaxation (17). A recent study by the same group indicated, however, that the rate of intracellular Ca2+ decline was indeed increased in PLB knockout animals (18).

The availability of specific antibodies against PLB and site-specific phosphorylated PLB phosphopeptides allowed us in a previous study to identify PLB in cat aortic smooth muscle and to directly determine that stimulation of the PKG cascade by sodium nitroprusside (NP) evoked an increase in Ser16 phosphorylation of PLB (41). In the present experiments, these specific antibodies, in association with measurements of mechanical parameters, will be used to gain further insights into the possible role of the PKA- and PKG-dependent cascades of PLB phosphorylation in the relaxation of vascular smooth muscle.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanical Studies

Cats were anesthetized with pentobarbital sodium (50 mg/kg ip). The aorta was excised and cleaned of adherent connective tissue, and the entire vessel was cut into a large helical strip 4-5 mm wide and 0.3-0.5 g wet wt. The strips were then divided into halves. Both ends of each half were tied together, forming a large ring that was suitable for use in mechanical and biochemical studies. Contractile responses were carried out as previously described (33) in a water-jacketed organ bath filled with a modified Krebs solution of the following composition (in mM): 130 NaCl, 4.7 KCl, 0.4 Na2HPO4, 1.16 MgSO4, 20.2 NaHCO3, 1.35 CaCl2 and 11.0 glucose. The organ bath was kept at 37°C and equilibrated with 5% CO2-95% O2 to give a pH of 7.40. The ring was gently suspended between two stainless steel wires that could be separated with a micrometer to achieve the desired length and/or passive force. The lower wire was fixed to a vertical plastic rod immersed in the organ bath, and the upper wire was connected to a force transducer (model FT.03D, Grass). The output of the transducer was amplified and fed into an analog-digital board (model DT16EZ, Data Translation, Marlboro, MA) mounted in a desktop computer. On-line recordings and files for later processing were obtained with an appropriated software (Labtech Notebook Pro, Laboratory Technology, Wilmington, MA). After the ring was suspended in the organ bath, a passive force of 4-5 g was imposed, and the preparation was stabilized over 1 h, with washing and readjustment of the force every 20 min. At the end of the stabilization period, the baseline was regained electronically, and the strip was contracted by the addition of 10 µM norepinephrine (NE) or high KCl (80 mM). In high-KCl solution, NaCl was lowered to prevent changes in osmolarity. Preliminary experiments determined that the contracture evoked by this KCl concentration was 84 ± 1% of the maximal contracture that was obtained at 100-125 mM KCl. When a relaxant agent was employed, it was added to the bath after the agonist-induced contraction reached steady state. Similarly, the relaxant agents were removed or the concentration was changed after a steady-state effect was reached. Tension was considered to reach steady state when it did not change by >2% in the last 2 min before a new drug application or a change in concentration. The relaxant effect of the different agents was expressed as the percent decrease of the NE- or KCl-induced contraction. When indicated in the respective protocols, the strips were freeze-clamped and stored at -70°C until biochemical assays were performed.

To evaluate the presence of a functional endothelium, control experiments were performed with the NO synthase inhibitor Nomega -nitro-L-arginine methyl ester (L-NAME). The contractile and relaxant responses to the different agents did not differ in the presence and absence of L-NAME. Therefore, experiments were performed in the absence of L-NAME.

Preparation of Microsomes

Microsomes were prepared as described by Levitsky et al. (19), except the pulverized tissue was homogenized in four volumes of ice-cold homogenization buffer containing 30 mM KH2PO4 (pH 7.4), 5 mM EDTA, 2 mM EGTA, 25 mM NaF, 250 mM sucrose, 0.1% beta -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. The microsomal pellet was resuspended in 30 mM histidine (pH 7.4), 10 mM NaF, 5 mM EDTA, and 250 mM sucrose. Protein was measured by the method of Bradford (3), with BSA as standard. The yield was 0.8-1.5 mg of microsomal protein per gram of tissue.

Electrophoresis and Western Blot Analysis

For immunologic detection of site-specific phosphorylated PLB, microsomal proteins (30 µg/gel lane) were separated by SDS-PAGE with use of 10% slab gels and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore), as previously described (25). Blots were probed according to Drago and Colyer (8), with polyclonal antibodies raised to a PLB peptide (residues 9-19) phosphorylated at Ser16 or Thr17 (1:5,000; PhosphoProtein Research, Fluorescience). Immunoreactivity was visualized by peroxidase-conjugated antibodies with use of a peroxidase-based chemiluminescence detection kit (Boehringer-Mannheim). The signal intensity of the bands on the film was quantified by optical densitometric analysis.

Determination of PKG Activity

PKG activity was determined in cat aorta extracts according to Jiang et al. (14). Briefly, the pulverized tissue from each aortic strip (0.3-0.5 g) was homogenized in two volumes of ice-cold homogenization buffer containing (in mM) 10 KH2PO4 (pH 7.4), 10 EDTA, 10 beta -mercaptoethanol, 1 IBMX, and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 16,000 g for 15 min at 4°C, and the supernatant was assayed immediately for kinase activity. The reaction was started by the addition of 20 µl of the extracts to 50 µl of reaction mixture containing 150 µM BPDEtide (RKISASEFDRPLR, a PKG-specific substrate), 40 mM Tris (pH 7.4), 20 mM MgCl2, 200 µM [gamma -32P]ATP, 100 µM IBMX, and 2 µM cAMP-dependent protein kinase inhibitor- (5-24). The assay was performed in the absence and presence of 10 µM 8-bromo-cGMP (8-BrcGMP). The reaction was stopped after 10 min at 0°C by spotting 50-µl aliquots onto phosphocellulose paper (Whatman P81). After two washes in 75 mM H3PO4, the papers were dried and counted in a liquid scintillation counter. A no-substrate blank was necessary to correct for background phosphorylation of endogenous proteins in the samples. Enzyme activity was calculated as the activity ratio in the absence and presence of 10 µM 8-BrcGMP (-cGMP/+cGMP).

Statistics

Values are means ± SE of n preparations. Student's t-test for paired or unpaired observations, as appropriate, was used to test for statistical differences. P < 0.05 was considered statistically significant.

Drugs

H-89 was obtained from Seikagaku America (Rockville, MD), BPDEtide and cAMP-dependent protein kinase inhibitor-(5-24) from Calbiochem-Novabiochem (La Jolla, CA), [gamma -32P]ATP from NEN (Boston, MA), IBMX, protein kinase I, 8-BrcGMP, 8-bromo-cAMP (8-BrcAMP), L-NAME, NE, thapsigargin (Thaps), forskolin, isoproterenol (Iso), tetraethylammonium (TEA), and the rest of the chemicals from Sigma Chemical (St. Louis, MO).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of NP on Cat Aorta Relaxation: Role of Phosphorylation of Ser16 and Thr17 Residues of PLB

Figure 1A shows representative records of the relaxant effect of NP, an exogenous NO donor, on NE-induced contractures in the absence and presence of 10 mM TEA, a K+ channel blocker. This concentration of TEA has been shown to block the Ca2+-activated K+ (KCa) channels, the ATP-dependent K+ channels, and the voltage-activated K+ channels (27). In the absence of TEA, NP completely relaxed the NE-induced contracture. In the presence of TEA, the force of the NE contracture increased and NP was unable to produce full relaxation. Similar results were obtained in three other experiments of this type. The increase in developed tension observed in the presence of TEA suggests that, on blockade of K+ channels, the membrane depolarized and Ca2+ entry increased. The fact that NP caused complete relaxation of NE tone in the absence of TEA, but reversed only partially the NE-induced contracture in the presence of TEA, would indicate that the fraction of the NP-induced relaxation inhibited by TEA was mediated by the activation of K+ channels. Activation of K+ channels would hyperpolarize the muscle, inhibiting Ca2+ entry. The fraction of the NP-induced relaxation resistant to TEA may therefore be attributed to mechanisms other than K+ channel activation.


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  		Fig. 1.   A: superimposed records of force developed by 2 strips of cat aorta contracted with norepinephrine (NE) and then relaxed with sodium nitroprusside (NP). Addition of K+ channel blocker tetraethylammonium (TEA) to 1 strip produced an increase in developed force. In presence of TEA, NP was unable to fully relax aortic strip. NP evoked a decrease in maximal developed force (relaxant effect) that amounted to 46%. Similar results were obtained in 3 other experiments of this type. B: immunoblot of endoplasmic reticulum (ER) membrane vesicles obtained from 5 different aortic strips that were freeze-clamped at plateau of NE-induced contracture in absence (NE, considered as control) or presence of TEA (NE + TEA) and after relaxation induced by 100 µM NP in a contracture induced by NE (NE + NP) or NE + TEA (NE + TEA + NP). NP increased phosphorylation of Ser16 residue (PSer16) of phospholamban (PLB) in absence or presence of TEA. Similar results were obtained in 4 other experiments of this type.

Figure 1B shows an immunoblot of ER membrane vesicles from aortic strips freeze-clamped at the peak of the NE-induced contracture and after NP-induced relaxation in the absence or presence of TEA. In the absence and presence of TEA, NP increased Ser16 phosphorylation of PLB to a similar degree [81 ± 25 (n = 4) and 103 ± 40 (n = 5), respectively, expressed as percent change from control] without significant changes in the phosphorylation of Thr17 (data not shown). This suggests that phosphorylation of PLB may be involved in the relaxant effect of NP. However, phosphorylation of PLB might be one of the several mechanisms determining the NP-induced relaxant effect or an epiphenomenon not causally related to this effect. In an attempt to clarify this point, a new series of experiments was performed in which the effect of the SERCA2 inhibitor Thaps on the NP-induced relaxation was studied (24). Figure 2 shows typical recordings of experiments performed to test whether, in our experimental conditions, Thaps inhibited SERCA2 and prevented the ER Ca2+ refilling. Aortic strips were exposed to two successive NE pulses in a Ca2+-free medium. Between the NE pulses the strips were allowed to refill their Ca2+ stores in control Krebs solution (1.35 mM Ca2+). When 1 or 10 µM Thaps was present during the refilling period, the fast phase of the second NE contracture was suppressed (Fig. 2B).


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  		Fig. 2.   A: records of force developed by cat aortic strips exposed to 10 µM NE in a Ca2+-free solution (Ca = 0). NE induced a rapid and transient contraction due to release of Ca2+ from intracellular stores. After Ca2+ stores were refilled with 1.35 mM Ca2+ solution (Ca = 1.35), a 2nd exposure to NE in Ca2+-free solution evoked a similar fast response. B: when 1 or 10 µM thapsigargin (Thaps) was added during refilling period, 2nd NE-induced contracture was suppressed. Results indicate that 1 µM Thaps was enough to completely inhibit sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) and eliminate ER function. NE-induced contracture failed to relax in presence of Ca2+-free solution and relaxed toward basal values on addition of extracellular Ca2+. Although we did not address this point in detail, this behavior may be explained by inhibition and reactivation, respectively, of Na+/Ca2+ exchanger.

Figure 3A illustrates a typical response to increasing concentrations of NP on KCl-induced contractures in the absence (top) and presence (bottom) of Thaps. In the absence of Thaps, NP produced a relaxant effect that reached its maximum at 100 µM. This maximal effective concentration of NP failed to return developed force to baseline values. These results suggest, in agreement with the experiments with NE + TEA, that a fraction of the NP-induced relaxation was mediated by activation of K+ channels, which would be blocked by high external K+ or TEA. In the presence of the SERCA2 inhibitor, the relaxant effect of NP was reduced at all NP concentrations. Figure 3B shows the overall mechanical results of the effect of NP in the absence and presence of Thaps. The difference between both curves (dashed area) represents the participation of the ER in the relaxant effect of NP, which reached 23 ± 9% at 100 µM NP. The ER-mediated relaxant effect of 100 µM NP was similar in NE-induced contractures in the absence of TEA (24 ± 6%, n = 4) and in the presence of the K+ channel blocker (32 ± 3%, n =4). These results indicate that the ER contribution to the relaxant effect of NP was similar whether or not K+ channels were inhibited.



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  		Fig. 3.   A: relaxant effect of NP in 2 cat aortic strips precontracted with high external KCl (80 mM) in absence (top) and presence (bottom) of 1 µM Thaps, a SERCA2 inhibitor. In presence of Thaps, NP-induced relaxant effect was smaller than in its absence. B: average concentration-response curves of relaxant effect of NP in absence (-Thaps) and presence (+Thaps) of Thaps. Difference between NP-induced relaxant effect in absence and presence of SERCA2 inhibitor (dashed area) represents contribution of ER to relaxant effect of NP. Values are means ± SE of 4-7 experiments. * P < 0.05, -Thaps vs. +Thaps.

Figure 4A shows immunoblots of ER membrane vesicles obtained from experiments in which aortic strips precontracted with KCl were relaxed with a single NP concentration (1-100 µM) and freeze-clamped for biochemical assay. NP produced a dose-dependent increase in Ser16 phosphorylation of PLB without detectable changes in phosphorylation of the Thr17 residue. Figure 4B shows the overall biochemical results of these experiments. The filled circles represent the difference between the NP-induced relaxant effect in the absence and presence of Thaps (Fig. 3B), i.e., the contribution of the ER to the relaxant effect of NP at each NP concentration. There is a correlation between the relaxant action of NP attributed to the ER and the phosphorylation of the Ser16 residue of PLB.


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  		Fig. 4.   A: immunoblots of microsomes prepared from cat aortic smooth muscle (SM) to show that NP produced a dose-dependent increase in phosphorylation of Ser16 residue of PLB and did not affect phosphorylation of Thr17 residue. Sarcoplasmic reticulum membrane vesicles obtained from a rat heart (CM) perfused with 30 nM isoproterenol (Iso) are shown for comparison. B: average concentration-response curve of Ser16 phosphorylation (open circle ) and concentration-dependent contribution of ER to relaxant effect of NP (), calculated as difference between curves of Fig. 3B.

To test whether PKG was involved in the effect of NP, the activity of PKG was measured in aortic strips precontracted with KCl before (control) and after the addition of 10 and 100 µM NP. These NP concentrations increased the PKG activity ratio (-cGMP/+cGMP) from 0.11 ± 0.01 (n = 4) to 0.22 ± 0.03 (10 µM NP, n =4) and to 0.32 ± 0.03 (100 µM NP, n = 4). Further evidence that cGMP mediates the ER-induced relaxant effect and the increase in phosphorylation of the Ser16 residue of PLB produced by NP was provided by the experiments performed with the membrane-permeable cGMP analog 8-BrcGMP. This nucleotide was used at 1 mM. Preliminary experiments demonstrated that lower and more physiological concentrations of 8-BrcGMP failed to produce relaxant effects similar to that evoked by NP (data not shown). Externally applied 8-BrcGMP produced a relaxant effect of 62 ± 7% (n = 8) that was reduced to 35 ± 2% (n = 4) in the presence of Thaps. This relaxant effect was associated with an increase in Ser16 phosphorylation of 58 ± 12% (n = 4).

Effect of Interventions That Increase cAMP on Cat Aorta Relaxation: Role of Phosphorylation of Ser16 and Thr17 Residues of PLB

To explore the possible role of the cAMP-dependent pathway of PLB phosphorylation on cat aorta relaxation, a series of experiments were performed.

Addition of Iso. Figure 5 shows the effect of Iso on force developed by a strip of cat aorta precontracted with KCl and then exposed to increasing concentrations of Iso. Iso failed to relax the aortic strip even at the highest concentration used. Similar results were obtained in 10 additional experiments in which 1 µM (n = 3) or 100 µM Iso (n = 7) was added as a single dose at the peak of the KCl-induced contracture. Immunodetection of phosphorylation sites of PLB indicated that Iso did not produce any significant increase in the Ser16 residue of PLB (Fig. 5B, Table 1). The lack of effect of Iso on cat aorta may suggest a lack of beta -receptors or an uncoupling between beta -receptors and the corresponding downstream intracellular signaling in this type of muscle.


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  		Fig. 5.   A: record of force developed by a strip of cat aorta contracted with high KCl. Addition of <= 100 µM Iso failed to relax muscle. Relaxant effect of NP is illustrated for comparison. B: immunoblot of ER membrane vesicles obtained from 5 different aortic strips that were freeze-clamped at plateau of KCl-induced contracture (control) or after addition of 100 µM NP (NP) or 1 µM Iso. Iso failed to increase phosphorylation of Ser16 residue of PLB.


                              
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  		Table 1.   Phosphorylation of Ser16 residue of PLB induced by interventions that increased cAMP

Addition of forskolin. To bypass beta -receptors, the cAMP cascade was explored by the addition of the adenylate cyclase activator forskolin. In control experiments a concentration-response curve to forskolin from 0.5 nM to 50 µM revealed that only the higher forskolin concentrations produced an evident relaxant effect in cat aortic strips. Addition of 5 and 50 µM forskolin relaxed the aorta by 35 ± 1% (n = 3) and 64 ± 11% (n = 6), respectively. At <5 µM, forskolin produced only a modest relaxant effect (<10%). Therefore, 50 µM forskolin was chosen to explore the mechanisms of the relaxant effect of this compound. Figure 6A, top, shows the superimposed records of 50 µM forskolin on KCl-induced contractures in the absence and presence of 1 µM Thaps. The relaxant effect of forskolin was reduced in the presence of the SERCA2 blockade. Figure 6A, bottom, shows an immunoblot of ER membrane vesicles obtained from aortic strips frozen at the peak of the KCl-induced contracture and after forskolin-induced relaxation. Forskolin increased the phosphorylation of the Ser16 residue, whereas phosphorylation of the Thr17 residue was not affected (not shown). The overall results of these experiments are shown in Fig. 8 and Table 1.


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  		Fig. 6.   A: top, relaxant effect of forskolin (FK) on 2 different aortic strips precontracted with high KCl in presence and absence of Thaps. Contractures have been scaled to same maximal force to better show different relaxation pattern under 2 different experimental situations. Thaps was added 20 min before KCl contracture was elicited. In presence of Thaps, relaxant effect of forskolin was of a lower magnitude than in absence of Thaps. Bottom, immunoblot of ER membrane vesicles obtained from 3 different aortic strips that were freeze-clamped at plateau of KCl-induced contracture (control) or after addition of 50 µM forskolin (FK) or 100 µM NP (NP). Forskolin and NP increased phosphorylation of Ser16 residue of PLB to a similar extent. B: top, relaxant effect of 8-BrcAMP on 2 aortic strips precontracted with high KCl in presence and absence of Thaps. Thaps reduced relaxant effect of 8-BrcAMP. Bottom: immunoblot of ER membrane vesicles obtained from 3 different aortic strips that were freeze-clamped at plateau of KCl-induced contracture (control) or after addition of 50 µM forskolin (FK) or 1 mM 8-BrcAMP. Forskolin and 8-BrcAMP increased phosphorylation of Ser16 residue of PLB with respect to control.

The phosphorylation of the Ser16 residue of PLB and the relaxant effect produced by 50 µM forskolin were mimicked by the membrane-permeable cAMP analog 8-BrcAMP. 8-BrcAMP was used at 1 mM. Preliminary experiments demonstrated that lower concentrations of the nucleotide produced a relaxant effect of <10% (data not shown). Figure 6B shows that the relaxant effect of 1 mM 8-BrcAMP was reduced in the presence of 1 µM Thaps (top) and occurred in association with an increase in phosphorylation of the Ser16 residue of PLB (bottom). The overall results of these experiments showed that 1 mM 8-BrcAMP produced an increase in Ser16 phosphorylation of 119 ± 38% (n = 4; Table 1) and relaxed cat aorta by 49 ± 3% (n = 4) and 35 ± 3% (n = 4) in the absence and presence of 1 µM Thaps, respectively.

Is the relaxant effect of forskolin mediated by activation of PKA? Figure 7A compares the relaxant effect of 50 µM forskolin in the absence and presence of H-89 (5 µM), an inhibitor of PKA that suppresses the kinase activity by binding to the active site of the catalytic subunit, or H-89 + 1 µM Thaps. In the presence of H-89, the relaxant effect of forskolin was reduced. Higher concentrations of H-89 up to 20 µM failed to further inhibit the relaxant effect of forskolin. Figure 7A also shows that the relaxant effect of forskolin was further reduced in the presence of H-89 + Thaps. Figure 7B shows an immunoblot of ER membrane vesicles obtained from aortic strips freeze-clamped at the peak of the KCl-induced contracture and after forskolin-induced relaxation in the absence or presence of H-89. Forskolin produced an increase in phosphorylation of Ser16 that decreased in the presence of H-89. Overall phosphorylation results are presented in Table 1.


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  		Fig. 7.   A: relaxant effect of forskolin on 3 different aortic strips precontracted with high KCl. Forskolin was administered alone (solid line) or in presence of 5 µM H-89 (dashed line) or 5 µM H-89 + 1 µM Thaps (dotted line). Contractures have been scaled to same maximal force. Thaps was added as indicated in Fig. 6. H-89 was added 20 min before addition of forskolin. Relaxant effect of forskolin was diminished in presence of H-89 and further diminished in presence of H-89 + Thaps. B: immunoblot of ER membrane vesicles obtained from 3 different aortic strips that were freeze-clamped at plateau of KCl-induced contracture (control) or after addition of 50 µM forskolin alone or 50 µM forskolin + 5 µM H-89. Increase in phosphorylation of Ser16 residue of PLB produced by forskolin was partially reduced by H-89.

Figure 8 shows the overall mechanical results of the experiments in which the relaxant effect of forskolin was analyzed. Forskolin produced a significant relaxant effect that did not return to baseline levels (64 ± 8%, n = 12; Fig. 8A). This would indicate that ~40% of the relaxant effect of forskolin was mediated by activation of K+ channels that were blocked by the elevated external K+. In the presence of Thaps, the relaxant effect of forskolin was reduced to 26 ± 3% (n = 7). Thus the ER-dependent relaxant effect of forskolin was 38 ± 9%. Figure 8B illustrates the results of the experiments that aimed to dissect the PKA dependence of the ER-mediated relaxant effect of forskolin. The relaxant effect of forskolin on the KCl-induced contracture was reduced in the presence of H-89 from 64 ± 8% (forskolin, n = 12) to 25 ± 4% (forskolin + H-89, n = 14). These results suggest that a fraction of the relaxant effect of forskolin was mediated by PKA-dependent mechanisms, as expected, and the rest by PKA-independent mechanisms. The relaxant effect of forskolin resistant to H-89 (forskolin + H-89) was further reduced to 12 ± 1% (n = 6) by Thaps (forskolin + H-89 + Thaps). This reduction represents the forskolin-induced relaxant effect mediated by the ER through PKA-independent mechanisms. Therefore, of the total ER-mediated forskolin relaxant effect (Fig. 8A), approximately one-third appears to be independent of PKA (Fig. 8B); the remaining two-thirds would be mediated by PKA activation.


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  		Fig. 8.   Average of relaxant effect of forskolin under different experimental conditions. A: forskolin produced a relaxant effect that was significantly reduced in presence of Thaps (FK + Thaps). This reduction (38 ± 9%) represents contribution of ER (ERdep) to relaxant effect of forskolin. Remaining 26 ± 3% has to be explained by mechanisms independent of ER (ERindep). B: relaxant effect of forskolin was significantly diminished in presence of H-89 (FK + H-89). This reduction represents contribution of protein kinase A-dependent mechanisms (PKAdep) to relaxant effect of forskolin. Relaxant effect of forskolin resistant to H-89 (PKAindep) was further reduced in presence of Thaps (FK + H-89 + Thaps). This reduction represents contribution of ER to relaxant effect of forskolin through protein kinase A-independent mechanisms.

Additional experiments showed that 50 µM forskolin increased the activity ratio of PKG from 0.11 ± 0.01 (n = 4) to 0.24 ± 0.05 (n = 4). These results suggest that an increase in the activity of PKG may be involved in the relaxant effect of forskolin independent of PKA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the mammalian heart the SR Ca2+ uptake constitutes the main mechanism of relaxation. PLB, the regulatory protein of the SR Ca2+-ATPase, is the primary target of agents that modulate cardiac relaxation, e.g., beta -agonists (37). In contrast, in vascular smooth muscle, several mechanisms appear to be significantly involved in the relaxant effect of endogenous vasodilators, e.g., NO and beta -agonists (31, 35). One of these potential mechanisms is the enhanced Ca2+ sequestration by the ER due to phosphorylation of PLB. The main goal of the present experiments was to determine the physiological relevance of PLB phosphorylation, if any, in the relaxant effect of the PKG- and PKA-dependent cascades, the two major pathways that regulate smooth muscle relaxation.

The present experiments in cat aorta show that the ER-mediated relaxant effect of NP (~25% at 1-100 µM NP) was associated with a significant increase in Ser16 phosphorylation of PLB. These effects were accompanied by an increase in the activity of PKG and were mimicked by 8-BrcGMP. Taken together, these results suggest that the relaxant effect of NP related to the ER occurred via an increase in Ca2+ uptake due to PKG phosphorylation of the Ser16 residue of PLB.

The forskolin-induced relaxant effect associated with the ER amounted to 38 ± 9% of the overall forskolin relaxant action and was associated with a significant increase in Ser16 phosphorylation. Both effects were mimicked by 8-BrcAMP. Part of the ER-mediated relaxant effect and of the increase in Ser16 phosphorylation produced by forskolin was blocked by H-89. The ER-dependent PKA-independent effect might be produced by a PKG-dependent mechanism. This conclusion was supported by the increase in PKG activity produced by forskolin described in these experiments. The results are in agreement with other evidence showing cross- activation of PKG by agents that elevate cAMP (9, 14, 21).

The approach used in the present study allowed delineation of some but not all of the possible mechanisms involved in the NP- and forskolin-induced relaxant effects. The failure of NP and forskolin to completely relax the muscle precontracted with NE + TEA or high external K+ would indicate that a component of the relaxant effect of both compounds (~40%) may be mediated by membrane repolarization due to the activation of K+ channels. This observation is consistent with the results showing that, at least in some smooth muscles, different types of K+ channels can be activated by cGMP- or cAMP-dependent cascades (1, 28). It has also been shown that NO can activate KCa channels by cGMP-independent mechanisms (2). Experimental evidence has been presented in rat cerebral and coronary arteries supporting a mechanism for cyclic nucleotide-mediated relaxation of vascular smooth muscle, which couples the ER to KCa channels (28). By this mechanism the increase in ER Ca2+ load would produce an increase in the frequency of Ca2+ sparks, which would in turn increase the frequency of spontaneous outward currents produced by activation of KCa channels. This would cause membrane hyperpolarization, closure of L-type Ca2+ channels, decrease in internal Ca2+ concentration ([Ca2+]i), and vasodilatation. The present experiments in cat aortic strips showed that the effects of Thaps on the relaxant effect of NP were additive to the effect of K+ channel blockade (TEA or high external K+). In addition, the ER contribution to the relaxant effect of NP was similar whether or not K+ channels were inhibited. These results indicate that the coupling between ER Ca2+ loading and KCa channel activation is not the main mechanism of the ER-mediated relaxant effect of cyclic nucleotides in cat aorta.

A sequential coupling of SERCA2, Ca2+ release channels, and the Na+/Ca2+ exchanger seems also to take place in some types of smooth muscle (26). According to this model, the peripheral ER would generate and maintain a Ca2+ gradient through a regulated vectorial release of its Ca2+ content toward the adjacent sarcolemma, inducing higher [Ca2+]i in the subsarcolemmal space. The excess [Ca2+]i would be then extruded from the cell through the Na+/Ca2+ exchanger. Although we did not explore this point, it is tempting to speculate that in cat aortic strips part or all of the Ca2+ accumulated in the ER by the action of NP or forskolin might be then extruded to the extracellular space by the Na+/Ca2+ exchange mechanism.

The relaxant effect of NP and forskolin resistant to Thaps and to blockade of K+ channels might be produced by different mechanisms: a direct increase in Ca2+ efflux by plasma membrane Ca2+-ATPase (10) and/or the Na+/Ca2+ exchanger (11), a decrease in Ca2+ influx by a direct action on L-type Ca2+ channels (5), or a decrease in Ca2+ release by the ER (30). Factors downstream from [Ca2+]i mobilization, e.g., a decrease in Ca2+ sensitivity of myosin phosphorylation or an uncoupling of force from myosin phosphorylation, may also play a role (4, 38).

Our results differ from previous studies showing that NO and forskolin mainly relaxed vascular smooth muscle by decreasing the Ca2+ concentration sensitivity of force with a minimal (4, 32) or null participation (38) of membrane repolarization. More important to the present discussion is the fact that, in the above-mentioned experiments, no effect of NO or forskolin on Ca2+ efflux or Ca2+ sequestration was detected (4, 32, 38). In a recent study in PLB knockout mice, it was shown that PLB played a minor role, if any, in cyclic nucleotide-mediated aortic relaxation (18). These differences may be due to the different types of smooth muscle and/or different species used (rat tail artery or mouse aorta vs. cat aorta). However, other experiments in rabbit and mouse aorta emphasized the crucial role of the ER in the relaxant effect of NO (6). The contribution of the ER to the relaxant effect of NP and forskolin shown in the present results is also supported by previous findings in primary cultures of rat aortic smooth muscle showing that cGMP and cAMP increased Ca2+ uptake by the ER (39) and ER Ca2+-ATPase activity (7, 20). Finally, in pig coronary arteries, a good correlation between the relaxant effect of agents that increase cGMP and the phosphorylation of PLB has been shown (15). However, no attempts were made in these experiments to establish whether the ER did actually participate in the relaxant effect observed.

In summary, the present experiments have demonstrated in cat aorta that the relaxant effect of NP and forskolin, two activators of the major cascades that regulate smooth muscle relaxation, can be separated into several mechanisms. It was shown that the relative contribution to this effect of the ER was closely associated with an increase in phosphorylation of the Ser16 residue of PLB. This contribution represents approximately one-third of the total relaxant effect of both compounds, which is consistent with the multiple mechanisms responsible for relaxation in smooth muscle.


    ACKNOWLEDGEMENTS

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). M. Said is a fellow from Universidad Nacional de La Plata. C. Mundiña-Weilenmann, L. Vittone, G. Rinaldi, G. Chiappe de Cingolani, and A. Mattiazzi are established Investigators of Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina).


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Mattiazzi, Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, 1900 La Plata, Argentina (E-mail: ramattia{at}atlas.med.unlp.edu.ar).

Received 9 June 1999; accepted in final form 17 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 278(6):H1856-H1865
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