Involvement of intracellular Ca2+ stores in inhibitory effects of NO donor SIN-1 and cGMP

Hartmut Franck, Martin Storr, Andreas Puschmann, Volker Schusdziarra, and Hans-Dieter Allescher

Department of Internal Medicine II, Technical University of Munich, 81675 Munich, Germany

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We investigated the role of K+ channels and intracellular Ca2+ stores in the relaxations induced by the NO donor 3-morpholinosydnonimine (SIN-1) and 8-bromo-cGMP (8-BrcGMP), 8-(4-chlorophenylthio)-cGMP (pCPT-cGMP), and alpha ,beta -methylene-ATP in isolated segments of rat ileum. The inhibitory responses to SIN-1 and the cGMP analogs were not influenced by the K+ blockers apamin, charybdotoxin, iberiotoxin, or glibenclamide, whereas relaxations induced by alpha ,beta -methylene-ATP were abolished by apamin and tetraethylammonium. The NO-donor SIN-1 and the cGMP analogs were able to inhibit contractions induced by activation of L-type Ca2+ channels (BAY-K-8644), by carbachol (CCh), and by cyclopiazonic acid (CPA), a blocker of sarcoplasmic Ca2+-ATPase. However, the inhibition of the combined CPA and CCh response was reduced and the dose-response curve of SIN-1 shifted to the right. Intracellular Ca2+ stores were emptied by incubation in Ca2+-free buffer and repetitive stimulation with CCh or BAY-K-8644. After restoration of extracellular Ca2+, the inhibitory effect of SIN-1 and pCPT-cGMP was only attenuated, whereas in the additional presence of CPA, the inhibitory effect of SIN-1 was blocked and the effect of 8-BrcGMP reduced. Thus depleting intracellular Ca2+ stores attenuated the effect of SIN-1 and 8-BrcGMP, suggesting an involvement of functional Ca2+ stores.

apamin; charybdotoxin; iberiotoxin; cyclopiazonic acid

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

A SUBSTANTIAL PART of the neuromuscular regulation of the enteric nervous system is the nonadrenergic noncholinergic (NANC) inhibitory neurotransmission of gastrointestinal (GI) smooth muscle. There is strong evidence indicating that nitric oxide (NO) or a related NO-donating substance and ATP are major candidate transmitters of NANC innervation in the GI tract (18, 19, 30). Because NO is an unstable gaseous agent, NO donors such as glyceryl trinitrate, sodium nitroprusside, and 3-morpholinosydnonimine (SIN-1) (33) have been used to study the effects of NO. NO is known to activate soluble guanylate cyclase with a subsequent increase in cGMP levels (16), which in turn causes activation of G kinase (33). This can be achieved directly by the stable analogs of cGMP, 8-bromo-cGMP (8-BrcGMP) (4, 26) and the more membrane-permeable compound 8-(4-chlorophenylthio)-cGMP (pCPT-cGMP) (28). The mechanism of action of NO and cGMP to induce smooth muscle relaxation is not fully understood. Several possibilities are a matter of debate: 1) cell membrane hyperpolarization (31), 2) sequestration of Ca2+ with lowered cytosolic Ca2+ concentration ([Ca2+]i) (22), 3) reduced sensitivity of the contractile apparatus, i.e., of myosin light-chain kinase (23), and 4) reduced activation of second messengers involved in the excitatory pathway.

ATP is known to induce relaxation via hyperpolarization of the cell membrane (19). alpha ,beta -Methylene-ATP has been shown to be a useful stable ATP agonist (32).

Electrophysiological studies suggest that NO, similar to other inhibitory NANC mediators, also induces hyperpolarization of the cell membrane (10, 30). In the pulmonary artery, aorta, and tracheal muscle of the guinea pig, blockade of Ca2+-dependent K+ channels has been demonstrated to decrease responses to NO donors such as SIN-1 (5). In GH4C1 cells, activation of a cGMP-dependent protein kinase stimulated large- conductance Ca2+-dependent K+ channel activity (35). In 1994, Bolotina et al. (6) presented evidence that NO directly activated charybdotoxin-sensitive Ca2+ channels in cell-free membrane patches of rabbit aorta.

Hyperpolarization of the cell membrane and receptor-bound mechanisms are able to regulate [Ca2+]i, which is a principal regulator of contraction in smooth muscle (11). The [Ca2+]i concentration is regulated by the sarcoplasmic reticulum on the one hand and influx of Ca2+ via cell membrane-bound voltage-operated and receptor-operated Ca2+ channels on the other hand (11). Particularly via voltage-operated L-type channels will [Ca2+]i increase with depolarization and decrease with hyperpolarization (11). Cell membrane potential is known to be modulated to a great extent by the large family of K+ channels, some of them having particular dependence on [Ca2+]i in regulating intracellular Ca2+.

Thus the aims of the present study were to investigate 1) the role of K+ channels and 2) the role of sarcoplasmic Ca2+ pumps responsible for Ca2+ sequestration in intracellular Ca2+ stores in NO donor- and G kinase activator-induced responses.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

The experimental model and protocol designed to investigate inhibitory responses were used as previously described (1). In brief, male Wistar rats (400-500 g) were killed by intraperitoneal injection of pentobarbital sodium (100 mg/kg). The terminal ileum was immediately removed, and six full-thickness gut segments were cut from it (length 1.5 cm). These were orientated longitudinally and attached to an isometric force transducer (Swegma force displacement transducer SG 4-500). The tissue was maintained in Krebs-Ringer-bicarbonate solution (KRS) (115.5 mM NaCl, 1.16 mM MgSO4, 1.16 mM NaH2PO4, 11.1 mM glucose, 21.9 mM NaHCO3, 2.5 mM CaCl2, and 4.16 mM KCl) gassed with 95% O2-5% CO2 at 37°C. A resting tension of 1 g was applied to the muscle before equilibrating for 30 min. Changes in tension were amplified by a Hellige coupler and recorded on a Rikadenki chart recorder.

At the beginning and the end of each experiment, the response to a submaximal contracting dose of carbachol (CCh) (10-6 M) [or the activator of L-type Ca2+ channels BAY-K-8644 (10-7 M)] was tested as a control. CCh produced submaximal contractions; the maximum response was obtained with 10-5 M CCh, but at a concentration of 10-6 M the plateau of the contractile response was more stable. BAY-K-8644 (10-7 M) produced maximal contractions, whereas further increase of BAY-K-8644 concentration produced no higher contractile response. CCh (or BAY-K-8644) was washed out after 5 min by three consecutive washes at 5-min intervals. The tissue was then incubated for 5 min in buffer before application of the next stimulus.

SIN-1-, 8-Br-cGMP-, pCPT-cGMP-, and alpha ,beta -methylene-ATP-induced inhibition. After washout and incubation, tissues were precontracted using CCh or BAY-K-8644. When the CCh- or the BAY-K-8644-induced response had reached a plateau, usually 30 s after addition of the stimulus, the inhibitory mediator was added. Concentration-response curves were constructed using separate applications of a single concentration of the inhibitory mediator, with washouts in between. In separate experiments, the inhibitory effect of a given concentration of the inhibitor on CCh- or BAY-K-8644-induced contraction was tested for stability by repeating this protocol two times. In these experiments, SIN-1 was used at a concentration of 5 × 10-4 M, which was approximately the EC50 of the dose range tested. In time controls, the inhibitory effect of SIN-1, pCPT-cGMP, and alpha ,beta -methylene-ATP remained stable for >3 h with this protocol. As far as 8-BrcGMP is concerned, the response to a second application of the inhibitor was significantly decreased up to 1 h after the first application. For this reason, 8-BrcGMP was applied only once per single preparation in the following experiments. Each segment of rat ileum was used only for a single concentration-response curve for agonist or blocker, respectively.

The neurotoxin TTX (10-6 M) was incubated with the tissue 5 min before contractile stimulation.

Effect of K+ channel blockers on inhibitor-induced response. After the initial stimulation with CCh, the inhibitory mediator was added as described above, with the inhibitory response serving as a control. After the respective washes, the blocker was added to the bath 5 min before stimulation with CCh. In the presence of the respective blocker, the effect on the inhibitor-induced response was tested. SIN-1 was used at the concentration of 5 × 10-4 M, which was approximately the EC50 of the dose range tested. 8-BrcGMP (10-3 M) and alpha ,beta -methylene-ATP (10-4 M) were used to compare a similar degree of relaxation. After repeated washes, the next concentration of the blocker was applied. Each segment was used only for a single concentration-response curve of a single blocker.

Experiments using cyclopiazonic acid. Application of cyclopiazonic acid (CPA), a blocker of the sarcoplasmic Ca2+-ATPase, was used to test participation of intracellular Ca2+ stores. CPA (10-5 M) induced a slow contractile response in the unstimulated muscle and was used as prestimulation similar to CCh or BAY-K-8644 to assess the inhibitory response of SIN-1, the cGMP analogs, and alpha ,beta -methylene-ATP. Second applications of CPA elicited only ~60% of the first contractile response in 12 of 20 preparations. Therefore CPA was applied only once per preparation, when CPA was used as the only contractile stimulus. In contrast, when the tissue was stimulated with CPA and CCh, the resulting contractile response remained stable in repeated time controls for at least 2 h. In this set of experiments, the second contractile stimulus (CCh) was applied 4 min after CPA. The inhibitory mediator was added as soon as the CCh-induced response had reached a plateau, usually 30 s after addition of this stimulus. A concentration-response curve was constructed using separate applications of a single concentration of the NO donor SIN-1 on combined prestimulation with CPA (10-5 M) and CCh (10-6 M), which were added as described above.

Ca2+-depleting protocol. In a different series of experiments, the tissue was incubated in a Ca2+-free Krebs-Ringer buffer containing 0.25 mM EGTA and CPA (10-5 M) to empty intracellular Ca2+ stores and stimulated with CCh (10-6 M) or BAY-K-8644 (10-7 M). After washout with Ca2+-free buffer containing CPA, this procedure was repeated twice until application of CCh (or BAY-K-8644) had virtually no contractile effect. After washout the tissue was incubated in Ca2+-free Krebs-Ringer buffer without EGTA. This buffer was prepared omitting CaCl2. After application of CPA and CCh (or BAY-K-8644), restoration of extracellular Ca2+ caused a contraction, which reached an instant plateau. The respective inhibitory agent was added within <10 s after restoration of extracellular Ca2+. Two sets of controls were performed. In the first control, an identical protocol was used, but CPA was omitted (see Table 3). In the second, the contractile response was tested, applying KRS instead of the inhibitory agent (see Fig. 4).

Data analysis and statistics. For data analysis the contraction level induced by the stimulus before the addition of the inhibitory mediator was determined as control. To study the fast component of relaxation, we defined the inhibitory response that reached a maximum within 20 s after application of SIN-1 and alpha ,beta -methylene-ATP analogs and within 40 s after application of the cGMP analogs as the remaining contraction after application of the inhibitory mediator. Contractile responses were given as absolute values, and the inhibition was expressed as a percentage of the control contraction. Data are given as means ± SE, and n indicates the number of independent observations in different muscle strips. Each protocol was repeated in ileal segments of at least two different animal preparations. When a statistical difference of two means was determined, we performed a paired two-tailed Student's t-test. For comparisons of more than two means one-way ANOVA, followed by post hoc test with Bonferroni correction for multiple comparisons, was carried out to determine statistical difference. Values of P < 0.05 were considered significant.

Drugs. The following drugs were used in this study. SIN-1 was obtained from Cassella-Riedel (Frankfurt, Germany), and pCPT-cGMP was from Biolog (Bremen, Germany). CCh and BAY-K-8644 were procured from Bayer (Heidelberg, Germany), and iberiotoxin was from Research Biochemicals (Natick, MA). Tetraethylammonium (TEA) was from Aldrich (Milwaukee, MN), and glibenclamide was from Boehringer Mannheim. 8-BrcGMP, alpha ,beta -methylene-ATP, TTX, apamin, charybdotoxin, CPA, and EGTA were all obtained from Sigma (Munich, Germany). The drugs were freshly dissolved in saline and further diluted with KRS. Glibenclamide was dissolved in N-methylformamide. The drugs were added to the bath in microliter volumes, and experiments were controlled for the effects of the drug solvents.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of SIN-1 on CCh- and BAY-K-8644-induced contraction. The muscarinic agonist CCh (10-6 M) caused a tonic contractile response of the longitudinal smooth muscle. As soon as the contractile response of CCh reached a plateau, application of SIN-1 (10-6 to 3 × 10-3 M) caused an instantaneous and concentration-dependent relaxation. The maximal inhibitory effect obtained at the highest concentration of SIN-1 tested (3 × 10-3 M) was 93.3 ± 3.8% of the contraction induced by CCh (10-6 M) (Fig. 1A). Repetitive application of SIN-1 in a single preparation revealed no change in relaxation when the preparation was washed with KRS periodically [n = 8; not significant (NS)]. The approximate EC50 of 5 × 10-4 M was used to compare the response of SIN-1 with the other inhibitors (Tables 1 and 2). The Ca2+ channel activator BAY-K-8644 was used to stimulate smooth muscle cells directly by inward Ca2+ current, which is independent from muscarinic receptor activation. BAY-K-8644 caused a tonic contraction of the longitudinal smooth muscle segments, which reached 25% of the contractile CCh (10-6 M) response at an optimum concentration of 10-7 M. Higher concentrations of BAY-K-8644 caused a decrease in contractile response. Application of SIN-1 (5 × 10-4 M) on the plateau of the BAY-K-8644-induced response caused an instantaneous relaxation reaching 100.2 ± 4.6% of the BAY-K-8644 response (n = 8) (Table 1).


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Fig. 1.   Concentration-dependent inhibitory effect of 3-morpholinosydnonimine (SIN-1) (A; n = 8), 8-bromo-cGMP (8-BrcGMP) (B; n = 12), pCPT-cGMP (C; n = 8), and alpha ,beta -methylene-ATP (D; n = 8) on longitudinal smooth muscle precontracted with 10-6 M carbachol (CCh). Values are means ± SE.

                              
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Table 1.   Effect of SIN-1, 8-BrcGMP, CPT-cGMP, and alpha ,beta -methylene-ATP on CCh- or BAY-K-8644-induced contraction

                              
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Table 2.   Effect of various K+ channel blockers on relaxation induced by SIN-1, 8-BrcGMP, and alpha ,beta -methylene-ATP

The neurotoxin TTX affected neither the contractile responses to CCh or BAY-K-8644 prestimulation nor the SIN-1-induced inhibition of CCh- or BAY-K-8644-induced responses (n = 8; NS; Table 1), indicating a nerve-independent action.

Effect of 8-BrcGMP and pCPT-cGMP on CCh- and BAY-K-8644-induced contraction. Application of 8-BrcGMP (10-5 to 10-3 M) and pCPT-cGMP (10-6 to 3 × 10-4 M) on the plateau of the CCh-induced response caused a sustained concentration-dependent relaxation (Fig. 1, B and C). The onset of action of pCPT-cGMP appeared to be faster than that of 8-BrcGMP, which in some tissues took >1 min to reach its maximal effect. The inhibitory effect obtained at the highest concentration of 8-BrcGMP tested (10-3 M) was 49.4 ± 6.6% of the contraction induced by CCh (10-6 M) (n = 12). Repetitive application of 8-BrcGMP (10-3 M) on a single preparation revealed a marked tachyphylaxis (1st application: relaxation 49.4%; 2nd application: 17% of the CCh-induced response) (n = 9). For this reason 8-BrcGMP was applied only once per preparation in the following experiments. Application of 8-BrcGMP (10-3 M) on the plateau of the BAY-K-8644-induced response caused a sustained relaxation reaching 100.0 ± 7.8% of the BAY-K-8644 response (n = 9; Table 1).

The inhibitory effect of pCPT-cGMP increased concentration dependently but showed no tachyphylaxis. This fact may be due to the greater membrane permeability of pCPT-cGMP. The maximal inhibitory effect obtained at the highest concentration of pCPT-cGMP tested (3 × 10-4 M) was 58.1 ± 8.0% of the contraction induced by CCh (10-6 M) (n = 8). Repetitive application of pCPT-cGMP (10-4 M) on a single preparation revealed no change in relaxation when the preparation was washed with KRS periodically (n = 9; NS). TTX did not affect the 8-BrcGMP- and pCPT-cGMP-induced inhibition, again indicating a neurally independent action (n = 9) (Table 1).

Effect of alpha ,beta -methylene-ATP on CCh- and BAY-K-8644-induced contraction. Application of alpha ,beta -methylene-ATP (10-8 to 10-4 M) on the plateau of the CCh-response caused an instantaneous concentration-dependent relaxation (Fig. 1D). The inhibitory effect of alpha ,beta -methylene-ATP (10-4 M) was 53.1 ± 5.7% (n = 9) (Table 1). Repetitive application of alpha ,beta -methylene-ATP (10-4 M) on a single preparation revealed no desensitization (1st application: relaxation 49.8%; 2nd application: 40.5% of the CCh-induced response; n = 9).

Application of alpha ,beta -methylene-ATP (10-4 M) on the plateau of the BAY-K-8644-induced response caused an instantaneous relaxation reaching 76.2 ± 8.5% of the BAY-K-8644 response (n = 6). TTX showed no effect on the alpha ,beta -methylene-ATP-induced inhibition of CCh- or BAY-K-8644-induced responses (n = 6) (Table 1).

Effect of K+ channel blockers on SIN-1-, 8-BrcGMP-, and alpha ,beta -methylene-ATP-induced inhibition. A variety of K+ channel blockers were tested against SIN-1 and 8-BrcGMP to gain insight into a possible K+ channel type being functional during muscle relaxation. Apamin (10-7 M; n = 9; NS), charybdotoxin (10-7 M; n = 9; NS), and iberiotoxin (10-8 M; n = 6; NS), specific blockers of Ca2+-dependent K+ channels, had no influence on the effect of SIN-1 (5 × 10-4 M) and 8-BrcGMP (10-3 M) (Table 2), as reported previously for NO-dependent relaxations in the canine ileocolonic junction (9). Iberiotoxin applied at higher concentrations of up to 10-6 M failed to exert any influence on the SIN-1-induced relaxation (Table 2). Furthermore, neither the nonspecific K+ channel blocker TEA (10-4 to 10-1 M) nor glibenclamide (10-5 M; n = 8) (Table 2), a specific blocker of ATP-dependent K+ channels, modified the inhibitory action of SIN-1 and 8-BrcGMP. The inhibitory response of alpha ,beta -methylene-ATP (10-4 M) was unaffected by charybdotoxin (10-7 M) and glibenclamide (10-5) (Table 2), whereas the Ca2+-activated K+ blocker apamin (10-7 M) significantly reduced the alpha ,beta -methylene-ATP-induced inhibition (Table 2). Application of TEA (10-1 M) induced a contractile response and reduced the subsequent CCh contraction. Thus, in comparison with controls, a significant baseline shift of contractile response occurred. Application of alpha ,beta -methylene-ATP (10-4 M) on this contractile response failed to exert any relaxation. Hence TEA (10-1 M) abolished the alpha ,beta -methylene-ATP (10-4 M)-induced inhibition (0.1 ± 1.4% of the contractile response; n = 8).

Effect of CPA on SIN-1-, pCPT-cGMP-, and alpha ,beta -methylene-ATP-induced responses. CPA (10-5 M), which is known to block the sarcoplasmic Ca2+-ATPase (34) and thereby to increase [Ca2+]i, caused a slowly developing tonic contraction that reached a plateau and increased to 15.1 ± 2.2 mN (n = 20). A second administration of CPA revealed a decrease to 60% of the first contractile response in 12 of 20 preparations. Therefore CPA was applied only once as a single contractile stimulus. Application of SIN-1 (10-3 M) or pCPT-cGMP (10-4 M) on this plateau caused a prompt relaxation, reaching 41.2 ± 4.4% (SIN-1; n = 15) or 88.2 ± 5.7% (pCPT-cGMP; n = 11) of the CPA-induced response (Fig. 2). Because of the attenuation of the SIN-1-induced inhibition in the presence of CPA, for the following experiments 10-3 M SIN-1 was used.


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Fig. 2.   A: representative tracing showing inhibitory response of SIN-1 (10-3 M) on smooth muscle prestimulated by cyclopiazonic acid (CPA; 10-5 M). Top: contraction induced by CPA (10-5 M) and combined prestimulation with CPA and CCh (10-6 M). Bottom: SIN-1 (10-3 M) induced responses on both prestimulations. B: representative tracing showing inhibitory response of pCPT-cGMP (10-4 M) on smooth muscle prestimulated by CPA (10-5 M). Top: contraction induced by CPA (10-5 M) and combined prestimulation with CPA and CCh (10-6 M). Bottom: pCPT-cGMP (10-4 M) induced responses on both prestimulations.

Application of alpha ,beta -methylene-ATP (10-4 M) on the CPA-induced contraction caused a prompt relaxation of 27.9 ± 5.0% of the CPA-induced response as well.

Effect of CPA on SIN-1- and pCPT-cGMP-induced responses after prestimulation with CCh. Preincubation with CPA (10-5 M) and subsequent application of CCh (10-6 M) induced a combined contractile response that did not exceed the contractile response of CCh (10-6 M) alone (35.1 ± 5.3 vs. 36.3 ± 4.6 mN, n = 8) (Fig. 2). When a concentration-response curve of SIN-1 was carried out on this combined contractile response, the curve was shifted to the right compared with control stimulation with CCh (10-6 M) alone (Fig. 3). The inhibitory effect of SIN-1, applied in a concentration of 10-3 M, was significantly reduced after combined stimulation with CPA and CCh (39.5 ± 7.4% of combined contraction vs. 82.9 ± 5.8% of CCh-induced contraction alone; P < 0.05; n = 8; Fig. 2A).


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Fig. 3.   Concentration-dependent inhibitory effect of SIN-1 on longitudinal smooth muscle precontracted by CPA (10-5 M) + CCh (10-6 M). Controls were precontracted with CCh (10-6 M) alone. Values are means ± SE (n = 8).

Because the combined tonic response to CPA and CCh was not well sustained, we used pCPT-cGMP instead of 8-BrcGMP. As mentioned, pCPT-cGMP showed a more rapid onset of action. Similar to that of SIN-1, the inhibitory response of pCPT-cGMP (10-4 M) was blocked after combined prestimulation with CPA and CCh (-0.7 ± 3.1% of combined contraction vs. 44.0 ± 9.8% of CCh-induced contraction alone; P < 0.05; n = 8; Fig. 2B).

The fact that simultaneous application of CPA and CCh is able to reduce the SIN-1- and G kinase activator-induced relaxation suggests a possible role of functional intracellular Ca2+ stores. For this reason, the influence of CPA and CCh was tested in Ca2+-free preparations.

Effect of depleting intracellular Ca2+ stores on SIN-1-, 8-BrcGMP-, and alpha ,beta -methylene-ATP-induced responses. Intracellular Ca2+ stores were depleted by repetitive stimulation of the smooth muscle with CCh (10-6 M) in Ca2+-free buffer prepared with EGTA either in the presence or absence of CPA. After several cycles, the medium was changed to Ca2+-free buffer without EGTA in the presence or absence of CPA (10-5 M). Addition of CCh (10-6 M) or BAY-K-8644 (10-7 M) under these conditions elicited no response but induced an immediate contraction after subsequent restoration of external Ca2+ (Fig. 4). The inhibitory effects of SIN-1, 8-BrcGMP, or alpha ,beta -methylene-ATP were tested immediately after the contraction had reached a plateau level.


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Fig. 4.   Representative tracing showing effect of depleting intracellular Ca2+ stores with CPA in Ca2+-free extracellular medium. Ca2+ was depleted with CPA (10-5 M) and EGTA in Ca2+-free medium. After washout tissue was incubated in Krebs-Ringer buffer omitting Ca2+ (without EGTA). CPA, CCh, and CaCl2 were added as indicated. Top: contraction in 1 set of control experiments. Bottom: SIN-1 (10-3 M)-induced responses. Combined prestimulation by adding CPA (10-5 M) and restoring Ca2+ in presence of CCh (10-6 M) reduced inhibitory response of SIN-1 (10-3 M).

After prestimulation with CCh (10-6 M), the inhibitory effect of SIN-1 was significantly reduced compared with control conditions in KRS (29.7 ± 3.7% vs. 62.1 ± 4.3%; Table 3) and was virtually abolished after Ca2+ depletion in the presence of CPA (10-5 M) to block the sarcoplasmic Ca2+-ATPase (-1.1 ± 1.2%; Table 3). The effect of 8-BrcGMP remained unchanged when Ca2+ stores were depleted in the absence of CPA. In presence of CPA, however, 8-BrcGMP-induced inhibition was significantly reduced (Table 3). The inhibitory effect of alpha ,beta -methylene-ATP on CCh-prestimulated smooth muscle also remained unchanged after intracellular Ca2+ depletion omitting CPA but was completely abolished after intracellular Ca2+ depletion in the presence of CPA (Table 3; Fig. 4).

                              
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Table 3.   Inhibitory effect of SIN-1, 8-BrcGMP, and alpha ,beta -methylene-ATP on longitudinal smooth muscle precontracted with CCh or BAY-K-8644

When the muscle was prestimulated with BAY-K-8644, the inhibitory effects of SIN-1 and 8-BrcGMP were significantly reduced compared with control conditions and further reduced after emptying the intracellular Ca2+ stores with CPA (Table 3). The inhibitory effect of alpha ,beta -methylene-ATP on BAY-K-8644 prestimulation was also significantly attenuated by depletion of intracellular Ca2+ stores, but this effect was not enhanced further in the presence of CPA (Table 3).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Application of SIN-1 on longitudinal smooth muscle of rat ileum precontracted with CCh caused a concentration-dependent relaxation. Contractions that involve protein kinase C (29) and intracellular Ca2+ stores have previously been shown to be inhibited by NO donors and cGMP analogs (34). The concentration range of the inhibitory action of SIN-1 is in good agreement with other reports on the action of SIN-1 in ventricular myocytes of the frog (21) and in isolated leukocytes (24). In the above-mentioned studies (21, 24), there was a biphasic effect with an excitatory portion at concentrations of SIN-1 in the nanomolar range. A similar response to NO donors was reported in the rat small intestine (3). The fact that we did not find any excitatory effect seems most likely to be due to the submaximal prestimulation before SIN-1 application. The cell membrane-permeable and stable cGMP analogs 8-BrcGMP and pCPT-cGMP showed a concentration-dependent inhibitory effect. The concentration range of the inhibitory action is in line with other reports on the action of 8-BrcGMP in opossum esophageal longitudinal muscle (26) and in the canine pyloric sphincter (4). Neural blockade by TTX (10-6 M) did not modify the SIN-1- and cGMP-induced relaxation, demonstrating an inhibitory action independent from nerves.

In GI smooth muscle there is ample evidence that NO-mediated relaxations involve hyperpolarization (22, 31), and it is most likely that this change in membrane potential is induced by K+ channels, which are activated via cGMP (17). Opening of these channels would hyperpolarize the membrane and relax smooth muscle. However, our results show that apamin (10-7 M), charybdotoxin (10-7 M), iberiotoxin (10-6 M), and glibenclamide (10-5 M), known blockers of Ca2+- and ATP-dependent K+ channels, and TEA (10-1 M) were without effect on the SIN-1- or the 8-BrcGMP-induced relaxation of rat ileum longitudinal muscle. These results are in clear contrast to several reports (5, 25) on vascular and tracheal smooth muscle that cGMP-dependent protein kinases activate Ca2+-dependent K+ channels.

Thus the inhibitory response induced by NO donors and cGMP-dependent mechanisms does not depend on K+ channels sensitive to the blockers tested. However, the ineffectiveness of all the K+ channel blockers used in our experiments does not exclude a role of other K+ channels for the effect of SIN-1 and cGMP analogs in this tissue, since it has been reported that NO-induced K+ channel activation in GI smooth muscle is not affected by any of the specific blockers used in this study (8, 14, 17).

The inhibitory effect of alpha ,beta -methylene-ATP, a stable analog of ATP, has been described previously (32). In contrast to SIN-1 and 8-BrcGMP, the inhibitory response of alpha ,beta -methylene-ATP was blocked by apamin and TEA, indicating that different cellular mechanisms were involved. A similar influence of apamin on ATP-induced inhibition has been reported in previous studies (2) and is thought to support a participation of Ca2+-dependent K+ channels in mediating ATP-induced relaxation. In patch-clamp experiments on smooth muscle of chicken rectum, alpha ,beta -methylene-ATP induced an inward current followed by a K+ outward current (20). Both currents were unchanged after depletion of intracellular stores with caffeine and blockade of L-type Ca2+ channels with nifedipine. Finally, removal of extracellular Ca2+ abolished the outward current (20).

To test the involvement of functional intracellular Ca2+ stores, we used CPA, a blocker of the Ca2+-ATPase of the sarcoplasmic reticulum. Depleting intracellular Ca2+ stores with CPA and Ca2+-free extracellular medium attenuated the alpha ,beta -methylene-ATP-induced inhibition in the presence of CCh. Consequently, CPA-dependent intracellular Ca2+ stores participate in the alpha ,beta -methylene-ATP-induced relaxation.

CPA blocks the sarcoplasmic Ca2+-ATPase and thereby is thought to increase [Ca2+]i and induce contraction (34). Thapsigargin, another blocker of sarcoplasmic Ca2+-ATPase, was not used because of a postulated direct interaction with voltage-dependent Ca2+ channels (7). The attenuation and rightward shift of the SIN-1-induced effect on the combined contractile response of CPA and CCh cannot be explained by a baseline shift of precontraction, because CCh (10-6 M) caused a submaximal response and the combined response of CPA plus CCh did not exceed the contractile response of CCh alone. However, we cannot exclude a shift of [Ca2+]i and/or intracellular protein kinases at this identical level of contraction. As the combination of CCh prestimulation and presence of CPA also reduced the inhibitory effect of the selective cGMP analog pCPT-cGMP, it can be speculated that the cGMP-dependent mechanisms inhibiting smooth muscle contraction are also dependent on functional intracellular Ca2+ stores.

To investigate a possible involvement of intracellular Ca2+ changes, we used repetitive stimulation with CCh in Ca2+-free buffer in the presence and absence of CPA to deplete the intracellular Ca2+ stores.

After emptying the intracellular Ca2+ stores with CPA, which was present to block the sarcoplasmic Ca2+-ATPase, we found that the inhibitory effects of all three inhibitors were either significantly reduced or abolished compared with control conditions without depleting the Ca2+ stores. The fact that this difference in the inhibitory effect occurred in CCh- as well as in BAY-K-8644-prestimulated tissue suggests that intracellular Ca2+ stores seem to be of importance in the inhibitory responses elicited on BAY-K-8644-induced contraction. This finding supports the idea that even with pure stimulation of Ca2+ influx through L-type Ca2+ channels, the inhibitory effect of all three inhibitors involves functional internal Ca2+ stores, regardless of the Ca2+ source for the contraction. It is possible that CPA treatment depletes intracellular Ca2+ stores involved in both contractile and inhibitory responses. In contrast, other strategies used to deplete Ca2+ (e.g., use of Ca2+-free medium and repetitive stimulation with CCh or BAY-K-8644 in the absence of CPA) may selectively remove stores responsible for the contractile responses.

The inhibitory effect of 8-BrcGMP showed less sensitivity to depletion of intracellular Ca2+ stores than the inhibitory responses to SIN-1. This effect was more pronounced after prestimulation with CCh than with BAY-K-8644. In general, 8-BrcGMP showed a smaller inhibitory response compared with SIN-1, and the onset and time course of this inhibition was slower than the immediate inhibitory effect of SIN-1. This prolonged time course of the cGMP-induced inhibition may have led to partial refilling of intracellular Ca2+ stores, especially when the Ca2+-ATPase was not blocked by CPA. Additionally, it may be possible that the high concentration of 8-BrcGMP causes a small degree of cross-activation of other second messenger mechanisms, such as protein kinase A (15). This could explain why the inhibitory effect of 8-BrcGMP was not completely abolished after Ca2+ depletion in the presence of CPA.

Altogether, contractions induced by CPA alone were inhibited ~50% by SIN-1. This inhibition is significant and similar to the SIN-1-induced inhibition of CCh responses. The combined presence of CPA and CCh reduced the SIN-1-induced effect substantially, and SIN-1-responses were abolished after depleting the stores in Ca2+-free medium. Thus strategies such as the presence of CCh and exposure to Ca2+-free medium appear to be necessary in addition to CPA to reveal an attenuation or block of the SIN-1-induced inhibition.

Possible targets of SIN-1 and cGMP analogs in smooth muscle cells of rat ileum include the following: 1) K+ channels or other ion channels not sensitive to the blockers tested in this study; 2) the sarcoplasmic reticulum, in which sequestration of Ca2+ may be activated, thereby decreasing [Ca2+]i and causing relaxation (under this scheme, sequestration mechanisms would have to be at least partially CPA insensitive, because SIN-1 and pCPT-cGMP caused significant inhibition of CPA-induced contractions); 3) Ca2+ channels, in which influx of extracellular Ca2+ may be reduced, which has been reported for SIN-1 in cardiac muscle (21); 4) pathways independent of cytosolic Ca2+ as reported for sodium nitroprusside in vascular muscle by Sato et al. (27), when the tissue is relaxed by SIN-1 or the cGMP analogs. The relative role of these different mechanisms in GI muscle is not yet known.

We conclude that SIN-1 and cGMP analogs inhibit contractile responses to isolated muscarinic stimulation, isolated activation of Ca2+ influx via L-type channels (BAY-K-8644), and isolated blockade of sarcoplasmic Ca2+-ATPase (CPA). K+ channels sensitive to apamin, charybdotoxin, iberiotoxin, and glibenclamide are not involved in mediation of the relaxation. Depletion of intracellular Ca2+ stores in the presence of CPA attenuated the effect of the inhibitors substantially, suggesting an involvement of functional sarcoplasmic reticulum Ca2+ stores.

    ACKNOWLEDGEMENTS

We acknowledge the courtesy of Cassella-Riedel GMBH (Frankfurt, Germany). We thank C. Fleischer for laboratory work, L. Kots for proofreading the manuscript, and Dr. C. W. R. Shuttleworth and Dr. S. D. Koh for numerous discussions.

    FOOTNOTES

A portion of this study was presented at the annual meeting of the American Gastroenterological Association in San Diego, CA, on May 14-17, 1995, and has been published previously in abstract form (see Ref. 12).

Address for reprint requests: H.-D. Allescher, II. Medizinische Klinik und Poliklinik der TU München, Ismaningerstr. 22, 81675 München, Germany.

Received 26 February 1997; accepted in final form 25 March 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(1):G159-G168
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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