Role of protein kinase C in calcium sensitization during muscarinic stimulation in airway smooth muscle

Dorothee H. Bremerich, David O. Warner, Robert R. Lorenz, Robin Shumway, and Keith A. Jones

Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Muscarinic receptor stimulation increases Ca2+ sensitivity, i.e., the amount of force produced at a constant submaximal cytosolic Ca2+ concentration ([Ca2+]i), in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+ sensitivity is in part mediated by protein kinase C (PKC). With the use of a beta -escin permeabilized canine tracheal smooth muscle (CTSM) preparation, the effect of four putative PKC inhibitors {calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC [PKC peptide-(19---31)], and staurosporine} on Ca2+ sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+ sensitization induced by muscarinic receptor stimulation in the presence of a constant submaximal [Ca2+]i, neither did any of these compounds reverse the increase in Ca2+ sensitivity induced by ACh plus GTP. Administration of a 1,2-diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, did not induce Ca2+ sensitization at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity produced by muscarinic receptor stimulation in permeabilized CTSM.

beta -escin; calcium sensitivity; lung; trachea; canine; protein kinase C inhibitors; activator; second messenger systems

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IN AIRWAY SMOOTH MUSCLE, contraction is mediated by an increase in the concentration of cytosolic Ca2+ ([Ca2+]i). Ca2+ binds to calmodulin and subsequently increases myosin light chain kinase (MLCK) activity and phosphorylation of the 20-kDa regulatory myosin light chain (rMLC; see Ref. 8). rMLC phosphorylation allows the binding of myosin to actin, which increases actomyosin adenosinetriphosphatase activity and causes smooth muscle contraction. However, contractile force is not determined by [Ca2+]i alone (19), as membrane receptor stimulation with various agonists increases force developed at a constant submaximal [Ca2+]i, i.e., the Ca2+ sensitivity (1, 19).

In vascular (14) and colonic (24) smooth muscle, evidence exists that protein kinase C (PKC) may act as a second messenger mediating increases in Ca2+ sensitivity. In tracheal smooth muscle, muscarinic receptor stimulation causes translocation of PKC from the cytosol to the membrane, where it is thought to be active (30). Both the putative PKC agonists phorbol esters and muscarinic receptor stimulation increase Ca2+ sensitivity in beta -escin permeabilized airway smooth muscle preparations (1, 3). However, other studies in a variety of smooth muscles have not demonstrated an involvement of PKC in the signal transduction pathway during membrane receptor stimulation (7, 11, 23). The physiological role of PKC in mediating membrane receptor agonist-induced increases in Ca2+ sensitivity in airway smooth muscle is unknown.

The purpose of the current study was to determine the role of PKC in the regulation of Ca2+ sensitivity in canine tracheal smooth muscle (CTSM) during muscarinic receptor stimulation. We used a beta -escin permeabilized CTSM preparation in which pores are produced in the cell membrane. In this way, [Ca2+]i can be controlled by manipulating the extracellular Ca2+ concentration, yet the membrane-associated second messengers, including PKC, remain intact and can be investigated (1, 3). We studied the effect of four putative PKC inhibitors, calphostin C, chelerythrine chloride, a PKC pseudosubstrate inhibitor [PKC peptide-(19---31) (PSSI)], and staurosporine on Ca2+ sensitization produced by acetylcholine (ACh) in the presence of GTP. We also examined the ability of a specific PKC activator, the 1,2-diacylglycerol (DAG) analog 1-oleoyl-2-acetyl-sn-glycerol (OAG), to increase Ca2+ sensitivity at a constant submaximal [Ca2+]i.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Experimental Techniques

After Institutional Animal Care and Use Committee approval, and in conformance with the Guiding Principles for Research Involving Animals as approved by the Council of the American Physiological Society, mongrel dogs (n = 30; 15-23 kg) of either sex were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated. The trachea was excised and immersed in chilled physiological salt solution (PSS) of the following composition (in mM): 110.5 NaCl, 25.7 NaHCO3, 5.6 dextrose, 3.4 KCl, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 MgSO4. Fat, connective tissue, and the epithelium were removed with tissue forceps and scissors under microscopic observation.

Permeabilized CTSM. Muscle strips (width 0.1-0.2 mm, length 3-5 mm, wet weight 0.05-0.1 mg) were mounted in 0.1-ml cuvettes and were continuously superfused at 2 ml/min with PSS (37°C) aerated with 94% O2 and 6% CO2, providing a pH of approx 7.4, PO2 of approx 400 mmHg, and PCO2 of approx 39 mmHg in the PSS. One end of the strips was anchored via stainless steel microforceps to a stationary metal rod, and the other end was anchored via stainless steel microforceps to a calibrated force transducer (model KG4; Scientific Instruments, Heidelberg, Germany). Optimal length (Lo) of each strip was achieved as previously described in detail (1, 3). The remainder of the experiment was performed at 25°C.

The strips were permeabilized with beta -escin (18), a method validated for CTSM in our laboratory (1). beta -Escin creates pores in the cell membrane, thus allowing substances of small molecular weight, such as Ca2+, to freely diffuse across the cell membrane. Accordingly, [Ca2+]i can be manipulated by changing the extracellular Ca2+ concentration with ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-buffered solutions in the bathing media. Larger cellular proteins necessary for contraction as well as the membrane receptor-coupled second messenger systems are preserved after this permeabilization procedure (1).

Muscle strips were permeabilized for 20 min with 100 µM beta -escin in relaxing solution. The composition of the relaxing solution was as follows: 7.5 mM ATP disodium salt, 4 mM EGTA, 20 mM imidazole, 1 mM dithiothreitol, 1 nM free Ca2+, 10 mM creatinine phosphate, and 0.1 mg/ml creatinine phosphokinase. The pH was buffered to 7.0 at 25°C with KOH; ionic strength was kept constant at 0.20 M by adjusting the concentration of potassium acetate. After permeabilization, strips were superfused with relaxing solution for 10 min to wash out the excess beta -escin. The Ca2+ ionophore A-23187 (10 µM) was added to the relaxing solution and all subsequent experimental solutions to deplete the sarcoplasmatic reticulum Ca2+ stores (16). Then, strips were maximally contracted with 10 µM free Ca2+; all subsequent isometric force measurements were normalized to these contractions. After this determination, strips were again superfused with relaxing solution including Pi (5 mM) for 10 min to reduce the time required for relaxation by accelerating the rate of cross bridge detachment. To remove the Pi, strips were superfused with relaxing solution for 10 min, and the experimental protocol was started. Solutions of varying free Ca2+ concentrations were prepared as previously described (1).

Intact CTSM. To determine the effects of the PKC inhibitors in intact CTSM, muscle strips (width 0.5-1 mm, length 1.0-1.5 cm, weight 0.8-3 mg) were suspended in 25-ml water-jacketed tissue baths filled with PSS (37°C). PSS was aerated with 94% O2 and 6% CO2, providing a pH of approx 7.4, PO2 of approx 400 mmHg, and PCO2 of approx 39 mmHg in the PSS. One end of the strips was anchored to a metal hook at the bottom of the tissue bath; the other end was attached to a calibrated force transducer (model FT03D; Grass Instruments, Quincy, MA). During a 1-h equilibration period, the strips were repeatedly contracted isometrically for 30 s every 5 min by supramaximal electrical field stimulation (EFS; 400 mA, 15 V, 25 Hz, 0.5-ms pulse duration). EFS was triggered by a stimulator (model S88D; Grass Instruments) and was delivered by a direct current amplifier (Section of Engineering, Mayo Foundation). The length of the strips was increased after each stimulation until Lo was achieved. Each strip was maintained at Lo for the rest of the experiment. During this equilibration period, the strips were washed with fresh PSS every 10 min.

Experimental Protocols

Effect of PKC inhibitors on ACh-induced Ca2+ sensitization in permeabilized CTSM. We determined whether any of the four PKC inhibitors could prevent or reverse the increase in Ca2+ sensitivity induced by muscarinic receptor stimulation. These experiments included four tissue sets, one for each inhibitor. For each experiment, two permeabilized CTSM strips were prepared and studied concomitantly.

The two strips were superfused with relaxing solution alone (control) or in the presence (preincubation) of either 1 µM calphostin C (n = 5), 40 µM chelerythrine chloride (n = 5), 3 µM PSSI (n = 5), or 0.1 µM staurosporine (n = 5) for 10 min. Both strips were contracted with 0.3 µM free Ca2+ (equivalent to the free Ca2+ concentration producing 10% of isometric force in this preparation; see Ref. 3), one in the absence (control) and one in the presence (preincubation) of the PKC inhibitor for 10 min, the time required for stable contractile response. Then, both strips were contracted with 3 µM ACh plus 10 µM GTP for an additional 10 min, and force responses were measured. As previously determined (1), this ACh concentration produces 80% of its maximal effect on isometric force development at a constant [Ca2+]i in this preparation.

To determine whether any of the four PKC inhibitors could reverse the increase in Ca2+ sensitivity induced by muscarinic receptor stimulation, the control CTSM strip was exposed to either 1 µM calphostin C (n = 5), 40 µM chelerythrine chloride (n = 5), 3 µM PSSI (n = 5), or 0.1 µM staurosporine (n = 5) for 10 min after contraction induced by 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP had stabilized. The isometric force in the presence of the PKC inhibitor was measured and was compared with the value immediately before the addition of inhibitor. At the concentrations of free Ca2+ and ACh plus GTP chosen, contraction remains stable for the entire time course of the experimental protocol (unpublished observation).

Effect of OAG on Ca2+ sensitivity in permeabilized CTSM. This experimental protocol was conducted to determine if OAG, a direct PKC activator, increases isometric force at a constant submaximal [Ca2+]i in a permeabilized smooth muscle preparation. Strips were contracted with 0.3 µM (n = 2) or 0.6 µM (n = 2) free Ca2+ for 10 min. Then, 100 µM OAG was added to the experimental solutions, and isometric force was continuously recorded for an additional 10 min. Subsequently, strips were stimulated with 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP to demonstrate that the second messenger pathways mediating Ca2+ sensitivity were intact.

Effect of PKC inhibitors on ACh-induced contraction in intact CTSM. To determine if calphostin C, chelerythrine chloride, and staurosporine have any effects on CTSM, we performed additional experiments investigating their effect on intact tissues. Because the synthetic peptide PSSI cannot penetrate intact cells due to its polarity and size, it was not investigated in this protocol. Three muscle strips obtained from a single dog (n = 6) were studied to determine concentration-response curves to cumulative concentrations of staurosporine, chelerythrine chloride, and calphostin C. After each muscle strip was set at Lo, force was induced by 0.1 µM ACh (equivalent to the ACh concentration producing 50% of its maximal effect in isometric force development in intact CTSM) for 20 min, the time required for stable contractile response. The force developed was defined as the initial force response, and subsequent contractions recorded were normalized to this initial response (%initial force). Then, one muscle strip was exposed to 0 (control), 0.03, 0.1, 0.3, 1.0, 3.0, 6.0, and 10.0 µM staurosporine, the second strip was exposed to 0 (control), 1.0, 3.0, 6.0, 10.0, and 30.0 µM chelerythrine chloride, and the third strip was exposed to 0 (control), 0.1, 1.0, 10.0, and 100.0 µM and 1 mM calphostin C. For each concentration studied, stable relaxation was achieved in ~4 min.

Additional strips were loaded with the Ca2+ fluorescent probe fura 2 acetoxymethyl ester (AM; n = 4) to investigate the effect of PKC inhibitors on [Ca2+]i in intact tissue. The emission fluorescence intensities due to excitation at 340 nm (F340) and 380 nm (F380) were measured, and the F340-to-F380 ratio was used as an index of [Ca2+]i. The effect of 0.1-3 µM staurosporine added to the perfusate on both F340/F380 and isometric force was studied. Due to the fluorescence properties of chelerythrine chloride and calphostin C, fura 2-AM fluorescence measurements could not be performed in our photometric system using these compounds.

Materials. Fura 2-AM was purchased from Molecular Probes (Eugene, OR). ATP disodium salt was purchased from Research Organics (Cleveland, OH). PSSI was purchased from Boehringer Mannheim Biochemica (Indianapolis, IN). All other drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO). A-23187, fura 2-AM, calphostin C, chelerythrine chloride, and staurosporine were dissolved in dimethyl sulfoxide (DMSO); in all experimental solutions, the final concentration of DMSO did not exceed 0.1%. At this concentration, DMSO had no effect on isometric force (data not shown). All other drugs and chemicals were prepared in distilled water.

Statistical analysis. Data are expressed as mean values ± SD; n represents the number of dogs. To test for differences in the experimental groups, isometric force measurements were compared by paired Student's t-test. A P value <= 0.05 was considered statistically significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of PKC Inhibitors on ACh-Induced Ca2+ Sensitization in Permeabilized CTSM

Table 1 shows the effect of preincubation with 1 µM calphostin C, 40 µM chelerythrine chloride, 3 µM PSSI, and 0.1 µM staurosporine on isometric force induced by 0.3 µM free Ca2+ alone or by 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP. Staurosporine in concentrations higher than 0.1 µM were not studied, since it attenuated force induced by 0.3 µM free Ca2+ alone (data not shown). This finding indicates that staurosporine at these concentrations has nonspecific effects other than inhibiting PKC. None of the PKC inhibitors significantly affected isometric force development induced by 0.3 µM free Ca2+ alone, nor did they prevent or attenuate Ca2+ sensitization induced by muscarinic receptor stimulation.

                              
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Table 1.   Effect of preincubation with PKC inhibitors on ACh plus GTP-induced Ca2+ sensitization

Table 2 shows the effect of each of the four PKC inhibitors when added to strips in which Ca2+ sensitization was induced by 3 µM ACh plus 10 µM GTP at a constant submaximal [Ca2+]i of 0.3 µM. None of the PKC inhibitors reversed the increase in Ca2+ sensitivity induced by muscarinic receptor stimulation (Fig. 1).

                              
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Table 2.   Effect of PKC inhibitors on ACh plus GTP-induced Ca2+ sensitization


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Fig. 1.   Representative tracing of the effect of 40 µM chelerythrine chloride on Ca2+ sensitivity in a beta -escin permeabilized canine tracheal smooth muscle strip contracted with 0.3 µM free Ca2+ and subsequently with 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP. After contraction induced by Ca2+ alone, ACh plus GTP induced an increase in isometric force, i.e., increased Ca2+ sensitivity. Addition of the putative protein kinase C inhibitor chelerythrine chloride did not reverse the Ca2+ sensitization induced by muscarinic receptor stimulation.

Effect of OAG on Ca2+ Sensitivity in Permeabilized CTSM

Adding 100 µM OAG to the permeabilized CTSM strips precontracted with either 0.3 or 0.6 µM free Ca2+ did not increase isometric force, i.e., did not increase Ca2+ sensitivity at a constant submaximal [Ca2+]i (Fig. 2). The increase in isometric force subsequently induced by 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP was equivalent to the magnitude observed in the control experiments at a constant submaximal [Ca2+]i of 0.3 µM and muscarinic receptor stimulation.


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Fig. 2.   Representative tracing of the effect of 100 µM 1-oleoyl-2-acetyl-sn-glycerol (OAG) on Ca2+ sensitivity in a beta -escin permeabilized canine tracheal smooth muscle strip contracted with 0.6 µM free Ca2+. After contraction induced by Ca2+ alone, OAG induced no further increase in isometric force, i.e., did not increase Ca2+ sensitivity. Furthermore, addition of OAG had no effect on subsequent Ca2+ sensitization induced by 3 µM ACh plus 10 µM GTP, indicating that the second messenger pathways mediating this agonist-induced increase in Ca2+ sensitivity are intact.

Effect of PKC Inhibitors on ACh-Induced Contraction in Intact CTSM

Based on the lack of effect in the use of the PKC inhibitors in beta -escin permeabilized CTSM, we investigated their effect in intact CTSM during muscarinic receptor stimulation to exclude a technical error in the application of these compounds. Staurosporine (0.03-10 µM) and chelerythrine chloride (1-30 µM) caused relaxation of the ACh-induced contraction in a concentration-dependent manner (Fig. 3). Calphostin C (0.1 µM-1 mM) had no effect on ACh-induced contraction (data not shown).


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Fig. 3.   Concentration-dependent effect of cumulative doses of staurosporine (0.03-10 µM; A) and chelerythrine chloride (1-30 µM; B) on isolated strips of canine tracheal smooth muscle. Contraction induced by 0.1 µM ACh [half-maximal effective concentration (EC50) in intact tissue] was defined as initial force, and subsequent contractions recorded were expressed as a percentage of this initial response (%initial force).

The effect of staurosporine on [Ca2+]i in intact CTSM was demonstrated in experiments measuring fura 2-AM fluorescence. ACh (0.1 µM) increased both F340/F380 and isometric force. Subsequent addition of staurosporine (0.1-3 µM) consistently caused an irreversible decrease in both F340/F380 and isometric force (Fig. 4).


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Fig. 4.   Representative tracing of the effect of 3 µM staurosporine on the ratio of fluorescence at 340 nm to fluorescence at 380 nm (F340/380; top) and isometric force (bottom) in a canine tracheal smooth muscle strip loaded with fura 2-acetoxymethyl ester. Contraction was induced by 0.1 µM ACh (EC50 in intact tissue) for 10 min, the time for stable contraction. Then, 3 µM staurosporine was added to the perfusate for 10 min. Both, F340/380 and isometric force decreased, demonstrating staurosporine's effect on cytosolic Ca2+ concentration. PSS, physiological salt solution.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The major findings of this study are that, in beta -escin permeabilized CTSM 1) preincubation with each of the four PKC inhibitors studied did not inhibit the increase in Ca2+ sensitivity induced by muscarinic receptor stimulation at a constant submaximal [Ca2+]i, 2) administration of each of the four PKC inhibitors did not reverse the increase in Ca2+ sensitivity at a constant submaximal [Ca2+]i, and 3) direct stimulation of PKC with a DAG analog, OAG, did not increase Ca2+ sensitivity at a constant submaximal [Ca2+]i.

beta -Escin permeabilized smooth muscle preparations have been used as a tool to investigate the mechanisms regulating Ca2+ sensitivity (1, 3, 18). The creation of pores in the plasma membrane permits control of the [Ca2+]i by changing the extracellular Ca2+ concentrations in the solution superfusing the smooth muscle while preserving large cellular proteins such as calmodulin, MLCK, rMLC, actin, and myosin. With the use of this method, coupling of membrane receptors to second messenger systems mediating Ca2+ sensitivity is retained and can be investigated (1, 3).

In our CTSM preparation, muscarinic receptor stimulation increases Ca2+ sensitivity (1, 3). This increase in Ca2+ sensitivity requires GTP, is mimicked by guanosine 5'-O-(3-thiotriphosphate), and is blocked by guanosine 5'-O-(2-thiodiphosphate), providing evidence for the involvement of GTP-binding proteins in this signal transduction pathway (3). The increase in Ca2+ sensitivity during membrane receptor stimulation is produced by the inhibition of rMLC phosphatase and increased rMLC phosphorylation (16). In vascular (14, 21) and colonic (24) permeabilized smooth muscle preparations, it has been suggested that membrane receptor agonists acting via GTP-binding proteins increase Ca2+ sensitivity in part by activating PKC. After receptor binding, phospholipase C is activated via a GTP-dependent process, producing DAG, a physiological activator of PKC, from phoshatidylinositol bisphosphate and phosphatidylcholine. It is thought that, once activated, PKC may directly or indirectly inhibit the rMLC phosphatases, increasing rMLC phosphorylation and isometric force at a constant submaximal [Ca2+]i (16). Increases in Ca2+ sensitivity induced by PKC activation might also be mediated by pathways not associated with increases in rMLC phosphorylation (15).

A role of PKC in agonist-induced Ca2+ sensitization is supported by the observation that phorbol esters such as phorbol 12,13-dibutyrate (PDBu; see Ref. 9) increase Ca2+ sensitivity in permeabilized smooth muscle preparations (3, 7, 24). Further evidence for the involvement of PKC in agonist-induced Ca2+ sensitization has been provided using PKC antagonists to reverse (4, 5, 21) or inhibit (5, 21) agonist-induced increases in Ca2+ sensitivity in vascular smooth muscle. For example, 100 nM staurosporine relaxed endothelin-1 (ET-1)-induced increases in isometric force at a constant submaximal [Ca2+]i in alpha -toxin permeabilized vascular smooth muscle but had little effect on contractions elicited by Ca2+ alone (21). Chelerythrine chloride completely blocked the increase in isometric force in response to ET-1 stimulation but could not reverse it when added during steady-state agonist-induced force. Nishimura et al. (21) concluded that the regulatory events important for the maintenance of agonist-induced Ca2+ sensitization occur between PKC activation and the interaction of actin and myosin. However, there is controversy about the involvement of PKC in mediating the agonist-induced Ca2+ sensitization in different types of smooth muscle (7, 11, 23). In a study using alpha -toxin permeabilized guinea pig stomach smooth muscle, calphostin C (1 µM) and another PKC inhibitor, K-252b, had no effect on ACh-induced Ca2+ sensitization (23). Similarly, in beta -escin permeabilized guinea pig vas deferens smooth muscle, PSSI did not affect Ca2+ sensitization induced by norepinephrine or AlF<SUP>−</SUP><SUB>4</SUB> (7).

This is the first study to investigate the role of PKC in mediating agonist-induced Ca2+ sensitization in permeabilized airway smooth muscle. Two different classes of PKC antagonists were examined, which should inhibit the activity of most of the isoforms of PKC present in CTSM [the Ca2+-dependent isoforms beta I and beta II and the Ca2+-independent isoforms delta , epsilon , theta , and xi  (6)]. Two domains within PKC are targets for the action of pharmacological inhibitors, a catalytic domain that binds ATP and a regulatory domain that controls kinase activity via binding of second messengers (e.g., adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and DAG). The interpretation of our results of course assumes that the appropriate pharmacological inhibitors were examined in the appropriate concentrations. We utilized the four inhibitors up to the highest concentrations reported (4, 21, 23), well above published half-maximal inhibitory concentration (IC50) values. Staurosporine, a microbial product of Streptomyces staurosporeus (IC50 for PKC = 2.7 nM; see Ref. 29), and chelerythrine chloride, a naturally occurring alkaloid (IC50 for PKC = 660 nM; see Ref. 10), both act on the catalytic domain. This ATP-binding site exhibits considerable homology with other serine- and threonine-specific kinases and tyrosine-specific kinases (28) so that compounds acting at this site are considered to be rather nonselective. Calphostin C, derived from Cladosporium cladosporioides (IC50 for PKC = 50 nM; see Ref. 17) and the pseudosubstrate inhibitor PSSI (directed toward a 19 amino acid consensus sequence for PKC, IC50 = 147 nM; see Ref. 12) both act at the regulatory site and are more selective.

None of the PKC inhibitors studied prevented or reversed ACh plus GTP-induced increases in Ca2+ sensitivity (Fig. 1). Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity during muscarinic receptor stimulation in permeabilized CTSM. It must be acknowledged that an isoform of PKC insensitive to inhibition by the compounds studied may mediate agonist-induced Ca2+ sensitivity. However, OAG, a DAG analog, stimulates Ca2+-activated, phospholipid-dependent isoforms of PKC, such as beta I and beta II, which are saliently expressed in CTSM (6) directly through coupling to the binding site for activator molecules (20). The lack of effect of OAG on Ca2+ sensitivity argues against a physiological role of PKC in agonist-induced Ca2+ sensitization.

The addition of the phorbol ester PDBu to unstimulated intact tracheal smooth muscle does not induce significant contraction (Ref. 2; unpublished observations). However, we found that PDBu increases Ca2+ sensitivity in a concentration-dependent manner in our beta -escin permeabilized CTSM preparation (1, 3). Because PDBu increases isometric force produced at a given submaximal [Ca2+]i in permeabilized CTSM, it would appear that activation of PKC can, under these circumstances, increase Ca2+ sensitivity (1, 3). In agreement with reports of alpha -toxin permeabilized rabbit mesenteric artery smooth muscle (21), isometric force induced by PDBu developed only in the presence of >= 0.1 µM [Ca2+]i, demonstrating that the PKC isoforms mediating Ca2+ sensitivity appear to be primarily Ca2+ dependent. This Ca2+ requirement might explain why PDBu does not induce contraction in intact tracheal smooth muscle if PDBu does not also significantly increase [Ca2+]i in this tissue. The effects of PDBu should be interpreted with caution because the phorbol esters may activate PKC by mechanisms unlike those involved during agonist stimulation, e.g., by forming complexes with PKC (9). In vascular smooth muscle, it has been postulated that there are at least two different pathways for Ca2+ sensitization, a PKC-dependent pathway that is activated by phorbol esters and a PKC-independent pathway that is activated by receptor agonists (11); similar mechanisms may be present in CTSM.

Taken together, these previous studies and the current results suggest that the involvement of PKC in agonist-induced increases in Ca2+ sensitivity depends on the type of smooth muscle studied, and, perhaps, the type of membrane receptor agonist stimulation. This may reflect the fact that many isoforms of PKC are present in smooth muscle, and their distribution varies with species and smooth muscle type. With the use of rat mesenteric small arteries, it has been reported that different PKC isoforms in the same tissue mediate the contractile response to different agonists (22). Additionally, it has been shown that the distribution of PKC isoforms in ocular smooth muscle is not only species specific but that each PKC isoform might have a specific physiological function in mediating the contractile response (13). However, the physiological role of each isoform remains unclear. In CTSM, it has been suggested that the Ca2+-dependent isoforms PKC-beta I and -beta II regulate the phosphorylation of MLCK (26).

Given our negative results of PKC inhibitors on ACh-induced Ca2+ sensitization, we were compelled to investigate whether they had any effect on intact CTSM. Both staurosporine and chelerythrine chloride caused concentration-dependent relaxation in intact CTSM (Fig. 3). These findings suggest that the lack of effect of these compounds in the beta -escin permeabilized CTSM preparation is unlikely to be the result of a technical error. In prior studies, we found that staurosporine also relaxed KCl-induced contractions in a concentration-dependent manner (data not shown), which suggests that this compound has effects independent of second messenger systems activated by membrane receptor stimulation. Consistent with our findings, staurosporine also inhibits contractile responses to KCl in vascular smooth muscle (25). Measuring fura 2-AM fluorescence, we demonstrated that the staurosporine-induced relaxation was associated with a decrease of [Ca2+]i (Fig. 4). These findings imply that PKC may be primarily involved in the regulation of [Ca2+]i in CTSM. Exploration of the mechanisms by which PKC affects [Ca2+]i are beyond the scope of this study. However, in other types of smooth muscle, evidence exists for a role of PKC in regulating ion channels (27).

In summary, none of the four pharmacological PKC inhibitors prevented or reversed ACh plus GTP-induced increases in Ca2+ sensitivity in beta -escin permeabilized CTSM. Additionally, OAG, a direct DAG activator, did not increase Ca2+ sensitivity at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity during muscarinic receptor stimulation in canine airway smooth muscle.

    ACKNOWLEDGEMENTS

We thank Kathy Street and Rosimar Torres-Lèon for expert technical assistance and Janet Beckman and Cathy Nelson for preparing the manuscript.

    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Research Grants HL-45532 and HL-54757.

Address for reprint requests: K. A. Jones, Mayo Clinic and Mayo Foundation, 200 First St. SW, Rochester, MN 55905.

Received 26 March 1997; accepted in final form 23 June 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Akao, M., A. Hirasaki, K. A. Jones, G. Y. Wong, D. H. Bremerich, and D. O. Warner. Halothane reduces myofilament Ca2+ sensitivity during muscarinic receptor stimulation of airway smooth muscle. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L719-L725, 1996[Abstract/Free Full Text].

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3.   Bremerich, D. H., A. Hirasaki, K. A. Jones, and D. O. Warner. Halothane attenuation of calcium sensitivity in airway smooth muscle. Anesthesiology 87: 94-101, 1997[Medline].

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