The unimportance of being (protein kinase C) epsilon¹

^a Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22906-0011, USA
^b Department of Medicine, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22906-0011, USA
^c Department of Pathology, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22906-0011, USA
^d Department of Physiology, Catholic University of Louvain, Brussels, Belgium
^e School of Nursing, Aarhus, Denmark

	ABSTRACT

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The purpose of our study was to determine the mechanism through which phorbol esters and smooth muscle myosin phosphatase inhibitors can induce contraction of smooth muscle in the absence of Ca²⁺. Protein kinase C- (PKC-) was previously implicated in this process based largely on its supposed absence in the ferret portal vein, and a correlation was drawn between the presence of this isoform and the ability of smooth muscle to contract independently of Ca²⁺ and phosphorylation of the 20 kDa regulatory light chains of myosin (MLC₂₀). We demonstrate here, with two antibodies, one to the NH₂ terminus and the other to the COOH terminus of PKC-, that is present in both ferret portal vein and rabbit portal vein smooth muscle, neither of which exhibits phorbol ester-induced contraction in the absence of Ca²⁺. However, in the presence of clamped submaximal Ca²⁺, phorbol ester increased MLC₂₀ phosphorylation from 17.7 ± 1.7% to 46.4 ± 3.6% in ferret portal vein smooth muscle and evoked an increase in force. Prolonged (48 h) incubation of ferret portal vein with phorbol esters completely down-regulated PKC-, as shown by Western blots, and abolished the phorbol ester-evoked contraction at submaximal Ca²⁺, but not Ca²⁺-independent, contractions induced by the phosphatase inhibitor microcystin. Contractions induced by microcystin in Ca²⁺-free solution were associated with increased phosphorylation of myosin light chain kinase (MLCK). Activation of MLCK by autophosphorylation in the absence of Ca²⁺ occurs in vitro (1). We conclude that PKC- is neither necessary nor sufficient for Ca²⁺-independent regulation of myosin II in smooth muscle, but contractions induced by agents that inhibit smooth muscle myosin phosphatase in the absence of Ca²⁺ may be mediated by MLCK autophosphorylated or activated by another Ca²⁺-independent kinase.—Walker, L. A., Gailly, P., Jensen, P. E., Somlyo, A. V., Somlyo, A. P. The unimportance of being (protein kinase C) epsilon. FASEB J. 12, 813–821 (1998)

Key Words: calcium sensitization • PKC • myosin light chain kinase • phosphorylation

	INTRODUCTION

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IT IS NOW GENERALLY RECOGNIZED that contraction of smooth muscle is regulated not only by cytoplasmic [Ca²⁺], but also by agents that sensitize or desensitize the contractile regulatory proteins to calcium (2). Phorbol esters, activators of protein kinase C (PKC)³, were among the earliest agents shown to Ca²⁺-sensitize smooth muscle, as indicated by their ability to increase force at constant [Ca²⁺] (3–5), but there is still little direct information about the specific PKC isoform (or isoforms) involved in this process. The more than ten identified PKC isoforms fall into three categories (6–8): 1) the conventional PKCs (, ß1, ß2, ), which are Ca²⁺ dependent and activated by phorbol esters; 2) the novel PKC isoforms (, , , ), which are also activated by phorbol esters but are Ca²⁺ independent, and 3) the atypical PKCs (, ), which are Ca²⁺ independent and phorbol ester insensitive.

The multiplicity of PKC isoforms leaves considerable uncertainty about the identity of the specific isoforms involved in Ca²⁺ sensitization. One laboratory claimed a special role for PKC- in mediating calcium-independent contraction in vascular smooth muscle (9, 10) based largely on their inability to detect PKC- in ferret portal vein (PV), a smooth muscle that fails to contract in the absence of Ca²⁺, whereas they detected PKC- in ferret aortic smooth muscle, which can contract in the absence of extracellular calcium in response to agonist and phorbol esters. This contraction was associated with a translocation of PKC- to the membrane (11, 12). The same laboratory reported that phorbol ester-induced Ca²⁺ sensitization of force occurred without an increase in the phosphorylation of the 20 kDa regulatory myosin light chains (MLC₂₀) (9), the physiological mechanism of G-protein-coupled Ca²⁺ sensitization (2). However, we recently found that PKC- is present in rabbit PV, a smooth muscle that, like ferret PV, does not contract in the absence of calcium (13), indicating that PKC- is not sufficient as a mediator of Ca²⁺-independent contraction.

The purpose of the present study was to examine the two basic premises on which the PKC- hypothesis was predicated: the absence of PKC- in ferret PV and the requirement to activate PKC- in order to induce Ca²⁺-independent contraction. We have previously shown that neither conventional nor novel PKCs are necessary for agonist-induced, G-protein-coupled Ca²⁺ sensitization (13), and our present results specifically eliminate PKC- as a necessary messenger of Ca²⁺-independent contraction. We also suggest an alternative mechanism of activation of smooth muscle in the absence of calcium.

	METHODS

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Isometric tension measurement
Male ferrets (12–15 wk, 850–1200 g) were killed with halothane and exsanguinated in accordance with institutional policies and Public Health Service guidelines regarding humane use of laboratory animals. The PV was removed, cleaned of adventitia, and longitudinal muscle strips (250–300 µm wide and 3–4 mm long) were dissected. Strips were attached to a force transducer (AE801; AME, Horten, Norway) and incubated at 21°C in a well on a bubble plate for measurement of isometric tension (14). Strips were stretched to 1.3x resting length prior to stimulation. After obtaining contractions evoked by high K⁺, strips were incubated in relaxing solution [Ca²⁺ free, 1 mM EGTA (G1)] and permeabilized with 17.5 µg/ml -toxin (List Biological Laboratories, Inc., Campbell, Calif.) for 30 min. The sarcoplasmic reticulum was depleted of calcium by incubation of strips with 10 µM A23187 (Calbiochem, La Jolla, Calif.) for 10 min in relaxing solution. For experiments requiring the use of microcystin, strips were permeabilized with 50 µM ß-escin for 15 min in relaxing solution and incubated with A23187 to deplete calcium from the sarcoplasmic reticulum.

MLC₂₀ phosphorylation
Permeabilized muscle strips were incubated with either relaxing solution (G1), maximal calcium (pCa 4.5), submaximal calcium (pCa 6.5), or submaximal calcium and phorbol 12, 13-dibutyrate (PDBu) (1 µM) containing solutions for predetermined times. Individual strips were rapidly frozen in liquid freon precooled with liquid nitrogen, removed from the transducer, and freeze substituted in 10% trichloroacetic acid/acetone. MLC₂₀ phosphorylation was determined with 2-dimensional isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (15) and quantitated by densitometry using a Bio-Rad GS-670 imaging densitometer.

Western blotting and immunoprecipitation
Tissues were homogenized in small glass on glass homogenizers in buffer containing 150 mM NaCl, 50 mM Tris, pH 7.0, 1% NP-40 and the protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride (10 µM), leupeptin (100 µg/ml), and aprotinin (1 µg/ml). Extracts were centrifuged at 600 g for 5 min and the supernatant was collected. For Western blotting, extracts were subjected to SDS-polyacrylamide gel electrophoresis (10% acrylamide) and transferred to poly(vinylidene difluoride) membranes. Nonspecific binding sites were blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) containing 0.5% Tween-20 and the membranes were incubated with primary monoclonal antibody to PKC- for 3 h at room temperature. The blots were washed and incubated with anti-mouse antibody conjugated to horseradish peroxidase (1:65,000) for 1 h. Proteins were visualized by chemiluminescence (ECL, Amersham, Arlington Heights, Ill.).

For immunoprecipitation, a minimum of 20 small strips were used for each condition. Extracts were prepared as described above and precleared with protein A-agarose for 1 h at room temperature. The protein A-agarose used to preclear samples was saved and blotted to exclude the possibility that the proteins of interest were binding nonspecifically to the protein A-agarose. Extracts were incubated with primary antibody overnight at 4°C. Secondary rabbit anti-mouse antibody (10–20 µg/ml) was added for 1 h, followed by the addition of protein A-agarose (1 h, 1:100). Immunoprecipitates were collected by centrifugation, and washed once with homogenization buffer and twice with PBS. Laemmli sample buffer was added and immunoprecipitates were heated for 5 min at 85°C. Mouse monoclonal antibody to PKC- was used at a concentration of 0.5 µg/ml for Western blotting and 10 µg/ml for immunoprecipitation. Mouse monoclonal antibody to myosin light chain kinase (MLCK) was used at a concentration of 2 µg/ml for Western blotting and 20 µg/ml for immunoprecipitation.

Down-regulation of PKCs
Tissues were dissected and prepared as for tension measurements. Muscle strips were incubated at 37°C for 48 h in sterile Hepes-buffered Krebs solution containing PDBu (20 µM). Control strips were incubated under similar conditions in solutions containing only the vehicle (0.1% DMSO). All solutions used to incubate tissues contained 1% penicillin/streptomycin and were changed twice daily.

In situ phosphorylation of myosin light chain kinase with [³²P]ATP
Twenty-five PV strips were used for each condition. ß-Escin-permeabilized strips were washed with G1 and incubated in G10 solution containing 0.5 mM ATP and no creatinine phosphate. After 5 min, strips were changed to the same G10 solution containing 2.5 mCi [³²P]ATP. Microcystin was added appropriately and strips were incubated (rotating) for 60 min. Strips were removed, homogenized, and processed for immunoprecipitation and Western blotting as described above.

Materials
Monoclonal anti PKC- generated against NH₂-terminal residues 1-175 was from Trandsuction Laboratories (Lexington, Kentucky). A second (polyclonal) antibody to a COOH-terminal epitope of PKC- used to confirm the identity of the PKC isoform and protein A-agarose were from Santa Cruz Biotechnologies (Santa Cruz, Calif.). Monoclonal anti-MLCK was from Sigma (St. Louis, Mo.). Anti-rabbit antibody conjugated to horseradish peroxidase was from Amersham, and anti-mouse antibody conjugated to horseradish peroxidase was from Goldmark, Inc. (Phillipsburg, N.J.) [³²P]ATP (150 mCi/ml) was from Dupont, NEN (Wilmington, Del.).

	RESULTS

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Identification of PKC- in ferret portal vein
It has been reported that ferret PV does not contain PKC- (12), even though we detected this isoform in rabbit PV (13), and so we examined the distribution of PKC- in ferret smooth muscle. As shown in Fig. 1A, PKC- was detected by Western blot in ferret PV, aorta, femoral artery, and ileum. In each tissue examined, a doublet was apparent at approximately 92 kDa; a band of 88 kDa was also detected in ileum. The 92 kDa band (or bands) was not detected if the antibody was preincubated (adsorbed) with the peptide (data not shown), confirming the presence of PKC- in ferret PV. Furthermore, the immunoreactive band in portal vein was immunoprecipitated and the immunoprecipitated protein was recognized by an anti-PKC- antibody ( Fig. 1B). The specificity of this antibody was tested against recombinant PKC- and -ß, and no cross-reactivity was seen at the concentrations used (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 1. Distribution of PKC- in ferret smooth muscles. A) Western blots of tissue homogenates of aorta (A), portal vein (PV), femoral artery (FA), and ileum (I). All lanes contain 50–100 µg total protein. B) Western blot of portal vein after immunoprecipitation of PKC-. WE, whole extract (before immunoprecipitation); S, supernatant (after immunoprecipitation); P, precipitate; B, rat brain extract (control) Representative of six experiments.

Phorbol ester- and agonist-induced calcium sensitization
The effect of PDBu on the Ca²⁺ sensitivity of contraction of ferret PV was assessed in smooth muscle strips permeabilized with Staphylococcus aureus -toxin. Representative recordings of the effects of PDBu on the pCa tension curve are shown in Fig. 2A. The vehicle for PDBu, 0.1% DMSO, had a minor relaxing effect on submaximally contracted strips. PDBu (1 µM) shifted the pCa tension curve to the left ( Fig. 2B) and also increased the maximal force produced by cumulative additions of calcium by 2.1-fold. The effect of phenylephrine and GTP on the pCa tension curve in PV was similar but much smaller (data not shown), i.e., shifting the pCa tension curve to the left and increasing the maximal Ca²⁺-activated force by only 1.2-fold.

View larger version (26K): [in this window] [in a new window]   Figure 2. PDBu-induced calcium sensitization of -toxin permeabilized ferret portal vein smooth muscle. After permeabilization, strips were contracted once with CaG, relaxed, and contracted with increasing amounts of calcium. Note the desensitization of force to calcium during the cumulative calcium response curve (-PDBu) (41). To assess the effect of PDBu on PV strips, the muscles were incubated in 1 µM PDBu for 15 min and a second pCa-tension curve was obtained. A) Representative trace showing PDBu-induced increases in calcium sensitivity. B) Force expressed as percent of the control CaG response. n = seven experiments; vertical bars represent 1 SEM.

To assess the calcium requirement of PDBu-induced Ca²⁺ sensitization, we incubated the ferret portal vein strips in Ca²⁺-free solutions [10 mM EGTA (G10)] with PDBu. In the absence of calcium, PDBu (1 µM) had no effect on smooth muscle tension. Even prolonged incubations (12–16 h) with 10 µM PDBu failed to evoke contractions under calcium-free (G10) conditions ( Fig. 3A). Similar results were obtained with rabbit PV: 10 µM PDBu did not cause contraction in Ca²⁺-free medium even after 15 h incubation. In contrast, rabbit femoral artery incubated with PDBu (1 µM) under Ca²⁺-free conditions (G10) exhibited very slow force development, reaching 44 ± 2% (n=7) of the maximal response to pCa 5 after 12–16 h ( Fig. 3B). Furthermore, in rabbit ileal smooth muscle, no force was elicited by PDBu under Ca²⁺-free conditions, consistent with the low Ca²⁺ sensitivity of this phasic smooth muscle (15).

View larger version (12K): [in this window] [in a new window]   Figure 3. The absence of PDBu-induced increases in force in the absence of calcium. Ferret portal vein (A) was incubated in calcium-free solution containing 10 mM EGTA (G10) and stimulated with 10 µM PDBu. No change in force was elicited, even during prolonged exposure. In contrast, rabbit femoral artery (B) treated in the same way developed a slow contraction with exposure to 10 µM PDBu, even in the absence of calcium.

MLC₂₀ phosphorylation
The effect of 1 µM PDBu on the extent of phosphorylation of the 20 kDa light chains of myosin was determined at a submaximal concentration of calcium. As shown in Fig. 4A, ferret PV strips were contracted with submaximal calcium and further sensitized with 1 µM PDBu. At the times indicated by asterisks, muscle strips were rapidly frozen and MLC₂₀ phosphorylation was measured (see Methods). Figure 4B shows that in relaxing solution (1 mM EGTA, G1), MLC₂₀ phosphorylation was 2.9 ± 1.7% (n=3). Maximal calcium (CaG) solution caused MLC₂₀ phosphorylation to increase to 54.7 ± 6.4% (n=3). Incubation of the muscle with submaximal calcium (pCa 6.5) resulted in an increase in MLC₂₀ phosphorylation to 17.7 ± 1.7% (n=8) and addition of 1 µM PDBu further increased phosphorylation to 46.4 ± 3.6%.

View larger version (25K): [in this window] [in a new window]   Figure 4. PDBU-induced increases in phosphorylation of the 20 kDa light chains of myosin. Ferret portal vein strips were contracted with submaximal calcium and sensitized with 1 µM PDBu. At predetermined times, the muscles were rapidly frozen and MLC20 phosphorylation was measured. A) Typical force trace showing the sensitization protocol and the times at which the muscles were frozen (asterisks). B) Summary of at least three experiments (see Results).

The lack of effect of down-regulation of PKC- on contraction induced by microcystin, an inhibitor of myosin light chain phosphatase
As in femoral artery (16), incubation of permeabilized PV strips with 1 µM microcystin ( Fig. 5) under calcium-free conditions (G10) resulted in a slow increase in force that reached 24.4 ± 3.7% of the maximal force elicited by prior incubation with CaG. Addition of 10 µM PDBu or 50 µM GTPS at the plateau of microcystin-induced contraction caused no further increase in force. The force elicited by CaG after incubation with microcystin was slightly lower than in controls, suggesting that microcystin also had a small inhibitory effect on the actomyosin-ATPase. Furthermore, down-regulation of PKC- by chronic exposure to phorbol esters (48 h, 20 µM PDBu) had no effect on microcystin-evoked contraction in the absence of calcium ( Fig. 6), with microcystin-evoked contraction reaching 29.8 ± 3.7% of the CaG contraction. The absence of PKC- on Western blots ( Fig. 6B) demonstrates the effectiveness of the down-regulation protocol.

View larger version (11K): [in this window] [in a new window]   Figure 5. Force development induced in Ca2+-free solution by the myosin light chain phosphatase inhibitor microcystin. Permeabilized ferret portal vein strips were incubated with 1 µM microcystin in calcium-free solution (10 mM EGTA, G10). Addition of 10 µM PDBu or 50 µM GTPS caused no further increase in force. Representative of three experiments.

View larger version (8K): [in this window] [in a new window]   Figure 6. Microcystin-evoked increases in force under calcium free conditions in PKC down-regulated rabbit portal vein. Rabbit portal vein strips were incubated with 20 µM PDBu for 48 h at 37°C. Western blots confirm the complete absence of PKC-. In these tissues, where PKC- is absent, tissues still exhibit increases in force upon inhibition of the smooth muscle phosphatase by microcystin. Inset: Western blot of for PKC- in control (C) and down-regulated (D) tissues.

Myosin light chain kinase is phosphorylated during microcystin-evoked contraction in both control and down-regulated tissues
Autophosphorylated MLCK is active in solution even in the absence of calcium (1; J. E. Andrea and M. P. Walsh, personal communication). Therefore, to determine whether autophosphorylation of MLCK could be a mechanism of Ca²⁺-independent activation of myosin kinase activity in situ, we examined MLCK immunoprecipitates of control and PDBu down-regulated tissues for evidence of MLCK phosphorylation during exposure to microcystin under calcium-free conditions. Tissues were incubated in G10 solution (calcium free) containing 2.5 mCi [³²P]ATP and stimulated with microcystin (1 µM) for 1 h. In both control and down-regulated tissues, microcystin increased the incorporation of ³²P into several substrates (data not shown) and caused increased incorporation of ³²P into immunoprecipitates of MLCK ( Fig. 7A). Western blots of the same membrane verified the presence of MLCK in the immunoprecipitates ( Fig. 7B).

View larger version (46K): [in this window] [in a new window]   Figure 7. Microcystin-evoked increases in phosphorylation of myosin light chain kinase in control and PDBu down-regulated tissues. Control and down-regulated tissues were stimulated with 1 µM microcystin for 1 h in the presence of [32P]ATP, homogenized, and subjected to immunoprecipitation with an antibody against MLCK. A) Autoradiograph of the immunoprecipitate of control (C) and down-regulated tissues (D). B) Western blot of the same membranes for MLCK. Representative of three experiments.

	DISCUSSION

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Phosphorylation of the regulatory myosin light chain (MLC₂₀) by a calcium-calmodulin (CaCaM)-dependent myosin light chain kinase is the primary mechanism of initiating contraction of vertebrate smooth muscle (reviewed in refs 2, 17, 18). However, the existence of a Ca²⁺-independent MLC₂₀ kinase has been inferred from the ability of a variety of agents to induce contraction in the absence of Ca²⁺ in certain, but not all, smooth muscles. These agents include the phosphatase inhibitors okadaic acid (19), calyculin (20, 21), microcystin (16), tautomycin (16), GTPS (14), and phorbol esters (3, 5). One laboratory has focused on protein kinase C- (PKC) as a possible mediator of Ca²⁺-independent contraction via a MAP-kinase cascade (10). This hypothesis is based on 1) an apparent correlation between the presence of PKC- in a given smooth muscle and the ability to contract in the absence of Ca²⁺ (see opening paragraphs) and 2) the ability of a constitutively active exogenous PKC- to induce Ca²⁺-independent contraction (10, 22, 23). It has also been suggested by these (9) and other (24–27) investigators that contractions induced by phorbol esters (and, by implication, PKC) are not accompanied by or due to phosphorylation of MLC₂₀. Our present findings and reports in the literature now allow us to evaluate whether PKC- is necessary and/or sufficient for inducing Ca²⁺-independent contractions in smooth muscle and to consider alternative mechanisms.

In contrast to previous studies (10), we found that ferret PV, like rabbit PV (13), does indeed contain PKC-, whereas none of these smooth muscles contract in the absence of Ca²⁺. We detected PKC- in ferret and rabbit PV with two different antibodies: one directed to the NH₂ terminus ( Fig. 1A) and the other to the COOH terminus of this isoform (data not shown). In view of the failure of PDBu to stimulate a Ca²⁺-independent contraction ( Fig. 3A) despite the presence of PKC- in the portal vein of both species, we conclude that PKC- is not a sufficient mediator of Ca²⁺-independent contraction.

Turning to the question of whether PKC- is necessary for Ca²⁺-independent contractile responses, we find that inhibition of myosin phosphatase by microcystin can induce Ca²⁺-independent contractions even in smooth muscles in which PKC- (together with other conventional and novel PKCs) was down-regulated through chronic treatment with phorbol ester ( Fig. 6; refs 13, 28). Thus, inhibition of smooth muscle myosin light chain phosphatase reveals that the activity of a Ca²⁺-independent MLC₂₀ kinase does not require PKC-.

Phosphorylation of MLC₂₀ increased during PDBu-induced contraction at pCa 6.5 in ferret PV ( Fig. 4B). Several independent laboratories have shown that the sites phosphorylated in response to phorbol esters in vivo are the same (Thr¹⁸, Ser¹⁹) as the sites physiologically phosphorylated by MLCK (29, 30). These findings suggest that the primary mechanism of phorbol ester-induced Ca²⁺ sensitization is through MLC₂₀ phosphorylation, although they do not exclude the existence of ancillary phosphorylation-independent mechanisms. This increased phosphorylation is thought to be the result not of a shift of PKC substrate specificity, but due to activating phosphorylation of a phosphatase inhibitor that can respond in this fashion (31).

We also question arguments based on the contractile effect of constitutively active PKC- (22, 23), because the substrate specificity of constitutively active PKCs is altered (32) and such enzymes can phosphorylate the MLCK sites of MLC₂₀ (33).

Inhibitors of neither PKC nor CaCaM-dependent protein kinase II inhibit Ca²⁺-independent contraction induced by calyculin (21), in contrast to the inhibitory effect of ML-9, a MLCK inhibitor (16, 21). These findings suggested that MLCK or a related kinase mediates Ca²⁺-independent phosphorylation and contraction, although the Ca²⁺-independent MLCK activity in permeabilized smooth muscle is considerably greater than the in vitro activity of MLCK in the absence of Ca²⁺ (16). However, it was recently shown that MLCK is autophosphorylated (on Thr⁸⁰³) and, as a result, becomes active under Ca²⁺-free conditions (1; J. E. Andrea and M. P. Walsh, personal communication). The activity of auto-activated MLCK may be sufficient to cause MLC₂₀ phosphorylation and contraction in the absence of Ca²⁺ when dephosphorylation of MLC₂₀ is prevented by an exogenous phosphatase inhibitor, such as microcystin, or by a kinase C or G-protein-mediated process (2, 13, 16). Our finding of increased MLCK phosphorylation during microcystin-induced contraction ( Fig. 7) is consistent with autophosphorylated MLCK being a mediator of calcium-independent contraction. However, the small amount of permeabilized tissue available precluded our identifying the site of phosphorylation on MLCK, and further study is required to definitively establish the probable role of activating phosphorylation of MLCK in Ca²⁺-independent MLC₂₀ phosphorylation and contraction in situ.

The use of microcystin in the present study allowed the unmasking of a low activity, calcium-independent kinase that our current data suggest may be MLCK. The differences between the ability of femoral artery and portal vein to contract in the absence of calcium ( Fig. 3) may be due to the much higher phosphatase activity in portal vein than in femoral artery (16), as the ratio of kinase to phosphatase activity may be one determinant of sensitization. Telokin, which is thought to activate the phosphatase, is abundant in phasic but not in tonic smooth muscles like the femoral (34A), and the presence or absence of telokin probably affect the balance of phosphatase to kinase activity (34A).

Thus, in spite of the well-documented Ca²⁺-sensitizing activity of activators of conventional and novel PKCs, phorbol esters, and diacylglycerol (DAG; refs 3–5, 13, 29), the physiological significance of conventional and novel kinase Cs in agonist-induced G-protein-coupled Ca²⁺-sensitization is open to question (13, 35). The upstream components of the major G-protein-coupled and phorbol ester-activated pathways are different; consequently, the two mechanisms of Ca²⁺ sensitization are additive (36). Furthermore, down-regulation of novel and conventional PKCs by chronic treatment with phorbol esters abolishes phorbol ester-induced (but not agonist-induced) G-protein-coupled Ca²⁺ sensitization (13, 35). The effects of DAG released from phosphatidylinositol by phospholipase C are likely to be transient, as a result of rapid metabolism of this lipid messenger (37), whereas the DAG released from phosphatidylcholine by phospholipase D has a different fatty acid composition and, at least in some cells, does not activate PKCs (38). Down-regulation of G-protein-coupled Ca²⁺ sensitization does not inhibit PDBu-induced Ca²⁺ sensitization (28); inhibition of RhoA, a low molecular weight G-protein that mediates Ca²⁺ sensitization, does not inhibit Ca²⁺ sensitization by phorbol esters or the translocation of PKCs (39). Activated MAP kinase, claimed to be a downstream effector of kinase C--mediated Ca²⁺ sensitization, failed to have a Ca²⁺-sensitizing effect on permeabilized smooth muscle, although it did phosphorylate endogenous caldesmon (40).

We conclude that both G-protein-coupled and phorbol ester-induced Ca²⁺ sensitization are primarily due to inhibition of smooth muscle myosin phosphatase and that PKC- is neither necessary nor sufficient for this process; the presence or absence of Ca²⁺-independent contraction depends on the balance of the myosin light chain kinase/myosin phosphatase activities in different (e.g., tonic and phasic) smooth muscles (14, 16). In the absence of Ca²⁺ and with a phosphatase inhibitor present, a condition favoring activating autophosphorylation of MLCK (1), Ca²⁺-independent contractions are probably the result of MLC₂₀ phosphorylation by the autoactivated enzyme unopposed by the inhibited myosin phosphatase or to MLCK activation by another Ca²⁺-independent kinase.

	ACKNOWLEDGMENTS

 
This work was supported by NIH PO1 HL48807 and AHA VA96-F-02 (L.A.W.).

	FOOTNOTES

 
¹ With apologies to Oscar Wilde.

¹ Correspondence: Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, P.O. Box 10011, Charlottesville, VA 22906-0011, USA. E-mail: aps2n{at}aemsun.med.virginia.edu

³ Abbreviations: PKC, protein kinase C; MLC₂₀, 20 kDa regulatory myosin light chains; G1, relaxing solution (Ca²⁺ free, 1 mM EGTA); PDBu, phorbol 12, 13-dibutyrate; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; MLCK, myosin light chain kinase; PV, portal vein; CaG, maximal calcium; CaCaM, calcium-calmodulin; DAG, diacylglycerol.

Received for publication December 12, 1997. Accepted for publication February 2, 1998.

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