Introduction

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The lipid second messenger diacylglycerol (DAG) and phorbol esters regulate the activity of Ca²⁺-dependent (, I, II, ) and Ca²⁺-independent (, , , , µ) isoforms of protein kinase C (PKC) (Nishizuka, 1995; Ron and Kazanietz, 1999). DAG and phorbol esters bind to the regulatory C1 domain of PKC and increase the affinity of PKC for membrane and other target proteins. A Ca²⁺-binding region in the regulatory C2 domain is present only in Ca²⁺-dependent PKC isozymes. Binding of PKC to the membrane and enzyme activation are differentially regulated by Ca²⁺: low concentrations of Ca²⁺ increase the affinity for binding, whereas higher concentrations are required for PKC activation.

Several studies have examined the requirement of Ca²⁺ for phorbol-stimulated, PKC-dependent contraction of smooth muscle. Both phorbols and DAG analogs are known to cause contraction of vascular and visceral smooth muscle that is characteristically slow in developing and is preceded by a prolonged lag period (Chatterjee and Tejada, 1986; Nakajima et al., 1991). Only a few smooth muscle tissues, for example, bovine trachea, guinea pig tenia coli, and rat anococcygeus, do not respond directly to phorbols, but even in these, phorbols can potentiate the response to agents, such as extracellular K⁺ and Ca²⁺ ionophores, that increase intracellular Ca²⁺ levels (Mitsui and Karaki, 1993; Kaneda et al., 1995; Tajimi et al., 1997). In other muscle tissues, phorbol-induced contraction is sensitive to the absence of Ca²⁺ from the extracellular medium and can be either partly or completely inhibited by L-type Ca²⁺ channel blockers, implying a phorbol-stimulated increase in Ca²⁺ influx (Gleason and Flaim, 1986; Chiu et al., 1987, 1988; Nakajima et al., 1991; Hattori et al., 1995; Masui and Wakabayashi, 1997). An increase in Ca²⁺ influx has been directly measured in rabbit and rat aorta and dog splenic artery (Gleason and Flaim, 1986; Chiu et al., 1987; Khoyi et al., 1999). Measurements of cytosolic free [Ca²⁺]_i have yielded conflicting results: an increase in [Ca²⁺]_i has been reported in some studies of rat aorta and porcine carotid arteries (Rembold and Murphy, 1988; Nakajima et al., 1993; Kaneda et al., 1995), but not in other studies of rat and ferret aorta, and canine trachea (Jiang and Morgan, 1987; Ozaki et al., 1990).

In this study we have examined the ability of phorbol esters to mobilize Ca²⁺ and cause contraction of smooth muscle cells isolated separately from the circular and longitudinal muscle layers of guinea pig intestine. Smooth muscle cells from the two layers differ in the mechanisms they use to mobilize Ca²⁺ in response to activation of G protein-coupled receptors. Ca²⁺ mobilization in circular muscle is initiated by activation of phospholipase C-1 or -3 and is mediated by inositol 1,4,5-triphosphate-dependent Ca²⁺ release (Murthy et al., 1991; Makhlouf and Murthy, 1997), whereas Ca²⁺ mobilization in longitudinal muscle is initiated by activation of phospholipase A₂ (PLA₂) and arachidonic acid-mediated Ca²⁺ influx that triggers both Ca²⁺- and cyclic ADP ribose-induced Ca²⁺ release (Kuemmerle et al., 1994; Murthy et al., 1995). The results indicate that phorbol esters induce rapid, sustained contraction and increase [Ca²⁺]_i in both circular and longitudinal muscle cells. The increase in [Ca²⁺]_i is essential for contraction and is mediated by Ca²⁺ influx in longitudinal muscle and Ca²⁺ release in circular muscle. Phorbol-induced contraction, however, is entirely mediated by one or more Ca²⁺-dependent PKC isozymes.

	Experimental Procedures

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Preparation of Dispersed Intestinal Smooth Muscle Cells. Muscle cells were isolated separately from the circular and longitudinal muscle layers of guinea pig intestine by sequential enzymatic digestion, filtration, and centrifugation as described previously (Murthy et al., 1991). Briefly, muscle strips were incubated at 31°C for 30 min in HEPES medium with type II collagenase (0.1%) and soybean trypsin inhibitor (0.1%). The partly digested strips were washed and muscle cells allowed to disperse spontaneously for 30 min. The cells were harvested by filtration through 500-µm Nitex (Tetko Inc., Briarcliff Manor, NY) and centrifuged twice at 350g for 10 min.

Measurement of Muscle Cell Contraction by Scanning Micrometry. Contraction was measured in intact and permeabilized muscle cells by scanning micrometry as described previously (Murthy et al., 1991). An aliquot containing 10⁴ cells/ml was added to 0.1 ml of medium containing various concentrations of phorbol-12-myristate-13-acetate (PMA), or phorbol 12,13-dibutyrate (PDBu) and the reaction terminated at various intervals with 1% acrolein. In some experiments, the cells were incubated in Ca²⁺-free medium (0 Ca²⁺ plus 1 mM EGTA) or were incubated in Ca²⁺-containing medium and treated for 10 min with methoxyverapamil (D600, 1 µM) or caffeine (10 mM) before addition of PMA. The effect of PKC antibodies was determined in permeabilized muscle cells after preincubation for 1 h with 1 µg/ml of each antibody separately. The mean length of muscle cells treated with phorbol esters was compared with the mean length of untreated cells and contraction was expressed as percentage of decrease in mean cell length.

Measurement of [Ca²⁺]_i in Dispersed Smooth Muscle Cells. [Ca²⁺]_i was measured in suspensions of muscle cells with the Ca²⁺ fluorescent dye fura 2 as described previously (Murthy et al., 1991; Kuemmerle et al., 1994). Muscle cells were suspended in a medium containing 10 mM HEPES, 125 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 0.5 mM MgSO₄, 5 mM glucose, 20 mM taurine, 45 mM sodium pyruvate, and 5 mM creatine, and incubated with fura 2/AM (2 µM) for 20 min at 31°C. After centrifugation at 350g for 20 min, the cells were incubated in fura 2-free medium for immediate measurement of Ca²⁺. Fluorescence was monitored at 510 nm, with excitation wavelengths alternating between 340 and 380 nm, and the measurements were corrected for autofluorescence of unloaded cells. An estimate of [Ca²⁺]_i was obtained from observed, maximal, and minimal fluorescence ratios.

Measurement of PKC Activity. PKC activity was measured in the particulate and cytosolic fractions by an adaptation of the method of Takai et al. (1979). One milliliter of cell suspension (2 × 10⁶ cells/ml) was incubated with 1 µM PMA for 60 s, and the reaction was terminated by rapid freezing in a dry ice/acetone slurry. The cell suspension was thawed and centrifuged at 1000g for 15 min, and the cells were resuspended in ice-cold 20 mM Tris-HCl medium, pH 7.5, containing 250 mM sucrose, 1 mM EGTA, 10 mM mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride and were then homogenized. Particulate and cytosolic fractions were separated by centrifugation and purified on DEAE-cellulose columns. PKC activity was measured by Ca²⁺/phospholipid-dependent phosphorylation of histone-1 and was expressed as picomoles of phosphorus transferred to histone per minute per milligram of protein.

Materials. [-³²P]ATP was obtained from NEN Life Sciences Products (Boston, MA); HEPES from Research Organics (Cleveland, OH); soybean trypsin inhibitor and collagenase (type II) from Worthington Biochemical (Lakewood, NJ); fura 2/AM from Molecular Probes (Eugene, OR); calphostin C from Calbiochem (La Jolla, CA); and KT5926 and AACOCF₃ from Biomol (Plymouth Meeting, PA). PKC- antibody and a common PKC-,, antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were obtained from Sigma (St. Louis, MO).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Time Course and Stoichiometry of Phorbol-Stimulated Contraction in Intestinal Smooth Muscle. PMA (1 nM) caused a rapid contraction of intestinal muscle cells that rose to a peak within 90 s, and was well sustained thereafter (Fig. 1). The response was instantaneous with no evidence of the prolonged lag period characteristic of the response to phorbols in smooth muscle strips. Similar results were obtained with PDBu. The effect of PMA and PDBu in longitudinal muscle cells was concentration-dependent with an EC₅₀ of 0.5 nM and a maximal response at 1 µM (Fig. 1). The responses of circular muscle cells measured at 1 nM and 1 µM PMA were very similar to the corresponding responses of longitudinal muscle cells. The maximal responses elicited by PMA (27.2 ± 1.4% decrease in cell length) and PDBu (25.6 ± 1.1%) were similar to those elicited by the receptor-linked agonist CCK-8 (25.5 ± 2.0%). There was no contractile response to the inactive phorbol 4- phorbol 12,13-didecanoate.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Time course and concentration dependence of phorbol-stimulated contraction of dispersed intestinal smooth muscle cells. Bottom, PMA (10 nM) was added to isolated longitudinal muscle cells for periods ranging from 15 s to 10 min. Contraction was instantaneous, attained a peak in 90 s, and was sustained for 10 min. Top, PMA was added to longitudinal muscle cells at concentrations ranging from 10 pM to 10 µM for a period of 90 s.  denotes identical responses of circular muscle cells. Maximal response to PMA was similar to that elicited by agonists. Contraction was measured by scanning micrometry and expressed as percentage decrease in control cell length. Values are mean ± S.E. of three experiments.

Phorbol-Stimulated Increase in [Ca²⁺]_i. PMA caused similar concentration-dependent increases in free cystolic [Ca²⁺]_i in longitudinal and circular muscle cells with an EC₅₀ of about 50 nM (Fig. 2). The concentration-[Ca²⁺]_i curves were shifted considerably to the right of the concentration-contraction curves (Figs. 1 and 2). The maximal increases in [Ca²⁺]_i elicited by PMA in circular and longitudinal muscle cells (208 ± 13 and 257 ± 12 nM, respectively) were significantly lower than the maximal increases elicited by CCK-8 (459 ± 56 and 587 ± 49 nM, respectively). The effect of a combination of PMA and CCK-8 was not additive, and did not exceed the maximal increase in [Ca²⁺]_i induced by CCK-8 alone (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent stimulation of [Ca2+]i by PMA and CCK-8 in dispersed intestinal circular and longitudinal muscle cells. [Ca2+]i was measured in fura-2-loaded intestinal circular (closed symbols) and longitudinal (open symbols) muscle cells as described under Experimental Procedures and expressed in nanomolar concentration above basal level (basal: 63 ± 4 and 68 ± 5 nM in circular and longitudinal muscle cells, respectively). The increase in [Ca2+]i was immediate after addition of agonist or PMA. Comparison with Fig. 1 shows that the concentration-[Ca2+]i curves for PMA were shifted to the right by comparison with concentration-contraction curves. Values are mean ± S.E. of six experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of D600 on [Ca2+]i stimulated by PMA in dispersed intestinal circular and longitudinal smooth muscle cells. [Ca2+]i stimulated by maximal concentrations of PMA (1 µM) or CCK-8 (1 nM) was expressed in nanomolar concentration above basal levels. D600 (1 µM) abolished the increase in [Ca2+]i in longitudinal muscle cells only. The effect of adding PMA (1 µM) after CCK-8 (1 nM) was similar to that of CCK-8 alone. Values are mean ± S.E. of six experiments.

Dependence of Contractile Response to Phorbol Esters on [Ca²⁺]_i. Contraction induced by PMA in longitudinal muscle cells was abolished by D600 and by incubation in Ca²⁺-free medium (0 Ca²⁺/1 mM EGTA; Fig. 4). A similar dependence on extracellular Ca²⁺ was observed with the DAG analog 1-oleoyl-2-acetyl-rac-glycerol (1 µM; control longitudinal muscle cell contraction: 28.5 ± 0.6% decrease in cell length versus 2.0 ± 1.7 and 3.5 ± 0.6% with 0 Ca²⁺ and D600, respectively). A similar dependence of longitudinal muscle contraction on extracellular Ca²⁺ also was observed in human intestinal muscle cells [control longitudinal muscle cell contraction: 24.0 ± 1.0 versus 0.1 ± 1.0 and 1.5 ± 0.7% with 0 Ca²⁺ and D600, respectively (n = 5); control circular muscle cell contraction: 24.3 ± 1.1 versus 23.1 ± 1.3 and 22.5 ± 1.3% with 0 Ca²⁺ and D600, respectively (n = 5)].

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of Ca2+-free medium, D600, and caffeine on contraction induced by PMA in dispersed intestinal smooth muscle cells. Muscle cell were treated with PMA for 90 s in the presence or absence of extracellular Ca2+ (0 Ca2+/1 mM EGTA). In experiments with D600 (1 µM) and caffeine (10 mM), the agents were added 10 min before PMA. D600 and Ca2+-free medium abolished the response to PMA in longitudinal muscle cells only, whereas pretreatment with caffeine abolished the response to PMA in circular muscle cells only. Values are mean ± S.E. of four to six experiments.

Mechanism of Phorbol-Stimulated Ca²⁺ Mobilization and Contraction. We have previously shown that Ca²⁺ mobilization induced by receptor-linked agonists is initiated by activation of PLA₂ and Ca²⁺ influx in intestinal longitudinal muscle, and by activation of phospholipase C-1 or -3 and Ca²⁺ release in circular muscle (Makhlouf and Murthy, 1997). However, neither inhibition of PLA₂ activity with AACOCF₃ (10 µM), nor inhibition of phosphoinositide (PI) hydrolysis with U73122 (10 µM) affected PMA-induced contraction in circular or longitudinal muscle cells (Fig. 5). The possibility that the phorbol-induced increase in [Ca²⁺]_i could contribute directly to contraction by activating Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) was examined with the selective kinase inhibitor KT5926 (1 µM): this inhibitor, however, had no effect on PMA-induced contraction of circular or longitudinal muscle cells (Fig. 5). In contrast, contraction of both cell types was strongly inhibited by calphostin C (1 µM), which blocks the phorbol/diacylglycerol-binding site of PKC, whether at 30 s during the peak increase in [Ca²⁺]_i or at 300 s during maximally sustained contraction (Fig. 5). PMA (1 µM) stimulated PKC activity by 570 ± 30 and 320 ± 50 pmol/mg of protein · min above basal levels (135 ± 19 to 152 ± 13 pmol/mg · min) in circular and longitudinal muscle cells, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Selective inhibition of PMA-stimulated contraction by calphostin C in dispersed intestinal circular and longitudinal muscle cells. The PLA2 inhibitor AACOCF3 (10 µM), the PI hydrolysis inhibitor U73122 (10 µM), the MLCK inhibitor KT5926 (1 µM), and the PKC inhibitor calphostin C (1 µM) were added separately for 10 min before addition of PMA. Values are mean ± S.E. of three experiments.

Phorbol-Induced Contraction Mediated by Ca²⁺-Dependent PKC Isozymes. Our earlier studies showed that sustained contraction of intestinal muscle cells induced by receptor-linked agonists was mediated by the Ca²⁺-independent PKC isozyme PKC-, whereas contraction induced by epidermal growth factor and by Ca²⁺ in permeabilized muscle cells was mediated by one or more Ca²⁺-dependent PKC isozyme(s) (Murthy et al., 2000). Consistent with this pattern, incubation of permeabilized circular muscle cells with a common PKC-,, antibody (1 µg/ml) abolished PMA-induced contraction, whereas incubation with a PKC- antibody (1 µg/ml) had no effect (Fig. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Inhibition of PMA-stimulated contraction of dispersed intestinal circular and longitudinal muscle cells by a common antibody to PKC-,,. Bottom, saponin-permeabilized muscle cells were incubated for 60 min with a specific PKC- antibody (1 µg/ml) or a common PKC-,, antibody (1 µg/ml) before addition of PMA (1 µM); contraction was measured at 90 s. Contraction was expressed as percentage of decrease in mean cell length from control. PKC-,, antibody but not PKC- antibody inhibited sustained contraction induced by PMA. Top, concentration-response curves demonstrating the effectiveness of the PKC-,, antibody (IC50 = 50 ng/ml). Values are mean ± S.E. of three or four experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This study showed that phorbol esters induce rapid, sustained contraction and an increase of [Ca²⁺]_i in smooth muscle cells isolated from the circular and longitudinal muscle layers of guinea pig intestine. The increase in [Ca²⁺]_i was mediated by Ca²⁺ influx in longitudinal muscle cells and Ca²⁺ release in circular muscle cells and was essential for phorbol-induced contraction. In both muscle cell types, the increase in [Ca²⁺]_i did not contribute directly to contraction by activating Ca²⁺/calmodulin-dependent MLCK. Phorbol-induced contraction was entirely mediated by one or more Ca²⁺-dependent PKC isozymes.

A phorbol-stimulated increase in [Ca²⁺]_i was not detected in early studies of ferret and rat aortic muscle strips (Jiang and Morgan, 1987), although more recent studies of rat aortic muscle strips and aortic smooth muscle cell lines have demonstrated an increase in [Ca²⁺]_i (Nakajima et al., 1993; Kaneda et al., 1995). The increase in [Ca²⁺]_i could not be detected in Ca²⁺-free medium and appeared to reflect stimulation of Ca²⁺ influx. An increase in Ca²⁺ influx was suggested by previous studies showing that phorbol-stimulated contraction of smooth muscle, including rat and rabbit aortic muscle, was partly or completely inhibited by Ca²⁺ channel blockers and in Ca²⁺-free medium (Gleason and Flaim, 1986; Chiu et al., 1987, 1988; Nakajima et al., 1991; Hattori et al., 1995; Masui and Wakabayashi, 1997). The results obtained in guinea pig intestinal longitudinal muscle cells conformed to this pattern in that PMA-stimulated contraction and increase in [Ca²⁺]_i were completely inhibited by the Ca²⁺ channel blocker D600, and in Ca²⁺-free medium (0 Ca²⁺/1 mM EGTA). Identical results were obtained in human intestinal longitudinal muscle cells.

In intestinal circular muscle cells, however, neither contraction nor the increase in [Ca²⁺]_i induced by PMA was affected by D600 or in Ca²⁺-free medium. Contraction induced by PMA in human intestinal circular muscle cells also was not affected by D600 or in Ca²⁺-free medium. The pattern implied that the increase in Ca²⁺ in circular muscle cells was mediated by Ca²⁺ release from intracellular stores. The stores appeared to be those mobilized by receptor-linked agonists because the increase in [Ca²⁺]_i induced by a maximal concentration of CCK-8 was not augmented by PMA. Depletion of Ca²⁺ stores by preincubation of circular muscle cells with caffeine abolished PMA-stimulated contraction, implying that in these cells also contraction was dependent on an increase in [Ca²⁺]_i. A PMA-induced Ca²⁺ release and a dependence of PMA-stimulated contraction on Ca²⁺ release has not been previously reported.

The mechanisms by which phorbol esters mobilize Ca²⁺ remain elusive. We have previously shown that in intestinal longitudinal muscle cells, Ca²⁺ mobilization by receptor-linked agonists involves sequential activation of PLA₂ and generation of arachidonic acid; the latter activates Cl channels, which causes depolarization of the plasma membrane and activation of voltage-sensitive Ca²⁺ channels, resulting in Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release (Kuemmerle et al., 1994; Murthy et al., 1995; Makhlouf and Murthy, 1997). A selective PLA₂ inhibitor, AACOCF₃ (Kim et al., 1997), however, had no effect on PMA-induced contraction in longitudinal muscle cells, and it appeared more likely that PMA-stimulated Ca²⁺ influx reflected the ability of PKC to phosphorylate and thus, activate voltage-sensitive Ca²⁺ channels (Fish et al., 1988; Oberjero-Paz et al., 1998). In intestinal circular muscle cells, Ca²⁺ mobilization by receptor-linked agonists is mediated by PI hydrolysis and inositol 1,4,5-triphosphate-dependent Ca²⁺ release (Murthy et al., 1991). An inhibitor of PI hydrolysis, U73122 (Murthy and Makhlouf, 1998), however, had no effect on PMA-stimulated contraction, suggesting that Ca²⁺ release, a requirement for PMA-stimulated contraction in this cell type, was not dependent on PI hydrolysis. It is possible that small increases in [Ca²⁺]_i induced by low concentrations of PMA occurred that were not detected by measurement of [Ca²⁺]_i in suspensions, which could explain the difference in the EC₅₀ for PMA-induced contraction and [Ca²⁺]_i.

KT5926, an inhibitor of Ca²⁺/calmodulin-dependent MLCK activity (Nakanishi et al., 1990) had no effect on phorbol-stimulated contraction in circular or longitudinal intestinal muscle cells, suggesting that Ca²⁺/calmodulin-dependent activation of MLCK was suppressed either directly by PMA or via PKC (Ikebe et al., 1985).

In both muscle cell types, PMA-induced contraction was mediated by one or more Ca²⁺-dependent PKC isozymes. A common antibody to PKC-,, abolished PMA-induced contraction in circular and longitudinal muscle cells, whereas a specific antibody to Ca²⁺-independent PKC- had no effect. Similar results were obtained by Sohn et al. (1997) in muscle cells isolated from the cat esophagus with a DAG analog. Our recent studies have shown a differential involvement of PKC isozymes in muscle contraction (Murthy et al., 2000): the same PKC-,, antibody used in this study abolished contraction induced by epidermal growth factor, whereas the selective PKC- antibody and a myristoylated peptide pseudosubstrate inhibitor of PKC- abolished PKC-mediated sustained contraction induced by receptor-linked agonists. Differential involvement of PKC isozymes in agonist-induced contraction of vascular muscle cells has been recently reported by Lee et al. (1999). In permeabilized intestinal muscle cells exposed to high concentrations of Ca²⁺ (400 nM), initial contraction is mediated by Ca²⁺/calmodulin-dependent activation of MLCK, whereas sustained contraction is mediated by Ca²⁺-dependent PKC isozymes (Murthy et al., 2000). This may provide an explanation for the requirement of Ca²⁺ and the involvement of Ca²⁺-dependent PKC isozymes in phorbol-stimulated smooth muscle contraction. It should be emphasized, however, that phorbol-stimulated contraction, although PKC-dependent, is mediated by myosin light chain phosphorylation at serine 19, and results from inhibition of myosin light chain phosphatase (Masuo et al., 1994; Jensen et al., 1996; Walker et al., 1998). A pathway linking PKC to activation of an endogenous 17-kDa inhibitor of myosin light chain phosphatase (Eto et al., 1997; Li et al., 1998) has been identified that could serve as a link between receptor- or PMA-mediated activation of PKC and sustained myosin light chain phosphorylation and contraction.