Introduction

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Mobilization of intracellular Ca²⁺ ([Ca²⁺]_i) is the earliest event in both receptor-mediated and nonreceptor-mediated responses of a wide variety of cells to various stimuli. Thus, stimulation of smooth muscle results in an increase in [Ca²⁺]_i and the rapid activation of multiple protein kinases, such as Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), myosin light chain (MLC) kinase, protein kinase C, and mitogen-activated protein (MAP) kinases. These are involved in phosphorylation and activation of their downstream targets, which play an essential role in the regulation of cellular functions (Zhang et al., 1998). The most widely studied pathway in smooth muscle is the Ca²⁺ and calmodulin-dependent MLC kinase, which catalyzes phosphorylation of the 20-kDa MLC and initiates contraction (Kamm and Stull, 1985). Activation of myosin light chain kinase and the resultant phosphorylation of MLC are considered key events in the initiation of smooth muscle contraction. The phosphorylation triggers cycling of myosin cross-bridges along actin filaments and force development in smooth muscle cells. Several laboratories have suggested that CaMKII might play a role in the regulation of vascular smooth muscle contractility (for review, see Kim et al., 2000) and several reports have suggested that CaMKII might modulate the Ca²⁺ sensitivity of the contractile apparatus by either the phosphorylation (and consequent desensitization to Ca²⁺-calmodulin) of myosin light chain kinase (Tansey et al., 1992) or the direct phosphorylation (and consequent sensitization to Ca²⁺-calmodulin) of MLC₂₀ (Edelman et al., 1990). These reports have remained controversial, in part because key experiments in which CaMKII activity was blocked were performed only in cultured smooth muscle cells, which are noncontractile (Tansey et al., 1992). More recently, Kim et al. (2000) provided evidence for a CaMKII-dependent component of contractile force in ferret aorta smooth muscle. Furthermore, they showed that MAP kinase activation as well as phosphorylation of MLC₂₀ are linked to CaMKII as downstream events in a signaling cascade. There is accumulating evidence that indicates that in addition to the MLC kinase pathway, tyrosine kinase pathways play an important role in the regulation of smooth muscle contraction (Di Salvo et al., 1997; Somlyo et al., 1999; Abdel-Latif, 2001). Protein kinase C and MAP kinase are two families of kinases that are present in smooth muscle cells and are believed to play important roles in smooth muscle regulation. MAP kinases are serine/threonine kinases that are activated by phosphorylation on both threonine and tyrosine residues through an upstream MAP kinase kinase. Activation of MAP kinase in response to mechanical and pharmacological stimulation has been reported in vascular smooth muscle (Adam et al., 1995; Katoch and Moreland, 1995; Watts, 1996; Dessy et al., 1998; Kim et al., 2000) and in nonvascular smooth muscle (Gerthoffer et al., 1996, 1997; Nohara et al., 1996; Ohmichi et al., 1997; Yousufzai et al., 2000). Prostaglandin F₂ (PGF₂) was reported to activate MAP kinase and MAP kinase kinase in cultured rat puerperal uterine myometrial cells (Ohmichi et al., 1997).

Several studies have shown that pharmacological elevation of [Ca²⁺]_i activated p42/p44 MAP kinases in a number of cells, including human epidermal carcinoma and human foreskin fibroblasts (Chao et al., 1992), human polymorphonuclear neutrophils (Zhang et al., 1998), B lymphocytes (Franklin et al., 1994), and MCF-7 breast cancer cells (Malaguti et al., 1999). Importantly, CaMKII has been demonstrated to activate all major members of the MAP kinase family (c-Jun NH₂-terminal kinase, stress-activated protein kinase, p38, and ERK) (Zhang et al., 1993; Enslen et al., 1996). These findings suggest cross talk between Ca²⁺/calmodulin-dependent protein kinases and p42/p44 MAP kinases in these tissues. In cat iris sphincter, a nonvascular smooth muscle, protein tyrosine phosphorylation is involved in the mechanism of PGF₂-induced contraction, inositol phosphates accumulation, and Ca²⁺ mobilization (Yousufzai and Abdel-Latif, 1998). More recently, we reported that MAP kinase phosphorylation is involved in the mechanism of PGF₂-induced MLC phosphorylation and contraction in this smooth muscle (Yousufzai et al., 2000). In this study we reported that PD98059, a specific inhibitor of p42/p44 MAP kinase, and KN-93, a specific inhibitor of CaMKII, inhibited PGF₂-induced contraction by 94 and 80%, respectively. PGF₂ increased MAP kinase phosphorylation in a concentration-dependent manner with an EC₅₀ value of 1.1 × 10⁸ M and increased contraction with EC₅₀ of 0.92 × 10⁹ M. To further investigate the cross talk between the Ca²⁺ mobilization pathway and the MAP kinase pathway and contraction in this smooth muscle we used three Ca²⁺-mobilizing agonists, namely, PGF₂ (a Ca²⁺-mobilizing agonist that activates a G protein-coupled receptor), ionomycin (an agent that increases the influx of extracellular Ca²⁺), and thapsigargin (an agent that increases [Ca²⁺]_i by inhibiting the microsomal Ca²⁺-pump); and two specific inhibitors, PD98059 and KN-93. In addition, we used isoproterenol, a cAMP-elevating agent, which inhibits [Ca²⁺]_i mobilization (Ding et al., 1997) and contraction (Tachado et al., 1989) in this smooth muscle.

	Experimental Procedures

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Materials. Reagent sources were as follows: prostaglandin F₂ from Cayman (Ann Arbor, MI); 2'-amino-3'-methoxyflavone (PD98059) from BIOMOL (Plymouth Meeting, PA); isoproterenol and carbachol from Sigma Chemical Co. (St. Louis, MO); ionomycin, thapsigargin, and 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzene sulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93) from Calbiochem (La Jolla, CA); and polyclonal anti-MAP kinase [ERK-1 (C-16)] antibodies recognizing p42/p44 MAP kinases, anti-phospho-p42/p44 MAP kinase (E-4), and anti-mouse IgG-horseradish peroxidase and anti-rabbit IgG-horseradish peroxidase secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of reagent grade.

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Preparation of the Iris Sphincter for Muscle Contraction. Cat eyes were obtained through the courtesy of the Augusta-Richmond County Animal Control. Eyes were enucleated immediately after death and were transported to the laboratory packed in ice. The iris sphincter was dissected out and placed in Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 10 mM D-glucose, and 1.25 mM CaCl₂. Indomethacin (1 µM), a cyclooxygenase inhibitor, was added to the incubation medium in all of the experiments to prevent the formation of endogenous prostaglandins. The KRB buffer was used as the incubation medium in the following studies. pH of the buffer was adjusted and maintained at 7.4 with 97% O₂, 3% CO₂. In general, the whole sphincter from one of the pair of eyes served as control, and the other from the fellow eye was used as experimental. To test the viability of the tissue, we routinely monitored the contractility of this cholinergically innervated smooth muscle with carbachol. The methods for securing animal tissue were humane and complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Measurement of Agonist-Induced Muscle Contraction in Iris Sphincter. For measurements of the contraction response, the sphincters were mounted individually in separate organ baths (10 ml) containing KRB buffer. A mixture of 97% O₂, 3% CO₂ was bubbled continuously through the buffer, which was maintained at 37°C. The tissue was allowed to equilibrate for 90 min under a resting tension of 50 mg. After equilibration of the tissue, the agonist was added and changes in tension were monitored continuously with a Grass FT-03 force transducer connected to a Grass DC amplifier (Astro-Med, West Warwick, RI) as previously described (Howe et al., 1986).

Western Blotting and Measurement of p42/p44 MAP Kinase Activation. Phosphorylated species of p42/p44 MAP kinases were detected by anti-phospho-p42/p44 MAP kinase antibodies as described previously (Husain and Abdel-Latif, 1999). Briefly, the muscles were first preincubated for 75 min at 37°C in 1 ml of KRB buffer, pH 7.4. At this time the tissues were transferred to 1 ml of fresh KRB buffer and incubation continued for an additional 15 min to give a total preincubation time of 90 min. The tissues were then incubated in the absence or presence of the Ca²⁺-mobilizing agonists for 5 min. Inhibitors were added 15 min prior to the addition of the agents. The reaction was stopped by immersing the tissue in a methanol-dry ice slurry at 80°C.

	Results

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Ca²⁺-Mobilizing Agonist Stimulation of Contraction and p42/p44 MAP Kinase Phosphorylation. Preliminary experiments were carried out to define the concentrations of agonists and inhibitors and the times of incubation to use in our study. The results we obtained showed that addition of 50 nM PGF₂, 1 µM ionomycin, or 1 µM thapsigargin for 5 min resulted in maximal stimulation of contraction and MAP kinase phosphorylation, and preincubation of the muscles with 10 µM inhibitors resulted in maximal inhibition of the agonist-induced responses (Yousufzai et al., 2000).

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View this table: [in this window] [in a new window]   TABLE 1 Effects of PGF2, ionomycin, and thapsigargin on contraction and p42/p44 MAP kinase activation in cat iris sphincter smooth muscle For measurement of the contractile response the muscles were pre-equilibrated in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 1 µM indomethacin for 90 min. PGF2 (50 nM for contraction, 1 µM for the MAP kinase assay), ionomycin (1 µM), and thapsigargin (1 µM) were then added as indicated and changes in tension responses were monitored as described under Experimental Procedures. The data are expressed as percentage of maximal contraction by 100 nM carbachol (19.4 ± 1.7 mg of tension/mg of wet weight tissue). The data are the mean ± S.E.M. of three to five different experiments. In the studies on agonist-induced MAP kinase phosphorylation in the iris sphincter, the muscles were equilibrated in KRB buffer, pH 7.4, for 90 min, the agonists were added as indicated for 5 min, and the phosphorylated MAP kinase was determined by immunoblotting as described under Experimental Procedures. Phosphorylation of MAP kinase in the iris sphincter was quantitated by densitometry. The autoradiographs were analyzed by densitometry, and the values are expressed in arbitrary units as mean ± S.E.M. of three independent experiments performed in duplicate.

PD98059 Inhibits Ca²⁺-Mobilizing Agonist-Induced Contraction and p42/p44 MAP Kinase Phosphorylation. There is evidence that cross talk occurs between Ca²⁺ and the MAP kinase cascade in a wide variety of tissues (see Introduction). Elevation of [Ca²⁺]_i may lead to the activation of CaMKII, and probably other protein kinases that could result in MAP kinase activation. To investigate the role of p42/p44 MAP kinase in PGF₂-, ionomycin-, and thapsigargin-induced contraction, we used a specific inhibitor of p42/p44 MAP kinase, PD98059. Figure 1 shows typical recordings of mechanical responses of the iris sphincter to PGF₂ alone (Fig. 1A) and to PGF₂ (Fig. 1B), ionomycin (Fig. 1C), and thapsigargin (Fig. 1D) in the absence and presence of PD98059. The p42/p44 MAP kinase inhibitor PD98059 inhibited PGF₂-, ionomycin-, and thapsigargin-induced contraction by 76, 60, and 57%, respectively. These results demonstrate that p42/p44 MAP kinases play an important role in the action of the Ca²⁺-mobilizing agonists on contraction in this smooth muscle. Similarly, when MAP kinase phosphorylation and activation were measured in the iris sphincter after 5-min exposure to the agonists in the absence and presence of various concentrations of PD98059, phosphorylation of MAP kinase was inhibited in a concentration-dependent manner (Fig. 2). As shown in Fig. 2, A and C, PGF₂, ionomycin, and thapsigargin increased the MAP kinase phosphorylation by 220, 188, and 280%, respectively. In the presence of 0.1, 1, and 10 µM PD98059, stimulation of MAP kinase phosphorylation by PGF₂ was inhibited by 13, 33, and 100%, respectively; the stimulation by ionomycin was inhibited by 14, 30, and 100%, respectively; and the stimulation by thapsigargin was inhibited by 23, 46, and 80%, respectively. To rule out the possibility of variation in the levels of p42/p44 MAP kinases, equal amounts of all samples were loaded on SDS-PAGE and immunoblotted with anti-p42/p44 MAP kinase antibodies. As can be seen in Fig. 2B, all samples contained comparable amounts of the p42/p44 MAP kinases. These results demonstrate that elevation of [Ca²⁺]_i by the Ca²⁺-mobilizing agonists is linked to the downstream activation of the p42/p44 MAP kinases.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Representative recordings of mechanical responses of iris sphincter muscle to PGF2 alone (A) and to PGF2 (B), ionomycin (C), and thapsigargin (D) in the absence and presence of PD98059. The muscles were pre-equilibrated in KRB buffer containing 1 µM indomethacin for 90 min. PGF2 (50 nM), ionomycin (1 µM), and thapsigargin (1 µM) were then added as indicated for 3 to 5 min followed by the addition of PD98059 (10 µM).

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent effects of PD98059 on PGF2-, ionomycin-, and thapsigargin-induced MAP kinase activation in iris sphincter muscle. After 90 min of equilibration in KRB buffer, pH 7.4, iris sphincter muscles were pretreated for 15 min with different concentrations of PD98059 prior to stimulation with PGF2 (1 µM), ionomycin (1 µM), or thapsigargin (1 µM) for 5 min. Phosphorylated p42/p44 MAP kinase was determined by using specific anti-phospho p42/p44 MAP kinase antibodies as described under Experimental Procedures. A, phosphorylated p42/p44 MAP kinase. B, Western blot analysis of the tissue extract. C, quantitation of p42/p44 MAP kinase phosphorylation. Phosphorylation of MAP kinases in the iris sphincter was quantitated by densitometry. The autoradiographs were analyzed by densitometry, and the values are expressed in arbitrary units. Results are from one experiment that is a representative of three independent experiments.

PD98059 Inhibits Ca²⁺-Mobilizing Agonist-Induced p42/p44 MAP Kinase Activation. The purpose of this experiment was to determine whether activation of p42/p44 MAP kinase is inhibited by PD98059. As shown in Fig. 3, A and B, the activities of p42/p44 MAP kinases were increased in the presence of PGF₂, ionomycin, and thapsigargin by 212, 191, and 162%, respectively, and these increases were reduced to 7, 16, and 10%, respectively, in the presence of PD98059. To rule out the possibility of variation in the levels of p42/p44 MAP kinases, equal amounts of all samples were loaded on SDS-PAGE and immunoblotted with anti-MAP kinase polyclonal antibodies. As can be seen from Fig. 3C all samples contained comparable amounts of p42/p44 MAP kinase. During the determination of p42/p44 MAP kinases by in-gel kinase assay, we routinely get two additional bands of molecular weights approximately 65 and 110 kDa. However, in the phosphorylation studies performed by using phospho-p42/p44 MAP kinase antibodies we find only two bands corresponding to p42 and p44 MAP kinases. These data provide evidence that in addition to their stimulatory effects on MAP kinase phosphorylation (Fig. 2) the Ca²⁺-mobilizing agonists also stimulate MAP kinase activation (Fig. 3). Furthermore, the data show that p42/p44 MAP kinase activation is abolished by the MAP kinase inhibitor.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of PD98059 on PGF2-, ionomycin-, and thapsigargin-induced p42/p44 MAP kinase activation in iris sphincter muscle. A, effects of PD98059 on p42/p44 MAP kinase activation. The p42/p44 MAP kinase activity was determined by the in-gel renaturation kinase assay as described under Experimental Procedures. B, densitometric analysis: intensity of p42/p44 MAP kinase bands was determined by densitometry. The autoradiographs were quantitated by Alpha Imager TM 2200 documentation and analysis system and expressed as arbitrary units. C, Western blot analysis: the same amounts of proteins (as loaded in A) were separated on 10% SDS-PAGE and the proteins were transferred to nitrocellulose membranes. The membranes were probed with anti-p42/p44 MAP kinase antibodies as described under Experimental Procedures. Results shown are from one experiment that is representative of three independent experiments.

KN-93 Inhibits Ca²⁺-Mobilizing Agonist-Induced Contraction and p42/p44 MAP Kinase Phosphorylation. To investigate the role of CaMKII in PGF₂-, ionomycin-, and thapsigargin-induced contraction and its correlation with p42/p44 MAP kinase activation, we used a specific pharmacological inhibitor of CaMKII, KN-93. KN-93, like KN-62, acts via competition of calmodulin binding to CaMKII (K_i = ~0.37 µM in vitro) (Sumi et al., 1991) but has superior water solubility relative to KN-62. As shown in Fig. 4, KN-93 inhibited PGF₂-, ionomycin-, and thapsigargin-induced contraction by 75, 50, and 55%, respectively. Addition of KN-93 (10 µM) and PD98059 (10 µM) together inhibited PGF₂-induced contraction by about 83% (data not shown). These data indicate that CaMKII plays an important role in PGF₂-, ionomycin-, and thapsigargin-induced contraction. Similarly, when MAP kinase phosphorylation and activation were measured in the iris sphincter after 5-min exposure to the Ca²⁺-mobilizing agonists in the absence and presence of KN-93, the PGF₂-, ionomycin-, and thapsigargin-induced p42/p44 MAP kinase phosphorylation and activation were inhibited by 80, 76, and 75%, respectively (Fig. 5, A and C). To rule out the possibility of variation in the loaded proteins, equal amounts of all samples were loaded on SDS-PAGE and immunoblotted with anti-p42/p44 MAP kinase antibodies. As can be seen in Fig. 5B, all samples contained comparable amounts of p42/p44 MAP kinases. These data support the concept that CaMKII is an upstream regulator of p42/p44 MAP kinase in this smooth muscle.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Representative recordings of mechanical responses of iris sphincter muscle to PGF2 (A), ionomycin (B), and thapsigargin (C) in the absence and presence of KN-93. Experimental conditions were the same as described in Fig. 1 except that PD98059 was replaced with KN-93 (10 µM).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effects of KN-93 on PGF2-, ionomycin-, and thapsigargin-induced p42/p44 MAP kinase activation in iris sphincter muscle. Experimental conditions were the same as described in Fig. 2 except that PD98059 was replaced with KN-93 (10 µM). Results are from one experiment that is a representative of three independent experiments.

Isoproterenol Inhibits Ca²⁺-Mobilizing Agonist-Induced Contraction and p42/p44 MAP Kinase Phosphorylation. Isoproterenol, a -adrenergic agonist, increases intracellular cAMP accumulation and induces relaxation in the iris sphincter (Tachado et al., 1989). As shown in Fig. 6, isoproterenol inhibited PGF₂-, ionomycin-, and thapsigargin-induced contraction by 70, 96, and 78%, respectively (Fig. 6). When isoproterenol was added prior to the addition of the agents there was a complete inhibition of the agonist-induced contraction (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Representative recordings of mechanical responses of iris sphincter muscle to PGF2 (A), ionomycin (B), and thapsigargin (C) in the absence and presence of isoproterenol (ISO). Experimental conditions were the same as described in Fig. 1 except that PD98059 was replaced with ISO (1 µM).

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View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effects of isoproterenol (ISO) on PGF2-, ionomycin-, and thapsigargin-induced MAP kinase activation in iris sphincter muscle. Experimental conditions were the same as described in Fig. 2 except that PD98059 was replaced with ISO (1 µM). Results are from one experiment that is a representative of three independent experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The main findings of the present study are that 1) stimulation of cat iris sphincter smooth muscle with the Ca²⁺-mobilizing agonists PGF₂, ionomycin, or thapsigargin resulted in rapid phosphorylation and activation of p42/p44 MAP kinase and contraction; and 2) treatment of the muscles with PD98059, KN-93, or isoproterenol resulted in inhibition of the Ca²⁺-mobilizing agonist-induced responses. These findings, coupled with the observation that the combined stimulatory effects of PGF₂ and ionomycin, or PGF₂ and thapsigargin, or PGF₂ + ionomycin + thapsigargin on MAP kinase phosphorylation are not additive (Table 1), suggest that the observed biochemical actions of these agents may be mediated through a common signaling pathway. The finding in the present study that PD98059 also inhibited ionomycin- and thapsigargin-induced MAP kinase phosphorylation and activation (Figs. 1-3) indicates that activation of MAP kinase in this tissue requires Ca²⁺ and that the Ca²⁺ could come either from internal stores or from extracellular sources. Although Ca²⁺ requirement for MAP kinase phosphorylation has been reported in a wide variety of cells (see Introduction) there is little work on Ca²⁺ requirement for MAP kinase activation in intact tissues. The Ca²⁺ requirement for MAP kinase activation in the iris sphincter is further supported by the finding that KN-93, a CaMKII inhibitor, markedly inhibited PGF₂-, ionomycin-, and thapsigargin-induced MAP kinase phosphorylation and contraction (Figs. 4 and 5). These data clearly indicate that in this tissue CaMKII plays an important role in the regulation of contraction and suggest that this enzyme could act as an upstream regulator of p42/p44 MAP kinase. These observations are consistent with the findings of Kim et al. (2000) who reported that in the ferret aorta inhibition of CaMKII results in inhibition of contraction. This conclusion in the ferret aorta was based on the findings that 1) down-regulation of CaMKII by treatment with both KN-93 and antisense oligonucleotides resulted in a decrease in force in the aortic strips, and 2) CaMKII autophosphorylation increases with a time course comparable with that of the contraction (Kim et al., 2000). Furthermore, Kim et al. (2000) concluded that the ability of CaMKII to influence contraction may be mediated by MAP kinase. This conclusion was based on the following findings by these authors: 1) depletion of Ca²⁺ significantly decreased activation of MAP kinase, 2) KN-93 blocked activation of MAP kinase contraction and phosphorylation of LC20, and 3) PD98059 inhibited contraction and phosphorylation of LC20 in this tissue. Kim et al. (2000) suggested that a plausible sequence of signaling events in KCl-stimulated ferret aorta would be: Ca²⁺   CaMKII  ?  MAP kinase  MLC kinase  LC20. More recently Kamm and Stull (2001) suggested that the presumed decrease in MLC kinase activity shown by Kim et al. (2000) may be explained by CaMKII effects on calcium levels. In aortic vascular smooth muscle cells PD98059 markedly inhibited platelet-derived growth factor (PDGF)-stimulated CaMKII activity, suggesting that MAP kinase may act upstream of CaMKII to control its activation in response to PDGF (Lundberg et al., 1998). However, in this study although both KN-62, a CaMKII inhibitor, and PD98059 inhibited PDGF-stimulated CaMKII activity, only KN-62 inhibited ionomycin (1 µM)-stimulated CaMKII activity. MAP kinase has been shown to be activated by CaMKII in both cultured rabbit aortic smooth muscle tissue (Katoch et al., 1999) and in cultured rat aortic vascular smooth muscle cells (Abraham et al., 1997). Recently, Watt and Storm (2001), working with primary culture of neonatal rat olfactory sensory neurons, reported that odorant stimulation of ERK phosphorylation was ablated by inhibition of CaMKII, suggesting that odorant activation of ERK is mediated through this kinase.

The finding that isoproterenol inhibits PGF₂-, ionomycin-, and thapsigargin-induced contraction and MAP kinase phosphorylation (Figs. 6 and7) indicates that the cAMP-elevating agent may exert its inhibitory effect in part at the level of [Ca²⁺]_i mobilization and activation of CaMKII, which precedes the activation of MAP kinase. The mechanism underlying the relaxing effects of cAMP-elevating agents on smooth muscle contraction remain unresolved (for review, see Abdel-Latif, 2001). Although it is well established that cAMP- and cGMP-elevating agents reduce agonist-induced [Ca²⁺]_i mobilization and relax smooth muscle, the precise sites and mechanisms underlying cyclic nucleotide inhibition of stimulated polyphosphoinositide hydrolysis and Ca²⁺ mobilization remain to be clarified. Cyclic nucleotide-elevating agents may relax smooth muscle by other mechanisms in addition to a decrease in activator calcium concentrations (for review, see Abdel-Latif, 2001). These mechanisms include protein kinase A phosphorylation of MLC kinase (De Lanerolle et al., 1984); phospholipase C (Liu and Simon, 1996); Raf, which activates the MAP kinases (Cospedal et al., 1999); Rho kinase (Wu et al., 1998); and myosin phosphatase (Wu et al., 1996). Studies designed to elucidate molecular mechanisms underlying interactions between cyclic nucleotides and agonist-induced contraction are important in our understanding of the regulation of smooth muscle tone, which is central to the development of novel therapeutic agents for the treatment of diseases such as asthma, hypertension, and glaucoma where cAMP-elevating drugs are used routinely. The scheme given in Fig. 8 is a plausible sequence of events in the signal transduction pathway in the iris sphincter, which begins with the stimulation of Ca²⁺ mobilization by the Ca²⁺-mobilizing agonists, followed by activation of both CaMKII and MLC kinase, and subsequently MLC phosphorylation and contraction. The steps between CaMKII and p42/p44 MAP kinase phosphorylation and the phosphorylation and activation of MLC kinase by the MAP kinase remain to be established (Fig. 8). In support of the latter, Morrison et al. (1996) have shown that MAP kinase can directly activate smooth muscle MLC kinase. Gerthoffer et al. (1996) demonstrated that the addition of activated MAP kinase to a permeabilized fiber resulted in a contraction. In addition, the studies of Kim et al. (2000) on the ferret aorta discussed above support the scheme given in Fig. 8. In general the observations in the present work that PD98059, KN-93, and isoproterenol produced inhibition of both the biochemical and physiological responses are in accord with this scheme. There is little known about the direct actions of these inhibitors on the activities of CaMKII and MLC kinase in smooth muscle, and the relative contributions of these enzymes to the signal transduction pathway that leads to the contractile response remain to be determined. Although activation of MAP kinase in response to mechanical and pharmacological stimulation has been extensively studied in both vascular and nonvascular smooth muscle (see Introduction) it must also be mentioned that whether or not MAP kinase activation is important in the actual contractile event is controversial (for review, see Somlyo et al., 1999). Watts (1996) and Dessy et al. (1998) have suggested that a small but significant proportion of force development is sensitive to inhibition of MAP kinase. In contrast, Gorenne et al. (1998) reported that inhibition of p42/p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery. Caldesmon is one of the substrates of MAP kinase, but exposure of Triton X-100-permeabilized smooth muscles to activated MAP kinase at concentrations sufficient to near-stoichiometrically phosphorylate endogenous caldesmon (Nixon et al., 1995) did not Ca²⁺-sensitize vascular smooth muscle (Nixon et al., 1995). According to another report, MAP kinase has no effect on contractility of rabbit colonic smooth muscle, but enhances the contraction of canine trachealis (Gerthoffer et al., 1997). The differences observed in these studies could be due in part to the fact that investigators in the past have used in their work different species, different types of smooth muscles (vascular versus nonvascular), intact muscle, isolated cells, permeabilized cells, permeabilized muscle fibers, and lastly, various agonists.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Schematic representation of the role of Ca2+ and CaMKII in the biochemical actions of PGF2, ionomycin, and thapsigargin on MAP kinase phosphorylation and contraction in cat iris sphincter smooth muscle. The relative contributions of the CaMKII and MLC kinase pathways to the contractile pathway remain to be determined. The scheme shown in this figure is based on data reported here as well as data reported by other investigators (see text for references).

In summary, the results presented here indicate that in cat iris sphincter smooth muscle, activation of p42/p44 MAP kinases by PGF₂, ionomycin, or thapsigargin requires Ca²⁺ either from extracellular sources or from internal stores; that CaMKII plays an important role in the regulation of contraction; that CaMKII acts upstream of MAP kinase to control its activation; and that the MAP kinase signaling pathway can play a significant role in mediating the cellular effects of these Ca²⁺-mobilizing agonists.