Contraction of esophageal (Eso) and lower esophageal sphincter (LES) circular muscle depends on distinct signal-transduction pathways. ACh-induced contraction of Eso muscle is linked to phosphatidylcholine metabolism, production of diacylglycerol and arachidonic acid (AA), and activation of the Ca2+-insensitive PKCε. Although PKCε does not require Ca2+ for activation, either influx of extracellular Ca2+ or release of Ca2+ from stores is needed to activate the phospholipases responsible for hydrolysis of membrane phospholipids and production of second messengers, which activate PKCε. In contrast, the LES uses two distinct intracellular pathways: 1) a PKC-dependent pathway activated by low doses of agonists or during maintenance of spontaneous tone, and 2) a Ca2+-calmodulin-myosin light chain kinase (MLCK)-dependent pathway activated in response to maximally effective doses of agonists during the initial phase of contraction. The Ca2+ levels, released by agonist-induced activity of phospholipase C, determine which contractile pathway is activated in the LES. The Ca2+-calmodulin-MLCK-dependent contractile pathway has been well characterized in a variety of smooth muscles. The steps linking activation of PKC to myosin light chain (MLC20) phosphorylation and contraction, however, have not been clearly defined for LES, Eso, or other smooth muscles. In addition, in LES circular muscle, a low-molecular weight pancreatic-like phospholipase A2 (group I PLA2) causes production of AA, which is metabolized to prostaglandins and thromboxanes. These AA metabolites act on receptors linked to heterotrimeric G proteins to induce activation of phospholipases and production of second messengers to maintain contraction of LES circular muscle. We have examined the signal-transduction pathways activated by PGF2α and by thromboxane analogs during the initial contractile phase and found that these pathways are the same as those activated by other agonists. In response to low doses of agonists or during maintenance of tone, presumably due to low levels of calcium release, a PKC-dependent pathway is activated, whereas at high doses of PGF2α and thromboxane analogs, in the initial phase of contraction, calmodulin is activated, PKC activity is reduced, and contraction is mediated, in part, through a Ca2+-calmodulin-MLCK-dependent pathway. The PKC-dependent signaling pathways activated by PGF2α and by thromboxanes during sustained LES contraction, however, remain to be examined, but preliminary data indicate that a distinct PKC-dependent pathway may be activated during maintenance of tonic contraction, which is different from the one activated during the initial contractile response. The initial contractile response to low levels of agonists depends on activation of Gq. Sustained contraction in response to PGF2α may involve activation of the monomeric G protein RhoA, because the contraction is inhibited by the RhoA-kinase antagonist Y27632. This shift in signal-transduction pathways between initial and sustained contraction has been recently reported in intestinal smooth muscle.
- integrin-linked kinase
- myosin light chain kinase
- protein kinase C
- zipper-interacting kinase
the lower esophageal sphincter (LES) maintains a sustained pressure and relaxes to allow the passage of a bolus, whereas the body of the esophagus (Eso) is normally relaxed and contracts only briefly when required to produce peristalsis. By virtue of their function, these muscles may thus be defined as tonic (LES) and phasic (Eso).
Differences in the signal-transduction pathways mediating the initial and the sustained phases of contraction have recently been demonstrated (56, 57). These differences are particularly relevant to LES function, where both initial and the sustained contraction may occur. Because of the relative brevity of esophageal (Eso) contraction, however, considering only the initial contractile phase may be sufficient for Eso muscle.
The neuromuscular mechanisms that participate in the physiological regulation of these functions are not well understood, but it is thought that LES tone is spontaneous and regulated mostly through myogenic mechanisms, whereas LES relaxation and Eso contraction are induced by neural mechanisms. In recent years, considerable attention has been given to neural mechanisms regulating gastrointestinal motor function, but relatively little information is available on the intracellular signal-transduction pathways responsible for contraction of Eso and LES smooth muscle.
ESO AND LES INITIAL CONTRACTION
Initial contraction: myosin light chain kinase- and PKC-dependent contractile pathways in LES and Eso circular smooth muscle.
In smooth muscle cells enzymatically isolated from the circular layer of LES, initial contraction in response to a maximally effective dose of ACh is mediated through 1,4,5-inositol trisphophate (IP3)-induced Ca2+ release from intracellular stores, and activation of a calmodulin-dependent pathway (7) as shown in Fig. 1. Calmodulin, in the presence of elevated calcium concentrations, inhibits PKC and activates myosin light chain kinase (MLCK), resulting in phosphorylation of myosin light chains and contraction (45). This MLCK-dependent pathway has been well described for other smooth muscles (1, 75).
In contrast, in Eso circular muscle (Fig. 2), ACh-induced contraction is IP3- and calmodulin-independent and mediated by muscarinic M2 receptors linked to Gi3-type G proteins, which activate phosphatidylcholine (PC)-specific phospholipase (PL)C and PLD to produce diacylglycerol (DAG) (66).This DAG and arachidonic acid (AA) generated by a cytosolic PLA2 (67) interact to activate a calcium-insensitive PKCε (69). Although PKCε is Ca2+ independent, Ca2+ is required for activation of the phospholipases and production of the second messengers DAG and AA (64, 66, 68). When the second messengers are present, activation of PKCε and contraction proceed, even in the absence of intracellular calcium, and contraction is not affected by calmodulin and MLCK antagonists (68). For instance, in Eso muscle cells permeabilized to allow control of cytosolic Ca2+, clamping Ca2+ concentration at specified levels (0.2–1.0 μM) produced a concentration-dependent contraction that was antagonized by PL antagonists and by antibodies against PKCε but not by calmodulin inhibitors or by MLCK inhibitors. Similarly, addition of Ca2+ to permeable cells caused myosin light chain phosphorylation, which was inhibited by the PKC inhibitor chelerythrine and by phospholipase inhibitors, suggesting that Ca2+ may directly activate PLs producing DAG and AA and activation of PKCε and resulting in phosphorylation of myosin light chain.
In addition, direct G protein activation by GTPγS augmented Ca2+-induced contraction and caused dose-dependent production of DAG, which was antagonized by the same PL inhibitors that blocked Ca2+-induced contraction, suggesting that the same PLs are equally well activated by G protein activation or by increased cytosolic Ca2+ concentration. Activation of the same contractile pathways by Ca2+ or by G proteins may explain the so-called “Ca2+ sensitization” induced by GTPγS (35, 42, 46).
Thus agonist (ACh)-induced contraction may be mediated by activation of PLs through two distinct mechanisms (increased intracellular Ca2+ and G protein activation) producing second messengers such as DAG and AA and activating PKCε-dependent mechanisms to cause contraction.
Role of MAPK in PKC-dependent contraction.
MAPKs comprise a family of serine threonine kinases, which includes ERK1 and ERK2, the Jun NH2-terminal kinase/stress-activated protein kinase, and p38 kinase (20). ERK1 and ERK2 are activated by diverse extracellular stimuli and by protooncogene products that induce proliferation or enhance differentiation (20, 37, 43, 73). The p38 kinase is thought to be activated by inflammatory cytokines and environmental stress; it was identified as part of a protein kinase cascade activated by interleukin 1β or physiological stress and ending in activation of MAPKAPK-2 and phosphorylation of HSP25 and 27 (28, 61), because MAPKAPK-2 phosphorylates the HSP25/HSP27 in a cell-free preparation at the sites phosphorylated in intact cells in response to stress (71).
A connection between MAPK activation and PKC-mediated smooth muscle contraction has been proposed for some time (40, 52, 76). In ferret aorta, the MAPKs ERK1, ERK2, and possibly p38 kinase have been shown to be activated in response to phenylephrine (52). In colonic smooth muscle, it has been demonstrated that MAPK is activated during PKC-dependent contraction and cotranslocated with HSP27 (76).
We have shown that PKC-dependent contraction, such as ACh-induced contraction of Eso, may be associated with activation of MAPKs (17, 68). For example, ACh and DAG-induced contraction of Eso cells is reduced by ERK1 and ERK2 antibodies or by the MAPK kinase (MEK) inhibitor PD98059, as well as by p38 kinase antibodies and by the p38 kinase inhibitor SB203580. PD98059 and SB203580, in combination, nearly abolish the contraction, suggesting that Eso contraction is mediated by activation of a dual pathway, one dependent on activation of ERK1/ERK2 and the other dependent on p38 kinase.
In addition, antibodies against the 27-kDa HSP27 reduced ACh and DAG-induced contraction of Eso cells. HSP27 and p38 MAPK antibodies in combination caused no greater inhibition than either one alone, and p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK.
These data indicate that PKC-dependent contraction, such as ACh-induced contraction of ESO, may be associated with activation of two parallel MAPK contractile pathways: an ERK MAPK pathway and an HSP27-linked p38 kinase pathway (Fig. 2). PKC may activate Raf, causing it to phosphorylate MEK, which in turn, may phosphorylate MAPK (22). MAPK may then phosphorylate calponin or caldesmon and other intermediate proteins. It is possible that two distinct parallel pathways may contribute to contraction, one involving p38 kinase and HSP27 and the other one involving ERKs and calponin/caldesmon.
One of the intermediate proteins in the PKC-MAPK contractile pathway may be integrin-linked kinase (ILK) (23). The Ca2+ independence of ILK activity makes it eligible as a candidate intermediate protein in the Ca2+-independent PKCε-mediated contraction. ILK interacts with various proteins, several of which are connected to the actin cytoskeleton, and anchors it to the extracellular matrix. ILK is expressed in most mammalian cells with high expression levels in cardiac and skeletal muscle, and its sequence is highly conserved across species.
Two distinct populations of ILK are present in smooth muscle. Some ILK are linked to integrins localized to membrane-associated dense plaques, which are structurally similar to the focal adhesion sites of cultured cells (10). A second population of ILK is associated with myofilaments and may be responsible for Ca2+-independent phosphorylation of myosin in smooth muscle preparations (23). The report that purified myosin contains a low level of Ca2+-independent MLC20 kinase activity that is resistant to the MLCK inhibitor AV25 supports the association of ILK with myosin filaments (23). ILK directly phosphorylates myosin light chain on serine 18 and threonine 19, which are also phosphorylated by MLCK (23).
We have identified ILK by Western blot analysis in both Eso and LES circular muscle. ACh increases ILK activity in Eso smooth muscle, and the increase was inhibited by the ERK1/ERK2 kinase inhibitor PD98059 and not by the p38 MAPK inhibitor SB203580, suggesting that ILK is activated (perhaps indirectly) by ERK1/ERK2 and not by p38 kinase (12). ILK and ERK1/ERK2 are likely to be in the same pathway, because inhibition produced by an ILK antibody was not increased by the ERK1/ERK2 kinase inhibitor PD98059 but was increased by the p38 MAPK inhibitor SB203580. These data suggest that ILK and the p38 MAPK are not in the same contractile pathway (12). Similarly, we have previously shown that HSP27 is linked with p38 kinase (17). The HSP27 antibody partially reduced ACh-induced contraction. HSP27 inhibition was increased by the ERK1/ERK2 kinase inhibitor PD98059, suggesting that HSP27 and the ERK1/ERK2 MAPK are not in the same contractile pathway. HSP27 inhibition was not increased by the p38 MAPK inhibitor SB203580, because p38 and HSP27 may be in the same pathway. Finally, antibodies against HSP27 and ILK, when used in combination, almost abolished ACh-induced contraction, suggesting that the contraction is mediated through a dual pathway, one involving ILK, the other involving HSP27 (12).
Zipper-interacting protein kinase.
A second intermediate protein may be zipper-interacting protein (ZIP) kinase (ZIPK), which was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a ZIPK, which mediates apoptosis. ZIPK phosphorylated the regulatory light chain of myosin II at both serine 19 and threonine 18 in a Ca2+/calmodulin-independent manner (55) and thus may be a candidate as an intermediate protein in the Ca2+-independent PKCε-mediated contraction. These results have been confirmed in smooth muscle by Niiro and Ikebe (60), who found that ZIPK is expressed in smooth muscle tissues and that it can phosphorylate myosin in a Ca2+-independent manner and thereby induce Ca2+-independent contraction of permeabilized smooth muscle.
In a Western blot analysis using an antibody raised against the COOH terminal of ZIPK in Eso and LES circular muscle, we found a band at 52 kDa, which is the appropriate molecular mass, and a ZIPK antibody partially inhibits ACh-induced contraction. Preliminary data demonstrate that ILK and ZIPK antibodies, when used in combination, almost abolish ACh-induced contraction of permeable Eso cells, suggesting that ILK and ZIPK are not in the same contractile pathway. In addition, ZIPK antibodies caused the same inhibition as HSP27 antibodies or a combination of HSP27 and ZIPK antibodies, suggesting that HSP27 and ZIPK may be part of the same contractile pathway (W. Cao, unpublished observations).
These data suggest the working hypothesis illustrated in Fig. 3. It is thought that the contractile state is regulated by the level of phosphorylation of myosin light chain (MLC20) and that this level is regulated by the interaction of kinases and phosphatases. Contraction may be induced either by increased activation of kinases or by decreased activation of phosphatase. Phosphatases were thought to be constitutively active and unregulated, with regulation occurring only for myosin kinase activity. Recent studies (32), however, have focused on mechanisms of phosphatase regulation. Myosin phosphatase isolated from smooth muscle is a holoenzyme consisting of three subunits (shown in gray in Fig. 3): a small 20-kDa subunit of uncertain function (M20), a 38-kDa catalytic subunit, which is a type 1 phosphatase (PP1), and a myosin-targeting subunit (MYPT1) of 110–133 kDa (9, 32). MYPT1 may be phosphorylated by ILK (8), by ZIP-like kinase(8), and in some muscles, MYPT1 may be phosphorylated by Rho kinase (RhoK), inhibiting phosphatase activity and promoting contraction. RhoK, however, plays no role in ACh-induced contraction of Eso, because the contraction is not affected by either C3, an exoenzyme of Clostridium botulinum that is a specific RhoA inhibitor (14), or by the RhoK inhibitor Y27632 (W. Cao, unpublished observations).
In addition, CPI-17 (PKC-dependent phosphatase inhibitor of 17 kDa) is a specific inhibitor protein for myosin phosphatase predominantly expressed in smooth muscle tissues (26). When phosphorylated by PKC, CPI-17 becomes a potent inhibitor of type 1 protein Ser/Thr phosphatases, such as myosin phosphatase PP1. CPI-17 may also be phosphorylated by RhoK (31), by ILK (24), and by ZIP-like kinase (8).
We propose that PKCε-mediated contraction of Eso circular muscle is mediated by activation of at least two pathways: an ERK1/ERK2 MAPK pathway that may be linked to the Ca2+-independent ILK and an HSP27-p38 kinase pathway (Fig. 3). Preliminary data support the association of a Ca2+-independent ZIPK (55) with HSP27-p38 kinase. In addition, ZIPK or ZIP-like kinase may phosphorylate the targeting subunit of myosin phosphatase (MYPT1) also in a Ca2+-independent manner (8).
A ZIP-like kinase that is closely related to ZIPK has been recently identified (51) and shown to phosphorylate the targeting subunit of myosin phosphatase (MYPT1). This kinase, which is also referred to as MYPT1 kinase (8), may be a truncated version of ZIPK or an independent relative. If not identical, the two kinases are closely related, as evidenced by cross-reactivity of ZIP-like kinase with some ZIPK antibodies and by similarities in enzyme properties (8). ZIPK, which is detected by Western blot analyses at 54–59 kDa, contains an NH2-terminal catalytic domain of 263 residues and three leucine zipper sequences at its COOH terminus. Because the mass of the ZIP-like kinase is ∼32 kDa, it may contain only ∼60% of the ZIPK structure. The catalytic domain may account for ∼30 kDa and thus constitute most of the protein. The molecular basis underlying the interaction of ZIP-like kinase with the targeting subunit of myosin phosphatase (MYPT1) is not known, but it is thought that ZIP-like kinase is cytosolic and associated with MYPT1 and myosin (51). This ZIP-like kinase phosphorylates MYPT1 and inhibits phosphatase activity, promoting contraction (8).
PHOSPHATASES AND PHOSPHATASE INHIBITION IN ESO CONTRACTION
Phosphatase activity is present in Eso cells as the cells contract in the presence of phosphatase inhibitors and in the presence of antibodies against the catalytic subunit of myosin phosphatase (PP1). In addition, it has been demonstrated that phosphorylation of CPI-17 and of phosphatase holoenzyme inhibitor-1 results in phosphatase inhibition and contraction of rat tail artery muscle (24). Thus inhibition of phosphatases is a regulatory mechanism of smooth muscle contraction. Inhibition of phosphatases in response to appropriate stimuli may potentiate myosin phosphorylation, either by MLCK or by other kinases in the PKC-dependent pathway, such as ILK, ZIPK, and/or ZIP-like kinase.
We have reported that the phosphatase inhibitors microcystin-LR (36) and okadaic acid cause contraction of LES and Eso muscle cells. LES contraction induced by the phosphatase inhibitors is reduced by the MLCK inhibitors ML7 and ML9, but contraction of Eso is not. Eso contraction induced by the phosphatase inhibitors is reduced by the PKC inhibitor chelerythrine and by PKCε antibodies. Thus, in Eso, phosphatase inhibition does not produce contraction by potentiating MLCK but possibly through an MLCK-independent, PKCε-dependent pathway. For example, okadaic acid stimulates PKC activity of Eso smooth muscle. The ratio of PKC activity (membrane/cytosolic) was significantly increased 1, 2, and 5 min after okadaic acid stimulation. In addition, contraction of Eso circular smooth muscle cells induced by okadaic acid, by microcystin-LR, and by an antibody against the muscle-specific catalytic subunit of phosphatase (PP1) were blocked by PKCε antibodies, by MAPK (ERK1 and ERK2) antibodies, and by the MEK antagonist PD98059, supporting the conclusion that inhibition of Eso smooth muscle phosphatases activates a PKC-MAPK-dependent contractile pathway(41).
Although smooth muscle-specific phosphatases have been identified, the selectivity of the phosphatase within the cell is due to its targeting subunit. Exogenously added phosphatase inhibitors or antibodies may reduce phosphatase activity throughout the cell and release all inhibited kinases. Nevertheless the data confirm that inhibition of a phosphatase linked to MLCK and increased MLCK activity do not play a role in contraction of Eso, which may instead be linked to inhibition of phosphatases in the PKC-dependent contractile pathway (41).
These data suggest that two distinct parallel pathways may contribute to PKC-mediated contraction, one involving p38 kinase and HSP27, and the other involving ERKs and ILK (Fig. 3) (23, 24, 54). In addition, other kinases, such as ZIPK or ZIP-like kinase (8, 50), may participate in the PKC contractile pathway (60). Both ILK and ZIPK can directly phosphorylate the 20-kDa regulatory myosin light chain (MLC20) and cause contraction in a Ca2+/calmodulin-independent manner (55) and thus are suitable candidates as mediators of PKCε-induced contraction. ILK and ZIP-like kinase may also phosphorylate the regulatory unit of myosin phosphatase (MYPT1), inhibiting the phosphatase and allowing phosphorylation of the regulatory MLC20.
CONTRACTILE PATHWAYS IN MAINTENANCE OF LES TONE
LES tone, similarly to Eso contraction, is maintained through a PKC-dependent pathway. In the LES, however, this pathway may involve the Ca2+-sensitive PKCβ (69) and depends on activation of at least one (a pancreatic-like or group I) secreted PLA2 (13, 18), resulting in production of AA (Fig. 4). This hypothesis is supported by the finding that when LES circular muscle squares are incubated (4 h) in Krebs solution containing [3H]AA and then washed, the muscle released radioactivity into the medium in a linear fashion as a function of time, presumably due to PLA2 activity.
Several secreted PLA2 have been recently discovered. Group I, group II, group V, and group X PLA2s are structurally related and may participate in AA production in LES circular muscle. Whether one or more of these PLs is important in maintenance of LES tone remains to be determined. AA produced by one or more secreted PLA2s (sPLA2) is metabolized to prostaglandin PGF2α and thromboxane A2 (TXA2), which are membrane permeable and can act on receptors linked to trimeric G proteins to induce activation of PLs and production of second messengers (Fig. 4). In the initial phase of the contractile response to these and other agonists, interaction of DAG with low levels of Ca2+ released from intracellular stores (7, 34) causes contraction of LES circular muscle through a PKCβ-dependent pathway (69). The steps mediating PKCβ-dependent contraction, however, have not been explored in detail.
ACTIVATION OF PLA2S
In mammalian cells, three types of PLA2s have been distinguished: an ubiquitously expressed high molecular mass (85 kDa) PLA2 (cPLA2) with a high specificity for AA-containing phospholipids (19, 63), a 40-kDa Ca2+-independent enzyme that prefers sn-2 arachidonoyl plasmalogens as substrate (33), and the low molecular mass (14–16 kDa), Ca2+-dependent sPLA2 (21, 38, 44). Among PLA2s, cPLA2 has received much attention as a key regulator of stimulus-initiated eicosanoid biosynthesis, because it selectively releases AA, shows submicromolar Ca2+ sensitivity, and is activated by MAPK-directed phosphorylation (5, 19). cPLA2 undergoes Ca2+-dependent translocation from the cytosol to perinuclear and endoplasmic reticular membranes (30, 62), where several downstream eicosanoid-generating enzymes, including two cyclooxygenase (COX) isozymes, are localized (70).
The sPLA2s have several features distinct from other PLA2 families, such as a high disulfide bond content, a requirement for millimolar concentration of Ca2+ for catalysis, and a broad specificity for phospholipids with different polar head groups and fatty acyl chains (39, 72). The sPLA2 family comprises 14- to 16-kDa calcium-dependent interfacial enzymes that liberate sn-2 fatty acids including AA for the biosynthesis of eicosanoids (4, 29, 47, 48). Ten sPLA2 enzymes have been described in mammals that currently include groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII sPLA2 (6, 72). These enzymes exert biological effects through multiple mechanisms, including release of AA, which may be metabolized to leukotrienes and PGs (3, 53), bactericidal activity via hydrolysis of the outer membrane of Gram-positive bacteria (Foreman-Wykert, 2000 #1368; Adachi, 1998 #1512), and binding to specific sPLA2 receptors (2, 27, 49).
We have cloned, by RT-PCR, groups IB, IIA, and V sPLA2 from the human LES circular muscle layer. In addition to the LES, group I is present in the cat internal anal sphincter, the pyloric sphincter, and the colon, suggesting that sPLA2 may be present in sphincters and other gastrointestinal smooth muscles (16). sPLA2s belonging to groups I, II, V, and X are closely related and have a highly conserved Ca2+-binding loop (XCGXGG) and catalytic site (DXCCXXHD). The presence of multiple PLA2 in the same tissue has been reported in duodenum, jejunum, and lung (59).
Groups I, II, V, and X sPLA2 may all be present and contribute to AA release in LES circular muscle. We have demonstrated the presence and function of a pancreatic (group I) sPLA2, and we have cloned, by RT-PCR, the mRNA for groups I, IIA, and V in human LES muscle. mRNA for group IIA PLA2 is present in the LES at higher density than group I PLA2. We have not yet tested for the presence of a group X mRNA.
Whether the group IIA, V, and X enzymes have a physiological contractile function in LES muscle remains to be demonstrated.
IDENTIFICATION OF SECRETED PLA2 IN MAINTENANCE OF LES TONE
We have shown that LES tone may be mediated by the activity of a group I (secreted) sPLA2 because 1) unstimulated LES circular smooth muscle has higher AA levels than Eso, which does not maintain tone; 2) MJ33, a selective inhibitor of group I sPLA2, significantly reduced AA content and spontaneous tone of LES circular muscle strips, whereas the group II sPLA2 antagonist MJ45 and the cPLA2 inhibitor AACOCF3 had no effect on LES tone; and 3) cobra venom (group IA) and purified porcine pancreatic (group IB) sPLA2 caused dose-dependent contraction of LES strips (18).
These data suggest that AA production through group I secreted PLA2 participates in maintenance of LES tone.
To examine the origin of this group I sPLA2, we developed rabbit polyclonal antibodies against purified porcine pancreatic (group 1B) PLA2. On Western blot analysis, the antiserum cross-reacted with a 13.6-kDa band in human and cat pancreas and LES and at a 1:400 dilution significantly reduced in vitro cat and human LES tone.
We used degenerate primers, taken from mostly conserved regions of the published pancreatic PLA2 sequences of human, pig, bovine, mouse, dog, and rat, to amplify by RT-PCR and then sequence cat pancreatic PLA2. The resulting mRNA of cat pancreatic sPLA2 had 555 bases and 89% identity with dog, 84% with human and pig, 81% with rat, and 80% with mouse. Primers for human and cat pancreatic PLA2s were then used to obtain human and cat LES PLA2s, respectively. A group IB PLA2 was found in the circular smooth muscle layer of cat and human LES and exhibited complete mRNA identity with pancreatic PLA2.
These data suggest that a group I sPLA2 may exist in the circular muscle layer of cat and human LES, supporting our view that group I sPLA2 may contribute to LES tone.
In addition, preliminary data in human LES indicate that mRNA for group IIA and group V PLA2 is also present in the LES circular muscle layer. The presence of the related group X PLA2 has not yet been tested. The cellular localization of these enzymes remains to be established, and their relative importance for production of AA in maintenance of tone remains to be determined.
ROLE OF AA METABOLITES IN MAINTENANCE OF LES TONE
The AA produced by sPLA2 in the LES is metabolized to PGs, such as PGF2α and thromboxanes, which, in turn, contribute to maintaining tone because the COX inhibitors indomethacin and aspirin and not the lipoxygenase inhibitor nordihydroguaiaretic acid, dose dependently inhibit LES tone (18). In addition, PGF2α content is significantly higher in LES than in Eso, and PGF2α dose dependently contracts LES strips and single cells. TXA2 and thromboxane B2 (TXB2) may also be involved in LES tone. TXA2 could not be tested because of its short half-life, but the thromboxane analog U46619 and the stable TXB2 dose dependently contracted LES strips, and the TXA2 antagonist SQ29548 dose dependently reduced LES tone (18).
In addition, in LES muscle, several G proteins are spontaneously active, i.e. bound to GTP, in the absence of exogenously added excitatory neurotransmitters. In unstimulated LES smooth muscle, [35S]GTPγS binding to Gi3, Gi1/2, and Gq is higher than in Eso smooth muscle, suggesting that these G proteins may be in an active state in the LES. [35S]GTPγS binding is significantly reduced by indomethacin (10 μM), suggesting that G protein activity is due to COX-dependent production of AA metabolites. PGF2α and the TXA2 analog U46619 significantly increase the [35S]GTPγS binding of the same Gi3, Gi1/2, and Gq that are active in basal conditions in LES circular muscle membranes (18).
These data, initially obtained in the cat, were confirmed for human LES circular muscle from organ donors (13). They support the hypothesis, illustrated in Fig. 4, that spontaneous activation of a group I sPLA2 causes production of AA and AA metabolites such PGF2α and thromboxanes, which maintain activation of G proteins such as Gi3, Gi1/2, and Gq. These G proteins are linked to PLs such as phosphatidylinositol (PI)-specific PLC and PC-PLC, which, in turn, produce diacyglycerol and IP3, to synergistically activate PKCβ (66, 69).
These data suggest a new and quite unexpected mechanism for maintenance of LES tone and may apply to other sphincters as well (74).
SINGLE-CELL CONTRACTION BY sPLA2 OR AA METABOLITES AS A MODEL OF LES TONE
Unlike circular LES muscle strips, which maintain spontaneous tone, LES single cells exhibit no evidence of spontaneous contraction in the absence of exogenously added agonists. For instance, LES cell length is not significantly different from Eso cell length and changes little in the presence of inhibitory neurotransmitters such as VIP or nitric oxide, suggesting that no spontaneous contraction is present in these muscle cells.
We have previously examined LES cell contraction in response to maximally effective doses of agonists (e.g., ACh) and found it to be mediated through a calmodulin-MLCK-dependent pathway that is quite different from the PKC-dependent pathway used to maintain tone. To “mimic” LES tone by activating a PKC-dependent pathway, it is necessary to use low doses of agonist, such as ACh, that may not be involved in maintenance of tonic contraction or to directly activate a PKC-dependent pathway with DAG analogs, such as 1–2-dioctanoyl-glycerol or phorbol esters. Direct activation of PKC by DAG analogs, however, may not include other events (e.g. IP3-induced Ca2+ release), which are also associated with maintenance of LES tone (7).
Because we established that PGF2α and TXA2 may be involved in maintenance of LES tone, it was reasonable to examine the signal-transduction pathways activated by these agonists in LES circular muscle. We therefore examined PGF2α signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus and LES.
SINGLE-CELL CONTRACTION BY PGF2α
In LES, PGF2α-induced contraction was inhibited by antibodies against the α-subunit of Gq and Gi3, and a [35S]GTPγS binding assay confirmed that Gq and Gi3 were activated by PGF2α (14). PGF2α-induced contraction of LES was reduced by U73122 and D609, and unaffected by propranolol. At low PGF2α concentration, contraction was blocked by chelerythrine alone, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in LES circular muscle, PGF2α receptors are coupled to Gq and Gi3, activating PI-PL and PC-PLC. At low concentrations, PGF2α activates PKC; at high concentration, it activates both PKC- and calmodulin-dependent pathways (14). Sustained contraction in response to PGF2α remains to be examined.
sPLA2 OR AA METABOLITES IN GUT MOTOR FUNCTION
We have considered the possibility that sPLA2 may participate in contraction of other portions of the gastrointestinal tract. For example, sPLA2 may participate in maintenance of internal anal sphincter tone in the guinea pig (74). We find that guinea pig sigmoid circular muscle spontaneous activity may be inhibited by indomethacin and by sPLA2 inhibitors. Whether this applies to the human colon remains to be determined, but RT-PCR amplification detects the presence of group I PLA2 mRNA in human colonic circular muscle.
SUSTAINED LES CONTRACTION
LES tone may be mediated by AA and its metabolites PGFα and TXA2/B2 (11, 18). Contractile pathways activated by these agonists may provide models of LES tone. It has recently been shown (56) that the contractile pathways that mediate the initial phase of contraction may be different from those activated as contraction persists. It is thus likely that sustained LES tone may be maintained through contractile pathways that are quite distinct from those activated immediately after addition of AA and its metabolites PGFα and TXA2/B2. We have examined initial contraction of LES muscle cells in response to PGFα (14). PGF2α-activated mechanisms during sustained contraction remain to be defined, but preliminary data indicate that they are different from those activated in the initial phase of contraction (Figs. 5 and 6). Spontaneous tone as well as sustained contraction in response to PGFα may depend, at least in part, on monomeric G proteins such as RhoA (Figs. 5 and 6), whereas initial contraction depends on activation of the trimeric G proteins Gq and Gi3 (18), in agreement with contraction of intestinal smooth muscle, as described by Murthy et al. (56). We know that LES tone is associated with activation of pertussis toxin-sensitive G proteins (11, 18) linked to PI-PLC and PC-PLC and activation of PKC.
The data in Fig. 5 demonstrate that RhoA participates in maintenance of spontaneous LES tone, because the RhoK inhibitor Y27632 reduced tone of in vitro LES circular muscle strips. Similarly, the RhoK inhibitor reduced sustained PGF2α-induced contraction of LES circular muscle strips. Initial contraction of LES muscle cells in response to PGF2α is mediated by activation of Gq and Gi3(18) and not by activation of Gi3 or RhoA (14). Thus different contractile pathways are activated in these two phases of contraction.
Preliminary data (15)indicate that sustained contraction of LES cells in response to PGF2α is mediated by activation of the same RhoA that is involved in maintenance of LES tone. In LES circular muscle cells isolated by enzymatic digestion, PGF2α caused a relatively rapid contraction that achieved maximum shortening in ∼30 s, similar to other agonists. The shortening, however, persisted for >20 min after exposure to PGF2α. Cells incubated in the RhoK inhibitor Y27632 achieved the same maximum initial shortening as untreated cells but did not remain contracted and returned to their unstimulated length within ∼10 min. This inhibition of sustained contraction by the RhoK inhibitor in cells mimics the inhibition of tone and of sustained PGF2α-induced contraction in strips and strongly supports the proposition that PGF2α-induced sustained contraction of cells may be a useful model for LES tone, because both are mediated through the same intracellular mechanisms. This view was further supported by the finding that in permeable cells, where PGF2α-induced shortening was the same as in intact cells, Gi3 and RhoA antibodies reduced and C3 abolished sustained contraction (measured at 10 min) after addition of PGF2α.
These data suggest that tone and sustained PGF2α-induced contraction depend on activation of Gi3 and RhoK. In sustained contraction of muscle cells from the small intestine, Murthy et al. (56) proposed, as shown by the interrupted gray arrows in Fig. 6, that agonist binding to receptors activates Gi3, which initiates a cascade involving sequential activation of a Rho-specific, guanine nucleotide exchange factor, RhoA, and PLD. In this pathway PLD produces phosphatidic acid, which is dephosphorylated to DAG (25). DAG, in turn, activates PKC, which causes contraction and may also enhance DAG production by further potentiating PLD activation. PLD is also activated by an ADP ribosylation factor (ARF)-dependent pathway, but the mechanism of activation of ARF is unknown (14, 58). In addition, as shown by the solid black arrows in Fig. 6, we know from our own work that maintenance of LES tone is PKC dependent (34), that Gq and Gi3 are active(18), that Gq is linked to PI-PLC and Gi3 to PC-PLC (65), and that PI-PLC and PC-PLC are also active (34) as tone is maintained. The interrupted gray arrows represent an additional likely contractile pathway for maintenance of LES tone and of sustained contraction in response to PGF2α. This pathway is also activated during sustained contraction of intestinal smooth muscle cells in response to CCK (58) and during initial contraction of Eso in response to PGF2α (14) and is likely to be activated during maintenance of LES tone and during sustained contraction induced by PGF2α.
In conclusion, we propose that ACh-induced phasic contraction of Eso circular smooth muscle depends on activation of PC-PLC, PLD, and cPLA2, resulting in formation of DAG and AA, which interact to activate the Ca2+-insensitive PKC-ε. PKC-ε-mediated contraction is Ca2+ and MLCK independent. It is associated with Ca2+-independent phosphorylation of MLC through activation of MAPKs and of intermediate Ca2+-independent kinases such as ILK and ZIPK, which may directly phosphorylate MLC as well as inhibit MLC phosphatase activity.
LES tone depends on sustained contractile mechanisms, which are distinct from those activated in the initial phase of contraction. LES tone depends on sustained production of AA by a group I PLA2 and, possibly, by other sPLA2s. AA, in turn, is metabolized to PGF2α and thromboxanes, which maintain tone by inducing sustained contraction through activation of PGF2α and thromboxane receptors. The mechanism of receptor-induced sustained contraction has not been completely elucidated for LES circular muscle but is likely to depend on activation of PKCβ by several pathways, including activation of PI-PLC, PC-PLC, and RhoA.
The mechanisms for PKCβ-induced sustained contraction remain to be established.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-28614.
- Copyright © 2005 the American Physiological Society