Differential Regulation of Phospholipase A 2 (PLA 2 )-dependent Ca 2 1 Signaling in Smooth Muscle by cAMP- and cGMP-dependent Protein Kinases INHIBITORY PHOSPHORYLATION OF PLA 2 BY CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES*

Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C- b (PLC- b ) activity and inositol 1,4,5-trisphosphate-dependent Ca 2 1 release in vascular and visceral smooth muscle. In smooth muscle of the intestinal longitudinal layer, how-ever, the initial steps in Ca 2 1 mobilization involve activation of cytosolic PLA 2 (cPLA 2 ) and arachidonic acid (AA)-dependent stimulation of Ca 2 1 influx. The present study examined whether cAMP- and cGMP-dependent protein kinases are capable of regulating these processes also. Agents that activated cAMP-dependent protein kinase (5,6-dichloro-1- b - D -ribofuranosylbenzimida-zole 3 * ,5 * -cyclic monophosphothioate (Sp-isomer) and isoproterenol), cGMP-dependent protein kinase (8-(4-chlorophenylthio)-guanosine 3 * ,5 * -cyclic monophos-phate and Na nitroprusside), or both kinases (vasoactive intestinal peptide and isoproterenol > 1 m M ) induced phosphorylation of cPLA 2 and inhibition of agonist- stimulated cPLA 2 activity. Phosphorylation and inhibi- tion of cPLA 2 activity by cAMP- and cGMP-dependent protein kinases were blocked by the corresponding selective inhibitors (cAMP-dependent protein kinase, tivity in the band was measured. Agonist-mediated Stimulation of cPLA 2 Activity— cPLA 2 activity in dispersed muscle cells was measured by an adaptation of the method of Damron et al. (33) as described previously (22). Twenty ml of cell suspension (10 6 cells/ml) were incubated with [ 3 H]AA (1 m Ci/ml) at 31 °C for 3 h. The cells were diluted with 50 ml of HEPES medium, centrifuged at 350 3 g for 15 min, and then resuspended in 10 ml of fresh medium containing 10 m M indomethacin and 10 m M nordihydroguaiaretic acid to inhibit AA metabolism via the lipoxygenase and cyclooxygenase pathways. Duplicate samples (10 6 cells/0.5 ml) were incubated at 31 °C with 1 n M cholecystokinin octapeptide (CCK-8) for 30 s, and the reaction was terminated with 1.8 ml of chloroform/ methanol/HCl (100:200:2, v/v/v). The phases were separated with 0.6 ml of chloroform and 0.6 ml of 2 m M HCl. The organic phase was dried under nitrogen, resuspended in 50 m l of chloroform/methanol (9:1), and spotted on silica gel plates for thin layer chromatography using hexane/ ethylether/acetic acid (70:30:3.5). The radioactivity in spots correspond- ing to AA was counted, and the results were expressed as cpm/10 6 cells above basal levels.


N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89) and myristoylated protein kinase inhibitor
Phospholipases A 2 (PLA 2 s), 1 , which catalyze the hydrolysis of the Sn2 fatty acyl bond of phospholipids to yield free fatty acids and lysophospholipids, have been divided into two main categories comprising up to nine groups (1,2). One category (Groups I, II, III, V, VII, and IX) includes the small molecular weight, secretory PLA 2 s, which are Ca 2ϩ -dependent except for Group VII PLA 2 (3)(4)(5)(6). The other category includes large molecular weight, Ca 2ϩ -dependent (Group IV) and Ca 2ϩ -independent (Groups VI and VIII) cytosolic PLA 2 s (7-9). The Ca 2ϩindependent, Group VII secretory PLA 2 and the Ca 2ϩdependent, Group VIII cytosolic PLA 2 s are specific for plateletactivating factor and may be viewed as platelet-activating factor acetyl hydrolases (2,10). The Ca 2ϩ -independent, Group VI cytosolic PLA 2 is involved in the continuous recycling of phospholipids and incorporation of arachidonic acid (11,12).
Group IV cytosolic PLA 2 (cPLA 2 ) possesses several distinctive features, including a dependence on submicromolar concentrations of Ca 2ϩ essential for translocation of the enzyme via a Ca 2ϩ and phospholipid binding domain, a preference for hydrolysis of arachidonate-containing phospholipids, and a susceptibility to regulatory phosphorylation by mitogen-activated protein kinase and protein kinase C (13)(14)(15)(16)(17)(18). There is evidence that phosphorylation by protein kinase C may be mediated indirectly by mitogen-activated protein kinase (19). Phosphorylation by mitogen-activated protein kinase on serine residues (chiefly Ser 505 ) or tyrosine residues is associated with increase in cPLA 2 activity (14,17,18,20,21). Phosphorylation by cAMP-or cGMP-dependent protein kinase in vivo has not been characterized.
The initial steps in Ca 2ϩ mobilization in vascular and intestinal circular smooth muscle (i.e. activation of PLC-␤ and stimulation of Ca 2ϩ release) are inhibited by cAMP-and cGMP-dependent protein kinases (27)(28)(29)(30)(31)(32). It is not known, however, whether the initial steps in Ca 2ϩ mobilization in intestinal longitudinal smooth muscle (i.e. activation of cPLA 2 and stimulation of Ca 2ϩ influx) are influenced by cAMP-and cGMP-dependent protein kinases. In this study, we have examined the initial steps in Ca 2ϩ signaling in longitudinal intestinal smooth muscle to determine the ability of cAMP-and cGMP-dependent protein kinases (a) to phosphorylate cPLA 2 and influence its activity and (b) to modulate AA-induced stimulation of Ca 2ϩ influx. The results provide the first evidence of inhibitory phosphorylation of cPLA 2 by both cAMP-and cGMP-dependent protein kinases and demonstrate their ability to regulate the initial step in Ca 2ϩ signaling. The next step, i.e. AA-stimulated Ca 2ϩ influx, is selectively inhibited by cGMP-dependent protein kinase.

Dispersion of Intestinal Smooth Muscle Cells-Smooth muscle cells
were isolated from the longitudinal muscle layer of rabbit intestine by sequential enzymatic digestion, filtration, and centrifugation as described previously (22)(23)(24). Muscle strips were incubated for 30 min at 31°C in 15 ml of HEPES medium containing 0.1% collagenase (type II) and 0.1% soybean trypsin inhibitor with no added Ca 2ϩ . The composition of the medium was 120 mM NaCl, 4 mM KCl, 2.6 mM KH 2 PO 4 , 0.6 mM MgCl 2 , 25 mM HEPES, 14 mM glucose, and 2.1% Eagle's essential amino acid mixture. The partly digested tissue was washed with 100 ml of enzyme-free medium and reincubated for 30 min to allow spontaneous dispersion of muscle cells. The cells were harvested by filtration through 500-m Nitex mesh, centrifuged twice for 10 min at 350 ϫ g, and resuspended in HEPES medium containing 2 mM Ca 2ϩ .

AA-induced Contraction of Dispersed Smooth
Muscle Cells-Contraction of dispersed muscle cells was measured by scanning micrometry as described previously (22)(23)(24). An aliquot (0.5 ml) of cells (10 4 cells/ml) was added to 0.2 ml of medium containing 1 M AA in the presence of the cyclooxygenase inhibitor indomethacin (10 M) and the lipoxygenase inhibitor nordihydroguaiaretic acid (10 M). The reaction was terminated after 30 s with acrolein. Inhibition of contraction (i.e. relaxation) was measured in muscle cells maximally contracted for 30 s with AA (1 M). Relaxation was expressed as the increase in the length of AA-contracted muscle cells (mean resting muscle cell length, 118 Ϯ 5 m; mean length of AA-contracted muscle cells, 83 Ϯ 2 m).
AA-induced Ca 2ϩ Influx-Ca 2ϩ influx in dispersed muscle cells was measured from the initial uptake of 45 Ca 2ϩ in the presence of antimycin and thapsigargin to prevent uptake into mitochondrial and sarcoplasmic Ca 2ϩ stores. Dispersed muscle cells were suspended in 10 ml of HEPES medium containing 2 mM Ca 2ϩ and 45 Ca 2ϩ (10 Ci/ml), with thapsigargin (2 M) and anti-mycin (10 M). The muscle cells were treated for 60 s with a relaxant agent (Na nitroprusside (SNP) and isoproterenol) or protein kinase activator (cBIMPS and 8-pcCPT-cGMP), followed by addition of 1 M AA for 2 min. Samples (0.5 ml) were withdrawn at intervals for measurement of 45 Ca 2ϩ cell content. The muscle cells were centrifuged and washed twice with HEPES medium, and the 45 Ca 2ϩ cell content was measured and expressed as cpm/10 6 cells.
Phosphorylation of cPLA 2 -Phosphorylation of cPLA 2 was measured from the amount of [ 32 P]ATP incorporated into the enzyme after immunoprecipitation with specific cPLA 2 antibody. Dispersed smooth muscle cells (10 ml, 4 ϫ 10 6 cells/ml) were prelabeled with 0.5 mCi/ml [ 32 P]orthophosphate for 3 h. Samples (0.5 ml) were incubated with various relaxant agents for 60 s in the presence or absence of cAMPdependent protein kinase inhibitors (H-89 or myristoylated PKI) or cGMP-dependent protein kinase inhibitor (KT-5823), and the reaction was terminated with an equal volume of lysis buffer (final concentrations, 1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 100 g/ml aprotinin, 10 mM Na 4 P 2 O 7 , 50 mM NaF, 0.2 mM Na 3 VO 4 ) and placed on ice for 30 min. The cell lysates were separated from the insoluble material by centrifugation at 13,000 ϫ g for 15 min at 4°C, precleared with 40 l of protein A-Sepharose CL-4B, and incubated with polyclonal rabbit cPLA 2 antibody for 2 h at 4°C and with 40 l of protein A-Sepharose CL-4B for another 1 h. The immunoprecipitates were collected, washed five times with 1 ml of wash buffer (0.5% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4), extracted with Laemmli sample buffer, boiled for 15 min, and separated on 10% SDS-polyacrylamide gel electrophoresis. After transfer to polyvinylidene difluoride membranes, 32 P-labeled cPLA 2 was visualized by autoradiography, and the amount of radioac- Agonist-mediated Stimulation of cPLA 2 Activity-cPLA 2 activity in dispersed muscle cells was measured by an adaptation of the method of Damron et al. (33) as described previously (22). Twenty ml of cell suspension (10 6 cells/ml) were incubated with [ 3 H]AA (1 Ci/ml) at 31°C for 3 h. The cells were diluted with 50 ml of HEPES medium, centrifuged at 350 ϫ g for 15 min, and then resuspended in 10 ml of fresh medium containing 10 M indomethacin and 10 M nordihydroguaiaretic acid to inhibit AA metabolism via the lipoxygenase and cyclooxygenase pathways. Duplicate samples (10 6 cells/0.5 ml) were incubated at 31°C with 1 nM cholecystokinin octapeptide (CCK-8) for 30 s, and the reaction was terminated with 1.8 ml of chloroform/ methanol/HCl (100:200:2, v/v/v). The phases were separated with 0.6 ml of chloroform and 0.6 ml of 2 mM HCl. The organic phase was dried under nitrogen, resuspended in 50 l of chloroform/methanol (9:1), and spotted on silica gel plates for thin layer chromatography using hexane/ ethylether/acetic acid (70:30:3.5). The radioactivity in spots corresponding to AA was counted, and the results were expressed as cpm/10 6

Phosphorylation of cPLA 2 in Intestinal Smooth Muscle by cAMP-and cGMP-dependent Protein
Kinases-Selective activators of cGMP-dependent protein kinase (8-pcCPT-cGMP) and cAMP-dependent protein kinase (cBIMPS) and three relaxant agents were used to determine the ability of cAMP-and cGMPdependent protein kinases to phosphorylate cPLA 2 in smooth muscle: SNP, which stimulates cGMP and selectively activates cGMP-dependent protein kinase at concentrations Ͻ1 M; isoproterenol, which stimulates cAMP and preferentially activates cAMP-dependent protein kinase at low concentrations (Ͻ1 M) but can cross-activate cGMP-dependent protein kinase at higher concentrations; and VIP, which stimulates both cAMP and cGMP in gastric smooth muscle and activates cAMP-and cGMP-dependent protein kinases at all concentrations (32, 34 -36). The cAMP-dependent protein kinase inhibitors H-89 and myristoylated PKI and the cGMP-dependent protein kinase inhibitor KT-5823 were used to evaluate the involvement of each kinase in cPLA 2 phosphorylation. Previous studies (32) of cAMP-and cGMP-dependent protein kinase activity in dispersed muscle cells had shown that at concentrations of Յ1 M, H-89 and KT-5823 were selective inhibitors of cAMP-and cGMP-dependent protein kinases, respectively.
Inhibition of cPLA 2 Activity by cAMP-and cGMP-dependent Protein Kinases-Previous studies have shown that activation of cPLA 2 by contractile agonists (e.g. CCK-8) is biphasic, with an initial peak of activity during the first minute followed by a sustained phase of activity that is partly mediated by protein kinase C (22). Phosphorylation of cPLA 2 was not present during the initial phase (1 min) of cPLA 2 activity but increased significantly during the sustained phase (10 min after treatment with CCK-8). Preincubation of the cells with the protein kinase C inhibitor calphostin C (1 M) abolished the increase in cPLA 2 phosphorylation during the sustained phase and, as shown previously (22), inhibited cPLA 2 activity by 40% (Fig. 3).
Selective Inhibition of AA-induced Ca 2ϩ Influx by cGMP-dependent Protein Kinase-We have previously shown that endogenous formation of AA stimulates Ca 2ϩ influx in intestinal longitudinal muscle cells; the effect could be reproduced by addition of nanomolar concentrations of AA (22). In the present study, the rate of Ca 2ϩ influx was measured from the initial uptake of 45 Ca 2ϩ (2869 Ϯ 510 cpm/10 6 cells) induced by exogenous AA in the presence of cyclooxygenase and lipoxygenase inhibitors. AA-induced Ca 2ϩ influx was not affected by selective activation of cAMP-dependent protein kinase with cBIMPS (3115 Ϯ 321 cpm/10 6 cells) or 1 M isoproterenol (2816 Ϯ 636 cpm/10 6 cells). A higher concentration of isoproterenol (100 M) inhibited AA-induced Ca 2ϩ influx by 84 Ϯ 4% (p Ͻ 0.001); the inhibition was completely reversed by KT-5823 but was not affected by H-89 (Fig. 7), implying that inhibition was dependent on cross-activation of cGMP-dependent protein kinase by higher concentrations of isoproterenol.
Inhibition of AA-induced Muscle Contraction by cAMP-and cGMP-dependent Protein Kinases-AA-stimulated Ca 2ϩ influx in intestinal longitudinal smooth muscle cells leads to Ca 2ϩinduced Ca 2ϩ release from sarcoplasmic stores in intestinal longitudinal smooth muscle cells; the resultant increase in [Ca 2ϩ ] i triggers an initial muscle contraction (22)(23)(24). AA-induced muscle contraction (36.2 Ϯ 2.9 m decrease in muscle cell length) was inhibited by activators of cGMP-dependent protein kinase (SNP and 8-pcCPT-cGMP) and cAMP-dependent protein kinase (1 M isoproterenol and cBIMPS) (Figs. 9 and 10). The inhibition of contraction (i.e. relaxation) induced by SNP and 8-pcCPT-cGMP was selectively blocked by KT-5823 (Fig. 9), whereas the inhibition of contraction induced by 1 M isoproterenol and cBIMPS was selectively blocked by H-89 (Fig. 10). Thus, although cAMP-dependent protein kinase had no effect on AA-induced Ca 2ϩ influx, it inhibited contraction by acting at one or more loci distal to AA-induced Ca 2ϩ influx. DISCUSSION Agonist-induced activation of PLC-␤ and generation of IP 3 are the initial steps in Ca 2ϩ mobilization in most cell types. In vascular and visceral smooth muscle, including smooth muscle of the intestinal circular layer (26 -30, 37), relaxant agents inhibit Ca 2ϩ mobilization by activating cAMP-dependent protein kinase and/or cGMP-dependent protein kinase. The kinases act on various molecular targets to inhibit Ca 2ϩ release from sarcoplasmic stores and Ca 2ϩ influx into the cell, and to stimulate Ca 2ϩ efflux from the cell. The targets include the effector enzyme PLC-␤, the sarcoplasmic IP 3 receptor/Ca 2ϩ channel, plasmalemmal, and sarcoplasmic Ca 2ϩ -ATPase pumps, and plasmalemmal Ca 2ϩ and K ϩ channels, all of which are affected by both cAMP-and cGMP-dependent protein kinases, except for the sarcoplasmic Ca 2ϩ -ATPase pump, which is selectively inhibited by cGMP-dependent protein kinase (31, 32, 38 -42). Both PLC-␤ and IP 3 receptors/Ca 2ϩ channels are readily phosphorylated by cGMP-and cAMP-dependent protein kinases, resulting in inhibition of IP 3 formation and IP 3dependent Ca 2ϩ release (30 -32, 42).
Ca 2ϩ mobilization in smooth muscle from the intestinal longitudinal layer, however, differs markedly in that it is initiated by G protein-dependent activation of cPLA 2 and generation of arachidonic acid; the latter triggers Ca 2ϩ influx and induces Ca 2ϩ release from sarcoplasmic stores via ryanodine receptors/ Ca 2ϩ channels (22,23). Minimal amounts of IP 3 are produced in this smooth muscle, which is virtually devoid of IP 3 receptors/Ca 2ϩ channels (23,26). In this study we show that by analogy with other types of smooth muscle, the initial steps in Ca 2ϩ mobilization in longitudinal smooth muscle, i.e. activation of cPLA 2 and arachidonic acid-induced Ca 2ϩ influx, are inhibited by cAMP-or cGMP-dependent protein kinase. It is possible that more distal targets involved in Ca 2ϩ mobilization in intestinal longitudinal muscle, such as ryanodine receptors/ Ca 2ϩ channels, Ca 2ϩ -ATPase pumps, and plasmalemmal Ca 2ϩ and K ϩ channels, are also susceptible to regulatory phosphorylation by cAMP-and cGMP-dependent protein kinases, but these were not examined in the present study.
Agonist-induced phosphorylation of cPLA 2 in various cell types is accompanied by increase in cPLA 2 activity, which appears to be mediated by protein kinase C-dependent and -independent activation of mitogen-activated protein kinase (19 -21, 43-45). In the present study we show a delayed, protein kinase C-dependent, stimulatory phosphorylation of cPLA 2 induced by the contractile agonist CCK and demonstrate for the first time an inhibitory phosphorylation induced by relaxant agonists (SNP, isoproterenol, and VIP) and activators of cAMP-dependent protein kinase (cBIMPS) and cGMPdependent protein kinase (8-pcCPT-cGMP). The different patterns of phosphorylation by contractile and relaxant agonists suggest that the residues phosphorylated by cAMP-and cGMPdependent protein kinases are distinct from the residues (chiefly Ser 505 ) phosphorylated by protein kinase C-dependent mechanisms (14,(17)(18)(19)(20). Phosphorylation of cPLA 2 induced by relaxant agents that preferentially activated cAMP-dependent protein kinase (i.e. low concentrations of isoproterenol) was blocked by the selective cAMP-dependent protein kinase inhibitors H-89 and myristoylated PKI, whereas phosphorylation induced by relaxant agents that preferentially activated cGMP-dependent protein kinase (i.e. SNP) was blocked by the selective cGMP-dependent protein kinase inhibitor KT-5823. Phosphorylation induced by agents that activated both cAMP-and cGMP-dependent protein kinases (i.e. VIP) was partially blocked by cAMP-and cGMP-dependent protein kinase inhibitors and abolished by a combination of both inhibitors (32).
The pattern of inhibition of cPLA 2 activity by cAMP-and cGMP-dependent protein kinases paralleled the pattern of phosphorylation of cPLA 2 , suggesting that inhibition of activity was probably mediated by phosphorylation. The initial peak (1 min) of cPLA 2 activity elicited by the contractile agonist CCK-8 was inhibited by both cAMP-and cGMP-dependent protein kinases. The inhibition induced by cBIMPS and low concentrations of isoproterenol was selectively blocked by the cAMP-dependent protein kinase inhibitors H-89 and myristoylated PKI, whereas the inhibition induced by 8-pcCPT-cGMP and SNP was selectively blocked by the selective cGMP-dependent protein kinase inhibitor KT-5823. Inhibition induced by VIP and high concentrations of isoproterenol, which activate both cAMP-and cGMP-dependent protein kinases, was partially blocked by cAMP-and cGMP-dependent protein kinase inhibitors and completely blocked by a combination of both inhibitors (32). The next step in Ca 2ϩ mobilization in intestinal longitudinal smooth muscle, i.e. arachidonic acid-induced Ca 2ϩ influx (22)(23)(24), was inhibited by agents that activated cGMP-dependent protein kinase only (i.e. SNP, 8-pcCPT-cGMP, and high concentrations of isoproterenol). The molecular target of cGMP-dependent protein kinase in this instance could be either Cl Ϫ channels or voltage-sensitive Ca 2ϩ channels. Previous studies (46) have shown that arachidonic acid activates primarily Cl Ϫ channels, resulting in membrane depolarization and opening of voltage-sensitive Ca 2ϩ channels. Consistent with this notion, depolarization and Ca 2ϩ influx induced by contractile agonists was abolished by cPLA 2 inhibitors and stilbene Cl Ϫ channel blockers; nifedipine, on the other hand, abolished Ca 2ϩ influx but only partially inhibited depolarization, implying that Ca 2ϩ influx was only a minor contributor to membrane depolariza-tion. Depolarization and Ca 2ϩ influx induced by nanomolar concentrations of exogenous arachidonic acid was also abolished by stilbene Cl Ϫ channel blockers (46).
The present study addressed only the initial steps in Ca 2ϩ mobilization and showed that inhibition of the effector enzyme cPLA 2 , which mirrors inhibition of PLC-␤ in other smooth muscle cell types, was mediated by cAMP-and cGMP-dependent protein kinases, whereas inhibition of AA-induced Ca 2ϩ influx was exclusively mediated by cGMP-dependent protein kinase. Blockade of either step by cAMP-dependent protein kinase and/or cGMP-dependent protein kinase should lead to a decrease in [Ca 2ϩ ] i and inhibition of muscle contraction. A decrease in [Ca 2ϩ ] i , however, could also result from inhibition of Ca 2ϩ -induced Ca 2ϩ release, stimulation of Ca 2ϩ uptake into intracellular stores, and Ca 2ϩ extrusion from the cell. Furthermore, inhibition of muscle contraction could also reflect phosphorylation by cAMP-and cGMP-dependent protein kinases of targets distal to those involved in Ca 2ϩ mobilization, such as myosin light chain kinase and/or phosphatase (37,47). The importance of the inhibitory processes examined in the present study resides in their location at the start of the signaling cascade.