suspensions were treated at 4 "C with 6 strokes (5 s, 20 watts) of a. Heat-Systems ...... Schell-Frederick, E. (1984) Cell Calcium 5, 237-251. 43. Danthuluri, N. R.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry andMolecular Biology, Inc
Vol. 264, No. 12. Issue of April 25, pp. 68364343,1989 Printed in U.S.A .
Bidirectional Effects of Protein KinaseC Activators STUDIESWITH
HUMAN NEUTROPHILS AND PLATELET-ACTIVATING FACTOR* (Received for publication, April 29, 1988)
Joseph T. O'FlahertyS, David P. Jacobson, and JimmyF. Redman From the Bowman Gray Schoolof Medicine, Wake Forest University, Winston-Salem,North Carolina 27103
Protein kinase C (PKC) (Ca'+/phospholipid-depend- binds to specific receptors (2-4) that indirectly induce cells ent enzyme) activators stimulated human neutrophils (e.g. platelets, PMN, endothelium, fibroblasts, etc.) to cleave to reduce the availabilityof high affinity receptors for plasmalemma1 phosphatidylinositol diphosphate into inositol platelet-activating factor. These effects were concen- triphosphate and diacylglycerol (5-13). The former product tration dependent, irreversible, temperature sensitive, and antagonized by a PKC blocker. The activatorsalso raises cytosolic Ca2+by releasing the cation from subcellular inhibited 1-0-alkyl-65% hexadecyl, 25% octadecyl- storage pools,while the latter product remains in plasma 2-acetyl-sn-glycero-3-phosphocholine (PAF)-induced membrane where it binds with, and activates, PKC through Ca2+ transients;this inhibition correlated precisely a Ca2+-enhanced reaction (14, 15). Ca2+ and diacylglycerol, with receptordepletion. Contrastingly, PKC activators then, cooperate synergistically in moving PKC from cytosol could enhance as well as inhibit PAF-induced degran- to plasmalemma (15-21). The membrane-adherent, diacylulation. Inhibition of degranulation occurred only at glycerol-bound PKC proceeds to phosphorylate elements of concentrations of the activatorswhich depressed high the response mechanism (15). This triggers, e.g. degranulation affinity PAF binding by >75%. Cells treatedwith responses. It may also dampen cellular function. Thus, direct lesser activator concentrationsresponded to PAF with activators of PKC ( e g . diacylglycerol, PMA, and MEZ) can reduced but still substantial rises in cytosolic Ca'+, enhance (22-39) or inhibit (34-60) the bioactions of PAF as markedly increased degranulation, and markedly increased PKC mobilization. Thelast two responses, well as various other stimuli. These bidirectional influences however, failed to occur in cells that were (a)calcium of PKC are incompletely understood although they may be critical to stimulus-response coupling, cellular priming, limidepleted, ( b ) treated with high activator concentrations (which inhibited virtually all PAF binding and tation of cellular responses, down-regulation, and desensitia system PAF-induced Ca2+transients), or(c) treated withPAF zation. Here, we use PAF and human PMN as model 5 min before a PKC activator (PAF-induced rises in to investigate the opposing, seemingly contradictory effects cytosolic Ca2+ reversed in 95% PMN; 0.92). The resulting curves of low affinity binding were used to estimate low affinity receptor binding occurring at lower (10 pM to 3.16 nM) PAF concentrations. These estimates were subtracted from binding observed at 10 PM to 3.16 nM PAF concentrations to obtain data for high affinity binding isotherms, which also were fit by the method of least squares (coefficients of linearity >0.95). Our analyses are more fully described (3, 61). They gave R, and k d estimates for high and low affinity binding sites as recorded in Table 11. Because these estimates are appreciably higher than those obtained with filtration assays (3), we duplicated all studies using filtration techniques and found that PMA had qualitatively similar effects on PAF binding as observed with oil-centrifugation assays. The actions of PMA on binding were not assay-specific. PHIPMA Binding--1.5 X lo7 PMN in 1.5 mlof buffer were incubated at 37 "C for 20 min and then sequentially exposed to PKC activator, cytochalasin B, and PAF, as indicated in individual experiments. Suspensions were centrifuged (12,000 X g, X 1 min, 23 "C) and thepelleted cells twice washed with and resuspended (lo7PMN/ ml) in 1 mlof calcium-free buffer (4 "C, EGTA = 100 p ~ ) The . suspensions were treated at 4 "C with 6 strokes (5 s, 20 watts) of a Heat-Systems Ultrasonic, Inc., sonicator. The sonicates, which contained 0.2, Student's paired t test) different from MeaSO-treated controls.
25
9v)
tlm
+
experiments), 162 f 10,142 f 14, and 148 f 18 nM in PMN preincubated with MezSO or 0.5, 5 , or 50 nM PMA, respectively, for 5 min. The PKC activators, therefore, did not dampen the actions of PAF simply by stimulating [Ca2+];extrusion from cytosol. PMA had strikingly different influences on degranulation. At 0.16-1.6 nM, it enhanced, but at 2 5 nM it inhibited, PAFinduced release of vitamin B-12-binding protein (a specific granule marker), myeloperoxidase and p-glucuronidase (azurophilic granule markers), and lysozyme (contained in both granule types) (Fig. 2 and lower two panels of Fig. 6). Again, MEZ and MAG acted like PMA while HEG had little influence on these responses (Table I). We note that PKC activators, by themselves, stimulated some enzyme release (see legends to Figs. 2 and 6). These direct actions of the PKC activators were subtracted frpm all presented data on PAFinduced responses. We conclude that PKC activators have assay-specific effects. They weakly stimulate degranulation, fail to mobilize calcium, inhibit PAF-induced calcium transients, and can enhance as well as inhibit PAF-induced degranulation. Indeed, low concentrations of the activators partially inhibit calcium transients while concurrently priming PMN to degranulate. We related the above findings to PAF receptors using the following protocol. PMN were pretreated with PMA at 37 "C exactly as in functional assays. Cells were then washed and resuspended in 4 "C buffer before incubating with [3H]PAF at 4 "C for 60 min. PMA, at 0.16-1.6 nM, enhanced, but at 2 5 nM inhibited, the specific binding of PAF to PMN (Fig. 3). The phorbol diester's effects were temperature dependent: PMN incubated with 0.5 or 50 nM PMA for 5 or 40 min at 4 "C bound PAF normally (data notshown). Furthermore, cI, a PKC blocker (63), antagonized the actions of PMA (Fig.
0 0
0.05
0.5 PMA(nM)
5
50
FIG. 2. Effects of PMA on PAF-induced release of myeloperoxidase (azurophilic granule marker, upper panel) and vitamin B-12-binding protein (specific granule marker, lower panel). PMN (lO'/ml) were incubated with 0-50 nM PMA for 4.5 min, treated with 5 pg/ml cytochalasin B for 0.5 min, and challenged with 100, 10, or 1 nM PAF (solid,interrupted, and dottedlines, respectively) for 1.5 min. All results are corrected for PMA-induced enzyme release. (Cells incubated with 0.05,0.16,0.5, 1.58,5, or 50 nM PMA and thenchallenged with BSA exhibited the following amounts of net myeloperoxidase release: 1.3, 0.6, -0.3, 2.3, 3.3, and 10.1. For vitamin B-12-binding protein release these respective values were: -0.3, 0.4, 3.2, 6.9, 13.5, and 18.3.) Data points are the mean of six (upper panel)or four (lower panel)experiments.
4). Finally MEZ and, to a lesser extent, MAG, but not HEG, also showed biphasic influences on PAF binding (Table I). Apparently, the activators altered PAF binding by an indirect, PKC-dependent mechanism. Changes in PAF-specific binding corresponded remarkably well with changes in PAF-induced degranulation (compare Fig. 2 with the lower panel of Fig. 3). Scatchard analyses of binding data, however, suggested that this correspondence needed to be interpreted with caution. Resting PMN had high and low affinity binding sites for PAF (Fig. 5 , upper panel). PMA influenced these two classes of binding sites differently. It reduced the number of high affinity receptors but did not appreciably alter these receptors' affinity for ligand (Fig. 5 , lower two panels; Table 11). The PMA-induced high affinity receptor losses correlated precisely with PMA-induced inhibition of calcium transients (Fig. 6, upper two panels). In contrast to these results, PMA enhanced the ligand affinities of low affinity (or, nonspecific) binding sites without consistently influencing their numbers (Fig. 5 , lower twopanels; Table 11).The latterchanges could not be related to function. For instance, 0.16 and 50 nM PMA had about equal effects on low affinity binding site K d values (Table 11) but opposite effects on PAF-induced degranulation (Fig. 6, lower two panels). We did note, however, a subtle relationship between
6839
Protein KinaseC, PAF, and Neutrophils
PMA 1.58 nM
I'"- 1
y-"
" " "
~"""""""""
-1
" 1.58
t
1
I
5.0
v'
PMA 5 nM PMA 15.8 nM PMA 50 nM
ii 0.5 nM
r' 0.158
1.sa
15.8
158
l&
PMA (nM)
FIG. 3. Effects of PMA on the binding of PAF. PMN (lo7/ ml) were exposed to theindicated concentration of PMA or itsvehicle (Me,SO) at 37 "C for the indicated time (upper panel)or for 5 min (lower panel).The cells were rapidly washed X 2 and resuspended (5 X 106/ml) in 4 "C buffer, and then accessed for the binding of 31.6 p~ PAF, & 200 nM PAF, a t 4 "C over 60 min. Results are reported as the percentage change in specific binding compared to simultaneously treated Me2S0controls. Each point is the mean of 7-10 experiments.
the effects of PMA on degranulation and high affinity receptors (refer to Fig. 6). Thephorbol diester's enhancing actions on degranulation abruptly switched to almost complete inhibition at a critical PMA concentration,5 nM. This PMA concentration virtually abolished PAF binding to high affinity receptors and PAF-induced calcium transients. It therefore seemed possible that thehigh affinity receptors regulate some process crucial to enhanced degranulation responses. Given the established synergy between ambient Ca2+and PKC activators (14-21), we decided to evaluate PAF-induced [Ca2+]i and PKC in PMNsequentially exposed to PMA and PAF. During its activation, PKC moves from cytosol to plasmalemma. This mobilization can be quantitated by enumerating PKC-associated PMA receptors in thesoluble and particulate fractions of cells (15,20,62,66-67). PKC mobilization occurs in PMN exposed to >0.5 nM PMA or >1 nM PAF (62, 68, 69). Hence, cells incubated with these subthreshold concentrations of stimuli did not measurably decrease their soluble PMA receptors (Fig. 7, two upper and left center panels) or increase their particulate receptors above 8,00O/cell (data not shown). However, PMN treatedwith 0.5 nM PMA X 4.5 min, cytochalasin B X 0.5 min, and 1nM PAF X 1.5 min (the same sequence was used in functional assays) reduced soluble PMA receptors by >50% (Fig. 7, right center panel) and increased particulate receptors to -14,00O/cell. This response appeared to depend on appropriately timed, PAF-induced rises in [Ca2+]i.Thus, PMN incubated with 5 nM PMA neither mo-
FIG. 4. Effect of the proteinkinase C blocker, C-I, on PMAinduced changes in PMN binding of ['HIPAF. PMN (107/ml) were incubated with 200 PM C-I (shaded bars) or buffer (open bars) for 20 min and treated with PMA or Me2S0 for 5 min at 37 "C. The cells were rapidly washed X 2, resuspended (5 X 106/ml) in 4 "C buffer, and accessed for the binding of 31.6 PM [3H]PAF, & 200 nM PAF, at 4 "C over 60 min. Results are reported as the percentage change in specific binding compared to identically (including C-I preincubation) treated Me2S0 controls. Specific binding of PAF in C-I pretreated (0.112 & 0.017) and C-I untreated (0.116 & 0.0210) Me2S0 controls were similar. Each bar graph is the mean of six experiments.
bilized calcium (Fig. 1)nor increased PKC mobilization (Fig. 7,lowertwo panels) when challenged with PAF. Calciumdepleted PMN likewise did not raise [Ca2+]ior mobilize PKC in response to PMA andPAF(Table 111). Finally, PMN stimulated with PAF 5 min before PMA exposure had baseline [Ca"]i concentrations duringphorbol diester stimulation. These cells also did not mobilize PKC (Table111).Under each of these three stimulatingconditions, degranulation failed to be enhanced (Table 111). There was, then, a strong relationship between PAF-induced [Ca2+]irises, enhanced PKC mobilization, and enhanced degranulation responses in PMAtreated PMN. DISCUSSION
PKC is a central element in cellular physiology. Many cell types, when stimulated, raise [Ca2']i, produce diacylglycerol, and use these two signals synergistically to mobilize the effector enzyme. Based on the bioactivities of PKC activators (22-60), the mobilized PKC may then regulate events that stimulate, enhance, or inhibit function. For example, PKC
Protein KinaseC, PAF, and Neutrophils
6840
Control
0.15
0.10
i ,
0.05
0.20
8
#
0.15 I
0.10
3 0.05
PMA 5 nM
0.15
0.10
mx,
lo00
3ooo
Bound (fmollml)
FIG. 5. Scatchard plots of PAF binding to MeSSO- and PMA-pretreatedPMN. PMN (107/ml)were incubated with Me2S0 or PMA at 37 "C for 5 min, washed X 2, and resuspended (5 X lo6/ ml) in 4 "C buffer, and accessed for the specific binding of PAF (10 pM-150nM) over 60 min a t 4 "C. Each point is the mean of nine experiments.
I v)
tl 20-
i @
15-
TABLE I1 Effect of PMA on neutrophil bindingof PAF PMN (1 X 107/ml)were exposed to Me2S0 or PMA for 5 min at 37 "C in Hanks' buffer (CaZ+= 1.4 mM), washed X 2 in calcium-free buffer (4 "C), and resuspended at 5 X lo6 cells/ml in Hanks' buffer (4"C, Ca2+= 1.4 mM). Suspensions were then incubated with 0.01150 nMof [3H]PAF PAF for 60 min before centrifuging through silicone oil. Binding data were corrected for nonspecific uptake (see "Experimental Procedures") and analyzed by Scatchard plots (see Fig. 5). Each plot was hyperbolic except that for 50 nM PMApretreated cells which appeared rectilinear (correlation coefficient of linearity = 0.98). Data are from more than eight experiments in each treatment erouu.
+
Receptor type (parameter)
PMA
flM
0 0.05
0.158 0.5 5 50
High affinity
flM
0.97 1.10 0.77 0.74 1.20 8.0
Low affinity
XIO'
flM
x106
1.2 1.2 0.8 0.6 0.3 -0
700 330 290 68 160 310
8.9 9.3 8.6 8.3 6.8
activators weakly stimulated PMN degranulation and, depending upon their concentration, either enhanced or inhibited the degranulating actions of PAF (Figs. 2 and 6, Table I). Under very similar assay conditions, however, the activators failed to raise [Ca2+]iand inhibited, but never enhanced,
0
5
0.05
PMA (nM)
FIG. 6. Effects of PMA on neutrophil binding of, and responses to, PAF. PMN (10'/ml) were treated with the indicated PMA concentration or MezSOfor 5 min and accessed for high affinity PAF-binding sites (upper panel)as in Fig. 4. Alternately, PMN were incubated with PMA or Me2S0for 4.5 min, treated with cytochalasin B (5 pg/ml) for 0.5 min, and challenged with PAF for 1.5 min. Center panel, change (nM) in [Ca2+Ii15 s after challenge with 1 nM PAF (PMA had virtually identical inhibitory effects on 10 and 100 nM PAF). Lower panel, release of lysozyme by cells challenged with PMA plus 100 (solid line) or 10 (interrupted line)nM PAF minus the release of lysozyme in PMA-pretreated cells challenged with BSA instead of PAF. PMA had similar dose-dependent enhancing and inhibitory effects on 1 nM PAF. Values for PMA-induced lysozyme release were -0.2 f 0.8, -0.6 f 1.2, 1.0 f 1.1, 3.2 f 1.1, 7.3 f 2.0, and 18.6 f 2.6 (mean f S.E.) for 0.05,0.15,0.5,1.58,5, and 50 nM PMA, respectively. Release of (3-glucuroidase wassimilarly effected by PMA. Each point is the mean of at least 6 experiments.
PAF-induced calcium transients (Fig. 1 and Table I). Such paradoxic effects, while repeatedly observed (22-60), are incompletely understood. We suggest that they result from the operation of two opposing mechanisms, receptor down-regulation and synergistic PKC mobilization. PMA, MEZ, and MAG altered the specific binding of PAF to PMN (Table I and Fig. 3). Their effects proved to be
Protein Kinase C, PAF, and Neutrophils structurally specific (i.e. not produced by HEG (Table I)), temperature sensitive (PMN treatedwith PMA at 4 "C bound PAF normally), and inhibited by a PKC blocker (Fig. 4). Scatchard analyses revealed that PMA ( a ) depleted PMN of high affinity receptors and ( b ) increased the affinity oflow 024
h
0.12
028
PMA (0.5 nM) + PAF (1nM) K.j = 0.71 nM Rt = 0.74
PMA (0.5 nM) & = 0.84nM
si 0.14
+
PMA (3") PAF (1nM) & = 0.75 nM @
= 0.44
\*
100
100
200
200
Bound (fmolll5Opg protein)
FIG. 7. Scatchard plots of PMA binding to soluble fractions of variably stimulated PMN. PMN (107/ml) were incubated at 37 'C with PMA (0.5 or 5 nM) or MepSO X 4.5 min, cytochalasin B (5 pg/ml) X 0.5 min, and PAF (1nM) or BSA X 1.5 min. The cells were then rapidly washed and resuspended (107/ml) in 4 "C buffer, placed on ice and sonicated, and separated intosupernatant fluid and pelleted material by centrifugation, Isolated soluble fractions (100 pl) were assayed for the binding of 250 PM-8.25 nM 13H]PMA + PMA at 23 "C over 20 min. Each point is the mean of >8 experiments. Rt is in IO6 receptor/PMN.
6841
affinity (or nonspecific) binding sites (Table I1 and Fig. 5). The former effect paralleled inhibition of PAF-induced calcium transients (Fig. 6, upper two panels) suggesting that losses in high affinity receptors were responsible for reducing the calcium mobilization response. Down-regulation of these receptors may also have contributed to inhibiting the degrandating actions of PAF. Thus, PMA blocked PAF-induced degranulation precisely at thoseconcentrations ( 2 5 nM) where it virtually abolished high affinity PAF binding (Fig. 6). We do not suggest that thelow affinity binding sites either reflect true receptors or, alternatively, are without function. Similar sites in rabbit platelets have been viewed as nonspecific sinks that merely absorb PAF (64).Nevertheless, nothing in our data discounts a possibility that these sites areinvolved in transducing, e.g. degranulation responses. We only comment, therefore, that thereappeared to be no simple relationship between the affects of PMA on low affinity binding and function. In any event, the hypothesis that high affinity receptors contribute to degranulation must consider how PMA could down-regulate these receptors and inhibitcalcium mobilization yet still enhance degranulation responses. In cell-free systems, Ca2+ and PKC activators cooperate synergistically to mobilize and activate PKC (16, 17). A similar synergy may occur within the cytosolic environment of cells and lead to enhanced PKC mobilization and, thereby, enhanced functional responses (15,18,20).These interactions seemed to have occurred in PMN challenged with PMA and PAF. At a concentration(i.e.0.5 nM) where it enhanced PAFinduced degranulation, PMA enhanced the PKC mobilizing actions of PAF (Fig. 7, upper four panels). Enhanced degranulation and PKC mobilization responses, however, failed to develop in PMN that were calcium-depleted, treated with 5 nM PMA, or challenged with PAF before PMA (Table 111). Each of these maneuvers interfered with the extentor timing of PAF-induced calcium transients (Table 111).These results suggest that PAF-induced rises in [Ca2+]icooperated with PKC activators to promote both PKC mobilization and degranulation. However, we cannot exclude a possibility that other events regulated by high affinity PAF receptors were responsible for the enhancement of function. Such events would have to be associated closely with calcium transients and, like the rises in [Ca2+]i,act only over short time periods (i.e.
