Journal of Neurochemislry Raven Press, Ltd., New York 0 1992 International Society for Neurochernistry
Tachykinin-Stimulated Inositol Phospholipid Hydrolysis and Taurine Release from Human Astrocytoma Cells C . M. Lee, W. L. Tung, and *J. D. Young Department of Biochemistry, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; and *Department of Physiology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada
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Abstract: The activation of NKI receptors on U373 MG human astrocytoma cells by substance P (SP) and related tachykinins was accompanied by an increase in taurine release and an accumulation of inositol phosphates. Both of these effects could be inhibited by spantide, a SP receptor antagonist. The relative potency of tachykinins in stimulating 3H-inositol phosphate accumulation correlated very well with their effects in stimulating the release of [3H]taurine and inhibiting 1251-Bolton-Hunterreagent-conjugated SP binding. The effect on [3H]taurinerelease was mimicked by a protein kinase C (PKC) activator, phorbol 12myristate 13-acetate (PMA). The inactive phorbol ester analogue 4-a-phorbol 12,13-didecanoate, however, was without effect. Both SP- and PMA-induced releases of [3H]taurine were markedly inhibited by staurosporine, a po-
tent PKC inhibitor. Pretreatment of U373 MG cells with 10 pMPMA for I9 h to down-regulate PKC activity also markedly inhibited both SP- and PMA-induced releases of [3H]taurine. Treatment of cells with 100 M S P induced a timedependent translocation of PKC from the cytosolic fraction to the membrane fraction. These findings are consistent with the hypothesis that an activation of NKl receptors on U373 MG cells results in the release of inositol phosphates and activation of PKC, which in turn may regulate the release oftaurine. Key Words: Substance P-Neurokinin ANeurokinin B-Spantide-Protein kinase C-Staurosporine-Taurine-Astrocytoma cells. Lee C. M. et al. Tachykinin-stimulated inositol phospholipid hydrolysis and taurine release from human astrocytoma cells. J. Neurochew. 59,406-4 14 ( 1992).
Fibers containing substance P (SP)-like immunoreactivity have been demonstrated to form synaptoid contacts with astrocytic processes around blood vessels in the dorsal horn of the spinal cord (Barber et al., 1979), suggesting that glial cells may receive SP as a chemical messenger from neurons. This signaling system may be particularly important under certain pathological conditions because an increase in SP receptor expression in vivo occurs after neuronal injury (Mantyh et al., 1989).Glial cells contain high levels of taurine, which has been suggested to act as an osmolyte to be shuttled in and out of glial cells to maintain the normal cell volume and intracellular osmolarity (Walz and Allen, 1987; Pasantes-Morales and Schousboe, 1988). Astrocytes are swollen in many disease states, including traumatic (stab wound) brain
edema (Gerschenfeld et al., 1959). It is possible that the stimulation of taurine release from astrocytes by SP plays a role in the regulation of astrocytic swelling after neuronal injury (Tung and Lee, 1991). Three types of tachykinin receptors-NKl, NK2, and NK3-have been identified in the CNS and in peripheral tissues (Lee et al., 1986; Quirion and Dam, 1988). SP is the preferred endogenous tachykinin at the NK1 site and exhibits activity at nanomolar concentrations (Lee et al., 1986). In addition, SP methyl ester, a selective NKl receptor agonist, is approximately equipotent with SP at NKl receptors but 1001,000-foldless active than SP at NK2 and NK3 sites (Watson et al., 1983;Lee et al., 1986). Multiple tachykinin receptors can also be differentiated on the basis of the relative activities of two hexapeptide analogues
Received July 3, 199 I ; revised manuscript received November 6, 1991; accepted January 13, 1992. Address correspondenceand reprint requests to Dr. C. M. Lee at 47H, APIO, Abbott Laboratories, Abbott Park, IL 60064, U.S.A. Abbreviations used: BHSP, Bolton-Hunter reagent-conjugated substance P DAG, diacylglycerol;DiC8, 1,2-sn-dioctanoylglycerol; DMSO, dimethyl sulfoxide; G protein, guanine nucleotide binding
protein; IP, inositol phosphate; IP, , inositol monophosphate; IP,, inositol bisphosphate; IP,, inositol trisphosphate; MEM, modified Eagle’s minimal essential medium; NKA, neurokinin A; NKB, 4a-PDD, 4-aneurokinin B OAG, 1-0leoy1-2-acetyl-rac-glycerol; phorbol 12,13-didecanoate;PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PSS, physiological salt solution; SP, substance P.
406
PROTEIN KINASE C AND TAURINE RELEASE
407
of SP, [Glp6,~-Pro9]-SP(6-11) and [Glp6,~-Pro9]- Dr. B. J. Williams, Merck, Sharp and Dohme Neuroscience Research Centre, Harlow, U.K.). [3H]Taurine(specific acSP(6-11) (Lee et al., 1986; Suman-Chauhan et al., tivity, 35 Ci/mmol), [3H]inositol(10-20 Ci/mmol), and [y1990). [Glp6,~-Pro9]-SP(6-11) is one to two orders of 32P]ATP(3,000Ci/mmol) were purchased from Amersham magnitude more active at NKl sites but two orders of International, Bucks, U.K. magnitude less active at NK2 sites than [ G l p 6 , ~ Phorbol 12-myristate 13-acetate (PMA), 4-a-phorbol Pro9]-SP(6- 11). They are, however, approximately 12,13-didecanoate (4a-PDD), 1-oleoyl-2-acetyl-ruc-glycequipotent at N K 3 sites. erol (OAG), 1,2-sn-dioctanoylglycerol(DiCJ, tri-n-octylAll three types of tachykinin receptors have been amine, trichlorotrifluoroethane, HEPES, histone 111-S reported to be associated with an enhanced phospha(from bovine brain), phosphatidylserine,isoproterenol,and bovine serum albumin were from Sigma (St. Louis, MO, tidylinositol turnover on activation in different tisU.S.A.). Staurosporinewas obtained from Biomol Research sues, including rat salivary glands (NKl), hamster uriLaboratories (Plymouth, PA, U.S.A.) and Calbiochem nary bladder (NK2),and guinea pig myenteric plexus Corp. (San Diego, CA, U.S.A.). (NK3) (HanIey et al., 1980; Bristow et al., 1987; Guard et al., 1988; Suman-Chauhan et al., 1990). In Culture of human astrocytoma U373 MG cells various cell types, it has been demonstrated that the Human astrocytoma cells were cultured at 37°C in a huhydrolysis of phosphatidylinositol 4,5-bisphosphate midified incubator under an atmosphere of 5% COJ95% generates two intracellular messengers, inositol 1,4,5air, in a modified Eagle’s minimal essential medium trisphosphate and diacylglycerol (DAG) (Bemdge, (MEM)as previously described (Lee et al., 1989).They were 1984). Both inositol 1,4,5-trisphosphate and DAG plated at a density of 1 X lo4 cells/cm2 in 75-cm2 tissue culture flasks (Falcon; Becton Dickinson & Co., Oxnard, may play an important role in initiating a biological CA, U.S.A.) and subcultured once a week with one change response via elevation of the cellular free calcium conof medium during the week. Cells were counted with a hecentration and activation of protein kinase C (PKC), mocytometer (Neubauer improved) under a microscope. respectively (Nishizuka, 1984; Berridge, 1987). PKC, a Ca’+/phospholipid-dependent protein kinase, is inMeasurement of tachykinin-induced inositol volved in the control of a wide variety of biological phosphate (IP) accumulation processes, including secretion, contraction, and cell The hydrolysis of prelabeled inositol phospholipid indifferentiation (Nishizuka, 1986). The Ca2+/calmoduced by tachykinins was measured by determiningthe levdulin system and the PKC pathway may contribute to els of 3H-IPs according to the procedure described by the final response by acting either cooperatively or Downes et al. (1986) with some modifications.In brief, cells synergistically (Nishizuka, 1986; Berridge, 1987). We have previously demonstrated the presence of NK1 tachykinin receptors on a human astrocytoma cell line (U373 MG) by radioligand binding experiments (Lee et al., 1989). Because the specific binding of ‘251-Bolton-Hunter reagent-conjugated SP (12% BHSP) to N K l receptors on U373 MG cells was inhibited by guanine nucleotides, we suggested that these receptors may be coupled with a guanine nucleotide binding protein (G protein)-regulated biochemical process. More recently, we showed that tachykinins, similar to isoproterenol, can stimulate the release of taurine from these cells, but unlike the /3-adrenergic agonist, SP has no effect on the level of cyclic AMP (Tung and Lee, 1991). In the present study, we have characterized the effect of tachykinins on inositol phospholipid hydrolysis and examined the role of PKC in the tachykinin-
induced taurine release from U373 MG cells. MATERIALS AND METHODS Materials Human astrocytoma cells (U373 MG) were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). All cell culture-related chemicals were obtained from GIBCO (Grand Island, NY, U.S.A.). All tachykinin-relatedpeptideswere purchased from PeninsulaLaboratories (San Carlos, CA, U.S.A.) except for SP methyl ester (Cambridge Research Biochemicals, Cambridge, U.K.), [Glp6,~-Pro9]-SP(61 1), and [Glp6,~Pro9]-SP(61 1 ) (gift of
at confluence were harvested from 75-cm2 tissue culture flasks and plated at 2-4 X lo5cells per well on 24-well flatbottomed plastic trays (Nunclon, Naperville, IL, U.S.A.). They were cultured at 37°C for 24 h and then incubated with 3.5-4 pCi of [3H]inositol/mlper well in MEM for 19 h. Extracellular [3H]inositol was then washed away, and the cells were equilibrated in physiological salt solution (PSS) [containing118 mMNaC1, 1.2 mMKH2P04,4.7 mMKC1, 1.2 mM MgSO,, 3 mM CaCl,, 20 mM glucose, 20 mM HEPES, and 0.05% (wt/vol)bovine serum albumin, pH 7.41 for 30 min. To inhibit inositol monophosphatase,cells were preincubated for 15 rnin in 0.5 ml of PSS containing 10 mM LiCl before the addition of various tachykinins. The cells were incubated at 37°C for another 30 rnin unless otherwise mentioned. The reaction was terminated by removing the medium and adding 1 ml of ice-cold 5% perchloric acid. The mixture was kept on ice for 15 rnin before the supernatant was collected. One milliliter of a mixture of tri-n-octylamine and trichlorotrifluoroethane(1: 1 vol/vol) was added to the supernatant, followed by 100 p1 of 100 mMEDTA. The mixture was vortex-mixed vigorously and then centrifuged at 1 10 g for 1 min at room temperature. The upper aqueous layer was collected and diluted to 5 ml with distilled water. The pH of the sample was adjusted to 7.0 with 50 mn/i NaOH before loading onto a Dowex anion exchange column (Bio-Rad AGl -X8, formate form, 200-400 mesh; bed volume, 1.5 ml). After loading, the column was washed twice with 5 ml each of distilled water to remove free [3H]inositol. Glycerol phosphoinositol was eluted by adding two 5-ml aliquots of a mixture of 60 mM sodium formate/5 mM sodium tetraborate. Total 3H-IPs were eluted by adding 5 ml of a mixture of 1 M ammonium formate/ 100 mMformic acid. The anion exchange columns J. Neurochem., Vol. 59, No. 2, 1992
C.M, LEE ET AL.
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were regenerated by adding 10 ml of a mixture of 2 M ammonium formate/ 100 mM formic acid and then washed with 10 ml of distilled water. To measure ['H]inositol trisphosphate (['H]IP3) accumulation, cells were labeled with 8 pCi/ml of ['H]inositol for 17-20 h. After a wash and preincubation for 30 rnin in PSS without LiCl, cells were stimulated with 1 pM SP in a final volume of 0.5 ml for 15-45 s. The reaction was terminated by adding 0.5 ml of ice-cold 10%trichloroacetic acid. The mixture was left on ice for 15 rnin and then centrifuged at 1 10 g to bring down the precipitate. The trichloroaceticacid in the supernatant was removed by diethyl ether extraction (four 2-ml aliquots), and then the residual traces of ether were blown off. [3H]Inositoland ['Hlglycerol phosphoinosito1 were eluted from the Dowex column as described above. ['H]Inositol monophosphate (['HIIP,), ['H]inositol bisphosphate (['HIIP,), and ['HIIP, were eluted with two 5-ml aliquots of a mixture of 150 mMammonium formate/ 100 mM formic acid, 400 mM ammonium formate/100 m M formic acid, and 800 mM ammonium formate/100 mM formic acid, respectively. Radioactivity in these fractions was determined by liquid scintillation spectrometry.
containing 50 mM NaCl. An aliquot ( 15 pl) of the eluate was incubated in a reaction mixture (final volume, 50 p1) containing 50 mM Tris-HC1 (pH 7.4, 10 mM MgCl,, 2 mMCaCl,, 20 pg/ml of phosphatidylserine, and 400 pg/ml of histone 111-S for 5 min at 30°C. The reaction was initiated by adding 1 pCi of [y-"P]ATP. After incubation for 20 rnin at 30°C, the reaction was terminated by adding 1 ml of ice-cold 10%trichloroacetic acid, and the mixture was then filtered through GF/C filters. Preliminary experiments indicated an increase in enzyme activity when the calcium concentration was increased from 0.5 to 2 mM. Therefore, 2 mMCaz+was used in all subsequent experiments. The ability of the cytosolic and particulate fractions to catalyze the incorporation of the y-phosphate group of [y32P]ATPinto lysine-rich histone 111-S was measured with and without the addition of Ca2+(2 mM)and phosphatidylserine (20 pglml). PKC activity was defined as the amount of radioactivity incorporated into histone in the presence of both Ca2+and phosphatidylserine minus that in their absence.
RESULTS
Measurement of ['Hltaurine release For release experiments, cells at confluence were harvested and plated at 2-4 X lo5 cells per well on 24-well plates. They were cultured at 37°C for 24-28 h before incubation with ['Hltaurine (20-30 nM).Uptake of ['Hltaurine and hypoosmotic medium- or drug-induced [3H]taurinerelease were determined as described previously (Tung and Lee, 1991). The percentage of fractional release of ['HItaurine was calculated from the expression { ['Hltaurine in medium/(['H]taurine in medium [3H]taurinein cell)) X 100, whereas the percentage of basal release of ['Hltaurine was calculated from the expression (% fractional release in the presence of drug/% fractional release in buffer or vehicle) x 100.
+
Down-regulation of PKC by prolonged PMA pretreatment U373 MG cells were plated at 2-4 X lo5cells per well on 24-well plates or 1 X lo4cells/cm2in 75-cmZculture flasks. The growth medium was replaced after 24 h with fresh MEM (control) or 10 pMPMA in MEM (sterilizedby filtering through a Millipore filter with 0.2-pm pores). The cells were incubated for 19 h with PMA, washed twice with PSS, and then loaded with ['Hltaurine for 1 h before drug-induced ['Hltaurine release was determined. The PKC activity of U373 MG cells was determined as previously described (Xu et al., 1989). In brief, the cells were collected, washed three times with ice-cold phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 8 mM Na2HP04, and 1.5 mM KHZPO,, pH 7.4), and then homogenized in 3 ml of ice-cold solution A (20 mMTris-HC1,2 mMEDTA, 2 mM EGTA, 5 mMdithiothreitol, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mMleupeptin, pH 7.4). The homogenate was centrifuged at 100,000g for 1 h at 4°C. The supernatant was saved and represented the cytosolic fraction. The pellet was resuspended in solution A (containing 1% Triton X-loo), incubated for 1 h at 4"C, and centrifuged as above to obtain a soluble membrane fraction. These fraction were applied to diethylaminoethyl-cellulose columns (1 ml), which were preequilibrated in solution A without leupeptin. The columns were washed with 10 ml of solution A without leupeptin, and PKC was eluted with 1.5 ml of solution A J. Neurochem.. Vol. 59, No. 2, 1992
Effects of tachykinins on accumulation of IPS In the presence of 10 m M LiCl, SP (100 nM) induced a time-dependent accumulation of Ips in U373 MG cells. As illustrated in Fig. 1, the level of 'H-IPS increasedgradually after exposure for 1 rnin to SP and reached a plateau after 30 min. Increasing the incubation time to 60 rnin did not increase the level of 'HIPSfurther. Therefore, a 30-min incubation time was adopted in all subsequent experiments. Typically, 100 nMSP increased the level of 'H-IPS by two- to threefold over the basal level under these experimental conditions. SP and its related analogues increased the accumulation of 3H-IPs in a concentration-dependent manner (Fig. 2). Their rank order of potency was SP >> neurokinin A ( N U ) > neurokinin B (NKB), and 1 1) was more active than [ G l p 6 p [Glp6,~-Pro9]-SP(6Pro9]-SP(6-11). This pattern is very similar to their abilities in inhibiting I2%BHSPbinding and stimulat-
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60
lime (mid
FIG. 1. Time-dependent accumulation of 3H-IPsin U373 MG cells induced by 100 nM SP (0)comparedwith buffer control (0).Data are mean f SD (bars) values from three different wells on the same tray. Similar results were obtained in two separate experiments.
PROTEIN KINASE C AND TAURINE RELEASE 120 r
In the absence of LiCl, SP (1 p M ) caused a significant increase in accumulation of 'H-IPS in U373 MG cells (Table 3). The effect was more pronounced after incubation for 15 s (200%of control) than after 45 s (1 36% of control) (Table 3).
80
60
.
40 20 1
0.1
409
1
10
100
1000
10000
100000
Peptide Conc. (nM)
FIG. 2. Accumulation of 3H-IPs in U373 MG cells induced by 1) tachykinins and related analogues: SP (0),[Glps,~-ProQ]-SP(6-1 (A), NKA (A),NKB (O), and [Glp6,0-Pros]-SP(6-11) (0).Data are mean SD (bars) values from three different wells on the same tray and are those of a typical experiment. Similar results were obtained in two to six separate experiments. The accumulation of 3H-IPs induced by 100 nM SP was 3,206 -+ 1,426 cprn (mean SD, n = 6).
*
*
ing ['Hltaurine release from U373 MG cells (Table 1). The potencies of the three mammalian tachykinins in these three test systems were very similar, but [Glp6,~Pro9]-SP(6-1 1) was 160-180 times more active than 1 1) in stimulating the accumula[Glp6,~Pro9]-SP(6tion of 'H-IPS and the release of ['H]taurine, but only 26 times more potent in inhibiting '251-BHSPbinding. Spantide, a SP receptor antagonist, inhibited the effect of SP on accumulation of IPS. The effect of spantide was concentration dependent (Fig. 3). The presence of 0.1 and 1 pMspantide increased the EC5o (concentration of drug that produced 50%of the maximal response) of SP from 0.54 nM (control) to 1.28 and 1.73 nM, respectively. At these concentrations spantide had minimal effects on the basal level of 'HIPS. The levels of 'H-IPS in the buffer control and 0.1 and 1 pMspantide were 586 f 19,729 f 23, and 737 f 3 1 cpm (mean f SD, n = 3), respectively. Isoproterenol, a p-adrenergic agonist, which stimulates ['HItaurine release from U373 MG cells (Tung and Lee, 199I), did not alter the basal or the SP-induced accumulation of 3H-IPs (Table 2).
Effect of PMA on [3H]taurinerelease To examine the role of PKC in SP-stimulated taurine release, we studied taurine release in the presence of PMA, which mimics the action of DAG on this enzyme (Nishizuka, 1984). As illustrated in Fig. 4, PMA increased the release of ['Hltaurine from U373 MG cells with nanomolar potency. The EC5o was estimated to be 7.4 f 2.8 &(mean f SD, n = 5). At 100 nM, PMA produced a fourfold increase in ['HItaurine release, which is comparable to that observed for SP and related tachykinins. On the other hand, two synthetic analogues of DAG, OAG and DiCs, as well as the inactive phorbol ester analogue 4-a-PDD did not stimulate ['Hltaurine release significantly when tested from 1 nMto 30 pM (Fig. 4). The involvement of PKC activation in PMA-stimulated taurine release was further examined by studying the effect of staurosporine, a potent PKC inhibitor. Preliminary studies revealed that a 30-min preincubation of U373 MG cells with staurosporine was necessary to obtain maximal inhibition of the PMAinduced effect. Under these conditions, staurosporine shifted the dose-response curve of PMA to the right in a concentration-dependent manner (Fig. 5). At 10100 nM, staurosporinehad no significant effect on the basal release of [3H]taurine.The same concentrations of staurosporine, however, markedly inhibited the SP-induced release of ['Hltaurine (Fig. 6). Unlike the inhibitory effect of staurosporine on PMA-induced ['Hltaurine release, staurosporine (10- 100 nM) inhibition of SP-induced ['Hltaurine release could not be overcome with higher concentrationsof the stimulant (Fig. 6). Higher concentrations of staurosporine were not tested in these experiments because staurosporine itself stimulated ['Hltaurine release to 228 f 39% (mean f SD, n = 5) of basal values at 1 pM.
TABLE 1 . Comparison of potencies of mammalian tachykinins and analogues as inhibitors of specifrc '251-BHSPbinding and in stimulating accumulation of "H-IPSand ["Hltaurinerelease from human astrocytoma (U3 73 MG) cells
Peptides
ICm of'251-BHSP binding
(w
ECso of ['Hltaurine release (nM)'
SP NKA NKB [Glp6,~Pro9]-SP(6-lI ) [Glp6,~-Pro9]-SP(6-l 1) Spantide
0.33 f 0.09 (3) 52.6 f 12.3 (3) 308.0 -I: 82.1 (3) 2,770.0 70.0 (2) 104.0 4.7 (2) 384.0 f 27.3 (3)
0.31 0.06 (6) 20.0 f 5.0 (2) 200.0 2 35.0 (2) 3,630.0 480.0 (4) 20.0 f 10.0 (3)
* *
* *
-
ECm of accumulation of 'H-IPS (nM)
*
0.43 0.28 (6) 46.5 k 4.9 (2) 355.0 & 77.8 (2) 3,000.0 & 2,800.0 (2) 18.5 f 0.7 (2)
-
Data are mean f SD values (no. of separate experiments performed in triplicate).ICsoand ECsoare the concentration of peptide required to give 50%inhibition of specific "'I-BHSP binding and half-maximal stimulation of ['Hltaurine release and accumulation of 'H-IPS, respectively. Data from Lee et al. (1989). Data from Tung and Lee ( 199 1).
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J. Neurochem.. Vol. 59, No. 2, 1992
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TABLE 3. Efect of SP on accumulation of 'H-IPS in U373 MG astrocvtoma cells Radioactivity (cpm) Incubation time (s), treatment
t'HJIP1
t3~11p3
15 Control ann I ..-0.1
SP(1 PW 1
10
499 k 33 544 f 16
86 k 8 174 k 7"
81 t 4 160 7b
386 f 84 469 f 44
91 k 1 1 123 f 7
85 k 3 116+7"
*
45
100
Control
Substance P Conc. (nM)
SP(1 U M )
FIG. 3. Effect of spantide on SP-induced accumulationof 3H-IPs. Cells were pretreated with buffer (0)or 100 (0)or 1 (A) pA4 spantide for 15 min before the addition of SP. Data are mean SD (bars) values from three different wells on the same tray. Similar results were obtained in two separate experiments.
*
Effect of prolonged PMA pretreatment on ['Hltaurine release PMA was dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO in 10 pM PMA was 0.1% (vol/vol). This concentration of DMSO had no effect on the basal release of [3H]taurineduring a 15-min exposure (Table 4). Moreover, pretreating U373 MG cells with medium containing0.1%DMSO for 19 h had no effect on either basal or SP-stimulated [3H]taurinerelease (Table 4).Unlike DMSO, chronic PMA pretreatment caused a 5943% increase in the basal [3H]taurine release (Table 4). However, prolonged exposure of cells to 10 pM PMA for 19 h completely abolished the subsequent [3H]taurinerelease response to PMA (Fig. 7) and markedly inhibited SPstimulated [3H]taurine release (Table 5). The same pretreatment protocol, however, did not affect the release of [3H]taurineinduced by hypoosmotic medium (Table 5). Measurement of PKC activity As shown in Table 6, -55% of the PKC activity in U373 MG cells was found in the cytosolic fraction under normal culture conditions. After pretreatment with 10 pM PMA for 19 h, 95% of the cytosolic PKC activity was lost, and -45% of the original particulate PKC activity remained in the membrane fraction (Table 6). The ability of staurosporine to inhibit both cytosolic and particulate PKC activities was also con-
Reactions were terminated after the cells had been incubated with 1 p M SP or buffer for 15 or 45 s. The radioactivity associated with IPS was determined after separation on an anion exchange column. Data are mean k SD values of triplicate determinations. " p i0.05,b p < 0.005when compared with controls using Student's t test.
firmed when 1 pM staurosporine gave complete inhibition of PKC activities (Table 6). SP (100 nM)induced a time-dependent increase in the particulate PKC activity, which peaked after 1 min and gradually returned to the basal level after 5 min (Fig. 8). On the other hand, there was a slight decrease in cytosolic PKC activity on exposure of U373 MG cells to 100 nMSP, with the largest effect at 30 s after drug treatment (Fig. 8). DISCUSSION
The primary structures of the NK1 (Yokota et al., 1989; Hershey and Krause, 1990), NK2 (Masu et al., 1987), and NK3 (Shigemoto et al., 1990) tachykinin receptors have recently been deduced from their cDNA sequences. They all share a high degree of homology with other members of G protein-coupled receptors with seven transmembrane segments. Although the NKl receptor on the human astrocytoma cell line (U373 MG) has not been cloned, we have previously suggested that it may be a member of the G
500
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400
TABLE 2. Effect of isoproterenol on the basal and 100 nM SP-induced accumulation of 'H-IPS in U373 MG cells 200
Treatment
'H-IPS (cum)
Control Isoproterenol ( 100 nM)
744 t 33 751 k 4 1,750k 124 1,735 rt 113
SP(1OnM)
SP (10nM)+ isoproterenol(100nM)
Data are mean k S D values of triplicate determinations from a typical experiment. Similar results were obtained in two separate experiments.
J. Neurochem., Val. 59, No. 2, 1992
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10
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Drug Conc. (nM)
FIG. 4. Effects of phorbol esters and DAG analogues on [3H]taurine release from U373 MG cells: PMA (0).401-PDD (A),OAG (O),and DiC, (A). Data are mean f SD (bars) values from three different wells on the same tray. Similar results were obtained in three separate experiments.
PROTEIN KINASE C AND TAURINE RELEASE
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TABLE 4. Effects of DMSO and PMA pretreatment on basal and SP-stimulaied (3H]taiirine release from U373 MG cells ['HITaurine release (% of basal release)
FIG. 5. Effect of staurosporine on PMA-induced [3H]taurine release from U373 MG cells. Cells were pretreated with buffer (0) or 30 (0)or 100 (A) nM staurosporine for 30 min before addition of PMA. Data are mean k SD (bars) values from three different wells on the same tray. Similar results were obtained in two separate experiments. ' p < 0.05 when compared with buffer control using Student's t test.
protein-coupled receptorfamilybecauseagonist binding can be inhibited by guanine nucleotides(Lee et al., 1989). G protein-coupled receptors have been shown to regulate several transmembrane signaling processes, including the enzymatic activity of adenylate cyclase, cyclic GMP-specific phosphodiesterase, phosphoinositide-specific phospholipase C, and the permeability of potassium and calcium channels (Freissmuth et al., 1989).In spite oftwo earlier studies that reported a stimulatory effect of SP on cyclic AMP levels in cultured neuroblastoma cells (Narumi and Maki, 1978) and an enhancement of noradrenalinestimulated cyclic AMP levels in astrocytes (Rougon et al., 1983), a direct adenylate cyclase response to tachykinins has not been demonstrated (Lee et al., 1983; Tung and Lee, 1991). On the other hand, SP has been shown to activate phosphoinositide-specific phospholipase C in the guinea pig ileum, hamster urinary bladder, rat salivary glands and hypothalamus, and mouse cerebral cortical and rat spinal cord astrocytes (Hanley et al., 1980; Watson and Downes, 1983;
1
10
Buffer
100 nM SP
Normal PSS PSS 0.1% DMSO Pretreatment for 19 h 0.1% DMSO 10 pM PMA
loo+ 1 l o o k 10
351 k 33
107 ? 14 171 + 12"
308 ? 21 153 ? 8b
+
PMA conc. (nM)
0.1
Treatment
100
Substance P conc. (nM)
ND
Data are mean ? SD values of triplicate determinations. Similar results were obtained in two separate experiments. ND, not determined. p < 0.0 1, p i0.05 when compared with the respective normal PSS treatment using Student's t test.
Mantyh et al., 1984; Bristow et al., 1987; Guard et al., 1988; Torrens et al., 1989; Beaujouan et al., 1990; Suman-Chauhan et al., 1990; Mamott et al., 1991). In general, an activation of phosphoinositide-specific phospholipase C can lead to the hydrolysis of inositol phospholipids to produce DAG and IPS (Berridge, 1984, 1987). In the present study, we have demonstrated a clear stimulatory effect of SP on the accumulation of 3H-IPs and a transient increase in [3H]IP3concentration in U373 MG cells, presumably as a result of phosphatidylinositol 4,5-bisphosphate hydrolysis. Among the mammalian tachykinins tested, SP was the preferred ligand with an ECSoof 0.43 & 0.28 nM(n = 6), followed by NKA and NKB, and [Glp6,~-Pro9]-SP(61 1)was 160times more ac1 1). This pattern corretive than [Glp6,~-Pro9]-SP(6lates very well with their abilities to stimulate [3H]taurine release (Tung and Lee, 1991) and to inhibit I2%BHSP binding to NKl receptors on intact astrocytoma cells (Lee et al., 1989). Moreover, spantide, a SP receptor antagonist, shifted the dose-response curves of SP on 3H-IPaccumulation and [3H]taurine release to the right with comparable potency (com-
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roo!-
0.01
I
0.1
1
5
10
PMA Conc. (pM)
FIG. 6. Effect of staurosporine on SP-induced [3H]taurine release from U373 MG cells. Cells were pretreated with buffer (0) or 10 (O),30 (A), or 100 (A)nM staurosporine for 30 min before addition of SP. Data are mean k SD (bars) values from three different wells on the same tray. Similar results were obtained in two separate experiments. ' p < 0.05 when compared with buffer control using Student's t test.
FIG. 7. Effect of chronic PMA pretreatment (10 pM, 19 h) on PMA-induced [3H]taurine release from U373 MG cells: control (0) and PMA-treated(0)cells. Chronic PMA pretreatment increased the basal release by 55 +- 0.6%. Data are mean f SD (bars) values from triplicate determinations. Similar results were obtained in three separate experiments.
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TABLE 5 . Efects of SP- and hypoosmotic mediuminduced (3H]taurine releasefrom U373 MG astrocytoma cells
80 r
I
Net percentage of [3H]taurinerelease Treatment SP 0.1 nM InM 100 nM Hypoosmotic medium 100 mMNaCl 80 mM NaCl
Control 0.23 ? 0.02 1.30 k 0.10 1.96 2 0.08
3.57 f 0.88 44.92 ? 5.68
Prolonged PMA pretreatment % of control 0 0.29 f 0.070 0.60 f 0.09
4.21 f 0.14 42.59 f 1 . 1 1
(0) (22) (31) (1 18)
(95)
Data are mean f SD values of triplicate determinations. The net percentage of [3H]taurinerelease was defined as fractional release in the presence of SP or hypoosmotic medium minus basal fractional release in the presence of control buffer containing 1 18 mMNaCI. The basal fractional release with and without prolonged (19-h) 10 p M PMA pretreatment was 0.65 f 0.02 and I . 1 1 k 0.08%, respectively.
pare Fig. 3 with Fig. 2 in Tung and Lee, 1991). These results support the notion that the NKl receptors on U373 MG cells are coupled to a phosphoinositidespecific phospholipase C. Activation of NKl astrocytoma receptors by SP will hydrolyze inositol lipids to liberate DAG in addition to IPS. DAG in turn can stimulate the activity of PKC. Therefore, PKC may be involved in the regulation of taurine release by SP. Because both DAG and the tumor-promoting phorbol ester PMA can stimulate PKC activity directly by increasing the affinity of the enzyme for Ca*+and phospholipids (Nishizuka, 1984),we examined the effect of PMA on [3H]taurine release. Indeed, PMA induced [3H]taurine release with a nanomolar potency comparable to its binding affinity for PKC. On the other hand, the phorbol ester TABLE 6 . Effects of prolonged PMA pretreatment and acute treatment with staurosporine on cytosolic and particulate PKC activity of U373 MG astrocytoma cells PKC activity (cpm of 32Piincorporated into histone) Drug treatment
Cytosolic
Particulate
Control 37,339 2 15,471 30,446 f 4,556 1,653 2 2,863 13,835 f 4,217 PMA(IOfiM), 19 h 96 f I67 0 Staurosporine ( 1 pM), 20 min PKC activity was defined as the amount of ’*Pi radioactivity incorporated into histone in the presence of Ca2+and phosphatidylserine minus that in their absence. In the absence of Ca” and phosphatidylserine, and cytosolic and particulate PKC activities were 4,541 f 1,919 and 7,239 f 1,824 cpm, respectively.Data are mean f SD values from three separate experiments performed in duplicate.
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0
1
2
3
4
5
Time ( m i d
FIG. 8. Translocation of PKC induced by SP (100 nM) in supernatant (0)and particulate (0)fractions. PKC activity was defined as the amount of 32Piradioactivity incorporated into histone in the presence of Ca2+and phosphatidylserineminus that in their absence. In the absence of Ca2’ and phosphatidylserine, the cytosolic and particulate PKC activities were 6,237 f 1,879 and 6,423 710 cpm, respectively. Data are mean f SD (bars) values from quadruplicate determinations. Similar results were obtained in three separate experiments. *p < 0.05 when compared with zerotime control using Student’s t test.
*
analogue 4a-PDD which does not stimulate PKC, was without effect on taurine release. The lack of effect of two DAG analogues, DiCs and OAG, may be related to their rapid metabolism. The involvement of PKC is further supported by the inhibitory effect of staurosporine, a potent PKC inhibitor (Tamaoki et al., 1986), on PMA-induced taurine release. Moreover, the effect of PMA on [3H]taurine release was abolished in cells that had been depleted of PKC by prior exposure to PMA ( 10 p M ) for 19 h. The same treatment also markedly reduced the effect of SP on [3H]taurinerelease. On the other hand, the prolonged pretreatment of U373 MG cells with PMA did not affect the release of [3H]taurineinduced by decreasing the concentration of NaCl in the medium from 118 mM to 100 or 80 mM (Table 5). This observation argues against a nonspecific interference with cell function after PMA pretreatment. Moreover, pretreating cells with staurosporine also inhibited the effect of SP. These results support the involvement of PKC activation in both PMA- and SP-stimulated [3H]taurinerelease. In several cell types, including astrocytes and C6 glioma (Fishman et al., 1987;Neary et al., 1988),activation of PKC is associated with translocation of the cytosolicPKC to the membrane, resulting in the eventual proteolytic degradation or “down-regulation” of the enzyme (Kraft and Anderson, 1983; RodriguezPena and Rozengurt, 1984; Wooten and Wrenn, 1984; Young et al., 1987; Snolk et al., 1988). SP has been reported to stimulate the translocation of PKC from the cytosolic to the membrane fraction in bovine brain microvessels (CatalAn et al., 1989). In the present study, we observed a marked loss in the total activity of the calcium-sensitive,phospholipid-dependent PKC in U373 MG cells after they had been ex-
PROTEIN KINASE C AND TAURI" RELEASE
posed to PMA (10 p M ) for 19 h, presumably the result of an accelerated proteolytic degradation. We also observed a significant increase in membrane-associated PKC activity in U373 MG cells after they have been exposed to 100 nMSP for 1 min, and there was a concomitant decrease in the cytosolic PKC activity. This further supportsthe involvement of PKC activation on activation of NKl receptors on U373 MG cells. In summary, we have presented data to support the notion that the NK1 receptors on U373 MG cells are coupled with a G protein-regulated, phosphatidylinositol bisphosphate-specificphospholipase C. The activation of these receptors releases IP, and activates PKC. Although the effect of IP, on the cytosolic calcium level and release of taurine remains to be elucidated, the activation of PKC seems to be involved in the regulation of taurine release. Acknowledgment: This work was supported by a grant from the University and Polytechnics Grant Committee, Hong Kong and by the Alberta Heritage Foundation for Medical Research. J. D. Young is a Heritage Medical Scientist. We thank Mrs. Flora Tang and Mrs. MaryAnn Lam for their efficient preparation of this manuscript.
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