FEBS 10522 Volume 295, number lA3, 107-109 C 1991 Federation or Europcan Biochemical Socictics 00145793/9liS3.50
December
J99!
Delayed activation of phospholipase D by gonadotropin-releasing hormone in a clonal pituitary gonadotrope cell line (aT3-1) Einat Netiv’, ‘Dqw~rtterr~
Mordechai
Liscovitch’
and Zvi Naor’
of Bivcltcrtrisny, George S. Nk’iscFunrlty oJ L$e Scicwcc, Td Aviv Uttiwrsit_v, Runtar Aviv 69978. huel of Hortttotte Rcseurclt. The CVeiztttutrrt ittstitrrtc of Sciettcc, PO 60s 26, Reltovo~ 76IOll. I,sruel Received
6 October
and ‘Dqturttnent
1991
Slim&ion orcuhurcd pituitary cells from a gonadolropc lineage (aT3-I) by the gonadotropin-rclcasing hormone agonist ana!og [D-Trpb]GnRH (GnRH-A) rcsuhcd in a manirold incrcasc in accumulalion oTphosphalidylclhanol. a specilic product ofphospholipase D phospharidyl transfcrasc activity when ethanol is the phosphatidyl group acceptor. Levels ofthc natural lipid producl ofphospholipssc D. phosphatidic acid, wcrc incresscd 2-3-Told. Activation or phospholipasc D by GnRH-A was dose- and ;imc-dcpcndcnt and was blocked by a GnRH receptor antagonist [DpCIPhc’.D-Trpj.“]GnRH. GnRH-A stimulated phospholipdsc D activity arlcr a lag or l-2 min. WC conclude that in rrT3-I gonadotropcs GnRH receptor occupancy results in delayed aclivalion of phospholipasc D which could parlicipalc in la~c phases of gonadotropc regulation by the ncurohormonc. Diacylglyccrol;
GnRH; Phosphatidylcholinc; Phosphalidic acid; Protein kinasr: C; Signal transduction
I. INTRODUCTION Gonadotropin-releasing hormone (GnRH) is the first key hormone of the reproductive system. Nevertheless. its mechanism of action is not fully understood [I]. Previous studies have shown ihat GnRH stimulates a phosphoinositide-specific phospholipase C activity [2,3], mobilizes cellular and extracellular Ca” [4-61, activates protein kinase C [7,8] and induces the release of arachidonic acid [9]. More recently it was shown that a GnRH agonist analog stimulates phospholipase D activity in cultured rat granulosa cells, and evidence implicating phospholipase D activation in signaling granulosa cell differentiation was presented [IO]. Yet, the role of phospholipase D in GnRH action on its classical target cells, the pituitary gonadotropes, remained unexplored. The recent recognition of the potential importance of phospholipase D in signal transduction [I I] prompted us to examine the effect of GnRH upon phospholipase D activity in cultured piluitary &lb. Pituitary cells are heterogeneous and only about i’3% are GnRH-responsive cells (gonadotropes). We have therefore utilized here a clonal pituitary cell line of the gonadotrope lineage ocT3-I which was obtained by targeted oncogencsis in transgenic mice [ 121. 2. EXPERIMENTAL The xT3-I
cell line was kindly provided by Dr. P. Mellon (Salk
Cmx~pottr/ctrce ddrcss: 2. Naor. Dcparuncnt or Biochemistry, Tel Aviv University, Ramal Avid 69978, Israel.
Inslitulc, La Jolla, CA) and was cuhurcd as described by Mellon el al. [ 12,131. To prcfcrcnlially label the phosphatidyl moiety orphosphalidylcholinc, cells (1.5-3 x 10b/35-mm dish) were routinely prcincubalcd overnight (16-24 h) whh [‘Hlmyrislic acid (5 PCilmlldish) in DMEM containing fatty acid-rree bovine serum albumin (BSA; 0.18X). Stimulants wcrc then added in 100 ~1 or the above medium in Ihc presence or absence of ethanol (0.5%) for the indicated lime intervals and incubalions were terminated by placing rhc dishes on ice and removal oT medium. Cold methanol (I ml) was added and the cells were scraped and iransfcrrcd to 12x75mm glass tubes. Chlorolbrm (I ml) and a mixture oTO.1 N HCI. I mM EGTA (I ml) were added. The lubes wcrc vigorously mixed, ccn~riliqcd (2000 rpm, IO min) and the lower chloroform phase was collcclcd and dried under vacuum. rH]Phosphalidic acid and [‘H]phosphatidylethanol were separated by thin layer chromatography on oxalalc-impregnated Whatman LK6 plates dcvclopcd with the organic phascof a mixlurc orclhyl acetalc/ 2.2.4-trimcthylpcnlanc/acctic acid/water (13:2:3:10) as previously dcscribed [lO,l4]. The regions corresponding lo the appropriate standards wcrc scraped. cxlracrcd with I ml ol’ methanol/HCl (I 5O:l) and counted ancr addilion oT 9 ml of Insla-Mix (Packard). [9,IO-“H(N)]Myristic acid (33.9 Ci/mmol) was purchased from Dupom-New England Nuclear (Boston. MA). GnRH agonist and antagonist wcrc kindly provided by Dr. D. Coy (Tulane University, LA).
3. R’5SULTS AND DISCUSSION In a preliminary study we examined the effect ol GnRH and GnRH-A on phospholipase D activity in cultured, primary rat pituitary cells. While GnRH-A caused some stimulation of phospholipase D in most experiments, the response was weak, most likely due to the fact thal GnRH-responsive cells (gonadotropes) constitute only about 10% of the total pituitary ccl1 population. We next examined the effect of GnRH-A on phospholipase D activity in the clonal aT3-I cell line which
107
Volume
295.
number
FEBS LETTERS
I,?,3
December
199 I
is of gonadotrope lineage and was derived by targeted oncogenesis in transgenic mice [IZ]. GnRH binds with high-affinity to specific binding sites in aT3- 1 cells and stimulates phosphoinositide-specific phospholipase C activity within 30 s of incubation [ 131. GnRH-A stimulated the accumulation of orT3- I cells of both the natural lipid product of phospholipasc D. phosphatidic acid and. in the presence of ethanol. of the specific phospholipasc D product, phosphatidylethanol. in a dose-dependent manner (Fig. I ). EC,,, \Ii\lues for [“Hlphosphatidie acid and Kd of GnRH-A binding to the GnRH receptor (ca. 3 nM: [lj]) but are much higher than the ECs, value for LH release (ca. 0.03 nM; [l5]). The results imply that activation of phospholipase D is tightly COLIpled to receptor occupancy, wllile LH release requires occupancy of only a fraction of the receptors (ca. 20%: [ 161). Interestingly, whereas niaximal formation of vH]phosphatidic acid (achieved at IO nM of GnRH-A) was followed by a decrease at higher concentrations. there was no comparable decrease in formation of ~‘H]phosphatidylethanol at concentrations above IO nM of GnRH-A (Fig. I). Similar results were obtained in GnRH-A-stimulated granulosa cells [IO]. The results suggest the interesting possibility that, at high concentrations. GnRH stimulates the activity of a signal termination pathway. possibly involving phosphatidic acid degradation into diacylglycerol or lysophosphatidic acid. The stimulatory effect of GnRH-A upon phospholipase D activity is a receptor-mediated response as co-
GnRH-Ag
-
GnRH-Ant
_
Fig. I. Slimulalion of phospholipasc D nctivilf by GnRH-A in aT3-I gonadotropcs. Cells wcrc prchlbclcd wilh [‘H]myrisiic acid and incubated for 60 min whh ~hc indicakd conccnlrations or ihc GnRH agonist amdog [D-Trp”]GnRH. Incubalions wcrc lcrminarcd, lipids wcrc cxlreclcd ;Ind rH]phospha\idic acid (PA) and [!H]phosph;uidyIclhanol (WI) wcrc quomiraicd as dclililcd in section 7. Rcsuhs arc IIIC mciin 0r dllpliC;~lC dclcrminiili0ns from il rcprcscnliilivc cxpcrinicnl.
I08
_
_
+
+
+
Fig. 2. Inhibition orGnRH-A-induccdactkiion ~Tphospholipasc D by ;I specific GnRH rcccplor antagonisl. Cells wcrc prclabelcd whh [“H]myrislic acid nnd incubated ror GO tnin with GnRH-A (I nM) alone.or together wiih 100 n&f of 111~GnRH nnlagonist analoy [DpCIPhc:. D-Trp’.“]GnRH (GnRH-An~)x indicated. Incubalions u’crc tcrrninatcd. lipids wcrc cswacwd ;IIKI [‘H]phospha~idylc~h:inol WBS quiinlilattd as dcceilcd in scclion 2. Results arc lhc mean ol‘duplicatc dckrmimuions rrom a rcprewtaivc cxpcrimcni.
with a potent GnRH receptor antagonist [D-pclPhe’. D-Trp’*“]GnRH nearly abolished the response (Fig. 2). The time-course of the effect of GnRHA on phospholipase D activity is shown in Fig. 3. There w;is a lag of l-2 min before detectable accumulation of incubation
“.“-
[GnAH-A] (nM)
+
0
50 Time (min)
100
Fig. 3. Time-course ol’ ~Pl]phospli;llidylcl~i~inol wxmuh~~ion in GnRH-A-sIimul;i\cd aT3-I gonadoiropcs. Cells vIerc prclabclcd with [‘I-l]myrisIic acid and incub;ucd for lhc indicated lime with GnRH-A (IO n&l). Incubalions wcrc tcrr.L~il~cd, lipids wcrc cxir;lckd and [“I-ljphosphalidylc~hanol was quilnlii;lltd 21s dcwilcd in scciion 2. RcSIllIS iirc cxprcsscd iis llic pCrCClllilgC 0r cpm incOrp0relcd into lotal lipids imd iirc rhc nican ol’duplicarc dcwminations Tram u rcprcscmalive cxpcrimcnl. The i~rwr shows ;I dc\;\ilcd view or the lirsl IO min or ~hc incubulion.
Volume
295. number 1.2.3
FEBS LETTERS
could be observed. Maximal rates of PLD activity were observed between 10 and 60 min of incubation, and no further increase of rH]phosphatidylethanol accumulation could be observed between 90 and 120 min. The delayed activation of phospholipase D by GnRH-A in aT3-I cells suggest that the stimulation of phospholipase D activity is not a primary response to receptor stimulation. Stimulation of gonadotropin secretion and gene expression by CnRH is a complex signaling process involving phosphoinositide-specific phospholipasc C. Ca”, protein kinase C and arachidonate and its products (see [I] for review). In addition, gonadotropin secretion induced by GnRH is a biphasic process which might require diacylglycerol in an immediate as well as 2 delayed kinetics for biphasic activation of protein kinase C [ l7- 191. As phosphatidylinositol 4.5-bisphosphate and even phosphatidylinositol are minor phospholipids as compared to phosphatidylcholinc, generation of diacylglycerol from phosphatidylinositol and phosphatidylinositol 4.5-bisphosphate (via phospholipase C) might represent an initial rapid response, while the bulk of diacglgiycerol might be derived from pbosphatidylcl~olinc via phospholipase D to serve as a protein kinase C activator during the sustained phase ofhormone action. Since protein kinase C is implicated also in gonadotropin gene expression (see [I] for review), delayed supply of diacylglycerol (via phospholipase D) might distinguish between immediate and sustained responses to the neurohormone. Indeed, kinetic analysis reveals that while GnRH-stimulated phospholipase C activity is detected within 5-10 s in normal pituitary cells [2] or within 30 s in aT3-I cells [13]. stimulation of phospholipase D activity was observed in this study only after a lag of l-2 min. Thus, activation of phospho!ipase D by GnRH in u.T3-I cells follows the activation of phospholipase C, and might be dependent on this initial response. Both Ca” and protein kinasc C were reported to be involved in phospholipase D activation [20-221, although the extent of their involvement may vary widely in different cell types (cf. [ lO,23]). It is therefore possible that initially, GnRH activates phospholipnsc C to generate a transient rise of Ca” and a iimited amount of diacyigiyceroi, and consequentiy, a rapid activation of protein kinase C. This would lead to stimulation of phospholipase D activity wb~ch might generate 2 second phase of diacylglycerol for sustained activation of protein kinase C. Further studies are required in order to fully evaluate this proposed sequence of signaling. [JE]phospl~atiilylethanol
December 1991
,l~~rro~rl~rl~~t,rr,lrs: WC thank Dr. kilnda L. Mellon for kindly providing the (zT3-I cell lint. This work was supported by the Gcrmanlsracli Foundation for Rcscarch and Dcsclopmcnc (Z.N.). the Fund for Basic Rcscarch adminiswrcd by the isracl Academy of Scicnccs, Ihc Irwin Green Rcscarch Fund in lhc Ncuroscicnccs and Ihc Chief Scicnlisl’s Office. Minislry of Health. krusdh (M.L.). M.L. is a rccipisnl of an Yigal Allon Fellowship and the incumbcni of the Shloimo and Michla Tomarin Career Dcvclopmcnl Chair in Mcmbranc Physiology.
REFERENCES [I] Naor. 2. (1990) Endocrine Rev. I I. 3X-353. (2: Naor. Z.. Aural. A.. Limor. R.. Zakut. H. and LoIan. M. (1986) J. Biol. Chcm. 261. 12506-12512. [3] Morgan. R.O., Chang. J.P. and Call. K.J. (1987) J. Biol. Chcm. 262. 1166-l 171. [4] Limor. R.. Ayillon. D.. Capponi. A.M.. Child% G.V. and Naor. Z (1987) Endocrinology l-90. 497-503. [5] Chang. J.P.. McCoy. E.E.. Graclcr. J..Tasaka. K.andCall. K.J. (1986) J. Biol. Chcm. 261. 9105-9108. 161 Naor. Z.. Capponi. A.M.. Rossier. M.F.. Aydlon. D. and Limor. R. (1988) Mol. Endocrinol. 2. 511-520. [7] Hirota. K.. Hirola, T.. Agui1cra.G. and Call. K.J. (1985) J. Biol. Chcm. 260. 3243-3246. PI Nilor. 2.. Zcr. J.. Zakul. H. and Hermon. J. ( 1985) Proc. Nail. Ac;~d. Sci. USA 82. 8203-8207. 191 Naor. Z. and MI. K.J. (19811 J. Biol. Chcm. 256. 2726-2119. A. (1989) J. Biol. Chcm. 261. [W Lisco&ch. hl. and Am&dam. 11761-l 1767. [I 11 Liscovitch. M. (1991) Biochcm. Sec. Trans. 19. 402107. [I’1 Windlc. J.J., Wcincr. R.I. and Mcilon. P.L. (1990) Mol. Endocrinol. 4. 597-603. [I31 Horn. F.. Bilcrikjian. L.M.. Pcrrin. M.H., Bosma. M.M.. Wind!& J.J., Habcr. K.S.. Blounc. A.L.. Hillc. B.. Vale, W. and Mellon. P.L. (1991) Mol. Endocrinol. 5. 347-355. [I41 Chalifa. V.. Xlbhn. H. and Liscovhch. M. (1990) J. Biol. CIICIII. 265. 17512-17519. Iljl Lournayc. E.. Naor. Z. and Call. KJ. (1982) Endocrinology I I I. 730-736. II61 Naor, Z.. Clsylon. R.N. and Cat. K.J. (1980) Endocrinolog 107. 1144-l IS. D7l Nuor.Z.. Kahikincni. %I.. Loumayc. E.. Garcia Vc!la. A.. Dufau. M.L. and &I. K.J. (1982) Mol. Cell. Endocrinol. 17. 113220. iI81 Ch;rng, J.P.. Graclcr, J. anb Ca~r. K.J. ( 1987) Endocrinology 87, 1837-1845. 1191 Davidson. L.S.. Wakciicld. I.K., King. J.A., Mulligan. G.P. and Millar, R.P. (1988) Mol. Endocrinol. 2. 382-390. WI HtIdng. R.. Kuccra. G.L. and Rittcnhousc, SE. (i991) J. Biol. Chum. 266. 1652-1655. WI Pai, J.-K.. Pachtcr, J.A.. Wcinslein, B. and Bishop, W.R. (1991) Proc. Nail. Aced. Sci. USA 88. 598-602. [73] Billah, M.M.. Pai. J.K., Mul1mann.T.J.. Egan. R.W. and Sicgc’l. M.I. (1989) J. Uiol. Chcm. 26% 9069-9076. P31 Liscovilch. M. end Eli, Y. (1991) Cell Rcgulalion 2 (in press).
I09
December
J99!
Delayed activation of phospholipase D by gonadotropin-releasing hormone in a clonal pituitary gonadotrope cell line (aT3-1) Einat Netiv’, ‘Dqw~rtterr~
Mordechai
Liscovitch’
and Zvi Naor’
of Bivcltcrtrisny, George S. Nk’iscFunrlty oJ L$e Scicwcc, Td Aviv Uttiwrsit_v, Runtar Aviv 69978. huel of Hortttotte Rcseurclt. The CVeiztttutrrt ittstitrrtc of Sciettcc, PO 60s 26, Reltovo~ 76IOll. I,sruel Received
6 October
and ‘Dqturttnent
1991
Slim&ion orcuhurcd pituitary cells from a gonadolropc lineage (aT3-I) by the gonadotropin-rclcasing hormone agonist ana!og [D-Trpb]GnRH (GnRH-A) rcsuhcd in a manirold incrcasc in accumulalion oTphosphalidylclhanol. a specilic product ofphospholipase D phospharidyl transfcrasc activity when ethanol is the phosphatidyl group acceptor. Levels ofthc natural lipid producl ofphospholipssc D. phosphatidic acid, wcrc incresscd 2-3-Told. Activation or phospholipasc D by GnRH-A was dose- and ;imc-dcpcndcnt and was blocked by a GnRH receptor antagonist [DpCIPhc’.D-Trpj.“]GnRH. GnRH-A stimulated phospholipdsc D activity arlcr a lag or l-2 min. WC conclude that in rrT3-I gonadotropcs GnRH receptor occupancy results in delayed aclivalion of phospholipasc D which could parlicipalc in la~c phases of gonadotropc regulation by the ncurohormonc. Diacylglyccrol;
GnRH; Phosphatidylcholinc; Phosphalidic acid; Protein kinasr: C; Signal transduction
I. INTRODUCTION Gonadotropin-releasing hormone (GnRH) is the first key hormone of the reproductive system. Nevertheless. its mechanism of action is not fully understood [I]. Previous studies have shown ihat GnRH stimulates a phosphoinositide-specific phospholipase C activity [2,3], mobilizes cellular and extracellular Ca” [4-61, activates protein kinase C [7,8] and induces the release of arachidonic acid [9]. More recently it was shown that a GnRH agonist analog stimulates phospholipase D activity in cultured rat granulosa cells, and evidence implicating phospholipase D activation in signaling granulosa cell differentiation was presented [IO]. Yet, the role of phospholipase D in GnRH action on its classical target cells, the pituitary gonadotropes, remained unexplored. The recent recognition of the potential importance of phospholipase D in signal transduction [I I] prompted us to examine the effect of GnRH upon phospholipase D activity in cultured piluitary &lb. Pituitary cells are heterogeneous and only about i’3% are GnRH-responsive cells (gonadotropes). We have therefore utilized here a clonal pituitary cell line of the gonadotrope lineage ocT3-I which was obtained by targeted oncogencsis in transgenic mice [ 121. 2. EXPERIMENTAL The xT3-I
cell line was kindly provided by Dr. P. Mellon (Salk
Cmx~pottr/ctrce ddrcss: 2. Naor. Dcparuncnt or Biochemistry, Tel Aviv University, Ramal Avid 69978, Israel.
Inslitulc, La Jolla, CA) and was cuhurcd as described by Mellon el al. [ 12,131. To prcfcrcnlially label the phosphatidyl moiety orphosphalidylcholinc, cells (1.5-3 x 10b/35-mm dish) were routinely prcincubalcd overnight (16-24 h) whh [‘Hlmyrislic acid (5 PCilmlldish) in DMEM containing fatty acid-rree bovine serum albumin (BSA; 0.18X). Stimulants wcrc then added in 100 ~1 or the above medium in Ihc presence or absence of ethanol (0.5%) for the indicated lime intervals and incubalions were terminated by placing rhc dishes on ice and removal oT medium. Cold methanol (I ml) was added and the cells were scraped and iransfcrrcd to 12x75mm glass tubes. Chlorolbrm (I ml) and a mixture oTO.1 N HCI. I mM EGTA (I ml) were added. The lubes wcrc vigorously mixed, ccn~riliqcd (2000 rpm, IO min) and the lower chloroform phase was collcclcd and dried under vacuum. rH]Phosphalidic acid and [‘H]phosphatidylethanol were separated by thin layer chromatography on oxalalc-impregnated Whatman LK6 plates dcvclopcd with the organic phascof a mixlurc orclhyl acetalc/ 2.2.4-trimcthylpcnlanc/acctic acid/water (13:2:3:10) as previously dcscribed [lO,l4]. The regions corresponding lo the appropriate standards wcrc scraped. cxlracrcd with I ml ol’ methanol/HCl (I 5O:l) and counted ancr addilion oT 9 ml of Insla-Mix (Packard). [9,IO-“H(N)]Myristic acid (33.9 Ci/mmol) was purchased from Dupom-New England Nuclear (Boston. MA). GnRH agonist and antagonist wcrc kindly provided by Dr. D. Coy (Tulane University, LA).
3. R’5SULTS AND DISCUSSION In a preliminary study we examined the effect ol GnRH and GnRH-A on phospholipase D activity in cultured, primary rat pituitary cells. While GnRH-A caused some stimulation of phospholipase D in most experiments, the response was weak, most likely due to the fact thal GnRH-responsive cells (gonadotropes) constitute only about 10% of the total pituitary ccl1 population. We next examined the effect of GnRH-A on phospholipase D activity in the clonal aT3-I cell line which
107
Volume
295.
number
FEBS LETTERS
I,?,3
December
199 I
is of gonadotrope lineage and was derived by targeted oncogenesis in transgenic mice [IZ]. GnRH binds with high-affinity to specific binding sites in aT3- 1 cells and stimulates phosphoinositide-specific phospholipase C activity within 30 s of incubation [ 131. GnRH-A stimulated the accumulation of orT3- I cells of both the natural lipid product of phospholipasc D. phosphatidic acid and. in the presence of ethanol. of the specific phospholipasc D product, phosphatidylethanol. in a dose-dependent manner (Fig. I ). EC,,, \Ii\lues for [“Hlphosphatidie acid and Kd of GnRH-A binding to the GnRH receptor (ca. 3 nM: [lj]) but are much higher than the ECs, value for LH release (ca. 0.03 nM; [l5]). The results imply that activation of phospholipase D is tightly COLIpled to receptor occupancy, wllile LH release requires occupancy of only a fraction of the receptors (ca. 20%: [ 161). Interestingly, whereas niaximal formation of vH]phosphatidic acid (achieved at IO nM of GnRH-A) was followed by a decrease at higher concentrations. there was no comparable decrease in formation of ~‘H]phosphatidylethanol at concentrations above IO nM of GnRH-A (Fig. I). Similar results were obtained in GnRH-A-stimulated granulosa cells [IO]. The results suggest the interesting possibility that, at high concentrations. GnRH stimulates the activity of a signal termination pathway. possibly involving phosphatidic acid degradation into diacylglycerol or lysophosphatidic acid. The stimulatory effect of GnRH-A upon phospholipase D activity is a receptor-mediated response as co-
GnRH-Ag
-
GnRH-Ant
_
Fig. I. Slimulalion of phospholipasc D nctivilf by GnRH-A in aT3-I gonadotropcs. Cells wcrc prchlbclcd wilh [‘H]myrisiic acid and incubated for 60 min whh ~hc indicakd conccnlrations or ihc GnRH agonist amdog [D-Trp”]GnRH. Incubalions wcrc lcrminarcd, lipids wcrc cxlreclcd ;Ind rH]phospha\idic acid (PA) and [!H]phosph;uidyIclhanol (WI) wcrc quomiraicd as dclililcd in section 7. Rcsuhs arc IIIC mciin 0r dllpliC;~lC dclcrminiili0ns from il rcprcscnliilivc cxpcrinicnl.
I08
_
_
+
+
+
Fig. 2. Inhibition orGnRH-A-induccdactkiion ~Tphospholipasc D by ;I specific GnRH rcccplor antagonisl. Cells wcrc prclabelcd whh [“H]myrislic acid nnd incubated ror GO tnin with GnRH-A (I nM) alone.or together wiih 100 n&f of 111~GnRH nnlagonist analoy [DpCIPhc:. D-Trp’.“]GnRH (GnRH-An~)x indicated. Incubalions u’crc tcrrninatcd. lipids wcrc cswacwd ;IIKI [‘H]phospha~idylc~h:inol WBS quiinlilattd as dcceilcd in scclion 2. Results arc lhc mean ol‘duplicatc dckrmimuions rrom a rcprewtaivc cxpcrimcni.
with a potent GnRH receptor antagonist [D-pclPhe’. D-Trp’*“]GnRH nearly abolished the response (Fig. 2). The time-course of the effect of GnRHA on phospholipase D activity is shown in Fig. 3. There w;is a lag of l-2 min before detectable accumulation of incubation
“.“-
[GnAH-A] (nM)
+
0
50 Time (min)
100
Fig. 3. Time-course ol’ ~Pl]phospli;llidylcl~i~inol wxmuh~~ion in GnRH-A-sIimul;i\cd aT3-I gonadoiropcs. Cells vIerc prclabclcd with [‘I-l]myrisIic acid and incub;ucd for lhc indicated lime with GnRH-A (IO n&l). Incubalions wcrc tcrr.L~il~cd, lipids wcrc cxir;lckd and [“I-ljphosphalidylc~hanol was quilnlii;lltd 21s dcwilcd in scciion 2. RcSIllIS iirc cxprcsscd iis llic pCrCClllilgC 0r cpm incOrp0relcd into lotal lipids imd iirc rhc nican ol’duplicarc dcwminations Tram u rcprcscmalive cxpcrimcnl. The i~rwr shows ;I dc\;\ilcd view or the lirsl IO min or ~hc incubulion.
Volume
295. number 1.2.3
FEBS LETTERS
could be observed. Maximal rates of PLD activity were observed between 10 and 60 min of incubation, and no further increase of rH]phosphatidylethanol accumulation could be observed between 90 and 120 min. The delayed activation of phospholipase D by GnRH-A in aT3-I cells suggest that the stimulation of phospholipase D activity is not a primary response to receptor stimulation. Stimulation of gonadotropin secretion and gene expression by CnRH is a complex signaling process involving phosphoinositide-specific phospholipasc C. Ca”, protein kinase C and arachidonate and its products (see [I] for review). In addition, gonadotropin secretion induced by GnRH is a biphasic process which might require diacylglycerol in an immediate as well as 2 delayed kinetics for biphasic activation of protein kinase C [ l7- 191. As phosphatidylinositol 4.5-bisphosphate and even phosphatidylinositol are minor phospholipids as compared to phosphatidylcholinc, generation of diacylglycerol from phosphatidylinositol and phosphatidylinositol 4.5-bisphosphate (via phospholipase C) might represent an initial rapid response, while the bulk of diacglgiycerol might be derived from pbosphatidylcl~olinc via phospholipase D to serve as a protein kinase C activator during the sustained phase ofhormone action. Since protein kinase C is implicated also in gonadotropin gene expression (see [I] for review), delayed supply of diacylglycerol (via phospholipase D) might distinguish between immediate and sustained responses to the neurohormone. Indeed, kinetic analysis reveals that while GnRH-stimulated phospholipase C activity is detected within 5-10 s in normal pituitary cells [2] or within 30 s in aT3-I cells [13]. stimulation of phospholipase D activity was observed in this study only after a lag of l-2 min. Thus, activation of phospho!ipase D by GnRH in u.T3-I cells follows the activation of phospholipase C, and might be dependent on this initial response. Both Ca” and protein kinasc C were reported to be involved in phospholipase D activation [20-221, although the extent of their involvement may vary widely in different cell types (cf. [ lO,23]). It is therefore possible that initially, GnRH activates phospholipnsc C to generate a transient rise of Ca” and a iimited amount of diacyigiyceroi, and consequentiy, a rapid activation of protein kinase C. This would lead to stimulation of phospholipase D activity wb~ch might generate 2 second phase of diacylglycerol for sustained activation of protein kinase C. Further studies are required in order to fully evaluate this proposed sequence of signaling. [JE]phospl~atiilylethanol
December 1991
,l~~rro~rl~rl~~t,rr,lrs: WC thank Dr. kilnda L. Mellon for kindly providing the (zT3-I cell lint. This work was supported by the Gcrmanlsracli Foundation for Rcscarch and Dcsclopmcnc (Z.N.). the Fund for Basic Rcscarch adminiswrcd by the isracl Academy of Scicnccs, Ihc Irwin Green Rcscarch Fund in lhc Ncuroscicnccs and Ihc Chief Scicnlisl’s Office. Minislry of Health. krusdh (M.L.). M.L. is a rccipisnl of an Yigal Allon Fellowship and the incumbcni of the Shloimo and Michla Tomarin Career Dcvclopmcnl Chair in Mcmbranc Physiology.
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