Novel Aspects of Gonadotropin-releasing Hormone Action on Inositol Polyphosphate Metabolism in Cultured Pituitary Gonadotrophs

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 262, No. 3, Issue of January 25, pp Printed in h.a. Novel Aspects of Gonadotropin-releasing Hormone Action on Inositol Polyphosphate Metabolism in Cultured Pituitary Gonadotrophs (Received for publication, May 14, 1986) Reginald 0. Morgan, John P. Chang, and Kevin J. Catt From the Endocrinology and ReDroduction Research Branch, National Institute of Child Health and Human Development, National Znstitutes of Health, Bktksda, Maryland The hypothalamic neuropeptide gonadotropin-re- lease from pituitary gonadotrophs by a calcium-dependent leasing hormone (GnRH) stimulates luteinizing hor- mechanism that is known to involve increased phosphoinomone secretion via receptor-mediated activation of sitide turnover. Thus, GnRH stimulates [3H]inositol phosphosphoinositide hydrolysis to yield inositol phos- phate accumulation in pituitary gonadotrophs (3-5), granuphates and diacylglycerol. Application of anion-ex- losa cells (6), and hemipituitary glands (7) and promotes [3H] change high-performance liquid chromatography to- arachidonate release (8) and [32P]phosphate uptake into phosgether with absorbance and radiochemical flow detec- phoinositides (9-13). Also, translocation and activation of tion has enabled both the characterization and quanprotein kinase C by endogenous diacylglycerol are believed to titative estimation of pituitary cell inositol phosphates occur during stimulation of gonadotropin secretion by GnRH and phosphoinositides. In cultured pituitary cells, GnRH caused a rapid and progressive rise in the for- (14) and phorbol esters (15). mation of inositol 1,4,5-trisphosphate and of higher Although GnRH action is clearly accompanied by increased polyphosphoinositols corresponding to inositol tetra- phospholipid turnover, evidence for the occurrence and role kisphosphate, pentakisphosphate, and hexakisphos- of rapid, selective changes in IP3 formation is still lacking, phate. The inositol 1,4,5-trisphosphate formed during and the relative contributions of the major phosphoinositides GnRH action was dephosphorylated predominantly via to inositol phosphate production have not been defined. Thus, inositol 4-monophosphate rather than the expected me- there is no tangible evidence that PIPz and PIP remain the tabolite, inositol 1-monophosphate. The catabolism of major targets of ligand-activated phospholipase C, i.e. that inositol 4-monophosphate, like that of inositol l-mon- Ins-1,4,5-P3 as well as diacylglycerol continue to be produced ophosphate, was inhibited by lithium. For these rea- so as to maintain regulatory influences on both intracellular sons and because it was the major metabolite of [3H] calcium and protein kinase C activity. The present study inositol 1,4,5-trisphosphate in permeabilized gonado- focuses on the questions of whether GnRH does indeed trigger trophs, inositol 4-monophosphate appears to represent a rapid, initial release of Ins-1,4,5-P3 and whether this is a a specific marker for ligand-stimulated inositol poly- transient or a sustained response. The identification of several phosphate formation and metabolism. The marked and novel products of IP3 metabolism underscores the pivotal role sustained elevations of inositol 4-monophosphate and of this intracellular messenger in phosphoinositide turnover inositol 1,4-bisphosphate in GnRH-stimulated gonad- and calcium mobilization. otrophs indicate that polyphosphoinositides rather than phosphatidylinositol are the preferred substrates MATERIALS AND METHODS of phospholipase C during GnRH action. Anterior pituitary cells were prepared from adenohypophyses of adult, female rats by trypsin treatment and mechanical dispersion. For certain experiments, a 4-5-fold enriched population of gonadotrophs was obtained by centrifugal elutriation of the dispersed cells (16). Cells were cultured on Cytodex-1 beads for 2 days in Medium 199 with Earle s salts containing 10% horse serum plus 25 mm Phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is a primary cell membrane event that is initiated by specific hormone-receptor binding and generates the intracellular messengers inositol 1,4,5-trisphosphate (IP,) and diacylglycerol (1, 2). Gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone re- * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: PIPz, phosphatidylinositol 4,5-bisphosphate; GnRH, gonadotropin-releasing hormone; GPI, glycerophosphoinositol; GPIPz, glycerophosphoinositol 4,5-bisphosphate; Ins-1-P, myo-inositol 1-monophosphate; Ins-4-P, myo-inositol 4- monophosphate; Ins-1,4-P~, myo-inositol1,4-bisphosphate; Ins-1,4,5- PB, myo-inositol 1,4,5-trisphosphate; IP, inositol phosphate; IP1, IPz, and IP3, inositol mono-, bis-, and trisphosphate; IP,,IPS, and IPg, myo-inositol tetrakis-, pentakis-, and hexakisphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; SAX, strong anion-exchange; WAX, weak anion-exchange; HPLC, high-performance liquid chromatography; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. HEPES at ph 7.4. They were then incubated in fresh medium containing 3 pci of [3H]inositol, [32P]phosphate, and/or [33P]phosphate/l million cells for 3 days in order to achieve isotopic equilibrium. Aliquots of the washed cell suspension were placed in glass tubes maintained at 37 C and exposed to test agents for various times up to 30 min. This system is characterized by prompt luteinizing hormone secretory responses to GnRH, in contrast to plated cells, with changes in radiolabeled inositol phosphate levels that closely reflect actual mass changes. Cell stimulations were performed with native GnRH (Peninsula Laboratories, Inc.); and in some experiments, blockade of GnRH action was produced by subsequent addition of a potent antagonist derivative, [N-acetyl-D-p-chloro-Phe -,D- Trp3,~-Lys6,~-Ala o]gnrh, with high affinity for GnRH receptors (17). Incubations of prelabeled cells were terminated by addition of trichloroacetic acid to 5% (w/v), followed by removal of the supernatant which was extracted with ether and neutralized. Lipids were extracted from the corresponding cell pellets (181, deacylated (19), and analyzed as water-soluble glycerophosphodiesters by high-performance liquid chromatography (HPLC). For studies in permeabilized cells, aliquots of 5 million cells in 0.1 ml of medium containing 200 pg/ml saponin and 10 mm LiCl were incubated at 37 C for

2 GnRH Metabolism and Phosphate Inositol 1167 min prior to the addition of 50,000 dpm of [3H]Ins-1,4,5-P3(New England Nuclear). After a further 60-min incubation, the reaction was stopped by ice-cold acidic extraction with methanol, chloroform, 0.1 N HCl containing 1 mm EDTA (1.25:2.5:0.65). Neutralized aqueous samples were analyzed using newly developed procedures for strong anion-exchange (SAX) and weak anion-exchange (WAX) HPLC of inositol phosphates (20). SAX-HPLC resolution of IP, to IPS and of the glycerophosphoinositols (GPIs) was achieved by elution from an Adsorbosphere (5-pm) 4.6 X 250-mm SAX column (Alltech/Applied Science) with a linear gradient of aqueous ammonium phosphate (ph 3.35) from 0 to 0.9 M between 5 and 65 min. Ins-1-P, Ins-l,4-Pz, and Ins-1,4,5-P3 were identified by coelution with tritiated standards (New England Nuclear) from a system which resolved all known isomers of these compounds, including myo-inositol 1:2-cyclic monophosphate, Ins-4-P, Ins-4,5-P,, Ins- 1,3,4-P3 and Ins-2,4,5-IP3 (20). The identification of inositol monophosphate isomers was based on comparison with standard [3H]Ins-l-P(New England Nuclear), with [3H]Ins-2-P prepared by acidic phospholipase C treatment of [3H]PI followed by acid hydrolysis of myo-inositol l:2-cyclic monophosphate (21), and with [3H]Ins-4-P prepared from [3H]PIP(22). These were resolved by weak anion-exchange using a WAX-HPLC system with an Adsorbosphere (5-pm) 4.6 X 250-mm NH, column (Alltech/Applied Science) eluted by ammonium phosphate (ph 3.35) from 0 to 12.5 mm over 0-50 min, followed by mm between 50 and 75 min. Ins-4-P was retained for 7 min longer than authentic [3H]Ins-l-P and 2 min longer than [3H]Ins-2-P. Sample peaks were identified by comparison with external standards in adjacent runs and with internal standards in replicate sample runs. Radioactivity measurements were made with an in-line radioactivity flow detector (Flo-One Beta IC, Radiomatic Instruments & Chemical Co.) by liquid scintillation counting after mixture of the HPLC eluant with Flo-Scint I11 scintillator (1:6, v/v). Calculations of disintegrations/minute were corrected for background and gradientrelated changes in counting efficiencies and channel crossover by an internal computer using program software version 4e and high-level integration software version 4e (Radiomatic Instruments & Chemical Co.). Each recorded point represented a 6-s count and is therefore expressed in units of dpm/lo to reflect the 10-fold diminution in scale due to the use of 10 X 0.1-min counting periods. RESULTS An example of the SAX-HPLC elution profile of [3H] inositol [32P]phosphates extracted from 30-min control and GnRH-stimulated gonadotrophs is shown in Fig. 1. TWO features of particular interest are the relative prominence of Ins- 4-P uers sus Ins-1-P in the inositol monophosphate fraction of GnRH-stimulated cells and the existence of late-eluting, 32P/ 3H-double-labeled inositol polyphosphates including IP4, Ips, and IPS. Ins-1-P, Ins-4-P, and Ins-l,4-P2 were increased by factors of 1.3, 4.6, and 7.5, respectively, during 30-min stimulation with GnRH in the absence of Li. The significant accumulation of Ins-4-P and Ins-1,4-P2 provides indirect evidence that polyphosphoinositides rather than PI are the chief targets of GnRH-activated phospholipase C and that the enzymatic capacity for catabolism of these substances may become limiting. The identities of the IPS eluting after known Ins-1,4,5-P3 were ascertained through additional studies (Fig. 2) using [3H] inositol and ~rtho[~~p]phosphate double-labeling of cellular phospholipids to equilibrium, with calculation of 33P/3H ratios in the SAX-HPLC-separated glycerophosphoinositoi derivatives and comparison by linear regression analysis with the observed ratios in the peaks designated IP4, IPS, and IP,. For example, in one sample from the experiment in Fig. 2, the 33P/3H ratios in lipids were for GPI and for GPIPP and among the IPS were for IP4, for Ips, and for IP, (r = 0.99). Typical sample calculations showed a fit of r2 > 0.9 using either 32P/3H (Fig. 1) or 33P/3H (Fig. 2). The experiments shown in Figs. 1 and 2 differed in that the latter employed a lower concentration of gonadotrophs (12% in mixed pituitary cells uersus 40% in elutriated cells) and that stimulation by GnRH was performed in the presence of Li. Both experiments nevertheless showed identical 4.6-fold increases in Ins-4-P with GnRH, apparently because the expected decrease in specific productions of Ins-4-P was com- SAX-HPLC Elution Time (mid FIG. 1. SAX-HPLC resolution of water-soluble [SH]inositol [azp]phosphate metabolites from prelabeled rat pituitary gonadotrophs incubated 30 min without (A) or with GnRH (B). Each of the two samples represented 1 million elutriated cells comprising 40% gonadotrophs, which were labeled to equilibrium during 3 days of incubation with 3 pci of each tracer. Cells were incubated for 30 min in glass tubes in a 37 C water bath with 0.15 ml of Medium 199 (Li+-free) in the absence or presence of 100 nm GnRH. A (control), inset AI, 150,000 dpm mainly in 32P-labeled nucleotides with resolution of several inositol polyphosphates, e.g. Ins-4-P, IPS, and IPS. A2 and B, 87,000 dpm (control) and 206,000 dpm (+GnRH), respectively, in [3H]inositol polyphosphates (excluding inositol, which was 1,640,000 dpm in both samples). Identification of GPI, Ins-1-P, Ins-1,4-P2, and Ins-1,4,5-P3 was based on their coelution with authentic tritiated standards, whereas the designations of Ins- 1,3,4-P3, IP,, IPS, and IPS were based on their relative retention times compared to known (3H]inositol polyphosphate and nucleotide (A2w) standards and on the estimated phosphate content derived from 32P/3H double-label analyses (see text).

3 1168 GnRH and Inositol Phosphate Metabolism SO 40 SO 60 SAX-HPLC Elution Time (mi) FIG. 2. Double-label and ultraviolet absorbance detection of SAX-HPLC-resolved [SH]inositol and [SsP]phosphate metabolites from primary cultures of rat pituitary cells stimulated by GnRH in the presence of Li+. The sample comprised 5 million mixed pituitary cells prelabeled for 3 days with 5 pci of myo- [3H]inositol plus 10 pci of [33P]phosphoric acid in "inositol/phosphate-free" Medium 199. Washed cells were resuspended in medium containing 10 mm LiCl and exposed to 100 nm GnRH for 30 min at 37 "C. The lower panel shows the distribution of 2.6 million dpm in free [3H]inositol at 3.5 min, followed by 1.1 million dpm in [3H] inositol phosphates. Among the latter, Ins-1-P, Ins-4-P, Ins-1,4-P2, and Ins-1,4,5-P3 (later of two IP3 peaks) were identified by coelution with authentic standards and quantitated, respectively, at 25.5,49.3, 20.9, and 0.53% of total phosphoinositol disintegrations/minute. Ins- 1,3,4-P3 (0.67%) (first IP3), IP, (0.22%) (doublet), IP, (0.71%), and IPS (0.45%) were identified on the basis of their elution times in relation to known standards (e.g. ATP), their lack of UV absorbance, and their 33P/3H ratios compared (by linear regression analysis) to those measured in the corresponding cellular deacylated phosphoinositides (see text). Insets in the two lower panels depict a 40-fold scale expansion of sample peaks barely visible immediately below. pensated by the decrease in catabolism of Ins-4-P due to Li'. GnRH alone produced only a 34% increase in Ins-1-P accumulation (Fig. l), whereas the inclusion of Li', with or without GnRH (experiment of Fig. 2), enhanced Ins-1-P and Ins-4-P to a similar degree. This indicated that basal, relatively hormone-insensitive phospholipase C-mediated hydrolysis of PI was continuously producing Ins-1-P, that GnRH stimulated selective production of Ins-4-P, and that Li' inhibited the dephosphorylation of both inositol monophosphates. The accumulation of Ins-1,4-P2 was stimulated 7.5-fold by GnRH alone (Fig. 1) compared to a 27-fold stimulation by GnRH in the presence ofli' (Fig. 2, Li' controls not shown). This indicated that the lithium-induced accumulation of Ins-1-P and/or Ins-4-P may have contributed to product inhibition of Ins-1,4-P2 catabolism. The impressive accumulation of IP2 in either case points to substantial PIP hydrolysis and/or IP, catabolism during the gonadotroph response to GnRH. Calculation of the specific activity and mass of individual inositol phosphates could be based on absorbance integration data for ATP and on measurements of 33P and 3H radioactivity which, at isotopic equilibrium, are directly proportional to actual mass levels. It was confirmed experimentally that gonadotrophs cultured and radiolabeled for 3 days under the conditions employedwere at isotopic equilibrium, and the constancy of the 33P/3H ratio under our test conditions was verified, e.g. 33P/3H for PIP2 from the experiment of Fig. 1 was f (mean f S.E.) at zero time and f after 30 min with GnRH. Integration data obtained from the sample chromatograms in Fig. 2 and empirical determinations of ATP standard absorbances under the same HPLC conditions permitted calculation of the sample ["PI ATP specific activity as 12,870 dpm/pmol. PIP, from the same sample was analyzed as GPIP, by double-label SAX- HPLC and assumed to have the same 33P specific activity (23). The 10,810 dpm of 33P in GPIPz therefore corresponded to 0.84 pmol/5 million cells. The constancy of the "P/'H ratio permitted calculation of the [3H]inositol specific activity in GPIP, as 17,260 dpm/pmol, which represents approximately a 2-fold dilution of the original [3H]inositol radiolabel in unlabeled intracellular inositol. The above calculations were extended to determine the masses of other phosphoinositides and inositol phosphates from SAX-HPLC chromatograms of the sample in Fig. 2. Thus, the specific activity of the total phosphoinositide pool was 518,000 dpm/30 pmol of lipid/l million cells, distributed as PI (97.8%), PIP (1.4%), and PIP, (0.8%). The stimulated levels of certain phosphoinositols in Fig. 2 were estimated, in pmol/million cells, at 3.0 for Ins-1-P, 5.8 for Ins-4- P, 2.5 for Ins-1,4-P2, and for Ins-1,4,5-P3. A minimum estimate for the concentration of Ins-1,4,5-P3 in the stimulated cells of Fig. 2 (assuming uniform distribution in the cell volume) was calculated to be 56 nm based on values of 1,071 dpm of [3H]inosito1/0.062 pmol/l million cells, having an average diameter of14.1pm (16) and a volume of 1.1 pl/ million cells. Tissue contents of the inositol monophosphates may vary considerably (24), but the level of IP, in stimulated gonadotrophs is similar to that in stimulated platelets (23). The chief value in studying cells labeled to equilibrium has been the use of radioactivity changes to approximate the magnitude of actual mass changes during metabolism of specific phosphoinositols. The specificity and temporal sequence of changes in levels of individual inositol phosphates were assessed by stimulation of gonadotrophs in timed incubations with GnRH, followed at 15 min by addition of a GnRH antagonist (Fig. 3). GnRH produced a prominent, initial rise in Ins-1,4,5-P3 at 15 s, followed by progressive and antagonistreversible increases in Ins-1,4,5-P3, Ins-1,4-P2, and Ins-4-P. Note that the antagonist reversal was most rapid for Ins- 1,4,5-P3, slower for Ins-1,4-Pz, and slowest for Ins-4-P, suggestive of a catabolic relationship. No significant changes were observed for GPI, Ins-1-P (the putative product of PI hydrolysis), or Ins-1,3,4-P3. The rapid rise and sustained elevation of Ins-1,4,5-P3 caused by GnRH was consistently observed, but the biphasic profile seen in Fig. 3 was not always evident. Complete separation of IP, isomers during HPLC analysis (Fig. 2) excluded the possibility that the later rise simply represented Ins-1,3,4- P3 accumulation, although a secondary production of Ins- 1,4,5-P3 (or an unknown isomer which coelutes with it) from higher inositol phosphates remains a possibility. Since PIP, is a known precursor of Ins-1,4,5-P3, the corresponding parent phosphoinositides were analyzed by SAX-HPLC as their deacylated glycerophosphoinositols to determine whether concomitant changes had occurred. As shown in Fig.4, PIP, exhibited a monophasic decrease in radioactivity (and mass) between 15 s and 5 min with a loss of radioactivity of 1200 dpm from PIP, which approximated the rise of 1530 dpm in Ins-1,4,5-P3 at 15 s. At later times, the elevation of radioactivity in [3H]inositol phosphates (Fig. 3) was not matched by equivalent losses from the phosphoinositides (Fig. 4), presum- ably due to rapid resynthesis of the latter from available precursors. The transient initial drop in PIP, and the relative constancy of PI have similarly been observed in thyrotrepin-

4 GnRH Metabolism and Phosphate Inositol 1169 * II, &-"-- 0 IO 20 x Incubation I60 30 FIG. 3. Extended time course of GnRH-induced changes in Ins-1,4,S-Ps, In~-l,4-Pa, and Ins-4-P in rat pituitary gonadotrophs and their reversibility by a GnRH antagonist. Cell prelabelingandanalysis of the [3H]inositolpolyphosphates were performed as described under "Materials and Methods." Test incubations were in 0.15 ml of Medium 199 (Li+-free)(O), 100 nm native GnRH alone (O), or the latter with addition of 10 p~ GnRH antagonist at 15 min (m). No treatment-related changes occurred in GPI or Ins-1-P, which were all well resolved (Fig. 1). All compounds were f r; """_ 5ilp:z:"""il I l l * I- """-~-""""""" O-0 6 io 1 5 io 25 io Incubation Time (mid FIG. 4. Time course of changes in levels of the phosphoinositides PI, PIP, and PIP2 extracted from [SH]inositol-prelabeled rat pituitary gonadotrophs following exposure to GnRH and a GnRH antagonist. Elutriated gonadotrophs were prelabeled during 3 days of incubation with 3 pci of my~-[~h]inositol/l million cells. Test incubations were in 0.15 ml of Medium 199 (Li+-free)(O), 100 nm native GnRH alone (O), or the latter with addition of 10 PM GnRH antagonist at 15 min (m). Lipids corresponding to the samples in Fig. 3 were extracted and deacylated and then analyzed by SAX- HPLC as described under "Materials and Methods." All compounds were identified by coelution with authentic, radiolabeled standards. The star indicates significant decreases at 15 s and 1 min in PIP2 below control and GnRH antagonist levels (dashed Eines). PIP levels were slightly elevated at times beyond 5 min. GPZP, glycerophosphoinositol4-phosphate. identified by coelution with authentic, radiolabeled isomer standards on SAX-HPLC and WAX-HPLC. We have also observed an early eluting IP3, presumably Ins- 1,3,4-P3, coinciding with ATP but eluting 3-4 min before Insreleasing hormone-stimulated GH3 pituitary cells labeled to 1,4,5-P3 and GTP. However, the low levels of Ins-1,3,4-P3 and equilibrium with 32P or [3H]inositol, in which PIP also exhib- its lack of accumulation during GnRH stimulation may indiited a smaller initial decline (25, 26). cate that it is formed in low abundance from IP4, or simply To examine the origin of Ins-4-P from higher inositol that it is catabolized very rapidly. phosphates, we analyzed the dephosphorylation products of authentic [3H]Ins-1,4,5-P3 in saponin-permeabilized pituitary DISCUSSION cells (Fig. 5). The notable finding was that Ins-4-P was The identification of Ins-4-P as the principal metabolite of virtually the only monophosphate product of exogenous IPS IPS in gonadotrophs and the finding that it accumulated a to catabolism, with barely a trace of the expected metabolite, much greater extent than Ins-1-P in response to GnRH Ins-1-P. Control samples employing nonpermeabilized cells represent new evidence that PIP and PIPz rather than PI or including CdC1, with permeabilized cells showed no metab- (which has only a 1-phosphate ester linkage) remain the olism of the exogenous [3H]Ins-1,4,5-P3. It is noteworthy that the inclusion of Li' in this sample did block not IP3 conversion to Ins-4-P but did partially inhibit the dephosphorylation of Ins-4-P to inositol. The higher IPS which were characterized by their relative phosphate contents (and the possible existence of isomers in the double peak for IP,) were also shown to be components principal targets of hormone-activated phospholipase C during stimulation by GnRH. This conclusion is strongly supported by the specificity and temporal relationships of GnRHstimulated and GnRH antagonist-reversible changes in Ins- 1,4,5-P3, Ins-1,4-Pz, and Ins-4-P, as distinguished from Ins-l- P and glycerophosphoinositol. The unexpected ability of Li' to enhance the accumulation of both Ins-4-P and Ins-1-P may of the response to GnRH. Thus, IP4, IPS, and/or IP6 all account for the apparent product inhibition of Ins-1,4-Pz displayed a similar rapid elevation as Ins-1,4,5-P3 within 15 s degradation as well. The selective accumulation of Ins-4-P of GnRH addition, in contrast to the progressively delayed over Ins-1-P appears to represent a unique product marker responses of IPz and total IP, (Fig. 6). The polyphosphate IP4 for either 1-phosphatase action on Ins-l,4-Pz (major interwas invariably the least abundant IP in gonadotrophs, but mediate) or 5-phosphatase action on Ins-4,5-Pz (minor), both showed the greatest relative increase in one experiment (Fig. 6). The elevations of IP4, IPS, and IP, to higher steady-state levels over min were observed in three experiments. of which are derived from Ins-1,4,5-P3 (22). The existence of Ins-4-P in brain, kidney, testis, and platelets has only recently been reported (24, 27), although no selective stimulation of

5 1170 GnRH and Inositol Phosphate Metabolism r I : s p3 1ETCIBOL I TE Ins. I P2/3 io so eo WAX-HPLC ELUTION TIME (mid FIG. 5. Weak anion-exchange analysis of inositol monophosphate(s) produced during incubation of authentic [3H]Ins- 1,4,5-& with saponin-permeabilized rat anterior pituitary cells. Aliquots of 5 million cells in 0.1 ml of Medium 199 (ph 7.3) containing 200 pg/ml saponin and 10 mm LiCl were incubated at 37 C for 15 min prior to the addition of 50,000 dpm of [3H]Ins-1,4,5-P3(New England Nuclear). After a further 60 min of incubation, the extracted, neutral aqueous layer was analyzed on an Adsorbosphere (5-pm) NH, column eluted with a gradient of aqueous ammonium phosphate as described under Materials and Methods. Elution positions for [3H]Ins-l-P(New England Nuclear) and [3H]Ins-4-P were determined both as external standards in adjacent runs and as internal standards in replicate sample runs. Note that the presence of Li+ did not block dephosphorylation of the IPz intermediate. but that the absence of Li+ (not shown) permitted -10% greater conversion of the Ins-4-P product to inositol. a I I Incubation Time (sec) FIG. 6. Early time course of GnRH-induced changes in [3H] inositol polyphosphate production and metabolism in gonadotrop-enriched rat anterior pituitary cells. Elutriated cells consisting of 40% gonadotrophs were prelabeled during 3 days of culture on Cytodex-1 beads in serum-containing inositol-free Medium 199 with 4 pci of my0-[~h]inositol/l.5 million cells. Aliquots of the washed cell suspension were then incubated for 0, 15, or 60 s in a 37 C water bath with 0.15 ml of Medium 199 containing 10 mm LiCl Ins-4-P over Ins-1-P was apparent in these studies, and its origin as a product of IP, or IP, dephosphorylation was inferred. The time course of changes in individual IPS in GnRHtreated gonadotrophs suggests that Ins-1,4,5-P3 release represents both a primary and sustained response to hormonal stimulation, initiated by an early monophasic decrease in PIP2. The possible occurrence of different phases of IP, generation and additional sources of IP3 such as higher inositol phosphates require further investigation. The ongoing release of Ins-1,4,5-P3 together with that of arachidonic acid (8) may contribute to the mobilization of intracellular Ca2+ in gonadotrophs (28, 29), thus initiating luteinizing hormone release (30). Although there is an initial lag time for IP, production from IP3 and significant early hydrolysis of PIP was not detectable, a secondary contribution from PIP hydrolysis may be responsible for the generation of comparatively greater amounts of IP, and diacylglycerol, the latter serving to sustain protein kinase C activation and luteinizing hormone release (14, 31). Double-labeling studies of inositol phosphates with 32P/3H have not been reported previously because of the difficulty in resolving these components from 32P-nucleotides. The HPLC methodology employed here, combined with computer-assisted two-channel radioactivity counting and absorbance detection, permitted improved resoluton of these species and unequivocal identification of higher inositol phosphates. In the absence of any evidence in our double-label analyses for alone (0), 10 mm LiCl plus 100 nm GnRH (a), 10 mm LiCl plus 10 p~ GnRH antagonist (U), or LiCl plus GnRH plus GnRH antagonist (m). Reactions were terminated, processed, and analyzed by SAXscribed under Materials and Methods. Results are expressed in disintegrations/minute as the mean -+ S.E. of three replicates. The responses to GnRH in Ins-1,4,5-P3, IP,, IPS, and IPB were significant (denoted by stars) at 15 s (p < 0.05, by two-tailed t test against timematched controls) and 60 s (p < 0.01), as was the 60-s increase in Ins-l,4-Pn (p e 0.001). No treatment-related changes occurred in GPI, Ins-1,3,4-P3, or total IPI within 60 s.

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