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PYRULARIA THIONIN INCREASES ARACHIDONATE

LIBERATION AND PROLACTIN AND GROWTH HORMONE RELEASE FROM ANTERIOR PITUITARY CELLS ALLAN M. Junn,'

Lao P. Va~txoN~ and ROBERT M. MACLaon'

rDepartmcnt of Internal Medicine, University of Virginia Health Sciences Centet, Charlottesville, VA 22908; and ~Departmcnt of chemistry, Brigham Young University, Provo, UT 84602, U.S .A . (Rccdved

18

March

1992; accepted 31 July 1992)

A. M. JUDD, L. P. VaRNON and R. M. MACLEOD. Pyrularia thionin increases arachidonate liberation and prolactin and growth hormone release from anterior pituitary cells. Toxicon 30, 1563-1573, 1992 .-Pyrularia thionin is a 47 amino acid peptide isolated from the nuts of Pyrularia pubes. This peptide does not have intrinsic phospholipase AZ activity, but it increases the liberation of arachidonate from several tissues. Exposure of anterior pituitary cells to this toxin increases the liberation of arachidonate, increases the cellular levels of lysophospholipids, and decreases cellular phospholipids. Thus, phospholipase AZ is involved in the liberation of arachidonate stimulated by this peptide. Because this toxin also increases stearate liberation from the pituitary cells, either diacylglycerol lipase, phospholipase A, or lysophospholipase may be directly or indirectly activated by this toxin. In addition to increasing fatty acid liberation, Pyrttlaria thionin increases the release of prolactin and growth hormone from anterior pituitary cells over the identical concentration ranges that this toxin liberates the fatty acids. Pyrularia thionin increased arachidonate liberation and prolactin release from perifused pituitary cells within 2 min, and following withdrawal of the toxin, arachidonate liberation and prolactin release returned to near basal levels within 6 min. Dopamine, a physiological inhibitor of prolactin release that closes calcium channels, decreased prolactin release stimulated by Pyrularia thionin. However, dopamine had no effect on the arachidonate liberation stimulated by this peptide. Similarly, D-600, an organic calcium channel blocker, decreased the prolactin and growth hormone release stimulated by the toxin without affecting the toxin-stimulated arachidonate liberation . Therefore, Pyrularia thionin increases arachidonate liberation through the rapid activation of phospholipase A2 by a mechanism that is not dependent on calcium uptake via D-600-inhabitable calcium channels . In contrast, the prolactin and growth hormone release stimulated by this toxin requires calcium uptake via D-600 inhabitable calcium channels . INTRODUCITON

Pyrularia thionin (P. thionin) is a 47 amino acid peptide isolated from the nuts of Pyrularta pubes (VaRrroN et al., 198 . This basic peptide binds to sites on cell membranes and has hemolytic, cytotoxic, and neurotoxic properties (Evsrr et al., 1986 ; OsoRIO E 1563

1564

A. M . JUDD et aJ.

C~sTtto et al., 1989 ; OsoRlo E C~sTxo and VHtNON, 1989). The biological activity of P. thionin is mediated at least in part by the activation of phospholipases that liberate fatty acids from cellular lipids . In particular, this peptide releases arachidonate from cellular phospholipids through the activation of phospholipase Az (ANG>~ixoi:FEtt et al., 1990). The peptide also increases intracellular free calcium concentration by increasing calcium uptake into the cell (EVANS et al., 1989). Although the effects of P. thionin on cellular function have been studied in several cell types, its effects on the release of peptides from secretory cells are unknown. Arachidonate and arachidonate metabolites have profound effects on the release of peptide hormones. In the anterior pituitary, this fatty acid and its metabolites stimulate the release of prolactin (PRL) (GRANDISOIV, 1984; CANOHICO et al., 1985 ; CAMORATlb and GRANDISON, 1985 ; IC»~. et al., 1987 ; Juan et al., 1988) and growth hormone (GH) (June et al., 1985). Furthermore, exogenous phospholipase AZ or pharmacological activators of this enzyme increase prolactin release (CANONI00 et al., 1983, 1985 ; GRANDISON, 1984 ; CAIKOxATro and GRANDISON, 1985 ; OFnucIII et al., 1990). Therefore, we initiated studies to determine whether P. thionin is a phospholipase A2 activator in pituitary tissue and whether this toxin stimulates the release of PRL and GH from pituitary cells. MATERIALS AND METHODS Pluumacologicol agents Thionin was purified from the nuts of Pyrularia pubes by ammonium sulfate fractionation followed by cation~xchange chromatography (VSertox et al., 1985) . The P . thionin was dissolved in water at 2 mg/ml and stored in aliquots at -20°C until utilized for an experiment . On the day of an experiment, the P . thionin stock solutions were diluted with incubation medium shortly before the start of the experiment. Dopamine, purchased from Sigma (St. Louis, MO, U .S.A .), was dissolved in incubation medium immediately before the start of an experiment to minimize oxidation of this monoamine . D-600 (methoxyverapamil), a gift from Knoll AG (Ludwigshaven, F .R .G.), was dissolved directly into the incubation medium by gently stirring the incubation medium and D-600 mixture with a magnetic stirrer for 3 hr. Preparation ojanterior pituitary cells Anterior pituitary ghmda from female Sprague-Dawley rats (225-250 g, Domion Labs, Dublin, VA, U.S .A.) were dispersed as described previously (Cnnor~nco et al., 1985) . This involved decapitating the rats, removing the pituitary, separating the anterior pituitary from the posterior pituitary, and then placing the anterior pituitary in RPMI-1640 medium (Gibco, Grand Island, NY, U .S.A.) supplemented with serum and antibiotics. Under sterile conditions, the anterior pituitaries were diced with a scalpel blade and the fragments rinsed with emus-frce RPMI . The fragments were then exposed to trypsin (2 mg/ml in serum-free RPMn (Worthington Hi Mhe , ^:..oi Corporation, Freehold, NJ, U .S.A.) for 30 min at 37°C . After centrifugation (200 x a, 5 min), the fragments wero resuspended in grade II pancreatin (2 .5 mg/ml is scrum-free RPMn (Sigma), incubated at 37°C for 10 min, and again centrifuged. The enzyme solution was then decanted, the fragments resuspended in suspension minimum essential medium (SMEM, Giboo) containing 10°h horse serum, and the fragments incubated at 23°C for 30 min . The fragments were again centrifuged, resuapended in scrum-free SMEM, and dispersed into a monocellular auspmsion using gentle titration. The sells wen then either transferrod (400,000/well) to 24-well culture dishes (static incubation experiments) containing 1 .5 ml RPMI supplemented with horse serum, fetal calf serum and antibiotics, or tn~nsferred to 75 cm= t-fiaaka (10 million oell/8ask) and cultured with 30 ml of serum supplemented RPMI (perifusion experiments) . Cells wen incubated (37°C) for 72-96 hr under an atmosphere of 95% air.5%

Mearracnrcnt ojphospholipidr and lysophospholiplds Primary cultures of anterior pituitary cells wen prepared as explained above. Following 4 days in culture, the RPMI-1640 medium was removed from the cells and replaced with 500 irl of medium containing 2 ieCi L-myo-[2,3-~H]inositol (37 Ci/mmole, New England Nordcar, Boston, MA, U .S.A.) or (methyl-'H] choline chloride (90 Ci/mmole, New England Nuclear) . The medium utilized during this 4S hr labeling period was inositol-froe Ham's F-10 supplemented with 2 .5% fetal bovine serum and 7.5°/, horse senmi. This serum had been extensively dialyzed against inoaitol-free Ham's F-10 to reduce its choline and inasitol content (Jeevrs et al.,

Pyrularia Thionin

Stimulates Pituitary Cells

1565

1988). On the day of the experiment, the labeling medium was removed and rephuxd with 1 ml RPMI-1640 medium containing 0.2% bovine serum albumin (BSA, 3igma). This medium was then removed and replaced with 1 ml RPMI-1640 medium containing 0.2°~ BSA and either vehicle (2 Kl distilled water) or S ltg thionin toxin. The cells were then incubated for 2 min, the medium removed, and the cells exposed to 500pl 0°C methanol:chloroform (2 :1 v/v). The cells were then scraped from the cell culture plastic, and the organk solvent and cell fragments were transferred to a glass 12 x 75 mm test tube and stored at -20°C until the phosphoGpids were analysed by thin layer chromatography (TL.C). On the day of analysis 100 p10.9% KCl was added to each glass test tube that contained the cellular extract. The test tubes were vortexed for 30 sec, and the organic and aqueous layers separated by centrifugation at 500 x!t for 10 min. The organic layer was removed from the test tube with a Pasteur pipette, transferred to another 12 x 75 mm glass test tube, and the organic solvents evaporated with a stream of nitrogen gas. The lipid residue was then dissolved in 100 ul chloroform containing 30 pg phospholipid standard, i.e . either phosphatidylcholine and lysophosphatidykholine, or phoaphatidylinositol and lysophoaphatidylinositol . The cell extract and lipid standards were then spotted onto an oxalate coated silica ge160 TLC plate (Mark, Darmstadt, F.R .G .) and the plate developed to a height of 17 cm in a solvent of chloroform :methanol:acetic acid :water (50:37.5 :3 .5 :2 v/v/ v/v). The plate was then exposed to iodine vapors to visualise the lipid spots. Following decolorization of the spots, the silica gel from each spot was scraped from the plates and the radioactivity in each spot determined by standard scintillation techniques. Measurement of arachidonate Ilberatiort in static incubation

On the day of the experiment, the culture medium was removed, and the cells were incubated in 0.5 ml serum-free RPMI containing 0.25 pCi/ml ['H]arachidonate (180-240 Ci/mmok: New England Nuclear) . The cells were then incubated at 37°C for 1.5-2 hr, the medium removed, and the cells rinsed three times with 1 ml 0.2% HSA-RPMI. Following the final rinse, the cells were exposed for 10 min to 0.2% BSA-RPMI containing vehicle (medium), D-600, or dopamine. The cells then wen exposed for 30 min to 1 ml 0.2% HSA-RPMI (control) or 0.2% HSA-RPMI containing P. thionin, with or without dopamine or D-600. The incubation medium was then removed, a 100pl aliquot of the medium utaized in the radioimmunoassay for PRL, and the radioactivity in the remaining medium determined using liquid acintillometry. Measurement of stearate and arac~idonatt liberation in static 6~cubatiore

Anterior pituitary cells were dispersed and phuxd into 24well plates as explained in the previous extions. Twenty-four hours before the start of an experiment, the medium was removed from the wells and replaced with 1.0 ml sterile RPMI containing 0.2% BSA, 1 uCi/ml ['i']stearate, and 0.5°A pCi ['Ii]arechidonate/ml. The cells were then incubated for 24 hr in a 95% sir:5% CO, atmosphere . The ells were then rinsed and the experiments carried out as explained for the arachidonate release experiments in static incubation. The ['Hj and ['~cJ content of the incubation medium was determined by standard dual channel counting techniques . Measwement of arachidonate liberation in perifusedpituitary celLr

Perifused anterior pituitary cells were utiliad to determine the dynamics of P. thionin-stimulated arachidonate liberation . On the day of the experiment, the cells were harvested from the t-basks by a mild exposure to trypsin (Ross et aJ., 1988). The cells were then resuapended in a serum-free balanced salt solution (Ross et al., 1988) and incubated for 1.5-2 hr with 1 pCi/ml ['H]arachidonate. The cells were mixed with preswolkn BioGel P-2 1plY~Y~~ B~ (Bio-Rad Laboratories, Richmond, CA, U.S .A .), transferred to a perifusioa chamber (Ross et al., 1988), and perifused (0.55 ml/min) for 45 min with a modified balanced salt solution (MBSS) containing 0.2% BSA. After this rinse period, five 2-min fractions of chambar eluate were collected and the cells exposed to vehicle or P. thionin in MHSS for 20 min. The cells then were returned to tl~ original medium for a final 18 min. Two-minute fractions of all column eluate were collected throughout the experimental period . Column eluate fractions were split with 100Kl being used for the radioimmunoassay of prolactin and 1 ml used for the determination of radioactivity in the sample. In previous experiments it has ban determined that over 90% of the ('Hj counU in the medium are associated with arachidonate (June et aJ., 1988). Fractional araehidonate e®ux was cakulated as explained previously (Roes et al., 1988). At the termination of the experiment, the total [rH] label incorporated into the cells was measured by vortexing the all-Hio-Gel mixture in 1°k Triton x-100 (Sigma). The all fragments were incubated in this solution overnight at 4°C and the liquid separated from the Bio-Gel by centrifugation at 800 x a for 5min. The supernatant was then removed from the Bio-Gd and the radioactive content of the supernatant determined by scintillation counting . Prolactin radioitnnparoavay

The PRL content of the incubation and perifusion medium was determined with a standard double antibody assay for PRL. The reagents for the sassy ware kindly provided by the NIDDK Rat Pituitary Hormone

1566

A. M. JUDD et al.

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Pyrularia THIONIN ON PH06PHOLIPID AND LY30PHOSPHOLIPID LEVELS IN ANTERIOR PTfUITARY CELL3 IN PRIMARY CULTURE .

Primary cultures of anterior pituitary cells were labeled with [3Hlcholine or [3H]inositol and the lipids separated as described in the Methods section of this study. Pyrularia thionin significantly (P < 0.05) decreased phosphatidylinositol (PI) and phosphatidylcholine (PC) levels and increased lysophosphatidylcholine and lysophosphatidylinositol (LPI) levels . The incubation period was 2 min. Distribution Program. Results are expressed in terms of NIDDK rat PRL RP-3 standard (intraassay variability, < 0.06% ; interassay variability, < 10%) . Statistical analysis In the static incubation experiments, data are expressed as ng PRL/well and cpm [Î-Ilarachidonate/well . In the perifusion experiments, data are expressed as ng PRL/min/10' cells and [~Ii]arachidonate efflux/min . Data were analysed with a one-way analysis of variance and the Bonferroni analysis for multiple comparisons (WALLENS1FrN et al., 1980). A value of P < 0.05 was considered significant. RESULTS

Pyrularia thionin should increase the cellular levels of lysophospholipids and decrease cellular phospholipids if P. thiotoxin is a phospholipase AZ activator in pituitary cells. In primary cultures of anterior pituitary cells P. thionin (2 ~g/ml) increased lysophosphatidylcholine and lysophosphatidylinositol levels and decreased the levels of phosphatidylinositol (Fig. 1). This peptide had no effect on inositol phosphate production by pituitary cells at this same concentration (control, 8911 ± [ 17 cpm total inositol phosphate per well; 2 hg P. thionin/ml, 9299 ± 317 cpm total inositol phosphate per well).

Pyrubra Thionin Stimulates Pituitary Celle 3500

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FYo . 2. TILE >~t+ecls of Pyrularia TEnoNna oN FROLACInv (PRL) AND oaowTll IIORr"oNE (GH) IIlif P"e7 AND ARACI~ONATE (AA) AND STÉARATE (SA) LIBFFRATION FROM PAI!(ARY CULTURES OF RAT ANTERIOR PrIVirAAY Cl~.L4 .

Anterior pituitary cultures that had previously been incubated with [3H] arachidonate and f ~C]stearate for 24 hr were exposed to the Pyrularia thionin for 15 min and the radioactivity in the incubation medium determined by standard scintillation techniques. The medium GH and PRL content were determined by radioimmunoassays . Pyrularia thionin of 0.5 pg/ml and greater increased (P < 0.05) arachidonate and stearate liberation, and 1.0 i+g/ml and greater increased (P < 0.01) PRL and GH release .

Therefore, P. thionin increased the phosopholipase A2 activity of pituitary cells without affecting the activity of the phospholipase C that hydrolyzes phosphatidylinositol. The activation of phospholipase AZ in the pituitary may be accompanied by the activation of phospholipase A, or lysophospholipase (SPANGI~.O et al., 1991) . Arachidonate is predominantly esterified to the 2 position of the diacylglycerol backbone of phospholipids. In contrast, stearate is predominantly esterified to the 1 position . Therefore, phospholipase AZ which cleaves fatty acid from the 2 position releases predominantly arachidonate, whereas phospholipase A, which cleaves fatty acids from the 1 position releases primarily stearate . Lysophospholipase cleaves stearate from the 1 position of lysophospholipids. In Fig. 2, P . thionin increased both arachidonate and stearate liberation in a concentration-related manner; it also increased the release of PRL and GH from the anterior pituitary cells. Pyrularia thionin had no effect on cell morphology at concentrations less than 20 hg/ml; however, 20 pg/ml modified cellular morphology in that the cells lost their projections and took on a more rounded appearance. Furthermore, the pituitary cells exposed to this high concentration of toxin did not adhere tightly to the cell culture plastic. The primary cultures of anterior pituitary cells utilized in these experiments responded to thyrotropin-releasing hormone (TRi~ (control 950 f 41 ng prolactin/well; 100 mM TRH, 2372 f 92 ng prolactin/well) and growth hormone releasing hormone (GRH) (control 300 f 32 ng growth hormone/well; 10 nM GRH, 850 f 65 ng growth hormone/well) in the manner previously reported (June et al., 1985, 1986, 1988).

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1568

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ON ARACF®ONA78 (AA) LIBERA710N AND PROLACIIId Rmp~~ STAtULA1ED BY Pyrularia i'IDONIN .

(PRL)

Primary cultures of anterior pituitary cells that had been incubated with [ 3Hlalachidonate for 90 min wen incubated with Pyndaria thionin and vehicle (control) or dopamine (DA) for 15 min and the [ 3Iilarachidonate and PRL content of the incubation medium determined . Pyrularia thionin at 1 and 5 pg/ml increased (P < 0 .01) the prolactin (PRL) and arachidonate (AA) content of the incubation medium . Dopamine decreased (P < 0 .05) the prolactin please stimulated by the toxin, but had no effect on the toxin-stimulated arachidonate liberation.

PRL release from the anterior pituitary is inhibited by the monamine dopamine (Lw1~sRTS and MwcLEOD, 1990) . Therefore, the effects of dopamine on P. thioninstimulated PRL release and arachidonate liberation were investigated . Dopamine decreased basal and P. thionin-stimulated PRL release without altering P. thioninstimulated arachidonate liberation (Fig. 3). At higher concentrations of P. thionin (5 ~g/ml), the dopamine inhibition of prolactin release was less pronounced than at lower concentrations . Calcuim has a key role in prolactin release and phospholipase A2 activation. Therefore, the effects of D-600, a potent calcium channel blocker (Login et al., 1985), on P. thioninstimulated arachidonate liberation and prolactin release were determined. D-600 had no effect on P. thionin-stimulated arachidonate liberation, but this agent inhibited the PRL and GH release stimulated by the toxin (Fig. 4). Pyrularia thionin produced slow effects (20 min) on arachidonate liberation in NIH 3T3 fibroblasts (ANGERHOFFF1t et al., 1990); however, these effects were rapid in other tissues (Evwxs et al., 1989). Therefore, the dynamics of P. thionin-stimulated arachidonate liberation and PRL release were determined with perifused anterior pituitary cells. Pyrularia thionin at both 1 and 5 ~g/ml increased arachidonate liberation and PRL release within 2 min (Fig. 5). The arachidonate liberation and PRL release reached a plateau within 4 min and the PRL release and arachidonate liberation was maintained at this level throughout the 20 min exposure to the toxin . Following withdrawal of the toxin, arachi-

Pyrutarta Thionin Stimulates Pituitary Cells

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A CALCIUèI CHATINHI. BLOCYEA, ON PynlIQrlo l'1flONIN-STIMITLAlED GROWTH FiORNONE (GI-n Ra, u~et ANp AAACHIDONATE (pA) L®ERATION FAON PAMARY CULTURES OF ANTBigOA PrIVITAAY CBLLS .

EPFF.~,TS OF

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D~00 decmasod (P < 0.01) basal and toxin-stimulated PRL and GH release, but had no effect on basal or toxin-stimulated arachidonate liberation . The incubation period of these experiments was 15 min.

donate liberation returned to basal levels within 4 min. PRL release decreased following withdrawal of the toxin, but did not attain basal levels over the time course of the experiment . DISCUSSION

Many secretagogtles that stimulate PRL release from the anterior pituitary also increase the liberation of arachidonate from the phospholipids of the pituitary cells. In contrast, dopamine and somatostatin inhibit secretagogue-stimulated arachidonate liberation and PRL release (JuDD et al., 1986 ; Ross et al., 1988 ; CANONICO, 1989 ; Ol~nucl~II et al., 1990). Similarly, growth hormone releasing hormone stimulates GH release and arachidonate liberation and somatostatin blocks these effects (JUDD et al., 1985). Exposing pituitary cells to exogenous arachidonate, arachidonate metabolites, phospholipase A2, or agents that activate the endogenous phospholipase A2 increases PRL and GH release. In contrast, pharmacological agents that decrease arachidonate liberation or the metabolism of arachidonate, decrease PRL and GH release from pituitary cells. Therefore, arachidonate may have a role in regulating PRL and GH release from anterior pituitary cells (CANONICO et al., 1983, 1985 ; GRANDISON, 1984; JuDD et al., 1986). It is of interest that the phospholipase AZ hydrolysis of phospholipids produces free arachidonate and lysophospholipids. Lysophospholipids are also potent stimulators of PRL and GH release (JUDD and KUAN, 1990), and thus these molecules may have some role in regulating the release of these hormones from the pituitary.

1570

A. M. JUDD et al.

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FIG. S. Pj'!'ujal(a TFDONIN REVER~BLY INCRâA .~4 PROLACITN (PRL) Rrru " ~ AND ARACInuoNATe (AA) LIHFRATION FROAf PERIFIJS® ANTERIOR PITUITARY CELL3.

Pyrularia thionin increases arachidonate liberation from several cell types; in the anterior pituitary, the liberation of arachidonate is accompanied by the production of lysophospholipids and a decrease in the levels of phosphatidylcholine and phosphatidylinositol. Therefore, P. thionin is stimulating the phospholipase AZ hydrolysis of phospholipids . The resulting increase in free arachidonate and lysophospholipids may be responsible for the P. thionin stimulation of PRL and GH release. Phospholipase AZ activity in the pituitary may be accompanied by phospholipase A, or lysophospholipase activity (SPANGELO et al., 1991). Stearate is the primary fatty acid esterified to position 1 of the diacylglycerol backbone of phospholipids. Pyrularia thionin increases stearate liberation over the same concentration range as it increases arachidonate liberation. Therefore, P. thionin may activate phospholipase A the enzyme that hydrolyzes fatty acids from position 1 of the diacylglycerol backbone of phospholipids, or lysophospholipase either directly or indirectly by the production of arachidonate or lysophospholipids . An indirect activation of lysophospholipase by P. thionin-induced increase on lysophospholipids may be the mechanism whereby this toxin caused a decrease in phosphatidylcholine production, but caused only a slight increase in lysophosphatidylcholine production in the present study. Pyrularia thionin may also stimulate diacylglycerol lipase, an enzyme that removes both arachidonate and stearate from diacylglycerol (LAI~~RTS and MACLi~on, 1990). This hypothesis is supported by the P. thionin stimulation of stearate release. For diacylglycerol lipase to play a role in arachidonate liberation, P. thionin must increase the phospholipase C hydrolysis of phospholipids to produce diacylglycerol. Pyrularia thionin has no effect on inositol phosphate production and thus does not stimulate the phospho-

Pyrularia Thionin stimulates Pituitary cous

1 s71

lipase C hydrolysis of phosphatidylinositol . However, it is possible that P, thionin may stimulate the phospholipase C hydrolysis of other phospholipids. The effects of P. thionin toxin on both arachidonate e~ux and PRL release are rapid, and furthermore, are rapidly reversible, because arachidonate efliux returns to basal levels within 4 min, and PRL release reaches near basal levels within 6 min. We cannot determine if the mild continued elevation of PRL release following withdrawal of the toxin represents damage to the cellular integrity or some physiological process activated by the elevated levels of free arachidonate or lysophospholipids . It has been hypothesized for other tissues that P. thionin activates a calcium channel, and the resulting increase in intracellular calcium activates phospholipase Az (EVANS et al., 1989). Dopamine decreases basal calcium uptake into pituitary cells and blunts the increase in calcium uptake stimulated by the calcium channel activators neurotensin, maitotoxin or BAY K8644 (LoGnv et al., 1990a,b) . If P. thionin is directly activating the calcium channel of the pituitary cells, dopamine should inhibit P. thionin-stimulated PRL release and arachidonate liberation if the latter reaction is calcium dependent. However, dopamine decreases P. thionin-stimulated PRL release, but has no effect on arachidonate liberation. Similarly, D-600, a potent pharmacological blocker of calcium channels, inhibits P. thionin-stimulated PRL and GH release, but has no effect on arachidonate liberation . Therefore, in anterior pituitary cells, P. thionin is probably not stimulating arachidonate liberation by activating D-600-inhibitable calcium channels ; however, its effects on PRL and GH release may involve an increase in calcium uptake through these calcium channels . The calcium uptake that is necessary for the stimulation of anterior pituitary hormone release may be activated directly by P. thionin or may be mediated by arachidonate or arachidonate metabolites. Arachidonate increases calcium entry through voltage-dependent calcium channels in GH, cells, a PRL and GH-secreting tumor cell line (VAl',EIER et al., 1989). This fatty acid also increases the intracellular calcium levels of normal anterior pituitary cells (KNEBEL, 1988) and GH3 cells (KOLESNICK et al., 1984). Furthermore, S-hydroxyeicosatetraenoic acid, a metabolite of arachidonate, stimulates PRL release from pituitary cells and this stimulation is blocked by D-600 (KOixE et al., 1985). Although it is unknown how P. thionin stimulates phospholipase AZ in pituitary cells, it may affect some intracellular system that in tum stimulates the phospholipase AZ. In contrast, P. thionin may stimulate the phospholipase A2 directly, affect the G protein that may be associated with some forms of this enzyme (COCKCROFr' et al., 1991), or affect the phospholipid structure in the cellular membrane to increase its susceptibility to phospholipase AZ hydrolysis . The latter mechanism is favored (Evnxs, J. G., Thesis, Brigham Young University, 1990). It is of interest that the low concentrations of P. thionin used in this study do not appear to be cytotoxic. In support of this hypothesis, the effects of the toxin are reversed by dopamine and D-600. Furthermore, the cellular morphology of the pituitary cells is not modified by low concentrations of the toxin and the effects of tl~e toxin are reversible following its withdrawal . Therefore, P. thionin used at low concentrations may serve as a useful tool in studying the intracellular mechanisms governing the control of cellular function . Acknowkdgamnt.~We would like to thank Y~-RoNa Cie and Xuo-Yexc+ Jnv for their excellent technical support. This work was supported by a research great from the National Cancer Institute (CA-07s3s) to R . M . MecLeon and a grant from the Foundation for the Control of Cancer to L . P. VlflarooN.

1572

A. M. JUDD et al. REFERENCES

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Pyrularia Thionin Stimulates Pituitary Cells

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Pyrularia thionin increases arachidonate liberation and prolactin and growth hormone release from anterior pituitary cells.

Pyrularia thionin is a 47 amino acid peptide isolated from the nuts of Pyrularia pubera. This peptide does not have intrinsic phospholipase A2 activit...
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