Bombesin-like peptides stimulate phosphatidylinositol turnover in rat brain slices

Pepttdes, Vol 9, pp 1345-1349 ©PergamonPress plc, 1989 Pnntedm the U S A

0196-9781/88$3 00 + 00

Bombesin-Like Peptides Stimulate Phosphatidylinositol Turnover in Rat Brain Slices V. N. H A R I P R A S A D A N D T E R R Y W. M O O D Y 2

Department of Biochemistry, The George Washington University School of Medwme and Health Sciences Washington, DC 20037 R e c e i v e d 14 M a r c h 1988 HARI PRASAD, V N AND T W MOODY Bombesm-hkepepttdes sttrnulatephosphat:dyhnosttol turnover :n rat brain shces PEPTIDES 9(6) 1345-1349, 1988 --The abdlty of bombesm (BN)-hke peptldes to stimulate phosphatldyhnosltol turnover in rat brain shces was investigated BN (I /xM) slgmficantly stimulated lnosltol-l-phosphate (IP1) but not inositol-4,5-blphosphate (IP2) or mosltol-1,4,5-tnsphosphate (IP3) production using frontal cortex shces m the presence of LICi (7 5 mM), BN had no effect on cAMP or cGMP levels BN and the structurally-related gastnn releasing peptlde (GRP) elevated IPI levels in a dose-dependent manner S~mllarly,nanomolar concentraUons of the GRP fragment (Ac-GRPs°-z~) significantly elevated IP~ levels, whereas mlcromolar concentraaons of the mact~ve GRP1-18 did not BN signLficantly elevated IP~ levels m those brmn regions ennched m BN receptors such as the olfactory bulb, hlppocampus, stnatum, thalamus and frontal cortex, whereas IP1 levels were not stgmficantly increased m areas which have a low density of BN receptors such as the cerebellum, medulla/pons and mldbram These data suggest that CNS BN receptors may utlhze phosphatldyllnositol as a second messenger CNS

Neuropepttdes

Bombesm

GRP

Phosphat~dyhnosltol turnover

BOMBESIN (BN)-hke peptldes are biologically active in the mammalian CNS. Endogenous BN-hke peptides are present m discrete rat brain areas (20, 23, 24) They are released from rat brain shces in depolarizing stimuli (19) and diffuse and activate receptors for BN-like pepttdes which are locahzed m discrete brain areas (18,32). Bombesm and the structurally-related mammahan peptldes, GRP and GRP ls-zr (13,17), are biologically active in the CNS where they cause hypothermla (2), hyperglycemm (3), satiety (25,29), altered gastric a o d secretion (26) and groommg (5, 7, 11, 16). The second messengers which BN utdlzes have only recently been explored. In Swiss 3T3 cells, which have 100,000 BN receptors/cell (30), BN stimulates phosphatldyhnosltol turnover (4,9) The phosphattdyhnosltol-4,5-blphosphate generated by BN may be metabohzed to dmcylglycerol and inos~tol-l,4,5-tnsphosphate. The former agent may activate protein kmase C and BN stimulates phosphorylaUon of an 80,000 dalton protein (31) The latter agent may release calcium from mtracellular pools and BN elevates cytosolic Ca 2+ levels in Swiss 3T3 cells (15) and small cell lung cancer (SCLC) cells (21). In dispersed guinea pig pancreatic aclm, BN stimulates 45Ca efflux and elevates intracellular cGMP levels (11) Also, BN stimulates phosphatldyhnosltol turnover m pancreatic cells (22). These data suggest that peripheral BN receptors may utilize phosphatidyhnositol as a sec-

ond messenger. Here the effects of BN-hke peptldes on mosltol-l-phosphate levels in the CNS were investigated. METHOD Male Sprague-Dawley rats (200-300 g) were decapitated and their brmns rapidly removed and dissected over ice Cross-chopped slices (250/xm) were then prepared usmg a McIlwam tissue chopper. For the cychc nucleotide studies, the slices were incubated m Kreb's Ranger buffer which contamed l0 mM glucose and l0 mM theophyllme for 20 min with two changes of buffer. Peptldes were added for 20 mm, and after incubation for 20 mm at 37°C, the reaction was stopped by the addition of an equal volume of iced ethanol. Cychc GMP and clychc AMP were determined by radlolmmunoassay as descnbed previously (8). For the phosphatidyhnosltol studies, the slices were reactlved by gently agitating in Kreb's buffer contaimng 10 mM glucose at 37°C for 30 rain with two changes of buffer aH-Inosltol (14 C1/mmol) was purified on a 0.75 ml Dowex-1 formate column and eluted with 1 ml of water before use. Then 75/zl ahquots of brain slices were added to 215/~1 of Kreb's buffer which contained 7.5 mM LtCI and 3 /~C1 of purified 3H-inositol. The solutions were gassed with 95% OJ5% COz and shaken at 37°C for 30 mm. The peptldes were

~Thls paper was presented at the 9th Winter Neuropept~de Conference m Breckenndge, CO 2Requests for repnnts should be addressed to Dr Terry W Moody, Department of Biochemistry, The George Wasbangton University Medical Center, 2300 Eye Street, N W , Washington, DC 20037

1345

1346

HARI P R A S A D AND MOODY 600

L =

40(

300

? o. o

20C

B G C •

Glycerol

IP

IP 2

45

IP 3

FIG 1 FracUonaUon of phosphatldyhnos~tol metabohtes using column chromatography methods The cpm/mg protein was determined m the presence of 10 ttM GRP (G), I mM carbachol (C) and basal (B) condlt~ons The mean value-S D of 4 determinations ~s indicated (*p<0 05, **p<0.0~)

added and incubated for 120 mln. The reaction was terminated by the addition of 940/~l of chloroform/methanol (12:vol/vol) and the water soluble mositol phosphates were extracted after 16 hr. Three hundred and ten /xl each of chloroform and water were added, the sample vortexed and centrifuged at 1,000xg for 15 mm to separate the layers. Seven hundred /zl o f the aqueous layer was apphed to a Dowex-1 formate column (0.5 ml) and the unreacted 3Hmosltol was removed by 5x3 ml washes with 5 mM cold myolnositol. 3H-Glycerol, ~H-IP~, 3H-IPz and aH-IP3 were eluted by 5 mM sodium tetraborate/60 mM sodium formate, 0.2 M ammomum formate/0.1 M formic acid, 0.4 M ammonium formate/0.1 M formic acid and 1 M ammonium formate/0.1 M formic acid respecUvely as described previously (1). Twelve ml of scmtdlation fired (aquasol-2) was added and the vtals counted at 40% efficiency. The protein content of the rat brmn slices was determmed using the Lowry method (12) Peptides were purchased from Pemnsula L a b o r a t o n e s Inc. (San Carlos, CA). Dowex-1 formate was purchased from Biorad Laboratories (Richmond, CA). Myomositol was purchased from Sigma Chemical Co. (St. Louis, MO). Myo-2-(aH)-mositol was obtained from New England Nuclear Corp. (Boston, MA) RESULTS Prewous studies mdlcated that CNS muscanmc chohnerglc receptors stimulate phosphatldyhnosltol turnover (6) Figure 1 shows that using rat brain frontal cortex shces, various phosphatidylinosltol metabolites were separated usmg a Dowex-1 resin The density of radmactive glycerol was very low (<30 cprrdmg protein) m all fractions. In contrast, most of the rad~oact~wty was present m the IP1 fraction. The basal cpm/mg protein was 135, whereas GRP (10/~M) signh'icantly increased the IP~ levels by 37% and 1 mM carbachol, a muscarlnic cholmergdc agomst, caused a 300% increase. Less radloacUwty was presented m the IP2 and 11)3 fractions; the basal levels were 128 and 46 corn/rag protein respectwely In each of these fractions carbachol

I 135

80

Time, mln

FIG 2 T~me course of GRP mcubauon The IP1 concentration was determined m the presence (©) or absence (e) of 10 t~M GRP as a function of Ume The mean value-+S D of 4 determinations ~s indicated (*p<0 05)

20C B

150

• A

•

S~

~ 150 e~5

~=

o

g 10

10~

~L

~a

(QRP), Log M

I

-a

_l7

I

-e

(BN), Log M

FIG 3 Dose response curve for GRP and BN (A) The abdlty of GRP to sUmulate phosphatldylmositol turnover at &fferent concentrations m the presence (e) or absence (©) of insulin (2 ~g/ml) is shown (B) The abthty of different doses of BN to elevate IP~ levels was determined m the presence (e) or absence (©) of msuhn (2 /~g/ml) The mean value+_S D of 4 determmauons is indicated The 100% value represents basal acUwty *p<0 05, **p<0 01) significantly elevated (by approximately 70%) the values above basal levels, whereas GRP was not significantly different from the control Because GRP caused a small but significant increase m the IP1 levels, the time course of incubation was determined. Figure 2 shows that after a 45, 90 and 135 rain incubation with 10 /~M GRP, the levels o f IP1 were significantly mcreased by 35, 61, and 42% respectwely above basal values. Routmely, a 120 rain incubation period was used in all subsequent studies. Previous studies mchcated that BN alone caused a shght increase m phosphatidylinositol turnover, whereas when insulin was added wzth BN a dramatic increase was observed, msuhn alone had no effect on phosphatldyhnositol turnover (9). Therefore, we investigated the ability o f BNdike pepudes to sttmulate phosphatidylinoaitol turnover in the presence or absence of insulin. Figure 3A shows that G R P only

BOMBESIN AND P H O S P H A T I D Y L I N O S I T O L T U R N O V E R

1347 TABLE 1

140

REGIONAL DISTRIBUTION OF BOMBESIN-STIMULATED PHOSPHATIDYL INOSITOL TURNOVER

Region

% St~mulatmn Relative to Control

II

_E

120

¢D

100q T -9

i -8

I

l

J

-7

-6

-5

( P e p t i d e ) , Log M

FIG 4 Dose response curve for (Ac-GRtn°-~7) and GRPH6 The abdlty of (Ac-GRP2°-2r) (@) and GRP~-~6((3) to elevate IP~ levels in the presence of msuhn (2/zg/ml) was investigated as a function of pept~de concentration The mean value_+S D of 4 determinations is lndtcated The 100% value represents basal activity (*p<0 05 relative to control)

shghtly increased IPi levels at a 100 and 1000 nM concentration In the presence of lnsuhn, GRP significantly increased IPI levels at a 10, 100, 1000 and 10000 nM, whereas m the absence of msuhn GRP only slightly increased IP1 levels. BN, m the presence of msuhn, stimulated phosphatidyhnomtol turnover by 26, 62, 64 and 83% at a 1, 10, 100 and I000 nM concentration respectively (Fig. 3B). These data suggest that BN-hke peptides and msuhn are synergistic in stimulating phosphatidyhnomtol turnover The effects of other GRP analogues on phosphatldyhnomtol turnover were studied. Figure 4 shows that (Ac-GRP 2°-27) but not GRP H e mgnlficantly stimulated phosphatldyhnomtol turnover The effect was dose-dependent and 10, 100, 1,000 and 10,000 nM (Ac-GRP2°-2r) mgnlficantly stimulated phosphatldyhnomtol turnover by I I, 23, 34 and 24% respectively In contrast, low doses of (Ac-GRP2°-27) and GRP H e at any dose tested did not significantly increase the IPi concentration. Therefore the C-terminal of GRP is essentml to stmaulate phosphatidyllnomtol turnover. The ability of BN to stimulate PI turnover was investigated m different brain regmns. Table 1 shows that 1/xM BN significantly increased IPi levels by 70% in the olfactory bulb Also, BN stimulated IP~ production m the hippocampus, stnatum, thalamus and frontal cortex but not the m~dbrain, hlndbram or cerebellum. Due to the large tissue requirements of the assay, the frontal cortex was routinely studied in other experiments BN or GRP had no effect on the cAMP or cGMP levels (Table 2). As a positive control, VIP increased the cAMP levels approxnnately 9-fold from 54 to 471 pmol/mg protein. VIP had no effect on the cGMP levels. Therefore BN-hke peptides have no effect on the cAMP or cGMP levels in the rat brain but do stimulate phosphatidyhnositol turnover DISCUSSION

It is generally accepted that CNS neuropept~de receptors, when activated, stimulate second messenger production. In this regard, VIP and the structurally-related secretin elevate cAMP levels using rat frontal cortex shces (8). These actions

Olfactory bulb Hippocampus Stnatum Thalamus Frontal cortex Cerebellum Medulla/Pons M~dbram

170 -+ 32 134 _+ 24 126 -+ 16 125 -+ 16 124 _+ 12 115 - 11 108 -+ 15 107 -+ 7

The abihty of BN (1 /zM) to stimulate phosphatldyhnomtol turnover m different brain regions was determined The mean value _+ S D of 3 determinations each repeated in quadruphcate ~s mdtcated

TABLE 2 EFFECTS OF BN-LIKE PEPTIDES ON CYCLIC NUCLEOTIDE PRODUCTION

Peptide None BN(10/.~M) GRP(10p.M) VIP (10 p.M)

cAMP 54-+ 50-+ 58_+ 471 +

4 5 3 53

cGMP 025_+ 003 022_+ 004 026 +_ 002 0 30 _+ 0 05

The mean value _+ S D of 4 detenmnatmns Is indicated The cAMP and cGMP (pmol/mg protein) was determined by radlolmmunoassay.

may be mediated by the distinct VIP and secretin receptors which have been characterized m the CNS (8). Therefore, second messenger assays are useful to evaluate the biological activity of neuropeptides in the CNS. Previously we found that the C-terminal of BN or GRP was essential for thew high affimty binding to CNS BN receptors (18) BN and GRP, which have 7 of the 8 same C-terminal amino acid remdues, inhibited specific (t25ITyr4)BN binding to rat brain shces with ICs0 values of l0 and 20 nM respectively Similarly, the GRP fragment (AcGRP 2a-~7) but not GRP 1-16 inhibited specific binding to BN receptors with high affinity. Here BN, GRP and Ac-GRP2°-~7 but not GRP 1-ie stimulated phosphatldyhnositol turnover The half maximal effective dose for BN, GRP and AcGRP 2°-27 to stimulate phosphatidyhnomtol turnover was approximately 10 nM. These data suggest that BN receptors, when actwated by appropriate agonists, may stimulate phosphatidyhnomtol turnover. Other CNS receptors such as muscarimc cholinerglc receptors (6) and substance P (28) receptors stimulate phosphat~dyllnomtol turnover. As a positive control we found that carbachol routinely stimulated IP1 levels by approximately 3-fold using frontal cortex slices Using rat hypothalamic

1348

HARI PRASAD AND MOODY

slices SP and its C-terminal analogues increased IPt levels by approximately 50%. Further studies indicated that SP stimulated phosphatldyhnositol turnover in those brain regions which had a high receptor density such as the hypothalamus and strlatum but not the cerebellum (14) The density of BN receptors in various brain regions correlates with the ability of BN to stimulate phosphatidyhnositol turnover. Previously we found that there was a high density of BN receptors in the olfactory bulb, hippocampus, hypothalamus, frontal cortex, thalamus and stnatum (32). Similarly, BN significantly stimulates phosphatidyllnositol turnover m slices derived from these brain regions. In contrast, the density of BN receptors is low in the midbrain, medull,a/pons and cerebellum and BN does not significantly stimulate phosphatidylinositol turnover in these brain regions. Due to the large quantity of available tissue, we routinely used frontal cortex slices in our experiments, however, similar data were obtained using olfactory bulb slices The effect of BN on other second messengers was investigated BN had no effect on cAMP or cGMP levels using rat brain slices. In contrast, BN (1 nM) increased cGMP levels 23-fold using dispersed pancreatic aclni (10) Therefore, peripheral but not central CNS BN receptors may positively affect guanylate cyclase activity BN (1/zM) increased IP~ but not IP2 or IP~ levels using rat brain slices and LICI (7 5 mM) Previously using Swiss 3T3 cells, BN increased IP~ production 2-fold in the absence, 8-fold m the presence of 10 mM LICI and 14-fold when LIC1 was present with insuhn (9) Also, m Swiss 3T3 cells, nanomolar concentrations of BN stimulate ZH-thymidme uptake, however, the cpm incorporated into the DNA by BN approximately double when insulin is added (4,9) Similarly,

in the rat CNS, BN-hke pepttdes and insulin were synergistic with regards to stimulating phosphattdylmosRol turnover The mechanism for the synergistic action between insulin and BN remaans unknown For Swiss 3T3 cells, IP3 levels significantly increase within 30 sec after the addition of BN (9) The IP3 produced causes release of Ca 2+ from intracellular stores. Nanomolar concentrations of BN or GRP but not GRP H 6 increase the lntracellular concentrations of Ca z+ from approximately 150 to 350 nM within 15 sec after the addition of peptide (15). Similarly, using SCLC cells, BN or GRP but not GRP H 6 increased the lntracellular Ca 2+ levels and the Ca 2+ released was from lntracellular stores (21) In Swiss 3T3 cells BN elevates the dlacylglycerol levels after 30 sec (27) This diacylglycerol may activate protein kinase C resulting in mcreased Nat/K + exchange and alkahmzation of the cytoplasm (15) Previously, the biological activity of CNS BN-hke peptides was evaluated using biological assays (hypothermia or grooming) While these assays have indicated that the C-terminal of BN is essential for biological activity they are unfortunately very time consuming. The results presented here indicate that BN or GRP increase IP~ levels using rat brain shces These phosphatldylinositol assays may prove useful m evaluating the biological activity of BN-hke peptides In mammalian brain

ACKNOWLEDGEMENTS The authors thank Drs Gary Flskum, Kenneth J Kellar and Barry B Wolfe for helpful discussions This research is supported by NSF grant BNS 8500552

REFERENCES 1 Bemdge, M J , Downes, C P , Hanley, M R Llthmm amphfies agomst dependent phosphatidyhnomtol responses m brmn and salivary glands Bmcbem J 206 587-195, 1982 2 Brown, M ; Ravler, J Vale, W Bomesm Potent effects on thermoregulatmn m rat Science 196"998-1000, 1978. 3 Brown, M. R , Rlvmr, J , Vale, W Bombesm affects the central nervous system to produce hyperglycemm m rats. Life Sol 21 1729-1734, 1978 4 Corps, A N., Rees, L. H., Brown, K. D A peptlde that mhlblts the mltogemc stimulation of Swiss 3T3 cells by bomesm or vasopressm. Blochem J 231 781-784, 1985 5 Crawley, J N , Moody, T W Anxmlyttcs block excessive grooming behavior induced by ACTH1-24and bombesm Brain Res. Bull 10'399-401, 1983. 6 Gll, G W , Wolfe, B. B Ptrenzepme distinguishes between muscanmc receptor mediated phosphomosR,,de breakdown and mhlbmon of adenylate cyclase J Pharmaeol Exp Ther 232"608-616, 1985 7 Gmerek, D. E., Cowan, A Classification of opmlds on the basis of their abihty to antagomze bombesm-mduced grooming m rats Life So 31"2229-2232, 1982. 8 Fremeau, R. T , Korman, L Y., Moody, T W Secretm sUmulates cAMP formataon m ratbraan J Neuroehem 46:1947-I955, 1986 9 Heslop, J P., Blakeley, D M , Brown, K D., Irvme, R F , Berridge, M. J. Effects of bombcsm and insulin on mosttol (1,4,5)tnsphosphate and mosttol (l,3,4)tnsphosphate formation in Swiss 3T3 cells Cell 47"703-709; 1986 ,

10 Jensen, R T , Moody, T , Pert, C , Rlvler, J E , Gardner, J D Interaction of bombesin and htonn wRh specific membrane receptors on pancreaUc acmar cells Proc Natl Acad So USA 12 6139-6143, 1978 11 Katz, R Grooming ehclted by mtraventncular bomtmsm and eledmsm m the mouse Neuropharmacology 19' 143-146, 1980 12 Lowry, O. H , Rosebrough, J J , Farr, L., Randall, R J Protein measurement with the Fohn Phenol reagent J Biol Chem. 193"265--275, 1951 13 McDonald, T J , Jornvall, J , Nllsson, G , Vagne, M , Ghatel, M , Bloom, S R , Mutt, V Charactenzatton ofgastnn releasing peptIde from porcme non-antral gastric t~ssue BlOebem Biophys Res Commun 90 227-233, 1979 14 Mantyh, P W , Plnnock, R D , Downes, C P , Goedert, M., Hunt, S P Correlation between mosRol phosphohptd hydrolysis and substance P receptors m rat CNS Nature 309.795-797, 1984 15 Mendoza, S A , Schneider, J A , Lopez-Rtvas, A , SlrmetSmith, J W., Rozengurt, E Early events ehcited by bombesm and structurally related peptides m quiescent swiss 3T3 cells. II Changes m Na + and Cat+ fluxes, Na +, K + pump act~wty, and lntra-cellular pH J. Cell Biol. 102"2223-2233; 1986 16 Merah, Z , Johnston, S , Zalcman, S Bombesm-mduced behavmural changes Antagomsm by neuroleptaes Pept~des 4-693-697, 1983 17 Minamino, N ; Kangawa, K , Matsuo, H. Neuromedm C A novel bombzsm-hke peptide identified in porcine spinal cord Biochem Biophys Res Commun 119"14-20, 1984.

BOMBESIN AND PHOSPHATIDYLINOSITOL

TURNOVER

18 Moody, T W , Pert, C B , Ravler, J , Brown, M R Bombesm Specafic binding to rat brain membranes Proc Natl Acad S o USA 75 5372-5376, 1978 19 Moody, T W , T h o a , N V , O'Donohue, T L , P e r t , C B Bombesm-hke peptades m rat brain Locahzauon m synaptosomes and release from hypothalamac shces Life Sca 26 17011712, 1980 20 Moody, T W , O'Donohue, T L , Jacobowatz, D M Baochemacal Iocahzatlon and charactenzataon of bombesln-hke pepudes in dascrete regaons of rat brmn Peptades 2 75-79, 1981 21 Moody, T W , Murphy, A , Mahmoud, S , Faskum, G Bombesm-hke peptldes elevate cytosohc calcmm in small cell lung cancer cells Biochem Baophys Res Commun 147 189195, 1987 22 Pandol, S J Role of calcmm flux m the mechanism of action of exocnne pancreatac secretagogues Regul Pept 19 131, 1987 23 Panula, P , Yang, H Y T , Costa, E Neuronal locahzataon of the bombesm-hke lmmunoreactavlty m the central nervous system of the rat Regul Pept 4'275-283, 1983 24 Roth, K A , Weber, E , Barchas, J D D~stnbuaon of gastnn releasmg peptade/bombesln-hke immunostalnmg m rat brain Brain Res 251 277-282, 1982 25 Stucky, J A , Gibbs, J Smath, G P Lateral hypothalamac mjectaon of bombesm decreased food retake m rats Brain Res Bull 8 617-622, 1982 ,

1349

26. Tache, Y , Gumon, M Central nervous system action of bombesm to mlublt gastric acid secreaon Life SOl 37 115-123, 1985 27 Takuwa, N , Takuwa, Y , Bollag, W E , Rasmussen, H The effects of bombesln on polyphosphomos~tide and calcmm metabohsm m Swiss 3T3 cells J Biol. Chem 262 182-188, 1987 28 Watson, S P , Downes, C P Substance P induced hydrolysis of mosltol phosphohplds in guinea-pig ileum and rat hypothalamus Eur J Pharmacol 93 245-253, 1983 29 Willis, G L , Hansky, J , Smith, G C. Ventncular, paraventncular and clrcumventncular structures revolved m peptldereduced sataety Regui Pept 9 87-93, 1984 30 Zachary, I , Rozengurt, E High affimty receptors for pept~des of the bombesln famdy an Swiss 3T3 cells Proc Natl Acad Sca USA 82 7616--7620, 1985 31 Zachary, I , Smnett-Smath, J W , Rozengurt, E. Early events ehcated by bombesln and structurally related peptldes m qmescent swass 3T3 cells I Actwataon of protein kmase C and mbabataon of epadermal growth factor binding J Cell Blol 102 2211-2222, 1986 32 Zarbm, M A , K u h a r , M J , O ' D o n o h u e , T L , W o l f , S . S , Moody, T W Autoradaographac localazatlon of (l~SI-Tyr4) bombesm binding sates m rat brain J Neuroscl 5 429-437, 1985