dopamine d, receptors modulate neurotransmitter-regulated calcium ...

aliqnot of homogena,te corresponding to I mg offresh iveight tisme. The mixtures wer' incubatedwith or without the peptides for 5 minutes at 30 T.The reaction was stopped hy adding 500 ~1 of asolut,ion containing 2 mM cyclic AMP. The amountof cyclic AMP formed was measured by using subsequentohromatography on alnmina oxiclc andDowex 50 W X4. The recovery was 70-80 %. Tuavoid peptide degradation, neurotensin and VIPwere dissolved in TrisHC1 buffer eontaining 5.10-5Nbacitracine.Ca++ inpux measwernent. - Rat emipituitary werepreincubated for 30' in Krebtibicarhonate bufferpH 7.4 supplemented with 10 mM sucrose and 1.14 mMascorbic acid at 37 OC. Thereafter, different concentrationsof neurotensin were added and incnhationwas continued for an additional 30min. Then,0.02 mCi 45Ca++ was included in the incubationmixture and l min after tissue wan removed fromincnhation medium and repeatedly washed in aMillipore apparatus under vacuum.tissue from lactating female rats is about 35 %of the total. Tbis is 7 fold greater than thenumber of PRIrsecrcting celln in male, rat pituitary(5 % of the total). As shown in Tahle 1, VIPstimulated cyclic AMP formation in homogenat,esof anterior pituitary from lactat,ing femalerats, while NT was inactive. Sinee secretion from avariety of endocrine tissues, ineluding anterior pituitary.is linked to elevation of cytosolic concentrationof Ca+f, the influence of NT on Ca+* influx wasinvestigated.As shown in Fig. 1, NT induced a significant increasoof "Gai-+ influx in the anterior pituitary in comparisonwith the tissue incubated without peptide. Tbedose-response relationship indicate6 an EC,, of15 I 2 nM (Fig. 1A). The effect was timdependent(Fig. lB), being measurahle after 10 min of preinciihstionwith NT and reaching the plateon after 20 min.Contrary to VIP, NT is unable to stimulate adenylatecyclase activity in homogenates prepared from ratanterior pituitary (Table 1). As previously describedby different groupn 115, 161, 104M VIP stimulatesadenylate oyclase activity hy 140 % in reiipeet tothe hssal values. This effect was dosdependentindieating an EC,, of 25 nM (data not shown). NTwas completely devoid of adenylate cyclase stimulatoryproperties np to the concentration of 10-601.The same pattern of resultn was obtained whenadenylate cyclase activity was measured in homogenatesof anterior pituitary from lactating femalerats. This experimental modcl was chosen heeausethe coutent of PRL secreting ceUs in pituitaryTable 1. - Effect o/ neurolonsin and VIP on adenylatecplase adivify in hornogmate of rat pituitary gland.UYdILONT CONCENTRATION- 43&8 65+8Neurotonsin 1 WM ............. 40 i 5 70 & 7VIP 1 pM. .................... 105 i 10 110 Q(a)(a) p < 0.01 when oompared io the oorrispondent basa1vdues.Kumbers are the mem f SEM of four different experimentamd reprcsent the pmolen af cyolic &W formad/rnin/mgprot.Prokin rontont wns nxasiired as desciibed in the litern.tire [27].T I M E (min.)Fm. 1. - InUuence of neurotensin on C&++ influx in ratemipituitmy. A = Dose-responno relationship of neurotensin-inducedincrease of C&++ inhx; B = Time-ooursw ofneurotensin increase of Ca'+ influx. '%++ inUux inabsenceof nourotonsin was 8.3 5 0,4 pmollgland. Eaeh point representathe mean + SEM af four similar eexpeiment,~.

stance P in rabbit ileum 1221, fieeni to iuvolve anincrease of Ca++ influx across the plasma membrane.Furthemore, D-4 per se does not affwt basd adenylatecyclase activity in anterior pituitary cells froni malesats 1231. Theu, our results suggest that 110th KTand DA regulate PRL releane through a modificationof Ca++ permeability across the membrane and thiseffect is completely independent from adenylatecyclase system.The existence of a particular typc of DA receptornnt couplcd in excitatory or inhihitory way withadenylate cyclane has heen already proposed [17,24, 251. According to pharmacological and anatomica1criteria thene putativc DA rcceptors can be ascribedto the I), type.We propose that in the anterior pituitary arcpresent specific ~ubpopulations of D, dopamine re-ceptorr, which are coupled in ai1 inhibitory way withdifferent effectors (ie. adenylate cyclase enzyme andCa++ channels) and regulate t'lie physiological act,ivityof differcnt neurotransmittew, VIP and NT, respecti-vely.1. MACLEOD, R. M. 1976. Raguletion of prolactin aecretion. In: Frmtiers in nesroeadocri~iology. L. Ilfartini & W. P. Ganon(Eds). Raven Pres~, Nnw York. pp. 169-194.2. CHEN, H. J. & MEITES, J. 1975. Effwcts af biogenic arniner and TRH oli xlenne of pmlnetin nnd TSH in tlie rat.Bnàocrinoloyy 9k 10-14.3. Hm-Snmr, M. & MACLEOD, R. Al. 1974. Intsract,ion of thyrotropin-relea~ing horrnonc and dopsmine aa the releaae ofprolaotin from the rat snteriar pituitary. Ewimrinology 95: 1189-1193.4. R~mEno, M.. ROTSZTEJN, W. H., ARANCIBIA, S., B~sso~. J. & ENJALBERT, A. 1978. Stiniuiation af prolnetin raleaae hyvaaowtive intestind peptlde (VIP). E,ur J. Plmrnauil. 51: 319-320.5. VIJAYAN, E. & MACCANN, S. M. 1980. In uiuo snd in, oitm sfferb~ of snbstanro P and naiirotensin on gonadotrnpin andprolaotin releaso. Enàorrinology 105: 64-68.6. ENJALBERT, A., ARANCIBIA, S., PRLV~ M., BLUET-PAJOT, M. T. & KORDOK, C. 1RR2. ~~lir~t~n~in stimulation of prolactinssoretion in vilro. Neuroend(>cirinology 34: 96-98.7. CA~AWAY, R,. & LEEUN, S. E. 1973. The inolation of a new hypotennive poptide, neurotemin, from bovine hypothalsmi.3. Biol. Chem. 248: 686P6861.8. CARR.~WAY, R. & LEEMAN, S. E. 1979. Chmacterizetion of radioimmunoassayable neurntenein in the rat. Its differentialdistribution in the eentd nervoiin nystem, small intesbine and storneili. I. Biol. Chmn. 251: 7046-7052.9. IwnsEN, L. L,, IVE~EN, S. D,, BLOOM, F. E., DouoLAS, C., Bnom, M. & VALE, W. 1978. Calnium-dependent releaseof somstostatin and neuroteuain from rat brain in uilro. Nulure (hdon) 273: 161-163,10. GOEDERT, M., LIQRTMAN, S. Li, hTACY, J. I., MARLEY, P. D. & EMSON, P. C. 1982, Neuiobnsin in the rat nnteriorpitnitmy gland. Nature (LoluEm) 298: 163-165.11. MncLmm, R. M. 8: BONT~M, E. H. 1970. InAuenoe of ianie environment. on tlie in vitrn sptlissia and rtllcase of pituitaryhormones. Enàoerinology 86: 863-869.12. THORNER, M. O., HAOKETT, J. T., MURAD, F. & MACLEOD, R. M. 1980. Calcium rather than cyolio AMP is tho physiologicnlintracellular regidatar of prolactin rolaase. Neuroen

18. Bnrsso~, R. G., &uss~-L*o~~, F. & ~AISSE, W. J. 1972. The atimulueseeretion ronpling of glueos+induced insulinrelease. J. Clin. Inueat. 51: 230-241.19. MEMO, M., CARRONI, E., TRABUCCHI,M. & SPANO, P. F. 1984. In: iYeumm&lutim ami hin funetion. G. Biggia, P. F. Spano,(4. Toffana & E. Conta (Eds). Pergamon Presa pp. 165-161.20. TAM, S. W. & DAmEs, P. S. 1980. Dopaminergic inhibition of ionophore A 23187- stimulsted relesse of prolactin fmm ratanterior pituitmy cella. J. Biol. Chen. 255: 6595-6599.21. Scn~nm~, G., Cao~w, M. J. & MACLEOD, R. M. 1983. Adenosine 3',5'-monopohsphate (eA4DIP) and cdeium-cslmodolininterrelatian in the eontml of pmlactin mcretion: svidence for dopemine inhihition of cAMP d~aocumulstion and prolsotin releasenfter ritloium mabilizetion. Endacrinology 112: 1801-1807.2. Do~owrrz, M., FO~EL, R., BATTISTI, L. & ASARKOP, N. 1982. The neurohumorsl negretagogues carbechol, substance Pand neurotensin incresse C*+< influx and calcium content in rahbit ileum. Life S'ci. 31: 1929-1937.23. DE CAMILLI, P,, MACCONK, D. 8C SPADA, A. 1979. Dopemine inhibita adenylate cyclaaa in human pralaetin secreting adeno.mas. Nature (Lmda) 278: 252-254.24. CREEESE, I., USDIN, T. & SNYDER, S. H. 1979. Guanine nucleotides distinguish between two dopamine receptors. Nr'iwe(London) 278: 577-678.x.,26. &ANO, P.F., GOVOKI, S., UZUMAKI, BOSIO, A., MEMO, M., Luccm, L., CARRWA,M. & TRABDCCHI, M. 1983. Stimulationof D,-dopamine receptors by dopaminergic srgot slksloids: studies on the mechaniam of action. Aging 23: 165-177.26. CLEXENT-COME~, Y. C,, KEBAEIAN, J. W., PETZOLD, G. L. & GREENQARD, P. 1974. Dopamine sensitive adenylate cyolssein mammalian brain: e possible site of action af antipvahatic druga. Pmc. Nutl. &i. UBA 71: 1113-1117.27. LOWXY, O. H., RosENBROU~~, M. J., FARE, A. C. & RANDALL, R. J. 1961. Protein measurement with the Fdin phenolreagent. J. Biol. Chem. 193: 265-275.