Ann. I d . A V ~ L T . Snniiù Vai. 20, N. 1 (1984) pp. 5-10

DOPAMINE D, RECEPTORS MODULATE NEUROTRANSMITTER-REGULATED CALCIUM ION GATES

N. MEMO and P. F. SPANO

Institute of Pharwacology and Ezperimental Therapeutics, University of Brescia, I t d y

Summary. - I n this paper, Tue report that th,e intra- cellular mechanism elicited by neurotensin in stim~c- lating prolactin release involves a n increased Ca++ uptalie by pituitary cells rather tlian afecting adenylat*? cyclase system. I n addition, d o p m i n e i s able to prevent neurotensin-induced caleium inflzrz by interactinq with a particular subtype of D% receptors urhich appenr to be completely independent fromn adenylate cyclase mmety &t are coupled to colcium cl~annels.

Riassunto (I reccttori dopaminergici D, niodulano i canali del calcio dipendenti dai neurotrasmettitori). - I n questo lavoro si dimostra che i m*?ccan.ismi intra- cellnlari indotti dalla newotensina, i n relazione alla sua attività prolattin+secernente, coinvolqono un uu- nnento dell'influsso d i calcio nelle cellule ipofismrie piutlo- sto che una modificazione della attività adenil ciclasim. Inoltre la dopamina è in grado d i prevenire l'aumento dell'influsso d i calcio indotta dalla neurotensina attra- verso I'interazione con recettori D,. Questi recettori dopaminerqioi sembrano essere oompletamente indz- pendenti dall 'enaim adenil ciclasi bensi deragent i con specifici canali del calcio.

Secretory activity of the anterior pituitary glancl is regulated by the brain through stiniulatory and inhibitory siibntances released from nerve endings in the median eminence. I n particular, prolactin (PRL) secreting cells are inainly controlled hy inhihitory factors, among which dopamine (DA) is one of the most potent in vivo as well i n nitvo [l]. On the othcr hand PRL secretion is, a t least partially, iinder the ntimulatory influence of different polypeptides includ- ing Tyrotropin-Rcleasiiig Horiiione (TRII) p, 31 ancl Vasoactive Intestina1 ~ & t i d e (VIP) [4]. Recently,

neurotensin (NT) has been found to stiiiiulate PRL sccretion in vitro [5, 61. JST, originally isolated from bovine hypothalamua [l], is widely distributed in the CNS and the highest levels of this peptide were found in the median eminence [E]. In addition, liypothalamic NT is concentratcd in nerve terminals and can be released upon depolarization [9]. These finding suggest that NT might play a role as neuro- transmitter or neuromodulator in regulating the physiological PRL secretion. The presence of NT in the anterior pituitary gland [IO] and its PRL releasing properties [5, 61 prompted us to investigate the intracelliilar events indnced by this peptide.

At least two secondary messengers seem to regu- late PRL release: cyclic AMP and CA++. Increased concentrations of C&++ enhance PRL release, whe- reas facturs which suppress Ca++ activation inhibit PRLrelease [I l , 121. Cyclic AMP involvement in the contro1 of PRL aecretion has been suggested since compounds that affect intracellular cyclic AMP levels also modify PRL release 113, 141. In this respect, the close symmetry between PRL release and adenylate cyclase activity in response to VIP strongly supports the hypothesis of an involvement of cyclic AMP in the intracellnlar mechanisms by which VIP regiilated PRL release [151.

Adenylutt. cyclase assay. - Pituitaries were dissected nnd gently homogenized in 10 v01 (wtlvol) of 10 mliI Tris-HC1 pH 7.5 containing 1.2 mM EGTA. Adeny- late cyclase activity was measured according to Clement-Cormier at al. [%] with minor iiiodificat,ions. The incubation niedium contained 82.5 mM Tris- HCI buffer (pH 7.4), 8 mM Mg804, 5 n131 teophilline, 0.3 niM EGTA (carricd river from t,isniie prep:bration). 2 mX "C!-ATP (58 mCi/inmol, dniersham) and an

aliqnot of homogena,te corresponding to I mg of fresh iveight tisme. The mixtures wer' incubated with or without the peptides for 5 minutes a t 30 T. The reaction was stopped hy adding 500 ~1 of a solut,ion containing 2 mM cyclic AMP. The amount of cyclic AMP formed was measured by using sub- sequent ohromatography on alnmina oxiclc and Dowex 50 W X4. The recovery was 70-80 %. Tu avoid peptide degradation, neurotensin and VIP were dissolved in TrisHC1 buffer eontaining 5.10-5N bacitracine.

Ca++ inpux measwernent. - Rat emipituitary were preincubated for 30' in Krebtibicarhonate buffer p H 7.4 supplemented with 10 mM sucrose and 1.14 mM ascorbic acid a t 37 OC. Thereafter, different concen- trations of neurotensin were added and incnhation was continued for an additional 30min. Then, 0.02 mCi 45Ca++ was included in the incubation mixture and l min after tissue wan removed from incnhation medium and repeatedly washed in a Millipore apparatus under vacuum.

Contrary to VIP, NT is unable to stimulate adenyla- t e cyclase activity in homogenates prepared from rat anterior pituitary (Table 1). As previously described by different groupn 115, 161, 104M VIP stimulates adenylate oyclase activity hy 140 % in reiipeet to the hssal values. This effect was dosdependent indieating an EC,, of 25 nM (data not shown). NT was completely devoid of adenylate cyclase stimu- latory properties np to the concentration of 10-601.

The same pattern of resultn was obtained when adenylate cyclase activity was measured in homo- genates of anterior pituitary from lactating female rats. This experimental modcl was chosen heeause the coutent of PRL secreting ceUs in pituitary

Table 1. - Effect o/ neurolonsin and VIP on adenylate cplase adivify in hornogmate of rat pituitary gland.

- 4 3 & 8 6 5 + 8

Neurotonsin 1 WM ............. 40 i 5 70 & 7

VIP 1 pM. .................... 105 i 10 110 Q(a )

(a) p < 0.01 when oompared io the oorrispondent basa1 vdues.

Kumbers are the mem f SEM of four different experi- menta m d reprcsent the pmolen af cyolic &W formad/rnin/mg prot.

Prokin rontont wns nxasiired as desciibed in the litern. tire [27].

tissue from lactating female rats is about 35 % of the total. Tbis is 7 fold greater than the number of PRIrsecrcting celln in male, rat pi- tuitary (5 % of the total). As shown in Tahle 1, VIP stimulated cyclic AMP formation in homoge- nat,es of anterior pituitary from lactat,ing female rats, while NT was inactive. Sinee secretion from a variety of endocrine tissues, ineluding anterior pitui- tary. is linked to elevation of cytosolic concentration of Ca+f, the influence of NT on Ca+* influx was investigated.

As shown in Fig. 1, NT induced a significant increaso of "Gai-+ influx in the anterior pituitary in comparison with the tissue incubated without peptide. Tbe dose-response relationship indicate6 an EC,, of 15 I 2 nM (Fig. 1A). The effect was t imdependent (Fig. lB), being measurahle after 10 min of preincii- hstion with NT and reaching the plateon after 20 min.

UY d NT CONCENTRATION

IL O

T I M E (min.)

Fm. 1. - InUuence of neurotensin on C&++ influx in rat emipituitmy. A = Dose-responno relationship of neuro- tensin-induced increase of C&++ inhx ; B = Time-ooursw of neurotensin increase of Ca'+ influx. '%++ inUux inabsence of nourotonsin was 8.3 5 0,4 pmollgland. Eaeh point repre-

senta the mean + SEM af four similar eexpeiment,~.

The results illustrated in Fig. 2 sliow that prein- cuhation of anterior liemipituitary for 30 min with DA dose-dependently prevented the NT-induced increase of Ca*+ influx. Like DA, apomorphine and bromocriptine inhibit NT-induced increase of Ca++ influx (Table 2). The inhibitory effect of DA was reversed by (-) sdpiride, the pharmacological active form of the dnig, while (+) siilpiride, a t the concen- tration used, was unable to counteract the effect of DA (Table 2).

&o. 2. - Dose-depndent inhibition of neurotensin-indiiced inereese in C%++ influx by dopamine. Dopamine w m added 30 min hefars the inoluaion in the medium of IOW7M neuro- tensin. Then, inoubation was oontinued for an ndditional period of 30 min C&++ influx was messiirod a8 desoribed in Fig. 1. C&++ influx in absence of either neiirotonsin or

dopamine was 7.9 + 0.4 pmal/glmd.

Two major points arise from our data: first, NT induces an increase of Ca++ influx onto pituitary ceils without affecting the adenylate cyclase system; second, DA, by a receptor-mediated mechanism, is able to prevent the increased entcy of Ca++ induced by NT.

Since PRL release appears to be regulated hy intracellular free Ca++ content, it might be snggested that intracellular ionized Calcium concentrations are modified by a mechanism which involves either cyclic AMP, possibly through a mobilization of intracellular Ca++ from mithocondria [la], or cell depolarization, with subsequent opening of Ca+'channels. According to recent data from our laboratory, DA inhibits either the activation of adenylate cyclase by VIP and the entry of Ca++ onta the cells induced by NT 1191.

Tahle 2. - Effect o/ l~arious dopaminergic agonists and nntayonists on neurotensin (NT)-indz~ced Ca+f inpus increase in rat emipituilary.

Drug *'C& lnuux (pnioljgland)

8 . 6 i 0 . 6 NT ...................................... 1 2 . 2 + 0 . 7

N T , D A .................................. 7 . 9 1 0 . 3

NT, Apomorphine ........................ 8 . 1 h 0 . 4

S T , Bromooriptine . . . . . . . . . . . . . . . . . . . . . . . . 8 . 2 1 0 . 4

NT, DA, ( - ) Sulpirido . . . . . . . . . . . . . . . . . . . 1 1 . 8 1 0. G

XT, DA, (+) Sulpiride ................... 8 . 5 0 . 6

Neiirotensin, DA, apornorphine and bromocriptine were added at the ooncentration of 10-'M. (+) and (-) siilpiride \vere added at the conoentri~tion of 1 V M .

DA, DA-agonista snd DA-mtaganists were included in tho mwdium for the entireinciibatition period (30 min). C&++ infliix wes meilsured aa described in Fig. 1. The two isorneric forms of aiilpiride did not modify either bssal or neurotensin- indiioed Ce++ influx.

Both inhibitory actions may lead to decreased intraceilular concentrations of free ionized calcium and dccreased PRL secretion. The pharmacological characterization of the phenomenon indicates that DA inhibits NT-induced increase of C&++ influx by interacting with a D, receptor type. Therefore, our results suggest that in anterior pituitary a subset of D, receptors i8 completely independent from the adcnylate cyclase m i e t y but coupled with Ca++ channels.

It has been reported that the dopaminergic agonist bromocriptine does not affect spontaneously and A23187-activated Ca++ uptake by pituitary cells, suggesting that the inhibition of PRL release by DA occurs after calcium mobilization [20]. Moreover, Schettini et al. provided evidence that enhanced Ca++ mobilization by ionophore A23187 stimulates cyclic A3fP accumiilation in pituitary cells suggesting that the stimulus-secretion coupling of PRL rnay be due to an increase in adenylate cyclase activity 1211.

The authors propose t,hat the inhibition of PRL release by DA occurs after Ca++ is mohilized although they do not exclude the possibility that DA receptor- induced reduction in Ca++ influx is the primary temporal event. Our results support the latter hypo- thesis as the mechanism by which DA inhibits NT- induced Catf influx. AS reported by different groups, NT does not affect adenylated oyclase activity in homogenates prepared from rat pituitary gland nor modify the intracellùlar concentration of cyclic AXP in intact cells. The mechanism of action of NT, like other iiecretagognes such as carbachol and sub-

stance P i n rabbit ileum 1221, fieeni t o iuvolve an

increase of Ca++ influx across the plasma membrane.

Furthemore, D-4 per se does not affwt basd adenylate

cyclase activity i n anterior pituitary cells froni male

sats 1231. Theu, our results suggest tha t 110th KT and DA regulate PRL releane through a modification

of Ca++ permeability across the membrane and this effect is completely independent from adenylate

cyclase system.

The existence of a particular typc of DA receptor

nnt couplcd in excitatory or inhihitory way with

adenylate cyclane has heen already proposed [17, 24, 251. According t o pharmacological and anatomica1

criteria thene putativc DA rcceptors can be ascribed

t o the I), type.

We propose that i n the anterior pituitary arc

present specific ~ubpopulations of D, dopamine re-

ceptorr, which are coupled in ai1 inhibitory way with

different effectors (ie. adenylate cyclase enzyme a n d

Ca++ channels) and regulate t'lie physiological act,ivity

of differcnt neurotransmittew, VIP and NT, respecti-

vely.

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