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Regulation of the intracellular calcium concentration in MMQ pituitary cells by dopamine and protein kinase C IS. LOGIN’, S-l. KUAN*, A.M. JUDD* and R.M. MACLEOD* Departments of Neurology’ and Internal Medicing, and Center for Cancer Research, University of Virginia School of Medicine, Charlottesville, Virginia, USA Abstract - The MMQ pituitary cell line, which expresses a membranal dopamlne receptor, was used to examine the individual contributions of dopamine and protein klnase C (PKC) to control of the intracellular calcium concentration. The calcium concentration, monitored with the fluorescent dye Indo-l, increased in response to elevated K+, BAY K8844, and maitotoxin, implicating the presence of voftage-dependent calcium channels. Dopamine had no detectable independent effect, but significantly inhibited the rise in intracellular calcium mediated by activation of voltage-dependent calcium channels; this dopaminergic action was prevented by haloperidol. Acute pharmacological activation of PKC for 88 s inhibited the stimulatory effects of these calcium channel activators, and this acute inhibitory action was abolished by prior depletion of PKC. In contrast, however, PKC depletion did not alter the calcium response to BAY K8844 or maitotoxin. Thus, MMQ cells appear to have voltage-dependent calcium channels which, at rest, are either at low density or in a closed state. The rise in intracellular calcium resulting from stimulation of the channels is under inhibitory control by an apparent 5-2 dopamine receptor. When pharmacologically activated, phorbol diester-sensitive PKC limits the rise in the cellular calcium level associated with calcium uptake. In the absence of pharmacological activation, however, this enzyme system does not appear to play a role in the cellular calcium response to BAY K8844 or maitotoxin. A prolactin-secreting clonal pituitary cell line that expresses functional dopamine receptors has been developed and designated the MMQ line [l]. The homogeneity of this cell line presents a unique opportunity to further our understanding of biochemical responses to receptor activation. The MMQ line was derived from the transplantable rat pituitary tumor 7315a. which exhibited abnormal mobilization of intracellular calcium in response to thyrotropiaGeleasing hormone [21, and

dopaminergic control of 45Ca2+ fluxes only under stimulated conditions 131. The MMQ cells respond to phorbol die&r-induced activation of PKC with increased prolactin release, and demonstrate dopaminergic inhibition of 45Ca2’ flux only when stimulated [l]. We focused this study on control of the intracellular calcium concentration by dopamine and phorbol dies&r-sensitive PKC using the intracellular calcium-sensitive fluorescent dye, Indo-1. 525

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stirring. After excitation at 339 nm the Indo-l emission spectra at 400 and 480 nm were monitored each second simultaneously in an SLM 8000 Spectrofluorometer (SLM Instruments, Inc., Urbaua, lL, USA). The baseline was mcorded for 30-60 s, after which test agents were injected by Hamilton syringe into the stirred cell suspension aud recording continued as necessary. The signals were corrected for autofluorescence and dye leakage to yield a final corrected value for the ratio of 400/480. The fluorescent ratio of a series of calcium-l3GTA standards was measured. A standard curve was fitted to au equation for one binding site and was used to derive the corresponding calcium concentrations for the sample ratios. Eighteen individual preparations of MMQ cells were used to generate these data. To deplete PKC, 1 p&I phorbol my&ate acetate (PIvIA), a potent PKC activator, was added to the cells for 12 h, starting 24 h before the experiment

The derivation and maintenance of the MMQ cell line in culture have been described [l]. In each experiment, the MMQ cells cultured in complete RPM&1640 medium were incubated with 2.5 p.M Indo-l/AM for 30 min. The dye was trapped intracell~arly by esterase cleavage. Then the cells were washed and brought to a density of 1.5 million cells per ml in a balanced salt solution containing 1.5 mM calcium. The cells accumulated about 40% of the dye, which did not affect celhrlar viability as judged by trypan blue exclusion. All manipulations were performed in subdued light and, following incubation, the cells were maintained on ice to retard dye leakage. Prior to placement in the fluorometer, the cells were brought to 37°C during a 30 min incubation and maintained at this temperahue during the experiment. The cell suspension was subjected to continuous magnetic 700 r 600 500

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intracellular calcium level by BAY K8644.

Bottom

the absence or presence of haloperidol when BAY K8644 was added

[4, 51. After 12 h, this medium was replaced with fresh medium containing no PMA for another 12 h until incubation with Indo-l/AM. Although not directly measured in this work, a similar protocol for PKC depletion in normaI pituitary cells resulted in virtually complete elimination of phorbol diester-sensitive PKC activity [5]. We have previously reported [4] that this treatment protocol for PKC depletion abolished the acute effects of phorbol diesters to stimulate prolactin secretion and arachidonate release from pre-labeled cells which provided a biological test to demonstrate the extent of PKC depletion. Dopamine, haloperidol, and PMA were purchased from Sigma. Indo-l/AM was obtained from Molecular Probes, Inc., Eugene, OR, USA. Maitotoxin was supplied to us by Professor T. Yasumoto, Tohoku University, Sendai, Japan, and BAY K8644 was a kind gift from Miles Laboratory, West Haven, CT, USA.

Results

The resting concentration of intracellular calcium in MMQ cells varied between 25 and 300 nM, with a mean + SE of 158 k 23 nM. There was no obvious explanation for differences in resting intracelhrlar calcium concentrations among the different preparations; they may reflect the distribution of cells at different phases in the growth cycle. After 30 s of baseline fluorometric recording, increasing the potassium concentration acutely from 5.9 mM to 20 or 50 mM caused the intracellular calcium concentration to increase, with the magnitude of this response dependent upon the K+ concentration (Fig. 1, top). We examined the effect of a similar osmotic stimulus by exposing cells to identical concentrations of NaCl (as shown in Fig. 1, bottom): this treatment had no effect. These data suggested the presence of voltage-dependent calcium channels in MMQ cells.

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This premise was further examined by assessing the Mh4Q response to BAY K8644, a dihydropyridine calcium-channel activator (Fig. 2, top). MMQ ceils demonstrated a concentration-dependent increase in the intracellular calcium concentration in response to 0.01-l @VIBAY. This response occurmd within 10 s and was sustained for several minutes. The dopamine receptor in normal pituitary cells has been shown to modulate transmembranal calcium flux [6] and the intracellular calcium concentration [7, 81. We therefore examined the effect of dopamine on the control of basal and stimulated intracellular calcium levels in the MMQ cells. Dopamine (1 p&l) had no effect on the resting level of intracellular calcium, either immediately upon addition or after a 30 min exposure (data not shown). F’retmatmentof the cells with 1 pM dopamine, however, had a significant inhibitory effect on the ability of BAY K8644 to increase the intracellular calcium concentration. As shown in Figure 2 (bottom), BAY K8644 increased

After a 30 min exposure to 1 @VIdopamine the rate of response to BAY K8644 was reduced and the maximum effect of BAY K8644 diminished by approximately 50%. Coincubation of 1 @I dopamhre with 100 nM haloperidol, a dopamine receptor antagonist, abolished the inhibitory effect of dopamine on BAY K8644. In 3 experiments the values for the increment in the intracellular calcium level (peak minus basal) were: BAY K8644,98 -+4 nM Ca2’; BAY K8644 in the presence of dopakne, 54 + 7 nM (RO.01); and BAY K8644 in the presence of haloperidol plus dopamine, 108 + 8 nM. Maitotoxin is another agent that increases transmembranal cakium influx in pituitary cells [7, 91 through unclear mechanisms that may include activation of calcium channels [lo, 1l] or phosphoinositide breakdown 1121. The data in Figure 3 (top) show that ma&toxin stimulated a dose-dependent increase of the intracellular calcium concentration. Unlike BAY K8644, maitotoxin

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K8644 or by maitotoxin in control cell8, but not in In these expekmnts with PKCdepleted tXu8. PMA a different preparation of maitotoxin demonstrated greater potency compared with the datainPigure3.

1pM

Discussion

,

These data reveal that MMQ cells can be labeled with Indo-l successfully, facilitating the study of intracellular calcium concentration by fluorescence (B) MAITOTOXIN 0.05 ng/ml spectroscopy. MMQ cells appear to POSseSS voltage-dependent calcium channels that can be activated by either BAY K8644 or depolarizing concentrations of K’. The D-2 dopamine receptors expressed on MMQ cells can inhibit the change in cellular calcium levels resulting from activation of these calcium channels under stimulated (but not basal) conditions, as in the parent tumor, 7315a [33. The lack of an independent effect of dopamine, 100 200 300 400 0 100 200 300 4ao despite its clear ability to modulate the actions of 0 BAY K8644, suggests that voltage-dependent SECONDS calcium channels in MMQ cells may exist at a low lrdluence of the PKC system on stimulation of Fig. 4 density or, perhaps, in a closed state under basal intracellular calcium levels by BAY KS644 (top) or maitotoxiu conditions. Dopamine stimulates a (bottom). The control (left) and PKC-dep1e-te-d(right) cellswere voltage-dependent transient K’ current, the IA stimulated with BAY KS644 or maitotoxin in the absence (upper current, in these h4MQ cells 1133 that theoretically trace) or presence (lower trace) of 100 nM PMA. PMA was could contribute to dopaminergic inhibition of added 30 s, and test agents 90 s, a&r the start of the mcording. calcium influx; but such a link remains speculative With PKC-depleted cells. traces with and without PMA were at this time. superimposed Phorbol die&r-sensitive PKC does not appear to became effective over about 1 min following its participate in the increased calcium concentration iUttDdUCtiOn t0 tie ceil SUSpfZlSiOn. Like BAY Other caused by BAY K8644 or maitotoxin. K8644, however, dopamine inhibited the effect of species of PKC may still be involved [14], however. maitotoxin in a Moperidol-reversible manner (Pig. It is evident that a phorbol diester-sensitive PKC 3. bottom). could have a potential role in regulating the We Studied the effects of PKC depletion to test intracellular calcium level as seen in the the hypothesis that PKC contributes to the control of expeiimcnts using acute administration of PMA. intracellular calcium levels. Control and These data suggest that PKC may inhibit calcium PKC-depleted h4MQ cells (Pig. 4) exhibited similar uptake, but several different actions of PKC on patterns of response to the calcium channel calcium regulatory systems have been observed [15, activators. 161. Acute treatment with PMA had no effect on Although PMA has no effect on the resting level resting [Ca2+]i in either control or PKC-depleted of cellular calcium, the phorbol diesters stimulate MIvIQ cells (data not shown). In contrast, 100 nM basal prolactin release [l]. This dissociation PMAtreatment for 60 s inhibited the stimulation of suggests that phorbol diester-stimulated hormone the intracellular calcium concentration by BAY release does not require a rise in the intracellular 100

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that PMA and maitotoxin actually have a synergistic action to increase release of prolactin [4] despite the inhibition of maitotoxin-stimulated intracellular calcium levels by PMA (Fig. 4). The unique temporal patterns of response to BAY K8644 and maitotoxin suggest a difference in the underlying mechanisms of action by which these agents stimulate calcium influx. However, the similarity of the dopaminergic inhibition of each stimulant, and the similarity of their interactions with PMA and the PKC systems, may imply that there are some common mechanisms. The MMQ cell line will be valuable in elucidating the subcellular mechanisms that regulate secretory physiology. Acknowledgments M. Kathleen Borland assisted with cell culture, and we appreciate the editorial review of Boyd Zenner and the work of Rose Powell in preparing the manuscript. This work was supported in part by Biomedical Research Grsnt 5-SO7 RR05431 to ISL. and by NIH grants CA-38228 to ISL and CA-07535~28 to RMM.

References Judd AM. Login IS. Kovacs K. et al. (1988) Characterixation of the MMQ cell, a prolactin-secreting clonal cell line that is rqonsive to dopamine. Endocrinology, 123,2341-2350. Judd AM Login IS. Jarvis WD. Mackod RM. (1987) Impaired calcium mobilization in the 7315a prolactin-secreting pituitary tumour. Cell Calcium, 8, 189-196. Login IS. Judd AM. MacLeod RM. (1988) Dopamina inhibits calcium flux in the 73 15s prolactin-secreting pituitary tumour. Cell Calcium, 9.27-31. Judd AM. Login IS. MacLeodRM. (1989) Evidence that phorbol diester-sensitive protein kinase-C(s) may not be directly involved in secretagogue-stimulated prolactin release and ar&idonate liberation. Endocrinology, 125, 11361141.

S. McArdle CA. Huckle WR. Cmn PM. (1987) Phnbol esters mduce gonadotrope ~msiveness to protein kinase C activators but not to Ca -mobilizing secretagogues. J. Biol. Chem., 262.5028-5035. 6. Login Is. Judd AM. MacLeod RM. (1988) Dopamineqic leducthm of illtr&eIIular CaIciumz the role of caIcium infla. B&hem. Biophys. Res. Commun., 151.913-918. 7. Anderson JM, Yasumoto T, Croniu MJ. (1987) Intracellular fke cakium in rat anterior pituitary calls monitored by fura-2. Life Sci., 41, 519-526. 8. Malgaroli A. VaIlar L. Elahi FR. Pozxan T. Spada A. Meldolesi J. (1987) Dopamine inhibits cytosohc CI? increases in rat lactotroph cells: evidence of a dual mechanism of action. J. Biol. Chem.. 262,1392O-13927. 9. Login IS, Judd AM, Ma&sod RM. (1987) Activation of calcium channels by maitotoxin. Meth. Enzymol., 141, 63-79. 10. Login IS. Judd AM. Ctonin MJ. et al. (1985) Tha effects of maitotoxin on “Ca2+ fhu and hormone release in ClI-Israt pituitary cells. Endocrinology, 116.622627. 11. Takahashi M. Tatwmi M. Ohixumi Y. Yasumoto T (1983) Ca2+channel-activating function of maimtoxin, the most potent marine toxin knowq in clonal rat phecchromocytoma cells. J. Biol. Chem., 258, 1094410949. 12. Gusovsky F. Bitmn JA. Yasumoto T. Daly JW. (1990) Mechanism of maitotoxin-stimulated phosphoinositide breakdown in I-IL60 cells. J. Pharmacol. Exp. Ther., 252, 466-473. 13. Login IS. Panctaxio JJ. Kim YI. (1990) Dopamine enhanws a vohage-dependent transient Kt current in the MMQ cell, a clonal pituitary line expressing fbnctionaI Dz dopamine receptors. Brain Res., 506,33 l-334. 14. Naor 2. (1990) Further chamcterixation of protein lcinase-C subspecies in the hypothalamo-pituitary axis: differential activation by phorbol esters. Endocrinology, 126, 1521-1526. 15. Lagast H. Pozran T. Waldvogel FA. Law PD. (1984) Phorbol my&ate acetate stimulates ATP-dependent caIcium transport by the plasma membrane of neutrophils. J. Clin. Invest., 73, 878-883. 16. Di Virgilio F. Pozzan T. Wollheim CB. Vicentini LM. Meldolesi J. (1989 Tumor promoter phorbol myristaE acetate inhibits Ca ’ influx through voltagegated Ca channels in two secretory cell lines. PC12 and RlNm5F. J. Biol. Chem., 261, 32-38. Please send reprint requests to : Dr IS. Login, Department of Neurology, University of Virginia School of Medicine, Charlottesville. VA 22908, USA Received : 26 June 1990 Revised : 25 July 1990 Accepted : 27 August 1990