the blockers of the Ca2+ ATPase, thapsigargin and cyclopia- zonic acid. Since the present work is focused on intracellular stores, experiments were carried out ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 266, No. 30, Issue of October 25, pp. 20152-20158.1991 Printed in U.S.A.
Intracellular Ca2+Pools in PC12 Cells A UNIQUE, RAPIDLY EXCHANGING POOL ISSENSITIVE AND CAFFEINE-RYANODINE*
TO BOTHINOSITOL1,4,5-TRISPHOSPHATE
(Received for publication, February 21,1991)
Daniele ZacchettiS, Emilio ClementiS, Cristina Fasolatog, Paola LorenzonS, Michela Zottinig, Fabio GrohovazS, GuidoFumagalliS, Tullio PozzanEj, and JacopoMeldolesiSIl From the $Departmentof Pharmacology, Consiglio Nazionale delle Ricerche, Cytopharmacology and Bruno Ceccarelli Centers, Scientific Institute S a n Raffaele, University of Milano, 20132 Milano and the §Instituteof General Pathology, Consiglio Nazionale delle Ricerche Centerof Biomembranes, University of Padova, Padova 35100, Italy
Release of Ca2+from intracellular stores was studied proaching, andin some cases exceeding, the M level (1, in the parent PC12 cell line and in recently isolated 2). Two different Ca2+sources exist to sustain these events: clones sensitive or insensitive to caffeine. In the caf- the extracellular space, dynamically connected to the cytosol feine-sensitive cells the cytosolic free Ca2+concentra- via various types of Ca2+-permeablechannels (3-5), and raption ([Ca2+Ii)responses by the xanthine drug and by idly exchanging intracellular stores,capable of releasing their stimulants of receptors coupled to inositol 1,4,5-tris- segregated Ca2+in response to appropriate stimuli (5-9). Until phosphate (Ins-P3) generation (bradykinin, ATP) de- 1982, the only such store known in some detail was the pend on separate pathways because l ) caffeine does sarcoplasmic reticulum (SR) of striated muscle fibers, a comnot stimulate the hydrolysis of phosphatidylinositol 4,s-bisphosphate and2) Ca2+-inducedCa2+release, the plex system of anastomized tubules and cisternae endowed process activated by caffeine, plays no major role in with high conductance, strategically located channels. These the Ins-Pa-induced Caz+ mobilization. Although dis- channels are specifically affected and maintained open in a tinct, these two mechanisms converge onto the same low conductance substate, by the plant alkaloid, ryanodine, Ca2+ store. In fact 1) the [Ca2+]iresponses by receptor and are therefore referred to asthe ryanodine receptors agonists andcaffeine were not additive; 2) either type (RyRs). Activation of these channels can be triggered by an of agent reduced (up to complete inhibition) the re- increase in [Ca2+Ii(calcium-induced Ca2+release, CICR), a sponse to a subsequent administration of the same or process stimulated by another alkaloid, caffeine (5, 6, 8, 10). In non-muscle cells, the development of knowledge regardthe other agent; 3)all these responses were prevented by selective Ca2+ ATPase blockers; 4) ryanodine, ing Ca2+ stores has been slow compared to muscle fibers. which affects the intracellularCa2+channel sensitive Except for a few reports on the effects of caffeine and ryanoto caffeine, also induced depletion of the receptordine in neurons (11, 12), intense interest has in fact begun sensitive Ca2+pool; 5) in the 10 PC12 clones tested, only with the discovery of inositol 1,4,5-trisphosphate (Inssensitivity tocaffeine paralleled ryanodine sensitivity. P3) (13). Presently, the intracellular Ca2+release induced by Therefore, PC12 cells, similar to some smooth muscle Ins-P3 is believed to account for part of the [Ca2+Ii-dependent fibers but at variance with neurons and other secretory effects elicited by the activation of surface receptors coupled cells, express a single, rapidly exchanging Ca2+store with the hydrolysis of phosphatidylinositol 4,5-bisphosphate in which two distinct intracellular Ca2+channels, Le. (PtIns-Pz) (7).During the last few years, however, interest in the receptors for caffeine-ryanodine and Ins-P3, apthe caffeine- and ryanodine-sensitive release has gained mopear to be colocalized. mentum. Clear responses to these two alkaloids have been demonstrated in a variety of cell types such as smooth muscle cells (14-17), various types of neurons (18-23), endocrine cells In eukaryotic cells, a variety of functions are regulated by (24-26), and theacinar cells of the pancreas and parotidgland the rapid increase of the cytosolic free Caz+ concentration (27, 28). ([CaZ+li)’from resting levels of around M to values apThe existence of two rapidly exchanging Ca2+release processes within individual cells raises questions about their sub* This work was supported in part by grants from the Consiglio cellular localization and functional interconnection. In Nazionale delle Ricerche, the Target Project on Biotechnology and Biostrumentation, and the Biotechnology-Molecular Biology Com- smooth muscle cells, results have been reported suggesting mittee Project on the Biology and Pathology of Ca2+.The costs of the coexpression of both RyRs and Ins-P3 receptors (Inspublication of this article were defrayed in part by the payment of P3Rs) in single a organelle which, in analogy with the striated page charges. This article must therefore be hereby marked “aduer- muscle, is also referred to as the SR(15, 16; see however 29). tisement” in accordance with 18U.S.C. Section 1734 solelyto indicate In contrast, in neurons (19, 30) and secretory cells (24-28), this fact. n To whom correspondence should be addressed Dept. of Phar- extensive evidence has been interpreted to indicate the existmacology, San Raffaele Institute, Via Olgettina, 60, 20132 Milano, ence of two distinct Ca2+stores. The intracellular organelles corresponding to these stores have not yet been clearly idenItaly. The abbreviations used are: [Ca2+],,cytosolic free Ca2+concentra- tified. tion; SR, sarcoplasmic reticulum; RyR, ryanodine receptor; CICR, In order to investigate cellular Ca2+pools, we have employed Caz’-induced Ca2+ release; Ins-Ps, Ins-PsR, inositol 1,4,5-trisphos- a cell line, PC12, which expresses properties common to both phate and its receptor; PtIns-P2, phosphatidylinositol 4,5-bisphosphate; Bk, bradykinin; [Ca2+l0,extracellular Ca2+ concentration; neurons andneurosecretory cells (31), andwhich is one of the EGTA, [ethylenebix(oxyethylenenitrilo)]tetraaceticacid; Hepes, 4- experimental models most often employed. A clear advantage in our studies was the availability of not only the parent line, (2-hydroxyethyl)-l-piperazineethanesulfonic acid.
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A Ca2+Pool Sensitive to Ins-P3 and Caffeine-Ryamdine which is known to be heterogeneous (32), but also of recently isolated PC12 clones differing, among other features, in their responsiveness to caffeine and ryanodine. The results we have obtained unambiguously demonstrate that in PC12 cells the Ca2+release responses elicited by caffeine-ryanodine and InsP3 are due to the discharge of one and the same Caz+ pool. This pool can be readily distinguished from the other, slowly turning over pools of PC12 cells (described in the accompanying paper, Ref. 33) because of its kinetic and physiopharmacological properties. EXPERIMENTAL PROCEDURES
The PC12 cell line, initially established by Greene and Tischler (31), hasbeen extensively employed in the laboratory. In our experience and that of others (see Ref. 34), conventional procedures for clone isolation from this line have often been unsuccessful. In order to develop a large panel of clones, we have therefore relied on exogenous gene expression followed by antibiotic resistance selection. Cells of the parent line were transfected with the pMV7 vector containing the neomycin resistance gene (generous gift of Dr. H. Bourne, San Francisco, CA), as described by Schweitzer and Kelly (35). Three X lo6 cells were plated on 10-cm polyornithine-coated plastic tissue culture dishes. One day later, the plasmid DNA (20 pg) was added to the cells prerinsed with the HeBS medium in the form of a calcium phosphate precipitate. After 20 min of incubation at room temperature, cells were supplemented with Dulbecco's modified Eagle's medium containing 5% fetal calf serum, incubated a t 37 "C for 6 h, osmotically shocked with 25% glycerol in Dulbecco's modified Eagle's medium for 1 min, and then rapidly washed. The entire population of cells was grown in standard culture medium for 72 h before adding 0.8mg/ml of G418 (Geneticin) to select for stable transfectants. These cells were then collected as individual clones. The parent cell line and theclones were cultured a t 37 "C in Dulbecco's modified Eagle's medium containing 2mM glutamine, 10% horse serum, and 5% fetal calf serum, in a humidified atmosphere with 5% CO,. Cells were plated weekly 1:4 in 10-cm Petri dishes. [Ca*+]i Measurements-At the beginning of the experiment, cell monolayers were detached from Petri dishes by applying a gentle flow of incubation medium (Krebs-Ringer-Hepes (KRH)) containing (in mmol/liter): NaCl, 125; KC], 5; KHzP04, 1.2; MgS04, 1.2;CaCl,,2; glucose, 6; Hepes-NaOH buffer, pH 7.4, 25. Cells were then washed in the same medium and loaded for 30 min at 37 "C with the Ca2+ sensitive dyes (fura-2 and, in a few experiments, quin2) added as acetoxymethylesters at final concentrations of2-5 and 10-20 p ~ respectively. After rinsing once with KRH medium, the loaded cell suspensions were divided into aliquots of -0.5-1 X lo6cells and were kept at room temperature until use (maximum 2.5 h). For [Ca2+Ii measurement these aliquots were resuspended by gentle swirling in 1.5 ml of the incubation medium supplemented with 250 p~ sulphynpirazone (to prevent dye leakage), transferred to a thermostatted cuvette (37 "C), maintained under continuous stirring, and analyzed in a Perkin Elmer LS-5B fluorimeter as recommended by Grynkiewicz et al. (36). In most experiments, 1-2 min before the addition of stimulants, excess EGTA (usually 3 mM) was added to the cell suspension to convert the complete Ca2+-containingKRH into the Ca*+-freemedium referred to in the text. In the experimental conditions used, this addition caused only small changes of the medium pH (maximum 0.15 units). At the fura-2 wavelength, caffeine is weakly fluorescent. This artifact (2-10% of the total signal) was thoroughly evaluated and corrected graphically. All [Caz+]; results presented in this paper are representative of at least three different experiments with results varying by 4 5 % . Zns-P3 Generation-Monolayer cultures were first labeled for 24 h with [3H]myoinositol (1 pCi/ml), then the cells were detached, and the suspensions exposed to the treatmentsdescribed in Fig. 3. Reactions were stopped with trichloroacetic acid, and radioactive inositol phosphates were separated by ion-exchange and high performance liquid chromatography (37) columns. Materials-Fura-2, quin2, and ryanodine were purchased from Calbiochem, San Diego, CA, and thapsigargin from LC Services Corp., Woburn, MA. The Bz receptor inhibitor, Ac-D-Ar$[Hyp3,~Phe',Leua]bradykinin was fromNovabiochem, Laufelfingen, Switzerland; [3H]myo-inositolfrom Amersham Int., United Kingdom; geneticin, culture sera, and media from GIBCO. Caffeine, bradykinin
20153
(Bk), ATP, carbachol, forskolin, phorbolmyristate acetate, cyclopiazonic acid, and theremaining chemicals were purchased from Sigma. RESULTS
As demonstrated by previous studies (38, 39), the [Ca2+]; increases elicited in PC12 cells by the activation of receptors coupled to PtIns-Pz hydrolysis are the result of two components. The first, originating from intracellular stores, is responsible for the bulk of the initial peak, and thesecond, from the extracellular space, for the subsequent, long lasting plateau. Recent evidence (reviewed in Ref. 40) demonstrates that the dual (intra- and extracellular) origin of the [Ca2+];responses is valid also for other substances employed here: the alkaloids addressed to the RyR, ryanodine and caffeine, and the blockers of the Ca2+ATPase, thapsigargin and cyclopiazonic acid. Since the present work is focused on intracellular stores, experiments were carried out with cells suspended in a medium supplemented with excess EGTA shortly before drug addition, i.e. under conditions in which [Ca2+l0is very low (
