Vol. 188, No. 3, 1992 November
BIOCHEMICAL
AND BIOPHYSICAL
16, 1992
ATP-INDUCED
Ca2+
INFLUX
SENSITIVE Emilio
IS
REGULATED
VIA
I RESEARCH COMMUNICATIO,NS Pages 1184-1190
A PERTUSSIS
TOXIN-
MECHANISM IN A PC12 CELL CLONE
Clementi,
Heimo
Scheer+,
and Jacopo
Martha
Raichman
Meldolesi
Department of Pharmacology, CNR Center of Cytopharmacology, B.Ceccarelli Center and S.Raffaele Scientific Institute, University of Milano, Italy 20132 .tDepartment
of
Pharmacology, University Canada H3C 357
of Montreal,
Received September 30, 1992 SUMMARY A PC12 cell clone that responds to ATP with polyphosphoinositide h%d+rolysis and with a m?qked, biphasic intracellular free Ca concentration ([Ca Ii) response (composed by release from intracellular stores accompanied by stimulated influx from the medium), was pretreated with pertussis toxin. In the pretreated cells the responses induced by ATP we e differently modified. Polyphosphoinositide hydro$y+sis and Ca Z+ release were moderately inhibited whereas Ca influx was enhanced. Pharmacological experiments revealed the influx voltage-gated nor second enhancement to be sust ined by neither messenger-operated CaaL+ channels. a channel of the Rather, receptor-operated type activated by ATP (Pzw receptor) appears to work under the negative control of a pertussis toxin-sensitive G protein, acting presumably by direct interaction with the channel in the plane of the plasma membrane. 0 1992 Academic Press, Inc.
A variety of transmembrane signalling events are known to be regulated by heterotrimeric GTP binding proteins (G proteins) that couple activated receptors to their functional effecters in the plasma membrane. Among the G protein-regulated effecters (l4) are enzymes, such as adenylate and various cyclase phospholipases (C,D,A2), and ion channels (1,2). These include K+ and Ca2+ channels sensitive to membrane potential (voltageoperated channels, VOCs) and possibly also a subset of the cation channels activated by receptors that induce hydrolysis of polyphosphoinositides (PPI) (2-4). These last channels, also referred to as second messenger operated channels, SMOCs (5), are distinct from an additional class (receptor-operated channels, ROCs) in which the channel and receptor activities are combined in a single molecule (3,6). up until now the members of the ROC class (including the various forms of the nicotinic and aminoacid 0006-291X/92 $4.00 Copyright All rights
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
1184
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188,
No.
receptors; receptor proteins. the case ROC, in proteins therefore MATERIALS
3, 1992
BIOCHEMICAL
the 5HT3 receptor of ATP) had never The evidence herewith for a Ca2+ influx a recently isolated in the regulation wider than previously
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of serotonin and the P2w purinergic been shown to be regulated via G reported shows however this to be activated by ATP, apparently via a The role of G PC12 cell clone. of transmembrane signalling appears envisaged.
AND METHODS
Measurements of [Ca2+ Ii. The ~~12 clone investigated, denominated employed for its isolation and culturing 15, and the procedures have been described elsewhere (7,8). At the beginning of the experiments the ~~12 cell monolayers were detached and the cells dissociated mechanically by gentle flow of incubation medium (KRH, containing in mmoles/liter: NaCl, 125; KCl, 5; MgSO4, 1.2; KH PO 1.2; CaC12, 2; glucose, 6; Hepes-NaOH buffer, pH 7.4, 257. f?;, e cell suspensions thus obtained were loaded with furafuraby incubation with the membrane-permeant precursor, acetoxymethylester (2-5 uM, 30 min, 37" C), followed by thorough washing. Aliquots of 0.5-1~10~ loaded cells were suspended by gentle swirling in 250 ,uM 1.5 ml of KRH (containing sulfinpyrazone, either complete or free to reduce dye leakage), of added CaC12 and supplemented with 1 mM EGTA. They were then transferred to a thermostatted (37 "c) cuvette, maintained under continuous stirring, and analyzed in a Pelkin-Elmer LS 5B fluorimeter as described elsewhere (7,8). Measurements of IP3 generation. Cells suspended in Basal Medium Eagle supplemented with 1% dialysed geta. calf serum, were labeled for 24 h with 2 uCi/ml of myo[2H] inositol, then gently detached from the petri dishes, washe 3 times in the KRH medium and warmed at 37" C. Aliquots of 2x10 % cells were then incubated for 10 set with either ATP or GTP as described in the results. The reaction was stopped by addition of 50% trichloroacetic acid, samples were centrifuged and the supernatants extracted 3 times with diethylether. Radioactive inositol phosphates, separated by ion-exchange columns, were counted in a Beckman LS7500 counter (see ref. 9 for details). Materials. Pertussis toxin (PTx) was the kind gift of Dr R, Rappuoli, IRIS, Siena, Italy; and SC38249 of Dr. H. Ruegg, Sandoz AG Basel, Switzerland. Furaacetoxymethylester was purchased from Calbiochem, San Diegy, CA, U.S.A.; w-conotoxin from Bachem, Bubendorff, Switzerland; H-inositol from Amersham, U.K.; culture sera and media from GIBCO, all other chemicals from Sigma. RESULTS
The PC12 clone we have employed (j/15) differs from other clones and from the mixed parent line employed in previous studies (7,lO) because it responds to ATP with a marked [Ca2+]i response, which in its initial phase relies primarily on inositol 1,4,5-trisphosphate (IP3)-sustained Ca2+ release from intracellular stores. The continuous traces in Fig. 1, a and b, illustrate this property as revealed by parallel furaexperiments carried out in Ca2+-containing and Ca2+-free medium, 1185
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BIOCHEMICAL
AND BIOPHYSICAL
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42s 298
106-
4
ATP
b
448 340155118109
1dIi -
1. [Ca2+li responses induced by ATP (100 UM) in PC12 cell clone 15, naive or pretreated with PTx (300 rig/ml, overnight) (continuous and dashed lines, respectively). The ifaces shown in a) were obtained with ce l+s suspended in the Ca -containing, those in b) in tp+e Ca 3 -free, EGTAcontaining KRH medium. Where indicated, Ca was reintroduced to the latter medium at the final free concentration of 2 mu. Fig.
respectively. Notice that the initial peak is almost completely maintained in the Ca2+-free medium, indicating its intracellular origin. In the same panels the dashed lines show that the initial peak was partially reduced (on the average - 44.3 5 2.2%; n=6) when the cell monolayers were pretreated with PTx (300 rig/ml; overnight). A similar degree of inhibition was observed with the ATP-induced IP3 accumulation (Table I). The subsequent phase of the [Ca2+li response to ATP was differently modified by PTx. In fact, the slowly declining visible when the cells were incubated in the [Ca'+]i plateau, trace b) medium, was Ca2+-containing (but not in the Ca2+-free, not decreased but rather enhanced for up to 6 min after ATP in the PTx-pretreated cells (t 57.2 t 3.8%; n=6). Also enhanced (+ 79.5 t- 7.0%; n=5) was the [CaZ+]i increase induced by the reintroduction of CaC12 in the Cazt-free medium, 2-3 min after the application of ATP (Fig. lb). Based on extensive previous evidence in PC12 cells (8,10), the persistent phase of the [Ca2+] i responses triggered by ATP depends not on intracellular however stores but on the influx of extracellular Ca 2+ , occurring via multiple pathways: Ca2+ VOCs, activated as the consequence of follows the plasma membrane depolarization; SMOCs, whose opening such as the activation of the PPI hydrolysis-coupled receptors, 1186
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TABLE I
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: ATP and GTP-induced IPg generation and untreated Pc12 clone 15 cells
in
PTx-
treated
Treatment
IP3 generation (% increase over
PTX
ATP 100 FM +
ATP 100 FM GTP
1 mM
GTP
1 mM
+
basal)
25.84
-+ 2.0
17.5
& 1.1
13.73
-+ 1.8
9.25
-+ 0.9
Parallel aliquots of cells were incubated for 10 set Results are averages of 3 experiments + SD. Basal IP3 radioactivity was 1529 -+ 243 cpm/sample.receptor; and ROCs (Pzw receptors) (10-12). The experiments p2Y of Fig. 2 were therefore carried out to identify which of these for the PTx-induced enhancement. components is responsible Pretreatment of our ~~12 cell clone with a mixture of blockers
a 385-
r ‘, ‘. 1 , loo-
-..
-r.-
I -.-
r -_- -,_
J---AP
291-
104-
Jbz?:
4
ATP
Fig. 2. Effects of channel blockers and PMA on the [Ca2+]i responses induced by ATP (100 PM) in PC12 cells clone 15 without (a) or with (b) PTx pretreatment (300 n /ml, overnight). Continuous traces illustrate the [Ca s +I1 responses in parallel suspensions of cells additioned with a mixture of verapamil (20 PM) and O-conotoxin (1 PM) 2 min before ATP (100 FM); dashed lines illustrate similar experiments in which however SC38249 (60 FM) was also added together with the other blockers. The combined effect of pretreatment with SC38249 and PMA (100 nM) is also shown (dotted lines). 1187
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AND BIOPHYSICAL
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144101-
J+B
b
4
GTP
lmin Fig. 3. [Ca2+li responses induced by GTP (1 mM) in PC12 cell clone 15, naive (a) or pretreated with PTx (b). The effects of pretreatment with sC38249, without and with PMA, are shown by the dashed and dotted lines, respectively. Experimental conditions as in Fig. 2,
(verapamil and o--conotoxin) at concentrations known to inhibit completely the Ca2+ VOCs (of the L and N type) expressed in PC12 cells (13,14), decreased only moderately the ATP-induced, influxsustained [Ca2+li response, both in PTx-treated and untreated Cells (-9.3 + 0.9 and -13.8 + l.O%, respectively; n=El; compare Fig. la anb2). In contrast these blockers were without appreciable effect on the PTx-sustained enhancement. On the other hand, with the imidazole derivative X38249. a blocker of both Ca2+ VOCs and SMOCs which however does not affect ROCs the ATP-induced influx response was inhibited almost (10,15,16), completely, however only in naive cells (trace a, dashed line). In fact, in the cells pretreated with PTx part of the ATP-induced influx response was maintained and could be eliminated only when phorbol myristate acetate (PMA) was administered combined to SC38249 (trace b, dotted line). In a final group of experiments (Fig. 3) ATP was replaced by GTP, a nucleotide known to activate PPI hydrolysis-coupled P2 but not the ROC P2w receptor (10). As shown by traces a and b 0 f Fig. 3 the influx responses induced by GTP differed from those by ATP because, they were completely blocked by on the one hand, SC38249; on the other hand, they were not enhanced by the PTx pretreatment of the cells. DISCUSSION
The simplest explanation of our results clone 15 expresses at least two types induces PPI hydrolysis, and the ROC Pzw, 1188
is as follows. of receptors: activated
The PC12 P2yr that
simultaneously
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AND BIOPHYSICAL
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C after addition of ATP. Coupling of P2y to its phospholipase effector appears to be mediated by at least two G proteins, one insensitive the other sensitive to PTx. PPI hydrolysis and the ensuing [Ca2+]. responses, i.e. intracellular Ca2+ release and Ca2+ influx thcough the SMOCs, are stimulated primarily via the first G protein, however with a minor contribution of the second. ATP stimulates Ca2+ influx via its interaction Concomitantly, with the P2w ROC, and this latter channel is negatively controlled (desensitized) by the PTx-sensitive G protein activated by the P2y receptor. When in fact the G protein is influx inactivated by pretreatment of the cells with the toxin, persists several minutes longer than in naive cells. The involvement of the P2w receptor in the PTx- enhanced Ca2+ influx response is indicated by the insensitivity of this toxin effect to the blockers of both VOcs and SMOCs, and by the lack of activation by GTP. Both these properties were previously reported for the ATP-activated ROC of PC12 cells (10). As far as the mechanism of the ROC desensitization, we favor the direct interaction of the channel with the PTx-sensitive G protein subunit in the plane of the plasmalemma, analogously to what previously suggested for both L and N type Ca2+ VOCs (2)” An indirect mechanism via the generation of diacylglycerol and activation of protein kinase C appears in fact unlikely because in our experimental conditions these processes are expected to depend primarily on PPI hydrolysis which is moderately inhibited (and not enhanced) by the toxin pretreatment of the cells. Two possible hypotheses can be proposed to explain why the now observed enhancement by PTx of the ATP-induced Ca 2+ influx in PC12 cell clone 15, had not been observed in a' previous study On the one hand, the P responses in the mixed PC12 (10). 2Y population employed in ref. 10 were much weaker than in our clone. The G protein mediated ROC inhibition, although presumably present, might have thus been too small to be detected. Alternatively, the P2w ROC expressed in our clone might be different from the form predominating in the mixed population, as suggested also by its sensitivity to PMA, that was not observed in the previous study (10). Indeed, heterogeneity of Pzw receptors has been reported (see 6). The possibility, recently suggested in Pc12 cells, of an identity of the ATP and nicotinic ROCs (17) appears unlikely because our clones do not express a functioning nicotinic receptor (7). Whatever the explanation, our present results provide evidence for a new ROC regulatory mechanism that deserves to be specifically investigated also with other channels of the family, different from the P2w receptor activated by ATP. ACKNOWLEDGMENTS
secretarial acknowledged. advice and
The assistance We thank discussions.
technical help of G. Racchetti and of L. Di Giorgio are gratefully T. Pozzan, University of Padova, Supported in part by grants from 1189
the for the
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1992
Consiglio Nazionale and Bioinstruments
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