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Protein kinase C differentially inhibits muscarinic receptor operated Ca2+ release and entry in human salivary cells
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 1062-1069
Vo1..152, No. 3,1988 May 16,1988
PROTEIN KINASE C DIFFERENTIALLY INHIBITS MUSCARINIC RECEPTOR OPERATEDCa2+ RELEASE AND ENTRY IN HUMAN SALIVARY CELLS Xinjun He, Xiaozai Wu and Bruce J. Baum* Clinical Investigations and Patient Care Branch, National I n s t i t u t e of Dental Research, National I n s t i t u t e s of Health Bethesda, Maryland 20892 Received March 28, 1988
We investigated the effects of phorbol myristate acetate on muscarinic receptor-induced Caz+ release from i n t r a c e T l u l a r stores and e x t r a c e l l u l a r entry_in a human salivary duct cell Iine,^HSG-PA. Phorbol myristate acetate (~IO-/M) blocked both Ca~+ release and Caz+ entry induced by the muscarinic agonist carbachol. This blockade was the result of the activation of protein kinase C since 4~-phorbol 12,13-didecanoate, which lacks the a b i l i t y to activate protein kinase C, did not i n h i b i t Caz+ mobilization responses to carba~hol. Importantly, at lower phorbol myristate acetate concentrations (~IO-~M), carbachol-induced Caz+ release was blocked, but carbachol-induced c~Z+ entry was maintained. These results show that carbachol-induced Caz+ entry does not occur via an i n t r a c e l l u l a r store and that protein kinas~±C plays a role in a feedback control mechanism for muscarinic-induced CaLT mobilization at d i f f e r e n t levels. © 1988AcademicP..... Znc.
Many reports have demonstrated recently that phorbol esters can i n h i b i t receptor-induced Ca2+ mobilization via activation of PKC in various cell types (for reviews, see r e f .
1-3).
Receptor-induced Ca2+ mobilization is the result
of two phases, release of Ca2+ from an i n t r a c e l l u l a r e x t r a c e l l u l a r Ca2+ across the plasma membrane (3,4).
store and entry of To our knowledge, there
are only two reports showing that phorbol esters p r e f e r e n t i a l l y agonist-induced Ca2+ release (5,6).
inhibit
The two phases of receptor-induced Ca2+
*To whom correspondence should be addressed Abbreviations used: cytosolic free Ca2+ concentration ([Ca2+]i); phosphatidylinositol 4,5-bisphosphate (PIP?); i n o s i t o l trisphosphate (IP3); protein kinase C (PKC); phospholipase C (P[C); phorbol myristate acetate (PMA); 4~-phorbol 12,13-didecanoate (4~-PDD); carbachol (Cch); balanced salt solution (BSS); bovine serum albumin (BSA); 4 - ( 2 - h y d r o x y e t h y l ) - l piperazineethane-sulfonic acid (HEPES); ethylene glycol bis (~-aminoethyl ether)-N,N,N', N ' - t e t r a a c e t i c acid (EGTA); the acetoxymethyl ester of quin-2 (Quin 2/AM); GTP bi~ding protein coupled to phospholipase C (Np); GTP binding protein mediating Ca~T i n f l u x (Ni); Eagle's minimal essential medium (EMEM). 0006-291X/88
$1.50
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m o b i l i z a t i o n can be independently examined by stimulating c e l l s with agonists in Ca2+-free media (measurement of i n t r a c e l l u l a r
Ca2+ release) and t h e r e a f t e r
reintroducing Ca2÷ to the incubation media (measurement of e x t r a c e l l u l a r Ca2+ entry)
(7-10).
We have used t h i s approach to examine the e f f e c t s of PMA on
muscarinic receptor induced Ca2+ release and entry in a human s a l i v a r y duct cell l i n e ,
HSG-PA ( i i ) o
We report here that PMA (~10-7MI blocks both the Ca2+
release and entry induced by the muscarinic agonist Cch via a c t i v a t i o n of PKCo However, at lower PMA concentrations (~IO-9M), Cch-induced Ca2+ release is blocked, but Cch-induced Ca2+ entry is maintained.
These results show that
Cch-induced Ca2+ entry does not occur via an i n t r a c e l l u l a r
store and that PKC
plays a role in a feedback control mechanism for Cch-induced Ca2÷ m o b i l i z a t i o n at d i f f e r e n t
levels. MATERIALS AND METHODS
Materials - Eagle's minimal essential medium, newborn c a l f serum, p e n i c i l l i n G and streptomycin s u l f a t e were purchased from B i o f l u i d s . Hank's balanced s a l t solution was purchased from GIBCO and Quin 2/AM was purchased from Calbiochem. The f o l l o w i n g compounds were purchased from Sigma: EGTA, HEPES, 6-D(+)glucose, BSA, Cch, atropine s u l f a t e , PMA and 4~-PDD. Cell Culture - Experiments were performed on human submandibular duct c e l l s , HSG-PA, a kind g i f t from Dr. Mitsunobu Sato (11). HSG-PA c e l l s were cultured in EMEM, supplemented with 10% newborn c a l f serum, 100 ~g ml -~ p e n i c i l l i n G and streptomycin s u l f a t e , at 37°C in a humidified 5% CO2 atmosphere• HSG-PA c e l l s used in these studies were at passages 6 to 36. Measurement of I n t r a c e l l u l a r Ca2+ Concentration - Cells were grown to confluence and detached with Ca2+, Mg~+-free Hank's balanced s a l t solution containing 4 mM EGTA and 10 mM HEPES, pH 7.4 f o r 10 min at 37%. The c e l l s were then c o l l e c t e d by b r i e f c e n t r i f u g a t i o n (15 sec) and resuspended in BSS (NaCl 130; KCI 5; MgCl2 1 O; CaCl~ 1 5; HEPES 20 buffered to pH 7 4 with Tris base; In ~M) contalnlng 10 mM glucose. The cell suspensions (about 5x106 c e l l s ml -z) were preincubated at 37°C f o r 5 min and then were incubated with 50 ~M Quin 2/AM for a f u r t h e r 20 min. They were washed twice by b r i e f c e n t r i f u g a t i o n in BSS• Thereafter, t~e c e l l s were resuspended in BSS containing 10 mM glucose and I mg ml -~ BSA and kept at room temperature• Just before fluorescence measurements were performed, the c e l l s were again centrifuged and resuspended (about lx106 c e l l s ml - I ) in Ca2+-free BSS containing glucose plus BSA. Caz+ was added as indicated• Fluorescence was measured at 37°C in a SLM-8000 s p e c t r o f l u o r i m e t e r as previously described (12). C a l i b r a t i o n of the signal and c a l c u l a t i o n of c y t o s o l i c Caz+ were as described by Tsien et al (13).
RESULTS AND DISCUSSION HSG-PA c e l l membranes possess a single class of high a f f i n i t y
muscarinic
receptors (Kd=O.17±O.08 nM, Bmax=38±3 fmol/mg protein) as measured using a [3H]-quinuclidinylbenzilate
binding assay (not shown)•
The muscarinic agonist
Cch increases production of IP 3 and elevates [Ca2+]i in HSG-PA c e l l s (unpublished data).
The two phases of Ca2+ m o b i l i z a t i o n were observed
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separately when HSG-PA cells were exposed to Cch in Ca2+ free media and then Ca2+ was reintroduced (Fig. l a ) . from an i n i t i a l
Cch resulted in a rapid increase of [Ca2+]i
value of 117±11 to 226±24 nM (mean±SEM; n=13).
A second,
slower increase of [Ca2+]i was evoked when Ca2+ was reintroduced into the media (final
[Ca2+], 1.5 mM) 5 min after stimulation with Cch (Fig. l a ) , by
which time [Ca2+]i had returned to i n i t i a l
levels.
This second increase of
[Ca2+]i reached a maximum (277±24 nM;n=ll) at about 2 min a f t e r Ca2+ readdition and was completely blocked when the muscarinic antagonist atropine was added j u s t before reintroduction of Ca2+ (Fig. i b ) .
Both [Ca2+]i
responses were blocked i f atropine was added before the cells were exposed to Cch (Fig. I c ) .
The f i r s t
peak of the [Ca2+]i rise was not affected by the
inorganic Ca2+ channel blocker La 3+ (25 ~M; not shown), and represents agonist-induced Ca2+ release from the i n t r a c e l l u l a r
store.
The second peak
was abolished by La 3+, but was not affected by blockers of voltage-operated Ca2+ channels such as verapamil, diltiazem and nifedipine
(IO-6M).
This
response was also dependent on Ca2+ concentration in the media (not shown). Therefore, the second peak of the [Ca2+]i rise presumably represents Ca2+ entry across the plasma membrane, the so-called receptor-operated Ca2+ channel (3).
When HSG-PA cells were exposed to PMA (10-7M, 5 min p r i o r to Cch
a d d i t i o n ) , there was an almost complete i n h i b i t i o n of Cch-induced i n t r a c e l l u l a r Ca2+ release and e x t r a c e l l u l a r Ca2+ entry (Fig. l d ) .
4~-PDD,
which lacks the a b i l i t y to activate PKC, did not i n h i b i t any responses to Cch (Fig. l e ) .
Several investigators (5,6,14-16) have observed similar results
with other c e l l s , i . e . ,
phorbol esters can block both phases of agonist-
induced Ca2+ mobilization. We next examined the effects of d i f f e r e n t
[PMA] on both the Ca2+ release
and Ca2+ entry induced by Cch. The resulting i n h i b i t i o n patterns indicate differential
effects of PMA on these two responses (Fig. 2).
At low [PMA],
Cch-induced i n t r a c e l l u l a r Ca2+ release was s u b s t a n t i a l l y blocked and t h i s i n h i b i t o r y action was r e l a t i v e l y insensitive to increasing concentrations of PMA (from 5xlO -10 to 5x10 -5 M).
In contrast, the i n h i b i t i o n by PMA of Cch1064
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2
Cch
Atro Caz+
Atro Cch
Ca2+ 175150-
T
125. C~a2÷
Ceh
n-6 .E
e
10075.
2
50-
I
25,
0t~
C)
100
Fig. i
I 200
300
i 400
0 -10
500
Time, Sec
6
1
I
I
I
I
I
I
-g
-8
-7
-6
-5
-4
@
Log [PMA], M
Carbachol-induced i n t r a c e l l u l a r Ca2+ transients and the effects of PMA. a. Control c e l l s were preincubated with 0.01% dimethylsulfoxide (the vehicle used with PMA, below) for 5 min; Cch (10 -4 M) and Caz+ (1.5 mM) were added at arrows, b. The experimental conditions were • ii the same as In a 11 but atrop]ne (Atro, i0 -~~ M) was added 20 sec b e f o r e a d d i t i o n o f CaZ+ . c . A t r o p i n e ( I 0 -D M) was added 20 s e c . b e f o r e t h e ~ d d i t i o n o f Cch. d and e . C e l l s were t r e a t e d w i t h PMA o r 4~-PDD (10 - I M) f o r 5 m i n , r e s p e c t i v e l y , t h e n Cch and Ca 2+ were added as i n " a " .
Fig. 2
I n h i b i t i o n o£ the Cch-induced Ca2+ release from an i n t r a c e l l u l a r store and CaZ+ entry across the plasma membrane by d i f f e r e n t [ P ~ ] . Experimental conditions were as described in the t e x t . The [Ca:T]i response was calculated for each phase as a percentage of the maximal Cch-induced [CaZ+]i rise, i . e . , that observed without PMA present. Each data point is the mean±SEMof at least 3 separate experiments performed on d i f f e r e n t cell preparations. (o o) refers to i n t r ~ c e l l u l a r calcium release at 20 sec a f t e r addition of Cch (IO-~M), (~ e) refers to e x t r a c e l l u l a r calcium entry at 2 min a f t e r reintroduction of CaLT (1.5 mM).
induced e x t r a c e l l u l a r
Ca2+ e n t r y showed a d i f f e r e n t
concentration-dependence
p a t t e r n ( F i g . 2), w i t h h a l f - m a x i m a l and maximal i n h i b i t i o n IO-7M PMA, r e s p e c t i v e l y .
at about 10 -8 and
This PMA c o n c e n t r a t i o n range is considered s p e c i f i c
f o r the a c t i v a t i o n o f PKC (17).
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A p a r t i c u l a r l y interesting finding in these experiments is that at lower concentrations of PMA (~IO-9M), Cch-induced Ca2+ release is blocked substantially,
but Cch-induced Ca2+ entry is e s s e n t i a l l y unchanged (Fig. 2).
Valone and Johnson have recently shown in platelets that agonist-induced intracellular
Ca2+ release and divalent cation i n f l u x are d i f f e r e n t i a l l y
affected by PMA (5).
Also, Owen has shown in murine lymphocytes that phorbol
esters p r e f e r e n t i a l l y
i n h i b i t anti-immunoglobulin triggered Ca2+ release
(6).
In addition,
i t has been observed that pertussis toxin blocks
angiotensin I I induced Ca2+ i n f l u x without affecting Ca2+ release in adrenal glomerulosa cells (7).
These observations indicate that receptor-induced Ca2+
entry into the cytosol does not occur through an i n t r a c e l l u l a r proposed by Putney (18).
store, as
These data also suggest that PKC may regulate
receptor-induced Ca2+ release and entry via feedback control mechanisms at different
sites.
Figure 3 shows a model for receptor-induced Ca2+ mobilization and i t s feedback control by PKC that has been modified from that of Putney (18).
In
addition to the findings described above, three other lines of evidence support this model.
F i r s t , the r e f i l l i n g
of the receptor-regulated
i n t r a c e l l u l a r Ca2+ store proceeds slowly and is dependent on e x t r a c e l l u l a r Ca2+ (8,9,19). stimulation
Significantly,
(8).
i t occurs j u s t a f t e r termination of agonist
Since receptor-induced Ca2+ entry is absolutely blocked by
antagonists but the r e f i l l i n g
pathway is not blocked by these agents (in f a c t ,
such a blockade is a prerequisite for the opening of the r e f i l l i n g
pathway),
i t seems unlikely that e x t r a c e l l u l a r Ca2+ enters the cytosol via an intracellular
store during agonist stimulation.
Second, receptor-induced Ca2+
release and entry have a requirement for the second messengers (1,4,5) IP 3 and (1,3,4,5)
IP 4 (4,20).
feedback i n h i b i t i o n
It has been postul ated that the site of the PKC
is at the generation of these second messengers, possibly
between the receptor and PIP 2 hydrolysis (14), and many reports support t h i s contention (5,15,16,21-24).
The receptor-induced activation of PIP 2
hydrolysis by PLC appears coupled to a GTP binding protein ('25,26) (designated 1066
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Ca2+
/ ~
Fig. 3
"(1'4'5IP3~ )
A model for recept0r-induced Ca2+ release and Ca2+ entry. Agonist binding to i t s receptor (RA) leads to phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown into diacylglycerol (DG) and inositol 1,4,5-trisphosphate [(1,4,5) IP 3] via activation of phospholipase C (PLC) coupled with a GTP binding protein (Np). (1,4,5) IP 3 bind~ to a receptor (RI) on the agonist regulated ~ intracelluTar Ca~T store. This IP~ binding activates release of Ca~ into the cytosol (Cai I from the i n t r a c e l l u l a r Ca store (the Ca release phase of t ~ CaZ+ mobilization) and closes the r e f i l l i n g ~ + pathway to this CaL store. Simultaneously a plasma membrane Ca: entry pathway opens and extrac~!lula r CaZ÷ goes d i r e c t l y into the cy~sol rather than via the Ca~T store (the Caz+ entry phase of the Ca~ mobilization). Inositol 1,3,4,5-tetrakisphosphate [(1,3,4,5) IP 4] and/or another GIP-binding protein (Ni) may be responsible for the opening of the Caz+ entry pathway. The activation of protein kinase C (PKC) by DG blocks the receptor-induced CaZ+ release and Caz+ entry responses by i n h i b i t i n g the production of (1,4,5)_IP 3 and (1,3,4,5) IP 4 via phosphorylation of Np and/or closes the Caz+ entry pathway by phospborylation of Ni . The r e f i l l i n g pathway of the i n t r a c e l l u l a r CaZ+ store w i l l open again after the termination of agonist stimulation.
Np as by Blackmore and Exton ( 2 7 ) ) .
Considerable evidence i n d i c a t e s t h a t the
r e g u l a t o r y s i t e a f f e c t e d by PKC i s located on Np (27-30).
Third,
receptor-induced Ca2+ e n t r y may also be governed by separate undefined s i g n a l s (i0).
For example, in mouse B lymphocytes, phorbol esters not only i n h i b i t e d
PIP 2 breakdown and Ca2+ m o b i l i z a t i o n s t i m u l a t e d by anti-immunoglobulin a n t i b o d i e s , but also blocked concanavalin A evoked Ca2+ i n f l u x does not induce production of IP 3) (31).
(concanavalin A
As mentioned above, in adrenal
glomerulosa c e l l s p e r t u s s i s t o x i n blocked agonist-induced Ca2+ e n t r y w i t h o u t affecting
IP 3 production and Ca2+ release ( 7 ) .
Receptor-induced Ca2÷ e n t r y
thus may be regulated by another GTP binding p r o t e i n (designated Ni f o r influx)
as well as by Np, and PKC s t i m u l a t i o n would cause feedback i n h i b i t i o n 1067
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of receptor-induced Ca2+ entry not only by suppressing PIP2 degradation (via Np) but also by exerting additional effects (via Ni). The specific mechanism(s) by which Ca2+ enters c e l l s via receptoroperated Ca2+ channels is s t i l l
not clear (3).
The model for t h i s event
proposed by Putney (18), while useful for understanding the process of refilling
the IP3-sensitive i n t r a c e l l u l a r Ca2+ store, does not adequately
accomodate our data and that of other investigators (5-8,31).
Such data
indicate the existence of a d i r e c t Ca2+ entry pathway to the cytosol.
Our
modification of Putney's model should permit f u r t h e r , more specific investigations of t h i s key regulatory process.
ACKNOWLEDGEMENTS We thank Drs. Indu S. Ambudkar, Valerie J. Horn, R. James Turner and Robert B. Wellner for helpful discussions and c r i t i c a l reading of the manuscript. We also appreciate Dr. Wellner's guidance with cell culture procedures.
REFERENCES 1. 2. 3. 4. 5, 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Drummond, A.H., and Macintyre, D.E. (1985) Trends Pharmacol. Sci. 6, 233-234. Nishizuka, Y. (1986) Science 233, 305-312. Berridge, M.J° (1987) Ann. Rev. Biochem. 56, 159-193. Berridge, M.J., and I r v i n e , R.F. (1984) Nature 312, 315-321. Valone, F.H., and Johnson, B. (1987) Biochem. J. 247, 669-674. Owen, C.S. (1988) J. Biol. Chem. 263, 2732-2737. Kojima, I . , Shibata, H., and Ogata, E. (1986) FEBS Lett. 204, 347-351. Muallem, S., Fimmel, C.J., Pandol, S.J., and Sachs, G. (1986) J. Biol. Chem. 261, 2660-2667. M e r r i t t , J.E., and Rink, T.J. (1987) J. Biol. Chem. 262, 17362-17369. Pandiella, A., Malgaroli, A., Meldolesi, J., and V i c e n t i n i , L.M. (1987) Exp. Cell Res. 170, 175-185. Shirasuna, K., Sato, M., and Miyazaki, T. (1981) Cancer 48, 745-752. Helman, J., Ambudkar, I°S., and Baum, B.J. (1987) Eur. J. Pharmacol. 143, 65-72. Tsien, R.Y., Pozzan, T., and Rink, T.J. (1982) J. Cell Biol. 94, 325-334. Lynch, C.J., Charest, R., Bocckino, S.B., Exton, J.H., and Blackmore, P.F. (1985) J. Biol. Chem. 260, 2844-2851. Naccache, P.H., Molski, T.F.P., Borgeat, P., White, J.R., and Sha'afi, R.I. (1985) J. Biol. Chem. 260,2125-2131. V i c e n t i n i , L.M., V i r g i l i o , F.D., Ambrosini, A., Pozzan, T., and Meldolesi, J. (1985) Biochem. Biophys. Res. Commun. 127, 310-317. Nishizuka, Y. (1984) Nature 308, 693-698. Putney, J.W. Jr. (1986) Cell Ca 7,1-12. Pandol, S,J., Schoeffield, M.S.,Fimmel, C.J., and Muallem, S. (1987) J. Biol. Chem. 262, 16963-16968. Morris, A,P., Gallacher, D.V., I r v i n e , R.F., and Petersen, O.H. (1987) Nature, 330, 653-655. 1068
Vol. 152, No. 3, 1 9 8 8 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Orellana, S.A., Solski, P.A., and Brown, J.H. (1985) ~ J. Biol. Chem. 260, 5236-5239. Rittenhouse, S.E., and Sasson, J.Po (1985) J. Biol. Chem. 260, 8657- 8660. Watson, S.P., and Lapetina, E.G. (1985) Proc. Natl. Acad. Sci. USA 82, 2623-2626. Zavoico, G.B., Halenda, S.P., Sha'afi, R . I . , and Feinstein, M.B. (1985) Proc. Natl. Acad. Sci. USA 82, 3859-3862. Cockcroft, S., and Gomperts, B.D. (1985) Nature, 314, 534-536. Smith, C.D., Cox, C.C., and Snyderman, R. (1986) Science, 232, 97-100. Blackmore, P.F., and Exton, J.H. (1986) J. Biol. Chem. 261, 11056-11063. Llano, I . , and Marty, A. (1987) J. Physiol. 394, 239-248. Muldoon, L.L., Jamieson, G.A. J r . , and V i l l e r e a l , M.L. (1987) J. Cell. Physiol. 130, 29-36. Osugi, T., Imaizumi, T., Mizushima, A., Uchida, S., and Yoshida, H. (1987) J. Pharm. Exp. Ther. 240, 617-622. Bijsterbosch, M.K., and Klaus, G.G.B. (1987) Eur., J. Immunol. 17,113-118.
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PROTEIN KINASE C DIFFERENTIALLY INHIBITS MUSCARINIC RECEPTOR OPERATEDCa2+ RELEASE AND ENTRY IN HUMAN SALIVARY CELLS Xinjun He, Xiaozai Wu and Bruce J. Baum* Clinical Investigations and Patient Care Branch, National I n s t i t u t e of Dental Research, National I n s t i t u t e s of Health Bethesda, Maryland 20892 Received March 28, 1988
We investigated the effects of phorbol myristate acetate on muscarinic receptor-induced Caz+ release from i n t r a c e T l u l a r stores and e x t r a c e l l u l a r entry_in a human salivary duct cell Iine,^HSG-PA. Phorbol myristate acetate (~IO-/M) blocked both Ca~+ release and Caz+ entry induced by the muscarinic agonist carbachol. This blockade was the result of the activation of protein kinase C since 4~-phorbol 12,13-didecanoate, which lacks the a b i l i t y to activate protein kinase C, did not i n h i b i t Caz+ mobilization responses to carba~hol. Importantly, at lower phorbol myristate acetate concentrations (~IO-~M), carbachol-induced Caz+ release was blocked, but carbachol-induced c~Z+ entry was maintained. These results show that carbachol-induced Caz+ entry does not occur via an i n t r a c e l l u l a r store and that protein kinas~±C plays a role in a feedback control mechanism for muscarinic-induced CaLT mobilization at d i f f e r e n t levels. © 1988AcademicP..... Znc.
Many reports have demonstrated recently that phorbol esters can i n h i b i t receptor-induced Ca2+ mobilization via activation of PKC in various cell types (for reviews, see r e f .
1-3).
Receptor-induced Ca2+ mobilization is the result
of two phases, release of Ca2+ from an i n t r a c e l l u l a r e x t r a c e l l u l a r Ca2+ across the plasma membrane (3,4).
store and entry of To our knowledge, there
are only two reports showing that phorbol esters p r e f e r e n t i a l l y agonist-induced Ca2+ release (5,6).
inhibit
The two phases of receptor-induced Ca2+
*To whom correspondence should be addressed Abbreviations used: cytosolic free Ca2+ concentration ([Ca2+]i); phosphatidylinositol 4,5-bisphosphate (PIP?); i n o s i t o l trisphosphate (IP3); protein kinase C (PKC); phospholipase C (P[C); phorbol myristate acetate (PMA); 4~-phorbol 12,13-didecanoate (4~-PDD); carbachol (Cch); balanced salt solution (BSS); bovine serum albumin (BSA); 4 - ( 2 - h y d r o x y e t h y l ) - l piperazineethane-sulfonic acid (HEPES); ethylene glycol bis (~-aminoethyl ether)-N,N,N', N ' - t e t r a a c e t i c acid (EGTA); the acetoxymethyl ester of quin-2 (Quin 2/AM); GTP bi~ding protein coupled to phospholipase C (Np); GTP binding protein mediating Ca~T i n f l u x (Ni); Eagle's minimal essential medium (EMEM). 0006-291X/88
$1.50
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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m o b i l i z a t i o n can be independently examined by stimulating c e l l s with agonists in Ca2+-free media (measurement of i n t r a c e l l u l a r
Ca2+ release) and t h e r e a f t e r
reintroducing Ca2÷ to the incubation media (measurement of e x t r a c e l l u l a r Ca2+ entry)
(7-10).
We have used t h i s approach to examine the e f f e c t s of PMA on
muscarinic receptor induced Ca2+ release and entry in a human s a l i v a r y duct cell l i n e ,
HSG-PA ( i i ) o
We report here that PMA (~10-7MI blocks both the Ca2+
release and entry induced by the muscarinic agonist Cch via a c t i v a t i o n of PKCo However, at lower PMA concentrations (~IO-9M), Cch-induced Ca2+ release is blocked, but Cch-induced Ca2+ entry is maintained.
These results show that
Cch-induced Ca2+ entry does not occur via an i n t r a c e l l u l a r
store and that PKC
plays a role in a feedback control mechanism for Cch-induced Ca2÷ m o b i l i z a t i o n at d i f f e r e n t
levels. MATERIALS AND METHODS
Materials - Eagle's minimal essential medium, newborn c a l f serum, p e n i c i l l i n G and streptomycin s u l f a t e were purchased from B i o f l u i d s . Hank's balanced s a l t solution was purchased from GIBCO and Quin 2/AM was purchased from Calbiochem. The f o l l o w i n g compounds were purchased from Sigma: EGTA, HEPES, 6-D(+)glucose, BSA, Cch, atropine s u l f a t e , PMA and 4~-PDD. Cell Culture - Experiments were performed on human submandibular duct c e l l s , HSG-PA, a kind g i f t from Dr. Mitsunobu Sato (11). HSG-PA c e l l s were cultured in EMEM, supplemented with 10% newborn c a l f serum, 100 ~g ml -~ p e n i c i l l i n G and streptomycin s u l f a t e , at 37°C in a humidified 5% CO2 atmosphere• HSG-PA c e l l s used in these studies were at passages 6 to 36. Measurement of I n t r a c e l l u l a r Ca2+ Concentration - Cells were grown to confluence and detached with Ca2+, Mg~+-free Hank's balanced s a l t solution containing 4 mM EGTA and 10 mM HEPES, pH 7.4 f o r 10 min at 37%. The c e l l s were then c o l l e c t e d by b r i e f c e n t r i f u g a t i o n (15 sec) and resuspended in BSS (NaCl 130; KCI 5; MgCl2 1 O; CaCl~ 1 5; HEPES 20 buffered to pH 7 4 with Tris base; In ~M) contalnlng 10 mM glucose. The cell suspensions (about 5x106 c e l l s ml -z) were preincubated at 37°C f o r 5 min and then were incubated with 50 ~M Quin 2/AM for a f u r t h e r 20 min. They were washed twice by b r i e f c e n t r i f u g a t i o n in BSS• Thereafter, t~e c e l l s were resuspended in BSS containing 10 mM glucose and I mg ml -~ BSA and kept at room temperature• Just before fluorescence measurements were performed, the c e l l s were again centrifuged and resuspended (about lx106 c e l l s ml - I ) in Ca2+-free BSS containing glucose plus BSA. Caz+ was added as indicated• Fluorescence was measured at 37°C in a SLM-8000 s p e c t r o f l u o r i m e t e r as previously described (12). C a l i b r a t i o n of the signal and c a l c u l a t i o n of c y t o s o l i c Caz+ were as described by Tsien et al (13).
RESULTS AND DISCUSSION HSG-PA c e l l membranes possess a single class of high a f f i n i t y
muscarinic
receptors (Kd=O.17±O.08 nM, Bmax=38±3 fmol/mg protein) as measured using a [3H]-quinuclidinylbenzilate
binding assay (not shown)•
The muscarinic agonist
Cch increases production of IP 3 and elevates [Ca2+]i in HSG-PA c e l l s (unpublished data).
The two phases of Ca2+ m o b i l i z a t i o n were observed
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separately when HSG-PA cells were exposed to Cch in Ca2+ free media and then Ca2+ was reintroduced (Fig. l a ) . from an i n i t i a l
Cch resulted in a rapid increase of [Ca2+]i
value of 117±11 to 226±24 nM (mean±SEM; n=13).
A second,
slower increase of [Ca2+]i was evoked when Ca2+ was reintroduced into the media (final
[Ca2+], 1.5 mM) 5 min after stimulation with Cch (Fig. l a ) , by
which time [Ca2+]i had returned to i n i t i a l
levels.
This second increase of
[Ca2+]i reached a maximum (277±24 nM;n=ll) at about 2 min a f t e r Ca2+ readdition and was completely blocked when the muscarinic antagonist atropine was added j u s t before reintroduction of Ca2+ (Fig. i b ) .
Both [Ca2+]i
responses were blocked i f atropine was added before the cells were exposed to Cch (Fig. I c ) .
The f i r s t
peak of the [Ca2+]i rise was not affected by the
inorganic Ca2+ channel blocker La 3+ (25 ~M; not shown), and represents agonist-induced Ca2+ release from the i n t r a c e l l u l a r
store.
The second peak
was abolished by La 3+, but was not affected by blockers of voltage-operated Ca2+ channels such as verapamil, diltiazem and nifedipine
(IO-6M).
This
response was also dependent on Ca2+ concentration in the media (not shown). Therefore, the second peak of the [Ca2+]i rise presumably represents Ca2+ entry across the plasma membrane, the so-called receptor-operated Ca2+ channel (3).
When HSG-PA cells were exposed to PMA (10-7M, 5 min p r i o r to Cch
a d d i t i o n ) , there was an almost complete i n h i b i t i o n of Cch-induced i n t r a c e l l u l a r Ca2+ release and e x t r a c e l l u l a r Ca2+ entry (Fig. l d ) .
4~-PDD,
which lacks the a b i l i t y to activate PKC, did not i n h i b i t any responses to Cch (Fig. l e ) .
Several investigators (5,6,14-16) have observed similar results
with other c e l l s , i . e . ,
phorbol esters can block both phases of agonist-
induced Ca2+ mobilization. We next examined the effects of d i f f e r e n t
[PMA] on both the Ca2+ release
and Ca2+ entry induced by Cch. The resulting i n h i b i t i o n patterns indicate differential
effects of PMA on these two responses (Fig. 2).
At low [PMA],
Cch-induced i n t r a c e l l u l a r Ca2+ release was s u b s t a n t i a l l y blocked and t h i s i n h i b i t o r y action was r e l a t i v e l y insensitive to increasing concentrations of PMA (from 5xlO -10 to 5x10 -5 M).
In contrast, the i n h i b i t i o n by PMA of Cch1064
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2
Cch
Atro Caz+
Atro Cch
Ca2+ 175150-
T
125. C~a2÷
Ceh
n-6 .E
e
10075.
2
50-
I
25,
0t~
C)
100
Fig. i
I 200
300
i 400
0 -10
500
Time, Sec
6
1
I
I
I
I
I
I
-g
-8
-7
-6
-5
-4
@
Log [PMA], M
Carbachol-induced i n t r a c e l l u l a r Ca2+ transients and the effects of PMA. a. Control c e l l s were preincubated with 0.01% dimethylsulfoxide (the vehicle used with PMA, below) for 5 min; Cch (10 -4 M) and Caz+ (1.5 mM) were added at arrows, b. The experimental conditions were • ii the same as In a 11 but atrop]ne (Atro, i0 -~~ M) was added 20 sec b e f o r e a d d i t i o n o f CaZ+ . c . A t r o p i n e ( I 0 -D M) was added 20 s e c . b e f o r e t h e ~ d d i t i o n o f Cch. d and e . C e l l s were t r e a t e d w i t h PMA o r 4~-PDD (10 - I M) f o r 5 m i n , r e s p e c t i v e l y , t h e n Cch and Ca 2+ were added as i n " a " .
Fig. 2
I n h i b i t i o n o£ the Cch-induced Ca2+ release from an i n t r a c e l l u l a r store and CaZ+ entry across the plasma membrane by d i f f e r e n t [ P ~ ] . Experimental conditions were as described in the t e x t . The [Ca:T]i response was calculated for each phase as a percentage of the maximal Cch-induced [CaZ+]i rise, i . e . , that observed without PMA present. Each data point is the mean±SEMof at least 3 separate experiments performed on d i f f e r e n t cell preparations. (o o) refers to i n t r ~ c e l l u l a r calcium release at 20 sec a f t e r addition of Cch (IO-~M), (~ e) refers to e x t r a c e l l u l a r calcium entry at 2 min a f t e r reintroduction of CaLT (1.5 mM).
induced e x t r a c e l l u l a r
Ca2+ e n t r y showed a d i f f e r e n t
concentration-dependence
p a t t e r n ( F i g . 2), w i t h h a l f - m a x i m a l and maximal i n h i b i t i o n IO-7M PMA, r e s p e c t i v e l y .
at about 10 -8 and
This PMA c o n c e n t r a t i o n range is considered s p e c i f i c
f o r the a c t i v a t i o n o f PKC (17).
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A p a r t i c u l a r l y interesting finding in these experiments is that at lower concentrations of PMA (~IO-9M), Cch-induced Ca2+ release is blocked substantially,
but Cch-induced Ca2+ entry is e s s e n t i a l l y unchanged (Fig. 2).
Valone and Johnson have recently shown in platelets that agonist-induced intracellular
Ca2+ release and divalent cation i n f l u x are d i f f e r e n t i a l l y
affected by PMA (5).
Also, Owen has shown in murine lymphocytes that phorbol
esters p r e f e r e n t i a l l y
i n h i b i t anti-immunoglobulin triggered Ca2+ release
(6).
In addition,
i t has been observed that pertussis toxin blocks
angiotensin I I induced Ca2+ i n f l u x without affecting Ca2+ release in adrenal glomerulosa cells (7).
These observations indicate that receptor-induced Ca2+
entry into the cytosol does not occur through an i n t r a c e l l u l a r proposed by Putney (18).
store, as
These data also suggest that PKC may regulate
receptor-induced Ca2+ release and entry via feedback control mechanisms at different
sites.
Figure 3 shows a model for receptor-induced Ca2+ mobilization and i t s feedback control by PKC that has been modified from that of Putney (18).
In
addition to the findings described above, three other lines of evidence support this model.
F i r s t , the r e f i l l i n g
of the receptor-regulated
i n t r a c e l l u l a r Ca2+ store proceeds slowly and is dependent on e x t r a c e l l u l a r Ca2+ (8,9,19). stimulation
Significantly,
(8).
i t occurs j u s t a f t e r termination of agonist
Since receptor-induced Ca2+ entry is absolutely blocked by
antagonists but the r e f i l l i n g
pathway is not blocked by these agents (in f a c t ,
such a blockade is a prerequisite for the opening of the r e f i l l i n g
pathway),
i t seems unlikely that e x t r a c e l l u l a r Ca2+ enters the cytosol via an intracellular
store during agonist stimulation.
Second, receptor-induced Ca2+
release and entry have a requirement for the second messengers (1,4,5) IP 3 and (1,3,4,5)
IP 4 (4,20).
feedback i n h i b i t i o n
It has been postul ated that the site of the PKC
is at the generation of these second messengers, possibly
between the receptor and PIP 2 hydrolysis (14), and many reports support t h i s contention (5,15,16,21-24).
The receptor-induced activation of PIP 2
hydrolysis by PLC appears coupled to a GTP binding protein ('25,26) (designated 1066
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Ca2+
/ ~
Fig. 3
"(1'4'5IP3~ )
A model for recept0r-induced Ca2+ release and Ca2+ entry. Agonist binding to i t s receptor (RA) leads to phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown into diacylglycerol (DG) and inositol 1,4,5-trisphosphate [(1,4,5) IP 3] via activation of phospholipase C (PLC) coupled with a GTP binding protein (Np). (1,4,5) IP 3 bind~ to a receptor (RI) on the agonist regulated ~ intracelluTar Ca~T store. This IP~ binding activates release of Ca~ into the cytosol (Cai I from the i n t r a c e l l u l a r Ca store (the Ca release phase of t ~ CaZ+ mobilization) and closes the r e f i l l i n g ~ + pathway to this CaL store. Simultaneously a plasma membrane Ca: entry pathway opens and extrac~!lula r CaZ÷ goes d i r e c t l y into the cy~sol rather than via the Ca~T store (the Caz+ entry phase of the Ca~ mobilization). Inositol 1,3,4,5-tetrakisphosphate [(1,3,4,5) IP 4] and/or another GIP-binding protein (Ni) may be responsible for the opening of the Caz+ entry pathway. The activation of protein kinase C (PKC) by DG blocks the receptor-induced CaZ+ release and Caz+ entry responses by i n h i b i t i n g the production of (1,4,5)_IP 3 and (1,3,4,5) IP 4 via phosphorylation of Np and/or closes the Caz+ entry pathway by phospborylation of Ni . The r e f i l l i n g pathway of the i n t r a c e l l u l a r CaZ+ store w i l l open again after the termination of agonist stimulation.
Np as by Blackmore and Exton ( 2 7 ) ) .
Considerable evidence i n d i c a t e s t h a t the
r e g u l a t o r y s i t e a f f e c t e d by PKC i s located on Np (27-30).
Third,
receptor-induced Ca2+ e n t r y may also be governed by separate undefined s i g n a l s (i0).
For example, in mouse B lymphocytes, phorbol esters not only i n h i b i t e d
PIP 2 breakdown and Ca2+ m o b i l i z a t i o n s t i m u l a t e d by anti-immunoglobulin a n t i b o d i e s , but also blocked concanavalin A evoked Ca2+ i n f l u x does not induce production of IP 3) (31).
(concanavalin A
As mentioned above, in adrenal
glomerulosa c e l l s p e r t u s s i s t o x i n blocked agonist-induced Ca2+ e n t r y w i t h o u t affecting
IP 3 production and Ca2+ release ( 7 ) .
Receptor-induced Ca2÷ e n t r y
thus may be regulated by another GTP binding p r o t e i n (designated Ni f o r influx)
as well as by Np, and PKC s t i m u l a t i o n would cause feedback i n h i b i t i o n 1067
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of receptor-induced Ca2+ entry not only by suppressing PIP2 degradation (via Np) but also by exerting additional effects (via Ni). The specific mechanism(s) by which Ca2+ enters c e l l s via receptoroperated Ca2+ channels is s t i l l
not clear (3).
The model for t h i s event
proposed by Putney (18), while useful for understanding the process of refilling
the IP3-sensitive i n t r a c e l l u l a r Ca2+ store, does not adequately
accomodate our data and that of other investigators (5-8,31).
Such data
indicate the existence of a d i r e c t Ca2+ entry pathway to the cytosol.
Our
modification of Putney's model should permit f u r t h e r , more specific investigations of t h i s key regulatory process.
ACKNOWLEDGEMENTS We thank Drs. Indu S. Ambudkar, Valerie J. Horn, R. James Turner and Robert B. Wellner for helpful discussions and c r i t i c a l reading of the manuscript. We also appreciate Dr. Wellner's guidance with cell culture procedures.
REFERENCES 1. 2. 3. 4. 5, 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Drummond, A.H., and Macintyre, D.E. (1985) Trends Pharmacol. Sci. 6, 233-234. Nishizuka, Y. (1986) Science 233, 305-312. Berridge, M.J° (1987) Ann. Rev. Biochem. 56, 159-193. Berridge, M.J., and I r v i n e , R.F. (1984) Nature 312, 315-321. Valone, F.H., and Johnson, B. (1987) Biochem. J. 247, 669-674. Owen, C.S. (1988) J. Biol. Chem. 263, 2732-2737. Kojima, I . , Shibata, H., and Ogata, E. (1986) FEBS Lett. 204, 347-351. Muallem, S., Fimmel, C.J., Pandol, S.J., and Sachs, G. (1986) J. Biol. Chem. 261, 2660-2667. M e r r i t t , J.E., and Rink, T.J. (1987) J. Biol. Chem. 262, 17362-17369. Pandiella, A., Malgaroli, A., Meldolesi, J., and V i c e n t i n i , L.M. (1987) Exp. Cell Res. 170, 175-185. Shirasuna, K., Sato, M., and Miyazaki, T. (1981) Cancer 48, 745-752. Helman, J., Ambudkar, I°S., and Baum, B.J. (1987) Eur. J. Pharmacol. 143, 65-72. Tsien, R.Y., Pozzan, T., and Rink, T.J. (1982) J. Cell Biol. 94, 325-334. Lynch, C.J., Charest, R., Bocckino, S.B., Exton, J.H., and Blackmore, P.F. (1985) J. Biol. Chem. 260, 2844-2851. Naccache, P.H., Molski, T.F.P., Borgeat, P., White, J.R., and Sha'afi, R.I. (1985) J. Biol. Chem. 260,2125-2131. V i c e n t i n i , L.M., V i r g i l i o , F.D., Ambrosini, A., Pozzan, T., and Meldolesi, J. (1985) Biochem. Biophys. Res. Commun. 127, 310-317. Nishizuka, Y. (1984) Nature 308, 693-698. Putney, J.W. Jr. (1986) Cell Ca 7,1-12. Pandol, S,J., Schoeffield, M.S.,Fimmel, C.J., and Muallem, S. (1987) J. Biol. Chem. 262, 16963-16968. Morris, A,P., Gallacher, D.V., I r v i n e , R.F., and Petersen, O.H. (1987) Nature, 330, 653-655. 1068
Vol. 152, No. 3, 1 9 8 8 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Orellana, S.A., Solski, P.A., and Brown, J.H. (1985) ~ J. Biol. Chem. 260, 5236-5239. Rittenhouse, S.E., and Sasson, J.Po (1985) J. Biol. Chem. 260, 8657- 8660. Watson, S.P., and Lapetina, E.G. (1985) Proc. Natl. Acad. Sci. USA 82, 2623-2626. Zavoico, G.B., Halenda, S.P., Sha'afi, R . I . , and Feinstein, M.B. (1985) Proc. Natl. Acad. Sci. USA 82, 3859-3862. Cockcroft, S., and Gomperts, B.D. (1985) Nature, 314, 534-536. Smith, C.D., Cox, C.C., and Snyderman, R. (1986) Science, 232, 97-100. Blackmore, P.F., and Exton, J.H. (1986) J. Biol. Chem. 261, 11056-11063. Llano, I . , and Marty, A. (1987) J. Physiol. 394, 239-248. Muldoon, L.L., Jamieson, G.A. J r . , and V i l l e r e a l , M.L. (1987) J. Cell. Physiol. 130, 29-36. Osugi, T., Imaizumi, T., Mizushima, A., Uchida, S., and Yoshida, H. (1987) J. Pharm. Exp. Ther. 240, 617-622. Bijsterbosch, M.K., and Klaus, G.G.B. (1987) Eur., J. Immunol. 17,113-118.
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