mediated by a-adrenergic receptors helps maintain the elevated cytosolic [Ca2+]i ... [Ca2+];.3 Evidence has recently been presented which shows that the (M$+ ...
Communication
THEJOURNALOF BIOLOGICALCHEMISTRY Vol. 259 No. 20 Issue of October 25 pp. 12322-12325 1984 0 1984 b; The American Society of B\ological Chemisi, Inc. Printed in U.S.A.
and &Adrenergic Stimulation of Parenchymal Cell Ca2+Influx
cellular CAMPlevels, the Ca2+mobilizing hormones promoted a net influx of Ca2+into parenchymal cells (12). This study clearly showed that the parenchymal cell did contain a Ca2+ entry mechanism which was regulated by hormones. A survey INFLUENCEOFEXTRACELLULARpH* of the literature indicates that very little information existed (Received for publication, April 2, 1984) about Ca2+ influx mechanisms in nonexitable cells such as parenchymal cells. It has been proposed that a Ca2+/Na+ Peter F. Blackmore$, Laura E. Waynick, antiport operates; however, due to the Na’ gradient, electroGregory E. Blackman, ChristopherW. Graham, and genic Ca2+efflux would probably occur by that mechanism (4, Richard S . Sherry 13). A Ca2+/H+antiport has also been postulated and some From the Laboratories for the Studies of Metabolic evidence for its existence has been presented (13, 14). It was Disorders, Howard Hughes Medical Institute, and the the purpose of this study to examine such a mechanism. By Department of Physiology, Vanderbilt University School of raising the extracellular pH to8.1, which would make the pH Medicine, Nashuille, Tennessee 37232 gradient across the plasma membrane more favorable for Stimulation of parenchymal cell a- and &adrenergic Ca2+/H+exchange (more alkaline outside),it was found that receptors, with either epinephrine or the specific a- a- and P-adrenergic stimulation resulted in a net Ca2+influx. This result suggested that parenchymal cells contain a horagonist phenylephrine or the &agonist isoproterenolol, promoted a net Ca2+ influx when thepH of theexternal monally responsive Ca2+/H+antiport or a Ca2+/0H- symport, medium was greater thanpH 7.7. The effect was both which together with the inhibition of the plasma membrane time- and dose-dependent. At the elevated pH of 8.1, (Mg“‘ - Ca2+)-ATPasepump (8) would act to maintain an a-adrenergic receptors were still able to mobilize in- elevated [Ca2+];after a-adrenergic stimulation tracellular Caz+,since when the externalCa” concentration was reduced to 0.05 mM, epinephrine promoted EXPERIMENTAL PROCEDURES net Ca2+ efflux. The data suggest the existence of a Hepatocyte Isolation and Incubation-Isolated liver parenchymal a Ca2+/0H- symport in the plasma cells Ca2+/H+ antiport or were prepared from 200-250-g(body weight) male Spraguemembrane which can be stimulated by either a- or 8- Dawley rats (Harlan Industries, Indianapolis, IN) as previously deadrenergic receptors.It is proposed that the Ca2+ influx scribed (15).Cell suspensions were either incubated in 25-or 250-ml mediated by a-adrenergic receptors helps maintain the Erlenmeyer flasks and constantly gassed with 95% 02:5% COz. The elevated cytosolic [Ca2+]i for longer time periods afterbuffer used was Krebs-Henseleit bicarbonate buffer (16); the pH was the initial mobilization from intracellular Ca2+ stores. adjusted by altering the bicarbonate concentration. When the bicarCY-
Many studies have clearly shown that in rat liver parenchymal cells a-adrenergic agonists, vasopressin, angiotensin 11, and glucagon initially mobilize intracellular Ca2+ which raises the free cytosolic Ca2+ ([Ca”];) (e.g.Refs. 1-4). The second message for this mobilization in rat parenchymal cells is probably IP3’ (5-7).‘r3 Since the intracellular pools are of limited magnitude, and since Ca2+is extruded from the cell by a plasma membrane (M$+ - Ca2+)-ATPase, one must postulate that either the Ca2+ extrudingmechanism is inhibited and/or Ca2+influx is stimulated to maintain the elevated [Ca2+];.3Evidence has recently been presented which shows that the (M$+ - Ca2+)-ATPasepump is inhibited (8) and there is also evidence to show that these same hormones promote 45Ca2+influx (e.g. Refs. 9-11). More recently we demonstrated that by raising the intra-
bonate concentration was 80 or 24 mM, the pH was 8.1 and 7.4, respectively. The total Ca2+ concentration in the medium was 2.4 mM. Analytical Methods and Materials-Methods for the determination of phosphorylase a activity, total cell Ca2+ and [Ca”], have been previously described (1-3, 15). Expression of Data-Representative experiments are shown. Each value is the mean f S.E. from triplicate flasks, assayed in duplicate. Each experiment was performed at least three times. Materials-Verapamil HCl was obtained from Knoll Pharmaceutical Co., Whippany, NJ; Diltiazem HCI was from Marion Laboratories, Inc., Kansas City, MO; and Nifedipine was from Pfizer Inc., Brooklyn, NY. Sources of other drugs and hormones have been described previously (1-3). RESULTS
Effect of Hormones on Cell Ca2+ Content at Various Extracellular p H Values-The data inFig. 1 show the effect of vasopressin, epinephrine,angiotensin 11, and glucagon on total cell Ca2+as a function of extracellular pH. Allof the * This work wassupported inpart by National Institutes of Health hormones promoted a net loss of Ca2+when the pH was less Grant AM 20593.An account of this work waspresented as amedical than pH 7.7. Above pH 7.7 a net increase in cell Ca2+content student research project in the Department of Physiology, Feb. 23, was observed, with glucagon producing by far the largest 1984 and at the Annual Meeting of the American Society of Biological Chemists, St. Louis, MO, June 3-7, 1984. The costs of publication of increase. Since our primary interest is in the mechanism of this article were defrayed in part by the payment of page charges. a-adrenergic action in the liver (1-3), the rest of the data This article must therefore be hereby marked “aduertisement” in presented in this communication will be concerned with this accordance with 18 U.S.C. Section 1734 solely to indicate this fact. catecholamine; the effects of theother hormones willbe $ Associate Investigator, Howard Hughes Medical Institute. presented elsewhere: The abbreviations used are: IPB,myo-inositol1,4,5-trisphosphate; It is assumed that the adrenergic agonists and antagonist Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. P. Thiyagarajah, R. Charest, J. H. Exton, and P. F. Blackmore, used in this study retain their same specificity at pH8.1 as at pH 7.4. Epinephrine was used instead of norepinephrine, since submitted to J . Bwl. Chem. R. Charest, V. Prpik, J. H. Exton, and P. F. Blackmore, manuP. F. Blackmore, manuscript in preparation.
script submitted to J. Biol. Chem.
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FIG. 1. Effect of varying extracellular pH on the ability of epinephrine (Epi)(lo-' M), vasopressin (Vmo) (lo-' M), angiotensin I1 (Angio) (lo-' M), and glucagon (Gh)(lo-' M) to alter total cell CaZ+.The pH of the medium was varied by altering
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FIG. 2. Effect of adrenergicblockade on epinephrine (Epi)mediated changes in cell Cas+ andphosphorylase activity together with the effects of phenylephrine (Phe) and isoproterenolol (Zso) at pH 7.4 ( A and B ) and pH 8.1 (Cand D ) .The concentration of epinephrine was 1O"j M,while that of isoprotereno101, phenylephrine, phentolamine (Phentol),and propranolol (Prop) were M. Antagonists and agonists were added simultaneously. Duplicate samples were removed after 5 min of incubation for the measurement of cell ea2+ and phosphorylase.
content and phosphorylase. In Fig. 2A it can be seen, in confirmation of our earlier studies,that theeffect of epinephrine to promote Ca2+ efflux was mediated by a-adrenergic receptors at pH 7.4 (17, 18). The Ca2+ efflux can also be mimicked by the specific a-adrenergic agonist phenylephrine norepinephrine was less effective at pH8.1 (data not shown). but not by the P-agonist isoproterenolol (Fig. 2 A ) . The actiWhen the extracellular pH was 7.4 or 8.1, total cell Ca2+ vation of phosphorylase at pH 7.4 was also mediated by acontent was 0.39 f 0.02 and 0.54 f 0.07 nmol. mg wet weight, adrenergic receptors, whereas @-adrenergicreceptors only prorespectively, while basal phosphorylase a was 12.1 f 0.8 and duced a small activation at 5 min (Fig. 2B). A slightly larger 19.1 f 0.6 units.g wet weight, respectively (mean k S.E. from increase in phosphorylase was observed at 2 min (data not 10 separate experiments). For purposes of comparison, how- shown). In Fig. 2C, both a- and @-adrenergicstimulation can ever, the results herein are expressed as per cent change from mediate the Ca2+ influx at pH 8.1 since neither a- nor 0the appropriate control. When 20 mM Hepes buffer at pH8.1 adrenergic blockade alone could inhibit the influx induced by was used instead of 80 mM bicarbonate buffer, then vasopres- epinephrine. In confirmation of this, the a-agonist phenylsin, angiotensin 11, epinephrine, and glucagon all increased ephrine and the P-agonist isoproterenol also promoted Ca2+ the Ca2+ content of hepatocytes by32, 34, 63, and 118%, influx at pH 8.1, to an even greater extent to that observed respectively (means from two separate experiments, each with epinephrine alone. Phosphorylase activation (Fig. 2 0 ) performed in triplicate).If the hepatocytes were isolated from however, was mediated predominantly by a-adrenergic recepovernight starved animals, then the pHdecreased by approx- tors even though @-adrenergicstimulation promoted a large imately 0.3 pH units in 10 min while hepatocytes from fed influx of Ca2+(Fig. 2C). This result suggested that the mobianimals produced a fall in pHof approximately 1.0 unit in 10 lization of intracellular Ca2+ was still the most important min. This fall in pH is most likely due to the production of determinant of [Ca2+Ii,since @-adrenergicstimulation only e.g. lactic and pyruvic acids by the hepatocytes. For this promotes a very small mobilization of intracellular Ca2+at reason, bicarbonate buffer was routinely used, with constant pH 7.4 (Fig. W ) . Thus, it is postulated that the Ca2+that gassing, instead of e.g. Hepes. enters the cell, induced by @-adrenergicstimulation, at pH Effect of Adrenergic Antagonists on Epinephrine-induced 8.1, does not reside in the cytoplasm but enters themitochonChanges in Ca2+ Content and Phosphorylase Activation at pH dria. @-Adrenergicstimulation does not increase the level of 7.4 and 8.1-The data in Fig. 2 show the effect of the a - the second message IPS (data not shown). Subcellular fracadrenergic antagonist phentolamine and the@-adrenergican- tionation studies (2,12) have confirmed that thebulk of Ca2+ tagonist propranolol on epinephrine-inducedchanges in Ca2+ taken up into these cells at pH 8.1 by epinephrine is located
the bicarbonate concentration as indicated. Cells were continuously gassed with 95% 0,:5% CO,. After incubation with the various hormones for 5 min, duplicate 0.5-ml aliquots of cell suspension were removed and total cell Ca" concentration was determined.
Adrenergic Control of Ca2+Influx
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FIG. 3. Influence of extracellular Caz+ onthe ability of epinephrine (lo-' M) to alter cell Ca2' and activate phosphorylase a at pH 7.4 ( A and B ) and 8.2 (Cand D ) . Aliquots of cell suspension were removed at theindicated times for the determination of cell Ca2+and phosphorylase a. The extracellular Ca2+concentration was either 2.4 mM (+Ca) or 0.05 mM (-Ca).
in the mitochondrial fractionwith a 54% increase (mean from triplicate experiments) in Ca2+content being observed after 5 min. Role of Extracellular Ca" in the Epinephrine-induced Changes in Cell ea2+ atpH 7.4 and 8.1 -The data in Fig. 3A show that epinephrine promotes a mobilization of intracellular Ca2+when the extracellular Ca2+concentration was either 2.4 or 0.05 mM, although a greater efflux was observed when the extracellular Caz+was reduced to 0.05 mM, probably due to therebeing less Ca2+influx at thislower extracellular Ca2+ concentration; also consistent with this is the fact that phosphorylase a activation was more transient at thelower extracellular Ca2+concentration (Fig. 3B).3 A t pH 8.1,epinephrine either promoted Ca2+influx or efflux depending on the Ca2+concentration in the medium. When the extracellular Ca2+ was reduced to 0.05 mM, epinephrine caused an efflux comparable to that observed at pH 7.4 (Fig. 3 A ) . This suggests that at pH8.1 the intracellular Ca2+pools, which are hormone responsive, are mobilized normally, and it is only when extracellular Ca2+is physiological (i.e. 2.4 mM total) that net Ca2+influx was observed. The ability of epineDhrine to activate phosphorylase a was not modified at either extracellular C$+ concentration (Fig. 3 0 ) . Since phosphorylase activation was not transient at pH8.1, when extracellular Ca2+was 0.05 mM, then there was probably some Ca2+ influx even at this low extracellularCa2+concentration.
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FIG. 4. Dose response of epinephrine on phosphorylase a levels ( A ) and cell Caz+content ( B )as a function of pH. After incubation for 5 min with epinephrine, cell Ca2+and phosphorylase a activity were determined.
Dose Response of Epinephrine to Activate Phosphorylase and Mobilize Ca2+ at pH 7.4 and 8.1 -The results in Fig. 4 show that theability of epinephrine to activate phosphorylase at pH 8.1 was not altered significantly, when dose response relationships were considered (Fig. 4A). The ability of epinephrine to promote Ca" influx at pH 8.1 was slightly less sensitive than epinephrines' ability to cause Ca" efflux at pH 7.4 (Fig. 4B). The half maximally effective doses at pH 8.1 and 7.4 were approximately 80 and 50 nM, respectively. When [Ca2+];was measured at pH7.4 and 8.1 ( 3 , 15), there was no difference in the resting level, also M epinephrine was able to increase [Ca'+]; to the same extent and at the same rate, regardless of whether there was net Ca2+influx or net Ca2+efflux (data not shown). DISCUSSION
The data presented herein clearly show that ratliver parenchymal cells possess aCa2+ entry mechanism which is pH
Control Adrenergic
of Ca2+Influx
12325
sensitive and which can be controlled by hormones such as receptors to the Caz+channel maybe favored at the more a- and @-adrenergicagonists, vasopressin, angiotensin 11, and alkaline pH. Also, since proteins becomemorenegatively glucagon (Fig. 1).The data in Fig. 2 show that both a- and @- charged at alkaline pH, Ca2+binding may be increased to the adrenergic stimulation can increase Ca2+influx. This result Ca2+binding site of the Ca2+channel and thusfacilitate Ca2+ suggests that both theCa2+ mobilizing receptor (1-3), namely entry. It is appropriate here to make a cautionary note regarding the al-adrenergic receptor whichprobably uses IPS as its intracellular 2nd message for Ca2+mobilization (6)2*3 and thethe use of Krebs-Henseleit bicarbonate buffer (16). It is @-adrenergicreceptor which usescAMP as itssecond message essential that thisbuffer be continuously gassed with5% CO, (19), are capable of causing Ca2+ influx. It must be emphasized in order to maintain the correct physiological pH of 7.4, for if that al-adrenergic receptor stimulation also causes an eleva- it is not, the pH rises to a maximumvalue of8.2 within tion of cAMP (19), thus theCa2+influx may partly be due to several minutes. The rate of pH rise is dependent on the cell this increase. Consistent with this is the fact that glucagon density and the volume of cell suspension in the flask (data can promote Ca2+ influx (Fig. 1) as well as @-adrenergic not shown). This fact may account for somediscrepant results stimulation (Fig. 2); both receptors increase cAMP (e.g. 12, which have appeared regarding the direction of Ca2+move19). However, since angiotensin I1 and vasopressin also pro- ments in hepatocytes (e.g. Refs. 23 and 24). In conclusion, direct evidence is presented which supports mote Ca2+ influx (Fig. l), independent of any detectable the existence of a ligand gated Ca2+channel in the plasma cAMP increase (see Ref. 4 for references), then the influx mechanism can be stimulated independently of any cAMP membrane of rat liver parenchymal cells. This Ca2+ influx increase. The effect of glucagon plus vasopressin, or glucagon mechanism is pH sensitive and is controlled by a- and @plus epinephrine, were synergistic on Ca2+ influx at pH 8.1 adrenergic receptors, vasopressin, angiotensin 11, and glucagon either directly or indirectly through the second messages (data not shown). Presently it is not known if IP3 andcAMP act directly on Ca2+,IP3(5-7); and CAMP. It is possible that the ligand the plasma membrane channel or whether the change inCa2+ gated channel is controlled by phosphatidylinositol 4,5-bisthe largest influx is secondary to phosphorylation by a Ca2+-dependent phosphate hydrolysis, however, glucagon produces net Ca2+influx (Fig. 1)but is unable to cause phosphatidyliprotein kinase or the CAMP-dependent protein kinase. The nositol 4,5-bisphosphate hydrolysis as indicated by no inability of epinephrine to increase cAMP via a*- and @adrecrease in IP3 level^,^ thus at least two separate mechanisms nergic receptors and to increase IPS via al-adrenergic recep- appear to control Ca2+entry, one of which is CAMP-dependent tors (19)3is not altered at pH 8.1 from that observed at pH and the other CAMP-independent.The pH sensitivity of the 7.4 (data not shown). influx process suggests the operation of a Ca2+/H+ antiport Evidence to support the existence of a Ca" channel comes or a Ca2+/0H-symport. from the fact that the Ca2+channel blockers diltiazem, nifedipine, and verapamil block vasopressin-induced Ca2+entry Acknowledgment-I am indebtedto Dr. John H. Exton for providat pH 8.1, with half-maximal effective concentrations of 5 X ing access to equipment and materials vitalto this project. lod4, 3 X and5 X low6M, respectively. The bivalent REFERENCES cations Co2+,Mn2+,and Ni2+ (20) block Ca2+influx induced 1. Blackmore, P. F., Brumley, F. T., Marks, J. L., and Exton, J. H. (1978) J. by all of the hormones with equal potency and with a half Biol. Chem. 253,4851-1858 2.Blackmore, P. F., Dehaye, J.-P., and Exton, J. H. (1979) J. Bwl. Chem. maximallyeffective concentration of 3 X M. 264,6945-6950 It was reported previously that when hepatocytes were 3. Charest, R., Blackmore, P. F., Berthon, B., andExton, J. H. (1983) J. Biol. Chem. 268,8769-8773 incubated in Krebs-Henseleit bicarbonate buffer at pH 7.4, 4. Williamson, J. R., Cooper, R. H., and Hoek, J. B. (1981) Biochim. Biophys. the cytosolic pH was 7.2 (21). Raising the extracellular pH to Acta 639,243-295 H., Imine, R. F., Berridge, M.J., and Schulz, 1. (1983) Nature (Land.) 8.1 only raises the cytosolic pH by approximately 0.3 pH units 5. Streb, 306,6749 to 7.5 (21). Thusthe ApH across the plasma membrane 6. Joseph S. K. Thomas A. P Williams R. J. Imine, R. F., and Williamson, J. R.'(1984; J. Biol hem.' 2 5 9 , 307?-30~1 increases from 0.2 to 0.6 units when the extracellular pH is 7. Burgess, G.M., Godfre P P , McKinney, J. S., Berridge, M. J., Imine, R. F., and Putney, J. $\1984j Nature ( L o n d . ) 3 0 9 , 6 3 4 6 raised from 7.4 to 8.1. When the external pH was raised to V., Green, K. C., Blackmore, P. F., and Exton, J. H.(1984) J. Biol. 8.0 from 7.4 in the present study, the internal pH, which was 8. PrpiC, Chem. 259,1382-1385 9. Assimacopoulos-Jeannet, F. D., Blackmore, P. F., and Exton, J. H. (1977) 7.1 determined by the null point method (22), remained J. Biol. Chem 2 5 2 , 2662-2669 constant; thus the ApH increased from 0.3 to 0.9 when the 10. Keppens, S., Vandenheede,J. R., and DeWulf, H. (1977) Biochim. Biophys. Acta 496,448-451 external pH was elevated. It should be emphasized that the 11. Parker, J. C., Barritt, G. J., and Wadsworth, J. C. (1983) Biochem. J. 2 1 6 , pH gradient across the inner mitochondrial membrane is 514 9 N. G., Blackmore, P. F., and Exton, J. H. (1983) J. Bwl. Chem. normally approximately 1pH unit,which is sufficient to drive 12. Morgan, 258,5110-5116 ATP synthesis by the FoFl-ATPase. This pHgradient across 13. Racker, E. (1980) Fed. Proc. 39,2422-2426 N., Biber, J., Murer, H., and Carafoli, E. (1982) Eur. J. the plasma membrane may be partly responsible, together 14. Kraus-Friedman Bwchem 129; 7-12 with the transmembrane electrical potential, for the Ca2+ 15. Blackmore, P. F., and Exton, J. H.(1984) Methods Enzymol. 109 i H. A., and Henseleit, K. (1932) Hoppe-Seylers 2. Physz&n8zE influx by either a Ca2+/H+ antiportor a Ca2+/0H- symport. 16. Krebs, 210,33-66 Consistent with this proposal that the proton motive force 17. Dehaye, J.-P., Blackmore, P. F., Venter, J. C., and Exton, J. H. (1980) J. BwL Chem. 256,3905-3910 across the plasma membrane is responsible for the Ca2+influx 18. Blackmore, P. F., and Exton, J. H. (1981) Biochem. J. 198,379-383 is the fact that M carbonyl cyanide m-chlorophenylhy- 19. Mor an, N G , Blackmore, P. F., and Exton, J. H. (1983) J. Biol. Chum. z t . mhx-ilns "_, drazone completely suppresses the Caz+ influx at pH 8.1 20. Kohlhardt, M.,Bauer, B., Krause, H., and Fleckenstein, A. (1973) Pfluegers Arch. Eur. J. Physwl. 3 3 8 , 115-123 induced by vasopressin, epinephrine, angiotensin 11, and glu21. Kaahiwagura, T.,Deutsch, C. J., Taylor, J., Erecinska, M., and Wilson, D. cagon (data not shown). It must also be mentioned that F. (1984) J. Biol. Chem. 269,237-243 Rink, T. J., Tsien, R. Y., and Pozzan, T. (1982) J. Cell Biol. 95,189-196 uncouplers act on mitochondria and produce Ca" release. 22. 23. Poggioli, J. Berthon B., and Claret M. (1980) FEBS Lett. 115, 243-246 Alternatively, the pHoptima for the coupling ofthe hormone 24. Shears, J. $., and Kirk, C. J. (1984)'Biochem.J. 220,417-421 " "
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