Mar 4, 1991 - modified Hank's buffer consisting of 20 mM Hepes, 137 m M NaC1,. 5.4 m M ...... Joseph, S. K., Thomas, A. P., Williams, R. J., Irvine, R. F., and ...
THEJOURNAL OF BIOLOGICAL IC'
Vol. 266. No. 28, Issue of October 5, pp. 18573-18579,1991 Printed in U. S A.
CHEMISTRY
1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanisms of Receptor-mediated Ca2+Signaling inRat Hepatocytes" (Received for publication, March 4, 1991)
Carl A. HansenS, Lijun Yang,and JohnR. Williamson$ From the DeDartment of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia; Pennsyluania 19104 " "
The Ca2+signal observed in individual fura-2-loadedlular messengers, Ins-1,4,5-P3' and diacylglycerol (1, 2). The hepatocytes stimulated with the al-adrenergic agonistensuing rise in cytosolic free Ca2+ results from an Ins-1,4,5phenylephrine consisted of a variable latency period, P3-mediated release of Ca2+from internal stores and from an a rapid biphasic increase in the cytosolic free Ca2+, activation of Ca2+ influx (2, 3). The mechanism of agonistyet resolved, followed by a period of maintained elevated cytosolic stimulated extracellular Ca2+ entry has not been but in many cell types it is distinct fromvoltage-operated Ca2+ (plateau phase) that depended on the continued presence of both agonist and external Ca". Microin- Ca2+ channels (4). Current experimental datasuggests three jection of guanosine-5'-0-(3-thiophosphate)elicited a possible mechanisms: 1) receptor-activated Ca2+channels, in which receptor activation and channelopening are intimately Ca2+ transient with the same basic features. The Ca2+ transient resulting from microinjectinginositol 1,4,5- interconnected (5,6), perhaps via a G protein (5,7),2) second trisphosphate (Ins-1,4,5-P3) occurred with essentially messenger operated Ca2+ channels,which are gated either by no latency period and consisted of a rapid spike that Ins-1,4,5-P3 (8-lo), Ins-1,3,4,5-P4(11,12), or Ca2+itself (13), decayed back to preinjection levels within 15 s. Mi- and 3) capacitativecalcium entry, inwhich activation of Ca2+ croinjection of inositol 1,4,5-trisphosphorothioate entry is linked to emptying of the internal Ins-1,4,5-P3-sen(thio-Ips), a nonmetabolizable analog of Ins-1,4,5-P3, sitive Ca2+ store(14, 15). It is likely that the various mechaelicited a Ca2+ transient that was initially identical tonisms operate to different extents in different cell types. In that observed with Ins-1,4,5-P3, except that the cyto- this study, we have characterized the Ca" transient elicited solic Ca2+remained elevated. The maintained thio-Ips-by phenylephrine in hepatocytes at the single cell level and induced Ca2+increase was dependent on the presence subsequently attempted to determine the essential intracelof external Ca2+, suggesting an activationof Ca2+in- lular signals required to generate the various aspects of the was accomplished by activating thesignal flux. Reintroduction of external Ca2+ in the presence Ca2+ transient. This of 5 p~ phenylephrine to Ca2+-depletedcells resulted transduction process distal to receptor activation by microinmein a 2-fold greater rate of rise in the cytosolic Ca2+ jecting specific compounds that are currently thought to compared to the rate observed upon Ca2+ addition to diate aspects of this process. cells Ca2+-depleted by preatement with thapsigargin. EXPERIMENTALPROCEDURES The rate of Ca2+ rise upon Ca2+ addition to cells microinjected with thio-IP3 was similar to that observed Hepatocyte Preparation-Hepatocytes were isolated from livers of with phenylephrine. Coinjection of the cells with thio- fed, male Sprague-Dawley rats weighing 180-220 gbycollagenase IPS plus heparin reduced the rate of Ca2+ rise upon digestion as described previously (16). Cells were resuspended at 2 X Ca2+ addition tothat observed in thapsigargin-treated lo5cells/ml in Leibowitz-15 media supplemented with 20 m M Hepes cells. These data indicate that themechanism respon- and 5.5 mM glucose. Cells were attachedto poiy-D-Iysine coated coverslips by incubating 3 ml of cell suspension for 1 h at 37 "C, at sible for receptor-mediated stimulation of Ca2+ entry which point the media was changed and unattached cells removed. into hepatocytes involves not only capacitative Ca2+ Cells were used from 1.5 to 6 h post-plating. entry but also an additional component mediated diMeasurement of Cytosolic Free Ca""Ce1ls attached to coverslips were transferred to a Leiden cell chamber and incubated in 3 ml of rectly by Ins- 1 ,4,5-P3.
The concentration of cytosolicfree Ca2+ regulates many intracellular events. Theseinclude the controlof metabolism, secretion, and muscle contraction, as well as other functions mediated by hormones, neurotransmitters, and growth factors.Hormone-mediatedCa2+signalingisinitiated by a n agonist-induced activation of phospholipase C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate intotwo intracel*This study was supported by an American Heart Association Grant-in-aid and National Institutesof Health Grant DK15120. The costs of publication of thisarticle were defrayed inpart by the payment of page charges. Thisarticlemusttherefore be hereby marked "aduertlsement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Presentaddress: GeisingerClinic, WeisCenter for Research, Danville, PA 17822-2619. To whom requests for reprints should be addressed.
modified Hank's buffer consisting of 20 mM Hepes, 137 m M NaC1, 5.4 m M KC1, 4.2 mM NaHCO:
