Characterization of inositol 1, 4, 5-trisphosphate receptors and calcium ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 263, No. 10,Issue of April 5. pp. 4541-4548,1988 Printed in U.S.A.

Characterization of Inositol 1,4,5-Trisphosphate Receptors and Calcium Mobilization in a Hepatic PlasmaMembrane Fraction* (Received forpublication, May 7,1987)

Gaetan GuillemetteS, Tam& Balla, Albert J. Baukal, andKevin J. Catt From the Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 and the Department of Pharmacology, Schoolof Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J l H 5N4

The distribution of hepatic binding sites for the cal-stimulated phosphodiesteratic cleavage of phosphatidylinosicium-mobilizing second messenger, inositol 1,4,5-tris- to1 4,5-bisphosphate is a major intracellular messenger which phosphate (Ips),was analyzed in subcellular fractions serves to increase the cytosolic Ca2+ concentration during of the rat liver by binding studies with ['"P]IP3 and hormone action. Recently, IP, has been shown to release compared with the Ca2+release elicited by IPSin each calcium from non-mitochondrial stores in a wide variety of fraction. Threemajor subcellular fractions enriched in cells including hepatocytes (1-5), smooth muscle cells (6-9), plasma membrane, mitochondria, and endoplasmic re- neuronal cells (10, l l ) , pituitary cells (12, 13),and many ticulum were characterized for their 5'-nucleotidase, others (for review, see Ref. 14). IP, acts through specific glucose-6-phosphatase, succinate reductase, and angiotensin I1 binding activities. The fraction enriched intracellular receptors that have been demonstrated in the in plasma membrane showed 7- and 20-fold increases adrenal cortex (15, 16), macrophages (17), liver (18), neutroin IPS binding capacity over those enriched in endo- phils (19), brain (20) and anterior pituitary gland (21) by plasmic reticulum andmitochondria, respectively, and direct ligand-binding studies. An important question about contained a single class of high-affinity binding sites the mechanism of action of IP3 is the nature and location of with Kdof 1.7 f 1.0 nM and concentrationof 239 f 91 the pool from which Ca2+is mobilized. Currently, the endofmol/mg protein. IPS binding reached equilibrium in plasmic reticulum is believed to be the major source for release 30 min at 0 "C, and the half-time of dissociation was of calcium from intracellular stores. However, IPS may also about 15 min. The specificity of the IP3 binding sites promote calcium mobilization and/or calcium influx at the was indicated by their markedly lower affinities for level of the plasma membrane during its action on the redisinositol 1-phosphate, phytic acid, fructose 1,6-bisphos- tribution of intracellular calcium. We have recently observed phate, 2,3-bisphosphoglycerate,and inositol 1,3,4,5- that IP3 binding sites are relatively abundant in a plasma tetrakisphosphate. The Ca"+-releasing activity of IP3 membrane-enriched preparation, consistentwith an action of in the subcellular fractions was monitored with the fluorescent indicator,Fura-2. All three fractions IP3 at this location (16). Since liver is readily fractionated into enriched organelles, and has been widely used for the showed ATP-dependent Ca2+ uptake and rapidly released Ca2+ in response in IPS. The fraction enriched study of calcium-mobilizinghormones (22-26 ; for review, see in plasma membrane was the most active in this regard,Ref. 27), it was employed for a more detailed analysis of the releasing 174 f 67 pmol Ca2'/mg of protein compared subcellular distribution of IPSbinding sites. In thisstudy, the to 46 f 10and 48 f 7 pmol/mg protein for the fractions fraction enriched in plasma membrane was found to be highly enriched in endoplasmic reticulum and mitochondria, enriched in IP, receptors with binding properties similar to respectively. These data suggest that the[""PIIP, bind- those recently described in adrenal cortex (16) and anterior ingsitesrepresent specific intracellularreceptors pituitary gland (21). Significantly, the abundance of IP, recepthrough which IPSmobilizes Ca2+from a storage site tor sites in the plasma membrane fraction was correlated with associated (or co-purifying) with plasma the membrane high IP3-induced Ca2+release in this fraction. These results of the rat liver. It is likelythat a specialized vesicular indicate that the vesicular system to which IP, binds and system (to which IP, can bind and trigger the release releases Ca2+is closely associated with the plasma membrane of Ca2+)is located in close proximity with the plasma and co-purifies with it during subcellular fractionation proat which IP, membrane and is thus adjacent to the site is produced during stimulation of the hepatocyte by cedures. Ca"+-mobilizinghormones. EXPERIMENTALPROCEDURES Inositol 1,4,5-trisphosphate (IP,)' generated from agonist-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a Medical Research Council of Canada Postdoctoral fellowship. To whom correspondence should be addressed National Institutes of Health, Bldg. 10, Rm. 8C-407, Bethesda, MD 20892. The abbreviations used are: IP,, inositol 1,4,5-trisph.osphate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; PEG, polyethylene glycol 6000; Gpp(NH)p, guanyl-5"yl imidodiphosphate.

Materials-Inositol 1,4,5-trisphosphate and inositol 1-phosphate were obtained from Amersham Corp. ["P]IP, (20-200 Ci/mmol) and ['HIIP, (3.6 Ci/mmol) were obtained from New England Nuclear. Fura-2 (pentasodium salt) was from Behring Diagnostics. Monoiodinated 1261-angiotensinI1was prepared by a modified IODO-GEN (Pierce Chemical Co.) method (28) (Meloy Laboratories, Springfield, VA) and had a specific radioactivity of 1500 Ci/mmol. All other reagents were from Sigma or Boehringer Mannheim. Preparation of Subcellular Fractions-The three subcellular fractions were prepared following a combination of Neville's (29) and Dawson's (4) procedures. Livers from 250-400-g male albino rats were minced and homogenized with 10 strokes of a Dounce homogenizer (loose pestle) in a buffer containing: 20 mM HEPES/KOH, pH 7.2, 110 mM KCl, 10 mM NaCl, 2 mM MgC12, 5 mM KH2P0,, 2 mM EGTA, and 1 mM dithiothreitol. After stirring for 5 min, filtration

4541

IP3 Receptors and Calcium Release Liver in

4542

Fractions

through cheesecloth, and centrifugation a t 1500 X g for 20 min, the treatment with inomycin (1p ~ )The . equation used was [Ca'+] = 224 pellet was resuspended in homogenization buffer and adjusted to 44% nM ( F - F-)/(F',,,- - F ) (34). in sucrose. From that point, the plasma membrane fraction was purified according to the method of Neville (29) up to step 11. This RESULTS fraction was washed and resuspended in Buffer A (the homogenizaCharacterization of Hepatic Subcellular Fractions-Since tion buffer without EGTA) a t a concentration of 20-30 mg ofprotein/ modifications of the previously described procedures (4, 29) ml(30). The mitochondrial fraction was prepared by centrifugation of the were employedfor separationof the liver subcellular fractions, 1,500 X g supernatant at 8,000 X g for 10 min. The pellet was then enzymatic characterization of each preparation was perresuspended in Buffer A, sedimented at 8,000 X g for 10 min and formed. As shown in Table I, the plasma membrane fraction taken up a t a high protein concentration (-30 mg/ml) in Buffer A. was the most enriched in 5'-nucleotidase activity (a marker The microsomal fraction was prepared by centrifugation of the 8,000 for plasma membrane) and the least enriched in glucose-6X g supernatant at35,000 X g for 20 min. This pellet was resuspended in Buffer A, recentrifuged at 35,000 X g for 20 min and taken up in phosphatase activity (a marker for endoplasmic reticulum). expected, the microsomal fraction Buffer A. The subcellular fractions were used freshly for Ca2+-mobi- On the otherhand,as lization studies or were frozen in small aliquots for binding or enzy- showed an exactly opposite pattern of enrichment. The mimatic analysis. tochondrial fraction, although the most enriched in succinate Churacterization of SubceUular Fractions-Glucose-6-phosphatase reductase activity, showed substantial contamination with was measured a t 37"Cby the method of Salomon et al. (31), 5'- both glucose-6-phosphatase and 5"nucleotidase activities. nucleotidase by the method of Bramley and Ryan (32). and succinate- The angiotensin I1 binding capacity, which provided a further reductase according to Pennington (33). lZ6I-AngiotensinI1 binding marker of the plasma membrane, was consistent with the data was performed for 30 min a t room temperature ina buffer containing: 20mM Tris/HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl,, 0.2% bovine obtained by enzyme assays. The three subcellular fractions were analyzed for their IP3 serum albumin, and 0.5 mM dithiothreitol. The bound and free ligand were separated by vacuum filtration through glass-fiber (GF/c) mem- binding properties. As shown in Fig. 1 (see also Table I), the branes. plasma membrane fraction showed a much higher binding IPS Binding Studies--Rat liver fractions containing 200- OC3 pg capacity for IPS (6 and 20 times higher than the fractions of protein were incubated in a medium of the following composition: enriched in endoplasmic reticulum and mitochondria, respec25 mM NazHPO,, pH 7.4, 100 mMKC1,20mM NaCl, 0.1% bovine tively). The upper leftpanel shows typical displacement curves serum albumin, and 1mM EDTA. Incubations were performed, unless obtained during inhibition of tracer ["P]IP, binding by inotherwise specified, for 30 min a t 0 "C in a final volume of 500 pl creasing concentrations of unlabeled IPS.In this experiment, with ["P]IP3 (-20,000 cpm 100 fmol 0.3 nM).Nonspecific binding was determined in the presence of 1 p~ IP3. Incubations were ter- the IP3 binding capacity of the plasma membrane fraction minated by filtration through presoaked glass-fiber filters (Whatman was6.3 and 13 times higher than those of the fractions G F P ) and rapid washing with 2.5 ml of ice-cold incubation medium. enriched in endoplasmic reticulum and mitochondria, respecThe receptor-bound radioactivity was analyzed by liquid scintillation tively. In the upper right panel, thedata from the same spectrometry. experiment are normalized to show the similar binding affinCa'+-mobilization Studies-Subcellular fractions (3-10 mg of pro- ities of the three fractions for IPS.Scatchard analyses of the tein) were incubated in Ca'+-mobilization buffer (Buffer A complesame data (lowerpanel) also indicate the similar binding mented with an ATP-regenerating system: 10 mM creatine phosphate and 20 IU/ml creatine phosphokinase). The free Ca" concentration affinities (0.8, 0.9, and 0.8 nM) and the marked difference of the medium was monitored using Fura-2 free acid (3 p h i ) in a between the binding capacities (146, 23, and 11 fmol/mg Perkin-Elmer LS-5 fluorescence spectrophotometer; the excitation protein) of the plasma membrane, endoplasmic reticulum, wavelength was 340 nm (slit 10 nm) and the emission was recorded and mitochondria-enriched fractions, respectively. a t 500 nm (slit 10 nm). Ca2+uptake and release were measured under To define better the subcellular location of the binding sites constant agitation at 37 "C in a final volume of 2 ml. Oligomycin (2.5 for IPS, the plasma membrane fraction was further purified pg/ml) was added to block mitochondrial ATPase. ATP, IP3, and by application to a 13-37% sucrose gradient overlaying a 50% other effectors were added in small volumes. Each record was calibrated by the addition of known amounts ofCa'+ (CaC03) to the sucrose cushion. After centrifugation for 60 min at 550 X g, mixture. The actual Ca2+concentration of the medium wascalculated the material at thecushion interface was collectedand washed from the maximal fluorescence (Fmm)and autofluorescence (Fmin) in Buffer A. The 5"nucleotidase activity and IPS binding values obtained by adding excess Ca2+and Mn2+,respectively, after capacity of this more purified membrane fraction were further

-

-

TABLE I Churacterization of subcellularfractions prepared from rat liver Angiotensin I1 binding was estimated by incubating 25 pg of each fraction with '"I-angiotensin I1 (200,000 cpm -60 fmol -0.5 nM) in the presence and absence of M unlabeled ligand. For the determination of 5"nucleotidase and glucose-6-phosphatase activities, 50 pg of each fraction was used; the amount of organic phosphate formed was measured by the method of Fiske and Subbarow (35). Mitochondrial succinate-tetrazolium reductase activity was evaluated with 50 pg of each fraction; formazan production was calculated from a value of 20.1 X lo3 for the molar extinction coefficient (490 nm) of the formazan dissolved in ethyl acetate (33). IP3 binding was evaluated by Scatchard analysis of competitive dose-displacement experiments using, respectively, 500 pg, 1mg, and 2 mg of protein for the plasma membrane, the endoplasmic reticulum and the mitochondria-enriched fractions. These results represent the mean f standard deviation of triplicate determinations for the different activities in a single preparation. Similar results were obtained in five other preparations. Plasma membrane fraction

Angiotensin I1 binding (cpm/25 pg protein) 5'-Nucleotidase (pmol P/mg protein/20 min) Glucose-6-phosphatase (pmol P/mg protein/30 min) Succinate reductase (mmol Formazan/mg/lS min) IP3 binding (fmol/mg protein)

6954 f 416 2.10.9 f 0.1 3.3 f 0.1 1.2 f 0.1 289 56

Microsomal fraction (endoplasmic reticulum)

1933 f 110 1.3 f 0.1 9.5 f 0.2 0.1 f 0.05

Mitochondrial fraction

1882 f 50 f 0.2 5.9 f 0.1 1.8 f 0.2 8

1543

IPS Receptors and Calcium Release in LiverFractions

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FIG. 1. IPSbinding capacities ofliver subcellular fractions. Liver particles (500pg of protein) were incubated at 0 "C in medium containing 20,000 cpmoflabeledligand [32P]IP3andincreasing concentrations of unlabeledIPS.After 30 min the sampleswere

FIG. 2. Association and dissociation of [""PIIP, with liver binding sites as a function of time. Binding was initiated at 0 "C by addition of the membrane suspension (300 pg of protein) to assay buffer containingthe radioligand (28,000 cpm), and dissociation was initiatedby adding M (final concentration) unlabeled IP3(arrow).

At the indicated times, aliquots were removedand filtered as described under "Experimental Procedures." The dissociation data expressed on a logarithmic scale are shown inthe lower panel. This representativeexperimentperformedinduplicate wasrepeatedwith three different membrane preparations.

filtered, washed, and analyzed by scintillation spectrometry. In the upper leftpanel the results are expressed as specific counts/min bound M unlabeled IP3 (ordinate)as a function of unlabeled ligand concentration. Each valueBinding was reversible, andaddition of was corrected for nonspecific binding (plasma membrane, 259 cpm; was followed by rapid dissociation of the boundligand with a microsomes, 321 cpm;mitochondria, 328 cpm)determinedin the half-time of about 15 min, according to a single exponential presence of M unlabeled ligand. In the upper right panel the data function asshown in thelower panel of Fig. 2. The association are expressed as values relativeto the total binding observed without rate data fitted a second-order equation as shown in Fig. 3, unlabeled ligand and corrected for nonspecific binding. Scatchard plots of the same binding data are shown in the lower panel. This upper panel. The association rate constant ( k + l ) ,calculated experiment performed in duplicate is representative of similar obser- from the slope of the curve, was 2.1 X lo7 M" .min-'. The vations with six different liver preparations. 0, plasma membrane dissociation rate constant (12-J estimated by fitting the bindfraction; 0, microsomal fraction; 0,mitochondrial fraction. ing data to a first-order rate equation (Fig. 3, lower panel) was 0.02 min-'. The Kdcalculated from the ratio of the rate enriched by comparison with the original plasma membrane constants for dissociation and association was 0.95 nM, in fraction and the homogenate. In a typical preparation, the 5'- agreement with thevalue obtained from steady state binding nucleotidase enzyme activity was 7.6 and 10.5 times higher in experiments. the original membranefractionandtheenrichedplasma In saturation studies (Fig. 4), tracer binding was reduced membrane fraction, respectively, as compared to thehomog- by about 10% in the presenceof 0.2 nM unlabeled ligand and enate. On the other hand,glucose-6-phosphatase activity was was progressively inhibited by increasing concentrations up only 1.8 and 1.3 times as active in the same fractions as to 100 nM IPS(upper panel). Scatchard analysis of the binding compared to the homogenate. The IP, binding capacityof the data was consistent with a single set of high-affinity sites enriched plasma membrane fraction was 757 fmol/mg protein, (upper panel, inset) with K d of 1.7 f 1.0 nM and maximal almost 3 times that of the original membrane fraction (264 binding capacityof 239 f 91 fmol/mg protein ( n = 12). Since fmol/mg protein) and about 30-fold higher than that of the two classesof binding sitesfor IP, have been observed in liver homogenate (25 fmol/mg). Similar results were obtained in microsomes (la), and to complement the data obtained by three different preparations, confirming the co-purification of competition analysis (in which low-affinity binding of ["PI IP, binding sites with plasma membrane components. IPSmay be obscured as themeasured ligandbinding decreases Characterization of IP, Binding Sites-Since the plasma toward the nonspecific value), we also performed saturation membrane fraction was highly enriched in IP, binding sites, analysis with increasing concentrations of the low specific subsequent studies t o characterize the sites were performed radioactivity radioligand ['HIIP, (3.6 Ci/mmol). This method with this subcellular fraction. Kinetic studies exemplified in provides a more accurate determination of the receptor bindFig. 2 revealed that specific binding of IP, at 0 "C was half- ing profile at high ligand concentrations, where low-affinity maximal in about5 min and reacheda plateau within 30 min. sites would be detected. As shown in Fig. 4 (lower panel),

4544

IP3 Receptors and Calcium Release in Liver Fractions

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FIG. 4. Saturation of IPS binding sites in liver plasma memMinutes brane fraction. Upper panel,liver particles (250 pg of protein) were FIG. 3. Association and dissociation rate constants for bind- incubated at 0 "C in medium containing [32P]IP3(34,000cpm) and ing of [SaP]IPs to liver plasma membrane fraction. In theupper increasing concentrations of unlabeled ligand. After 30 min the inpanel, the data from the experiment depicted in Fig. 2 (upper panel) cubation was stopped as indicated under "Experimental Procedures." The dataare expressed as values relative to the total binding observed have been fitted to thesecond-order rate equation: without unlabeled ligand (4,474cpm) and corrected for nonspecific M N o- Lo) In (NO- LJ/(L, - LC) binding (403cprn). This experiment performed in duplicate is repreas a function of time, where No is the total number of binding sites; sentative of 12 such observations. The Scatchard plot of the same is the totalligand present at time 0; L,is the ligand bound at time binding data is shown in the inset: this experiment gave a Kd of 1.4 ( t ) .The rate constant of association was calculated from the slope of nM and maximal binding capacity of 333 fmol/mg protein. Lower this curve. In the lower panel, the datafrom Fig. 2 (lower panel)were panel, aliquots (500 pg of protein) of the same liver particle preparafitted to a first-order rate equation: In BIB, as a function of time, tion were incubated a t 0 "C for 30 min with increasing amounts of where B is the ligand bound a t time ( t ) and Bo is the ligand bound a t [3H]IP3(3,000-100,000cprn). For each [,H]IP3 concentration, nonthe moment when the unlabeled IP3 was added. The rateconstant of specific binding was evaluated in the presence of 20 p~ IP,. The dissociation was calculated from the slope of this curve. The Kd incubations were stopped by filtration and the data were expressed calculated from the ratio of the rate constants for dissociation and as theamount of ['HIIP, specifically bound as a function of the total amount of labeled ligand present in the incubation. The Scatchard association was 0.95 nM. plot of the same binding data (shown in inset) revealed a high-affinity binding site with Kd of 1.4 nM and maximal binding capacity of 347 saturation of IP, receptors was observed in the presence of fmol/mg protein. This experiment was performed in duplicate.

about 30,000 cpm (-20 nM) [,H]IP3, with maximum specific binding of 675 cpm (plateau value) corresponding to 174 fmol [,H]IP3. Scatchard analysis of these data was consistent with a single class of high-affinity sites with Kd of 1.4 nM and maximal binding capacity of347 fmol/mg protein (Fig. 4, lower panel, inset). In the same membrane preparation, the binding capacity estimated from binding-inhibition analysis with ["P]Ip3 was 352 fmol/mg protein and the Kd was 1.4 nM . The specificity of the IP, binding sites was analyzed in competitive binding experiments performed with inositol phosphates and related compounds. As shown in Fig. 5,phytic acid and inositol 1-phosphate showed extremely low affinities for the binding site (at least 10,000-fold less than thehomologous ligand). Other compounds bearing phosphate groups on vicinal carbon atoms, such as fructose 1,6-bisphosphate and 2,3-bisphosphoglycerate (an inhibitor of the enzyme IP3 5'phosphatase) (36, 37), showed extremely low binding affinities. The most closely similar compound, inositol 1,3,4,5tetrakisphosphate, showed relatively low affinity (about 5% compared with IP,) for the binding site. The shapes of the displacement curves and their parallelism with the IPSbinding-inhibition curve were consistent with competitive interaction of these compounds with the IPSbinding site. Ca2+-rekasingActivity of IP3-Ca2+ uptake and release in response to IP3 were determined in each of the subcellular

fractions. The plasma membrane fraction showed ATP-dependent Ca2+sequestering activity (which was inhibited by vanadate), and decreased the ambient Ca2+concentration to about 500 nM (Fig. 6, panel A). This relatively high value should not be considered aa the "set point" but was probably due to thelimited capacity of the vesicular system in comparison to the large amount of Ca2+in the medium. When the ambient calcium concentration was reducedby adding a small amount of EGTA (-10 PM) or if the plasma membrane preparation was added in higher concentration (10 mgof protein), the ATP-dependent Ca2+uptake process lowered the Ca2+level to below 200 nM (data not shown). Addition of IP, (2.5 PM) caused immediate release of 0.4 nmol of Ca2+followed by slow re-uptake. Subsequent stimulation with IP, evoked a comparable response, although with repeated additions the response diminished in magnitude. Addition of 1 PM ionomycin immediately released the accumulated Ca2+, indicating the vesicular nature of the Ca2+ sequestering process. The fractions enriched in endoplasmic reticulum (Fig. 6,panel B ) and mitochondria (Fig. 6,paneE C) also showed highATP-dependent Ca2+sequestering activities, decreasing the ambient Ca2+concentration to about 200 nM. However, these preparations showed smaller responses to IP3 (2.5 FM) stimulation, releasing 0.13 and 0.07 nmol of Ca2+,

4545

IPaReceptors and CalciumRelease in LiverFractions

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FIG. 5. Competitive binding of IPS and related compounds in liver plasma membrane fraction. Liver particles (500 pgof protein) were incubatedat 0 “C in medium containing [3zP]IP3(14,000 cpm) and increasing concentrations of unlabeled compounds. After 30 min the incubations were stopped and analyzed as indicated under “Experimental Procedures.” The data are expressed as values relative to the total binding observed without unlabeled ligand (2,482 cpm) and corrected for nonspecific binding (192 cpm). IPS,phytic acid; IP, inositol 1-phosphate; FPz, fructose 1,6-bisphosphate; BPG, 2,3-bisphosphoglycerate; ZP,,inositol 1,3,4,5-tetrakisphosphate. IP, was prepared by phosphorylation of IP3 using partially purified IP3 kinase from the bovine adrenal cortex and was purified by ion exchange high performance liquid chromatography (38). This experiment performed in duplicate is representative of two such observations.

respectively. Both fractions again released sequestered Ca2+ upon addition of ionomycin. The average values for maximal IP3-induced Ca2+release were 174 f 67 pmol of Ca2+/mgof protein ( n = 8) in the plasma membrane fraction, 45 f 10 pmol of Caz+/mg of protein ( n = 3) in the endoplasmic reticulum-enriched fraction, and 48 & 7 pmol of Ca2+/mgof protein ( n= 3) in the mitochondria-enriched fraction. Again, since the plasma membrane preparation was the most active, this fraction was used to furthercharacterize the action of IP, in the liver. The dose-response relationship between IPS and Ca2+mobilization is shown in Fig. 7. The threshold response was elicited by 50 nM IP, and the EDsofor Ca2+mobilization was 257 f 61 nM ( n = 4), comparable to values observed in other laboratories (1,4). To determine whetherthe effect of IP, on Ca2+release was influenced by GTP, as previously observed in liver microsomes (39), we also examined the influence of guanyl nucleotides on IP, action and Ca2+release in theplasma membrane ) no effect on the fraction. In our hands, GTP (100 p ~ had IP3-induced Ca2+release and was unable by itself to release Ca2+from the liver plasma membrane preparation (see Fig. 8, panel A ) . However, whenthe incubation medium was complemented with 3% PEG(Fig. 8,panel B ) , GTP alone was found to release Ca2+.The amount of Ca2+released (467 pmol/mg protein) by a maximal dose of GTP (100 p ~ was ) larger than the amount released by IPS (250 pmol/mg protein in the experiment shown in panel A ) , although the rate of release was much slower (a few seconds to reach the peak response with IPS compared to about 5 min with GTP). Thethreshold response was elicited by 1 p~ GTP and the EDb0for Ca2+ mobilization was about7 p~ (not shown). Gpp(NH)p,a nonhydrolyzable analog of GTP, did not release Ca2+ at concentrations up to 250 p ~ but , was able to block the effect of low doses of GTP (not shown). In the presence of PEG, doses of GTP that elicited small increments in Ca2+release did not influence the effects of low doses of IPS. As shown in Fig. 8 (panel C ) , 0.25 p~ IP3 released 120 pmol of Ca2+in the presence of GTP (2 p ~ and ) released 130 pmol of Ca2+in its

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.24

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FIG. 6. IP,-induced Caz+release in liver subcellular fractions. Liver particles (3 mg of protein) were incubated at 37 “C and their Caz+uptake and release activities were monitored with Fura-2 (free acid) as described under “Experimental Procedures.” Panels A , B, and C show typical traces derived with plasma membrane, endoplasmic reticulum, and mitochondria-enriched fractions, respectively. c, 0.2 nmol of Ca2+;I , 2.5 p M IP3; lo, 1 pM ionomycin. These traces are representative of several such observations in at least 1three different liver preparations.

0.1

1 .o

10

IPR (!AM)

FIG. 7. Dose-dependent effects of IP, on release of Ca” from the liver plasma membrane fraction. The experiment was performed as described in the legend of Fig. 5. Ordinate, amount of CaZfreleased expressed as a ratioof the maximal response (290 pmol/ mg protein). Abscissa, concentration of IP3 added. Similar results were obtained with four different liver preparations.

IP3 Receptors and CalciumRelease in LiverFractions

4546

pituitary gland (21).Our finding that the plasma membrane fraction contains the highest level of IP,-induced Ca2+release activity also lends further significance to the presence of membrane binding sites for IP,. Comparison of the subcellular distributions of IP, binding 4 activity and IP3-induced calcium-release activity required analysis of each of these functions by procedures that differed widely in their requirements for optimal assay conditions. Also, the conditions required for the preparation of optimally 8 responsive microsomes for Ca2+-releasestudies differ considerably from those employed for liver fractionation. The ho2: +PEG AB mogenization medium is iso-osmotic,containing Hepes buffer, EGTA, and dithiothreitol, whereas a hypotonic medium containing 1 mM NaHC0, is used for plasma membrane purification by the Neville procedure (29). Furthermore, the sub4: cellular preparations are relatively unstable and rapidly lose their Ca2+mobilizing properties. For these reasons, we employed methods that allow rapid fractionation of the liver tissue, and the purity of our routine subcellular fractions was somewhat less than thatattainable by more rigorous fraction9’ J ation schemes. However, when additional purification of the ATP I plasma membrane fraction was performed by sucrose density 2E gradient centrifugation, concomitant enrichment of 5”nucleotidase activity and IPSbinding capacity was again observed. I ci A current question of major interest is whether IPS serves to control Caz+influx directly across the plasma membrane, 47 and/or its release from a calcium-containing structure associated with the plasma membrane, as well as from the endoi plasmic reticulum. IP, was recently found to activate Ca2+ 89 channels in the plasma membrane of T-lymphocytes (42), i ATP consistent with Michell’s (43)original proposal that phospholipid breakdown could regulate the influx of external Ca2+ 5 MIN through transmembrane channels. In addition to its possible FIG. 8. Effect of GTP on Caa+release in the liver plasma entry through IP,-regulated calcium channels, our data show membrane fraction. The experiment was performed as described that Ca2+is released by IP3 from vesicular structures after in the legend of Fig. 5. C, 0.2 nmol of Ca2+;G, 100 p~ GTP G ’ , 2 PM uptake by an ATP-dependent process (which is inhibited by GTP; I,2.5 p M IPS;I ‘ , 0.25 p M IPS;l o , 1p M ionomycin. Panels B and vanadate and can be completely reversed upon addition of a C, the incubation medium contained 3% PEG. These typical traces calcium ionophore). Although such structures could represent are representative of several similar observations made with three inside-out vesicles originating from the plasma membrane different liver preparations. during homogenization, this would not account for the Ca2+absence. This observation, together with the finding that IP3 releasing activity of IP, in a wide variety of permeabilized which do not could still release substantial amounts of Ca2+after GTP has cells (8, 44-46) including hepatocytes (1-3, 47) contain inside-out vesicles. The most likely explanation is produced its maximal effect (see panel B ) , suggests that IP, and GTP exert their actions through distinct mechanisms, as that IP, binds to and triggers the release of CaZ+from a vesicular system that is closely associated with the plasma also indicated by other recent studies(11,40,41). membrane, and which purifies with the membrane when DISCUSSION subcellular fractions are prepared. Several recent reports have provided evidence for a mechThe messenger action of IP, is expressed through its interanism whereby direct entry of extracellular calcium into an action with intracellular receptors that have recently been characterized in several different tissues. The intracellular IP3-sensitive pool contributes to receptor-operated calcium location of this receptor is an important aspect of the mech- influx and is responsible for sustained calcium entry in agoanism of action of IP,. This question was addressed by Daw- nist-stimulated cells (48-52). The IP3-sensitive pool, with its son and Imine (4)in the liver by studies which showed that associated IPS receptors and calcium releasing mechanism, IPS releases Ca2+from a vesicular system other than mito- would thus be expected to be adjacent to the plasma memchondria. Attempts to purify a microsomal fraction caused brane. At least three current models of receptor-activated loss of the IPS effect, suggesting that the IP,-sensitive Ca2+ calcium entry involve a close apposition between IP,-sensitive pool is a specialized part of the endoplasmic reticulum which regions of endoplasmic reticulum and theplasma membrane. co-purifies with heavier cellular organelles. Similar experi- In Putney’s (52) capacitative scheme, agonist-induced empments by Joseph et al. (1)led to the same conclusion. Subse- tying of calcium from a component of the endoplasmic reticquent binding studies by Spat et al. (18)showed enrichment ulum (the receptor-regulated calcium pool) serves as a signal of IP, binding sites in the microsomal as compared to the for calcium entry by promoting its transport from the subplasmalemma1 space into the endoplasmic reticulum. A more cytosolic and mitochondrial fractions of rat liver, but the plasma membrane fraction was not examined. The present complex model developed by Gill and colleagues (53) to exdata clearly demonstrate that theplasma membrane fraction plain the role of GTP in calcium movements involves the is highly enriched in IP, receptors with binding properties formation of junctional processes between adjacent memsimilar to those present inthe adrenal cortex (16)and anterior branes, including connections between the plasma membrane 2

/‘\

IP3 Receptors a n d Calcium Release in Liver Fractions and theendoplasmic reticulum. Likewise, the model proposed by Irvine and Moor (54) for the conjoint actions of IP, and IP, on calcium entry also includes functional coupling between plasma membrane and endoplasmic reticulum, in this case promoted by Ins-1,3,4,5-P4, to permit direct entry of external calcium into theendoplasmic reticulum. It hasnot been established whether distinctstructural associations between the plasma membrane and endoplasmic reticulum, which could represent the membrane-associated vesicular system implicit in the above models and suggested by the present findings, are present in non-excitable cells. However, the presence of associations between the plasma membrane and sarcoplasmic reticulum in smooth muscle (55) and endoplasmic reticulum in platelets (56), as well as the recent proposal that specialized organelles (calciosomes ) are involved in calcium mobilization (57), suggest that discrete perimembrane structures may play a general role in the process of calcium entry and distribution within the cell. Such structures may be present in plasma membrane vesicle fractions from human platelets (56) and rat parotid acini (58), both of whichhave been shown to release Ca'+ in an IP,-sensitive manner. The IP, binding sites observed in the plasma membrane fraction of the ratliver possess all the characteristics of a true receptor. IP, binds to a particulate component and the level of binding is proportionalto the amount of membrane protein. The binding is rapidly reversible as expected for a normal ligand-receptor interaction, and this would permit rapid termination of the response. The rapid rates of association and dissociation are consistent with the calcium mobilizing activity of IP,. The binding sites are saturable andbinding of the ligand is competitive, being inhibited by increasing concentrations of unlabeled ligand. The binding is also highly specific for IP,; inositol-derived compounds and organic compounds bearing phosphoryl groups on vicinal carbons showed weaker affinities for the binding site. Finally, the important criterion of a functional response is demonstrated by the ability of IP, to release Caz+from the same membrane preparation used for the binding experiments. One question arising from our study is the high affinity of the binding site by comparison with the potency of IP, for releasing Ca*+.Such a discrepancy was also observed in the adrenal cortex (16) and anterior pituitarygland (21), andmay be largely attributable to the divergent experimental conditions necessary for the two assays. In particular, the optimal conditions for IP, binding arevery different from those within the cell. Also, the degradation of added IP, by the plasma membrane fraction at 37 "C isvery rapid and could lower the actual IP3concentration during Ca2+ release studies. Furthermore, ATP inhibits the binding of IP, by a mechanism that is not yet clear, and its presence at millimolar concentration in the Ca2' release assays could substantially lower the potency of IP,. Thus, thediscrepancy probably results from the combination of higher apparent affinity in the binding assay, due to choice of optimal binding conditions, and lower apparent affinity in theCa'+ release assays, due to rapid degradation of IP, at 37 "C in the presence of Mg2+ together with the competitive effect of ATP. The difference might also reflect decreased efficiency of the calcium-gating mechanism due to loss or impairment of a putativeregulatory component during preparation of the subcellular fractions. One such component could be a guanine nucleotide regulatory protein associated with the calcium release mechanism. However, the effect of GTP on Caz+ release from the liver plasma membrane fraction was independent of IP,, as previ-

4547

ously observed in the adrenal cortex' and the anterior pituitary gland (21). In several tissues, GTP seems to actthrough a mechanism distinct from IP3, and in parotid acini the GTPsensitive and IP,-sensitive calcium pools appear to reside in different regions of the endoplasmic reticulum (58). Thus, further studies are needed to clarify the importance of this effect of GTP in the regulation of Ca'+ mobilization during hormonal stimulation. It should be noted that two previous studies using subcellular fractions of rat pancreas (59) and rat adipocytes (60) showed that IP, mobilized Ca2+from endoplasmic reticulum but not from plasma membrane. Whether this is a reflection of differences between specific tissues or among fractionation procedures remains to be clarified. We are attempting to answer this question by analysis of IP3 binding in adrenal cortex and anterior pituitary plasma membranes to establish the intracellular site of action of IP, in these tissues. However, the presence of IPS binding and/or IPS-induced calcium mobilization in hepatic, platelet (56), and parotid acinar (58) plasma membrane fractions suggests that the IP,-sensitive calcium pool may be closely related to the plasma membrane in a wide variety of target cells that respond to calciummobilizing stimuli. REFERENCES 1. Joseph, S. K., Thomas, A. P., Williams, R. J., Irvine, R. F., and Williamson, J. R. (1984) J. Bwl. Chern. 259, 3077-3081 2. Burgess, G. M., Godfrey, P. P., McKinney, J. S., and Putney, J. W. (1984) Nature 309,63-66 3. Burgess, G . M., Irvine, R. F., Berridge, M. J., McKinney, J. S., and Putney, J. W., Jr. (1984) Biochern. J. 224,741-746 4. Dawson, A. P.,and Irvine, R. F. (1984) Biochem. Biophys. Res. Commun. 120,858-864 5. Muallem, S., Schoeffield, M., Pandol, S., and Sachs, G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,4433-4437 6. Bitar, K. N., Bradford, P. G., Putney, J. W., and Makhlouf, G. M. (1986) J. Biol. Chem. 261,16591-16596 7. Hashimoto, T., Hirata, M., Itoh, T., Kanmura, Y.,and Kuriyama, H. (1986) J. Physiol. 3 7 0 , 605-618 8. Smith, J. B., Smith, L., and Higgins, B. L. (1985) J. Biol. Chem. 260,14413-14416 9. Somlyo, A. V., Bond, M., Somlyo, A. P., and Scarpa, A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5231-5235 10. Chueh, S.-H., and Gill, D. L. (1986) J. Bwl. Chem. 2 6 1 , 1388313886 11. Jean, T., and Klee, C. B. (1986) J . Biol. Chern. 261,16414-16420 12. Gershengorn, M. C., Geras, E., Purrello, V. S., and Rebecchi, M. J. (1984) J. Biol. Chern. 269,10675-10681 13. Biden, T.J., Wollheim, C. B., and Schlegel, W. (1986) J. Biol. Chern. 2 6 1 , 7223-7229 14. Abdel-Latif, A. A. (1986) Pharmacal. Rev. 3 8 , 227-272 15. Baukal, A. J., Guillemette, G., Rubin, R., Spat, A,, and Catt, K. J. (1985) Biochern. Biophys. Res. Commun. 133,532-538 16. Guillemette, G., Balla, T., Baukal, A. J., Spat, A., and Catt, K. J. (1987) J. Bwl. Chern. 2 6 2 , 1010-1015 17.Hirata, M., Sasaguri, T., Hamashi, T., Hashimoto, T., Kukita, M., and Koga, T. (1985) Nature 317, 723-725 18. Spat, A., Fabiato, A., and Rubin, R. P. (1986) Biochern. J. 2 3 3 , 929-932 19. Spat, A., Bradford, P. G., McKinney, J. S., Rubin, R. P., and Putney, J. W. (1986) Nature 319,514-516 20. Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161 21. Guillemette, G., Balla, T., Baukal, A. J., and Catt, K. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8195-8199 22. Creba, J. A., Downes, C. P., Hawkins, P. T.,Brewster, G., Michell, R. H., and Kirk, C. J. (1983) Biochern. J. 212,733-747 23. Thomas, A. P.,Marks, J. S., Coll, K. E., and Williamson, J. R. (1983) J . Bid. Chern. 258, 5716-5725 G. Guillemette, T. Balla, A. J. Baukal, and K. J. Catt, unpublished observations.

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