Distribution of endoplasmic reticulum and calciosome markers in membrane fractions isolated from different regions of the canine brain

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 272, No. 1, July, pp. 162-174,1989

Distribution of Endoplasmic Reticulum and Calciosome Markers in Membrane Fractions Isolated from Different Regions of the Canine Brain’ BARBARA

H. ALDERSON

Department of Physiology and Bioph@cs,

AND POMPEO

VOLPE

The University of Texas Medical Branch, Galveston, Texus ?XXI

Received January 13,1989

Four regions of the canine brain (frontal lobe, parieto-occipital lobe, brainstem, and cerebellum) were each fractionated by differential centrifugation into a crude mitochondrial pellet (Ps) and a crude microsomal pellet (Ps). Markers of endoplasmic reticulum (glucose-6-phosphate phosphatase and rotenone-insensitive NADPH cytochrome c reductase) and markers of the 1,4,&trisphosphate (IP&sensitive Cazf store ([3H]IPs binding and IP3-induced Ca2+ release) were measured. No correlation was found between the two classes of markers, which suggests that the IP3 receptor does not belong to the endoplasmic reticulum in canine brain. Cerebellum Ps and P3 fractions displayed levels of c3H]IP3 binding lo- to 30-fold higher, and rates of IP3-induced Ca2+ release =-l&fold faster than the homologous cerebrum and brainstem fractions. Actively accumulated Ca2+ was only partially released by IP3, both before and after saponin disruption of the plasma membrane compartment. The proportion of the IP3-sensitive Ca2+ store relative to that of the total (IP3-sensitive and IP3-insensitive) Ca2+ store was variable; i.e., it was larger in cerebellum P2 (approximately 90%) than in cerebrum fractions (~30%). Cerebellum fractions constitute the best source from which an IP3-sensitive Ca2+ storing organelle can be purified. o 1989Academie Press, Inc.

Eukaryotic cells share the ability to accumulate Ca2’ within discrete, intracellular membrane-bound compartments, and to release it rapidly into the cytosol in response to adequate stimuli. Several neurotransmitters, hormones, agonists, and growth factors act at plasma membrane receptors to stimulate the breakdown of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and IP33 (1,2). The link i This work was supported by NIH Grant ROl GM4006801. ’ Abbreviations used: IP3, inositol 1,4,5-trisphosphate; Pz, crude mitochondrial pellet; P1, crude microsomal pellet; CBL, cerebellum; POL, parieto-occipital lobe; FL, frontal lobe; BS, brainstem; PMSF, phenylmethylsulfonyl fluoride; ER, endoplasmic reticulum; Mops, 3-(N-morpholino)propanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, r, linear regression correlation coefficient; S, supernatant. 0003-9861/89 $Z%OO Copyright All rights

0 1989 by Academic Press. Inc. of reproduction in any form reserved.

between receptor activation and Ca2+ release from intracellular store(s) has been shown to be IP3 (1). The ability of IPs to act as an intracellular messenger has been described in a large number of cell types (3), including neurons and model neurotumor cells (4, 5). Hydrolysis of phosphatidylinositol4,5-bisphosphate might serve a number of functions in nerve cells, e.g., excitability, secretion of neurotransmitters, post-tetanic potentiation, and differentiation. The physiological role of IPs-induced Ca2+ release in nerve cells remains to be fully elucidated. The identification of the intracellular membrane target of IPs has become controversial in the last 2 years. The tentative identification of the IPa-sensitive Ca2+ store with the endoplasmic reticulum as a whole (6-10) has been challenged. The relevant argument was that in neutrophils (ll), hepatocytes (12, 13), and HL-60 cells 162

IP,-SENSITIVE

Ca2+ STORE OF CANINE

(14) no correlation was found between known ER markers and markers of the IPa-sensitive Ca2+ store. On the basis of biochemical (14) and immunocytochemical (14-16) data it has been proposed that the IP,-sensitive Ca2+ store of nonmuscle cells is distinct from the ER and might reside, instead, in a population of vesicles and small vacuoles distributed throughout the cytoplasm and collectively termed calciosome3(14). As a preliminary step toward the purification and characterization of the IP3sensitive Ca2+ store of nerve cells we have fractionated different regions of the canine brain. We report here that the cerebellum is the richest source of the IP,-sensitive Ca2+ storing organelle (see Ref. (1’7)) and that bona&de ER markers are not associated with such an organelle. EXPERIMENTAL

PROCEDURES

Isolation of P2 and Ps Fractions from Canine Brain Brains were obtained from mongrel dogs of either sex weighing lo-15 kg. The dogs were anesthetized by intravenous injection of a mixture of chloralose (0.1 g/kg) and urethane (1 g/kg) and later sacrificed with a lethal dose of the anesthetic mixture. Immediately following exsanguination, the skull was opened using an osteotome and the meninges surrounding the brain were cut away. The brain was removed and immediately sealed in a plastic bag on ice from which it was transferred to a -80°C freezer and stored until needed. In order to fractionate the brain, the frozen brain was initially thawed in a plastic bag in a beaker of cold water on ice. Then, if not already separated prior to freezing, four brain regions were sectioned: the frontal lobe region (FL) corresponding to the area from the cruciate sulcus to the prorean gyrus near the olfactory sulcus and anterior, the parieto-occipital lobe region (POL) corresponding to that area extend-

3 The calciosome has been identified as the intracellular membrane-bound compartment labeled with anti-(sarcoplasmic reticulum Ca’+-ATPase) and anti(skeletal muscle calsequestrin) antibodies (14, 15). Throughout the text, we shall refer to either calciosome or IP,-sensitive Cazf store solely to indicate the finding that the IP,-sensitive Ca*+ store and endoplasmic reticulum are biochemically different and perhaps separate cytological entities (15). No immunocytochemical data are shown in this paper.

BRAIN

163

ing from the region near the entolateral sulcus to the posterior suprasylvian gyrus and posteriorly, the brainstem (BS), and the cerebellum (CBL). In preliminary experiments, we compared Pz and P3 fractions obtained from either frozen or fresh brain: no appreciable differences were detected in the activity of enzymatic markers (glucose-6-phosphate phosphatase, rotenone-insensitive NADPH cytochrome c reductase, and succinate cytochrome c reductase), IPa-induced Ca2+ release, or [aHlIP binding. The procedure used for isolating the four brain regions into their respective crude mitochondrial pellet (Pz) and crude microsomal pellet (P3) was based on that outlined by Edelman et al. (18) with a few minor modifications. The following changes were introduced: (a) the brain regions were initially minced with a scalpel on a chilled plate glass surface prior to the first homogenization step. (b) The minced brain regions were homogenized at 4°C using a wide-clearance glass/Teflon Potter-Elvehjem homogenizer in 10 vol chilled Buffer A consisting of 0.32 M sucrose, 5 mM Hepes, 0.1 mM PMSF, pH 7.4. Homogenization was accomplished with five high-speed strokes of the pestle attached to an electric drill. Following the first spin at 9OOg,the pellets (Pi) were resuspended in 5 vol Buffer A and homogenized again using 3 strokes of the homogenizer. (c) The supernatants (S,) collected from the first and second centrifugation steps were poured through six layers of cheesecloth prior to the 17,OOOgspin from which the P2 fractions were obtained. P3 fractions were obtained by centrifugation of Sz supernatants at 100,OOOg.All centrifugations were carried out at 4°C. The Pz and P3 fractions obtained from each brain region were resuspended in a small volume of Buffer A and stored in 0.5- to l.O-ml aliquots in liquid nitrogen until used. The yield of the several Pz and P3 fractions is reported in Table I. The protein concentration of each fraction was determined as described by Lowry et al. (19) using bovine serum albumin as a standard. When osmotically shocked preparations were required, each brain fraction was diluted approximately l&fold in chilled distilled water and then concentrated by centrifugation at 100,OOOgfor 1 h. The pellet obtained was resuspended in Buffer A. CBL Pz was subfractionated on a discontinuous sucrose gradient consisting of (in M) 0.8,0.9,1.1,1.3, and 1.6 sucrose steps from the top to the bottom of the gradient. All of the sucrose solutions also contained 5 mM Hepes buffer, pH 7.4, and 0.1 mM PMSF. Between 20 and 35 mg of protein was loaded onto each gradient which was then spun in a Beckman SW-28 rotor at 20,000 rpm overnight. Four fractions were collected: fraction 1 from the 0.8 M step; fraction 2 from the upper half of the 1.1 M step; fraction 3 from the lower half of the 1.1 M step; and fraction 4 from the 1.3/1.6 M interface. Fractions corresponding to various bands in the sucrose gradient were collected, diluted

164

ALDERSON TABLE

MEMBRANE

AND

I

PROTEIN RECOVERY FROM DIFFERENT REGIONS OF THE CANINE BRAIN Membrane fractions (mg of protein/g wet wt)

Brain

regions

Cerebellum Parieto-occipital Frontal lobe Brainstem

P2

lobe

27.9 36.5 40.3 25.8

f f + f

3.5 4.3 3.3 5.1

P3 (5) (4) (4) (4)

2.9 2.9 4.2 4.4

f + + +

0.2 0.3 0.2 0.7

(5) (4) (4) (4)

Note. Pz and P3 fractions were isolated as described under Experimental Procedures. Data are means f SE for the number of preparations shown in parenthesis. Data are comparable to those reported by Edelman et al. (18) for membrane fractions from rat brain.

two to crose, 30,000 pended used.

three times with chilled Buffer A without suand centrifuged in a Beckman Type 35 rotor at rpm for 1 h. The resulting pellets were resusin Buffer A and stored in liquid nitrogen until

Biochemical

Assays

[sHyp, binding. [‘H]IP3 binding was carried out at 4°C in a medium containing 50 mM Tris-HCl, pH 8.3, 1 mM EDTA, and 100 mM KCI, in a final volume of 0.5 ml. Total pH]IPB binding was measured in the presence of 40 nM [3H]IP, only, whereas nonspecific binding was measured in the presence of 40 nM [3H]IPs and 4 PM nonradioactive IP,. Specific IP3 binding was determined as the difference between total and nonspecific binding. Brain fractions were added in a final bath concentration of 0.6 to 1.0 mg/ml. In preliminary experiments, specific [3H]IP3 binding was found to be linear in the protein range 0.2-1.0 mg/ml (not shown). The fractions were incubated in the binding baths on ice for 30 min with occasional vortexing. Immediately following the introduction of the brain protein to the binding bath, a 20-pl aliquot of each bath was removed and analyzed for total radioactivity. After the 30-min incubation period, 0.43 ml of each IPI binding bath was transferred to an ultraclear Airfuge centrifuge tube which was spun at high speed in a Beckman Airfuge for 10 min at 23°C. The brain protein formed a hard pellet following centrifugation. The colorless supernatant was carefully removed from the Airfuge tube and discarded. The pellet was solubilized by adding 0.43 ml of 10% (w/v) glycerol, 5% (v/v) 2-mercaptoethanol, 2.3% (w/v) sodium dodecyl sulfate, and

VOLPE

62.5 mM Tris-HCl, pH 6.3. After a minimum of 3 h, the entire tube containing the pellet and solubilizing buffer was placed in a scintillation vial. Both the pellets and aliquots for total radioactivity were analyzed by liquid scintillation spectrometry at a counting time of 5 min. Apparent dissociation constants (Ka) for rH]IPs binding to CBL fractions were obtained by Scatchard plot analysis. [3H]IP3 binding was carried out as described above, varying the rH]IPa concentration from 5 to 160 nM. Caz’ uptake and IP&duced Cap’ release. Caa+ uptake and IP3-induced Ca2’ release were measured using a slightly modified assay developed by C. A. Dettbarn and P. Palade (unpublished results). The assay was carried out at 37°C in a medium containing 40 mM KCI, 62.5 mM K-phosphate, 8 mM K-Mops, pH 7.0, 0.04 mg/ml creatine phosphokinase, 0.2 mM phosphocreatine, 2 mM NazATP, 2 mM MgC12, and 162.5 PM antipyrylazo III, in a final volume of 1 ml. Ca2+ fluxes were monitored spectrophotometrically in a Hewlett-Packard 3451A spectrophotometer following the differential absorbance (790-710 nm) of the Ca2+ sensitive dye antipyrylazo III. Each brain fraction (0.5 mg of protein) was added to the uptake/release medium and allowed to equilibrate to 37°C for 10 min. Following this, Caz+ was administered in two or three lo-nmol aliquots. After the administered Ca*+ was accumulated by the preparation, 10 pM IP, was added to the bath. At the end of each experiment, 10 nmol CaClz was added to recalibrate the antipyrylazo III response. The free Ca*’ concentration of the uptake/release medium was calculated using a modified computer program (IONS) originally designed by Dr. Alexandre Fabiato (Medical College of Virginia, Richmond, VA). The maximal free Ca” concentration, after each lo-nmol Ca” increment, was estimated to be 1.7 PM. Incubation of brain fractions with saponin was carried out essentially as described by Michaelis et al. (20). Saponin was added to a final concentration of 0.01 to 0.3% (w/v) and the brain fractions were incubated for 3 min at room temperature and then diluted 50-fold into the uptake/release medium. Glucose-6-phosphate phosphatase. Glucose-6-phosphate phosphatase activity was measured at 37°C for 30 min in a medium containing 30 mM imidazole, 30 mM histidine, 100 mM KCl, 30 mM glucose 6-phosphate, pH 6.8, and 0.15-0.3 mg of protein, in a final volume of 0.5 ml (21). The reaction was found to be linear up to 30 min (not shown). Phosphate production was measured using a modification (22) of the method of Ottolenghi (23).

Rotenone-insensitive

NADPH

cgtochmme c reduc-

tase. Rotenone-insensitive NADPH cytochrome reductase activity was measured spectrophotometritally at 30°C for 5 min as described by Sottocasa

d (24

c

et

IP,-SENSITIVE

Ca2+ STORE

OF

CANINE

BRAIN

165

= 0.852, n = 56) which indicates that both enzymes belong to the same membrane, i.e., ER, as expected. On the contrary, a plot of glucose-6-phosphate phosphatase activity versus rH]IP, binding (Fig. 1B) Materials shows no correlation (r = 0.333, n = 56) and this suggests that the IP3 receptor does not IPa, Hepes, and Mops were obtained from Calbiobelong to the ER in the canine brain. Two them; [3H]IP3 was from New England Nuclear; CaClz additional lines of evidence support the stock solutions, saponin, glucose-&phosphate, and latter suggestion: (i) As shown in Table IV, antipyrylazo III were from Sigma; and ATP was from Pharmacia. Opti-Fluor scintillation fluid was from CBL fractions accounted for about 16% of Packard Instrument Co., and ultrapure grade sucrose the total protein recovered and more than was from Schwarz/Mann Biotech. All other chemi90% of the specific [3H]IP3 binding. On the cals were analytical or higher grade. other hand, distribution of glucose-6-phosphate phosphatase largely paralleled that RESULTS of protein recovery in cerebellum and in combined cerebrum and brainstem fracDistribution of ER Markers and [‘HYP, tions. (ii) Subfractionation of CBL PZ on a Binding discontinuous sucrose gradient yielded It is known (18, 29) that PZ fractions four fractions of increasing buoyant denare heterogeneous mixtures of different sity (l-4, from top to bottom). Figure 2 membrane fragments, e.g., mitochondria, shows that [3H]IP3 binding increased twoto threefold in fractions 2 and 3, whereas synaptosomes, synaptosomal components (plasma membrane, synaptic vesicles, ER, glucose-6-phosphate phosphatase was not etc.), and myelin. P3 fractions are referred appreciably enriched. Least-squares regression analysis also indicated lack of to as crude microsomes and contain mostly ER, Golgi membranes, and plasma mem- correlation between the two markers (r brane (18, 20, 30). Table II shows that P2 = 0.44, n = 4) in subfractions derived from fractions from either CBL, POL, FL, or BS cerebellum membranes. contained remarkably similar amounts of the mitochondrial marker enzyme succi- Ca2’ Uptake and IP,-Sensitive Ca2’ Release nate cytochrome c reductase. P3 fractions contained less than one-tenth of the mitoSince the free Ca2+ concentration of the chondrial activity of the corresponding PZ uptake medium was always ~1.7 PM, low fractions, and were two- to threefold en- affinity Ca2+ loading into mitochondria riched with respect to two ER markers, should have been negligible (32). Table III glucose-6-phosphate phosphatase (9, 26) shows that all brain fractions displayed and rotenone-insensitive NADPH cytohigh affinity, ATP-dependent Cazt uptake, chrome c reductase (27, 28). Taken to- which is due to, at least, two different Ca2+ gether, Tables I and II indicate that cere- pumps localized on two distinct membrum, brainstem, and cerebellum fractions branes: one is on the plasma membrane and the second is on the intracellular memcontain similar amounts of ER and mitochondria. brane(s) (cf. Refs. (20,29,32)). Ca2+uptake Table III shows, on the other hand, that rates of P3 fractions were two- to threefold higher than those of P2 fractions, and did both PZ and P3 fractions from cerebellum had levels of specific [3H]IP3 binding lo- to not vary significantly among cerebrum, 35-fold higher than those of corresponding brainstem, and cerebellum fractions. fractions from either POL, FL, or BS (cf. The IP3-sensitive Ca2+store is viewed as Refs. (17,31,33)). a vesicular compartment (2,6-S) endowed A plot of glucose-gphosphate phospha- with a Ca2+pump and an IP3-sensitive Ca2+ tase activity versus rotenone-insensitive efflux pathway, which is likely to be a Ca2’ channel (34, 35). Thus, actively accumuNADPH cytochrome c reductase activity (Fig. 1A) shows a positive correlation (r lated Ca2’ could be released by IP3. The

,%.&nate cytochrome c redudase. Succinate cytochrome c reductase activity was measured spectrophotometrically at 30°C for 5 min as described by Fleischer and Fleischer (25).

166

ALDERSON

AND TABLE

DISTRIBUTION

OF ENDOPLASMIC

Markers

CBL

Endoplasmie reticulum Glucose-Bphosphate phosphatase (nmol Pi/min/mg protein) Rotenone-insensitive NADPH cytochrome c reductase (nmol cytochrome c/ min/mg protein) Mitochondrial Succinate eytochrome e reductase (nmol eytochrome c/ min/mg protein)

5.6+

VOLPE II

RETICULUM AND MITOCHONDRIAL MARKERS FROM DIFFERENT REGIONS OF THE BRAIN

POL

FL

BS

CBL

0.4

4.0*

0.3

5.4 -+ 0.5

5.0 + 0.7

47.4 -+ 4.8

33.8+

4.2

38.3 + 5.4

103.7 f 10.5

33.4 f 6.6

106.5 f 12.2

Note. Ps and Ps fractions were isolated and assays were dures. Data are means k SE for four different preparations. a After osmotic shock, Ps fractions displayed unchanged chondrial marker activity.

density of IP3 binding sites would be expected to correlate with rates of IP3-induced Ca2+ release, whereas the extent of IP3-induced Ca2+ release should be related to the capacity and degree of filling of the IPrsensitive Ca2+ store. Cerebellum fractions displayed rates of IP&nduced Ca2’ release that were faster than those of cerebrum fractions (Table III), in accordance with higher levels of rH]IP3 binding. Figure 3 shows, in more detail, the effect of IP3 on membrane fractions from CBL and FL after active preloading of 60 nmol Ca2+/mg of protein. Upon addition of 10 pM IP3, cerebellum P3 rapidly released about 25% of the accumulated Ca2+ (Fig. 3A). FL P3 released only about 6% of the accumulated Ca2’ (Fig. 3B; see also Table III). In all cases, released Ca2+ was slowly reaccumulated (not shown). The cerebellum P2 fraction released a larger proportion (approx 45% ) of accumulated Ca2+ (Fig. 3C and Table III) as compared to the cerebellum P3 fraction, even though it had a slightly

IN Ps AND Ps FRACTIONS

FL

0.9

8.0 k 0.8

37.3 3~5.0

121.1 f 14.4

71.8* 6.6

30.9 + 7.4

6.2 + 1.2

5.9 * 1.3

carried

9.7 f

POL

out as described

ER activities

under

and a twofold

ISOLATED

BS

8.7 f 0.9

74.2k8.2

80.3k5.2

4.6 f 0.6 Experimental

increase

9.7 + 1.5

6.5 k 0.7 Proce-

of the mito-

lower level of specific [3H]IP3 binding and a slightly lower rate of IP,-induced Ca2+ release. IPrSensitive and IPgInsensitive Ca2’ Stores These apparent discrepancies, namely the lack of a direct relationship between IPB binding and extent of IP,-induced Ca2+ release and between Ca” uptake and IPBinduced Ca2+ release, can be partially explained assuming that there are at least three high-affinity Ca2+ stores: one sensitive to IP3 and another insensitive to IP3, both being derived from an intracellular compartment(s), and a third insensitive to IP3 but derived from the plasma membrane. The relative proportion, i.e., capacity, of the Ca2+ stores might be different in the several brain fractions investigated in the present study, and thus, the amount of Ca2+ accumulated in the IPs-sensitive store might be variable. In order to address this

IP,-SENSITIVE

Ca2+ STORE OF CANINE

167

BRAIN

TABLE III DISTRIBUTION

OF

Ca2+ UPTAKE

RATE, IPs-INDUCED P3 FRACTIONS ISOLATED FROM

AND

Ca2+ RELEASE DIFFERENT

(3H)IP3

AND

REGIONS

BINDING OF THE BRAIN

p2 Markers

CBL

ACTIVITIES

IN

P2

Pa

POL

FL

BS

CBL

POL

BS

FL

Ca’+ uptake rate (nmol W+/min/

mg protein) [8H]IP8 binding” (pm01 IPJmg protein) Rate of IP8-induced Ca2+ release (nmol Ca’+/ min/mg protein) Extent of IPsinduced Ca” releaseb (nmol Ca’+/ mg protein) Extent of IPsinduced Ca2+ release (% of total

7.1

f

4.18+

133.6

0.6

5.8

f0.4

1.30

0.24+

0.07

6.2

kO.6

0.15+0.08

2.1

+0.3

0.17+0.06

12.3

f

5.40+

0.9

1.56

17.6

k2.0

0.28+

0.13

15.1

f 1.4

0.51 +0.23

6.4

f0.9

0.15kO.06

k32.3

O-4.8

o-9.9

0

12.9

*

1.3

O-3.6

O-5.0

0

20.0

f

1.9

O-6.0

O-8.9

0

k25.1

o-7.4

O-6.0

0

154.2

24.5

IL 2.5

o-5.9

o-5.4

0

42.5

+

O-9.8

O-4.0

0

Ca2+ accumulated)

4.5

Note. P2 and P3 fractions were isolated and assays were carried out as described under Experimental Procedures. Data are means & SE for five cerebellum and four brainstem and cerebrum preparations. Since the free Ca2+ concentration of the uptake/release medium was 4.7 PM, Ca2+ loading into mitochondria was negligible (32) as also indicated by the lack of effect of mitochondrial inhibitors, e.g., 0.1 mMNaN, and 0.7 pg/ml oligomytin (not shown). rH]IP3 binding data were obtained using 40 nM IP3 and do not represent B,,,, values. One single class of high affinity pH]IP3 binding sites was detected, and the apparent KD was found to be 24 nM for CBL fractions as previously reported (31,33). In some cases, IP,-induced Ca2’ release from certain fractions could not be detected because Ca2+ uptake rates were probably fast enough to offset releases, and, thus, a range of values is given. a One P2 preparation was osmotically shocked (see Experimental Procedures for details); specific IP3 binding values were not modified. Preincubation with saponin (0.05-0.3%), however, increased rH]IP3 binding of CBL P2 by 10-12s suggesting that a small proportion of IP3 binding sites are either inaccessible and/or contained within intact synaptosomes. This result is in good agreement with a previous report that PH]IPs binding of crude cerebellum membranes of the rat increased modestly after Triton X-100 solubilization (60). * The extent of IP3-induced Ca” release from CBL fractions is the highest so far reported in the literature; values of l-3 nmol Ca’+/mg of protein have been reported for rat brain fractions in the absence of Ca-precipitating anions (54,61).

question, we measured IPg-induced Ca” release from brain fractions after preincubation with saponin. Saponin, a plant glycoside, interacts with cholesterol and makes plasma membranes permeable to macromolecular tracers without major effects on cholesterol-free membranes, as intracellular Ca2+ stores are thought to be (36). Figure 4A shows that saponin, in the

concentration range O.Ol-0.3%, decreased Ca2+ uptake of both CBL and FL P3 fractions with an I&,, of approximately 0.15% (cf. Ref. (20)). On the other hand, the extent of IP,-induced Ca2+ release increased, as shown in Fig. 4B: from 9 to 14 nmol Ca2+/mg of protein in CBL Ps, and from 2.5 to 6 nmol Ca2+/mg of protein in FL P3. The amount of Ca2+ released by IP3 expressed

168

ALDERSON

0

4.0

6.0

Glucose-6-phosphate

10.0 8.0

E -5 $

10.0

8.0

12.0

phosphotose

B

0 0 0

6.0

Glucose-6-phosphate

phosphatase

FIG. 1. IPa binding activity does not belong to the endoplasmic reticulum membrane. Glucose-6-phosphate phosphatase, rotenone-insensitive NADPH cytochrome c reductase, and specific [3H]IPa binding activities were measured as described under Experimental Procedures, and are expressed as nmol Pi/ min/mg protein, nmol cytochrome c reduced/min/mg protein, and pmol IPdmg protein, respectively. Twenty-eight pairs of data from four different preparations are plotted. (A) Experimental points were fitted by linear regression analysis; correlation coefficient was 0.352 (n = 56). (B) The correlation coefficient of the experimental points was 0.333 (n = 56).

as a percentage of the accumulated Ca2’ was also increased, as shown in Fig. 4C. Above 0.1% saponin, the extent of IPs-induced Ca2+ release decreased (Fig. 4B) and this might indicate that some of the vesicles derived from intracellular compartments became disrupted. Cerebellum P2 was similarly affected by saponin: Ca2’ uptake was inhibited (I&,, about 0.12%; Fig. 4A) and the percentage of Ca2+ released by IP3 rose from 48 to 100% (Fig. 4C). This finding is further illustrated in Fig. 5B: after preincubation with 0.12% saponin, CBL P2 accumulated

AND VOLPE

less Ca2+ than the control (cf. Fig. 5A) but released it almost completely upon addition of IP3. If 0.1% saponin completely inhibits Ca2’ uptake by inside-out plasma membrane vesicles (20), approximately 30% of total Ca2+ uptake was due to plasma membrane vesicles (cf. Fig. 4A). This assumption is corroborated by the experiment of Fig. 5C in which CBL P2 was allowed to accumulate 40 nmol Ca2+/mg of protein. The addition of saponin to the medium (15 hg/ml, final concentration) released about 27% of the accumulated Ca2+, yet did not prevent the action of IPQ which released 44% of the accumulated Ca2’. A plausible interpretation of the data of Figs. 4 and 5 is as follows: In the absence of saponin, Cap+ is accumulated by distinct Ca2+ stores and Ca2+ released by IPa can be also actively rear-cumulated by IPa-insensitive Ca2+ stores. After preincubation with saponin, Ca2’ is no longer accumulated by the saponin-sensitive, IPa-insensitive Ca2’ store; thus, more Ca2+ is accumulated in the IP,-sensitive Ca2+ store and can be released by IP3. Moreover, the existence of multiple high affinity Ca2+ stores renders Ca2’ uptake an unreliable marker of the IPs-sensitive Ca2’ store in crude membrane preparations. Both the percentage of releasable Ca2’ and the extent of IP,-induced Ca2+ release (Table III and Figs. 4B and 4C) are larger in the CBL fractions than those in the cerebrum fractions, and this seems to correlate with the proportion of the IPs-sensitive Ca2+ store relative to that of the IPs-insensitive Ca2’ stores. As shown in Fig. 4D, the saponin-insensitive Ca2+ store of CBL Pa was almost entirely sensitive to IP3 (91% ), whereas 72% of the saponin-insensitive Ca2+ store of FL P3 was also insensitive to IPB. Figure 4D indicates that Ca2” accumulation into the IPs-sensitive Ca2+ store of CBL P2 was twice that of CBL P3 and this might explain why the extent of IPa-induced Ca2’ release from CBL P2 was larger than that of CBL P3 (Table III). DISCUSSION

Preliminary Observations on the Nature of the IP&‘ensitive Ca*’ Store In neurons, the resting free Ca2+ concentration is maintained around ~O-?M by sev-

IPa-SENSITIVE

Ca”

STORE TABLE

RECOVERY

AND

DISTRIBUTION OF [3H]IP3 IN Pz AND Pa FRACTIONS

BINDING ISOLATED

OF

fractions

Cerebellum Pz Combined cerebrum brainstem Pzs Cerebellum P3 Combined cerebrum brainstem Pas

mg protein

AND GLUCOSE-6-PHOSPHATE FROM DIFFERENT REGIONS

[3H]IP3

200

pm01

Pi

% of total

252.8

14.4

435.1

80.4

1.5

18.1

1363.1 23.8

77.8 1.4

41.5 55.0

7.7 10.2

5.7 0.2

69.4 2.8

111.5

6.4

9.4

1.7

0.8

9.7

% of total

and

experiment cerebrum

are shown. Values and brainstem.”

for FL, POL,

and BS fractions

were

summated

and

Ca2+ pumps (40) and Na+-Ca2+ exchangers (41). The nonmitochondrial, high affinity Ca2+ store seems to be ideally suited for short-term regulation of cytoplasmic free Ca2+ in the physiological range of 0.1-5 PM

AL(---

400 300

Glucose-g-phosphate phosphatase

binding

pm01 IPe

‘G -G 2 :

ACTIVITIES

% of total

era1 mechanisms which buffer cytoplasmic Ca2+ via Ca2’ binding proteins such as calmodulin (37) and parvalbumin (38), sequester Ca2+ into mitochondrial and nonmitochondrial membrane stores (29, 32, 39), or extrude Ca2+ via plasma membrane

::

PHOSPHATASE OF THE BRAIN

and

Note. Results of a typical are referred to as “combined

x .$

169

BRAIN

IV

Recovery Membrane

CANINE

t3rd

” % x

100 0 1

FIG. 2. Fractionation

2

3

4

of CBL Pz on a discontinuous sucrose gradient. Distribution of glucose-6-phosphate phosphatase, succinate cytochrome c reductase, and specific rH]IP3 binding activities. Four fractions of increasing buoyant density were obtained by sucrose gradient centrifugation (l-4 from top to bottom; see Experimental Procedures for details). Activities were measured as described under Experimental Procedures, and are expressed as percentage of the specific activity of unfractionated (control) CBL Pz loaded onto the gradient. Control activities were 6.3 nmol PJmin/mg protein, 103 nmol cytochrome c reduced/min/mg protein, and 3.8 pmol IP,/mg protein, for glucose-g-phosphate phosphatase (hatched bars), succinate cytocrome c reductase (solid bars), and [3H]IP8 binding (blank bars), respectively.

FIG. 3. IPa-induced Ca2+ release from cerebellum and frontal lobe fractions. Ca2+ loading and Ca2+ release were measured as described under Experimental Procedures, using antipyrylazo III as the Ca*+ indicator. The assay was started by adding 500 pg of CBL Pa (A), FL P3 (B), or CBL Pz (C). Three consecutive lo-nmol CaClz pulses were administered and the third (3rd) CaCl, pulse is shown in A (arrow). After completion of Ca2+ uptake, 10 PM IPa was added (arrowhead). Data points were stored on Hewlett-Packard microflexible disks and each file was made up of 300 data points aquired over either 150 s (e.g., C) or 300 s (e.g., B). Tracings were electr r4nically scaled so that releases are directly comparable. A downward deflection of the absorbance tracing is indicative of Ca” uptake and an upward deflection corresponds to Ca2+ release.

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AND VOLPE 25 r

0.1 Saponin

0.2

0.1

(%)

Saponin

5

0.2 (%)

15

;: s B s 9 E : P

10 5 0 CBL P3

Saponin

FL P3

(%)

FIG. 4. Effect of saponin on Ca” uptake and IPs-induced Ca’+ release. Ca2+ loading and Ca2+ release were measured as described under Experimental Procedures and the legend to Fig. 3. All fractions (CBL Ps, filled circles; FL Ps, triangles; and CBL Pz, empty circles) were preincubated for 3 min in the absence (control) and presence of saponin at concentrations specified on the abscissa. Experiments were carried out on two different preparations and data averaged. (A) Effect of saponin on the extent of Ca2+ uptake. Initial rates of Ca2+ uptake were also affected by saponin: ICW was approximately 0.1 and 0.05% for P3 and Pa fractions, respectively. POL and BS fractions were similarly affected by saponin (not shown). (B and C) Effect of saponin on the extent of IPs-induced Ca2+ release expressed as nmol Ca’+/mg protein (B) and percentage of accumulated Ca2+ (C). (D) Histograms representing the extent of Ca2+ accumulation by the saponin-sensitive (blank bars), IPg-sensitive (solid bars), and IP8-insensitive (cross-hatched bars) Caa’ stores. The capacity of the saponin-sensitive Ca2+ store is derived from the data of A, and corresponds to the amount of Ca2+ uptake inhibited after preincubation with 0.1% saponin, a concentration which should inhibit Ca2+ uptake by insideout plasma membrane vesicles (20). The capacity of the IPa-sensitive Ca2+ store is derived from the data of B, and corresponds to the amount of Ca*+ released by IP3 after preincubation with 0.1% saponin. The capacity of the saponin-, IPs-insensitive Ca” store is estimated as the difference between the saponin-insensitive and IPs-sensitive Ca*+ stores.

(29). Following cell activation, ATP-dependent Ca2+ sequestration, along with Ca2+ binding to parvalbumin (where present; Ref. (38)) and Ca2+ extrusion through the plasma membrane, would very rapidly restore the resting free Ca2+ concentration. The high-affinity Ca2+ store, or part of it (see below), is also responsive to IPa and is able to release Ca2+ into the cytoplasm when the pathway(s) leading to IPa generation is activated.

There is evidence supporting the involvement of smooth-surfaced vacuoles, tubules, and cisternae, usually defined together as the “smooth ER,” in the control of Ca2+ homeostasis. By electron probe Xray microanalysis, smooth ER elements have been tentatively identified as Ca2+sequestering organelles in rat brain presynaptic terminals (42), in squid giant axon (43), and in mouse cerebellum preand postsynaptic areas (44). By the same

IP,-SENSITIVE

Ca2+ STORE OF CANINE

FIG. 5. Saponin- and IPs-induced Ca*+ release from CBL Pz. Ca2+ loading and Ca2+ release were measured as described under Experimental Procedures and the legend to Fig. 3. (A) CBL P2 (500 pg) actively accumulated two consecutive lo-nmol CaCI, pulses, the second (2nd) of which is shown (arrow), and was then challenged with 10 pM IP3 (arrowhead). (B) CBL Pz (500 fig) was preincubated with 0.12% saponin for 3 min, and was then allowed to accumulate one lo-nmol CaClz pulse (arrow). Both the extent and rate of Ca2+ uptake were reduced. Ten micromolar IP3 (arrowhead) was able to release almost all of the aceumulated Ca2+. (C) CBL P2 (500 fig) actively accumulated two consecutive lo-nmol CaCl, pulses (not shown). Saponin (15 rg/ml, final concentration; sap) induced partial Ca2+ release (27% of accumulated Ca’+). When a new steady state was attained, addition of 10 pM IPB (arrowhead) released 44% of accumulated Ca’+.

methodology, mouse cerebellum neurons showed discrete compartments that accumulate Ca2+in an activity-dependent fashion and are less widely distributed than the entire ER (45). Moreover, specialized structures, such as subsurface cisterns (46) and sacs of the dendritic spine apparatus (47), have been observed in mouse cerebellum neurons and their role in Ca2+homeostasis has been implied on the basis of striking morphological similarities with the junctional sarcoplasmic reticulum of striated muscle. The present results (Tables II-IV and Figs. 1 and 2) clearly indicate that the IPBsensitive Ca2+ store of canine brain is biochemically distinct from the ER and imply, together with previous findings (4547), that smooth-surfaced elements of ER, despite their overall morphological similarity, might be heterogeneous in terms of

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function and protein composition. Thus, some elements of the “smooth ER” do not contain bonajide ER markers and contain the IPB receptor. At present, our data are compatible with either the IPs-sensitive Cazf store being a subcompartment of the ER or the IPa-sensitive Ca2+ store and ER being separate cytological entities (14,15). They are not compatible with the notion that smooth ER as a whole is the IPa-sensitive Ca2+ store. Brain membrane vesicles containing the IP3 receptor must be specialized to accomplish rapid uptake and release of Ca2+. Further purification and study of their protein composition and properties are likely to unfold additional, marked differences with respect to ER. In nonnerve cells, it has been proposed that the IPs-sensitive Ca2+ store (“calciosome”) is different from the ER, resembles the sarcoplasmic reticulum of striated muscle, and is at a minimum endowed with a Ca2+ pump, an IPs-sensitive Ca2+ channel, and an intraluminal Ca2+ binding protein, analogous to striated muscle calsequestrin, which serves as a Ca2+storage site (14, 15). A cardiac/slow-twitch muscle-like isoform of the Ca2+ pump has been very recently identified in both rat and rabbit brain by cDNA cloning (48,49) and has been detected immunologically in “microsomal” fractions (49). A calsequestrin-like protein has been partially purified from bovine brain (50). Preliminary immunofluorescence experiments have indicated that a calsequestrin-like protein is present in rat neurons (16) and the study of its distribution in canine brain fractions is in progress.

Regional Distribution of the IP,-Sensitive Ca2’ Store: Neuron-Type SpeciTcity? The present results extend previous observations on brain homogenates (17, 31), and show that cerebellum fractions are enriched in IP3 binding sites and contain vesicles which are able to accumulate Ca2+ and to release it upon addition of IP3. If the K. for IP3 binding is similar in cerebellum and cerebrum fractions, and if the IP3 receptor density per membrane area unit is the same in all brain regions, the overall

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development of the IP,-sensitive Ca2+ store of cerebellum nerve cells should be on average lo-30 times that of cerebrum and brainstem nerve cells. Given the neuronal heterogeneity of the brain regions investigated, this conclusion must be taken cautiously (see below). Cerebellum fractions, nonetheless, lend themselves as the best source from which an lP,-sensitive Ca2+ storing organelle can be isolated and characterized. Several different receptors, e.g., muscarinic, ai-adrenergic, serotonin, etc. (for a review see Refs. (3-5)), are coupled to phosphatidylinositol4,5-bisphosphate hydrolysis and IPa generation in the nervous tissue. Since most of the studies have been carried out on brain slices (4, 5) and only recently on primary cultures of neurons (51,52), the specific pattern of IP3 response for each type of neuron is far from known. The IP3 pathway seems to be both pre- (53, 54) and postsynaptic (4, 5) and the Ca2+ store sensitive to IP, should be localized accordingly, i.e., in dendrites, soma, and nerve terminals. It is not known, however, whether the distribution of the organelle is constant throughout the soma and its processes or whether discrete intraneuronal areas exist. Autoradiographic studies of rat brain sections (17) have indicated striking regional variations in the density of IP3 binding sites, i.e., higher in the cerebellum than in the cerebrum, as well as marked differences within layers of the cerebellum cortex, i.e., high in the molecular layer and negligible in the granule cell layer. All of the above observations, together with the present results, suggest that the IP,-sensitive Ca2+ store might have a specific architecture within each type of neuron. Indeed studies on homogeneous populations of neurons become imperative to assess with certainty the characteristics, distribution, and relative development of the IPa-sensitive Ca” store of each neuron. IP,-Sensitive an& IP,-Insensitive Ca2+ Stores Studies with permeabilized NlE-115 neuroblastoma cells (55) have shown that

AND

VOLPE

IPB releases only part, i.e., 30-50%, of the Ca2+ accumulated by the intracellular, high-affinity Ca2+ stores. The nature of the IP,-insensitive Ca2+ store in nerve cells is unknown and might be either a unique membrane compartment or, in part, a GTP-modulated reservoir for the IP,-sensitive Ca2+ store (55). If it is a store distinct from the IPs-sensitive one, part of it might be acted upon by either GTP (9) or caffeine (56-53) since both agents have been shown, under specific conditions, to release Ca2’ independently of IP3. In this context, it is noteworthy that in mammalian sensory neurons the caffeine- and IPs-sensitive Ca2+ stores have been reported to be preferentially localized in the soma and in the processes, respectively, and to be separate cytological entities (57, 59). At least two Ca2+ pump isoforms are expressed in rat and rabbit brain (48, 49) and might be either localized in different Ca2+ pools within the same neuron or segregated in specific neuron types. Figure 4 shows that brain fractions eontain a saponin-insensitive Ca2+ store which is also insensitive to IP3. The rate of Ca*+ uptake of cerebrum, brainstem, and cerebellum fractions is of similar order of magnitude irrespective of the brain region being analyzed (Table III), and this suggests that there are vesicles (especially in cerebrum and brainstem fractions) which contain Ca2+ pump units but do not contain IPB receptor sites. Thus, the finding that the relative proportion of the IPs-sensitive and IP,-insensitive Ca2+ stores is different between cerebellum and other fractions of the brain (Fig. 4D and Table III), might be taken as indirect evidence that different neurons are variably endowed with several types of Ca2+ store.4 It would be of interest 4 The extrapolation from IP,-insensitive vesicles to intracellular IPa-insensitive stores is justified since IPa-insensitive Ca2+ stores are detected in intact and permeabilized cells. During fractionation, vesicles derived from IPhsensitive stores but lacking the IPa receptor might be generated. Thus, if a similar fraction of false-negative IPa-insensitive vesicles is present in both cerebellum and cerebrum fractions, the observed differences between cerebellum and cerebrum would persist. However, if the density of the IPa re-

IP,-SENSITIVE

Ca’+ STORE OF CANINE

to investigate the effect of caffeine on the several brain fractions used in the present study, and to ascertain whether part of the IP,-insensitive Ca2+ store is caffeine-sensitive, and whether the caffeine-sensitive Ca2+ store overlaps, if any, with the IPSsensitive Ca2+ store. ACKNOWLEDGMENTS We thank Dr. Philip Palade for comments on the manuscript, and Drs. Giuseppe and Franca B. Sant’Ambrogio for providing canine brains. Thanks are also due to Monica “LG” Tzinas for excellent and lively assistance.

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21. LOWREY, K., GLENDE, E.A., JR.,ANDRECKNAGEL, R. D. (1981) B&hem. Pharmacol30,135-140.

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