CELL BIOCHEMISTRY AND FUNCTION

VOL.

8: 191-198 (1990)

Effects of Inositol 1,425-Trisphosphateon Calcium Release from the Endoplasmic Reticulum and Golgi Apparatus in Mouse Mammary Epithelial Cells: A Comparison During Pregnancy and Lactation AKIO YOSHIMOTO, KEIKO NAKANISHI, TADASHI ANZAI AND TSENICHI KOMINE Department of Biochemistry, School of Medicine, Kyorin University, Mitaka, Tokyo 181, Japan

It has been established that inositol 1,4,5-trisphosphate(IP3)is responsible for the mobilization of calcium(Ca’ ) from intracellular locations in a wide variety of tissues, and that this response triggers the stimulation of several hormones and neurotransmitters. However, these phenomena have yet to be examined in the mammary epithelium. Ca2+uptake from the medium into the endoplasmic reticulum(ER)and Golgi apparatus in uitro in both pregnant and lactating mouse mammary epithelial cells was studied and a strong CaZ+release from these organelles into the medium with the use of IP3 was shown. The Ca2+uptake and its release due to IP3 was also usually greater during +

pregnancy than lactation. K E Y WORDS

Inositol 1,4,5-trisphosphate; CaZ+;mammary epithelial cell; endoplasmic reticulum; Golgi apparatus.

INTRODUCTION It has been established that insulin, prolactin and MATERfALS AND METHODS hydrocortisone are needed for the development of the mammary gland and for lactation.”’ However, Animals und Tissues the mechanism of action of these hormones in mammary epithelial cells still remains unknown. Lactating DDY mice, 5 days from the delivery of Pizzaro and Sapag-Hagar3 observed a change in the first pregnancy, and the same strain, at 14 days the concentration of calmodulin during pregnancy of the first gestation were decapitated. Their and lactation in rats, and found that its maximum mammary glands were isolated, minced, and level was reached at parturition compared with the treated for 60 min at 37°C with 5 volumes of 0.2 M amount present during lactation. They thus con- Tris-HC1 buffer (pH 7.4) containing 0.1 per cent cluded that calmodulin (including the intracellular collagenase. After 5 min centrifugation at 800 g, the Ca2 ) plays a regulatory role during lactogenesi~.~ epithelial cells were collected. It is known that inositoll,4J-triphosphate (IP3), formed by phospholipase C hydrolysis of phosphaSubcellular Fractionation of Golgi Apparatus and tidylinositoI,4,5-bisphosphate,is responsible for the E R mobilization of Ca2 from the intracellular areas in a wide variety of tissues, moreover, this response As referred to in the methods of Jarasch et al.’g6 triggers the stimulation of several hormone^.^ and Anderson et al.’ the epithelial cells were However, these phenomenas have yet to be exam- homogenized in 5 volumes of 20 mM Tris-malate ined in the mammary epithelium. Thus, we studied buffer (pH 6.5,containing 0.3 M sucrose and 5 mM C a 2 + uptake from the medium into the endop- MgCI,), with a glass-Teflon Potter Elvehjem delasmic reticulum (ER) and the Golgi apparatus and vice, and then centrifuged at 10 000 g for 20 min to C a 2 +release from these organelles into the medium remove cell debris, nuclei and mitochondria. The with the use of IP3 in uitro in both pregnant and supernatant was mixed with 2.5 M sucrose (same lactating mouse mammary epithelial cells. buffer) to a final concentration of 2-0 M sucrose and +

+

0263-6484 90/040191-08 $05.00 ( 1990 by John Wiley & Sons, Ltd

192

A. YOSHIMOTO E T A L

C,CI was added to a concentration of 10 mM. The solution was layered on the bottom of a centrifuge tube (SM 27 rotor, Beckman ultracentrifuge) and overlayed with 8 ml each of 1.3 M, 1.15 M and 0.3 M sucrose (same buffer) and centrifuged at 130 000 g for 4 h. The band that formed between the 0.3/ 1.15 M sucrose level, the band that formed between the 1.15/1-3M sucrose level, and the band that formed between the 1.3/2.0 M sucrose level were collected individually and diluted with 0.3 M sucrose, then recentrifuged at 105 000 g for 90 min. The pellet obtained from the 0.3/1.15 M band was used for the Golgi apparatus enrichment fraction, the pellet obtained from the 1.15/1.3 M band was used for smooth ER (SER), and the pellet formed by the 1 - 3 / 2 . 0 ~band was used for rough ER (RER). Ca2+ Uptake into the Organelles

The Golgi apparatus and ER, each of 40pg protein, were suspended in 3 ml of a HEPES buffer 2 5 m ~ pH , 7.4 (Ca2+ concentration was 1.1 x lo-' M, which was made up from CaCl, and EGTA solution by the method of O g a ~ a * *con~) taining 6 mM MgCl,, 110 mM KCI, 10 mM sodium oxalate, 5 mM sodium succinate, 5 mM sodium pyruvate, and 75 pMof Quin 2.

The C a 2 +uptake from the buffer into the organelles was estimated by the decrease in fluorescence intensity at 493nm (excitation at 340nm) by a fluorimeter (Hitachi-204) at 37"C, following the method of Tsien et aL" After no further decrease was observed, 10 mM creatine phosphate, 5 mM ATP, and 10 units of creatine phosphokinase were added to the medium, and the further decrease of the fluorescence intensity followed. Ca2+ Release from the Organelles

When Ca2+ uptake into the organelles had ceased, IP3 was added to the suspension, and the fluorescence intensity was observed. The released C a 2 + due to the addition of IP3 was again completely taken up into the organelles within a few minutes. The amount of added IP3 was increased and the level of released Ca2' then rose and reached a maximum. After the releasing effect of the IP3 had concluded, the C a 2 + ionophore A23187 was added to the suspension until a further Ca2+ release was effected (refer to Figure 2). Chemical Analyses

Protein was determined by the procedure of Lowry ef al." with bovine serum albumin as stan-

Table 1. The distribution and specific activity of enzymes, and the contents of protein, DNA, and RNA in various fractions obtained from lactating mouse mammary epithelial cells.

Isocitrate dehydrogenase* Acid phosphataset

Glucose-6phosphataset Thiamine pyro phosphataset ATPaset Protein mg g.-' wet cell DNA pg mg- protein RNA pg mg-' protein

'

Homogenate

Post-Mitochon drial fraction

RER

67.2 f 35.7 (21 10 f 1107) 17.8 f 8.4 (559 f 264) 4.5 f 3.8 (141 f 119) 11.6 f 9.6 (364 f 301) 13.5 f 12.2 (427 f 383) 31-4 f 5.1

38.7 f 30.3 (136 f 105) 15.9 f 4.4 (55.7 f 15.4) 4.2 f 3.7 (14.7 f 13.0) 10-4 f 9.3 (36.4 f 32.6) 9.7 f 6.5 (34.0 f 22.8) 3.5 f 2.7

6-5 f 5.2 (0.9 f 0.7) 2.6 f 0.9 (0.37 f 0.13) 10.8 f 8-3 (1-5 f 1-2) 4.7 f 4.3 (0.66 f 0.60) 9.6 f 5.1 1.3 f 0.7 0.14 f 0.1

52.4 f 19.1 (1645 f 600) 153 k 35 (4804 f 1099

12-0 f 9.7 (42 f 34) 65 f 52 (217 f 182)

7.8 f 5.6 (1.1 f 0.8) 128 f 80 (18 f 11)

Golgi Apparatus

SER

N.D.

N.D

N.D.

N.D.

11.4 f 7.5 (0.3 f 0.2) 27.3 f 24-9 (0.82 f 0.75) 14-4 f 9.2 (0.43 f 0.28) 0-03 2 0.02

3.9 f 1.9 (0.4 f 0-2) 35.6 f 16.3 (3.6 f 1-6) 34.6 f 11.1 (3.5 f 1.1) 0.10 f 0-03

N.D.

N.D.

+

N.D.

46 19 (1.4 f 0.5)

*Specific activity: the amount which causes an increase in optical density (at 340 nm) of 0.01 min.-* mg-' protein x 100. Wpecific activity: Released inorganic phosphate (pg) from the substrate at 37°C min-' mg-' protein x 10. ( ) = Total activity or the content in 1 (wet weight) of the epithelial cells. N.D. = Not detected. The values were the average of five samples and their standard deviations.

193

EFFECT OF IP3 ON CA2' RELEASE FROM THE MAMMARY CELL

dard. DNA was measured using the diphenylamine reagent" and RNA was analysed using the orcinol reagent," using deoxyribose and ribose as standards respectively. Enzyme Assays

Isocitrate dehydrogenase was measured by the method of Ochoa.' Glucose 6-phosphatase,14 acid phosphatase,' ATPaseI5 and .thiamine pyrophosphatase16 were estimated by the measurement of inorganic phosphate which was released from each substrate. RESULTS Purity and Yield of Organelles

Trypan blue stainingI7 indicated that 80 per cent or more of the epithelial cells remained intact while being treated with collagenase. One gram of the epithelial cells(wet weight) contained 5 x lo7 cells approximately. The protein content was approximately 30mg and 1 0 m g g - ' of the wet cells in lactating and pregnant cells respectively. Table 1 shows the distribution and specific activity of enzymes, and the contents of protein, DNA, and RNA in the various fractions. Morphology of the Golgi apparatus and ER fractions were examined by electron microscopy as shown in Figure 1. From Table 1 and Figure 1, it can be concluded that the fractions of RER, SER, and Golgi apparatus attained by these methods are satisfactorily preserved. The wet weight of the mammary epithelial cells, and the contents of Golgi apparatus, SER, and RER per mouse differed during lactation and in pregnancy. The weight of lactating mammary cells per mouse was about 3.5 g, which was about eight times the weight during pregnancy. As compared by the protein content, the content of lactating organelles (Golgi apparatus and ER) per mouse was approximately 30-40 times more than that in pregnant mice (Table 2). The content of RER and Golgi apparatus increased remarkably in lactating cells. In both cases (whether lactating or pregnant),

Figure 1 . Electron micrographs of SER, RER, and Golgi apparatus fractions obtained from lactating mouse mammary epithelial cells. A: SER rich fraction, x 32 OOO. B RER rich fraction, x 2 8 800. C: Golgi apparatus rich fraction, x 2 2 4000. Fixed with glutaraldehyde and OsO,, and stained with acetate and lead citrate.

A

194

A. YOSHIMOTO E T A L

Table 2. Contents of Epithelial Cells and Organelles (per Mouse). Epithelial Cells (g)* Cmglt ~~

~~~

~~

~

~

Golgi apparatus (mg)t

~~~

~

Lactating

3.66 & 0.87 1114.9 f 18.71t

2.90 2 0.89

(100%)

Pregnant

0.44 f 0.08 111.0 I.S]t (loo”/,)

(2.5 73 0.087 0.018

*

*

(0.7 2,)

SER (mg)t

~~~

1.70

* 0.62

RER

(mdt 6. I3

2.34

0.067 f 0.034

(5.3 XJ 0.154 0.066

(0.6 73

(l.422

(1.8%)

*Wet weight/mouse. tProtein mg/mouse. The values were the average of five samples and their standard deviations.

the RER content was greater than that of the Golgi apparatus, and SER content was the lowest among these three kinds of organelle. Ca2+ Uptake into the Intact Ce1l.s from the Medium At first we tried to use intact epithelial cells for Ca” uptake from the medium. A t a temperature of 37”C, and 1/4 of the C a 2 + contained in 4 ml of the medium, in which the Ca” concentration was 1.1 x M, was absorbed within 10min into 40 mg of the intact lactating mammary epithelial cells. This Ca” uptake was of the same magnitude in both intact cells and in cells that had been permeabilized by incubation with digitonin. Further, the Ca2+ uptake did not change when 2 pg ml- of oligomycin was added to the medium to inhibit mitochondria or when 100 ,UM of orthovanadate was added to inhibit ATPase.I8 Even when ATP, creatine phosphate, and creatine phosphokinase were added to the medium, the C a 2 + uptake was not enhanced.

Cu2+ Uptake into the Orgunellc~sfrom the Medium and Cu‘ Reltase front the Organelles Induced by IP3 and the Ca’ + Ionophore A23187 +

Figure2 is a model chart of the fluorescence intensity pattern with Quin 2 in the medium during Ca” uptake into the organelle and Ca” release from the organelle. As seen in Table 3, Ca” release from the pregnant SER and RER induced by the Ca” ionophore A23187 was 166 per cent and 289 per cent respectively, which means that the SER and RER contained an excess of 66 per cent and 189 per cent of the Ca’+ content in the suspension medium, so it might be considered that these extra quantities of Ca” were stored in the ER in the cell. The stored Ca” in the lactating ER was about

20-fold over that in the pregnant ER. The stored Ca” in the lactating Golgi apparatus was about 2.5-fold more than that in pregnancy. Spontaneous C a 2 + uptake of the pregnant ER was more active than that of the lactating ER, so that the percentage uptake was higher. Spontaneous Ca2 uptake into the lactating RER was less than that into SER and Golgi apparatus (Table 3, Ca2+ uptake A). However, with the addition of ATP, creatine phosphate, and creatine phosphokinase, about 95 per cent of the Ca” in the medium was absorbed into all organelles within 7 to 8 min (Table 3, Ca2+ uptake B). The C a 2 + release induced by TP3 was more responsive in the lactating organelles, so that the concentration of I P3 required to achieve maximum release was less than in the pregnant organelles (lactating < 3 ,UM, pregnant > 3 p ~ ) . The C a 2 +content in the lactating ER was about 20-fold greater, and the content in the lactating Golgi apparatus was also 2.5-fold more than that of the pregnant cell as has been previously mentioned. Thus, the Ca2+ release percentage due to IP3 from stored Ca2+ in the lactating organelles was very small as compared with that from the pregnant organelles. A larger volume of A23 187 was needed to release the Caz+ from the pregnant organelles than that required to achieve this release from the lactating organelles (pregnant 20 nM, lactating 25-45 nM; Table 3, Ca’ release, A23 187). +

+

DISCUSSION Reports have revealed that many secretory cells, such as in the l i ~ e r , ’ ~ . ~ the ’ i n pituitary cell,” in the uterine sarcoplasmic reticulum2’ and the adipocyte~ ’have ~ shown an uptake of Ca” from the medium. This Ca2’ uptake from the medium also occurs in lactating mammary epithelial cells.

195

EFFECT OF IP3 O N C A 2 + RELEASE FROM THE MAMMARY CELL

Fluorescence Intensity at 493 n m (Exciting a t 340-nm)

//I-4

>

.-

?a

n

Figure 2. A model chart of the fluorescence intensity pattern with Quin 2 in the medium during Ca2+ uptake into the organelle and Ca2+ release from the organelle. [Legends]: (1) Fluorescence intensity (FI) in the presence of 3 mM EGTA with Quin 2 was adjusted to 0 on the meter. (2) FI in the medium plus Quin 2 was then estimated against 0. (3) 400 p g (protein) of the organelle (SER, RER, or Golgi apparatus) was added to the medium. (4)FI of three was set at 100 per cent. (5) At the end of the spontaneous FI decrease, ATP, creatine phosphate (CP) and creatine phosphokinase (CK) were added to the medium. ( 6 ) At the end or the FI decrease following ATP, CP, and CK addition, IP3 was added to the suspension until the Ca2+ releasing effect reached a maximum. (7) Ca2+ ionophore A23187 was then added to the suspension until complete Ca2+ release was cffected. A: Spontaneous Ca2+ uptake into the organelle. B: C;;'+ uptake with the addition of ATP, CP, and CK. (min): duration of the uptake. a and b % : Percentage uptake of C a 2 + into the organelle compared with c%: Percentage release of Ca2+ from the organelle with the minimum concentration of IP3 which effected C a 2 + release. d%: Percentage release of C a 2 + from the organelle with the concentration of IP3 which effected the maximum release of C a 2 + .e x : Complete percent release of CaZ from the organelle induced by A23187. IP3 had no influence on the fluorescence intensity with the amount used. The Ca2- ionophore A23187 also had no effect on the change of the intensity. +

-

phenomena uncovered by our results may mean, therefore, that Ca2 uptake into the mitochondria may not greatly affect total Ca2+ uptake into the cell, which may be affected by the strong activity of Ca2+ -ATPase in the Golgi apparatus, so that cell activity might not be inhibited by the addition of 100 .DM orthovanadate. Self Ca2+uptake into the pregnant ER was more active than that into the lactating ER. In contrast, the Ca2' content in the lactating ER was about 20-fold greater than in pregnant ER, so that the C a 2 + concentration in the lactating ER was far higher than that in the medium. Therefore, self C a 2 + uptake into the lactating ER seemed to lack certain substances such as ATP, exhibited by the C a 2 + uptake into the pregnant ER, so that the activity of C a 2 + uptake into the lactating ER was weaker, as was evidenced with the ATP, creatine phosphate, and creatine phospokinase, even In like manner, the involvement of C a 2 + uptake though uptake in both the lactating and pregnant did not differ when cells were treated with digitonin ER accounted for more than 90 per cent of Ca2' in for permeabilisation. Also, no changes were found the medium. Similarly, the C a 2 + uptake into the when oligomycin was added to the medium for lactating RER was remarkably weaker than in the mitochondria1 inhibition, or when orthovanadate other ERs, while the Ca2+ content in the lactating was added to inhibit C a 2 +-ATPase in the Golgi RER was about two fold that of the lactating SER, apparatus. The Ca2+ concentration in milk is ap- this content ratio being almost the same in the proximately 30mM, whereas in the blood it is lactating Golgi apparatus. This phenomenon may 2 . 5 - 3 . 0 m ~ .In the transition from blood to milk, be accounted for by the same reasons as already some mechanism is thought to exist to account for mentioned. SDontaneous Ca2 uDtake into the this 10-fold increase in C a 2 +c o n c e n t r a t i ~ nThe . ~ ~ Golgi apparatus did not greatly differ between the +

+

196

A. YOSHIMOTO E T A L

Table 3. The results of Ca2+ uptake into the organelles from the medium.

Sample Pregnant SER RER Golgi Lactating SER RER Golgi

Ca2+ Uptake A a%* (A min*j

b%* (B min*)

Ca2+ Release A23187 Total nM (e %*)

62 f 34 (3.4 f 1.1) 61 f 29 (3.4 f 1.1) 58 f 39 (5.0 f 3.2)

97 f 3 (6-6 i 1.6) 93 f 7 (7.0 3.9) 94 f 2 (6.3 f 3.9)

21 f 5.5 (166 f 150) 21 f 0.5 (289 f 109) 19 f 2.1 (2438 f 1713)

46 f 31 (4.0 f 3.0) 18 f 18 (2.6 f 2.5) 64 f 25 (5.0 f 2.2)

95 f 9 (7.0 f 2.7) 88 f 13 (6.5 f 1.0) 95 f 7 (8.5 & 1.5)

25 k 1 (3223 f 3719) 46 f 17 (5843 & 5710) 34 f 23 (5957 f 4036)

B

*As described in the legends of Figure 2. The values were the average of five samples and their standard deviations.

Table 4. The results of C a 2 + release from the organelles into the medium induced by IP3 and Ionophore A23187. CaZ+ Release

IP3 Sample Pregnant SER RER Golgi Lactating SER RER Golgi

C C%* ( c pM IP3*)

D d%* (D p~ IP3*)

A23187 Total nM ( e x * )

6-7 f 4.6 (1.6 f 0-2) 14.8 f 12.2 (2.9 & 1.7) 1.4 f 1.3 (2.8 f 2.0)

32 f 30 (3.4 f 1.5) 33 f 3 (4.0 f 1-6) 4.5 & 4.4 (4.3 f 3.4)

21 f 5.5 (166 f 150) 21 f 0.5 (289 f 109) 19 f 2.1 (2438 f 1713)

3.0 f 1.7 (1.8 f 0.3) 2.1 f 2.7 (2.1 _+ 0.6) 0.3 f 0.4 (2-0 f 0-7)

7-3 f 9.9 (2-6 f 0.5) 6.5 f 11 (2.9 f 0.5) 0.4 f 0.5 (2.8 f 0.6)

2.5 f 1 (3223 f 3719) 46 f 17 (5843 & 5710) 34 f 23 (5957 f 4036)

*As described in the legends of Figure 2. The values were the average of five samples and their standard deviations.

pregnant and lactating cells. This would seem to shows a very high Ca2+-ATPase activity during ~ .that ~ ~ ,the releasing effect by IP3 confirm reports that the Golgi apparatus of the l a ~ t a t i o n ~so mammary epithelial cell shows considerable may have been considerably negated by this Ca2 -ATPase activity. Ca2 -ATPase activity a ~ t i v i t y . ~ ~ - ~ ~ As for the C a 2 + release induced by IP3, the sensitivity was greater in the lactating than in the pregnant ER and Golgi apparatus, indicating that CONCLUSION the membrane of the organelle may undergo a change from pregnancy to lactation, or that the (1) There was a Ca2+ uptake from the medium into the lactating mouse mammary epithelial Ca2+ content in the organelles may be influence by cells. IP3. Riden et aL2*have reported, however, that the change of C a 2 + content in the organelle did not (2) There was also spontaneous C a 2 + uptake from the medium into the organelles of the SER, exhibit any sensitivity for IP3, so it may be that a RER, and Golgi apparatus which were obsensitivity for IP3 develops as a change in the cells tained from the lactating and pregnant mouse occurs during pregnancy to lactation. As for the mammary epithelial cells. Golgi apparatus, the C a 2 +release caused by IP3 in the lactating Golgi apparatus was very weak. It has (3) The spontaneous Ca2+ uptake into pregnant ER was more active than into lactating ER. been found, however, that the Golgi apparatus +

+

197

EFFECT OF IP3 ON CAZ+RELEASE FROM THE MAMMARY CELL

The spontaneous Ca2+ uptake into lactating RER was less than into SER and the Golgi apparatus. On the addition of ATP, creatine phosphate, and creatine phosphokinase, in all cases about 95 per cent of C a2 + in the medium was taken up into the organelles within 7 to 8 min. The Ca2 release induced by IP3 was less in the lactating organelles than in pregnant organelles, while the Ca2+ content in the lactating ER was about 20-fold greater than that of the pregnant ER. Thus the real volume of released C a 2 +from lactating ER was very little less than that from pregnant ER. In the lactating Golgi apparatus, the amount of Ca2 release induced by IP3 was small. Because of these variations in the C a z + uptake and release caused by IP3, it was considered that the ER and Golgi apparatus may undergo a change in their functions during the transition from pregnancy to lactation. +

+

ACKNOWLEDGEMENT In then electron micrographs, we thank Dr K. Akai (Professor of pathology of this medical school) for kindly instruction, and are also grateful to Mr M. Asami for skillful technical assistance. REFERENCES I . Ichinose, R. and Nandi, S. (19661. Influence of hormones on Iubulo-albeolar development ofmouse mammary gland in vitro. f. Endocrinol., 35, 331 -340 Topper, Y. J. (1968). Multiple hormone interaction related to the growth and differentiation of mammary gland in vitro. Trans. N. Y. Acad. Sci., 30, 869-874. Pizarro M. and Sapag-Hager, M. (1981). Calmodulin and cyclic nucleotide-phosphodiesteraseactivities in rat mammary gland during the lactogenic cycle. FEBS Letts. 136, 127- 130. Berridge, M. J. and Irvine, R. F. (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature, 312, 315-320. Jarasch, E-D., Bruder, G., Keenan, T. W. and Franke, W. W. (1977). Redox constituents in milk fat globe membrane and rough endoplasmic reticulum from lactating mammary gland. J. Cell. Biol., 73, 223-241. Jarasch, E-D., Kartenbeck, J., Bruder, G., Fink, A., Morre, D. J. and Franke, W. W. (1979) fi-type cytochromes in plasma membranes isolated from rat liver, in comparison with these of endomembrane. J. Cell Biol., 80, 37-52. Andersson, G. N., Torndal, U-B. and Eriksson, L. C. (1978). Sequential preparation of rat liver microsomal and Golgi membranes. Biochim. Biophys. Acta, 512, 539-549. Ogawa, Y. (1968). The apparent binding constant of glycoletherdiamine-tetraacetic acid for calcium at neutral pH. J. Biochem., 64, 255-257.

9. Ogawa, Y. (1975) Calcium buffer. In: Guide Book for Biochemical Experiments, Vol. 15. (Yamakawa, T., ed.) Tokyo Chemical Group Press, pp. 325-327 (Japan). 10. Tsien, R. Y., Pozzan, T. and Rink, T. J. (1982). Calcium homeostasis in intact lympyocytes: Cytoplasmic free calcium monitored with a new intracellularly trapped fluorescent indicator. J. Cell Biol., 94, 325-334. 11. Lowry, 0.H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurements with the folin phenol reagent. f. Biol Chem., 193,265-275. 12. Ashwell, C . (1957). Colorimetric analysis of sugar. In: Method in Enzymology, Vol. 3. (Colowick, S. P. and Kaplan, N. O., eds.) Academic Press: New York, p.78. 13. Ochoa, S. (1955). Isocitrate dehydrogenase system (TPN) from pig heart. In: Methods in Enzymology, Vol. 1. ((2010wick, S. P. and Kaplan, N. O., eds.) Academic Press: New York, pp. 699-700. 14. Nordlie, R. C. and Arion, W. J. (1966). Glucose-6-phosphatase. In: Methods in Enzymology, Vol. 9. (Wood, W. A., ed.) Academic Press: New York, pp. 619-620. 15. Lowry, 0. H. (1957). Micromethods for the assay of enzymes. 11. Specific procedures: alkaline phosphatase, adenine triphosphatase. In: Methods in Enzymology, Vol. 4. (Colowick, S. P. and Kaplan, N. O., eds.) Academic Press: New York, pp. 371-373. 16. Morre. J. D. (1971). Isolation of Golgi apparatus. In: Methods in Enzymology, Vol. 22. (Jakoby, W. B., ed.) Academic Press: New York, pp. 138-139. 17. McLimans, W. F., Davis, Y. E., Glover, L. F. and Rake, W. G. (1955). The submerged culture of mammalian cells: The spinner culture. f. Immol., 78,428-435. 18. Streb, H., and Schulz, I. (1983). Regulation of cytosolic free Ca” concentration in acinar cells of rat pancreas J . Physiol., G-347-357. 19. Dawson, A. P. and Irvine, R. F. (1984). Inositol (1,4,5) triphosphate-promoted Caz+ release from microsomal fraction of rat liver. Biochem. Biophys. Res. Cornmun., 120, 853-864. 20. Da-son, A. P. (1985). G TP enhances inositol trisphosphate stimulated Ca2 + release from rat liver microsomes. FEBS Lefters, 185, 147-150. 21. Gershengorn, M. C., Geras, E., Purrello, V. S. and Rebecchi, M. J. (1984). Inositol trisphosphate mediates thyrotropinreleasing hormone mobilization of non-mitochondria1 calcium in rat mammotropic pituitary cells. J. Biol. Chem.,259, 10674- 10681. 22. Carsten, M. E. and Miller, J. D. (1985). Ca2+ release by inositol trisphosphate from CaZ+transporting microsomes derived from uterine sarcoplasmic reticulum. Biochem. Biophys. Res. Commun., 130, 1027-1031. 23. Deffert, D. M., Hills, S., Pershadsingh, H. A. and Sherman, W. R. (1986). Myoinositol 1,4,5-trisphosphate mobilizes Ca2 from isolated adipocyte endoplasmic reticulum but not from plasma membranes. Biochem. J., 236, 37-44. 24. Baumrucker, C. R. (1978). Calcium transport in lactation. In: Lactation, A Comprehensive Treatise, Vol. 4. (Larson, B. L., ed.) Academic Press, New York, pp. 463-474. 25. Virk, S. S., Krik, C. J. and Shears, S. B. (1985). Caz+ transport and CaZt dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands. Biochem. f., 226, 741 -748. 26. Watters, C. D. (1984). A Ca2 + stimulated adenosine triphosphate in Golgi entriched membranes of lactating murine mammary tissue. Biochem. J., 224, 39-45. +

198 27. Wilde, C. J., Hasan, H. R., White, D. A. and Mayer, R. J. (1981). The effect of CaZ+on synthesis and degradation of mammary cytosolic proteins and casein. Biochem. Biophys. Rex Commun., 103, 934-942. 28. Riden, T. J., Prenki, M., Irvine, R. F., Berridge, M.J., and Wollheim, C. B. (1984). Inositol 1,4,5-triphosphate mobi-

A. YOSHIMOTO E T A L

lizes intracellular Ca” from permeabilized insulin secreting cells. Biochem. J., 223,467-473. Receiued in revised form 14 January 1990 Accepted 25 February 1990