J Mol

Cell

Cardiol

24, 1003-1010

(1992)

Polyphosphoinositide Hideaki Hirofumi

Metabolism

Kawaguchi,* Sawa, Naoki

Department

in Hypertrophic

Mikako Shoki, Hitoshi Sano, Mochizuki, Hiroshi Okamoto, Akira Kitabatake

of Cardiovascular

Rat

Heart

Toshiyuki Kudo, Yuka Endo and

Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan

(Received 13 December 1990; accepted

in revisedform 31 March

1992)

TOSHIYUKI KUDO, HIROFUMI SAWA, NAOKI MOCHIZUKI, Polyphosphoinositide Metabolism in Hypertrophic Rat Heart. Journal oJ Molecular and Cellular Cardiology (1992) 24, 1003-1010. The accumulations of inositol-1,4,5trisphospate (IP,) and inositol-1,3,4,5-tetrakisphosphate (IP,) after hormonal stimulation may have a physiotissue. But the accumulation of inositol logical role, possibly by alteration of Ca?’ levels in cardiac polyphosphate in a pathophysiological condition has not been studied. We investigated phosphatidylinositol-4,5-bisphosphate (PIP,) metabolism in hypertrophic cardiac myocytes, and clarified that the accumulations of IP,, IP, and diacylglyceride after stimulation with norepinephrine were significantly enhanced in isolated myocytes from spontaneously hypertensive rat heart. Phospholipase C activity increased with age in SHRSP heart cells. These data suggest that PI turnover pathways, which can be mediated by both phosphatidylinositol-4,5bisphosphate and diacylglyceride, may play an important role in development of hypertrophy in the hearts of rats with spontaneous hypertension. HIDEAKI HIROSHI

KEY

KAWAGUCHI, OKAMOTO,

WORDS:

phate kinase;

MIKAKO SHOKI, HITOSHI SANO, YUKA ENDO, AKIRA KITABATAKE.

Phosphatidylinositide-specific phospholipase Inositol trisphosphate phosphatase.

Introduction The primary event in the mechanism of action of many different hormones and neurotransmitters involves receptor-mediated stimulation of the breakdown of plasma membrane inositol phospholipids (Brown et al., 1988; Baker and Singer, 1988; Dubyak et al., 1988; Fasolato et al., 1986). This so-called phosphatidylinositol (PI) turnover pathway generates two second messengers, inositol 1,4,5-trisphosphate (IP,) and 1,2-diacylglycerol (DAG) (Berridge, 1984; Williamson et al., 1985). DAG stimulates membrane-bound phospholipid-dependent, Ca’+-dependent protein kinase C (Nishizuka, 1983), while IP, releases Ca2+ from endoplasmic reticulum stores (Putney, 1987; Ehrlich and Watras, 1988). The possibility that catecholamines may be one of the molecular signals linking increased circulatory demand to myocardial hypertrophy has been raised (Simpson et al., 1982; *To

whom

correspondence

0022-2828/92/091003+08

should

$08.00/O

C; Inositol-1,4,5-trisphosphate:

Inositol

trisphos-

Simpson, 1983). Recently, it is reported, using neonatal rat heart cell cultures, that norepinephrine (NE)-stimulated hypertrophy is mediated through an a,-adrenergic receptor, and this a,-adrenergic stimulation by NE, phorbol esters and serum induces an increase in the cell size of cardiac myocytes (Simpson, 1985; Starksen et al., 1986). The purpose of these experiments was to study the polyphosphoinositide metabolism in the hypertrophic adult rat heart to clarify the relationship between PI-response and cardiac hypertrophy.

Materials

and Methods

Experimental

protocol

Experiments were carried out on male strokeprone spontaneously hypertensive rats (SHRSP) aged 5, 10,20,30 and 40 weeks and

be addressed. $31992

Academic

l’rrss

Limited

1004

H. Kawaguchi

age-matched male Wistar-Kyoto rats (WKY). Each age group comprised six animals. The left ventricle was excised from the rat heart and blood was carefully washed out. Then its weight was measured. Six experiments with cells from different hearts were used for each assay at each age. Each assay was done in triplicate. Cell preparation Cardiac myocytes of SHRSP and WKY were prepared according to a previously reported method in phosphate buffer (PB) (Glick et al., 1974), and cultured in Ham F-10 medium with 10% fetal calf serum (FCS) and used within 2 h. There were no differences in yield of myocytes between SHRSP and WKY. (We can prepare about 10’ cells/heart at an age of 5 weeks). The freshly prepared cells were maintained at 37°C in a humidified 5% CO,/ 95% air atmosphere (Kawaguchi and Yasuda, 1986a). In this medium (0.3 mM CaCI,), more than 90% of cells were rodshaped, and they were not beating until the addition of NE and 1 mM CaCl, (final concentration). They were lightly attached to dishes. Cellular For determination

response to norepinephrine of the cellular

response

of

Fraction

et al. PLC activity to NE, myocytes (3 x 106) were prelabeled with [3H]myoinositol (5OpCi) for 24 h in phosphate buffer with 0.3% fetal calf serum in 10 cm dishes, then the cells were washed with PB three times in 50 ml Falcon centrifuge tubes. Then cells were subcultured at 3 x lo5 cells/dish in 1 ml of PB containing 1 mM CaCI, in 35-mm dishes for assays. The incorporation rates of [3H]myoinositol into SHRSP and WKY were 4.6f 1.0% and 4.9 f 1 .O%, respectively. Cells were incubated with the indicated concentrations of NE, 5 mM 2,3-DPG (2,3-DPG; this concentration of 2,3-DPG inhibited the dephosphorylation of IP, and IP., by 98%) (Dowens et al., 1982), and 10 mM LiCl for the indicated periods in the presence of 1 pM metoprolol (Japan CIBA-GEIGY, Osaka, Japan) to exclude the effect of j?,-adrenergic receptor stimulation, then terminated with chloroform/methanol 2:1, v/v) (Folch et al., 1957). The aqueous phase was applied to an AG 1 x 8 column. Inositol monophosphate, IP,, IP, and IP, were separated by elution from AG 1 x 8 columns in formate form (lo&200 mesh) by a gradient of ammonium formate (0.2-1.2 M) plus 0.1 M formic acid (Downes et al., 1986; Merritt et al., 1986) (Fig. 1). For a more detailed analysis, including the separation of inositol phosphate isomers, samples were filtered and separated by high-performance

number

FIGURE 1. Elution pattern ofinositol phosphates. IP,, IP, and &ted as described in Materials and Methods in 1 ml fraction/tube. ammonium formate plus 0.1 M formic acid, respectively.

IP, (50 000 DPM each) IP,, IP, and IP, were

were &ted

applied to column 0.4 M, 0.8 M and

then 1.2 M

Increased

Inositol

Trisphosphate

liquid chromatography (Whatman Partisil 10 SAX anion-exchange column with a guard column) with a gradient of ammonium formate and phosphate (Bradford and Irvine, 1987; Irvine and Moor, 1986). The migration of IP, or IP, eluted from AG I x 8 column to each other fraction is negtigible. The released IP, was also determined by using IP, binding protein system (myo-inositol 1,4,5-trisphosphate assay system, Amersham, UK) (Podzuweit et al., 1989). For determination of diacylglyceride release, cardiac myocytes (3 x lo” tells/35-mm dish) were incubated at 37°C for up to 2 min with NE in 1 ml of PB containing 1 mM CaCI,. The reactions were terminated and lipids were extracted with 4 ml of chloroform/methanol (1: 2, v/v) (Kawaguchi and Yasuda, 198613,1989). The released diacylglyceride (DAG) was determined by DAG assay system (Amersham, UK) j Fukami and Takenawa, 1989). IP, kinase activity after stimulation by NE was determined in permeabilized cells with saponin (Tennes st at., 1987) (lO~g~m1, this concentration of saponin did not affect IP, kinase activity. LJp to lOO~g/ml, saponin IP, kinase activity was not altered. But cells burst at more than 150pug/ml saponin). Cells were preincubated with 1 PM of NE for 2 min, then saponin and IP, (1~~~50 000 DPM, preliminary experiments determined Km 1PM) were added and incubated for the indicated periods in 1 ml cytosolic buffer (KC1 120 mM, NaCl 10mM, KH,PO, 1 mM, NaHCO, 5 mM, HEPES 10 mM, pH 7.1), EGTA 0.2 mM and CaCI, 150 nM. Incubation was terminated with ~hIoroform/methanol (2 :I, v/v). Inositol phosphateswere separated asdescribed above by an AGl x 8 column and ammonium formate gradient system.

Release

in Heart

1905

inositol ( 14.6 Ci/mmol) were obtained from New England Nuclear (Boston, MA, USA). [5,6,8,9,1 1,12,14,15-3H]arachidonic acid (163 Ci/mmol) was purchased from Amersham International (Amersham, UK). Ham F-IO medium was obtained from Flow Laboratories, Inc. (McLean, VA, USA). IP, and IP, were purchased from DOJINDO Laboratories (Kumamoto, Japan). Diphospho ( 2,3)-Dglyceric acid (2,3-DPG) was obtained from Sigma (St Louis, MO, USA). AG 1 X 8 was obtained from RIO-RAD Laboratories I Richmond, CA, USA). Statistical analysis

Six experiments in triplicate were analyzed in all experiments. Results are expressed as mean f S.E.M.Statistical significance was estimated using Student’s t test taking P< 0.05 3s the limit of significance. Results Left ventricular weight increased with age. This weight in SHRSP was markedly increased compared to WKY (Fig. 2). The releaseof IP, and IP, from [‘H]myoinositol prelabeled myocytes was markedly enhanced by 1PM NE in SHRSP (IO weeks old) compared with age-matched WKY [Figs 3(a) and 3(b)]. DAG release from SHRSP myocytes was markedly increased by the stimulation of NE (Fig. 4). The median effective concentrations (EC50) of NE for DAG

Protein was determined by the method of Lowry et al. ( 1951) with bovine serum albumin as the standard.

D-[inositoI-2-3H]-( 1,4)-bisphosphate(Z-10 Ci/ mmol), D-[inositol-13H]-( 1,4,5)-trisphosphate ( 17 Ci/mmol), [inositol- 1-3H]-( 1,3,4,5)-tetrakisphosphate ( I7 Ci/mmol) and myo[2-3H]-

FIGURE 2. Increase of left ventricularweights of SHRSP and WKY. Left ventricular weight was determined as described in Materials and Methods. SHRSP (@) and WKY (A). Points represent meansfs.E.y. **P