Vol. 179, No. 2, 1991 September

BIOCHEMICAL

AND BIOPHYSICAL

16, 1991

PHOSPHOUPASE

RESEARCH COMMUNICATIONS Pages 1070-l 076

D IN CULTURED RAT VASCULAR SMOOTH MUSCLE CELLS AND ITS ACTIVATION BY PHORBOL ESTER

Fumiko Konishi, Takao Kondo and Tadashi lnagaml Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232 Received

July

31,

1991

Summary: We determined the phosphokpase D (PLD) actlvii in rat vascular smooth muscle cells by the formation of phosphatidylethanol in cells prelabeled with [3H] myristic acid. The enzyme was markedly activated by a phorbol ester (TPA). Down regulation of protein kinase C (PKC) resulted in almost complete inhibition indicating PKCdependent mechanism of its activation. Depletion of calcium by EGTA and TMB-6 caused 53% inhibition. Chelator-stable association of PKC to membrane by TPA was observed in the absence of extracellular Ca2+. The mitogenic peptide PDGF also caused a marked stimulation of PLD. These results indicate that PLD in vascular smooth muscle cells is stimulated by TPA through the activation of PKC both by calciumdependent and independent mechanisms. 0 1991 Academic Press, bc.

The unique roles of phospholipase agonists are well recognized. fibroblastst’s).

D (PLD) in signal transduction in response to calcium mobilizing

PLD generates phosphatidic

acid (PA) from phosphatidyfcholine

(PC) in

PA was reported to cause calcium influx across the plasma membrane of the fibroblasts,

triggers DNA synthesis and cdl prollferation(4s).

Prolonged formation of diacylglycerol, the natural protein

kinase C (PKC) activator, after calcium mobilizing stimuli is considered to be due to PLD rather than PLC. These observations suggest that the slow and chronic mechanisms involved in the regulation of vascular tone may be dependent

more on PLD rather than the rapid and transient reaction of PLC. However, there

is little evidence of PLD in vascular smooth muscle cells. In view of potentially important roles of PLD in contractile tissues, we investigated the presence of PLD in cultured vascufar smooth cells by employing a transphosphatldylation activll

reaction in the presence of ethanol and obtained evidence for the regulation of its

by PKC dependent

mechanisms.

MATERIALS

AND METHODS

Materials: The sources of the materials are fisted as follows: 13H] myristic acid (53 Ci/mmol) and adenosine 5-[r-32P] triphosphate (> 6006 Ci/mmol) from Amersham, phosphatidytethanol (PEt) and phosphatidic acid (PA) from Avanti Pdar Lipids, precoated silica-gel TLC plates (LKGDF) from Whatman, tissueculture media from Gibco, other biochemicals from Sigma. Abbreviations used: PLD: phospholipase D; PLC: phospholipase C; PEt: phosphatidylethanol; PA: DhosDhatidic acid: PKC: protein kinase C; VSMCs: vascular smooth muscle cells: TLC, thin laver chromatography; &A: bovine serum albumin; PDGF: platelet-derived growth factor; ET: endothelin; TPA: 120tetradecanoylphorbol 12-myristate 13-acetate; Ang II: angiotensin II; AVP: vasopressin; BK: bradykinin; EGF: epidermal growth factor; EGTA: [ethylene bis (oxyethylenitrolo)] tetraacetic acid; TMB-9: [a(diethylamino)-octyf-3,4,5-trimethoxybenzoate],HCL; HEPES: 4-(2-hydroxyethy)-1-piperazineethanesulfonic acid. 0006-291X/91

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1070

Vol. 179, No. 2, 1991

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

Cell culture: Vascular smooth muscle cells (VSMCs) were isdated enzymatically from thoracic aorta of Wistar-Kyoto rats and were grown in Dulbeco’s modifii Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) as previously described(‘). The cells between 7 and 15 passages were used. Labeling of VSMCs with PH] myristic acid. VSMCs seeded into 35-mm dishes at 2 x lo5 cells/dish were cultured for 3 days in 2.0 ml DMEM supplemented with 10% FCS. Then, the cells were cultured in 2.0 ml of serum-free DMEM for 24 hrs. The medium was replaced by 1 .O ml of DMEM containing 0.5 mg/ml fatty acid-free BSA and 2 &i 13H] myristic acid for 20 hrs. By this procedure, more than 30% of added radloactivii was incorporated into phospholipids fraction, mainly phosphatidylchdine and phosphatidylinositol. Incubation procedures and phospholipids extraction. Prelabeled cells were washed 3 times with 2.0 ml 4-(2-hydroxyethyf)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) containing 130 mM NaCI, 5 mM KCI, 1 mM MgCI, and 1.5 mM CaCl, (buffer A). After equitibration for 15 min in buffer A, the cells were stimulated with TPA. The reaction was initiated by replacing the equilibration buffer with 1 .O ml of buffer A containing ethanol and TPA (added as DMSO so&ion). Final concentration of DMSO in the reaction mixture was 0.2%, which showed no effect. In some experiments, growth hormones and vasoconstrictors were added into the reaction mixture instead of TPA. These cell treatments were done at 37°C. The reaction was terminated by rapid aspiration of the incubation medium and immediate addition of 0.5 ml methanol. Phospholipids were extracted by the method of Bligh and Dyer(s) under acidic condition and were taken to dryness under reduced pressure. Separation of r3H] myristic acid labeled metabolities. Phospholipids extract was redissolved in 40 3 of chloroform/methanol (2:1, v/v). An aliquot (5 d) of the redissotved phosphdipids extract was used for counting by a liquid scintillation counter to estimate total radioactivii incorporated into phospholipids fraction. Another aliquot (20 d) was applied on a silica gel TLC plate, which was developed in chloroform/methanol/acetic acid (65:15:2, v/v) to separate PEt. The organic phase of ethytacetate/isooctane/acetic acid/water (13:2:3:10, v/v) was used to separate PA from other phospholipids. The plates were air dried, and the comigrated standards were visualized in the vapor of iodine. The areas corresponding to PEt and PA were scraped and transferred into scintillation vials. Radioactivity was counted by a liquid scintillation counter. The amount of labeled metabolites were translated into the percentage of the total incorporated radioactivity in phospholipids fraction in order to eliminate the effect of differential uptake of radioactivity among individual dishes. Assay of chelator-stab/e protein kinase C translocation. VSMCs seeded into lOO-mm dishes at 106 cells per dish were cultured for 3 days in DMEM supplemented with 10% FCS. And then, the cells were cultured in serum-free DMEM for 43 hrs. The cells were equilibrated for 15 min in 5 ml of buffer A or &?-free buffer A containing 2 mM EGTA. The stimulation with TPA was initiated by replacing equilibration buffer with the same buffer containing TPA (100 nM). After 15 min, the reaction was terminated by rapid aspiration of incubation medium and washing with ice-cold 20 mM Tris-HCI (pH 7.5) containing 330 mM sucrose, 2 mM EDTA, 0.5 mM EGTA, 2 mM PMSF and 25 m/ml leupeptin (buffer 6). The cells were scraped into 1.5 ml of buffer B and homogenized by a Dounce homogenizer (40 strokes) at 0” C. By centrifugation (14,000 x g, 10 min at 4” C), cytosol fraction and membrane fraction were separated. From membrane fraction, PKC was extracted with the aid of detergent (Nonidet P-40) and then PKC from both fractions were partially puriiied by the previously established DEAE-cellulose chromatography(g). PKC activity was determined by measuring the transfer of 32P from [r-32P]ATP to histone by a previously described procedure(‘o) and revealed as the percent distribution in cytosol and membrane. Data and statistics. Values are expressed as mean + standard error of means. Statistical significance of difference was estimated by the Student’s t-test. RESULTS Cultured VSMCs, prelabeled with [3H] myristic acid, were incubated with 100 nM TPA for varying intervals. In the presence of 400 mM ethand TPA stimulated time-dependent

formation of [3H] PEt (Fig. 1).

When the cells were incubated for 30 min with different doses of TPA [3H] PEt formation was stimulated in a dose dependent

manner (Fig. 2). This stimulation was clearly visible at relatively low concentration

of TPA

(10 nM). VSMCs, prelabeled with [3H] myristic acid and stimulated with 100 nM TPA, produced an increasing amount of [3H] PEt in the presence of increasing concentrations

of ethand

consistent with the formation of [3H] PEt through the transphosphatidytation (PLD) 1071

(Fig. 3).

This data was

reaction by phosphdipase

D

Vol.

179,

No.

kidney

BIOCHEMICAL

2, 1991

microsomes

antibodies that against

and ribosomes

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with

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COMMUNICATIONS

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RESEARCH

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BIOPHYSICAL

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AND

There

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Vol.

179,

No.

BIOCHEMICAL

2, 1991

COnlrOl

Fig.5.

results

AND BIOPHYSICAL

TPA

EGTA+TPA

RESEARCH COMMUNICATIONS

EGTA+TMBI+TPA

Effects of EGTA and TM58 on TPA-stimulated [%I PE1 formation. Vascular smooth muscle cells prslabeled wirh i3H] myristic acid (2.0 &i/dish) were pretreated with EGTA (2mM) or EGTA plus TMB-8 (100 @) for 30 min followed by stimulation by TPA (100 r&l) in the presence of ethand (400 mM). PEt was analyzed as described in Materials and Methods. The results are means f SE (n=5). Statistical significance of differences: l p