Vasopressin via receptor-stimulated phospholipase D: Differential regulation of transphosphatidylation and phospholipid hydrolysis by protein kinase C

Neuropeptides (1995) 28, 277-285 © Pearson Professional Ltd 1995

Vasopressin via Receptor-stimulated Phospholipase D: Differential Regulation of Transphosphatidylation and Phospholipid Hydrolysis by Protein Kinase C Y. GARCES*, E. M. BRILEYt and C. C. FELDERt

Howard Hughes Medical Scholars Program, National Institutes of Health, and ? Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD, USA

Abstract-- Phospholipase D belongs to a group of membrane associated phospholipases which have been shown to be activated by G-protein coupled neurotransmitter receptors. Phosphatidylcholine is the primary substrate for phospholipase D generating phosphatidic acid (PA) and choline. In the presence of 1% ethanol, phospholipase D catalyzes a transphosphatidylation reaction generating phosphatidylethanol (PEt) which is an indicator of phospholipase D activation. In the present study, we utilized Chinese hamster ovary (CHO) cells stably transfected with and expressing a rat Vla vasopressin receptor to study the regulation of phospholipase D by protein kinase C and calcium. Arginine-vasopressin (AVP) stimulated the release of 3H-PEt and 3H-PA in cells pre-labelled overnight with 3H-palmitic acid. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated the release of PEt and PA that was additive with AVP over 15 min. However, long-term stimulation with PMA, which desensitizes protein kinase C, decreased PEt production while simultaneously increasing PA production. Differential regulation of PEt and PA production by PMA suggests the existence of more than one phospholipase D isoenzyme. Though differentially regulated by protein kinase C, both AVPstimulated PEt and PA production required extracellular and not intracellular calcium.

Abbreviations: PEt: phosphatidylethanol; PA: AVP: arginine%vasopressin;

phosphatidic acid; R59022: 6-(2-(4((p-fluorophenyl)-phenylmethylene)- l -piperidinyl)-

Date received 12 September 1994 Date accepted 14 September 1994 Correspondence to: Dr Christian C. Felder, Laboratory of Cell Biology, National Institute of Mental Health, Bldg. 36, Rm 3A15, Bethesda, MD 20892, USA.

ethyl)-7-methyl-5-H-thiazole-(3,2-a)-pyrimidine5-one: R59949: 3-(2-(4(bis-(4-fluorophenyl)-methylene)- 1-piperidinyl)-ethyl)-2, 3-dihydro-2- thioxo4(1H)-quinazolinonethimerosal, ethylmercurithiosalicylic acid; rnVP: [1-(beta-mercapto-beta, betacyclopentamethylene proprionic acid), 2-(0methyl)tyrosine]-ArginineS-vasopressin; B A P T A AM: bis-(o-aminophenoxy)-ethane-N,N,N',N',tetraacetic acid, tetra(acetoxymethyl)-ester; EGTA: ethyleneglycol-bis-(/%aminoethyl)-N,N,N',N',-tetraacetic acid.

277

278 Introduction

The pressor and antidiuretic actions of vasopressin are activated through vasopressin receptors of which three subtypes (Vla, Vlb, V2) are currently known, 1 two of which (Vla, V2) have been cloned. 2'3 Vasopressin-stimulated vasoconstriction is mediated through Vla or Vlb receptors while its antidiuretic effects occur through V2 receptors. As members of the super family of G protein coupled receptors, vasopressin receptors transduce their signals through the activation of effector enzymes or channels resulting in the release of second messenger molecules. V1 receptors activate phospholipases and intracellular calcium transients while V2 receptors activate adenylate cyclase and the production of cAMP. 4 Phospholipase D is one of several phospholipases, such as phospholipase A2 and phospholipase C, which act as effector enzymes in V1 receptor-mediated signal transduction. Receptor-dependent activation of phospholipase D was first demonstrated by vasopressin in liver cells, 5 leading to the discovery that a diverse array of agonists utilize this signalling pathway including hormones, neurotransmitters, cytokines and immune mediators. 6'7 Phosphatidylcholine serves as the primary substrate for phospholipase D, generating phosphatidic acid (PA) and free choline, both of which may act as second messengers following their intracellular release, s In the presence of a primary alcohol, phospholipase D can also catalyze a unique transphosphatidylation reaction generating a phosphatidylalcohol. This reaction has been shown to be a specific index of phospholipase D activation. 9'~° It is not clear however, if separate subtypes of phospholipase D are responsible for PA release and transphosphatidylation, or if one enzyme can catalyze both reactions. Little is currently known about the regulation of phospholipase D due in part to the lack of purified functional protein or clone of the enzyme for expression studies. Modulation ofphospholipase D has been demonstrated with agents that stimulate or inhibit protein kinase C. Phorbol esters and diacylglycerol, which activate protein kinase C, also activate phospholipase D in most cells tested. 6'7 Inactive phorbol esters are without effect, and down regulation of protein kinase C, following long-term phorbol treatment, results in loss of phospholipase D activation.

NEUROPEPTIDES In this study, vasopressin Vla receptor cDNA was stably expressed in Chinese hamster ovary (CHO) cells to provide single receptor subtypemediated activation of phospholipase D. Regulation of phospholipase D-mediated release of phosphatidic acid and transphosphatidylation was investigated following modulation of protein kinase C and calcium. Differential regulation of phorbol ester and vasopressin-stimulated phosphatidic acid and phosphatidylethanol formation was observed after phorbol ester treatment, suggesting that independent phospholipase D isoenzymes regulate the release of these products. Materials and methods

[9, 10-3H(N)] palmitic acid was obtained from New England Nuclear (Boston, MA, USA) and [3H]inositol was obtained from American Radiolabeled Chemicals (St. Louis, MO, USA). Pertussis toxin, R59022, R59949, and phorbol 12-myristate 13acetate (PMA) were obtained from Calbiochem (La Jolla, CA, USA). Calphostin C was obtained from Kamiya Biochemical (Thousand Oaks, CA, USA). Chelerythrine chloride was obtained from LC Services Corp. (Wolburn, MA, USA). Phospholipid standards were obtained from Avanti Polar Lipids (Pelham, AL, USA). ArginineS-vasopressin (AVP) and mVP were obtained from Peninsula Laboratories (Belmont, CA, USA). All other reagents were obtained from Sigma Chemical Company (St. Louis, MO, USA).

Cell culture CHO cells were transfected with the rat vasopressin Via receptor as previously described 2 and a clonal line was selected that stably expressed physiological levels of receptor (558 fmol/mg protein). CHO cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in Alpha modified Eagle's medium (Whittaker M.A. Bioproducts) with 10% fetal calf serum, 4 mM Lglutamine, 100 units/ml penicillin, and 100/~g/ml streptomycin, under an atmosphere of 5% CO2. Measurement of phospholipase D activity Cells were incubated with [3H]palmitic acid (4/~Ci/well) in 6-well Costar plastic plates for 18-

VASOPRESSIN VIA RECEPTOR-STIMULATED PHOSPHOLIPASE D

24 h. Cells were washed once with assay medium (Eagle's #2 medium containing 20 mM HEPES and fatty acid free BSA (1 mg/ml)) and allowed to incubate for 30 min. Cells were washed once more with the same media containing 1% ethanol. Experimental agents were then added and followed by a 15-rain incubation period. The reaction was stopped with the removal of the incubation medium followed by the addition of 2 ml ice cold methanol. Cells were scraped off the plate and the lipids were extracted as described 11 and analyzed by thin layer chromatography (TLC) using Whatman LK6D silica TLC plates with a mobile phase consisting of chloroform/pyridine/formic acid (50:30:7). The phospholipids of interest corresponding to authentic phospholipid standards were scraped and measured for radioactivity in a scintillation spectrophotometer. Measurement of intracellular free calcium concentration

Cells grown on glass cover slips coated with Vitrogen (300 /~g/ml; Collagen Corp., Palo Alto, CA, USA) were loaded with 5 /~M FURA-2-acetoxy methyl ester(FURA-2-AM) (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C in growth media, washed once and stored in Eagles #2 medium containing 1 mg/ml BSA and 20 mM HEPES buffer pH 7.4 at 25°C for no more than 45 rain before the experiment. FURA-2 fluorescence was measured at an emission wavelength of 510 nm in single cells mounted on a Nikon Diaphot microscope illuminated alternately with 350 nm and 380 nm light (bandpass 4 nm) using a SLM-Aminco DMX-1000 spectrofluorometer with an intensifiedCCD video camera as the emission detector (SLMAminco, Urbana, IL, USA) and imaging analysis software to acquire video images (Universal Imaging Corp., West Chester, PA, USA). 350 ran/380 nm ratios were converted to calcium concentrations using a previously described procedure.12

Results CHO cells, stably expressing the vasopressin Vla receptor, were pre-labelled overnight with 3H-palmitic acid and then exposed to AVP and the release of [3H]-phosphatidylethanol (PEt) and [3H]-phos-

279

phatidic acid (PA) was examined. PEt and PA release were used as an index of phospholipase D activation. AVP stimulated a concentration dependent increase in PEt production that increased with added ethanol indicating the requirement of ethanol for PEt formation (Fig. 1). There was a concomitant decrease in PA production with added ethanol (data not shown). AVP-stimulated PEt formation was blocked by the vasopressin receptor antagonist mAVP, indicating a Vla receptormediated response. No AVP-stimulated increase in either PEt or PA was detectable in control CHO cells similarly transfected with and expressing a muscarinic m3 receptor (data not shown) indicating a Vla receptor-mediated response. Both AVPstimulated PEt and PA production increased over time with maximal levels occurring at 5 rain and significant levels still measurable at 3 h (Fig. 2). The role of protein kinase C in the regulation of Vla receptor-stimulated PA and PEt production was investigated using the phorbol ester, PMA. Short-term (15 min) treatment of CHO cells with PMA results in maximal stimulation of protein kinase C while long-term (> 2 h) PMA treatment results in a down-regulation of protein kinase C activity?3 Short-term (15 min) treatment of CHO cells expressing the Vla receptor, with a saturating concentration of PMA, stimulated PEt production to a similar extent as a saturating concentration of AVP (Fig. 3A). When combined, AVP- and PMAstimulated PEt were essentially additive, suggesting different mechanisms of activation. Similar additivity was observed for the production of PA (Fig. 3B) except that PMA-stimulated PA production was modest when compared to PEt production. In contrast to the 15 min PMA treatment (Fig. 4A), 3 h PMA treatment (Fig. 4B) resulted in an inhibition of AVP- and PMA-stimulated PEt production to almost basal levels, while PA production was slightly elevated. Four-alpha phorbol, an inactive analogue of phorbol ester, was without effect on PEt or PA formation (data not shown). A role for protein kinase C in the regulation of AVP- and PMA-stimulated PA and PEt production was verified when the protein kinase C inhibitor, calphostin C, blocked both AVP- and PMA-stimulated PEt production and PMA stimulated PA release (Fig. 5). In contrast, calphostin C had essentially no effect on AVP-stimulated PA release indicating

280

NEUROPEPTIDES

-i--l.O

-1..0.5

r,

---0-- 0 . 2 5 N

~E

EtOH

~

EtOH EtOH

,

•

0. 0

ui

l Itll/~ I " * " -- -- ~--L

(/I < hi M td n'

--

/

.I

,"li //~

n

/..'

BSL-I2

-11 -iO

-'9

-B

[AVP],

M

-7

-'6 B S I

AVP

mVP

AVP ÷

log

mVP

Fig. l Effect of increasing ethanol concentration and addition of a V1 receptor antagonist on (PEt) formation. Increasing concentrations of AVP were used to stimulate [3H]PEt release in CHO cells expressing the cloned V la receptor over 15 min in the presence of increasing concentrations of ethanol (left panel)• AVP (100 nM)-stimulated [3H]PEt release was measured in the absence or presence of the V] receptor antagonist, mVP (10 nM)(right panel). Data are the mean _+SEM of three independent experiments, each performed in triplicate.

30000 .I.PA +PEt

2500020000-

! a_ 15000ii

10000-

1

5000- t ° t ° ~

~o I

0

180 (Seconds)

'

I

I

360"15 6o Time

|

I

120 iso

(Minutes)

Fig. 2 Time course of A V P (100 nM) P A and PEt production in C H O cells expressing the Vla receptor. Basal release was subtracted at every time point. Data are the mean_+ S E M of triplicatedeterminations from one representativeexperiment of three.

281

VASOPRESSINVIA RECEPTOR-STIMULATED PHOSPHOLIPASED 2000

25000

--I--AVP -~"AVP • PMA

20000-

+ P M A 1 X 10 - 7

M

15 rain

3 h

& 0 15ooo< W J

0

I0000-

0.. 5000-

t B~;L

0

A

0 ......

PI~A - i 2

O.

• ' " " 0--

-[1

12000-

+

AVP

• -o.

lO000-

•

+

-'9

-]0

io-7

-'8

PMA

I X

10 - 7

8000-

m <

6ooo-

"AVP

AVP

PMA

PMA

AVP

PMA

PMA

+

+

AVP

AVP

T

PMA

~

..{ .....

'o

. 700-

15 rain

3 h

-o 600soo-

d

•

@

0 .....

<3 . . . . .

.

.

[]

< &

--'6

M

&

O

--L7

log I'AVP], M

4000-

< 400-

0""

0..

2000m

0 B

B@L P # A 1°-7

-i2

-[I

-iO

-b Ioa

-'8 FAVPl,

-'7

-'6

M

Fig. 3 Effect of PMA on AVP concentration-dependentrelease of PEt (A) and PA (B) over 15 rain. Data are the mean + SEM of three experiments, each performed in triplicate.

that protein kinase C is not required for this reaction. It is not clear if extracellular or intracellular calcium, or both, are required for vasopressinmediated phospholipase D activation. The source of calcium regulating phospholipase D and the generation of PA or PEt was determined by incubating the cells in either E G T A containing media to remove extracellular calcium or BAPTA-AM to chelate intracellular calcium (Fig. 6). AVP-stimulated PA and PEt production were abolished in the absence of extracellular calcium. BAPTA-AM did not effect the formation of either PEt or PA. Similar results were observed following 15 rain and 3 h PMA pre-treatment (Fig. 6). In FURA-2 loaded cells, AVP stimulated a rapid spike followed by a sustained phase in intracellular free calcium concentration (Fig. 7). BAPTA-AM lowered the resting calcium level and prevented an increase in intracellular calcium following stimulation with AVP. The sustained phase was eliminated in the

----'r---- - -

AVP

PMA

PMA

AVP

PMA

PMA

+

+

AVP

AVP

Fig. 4 Effect of short-term (15 min) and long-term (3 h) PMA (t00 nM) pre-treatment on AVP (100 nM)-stimulated PEt release (A) and PA release (B) measured over 15 rain. Data are the mean--SEM of three experiments, each performed in triplicate.

absence of extracellular calcium indicating a calcium influx requirement for the sustained phase. These results suggest that calcium influx, and not intracellular calcium release, was essential for phospholipase D activation in the CHO cell. PA may arise from the action of diglyceride kinase on diacylglycerol. To rule out this possibility, two diglyceride kinase inhibitors were tested for their effect on AVP and P M A stimulated PA and PEt release. R59022, up to 1 mM, and R59949, up to 50 #M, had no effect on AVP and PMA stimulated PA or PEt release (data not shown). Alternatively, PA may increase following the action of phospholipase A2 and release of lysophosphatidic acid and its reacylation to PA. The acyltransferase inhibitor, thimerosal, had no effect on Vla-stimu-

282

NEUROPEPTIDES

10000 PEt RELEASE

PA RELEASE

8000-

60(

,.T,, ,.'.-::.v.'.-: .:.-.v.v.'.': ":.-.v,'.v,'. :w.w.:: v.v.-.-.'.'.': •:...:..::.-,. .:... •.......,

EL C._)

-::.'.v:::.

.,w:.v.':.'

J

20[

ii!iiiiiii:;iii!ii ..'.'.',v.v.'.' ...:.v.v:,-.. v.'.'.w.-.-,'

0

~":."i:i BSL

PMA

P I','1A

AVP

iii!i!i!!!ii!~i!

I AVP

BSL

PMA

PI'4A

AVP

AVP

+

+

+

+

CaI-C

CaI-C

CaI-C

CaI-C

Fig. 5 Effect of the protein kinase C inhibitor, calphostin C (1 #M), on PMA (100 nM)- and AVP (100 nM)-stimulated PEt (left panel) and PA (right panel) release. Cells were pre-incubated with calphostin C for 15 rain before exposure to AVP for 15 more rain. CHO cells were exposed to strong fluorescent light during the entire 30-rain incubation period to ensure calphostin C activity. Data are the mean 4- SEM of three experiments, each performed in triplicate.

Discussion

AVP

AVP +

AVP +

BAPTA EGTA

AVP

AVP +

AVP +

BAPTA EGTA

Fig. 6 Calcium dependency of AVP (100 nM)-stimulated release of PEt (left panel) and PA (right panel). 10 #M BAPTAAM was incubated with the CHO cells for 30-45 rain to ensure adequate loading. After washing away unincorporated BAPTA, cells were stimulated with 100 nM AVP for 15 min. EGTA (5 mM) was added to cells 10 rain before performing the 15-min AVP stimulation. Data are the mean_ SEM of three experiments, each performed in triplicate. lated P A release u p to 1 m M , the highest conc e n t r a t i o n tested ( d a t a n o t shown).

V 1a r e c e p t o r - m e d i a t e d a c t i v a t i o n o f p h o s p h o l i p a s e D catalyzes b o t h the h y d r o l y s i s o f p h o s p h o l i p i d to generate P A a n d a t r a n s p h o s p h a t i d y l a t i o n r e a c t i o n to generate, in the presence o f e t h a n o l , PEt. Shortt e r m t r e a t m e n t with P M A , c o n d i t i o n s u n d e r which p r o t e i n kinase C was stimulated, caused an increase in b o t h P E t a n d P A release a n d was essentially a d d i t i v e with v a s o p r e s s i n stimulation, suggesting independent mechanisms of activation by protein kinase C. H o w e v e r , after 3-hour e x p o s u r e to P M A , c o n d i t i o n s u n d e r which p r o t e i n kinase C is d o w n r e g u l a t e d in the C H O cell, ~3 b o t h b a s a l a n d A V P s t i m u l a t e d P E t release was a t t e n u a t e d , while A V P s t i m u l a t e d P A p r o d u c t i o n was slightly increased. V l a r e c e p t o r - s t i m u l a t e d p r o t e i n kinase C activity a p p e a r s to enable t r a n s p h o s p h a t i d y l a t i o n , b u t is n o t r e q u i r e d for P A release b y p h o s p h o l i p a s e D. These conclusions are s u p p o r t e d b y the inability o f c a l p h o s t i n - C , a specific p r o t e i n kinase C inhibitor, to significantly inhibit V 1a r e c e p t o r - s t i m u l a t e d P A release yet c o m p l e t e l y b l o c k P E t p r o d u c t i o n .

283

VASOPRESS1N VIA RECEPTOR-STIMULATED PHOSPHOLIPASE D

~,-6

~

--

II ~,

F'

$

-8-

Ca *+ ~

'

.

0

.

.

.

-AT. 100

.u~

-

.

.

"

.

.

.

...............

.

.

.

.

.

.

.

"

.

.

.

"

'

.

+BAPTA

200

TIME

300

400

500

(seconds)

Fig. 7 Effect of B A P T A and E G T A on intracellular calcium levels in F U R A - 2 loaded ceils. 5 # M F U R A - 2 - A M was incubated with the C H O celIs for 30-45 rain to ensure adequate loading and excess F U R A - 2 - A M removed by perfusion. Cells were similarly treated with 1 0 / I M B A P T A - A M . Once placed in an inverted microscope, cells were perfused with medium containing 1 m M

calcium alone or in the presence of 5 mM EGTA and imaged with an intensifiedCCD camera. AVP (100 nM) was added where indicated. Data are representativeof three similar experiments. Differential regulation of transphosphatidylation and phospholipid hydrolysis by protein kinase C suggests that two isoenzymes of phospholipase D exist within the same cell. PEt formation, following addition of ethanol to the incubation media, has been well-documented to be the result of phospholipase D-mediated transphosphatidylation. 9'l° PEt is not readily metabolized by the cell, allowing it to accumulate to high levels. However, PA is rapidly removed and reutilized by the cell which may account for the lower levels observed compared to PEt. Its formation may result from either direct release from phospholipids following activation of phospholipase D, or by phosphorylation of phospholipase C-derived diacylglycerol. 14'~s This route is unlikely to be the primary source of V l a receptorstimulated PA formation since P M A inhibits phospholipase C by at least 70-80% at 15 rain in the CHO cell (unpublished observations). Similar PMA-mediated inhibition of phospholipase C has

been shown previously for several cell types.16'i7 In addition, the diglyceride kinase inhibitors R59022 and R59949 had no effect on PA formation. Alternatively, PA formation may arise from the action of acyltransferase on lysophosphatidic acid. Previous reports have demonstrated V1 receptor stimulation of phospholipase A2 with subsequent release of arachidonic acid and presumably lysophospholipids. Is'~9 However, no effect on Via-mediated PA production was seen with addition of the acyltransferase inhibitor thimerosal, making this pathway unlikely to be a major source of PA as well. Receptors modulate intracellular calcium through the opening of calcium channels in the plasma membrane or by inducing IP3-mediated release from intracellular stores. Phospholipases have shown differential requirement for calcium which may arise from either or both of these sources. Previous reports have demonstrated extracellular independent 2°,2~ and dependent ~°,2~'23 phospholipase D activation. V l a receptor-stimu-

284 lated P E t a n d P A release in the C H O cell were d e p e n d e n t o n extracellular calcium a n d n o t intracellular calcium for activity, suggesting that extracellular calcium is the first messenger in this cascade. Similar results have been s h o w n for receptor-stimulated phospholipase A224 a n d phospholipase C-y 2s in the C H O cell. The receptorstimulated calcium influx was a t t r i b u t e d to activ a t i o n of voltage i n d e p e n d e n t receptor-operated calcium channels that were activated i n d e p e n d e n t of intracellular calcium release or second messengers. 17 V1 receptor-stimulated v a s o c o n s t r i c t i o n m a y be m e d i a t e d t h r o u g h several possible signal transd u c t i o n p a t h w a y s s h o w n to be activated by this receptor, i n c l u d i n g phospholipase D. Previous reports i n v o l v i n g characterization of this receptor, e n d o g e n o u s l y expressed in various tissues or cell lines, could n o t rule out the possible presence of related, b u t f u n c t i o n a l distinct subtypes. The recent c l o n i n g of the vasopressin V l a receptor p r o v i d e d the o p p o r t u n i t y to d e m o n s t r a t e signal t r a n s d u c t i o n t h r o u g h phospholipase D activation stimulated by this single receptor subtype. A m a m m a l i a n cell line expressing physiologically relevant levels o f V l a receptor was selected to avoid i n d i s c r i m i n a n t activ a t i o n of signalling pathways. S i m u l t a n e o u s expression o f m a m m a l i a n cells single receptor subtypes a n d p h o s p h o l i p a s e D(s), w h e n they are eventually purified a n d cloned, should provide a n excellent m o d e l system in which to study receptorp h o s p h o l i p a s e D interactions.

References 1. Michell, R. H., Kirk, C. J. and Billiah, M. M. Hormonal stimulation of phosphatidylinositol breakdown with particular reference to the hepatic effects of vasopressin. Biochem. Soc. Trans. 1979; 7: 861-865. 2. Morel, A., O'Carroll, A. M., Brownstein, M. J. and Lolait, S. J. Molecular cloning and expression of a rat V1a arginine vasopressin receptor. Nature 1992; 356: 523-526. 3. Lolait, S. J., O'Carroll, A. M., McBride, O. W., Konig, M., Morel, A. and Brownstein, M. J. Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus. Nature 1992; 357: 336-339. 4. Thibonnier, M. Signal transduction of Vl-vascular vasopressin receptors. Regul. Pept. 1992; 38:1-11. 5. Bocckino, S. B., Blackmore, P. F., Wilson, P. B. and Extort, J. H. Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. J. Biol. Chem. 1987; 262: 15309-15315.

NEUROPEPTIDES 6. Extort, J. H. Signaling through phosphatidylcholine breakdown. J. Biol. Chem. 1990; 265: 1-4. 7. Liscovitch, M. Signal-dependent activation of phosphatidylcholine hydrolysis:role of phospholipase D. Trends Pharmacol. Sci. 1991; 19: 402-407. 8. Billah, M. M. and Anthes, J. C. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem. J. 1990; 269: 281-291. 9. Pal, J-K., Siegel, M. I., Egan, R. W. and Billah, M. M. Activation of phospholipase D by chemotactic peptide in HL-60 granulocytes. Biochem. Biophys. Res. Commun. 1988; 150: 355-364. 10. Pal, J-K., Siegel, M. I., Egan, R. W. and Billah, M. M. Phospholipase D catalyzes phospholipid metabolism in chemotacticpeptide-stimulated HL-60 granulocytes,J. Biol. Chem. 1988; 263:12472 -12477. 11. Bligh, E. G. and Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959; 37: 911-917. 12. Grynkiewics, G., Poenie, M. and Tsien, R. Y. A new generation of calcium indicators with greatly improved fluorescence properties. J. Biol. Chem. 1986; 260: 3440-3450. 13. Felder, C. C., Kanterman, R. Y., Ma, A. L. and Axelrod, J. A transfected ml muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis. J. Biol. Chem. 1989;264: 20356-20362. 14. MacDonald, M. L., Mack, K. F., Richardson, C. N. and Glomset, J. A. Regulation of diacylglycerolkinase reaction in Swiss 3T3 cells. Increased phosphorylation of endogenous diacylglycerol and decreased phosphorylation of didecanoylglycerol in response to platelet-derived growth factor. J. Biol. Chem. 1988; 263: 1575-1583. 15. MacDonald, M. L., Mack, K. F., Williams, B. W., King, W. C. and Glomset, J. A. A membrane-bound diacylglycerol kinase that selectivelyphosphorylates arachidonoyl-diacylglycerol. Distinction from cytosolic diacylglycerol kinase and comparison with the membrane-bound enzyme from Escherichia coli. J. Biol. Chem. 1988; 263: 1584-1592. 16. Ryu, S. It., Kim, U-H., Wahl, M. I. et al. Feedback regulation of phospholipase C-l? by protein kinase C. J. Biol. Chem. 1990;265: 17941-17945. 17. Felder, C. C., Poulter, M. O. and Wess, J. Muscarinic receptor-operated Ca2+ influx in transfected fibroblast cells is independent of inositol phosphates and release of intracellnlar Ca2+. Proc. Natl. Acad. Sci. USA 1992; 89: 509-513. 18. Hassid, A. and Williams, C. Vasoconstrictor-evoked prostaglandin synthesis in cultured vascular smooth muscle. Am. J. Physiol. 1983;245: C278-C282. 19. Grillone, L. R., Clark, M. A., Godfrey, R. W., Stassen, F. and Crooke, S. T. Vasopressin induces V1 receptors to activate phosphatidylinositol- and phosphatidylcholinespecific phospholipase C and stimulates the release of arachidonic acid by at least two pathways in the smooth muscle cell line, A-10. J. Bio. Chem. 1988; 263: 658-2663. 20. Welsh, J. W., Schmeichel, K., Cao, H.-T. and Chabbott, H. Vasopressin stimulates phospholipase D activity against phosphatidylcholine in vascular smooth muscle cells. Lipids 1990; 25: 675-684. 21. Thibonnier, M., Bayer, A. L., Simonson, M. S. and Kester, M. Multiple signaling pathways of V1-vascularvasopressin receptors of ATr5cells. Endocrinology 1991; 129:2845-2856. 22. Cockroft, S. Ca2+-dependent conversion of phosphatidylinositol to phosphatidate in neutrophils stimulated with fIVIet-Leu-Phe or ionophore A23187. Biochem. Biophys. Acta 1984; 795: 37-46.

VASOPRESSIN VIA RECEPTOR-STIMULATED PHOSPHOLIPASE D

23. Biilah, M. M., Eckel, S., Mullmann, T., Egan, R. W. and Siegel, M. I. Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction. J. Biol. Chem. 1989; 264: 17069917077. 24. Felder, C. C., Dieter, P., Kinsella, J., Tamura, K., Kanter-

285

man; R. Y. and Axelrod, J. A transfected m5 muscarinic acetylcholine receptor stimulates phospholipase A2 by inducing both calcium influx and activation of protein kinase C. J. Pharmacol. Exp. Ther. 1990; 255: 1140-l 147. 25. Gusovsky, F., Leuders, J. E., Kohn, E. C. and Felder, C. C. Muscarinic receptor-mediated tyrosine phosphorylation of phospholipase C-y. J. Biol. Chem. 1993;‘268: 77687772.