Prostaglandin F2a stimulates tyrosine phosphorylation ofphospholipase C-c1

Shahid Husaina,* and Farahdiba Jafrib

a Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USAb Department of Clinical Pharmacy, Medical College of Georgia, Augusta, GA 30912, USA

Received 4 September 2002

Abstract

In this study, we investigated the ability of prostaglandin F2a (PGF2a) to induce tyrosine phosphorylation of phospholipase C-c1(PLC-c1) in cat iris sphincter smooth muscle (CISM) cells. PGF2að1lMÞ-stimulated PLC-c1 tyrosine phosphorylation in a time- anddose-dependent manner with a maximum increase of 3-fold at 0.5min. The protein tyrosine kinase inhibitors, genistein, and tyr-

phostin A-25, blocked the stimulatory effects of PGF2a, suggesting involvement of protein tyrosine kinase activity in the physio-

logical actions of the PGF2a. Furthermore, PGF2a-induced p42/p44 MAP kinase activation was also completely blocked by protein

tyrosine kinase inhibitors. In summary, these findings show that PGF2a stimulates tyrosine phosphorylation of PLC-c1 in CISM

cells and indicate that PGF2a-stimulated tyrosine phosphorylation is responsible for an early signal transduction event.

� 2002 Elsevier Science (USA). All rights reserved.

Keywords: Phospholipase C-c1; Tyrosine phosphorylation; Prostaglandin F2a; Non-vascular smooth muscle

Prostaglandins (PG) exert a broad range of physio-logical and pharmacological actions in a wide variety oftissues through interaction with specific cell-surfacereceptors. All PG receptors identified to date are seven-transmembrane proteins that couple to specific G-proteins mediating the formation of cAMP or inositolphosphate/diacylglycerol second messengers [1]. In theeye, prostaglandinF2a ðPGF2aÞand its analog latanoprost(PhXA41) mediate, through prostaglandin F2a (FP) re-ceptors, a broad range of biological effects includingsmooth muscle contraction and reduction of intraocularpressure (IOP) in glaucoma patients [2]. FP receptors ac-tivation in a number of cell types has been shown to resultin the generationof inositol phosphates via phospholipaseC (PLC) activation with a subsequent mobilization ofintracellular Ca2þ [3] and activation of a number of pro-tein kinases such as Ca2þ-calmodulin-dependent proteinkinase II, myosin light chain kinase, protein kinase C, andmitogen-activated protein (MAP) kinase [4,5].

Three families of mammalian PLC isozymes desig-nated b, c, and d have been described based on theirmolecular structure and mechanisms of regulation [6].The PLC-b group has been shown to be regulated by theGq-class of heterotrimeric GTP-binding proteins [7,8].The PLC-c family of isozymes appears to be regulatedby tyrosine phosphorylation [9]. The regulatory mech-anisms of PLC-d has not yet been elucidated althoughthey do not appear to be stimulated by G-proteins ortyrosine phosphorylation [6]. Little information isavailable concerning PGF2a-induced PLC-c1 phos-phorylation and down-stream signaling pathways inocular smooth muscle. Therefore, the purpose of thepresent study was to determine the effects of PGF2a ontyrosine phosphorylation of PLC-c1 and identificationof down-stream signaling targets in cat iris sphinctersmooth muscle (CISM) cells. We show that PGF2a in-duces transient phosphorylation of PLC-c1, which leadsto the activation of mitogen-activated protein (MAP)kinases in CISM cells. Furthermore, PGF2a-inducedphosphorylation of PLC-c1 and p42/p44 MAP kinasewere completely inhibited in the presence of proteintyrosine kinase inhibitors.

Biochemical and Biophysical Research Communications 297 (2002) 1102–1107

www.academicpress.com

BBRC

* Corresponding author. Fax: 1-706-721-6608.

E-mail address: shusain@mail.mcg.edu (S. Husain).

0006-291X/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

PII: S0006 -291X(02 )02347 -1

Materials and methods

Materials

Anti-PLC-c1 and anti-p42/p44 MAP kinase antibodies were ob-

tained from Santa Cruz Biotechnologies (Santa Cruz, CA). [c-32P]ATP(specific activity, 3000Ci/mmol) was obtained from Amersham Life

Science (Arlington Height, IL). PGF2a, genistein, and herbimycin A,

were obtained from Calbiochem (La Jolla, CA). Mylein basic protein,

protein A–Sepharose, and tyrphostin A-25 were purchased from Sigma

Chemical, (St. Louis, MO). Fetal bovine serum was obtained from

Hyclone (Logan, UT) and all other cell culture supplies were obtained

from Cell gro (Herndon, VA).

Methods

Cell culture. Iris sphincter smooth muscle cells were isolated from

4- to 6-months-old cats as described previously [10]. Briefly, the eyes

were enucleated immediately after the death of the animal and

sphincter muscle was dissected out, further cleaned and cut into 1–

2mm2 pieces. The explants were placed in Dulbecco’s modified Eagle’s

medium (DMEM) containing 2mgml�1 collagenase type IA, 10% fetal

bovine serum (FBS) and 50lgml�1 gentamicin and then incubated for1–2 h at 37 �C with occasional gentle shaking. The cell suspension was

centrifuged at 200g and resuspended in DMEM/F-12 supplemented

with 10% (v/v) FBS and 100Uml�1 penicillin, 100lgml�1 streptomy-cin, and 0.25lgml�1 amphotericin B in 5% CO2 humidified atmo-

sphere. The contaminating fibroblasts were removed as previously

described [10]. After 3 days, one third of the culture medium was re-

placed with fresh medium. The smooth muscle cells were subcultured

at a split ratio of 1:3 using 0.05% trypsin and 0.02% EDTA. The cells

of 3–7 passages were used in this study.

Immunoprecipitation of PLC-c1 and Western blotting. CISM cells

were starved in serum-free DMEM for 24 h and treated with PGF2a for

indicated time periods. The inhibitors were added 15min prior to the

addition of the PGF2a. After treatment, cells were lysed in 50mM Tris–

HCl buffer, pH 8.0, containing 100mM NaCl, 1mM EDTA, 1%

Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 50mM NaF, 1mM

Na3VO4, 5mM PMSF, 10 lg ml�1 leupeptin, and 50 lgml�1 aprotininfor 20min on ice. Cell lysates were centrifuged at 2500g for 10min andthe supernatant was used either for immunoprecipitation of PLC-c1 orfor immuno-detection of p42/p44 MAP kinase. PLC-c1 was isolated byimmunoprecipitation using rabbit anti-PLC-c1 polyclonal antibodies

for 2 h followed by an incubation for 12 h with protein A–Sepharose at

4 �C. The immunoprecipitates were washed and extracted with Lae-

mmli buffer as described earlier [10]. The proteins were resolved on

10% SDS–polyacrylamide gels and then transferred to nitrocellulose

membranes. The membranes were probed with anti-phospho-tyrosine,

anti-PLC-c1, or anti-p42/p44 MAP kinase antibodies followed by in-

cubation with secondary antibodies (HRP-conjugated goat anti-rabbit

IgG at 1:3000 dilution) for 1 h at 20 �C as previously described [4,10].

For chemiluminescent detection, the membranes were treated with

ECL reagent for 1min and subsequently exposed to ECL hyperfilm for

1–2min.

Measurement of p42/p44 MAP kinase activity in CISM cells. MAP

kinase activity was characterized by the in situ myelin basic protein

(MBP) phosphorylation assay as described earlier [4,10]. Briefly, qui-

escent cells were stimulated with 1 lM PGF2a and scraped into ice-cold

extraction buffer (20mM b-glycero-phosphate, 20mM NaF, 2mM

EDTA, 0.2mM sodium vanadate, 1mM PMSF, 25lgml�1 leupeptin,10lgml�1 aprotinin, and 0.3% (v/v) b-mercaptoethanol, pH 7.5). The

cell extracts were centrifuged at 10,000g for 10min at 4 �C, and the

supernatant was resolved on a 10% SDS–polyacrylamide gel co-poly-

merized with 0.5mgml�1 MBP. After electrophoresis, the gels were

washed with 50mM Tris–HCl buffer, pH 8.0, containing 20% (v/v)

propanol to remove SDS, then washed with denaturing buffer (50mM

Tris–HCl, pH 8.0, containing 6M guanidine hydrochloride and 5mM

b-mercaptoethanol). The enzymes on the gel were then renatured by

washing with 50mM Tris–HCl buffer, pH 8.0, containing 0.04% (v/v)

Tween 40 and 5mM b-mercaptoethanol at 4 �C for 21 h. The gel was

then preincubated with assay buffer containing 40mM Hepes, pH 8.0,

10mM MgCl2, 2mM dithiothreitol, and 0.1mM EGTA at 30 �C for

30min. The MAP kinase activity was determined by incubating the gel

with 20ml of the assay buffer, which contained 20 lM ATP and

100lCi [c-32P]ATP, at 30 �C for 1h. After extensive washing in 5% (w/v)

trichloroacetic acid containing 10mM sodium pyrophosphate, the gel

was dried and autoradiographed at )70 �C.

Results and discussion

Results

Immunochemical identification of PLC-c1We examined the presence of PLC-c1 in cat iris

sphincter smooth muscle (CISM) cells by polyclonalantibodies specific for PLC-c1 isoform. The anti-PLC-c1 antibodies reacted with a 145-kDa protein. As shownin Fig. 1, PLC-c1 is predominantly present in cytosolicfraction. Cytosolic and membrane fractions were ob-tained by centrifugation at 100,000g. The specificity ofPLC-c1 band was confirmed by loss of the immunore-active band upon incubation with the appropriateblocking peptide (i.e., an antigenic peptide that was usedto produce PLC-c1 antibodies). PLC-c1 band wascompletely disappeared when 1:1 ratio of antibodies andblocking peptides were used, suggesting that 145-kDaband correspond to PLC-c1 isoform.

Time course study of PGF2a-induced tyrosine phosphory-lation of PLC-c1

To investigate the effects of PGF2a on PLC-c1phosphorylation, PLC-c1 was immunoprecipitated fromcrude homogenates of PGF2a treated and untreated(control) CISM cells with polyclonal antibodies specificto PLC-c1. PGF2a-stimulated tyrosine phosphorylationof PLC-c1 in CISM cells was analyzed by two differentprotocols. First, anti-PLC-c1 antibodies were used toimmunoprecipitate the PLC-c1 from PGF2a treated and

Fig. 1. Immunochemical indentification of PLC-c1 in CISM cells.

Western blot analysis of PLC-c1 in cytosol (3), membrane (2), and

total homogenate (1) of CISM cells. Appropriate amounts (10–15lgprotein) were subjected to 10% SDS–PAGE. Proteins were transferred

to nitrocellulose membranes and immunoblotted with antibodies spe-

cific to PLC-c1 as described in Materials and methods. Results given

are from a representative experiment of three separate experiments.

S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107 1103

untreated homogenates. Western blot analysis of PLC-c1 immunoprecipitates with anti-phospho-tyrosine an-tibodies showed an increase in tyrosine phosphorylationof PLC-c1 by 3-fold relative to control levels (Fig. 2A).The peak response was seen at 0.5min and returned tonear control levels by 5min. Similar results were seenwhen tyrosine-phosphorylated protein were immuno-precipitated using anti-phospho-tyrosine antibodiesfollowed by detection of PLC-c1 by Western blot ana-lysis with anti-PLC-c1 antibodies (data not shown).

The above data clearly show an increased in tyrosinephosphorylation of PLC-c1 with maximal phosphory-lation observed approximately 0.5min after addition ofPGF2a. To rule out the possibility of variation in theloaded amounts of protein, immunoprecipitates were

probed with anti-PLC-c1 antibodies. These antibodiesselectively recognized one band of 145 kDa and all thesamples showed a comparable amounts of immuno-precipitated PLC-c1 in these cells (Fig. 2B).

Dose-dependence of PGF2a on tyrosine phosphorylation ofPLC-c1

CISM cells were treated with indicated concentra-tions of PGF2a and PLC-c1 was immunoprecipitatedfrom homogenates as described in Fig. 2. As shown inFig. 3A, PGF2a stimulates tyrosine phosphorylation ofPLC-c1 in a dose-dependent manner. The maximumincrease in tyrosine phosphorylation of PLC-c1 wasobserved at 1 lM of PGF2a. All the samples containedcomparable amounts of immunoprecipitated PLC-c1(Fig. 3B).

Effects of tyrosine kinase inhibitors on PGF2a-inducedphosphorylation of PLC-c1

To investigate the role of protein tyrosine kinase inPGF2a-induced PLC-c1 phosphorylation, we used ty-rosine kinase inhibitors, genistein, and tyrphostin A-25.At 1 lM, both compound blocked PGF2a-induced ty-rosine phosphorylation of PLC-c1 completely (Fig. 4A).These findings suggest that a tyrosine kinase mediatesPGF2a-stimulated phosphorylation of the PLC-c1 inCISM cells. In all the samples a comparable amounts ofPLC-c1 was immunoprecipitated (Fig. 4B).

Effects of PGF2a on p42/p44 MAP kinase activationMAP kinases are one of the most important signaling

components involved in the regulation of cytoslic

Fig. 2. Time course study for PGF2a-stimulated tyrosine phosphory-

lation of PLC-c1. Starved CISM cells were treated for the indicated

times periods with 1lM PGF2a and harvested with lysis buffer as de-

scribed in Materials and methods. In panel A, proteins were immu-

noprecipitated with an anti-PLC-c1 antibodies and developed with

anti-phospho-tyrosine antibodies. Panel B shows Western blot analysis

of immunoprecipitates that were probed with anti-PLC-c1 antibodies.Results given are from one experiment that is a representative of five

separate experiments (n ¼ 5).

Fig. 3. Dose dependence of PGF2a-stimulated tyrosine phosphoryla-

tion of PLC-c1. CISM cells were starved and incubated with indicated

concentrations of PGF2a for 0.5min and harvested with lysis buffer as

described in Materials and methods. (A) Tyrosine phosphorylation of

PLC-c1 in immunoprecipitates, (B) Western blot analysis of PLC-c1 asdescribed in legend to Fig. 2. Results given are from one experiment

that is a representative of five separate experiments (n ¼ 5).

Fig. 4. Effects of protein tyrosine kinase inhibitors on PGF2a-stimu-

lated tyrosine phosphorylation of PLC-c1. CISM cells were incubated

in serum-free DMEM with 1lM PGF2a in the absence or presence of

1 lM genistein or tyrphostin A-25. Inhibitors were added 15min prior

to the addition of PGF2a. (A) Tyrosine phosphorylation of PLC-c1 inimmunoprecipitates, (B) Western blot analysis of PLC-c1 as describedin legend to Fig. 2. Results given are from one experiment that is a

representative of five separate experiments (n ¼ 5).

1104 S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107

phospholipase A2, arachidonic acid (AA) release, andmatrix metalloproteinases (MMPs) secretion in varioustissues. Several lines of evidence suggest that MMPs areinvolved in the regulation of intraocular pressure (IOP)in normal and glaucoma human eyes. Since, PGF2a andits analogs are widely used to lower IOP, it is importantto understand PGF2a-induced signaling mechanisms.Recently, we have shown that PGF2a activates p42/p44MAP kinase through a PKC-dependent pathway inCISM cells [4]. However, their up-stream regulationremained unclear in these cells. To answer these ques-tions, CISM cells were stimulated with 1 lM PGF2a forvarious times of incubation and p42/p44 MAP kinaseactivity was measured by in-gel renaturation kinase as-say as described in Materials and methods. As shown inFig. 5A PGF2a stimulates p42/p44 MAP kinase activityin a time-dependent manner. There is a significant in-crease in the activity of p42/p44 MAP kinase after0.5min of PGF2a treatment, which was sustained upto5min. To establish a correlation between PLC-c1 andp42/p44 MAP kinase, cells were stimulated with PGF2a

in the absence or presence of protein tyrosine kinaseinhibitors and MAP kinase activity was measured. Asshown in Fig. 6A, PGF2a-induced p42/p44 MAP kinaseactivation was significantly inhibited in the presence of1 lM of genistein, herbimycin A, and tyrphostin A-25.Furthermore, PGF2a-induced p42/p44 MAP kinase ac-tivation was completely inhibited in the presence of10 lM of these compounds (data not shown). A specificinhibitor of p42/p44 MAP kinase, PD-98059, also in-hibited PGF2a-induced p42/p44 MAP kinase activation.To rule out the possibility of variation in the amounts ofloaded protein samples on the gels, protein were trans-ferred to nitrocellulose membranes and immunoblottedwith anti-p42/p44 MAP kinase antibodies. All samplescontained comparable amounts of p42/p44 MAP kinase(Figs. 5B and 6B).

Discussion

Glaucoma is a group of human disorders character-ized by progressive loss of retinal ganglion cells with anassociated loss of vision. Derivatives of prostaglandinF2a (i.e., activate the FP-prostanoid receptors) are themost potent and effective topical ocular hypotensiveagents currently known. Physiological studies in ani-mals, normal subjects [11,12], and glaucoma patients[13] indicate a remarkable reduction in intraocularpressure (IOP) in the presence of PGF2a and its analoglatanoprost (PhXA41). The underlying molecularmechanisms of action of anti-glaucoma drugs and cau-ses of glaucoma are still unknown. It is important toinvestigate signaling mechanisms induced by anti-glau-coma drugs such as PGF2a in ocular smooth muscle,which could shed more light on the molecular mecha-nisms underlying the IOP-lowering effects of PGF2a inglaucoma patients.

The major finding of this study is that PGF2a stim-ulates tyrosine phosphorylation of PLC-c1 (Figs. 2 and3). This is the first study to show a relationship betweenPGF2a-stimulated tyrosine phosphorylation of PLC-c1and p42/p44 MAP kinase in CISM cells. Both PLC-c1phosphorylation and p42/p44 MAP kinase activationwere inhibited in the presence of protein tyrosine kinaseinhibitors (Figs. 4 and 6). In addition, data also suggestthat G-protein-coupled receptors such as the FP-recep-tors, which have been thought to activate only PLC-b[14], can also stimulate PLC-c1 in CISM cells. Several

Fig. 5. Dose dependence of PGF2a on p42/p44 MAP kinase activation.

Starved CISM cells were incubated in the absence (control) or presence

of 1 lM PGF2a for indicated time periods. (A) p42/p44 MAP kinase

activity as described in Materials and methods, (B) Western blot

analysis by anti-p42/p44 MAP kinase antibodies. Results given are

from one experiment that is a representative of five separate experi-

ments (n ¼ �5).

Fig. 6. Effects of protein tyrosine kinase inhibitors on PGF2a-stimu-

lated p42/p44 MAP kinase activation. Starved CISM cells were incu-

bated with 1 lM PGF2a in the absence or presence of 1lM genistein,

tyrphostin A-25, herbimycin A, or PD-98059. Inhibitors were added

15min prior to the addition of PGF2a. (A) p42/p44 MAP kinase ac-

tivity as described in Materials and methods, (B) Western blot analysis

by anti-p42/p44 MAP kinase antibodies. Results given are from one

experiment that is a representative of five separate experiments (n ¼ 5).

S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107 1105

receptors that stimulate PLC are thought to act viaG-proteins [15], because their activation of PLC ismodulated by bacterial toxins, aluminum fluoride, andanalogs of GTP, which are known to modify the actionsof G-proteins.

At present, the biochemical link between the FP-re-ceptors and tyrosine phosphorylation of PLC-c1 is notknown in CISM cells. The tyrosine phosphorylation hasbeen reported for growth factor receptors that arethemselves kinases such as PDGF and epidermal growthfactors [16,17]. However, tyrosine phosphorylation hasalso been reported for receptors that lack kinase activity[18–20]. Previous studies have shown that the actions ofPGF2a on inositol-1,4,5-trisphosphate (IP3) productionand Ca2þ-mobilization, and contraction were inhibitedby protein tyrosine kinase inhibitors in cat iris sphinctersmooth muscle [14]. Angiotensin II stimulated PLC-c1tyrosine phosphorylation by 4.5-fold at 0.5min in rataortic vascular smooth muscle cells [21]. Palmier et al.[22] reported that orthovanadate, a potent phosphataseinhibitor, mediated an increased generation of inositolphosphates and contraction in rat myometrium throughphosphorylation and activation of PLC-c1. Gould et al.[23] reported that genistein inhibited both contractionand Ca2þ-mobilization induced by activation of recep-tors for histamine in porcine carotid arterial smoothmuscle.

In conclusion, the data obtained suggest that PGF2a-induced transient tyrosine phosphorylation of PLC-c1.Furthermore, data of protein tyrosine kinase inhibitorssuggest an important role of protein tyrosine kinase inPGF2a-induced phosphorylation and activation of PLC-c1. One or more intracellular tyrosine kinases must bestimulated by the PGF2a–FP-receptor interaction.Whichinitiates a signaling cascade and leads to the activation ofprotein kinases including p42/p44 MAP kinase. The un-derstanding of the signaling mechanisms of actionof PGF2a in the smooth muscle of iris-ciliary body couldlead to the development of rational and more effectiveanti-glaucoma drugs.

Acknowledgment

A Combined Intramural Research Grant awarded to S. Husain

from Medical College of Georgia supported this work.

References

[1] R.A. Coleman, W.L. Smith, S. Narumiya, International union of

pharmacology classification of prostanoid receptors: Properties,

distribution, and structure of the receptors and their subtypes,

Pharmacol. Rev. 46 (1994) 205–229.

[2] L.Z. Bito, J. Stjernschantz, B. Resul, O.C. Miranda, S. Basu, The

ocular effects of prostaglandins and the therapeutic potential of a

new PGF2a analog, PhXA41 (latanoprost), for glaucoma man-

agement, J. Lipid Mediators 6 (1993) 535–543.

[3] B.W. Griffin, P.E. Magnino, I.-H. Pang, N.A. Sharif, Pharmaco-

logical characterization of an FP prostaglandin receptor on rat

vascular smooth muscle cells (A7r5) coupled to phosphoinositide

turnover and intracellular calcium mobilization, J. Pharmacol.

Exp. Ther. 286 (1998) 411–418.

[4] S. Husain, A.A. Abdel-Latif, Effects of PGF2a and carbachol on

MAP kinases, cytosolic phospholipase A2 and arachidonic acid

release in cat iris sphincter smooth muscle cells, Exp. Eye Res. 72

(2001) 581–590.

[5] H.R. Ansari, S. Husain, A.A. Abdel-Latif, Role of p42/p44 MAP

kinases and calcium-dependent calmodulin kinases II in PGF2a,

ionomycin, and thapsigargin-induced muscle contraction in cat

iris sphincter smooth muscle, J. Pharmacol. Exp. Ther. 299 (2001)

178–186.

[6] S.G. Rhee, K.D. Choi, Regulation of inositol phospholipid-

specific phospholipase C isozymes, J. Biol. Chem. 267 (1992)

12393–12396.

[7] A.V. Smarka, J.R. Hepler, K.O. Brown, P.C. Sternweis, Regula-

tion of polyphosphoinositide-specific phospholipase C activity by

purified Gq, Science 251 (1991) 804–807.

[8] S.J. Taylor, H.Z. Chae, S.G. Rhee, J.H. Exton, Activation of the

b1 isozyme of phospholipase C by alpha subunits of the Gq class

of G-proteins, Nature 350 (1991) 516–518.

[9] H.K. Kim, J.W. Kim, A. Zilberstein, B. Margolis, C.K. Kim, J.

Schlesinger, S.G. Rhee, PDGF stimulation of inositol phospho-

lipid hydrolysis requires PLC-c 1 phosphorylation on tyrosine

residues 783 and 1254, Cell 65 (1991) 435–441.

[10] S. Husain, A.A. Abdel-Latif, Role of protein kinase C-a in

endothelin-1 stimulation of phospholipase A2 and arachidonic

acid release in cultured cat iris sphincter smooth muscle cells,

Biochim. Biophys. Acta 1392 (1998) 127–144.

[11] J.R. Kerstetter, R.F. Brubaker, S.E. Wilson, L.J. Kullerstrand,

Prostaglandin F2a-1-isopropylester lowers intraocular pressure

without decreasing aqueous flow, Am. J. Ophthalmol. 105 (1988)

30–34.

[12] P.Y. Lee, H. Shao, L. Xu, C.-K. Qu, The effect of prostaglandin

F2a on intraocular pressure in normotensive human subjects,

Invest. Ophthalmol. Vis. Sci. 29 (1988) 1474–1477.

[13] J. Stjernschantz, G. Selen, B. Sjoquist, B. Resul, Preclinical

pharmacology of latanoprost, a phenyl-substituted PGF2a analog.

Ad. in prostaglandin, Thrombox. Leukotr. Res. 23 (1995) 513–

518.

[14] S.Y.K. Yousufzai, A.A. Abdel-Latif, Tyrosine kinase inhibitors

suppress prostaglandin F2a-induced phosphoinositide hydrolysis,

Ca2þ elevation and contraction in iris sphincter smooth muscle,

Eur. J. Pharmacol. 360 (1998) 185–193.

[15] S.G. Rhee, K.D. Choi, Multiple forms of phospholipase C

isozymes and their activation mechanisms, Adv. Second Messen-

ger-phosphoprotein Res. 26 (1992) 35–61.

[16] B. Margolis, S.G. Rhee, S. Felder, M. Mervie, R. Lyall, A.

levitzki, A. Ullrich, A. Zilberstein, J. Schlessinger, EGF induces

tyrosine phosphorylation of phospholipase C-II: a potential

mechanism for EGF receptor signaling, Cell 57 (1989) 1101–

1107.

[17] D.A. Kumjian, A. Barnstein, S.G. Rhee, T.O. Daniel,

Phospholipase C-c complexes with ligand-activated platelet-

derived growth factor receptors. An intermediate implicated in

phospholipase activation, J. Biol. Chem. 266 (1991) 3973–

3980.

[18] T. Tsuda, Y. Kawahara, K. Shii, M. Koide, Y. Ishida, M.

Yokoyama, Vasoconstrictor-induced protein-tyrosine phosphory-

lation in cultured vascular smooth muscle cells, FEBS Lett. 285

(1991) 44–48.

[19] D.J. Park, H.W. Rho, S.G. Rhee, CD3 stimulation causes

phosphorylation of phospholipase C-c1 on serine and tyrosine

residues in a human T-cell line, Proc. Natl. Acad. Sci. USA 88

(1991) 5453–5456.

1106 S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107

[20] R.H. Carter, D.J. Park, S.G. Rhee, D.T. Fearon, Tyrosine

phosphorylation of phospholipase C induced by membrane

immunoglobulin in B lymphocytes, Proc. Natl. Acad. Sci. USA

88 (1991) 2745–2749.

[21] M.B. Marrero, W.G. Paxton, J.L. Duff, B.C. Berk, K.E. Bern-

stein, Angiotensin-II stimulates tyrosine phosphorylation of PLC-

c1 in vascular smooth muscle cells, J. Biol. Chem. 269 (1994)

10935–10939.

[22] B. Palmier, D. Leiber, S. Harbon, Pervanadate mediated an

increased generation of inositol phosphates and tension in rat

myometrium. Activation and phophorylation of PLC-c1, Biol.Reprod. 54 (1996) 1383–1389.

[23] E.M. Gould, C.M. Rembold, R.A. Murphy, Genistein a tyrosine

kinase inhibitor reduces Ca2þ-mobilization in swine carotid artery,

Am. J. Physiol. 268 (1995) 1425–1429.

S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107 1107