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Activation of phospholipase D by endothelin-1 and other pharmacological agents in rabbit iris sphincter smooth muscle
Cellular Signalling Vol. 4, No. 6, pp. 777-786, 1992. Printed in Great Britain.
0898--6568/92 $5.00 + 0.00 © 1992PergamonPressLtd
ACTIVATION OF PHOSPHOLIPASE D BY ENDOTHELIN-1 AND OTHER PHARMACOLOGICAL AGENTS IN RABBIT IRIS SPHINCTER SMOOTH MUSCLE YAWEN ZHANG and ATA A. ABDEL-LATIF* Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100, U.S.A.
(Received 17 July 1992; and accepted 10 August 1992) Almtraet--The stimulation of phospholipase D (PLD) activity by endothelin-1 (ET1) was investigated in rabbit iris sphincter prelabelled with [3H]myristic acid. In the presence of 0.5% ethanol, ETI caused a time- and dose-dependent increase in the production of [3H]phosphatidylethanol ([3H]PEt). Within 30 s the peptide increased PEt formation by 30% and after 5 min increased it by 140%. The Ecs0value for ETl-stimulated PEt formation was found to be 30 nM. This value is appreciably lower than the Ecs0 we previously obtained for ETl-induccd inositol trisphosphate production (45 nM), but considerably higher than that for arachidonic acid release (1 nM). PEt formation was significantly stimulated by prostaglandin F20, phorbol 12,13-dibutyrate (PDBu), chloroform, A23187 and A1F~', but it was not affected by carbachol or the platelet-activating factor. PDBu-stimulated PEt formation was blocked by staurosporine and it was not potentiated by A23187. Staurosporine had no effect on ETl-stimulated PEt formation. Our data indicate that ET1 stimulation of PLD occurs independently of protein kinase C activation, phospholipase C activation and intraccllular Ca2+ mobilization, and phospholipase A2 activation. In this tissue the ET1 receptor is probably coupled to the three phospholipases through several G-proteins, and this appears to be species and receptor type specific.
Key words: Rabbit iris sphincter, smooth muscle, endothelin-1, phospholipase D, phosphatidylcholine, phosphatidylethanol, protein kinase C, chloroform, phorbol ester. INTRODUCTION
lying the ETl-induced responses are not well understood. In smooth muscle, ETl-induced contractile responses are associated with stimulation of phosphoinositide hydrolysis, via phospholipase C (PLC), and elevation of intracellular Ca 2+ in vascular smooth muscle cells [6-12], rat aorta [13-16], rat tracheal smooth muscle [17], porcine coronary artery [18] and rabbit iris sphincter [19]. In addition, ET1 has been reported: (i) to stimulate phospholipase A2 (PLA2) and liberate arachidonic acid, from membrane phospholipids, for eicosanoid biosynthesis, in cultured vascular smooth muscle cells [20,21] and rabbit iris sphincter [22]; and (ii) to stimulate phospholipase D (PLD) to produce phosphatidic acid (PA) from phosphatidylcholine (PC), which in the presence of ethanol can be measured as phosphatidylethanol (PEt), and free choline in vascular smooth muscle cells [23-25] and rat aorta [26].
E1',-oo'rr~L~- 1 (ET1) is a 21-amino acid peptide isolated from vascular endothelial cells and shown to be the most potent vasoconstrictor known so far [1]. Several studies have demonstrated that in addition to its vasoconstrictive effects, ET1 possesses various pharmacological activities in both vascular and nonvascular tissues (for reviews see [2-5]). The peptide elicits biological responses by binding to specific cell surface receptors on the plasma membrane of the cell. The biochemical mechanisms under-
* Author to whom correspondence should be addressed. Abbreviations: BSA--bovine serum albumin; CCh--car-
bachol; DAG--l,2-diacylglycetol; ETl--cndothelin-1; IP~--inositol 1,4,5-trisphosphate; PA--phosphatidic acid; PC--phosphatidylcholine; PEt--phosphatidylethanoi; PDBu-phorbol 12,13-dibutyrate; PLA2--phospholipase A2; PLC--phospholipase C; PLD--phospholipas¢ D. 777
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YAWEN ZHANG
and A. A. ABDEL-LATIF
Activation o f these phospholipases may also lead to the stimulation of protein kinase C (PKC). The role of P L D in cell signalling and its relationship to phospholipases A2 and C has yet to be defined (for reviews see [27-29]). In the iris sphincter, ET1 is a potent agonist for the production of inositol trisphosphate (IP3) and 1,2-diacylglycerol (DAG) from polyphosphoinositides in rabbit, dog, cat and pig, and for cAMP formation in rabbit, dog, cat, pig, bovine, monkey and human [19], ET1 also stimulated the release o f arachidonic acid and prostaglandins, through activation of PLA2, in the rabbit iris sphincter [22]. In the past few years the agonist-induced stimulation o f PLD has emerged as an important mechanism in cellular signalling in a wide variety of tissues [27-29]. The mechanism that underlies this agonist-induced P L D activation is not well understood. In the studies reported here we have investigated the effects o f ET1 and other pharmacological agents on P L D activity in the rabbit iris sphincter. We find that ET1, as with its actions on PLC [19] and PLA2122], is a potent activator of PLD activity in this tissue. The ET1 effects are independent o f PKC and Ca 2÷ mobilization. MATERIALS AND METHODS
Materials [3H]Myristic acid (39.3 Ci/mmol) was purchased from New England Nuclear (Boston, MA) and myo-[3H]inositol (80--120 Ci/mmol) from Amersham Corp. (Arlington Heights, IL). Endothelin-I (human, porcine) was purchased from Peptides International (Louisville, KY). PEt and PA were purchased from AVANTI Polar Lipids, Inc., Alabaster, AL. Phorbol esters, carbachol (CCh) and other pharmacological agents were obtained from Sigma Chemicals Co. (St Louis, MO). All other chemicals were of reagent grade. Preparation of iris sphincter muscle Rabbit eyes packed in ice were obtained from a local abattoir. The iris sphincter muscle was dissected out and placed in modified Krebs-Ringer bicarbonate buffer of the following composition (in raM): NaCI, 118; NaHCO3, 25; KCI, 4.7; KH2POo 1.2; MgSOo 1.2; CaCI2, 1.25; D-glucose, 10; and 1 /~M
indomethacin. The pH of the buffer was adjusted and maintained at 7.4 with 97% 02-3% CO2. In general, of the pair one sphincter served as a control and the other as experimental.
Incubation of sphincter muscle with [3H]myristic acid and assay for PLD activity PLD is known to catalyse the transfer of phosphatidyl groups to various acceptors; in the presence of an alcohol, this transphosphatidylation produces phosphatidylalcohol [30]. The production of PEt is considered a good indication of PLD activity. The assay for PLD activity was as described by Liscovitch and Amsterdam [30]. Briefly, the paired sphincter strips were incubated for 90 min at 37°C in 1 ml Krebs-Ringer bicarbonate buffer containing 0.05% bovine serum albumin (BSA) and 5/~Ci [3H]myristic acid. At this time the sphincters were washed four times with 3 ml buffer that contained 0.05% BSA. The sphincters were preincubated singly in I ml buffer containing 0.5% (v/v) ethanol at 37°C for 5 min. The agonist was then added as indicated and incubation continued for an additional 5 min. Reactions were terminated by addition of 2 ml of icecold chloroform-methanol (1:2, v/v). Phospholipids were extracted according to the procedure of Bligh and Dyer [31]. The phospholipids in the lipid extract were separated by one-dimensional thin layer chromatography (TLC) using silica gel K6 plates (Whatman) and a solvent system consisting of the organic phase of ethylacetate--isooetane--acetic acidwater (13:2:3:10, by volume). Individual lipids were localized by iodine staining and identified by comigration with standards. The spots corresponding to PEt and PA were scraped into scintillation vials and the 3H-radioactivity measured by liquid scintillation counting. Data are expressed as d.p.m.//zmol lipid phosphorus. Incubation of sphincter muscle with myo[aH]inositol, [3H]myristic acid and analysis of inositol phosphates and PEt To double label the tissue with myo[3H]inositol and [3H]myristic acid, the paired sphincter strips were incubated for 90 min at 37°C in l ml Krebs-Ringer bicarbonate buffer containing 10 #Ci of myo[3H]inositol and 5/~Ci [aH]myristic acid. At this time the sphincters were washed four times with 3 ml non-radioactive Krebs-Ringer bicarbonate buffer and then suspended individually in 1 ml fresh non-radioactive buffer. LiCI (10mM, final concentration) was added to each incubation and 10 rain later the agonists were added and incubations continued for the time indicated. The incubations were terminated by the addition of l ml 10% (w/v)
Endothelin and phospholipases in iris sphincter TABLE 1. PREFERENTIAL INCORPORATION OF [3H]MYRISTIC ACID INTO PHOSPHATIDYLCHOLINE IN IRIS SPHINCTER
3H-Radioactivity (% of total)
Phospholipids PA CDP-DAG Phosphatidylinositol Polyphosphoinositides Phosphatidylserine Phosphatidylethanolamine PC
1.68_ 0.02 0.20_ 0.01 2.38 __0.40 0.18 ± 0.03 0.31 + 0.03 9.60 + 0.90 85.0+ 1.16
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Rabbit iris sphincters were incubated in Krebs-Ringer bicarbonate buffer containing [3H]myristic acid (5 pCi/ml) for 90 min at 37°C. Phospholipids were extracted and then separated by means of two-dimensional TLC as described previously [32]. Total 3H-incorporated was 3.53 x l0 s d.p.m./nmol lipid P. The data represent means +S.E.M. from three separate experiments.
trichloroacetic acid. The tissues were homogenized, centrifuged and the supernatant extracted with 4 x anhydrous diethyl ether. The precipitate was extracted with chloroform-methanol-HC i (300:300:1.5, by volume) and PEt separated from the lipid extract by one-dimensional TLC [30]. Phospholipids were separated by means of twodimensional TLC as previously described [32]. The water-soluble tissue extract was neutralized to pH 7.0 with NaOH and myo[3H]inositol phosphates were analysed by anion exchange chromatography using Biorad 1 x 8 resin (formate form, 200--400 mesh) as described previously [33].
Calculations and statistical analysis of the data To correct for variation in tissue size, the data for phospholipids and [3H]inositol phosphates were normalized to tissue lipid phosphorus. Data are expressed as the mean + S.E.M. RESULTS
Preferential incorporation of [3H]myristic acid into PC of iris sphincter In order to investigate the hormonal activation of PC hydrolysis, we first carried out experiments on the preferential radiolabelling of this phospholipid using [3H]myristic acid, [~H]oleic acid and [3H]arachidonic acid as
Time (Minutes)
FIG. 1. Time-course of ETl-induced [3H]PEt formation in rabbit iris sphincter. Muscles were prelabelled with [3H]myristic acid (5pCi/ml) in Krebs-Ringer bicarbonate buffer for 90 rain. The labelled muscles were washed with buffer and incubated in buffer containing 0.5*/0 ethanol with or without 0.1/aM ETI for the indicated times. [3H]PEt was analysed by one-dimensional TLC as described under Materials and Methods. The basal level of [3H]PEt formation was 2720+167d.p.m./pmol lipid P. Each point represents mean+S.E.M, that were obtained from four experiments, each run in triplicate.
precursors. We found [3H]myristic acid to be the most effective precursor for selectively labelling PC. Table I shows the radioactivity profiles of the various iris sphincter phospholipids after radiolabelling with [3H]myristic acid. Under basal conditions, i.e. in the absence of ET1 and ethanol, about 85% of the total radioactivity was detected in PC, and this was followed by 9.6% in phosphatidylethanolamine and 2.4% in phosphatidylinositol. Therefore for the PEt and PA assays in the present work we used [3H]myristic acid to label the iris sphincter phospholipids.
Time-course of ETl-induced [3H]PEt formation ETI stimulated PEt formation in a timedependent manner. ET! (0.1pM) induced a 30% increase in PEt formation by 30 s, reached a 140% increase within 5 min and plateaued by 10 min after addition of the agonist (Fig. 1). These data demonstrate that ET1 rapidly
780
YAWEN ZHANG and A. A. ABDEL-LATIF
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FIG. 2. Ethanol dependence of ETl-stimulated [ill]PEt formation in iris sphincter. Rabbit iris sphincters were prelabelled with [3H]myristic acid, washed with buffer, and then incubated in the presence of increasing concentrations of ethanol with and without 0.1 pM ET1 for 5 rain. [~I-I]PEt and [3H]PA were analysed by one-dimensional TLC as described under Materials and Methods. Basal levels of [3H]PA and [3H]PEt, in the absence of ethanol, were 11,950+950 and 1320+200 d.p.m.//anol lipid P, respectively. Each point represents mean+S.E.M, from four to five experiments, each run in triplicate.
stimulates, through activation of PLD, PEt formation in this tissue. Based on these findings, we employed 5-min incubations with the agonists in the experiments described below.
Ethanol dependence of ETl-stimulated [3H]PEt formation The effect of ethanol concentration on ETl-stimulated PET formation is shown in Fig. 2. ETl-stimulated PEt formation is completely dependent on the presence of ethanol. Ethanol, at 25mM, increased the ETl-induced PEt formation by 100%. Maximal stimulation, 320% increase, was obtained at about 85 mM (0.5% ethanol). PA production was significantly attenuated in the presence of ethanol. This is due to the competition between ethanol and water for the phosphatidyl group of PC. Higher concentrations of ethanol were inhibitory (Fig. 2). In the following experiments we employed an ethanol concentration of 0.5%.
Concentration dependence of ETl-stimulated [3H]PEt and [3H]PA formation The concentration dependence of ETIstimulated PEt and PA formation is shown in Fig. 3. ETl-stimulated PEt and PA formation were dose dependent with ECs0values of 3 x I 0"s and 2.6x 10"SM, respectively. The maximal
increases for PEt and PA formation due to 1/~M ET1 were 215 and 50%, respectively.
Effects of Ca2+-mobilizing agonists, phorbol esters and other pharmacological agents on [3H]PEtformation PLD can be activated by a variety of agonists in various tissues. The effects of typical concentrations of various agonists and phorbol esters on PEt formation are shown in Table 2. ET1 was the most potent Ca2+-mobilizing agonist. ETI increased PEt formation by 222% and prostaglandin F2= increased it by about 84% over that of the control. In contrast, CCh and the platelet-activating factor had no effect on the PLD activity. The iris sphincter is enriched with muscarinic receptors and CCh is a potent activator of PLC activity and contraction in this tissue [32,33]. The platelet-activating factor stimulates PLA 2, but not PLC, in the rabbit iris [34]. Further comparative studies on the effects of ETI and CCh on PLD and PLC activities were carried out in iris sphincter which was double labelled with [3H]myristic acid and [3H]inositol. ETI increased PEt formation and IP3 production by 176 and 252%, respectively, and CCh increased their formation by 9 and 109%, respectively (Fig. 4). These data suggest that in
Endothelin and phospholipases in iris sphincter
781
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FIG. 3. Concentration dependence of ETl-stimulated [3H]PEt and [3H]PA formation in rabbit iris sphincter. Rabbit iris sphincters were prelabelled with [3H]myristic acid for 90 min, washed with buffer, then exposed to different concentrations of ETI for 5 min. [3H]PEt and [3H]PA were analysed by means of one-dimensional TLC as described under Materials and Methods. Each point represents mean +_S.E.M. from two experiments, each run in triplicate. the rabbit iris sphincter muscarinic receptors are coupled to the activation of PLC but not to that of PLD. In addition, they provide evidence that the P L C and P L D pathways are dissociated.
Phorbol esters have been shown to cause stimulation of P L D in various tissues, suggesting a role for P K C in the regulation of P L D activity [35-38]. PDBu increased PEt formation in the iris sphincter by 194%, while
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FIG. 4. Comparative effects of ETI and CCh on [3H]PEt formation and [3H]IP~ production in iris sphincter. Rabbit iris sphincters were double labelled with [3H]myristic acid and [3H]inositoi, washed with buffer, and then incubated without or with ET! (0. i/JM) and CCh (25/~M) as indicated for 5 rain, The reactions were stopped with TCA and [3H]PEt and [3H]IP3 were analysed by TLC and ion-exchange chromatography, respectively, as decribed under Materials and Methods. The results are means _+S.E.M. from two experiments, each run in triplicate.
782
YAWEN ZHANG and A. A. ABDEL-LATIF
TABLE 2.
EFFECTS OF Ca2+-MOBILIZING AGONISTS, PHORBOL ESTERS AND OTHER PHARMACOLOGICAL AGENTS ON PEt FORMATION IN IRIS SPHINCTER
Addition ET1 Prostaglandin F2, CCh Platelet-activating factor PDBu 4~-Phorbol 4~-Phorbol 12,13-didecanoate A23187 PDBu (0.I /~M)+A23187 (1 vM) AIF~-
Concentration (/zM)
[3H]PEt (% of control)
0.1 (0.01-1)* 0.1 (0.01-1) 25 (1-50) 0.1 (0.001-1 ) 0.1 (0.01-0.1) 0.1 (0.01-0.1) 0.01 (0.01-0.1) I (0.1-10)
322+26 184+ 10 1144- 9 i 05 _+ 6 294+ I l 109+ 4 1034- 5 1754- 10 3104- 15
10 #M AICI3+ 10 mM NaF
1744- 4
Rabbit iris sphincters were prelabeUed with [3H]myristic acid, washed with buffer and then incubated in buffer containing 0.5% ethanol in the absence and presence of the agonist for 5 min. Jail]PEt in the lipid extract was analysed by means of one-dimensional TLC as described under Materials and Methods. The average ~H-radioactivity recovered in basal PEt was 2650+185d.p.m.//tmol total lipid P. The data represent means+ S.E.M. from two separate experiments, each run in triplicate. * Range of concentrations of agonists investigated. the biologically inactive analogue, 4~-phorbol and 4~-phorbol 12,13-didecanoate, exerted no effect on PEt formation. These data suggest a role for PKC in this tissue. The Ca2+-ionophore A23187 increased PEt formation by 65%. However, the ionophore was without effect on PDBu-induced PEt formation (Table 2). This could suggest that PEt formation stimulated by PDBu is Ca 2+ independent. A1F4-induced a 74% increase in PEt formation. This is in accordance with the work of others who have reported that P L D is regulated by G-proteins [28].
Effects of staurosporine on ET1- and PDBu-induced [3H]PEtformation To determine whether or not PKC is involved in PLD activation by ETI we investigated the effects of staurosporine, a potent inhibitor o f PKC, on ETI- and PDBu-induced PEt formation. Staurosporine had no effect on ETl-stimulated PEt formation, but it completely blocked the PDBu stimulation (Table 3). This demonstrates that ETl-stimulation of PLD activity is independent of PKC.
Differential effects of PDBu on ETl-induced formation of [3H]PEt and production of [3H]IP3 Further evidence that in this tissue the PLD and PLC pathways are dissociated is supported by the studies on the effects of PDBu on ETl-induced formation of PEt and IP 3 in iris sphincter which was double labelled with [3H]myristic acid and [3H]inositol. PDBu increased PEt formation by 213%, but it had no effect on IP 3 production (Table 4). In addition, while the stimulatory effects of PDBu on the production of PEt and IP 3 were additive, IP 3 production due to ETI was significantly attenuated in the presence of the phorbolester.
Dose-response effects of chloroform on [3H]PEt formation Sphingosine has recently attracted considerable attention because it inhibits the activity of PKC (for reviews see [39,40]). Sphingosine has also been reported to activate PLD in neuralderived N G 108-15 cells [41]. We found that when we dissolved the sphingolipid in ethanol,
Endothelin and phospholipases in iris sphincter TABLE 3. EFFECTS OF STAUROSPORINE ON E T 1 - AND PDBu-INDUCED
783
[3H]PEtFORMATION IN IRIS SPHINCTER
[3H]PEt (d.p.m./#mol of total lipid P)
% of control
None ETI (0.1 #M) ET 1 (0.1 #M) + staurosporine (5 #M)
2950 +_105 8850+ 150 9766 _ 164
100 300 331
None PDBu (0.1 #M) PDBu (0.1 /tM)+staurosporine (5 #M)
2620+ 110 8096+ 135 2710+95
!00 309 103
Addition Experiment 1
Experiment 2
Rabbit iris sphincters were prelabelled with [3H]myristic acid, washed with buffer, then pretreated with staurosporine for 10 min as indicated. The labelled tissues were then incubated in buffer containing 0.5% ethanol in absence and presence of ETI and PDBu for 5 and 15 min, respectively. The phospholipids were extracted and [3H]PEt separated by one-dimensional TLC and analysed as described under Materials and Methods. The data represent means + S.E.M. from two separate experiments, each run in triplicate. isopropanol, or dimethyl sulphoxide (DMSO) there was no significant stimulatory effect on P L D activity (data not shown). However, when sphingosine was dissolved in chloroform we found an appreciable amount of stimulation of P L D activity. This observation prompted us to investigate the dose-response effects of chloroform on PEt formation. Chloroform increased PEt formation in a dose-dependent manner (Fig. 5). Half maximal stimulation of PEt occurred at 30 mM, and the stimulatory effects of chloroform levelled off between 75 and 125 mM. Maximal stimulation (310% increase)
was reached at 75 mM chloroform. At 5-10 #M staurosporine had no inhibitory effect on chloroform stimulation o f PEt formation, suggesting that PKC is not involved in the mechanism of P L D activation (data not shown). This is in contrast to rabbit platelets where chloroform (250mM) was reported to activate PKC activity [42]. DISCUSSION Previously, we reported that in the rabbit iris sphincter ET1 is a potent agonist for: (i) PLC
TABLE 4. DIFFERENTIAL EFFECTS OF P D B u ON ETI-INDUCED [3H]PEtFORMATION AND [3H]IP3 PRODUCTION IN IRIS SPHINCTER
Addition ETI (0.1 /~M) PDBu (0.1 #M) ETI (0.1 #M)+PDBu (0.1 /~M)
[3H]PEt (% of control)
[3H]IP3 (% of control)
339+23 313_ 17 529±28
379_+ 15 108+_ il 199_+ 13
Rabbit iris sphincters were double labelled with [3H]myristic acid and [3H]inositol, washed with buffer, and then preincubated with PDBu (0.1 gM) in buffer containing 0.5% ethanol for 15 rain as indicated. ETI was then added and after 5 min the reactions were stopped with TCA. [3H]PEt and [3H]IP3 were analysed by means of one-dimensional TLC and ion-exchange chromatography, respectively, as described under Materials and Methods. 3H-Radioactivity recovered in basal [3H]IP3 was 3715_+308 d.p.m./mg protein. The results are means_+S.E.M, from four to five experiments, each run in triplicate.
784
YAWEN ZHANG and A. A. ABDEL-LATIF 400-
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300-
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~_.~200100"
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FIG. 5. Dose-response effects of chloroform on [3H]PEt formation in iris sphincter. Rabbit iris sphincters were prelabeUed with [3H]myristic acid, washed with buffer, then exposed to different concentrations of chloroform for 5 rain. Each point represents mean_ S.E.M. from two experiments, each run in triplicate. activation, IP3 production, muscle contraction and cAMP formation [19], and (ii) PLA2 activation, arachidonic acid release and prostaglandin synthesis [22]. In the present study, we have shown that ETI is also a potent agonist for PLD activation in this tissue. In the presence of ethanol, ET1 stimulates PLD to hydrolyse [3H]myristate PC in the iris sphincter to produce [all]PEt. Maximal stimulation of PLD due to 1/~M ET1 was obtained at about 85 mM (0.5% ethanol). This concentration of ethanol is optimum for studies on agonist-induced PEt formation in other systems [43]. Under basal conditions about 85% of the incorporated [~H]myristic acid was recovered in PC (Table 1). The peptide stimulated PEt formation in a time- and dose-dependent manner. Within 30 s ETI increased PEt formation by 30% and after 5 min it increased it by 140% (Fig. 1). ETl-stimulated PEt formation is dose-dependent with an ECs0of about 30 nM (Fig. 3). This value is appreciably lower than the Ecs0 we reported previously for ETl-induced IP3 production (45nM) [19], but considerably higher than that for ETI-induced arachidonic acid release (1 nM) [22]. Thus, in the rabbit iris sphincter the ECs0values for ETi-stimulation of PLA2, PLC and PLD are I, 45 and 30nM, respectively. The ECs0 for ETI-induced cAMP
formation in the bovine iris sphincter is 28 nM [19]. The ECs0 for ETl-induced contraction in rabbit iris sphincter is 46 nM [19]. These multi effects of ET1 on the second messenger systems in the iris sphincter could explain our previous observation that as compared to the rapid action of CCh, the contractile response to ETI in this tissue has a slow onset and is sustained for 6-20 min [19]. The maximal increase in PEt and PA formation due to 1/zM ETI was 215 and 50%, respectively (Fig. 3). This finding, coupled with the observation that PEt formation due to ET1 increases with ethanol concentration, while that of PA decreases (Fig. 2), suggests that the peptide activates PLD to hydrolyse PC into PA and free choline. Therefore, both hydrolysis of PC into PA and free choline and transphosphatidylation to produce PEt occur in the presence of ethanol. In several tissues, PLD activity may be induced by PKC activators, such as phorbol esters [35-38,44,45] and Ca 2+ ionophores [44,45]. Reinhold et al. [45] postulated that three separable but interacting routes lead to the activation of PLD: (i) direct activation by PKC in cells stimulated with phorbol esters; (ii) Ca2+-dependent activation in cells treated with Ca 2+ ionophore; and (iii) an undefined route stimulated by receptor-dependent agonists. In the rabbit iris sphincter both direct activation of PKC and an increase in Ca 2+ influx by A23187 stimulated PLD activity (Table 2). PDBu increased PEt formation by more than 200% (Tables 2-5). As expected, the inactive stereoiosomers 4g-phorbol and 4~-phorboi 12,13-didecanoate were ineffective in activating PLD (Table 2). The finding that the PDBu effect was not potentiated by A23187 could indicate that PEt formation stimulated by PDBu is Ca 2+ independent. Staurosporine, a potent PKC inhibitor, blocked PDBu-but not ETl-stimulated PEt formation. These data suggest that in the rabbit iris sphincter: (i) PKC is involved in PDBu stimulation of PLD activity; and (ii) that ETI stimulation of PLD is independent of PKC activation. Chloroform, a tumour promoter and a potent activator of PKC in platelets [42], stimulated PEt formation
Endothelin and phospholipasesin iris sphincter in the iris sphincter in a dose-dependent manner (Fig. 5). However, the finding that the stimulatory effect of chloroform on PLD activity was not influenced by pretreatment of the muscle with staurosporine suggests that the chloroform stimulation occurs independently of PKC activation. It is possible that chloroform acts by perturbation of the phospholipids of the plasma membrane. ETl-induced PLD activation is not dependent on PLC activation and subsequent increase in intracellular Ca 2+. This conclusion is supported by the following findings in the present work: (i) in iris sphincter muscle which was double labelled with [3H]myristic acid and [3H]inositol, PDBu potentiated ETl-induced PEt formation but attenuated that of ETl-induced IP 3 production (Table 4). This suggests that an increase in intracellular Ca 2+ concentration by IP 3 is not involved in PLD activation; (ii) CCh, a potent agonist for PLC activation in the iris sphincter [32,33] had no effect on PEt formation (Table 2, Fig. 4). There is evidence that the human m l acetylcholine receptor is linked to the signal transduction mechanism of PLD activation, whereas the human M2 receptor interacts with a different G-protein which does not cause the activation of PLD or the formation of PEt [46]. The iris sphincter contains mainly m3 (about 90%) and small amounts of M2 receptor subtypes [47]. ET1 is a potent activator of PLA2 in the rabbit iris sphincter [22]. The platelet-activating factor, which is an activator of PLA 2 but not PLC in this tissue [34], had no effect on PLD activity (Table 2). This could suggest that ETl-induced PLD activation is not dependent on PLA2 activation in this tissue. There is accumulating experimental evidence which suggests that the membrane-bound PLD, as with PLC and PLA 2 is linked to cell surface receptors through GTP-binding proteins [2729,38,48-50]. In the rabbit iris sphincter, AIF~- (10#M), which interacts directly with G-proteins and activates both Gs and Gi [51], induced a 74% increase in PEt formation (Table 2). It is possible that in this tissue the ET1 receptor is coupled to PLD, PLC and
785
PLA2 through several G-proteins, and this appears to be species and receptor type specific. Acknowledgements--This work was supported by National Institutes of Health Grants R37-EY-04171 and EY-04387.
I. 2. 3. 4. 5.
6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19.
REFERENCES Yanagisawa M., Kurihara H., Kimura S., Tomobe Y., Kobayashi M., Mitsui Y., Goto K. and Masaki T. (1988) Nature 332, 411-415. De Gouville A.-C., Lipton H. L., Cavero I., Summer W. R. and Hyman A. L. (1989) Life Sci. 45, 1499-1513. Lovenberg W. and Miller R. C. (1990) Neurochem. Res. 15, 407-417. Rubanyi G. M. and Botelho L. H. P. (1991) FASEB J. 5, 2713-2720. Sakurai T., Yanagisawa M. and Masaki T. (1992) Trends pharmac. Sci. 13, 103-108. Marsden P. A., Danthuluri R., Brenner B. M., Ballermann B. J. and Brock T. A. (1989) Biochem. biophys. Res. Commun. 158, 86-93. Xuan Y.-T., Whorton A. R. and Watkins W. D. (1989) Biochem. biophys. Res. Commun. 160, 758-764. Griendling K. K., Tsuda T. and Alexander R. W. (1989) J. biol. Chem. 264, 8237-8240. Muldoon L. I., Rodland K. D., Forsythe M. L. and Magun B. E. (1989) J. biol. Chem. 264, 8529-8536. Sunako M., Kawahara Y., Hirata K.-I., Tsuda T., Yokoyama M., Fukuzaki H. and Taki Y. (1990) Hypertension 15, 84-88. Danthuluri N. R. and Brock T. A. (1990) J. Pharmae. exp. Ther. 254, 393-399. Resink T. J., Scott-Burden T. and Buhler F. R. (1990) Fur. J. Biochem. 189, 415-421. Topouzis S. and Miller R. C. (1989) Br. J. Pharmac. 98, 669-677. Auget M., Delaflotte S., Chabrier P. E. and Braquet P. (1989) Life Sci. 45, 2051-2059. Huang X.-N., Takanayagi I. and Hisayama T. (1990) Gen. Pharmac. 21, 893-898. Hay D. W. P. (1990) Br. J. Pharmac. I00, 383-392. Henry P. J., Rigby P. J., Self G. J., Preuss J. M. and Goldie R. G. (1992) Br. J. Pharmac. 105, 135-141. Kasuya Y., Takuwa Y., Yanagisawa M., Kimura S., Goto K. and Masaki T. (1989) Biochem. biophys. Res. Commun. 161, 1049-1055. Abdel-Latif A. and Zhang Y. (1991) Invest. Ophthal. vis. Sci. 32, 2432-2438.
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20. Resnick T. J., Scott-Burden T. and Buhler F. R. (1989) Biochem. biophys. Res. Commun. 158, 279-286. 21. Reynolds E. T., Mok L. S. and Kurokawa S. (1989) Biochem. biophys. Res. Commun. 160, 808-813. 22. Abdel-Latif A. A., Zhang Y. and Yousufzai S. Y. K. (1991) Curr. Eye Res. 10, 259-265. 23. Konishi F. and Inagami T. (1991) Biochem. biophys. Res. Commun. 179, 1070-1076. 24. Lassegue B., Alexander R. W., Clark M. and Griendling K. K. (1991) Biochem. J. 276, 19-25. 25. Welsh C. J., Schmeichel K., Cao H.-T. and Chabbott H. (1990) Lipids 25, 675-684. 26. Liu Y., Geisbuhler B. and Jones A. W. (1992) Am. J. Physiol. 2 6 2 , (Cell Physiol. 31), C941--C949. 27. Shukla S. D. and Halenda S. P. (1991) Life Sci. 48, 851-866. 28. Exton J. H. (1990) 3". biol. Chem. 266, 1-4. 29. Billah M. M. and Anthes J. C. (1990) Biochem. J. 269, 281-291. 30. Liscovitch M. and Amsterdam A. (1989) J. biol. Chem. 264, 11,762-11,767. 31. Bligh E. G. and Dyer W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917. 32. Abdel-Latif A. A., Akhtar R. A. and Hawthorne J. N. (1977) Biochem. J. 162, 61-73. 33. Howe P. H., Akhtar R. A., Naderi S. and Abdel-Latif A. A. (1986) J. Pharmac. exp. Ther. 239, 574-583. 34. Yousufzai S. Y. K. and Abdei-Latif A. A. (1985) Biochem. 228, 697-706. 35. Daniel L. W., Waite M. and Wykle R. L. (1986) J. biol. Chem. 261, 9128-9132. 36. Cabot M. C., Welsh C. J., Zhang Z.-C. and Cao H.-T. (1989) FEBS Left. 245, 85-90.
37. Martin T. W., Feldman D. R. and Michaelis K. C. (1990) Biochim. biophys. Acta 1053, 162-172. 38. Meulen J. V. D. and Haslam R. J. (1990) Biochem. J. 271, 693-700. 39. Hannun Y. and Bell R. M. (1989) Science 243, 500-507. 40. Merrill A. H. and Stevens V. L. (1989) Biochim. biophys. Acta 1010, 131-139. 41. Lavie Y. and Liscovitch M. (1990) J. biol. Chem. 265, 3868-3872. 42. Roghani M., Da Silva C. and Castagna M. (1987) Biochem. biophys. Res. Commun. 142, 738-744. 43. Horwitz J. (1991) J. Neurochem. 56, 509-517. 44. Billah M. M., Pai J.-K., Mullmann T. J., Egan R. W. and Siegel M. I. (1989) J. bioL Chem. 7,64, 9069-9076. 45. Reinhold S. L., Prescott S. M., Zimmerman G. A. and Mclntyre T. M. (1990) FASEB J. 4, 208-214. 46. Pepitoni S., Mallon R. G., Pai J.-K., Borkowski J. A., Buck M. A. and McQuade R. D. (1991) Biochem. biophys. Res. Commun. 176, 453-458. 47. Honkanen R. E., Howard E. F. and AbdeI-Latif A. A. (1990) Inv. Ophthal. vis. Sci. 31, 590-593. 48. Bocckino S. B., Wilson P. B. and Exton J. H. (1987) FEBS Lett. 225, 201-204. 49. Anthes J. C., Wang P., Siegel M. I., Egan R. W. and Billah M. M. (1991) Biochem. biophys. Res. Commun. 175, 236-243. 50. Kanaho Y., Kanoh H. and Nozawa Y. (1991) FEBS Lett. 279, 249-252. 51. Gilman A. F. (1987) A. Rev. Biochem. 56, 615-649.
0898--6568/92 $5.00 + 0.00 © 1992PergamonPressLtd
ACTIVATION OF PHOSPHOLIPASE D BY ENDOTHELIN-1 AND OTHER PHARMACOLOGICAL AGENTS IN RABBIT IRIS SPHINCTER SMOOTH MUSCLE YAWEN ZHANG and ATA A. ABDEL-LATIF* Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100, U.S.A.
(Received 17 July 1992; and accepted 10 August 1992) Almtraet--The stimulation of phospholipase D (PLD) activity by endothelin-1 (ET1) was investigated in rabbit iris sphincter prelabelled with [3H]myristic acid. In the presence of 0.5% ethanol, ETI caused a time- and dose-dependent increase in the production of [3H]phosphatidylethanol ([3H]PEt). Within 30 s the peptide increased PEt formation by 30% and after 5 min increased it by 140%. The Ecs0value for ETl-stimulated PEt formation was found to be 30 nM. This value is appreciably lower than the Ecs0 we previously obtained for ETl-induccd inositol trisphosphate production (45 nM), but considerably higher than that for arachidonic acid release (1 nM). PEt formation was significantly stimulated by prostaglandin F20, phorbol 12,13-dibutyrate (PDBu), chloroform, A23187 and A1F~', but it was not affected by carbachol or the platelet-activating factor. PDBu-stimulated PEt formation was blocked by staurosporine and it was not potentiated by A23187. Staurosporine had no effect on ETl-stimulated PEt formation. Our data indicate that ET1 stimulation of PLD occurs independently of protein kinase C activation, phospholipase C activation and intraccllular Ca2+ mobilization, and phospholipase A2 activation. In this tissue the ET1 receptor is probably coupled to the three phospholipases through several G-proteins, and this appears to be species and receptor type specific.
Key words: Rabbit iris sphincter, smooth muscle, endothelin-1, phospholipase D, phosphatidylcholine, phosphatidylethanol, protein kinase C, chloroform, phorbol ester. INTRODUCTION
lying the ETl-induced responses are not well understood. In smooth muscle, ETl-induced contractile responses are associated with stimulation of phosphoinositide hydrolysis, via phospholipase C (PLC), and elevation of intracellular Ca 2+ in vascular smooth muscle cells [6-12], rat aorta [13-16], rat tracheal smooth muscle [17], porcine coronary artery [18] and rabbit iris sphincter [19]. In addition, ET1 has been reported: (i) to stimulate phospholipase A2 (PLA2) and liberate arachidonic acid, from membrane phospholipids, for eicosanoid biosynthesis, in cultured vascular smooth muscle cells [20,21] and rabbit iris sphincter [22]; and (ii) to stimulate phospholipase D (PLD) to produce phosphatidic acid (PA) from phosphatidylcholine (PC), which in the presence of ethanol can be measured as phosphatidylethanol (PEt), and free choline in vascular smooth muscle cells [23-25] and rat aorta [26].
E1',-oo'rr~L~- 1 (ET1) is a 21-amino acid peptide isolated from vascular endothelial cells and shown to be the most potent vasoconstrictor known so far [1]. Several studies have demonstrated that in addition to its vasoconstrictive effects, ET1 possesses various pharmacological activities in both vascular and nonvascular tissues (for reviews see [2-5]). The peptide elicits biological responses by binding to specific cell surface receptors on the plasma membrane of the cell. The biochemical mechanisms under-
* Author to whom correspondence should be addressed. Abbreviations: BSA--bovine serum albumin; CCh--car-
bachol; DAG--l,2-diacylglycetol; ETl--cndothelin-1; IP~--inositol 1,4,5-trisphosphate; PA--phosphatidic acid; PC--phosphatidylcholine; PEt--phosphatidylethanoi; PDBu-phorbol 12,13-dibutyrate; PLA2--phospholipase A2; PLC--phospholipase C; PLD--phospholipas¢ D. 777
778
YAWEN ZHANG
and A. A. ABDEL-LATIF
Activation o f these phospholipases may also lead to the stimulation of protein kinase C (PKC). The role of P L D in cell signalling and its relationship to phospholipases A2 and C has yet to be defined (for reviews see [27-29]). In the iris sphincter, ET1 is a potent agonist for the production of inositol trisphosphate (IP3) and 1,2-diacylglycerol (DAG) from polyphosphoinositides in rabbit, dog, cat and pig, and for cAMP formation in rabbit, dog, cat, pig, bovine, monkey and human [19], ET1 also stimulated the release o f arachidonic acid and prostaglandins, through activation of PLA2, in the rabbit iris sphincter [22]. In the past few years the agonist-induced stimulation o f PLD has emerged as an important mechanism in cellular signalling in a wide variety of tissues [27-29]. The mechanism that underlies this agonist-induced P L D activation is not well understood. In the studies reported here we have investigated the effects o f ET1 and other pharmacological agents on P L D activity in the rabbit iris sphincter. We find that ET1, as with its actions on PLC [19] and PLA2122], is a potent activator of PLD activity in this tissue. The ET1 effects are independent o f PKC and Ca 2÷ mobilization. MATERIALS AND METHODS
Materials [3H]Myristic acid (39.3 Ci/mmol) was purchased from New England Nuclear (Boston, MA) and myo-[3H]inositol (80--120 Ci/mmol) from Amersham Corp. (Arlington Heights, IL). Endothelin-I (human, porcine) was purchased from Peptides International (Louisville, KY). PEt and PA were purchased from AVANTI Polar Lipids, Inc., Alabaster, AL. Phorbol esters, carbachol (CCh) and other pharmacological agents were obtained from Sigma Chemicals Co. (St Louis, MO). All other chemicals were of reagent grade. Preparation of iris sphincter muscle Rabbit eyes packed in ice were obtained from a local abattoir. The iris sphincter muscle was dissected out and placed in modified Krebs-Ringer bicarbonate buffer of the following composition (in raM): NaCI, 118; NaHCO3, 25; KCI, 4.7; KH2POo 1.2; MgSOo 1.2; CaCI2, 1.25; D-glucose, 10; and 1 /~M
indomethacin. The pH of the buffer was adjusted and maintained at 7.4 with 97% 02-3% CO2. In general, of the pair one sphincter served as a control and the other as experimental.
Incubation of sphincter muscle with [3H]myristic acid and assay for PLD activity PLD is known to catalyse the transfer of phosphatidyl groups to various acceptors; in the presence of an alcohol, this transphosphatidylation produces phosphatidylalcohol [30]. The production of PEt is considered a good indication of PLD activity. The assay for PLD activity was as described by Liscovitch and Amsterdam [30]. Briefly, the paired sphincter strips were incubated for 90 min at 37°C in 1 ml Krebs-Ringer bicarbonate buffer containing 0.05% bovine serum albumin (BSA) and 5/~Ci [3H]myristic acid. At this time the sphincters were washed four times with 3 ml buffer that contained 0.05% BSA. The sphincters were preincubated singly in I ml buffer containing 0.5% (v/v) ethanol at 37°C for 5 min. The agonist was then added as indicated and incubation continued for an additional 5 min. Reactions were terminated by addition of 2 ml of icecold chloroform-methanol (1:2, v/v). Phospholipids were extracted according to the procedure of Bligh and Dyer [31]. The phospholipids in the lipid extract were separated by one-dimensional thin layer chromatography (TLC) using silica gel K6 plates (Whatman) and a solvent system consisting of the organic phase of ethylacetate--isooetane--acetic acidwater (13:2:3:10, by volume). Individual lipids were localized by iodine staining and identified by comigration with standards. The spots corresponding to PEt and PA were scraped into scintillation vials and the 3H-radioactivity measured by liquid scintillation counting. Data are expressed as d.p.m.//zmol lipid phosphorus. Incubation of sphincter muscle with myo[aH]inositol, [3H]myristic acid and analysis of inositol phosphates and PEt To double label the tissue with myo[3H]inositol and [3H]myristic acid, the paired sphincter strips were incubated for 90 min at 37°C in l ml Krebs-Ringer bicarbonate buffer containing 10 #Ci of myo[3H]inositol and 5/~Ci [aH]myristic acid. At this time the sphincters were washed four times with 3 ml non-radioactive Krebs-Ringer bicarbonate buffer and then suspended individually in 1 ml fresh non-radioactive buffer. LiCI (10mM, final concentration) was added to each incubation and 10 rain later the agonists were added and incubations continued for the time indicated. The incubations were terminated by the addition of l ml 10% (w/v)
Endothelin and phospholipases in iris sphincter TABLE 1. PREFERENTIAL INCORPORATION OF [3H]MYRISTIC ACID INTO PHOSPHATIDYLCHOLINE IN IRIS SPHINCTER
3H-Radioactivity (% of total)
Phospholipids PA CDP-DAG Phosphatidylinositol Polyphosphoinositides Phosphatidylserine Phosphatidylethanolamine PC
1.68_ 0.02 0.20_ 0.01 2.38 __0.40 0.18 ± 0.03 0.31 + 0.03 9.60 + 0.90 85.0+ 1.16
779
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60-
~
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Rabbit iris sphincters were incubated in Krebs-Ringer bicarbonate buffer containing [3H]myristic acid (5 pCi/ml) for 90 min at 37°C. Phospholipids were extracted and then separated by means of two-dimensional TLC as described previously [32]. Total 3H-incorporated was 3.53 x l0 s d.p.m./nmol lipid P. The data represent means +S.E.M. from three separate experiments.
trichloroacetic acid. The tissues were homogenized, centrifuged and the supernatant extracted with 4 x anhydrous diethyl ether. The precipitate was extracted with chloroform-methanol-HC i (300:300:1.5, by volume) and PEt separated from the lipid extract by one-dimensional TLC [30]. Phospholipids were separated by means of twodimensional TLC as previously described [32]. The water-soluble tissue extract was neutralized to pH 7.0 with NaOH and myo[3H]inositol phosphates were analysed by anion exchange chromatography using Biorad 1 x 8 resin (formate form, 200--400 mesh) as described previously [33].
Calculations and statistical analysis of the data To correct for variation in tissue size, the data for phospholipids and [3H]inositol phosphates were normalized to tissue lipid phosphorus. Data are expressed as the mean + S.E.M. RESULTS
Preferential incorporation of [3H]myristic acid into PC of iris sphincter In order to investigate the hormonal activation of PC hydrolysis, we first carried out experiments on the preferential radiolabelling of this phospholipid using [3H]myristic acid, [~H]oleic acid and [3H]arachidonic acid as
Time (Minutes)
FIG. 1. Time-course of ETl-induced [3H]PEt formation in rabbit iris sphincter. Muscles were prelabelled with [3H]myristic acid (5pCi/ml) in Krebs-Ringer bicarbonate buffer for 90 rain. The labelled muscles were washed with buffer and incubated in buffer containing 0.5*/0 ethanol with or without 0.1/aM ETI for the indicated times. [3H]PEt was analysed by one-dimensional TLC as described under Materials and Methods. The basal level of [3H]PEt formation was 2720+167d.p.m./pmol lipid P. Each point represents mean+S.E.M, that were obtained from four experiments, each run in triplicate.
precursors. We found [3H]myristic acid to be the most effective precursor for selectively labelling PC. Table I shows the radioactivity profiles of the various iris sphincter phospholipids after radiolabelling with [3H]myristic acid. Under basal conditions, i.e. in the absence of ET1 and ethanol, about 85% of the total radioactivity was detected in PC, and this was followed by 9.6% in phosphatidylethanolamine and 2.4% in phosphatidylinositol. Therefore for the PEt and PA assays in the present work we used [3H]myristic acid to label the iris sphincter phospholipids.
Time-course of ETl-induced [3H]PEt formation ETI stimulated PEt formation in a timedependent manner. ET! (0.1pM) induced a 30% increase in PEt formation by 30 s, reached a 140% increase within 5 min and plateaued by 10 min after addition of the agonist (Fig. 1). These data demonstrate that ET1 rapidly
780
YAWEN ZHANG and A. A. ABDEL-LATIF
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.160 .t40 .-~ "120 E ;-100
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150
200 250 Ethanol,mM
300
350 II SSO0
FIG. 2. Ethanol dependence of ETl-stimulated [ill]PEt formation in iris sphincter. Rabbit iris sphincters were prelabelled with [3H]myristic acid, washed with buffer, and then incubated in the presence of increasing concentrations of ethanol with and without 0.1 pM ET1 for 5 rain. [~I-I]PEt and [3H]PA were analysed by one-dimensional TLC as described under Materials and Methods. Basal levels of [3H]PA and [3H]PEt, in the absence of ethanol, were 11,950+950 and 1320+200 d.p.m.//anol lipid P, respectively. Each point represents mean+S.E.M, from four to five experiments, each run in triplicate.
stimulates, through activation of PLD, PEt formation in this tissue. Based on these findings, we employed 5-min incubations with the agonists in the experiments described below.
Ethanol dependence of ETl-stimulated [3H]PEt formation The effect of ethanol concentration on ETl-stimulated PET formation is shown in Fig. 2. ETl-stimulated PEt formation is completely dependent on the presence of ethanol. Ethanol, at 25mM, increased the ETl-induced PEt formation by 100%. Maximal stimulation, 320% increase, was obtained at about 85 mM (0.5% ethanol). PA production was significantly attenuated in the presence of ethanol. This is due to the competition between ethanol and water for the phosphatidyl group of PC. Higher concentrations of ethanol were inhibitory (Fig. 2). In the following experiments we employed an ethanol concentration of 0.5%.
Concentration dependence of ETl-stimulated [3H]PEt and [3H]PA formation The concentration dependence of ETIstimulated PEt and PA formation is shown in Fig. 3. ETl-stimulated PEt and PA formation were dose dependent with ECs0values of 3 x I 0"s and 2.6x 10"SM, respectively. The maximal
increases for PEt and PA formation due to 1/~M ET1 were 215 and 50%, respectively.
Effects of Ca2+-mobilizing agonists, phorbol esters and other pharmacological agents on [3H]PEtformation PLD can be activated by a variety of agonists in various tissues. The effects of typical concentrations of various agonists and phorbol esters on PEt formation are shown in Table 2. ET1 was the most potent Ca2+-mobilizing agonist. ETI increased PEt formation by 222% and prostaglandin F2= increased it by about 84% over that of the control. In contrast, CCh and the platelet-activating factor had no effect on the PLD activity. The iris sphincter is enriched with muscarinic receptors and CCh is a potent activator of PLC activity and contraction in this tissue [32,33]. The platelet-activating factor stimulates PLA 2, but not PLC, in the rabbit iris [34]. Further comparative studies on the effects of ETI and CCh on PLD and PLC activities were carried out in iris sphincter which was double labelled with [3H]myristic acid and [3H]inositol. ETI increased PEt formation and IP3 production by 176 and 252%, respectively, and CCh increased their formation by 9 and 109%, respectively (Fig. 4). These data suggest that in
Endothelin and phospholipases in iris sphincter
781
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8
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• PEt ECso=3xlOSM o PA ECso=2.6xlO'e
g = 150-
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o
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0
10 -Log Endothelin-1, M
FIG. 3. Concentration dependence of ETl-stimulated [3H]PEt and [3H]PA formation in rabbit iris sphincter. Rabbit iris sphincters were prelabelled with [3H]myristic acid for 90 min, washed with buffer, then exposed to different concentrations of ETI for 5 min. [3H]PEt and [3H]PA were analysed by means of one-dimensional TLC as described under Materials and Methods. Each point represents mean +_S.E.M. from two experiments, each run in triplicate. the rabbit iris sphincter muscarinic receptors are coupled to the activation of PLC but not to that of PLD. In addition, they provide evidence that the P L C and P L D pathways are dissociated.
Phorbol esters have been shown to cause stimulation of P L D in various tissues, suggesting a role for P K C in the regulation of P L D activity [35-38]. PDBu increased PEt formation in the iris sphincter by 194%, while
100"
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Control CCh
E
~
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FIG. 4. Comparative effects of ETI and CCh on [3H]PEt formation and [3H]IP~ production in iris sphincter. Rabbit iris sphincters were double labelled with [3H]myristic acid and [3H]inositoi, washed with buffer, and then incubated without or with ET! (0. i/JM) and CCh (25/~M) as indicated for 5 rain, The reactions were stopped with TCA and [3H]PEt and [3H]IP3 were analysed by TLC and ion-exchange chromatography, respectively, as decribed under Materials and Methods. The results are means _+S.E.M. from two experiments, each run in triplicate.
782
YAWEN ZHANG and A. A. ABDEL-LATIF
TABLE 2.
EFFECTS OF Ca2+-MOBILIZING AGONISTS, PHORBOL ESTERS AND OTHER PHARMACOLOGICAL AGENTS ON PEt FORMATION IN IRIS SPHINCTER
Addition ET1 Prostaglandin F2, CCh Platelet-activating factor PDBu 4~-Phorbol 4~-Phorbol 12,13-didecanoate A23187 PDBu (0.I /~M)+A23187 (1 vM) AIF~-
Concentration (/zM)
[3H]PEt (% of control)
0.1 (0.01-1)* 0.1 (0.01-1) 25 (1-50) 0.1 (0.001-1 ) 0.1 (0.01-0.1) 0.1 (0.01-0.1) 0.01 (0.01-0.1) I (0.1-10)
322+26 184+ 10 1144- 9 i 05 _+ 6 294+ I l 109+ 4 1034- 5 1754- 10 3104- 15
10 #M AICI3+ 10 mM NaF
1744- 4
Rabbit iris sphincters were prelabeUed with [3H]myristic acid, washed with buffer and then incubated in buffer containing 0.5% ethanol in the absence and presence of the agonist for 5 min. Jail]PEt in the lipid extract was analysed by means of one-dimensional TLC as described under Materials and Methods. The average ~H-radioactivity recovered in basal PEt was 2650+185d.p.m.//tmol total lipid P. The data represent means+ S.E.M. from two separate experiments, each run in triplicate. * Range of concentrations of agonists investigated. the biologically inactive analogue, 4~-phorbol and 4~-phorbol 12,13-didecanoate, exerted no effect on PEt formation. These data suggest a role for PKC in this tissue. The Ca2+-ionophore A23187 increased PEt formation by 65%. However, the ionophore was without effect on PDBu-induced PEt formation (Table 2). This could suggest that PEt formation stimulated by PDBu is Ca 2+ independent. A1F4-induced a 74% increase in PEt formation. This is in accordance with the work of others who have reported that P L D is regulated by G-proteins [28].
Effects of staurosporine on ET1- and PDBu-induced [3H]PEtformation To determine whether or not PKC is involved in PLD activation by ETI we investigated the effects of staurosporine, a potent inhibitor o f PKC, on ETI- and PDBu-induced PEt formation. Staurosporine had no effect on ETl-stimulated PEt formation, but it completely blocked the PDBu stimulation (Table 3). This demonstrates that ETl-stimulation of PLD activity is independent of PKC.
Differential effects of PDBu on ETl-induced formation of [3H]PEt and production of [3H]IP3 Further evidence that in this tissue the PLD and PLC pathways are dissociated is supported by the studies on the effects of PDBu on ETl-induced formation of PEt and IP 3 in iris sphincter which was double labelled with [3H]myristic acid and [3H]inositol. PDBu increased PEt formation by 213%, but it had no effect on IP 3 production (Table 4). In addition, while the stimulatory effects of PDBu on the production of PEt and IP 3 were additive, IP 3 production due to ETI was significantly attenuated in the presence of the phorbolester.
Dose-response effects of chloroform on [3H]PEt formation Sphingosine has recently attracted considerable attention because it inhibits the activity of PKC (for reviews see [39,40]). Sphingosine has also been reported to activate PLD in neuralderived N G 108-15 cells [41]. We found that when we dissolved the sphingolipid in ethanol,
Endothelin and phospholipases in iris sphincter TABLE 3. EFFECTS OF STAUROSPORINE ON E T 1 - AND PDBu-INDUCED
783
[3H]PEtFORMATION IN IRIS SPHINCTER
[3H]PEt (d.p.m./#mol of total lipid P)
% of control
None ETI (0.1 #M) ET 1 (0.1 #M) + staurosporine (5 #M)
2950 +_105 8850+ 150 9766 _ 164
100 300 331
None PDBu (0.1 #M) PDBu (0.1 /tM)+staurosporine (5 #M)
2620+ 110 8096+ 135 2710+95
!00 309 103
Addition Experiment 1
Experiment 2
Rabbit iris sphincters were prelabelled with [3H]myristic acid, washed with buffer, then pretreated with staurosporine for 10 min as indicated. The labelled tissues were then incubated in buffer containing 0.5% ethanol in absence and presence of ETI and PDBu for 5 and 15 min, respectively. The phospholipids were extracted and [3H]PEt separated by one-dimensional TLC and analysed as described under Materials and Methods. The data represent means + S.E.M. from two separate experiments, each run in triplicate. isopropanol, or dimethyl sulphoxide (DMSO) there was no significant stimulatory effect on P L D activity (data not shown). However, when sphingosine was dissolved in chloroform we found an appreciable amount of stimulation of P L D activity. This observation prompted us to investigate the dose-response effects of chloroform on PEt formation. Chloroform increased PEt formation in a dose-dependent manner (Fig. 5). Half maximal stimulation of PEt occurred at 30 mM, and the stimulatory effects of chloroform levelled off between 75 and 125 mM. Maximal stimulation (310% increase)
was reached at 75 mM chloroform. At 5-10 #M staurosporine had no inhibitory effect on chloroform stimulation o f PEt formation, suggesting that PKC is not involved in the mechanism of P L D activation (data not shown). This is in contrast to rabbit platelets where chloroform (250mM) was reported to activate PKC activity [42]. DISCUSSION Previously, we reported that in the rabbit iris sphincter ET1 is a potent agonist for: (i) PLC
TABLE 4. DIFFERENTIAL EFFECTS OF P D B u ON ETI-INDUCED [3H]PEtFORMATION AND [3H]IP3 PRODUCTION IN IRIS SPHINCTER
Addition ETI (0.1 /~M) PDBu (0.1 #M) ETI (0.1 #M)+PDBu (0.1 /~M)
[3H]PEt (% of control)
[3H]IP3 (% of control)
339+23 313_ 17 529±28
379_+ 15 108+_ il 199_+ 13
Rabbit iris sphincters were double labelled with [3H]myristic acid and [3H]inositol, washed with buffer, and then preincubated with PDBu (0.1 gM) in buffer containing 0.5% ethanol for 15 rain as indicated. ETI was then added and after 5 min the reactions were stopped with TCA. [3H]PEt and [3H]IP3 were analysed by means of one-dimensional TLC and ion-exchange chromatography, respectively, as described under Materials and Methods. 3H-Radioactivity recovered in basal [3H]IP3 was 3715_+308 d.p.m./mg protein. The results are means_+S.E.M, from four to five experiments, each run in triplicate.
784
YAWEN ZHANG and A. A. ABDEL-LATIF 400-
g ~g ,9
300-
e~
~_.~200100"
0,
2's
5'o
;5
160
1~s
Chloroform, mM
FIG. 5. Dose-response effects of chloroform on [3H]PEt formation in iris sphincter. Rabbit iris sphincters were prelabeUed with [3H]myristic acid, washed with buffer, then exposed to different concentrations of chloroform for 5 rain. Each point represents mean_ S.E.M. from two experiments, each run in triplicate. activation, IP3 production, muscle contraction and cAMP formation [19], and (ii) PLA2 activation, arachidonic acid release and prostaglandin synthesis [22]. In the present study, we have shown that ETI is also a potent agonist for PLD activation in this tissue. In the presence of ethanol, ET1 stimulates PLD to hydrolyse [3H]myristate PC in the iris sphincter to produce [all]PEt. Maximal stimulation of PLD due to 1/~M ET1 was obtained at about 85 mM (0.5% ethanol). This concentration of ethanol is optimum for studies on agonist-induced PEt formation in other systems [43]. Under basal conditions about 85% of the incorporated [~H]myristic acid was recovered in PC (Table 1). The peptide stimulated PEt formation in a time- and dose-dependent manner. Within 30 s ETI increased PEt formation by 30% and after 5 min it increased it by 140% (Fig. 1). ETl-stimulated PEt formation is dose-dependent with an ECs0of about 30 nM (Fig. 3). This value is appreciably lower than the Ecs0 we reported previously for ETl-induced IP3 production (45nM) [19], but considerably higher than that for ETI-induced arachidonic acid release (1 nM) [22]. Thus, in the rabbit iris sphincter the ECs0values for ETi-stimulation of PLA2, PLC and PLD are I, 45 and 30nM, respectively. The ECs0 for ETI-induced cAMP
formation in the bovine iris sphincter is 28 nM [19]. The ECs0 for ETl-induced contraction in rabbit iris sphincter is 46 nM [19]. These multi effects of ET1 on the second messenger systems in the iris sphincter could explain our previous observation that as compared to the rapid action of CCh, the contractile response to ETI in this tissue has a slow onset and is sustained for 6-20 min [19]. The maximal increase in PEt and PA formation due to 1/zM ETI was 215 and 50%, respectively (Fig. 3). This finding, coupled with the observation that PEt formation due to ET1 increases with ethanol concentration, while that of PA decreases (Fig. 2), suggests that the peptide activates PLD to hydrolyse PC into PA and free choline. Therefore, both hydrolysis of PC into PA and free choline and transphosphatidylation to produce PEt occur in the presence of ethanol. In several tissues, PLD activity may be induced by PKC activators, such as phorbol esters [35-38,44,45] and Ca 2+ ionophores [44,45]. Reinhold et al. [45] postulated that three separable but interacting routes lead to the activation of PLD: (i) direct activation by PKC in cells stimulated with phorbol esters; (ii) Ca2+-dependent activation in cells treated with Ca 2+ ionophore; and (iii) an undefined route stimulated by receptor-dependent agonists. In the rabbit iris sphincter both direct activation of PKC and an increase in Ca 2+ influx by A23187 stimulated PLD activity (Table 2). PDBu increased PEt formation by more than 200% (Tables 2-5). As expected, the inactive stereoiosomers 4g-phorbol and 4~-phorboi 12,13-didecanoate were ineffective in activating PLD (Table 2). The finding that the PDBu effect was not potentiated by A23187 could indicate that PEt formation stimulated by PDBu is Ca 2+ independent. Staurosporine, a potent PKC inhibitor, blocked PDBu-but not ETl-stimulated PEt formation. These data suggest that in the rabbit iris sphincter: (i) PKC is involved in PDBu stimulation of PLD activity; and (ii) that ETI stimulation of PLD is independent of PKC activation. Chloroform, a tumour promoter and a potent activator of PKC in platelets [42], stimulated PEt formation
Endothelin and phospholipasesin iris sphincter in the iris sphincter in a dose-dependent manner (Fig. 5). However, the finding that the stimulatory effect of chloroform on PLD activity was not influenced by pretreatment of the muscle with staurosporine suggests that the chloroform stimulation occurs independently of PKC activation. It is possible that chloroform acts by perturbation of the phospholipids of the plasma membrane. ETl-induced PLD activation is not dependent on PLC activation and subsequent increase in intracellular Ca 2+. This conclusion is supported by the following findings in the present work: (i) in iris sphincter muscle which was double labelled with [3H]myristic acid and [3H]inositol, PDBu potentiated ETl-induced PEt formation but attenuated that of ETl-induced IP 3 production (Table 4). This suggests that an increase in intracellular Ca 2+ concentration by IP 3 is not involved in PLD activation; (ii) CCh, a potent agonist for PLC activation in the iris sphincter [32,33] had no effect on PEt formation (Table 2, Fig. 4). There is evidence that the human m l acetylcholine receptor is linked to the signal transduction mechanism of PLD activation, whereas the human M2 receptor interacts with a different G-protein which does not cause the activation of PLD or the formation of PEt [46]. The iris sphincter contains mainly m3 (about 90%) and small amounts of M2 receptor subtypes [47]. ET1 is a potent activator of PLA2 in the rabbit iris sphincter [22]. The platelet-activating factor, which is an activator of PLA 2 but not PLC in this tissue [34], had no effect on PLD activity (Table 2). This could suggest that ETl-induced PLD activation is not dependent on PLA2 activation in this tissue. There is accumulating experimental evidence which suggests that the membrane-bound PLD, as with PLC and PLA 2 is linked to cell surface receptors through GTP-binding proteins [2729,38,48-50]. In the rabbit iris sphincter, AIF~- (10#M), which interacts directly with G-proteins and activates both Gs and Gi [51], induced a 74% increase in PEt formation (Table 2). It is possible that in this tissue the ET1 receptor is coupled to PLD, PLC and
785
PLA2 through several G-proteins, and this appears to be species and receptor type specific. Acknowledgements--This work was supported by National Institutes of Health Grants R37-EY-04171 and EY-04387.
I. 2. 3. 4. 5.
6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19.
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