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The effects of flosequinan on endothelin-l- induced changes in inositol 1,4,5-trisphosphate levels and protein kinase C activity in rat aorta

Derek Lang and Malcolm J. Lewis

Ih'l;artm~'t~t n/I~t~armacoh;gv and 77wrapt'.lic,~, Utlll er~ily o]' Wah'~ ('oll¢,ge o] Medwim', ("ardt]]' ("k'4 4XN, UK

]~.cccJvcd 18 Scplcmhcr 1991, revised MS received 13 March t992. accepted 24 March 1992

In ral aorta cndolhclin-I (10 ~ M) induces significant increases in inositol 1.4,5-trisphosphatc (IP 0 levels after a 3(I s exposure. An increase in parliculalc protein kinasc (' activity is also observed at 3fl s with a second peak of activity occurring aflcr 1{I rain. I:loscquinan, al c~mccntrations c~t 10 r, M or greater, inhibits these cndothclin-l-induccd changes in both IP~ and parliculalc I":otcin kinasc (' activity in the absence of changes in cithcr cyclic GMP or cyclic AMP. It is likcly therefore thai floscquinan inhibits the transduction mechanisms between the cndothclin-I receptor and hydroly,;h of phospilalidylinositol 4,5-bisphosphatc, po~,~,ibly ;11 the level el a (i-protein. thcsc results provide a mcctlani'~m to explain ttac vas~diialor effects of floscquinan ol~scrvcd ill vitro.

F.ndolhclin-t; Inosilol 1,4,5-tri.',ph~sphatc; Protein kinasc C; Floscquinan

I. Introduction 2. Materials and methods

Floscquinan (7-11uoro-l-methyl-3-mcthylsulphinyl- 2.1. Tissue preparation 4-quinohmc) is a novel artcriovcnous dilator agent which is currcntly i,ndcr clinical trial for usc in heart Malc Wistar rats (25(I-300 g) wcre kil!cd by cervical faihtrc (Kessler ar|d Packer, 1987; Sim ctal . , 1 9 8 8 ) . dislocation and their thoracic aortac removed and

In in vitro studics it has been shown to induce placed into Krebs-Ringer hicarbonate (KRB)solution rclaxation of phorbol ester-contracted aortae of the rat of the following composition (raM): NaCI 95.5, KCI 4.8, (Yalcs ;and llolmcs, 1¢/88) and phcnylcphrinc-con- MgSO~ 1.2, CaCI z 2.5, KH2PO 4 1.2, NaHCO 3 25, Iraclcd aortac of the ferret (Rcsnick ct al., 1991). Thc glucose I 1 and indomethacin (1.001. The vessels were inlracclhdar mcch,'misms rcsponsibIc for tiffs vasorclax- cleaned of all fat and connectivc tissue and each di- ant cffcc! ill' floscquinan have bccn variously attributed vidcd into four equal Icngths. Single lengths wcrc used to inhibition ill" protein kinasc (" actiwttion secondary for IP 3, cyclic GtvlP and cyclic AMP measurement and to clcwllion of cyclic ( iMP (Allcock ct ill., 1988; Yatcs paircd lengths wcrc used lor measurement of protein and llolmcs, 1988), a direct antagonism of protein kinasc C activiw.. The cntlotnc~ . . . . . . . . . LIIII was removed from kinasc (" (Rcsnick ct al., 1'-)89), and inhibition o[ phos- the vessels by gentle rubbing of the intimal surfacc with phoinositidc (i;i) mctabolisnl (Yatcs c tal . , ,o,-,,~ ,,,,-,~. a woodcn stick. The sections were placcd in KRB

None of these previous studies has however in- gassed with 95% O, :5 '% CO~ and left to equilibrate cludcd direct mcasurcnlcnts of protein kinasc ( ' activ- for at least 611 rain at 37°C. |:or protein kinasc C ity. In ',he present study therefore we havc investigated mcasuremcnt experiments wcrc carried out in KRB the effects of floscqttinan on the levels of inositol under these conditions. For mcasurcmcnt of IP~ tissues 1,4,5-trisl)hOsl~hatc (IP0, cyclic GMP, cyclic AMP and wcre incubated for a further 20 rain in KRB with !0 protein kinasc C activity in rat aortae stimulated with mM LiCI, and the IP.~ experiments then carried out ill Ihc vasoconslrictor pcptidc cndolhclin-I, ibis LiCl-containing solution.

2.2. ]t,h'asto'ement of IP¢, cydic GMP, cyclic AMP and protein ('o,'llcHl

('tlll"t-'StlOIIl.tl211f¢ Ill: l)r. M.J . l .cwis . I)cl~arinlcnl o1 t'harn~aeol,~gy and Tht'r:W, cutics. I Inivctsity of Wales ( 'ollcgc of Mcdiciiw. l loath Following c×l )Os t l rc o f t h e tissues to tile appropriate Park.(';;rditl('F.l ,IXN, UK. Tcl. 0__.-74_0(~h. Fax0222-7474S4. agents (scc abovc), each artery segment was placed

26O

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70 ~ * 25

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" ~ ~ A V / # / ¢

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10 r,~ " I

0 -" 0.0 " " 0 B0.5 2 5 10 B 0 . 5 1 2 5 10 B 0 . 5 1 2 5 10

Time (min) Time (min) Time (min)

(a) (b) (c) io] 70 ]1~ 25

= 60 '~z "~ 1.o T o,:,.. ~ 4 ~

++o: ++++I] l i b + +o..+z+ + ~ + ..,. ¢ 0.5 ~ +o 20 - ~ /~

5 1

. . . . . . o.o . . . . . &' o B 0 . 5 1 2 5 1 0 B 0 . 5 1 2 5 1 0 B 0 . 5 1 2 5 1 0

Time (rain) Time (min) Time (min)

$ 80" _~ ~ (a)

(b) (c) 1.5 ! 30] ,

lo ::. E /: +- 20 5 i: " ?!~}

1 , , I , 0.0 0 B 8 7 6 5 4 8 8 7 6 5 4 B 8 7 6 5 4

Flosequinan Flosequinan Flosequinan [Log molar] [Log molar] [Log -.-.,~.1 I I I V I O l I

Fig, 1. Bar diagram showing changes in lP.t (open columns), cyclic G M P (hatched columns) and +c l io A M P (shaded columns) in segments oi endothel ium-denuded rat aorta, The upper panel shows the effects of endothel in-I (10 -'~ M) exposure alone for the times indicated. The middle panel shows the effects of pro-incubation with flosequinan (10 3 M) for the times indicated before exposure to endothel in-I for 30 s. The lower panel shows the effects of pre- incubat ion with f loscquinan (10 - s to 10 -4 M) for 2 rain before exposure to endothel in-I ( I0 - s M) for 30 s. Control tissues were included for each experiment to provide basal values (B). Data are expressed as mean _+_ S.E.M. (n = 6); * P < 0.05;

"" P < 0.01 cf. basal values: ~ P < 0.05 cf. endothel in- t alone for 30 s.

261

into 0.5 ml of ice-cold 5% (v/v) perchloric acid (PCA). Having determined the optimum exposure time for the Each vessel was homogenised at 4°C using a 3 ml inhibitory effect of flosequinan (10 -s M) on the IP s "Jencons' glass mini-homogenising tube. The resulting response to endothelin-l , tissues were pre-incubated homogenate was centrifuged at 13,000 x g for 2 min at with varying concentrations of flosequinan from 10 -s 4°C. The supernatant was aspirated into cooled plastic to 10 -4 M, for this time (30 s) before exposure to tubes and together with the tissue pellet was stored at endothelin-I for 30 s. - 2 0 ° C for up to 2 weeks before assay.

The iP3, cyclic GMP and cyclic AMP contents of the Z5. Protein kinase C acHHty supernatant were measured using commercially avail- able kits (Amersham International, UK (IP3); New Tissues were initially incubated with endothelin-1 England Nuz:v,,r Research Products, Germany (cyclic (10 - s M) for 30 s, 1 2, 5, 10, 20 and 30 rnin to nucieotides)), determine its effects on protein kinase C activity. To

The protein content of the pellet was measured as investigate the effects of flosequinan (10 -3 M) on described previously (Collins et al., 1988). IP 3, cyclic protein kinase C activity, artery segments were then GMP and cyclic AMP levels are expressed as fmol/~zg pre-incubated with this agent for 30 s, 1. 2, 5 and 10 protein, min before being exposed to endothelin-t (10 -8 M)for

30 s (the time lbund to maximally induce an increase in Z3. Measurement of protein kinase C actiuity particulate protein kinase C activity). Having deter-

mined the optimum exposure time for the inhibitory The protein kinase C activity was measured using a effect of flosequinan (10 -3 M) on protein kinase C

commerciaity available kit ~ n e ~ s n a m . . . . ~;.,,,~' . . . . . . . . . .a,~,~,,,,-" ..... ' activity," " t~3~ucs" wcrc :,n,.,o,,,,.~"r'~'"" . . . . -""~',, var~c,u~" conccntra UK). The assay is dependent on protein kinase C tions of flosequinan from 10 - s to 1 0 - 4 M for this time catalysing the transfer of radiolabelled 32p of ATP to (2 min)before being stimulated with endothclinot (10 -s the threonine group of a peptide (a hist&le) which is M) for 30 s. specific for protein kinase C. A linear incorporation of 32p into the substrate peptide is observed. The assay is Z6. Drugs a modification of a previously described technique (Hannun et al., 1985). Flosequinan was supplied by Boots Pharmaceuticals,

The tissue samples were homogenised as for the IP 3 Nottingham, UK. Endothelin-1 was obtained from assay but using a hypotonic lysis buffer of the following Sigma Chemical Co., Dorset, UK. All chemicals used composition (mM): T r i s / H C l 20, sucrose 300, EGTA were Analar grade. 2, EDTA 5, dithiothreitol 5, phenylmethylsulphonyl fluoride 1, and leupeptin 25 p.g/ml , pH 7.4. Following 2. 7. Statistics centrifugation at 30,000 × g at 4°C for 20 min, the supernatant contained the cytosolic cell fraction of All values are expressed as mean_+ standard error protein kinase C and the pellet the particulate fraction, of the mean (S.E.M.). A one-way analysis of variance The pellet was resuspended in the above buffer supple- was used followed by Dunnett 's multiple range test to mented with 1% Nonidet P40. This suspension was identify significant differences at the 0.05 level. then briefly sonicated (5 × 5 s) before the ccntrifuga- Since the measurement of protein kina~e C activity tion procedure was repeated. The protein contents of was not based on standard curves for the enzyme, it the cytosolic and particulate fractions were also mea- was not possible to correct for day-to-day variability of sured (Collins et al., 1988). Protein kinase C activity is the assay. For this reason baseiine measurements were expressed as fmol phosphate transferred/min/p.g pro- included as standards for each assay. tei,a.

2.4. Lez'els of IP~, cyclic GMP, cyclic AMP 3. Results

To study the effects of endothelin-1 (10 -s M) on 3.1. IP s tissue levels of IP 3, and the cyclic nucleotides, artery. segments were exposed to this constrictor agonist for Endothelin-I induced a significant increase in IP 3 30 s, 1, 2, 5 and 10 min. Having determined the peak levels after 30 s incubation. Levels of IP 3 were not IP 3 response to endothelin-1, tissues were exposed to different from baseline however at 1, 2, 5 and 10 rain flosequinan (10 -3 M) for 30 s, 1, 2, 5 and 10 rain incubation times (fig. 1. upper panel, a). before the addition of endothelin-1 for 30 s (the time In the presence of flosequinan (10 -~ M), the IP 3 which produced the peak IP 3 response). Tissue levels response following a 30 s incubation with endothelin-I of IP 3 and cyclic nucleotides were then measured, was completely inhibited. This was the case for all

j -

262

incubalion times of flosequinan studied (fig. 1, middle followed by a 2 rain incubation with endotilelin-1, no panel, a). significant char, ges were observed in cyclic GMP at any

Following incubation with floscquinan for 2 rain at of the concer~tretions studied (fig. 1, lower panel, b) or concentrations from 10 -s to l0 -~ M, the IP 3 response in cyctic AMP levels down to 10 -7 M flosequinan (fig. to endothelin-I was significarltly inhibited by concert- i. lower panel, c). Curiously, 10 -s M flosequinan in- trations of flosequinan of 10-" M and greater (fig. 1, duced a small but significant increase in cyclic AMP. lower panel, a). Concentrations of t0 7 and 10 -s M Tiffs effect was uncxpcctcd sincc conccntrations as flosequinan did not inhibit IP~ levels in response to high as 1,t} -3 M caused no change in cyclic AMP levels. endothelin-l°

,. ~ o 3.3. l¥o te in kinase C 3.2. Cycfic G M P and cy, tc Az ~

Endothelin-1 alone caused a small but significant Incubation of tissues with endothelin-I for up to 30 increase in cyclic ,GMP levels after 5 and 10 min min resulted in two temporally separated increases in incubation (fit;. l, upper panel, b). No changes in cyclic protein kinase C activity in the particulate fraction (fig. GMP levels were seen following 30 s, 1 and 2 min 2a). the first occurring at 30 s and remaining elevated incubatkm with endothelin-l . No significant change in at 1 miv,, the second occurring at l0 min. Activity of cyclic AMP levels was observed at any of the incuba- the cytosolic fraction fell significantly below baseline at tion times studied with endothclin-I alonc (fig. 1. upo thc I. ,," 5 ,,,,~"~a l0 rain incubation times (fig. 3a). Unlike per panel, c). with the particulate activity however, no change oc-

Incubation of tissues with flosequinan (10 -3 M) for currcd following 30 s incubation with endothelin-1. various times followed by exposure to endothelin-1 for Incubation of tissues with 10 -3 M flosequinan for 30 s resulted in a gradual increase in cyclic GMP levels 30 s to 10 min followed by a 30 s exposure to cndothe- over the l0 rain period (fig. 1, middle panel, b). The lin-I inhibited the biphasic increase in particulate pro- increases in cGMP are significant at 2, 5 and 10 min. tein kinase C activity in response to endothelin-! up to Levels of cyclic GMP at 10 min wcre approximately 2 min exposure. In fact, endothelin-I produced a de- twice that of basal values. No changes in cyclic AMP crease in particulate protein kinase C activity at 0.5, 1 levels were observed at any of the incubation times and 2 rain in the presence of fiosequinan. Activity studied (fig. 1, middle panel, c). tended to increase after 5 and 1(1 rain exposure but

When tissues were incubatcd with various concen- remained below the levcl observed with_ endothelin-I trations of flosequinan from It) -~ to 10 -4 M for 3(1 s alone (fig. 2b).

* (al (b) (c) 100 1°°I lOO 1

° " I t ' " * * , , i 80 80 TT

:= T _ ~ eo ~ o T . , 60

. . . . . I

o B o;s ; 2 s lo 20 30 ~ 8 o.s ~, s 1'o o e 8 7 6 s

Time (rain) Time (min) Flosequinan - [Log molar]

Fig. 2. Bar diagram showing changes in particulate protein kinase C activity in segments of endothelium-denuded rat aorta; (a) following exposure to endothelin-I (10 -'s M) alone tk)r the times indicaled: (bl pre-incuba.:ion with flosequinan (10 " 3 M) for the times indicated before exposure to endothelin-I (ll) "~ M) for 30 s; (c} pre-incubation with flosequinan (10- ~ to 10 -'~ M) for 2 rain before exposure to endothelin-I (10- s M} for 3() s. Control tissues were included for each experiment to provide basal values (B). Data are expressed as mean-4-S.E.M, (n = 6);

• P < 0.i15: "" P < 0.01 cf. basal values; " P < 0.05 cf. endothelin-I alone for 3(1 s.

263

(a ) (b) (c)

300 21., ' * ~ * * at

g II - - T T - , -

~100 100 1 0 0

o • 0 , . , 0 B 0:5 2 5 Io 2'0 3.O 8 o.5 ~ ; s 1'o B 8 ~ 6 s 4

Time (rain) T ime (rain) F losequinan - [Log molar]

Fig. 3. Bar diagram showing changes in cytosolic protein kinase C activity in segments of cndothelium-denuded rat aorta: (a) following exposure to endothelin-I (10 - s M) alone for the times indicated; (b) pre-incubation with flosequinan ( I 0 - 3 M) for the times indicated before exposure to endothelin-I (10 - s MJ for 30 s; (c) pre-incubation wilh flosequinan (10 - s to 10 -4 M) for 2 rain before exposure to endothelin-I (10-s M) for 30 s. Control tissues were included for each experiment to provide basal values (B). Data are expressed as mean_+S.E.M, (n < 6); * P < 0.05,

• *P < 0 01 ef. hasM values.

Pre-incubation of the artery segments with various kinase C responses to endothelin-1. The data do show concentrations of flosequinan (10 -8 to 10 -4 M) for 2 however that a high concentration of flosequinan ( 10 -3

min caused progressive inhibition of the endothelin-1- M) can induce an increase in cyclic GMP when incuba- induced increase in particulate protein kinase C activ- tion periods of greater than 2 min are used. These ity (fig. 2c). Concentrations of flosequinan of 10 -6 M changes are unlikely to account for the observed ef- and greater produced significant inhibition of this en- fects of this agent seen following an incubation period dothe!in-1 respoose At !1'1-7 and 10 -8 M, no inhibi- of only 2 min and in the presence of lower concentra- tion of particulate activity was observed (fig. 2c). tions of flosequinan when no changes in cyclic GMP

At all concentrations of flosequinan stvdied the were observed. cytosolic protein kinase C activity remained unaffected In the light of these data, and since there is evi- by subsequent exposure to endothelin-1, though a small dence from ligand binding studies in rat ventricle that but significant decrease in activity was seen at 10 -8 M flosequinan does not antagonise the receptor for en- flosequinan (fig. 2c). dothelin-1 directly (Ruck, A., personal communication),

it is likely that it inhibits the transduction mechanism between this receptor and phosphatidylinositol 4,5-bis-

4. Discussion phosphate (PIP2), the intracellular substrate for IP 3 and diacyiglyceroi (DAG) formatiot~. Whether thb in-

The data show that exposure of rat aortic smooth hibition lies at the level of a G-protein or phospholi- muscle to endothelin-1 results in two temporally sepa- pase C is not known. In this respect the action of rated, transient increases in partio~h~te protein kinase flosequinan resembles that of cyclic GMP which has C activity occurring at 30 s and 10 mm, confirming an been shown to inhibit agonist-induced IP 3 formation in earl ier report by Lang and Lewis (1991a). The results vascular smooth muscle (Lang and Lewis, 1989) and also show conclusively that flosequinan, at a concentra- endothelium (Lang and Lewis, 1991a) and more re- tion of 10 -6 M, can inhibit endothel in-l- induced in- cently endothelin-l- induced activation of protein ki- creases in IP 3 levels and particulate protein kinase C nase C in vascular smooth muscle (Lang and Lewis, activity in the absence of changes in either cyclic GMP 1991b). From recent studies by Hirata and colleagues it or cyclic AMP over the time course studied. Previous is likely that this effect of cyclic GMP is through studies with flosequinan have suggested that this agent inhibition of G-protein activation (Hirata et at., 1990). induces vasorelaxation by elevation of intracellular Further studies will however be necessary to elucidate cyclic GMP (Allcock et al., 1988; Yates et al., 1989). the exact mechanism by whicb flosequinan inhibits IP 3 This finding is not confirmed by the present study since formation and protein kinase C activation. 10 -4 M flosequinan caused no change in cyclic GMP Flosequinan is known to inhibit the tonic phase of levels up to 2 min incubation time but 10 -`6 M flose- endothelin-l- induced vasoconstriction in rat aorta quinan significantly inhibited both the 1P 3 and protein (Yates, D., personal communication). In the present

264

s t u d y its e f fec t o n t he la te p h a s e o f p r o t e i n k i n a s e C Collins, P.. A.}t. Henderson, D. Lung and M.J. Lewis, 1988, En- activity (i.e. 10 rain)was not studied so it is not possible dothelium-derivcd relaxing factor and nitroprusside compared in

noradrenaline- and K*-contracted rabbit and rat aortae. , l , Phys- to provide a definite correlation between the mechani- iol. 4(10, 395.

ca[ a n d b i o c h e m i c a l e f f ec t s o f f l o s e q u i n a n . If t he a c t i o n l tannun, Y.A., C.R. Loomis and R.M. Bell, 1985, Activation of of flosequinan is to inhibit the'transduction mechanism protein kinase C by Triton X-100 mixed micelles containing between the receptor for endothelin-1 and P1P~, it is diacylglycerol and phosphatidylserine, J. Biol. Chem. 260, 1(X139. l ikely t ha t it will a l so inh ib i t t he la te p h a s e o f p r o t e i n Hirata, M., K.P. Kohse, C.It. Chang, T. lkebe and F. Murad, 1990.

Mechanism of cyclic GMP inhibition of inositol phosphate forma- k i n a s e C ac t iva t ion . F u r t h e r work will be n e e e s s a D , tion in rat aorta segments and cultured bovine aortic smooth h o w e v e r to a n s w e r th is q u e s t i o n , muscle cells, J. Biol. Chem. 265. i2(~8.

In c o n c l u s i o n , f l o s e q u i n a n i n h i b i t s e n d o t h c l i n - l - i n - Kessler, P.D. and M. Packer, 1987, Haem~dynamic effects of BTS 49 d u c e d lIP 3 f o r m a t i o n a n d p r o t e i n k i n a s c C act iv i ty ill 465, a new long acting systemic vasodilator drug in patients with rat aorta by a mechanism which does not involve cyclic severe congestive heart failure. Am. ltcart. J. 113, 137. GMP or cyclic AMP. It is likely that this effect involves Long, D. and M.J. Lewis, 1989. En:.k~thelium-derived relaxing factor

inhibits the formation of hlositol trisphosphate by r;!hhit ~orta. J, inhibition of the transduetion mechanisms between the Physiol. 41 I. 45.

e n d o t h e l i n - I r e c e p t o r arid IP 3 p r o d u c t i o n , poss ib ly at Lang. D. and M.J. Lewis, 1991a, inhibition of inositol 1.4,5-tris- t he level o f a G - p r o t e i n o r p h o s p h o l i p a s e C. phosphate formation by cyclic GMP in cultured aortic endothelial

cells of the pig, Br. J. Pharmacol. 102, 277. Lang, D. and M.J. Lewis, 1991b, Endothelium-derived relaxing factor

inhibits protein kinase C activation by endothclin-I in rat aorta. A c k n o w l e d g e m e n t s Br. J. Pharmacol. 104, 139.

Resnick, M.S_ L.A. Maitland and K.G. Morgan, 1991. Flosequinan. Thanks are due to Boots Pharmaceuticals for providing financial a vasodilator with a novel mechanism of action, Br. J. Pharmacol.

support for this study. 1(12, 974.

Sim, M.F., D.B. Yates, R. Parkinson and M.J. Cooling, 1988, Cardio- vascular effects of the novel arteriovenous dilator agent, flose-

R e f e r e n c e s quinan in conscious dogs and cats, Br. J. Pharmacol. 94, 371. Yates. D.B. and J.R. Holmcs, 1988, Effects of flosequinan and other

vasortqaxants oll rat aortic contractions stimulated by nor- AUcock, A.A., G. Frodsham and M.F. Sire, 1987, Effects of llosc- adrenaline and phorbol ester, Br. J. Pharmacol. 95, 819P.

quinan, a novel arteriovenous dilating agent, on cGMP levels in Yates. D.B., G. lticks and M.F. Sim, 1989, The effect of vasorelax- rat isolated aortic strips. Br. J. Pharmacol. 94. 430P. ants which increase cyclic nucleotides on phosphoinositide

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Nishizuka. 1082. Direct activation of calcium activated, phospho- lipid dependent protein kinase by tumour promoting phorboi esters, J. Biol. Chem. 251,781.