Effects of tyrosine kinase inhibitors on the contractility of

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1 British Journal of Pharmaology (1995) B 1995 Stokton Press All rights reserved /95 $9. x Effets of tyrosine kinase inhibitors on the ontratility of rat mesenteri resistane arteries 'atalin Toma, Peter E. Jensen, 2Dolores Prieto, 3Alun Hughes, Mihael J. 4hristian Aalkjoer Mulvany & Department of Pharmaology and Danish Biomembrane enter, The Bartholin Building, Aarhus University, 8 Aarhus, Denmark 1 A pharmaologial haraterization of tyrosine kinase inhibitors (TKI) belonging to two distint groups (ompetitors at the ATP-binding site and the substrate-binding site, respetively) was performed, based on their effets on the ontratility of rat mesenteri arteries. 2 Both the ATP-site ompetitors (genistein and its inative analogue, daidzein) and the substrate-site ompetitors (tyrphostins A-23, A47 and the inative analogue, A-1) reversibly inhibited noradrenaline (NA, (1 JM)) and Kl (125 mm) indued ontrations, onentration-dependently. Genistein was slightly but signifiantly more potent than daidzein; the tyrphostins were all less potent than genistein, and there were no signifiant differenes between the individual potenies. The tyrosine kinase substratesite inhibitor bis-tyrphostin had no inhibitory effet. 3 Genistein, daidzein, A-23 and A47 eah suppressed the ontration indued by a2+ (1 gam) in a-toxin permeabilized arteries. A-I and bis-tyrphostin had little or no effet on ontration of the permeabilized arteries. 4 Genistein was signifiantly more potent than daidzein with respet to inhibition of the ontration in the presene of NA (1 JAM) and GTP (3 JAM). The effet of A-23, A47, A-I indued by 2 nm a2" and bis-tyrphostin was similar in permeabilized arteries ativated with a2+ (2 nm) + NA (1IM) + GTP (3 JM) and permeabilized arteries ativated with 1 jam a2+. 5 Genistein (3 JAM) redued the fura-2 measured intraellular alium ativity ([a2+]j) in arteries stimulated with NA but had no effet on [a2+]i in arteries stimulated with Kl (125 mm). 6 The potent effet of the TKIs in this study is onsistent with a role for tyrosine kinases in the mehanisms whih regulate both ytoplasmi a2" levels and the effet of a2` on the ontratile apparatus in smooth musle ells in resistane arteries. However, the results must be interpreted autiously beause the enzyme inhibitors may have a poor speifiity in intat tissues and beause the presumed inative analogues had potent effets. Keywords: Tyrosine kinase inhibitors: vasular smooth musle; tyrphostin; genistein; daidzein; rat mesenteri resistane arteries Introdution During the last deade it has beome lear that tyrosine phosphorylation of proteins plays a major role in the diverse ellular signalling pathways whih are involved in ell proliferation and transformation (Bishop, 1987; Draetta et al., 1988), ellular interations with the extraellular matrix (Shaller & Parsons, 1993) and the regulation of neurotransmitter reeptors (O'Dell et al., 1991): for review see Glenney (1991). Against this bakground, tyrosine kinase inhibitors (TKIs) have reently been developed. Two groups of TKIs have been desribed: ompounds interating with the ATP binding site, suh as genistein, a queretin derivative (Akiyama et al., 1987; asnellie, 1991) and those whih interat with the substrate binding site, suh as the tyrphostins, whih are syntheti analogues of erbstatin (Levitzki & Gilon, 1991; asnellie, 1991). Inative analogues with respet to the epidermal growth fator reeptor kinase are also now available for eah group, namely daidzein and tyrphostin A-1, respetively. In smooth musle ells, growth fators ativate tyrosine kinases (Auger et al., 1989; Yang et al., 1992; Weiss & Nuitelli, 1992), and also have agonisti effets with respet Present address: ' Department of Physiology, University of Mediine and Pharmay Isai, Universitatii Str. 16, Isai 66, Romania. 2 Departmento de Fisiologia, Faultad de Veterinaria, Universidad omplutense, iudad Universitaria, 284 Madrid, Spain. 3 Department of linial Pharmaology, St Mary's Hospital Medial Shool, London W2 1NY. Author for orrespondene. to fore development (Berk et al., 1986; Yang et al., 1992). onversely, lassial onstritor agonists like angiotensin II, vasopressin and arbahol have been shown to ativate smooth musle tyrosine kinase and ause tyrosine phosphorylation (Tsuda et al., 1991), and have growth promoting effets. Thus, the tyrosine phosphorylation assoiated with the appliation of these hormones ould be of importane for both the growth response and the ontratile response of these ells. Muh of the evidene indiating that tyrosine kinase ativity influenes fore development is based on the potent antagonisti effet of TKIs against growth fator-indued fore development in smooth musles (see, Hollenberg, 1994). For the lassial onstritor agonists suh evidene is less lear. For some agonists, like arbahol and bradykinin, the inhibitory poteny of the TKIs is reported to be low (Yang et al., 1992; 1993), while for others, like angiotensin II, the poteny is high (Yang et al., 1993). Moreover, in a reent study, TKIs were shown to have an inhibitory effet in moderately high onentrations against arbahol- and noradrenaline (NA)-indued ontration (DiSalvo et al., 1993), while in another study they were without effet against phenylephrine- and phorbol ester-indued ontration (Sauro & Thomas, 1993). This prompted us to investigate a range of TKIs for their onentration-dependent effet on the aliumdependent and independent regulation of tone in rat isolated small mesenteri arteries. Experiments were also performed to determine the effet of TKIs on the intraellular alium ativity ([a2+]i) in these vessels.

2 Methods Preparation Mesenteri resistane arteries (seond or third order branhes of the superior mesenteri artery) from male Wistar rats (12-15 weeks old) killed with O2 were used for all experiments. The proedures used to isolate the arteries and the myograph for isometri fore measurements have been desribed previously (Mulvany & Halpern, 1977). Briefly, the arteries were disseted free from the surrounding onnetive tissue. Segments, approximately 2 mm long, were mounted in an isometri myograph (JP-trading, Denmark), as ring preparations. The internal irumferene of the vessels was set to.9 times the irumferene the vessels have at 1 mmhg, based on the passive length-tension urve (Mulvany & Halpern, 1977). At this setting, the arteries had an internal diameter of about 2 ;Lm and develop near maximal ative tension during stimulation (Mulvany & Halpern, 1977). In all experiments, exept those where the arteries were permeabilized, pretreatment with 6-hydroxydopamine (Aprigliano & Hermsmeyer, 1976), or 1 pm guanethidine for 2 min, was used to eliminate the effet. Toma et al Tyrosine kinases and smooth musle ontration of endogenous NA. NW-nitro-L-arginine methyl ester (L-NAME, 1 im) was added 3 min before the experimental proedure and during experiments to avoid possible interferene from NO release from endothelial ells. All experiments were started by repetitively stimulating vessels with a solution of 125 mm K+ (KPSS, for omposition see below) ontaining 1lM NA for 2 min with 1 min between stimulations, until reproduible ontrations were eliited. Experimental proedure The effets of different TKIs on ontrations indued by IO M NA or KPSS were assessed 1 min after induing ontrations. umulative onentration-response urves were obtained for tyrphostin A-1, A-23, A47, bis-tyrphostin, genistein and daidzein. All TKIs were dissolved in dimethylsulphoxide (DMSO). Time ontrol experiments where the vehile was added, were made before and after the appliation of TKI. The maximal DMSO onentration in the bath was 1.27% (for 1#LM TKI). Permeabilization with a-toxin The proedure used to permeabilize these vessels with a-toxin was desribed by Jensen (1994). The arteries were mounted as desribed above and stimulated one with KPSS at room temperature (experiments with permeabilized preparations were always onduted at room temperature). The bubbling was hanged to 1% 2 and the arteries were inubated for 15-2 min in relaxing solution (for omposition see below). Permeabilization was made by inubating the arteries 1-15 min in 1 pl relaxing solution with 1 glm free a2' and 1 u ml-' a-toxin. After permeabilization, the arteries were held in relaxing solution. They were stimulated twie with 1O PM free a2+ before further experimental proedures. The effet of the different TKIs was assessed on ontrations indued with 1 JLM free a2+ to give near maximal ontration or with 2 nm free a2+ after pretreatment of the arteries with 1 1M NA and 3 jam GTP for 1 min, whih gave the same ontration as 1 tm free a2+. Time ontrol experiments where the vehile was added, were arried out before and after the appliation of TKI. Simultaneous measurements of ytoplasmi free alium andfore We have previously desribed this tehnique and the alibration of the fluoresene signal in detail (Jensen et al., 1992; 1993). Briefly, arteries mounted in the myograph were loaded with fura-2. The arteries were stimulated every 1 s with 347 and 38 nm light. The emitted light was measured by a photomultiplier through a 5-53 nm filter and a ut-off (<72 nm) filter. The system was ontrolled by a omputer, and both fore and the two emission signals (F347 and F38) were stored for subsequent analysis. At the end of the experiment fura-2 signals were alibrated, based on the determination of the maximal (Rm.) and minimal (Rni,,) fluoresene ratio and the ratio (P) between the maximal and minimal fluoresene at 38nM (Grynkiewiz et al., 1985; Jensen et al., 1992). Bakground fluoresene was determined after quenhing with 2 mm MnI2. With this setup the only inhibitor we ould use was genistein, beause the other TKIs quenhed more than 9% of the fura-2 signals. Solutions The physiologial saline solution (PSS) had the following omposition (mm): Nal 119, Kl 4.7, KH2PO4 1.18, MgSO4 1.17, NaHO3 25, al2 2.5, ethylenediaminetetraaeti aid (EDTA).26 and gluose 5.5. The PSS was bubbled with 95% 2 and 5% O2 (ph = at 37 ). PSS ontaining high K+ (KPSS, 125 mm K+) was prepared by iso-osmolar substitution of Nal with KL. Relaxing solution had the following omposition (mm): EGTA 2, potassium methane sulphonate 13, Mgl2 4, Tris maleate 2, Na2ATP 4, reatine phoshate 1 and reatine phosphokinase 1 mg ml-'. ph was adjusted to 7.15 with KOH and the solution was gassed with 1% 2- hemials Fura-2AM and pluroni F127 (Moleular Probes, Oregon, U.S.A.), a-toxin (GIBO, U.S.A.), tyrphostins, genistein and daidzein (albiohem, alifornia, U.S.A.), remophor EL, noradrenaline, nigeriin, ionomyin, guanethidine, L-NAME and 6-hydroxydopamine (Sigma hemials, Poole, Dorset, U.K.) were used. The high purity EGTA (>99%) used in solutions for in situ alibration of fura-2 was from Fluka (Buhs, Switzerland). The TKIs were prepared as 1 mm stok solutions in DMSO. Statistis All results are shown as arithmetri means ± s.e.mean, n is the number of arteries, exept for the mean [a2j]i values, whih are given as geometri means. Student's two-tailed t test or one way ANOVA test followed by Bonferroni test were used for statistial omparisons. P <.5 was onsidered as a signifiant differene. Results Effets of TKIs on ontration indued with NA and KPSS 1267 The effets of TKIs on toni ontrations indued with either IO M NA or KPSS were assessed. The results are presented in two main groups, aording to the two different mehanisms for inhibition of the tyrosine kinases. In the first group (Figure la and b), genistein and daidzein aused similar onentration-dependent relaxations of the ative tension indued with both NA and KPSS. The pims (- logi5, where IM is the onentration of the ompounds ausing a 5% inhibition of the ontration) of genistein and daidzein in arteries ativated with KPSS were 4.65 ±.1 and 4.47 ±.3, n = 4, respetively (P<.5). The pims of genistein and daidzein in arteries ativated with 1 gm NA were 4.86 ±.1 and 4.56 ±.8, n = 4, respetively (P <.5). A slight but signifiantly greater poteny of genistein ompared to daidzein was thus observed both in vessels ativated with NA and with KPSS.

3 1268 In the seond group, the effets of four tyrphostins were assessed. Tyrphostin A-23, A-47 and A-1 aused onentration-dependent relaxations of ontration indued with both NA and KPSS (Figure I and d), but bistyrphostin had no signifiant effet on ontrations. Tyrphostin A-23 in a onentration of 1 gm aused a omplete inhibition of the fore development and pi5o was 4.3 ±.6, n = 6 and 4.43 ±.13, n = 6 for arteries._4 ) : 1.4a 1.2F 1.o F a 1. O ).4._ In a) 1 4) u a._a a) ) Z a) [ o.8 F.6 F.4 F I!t~~~i j- ~ AA * s ; S \ 1~~~ 44~~~~~~ d F 1. F.8F.6 F.4 F.2. A A ----~~~~ log (Inhibitor, [M]). Toma et al Tyrosine kinases and smooth musle ontration -4. Figure 1 Effets of tyrosine kinase inhibitors (TKIs) on potassiumindued ontration (a and ) and noradrenaline-indued (1 AM) ontration (b and d) in intat arteries. The responses are expressed relative to the ontration immediately before the first onentration of drug or vehile was added. Eah point represents mean (with s.e.mean of 6 vessels where they exeed the size of the symbols). (a and b): () Vehile; () genistein; (A) daidzein. ( and d): () Vehile; (A) bis-tyrphostin; (*) A47; (*) A-i; (U) A-23. ativated with KPSS and 1 pm NA, respetively. The inhibitory effet of tyrphostin A-47 and A-I was too small, in the onentration-range tested, to alulate the Im. After washout of genistein, daidzein, tyrphostin A-23, A-47 and A-i the response to NA and KPSS almost ompletely reovered (data not shown exept for genistein see Figure 2). Effets of genistein on fore and [a2-]i during stimulation with NA and KPSS Arteries were pretreated for 1 min with 3 tm genistein before stimulation with NA or KPSS. When genistein was added to the medium, both the F347 and the F38 signals 2 z a b 1.4r M 1. 4 J i u )._o z a) u 5 1 min 1 min - ~Noradrenaline (1 gm) Genistein (3 gm) A KPSS Genestein (3 gm) Figure 2 Reording of measurements of ontration and [a2+] during ativation with noradrenaline (NA) (a) and high K+ physiologial saline solution (KPSS) (b). Where indiated 3 jm genistein was present. In (a) the middle trae shows the ratio of fura-2 fluoresene before orretion for genistein-indued quenhing of the fluoresene.

4 . Toma - -- et - - al -- Tyrosine -I- kinases and ---- smooth musle ontration TyrphostinA23 I I 1 jm a2+ 1 gim a Tyrphostin A23 I I I 1 2 nm a2, 2 nm a gm GTP + 1 gm NA 2 N m-l L~ 15 min I Figure 3 Trae showing the ontration in a-toxin-permeabilized mesenteri small arteries during stimulation with I AM a2l and with 2 nm a2l in the presene of I1 im noradrenaline (NA) and 3 JM guanosine S'-triphosphate (GTP). Where indiated, tyrphostin A-23 was added in inreasing onentrations. dropped signifiantly, and also the ratio was redued (Figure 2a) as previously reported (Sargean et al., 1993). The effet was quikly reversible after washout of genistein. At the end of experiments after fura-2 signals had been quenhed by Mn2", 1 gm genistein had little effet on the remaining bakground signals (data not shown). This indiated that genistein quenhed some of the a2"-sensitive signals of fura-2, and ould also reflet an effet of genistein on the IKd of fura-2 for [a2+]i. However, if 3 JLM genistein was added to the aid form of fura-2 in PSS, there was little effet on fura-2 signals, indiating that genistein affeted only the fura- 2 signals from the ells. To irumvent these problems, the a2"-sensitive fluoresene signals in the presene of genistein were alibrated using a Rm., Rin and P whih were determined in the presene of genistein. Seondly, the KAd of the fura-2 a2" omplex in the presene of genistein was determined in the ells as desribed previously (Jensen et al., 1993) and found not to be different from the Kd in the absene of genistein. For alibration of the fura-2 signals we therefore used the previously determined Kd of 342 nm (Jensen et al., 1993). Figure 2 depits the effet of 3 gm genistein on ative tension and [a2j]i during stimulation with NA, (Figure 2a) and during stimulation with KPSS (Figure 2b). In the presene of genistein, ative tension after 1 min stimulation was signifiantly lower with both NA ativation (2.35 ±.15 Nm-' and.96 ±.21 Nm-', n = 6; ontrol and genistein treated, respetively) and KPSS ativation (2.72 ±.4 Nm-' and 1.27 ±.32 Nm-', n = 4; ontrol and genistein-treated, respetively). However, [a2+] at this time was signifiantly affeted by genistein only during stimulation with NA (p([a2+]j): ±.48 and ±.127, n = 6; ontrol and genistein-treated, respetively); during KPSS ativation no differene in [a2]j was observed (p([a2`]j): ±.37 and ±.43, n = 4; ontrol and genistein, respetively). Effets of TKI on tension of t-toxin permeabilized arteries Figure 3 illustrates the protool used in the experiments with a-toxin permeabilized preparations. After a-toxin permeabilization, the arteries developed an ative tension of 1.81 ±.7 N m'l (n = 5) when maximally stimulated with 1 ytm free a2". This response was 52 ± 2% of the response to KPSS (3.53 ±.12 N m-', n = 5) in the preparations before permeabilization. Sine the response of rat mesenteri small arteries to KPSS is redued to about 55% with denervation or in the presene of an ax-adrenoeptor bloker (see e.g. Jensen et al., 1992) this suggests that probably all of the smooth musles in the preparation are ativated by the 1 pm free a2+ and therefore presumably permeabilized. The effets of the two groups of TKIs on the ontration indued by a submaximal free a2" onentration (I tlm) are shown in Figure 4a and b, and their inhibitory potenies are given in Table 1. a2" (1 JLM) eliited, in most preparations, a biphasi response with an initial transient and a seond sustained phase whih reahed a plateau after 1-15 min (Figure 3). The plateau tension was 1.9 ±.9 N m' (n = 28), and this response was 6.1 ±.5% of the maximal tension eliited by 1 AM free a2+. Tyrosine kinase inhibitors relaxed in a onentrationdependent and reversible manner the ontration indued by a2+. At the highest onentration used (1 pm), the maximal inhibitions eliited by genistein and daidzein were 93 ± 2% (n = 5) and 9+ 4% (n = 5), respetively, of the a2"-indued ontration (Figure 4a). The relaxations evoked by tyrphostin A-23 and tyrphostin A-47 were 62± 8% (n = 6) and 8 + 4% (n = 6) (Figure 4b), respetively. In ontrast, 1 tim tyrphostin A-I indued only a slight inhibition of the ontration indued by a2, whereas the effet of bis-tyrphostin was not signifiantly different from that of the vehile. There were no signifiant differenes among the inhibitory potenies of the ative ompounds on the ontrations indued by 1 JiM free a2+ (Table 1). Effets on ontration indued with a2- of NA and GTP in the presene Addition of 1 1M NA and 3 ptm GTP 1 min before stimulation, inreased the ontration to 2 nm a2" from.54 ±.6 N m-' (n = 26) to 1.31 ±.13 N m-' (n = 26). These responses were 29 ± 6% and 7 ± 3% of the maximal tension eliited by 1 tlm free a2", respetively. Figure 4 and d shows the effets of TKIs on ontration indued by a2" in the presene of NA and GTP. The maximal relaxations evoked by genistein and daidzein were % (n = 5) and 93 ± 2% (n = 5) of the initial ontration, respetively (Figure 4). The maximal relaxations eliited by tyrphostin A-23 and tyrphostin A-47 were 68 ± 2% (n =4) and 87 ± 2% (n = 6), respetively (Figure 4d). The effet of bistyrphostin on ontration to a2" in the presene of NA and GTP was not signifiantly different from that of the vehile. Tyrphostin A-1 evoked a slight inhibitory effet at the highest onentrations used (Figure 4d). Genistein was more potent in inhibiting ontrations eliited by a2" in the presene of NA and GTP, than ontration eliited by a2' alone (Table 1), while for the other TKIs no signifigant differene was observed. Disussion In the present study, the effets of two struturally and pharmaologially distint groups of TKIs on ative tension

5 127 in mesenteri small arteries have been haraterized. In the disussion we first fous on the poteny of these substanes and seondly we disuss the possible role of tyrosine kinases for the exitation-ontration oupling in small arteries. Ċ _ u ad.r4 a1) U.._ ) )._ 1) nra) >._ 1.2a 1. F.8F.6.2 F F.6 ;.4 F.2 r d F \* * ,~~~~~ A ~ 7 :~=:. Toma et al Tyrosine kinases and smooth musle ontration 4 4 * log (Inhibitor, [M]) lb U -4. Figure 4 Effets of tyrosine kinase inhibitors (TKI) on a2l (a, b) and a2l + noradrenaline (NA) + guanosine 5'-triphosphate (GTP) (, d) indued ontration of a-toxin-permeabilized mesenteri small arteries. onentration-response urves for the inhibitory ation of genistein and daidzein (a, ) and tyrphostins (b, d) are shown. Responses are expressed as perentage of the ontration just prior to the addition of the inhibitor or vehile. Eah point represents mean (with s.e.mean where they exeed the size of the symbols) of 1 vessels. (a and ): () Vehile; () genistein; (A) daidzein. (b and d): () Vehile; (A) bis-tyrphostin; (*) A-47; (*) A-1; (O) A-23. Poteny of tyrosine kinase inhibitors in resistane arteries Genistein, whih binds to the ATP binding site of tyrosine kinase, inhibited ontration with a poteny that is onsistent with it ating through inhibition of tyrosine kinase. Partiularly in the m-toxin-permeabilized preparation, the poteny of genistein (Im a. 6 JAM) is in the same range or lower than that reported for the inhibitory effet of genistein on angiotensin II (Yang et al., 1993) or epidermal growth fator (EGF) indued ontration (Yang et al., 1992) in guinea-pig stomah musle. However, in the intat preparation, genistein was also potent with an I5 of about 2 jam. On the other hand, the supposedly inative analogue of genistein, daidzein, also potently inhibited the ontration both in the intat preparation and in the m-toxin-permeabilized preparation. Therefore, although the poteny of daidzein was about two times less than that of genistein towards NA ativated vessels, it is possible that genistein and daidzein also affet a tyrosine kinase independent pathway in these vessels, or that the tyrosine kinase in the smooth musle ells of the resistane arteries is suseptible to daidzein in low onentrations. Of the four tyrphostins tested in this study, A-1 is the supposedly inative analogue and was indeed inative in the permeabilized preparation in the sense that it had no effet at onentrations up to 1 ;M. However, in the intat preparation 1 JAM A-1 did ause an inhibition of both the K+- and the NA-indued fore development. This ould be aounted for by an inhibitory effet of tyrphostin A-1 on the voltageativated alium hannels, whih we have previously reported (Wijetunge et al., 1992). Bis-tyrphostin, whih has a speifiity towards EGF-reeptor tyrosine kinase, had no effet on either K+-indued or NA-indued responses, and was also inative in the permeabilized preparation in onentrations up to 1I JM. This suggests that bis-tyrphostin may be used as an inative analogue in the vessels studied here. In the rabbit ear artery though, one of us (Hughes, 1994) has shown, that 5 JAM bis-tyrphostin inhibits the PDGF-indued tension. With respet to the ative analogues, both A-23 and A-47 aused an inhibition of fore development. In the a- toxin-permeabilized preparation, the I5 of these two drugs was about JAM. This indiates a slightly less potent effet than reported for the EGF-ativated guinea-pig gastri musle (Yang et al., 1992), for the EGF-ativated rabbit Table 1 Inhibitory potenies of tyrosine-kinase inhibitors on the ontrations indued by I nm a2+ and by 2 nm a2" in the presene of noradrenaline (NA) plus guanosine 5'-triphosphate (GTP) in a-toxin-permeabilized mesenteri resistane arteries a2+(1 pi5o AM) a2+ (.2 gm) + GTP (3 jam) +NA (1 1M) n pi5 n Genistein 4.82 ±.6 (5) (5) Daidzein 4.75 ±.8 (6) 4.83 ±.5 (5) Tyr A-I N (7) N (6) Tyr A ±.19 (6) 4.74 ±.7 (4) Tyr A ±.13 (6) 4.76±.11 (6) Bis-Tyr N (4) N (4) Values are expressed as means ± s.e.mean. n indiates the number of vessels, N means not alulated. The inhibitory poteny of eah ompound is expressed as pim = - logim, where I5 is the onentration of the ompounds ausing a 5% inhibition of the ontration indued by either a2l alone or a2l plus NA and GTP. 'Indiates signifiant differenes between the plso for genistein and daidzein (one-way ANOVA, posterio Bonferroni test). 2Indiates signifiant differenes between the pl5 of genistein towards a2l and a2l plus NA and GTP ativated vessels.

6 aorta (Merkel et al., 1993) and for the PDGF-ativated rat aorta (Sauro & Thomas, 1993), but in the same range as that reported for the angiotensin II-ativated guinea-pig gastri musle (Yang et al., 1993) and probably onsistent with these drugs ating through inhibition of tyrosine kinase. In the intat preparation, the poteny of the TKIs was less than in the x-toxin-permeabilized preparations. It has been suggested (Lyall et al., 1989) that it may take up to 16 h for the full effet of the tyrphostins to develop in intat ells. It is possible therefore that the lower apparent poteny in the intat preparation, where the effet was assessed a few minutes after the TKIs were added, refleted poor penetration of the TKIs. This may point to diffiulties in using TKIs for aute pharmaologial experiments in membrane-intat preparations. Effet of tyrosine kinase inhibitors on alium-mediated ontration One of the important aspets of this study was to assess whih parts of the exitation-ontration oupling was potentially dependent on tyrosine phosphorylation. One way this was addressed was by omparing the effet of the TKIs on ontration indued by K+ and by NA. It has previously been reported (DiSalvo et al., 1993) that 5j1M tyrphostin A-47 inhibits the ontration of guinea-pig mesenteri small arteries to NA by about 8%, while the K+-indued ontration was inhibited only by 2%. An apparent seletivity for the NA-indued ontration was not onfirmed in this study, where the NA-indued response and the K+-indued response was inhibited with almost the same poteny and maximal effet by the tyrphostins. This observation was supported by the similar poteny of the tyrphostins against the ontration indued by alium and by alium plus GTP and NA in the x-toxin-permeabilized preparation. It may not be surprising that agonist independent ontration (K+-indued ontration of the intat arteries and alium-indued ontration of the a-toxin permeabilized preparation) was inhibited by TKIs, sine it has been shown in ultured vasular smooth musle ells (Tsuda et al., 1991) that the alium ionophore, ionomyin, an indue tyrosine phosphorylation. In fat, the tyrosine phosphorylation pattern indued by the ionophore was not dissimilar to that indued by vasoonstritors inluding NA (Tsuda et al., 1991). There seems therefore good evidene that alium an indue a tyrosine kinase ativity in vasular smooth musles. Also genistein had a potent effet on the KPSS-indued ontration and the alium-indued ontration in the permeabilized arteries whih supports this suggestion. In ontrast to the tyrphostins, genistein was, however, more potent towards the NA-indued than to the KPSS-indued ontration. This would suggest that in addition to the alium-indued tyrosine kinase ativity, NA may indue a tyrosine kinase ativity with a slightly higher suseptibility to genistein. The finding that genestein was also more potent towards the permeabilized arteries ativated with alium in the presene of NA and GTP ompared to the arteries ativated with alium alone might suggest that the additional tyrosine kinase ativity may play a role in the. Toma et al Tyrosine kinases and smooth musle ontration 1271 steps involved in agonist-indued modulation of the effetiveness of [a2+]j. On the other hand, the observation that the relaxant effet of genestein was assoiated with a derease of [a2+]i in NA-ativated arteries, but not in potassium-ativated arteries, indiates that the additional tyrosine kinase ativated by NA may also be involved in regulation of [a2+]i (see below). Effet of genistein on the ontrol of [a2+i We have previously shown that the alium urrent in rabbit ear artery smooth musle ells an be inhibited by genistein (Im, 36 glm), tyrphostin A-23 (I5, 88 gm) and tyrphostin A-I (I5, 11 in on- IOM), although daidzein had no effet entrations below 3 FM (Wijetunge et al., 1992). As pointed out above, the relaxant effet of tyrphostin A-l in the nonpermeabilized preparation ould be explained by the inhibition of the potential sensitive alium hannels. However, it was unexpeted that 3 gm genestein did not redue [a2+]i in potassium-ativated vessels, in view of the above findings on the rabbit ear artery smooth musle ells. Although we have no explanation for this, it is possible that the poteny of genestein is different in the rabbit ear artery smooth musle and in the rat mesenteri resistane arteries, either as a onsequene of speies differenes or due to the fat that the voltage lamp experiments were done on isolated ells while the [a2j]i measurements were made in whole tissue. In ontrast, [a2+j was redued by 3 gm genestein in NAativated rat mesenteri arteries. In these vessels, the effet of NA on [a2+], is thought to be mediated almost exlusively by depolarization with a onsequent opening of voltagedependent alium hannels (Nilsson et al., 1994). It is therefore a possibility that a tyrosine kinase is affeting the signal leading from stimulation of the adrenoeptor to membrane depolarization. In onlusion, these data support a role for tyrosine kinases in the fore development to lassial vasoonstritors. 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