E ects of the antihypertensive agent, cicletanine, on noradrenaline release and vasoconstriction in perfused mesenteric artery of SHR

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1 British Journl of Phrmcology (1998) 123, 427 ± Stockton Press All rights reserved 7 ± 1188/98 $12. E ects of the ntihypertensive gent, cicletnine, on nordrenline relese nd vsoconstriction in perfused mesenteric rtery of SHR 1 Yoshihis Ns, Hiroyuki Yoshid, Mki Urt, Kumiko Uchiyshi, Yumi Tsunod, Keiko Kmigt & Stoshi Tkeo Deprtment of Phrmcology, Tokyo University of Phrmcy nd Life Science, Horinouchi, Hchioji, 192-3, Jpn 1 The mechnism y which cicletnine () exerts its ntihypertensive e ects hs not een fully elucidted. The present study ws undertken to exmine the e ects of in vivo nd in vitro tretment with on the pressor response nd nordrenline (NA) over ow during perirteril nerve stimultion (PNS) in perfused mesenteric rteril eds isolted from spontneously hypertensive rts (SHR). 2 t dose of 5 mg kg 71 dy 71 ws dministered orlly to oth SHR nd normotensive Wistr- Kyoto rts (WKY) from the 6th to 1th week of ge. At the 1th week, the isolted mesenteric rteril ed ws perfused with Kres-Henseleit u er nd chnges in perfusion pressure nd NA over ow during PNS were mesured. 3 Chronic tretment with suppressed the ge-relted elevtion of systemic lood pressure in SHR ut not in WKY. 4 The PNS (2 Hz)-induced mesenteric vsoconstrictor response nd NA over ow were greter in SHR thn in WKY. In the vsculture of SHR chronic tretment with resulted in signi cnt ttenution of the vsoconstriction nd the NA over ow during PNS, wheres it did not lter vsoconstrictor responses to olus injections of KCl nd phenylephrine. 5 Tretment with 3 mm in vitro diminished the PNS-induced vsoconstriction nd NA over ow ut not the NA- nd KCl-induced vsoconstriction in the vsculture of untreted SHR. 6 In the vsculture of SHR PNS-induced NA over ow ws ttenuted y prostglndin E 2 (.5 mm), wheres it ws ugmented y the cyclo-oxygense inhiitor diclofenc-n (3 mm). In the presence of diclofenc, in vitro tretment with did not ttenute the NA over ow during PNS. 7 The results suggest tht the ntihypertensive e ect of in SHR is prtilly due to the presynptic inhiition of NA relese during sympthetic nerve ctivtion. Trnsjunctionl inhiition of NA relese y prostglndins my contriute to the inhiitory ction of on NA relese in the vsculture of SHR. Keywords: Cicletnine; perfused mesenteric rtery; perirteril nerve stimultion; nordrenline over ow; prostglndin; spontneously hypertensive rts Introduction The ntihypertensive gent cicletnine (), furopyridine derivtive, exhiits severl phrmcologicl ctions on vsculr vessels including ctivtion of prostcyclin synthesis (Dorin et l., 1984; 1988; Clder et l., 1992,), histmine H 1 -receptor lockde (Schoe ter et l., 1987; Schoe ter & Godfrind, 1988), 1 -drenoceptor lockde (Chrier et l., 1988), clcium chnnel lockde (Nock & Deitmer, 1993), opening of potssium chnnels (Kolti et l., 199), inhiition of gunosine 3':5'-cyclic monophosphte (cyclic GMP) phosphodiesterse (Silver et l., 199; 1991), nd direct relxtion of vsculr smooth muscle (Auguet et l., 1988). The mechnism underlying the ntihypertensive ction of is complex nd not fully understood. Long-term tretment with reduces systemic lood pressure in spontneously hypertensive rts (SHR) (Auguet et l., 1988; Jin et l., 1991; Ando et l., 1994) nd deoxycorticosterone cette (DOCA)-slt hypertensive rts (Fuentes et l., 1989; Cstro et l., 199) in which sympthetic nerve ctivity is incresed (Judy et l., 1976; Fujit et l., 1983). Acute tretment with lso reduced lood pressure in rts with stress induced y socil deprivtion (Cstro et l., 1988). 1 Author for correspondence. Sympthetic nerve ctivity of these rts is lso considered to e elevted. In ddition, Ando et l. (1994) hve shown tht reduced lood pressure of SHR fed with high slt diet, which exhiited incresed sympthetic nerve ctivity. The reduction ws ccompnied y decrese in the plsm concentrtion of ctecholmines (Ando et l. 1994). These ndings suggest tht exerts its ntihypertensive e ect through suppression of n elevted sympthetic tone. However, it is not known whether cts directly on sympthetic nerve terminls, resulting in inhiition of nordrenline (NA) relese upon nerve stimultion. The present study ws undertken to elucidte the possile mechnism of the inhiition of sympthetic tone y. For this purpose, the ex vivo nd in vitro e ects of on vsoconstriction nd NA over ow during perirteril nerve stimultion (PNS) in the mesenteric rteril ed isolted from SHR were exmined. In this preprtion, electricl nerve stimultion results in n over ow of NA tht is greter in SHR thn in Wistr-Kyoto rts (WKY) (Eks & Lokhndwl, 1981; Tsud et l., 1984). Furthermore, vsculr rectivity to exogenous NA is enhnced in the mesenteric rteril ed in SHR (Eks et l., 1983). Thus, the preprtion is pproprite for studying the e ect of on the sympthetic nerve system. If n inhiitory ction of on sympthetic trnsmitter

2 428 Y. Ns et l relese contriutes to its ntihypertensive ction, oth vsoconstriction nd NA over ow during PNS should e reduced in vessels from nimls treted with. In ddition, since the 1 -drenoceptor locking ction of hs een shown to e wek (Auguet et l., 1988), the vsoconstriction to PNS should e reduced y more e ectively thn tht to exogenous vsoconstrictor gents. It hs een shown tht increses the plsm concentrtions of prostglndin E 2 (PGE 2 ) nd prostglndin I 2 (Ysu et l., 1995), nd PGE 2 is known to inhiit NA relese vi the ctivtion of presynptic PGE 2 receptors (Tsud et l., 1987; Rump et l., 199). For this reson, the possile involvement of prostglndinmedited inhiition of NA relese in the ction of ws lso exmined. Methods Animls Six week-old mle SHR nd normotensive WKY (Chrles River Jpn, Atsugi, Jpn) were used in the present study. The rts were fed with regulr diet, llowed free ccess to tp wter nd cclimtized in qurntine room for t lest 1 week efore experiments. The niml protocol ws designed ccording to the Guideline of Experimentl Animl Cre issued from the Prime Minister's O ce of Jpn nd pproved y the Animl Cre nd Use Committee of the University. The SHR were rndomly divided into three groups: (1) SHR treted with for 4 weeks to ssess the chronic e ect, (2) SHR treted with the vehicle for 4 weeks, nd (3) gemtched untreted SHR to exmine the direct e ect of on the mesenteric rteril ed nd the possile involvement of prostglndins in modulting NA over ow. The WKY were lso divided into three groups, -treted WKY, vehicletreted WKY, nd ge-mtched untreted WKY. Mesurement of lood pressure Systolic lood pressure nd hert rte of conscious rts were determined y the til-cu method y use of lood pressure nlyser (BP-98A, Softron, Tokyo, Jpn) connected to personl computer (PC981 series, NEC, Jpn). An verge of three successive recordings ws mde once week, 3 ± 4 h fter the orl dministrtion of the gent. In preliminry study, we found tht the ntihypertensive e ect of ws mximl 3 ± 4 h fter dministrtion. Perfused mesenteric rteril preprtion The perfused mesenteric rteril ed ws prepred ccording to McGregor (1965). The rts were nesthetized with nitrous oxide, oxygen (3:1) nd 2.% hlothne. After lprotomy, the superior mesenteric rtery ws dissected nd clened of surrounding tissue. A stinless-steel cnnul (21G syringe, externl dimeter.8 mm, Terumo, Tokyo, Jpn) ws inserted into superior mesenteric rtery, nd the preprtions were perfused with Kres-Henseleit solution descried elow. After cnnultion, rnches of mesenteric rteries to the descending colon proximl to the rectum, those to the duodenum proximl to the stomch, those to the cecum nd the inferior mesenteric rtery rnches were tied o. Then, the entire mesenteric vsculture ws seprted from the smll intestine. The preprtion ws plced in chmer with wter jcket mintined t 378C nd covered with cotton guze to keep it moist. The preprtion ws perfused with Cicletnine nd nordrenline relese Kres-Henseleit solution equilirted with mixture of 95% O 2 nd 5% CO 2 (PO 2 46 mmhg). The Kres-Henseleit solution consisted of (mm): NCl 118, KCl 4.7, MgSO 4 1.2, KH 2 PO 2 1.2, CCl 2 2.5, NHCO 3 25, EDTA-N 1.25 nd glucose 11 (ph 7.4). The perfusion ow rte ws mintined t 2. ml min 71 with peristltic pump (Perist Pump, SJ-1211, Atto, Co., Ltd., Tokyo, Jpn). Perfusion pressure ws mesured through rnch of the perfusion cnnul y mens of pressure trnsducer (TP-4T, Nihon Kohden, Tokyo, Jpn) connected to crrier mpli er (AP-621G, Nihon Kohden, Tokyo, Jpn) nd recorded on therml penrecorder (WT-645G, Nihon Kohden, Tokyo, Jpn). The preprtion ws equilirted for 3 min efore the onset of ech experiment. Perirteril nerve stimultion (PNS) Two pltinum electrodes were used to pply perirteril nerve stimultion (PNS); one ws plced t the cnnul inserted into the superior mesenteric rtery nd the other t the peripherl prt of the mesenteric rteril ed. PNS ws pplied with n electricl stimultor (SEN-33, Nihon Kohden, Tokyo, Jpn) connected to n electricl isoltor (SS-32J, Nihon Kohden, Tokyo, Jpn). For pressor responses to PNS, rectngulr pulses (mplitude of 6 V nd pulse durtion of 3 ms) were pplied t frequency of either 5, 1, 2 or 4 Hz. The preprtion ws stimulted for 3 s t 5 min intervls. To determine the over ow of NA during PNS the mesenteric rteril ed ws stimulted t 2 Hz for 3 s (rectngulr pulses of 3 ms durtion) nd the perfuste ws collected. In preliminry study, we oserved tht frequencydependent vsoconstriction in the vsculture of SHR ws sustntilly locked y.3 mm tetrodotoxin nd 1 mm gunethidine (dt not shown). This is in greement with previous oservtions y others (Kwski & Tkski, 1984; Tsud et l., 1984; Li & Duckles, 1992). The concentrtions of tetrodotoxin nd gunethidine employed here did not lock the pressor response to exogenous NA. Although in the rt mesentric vsculture ATP my contriute to the pressor response, we determined only NA over ow during PNS. Chronic tretment with cicletnine Cicletnine (, 5 mg kg 71 ), suspended in.25% sodium croxymethyl cellulose (CMC-N) solution with the id of sonictor, ws dministered orlly through sonde (.2 ml 1 g 71 rt ody weight) t noon of ech dy from the 6th to 1th week of ge. Systolic lood pressure ws mesured once week, 3 ± 4 h fter dministrtion of. As vehicle-treted groups, vehicle suspended with.25% CMC-N (.2 ml 1 g 71 rt ody weight) ws dministered. Four weeks lter, the perfused mesenteric vsculr ed ws prepred. In these preprtions, pressor response nd NA over ow cused y PNS t 2 Hz were mesured, nd then pressor responses to KCl nd phenylephrine were determined. In vitro exposure to cicletnine Ten week-old, untreted SHR nd WKY were used. To ssess the in vitro e ects of, the mesenteric rteril ed ws perfused with u er contining either 3 or 1 mm or vehicle (.2% dimethylsulphoxide, DMSO). According to the results of Bukoski et l. (1993), t the concentrtion of 1 mm or more elicits signi cnt vsorelxtion of mesenteric resistnce rteries of SHR precontrcted with NA. In contrst,.1 to 1 mm of did not signi cntly ect the vsculr

3 response to NA nd other vsoconstrictors (Silver et l., 1991; Deitmer et l., 1992; Bukoski et l., 1993). Thus, we employed two concentrtions of, 3 nd 1 mm, in the in vitro study. In the rst series of experiments, pressor response nd NA over ow cused y PNS t 2 Hz were mesured in the presence of 3 mm, nd then pressor responses to KCl nd NA were determined. In the second series of experiments, frequency-response curve to PNS nd dose-responses curve to exogenous KCl nd NA were determined in the presence of 1 mm. ws perfused from 3 min efore the rst ppliction of PNS to the end of the experiment. Potssium chloride, phenylephrine nd NA solutions were injected in the perfusion u er t volume of 5 ml. The olus injection of 5 ml of vehicle into the perfusing solution produced little e ect on perfusion pressure. Role of prostglndins To exmine the possile involvement of prostglndins in the response to, the e ects of PGE 2 nd diclofenc sodium (DCF), cyclo-oxygense inhiitor, were studied in mesenteric rteril eds isolted from 1 week-old SHR. In preliminry study, ttenution of NA over ow y PGE 2 nd enhncement y DCF were not oserved when no NA trnsport inhiitor ws in the perfuste. This oservtion is in greement with previous one (Tsud et l., 1987). Thus, this series of experiments ws performed in the presence of.2 mm nisoxetine, NA trnsport inhiitor (Richelson & Nelson, 1984; Tejni-Butt, 1992). In these studies the mesenteric preprtions were stimulted once t 2 Hz (S1) to stilize further response to PNS. After 3 min equilirtion period, PNS-induced NA over ow ws determined (S2) in the sence of PGE 2 or DCF. The preprtions were then treted with.5 mm PGE 2 or 3 mm DCF for 1 h, nd NA over ow during PNS ws determined (S3) in the presence of PGE 2 or DCF. % chnges in NA over ow ((S3 ± S2)/S261) were clculted. As time-mtched control, NA over ow in the sence of PGE 2 or DCF ws lso determined. In nother set of experiments, the e ect of on PNSinduced NA over ow ws studied in vsculr eds from SHR in the presence or sence of 3 mm DCF. The perfusion protocol ws the sme s in the PGE 2 nd DCF experiments. Thus, the e ect of on PNS-induced NA over ow ws studied in one preprtion. NA over ow ws determined (S2) efore tretment, nd then t 3 mm ws pplied to the preprtion for 1 h followed y determintion of NA over ow in the presence of (S3). Similrly, the e ect of on NA over ow ws studied in the presence of DCF, which ws pplied during the whole period of the experiment. Both nisoxetine nd DCF were present in the Kres-Henseleit u er. % chnges in NA over ow ((S3 ± S2)/S261) were clculted. As time-mtched control, NA over ow in the sence of ws lso determined. Determintion of NA Y. Ns et l The perfuste through the mesenteric vsculture ws collected in tue contining.1 mg ml 71 glutthione s n ntioxidnt. NA ws sored on ctivted lumin t ph 8.3 ± 8.5 which ws otined y ddition of 2 M mmonium cette. The lumin ws wshed twice with distilled wter. NA ws then extrcted from the lumin with 2 ml of.2 M perchloric cid contining.1% EDTA-N 2. An liquot ws pplied to high-performnce liquid chromtogrphy (h.p.l.c.) system with n electrochemicl detector (ECD-1, Eicom, Kyoto, Jpn) (Hjemdkl et l., Cicletnine nd nordrenline relese ). 3,4-Dihydroxyenzylmine ws used s n internl stndrd. NA levels were normlized to g wet tissue weight for ech preprtion. NA over ow induced y PNS ws de ned s the di erence etween the sl NA e ux detected for 3 min efore stimultion nd the NA level detected during the 3 s of PNS nd the susequent 2.5 min period. Drugs The following drugs were used: (7)-phenylephrine hydrochloride, gunethidine monosulphte, 3,4-dihydroxyenzylmine hydroromide, prostglndin E 2, diclofenc-n (Sigm, MO, U.S.A.), nisoxetine hydrochloride (Funkoshi Ykuhin, Tokyo, Jpn), lumin ctivted 2 for determintion of ctecholmine (Ncli Tesque, Kyoto, Jpn), dimethylsulphoxide (Knto Chemicl, Tokyo, Jpn), (7)-nordrenline itrtrte, tetrodotoxin (Wko Pure Chemicl Industries, Osk, Jpn). Cicletnine hydrochloride ws generous gift from Nippon Roussel (Tokyo, Jpn). All gents except were dissolved in distilled wter just efore use. For the in vitro experiment, ws dissolved in.2% DMSO. Dt nlysis Results re expressed s the mens+s.e.men. Two-wy nlysis of vrince (ANOVA) for repeted mesures ws pplied to determine whether systolic lood pressure nd hert rte were ected y ge nd/or tretment. Two-wy ANOVA (repeted mesures) ws lso pplied to determine whether the pressor response to PNS or vsoconstrictors ws ected y frequency or dose nd/or tretment. In some cses two-wy fctoril ANOVA ws used to determine whether ech prmeter ws ected y the strin (SHR vs WKY) nd/or tretment. Post-hoc comprisons were performed y Sche e's test. Where pproprite, unpired twotiled Student's t test nd pired t test were used. In ll cses di erences with proility of 5% or less (P5.5) were considered to e sttisticlly signi cnt. Sttisticl nlysis ws performed with SttView softwre (Acus Concepts, Berkeley, CA, U.S.A.). Results Chronic tretment with Tle 1 shows chnges in systolic lood pressure nd hert rte in SHR nd WKY treted with or vehicle from the 6th to 1th week of ge. Chronic tretment with signi cntly ttenuted the rise in systolic lood pressure of the SHR. did not ect systolic lood pressure in WKY. Hert rte in oth groups ws not ected y. The e ects of chronic tretment with on PNS-induced vsoconstriction nd NA over ow re shown in Figure 1. The vsoconstrictor response to PNS t 2 Hz ws signi cntly greter in preprtions of SHR thn in preprtions of WKY (P=.4, two-wy ANOVA). The greter response ws ssocited with signi cntly greter NA over ow in SHR (P=.1, two-wy ANOVA). In the SHR, chronic tretment with resulted in signi cnt ttenution in oth vsoconstriction nd NA over ow. In the WKY, tretment with did not lter the PNS-induced vsoconstriction nd NA over ow. The e ects of chronic tretment with on KCl- nd phenylephrine-induced vsoconstriction re shown in Figure 2. A olus injection of KCl or phenylephrine resulted in dose-

4 43 Y. Ns et l Cicletnine nd nordrenline relese Tle 1 Chnges in systolic lood pressure (SBP) nd hert rte (HR) in spontneously hypertensive rts (SHR) nd Wistr-Kyoto rts (WKY) treted with cicletnine (, 5 mg kg 71 dy 71 ) or vehicle from the 6th to 1th week of ge SBP (mmhg) HR (ets min 71 ) Group n 6th week 1th week P (ANOVA) 6th week 1th week P (ANOVA) SHR vehicle P (vehicle vs ) Age, 5.1 Tret, Age,.342 Tret,.65 WKY vehicle P (vehicle vs ) Age,.1 Tret, Age,.1 Tret,.45 Ech vlue represents the men+s.e.men. Two-wy ANOVA (repeted mesures) ws used to determine whether SBP or HR ws ected y ge or tretment (Tret) with vehicle or. Men vlues for SBP nd HR t ech ge point were tested for di erences etween tretments y post-hoc comprison with Sche e's method. Chnges in perfusion pressure (mmhg) NA overflow (pg g 1 wet wt) dependent increse in perfusion pressure. These pressor responses were greter in SHR thn in WKY (P=.1 nd P=.1, KCl- nd phenylephrine-induced responses, respectively, two-wy ANOVA). In contrst to the pressor response to PNS, the rectivity of the mesenteric rteril ed of SHR to KCl nd phenylephrine ws not in uenced y chronic tretment with. Similrly, in WKY neither KCl- nor phenylephrine-induced vsoconstriction ws reduced fter tretment. In vitro e ects of (6) (6) SHR The in vitro e ects of (3 mm) on vsoconstrictor response to PNS, KCl nd exogenous NA re shown in Figure 3. (5) (5) WKY 1 (6) (6) (5) (5) SHR WKY Figure 1 Perirteril nerve stimultion (PNS)-induced pressor response () nd nordrenline (NA) over ow () in the perfused mesenteric rteril ed of SHR nd WKY treted with cicletnine (, 5 mg 71 kg 71 dy) or vehicle for 4 weeks. PNS t 2 Hz ws pplied to the vsculr eds. Dt re shown s the mens+s.e. men. Numers in prentheses represent the numers of nimls *P5.5 vs corresponding vehicle-treted groups (post-hoc comprisons were performed y Sche e's method). Chnges in perfusion pressure (mmhg) KCI (µmol) Phenylephrine (nmol) Figure 2 Potssium chloride- nd phenylephrine-induced pressor response in the perfused mesenteric rteril ed of SHR () nd WKY () treted with cicletnine (, 5 mg 71 kg 71 dy) or vehicle for 4 weeks. Potssium chloride (KCl) or phenylephrine t doses of 75 ± 15 mmol or 1 ± 5 nmol, respectively, ws injected into the perfuste in volume of 5 ml. Dt re shown s the mens+s.e. men of 5 ± 6 nimls for ech group. There were no signi cnt di erences etween vehicle nd -tretment (two-wy ANOVA). Electricl stimultion resulted in frequency-dependent pressor response. Infusion of (3 mm) per se did not ect the sl perfusion pressure. This concentrtion of suppressed the PNS-induced vsoconstriction ut did not ect the KCl- nd exogenous NA-induced vsoconstriction in mesenteric rteril eds of SHR nd WKY. The in vitro e ects of 3 mm on pressor response nd NA over ow of the mesenteric rtery of untreted SHR nd WKY t the stimultion frequency of 2 Hz re shown in Tle 2. The sl e ux of NA ws not ected y in either group (dt not shown). Two-wy ANOVA showed tht oth pressor response nd NA over ow were ected y the strin nd tretment. In the mesenteric preprtion of SHR, PNS-induced NA over ow ws signi cntly reduced y, n e ect ssocited with mrked reduction in the pressor response to PNS. In the vsculture of WKY, the pressor response only tended to e decresed y tretment with. PNS-induced NA over ow ws not ected y in WKY. The in vitro e ects of higher concentrtion (1 mm) of on pressor responses to PNS, KCl nd NA in

5 Y. Ns et l Cicletnine nd nordrenline relese 431 Chnges in perfusion pressure (mmhg) c d PNS (Hz) KCI (µmol) NA (nmol) Figure 3 E ects of 3 mm cicletnine () on pressor responses to PNS (, c), KCl nd nordrenline (, d) in the perfused mesenteric rteril eds of (, ) SHR nd (c, d) WKY. The ge of the SHR nd WKY (1 weeks old) ws the sme s tht of the nimls used in Figures 1 nd 2. Either or its vehicle ws perfused from 3 min efore PNS to the end of the experiment. The mesenteric rteril preprtion ws electriclly stimulted t frequencies rnging from 5 to 4 Hz for 3 s. Potssium chloride or nordrenline t nl doses of 75 ± 15 mmol or.1 ± 1 nmol, respectively, ws injected into the perfuste. Dt re shown s the mens+s.e.men of 5 nimls for ech group. *P5.5 vs corresponding vehicle-treted groups. In () nd (c), two wy fctoril ANOVA of the sum of the vlues of pressor response in oth groups showed tht PNS-induced pressor response ws ected y the strin (P =.33) nd y (P =.1) wheres two-wy ANOVA for repeted mesures showed tht the PNS-induced pressor response ws ected y in SHR (P5.1) ut not in WKY (P =.283). In (), two-wy ANOVA for repeted mesures showed tht pressor response to NA ws not ected y 3 mm (P =.279). Tle 2 In vitro e ects of 3 mm cicletnine () on pressor response nd nordrenline over ow during perirteril nerve stimultion (PNS) t 2 Hz in mesenteric rteril eds isolted from untreted SHR nd WKY Pressor response (mmhg) Nordrenline over ow (pg g 71 wet wt) Tretment SHR WKY P (ANOVA) SHR WKY P (ANOVA) Strin, (5) (7) Tret,.1 (5) (6) (6) (7) (5) (6) P (vehicle vs ) Strin, 5.1 Tret,.1 Either (3 mm) or its vehicle ws infused from 3 min efore PNS to the end of the experiments. Ech vlue represents the men+s.e.men. Numers in prentheses indicte the numers of nimls. Two-wy fctoril ANOVA ws used to determine whether presssor response or nordrenline over ow ws ected y the strin (Strin) or tretment with vehicle or (Tret). Men vlues for pressor response nd nordrenline over ow in ech strin, di erences etween vehicle nd tretment were tested y post-hoc comprison with Sche e's method. mesenteric rteril eds of the untreted SHR re shown in Figure 4. In the presence of 1 mm, the pressor responses to olus injection of KCl nd NA s well s those to PNS were signi cntly ttenuted in mesenteric rteril eds of the SHR. Role of prostglndins The e ects of PGE 2 nd DCF on PNS-induced NA over ow in the untreted SHR re shown in Figure 5. PNS-induced NA over ow ws signi cntly ttenuted y PGE 2 wheres it ws signi cntly ugmented y DCF. The result of time-mtched control experiments indicted tht the e ects of PGE 2 nd DCF were not due to time trends. The in vitro e ects of on NA over ow in the presence of DCF re shown in Figure 6. The inhiitory e ect of 3 mm on NA over ow ws not oserved in the presence of DCF. Discussion Hyperctivity of the sympthetic nervous system in SHR is well-known. In the isolted, perfused mesenteric rteril eds, PNS results in frequency-dependent increse in perfusion pressure, which is greter in SHR thn in WKY (Eks & Lokhndwl, 1981; Tsud et l., 1984). PNS-induced vsoconstriction is recognized minly to e due to endogenous NA relesed from sympthetic nerve endings

6 432 Y. Ns et l during PNS (Kwski & Tknshi, 1984 Tsud et l., 1984; Li & Duckles, 1992), which ctivtes the postsynptic 1 - drenoceptor on the vsculr smooth muscle. In ddition, the rectivity of vsculr smooth muscle to vsoconstrictor gents is lso elevted in SHR (Eks & Lokhndwl, Cicletnine nd nordrenline relese 1981; Tsud et l., 1984). Thus, the vsculr rectivity of SHR to sympthetic nervous stimultion is ugmented t oth presynptic nd postsynptic sites nd the incresed vsculr rectivity contriutes to the development of hypertension in SHR. Chnges in perfusion pressure (mmhg) PNS (Hz) KCI (µmol) NA (nmol) Figure 4 E ects of 1 mm cicletnine () on pressor responses to PNS (), KCl nd nordrenline () in the perfused mesenteric rteril eds of SHR. The ge of the SHR (1 weeks-old) ws the sme s tht of nimls used in Figure 3. Either or vehicle ws perfused from 3 min efore PNS to the end of the experiment. Experimentl protocol ws the sme s tht in Figure 3. Dt re shown s the mens+s.e.men of 5 nimls for ech group. *P5.5 vs corresponding vehicle-treted groups. % chnges in NA overflow NA overflow (pg g 1 wet wt) S2 S3 S2 S3 PGE 2 +PGE 2 DCF +DCF Figure 5 E ects of prostglndin E 2 (PGE 2 ) nd diclofenc sodium (DCF) on nordrenline (NA) over ow during PNS in the mesenteric rteril ed of SHR. The over ow of NA during PNS in the sence nd presence of PGE 2 or DCF (S2 nd S3, ) nd % chnges in NA over ow ((S3 ± S2)/S261, ) re shown. Ech preprtion ws perfused with Kres-Henseleit u er contining.2 mm nisoxetine nd sujected to PNS t 2 Hz 3 times. At 3 min fter the 1st PNS (S1), NA over ow during PNS in the sence of PGE 2 or DCF ws determined (S2). Then, the preprtion ws treted with either.5 mm PGE 2 or 3 mm DCF for 1 h efore the 3rd PNS (S3). In time-mtched control experiments (no PGE 2, no DCF), there ws no signi cnt di erence etween S2 ( pg g 71, n = 5) nd S3 (477+6 pg g 71, n = 5). Dt re shown s the mens+s.e.men of 6 ± 7 nimls for ech group. *P5.5 vs corresponding control. % chnges in NA overflow NA overflow (pg g 1 wet wt) S2 S3 S2 S3 + + DCF + + Figure 6 E ects of cicletnine () on nordrenline (NA) over ow during PNS in the sence or presence of 3 mm diclofenc sodium (DCF). The over ow of NA during PNS efore (S2) nd fter tretment with 3 mm (S3) in the sence nd presence of DCF () nd % chnges in NA over ow ((S3 ± S2)/S261, ) re shown. Ech preprtion ws perfused with Kres-Henseleit u er contining.2 mm nisoxetine nd sujected to PNS t 2 Hz 3 times. At 3 min fter the 1st PNS (S1), NA over ow during PNS in the sence of ws determined (S2). Then, the preprtion ws treted with for 1 h efore the 3rd PNS (S3). In time-mtched control experiments (no ), there ws no signi cnt di erences etween S2 ( pg g 71, n = 5) nd S3 ( pg g 71, n = 5). Dt re shown s the mens+s.e.men of 5 ± 7 nimls for ech group. *P5.5 vs corresponding control.

7 Y. Ns et l To exmine the e ect of on the sympthetic nervous system, we mesured PNS-induced pressor responses of mesenteric rteril eds in SHR nd WKY. We lso mesured NA levels in the e uent during PNS. Our hypothesis is tht, if possesses direct inhiitory ction on NA relese upon stimultion, oth pressor response nd NA over ow would e reduced in SHR treted with. The mjor nding of the present study ws tht oth pressor response nd NA over ow during PNS of the mesenteric rteril eds were ttenuted y long-term tretment with in SHR, ut not in WKY. This ws lso ssocited with suppression of the elevtion in systolic lood pressure only in SHR. hs een shown to exert n ntihypertensive e ect in SHR in which sympthetic nerve ctivity is high (Jin et l., 1991; Ando et l., 1994). Our results suggest tht long-term tretment with suppresses the elevted NA relese in peripherl vsculr eds, nd this ction of contriutes to its ntihypertensive e ect in SHR. High doses of KCl ct directly on vsculr eds nd depolrize them followed y vsoconstriction. Phenylephrine is n 1 -drenoceptor gonist. The present study showed tht pressor responses to KCl nd phenylephrine in the vsculture of SHR were greter thn those of WKY, nd these responses were not ttenuted y chronic tretment with. Thus, it is suggested tht long-term tretment with does not ect the incresed rectivity of the vsculr smooth muscle in SHR to these vsoconstrictors. Acute in vitro tretment with t concentrtion of 3 mm inhiited PNS-induced vsoconstriction more e ectively thn KCl- or exogenous NA-induced vsoconstriction. This suggests tht preferentilly suppresses the elevted sympthetic nerve ctivity in the SHR s compred with its e ects on the enhnced rectivity of vsculr smooth muscle. The results further suggest tht the sympthetic nervous system is trget for the -induced ntihypertensive ction in SHR. This is in greement with the hypothesis of other investigtors, tht exhiits potent ntihypertensive e ect in nimls with enhnced sympthetic nerve ctivity such s slt-sensitive SHR (Jin et l., 1991; Ando et l., 1994). Since neither KCl- nor phenylephrine-induced vsoconstriction ws ttenuted y cute ( t 3 mm) or chronic tretment with, the gent ppers to hve only minor e ects on depolriztion-induced or 1 -drenoceptor-medited pressor responses in the vsculture of SHR. A higher concentrtion (1 mm) of reduced vsoconstrictor responses to KCl nd NA s well s those to PNS. Auguet et l. (1988) showed tht 1 mm reduced oth NA- nd ngiotensin II-induced vsoconstriction, suggesting direct vsodiltor ction of on vsculr smooth muscle. As mentioned in Methods, t the concentrtion of 1 mm or more exerts direct vsodiltor e ect in the mesenteric rtery of SHR precontrcted with NA (Bukoski et l., 1993). Therefore, high concentrtions of my exert direct vsorelxnt e ects in SHR vessels, possily through mechnisms other thn n inhiitory ction on NA relese. NA relese from sympthetic nerve endings is loclly regulted y vriety of endogenous sustnces, such s prostglndins, ngiotensin II, denosine nd relesed NA itself. These vsoctive sustnces ctivte presynptic receptors. Altered prejunctionl control mechnisms my modify NA relese in the peripherl vsculr eds of SHR nd this my contriute to the pthogenesis of hypertension nd the ntihypertensive mechnism of. It hs een shown tht plsm levels of PGE 2 nd the urinry level of PGE 2 re Cicletnine nd nordrenline relese 433 incresed fter dministrtion in ptients with essentil hypertension (Ysu et l., 1995; Guinot & Frolich, 1985). Rump et l. (199) suggested tht in rt kidney there is trnsjunctionl PGE 2 inhiition of NA relese, i.e., NA relesed from nerve endings my induce the locl formtion nd relese of PGE 2 which then trnsjunctionlly inhiits further NA relese vi ctivtion of presynptic PGE 2 receptors. Thus, it is possile tht reduces NA over ow through the incresed production of PGE 2. In the present study, we showed tht the over ow of NA ws reduced y exogenously dministered PGE 2 while it ws enhnced y the cyclo-oxygense inhiitor DCF. The ltter nding suggests tht endogenous prostglndins, which cn modulte NA over ow during PNS, re produced in the isolted mesenteric rteril ed. In fct, it hs een oserved tht considerle mounts of PGE 2 re produced spontneously within this preprtion nd tht the production is incresed y exogenous NA (Pipili & Poyser, 1982). Thus, it is likely tht enhnced production nd relese of prostglndins such s PGE 2 ply role in the inhiitory e ect of on NA over ow. This is supported y the nding tht the inhiitory e ect of in vitro on NA over ow during PNS ws olished y DCF, consistent with the results of Cstro et l. (1989) tht pretretment with indomethcin lunted the ntihypertensive e ect of. In ddition, hs een shown to increse prostcyclin production in vsculr smooth muscle cells (Dorin et l., 1984; 1988; Clder et l., 1992; 1992). However, the inhiitory e ect of prostcyclin on NA relese is less potent thn tht of PGE 2 (Weitzell et l., 1978). Thus, the contriution of prostcyclin to the e ect of on NA relese my e smll. It must e noted tht lthough exogenously dministered PGE 2 diminished NA over ow during PNS in the mesenteric rteril preprtion, PGE 2 enhnces pressor responses of mesenteric rteril eds to NA nd PNS (Jckson & Cmpell, 198). The mechnism of the ltter phenomenon remins uncler. Since diminished oth vsoconstriction nd NA over ow during PNS, the mechnism y which inhiits NA relese cnnot e explined merely y the enhnced production of PGE 2. Severl other mechnisms y which reduces the sympthetic nerve ctivity hve een suggested, including inhiition of cyclic GMP phosphodiesterse (Silver et l., 199; 1991), opening of potssium chnnels (Clder et l., 1992) nd clcium chnnel lockde (Nock & Deitmer, 1993) in sympthetic nerve endings. Silver et l. (1991) demonstrted tht the potentiting e ect of on sodium nitroprusside-induced vsorelxtion ws not ltered in the presence of cyclo-oxygense inhiitor. This cyclo-oxygense inhiitor-resistnt component my lso contriute to the ntihypertensive mechnisms of. In conclusion, we demonstrted tht oth in vivo nd in vitro tretment with reduced NA over ow during sympthetic nerve stimultion. The results suggest tht chronic tretment with diminishes the incresed sympthetic nerve ctivity through inhiition of NA relese from nerve endings. Enhnced production nd relese of prostglndins my contriute to the mechnism y which reduced NA relese from sympthetic nerve endings of vsculr eds in SHR. This ction my ply n importnt role in the ntihypertensive e ect of in hypertensive nimls. The uthors wish to thnk Nippon Roussel Co., Ltd. (Tokyo, Jpn) for providing cicletnine.

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