Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature

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1 BrItIsh Journal of Pharmaology (1996) 117, B 1996 Stokton Press All rights reserved /96 $12. P Vasorelaxant properties of norbormide, a seletive vasoonstritor agent for the rat mirovasulature 'Sergio Bova, Luia Trevisi, Patrizia Debetto, Lorenzo Cima, *Maurizio Furnari, Sisto Luiani, Roberto Padrini & Gabriella Cargnelli Department of Pharmaology, University of Padova, Italy and *Division of Anaesthesiology, Hospital of Camposampiero, Italy 1 The effets of norbormide on the ontratility of endothelium-deprived rat, guinea-pig, mouse, and human artery rings, and of freshly isolated smooth musle ells of rat audal artery were investigated. In addition, the effet of norbormide on intraellular alium levels of A7r5 ells was evaluated. 2 In resting rat mesenteri, renal, and audal arteries, norbormide (.5-5 gm) indued a onentration-dependent ontratile effet. In rat audal artery, the ontration was very slowly reversible on washing, ompletely abolished in the absene of extraellular alium, and antagonized by high onentrations (1-8 ym) of verapamil. The norbormide effet persisted upon removal of either extraellular Na+ or K+. The ontratile effet of norbormide was observed also in single, freshly isolated smooth musle ells from rat audal artery. 3 In resting rat and guinea-pig aortae, guinea-pig mesenteri artery, mouse audal artery, and human subutaneous resistane arteries, norbormide did not indue ontration. When these vessels were ontrated by 8 mm KCl, norbormide (1-1 um) aused relaxation. Norbormide inhibited the response to Ca2l of rat aorta inubated in 8 mm KCl/Ca2+-free medium. Norbormide (up to 1 gm) was ineffetive in phenylephrine-ontrated guinea-pig and rat aorta. 4 In A7r5 ells, a ell line from rat aorta, norbormide prevented high K+- but not 5-hydroxytryptamine-indued intraellular alium transients. 5 These findings indiate that in vitro, norbormide indues a myogeni ontration, seletive for the rat small vessels, by promoting alium entry in smooth musle ells, presumably through alium hannels. In rat aorta and arteries from other mammals, norbormide behaves like a alium hannel entry bloker. Keywords: Norbormide; vasular smooth musle rings; vasular smooth musle ells; Ca2l influx; intraellular Ca2" levels; alium hannel entry bloker Introdution Norbormide, 5-(a-hydroxy-a-2-pyridylbenzyl)-7-(a-2-pyridylbenzylidene) 5-norbornene-2,3-diarboximide, is a rat toxiant desribed for the first time by Roszkowski (Roszkowski et al., 1964; Poos et al., 1966). Its toxiologial profile is very peuliar, for norbormide is the most seletive toxiant so far known in mammals (Roszkowski, 1965), its toxiity being onfined to a single genus, viz. Rattus. Data from in vivo experiments indiate that the toxiity of norbormide in rat is aounted for by tissue ishaemia due to a generalized vasoonstrition (Roszkowski, 1965). Furthermore, the vasospasti effet of norbormide ours only in rat vessels of relatively small alibre (i.e. mesenteri, ear, and oronary arteries) sine the drug does not ontrat rat aorti segments (Roszkowski, 1965). Thus, norbormide seems to be a vasoative agent, very seletive for the mirovessels of the rat. Neither the mehanism(s) underlying the vasoonstritor effet of norbormide nor the basis of its high seletivity for the rat mirovasulature have been ever investigated. Furthermore, reported in vivo experiments give no indiation as to whether norbormide exerts other effets different from vasoonstrition on vessels of other speies. This in vitro study has been undertaken to investigate the mehanism(s) of the vasospasti effet of norbormide on rat mirovasulature and its speies seletivity. In addition, we have investigated whether norbormide ould affet the ontratile funtion of vessels different from rat mirovasulature. For this purpose, norbormide has been tested on rings of both ondutane and resistane vessels isolated from rat (aorta, mesenteri, renal, and audal arteries), guinea-pig (aorta and 1 Author for orrespondene at: Department of Pharmaology, L.go E. Meneghetti, 2, Padova, Italy. mesenteri artery), mouse (audal artery), and man (subutaneous arteries), on freshly isolated smooth musle ells of rat audal artery, and on intraellular alium levels of A7r5 ells. Our results onfirm the seletivity of the vasospasti effet of norbormide for the rat mirovasulature and demonstrate that the drug ats as a vasodilator on all the other arteries tested. Methods Arterial rings Male Sprague-Dawley rats (25-3 g), guinea-pigs (3-35 g) and mie (4-5 g) were killed by deapitation, exsanguinated, and the thorai aorta (rat and guinea-pig), the ventral audal artery (rat), the renal (rat), and the first branh of the mesenteri artery (rat and guinea-pig) were removed. Human subutaneous resistane arteries were obtained from biopsies of anterior abdominal wall fat taken during routine abdominal surgery on patients who were reeiving no mediation. All the vessels were leaned of onnetive tissue and ut into rings of 2 mm length. The endothelium was removed by gently rubbing the lumen of the rings with a round wooden stik (aorta) or a very thin rough-surfaed tungsten wire (audal, mesenteri, renal, and subutaneous arteries). The rings were vertially suspended between steel hooks (aorti rings) or tungsten wires (audal, mesenteri, renal, and subutaneous artery rings) in an organ bath filled with 18 ml of physiologial salt solution (PSS). The PSS was of the following omposition (mm): NaCl 125, KCl 5, CaC12 2.7, MgSO4 1, KH2PO4 1.2, NaHCO3 25, and gluose 11, at ph 7.35, main-

2 142 tained at 37C and bubbled with 95% 2/5% Co2. Tension was reorded on a pen reorder via an isometri fore displaement transduer. Rings were strethed passively to impose a resting tension (1.5 g for the aorti rings and.5-1 g for the audal, mesenteri, renal, and subutaneous artery rings) and were allowed to equilibrate for 6 min. After equilibration, eah ring was repeatedly stimulated with 1 Mm phenylephrine until a reproduible response was obtained. To verify the absene of the endothelium, rings ontrated with 1 tim phenylephrine were exposed to 1-2 gm arbamylholine. The absene of the endothelium was revealed by the lak of arbamylholine-indued relaxation. All experiments were performed in the presene of 1 gm indomethain, to avoid any possible release of ylo-oxygenase produts. Some experiments were performed on arterial rings bathed in modified physiologial salt solutions: Na+-free solution NaCl was omitted, surose was added to maintain osmolarity, and KHCO3 was substituted for NaHCO3 in the PSS. KV-free solution KC1 was omitted and NaH2PO4 was substituted for KH2PO4 in the PSS. Ca2"-free solution CaCl2 was omitted and 1 mm ethylene glyol bis (P-aminoethyl ether) N,N'-tetraaeti aid (EGTA) was added to the PSS. High-K+ solution in the PSS. A7rS ells KCl onentration was inreased to 8 mm Cell ulture The A7r5 ell line was obtained from Aviano Onology Centre (Aviano, Italy). The ells were grown in Dulbeo's Modified Eagle's Medium (DMEM) (Sigma Chemial Co. St. Louis, MO, U.S.A.) ompleted with 1 units ml-' peniillin, 1 gg ml-' streptomyin, 2.5 yg ml-' fungizone, and 1% foetal bovine serum (FBS) (Sigma Chemial Co. St. Louis, MO, U.S.A.) at 37C under an atmosphere of 95% air/5% CO2. Before use, ells were allowed to reah onfluene, after whih they were maintained in.5% FBS for 7-12 days. Cells were then trypsinized and replated in 8 x 3-mm glass overslips at a density of 15, ells ml1-. The overslips were inubated overnight with DMEM plus 1% FBS to allow a better attahment of the ells; afterwards FBS onentration was again redued to.5% until the end of the experiment. Measurement of intraellular Ca2" levels in A7r5 ells [Ca21+] was measured by fluorometry in a double-wavelength mode in a Perkin Elmer LS-5B spetrofluorometer equipped with a magneti stirrer and a temperature regulator. Cells were loaded with fura-2 aetoxymethyl ester (fura-2/am) (Moleular Probes In., Eugene, OR, U.S.A.) as previously desribed (Goldman et al., 199). Briefly, A7rS ells grown as monolayers on glass overslips were inubated with 2 ml of omplete DMEM ontaining 1 gm fura-2/am and 2.5 y1 of 25% w/w pluroni F-127 for 12 min at room temperature. Coverslips were then transferred to a ustomized holder, inserted in a quartz uvette, and plaed in the spetrofluorometer where they were superfused at a rate of 5 ml min-' for 3-4 min with modified Krebs solution (MKS) of the following omposition (mm): NaCl 14, KCl 5.9, NaH2PO4 1.2, MgCl2 1.4, CaCl2 1.8, [4-(2-hydroxyethyl)-l-piperazine ethane sulphoni aid] (HEPES) S and gluose 11.5, at ph 7.4 and 37 C. [Ca2"1i was alulated by the formula of Grynkiewiz et al. (1985): [Ca2+]i = R x sf X Kd S. Bova et al Vasorelaxant and vasoonstritor effts of norbormide where Kd is the dissoiation onstant of fura-2 and was assumed to be 225 nm, R is the 34/38 nm ratio of fura-2 fluoresene measured in the ells, Rma, is the 34/38 nm ratio in the presene of a saturating alium onentration, Rmin is the 34/38 nm ratio of fura-2 fluoresene in a Ca2+-free solution ontaining K2H2-EGTA, and Sf/Sb is the ratio of Ca2"-free to Ca2+-bound fura-2 at 38 nm. Calibration was performed by using fura-2 free aid in 1% glyerol, that simulates the ell ytosol. R n was determined in a solution ontaining (in mm): KCl 115, NaCl 1, MgSO4 2, K2H2- EGTA 1, and K2-3-(N-morpholino) propanesulphoni aid (MOPS) 1 at ph 7.2. For the determination of Rma,,, 2 mm CaCl2 was added to the medium and CaK2-EGTA was substituted for K2H2-EGTA. Rm., Rmj,,, and Sf/Sb values were found to be 17.36, 1, and 7.81, respetively. In some experiments, A7r5 ells were depolarized by high- K+ solution (6 mm KCl), made by substituting KCl for equimolar NaCl in the MKS. Rat audal artery ells Isolation proedure Single smooth musle ells from rat audal artery were obtained from adult male Sprague-Dawley rats (25-3 g) as desribed by Furspan (1992). After exision of the ventral audal artery, proximal segments (about 6 mm long) were arefully leaned and inubated in a digestion medium ontaining.3 units ml-' ollagenase B (Boehringer Mannheim GmbH, Biohemia, Germany),.3 mg ml-' trypsin inhibitor,.4 mg ml-' papain,.3 mg ml-' dithiothreitol, and 7.5 mg ml-' bovine serum albumin (fatty aid free) for 9 min at 37 C. The segments were then transferred to a 35 mm ulture dish and gently shaked to obtain isolated smooth musle ells. Evaluation of ell ontration Culture dishes, superfused with MKS (see above) at a rate of 5 ml min'- at room temperature, were mounted on the stage of an inverted mirosope (Nikon TMD) onneted to a video imaging system able to display images of single ells. Drugs Norbormide was a kind gift of India S.p.A. (Padova, Italia). Phenylephrine hydrohloride, yohimbine hydrohloride, arbamylholine hloride, verapamil hydrohloride, 5-hydroxytryptamine hydrohloride, and indomethain were from Sigma Chemial Co. (St. Louis, MO, U.S.A.). Indomethain was dissolved in ethanol and norbormide in dimethylformamide. At the onentrations reahed in the medium, ethanol and dimethylformamide had no effet either on resting or stimulated tissue tone. All the other drugs were dissolved in twie-distilled water. Data and statistis Contratile responses of arterial rings were measured as mg of developed tension. The onentrations of norbormide ausing the half-maximal ontration of rat audal, mesenteri, and renal artery rings or the half relaxation of 8 mm KCl-ontrated rat and guinea-pig aorti rings (EC5 values) were alulated for eah experiment. Data are expressed as the mean + s.e. mean. Results Vasoonstritor effet of norbormide Rat audal, mesenteri, and renal artery rings responded similarly to norbormide (.5-5 gm) with onentration-dependent ontrations (ECms gm, n = 8; ym, n = 3;.9 ±.33 gm, n = 3, respetively) (Figure 1), in agreement with Roszkowski's (1965) observations in vivo.

3 The effet of eah drug onentration reahed equilibrium within 15-2 min and was slowly reversible on drug washout, the maximally ontrated rings taking about 9 min to relax. To investigate further the ontratile effet of norbormide, the rat audal artery was hosen as an experimental model in the following experiments. In the absene of external alium, norbormide was unable to indue ontration, while a transient ontration was eliited by phenylephrine (Figure 2). Pretreatment (3 min) of the artery rings with the seletive a2- adrenoeptor antagonist, yohimbine (1 gm) did not affet the onentration-response urve to norbormide (data not shown). The rat audal artery rings maximally ontrated by norbormide were relaxed by verapamil (1-8 gm) (Figure 3). Norbormide ontrated rat audal artery rings inubated either in Na'- or K+-free medium (Figure 4). The ontratile effet of norbormide, evaluated as shortening of the ell, was also observed in vasular smooth musle ells freshly isolated from rat audal artery (Figure 5b) and was not reversed by washout (Figure 5). Vasorelaxant effet of norbormide Norbormide did not affet the resting tone of rings from rat and guinea-pig aorta, guinea-pig mesenteri artery, mouse S. Bova et al Vasorelaxant and vasoonstritor effets of norbormide 143 audal artery and human subutaneous arteries. However, when these vessels were ontrated by 8 mm KCl, norbormide (1-1 pm) indued a onentration-dependent relaxation. Figure 6 shows the onentration-response urves to norbormide of 8 mm KCl-ontrated rat and guinea-pig aorti rings. Norbormide onentrations induing half relaxation (EC,) were gm and YM for rat and guinea-pig aorti rings, respetively. The same results were obtained when verapamil (.1-1 Mm) was used instead of norbormide (not shown). Furthermore, norbormide antagonized the ontratile responses to alium of rat aorti rings depolarized with 8 mm KC1 (Figure 7). In rat and guinea-pig aorti rings ontrated with 1 gm phenylephrine, 1 gm norbormide was ineffetive. Effet of norbormide on intraellular alium transients in stimulated A7rS ells Exposure of resting A7r5 ells to 6 mm KCl or 1 Mm 5-hydroxytryptamine raised [Ca2+]i from nm (n = 18) to nm (n = 8) or nm (n = 6), respetively. Pretreatment of the ells with 5 gm norbormide ompletely 1 I-) r., 5-2- a C U._ Norbormide (gim) Figure 1 Cumulative onentration-response urves to norbormide of resting rat audal (), mesenteri (Oi), and renal () artery rings. Eah point is the mean + s.e. mean of eight, three, and three separate experiments, respetively. Responses are normalized for the maximal response to norbormide ( mg, mg, and mg, for rat audal, mesenteri, and renal artery rings, respetively). 1 min Verapamil (gm) Figure 3 Conentration-response urve to verapamil of rat audal artery rings ontrated by 5 gm norbormide. Eah point is the mean + s.e. mean of six separate experiments. The tension developed by the rings in response to 5OtsM norbormide was mg. 1 min. < a.5 g i A A A A A =- E ^ E 2 ± E o _~ O LU) <".. C 2 C C - z. + C C~~~~~~~~~~C ~~~~~ 4 o Figure 2 Reording from an experiment on one rat audal artery ring. The ring was first stimulated with 1OyM phenylephrine in the PSS and then in Ca2+-free/1mm EGTA medium. After a 6min washout in the PSS, the ring was restimulated with phenylephrine (not shown), re-exposed to Ca2+ -free solutions (Ca2+ -free/i mm EGTA medium for 5min and Ca2+-free medium for lmin), and treated with 5M norbormide for 2min. Then CaCl2 (2.7mM, final onentration) was added. b A ~~~ A Norbormide Na+- Norbormide 5 gm free 5 gim Ir~~~~~~~~~ Norbormide K+- A A A Norbormide 5 jim free 5 gm Figure 4 Reordings of the ontratile responses indued by 5M norbormide in two separate rat audal artery rings inubated in Na+-free (a) or K+-free solution (b). Note that the Na+-free solution eliits a ontratile response, while the K+-free solution does not.

4 144 prevented the inrease in [Ca2+]i indued by 6 mm KCl (Figure 8), but did not affet the response to 5-hydroxytryptamine (not shown). The same results were obtained when 1 klm verapamil was used instead of norbormide. Disussion Vasoonstritor effet of norbormide This study shows that norbormide seletively ontrats rat audal, mesenteri, and renal artery rings in vitro, in agreement with the vasoonstrition of rat small vessels indued by the drug in vivo (Roszkowski, 1965). This effet is neither mediated S. Bova et a! Vasoreluaant and vasoonstritor effets of norbormide by a release of noradrenaline from nerve endings, sine norbormide also indues ontration of single ells isolated from rat audal artery, nor by a release of produts of ylo-oxygenase ativity from the vasular myoytes, as the ontration also ours in the presene of indomethain. Therefore, the ontratile effet of norbormide is probably due to an ation on the vasular smooth musle ells. Rat audal artery rings bathed in alium-free medium do not respond to norbormide, while they respond to phenylephrine with a transient ontration. Thus norbormide, unlike agonists ating by mobilizing intraellular Ca2", eliits a ontratile response entirely dependent on external alium. This observation is not onsistent with a alium release from S1_v Ca2+ (mm) Figure 7 Conentration-response urves to alium of rat aorti rings depolarized by 8mM KCl in the absene () and presene of 25Mm (@) or 5Mm (U) norbormide. Eah point is the mean ± s.e. mean of at least six separate experiments. In eah ring, two umulative onentration-response urves to CaCl2 were obtained first in the absene and then in the presene of norbormide. Data are normalized, so that ontration of untreated ring in 2.7mM CaCl2 (final onentration in the PSS) is taken as the 1% ontration. The atual value of developed tension of the untreated rings in 2.7mM CaCl2 was 163 ± 119mg (n = 11). 3 a Figure 5 Video images of a rat audal artery ell in resting state (a), after 1 min exposure to 5 gm norbormide (b), and after 15 min washout (). r._ t 1 r- 51- v Norbormide (gm) Figure 6 Conentration-response urves to norbormide of rat () and guinea-pig () aorti rings ontrated by depolarization (8mM KC1). Eah point is the mean s.e.mean of at least seven separate experiments. The tensions developed by the aorti rings from rat and guinea-pig in response to 8 mm KC1 were mg (n = 7) and 137 ± 84 mg (n = 1), respetively. i C * t KCI 6 mm b A Norbormide KCI gm ao mm Time (s) Reordings of [Ca2+]1 inrease indued by 6mM KCl (a) Figure 8 and prevention of this inrease by 5Mm norbormide (b) in A7r5 ells. [Ca2+ ]i was determined in fura-2-loaded A7r5 monolayers as desribed under Methods. The traes are representative of at least five similar experiments.

5 L- U+3 - o 1 5 II Norbormide (gm) Figure 9 Vasoonstrition of rat audal artery rings () and vasorelaxation of depolarization-ontrated rat aorti rings () indued by norbormide. S. Bova et al Vasorelaxant and vasoonstritor effts of norbormide 5 o ~x o the saroplasmi retiulum indued by the drug, either through stimulation of reeptors oupled to IP3 (van Breemen & Saida, 1989; Khalil et al., 199), or by induing IP3 prodution via ativation of some step beyond the agonist reeptors, or by a affeine-like ation. Therefore, it appears that norbormide ativates ontration by promoting alium influx into vasular smooth musle ells. It is well established that alium enters vasular smooth musle ells through alium hannels and, under some irumstanes, through a Na+/Ca2+ exhange mehanism (Ashida & Blaustein, 1987; Carafoli et al., 199). It has been reported that alium hannel ativation is primarily involved in the ontration of vasular smooth musle indued by postjuntional x2-adrenoeptor stimulation (Langer & Shepperson, 1982; Jim & Matthews, 1985). An interation of norbormide with these reeptors an be ruled out, sine the ontratile effet of the drug is not inhibited by the seletive a2-adrenoeptor antagonist, yohimbine. We suggest that norbormide may operate as a alium hannel opener, although a funtional approah provides only indiret evidene on the mehanism of ation of a drug. Rat audal artery rings ontrated by norbormide are relaxed by verapamil at onentrations higher than those antagonizing the ontration onsequent to the opening of alium hannels by high potassium. As high onentrations of alium entry blokers may have intraellular effets (Cauvin et al., 1983), it ould be that verapamil reverses the norbormide effet through an intraellular mehanism. On the other hand, the ontrated audal artery rings take a long time to relax when norbormide is removed from the medium, indiating a long lasting ation and suggesting that the drug dissoiates very slowly from its binding site(s). This ould explain why high onentrations of verapamil, ating as a alium entry bloker, are needed to ounterat the norbormide ation, whatever the mehanism of alium hannel ativation by norbormide and whihever the site(s) of norbormide binding. Furthermore, the proposal that norbormide-indued ontration is due to the ativation of alium hannels ould be onsistent with the large doses of vasodilators suh as organi nitrates or isoprenaline needed to alter the threshold of norbormide vasoonstrition in vivo (Roszkowski, 1965), as these vasodilators are poorly effetive on the ontration dependent on alium hannel ativation by high potassium (Shultz et al., 1979; Linoln, 1983; Jones et al., 1984). An alternative explanation has been onsidered, taking into aount that the inrease in intraellular sodium onentration indues myogeni ontrations in several isolated vasular smooth musle preparations as a onsequene of alium entry through a Na+/Ca2+ exhange (Karaki et al., 1978; Ozaki et al., 1978; Bova et al., 1988; Nabel et al., 1988; Woolfson et al., 199). Intraellular sodium an be inreased by inhibiting the Na + /K+ ATPase ativity of vasular smooth musle ell plasmalemma. However, the ontratile response of rat audal artery to norbormide annot result from this mehanism, sine rat audal artery rings do not ontrat when the Na+/K+ ATPase ativity is inhibited by means of extraellular potassium removal and norbormide is still effetive in K+-free medium. Intraellular sodium onentration an also be inreased by promoting sodium entry (Shinjoh et al., 1991), but it is unlikely that norbormide ontrats rat audal artery rings by this mehanism, as the effet of the drug is still evident in the absene of external sodium. Vasorelaxant effet of normide 145 This study shows that norbormide behaves like the alium entry bloker verapamil in vessels other than the small vessels of the rat (e.g. audal artery), sine it relaxes rat aorta and both large and small vessels from other speies (aorta and mesenteri artery from guinea-pig, audal artery from mouse, and subutaneous arteries from humans) ontrated by depolarization. Norbormide, although less potent than verapamil, is as effetive as verapamil in relaxing these vessels. In guinea-pig and rat aorta, norbormide, at onentrations effetive against the ontration indued by depolarization, is ineffetive on the ontration indued by phenylephrine, a feature that has been observed also for verapamil (Bova et al., 1994) and diltiazem (Cauvin et al., 1984). Likewise, in A7r5 ells both norbormide and verapamil inhibit intraellular alium transients indued by depolarization, while they do not affet the inrease in [Ca2+]i eliited by 5-hydroxytryptamine. The similarity between the effets of verapamil and norbormide suggests that the two drugs share a ommon mehanism of ation. However, the patterns of ation of norbormide and verapamil do not overlap ompletely. A simple ompetition mehanism between alium and verapamil has been reported (Asano & Hidaka, 1984), while norbormide inhibits the maximal response to alium, suggesting a non-surmountable antagonism. Altogether these data indiate that norbormide an eliit opposite effets in isolated vasular smooth musle from different speies and, within the same speies, in different vasular areas (Figure 9). The finding that norbormide in vitro ontrats small alibre vessels from rat but not from guinea-pig, mouse, or man is onsistent with the seletivity of its toxi ation in vivo and indiates the myogeni nature of this ation. The in vitro vasorelaxant effet of norbormide ould aount for the hypotension observed in human subjets treated with norbormide (see Pelfrene, 1991). Present data suggest that the vasorelaxant and vasoonstritor properties of norbormide are mediated by an ation on alium movements aross the sarolemma, most likely through alium hannels, without affeting the intraellular alium stores. In this ontext, it is noteworthy that the vasorelaxant effet of norbormide ours in a range of onentrations very lose to those induing vasoonstrition (Figure 9). In onlusion, the in vitro results onfirm in vivo observations (Roszkowski, 1965) and show that norbormide has a seletive, myogeni, vasoonstritor effet on rat mirovasulature by promoting alium entry. On the other hand, in rat aorta, guinea-pig vessels, mouse audal artery as well as human subutaneous arteries, norbormide behaves as a alium entry bloker. The seletivity of norbormide vasoonstrition indiates that the mehanisms regulating alium handling exhibit some anomalous features in rat mirovasulature like those that our in rat heart (Noble & Powell, 1985). Furthermore, one an wonder whether some relationship exists between the peuliar behaviour of rat mirovasulature and the fat that mammalian models of primary hypertension have been developed only in the rat. The authors wish to thank Mr G. Greggio and R. Varotto for skilful tehnial assistane. This work was supported by grants from CNR (FATMA PF41; CT4; FATMA '8' PF ) and MURST (4% and 6%).

6 146 S. Bova et al Vasorelaxant and vasoonstritor effets of norbormide Referenes ASANO, T. & HIDAKA, H. (1984). Vasodilatory ation of HA 14 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], a novel alium antagonist with no effet on ardia funtion. J. Pharmaol. Exp. Ther., 231, ASHIDA, T. & BLAUSTEIN, M.P. (1987). Regulation of ell alium and ontratility in mammalian arterial smooth musle. The role of sodium-alium exhange. J. Physiol., 392, BOVA, S., CARGNELLI, G. & LUCIANI, S. (1988). Na/Ca exhange and tension development in vasular smooth musle: effet of amiloride. Br. J. Pharmaol., 93, BOVA, S., ROSSI, G.P., LUCIANI, S., DEBETTO, P., PESSINA, A.C. & CARGNELLI, G. (1994). Effet of subthreshold ouabain on the tone of guinea-pig aorti strips following repeated noradrenaline stimulation. Br. J. Pharmaol., 11, CARAFOLI, E., CHIESI, M. & GAZZOTTI, P. (199). The membrane arriers related to intraellular alium regulation. In Hypertension: Pathophysiology, Diagnosis, and Management. ed. Laragh, J.H. & Brenner, B.M. pp New York: Raven Press. CAUVIN, C., LOUTZENHISER, R. & VAN BREEMEN, C. (1983). Mehanisms of alium antagonist-indued vasodilation. Annu. Rev. Pharmaol. Toxiol., 23, CAUVIN, C., SAIDA, K. & VAN BREEMEN, C. (1984). Extraellular Ca2+ dependene and diltiazem inhibition of ontration in rabbit onduit arteries and mesenteri resistane vessels. Blood Vessels, 21, FURSPAN, P.B. (1992). WAY 12,491 ativates ATP-sensitive potassium hannels in rat audal artery. Eur. J. Pharmaol., 223, GOLDMAN, W.F., BOVA, S. & BLAUSTEIN, M.P. (199). Measurement of intraellular Ca2+ in ultured arterial smooth musle ells using Fura-2 and digital imaging mirosopy. Cell Calium, 11, GRYNKIEWICZ, G., POENIE, M. & TSIEN, R.Y. (1985). A new generation of Ca2 + indiators with greatly improved fluoresene properties. J. Biol. Chem., 26, JIM, K.F. & MATTHEWS, W.D. (1985). Role of extraellular alium in ontrations produed by ativation of postsynapti alpha-2 adrenoeptors in the anine saphenous vein. J. Pharmaol. Exp. Ther., 234, JONES, A.W., BYLUND, D.B. & FORTE, L.R. (1984). AMP-dependent redution in membrane fluxes during relaxation of arterial smooth musle. Am. J. Physiol., 246, H36 - H31 1. KARAKI, H., OZAKI, H. & URAKAWA, N. (1978). Effets of ouabain and potassium-free solution on the ontration of isolated blood vessels. Eur. J. Pharmaol., 48, KHALIL, R.A., LODGE, N.J., SAIDA, K., GELBAND, C.H. & VAN BREEMEN, C. (199). Calium mobilization in vasular smooth musle and its relevane to the etiology of hypertension. In Hypertension: Pathophysiology, Diagnosis, and Management. ed. Laragh, J.H. & Brenner, B.M. pp New York: Raven Press. LANGER, S.Z. & SHEPPERSON, N.B. (1982). Reent developments in vasular smooth musle pharmaology: the post-synapti a2- adrenoeptor. Trends Pharmaol. Si., 3, LINCOLN, T.M. (1983). Effets of nitroprusside and 8-bromo-yli GMP on the ontratile ativity of the rat aorta. J. Pharmaol. Exp. Ther., 224, NABEL, E.J., BERCK, B.C., BROCK, T.A. & SMITH, T.W. (1988). Na- Ca exhange in ultured vasular smooth musle ells. Cir. Res., 62, NOBLE, D. & POWELL, T. (1985). Calium urrents and aliumdependent inward urrent. In Control and Manipulation of Calium Movement. ed. Parret, J.R., pp New York: Raven Press. OZAKI, H., KARAKI, H. & URAKAWA, N. (1978). Possible role of Na- Ca exhange mehanism in the ontrations indued in guineapig aorta by potassium free solution and ouabain. Naunyn- Shmied. Arh. Pharmaol., 34, PELFRENE, A.F. (1991). Syntheti organi rodentiides. In Handbook ofpestiide Toxiology, Volume 3. ed. Hayes, W.J. & Laws, E.R. pp New York: Aademi Press. POOS, G.I., MOHRBACHER, R.J., CARSON, E.L., PARAGAMIAN, V., PUMA, B.M., RASMUSSEN, C.R. & ROSZKOWSKI, A.P. (1966). Struture-ativity studies with the seletive rat toxiant norbormide. J. Med. Chem., 9, ROSZKOWSKI, A.P. (1965). The pharmaologial properties of norbormide, a seletive rat toxiant. J. Pharmaol. Exp. Ther., 149, ROSZKOWSKI, A.P., POOS, G.I. & MOHRBACHER, R.J. (1964). Seletive rat toxiant. Siene, 144, SCHULTZ, K.D., BOHME, E., KREGE, V. & SCHULTZ, G. (1979). Relaxation of hormonally stimulated smooth musle tissues by the 8-bromo-derivative yli GMP. Naunyn-Shmied. Arh. Pharmaol., 36, 1-9. SHINJOH, M., NAKAKI, T., OTSUKA, Y., SASAKAWA, N. & KATO, R. (1991). Vasular smooth musle ontration indued by Na' hannel ativators, veratridine and batrahotoxin. Eur. J. Pharmaol., 25, VAN BREEMEN, C. & SAIDA, K. (1989). Cellular mehanisms regulating [Ca2+], smooth musle. Annu. Rev. Physiol., 51, WOOLFSON, R.G., HILTON, P.J. & POSTON, L. (199). Effets of ouabain and low sodium on ontratility of human resistane arteries. Hypertension, 15, (Reeived June 6, 1995 Revised September 12, 1995 Aepted November 9, 1995)