Vasodilatation of canine cerebral arteries by nicorandil, pinacidil and lemakalim

Gen Pharmac Vol 23, No 2, pp 197-201,1992 Printed m Great Britain All rights reserved

0306-3623/92$5 00 + 0 00 Copyrtght© 1992PergamonPress pie

VASODILATATION OF CANINE CEREBRAL ARTERIES BY NICORANDIL, PINACIDIL AND LEMAKALIM H ZHANG, 1 N. STOCKBRIDGE, 1 B WEre, 1 B VOLLRATH2 and D. COOK2. Departments of ISurgery and 2pharmacology, Umverstty of Alberta, Edmonton, Canada T6G 2H7 [Tel (403) 492-3575, Fax (403) 492-4325] (Recewed 22 July 1991) Abstract--1 Nlcorandfl, pmactdll and lemakahm relaxed precontracted nngs of canme cerebral artery 2 The order of potency was lemakahm > mcorandfl -~ pmacldfl, but all these agents were less effectwe than mmodtpme 3 The effects of mcorandd were mhtblted by methylene blue but not by ghbenclamlde,wlule the effects of pmacldfl and lemakahm were tnhthted by ghbenclamlde but not by methylene blue 4. Thus mcorandfl probably causes relaxation mosdy by effects on guanylate cyclase whtle lemakahm and pmac~dfl produce the same effect by action at ATP-dependent potassmm channels

INTRODUCTION

Vasorelaxants nicorandil, pmacidfl and cromakahm hyperpolarize smooth muscle cells towards the potassmm eqmlibrium potential (Furukawa et al., 1981, Hamilton et a l , 1986, Southerton et al., 1988). These drugs increase SeRB+ or 42K+ efflux and reverse or attenuate the effects of a wide variety of directlyacting and endothehum-dependent vasoconstrictors (Hamilton and Weston, 1989). Whole-cell and singlechannel studies show that these drugs act on several types of potassium channels. In cardmc myocytes, these agents usually actwate a background, twae- and voltage-independent potassmm current which is blocked by physiological levels of ATP (Hiraoka and Fan, 1989; Sangumetti et a l , 1988; Arena and Kass, 1989). In peripheral artenal and venous smooth muscle, these drugs activate either a large-conductance calcmm-dependent potassmm channel (Gelband et a l , 1989, Hu et al., 1990; K16ckner et al., 1989, Kajloka et al., 1990) or ATP-dependent potassmm current (Standen et a l , 1989) There have been few studms of these agents in cerebral artenes and their mechanism of action m th~s system ~s not clear. High concentraUons of cromakahm and pmacldd increased the acuvmes of a large-conductance calcmm actwated potassium channels in rat basflar artery (Zhang et al., 1991a), but cromakahm opened ATP-dependent potassmm channels in canine m~ddle cerebral artery (Masuzawa et al., 1990) In the present study, we have examined the relaxatlon caused by mcorandll, pmacidfl and lemakahm (BRL 38227), the vasorelaxant ( - ) - t r a n s enantiomer of cromakahm (Edwards and Weston, 1990), m nngs of canine basdar and waddle cerebral arteries precontracted w~th prostaglandm F:~, and compared the effects of these drugs wtth those of mmodipine, an agent which blocks the L-type calcium channel and which has been extensively investigated in camne cerebral arteries (Nosko et aL, 1986). The mechanism of these drugs in canine basdar arteries was mvesti*To whom aU correspondence should be addressed OP 23/2--D

gated usmg the potassmm channel antagonist, glibenclamide. This drug blocks the ATP-dependent potassium channels in pancreaUc ~-cells (Fosset et al., 1988), and probably m cardiac myocytes (Escande et al,, 1988, Sangumetti et al., 1988) and rabbit mesentenc arteries (Standen et al., 1989). In addition, it has been suggested that, at least in the case of mcorandfl, the relaxaUon may also revolve effects on the soluble guanylate cyclase of vascular smooth muscle (Holzmann, 1983). We have exammed this further by usmg methylene blue, wlueh is an antagonist of guanylate cyclase (Greutter et al., 1981). MATERIALS AND METHODS

Preparatwn of arterial rings Mongrel dogs of eRher sex, 14-25kg m weight, were killed with an mtravenous overdose ofsodmm pentobarbttal (60 mg/kg) The brain was rapidly removed The basdar and n'addle cerebral arteries were tsolated from the brains and quickly immersed m a Krebs' phymologlcalsalme soluUon equthbrated ~ t h 95% 02 and 5% CO: at room temperature The composRton of the medium was (raM) NaC1 1137, NaHCO3 250, KC1 47, CaCI2 25, MgSO4 1.2, KH2PO4 1 2 and dextrose 10.1 The artenes were cleaned of connecttve tissue and stde branches and cut mto nngs 2 mm long Artenes were mounted vertically between small hooks m a water-jacketed tissue bath of 10 ml working volume mamtamed at 37°C and bubbled wRh 95% 02 and 5% COz (pH 7 4) No attempt was made to remove the endothehum Artertal tenswn recording At the begmmng of each experiment, the nngs were stretched to an mlttal tension of 500 mg, and allowed to eqmhbrate for approximately 2 hr. Dunng the eqmhbration period, the batlung medium was changed at 15-20ram intervals to prevent the accumulatton of metabohtes. Contracttons and relaxattons were recorded tsometncally nsmg strata gauges (model FTO 3, Grass Instrument Co.) connetted to a polygraph (model 7D, Grass Instrument Co.). Maxamum contra~ions were obtained w~th prostaglandm F2~ (10/~M) and then the tmsues were washed and allowed to recover. After a 1 hr recovery, the rings were contracted with prostaglandm F2, (3/JM) to produce approximately 50% of the maxtmum response. When a stable plateau 197

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contraction had been obtained, cumulative doses of the relaxant drugs were administered The relaxation response to each concentration was allowed to reach a plateau before the next addition was made At the end of each senes of expenments, papaverlne m a concentration of 100/~ M was applied to produce the maximum relaxation Experiments in which relaxations were produced with nlmodlpme were carried out under low-light conditions Experiments examining the effects of methylene blue was carried out as follows basilar arterial nngs were precontracted with prostaglandin F2~ and dose-response curves for relaxation were established as described above The arteries were then washed at 10 mln intervals over a 90 mln period, during which the basehne tension was continuously readjusted Methylene blue (3 #M) was then added to the bath for 20-30 mm Artenes were again contracted with prostaglandin F2~ and the dose-response curve was again obtained m the presence of methylene blue A similar protocol was used to assess the effects of ghbenclamide (10 # M) on relaxation by nicorandll, pmacidiI and lemakahm In these experiments, 5-HT was used to produce contraction since ghbenclamlde inhibits contractmn by prostaglandm F2~ (Zhang et al, 1991b)

Drugs and solutions Drugs used were plnacldd (Leo), nicorandll (Upjohn), lemakahm (Beecham), nlmodipme (Miles), ghbenclamlde (Sigma), papaverIne (Sigma), prostaglandm Fz~ (Sigma) and methylene blue (Fisher) Stock solutions of 10mM nIcorandi1, pInacldll, lemakahm and mmodlpine were prepared in 95% ethanol Ghbenclamide (10 mM) was dissolved in dimethyl sulfoxIde Control expenments established that effects were not the result of vehicles Stattsncal analysts Dose-response results have been expressed as a percentage of the maximum possible relaxation, i e the relaxatmn caused by 100 p M papaverme The results were expressed as

mean + standard error of the mean and compared using one-tailed t-test A probability of < 0 05 was considered significant The p D 2 values and maximum relaxations were calculated by the methods prescnbed previously (Zhang et al, 1991b)

RESULTS

The relaxant effects of mcorandd, pmacldd, lemakahm and mmodlpme in canine basdar and middle cerebral arteries Nlcorandfl, p m a o d d a n d l e m a k a h m each p r o d u c e d relaxatmn m canine basllar a n d middle cerebral arteries p r e c o n t r a c t e d with p r o s t a g l a n d m F2~ (Fig la, b , c ) Nlcorandfl a n d p m a o d f l p r o d u c e d slmdar r e l a x a t m n m b o t h b a s d a r a n d middle cerebral arteries. A t 100 # M , m c o r a n d d a n d p m a o d d relaxed canine basflar a n d m]ddle cerebral a r t e n e s close to the m a x i m u m relaxation reduced by 1 0 0 # M pap a v e r m e L e m a k a h m p r o d u c e d its m a x i m u m relaxa t m n s at 3 - 1 0 / ~ M a n d a l t h o u g h it a p p e a r e d to produce greater relaxations m middle cerebral artery t h a n m b a s d a r artery, this d~d n o t reach statistical slgmficance. T h e p D 2 values were m the order o f l e m a k a h m > m c o r a n d l l ~ p m a o d l l (Table l) The relaxant effects o f mcorandfl, p m a o d f l a n d l e m a k a h m m canine cerebral arteries were c o m p a r e d wRh nlmod]pm¢ F]gur¢ l d shows t h a t canine cerebral a r t e n e s are more sensitive to mmod~pm¢ t h a n to any o f the p o t a s s m m channel openers T h e p D : values for m m o d l p m e were 6.6 for b a s d a r artery a n d 7 7 for middle cerebral artenes; these values are n o t slgmficantly different

(o)

(b)

.!

,. -Loo(Nl©orandll) [M]

-Log(Plnacldll) [M]

(c)

(d)

100

100

i

~ i -[.og(~makallm) [M]

i

i

i "~ i -Lo~Nlmodlplne) [M]

i

Fig 1 Relaxant effects of nlcorandil (a, n = 9, 5, for basilar and middle cerebral artenes respectively), pinacldI1 (b, n = 12, 4), lemakalIm (c, n = 12, 6) and nimodlpme (d, n = 4, 6) In canine basdar (circle) and middle cerebral artenes (square) precontractexl by prostaglandm F~ (3/~M) Error bars indicate one standard error of the mean Relaxation was expressed as percentage of the relaxation by papaverlne

(100 ~M)

Potassium channel openers and canine cerebral arteries Table I The maximum relaxations and pD 2 values of mcorandd, pmacldd and lemakahm m canine basdar and middle cerebral arteries NIcorandd BA MCA

lhnacldll

pD 2 n

9 5

54 54

58 54

12 90 4 93

(o)

Lemakahm

n Rm~ pD2 n ~ 77 80

199

Rm.x pD2

12 61 6 75

61 64

Ba

Methy8 89 55 5 93 51 6 52 60 Methy+ 9 90 40 5 99 50 6 46 61 BA Ghbcn4 80 58 4 99 52 4 73 60 Ghben+ 4 80 58 4 99 48 4 57 43 BA = basdar artery, MCA = middle cerebral artery, Methy-=w~thout methylene blue, Methy+ =with methylene blue, Ghben-=without ghbenclamlde, Ghben + = with ghbenclamtde, Rr~ax ~ maximum relax* atmn (the relaxation by 100/tM papaverme as 100%), pD2= - log EDs0

-Log(Nloorandll)

(b)

5o

The relaxant effects o f mcoran&l, pmaczdd and lemakalon in camne basdar artery m the presence o f methylene blue Methylene blue (3 # M ) shghtly e n h a n c e d the resting tension a n d the a r t e n a l responses to prostaglandin F2= (data n o t shown), b u t h a d n o effects o n

-Log(Plnaoldll) [M]

(c)

(o)

.L~(Lmak-IIm) [~1 Fig 3 Relaxant effects of mcorandfl (a), Nna~dd (b) and lemakahm (c) m the absence (circle) and presence (square) of ghbenclamtde (10 #M) m canine basdar arteries precontracted by 5-HT (0 3/zM) Each data point represent 4 nngs Error bars indicate one standard error of the mean RelaxaUon was expressed as percentage of the relaxatmn by papavenne (100/aM) Asterisks represent the slgmficant differences (P < 0 05) from control

-Log(Nloorandll) [M]

(b)

_I

s

the relaxauons p r o d u c e d by l e m a k a h m or pinacidil (Fig 2b, c, Table I) Methylene blue a p p e a r e d to inhibit comparatively" the relaxation to m c o r a n d i l (Fig. 2a).

~ ~ .~ .Log(Plnacidll) [M]

4

(c)

Glibenclamlde reduced testing tension b u t did n o t inhibit the contractile responses o f canine basilar artery to 5-HT (0.3 # M ) A t 1 0 # M , ghbenclamide faded to m h l b R relaxaUon induced by nicorandil (Fig 3a), b u t did inhibit the effects o f lemakalim (Fig 3c) and, to a less degrees pmacidil (Fig. 3b) (Table 1).

0

i

The relaxant effects o f mcorandd, pinaczdil and lemakahm in camne basdar artery m the presence o f ghbenclamMe

~

~

-Log(Lemakallm) [M]

L

s

Fig 2 Relaxant effects of mcorandil (a, n = 8), pmacldd (b, n = 5) and lemakahm (c, n = 6) in the presence (circle) and absence (square) of methylene blue (3 #M) m canine basllar arteries precontracted by prostaglandm F2~ (3/zM). Error bars inchcate one standard error of the mean. Relaxatmn w a s expressed as percentage of the relaxataon by papavenne (100/aM). Asterisks represent the significant differences (P < 0.05) from control.

D~CUSSION T h e effects o f nicorandil, pinacidil a n d c r o m a k a l i m have been extensively stu&ed m vascular s m o o t h muscle in different a m m a l a n d tissue p r e p a r a t i o n ( H a n u l t o n a n d Weston, 1989). T h e results showed t h a t these drugs hyperpolarized the s m o o t h muscle, driving the m e m b r a n e potential further away from the threshold o f voltage-dependent calcium channels,

200

H ZHANGet al

resulting m muscle relaxation (Weston, 1988) However, there have been few studies of these drugs m cerebral arteries Since cerebral arteries may behave differently from penpheral arteries (Toda, 1977), it is ~mportant to know whether these drugs work the same way m cerebral arteries as they do m peripheral arteries, and by what mechamsms Since prostaglandins were rarely used as agonlsts m most of the studies of potassmm channel openers, and since prostaglandms play an important role m the control of cerebral c~rculatlon (Wahl et al, 1989), prostaglandm F2~ was employed as the agomst m the first part of th~s study The effects of mcorandd, pmac~dll and lemakallm in canine cerebral arteries were assessed using ~sometnc tension recordings All of these drugs s~gnlficantly relaxed the contractions reduced by prostaglandm F2~ In this study there were no slgmficant d~fferences between the basllar and m~ddle cerebral artenes m the relaxant effects of mcorandd and pmac~dll Lemakahm appeared to achieve half its peak effectweness at lower dose than pmac~dll and mcorandd m both basflar and middle cerebral arteries, but this difference ~s not statistically s~gn~ficant. Since some calcmm channel blockers were chmcally used against vasospasm after subarachnotd hemorrhage, the effects of potassmm channel openers m cerebral arteries have been compared w~th them m some studies The actions of pmacldd was found more pronounced m canine peripheral arteries than in cerebral arteries, whde nffedlpme, a calcium channel blocker, relaxed cerebral arteries better than peripheral arteries (Toda et al., 1985) In contrast, the effects of pmac~dfl m fehne pml arteries of the parietal cortex ~s about 100 t~mes greater than that of the calcium entry blocker mmod~pme (Wahl, 1989) Cam and N~cholson (1989) compared the effects of cromakahm and mmod~plne m nngs from rabb~t basflar arteries 5-HT reduced two components of contracUon m basdar artery, and only the first component was cromakahm sensmve, whde mmod~pme depressed both components of the contracUon The effects of potassmm channel openers m this study in canme cerebral arteries were compared w~th those of mmodlpme, a d~hydropyndme calcium channel antagomst N~modlpme at lower concentraUons produced more relaxation of prostaglandln F2~-lnduced contractions than any of these potassmm channel openers In the present study, the effects of mcorandd, but not pmac~dfl and lemakahm, were blocked by methylene blue, a inhibitor of gnanylate cyclase (Greutter et al, 1981) It ~s hkely that the effects of mcorandd in canine cerebral arteries are mainly mediated by Rs actwat~on of gnanylate cyclase, as its effects were attenuated by methylene blue In examining the effects of these agents on ATP-dependent potassmm channels, ghbenclam~de was used as the antagomst Prostaglandm F2~ is not an approprmte agomst for these studies, since ghbenclam~de ~s beheved to have a d~rect effect on the action of prostaglandm F2~ m canme cerebral artenes (Zhang et al., 1991b) Thus 5-HT was used to reduce the contraction for these studies. Ghbenclam~de was w~thout significant effects on the relaxatmn reduced by nicorandfl, and ~t thus seems ~mprobable that nicorandil owes Rs action in

cerebrovascular smooth muscle to effects on ATPdependent potassmm channels On the other hand ghbenclam~de but not methylene blue slgmficant mh~b~ted the effects of lemakahm, and th~s suggests that, in contrast to mcorandd, lemakahm promotes relaxation not through effects on guanylate cyclase, but from ~ts known action on potassmm conductance (Hamdton and Weston, 1989, Standen et al, 1989) A recent study (Masuzawa et al, 1990) reported that cromakahm relaxed the contraction of canine m~ddle cerebral artery reduced by low KC1 (20 mM) and this effect was antagonized by ghbenclamlde However cromakahm also actwates calcmm-dependent potassmm channels m vascular smooth muscles (Gelband et al, 1989, Zhang et al, 1991a, Hu et al, 1990) and ghbenclamlde also inhibits calcmm-dependent potassmm channels in vascular smooth muscles (Gelband et al, 1989, Hu et al, 1990; Tseng et al, 1990) Therefore, we could not exclude the poss~bihty that cromakahm (or lemakahm) may open two types of potassmm channels m cerebral arteries or open one type of potassmm channel which is regulated both by mtracellular ATP and calcmm concentrataons. Since the effects of pmac~dd were not affected by methylene blue but were somewhat inhibited by ghbenclamide and the effects of pmac~dd are reported to be sensmve to tolbutam~de, another ATP-dependent potassmm channel blocker, m fehne pml arteries m sztu (Wahl, 1989), we beheve that the effects of pmacldfl are at least partly medmted by opening potassmm channels in cerebral arteries Our results are substantmily m agreement w~th a recent study using canine coronary arterial smooth muscle (Yanaglsawa et al, 1990) Ghbenclamlde abolished the relaxant effect of cromakahm, but only shghtly attenuated the relaxant effects of pmac~dil and mcorandfl They beheve that cromakahm ~s a more specific potassmm channel opener than pmac~dd and mcorandd Although the effects of these potassmm channel openers m cerebral arteries were stud~ed in d~fferent ammals and tissues, by different methods, vath different agomsts, and w~th d~fferent results, mcorandd (Harder et al, 1987), pmacldd (Wahl, 1989) and cromakahm (Cam and N~cholson, 1989) have all been suggested as being potentmlly useful in the treatment of cerebral vasospasm after subarachnold hemorrhage by hyperpolarlzmg the smooth muscle membrane An m wee study that showed that mcorandfl partly reversed expenmentral cerebral vasospasm m the camne basflar artery (Harder et al, 1987) Whether any of these potassmm channel openers could play a role m the treatment of cerebral diseases, especmlly cerebral vasospasm after subarachnold hemorrhage deserves further study SUMMARY

In this study the effects of the three potassium channel openers, lemakahm, mcorandd and pinacldil were mvesUgated m nng preparations from camne cerebral artenes All agents produced a relaxation of arteries which had been precontraeted with prostaglandin F2~ or with 5-HT. In the ease of mcorandil the relaxation was sigmficantly attenuated by methylene blue, an agent which interferes with soluble gnanylate cyelase, and this supports the idea that nicorandil

Potassium channel openers and canine cerebral arteries owes its action to effects which revolve this enzyme. The relaxations produced by lemakahm or pmacidd were not affected by methylene blue. Glibenclamide, a drug which blocks ATP-dependent potassmm channels, was also used to help to elanfy the mechamsm o f action o f these agents. Ghbenclam~de antagonized the relaxaUon produced by lemakalim and, to a lesser extent, the relaxaUon produced by pmac~dfl. It was, however, devoid of s~gmficant effects on the relaxaUon produced by nicorandil, and it thus seems reasonable to suppose that these agents work by different mechamsms, lemakahm and pmacidtl causing their effects by action on potassium conductance and mcorandll by action on soluble guanylate cyclase Acknowledgements--We wish to express our thanks to Beecham Pharmaceuticals, Leo Laboratories, Miles Inc and Upjohn Co for providing potassium and calcium channel drugs This work was supported by two grants from the Canadmn Heart and Stroke Foundation (to N.S and D C ) and by NIH grant R01 NS25957-01 (to B W )

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