Inhibition by glibenclamide of the effects of cromakalim on responses of rat vas defererns to naphazoline

European Journal of Phnrmacology, 202 ( 1991) 93-96 r% 1991 Elsevier Science Fublishers B.V. All rights reserved ADONIS 001429999100604F

93

0014-2999/91/$03.50

EJF 20900

Short communication io

ses

Enzo Grana, A.nnalisa Barbieri

S

and Franc0 Zonta

Istituto di Farmacologia, Facoltri di Farmacia. Utziwrsitridi Paria, fiale Taramelli 14, Pavia, Iralia

Received 21 June 1991, accepted 7 July 1991

Cromakalim has been shown to inhibit naphazoline-induced contrasticns and spontaneous activity induced by exposure to naphazoline in the rat isolated vas deferens. Glibenclamide IO-’ M blocked both these effects of cromakalim. Our data add to the list of data derived mainly from experiments on vascular smooth muscle; they suggest that the same glibenclamide-sensitive K+ channel is present in vascular and non-vascular smooth muscle and that it may be involved in the relaxant actions ,of cromakalim.

Vas deferens (rat); Naphazoline;

1. Introduction Potassium channel openers are a new pharmacological class of powerful smooth muscle relaxants typified by cromakalim; this compound has been extensively studied on a variety of smooth muscles (for reviews see Quast and Cook, 1989; and Cook and Quast, 1990). In a previous work we studied the effects of cromakalim on mechanical responses of rat vas deferens to naphazoline, an imidazoline derivative (Grana et al., 1991). Naphazoline produced concentration-related contractions of the rat isolated vas deferens with prominent spontaneous contractile activity being superimposed on these contractions. The cumulative concentration-response curve for naphazoline (l-300 x lo-’ MJ was progressively shifted to the right in the presence of cromakalim l-10 X lo-’ M. After removal of naphazoline (3 X 10mh MI, the rat vas deferens, a preparation which is usually quiescent, displayed periodic spontaneous activity; cromakalim l-10 x lo-’ M inhibited this naphazoline-induced activity. The sulphonylurea glibenclamide, a potent blocker of the ATP-sensitive K’ channel, has been shown to inhibit the effects of cromakalim on vascular smooth muscle (for review see Cook and Quast, 1990). Our experiments were designed to investigate the effect of glibenclamide on the antagonism exerted by cromakalim towards the naphazoline-induced contrac-

Correspondence to: E. ‘Rw~melli 13. P;wi;l. Itah.

Istituto di Grima. Fax 39.382.3924(15.

Formacologh.

Viole

Cromakalim; Glibenclamide

tion and the spontaneous activity induced by exposure to naphazoline in the isolated rate vas deferens. The aim of this study was to investigate the possible involvement of a glibenclamide-sensitive K’ channel in this particular preparation.

2. Materials and methods Male Wistar-Morini rats (140-190 g) were killed by a blow to the head an exsanguinated. Whole vasa deferentia were dissected, isolated and placed in a IO-ml organ bath containing Krebs-Henseleit solution fcomposition in mmol/l: NaCl 118; KCI 5.6; CaClz 2.5; MgSO, 1.19; NaH?PO, 1.3; NaHCO, 25 and glucose IO) gassed with 5% CO, in 0, (pH 7.4) and kept at 37°C. Procedural details have been published previously (Grana et al.. 1991). Cumulative concentrationresponse curves for naphazoline were obtained by increasing the concentration by 0.5 logarithmic units. When cromakalim was tested as antagonist. it was allowed to equilibrate for 20 min before challenge with naphazoline. Results are expressed as percentage of the maximum effect of agonist in the absence of the antagonist. Paired preparations were used as cumulative-concentration response curves for nayhazoline arc not reproducible. Only one dose of antagonist was tested per preparation. The results are given as means + SE; S.E.s are shown by vertical bars. -Log ECS,, values (molar conccntrations producing 50% of maximal contractile rc-

sponsel were evaluated graphically from mean concentration-response curves. Glihcnclan~ide (Sigma) was dissolved in dimethylsulfoside (fins1 concentration of solvent less than 0.5%’ and without effect) and was allowed to equilibrate for -IO min before challenge with naphazoline. Glibenclamide was tested to study its effect on the antagonism eserted by cromakalim towards napharoline-induced contractions and the spontaneous activity induced by exposure to naphazoline. The experiments were performed on different preparations and repeated at least six times. Statistical evaluation was done with Student’s t-test; the P < 0.05 level of probability was regarded as statistically significant. 3. Results

Glibenclamide concentrations of 3 x lo-’ and IO-” M had no effect on the basal tone of the rat vas 1 %contract,on 100

a0 60

40

20

C

100

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t-tog molart

%contractron

Fig. 2. Typical recordings of naphazoline-induced spontaneous activity 4lowing: (A) The ‘control’ isolated rat vas deferens (only treated with naphazoline). (BJ After exposure for 20 min :o glibenclamide 3 x IO-” M. !CJ The effect of cromakalim IO-” M ( 1) in the control. (Ut The reduction of the effect of cromakalim IO-” M CL 1 after exposure to gliben~lamide IO-” M. (EJ The ~tbolition of the effect of ~r[)mttkalim 10 _” M f 1 f after exposure to glibenclamid~ 3 x IW” M. Mechanical activity was recorded isotonically.

a0 60

40

20

0

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i (-log molar) Fig. I. Effect of glihenclamidc 3 x 10 7 M (AJ and 10 ” M (BJ. Glibrnclamide ~3s allowed to eyui2ihrate for 41) min before challenge *iAh naphazoline. (aJ C‘oncentration-respr,nhr curves of naphazolinc. (=) C’onceotr~tion-response curves of naphazoline in the prr~nce of ~r~~mak~lirn Ill ” J Ct,nccntr;ttion-respctnse curves $11’naphazolinc in the presence of crormtkalim 10 ” M, made after the addititm of’ glihcnc4umide. Each point i+ the mean dcrivrtl from \i: expcrimcnts. Vertical lines \htr\cn SE.; when not visihlc the S.E. bar\ lie within

the \ymholr.

deferens or the subsequent concentration-response curve of naphazoline (not shown). When glibenclamide 3 x IO-’ M was tested, the cumulative concentration-response curves of naphazoline in the presence of cromakalim ltl-h M (fig. IA) were almost the same (P < 0.05) as those produced in the absence of glibenclamide: the negative logarithm of the molar concentration of cromakalim that produced a Z-fold shift tpA,) was 6.60, not significantly different from that reported previously (Grana et al., 1991). However, when glibenclamide lo-” M was used, the cromakalim-induced shift to the right of the naphazoline dose-response curve was abolished and the naphazolinc curve was superimposable upon that obtained in the absence of cromakalim (fig. 1B). Glibenclamidc (3-30 x IO-’ Ml was also tcstcd on the naphazolinc-induced periodic spontaneous activity

and was found to be without appreciable activity (fig. 2A,B), whereas cromakalim (lo-’ MI abolished the spontaneous activity. Glibenclamide 3 X lo-’ M left in contact with rat vas deferens for 20 min before the administration of cromakalim lo-’ M had no effect on the cromakaliminhibition of naphazoline-induced spontaneous activity (not shown). When glibenclamide 10m6 M was used, there was a pronounced decrease (40-70%) in the inhibitory effect of cromakalim (fig. 2C,D), and with glibenclamide 3 X lo-” M the action of cromakalim was suppressed (fig. 2E). The ability of glibenclamide to invoke the reappearance of spontaneous activity which had previously been suppressed with lo-’ M cromakalim was also tested in a few experiments. Glibenclamide IO-’ M (six out seven experiments: not shown) was required for a full restoration of naphazoline-induced spontaneous activity abolished by cromakalim.

4. Discussion Over the last few years pharmacological data have accumulated demonstrating that glibenclamide antagonizes the vascular relaxant responses of cromakalim in a competitive fashion (Cavero et al., 1989; Buckingham et al., 1989; Quast and Cook, 1989). These studies clear& indicate that cromakalim produces vasodilatation by opening the ATP-sensitive K+ channel in vascular smooth muscle cells (for review see Cook and Quast, 1990). In the present study we used glibenclamide to investigate the possible involvement of an ATP-sensitive K’ channel in the inhibitory effect of cromakalim on naphazoline-induced responses of the rat isolated vas deferens. Glibenclamide (3 X lo-’ and lO-h MI had no effect on the basal tone of rat vas deferens or on the subsequent concentration-response curve for naphazoline. These results are in agreement with those obtained under other experimental conditions: in rabbit aorta glibenclamide l-10 X lU_” M had no effect on the concentration-response curve for angiotensin II (Quast and Cook, 1989) and addition of glibenclamide l-10 x lo-” M to noradrenaline-contracted rat aortic rings produced no change in tension (Buckingham et al., 1989). The cumulative concentration-response curves for naphazoline obtained as control responses and those obtained in the prescncc of cromakalim IO-” M were practically identical to those obtained in a previous study (Grana ct al., 1991, fig. 4). The antagonistic cffcct cxcrtcd by cromakalim towards the contractions

induced by naphazoline was unaffected when glibenclamide 3 X lo-’ M was tested. However, when glibenclamide l-3 X lo-’ M was used, the antagonistic effect of crnmakalim was partially or fully suppressed. showing that glibencla.mide exerted a strong and concentration-related inhibitory effect towards cromakalim. Glibenclamide (3-30 X lo-’ MI was found to be without appreciable activity on the naphazoline-induced periodic spontaneous activity. Contrasting descriptions of the effects of glibenclamide on the spontaneous contractile activity of rat isolated portal veins have been reported (Quast and Cook, 1989; Buckingham et al., 1989; Longmore et al., 1990). Our data clearly demonstrate that glibenclamide was very active in counteracting the action of cromakalim on naphazoline-induced spontaneous activity. The antagonism of naphazoline by verapamil (Grana et al., 1991) was unaffected by glibenclamide l-3 x lo-’ M (Grana, unpublished observations), demonstrating that glibenclamide has a selective action, which was suggested earlier by binding studies with [‘Hlglibenclamide (Kramel nt a!., 1988). On the whole, our data obtained with a non-vascular smooth muscle add to the growing list of data, derived mainly from experiments on vascular smooth muscle. showing that glibenclamide at 0.1-3 PM inhibits the effects of cromakalim (McPherson and Angus. 1990; Cook and Quast. 1990). These results suggest that a similar glibenclamide-sensitive K’ channel is present on vascular and non-vascular smooth muscle and that it may be involved in the relaxant actions of cromakalim.

Acknowledgements Cromakalim was generously prtwidrd by Brecham Phwmacruticals. This study was supported by thr Italian M.U.R.S.T. The authora wish to thank Miss F. Molinari l’or technical assist:mce.

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