Influence of T-type Ca 2+ (mibefradil) and Cl - (indanyloxyacetic acid 94) channel antagonists on α...

Influence of T-type Ca2+ (mibefradil) and Cl–

(indanyloxyacetic acid 94) channel antagonistson 1-adrenoceptor mediated contractions inrat aorta

Jennifer A. Duggan and Reza Tabrizchi

Abstract: The effects of the T-type and L-type Ca2+ channel antagonists, mibefradil and nifedipine, respectively, andthose of a Cl– channel antagonist, indanyloxyacetic acid 94, on mechanical responses elicited by selective activation ofα1-adrenoceptors using cirazoline were examined in rat isolated aortic rings. The presence of mibefradil (300 nM),indanyloxyacetic acid, 94 (30µM) and nifedipine (300 nM) alone inhibited mechanical responses elicited by cirazoline.The concentration-response curves to cirazoline were displaced to the right with significant increases in the EC50 andsignificant depressions of the maximal responses in the presence of the individual agents mibefradil, indanyloxyaceticacid 94, or nifedipine. A combination of mibefradil and indanyloxyacetic acid 94 further inhibited the mechanicalactivity produced by cirazoline. The further reduction in the maximal response to cirazoline, in the presence ofmibefradil and nifedipine, was insignificant when compared with the effects of nifedipine alone. In addition, maximalmechanical responses produced by cirazoline were not significantly affected by a combination of nifedipine andindanyloxyacetic acid 94 when compared with either nifedipine alone or mibefradil and indanyloxyacetic acid 94combined. Our current findings indicate that mibefradil, indanyloxyacetic acid 94, and nifedipine can inhibit cirazoline-induced contractions to a varying degree. Moreover, based on our present data it would be reasonable to suggest thatthe contribution of T-type versus L-type Ca2+ channels to contractile responses obtained with cirazoline areapproximately 21% and 35%, respectively, of the Emax. It would appear that L-type Ca2+ channels play a greater role inprocesses that are involved in excitation–contraction coupling subsequent to stimulation ofα1-adrenoceptors. Inaddition, Cl– channels also appear to be involved in the process of contraction followingα1-adrenoceptor activation.

Key words: T-type Ca2+ channels, L-type Ca2+ channels, Cl– channels, isolated aortic rings.

Duggan and TabrizchiRésumé: Les effets des antagonistes des canaux Ca2+ de type T et de type L, mibéfradil et nifédipine, respectivement,et ceux d’un antagoniste des canaux Cl–, acide indanyloxyacétique 94, sur les réponses induites par l’activation sélec-tive des récepteurs-α1 adrénergiques au moyen de cirazoline ont été examinés dans des anneaux aortiques isolés derats. La présence de mibéfradil (300 nM), d’acide indanyloxyacétique 94 (30µM) et de nifédipine (30 nM) a aussi in-hibé les réponses mécaniques induites par la cirazoline. En présence de chacun de ces agents, les courbes concentra-tion-réponse à la cirazoline ont été déplacées vers la droite, et des augmentations significatives de la CE50 ainsi quedes diminutions significatives des réponses maximales à la cirazoline ont été observées. L’emploi combiné de mibéfra-dil et d’acide indanyloxyacétique 94 a inhibé davantage l’activité mécanique produite par la cirazoline. La diminutionsubséquente de la réponse maximale à la cirazoline, induite par l’emploi combiné de mibéfradil et de nifédipine, a éténégligeable par comparaison aux effets de la nifédipine seule. De plus, les réponses mécaniques maximales induites parla cirazoline n’ont pas été affectées par l’emploi combiné de nifédipine et d’acide indanyloxyacétique 94 comparative-ment à ce qui a été observé lors de l’emploi de nifédipine seule ou de mibéfradil et d’acide indanyloxyacétique 94combinés. Nos résultats indiquent que le mibéfradil, l’acide indanyloxyacétique 94 et la nifédipine peuvent inhiber à undegré variable les contractions induites par la cirazoline. De plus, selon les présents résultats, il serait raisonnable depenser que la contribution des canaux Ca2+ de type T et de type L aux réponses contractiles induites par la cirazolineest d’environ 21 % et 35 %, respectivement, du Emax. Les canaux Ca2+ de type L joueraient un plus grand rôle dansles processus mis en jeu dans le couplage excitation-contraction qui suit la stimulation des récepteurs-α adrénergiques.Les canaux Cl– interviendraient aussi dans le processus contractile qui suit l’activation des récepteurs-α1 adrénergiques.

720

Can. J. Physiol. Pharmacol.78: 714–720 (2000) © 2000 NRC Canada

714

Received April 24, 2000. Published on the NRC Research Press web site on August 14, 2000.

J.A. Duggan and R. Tabrizchi.1 Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland,St. John’s, NF A1B 3V6, Canada.

1Author for correspondence (e-mail: [email protected]).

I:\cjpp\Cjpp-78\Cjpp-09\Y00-049.vpTuesday, August 08, 2000 10:56:36 AM

Color profile: DisabledComposite Default screen

Mots clés: canaux Ca2+ de type T, canaux Ca2+ de type L, canaux Cl–, anneaux aortiques isolés.

[Traduit par la Rédaction]

Introduction

In recent years there has been great interest in the involve-ment of Ca2+-activated Cl– currents and subsequent events thatresult in excitation–contraction coupling in vascular smoothmuscle. The current proposed model is that agonist-activatedCl– currents cause membrane depolarization, producing theopening of voltage-gated Ca2+ channels and subsequent Ca2+

influx which, in turn, contributes to the mechanical response inblood vessels (Byrne and Large 1988a, 1988b; Greenwoodand Large 1995). The evidence in the current literature sup-ports the view that two types of voltage-gated Ca2+ channels(L- and T-type) seem to participate in the process that in-volves contraction of blood vessels after depolarization(Bean et al. 1986; Benham et al. 1987). Activation of L-typeCa2+ channels in blood vessels following agonist-receptor in-teraction has been reported to occur subsequent to changes inthe membrane potential following the opening of Cl– channels(Pacaud et al. 1989a; Amédée et al. 1990; Hogg et al. 1993).Furthermore, there is evidence in the literature that closelylinks the stimulation of post-junctionalα-adrenoceptors inblood vessels and smooth muscle depolarization to Cl–

efflux (Pacaud et al. 1989b; Hogg et al. 1993).It is clear that such a view receives support from a number

of pharmacological studies in the published literature. Forexample, Cl– channel blockers have been reported to blockdepolarization of smooth muscle cells, thus inhibiting Ca2+

currents (Hogg et al. 1994; Greenwood and Large 1995).Furthermore, in isolated blood vessel preparations, Cl– channelblockers have been reported to inhibit agonist-mediatedcontractions and vasoconstriction but not potassium-induced contractions (Carmines 1995; Criddle et al. 1996,1997; He and Tabrizchi 1997). We recently reported thatphenylephrine-induced Ca2+ influx could be impaired inthe presence of a Cl– channel blocker (Lum Min et al.1999). Moreover, we reported that removal of Cl– was foundto impair the inhibition produced by a Cl– channel antagonist.Also, we found that agonist-mediated Ca2+ influx in isolatedvessels was inhibited in Cl–-free medium (Lum Min et al.1999).

The data in the current literature appears to support theview that differences exist between the behavior of L-typeand T-type Ca2+ channels during the process of excitation–contraction coupling in vascular muscle (Sperelakis 1990;Bean 1991). Their relatively long time of opening (300–600 ms) and activation from –40 mV to a positive voltagerange (+10 to +20 mV) characterize the L-type Ca2+ chan-nels. In contrast, the T-type Ca2+ channels have a muchshorter time of opening (20–60 ms) and a relatively loweractivation threshold (–70 to –30 mV) that displays rapid in-activation (Sperelakis 1990; Orallo 1996). Based on differ-ences that exist in the electrophysiological properties of T-type and L-type Ca2+ channels in the smooth muscle, it ispossible that they each contribute in varying degrees to pro-cesses that lead to changes in mechanical forces after the ac-tivation of membrane bound receptors in blood vessels. Inthis investigation, we attempted to explore the interrelation-ship between the actions of T-type and L-type Ca2+ channel

antagonists, mibefradil and nifedipine, respectively, andthose of a Cl– channel antagonist, indanyloxyacetic acid 94,on mechanical responses elicited by selective activation ofα1-adrenoceptors by cirazoline in rat isolated aortic rings.

Materials and methods

Tissue isolationMale Sprague-Dawley rats (230–260 g) were anaesthetized with

sodium pentobarbital (65 mg/kg ip). The thoracic aorta was re-moved and dissected free of connective tissue at room temperaturein Krebs buffer of the following composition (in mM): NaCl, 120;KCl, 4.6; glucose, 11; MgCl2, 1.2; CaCl2, 2.5; KH2PO4, 1.2;NaHCO3, 25.3. The pH of the buffer following saturation with a95% O2–5% CO2 gas mixture was 7.4. These experiments conformto CCAC guidelines.

Experimental protocolsAortic rings (~2 mm in length) were mounted in 20 mL organ

baths at 37°C under a basline force of 19.6 mN and gassed contin-uously with a mixture of 95% O2–5% CO2. The endothelial cellswere not removed from these blood vessels. The tissues wereequilibrated for 60 min and isometric tension was measured usingforce displacement transducers (Model FT03, Grass InstrumentsCo., Mass.) connected to a polygraph (Model 7PCPB, Grass Instru-ments Co., Mass.). Tissues were initially contracted with cirazoline(300 nM) and then washed with Krebs solution and left for an ad-ditional 60 min (washed once at 30 min with Krebs) before a con-trol concentration response-curve was constructed to cirazoline(1 nM–3.0µM). The tissues were then washed with Krebs and al-lowed to equilibrate for 30 min. After this period, each tissue waswashed again. Ten minutes after this wash, chemicals were addedto each tissue bath: vehicle (6µL; 70% alcohol solution),mibefradil (300 nM), indanyloxyacetic acid 94 (30µM), nifedipine(300 nM), or a combination of mibefradil (300 nM) andindanyloxyacetic acid 94 (30µM), mibefradil (300 nM) andnifedipine (300 nM), or indanyloxyacetic acid 94 (30µM) andnifedipine (300 nM) for 20 min. Subsequently, in the presence ofthese chemicals, a second concentration-response curve to cirazolinewas constructed.

In two additional groups of experiments, tissues that had equili-brated for 60 min were initially contracted with cirazoline(300 nM) and then washed with Krebs solution and left for an ad-ditional 60 min (washed once at 30 min with Krebs) before eachtissues was exposed to high K+ (80 mM). The aortic rings werethen washed with normal Krebs solution and allowed to equilibratefor 30 min. After this period, each tissue was washed again withnormal Krebs. Ten minutes after this wash, indanyloxyacetic acid94 (30µM) or phentolamine (10µM) was added to separate bathsand 20 min later tissues were again exposed to high K+ solutionscontaining either indanyloxyacetic acid 94 (30µM) or phento-lamine (10µM). In experiments involving high K+, the appropriatequantities of K+ replaced Na+.

Data and statistical analysisResults from the contraction studies were calculated as a per-

centage of the maximum contraction of the control curve. Percentmaximum, Hill coefficient (nH) and EC50 values were calculatedfor individual curves using a program executed on an IBM compat-ible microcomputer (Wang and Pang 1993). These parameters weredetermined by fitting the percent contractile response at increasingconcentrations of agonist ([A]) by non-linear least squares to the

© 2000 NRC Canada

Duggan and Tabrizchi 715



relation Y = a + bX, where Y = response and X = [A] n / ([A] n +[EC50]

n) with n (nH) fixed at ‘floating’ integral values to obtain thebest fit. Pairedt-test was employed for comparisons between con-trol and treatment of various parameters of the concentration-response curves (percent maximum,nH and EC50) within eachgroup. An analysis of variance was used for comparisons of per-cent maximum,nH and EC50 between the groups. For multiplecomparisons, Duncan’s multiple range test was used to comparebetween means. A probability of error of less than 0.05 was se-lected as the criterion for statistical significance.

ChemicalsStock solutions of all drugs, except for nifedipine, were made in

double distilled water. Stock solutions of nifedipine (1.0 mM) wereprepared in a 70% alcohol solution. Nifedipine and indanyloxy-acetic acid 94 were purchased from Research Biochemical Interna-tional (Mass.). Mibefradil was a gift from Hoffmann-La Roche(Canada).

Results

A comparison between the control concentration-responsecurve to cirazoline and that following treatment with vehicleindicated no significant differences between maximal re-sponse,nH and EC50 values (Table 1). However, the presenceof mibefradil (300 nM), indanyloxyacetic acid 94 (30µM)and nifedipine (300 nM) alone inhibited mechanical re-sponses elicited by cirazoline (Fig. 1). The concentration-response curves to cirazoline were displaced to the rightwith significant increases in the EC50 and significant depres-sions of maximal response in the presence of the individual

agents mibefradil, indanyloxyacetic acid 94 or nifedipine (Ta-ble 1). However, it is interesting thatnH for the concentration-response curve to cirazoline was significantly reduced only inthe presence of nifedipine. Neither mibefradil norindanyloxyacetic acid 94 influencednH (Table 1). The de-pression of the maximal response produced by nifedipinealone was significantly greater when compared to that pro-duced by either mibefradil alone or indanyloxyacetic acid 94alone (Table 1).

A combination of mibefradil and indanyloxyacetic acid 94further inhibited the mechanical activity produced bycirazoline (Fig. 2A). Mibefradil and indanyloxyacetic acid 94combined significantly reduced the maximal response pro-duced by cirazoline when compared to the effects of eithermibefradil alone or indanyloxyacetic acid 94 alone (Table 1).The combined effects of mibefradil and indanyloxyacetic acid94 did not further affect the EC50 value when compared to theeffect of either drug alone. Moreover, the combined effects ofmibefradil and indanyloxyacetic acid 94 did not affectnHwhen compared to the effect of either drug alone (Table 1).

The further reduction in the maximal response tocirazoline, in the presence of mibefradil and nifedipine com-bined, was insignificant when compared to the effects ofnifedipine alone (Fig. 2B). However, a comparison betweenthe effects of mibefradil alone and that of mibefradil andnifedipine on the maximal response produced by cirazolinereveals that nifedipine did produce significant and additiveinhibition of this response (Table 1). In contrast, the maxi-mal mechanical responses produced by cirazoline were notsignificantly affected by a combination of nifedipine and

© 2000 NRC Canada

716 Can. J. Physiol. Pharmacol. Vol. 78, 2000

Groups EC50 nH % maximum

Control 54 ± 8.0 3.1 ± 0.1 96 ± 2.0Vehicle 57 ± 6.0 3.2 ± 0.2 96 ± 1.0

Control 57 ± 10 2.8 ± 0.2 97 ± 1.0Mibefradil 116 ± 32a 2.1 ± 0.3 79 ± 3.0a, c

Control 59 ± 8 2.9 ± 0.2 96 ± 1.0Indanyloxyacetic acid 94 154 ± 50a 3.3 ± 0.4 67 ± 4.0a, c, d

Control 58 ± 15 3.3 ± 0.4 96 ± 1.0Nifedipine 133 ± 30a 1.4 ± 0.1a, b 44 ± 4.0a, b

Control 56 ± .09 3.0 ± 0.2 96 ± 1.0Mibefradil + indanyloxyacetic acid 94 101 ± 16a 2.2 ± 0.3 50 ± 4.0a

Control 54 ± 9 3.2 ± 0.2 96 ± 1.0Mibefradil+Nifedipine 152 ± 26a 1.2 ± 0.1a,b 42 ± 2.0a,b

Control 51 ± 7.0 3.0 ± 0.2 96 ± 1.0Nifedipine+Idanyloxyacetic acid 94 169 ± 30a 1.2 ± 0.1a,b 45 ± 5.0a,b

aSignificantly different from control,P < 0.05.bSignificantly different from mibefradil,P < 0.05.cSignificantly different from mibefradil + indanyloxyacetic acid 94,P < 0.05.dSignificantly different from nifedipine,P < 0.05.

Table 1. EC50 (nM), Hill coefficient (nH), and % maximum response values obtained from individualconcentration response curves in aortic ring preparations. Each value represents the mean of 6 experi-ments ± SE.



indanyloxyacetic acid 94 when compared with eithernifedipine alone or mibefradil and indanyloxyacetic acid 94combined (Fig. 2C, Table 1).

Contractions produced by high K+ (80 mM) were not sub-stantially affected by the presence of indanyloxyacetic acid94, taking into account that adrenergic nerves also contributeto mechanical responses that are induced by depolarization.Essentially, the presence of indanyloxyacetic acid 94 insig-nificantly reduced contractions to high K+ by 10% ± 3% (n= 6; mean ± SE) when compared with phentolamine (non-selectiveα-adrenoceptor antagonist) which reduced it by14% ± 3% (n = 6; mean ± SE; Fig. 3). Neither the presenceof phentolamine nor indanyloxyacetic acid 94 significantlyreduced K+-induced contractions when compared with thecontrol (Fig. 3).

Discussion

It is evident from our present findings that mibefradil com-bined with indanyloxyacetic acid 94 resulted in additive inhi-bition when compared with the effects of mibefradil alone. Incontrast, a combination of nifedipine and indanyloxyaceticacid 94 did not result in additive inhibition when comparedwith nifedipine alone. Moreover, nifedipine and mibefradildid appear to further inhibit cirazoline mediated contractionswhen compared with the actions of mibefradil alone but notwhen compared with the actions of nifedipine alone.

Current evidence in the literature supports the view thatα1-adrenoceptors that predominantly mediate contractions(phasic and tonic) in the rat aorta are of theα1D sub-type(Buckner et al. 1996; Castillo et al. 1997). Based on pharma-cological and biochemical evidence, there are at least threedifferent subtypes ofα1-adrenoceptors (α1A, α1B, α1D) thatseem to contribute to contractile responses in smooth muscle(Lomasney et al. 1991; Schwinn and Lomasney 1992; Fordet al. 1996). It seems that cirazoline has a higher affinity fortheα1A-adrenoceptor andα1D-adrenoceptor subtypes than theα1B-adrenoceptors (Buckner et al. 1996). Most likely, the ac-tions of cirazoline in the rat aorta are mediated via the acti-vation ofα1D-adrenoceptors.

Mibefradil is considered to be a selective inhibitor of T-type Ca2+ channels. Mishra and Hermsmeyer (1994) exam-ined the selectivity of mibefradil on T-type versus L-typeCa2+ channels in primary cultures of vascular muscle cells.This study revealed that at concentrations of 100 nM and1 µM, mibefradil was able to block 50% and 85% of T-typeCa2+ channels, respectively, and 0% and 25% of L-type Ca2+

channels, respectively (Mishra and Hermsmeyer 1994). It islikely that a concentration of 300 nM mibefradil, which weused in the present study on isolated aortic rings, would haveprimarily inhibited T-type rather than L-type Ca2+ channels.We also tested the effect of mibefradil at various other con-centrations (10, 30, 100, and 1000 nM) on cirazoline-evokedcontractions. It was apparent that at 10 nM and 30 nM,mibefradil had negligible inhibitory effects on cirazoline-mediated contractions. However, mibefradil at 100 nM and1000 nM reduced the maximal responses to cirazoline by 11± 3% (n = 6; mean ± SEM) and 60 ± 7% (n = 6; mean ±SEM), respectively, (Tabrizchi and Duggan 1999, unpub-lished observations). The reported IC50 for mibefradil

© 2000 NRC Canada


Fig. 1. Concentration-response curves to cirazoline (A) in the ab-sence (closed circles) or presence (opened circles) of mibefradil(300 nM), (B) in the absence (closed circles) or presence(opened circles) of indanyloxyacetic acid 94 (30µM), and (C) inthe absence (closed circles) or presence (opened circles) ofnifedipine (300 nM). Each point represents the mean of 6 experi-ments ± SE.



against K+-evoked contractions in isolated blood vessels ap-pears to be of the order of 70 nM (Lam et al. 1998; van derLee et al. 1999). Therefore, based on the ability ofmibefradil to inhibit mechanical activity that was initiatedby the stimulation ofα1-adrenoceptors, it would be prudentto suggest thatα1-adrenoceptor mediated responses in rataortic ring preparations are, in part, produced as a result ofan influx of Ca2+ via the T-type Ca2+ channels. Such a viewis consistent with the finding that T-type Ca2+ channelblockade in vivo results in a reduction in blood pressure(Ernst and Kelly 1998).

A role for agonist-induced Ca2+-activated Cl– channels hasbeen identified on the basis of electrophysiological evidencein a variety of blood vessels. These include rat and rabbit por-tal vein (Pacaud et al. 1989b; Byrne and Large 1988a, b), rab-bit ear artery (Amédée et al. 1990), human mesenteric artery(Klöckner 1993), and rabbit coronary artery (Lamb et al.1994). Currently, it is believed that neurotransmitters, suchas noradrenaline, that act onα1-adernoceptors mediate the re-lease of Ca2+ from intracellular stores. This subsequently acti-vates Cl– channels resulting in an increase in Cl– conductanceand the opening of voltage-gated Ca2+ channels (Pacaud et al.1989a, 1989b; Amédée et al. 1990). Ca2+-activated Cl– chan-nels have been found to be blocked by 4’,4’-diisothiocyanostillbene-2,2-disulfonic acid, niflumic acid, 5-nitro-2-(3-phenylpropylamino) benzoic acid, and indanyloxy-acetic acid 94 (Pacaud et al. 1989a; Kokubun et al. 1991;Nelson et al. 1997; Greenwood and Large 1998). Reportedly,indanyloxyacetic acid 94 is a very selective blocker of Cl–

channels in the epithelial cells (Landry et al. 1987). More-over, experiments performed using cultured vascular smoothmuscle have revealed that indanyloxyacetic acid 94, at thesame concentration used in the present study, was capable ofinhibiting endothelin-induced depolarization (Takenaka et al.1992). In addition, endothelin-mediated vasoconstriction wasalso found to be impaired by indanyloxyacetic acid 94(Takenaka et al. 1992). Recently, it was also reported thatthe vasoconstrictor actions of angiotensin II on the renal af-ferent arterioles were attenuated by the presence of

© 2000 NRC Canada


Fig. 2. Concentration-response curves to cirazoline (A) in the ab-sence (closed circles) or presence (opened circles) of mibefradil(300 nM) + indanyloxyacetic acid 94 (30µM), (B) in the ab-sence (closed circles) or presence (opened circles) of mibefradil(300 nM) + nifedipine (300 nM), and (C) in the absence (closedcircles) or presence (opened circles) of nifedipine (300 nM) +IAA-94 (30 µM). Each point represents the mean of 6 experi-ments ± SE.

Fig. 3. High K+ (80 mM) induced contractions in the absence(open bars) or presence (closed bars) of indanyloxyacetic acid 94(IAA-94) (30 µM) or phentolamine (10µM). Each point repre-sents the mean of 6 experiments ± SE.



indanyloxyacetic acid 94 (same concentration as the presentstudy) while potassium-induced responses were not affectedby indanyloxyacetic acid 94 (Carmines 1995). Moreover,indanyloxyacetic acid 94 (30µM) has also been reported toinhibit myogenic vasoconstriction in isolated cerebral bloodvessels without affecting potassium-induced vasoconstrict-ion in the same vessels (Nelson et al. 1997). We also testedthe impact of IAA94 on cirazoline-evoked contractions (3and 10µM) at two lower concentrations. It was apparent thatwhile IAA-94 did not affect cirzaoline-evoked contraction at3 µM, it did reduce the maximal response to cirazoline by~10% at 10µM (Tabrizchi and Duggan 1999, unpublishedobservations).

The evidence from the present study indicates thatindanyloxyacetic acid 94, at the concentration used, does notaffect potassium-induced contractions. This suggests thatvoltage-gated Ca2+ channel are not affected directly by thisdrug. It is also apparent that indanyloxyacetic acid 94, incombination with mibefradil, produced additive inhibition ofthe mechanical responses produced by cirazoline. We thinkthat the greater inhibitory effects of the combination ofmibefradil and indanyloxyacetic acid 94 on cirazoline-induced contractions can be interpreted in two ways. The in-terpretation is dependent on whether the processes involvedwere affected in series or in parallel. It could be argued thatby blocking Cl– channels, indanyloxyacetic acid 94 inhibitedthe residual Ca2+ influx via the L-type Ca2+ channels, addingto the inhibitory effects of mibefradil which, itself, was act-ing on the T-type Ca2+ channels. This would imply that theprocesses inhibited by mibefradil and indanyloxyacetic acid94 were occurring in series. Equally, it is possible that T-type Ca2+ channel activation may occur independent of Cl–

efflux, and therefore indanyloxyacetic acid 94 and mibe-fradil inhibited two different process independently and pro-duced additive inhibition of the mechanical response. Such aview would support the idea that the processes that were in-hibited by mibefradil and indanyloxyacetic acid 94 were oc-curring in parallel. Based on the current experiments, thetwo processes can not be distinguished and further studiesare needed to explore the mechanism involved in more de-tail.

Although the effects of nifedipine and mibefradil were dis-covered to be substantially additive in comparison to those ofmibefradil alone, they were found to be insignificantly addi-tive when compared to those of nifedipine alone in the pres-ent investigation. This discrepancy may be due to the fact thatmibefradil, at the concentration used in the present study, wasacting more discriminantly and affecting a more selectiveclass of Ca2+ channels (T-type) while nifedipine was not. Re-portedly, a number of dihydropyridines can affect the functionof not only the L-type Ca2+ channels but also of the T-typeCa2+ channels, and nifedipine has been reported to be amongsuch drugs. An earlier report by Aaroson and co-workers(1988) had indicated that nifedipine was capable of inhibitingall Ca2+ currents in smooth muscle cells from rabbit ear ar-tery, a tissue that contains both L-type and T-type Ca2+ chan-nels (Benham et al. 1987). It is certainly possible that theactions of nifedipine on T-type Ca2+ channels may, in part,offer an explanation for the lack of a significant additive in-hibition observed with the combination of nifedipine andmibefradil when compared to those of nifedipine alone in

the present investigation. The additional inhibitory effects ofnifedipine on T-type Ca2+ channels may explain the smallyet significant additive inhibition observed for the combina-tion of indanyloxyacetic acid 94 and nifedipine, when com-pared to indanyloxyacetic acid 94 alone. However, thiswould imply that in the rat aortic ring preparation T-typeCa2+ channel activation occurs independently of Cl– efflux.Indanyloxyacetic acid 94 primarily inhibited the activationof L-type Ca2+ channels secondary to inhibiting the Ca2+-activated Cl– channels. In the present investigation we findthat the Hill coefficient calculated for the concentrationresponse curve to cirazoline was significantly reduced by thepresence of nifedipine. The Hill coefficient reflects agonist-receptor cooperativity. One reason that nifedipine wasfound to influence the Hill coefficient in the present inves-tigation may be attributed to its multiple sites of actions.

The inhibitory effects of mibefradil, indanyloxyacetic acid94 and nifedipine alone on the Emax. were 21%, 33%, and56%, respectively. Based on the present data, the inhibitoryeffect of mibefradil and indanyloxyacetic acid 94 combinedon Emax. was found to be 50% experimentally. Theoretically,a simple addition of the inhibitory effects of mibefradil(21%) and indanyloxyacetic acid 94 (33%) yields a value of54%, which is very close to the value obtained experimen-tally. A parallel comparison between the inhibitory effects ofnifedipine and mibefradil combined (58%) on Emax to that ofeither mibefradil (33%) or nifedipine (56%) alone wouldsuggest that nifedipine and mibefradil are not producingsubstantive additive inhibition. Theoretically, the combina-tion of mibefradil and nifedipine would have been expectedto result in a reduction in Emax. greater than 55%. It is evi-dent that the inhibition produced by nifedipine alone was56%. As suggested earlier, the most reasonable explanationfor this is an overlap between the inhibitory effects ofnifedipine and mibefradil. Based on the data obtained in thepresent investigation it may be reasonable to suggest that thecontribution of T-type versus L-type Ca2+ channels to con-tractile responses obtained with cirazoline are approximately21% and 35% of the Emax.

In summary, our findings seem to indicate that mibefradil,indanyloxyacetic acid 94 and nifedipine inhibit cirazoline-induced contractions to varying degrees. Moreover, both T-type and L-type Ca2+ channels appear to play a role inα1-adrenoceptor mediated contractions in isolated rat aorta.However, it would appear that L-type Ca2+ channels play agreater role in processes that involve excitation–contractioncoupling subsequent to stimulation ofα1-adrenoceptors.

Acknowledgement

This work was supported by a grant from the Natural Sci-ences and Engineering Research Council of Canada. Wethank Hoffmann-La Roche for providing us with a sample ofmibefradil.

References

Aaroson, P.I., Bolton, T.B., Lang, R.J., and MacKenzie, I. 1988.Ca2+ currents in single isolated smooth muscle cells from therabbit ear artery in normal- Ca2+ and high-barium solutions. J.Physiol.405: 57–75.

© 2000 NRC Canada




© 2000 NRC Canada


Amédée, T., Large, W.A., and Wang, Q. 1990. Characteristics ofCl– currents activated by noradrenaline in rabbit ear artery cells.J. Physiol.428: 501–516.

Bean, B.P., Sturek, M., Puga, A., and Hermsmeyer, K. 1986. Ca2+

channels in vascular muscle cells isolated from rat mesentericarteries: modulation by dihydropyridine drugs. Circ. Res.59:229–235.

Bean, B.P. 1991. Pharmacology of Ca2+ channels in cardiac mus-cle, vascular muscle, and neurons. Am. J. Hypertens.4(7 Pt 2):406S–411S.

Benham, C.D., Hess, P., and Tsien, R.W. 1987. Two types of Ca2+

channels in single smooth muscle cells from rabbit ear arterystudied with whole-cell and single-channel recordings. Circ.Res.61(Suppl. I): I10–I16.

Buckner, S.A., Oheim, K.W., Morse, P.A., Knepper, S.M., andHancock, A.A. 1996.α1-Adrenoceptor-induced contractility inrat aorta is mediated by theα1D subtype. Eur. J. Pharmacol.297:241–248.

Byrne, N.G., and Large, W.A. 1998a. Mechanism of action ofα-adrenoceptor activation in single cells freshly dissociated fromthe rabbit portal vein. Br. J. Pharmacol.94: 475–482.

Byrne, N.G., and Large, W.A. 1988b. Membrane ionic mechanismsactivated by noradrenaline in cells isolated from the rabbit portalvein. J. Physiol.404: 557–573.

Carmines, P.K. 1995. Segment-specific effect of Cl– channel block-ade on rat renal arteriolar contractile responses to angiotensin II.Am. J. Hypertens.8: 90–94.

Castillo, E.F., Valencia, I., Bobadilla, R.M., Villalon, C.M., andCastillo, C. 1997.α-Methylnoradrenaline-induced contractionsin rat aorta are mediated viaα1D-adrenoceptors. Fundam. Clin.Pharmacol.11: 339–345.

Criddle, D.N., de Moura, R.S., Greenwood, I.A., and Large, W.A.1996. Effect of niflumic acid on noradrenaline-induced contrac-tions of the rat aorta. Br. J. Pharmacol.118: 1065–1071.

Criddle, D.N., de Moura, R.S., Greenwood, I.A., and Large, W.A.1997. Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolatedmesenteric vascular bed of the rat. Br. J. Pharmacol.120: 813–818.

Ernst, M.E., and Kelly, M.W. 1998. Mibefradil, a pharmacologi-cally distinct Ca2+ antagonist. Pharmacotherapy,18: 463–485.

Ford, A.P., Arredondo, N.F., Blue, D.R. Jr., Bonhaus, D.W., Jasper, J.,Kava, M.S., Lesnick, J., Pfister, J.R., Shieh, I.A., Vimont, R.L., Wil-liams, T.J., McNeal, J.E., Stamey, T.A., and Clarke, D.E. 1996. RS-17053 (N-[2-(cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethanamine hydrochloride), a selec-tive α1A-adrenoceptor antagonist, displays low affinity for func-tional α1-adrenoceptors in human prostate: implications foradrenoceptor classification. Mol. Pharmacol.49: 209–215.

Greenwood, I.A., and Large, W.A. 1995. Comparison of the effectsof fenamates on Ca-activated Cl– and potassium currents in rab-bit portal vein smooth muscle cells. Br. J. Pharmacol.116:2939–2948.

Greenwood, I.A., and Large, W.A. 1998. Properties of a Cl-currentactivated by cell swelling in rabbit portal vein vascular smoothmuscle cells. Am. J. Physiol.275: H1524–H1532.

He, Y., and Tabrizchi, R. 1997. Effects of niflumic onα1-adrenoceptor-induced vasoconstriction in mesenteric artery invitro and in vivo in two-kidney one-clip hypertensive rats. Eur.J. Pharmacol.328: 191–199.

Hogg, R.C., Wang, Q., and Large, W.A. 1993. Time course ofspontaneous Ca2+-activated Cl– currents in smooth muscle cellsfrom the rabbit portal vein. J. Physiol.464: 15–31.

Hogg, R.C., Wang, Q., and Large, W.A. 1994. Action of niflumicacid on evoked and spontaneous Ca2+-activated Cl– and potas-sium currents in smooth muscle cells from rabbit portal vein. Br.J. Pharmacol.112: 977–984.

Klöckner, U. 1993. Intracellular Ca2+ ions activate a low-conductance Cl– channel in smooth-muscle cells isolated fromhuman mesenteric artery. Pflügers. Arch.424: 231–237.

Kokubun, S., Saigusa, A., and Tamura, T. 1991. Blockade of Cl–

channels by organic and inorganic blockers in vascular smoothmuscle cells. Pflügers. Arch.418: 204–213.

Lam, E., Skarsgard, P., and Laher, I. 1998. Inhibition of myogenictone by mibefradil in rat cerebral arteries. Eur. J. Pharmacol.358: 165–168.

Lamb, F.S., Volk, K.A., and Shibata, E.F. 1994. Ca2+-activated Cl–

current in rabbit coronary artery myocytes. Circ. Res.75: 742–750.

Landry, D.W., Reitman, M., Cragoe, E.J. Jr., and Al-Awqati, Q.1987. Epithelial Cl– channel. Development of inhibitory ligands.J. Gen. Physiol.90: 779–798.

Lomasney, J.W., Cotecchia, S., Lefkowitz, R.J., and Caron, M.G.1991. Molecular biology ofα-adrenergic receptors: implicationsfor receptor classification and for structure-function relation-ships. Biochim. Biophys. Acta,1095: 127–139.

Lum Min, S.A., Stapleton, M.P., and Tabrizchi, R. 1999. Influenceof Cl– ions on α1-adrenoceptor mediated contraction and Ca2+

influx in rat caudal artery. Life Sci.64: 1631–1641.Mishra, S.K., and Hermsmeyer, K. 1994. Selective inhibition of T-

type Ca2+ channels by RO 40–5967. Circ. Res.75: 144–148.Nelson, M.T., Conway, M.A., Knot, H.J., and Brayden, J.E. 1997.

Cl– channel blockers inhibit myogenic tone in rat cerebral arter-ies. J. Physiol.502: 259–264.

Orallo, F. 1996. Regulation of cytosolic Ca2+ levels in vascularsmooth muscle. Pharmacol. Ther.69: 153–171.

Pacaud, P., Loirand, G., Lavie, J.L., Mironneau, C., andMironneau, J. 1989a. Ca2+-activated Cl– current in rat vascularsmooth muscle cells in short-term primary culture. Pflügers.Arch. 413: 629–636.

Pacaud, P., Loirand, G., Mironneau, C., and Mironneau, J. 1989b.Noradrenaline activates a Ca2+-activated Cl– conductance and in-crease the voltage-dependent Ca2+ current is cultured singlecells of rat portal vein. Br. J. Pharmacol.97: 139–146.

Schwinn, D.A., and Lomasney, J.W. 1992. Pharmacological char-acterization of cloned alpha 1-adrenoceptor subtypes: selectiveantagonists suggest the existence of a fourth subtype. Eur. J.Pharmacol.227: 433–436.

Sperelakis, N. 1990. Properties of Ca2+ channels in cardiac muscleand vascular smooth muscle. Mol. Cell. Biochem.99: 97–109.

Takenaka, T., Epstein, M., Forster, H., Landry, D.W., Iijima, K.,and Goligorsky, M.S. 1992. Attenuation of endothelin effects bya Cl– channel inhibitor, indanyloxyacetic acid. Am. J. Physiol.262: F799–F806.

van der Lee, R., Pfaffendorf, M., and van Zwieten, P.A. 1999.Comparative effects of mibefradil and other Ca2+ antagonists onresistance arteries of different end organs. Fundam. Clin.Pharmacol.13: 198–203.

Wang, X.Y., and Pang, C.C.Y. 1993. Functional integrity of thecentral and sympathetic nervous system is a prerequisite forpressor and tachycardic effects of diphenyleneiodonium, a novelinhibitor of nitric oxide synthase. J. Pharmacol. Exp. Ther.265:263–272.



Influence of T-type Ca 2+ (mibefradil) and Cl - (indanyloxyacetic acid 94) channel antagonists on α...

Documents

Transcript of Influence of T-type Ca 2+ (mibefradil) and Cl - (indanyloxyacetic acid 94) channel antagonists on α...