aminophylline preferentially inhibits chloroethylclonidine-insensitive α-adrenoceptor-mediated...

Gen. Pharmac. Vol. 24, No. 6, pp. 1359-1364, 1993 0306-3623/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd

AMINOPHYLLINE PREFERENTIALLY INHIBITS CHLOROETHYLCLONIDINE-INSENSITIVE

oc-ADRENOCEPTOR-MEDIATED CONTRACTIONS IN RAT AORTA

JUAN DUARTE, l FRANCISCO PI~REZ-VIZCAiNO, l* ANTONIO ZARZUELO, 2 JOS]~ JIMI~NEZ 2 and JUAN TAMAROO 1

IDepartment of Pharmacology, School of Medicine, Universidad Complutense, 28040 Madrid, Spain [Tel. 34-1-394-14-72; Fax 34-1-394-14-70]

'Department of Pharmacology, School of Pharmacy, Universidad de Granada, 18071 Granada, Spain [Tel. 34-58-24-38-88, Fax 34-58-243893]

(Received 7 April 1993)

Abstract--l. In rat thoracic aortae, contractions induced by methoxamine were inhibited by chloroethylclonidine, whereas oxymetazoline-induced contractions, which were more dependent on Ca2+-entry, were insensitive to chloroethylclonidine.

2. Aminophylline inhibited the contractions and 45Ca2+-uptake induced by both methoxamine and oxymetazoline. However, oxymetazoline-induced contractions were more sensitive to inhibition by aminophylline and D600.

3. Thus, the partial selectivity of aminophylline for the chloroethylclonidine-resistant, highly dependent on extracellular Ca 2+, oxymetazoline-mediated responses may be explained by a preferential inhibition of agonist-induced Ca 2+ entry as compared to inhibition of other transduction pathways.

INTRODUCTION

In vascular smooth muscle, methylxanthines, such as caffeine and aminophylline, at concentrations in the range of mM, exert transient contractions in Ca 2+- free media due to the release of Ca '+ from intracellular stores (Manzini et al., 1982; Leijten and Van Breemen, 1984). Besides these contractile effects, at lower concentrations, these drugs exert potent relax- ing properties on vascular smooth muscle (Casteels et al., 1977; Leijten and Van Breemen, 1984; Ahn et al., 1988; Sato et al., 1988; Duarte et aL, 1992). The mechanism of the vasodilatory actions of these drugs is not fully understood as methylxanthines have been found to inhibit 4SCa2+ uptake stimulated by

both noradrenaline and high K + (Leijten and Van Breemen, 1984; Ahn et al., 1988; Duarte et al., 1992) as well as the inward current carried by barium ions through voltage-dependent Ca ~+ channels in single vascular smooth muscle cells (Hughes et al., 1990), increase cytosolic cyclic AMP by inhibiting phosphodiesterases (Hitchcock, 1973; Poison et al., 1978) and possibly produce a direct inhibition of the contractile apparatus (Sato et al., 1988; Ozaki et al., 1990; Van der Brent and B6ny, 1991).

The a-adrenoceptor system in rat vascular smooth muscle has been a major focus of attention for the

*To whom all correspondence should be addressed.

action of agents because of its contribution to vascular tone in both normal and hypertensive states (Lefevre-Borg et al., 1988). It has become clear in the last few years that a large heterogeneity exists upon postjunctional a-adrenoceptors (Van Zwieten and Timmermans, 1987; McGrath and Wilson, 1988; Minneman, 1988; Ruffolo et al., 1991; Bylund, 1992). ~-Adrenoceptors present in rat aorta differ from those of other species and classification of these receptors is still a matter of controversy (Ruffolo et al., 1982, 1991; Oriowo and Ruffolo, 1991). Very recently, Duarte et al. (1992) have reported that aminophylline inhibited noradrenaline-induced contractions and 45Ca2+ uptake. In the present paper, we

have analysed the inhibitory effects of aminophylline on the contractile responses and 4SCa2+ uptake induced by different a-adrenoceptor agonists in isolated rat aorta.

MATERIALS AND METHODS

Experimental procedure Thoracic aortae from Sprague-Dawley rats of either sex

weighing 250-350 g were dissected and placed immediately in a physiological saline solution (PSS) of the following composition (mM): NaClll8, KC14.75, NaHCO2 25, MgSO4 1.2, CaCI 2 2, KH2PO 4 1.2 and glucose I I. The tissue bath was maintained at 37°C and bubbled with 95% 02-5% COs gas mixture. After surrounding connective tissue and endothelium were removed, the aortae were cut into rings about 4-5 mm in length. The absence of functional endothelium was confirmed at the end of each experiment by the

1359

1360 JUAN DUARTE et al.

inability of the preparation precontracted with methoxamine or oxymetazoline to relax in response to acetylcholine (10 -5 M). Aortic rings were mounted under the tension of 2 g by two parallel L-shaped stainless-steel holders inserted into the lumen and connected to a Grass FT03 force-displacement transducers as previously described (Perez-Viz- caino et al., 1991; Duarte et al., 1992). Each preparation was allowed to equilibrate for 2 hr before exposure to drugs.

Characterization of methoxamine- and oxymetazoline-induced contractions

Aortic rings were exposed to 10-4M methoxamine or 10 -5 M oxymetazoline in normal PSS until they reached a steady-state contraction and then washed in normal PSS to reach again baseline tension. Then the rings were incubated in CaE+-free PSS containing 0.1 mM EDTA for 10 min and, at this time, addition of 10 -4 M methoxamine or 10 -5 M oxymetazoline induced a phasic contraction. 5 min later 2 mM CaCI2 was added to the bathing media to induce a tonic contraction.

In another group of experiments aortic rings were exposed to cumulative concentrations of methoxamine (10-7-10-3 M) and oxymetazoline (10-7-10-5M) in the presence or absence of 5 x 10 -6 M chloroethylclonidine added 30 min before and washed just prior to the initiation of the concentration-response curve. The results of these experiments are expressed as a percentage of the maximal methoxamine-induced contractile responses in the absence of chloroethylclonidine.

Effects of aminophylline on contractions induced by methoxamine and oxymetazoline

Aortic rings were contracted by addition of single maximal concentrations of methoxamine (10-4 M) and oxymetazoline (10-SM) until they reached steady-state and then washed with PSS for 30-45 min. Control contractile responses were obtained at the beginning of the experiment until two successive responses were almost identical in height. Afterwards rings were exposed to aminophylline for a 10 min period and the contractions were repeated in the continuing presence of the aminophylline. Only one agonist was tested in each experiment. Since aminophylline is a stable mixture of 85% anhydrous theophylline and 15% ethylenediamine, the same protocol was employed using ethylenediamine instead of aminophyUine. At concentrations equivalent to those of aminophylline, ethylenediamine did not modify the contractions to methoxamine and oxymetazoline.

To determine whether aminophylline and D600 could relax an existing contraction, aortic rings were contracted by single concentrations of methoxamine (10-4 M) or oxymetazoline (10-SM). When the contractile response to either agonist was maximum, aminophylline (10-6-I0 -~ M) or D600 (10-8-10 -5 M) were added in progressively increasing cumulative concentrations. The results were expressed as a percentage of the maximal control agonist-induced responses.

Effects of aminophylline on 4SCa2+ fluxes The 45Ca2+ uptake was estimated by measuring the

changes in the specific activity of the 45Ca2+ fraction resistant to displacement by lanthanum as previously described (Tamargo and Tejerina, 1989; Prrez-Vizcaino et aL, 1991). Aortic tings were equilibrated in PSS of the following composition (mM): NaC1 122, KC1 5, NaHCO 3 25, CaC12 1.25, MgSO4 1.2 and glucose 11.5, gassed with 95% 02-5% CO2. After equilibration experimental rings were treated with aminophylline for 10 min. Then both experimental and control tings were incubated for 5 min in PSS containing 45Ca2+ (sp. act. 1 #Ci/ml; ICN Biomedicals, Calif.) in the presence or absence of aminophyltine and then for another 2 min in the same solution with or without the

addition of 10 -4 M methoxamine or 10- ~ M oxymetazoline. At this time rings were washed for 5 min in 500 ml of a lanthanum solution of the following composition in mM: NaCII22, KCI 5.9, MgCI 2 1.25, glucose 11, LaC13 50 and Tris maleate 15 (pH6.8) at 0°C. Afterwards rings were removed, pressed with a roller and weighed. The rings were then placed in scintillation vials and 0.5 ml of Protosol (Dupont) added and the rings digested overnight at 60°C. Radioactivity was assayed in a liquid scintillation counter (LKB Wallac 1211 Rack).

Drugs

The following drugs were used: aminophylline (Elmu S.A., Madrid), methoxamine hydrochloride, oxymetazoline hydrochloride, acetylcholine chloride (Sigma Chemical Co., London), chloroethylclonidine dihydrochloride (ICN Biomedicals Inc., Calif., U.S.A.) and D600 (Knoll AG, Ludwigshafen/Rhein, Germany). Stock solutions (10 -2 M) of all drugs were prepared in distilled deionized water, further dilutions were made in PSS.

Statistics

Throughout the paper values are expressed as the mean + SEM and statistical analysis was performed with paired or unpaired Student's t-test. The concentrations of aminophylline producing a 50% inhibition of the maximal contractile response (IC50) were calculated from computer fitted concentration-response curves.

RESULTS

Effects o f methoxamine and oxymetazoline on Ca 2÷-

free P S S

As shown in Fig. 1, methoxamine (10 -4 M) and

oxymetazoline (10 -5 M) induced a tonic contract ion

in aortic rings in normal PSS averaging 2850 ___ 278

and 2120 _ 32 mg, respectively (n = 5, P < 0.05

methoxamine vs oxymetazoline). After washing in

Ca-free PSS, the phasic contractions induced by both

t-

2

P f

a . . _ . . / l ~ ~ j

2 mM Ca Ca-free 2 mM Ca

Fig. 1. Ca2+-dcpendence of the contractile responses induced by 10 -4 M methoxamine (open columns) and 10 -5 M oxymetazoline (solid columns). Aortic tings were initially incubated in Ca2+-containing PSS and a tonic contraction was elicited by addition of the appropriate agonist (left columns). After washing, tings were incubated in Ca2+-free medium containing 0.1 mM EDTA, addition of the agonists evoked a phasic contraction (middle columns) and then Ca 2+ (2 mM) was added to the medium to restore the tonic contraction (right columns). Ordinate: grams of tension. Each column represents the mean + SEM of five exper-

iments.

(.}

M 0

i I i

- 7 - 8 - S

Log [oxymetazoline]

100

(b)

I =

.o 100 o g

o

Ig

.~ 5 0 X IW

i !

- 4 -3

@ "M- "K. @

- 7 - 6 - 5 - 4 - 3

Log [methoxamine]

Fig. 2. Concentration-responses curve to (a) oxymetazoline and (b) methoxamine in rat aortae in the absence (O) and in the presence (@) of chloroethylclonidine (3 x 10 -6 M). Ordinate: percentage of the maximum control contractions obtained at the highest concentration of methoxamine. Abscissa: methoxamine and oxymetazoline concentration (log[M]). Each point represents the mean +_ SEM of six

experiments. *P < 0.05 compared to controls in the absence of chloroethylclonidine.

agonists averaged 22.3+_3.7% and 5.9+_1.3%,

respectively, of the previous contractions in

normal PSS (P < 0.01 methoxamine vs oxy-

metazoline). Further addition of 2 m M Ca 2÷

to the bathing media induced a tonic con-

traction of a similar magnitude of the initial con-

traction (96.4 + 4.2% and 96.1 _ 14.9%,

respectively).

O L -

c- O o 50 0

N

I I " r I I I I I

- 5 - 4 - 3 - 8 - 7 - 6 - 5 - 4

a b

I

-6 Log [Aminophylline] Log [D600]

Fig. 3. Relaxant effects of (a) aminophyhine and (b) D600 on methoxamine- (O, 10 -4M) and oxymetazoline- (0 , 10 -5 M) induced contractions. Ordinate: % of the control contractions. Abscissa: aminophylline and D600 concentration (Log[M]). Each point represents the mean + SEM of 5-8

experiments. *P < 0.05 methoxamine vs oxymetazoline.

Aminophylline and a-adrenoceptors 1361

OP 24/6---E

1362 JUAN DUARTE et al.

Effects o f chloroethylclonidine on methoxamine and oxymetazoline concentration-response curves

The concentration-response curves to methoxamine and oxymetazoline are shown in Fig. 2. Addition of 3 x 10 -6 M chloroethylclonidine to resting tings

induced a contraction averaging 486+175mg (n = 12). In the presence of 5 × 10 -6 M chloroethyl-

clonidine, there was only a minor, non-significant, shift in the curve to oxymetazoline. In contrast, this concentration of chloroethylclonidine markedly shifted to the tight the concentration-response curve to methoxamine. This inhibition cannot be attributed to the partial agonist effect of chioroethylclonidine since in the same conditions it produced almost no change in the curve to oxymetazoline.

Effects o f aminophylline and D600 on the contractile responses to oxymetazoline and methoxamine

In aortic tings previously contracted with methoxamine (10 -4 M) or oxymetazoline (10 -5 M), cumulative increases in aminophylline concentration (10-6-10 -3 M) produced a concentration-dependent

relaxation, the ICs0s being 1.2+0.1 x 10-4M and 4.4 + 0.7 × 10 -5 M, respectively [P < 0.05 methoxamine vs oxymetazoline, Fig. 3(a)]. In methoxamine- and oxymetazoline-precontracted rings, cumulative addition of D600 (10-g-10 -5 M) produced a concentration-dependent relaxation, the ICs0s being 2.5 _ 0.9 × 10 -5 M and 3.1 _ 0.5 x 10 -7 M, respectively [P <0.05 methoxamine vs oxymetazoline, Fig. 3(b)].

Figure 4 shows the inhibitory effects of aminopbylline (10-5-4 x 10 -5 M) on the contractions

100

o , m

o a

g =

o o

5 0

I =

o o

0 1 0 - S M 2 x 10"SM 4 x l O ' S M

Fig. 4. Effects of aminophylline on the contractions induced by methoxamine (10 -4 M, open columns) and oxymetazoline (10 -5 M, solid columns). Aminophylline was added 10 min before addition of the agonist. Ordinate: % of the control contractions. Abscissa: aminophyUine concentration. Each column represents the mean + SEM of five to eight experiments. *P < 0.05 methoxamine vs oxymetazo-

line.

8O0

~ 600 ,oo " 200

0 Resting MET OXY

Fig. 5. Effects of aminophyiline on the La3+-resistant 4SCa~+ uptake in resting and in aortic rings stimulated by 10 -4 M methoxamine (MET) and 10 -s M oxymetazoline (OXY). Results were obtained in the absence (open columns) and in the presence of aminophylline, 5 x 10 -5 M (hatched columns) and 5 x 10 -4 M (solid columns). Each column represents the mean of eight to ten aortic rings; vertical lines indicate SEM *P < 0.05 compared to controls

in the absence of aminophylline.

induced by methoxamine (10 -4 M) and oxymetazoline (10 -5 M). Aminophylline inhibited in a concen-

tration-dependent manner the responses induced by both agonists. However, aminophylline was significantly less potent in inhibiting methoxamine- than oxymetazoline-induced responses, the ICs0 for oxymetazoline-evoked contractions being 2.9 + 0.8 × 10 -5 M, whereas at the higher concentration tested (4 × 10 -5 M) aminophylline only inhibited by 32.4 + 4.9% the maximum tension elicited by 10 -4 M methoxamine.

Effects of aminophylline on '5Ca2+ uptake

The effects of aminophylline were studied on the La3+-resistant 45Ca2+ uptake in resting as well as in

aortic rings stimulated by methoxamine and oxymetazoline (Fig. 5). Aminophylline, 5 x 10 -5 and 5 x 10 -4 M, had no significant effects on 4SCa2+ up-

take in resting aortic tings. Addition of methoxamine and oxymetazoline significantly increased 45Ca2+ uptake. Aminophylline, 5 x 10 -5 and 5 x 10-4M,

inhibited, in a concentration-dependent manner, the increase in 45Ca2+ uptake induced by both agonists.

DISCUSSION

Findings from several laboratories suggest that the • ~-adrenoceptors can be pharmacologically subdi- vided further into at least three subtypes, namely ~^, ~,B and ~'tc (Minneman, 1988; Ruffolo et aL, 1991; Bylund, 1992). This subdivision is based on the differential sensitivity of ~-adrenoceptor-mediated

Aminophylline and ~-adrenoceptors 1363

responses to blockade by antagonists and the different requirements for extracellular Ca 2+ for contraction. Han et al. (1987a, b) reported that in different rat tissues stimulation of ~lB-adrenoceptor subtype induces inositol 1,4,5-triphosphate formation and causes contractions independent of extracellular Ca 2+ and insensitive to Ca 2+ antagonists, while the ~l^ subtype appears to control the opening of dihydro- pyridine-sensitive membrane channels.

Since we have previously demonstrated that the vasodilatory effects of aminophylline are independent of the presence of endothelium (Duarte et al., 1992), this structure was mechanically removed in order to avoid the interference of endothelial x-adrenoceptor- mediated responses.

In the present experiments both oxymetazoline and methoxamine produced a contractile response both in normal PSS and in Ca-free PSS. However, methoxamine-induced phasic contractions in the absence of external Ca 2+, reflecting Ca 2+ mobilization from intracellular stores, were significantly greater than those induced by oxymetazoline. Furthermore, oxymetazoline-induced contractions were more sensitive than methoxamine-induced contractions to inhibition by D600, a specific calcium channel blocker. These results suggested that oxymetazoline-induced contractions were more dependent on Ca2+-entry than those induced by methoxamine. Chloroethyl- clonidine, an irreversible alkylating agent, has been reported to specifically block ~m-adrenoceptors (Han et al., 1987a; Minneman, 1988). In the presence of chloroethylclonidine, the concentration-response curves to methoxamine were markedly shifted to the right, whereas those of oxymetazoline remained almost unchanged. ~-Adrenoceptors present in rat aorta differ from those of other species and classification of these receptors is still a matter of controversy (Ruffolo et al., 1982, 1991; Oriowo and Ruffolo, 1991). However, following the above scheme for ~-adrenoceptor subclassification, it is tempting to attribute methoxamine-induced contractions to an activation of ~tB-adrenoceptors which increased the release of Ca 2+ from intracellular stores, whereas contractions induced by oxymetazoline can be re- garded as independent of ~m-adrenoceptor activation and related to Ca 2+ entry through channels sensitive to D600.

Aminophylline decreased, in a concentration- dependent manner, the contractile responses and the increase in 45Ca2+ uptake induced by both methoxamine and oxymetazoline. However, aminophylline inhibited to a greater extent the contractile responses induced by oxymetazoline than those evoked by methoxamine. In isolated rat aorta, aminophylline has been found to inhibit the contractile responses

and 45Ca2+ uptake stimulated by both noradrenaline and high K + (Duarte et al., 1992). Furthermore, caffeine inhibited the inward current carried by barium ions through voltage-dependent Ca 2+ channels in single vascular smooth muscle cells (Hughes et al., 1990). However, aminophylline also affects other intracellular events. In fact, it inhibits the phasic contractions induced in Ca2+-free medium (Duarte et al., 1992), it increases cytosolid cyclic AMP by inhibiting phosphodiesterases (Hitchcock, 1973; Poison et al., 1978) and as reported with caffeine, it possibly produces a direct inhibition of the contractile apparatus (Sato et al., 1988; Ozaki et al., 1990; Van der Bent and B6ny, 1991). Since oxymetazoline- induced contractions were highly dependent on Ca z+- entry whereas contractions evoked by methoxamine were relatively more dependent on Ca2+-release, the results of this paper suggest that, in rat aorta, aminophylline exerts its vasodilatory effects on ~- adrenoceptor-mediated contractions mainly through inhibition of Ca x+ entry.

In conclusion, the partial selectivity of aminophylline for the chloroethylclonidine-resistant, highly dependent on extracellular Ca 2+, oxymetazoline-mediated responses may be explained by a preferential inhibition of agonist-induced Ca z+ entry as compared to inhibition of other transduction pathways.

SUMMARY

The present experiments indicated that in isolated rat aorta aminophylline decreased in a concentration- dependent manner the contractile responses and the increase in 45CaX+ uptake induced by methoxamine and oxymetazoline, but it inhibited to a greater extent the responses induced by oxymetazoline than by methoxamine. Since methoxamine-induced contractions were inhibited by the selective am-adrenoceptor antagonist chloroethylclonidine and were less dependent on extracellular Ca 2+ than contractions induced by oxymetazoline, these contractions can be attributed to an activation of ~ts-adrenoceptors. In contrast, contractions induced by oxymetazoline were highly dependent on Ca x+ entry and were resistant to blockade by chloroethylclonidine. There- fore, the preferential inhibition of aminophylline on chloroethylclonidine-insensitive contractions can be attributed to its ability to inhibit Ca 2+ entry into vascular smooth muscle cells.

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aminophylline preferentially inhibits chloroethylclonidine-insensitive α-adrenoceptor-mediated...

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