the role of endothelium in the calcium-induced reduction of the contractile response of the rabbit...

Gen. Pharmac. Vol. 28, No. 5, pp. 745-752, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA.

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the

The Role of Endothelium in the Calcium-Induced Reduction of Contractile Response of the Rabbit Aorta

Ana Ortega, Miguel Puerro, Visitaci6n Lopez-Miranda and Amaya Aleixandre* DEPARTMENT OF PHARMACOLOGY, FACULTY OF MEDICINE,

COMPLUTENSE UNIVERSITY, 28040, MADRID, SVAIN [TEL: 34-1-394-14-75; FAX: 34-1-394-14-63]

ABSTRACT. 1. Increase of Ca 2+ concentration in the bath solution diminishes the contractile response of isolated rabbit aorta rings to ~l-adrenoceptor agonists and KCI.

2. In intact preparations the contractions of methoxamine and phenylephrine were maximal when a 0.3- to 0.6-mM Ca 2+ bath solution was used, and those of KCI were maximal with a 2.5-mM Ca 2+ concentration.

3. The contractions of methoxamine and phenylephrine also were decreased by increasing the Ca 2+ concentration above 1.25 mM in disrupted endothelium preparations and in those incubated in indomethacin (10 -5 M), NO-nitro-L-arginine methyl ester (10 -4 M), or methylene blue (10 -6 M).

4. High organ bath Ca ~+ concentrations also caused a decrease in KCI contractions using endothelium- denuded and the treated preparations, the responses being similar with 1.25 mM and 2.5-mM Ca 2+ in the methylene blue-treated preparations, whereas they were greater with 1.25 mM Ca 2+ in the others. CEN PHAPa~AC 28;5:745--752, 1997. © 1997 Elsevier Science Inc.

KEY WORDS. High calcium, rabbit aorta contractility, endothelium, nitric oxide, prostacyclin

INTRODUCTION

The increase in the levels of free ionic Ca 2 + within vascular smooth- muscle cells regulates their contractile activity. However, it must be taken into account that the extracellular level of calcium always is physiologically much greater than the intracellular level and that an increase in the Ca 2+ concentration in the external medium does not necessarily imply an intracellular increase in this ion. Furthermore, the ability of elevated levels of external calcium to reduce the contractile response of vascular smooth muscle has been recognized for a long time (Bohr, 1963; Weeb and Bohr, 1978).

A role for calcium in the release of endothelium-derived relaxing factors has been demonstrated (Furchgott et al., 1981; Liikhoff et al., 1988; Singer and Peach, 1982). The first endothelium-derived relaxing substance described was prostacyclin, which is produced by the action of the cyclo-oxygenase enzyme (Vane et al., 1987). Nitric oxide (NO) was later identified as another important endothelial- derived relaxing factor (Moncada et al., 1987, 1988); Moncada's group also observed that alterations in the calcium content of the perfusion medium can modulate NO production in the rabbit aorta (L6pez-Jaramillo et al., 1990).

The present study was designed to study in the rabbit aorta contractile responses to cxl-adrenoceptor agonists and to KC1 with different extracellular calcium concentrations and to test whether the release of endothelial-derived relaxing factors is important in modulating the inhibition of the contractile activity caused by the increase in external calcium in this tissue. Therefore, the contractions of the isolated rabbit aorta elicited by c~l-adrenoceptor agonists and KCI, using different calcium concentrations in the bathing solution, were studied in intact and endothelium-disrupted preparations. We also studied these contractions with different calcium concentra-

*To whom correspondence should be addressed. Received 22 February 1996; revised 24 June 1996; accepted 8 July 1996.

tions in the presence of the cyclooxygenase inhibitor indomethacin and in the presence of a drug described as an in vitro and in vivo inhibitor of endothelial NO-synthase, N~-nitro-L-arginine methyl ester (L-NAME) (Moncada et al., 1991; Rees et al., 1990a, 1990b). On the other hand, the mechanism by which NO is thought to me- diate vascular relaxation is by the activation of guanylate cyclase, leading to an elevation of smooth-muscle cyclic guanosine 3':5'- monophosphate (cGMP) (Diamond and Chu, 1983; Holzmann, 1982; Kukovetz et al., 1979; Rapoport and Murad, 1983; Rapoport et al., 1983). Because the vital stain, methylene blue, blocks the guanylate cyclase enzyme (Ignarro and Kadowitz, 1985), we also studied the effect of an increase in extracellular calcium on these contractions in preparations incubated with methylene blue.

METHODS P r e p a r a t i o n

Adult New Zealand white rabbits were sacrificed with 40 mg/kg IV pentobarbital sodium injected in the ear. The thorax was opened, and the aorta from the aortic arch to the diaphragm was rapidly ex- cised and transferred to a beaker containing a low-bicarbonate (to pre- vent calcium precipitation when using salt solutions with high concentrations of this ion) physiological salt solution of the following composition (raM): 118.2 NaC1; 4.7, KC1; 2.5, CaClz; 6.25, NaHCO; and 10.0 glucose. This portion of the aorta was cleaned of surrounding connective and fat tissue and cut into rings about 3 to 5 mm in width. The denuded endothelium preparations were prepared by gently rub- bing the tissue before it was cut into rings. The aorta rings were sus- pended between two stainless steel hooks immersed in 40-ml organ baths, that also contained the same medium kept at 37°C, and con- stantly bubbled with 95% 02 and 5% CO2 (pH 7.3). The preparations were mounted with a resting tension of 5 g and allowed to equilibrate for a 90-min period. During the equilibration period, the bathing solu-

746 A. Ortega et al.

140.

120.

zlO0 0 g 80

z 60

40

20

140

120

zlO0 0 m

. 0

~ e0 o o 40

2O

0

140

120 •

~100 •

g 80.

~ 6 0

~ 4 0

20.

7 6 5 ,$ METHOXAMINE -log {M)

140

B ~ 120

zlO0 _o

80

_ _ e0

o o 4o

2o

o

METHOXAMINE -log (M]

FIGURE 1. Cumulative dose-response curve of methoxamine in intact (A), endothelium-denuded (B), 10 -s M indomethacin-treated (C), 10 -4 M L-NAME-treated (D), and 10 6 M methylene blue-treated (E) rabbit aorta ring preparations with different bathing solution Ca 2+ ( ~ ) concentrations: • 0.3; • 0.6; • 1.25; O 2.5; • 5; and • 10. The data represent the mean+SEM for 7-9 experiments,

6 5 4 taking the 80-mM KCl-induced METHOXAMINE -log IMI contraction in 2.5 mM Ca 2+ as 100.

140 -

120 -

z l O 0 -

~ 80-

~ 60- Z o o 40-

20-

METHOXAMINE -log (M) METHOXAMINE -log (M]

tion was changed every 15 min: it was sucked out with a water pump and then rapidly replaced in the organ bath. The resting tension was frequently adjusted to 5 g during this period.

The efficacy of the procedure used to remove the endothelial cells was judged by the loss of acetylcholine-induced relaxation in different rabbit isolated aorta preparations precontracted by 30 mM KCI or by 10 -5 M methoxamine, as these cells, according to Furch- gott and Zawadzki (1980), are necessary for this response.

Pharmacological studies

EXPERIMENTAL PROTOCOLS. Experiments were carried out with selective oq-adrenoceptor agonists (methoxamine and phenylephrine) and KCl. The designs for these experiments were distinct and are described in what follows. This was necessary because of the different tissue responses to repeated administrations of the oq-adrenoceptor agonists on the one hand, and KC1 on the other. Identical

successive doses of Cxl-adrenoceptor agonists to a single aorta prepa- ration do not produce the same response; a clear potentiation of the effect, particularly with the first administrations, is evident. Re- peated doses of KC1, however, produced similar responses.

~x~-ADRENOCEPTOR AGONIST EXPERIMENTS. As mentioned, the aorta rings were mounted and kept for the equilibration period in a 2.5-mM Ca 2+ bath solution. At the end of this period, they were first contracted by 80 mM KC1. When the contraction had reached the steady state (about 10 minutes after the administration), the preparations were washed with the same 2.5-mM Ca 2+ solution un- til the basal tension was reestablished. Then the preparations were incubated for 15 min in the solution in which the c~l-adrenoceptor agonist responses were to be evaluated. Saline solutions with Ca 2+ (mM) concentrations of 0.3, 0.6, 1.25, 2.5, 5, and 10 were used for these evaluations. After the 15-min incubation period the c~vadre- noceptor agonist was administered in increasing cumulative doses;

High Ca 2+ and Vascular Contractility 747

140 1 F I G U R E 2. Cumulative dose-re- /

curve of phenylephrine i n 1 2 0 ] " sponse intact (A), endothelium-denuded ~100q / / (B), 10 -5 M indomethacin-treated _ (C), 10 4 M L-NAME-treated (D), ~ 8 0 and 10 -6 M methylene blue-treated (E) rabbit aorta ring preparations ~z 6 0 with different bathing solution o

o 40- Ca z+ (mM) concentrations: • 0.3; • 0.6; • 1.25; O 2.5; • 5; and • 2 0 10. The data represent the mean-+SEM for 7 -9 experiments, taking the 80-raM KCl-induced contraction in 2.5 mM Ca 2+ as 100.

140 -

120 •

z l O 0 - _o ~ 80.

~ 60" z 0 o 40-

2 0

140

120

zlO0 0 ~ 80

~ so z o o 4o

20

A A A A

t PHENYLEPHRINE - log [M}

140

S 12o

z l O O _o ~ 80

6o i,o 2O

PHENYLEPHRINE - log (M]

140

D 120

z lOO - 0

80 -

• ~ 6 0

0 0 4 0

20-

0

PHENYLEPHRINE - log (M)

C

PHENYLEPHRINE - log (M}

K

PHENYLEPHRINE - log (M}

we obtained only one methoxamine or phenylephrine dose-response curve for each aorta ring. The contraction produced by 80 mM KC1 in 2.5-mM Ca 2+ (the standard calcium concentration for all our in

vitro rabbit aorta studies) was taken as the 100% response and served to evaluate subsequent cq-adrenoceptor agonist responses.

The previously described procedure was applied to intact and endothelium-disrupted tissue, but in the latter case only saline solutions with Ca 2+ (mM) concentrations between 1.25 and 10 were used. It also was applied to another 3 groups of intact preparations; one with the addition of indomethacin (10 -5 M), one with the addition of L-NAME (10 -4 M), and the last with the addition of methylene blue (10 6 M) to the incubation salt solutions in which the effect of the oq-adrenoceptor agonist was to be measured. To evaluate the treated intact preparations we also used only saline solutions with Ca 2+ (mM) concentrations between 1.25 and 10.

KCl EXPERIMENTS. For these experiments the aorta rings also were mounted and kept for the equilibration period in a 2.5-mM Ca 2+ bath solution. At the end of this period, successive contractions were induced by KC1 (at cumulative doses of 30 and 80 raM) in solutions with increasing C a 2+ concentrations of 0.3, 0.6, 1.25, 2.5, 5, and 10 mM. The tissue was always relaxed by washing with salt solution of the same Ca 2+ concentration used to generate its earlier contraction. Thus, the first 30- and 80-mM KCI contractions occurred after a 15-min incubation period in a 0.3-mM Ca 2+ solution following the equilibration period. The preparations were simi- larly incubated for 15 min in the salt solution in which the effect of KCl was to be evaluated before each of the remaining cumulative administrations. The response to 80 mM KCI in the 2.5-mM Ca > solution was always taken as the 100% response, serving to quantify the remaining responses.


160 A

120

~ * * * * *

80 * *

0.3 0.6 1.25 2.5 5 10 Ca 2+ [mM]

160 * 160

- 1 i lCt i 12o p120

l i i -- : o 40 o 4oJ

0 0 J 1.25 2.5 5 10 1.25 2.5 5 10

Ca 2+ [mM] Ca 2+ [raM]

160

i 120

80

4 0

0

* 1601 D E

~ 120

, - - , * * =<80 t

"11 °t " . ° 4O

o j 1.25 2.5 5 10 1.25 2.5 5 10

Ca 2+ [mM] Ca 2+ [raM]

F I G U R E 3. Histograms of the contractions produced by 30 mM KCI (D) and 80 m M K C I (11) in intact (A), endothelium- denuded (B), 10 -5 M indomethacin-treated (C), 10 -4 M L-NAME-treated (D), 10 -6 M methylene blue-treated (E) rabbit aorta ring preparations with different bathing solution C a 2+

concentrations. The data represent the mean+SEM for 7-9 experiments, taking the 80-mM KC1- induced contraction in 2.5 mM Ca 2+ as 100. The asterisks indicate significant differences from the contractions obtained in the 2.5- mM Ca 2+ solution: * P~< 0.05; ** P~< 0.01; *** P~< 0.001.

The previously described procedure was applied to intact and endothelium-disrupted tissue, but in the latter case only saline solutions with Ca 2+ concentrations between 1.25 and 10 mM were used. It also was applied to another 3 groups of intact preparations; one with the addition of indomethacin (10 -5 M), one with the addition of L-NAME (10 -4 M), and the last with the addition of methylene blue (10 6 M) to the incubation salt solutions in which the effect of KCl was to be measured. To evaluate the treated intact preparations, we also used only saline solutions with Ca 2+ concentrations between 1.25 and 10 mM.

Statistical procedure

The results are always expressed as mean values _+ SEM for 7-9 experiments. The Student's t-test was used to compare mean values. P~<0.05 indicated statistical significance.

Drugs

The following drugs were used in this study: methoxamine HCI, L-phenylephrine HC1, indomethacin, N~-nitro-L-arginine methyl ester, and methylene blue (Sigma, St. Louis, MO, USA). They were dissolved and diluted daily in distilled water, except indomethacin, which was dissolved in distilled water containing 50% ethanol. The concentrations of drugs are expressed as final molar concentrations in the bath solution.

R E S U L T S

Methoxamine and phenylephrine provoked maximal contractions in the intact rabbit aorta rings when bathing solutions with 0.3 to 0.6 mM Ca 2+ concentrations were used (Figs. 1 and 2). In the intact preparations the responses of KCl were maximal with the 2.5-mM C a 2+ concentration (Fig. 3). In any case, the increase in Ca 2+ con-

High Ca z+ and Vascular Contractility 749

TABLE 1. Area under the dose-response curve (AUC) , ~ maximal effect (ME), ~ and PD2 values of methoxamine in different rabbit aorta ring preparations and different Ca z+ conditions

Endothelium Indomethacin L -NAME Methylene Ca 2+ (mM) Intact denuded treated treated blue treated

10 A U C 51.6 -+ 3.9*** ME 44.1 -+ 3.3*** PD2 6.1 -+ 0.1

5 A U C 78.8 +- 2.8*** ME 72.6 -+ 5.3* PD2 6.0 -+ 0.1

2.5 A U C 100.0 -+ 3.3 ME 86.8 -+ 4.0 PD2 6.1 -+ 0.1

1.25 A U C 135.1 -+ 10.2" ME 118.8 -+ 11.0" PD2 6.0 -+ 0.1

0.6 A U C 153.7 -+ 5.1"** ME 129.2 -+ 4.2*** PD2 6.1 -+ 0.1

0.3 A U C 156.8 -+ 8.3*** ME 128.1 -+ 3.7*** PD2 6.1 -+ 0.1

58.2 -+ 3.2*** 66.1 -+ 4.1"** 53.3 +_ 5.5*** 71.9 -+ 8.7* 85.7 -+ 9.9 79.5 + 7.6 65.9 _+ 6.0** 96.3 -+ 10.6"

5.6 _+ 0.1 5.9 +_ 0.1 5.5 -+ 0.1 5.5 -+ 0.1

87.4 -+ 7.1 73.7 -+ 5.3** 84.9 -+ 7.1 80.1 -+ 9.5 91.5 _ 7.5 92.9 -+ 6.8 83.0 -+ 6.9* 104.6 -+ 10.6

5.8 -+ 0.1 5.6 + 0.1 5.9 _+ 0.1 5.6 -+ 0.9

100.0 -+ 4.6 100.0 -+ 5.8 100.0 -+ 5.4 100.0 -+ 6.2 104.1 -+ 7.9 109.1 + 6.0 103.8 _+ 3.8 126.2 _ 5.8

5.9 -+ 0.1 6.0 -+ 0.2 5.8 -+ 0.1 5.7 -+ 0.1

127.7 -+ 4.2*** 118.5 -+ 5.6* 121.9 -+ 5.6* 117.9 -+ 5.9 125.0 _+ 2.8* 140.7 -+ 9.8* 123.3 + 2.8** 125.6 -+ 2.1

5.9 -+ 0.1 6.2 -+ 0.2 5.9 _+ 0.1 6.0 _+ 0.1

m

m

m

m

m

m

m

m

m

m

m

m

m

m

Taking the area under the curve for the mean values in 2.5 mM Ca 2+ as 100. b Taking the 80 mM KCl-induced contraction in 2.5 mM Ca 2+ as 100. The data represent the mean +- SEM for 7-9 experiments, and the asterisks indicate significant differences from the AUC, the ME, or the PD2 value

in the 2.5 mM Ca 2+ solution: * P ~< 0.05; ** P ~< 0.01; *** P ~< 0.001.

centration in the bathing solution above 2.5 mM caused a diminu- tion in the methoxamine, phenylephrine, and KCI contractile responses.

With a 2.5-mM bathing solution Ca 2+ concentration, the contractions caused by 80 mM KC1 (100% in all the experiments) were similar in both intact (4.87-+0.30 g, n=25) and endothelium-disrupted preparations (5.24-+0.23 g, n=25). These measurements of the 80-mM KCI contraction with 2.5-mM Ca 2+ (expressed in grams) were evaluated in the tests with the c~l-adrenoceptor agonists in which KCI was administered in a single dose before the dose-response curves were obtained. Neither the presence of indomethacin (10 -s M) nor that of L-NAME (10 -4 M) or of methylene blue (10 -6 M) caused any alteration in the basal contractile tone of the preparations. Moreover, the 80-mM KC1 contractions with 2.5 mM Ca 2+ in preparations incubated in L-NAME (4.30-+0.46 g, n=8), in methylene blue (4.53-+0.39 g, n=7), or in indomethacin (6.55+0.59 g, n=6), also were similar to those produced in intact untreated preparations (5.73-+ 0.47 g, n= 7). These measurements of the 80-mM KCI contractions with 2.5 mM Ca 2+ (expressed in grams) were evaluated in the tests with cumulative KC1 administrations. No statistically significant differences were noted between these measurements using the Stu- dent's t-test for comparison of mean values (P<0.05 indicating statistical significance).

On the other hand, the contractions of (xl-adrenoceptor agonists were decreased by increasing the Ca 2+ concentration above 1.25 mM in the disrupted endothelium preparations and in those incubated in indomethacin, L-NAME, or methylene blue. This also happened in intact untreated preparations (Figs. 1 and 2, Tables 1 and 2). High organ bath Ca 2+ concentrations also caused a decrease in KC1 contractions

using endothelium-denuded and L-NAME-, methylene blue-, or indomethacin-treated preparations. In the preparations treated with methylene blue, the KC1 responses were similar with 1.25 and 2.5 mM Ca 2+, whereas in all other preparations, they were greater with 1.25 mM Ca 2+ (Fig. 3).

DISCUSSION

Our results show that rabbit aorta contraction in response to cq-adrenoceptor agonists, methoxamine, and phenylephrine, are inhibited when there is an increased extraceUular calcium concentration. Bohr (1963) described the decrease in noradrenaline contractions in the rabbit aorta at high extracellular calcium concentrations. The postsynaptic c~-adrenoceptor in the rabbit aorta has been identified as belonging to the ~l-adrenoceptor subtype (Docherty e t a l . , 1981; Docherty and Starke, 1982; Oriowo et a l . , 1987; Van Breemen et

a l . , 1982). The contractile responses elicited in this tissue by nonse- lective agonists such as noradrenaline are consequently a result of the stimulation of the a~-adrenoceptors situated there. It is therefore not surprising that the responses to the selective cq-adrenoceptor agonists methoxamine and phenylephrine also are reduced at high extracellular calcium concentrations.

It also should be borne in mind that in our experiments KC1- induced contractions also were decreased by increasing the calcium concentration in the bathing solution. It is now known that the rabbit aorta contractile responses to noradrenaline, previously referred to as a consequence of cq-adrenoceptor stimulation, are primarily due to calcium release from the sarcoplasmic reticulum (Van Bree-


T A B L E 2. Area under the dose-response curve (AUC)," maximal effect (ME), b and PD2 values of phenylephrine in different rabbit aorta ring preparations and different Ca 2+ conditions

Endothelium Indomethacin L.NAME Methylene Ca 2+ (mM) Intact denuded treated treated blue treated

I0 A U C 64.2 ± 3.1"** ME 70.6 ± 3.5*** PDz 6.3 ± 0.1

5 A U C 79.5 ± 5.3 ME 84.1 ± 7.1" PD2 6.7 -+ 0.2

2.5 A U C 100.0 ± 5.0 ME 107.7 ± 6.4 PD2 6.6 ± 0.1

1.25 A U C 104.1 ± 5.1 ME 106.8 +_ 5.4 PD2 6.5 ± 0.1

0.6 A U C 136.7 ± 4.6** ME 130.0 _+ 7.3* PDz 7.7 ± 0.5

0.3 A U C 132.5 ± 4.6 ME 118.8 _+ 2.0 PD2 7.1 _+ 0.2

64.1 ± 4.8*** 60.7 ± 5.8*** 55.1 ± 4.5*** 60.0 ± 4.9*** 76.3 _+ 6.4*** 71.1 ± 5.0*** 58.7 ± 2.9*** 82.0 ± 6.7*

6.2 ± 0.1 6.3 ± 0.1 6.3 _+ 0.1 6.4 ± 0.1

70.8 ± 2.4** 74.9 ± 7.2* 73.4 ± 3.2*** 80.1 ± 3.5** 88.4 _+_ 3.7*** 87.2 ± 7.5** 78.0 _+ 1.3"* 96.5 ± 5.0

6.6 ± 0.1 6.3 _+ 0.1 6.2 ± 0.1 6.4 ± 0.2

100.0 + 5.7 100.0 ± 4.1 100.0 + 4.6 100.0 _+ 3.0 118.8 ± 5.9 111.2 ± 6.5 103.0 ± 5.7 115.7 ± 10.3

6.3 ± 0.1 6.3 ± 0.1 6.4 ± 0.1 6.8 _+ 0.1

106.5 ± 6.7 109.5 -- 4.1 120.5 ± 8.1 123.3 _+ 9.4 116.5 ± 5.8 119.8 ± 5.5 112.6 ± 4.6 134.9 ± 12.9

6.5 _+ 0.1 6.5 ± 0.2 6.5 _+ 0.1 6.6 ± 0.1

m

m

m

m

aTaking the area under the curve for the mean values in 2.5 mM Ca z+ as 100. b Taking the 80 mM KCl-induced contraction in 2.5 mM Ca > as 100. The data represent the mean -+ SEM for 7-9 experiments, and the asterisks indicate significant differences from the ME or the PD2 value in the 2.5

mM Ca 2+ solution: * P ~< 0.05; ** P ~< 0.01; *** P ~< 0.001.

men et al. , 1986). The KCl-evoked contractions are, however, mainly the consequence of the influx of external calcium (Hudgins and Weiss, 1968). Therefore, high external calcium concentrat ions cause a reduction in the contract ion provoked both by agonists that mainly use the calcium from internal pools (methoxamine and phenylephrine) and those that mainly use the extracellular calcium (KCl) to increase the intracellular concentrat ion of this ion in the vascular smooth-muscle cells. We have nevertheless observed that the optimum extracellular calcium levels to obtain contractile responses caused by oq-adrenoceptor agonists in the vascular smooth muscle is less than those needed by depolarizing agents such as KC1 that fundamentally use extracellular calcium to contract the vascular smooth muscle. In the case of phenylephrine, when the extracellular calcium level was very low (0.3 mM), the area under the 0q-adrenoceptor agonist dose-response curve decreased also as a consequence of the lessening of the contract ion caused by the higher doses. Given that the 0q-adrenoceptor agonists also use extracellular calcium (although less than KC1) to contract the smooth-muscle cells, this is not surprising. Therefore, there always exist optimum extracellular calcium levels for vascular smooth-muscle contraction, and when these levels are exceeded, intracellular free calcium paradoxically diminishes, or else some basic mechanism always essential in activating the vascular smooth-muscle contractile process could be affected.

As we have proposed, maybe an increase in extracellular calcium causes a greater release of endothelial-derived relaxing factors that counteract, in part, the increase in arterial tone when an agonist ca- pable of contracting this tissue is administered. These factors also are released in endothelial cells in response to an increase in intra-

cellular calcium (Ltickhoff et al., 1988; Whi te and Martin, 1989; Williams et al., 1987). It has been suggested that there is an important difference in the membrane properties of endothelial and of smooth-muscle cells (Wu and Bohr, 1991). According to Wu and Bohr (1991), an increment in extracellular calcium concentrat ion apparently increases calcium influx in endothelial cells, whereas it does not increase calcium influx in other tissues, such as vascular smooth muscle. These authors examined the mechanisms of relaxation in intact and endothelium-denuded rat thoracic aorta rings in response to various concentrations of calcium (from 1.6 to 10.1 mM) after 30-mM KCl-induced contraction. The relaxation with calcium was concentrat ion-dependent in intact and denuded rings, but this effect was greater in the former. It also was greater in intact untreated preparations than in intact preparations treated with methylene blue (10 -5 M). This suggested that the relaxation with calcium could be partially mediated by the release of endothelium- derived relaxing factors.

Our results with rabbit aorta rings show significative inhibitions of the contractions to ~x~-adrenoceptor agonists and KCI in endothelium-denuded rings and in preparations incubated with indomethacin, L-NAME, or methylene blue when the calcium concentrat ion is increased in the physiological salt solution. It should be pointed out that the methylene blue concentrat ion used in this study (10 6 M), although somewhat lower than that used by Wu and Bohr (1991), is similar to that used by Alosachie and Godfraind (1986) (3×10 -6 M) to inhibit the production of cGMP in isolated rat aorta. As can be seen in Figs. 1 and 2, the inhibi t ion of the oq-adrenoceptor agonist responses was always pronounced, and Tables 1 and 2 show that the areas under the dose-response curves were less

High Ca 2+ and Vascular Contracti l i ty 751

when the Ca 2+ concentra t ion in the organ bath was increased. Ta- bles 1 and 2 also show similar variations in the maximal effects in all the different types of preparations used whenever the extracellular Ca 2+ concentrat ion is increased. O n the other hand, the PD2 values (-log of the dose producing 50% of the maximal effect) were always similar in these tables because a nonparallel displacement corresponding to a noncompet i t ive antagonism was always observed when the extracellular Ca 2+ concentra t ion increased.

O n the other hand, L6pez-Jaramillo et al. (1990) showed that the basal and acetylcholine-induced release of N O from the rabbit aorta was maximal when the concentra t ion of Ca 2+ perfusing the tissue was 1.25 mM. W h e n the Ca 2+ concentra t ion was further increased, a decline in the release of N O was observed. Therefore, these authors suggested that concentrat ions of Ca > above the physiological range suppress, rather than stimulate, N O production.

In conclusion, we therefore th ink that an increased release of the endothelium-derived relaxing factors in the rabbit aorta is not the main mechanism implicated in the inhibi t ion of contractile responses of this tissue in high extracellular Ca 2+ conditions.

S U M M A R Y

The ability of elevated levels of external Ca 2÷ to reduce the contractile response of vascular smooth muscle has been recognized for a long time. A role for calcium in the release of endothelium-derived relaxing factors as prostacyclin and nitric oxide (NO), has also been demonstrated.

The aim of this article was to study the decrease in the contractile activity of the rabbit aorta when the extracellular calcium concentrat ion is increased and to see whether endothel ium plays a role in the Ca>- induced reduction of the contractile response of this tissue. We investigated the depressant effects produced by the increase in the Ca 2÷ concentra t ion in the bathing solution (0.3-10 mM) on the contractile response of the rabbit aorta rings to KCI (30 and 80 raM), methoxamine (10 -7- 10 -4 M), and phenylephrine (10 7_ 10 -4 M). Trials were carried out in intact and endothelium-denuded preparations. They also were carried out in preparations incubated in the cyclooxygenase inhibitor indomethacin (10 -5 M), in the N©-synthase inhibitor N~-nitro-L-arginine methyl ester (10 4 M), or in the guanylate cyclase inhibitor methylene blue (10 6 M).

The contractile responses of all the agonists in the intact preparations were significantly and progressively reduced by increasing concentrat ions of Ca 2÷ in the bathing solution. Nevertheless, the optimum extracellular calcium level to obtain contactile responses with ~xl-adrenoceptor agonists was less (0.3-0.6 mM Ca 2+) than that needed to obtain the greatest contractions with KC1 (2.5 mM Ca2+). A Ca2+-induced reduction also was observed in endothe- l ium-denuded-, indomethacin- , L-NAME-, and methylene blue- treated preparations. Therefore, the release of endothelium-derived relaxing factors in the rabbit aorta seems not to be the main mechanism implicated in the Ca2+-induced reduction of its contractile activity.

This work was supported by the Complutense University, Madrid (PR 188/92- 4064) and by a grant (PB93-0065) from the DGICYT, Ministry of Education and Science, Spain.

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by endothelium of the adrenolytic action of prazosin in the rat isolated aorta. Br. J. Pharmac. 227, 525-532.

Bohr D. F. (1963) Vascular smooth muscle: dual effect of calcium. Sci. Wash. 39, 597-99.

Diamond J. and Chu E. B. (1983) Possible role of cyclic GMP in endothelium dependent relaxation in rabbit aorta by acetylcholine. Comparison with nitroglycerin. Res. Commun. Chem. Pathol. Pharmacol. 41, 369- 381.

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the role of endothelium in the calcium-induced reduction of the contractile response of the rabbit...

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