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Vasoconstrictor effect of the Ca2+ agonist Bay K 8644 on human cerebral arteries
Brain Research, 490 (1989) 133-140 Elsevier
133
BRE 14579
Vasoconstrictor effect of the Ca 2+ agonist Bay K 8644 on human cerebral arteries Mercedes Salaices, Maria J. Alonso, Inmaculada Rico, Maria S. Fern~indez-Alfonso and Jestis Marin Departamento de Farmacologia y Terap~utica, Facultad de Medicina, Universidad Aut6noma, Madrid (Spain) (Accepted 29 November 1988) Key words: Human cerebral artery; Bay K 8644; 45Ca2+ uptake; Nifedipine; Verapamil
The effects of Bay K 8644 on the reactivity and 45Ca2+ uptake in segments from human cerebral arteries were studied. Bay K 8644 induced concentration-dependent contractions up to 10-6 M; 10-5 M produced a reduction of the maximal response. The Ca 2÷ agonist elicited these contractions by itself, and no previous depolarization was needed. The response to Bay K 8644 was antagonized competitively by nifedipine (5 x 10-s and 10-7 M, pA 2 value of 8.17) and non-competitively by verapamil (10 -6, 5 × 10 -6 and 10-5 M). The contraction induced by 10 7 M Bay K 8644 was inhibited by a Ca2+-free medium containing 1 mM EGTA. The subsequent cumulative Ca 2÷ addition, caused concentration-dependent contractions up to 2.5 mM Ca2÷, which were reduced by nifedipine (10-s and 10-7 M) or verapamil (5 x 10-6 and 10-5 M). When the EGTA concentration in the CaZ+-free solution was reduced to 0.1 mM, contractions induced by Ca 2÷ up to 5 mM, including 0 Ca2÷, were increased with respect to those obtained in the presence of 1 mM EGTA. Basal 45Ca2+ uptake was not modified with Bay K 8644 (10-6 M) or nifedipine (10-6 M). K + (25 and 50 mM) produced an increase on 45Ca2+ uptake, which was potentiated by Bay K 8644 (10~ M) and antagonized by nifedipine (10 -6 M); this latter agent reduced the potentiation elicited by the Ca 2÷ agonist. These results suggest that contractions caused by Bay K 8644 in human cerebral arteries are produced by the opening of the voltage-dependent Ca 2÷ channels present in vascular smooth muscle cells, which appear to be preactivated in a basal situation, facilitating Ca 2÷ influx. Nevertheless, Bay K 8644 needed a previous cell depolarization to produce an increase in 45Ca2+ uptake.
INTRODUCTION Ca 2÷ entry into cells is thought to play an i m p o r t a n t role in the regulation of some processes, among them the e x c i t a t i o n - c o n t r a c t i o n coupling in vascular smooth muscle cells 7'8'25. The discovery of the existence of Ca 2÷ channels ( r e c e p t o r - o p e r a t e d ( R O C s ) , and p o t e n t i a l - d e p e n d e n t (PDCs)) has allowed analysis of the function of Ca 2+ in this coupling 7'8'25. Ca 2+ entry blockers, also t e r m e d Ca 2+ antagonists, are useful tools to investigate the role of Ca 2÷ in this coupling, because they have the ability to reduce Ca 2÷ influx into the cell7"8'25. D i h y d r o p y r i d i n e s ( D H P s ) , typified by nifedipine, r e p r e s e n t the most selective and p o t e n t group among these drugs 13. Recently, new D H P s , such as Bay K
8644 (a nifedipine analogue), t e r m e d Ca 2+ agonist have been synthesized. These drugs exhibit opposite effects to D H P s antagonists, i.e. they facilitate the Ca 2÷ entry into the cells, mainly through P D C s 24' 33,35
DHPs, included the most potent Ca 2÷ agonist Bay K 8644, appear to interact with a specific site of Ca 2÷ channels. Other Ca 2+ antagonists, for example diltiazero, interact with different sites of these channels, and they can regulate the binding of D H P s 11,16AT'z2. The interaction of D H P antagonists with their specific site of Ca 2÷ channels, specially PDCs, blocks the Ca 2÷ influx, probably by increasing their closure intervals; on the other hand, D H P agonists facilitate this influx by promoting a prolongation of mean open times of these channels 21'23.
Correspondence: J. Mar/n, Departamento de Farmacologla y Terap6utica, Facultad de Medicina, Universidad Aut6noma, C/Arzobispo Morcilio, 4, 28029 Madrid, Spain. 0006-8993/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
134 Cerebral arteries appear to be very sensitive to Ca 2+ antagonistsl'2"~7; in addition, a direct vasoconstrictor effect to Ca 2+ agonists has been observed in cat cerebral arteries a2'32. Nevertheless, in our knowledge, there is not a detailed analysis of the action of Ca 2+ agonists in human cerebral arteries. In the present study, the actions of Bay K 8644 on the vascular tone, and on basal and stimulated 45Ca2+ uptake were performed in segments of these arteries in order to further delineate its mechanism of action, as well as the properties of Ca 2+ channels present in the smooth muscle cells of human cerebral vessels. MATERIALS AND METHODS
Collection of the arteries The human cerebral vessels used in the present study were branches of the middle cerebral arteries (1-1.5 mm, o.d.) from dead individuals 6-8 h following acute myocardial infarction and small bowel infections without apparent alterations of brain vessels. Precautions were taken against possible brain viral contamination. Immediately following the autopsy in the hospital, the arteries were carefully isolated and immersed in a beaker containing Krebs-Henseleit solution (KHS) at 4 °C and transported to the laboratory. Afterwards, they were divided in cylindrical segments of 5 mm in length in a Petri dish containing this cold solution and cleaned of blood traces and adhered tissues.
Recording of changes in vascular tone For isometric tension recording, each arterial cylinder was set up in an organ bath according to the method described elsewhere 32. The organ bath contained 6 ml of KHS at 37 °C continuously bubbled with a 95% 0 2 - 5 % CO 2 mixture, which gave a pH of 7.4. Two stainless-steel pins, 150/~m diameter, were passed through the lumen of the arterial cylinder. One pin was fixed to the organ bath wall while the other one was connected to a strain gauge for isometric tension recording. The latter pin was in parallel position with the former and was movable, thus permitting the application of resting tension in a perpendicular plane to the long axis of the vascular cylinder. The isometric contraction was recorded through a force-displacement transducer (Grass FTO3C) connected to a Grass model 7D
poligraph. The segments were submitted to a tension of 1 g (optimal resting tension), which was readjusted every 15 min during a 120 min equilibration period before the administration of drugs were made. At the beginning of the experiment, the vessels were exposed to 75 mM K + to check their functional integrity. Thereafter, the bath medium was changed several times until the resting tone was recovered and then Bay K 8644 was added. When Ca 2+ antagonists, nifedipine or verapamil, were used to analyze their capacity to interfere with the contractions elicited by Bay K 8644, they were added to the bath 15 min prior to and during the administration of this Ca 2+ agonist. The influence of the extracellular Ca 2+ concentration on the contractile responses caused by Bay K 8644 was investigated. For this purpose, the arteries were exposed for 30 min to a CaZ+-free medium and then Bay K 8644 (10 -7 M) was applied; once the response developed, Ca 2+ (0.1, 0.5, 1, 2.5, 5, 8 and 11 mM) was added cumulatively. To analyze the action of Ca 2+ antagonists on the contractions elicited by Ca 2+ addition, they were added 15 min prior to and during the determination of the concentration-response curve to Ca 2+.
45Ca2+ uptake 45Ca2+ uptake in segments of 5 mm in length of cerebral arteries was basically performed according to the La 3+ method of Godfraind TM. Briefly, one end of the segments was carefully tied with a single 000 silk thread to glass rod, permitting that the lumen remained opened. Thereafter, they were immersed in 4 ml of oxygenated (95% 0 2 - 5 % CO2) KHS at 37 °C during 30 min (stabilization period) and then placed for 30 s in KHS at 37 °C containing 45Ca2+ (0.6 pCi/ml). Thereafter, the segments were washed with KHS at 4 °C for 10-15 s (to remove the 45Ca2+ slightly adhered) and then with 200 ml of a Ca2+-free solution containing La 3+ (50 mM) for 5 min. La 3÷ displaces the extracellular Ca 2÷ and blocks the Ca 2÷ fluxes 18"39, permitting measurement of the intracellular 45Ca2÷ uptake. Finally, the segments were blotted, weighed and digested in vials containing 1 ml of H20 2 (30% w/v) at 100 °C for 5 h, then, 2 ml of Ready-Solv HP (Beckman) were added, and the radioactivity present in the vials measured in a
135 scintillation counter (Beckman LS 2800). 45Ca2+ uptake was calculated from the formula 38. 45Ca2+ (mmol/kg wet wt.) =
cpm in muscle mmol Ca2÷/liter medium x wet wt. (kg) cpm/liter medium When Ca 2÷ antagonists were used to investigate their interference with the basai or K ÷- or Bay K 8644-stimulated 45Ca 2+ influx, they were administered 15 min prior to and during the incubation period. Solutions, drugs and statistical evaluation The composition of KHS was (mM): NaCI 115, KC1 4.6, CaCI 2 2.5, KHzPO4 1.2, NaHCO 3 25, MgSOa.7H20 1.2, glucose 11.1, Na2EDTA 0.03. Ca2+-free KHS was prepared by omitting CaC12 and Na2EDTA and adding 1 mM ethyleneglycol-bis (fl-aminoethyl ether) N,N'-tetraacetic acid (EGTA). La 3÷ solution composition was (mM)38: NaCI 118, KC1 5.9, MgSO4.7H20 1.2, glucose 10, Tris hydroxymethyl-aminomethane 5, LaCI3-7H20 50. The final pH was adjusted to 7, due to the acidity of LaC13. Stock solutions (10 -z M) of nifedipine, verapamil and Bay K 8644 were prepared in ethanol 99.5% and protected from the light, and they were used, at the appropriate concentrations in KHS, under sodium vapor light. The stock solutions were kept at -20 °C. The segments exposed to these drugs were used once because they did not disappear from them after several washing periods. Drugs used were: 45calcium chloride (spec. act. 20 mCi/ml, New England Nuclear), potassium chloride (Merck), lanthanum chloride (Sigma), nifedipine hydrochloride (Bayer), verapamil hydrochloride (Knoll) and Bay K 8644 (Bayer). Results are given as mean + S.E.M. Statistical significance was evaluated by the Student's t-test for paired or unpaired experiments, and P values of 0.05 or less were considered significant. Contractile responses induced by Bay K 8644 were expressed in percentage of the response induced by previous administration of 75 mM K ÷. Concentrations of Bay K 8644 (in absence or in presence of nifedipine or verapamil) producing 60% (EC6o) of the response induced by a previous tone with 75 mM K ÷ were determined. Concentrations producing 50% of max-
imum contractile responses (ECs0) were not calculated because in some cases this maximum was not reached. Log10 of molar concentrations (M) of Bay K 8644 were placed in the abscissa of the curves to this Ca 2÷ agonist. In addition, whether the presence of nifedipine or verapamil produces a parallel shift of the contraction-response curve to Bay K 8644 to the right (competitive antagonism) was tested. For this purpose, it was considered that normally the responses in the middle portion of concentrationresponse curves to different drugs (as occurs in our case with Bay K 8644), appear to be linearly related to log M, and this relationship can be calculated by a linear regression equation. Therefore, this equation was determined, as well as its slope, by means of Macintosh Plus computer with its corresponding statistical program. The apparent dissociation constant (Kd) for Ca 2÷ antagonists, assuming a competitive antagonism, can be calculated according to the equation6: Kd =
[B] (dose ratio-l)
Dose ratio is represented by A "/.4, i.e. the ratio between equieffective concentrations of agonist in the presence of antagonist [B]. p A z is defined as negative logarithm of [B] that produces 2-fold shift in the agonist concentration-response curve (A "/A = 2). RESULTS Vascular motor effects Bay K 8644 elicited concentration-dependent contractions in segments of human cerebral arteries up to 10 -6 M; administration of 10-5 M produced a reduction of the maximal contraction (Figs. 1 and 2). Preincubation of the arteries with nifedipine (5 x 10 -8 or 10 -7 M) during 15 min produced a parallel shift of the concentration-response curve to the agonist to the right (Table I). Verapamil (10 -6, 5 × 10 -6 and 10-5 M) did not cause this type of displacement (except 10 -6 M), but elicited a reduction of the contractions evoked by Bay K 8644 (Fig. 2). The actions of Ca 2÷ antagonists on the EC6o, as well as the slopes of the curves to Bay K 8644 in presence and absence of nifedipine and verapamil
136
400m15m' 0
TABLE I
Concentrations of Bay K 8644 (in absence or in presence of nifedipine. Nil., and verapamil, Ver.) producing 60% (EC~)) of the contraction induced by a previous tone with 7,5 mM K +, as well as the slopes of the regression lines corresponding to concentration-response curves to Bay K 8644 in the middle portion of them N.D., not determined, because it did not reach the 60% (see Fig. 2). n = number of segments used.
Drugs
ECho (M) (95% confidence interval)
Slope (mean -+ S.E.M. )
n
Bay K 8644 + Nif. (0.051tM) + Nif. (0.11~M) + Ver. (lllM) + Ver. (51~M) + Ver. (101~M)
3.4 (0.1-9.2) 10 '~ 1.5 (0.3-7.4) 10 x 8.9(3.7-21.4) 10 8 1.6(0.3-9.1)10 -7 9.9 (2.0-50.1) 10 8 N.D.
34.1 + 2.8 42.2+6.9 46.8+7. 6 31.7-+7.2 49.3 + 7.5* N.D.
16 7 10 6 6
U""Tt
11 10
~
BAYK~4,-log CM)
Nif. (0.11~M)
~' ~
J
Vet
~
,
,~
~
(I~M)
,...~,_..__.....~ Fig. 1. Typical records showing the effects of 15 min preincubation with nifedipine (Nif.) or verapamil (Ver.) on the concentration-response curve to Bay K 8644 performed in segments of human cerebral arteries.
*P < 0.05.
are shown in Table I. The K d value for the apparent competitive antagonism nifedipine-Bay K 8644 was of (9.96 + 4.01) x 10-9 M, and the corresponding p A 2 v a l u e was o f 8.17 + 0.17. T h e C a 2÷ o m i s s i o n f r o m a m e d i u m c o n t a i n i n g 10 -3 M E G T A p r a c t i c a l l y a b o l i s h e d t h e r e s p o n s e s e v o k e d b y B a y K 8644 (10 7 M ) , a n d t h e s u b s e q u e n t c u m u l a t i v e C a 2÷ a d d i t i o n elicited concentration-dependent
(Figs. 3 and 4). When E G T A concentration in the 0 medium was reduced from 10-3 to 10 -4 M, the responses induced by Ca 2÷ up to 5 mM, including 0 Ca 2+, were increased with respect to those obtained in the presence of 1 mM E G T A (Fig. 5). C a 2+
c o n t r a c t i o n s u p to
2,5 m M ; h i g h e r c o n c e n t r a t i o n s i n d u c e d r e l a x a t i o n .
4SCae + u p t a k e
Preincubation
To analyze the action of K + (25 and 50 mM) and Bay K 8644 (10 -6 M) o n 46Ca 2+ uptake, a short time incubation (0.5 min) was used in which Ca 2÷ influx predominates over other processes 26. In addition,
of the
segments
for
15 rain w i t h
n i f e d i p i n e (10 -8 o r 10 -7 M ) o r v e r a p a m i l (5 x 10 -6 or
10 5 M )
produced
a concentration-dependent
i n h i b i t i o n o f t h e r e s p o n s e s i n d u c e d b y C a 2+ a d d i t i o n
200-
200 4 - B A Y K 8 6 4 4 (16) "-~ " + 0.05 p,M Nil. (7) . . ~
,
.
'
,
4 - B A Y K 8 6 4 4 (16) "*- " + 1 pM Ver. (6)
fl
.
.-o- ,, + 10 p.M Ver. (8)
g 1O0
100 o
) -12
0 -11
-10
-9
-8
-7
-6
-5
-1
. -11
. . . -10 -9
. -8
. -7
. -6
-5
BAY K 8644 log (M) Fig. 2. Effects of 15 min preincubation with nifedipine (Nif.) and verapamil (Ver.) on the contractile responses elicited by Bay K 8644 in segments of human cerebral arteries. Responses were expressed as mean _+ S.E.M. and were calculated as % of the contraction elicited by a previous tone with 75 mM K +, which was of 1408 +_ 118 mg. Number of segments used are in parentheses. *P < 0.05. **P < 0.001.
137
WOCa
2+
uptake, which was further increased by the administration of Bay K 8644. Both increases were reduced by nifedipine (10 -6 M) (Fig. 6).
5 rain
DISCUSSION m.
~
~YK
The present results show that Bay K 8644 induces concentration-dependent contractions in human cerebral arteries up to 10 -6 M, whereas 10 -5 M produced a reduction of maximal contraction. This biphasic effect suggests that the drug acts not only as a Ca 2+ entry promoter, but also as a Ca 2÷ antagonist at high concentrations. Similar finding has been obtained in other tissues 15"3°'33'34. It has been reported that Bay K 8644 facilitates Ca 2÷ entry mainly through PDCs; in some tissues, a moderate depolarization with K ÷ is needed in order that this drug causes stimulatory effects, e.g., cat adrenal glands 15, rabbit aortic strips 33 and cat femoral arteries 12"32. Bay K 8644, however, produces direct contractile responses without previous depolarization with K ÷ (as it occurs in our case), in several vessels, such as cat cerebral arteries t2.32, rat thoracic aorta 27 and rabbit aorta 1°. The ability of Bay K 8644 to produce contractions by itself in human cerebral arteries suggests the PDCs are activated under basal situation or are directly so by the Ca 2+ agonists. Nifedipine, but not verapamil (except at 10-6 M), produced a parallel shift of the concentrationresponse curve to Bay K 8644 to the right, suggestive of a competitive antagonism. Other authors have also obtained similar results when they used D H P s
~
j
'/C,J
'
' Ver.
J
BAYK
I ( l o ~ ) (0.1~)
t'
~
BAYK (0.11dVl)
t
~Ts
0.1 0.5 1
5 Ca 2+(mM)
Fig. 3. Typical records showing the effects o f Ca 2+ addition
after the exposure of segments of human cerebral arteries for 30 min to a Ca2÷-free medium containing Bay K 8644 and EGTA (10-3 M), as well as the action of 15 min preincubation of the segments with nifedipine (Nil.) or verapamil (Ver.) on the response induced by the Ca2÷ addition. W, washed.
this short interval was chosen because during this period K ÷ induced the maximal response. K ÷, which opens PDCs, was used in these experiments for checking the action of Bay K 8644 in these channels, since this Ca 2÷ agonist appears to act mainly in them 33. Bay K 8644 (10 -6 M, 665.9 + 100 pmol/kg; n = 10), and nifedipine (10 -6 M, 303.4 + 95.7 pmmol/kg; n = 5) did not modify the basal 45Ca2÷ uptake (435.7 + 50.3 pmol/kg; n = 25). K ÷ (25 and 50 raM) significantly and similarly enhanced 45Ca 2+
200"
•.~ 0.1plVl BAYK8644 (13)
200- ' ~ 0.11dVlBAYK8644 (13) •-*- " + 5 p M V e r . (10) - o . . + 10 p.M Ver. (7)
" + 0 . 0 1 laM N i l . (15) -o- ,, + 0.1 I.tM Nil • (7)
.9o 100
100
o 0
-
,
2
-
,
4
.
,
6
-
,
8
-
,
0
-
10
Ca
2+
•
.
,
2
.
,
4
.
,
6
-
,
8
.
,
•
10
(mM)
Fig. 4. Inhibitory effects of 15 min preincubations with nifedipine (Nif.) and verapamil (Ver.) on the contraction induced by Ca2÷ addition in segments of human cerebral arteries exposed for 30 min to a Ca2+-free medium containing Bay K 8644 and EGTA (10 -3 M). Responses (mean + S.E.M.) were expressed as % of the contractions elicited by a previous tone with 75 mM K÷, which was of 1610 + 228 mg. Number of segments used are in parentheses. *P < 0.05, **P < 0.001.
138
250-
0.1 gM BAY K 8644 0.1 mMEGTA (13) 1 mMEGTA (7)
250 200
200-
150
g *
*~
8
E
0 {.}
100
d2)
(d,}
100' 50"
50 0
150
T
+
2
4
6
8
350
2+ Ca
+
(raM)
Fig. 5. Effect of EGTA on the contractions elicited by Ca z~ addition in segments of human cerebral arteries exposed for 30 min to a Ca2+-free medium containing 0.1/zM Bay K 8644. Responses (mean + S.E,M.) were expressed as % of contractions induced by 75 mM K ÷, which was of 1565 + 143 mg. Number of segments used are in parentheses. *P < 0.05.
300 • e,,
250"
5_
200" 150" 100"
50and other Ca 2+ blocking agents for antagonizing the contraction caused by this agent 32"33. The p A 2 value for the competitive antagonism between Bay K 8644 and nifedipine in human cerebral arteries (8.17) was similar to those obtained in dog basilar (8.17) 5, dog mesenteric (8.39) 5, rat femoral (8.36) 3 and rat tail (8.27) 36 arteries. These findings indicate that the affinity of nifedipine for D H P binding site associated to Ca 2÷ channels is similar in these vascular preparations, as well as the resemblance among the properties of the populations of these channels. The existence of specific D H P receptors associated to Ca 2÷ channels have been described in some preparations; the agonists and antagonists of the DHP type may interact with these receptors in a competitive w a y 1°'14-16'31. The antagonists appear to increase the closure periods of Ca 2÷ channels, whereas the agonists produce an increase of their mean open times21,23. On the other hand, the antagonism of Bay K 8644 contractions by verapamil seems to be non-competitive, likely it is due to this drug acts in a site of Ca 2÷ channels distinct to the corresponding of Bay K 8644 (see Introduction). Contractions induced by Bay K 8644 in human cerebral arteries were practically abolished in a Ca2+-free medium containing 10 -3 M EGTA. Concentration-dependent responses elicited by subsequent Ca 2÷ addition were reduced in a concentration-dependent way by nifedipine and verapamil.
Fig. 6. Effect of Bay K 8644, nifedipine (Nif.) and Bay K 8644 plus nifedipine on the 45Ca2+ uptake induced by K + (25 or 50 mM) in segments of human cerebral arteries. Results (mean + S.E.M.) were expressed as % of the basal 4SCa2+ uptake, which was of 435.7 + 50.3/zmol/kg. Number of segments used are in parentheses. *P < 0.05.
These results indicate the C a 2+ extracellular dependence of the contractions elicited by this Ca 2+ agonist, and further confirms its mechanism of action, i.e., the promotion of Ca 2+ influxZ°'Z8'32'4Q The relaxation found at high Ca 2+ concentrations may be due to the suggested membrane-stabilizing effects of elevated concentrations of this ion 19"4°. The reduction of E G T A concentration in the medium to 10 -4 M increased the responses induced by Bay K 8644 at different C a 2+ concentrations, including those obtained at 0 C a 2+. It is known that this C a 2+ complexing agent removes the extracellular and superficial C a 2+ and is confined to extracellular space 9. These facts indicate that the superficial Ca 2+ is used by this Ca 2+ agonist to produce contractile responses, as it was observed for noradrenaline and other agonists 8'9. Basal 45Ca2+ uptake was not modified either by nifedipine or Bay K 8644, which coincides with the results obtained in other vessels a°'42. However, 45Ca2+ accumulation induced by K + (25 and 50 mM) was increased by Bay K 8644, mainly that induced by
139
25 mM. This effect was r e d u c e d by nifedipine. These e x p e r i m e n t s agree w.ith those o b t a i n e d in the reactivity e x p e r i m e n t s and confirm that this agent facilitates Ca 2÷ influx into the cell. The ability of Bay K 8644 to p r o d u c e 45Ca2+ influx in different tissues, mainly in those partially d e p o l a r i z e d with K ÷, has b e e n o b s e r v e d 1°'15'34"35, as well as the capacity of nifedipine to inhibit this effect 1°'34. The fact that the greater increase on 45Ca 2÷ u p t a k e was p r o d u c e d at lower K ÷ concentrations, indicates that Bay K 8644 is able to increase the level of activation of the PDCs when it is m o d e r a t e , but not when it is maximal, as occurs at very high K ÷ concentrations. In addition, 45Ca2÷ u p t a k e induced by K ÷ (25 and 50 m M ) , but
to modify the basal 45Ca2+ accumulation has been observed in o t h e r vessels 29m. In conclusion, B a y K 8644 p r o d u c e s direct conc e n t r a t i o n - d e p e n d e n t contractions in segments of branches of h u m a n middle cerebral artery, which are r e d u c e d by the Ca 2÷ antagonists, nifedipine and verapamil, and abolished by Ca 2÷ omission from the medium. The Ca 2+ agonist increases 45Ca 2+ accumulation elicited by K ÷. These facts suggest that Bay K 8644 produces its stimulatory effects p r o b a b l y by acting on P D C s of vascular smooth muscle cells of these arteries, facilitating Ca 2÷ influx; these channels a p p e a r to be preactivated.
not basal one, was reduced by nifedipine. The ability of K ÷ to p r o d u c e direct depolarization of smooth muscle cells, opening P D C s and enhancing the intracellular free Ca 2÷ level, as well as the capacity of Ca 2÷ antagonists to block this Ca 2+ influx has been r e p o r t e d 7"8'25. The inability of Ca 2+ antagonists
ACKNOWLEDGEMENT
REFERENCES
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1 Allen, G.S, and Bahr, A.L., Cerebral arterial spasm. Part 10. Reversal of acute and chronic spasm in dogs with orally administered nifedipine, Neurosurgery, 4 (1979) 43-49. 2 Allen, G.S. and Banghart, S.B., Cerebral arterial spasm. Part 9. In vitro effects of nifedipine on serotonin-, phenylephrine-, and potassium-induced contractions of canine basilar and femoral arteries, Neurosurgery, 4 (1979) 37-42. 3 Aoki, K. and Asano, M., Effects of Bay K 8644 and nifedipine on femoral arteries of spontaneously hypertensive rats, Br. J. Pharmacol., 88 (1986) 221-230. 4 Arunlakshana, O. and Schild, H.O., Some quantitative uses of drug antagonists, Br. J. Pharmacol., 14 (1959) 48-58. 5 Asano, M., Aoki, K., Suzuki, Y. and Matsuda, T., Effects of Bay K 8644 and nifedipine on isolated dog cerebral, coronary and mesenteric arteries, J. Pharmacol. Exp. Ther., 243 (1987) 646-656. 6 Besse, J.C. and Furchgott, R.E, Dissociation constants and relative efficacies of agonists acting on alpha adrenergic receptors in rabbit aorta, J. Pharmacol. Exp. Ther., 197 (1976) 66-78. 7 Bolton, T.B., Mechanism of action of transmitters and other substances on smooth muscle, Physiol. Rev., 59 (1979) 606-718. 8 Cauvin, C., Loutzenhiser, R. and Van Breemen, C., Mechanisms of calcium antagonist-induced vasodilation, Annu. Rev. Pharmacol. Toxicol., 23 (1983) 373-396. 9 Daniel, E.D., Cranksham, D.J. and Kwan, C.Y., Intracellular sources of calcium for activation of smooth muscle. In S. Kalsner (Ed.), Trends in Autonomic Pharmacology, Vol. 1, Urban and Schwarzenberg, Baltimore, 1979, pp. 195-249.
We thank Ms. Natividad Tera for typing the manuscript. This work was s u p p o r t e d by grants from C . A . I . C . Y . T , (327/24) and F.I.S.S. (87/1666 and 87/1528).
140 19 Godfraind, T., Actions of nifedipine on calcium fluxes and contraction in isolated rat arteries, J. Pharmacol. Exp. Ther., 224 (1983) 443-450. 20 Gopalakrishnan, V., Park, L.E. and Triggle, C.R., The effect of the calcium channels agonists, Bay K-8644 on human vascular smooth muscle, Eur. J. Pharmacol., 113 (1985) 447-451. 21 Hess, P., Lansmann, J.B. and Tsien, R.W., Different modes of calcium channel gating behaviour favoured by dihydropyridine Ca agonists and antagonist, Nature (Lond.), 311 (1984) 538-544. 22 Janis, R.A. and Triggle, D.J., 1,4-Dihydropyridine Ca 2~ channels antagonists and activators: a comparison of binding characteristics with pharmacology, Drug Dev. Res., 4 (1984) 257-274. 23 Kokubun, S. and Reuter, H., Dihydropyridine derivatives prolong the open state of Ca channels in cultured cardiac cells, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 482-527. 24 Loutzenhiser, R.D., Rfiegg, U.T., Hof, A. and Hof, R.P., Studies on the mechanism of action of the vasoconstrictive dihydropyridine, CGP 28392, Eur. J. Pharmacol., 105 (1984) 229-237. 25 Matin, J., Vascular effects of calcium antagonists. Uses in some cerebrovascular disorders, Gen. Pharmacol., 19 (19881 295-306. 26 Meisheri, K.D., Hwang, O. and Van Breemen, C., Evidence for two separate Ca 2+ pathway in smooth muscle plasmalemma, J. Membr. Biol., 59 (1981) 19-25. 27 Mikkelsen, E.O. and Nyborg, N.C.B., Comparison of the effects of the vasoconstrictive dihydropyridines Bay K 8644 and CGP 28392 on isolated rat aorta: different sensitivities to ultraviolet radiation, J. Cardiovasc. Pharmaeol., 8 (1986) 476-482. 28 Montiel, C., Artalejo, A.R. and Garcfa, A.G., Effect of the novel dihydropyridine Bay-K-8644 on adrenomedullary catecholamine release evoked by calcium reintroduction, Biochem. Biophys. Res. Commun., 120 (19841 851-857. 29 Ozaki, H., Shibata, S., Kitano, H., Matsumoto, P. and Ishida, Y., A comparative study of the relaxing effect of nitroprusside and verapamil on umbilical vessels, Blood Vessels, 18 (1981) 321-329. 30 Perlmutter, B., Kennedy, R.M., Seifen, E. and Soulby, M., Effects of norepinephrine on Bay-K-8644-induced contraction in porcine coronary artery, Arch. Int. Pharmacodvn. Ther., 285 (1987) 80-86. 31 Rogg, H., Criscione, L., Truog, A. and Meier, M., In vitro
32
33
34
35
36
37
38
39
40
41
42
comparative studies of the calcium-entry activators YC170, CGP 28392, and Bay K 8644, J. Cardiovasc. Pharmacol., 7 (Suppl. 6) (1985) 531-537. Salaices, M., Marfn, J., S~nchez-Ferrer, C.F. and Reviriego, J., The effects of Bay-K-8644 on the contraction of cat middle cerebral and femoral arteries, Biochem. Pharmacol., 34 (1985) 3131-3135. Schramm, M., Thomas, G., Towart, R. and Franckowiak, G., Novel dihydropyridines with positive inotropic action through activation of Ca 2~ channels, Nature (Lond.), 303 (1983) 535-537. Schramm, M., Towart, R., Lamp, B. and Thomas, G., Modulation of calcium ion influx by the 1,4-dihydropyridines nifedipine and Bay K 8644, J. Cardiovasc. Pharmacol., 7 (1985) 493-496. Scriabine, A., Andersson, G.L. and Janis, R.A., Bay K 8644 induced enhancement of 45Ca uptake by rabbit aortic rings, J. Cardiovasc. Pharmacol., 9 (1986) 665-669. Su, C.M., Swamy, V.C. and Triggle, D.J., Calcium channel activation in vascular smooth muscle by Bay K 8644, Can. J. Physiol. Pharmacol., 62 (1984) 1401-141(/. Towart, R., The selective inhibition of serotonin-induced contractions of rabbit cerebral vascular smooth muscle by calcium-antagonistic dihydropyridines. An investigation of the mechanism of action of nimodipine, Circ. Res., 48 (1981) 650-657. Turlapaty, P.D.M.V., Altura, B.T. and Altura, B.M., Ethanol reduces Ca 2+ concentrations in arterial and venous smooth muscle, Experientia, 35 (1979) 639-640. Van Breemen, C., Farinas, B.R., Gerba, P. and McNaughton, E.D., Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx, Circ. Res., 30 (1972) 44-54. Van Breemen, C., Hwang, O. and Meisheri, K.D., The mechanism of inhibitory action of diltiazem on vascular smooth muscle contractility, J. Pharmacol. Exp. Ther., 218 (1981) 459-463. Wendling, W.W. and Harakal, C., Effects of calcium antagonists on isolated bovine cerebral arteries: inhibition of constriction and calcium-45 uptake induced by potassium or serotonin, Stroke, 18 (1987) 591-598. Yamamoto, It., Hwang, O. and Van Breemen, C., Bay K 8644 differentiates between potential and receptor-operated Ca 2+ channels, Eur. J. Pharmacol., 102 (1984) 555-557.
133
BRE 14579
Vasoconstrictor effect of the Ca 2+ agonist Bay K 8644 on human cerebral arteries Mercedes Salaices, Maria J. Alonso, Inmaculada Rico, Maria S. Fern~indez-Alfonso and Jestis Marin Departamento de Farmacologia y Terap~utica, Facultad de Medicina, Universidad Aut6noma, Madrid (Spain) (Accepted 29 November 1988) Key words: Human cerebral artery; Bay K 8644; 45Ca2+ uptake; Nifedipine; Verapamil
The effects of Bay K 8644 on the reactivity and 45Ca2+ uptake in segments from human cerebral arteries were studied. Bay K 8644 induced concentration-dependent contractions up to 10-6 M; 10-5 M produced a reduction of the maximal response. The Ca 2÷ agonist elicited these contractions by itself, and no previous depolarization was needed. The response to Bay K 8644 was antagonized competitively by nifedipine (5 x 10-s and 10-7 M, pA 2 value of 8.17) and non-competitively by verapamil (10 -6, 5 × 10 -6 and 10-5 M). The contraction induced by 10 7 M Bay K 8644 was inhibited by a Ca2+-free medium containing 1 mM EGTA. The subsequent cumulative Ca 2÷ addition, caused concentration-dependent contractions up to 2.5 mM Ca2÷, which were reduced by nifedipine (10-s and 10-7 M) or verapamil (5 x 10-6 and 10-5 M). When the EGTA concentration in the CaZ+-free solution was reduced to 0.1 mM, contractions induced by Ca 2÷ up to 5 mM, including 0 Ca2÷, were increased with respect to those obtained in the presence of 1 mM EGTA. Basal 45Ca2+ uptake was not modified with Bay K 8644 (10-6 M) or nifedipine (10-6 M). K + (25 and 50 mM) produced an increase on 45Ca2+ uptake, which was potentiated by Bay K 8644 (10~ M) and antagonized by nifedipine (10 -6 M); this latter agent reduced the potentiation elicited by the Ca 2÷ agonist. These results suggest that contractions caused by Bay K 8644 in human cerebral arteries are produced by the opening of the voltage-dependent Ca 2÷ channels present in vascular smooth muscle cells, which appear to be preactivated in a basal situation, facilitating Ca 2÷ influx. Nevertheless, Bay K 8644 needed a previous cell depolarization to produce an increase in 45Ca2+ uptake.
INTRODUCTION Ca 2÷ entry into cells is thought to play an i m p o r t a n t role in the regulation of some processes, among them the e x c i t a t i o n - c o n t r a c t i o n coupling in vascular smooth muscle cells 7'8'25. The discovery of the existence of Ca 2÷ channels ( r e c e p t o r - o p e r a t e d ( R O C s ) , and p o t e n t i a l - d e p e n d e n t (PDCs)) has allowed analysis of the function of Ca 2+ in this coupling 7'8'25. Ca 2+ entry blockers, also t e r m e d Ca 2+ antagonists, are useful tools to investigate the role of Ca 2÷ in this coupling, because they have the ability to reduce Ca 2÷ influx into the cell7"8'25. D i h y d r o p y r i d i n e s ( D H P s ) , typified by nifedipine, r e p r e s e n t the most selective and p o t e n t group among these drugs 13. Recently, new D H P s , such as Bay K
8644 (a nifedipine analogue), t e r m e d Ca 2+ agonist have been synthesized. These drugs exhibit opposite effects to D H P s antagonists, i.e. they facilitate the Ca 2÷ entry into the cells, mainly through P D C s 24' 33,35
DHPs, included the most potent Ca 2÷ agonist Bay K 8644, appear to interact with a specific site of Ca 2÷ channels. Other Ca 2+ antagonists, for example diltiazero, interact with different sites of these channels, and they can regulate the binding of D H P s 11,16AT'z2. The interaction of D H P antagonists with their specific site of Ca 2÷ channels, specially PDCs, blocks the Ca 2÷ influx, probably by increasing their closure intervals; on the other hand, D H P agonists facilitate this influx by promoting a prolongation of mean open times of these channels 21'23.
Correspondence: J. Mar/n, Departamento de Farmacologla y Terap6utica, Facultad de Medicina, Universidad Aut6noma, C/Arzobispo Morcilio, 4, 28029 Madrid, Spain. 0006-8993/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
134 Cerebral arteries appear to be very sensitive to Ca 2+ antagonistsl'2"~7; in addition, a direct vasoconstrictor effect to Ca 2+ agonists has been observed in cat cerebral arteries a2'32. Nevertheless, in our knowledge, there is not a detailed analysis of the action of Ca 2+ agonists in human cerebral arteries. In the present study, the actions of Bay K 8644 on the vascular tone, and on basal and stimulated 45Ca2+ uptake were performed in segments of these arteries in order to further delineate its mechanism of action, as well as the properties of Ca 2+ channels present in the smooth muscle cells of human cerebral vessels. MATERIALS AND METHODS
Collection of the arteries The human cerebral vessels used in the present study were branches of the middle cerebral arteries (1-1.5 mm, o.d.) from dead individuals 6-8 h following acute myocardial infarction and small bowel infections without apparent alterations of brain vessels. Precautions were taken against possible brain viral contamination. Immediately following the autopsy in the hospital, the arteries were carefully isolated and immersed in a beaker containing Krebs-Henseleit solution (KHS) at 4 °C and transported to the laboratory. Afterwards, they were divided in cylindrical segments of 5 mm in length in a Petri dish containing this cold solution and cleaned of blood traces and adhered tissues.
Recording of changes in vascular tone For isometric tension recording, each arterial cylinder was set up in an organ bath according to the method described elsewhere 32. The organ bath contained 6 ml of KHS at 37 °C continuously bubbled with a 95% 0 2 - 5 % CO 2 mixture, which gave a pH of 7.4. Two stainless-steel pins, 150/~m diameter, were passed through the lumen of the arterial cylinder. One pin was fixed to the organ bath wall while the other one was connected to a strain gauge for isometric tension recording. The latter pin was in parallel position with the former and was movable, thus permitting the application of resting tension in a perpendicular plane to the long axis of the vascular cylinder. The isometric contraction was recorded through a force-displacement transducer (Grass FTO3C) connected to a Grass model 7D
poligraph. The segments were submitted to a tension of 1 g (optimal resting tension), which was readjusted every 15 min during a 120 min equilibration period before the administration of drugs were made. At the beginning of the experiment, the vessels were exposed to 75 mM K + to check their functional integrity. Thereafter, the bath medium was changed several times until the resting tone was recovered and then Bay K 8644 was added. When Ca 2+ antagonists, nifedipine or verapamil, were used to analyze their capacity to interfere with the contractions elicited by Bay K 8644, they were added to the bath 15 min prior to and during the administration of this Ca 2+ agonist. The influence of the extracellular Ca 2+ concentration on the contractile responses caused by Bay K 8644 was investigated. For this purpose, the arteries were exposed for 30 min to a CaZ+-free medium and then Bay K 8644 (10 -7 M) was applied; once the response developed, Ca 2+ (0.1, 0.5, 1, 2.5, 5, 8 and 11 mM) was added cumulatively. To analyze the action of Ca 2+ antagonists on the contractions elicited by Ca 2+ addition, they were added 15 min prior to and during the determination of the concentration-response curve to Ca 2+.
45Ca2+ uptake 45Ca2+ uptake in segments of 5 mm in length of cerebral arteries was basically performed according to the La 3+ method of Godfraind TM. Briefly, one end of the segments was carefully tied with a single 000 silk thread to glass rod, permitting that the lumen remained opened. Thereafter, they were immersed in 4 ml of oxygenated (95% 0 2 - 5 % CO2) KHS at 37 °C during 30 min (stabilization period) and then placed for 30 s in KHS at 37 °C containing 45Ca2+ (0.6 pCi/ml). Thereafter, the segments were washed with KHS at 4 °C for 10-15 s (to remove the 45Ca2+ slightly adhered) and then with 200 ml of a Ca2+-free solution containing La 3+ (50 mM) for 5 min. La 3÷ displaces the extracellular Ca 2÷ and blocks the Ca 2÷ fluxes 18"39, permitting measurement of the intracellular 45Ca2÷ uptake. Finally, the segments were blotted, weighed and digested in vials containing 1 ml of H20 2 (30% w/v) at 100 °C for 5 h, then, 2 ml of Ready-Solv HP (Beckman) were added, and the radioactivity present in the vials measured in a
135 scintillation counter (Beckman LS 2800). 45Ca2+ uptake was calculated from the formula 38. 45Ca2+ (mmol/kg wet wt.) =
cpm in muscle mmol Ca2÷/liter medium x wet wt. (kg) cpm/liter medium When Ca 2÷ antagonists were used to investigate their interference with the basai or K ÷- or Bay K 8644-stimulated 45Ca 2+ influx, they were administered 15 min prior to and during the incubation period. Solutions, drugs and statistical evaluation The composition of KHS was (mM): NaCI 115, KC1 4.6, CaCI 2 2.5, KHzPO4 1.2, NaHCO 3 25, MgSOa.7H20 1.2, glucose 11.1, Na2EDTA 0.03. Ca2+-free KHS was prepared by omitting CaC12 and Na2EDTA and adding 1 mM ethyleneglycol-bis (fl-aminoethyl ether) N,N'-tetraacetic acid (EGTA). La 3÷ solution composition was (mM)38: NaCI 118, KC1 5.9, MgSO4.7H20 1.2, glucose 10, Tris hydroxymethyl-aminomethane 5, LaCI3-7H20 50. The final pH was adjusted to 7, due to the acidity of LaC13. Stock solutions (10 -z M) of nifedipine, verapamil and Bay K 8644 were prepared in ethanol 99.5% and protected from the light, and they were used, at the appropriate concentrations in KHS, under sodium vapor light. The stock solutions were kept at -20 °C. The segments exposed to these drugs were used once because they did not disappear from them after several washing periods. Drugs used were: 45calcium chloride (spec. act. 20 mCi/ml, New England Nuclear), potassium chloride (Merck), lanthanum chloride (Sigma), nifedipine hydrochloride (Bayer), verapamil hydrochloride (Knoll) and Bay K 8644 (Bayer). Results are given as mean + S.E.M. Statistical significance was evaluated by the Student's t-test for paired or unpaired experiments, and P values of 0.05 or less were considered significant. Contractile responses induced by Bay K 8644 were expressed in percentage of the response induced by previous administration of 75 mM K ÷. Concentrations of Bay K 8644 (in absence or in presence of nifedipine or verapamil) producing 60% (EC6o) of the response induced by a previous tone with 75 mM K ÷ were determined. Concentrations producing 50% of max-
imum contractile responses (ECs0) were not calculated because in some cases this maximum was not reached. Log10 of molar concentrations (M) of Bay K 8644 were placed in the abscissa of the curves to this Ca 2÷ agonist. In addition, whether the presence of nifedipine or verapamil produces a parallel shift of the contraction-response curve to Bay K 8644 to the right (competitive antagonism) was tested. For this purpose, it was considered that normally the responses in the middle portion of concentrationresponse curves to different drugs (as occurs in our case with Bay K 8644), appear to be linearly related to log M, and this relationship can be calculated by a linear regression equation. Therefore, this equation was determined, as well as its slope, by means of Macintosh Plus computer with its corresponding statistical program. The apparent dissociation constant (Kd) for Ca 2÷ antagonists, assuming a competitive antagonism, can be calculated according to the equation6: Kd =
[B] (dose ratio-l)
Dose ratio is represented by A "/.4, i.e. the ratio between equieffective concentrations of agonist in the presence of antagonist [B]. p A z is defined as negative logarithm of [B] that produces 2-fold shift in the agonist concentration-response curve (A "/A = 2). RESULTS Vascular motor effects Bay K 8644 elicited concentration-dependent contractions in segments of human cerebral arteries up to 10 -6 M; administration of 10-5 M produced a reduction of the maximal contraction (Figs. 1 and 2). Preincubation of the arteries with nifedipine (5 x 10 -8 or 10 -7 M) during 15 min produced a parallel shift of the concentration-response curve to the agonist to the right (Table I). Verapamil (10 -6, 5 × 10 -6 and 10-5 M) did not cause this type of displacement (except 10 -6 M), but elicited a reduction of the contractions evoked by Bay K 8644 (Fig. 2). The actions of Ca 2÷ antagonists on the EC6o, as well as the slopes of the curves to Bay K 8644 in presence and absence of nifedipine and verapamil
136
400m15m' 0
TABLE I
Concentrations of Bay K 8644 (in absence or in presence of nifedipine. Nil., and verapamil, Ver.) producing 60% (EC~)) of the contraction induced by a previous tone with 7,5 mM K +, as well as the slopes of the regression lines corresponding to concentration-response curves to Bay K 8644 in the middle portion of them N.D., not determined, because it did not reach the 60% (see Fig. 2). n = number of segments used.
Drugs
ECho (M) (95% confidence interval)
Slope (mean -+ S.E.M. )
n
Bay K 8644 + Nif. (0.051tM) + Nif. (0.11~M) + Ver. (lllM) + Ver. (51~M) + Ver. (101~M)
3.4 (0.1-9.2) 10 '~ 1.5 (0.3-7.4) 10 x 8.9(3.7-21.4) 10 8 1.6(0.3-9.1)10 -7 9.9 (2.0-50.1) 10 8 N.D.
34.1 + 2.8 42.2+6.9 46.8+7. 6 31.7-+7.2 49.3 + 7.5* N.D.
16 7 10 6 6
U""Tt
11 10
~
BAYK~4,-log CM)
Nif. (0.11~M)
~' ~
J
Vet
~
,
,~
~
(I~M)
,...~,_..__.....~ Fig. 1. Typical records showing the effects of 15 min preincubation with nifedipine (Nif.) or verapamil (Ver.) on the concentration-response curve to Bay K 8644 performed in segments of human cerebral arteries.
*P < 0.05.
are shown in Table I. The K d value for the apparent competitive antagonism nifedipine-Bay K 8644 was of (9.96 + 4.01) x 10-9 M, and the corresponding p A 2 v a l u e was o f 8.17 + 0.17. T h e C a 2÷ o m i s s i o n f r o m a m e d i u m c o n t a i n i n g 10 -3 M E G T A p r a c t i c a l l y a b o l i s h e d t h e r e s p o n s e s e v o k e d b y B a y K 8644 (10 7 M ) , a n d t h e s u b s e q u e n t c u m u l a t i v e C a 2÷ a d d i t i o n elicited concentration-dependent
(Figs. 3 and 4). When E G T A concentration in the 0 medium was reduced from 10-3 to 10 -4 M, the responses induced by Ca 2÷ up to 5 mM, including 0 Ca 2+, were increased with respect to those obtained in the presence of 1 mM E G T A (Fig. 5). C a 2+
c o n t r a c t i o n s u p to
2,5 m M ; h i g h e r c o n c e n t r a t i o n s i n d u c e d r e l a x a t i o n .
4SCae + u p t a k e
Preincubation
To analyze the action of K + (25 and 50 mM) and Bay K 8644 (10 -6 M) o n 46Ca 2+ uptake, a short time incubation (0.5 min) was used in which Ca 2÷ influx predominates over other processes 26. In addition,
of the
segments
for
15 rain w i t h
n i f e d i p i n e (10 -8 o r 10 -7 M ) o r v e r a p a m i l (5 x 10 -6 or
10 5 M )
produced
a concentration-dependent
i n h i b i t i o n o f t h e r e s p o n s e s i n d u c e d b y C a 2+ a d d i t i o n
200-
200 4 - B A Y K 8 6 4 4 (16) "-~ " + 0.05 p,M Nil. (7) . . ~
,
.
'
,
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fl
.
.-o- ,, + 10 p.M Ver. (8)
g 1O0
100 o
) -12
0 -11
-10
-9
-8
-7
-6
-5
-1
. -11
. . . -10 -9
. -8
. -7
. -6
-5
BAY K 8644 log (M) Fig. 2. Effects of 15 min preincubation with nifedipine (Nif.) and verapamil (Ver.) on the contractile responses elicited by Bay K 8644 in segments of human cerebral arteries. Responses were expressed as mean _+ S.E.M. and were calculated as % of the contraction elicited by a previous tone with 75 mM K +, which was of 1408 +_ 118 mg. Number of segments used are in parentheses. *P < 0.05. **P < 0.001.
137
WOCa
2+
uptake, which was further increased by the administration of Bay K 8644. Both increases were reduced by nifedipine (10 -6 M) (Fig. 6).
5 rain
DISCUSSION m.
~
~YK
The present results show that Bay K 8644 induces concentration-dependent contractions in human cerebral arteries up to 10 -6 M, whereas 10 -5 M produced a reduction of maximal contraction. This biphasic effect suggests that the drug acts not only as a Ca 2+ entry promoter, but also as a Ca 2÷ antagonist at high concentrations. Similar finding has been obtained in other tissues 15"3°'33'34. It has been reported that Bay K 8644 facilitates Ca 2÷ entry mainly through PDCs; in some tissues, a moderate depolarization with K ÷ is needed in order that this drug causes stimulatory effects, e.g., cat adrenal glands 15, rabbit aortic strips 33 and cat femoral arteries 12"32. Bay K 8644, however, produces direct contractile responses without previous depolarization with K ÷ (as it occurs in our case), in several vessels, such as cat cerebral arteries t2.32, rat thoracic aorta 27 and rabbit aorta 1°. The ability of Bay K 8644 to produce contractions by itself in human cerebral arteries suggests the PDCs are activated under basal situation or are directly so by the Ca 2+ agonists. Nifedipine, but not verapamil (except at 10-6 M), produced a parallel shift of the concentrationresponse curve to Bay K 8644 to the right, suggestive of a competitive antagonism. Other authors have also obtained similar results when they used D H P s
~
j
'/C,J
'
' Ver.
J
BAYK
I ( l o ~ ) (0.1~)
t'
~
BAYK (0.11dVl)
t
~Ts
0.1 0.5 1
5 Ca 2+(mM)
Fig. 3. Typical records showing the effects o f Ca 2+ addition
after the exposure of segments of human cerebral arteries for 30 min to a Ca2÷-free medium containing Bay K 8644 and EGTA (10-3 M), as well as the action of 15 min preincubation of the segments with nifedipine (Nil.) or verapamil (Ver.) on the response induced by the Ca2÷ addition. W, washed.
this short interval was chosen because during this period K ÷ induced the maximal response. K ÷, which opens PDCs, was used in these experiments for checking the action of Bay K 8644 in these channels, since this Ca 2÷ agonist appears to act mainly in them 33. Bay K 8644 (10 -6 M, 665.9 + 100 pmol/kg; n = 10), and nifedipine (10 -6 M, 303.4 + 95.7 pmmol/kg; n = 5) did not modify the basal 45Ca2÷ uptake (435.7 + 50.3 pmol/kg; n = 25). K ÷ (25 and 50 raM) significantly and similarly enhanced 45Ca 2+
200"
•.~ 0.1plVl BAYK8644 (13)
200- ' ~ 0.11dVlBAYK8644 (13) •-*- " + 5 p M V e r . (10) - o . . + 10 p.M Ver. (7)
" + 0 . 0 1 laM N i l . (15) -o- ,, + 0.1 I.tM Nil • (7)
.9o 100
100
o 0
-
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2
-
,
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,
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,
8
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,
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Ca
2+
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.
,
2
.
,
4
.
,
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-
,
8
.
,
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10
(mM)
Fig. 4. Inhibitory effects of 15 min preincubations with nifedipine (Nif.) and verapamil (Ver.) on the contraction induced by Ca2÷ addition in segments of human cerebral arteries exposed for 30 min to a Ca2+-free medium containing Bay K 8644 and EGTA (10 -3 M). Responses (mean + S.E.M.) were expressed as % of the contractions elicited by a previous tone with 75 mM K÷, which was of 1610 + 228 mg. Number of segments used are in parentheses. *P < 0.05, **P < 0.001.
138
250-
0.1 gM BAY K 8644 0.1 mMEGTA (13) 1 mMEGTA (7)
250 200
200-
150
g *
*~
8
E
0 {.}
100
d2)
(d,}
100' 50"
50 0
150
T
+
2
4
6
8
350
2+ Ca
+
(raM)
Fig. 5. Effect of EGTA on the contractions elicited by Ca z~ addition in segments of human cerebral arteries exposed for 30 min to a Ca2+-free medium containing 0.1/zM Bay K 8644. Responses (mean + S.E,M.) were expressed as % of contractions induced by 75 mM K ÷, which was of 1565 + 143 mg. Number of segments used are in parentheses. *P < 0.05.
300 • e,,
250"
5_
200" 150" 100"
50and other Ca 2+ blocking agents for antagonizing the contraction caused by this agent 32"33. The p A 2 value for the competitive antagonism between Bay K 8644 and nifedipine in human cerebral arteries (8.17) was similar to those obtained in dog basilar (8.17) 5, dog mesenteric (8.39) 5, rat femoral (8.36) 3 and rat tail (8.27) 36 arteries. These findings indicate that the affinity of nifedipine for D H P binding site associated to Ca 2÷ channels is similar in these vascular preparations, as well as the resemblance among the properties of the populations of these channels. The existence of specific D H P receptors associated to Ca 2÷ channels have been described in some preparations; the agonists and antagonists of the DHP type may interact with these receptors in a competitive w a y 1°'14-16'31. The antagonists appear to increase the closure periods of Ca 2÷ channels, whereas the agonists produce an increase of their mean open times21,23. On the other hand, the antagonism of Bay K 8644 contractions by verapamil seems to be non-competitive, likely it is due to this drug acts in a site of Ca 2÷ channels distinct to the corresponding of Bay K 8644 (see Introduction). Contractions induced by Bay K 8644 in human cerebral arteries were practically abolished in a Ca2+-free medium containing 10 -3 M EGTA. Concentration-dependent responses elicited by subsequent Ca 2÷ addition were reduced in a concentration-dependent way by nifedipine and verapamil.
Fig. 6. Effect of Bay K 8644, nifedipine (Nif.) and Bay K 8644 plus nifedipine on the 45Ca2+ uptake induced by K + (25 or 50 mM) in segments of human cerebral arteries. Results (mean + S.E.M.) were expressed as % of the basal 4SCa2+ uptake, which was of 435.7 + 50.3/zmol/kg. Number of segments used are in parentheses. *P < 0.05.
These results indicate the C a 2+ extracellular dependence of the contractions elicited by this Ca 2+ agonist, and further confirms its mechanism of action, i.e., the promotion of Ca 2+ influxZ°'Z8'32'4Q The relaxation found at high Ca 2+ concentrations may be due to the suggested membrane-stabilizing effects of elevated concentrations of this ion 19"4°. The reduction of E G T A concentration in the medium to 10 -4 M increased the responses induced by Bay K 8644 at different C a 2+ concentrations, including those obtained at 0 C a 2+. It is known that this C a 2+ complexing agent removes the extracellular and superficial C a 2+ and is confined to extracellular space 9. These facts indicate that the superficial Ca 2+ is used by this Ca 2+ agonist to produce contractile responses, as it was observed for noradrenaline and other agonists 8'9. Basal 45Ca2+ uptake was not modified either by nifedipine or Bay K 8644, which coincides with the results obtained in other vessels a°'42. However, 45Ca2+ accumulation induced by K + (25 and 50 mM) was increased by Bay K 8644, mainly that induced by
139
25 mM. This effect was r e d u c e d by nifedipine. These e x p e r i m e n t s agree w.ith those o b t a i n e d in the reactivity e x p e r i m e n t s and confirm that this agent facilitates Ca 2÷ influx into the cell. The ability of Bay K 8644 to p r o d u c e 45Ca2+ influx in different tissues, mainly in those partially d e p o l a r i z e d with K ÷, has b e e n o b s e r v e d 1°'15'34"35, as well as the capacity of nifedipine to inhibit this effect 1°'34. The fact that the greater increase on 45Ca 2÷ u p t a k e was p r o d u c e d at lower K ÷ concentrations, indicates that Bay K 8644 is able to increase the level of activation of the PDCs when it is m o d e r a t e , but not when it is maximal, as occurs at very high K ÷ concentrations. In addition, 45Ca2÷ u p t a k e induced by K ÷ (25 and 50 m M ) , but
to modify the basal 45Ca2+ accumulation has been observed in o t h e r vessels 29m. In conclusion, B a y K 8644 p r o d u c e s direct conc e n t r a t i o n - d e p e n d e n t contractions in segments of branches of h u m a n middle cerebral artery, which are r e d u c e d by the Ca 2÷ antagonists, nifedipine and verapamil, and abolished by Ca 2÷ omission from the medium. The Ca 2+ agonist increases 45Ca 2+ accumulation elicited by K ÷. These facts suggest that Bay K 8644 produces its stimulatory effects p r o b a b l y by acting on P D C s of vascular smooth muscle cells of these arteries, facilitating Ca 2÷ influx; these channels a p p e a r to be preactivated.
not basal one, was reduced by nifedipine. The ability of K ÷ to p r o d u c e direct depolarization of smooth muscle cells, opening P D C s and enhancing the intracellular free Ca 2÷ level, as well as the capacity of Ca 2÷ antagonists to block this Ca 2+ influx has been r e p o r t e d 7"8'25. The inability of Ca 2+ antagonists
ACKNOWLEDGEMENT
REFERENCES
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