Modulation of 3H]dihydropyridine binding by activation of protein kinase C in vascular smooth muscle

European Journal of Pharmacology - Molecular Pharmacology Section, 208 (1991)223-230 © 1991 ElsevierSciencePublishersB.V. All rights reserved 0922-4106/91/$03.50

223

EJPMOL 90235

Modulation of [ SH]dihydropyridine binding by activation of protein kinase C in vascular smooth muscle C h a n t a l M i r o n n e a u , Lala R a k o t o a r i s o a , I r e n e Sayet a n d J e a n M i r o n n e a u Laboratoire de Physiologie Celhdaire et Pharmacologie Moldeulaire, Unit,ersitdde Bordeaux II, INSERM CJF 88-13, 33076 Bordeattr Cedex, France

Received 1 July 1991,accepted 30 July 1991

The influence of noradrenaline and protein kinase C modulators on ( + )-[3H]isradipine binding to voltage-dependent calcium channels has been studied in membranes of equine portal vein smooth muscle and intact strips isolated from rat portal vein. Specific (+)-[3H]isradipine binding to intact strips was increased by noradrcnaline, a combination of aluminium and fluoride, and phorbol esters. The increase in isradipine binding induced by noradrenaline was inhibited by 1 p.M prazosin while that induced by phorbol esters was inhibited by H7 (a protein kinase C inhibitor). In strips pretreated 6 h with 10 txg- ml- ~ pertussis toxin, the noradrenaline-inducedincrease in isradipine binding was unchanged. In contrast, isradipine binding to membranes was unaffected by noradrenaline or GTP-y-S. Only phorbol esters had a stimulator), effect on isradipine binding when.membranes were incubated in a medium containing 10/~M ATP and 5 mM Mg 2÷. Scatchard plot analysis reveals that the stimulation of isradipine binding by both noradrenaline and phorbol esters appears to result from a decrease in K D rather than an effect on the maximal binding capacity. Contractions evoked by noradrenaline were concentration-dependentlydepressed by isradipine. About 30% of the response was resistant to inhibition, while KCI-induced contractions were completely blocked. However, noradrenaline-induced contractions were more sensitive to isradipine inhibition than were KCl-induced contractions. These results suggest that activation of protein kinase C modulates isradipine binding to voltage-dependent Ca2+ channels indelaendently of a separate modulation by membrane depolarization. Dihydropyridine receptor;

Ca 2 4

channels; Protein kinase C; Smooth muscle (venous)

1. Introduction Dihydropyridine derivatives are potent inhibitors of voltage-dependent calcium channels in many excitable tissues including vascular smooth muscle. Radioligands of 1,4-dihydropyridines have been shown to be useful for the identification, purification and molecular characterization of L-type Ca 2÷ channels (Janis and Triggle, 1984; Catterall, 1988; Hosey and Lazdunski, 1988). Voltage-dependent Ca 2÷ channels play a key role in excitation-contraction coupling of smooth muscle (Mironneau, 1973). In vascular smooth muscle, both noradrenaline and phorbol esters increase the Ba 2+ current through L-type Ca z+ channels (Fish et al., 1988; Nelson et al., 1988; Loirand et al., 1990). Stimulation of Ca 2÷ channels by noradrenaline is mediated through activation of a pertussis toxin-insensitive G protein. It has been shown that this effect involves activation of phospholipase C enzyme and production

Correspondence to: Jean Mironneau, Laboratoire de Physiologie Cellulaireet PharmacologieMol~culaire,Universit~de Bordeaux II, INSERM CJF 88-13. 3 Place de la Victoire, 33076 Bordeaux Cedex, France.

of both D-myo-l,4,5-triphosphate which depletes Ca 2+ stores and diacylglycerol which activates protein kinase C (Loirand et al., 1990). The present study investigates the effects of noradrenaline and protein kinase C modulators on ( + ) [3H]isradipine binding to membranes and intact strips of portal vein smooth muscle. These data provide evidence that both noradrenaline and phorbol esters increase the binding affinity of (+)-[3H]isradipine to Ca 2+ channels through a mechanism that probably involves phosphorylations by protein kinase C. The higher sensitivity to ( + )-isradipine of noradrenaline-induced contractions compared to contractile responses induced by KCI depolarization may be accounted for by the sensitivity of dihydropyridine binding site to protein kinase C-induc=d phosphorylation.

2. Materials and methods 2.1. Measurement o f (+)-[SH]isradipine binding to portal t'ein smooth muscle membranes

Microsomes from fresh equine portal veins were prepared as previously described (Dacquet et al., 1988)

224 and protein concentration was determined according to Bradford (1976). Membrane protons ((l.1-0.2 rag" m l - t ) were incubated for 6(1 rain at 25 ° C with various concentrations of ( + )-[3H]isradipine in 2 ml of 20 mM HEPES buffer (pH 7.4) containing 0.19f bovine serum albumin. In some experiments, various concentrations of ATP and Mg :+ were added to thc incubation medium. Non-specific binding was defined as the amount of radioligand bound in the presence of 2/xM unlabelled nifedipine and subtracted from thc total binding.

France). Nifcdipine was from Bayer (Puteaux, France). Phorbol 12,13-dibutyrate (PDB), phorbol 12-myristate 13-acetate (PMA), adenosine 5'-triphosphate (ATP), guanosinc 5'-O-(-y-thio)triphosphate (GTP-y-S), guanosine 5'-O-(/3-thio)diphosphate (GDP-fl-S), pertussis toxin and 1-(5-isoquinolinesulfonyl)-2-methy[pipcrazinc (H7) were from Sigma (St. Louis, MO, U.S.A.). Cromakalim was a gift from Rh6ne-Poulenc (Vitry, France).

2.2. Measurement of (+)-/+tt]isrado~ine binding to intact portal t'ein smooth muscle strips

Scatchard plots for binding of (+)-[3H]isradipine wcrc obtained by a non-linear least-square ligand program (Munson and Rodbard, 1980) with a microcomputer. The experimental results were expressed as means + S.E. and significance was tested by Student's t-test; P values smaller than 0.05 were considered to be significant.

Studies of binding to intact strips of rat portal vein (0.5-1 mg wet weight) were oerformed under conditions similar to thosc described previously (Dacquet et al., 1989). Strips were incubated for 90 min at 3 7 ° C in a phy.'+iological solution (mM): NaCI 130, KCI 5.6, CaCI_, 2, MgCI 2 0.24, HEPES 8.3, glucose 11, pH 7.4, with various concentrations of the radioligand. At the end of the incubation period each strip was dried on filter paper, and then weighed. Radioactivity was measured by dissolving the vein strips in 100 p.I of a mixture of perchloric acid and H 2 0 2 (1: 1). Nonspecific binding was defined as the amount of radioligand bound in the presence of 2 p.M unlabelled nifedipine and subtracted from the total binding. All binding experiments were performed under a dim light.

2.3. Membrane potential meastlremetlts Membrane potential was measured with conventional micro-electrodes filled with 3 M KCI (resistance 40-50 MI2). Short muscle segments obtained by crushing the portal vein tmder a grid of five silver wires were used for electrical measurements (Mironueau et al., 1982).

2.4. Contraction experiments Isometric contractions of longitudinal strips from rat portal vein were recorded in an experimental chamber described previously (Mironneau et al., 1980) by means of a highly sensitive isometric force transducer (Akers 801 AME, Norten, Norway). The reference solution contained (mM): NaCI 130, KCI 5.6, CaCI 2, MgCl 2 0.24, HEPES 8.3, glucose 11, pH 7.4. In 60.6 mM K+-containing solution NaCI was substituted with KCI. The circulating solution was maintained at 36 +_ I°C.

2.5. Chemicals (+)-[3H]lsradipine (specific activity 80-85 C i . mmol -~) was obtained from Amersham (Les Ulis,

2.6. Statistical analysis

3. Results

3.1. Temperature and cation dependeno, of (+)[~H]isradipine binding in portal vein smooth muscle membranes The temperature dependence of the interaction of (+)-[3H]isradipine with the binding site on Ca+-'+ channels was determined by equilibrium binding at 0, 15, 25 and 37 ° C. Scatchard plots were constructed from saturation isotherms and parameters of (+)-[3H]isradipine binding are listed in table 1. The results showed that temperature had significant effects on K D values but very little effect on B,,,x values. The affinity of the receptor for ( + )-[3H]isradipine increased with temperature and showed a maximal value at 25°C. These data are in good agreement with those of Maan and Hosey (1987) in chick heart membranes. When binding was determined in the presence of 0.1 mM EGTA (in the absence of CaCI,) Bm~,xwas strongly reduced to 20 + 3 fmol.mg-~ protein (table 2). Spe-

TABLE 1 Parameters of (+)-[3H]isradipinebindingto portal vein microsomes as a functionof temperature. Incubation solution conlained 10 p.M CaCI2 and 10 #M MgCI2. Each value is the mean_+S.E. of a number(n) of experiments. Temperature 0°C 15°C 25°C 37°C

KD (pM) 533_+24 215+_ 8" 78_+ 6" 97+_ 5"

* Significantchange (P < 0.05).

Bma x

n

(fmnl.mg t protein) 67_+4 69_+4 66_+5 6b_+5

4 4 6 5

225 TABLE 2 Parameters of (+)-[3tt]isradipine binding to portal vein microsomes in the presence of various concentrations of Ca ~ and Mg 2..

~ ® o~

~

• PDB & PDB + H7 I NA

20001 /

Each value is the mean + S.E. of a number (n) of experiments. Ligand

Bin., (fmol.mg i protein)

n

99± 3 97± 5 86_+ 4 * 65_+10 * 73_+ 7 " 60_+ 6 *

20_+3 71 ± 5 * 122_+4" 69_+6 * 110±4" 123±3"

4 4 4 6 6 4

(P < (i.05).

cific binding of (+)-[3H]isradipine was restored by progressive addition of CaCI 2 or MgCI 2 (fig. I). Halfmaximal binding was achieved at 7 _+ 3/.tM Ca 2÷ (n = 6) and 9 0 + 15 /xM Mg 2÷ (n =4). In order to analyze more thoroughly the effects of Ca -'+ and Mg 2÷ on (+)-[3H]isr.,dipine binding, saturation isotherms were constructed in the presence of various concentrations of CaCI 2 and MgCi 2. At concentrations between 0.01 and 5 mM, both CaCI2 and MgCI2 increased significantly B,,~x and reduced KD (table 2).

3.2. Effects of protein kinase C modulators on ( + )[¢H]isradipine binding to portal t'ein smooth muscle membranes Addition of PMA or PDB to the incubation medium containing both 10/.tM ATP and 5 mM Mg 2+ shewed a concentration-effect relationship (fig. 2). The effects were similar when the incubation medium con.rained a lower concentration of Mg 2+ (10 p.M). Both PMA and PDB had no effect on non-specific binding and stimulated specific isradipine binding by 41 +_4% at 0.1 /.tM (n = 4). In contrast, H7 (an inhibitor of protein kinase C) and cromakalim (a K ÷ channe! activator) h~d no important effect on specific isradipine binding which

1

.~ [

i* + - Itl"= t -Ill-- 4'--Itl-- 4'--Ill

~-~ "°

Without CaCI 2 + E G T A ( 0 . 1 mM) CaCI2 (0.01 mM) CaCI2 (1 mM) MgCI~ (0.1 raM) MgCI 2 ( l m M ) MgCI2 (5 mM) * Significant change

Ki) (pM)

cm 1000-J.

L :6

lO

1500,]

.-

~~ x ~

~:~ +"+"~

750

/

o ~:g=+

r'

6.1i,i

6 6

.i IY 6"6, n/gr

8

7

6

-log

(¢

5

4

3

2

1

Fi~. 1. Effects of calcium and magnesium on dihydropyridine binding to equine portal vein smooth muscle membranes. Binding studies were performed with 0.1 n M ( + )-[3H]isradipine and varying concentrations of free Ca 2. (e) or Mg ~-* (o). The protein concentration was 0.1 m g . m l - t . Non-specific binding was less than 150 dpm/0.1 mg protein. Data shown are means + S.E. of 4 - 6 experiments.

a

7

[drug]
reached 96.6 _+ 2.5% of control (n = 4). When PMA or PDB was added in the presence of 10 /J.M H7, ( + ) [~H]isradipine binding was not affected ([.8_+ 1.1%, n = 4). Both GDP-~-S, GTP-7-S and noradrenaline (30 nM to 3 /.tM) had no significant effect on specific isradipine binding (2.1 _+ 1.2%, n = 4). The effects of phorbol esters on specific isradipine binding were not dependent on the cation concentrations as similar results were obtained in incubation solutions containing either 1 mM Ca 2+ and 5 mM Mg 2+ or 10 /zM Ca 2+ and 10/.tM Mg 2+ (n = 4).

3.3. Effects of proteht khlase C modulators on (i-J[¢Hiisradipine binding to intact portal vein strips Characteristics of (+)-[3H]isradipine binding to intact strips of rat portal vein have been reported previously (Dacquet et al., 1989). In physiological solution, both PMA and PDB increased the (+)-[SH]isradipine binding and the concentration-effect curves were bellu

'~

• PDB 'OA PDB+HIH7

~,.~"//"~1--1

...,1'

=._ '5°°l _g

÷' 15

[cation] (M)

.

Fig. 2. Effects of phorbol esters and noi'adrenaline on ( + ) [3H]isradipine binding to portal vein smooth muscle membranes with 5 mM Mg 2+. Binding studies were perf(irmed with 0.15 nM ( + ) [~H]isradipine specific in the presence of PDB ( • l, noradrenaline ( • ), or PDB + 10 g M 1t7 ( A ). The incubation medium contained 10 /zM ATP, The specific binding in the absence of drugs, determined for tit least four experiments, was 1250±60 dpm/0.1 mg protein. Non-specific binding was not changed in the presence of the different compounds and was les,; ti)an 150 dpm/0.1 mg protein. Data shown are means±S.E, of four experiments.

T=

"<>

0=0--~ ~ 9 9 9 9

9

.E E

Ca''+

t

I

.

-log

~ "~m 2 0 0 0 ] ¢o~3 -•~, "5 ¢~

.

~,

,ooo, ~t. , 10

t

9

. . 8

, 7

6

-log [drug] (M)

Fig. 3. Effects of phorbol esters on ( + )-[3H]isradipine-specific binding to intact strips of rat portal vein. Binding studies were performed with 0.15 nM (+)-13H]isradipine in the presence of PDB ( i ) , H7 ( * ) and PDB + 10 ,aM H7 ( A ). The specific binding in the absence of drugs, determined for at least four experiments, was 1150+_50 d p m / m g wet weight. Non-specific binding w;',s not changed in the presence of the different compounds and was less than 500 d p m / m g wet weight. Data shown are means + S.E. of 3 - 6 experiments.

226 TABLE 3

A

Parameters of ( + )-[3tl]isradipinc binding to intact portal vein strips in the presence of phorbol esters and phorh,q ester inhibitor.

~

"~ ~: 2000"1 • I

II

NA

• 0

NA ÷ PRAZOSIN

NA+PTX ~111-i

Each value is the mean + S.E. of a number (n) of experiments. Ligand

Control PDB(0.I`aM)

PMA (0.1`aM) PMA 111.1 ,aM) +H7(10`aM)

KI):mp (pM)

(fmol'mg I wclwcight)

140± 9 87± g * 75_+. 5 "

14.4+ 1.2 16.5+ 1.2 16.1+0,8

~ 6 6

147+14

14.1~ 1.2

4

[1 m,~

o_ -~. ~, E o~

n

m

E

~

"~ c

"~5

, 8

,

9

B

o

3.4. Effects of" noradrenaline and fluoroahmthtates on (+)-[~H]isradipine binding to intact portal cein strips

1000

a'/ii/

,

,

7

6

, 5

+

-log

* Significant change (P < (U)5).

shaped (fig. 31. Fur e x a m p l e , t h e m a f i m a t s t i m u l a t o r y effect of PDB was obtained at 1 # M and rcpresentcd an increase in isradipine binding of 75 _+ 3% (n = 6). In the presence of 1-10 ~M H7 applicatiens of PMA or PDB were without effect on specific isradipinc binding (3.1 + 2.1%, n = 4). To elucidate the mode of binding action of phorbol esters on specific isradipinc binding in intact portal vein strips, Scatchard analysis of the ( + )-[3H]isradipine binding was carried out in the presence or absence of phorbol esters and H7 (table 3). Both PDB and PMA (0.1 /.tM) significantly increased the affinity of isradipine to its binding receptors without affecting the binding capacity. Characteristics of isradipine binding were restored to control when phorbol esters were added in the presence of 10 /.tM H7. These findings support the hypothesis that increase in isradipine binding is mediated through activation of protein kinase C.

150o l

-~

[d,u0]

(M)

2°°°l

C E

/

1500

e~

~

c.

~c

1000

, 5

,

4 -1o 0

, 3 [drug]

2

"~

(M)

Fig. 4. Efft:cts of noradrenaline and sodium fluoride on ( + ) [~lf]isradipine-specific binding to intact strips of rat portal vein. Binding studies were performed with 0.15 nM (+)-[3H]isradipine, (A) in the presence of increasing concentrations of noradrena[ine (NA. II), noradrenaline+10 # M prazosin (O) and in strips pretreated with pertussis toxin (I /.tg.ml - l ) for 6 h ([3); (B) in the presence of increasing concentrations of NaF with 10 # M AICI 3 (e). The specific binding in the absence of drugs, determined for at least four experiments, was 11511_+50 d p m / m g wet weight. Non-specific binding was not changed with the different compounds and was less than 5110 d p m / m g wet weight. Data shown are means_+S.E, of 3 - 6 experiments.

its binding sites without affecting the Bmax value. The noradrenali,e increase in isradipine affinity was similar to that obtained in the presence of phorbol ester (table 3).

Addition of increasing concentration~ of noradrenaline (fig. 4A) produced an increase of ( + ) [3H]isradipine binding. At 3 p.M noradrenaline caused an increase in isradipine binding of 63 + 7% (n = 6). At higher concentrations of noradrenaline (10/.t M) the stimulation of isradipine binding began to decrease. Non-specific binding of (+)-[3H]isradipine was unaffected by noradrenaline. In strips pretreated with 1 g M prazosin, noradrena!ine had no effect on specific isradipine binding 11.7 + 0.9%, n = 4). A similar absence of effect of noradrenaline was observed when the strips were pretreated with 10 g M H7 (2.4 + 0.9%, n = 4). In contrast, incubation with 1 /.tg. m l - t pertussis toxin in the presence of 10 mM dithiothreitol for 6 h did not significantly modify the increase in isradipine binding induced by noradrenaline (62_+ 5%, n = 3). Scatchard analysis of (+)-[3H]isradipine saturation binding was carried out in the presence of noradrenaline and noradrenaline plus H7 (table 4). Noradrenaline significantly increased the affinity of isradipine to

Combination of aluminium and fluoride is believed to stimulate G proteins by mimicking the effect of GTP-y-S on the a-subunit (Cockroft, 1987). The effects of increasing concentrations of NaF (in the presence of 10 /xM AICI 3) resulted in an increase in specific isradipine binding of 52 _+ 7% (n = 6) at an NaF concentration of 10 mM (fig. 4B). At higher

TABLE 4 Parameters of (+)-[3H]isradipine binding to intact rat portal vein strips in the presence of noradrenaline and phorbol ester inhibitor. Each value is the mean +_S.E. of a number (n) of experiments. Ligand

KDdpp (pM)

Bmax ( f m o l . m g - 1 wet weight)

n

Control Noradrenaline (10 `aM) Noradrenaline (10 `aM) +H7(10`aM)

146+ 19 67_+16 *

14,4_+0.6 14,9_+0.6

6 6

124_+11

14,8_+1,2

6

• Significant change (P < 0.05).

227 concentrations of NaF, the stimulation of isradipine binding began to decrease. Non-specific binding was unaffected in the presence of a combination of a!uminium and fluoride. At 10 p-M, AICI 3 had no effect on (+)-[3H]isradipine binding. As N a F alone did not stimulate specific isradipine binding, it is suggested that fluoroaluminate complexes rather than F - or AI ~+ alone are involved in the stimulation of specific isradipine binding.

3.5. Membrane potential dependence of ( + )-[-~H/isradipine bbtding In intact strips of portal vein smooth muscle, the affinity of (+)-[3H]isradipine increased without variations in the maximal binding capacity as the m e m b r a n e depolarized suggesting that isradipine bound to Ca 2+ channels in the inactivated state with a higher affinity than in the resting state ( D a c q u e t et al., 1988; D a c q u e t et al., 1989). T o test the effect of the KCi concentration on (+)-[3H]isradipine binding, specific binding was d e t e r m i n e d in the presence of a fixed concentration of (+)-[3H]isradipine (0 1 nM) in solutions containing various concentrations of KC1. T h e NaCI concentration was correspondingly modified. As illustrated in fig. 5, specific binding of isradipine increased when external K + concentration was e n h a n c e d from 5.6 m M (physiological solution) to 135.6 mM. At 135.6 m M K +, the increase in isradipine binding was 81 +_ 8% (n = 6). A variation in external K + concentration between 5.6 and 40 m M induced a limited increase in specific isradipine binding (18 + 7%, n = 6). W h e n intact strips were incubated in the presence of cromakalim, a K + channel o p e n e r (Weir and Weston, 1986), the m e m brane potential was hyperpolarized by 25 + 3 m V (n = 5). As indicated in table 5, the apparent K D was significantly increased while the Bm~~ was unchanged. In the presence of cromakalim, addition of noradrena-

ta

.E E

,¢¢

[

z ,ooo

[

,

4-

-

Parameters of (+)-[~H]isradipine binding to intact rat portal vein strips in the presence of cromakalim, noradrcnaline and a combination of aluminium and fluoride. Each value is the mean ~ S.E. of a number (n) of experiments. Ligand

K J~,,w> (pM) 149+_ 16 265+21 ~

B,,,~ (fmol- mg i wet weight) 14.2_+ 1.4 13.8 +-0.~

Control Cromakalim (10 # M) Cromakalim (10 ,uM) + noradrenaline (3 ~.M) Cromakalim ( l0 #M) + AICI~(10 p.M) + NaF (10 raM) Cromakalim ( 10 t~M) + noradrenaline (3 #M) +H7 (10/~M)

n

6 4

133 + 16

14.6± 1.1

4

137 +_23

13.8 +-0.8

4

224+_20 *

14.0+1.1

4

* Significant change (P < (I.05).

line (3 p-M) or a combination of aluminium and fiforide significantly decreased the K D value. In portal vein smooth muscle cell, noradrenaline produced a c o n c e n t r a t i o n - d e p e n d e n t m e m b r a n e depolarization (By~-ne and Large, 1988; Pacaud et al., 1989) which was the trigger for Ca z+ channel opening and further depolarization. In contrast, the combination of aluminium and fluoride had no effect on the resting m e m b r ; m e potential (2-t- 1.5 mV, n = 8). T o confirm that these effects of noradrenaline on specific isradipine binding in the presence of cromakalim were due to activation of pr6tein kinase C rather than a depolarization induced by noradrenaline (which was not large enough to reach the threshold for Ca 2+ channel activation), specific binding of (+)-[3H]isradipine was d e t e r m i n e d in the presence of both cromakalim ( i 0 p.M), noradrenaline (3 p-M) and H7 (10 p-M). As indicated in table 5, the decrease in a p p a r e n t K o induced by noradrenaline was completely removed in the presence of H7.

A

~ 2ooo]

I..--)

TABLE 5

-mog

I-K4-]tM)

Fig. 5. Influence of the KCI concentration on (+)-[3H]isradipinespecific binding. Specific binding was measured after 60 min of incubation in physiological solution containing 0.1 nM (+l[3H]isradipine followed by 30 rain incubation in solutions containing different concentrations of KCI and the same concentration of the radioligand. Non-specific binding was not changed by increasing the KCI concentration and was less than 500 dpm/mg wet weight. Data are means ~+S.E. of six experiments,

3.6. lsradip#~e effect on contractions induced by noradrenaline an,l KCI At the beginning of each experiment, three successive applications of 60.6 mM K + or I0 p-M noradrenaline were made in o r d e r to obtain at least two contractions similar in amplitude. T h e contraction produced by the application of 1() p-M noradrenaline was not significantly different from that produced by 60.6 m M K + (125 + 17 mg and 137 + 23 mg, n = 8, respectively, P > 0.05). Then, isradipine was added for a period corresponding to its equilibrium time (10 rain) before application of KCI or noradrenaline. After 30 rain of washing in reference solution complete recovery of contractions was obtained. Non-cumulative appliea-

228 100"10~>~

•

60.6 mMK +

'~ 5 0 -

E

13

12 -

11

10

9

Io9 [isradlplne]

8

7

(M)

Fig. 6, Effecls of ( + )isradipine on the conlraction of inlact strips of ral portal vein exposed to noradrcnalinc ( , . Ill p.M) or to 60.6 mM KC[ solulion (e). Contraction is expressed as a percentage of the conlraclion measured in the absence of isradipine. Data are means_+ S.E. of 4 - 7 dctermimttions.

tions of isradipine produced a conccntration-depcndent depression of the peak contraction evoked by 10 /.tM noradrenaline (fig. 6). A part of the contraction was resistant to isradipine. The isradipine-resistant fraction, measured in the presence of 100 nM isradipine, was equal to 29 _+6% of the control contraction (n = 7). The conccntration of isradipine required to inhibit the noradrenaline-induced contraction to 50% of its maximal value (IC5~~) was 62 +_ 8 pM (n = 4). The peak contraction induced by 60.6 mM K + was concentration-dependently inhibited by isradipine (fig. 6). Complete inhibition was obtained with 100 nM isradipine or less. The IC.s0 value was estimated to be 210+ 18 pM (n = 4) indicating that the potency of isradipine was lower on KCl-induced contraction than on noradrenaline-induced contraction in rat portal vein smooth muscle.

4. Discussion

Several properties of the binding of ( + )-[3 H]isradipine to vascular smooth muscle membranes have been characterized, including sensitivity to temperature and divalent cation modulation. The affinity of ( + ) [3H]isradipine is optimal at temperatures (25-37°C) which are generally used for functional data. We also show that isradipine binding to smooth muscle microsomes is calcium- and magnesium-dependent, lsradipine binding is inhibited by EGTA and is half-maximally restored by 7-10/.tM Ca 2+ and by 90-100 p,M Mg 2+. In transverse tubule membrane vesicles, it has been recently shown that Ca 2+ ions do not stimulate dihydropyridine binding if the vesicle preparation contains mainly dihydropyridine receptors in an inside-out configuration (Kanngiesser and Pongs, 1989; Ebata et al., 1990). Consistent with this, it is assumed that the extraeellular side of the membrane has high-affinity Ca 2+ binding sites which are responsible for regulating the Ca 2+ and Mg 2+ dependence of dihydropyridine binding. Our results suggest that the vascular smooth

muscle microsomes are mainly in both outside-out and leaky configurations and that the apparent dissociation constant for binding Ca 2+ to this Ca2+-selective binding site is in the micromolar range. It is worth noting that electrophysiological data in smooth muscle have indicated a high-affinity regulatory Ca 2+ binding site at the extraccllular side of the Ca 2+ channel. Binding of Ca 2+ to this side blocks Na + current and renders the Ca -'+ channel CaZ~-selective (Jmari et al., 1987). The dissociation constant for binding to this site was determined to be about 1 ~M. Presumably, there is only one high-affinity binding site on dihydropyridine receptor for Ca -'+. Hence, the Ca 2+ binding site which is involved in dihydropyridine binding would be the one which is important for Ca 2+ channel selectivity (Hess and Tsien, 1984). Activatim of protein kinase C has been recently associated with stimulation of voltage-dependent Ca 2+ channels in vascular smooth muscle cells (Fish et al., 1988; Loirand et al., 1990). Stimulation of a r a d r e n o ceptors produces activation of phospholipase C and production of second messengers. One of the products of the reaction catalyzed by phospholipase C is diacylglycerol, the natural activator of protein kinase C. It has been shown that a pertussis toxin-insensitive G protein is involved in regulating the phospholipase C activity in the electrophysiological effects of noradrenaline in rat portal vein smooth muscle cells (Loirand et al., 1990). In this paper, we have explored the role of the transduction way detailed by electrophysiological data in the regulation of dihydropyridine binding in both membranes and intact strips of portal vein smooth m~z ~!es. We have found that in intact strips both noradrenaline, a combination of aluminium and fluoride activating G proteins and phorbol esters caused an increase in isradipine affinity with little change (if at all) in the number of dihydropyridine binding sites. These results are in concordance with electrophysiological data obtained in vascular smooth muscle cells which show that noradrenaline increases the open-state probability of calcium channels without modifying either the number of functional channels or the amplitude of the single channel current (Nelson et al., 1988; Pacaud~ Loirand, Baron, Mironneau and Mironneau, unpublished data). Therefore, the present data confirm that during noradrenaline stimulation the number of dihydropyridine binding sites and consequently the number of L-type Ca 2+ channels remain unchanged. Increase in isradipine affinity to binding sites in Ca -'+ channels is associated with direct activation of protein kinase C by phorbol esters as well as by activation of oq-adrenoceptors by noradrenaline. In membranes, phorbol esters produce an increase in isradipine affinity to its binding sites when the incubation medium contains both ATP and MgCl 2 and this effect is independent of the divalent cation concentration in the

229

incubation medium. More experiments are needed to explain the larger increase of isradipine binding in intact strips compared to microsomes. Thus, the regulation of dihydropyridine receptors by protein kinase C seems to involve a phosphorylation process. Whether this phosphorylation directly affects Ca 2+ channels or an intermediary intracellular protein remains to be determined. In vascular smooth muscle cells, membrane depolarization increases the affinity of dihydropyridine binding without affecting the number of dihydropyridine receptors (Morel and Godfraind, 1987; Sumimoto et al., 1988; Dacquet et al., 1989). Our results demonstrate that the effects of phorboi esters on isradipine binding are independent of the membrane potential and are selectively blocked by H7, a protein kinase C inhibitor. These observations are therefore consistent with the idea that protein kinase C may increase the affinity of dihydropyridine binding to Ca 2+ channels independently of a separate modulation by membrane potential. A similar modulation of the binding sites of dihydropyridines by noradrenalinc ~s confirmed by functional data. Analysis of the effects of isradipine shows that noradrenaline- and depolarization-induced contractile responses have different sensitivities to Ca 2+ entry blockers. We report that in rat portal vein ( + ) isradipine is a more potent inhibitor of the contraction induced by noradrenaline than by depolarization, in contrast to data obtained in arteries (Morel and Godfraind, 1991). Our observations suggest that in vein smooth muscle ion permeation through voltage-dependent Ca 2+ channels can be modolated by phosphorylation dependent on protein kinase C activation. Further biochemical experiments are required in order to characterize Ca 2 + channel phosphorylation in smogth muscle.

Acknowledgements This work was supported by grants from Etablissement Public R~gional d'Aquitaine, Minist~re de la Recherche et de la Technologic (89.H.0082), Minist~re de I'Education Nationale, Centre National des Etudes Spatiales and Fondation pour la Recherche M~dicale, France. We thank Ms. Nathalie Biendon for excellent assistance.

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