Short Term and Long Term Effects of &Adrenergic

Vol. 260, No. 24, Issue of October 25, pp. 13041-13046,1985 Printed inU.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY The American Society of Biological Chemists, Inc.

@ 1985 hy

Short Term and Long Term Effects of &Adrenergic Effectors and Cyclic AMP on Nitrendipine-sensitive Voltage-dependent Ca2+ Channels of Skeletal Muscle* (Received for publication, April 26, 1985)

Annie Schmid, Jean-Franwis Renaudg and MkhelLazdunski From the Centre de Biochimie, Centre National de la Recherche Scientihue, Facult6 des Sciences, Parc Valrose, 06034 Nice Cedex, France

The effects of short termstimulation of &adrenergic verapamil (10-13), [3H]D888 (14), or [3H]diltiazem (15) have receptors and elevations in intracellular cyclic AMP shown that receptor sites for these different molecules are on nitrendipine-sensitivevoltage-dependent Caz+ present in high density in T-tubule membranes where they channels of skeletal muscle cells in vitro has been are presumably associated with voltage-dependent Ca2+chanstudied using both the 4sCa2+flux technique and [’HI nels. Because of the high density of dihydropyridine receptor nitrendipine-binding experiments. Isoproterenol in- sites in skeletal muscle T-tubule membranes as compared to creased the nitrendipine-sensitive 4sCa2+influx under other membranes (heart and smooth muscle) (16, 17), these depolarizing conditions. The effects of isoproterenol membranes have been used in the first studies to purify and were additive tothose of depolarization and were an- label the putative dihydropyridine-sensitivevoltage-dependtagonized by alprenolol. Half-maximal inhibition of ent Ca2+channel (18-20). 46Ca2+influx induced both by depolarization and by Developmental properties of voltage-sensitive Ca2+ chanisoproterenol occurred at a nitrendipine concentration nels duringin vivo myogenesis of chick muscle cells have been of 1nM. Treatments that resulted in an increased level of intracellular cyclic AMP, such as treatment with assessed using 45Ca2+flux experiments and [3H]nitrendipinel-methyl-3-isobutylxanthine,theophylline, dibutyryl binding assays (3). Both approaches have shown that [3H] cyclic AMP, or 8-bromocyclic AMP also resulted in an nitrendipine receptors, as well as functional dihydropyridineincreased rate of 46CaZ+entry via nitrendipine-sensi- sensitive and voltage-sensitive Ca2+channels, are absent at the myoblast stage. However, both [3H]nitrendipine receptors tive Ca” channel. In contrast, long term treatmentof myotubes in cul- and nitrendipine inhibitable Ca2+flux appear in parallel with ture with isoproterenol and other compounds that in- the fusion of myoblasts into myotubes (3). There are two distinct phases in the appearance of [3H] creased intracellular cyclic AMP led to a large increase in the number of nitrendipine receptors. This increase nitrendipine receptors during myogenesis in vivo (3). The first was accompanied bya 4-10-fold decrease in the affin- one is observed during embryonic life and corresponds to the ity of the receptors fornitrendipine. Alprenolol inhib- appearance of [3H]dihydropyridine sites during fusion of myited the long term effects of isoproterenol. oblasts into myotubes. The second phase occurs near hatching In vivo treatment of 7-day-old chicks with reserpine and corresponds to an increased maximum binding capacity and alprenolol produced a decrease in the number of for [3H]nitrendipine by a factor of 7. This increased number skeletal muscle nitrendipine receptors. This decrease of [3H]nitrendipine-binding sites is accompanied by a dein receptor number was accompanied by an increase in crease of affinity of nitrendipine for its receptor by a factor the affinityof nitrendipine forits receptor by a factor of 4-10. Ca2+ channels in other tissues, notably in cardiac of 4 to 5. These effects on the nitrendipine receptor were preventedby simultaneous injection of isoproter- muscle cells, are known to be regulated by cyclic AMP (2124). In order to gain a further understanding of the skeletal enol. muscle Ca2+ channel, wenow have analyzed the effects of The resultsare discussed in relation to the role of 8adrenergic receptors and intracellular cyclic AMP in short term and long term elevations in cyclic AMP on both the activity and expression of dihydropyridine-sensitive Ca2+ the regulation of skeletal muscle Ca” channels. channels and dihydropyridine receptors in skeletal muscle cells. The skeletal muscle cell contains voltage-dependent Ca” channels that are inhibitable by dihydropyridines such as nifedipine or nitrendipine (1-3). These Ca” channels appear to beprimarilylocated in the T-tubular system (1, 4-6). Binding studies using [3H]dihydropyridines (3, 7-9) as well as with other tritiated Ca2+ channel blockers such as [3H]

* This work was supported by the Centre National de la Recherche Scientifique and the Association Franpise de Lutte contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom reprint requests shouldbe addressed.

MATERIALS AND METHODS

Cell Culture-Primary cultures of skeletal muscle cells fromchick embryos were prepared according t o the method previously described (3,251 using 11-day-oldchick embryonic breast and leg muscles. Cells were grown in Dulbecco’s modified Eagle’s mimimal essential medium and M199 medium (3:l)supplemented with 5% fetal calf serum, 200 units/ml penicillin, and 50 pg/ml streptomycin. Cells were plated either in gelatin-coated 60-mm diameter dishes (Corning tissue culture dish) or 24-well tissue culture plates (Nunc) at a density of 1.5 X lo5 cells/cm*.Cultures were maintained at 37 “C in a watersaturated atmosphere of air/COz (95/5). Differentiated myotubes were used for 45Ca*+ flux experiments and binding assays. 45Ca2+Flux Experiments-The determination of nitrendipine-sensitive and -insensitive rates of &Ca2+uptake by myotubes in culture was carried out ingelatin-coated 24-well tissuecultureplates as

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,&Adrenergic Effects on Ca" Channels in Skeletal Muscle

previously described (3). Cells were cultured between 48 and 100 h RESULTS before flux measurements were performed. The determination of rate Evidence for a Role of &Adrenergic Receptors and cAMP in of 45Ca2+uptake first involved a preincubation of the cells for 15 min a t 37 "c in a medium containing 40 mM K+, 105 mM Na+, 5 mM Regulating Dihydropyridine-sensitive45Ca2+Uptake in Chick glucose, 25 mM Hepesl-Tris at pH 7.4 in the presence or absence of Myotubes in Culture-The effect of isoproterenol on the ninitrendipine and other molecules such as (-)-isoproterenol, N6,02- trendipine-sensitive component of 45Ca2+ uptake in chick mydibutyrylcyclic AMP (BtZcAMP), 8-bromocyclic AMP, l-methyl-3otubes in culture has been investigated using a high external isobutylxanthine (MIX), andtheophylline as specified. The medium was then removed by aspiration and the cells were incubated in a K+ medium containing 40 mMKC1. Under these conditions medium containing 40 mM K', 105 mM Na+, 5 mM glucose, 1.8 mM cells are depolarized to -25 mV (3). Fig. 1 shows that in the presence of 10 pM isoproterenol, a concentration increasing CaC12, 5 pCi/ml &Ca2+,and 25 mM Hepes-Tris at pH 7.4 in the presence of the same concentrations of nitrendipine and of the other the cAMP level in avian myotubes by approximately &fold molecules that were used in the preincubation. Kinetics of"Ca2+ (27), the &Ca2+uptake was higher than in the absence of uptake were followed by removing the medium a t different times of isoproterenol. The same results were obtained with 3 ~ L M incubation between 0 and 1min. In experiments done to obtain doseisoproterenol (not shown). T!a2+ uptake in the presence or response curves for nitrendipine action on &Ca2+uptake, the initial rate of &Ca2+uptake was measured after 20 s of incubation. At the in theabsence of isoproterenol was inhibited by nitrendipine. end of the incubation, cells were washed three times in less than 5 s Addition of 10 p~ alprenolol to the incubation medium preof &Ca2+uptake. with 3 ml of washing medium containing 140 mM choline, 1mM La3+, vented the effect of isoproterenol on the rate Under the laterconditions, the kinetics of 45Ca2+ uptake were and 25 mM Hepes-Tris at pH7.4. After the thirdwash, 2 ml of 0.1 N NaOH were added to each well and the radioactivity that was incor- identical to those obtained in theabsence of both alprenolol porated by the cells was counted. Protein concentrations were mea- and isoproterenol (Fig. 1). Dose-response curves for the action sured according to Hartree (26). of nitrendipine on the initial rate of %a2+ uptake in the Treatment with Reserpine, Isoproterenol, and Alprenolol-Onepresence and absence of isoproterenol are shown in Fig. 1, week-old chicks (50-60 g, body weight) received either intraperitoneal inset. The half-maximum effect of nitrendipine on the inhior intramuscular injections of 5 mg/kg of reserpine on day 0, 2, 4, and 6 were killed either on day 2, 3, 5, or 7. Some chicks were also bition of 45Ca2+flux was observed at 1 nM in both cases, injected either with (-)-isoproterenol a t a concentration of 10 mg/kg indicating that theinhibition of 45Ca2+ uptake by nitrendipine or with reserpine and isoproterenol under the same conditionsas for was not affected by isoproterenol. We also tested the effects reserpine alone. Other chicks were injected with alprenolol at concen- of other molecules that are known to elevate cAMP on the trations ranging between 0.5 to 10 mg/kg on day 0 and 2 and were activity of the skeletal muscle Ca2+channel. Fig. 2 compares killed on day 3. Control chicks received injections of vehicles only. Tissue Preparation-Skeletal muscles from legs of 7-day-old chicks the effect of isoproterenol with the effects of BhcAMP, MIX, were obtained from treated andnontreated animalsa t different stages and theophylline on the initial rate of nitrendipine-sensitive of treatment. In all cases, muscles were removed and washed in an &Ca2+uptake in myotubes in culture. Dibutyryl CAMP inuptake (measured at 20 s) by ice-cold buffer containing 20 mM Tris-C1, 0.25 M sucrose, and 1 mM creased the initial rateof 45Ca2+ EDTA at pH 7.4 (TSE buffer). Subsequently the muscles were 60%,MIX increased the initial rateof 45Ca2+ by 47%,whereas dissected, minced, rinsed once with the TSEbuffer, and h~mogenized theophylline increased the rate of &Ca2+uptake by 56% as a t setting 5 with a Polytron apparatus using 5-s burstsseparated by compared to the control. Under the same conditions, isopro30-s pauses. Crude membranes were prepared from the muscle hoterenol increased the initial rate of 45Ca2+uptake by 65% as mogenates by two centrifugations a t 20,000 X g for 10 min. The twiceuptake was inhibitable washed pellets were resuspended in fresh TSE buffer to a concentra- compared to control. In all cases 45Ca2+ tion of 100 mg of original wet tissue weight/ml of buffer. Subsequently by nitrendipine (Fig. 2). Long Term Effects of Isoproterenol Treatment on Properties the pellets were filteredthrough four layers of cheesecloth. For binding assays, skeletal muscle cells in culture were washed with an of [3H]Nitrendipine Binding to Membranes from Embryonic ice-cold TSE buffer, scraped, homogenized with a Potter-Elvejhem apparatus, centrifuged, and washed as described for leg muscles. The final pellets were suspended in the TSE buffer and assayed for binding with [3H]nitrendipine. Binding Assays-Binding assays were carried out asfollows. 0.5-1 mgof crude membrane protein (either from leg muscles or from cultured cells) was equilibrated in 1 ml of the standard incubation medium containing 20 mM Tris-C1 and 50 mM choline chloride at pH 7.4 and 4 "C in the absence (total binding) or in the presence (nonspecific binding) of 1p~ unlabeled nitrendipine for 60 min a t 4 "C. Incubations were stopped by filtering in duplicate 400-pl aliquots of the incubation medium through Whatman GF/B glass fiber filters under reduced pressure. Filters were immediately washed twice with 5 ml of an ice-cold solution containing 200 mM choline chloride and 20 mM Tris-C1 buffer at pH 7.4. Aliquots (100 pl) of the incubation mixture were taken for measurement of the amount of [3H]nitrenditime (sed -logCnitrendipine] (MI pine present.All experiments were carried out under dim light because of the light sensitivity of the dihydropyridine derivatives. Competition FIG. 1. Short term effect of isoproterenol on nitrendipineexperiments between [3H]nitrendipine and unlabeled nitrendipine sensitive rate of4aCaz+ uptake by myotubes differentiated in were done under equilibrium conditions at 4 "C as described above, culture for 50 h. Main panel, time course of 45Caz+uptake was using fixed concentrations of [3H]nitrendipine. Radioactivity bound measured under depolarizing conditions (40 mM K+) in thepresence to filters was measured using Biofluor (New England Nuclear) in a (A)and theabsence (0)of 10 p~ isoproterenol and 1p~ nitrendipine Kontron MR 300 scintillation spectrometer. (A and O), and in presence of both 10 p~ isoproterenol and 1 p M Chemicul~-[5-methyl-~H]Nitrendipine at 88 Ci/mmol was ob- alprenolol (0 and in the presence (0)and absence).( of 1 p M tained from New England Nuclear. 45CaC12was obtained from c. E. nitrendipine. Inset, dose-response curves for nitrendipine inhibition A. Nitrendipine was from Bayer Pharma. All other compounds used of the initial rate of 45Ca2+uptake under depolarizing conditions in the presence (A)and in the absence (0)of 10 p M isoproterenol. In were from standard commercial sources. this representation, nitrendipine-sensitive rates of &CaZ+uptake were The abbreviations used are: Hepes, 4-(2-hydroxyethyl ether)-1- obtained from 3 differents experiments. Rates were calculated by piperazineethanesulfonic acid; MIX, 1-methyl-3-isobutylxanthine; subtracting the value obtained in the presence of 1p~ nitrendipine from the initial rate of &Ca2+uptake. Bt2cAMP, dibutyryl cyclic AMP.

.)

/3-Adrenergic Effects on Ca2' Channels in Skeletal Muscle

13043

isoproterenol for 37 h indicated that isoproterenol caused a large increase in themaximum binding capacity and a change in Kd value as compared to the control. After the chronic isoproterenol treatment, the Bmaxwas 427 fmol/mg protein and theKd value was 1.5 nM. The Scatchardplots were linear for both the control and isoproterenol-treated myotubes in8 dicating the presence of a single class of binding sites. The nitrendipine 1pM T inset of Fig. 3 shows the time course for the isoproterenolg 10 induced changes in [3H]nitrendipine-bindingproperties. The 5 level of 13H]nitrendipine receptors remained relatively stable Q, x Q at a value of 130 k 10 fmol/mg protein during the first 20 h - 5 4 of treatment with isoproterenol. Then the receptor level in+, creased to reach a new plateau at 500 fmol/mg protein after (D 30 h of treatment. These variations in the number of [3H] x 0 FIG.2. Influence of molecules known to increase intracel- nitrendipine-binding sites were accompanied by variations in lular CAMPlevels on *'Ca2+ uptake under depolarized condi- the Kd values. The Kd was 0.4 k 0.1 nM during the first 20 h tions by myotubes differentiated in culture50for h. Uptake of of treatment, changed to 1.6 f 0.2nM at 30 h, and then &Ca2+was measured for 20 s at 37 "C under the indicated conditions remained essentially unchanged for the remaining period. in the absence and presence of 1p~ nitrendipine. Eachbar gives the Untreated myotubes have a level of [3H]nitrendipinereceptors mean value +- S.E. of 4 experiments done in duplicate. db CAMP, and a Kd value that remain stable at a value of 140 +- 10 fmol/ dibutyryl CAMP. mg protein and 0.4 k 0.1 nM, respectively, during the time time of culture (dam) course of the culture (not shown). 2 3 4 The chronic effects of isoproterenol were dependent onthe concentrations of isoproterenol used. Fig. 4, A and B, shows **e%" the dose-response curve for an isoproterenol treatment of 42 h on 92 h-old-myotubes. Isoproterenol was added every 12 h at concentrations rangingbetween 10 nM and 30 p~ and once more 2 h before takingthe cells for binding studies. At concentrations between 10 and 100 nM, isoproterenol did not induce significant changes in Kd and B,,, values for [3H] nitrendipine which remained at 0.4 zk 0.1 nM and 140 f 15 M fmol/mg protein, respectively. At concentrations of 3 ~ L and higher, isoproterenol induced changes both in Kd and 3values that reached 1.6 -+ 0.2 nM and 425 f 22 fmol/mg protein, respectively. Alprenolol(10 p ~ produced ) a complete blockade of changes in [3H]nitrendipine-bindingproperties 0 control E 4 isoproterenol lOpM DBt2cAMP lw IN theophylline l0m

-

-0-

J_-

PHI nitrendipine specifically bound (fmol/mg protein)

FIG.3. Long term effects of isoproterenol treatment on the properties of ['Hlnitrendipine to membranes from skeletal muscle cells in culture. Fused myoblasts were treated 48 h after plating with 10 p~ isoproterenol as described under "Results." Main panel, Scatchard plots for specific [3H]nitrendipine binding memto branes fromof myotubes treated for 37 h (0)and to membranes from untreated cells (0)of the same age. Inset, variations of maximum bindingcapacities (A) and Kd values (0)for['Hlnitrendipine on isoproterenol treated as a function of the time of treatment with 10 pM isoproterenol. Chick Skeletal Muscle Cells in Culture: the Role of Cyclic AMP-Isoproterenol (10 p ~ was ) added to cultures of chick skeletal myotubes two days after plating, at the time when the maximum extent of fusion reaches 80% (3). This isoproterenol treatmentwas repeated every 12 h. Then themyotubes were used at different lengths of the chronic isoproterenol treatment ranging between 1 and 50 h. Myotubes cultured in the presence of isoproterenol did not show any detectable morphological change as compared to control untreated cells. Fig. 3 shows typical Scatchard plots for specific [3H]nitrendipine binding to crude membranes from differentiated myotubes cultured for a total length of 85 h in the presence or absence of isoproterenol for 37 h. Membranes from control myotubes had a Kd value of 0.4 nM and a binding capacity of 137 fmol/mg protein. Scatchard plotsfor the specific binding of [3H]nitrendipine to myotubes treated chronically with

-~ogCi~erenoll(M)

-log[n4trendipinel(M)

FIG.4. The influence of varying concentrations of isoproterenol on changes in binding properties at ['Hlnitrendipine A, gives mean receptors membranes from myotubes in culture. K d values (0 and A) for the nitrendipine receptor andB gives mean B , values ( and .A) for the nitrendipine receptors of cells treated by isoproterenol alone (0and . ) or isoproterenol +10 p~ alprenolol (A and A). Results are the average of 3 experiments. C, competition between [3H]nitrendipineand unlabeled nitrendipine for binding to membranes of 42-h isoproterenol-treated ).( and untreated (0)myotubes taken at the same age of culture. These curves are the mean of 4 experiments. The bindingconstant (Kd)in conditions of competitive inhibition was calculated Using KO,= K d (1 + L*/&*), where L* is the concentration of free ['Hlnitrenedipine at half-dissociation, Kd* is the dissociation constant of the nitrendipine receptor complex (0.4 or 1.7 nM) determined from the direct equilibrium constant, KO, is the concentrationof unlabeled nitrendipine usedto obtain the halfmaximum inhibition of [3H]nitrendipine at given concentration of

[3H]nitrendipine(0.2 nM).

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,&Adrenergic Effects on ea2+Channels in Skeletal Muscle

induced by the highest dose of isoproterenol (Fig. 4,A and B ). Fig. 4C shows the results of competition experiments between unlabeled and labeled nitrendipine performed on membranes from myotubes cultured with and without isoproterenol. Half-maximal inhibition for unlabeled nitrendipine is observed at 0.3 nM for untreated cells and at 3 nM for treated cells. The Kd values of the nitrendipine-receptor complex calculated from these experiments were 0.1 and 4.6 nM for myotubes cultured withoutand with isoproterenol, respectively. In other experimentswe determined the effects of long term treatment of myotubes with a series of molecules that lead to elevations in intracellular CAMP, either by inhibiting phosphodiesterase activity (like MIXand theophylline) or by activating adenylate cyclase activity (like isoproterenol). Molecules like Bt,cAMP and 8-bromo-CAMP also were tested. Results presented in Table I summarize the effects of these agents on specific [3H]nitrendipine binding to membranes from cultured myotubes. All these compounds caused a decrease in the Kd values, as compared to the control, and a parallel increase in themaximum binding capacity. The effect of isoproterenol on myotubes is given for comparison. Effects of Treatments with Reserpine and Isoproterenol on the Properties of [3HJNitrendipine-binding Properties in Chick Skeletal Muscle-Chicks (7-day postnatal) were treated with reserpine, which depletes norepinephrine and epinephrine and inhibits their synthesis (28). Chicks were divided into two different groups. The first group received injections of reserpine (5 mg/kg) on day 0 and day 2 and the second group received only the vehicle. Under these conditions, survival of the reserpine-treated group was 80%.Reserpine treatment caused a decrease in the maximum number of [3H] nitrendipine-binding sites from a control value of 885 to 565 fmol/mg protein (Table 11).Under theseconditions Kd values were 0.4 and 1.8 nM for the reserpine treated and for control, respectively. The decreases in maximum binding capacity and Kd value remained stable for 7 days under the treatment schedule used (not shown). Reserpine (10 p ~ was ) found to have no direct effect by itself on [3H]nitrendipine binding. Another series of experiments was carried out using membranes from chicks that received injections of isoproterenol alone (10 mg/kg) or isoproterenol plus reserpine (5 mg/kg) according to theprotocol used for reserpine. Treatment with isoproterenol alone did not produce significant changes on B,, or Kd values for t3H]nitrendipine binding as compared to control value (Table 11). However, as shown in Table I1

TABLE I1 Maximum binding capacities and Kd values for pH]nitrendipine binding to skeletal muscle membranes from 7-day-old chicks treated with reserpine andlor isoproterenol and alprenolo1for 3 days Values are mean f S.E. of the number of determinations shown in parentheses. Each Kd and B , value was derived fromScatchard plot analysis.

Control (10 p ~ ) Reserpine (10 PM) Isoproterenol (10PM) Reserpine (10PM) isoproterenol (10 PM) Alprenolol (10PM)

+

B, fmol/mgprotein 880 f 40 ( n = 12) 565 f 35 ( n= 3) 890 +- 45 ( n = 2) 900 f 50 ( n = 3)

1.8 -C 0.2 0.4 -C 0.1 1.8 f 0.2 1.7 f 0.2

330 f 30 ( n = 3)

0.5 f 0.1

Kd

nM

simultaneous treatment with isoproterenol and reserpine prevented the effects caused by reserpine alone. Under these conditions the Kd value for [3H]nitrendipine binding was 1.7 nM and the B,, was 900 fmol/mg protein, similar to the values obtained for control membranes. Effects of Alprenolol Treatment on the Properties of r3HJ Nitrendipine Binding to Chick Skeletal Muscle MembranesTwo groups of chicks received three successive injections of alprenolol at concentrations of either 1 or 10 mg/kg on day 0, 1, and 2, and were killed on day 3. Controls received the vehicle only. Results presented in Table I1 show that alprenolol, at 10 mg/kg, caused a decrease in themaximum binding capacity from a control value of 900 to 330 fmolfmg protein. This change in B,, was accompanied by a decrease in theKd value from a controlvalue of 1.8 to 0.5 nM. Concentrations of alprenolol of 1 mg/kg or lower were without effect on the pioperties of nitrendipine binding (not shown). Competition experiments between unlabeled and labeled nitrendipine were performed on nontreated 7-day-old chick skeletal muscle membranes in the presence or absence of 10 PM alprenolol. The results of this series of experiments gave Kd values for nitrendipine of 6 nM, in the absence and presence of alprenolol, respectively. These results indicated that alprenolol had no direct effect itself on [3H]nitrendipinebinding. DISCUSSION

The effects of @-adrenergicagonists and of other agents modulating intracellular cAMP levels on the activity of the voltage-dependent Ca2+channel in heart cells have been extensively studied using electrophysiological techniques (23, 29-34). The mechanism of action of /?-adrenergicagonists on cardiac Ca2+channels can be summarized as follows. After binding of adrenergic agents to the @-receptor,the adenylate TABLEI cyclase is activated via the guanine nucleotide-regulated N, Long term effects of molecules that lead to an elevation of unit leading to an increase in intracellular cAMP levels (35). intracellular cyclic AMP on PHlnitrendipine-binding properties to Cyclic AMP binds to the regulatory subunit of the CAMPmembranes from myotubes in culture dependent protein kinase, thereby liberating the catalytic Chick skeletal myotubes received the different molecules for the subunit of the enzyme. Then itis assumed that phosphorylafirst time 48 h after plating, and treatments were repeated every 12 h for two days. Values are mean C S.E. of the number of determina- tion of the ion channels or of a protein closely associated with tions given in parentheses. Each K d and B,, value was derived from the channel occurs (23, 33,36-38). This presumably causes a Scatchard plot analysis. conformational change in the channel, the consequence of which is that there is a higher probability that the channel [3H]Nitrendipinebinding Treatment will open on membrane depolarization than in the dephosKd phorylated state (21, 23). As a result, @-adrenergicagents nM fmollmgprotein increase I,, in cardiac cells (29-32). This causes an elevation 0.4f 0.1 140 f 15 ( n = 17) Control of the plateau height of the cardiac action potential (39, 40). 1.6 f 0.2 425 f 22 ( n= 10) (-) Isoproterenol (10pM) Kinetic analyses of I,. in the presence of adrenergic agonists Dibutyryl cyclic AMP (10p ~ ) 1.3 f 0.1 346 2 30 ( n = 17) have shown that neither the voltage-dependent kinetics of the 1.4 f. 0.2 374 f 28 ( n = 6) 8-Bromocyclic AMP (10PM) 1.6 f 0.1 432 ? 34 ( n = 7) Ca2+current nor the reversal potential are affected by the 1-Methyl-3-isobutylxanthine (10P M ) adrenergic agonists (31, 32, 41). The effects of @-adrenergic Theophylline (10PM) 1.5 f 0.2 422 +. 41 ( n = 4) agents can be mimicked by the injection of cAMP into my-

P-Adrenergic Effects on Ca2' Channels in Skeletal Muscle ocardial cells (42-44), by the application of BtzcAMP and 8bromo-CAMP, bythe use of phosphodiesterase inhibitors (22, 44, 45), and by injection of catalytic subunit ofCAMP-dependent protein kinase into cells (21). The catecholamine effect on both Ca2+current and plateau height of the action potential can be inhibited by @-receptorantagonists (36,441. @-Adrenergicreceptors have been identified in avian embryonic skeletal muscle cellsboth in uivo and in vitro (27,46). Occupancy of these receptors by p-adrenergic agonists increases adenylate cyclase activity as well as cytoplasmic levels of CAMP (27,46). The first part of the work presented in this paper has shown that isoproterenol, a well-known @-adrenergic agonist, increased the rateof nitrendipine-sensitive 45Ca2+ entry into depolarized chick skeletal muscle cells in culture. Depolarization by itself opens these Ca2+channels (3). The effects of isoproterenol were additive to those of depolarization. Alprenolol, a potent @-adrenergicantagonist, prevented the effect of isoproterenol on 45Ca2+uptake (Fig. 1).The halfmaximum inhibition of &Ca2+influx induced by depolarization or by depolarization plus isoproterenol was observed at the same concentration of nitrendipine, = 1 nM). This K , , & value is very similar to the dissociation constant of 0.4 nM previously found for specific [3H]nitrendipine binding to membranes from chick skeletal muscle cells differentiated in vitro and in ouo (3) (of course, isolated membranes are depolarized). The increased Ca2+-channelactivity due to the short term effect of @-adrenergicagonists on skeletal muscle cells seems to occur by the same mechanism as that found for cardiac cells, i.e. the effect appears to be mediated via CAMP. Furthermore, treatments that resulted in an increased level of cAMP in cultured skeletal musclecells, such asthose caused by an inhibition of phosphodiesterase activity with MIX and theophylline, also resulted in an increased rate of 45Ca2+entry via nitrendipine-sensitive Caz+channels (Fig. 2). Similar results were obtained with analogues of cAMP such as Bt2cAMP. All these compounds caused results similar to those observed with isoproterenol. Isoproterenol at a concentration of 1-3 PM caused an increase in the contractile force of isolated tibia skeletal muscle fibers from 5-day-old chicks. The development of the tension phase was faster than in the absence of isoproterenol and the peak tension was increased by 30-45% (not shown). In addition to their shortterm effects, @-adrenergicagents also exerted long term effects. The long term effects may be directly related to the changes in [3H]nitrendipine-binding properties that occur in ouo near hatching (3). Binding characteristics of [3H]nitrendipine at two stages of development are summarized Table 111.The postnatalform of nitrendipine receptors is characterized by a high binding capacity (880 fmol/mg protein) and a K d value of 1.8 nM (3), whereas the fetal form is characterized bylower binding capacity (130 fmol/mg protein anda K d value of 0.4 nM (3). The hypothesis, suggesting a role of neurotransmitters on putative nitrendipine-sensitive Ca2+channels via the /?-adrenergicsystem during chick skeletal muscle development, has been tested in TABLE I11 Summary of PHInitrendipine-binding properties inmembranes of chick skeletal muscles at two stages of development and under different treatments stage

Ki nM

Fetal (11 to 15 days in ouo) Postnatal (7 days) Reserpine + Alprenolol

+

0.4

1.7-1.8 0.4

0.4

B, fmol/mg protein

130 880-900 565 330

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uivo. Two different chronic treatments, that affect the 0adrenergic,system carried out on 7-day-old chicks, support this conclusion, The firstone was with reserpine, a molecule that depletes norepinephrine storage and inhibitsnorepinephrine and epinephrine synthesis (28). The second treatment was with alprenolol, a well-known @-adrenergic receptor blocking agent. Both treatmentswill of course suppress, or at least strongly decrease, @adrenergic stimulation of the chick skeletal musclecells. Reserpine decreased the number of receptors for [3H]nitrendipineby 64% and alprenolol by 40%. Both drugs also changed the affinity of the receptor for nitrendipine (Table 111).The Kd values decreased by a factor of 4-5 in reserpine- and alprenolol-treated animals ( K d = 0.4 & 0.1 nM) compared to control animals ( K d = 1.7 f 0.1 nM). Therefore, skeletal muscle nitrendipine receptors in reserpine- and alprenolol-treated animals are more similar to "fetal" nitrendipine receptors than to the postnatal nitrendipine receptors. These results are clearly in favor of the conclusion that the physiological stimulation of @-adrenergicreceptors regulate both the number of specific nitrendipine receptors and the affinity of the receptor for dihydropyridines. This conclusion is supported by the fact that if reserpine treatment was accompanied by repetitive isoproterenol injections then the reserpine effects on [3H]nitrendipine-bindingproperties were not seen (Table 11). Although functional /?-adrenergic receptors are present in chick skeletal myotubes in culture (27,46),treatment of these cells with isoproterenol for periods of time shorter than 20 h modified neither K d nor B,, values for nitrendipine receptors. More prolonged treatments (30 h) with isoproterenol at concentrations higher than 1 PM induced an increase in BmXvalues by a factor of 4 and a change in Kd values from 0.4 t 0.1 nM in the control to 1.6 k 0.2 nM in the isoproterenol-treated cells. The @-adrenergicantagonist alprenolol blocked the effects of the ,&adrenergic agonist isoproterenol (Fig. 4). The long term @-adrenergiceffects observed in vitro are clearly linked to changes in cytoplasmic levels of cAMP since they can be mimicked by inhibitors of phosphodiesterases such as MIX and theophylline, and by analogs of cAMP like Bt2cAMP and 8-bromo-CAMP (Table

1). The results that are found here with skeletal muscle cells in culture have similarities with recent results obtained with different neuronal clonal cell lines (48). These cell lines have little or no voltage-sensitive Ca2+channels under standard cell culture conditions. However, when their cellular level of CAMP is increased by using agents, like prostaglandin, BtZcAMP, forskolin, cholera toxin, etc., they acquire functional voltage-sensitive Ca2+ channels that can be blocked with dihydropyridines (47, 48). This effect of CAMP on the expression of voltage-sensitive Ca2+channels in neurones has been associated with a possible roleof CAMPin the regulation in synaptogenesis (47). Finally, it has beenproposed that CAMPaffects post-translational modifications of some species of glycoproteins (47), and it is now known that the dihydropyridine receptor from skeletal muscle is a glycoprotein since the solubilized receptor is retained on wheat germ agglutinin columns (18). Acknowledgments-We thank M. T. Ravier and N. Boyer for expert technical assistance, M. Valetti for skillful technical help, and Dr.M. Hosey for fruitful discussions and for a careful reading ofthe manuscript.

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