Ca2+ channels in chick neural retina cells

caZ+channels in chick neural retina cells characterized by 1,4-dihydropyridine

antagonists and activators X. Y. WEI, A. RUTLEDGE,Q . ZHBNG,J. IFERRANTE,

AND

D. J.

TRIGGLE'

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 216.208.156.69 on 06/06/13 For personal use only.

Department of Biochemical Phrmacology, School ofPhrma~'g.1, State University of New York at Bufalo, 126 Cooke Had&, Bufalo, Wry 14260, U.S.A. Received December 31, 1988

WEE,X. Y.,RUTLEDGE,A., ZHONG,Q . , FERRANTE, J . , and TRHGGLE, D. J. 1989. Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridineantagonists and activators. Can. 3. Physiol. % m a c o l , 67: 506-514. The voltage-sensitivecalcium c h m e l in cultured chick neural retina cells was characterized by the actions of tbe enantiomen of Bay K $644 and 2632-791 and other 1,4-dihydropyridines. These cells showed t h e - and voltage-dependentCa2+ uptake that was stimulated by Kt depolarization and blocked by the inorganic calcium channel blockers Cd2+ m d @202+. A small fraction only (15% maximum) ofthe uptake was inactivated by predepolarization of the cells with $8 rdvf Kt. Ca2+uptake was sensitive to the I ,4-dihydlropyridiinecalcium channel antagonists and activators. ($)-Bay K 864.4 and (S)-202-791 stimulated the Ca2+uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 26BO-118 inhibited Ca2+uptake stimulated by #+ &pol&zation or channel activators. The K+ depl~zation-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blmked by the 1,4-dihydropyridineantagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the s m e cells or their binding and phadganacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 4 5 ~ a 2 + uptake was not observed in a glial cell cultwe. ['H~~iapendipine and E~H]PN 2W-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site. ['HIPN 200- 110 bound with higher affinity (KD = 7.09 k 0.W x 10- I' M) than did ['I3]nitrendipine (KD= 4. IQ k 0.92 x 10- M), but the B,,, values were similar for the two ligmds (98.9 9 4.1 and 99.4 k 6.58 frnoB/mg protein, respectively). The discrepancy between binding and phmacologic activities sf the antagonist Iigmds does not appear to be due to the presence of 1,4-dihy&opy9idine-insensitive ca2+ channels, but may relate to the inability of these agents to access a high affinity inactivated state in the intact cell or to the presence of discrete categories of binding sites. This marked discrepancy between affinities does not exist for the activator ligmds studied. 'This study c o d m s the presence of voltage-dependent 1,4-dihydrspyidine sensitive Ca" channels in chick neural retina cells. Key w ~ r d sca2+ : channels, 1,4-dihydropyridine~,chick neural retina, retinal neurons. WEI, X. Y., RUTEEWE, A., ZHQNG,Q., F E ~ A N T E I.,, et TRIGGEE,D. 3. 1989. @a2+channels in chick neural retina cells characterized by I ,4-dihydropyridine antagonists and activators. Can. J. Physiol. Phmacol. 67 : 506-514. On a ca-act6risC He cand calcium sensible A la tension dms les cellules rttiniennes neuronales cultivtes de pussins en Gtudiant les efets des knantiom2res du Bay K 8644 et du 202-791 et d'autres I ,4-dihydropyridines.Ces cellules ont montrk une capture de Ca2+ fonction du temps et de la tension qui a 6tC stimul$e par une depolarisation K+ et inhibh par les bloqueurs de canaux calcium inoganiques Cd" et Co2+. Seule une petite fraction (15% au maximum) de la capture kt6 inactivke par la pr$-dkpl~saeiondes cellules avec 8Q mM & #+. La capture de Cae' a kt$ sensible aux activateurs et antagonistes 1,4-dihydropyridine~des cmaux calcium, Le ($)-Bay K $644 et le (5')-202-791 ont stimulG la capture de ca2+ et le (R)-Bay K 8644, le (W)-202-791 ainsi que la nitrendipine et He PN 200- 1I0 ont inhib6 la capture de Ca2+sthulde par la d6polaisation K+ ou par les activateurs de canaux. La capture stimulke par la dkpolarisation K+ a kt6 inhibee de 90%, mais la capture stlmulde par B'activateur a kt6 compl$tement bloquee pa- les antagonistes 1,4-dihy&opyridines. Les puissances de ces agents en tant qu9i&ibiteurs de capture dn c$+ont Ctt significativement plus faibles que Ies affinitds de fixation dans les prkparations de membranes des d m e s cellules ou que leurs affinitks p acologiques et de fixation dans le muscle lisse vasculake. La capture de 4"a2+ induite par le (3)-Bay K 8644 ou par la deplksation K+ n'a pas ttC observde &ins une culture de cellules gliales. La ['~lnitrendipineet le ['HIPN 200-110 se sont fixis aux prbpaations mernbranaires des cellules en accord avec la presence d'un seul type de site de fixation de haute affhitd. Le ['HIPN 280-1 10 s'est fix6 avec une plus haute affinitC (KD = 7,09 k 8,W X 10s" M) que la ['Hlnitrendipine (KD = 4'10 +. 0,92 x 10-lo M), mais les valeurs de B,,, on Ctk similaires pow les deux ligands (98,9 k 4'1 et 99,4 k 4'58 fmol/mg de protdine, respectivement). La difftrence entre les activitCs p h m a cologiques et de fixation des ligands antagonistes ne semble pas Ctre due A la prdsence des canaux ca2+ insensibles aux 1,4-dihydropfldines, mais pounait Ctre associde h l'incapacitk de ces agents d'atteindre un Ctat d9inactivationde haute affinitd dans la cellule intacte ou A la prCsence & groups disthcts de sites de fixation. Cette ddiiff6~ncemarqu6e entre les affinitks n'existe pas pour les Bigands activateurs examinks. Cette Ctude confime la presence de canaux Ca" sensibles aux 1,4-dihydmpyridines, dCpndamts de la tension, dans les cenules rktiniemes neuronales des poussins. [Traduit par la revue]

Introduction Voltage-sensitive calcium channels are a major mechanism for the control of the plasmalemmal e a 2 + movements in excitable cells. Several structural classes sf organic ligands , including the clinically available vempamil, Qiltiazem, and nifedipine, are available that act at specific sites to modulate ca2+channel function (Tiggle and dank 1984a, 19846;Janis et al. 1887). Of particular importance is the 1,4-dihy&spgr~dine ' ~ u t h o for r comesponderace. Printed in Canda i Imprim6 au Canada

class containing both antagonists and activators that serve as useful probes to elucidate the functions and structure of the It-type calcium channel. Structure-activity analyses and the close correlation sf phmacological md receptor binding activities in several tissues indicate that the binding sites for these compounds represent functional calcium channels (Janis and Triggle 1984; Thiggle and Janis 1984a, 1987). Ca2+ is known to be involved in several neursnal functions including the control of neurotransmitter release, regulation of membrane excitability, and neuronal growth m d development

WE1 ET AE.

569'9


(Miller 1986, 1987). Influx of c a 2 + ions through the voltagesensitive calcium channels is thus an important process by which these and other neuronal ca2+-dependent functions may be regulated. However, neuronal calcium channels are frequently insensitive or apparently insensitive to calcium channel antagonists and activators (Miller and Freedman 1984; Rampe et al. 1984; Suszkiw et al. 1986), although specific high affinity binding sites for the channel ligands have been identified in neuronal tissues and have identical or similar binding properties to those found in smooth m d cardiac muscle where functions are well demonstrated (Bolger et al. 1983; Janis et al. 1984.~).Such discrepancies between binding and phamacological activities have presented a major problem in determining the functions of neuronal calcium channel binding sites. However, functional effects of Ca2+ channel ligands have been reported in a number of neuronal systems both in vitro and in vivo. Thus, 1,4dihydropyridine sensitive Ca2+ uptake and neurotransmitter release in brain synaptosomes have been reported by some authors (Turner and Goldin 1985; Creba and Karobath 1986; White and Bradford 1986). Furthermore, sensitivity to 1,4dihydropyridines and other Ca2+ channel ligands has been demonstrated in a number of cultured neuronal cell lines (Toll 1982; Takahashi and Bgura 1983; Freedman et al. 1984; Perney et al. 1986; Thayer et al. 1986), and behavioral responses following in vivo administration have also been described (Bolger et al. 1985). The insensitivity of some neurons to c a 2 + channel ligands may be due to a number of factors including cell differentiation and the presence of multiple types of voltage-sensitive calcium channels (McCleskey et al. 1986; Creba and Karobath 1986; Perney et al. 1986). Because of the importance of Ca2+channels to neuronal function and the therapeutic significance of c a 2 + channel drugs, it is important to understand the basis for the neuronal effects, or lack thereof, of ca2+ channel ligands. The (S)- and (R)-enantiomers of Bay K 8644 (2,6-dimethyl3-carbornethoxy-5-nitro-4-(2-t~fluoromethylphenyl1,4-dihydropyridine) and 202-79 1 (2,6-dimethyl-3-carbo-methoxy-5nitro-4-(2,1,3-benzoxadiazol-4-yo1,4-dihydropyridine) have been reported to have the opposing effects of cdcium channel activation and antagonism, respectively (Franckowidc et al. 1985; Hof et al. 1985; Kongsamut et al. 1985). We have characterized previously the actions of these enantiomeric pairs in vascular and nonvascular smooth muscle preparations (Wei et al. 1986). In the present study we have used these enantiomeric pairs to characterize by ph acslogicd and radisligand binding methods the calcium channel in chick neural retina cells, a preparation reported to possess l,4-dihydropyridine FIG. 1. Cultured chick neural retina cells. ( a ) The regular neuronal sensitive calcium channels (Ray and Clark 1985). culture in horse serum containing medium, 4 days. (6) The 21-day

Materials md methods Cell cukdl~re Neural retina cells were cultured by combining the methods of Sheffield and Moscona (1970) and Brackenbury et al. (1977). Neural retinas were removed from the eyes of 10-day-old chick embryos and dissected free from the surrounding tissues. The retinas were then incubated with 0.5% Qpsin (type II crude, Sigma Chemical Company, St. Louis, MO), approximate1 0.25 mL r retina at 37'C for 28 inin. Trypsin was prepared in a CaL- and MgK-free saline solution of the following composition (mM): NaCl, 127; KC I , 4.56; KW2PB4,8.44; NaHC03, 4.16; Na2HPQ4,0.63; glucose, 5.56; and Wepes, 20; pH 7.4. After trypsinization the tissues were gently washed 5 times with minimum essential medium (MEM) with Earle's salt (GIBCO Labsrabries, Grand Island, NY), and dispersed into single cells by pipetting in and out of a Pasteur pipette. The cell suspension was centrifuged at

culture in fetal bovine serum containing medium before the removal of neurons. ( c ) The glial culture of 21 days in fetal bovine serum containing medium after washout of neurons. 150 X g for 8 min, and the pellet was resuspended in culture medium that contained MEM, 80%; horse serum, 18%; fetal bovine serum, 10%;L-glutamine, 4 M;penicillin, 180 units/mL; streptomycin, 1 0 0 p@mL; and arnphotericin B, 258 ng/mL. Viability, which was normally wound 9 5 8 , was assayed by Trypan blue exclusion. Cell density was adjusted to 4-5 million viable cells per millilitre of medium. Cells were plated in Cornin 35-mm plastic tissue culture dishes (2 mL per dish) for studying CaF+ influx and in 100-mm dishes (15 mL per dish) for radioligand binding studies. Culture dishes were maintained under an atmosphere of 5% C 0 2 and 95% air at 37°C for 4-6 days before use. The typical msfphslogy of such cultured cells is shown in Fig. 1. The majority of our studies used these mixed cultures.


508

CAN. J . PHYSIBE. PHARMACOL. VOL. 67, 1989

Glial cells were cultured according to the method of Adler et al. (19'79, 1982). Cells were prepared as described above and maintained for 3 weeks in a medium containing only fetal bovine semm but not horse semm with a medium change every 3-4 days. The glial cells formed a confluent "carpet" on which was attached a network of neurons bearing processes. The neurons were washed out thoroughly with a gentle stream of medium using a Pasteur pipette, producing a culture containing only glial cells (Fig. 4). Cultures consisting of neurons only were prepared by plating the cells on dishes precoated with polyomithine (0.1 mg1mL) and growing them in a medium containing horse serum only (Adler et al. 19'79, 1982). Under these conditions the neurons (Fig. 1) have a lifetime of 3-6 days. - [ 3 ~ ]200-1 ~ ~ 10 binding Membrane preparations for radioligand binding were made from the cells grown in 100-mm culture dishes. Medium was removed anad the culture was rinsed with ice-cold Tris buffer (50 KIM,pH 7.2). The cells weE scraped from the plate amd homogenized by six passes of a motor-driven (TRI-R-stirrer, setting 4) glass-Teflon pestle homogenizer (nominal clearance, 0.13-0.18 m). The homogenate was centrifuged at 45 000 x g for 45 raain, and the pellet was suspended in ice-cold 58 mhl Tris buffer for binding studies at a concentration of 150-200 kg protein15 mL assay volume. Protein was determined by the method of Bradford (1976). The binding assay was conducted essentially by the method established previously in our laboratory (Bolger et d. 1983). Membrane protein was incubated with various concentrations of ( + ) - [ 3 ~ ] 200-1 ~ ~ 10 in 5 d of Tris buffer (50 M,pH 7.2) for 98 min at 25°C. Nonspecific binding was determined by incubation in the presence of M nonlabeled (+)-PN 200-1 10. Incubation was terminated by rapid filtration under vacuum through Whatman GFlB filters followed by two washes (total 10 d ) with ice-cold Tris buffer using a cell harvester (model M-24R, Brandel Instmments, Gaithersberg, MD). Radioactivity was determined by liquid scintillation s ectrometry. ( + ) - [ 3 ~ ] 200P ~ 110 at a concentration of 1.14 x lo-' i? M was used for competition binding studies.

(+ )

"'cd' uptake Ga" uptake studies were carried out at 30°G by replacing the culture d i u m with 2 dbuffer contabing 4"a2f (0.4-0.5 $ilpmol Ca2') (1 Ci = 37 GBq). Uptake was allowed for 15 s unless otherwise indicated and terminated by rapidly removing the "ca2+ -containing buffer by aspiration md washing the culture with ice-cold resting buffer 3 times within 15 s. Cells were extracted overnight with 1 IHlL 0.5 M NaOH, and radioactivity was determined by liquid scintillation counting. Prokin was assayed by the method of Lowry et a%.(1951) using bovine serum albumin as the standard. The resting buffer contained the following (d): NaCB, 132; KC], 5; CaC12, 1.2; MgC12, 1.3; glucose, 10; and Tris, 25; pH 7.2. Equimolar substitution of NaCl with KCI was made when elevated K ' concentrations were desired. In Na+-freebuffer NaCl was replaced with equhnolar amounts of choline chloride. Triplicate measurements were made in each experiment. uptake, cells were preTo observe the effects of a drug on 4"a'+ incubated with the dmg at the indicated concentration in the culture medium for 36) min, and "ca2+ uptake was performed in the presence of the same concentration of the h g . In separate experiments it was found that partial deplaization with 25 mM K+ was optimum for calcium channel activation by the 1,4-dihydropyridine Ca2+ channel activators and this concentration was empIoyeQ in experiments involving the activators. Inactivation of the ca2+uptake process was studied by predepolarizing the cells with K+-enriched buffer for 5, 10.20, or 30 min. Control experiments were established in which cells were incubated in the same buffer of n o m d K+ concentration for the same amount of time.

England Nuclear ((Boston, MA). Tissue culture medium, L-glutamine, semm, and antibiotics were obtained from GIBCB laboratories (Grand Island, NY). The enantiomers of Bay K 8644 were the generous gift of Dr. A. Scriabine (Miles Institute for Preclinical Pharmacology, New Haven, CT) and the enantiomers of 202-791 were the generous gift of Dr. P. R. Hof (Samdoz, Basle, Switzerland). Analysis sf data Data were processed using an IBM personal computer. P h m a c s logical data and significance tests were analyzed using the p h m a c o logical programs of Tallarida and Murray ( 1984 ) . Radioligand binding data were analyzed with an iterative curve-fitting program (BDATA, EMF Software, Knoxville, TN).

Results R+ cfeaolarization stirnu!abed 4 5 ~ a 2 uptake + 4 5 ~ a 2influx + into the neural retina cells was time dependent (Fig. 2). The influx tended to plateau at about 1 min, and at 15 s the uptake was 60%of that at 1 mim. The 15-s uptake was used in all other experiments. Substitution of Na+ by choline did not affect the uptake at any time up to 1 min (Fig. 2). 4 5 ~ a 2uptake + was stimulated by increasing concentrations of K + . Concentrations of K+ below 10 mM did not stimulate uptake compared with the resting level at 5 mM. The EG50 value for K+ stimulation was 34.6 mM and maximum uptake was observed at $8 mM KCl. As shown in Fig. 3, 45Ca2+uptake at $0 mM K+ was 2.2 1 + 0.I I nmol /mg protein, which is some fivefold greater than uptake at 25 + was blocked The K+ depolarization-induced 4 5 ~ a 2uptake by the inorganic divdent cations, cd2+ and Co2+ (Fig. 3). When included in the uptake buffer, cdZ+( l W 4 M) and coZ+ (1W3 M) inhibited ca2+ uptake (80 mM K+ stimulated) by 74 and 6996, respectively. Reincubation of the cations with the cells did not increase the extent of inhibition. Predepolarization of the cells with elevated K+ ($0d) for 5,10,20, or 30 min produced an approximate 15% loss of Ca2+ uptake compared with control. This inactivation of uptake did not increase with time after 5 min and was not different when cells were predepolarized in ca2+-containing or ca2+-free buffer. Potentiation of C d + uptake by C d + channel activators At the resting concentration of KC1 (5 ), the ~ a ' +channel activators (S)-Bay K 86-44 and (S)-282-791 did not increase ca2+ uptake into the chick neural retina cells. At elevated KC1 concentrations, however, significant potentiation of CaZ+ uptake by the activators could be observed. Optimum potentiation was obtained at 25 mM KCl; under this condition, (S)-Bay K 8644 (10-%) and (S)-202-791 (1W5 M) enhanced Ca2' uptake by 7- and 6.7-fold, respectively (Fig. 3). The doseresponse relationships for (S)-Bay K 8644 and (S)-202-791 are shown in Fig. 4; the ECm values were 1.18 x and 1.18 x M, respectively (Table 1).

+ antagonists Inhibition of c a 2 + uptake by ~ dchannel The inhibitory effects of Ca2+ channel antagonists on CaZ+ uptake into the chick neural retina cells were determined against K+-stimulated uptake and activator-stimulated uptake. (R)-Bay K 8644 (Fig. 5a) dose-dependently inhibited 80 d Ktstimulated caZ+uptake and M (S)-Bay K 8644-stimulated ca2+uptake, with ICSovalues of 4.00 x and 5 -23 x Sources of ckemicaIs and drugs M, the K+-stimulated ca2+ uptake M, respectively. At [3~]~itrendipine (2,6-dimethyl-3-c~b~thoxy-5-cxbometRoxy-4was inhibited by 68% and the @)-Bay K $644-stimulated uptake (3-nitmphenyl)-l,4-dhydropyridine;specific activity, 79.5 C i l m o l ) , by 7395, but higher cancentratisns of this drug could not be [ 3 ~288-1 ] 10 ~ (specific ~ activity, 70.0 C i l m o l ) , and 4 5 ~ a "(CaC12; reached because of solubility limitations. At concentrations specific activity, 23.8 mCi/mg) were p u ~ h a s e dfrom DuPont-New


WE1 ET AL.

FIG.2. Time course of 4 5 ~ a 2uptake + into chick neural retina cells. %he uptake was measured in both Na+-containing buffer (open bars) and &+-free buffer (hatched bars). Bars indicate standard error of mean (n = 6). The maximum uptake at 60 s is 3.68 nmol/rng protein.

K ' ( ~ M ) 80 Control

80 Cd2*

80 b2* ~ O - ~ R ~A o - ~ M

25 25 25 25 Control (S18644 (S18644 (S1202-791 IO-'M 40% IO-~M

FIG.3. Chmcteristics of @a2+uptake into chick neural retina cells.

De larimtion ($0 HglM K+) stimulated ca2+ uptake is blocked by Cd * and Go2+,and at 25 HglM K+ (S)-Bay K $644 and (S)-202-791

P"

stimulate @a2+uptake. Bars indicate standard error of the mean ( n = 6-9).

M, (R)-Bay K $644 inhibited K+lower than 3 x stimulated Ca2+ uptake more than (5)-Bay K 864-stimulated ca2+ uptake, but this difference in sensitivity was not seen at higher concentrations. Similarly (R)-202-79 1 (Fig. 5 b) inhibited both 80 ffnh.I K+-stimulated and 10-% (5)-202-791-stimulated caZ9 uptake, with IC50 values of 6.23 x and 2.82 X M, respectively. (R)-202-791 at low concentrations (below 3 x M) was more potent against K+ depolarization-induced uptake than against the activator-induced response. Moreover, the activator-induced ca2+ uptake was completely blocked by (R)-202-791 M), while the depolarization-induced response was inhibited by a maximum of 91% (significantly different, g < 0.01). Another calcium channel antagonist, (+)-Phi 200-110 inhibited both K+ depolarization- and M (9-Bay K 8644-stimulated Ca2+ uptake (Fig. 6), with ICSo values of 2.21 x and 3.16 x M, respectively. Similar to (R)-202-791, (+)-PN 200-1 10 completely blocked the activator-stimulated uptake but inhibited depolarizationstimulated uptake by 98% (significantly different, p < 0.05) at maximum. However, unlike (R)-202-79 1, (+)-PN 200- 1f 0 did not show any significant difference in potency against depolarization- or activator-induced "'ca2+ uptake. "'ca2+ uptake was also blocked by nitrendipine with an ICSoof 8.73 x 1OA8 M (Table 1). Antagonist effects of (R)-202-791 and nitrendipine were also determined by allowing cells to equilibrate with the drug under depolarizing conditions. (R)-202-791 M) was preincubat-

(SI-202-791 (MI R e . 4. Potentiation of &la2+uptake into chick neural retina cells by ( a ) (&')-BayK 8644, and ( b ) (5)-202-791. Effects were measured in a 25 HglM K+ buffer at 15 s. The maximum @a2+uptake (100% response) represents 3.05 2 0.24 runol/mg protein for @)-Bay K 8 6 4 and 2.96 -g 0.18 mol/rng protein for (S)-202-791, respectively. Bars indicate standard error of the mean ( n = 5).

ed with cells in culture medium for 20 min and then in buffer containing 25 or 80 mM K+ for 10 min; this treatment did not change the percentage of inhibition of the deplarizationstimulated Ca2+ uptake by (R)-202-791 (Fig. 7). When 4 5 ~ a 2 9 uptake stimulated by K9 (80 mM) was measured over 15 s, 1 min, or 5 min, the extent of inhibition by nitrendipine M) was not significantly different (from 53 to 6l%), although the amount of uptake approximately doubled between 15 s and 5 anin (Fig. 8). C d + uptake into glial cells ca2+ uptake in glial cells was measured under the same conditions as those employed with neurons in Nat-containing buffer. As shown in fig. 9, the uptake was not stimulated either by Kf (25 and 80 mM) induced depolarization or by the Ca2+ channel activator (5)-Bay K 8644 M) in the presence of 25 ffnh.I K+, which is the optimal condition for the action of the activators in the neurons. The antagonist, (R)-Bay K 8644 (1os7 M), did not inhibit Ca2+ uptake under depolarizing conditions.

Specific binding ~f[~?l]nitrendipineand ['H]PN 200-1PO The membrane preparation of the chick retina cells bound [3H]nitrendipine specifically (Fig. 10a). Specific binding was

CAN. B. PHYSIOL. PHAWMACOL. VOL. 67. 1983

TABLE1. The effects of 1,4-dihydropyridines on 45~a'+uptake and [ binding in chick neural retina cells

ca2+uptake

3

~ 200-1 ] ~10 ~

Radioiigand binding EC~~/K1


Ec5~

KI(M)

1650'

n~

(I~~o/KI)

NOTE:Values axe means (95% confidence limits); n = 5-7. "Against K+-induced uptake. bAgaimt activator-induced uptake.

55% of total binding at 4.6 x 10-I' ha and 37% at 5.75 x 10-" M [3H]nitrendipine, and revealed a single class of binding site with a & value of 4.18 + 0.92 x 10-'" M, a B,,, of 99.43 +

6.58 fmollmg protein, and a Hill coefficient of 1.02 + 0.8%. The binding properties of [ 3 ~ 2 0]- 1~10 (Fig. ~ 10b) were since [ 3 ~200] 110 ~ different from those of [3~~nitrendipine, had lower level of nonspecific binding and higher affinity. However, the B,,, for ['HJPN 200-1 10 was not significantly different from that for ['Hlnitrendipine. Specific binding rangedfrorn81%at5.68 x 10-"Mto64%at5.11 X 10-"M ['HIPN 200- 110, and revealed a single class of binding site with a K g value of 7.09 + 0.90.x 18-" M, B,, of 98.9 + 4.1 fmollmg protein, and a Hill coefficient of 1.03 + 0.02. The binding signal in mixed cultures of neurons and glia may be attributed predominantly to the neuronal elements, since mea] ~10 in surement of the binding site density of [ 3 ~ 200-1 membranes from glial preparations was much lower, 19.4 + 4 .0 fmollmg protein, but with the same Kg value of 5.98 k 0.17 X M. Inhibition of specfle ['H]PN 200-1 10 binding (S)-Bay K 864-4, (R)-Bay K $644, (S)-202-791, and (R)-282791 competitively inhibited the specific binding of ['H]PN 200118 to the rnicrosornd preparation from chick neural retina cells. The K1 values and pseudo-Hill coefficients, the latter ranging from 0.83 to 0.94, are sumarized in Table 1. These affinities differ markedly from those determined from the 45Ca2+uptake studies (see Discussion).

This study as well as the previous report by Ray and Clark (1985) show that the cultured chick neural retina cells are useful preparations for studying neuronal CaZ+ channels. The existence of voltage-sensitive Ca2+ channels in this preparation is ~ supported by the K+ concentration dependence of 4 5 ~ a 2 + uptake, the sensitivity of the uptake process to the inorganic ions cd2+ and Co2+, the sensitivity of "ca2+ uptake to the 1,Cdihy&opypidine ca2+ channel activators and antagonists, a d the presence of specific high affinity l,4-dihydropyridine binding sites in membrane preparations. The characteristics of the 1,4-dihydropyridine binding sites are very similar to those in smooth and cardiac muscle where binding and functional properties have been comlated (Bolger et al. 1983; Janis et al. 1984a, 1984b; Triggle and Jmis 1887). A major observation in this study is the significant discrepancy between the binding and ph acologic activities of the 1,4-dihydropyridine antagonists studied. As listed in Table 1 and depicted in Fig. 11, the binding affinities do not show a I :1 correlation with the ph acologic affinities for blocking 4 5 ~ a 2 'uptake. This discrepancy between binding and p h m a cologic activities is most obvious with the antagonist 1,4dihydropyridines and is very much reduced, to less than 10-fold, with the activator %,4-dihydropyridines(Table 1). However, there is essentially a 1:1 correlation between the binding activities for both activator and antagonist 1,4dihydropyridines in membrane preparations from retinal neurons and vascuHar smooth muscle (Fig. 12). Furthermore,


WI ET AL.

FIG.5. Inhibition of 5@la2+ uptake into chick neural retina cells by the (R)-enantiomers of Bay K 8644 and 202-791. (a) Inhibition of 80 dv! K+-stimulated ( a ) and l W 7 M (S)-Bay K 8644-stimulated (A) M are uptake by (R)-Bay K 8644. Data points below 3 x significantly different. ( b ) Inhibition of 80 mh4 K+-stimulated uptake (@) and lo-% (S)-202-791-stimulated uptake (A) by (I?)-202-791. Data points below 3 X M are significmt8y different. Bars indicate standard error of the mean (n = 5).

FIG. 7. Inhibition of "5@la2+uptake stimulated by 80 dv! K+ and measured at 15 s into chick neural retina cells by (R)-202-791 M) as a function of predepolarization. Open colums represent control uptdce, and the numbers above the (R)-202-791 columns indicate percentage of inhibition. Bars indicate standard error of the mean (n = 4).

Time $OmM#* N~BP. (x07

5 min

4 min

+ -

+

+

+

9

-

+

FIG. 8. Semsitivity of 4 5 ~ a 2uptake + stimulated by 80 mh4 K+ to nitrendipine ( l r 7 M, hatched colums) as a function of time in chick neural retina cells. The open columns represent control uptake, and the numbers above the hatched columns indicate percentage of inhibition. Bars indicate standard error sf the mean ( n = 4).

~ ' ( r n ~ )5

Drug

FIG. 6. Inhibition of 80 mM K+-stimulated (B) and M (S)-Bay K 8644-stimulated ( a ) 45~a'*uptake into chick neural retina cells by (+)-PN208-110. Bars indicate standard error of the mean (n = 5).

M)

!5 s + + - +

80

80 (19)BK

25

25

25

(S)BK

(S)BK+(W)BK

FIG. 9.Totd 4 5 ~ a 2uptake + into chick retinal glial cells. (S)BK is 1W7 M (9-Bay K 8644. (R)BK is M (R)-Bay K 8644. Bars indicate standard error of the mean (n = 4).

5 12

CAN. I. PHYSIOL. PHARMACOE. VOE. 67, 1989

60

80

400


g protein

6

7

8

9

10

-Log K t (Binding, Neuron)

FIG.12. Correlation between the binding constants of the enantiomers of Bay K $644 and 202-791 in chick neural retina cells and those in rat tail artery ( h mWei et al., 1986). The solid line represents linear regression and the broken line represents unit slope.

I./

B (frnollrng protein)

FIG.10. Specific binding s f (Q) [3~]nitrendipineand ( b ) ['HIPN 200-110 as a function of increasing concentration. Insets are the Scatchard plots of the corresponding data. For [3~]nit9-endipine,KD was 4.18 iZ 0.92 X lo-" M and B,, was 99.43 iZ 6.58 fmol/mg protein, m = 4; for ['HIPN 200-1 10, KD was 7.09 ? 0.90 X 1 0 - ~ ' M and B,, was 98.9 2 4. B fmol/rng protein ( m = 5).

-Log KI (Binding, Neuron)

FIG. I 1. Correlation of radioligand binding and @a" uptake data in chick newd retina cells. The -log IC50 values of (R)-Bay K $644, (R)-202-791, nibendipine, and PN 200-110 for inhibition of K+ &polarization-induced @a2+uptake are plotted against the -log KI values for inhibition of ['H]PN 2QM)-1I0binding. The solid line represents linear regression and the broken line represents unit slope.

despite the quantitative differences in activity measured in binding and phmacologic experiments, the rank orders of activities remain constant for activators, (S)-Bay K $644 > (S)-202-791, and differ only slightly for antagonists, PN 200-1 10 > nitrendipine > (R)-202-791 > (R)-Bay K $644. Although close correlations between binding and ph logical activities of Ca2+ channel drugs have been established fm both vascular and nonvascular smooth muscle (Bolger et al.

1983; Yousif and Triggle 1985; Wei et al. 1986), significant discrepancies occur in other systems including cardiac muscle and neuronal preparations. In principle, a variety of factors may underlie such discrepancies (for reviews see Tkggle and Janis, 1987; Janis et al. 19848). The existence of ea2+ channel of the L,T, and N categories (Miller 1985, 1986, 1987; Nowycky et al. 1985a, 1985b; McCleskey et al. 1986) will be owe important determinant. These subtypes of channel differ in their electroacologic properties. physiologic, ion permeation, and ph The L-type channel is sensitive to the 1,4-dihydropyridines and other existing CaZ+ channel ligands (Nowycky et al. 1985a, 1985b). The differential distribution sf discrete channel types in the cell body and processes will add a further level of selectivity (Miller 1987). Our data show that in the chick neural retina cells approxi+ is mately 90% of the K+ depolarization-induced 4 5 ~ a 2uptake blocked by the 1,4-dihydropykdine antagonists and that Ca2+ channel activator-induced uptake is completely blocked by these antagonists. These results indicate that K+ depolarization may activate more than one type of CaZ+channel in the neural retina cells, but that the 1,4-dihydropyidine sensitive L type of channel is dominant. m e e l l preparation routinely used in the present study contained a mixed population of glial cells and neurons (Adler et d. 1979, 1982). However, the preparation containing only glial cells did not exhibit K+ depolarizationsensitive ca2+uptake under the same conditions as employed in the neuronal culture. Furthermore, the binding site density of 1,4-dihydropyridine sites is very much less in the glial preparations. Such results suggest that the presence of glial cells is not likely to contribute to the observed discrepancies between binding and pharm~cology. Furthemore, depolarization+ is not altered when Na+ is replaced by induced 4 5 ~ a 2uptake choline thus indicating that participation of a Nat:Ca2+ exchange process is unlikely (Blaustein and Obom 1975; Wampe et al. 1986). If it is accepted that an L type of Ca2+ channel dominates this preparation, then the discrepancies between binding and pharmacologic activities may have several origins, alone or in combination. C o m p ~ s o n of s affinities may reflect interactions at different channel states whereby drug affinity varies according to channel equilibria between resting, open, or inactivated states (Smguinetti and Kass 1984; Hondeghem and Katzung


WEI ET AL.

1984; Bean 1984; Kokubun et al. 1986). According to this modulated receptor hypothesis, electrophysiologic evidence suggests that 1,4-dihydropyridine ligands bind with highest affinities as activators or antagonists to the open and inactivated states, respectively, of the c a 2 + channel (Hess et al. 1984; Sanguinetti and Kass 1984; Bean 1984; Sanguinetti et a1. 1986). Thus the high affinity binding interactions measured in the membrane fractions, neuronal and nonneuronal , likely represent interactions with the inactivated channel state (Bean 1984; Hess et d. 1984; Kokubun et al. 1986). State-dependent interactions of the channels with drugs may also be reflected by the observations that the (R)-enantiomers of Bay K 8644 and 282-79 1 at lower concentrations were more effective in inhibiting K+ depolarization-stimulated Ca2+ uptake than activatorstimulated ca2+ uptake. Similar observations were made by Enyeart et al. (1987) in the pituitary GH4Cl cell line where a number of c a 2 + channel antagonists were less potent against Bay K 8644 responses than against K+ responses. The lower affinities of 1,4-dihydropyridines derived from the pharmacologic experiments may reflect interactions of the antagonists with lower affinity states, resting or open, of the channel or interactions with the presumed high affinity inactivated state, the formation of which is kinetically unfavoured. Suppart for the latter view arises from the apparent inability or slowness of the Ca2+ channels in the intact neural retina cells to become inactivated during the course of the experimental protocols. Thus, constant depolarization by K+ produces only a 15% loss of induced c a 2 + uptake over 30 min; this stands in marked contrast with, for example, synaptosomes , where inactivation is very rapid and significant (Nachshen and Blaustein 1980, Nachshen 1986) and also with PC 12 cells (DiVirgilio et al. 1987). Alternatively, it may be argued that the 15-s depolarization over which 45Ca2+ uptake was routinely measured did not permit adequate time for 1,4-dihydropyridine antagonist equilibrium. This view is not supported by the observations showing that antagonist sensitivity was not significantly different following 60 s, 5 min, or 10 min deplarization and incubation with drug and c a 2 + uptake for 15 s, 1 min, and 5 min (depicted in Figs. 7 and 8). The absence of any significant discrepancy between binding and pharmacological activities for the two activator 1,stdihydropyridine molecules studied may reflect the suggestion by Williams et al. (1985) that the affinities of activators are relatively independent of membrane potential. Our previous studies with these activators in smooth muscle preparations also revealed a good accord between binding and pharmacologic activities similar to that reported here (Wei et al. 1986). Finally, the possibility must be acknowledged that the major discrepancy between binding and pharmacologic affinities for the antagonists and the significantly smaller discrepancy for the activators may arise because of the existence of discrete classes of b h h g sites for activator and antagonist 1,4-d&ydropyridhes. Evidence to support this has been advanced by Kokubun et al. (1986) and Lee et al. (1987). It is possible that the high affinity antagonist binding sites are not related to lower affinity sites at which pharmacological antagonism is exerted. Further work to explore this possibility is continuing using radioligand binding to intact functional neuronal cells.

Acknowledgements This work was supported by grants from the National Institutes of Health (HE 16803, HEBAH 31 178),

5 13

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