Stereoselective and non-stereoselective effects of D 600 (methoxyverapamil) in smooth muscle preparations

European Journal of Pharmacology, 76 ( 1981 ) 67-72 Elsevier/North-Holland Biomedical Press

67

STEREOSELECTIVE AND NON-STEREOSELECTIVE EFFECTS OF D 600 (METHOXYVERAPAMIL) IN S M O O T H MUSCLE PREPARATIONS K. JIM, A. HARRIS, L.B. ROSENBERGER and D.J. TRIGGLE *

Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo, Buffalo, N. K 14260, U.S.A. Received 14 May 1981, revised MS received 27 August 1981, accepted 7 September 1981

K. JIM, A. HARRIS, L.B. ROSENBERGER and D.J. TRIGGLE, Stereoselective and non-stereoselective effects of D 600 (methoxyverapamil) in smooth muscle preparations, European J. Pharmacol. 76 (1981) 67-72. Mechanical responses in guinea-pig ileal longitudinal, rat vasa deferentia and rat portal vein smooth muscle strips elicited by receptor activation (muscarinic or a-adrenergic) or by K + depolarization were blocked by D 600 (methoxyverapamil). Stereoselectivity was observed with the ( - ) - e n a n t i o m e r being more potent than the (+)-enantiomer (the ratio varying from 6 to 180). The tonic (slow) component of response was more sensitive than the phasic (fast) component. D 600 competitively blocked binding of ( - ) - [ 3 H ] Q N B and [3H]WB 4101 to muscarinic and a-adrenergic receptors respectively, but in marked contrast to the effects on mechanical responses antagonism of ligand binding was non-stereoselective. It is suggested that stereoselectivity of action of D 600 may be a useful criterion to distinguish between its several sites of action.

Muscarinic receptors D 600

D 600 stereoisomers Adrenergic receptors

Receptor binding Rat vas deferens

1. Introduction

D 600 (methoxyverapamil, a-isopropyl-a-[(Nmethyl-N-homoveratryl)-y-aminopropyl]-3,4,5trimethoxyphenylacetonitrile) and its analog verapamil have achieved some prominence as agents which interfere with Ca 2+ movements in excitable tissues (Fleckenstein, 1977; Rosenberger and Triggle, 1978; Triggle, 1981). This activity contributes to their therapeutic utility in certain cardiovascular disorders (Henry, 1979; Ellrodt et al., 1980). At concentrations of 1 0 - 6 M and less D 600 reduces cardiac contractility by inhibiting the voltage-sensitive slow inward Ca z+ current and has little effect on the fast tetrodotoxin-sensitive Na + current (Fleckenstein, 1977; Kohlhardt et al., 1972; Biiyer and Ehara, 1978). D 600 has been regarded * To whom all correspondence should be addressed: Department of Biochemical Pharmacology, 317 Hochstetter Hall, State University of New York at Buffalo, Buffalo, New York 14260, U.S.A.

Ca 2+ antagonists Guinea-pig ileum

Stereoselectivity

Smooth muscle

as a comparatively specific antagonist of slow Ca 2+ channels, which activity is suggested to be the basis of its inhibition of excitation-contraction coupling in many smooth muscles (Fleckenstein, 1977; Rosenberger and Triggle, 1978; Imai, 1979). However, D 600 has also been shown to exert other pharmacological effects including inhibition of Na + currents (B~iyer et al., 1975; Galper and Catterall, 1979), neurotransmitter-receptor binding (Cavey et al., 1977; Blackmore et al., 1979; Fairhurst et al., 1980) and high affinity neurotransmitter uptake (McGee and Schneider, 1979). It is also clear that D 600 is not an effective inhibitor of all Ca 2+ channel-mediated events (Rosenberger and Triggle, 1978; Triggle, 1981). Thus, many secretory processes are sensitive to D 600 only at high concentrations where it is likely that effects other than at a specific Ca 2+ channel are being observed (Haeusler, 1972; Nachsen and Blaustein, 1979). Racemic D600 has been used in the great majority of studies and it is possible that different activities of the two enantiomers contribute to

0014-2999/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press

68

these several pharmacological activities. Accordingly, we have compared the activities of D600 and its enantiomers on excitation-contraction coupling and receptor binding in smooth muscle systems, the guinea-pig ileal longitudinal smooth muscle, the rat vas deferens and the rat portal vein. These smooth muscles were selected because their responses were known to be highly dependent upon extracellular Ca 2+ and to be sensitive to D 600 and related Ca 2+ antagonists (Rosenberger et al., 1979; Triggle et al., 1979; Harris et al., 1980).

2. Materials and methods

Guinea-pig ileal longitudinal, rat vasa deferentia and rat portal vein smooth muscles were prepared for recording mechanical responses as previously described (Rosenberger et al., 1979; Triggle et al., 1979; Harris et al., 1980). Guinea pig ileal longitudinal muscle and rat vasa deferentia were incubated at 37°C in saline of the following composition: NaC1 127 mM; KC1 2.7 mM; CaCI 2 1.8 mM; MgC12 0.88 mM; NaH2PO 4 0.36 mM; N a H C O 3 12.0 mM; glucose 5.5 mM. Isotonic contractions were recorded with the ileal smooth muscle using a light lever (9:1 magnification) under a resting tension of 350 mg (Grass Polygraph 7B, FT03 transducer). Rat portal veins were incubated for isometric recording at 37°C in saline of the following composition: NaCI 118 mM; KC1 4.7 mM; MgCL 2 1.2 mM; K H 2 P O 4 1.2 mM; N a H C O 3 21.5 mM; CaCI 2 2.5 mM; glucose 11.1 mM. Tissues were aerated with 95% 02-5% CO 2 and were initially equilibrated for 60 min with several solution changes. Control maximum responses were generated with cis-2-methyl-4dimethylaminomethyl- 1,3-dioxolane methiodide (CD, 5 × 10 -7 M), a potent and stable muscarinic agonist (Rosenberger et al., 1979), in the guinea pig ileal longitudinal muscle, with norepinephrine (NE; 10-4 M) in the rat vasa deferentia and with norepinephrine (10 -5 M) or KC1 (80 mM, by replacement of NaC1) in the rat portal vein. The means of two such responses elicited at 60 min intervals, with several solution changes between responses, were taken to measure control phasic

(fast) and tonic (slow) responses (Rosenberger et al., 1979; Triggle et al., 1979; Harris et al., 1980). Inhibition of responses to maximal concentrations of CD and N E were determined because the use of these concentrations permits maximum discrimination of the phasic and tonic components of response. However, the effects of (-+)-D600 on the complete dose-response curves to CD and to K ÷ have been noted previously (Rosenberger et al., 1979; Triggle et al., 1979). After determination of control responses tissues were equilibrated with a concentration of D 600 for 10 min and the response to stimulant redetermined. Only one concentration of D600 was employed in any one tissue. IDs0 values for D 600 inhibition of response were determined by probit plots. To determine C a 2+ dose-response curves in a K +-depolarizing medium rat vasa deferentia and guinea-pig ileum were prepared as described and were incubated in Ca 2+ -free saline for 30 min with a change of solution every 5 min, and then in Ca2+-free K+-depolarizing solution (by replacement of 80 mM NaCI by 80 mM KC1) for 10 min. A cumulative dose-response curve to Ca 2+ (0.1 15.0 mM) was then determined. Tissues were then reincubated in the physiologic saline for 60 min with several solution changes and the above procedure repeated with D 600 being present in the 10 min tissue exposure to Ca 2+ -free K + -depolarizing media. Control experiments showed that the more prolonged incubation times did not produce further antagonism. Only one concentration of D 600 was used in any one tissue, pA 2 values for D 600 and its enantiomers were determined by linear regression analysis of the plot of log ( D R - 1 ) against log D 600. [3H]QNB binding was measured in guinea-pig ileal longitudinal smooth muscle by a modification of the method of Yamamura and Snyder (1974) as described by us previously (Aronstam et al., 1979). Tissue was homogenized in 10 vol of 50 mM Tris buffer pH 7.4, centrifuged at 1 0 0 0 × g / 1 5 min, the supernatant recentrifuged at 50000 × g / l 5 rain and the pellet resuspended in buffer. Aliquots of crude membrane (80-110 /~g protein) were incubated with varying concentrations of ( - ) [3 H]QNB (44.5 Ci/mol; Amersham Corp., Arling-

69 TABLE I IDs0 values for D 600 inhibition of smooth muscle responses. System, agonist

Ratio(

ID~o, M × 10 - s

G.p. ileal long., CD Phasic (P) Tonic (T) P/T ~ Rat vas def., NE Phasic (P) Tonic (T) P/T c Rat portal vein. NE Phasic (P) Tonic (T) P/T ~ Rat portal vein, K + Phasic(P) Tonic (T) p/T ~

( - ) - D 600

( + ) - D 600

62 (53-67; 10) 1.0 (0.7-1.4; 10) 62 d

150 (100-190; 10) 180 (90-230; 10) 0.85

2.5 d 180 a

29.5 (8.9-97; 12) 31.9 (9-102; 12) 1.0

139 (34-560; 12) 491 (141-1 900; 12) 0.3

5 15 d

7,1 (2.8-17.6: 21) 0.71 (0.2-2.0; 12) 8,0 d

44 (19-108; 21) 18.3 (5.5-61; 12) 2.4

6.2 d 25.8 a

14.5(4-51; 12) 1,21 (0.4-3.7; 12) 12.0 't

121 (55-266: 12) 20.2 (9,2-44; 12) 6.0 d

8.4 d 16.7 d

):(+)b

Concentration to reduce by 50% the control maximum response; 95% C.L. and number of observations. b Ratio of IDso concentrations for inhibition of phasic or tonic components, e Ratio of IDso concentrations for inhibition of phasic and tonic responses. d Significantly different from unity P<0.05.

TABLE 2 pA 2 values for D 600 inhibition of C a 2 + / K + responses in smooth muscle. System

G.p. ileal long. Rat vas deferens a b c d

pA 2 (slope)

Ratio(-):(+)

(_+)-D 600

( - ) - D 600

( + ) - D 600

8.8-+0.1 (0.90) a 7.7 -+ 0.15 (1.04) a

8.9-+0.1 (0.92) a 8.3 -+ 0.1 (0.66) b

7.9+0.1 (0.63) b 6.7 -+ 0.15 (1.06) a

10 c.d 40 c.a

Not significantly different from unity (P<0.01). Significantly different from unity (P<0.01). Based on 6~8 observations at each D 600 concentration. Ratio significantly greater than unity (P<0.01).

TABLE 3 K I values for D 600 inhibition of [3H]QNB and [3H]WB-4101 binding. System

( - ) - [ 3 H ] Q N B / G . p . ileal long. [3 H]WB-4101/Rat vas deferens

K I, M a ( --+)-D 600

( - )-D 600

6.9---0.9 × 10 6 2.2-+0.84× 10-6

8.4"- 1.0× 10 -6 1.5+0.2× 10-6

a Values reported are means -+ S.E.M. ( n = 5).

+ )-D 600 7.0_+0.9 x 10 -6 1.7-+0.33 × 10-6

70

ton Heights, Ill.) in the presence and absence of D 6 0 0 for 60 min at 25°C. Reactions were terminated by filtration through G F / B filters and the filters washed three times with 6 ml ice cold buffer. Nonspecific binding was defined by the inclusion of atropine (1/~M) in a duplicate set of tubes. Specific binding was > 90% total binding. The dissociation constant of [3H]QNB was 1.4 ÷ 0.06 X 10 l0 M and a Hill plot was linear with a slope of 0.99 consistent with binding at a single set of noninteracting sites. [3H]WB 4101 binding was determined by incubating a crude membrane fraction from rat vasa deferentia with [3H]WB 4101 (24.7 Ci/mmol; New England Nuclear Corporation, Boston). The crude membrane fraction was prepared exactly as described by U'Prichard and Snyder (1979) and the binding protocol, in Tris-HC1 buffer (pH 7.7; 50 mm) was identical. Specific binding, defined as that displaced by phentolamine (10 5 M) was 607.5%. The dissociation constant of [3H]WB 4101 was 3.1 ± 0.5 X 10- tOM and a Hill plot was linear with a slope of 1.0 indicating binding at a single set of sites. D600 and its stereoisomers were a generous gift from Knoll. A.G., Ludwigshafen, FRG. ( - ) - N o r e p i n e p h r i n e as the bitartrate salt was from Sigma.

3. Results

Stereoselectivity of antagonism by D 600 of the mechanical responses induced by agonist (NE or CD) or by K + -depolarization was observed (table 1). ( - ) - D 6 0 0 was found to be the more potent enantiomer, but the observed stereoselectivity was not constant, varying with both the tissue and the component of response. This stereoselectivity, whilst generally modest, was significant and was most pronounced with the tonic component of the guinea-pig ileal response. A similar stereoselectivity was seen in the pA 2 values of the D600 enantiomers determined from Ca 2+ dose-response curves (table 2). The Schild plots for D 600 action in the rat vas deferens are shown in fig. 1. The higher activity was again seen in the guinea-pig ileal longitudinal smooth muscle. Quantitative comparison of the stereoselectivity, as revealed by

2.0-

d:

~.o

0.5

0

I

5

6

I

7

8

9

- 109 (D-600) Fig. 1. Schild plot for antagonism of Ca 2+ responses in K depolarizing media in rat vas deferens. • ( + ) - D 600; O ( ")D 600; • ( )-D 600. Horizontal bars indicate standard error of the mean, n = 6 - 8 .

the pA 2 values is difficult, however, because the enantiomers do not exhibit, as judged by the slopes of the Schild plots, uniformly simple competitive behaviour. However, it is clear that, with only one apparent exception, the phasic component of the norepinephrine response in the rat vas deferens, the ( - ) - e n a n t i o m e r of D 600 is the more active against the Ca 2+ dependent processes summarized in tables 1 and 2. D 600 exhibits both reduced activity and absence of stereoselectivity against two receptor binding processes (table 3). Racemic D 600 and its enantiomers are competitive antagonists of ( - ) [3H]QNB binding to guinea-pig ileal muscarinic receptors and of [3H]WB 4101 binding to rat vasa deferentia a I -receptors. 4. D i s c u s s i o n

Similar stereoselective actions of D 600 (and verapamil) to those reported here have been noted

71 TABLE 4 Some effects of D-600 and verapamil on non-Ca2+-mediated systems. (A) Inhibition of receptor binding Agent +)-D -)-D +)-D + )-D )-D ±)-D ± )-D - )-D +)-D ±)-D + )-D --+)-D

600 600 600 600 600 600 600 600 600 600 600 600

Receptor

Ligand

K i, M

Reference

Muscarinic (g.p. ileum) Muscarinic (g.p. ileum) Muscarinic (g.p. ileum) Muscarinic (rat brain) a-Adrenergic (rat brain) a-Adrenergic (rat brain) a-Adrenergic (rat vas def) a-Adrenergic (rat vas def) a-Adrenergic (rat vas def) Opiate (rat brain) a-Adrenergic (rat brain) a-Adrenergic (rat brain)

[3H]QNB [3H]QNB [3H]QNB [ 3 H]QNB [3H]WB 4101 [3H]WB 4101 [3H]WB 4101 [ 3H]WB 4101 [3H]WB 4101 [3H]Naloxone [ 3H]Clonidine [ 3H]Prazosin

6.9× 10 6 8.4× 10 6 7.0x 10 6 8.8 × 10 --6 1. I × 10 -6 0.9x 10 6 2.2 X 10 -6 1.5 x I0 6 1.7× 10 6 4 X 10 -6 9.3 x 10 6 2.8 x 10 6

This work This work This work Fairhurst et al., 1980 Fairhurst et al., 1980 Atlas and Adler, 1980 This work This work This work Fairhurst et al., 1980 Glossman and Hornung, 1980 Glossman and Hornung, 1980

(B) Inhibition of ionic (non-Ca 2+) events Agent

System

ICs0, M

Reference

( ± ) - D 600 ( + ) - D 600 ( -4-)-Verapamil

Veratridine-stim. Na + uptake (heart and neuroblastoma cells) Veratridine depol, of synaptosomes Na +-dep. neurotransmitter uptake in rat brain

Galper and Catterall, 1979 Nachsen and Blaustein, 1979 McGee and Schneider, 1979

( + )-D 600

Na + current and slow outward K + current: Cardiac purkinje fibers

--3 × 10-6 ~ 5 × 10 s 2 × 10 - 6 1×10 4 ~ I × 10 -6

in cardiac (B~iyer et al., 1975; Ludwig and Nawrath, 1977; Satoh et al., 1980) and slow skeletal muscle (Kaumann and Uchitel, 1976). Both ( - ) - D 6 0 0 and ( - ) - v e r a p a m i l are more potent negative inotropic agents than their corresponding enantiomers. Furthermore, as judged by the different patterns of inotropic and electrophysiological events produced, B~iyer et al. (1975) have suggested that the ( - ) - and ( +)-enantiomers of D 600 and verapamil may act at different sites, the slow Ca 2+ channels and the fast Na ÷ channels respectively. With the data available such a conclusion is not possible for the smooth muscle systems under study. However, there is some evidence to suggest that ( - ) - and ( + ) - D 600 may function in qualitatively and quantitatively different fashion. The data of tablel show that save for the rat vas deferens, ( - ) - D 600 is more effective against the tonic component of response whilst ( + ) - D 6 0 0 does not, save for K + responses in the rat portal vein, show such discrimination. It has been suggested that distinct potential-dependent Ca 2+ channels may generate the phasic and tonic corn-

Kass and Tsien, 1975

ponents of response (Bolton, 1979; Hurwitz et al., 1980). It is possible, therefore, that the higher stereoselectivity exhibited against the tonic component of response, the discrimination by ( - ) - D 600 between the phasic and tonic components and the lack of such discrimination by ( + ) - D 600 reflect the operation of such distinct Ca 2÷ channels. The absence of stereoselectivity by the enantiomers of D 600 against ligand binding to muscarinic and a-adrenergic receptors stands in marked contrast to the stereoselectivity exhibited against Ca 2÷-mobilizing events and is clearly consistent with different sites of action. Since racemic D 600 and verapamil have now been shown to inhibit competitively with a K t range of 1 0 - 6 - 1 0 - 5 M, muscarinic, adrenergic a I and a 2 and opiate receptor binding, as well as to inhibit ionic events including Na ÷ and K ÷ currents (table 4) some caution is clearly necessary in considering the specificity of action of these compounds. This is particularly important when concentrations of D 600 and verapamil are being used that exceed 10 - 6 M.

72

The data that we have reported suggest that stereoselectivity of action may be a useful criterion in distinguishing the actions of verapamil and D 600 at Ca 2 + channel and other events.

Acknowledgements This work was supported by grants from the U.S. Public Health Service (HL 16003 and 2T32GM07145).

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