Chemistry and Physics of Lipids, 62 (1992) 11-17 Elsevier Scientific Publishers Ireland Ltd.
11
Perturbation effect of eight calcium channel blockers on liposomal membranes prepared from rat brain total lipids K a r o l O n d r i a g a, E l e n a O n d r i a ~ o v ~ b a n d A n d r e j Stas'ko c alnstitute of Experimental Pharmacology, Slovak Academy of Sciences, 84216 Bratislava, bFaculty of Pharmacy, Comenius University, Odbojtirov 10, 83232 Bratislava and CFaculty of Chemical Technology, Slovak Technical University, 81237 Bratislava, (CSFR) (Received November 4th, 1991; revision received January 15th, 1992; accepted March 20th, 1992)
EPR spectroscopy of phosphatidyicholine or stearic acid labeled at the doxyl group at the 16-carbon position was used to compare the perturbation effect of eight calcium channel blockers (CB) on overall dynamics/disorder of the hydrophobic part of iiposome membranes prepared from rat brain total lipids at the drug/lipid molar ratio of 1/2. Nifedipine (NIF), nimodipine, niludipine and nitrendipine had a minor effect on the dynamics/disorder of the liposome membranes, whereas the disordering effect of verapamil (VER), mepamil, galiopamil and diltiazem was more pronounced. Concentration dependence of the overall disordering effect of VER on liposomal membranes was found at the VER/lipid ratio >0.02 and for the tranquilliser thioridazine >0.005. VER exerted a disordering effect at the hydrophobic part of synaptosomal membranes at concentrations _>0.32 mmol/l, whereas NIF did not exhibit a disordering effect even at concentrations of 10-20 mmol/I.
Key words: calcium antagonists; membrane perturbation; EPR spectroscopy
Introduction
Calcium channel blockers (CB) play an important role in the excitation-contraction coupling mechanism of cardiac and smooth muscle by inhibiting the membrane influx of extracellular calcium. In addition to the inhibitory effect of VER on Ca 2+ channels, it has a wide spectrum of pharmacological activities not believed to be related to blockade of Ca 2+ channels. Verapamil (VER) (2.3 mmol/1) was found to inCorrespondence to: Karol Ondriag, Institute of Experimental Pharmacology, Slovak Academy of Sciences, 84216 Bratislava, CSFR. Abbreviations:Arnin, apparent inner hyperfine splitting (see Fig. 2); CB, calcium channel blockers; DIL, diltiazem; EPR, electron paramagnetic spectroscopy; GAL, gallopamil; MEP, mepamil; NIF, nifedipine; NIM, nimodipine; NIL, niludipine; NIT, nitrendipine; VER, verapamil; NTP, 2,6-dimethyl-4(2-nitrosophenyl)-3,5-pyridine-dicarboxylic acid dimethyl ester; 16-PC, l-palmitoyl-2-stearoyl phosphatidylcholine labeled at the doxyl group at the 16th carbon positon; S, apparent order parameter; 16-SA, steric acid labeled at the doxyl group at the 16th carbon position; TL, total lipids.
hibit both the early and late potassium outward currents in intact isolated fibers of crayfish muscle membrane [1]. It inhibited the uptake of serotonin, dopamine, norepinephrine and choline into rat forebrain synaptosomes. The concentrations of VER required for 50% inhibition of the four uptake systems were (in/~moFl) 3.2, 18, 32 and 150, respectively [2]. VER (0.175 mmol/1) inhibited the Na+-dependent Ca 2+ uptake system of synaptic plasma membrane vesicles [3]. In some non-specific membrane activities VER was found to be more effective than the dihydropiridine calcium entry blocker nifedipine (NIF). Stolc and Nem~ek [4] studied the effect of NIF and VER on calcium, sodium and two types of potassium voltage-dependent ion channels in the membrane of a single neuron of the rat dorsal root ganglion. They found that NIF had high affinity to the ion channels responsible for the calcium inward current, whereas it did not affect either the sodium inward current or the fast inactivating and the slow non-inactivating potassium outward currents even in the 15-/zmol/l concentration. On the
0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
12
other hand VER was found to be only a nonspecific inhibitor and equipotently blocked all the ionic currents tested. The authors supposed that the effects of VER could be accounted for by some non-specific mechanisms, such as membrane lipid disordering effect. Ru~Efik et al. [5] reported that VER, NIF and nimodipine (NIM) at 1 mmol/1 concentration inhibited Na+-Ca 2+ exchange in rat brain microsomal membranes with relative efficiency of VER > NIM = HIF and Kim et al. [6] found that VER and diltiazem (DIL) (1 mmol/l) inhibited the calmodulin-regulated plasma membrane Ca 2+ pump ATPase, whereas NIF (10 /~mol/l) was ineffective. Hay and Wadsworth [7] studied the local anaesthetic activity of organic calcium antagonists on the rat phrenic nerve. They found that verapamil (ICs0 63.7 ± 21/~mol/1) had 3.0 x the potency of the local anaesthetic lidocaine, whereas nifedipine was without effect. The mechanisms of the non-specific membrane activities of CB are not fully understood. Since some of the membrane activities of VER were observed at millimolar concentrations, we studied the perturbation effect of VER on liposomal and synaptosomal membranes in comparison to other CB.
~
C
H
R&
tl R 2
R3
H~C
H~ H
Name:
R1
R2
R3
R4
Nife~ipine Nimodlplne Nitr~ndipine
H NO 2 NO 2
NO 2 H H
-COOCH 3 -COOCH2CH2OCH3 ~COOCH 3
-COOCH 3 -COOCH(CH3) 2 -COOC2H 5
Niludipine
NO 2
H
-COOCH2CH2OC3H ?
-COOCH2CH2OC3H 7
R2
RI
R3
0CH3
CICH213NC I H2CH2
0CH3
'HC[
84 Name:
R1
R2
R3
R4
VerapaBil Gallopau/1 Mepamll
H H OCH 3
OCH 3 OCH 3 H
OCH 3 OCH 3 H
H OCH 3 H
H~ O C H S ' .H ~
N
~
OOCCH3
3 .HCI
CH 2 'CH2N(CH3)2 Diltiazem
Fig. 1. Chemical formulas of calcium channel blockers.
Materials and Methods NIF, VER, thioridazine and stearic acid labeled at the doxyl group at the 16th carbon position (16-SA) were from Sigma. NIM, niludipine (NIL), nitrendipine (NIT), gallopamil (GAL), were provided by the Inst. for Drug Research, Modra, (~SFR. Mepamil (MEP) was from Research Inst. for Pharmacy and Biochemistry, Praque, (~SFR. DIL was from Lachema, t~SFR. The high purity and pharmacological effects of these calcium antagonists have been reported [1,5,8,9]. The purity of the CB was determined by TLC and LC and their content was determined by LC and UV spectroscopy. The content ofNIM, MEP and DIL was not less than 99% and the content of NIT and NIL was not less than 98%. The purity of GAL was determinated by HPLC and the content was not less than 99%. The chemical structures of the CB are shown in Fig. 1. 1-Palmitoyl-2-stearoyl-phosphatidyl-choline
labeled at the doxyl group at the 16th carbon position (16-PC) was from Avanti Polar Lipids. NTP (2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester) was obtained by illumination of NIF (1 mg/ml ethanol) by daylight for 1 h as described in our previous study [10].
Liposomes Total lipids (TL) from rat brain were isolated according to Folch et al. [l 1]. TL (2 rag) from brain and spin probe (100:l molar ratio) and each particular drug were mixed in chloroform: methanol (2" 1). The solvent was evaporated under nitrogen followed by evacuation. The dry samples were hydrated with 30 ~1 of the buffer (in retool/l), pH 7.4, NaCI 145, KC1 5, MgCI2 1.4, CaCI2 1, Hepes.HC1 5. In order to attain equilibrium of the drugs in the liposomes the samples were subjected to vortex-freeze-thaw cycles several times.
13
Synaptosomal membranes Isolated synaptosomes [12] (3 mg of proteins) were incubated with or without the drug at 37°C for 60 min in 5 ml of the buffer. The samples were centrifuged at 10000 x g for 10 min and the pelleted synaptosomes (50 #1) were mixed with the dry spin probe at the ratio of 15/~g spin probe/3 mg of protein and vortexed for 3-5 min. The samples were further incubated for 30 min at 37°C and transferred into capillaries (1 mm i.d.). Assuming 1/zl of lipids per 1 mg of protein in the synaptosomes [13] and the molecular weight = 775 for TL, it can be calculated that the spin probe/ lipid molar ratio in synaptosomes was 0.01. EPR spectra were measured in the glass capillaries using a Bruker 200 D Spectrometer. Typical instrument settings were: 10 mW microwave power, modulation amplitude = 0.1 mT. To assess the relative efficiency of the drugs in perturbing the lipid membrane, the apparent order parameter S was calculated from the apparent outer (Amax) and inner (Amin) splittings [14] which were directly measured from the EPR spectra of the spin probe as shown in Fig. 2.
_/ _/
/
/ Fig. 2. EPR spectra of the 16-PC probe in TL liposomes. A, control; B, TL/NIF = 2:1 molar ratio; C, TL/VER = 2:1 molar ratio. Spectrum width 6 roT. Temperature 37°C. Arrows in spectrum A indicate Areax determination. Arrows in spectrum C indicate Amin determination.
Sapp = (Amax- B) * 0.5407/C B
= .4min + 1.4 * [1 - (Amax - Amin)/27.25]
C
= (Amax + 2 . B)/3
Since the motion of the probe was rather isotropic so that the Amax and .4mi n parameters were not well resolved, it should therefore be emphasized that the changes in the .4max and Amin parameters and the apparent parameter S were used for comparison of the relative perturbation effect of the drugs. Decrease of the apparent parameter S or increase of the parameter .4mi n indicates higher overall disorder and/or dynamics of the hydrophobic part of the membrane. The membrane order and dynamics were not distinguished from the EPR parameters obtained [14,15]. Results
An example of the EPR spectra of the 16-PC spin probe in TL liposomes is shown in Fig. 2. The EPR spectrum of the probe in liposomes treated with NIF (and with MIN, NIT and NIL, spectra not shown) was similar to the control spectrum. However, when the liposomes were treated with VER (and with GAL, MEP and DIL, spectra not shown) the spectra were 'more fluid' than the control spectrum. The effect of the CB drugs on the apparent parameter S of the 16-PC probe in the TL liposomes, measured at 25°C and 37°C is shown in Fig. 3. A qualitatively similar effect was found at both temperatures. The dihydropyridines, NIF, NIM, NIT and NIL, had a non-significiant or minor effect (NIF) on the apparent parameter S, NTP increased S, but VER, GAL, MEP and DIL pronouncedly decreased S. Qualitatively the same results were obtained when only parameter .4mi n was evaluated from the EPR spectra. The dynamics/disorder of the hydrophobic part of the liposomes treated with VER, GAL, MEP and DIL and measured at 25°C was pronouncedly higher than the dynamics/disorder of the control liposomes at 37°C (Fig. 3). Taking into account the temperature dependence in the range 20-50°C
14
0.26 ~0.23 |+
r-7 25% ~ 37%
00' 111111 o.14~ 0.11~
Co
NIF
NTP
NIM
NI T
NIL
VER
GAL g ~ . P
DI L
Fig, 3. Comparison of the perturbation effect of CB drugs on parameter S of 16-PC in liposomal membranes at 25 and 37°C, Co, control. For abbreviations see Material and Methods.
of the parameter S in the TL liposomes for the 16-PC probe which was determined to be -0.0034/ °C, it can be calculated that VER, GAL, MEP and DIL decreased the apparent parameter S similarly as did the increase of the temperature of the control liposomes by 27-35°C. Dependence of the effect of CB drugs on S of the liposomes hydrated with the buffer at pH 6.0, pH 7.4 and pH 9.0 is shown in Fig. 4. The effect of the drugs did not depend on the pH values of the hydrated buffer. NIM had no effect on S at either of the three pH values, whereas VER and DIL markedly decreased S. Qualitatively the same
results were obtained when only parameter Ami, was evaluated from the EPR spectra. It was of interest to determine whether the perturbation efficiency of the CB drugs was similar in biological membranes to that found in TL liposomes. We therefore studied the effect of the drugs on parameter Amin of the 16-SA probe in synaptosomal membranes (Fig. 5). VER at concentrations >0.32 retool/1 increased parameter Amin, whereas NIF had no effect even at concentrations of 10 and 20 mmoFl (Fig. 5). The results are qualitatively similar to those found in TL liposomes (Fig. 3). The further aim of the present study was to determine at what drug/lipid molar ratio the overall perturbation effect is detected by the 16-PC probe. The concentration dependence of the perturbation effect of VER on the liposomal membrane was compared with that of the drug thioridazine (Fig. 6 and 7). The parameters Ami~ and S were evaluated from the EPR spectra. There was a qualitative difference between their concentration dependence effect. The perturbation effect of thioridazine was linearly dependent on concentration and the effect started at concentrations >0.5%. The concentration effect of VER, on the other hand, was non-linear and started at concentrations > 2%.
1.25/
0.25 __
.0
1.20'
x7
0.200.15
S
i
I
"~ 1.15
0.10
0.05 0.00
, I
1.10
I
pH 7.4
I
I
I
I
I
20
25
30
35
40
45
T e m p e r a t u r e [°c]
Ls pH 6.0
15
)H 9.0
Fig. 4. Perturbation effect of the drugs on parameter S of 16-PC in liposomai membranes at pH 6.0, 7.4 and 9.0. Temperature 250C.
Fig. 5. Temperature dependence of the perturbation effect of VER ( - - - ) and NIF (....) on parameter Amin of 16-SA in synaptosomal membranes. Control (0); NI[F 10 mmol/l (Q) and 20 mmol/l (4); VER 0.32 mmolfl (O), 1 mmoFl (V) and 3.2 mmol/l (0).
15 1.15-
,~.I.14-
.< 1.13' -
1.12
I 0
-
]"
-
T I I I I 2 4 6 8 I0 Drug concentration [Z molar]
12
Fig. 6. Concentration dependence (molar percent of drug in lipids) of perturbation effect of VER (0) and thioridazine (O) on parameter Amin of 16-PC in liposomal membranes. Temperature 25°C. The points represent mean values and standard error from two experiments.
Discussion
Perturbation effect In our previous study we found that the perturbation effect of VER depended on the lipid composition of the liposomes [16] and was most pronounced in TL liposomes. Therefore in the present study we compared the perturbation effect of CB in TL liposomes. Since we were interested in the perturbation effect exerted in the proximity of the drug incor-
0.23 0.220.21
S 0.20
0.190.18
0
2 4 6 8 i0 Drug concentration [~ molar]
12
Fig. 7. Concentration dependence (molar percent of drug in
lipids) of perturbation effect of VER (S) and thioridazine (O) on parameter S of 16-PC in liposomal membranes. Temperature 25°C. The points represent mean values and standard error from two experiments.
porated in the liposomes, the drug/lipid molar ratio of 1/2 was chosen (Fig. 3). Presuming that two lipid molecules and one drug molecule are around the spin probe in the liposomes, at the drug/lipid molar ratio of 1/2 the drug is in the proximity of the spin probe, so that membrane perturbation around the drug can be detected. NTP decreased dynamic/disorder of the hydrocarbon part of the TL liposomes. This effect may be explained by its interaction with unsaturated fatty acid chains of lipids which was studied in our previous work [10]. NTP was found to form stable radicals mainly by interaction of its nitroso group with the unsaturated bonds of lipids in a pseudo Diels-Alder reaction. Therefore, the motion of the NTP-lipid complex in the lipid membrane is partially restricted resulting in a higher value of the S parameter. In the present study NIF, NIL, NIM and NIT had an insignificant or minor perturbation effect at the drug/lipid molar ratio of 1/2 in spite of their high partitioning into lipid and biological membranes [17]. This may be explained by spatial incorporation of the dihydropyridines in the lipid membrane. Herbette et al. [18], using a neutron diffraction technique, studied the incorporation of NIM in sarcoplasmic reticulum membranes. They reported that NIM was located in the protein knob region and at the water/hydrocarbon core interfaces of the bilayer. Seelig et al. [19] using the NMR method studied incorporation of NIM in lipid membranes. They reported that NIM as a non-charged molecule was strongly hydrophobic and was homogeneously distributed across the whole hydrocarbon layer of lipid membrane and did not interact with the lipid headgroup level. Since the dihydropyridines used in our study are uncharged molecules and they are strongly hydrophobic, in analogy with the results of Seelig et al. [19] obtained in lipid membranes, we supposed that the dihydropyridines were homogeneously distributed across the whole hydrocarbon layer of the lipid membrane and did not interact with the lipid headgroup level. Thus the spatial distribution of the dihydropyridine drugs may not appreciably perturb the lipid membrane. On the other hand, VER, GAL, MEP and DIL had a pronounced perturbation effect at the
16 drug/lipid molar ratio of 1/2 and the effect was similar to that of temperature increase of the control sample by 27-35°C. The perturbation effect of these drugs may be explained by a high partition coefficient and by the amphiphilic (charged) nature of the drugs' molecules, as similarly supposed for other amphiphilic drugs such as local anaesthetics or ~-adrenoceptor blocking drugs [18-20]. The amphiphilic molecule is supposed to be located in the lipid membrane with the polar part located at the membrane surface and the hydrophobic part intercalated between the lipid acyl chains [20,221. Such drug incorporation can create more molecular freedom for the lipid acyl chain at the hydrocarbon core.
Pharmacological concentrations We set out to determine at what VER concentration its overall perturbation effect can be detected by spin probes. The perturbation effect of VER in synaptosomes was observed at concentrations ~0.32 mmol/l, i.e., in the same range at which some of its in vitro biological membrane activities were reported [ 1,5,6]. The perturbation effect of VER in TL liposomes was detected at > 2 molar percent of VER in TL liposomes. Taking the average molecular weight of TL as 775, it can be calculated that VER perturbed liposomes at a membrane concentration >26 mmol/l. Since the partition coefficient in the TL liposomes/buffer system is unknown we cannot determine the buffer concentration of VER which produces a perturbation effect in TL liposomes. If the partition coefficient of 67 for VER found in the octanol/buffer [22] is considered, the buffer concentration of VER-perturbing liposomes would be >0.39 mmol/1, which lies in the range of some of its non-specific biological activities. It should be noted that the concentration dependence of the perturbation effect of VER in liposomes measured at 25°C was non-linear (Figs. 6 and 7). Among other implications this may indicate that either VER at low concentrations perturbed the membrane locally, leaving the bulk of the membrane unperturbed and/or that VER was saturably bound to TL liposomes. Concerning the first possibility, the following has to be considered: The EPR spectrum is a sum-
mary of the signals of all spin probes in the membrane. In our study the probe/lipid ratio of 0.01 was used. If at low drug/lipid concentrations the drug perturbation effect is a non-cooperative phenomenon, than the drug may induce local perturbation in the membrane, leaving the bulk of the membrane unperturbed. At a low drug/lipid ratio, the negligible amount of probes which are close to the drug reflect the perturbated membrane region, but they are covered by signals from probes incorporated in the unperturbed membrane bulk, resulting in non-significant changes of the EPR membrane parameters. Increasing membrane concentrations of drugs in the membrane lead to a non-linear increase of membrane perturbation. Concerning the second possibility, VER may bind specifically and saturably to TL membranes so that it does not perturb physical properties of the membrane. When the binding sites are saturated, after further addition the drug dissolves in membrane bilayers and induces membrane perturbation. In light of our experiments we cannot distinguish whether any of the described possibilities or even some other events were involved in drug concentration-dependent membrane perturbation. Pang and Sperelakis [23] compared the lipophilicity of CB drugs in the corn oil/buffer, octanol/buffer and chloroform/buffer systems. The order of lipophilicity in these systems was VER > NIT > NIF ~, DIL. The same order of the drugs was established for their uptake in isolated sarcoplasmic reticula from rat skeletal muscle [20]. Comparison of the perturbation efficiency of the CB tested in our study with their published lipophilicity, evidently shows that the perturbation potency of the drugs does not correlate with their liposolubility. In conclusion, there is a clear difference between the efficiency of CB to perturb the hydrophobic part of lipid or synaptosomal membranes. Dihydropyridine CB had a non-significiant or minor perturbation effect, whereas VER, GAL, MEP and DIL remarkably perturbed membranes, indicating that some of their non-specific biological activities may be mediated at least partially through their perturbation effect on biological membranes.
17
Acknowledgment We gratefully acknowledge Dr. Barbara E. Ehrlich (University of Connecticut, Farmington, CT) for her assistance and helpful discussion on the work.
References 1 2 3 4 5 6 7 8 9
I. Zfihradnik and J. Zachar (1983) Gen. Physiol. Biophys. 2, 181-192. R. McGee and J.E. Schneider (1979) Mol. Pharmacol. 16, 877-885. A. Erdeich, R. Spanier and H. Rahamimoff(1983) Eur. J. Pharmacol. 90, 193-202. S. Stoic and V. Nem6ek (1990) Eur. J. Pharmacol. 183, 1662-1663. M. Rug6~ik, O. Juhfisz, J. Orlicky and J. Zachar (1986) Gen. Physiol. Biophys. 5, 529-536. H.C. Kim and B.U. Raess (1988) Biochem. Pharrnacol. 37, 917-920. D.S.W.P. Hay and R.M. Wadsworth (1982) Eur. J. Pharmacol. 77, 221-228. P. Gibala, R. Somikovfi, V. Knezl and J. Dffmal (1991) Bratisl. Lek. Listy 92, 474-478 in Slovak. R. Sotnikov~i, V. Knezl, P. Gibala, J. Dfimal (1989) (:s. Fysiologie 38, 171 in Slovak.
10 !1 12 13 14 15 16 17 18 19 20 21 22 23
V. Migik, A. Stagko, D. Gergel and K. Ondriag (1991) Mol. Pharmacol. 40, 435-439. J.M. Folch, M. Lees and G.H.S. Stanley (1957) J. Biol. Chem. 226, 497-509. B.K. Krueger, R.W. Ratzlaff, G.R. Strichartz and M.P.J. Blaustein (1979) J. Membrane Biol., 50, 287-310. R.J. Hitzemann and P.A. Johnson (1983) Neurochem. Res. 8, 121-127. E. Grell (Ed.) (1981) Membrane Spectroscopy, SpringerVerlag, New York, pp. 51-142. K. Ondriag (1989) J. Pharm. Biomed. Anal. 7, 649-675. K. Ondriag, A. Stas'ko, V. Migfk, J. Reguli and E. Svajdlenka (1991) Chem.-Biol. Interact. 79, 197-206. L.G. Herbette, Y.M.H. Vanterve and D.G. Rhodes (1989) J. Mol. Cell. Cardiol., 21, 187-201. L.G. Herbette, D.W. Chester and D G . Rhodes (1986) Biophys. J. 49, 91-94. H.-D. B/iuerle and J. Seclig (1991) Biochemistry 30, 7203-7211. Y. Boulanger, S. Schreier and I.C.P. Smith (1981) Biochemistry, 20, 6824-6830. U Herbette, A.M. Katz and J.M. Sturtevant (1983) Mol. Pharmacol. 24, 259-269. K. Ondriag, A. Stagko, V. Jan~inov~i and P. Balgav~, (1987) Mol. Pharmacol. 31, 97-102. D.C. Pang and N. Sperelakis (1984) Biochem. Pharmacol. 33, 821-826.
11
Perturbation effect of eight calcium channel blockers on liposomal membranes prepared from rat brain total lipids K a r o l O n d r i a g a, E l e n a O n d r i a ~ o v ~ b a n d A n d r e j Stas'ko c alnstitute of Experimental Pharmacology, Slovak Academy of Sciences, 84216 Bratislava, bFaculty of Pharmacy, Comenius University, Odbojtirov 10, 83232 Bratislava and CFaculty of Chemical Technology, Slovak Technical University, 81237 Bratislava, (CSFR) (Received November 4th, 1991; revision received January 15th, 1992; accepted March 20th, 1992)
EPR spectroscopy of phosphatidyicholine or stearic acid labeled at the doxyl group at the 16-carbon position was used to compare the perturbation effect of eight calcium channel blockers (CB) on overall dynamics/disorder of the hydrophobic part of iiposome membranes prepared from rat brain total lipids at the drug/lipid molar ratio of 1/2. Nifedipine (NIF), nimodipine, niludipine and nitrendipine had a minor effect on the dynamics/disorder of the liposome membranes, whereas the disordering effect of verapamil (VER), mepamil, galiopamil and diltiazem was more pronounced. Concentration dependence of the overall disordering effect of VER on liposomal membranes was found at the VER/lipid ratio >0.02 and for the tranquilliser thioridazine >0.005. VER exerted a disordering effect at the hydrophobic part of synaptosomal membranes at concentrations _>0.32 mmol/l, whereas NIF did not exhibit a disordering effect even at concentrations of 10-20 mmol/I.
Key words: calcium antagonists; membrane perturbation; EPR spectroscopy
Introduction
Calcium channel blockers (CB) play an important role in the excitation-contraction coupling mechanism of cardiac and smooth muscle by inhibiting the membrane influx of extracellular calcium. In addition to the inhibitory effect of VER on Ca 2+ channels, it has a wide spectrum of pharmacological activities not believed to be related to blockade of Ca 2+ channels. Verapamil (VER) (2.3 mmol/1) was found to inCorrespondence to: Karol Ondriag, Institute of Experimental Pharmacology, Slovak Academy of Sciences, 84216 Bratislava, CSFR. Abbreviations:Arnin, apparent inner hyperfine splitting (see Fig. 2); CB, calcium channel blockers; DIL, diltiazem; EPR, electron paramagnetic spectroscopy; GAL, gallopamil; MEP, mepamil; NIF, nifedipine; NIM, nimodipine; NIL, niludipine; NIT, nitrendipine; VER, verapamil; NTP, 2,6-dimethyl-4(2-nitrosophenyl)-3,5-pyridine-dicarboxylic acid dimethyl ester; 16-PC, l-palmitoyl-2-stearoyl phosphatidylcholine labeled at the doxyl group at the 16th carbon positon; S, apparent order parameter; 16-SA, steric acid labeled at the doxyl group at the 16th carbon position; TL, total lipids.
hibit both the early and late potassium outward currents in intact isolated fibers of crayfish muscle membrane [1]. It inhibited the uptake of serotonin, dopamine, norepinephrine and choline into rat forebrain synaptosomes. The concentrations of VER required for 50% inhibition of the four uptake systems were (in/~moFl) 3.2, 18, 32 and 150, respectively [2]. VER (0.175 mmol/1) inhibited the Na+-dependent Ca 2+ uptake system of synaptic plasma membrane vesicles [3]. In some non-specific membrane activities VER was found to be more effective than the dihydropiridine calcium entry blocker nifedipine (NIF). Stolc and Nem~ek [4] studied the effect of NIF and VER on calcium, sodium and two types of potassium voltage-dependent ion channels in the membrane of a single neuron of the rat dorsal root ganglion. They found that NIF had high affinity to the ion channels responsible for the calcium inward current, whereas it did not affect either the sodium inward current or the fast inactivating and the slow non-inactivating potassium outward currents even in the 15-/zmol/l concentration. On the
0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
12
other hand VER was found to be only a nonspecific inhibitor and equipotently blocked all the ionic currents tested. The authors supposed that the effects of VER could be accounted for by some non-specific mechanisms, such as membrane lipid disordering effect. Ru~Efik et al. [5] reported that VER, NIF and nimodipine (NIM) at 1 mmol/1 concentration inhibited Na+-Ca 2+ exchange in rat brain microsomal membranes with relative efficiency of VER > NIM = HIF and Kim et al. [6] found that VER and diltiazem (DIL) (1 mmol/l) inhibited the calmodulin-regulated plasma membrane Ca 2+ pump ATPase, whereas NIF (10 /~mol/l) was ineffective. Hay and Wadsworth [7] studied the local anaesthetic activity of organic calcium antagonists on the rat phrenic nerve. They found that verapamil (ICs0 63.7 ± 21/~mol/1) had 3.0 x the potency of the local anaesthetic lidocaine, whereas nifedipine was without effect. The mechanisms of the non-specific membrane activities of CB are not fully understood. Since some of the membrane activities of VER were observed at millimolar concentrations, we studied the perturbation effect of VER on liposomal and synaptosomal membranes in comparison to other CB.
~
C
H
R&
tl R 2
R3
H~C
H~ H
Name:
R1
R2
R3
R4
Nife~ipine Nimodlplne Nitr~ndipine
H NO 2 NO 2
NO 2 H H
-COOCH 3 -COOCH2CH2OCH3 ~COOCH 3
-COOCH 3 -COOCH(CH3) 2 -COOC2H 5
Niludipine
NO 2
H
-COOCH2CH2OC3H ?
-COOCH2CH2OC3H 7
R2
RI
R3
0CH3
CICH213NC I H2CH2
0CH3
'HC[
84 Name:
R1
R2
R3
R4
VerapaBil Gallopau/1 Mepamll
H H OCH 3
OCH 3 OCH 3 H
OCH 3 OCH 3 H
H OCH 3 H
H~ O C H S ' .H ~
N
~
OOCCH3
3 .HCI
CH 2 'CH2N(CH3)2 Diltiazem
Fig. 1. Chemical formulas of calcium channel blockers.
Materials and Methods NIF, VER, thioridazine and stearic acid labeled at the doxyl group at the 16th carbon position (16-SA) were from Sigma. NIM, niludipine (NIL), nitrendipine (NIT), gallopamil (GAL), were provided by the Inst. for Drug Research, Modra, (~SFR. Mepamil (MEP) was from Research Inst. for Pharmacy and Biochemistry, Praque, (~SFR. DIL was from Lachema, t~SFR. The high purity and pharmacological effects of these calcium antagonists have been reported [1,5,8,9]. The purity of the CB was determined by TLC and LC and their content was determined by LC and UV spectroscopy. The content ofNIM, MEP and DIL was not less than 99% and the content of NIT and NIL was not less than 98%. The purity of GAL was determinated by HPLC and the content was not less than 99%. The chemical structures of the CB are shown in Fig. 1. 1-Palmitoyl-2-stearoyl-phosphatidyl-choline
labeled at the doxyl group at the 16th carbon position (16-PC) was from Avanti Polar Lipids. NTP (2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester) was obtained by illumination of NIF (1 mg/ml ethanol) by daylight for 1 h as described in our previous study [10].
Liposomes Total lipids (TL) from rat brain were isolated according to Folch et al. [l 1]. TL (2 rag) from brain and spin probe (100:l molar ratio) and each particular drug were mixed in chloroform: methanol (2" 1). The solvent was evaporated under nitrogen followed by evacuation. The dry samples were hydrated with 30 ~1 of the buffer (in retool/l), pH 7.4, NaCI 145, KC1 5, MgCI2 1.4, CaCI2 1, Hepes.HC1 5. In order to attain equilibrium of the drugs in the liposomes the samples were subjected to vortex-freeze-thaw cycles several times.
13
Synaptosomal membranes Isolated synaptosomes [12] (3 mg of proteins) were incubated with or without the drug at 37°C for 60 min in 5 ml of the buffer. The samples were centrifuged at 10000 x g for 10 min and the pelleted synaptosomes (50 #1) were mixed with the dry spin probe at the ratio of 15/~g spin probe/3 mg of protein and vortexed for 3-5 min. The samples were further incubated for 30 min at 37°C and transferred into capillaries (1 mm i.d.). Assuming 1/zl of lipids per 1 mg of protein in the synaptosomes [13] and the molecular weight = 775 for TL, it can be calculated that the spin probe/ lipid molar ratio in synaptosomes was 0.01. EPR spectra were measured in the glass capillaries using a Bruker 200 D Spectrometer. Typical instrument settings were: 10 mW microwave power, modulation amplitude = 0.1 mT. To assess the relative efficiency of the drugs in perturbing the lipid membrane, the apparent order parameter S was calculated from the apparent outer (Amax) and inner (Amin) splittings [14] which were directly measured from the EPR spectra of the spin probe as shown in Fig. 2.
_/ _/
/
/ Fig. 2. EPR spectra of the 16-PC probe in TL liposomes. A, control; B, TL/NIF = 2:1 molar ratio; C, TL/VER = 2:1 molar ratio. Spectrum width 6 roT. Temperature 37°C. Arrows in spectrum A indicate Areax determination. Arrows in spectrum C indicate Amin determination.
Sapp = (Amax- B) * 0.5407/C B
= .4min + 1.4 * [1 - (Amax - Amin)/27.25]
C
= (Amax + 2 . B)/3
Since the motion of the probe was rather isotropic so that the Amax and .4mi n parameters were not well resolved, it should therefore be emphasized that the changes in the .4max and Amin parameters and the apparent parameter S were used for comparison of the relative perturbation effect of the drugs. Decrease of the apparent parameter S or increase of the parameter .4mi n indicates higher overall disorder and/or dynamics of the hydrophobic part of the membrane. The membrane order and dynamics were not distinguished from the EPR parameters obtained [14,15]. Results
An example of the EPR spectra of the 16-PC spin probe in TL liposomes is shown in Fig. 2. The EPR spectrum of the probe in liposomes treated with NIF (and with MIN, NIT and NIL, spectra not shown) was similar to the control spectrum. However, when the liposomes were treated with VER (and with GAL, MEP and DIL, spectra not shown) the spectra were 'more fluid' than the control spectrum. The effect of the CB drugs on the apparent parameter S of the 16-PC probe in the TL liposomes, measured at 25°C and 37°C is shown in Fig. 3. A qualitatively similar effect was found at both temperatures. The dihydropyridines, NIF, NIM, NIT and NIL, had a non-significiant or minor effect (NIF) on the apparent parameter S, NTP increased S, but VER, GAL, MEP and DIL pronouncedly decreased S. Qualitatively the same results were obtained when only parameter .4mi n was evaluated from the EPR spectra. The dynamics/disorder of the hydrophobic part of the liposomes treated with VER, GAL, MEP and DIL and measured at 25°C was pronouncedly higher than the dynamics/disorder of the control liposomes at 37°C (Fig. 3). Taking into account the temperature dependence in the range 20-50°C
14
0.26 ~0.23 |+
r-7 25% ~ 37%
00' 111111 o.14~ 0.11~
Co
NIF
NTP
NIM
NI T
NIL
VER
GAL g ~ . P
DI L
Fig, 3. Comparison of the perturbation effect of CB drugs on parameter S of 16-PC in liposomal membranes at 25 and 37°C, Co, control. For abbreviations see Material and Methods.
of the parameter S in the TL liposomes for the 16-PC probe which was determined to be -0.0034/ °C, it can be calculated that VER, GAL, MEP and DIL decreased the apparent parameter S similarly as did the increase of the temperature of the control liposomes by 27-35°C. Dependence of the effect of CB drugs on S of the liposomes hydrated with the buffer at pH 6.0, pH 7.4 and pH 9.0 is shown in Fig. 4. The effect of the drugs did not depend on the pH values of the hydrated buffer. NIM had no effect on S at either of the three pH values, whereas VER and DIL markedly decreased S. Qualitatively the same
results were obtained when only parameter Ami, was evaluated from the EPR spectra. It was of interest to determine whether the perturbation efficiency of the CB drugs was similar in biological membranes to that found in TL liposomes. We therefore studied the effect of the drugs on parameter Amin of the 16-SA probe in synaptosomal membranes (Fig. 5). VER at concentrations >0.32 retool/1 increased parameter Amin, whereas NIF had no effect even at concentrations of 10 and 20 mmoFl (Fig. 5). The results are qualitatively similar to those found in TL liposomes (Fig. 3). The further aim of the present study was to determine at what drug/lipid molar ratio the overall perturbation effect is detected by the 16-PC probe. The concentration dependence of the perturbation effect of VER on the liposomal membrane was compared with that of the drug thioridazine (Fig. 6 and 7). The parameters Ami~ and S were evaluated from the EPR spectra. There was a qualitative difference between their concentration dependence effect. The perturbation effect of thioridazine was linearly dependent on concentration and the effect started at concentrations >0.5%. The concentration effect of VER, on the other hand, was non-linear and started at concentrations > 2%.
1.25/
0.25 __
.0
1.20'
x7
0.200.15
S
i
I
"~ 1.15
0.10
0.05 0.00
, I
1.10
I
pH 7.4
I
I
I
I
I
20
25
30
35
40
45
T e m p e r a t u r e [°c]
Ls pH 6.0
15
)H 9.0
Fig. 4. Perturbation effect of the drugs on parameter S of 16-PC in liposomai membranes at pH 6.0, 7.4 and 9.0. Temperature 250C.
Fig. 5. Temperature dependence of the perturbation effect of VER ( - - - ) and NIF (....) on parameter Amin of 16-SA in synaptosomal membranes. Control (0); NI[F 10 mmol/l (Q) and 20 mmol/l (4); VER 0.32 mmolfl (O), 1 mmoFl (V) and 3.2 mmol/l (0).
15 1.15-
,~.I.14-
.< 1.13' -
1.12
I 0
-
]"
-
T I I I I 2 4 6 8 I0 Drug concentration [Z molar]
12
Fig. 6. Concentration dependence (molar percent of drug in lipids) of perturbation effect of VER (0) and thioridazine (O) on parameter Amin of 16-PC in liposomal membranes. Temperature 25°C. The points represent mean values and standard error from two experiments.
Discussion
Perturbation effect In our previous study we found that the perturbation effect of VER depended on the lipid composition of the liposomes [16] and was most pronounced in TL liposomes. Therefore in the present study we compared the perturbation effect of CB in TL liposomes. Since we were interested in the perturbation effect exerted in the proximity of the drug incor-
0.23 0.220.21
S 0.20
0.190.18
0
2 4 6 8 i0 Drug concentration [~ molar]
12
Fig. 7. Concentration dependence (molar percent of drug in
lipids) of perturbation effect of VER (S) and thioridazine (O) on parameter S of 16-PC in liposomal membranes. Temperature 25°C. The points represent mean values and standard error from two experiments.
porated in the liposomes, the drug/lipid molar ratio of 1/2 was chosen (Fig. 3). Presuming that two lipid molecules and one drug molecule are around the spin probe in the liposomes, at the drug/lipid molar ratio of 1/2 the drug is in the proximity of the spin probe, so that membrane perturbation around the drug can be detected. NTP decreased dynamic/disorder of the hydrocarbon part of the TL liposomes. This effect may be explained by its interaction with unsaturated fatty acid chains of lipids which was studied in our previous work [10]. NTP was found to form stable radicals mainly by interaction of its nitroso group with the unsaturated bonds of lipids in a pseudo Diels-Alder reaction. Therefore, the motion of the NTP-lipid complex in the lipid membrane is partially restricted resulting in a higher value of the S parameter. In the present study NIF, NIL, NIM and NIT had an insignificant or minor perturbation effect at the drug/lipid molar ratio of 1/2 in spite of their high partitioning into lipid and biological membranes [17]. This may be explained by spatial incorporation of the dihydropyridines in the lipid membrane. Herbette et al. [18], using a neutron diffraction technique, studied the incorporation of NIM in sarcoplasmic reticulum membranes. They reported that NIM was located in the protein knob region and at the water/hydrocarbon core interfaces of the bilayer. Seelig et al. [19] using the NMR method studied incorporation of NIM in lipid membranes. They reported that NIM as a non-charged molecule was strongly hydrophobic and was homogeneously distributed across the whole hydrocarbon layer of lipid membrane and did not interact with the lipid headgroup level. Since the dihydropyridines used in our study are uncharged molecules and they are strongly hydrophobic, in analogy with the results of Seelig et al. [19] obtained in lipid membranes, we supposed that the dihydropyridines were homogeneously distributed across the whole hydrocarbon layer of the lipid membrane and did not interact with the lipid headgroup level. Thus the spatial distribution of the dihydropyridine drugs may not appreciably perturb the lipid membrane. On the other hand, VER, GAL, MEP and DIL had a pronounced perturbation effect at the
16 drug/lipid molar ratio of 1/2 and the effect was similar to that of temperature increase of the control sample by 27-35°C. The perturbation effect of these drugs may be explained by a high partition coefficient and by the amphiphilic (charged) nature of the drugs' molecules, as similarly supposed for other amphiphilic drugs such as local anaesthetics or ~-adrenoceptor blocking drugs [18-20]. The amphiphilic molecule is supposed to be located in the lipid membrane with the polar part located at the membrane surface and the hydrophobic part intercalated between the lipid acyl chains [20,221. Such drug incorporation can create more molecular freedom for the lipid acyl chain at the hydrocarbon core.
Pharmacological concentrations We set out to determine at what VER concentration its overall perturbation effect can be detected by spin probes. The perturbation effect of VER in synaptosomes was observed at concentrations ~0.32 mmol/l, i.e., in the same range at which some of its in vitro biological membrane activities were reported [ 1,5,6]. The perturbation effect of VER in TL liposomes was detected at > 2 molar percent of VER in TL liposomes. Taking the average molecular weight of TL as 775, it can be calculated that VER perturbed liposomes at a membrane concentration >26 mmol/l. Since the partition coefficient in the TL liposomes/buffer system is unknown we cannot determine the buffer concentration of VER which produces a perturbation effect in TL liposomes. If the partition coefficient of 67 for VER found in the octanol/buffer [22] is considered, the buffer concentration of VER-perturbing liposomes would be >0.39 mmol/1, which lies in the range of some of its non-specific biological activities. It should be noted that the concentration dependence of the perturbation effect of VER in liposomes measured at 25°C was non-linear (Figs. 6 and 7). Among other implications this may indicate that either VER at low concentrations perturbed the membrane locally, leaving the bulk of the membrane unperturbed and/or that VER was saturably bound to TL liposomes. Concerning the first possibility, the following has to be considered: The EPR spectrum is a sum-
mary of the signals of all spin probes in the membrane. In our study the probe/lipid ratio of 0.01 was used. If at low drug/lipid concentrations the drug perturbation effect is a non-cooperative phenomenon, than the drug may induce local perturbation in the membrane, leaving the bulk of the membrane unperturbed. At a low drug/lipid ratio, the negligible amount of probes which are close to the drug reflect the perturbated membrane region, but they are covered by signals from probes incorporated in the unperturbed membrane bulk, resulting in non-significant changes of the EPR membrane parameters. Increasing membrane concentrations of drugs in the membrane lead to a non-linear increase of membrane perturbation. Concerning the second possibility, VER may bind specifically and saturably to TL membranes so that it does not perturb physical properties of the membrane. When the binding sites are saturated, after further addition the drug dissolves in membrane bilayers and induces membrane perturbation. In light of our experiments we cannot distinguish whether any of the described possibilities or even some other events were involved in drug concentration-dependent membrane perturbation. Pang and Sperelakis [23] compared the lipophilicity of CB drugs in the corn oil/buffer, octanol/buffer and chloroform/buffer systems. The order of lipophilicity in these systems was VER > NIT > NIF ~, DIL. The same order of the drugs was established for their uptake in isolated sarcoplasmic reticula from rat skeletal muscle [20]. Comparison of the perturbation efficiency of the CB tested in our study with their published lipophilicity, evidently shows that the perturbation potency of the drugs does not correlate with their liposolubility. In conclusion, there is a clear difference between the efficiency of CB to perturb the hydrophobic part of lipid or synaptosomal membranes. Dihydropyridine CB had a non-significiant or minor perturbation effect, whereas VER, GAL, MEP and DIL remarkably perturbed membranes, indicating that some of their non-specific biological activities may be mediated at least partially through their perturbation effect on biological membranes.
17
Acknowledgment We gratefully acknowledge Dr. Barbara E. Ehrlich (University of Connecticut, Farmington, CT) for her assistance and helpful discussion on the work.
References 1 2 3 4 5 6 7 8 9
I. Zfihradnik and J. Zachar (1983) Gen. Physiol. Biophys. 2, 181-192. R. McGee and J.E. Schneider (1979) Mol. Pharmacol. 16, 877-885. A. Erdeich, R. Spanier and H. Rahamimoff(1983) Eur. J. Pharmacol. 90, 193-202. S. Stoic and V. Nem6ek (1990) Eur. J. Pharmacol. 183, 1662-1663. M. Rug6~ik, O. Juhfisz, J. Orlicky and J. Zachar (1986) Gen. Physiol. Biophys. 5, 529-536. H.C. Kim and B.U. Raess (1988) Biochem. Pharrnacol. 37, 917-920. D.S.W.P. Hay and R.M. Wadsworth (1982) Eur. J. Pharmacol. 77, 221-228. P. Gibala, R. Somikovfi, V. Knezl and J. Dffmal (1991) Bratisl. Lek. Listy 92, 474-478 in Slovak. R. Sotnikov~i, V. Knezl, P. Gibala, J. Dfimal (1989) (:s. Fysiologie 38, 171 in Slovak.
10 !1 12 13 14 15 16 17 18 19 20 21 22 23
V. Migik, A. Stagko, D. Gergel and K. Ondriag (1991) Mol. Pharmacol. 40, 435-439. J.M. Folch, M. Lees and G.H.S. Stanley (1957) J. Biol. Chem. 226, 497-509. B.K. Krueger, R.W. Ratzlaff, G.R. Strichartz and M.P.J. Blaustein (1979) J. Membrane Biol., 50, 287-310. R.J. Hitzemann and P.A. Johnson (1983) Neurochem. Res. 8, 121-127. E. Grell (Ed.) (1981) Membrane Spectroscopy, SpringerVerlag, New York, pp. 51-142. K. Ondriag (1989) J. Pharm. Biomed. Anal. 7, 649-675. K. Ondriag, A. Stas'ko, V. Migfk, J. Reguli and E. Svajdlenka (1991) Chem.-Biol. Interact. 79, 197-206. L.G. Herbette, Y.M.H. Vanterve and D.G. Rhodes (1989) J. Mol. Cell. Cardiol., 21, 187-201. L.G. Herbette, D.W. Chester and D G . Rhodes (1986) Biophys. J. 49, 91-94. H.-D. B/iuerle and J. Seclig (1991) Biochemistry 30, 7203-7211. Y. Boulanger, S. Schreier and I.C.P. Smith (1981) Biochemistry, 20, 6824-6830. U Herbette, A.M. Katz and J.M. Sturtevant (1983) Mol. Pharmacol. 24, 259-269. K. Ondriag, A. Stagko, V. Jan~inov~i and P. Balgav~, (1987) Mol. Pharmacol. 31, 97-102. D.C. Pang and N. Sperelakis (1984) Biochem. Pharmacol. 33, 821-826.
