L-type Ca2+ channel blockers inhibit the window contraction of mouse aorta segments with high affinity.

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

L-type Ca2 þ channel blockers inhibit the window contraction of mouse aorta segments with high affinity Cédéric F. Michiels a,n, Cor E. Van Hove b, Wim Martinet a, Guido R.Y. De Meyer a, Paul Fransen a a b

Laboratory of Physiopharmacology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium Laboratory of Pharmacology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium

art ic l e i nf o

a b s t r a c t

Article history: Received 4 December 2013 Received in revised form 20 May 2014 Accepted 21 May 2014

L-type calcium channel blockers (LCCBs) reduce blood pressure more effectively in hypertensive than in normotensive subjects and are more effective in vascular smooth muscle (VSM) than in cardiac muscle. This has been explained by the depolarized resting potential of VSM in comparison with heart muscle cells and by the hypertension because both favor the “high affinity” inactivated state of the L-type calcium channel (LCC). Depolarized resting potentials, however, also increase Ca2 þ influx via window, non-inactivating LCC. The present study investigated whether these channels can be effectively blocked by nifedipine, verapamil or diltiazem, as representatives of different LCCB classes. C57Bl6 mouse aortic segments were depolarized by 50 mM K þ to attain similar degree of inactivation. The depolarization evoked biphasic contractions with the slow force component displaying higher sensitivity to LCCBs than the fast component. Removal of the fast force component increased, whereas stimulation of Ca2 þ influx with the dihydropyridine BAY K8644, a structural analog of nifedipine, decreased the efficacy of the LCCBs. Addition of LCCBs during the contraction caused concentration-dependent relaxation, which was independent of the presence of a fast force component, but still showed lower sensitivity in the presence of BAY K8644. Our data suggest that steady-state contractions by depolarization with 50 mM K þ are completely due to window Ca2 þ influx, which is preferentially inhibited by LCCBs. Furthermore, results point to interactions between the LCCB receptors and Ca2 þ ions or BAY K8644. The high affinity for open, non-inactivating LCC may play a dominant role in the anti-hypertensive effects of LCCBs. & 2014 Published by Elsevier B.V.

Keywords: Vascular reactivity L-type calcium channel Calcium channel blocker Depolarization Contraction Chemical compounds studied in this article: Nifedipine (PubChem CID: 4485) Amlodipine (PubChem CID: 2162) Diltiazem HCl (PubChem CID: 62920) Verapamil HCl (PubChem CID: 62969) BAY K8644 (PubChem CID: 2303)

1. Introduction Ca2 þ influx via L-type Ca2 þ channels (LCCs) plays a dominant role in blood pressure regulation and development of hypertension (Moosmang et al., 2003; Pesic et al., 2004; Rhee et al., 2009; Zhou et al., 2008). Inhibition of this Ca2 þ influx with L-type Ca2 þ channel blockers (LCCBs) causes vascular smooth muscle (VSM)

Abbreviations: LCCBs, L-type calcium channel blockers; LCCs, L-type calcium channels; KR, Krebs Ringer; 0Ca, calcium-free solution; 0Ca/þ Ca, re-addition of Ω external Ca2 þ to calcium-free solution; L-NAME, N -nitro-L-arginine methyl ester; Ω L-NNA, N -nitro-L-arginine; NO, nitric oxide; IC50, drug concentration for 50% inhibition; VSM, vascular smooth muscle n Correspondence to: Universiteitsplein 1, University of Antwerp, CDE T2.26, 2610 Wilrijk, Belgium. Tel.: þ 32 32652562; fax: þ 32 32652567. E-mail addresses: [email protected] (C.F. Michiels), [email protected] (C.E. Van Hove), [email protected] (W. Martinet), [email protected] (G.R.Y. De Meyer), [email protected] (P. Fransen).

cell relaxation and consequential vasodilatation. Three classes of LCCBs with different chemical structures are clinically used: phenylalkylamines (e.g. verapamil), benz(othi)azepines (e.g. diltiazem) and dihydropyridines (e.g. nifedipine). They exhibit unique features by reducing blood pressure more effectively in hypertensive than in normotensive subjects (Leonetti et al., 1982; Godfraind et al., 1984; Godfraind, 2005; Striessnig et al., 1998). LCCBs decrease the aortic pulse wave velocity, central aortic pressure and augmentation index, suggesting that in baseline conditions L-type Ca2 þ influx contributes to compliance of conduit arteries (Koumaras et al., 2012; Kum and Karalliedde, 2010; Mackenzie et al., 2009; Williams et al., 2006; Zulliger et al., 2004). Several attempts have been made to explain the unique features of LCCBs in their action on blood pressure and arterial stiffness. The increased response to vasoconstrictors in hypertension may not only be due to an increased number of voltage-gated LCCs (Godfraind, 2005; Pesic et al., 2004; Pratt et al., 2002), but also due to depolarization of the resting potential of VSM cells (Morel and Godfraind, 1994; Pesic et al., 2004). Depolarization

http://dx.doi.org/10.1016/j.ejphar.2014.05.036 0014-2999/& 2014 Published by Elsevier B.V.

Please cite this article as: Michiels, C.F., et al., L-type Ca2 þ channel blockers inhibit the window contraction of mouse aorta segments with high affinity. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.036i

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C.F. Michiels et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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leads to an increased proportion of inactivated LCCs, which according to the “modulated receptor theory” may have higher affinity for LCCBs than channels in the resting state (Bean et al., 1986; Godfraind, 2005; Morel and Godfraind, 1987). Furthermore, depolarization of the VSM cell resting potential by hypertension not only favours the inactivated state of the channels, but is also expected to increase L-type Ca2 þ influx via the channel window (Fleischmann et al., 1994). Recently, we presented a new view on how contraction of depolarized mouse aortic segments is caused by a time-independent, non-inactivating Ca2 þ influx. Whenever the resting potential of VSM cells resides in the voltage window of the LCC, there is a continuous Ca2 þ influx that leads to a sustained, tonic contraction (Fransen et al., 2012a). Hence, depolarization of the resting potential by hypertension not only favours the inactivated state of the channels, but is also expected to increase window LCC Ca2 þ influx (Fleischmann et al., 1994). Therefore, the present study investigated whether nifedipine/amlodipine, verapamil or diltiazem as representatives of the different LCCB classes also block the window contraction with high affinity in depolarized mouse aortic segments.

2. Materials and Methods 2.1. Aortic segments The studies were approved by the Ethical Committee of the University of Antwerp, and conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85–23, revised 1996). C57Bl6 mice (food and water ad libitum, 12/12 light–dark cycle) were used at the age of 4 to 7 months. After anesthesia (sodium pentobarbital, 75 mg kg 1, i.p.) and sacrificing the animals by perforating the diaphragm, the thoracic aorta was carefully removed, stripped of adherent tissue and dissected systematically. Starting at the diaphragm, the descending thoracic aorta was cut in segments of 2 mm width (5–6 segments). Vessels were immersed in Krebs Ringer solution (KR solution, 37 1C, 95% O2/ 5% CO2, pH 7.4) with (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025, and glucose 11.1. 2.2. Isometric tension measurements Aortic segments were mounted in 10 ml organ baths as previously described (Van Hove et al., 2009). Isometric force was reported in mN. In all experiments, endothelial cells were present but basal NO formation was inhibited with a combination of Ω 300 μM N -nitro-L-arginine methyl ester (L-NAME) and 300 μM Ω N -nitro-L-arginine (L-NNA) and to avoid any vasomotor interference due to prostanoids, indomethacin (10 μM) was present. 2.3. Experimental conditions Before or after incubation with LCCB, window contractions were induced by depolarization of the aortic segments with 50 mM K þ in three different experimental conditions (Fransen et al., 2012a) (protocol, Fig. 1). In control conditions, contractions were elicited by replacing normal K þ with high K þ solution. Here, a transient increase of intracellular Ca2 þ due to activation and inactivation of a fast population of LCC causes transient contraction and is followed by a tonic contraction due to Ca2 þ influx via non-inactivating LCC (window contraction). Secondly, to avoid the fast component of contraction, segments were first depolarized in the absence of extracellular Ca2 þ (0Ca) and then contracted by addition of 3.5 mM Ca2 þ . In the third condition, the window

contraction was modulated by addition of the LCC agonist, 30 nM BAY K8644. These experiments were performed with preincubation of the segments with LCCBs and repeated for addition of cumulative concentrations of LCCBs on top of the 50 mM K þ induced contractions to induce relaxation (Fig. 1A–C versus D–F). High K þ -solution was prepared as KR solution, in which NaCl was replaced by KCl in equimolar concentrations. Ca2 þ -free solution (0Ca) was prepared by omitting Ca2 þ from the KR solution and adding 1 mM EGTA as chelator. To restore extracellular Ca2 þ , 3.5 mM Ca2 þ was added to 0Ca (0Ca/ þCa condition). To obtain Ca2 þ -free KR solution with different K þ concentrations, NaCl in the 0Ca solution was replaced by equimolar amounts of K þ . 2.4. Data analysis All results are expressed as mean 7S.E.M.; n represents the number of mice. Concentration–response curves were fitted with sigmoidal concentration–response equations with variable slopes, which revealed maximal contraction or relaxation responses (Emax) and the negative logarithm of the concentration resulting in 50% of the maximal contraction or relaxation (pEC50) for each vessel segment. Two-way ANOVA analysis with Bonferroni multiple comparison post-hoc test and paired or unpaired t-test (GraphPad Prism, version 5, GraphPad Software, San Diego, California, USA) were used to compare means of the different experimental groups. A 5% level of significance was selected. 2.5. Materials Sodium pentobarbital (Nembutals) was obtained from Sanofi (Brussels, Belgium), indomethacin from Federa (Belgium), L-NNA, L-NAME, and nifedipine from Sigma (Bornem, Belgium), verapamil hydrochloride, diltiazem hydrochloride, amlodipine, and (7) BAY K8644 from TOCRIS (Bristol, United Kingdom).

3. Results 3.1. Nifedipine, verapamil and diltiazem inhibit contractions elicited at depolarized membrane potentials Aortic segments were depolarized with 50 mM external K þ in control conditions, after stimulation of LCCs with 30 nM BAY K8644 or following re-addition of external Ca2 þ to segments depolarized with 50 mM K þ in 0Ca. In the absence of LCCB, basal force increased significantly with the addition of 30 nM BAY K8644 (þ2.147 0.99 mN, P o0.001, Fig. 1B), but decreased in 0Ca conditions ( 0.37 7 0.06, P o0.001, results not shown). Near steady-state contractions induced by depolarization with 50 mM K þ were 10.8 70.2 mN in control, 10.9 70.9 mN in BAY K8644 and 10.7 7 0.7 mN in 0Ca/ þCa and were not significantly different (n ¼4, P 40.05, Fig. 2). Pre-incubation of the aortic segments with 1 10–8 M nifedipine, 3 10–6 M verapamil or 1 10–6 M diltiazem inhibited K þ induced contractions, but the efficacy of the LCCBs was more dependent on the condition. In control conditions, LCCBs inhibited mainly the slow component of force development (Fig. 2A). The increase of basal force by 30 nM BAY K8644 was inhibited by preincubation of the segments with LCCB. After compensation for the BAY K8644-mediated increase in basal force (Fig. 2B, right axis), it was observed that none of the LCCBs significantly inhibited the subsequent, depolarization-induced contraction. Finally, when window contractions were elicited in the 0Ca/ þCa condition, inhibition of the depolarization-induced contraction was almost complete for the LCCBs (Fig. 2C). Hence, the inhibitory effects of


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Fig. 1. Representative examples of the inhibitory or relaxation effects by diltiazem of depolarization-induced contractions in three conditions. Contractions were measured in control conditions (A, D), after stimulation of LCCs with 30 nM BAY K8644 (B, E) and after re-addition of extracellular Ca2 þ to segments depolarized in Ca2 þ -free KRsolution with 50 mM K þ (C, F). In A–C, segments were incubated with diltiazem before eliciting the contraction. Horizontal bars indicate addition of 50 mM K þ (control, BAY K8644) or Ca2 þ in 50 mM K þ /0Ca. Asterisks indicate addition of diltiazem and arrows the application of 30 nM BAY K8644. In D–F diltiazem was applied on top of the respective contractions.

1 10–8 M nifedipine, 3 10–6 M verapamil or 1 10–6 M diltiazem ranged from zero (BAY K8644) over intermediate (control) to almost complete inhibition (0Ca/þ Ca). 3.2. Calcium channel blockers pre-incubated in control conditions Isometric contractions elicited by depolarization with 50 mM K þ displayed a double exponential time course with a fast component of 4.44 70.17 mN with time constant of 4.3 70.3 s and slow component of 9.87 70.73 mN with time constant of 284 7 10 s (n¼ 12). Both components were concentrationdependently inhibited by incubating the segments with the three classes of LCCBs (Fig. 3), suggesting that force development by depolarization is completely dependent on Ca2 þ influx via LCCs (Fransen et al., 2012a). The slow component of force development was, however, considerably more sensitive to the LCCB than the fast component. 3.3. Calcium channel blockers pre-incubated in the presence of 30 nM BAY K8644 Fig. 4 shows LCCB-inhibition curves after stimulation of LCC with 30 nM BAY K8644. In comparison with control conditions (Fig. 3), higher concentrations of the LCCB were needed to completely inhibit contraction. Also in the presence of BAY K8644, the three blockers displayed higher affinity for the slow than for the fast force component although this was less pronounced with verapamil.

3.4. Calcium channel blockers pre-incubated in 0Ca/50K When aortic segments were challenged with 0Ca to which 50 mM K þ was added, no contractions were observed. Subsequent addition of 3.5 mM Ca2 þ can only increase force via the LCC window Ca2 þ influx (Fransen et al., 2012a). In these conditions, force still developed in two phases, but the fast and slow components developed significantly slower than in control conditions (τ respectively 17.12 72.72 versus 5.63 70.33 s, P o0.05; 445727 versus 294 77 s, Po 0.01, n ¼4, Fig. 5). When LCCB were applied before the addition of 3.5 mM Ca2 þ , there was concentration-dependent inhibition of the contraction as in the other conditions, but in comparison with the control or BAY K8644 conditions lower LCCB concentrations were needed to get complete inhibition (Table 1). Moreover, in 0Ca/þ Ca conditions LCCB displayed equal affinity for the slow and fast force components (Figs. 5 and 6). 3.5. Fast and slow force components inhibition by LCCB in the different conditions Fig. 6 summarizes the concentration–response curves of the different drugs for the fast and slow force components in the different conditions. For both the fast and slow force components, LCCBs displayed the highest sensitivity for the LCCs in the 0Ca/ þCa condition, whereas sensitivities were the lowest after stimulation of LCCs with BAY K8644. Under control conditions and in the presence of BAY K8644, but not in 0Ca/þ Ca, LCCBs showed higher affinity for the slow than for the fast force component. The


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Fig. 2. Effect of LCCBs on the depolarization-mediated contraction. Effects of 1 10–8 M nifedipine, 3 10–7 M verapamil and 1 10–6 M diltiazem on contractions elicited by 50 mM K þ (white) in control conditions (A), after stimulation of LCCs with 30 nM BAY K8644 ((B) BAY K8644 data points are plotted against the right axis for each LCCB) and after re-addition of extracellular Ca2 þ to segments depolarized in Ca2 þ -free KR-solution with 50 mM K þ (C). *, **, ***: Po 0.05, 0.01, 0.001 presence versus absence of LCCB (n¼4).

respective shifts of log(IC50) for the fast and slow component in the BAY K8644 condition and for the slow component in the 0Ca/ þCa condition, with respect to log(IC50) in control conditions (Fig. 7), further revealed that the shifts were similar for the respective LCCBs. These results suggest that experimental conditions can be chosen in which a concentration of a LCCB has minimal effects on the fast component of contraction, whereas the slow component is severely compromised. This is illustrated in experiments in which Ca2 þ influx was maximized at the start of depolarization by 124 mM K þ (Fig. 8). Amlodipine was added in increasing concentrations (50 nM up to 500 nM) ten minutes before contraction of the segments with 124 mM K þ . At concentrations of 50, 100 and 250 nM, amlodipine affected only the slow phase of force development. The highest concentration (Fig. 8D) also partially inhibited the fast component of contraction.

3.6. LCCB-mediated relaxation of the various depolarization-induced contractions When LCCBs were added on top of the 50 mM K þ -contractions in the 0Ca/þCa condition, none of the LCCBs concentration– relaxation curves significantly shifted with respect to the control curves. However, in the presence of 30 nM BAY K8644, significantly higher concentrations of LCCB were needed to induce the same relaxing effect as in control or 0Ca/þCa (Fig. 9). Remarkably, while log IC50-values were similar for all LCCBs in control and 0Ca/ þCa2 þ conditions, Δlog IC50-values in the BAY K8644 condition

were significantly higher for nifedipine than for verapamil and diltiazem (see also Table 1).

4. Discussion As expected, representatives of the three LCCB classes i.e. nifedipine, verapamil and diltiazem inhibit contraction of aortic segments with different sensitivities. Remarkably, the sensitivity of each LCCB depended on the way by which Ca2 þ influx in the absence or presence of LCCB was elicited at constant depolarized voltage (50 mM K þ ). LCCBs block LCC in smooth muscle cells, neurons or cardiomyocytes with high affinity in a voltage- and frequency-dependent way. To explain and describe their effects, one often refers to the “modulated receptor theory” (Bean et al., 1986; Godfraind, 2005; Morel and Godfraind, 1987). According to this hypothesis, LCCBs have different affinities for LCCs in the resting (R), open (O) or inactivated (I) state. Hence, their apparent dissociation constant depends on membrane voltage. Because most LCCBs preferentially bind to LCCs in depolarized cells, they are supposed to have higher affinity for I than for R channels. To test this hypothesis in the present study, the relative amount of I versus R Ca2 þ channels was kept constant by clamping the aortic segments to a depolarized membrane potential of 20 mV (at 50 mM K þ ). Our results, however, showed remarkable changes of the apparent sensitivity of each drug in different experimental conditions. In control conditions depolarization of mouse aortic segments with extracellular K þ above 20 mM, caused force to develop with


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Fig. 3. Effect of LCCBs on the depolarization-mediated contraction in control conditions. Isometric contractions by 50 mM K þ in the absence and presence of increasing concentrations of nifedipine ((A) in nM, n ¼4), verapamil ((C) in mM, n¼ 4) and diltiazem ((E) in mM, n ¼4). The amount of inhibition by the different LCCBs was measured at 20 s (fast force component) and at 575 s (slow force component) and revealed the different [LCCB]-inhibition curves as shown in B, D and F. *, **, ***: P o0.05, 0.01, 0.001 fast versus slow force component.

Fig. 4. Effect of LCCBs on the depolarization-mediated contraction in the presence of BAY K8644. [LCCB]-inhibition curves after incubation of the aortic segments (n ¼4) with 30 nM BAY K8644 in the presence of increasing concentrations of nifedipine (A), verapamil (B) and diltiazem (C). The amount of inhibition by the different LCCBs was measured at 20 s (fast force component) and at 575 s (slow force component). **, ***: Po 0.01, 0.001 fast versus slow force component.

a bi-exponential time course. The fast phase of isometric contraction occurs by transient Ca2 þ influx via a population of LCCs which activated and subsequently inactivated rapidly, giving rise to a phasic, transient contraction. The slow phase is voltage-dependent

at membrane potentials where activation and inactivation curves of the LCCs overlap (window voltage range) and is due to a population of channels remaining in the open state (time-independent) (Fleischmann et al., 1994; Fransen et al., 2012a). The half-maximal


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Fig. 5. Effects of LCCBs on window contraction in 0Ca/Ca conditions. [LCCB]-inhibition curves after incubation of the aortic segments (n¼4) with 0Ca/50 mM K þ to which increasing concentrations of nifedipine (A), verapamil (B) and diltiazem (C) were added. The amount of inhibition by the different LCCB was measured at 20 s (fast force component) and at 575 s (slow force component). Table 1 IC50-values of LCCBs under different experimental conditions. Condition

PRI nifedipine (nM) POI nifedipine (nM) PRI verapamil (lM) POI verapamil (lm) PRI diltiazem (lM) POI diltiazem (lM)

Fast component

Slow component

BAY K8644

Control

0Ca/þ Ca

BAY K8644

Control

0Ca/ þ Ca

2457 57 – 3.7 7 0.2 – 11.1 7 2.5 –

237 3 – 0.647 0.07 – 1.737 0.25 –

2.8 70.9 – 0.05 70.01 – 0.247 0.04 –

57 79a 99 78 2.3 70.2a 1.31 70.10 4.4 71.0a 3.5 70.4

87 3b 3.8 7 2.1 0.417 0.11a 0.137 0.01 1.107 0.21a 0.65 7 0.09

3.2 7 0.9 3.7 7 1.7 0.06 7 0.02 0.26 7 0.08 0.247 0.09 0.96 7 0.09

IC50-values in nM (nifedipine, N) or mM (verapamil, V and diltiazem, D) for fast and slow force components during pre- (PRI) or post-incubation (POI) in the different experimental conditions of depolarization with 50 mM K þ . a, b: Po 0.05, 0.01 slow versus fast component IC50 (n¼ 4).

Fig. 6. Summary of the effects of LCCBs on the depolarization-mediated contraction in different conditions. [LCCB]-inhibition curves for fast (white) and slow (black) components of force developed in control (C), 30 nM BAY K8644 (B) and 0Ca2 þ /þ Ca2 þ (0) (n¼ 4 for each condition). The dotted horizontal lines show 50% inhibition, the vertical lines show the half maximal effective dose for the fast component.

inhibitory concentration (IC50) of the LCCBs was significantly lower for the slow than for the fast force component, suggesting a high affinity for open, non-inactivating LCCs. According to this view, LCCBs should have a certain, fixed affinity for open LCCs, but dependent on the duration of the open state they may have as many different apparent affinities as there are experimental conditions.

BAY K8644, a LCC agonist, strongly potentiates the contractile effects of moderate depolarization, but only slightly increases these of strong depolarization (30 and 60 mM K þ ) (Asano and Nomura, 1999; Fusi et al., 2003). Similar results were obtained in the present study at 50 mM K þ . Because BAY K8644 shifts the activation curve in the hyperpolarizing direction, but does not or only minimally affect the inactivation curve (Teramoto et al.,


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2005), the window Ca2 þ current and contraction voltage range are shifted in the hyperpolarizing direction. Sustained Ca2 þ influx via non-inactivating LCCs in basal conditions caused a basal contraction of about 2 mN in the present study, which was very sensitive to low LCCB concentrations (Fig. 2). Because in basal conditions the amount of channels in the I-state is not altered in the presence of BAY K8644, this cannot be explained in terms of the modulated receptor hypothesis. During depolarization with high K þ , however, BAY K8644 increased the log(IC50) of the different LCCBs by 0.8–0.9 log M (Fig. 6). The shift of the inhibition curves was

Fig. 7. Shift in log IC50-values of the different LCCBs under BAY K8644 and 0Ca/Ca conditions as compared to control conditions. Shifts (Δlog(IC50)) of the LCCB inhibition curves for fast and slow component in the BAY K8644 condition and for the slow component in the 0Ca/þ Ca condition for nifedipine, verapamil and diltiazem. *, ***: P o0.05, 0.001 versus control, ns: non-significant differences between the drugs.

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independent of the drug and the force component (fast or slow) and might be due to the fact that BAY K8644 and the LCCB bind to open channel receptor sites, which are in close proximity and allosterically linked (Hockerman et al., 1997). Nifedipine and BAY K8644 are both dihydropyridines and share the same binding site (Lacinova, 2005). This was evident as an increased IC50 of 99 nM compared with 57 nM (Table 1) when nifedipine was added after or before eliciting contraction with 50 mM K þ and BAY K8644. Elimination of the fast component of contraction by depolarizing segments in 0Ca2 þ resulted in steady-state contractions upon re-addition of Ca2 þ , which are due to Ca2 þ influx via open, noninactivating Ca2 þ channels only (Fleischmann et al., 1994; Fransen et al., 2012a). Force still followed a bi-exponential time course, but the fast and slow components were, in contrast to the control and BAY K8644 conditions, equally sensitive to inhibition by the LCCBs. Hence, they are due to Ca2 þ influx via the same population of noninactivating LCCs. LCCB pre-incubation inhibited these window contractions more effectively than in control although in both conditions window contractions in the absence of LCCB are equal. Hence, addition of the LCCBs on top of the isometric contractions, when steady-state window contraction is the same, revealed equal sensitivity to the different LCCBs. Also these results are not compatible with the modulated receptor hypothesis, but suggest a high affinity of the LCCBs for open, non-inactivating LCCs. Here, they bind within a region close to the pore of the channel where they may interact with Ca2 þ -binding sites (Catterall, 2011). This may explain their higher efficacy (0Ca/50K conditions) when present prior to contractions (pre-incubated) than when they were applied after attaining steady-state contractions (Table 1). Moreover, it may also explain why the fast component influences LCCB-affinity for the slow component in control conditions, where fast and transient Ca2 þ influx precede tonic window Ca2 þ influx (Fransen et al., 2012a).

Fig. 8. Effect of increasing concentrations of amlodipine on the depolarization-mediated contraction. Contraction by 124 mM K þ of control segments or segments incubated with amlodipine (50–500 nM, A–D). *, **, ***: P o0.05, 0.01, 0.001 versus control.


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Fig. 9. Shifts of relaxation curves for nifedipine and log(IC50)-values of the different LCCBs in control, BAY K8644 and 0Ca/ þCa conditions. Nifedipine concentration– relaxation curves for contractions induced by 50 mM K þ in control (C), 30 nM BAY K8644 (B) and 0Ca/ þCa (0) conditions (A). Cumulative concentrations of nifedipine were applied on top of the contractions. Shifts (Δlog(IC50)) of the LCCB relaxation curves in the BAY K8644 and 0Ca/ þ Ca conditions as compared with control conditions for nifedipine, verapamil and diltiazem (B). nnnPo 0.001 versus control, ns: non-significant difference versus control, #, ##: Po 0.05, 0.01 versus nifedipine (n ¼4).

LCCBs inhibit LCC-mediated contraction with higher sensitivity in the VSM cells than in cardiomyocytes and are more effective in hypertensive than in normotensive subjects. For example, nifedipine has higher affinity for LCCs in human coronary artery than in heart (IC50 respectively 5 and 70 nM) (Godfraind et al., 1984). Specific binding of amlodipine to hypertensive arteries was significantly higher than to normotensive at various K þ concentrations (Morel and Godfraind, 1994). These results were explained in terms of the modulated receptor model by preferential binding of LCCBs to I channels. At physiological pH of 7.2–7.4, verapamil, diltiazem and amlodipine are primarily ionized (þ ), whereas nifedipine, because of its lower pKa, is mostly present in uncharged form. According to the modulated receptor model, these differences in pKa-values and concomitant ionization of the drugs should determine the apparent affinity for the different channel states. Comparing the dihydropyridines, nifedipine and amlodipine, might suggest higher affinity of nifedipine than amlodipine for the closed inactivated channels state. In accordance, we demonstrated that pre-incubation with amlodipine (250–500 nM) completely inhibited the window contraction without significant effects on the fast contraction phase. Hence, apparent dissociation constants for inhibition of the fast or slow component were very different for amlodipine. Roughly estimated, the IC50 for the slow component (window contraction) is about 5 10–8 M, whereas this for the fast component is larger than 5 10–7 M (more than 10 times less effective). Nifedipine, verapamil and diltiazem were respectively 3 times, 1.5 times and 1.5 times more effective for the window contraction in control conditions. In the present study, LCCBs inhibited the depolarization-mediated contraction with different apparent affinities, which depended on the experimental condition, but not on the ionization of the drugs (compare amlodipine with nifedipine). Therefore, we suggest that LCCBs have the highest affinity for the open, non-inactivating LCCs residing in the window voltage range. We and others have shown that smooth muscle cells have less polarized resting membrane potential than cardiomyocytes and that VSM cells in baseline conditions display a continuous influx of Ca2 þ via LCCs (Cobine et al., 2007; Fransen et al., 2012a; Sonkusare et al., 2006; Zhang et al., 2007). The higher sensitivity of LCCB for window contractions might also have important implications for NO-dependent relaxation of the aortic segments. NO shifts the voltage-dependence of the window Ca2 þ influx and contraction to depolarized potentials (Fransen et al., 2012b). As a consequence, the amount of baseline Ca2 þ influx is inversely related to the amount of basal NO produced by the vascular

endothelium. Endothelial dysfunction, as occurring in many vascular diseases, is expected to increase window Ca2 þ influx and basal constriction of aortic segments (compare with BAY K8644) and to increase their sensitivity to LCC block (no interaction with LCCB binding site). Moreover, inhibition of the LCCs with nifedipine increased the sensitivity of the VSM cells to relaxation by NO (Van Hove et al., 2009), suggesting that NO and probably all classes of LCCB act in synergy to dilate the arterial segments. Recent reports demonstrated that expression of LCC isoforms strongly differs between aortic and cardiac tissue and that this differential expression also implicated alterations in LCCB affinity. (Zahradnikova et al., 2007). Following these observations, selective sensitivity of different voltage-dependent calcium channel subtypes to dihydropyridines (and other LCCB) may provide an alternative explanation for the preferential peripheral vascular selectivity of LCCB inhibition, the voltage-dependence of LCCB effects and the higher sensitivity of the slow than of the fast force component for the different LCCBs in the present study (Liao et al., 2005, 2007; Morel and Godfraind, 1987).

5. Conclusion The present study showed that the efficacy of the three classes of LCCBs in their inhibition of the depolarization-induced contraction of mouse aortic segments depends on the window Ca2 þ influx via LCCs. Promotion of the window Ca2 þ influx increased the efficacy of the different LCCBs, whereas stimulation of Ca2 þ influx with the LCC agonist BAY K8644 had the opposite effect. Our results indicate that LCCBs have a higher apparent affinity for noninactivating, open LCCs, which are of greater importance in vascular than in cardiac tissue and, as such, they do not support the modulated receptor model. Our results further suggest an important (patho)physiological role of the window LCC Ca2 þ influx for reactivity of blood vessels and, because of the increased sensitivity to LCCBs, for the management of hypertension and vascular compliance.

Acknowledgments Research was funded by the Fund for Scientific Research Flanders (FWO) and the University of Antwerp. C.M. Michiels is a fellow of the Agency for Innovation through Science and Technology (IWT).


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Antinociceptive effects of Ca2+ channel blockers.

Poly(ethylene glycol) as a scaffold for high-affinity open-channel blockers of the mouse nicotinic acetylcholine receptor.

[Central effect of Ca2+ channel blockers: multiple sites of action].

Cellular processes in atherogenesis: potential targets of Ca2+ channel blockers.

Role of Ca2+ channel in cardiac excitation-contraction coupling in the rat: evidence from Ca2+ transients and contraction.

Contraction of rat thoracic aorta strips by endothelin-1 in the absence of extracellular Ca2+.

Vasorelaxing effect in rat thoracic aorta caused by fraxinellone and dictamine isolated from the Chinese herb Dictamnus dasycarpus Turcz: comparison with cromakalim and Ca2+ channel blockers.

Nelumbo nucifera leaves extracts inhibit mouse airway smooth muscle contraction.

Calcium channel blockers inhibit retinal degeneration in the retinal-degeneration-B mutant of Drosophila.

Intracellular Ca2+ release by flufenamic acid and other blockers of the non-selective cation channel.

Modulation of the Ca2+ channel voltage sensor and excitation-contraction coupling by silver.

Basal activity of voltage-gated Ca(2+) channels controls the IP3-mediated contraction by α(1)-adrenoceptor stimulation of mouse aorta segments.

Blockers of platelet-derived growth factor-activated nonselective cation channel inhibit cell proliferation.

L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current.

Dibutyryl cyclic AMP and forskolin inhibit phosphatidylinositol hydrolysis, Ca2+ influx and contraction in vascular smooth muscle.

Selectivity of Ca2+ channel blockers in inhibiting muscular and nerve activities in isolated colon.

Structure-activity relationship of Ca2+ channel blockers: a study using conformational analysis and chemometric methods.

Analysis of the vasorelaxant action of jatrophone in the isolated aorta of the rat: influence of potassium channel blockers.

Ca2+ release from isolated sarcoplasmic reticulum of guinea-pig psoas muscle induced by K(+)-channel blockers.

Chloride channel blockers.

Monoamine NMDA receptor channel blockers inhibit and potentiate native and recombinant proton-gated ion channels.

Dissecting out the complex Ca2+-mediated phenylephrine-induced contractions of mouse aortic segments.

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High and low affinity Ca2+ binding to the sarcoplasmic reticulum: use of a high-affinity fluorescent calcium indicator.