L-type Ca2+ channels play a dominant role in blood pressure regulation as suggested by the fall in mean arterial blood pressure in mice with selective ablation of the channel gene (CACN1C) in vascular smooth muscle cells (VSMCs) (Moosmang et al., 2003), the increased CACN1C gene expression in VSMCs in rodent models of hypertension (Pesic et al., 2004; Rhee et al., 2009), the anti-hypertensive effect of L-type Ca2+ channel blockers (CaBs) (Mancia et al., 2007) and the relationship between an autoantibody against vascular L-type Ca2+ channels and clinical characteristics of hypertensive patients (Zhou et al., 2008). Transcripts and protein expression of CACN1C are found widely in the cardiovascular system, where the ion channels serve the time- and voltage-dependent influx of Ca2+ ions to initiate muscle contraction (Akata, 2007; Bers, 2002). It is now recognized, however, that not only peripheral resistance but also arterial compliance is of great importance, especially in old age (systolic) hypertension (Belz, 1995; Mitchell, 1999; Westerhof et al., 2009). In the large conduit arteries, L-type Ca2+ channels are important determinants of their mechanical properties and compliance, which are such that blood pressure and flow are propagated between the heart and arterioles and that thereby pulsatile flow is transformed into steady flow due to the “windkessel” effect (Westerhof et al., 2009). For example, CaBs increase vascular compliance of large elastic vessels and may be of importance for the pathogenesis and prognosis of cardiovascular complications such as atherosclerosis, left ventricular hypertrophy and heart failure (Bellien et al., 2010; Belz, 1995; Essalihi et al., 2007; Safar et al., 1989; Slama et al., 1995; Vayssettes-Courchay et al., 2011). Further evidence for a role of L-type Ca2+ channels in atherosclerosis was obtained in carotid and femoral VSMCs, where the L-type Ca2+ channel gene expression differs between atherosclerotic versus non-atherosclerotic regions (Tiwari et al., 2006). Not only Ca2+ channels but also endothelial released nitric oxide (NO) may be involved in these processes. Inhibition of endothelial NO synthase (eNOS) with NΩ-nitro-L-arginine methyl ester (L-NAME) or NΩ-nitro-L-arginine (L-NNA) causes hypertension and the decrease of basal endothelial NO release or availability may be at the basis of increased reactivity to vasoconstrictors in hypertension (Panza et al., 1993). Furthermore, in the beginning of plaque development in animal models and in patients with atherosclerotic symptoms or risk factors, eNOS activity and concomitant NO release is altered, especially in atherosclerosis-prone aortic segments (Fransen et al., 2008; Kauser et al., 2000; Vanhoutte et al., 2009). Recently, we showed that L-type Ca2+ influx and its inhibition with CaBs may also have consequences for the capacity of NO to relax constricted mouse aorta. The relaxing efficacy of NO in mouse aorta was dependent on the contractile agonist, and more specifically, decreased when the contraction was mainly elicited via L-type Ca2+ influx, but increased when Ca2+ influx was partially inhibited with CaBs (Van Hove et al., 2009). The above observations may suggest an interaction between basal NO and VSMC L-type Ca2+ channels. An inhibitory effect of NO on Ca2+ currents in vascular smooth muscle and cardiomyocytes has been described (Blatter & Wier, 1994; Fischmeister et al., 2005; Tsai & Kass, 2009), but has not been associated with L-type Ca2+ channel-mediated contractions in mouse aorta. This chapter will focus on the specific role of L-type Ca2+ channels in vasoconstriction and dilation and the interplay between NO and these L-type Ca2+ channels.
2. Interaction between endothelial and vascular smooth muscle cells in intact mouse aorta
It is generally assumed that the increase of intracellular Ca2+ in vascular endothelial cells results in release of NO via complex interactions with calmodulin, caveolin, endothelial NOS (eNOS) and tetrahydrobiopterin. NO released from the endothelial cells stimulates guanylate cyclase (sGC) to produce cGMP, which causes relaxation of the VSMCs via reduction of intracellular Ca2+ and the Ca2+-sensitivity of the contractile elements. Reduction of intracellular Ca2+ can occur via different mechanisms: inhibition of IP3-mediated Ca2+ release from intracellular Ca2+ stores; removal and sequestration of intracellular Ca2+ pump mechanisms and/or both direct and indirect inhibition of influx of extracellular Ca2+ through voltage-gated Ca2+ channels (Tsai & Kass., 2009). In baseline conditions, i.e. in the absence of receptor-stimulation with agonists such as acetylcholine (ACh), there is basal release of NO through constitutive activity of endothelial eNOS in the mouse aorta. In isolated arteries, the basal NO production can be assessed by comparing force development by the SMCs in response to a vasoconstrictor such as phenylephrine in the absence and presence of NOS inhibitors. On the other hand, receptor-stimulated eNOS activity can be determined based on its sensitivity for agonists like ACh to induce endothelium-dependent relaxation of pre-constricted segments. Remarkably, basal and stimulated eNOS activity are differentially regulated. In rat aorta, basal NO release is more sensitive to destruction by superoxide anion, and can be selectively inhibited by NΩ-monomethyl-L-arginine (L-NMMA) and asymmetric NΩ-dimethyl-L-arginine (ADMA) without ACh responses being affected (Al-Zobaidy et al., 2011; Frew et al., 1993; Mian & Martin, 1995).
Differences between basal and stimulated eNOS activity were also observed in apolipoprotein E-deficient (apoE-/-) mice which develop atherosclerotic lesions in the thoracic aorta spontaneously at the age of 6 months, a process, which can be effectively accelerated by feeding these mice a high cholesterol diet. The mice exhibit preferential lesion formation in the aortic root and arch, and the proximal and distal part of the thoracic aorta (Crauwels et al., 2003; Nakashima et al., 1998; Reddick et al., 1994). This makes the apoE-/- mouse an interesting model to study differences between atherosclerosis-prone and atherosclerosis-resistant regions. We have previously shown that at the age of 4 months, hence, before development of any visible lesions in the apoE-/- mouse model, basal and agonist-stimulated NO release decreased, respectively increased in atherosclerosis-prone aortic segments of apoE-/- in comparison with wild-type (WT) mice (Fransen et al., 2008). Because no differences in eNOS expression were found between aorta of WT and apoE-/- mice, this indicated that the release or efficacy of NO differed between both mouse strains. In non-stimulated conditions the compromised basal NO release in the apoE-/- mouse might be related to lower intra-endothelial Ca2+ concentrations because it could be restored to “normal” wild-type levels by experimentally increasing intracellular Ca2+. Agonist (ACh)-stimulated VSMC relaxation was temporally and dose-dependently related to the increase of endothelial Ca2+. Although the agonist-stimulated increase of endothelial Ca2+ was similar in both strains, apoE-/- segments were significantly more sensitive than WT segments in their relaxation to ACh. Results of these studies might suggest an important role of basal and stimulated eNOS activity in the pathophysiology of atherosclerotic lesions.
In functional and molecular biological studies we have further demonstrated that before development of any visible lesions, not only endothelial cells but also the SMCs of the thoracic aorta of apoE-/- mice displayed altered intracellular Ca2+ homeostasis in comparison with WT VSMCs (Van Assche et al., 2007) and that within the apoE-/- strain the smooth muscle transcriptome is altered at atherosclerosis-prone versus atherosclerosis-resistant locations (Van Assche et al., 2011). Hence, not only endothelial, but also VSMC function is altered in atherosclerosis-prone versus -resistant segments. Whether the cross-talk between both cell types is affected in both directions during the process of atherosclerosis, is far less studied. At least, we have shown before that NO-dependent vasodilation is dependent on the agonist causing contraction and whenever VSMC function alters during development of atherosclerosis, this is also expected to affect the efficacy of endothelial cell stimulation.
3. Relaxation of VSMCs depends on the agonist causing contraction
Contractions of isolated arteries are often studied by initiating VSMC intracellular Ca2+ increase with two widely used and different stimuli. On the one hand, elevated external K+ depolarises the VSMCs and elicits multiphasic Ca2+ signalling and force development through influx of extracellular Ca2+ via L-type Ca2+ channels. On the other hand, α1-adrenoceptor stimulation with phenylephrine also causes multiphasic Ca2+ signalling and force development, but the signalling is different from the depolarization-induced signalling. The vasodilator effects of NO differ significantly for both contractile agents. Bigger contractions produced by increasing concentrations of phenylephrine were all equally sensitive to relaxation by ACh (figure 1) or the NO donor DEANO. In contrast, when the VSMCs were clamped to depolarized membrane potential (Vm) with high extracellular K+ concentrations, relaxation by ACh or DEANO became attenuated as K+-induced force augmented (figure 1) (Van Hove et al., 2009).
Simultaneous measurement of intracellular Ca2+ and force development in SMCs of de-endothelialised aortic segments revealed important differences between Ca2+ signals in depolarization (50 mM K+)- or phenylephrine-constricted segments upon addition of DEANO, a donor of exogenous NO (figure 2). For phenylephrine-induced contractions and VSMC Ca2+ signals, addition of NO resulted in an abrupt decrease of intracellular Ca2+, which could be blocked by adding cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA), but not by inhibition of sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Results indicated a direct stimulation by NO of Ca2+ re-uptake to the SR by SERCA, which is in accordance with previous reports (Cohen et al., 1999; Cohen & Adachi, 2006; Van Hove et al., 2009). Contractions, but not Ca2+ signals of VSMCs, depolarized with 50 mM K+, declined upon addition of exogenous NO. Nevertheless, as seen for contractions by depolarization with elevated K+, also phenylephrine-induced contractions are associated with depolarization and opening of L-type Ca2+ channels (Akata, 2007; Plane et al., 1998; Quignard et al., 2000; Richards et al., 2001; Van Hove et al., 2009). If L-type Ca2+ influx is an important determinant of the vasodilator capacity of NO, why then is there an abrupt decrease of intracellular Ca2+ in the VSMCs after addition of exogenous NO to phenylephrine-elicited contractions and not to 50 mM K+-evoked contractions? Reduction of Ca2+ influx in depolarized segments with CaBs increased NO´s capacity to dilate the segments, indicating that not only the depolarized Vm of the VSMCs, but also the amount of Ca2+ influx determines the efficacy of NO to cause complete relaxation (Van Hove et al., 2009). Therefore, it was decided to investigate the phenylephrine- and K+-induced isometric contractions in more detail.
4. Phenylephrine-induced contractions and NO
Addition of phenylephrine causes intracellular Ca2+ release from intracellular SR Ca2+ stores via activation of IP3-receptors (Karaki et al., 1997). Indeed, in the absence of extracellular Ca2+ phenylephrine causes emptying of the SR Ca2+ stores and elicits a transient contraction (figure 3). This transient contraction in Ca2+-free conditions is higher with the constitutive activity of eNOS because inhibition of basal NO release decreased the IP3-mediated contraction by phenylephrine (figure 3). These results are compatible with previous observations that basal NO stimulates SERCA activity (Cohen et al., 1999; Cohen & Adachi., 2006; Van Hove et al., 2009). Thereby, the Ca2+ content of the SR stores of the VSMCs may be increased and may then lead to higher IP3-mediated contractions. The transient contractions by phenylephrine in the absence of extracellular Ca2+ were dose-dependently inhibited by exogenous NO (DEANO, figures 4A, B). This suggests that although basal NO might stimulate Ca2+ uptake to the SR via stimulation of SERCA, it inhibits the IP3-mediated release of Ca2+ or the concomitant transient contraction. When these experiments were repeated with addition of ACh to promote endothelial NO release, however, relaxation of phenylephrine-induced contractions by increasing concentrations of ACh in the absence of external Ca2+ was completely absent (figures 4C, D). These experiments demonstrate the absolute necessity of external Ca2+ to induce ACh-stimulated release of NO from the endothelial cells. Although ACh has been described to release Ca2+ from the SR (Fransen et al., 1998), this release does not stimulate eNOS to inhibit IP3-mediated contractions in mouse aortic SMCs. Hence, release of endogenous NO is dependent upon influx of extracellular Ca2+ into the endothelial cells. It has been observed before that Ca2+ influx into the endothelial cells stimulated NO production more potently than Ca2+ released from internal stores, which evokes little NO production (Isshiki et al., 2002; Isshiki et al., 2004). This is also consistent with old observations that the resting level of cGMP falls after removal of external Ca2+ (White & Martin, 1989). In other studies, it has been shown that the TRPV4-mediated Ca2+ signal was required for eNOS activation by ACh (Adapala et al., 2011; Zhang et al., 2009), also illustrating the necessity of Ca2+ influx for endothelial NO release. Isshiki et al. (Isshiki et al., 2004) suggested that agonist-stimulated eNOS activity in the endothelial cells was sensitive to external Ca2+-dependent acute changes in intracellular subcortical Ca2+ signals and that basal eNOS activity was maintained and regulated by subplasmalemmal Ca2+ equilibrated with extracellular Ca2+.
Concomitant with the transient contraction, due to IP3-mediated Ca2+ release, α1-adrenoceptor stimulation with phenylephrine causes also tonic contractions, which are measureable only in the presence of external Ca2+ (figure 5A). These contractions are mediated by Ca2+ influx from the extracellular medium via Ca2+-permeable channels (SOCE or store-operated Ca2+-entry). The Ca2+ entry occurs via L-type Ca2+ channels, which can be inhibited with CaBs (figure 5B) and via non-selective cation channels, which can be inhibited with 50 µM 2-aminoethoxydiphenylborane (2-APB) (Bootman et al., 2002; Peppiatt et al., 2003) (figure 5C). Addition of verapamil (CaB) or 2-APB between the phasic and tonic contraction (figure 5B, C) results in reduced SOCE and SOCE-related contraction. The SOCE contraction in the presence of 2-APB can be completely inhibited with CaBs, indicating it is mediated by L-type Ca2+ influx only, whereas the SOCE contraction in the presence of verapamil can be completely inhibited with 2-APB, indicating it is mediated by non-selective cation Ca2+ influx only. Figure 6 compares relaxation by ACh of phenylephrine-pre-constricted aortic segments in control conditions (contractions in the presence of external Ca2+), upon re-addition of external Ca2+ (SOCE contractions) and following inhibition of the cation channel-mediated Ca2+ influx with 2-APB.
Relaxation by ACh of these tonic phenylephrine-induced contractions were not different for control contractions (normal Krebs-Ringer solution with external Ca2+) or SOCE contractions (elicited by re-addition of external Ca2+ after emptying the stores with phenylephrine) (figure 6A). When Ca2+ influx via non-selective cation channels during the phenylephrine-induced SOCE contraction was inhibited with 2-APB, the tonic contraction induced by phenylephrine was significantly smaller and relaxation by ACh was reduced by about 50% (figure 6B). Results indicate that the relaxing capacity of NO is small when contractions are mediated by Ca2+ influx via L-type Ca2+ channels as shown before for depolarization-mediated contractions. These results further confirmed the hypothesis that the difference in NO-mediated relaxation of segments depolarized with K+ versus phenylephrine, as shown in figure 1, is due to the amount of Ca2+ influx via L-type Ca2+ channels and the membrane potential (Vm) of the VSMCs. Whereas relaxation of contractions elicited with high external K+ was not complete, was not associated with re-uptake of Ca2+ to the SR or with a significant decrease of intracellular Ca2+, relaxation of phenylephrine-induced contractions was accompanied by inhibition of IP3-mediated contractions, by NO-mediated re-uptake of Ca2+ to the SR, by repolarising the VSMC Vm and by inhibition of Ca2+ influx during the tonic contraction (Van Hove et al., 2009). To investigate whether the effects of NO on relaxation of VSMCs were voltage-dependent, the effects of NO were studied at different external K+ concentrations or Vm of the VSMCs.
5. Depolarization-induced contractions and NO
Aortic segments are unable to produce force upon depolarization with high K+ in 0 mM Ca2+. In the presence of extracellular Ca2+, depolarization-induced contractions could be completely inhibited with CaBs, but were not affected by blocking the ryanodine receptor (15 µM ryanodine) or IP3 receptor of the SR (50 µM 2-APB) (Peppiatt et al., 2003). These observations suggested that K+-induced contractions of mouse aortic segments are solely supported by influx of Ca2+ via L-type Ca2+ channels. Figure 1 showed that ACh-induced relaxations of contractions produced by 20 mM extracellular K+ (mild depolarization) were near complete and occurred with a sensitivity close to the ACh-sensitivity for relaxations of phenylephrine-induced contractions. However, for stronger depolarization, relaxation was severely compromised. The latter results suggest that relaxation of depolarization-induced contractions depended upon Vm of the VSMCs. Vm of the endothelial cells did not significantly contribute to the sensitivity to ACh because ACh-induced relaxations of phenylephrine-induced contractions at high extracellular K+ in the presence of CaB were not attenuated (Van Hove et al., 2009). Why are relaxations of depolarized segments dependent on Vm of the VSMCs, i.e. complete for phenylephrine-induced contractions, which are also accompanied by depolarization of the segments, nearly complete for mild K+-induced depolarization and severely attenuated for high K+-induced depolarization? To solve this question we investigated the depolarization-induced contraction in more detail.
5.1. Depolarization-induced window contractions
Electrophysiological studies in isolated SMCs revealed the occurrence of a voltage range (window), in which L-type Ca2+ channels do not inactivate, leading to a “time-independent” influx of Ca2+ ions (Curtis & Scholfield, 2001; Fleischmann et al., 1994; Ganitkevich & Isenberg, 1990; Matsuda et al., 1990; Smirnov & Aaronson, 1992). This window influx of Ca2+ ions has been shown to lead to an increase of intracellular Ca2+ within the SMCs (Fleischmann et al., 1994), but has never been associated with the tonus of blood vessels. By clamping Vm of the endothelial and SMCs in aortic segments to depolarized potentials by elevating extracellular K+, we were able to show a window contraction within the voltage range of overlap of activation and inactivation curves of the L-type Ca2+ channels (manuscript submitted, see also figure 8). In order to correlate the extracellular K+ with Vm of the VSMCs of the aortic segments, Vm was measured with sharp glass intracellular microelectrodes (filled with 2 M KCl, tip resistances between 65 and 90 MΩ, HEKA EPC9 amplifier in the zero current-clamp mode). Deviation of measured Vm from the K+ equilibrium potential (Nernstian VK) was largest at physiological K+ concentrations, indicating that in non-stimulated VSMCs other ions than K+ contribute in setting the resting Vm (figure 7A). However, by adding levcromakalim, a drug that sets Vm to VK by activating ATP-dependent K+ channels (Knot & Nelson, 1998; Weston et al., 2002), Vm of the VSMCs could be hyperpolarised to VK. Indeed, when measured with intracellular microelectrodes, the resting Vm of VSMCs of aortic segments was -60.1±2.6 mV (n=8), which could be repolarized with levcromakalim to -84 mV.
Taking into account the shift of the contraction curves at different K+ in the presence of levcromakalim (see figure 8), Vm of the VSMCs evoked by increasing extracellular K+ (Vclamp) could be estimated. Vclamp changed with K+ concentration according to Vclamp = 61*log(([K+]o + 6)/[K+]i)) with [K+]o the extracellular K+ concentration in mM, which is elevated by 6 mM as revealed by the shift induced by levcromakalim (figure 8), and [K+]i the intracellular K+ concentration (assumed to be 140 mM). Figure 7B shows that the relationship between the estimated and measured Vm was close to unity. This means that in the following graphs the K+ axis could be replaced by a Vclamp axis. This is shown in figure 8, where the effects of levcromakalim on K+-induced isometric contractions at different K+ concentrations are considered. By adding levcromakalim, the aortic segments were all set to the same Vm, i.e. VK.
Depolarization by elevated K+ causes the isometric contraction to increase until maximal force of 100% was attained at 50 mM K+. At higher K+, force decreased again, leading to a bell-shape of the force-K+ relationship, which agrees well with the bell-shaped voltage dependency of the L-type Ca2+ currents measured with voltage-clamp in single, isolated SMCs. The “steady-state” contractions observed at each K+ concentration are window contractions, which are due to influx of Ca2+ via non-inactivating L-type Ca2+ channels. Differentiation of these contractions (%force/mM K+ change) reveals a K+-dependent contraction curve, which fits very well with the intracellular Ca2+ mobilized during the depolarizing voltage clamp steps in single SMCs (Fleischmann et al., 1994). It is clear that levcromakalim, which causes repolarization of Vm from -60 to -80 mV, shifts the window contraction to higher extracellular K+, indicating that in the presence of levcromakalim higher extracellular K+ is needed to attain the same isometric contraction as in control. When contractions at different external K+ concentrations were plotted as a function of Vclamp, contraction curves in the absence and presence of levcromakalim were identical (figure 8C, D). Hence, levcromakalim shifted the K+-dependence of the isometric contraction, but not the voltage-dependence, suggesting that levcromakalim does not affect L-type Ca2+ channel gating properties. Results further suggest that at resting Vm of the VSMCs (-50 to -60 mV), Vclamp situates within the window voltage range, which results in continuous baseline Ca2+ influx and concomitant tonus via open L-type Ca2+ channels.
5.2. Depolarization-induced window contractions and NO
To investigate the effects of basal NO release on voltage-dependent L-type Ca2+ channel-mediated contractions, the contraction evoked by the increase of extracellular K+ was measured before and after inhibition of eNOS with 300 µM LNAME/LNNA. NO has been described to activate voltage-gated K+ channels, to hyperpolarize VSMCs and to decrease the intracellular Ca2+ concentration (Edwards et al., 2010; Quignard et al., 2000; Yuan et al., 1996). To compensate for the hyperpolarizing effects of NO or depolarizing effects of eNOS inhibition, 1 µM levcromakalim was added to set Vm of all the segments to VK (figure 9). Inhibition of basal NO release shifted the contraction window to hyperpolarized potentials. A possible explanation is that S-nitrosylation of cysteine residues of the channel changes the voltage-dependence of the L-type Ca2+ channel-mediated contraction. Effects of NO and cGMP on L-type Ca2+ current have been described before. They might occur via an indirect mechanism through a NO-cGMP-PKG pathway and/or a direct mechanism mediated by S-nitrosylation (Almanza et al., 2007). The α1C-subunit of the L-type Ca2+ channel contains more than 10 cysteine residues that modulate channel gating and is constitutively S-nitrosylated in the mouse heart (Sun et al., 2006; Tamargo et al., 2010). According to this hypothesis, basal NO release should diminish Ca2+ influx via the L-type Ca2+ window at physiological Vm, leading to vasodilation. It will be interesting to test this hypothesis with intracellular VSMC Ca2+ measurements. Is intracellular Ca2+ decreasing upon addition of NO to segments pre-contracted with different K+ (different Vclamp)? Indirect evidence for the hypothesis is provided by the experiments of figure 1, where it was shown that the relaxing capacity of NO increased when external K+ was decreased.
The shift of the contraction window by eNOS inhibition could be completely reversed by adding exogenous NO (100 nM DEANO) (figure 10). The addition of exogenous NO to segments, in which eNOS activity was inhibited, caused a rightward (depolarising) shift of the voltage-dependency of contraction. Also in this situation, effects of eNOS inhibition or exogenous NO addition on Vm were avoided by performing the experiments in the presence of 1 µM levcromakalim. Results confirmed that NO altered the voltage-dependency of the L-type Ca2+ channel-mediated contraction, probably by direct effects of NO on the channel´s gating properties. It should be noted that for strong depolarization above -20 mV (90 and 124 mM K+), addition of exogenous NO caused significant relaxation of the pre-constricted segments. Hence, the window contraction in the presence of NO is narrower than in the absence of NO (figure 10B). Whether these results can also be explained by NO-dependent changes of the voltage dependence of activation of the L-type Ca2+ channels needs further investigation.
The above data may provide an explanation for the absence of a decrease of intracellular VSMC Ca2+ with relaxation upon addition of NO to segments depolarized with 50 mM K+, whereas similar experiments in phenylephrine-constricted segments displayed relaxation with an abrupt decrease of intracellular Ca2+ upon addition of NO (see figure 2). Firstly, depolarization with elevated K+ did not empty the SR Ca2+ stores, and, hence, did not stimulate SOCE via NO-sensitive non-selective cation channels. Secondly, at 50 mM K+ Vm of the VSMCs was clamped at depolarized potentials and NO cannot elicit hyperpolarisation of Vm. Thirdly, at 50 mM K+, the window Ca2+ influx and concomitant contraction were maximal and addition of NO will only cause small relaxations, probably via effects on Ca2+-sensitivity and not on intracellular Ca2+ concentrations.
6. NO efficacy and VSMC L-type Ca2+ channels along the thoracic aorta
Increased stiffness of elastic arteries represents an early risk factor for cardiovascular diseases (O'Rourke & Mancia, 1999) and, therefore, the assessment of mechanical properties of the aorta is important to understand the mechanisms of cardiovascular disease. It has been shown that in the aorta of C57Bl6 mice, the circumferential modulus is greatest (most rigid) near the diaphragm, and that about 85% of volume compliance is in the thoracic compared with abdominal aorta (Guo & Kassab, 2003). Because as well NO as CaBs have been described to de-stiffen large arteries (Fitch et al., 2006; Safar et al., 1989; Safar et al., 2011), we wondered whether NO release (basal and stimulated) and L-type Ca2+ channel activity differed along the length of the thoracic aorta.
In this part of the chapter, we show some preliminary data on aortic segment differences with respect to NO and L-type Ca2+ channels. In order to test whether the release or efficacy of NO differs between different locations along the thoracic aorta of C57Bl6 mice, the aorta was divided into 5 segments (figure 11) and in each segment endogenous and exogenous NO efficacy was determined. Basal release of NO and its efficacy to counteract contraction was measured in each aortic segment by measuring isometric contractions to the α1-adrenoceptor agonist phenylephrine before and after inhibition of eNOS with 300 µM LNAME/LNNA. The increase of force (Δ force) and the shift of logEC50 (Δ logEC50) by eNOS inhibition are an index of the basal release of endogenous NO in each segment (figures 12A and B).
Although not significant, the effects of eNOS inhibition on phenylephrine-induced contractions were smaller in the atherosclerosis-resistant segments TA2, TA3 and TA4 as compared with the atherosclerosis-prone segments TA1 and TA5 (figure 12A). In accordance, inhibition of eNOS caused a significantly larger shift of the segment´s sensitivity to phenylephrine in the atherosclerosis-prone segment TA1 compared with the other segments (figure 12B). Results may point to a higher basal eNOS activity or release of basal NO in the atherosclerosis-prone segments TA1 and TA5. Subsequently, endogenous release of NO was evoked by adding increasing concentrations of ACh to segments pre-contracted with 1 µM phenylephrine. Figures 12C and D show maximal relaxation evoked by and sensitivity (logEC50) for ACh in each segment. Although TA1 seemed to have the highest NO efficacy without endothelial stimulation (basal NO, figures A and B), this atherosclerosis-prone segment caused only relaxations of about 65% and displayed the lowest sensitivity for ACh-induced relaxations. This was due to endothelial dysfunction because the effects of exogenous NO (DEANO) were not significantly different between the different aortic segments (figures 12 E and F). Results of these experiments show that endothelial function (basal and stimulated release of NO) may differ between atherosclerosis-prone and –resistant segments along the thoracic aorta of wild-type mice, which may have important consequences for the tonus and compliance of the thoracic aorta.
When the impact of ACh-stimulated eNOS activity on L-type Ca2+ channels was investigated further (see figure 6), but now in atherosclerosis-prone and -resistant segments, figures 13 A and C show that as well in the presence of external Ca2+ as after eliciting contraction by re-addition of Ca2+ to zero Ca2+ (SOCE contraction), relaxation in prone segments in comparison with resistant segments was attenuated. When SOCE via cation-selective channels was inhibited with 50 µM 2-APB, the SOCE contraction by phenylephrine was smaller in the resistant than in the prone segments (figure 13B, values at -9 logM ACh). Because this SOCE-contraction by phenylephrine is due to influx of Ca2+ via L-type Ca2+ channels only (see figure 5), this suggests that the L-type Ca2+ channel-mediated SOCE contraction in prone segments is larger than in resistant segments. This L-type Ca2+ channel-mediated contraction by phenylephrine in prone segments displayed attenuated relaxation to ACh (figure 13D), again pointing to the lower capacity of NO to relax contractions evoked by L-type Ca2+ influx.
The present chapter investigated the relaxing efficacy of NO for contractions induced by α1-adrenoceptor stimulation with phenylephrine or by depolarization with elevated extracellular K+ and confirmed previous observations that relaxation by NO is attenuated when the contraction was due to L-type Ca2+ influx (Van Hove et al., 2009). Contractions, which were initiated by Ca2+ release from the SR as with phenylephrine were very sensitive to endogenous or exogenous NO. Here, NO caused relaxation by inhibiting the IP3-mediated contraction, by stimulating Ca2+ re-uptake to the SR via stimulation of SERCA, by inhibiting SOCE via cation channels and by reducing SOCE via L-type Ca2+ channels. Although phenylephrine has been described to cause depolarization of the VSMCs, Vm is not clamped at depolarized values and addition of NO is expected to cause hyperpolarization, thereby reducing Ca2+ influx via L-type Ca2+ channels. By clamping Vm to depolarized values with elevation of K+, we were able to show contractions due to Ca2+ influx via L-type Ca2+ channels only and more specifically, to Ca2+ influx via non-inactivating Ca2+ channels (window contraction). This window contraction could be decreased by repolarizing Vm of VSMCs with levcromakalim (an opener of ATP-dependent K+ channels) or by adding exogenous NO as long as the extracellular K+ concentration was below 50 mM. Because NO also shifted the voltage-dependence of the window contraction in the presence of levcromakalim, it was hypothesized that NO, but not levcromakalim exerted a direct effect on the gating properties of the L-type Ca2+ channel. Although it has been described that NO affects L-type Ca2+ channels directly via S-nitrosylation (Almanza et al., 2007), an effect on the voltage-dependent contraction has not been directly demonstrated yet. Hence, it is hypothesized that in mouse aortic segments NO changed the voltage-dependence of the contraction mediated by L-type Ca2+ influx, probably via S-nitrosylation of the cysteine residues located in the gating part of the channel´s α1C-subunit.
An interplay between NO and L-type Ca2+ channels was also suggested by a number of other observations: a) When treated with the CaB, lacidipine, Western-type diet-evoked hypertension and atherosclerosis development in apoE-deficient mice was reduced, whereas endothelial function was preserved (Kyselovic et al., 2005), b) after partly inhibiting L-type Ca2+ influx induced by high K+ with the CaB nifedipine, relaxation by exogenous NO was ameliorated (Van Hove et al., 2009) and c) ApoE-/- mice, which spontaneously develop atherosclerotic lesions at the age of 6 months when fed a normal diet, displayed altered Ca2+ homeostasis at the age of 4 months, hence, before development of plaques. In comparison with wild-type C57Bl6 mice, they showed higher baseline intracellular Ca2+ in the SMCs (Van Assche et al., 2007), lower baseline Ca2+ in endothelial cells and decreased basal but normal or even enhanced agonist-evoked NO release and efficacy (Fransen et al., 2008).
The present study might also provide an explanation for the unique features of the antihypertensive classes of CaBs with different chemical structure (phenylalkylamines such as verapamil, benzothiazepines such as diltiazem and dihydropyridines such as nifedipine). They reduce blood pressure more effectively in hypertensive than in normotensive subjects (Leonetti et al., 1982) and they inhibit L-type Ca2+ influx more effectively in vascular tissue than in heart (Godfraind et al., 1984; Godfraind, 2005; Striessnig et al., 1998). Several attempts have been made to explain these unique features. In hypertension, there is an increase of reactivity to vasoconstrictors, which is not only due to a higher number of L-type Ca2+ channels (Godfraind, 2005; Pesic et al., 2004; Pratt et al., 2002), but also to depolarization of the resting potential of hypertensive VSMCs (Morel & Godfraind, 1994; Pesic et al., 2004). Depolarization leads to an increased proportion L-type Ca2+ channels in the inactivated state, which according to the “modulated receptor theory” may have higher affinity to CaBs than channels in the resting state (Bean et al., 1986; Godfraind, 2005; Morel & Godfraind, 1987). Cardiac muscle cells are hyperpolarized with respect to VSMC and hence less susceptible to block by CaBs. Another specific feature of L-type Ca2+ channels is that their population is not homogeneous because of the occurrence of alternatively spliced isoforms (Koch et al., 1990). Among the 55 known human L-type Ca2+ channel exons 19 undergo alternative splicing and display differences in tissue distribution, physiology, pharmacology and disease-related up- and/or down-regulation (Liao et al., 2005; Tang et al., 2007; Tiwari et al., 2006). Some of these isoforms were dominant in aorta (> 50%) and less abundant in heart (<5%). Moreover, the VSM-specific splice variant of the L-type Ca2+ channel displayed hyperpolarised window current at voltages where there is overlap between activation and inactivation curves and enhanced inhibition by nifedipine in comparison with the predominant cardiac isoform (Liao et al., 2007). The selective affinity of CaBs for the different L-type Ca2+ channel subpopulations may further contribute to the different susceptibility of VSMC or cardiac cells to CaBs. Because the L-type Ca2+ channel window in VSMCs may be responsible for “time-independent” baseline Ca2+ influx at normal resting or slightly depolarized membrane potentials (Fleischmann et al., 1994; Poburko et al., 2004), depolarization of the resting potential by hypertension not only favours the inactivated state of the L-type Ca2+ channel, but is also expected to increase L-type Ca2+ influx via the L-type Ca2+ channel window and to evoke inhibition by CaBs (Fleischmann et al., 1994).
Preliminary results of this chapter, finally, indicate that NO efficacy and L-type Ca2+ channel distribution may differ along the length of the thoracic aorta. Basal and stimulated eNOS activation or NO release occurred differently along the thoracic aorta. Close to the aortic arch, segments released more basal NO but less stimulated NO than segments close to the diaphragm. Similarly, L-type Ca2+ channel distribution and related window contraction were higher in atherosclerosis-prone than in -resistant segments. These preliminary data need to be further explored, but may have important consequences for the compliance of the different aortic segments and their susceptibility to the development of atherosclerosis.