Naunyn-Schmiedeberg’s Arch Pharmacol (2007) 375:73–80 DOI 10.1007/s00210-006-0129-3
ORIGINAL ARTICLE
Functional contribution of the endothelial component to the vasorelaxing effect of resveratrol and NS 1619, activators of the large-conductance calcium-activated potassium channels Vincenzo Calderone & Alma Martelli & Lara Testai & Enrica Martinotti & Maria C. Breschi
Received: 2 November 2006 / Accepted: 29 November 2006 / Published online: 4 January 2007 # Springer-Verlag 2007
Abstract Large-conductance calcium-activated potassium channels (BK) of smooth muscle play a role in the relevant modulation of vascular tone, due to their calcium- and voltage-dependent mechanisms of activation. A potential role of endothelial BK channels has also been suggested by approaches on endothelial cell cultures. However, no functional study, aimed at evaluating the contribution of endothelial BK channels to the effect of BK-openers, has been reported. Resveratrol and NS 1619, BK-openers, have been tested on endothelium-intact and -denuded aortic rings. Furthermore, the effects of high depolarisation of potassium channel blockers TEA (Tetraethylammonium), 4-AP ( 4-Aminopyridine) and IbTX (Iberiotoxin) and of inhibitors of NO-pathway (L-NAME and ODQ) have been evaluated. The presence of endothelium increased the vasorelaxing potency of BK-openers. This potentiation was eliminated by L-NAME and ODQ. TEA, 4-AP, IbTX and high depolarisation had modest or no antagonist influence on resveratrol in endothelium-denuded aortic rings. The effects of NS 1619 on endothelium-denuded aortic rings were not affected by IbTX, and were modestly antagonised by TEA, 4-AP and high depolarisation. In intact endothelium vessels, TEA, IbTX and 4-AP antagonised the vasorelaxing effect of the two BK-activators. A BK-mediated release of endothelial NO seems a very important factor, determining a strong influence on vasodilator profile of BK-openers. Therefore, an eventual therapy V. Calderone (*) : A. Martelli : L. Testai : E. Martinotti : M. C. Breschi Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy e-mail:
[email protected]
with a BK-opener could promote a series of cardiovascular impacts not confined to the only direct vasorelaxing effects, but also due to a significant contribution of endothelial NO. Keywords BK channels . Endothelium . Nitric oxide . Resveratrol . NS 1619
Introduction Large-conductance calcium-activated potassium channels (BK channels) present high levels of single-channel conductance, ranging between 100 pS and 300 pS (Latorre et al. 1989; Reinhart et al. 1989; McManus 1991), accounting for their strong influence on the membrane potential. Moreover, both their calcium-dependent and voltage-operated activation ensure an effective function of feed-back regulation, through a massive outward flow of K+ ions, leading to a membrane hyperpolarisation, which counteracts the membrane depolarisation and the entry of Ca2+ ions in excitable cells (Hille 1992). Indeed, BK channels are almost ubiquitously expressed in excitable and non-excitable cells, where they control many cell functions. Among these, BK channels play a relevant role on the modulation of the tone of vascular smooth muscle (VSM) (Perez and Toro 1994; Kume et al. 1995; Vogalis 2000), regulating the muscular response to vasocontractile stimuli (Nelson et al. 1995; Kuriyama et al. 1995; Quayle et al. 1997; Michelakis et al. 1997; Faraci and Heistad 1998; Jackson 2000). BK channels of VSM are composed by four poreforming α subunits and four β1 accessory subunits, ensuring an increased sensitivity to the rise of free Ca2+ ions (Meera et al. 1996; Cox and Aldrich 2000). Consis-
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tently, a link has been hypothesised between a downregulation of the β1 subunit and hypertension (Amberg et al. 2003). The voltage-operated and Ca2+-dependent mechanisms are not the only triggers involved in the opening of vascular BK channels. In fact, intracellular cGMP activates the BK channels of VSM, through a cGMP-dependent protein kinase (Williams et al. 1988; Taniguchi et al. 1993). In addition, their activation can be due to cAMP-dependent (Sadoshima et al. 1988; Kume et al. 1989; Yamaki et al. 2001), cAMP-independent (Yamaki et al. 2001), G-proteins (Scornik et al. 1993) and GMP (Williams et al. 1988). There is evidence that BK channels in many vascular districts can be activated by many endogenous agents, such as reactive oxygen species (Sobey et al. 1998), nitric oxide (NO) (Bolotina et al. 1994; Da Silva-Santos and Assreuy 1999; Sobey and Faraci 1999), dihydroxyeicosatrienoic acids (Lu et al. 2001) and probably the endotheliumderived hyperpolarising factor (Félétou and Vanhoutte 1999). The activation of BK channels can also be due to the stimulation of the AT2 receptor by the endogenous peptide angiotensin II, a potent vasoconstrictor, which, however, possesses AT2-mediated vasorelaxing properties on certain vascular beds (Dimitropoulou et al. 2001). Therefore, VSM BK channels can be viewed as a key element, modulating the mechanical and electrical state of blood vessels through endothelial chemical signalling (Tanaka et al. 2004). It is generally accepted that small synthetic or natural molecules, acting as BK-openers, can represent promising tools for the therapeutic approach to several disorders (Calderone 2002; Wu 2003; Wu et al. 2006; Nardi et al. 2003; Calderone et al. 2004; Nardi et al. 2006; Lawson and McKay 2006). In particular, because of the direct effects of BK-openers on VSM cells, evaluated in many vascular districts, this class of drugs has been viewed as a new group, which can be joined to the other available classes of vasodilators (i.e., calcium-antagonists, KATP-openers, etc.), in order to offer a wide choice of therapeutic approaches for the clinical management of pathological conditions, such as hypertension, erectile dysfunctions, ischemia and stroke (Balwierczak et al. 1995; Shieh et al. 2000; Gribkoff et al. 2001; Coghlan et al. 2001). Although the function played by BK channels expressed in the VSM cells accounts, in itself, for the purported therapeutic use of BK-openers as vasodilator drugs, it is noteworthy that, at the vascular level, these channels are also expressed in the endothelial cells. In particular, endothelial BK channels, which are composed of only four α subunits (Papassotiriou et al. 2000), have also been indicated as a fundamental element for the coupling between mechanical stimuli (such as the shear stress) and
Naunyn-Schmiedeberg’s Arch Pharmacol (2007) 375:73–80
the endothelial release of vasoactive compounds (such as NO and prostacyclin) (Rusko et al. 1992; Jacobs et al. 1995; Cooke et al. 1991; Sun et al. 2001). Indeed, BK currents have been recorded in endothelial cells (Li et al. 2000) and pharmacologically achieved; the release of NO by cultured endothelial cells exposed to a pharmacological treatment with well-known BK-openers has been reported (Kuhlmann et al. 2004). Of course, the involvement of such endothelial BK channels in the overall pharmacological response to BKactivating molecules could reasonably add a contribution, strengthening the primary vasorelaxing effect of these drugs (due to the endothelium-independent mechanism, i.e., the activation of VSM BK channel). Furthermore, an endothelial BK-induced release of endothelium-derived endogenous substances such as NO (if released in adequate and significant amounts) could confer to a BK-activating drug additional positive effects at the cardiovascular level, other than the vasorelaxing ones. Since, to our knowledge, there is a lack of functional studies of the endothelium-mediated effects of BK-openers, aimed at evaluating, qualitatively and above all quantitatively, the real contribution of the endothelial component in the vascular response to BK-activating drugs, we tested functionally two well-known BK activators (the synthetic benzimidazolone NS 1619 and the natural phenol derivative resveratrol), in order to evaluate and ponder the real importance of a possible endothelial contribution, through indirect pharmacological approaches.
Methods Pharmacological procedures All the experimental procedures were carried out following the guidelines of the European Community Council Directive 86–609. To determine a possible vasodilator mechanism of action, the compounds were tested on isolated thoracic aortic rings of male normotensive Wistar rats (250–350 g). The rats were sacrificed by cervical dislocation under light ether anaesthesia, and bled. The aortae were immediately excised and freed of extraneous tissues. When required by the experimental protocol, the endothelial layer was removed by gently rubbing the intimal surface of the vessels with a hypodermic needle. Conversely, when required, the endothelium was mantained. Five-mm wide aortic rings were suspended, under a preload of 2 g, in 20-ml organ baths containing Tyrode solution (composition of saline in mM: NaCl 136.8; KCl 2.95; CaCl2 1.80; MgSO4 7H2O 1.05; NaH2PO4 0.41; NaHCO3 11.9; Glucose 5.5), thermostated at 37°C and continuously gassed with a mixture of O2
Naunyn-Schmiedeberg’s Arch Pharmacol (2007) 375:73–80
(95%) and CO2 (5%). Changes in tension were recorded by means of an isometric transducer (Grass FTO3), connected with a preamplifier (Buxco Electronics) and with software for data acquisition (BIOPAC Systems Inc., MP 100). After an equilibration period of 60 minutes, the endothelial removal/integrity was confirmed by the administration of acetylcholine (ACh) (10 μM) to KCl (20 mM)precontracted vascular rings. A relaxation
