C J Garland

K + is an endothelium-derived hyperpolarizing factor in rat arteries

In arteries, muscarinic agonists such as acetylcholine release an unidentified, endothelium-derived hyperpolarizing factor (EDHF) which is neither prostacyclin nor nitric oxide. Here we show that EDHF-induced hyperpolarization of smooth muscle and relaxation of small resistance arteries are inhibited by ouabain plus Ba2+; ouabain is a blocker of Na+/K+ ATPase and Ba2+ blocks inwardly rectifying K+ channels. Small increases in the amount of extracellular K+ mimic these effects of EDHF in a ouabain- and Ba2+-sensitive, but endothelium-independent, manner. Acetylcholine hyperpolarizes endothelial cells and increases the K+ concentration in the myoendothelial space; these effects are abolished by charbdotoxin plus apamin. Hyperpolarization of smooth muscle by EDHF is also abolished by this toxin combination, but these toxins do not affect the hyperpolarizaiton of smooth muscle by added K+. These data show that EDHF is K+ that effluxes through charybdotoxin- and apamin-sensitive K+ channels on endothelial cells. The resulting increase in myoendothelial K+ concentration hyperpolarizes and relaxes adjacent smooth-muscle cells by activating Ba2+-sensitive K+ channels and Na+/K+ ATPase. These results show that fluctuations in K+ levels originating within the blood vessel itself are important in regulating mammalian blood pressure and flow.

Endothelium-dependent hyperpolarization: a role in the control of vascular tone

Endothelial-dependent relaxation of vascular smooth muscle cells evoked by a number of agonists, including cholinomimetics and substance P, is often accompanied by an increase (repolarization and/or hyperpolarization) in the membrane potential. This change in membrane potential appears predominantly to reflect the action of an endothelial-derived hyperpolarizing factor (EDHF), which is distinct from NO (or endothelial-derived relaxing factor), and is discussed in this article by Chris Garland and colleagues. In large conducting arteries, EDHF may provide a secondary system to NO, which assumes primary importance in some disease states such as pulmonary hypertension and atherosclerosis. However, in small resistance arteries (100-300 microns), EDHF appears to be a major determinant of vascular calibre under normal conditions, and may therefore be of primary importance in the regulation of vascular resistance.

Evidence that nitric oxide does not mediate the hyperpolarization and relaxation to acetylcholine in the rat small mesenteric artery

1 Acetylcholine caused a concentration‐dependent smooth muscle hyperpolarization and relaxation in rat small mesenteric arteries (diameter at 100 mmHg 250–450 mm) stimulated with noradrenaline (3 μm). 2 Nitric oxide (NO), generated from either NO‐gas or from acidified sodium nitrite, also induced smooth muscle hyperpolarization but only in the absence of active force. However, unlike the hyperpolarizations to acetylcholine, those to NO were abolished either by prior smooth muscle depolarization caused by noradrenaline, or by the K+ channel blocker, glibenclamide (3 μm). 3 Hyperpolarization and relaxation to acetylcholine were unaffected by prior exposure of the mesenteric artery to either the cyclo‐oxygenase inhibitor, indomethacin (10 μm), or the nitric oxide synthase inhibitor, NG‐nitro‐l‐arginine (l‐NNA, 100 μm). 4 Haemoglobin (1.5 μm), which binds and inactivates NO, blocked the hyperpolarizing and vasorelaxant response to NO, but did not alter either response to acetylcholine. 5 These data show that, in the rat small mesenteric artery, membrane hyperpolarizations to NO and acetylcholine are mediated by different mechanisms, and that the hyperpolarization induced by NO is not involved in the responses to acetylcholine. In addition, they provide evidence that the acetylcholine responses in this artery, which are endothelium‐dependent, are not mediated by the release of NO.

Nitric oxide (NO)-induced activation of large conductance Ca2+-dependent K+ channels (BKCa) in smooth muscle cells isolated from the rat mesenteric artery

1 To assess the action of nitric oxide (NO) and NO‐donors on K+ current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery. 2 Inside‐out patches contained channels, the open probability of which increased with [Ca2+]i. The channels had a linear slope conductance of 212±5 pS (n = 12) in symmetrical (140 mm) K+ solutions which reversed in direction at 4.4 mV. In addition, the channels showed K+ selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K+. Barium (1 mm) applied to the intracellular face of the channel produced a voltage‐dependent block and external tetraethylammonium (TEA; at 1 mm) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BKCa. 3 In five out of six inside‐out patches, NO (45 or 67 μm) produced an increase in BKCa activity. In inside‐out patches, BKCa activity was also enhanced in some patches with 100 or 200 μm 3‐morpholino‐sydnonimine (SIN‐1) (4/11) and 100 μm sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may reflect variability in the release of NO from these compounds. 4 In inside‐out patches, 100 μm SIN‐1 failed to increase BKCa activity (in all 4 patches tested), while at a higher (500 μm) concentration SIN‐1 had a direct blocking effect on the channels (n = 3). NO applied directly to inside‐out patches increased (P<0.05) BKCa activity in two patches. 5 In the majority of cells (6 out of 7), application of NO (45 or 67 μm) evoked an increase in the amplitude of whole‐cell currents in perforated patches. This action was not affected by the soluble guanylyl cyclase inhibitor, 1H‐[1,2,4] oxadiazolo [4,3‐a]quinoxalin‐1‐one (ODQ). An increase in whole‐cell current was also evoked with either of the NO donors, SIN‐1 or SNP (each at 100 μm). With SIN‐1, the increase in current was blocked with the BKCa channel blocker, iberiotoxin (50 nm). 6 With conventional whole‐cell voltage clamp, the increase in the outward K+ current evoked with SIN‐1 (50–300 μm) showed considerable variability. Either no effect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7±10.2% was recorded at 40 mV. 7 In cell‐attached patches, large conductance voltage‐dependent K+ channels were stimulated by SIN‐1 (100 μm) applied to the cell (n = 5 patches). 8 These data indicate that NO and its donors can directly stimulate BKCa activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BKCa channels could play an important functional role in NO‐induced relaxation of the vascular smooth muscle cells in this small resistance artery.

How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines

Given that cardiovascular safety liabilities remain a major cause of drug attrition during preclinical and clinical development, adverse drug reactions, and post‐approval withdrawal of medicines, the Medical Research Council Centre for Drug Safety Science hosted a workshop to discuss current challenges in determining, understanding and addressing ‘Cardiovascular Toxicity of Medicines’. This article summarizes the key discussions from the workshop that aimed to address three major questions: (i) what are the key cardiovascular safety liabilities in drug discovery, drug development and clinical practice? (ii) how good are preclinical and clinical strategies for detecting cardiovascular liabilities? and (iii) do we have a mechanistic understanding of these liabilities? It was concluded that in order to understand, address and ultimately reduce cardiovascular safety liabilities of new therapeutic agents there is an urgent need to:

Rapid Endothelial Cell–Selective Loading of Connexin 40 Antibody Blocks Endothelium-Derived Hyperpolarizing Factor Dilation in Rat Small Mesenteric Arteries

In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor (EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (≈15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40; residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra- and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs (43Gap 26, 40Gap 27, and 37,43Gap 27; 300 &mgr;mol/L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries.

EDHF: Spreading the influence of the endothelium

Our view of the endothelium was transformed around 30 years ago, from one of an inert barrier to that of a key endocrine organ central to cardiovascular function. This dramatic change followed the discoveries that endothelial cells (ECs) elaborate the vasodilators prostacyclin and nitric oxide. The key to these discoveries was the use of the quintessentially pharmacological technique of bioassay. Bioassay also revealed endothelium‐derived hyperpolarizing factor (EDHF), particularly important in small arteries and influencing blood pressure and flow distribution. The basic idea of EDHF as a diffusible factor causing smooth muscle hyperpolarization (and thus vasodilatation) has evolved into one of a complex pathway activated by endothelial Ca2+ opening two Ca2+‐sensitive K+‐channels, KCa2.3 and KCa3.1. Combined application of apamin and charybdotoxin blocked EDHF responses, revealing the critical role of these channels as iberiotoxin was unable to substitute for charybdotoxin. We showed these channels are arranged in endothelial microdomains, particularly within projections towards the adjacent smooth muscle, and close to interendothelial gap junctions. Activation of KCa channels hyperpolarizes ECs, and K+ efflux through them can act as a diffusible ‘EDHF’ stimulating Na+/K+‐ATPase and inwardly rectifying K‐channels. In parallel, hyperpolarizing current can spread from the endothelium to the smooth muscle through myoendothelial gap junctions upon endothelial projections. The resulting radial hyperpolarization mobilized by EDHF is complemented by spread of hyperpolarization along arteries and arterioles, effecting distant dilatation dependent on the endothelium. So the complexity of the endothelium still continues to amaze and, as knowledge evolves, provides considerable potential for novel approaches to modulate blood pressure.

Evidence that anandamide and EDHF act via different mechanisms in rat isolated mesenteric arteries

The endogenous cannabinoid, anandamide, has been suggested as an endothelium‐derived hyperpolarizing factor (EDHF). We found that anandamide‐evoked relaxation in isolated segments of rat mesenteric artery was associated with smooth muscle hyperpolarization. However, although anandamide‐evoked relaxation was inhibited by either charybdotoxin (ChTX) or iberiotoxin, inhibition of the relaxation to EDHF required a combination of ChTX and apamin. The relaxations induced by either anandamide or EDHF were not inhibited by the cannabinoid receptor (CB1) antagonist SRI41716A, or mimicked by selective CB1 agonists. Thus, anandamide appears to cause smooth muscle relaxation via a CB1 receptor‐independent mechanism and cannabinoid receptor activation apparently does not contribute to EDHF‐mediated relaxation in this resistance artery.

An indirect influence of phenylephrine on the release of endothelium-derived vasodilators in rat small mesenteric artery.

The possibility that stimulation of smooth muscle α1‐adrenoceptors modulates contraction via the endothelium was examined in rat small mesenteric arteries. Nω‐nitro‐L‐arginine methyl ester, (L‐NAME, 100 μM to inhibit NO synthase) increased contraction to single concentrations of phenylephrine (1–3 μM) by approximately 2 fold (from a control level of 14.2±3.0 to 34.1±4.2% of the maximum contraction of the artery, n=20). The action of L‐NAME was abolished by disrupting the endothelium. The subsequent addition of apamin (to inhibit small conductance Ca2+‐activated K+ channels, 50 nM) further augmented phenylephrine contractions, in an endothelium‐dependent manner, to more than 3 fold above control (50.4±5.3% of the maximum contraction, n=11). Charybdotoxin (non‐selective inhibitor of large conductance Ca2+‐activated K+ channels, BKCa, 50 nM) plus L‐NAME augmented the level of phenylephrine contraction to 4–5‐fold above control (64.1±3.1%, n=5), but this effect was independent of the endothelium. The potentiation of contraction by charybdotoxin could be mimicked with the selective BKCa inhibitor, iberiotoxin,. Apamin together with L‐NAME and charybdotoxin further significantly increased the phenylephrine contraction by 5–6‐fold, to 79.9±3.5% of the maximum contraction of the artery (n=13). Phenylephrine failed directly to increase the intracellular Ca2+ concentration in endothelial cells freshly isolated from the small mesenteric artery. Stimulation of smooth muscle α1‐adrenoceptors in the mesenteric artery induces contraction that is markedly suppressed by the endothelium. The attenuation of contraction appears to reflect both the release of NO from the endothelium and the efflux of K+ from both endothelial and smooth muscle cells. This suggests that the release of NO and endothelium‐derived hyperpolarizing factor can be evoked indirectly by agents which act only on the smooth muscle cells.

Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone.

Endothelial cell (EC) Ca2+-activated K channels (SKCa and IKCa channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IKCa channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca2+ events (Ca2+ puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (∼2 min−1) inositol 1,4,5-trisphosphate receptor-based Ca2+ events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca2+ events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet- and nonsparklet-evoked Ca2+ increases, which on occasion led to intracellular Ca2+ waves. The concurrent vasodilation associated with increases in Ca2+ event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IKCa channel blocker), but not by apamin (SKCa channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca2+ event frequency; at low pressures the consequence is activation of EC IKCa channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation.