Perivascular adipose tissue-derived adiponectin activates BK(Ca) channels to induce anticontractile responses.

This study aims to identify the potential mechanisms by which perivascular adipose tissue (PVAT) reduces tone in small arteries. Small mesenteric arteries from wild-type and large-conductance Ca(2+)-activated K(+) (BKCa) channel knockout mice were mounted on a wire myograph in the presence and absence of PVAT, and contractile responses to norepinephrine were assessed. Electrophysiology studies were performed in isolated vessels to measure changes in membrane potential produced by adiponectin. Contractile responses from wild-type mouse small arteries were significantly reduced in the presence of PVAT. This was not observed in the presence of a BKCa channel inhibitor or with nitric oxide synthase (NOS) inhibition or in BKCa or adiponectin knockout mice. Solution transfer experiments demonstrated the presence of an anticontractile factor released from PVAT. Adiponectin-induced vasorelaxation and hyperpolarization in wild-type arteries were not evident in the absence of or after inhibition of BKCa channels. PVAT from BKCa or adiponectin knockout mice failed to elicit an anticontractile response in wild-type arteries. PVAT releases adiponectin, which is an anticontractile factor. Its effect on vascular tone is mediated by activation of BKCa channels on vascular smooth muscle cells and adipocytes and by endothelial mechanisms.

Endothelium-derived hyperpolarising factors and associated pathways: a synopsis.

The term endothelium-derived hyperpolarising factor (EDHF) was introduced in 1987 to describe the hypothetical factor responsible for myocyte hyperpolarisations not associated with nitric oxide (EDRF) or prostacyclin. Two broad categories of EDHF response exist. The classical EDHF pathway is blocked by apamin plus TRAM-34 but not by apamin plus iberiotoxin and is associated with endothelial cell hyperpolarisation. This follows an increase in intracellular [Ca(2+)] and the opening of endothelial SK(Ca) and IK(Ca) channels preferentially located in caveolae and in endothelial cell projections through the internal elastic lamina, respectively. In some vessels, endothelial hyperpolarisations are transmitted to myocytes through myoendothelial gap junctions without involving any EDHF. In others, the K(+) that effluxes through SK(Ca) activates myocytic and endothelial Ba(2+)-sensitive K(IR) channels leading to myocyte hyperpolarisation. K(+) effluxing through IK(Ca) activates ouabain-sensitive Na(+)/K(+)-ATPases generating further myocyte hyperpolarisation. For the classical pathway, the hyperpolarising "factor" involved is the K(+) that effluxes through endothelial K(Ca) channels. During vessel contraction, K(+) efflux through activated myocyte BK(Ca) channels generates intravascular K(+) clouds. These compromise activation of Na(+)/K(+)-ATPases and K(IR) channels by endothelium-derived K(+) and increase the importance of gap junctional electrical coupling in myocyte hyperpolarisations. The second category of EDHF pathway does not require endothelial hyperpolarisation. It involves the endothelial release of factors that include NO, HNO, H(2)O(2) and vasoactive peptides as well as prostacyclin and epoxyeicosatrienoic acids. These hyperpolarise myocytes by opening various populations of myocyte potassium channels, but predominantly BK(Ca) and/or K(ATP), which are sensitive to blockade by iberiotoxin or glibenclamide, respectively.

Targeted deletion of the extracellular signal-regulated protein kinase 5 attenuates hypertrophic response and promotes pressure overload-induced apoptosis in the heart.

RATIONALE: Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase (ERK)5, an atypical MAPK is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating hypertrophic growth and hypertrophy-induced apoptosis is not completely understood. OBJECTIVE: Herein, we investigate the in vivo role and signaling mechanism whereby ERK5 regulates cardiac hypertrophy and hypertrophy-induced apoptosis. METHODS AND RESULTS: We generated and examined the phenotypes of mice with cardiomyocyte-specific deletion of the erk5 gene (ERK5(cko)). In response to hypertrophic stress, ERK5(cko) mice developed less hypertrophic growth and fibrosis than controls. However, increased apoptosis together with upregulated expression levels of p53 and Bad were observed in the mutant hearts. Consistently, we found that silencing ERK5 expression or specific inhibition of its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs) reduced myocyte enhancer factor (MEF)2 transcriptional activity and blunted hypertrophic responses. Furthermore, the inhibition of MEF2 activity in NRCMs using a non-DNA binding mutant form of MEF2 was found to attenuate the ERK5-regulated hypertrophic response. CONCLUSIONS: These results reveal an important function of ERK5 in cardiac hypertrophic remodeling and cardiomyocyte survival. The role of ERK5 in hypertrophic remodeling is likely to be mediated via the regulation of MEF2 activity.

Cardiac-specific deletion of mkk4 reveals its role in pathological hypertrophic remodeling but not in physiological cardiac growth.

Mitogen-activated protein kinase kinase (MKK)4 is a critical member of the mitogen-activated protein kinase family. It is able to activate the c-Jun NH(2)-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase in response to environmental stresses. JNK and p38 are strongly implicated in pathological cardiac hypertrophy and heart failure; however, the regulatory mechanism whereby the upstream kinase MKK4 activates these signaling cascades in the heart is unknown. To elucidate the biological function of MKK4, we generated mice with a cardiac myocyte-specific deletion of mkk4 (MKK4(cko) mice). In response to pressure overload or chronic beta-adrenergic stimulation, upregulated NFAT (nuclear factor of activated T-cell) transcriptional activity associated with exacerbated cardiac hypertrophy and the appearance of apoptotic cardiomyocytes were observed in MKK4(cko) mice. However, when subjected to swimming exercise, MKK4(cko) mice displayed a similar level of physiological cardiac hypertrophy compared to controls (MKK4(f/f)). In addition, we also discovered that MKK4 expression was significantly reduced in heart failure patients. In conclusion, this study demonstrates for the first time that MKK4 is a key mediator which prevents the transition from an adaptive response to maladaptive cardiac hypertrophy likely involving the regulation of the NFAT signaling pathway.

Evidence for the presence of GPRC6A receptors in rat mesenteric arteries.

In this study, the presence of GPRC6A receptors in rat mesenteric artery was investigated. In artery homogenates, GPRC6A mRNA was detected and Western blotting showed the presence of GPRC6A protein. Immunohistochemical studies revealed GPRC6A in both endothelial cells and myocytes. In whole vessel segments, the GPRC6A activators, 300 microM l-ornithine and 100 microM Al(3+), induced endothelium-dependent myocyte hyperpolarizations sensitive to 10 microM TRAM-34, a blocker of intermediate conductance, Ca(2+)-sensitive K(+) channels (IK(Ca)). Activation of IK(Ca) with calindol (300 nM; a positive allosteric Ca(2+)-sensing receptor - CaR - modulator) was inhibited by 500 nM ouabain (inhibition of rat type 2 and type 3 Na(+)/K(+)-ATPases) but unaffected by 30 microM Ba(2+) (blockade of inwardly rectifying K(+) channels). Neither l-ornithine nor Al(3+) activated CaRs heterologously expressed in CHO or HEK293 cells. In the presence of 300 microM l-ornithine or 100 microM Al(3+), myocyte hyperpolarizations to calindol were potentiated whereas this potentiation and hyperpolarizations to l-ornithine were lost following incubation with an anti-GPRC6A antibody. It is concluded that GPRC6A receptors are present on mesenteric artery endothelial cells and myocytes and that their activation selectively opens IK(Ca) channels. This triggers a ouabain-sensitive myocyte hyperpolarization suggesting a close functional relationship between GPRC6A, the IK(Ca) channel and type 2 and/or type 3 Na(+)/K(+)-ATPases.

Impaired small-conductance Ca2+-activated K+ channel-dependent EDHF responses in Type II diabetic ZDF rats.

We have examined the relative contributions of small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca) and IK(Ca)) to the endothelium-derived hyperpolarizing factor (EDHF) pathway response in small mesenteric arteries of Zucker Diabetic Fatty (ZDF) rats, before and after the development of Type II diabetes, together with Lean controls. Smooth muscle membrane potential was recorded using sharp microelectrodes in the presence of 10 microM indomethacin plus 100 microM N(omega)-nitro-L-arginine. SK(Ca) was selectively inhibited with 100 nM apamin, whereas IK(Ca) was blocked with 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile). Resting membrane potentials were similar in arteries from 17- to 20-week-old control and diabetic rats (approximately -54 mV). Responses elicited by 1 and 10 microM acetylcholine (ACh) were significantly smaller in the diabetic group (e.g. hyperpolarizations to -69.5 +/- 0.8 mV (ZDF; n = 12) and -73.2 +/- 0.6 mV (Lean; n = 12; P < 0.05) evoked by 10 microM ACh). The IK(Ca)-mediated components of the ACh responses were comparable between groups (hyperpolarizations to approximately -65 mV on exposure to 10 microM ACh). However, SK(Ca)-mediated responses were significantly reduced in the diabetic group (hyperpolarizations to -63.1 +/- 1.0 mV (ZDF; n = 6) and -71.5 +/- 1.2 mV (Lean; n = 6; P < 0.05) on exposure to 10 microM ACh. Impaired ACh responses were not observed in arteries from 5- to 6-week-old (pre-diabetic) animals. SK(Ca) subunit mRNA expression was increased in the diabetic group. The EDHF pathway, especially the SK(Ca)-mediated response, is impaired in Type II diabetic ZDF rats without a reduction in channel gene expression. These results may be particularly relevant to the microvascular complications of diabetes. The functional separation of SK(Ca) and IK(Ca) pathways is discussed.

Investigation of the role of TASK-2 channels in rat pulmonary arteries; pharmacological and functional studies following RNA interference procedures.

In the present study, we investigated the ability of RNA interference technology to suppress TASK-2 potassium channel expression in human embryonic kidney (HEK293) cells stably transfected with TASK-2 cDNA and in rat isolated intact pulmonary arteries. Lipofectamine-induced transfection of a specific siRNA sequence targeted against TASK-2 resulted in a dose- and time-dependent decrease in TASK-2 channel protein expression. In siRNA-transfected cells the TASK-2 peak currents were significantly smaller than in control cells at every investigated pH, while the pH sensitivity was not altered. Using scrambled siRNA as a negative control, there were no significant changes in TASK-2 protein expression or current compared to mock-transfected cells. In TASK-2 siRNA-transfected small pulmonary arteries, but not in scrambled siRNA-treated vessels, myocyte resting membrane potential at pH 7.4 was significantly less negative and the hyperpolarisations in response to increasing pH from 6.4 to 8.4 were significantly smaller compared with control. The application of levcromakalim (10 microM), NS1619 (33 microM) and a potassium channel inhibitor cocktail (5 mM 4-aminopyridine, 10 mM tetraethylammonium chloride, 30 microM Ba2+ and 10 microM glibenclamide) had similar effects in control and in siRNA-transfected vessels. The TASK-1 (anandamide-sensitive) contribution to resting membrane potential was comparable in each group. Clofilium (100 microM) generated significantly smaller responses in transfected artery segments. These results suggest that RNA interference techniques are effective at inhibiting TASK-2 channel expression in cultured cells and in intact vessels and that TASK-2 channels have a functional role in setting the membrane potential of pulmonary artery myocytes.

Reduced Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels from arteries of Type 2 diabetic Zucker diabetic fatty rats.

Although it is well established that diabetes impairs endothelium-dependent vasodilation, including those pathways involving vascular myocyte large-conductance Ca(2+)-activated K(+) channels (BK(Ca)), little is known about the effects of diabetes on BK(Ca) activation as an intrinsic response to contractile stimulation. We have investigated this mechanism in a model of Type 2 diabetes, the male Zucker diabetic fatty (ZDF) rat. BK(Ca) function in prediabetic (5-7 wk) and diabetic (17-20 wk) ZDF and lean control animals was assessed in whole arteries using myograph and electrophysiology techniques and in freshly dissociated myocytes by patch clamping. Log EC(25) values for phenylephrine concentration-tension curves were shifted significantly to the left by blockade of BK(Ca) with iberiotoxin (IBTX) in arteries from non- and prediabetic animals but not from diabetic animals. Smooth muscle hyperpolarizations of arteries evoked by the BK(Ca) opener NS-1619 were significantly reduced in the diabetic group. Voltage-clamp recordings indicated that IBTX-sensitive currents were not enhanced to the extent observed in nondiabetic controls by increasing the Ca(2+) concentration in the pipette solution or the application of NS-1619 in myocytes from diabetic animals. An alteration in the expression of BK(Ca) beta(1) subunits was not evident at either the mRNA or protein level in arteries from diabetic animals. Collectively, these results suggest that myocyte BK(Ca) of diabetic animals does not significantly oppose vasoconstriction, unlike that of prediabetic and control animals. This altered function was related to a reduced Ca(2+)-dependent activation of the channel not involving beta(1) subunits.

Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231.

Small increases in extracellular Ca2+ dilate isolated blood vessels. In the present study, the possibility that a vascular, extracellular Ca2+-sensing receptor (CaSR) could mediate these vasodilator actions was investigated. Novel ligands that interact with the CaSR were used in microelectrode recordings from rat isolated mesenteric and porcine coronary arteries. The major findings were that (1) raising extracellular Ca2+ or adding calindol, a CaSR agonist, produced concentration-dependent hyperpolarizations of vascular myocytes, actions attenuated by Calhex 231, a negative allosteric modulator of CaSR. (2) Calindol-induced hyperpolarizations were inhibited by the intermediate conductance, Ca2+-sensitive K+ (IKCa) channel inhibitors, TRAM-34, and TRAM-39. (3) The effects of calindol were not observed in the absence of endothelium. (4) CaSR mRNA and protein were present in rat mesenteric arteries and in porcine coronary artery endothelial cells. (5) CaSR and IKCa proteins were restricted to caveolin-poor membrane fractions. We conclude that activation of vascular endothelial CaSRs opens endothelial cell IKCa channels with subsequent myocyte hyperpolarization. The endothelial cell CaSR may have a physiological role in the control of arterial blood pressure.

Hydrogen peroxide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery.

This study was designed to determine whether or not endothelium-dependent hyperpolarizations evoked by acetylcholine in the isolated guinea-pig carotid artery involve hydrogen peroxide. Membrane potential was recorded in the vascular smooth muscle cells of that artery. Under control conditions, acetylcholine induced endothelium-dependent hyperpolarization of the vascular smooth muscle cells which was not affected by the presence of catalase, superoxide dismutase or their combination. Neither the superoxide dismutase mimetic, tiron nor the thiol-reducing agent N-acetyl-L-cysteine modified the hyperpolarization evoked by 0.1 microM acetylcholine but each produced a partial and significant inhibition of the hyperpolarization induced by 1 microM acetylcholine. Neither 10 nor 100 microM hydrogen peroxide influenced the resting membrane potential of the smooth muscle cells and the higher concentration did not significantly influence the hyperpolarization elicited by acetylcholine. These data indicate that, in the guinea-pig isolated carotid artery, hydrogen peroxide is unlikely to contribute to the endothelium-dependent hyperpolarization evoked by acetylcholine.

Bradykinin-induced, endothelium-dependent responses in porcine coronary arteries: involvement of potassium channel activation and epoxyeicosatrienoic acids.

In coronary arteries, bradykinin opens endothelial intermediate- and small-conductance Ca2+-sensitive K+ channels (IK(Ca) and SK(Ca)) and, additionally, releases epoxyeicosatrienoic acids (EETs) from the endothelium. To clarify the involvement of these pathways in endothelium-dependent myocyte hyperpolarization, bradykinin-induced electrical changes in endothelial cells and myocytes of porcine coronary arteries (following nitric oxide (NO) synthase and cyclooxygenase inhibition) were measured using sharp microelectrodes. Hyperpolarization of endothelial cells by bradykinin (27.0 +/- 0.9 mV, n = 4) was partially inhibited (74%) by blockade of IK(Ca) and SK(Ca) channels using 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile) plus 100 nM apamin (leaving an iberiotoxin-sensitive component), whereas the response to substance P was abolished. After gap junction blockade with HEPES, (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulphonic acid)) hyperpolarization of the endothelium by 100 nM bradykinin was abolished by TRAM-39 plus apamin, whereas myocyte hyperpolarization still occurred (12.9 +/- 1.0 mV, n=4). The residual hyperpolarizations to 100 nM bradykinin were antagonized by the EET antagonist, 14,15-EEZE (14,15-epoxyeicosa-5(Z)-enoic acid) (10 microM), and abolished by iberiotoxin. Bradykinin-induced myocyte hyperpolarizations were also reduced by 14,15-EEZE-mSI (14,15-EEZE-methylsulfonylimide) (5,6- and 14,15-EET antagonist), whereas those to exogenous 11,12-EET were unaffected. These data show that bradykinin-induced hyperpolarization of endothelial cells (due to the opening of IK(Ca) and SK(Ca) channels) is electrotonically transferred to the myocytes via gap junctions. Bradykinin (but not substance P) also hyperpolarizes myocytes by a mechanism (independent of endothelial cell hyperpolarization) which involves endothelial cell production of EETs (most likely 14,15- and/or 11,12-EET). These open endothelial IK(Ca) and SK(Ca) channels and also activate large-conductance calcium-sensitive K+ channels (BK(Ca)) on the surrounding myocytes.

Role of SK(Ca) and IK(Ca) in endothelium-dependent hyperpolarizations of the guinea-pig isolated carotid artery.

1. This study was designed to determine whether the endothelium-dependent hyperpolarizations evoked by acetylcholine in guinea-pig carotid artery involve a cytochrome P450 metabolite and whether they are linked to the activation of two distinct populations of endothelial K(Ca) channels, SK(Ca) and IK(Ca.) 2. The membrane potential was recorded in the vascular smooth muscle cells of the guinea-pig isolated carotid artery. All the experiments were performed in the presence of N(omega)-L-nitro arginine (100 microM) and indomethacin (5 microM). 3. Under control conditions (Ca(2+): 2.5 mM), acetylcholine (10 nM to 10 muM) induced a concentration- and endothelium-dependent hyperpolarization of the vascular smooth muscle cells. Two structurally different specific blockers of SK(Ca), apamin (0.5 microM) or UCL 1684 (10 microM), produced a partial but significant inhibition of the hyperpolarization evoked by acetylcholine whereas charybdotoxin (0.1 microM) and TRAM-34 (10 microM), a nonpeptidic and specific blocker of IK(Ca), were ineffective. In contrast, the combinations of apamin plus charybdotoxin, apamin plus TRAM-34 (10 microM) or UCL 1684 (10 microM) plus TRAM-34 (10 microM) virtually abolished the acetylcholine-induced hyperpolarization. 4. In the presence of a combination of apamin and a subeffective dose of TRAM-34 (5 microM), the residual hyperpolarization produced by acetylcholine was not inhibited further by the addition of either an epoxyeicosatrienoic acid antagonist, 14,15-EEZE (10 microM) or the specific blocker of BK(Ca), iberiotoxin (0.1 microM). 5. In presence of 0.5 mM Ca(2+), the hyperpolarization in response to acetylcholine (1 microM) was significantly lower than in 2.5 mM Ca(2+). The EDHF-mediated responses became predominantly sensitive to charybdotoxin or TRAM-34 but resistant to apamin. 6. This investigation shows that the production of a cytochrome P450 metabolite, and the subsequent activation of BK(Ca), is unlikely to contribute to the EDHF-mediated responses in the guinea-pig carotid artery. Furthermore, the EDHF-mediated response involves the activation of both endothelial IK(Ca) and SK(Ca) channels, the activation of either one being able to produce a true hyperpolarization.

Potassium and potassium clouds in endothelium-dependent hyperpolarizations.

A small increase in extracellular K(+) acts as a local, physiological regulator of blood flow to certain vascular beds. The K(+) derives from active tissues such as contracting skeletal muscle and brain and increases blood supply to these organs by the activation of Na(+)/K(+)-ATPases and/or inwardly-rectifying K(+) channels on the vascular myocytes. K(+) liberated from the vascular endothelium also acts as an endothelium-derived hyperpolarizing and relaxing factor within blood vessels. The K(+) effluxes from endothelial cell intermediate- and small-conductance, Ca(2+)-sensitive K(+) channels which open in response to stretch and local hormones. In many vessels, endothelium-derived hyperpolarizing factor (EDHF) seems identical to the K(+) derived from endothelial cells; it activates Na(+)/K(+)-ATPases (particularly those containing alpha2 and alpha3 subunits) and inward rectifiers (particularly Kir2.1) located on the vascular myocytes. Vasospastic agents generate "potassium clouds" around vascular smooth muscle cells via the efflux of this ion through large conductance, Ca(2+)-sensitive K(+) channels on the myocytes. These potassium clouds can reduce the hyperpolarizing actions of endothelium-derived K(+) by effectively saturating the Na(+)/K(+)-ATPases and inward rectifiers on the muscle cells and they may be of clinical significance in vasospastic conditions.

[Communication between endothelial and smooth muscle cells]. / Dialogue entre cellules endothéliales et cellules musculaires lisses.

Vascular endothelial cells play a fundamental role in the control of vascular tone, and therefore in the control of local blood flow, by releasing various contracting (endothelin, prostaglandins) and relaxing (prostacycline, NO) factors. An additional mechanism involving the hyperpolarization of the vascular smooth muscle cells is observed mainly in the coronary vascular bed and in the periphery. This phenomenon was attributed to an elusive endothelial factor called endothelium-derived hyperpolarizing factor (EDHF). This mechanism is now better understood. It involves first an increase in the endothelial intracellular concentration of calcium, the activation of endothelial potassium channels and the resulting hyperpolarization of the endothelial cells. The hyperpolarization of the endothelial cells is transmitted to the smooth muscle cells by different pathways. This hyperpolarization propagates along the vessels not only via the smooth muscle cells but also via the endothelial cells. Therefore, the endothelial layer can also be considered as a conducting tissue. The discovery of specific inhibitors of the endothelial cell hyperpolarization allows the assessment of the contribution of EDHF-mediated responses in the control of vascular tone.

Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF.

1. This study characterizes the K(+) channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. 2. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3. Single-channel, outside-out patch recordings revealed a 17.1+/-0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca(2+) demonstrated that the open probability was increased by Ca(2+). This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1+/-0.7 mV) were reduced by apamin (100 nM; 17.0+/-1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 microM, 21.0+/-0.3 mV) were unaffected (21.7+/-0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. 5 A porcine homologue of the intermediate-conductance Ca(2+)-activated K(+) channel (IK1) was identified in endothelial cells. 6. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca(2+)-activated K(+) channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response.

EDHF: bringing the concepts together.

Endothelial cells synthesize and release vasoactive mediators in response to various neurohumoural substances (e.g. bradykinin or acetylcholine) and physical stimuli (e.g. cyclic stretch or fluid shear stress). The best-characterized endothelium-derived relaxing factors are nitric oxide and prostacyclin. However, an additional relaxant pathway associated with smooth muscle hyperpolarization also exists. This hyperpolarization was originally attributed to the release of an endothelium-derived hyperpolarizing factor (EDHF) that diffuses to and activates smooth muscle K(+) channels. More recent evidence suggests that endothelial cell receptor activation by these neurohumoural substances opens endothelial cell K(+) channels. Several mechanisms have been proposed to link this pivotal step to the subsequent smooth muscle hyperpolarization. The main concepts are considered in detail in this review.