Endothelium-derived Hyperpolarizing Factor: A Cousin to Nitric Oxide and Prostacyclin

Vascular reactivity is often studied in isolated arteries and arterioles. The two most common methods are to directly measure isometric force generation of the vessel or to measure diameter changes of the vessel. For the studies involving force measurement, a decrease in force or relaxation of the smooth muscle is equivalent to vessel dilation. When endothelial receptors are exposed to certain agonists, a dose-dependent dilation or relaxation of the vessel occurs (figs. 2A–C, solid lines). Depending on the vessel size and vessel type, endothelium-dependent dilations can be elicited by a number of agonists, including acetylcholine, bradykinin, substance P, adenosine triphosphate (ATP), adenosine diphosphate, uridine triphosphate, vasopressin, and histamine. The endothelial-mediated dilations elicited by the above agonists often involve the production and release of nitric oxide, PGI², or a combination of both (fig. 1).

After complete inhibition of nitric oxide synthase (NOS) and COX to inhibit nitric oxide and PGI²production, respectively, residual dilation often remains (figs. 2A–C, dashed lines). Figures 2A–Cdepict models of the residual dilations based on published results.24–26The difference between the original dilatory curve (solid lines) and the curve after inhibition of NOS and COX (dashed lines) can be considered the portion of the dilation that is attributable to nitric oxide and COX metabolites. The residual dilatory curve after NOS and COX inhibition may be shifted to the right24,25(fig. 2A), shifted to the right with a suppression in the maximum response26(fig. 2B), or identical to the original curve before NOS and COX inhibition24(fig. 2C). The residual dilations after inhibition of NOS and COX in figure 2(dashed lines) are generally considered to be the EDHF component.

Endothelium-derived hyperpolarizing factor–mediated dilations hyperpolarize the vascular smooth muscle by 15–30 mV.24,25,27Note that smooth muscle hyperpolarization is not unique to EDHF. Nitric oxide and PGI²can also hyperpolarize vascular smooth muscle to varying degrees by activating potassium channels. The smooth muscle hyperpolarization elicits relaxation (or dilation) by decreasing the concentration of cytoplasmic free Ca²⁺through closure of voltage-operated Ca channels in the smooth muscle cell membrane. The cytoplasmic concentration of free Ca²⁺is a major determinant of the contractile state of smooth muscle. In general, increases in Ca²⁺concentration produce contractions, whereas decreases in Ca²⁺relax the smooth muscle cell. In addition to regulating Ca²⁺concentrations, the sensitivity to cytoplasmic Ca²⁺can be regulated by kinases and phosphatases to alter the contractile state of vascular smooth muscle. However, it is not known whether EDHF affects vascular smooth muscle sensitivity to Ca²⁺.

One of the defining characteristics of EDHF is that it is inhibited by blocking potassium channels. A combination of potassium channel blockers is often required to effectively block the response. Table 1shows the potassium channels most relevant to EDHF studies and their inhibitors. Depending on the mechanistic model (see “Mechanisms of EDHF Dilations”), the location of the potassium channels involved with the EDHF response can be on the endothelium, vascular smooth muscle, or both.

Because of the limited knowledge that we have regarding EDHF, its role remains to be fully elucidated. Nevertheless, one observation that may provide a significant clue as to its physiologic role is that EDHF seems to be more prominent in smaller arteries and arterioles than in larger arteries. This observation has been made in a number of vascular beds, including those from the mesenteric, cerebral, ear, and stomach.24,28–32In fact, control of vessel diameter in these smaller arteries and arterioles by EDHF may be more important than endothelium-derived nitric oxide. For example, proceeding from larger to smaller arteries and arterioles, the relative importance of EDHF increased while that of endothelium-derived nitric oxide decreased.24,32Because of the fundamental role of these smaller vessels in the control of vascular resistance, it would therefore seem that EDHF plays a significant role in the regulation of vascular resistance and thus in the control of blood flow during normal physiologic conditions. Although there are uncertainties regarding the relative contributions of endothelium-derived nitric oxide and EDHF, it is possible that EDHF may be the more important of the two in normal regulation of blood flow in some organs of the body.

Another physiologic role for EDHF may be in conducted dilations of arterioles. When an artery or arteriole is stimulated to dilate at a focal site, the dilation can be conducted several millimeters upstream and downstream from the foci. Micropipette application of certain substances onto the surface of arterioles induces both a local vasomotor response as well as a response that is propagated along the vessel, both upstream and downstream to the application. This phenomenon is termed conducted vasomotor response . This conducted dilation is involved with the spatial and temporal regulation of blood flow within a microvascular network. For example, optimum blood flow control in the exercising muscle requires an overall coordination of vascular resistances. Without a functional conducted dilator response, areas within the microvascular network could be at risk for insufficient delivery of oxygen during times of maximum exercise. The conducted dilation is an important aspect of this coordinated response and is required to maximize blood flow control.33 

In the intact hamster cheek pouch or cremaster microcirculatory beds, application of acetylcholine at a focal site produced a conducted dilation approximately 1 mm upstream of the application.34,35Inhibition of COX or NOS had little or no effect on the conducted dilation. However, inhibitors of P-450 epoxygenase, which inhibit EDHF dilations in some vessels, or blockers of K^Casignificantly suppressed the conducted vasodilation to acetylcholine.34,35Thus, EDHF seems to have a major role in conducted dilations and the coordination of vascular resistances within the microcirculation.34,35 

If EDHF has a widespread physiologic role, it follows that it should be found in a number of vessel types. Indeed, evidence for EDHF exists in a wide diversity of arteries from mammals. In humans, EDHF or EDHF-like dilations have been described in coronary arteries and arterioles,36,37cerebral arteries,38renal arteries,39interlobar arteries,40penile resistance arteries,41internal mammary arteries,42,43subcutaneous resistance arteries,44,45radial arteries,46gastroepiploic arteries,29mesenteric arteries,47and omental arteries.48,49The widespread existence of EDHF provides evidence for a significant physiologic role in the regulation of blood flow.

Most studies of EDHF have used isolated arteries and arterioles, i.e. , ex vivo  vessels studied in a dish or organ bath as mentioned previously. If EDHF is an important regulator of blood flow, it must also be functional in intact animals. EDHF or EDHF-like dilations have been demonstrated in vivo  in canine coronary and kidney arterioles,50–52hamster cremaster and cheek pouch arterioles,34,35rat cremaster arterioles,53and rat mesenteric, hind limb, and sciatic nerve circulations.54,55In humans, forearm blood flow shows an EDHF-like dilation with the administration of bradykinin or acetylcholine.56–58Therefore, EDHF has been reported in a wide diversity of vascular beds and in virtually all mammalian species studied, most important of which is the human.

Hormones seem to alter the EDHF response. Estrogen, the most studied of these hormones, seems to up-regulate EDHF in peripheral vessels and down-regulate EDHF in the cerebral circulation. Relaxation responses in the perfused mesenteric bed in male and female rats were similar. The addition of a NOS inhibitor attenuated the relaxation response in males but had no effect in females.59,60The authors suggested that EDHF is functionally more important in females than males in the mesenteric circulation. In mesenteric arteries from female rats, EDHF dilations were attenuated after ovariectomy when compared with intact rats.61Supplementing ovariectomized rats with estrogen rescued the EDHF response. Similarly, the EDHF response was reduced during diestrus, a time of low estrogen, when compared with estrus controls. Interestingly, supplementing male rats with 17β-estradiol or the phytoestrogen daidzein up-regulated EDHF in the aorta.62 

In contrast to the peripheral circulation, EDHF in cerebral arteries and arterioles is down-regulated by estrogen. The EDHF response in isolated rat middle cerebral arteries was dramatically reduced in female rats as compared with male rats. The EDHF response in ovariectomized females was identical to the response in male rats and could be reversed by estrogen replacement.63,64,In vivo  studies of pial arterioles in intact female rats, ovariectomized rats, and ovariectomized rats with estrogen replacement came to the same conclusion that estrogen down-regulates EDHF.65The up-regulation of EDHF after ovariectomy involves gap junctions66but does not seem to be related to a repressed endothelial NOS-derived nitric oxide–generating function.67 

Pregnancy is characterized by an increased sensitivity to endothelial dependent dilators.68,69A number of studies provide strong evidence that an up-regulated EDHF may be a major component to the vascular adaptations to pregnancy.68–73Interestingly, in preeclamptic patients, EDHF may not be up-regulated during pregnancy.73,74It is not known whether this failure to up-regulate EDHF is the cause or the result of the pathologic condition.

The male sex hormone, testosterone, seems to increase vascular tone or contractile state in cerebral arteries by suppressing EDHF.75An interesting twist involving this study is that the reported EDHF was not agonist induced but was present in the resting pressurized state.

Cortisol may also alter EDHF dilations. Exposure of the porcine coronary artery to cortisol for 24 h, but not 30 min, up-regulated EDHF-mediated dilations.76Concomitant with the up-regulation of the EDHF response, cytochrome P-450 2C expression was increased. The authors suggested that chronic cortisol exposure potentiates the EDHF response by up-regulating an epoxygenase that converts arachidonic acid to epoxyeicosatrienoic acids (EETs) that are putative EDHFs.76 

Although much of the mechanism is controversial, there is agreement that an increase of free Ca²⁺in endothelial cells is an initial step required for EDHF dilations (fig. 3). Interestingly, it is this Ca²⁺increase in endothelial cells that ultimately leads to the decrease in smooth muscle Ca²⁺and dilation of the vessel. Thus, during EDHF-mediated dilations, the concentrations of Ca²⁺in endothelial and smooth muscle cells change in opposite directions.

The evidence for a role of endothelial Ca²⁺is based on the following observations. (1) Direct measurements in endothelial cells showed that relatively large increases in Ca²⁺occurred with EDHF dilations.77(2) Ca²⁺ionophores, which directly increase endothelial Ca²⁺by selectively increasing the membrane conductance to Ca²⁺, elicited EDHF-mediated dilations without involvement of receptor stimulation.26,64,77–81(3) Cyclopiazonic acid, which stimulates Ca influx via  capacitative Ca entry, elicited an EDHF response.82(4) EDHF dilations are inhibited by preventing Ca²⁺influx through nonselective cation channels.83 

On stimulation of endothelial receptors, Ca²⁺is thought to be initially released from internal stores through activation of phospholipase C and inositol trisphosphate–gated Ca²⁺-release channels.26,84–87This initial Ca²⁺increase is sustained by an influx of Ca²⁺into the endothelial cell from the extracellular milieu.82,83,86,87In pressurized cerebral arteries, the resting Ca²⁺concentration in endothelial cells ranges from 130 to 160 nm.27,64,77,84,88,89On stimulation of endothelial receptors with ATP or uridine triphosphate (agonists for P2Y²receptors), endothelial Ca²⁺increased to 400– 700 nm and elicited an EDHF dilation27,64,77,84,90(fig. 3). The Ca²⁺threshold for eliciting an EDHF-mediated dilation is approximately 340 nm.77For comparison, the Ca²⁺threshold for activation of NOS in the same artery is approximately 230 nm.77 

The manner in which the increase in endothelial Ca²⁺transitions into the next step of the EDHF mechanism is a controversial point. It is at this step where the proposed mechanisms for EDHF diverge. The major mechanisms currently being considered to explain EDHF dilations are (1) arachidonic acid metabolites, (2) the monovalent cation, K⁺, (3) gap junctions, (4) and hydrogen peroxide. Other candidates for EDHF have been suggested, but evidence for these does not warrant discussion at this time.91–94The major proposed mechanisms of EDHF are discussed in greater detail below.

During EDHF-mediated dilations, endothelial Ca²⁺can increase to 400–700 nm.27,64,77,84,90At a Ca²⁺concentration of 450 nm, 70% of the phospholipase A²(PLA²) is translocated from the cytoplasm to cellular membranes.95PLA²is a lipase that hydrolyzes the linkage at the 2 position of the glycerophosphate backbone of membrane phospholipids. The major product of the hydrolysis is arachidonic acid. Because PLA²is constitutively active, the translocation to the membrane places it in contact with the phospholipid substrate and promotes the release of arachidonic acid within the cell. A role for PLA²involvement with the EDHF mechanism has been demonstrated by studies using pharmacologic inhibitors.26,84,96,97 

The liberated arachidonic acid has several possible fates. It can be reincorporated into the membrane phospholipids; it can act as a messenger; or it can be metabolized further by COX, epoxygenase, lipoxygenase, or Ω hydroxylase (fig. 4).

The first mechanism proposed for EDHF dilations involves the metabolism of arachidonic acid through the epoxygenase pathway to form epoxyeicosatrienoic acids. In this model (fig. 5), activation of the endothelial receptor increases cytoplasmic free Ca²⁺. The increase in Ca²⁺in turn elicits the translocation of PLA²to the membrane and the subsequent liberation of arachidonic acid from the membrane phospholipids. Arachidonic acid is metabolized by epoxygenase, an enzyme with a cytochrome P-450 moiety, to EETs. The EETs diffuse from the endothelium to the vascular smooth muscle, where they activate a large conductance calcium-activated K channel (BK^Ca). Opening of the BK^Cachannel results in K⁺efflux from the smooth muscle cell, hyperpolarization, and dilation as described previously.

The idea that EETs are EDHF is based on several observations. (1) Selective inhibition of cytochrome P-450 epoxygenases by pharmacologic means and by antisense oligonucleotides blocked EDHF dilations.98–102(2) An antagonistic EETs analog blocked EDHF-mediated dilations.103(3) EETs are produced by endothelium.101,102,104,105(4) EETs and their metabolites dilate vessels by increasing the open state probability of BK^Cachannels and hyperpolarizing vascular smooth muscle.102,105(5) EDHF-mediated dilations are enhanced by agents that increase expression of cytochrome P-450 epoxygenase.101,106(6) Endothelium releases a transferable factor similar to P-450 epoxygenase products.21,106,107 

A role for EETs as EDHF comes mostly from coronary and renal arteries,36,100,102,108,109although EDHF dilations in other vessels including skeletal muscle seem to involve a similar pathway.98,100In hepatic, cerebral, and mesenteric arteries, an involvement of this pathway could not be demonstrated using pharmacologic inhibitors of P-450 epoxygenase.110–112 

However, these latter studies using P-450 epoxygenase inhibitors do not necessarily exclude a potential role for previously synthesized and stored EETs. EETs may be stored in the phospholipid pool in the 2 position of the glycerophosphate backbone, the same location as stored arachidonic acid.113–117EETs could be liberated by the direct action of PLA²without the immediate need for P-450 epoxygenase. Therefore, the P-450 epoxygenase inhibitors would not be effective in inhibiting EDHF-mediated dilations until all stored EETs had been depleted.118 

If the EETs hypothesis (fig. 5) is valid, iberiotoxin, a specific inhibitor of BK^Ca(table 1), should block EDHF-mediated dilations. Iberiotoxin alone does inhibit EDHF dilations in coronary arteries21,119supporting the idea that EETs serve as an EDHF. However, iberiotoxin is not an effective inhibitor of EDHF in hepatic, cerebral, and mesenteric arteries.110–112,120Therefore, for these latter arteries, EETs do not seem to be an EDHF.

An alternative to the above idea involves EETs as key messengers, modulators, or amplifiers in the EDHF mechanism without being the actual EDHF, i.e. , a factor that diffuses from the endothelium to hyperpolarize vascular smooth muscle.121Metabolites of the cytochrome P-450 epoxygenase may regulate Ca²⁺entry into endothelial cells,82,122–124activate K^Cachannels on endothelium,125,126and regulate gap junctions.127As discussed previously, regulation of Ca²⁺in endothelium is a critical step in EDHF dilations. Activation of endothelial potassium channels and conduction through gap junctions are important steps in other models of EDHF (discussed below).

In summary, it is reasonable to consider that EDHF is a metabolite of arachidonic acid produced by the P-450 epoxygenase pathway in coronary, renal, and possibly skeletal muscle vascular beds. Alternatively, P-450 epoxygenase metabolites may serve as messengers or modulators in the EDHF pathway, but EETs per se  do not seem to be the EDHF. However, it must be emphasized that species differences, conditions (physiologic, pathologic, or both), methods for studying the isolated vessels, and even diet could have major impacts on the EDHF mechanism and the involvement of EETs.128 

In addition to the epoxygenase pathway, metabolism of arachidonic acid through the lipoxygenase pathway may also be involved with EDHF dilations. Several metabolites of arachidonic acid through the lipoxygenase pathway dilate arteries through activation of potassium channels.129–131At least one of these metabolites seems to be associated with EDHF dilations to acetylcholine in the rabbit aorta.132 

Vascular smooth muscle contains many types of potassium channels. One type commonly found in the membranes of smooth muscle is the inwardly rectifying potassium channel (K^ir; table 1). K^irs are responsive to increases in extracellular K⁺. When extracellular K⁺increases from approximately 4 mm during rest to approximately 8 mm, K^irs become activated.133–137Although the name of this potassium channel can be misleading, K⁺ions move in the same direction through the K^iras with other potassium channels. Thus, under physiologic conditions, the electrochemical gradient favors K⁺movement out of the smooth muscle cell. The loss of positively charged K⁺results in hyperpolarization and subsequent dilation of the artery. At extracellular K⁺of 20–30 mm, the depolarizing effect of K⁺begins to offset any hyperpolarizing effects of K^iractivation.

A second method whereby extracellular K⁺can hyperpolarize vascular smooth muscle is by activation of Na/K adenosine triphosphatase (ATPase). At the expense of ATP, this enzyme exchanges three intracellular Na⁺s for two extracellular K⁺s. This net loss of a positive charge from the cell results in hyperpolarization. Na/K ATPase is activated by a number of mechanisms, one of which is an increase in extracellular K⁺. Not all isoforms of Na/K ATPase can hyperpolarize vascular smooth muscle during physiologic conditions. One type of Na/K ATPase is fully activated at basal extracellular K⁺concentrations (approximately 4 mm). Therefore, any increase in extracellular K⁺could not further stimulate this isoform to hyperpolarize the vascular smooth muscle. However, two isoforms of Na/K ATPase that have a lower affinity for K⁺can be activated when K⁺increases above basal concentrations.138,139Dilations produced by increasing K⁺above basal concentrations require that the lower affinity Na/K ATPase isoforms are present.139,140 

The model for K⁺as an EDHF is based on studies by Edwards et al.  141(fig. 5). In this model, activation of endothelial receptors opens small and intermediate conductance calcium-activated potassium channels (SK^Caand IK^Ca) on endothelium by increasing cytoplasmic Ca²⁺. Intracellular K⁺moves down its electrochemical gradient through the open channels to the extracellular space. As a result of the ion movement, K⁺increases from approximately 4 mm to approximately 12 mm in the extracellular space located between endothelium and vascular smooth muscle. The increase in extracellular K⁺activates both the K^ir(table 1) and Na/K ATPase in the membrane of the vascular smooth muscle (fig. 5), resulting in hyperpolarization of the smooth muscle. Movement of K⁺to the extracellular space from the smooth muscle through the K^iralso helps to sustain increased extracellular K⁺concentrations. The hyperpolarization of the vascular smooth muscle by K^irand Na/K ATPase elicits dilation as described previously.

One major difference between the EETs model and the K⁺model for EDHF involves the cellular location of K^Cachannels. The K^Cas in the EETs model are located on the vascular smooth muscle, whereas the K^Cas are located on the endothelium for the K⁺model. Several studies have demonstrated that the IK^Caand SK^Cainvolved with EDHF dilations are located on the endothelium and that the hyperpolarization of the endothelium by activation of these channels is necessary for agonist-induced EDHF dilations.27,141–145Although the hyperpolarization of the endothelium by IK^Caand SK^Cais consistent with the K⁺model, it is also consistent with models involving gap junctions (see “Mechanism 3: Gap Junctions”) and is not necessarily inconsistent with the EETs model.

Inhibition of both K^irand Na/K ATPase with Ba²⁺(table 1) and ouabain, respectively, blocked EDHF dilations in rat hepatic arteries in addition to blocking the related hyperpolarization of smooth muscle.141Similarly, the same combination of Ba²⁺and ouabain blocked K⁺-induced hyperpolarizations and dilations when extracellular K⁺was increased from 5 to 10 mm. Thus, K⁺mimicked EDHF.

K⁺, measured in or near the myoendothelial space, increased from approximately 5 mm to 11 mm on the addition of acetylcholine.141A combination of charybdotoxin and apamin (table 1) blocked the hyperpolarization of the endothelium produced by acetylcholine and blocked the increase in K⁺in the myoendothelial space.141The above data reported by Edwards et al.  141and a number of subsequent studies provide evidence in support of the model shown in figure 5.

Other studies have disputed the finding that K⁺serves as an EDHF.45,146–152The conclusion of the above studies was based on the inability of Ba²⁺and ouabain to inhibit EDHF dilations and the fact that increasing extracellular K⁺did not mimic EDHF dilations.

In summary, there is good evidence in the literature supporting the idea that K⁺is an EDHF in some arteries. However, there are other studies that oppose the K⁺hypothesis. It must be noted that all of the above studies were conducted in ex vivo  vessels. Seemingly subtle differences in experimental conditions could alter the mechanism of the dilation and thus account for the differences between investigators.153,154Further studies are required to determine the role of K⁺as an EDHF in vivo .

Gap junctions are intercellular channels that allow passage of small water-soluble molecules (< 1,000 Da) including cyclic adenosine monophosphate, cyclic guanosine monophosphate, inositol triphosphates, and inorganic ions but do not allow proteins to pass from cell to cell. There is some selectivity for cations over anions.16,155,156Gap junctions consist of connexins, which are protein subunits with four transmembrane-spanning domains (fig. 6A). Six connexin subunits are required to form a connexon or hemichannel. Two adjacent cells each provide a hemichannel, and the hemichannels dock to form a complete gap junction. More than a dozen connexins have been identified, of which connexins 37, 40, 43, and 45 have been identified in vessels.157,158Gap junctions can be regulated by Ca²⁺, voltage, pH, phosphorylation, ATP, or EETs.127,159–162 

Although it has been known that gap junctions exist between endothelial cells and between vascular smooth muscle cells, they also seem to exist between endothelium and vascular smooth muscle (myoendothelial gap junctions).163Functional or indirect evidence, including electrical conductivity and transfer of dye between adjacent cells, supports the existence of myoendothelial gap junctions.158,164,165These myoendothelial gap junctions are thought to have a major role in the EDHF response by relaying the dilator signal from the endothelium to the vascular smooth muscle.

The study of gap junctions has been hampered in the past by a lack of specific inhibitors.16,166,167Recently, peptide inhibitors, which seem to have greater selectivity for gap junctions, have been used in the study of EDHF.167The peptide inhibitors, termed gap peptides , consist of amino acid sequences identical to portions of extracellular loops of the connexin proteins. It is thought that these gap peptides interfere with docking of hemichannels between adjacent cells. One gap peptide (11 amino acids), based on the extracellular loop of connexins 37 and 43, blocked dye transfer in cultured cells.168,169The same gap peptide inhibited EDHF-mediated dilations in isolated superior mesenteric artery and aorta of the rabbit and cerebral pial arterioles in vivo .66,167In the cerebral pial arterioles, antisense oligonucleotides directed against connexin 43 blocked the EDHF-like dilation.66The antisense study amplified the conclusion involving gap junctions and confirmed the efficacy of the gap peptide. The connexin composition of the gap junctions involved with EDHF dilations varies among arteries. Connexin 37, 40, and 43 have been identified as the protein building blocks of gap junctions important in EDHF dilations.66,167,168,170Therefore, it seems that there is heterogeneity of gap junction types involved with the EDHF mechanism in different arteries.

Sandow et al.  171conducted an elegant study comparing arteries that did and did not have an EDHF-mediated dilation. The rat mesenteric artery, which has EDHF-mediated dilations, contains anatomically identified myoendothelial gap junctions and tight electrical coupling between endothelial and vascular smooth muscle cells. On the other hand, the rat femoral artery, which does not have an EDHF dilation, contained no myoendothelial gap junctions and showed no electrical coupling between endothelial and smooth muscle cells. The data from the different arteries provides further evidence for a role of myoendothelial gap junctions in EDHF dilations.

The gap peptides have served as major tools in studies where a role for myoendothelial gap junctions in EDHF dilations has been implicated. However, the gap peptides have two limitations. First, evidence is beginning to emerge that unpaired connexons or hemichannels can function as cellular pores, in addition to acting as half of a gap junction. The gap peptides used for blocking gap junctions also seem to block the function of a hemichannel pore.172Therefore, if a single hemichannel is a functional pore and can be inhibited by the gap peptides, it is possible that the hemichannel, not the gap junction, is the structure involved with EDHF dilations. Further studies are required to determine the role of hemichannels in cellular regulation and EDHF-mediated dilations. Second, the gap peptides do not selectively block only those gap junctions between endothelium and vascular smooth muscle. The inhibitors also block the gap junctions between endothelial cells and those between smooth muscle cells. Without selectivity of the peptide, an absolute requirement for myoendothelial gap junctions is questioned.12,173 

Although there is evidence for gap junction involvement in many vessel types, a question remains as to what is conducted through the gap junctions for the endothelium to pass the appropriate signal to the smooth muscle. One possibility is that the gap junctions conduct the EDHF from endothelium to smooth muscle (fig. 6B). Another possible role for gap junctions is not the passage of EDHF per se  but the passage of electrical current in the form of ions. Electrophysiologic studies in arteries from guinea pigs, rats, and humans demonstrated that EDHF involves electrical spread of hyperpolarization from the endothelial cells to the smooth muscle cells.45,174One possibility is that K⁺carries the current as shown in figure 6B. That is, potassium movement from the vascular smooth muscle to the endothelium via  gap junctions would result in smooth muscle hyperpolarization. Figure 6Bshows two possible scenarios where myoendothelial gap junctions could be involved with EDHF dilations. Movement of K⁺out of the vascular smooth muscle through gap junctions to the endothelium and ultimately to the extracellular space would produce a net hyperpolarization of the vascular smooth muscle. The second scenario would be for the EDHF to move from the endothelium to the vascular smooth muscle by way of the myoendothelial gap junctions.

Hydrogen peroxide (H²O²) dilates a number of arteries and arterioles by hyperpolarizing the vascular smooth muscle through activation of K^Caor sometimes K^ATP.19,47,175–183H²O²has been reported to be an EDHF in a number of arteries.19,47,175–177One hypothesis is that superoxide is generated on activation of endothelial nitric oxide. The superoxide is converted to H²O²by the actions of superoxide dismutase.176The newly generated H²O²diffuses to the vascular smooth muscle, where it activates K^Caor K^ATP, hyperpolarizes the vascular smooth muscle, and elicits dilation.19,175 

Hydrogen peroxide can also be produced by the action of superoxide dismutase on superoxide generated from COX, lipoxygenase, epoxygenase, xanthine oxidase, NADPH oxidase, and sites along the mitochondrial respiratory chain.19,176,184,185 

The idea that H²O²is an EDHF is based on the facts that (1) catalase, an enzyme that catalyzes the decomposition of H²O²to H²O and O², attenuates EDHF dilations; (2) H²O²and EDHF dilate by a similar mechanism; and (3) H²O²production is increased with the EDHF response.19,175–177,185 

However, just as there is evidence for H²O²in the mechanism of EHDF, there is also evidence against H²O². A number of studies have not found catalase to effectively inhibit EHDF dilations.46,186–188Furthermore, dilations elicited by H²O²do not always mimic EDHF dilations.189 

After many pathologic conditions, dilation produced by endothelium-derived nitric oxide can be significantly attenuated. The primary reasons for the attenuated dilations include an excessive production of reactive oxygen species, which inactivate nitric oxide, and/or dysfunction in eNOS generation of nitric oxide.190–195In contrast, EDHF seems to be resistant to reactive oxygen species.196In fact, EDHF has been reported to be up-regulated after a variety of pathologic conditions when nitric oxide-mediated dilations have been attenuated. The up-regulation seems to occur after ischemia–reperfusion, traumatic injury, congestive heart failure, coronary artery disease, hypercholesterolemia, and angioplasty.13,19,81,90,197–202Of note, patients with congestive heart failure showed an up-regulated EDHF-like dilation in the forearm circulation after administration of acetylcholine.58,203In a rat model of hyperthyroidism, EDHF was up-regulated 36 h after triiodothyronine treatment in renal arteries, but it was down-regulated after 8 weeks.204,Figure 7shows dilations of branches of middle cerebral arteries taken from rats 1 day after a mild head injury or after sham injury.197The dilations were elicited by the P2Y²receptor agonist ATP. In sham-injured rats, inhibition of NOS (N  ^G-nitro-l-arginine methylester) and COX (indomethacin) shifted the response to ATP 10-fold to the right (fig. 7A). The dilation after NOS and COX was mediated by EDHF.24,197In arteries from injured rats, inhibition of NOS and COX had no effect on the dilation (fig. 7B). Figure 7Cshows the up-regulation of the EDHF response by a direct comparison in the two groups.

During hypertension, EDHF has been reported to be either enhanced or suppressed.205–211Pulmonary hypertension in sheep enhanced EDHF in the pulmonary artery.212Other conditions where EDHF has been reported to be suppressed include aging29,205,210,213and type I diabetes.214–219EDHF has been reported to be either enhanced or suppressed in animal models of type II diabetes.220,221 

The effect of the pathologic condition on the EDHF response could be a result of multiple factors. In some pathologic conditions, the metabolic pathways that regulate EDHF could be compromised (producing down-regulation), whereas in other cases, up-regulation of EDHF could be a response to the pathologic conditions.222In addition, the effect of the pathologic condition on the EDHF dilation could be related to the vessel size, the vascular bed being studied, or the severity and duration of the pathologic condition.

Although EDHF dilations are up-regulated in both large and small arteries in certain pathologic conditions, there seems to be a greater propensity for up-regulation in smaller vessels. EDHF responses in eNOS null mice support this idea. Larger conduit arteries typically showed no up-regulation of EDHF in eNOS null mice, whereas up-regulation of EDHF did occur in smaller “resistance-sized” arteries and arterioles.223–228 

In those pathologic conditions where EDHF is up-regulated, it is thought to be a protective mechanism that compensates for insufficient endothelium-derived nitric oxide. A number of studies suggest that there is a balance in the nitric oxide and EDHF response.197,199,200,229–231When the nitric oxide–mediated dilation is impaired during pathologic conditions, EDHF is up-regulated sufficiently to maintain near-normal dilation (fig. 8). Thus, the relative contributions of nitric oxide and EDHF to the overall dilation are adjusted accordingly for the response to remain relatively unchanged. An example during traumatic brain injury is shown in figure 7.

In disease states, activated eNOS can generate superoxide anions after depletion of tetrahydrobiopterin or l-arginine.19,176,190In the presence of superoxide dismutase, the superoxide anions are converted to H²O², a putative EDHF (see “Mechanism 4: Hydrogen Peroxide”). This scenario represents one of many ways that the contribution of endothelium-derived H²O²could be increased in disease states.19,190 

There is good agreement in the literature that anesthetics in general suppress EDHF dilations (tables 2 and 3). For the inhalation anesthetics, isoflurane and sevoflurane are more potent inhibitors than desflurane, enflurane, or halothane on a molar basis.232Ketamine, propofol, and all barbiturates, with the exception of phenobarbital, inhibited the EDHF dilations (tables 2 and 3). Etomidate (1 μm) enhanced EDHF dilations in arteries but suppressed it at greater concentrations.39,48,233Further evidence showed that isoflurane (1.4%) suppressed the EDHF dilation when compared with either halothane (1.2%) or ketamine in rat cremaster arterioles (150 mg/kg followed by an infusion of 1.5 mg · kg⁻¹· min⁻¹).53 

Lischke et al.  232,234demonstrated that all volatile anesthetics studied, etomidate, thiopental, and methohexital, but not phenobarbital, inhibited cytochrome P-450 activity in rabbit liver microsomes. The authors suggested that anesthetics inhibit the cytochrome P-450 epoxygenase, the enzyme family responsible for metabolizing arachidonic acid to EETs.232,234However, as the authors pointed out, the cytochrome P-450 in the liver microsomes is likely different from the cytochrome P-450 epoxygenase that synthesizes EETs.234 

Anesthetics in general seem to inhibit EDHF dilations. One possible mechanism for blocking EDHF could be through inhibition of cytochrome P-450 epoxygenase, a putative “EDHF synthase.” Although inhalation anesthetics could block EDHF dilations by their ability to block gap junction communication,235,236the concentration required to block gap junctions is above clinically relevant halothane concentrations.232,237In arteries where P-450 epoxygenase does not seem to be involved with EDHF dilations, a mechanism of inhibition by anesthetics is lacking. Although anesthetics seem to inhibit EDHF dilations, they do not abolish them. Therefore, there is the potential to augment or further inhibit EDHF dilations as necessary in the operating room (see “Clinical Implications”). More studies are needed to determine how anesthetics affect EDHF dilations and what this inhibition would mean to the clinical practice of anesthesiology.

Anesthesiologists and intensivists have to deal with two problems involving the cardiovascular system. First, blood pressure must to be maintained for perfusion of vascular beds. Second, anesthesiologists and intensivists must ensure that adequate blood flow to vital organs is maintained. Compared with other organs of the body, the brain, heart, and kidney are relatively more sensitive to interruptions in the blood supply. Although the study of EDHF is relatively new and is not currently a clinical consideration, manipulating it does have potential for controlling both blood pressure and maintaining blood flow to vital organs. As the future clinical potential is considered for EDHF, parallels to its cousin, nitric oxide, will provide some insight.

The nitric oxide–cyclic guanosine monophosphate system is used clinically to control blood pressure and to maintain blood perfusion to selected tissues, including heart, brain, and lung.238–249For example, inhaled nitric oxide is effective in reversing conditions affecting the pulmonary vasculature, including persistent pulmonary hypertension of newborns, hypoxia-induced pulmonary hypertension, and adult respiratory distress.239,250In addition, nitric oxide has also been used in the treatment of cerebral vasospasm by selectively applying nitric oxide donors to the vasospastic artery.240 

The potential does exist for EDHF to be used in similar ways as nitric oxide to control blood pressure and to maintain blood perfusion to vital organs. In fact, manipulations of EDHF in conjunction with nitric oxide could prove to be more effective during conditions where nitric oxide therapy alone has been met with limited success.250,251 

For controlling blood pressure, EDHF could be manipulated to increase or decrease the pressure as required. Enhancement of EDHF to globally dilate vessels would act to decrease blood pressure. Conversely, global inhibition of EDHF would constrict vessels and increase blood pressure. Major contributors to the maintenance of blood pressure are the small arteries and arterioles (< 300 μm).252Importantly, it is these vessels that seem to have a more pronounced EDHF response, possibly at the expense of endothelium-derived nitric oxide.24,28–32Manipulation of the EDHF system could therefore provide as great or even greater control of blood pressure than manipulation of the nitric oxide–cyclic guanosine monophosphate system. For example, overexpression in mice of SK3, the small conductance calcium-activated K channel involved with EDHF, hyperpolarized both the endothelium and vascular smooth muscle, dilated arteries in vivo  and in vitro , and decreased blood pressure.253Conversely, decreasing the expression of the SK3 had the opposite effects: vessel constriction and increased blood pressure.253Manipulation of EDHF could therefore be an important means to regulate systemic blood pressure. It follows that a better understanding of the mechanisms controlling EDHF is a necessary step to reach the important endpoint of manipulating EDHF therapeutically.23 

Manipulation of EDHF could be used to maintain blood flow to vital organs after compromise of the vascular system. There are two considerations regarding EDHF that are relevant. First, as stated previously, the smaller resistance-sized arteries and arterioles have a more pronounced EDHF response.24,28–32It is these smaller vessels where the major resistance to blood flow occurs and is thus the major control point of blood flow. Manipulation of these smaller sized vessels through EDHF could be an effective and efficient means to control blood flow in a vascular bed. Second, the mechanism for EDHF seems to be different in different vascular beds. This fact could be exploited to stimulate EDHF, vessel dilation, and blood flow to a selective organ without affecting resistance in the vascular beds of other organs. For example, pharmacologic agents could possibly stimulate the EDHF pathway in cerebral arteries and arterioles to selectively reduce the cerebrovascular resistance without altering the resistance in other vascular beds. Because global resistance would be minimally affected, cerebral perfusion would increase with no or very little change in blood pressure. Another example would be to selectively activate EDHF in the kidney to increase blood flow and restore urine output in a patient whose renal blood flow has been decreased to critical rates. Again, the selectivity of the dilation compared with a global dilation would ensure that kidney vascular resistance would be decreased without affecting the overall vascular resistance.

Interestingly, the anesthesiologist may already be manipulating EDHF during cardiac bypass when using pulsatile flow. Pulsatile flow decreases vascular resistance by enhancing the release of nitric oxide through mechanical deformation of endothelial cells.254–257Pulsatile flow also elicits EDHF dilations.21,258Perhaps enhanced EDHF dilations work in conjunction with endothelium-derived nitric oxide to improve tissue perfusion with pulsatile flow. During hypoxia, when dilations by endothelium-dependent nitric oxide are inhibited, EDHF, which is not affected by hypoxia,22may contribute more to the decreased vascular resistance than nitric oxide during bypass with pulsatile flow.

Often, it is very sick patients who come to the operating room or intensive care unit. As stated earlier, pathologic conditions often affect EDHF to either suppress or enhance the response. With knowledge of the mechanism of EDHF and how the EDHF response is affected by a particular condition, EDHF could be exploited in a beneficial way to provide a combination of desirable pressure maintenance and adequate flow to vital organs in individual disease states.

There is overwhelming evidence that an endothelial mechanism, other than nitric oxide or prostacyclin, exists for dilating arteries and arterioles. The third pathway is characterized by hyperpolarization of the vascular smooth muscle and involvement of potassium channels, most often small and intermediate conductance calcium-activated potassium channels (IK^Caand SK^Ca). EDHF is more prevalent in smaller resistance-sized arteries and arterioles than in larger conduit arteries. Because these resistance-sized vessels are more significant in the regulation of blood flow, EDHF may have a major but relatively unrecognized role in the control of flow during normal physiologic conditions. During some pathologic states, EDHF can be up-regulated. This up-regulation often occurs as the dilator effects of endothelium-derived nitric oxide are suppressed. The up-regulated EDHF may serve in a protective capacity to help maintain blood flow to organs and tissues during these stressful states.

The most controversial aspect of EDHF research is the mechanism. Arachidonic acid metabolism to EETs through the epoxygenase pathway, K⁺, gap junctions, and H²O²are the most widely studied mechanisms, although others do exist. There is evidence for and against each of the above mechanisms. These principal mechanisms are not necessarily mutually exclusive and could possibly coexist. However, there are likely multiple mechanisms for EDHF. In an individual vessel, the mechanism for EDHF likely depends on the species, organ, vessels size, diet, hormonal state, environmental conditions, and presence or absence of a pathologic condition. As a case in point, the P-450 epoxygenase pathway can be readily altered as a result of the above conditions.128 

The discovery of a new endothelial-mediated dilatory process is intriguing, but many questions must be answered before the true therapeutic potential of EDHF can be fully recognized. Currently, EDHF is almost exclusively studied after inhibition of both nitric oxide and the COX pathway. Only when the mechanism of EDHF-mediated dilations is elucidated can it be studied in the presence of nitric oxide and COX metabolites, and only then can its relative importance in the control of blood flow during normal physiologic conditions be determined. We as scientists are challenged to understand the mechanism, role, and clinical implications of EDHF.