|
|
Contents
|
§Smooth Muscle Research Group, Heritage Medical Research Building, University of Calgary, Calgary, Alberta T2N-4N1, Canada
1. Modulation of vascular cell calcium is critical for control of vascular tone, blood flow and pressure.
2. Specialized microdomain signalling sites associated with calcium modulation are present in vascular smooth muscle cells (SMCs), where spatially localized channels and calcium store receptors functionally interact. Anatomical studies suggest that such sites are also present in endothelial cells (ECs).
3. The characteristics of these sites near heterocellular myoendothelial gap junctions (MEGJs) are described, focussing on rat mesenteric artery. MEGJs enable current and small molecule transfer to coordinate arterial function, thus being critical for endothelium-derived hyperpolarization (EDH), diameter regulation in response to SMC contractile stimuli and vasomotor conduction over distance.
4. Whilst MEGJs occur on EC projections within internal elastic lamina (IEL) holes, not all IEL holes have MEGJ-related projections; 0 to ∼50% of such holes having MEGJ-related projections; variation occurring within and between vessels, species, strains, and disease.
5. In rat mesenteric, saphenous and caudal cerebellar artery, and hamster cheek pouch arteriole, but not rat middle-cerebral artery or cremaster arteriole, intermediate conductance calcium-activated potassium channels (IKCa) localize to EC projections.
6. Rat mesenteric artery MEGJ connexins and IKCa are in close spatial association with EC inositol 1,4,5-trisphosphate receptors and endoplasmic reticulum.
7. Data suggest a relationship between spatially associated EC ion channels and calcium stores in modulation of calcium release and action. Differences in spatial relationships between ion channels and calcium stores in different vessels reflect heterogeneity in vasomotor function, representing a selective target for control of endothelial and vascular function.
Control of vascular tone, and thus blood flow and pressure, is dependent on the balance between vasoconstrictor and vasodilator action, which in turn are dependent on neural, humoral and physical stimuli.1 Endothelium-derived vasoconstrictors include peptides such as endothelin-1 and angiotensin II, metabolites of arachidonic acid such as thromboxane A2 and superoxide anions,2 whilst endothelium-derived vasodilators include nitric oxide (NO), prostacyclin (PGI2) and the non-NO/PGI2, endothelium-derived hyperpolarization (EDH) mechanism.3-5 The relative contribution of these different vasoconstrictor and vasodilator mechanisms, varies within and between vascular beds, species, strains, sex, development, ageing and disease, as well as with experimental conditions.
In addition to the above, functional variability in arterial smooth muscle cells (SMCs) is associated with the separation of the signalling pathways involved in the control of tone,6 with electrical current and second messenger molecule movement between vascular cells via a gap junctions also being critical for the maintenance of tone. Changes in calcium in both SMCs and endothelial cells (ECs) are essential for the maintenance of vascular tone.1,3,4,6-9 Aspects of the specific mechanisms involved in arterial SMC function, and particularly those related to modulation of intracellular calcium, appear to depend on the spatial compartmentalization of ion channels and receptors and associated calcium stores at ‘microdomain’ sites.6,10,11 Such sites enable microdomain-specific channel and receptor activation,6,10 although the spatial arrangement and physiological role of such sites in ECs is a new area of study. Of interest, the connexins (Cxs) comprising the myoendothelial gap junctions (MEGJs) are in close spatial association with intermediate conductance calcium-activated potassium (IKCa) channels in rat mesenteric artery,12,13 potentially mediating specific functional aspects of EDH activity.14
The focus of this brief review is to describe the limited current knowledge on the microdomain signaling mechanisms associated with contact mediated gap junction communication between ECs and SMCs at MEGJs.
Consensus on the mechanism of EDH involves agonist-induced release of EC calcium, subsequent activation of EC small (S) KCa and IKCa channels, release of epoxyeicosatrienoic acids (EETs), K+ and/or current, which is transferred to the adjacent smooth muscle, with subsequent hyperpolarization, closure of voltage-dependent calcium channels and resultant vessel relaxation (Figure 1). A proposed role for C-type natriuretic peptide and hydrogen peroxide in EDH is not supported by current evidence, with the original studies being based on questionable experimental design and data.3,15-18
Figure 1. Endothelium-derived hyperpolarization (EDH) mechanism - endothelial to smooth muscle signaling in rat mesenteric artery. In response to agonist (R) intracellular endothelial cell (EC) calcium release occurs from inositol 1,4,5-trisphosphate receptor (IP3R)-mediated29,30 endoplasmic reticulum (ER) stores which are in close proximity to myoendothelial gap junction (MEGJ) connexins (Cxs) 37 and 40 (mechanism 1 32) and intermediate conductance calcium-activated potassium channels (IKCa; mechanism 2 32). The net result of such activity is hyperpolarization of the adjacent smooth muscle, closure of voltage-dependent calcium channels (VDCC), subsequent reduced smooth muscle cell (SMC) calcium and vessel relaxation. Mechanism 1 involves transfer of EC-derived hyperpolarization via MEGJ Cxs 37 and 40, whilst mechanism 2 involves localized potassium release from IKCa, activation of smooth muscle Na+/K+-ATPase, with endothelial (and perhaps smooth muscle) inward rectifying potassium channels (Kir)23,24 regulating such localized potassium activity. Replenishment of EC calcium putatively occurs via non-selective cation channel (NSCC; as transient receptor potential (TRP)82,104,105; see also Figure 4, lower right inset) or calcium-sensing receptor channels (CaSR106) activity. Endothelial to smooth muscle (or vice versa; Figure 4) movement of calcium and/or IP3 may also be involved in the modulation of mesenteric artery tone; 29,30,42 with such a suggestion being supported from culture studies showing Cx modulation of IP3 transfer.107 In addition, calcium modulation of gap junction Cx function,108,109 may also regulate current and/or IP3 transfer; thus also being critical for the control of vessel tone. Mechanisms 1 and 2 may operate independently or in a concomitant manner. The use of characterized antibodies to small conductance calcium-activated potassium channels (SKCa),12,110 demonstrate the presence of SK3 at adjacent EC-EC gap junctions, but not at MEGJs in rat mesenteric artery. The ‘space’ between EC and SMC membranes at the MEGJ-related site is ∼10-30 nm (*), although such an apparent distance will likely be in part dependent on the ultrastructural preparation methodology.
In a limited number of vascular beds, being coronary, renal and cerebral beds of some species, and in response to selected stimuli, EETs are synthesized by cytochrome P450 2C or 2J epoxygenases.4,5 EETs may then act to regulate gap junctions19 and/or be released from ECs to activate SMC large (B) KCa, although the precise mechanism of EETs-BKCa activation is not fully characterized.4 An EET-independent BKCa-dependent EDH activity has also been reported in mouse cremaster arteriole and SHR mesenteric artery.20,21
In regard to K+, it was originally proposed that K+ released from ECs during channel opening accumulated in the space occupied by the internal elastic lamina (IEL) and that surrounding SMCs, in a concentration sufficient to activate inward rectifying potassium channels (Kir) and the Na+/K+-ATPase (Na+-K+ pump) on SMCs.4,22 However, this scenario is implausible,23,24 as such a space is physically too large to achieve the dynamic K+ flux required for the characterized rapid EDH response. Alternatively, as outlined in this review, diffusible K+ may rather act at specialized microdomain signaling sites associated with MEGJs (Figure 1).
An ongoing problem within the EDH field is the restriction of studies to the use of pharmacological blockers of relevance only to certain pathways of specific interest, with the failure to consider the use of inhibitors that may implicate a role for alternative EDH mechanisms. Such behaviour results in bias in favour of one EDH mechanism over others that could otherwise be implicated. This situation is particularly prevalent in studies considering the role of CNP and hydrogen peroxide in EDH.3,16,17 A further ongoing issue with the EDH field (as with many fields) is the lack of specific characterized blockers. Indeed, 1-[(2-chlorophenyl)diphenyl-methyl]-1H pyrazole (TRAM-34), a key IKCa antagonist used in many EDH studies, has recently been suggested to have effects at non-selective cation channels (NSCCs; such as transient receptor potential (TRP) channels).25 Further, Cx-mimetic ‘Gap’ peptides, key blockers of electrically-mediated EDH, have additional effects on intracellular calcium stores.26 This latter effect may be related to the close spatial association of vascular Cxs and sites of calcium modulation, discussed below (Figures 1-4).12 Unfortunately, such observations complicate the interpretation of data in EDH studies.
Spatial and temporal modulation of calcium dynamics is critical for vascular function and anatomical and functional studies suggest an association between sites of calcium release, targets of calcium action and gap junction Cxs.12,13 In rat mesenteric artery, adjacent ECs are coupled by Cxs 37, 40 and 43,27 which are spatially close to densities of SKCa,12 whilst MEGJ Cxs 37 and 40 are spatially associated with IKCa,12,13 potentially corresponding to different facets of the functional EDH response.28 Other data demonstrate a close spatial relationship between Cxs, inositol 1,4,5-trisphosphate receptors (IP3Rs) and endoplasmic reticulum (ER) within EC projections through holes in the IEL, with MEGJs being located between ECs and SMCs on such EC projections. Intense IP3R labeling is present near the endothelial side of MEGJs in rat mesenteric artery (Figures 1,3A-E), consistent with the integral role for IP3 and IP3R in EDH.29,30 Such sites likely reflect IP3-mediated ‘calcium pulsars’ within IEL holes, at putative MEGJ sites, as are present in pressurized mouse mesenteric artery.31,32 The close spatial localization of sites of calcium release and vascular Cxs suggests the potential for a causal functional relationship in that these sites of current transfer and calcium modulation likely interact.
Figure 2. Distribution of intermediate conductance calcium-activated potassium channels (IKCa; red) at the internal elastic lamina (IEL; green) hole (dark spots) to smooth muscle cell interface, as potential myoendothelial gap junction (MEGJ) sites (examples arrowed), in selected resistance arteries. Confocal microscopy using antibodies to IK1 (SK4), developed and characterized by Neylon111 and Chen,110 demonstrate IKCa distribution corresponding to MEGJ density in rat primary mesenteric (A-C; 1o MA), juvenile and adult saphenous (D-F (juv SA), G-I (ad SA), respectively) and caudal cerebellar arteries (J-L; CCA). Approximately 40 and 46% of IEL holes are associated with localized IKCa densities and MEGJ incidence in rat primary mesenteric and caudal cerebellar arteries, respectively, and 46 and 4% of juvenile and adult saphenous arteries, respectively (MEGJ density from published studies.26,33,112 See also Table 6 of Sandow, Gzik & Lee38). Primary antibodies were conjugated to Alexa633 secondary, with immunohistochemistry and controls conducted as per standard.12,13 Endothelial cells are arranged left to right. Bar; 20 μm.
Of note, the relationship between IKCa and MEGJs differs between vascular beds and species (Figure 2A-L), with IKCa density corresponding to the density of MEGJ-related EC projections in some, but not all vascular beds. In rat mesenteric, saphenous and caudal cerebellar artery, IKCa are localized on EC projections (Figure 2A-L), corresponding to MEGJ incidence.26,33,34 Furthermore, such a relationship is present in hamster cheek pouch arteriole, but not rat middle cerebral artery or cremaster arteriole (Sandow, unpublished results). The hamster cheek pouch arteriole exhibits a high density of localized IKCa, consistent with the high MEGJ density in this vessel.35 Of note, whilst MEGJs are relatively common in rat cremaster arteriole and middle cerebral artery36,37 (∼25% of IEL holes have MEGJs in rat middle cerebral artery), IKCa densities at IEL holes are absent and thus not localized to MEGJs in these two vessels. Thus, whilst MEGJs generally occur in a similar location on EC projections which pass through holes in the IEL, not all such holes have EC projections with MEGJs passing through them. In fact, only 0 to ∼50% of such holes have projections with MEGJs, with the variation occurring within and between vessels, species, strains, and disease.38 In general, MEGJ density is decreased with increased vessel size,3 concomitant with the contribution of EDH to vasodilation.3 In this regard, MEGJs are rare to absent in adult male saphenous and femoral artery, respectively, where EDH is absent.33,34 However, even in these vessels, IEL holes still exist, and indeed are prevalent.38 In the absence of heterocellular coupling, these holes may act as low resistance pathways for diffusion of vasoactive substances between ECs and SMCs (or vice versa), or they may signify previous developmental history, or subsequent ageing and disease involving alterations to MEGJs and associated signaling mechanisms.
As outlined above, myoendothelial EC to SMC communication is essential for vasodilator control of arterial diameter and by association, blood flow and pressure. Studies have focused on how ECs regulate the diameter of underlying SMCs via the release diffusible factors such as nitric oxide (NO), and by direct electrical coupling via MEGJs. However, it is becoming evident that the converse pathway; being transfer of chemical and/or electrical signals from SMCs to ECs via MEGJs, may be important for the regulation of arterial diameter in response to stimuli that cause SMC contraction.
Constriction of resistance arteries in response to elevations in intraluminal pressure, stimulation of sympathetic nerves and application of contractile agonists is limited by endothelium-derived NO and EDH (Figure 4).39-41 In addition, changes in vessel diameter occurring when calcium oscillations become synchronized among adjacent SMCs, such as in vasomotion,26,42-45 is suggested to be modulated by NO and/or EDH in many vessels, such as rat mesenteric and basilar artery and hamster aorta.26,42-47 On the other hand, in contrast to this, synchronization of calcium oscillations and vasomotion is suggested to be dependent on EDH, but not NO in rat mesenteric artery,48,49 while yet other studies emphasize an essential role for a cGMP dependent chloride current.43 A question yet to be addressed is how does SMC contraction stimulate production of NO and/or EDH in ECs? - an MEGJ-mediated feedback mechanism being the most likely explanation.12
Generation of both NO and EDH depend on an increase in EC calcium levels.50,51 Agonist-induced SMC contraction, such as that caused by the α-adrenergic agonist phenylephrine, is associated with increased global EC calcium levels in intact arteries, leading to the suggestion that calcium may move from activated SMCs to adjacent ECs.42,52 However, movement of calcium from one cell type to another has yet to be demonstrated directly.
The movement of calcium within cells is slow and spatially restricted,53 which means that it is unlikely that bulk diffusion of this second messenger from SMCs can account for the increase in global EC calcium levels observed following contractile stimulation of intact vessels. Furthermore, discrete calcium changes can regulate signaling pathways independent of global calcium changes, which may occur at the same time.54-56 Thus, the role of the rise in EC bulk calcium levels in stimulating production of NO and/or EDH in response to SMC contraction needs to be defined.
Figure 3. Distribution of inositol 1,4,5-trisphosphate receptor (IP3R; red and Au particles, examples arrowed) and endoplasmic reticulum (ER) at potential myoendothelial gap junction (MEGJ) sites in rat mesenteric artery. Confocal microscopy using antibodies to IP3R (pan, Chemicon, AB1622, A-D; IP3R1, Alomone, ACC019, E), demonstrate IP3R distribution corresponding to MEGJ density (A-C) at the internal elastic lamina (IEL; blue) hole (dark spots, examples arrowed, A-C) to smooth muscle cell (SMC) interface, as potential myoendothelial gap junction sites. Ultrastructural data confirm the localization of IP3R to discrete regions of the endothelial cell (EC) projections (D,E; antibody conjugated to 5 and 10nm Au, respectively; high pressure frozen, freeze-substituted and low temperature embedded tissue12,13,26,113), previously shown to be associated with MEGJ connexins.12,13,26 Localized, but apparently sparse IP3R were also present in the adjacent smooth muscle (E, arrow with asterisk), suggesting sarcoplasmic reticulum (SR) localization. Conventional ultrastructural preparation33 with post-fixation in KFeCN/Os04 (in a similar manner SR staining114) show labeling of apparent ER (F-H, arrowed), consistent with IP3R localization (D,E) at such sites. Regions between arrowheads (F-H; being every second section of a series of sections) are small areas of pentalaminar membrane, consistent with the presence of gap junctions at such sites. No discernible space is present between the endoplasmic reticulum and the point of myoendothelial contact in one section (H; area adjacent to arrowheads). Of note, apparent such ER are only present in ∼14% of MEGJ-like projections (72 examined from 3 vessels, each from a different rat). The likely reason for this is that such structures do not consistently take up the label. For confocal and immunoelectron microscopy, respectively, primary antibodies were conjugated to Alexa633 and 5 and 10nm Au secondaries, and procedures conducted as per standard.12,13,26,113 Bar; A-C, 20 μm; D,F-H, 0.5 μm; D, inset, E, 100 nm.
Of note, the use of agonists that activate a global receptor population to elevate SMC calcium, such as those acting at extrajunctional and junctional α-adrenergic receptors,57 should be treated with caution, as their use may not necessarily reflect a physiological state. Thus, it is more pertinent, although technically more difficult, to examine such responses under conditions of basal myogenic tone, or in response to local application of vasoconstrictors that mimic discrete neurotransmitter release, and not in the presence of globally acting preconstricting agents, such as phenylephrine.
Figure 4. Smooth muscle cell (SMC) to endothelial cell (EC) signaling mechanism in rat mesenteric artery. Contractile agonist (R) activation results in increased SMC calcium and IP3 levels; movement through MEGJs leading to increased EC intracellular calcium ([Ca2+]i) within spatially restricted EC projections. Subsequent IKCa activation hyperpolarizes EC facilitating calcium entry through NSCCs (TRP channels) to refill stores and potentially activate eNOS. Spread of EC hyperpolarization back to SMCs limits further contractile activation. TRPC3 shows apparent localization to the MEGJ-related EC projection (inset lower right panel; Alomone, ACC-016; 1:200, with matching 10 nm Au secondary; arrowheads indicate position of Au label; high pressure frozen, freeze-substituted and low temperature embedded tissue,12,13,26,113 although note that characterization of this antibody has not been conducted. *, MEGJ-related EC projection. Bar; 100 nm.
SMC contraction is associated with membrane depolarization which can spread to ECs via MEGJs in rat basilar artery and juvenile (1-2 mth old) aorta.58,59 This mechanism may play a role in endothelium-dependent coordination of vasomotion,26 but is unlikely to contribute to increased EC calcium, as depolarization would be predicted (depending on its size) to decrease calcium through NSCC action; a major calcium entry route in arterial ECs.60 In support of this prediction, SMC depolarization following inhibition of voltage-dependent K+ channels causes EC depolarization and inhibition of acetylcholine (ACh)-evoked, endothelium-derived NO-mediated relaxation in rat basilar artery.58 As SMC contraction to depolarizing stimuli such as neurotransmitters is limited by generation of endothelium-derived NO and/or EDH, another mechanism must be activated to oppose the transfer of depolarization to ECs during SMC contraction.
In both SMCs and neurons, cellular compartmentalization provides for microdomain-specific activation of ion channels,6,61 although the spatial arrangement and physiological role of such signaling domains in ECs has received little attention. Selective activation of spatially distinct populations of KCa by vasodilators such as ACh in rat mesenteric artery,28,62 ATP and ACh in rat aorta,63 and bradykinin and substance P in porcine coronary artery,64 have been described. However, only recently have immunohistochemical studies provided a structural basis for these observations by revealing the specific localization of KCa subtypes in ECs of intact arteries (Figures 1,2A-L).12,26
Figure 5. Endothelium-dependent modulation of vasoconstriction in rat mesenteric artery is inhibited by TRAM-34. Representative traces of cumulative concentration-response curves to phenylephrine in segments of 3rd order rat mesenteric artery mounted in a wire myograph (A). Contractile responses of an endothelium-intact artery (black), an endothelium-denuded artery (red) and an endothelium-intact artery pre-incubated with TRAM-34 (1 μM; 10 min; an inhibitor of IKCa channels). Mean concentration–response curves for phenylephrine in endothelium-intact and denuded segments of 3rd order rat mesenteric artery (B) in the presence and absence of TRAM-34 (1 μM). TRAM-34 causes endothelium-dependent enhancement of contractile responses to phenylephrine (n=4).
As above, immunohistochemical studies show discrete and intermittent expression of IKCa and nearby IP3R within IEL holes, as potential MEGJ sites, in intact rat mesenteric artery (Figures 1-4). Ultrastructural studies confirm the presence of IP3R within EC projections in this vessel, and demonstrate the presence of ER within ∼14% of EC projections in the same vessels (Figure 3F-H). In a similar manner, Isakson et al. suggest that ER (as endoplasmic and smooth sarcoplasmic reticulum, respectively) is juxtaposed to the plasma membranes of both ECs and SMCs at MEGJs in mouse cremaster arteriole.65
As well as being involved in EDH-mediated signaling, we propose that the distribution of IKCa, IP3R and ER in close spatial association with MEGJ Cxs12,13 provides a mechanism for SMC to EC communication which underlies endothelium-dependent modulation of SMC contraction. The localization of IP3R within EC projections places them in an ideal position to be activated by small amounts of IP3 diffusing through MEGJs from contracting SMCs. The subsequent ER calcium release would then give rise to a local increase in calcium within the spatially restricted EC projections resulting in IKCa activation. Subsequent IKCa-mediated hyperpolarization of EC membrane potential would spread rapidly across and between ECs (via extensive gap junctions at such sites12,27,66) to facilitate calcium entry through NSCCs and also spread through MEGJs back to electrically coupled SMCs to limit further contraction (Figure 4). Further studies are required to determine the precise spatial arrangement of SMC sarcoplasmic reticulum, as a source of IP3, and MEGJs in this model.
Diffusion of IP3 is less spatially restricted than that of calcium53 and a role for this second messenger in SMC to EC communication is supported by observations that pharmacological inhibition of IP3 generation within SMCs or blockade of EC IP3R, prevents the vasoconstrictor-evoked increase in EC bulk calcium in both intact rat mesenteric artery and in mouse aortic EC-SMC co-culture, indicating that diffusion of IP3 through MEGJs may contribute to SMC to EC communication.65,67,68
Central to the smooth muscle to endothelial MEGJ communication model is the hypothesis that a localized increase in calcium at the MEGJ, within restricted EC sites, rather than a global increase in EC calcium, is crucial to SMC to EC communication and to endothelium-dependent modulation of SMC contraction. To date, studies of changes in EC calcium levels within intact arteries have almost exclusively focused on measurement of bulk calcium,42,52,69 a situation resulting mainly from technical limitations, which may hinder elucidation of mechanisms that underlie EC function, and in particular those relating to myoendothelial communication. For example, in contrast to cultured ECs,70-73 measurement of bulk calcium in ECs isolated from rat middle cerebral artery and cremaster arteriole74,75 suggest that agonist-evoked changes in EC calcium are not modulated by EC membrane potential. However, elevations in intracellular calcium, particularly in response to calcium influx, are rarely homogeneous throughout the cell. For instance, invoking store-operated calcium in SMC-derived A7r5 cells produces a modest increase in bulk calcium, while increasing sub-plasmalemmal calcium levels up to 300 fold.76 Furthermore, data suggests that cultured ECs expressing the calcium indicator cameleon, targeted to the plasma membrane, provide evidence for the spatial restriction of changes in calcium at the plasma membrane, rather than an increase in bulk calcium, which may be required for NO production.77 Thus, the use of more sophisticated imaging techniques is required to resolve the importance of localized changes in calcium for EC function.
In support of the proposal that specifically localized changes in calcium are key mediators of EC function, Ledoux et al. recently described spontaneous, IP3-mediated calcium release events (‘calcium pulsars’) within IEL holes, as putative MEGJ projection sites, in pressurized mouse mesenteric artery.31,32 Expression of IP3R and IKCa in MEGJ-related EC projections confirms previous reports of selective localization of these proteins to an area of the cell crucial to SMC to EC communication.12 Furthermore, inhibition of calcium pulsars provides evidence of a functional link between release of calcium from ER stores, the generation of pulsars and IKCa activation. The frequency of calcium pulsars is increased by the endothelium-dependent vasodilator ACh, indicating a potential role for these localized calcium events in EC to SMC signaling, although the effect of SMC contraction on the EC calcium pulsars was not investigated. The frequency of spontaneous local EC calcium events following contractile stimulation of SMCs in rat mesenteric artery is increased,67 although the location of these calcium events was not defined. Thus, the proposal that signaling microdomains at MEGJs provide a novel mechanism by which increased local calcium events link SMC contraction to EC function awaits further investigation. If local calcium events are key to SMC to EC communication then the relationship between these events, activation of IKCa and generation of NO and/or EDH needs further examination.
Functional evidence to support a role for IKCa in SMC to EC communication comes from preliminary studies which show that the IKCa inhibitor TRAM-34 (1 μM) blocks endothelium-dependent modulation of agonist-evoked rat mesenteric artery contraction (Figure 5),78 a vessel in which IKCa are localized to MEGJs12 (Figure 2A-C) and where IKCa are found only on ECs and not on SMCs.12 In contrast, apamin, an inhibitor of SKCa, was without effect on agonist-evoked contraction in endothelium-intact segments of the same vessel.78 Using characterized SK3 antibodies, SKCa are localized to EC-EC junctions, but not to MEGJs in this vessel, suggesting that they may play a different physiological role to IKCa.12 However, as mentioned above, TRAM-34 has been shown to block NSCCs in inflammatory cells25 and thus, further confirmation of IKCa involvement is required.
In addition to IP3R and IKCa, NSCCs are also potential contributors in SMC to EC signaling domains at MEGJs. SKF 96365 and NiCl2, inhibitors of NSCCs, and KBR 7943, an inhibitor of the sodium-calcium exchanger (NCX) reduce spontaneous, localized calcium events within ECs in pressurized rat mesenteric artery, suggesting that Na+ influx through NSCCs leads to activation of the reverse mode of the NCX to cause calcium influx; the process thus being necessary to maintain these events.67 However, interpretation of this finding is complicated by the observation that KBR 7943 can also block store-operated calcium channels79 and TRP channels.80
The molecular identity of EC NSCCs remains to be defined, but these channels are most likely composed of homo- and/or heteromultimers of TRP channels.81 Interestingly, as in cocultured mouse aortic ECs and SMCs,82 preliminary studies suggest that TRPC3 channels are localized to MEGJs in rat mesenteric artery (Figure 4) placing them an ideal position to participate in SMC to EC communication. However, further investigation of their functional role may be limited by the poor selectivity of putative TRP channel blockers and by the expression of these channels on SMCs, and thus molecular approaches such as the use of dominant negative strategies or siRNA may be required. In addition, apparent poor specificity and characterization of a number of the commercially available TRP antibodies currently precludes detailed examination of their distribution (Grayson, Sandow & Hill, unpublished observations).
In response to vasoconstrictor stimuli, SMC to EC communication likely plays an important role modulating changes in arterial diameter, and thus blood flow and pressure. We propose a model in which specific localization of IP3R and IKCa at MEGJs provides a signaling microdomain linking SMC contraction to EC activation,61,62 with the spatial complexity of EC signalling only now becoming apparent. Selective expression of ion channels and receptors at the luminal or abluminal EC surface may confer functional polarity, analogous to that in epithelial cells,83 permitting differential activation of signaling mechanisms by localized changes in calcium from spatially distinct sources, and thus providing an additional level of control of EC function.
Studies of intact vessels in chronically anaesthetized animals in vivo and of the same isolated pressurized vessels in vitro yield apparently contradictory results; being the absence of myoendothelial coupling in vivo and the presence of such coupling in vitro (hamster cheek pouch84,85 and mouse cremaster86,87 arteriole in vivo cf. in vitro, respectively), in spite of the demonstration of MEGJs in these vessels.35,87 This apparently contradictory observation is likely related to anaesthetic use in in vivo studies. Indeed the potential for anaesthetics to produce effects that inhibit specific aspects of dilator and constrictor function has considerable implications for the interpretation of data in many previous studies.
Anaesthetic effects on dilator or constrictor function generally result in an increase or decrease in blood pressure, with the currently poorly characterized etiology of these effects,88 likely being related to differences in anaesthetic action within and between vascular beds. Studies of vessel function in vivo require experimental animals to be chronically anaesthetized and the apparent lack of MEGJ coupling in this state is consistent with anaesthetics having effects on endothelium-dependent vasodilator activity (cf. data for hamster cheek pouch in84,85 and data for mouse cremaster in86,87). Indeed, in vivo use of isoflurane, halothane, ketamine, pentobarbitol and etomidate result in antagonism of NO and EDH.89,90 Under specific conditions, similar antagonism of NO and EDH occurs in vitro, via a mechanism that includes blocking intracellular EC calcium release,91 which underlies the predominant mechanisms of endothelium-dependent vasodilation. Thus, a primary underlying factor having an effect on the apparent myoendothelial coupling derived from in vivo versus in vitro studies, is the type of anaesthetic used in chronic in vivo experiments. Interestingly, in vivo urethane is without apparent effect on endothelium-dependent EDH and NO,90 and further studies are required to clarify the mechanisms that underlie anaesthetic action on endothelial function.
The focal application of ACh to small resistance vessels initiates a vasodilatory response that conducts robustly along the vessel wall.92,93 This so called ‘conducted’ response is present in a range of vascular beds and it begins with the initiation of hyperpolarization and its subsequent spread along ECs.84,94,95 At sites remote to the point of agent application, endothelial hyperpolarization is thought to effect SMC relaxation through one of two mechanisms. First, in similar studies on larger vessels, conducted hyperpolarization augments EC calcium, elevating the production and release of NO or EETs.36,93,96 Such factors would presumably induce smooth muscle relaxation by activating a K+ conductance.36,93,96 However, further studies suggest that paracrine agents are of limited importance in this process, and that remote smooth muscle relaxation reflects direct charge transfer between the two cell layers.94,97,98 While it is difficult to fully summarize the diversity of data and opinion, there is increasing consensus that charge transfer via MEGJs is the principal means of effecting smooth muscle relaxation at remote conducted sites. This consensus reflects findings from a range of studies which have cumulatively shown that: 1) NO synthase and EETs blockers have only a modest effect on conduction;36,93,96 2) MEGJ sites are present in many vascular beds;3,8,98,99 and 3) current can directly pass between the two cell layers.98
As a consequence of the preceding work, current investigations are increasingly examining how other elements of myoendothelial communication could shape the conducted response. One area receiving substantive attention is whether the spread of second messengers, such as calcium or IP3, through MEGJs, is sufficient to influence how electrical signals spread along an arterial wall.85 Duling first raised this idea as a means to explain why depolarizing responses initiated in smooth muscle fail to conduct like their endothelial counterparts.85,100 While an appealing concept, evidence of second messenger flux influencing conduction is limited and incomplete. Indeed, existing studies are noted for their limited presentation of controls and a host of conceptual and theoretical inconsistencies. Particularly problematic is whether limited second messenger flux from a small number of activated SMCs generate sufficient endothelial current to alter membrane potential. Another area of emergence centers on whether the Cx composition or phosphorylation state alters the conducted response.101,102 Testing this concept has proven difficult with the consequences of altered myoendothelial function not being entirely obvious. This is exemplified by work which has tried to link augmented conduction decay, in Cx 40 knockout mice, with either decreased cell coupling in the EC layer or at MEGJs.101,102 While elevated resistance of MEGJs would diminish the ability of the endothelium to drive smooth muscle membrane potential, it should not promote conduction decay.97 Indeed, by limiting charge loss to the smooth muscle, increased myoendothelial resistance would in theory induce the opposing effect. Thus, further work is required in this emerging field.
Heterocellular MEGJ coupling in arteries plays a key role in the maintenance of vascular tone, blood flow and pressure and thus has implications for the etiology of vascular disease.4,14 Potential selective bidirectional MEGJ signaling at local sites, and MEGJ signaling associated with conduction of responses over distance,9,103 are three key processes associated with such coupling, with heterogeneity in MEGJs and associated structures conferring additional functional specialization at these sites. Anatomical and functional studies support the proposition of a close spatial relationship between channels and the distribution of receptor-mediated calcium stores, and the related dynamic functional modulation of calcium release and action, as a key mechanism that underlies heterogeneity in arterial function, and thus represents a selective target for the control of endothelial and vasomotor function. The possibility that differential channel, receptor and store activity may underlie different aspects of MEGJ function, and in particular EDH, is being investigated in other resistance vessels and in disease, such as that associated with diet-induced obesity.14
This study was supported by grants from the National Health and Medical Research Council of Australia (ID 401112 and 455243 to SLS; ID 466009 to REH; ID 471420 to CEH), the Ramaciotti Foundation (to REH) and the Canadian Heart and Stroke Foundation (to FP).
1. Hill CE, Phillips JK, Sandow SL. Heterogeneous control of blood flow amongst different vascular beds Med. Res. Rev. 2001; 21: 1-60.
2. Vanhoutte PM, Feletou M, Taddei S. Endothelium-dependent contractions in hypertension. Br. J. Pharmacol. 2005; 144: 449-58.
3. Sandow SL. Factors, fiction and endothelium-derived hyperpolarizing factor. Clin. Exp. Pharmacol. Physiol. 2004; 31: 563-70.
4. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarizations: past beliefs and present facts. Annals of Medicine 2007; 39: 495-516.
5. Campbell WB, Gauthier KM. What is new in endothelium-derived hyperpolarizing factors? Curr. Opin. Nephrol. Hyperten. Res. 2002; 11: 177-83.
6. Poburko D, Kuo KH, Dai J, Lee CH, van Breemen C. Organellar junctions promote targeted Ca2+ signalling in smooth muscle: why two mechanisms are better than one. Trends Pharmacol. Sci. 2004; 25: 8-15.
7. Oishi H, Budel S, Schuster A, Stergiopulos N, Meister J, Beny J. Cytosolic-free calcium in smooth-muscle and endothelial cells in an intact arterial wall from rat mesenteric artery in vitro. Cell Calcium 2001; 30: 261-7.
8. McGuire JJ, Ding H, Triggle CR. Endothelium-derived relaxing factors: a focus on endothelium-derived hyperpolarizing factor(s). Can. J. Physiol. Pharmacol. 2001; 79: 443-70.
9. de Wit C, Wolfle SE. EDHF and gap junctions: important regulators of vascular tone within the microcirculation. Current Pharm. Biotechnol. 2007; 8: 11-25.
10. Bolton TB, Gordienko DV, Povstyan OV, Pucovsky V. Smooth muscle cells and interstitial cells of blood vessels. Cell Calcium 2004; 35: 643-57.
11. Poburko D, Liao CH, Lemos VS, et al. Transient receptor potential channel 6-mediated, localized cytosolic [Na+] transients drive Na+/Ca2+ exchanger-mediated Ca2+ entry in purinergically stimulated aorta smooth muscle cells. Circ. Res. 2007; 101: 1030-8.
12. Sandow SL, Neylon CB, Chen MX, Garland CJ. Discrete KCa localization in resistance vessels: Implications for endothelium-derived signaling? J. Anat. 2006; 209: 689-98.
13. Mather S, Dora KA, Sandow SL, Winter P, Garland CJ. Rapid endothelial cell-selective loading of connexin 40 antibody blocks EDHF dilation in rat small mesenteric arteries. Circ. Res. 2005; 97: 399-407.
14. Haddock RE, Howitt L, Morris MJ, Sandow SL. Changes in KCa subtype activity may underlie altered endothelium-derived hyperpolarization in diet-induced obese rat mesenteric artery. J. Vasc. Res. 2008; 45 (Suppl. 1): 63.
15. Sandow SL, Tare M. C-type natriuretic peptide: a new endothelium-derived hyperpolarizing factor? Trends Pharmacol. Sci. 2007; 28: 61-7.
16. Ellis A, Triggle CR. Endothelium-dependent reactive oxygen species: their relationship to endothelium-dependent hyperpolarization and the regulation of vascular tone. Can. J. Physiol. Pharmacol. 2003; 81: 1013-28.
17. Leuranguer V, Vanhoutte PM, Verbeuren T, Feletou M. C-type natriuretic peptide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery. Br. J. Pharmacol. 2008; 153: 57-65.
18. Dora KA, Gallagher NT, McNeish A, Garland CJ. Modulation of Endothelial Cell KCa3.1 Channels During Endothelium-Derived Hyperpolarizing Factor Signaling in Mesenteric Resistance Arteries. Circ. Res. 2008; 23:1247-55
19. Popp R, Brandes RP, Ott G, Busse R, Fleming I. Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid. Circ. Res. 2002; 90: 800-6.
20. Koeppen M, Feil R, Siegl D, et al. cGMP-dependent protein kinase mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo. Hypertension 2004; 44:952-5.
21. Ellis A, Goto K, Brackenbury T, Falck JR, Hill CE. Gap junction-dependent and independent mechanisms underlie EDHF dilation in mesenteric arteries from normotensive and hypertensive rats. J. Vasc. Res. 2008; 45: 4.
22. Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature 1998; 396: 269-72.
23. Smith PD, Brett SE, Luykenaar KD, et al. KIR channels function as electrical amplifiers in rat vascular smooth muscle. J. Physiol. 2008; 586: 1147-60.
24. Hill CE. Inward rectification and vascular function: as it was in the beginning. J. Physiol. 2008; 586:1465-7.
25. Schilling T, Eder C. TRAM-34 inhibits nonselective cation channels. Pflügers Arch. 2007; 454: 559-63.
26. Haddock RE, Grayson TH, Brackenbury TD, et al. Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins37 and 40. Am. J. Physiol. 2006; 291: H2047-H56.
27. Goto K, Rummery NM, Grayson TH, Hill CE. Attenuation of conducted vasodilatation in rat mesenteric arteries during hypertension: role of inwardly rectifying potassium channels. J. Physiol. 2004; 561: 215-31.
28. Crane GJ, Gallagher NT, Dora KA, Garland CJ. Small and intermediate calcium-dependent K+ channels provide different facets of endothelium-dependent hyperpolarization in rat mesenteric artery. J. Physiol. 2003; 553: 183-9.
29. Fukao M, Hattori Y, Kanno M, Sakuma I, Kitabatake A. Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. Br. J. Pharmacol. 1997; 120: 1328-34.
30. Cao YX, Zheng JP, He JY, Li J, Xu CB, Edvinsson L. Induces vasodilatation of rat mesenteric artery in vitro mainly by inhibiting receptor-mediated Ca2+-influx and Ca2+-release. Arch. Pharm. Res. 2005; 28:709-15.
31. Ledoux J, Taylor MS, Bonev AD, et al. Ca2+ pulsars: spatially restricted, IP3R-mediated Ca2+ release important for endothelial function. J. Vasc. Res. 2008; 45: 3.
32. Ledoux J, Taylor MS, Bonev AD, et al. Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections. Proc. Natl. Acad. Sci. 2008; 105:doi/10.1073/pnas.0801963105.
33. Sandow SL, Goto K, Rummery N, Hill CE. Developmental changes in myoendothelial gap junctions in the rat saphenous artery. J. Physiol. 2004; 556: 875-86.
34. Sandow SL, Tare M, Coleman HA, Hill CE, Parkington HC. Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor. Circ. Res. 2002; 90: 1108-13.
35. Sandow SL, Looft-Wilson RC, Grayson TH, Segal SS, Hill CE. Expression of homocellular and heterocellular gap junctions in hamster arterioles and feed arteries. Cardiovasc. Res. 2003; 60: 643-53.
36. McSherry IN, Sandow SL, Campbell WB, Falck JR, Hill MA, Dora KA. EDHF-like responses in rat cremaster muscle arterioles involve SKCa and IKCa channels, and Cyp P450 metabolites. Microcirculation 2006; 13: 119-30.
37. McNeish AJ, Sandow SL, Neylon CB, Chen MX, Dora KA, Garland CJ. Evidence for involvement of both IKCa and SKCa channels in hyperpolarizing responses of the rat middle cerebral artery. Stroke 2006; 37: 1277-82.
38. Sandow SL, Gzik DJ, Lee RMKW. Arterial internal elastic lamina holes: relationship to function? J. Anat. 2008; In Press.
39. Scotland RS, Chauhan S, Vallance PJ, Ahluwalia A. An endothelium-derived hyperpolarizing factor-like factor moderates myogenic constriction of mesenteric resistance arteries in the absence of endothelial nitric oxide synthase-derived nitric oxide. Hypertension 2001; 38: 833-9.
40. Ralevic V, Burnstock G. Mesenteric arterial function in the rat in pregnancy: role of sympathetic and sensory-motor perivascular nerves, endothelium, smooth muscle, nitric oxide and prostaglandins. Br. J. Pharmacol. 1996; 117: 1463-70.
41. Murphy TV, Kotecha N, Hill MA. Endothelium-independent constriction of isolated, pressurized arterioles by Nomega-nitro-L-arginine methyl ester (L-NAME). Br. J. Pharmacol. 2007; 151: 602-9.
42. Schuster A, Oishi H, Beny JL, Stergiopulos N, Meister JJ. Simultaneous arterial calcium dynamics and diameter measurements: application to myoendothelial communication. Am. J. Physiol. 2001; 280: H1088-H96.
43. Peng H, Matchkov V, Ivarsen A, Aalkjaer C, Nilsson H. Hypothesis for the initiation of vasomotion. Circ. Res. 2001; 88: 810-5.
44. Mauban JR, Lamont C, Balke CW, Weir WG. Adrenergic stimulation of rat resistance arteries affects Ca2+ sparks, Ca2+ waves, and Ca2+ oscillations. Am. J. Physiol. 2001; 280: H2399-H405.
45. Haddock RE, Hill CE. Differential activation of ion channels by inositol 1,4,5-trisphosphate (IP3)- and ryanodine-sensitive calcium stores in rat basilar artery vasomotion. J. Physiol. 2002; 545: 615-27.
46. Jackson WF, Mulsch A, Busse R. Rhythmic smooth muscle activity in hamster aortas is mediated by continuous release of NO from the endothelium. Am. J. Physiol. 1991; 260: H248-H53.
47. Gustafsson H, Mulvany MJ, Nilsson H. Rhythmic contractions of isolated small arteries from rat: influence of the endothelium. Acta Physiol. Scand. 1993; 148: 153-63.
48. Okazaki K, Seki S, Kanaya N, Hattori J, Tohse N, Namiki A. Role of endothelium-derived hyperpolarizing factor in phenylephrine-induced oscillatory vasomotion in rat small mesenteric artery. Anaesthesiology 2003; 98: 1164-71.
49. Mauban JR, Weir WG. Essential role of EDHF in the initiation and maintenance of adrenergic vasomotion in rat mesenteric arteries. Am. J. Physiol. 2004; 287: H608-H16.
50. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 1991; 43: 109-42.
51. Bryan RM, Jr., You J, Golding EM, Marrelli SP. Endothelium-derived hyperpolarizing factor: a cousin to nitric oxide and prostacyclin. Anesthesiology 2005; 102: 1261-77.
52. Budel S, Schuster A, Stergiopoulos N, Meister JJ, Beny JL. Role of smooth muscle cells on endothelial cell cytosolic free calcium in porcine coronary arteries. Am. J. Physiol. 2001; 281: H1156-H62.
53. Allbritton NL, Meyer T, Stryer L. Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 1992; 258: 1812-5.
54. McCarron JG, Chalmers S, Bradley KN, MacMillan D, Muir TC. Ca2+ microdomains in smooth muscle. Cell Calcium 2006; 40: 461-93.
55. Wang SQ, Wei C, Zhao G, et al. Imaging microdomain Ca2+ in muscle cells. Circ. Res. 2004; 94: 1011-22.
56. Berridge MJ. Calcium microdomains: organization and function. Cell Calcium 2006; 40: 405-12.
57. Hirst GDS, Neild TO. Evidence for two populations of excitatory receptors for noradrenaline on arteriolar smooth muscle. Nature 1980; 283: 767-8.
58. Allen T, Iftinca M, Cole WC, Plane F. Smooth muscle membrane potential modulates endothelium-dependent relaxation of rat basilar artery via myo-endothelial gap junctions. J. Physiol. 2002; 545:975-86.
59. Marchenko SM, Sage SO. Smooth muscle cells affect endothelial membrane potential in rat aorta. Am. J. Physiol. 1994; 267: H804-11.
60. Kohler R, Brakemeier S, Kuhn M, et al. Expression of ryanodine receptor type 3 and TRP channels in endothelial cells: comparison of in situ and cultured human endothelial cells. Cardiovasc. Res. 2001; 51: 160-8.
61. Raymond CR, Redman SJ. Spatial segregation of neuronal calcium signals encodes different forms of LTP in rat hippocampus. J. Physiol. 2006; 570: 97-111.
62. Doughty JM, Plane F, Langton PD. Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium. Am. J. Physiol. 1999; 276: H1107-H12.
63. Marchenko SM. Acetylcholine and ATP hyperpolarize endothelium via activation of different types of Ca2+-activated K+ channels. Bull. Exp. Biol. Med. 2002; 134: 422-4.
64. Frieden M, Sollini M, Beny JL. Substance P and bradykinin activate different types of KCa currents to hyperpolarize cultured porcine coronary artery endothelial cells. J. Physiol. 1999; 519: 361-71.
65. Isakson BE, Ramos SI, Duling BR. Ca2+ and inositol 1,4,5-trisphosphate-mediated signaling across the myoendothelial junction. Circ. Res. 2007; 100: 246-54.
66. Haddock RE, Hill CE. Rhythmicity in arterial smooth muscle. J. Physiol. 2005; 566: 645-56.
67. Kansui Y, Garland CJ, Dora KA. Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries. Cell Calcium 2008; 44: 135-46
68. Lamboley M, Pittet P, Koenigsberger M, Sauser R, Beny JL, Meister JJ. Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger. Cell Calcium 2005; 37: 311-20.
69. Lamboley M, Schuster A, Beny JL, Meister JJ. Recruitment of smooth muscle cells and arterial vasomotion. Am. J. Physiol. 2003; 285: H562-H9.
70. Adams DJ, Barakeh J, Laskey R, Van Breemen C. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB J. 1989; 3: 2389-400.
71. Schilling WP. Effect of membrane potential on cytosolic calcium of bovine aortic endothelial cells. Am. J. Physiol. 1989; 257: H778-84.
72. Luckhoff A, Busse R. Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential. Pflügers Arch. 1990; 416: 305-11.
73. Sheng JZ, Braun AP. Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells. Am. J. Physiol. 2007; 293: C458-67.
74. Cohen KD, Jackson WF. Membrane hyperpolarization is not required for sustained muscarinic agonist-induced increases in intracellular Ca2+ in arteriolar endothelial cells. Microcirculation 2005; 12: 169-82.
75. Marrelli SP, Eckmann MS, Hunte MS. Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations. Am. J. Physiol. 2003; 285: H1590-H9.
76. Marsault R, Murgia M, Pozzan T, Rizzuto R. Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells. EMBO J. 1997; 16: 1575-81.
77. Isshiki M, Mutoh A, Fujita T. Subcortical Ca2+ waves sneaking under the plasma membrane in endothelial cells. Circ. Res. 2004; 95: e11-21.
78. Kerr PM, Sandow SL, Baserman JN, Welsh DG, Cole WC, Plane F. Role of intermediate conductance Ca2+-activated potassium channels in NO-mediated modulation of vasoconstriction in rat mesenteric arteries. J. Vasc. Res. 2008; 45: 53.
79. Arakawa N, Sakaue M, Yokoyama I, et al. KB-R7943 inhibits store-operated Ca2+ entry in cultured neurons and astrocytes. Biochem. Biophys. Res. Commun. 2000; 279: 354-7.
80. Kraft R. The Na+/Ca2+ exchange inhibitor KB-R7943 potently blocks TRPC channels. Biochem. Biophys. Res. Commun. 2007; 361: 230-6.
81. Nilius B, Droogmans G, Wondergem R. Transient receptor potential channels in endothelium: solving the calcium entry puzzle? Endothelium 2003; 10: 5-15.
82. Isakson BE, Duling BR. Organization of IP3-R1 and TRPC3 at the myoendothelial junction may influence polarized calcium signaling. FASEB J. 2006; 20: A634
83. Thorn P. Spatial domains of Ca2+ signaling in secretory epithelial cells. Cell Calcium 1996; 20: 203-14.
84. Welsh DG, Segal SS. Endothelial and smooth muscle cell conduction in arterioles controlling blood flow. Am. J. Physiol. 1998; 274: H178-H86.
85. Yashiro Y, Duling BR. Integrated Ca2+ signaling between smooth muscle and endothelium of resistance vessels. Circ. Res. 2000; 87: 1048-54.
86. Siegl D, Koeppen M, Wolfle SE, Pohl U, de WC. Myoendothelial coupling is not prominent in arterioles within the mouse cremaster microcirculation in vivo. Circ. Res. 2005; 97: 781-8.
87. Potocnik SJ, McSherry IN, Ding H, et al. Endothelium-dependent vasodilatation in myogenically-active mouse skeletal muscle arterioles: Role of EDHF and K+ Channels. Microcirculation 2008; In Press.
88. Boillot A, Haddad E, Vallet B, Barale F. Effects of anesthetic agents on arterial reactivity. Ann. Fr. Anesth. Reanim. 1999; 18: 415-29.
89. Loeb AL, Godeny I, Longnecker DE. Anesthetics alter relative contributions of NO and EDHF in rat cremaster muscle microcirculation. Am. J. Physiol. 1997; 273: H618-H27.
90. de Wit C, Esser N, Lehr HA, Bolz SS, Pohl U. Pentobarbitol-sensitive EDHF comediates ACh-induced arteriolar dilation in the hamster microcirculation. Am. J. Physiol. 1999; 276: H1527-H34.
91. Ogawa K, Tanaka S, Murray PA. Inhibitory effects of etomidate and ketamine on endothelium-dependent relaxation in canine pulmonary artery. Anesthesiology 2001; 94: 668-77.
92. Segal SS, Duling BR. Flow control among microvessels coordinated by intercellular conduction. Science 1986; 234: 868-70.
93. Segal SS, Welsh DG, Kurjiaka DT. Spread of vasodilatation and vasoconstriction along feed arteries and arterioles of hamster skeletal muscle. J. Physiol. 1999; 516: 283-91.
94. Emerson GG, Segal SS. Endothelial cell pathway for conduction of hyperpolarization and vasodilation along hamster feed arteries. Circ. Res. 2000; 86: 94-100.
95. Segal SS, Beny JL. Intracellular recording and dye transfer in arterioles during blood flow. Am. J. Physiol. 1992; 263: H1-H7.
96. Doyle MP, Duling BR. Acetylcholine induces conducted vasodilation by nitric oxide-dependent and -independent mechanisms. Am. J. Physiol. 1997; 272: H1364-71.
97. Diep HK, Vigmond EJ, Segal SS, Welsh DG. Defining electrical communication in skeletal muscle resistance arteries: a computational approach. J. Physiol. 2005; 568: 267-81.
98. Emerson GG, Segal SS. Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries. Role in vasomotor control. Circ. Res. 2000; 87: 474-9.
99. Looft-Wilson RC, Payne GW, Segal SS. Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle. J. Appl. Physiol. 2004; 97: 1152-8.
100. Dora KA, Doyle MP, Duling BR. Elevation of intracellular calcium in smooth muscle causes cell generation of NO in arterioles. Proc. Natl. Acad. Sci. 1997; 94: 6529-34.
101. de Wit C, Roos F, Bolz SS, et al. Impaired conduction of vasodilation along arterioles in connexin40-deficient mice. Circ. Res. 2000; 86: 649-55.
102. Wolfle SE, Schmidt VJ, Hoepfl B, et al. Connexin45 cannot replace the function of connexin40 in conducting endothelium-dependent dilations along arterioles. Circ. Res. 2007; 101: 1292-9.
103. Welsh DG, Segal SS. Role of EDHF in conduction of vasodilation along hamster cheek pouch arterioles in vivo. Am. J. Physiol. 2000; 278: H1832-H9.
104. Kohler R, Heyken WT, Heinau P, et al. Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1495-502.
105. Firth AL, Remillard CV, Yuan JX. TRP channels in hypertension. Biochim. Biophys. Acta 2007; 1772 :895-906.
106. Weston A, Absi M, Harno E, et al. The expression and function of Ca2+-sensing receptors in rat mesenteric artery; comparative studies using a model of type II diabetes. Br. J. Pharmacol. 2008; 154: 652-66.
107. Carter TD, Chen XY, Carlie G, Kalapothakis E, Ogden D, Evans WH. Porcine aortic endothelial gap junctions: identification and permaeation by caged InsP3. J. Cell Sci. 1996; 109: 1765-73.
108. Dhein S, Polontchouk L, Salameh A, Haefliger JA. Pharmacological modulation and differential regulation of the cardiac gap junction proteins connexin 43 and connexin 40. Biol. Cell 2002; 94: 409-22.
109. Sosinsky GE, Nicholson BJ. Structural organization of gap junction channels. Biochim. Biophys. Acta 2005; 1711: 99-125.
110. Chen MX, Gorman SA, Benson B, et al. Small and intermediate conductance Ca2+-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum. Naunyn Schmiedebergs Arch. Pharmacol. 2004; 369: 602-15.
111. Neylon CB, Nurgali K, Humme B, et al. Intermediate-conductance calcium-activated potassium channels in enteric neurones of the mouse: pharmacological, molecular and immunochemical evidence for their role in mediating the slow afterhyperpolarization. J. Neurochem. 2004; 90: 1414-22.
112. Sandow SL, Hill CE. The incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat is suggestive of a role in EDHF-mediated responses. Circ. Res. 2000; 86:341-6.
113. Chen MX, Sandow SL, Doceul V, et al. Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature. BMC. Biotechnol. 2007; 7: 93.
114. Nixon GF, Mignery GA, Somlyo AV. Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle. J. Muscle Res. Cell Motil. 1994; 15: 682-700.