Myoendothelial Gap Junctions Research Articles

WNK kinase, ion channels and arachidonic acid metabolites choreographically execute endothelium-dependent vasodilation

The smooth muscle-walled blood vessels control blood pressure. The vessel lumen is lined by an endothelial cell (ECs) layer, interconnected to the surrounding smooth muscle cells (SMCs) by myoendothelial gap junctions. Gap junctions also maintain homo-cellular ECs-ECs and SMCs-SMCs connections. This gap junction network nearly equalises both cells' membrane potential and cytosolic ionic composition, whether in resting or stimulated conditions. When acetylcholine (ACh) activates ECs M3 receptors, a complex signalling cascade involving second messengers and ion channels is triggered to induce vasodilation.

α acid fraction from Hop extract exerts an endothelium-derived hyperpolarization vasorelaxant effect through TRPV4 employing the feedforward mechanism of PKCα.

Until now, the beneficial vascular properties of Hop reported in the literature have been mainly attributed to specific compound classes, such as tannins and phenolic acids. However, the potential vascular action of a Hop subfraction containing a high amount of α or β acids remains completely understood. Therefore, this study aims to investigate the vascular effects of the entire Hop extract and to fraction the Hop extract to identify the main bioactive vascular compounds. A pressure myograph was used to perform vascular reactivity studies on mouse resistance arteries. Phytocomplex fractionation was performed on a semi-prep HPLC system and characterized by UHPLC-PDA-MS/MS coupled to mass spectrometry. Western blot analysis was performed to characterize the phosphorylation site enrolled. The entire Hop extract exerts a direct dose-dependent endothelial vascular action. The B1 subfraction, containing a high concentration of α acids, recapitulates the vascular effect of the crude extract. Its vasorelaxant action is mediated by the opening of Transient Receptor Potential Vanilloid type 4 (TRPV4), potentiated by PKCα, and subsequent involvement of endothelial small-conductance calcium-activated potassium channels (SKCa) and intermediate-conductance calcium-activated potassium channels (IKCa) that drives endothelium-dependent hyperpolarization (EDH) through heterocellular myoendothelial gap junctions (MEGJs). This is the first comprehensive investigation of the vascular function of Hop-derived α acids in resistance arteries. Overall, our data suggest that the B1 subfraction from Hop extracts, containing only α acids, has great potential to be translated into the useful armamentarium of natural bioactive compounds with cardiovascular benefits.

Cooling-induced cutaneous vasodilatation is mediated by small-conductance, calcium-activated potassium channels in tail arteries from male mice.

Cooling causes cutaneous dilatation to restrain cold-induced constriction and prevent tissue injury. Cooling increases communication through myoendothelial gap junctions (MEGJs), thereby increasing endothelium-derived hyperpolarization (EDH)-type dilatation. EDH is initiated by calcium-activated potassium channels (KCa ) activated by endothelial stimuli or muscle-derived mediators traversing MEGJs (myoendothelial feedback). The goal of this study was to determine the individual roles of KCa with small (SK3) and intermediate (IK1) conductance in cooling-induced dilatation. Vasomotor responses of mice isolated cutaneous tail arteries were analyzed by pressure myography at 37°C and 28°C. Cooling increased acetylcholine-induced EDH-type dilatation during inhibition of NO and prostacyclin production. IK1 inhibition did not affect dilatations to acetylcholine, whereas SK3 inhibition inhibited dilatation at both temperatures. Cooling uncovered myoendothelial feedback to inhibit constrictions in U46619. IK1 inhibition did not affect U46619 constrictions, whereas SK3 inhibition abolished the inhibitory effect of cooling without affecting U46619 constriction at 37°C. Immunoblots confirmed SK3 expression, which was localized (immunofluorescence) to holes in the internal elastic lamina consistent with myoendothelial projections. Immunoblots and Immunofluorescence did not detect IK1. Studies in non-cutaneous arteries have highlighted the predominant role of IK1 in EDH-type dilatation. Cutaneous arteries are distinctly reliant on SK3, which may enable EDH-type dilation to be amplified by cooling.

The Transient Receptor Potential Vanilloid 4 Channel and Cardiovascular Disease Risk Factors.

Vascular endothelial cells regulate arterial tone through the release of nitric oxide and other diffusible factors such as prostacyclin and endothelium derived hyperpolarizing factors. Alongside these diffusible factors, contact-mediated electrical propagation from endothelial cells to smooth muscle cells via myoendothelial gap junctions, termed endothelium-dependent hyperpolarization (EDH), plays a critical role in endothelium-dependent vasodilation in certain vascular beds. A rise in intracellular Ca2+ concentration in endothelial cells is a prerequisite for both the production of diffusible factors and the generation of EDH, and Ca2+ influx through the endothelial transient receptor potential vanilloid 4 (TRPV4) ion channel, a nonselective cation channel of the TRP family, plays a critical role in this process in various vascular beds. Emerging evidence suggests that the dysregulation of endothelial TRPV4 channels underpins endothelial dysfunction associated with cardiovascular disease (CVD) risk factors, including hypertension, obesity, diabetes, and aging. Because endothelial dysfunction is a precursor to CVD, a better understanding of the mechanisms underlying impaired TRPV4 channels could lead to novel therapeutic strategies for CVD prevention. In this mini review, we present the current knowledge of the pathophysiological changes in endothelial TRPV4 channels associated with CVD risk factors, and then explore the underlying mechanisms involved.

18b-glycyrrhetinic acid increases blood pressure in rats through the action on myoendothelial gap junctions

18b-glycyrrhetinic acid (GA), a pentacyclic triterpenoid found in the roots of licorice plants (Glycyrrhiza glabra), posseses high biological activity, including anti-inflammatory and antimicrobial properties. Additionally, it effectively blocks myoendothelial gap junctions. Our experiments on adult Wistar rats have shown that GA administration via gavage in the dose 0.015 g per 100 g of body weight caused a significant increase in arterial blood pressure measured both invasively and non-invasively as early as on the 7th day after beginning of the treatment. Further administration of GA up to 21 days did not change blood pressure significantly. Thoracic aortic rings obtained from GA-treated animals demonstrated a decreased ability to relax in response to acetylcholine (ACh): the maximal dilatory response and the sensitivity of the vascular preparations to ACh measured as pD2 (-log ЕС50) were significantly suppressed compared to the relaxant responses of rings from untreated rats. GA externally applied to intact tissues at a concentration of 2×10-5 M inhibited the ACh-induced relaxation. The inhibition was more pronounced than that observed in vascular rings obtained from the GA-treated animals. Nevertheless, it was smaller than that observed under combined action of the nitric oxide synthase inhibitor, L-NAME, and indomethacin, indicating that GA affects preferentially EDHF-dependent component of the ACh-induced relaxation. These results may suggest that damage of myoendothelial gap junctions that provide electrical communication between the endothelium and the smooth muscle layers may, at least partially, cause the development of arterial hypertension under GA treatment.

Role of crosstalk between endothelial cells and smooth muscle cells in vascular calcification in chronic kidney disease.

Chronic kidney disease (CKD) is a severe health problem worldwide, and vascular calcification (VC) contributes substantially to the cardiovascular morbidity and high mortality of CKD. CKD is often accompanied by a variety of pathophysiological states, such as inflammation, oxidative stress, hyperglycaemia, hyperparathyroidism and haemodynamic derangement, that can cause injuries to smooth muscle cells (SMCs) and endothelial cells (ECs) to promote VC. Similar to SMCs, whose role has been widely explored in VC, ECs may contribute to VC via osteochondral transdifferentiation, apoptosis, etc. In addition, given their location in the innermost layer of the blood vessel lumen and preferential reception of various pro‐calcification stimuli, ECs can pass messages to vascular wall cells and communicate with them. Crosstalk between ECs and SMCs via cytokines through a paracrine mechanism, extracellular vesicles, miRNAs and myoendothelial gap junctions also plays a role in VC. In this review, we emphasize the role of intercellular crosstalk between ECs and SMCs in VC associated with CKD.

Control of Vasoconstriction by Myoendothelial Ca2+ Signaling Through Gap Junctions

In resistance arteries, smooth muscle cells (SMCs) and endothelial cells (ECs) are communicated through gap junctions (i.e. myoendothelial gap junctions), which provide a signaling pathway for control of vasomotor tone. In this context, the Ca2+ signaling associated to smooth muscle contraction might also be transmitted via myoendothelial gap junctions and activate a regulatory feedback mechanism (myoendothelial feedback) by the production of endothelium‐dependent vasodilators. It is thought that IP3 is the main Ca2+‐related signaling transmitted from smooth muscle cells to endothelial cells, but the direct contribution of Ca2+ to the myoendothelial feedback is not clear. We investigated the temporal pattern of vasoconstriction in mesenteric resistance arteries, and their temporal relationship with the increase of Ca2+ in SMC and EC. We used intact mesenteric resistance arteries of Sprague dawley male rats (≤ 10 mm in length) to measure the vasoconstriction and changes in [Ca2+]i of smooth muscle cells and endothelial cells observed in response to phenylephrine (PE) or KCl using the fluorescent indicators of Ca2+, Fluo‐4 and X‐Rhod‐1.Changes in [Ca2+]i were also evaluated in primary cultures of mesenteric endothelial cells. All procedures were approved by the local Bioethical Committee (ID:180806005). The vasoconstriction induced by PE and KCl was not only associated with an increase of [Ca2+]i in smooth muscle cells, but also in endothelial cells, which was prevented by the blockade of L‐type voltage‐dependent Ca2+ channels with 10 μM nifedipine or the inhibition of gap junctions with 50 μM 18‐b‐Glycyrrhetenic acid. Although the stimulation with PE or KCl did not evoke an increment of [Ca2+]i in primary cultures of endothelial cells, the response induced by these vasoconstrictors was larger after blocking nitric oxide (NO) production with NG‐nitro‐L‐arginine (100 μM). These results suggest that the direct diffusion of Ca2+ contributes to the NO‐mediated component of the myoendothelial feedback.Support or Funding InformationConicyt 21171840

Intrinsic regulation of microvascular tone by myoendothelial feedback circuits.

The endothelium is an important regulator of arterial vascular tone, acting to release nitric oxide (NO) and open Ca2+-activated K+ (KCa) channels to relax vascular smooth muscle cells (VSMCs). While agonists acting at endothelial cell (EC) receptors are widely used to assess the ability of the endothelium to reduce vascular tone, the intrinsic EC-dependent mechanisms are less well characterized. In small resistance arteries and arterioles, the presence of heterocellular gap junctions termed myoendothelial gap junctions (MEGJs) allows the passage of not only current, but small molecules including Ca2+ and inositol trisphosphate (IP3). When stimulated to contract, the increase in VSM Ca2+ and IP3 can therefore potentially pass through MEGJs to activate adjacent ECs. This activation releases NO and opens KCa channels, which act to limit contraction. This myoendothelial feedback (MEF) is amplified by EC Ca2+ influx and release pathways, and is dynamically modulated by processes regulating gap junction conductance. There is a remarkable localization of key signaling and regulatory proteins within the EC projection toward VSM, and the intrinsic EC-dependent signaling pathways occurring with this highly specialized microdomain are reviewed.

Increased inward rectifier K+ current of coronary artery smooth muscle cells in spontaneously hypertensive rats; partial compensation of the attenuated endothelium-dependent relaxation via Ca2+ -activated K+ channels.

Endothelium-dependent vasorelaxation is partly mediated by small-conductance (SK3) and intermediate-conductance Ca2+ -activated K+ channels (SK4) in the endothelium that results in endothelium-dependent hyperpolarization (EDH). Apart from the electrical propagation through myoendothelial gap junctions, the K+ released from the endothelium facilitates EDH by increasing inward rectifier K+ channel (Kir) conductance in smooth muscle cells. The EDH-dependent relaxation of coronary artery (CA) and Kir current in smooth muscle cells (CASMCs) of hypertensive animals are poorly understood despite the critical role of coronary flow in the hypertrophic heart. In spontaneously hypertensive (SHR) and control (WKY) rats, we found attenuation of the CA relaxation by activators of SK3 and SK4 (NS309 and 1-EBIO) in SHR. In isolated CASMCs, whole-cell patch-clamp study revealed larger IKir in SHR than WKY, whereas the myocytes of skeletal and cerebral arteries showed smaller IKir in SHR than WKY. While the treatment with IKir inhibitor (0.1mmol/L Ba2+ ) alone did not affect the WKY-CA, the SHR-CA showed significant contractile response, suggesting relaxing influence of the higher IKir in the CASMCs of SHR. Furthermore, the attenuation of NS309-induced relaxation of CA by the combined treatment with 0.1mmol/L Ba2+ was more prominent in SHR than WKY. Our study firstly shows a distinct increase of IKir in the CASMCs of SHR, which could partly compensate for the attenuated relaxation via endothelial SK3 and SK4.

Curcumin vasorelaxation in uterine artery of goat (Capra hircus) is mediated by differential activation of nitric oxide, prostaglandin I2, soluble guanylyl cyclase, and gap junction communication

Background: Curcumin is a principal active constituent of Curcuma longa and has potential therapeutic application in various disease states. In this study, we investigated the mechanism of vasorelaxatory effects of curcumin in middle uterine artery (MUA) of both pregnant (P) and nonpregnant (NP) Capra hircus (Ch). Materials and Methods: The middle uterine arterial rings (MUA) were mounted in an automatic organ bath attached to a PowerLab data acquisition system. We analyzed the effect of curcumin on signaling mediators of endothelium-derived hyperpolarizing factor: nitric oxide (NO), soluble guanylyl cyclase (sGC), prostaglandin I2(PGI2), and myoendothelial gap junctions (MEGJs). Results:The maximal vasorelaxation response to curcumin (1 ρM–100 μM) induced in phenylephrine- precontracted endothelium intact and denuded MUA rings were 42.58%, 25.12% in NP and 55.49%, 12.66%, in P Ch. In the presence of 1H-(1,2,4) oxadiazolo (4,3,-a) quinoxalin-1-one, the maximal curcumin-induced vasorelaxation (CVR) was inhibited to 20.65%, and 15.81% in MUA of NP and P Ch. In the presence of N.-nitro-L-arginine methyl, indomethacin and combination of both CVR were decreased to 20.49%, 12.47%, 12.82% in MUA ring of NP and to 40.60%, 52.55%, 46.53% in MUA ring of P Ch, respectively. The sensitivity of curcumin to MEGJ blockers carbenoxolone, 18β-glycyrrhetinic acid in causing vasorelaxation was 33.18%, 22.61% in MUA rings of NP, and to 15.76%, 18.54% in P Ch. Conclusion: In P Ch, endothelium-dependent vasorelaxation to curcumin is augmented by two-fold as that of MUA of NP. Endothelial vasorelaxation in MUA of NP is mediated through the activation of cyclooxygenase-PGI2-cyclic adenosine monophosphate and endothelial NO synthase (eNOS)-sGC-cyclic guanosine monophosphate (cGMP) signaling with low activation of sGC and MEGJ. In contrast, vasorelaxation to curcumin is mediated through increased activity of sGC and MEGJ with major involvement of eNOS-NO-sGC-cGMP in MUA of P Ch. These studies have strong implications in the therapeutic targeting for hypertensive disorders.

Acidosis potentiates endothelium-dependent vasorelaxation and gap junction communication in the superior mesenteric artery

Extracellular pH is an important physiological determinant of vascular tone that is normally maintained within 7.35–7.45. Any change outside this range leads to severe pathological repercussions. We investigated the unknown effects of extracellular acidosis on relaxation in the superior mesenteric artery (SMA) of goat. SMA rings were employed to maintain isometric contractions at extracellular pH (pHo) 7.4 and 6.8. We analyzed the effect of acidosis (pHo 6.8) compared to physiological pH (pHo 7.4) on three signaling mediators of endothelium-dependent hyperpolarization: nitric oxide (NO), prostaglandin I2 (PGI2), and myoendothelial gap junctions (MEGJ). NO and cyclic guanosine monophosphate (cGMP) levels were compared between normal and acidic pH. Quantitative real-time PCR (qPCR) studies determined the change in expression of vascular connexin (Cx), Cx37, Cx40, and Cx43. Under acidosis, acetyl choline-induced relaxation was augmented in an endothelium-dependent manner via eNOS-NO-cGMP signaling. Conversely, at normal pH, acetyl choline-induced vasorelaxation was mediated primarily via COX-PGI2 pathway. The functional activity of MEGJ was increased under acidosis as evident from increased sensitivity of connexin blockers and upregulated gene and protein expression of connexins. In conclusion, acetyl choline-induced augmented vasorelaxation under acidosis is mediated by NOS-NO-cGMP, with a partial role of MEGJ as EDH mediators in the SMA. Present data suggest a novel role of connexin as therapeutic targets to attenuate the detrimental effect of acidosis on vascular tone.

Myoendothelial coupling through Cx40 contributes to EDH-induced vasodilation in murine renal arteries: evidence from experiments and modelling.

To investigate the role of vascular gap junctions and potassium channels in the renal endothelial derived hyperpolarization. In interlobar arteries from wild-type and connexin40 knockout mice, we assessed the role of calcium-activated small (SK) and intermediate (IK) conductance potassium channels. The role of inward rectifier potassium channels (Kir) and Na+ /K+ -ATPases was evaluated as was the contribution from gap junctions. Mathematical models estimating diffusion of ions and electrical coupling in myoendothelial gap junctions were used to interpret the results. Lack of connexin40 significantly reduces renal endothelial hyperpolarization. Inhibition of SK and IK channels significantly attenuated renal EDH to a similar degree in wild-type and knockout mice. Inhibition of Kir and Na+ /K+ -ATPases affected the response in wild-type and knockout mice but at different levels of stimulation. The model confirms that activation of endothelial SK and IK channels generates a hyperpolarizing current that enters the vascular smooth muscle cells. Also, extracellular potassium increases sufficiently to activate Kir and Na+ /K+ -ATPases. Renal endothelial hyperpolarization is mainly initiated by activation of IK and SK channels. The model shows that hyperpolarization can spread through myoendothelial gap junctions but enough potassium is released to activate Kir and Na+ /K+ -ATPases. Reduced coupling seems to shift the signalling pathway towards release of potassium. However, an alternative pathway also exists and needs to be investigated.

Myoendothelial gap junctions mediate regulation of angiopoietin-2-induced vascular hyporeactivity after hypoxia through connexin 43-gated cAMP transfer.

Angiopoietin-2 (Ang-2) contributes to vascular hyporeactivity after hemorrhagic shock and hypoxia through upregulation of inducible nitric oxide synthase (iNOS) in a vascular endothelial cell (VEC)-specific and Ang-2/Tie2 receptor-dependent manner. While iNOS is primarily expressed in vascular smooth muscle cells (VSMCs), the mechanisms of signal transfer from VECs to VSMCs are unknown. A double-sided coculture model with VECs and VSMCs from Sprague-Dawley rats was used to investigate the role of myoendothelial gap junctions (MEGJs), the connexin (Cx) isoforms involved, and other relevant mechanisms. After hypoxia, VSMCs treated with exogenous Ang-2 showed increased iNOS expression and hyporeactivity, as well as MEGJ formation and communication. These Ang-2 effects were suppressed by the MEGJ inhibitor 18α-glycyrrhetic acid (18-GA), Tie2 siRNA, or Cx43 siRNA. Reagents antagonizing cAMP or protein kinase A (PKA) in VECs inhibited Cx43 expression in MEGJs, decreasing MEGJ formation and associated communication, after hypoxia following Ang-2 treatment. The increased cAMP levels in VSMCs and transfer of Alexa Fluor 488-labeled cAMP from VECs to VSMCs, after hypoxia following Ang-2 treatment, was antagonized by Cx43 siRNA. A cAMP antagonist added to VECs or VSMCs inhibited both increased iNOS expression and hyporeactivity in VSMCs subjected to hypoxia following Ang-2 treatment. Based on these findings, we propose that Cx43 was the Cx isoform involved in MEGJ-mediated VEC-dependent regulation of Ang-2, which induces iNOS protein expression and vascular hyporeactivity after hypoxia. Cx43 was upregulated by cAMP and PKA, permitting cAMP transfer between cells.

Endothelium-Dependent Hyperpolarization and Endothelial Dysfunction

The endothelium controls vascular tone not only by releasing various vasoactive substances but also by another pathway associated with the hyperpolarization of both endothelial and vascular smooth muscle cells and is termed endothelium-dependent hyperpolarization (EDH). These responses involve an increase in the endothelial intracellular Ca concentration by the activation of transient receptor potential channels (predominantly TRPV4) followed by the opening of Ca-activated K channels of small and intermediate conductance (SKCa and IKCa). These channels show a distinct subcellular distribution. SKCa are widely distributed over the plasma membrane but segregates at sites of homocellular endothelial junctions, whereas IKCa are preferentially expressed in the myoendothelial projections. Following KCa activation, smooth muscle hyperpolarization is evoked by electrical coupling through myoendothelial gap junctions and/or by the potassium efflux that subsequently activates smooth muscle Kir2.1 and/or Na/K-ATPase. Alteration of the EDH contributes to the endothelial dysfunctions observed in various pathologies or conversely compensates for the loss in NO bioavailability. A better characterization of EDH should allow determining whether new druggable targets can be identified for the treatment of cardiovascular diseases.

Contribution of K(+) channels to endothelium-derived hypolarization-induced renal vasodilation in rats in vivo and in vitro.

We investigated the mechanisms behind the endothelial-derived hyperpolarization (EDH)-induced renal vasodilation in vivo and in vitro in rats. We assessed the role of Ca(2+)-activated K(+) channels and whether K(+) released from the endothelial cells activates inward rectifier K(+) (Kir) channels and/or the Na(+)/K(+)-ATPase. Also, involvement of renal myoendothelial gap junctions was evaluated in vitro. Isometric tension in rat renal interlobar arteries was measured using a wire myograph. Renal blood flow was measured in isoflurane anesthetized rats. The EDH response was defined as the ACh-induced vasodilation assessed after inhibition of nitric oxide synthase and cyclooxygenase using L-NAME and indomethacin, respectively. After inhibition of small conductance Ca(2+)-activated K(+) channels (SKCa) and intermediate conductance Ca(2+)-activated K(+) channels (IKCa) (by apamin and TRAM-34, respectively), the EDH response in vitro was strongly attenuated whereas the EDH response in vivo was not significantly reduced. Inhibition of Kir channels and Na(+)/K(+)-ATPases (by ouabain and Ba(2+), respectively) significantly attenuated renal vasorelaxation in vitro but did not affect the response in vivo. Inhibition of gap junctions in vitro using carbenoxolone or 18α-glycyrrhetinic acid significantly reduced the endothelial-derived hyperpolarization-induced vasorelaxation. We conclude that SKCa and IKCa channels are important for EDH-induced renal vasorelaxation in vitro. Activation of Kir channels and Na(+)/K(+)-ATPases plays a significant role in the renal vascular EDH response in vitro but not in vivo. The renal EDH response in vivo is complex and may consist of several overlapping mechanisms some of which remain obscure.

Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo

Aging is associated with vascular dysfunction that impairs tissue perfusion, physical activity, and the quality of life. Calcium signaling in endothelial cells (ECs) is integral to vasomotor control, exemplified by localized Ca(2+) signals within EC projections through holes in the internal elastic lamina (IEL). Within these microdomains, endothelium-derived hyperpolarization is integral to smooth muscle cell (SMC) relaxation via coupling through myoendothelial gap junctions. However, the effects of aging on local EC Ca(2+) signals (and thereby signaling between ECs and SMCs) remain unclear, and these events have not been investigated in vivo. Furthermore, it is unknown whether aging affects either the number or the size of IEL holes. In the present study, we tested the hypothesis that local EC Ca(2+) signaling is impaired with advanced age along with a reduction in IEL holes. In anesthetized mice expressing a Ca(2+)-sensitive fluorescent protein (GCaMP2) selectively in ECs, our findings illustrate that for mesenteric arteries controlling splanchnic blood flow the frequency of spontaneous local Ca(2+) signals in ECs was reduced by ∼85% in old (24-26 mo) vs. young (3-6 mo) animals. At the same time, the number (and total area) of holes per square millimeter of IEL was reduced by ∼40%. We suggest that diminished signaling between ECs and SMCs contributes to dysfunction of resistance arteries with advanced age.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/aging-impairs-endothelial-ca2-signaling/.

EDH: endothelium-dependent hyperpolarization and microvascular signalling.

Endothelium-dependent hyperpolarizing factor (EDHF) is a powerful vasodilator influence in small resistance arteries and thus an important modulator of blood pressure and flow. As the name suggests, EDHF was thought to describe a diffusible factor stimulating smooth muscle hyperpolarization (and thus vasodilatation). However, this idea has evolved with the recognition that a factor can operate alongside the spread of hyperpolarizing current from the endothelium to the vascular smooth muscle (VSM). As such, the pathway is now termed endothelium-dependent hyperpolarization (EDH). EDH is activated by an increase in endothelial [Ca2+ ]i , which stimulates two Ca2+ -sensitive K channels, SKCa and IKCa . This was discovered because apamin and charybdotoxin applied in combination blocked EDHF responses, but iberiotoxin - a blocker of BKCa - was not able to substitute for charybdotoxin. SKCa and IKCa channels are arranged in endothelial microdomains, particularly within projections towards the adjacent smooth muscle, which are rich in IKCa channels and close to interendothelial gap junctions where SKCa channels, are prevalent. KCa activation hyperpolarizes endothelial cells, and K+ efflux through them can act as a diffusible 'EDHF' by stimulating VSM Na+ ,K+ -ATPase and inwardly rectifying K channels (KIR ). In parallel, hyperpolarizing current spreads from the endothelium to the smooth muscle through myoendothelial gap junctions located on endothelial projections. The resulting radial EDH is complemented by the spread of 'conducted' hyperpolarization along the endothelium of arteries and arterioles to affect conducted vasodilatation (CVD). Retrograde CVD effectively integrates blood flow within the microcirculation, but how the underlying hyperpolarization is sustained is unclear.

Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress

Background/Aims: Smooth muscle cells may dedifferentiate into the synthetic phenotype and promote atherosclerosis. Here, we explored the role of myoendothelial gap junctions in phenotypic switching of human coronary artery smooth muscle cells (HCASMCs) co-cultured with human coronary artery endothelial cells (HCAECs) exposed to shear stress. Methods: HCASMCs and HCAECs were seeded on opposite sides of Transwell inserts, and HCAECs were exposed to laminar shear stress of 12 dyn/cm² or 5 dyn/cm². The myoendothelial gap junctions were evaluated by using a multi-photon microscope. Results: In co-culture with HCAECs, HCASMCs exhibited a contractile phenotype, and maintained the expression of differentiation markers MHC and H1-calponin. HCASMCs and HCAECs formed functional intercellular junctions, as evidenced by colocalization of connexin(Cx)40 and Cx43 on cellular projections inside the Transwell membrane and biocytin transfer from HCAECs to HCASMCs. Cx40 siRNA and 18-α-GA attenuated protein expression of MHC and H1-calponin in HCASMCs. Shear stress of 5 dyn/cm² increased Cx43 and decreased Cx40 expression in HCAECs, and partly inhibited biocytin transfer from HCAECs to HCASMCs, which could be completely blocked by Cx43 siRNA or restored by Cx40 DNA transfected into HCAECs. The exposure of HCAECs to shear stress of 5 dyn/cm² promoted HCASMC phenotypic switching, manifested by morphological changes, decrease in MHC and H1-calponin expression, and increase in platelet-derived growth factor (PDGF)-BB release, which was partly rescued by Cx43 siRNA or Cx40 DNA or PDGF receptor signaling inhibitor. Conclusions: The exposure of HCAECs to shear stress of 5 dyn/cm² caused the dysfunction of Cx40/Cx43 heterotypic myoendothelial gap junctions, which may be replaced by homotypic Cx43/Cx43 channels, and induced HCASMC transition to the synthetic phenotype associated with the activation of PDGF receptor signaling, which may contribute to shear stress-associated arteriosclerosis.

Endothelium-Derived Hyperpolarization and Coronary Vasodilation: Diverse and Integrated Roles of Epoxyeicosatrienoic Acids, Hydrogen Peroxide, and Gap Junctions.

Myocardial perfusion and coronary vascular resistance are regulated by signaling metabolites released from the local myocardium that act either directly on the VSMC or indirectly via stimulation of the endothelium. A prominent mechanism of vasodilation is EDH of the arteriolar smooth muscle, with EETs and H(2)O(2) playing important roles in EDH in the coronary microcirculation. In some cases, EETs and H(2)O(2) are released as transferable hyperpolarizing factors (EDHFs) that act directly on the VSMCs. By contrast, EETs and H(2)O(2) can also promote endothelial KCa activity secondary to the amplification of extracellular Ca(2+) influx and Ca(2+) mobilization from intracellular stores, respectively. The resulting endothelial hyperpolarization may subsequently conduct to the media via myoendothelial gap junctions or potentially lead to the release of a chemically distinct factor(s). Furthermore, in human isolated coronary arterioles dilator signaling involving EETs and H(2)O(2) may be integrated, being either complimentary or inhibitory depending on the stimulus. With an emphasis on the human coronary microcirculation, this review addresses the diverse and integrated mechanisms by which EETs and H(2)O(2) regulate vessel tone and also examines the hypothesis that myoendothelial microdomain signaling facilitates EDH activity in the human heart.

Activation of endothelial IKCa channels underlies NO-dependent myoendothelial feedback

Agonist-induced vasoconstriction triggers a negative feedback response whereby movement of charged ions through gap junctions and/or release of endothelium-derived (NO) limit further reductions in diameter, a mechanism termed myoendothelial feedback. Recent studies indicate that electrical myoendothelial feedback can be accounted for by flux of inositol trisphosphate (IP3) through myoendothelial gap junctions resulting in localized increases in endothelial Ca2+ to activate intermediate conductance calcium-activated potassium (IKCa) channels, the resultant hyperpolarization then conducting back to the smooth muscle to attenuate agonist-induced depolarization and tone. In the present study we tested the hypothesis that activation of IKCa channels underlies NO-mediated myoendothelial feedback. Functional experiments showed that block of IP3 receptors, IKCa channels, gap junctions and transient receptor potential canonical type-3 (TRPC3) channels caused endothelium-dependent potentiation of agonist-induced increase in tone which was not additive with that caused by inhibition of NO synthase supporting a role for these proteins in NO-mediated myoendothelial feedback. Localized densities of IKCa and TRPC3 channels occurred at the internal elastic lamina/endothelial-smooth muscle interface in rat basilar arteries, potential communication sites between the two cell layers. Smooth muscle depolarization to contractile agonists was accompanied by IKCa channel-mediated endothelial hyperpolarization providing the first demonstration of IKCa channel-mediated hyperpolarization of the endothelium in response to contractile agonists. Inhibition of IKCa channels, gap junctions, TRPC3 channels or NO synthase potentiated smooth muscle depolarization to agonists in a non-additive manner. Together these data indicate that rather being distinct pathways for the modulation of smooth muscle tone, NO and endothelial IKCa channels are involved in an integrated mechanism for the regulation of agonist-induced vasoconstriction.