2.2.2. NO

In contrast to EDH, the role of localized Ca2+ signaling in agonist-evoked NO release has received little attention. NOS, TRPV4 and TRPC3 are located in caveolae microdomains, and deletion of either channel blunts acetylcholine-evoked NO release and NO-mediated relaxation in mouse mesenteric and carotid arteries [75, 99] suggesting they may provide a source of Ca2+ for agonist-driven NOS activation. Heteromultimers of TRPV4-TRPC1 channels mediate vasorelaxation of rabbit mesenteric arteries in response to stimulation of the Ca2+-sensing receptor through NO production [100] but the underlying Ca2+ dynamics were not assessed. A recent study has shown that TRPV4-mediated sparklets underlie ATP driven activation of endothelial NOS in mouse small pulmonary arteries. The resulting NO initiates vasodilation and also guanylyl cyclase-protein kinase G signaling in the endothelium that limits TRPV4 channel cooperativity and serves as a negative feedback signal to regulate TRPV4 channel function [17]. This description of ATP-evoked, spatially distinct TRPV4 sparklets and localized TRPV4-NOS signaling support a novel paradigm that NOS can be activated by spatially restricted Ca2+ signals, and identifies TRPV4 channels as a key regulator of NOS activity in the pulmonary microcirculation.

In contrast, in porcine isolated coronary arteries, substance P increased the occurrence of discrete InsP3-dependent endothelial Ca2+ events in a concentration-dependent manner; low concentrations primarily increased the number of Ca2+ events and at higher concentrations the number of Ca2+ events saturated while the magnitude of individual events increased [12]. This pattern correlated with a greater role for NO in vasorelaxation at lower concentrations suggesting subtle Ca2+ signal expansion at low stimulation levels may preferentially target NOS. A key finding of this study was that idiosyncratic Ca2+ signal expansion corresponded with coronary artery vasorelaxation whereas global changes in [Ca2+]i did not highlighting that frequency modulation of discrete Ca2+ signals is the primary driver of this functional response and that measurement of changes in bulk [Ca2+]i do not adequately describe the Ca2 <sup>+</sup> signaling pathways that underlie endothelium-dependent vasodilation.

#### 2.2.3. Membrane potential and Ca2+ microdomain signaling

Production of NO and stimulation of EDH have long been regarded as separate mechanisms for agonist-evoked vasodilation but several lines of evidence indicate that there may be a facilitatory relationship between endothelial SKCa and IKCa channel activity and NO. SKCa channel activity has been linked to NO-mediated vasodilation to agonists with deletion of these channels causing impaired NO-mediated dilation to acetylcholine in mouse carotid arteries and increased expression enhancing NO-mediated dilation of cremaster arterioles [32]. In rat mesenteric arteries, block of SKCa and IKCa channels reduces agonist-evoked, NOmediated vasorelaxation and NO release [101]. Conversely, activators of endothelial KCa channels can enhance NO release from cultured endothelial cells, enhance ATP-induced increases in cytosolic Ca2+ concentration and NO synthesis in rat cremaster arterioles, and elicit NOmediated relaxation in mesenteric arteries [102–104].

Lacking voltage-operated Ca2+ channels, endothelial Ca2+ influx is mediated by TRP channels and so membrane hyperpolarization may be required to maintain an appropriate electrochemical driving force for agonist-induced Ca2+ influx and also to prevent channel inactivation and/ or reduction in unitary conductance [105, 106]. Membrane depolarization does inhibit both agonist-induced increases in [Ca2+]i and NO release in cultured endothelial cells [107, 108], and in rat isolated basilar arteries, endothelial depolarization was associated with a reduction in NO-mediated relaxation to acetylcholine [109]. Nonetheless, the ability of hyperpolarization to regulate Ca2+ entry by increasing the electrical driving force is controversial. The large concentration gradient (20,000-fold for extracellular versus intracellular) [110] and driving force for Ca2+ entry raising the question of whether a small amplitude hyperpolarization will be insufficient to modulate Ca2+ entry. In rat mesenteric and cerebral arteries, that certainly appeared to be the case as changes in global endothelial [Ca2+]i were independent of changes membrane potential [89, 111]. However, more recent work with endothelial cell tubes isolated from resistance arteries has provided renewed support for hyperpolarization enhancing acetylcholine-evoked Ca2+ influx through TRPV4 [112] and indicate that pharmacological activation of SKCa and IKCa channel may not only enhance Ca2+ entry to further amplify KCa channel activity, but also boost NO production [113]. In mouse mesenteric arteries, acetylcholine-evoked TRPV4-dependent Ca2+ signaling was inhibited in arteries from mice lacking IKCa channels indicating that in these arteries, endothelial stimulation drives sufficient IKCa-dependent Ca2+ entry through TRPV4 to enhance dynamics [13]. IKCa channel activity modestly augmented Ca2+ event amplitude but the most notable impact was in recruiting new Ca2+ firing sites as well as increasing firing frequencies at pre-existing sites. In the same study, increasing or decreasing SKCa expression had little additional effect on the occurrence of Ca2+ events but did promote increased amplitudes and durations indicating that SKCa channels may play a role in positive feedback Ca2+ regulation by shaping the size and time course of individual events. In porcine coronary arteries stimulation of NOS by InsP3-dependent, large amplitude-low frequency Ca2+ waves [12], exactly the types of events which were lost in mesenteric arteries from mice with an endothelial specific knockout of SKCa channels [114], suggests that SKCa channels are required for their development. As mentioned above, deletion of SKCa channels impaired NO-mediated dilation to acetylcholine [32] and together, these findings support the notion that their role in protraction of Ca2+ events may be important in stimulation NOS.

#### 2.3. Myoendothelial feedback

appears that Ca2+ influx through TRP channels is the primary source of Ca2+ for agonist stimulation of endothelial SKCa channels [94, 98]. In mouse cerebral artery, ATP caused rapid trafficking of TRPC3 to the plasma membrane to provide Ca2+ influx to selectively activate SKCa channels to cause EDH [98] (Figure 2). As described earlier, TRPV4 are also associated with caveolae and are a source of Ca2+ for SKCa channel activation in response to increases in shear stress but whether this relationship accounts for engagement of SKCa channels by

In contrast to EDH, the role of localized Ca2+ signaling in agonist-evoked NO release has received little attention. NOS, TRPV4 and TRPC3 are located in caveolae microdomains, and deletion of either channel blunts acetylcholine-evoked NO release and NO-mediated relaxation in mouse mesenteric and carotid arteries [75, 99] suggesting they may provide a source of Ca2+ for agonist-driven NOS activation. Heteromultimers of TRPV4-TRPC1 channels mediate vasorelaxation of rabbit mesenteric arteries in response to stimulation of the Ca2+-sensing receptor through NO production [100] but the underlying Ca2+ dynamics were not assessed. A recent study has shown that TRPV4-mediated sparklets underlie ATP driven activation of endothelial NOS in mouse small pulmonary arteries. The resulting NO initiates vasodilation and also guanylyl cyclase-protein kinase G signaling in the endothelium that limits TRPV4 channel cooperativity and serves as a negative feedback signal to regulate TRPV4 channel function [17]. This description of ATP-evoked, spatially distinct TRPV4 sparklets and localized TRPV4-NOS signaling support a novel paradigm that NOS can be activated by spatially restricted Ca2+ signals, and identifies TRPV4 channels as a key regulator of NOS activity in

In contrast, in porcine isolated coronary arteries, substance P increased the occurrence of discrete InsP3-dependent endothelial Ca2+ events in a concentration-dependent manner; low concentrations primarily increased the number of Ca2+ events and at higher concentrations the number of Ca2+ events saturated while the magnitude of individual events increased [12]. This pattern correlated with a greater role for NO in vasorelaxation at lower concentrations suggesting subtle Ca2+ signal expansion at low stimulation levels may preferentially target NOS. A key finding of this study was that idiosyncratic Ca2+ signal expansion corresponded with coronary artery vasorelaxation whereas global changes in [Ca2+]i did not highlighting that frequency modulation of discrete Ca2+ signals is the primary driver of this functional response and that measurement of changes in bulk [Ca2+]i do not adequately describe the Ca2

Production of NO and stimulation of EDH have long been regarded as separate mechanisms for agonist-evoked vasodilation but several lines of evidence indicate that there may be a facilitatory relationship between endothelial SKCa and IKCa channel activity and NO. SKCa channel activity has been linked to NO-mediated vasodilation to agonists with deletion of these channels causing impaired NO-mediated dilation to acetylcholine in mouse carotid arteries and increased expression enhancing NO-mediated dilation of cremaster arterioles

<sup>+</sup> signaling pathways that underlie endothelium-dependent vasodilation.

2.2.3. Membrane potential and Ca2+ microdomain signaling

agonists has not been explored.

50 Calcium and Signal Transduction

the pulmonary microcirculation.

2.2.2. NO

The sympathetic nervous system regulates total peripheral resistance and is a key modulator of resistance artery diameter through release of noradrenaline and co-transmitters such as ATP and neuropeptide Y [115]. Noradrenaline causes vasoconstriction through activating α1 adrenoceptors on vascular smooth muscle cells, a process which is limited by engagement of endothelial mechanisms through myoendothelial feedback. The current model of myoendothelial feedback involves flux of InsP3 from smooth muscle to endothelial cells to elicit localized increases in Ca2+, activation of IKCa channels and possibly NOS, to limit smooth muscle contractility [11, 91, 116]. This model is supported by ultrastructural and histochemical studies showing that in rat mesenteric and basilar, and hamster retractor feed arteries, MEGJ connexins and IKCa channels are in close spatial association with ER and InsP3 receptors within endothelial projections that extend through the internal elastic lamina to make contact with smooth muscle cells [11, 55, 91, 94]. In hamster retractor feed arteries, myoendothelial feedback is fully accounted for by EDH. The α1-adrenoceptor agonist phenylephrine induced localized, InsP3-mediated Ca2+ signaling events within endothelial projections and block of endothelial IKCa channels enhanced smooth muscle depolarization and vasoconstriction [11]. In rat basilar arteries in which NO makes a major contribution to myoendothelial feedback, smooth muscle depolarization to 5-HT was accompanied by IKCa channel-mediated endothelial hyperpolarization. Inhibition of IKCa channels, gap junctional communication, TRPC3 or NOS potentiated smooth muscle depolarization to 5-HT in a non-additive manner indicating that rather being distinct pathways, NO and endothelial IKCa channel activity are part of an integrated mechanism for the regulation of agonist-induced vasoconstriction [91]. In the latter study, Ca2+ signaling was not investigated and the link between IKCa channel activity and NO production was not defined. However, NOS has now been localized close to MEGJs [117] and in co-cultures stimulation of smooth muscle cells with phenylephrine leads to MEGJ specific NOS phosphorylation within endothelial cells to increase NO [118]. Also, in mouse mesenteric vessels, phenylephrine stimulated endothelial TRPV4 sparklets in an InsP3-dependent manner, to engage SKCa and IKCa channels as well as, to a lesser extent, NOS [17]. Thus, given the ability of IKCa channels to modulate endothelial Ca2+ dynamics [12, 113, 114], it may be proposed that activation of IKCa channels at MEGJs following stimulation of smooth muscle cells by GPCR agonists, may amplify dynamic Ca2+ signals to enhance NO production.

4. Conclusion

Acknowledgements

Conflict of interest

Author details

Ran Wei1

It has become apparent over the past 15 years that endothelial Ca2+ signaling patterns underlie the engagement of effectors such as NOS and/or KCa channels. The physiological significance of these stimulus-specific signaling pathways is not just that they determine the mediator of vasodilation, but also the scope of the impact of each stimulus on blood flow. Stimuli which predominantly elicit release of diffusible mediators will elicit local vasodilation whereas those that initiate EDH have the potential to dilate multiple arterial segments and so affect tissue perfusion. Further work is required to determine if the patterns of Ca2+ signaling described here have widespread applicability, and how they are impacted by age, sex and cardiovascular risk factors. Investigation of how changes in the components of signaling microdomains contribute to the etiology of endothelial dysfunction in conditions such as diabetes and hyper-

The Endothelium: The Vascular Information Exchange http://dx.doi.org/10.5772/intechopen.79897 53

Work in the author's lab is supported by a grant-in-aid from the Heart and Stroke Foundation of Alberta, Nunavut and the North West Territories to F.P. (G130003076). P.M.K. received a RASCAF award from MacEwan University. Stipend support for graduate students was provided by the University of Alberta (QEII scholarships to S.E.L. and S.L.G., Walter H Johns Graduate Fellowship to S.E.L., 75th Anniversary award to R.W., Faculty of Medicine Recruitment Scholarship to S.L.G.), CIHR (Banting and Best Canada Graduate Scholarship, Masters

tension may lead to the identification of new therapeutic targets.

Award to S.E.L.) and AIHS (Doctoral Studentship to S.E.L.).

, Stephen L. Gust<sup>1</sup>

1 Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta,

2 Cardiovascular Research Centre, Faculty of Medicine and Dentistry, University of Alberta,

3 Faculty of Nursing, Robbins Health Learning Centre, MacEwan University, Edmonton,

, Paul M. Kerr<sup>3</sup> and Frances Plane1,2\*

The authors have declared no conflict of interest.

\*Address all correspondence to: fplane@ualberta.ca

, Stephanie E. Lunn<sup>1</sup>

Edmonton, Alberta, Canada

Edmonton, Alberta, Canada

Alberta, Canada

#### 3. Local versus conducted responses

The majority of studies described in this chapter have been conducted on isolated resistance arteries which in in vivo would be part of branching network of resistance vessels supplied by feed arteries in which effective control of blood flow requires coordinated behaviour amongst arterial segments [119]. As described above, diffusible mediators such as NO act locally to increase arterial diameter. In contrast, KCa channel-mediated hyperpolarization leads to both local dilation and conduction of the response through the endothelium for distances of several millimeters. This conduction allows for coordination of changes in arterial diameter in multiple vessel segments and so optimizes blood flow [4, 119, 120]. That is not to say that diffusible mediators do not play a role in global blood flow regulation within vascular beds. A recent study of the vascular bed of the mouse gluteus maximus muscle revealed that NO and EDH provide complementary endothelial pathways for ascending vasodilatation to optimize oxygen delivery to the muscle. EDH of downstream arterioles conducts along the endothelium into proximal feed arteries to cause dilation, and NO is released in response to luminal shear stress which increases secondary to downstream dilatation [120].
