**4.3 PO2**

*Basic and Clinical Understanding of Microcirculation*

Adenosine has been proposed as being the primary metabolite controlling metabolic regulation of cerebral vascular tone. It is a naturally occurring nucleoside produced as a byproduct of ATP metabolism; thus, its accumulation signals a need for increased blood flow to match the metabolic activity. This relationship with metabolism has widely implicated adenosine in local regulation of cerebral vascular tone during functional hyperemia, ischemia, or whenever PO2 becomes limited [89–91]. Adenosine has direct effects on the vasculature [92, 93] that can both vasodilate and hyperpolarize VSM and is therefore considered an EDHF [94]. There are four distinct subtypes of adenosine receptors; however, the A2A receptor appears to be of high importance in mediating vasodilation [95–97]. The A2A receptor is a purinergic P1 receptor that has been confirmed to be present in cerebral microvessels [98–101] on the VSM cells [102, 103]. It causes dilation in a concentration-dependent manner [101, 104] once bound to the A2A receptor by activation of adenylate cyclase [105] and therefore cAMP [106–108] which reduces cytosolic calcium and leads to vasodilation. The opening of KATP channels also occurs secondary to the increase in cAMP levels as a result of adenosine binding to its receptors on the cell membrane [109–112]. The contribution of opening KATP channels to dilation is likely substantial since during blockade of KATP channels with glibenclamide, adenosine-induced dilation was reduced by approximately 50% [113]. Although A2A receptors are generally considered the most important mediators of the effects of adenosine on vascular tone the A2B receptors are also proposed to cause dilation through similar mechanisms as A2A in addition to coupling to Gq proteins to produce Ca2+ mobilization by activation of phospholipase C and mitogen-activated protein kinase activation [98, 114, 115]. In addition to dilating the cerebral vasculature adenosine may also block vasoconstrictive signals in the parenchyma as evidenced by *in vitro* data from Gordon et al. [116]. When adenosine receptors are blocked with theophylline dilation was attenuated to arterial hypoxia [117]. Similarly the competitive adenosine receptor antagonist aminophylline causes a

20–30% decrease in CBF and cerebral oxygen delivery in normoxia [118].

Similar to many of the other metabolites discussed CO2 tends to increase under conditions of increased metabolism without adequate flow to eliminate it from the area of production and thus its accumulation leads to dilation of the vasculature. High sensitivity to PCO2 is unique to the cerebral circulation [119] causing approximately 3–6% increase and 1–3% decrease in flow per mmHg change in PaCO2 above or below eupnoeic PaCO2 respectively. This high sensitivity is seen throughout the arterial side of the vascular network including the large arteries in the neck [120] and large intracranial arteries [121–123] to the smallest pial arterioles [124] and parenchymal vessels [125–128]. There are likely several redundant mechanistic contributors to the sensitivity of the vasculature to PCO2 which may contribute to the debate as to whether the dilation is triggered by increased PCO2 or rather the accompanying increase H+ concentration from the carbonic anhydrase reaction. There is strong support that the change in PCO2 mediates at least in part alterations in cerebral vascular tone locally by changes in perivascular pH [107, 129–131] as evidenced by acidic and alkaline perfusate administered through an intracranial window. Experiments that have been able to alter pH and PCO2 independently have provided evidence of the dependence of altered pH to initiate a change in cerebral vascular tone [74]. In a cat pial arteriole cranial window preparation, lowering pH along with hypercapnia resulted in no difference in the magnitude of vasodilation when compared to acidic isocapnia [132]. Interestingly, vessel tone was unaltered in response to intraluminal CO2 change,

**4.1 Adenosine**

**28**

**4.2 PCO2**

The effects of oxygen are unique in that its availability is required for aerobic metabolism rather than a byproduct like some of the metabolic factors discussed. Therefore it is not surprising that its abundance leads to vasoconstriction and its relative shortage leads to dilation. Although its availability is tightly linked to that of PCO2 and H+ and other metabolic byproducts in a physiological setting, studies have been able to discern its independent effect in the presence of otherwise constant conditions. Data from isolated arteries/arterioles suggest there is in fact an oxygen sensor independent of other vasoactive metabolic byproducts within the vascular wall itself [137–142]. Once the change in oxygen is sensed there are various mediators of hypoxic dilation including endothelial-derived NO [138, 141], prostanoids [137, 140, 141, 143], 20-HETE [141] and EDHF [139]; however, the contribution of each factor appears to be dependent on the severity of hypoxia [141]. In skeletal muscle, dilation from mild hypoxia (15% O2) was mostly NO-dependent, while moderate (10% O2) was mediated by a combination of increased PGI2 and decreased 20-HETE, and severe (0% O2) was almost entirely accounted for by an increase in PGI2 [141]. In all cases, there appears to be significant involvement by the endothelium to mediate the dilation, which is further supported by a reduction in hypoxic dilation when isolated vessels were exposed to indomethacin (an inhibitor of AA metabolism and thus the production of PGI2) [137, 144, 145] and to a lesser degree by L-NAME (an inhibitor of NO production from NOS) [145]. Therefore, PGI2 is likely a substantial contributor to hypoxic dilation with a lesser but likely still significant role for NO. Human studies measuring CBF with pcMRI, instead of isolated vessel diameter as in the aforementioned studies, with and without administration of L-NAME suggest that hypoxic dilation is highly dependent on NO, with no change from baseline observed in hypoxia when L-NAME was administered [146]. These differences may be because of a species-specific response or differences in *ex vivo* and *in vivo* conditions.

Additional mechanisms of hypoxic dilation include adenosine whose action has previously been discussed. In hyperoxic conditions constriction is favored, mediated by a greater conversion of AA to 20-HETE as opposed to dilators such as PGI2 [147].

#### **4.4 Potassium and neurovascular coupling**

Although K+ is not directly a byproduct of a metabolite pathway it tends to increase in concentration when the frequency of neuronal depolarization is increased, and is therefore indicative of increased metabolism. K+ channels are present in cerebrovascular smooth muscle cells and are important regulators of tone because of their ability to alter membrane potential [148, 149]. Although K+ channels are present through the peripheral vasculature, the cerebral circulation has a unique anatomical feature that allows for intimate interaction of astrocytic endfeet and cerebral vessels. This tight interaction between the astrocytic endfeet and vasculature allows for precise localized changes in blood flow to match the site-specific neural activity in the brain [150, 151]. K+ can therefore be thought of as a direct link between neuronal activity and blood flow. This pairing of neuronal metabolism with appropriate blood flow and is termed neurovascular coupling (NVC). NVC forms the mechanistic basis for neuroimaging techniques that are able to map changes in neuronal activity based on vascular responses such as changes in blood flow or oxygen saturation [152]. As such, an understanding of NVC is not only crucial to understand the regulation of cerebral vascular tone but is also needed to interpret these neuroimaging techniques including functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and near-infrared spectroscopy [153].

NVC is dependent on the interactions of the neurovascular unit which is made up of three major components: the vascular smooth muscle, neuron, and astrocyte glial cell. Somewhat surprisingly, the initiation site of neurovascular coupling is at the level of capillaries which then leads to changes upstream; however, once the close anatomical locations of neurons to the capillaries (8–20 μm) are considered this becomes a logical point of initiation [154]. Not only does NVC rely on interactions apart from the vasculature but it also requires several structural components to facilitate cell-to-cell interactions of these different cell types. These components include gap junctions [155, 156], anchoring proteins [157] and specialized ion channels [158] expressed on cell–cell interface membranes. Neurons initiate NVC by generating direct signals that act on the vasculature and indirect signals that are transmitted through astrocytes and lead to increases in intracellular Ca2+ within the astrocyte. This is achieved by the glutamatergic synaptic activity initiating post-synaptic N-methyl-D-receptors (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The increase in intracellular Ca2+ activates Ca2+-dependent enzymes to produce vasodilators. Some of the enzymes activated include neuronal NOS (nNOS) and cyclooxygenase 2 (COX-2) [159, 160]. Glutamate also acts on metabotropic glutamate receptors in astrocytes increasing Ca2+ in these cells and leading to the production of vasoactive metabolites including adenosine, ATP, and K<sup>+</sup> that act of the VSMCs [161].

Astrocytes are anatomically well positioned to transmit signals from neurons to the vasculature because of their close proximity to the capillaries; however, in a vascular network both the downstream and upstream vasculature must work in concert in order to effectively regulate blood flow delivery. It appears that endothelial cells provide the crucial role of retrograde propagation of the vasomotor response allowing for coordination between up and downstream vasculature. Endothelial cells can produce numerous metabolites including NO, prostanoids, and endothelin to alter VSMCs constriction. Endothelial intermediate K+ channels (KIR) channels and small conductance K+ channels (KSK) have been implicated as a

**31**

*Cerebral Vascular Tone Regulation: Integration and Impact of Disease*

mechanistic contributor to the propagation of the vasodilatory signal upstream for a synchronized hemodynamic response between the capillaries and arterioles via myoendothelial gap junctions [162]. Secondary to the movement of hyperpolar-


inward rectifying potassium channels in the VSMC of cerebral arteries and arteri-

with closed voltage-gated calcium channels and therefore results in the relaxation

tion seen in experimental inhibition of the channels [163]. For example, 6–10 mM

and sustained dilation respectively through mechanisms previously discussed [165].

neighboring VSMC resulting in constriction; this response predominates at high [K+

at work in the VSMC is supported by Em measurement in VSMCs when a solution con-

signals generated by neurons, astrocytes, and endothelial cells must be received and integrated into a final level of tone by the VSMCs. A schematic representation of the impact of neurovascular coupling on cerebral vascular tone is presented in **Figure 1**.

Each component implicated in the regulation of tone has a multitude of signals being produce that is intended to affect a vasomotor response from the VSMCs. Signals from myogenic, shear, metabolic, and neurovascular influences may be additive or opposing in their effect on VSMCs and further combine with one another creating intricate and precise regulation of cerebral vascular tone. It is this intricacy however, that also introduces many possible steps in the pathway for an abnormal response to occur. As such changes to the regulation of tone may form the basis of several pathologies including MetS. The contributing risk factors associated with MetS alter the local regulation of cerebral vascular tone by inducing changes in both the structure and function of the vessels. These risk factors include hypertension, T2DM, and obesity

efflux as previously described. This description of the electrophysiology

] (<20 mM) is applied and produces VSM hyperpolarization, whereas at

concentrations depolarization predominates [166, 172, 178]. Ultimately the

 concentrations cause constriction; however, under normal physiological conditions this concentration is not reached and is only seen under pathological processes such as spreading depression and stroke [169, 170]. This response seen in isolated vessel has also been confirmed in vivo [151]. There is a combination of

for the upstream arterioles to be the effector of the response [161].

 applied to capillaries generated a hyperpolarizing response in endothelial cells which was transmitted upstream to penetrating arterioles hyperpolarizing and relaxing VSMCs [168]. This propagation of the vasodilation signal was blocked with inhibition of KIR with barium or endothelial deletion of KIR1.2 channels, supporting its role transmitting the hyperpolarizing current. Interestingly the capillaries

efflux from KIR and KSK channels generates

electrogenic pump and stimulation of

from VSMC [164–167] in conjunction

cloud" [163] that also hyperpolarizes neighboring

channels in dilating the cerebral vasculature

conductance, depolarization and ultimately constric-

suggesting they act as a sensor detecting the signal

tends to lead to vasodilation, the response

]. In isolated cerebral arterioles low (<7 mM) and

] result in endothelium-independent dilations

that may explain the opposing responses to

]o actually tends to depolarize

/K+

]o would by itself lead to constriction

ATPase [175–177]

].

*DOI: http://dx.doi.org/10.5772/intechopen.90404*

izing current through gap junctions, K<sup>+</sup>

VSMC through both the inducible Na+

of the VSMC. The importance of K+

is supported by reduced K+

themselves did not dilate to K<sup>+</sup>

K+

to K+

Higher K+

promoting K+

taining [K+

higher K+

oles. In both cases, there is a net efflux of K+

Although in a physiological setting K+

may be dependent on the [K+

moderate (8–15 mM) increases in [K+

mechanisms at work in response to K+

**5. Effect of diseased states**

different concentrations. Alone an increase in [K+

At low concentrations, the minor increase in [K+

but it also stimulates both KIR channels [150, 171–174] and Na+

what has been described as a "K<sup>+</sup>

#### *Cerebral Vascular Tone Regulation: Integration and Impact of Disease DOI: http://dx.doi.org/10.5772/intechopen.90404*

*Basic and Clinical Understanding of Microcirculation*

**4.4 Potassium and neurovascular coupling**

neural activity in the brain [150, 151]. K+

Although K+

Additional mechanisms of hypoxic dilation include adenosine whose action has previously been discussed. In hyperoxic conditions constriction is favored, mediated by a greater conversion of AA to 20-HETE as opposed to dilators such as PGI2 [147].

to increase in concentration when the frequency of neuronal depolarization is

because of their ability to alter membrane potential [148, 149]. Although K+

present in cerebrovascular smooth muscle cells and are important regulators of tone

are present through the peripheral vasculature, the cerebral circulation has a unique anatomical feature that allows for intimate interaction of astrocytic endfeet and cerebral vessels. This tight interaction between the astrocytic endfeet and vasculature allows for precise localized changes in blood flow to match the site-specific

between neuronal activity and blood flow. This pairing of neuronal metabolism with appropriate blood flow and is termed neurovascular coupling (NVC). NVC forms the mechanistic basis for neuroimaging techniques that are able to map changes in neuronal activity based on vascular responses such as changes in blood flow or oxygen saturation [152]. As such, an understanding of NVC is not only crucial to understand the regulation of cerebral vascular tone but is also needed to interpret these neuroimaging techniques including functional magnetic resonance imaging (fMRI), positron

NVC is dependent on the interactions of the neurovascular unit which is made up of three major components: the vascular smooth muscle, neuron, and astrocyte glial cell. Somewhat surprisingly, the initiation site of neurovascular coupling is at the level of capillaries which then leads to changes upstream; however, once the close anatomical locations of neurons to the capillaries (8–20 μm) are considered this becomes a logical point of initiation [154]. Not only does NVC rely on interactions apart from the vasculature but it also requires several structural components to facilitate cell-to-cell interactions of these different cell types. These components include gap junctions [155, 156], anchoring proteins [157] and specialized ion channels [158] expressed on cell–cell interface membranes. Neurons initiate NVC by generating direct signals that act on the vasculature and indirect signals that are transmitted through astrocytes and lead to increases in intracellular Ca2+ within the astrocyte. This is achieved by the glutamatergic synaptic activity initiating post-synaptic N-methyl-D-receptors (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The increase in intracellular Ca2+ activates Ca2+-dependent enzymes to produce vasodilators. Some of the enzymes activated include neuronal NOS (nNOS) and cyclooxygenase 2 (COX-2) [159, 160]. Glutamate also acts on metabotropic glutamate receptors in astrocytes increasing Ca2+ in these cells and leading to the production of vasoactive metabolites including

increased, and is therefore indicative of increased metabolism. K+

emission tomography (PET), and near-infrared spectroscopy [153].

that act of the VSMCs [161].

and endothelin to alter VSMCs constriction. Endothelial intermediate K+

Astrocytes are anatomically well positioned to transmit signals from neurons to the vasculature because of their close proximity to the capillaries; however, in a vascular network both the downstream and upstream vasculature must work in concert in order to effectively regulate blood flow delivery. It appears that endothelial cells provide the crucial role of retrograde propagation of the vasomotor response allowing for coordination between up and downstream vasculature. Endothelial cells can produce numerous metabolites including NO, prostanoids,

is not directly a byproduct of a metabolite pathway it tends

channels are

can therefore be thought of as a direct link

channels

channels

channels (KSK) have been implicated as a

**30**

adenosine, ATP, and K<sup>+</sup>

(KIR) channels and small conductance K+

mechanistic contributor to the propagation of the vasodilatory signal upstream for a synchronized hemodynamic response between the capillaries and arterioles via myoendothelial gap junctions [162]. Secondary to the movement of hyperpolarizing current through gap junctions, K<sup>+</sup> efflux from KIR and KSK channels generates what has been described as a "K<sup>+</sup> cloud" [163] that also hyperpolarizes neighboring VSMC through both the inducible Na+ -K+ electrogenic pump and stimulation of inward rectifying potassium channels in the VSMC of cerebral arteries and arterioles. In both cases, there is a net efflux of K+ from VSMC [164–167] in conjunction with closed voltage-gated calcium channels and therefore results in the relaxation of the VSMC. The importance of K+ channels in dilating the cerebral vasculature is supported by reduced K+ conductance, depolarization and ultimately constriction seen in experimental inhibition of the channels [163]. For example, 6–10 mM K+ applied to capillaries generated a hyperpolarizing response in endothelial cells which was transmitted upstream to penetrating arterioles hyperpolarizing and relaxing VSMCs [168]. This propagation of the vasodilation signal was blocked with inhibition of KIR with barium or endothelial deletion of KIR1.2 channels, supporting its role transmitting the hyperpolarizing current. Interestingly the capillaries themselves did not dilate to K<sup>+</sup> suggesting they act as a sensor detecting the signal for the upstream arterioles to be the effector of the response [161].

Although in a physiological setting K+ tends to lead to vasodilation, the response to K+ may be dependent on the [K+ ]. In isolated cerebral arterioles low (<7 mM) and moderate (8–15 mM) increases in [K+ ] result in endothelium-independent dilations and sustained dilation respectively through mechanisms previously discussed [165]. Higher K+ concentrations cause constriction; however, under normal physiological conditions this concentration is not reached and is only seen under pathological processes such as spreading depression and stroke [169, 170]. This response seen in isolated vessel has also been confirmed in vivo [151]. There is a combination of mechanisms at work in response to K+ that may explain the opposing responses to different concentrations. Alone an increase in [K+ ]o actually tends to depolarize neighboring VSMC resulting in constriction; this response predominates at high [K+ ]. At low concentrations, the minor increase in [K+ ]o would by itself lead to constriction but it also stimulates both KIR channels [150, 171–174] and Na+ /K+ ATPase [175–177] promoting K+ efflux as previously described. This description of the electrophysiology at work in the VSMC is supported by Em measurement in VSMCs when a solution containing [K+ ] (<20 mM) is applied and produces VSM hyperpolarization, whereas at higher K+ concentrations depolarization predominates [166, 172, 178]. Ultimately the signals generated by neurons, astrocytes, and endothelial cells must be received and integrated into a final level of tone by the VSMCs. A schematic representation of the impact of neurovascular coupling on cerebral vascular tone is presented in **Figure 1**.
